CN111479921B - Methods and compositions for genetically modifying and expanding lymphocytes and modulating their activity - Google Patents

Abstract

The present invention provides methods and compositions for genetically modifying lymphocytes and related methods including genetically modifying T cells and/or NK cells. The methods use replication defective recombinant retroviral particles comprising a pseudotyped component and optionally a membrane bound T cell activating component (such as anti-CD 3) on their surfaces and encoding one or more engineered signaling polypeptides that may include a lymphoproliferative component and/or a Chimeric Antigen Receptor (CAR). The method may comprise contacting the PBMCs with the replication defective recombinant retroviral particles for different exemplary periods of time, such as less than 24 hours or in some illustrative embodiments less than 15 minutes. An illustrative chimeric lymphoproliferative component is provided that is capable of promoting the survival and/or proliferation of T cells or NK cells in culture without the addition of IL-2. In addition, additional regulatory components, such as inhibitory RNA molecules, are provided.

Description

Methods and compositions for genetically modifying and expanding lymphocytes and regulating their activity

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of International Application No. PCT/US2018/020818, filed on March 3, 2018; and claims the benefit of U.S. Provisional Application Nos. 62/560,176, filed on September 18, 2017; 62/564,253, filed on September 27, 2017; 62/564,991, filed on September 28, 2017; and 62/728,056, filed on September 6, 2018; International Application No. PCT/US2018/020818 is a continuation-in-part of International Application No. PCT/US2017/023112, filed on March 19, 2017; International Application No. PCT/ Continuation-in-Part U.S. Application No. US2017/041277; Continuation-in-Part U.S. Application No. 15/462,855 filed on March 19, 2017; and Continuation-in-Part U.S. Application No. 15/644,778 filed on July 8, 2017; and claim the benefit of U.S. Provisional Application No. 62/467,039 filed on March 3, 2017; U.S. Provisional Application No. 62/560,176 filed on September 18, 2017; U.S. Provisional Application No. 62/564,253 filed on September 27, 2017; and U.S. Provisional Application No. 62/564,991 filed on September 28, 2017; International Application No. PCT/US2017/023112 claiming the benefit of U.S. Provisional Application No. 62/564,991 filed on March 28, 2016 No. 62/390,093, filed on March 19, 2017; No. 62/360,041, filed on July 8, 2016; and No. 62/467,039, filed on March 3, 2017; International Application No. PCT/US2017/041277 claims the benefit of International Application No. PCT/US2017/023112, filed on March 19, 2017; U.S. Patent Application No. 15/462,855, filed on March 19, 2017; U.S. Provisional Application No. 62/360,041, filed on July 8, 2016; and U.S. Provisional Application No. 62/467,039, filed on March 3, 2017; U.S. Application No. 15/462,855 claims the benefit of The present invention claims the benefit of U.S. Provisional Application No. 62/390,093, filed on March 19, 2016; U.S. Provisional Application No. 62/360,041, filed on July 8, 2016; and U.S. Provisional Application No. 62/467,039, filed on March 3, 2017; and U.S. Application No. 15/644,778 is a continuation-in-part of International Application No. PCT/US2017/023112, filed on March 19, 2017; and a continuation-in-part of U.S. Patent Application No. 15/462,855, filed on March 19, 2017; and claims the benefit of U.S. Provisional Application No. 62/360,041, filed on July 8, 2016, and U.S. Provisional Application No. 62/467,039, filed on March 3, 2017. These applications are incorporated herein by reference in their entirety.

Sequence Listing

This application hereby incorporates by reference the materials of the electronic sequence listing submitted with this application. The materials in the electronic sequence listing are submitted as a text (.txt) file (with a file size of 529KB) titled "F1_001_TW_04_Sequence_Listing_2018_09_17" created on September 17, 2018, and are incorporated herein by reference in their entirety.

Technical Field

The present invention relates to the field of immunology, or more specifically, to gene modification of T lymphocytes or other immune cells, and methods for preparing replication-deficient recombinant retroviral particles and controlling gene expression therein.

Background technique

Lymphocytes isolated from individuals (e.g., patients) can be activated in vitro and genetically modified to express synthetic proteins that can relocate and bind to other cells and the environment based on the genetic program incorporated. An example of this synthetic protein is a chimeric antigen receptor (CAR). A currently used CAR is a fusion of an extracellular recognition domain (e.g., an antigen binding domain), a transmembrane domain, and one or more intracellular signaling domains encoded by a replication-deficient recombinant retrovirus.

Although recombinant retroviruses have shown efficacy in infecting non-dividing cells, resting CD4 and CD8 lymphocytes are insensitive to gene transduction of these vectors. In order to overcome these difficulties, stimulators are usually used to activate these cells in vitro before genetic modification of CAR gene vectors can occur. After stimulation and transduction, the genetically modified cells are expanded in vitro and then reintroduced into patients with lympholysis. After antigen engagement in vivo, the intracellular signaling portion of CAR can start activation-related reactions in immune cells and release cytolytic molecules to induce tumor cell death.

Such current methods require extensive manipulation and the manufacture of proliferative T cells in vitro before T cells are reinfused into patients, as well as lympho-depleting chemotherapy to release cytokines and consume competitive receptors to facilitate T cell transplantation. Once introduced into the body, such CAR therapies can not control the rate of in vivo propagation, nor can they safely direct targets that are also expressed outside the tumor. Therefore, today's CAR therapy is generally a cell infusion with a dose of 1×10 ⁵ cells/kg to 1×10 ⁸ cells/kg ex vivo amplification for 12 to 28 days, and directed targets (e.g., tumor targets), which are generally acceptable for the CAR therapy to leave the tumor in terms of target toxicity. These relatively long ex vivo amplification times produce cell viability and sterility problems, as well as sample consistency problems in addition to the challenges of adjustability. Therefore, there is a significant need for a safer, more effective and adjustable T cell or NK cell therapy.

Since our understanding of the processes driving transduction, proliferation and survival of lymphocytes is the core of various potential commercial uses involving immune processes, improved methods and compositions are needed for studying lymphocytes. For example, it will help to identify methods and compositions that can be used to better characterize and understand how lymphocytes can be modified by genetic means and factors that affect their survival and proliferation. In addition, it will help to identify compositions that drive lymphocyte proliferation and survival. These compositions can be used to study the regulation of these processes. In addition to the methods and compositions for studying lymphocytes, improved virus packaging cell lines and methods of their manufacture and use are needed. For example, these cell lines and methods will be suitable for analyzing different components of recombinant viruses (such as recombinant retroviral particles), and methods for using packaging cell lines for producing recombinant retroviral particles.

Summary of the invention

Provided herein are methods, compositions and kits that help overcome the effectiveness and safety of transducing and/or genetically modifying lymphocytes (such as T cells and/or NK cells). Certain embodiments of these methods are applicable to performing adoptive cell therapy with these cells. Therefore, in some aspects, provided herein are methods, compositions and kits for genetically modifying and/or transducing lymphocytes (especially T cells and/or NK cells) and/or for regulating transduced and/or genetically modified T cells and/or NK cells. These methods, compositions and kits provide improved efficacy and safety that are superior to current technology, especially with respect to expressing chimeric antigen receptors (CAR) and T cells and/or NK cells that microenvironmentally restrict biological CARs in illustrative embodiments. Transduced and/or genetically modified T cells and/or NK cells produced using the methods provided herein and/or using the methods include, in illustrative embodiments delivered from a retroviral (e.g., lentiviral) genome via retroviral (e.g., lentiviral) particles, functionality and functional combinations that provide improved features for such cells and for methods utilizing such cells (such as research methods, commercial production methods, and adoptive cell therapy). For example, these cells can be generated ex vivo in a shorter time, and the cells have improved growth characteristics that can be better regulated.

In some aspects, there is provided herein a regulatory component for regulating CAR, mRNA, inhibitory RNA, and/or a lymphoproliferative component in lymphocytes (such as B cells, T cells and NK cells), such as a chimeric lymphoproliferative component. In addition, in some aspects, there is provided herein a recombinant retrovirus expressing various functional components and carrying various functional components on its surface, and a method and packaging cell line for producing the recombinant retrovirus. These recombinant retroviruses and methods and cells for producing them overcome the limitations of the prior art on the number and size of genomes, with different functional components that provide benefits when delivered to T cells and/or NK cells.

In some aspects, methods for transducing and/or genetically modifying lymphocytes (such as T cells and/or NK cells) are provided, and in illustrative embodiments, ex vivo methods for transducing and/or genetically modifying resting T cells and/or NK cells are provided. Some of these aspects can be performed more quickly than previous methods, which can contribute to more efficient research, more efficient commercial production, and improved patient care methods. In addition, provided herein are methods for utilizing in some embodiments the recombinant retrovirus and pharmaceutical agents provided herein in some aspects to provide an improved safety mechanism to help regulate the activity of transduced and/or genetically modified lymphocytes (such as T cells and/or NK cells). These methods, compositions, and kits can be used as research tools in commercial production and in adoptive cell therapy with transduced and/or genetically modified T cells and/or NK cells expressing CAR.

Additional details regarding aspects and embodiments of the invention are provided throughout this patent application.Chapters and section headings are for ease of reading and are not intended to limit the combinations of the invention, such as methods, compositions, and kits, or the functional components thereof throughout the sections.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows a schematic diagram of an illustrative composition including a packaging cell (100) and a replication-defective recombinant retroviral particle (200) produced by the packaging cell (100) according to an exemplary, non-limiting embodiment of the present invention. In Figure 1, various vectors capable of encoding aspects of the present invention, referred to as recombinant polynucleotides (110), are packaged into a recombinant retroviral particle (200) that includes in its genome a first engineered signaling polypeptide (which includes one or more lymphoproliferative elements) and in some embodiments a second engineered signaling polypeptide (which is a chimeric antigen receptor or CAR). The replication-deficient recombinant retroviral particles express on their membrane: pseudotype components that allow the replication-deficient recombinant retroviral particles to bind and fuse with target cells (in a non-limiting embodiment, measles virus hemagglutinin (H) polypeptide and measles virus fusion (F) polypeptide, or cytoplasmic domain deletion variants thereof) (240); activation components that can bind and activate resting T cells (in a non-limiting embodiment, activation components having a polypeptide capable of binding to CD28 and a polypeptide capable of binding to CD3) (210 and 220, respectively); and membrane-bound cytokines (in a non-limiting embodiment, IL-7DAF fusion polypeptide) (230). The parts marked as (250), (260), (270), (280) and (290) are Src-FLAG-Vpx, HIV gag matrix, HIV gag capsid, RNA and HIV pol, respectively.

FIG2 shows a schematic diagram of a non-limiting illustrative composition including replication-deficient recombinant retroviral particles (200) produced by packaging cells (100) and resting T cells (300) transfected by the replication-deficient recombinant retroviral particles (200). Components on the surface of the replication-deficient recombinant retroviral particles (200) bind to receptors and/or ligands on the surface of resting T cells. In non-limiting embodiments, the pseudotyped components may include binding polypeptides and fusogenic polypeptides that facilitate the binding and fusion of the replication-deficient recombinant retroviral particles (200) with T cells (in non-limiting embodiments, measles virus hemagglutinin (H) polypeptide and measles virus fusion (F) polypeptide, or cytoplasmic domain deletion variants thereof). In non-limiting embodiments, the replication-deficient recombinant retroviral particles (200) include on their surface an activation component that is capable of activating resting T cells by engaging the T cell receptor complex and optionally an auxiliary receptor (320) (in non-limiting embodiments, an activation component having a polypeptide capable of binding to CD28 and a polypeptide capable of binding to CD3). In addition, the membrane-bound cytokine (in a non-limiting embodiment, IL-7DAF fusion polypeptide) present on the surface of the replication-deficient recombinant retroviral particle (200) binds to IL-7Rα (310) on the surface of the resting T cell. The replication-deficient recombinant retroviral particle (200) fused with the T cell and the polynucleotide encoding the first engineered signaling polypeptide including the lymphoproliferative component (in an illustrative embodiment, the constitutively active IL-7Rα) (370) are reverse transcribed in the cytosol before migrating to the nucleus to be incorporated into the DNA of the activated T cell. Without being limited by theory, in some non-limiting embodiments, the virally packaged Src-FLAG-Vpx (250) enters the cytosol of the resting T cell and promotes the degradation of SAMHD1 (350), thereby increasing the cytoplasmic dNTP pool available for reverse transcription. In some embodiments, the polynucleotide may also encode a second engineered signaling polypeptide including CAR (360). In some embodiments, the lymphoproliferative component is expressed when the compound is bound to a control component that regulates its expression (in a non-limiting example, the control component is a riboswitch that binds a nucleoside analog). In some embodiments, the expression of CAR is also regulated by the control component. Portion (330) is SLAM and CD46. Portion (340) is CD3.

Figures 3A to 3E show schematic diagrams of non-limiting, exemplary vector constructs for transfecting packaging cells to produce replication-defective recombinant retroviral particles described herein. Figure 3A shows a construct containing a polynucleotide sequence encoding an FRB domain fused to the NFκB p65 activation subdomain (p65AD) and a ZFHD1 DNA binding domain fused to three constitutively expressed FKBP repeats. The construct in Figure 3A also includes HIV1 REV and Vpx as a fusion of SrcFlagVpx under the rapamycin-inducible ZFHD1/p65 AD promoter. Figure 3B shows a construct containing a polynucleotide encoding an rtTA sequence under the control of the ZFHD1/p65 AD promoter. Figure 3C shows a construct containing a polynucleotide encoding a puromycin resistance gene flanked by loxP sites and an extracellular MYC marker flanked by lox2272 sites. Both selectable markers are under the control of the BiTRE promoter, which is flanked by FRT sites. FIG. 3D shows a construct containing a polynucleotide encoding RFP flanked by loxP sites under the control of the TRE promoter and a single FRT site between the TRE promoter and the 5' loxP site of RFP. FIG. 3E shows a construct containing a polynucleotide encoding GFP flanked by loxP sites under the control of the TRE promoter and a single FRT site between the TRE promoter and the 5' loxP site of GEP. The constructs in FIG. 3C to FIG. 3E serve as landing points for insertion of other polynucleotide sequences into the genome of the packaging cell line.

Figures 4A to 4C show schematic diagrams of non-limiting, exemplary vector constructs used to transfect packaging cells to produce replication-defective recombinant retroviral particles described herein. Figure 4A shows a construct containing a tricistronic polynucleotide encoding an anti-CD3 (cloned UCHT1) scFvFc with a CD14 GPI anchor attachment site, a CD80 extracellular domain (ECD) capable of binding CD28 to a CD16B GPI anchor attachment site, and IL-7 fused to a decay accelerating factor (DAF) wherein transposon sequences flank the polynucleotide regions for integration into the HEK293S genome. Figure 4B shows a construct containing a polynucleotide with a BiTRE promoter and a polynucleotide region encoding a gag polypeptide and a pol polypeptide in one direction and a polynucleotide region encoding a measles virus FΔx protein and a HΔy protein in the other direction. Fig. 4C shows a construct containing a polynucleotide sequence encoding CAR and a lymphoproliferative component IL7Rα-insPPCL under the control of a CD3Z promoter (which is not active in HEK293S cells), wherein CAR and IL7Rα-insPPCL are separated by a polynucleotide sequence encoding a T2A ribosomal skipping sequence, and IL7Rα-insPPCL has a ribonuclease controlled by an acyclovir riboswitch. The construct containing CAR further includes a cPPT/CTS, an RRE sequence, and a polynucleotide sequence encoding HIV-1Psi (Ψ). The entire polynucleotide sequence on the construct containing CAR to be integrated into the genome is flanked by a FRT site.

FIG. 5 shows a schematic diagram of a lentiviral expression vector encoding GFP, anti-CD19 chimeric antigen receptor, and eTAG (referred to herein as F1-0-03).

Figures 6A and 6B show histograms of the percentage (%) of CD3+GFP+ cells in the total CD3+ population (Figure 6A) and histograms of the absolute cell counts per well of the CD3+GFP+ population (Figure 6B) on days 3, 6, 9, 13, and 17, 14 hours after transduction of freshly isolated and unstimulated PBMCs from donor 12M with VSV-G pseudotyped lentiviral particles encoding F1-0-03 and displaying GPI-anchored anti-CD3scFvFc on their surface as indicated. Each bar represents the mean +/- SD of two replicates.

Figures 7A and 7B show histograms of the percentage (%) of CD3+GFP+ cells in the total CD3+ population on days 3 and 6, 14 hours after transduction of freshly isolated, unstimulated PBMCs from donor 13F with the indicated lentiviral particles encoding F1-0-03 (Figure 7A) and histograms of the absolute cell counts per well of the CD3+GFP+ population (Figure 7B). Please note that "A" shows the results using VSV-G pseudotyped lentiviral particles (three replicates); "B" shows the results using VSV-G pseudotyped lentiviral particles (two replicates) with the addition of OKT3Ab (1 μg/mL) to the transduction medium; "C" shows the results using VSV-G pseudotyped lentiviral particles expressing GPI-anchored UCHT1scFvFc on its surface (three replicates); and "D" shows the results using VSV-G pseudotyped lentiviral particles displaying GPI-anchored UCHT1scFvFc and GPI-anchored CD80 on its surface (two replicates). Each bar represents the mean +/- SD of two or three replicates, as indicated in Figure 7A.

Figures 8A and 8B show histograms of the percentage (%) of CD3+GFP+ cells in the total CD3+ population (Figure 8A) and histograms of the absolute cell counts per well of the CD3+GFP+ population (Figure 8B) on days 3, 6, and 9 after transduction of freshly isolated and unstimulated PBMCs from donor 12M with VSV-G pseudotyped lentiviral particles encoding F1-0-03 and displaying GPI-anchored UCHT1scFvFc and GPI-anchored CD80 on their surface as indicated for the indicated exposure times (2h to 20h). Transductions were performed in plates or shake flasks as indicated. Each bar represents the mean +/- SD of two replicates of lentiviral particles pseudotyped with VSV-G ("VSV-G"); other experiments did not have replicates.

Figures 9A and 9B show a histogram of the percentage (%) of CD3+GFP+ cells in the live CD3+ population (Figure 9A) and a histogram of the absolute cell count per microliter of the total live population (Figure 9B) on day 3 after transduction of freshly isolated and unstimulated PBMCs from donor 18 with the indicated lentiviral particles encoding F1-0-03. Each bar represents the mean +/- SD of two replicates.

Figures 10A and 10B show histograms of the percentage (%) of GFP+ cells in the total viable cell population on day 6 after transduction of unstimulated PBMCS with different replication-defective lentiviral particles at 1 MOI for 4 hours (Figure 10A) and the number of GFP+ cells per microliter of culture (Figure 10B). VSV-G pseudotyped lentiviral particles encode F1-0-03 [VSV-G] and UCHT1 scFvFc-GPI [VSV-G+U] alone or with CD80-GPI [VSV-G+U+CD80] were visualized as indicated. Samples were treated with the antiretroviral drugs dapivirine and dolutegravir as indicated. "Cells only" (untransduced) were included as controls.

Figures 11A and 11B show a histogram of the percentage (%) of FLAG+ cells in the total viable cell population on day 6 after 4 hours of transduction of unstimulated PBMCs by different replication-deficient lentiviral particles (Figure 11A) and the number of FLAG+ cells per microliter of culture (Figure 11B). Lentiviral particles encoded F1-3-219 and were used at an MOI of 1. Lentiviral particles were pseudotyped with VSV-G [VSV-G], BaEV [BaEV], or BaEV in which the fusion inhibitory R peptides [BaEVΔR(HA)] and [BaEVΔR(HAM)] were deleted. Untransduced PBMCs ("cells") were included as controls.

Figures 12A and 12B show a histogram of the percentage (%) of CD3+FLAG+ cells in the total viable cell population on day 6 after 4 hours of transduction of unstimulated PBMCs by different replication-defective lentiviral particles (Figure 12A) and the number of CD3+FLAG+ cells per microliter of culture (Figure 12B). Lentiviral particles encoded F1-3-451 and were used at an MOI of 1. Lentiviral particles were pseudotyped with VSV-G [VSV-G], MuLV [MuLV], or a fusion of UCHT1 scFv and MuLV [U-MuLV]. Lentiviral [VSV-G+U] and [MuLV+U] were pseudotyped with VSV-G and MuLV, respectively, and UCHT1 scFv Fc-GPI was further visualized. Non-transduced PBMCs ("cells") were included as controls.

Figure 13 is a histogram showing the number of CD3+FLAG+ cells per microliter of culture on day 6 after transduction of unstimulated PBMCs with 1 MOI by different replication-deficient lentiviral particles for the indicated periods. F1-3-253 encodes anti-CD19 CAR and F1-3-451 encodes CLE in addition to the same CAR. Lentiviral particles were pseudotyped with VSV-G [VSV-G] and UCHT1ScFvFc-GPI [VSV-G+U] was developed as indicated. Samples were treated with dapline (inhibitor of reverse transcription (RT inb)) or dolutegravir (integration inhibitor (INT Inb)).

Figure 14A provides a schematic diagram of IL7Ra variants tested for lymphoproliferative/survival activity when expressed in PBMCs.Figure 14B provides a bar graph showing the percentage of survivability of PBMCs in the presence and absence of IL-2.

15 is a schematic diagram of a non-limiting exemplary transgenic expression cassette containing a polynucleotide sequence encoding a CAR and candidate CLEs of pools 3A, 3B, 3.1A, and 3.1B.

16 is a schematic diagram of a non-limiting exemplary transgene expression cassette containing polynucleotide sequences encoding CAR and candidate chimeric lymphoproliferative elements (CLEs) of pools 1A, 1.1A, and 1.1B.

FIG. 17 is a schematic diagram of a non-limiting exemplary transgenic expression cassette containing polynucleotide sequences encoding candidate CLEs of libraries 2B and 2.1B.

FIG. 18 is a schematic diagram of a non-limiting exemplary transgenic expression cassette containing polynucleotide sequences encoding candidate CLEs of libraries 4B and 4.1B.

FIG. 19 is a graph showing the fold expansion of PBMCs transduced with lentiviral particles encoding individual CLEs and cultured for 35 days in the absence of exogenous cytokines.

FIG. 20 is a graph showing the fold expansion of PBMCs transduced with lentiviral particles encoding anti-CD19 CAR constructs and individual CLEs and cultured for 35 days in the presence of donor-matched PBMCs but in the absence of exogenous cytokines.

Figure 21 is a graph showing the efficiency of the indicated lentiviral particles in transducing resting PBMCs within 4 hours. In the absence of exogenous cytokines, transduction efficiency was measured as CAR+PBMC% in culture after 6 days, as determined by FACS. Each lentiviral particle encodes CAR and CLE. Lentiviral particles F1-1-228U and F1-3-219U display UCHT1 scFvFc-GPI on their surface.

Figures 22A and 22B are graphs showing the time course of the total number of viable cells after resting PBMCs were transduced with the indicated lentiviral particles for 4 hours and cultured in vitro for 6 days in the absence of exogenous cytokines. Each lentiviral particle encodes CAR and CLE. Lentiviral particles F1-1-228U and F1-3-219U display UCHT1 scFv Fc-GPI on their surface.

Figures 23A, 23B and 23C are graphs showing the time course of lentiviral genome copies per microgram of genomic DNA from the blood of tumor-bearing NSG mice dosed with human PBMCs transduced for 4 hours with the indicated lentiviral particles and injected intravenously without ex vivo expansion of PBMCs. Each lentiviral particle encodes CAR. F1-1-228, F1-1-228U, F1-3-219 and F1-3-219U also encode CLE. Lentiviral particles F1-1-228U and F1-3-219U also display UCHT1 scFvFc-GPI on their surface.

Figure 24 is a graph showing the number of CAR+ cells per 200 μl of blood of tumor-bearing NSG mice dosed with human PBMCs transduced for 4 hours with the indicated lentiviral particles and injected intravenously without ex vivo expansion of PBMCs. Blood was sampled when the mice were euthanized. Each lentiviral particle encodes CAR. F1-1-228, F1-1-228U, F1-3-219, and F1-3-219U also encode CLE. Lentiviral particles F1-1-228U and F1-3-219U also display UCHT1 scFvFc-GPI on their surface.

Figure 25A is a graph showing the average volume of CHO-ROR2 tumors in NSG mice intravenously dosed with PBS or human PBMCs transduced for 4 hours with the indicated lentiviral particles encoding anti-ROR2 MRB CAR and CLE without ex vivo expansion of PBMCs. Figure 25B is a graph showing the average volume of Raji tumors in NSG mice intravenously dosed with PBS or human PBMCs transduced for 4 hours with the indicated lentiviral particles encoding anti-CD19 CAR and CLE without ex vivo expansion of PBMCs. Lentiviral particles F1-1-228U and F1-3-219U display UCHT1 scFvFc-GPI on their surface.

FIG26A is a schematic diagram of the lentiviral vector backbone F1-0-02, which includes a transgene expression cassette driving expression of GFP and eTag and a synthetic EF-1α promoter and intron A upstream of GFP. FIG26B shows the insertion of miRNAs into EF1α intron A of the F1-0-02 backbone. "1" indicates EF1α overlap; "2" indicates 5' arm; "3" indicates miRNA1 5' stem; "4" indicates loop; "5" indicates miRNA1 3' stem; "6" indicates 3' arm; "7" indicates connector; "8" indicates miRNA2 5' stem; "9" indicates miRNA2 3' stem; "10" indicates miRNA3 5' stem; "11" indicates miRNA3 3' stem; "12" indicates miRNA4 5' stem; and "13" indicates miRNA4 3' stem.

FIG. 27 is a graph showing that miRNA targeting CD3ζ located in the intron of the EF-1α promoter is able to block the expression of the CD3 complex.

Figure 28 is a histogram showing the ΔΔCt of samples transduced with miR-TCRα containing replication-defective lentiviral particles. The ΔΔCt values represent the amount of treated miR-TCRa miRNA in each transduced sample relative to the untransduced control.

Definition

As used herein, the term "chimeric antigen receptor" or "CAR" or "CARs" refers to an engineered receptor that transplants antigen specificity to cells, such as T cells, NK cells, macrophages, and stem cells. The CAR of the present invention includes at least one antigen-specific targeting region (ASTR), a transmembrane domain (TM), and an intracellular activation domain (IAD), and may include a handle and one or more co-stimulatory domains (CSD). In another embodiment, CAR is a bispecific CAR specific for two different antigens or antigenic determinants. After ASTR specifically binds to the target antigen, IAD activates intracellular signaling. For example, IAD can reset T cell specificity and reactivity to the selected target in a non-MHC restricted manner, thereby utilizing the antigen binding properties of antibodies. Non-MHC restricted antigen recognition gives CAR-expressing T cells the ability to recognize antigens independently of antigen processing, thereby bypassing the main mechanism of tumor escape. In addition, when expressed in T cells, CAR advantageously does not dimerize with endogenous T cell receptor (TCR) α chain and β chain.

As used herein, the term "microenvironment" means any part or region of a tissue or body that has constant or temporal, physical or chemical differences from other tissue regions or body regions. For example, as used herein, "tumor microenvironment" refers to the environment in which a tumor exists, which is a non-cellular region within the tumor, and the area just outside the tumor tissue does not belong to the intracellular compartment of the cancer cells themselves. The tumor microenvironment can refer to any and all conditions of the tumor environment, including conditions that create a structural and/or functional environment for malignant processes to survive and/or proliferate and/or spread. For example, the tumor microenvironment can include changes in conditions such as, but not limited to, pressure, temperature, pH, ionic strength, osmotic pressure, osmolality, oxidative stress, concentration of one or more solutes, concentration of electrolytes, concentration of glucose, concentration of hyaluronic acid, concentration of lactic acid or lactate, concentration of albumin, level of adenosine, level of R-2-hydroxyglutarate, concentration of pyruvate, concentration of oxygen, and/or the presence of oxidants, reductants or cofactors, as well as other conditions that will be understood by those skilled in the art.

As used interchangeably herein, the term "polynucleotide" and the term "nucleic acid" refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other natural, chemically or biochemically modified non-natural or derived nucleotide bases.

As used herein, the term "antibody" includes polyclonal antibodies and monoclonal antibodies, including intact antibodies and antibody fragments that retain specific binding to an antigen. Antibody fragments may be, but are not limited to, fragment antigen binding fragment (Fab) fragments, Fab' fragments, F(ab') ₂ fragments, Fv fragments, Fab'-SH fragments, (Fab') ₂ Fv fragments, Fd fragments, recombinant IgG (rIgG) fragments, single-chain antibody fragments, including single-chain variable fragments (scFv), bivalent scFv, trivalent scFv and single domain antibody fragments (e.g., sdAb, sdFv, nanobodies). The term includes genetically engineered and/or other modified forms of immunoglobulins, such as intracellular antibodies, peptide antibodies, chimeric antibodies, single-chain antibodies, fully human antibodies, humanized antibodies, fusion proteins including antigen-specific targeting regions of antibodies and non-antibody proteins, heterojunction antibodies, multispecific antibodies (e.g., bispecific antibodies, bifunctional antibodies, trifunctional antibodies and tetrafunctional antibodies), tandem bi-scFvs and tandem tri-scFvs. Unless otherwise stated, the term "antibody" should be understood to include functional antibody fragments thereof. The term also includes intact or full-length antibodies, including antibodies of any class or subclass, including IgG and its subclasses, IgM, IgE, IgA and IgD.

As used herein, the term "antibody fragment" includes a portion of an intact antibody, for example, an antigen binding region or variable region of an intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; bifunctional antibodies; linear antibodies (Zapata et al., Protein Eng. 8(10): 1057-1062 (1995)); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments (called "Fab" fragments, each with a single antigen-binding site) and a residual "Fc" fragment (an indicator of the ability to crystallize easily). Pepsin treatment produces F(ab') ₂ fragments that have two antigen-binding sites and are still able to cross-link antigens.

As used interchangeably herein, the terms "single-chain Fv,""scFv," or "sFv" antibody fragments include the _VH and _VL domains of an antibody, wherein these domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide further includes a polypeptide connector or spacer between the _VH and _VL domains that enables the sFv to form a desired structure for antigen binding. For a review of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994).

As used herein, "naturally occurring" _VH and _VL domains refer to VH and _VL domains that have been isolated from a _host without further molecular evolution to alter their affinity when produced in scFv format under specific conditions, such as those disclosed in U.S. Pat. No. 8,709,755B2 and application WO/2016/033331A1.

As used herein, the term "affinity" refers to the equilibrium constant for the reversible binding of two agents and is expressed as a dissociation constant (Kd). The affinity may be at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, or at least 1000-fold, or more, than the affinity of the antibody for an unrelated amino acid sequence. The affinity of the antibody for the target protein may be, for example, about 100 nanomolars (nM) to about 0.1 nM, about 100 nM to about 1 picomolar (pM), or about 100 nM to about 1 femtomolar (fM) or more. As used herein, the term "avidity" refers to the resistance of a complex of two or more agents to dissociate upon dilution. With respect to antibodies and/or antigen-binding fragments, the terms "immunoreactivity" and "preferential binding" are used interchangeably herein.

As used herein, the term "binding" refers to the direct association between two molecules due to, for example, covalent interactions, electrostatic interactions, hydrophobic interactions, and ionic interactions and/or hydrogen bonding interactions (including interactions such as salt bridges and water bridges). Non-specific binding shall refer to binding with an affinity less than about ^10-7 M, for example, binding with an affinity less than ^10-6 M, ^10-5 M, ^10-4 M, etc.

As used herein, reference to a "cell surface expression system" or "cell surface display system" refers to the display or expression of a protein or portion thereof on the surface of a cell. Typically, cells are generated that express a protein of interest fused to a cell surface protein. For example, a protein is expressed as a fusion protein with a transmembrane domain.

As used herein, the term "component" includes polypeptides (including fusions of polypeptides, regions of polypeptides, and functional mutants or fragments thereof) and polynucleotides (including microRNAs and shRNAs, and functional mutants or fragments thereof).

As used herein, the term "region" is any segment of a polypeptide or polynucleotide.

As used herein, a "domain" is a region of a polypeptide or polynucleotide that has functional and/or structural properties.

As used herein, the term "handle" or "handle domain" refers to a flexible polypeptide linking region that provides structural flexibility and is spaced from the flanking polypeptide regions, and may be composed of natural or synthetic polypeptides. The handle may be derived from the hinge or hinge region of an immunoglobulin (e.g., IgG1), which is generally defined as stretching from Glu216 to Pro230 of human IgG1 (Burton (1985) Molec. Immunol., 22: 161-206). Hinge regions of other IgG isotypes may be aligned with the IgG1 sequence by forming an inter-heavy chain disulfide bond (S-S) at the same position between the first cysteine and the last cysteine. The handle may be naturally occurring or non-naturally occurring, including, but not limited to, an altered hinge region, as disclosed in U.S. Pat. No. 5,677,425. The handle may include a complete hinge region derived from an antibody of any class or subclass. The stalk may also include regions derived from CD8, CD28, or other receptors that provide flexibility and provide similar functions in terms of spacing from the flanking regions.

As used herein, the term "isolated" means that the material is removed from its original environment (e.g., from the natural environment when it occurs in nature). For example, a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide separated from some or all of the coexisting materials in the natural system is isolated. Such a polynucleotide can be part of a vector, and/or such a polynucleotide or polypeptide can be part of a composition and still be isolated because the vector or composition is not part of its natural environment.

As used herein, a "polypeptide" is a single chain of amino acid residues linked by peptide bonds. A polypeptide is neither folded into a fixed structure nor has any post-translational modifications. A "protein" is a polypeptide folded into a fixed structure. "Polypeptide" and "protein" are used interchangeably herein.

As used herein, a polypeptide may be "purified" to remove impure components of the polypeptide's natural environment, e.g., materials that would interfere with the polypeptide's diagnostic or therapeutic use, such as enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. The polypeptide may be (1) purified to greater than 90%, greater than 95%, or greater than 98% by weight of the antibody as determined by the Lowry method, e.g., greater than 99% by weight, (2) purified by means of a spinning cup sequenator to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence, or (3) purified to homogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or non-reducing conditions using Coomassie blue or silver stain.

As used herein, the term "immune cell" generally includes white blood cells (leukocytes) derived from hematopoietic stem cells (HSCs) produced in the bone marrow."Immune cells" include, for example, lymphocytes (T cells, B cells, natural killer (NK) cells) and cells derived from the bone marrow (neutrophils, eosinophils, basophils, monocytes, macrophages, dendritic cells).

As used herein, "T cells" include all types of immune cells that express CD3, including helper T cells (CD4 ⁺ cells), cytotoxic T cells (CD8 ⁺ cells), regulatory T cells (Tregs), and γ-δ T cells.

As used herein, "cytotoxic cells" include CD8 ⁺ T cells, natural killer (NK) cells, NK-T cells, γδ T cells (a subset of CD4 ⁺ cells), and neutrophils (which are cells capable of mediating a cytotoxic response).

As used herein, the term "stem cell" generally includes pluripotent or multipotent stem cells."Stem cell" includes, for example, embryonic stem cells (ES), mesenchymal stem cells (MSC), induced pluripotent stem cells (iPS) and committed progenitor cells (hematopoietic stem cells (HSC), bone marrow-derived cells, etc.).

As used herein, the terms "treatment", "treating" and the like refer to obtaining a desired pharmacological and/or physiological effect. The effect may be preventive, in terms of completely or partially preventing a disease or its symptoms, and/or therapeutic, in terms of partially or completely curing a disease and/or adverse effects caused by a disease. "Treatment" as used herein encompasses any treatment of a disease in a mammal (e.g., in a human), and includes: (a) preventing the occurrence of a disease in an individual susceptible to the disease but not yet diagnosed as having it; (b) inhibiting the disease, that is, arresting its development; and (c) relieving the disease, that is, causing regression of the disease.

As used interchangeably herein, the terms "individual," "subject," "host," and "patient" refer to mammals, including but not limited to humans, murines (e.g., rats, mice), lagomorphs (e.g., rabbits), non-human primates, humans, dogs, cats, ungulates (e.g., horses, cattle, sheep, pigs, goats), and the like.

As used herein, the term "therapeutically effective amount" or "effective amount" refers to the amount of an agent or a combined amount of two agents that, when administered to a mammal or other individual for treatment of a disease, is sufficient to affect such treatment of the disease. The "therapeutically effective amount" will vary depending on the agent, the disease and its severity, and the age, weight, etc., of the individual to be treated.

As used herein, the terms "evolution" and "evolving" refer to the use of one or more mutational methods to generate different polynucleotides encoding otherwise improved polypeptides that are themselves improved biomolecules and/or contribute to the production of another improved biomolecule. "Physiological" or "normal" or "normal physiological" conditions are conditions such as, but not limited to, pressure, temperature, pH, ionic strength, osmotic pressure, osmolar osmotic concentration, oxidative stress, concentration of one or more solutes, concentration of electrolytes, concentration of glucose, concentration of hyaluronic acid, concentration of lactic acid or lactate, concentration of albumin, level of adenosine, level of R-2-hydroxyglutarate, concentration of pyruvate, concentration of oxygen, and/or presence of oxidants, reductants, or cofactors, and other conditions that would be considered to be within the normal range for an individual at the site of administration or at a tissue or organ at the site of action.

As used herein, "genetically modified cells" include cells that contain exogenous nucleic acid, whether or not the exogenous nucleic acid is integrated into the genome of the cell.

As used herein, a "polypeptide" may include a portion of a protein molecule or the entire protein molecule as well as any post-translational or other modifications.

As used herein, pseudotyping components may include: "binding polypeptides", which include one or more polypeptides (usually glycoproteins) that recognize and bind to target host cells; and one or more "fusogenic polypeptides", which mediate the fusion of the retrovirus with the target host cell membrane, thereby allowing the retroviral genome to enter the target host cell. As used herein, "binding polypeptides" may also be referred to as "T cell and/or NK cell binding polypeptides" or "target engagement components", and "fusogenic polypeptides" may also be referred to as "fusogenic components".

"Rest" lymphocytes (such as resting T cells) are lymphocytes in the G0 phase of the cell cycle that do not express activation markers (such as Ki-67). Resting lymphocytes can include naive T cells that have never been exposed to a specific antigen and memory T cells that have been altered by previous exposure to the antigen. "Rest" lymphocytes may also be referred to as "quiescent" lymphocytes.

As used herein, "lympholysis" refers to a method of reducing the number of lymphocytes in an individual (e.g., by administering a lympholysis agent). Partial body or whole body fractionated radiation therapy may also result in lympholysis. A lympholysis agent may be a chemical compound or composition that is capable of reducing the number of functional lymphocytes in a mammal when it is administered to the mammal. An example of such an agent is one or more chemotherapeutic agents. Such agents and dosages are known and may be selected by the treating physician depending on the individual to be treated. Examples of lympholysis agents may include, but are not limited to, fludarabine, cyclophosphamide, cladribine, denileukin diftitox, or a combination thereof.

RNA interference (RNAi) is a biological process in which RNA molecules inhibit gene expression or translation by neutralizing a target RNA molecule. The RNA target may be mRNA, or it may be any other RNA that is sensitive to the functional inhibition of RNAi. As used herein, "inhibitory RNA molecules" refer to RNA molecules whose presence produces RNAi in cells and causes the expression of transcripts targeted by the inhibitory RNA molecules to be reduced. Inhibitory RNA molecules as used herein have 5' stems and 3' stems that can form RNA double helices. Inhibitory RNA molecules may be, for example, miRNA (endogenous or artificial) or shRNA, a precursor of miRNA (i.e., Pri-miRNA or pre-miRNA) or shRNA, or as dsRNA that is directly transcribed or introduced into a cell or individual through an isolated nucleic acid.

As used herein, "double-stranded RNA" or "dsRNA" or "RNA duplex" refers to an RNA molecule consisting of two chains. Double-stranded molecules include those consisting of two RNA chains that hybridize to form a double-stranded RNA structure, or single-stranded RNA that folds itself to form a double-stranded structure. Most, but not necessarily, all bases in the double-stranded region are base-paired. The double-stranded region includes a sequence complementary to the target RNA. The sequence complementary to the target RNA is an antisense sequence, and is generally 18 to 29, 19 to 29, 19 to 21, or 25 to 28 nucleotides long, or in some embodiments, 18, 19, 20, 21, 22, 23, 24, 25 on the low end and 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 on the high end, wherein a given range generally has a low end lower than the high end. Such structures generally include a 5' stem, a loop, and a 3' stem connected by a loop adjacent to each stem (which is not part of the double helix). In certain embodiments, the loop comprises at least 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides. In other embodiments, the loop comprises 2 to 40, 3 to 40, 3 to 21 or 19 to 21 nucleotides; or in some embodiments, between 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 on the low end and 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35 or 40 on the high end, wherein a given range typically has a lower low end than a higher high end.

As used herein, the term "microRNA flanking sequence" refers to a nucleotide sequence comprising a microRNA processing component. The microRNA processing component is a minimal nucleic acid sequence that helps to produce a mature microRNA from a precursor microRNA. These components are typically located within 40 nucleotide sequences flanking the microRNA stem-loop structure. In some cases, the microRNA processing component is found within an extension of a nucleotide sequence flanking the microRNA stem-loop structure in a length between 5 and 4,000 nucleotides.

The term "linker" when used in reference to multiple inhibitory RNA molecules refers to a connecting component that joins two inhibitory RNA molecules.

As used herein, "recombinant retrovirus" refers to a non-replication or "replication-defective" retrovirus, unless it is explicitly indicated as a replication-competent retrovirus. The terms "recombinant retrovirus" and "recombinant retroviral particle" are used interchangeably herein. Such retroviruses/retroviral particles can be any type of retroviral particles, including, for example, gamma retroviruses and, in illustrative embodiments, lentiviruses. As is well known, such retroviral particles (e.g., lentiviral particles) are typically formed in packaging cells by transfecting the packaging cells with plasmids (which include packaging components such as Gag, Pol, and Rev) and envelope or pseudotyped plasmids (which encode pseudotyped components) and transfer, genomic or retroviral (e.g., lentiviral) expression vectors (which are typically plasmids encoding genes or other relevant coding sequences thereon). Thus, retroviral (e.g., lentiviral) expression vectors include sequences that facilitate expression and packaging after transfection into cells (e.g., 5'LTR and 3'LTR flanking, for example, psi packaging components and target heterologous coding sequences). The terms "lentivirus" and "lentiviral particle" are used interchangeably herein.

The "framework" of a miRNA consists of a "5' microRNA flanking sequence" and/or a "3' microRNA flanking sequence" surrounding the miRNA and (in some cases) a loop sequence separating the stem of a stem-loop structure in the miRNA. In some examples, the "framework" is derived from a naturally occurring miRNA, such as miR-155. The terms "5' microRNA flanking sequence" and "5' arm" are used interchangeably herein. The terms "3' microRNA flanking sequence" and "3' arm" are used interchangeably herein.

As used herein, the term "miRNA precursor" refers to an RNA molecule of any length that can be enzymatically processed into a miRNA, such as a primary RNA transcript, pri-miRNA, or pre-miRNA.

As used herein, the term "construct" refers to an isolated polypeptide or an isolated polynucleotide encoding a polypeptide. A polynucleotide construct may encode a polypeptide, such as a lymphoproliferative component. A skilled artisan will understand that whether a construct refers to an isolated polynucleotide or an isolated polypeptide depends on the context.

It should be understood that the present invention and the aspects and embodiments provided herein are not limited to the specific examples disclosed, and therefore, of course, variations are possible. It should also be understood that the techniques used herein are only for the purpose of disclosing specific examples and embodiments, and are not intended to be limiting, as the scope of the present invention will only be limited by the appended claims.

When providing a range of values, it is understood that each intermediate value between the upper and lower limits of the range (unless the context clearly indicates otherwise, one-tenth of the lower limit unit) and any other value or intermediate value in the stated range are included in the present invention. The upper and lower limits of these smaller ranges can be independently included in the smaller ranges, and are also included in the present invention, except for any specific excluded limits in the stated range. When the stated range includes one or two of the limits, the range excluding one or two of those included limits is also included in the present invention. When multiple low values and multiple high values that overlap are given for a range, the skilled person will recognize that the selected range will include a low value that is lower than the high value. All titles in this specification are for ease of reading and are not restrictive.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods similar to or equivalent to those described herein may also be used in the practice or testing of the present invention, preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe methods and/or materials related to the cited publications.

It must be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "chimeric antigen receptor" includes a plurality of such chimeric antigen receptors and equivalents thereof known to those skilled in the art, and so forth. It should be further noted that the claims may be drafted to exclude any optionally optional element. Therefore, this statement is intended to serve as an antecedent basis for the use of exclusive terminology such as "solely," "only," and the like in connection with the recitation of claim elements, or for the use of a "negative" limitation.

It should be understood that certain features of the invention described in the context of separate embodiments for clarity may also be provided in combination in a single embodiment. Conversely, various features of the invention described in the context of a single embodiment for brevity may also be provided individually or in any suitable sub-combination. All combinations of embodiments of the invention are specifically included in the present invention and are disclosed herein as if each and every combination were individually and clearly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically included in the present invention and are disclosed herein as if each and every such sub-combination were individually and clearly disclosed.

Detailed ways

The present invention overcomes prior art challenges by providing improved methods and compositions for genetically modifying lymphocytes (e.g., NK cells, and in illustrative embodiments, T cells). For example, some of these methods do not include pre-activating lymphocytes, and some of these methods are performed in less time than previous methods. In addition, compositions with many uses (including their use in these improved methods) are provided. Some of these compositions are genetically modified lymphocytes with improved proliferation and survival qualities, including when cultured in vitro, such as in the absence of growth factors. These genetically modified lymphocytes will have uses such as the following: as a research tool to better understand the factors that affect T cell proliferation and survival; and commercial production, such as the production of certain factors (such as growth factors and immunomodulators) that can be collected and tested or used in commercial products.

Some embodiments provided herein are methods for performing adoptive cell therapy, which include transducing T cells and/or NK cells ex vivo in a short period of time (e.g., 24 hours, 12 hours, or 8 hours or less), and in some embodiments, no prior ex vivo stimulation is required. These methods are preferably suitable for closed systems that process blood from an individual ex vivo, and can be performed for an individual present in the same room as some embodiments and/or an individual in some embodiments within their line of sight of their blood or its separated blood cells at all times during the performance of the method. More specifically, the aspects and embodiments of the disclosure herein overcome the problems associated with current adoptive cell therapy by providing a method for transducing resting T cells and/or resting NK cells, which methods generally utilize pseudotyped components that facilitate binding and fusion of replication-defective recombinant retroviral particles with resting T cells and/or resting NK cells to facilitate genetic modification of resting T cells and/or resting NK cells using replication-defective recombinant retroviral particles. Furthermore, the methods provided herein overcome problems in the art by utilizing, in illustrative embodiments, a chimeric antigen receptor and one or more lymphoproliferative components whose expression is under the control of a control component, such that exposure of an individual to a compound that binds the control component, or termination of such exposure, promotes expansion of genetically modified T cells and/or NK cells in vivo.

Due to these and other improvements disclosed in detail herein, in one aspect, a method for genetically modifying resting T cells and/or resting NK cells of an individual (such as a patient with a disease or condition) is provided herein, wherein blood from the individual is collected; resting T cells and/or NK cells are genetically modified by contacting them with replication-deficient recombinant retroviral particles; and the genetically modified cells are reintroduced into the individual, typically within a shorter period of time (e.g., within 24 hours and in some non-limiting embodiments, within 12 hours) than in prior art methods and/or without further ex vivo expansion of the population of genetically modified T cells and/or NK cells, such that the genetically modified resting T cells and/or NK cells do not undergo more than 4 ex vivo cell divisions. Therefore, the methods provided herein can be performed in a much shorter time than current CAR therapies, thereby providing a method in which an individual can stay in a clinic for the entire time of the ex vivo step. This helps to perform the ex vivo step in a closed system, which reduces the chance of contamination and mixing of patient samples and can be more easily performed by a clinical laboratory.

Thus, Figures 1 and 2 provide schematic block diagrams of illustrative compositions for use in the methods provided herein. Figure 1 provides a block diagram of a packaging cell (100) and a replication-defective recombinant retroviral particle (200) produced by such a packaging cell. The packaging cell (100) includes a recombinant polynucleotide (110) incorporated into its genome (which includes a recombinant transcriptional component that expresses retroviral proteins) and a variety of different membrane-bound polypeptides under the control of an inducible promoter (which is regulated by a transactivator that binds a ligand and is activated by the ligand). These transactivators, inducible promoters, and ligands are used to induce the sequential expression and accumulation of cell membrane-bound polypeptides that will be incorporated into the membrane of the replication-defective recombinant retroviral particle and into the retroviral components necessary for packaging and assembling the replication-defective recombinant retroviral particles.

As a result of the sequential induction of expression of various polynucleotides as discussed in detail below, the illustrative packaging cell (100) illustrated in Figure 1 is produced and can be used in an illustrative method to produce replication-defective recombinant retroviral particles for use in the methods of transfecting resting T cells and/or NK cells provided herein ((300) in Figure 2). In a non-limiting embodiment, the packaging cell (100) includes in its genome a nucleic acid encoding a packageable retroviral RNA genome that includes at least some of the components of the retroviral genome necessary for packaging and assembling replication-defective recombinant retroviral particles (as non-limiting illustrative examples, the retroviral psi component, the retroviral gag polypeptide, and the retroviral pol polypeptide).

Some membrane-bound polypeptides that are bound or associated with the cell membrane of the packaging cell will be incorporated or associated with the replication-defective recombinant retroviral particles, but are not encoded by the retroviral genome. For example, the packaging cell and the replication-defective recombinant retroviral particles formed in particular may include: a retroviral Vpx polypeptide (250), which in a non-limiting embodiment may be expressed as a membrane-associated fusion protein, such as a Src-Flag-Vpx polypeptide; a pseudotyped component (240) that may include a binding polypeptide and a fusogenic polypeptide, which in a non-limiting embodiment may include a measles virus hemagglutinin (H) polypeptide and a measles virus fusion (F) polypeptide, or a cytoplasmic domain-deficient variant thereof; one or more activation components (210, 220) that may be selected as appropriate, which in a non-limiting embodiment may include a membrane-bound polypeptide capable of binding to CD3 and a membrane-bound polypeptide capable of binding to CD28; and/or an optional membrane-bound cytokine (230), a non-limiting example of which is a fusion polypeptide comprising IL-7 fused to DAF or a fragment thereof. Various other specificities for these membrane-bound polypeptides are provided herein.

As a result of expressing the transcription components in sequence by the packaging cells, replication-deficient recombinant retroviral particles are produced. The RNA retroviral genome that becomes the genome of the packaging cells of the replication-deficient recombinant retroviral particles and is usually integrated therein includes the retroviral components (as non-limiting illustrative embodiments, retroviral Gag and Pol polynucleotides) necessary for retroviral production, infection and integration into the genome of the host cell (which is usually a resting T cell and/or NK cell). In addition, the retroviral genome further includes a polynucleotide encoding one or usually two engineered signaling polypeptides provided herein. One of the engineered signaling polypeptides usually encodes at least one lymphoproliferative component lymphoproliferative component (in a non-limiting example, a constitutive interleukin 7 receptor mutation) and other engineered signaling polypeptides usually encode chimeric antigen receptors.

Next, the replication-deficient recombinant retroviral particles (200) are used to transduce resting T cells and/or resting NK cells (300) using the methods provided herein. As shown in Figure 2, after resting T cells and/or NK cells (300) are contacted with the replication-deficient recombinant retroviral particles (200), the membrane polypeptides discussed above on the surface of the replication-deficient recombinant retroviral particles bind to receptors and/or ligands on the surface of the resting T cells and/or NK cells (300). For example, as indicated above, a pseudotyped assembly that may include binding polypeptides and fusogenic polypeptides that bind to molecules on the surface of resting T cells and/or resting NK cells facilitates the binding and fusion of replication-deficient recombinant retroviral particles (200) with T cell and/or NK cell membranes. The activation assembly (210, 220) activates resting T cells and/or NK cells (300) by engaging a T cell receptor complex (a process that occurs during the time course of contact or a subsequent culture). In addition, membrane-bound cytokines (230) may be present on the surface of the replication-deficient recombinant retroviral particles and bind to cytokine receptors (310) on the surface of resting T cells and/or NK cells (300), thereby further promoting binding and activation. Therefore, without being limited by theory, in the illustrative embodiments provided herein, due to one or more of the replication-deficient recombinant retroviral particles (200) components, there is no need to utilize ex vivo stimulation or activation of components that are not already in or on the replication-deficient recombinant retroviral particles (200). This in turn helps to reduce the ex vivo time required to complete the methods in these illustrative methods provided herein.

After binding to T cells and/or NK cells (200), the replication-deficient recombinant retroviral particles then fuse with T cells and/or NK cells (300), and the polypeptides and nucleic acids in the replication-deficient recombinant retroviral particles enter the T cells and/or NK cells (300). As indicated above, one of these polypeptides in the replication-deficient recombinant retroviral particles is the Vpx polypeptide (250). The Vpx polypeptide (250) binds to the SAMHD1 restriction factor (350) that degrades free dNTPs in the cytoplasm and induces its degradation. Therefore, the concentration of free dNTPs in the cytoplasm increases as Vpx degrades SAMHD1, and increases reverse transcription activity, thereby facilitating reverse transcription of the retroviral genome and integration into the T cell and/or NK cell genome.

After the retroviral genome is integrated into the T cell and/or NK cell (200), the T cell and/or NK cell genome includes a nucleic acid encoding a signaling polypeptide (which encodes a lymphoproliferative component lymphoproliferative component (370)) and a signaling polypeptide encoding a CAR (360) selected as appropriate. The expression of the lymphoproliferative component lymphoproliferative component and the CAR selected as appropriate is under the control of the control component. Exposure to a compound that binds to the control component (which can occur in vitro or in vivo by administering the compound to an individual who transduces their T cells and/or NK cells (300)) promotes the proliferation of T cells and/or NK cells (300) in vitro or in vivo by expressing the lymphoproliferative component lymphoproliferative component and, as appropriate, due to the expression of the CAR and the binding of the CAR to its target cell. Therefore, the T cells and/or NK cells transduced with replication-deficient recombinant retroviral particles herein have one or more signals that drive proliferation and/or inhibit cell death (in illustrative embodiments, this in turn avoids the need for a previous method to deplete host lymph before returning transduced T cells and/or NK cells to an individual). In illustrative embodiments, this in turn further reduces the number of days required for treatment before reintroducing transduced T cells and/or NK cells into an individual. Therefore, in illustrative embodiments, the time required for blood collection from an individual to reintroducing blood into an individual is no more than 36 hours, 24 hours, 12 hours, or even no more than 8 hours in some cases, which fundamentally changes the CAR-T method from the prior art method. In addition, the control component also provides one of the safety mechanisms provided herein. For example, stopping the administration of the compound can downregulate or even terminate the expression of the lymphoproliferative component lymphoproliferative component and the CAR selected as appropriate, thereby ending the proliferation and/or survival signals for transduced T cells and/or NK cells and their progeny.

Methods for transducing and/or genetically modifying lymphocytes

In certain aspects, provided herein is a method of transducing and/or genetically modifying peripheral blood mononuclear cells (PBMCs) or lymphocytes, typically T cells and/or NK cells, and in certain illustrative embodiments, typically resting T cells and/or resting NK cells, comprising contacting the lymphocytes with replication-defective recombinant retroviral particles, wherein the replication-defective recombinant retroviral particles typically comprise a pseudotyping component on their surface, wherein the contacting (and incubation under contacting conditions) facilitates transduction of resting T cells and/or NK cells by the replication-defective recombinant retroviral particles, thereby producing genetically modified T cells and/or NK cells. The pseudotyping component is typically capable of binding to resting T cells and/or NK cells and may facilitate its own membrane fusion or binding to other proteins of the replication-defective recombinant retroviral particles.

Various components or steps of such method aspects for transducing and/or genetically modifying PBMCs, lymphocytes, T cells, and/or NK cells are provided herein (e.g., in this section and in the illustrative embodiments section), and as further discussed herein, such methods include the embodiments provided throughout this specification, for example, embodiments of any of the aspects for transducing and/or genetically modifying PBMCs or lymphocytes (e.g., NK cells, or in illustrative embodiments, T cells) provided for the examples in this section and in the illustrative embodiments section may include any of the embodiments of replication-deficient recombinant retroviral particles provided herein, including those including: one or more lymphoproliferative components, CARs, pseudotype components, riboswitches, activation components, membrane-bound cytokines, miRNAs, Kozak-type sequences, WPRE components, triple stop codons, and or other components disclosed herein, and may be combined with the methods herein for producing retroviral particles using packaging cells. In certain illustrative embodiments, the retroviral particles are lentiviral particles. Such methods for genetically modifying and/or transducing PBMCs or lymphocytes (such as T cells and/or NK cells) can be performed in vitro or ex vivo. The skilled artisan will recognize that the details provided herein for transducing and/or genetically modifying PBMCs or lymphocytes (such as T cells and/or NK cells) can be applied to any aspect including such steps.

In some illustrative embodiments, in the case of no need for pre-in vivo, in vitro or ex vivo activation or stimulation, genetically modified and/or transduced cells. In some illustrative embodiments, cells are activated during contact, and are completely inactivated or activated for more than 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours or 8 hours before contact. In some illustrative embodiments, activation by components not present on the surface of retroviral particles does not require genetically modified and/or transduced cells. Therefore, before, during or after contact, except on retroviral particles, such activation or stimulation components are not required. Therefore, as discussed in more detail herein, these illustrative embodiments that do not require pre-activation or stimulation provide the ability to quickly perform in vitro experiments intended to better understand T cells and the biological mechanisms therein. In addition, such methods provide more effective commercial production of biological products produced using PBMC, lymphocytes, T cells or NK cells and the development of such commercial production methods. Ultimately, such methods provide for faster ex vivo processing of PBMC for adoptive cell therapy, such as by providing a point of care method to substantially simplify the delivery of such therapies.

The contact step for the method for transduction provided herein and/or for the method for genetically modifying generally includes an initial step, wherein retroviral particles (usually retroviral particle colonies) contact with cells (usually cell colonies) to form a transduction reaction mixture when in a liquid buffer and/or culture medium containing a suspension, followed by a cultivation period selected as appropriate in this reaction mixture of the retroviral particles and cells in the suspension. Can, for example, be contacted in a chamber suitable for the closed system for processing PMBC, as discussed in more detail herein. The transduction reaction mixture may include one or more buffers or ions, and in illustrative embodiments, includes culture medium, such as those culture mediums for in vitro treatment involving lymphocytes known in the art, as provided in further detail herein.

The transduction reaction mixture may be between 23°C and 39°C, and in some illustrative embodiments, cultivated at 37°C. In certain embodiments, the transduction reaction may be performed at 37°C to 39°C for faster fusion/transduction. When contacted in the transduction reaction mixture, cells and retroviral particles may be immediately processed to remove retroviral particles that remain free in the suspension and are not associated with the cells from the cells. Depending on the circumstances, cells and retroviral particles in the suspension, whether free in the suspension or associated with the cells in the suspension, are cultivated for different lengths of time, as provided herein for use in the contact steps in the methods provided herein. Before other steps, washing may be performed, such as washing in the culture medium used in the transduction reaction, regardless of whether such cells will be studied in vitro, in vitro, or introduced into an individual.

In some embodiments, the replication-defective recombinant retroviral particle may further include an activation module, which may be any activation module provided herein. In illustrative embodiments, the activation module may be anti-CD3, such as anti-CD3 scFv or anti-CD3 scFv Fc.

In some embodiments, the contacting step in the methods provided herein for transducing and/or genetically modifying PBMCs or lymphocytes (typically T cells and/or NK cells) can be performed (or can occur) between 1 and 24 hours, such as between 1 and 12 hours, or between 1 and 6 hours. In some illustrative embodiments, the contacting can be performed for less than 24 hours, such as less than 12 hours, less than 8 hours, less than 4 hours, less than 2 hours, less than 1 hour, less than 30 minutes, or less than 15 minutes, but in each case, there is at least one initial contacting step in which the retroviral particles are contacted with the cells in a suspension in the transduction reaction mixture. Such a suspension can include sedimentation of the cells and retroviral particles, or such sedimentation of the bottom of a container or chamber caused by the application of forces such as centrifugal forces. After such initial contact, additional optional cultivation can be performed in the reaction mixture (containing cells and retroviral particles in suspension in the reaction mixture) without removing the retroviral particles that remain free in the solution and are not associated with the cells. In illustrative embodiments, the contacting step may be performed (or occur) between 30 seconds or 1 minute, 2 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes, or 45 minutes at the low end of the range, or 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, or 8 hours at the high end of the range and 10 minutes, 15 minutes, 30 minutes, or 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours, and 72 hours at the high end of the range. In certain illustrative embodiments, the contacting step may be performed between 30 seconds, 1 minute, 5 minutes, 10 minutes, 15 minutes, or 30 minutes at the low end of the range and 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, or 12 hours at the high end of the range. In some embodiments, the contacting step is performed between 30 seconds, 1 minute, and 5 minutes at the high end of the range and 10 minutes at the high end of the range. In another illustrative embodiment, contacting is performed only between the initial contacting steps (without any additional incubation in the reaction mixture comprising free retroviral particles in suspension and cells in suspension) without any additional incubation in the reaction mixture, or between incubations of the reaction mixture for 5 minutes, 10 minutes, 15 minutes, 30 minutes or 1 hour.

The methods for genetic modification and/or transduction of cells provided herein generally include inserting into cells a polynucleotide comprising one or more transcriptional units encoding a CAR or a lymphoproliferative component, or in illustrative embodiments, encoding a CAR and a lymphoproliferative component according to any one of the CAR and lymphoproliferative component embodiments provided herein. As illustrated in the examples herein, the lymphoproliferative component promotes survival and/or proliferation, which causes the survival and/or expansion of genetically modified and/or transduced cells under appropriate conditions including, but not limited to, those provided in the examples provided herein. Thus, the PBMCs, lymphocytes, NK cells or T cells genetically modified herein with one or more nucleic acids encoding CAR or both CAR and a lymphoproliferative component are capable of, suitable for, possess and/or modified for improved survival or expansion in culture ex vivo or in vitro in the absence of one or more added cytokines (such as IL-2, IL-15 or IL-7) or added lymphocyte mitogens, as compared to control cells that are identical to the genetically modified and/or transduced cells prior to their genetic modification and/or transduction, or in some embodiments, as compared to control cells modified and/or transduced with the same control recombinant retroviral particles as those used to produce the genetically modified and/or transduced cells capable of improved survival or expansion, except that the control retroviral particles do not comprise a transcription unit encoding CAR, a lymphoproliferative component or an intracellular domain thereof, or both CAR and a lymphoproliferative component or an intracellular domain thereof. In illustrative examples performed in vitro or ex vivo, the control cells and conditions of culture are selected such that under these conditions, the survival and/or proliferation of the control cell(s) will be promoted by the addition of the one or more cytokines (such as IL-2, IL-15 or IL-7), or the lymphocyte mitogen to the culture medium, even though such cytokines or mitogens are not actually added for comparison.

Therefore, in illustrative embodiments, the method for genetically modifying and/or transducing herein provides a rapid means, which provides new functionality by inserting nucleic acids into cells, and the nucleic acids provide the functions of CAR and/or lymphoproliferative components as discussed herein when expressed. For example, as provided in the examples herein, such cells can better survive and/or amplify in vitro and in vivo compared to control cells, and the control cells are the same as genetically modified cells but do not include nucleic acids encoding lymphoproliferative components and optionally selected CARs. For example, the examples herein confirm that, compared to the same control cells that are not genetically modified, after contact in the culture for the first 7 days, PBMCs modified by genetic means to express CAR or express both CAR and lymphoproliferative components survive and/or proliferate more, wherein the culture is carried out in the absence of any added cytokines (such as but not limited to IL-2, IL-15 or IL-7) or added lymphocyte mitogens. Furthermore, this example demonstrates that T cells genetically modified to express a CAR and a lymphoproliferative component have increased survival between days 7 and 14 or 21 following contact under these conditions compared to the same control cells that have not been genetically modified. In fact, T cells genetically modified to express a CAR (e.g., an anti-CD19 CAR) and a driver (as exemplified for a number of drivers identified herein) have increased survival characteristics between days 7 and 21 following contact under these conditions compared to the same cells genetically modified to express a CAR without a lymphoproliferative component.

In certain embodiments, the genetically modified and/or transduced cells exhibit, are capable of, are suitable for, possess the following properties, or are modified for improved expansion in culture in the absence of any exogenously added T cell stimulator (e.g., IL-2, IL-7, IL-15, anti-CD3 or anti-CD28) or other exogenously added lymphocyte mitogens, compared to control cells that were genetically modified and/or transduced cells before they were genetically modified and/or transduced. In certain embodiments, the genetically modified cells exhibit, are capable of, are suitable for, possess the following properties, or are modified for better expansion in culture in the absence of exogenously added cytokines, in the absence of an antigen bound by a CAR encoded by the genome of a retroviral particle, which is optionally selected, and/or in the absence of a lymphocyte mitogen, compared to control cells between days 3 and 6 of ex vivo culture after contact. In certain embodiments, the genetically modified cells exhibit, are capable of, are suitable for, possess, or are modified to expand at least twice between days 3 and 6 of ex vivo culture after contacting in the absence of exogenously added cytokines and in the absence of an antigen bound by an optionally selected CAR encoded by the genome of a retroviral particle.

For example, when cells and retroviral particles are initially contacted with retroviral particles (including retroviral particles and cells in a medium containing a suspension) or thereafter contacted with them during an incubation period selected as appropriate, the medium that may be included in the contacting step or the medium that may be used during cell culture and/or during various washing steps may include a basal medium, such as a commercially available medium for ex vivo T cell and/or NK cell culture. Non-limiting examples of such culture media include: X-VIVO ^™ 15 Chemically Defined Serum-Free Hematopoietic Cell Medium (Lonza) (2018 Catalog Nos. BE02-060F, BE02-00Q, BE-02-061Q, 04-744Q or 04-418Q), ImmunoCult ^™ -XF T Cell Expansion Medium (STEMCELL Technologies) (2018 Catalog No. 10981), T cell expansion XSFM (Irvine Scientific) (2018 catalog number 91141), Culture medium CTS ^™ (Therapeutic Grade) (Thermo Fisher Scientific (referred to herein as "Thermo Fisher") or CTS ^™ Optimizer ^™ culture medium (Thermo Fisher) (2018 Catalog No. A10221-01 (basal culture medium (bottle)) and A10484-02 (supplements), A10221-03 (basal culture medium (bag)), A1048501 (basal culture medium and supplement kit (bottle)) and A1048503 (basal culture medium and supplement kit (bag)). Such culture medium can be a chemically defined serum-free formulation manufactured in compliance with cGMP. The medium can be xeno-free and complete. In some embodiments, the basal medium has been cleared by a regulatory agency for use in ex vivo cell processing, such as an FDA 510 (k) cleared device. In some embodiments, the medium is 2018 Catalog No. A1048501 (CTS ^™ Optimizer ™) with or without Optimizer ™, both available from Thermo Fisher (Waltham, MA). The basal medium supplemented with T cell expansion supplements is A1048503 ⁽ CTS ^™ OpTmizer ^™ T Cell Expansion SFM, bottle format) or A1048503 (CTS™ OpTmizer™ T Cell Expansion SFM, bag format). Additives such as human serum albumin, human AB+ serum, and/or serum derived from an individual may be added to the transduction reaction mixture. Supporting cytokines such as IL2, IL7, or IL15 or those found in human serum may be added to the transduction reaction mixture. dGTP may be added to the transduction reaction mixture in certain embodiments.

In an exemplary embodiment of such methods, genetically modified T cells or NK cells can be transplanted into mice in vivo and/or enriched in mice in vivo for at least 7 days, 14 days, or 28 days. The skilled artisan will recognize that such mice can be treated or otherwise genetically modified so that any immunological differences between the genetically modified T cells and/or NK cells do not result in an immune response in the mouse directed against any component of the lymphocytes transduced by the replication-defective recombinant retroviral particles.

The packaging cells (and in illustrative embodiments, the packaging cell lines, and in certain illustrative embodiments, the packaging cells provided in certain aspects herein) are used to produce replication-defective recombinant retroviral particles. In some embodiments, the packaging cell line can be a suspension cell line. In illustrative embodiments, the packaging cell line can be grown in serum-free medium. In some embodiments, the lymphocytes can be from an individual. In illustrative embodiments, the lymphocytes can be from the blood of an individual.

Thus, in one aspect, provided herein is a method for transducing (and/or genetically modifying) lymphocytes (generally resting T cells and/or resting NK cells from separated blood), the method comprising:

A. Collect blood from individuals;

B. isolating peripheral blood mononuclear cells (PBMCs) containing resting T cells and/or resting NK cells; and

C. contacting an individual's resting T cells and/or resting NK cells ex vivo with non-replicating recombinant retroviral particles, wherein the non-replicating recombinant retroviral particles contain a pseudotyped component on their surface that is capable of binding to resting T cells and/or resting NK cells and facilitating membrane fusion of the non-replicating recombinant retroviral particles with the resting T cells and/or resting NK cells, wherein the contact facilitates transduction of at least 5% of the resting T cells and/or resting NK cells using the non-replicating recombinant retroviral particles, thereby producing genetically modified T cells and/or NK cells, thereby transducing resting T cells and/or NK cells.

In some embodiments of any method herein including the step of transducing T cells and/or NK cells, cells can be contacted with retroviral particles without prior activation. In some embodiments of any method herein including the step of transducing T cells and/or NK cells, before transduction, T cells and/or NK cells are cultivated on a matrix adhered to a monocyte for more than 4 hours in one embodiment, or more than 6 hours in another embodiment, or more than 8 hours in another embodiment. In an illustrative embodiment, before transduction, T cells and/or NK cells are cultivated overnight on an adhesive matrix to remove monocytes. In another embodiment, the method may include cultivating T cells and/or NK cells on an adhesive matrix in conjunction with monocytes for no more than 30 minutes, 1 hour or 2 hours before transduction. In another embodiment, before the transduction step, T cells and/or NK cells are not exposed to the step of removing monocytes by cultivating on an adhesive matrix. In another embodiment, the T cells and/or NK cells are not cultured with or exposed to bovine serum (such as cell culture bovine serum, e.g., fetal bovine serum) prior to or during transduction.

Thus, in another aspect, provided herein is a method for genetically modifying or transducing lymphocytes (in illustrative embodiments, T cells and/or NK cells, or a population of T cells and/or NK cells) of an individual, the method comprising contacting T cells and/or NK cells of a general individual ex vivo with replication-defective recombinant retroviral particles, the replication-defective recombinant retroviral particles comprising a polynucleotide in their genome, the polynucleotide comprising one or more nucleic acid sequences operably linked to a promoter active in T cells and/or NK cells, wherein a first nucleic acid sequence of the one or more nucleic acid sequences encodes two or more inhibitory RNA molecules against one or more RNA targets, and a second nucleic acid sequence of the one or more nucleic acid sequences encodes a chimeric antigen receptor (CAR) comprising an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain, wherein the contacting facilitates transduction of resting T cells and/or NK cells or at least some of the resting T cells and/or NK cells using the replication-defective recombinant retroviral particles, thereby producing genetically modified T cells and/or NK cells.

In another aspect, provided herein is a method for genetically modifying or transducing lymphocytes (e.g., T cells and/or NK cells) or a population thereof of an individual, the method comprising contacting lymphocytes (e.g., T cells and/or NK cells) or a population thereof of an individual with a replication-defective recombinant retroviral particle ex vivo, the replication-defective recombinant retroviral particle comprising a polynucleotide in its genome, the polynucleotide comprising one or more nucleic acid sequences operably linked to a promoter active in lymphocytes (e.g., T cells and/or NK cells), wherein a first nucleic acid sequence in the one or more nucleic acid sequences encodes One or more (e.g., two or more) inhibitory RNA molecules directed against one or more RNA targets, and a second nucleic acid sequence among the one or more nucleic acid sequences encodes a chimeric antigen receptor (CAR) comprising an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain, wherein the contact facilitates genetic modification and/or transduction of lymphocytes (e.g., T cells and/or NK cells) or at least some of the lymphocytes (e.g., T cells and/or NK cells) using replication-defective recombinant retroviral particles, thereby producing genetically modified and/or transduced lymphocytes (e.g., T cells and/or NK cells).

In some embodiments of the methods provided immediately above, genetically modified and/or transduced lymphocytes (e.g., T cells and/or NK cells) or a colony thereof are introduced into an individual. In some embodiments, genetically modified and/or transduced lymphocytes (e.g., T cells and/or NK cells) or a colony thereof are subjected to 4 or fewer cell divisions in vitro before being introduced or reintroduced into an individual. In some embodiments, lymphocytes are resting T cells and/or resting NK cells that are contacted with replication-deficient recombinant retroviral particles for between 1 hour and 12 hours. In some embodiments, the time between the time of collecting blood from an individual and the time of reintroducing genetically modified T cells and/or NK cells into an individual is no more than 8 hours. In some embodiments, all steps after collecting blood and before reintroducing blood are performed in a closed system, and the closed system is monitored by a person during the entire treatment period.

In any of the method aspects including polynucleotides provided immediately above, the polynucleotide comprises one or more nucleic acid sequences operably linked to a promoter active in T cells and/or NK cells, wherein the first nucleic acid sequence in the one or more nucleic acid sequences encodes one or more (e.g., two or more) inhibitory RNA molecules for one or more RNA targets, and the second nucleic acid sequence in the one or more nucleic acid sequences encodes a chimeric antigen receptor (CAR) comprising an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain, and the polynucleotide may further include a third nucleic acid sequence encoding at least one lymphoproliferative component lymphoproliferative component that is not an inhibitory RNA molecule. In some embodiments, the lymphoproliferative component lymphoproliferative component may be a cytokine or a cytokine receptor polypeptide, or a fragment thereof comprising a signaling domain. In some embodiments, the lymphoproliferative component lymphoproliferative component is constitutively active. In certain embodiments, the lymphoproliferative component lymphoproliferative component may be an IL-7 receptor or a fragment thereof. In illustrative embodiments, the lymphoproliferative element The lymphoproliferative element can be a constitutively active IL-7 receptor or a constitutively active fragment thereof.

In any one of the method aspects including polynucleotides provided immediately above, the polynucleotides include one or more nucleic acid sequences operably connected to a promoter active in T cells and/or NK cells, wherein the first nucleic acid sequence in the one or more nucleic acid sequences encodes one or more (e.g., two or more) inhibitory RNA molecules for one or more RNA targets, and the inhibitory RNA molecules may include 5' chains and 3' chains partially or completely complementary to each other in some embodiments, wherein the 5' chain and the 3' chain can form 18 to 25 nucleotide RNA double helices. In some embodiments, the 5' chain length may be 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides, and the 3' chain length may be 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides. In some embodiments, the 5' chain and the 3' chain length may be the same or different. In some embodiments, the RNA double helix may include one or more mismatches. In alternative embodiments, the RNA double helix does not have a mismatch.

In any of the method aspects including polynucleotides provided immediately above, the polynucleotides include one or more nucleic acid sequences operably linked to a promoter active in T cells and/or NK cells, wherein the first nucleic acid sequence in the one or more nucleic acid sequences encodes one or more (e.g., two or more) inhibitory RNA molecules for one or more RNA targets, and the inhibitory RNA molecules may be miRNA or shRNA. In some embodiments, the inhibitory molecule may be a precursor of miRNA, such as Pri-miRNA or Pre-miRNA, or a precursor of shRNA. In some embodiments, the inhibitory molecule may be an artificially derived miRNA or shRNA. In other embodiments, the inhibitory RNA molecule may be a dsRNA (transcribed or artificially introduced) or siRNA itself processed into siRNA. In some embodiments, the inhibitory RNA molecule may be a miRNA or shRNA having a sequence not found in nature, or having at least one functional fragment not found in nature, or having a combination of functional segments not found in nature. In illustrative embodiments, at least one or all of the inhibitory RNA molecules are miR-155.

In any of the method aspects including a polynucleotide (comprising one or more nucleic acid sequences operably linked to a promoter active in T cells and/or NK cells) provided immediately above, wherein the first nucleic acid sequence in the one or more nucleic acid sequences encodes one or more (e.g., two or more) inhibitory RNA molecules for one or more RNA targets, in some embodiments, the inhibitory RNA molecule may be oriented from 5' to 3' to include: a 5' arm, a 5' stem, a loop, a 3' stem partially or completely complementary to the 5' stem, and a 3' arm. In some embodiments, at least one of the two or more inhibitory RNA molecules has this arrangement. In other embodiments, all of the two or more inhibitory RNA molecules have this arrangement. In some embodiments, the 5' stem length may be 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides. In some embodiments, the 3' stem length may be 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides. In some embodiments, the loop length may be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides. In some embodiments, the 5' arm, the 3' arm, or both are derived from naturally occurring miRNAs. In some embodiments, the 5' arm, the 3' arm, or both are derived from naturally occurring miRNAs selected from the group consisting of miR-155, miR-30, miR-17-92, miR-122, and miR-21. In illustrative embodiments, the 5' arm, the 3' arm, or both are derived from miR-155. In some embodiments, the 5' arm, the 3' arm, or both are derived from Mus musculus miR-155 or Homo sapiens miR-155. In some embodiments, the 5'-arm has a sequence as set forth in SEQ ID NO: 256 or is a functional variant thereof, such as being the same length as SEQ ID NO: 256, or being 95%, 90%, 85%, 80%, 75%, or 50% of the length of SEQ ID NO: 256, or being a sequence of 100 nucleotides or less, 95 nucleotides or less, 90 nucleotides or less, 85 nucleotides or less, 80 nucleotides or less, 75 nucleotides or less, 70 nucleotides or less, 45 nucleotides or less, 40 nucleotides or less, 35 nucleotides or less, 30 nucleotides or less, or 25 nucleotides or less; and being at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to SEQ ID NO: 256. In some embodiments, the 3' arm has a sequence set forth in SEQ ID NO: 260 or a functional variant thereof, such as a sequence that is the same length as SEQ ID NO: 260, or is 95%, 90%, 85%, 80%, 75%, or 50% of the length of SEQ ID NO: 260, or is 100 nucleotides or less, 95 nucleotides or less, 90 nucleotides or less, 85 nucleotides or less, 80 nucleotides or less, 75 nucleotides or less, 70 nucleotides or less, 65 nucleotides or less, 60 nucleotides or less, 55 nucleotides or less, 50 nucleotides or less, 45 nucleotides or less, 40 nucleotides or less, 35 nucleotides or less, 30 nucleotides or less, or 25 nucleotides or less; and is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to SEQ ID NO: 260. In some embodiments, the 3' arm comprises nucleotides 221 to 283 of the Mus musculus BIC.

In any of the method aspects provided immediately above including a polynucleotide (comprising one or more nucleic acid sequences operably linked to a promoter active in T cells and/or NK cells), wherein the first nucleic acid sequence in the one or more nucleic acid sequences encodes two or more inhibitory RNA molecules for one or more RNA targets, in some embodiments, the two or more inhibitory RNA molecules may be located continuously in the first nucleic acid sequence. In some embodiments, the inhibitory RNA molecules may be directly or indirectly linked to each other using a non-functional connector sequence. In some embodiments, the connector sequence length may be between 5 and 120 nucleotides, or between 10 and 40 nucleotides in length.

In any of the method aspects including polynucleotides (including one or more nucleic acid sequences operably linked to a promoter active in T cells and/or NK cells) provided immediately above, wherein the first nucleic acid sequence in the one or more nucleic acid sequences encodes two or more inhibitory RNA molecules for one or more RNA targets, in some embodiments, the first nucleic acid sequence encodes two to four inhibitory RNA molecules. In illustrative embodiments, the first nucleic acid sequence includes between 2 and 10, between 2 and 8, between 2 and 6, between 2 and 5, between 2 and 4, between 3 and 5, or between 3 and 6 inhibitory RNA molecules. In an illustrative embodiment, the first nucleic acid sequence includes four inhibitory RNA molecules.

In any of the method aspects provided immediately above including a polynucleotide (comprising one or more nucleic acid sequences operably linked to a promoter active in T cells and/or NK cells), wherein the first nucleic acid sequence in the one or more nucleic acid sequences encodes one or more (e.g., two or more) inhibitory RNA molecules for one or more RNA targets, the one or more (e.g., two or more) inhibitory RNA molecules may be in an intron. In some embodiments, the intron is in a promoter. In illustrative embodiments, the intron is EF-1α intron A. In some embodiments, the intron is adjacent to the promoter and downstream of the promoter, and in illustrative embodiments, the intron is inactive in a packaging cell for producing replication-defective recombinant retroviral particles.

In any of the method aspects including a polynucleotide (comprising one or more nucleic acid sequences operably linked to a promoter active in T cells and/or NK cells) provided immediately above, wherein the first nucleic acid sequence in the one or more nucleic acid sequences encodes two or more inhibitory RNA molecules for one or more RNA targets, in some embodiments, the two or more inhibitory RNA molecules may be directed to different targets. In an alternative embodiment, the two or more inhibitory RNA molecules are directed to the same target. In some embodiments, the RNA target is an mRNA transcribed from a gene expressed by a free T cell, such as, but not limited to: PD-1 (prevents inactivation); CTLA4 (prevents inactivation); TCRα (safely prevents autoimmunity); TCRb (safe-prevents autoimmunity); CD3Z (safe-prevents autoimmunity); SOCS1 (prevents inactivation); SMAD2 (prevents inactivation); miR-155 target (promotes activation); IFNγ (reduces CRS); cCBL (prolongs signaling); TRAIL2 (prevents death); PP2A (prolongs signaling); ABCG1 (increases cholesterol microcontent by limiting cholesterol clearance). In some embodiments, the RNA target is an mRNA transcribed from a gene encoding a component of the T cell receptor (TCR) complex. In some embodiments, at least one of the two or more inhibitory RNA molecules can reduce the expression of a T cell receptor (in illustrative embodiments, one or more endogenous T cell receptors of a T cell). In certain embodiments, the RNA target may be mRNA transcribed from an endogenous TCRα or TCRβ gene of a T cell whose genome comprises a first nucleic acid sequence encoding one or more miRNAs. In an illustrative embodiment, the RNA target is mRNA transcribed from a TCRα gene.

In any of the method aspects provided immediately above including a polynucleotide (comprising one or more nucleic acid sequences operably linked to a promoter active in T cells and/or NK cells), wherein the first nucleic acid sequence in the one or more nucleic acid sequences encodes one or more (e.g., two or more) inhibitory RNA molecules for one or more RNA targets, and the second nucleic acid sequence in the one or more nucleic acid sequences encodes a chimeric antigen receptor (CAR) comprising an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain, in some embodiments, CAR is a microenvironment-restricted organism (MRB)-CAR. In other embodiments, the ASTR of CAR binds to tumor-associated antigens. In other embodiments, the ASTR of CAR is a microenvironment-restricted organism (MRB)-ASTR. In any of the aspects and embodiments comprising MRB-ASTR or MRB-CAR disclosed herein, MRB-ASTR preferably or only binds to its cognate antigen under certain abnormal conditions, such as those present in the tumor microenvironment. MRB-ASTRs that bind better or exclusively under abnormal conditions of the tumor microenvironment can provide a reduced on-target off-tumor effect because binding to the antigen under normal physiological conditions is reduced, in some cases to levels below those detected by immunoassays. Normal physiological conditions may include those that are considered to be within the normal range for an individual at the site of administration or at the tissue or organ at the site of action, temperature, pH, osmotic pressure, weight molar osmotic concentration, oxidative stress, and electrolyte concentration. Abnormal conditions are conditions that deviate from the normal acceptable range. In some embodiments, MRB-ASTRs are reversibly or irreversibly inactivated under normal conditions. As used herein, MRB-ASTRs may be antibodies, antigens, ligands, receptor binding domains of ligands, receptors, ligand binding domains of receptors, or affinity bodies. In embodiments where MRB-ASTRs are antibodies, MRB-ASTRs may be full-length antibodies, single-chain antibodies, Fab fragments, Fab' fragments, (Fab')2 fragments, Fv fragments, bivalent single-chain antibodies, or double antibodies, wherein the ASTRs comprise heavy and light chains from antibodies. In some embodiments, the MRB-ASTR is a single chain variable fragment.

In any of the method aspects including polynucleotides provided above (including one or more nucleic acid sequences operably linked to a promoter active in T cells and/or NK cells), wherein the first nucleic acid sequence in the one or more nucleic acid sequences encodes one or more (e.g., two or more) inhibitory RNA molecules for one or more RNA targets, and the second nucleic acid sequence in the one or more nucleic acid sequences encodes a chimeric antigen receptor (CAR) comprising an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain, and in some cases, the third nucleic acid sequence in the one or more nucleic acid sequences encodes at least one lymphoproliferative component lymphoproliferative component that is not an inhibitory RNA molecule, in some embodiments, any one or all of the first nucleic acid sequence, the second nucleic acid sequence, and the third nucleic acid sequence are operably linked to a riboswitch. In some embodiments, a riboswitch is capable of binding nucleoside analogs. In some embodiments, a nucleoside analog is an antiviral drug.

In some embodiments, methods are provided for activating and/or genetically modifying, and typically transducing, resting T cells or NK cells (in illustrative embodiments, resting T cells) by contacting the cells with a retroviral particle disclosed herein and a soluble anti-CD3 antibody at 25 ng/ml to 200 ng/ml, 50 ng/ml to 150 ng/ml, 75 ng/ml to 125 ng/ml, or 100 ng/ml. In illustrative embodiments, these methods are performed without prior activation and can be performed, for example, for 8 hours or less, 4 hours or less, or between 2 hours and 8 hours, 2 hours and 4 hours, or between 2 hours and 3 hours.

In certain aspects, provided herein are methods for performing adoptive cell therapy on an individual. As an illustrative example, the method may include the following:

A. Collect blood from individuals;

B. Isolating peripheral blood mononuclear cells (PBMCs) containing resting T cells and/or resting NK cells;

C. contacting resting T cells and/or resting NK cells of the individual ex vivo with replication-defective recombinant retroviral particles, wherein the replication-defective recombinant retroviral particles comprise a pseudotyping component on their surface that is capable of binding to resting T cells and/or NK cells and facilitating membrane fusion of the replication-defective recombinant retroviral particles therewith, wherein the contacting facilitates transduction of resting T cells and/or NK cells with the replication-defective recombinant retroviral particles, thereby generating genetically modified T cells and/or NK cells; and

D. Reintroducing the genetically modified cells into the individual within 36 hours, 24 hours, 12 hours, or even 8 hours of collecting blood from the individual, thereby performing adoptive cell therapy in the individual.

In some aspects provided herein, methods with similar steps are referred to as methods for genetically modifying and expanding lymphocytes of an individual. A skilled artisan will appreciate that the discussion herein also applies to methods for genetically modifying and expanding lymphocytes of an individual when it applies to methods and compositions for performing adoptive cell therapy.

Generally, the adoptive cell therapy of the present invention is carried out by autologous transfer, wherein the cells are separated and/or otherwise prepared from the individual receiving cell therapy or from the sample derived from such individual. Therefore, in some aspects, the cells are derived from the individual (e.g., patient) in need of treatment and the cells after separation and treatment given to the same individual. In some embodiments of the methods and compositions disclosed herein, the individual suffering from disease or illness enters a medical institution where the blood of the individual is extracted using known methods (such as venipuncture). In certain embodiments, the volume of the blood extracted from the individual is 10ml, 15ml, 20ml, 25ml, 30ml, 35ml, 40ml, 50ml, 75ml or 100ml at the low end of the range and 200ml, 250ml, 300ml, 350ml, 400ml, 500ml, 750ml, 1000ml, 2000ml or 2500ml at the high end of the range. In some embodiments, blood between 10ml and 400ml is extracted from the individual. In some embodiments, between 20 ml and 250 ml of blood is drawn from the subject. In some embodiments, the blood is fresh when processed. In any of the embodiments disclosed herein, fresh blood can be blood drawn from the subject less than 15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, 120 minutes, 150 minutes, or 180 minutes ago. In some embodiments, blood is processed with the methods provided herein without storage.

Contact between T cells and/or NK cells and replication-deficient recombinant retroviral particles generally facilitates transduction of T cells and/or NK cells using replication-deficient recombinant retroviral particles. Throughout the present invention, transduced T cells and/or NK cells include at least some of the progeny of nucleic acids or polynucleotides incorporated into cells during ex vivo transduction of ex vivo transduced cells. In the methods herein in which "reintroduction" of transduced cells is cited, it should be understood that such cells are generally not in a transduced state when they are collected from the blood of an individual. The individual in any of the aspects disclosed herein may be, for example, an animal, a mammal, and in illustrative embodiments, a human being.

Without being limited by theory, in a non-limiting illustrative method, ex vivo delivery of a polynucleotide encoding a lymphoproliferative component (such as a constitutively active mutant of IL7) to resting T cells and/or NK cells, which can be integrated into the genome of T cells and/or NK cells, provides cells with drivers for in vivo expansion without subjecting the host to lymphodepletion. Thus, in illustrative embodiments, the individual is not exposed to a lymphodepletion agent within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 14 days, 21 days, or 28 days, or 1 month, 2 months, 3 months, or 6 months of performing the contact, during the contact, and/or within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 14 days, 21 days, or 28 days, or 1 month, 2 months, 3 months, or 6 months after the modified T cells and/or NK cells are reintroduced into the individual. Furthermore, in non-limiting embodiments, the methods provided herein can be performed without exposing the individual to a lymphodepleting agent during the step in which the replication-defective recombinant retroviral particles are contacted with resting T cells and/or NK cells of the individual or during the entire ex vivo method.

Thus, in vivo expansion of genetically modified T cells and/or NK cells in an individual is a feature of some embodiments of the invention.In illustrative embodiments, the methods are ex vivo transmission-free or substantially transmission-free.

In the non-limiting illustrative embodiments herein, this entire method/process of drawing blood from an individual to reintroducing the blood back into the individual after ex vivo transduction of T cells and/or NK cells can occur within a time period of less than 48 hours, less than 36 hours, less than 24 hours, less than 12 hours, less than 11 hours, less than 10 hours, less than 9 hours, less than 8 hours, less than 7 hours, less than 6 hours, less than 5 hours, less than 4 hours, less than 3 hours, 2 hours, or less than 2 hours. In other embodiments, this entire method/process of drawing/collecting blood from an individual to reintroducing the blood back into the individual after ex vivo transduction (in the non-limiting illustrative embodiments herein) of T cells and/or NK cells occurs within a time period of between 1 hour and 12 hours, or between 2 hours and 8 hours, or between 1 hour and 3 hours, or between 2 hours and 4 hours, or between 2 hours and 6 hours, or between 4 hours and 12 hours, or between 4 hours and 24 hours, or between 8 hours and 24 hours. , or between 8 hours and 36 hours, or between 8 hours and 48 hours, or between 12 hours and 24 hours, or between 12 hours and 36 hours, or between 12 hours and 48 hours; or occurs within the following time periods: between 15 minutes, 30 minutes, 60 minutes, 90 minutes, 120 minutes, 180 minutes and 240 minutes at the low end of the range and 120 minutes, 180 minutes, 240 minutes, 300 minutes, 360 minutes, 420 minutes and 480 minutes at the high end of the range. In other embodiments, this entire method/process from drawing/collecting blood from a subject to reintroducing the blood back into the subject after ex vivo transduction of T cells and/or NK cells occurs within a time period of between 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 8 hours, 10 hours, and 12 hours at the low end of the range and 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 18 hours, 24 hours, 36 hours, or 48 hours at the high end of the range. In some embodiments, the genetically modified T cells and/or NK cells are separated from the replication-defective recombinant retroviral particles after the time period of contacting occurs.

In some embodiments of any method herein (which includes the step of blood collection and the step of transducing lymphocytes (in illustrative embodiments, T cells and/or NK cells, including resting T cells and NK cells)), the method of collecting from blood to transducing T cells and/or NK cells does not include the step of removing monocytes by culturing on an adhesive matrix for more than 4 hours in one embodiment, or more than 6 hours in another embodiment, or more than 8 hours in another embodiment. In an illustrative embodiment, the method of collecting from blood to transducing T cells and/or NK cells does not include overnight culturing on an adhesive matrix to remove monocytes. In another embodiment, the method of collecting from blood to transducing T cells and/or NK cells includes the step of removing monocytes by culturing on an adhesive matrix for no more than 30 minutes, 1 hour, or 2 hours. In another embodiment, the method of collecting from blood to transducing lymphocytes (in illustrative embodiments, T cells and/or NK cells, including resting T cells and/or NK cells) from an individual does not include the step of removing monocytes by culturing on an adhesive matrix. In another embodiment, a method for collecting blood from an individual to transduce lymphocytes (in illustrative embodiments, T cells and/or NK cells, including resting T cells and/or NK cells) includes that the T cells and/or NK cells are not cultured with or exposed to bovine serum (such as cell culture bovine serum, e.g., fetal bovine serum) during the method.

In some embodiments of any method herein (which includes the step of blood collection and the step of transducing lymphocytes (in illustrative embodiments, T cells and/or NK cells, including resting T cells and NK cells)), the method of collecting blood from an individual to reintroducing T cells and/or NK cells into the individual does not include the step of removing monocytes by culturing on an adhesive matrix for more than 4 hours in one embodiment, or more than 6 hours in another embodiment, or more than 8 hours in another embodiment. In an illustrative embodiment, the method of collecting blood from an individual to reintroducing T cells and/or NK cells into the individual does not include culturing overnight on an adhesive matrix to remove monocytes. In another embodiment, the method of collecting blood from an individual to reintroducing T cells and/or NK cells includes the step of removing monocytes by culturing on an adhesive matrix for no more than 30 minutes, 1 hour, or 2 hours. In another embodiment, the method of collecting blood from an individual to reintroducing T cells and/or NK cells into the individual does not include the step of removing monocytes by culturing overnight on an adhesive matrix. In another embodiment, a method is provided for collecting blood from an individual and reintroducing T cells and/or NK cells into the individual, during which the T cells and/or NK cells are not cultured with or exposed to bovine serum (such as cell culture bovine serum, e.g., fetal bovine serum).

Because the methods for adoptive cell therapy provided herein, and the related methods for modifying the resting T cells and/or NK cells in vitro before amplifying T cells and/or NK cells in vivo, can be performed in a significantly shorter time than the previous method, making it possible to improve patient care and safety and product manufacturing. Therefore, in the view of the regulatory agencies responsible for approving such methods for therapeutic purposes in vivo, such methods are expected to be advantageous. For example, in a non-limiting example, before the modified T cells and/or NK cells are reintroduced into the patient, the individual can stay in the same building (e.g., infusion clinic) or room of the institution that processes its blood or sample during the entire time of sample processing. In a non-limiting illustrative embodiment, during the entire method/process of reintroducing blood into the individual after blood extraction/collection from the individual to the transduction of T cells and/or NK cells in vitro, the individual remains within the position line and/or within 100, 50, 25 or 12 feet or arm's distance of the blood or cells being processed. In other non-limiting illustrative embodiments, after blood is drawn/collected from an individual to transduce T cells and/or NK cells in vitro, blood is reintroduced into the whole method/process of the individual and/or continuously, the individual remains awake and/or at least one person can continue to monitor the blood or cells being processed of the individual. Because of the improvements provided herein, the whole method/process of blood is reintroduced into the individual after blood is drawn/collected from an individual to transduce T cells and/or NK cells in vitro for adoptive cell therapy and/or transduction of resting T cells and/or NK cells, can be performed together with the continuous monitoring of the human beings. In other non-limiting illustrative embodiments, after blood is drawn/collected from an individual to transduce T cells and/or NK cells in vitro, blood is reintroduced into any point of the whole method/process of the individual, blood cells will not be cultivated in a room without the presence of an individual. In other non-limiting illustrative embodiments, the entire method/process of reintroducing blood into an individual after blood extraction/collection to ex vivo transduction of T cells and/or NK cells is performed next to the individual and/or in the same room as the individual and/or next to the individual's bed or chair. Therefore, sample consistency confusion can be avoided, as well as long and expensive cultivation over days or weeks. The fact that the method provided herein is easily applicable to closed and automated blood processing systems further confirms this advantage, wherein the blood sample and its components reintroduced into the individual are only contacted with single, disposable components.

Methods for performing adoptive cell therapy provided herein generally include 1) a method for transducing lymphocytes (such as T cells and/or NK cells, which are resting T cells and/or NK cells in illustrative embodiments), and/or include 2) a method for genetically modifying lymphocytes (such as T cells and/or NK cells, which are resting T cells and/or NK cells in illustrative embodiments), both of which (1 and 2) themselves form different aspects of the present invention. Such methods can be performed with or without other steps identified herein for performing adoptive cell therapy. In methods for adoptive cell therapy and any methods provided herein including ex vivo transduction of resting T cells and/or resting NK cells, neutrophils/granulocytes are usually separated from blood cells before contacting the cells with replication-deficient recombinant retroviral particles. In some embodiments, peripheral blood mononuclear cells (PBMCs) including peripheral blood lymphocytes (PBL) (such as T cells and/or NK cells) are separated from other components of a blood sample using, for example, hemacytosis and/or density gradient centrifugation. In some embodiments, neutrophils are removed before PBMCs and/or T cells and/or NK cells are treated, contacted with replication-defective recombinant retroviral particles, transduced or transfected.With respect to the individual to be treated, the cells may be allogeneic and/or autologous.

As a non-limiting example, in some embodiments of the method for transduction or genetic modification herein, PBMCs are separated using Sepax or Sepax 2 cell processing systems (BioSafe). In some embodiments, PBMCs are separated using CliniMACS Prodigy cell processors (Miltenyi Biotec). In some embodiments, blood is collected from an individual using an automatic blood cell separator, the blood is passed through a device that selects peripheral cell types (such as PBMCs), and the remainder is returned to the individual. Density gradient centrifugation can be performed after blood cell separation. In some embodiments, PBMCs are separated using a filter device that removes leukocytes. In some embodiments, magnetic bead activated cell sorting is then used to purify specific cell populations from PBMCs (such as PBLs or their subsets) according to cell phenotypes (i.e., positive selection). Other methods for purification may also be used, such as matrix adhesion, which utilizes a matrix that simulates the environment encountered by T cells during supplementation, thereby causing them to adhere and migrate, or negative selection, wherein unwanted cells are removed using antibody complexes (targeting unwanted cells). In some embodiments, erythrocyte rosetting can be used to purify cells.

In some illustrative embodiments of any one of the related aspects herein, PBL includes T cells and/or NK cells. During certain embodiments herein, such as in methods for modifying lymphocytes and methods for performing adoptive cell therapy, the T cells and/or NK cells contacted with the replication-deficient recombinant retroviral particles of the present invention are mainly resting T cells. In some embodiments, T cells and/or NK cells include resting cells (Ki-67 ^- ) between 95% and 100%. In some embodiments, T cells and/or NK cells contacted with replication-deficient recombinant retroviral particles include 90%, 91%, 92%, 93%, 94% and 95% resting cells at the low end of the range and 96%, 97%, 98%, 99% or 100% resting cells at the high end of the range. In some embodiments, T cells and/or NK cells include protozoa.

In some embodiments of the methods and compositions disclosed herein, T cells and/or NK cells are contacted in vitro with replication-deficient recombinant retroviral particles to genetically modify T cells and/or NK cells to cause a targeted immune response in an individual when reintroduced into the individual. During the contact period, the replication-deficient recombinant retroviral particles recognize T cells and/or NK cells and bind to T cells and/or NK cells, at which point the retrovirus begins to fuse with the host cell membrane. Then, via a transduction procedure, the genetic material from the replication-deficient recombinant retroviral particles enters the T cells and/or NK cells and is incorporated into the host cell DNA. The method of lentiviral transduction is known. Exemplary methods are described in, e.g., Wang et al. (2012) J. Immunother. 35(9):689-701, Cooper et al. (2003) Blood. 101:1637-1644, Verhoeyen et al. (2009) Methods Mol Biol. 506:97-114, and Cavalieri et al. (2003) Blood. 102(2):497-505.

Many methods in the method provided herein include transducing T cells and/or NK cells. It is known in the art to use a method for transducing T cells and/or NK cells with replication-defective recombinant retroviral particles (such as replication-defective recombinant lentiviral particles) in vitro. In an illustrative embodiment, the method provided herein does not require ex vivo stimulation or activation. Therefore, this conventional step in the previous method can be avoided in the present method, but ex vivo stimulatory molecules (such as anti-CD3 and/or anti-CD28 beads) may be present during transduction. However, using the illustrative method provided herein, ex vivo stimulation is not required. In some exemplary methods, between 3 and 10 heavy infections (MOI), and in some embodiments, the replication-defective recombinant retroviral particles of the unit between 5MOI and 10MOI, such as lentivirus, can be used.

The transduction reaction can be performed in a closed system (such as the Sepax system as discussed herein), wherein the transduction reaction can be performed in a disposable bag loaded on the system. Once blood cells (such as PBMCs) from a blood sample collected from an individual are separated, isolated and/or purified from granulocytes (including neutrophils), these blood cells can be contacted with the replication-defective recombinant retroviral particles disclosed herein in the bag, wherein the granulocytes are generally not present during the contacting step (i.e., the transduction reaction).

The replication-defective recombinant retroviral particles can be reintroduced into the bag containing the isolated PBMCs, thereby contacting the PBMCs. The time from the time of blood collection from the individual to the time of adding blood cells (such as PBMCs) to the transduction reaction bag can be between 30 minutes and 4 hours, between 30 minutes and 2 hours, or in some examples about 1 hour.

In certain embodiments where cells are infused into an individual, after transduction, before the transduced T cells and/or NK cells are infused back into the individual, the cells are washed to remove the transduction reaction mixture prior to infusion back into the individual. For example, before the transduced cells are infused back into the individual, a system (such as a Sepax instrument) can be used to wash the cells, for example, with 10 ml to 50 ml of a washing solution. In some embodiments, neutrophils are removed prior to treatment of PBMCs and/or T cells and/or NK cells, contact with replication-defective recombinant retroviral particles, transduction, or transfection.

In an illustrative embodiment for performing adoptive cell therapy, blood is collected from an individual into a blood bag, and the blood bag is connected to a cell processing system, such as a Sepax cell processing system. The PBMC separated using the cell processing system is collected into a bag, and the PBMC is contacted with replication-deficient recombinant retroviral particles under conditions sufficient to transduce T cells and/or NK cells, and cultivated. After cultivation, a bag containing a mixture of PBMC and replication-deficient recombinant retroviral particles is connected to the cell processing system, and the PBMC is washed. The washed PBMC is collected into a bag and re-infused into an individual. In certain embodiments, the entire method from collecting blood to re-infusing transduced T cells and/or NK cells is performed within 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 15 hours, 18 hours or 24 hours. In an illustrative embodiment, the entire method is performed within 12 hours.

In some embodiments, the target cell of the replication-deficient recombinant retroviral particle is a PBL. In some embodiments, the target cell is a T cell and/or a NK cell. In some embodiments, the T cell is a helper T cell and/or a killer T cell.

In some embodiments, the replication-deficient recombinant retroviral particles provided herein have pseudotyped components on their surface, which can bind to T cells and/or NK cells and contribute to the membrane fusion of replication-deficient recombinant retroviral particles. In other embodiments, the replication-deficient recombinant retroviral particles have activation components on their surface that can bind to resting T cells and/or NK cells. In yet other embodiments, the replication-deficient recombinant retroviral particles have membrane-bound cytokines on their surface. In some embodiments, the replication-deficient recombinant retroviral particles include polynucleotides having one or more transcription units encoding one or more engineered signaling polypeptides, one or more of which include one or more lymphoproliferative components lymphoproliferative components. In other embodiments, when two signaling polypeptides are used, one includes at least one lymphoproliferative component lymphoproliferative component, and the other is generally a chimeric antigen receptor (CAR) including an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain. As indicated herein, the activation assembly that is usually associated with the surface of the replication-deficient recombinant retroviral particles provided herein is able to contact resting T cells and/or NK cells for a sufficient period of time, and due to the contact and activation of resting T cells and/or NK cells under appropriate conditions. It should be understood that this activation occurs over time during the contact step of the method herein. In addition, it should be understood that in some embodiments where the pseudotyped assembly is found on the surface of the replication-deficient recombinant retroviral particles that bind T cells and/or NK cells, in the method herein, activation can be induced by binding the pseudotyped assembly. The activation assembly is present as appropriate in those embodiments.

Additional descriptions of pseudotyping components, activation components, membrane-bound cytokines, engineered signaling polypeptides, lymphoproliferative components, lymphoproliferative components, and CARs are provided elsewhere herein.

In some embodiments of the methods and compositions disclosed herein, between 5% and 90% of the total lymphocytes collected from the blood are transduced. In certain embodiments, the percentage of transduced lymphocytes is between 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, and 60% at the low end of the range and 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, and 90% at the high end of the range. In some embodiments, the percentage of transduced lymphocytes is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, or at least 60%.

In some embodiments of the methods and compositions disclosed herein, genetically modified T cells and/or NK cells are introduced back, reintroduced or reinfused into an individual without additional ex vivo manipulation, such as stimulation and/or activation of T cells and/or NK cells. In prior art methods, ex vivo manipulation is used to stimulate/activate T cells and/or NK cells, and for amplifying genetically modified T cells and/or NK cells before genetically modified T cells and/or NK cells are introduced into an individual. In prior art methods, this usually takes several days or weeks, and requires individuals to return to the clinic for blood transfusion for several days or weeks after initial blood draw. In some embodiments of the methods and compositions disclosed herein, before T cells and/or NK cells contact with replication-deficient recombinant retroviral particles, T cells and/or NK cells are not stimulated in vitro by exposure to anti-CD3/anti-CD28 solid carriers (such as, beads coated with anti-CD3/anti-CD28). Therefore, a method for ex vivo transmission-free is provided herein. In other embodiments, the genetically modified T cells and/or NK cells are not expanded in vitro, or only a small number of cell divisions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 rounds of cell division) are expanded, but instead refer to in vivo (i.e., in the individual) expansion or mainly in vivo expansion. In some embodiments, no additional medium is added to allow additional expansion of the cells. In some embodiments, when the PBL is contacted with the replication-deficient recombinant retroviral particles, the cell manufacturing of the PBL does not occur. In illustrative embodiments, the cell manufacturing of the PBL does not occur when the PBL is ex vivo. In previous methods of adoptive cell therapy, the individual is subjected to lympholysis before reinfusion of genetically modified T cells and/or NK cells. In some embodiments, the patient or individual does not perform lympholysis before blood is drawn. In some embodiments, the patient or individual does not perform lympholysis before reinfusion of genetically modified T cells and/or NK cells. However, the embodiments of the methods and compositions disclosed herein can also be used for pre-activated or pre-stimulated T cells and/or NK cells. In some embodiments, T cells and/or NK cells can be stimulated in vitro by exposing them to anti-CD3/anti-CD28 solid supports before contacting them with replication-defective recombinant retroviral particles. In some embodiments, before contacting T cells and/or NK cells with replication-defective recombinant retroviral particles, T cells and/or NK cells can be exposed to anti-CD3/anti-CD28 solid supports for less than 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours or 24 hours. In illustrative embodiments, before contacting T cells and/or NK cells with replication-defective recombinant retroviral particles, T cells and/or NK cells can be exposed to anti-CD3/anti-CD28 solid supports for less than 1 hour, 2 hours, 3 hours, 4 hours, 6 hours or 8 hours.

In any of the embodiments disclosed herein, the number of T cells and/or NK cells to be reinfused into a subject can be between 1×10 ³ , 2.5×10 ³ , 5×10 3 , 1×10 ⁴ , 2.5×10 ⁴ , 5×10 ⁴ , 1×10 ⁵ , 2.5×10 ⁵ , 5×10 ⁵ , 1×10 ⁶ , 2.5×10 ⁶ , 5×10 6 , and 1×10 ⁷ cells/kg at the low end of the range and 5×10 ⁴ , 1×10 ⁵ , 2.5×10 ⁵ , 5×10 ⁵ , 1×10 ⁶ , 2.5×10 ⁶ , 5×10 ⁶ , 1×10 ⁷ , 2.5×10 ⁷ , 5×10 ⁷ , and 1× ^{10 8 cells} /kg at the high ^end of the range. In illustrative embodiments, the number of T cells and/or NK cells to be reinfused into an individual may be between 1×10 ⁴ , 2.5×10 ⁴ , 5×10 ⁴ , and 1×10 ⁵ cells/kg at the low end of the range and 2.5×10 ⁴ , 5×10 ⁴ , 1×10 ⁵ , 2.5×10 ⁵ , 5×10 ⁵ , and 1×10 ⁶ cells/kg at the high end of the range. In some embodiments, the number of PBLs to be reinfused into an individual may be less than 5×10 ⁵ , 1×10 ⁶ , 2.5×10 ⁶ , 5×10 ⁶ , 1×10 7 , 2.5×10 ⁷ , 5×10 ⁷ , and 1×10 ⁸ cells at the low end of the range, and 2.5×10 ⁶ , 5×10 ⁶ , 1×10 ⁷ , 2.5×10 ⁷ , 5×10 ⁷ , 1×10 ⁸ , 2.5×10 ⁸ , 5×10 ⁸ , and 1×10 ⁹ cells at the high end of the range. In some embodiments, the number of T cells and/or ^NK cells that can be used for reinfusion into a 70 kg individual or patient is between 7×10 ⁵ cells and 2.5×10 ⁸ cells. In other embodiments, the number of T cells and/or NK cells that can be used for transduction is approximately 7×10 ⁶ plus or minus 10%.

In the methods disclosed herein, the entire adoptive cell therapy procedure (from blood extraction to re-infusion of genetically modified T cells and/or NK cells) can be advantageously performed in a shorter time than previous methods. In some embodiments, the entire adoptive cell therapy procedure can be performed in less than 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 15 hours, 18 hours, or 24 hours. In illustrative embodiments, the entire adoptive cell therapy procedure can be performed in less than 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, or 12 hours. In some embodiments, the entire adoptive cell therapy procedure may be performed between 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours or 15 hours at the low end of the range and 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 15 hours, 18 hours or 24 hours at the high end of the range.

In some embodiments herein, a closed system is used to process PBMCs, for example, in a method comprising genetically modifying PBMCs, NK cells, and T cells in illustrative embodiments, for example, by transducing PBMCs or a subset thereof. These methods can be used to genetically modify lymphocytes to be used for scientific research, commercial production, or treatment methods. For example, these methods can include transferring peripheral blood mononuclear cells (PBMCs) (including NK cells, T cells, or both, and in some embodiments, resting T cells and/or resting NK cells) from a container to a transduction reaction mixture in a closed system, which therefore does not require environmental exposure. The container can be, for example, a tube, a bag, a syringe, or other container. In some embodiments, the container is a container for a research facility. In some embodiments, the container is a container for commercial production. In other embodiments, the container can be a collection container for a nighttime blood collection process. The method for genetically modifying herein generally involves a contact step in which lymphocytes are contacted with replication-deficient recombinant retroviral particles. In some embodiments, the contact can be performed in a container (e.g., in a blood bag). In other embodiments, PBMC or its sub-portion can be transferred from the container to another container in the closed system (e.g., from the first container to the second container) for contact. The second container can be a cell processing compartment of a closed device (such as a G-Rex device). In some embodiments, after contact, cells modified by genetic means (e.g., transduced) can be transferred to different containers in the closed system (i.e., without exposure to the environment). Before or after this transfer, cells are usually washed in a closed system to substantially remove all or all retroviral particles. In some embodiments, after contact, PBMC or its portion is transferred to a container in which contact (e.g., transduction) will occur via washing cells. The process disclosed in this paragraph is performed within 12 hours. In some embodiments, it is performed between 1 hour, 2 hours, 3 hours or 4 hours at the low end of the range and 4 hours, 8 hours, 10 hours or 12 hours at the high end of the range.

In some embodiments provided herein, the steps of extracting a blood sample from an individual, contacting T cells and/or NK cells with replication-deficient recombinant retroviral particles, and/or introducing genetically modified T cells and/or NK cells into an individual occur in a closed system. A closed system is a culture method that is usually closed or completely closed to contamination. Therefore, this system or process does not expose cells to the environment. An advantage of the present invention is that a method for performing CAR therapy in a closed system is provided herein. One of the highest risks for safety and regulation in cell processing procedures is the risk of contamination through frequent exposure to the environment, as found in traditional open cell culture systems. In order to mitigate this risk, especially in the absence of antibiotics, some commercial methods dedicated to the use of disposable (single-use) equipment have been developed. However, even when used under sterile conditions, there is always a risk of contamination when opening a flask to sample or add additional growth medium. In order to overcome this problem, a closed system method is provided herein, a method designed and operable so that the product is not exposed to the external environment. This is important because the external environment is usually not sterile. Material transfer is performed via a sterile connection or welded tube. Air for gas exchange is passed through a 0.2 μm filter via a breathable membrane or similar other additives to prevent environmental exposure.

In some embodiments, the closed system comprises an ex vivo circulatory system connected to the in vivo circulatory system of the individual so that blood is drawn and then circulated to the ex vivo circulatory system before being introduced back to the individual. In some embodiments, the ex vivo circulatory system comprises a system or device for isolating PBLs and/or a system or device for isolating T cells and/or NK cells in combination with a system or device for exposing cells to replication-defective recombinant retroviral particles. In some embodiments, the closed system does not allow T cells and/or NK cells to be exposed to air.

Such closed system methods can be performed using commercially available devices. For example, the method can be performed in a device suitable for closed system T cell production. Such devices include G-Rex ^™ , WAVE Bioreactor ^™ , OriGen PermaLife ^™ bags, and bag.

In some embodiments of the methods and compositions disclosed herein, genetically modified T cells and/or NK cells in an individual are exposed to a compound that binds to an in vivo control component present therein, wherein the control component is a portion of the genetic material introduced by a replication-deficient recombinant retroviral particle. In some embodiments, the control component may be a riboswitch, and the compound may bind to the aptamer domain of the riboswitch. In some embodiments, the control component may be a chaperone. A chaperone is a compound that directly affects the activity of other components of a lymphoproliferative component or the first or second engineered signaling polypeptide herein, usually by binding. In any of the embodiments disclosed herein, the compound may be a nucleoside analog. In some embodiments, the nucleoside analog may be a nucleoside analog antiviral drug, wherein the antiviral drug is a compound approved by the Food and Drug Administration for antiviral therapy or a compound in a U.S. antiviral clinical trial. In illustrative embodiments, the compound may be acyclovir or penciclovir. In some embodiments, the compound may be famciclovir (an oral prodrug of penciclovir) or valaciclovir (an oral prodrug of acyclovir). The combination of the compound and the control component affects the expression of the introduced genetic material and thus affects the propagation of the genetically modified T cells and/or NK cells.

In some embodiments, a nucleoside analog antiviral drug or prodrug (e.g., acyclovir, valacyclovir, penciclovir, or famciclovir) is administered to the subject before, simultaneously with, and/or after the PBLs are isolated from the subject's blood and before the T cells and/or NK cells are contacted with the replication-defective recombinant retroviral particles. In some embodiments, a nucleoside analog antiviral drug or prodrug is administered to the subject between 5 minutes, 10 minutes, 15 minutes, 30 minutes, and 60 minutes at the low end of the range and 1.5 hours, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 12 hours, or 24 hours at the high end of the range before the PBLs are isolated from the blood or before the T cells and/or NK cells are contacted with the replication-defective recombinant retroviral particles. In other embodiments, a nucleoside analog antiviral drug or prodrug is administered to a subject between 1.5 hours, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 12 hours or 24 hours at the low end of the range and 1/2 day, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 14 days, 21 days or 28 days at the high end of the range after PBLs are isolated from the blood and after T cells and/or NK cells are contacted with the replication-defective recombinant retroviral particles in the methods provided herein. In some embodiments, a nucleoside analog antiviral drug or prodrug is administered to a subject at least 1.5 hours, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 12 hours, or 24 hours, or at least 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 14 days, 21 days, or 28 days after PBLs are isolated from the blood and after the T cells and/or NK cells are contacted with the replication-defective recombinant retroviral particles in the methods provided herein. In some embodiments, the nucleoside analog antiviral drug or prodrug is administered to the subject at least 1 day, 2 days, 3 days, 4 days, 5 days, 7 days, 10 days, 14 days, 21 days, 28 days, 30 days, 60 days, 90 days, or 120 days, or 5 months, 6 months, 9 months, 12 months, 24 months, 36 months, 48 months, 60 months, 72 months, 84 months, 96 months, 120 months, or indefinitely after PBLs have been reinfused into the subject. In any of the embodiments disclosed herein, the nucleoside analog antiviral drug or prodrug may be administered before and/or during PBL reinfusion and/or after PBLs have been reinfused.

In some embodiments, the compound combined with the control component is administered to the individual once, twice, three times, or four times daily. In some embodiments, the daily dose of the compound is provided for 1 week, 2 weeks, 4 weeks, 3 months, 6 months, 1 year, until the individual is disease-free (such as cancer-free) or indefinitely. In illustrative embodiments, the drug is a nucleoside analog antiviral drug combined with a nucleoside analog, such as a riboswitch, as further disclosed in detail in WO2017/165245A2, WO2018/009923A1, and WO2018/161064A1.

Methods for delivering drugs are known in the art, whether small molecules or biologics and can be used in the methods provided herein. Any such method can be used to deliver drugs or candidate compounds or antibodies used in the methods of the present invention. For example, conventional routes of administration include non-invasive oral (via mouth), topical (skin), transmucosal (nasal, buccal/sublingual, vaginal, ocular and rectal) and inhalation routes. Many protein and peptide drugs (such as monoclonal antibodies) must be delivered by injection or nanoneedle arrays. For example, many immunizations are based on the delivery of protein drugs, and are usually performed by injection.

Engineered signaling peptides

In some embodiments, the replication-deficient recombinant retroviral particles for contacting T cells and/or NK cells have a polynucleotide having one or more transcription units encoding one or more engineered signaling polypeptides. In some embodiments, the engineered signaling polypeptide includes any combination of an extracellular domain (e.g., an antigen-specific targeting region or ASTR), a handle, and a transmembrane domain, with one or more intracellular activation domains, one or more regulatory domains (such as a co-stimulatory domain) selected as appropriate, and one or more T cell survival primitives selected as appropriate. In illustrative embodiments, at least one, both, or all of the engineered signaling polypeptides are chimeric antigen receptors (CARs) or lymphoproliferative components lymphoproliferative components lymphoproliferative components (LEs) (such as chimeric lymphoproliferative components (CLEs)). In some embodiments, when two signaling polypeptides are utilized, one encodes a lymphoproliferative component lymphoproliferative component and the other encodes a chimeric antigen receptor (CAR) including an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain. One of ordinary skill in the art will be able to configure the system for the methods and compositions disclosed herein to place the lymphoproliferative element, lymphoproliferative element and CAR on different polynucleotides using similar or dissimilar control elements. One of ordinary skill in the art will recognize that an engineered polypeptide may also be referred to as a recombinant polypeptide.

Extracellular domain

In some embodiments, the engineered signaling polypeptide includes an extracellular domain that is a member of a specific binding pair. For example, in some embodiments, the extracellular domain may be an extracellular domain of a cytokine receptor or a mutant thereof or a hormone receptor or a mutant thereof. This mutant extracellular domain is reported in some embodiments to be constitutively active when expressed in at least some cell types. In illustrative embodiments, this extracellular domain and transmembrane domain do not include a ligand binding region. It is believed that these domains do not bind ligands when present in an engineered signaling polypeptide and expressed in B cells, T cells and/or NK cells. Mutations in these receptor mutants may occur in the extracellular near-membrane region. Not limited by theory, mutations in at least some extracellular domains (and some extracellular-transmembrane domains) of the engineered signaling polypeptides provided herein are responsible for the signaling of the engineered signaling polypeptide in the absence of a ligand by bringing together activation chains that are usually not together. Additional embodiments of extracellular domains comprising mutations in the extracellular domain can be found, for example, in the lymphoproliferative component lymphoproliferative component section herein.

In certain illustrative embodiments, the extracellular domain comprises a dimerization motif. In an illustrative embodiment, the dimerization motif comprises a leucine zipper. In some embodiments, the leucine zipper is from a jun polypeptide, such as c-jun. Additional embodiments of extracellular domains comprising a dimerization motif may be found, for example, in the lymphoproliferative component lymphoproliferative component section herein.

In certain embodiments, the extracellular domain is an antigen-specific targeting region (ASTR), sometimes referred to herein as an antigen binding domain. Specific binding pairs include, but are not limited to, antigen-antibody binding pairs; ligand-receptor binding pairs; and the like. Thus, members of specific binding pairs suitable for use in the engineered signaling polypeptides of the present invention include ASTRs that are antibodies, antigens, ligands, receptor binding domains of ligands, receptors, ligand binding domains of receptors, and affibodies.

ASTRs suitable for use in the engineered signaling polypeptides of the present invention can be any antigen-binding polypeptides. In certain embodiments, ASTRs are antibodies, such as full-length antibodies, single-chain antibodies, Fab fragments, Fab' fragments, (Fab')2 fragments, Fv fragments, and bivalent single-chain antibodies or bifunctional antibodies.

In some embodiments, the ASTR is a single chain Fv (scFv). In some embodiments, the heavy chain is located at the N-terminus of the light chain in the engineered signaling polypeptide. In other embodiments, the light chain is located at the N-terminus of the heavy chain in the engineered signaling polypeptide. In any of the disclosed embodiments, the heavy chain and the light chain may be separated by a connector, as discussed in more detail herein. In any of the disclosed embodiments, the heavy chain or the light chain may be at the N-terminus of the engineered signaling polypeptide and typically at the C-terminus of another domain, such as a signal sequence or signal peptide.

Other antibody-based recognition domains (cAb VHH (camelid antibody variable domain) and humanized versions, IgNAR VH (shark antibody variable domain) and humanized versions, sdAb VH (single domain antibody variable domain) and "camelized" antibody variable domain) are suitable for use with engineered signaling polypeptides and are suitable for use in methods of using engineered signaling polypeptides of the invention. In some cases, it is also suitable to use T cell (TCR)-based recognition domains, such as single-chain TCR (scTv, a single-chain two-domain TCR containing VαVβ).

In some embodiments, ASTRs may be multispecific, such as bispecific antibodies. Multispecific antibodies have binding specificities for at least two different sites. In certain embodiments, one of the binding specificities is for one target antigen and the other is for another target antigen. In certain embodiments, bispecific antibodies may bind to two different antigenic determinants of a target antigen. Bispecific antibodies may also be used to localize cytotoxic agents to cells expressing target antigens. Bispecific antibodies may be prepared as full-length antibodies or antibody fragments.

ASTRs suitable for use in the engineered signaling polypeptides of the present invention may have a variety of antigen binding specificities. In some cases, the antigen binding domain is specific for an antigenic determinant present in an antigen expressed (synthesized) by a target cell. In one example, the target cell is an antigen associated with a cancer cell. Cancer cell-associated antigens may be antigens associated with, for example, breast cancer cells, B-cell lymphomas, Hodgkin's lymphoma cells, ovarian cancer cells, prostate cancer cells, mesothelioma, lung cancer cells (e.g., small cell lung cancer cells), non-Hodgkin's B-cell lymphoma (B-NHL) cells, ovarian cancer cells, prostate cancer cells, mesothelioma cells, lung cancer cells (e.g., small cell lung cancer cells), melanoma cells, chronic lymphocytic leukemia cells, acute lymphocytic leukemia cells, neuroblastoma cells, gliomas, glioblastomas, medulloblastomas, colon cancer cells, etc. Cancer cell-associated antigens may also be expressed by non-cancerous cells.

Non-limiting examples of antigens to which the ASTR of the engineered signaling polypeptide can bind include, e.g., CD19, CD20, CD38, CD30, ERBB2, CA125, MUC-1, prostate-specific membrane antigen (PSMA), CD44 surface adhesion molecule, mesothelin, carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR), EGFRvIII, vascular endothelial growth factor receptor-2 (VEGFR2), high molecular weight melanoma-associated antigen (HMW-MAA), MAGE-Al, IL-13R-a2, GD2, Axl, Ror2, and the like.

In some cases, members of the specific binding pairs suitable for use in engineered signaling polypeptides are ASTRs that are ligands of receptors. Ligands include, but are not limited to: hormones (e.g., erythropoietin, growth hormone, leptin, etc.); cytokines (e.g., interferons, interleukins, certain hormones, etc.); growth factors (e.g., regulatory proteins; vascular endothelial growth factor (VEGF); and the like); integrin binding peptides (e.g., peptides comprising the sequence Arg-Gly-Asp); and the like.

When the member of the specific binding pair in the engineered signaling polypeptide is a ligand, the engineered signaling polypeptide can be activated in the presence of a second member of the specific binding pair, wherein the second member of the specific binding pair is a receptor for the ligand. For example, when the ligand is VEGF, the second member of the specific binding pair can be a VEGF receptor including a soluble VEGF receptor.

As mentioned above, in some cases, the member of the specific binding pair included in the engineered signaling polypeptide is an ASTR, which is a receptor, such as a receptor for a ligand, a co-receptor, etc. The receptor may be a ligand binding fragment of a receptor. Suitable receptors include, but are not limited to, growth factor receptors (e.g., VEGF receptor); killer cell lectin-like receptor subfamily K; member 1 (NKG2D) polypeptide (receptor for MICA, MICB, and ULB6); cytokine receptors (e.g., IL-13 receptor; IL-2 receptor, etc.); CD27; natural cytotoxicity receptor (NCR) (e.g., NKP30 (NCR3/CD337) polypeptide (receptor for HLA-B associated transcript 3 (BAT3) and B7-H6), etc.), etc.

In certain embodiments of any of the aspects provided herein including ASTRs, the ASTRs may be positioned to intermediate proteins that connect the ASTRs to targeting molecules expressed on target cells. The intermediate proteins may be endogenously expressed or exogenously introduced, and may be naturally, engineered, or chemically modified. In certain embodiments, the ASTRs may be anti-labeled ASTRs, such that at least one labeled intermediate (usually an anti-labeled conjugate) is included between a label recognized by the ASTR and a targeting molecule (usually a protein target expressed on a target cell). Thus, in these embodiments, the ASTRs bind to labels and the labels are conjugated to antibodies against antigens on target cells (such as cancer cells). Non-limiting examples of labels include fluorescein isothiocyanate (FITC), streptavidin, biotin, histidine, dinitrophenol, peridinin chlorophyll protein complex, green fluorescent protein, phycoerythrin (PE), horseradish peroxidase, palmitoylation, nitrosylation, alkaline phosphatase, glucose oxidase, and maltose binding protein. Thus, the ASTRs include molecules that bind to labels.

handle

In some embodiments, the engineered signaling polypeptide includes a handle located in the portion of the engineered signaling polypeptide that is located outside the cell and inserted between the ASTR and the transmembrane domain. In some cases, the handle is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the wild-type CD8 handle region (TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGG AVHTRGLDFA (SEQ ID NO: 79)), at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the wild-type CD28 handle region (FCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 80)), or at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the wild-type immunoglobulin heavy chain handle region. In the engineered signaling polypeptide, the handle used allows the antigen-specific targeting region, and often the entire engineered signaling polypeptide, to retain increased binding to the target antigen.

The handle region may be about 4 to about 50 amino acids in length, e.g., about 4 amino acids to about 10 amino acids, about 10 amino acids to about 15 amino acids, about 15 amino acids to about 20 amino acids, about 20 amino acids to about 25 amino acids, about 25 amino acids to about 30 amino acids, about 30 amino acids to about 40 amino acids, or about 40 amino acids to about 50 amino acids.

In some cases, the handle of the engineered signaling polypeptide includes at least one cysteine. For example, in some cases, the handle can include the sequence Cys-Pro-Pro-Cys (SEQ ID NO: 62). If present, the cysteine in the handle of the first engineered signaling polypeptide can be capable of forming a disulfide bond with the handle in the second engineered signaling polypeptide.

The handle may include an immunoglobulin hinge region amino acid sequence known in the art; see, eg, Tan et al. (1990) Proc. Natl. Acad. Sci. USA 87:162; and Huck et al. (1986) Nucl. Acids Res. 14:1779. As non-limiting examples, an immunoglobulin hinge region can include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20 or all of the amino acids of any of the following amino acid sequences: DKTHT (SEQ ID NO:63); CPPC (SEQ ID NO:62); CPEPKSCDTPPPCPR (SEQ ID NO:64); (see, e.g., Glaser et al. (2005) J. Biol. Chem. 280:41494); ELKTPLGDTTHT (SEQ ID NO:65); KSCDKTHTCP (SEQ ID NO:66); KCCVDCP (SEQ ID NO:67); KYGPPCP (SEQ ID NO:68); EPKSCDKTHTCPPCP (SEQ ID NO:69); NO:69) (human IgG1 hinge); ERKCCVECPPCP (SEQ ID NO:70) (human IgG2 hinge); ELKTPLGDTTHTCPRCP (SEQ ID NO:71) (human IgG3 hinge); SPNMVPHAHHAQ (SEQ ID NO:72) (human IgG4 hinge); and the like. The handle may include a hinge region having the amino acid sequence of a human IgG1, IgG2, IgG3, or IgG4 hinge region. The handle may include one or more amino acid substitutions and/or insertions and/or deletions compared to a wild-type (naturally occurring) hinge region. For example, His229 of the human IgG1 hinge may be substituted with Tyr such that the handle includes the sequence EPKSCDKTYTCPPCP (see, e.g., Yan et al. (2012) J. Biol. Chem. 287:5891). The handle may include an amino acid sequence derived from human CD8; for example, the handle may include the amino acid sequence:

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 73), or a variant thereof.

Transmembrane domain

The engineered signaling polypeptide of the present invention may include a transmembrane domain for insertion into a eukaryotic cell membrane. The transmembrane domain may be inserted between the ASTR and the costimulatory domain. The transmembrane domain may be inserted between the handle and the costimulatory domain, so that the chimeric antigen receptor includes, from the amino terminus (N-terminus) to the carboxyl terminus (C-terminus), the ASTR, the handle, the transmembrane domain, and the activation domain.

Any transmembrane (TM) domain that provides for insertion of a polypeptide into the cell membrane of a eukaryotic (eg, mammalian) cell is suitable for use with the aspects and embodiments disclosed herein. Non-limiting examples of TM domains suitable for use in any of the aspects or embodiments provided herein include domains having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20 or all amino acids of any of the following TM domains or combined handle and TM domains: a) CD8α TM (SEQ ID NO:46); b) CD8β TM (SEQ ID NO:47); c) CD4 handle (SEQ ID NO:48); d) CD3Z TM (SEQ ID NO:49); e) CD28 TM (SEQ ID NO:50); f) CD134 (OX40) TM (SEQ ID NO:51); g) CD7 TM (SEQ ID NO:52); h) CD8 handle and TM (SEQ ID NO:53). NO:75); and i) CD28 handle and TM (SEQ ID NO:76).

As a non-limiting example, a transmembrane domain of one aspect of the present invention may have at least 80%, 90% or 95% or may have 100% sequence identity with the transmembrane domain of SEQ ID NO:46, or may have 100% sequence identity with any one of the transmembrane domains from the following genes, respectively: CD8β transmembrane domain, CD4 transmembrane domain, CD3ζ transmembrane domain, CD28 transmembrane domain, CD134 transmembrane domain, or CD7 transmembrane domain.

intracellular activation domain

The intracellular activation domains in the engineered signaling polypeptides suitable for use in the present invention generally induce the production of one or more cytokines upon activation; increase cell death; and/or increase the proliferation of CD8 ⁺ T cells, CD4 ⁺ T cells, NKT cells, γδT cells and/or neutrophils. The activation domain may also be referred to herein as the activation domain. The activation domain may be used in the CAR provided herein or in the lymphoproliferative component lymphoproliferative component.

In some embodiments, the intracellular activation domain includes at least one (e.g., one, two, three, four, five, six, etc.) ITAM motif as described below. In some embodiments, the intracellular activation domain may have at least 80%, 90%, or 95% or may have 100% sequence identity with a CD3Z, CD3D, CD3E, CD3G, CD79A, CD79B, DAP12, FCER1G, FCGR2A, FCGR2C, DAP10/CD28, or ZAP70 domain as described below.

Intracellular activation domains suitable for use in the engineered signaling polypeptides of the present invention include intracellular signaling polypeptides containing an immunotyrosine-based activation motif (ITAM). The ITAM motif is YX ₁ X ₂ L/I, wherein X ₁ and X ₂ are independently any amino acids. In some cases, the intracellular activation domain of the engineered signaling polypeptide includes 1, 2, 3, 4 or 5 ITAM motifs. In some cases, the ITAM motif is repeated twice in the intracellular activation domain, wherein the first instance and the second instance of the ITAM motif are separated from each other by 6 to 8 amino acids (e.g., (YX ₁ X ₂ L/I)(X ₃ ) _n (YX ₁ X ₂ L/I), wherein n is an integer from 6 to 8, and each of the 6 to 8 X ₃ can be any amino acid). In some cases, the intracellular activation domain of the engineered signaling polypeptide includes 3 ITAM motifs.

A suitable intracellular activation domain may be a portion containing an ITAM motif that is derived from a polypeptide containing an ITAM motif. For example, a suitable intracellular activation domain may be a domain containing an ITAM motif from any protein containing an ITAM motif. Therefore, a suitable intracellular activation domain does not need to contain the entire sequence of the entire protein from which it is derived. Examples of suitable polypeptides containing ITAM motifs include, but are not limited to: CD3Z (CD3ζ); CD3D (CD3δ); CD3E (CD3ε); CD3G (CD3γ); CD79A (antigen receptor complex-associated protein α chain); CD79B (antigen receptor complex-associated protein β chain) DAP12; and FCER1G (Fcε receptor Iγ chain).

In some embodiments, the intracellular activation domain is derived from the T cell surface glycoprotein CD3 zeta chain (also known as CD3Z, T cell receptor T3 zeta chain, CD247, CD3-zeta, CD3H, CD3Q, T3Z, TCRZ, etc.). For example, a suitable intracellular activation domain may include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in the following sequence, or to a continuous stretch of about 100 amino acids to about 110 amino acids (aa), about 110 aa to about 115 aa, about 115 aa to about 120 aa, about 120 aa to about 130 aa, about 130 aa to about 140 aa, about 140 aa to about 150 aa, or about 150 aa to about 160 aa:

MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQL[YNELNLGRREEYDVL]DKRRGRDPEMGGKPRRKNPQEGL[YNELQKDKMAEAYSEI]GMKGERRRGKGHDGL[YQGLSTATKDTYDAL]HMQALPPR(SEQ ID NO:11) or MKWK ALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQL[YNELNLGRREEYDVL]DKRRGRDPEMGGKPQRRKNPQEGL[YNELQKDKMAEAYSEI]GMKGERRRGKGHDGL[YQGLSTATKDTYDAL]HMQALPPR(SEQ ID NO:12), wherein the ITAM primitives are enclosed in brackets.

Likewise, suitable intracellular activation domain polypeptides may include portions of full-length CD3ζ amino acid sequences containing ITAM motifs. Thus, suitable intracellular activation domains may include a stretch of at least 10, 15, 20 or all of the amino acids in the following sequences or a continuous stretch of about 100 amino acids to about 110 amino acids (aa), about 110 aa to about 115 aa, about 115 aa to about 120 aa, about 120 aa to about 130 aa, about 130 aa to about 140 aa, about 140 aa to about 150 aa, or about 150 aa to about 160 aa having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of the following amino acid sequences:

RVKFSRSADAPAYQQGQNQL[YNELNLGRREEYDVL]DKRRGRDPEMGGKPRRKNPQEGL[YNELQKDKMAEAYSEI]GMKGERRRGKGHDGL[YQGLSTATKDTYDAL]HMQALPPR(SEQ ID NO:13);RVKFSRSADAPAYQQGQNQL[YNELNLGRREEYDVL]DKRRGRDPEMGGKP QRRKNPQEGL[YNELQKDKMAEAYSEI]GMKGERRRGKGHDGL[YQGLSTATKDTYDAL]HMQALPPR(SEQ ID NO:81); NQL[YNELNLGRREEYDVL]DKR(SEQ ID NO:14);EGL[YNELQKDKMAEAYSEI]GMK(SEQ ID NO: 15); or DGL[YQGLSTATKDTYDAL]HMQ (SEQ ID NO: 16), wherein the ITAM motif is enclosed in brackets.

In some cases, the intracellular activation domain is derived from the T cell surface glycoprotein CD3δ chain (also known as CD3D, CD3-δ, T3D, CD3 antigen, δ subunit, CD3δ, CD3d antigen, δ polypeptide (TiT3 complex), OKT3, δ chain, T cell receptor T3δ chain, T cell surface glycoprotein CD3δ chain, etc.). Thus, suitable intracellular activation domains may include domains having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in the following sequence, or to a continuous stretch of about 100 amino acids to about 110 amino acids (aa), about 110 aa to about 115 aa, about 115 aa to about 120 aa, about 120 aa to about 130 aa, about 130 aa to about 140 aa, about 140 aa to about 150 aa, or about 150 aa to about 160 aa of any of the following amino acid sequences:

or

MEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRTADTQALLRNDQV[YQPLRDRDDAQYSHL]GGNWARNK (SEQ ID NO: 18), wherein the ITAM motif is enclosed in brackets.

Likewise, suitable intracellular activation domain polypeptides may comprise a portion of the full-length CD3 delta amino acid sequence that contains an ITAM motif. Thus, suitable intracellular activation domains may include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20 or all of the amino acids in the following sequence: DQV[YQPLRDRDDAQYSHL]GGN (SEQ ID NO: 19), wherein the ITAM motif is enclosed in brackets.

In some cases, the intracellular activation domain is derived from the T cell surface glycoprotein CD3ε chain (also known as CD3e, T cell surface antigen T3/Leu-4ε chain, T cell surface glycoprotein CD3ε chain, AI504783, CD3, CD3ε, T3e, etc.). Thus, suitable intracellular activation domains may include domains having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in the following sequence, or to a continuous stretch of about 100 amino acids to about 110 amino acids (aa), about 110 aa to about 115 aa, about 115 aa to about 120 aa, about 120 aa to about 130 aa, about 130 aa to about 140 aa, about 140 aa to about 150 aa, or about 150 aa to about 160 aa of the following amino acid sequence:

MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPD[YEPIRKGQRDLYSGL]NQRRI (SEQ ID NO: 20), wherein the ITAM motif is enclosed in brackets.

Likewise, suitable intracellular activation domain polypeptides may comprise a portion of the full-length CD3ε amino acid sequence containing an ITAM motif. Thus, suitable intracellular activation domains may include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20 or all of the amino acids in the following sequence: NPD[YEPIRKGQRDLYSGL]NQR (SEQ ID NO: 21), wherein the ITAM motif is enclosed in brackets.

In some cases, the intracellular activation domain is derived from the T cell surface glycoprotein CD3γ chain (also known as CD3G, T cell receptor T3γ chain, CD3-γ, T3G, γ polypeptide (TiT3 complex), etc.). Therefore, a suitable intracellular activation domain may include a stretch of at least 10, 15, 20 or all of the amino acids in the following sequence or a continuous stretch of about 100 amino acids to about 110 amino acids (aa), about 110aa to about 115aa, about 115aa to about 120aa, about 120aa to about 130aa, about 130aa to about 140aa, about 140aa to about 150aa, or about 150aa to about 160aa of the following amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity:

MEQGKGLAVLILAIILLQGTLAQSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATISGFLFAEIVSIFVLAVGVYFIAGQDGVRQSRASDKQTLLPNDQL[YQPLKDREDDQYSHL]QGNQLRRN (SEQ ID NO: 22), wherein the ITAM motif is enclosed in brackets.

Likewise, suitable intracellular activation domain polypeptides may comprise a portion of the full-length CD3γ amino acid sequence containing an ITAM motif. Thus, suitable intracellular activation domains may include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20 or all of the amino acids in the following sequence: DQL[YQPLKDREDDQYSHL]QGN (SEQ ID NO: 23), wherein the ITAM motif is enclosed in brackets.

In some cases, the intracellular activation domain is derived from CD79A (also known as B-cell antigen receptor complex-associated protein alpha chain; CD79a antigen (immunoglobulin-related alpha); MB-1 membrane glycoprotein; Ig-alpha; membrane-bound immunoglobulin-related protein; surface IgM-associated protein, etc.). Thus, suitable intracellular activation domains may include domains having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in the following sequence, or to a continuous stretch of about 100 amino acids to about 110 amino acids (aa), about 110 aa to about 115 aa, about 115 aa to about 120 aa, about 120 aa to about 130 aa, about 130 aa to about 140 aa, about 140 aa to about 150 aa, or about 150 aa to about 160 aa of any of the following amino acid sequences:

MPGGPGVLQALPATIFLLFLLSAVYLGPGCQALWMHKVPASLMVSLGEDAHFQCPHNSSNNANVTWWRVLHGNYTWPPEFLGPGEDPNGTLIIQNVNKSHGGIYVCRVQEGNESYQQSCGTYLRVRQPPPRPFLDMGEGTKNRIITAEGIILLFCAVVPGTLLLFRKRWQNEKLGLDAGDEYEDENL[YEGLNLDDCSMYEDI]SRGLQGTY QDVGSLNIGDVQLEKP(SEQ ID [0136] SEQ ID NO: 24) or MPGGPGVLQALPATIFLLFLLSAVYLGPGCQALWMHKVPASLMVSLGEDAHFQCPHNSSNNANVTWWRVLHGNYTWPPEFLGPGEDPNEPPPRPFLDMGEGTKNRIITAEGIILLFCAVVPGTLLLFRKRWQNEKLGLDAGDEYEDENL[YEGLNLDDCSMYEDI]SRGLQGTYQDVGSLNIGDVQLEKP (SEQ ID NO: 25), wherein the ITAM motif is enclosed in brackets.

Likewise, a suitable intracellular activation domain polypeptide may comprise a portion of the full-length CD79A amino acid sequence containing an ITAM motif. Thus, a suitable intracellular activation domain may include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20 or all of the amino acids in the following sequence: ENL[YEGLNLDDCSMYEDI]SRG (SEQ ID NO: 26), wherein the ITAM motif is enclosed in brackets.

In some cases, the intracellular activation domain is derived from DAP12 (also known as TYROBP; TYRO protein tyrosine kinase-binding protein; KARAP; PLOSL; DNAX-activated protein 12; KAR-associated protein; TYRO protein tyrosine kinase-binding protein; killer-activated receptor-associated protein; killer-activated receptor-associated protein, etc.). For example, a suitable intracellular activation domain may include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in the following sequence, or to a continuous stretch of about 100 amino acids to about 110 amino acids (aa), about 110 aa to about 115 aa, about 115 aa to about 120 aa, about 120 aa to about 130 aa, about 130 aa to about 140 aa, about 140 aa to about 150 aa, or about 150 aa to about 160 aa of any of the following amino acid sequences (4 isoforms):

MGGLEPCSRLLLLPLLLAVSGLRPVQAQAQSDCSCSTVSPGVLAGIVMGDLVLTVLIALAVYFLGRLVPRGRGAAEAATRKQRITETESP[YQELQGQRSDVYSDL]NTQRPYYK(SEQ ID NO:27),MGGLEPCSRLLLLPLLLAVSGLRPVQAQAQSDCSCSTVSPGVLAGIVMGDLVLTVLIALAVYFLGRLVPRGRGAAEATRKQR ITETESP[YQELQGQRSDVYSDL]NTQ(SEQ ID NO:28),MGGLEPCSRLLLLPLLLAVSDCSCSTVSPGVLAGIVMGDLVLTVLIALAVYFLGRLVPRGRGAAEAATRKQRITETESP[YQELQGQRSDVYSDL]NTQRPYYK(SEQ ID NO:29) or MGGLEPCSRLLLLPLLLAVSDCSCSTVSPGVLAGIVMGDLVLTVLIALAVYFLGRLVPRGRGAAEATRKQRITETESP[YQELQGQRSDVYSDL]NTQRPYYK (SEQ ID NO:30), wherein the ITAM motif is enclosed in brackets.

Likewise, suitable intracellular activation domain polypeptides may comprise a portion of the full-length DAP12 amino acid sequence containing an ITAM motif. Thus, suitable intracellular activation domains may include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20 or all of the amino acids in the following sequence: ESP[YQELQGQRSDVYSDL]NTQ (SEQ ID NO: 31), wherein the ITAM motif is enclosed in brackets.

In some cases, the intracellular activation domain is derived from FCERIG (also known as FCRG; Fcε receptor I γ chain; Fc receptor γ chain; fc-εRI-γ; fcRγ; fceRIγ; high affinity immunoglobulin epsilon receptor subunit γ; immunoglobulin E receptor, high affinity γ chain, etc.). For example, a suitable intracellular activation domain may include a stretch of at least 10, 15, 20 or all of the amino acids in the following sequence or a continuous stretch of about 50 amino acids to about 60 amino acids (aa), about 60aa to about 70aa, about 70aa to about 80aa, or about 80aa to about 88aa of the following amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity:

MIPAVVLLLLLLVEQAAALGEPQLCYILDAILFLYGIVLTLLYCRLKIQVRKAAITSYEKSDGV[YTGLSTRNQETYETL]KHEKPPQ (SEQ ID NO: 32), wherein the ITAM motif is enclosed in brackets.

Likewise, a suitable intracellular activation domain polypeptide may comprise a portion of the full-length FCER1G amino acid sequence that contains an ITAM motif. Thus, a suitable intracellular activation domain may include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20 or all of the amino acids in the following sequence: DGV[YTGLSTRNQETYETL]KHE (SEQ ID NO: 33), wherein the ITAM motif is enclosed in brackets.

Intracellular activation domains suitable for use in the engineered signaling polypeptides of the present invention include DAP10/CD28 type signaling chains. An example of a DAP10 signaling chain is the amino acid SEQ ID NO: 34. In some embodiments, suitable intracellular activation domains may include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20 or all of the amino acids in SEQ ID NO: 34.

An example of a CD28 signaling chain is the amino acid SEQ ID NO: 35. In some embodiments, a suitable intracellular activation domain may include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20 or all of the amino acids in SEQ ID NO: 35.

Intracellular activation domains suitable for use in the engineered signaling polypeptides of the present invention include ZAP70 polypeptides. For example, suitable intracellular activation domains may include a stretch of at least 10, 15, 20 or all of the amino acids in SEQ ID NO: 36, or a continuous stretch of about 300 amino acids to about 400 amino acids, about 400 amino acids to about 500 amino acids, or about 500 amino acids to about 619 amino acids of the following amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

Regulatory domain

The regulatory domain can change the effect of the intracellular activation domain in the engineered signaling polypeptide, including enhancing or inhibiting the downstream effect of the activation domain or changing the nature of the reaction. The regulatory domain suitable for the engineered signaling polypeptide of the present invention includes a costimulatory domain. The length of the regulatory domain suitable for the inclusion body in the engineered signaling polypeptide can be about 30 amino acids to about 70 amino acids (aa), for example, the length of the regulatory domain can be about 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, about 45aa to about 50aa, about 50aa to about 55aa, about 55aa to about 60aa, about 60aa to about 65aa or about 65aa to about 70aa. In other cases, the length of the regulatory domain can be about 70aa to about 100aa, about 100aa to about 200aa, or greater than 200aa.

The co-stimulatory domain generally enhances and/or changes the nature of the response of the activation domain. The co-stimulatory domain suitable for use in the engineered signaling polypeptide of the present invention is generally a polypeptide derived from a receptor. In some embodiments, the co-stimulatory domain homodimerizes. An individual co-stimulatory domain may be an intracellular portion of a transmembrane protein (that is, a co-stimulatory domain may be derived from a transmembrane protein). Non-limiting examples of suitable co-stimulatory polypeptides include, but are not limited to, 4-lBB (CD137), CD27, CD28, CD28, ICOS, OX40, BTLA, CD27, CD30, GITR, and HVEM for Lck binding (ICΔ). For example, the co-stimulatory domain of the aspect of the present invention may have at least 80%, 90%, or 95% sequence identity with the co-stimulatory domain of 4-lBB (CD137), CD27, CD28, CD28, ICOS, OX40, BTLA, CD27, CD30, GITR, or HVEM for Lck binding (ICΔ). For example, the costimulatory domain of the aspect of the present invention may have at least 80%, 90% or 95% sequence identity with the costimulatory domain of a non-limiting example of a suitable costimulatory polypeptide, including, but not limited to, 4-1BB (CD137), CD27, CD28, CD28 for Lck binding (ICΔ) deletion, ICOS, OX40, BTLA, CD27, CD30, GITR and HVEM. For example, the costimulatory domain of the aspect of the present invention may have at least 80%, 90% or 95% sequence identity with the costimulatory domain of 4-1BB (CD137), CD27, CD28, CD28 for Lck binding (ICΔ) deletion, ICOS, OX40, BTLA, CD27, CD30, GITR or HVEM.

The length of the costimulatory domain suitable for inclusion in the engineered signaling polypeptide can be from about 30 amino acids to about 70 amino acids (aa), for example, the length of the costimulatory domain can be from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, or from about 65 aa to about 70 aa. In other cases, the length of the costimulatory domain can be from about 70 aa to about 100 aa, from about 100 aa to about 200 aa, or greater than 200 aa.

In some cases, the costimulatory domain is derived from the intracellular portion of the transmembrane protein CD137 (also known as TNFRSF9; CD137; 4-1BB; CDwl37; ILA, etc.). For example, a suitable costimulatory domain may include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20 or all of the amino acids in SEQ ID NO: 1. In some of these embodiments, the length of the costimulatory domain is about 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, about 45aa to about 50aa, about 50aa to about 55aa, about 55aa to about 60aa, about 60aa to about 65aa, or about 65aa to about 70aa.

In some cases, the costimulatory domain is derived from the intracellular portion of the transmembrane protein CD28 (also known as Tp44). For example, a suitable costimulatory domain may include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in SEQ ID NO: 2. In some of these embodiments, the length of the costimulatory domain is about 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, about 45aa to about 50aa, about 50aa to about 55aa, about 55aa to about 60aa, about 60aa to about 65aa, or about 65aa to about 70aa.

In some cases, the costimulatory domain is derived from the intracellular portion of the transmembrane protein CD28 deleted for Lck binding (ICΔ). For example, a suitable costimulatory domain may include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20 or all amino acids in SEQ ID NO: 3. In some of these embodiments, the length of the costimulatory domain is about 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, about 45aa to about 50aa, about 50aa to about 55aa, about 55aa to about 60aa, about 60aa to about 65aa, or about 65aa to about 70aa.

In some cases, the costimulatory domain is derived from the intracellular portion of the transmembrane protein ICOS (also known as AILIM, CD278, and CVID1). For example, a suitable costimulatory domain may include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in SEQ ID NO: 4. In some of these embodiments, the length of the costimulatory domain is about 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, about 45aa to about 50aa, about 50aa to about 55aa, about 55aa to about 60aa, about 60aa to about 65aa, or about 65aa to about 70aa.

In some cases, the co-stimulatory domain is derived from the intracellular portion of the transmembrane protein OX40 (also known as TNFRSF4, RP5-902P8.3, ACT35, CD134, OX-40, TXGP1L). OX40 contains a p85 PI3K binding motif at each residue 34 to 57 of SEQ ID NO: 504, and a TRAF binding motif at residues 76 to 102. In some embodiments, the co-stimulatory domain may include the p85 PI3K binding motif of OX40. In some embodiments, the co-stimulatory domain may include the TRAF binding motif of OX40. The lysine corresponding to amino acids 17 and 41 of SEQ ID NO: 504 is a potential negative regulatory site that acts as part of a ubiquitin targeting motif. In some embodiments, one or both of these lysines in the co-stimulatory domain of OX40 is a mutant arginine or another amino acid. In some embodiments, suitable costimulatory domains may include domains having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in SEQ ID NO: 5. In some of these embodiments, the length of the costimulatory domain is about 20aa to about 25aa, about 25aa to about 30aa, 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, about 45aa to about 50aa. In illustrative embodiments, the length of the costimulatory domain is 20aa to about 50aa, e.g., 20aa to 45aa or 20aa to 42aa.

In some cases, the costimulatory domain is derived from the intracellular portion of the transmembrane protein CD27 (also known as S152, T14, TNFRSF7, and Tp55). For example, a suitable costimulatory domain may include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in SEQ ID NO: 6. In some of these embodiments, the length of the costimulatory domain is about 30 aa to about 35 aa, about 35 aa to about 40 aa, about 40 aa to about 45 aa, about 45 aa to about 50 aa.

In some cases, the costimulatory domain is derived from the intracellular portion of the transmembrane protein BTLA (also known as BTLAl and CD272). For example, a suitable costimulatory domain may include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in SEQ ID NO:7.

In some cases, the costimulatory domain is derived from the intracellular portion of the transmembrane protein CD30 (also known as TNFRSF8, D1S166E and Ki-1). For example, a suitable costimulatory domain may include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a stretch of about 100 amino acids to about 110 amino acids (aa), about 110 aa to about 115 aa, about 115 aa to about 120 aa, about 120 aa to about 130 aa, about 130 aa to about 140 aa, about 140 aa to about 150 aa, about 150 aa to about 160 aa, or about 160 aa to about 185 aa in SEQ ID NO: 8.

In some cases, the costimulatory domain is derived from the intracellular portion of the transmembrane protein GITR (also known as TNFRSF18, RP5-902P8.2, AITR, CD357, and GITR-D). For example, a suitable costimulatory domain may include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in SEQ ID NO: 9. In some of these embodiments, the length of the costimulatory domain is about 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, about 45aa to about 50aa, about 50aa to about 55aa, about 55aa to about 60aa, about 60aa to about 65aa, or about 65aa to about 70aa.

In some cases, the co-stimulatory domain is derived from the intracellular portion of the transmembrane protein HVEM (also known as TNFRSF14, RP3-395M20.6, ATAR, CD270, HVEA, HVEM, LIGHTR, and TR2). For example, a suitable co-stimulatory domain may include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in SEQ ID NO: 10. In some of these embodiments, the length of the costimulatory domains of the first and second polypeptides is about 30 aa to about 35 aa, about 35 aa to about 40 aa, about 40 aa to about 45 aa, about 45 aa to about 50 aa, about 50 aa to about 55 aa, about 55 aa to about 60 aa, about 60 aa to about 65 aa, or about 65 aa to about 70 aa.

Joiner

In some cases, the engineered signaling polypeptide includes a connector between any two adjacent domains. For example, the connector can be between the transmembrane domain and the first stimulatory domain. As another example, the ASTR can be an antibody, and the connector can be between the heavy chain and the light chain. As another example, the connector can be between the ASTR and the transmembrane domain and the costimulatory domain. As another example, the connector can be between the costimulatory domain and the intracellular activation domain of the second polypeptide. As another example, the connector can be between the ASTR and the intracellular signaling domain.

The connector peptide may have any of a variety of amino acid sequences. Proteins may be connected by spacer peptides that are generally flexible, but other chemical bonds are not excluded. The connector may be a peptide between about 1 and about 100 amino acids in length, or between about 1 and about 25 amino acids in length. These connectors may be produced by coupling the protein using synthetic oligonucleotides encoding the connector. Peptide connectors with a certain degree of flexibility may be used. The connecting peptide may have virtually any amino acid sequence, given that a suitable connector will have a sequence that produces a generally flexible peptide. The use of smaller amino acids, such as glycine and alanine, is for creating flexible peptides. The creation of such sequences is conventional for those familiar with the art.

Suitable connectors can be readily selected and can be any of a variety of suitable lengths, such as 1 amino acid (e.g., Gly) to 20 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6 or 7 amino acids.

Exemplary flexible linkers include glycine polymers (G) _n , glycine-serine polymers (including, for example, (GS) _n , GSGGS _n , GGGS _n , and GGGGS _n , where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are of interest because both of these amino acids are relatively unstructured and therefore can serve as neutral links between components. Glycine polymers are of particular interest because glycine has significantly more phi-psi space than even alanine and is less constrained than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). Exemplary flexible linkers include, but are not limited to, GGGGSGGGGSGGGGS (SEQ ID NO: 53), GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 54), GGGGSGGGSGGGGS (SEQ ID NO: 55), GGSG (SEQ ID NO: 56), GGSGG (SEQ ID NO: 57), GGSSG (SEQ ID NO: 58), GSGGG (SEQ ID NO: 59), GGGSG (SEQ ID NO: 60), GSSSG (SEQ ID NO: 61), and the like. One of ordinary skill in the art will recognize that the design of peptide conjugation to any of the components described above may include linkers that are fully or partially flexible, such that a linker may include a flexible linker and one or more portions that impart a less flexible structure.

combination

In some embodiments, the polynucleotide provided by the replication-deficient recombinant retroviral particle has one or more transcription units encoding certain combinations of one or more engineered signaling polypeptides. In some methods and compositions provided herein, after the T cells are transcribed by the replication-deficient recombinant retroviral particles, the genetically modified T cells include a combination of one or more engineered signaling polypeptides. It will be understood that reference to the first polypeptide, the second polypeptide, the third polypeptide, etc. is for convenience, and the components on the "first polypeptide" and those on the "second polypeptide" mean that the components are on different polypeptides, and the polypeptides are referred to as the first or second to be generally used in other components or steps of specific polypeptides for reference and convenience only.

In some embodiments, the first engineered signaling polypeptide includes an extracellular antigen binding domain capable of binding to an antigen, and an intracellular signaling domain. In other embodiments, the first engineered signaling polypeptide also includes a T cell survival motif and/or a transmembrane domain. In some embodiments, the first engineered signaling polypeptide does not include a costimulatory domain, while in other embodiments, the first engineered signaling polypeptide does include a costimulatory domain.

In some embodiments, the second engineered signaling polypeptide comprises a lymphoproliferative gene product and an optional extracellular antigen binding domain. In some embodiments, the second engineered signaling polypeptide also comprises one or more of the following: a T cell survival motif, an intracellular signaling domain, and one or more co-stimulatory domains. In other embodiments, when two engineered signaling polypeptides are used, at least one is a CAR.

In one embodiment, one or more engineered signaling polypeptides are expressed under a T cell-specific promoter or a general promoter under the same transcript, wherein in the transcript, nucleic acids encoding the engineered signaling polypeptides are separated by nucleic acids encoding one or more internal ribosome entry sites (IREs) or one or more protease cleavage peptides.

In certain embodiments, the polynucleotide encodes two engineered signaling polypeptides, wherein the first engineered signaling polypeptide comprises a first extracellular antigen binding domain capable of binding to a first antigen, and a first intracellular signaling domain other than a co-stimulatory domain, and the second engineered signaling polypeptide comprises a second extracellular antigen binding domain capable of binding to VEGF, and a second intracellular signaling domain, such as a signaling domain of a co-stimulatory molecule. In certain embodiments, the first antigen is PSCA, PSMA, or BCMA. In certain embodiments, the first extracellular antigen binding domain comprises an antibody or fragment thereof (e.g., scFv), such as an antibody or fragment thereof specific for PSCA, PSMA, or BCMA. In certain embodiments, the second extracellular antigen binding domain that binds VEGF is a receptor for VEGF, i.e., VEGFR. In certain embodiments, VEGFR is VEGFR1, VEGFR2, or VEGFR3. In certain embodiments, VEGFR is VEGFR2.

In certain embodiments, the polynucleotide encodes two engineered signaling polypeptides, wherein the first engineered signaling polypeptide comprises an extracellular tumor antigen binding domain and a CD3 zeta signaling domain, and the second engineered signaling polypeptide comprises an antigen binding domain (wherein the antigen is an angiogenic or angiogenic factor), and one or more costimulatory molecule signaling domains. Angiogenic factors may be, for example, VEGF. One or more costimulatory molecule signaling motifs may include, for example, costimulatory signaling domains from each of CD27, CD28, OX40, ICOS, and 4-1BB.

In certain embodiments, the polynucleotide encodes two engineered signaling polypeptides, wherein the first engineered signaling polypeptide comprises an extracellular tumor antigen binding domain and a CD3ζ signaling domain, and the second polypeptide comprises an antigen binding domain capable of binding to an antigen binding domain of VEGF, and a co-stimulatory signaling domain from each of CD27, CD28, OX40, ICOS, and 4-1BB. In another embodiment, the first signaling polypeptide or the second signaling polypeptide also has a T cell survival motif. In some embodiments, the T cell survival motif is an intracellular signaling domain of an IL-7 receptor (IL-7R), an intracellular signaling domain of an IL-12 receptor, an intracellular signaling domain of an IL-15 receptor, an intracellular signaling domain of an IL-21 receptor, or an intracellular signaling domain of a transforming growth factor β (TGFβ) receptor or a TGFβ decoy receptor (TGF-β—dominant-negative receptor II (DNRII)) or derived therefrom.

In certain embodiments, the polynucleotide encodes two engineered signaling polypeptides, wherein the first engineered signaling polypeptide comprises an extracellular tumor antigen binding domain and a CD3 zeta signaling domain, and the second engineered signaling polypeptide comprises an antigen binding domain capable of binding to VEGF, an IL-7 receptor intracellular T cell survival motif, and a co-stimulatory signaling domain from each of CD27, CD28, OX40, ICOS, and 4-1BB.

In some embodiments, more than two signaling polypeptides are encoded by a polynucleotide. In certain embodiments, only one of the engineered signaling polypeptides includes an antigen binding domain that binds to a tumor-associated antigen or a tumor-specific antigen; each of the remaining engineered signaling polypeptides includes an antigen binding domain that binds to a non-tumor-associated antigen or a non-tumor-specific antigen. In other embodiments, two or more of the engineered signaling polypeptides include an antigen binding domain that binds to one or more tumor-associated antigens or tumor-specific antigens, wherein at least one of the engineered signaling polypeptides includes an antigen binding domain that does not bind to a tumor-associated antigen or a tumor-specific antigen.

In some embodiments, the tumor-associated antigen or tumor-specific antigen is Her2, prostate stem cell antigen (PSCA), PSMA (prostate-specific membrane antigen), B cell maturation antigen (BCMA) alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen-125 (CA-125), CA19-9, calretinin, MUC-1, epithelial membrane protein (EMA), epithelial tumor antigen (ETA), tyrosinase, melanoma-associated antigen (MAGE), CD34, CD45, CD99, CD117, chromogranin, cytokeratin, desmin, glial fibrillary acidic protein (GFAP), large cystic disease fluid protein (GCDFP-15), HMB-45 antigen, protein Melanin A (melanoma antigen recognized by T lymphocytes; MART-1), myosin-D1, muscle-specific actin (MSA), neuropil, neuron-specific enolase (NSE), placental alkaline phosphatase, synaptophysin, thyroglobulin, thyroid transcription factor-1, pyruvate kinase isozyme M2 (tumor M2-PK), CD19, CD22, CD27, CD30, CD70, GD2 (ganglioside G2), EphA2, CSPG4, CD138, FAP (fibroblast activation protein), CD171, kappa integrin, lambda integrin, 5T4 integrin, αvβ6 integrin, integrin αvβ3 (CD61), prolactin K-Ras (V-Ki-ras2 Kirsten rat sarcoma viral oncogene), Ral-B, B7-H3, B7-H6, CAIX, CD20, CD33, CD44, CD44v6, CD44v7/8, CD123, EGFR, EGP2, EGP40, EpCAM, embryonic AchR, FRα, GD3, HLA-A1+MAGE1, HLA-A1+NY-ESO-1, IL-11Rα, IL-13Rα2, Lewis-Y, Mu c16, NCAM, NKG2D ligand, NY-ESO-1, PRAME, ROR1, survivin, TAG72, TEMs, VEGFR2, EGFRvIII (epidermal growth factor variant III, sperm protein 17 (Sp17), mesothelin, PAP (prostatic acid phosphatase), prostate protein, TARP (T cell receptor gamma alternative reading frame protein), Trp-p8, STEAP1 (six transmembrane epithelial antigen of the prostate 1), abnormal mouse protein or abnormal p53 protein.

In some embodiments, the first engineered signaling polypeptide includes a first extracellular antigen binding domain that binds to a first antigen, and a first intracellular signaling domain; and the second engineered signaling polypeptide includes a second extracellular antigen binding domain that binds to a second antigen or a receptor that binds to a second antigen, and a second intracellular signaling domain, wherein the second engineered signaling polypeptide does not include a co-stimulatory domain. In a certain embodiment, the first antigen binding domain and the second antigen binding domain are independently an antigen binding portion of a receptor or an antigen binding portion of an antibody. In a certain embodiment, one or both of the second antigen binding domain or the second antigen binding domain are scFv antibody fragments. In certain embodiments, the first engineered signaling polypeptide and/or the second engineered signaling polypeptide additionally include a transmembrane domain. In a certain embodiment, the first engineered signaling polypeptide or the second engineered signaling polypeptide includes a T cell survival motif, such as any of the T cell survival motifs described herein.

In another embodiment, the first engineered signaling polypeptide comprises a first extracellular antigen binding domain that binds HER2, and the second engineered signaling polypeptide comprises a second extracellular antigen binding domain that binds MUC-1.

In another embodiment, the second extracellular antigen binding domain of the second engineered signaling polypeptide binds interleukin.

In another embodiment, the second extracellular antigen binding domain of the second engineered signaling polypeptide binds to a damage-associated molecular pattern molecule (DAMP; also known as alarmin). In another embodiment, the DAMP is a heat shock protein, a chromatin-associated protein high mobility group box 1 (HMGB1), S100A8 (also known as MRP8 or calgranulin A), S100A9 (also known as MRP14 or calgranulin B), serum amyloid A (SAA), deoxyribonucleic acid, adenosine triphosphate, uric acid or heparin sulfate.

In certain embodiments, the second antigen is an antigen on an antibody that binds an antigen presented by a tumor cell.

In some embodiments, the signal transduction activation via the second engineered signaling polypeptide is non-antigenic but is associated with hypoxia. In certain embodiments, hypoxia is induced by activation of hypoxia-inducible factor-1α (HIF-1α), HIF-1β, HIF-2α, HIF-2β, HIF-3α, or HIF-3β.

In some embodiments, expression of one or more engineered signaling polypeptides is regulated by control elements disclosed in more detail herein.

Additional Sequences

Engineered signaling polypeptides (such as CARs) may further include one or more additional polypeptide domains, wherein such domains include, but are not limited to, signal sequences, antigenic determinant tags, affinity domains, and polypeptides whose presence or activity can be detected, for example, by antibody analysis or because they are polypeptides that produce detectable signals (detectable markers). Non-limiting examples of additional domains for any of the aspects or embodiments provided herein include domains having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with any of the following sequences as described below: signal sequences, antigenic determinant tags, affinity domains, or polypeptides that produce detectable signals.

Signal sequences suitable for use in an individual CAR (e.g., the first polypeptide of an individual CAR) include any eukaryotic signal sequence, including naturally occurring signal sequences, synthetic (e.g., artificial) signal sequences, etc. In some embodiments, for example, the signal sequence may be the CD8 signal sequence MALPVTALLLPLALLLHAARP (SEQ ID NO: 74).

Suitable antigenic determinant tags include, but are not limited to, hemagglutinin (HA; e.g., YPYDVPDYA; SEQ ID NO: 37), FLAG (e.g., DYKDDDDK; SEQ ID NO: 38), c-myc (e.g., EQKLISEEDL; SEQ ID NO: 39), and the like.

Affinity domains include peptide sequences useful for identification or purification that can interact with a binding partner (e.g., one immobilized on a solid support). DNA sequences encoding multiple consecutive single amino acids (e.g., histidine) when fused to expressed proteins can be used for one-step purification of recombinant proteins by high affinity binding to resin columns (such as agarose gel). Exemplary affinity domains include His5 (HHHHH; SEQ ID NO:40), HisX6 (HHHHHH; SEQ ID NO:41), c-myc (EQKLISEEDL; SEQ ID NO:39), Flag (DYKDDDDK; SEQ ID NO:38), Strep tag (WSHPQFEK; SEQ ID NO:42), hemagglutinin (e.g., HA tag (YPYDVPDYA; SEQ ID NO:37)), GST, thioredoxin, cellulose binding domain, RYIRS (SEQ ID NO:43), Phe-His-His-Thr (SEQ ID NO:44), chitin binding domain, S peptide, T7 peptide, SH2 domain, C-terminal RNA tag, WEAAAREACCRECCARA (SEQ ID NO:45), WT3A194A (SEQ ID NO:46), WT3A242A (SEQ ID NO:47), WT3A243A (SEQ ID NO:48), WT3A244A (SEQ ID NO:49), WT3A245A (SEQ ID NO:50), WT3A246A (SEQ ID NO:51), WT3A247A (SEQ ID NO:52), WT3A248A (SEQ ID NO:53), WT3A249A (SEQ ID NO:54), WT3A249A (SEQ ID NO:55), WT3A243A (SEQ ID NO:56), WT3A244A (SEQ ID NO:57), WT3A245A (SEQ ID NO:58), WT3A246A (SEQ ID NO:59), WT3A247A (SEQ ID NO:5 NO:45), a metal binding domain, e.g., a zinc binding domain or a calcium binding domain, such as those from calcium binding proteins, e.g., calmodulin, troponin C, calcineurin B, myosin light chain, recoverin, S-regulin, cone opsin, VILIP, calcineurin, hippocampal calcium binding protein, aggrecin, caltractin, calpain large subunit, S100 protein, parvalbumin, calcium binding protein D9K, calcium binding protein D28K and calretinin, intein, biotin, streptavidin, MyoD, Id, leucine zipper sequence, and maltose binding protein.

Suitable detectable signal producing proteins include, for example, fluorescent proteins, enzymes that catalyze a reaction that produces a detectable signal as a product, and the like.

Suitable fluorescent proteins include, but are not limited to, green fluorescent protein (GFP) or variants thereof, blue fluorescent variant of GFP (BFP), cyan fluorescent variant of GFP (CFP), yellow fluorescent variant of GFP (YFP), enhanced GFP (EGFP), enhanced CFP (ECFP), enhanced YFP (EYFP), GFPS65T, Emerald, Topaz (TYFP), Venus, Citrine, mCitrine, GFPuv, destabilized EGFP (dEGFP), destabilized ECFP (dECFP), destabilized EYFP (dEYFP), mCFPm, Cerulean, T-Sapphire, CyPet, Ypet, mKO, HcRed, t-HcRed, DsRed, DsRed2, DsRed-monomer, J-Red, Dimer 2, t-Dimer 2 (12), mRFP1, coral, Renilla GFP, Monster GFP, paGFP, Kaede protein and kindling protein, phycobiliproteins and phycobiliprotein conjugates, including B-phycoerythrin, R-phycoerythrin and allophycocyanin. Other examples of fluorescent proteins include mHoneydew, mBanana, mOrange, dTomato, tdTomato, mTangerine, mStrawberry, mCherry, mGrapel, mRaspberry, mGrape2, mPlum (Shaner et al. (2005) Nat. Methods 2:905-909), and the like. Any of a variety of fluorescent and colored proteins from Anthozoan species are suitable for use, as described, for example, in Matz et al. (1999) Nature Biotechnol. 17:969-973.

Suitable enzymes include, but are not limited to, horseradish peroxidase (HRP), alkaline phosphatase (AP), β-galactosidase (GAL), glucose-6-phosphate dehydrogenase, β-N-acetamido glucosidase, β-glucuronidase, invertase, xanthine oxidase, firefly luciferase, glucose oxidase (GO), and the like.

Identify domains and/or eliminate domains

Any of the replication-deficient recombinant retroviral particles provided herein may include a nucleic acid encoding a recognition domain or an elimination domain, which is part of or separated from the nucleic acid encoding any of the engineered signaling polypeptides provided herein. Therefore, any of the engineered signaling polypeptides provided herein may include a recognition domain or an elimination domain. For example, any of the CARs disclosed herein may include a recognition domain or an elimination domain. In addition, the recognition domain or the elimination domain may be expressed together with any of the lymphoproliferative components disclosed herein, or even fused therewith. The recognition domain or the elimination domain is expressed on T cells and/or NK cells, but not on replication-deficient recombinant retroviral particles.

In some embodiments, the recognition domain or elimination domain may be derived from the herpes simplex virus-derived enzyme thymidine kinase (HSV-tk) or inducible caspase 9. In some embodiments, the recognition domain or elimination domain may include a modified endogenous cell surface molecule, such as disclosed in U.S. Pat. No. 8,802,374. The modified endogenous cell surface molecule may be any cell surface-related receptor, ligand, glycoprotein, cell adhesion molecule, antigen, integrin, or modified differentiation cluster (CD). In some embodiments, the modified endogenous cell surface molecule is a truncated tyrosine kinase receptor. In one aspect, the truncated tyrosine kinase receptor is a member of the epidermal growth factor receptor (EGFR) family, such as ErbB1, ErbB2, ErbB3, ErbB4. In some embodiments, the recognition domain may be a polypeptide recognized by an antibody that recognizes the extracellular domain of an EGFR member. In some embodiments, the recognition domain may be at least 20 consecutive amino acids of an EGFR family member, or, for example, between 20 and 50 consecutive amino acids of an EGFR family member. For example, SEQ ID NO: 78 is an exemplary polypeptide that is bound by an antibody that recognizes the extracellular domain of an EGFR member and is recognized under appropriate conditions. Such extracellular EGFR antigenic determinants are sometimes referred to herein as eTags. In illustrative embodiments, such antigenic determinants are recognized by commercially available anti-EGFR monoclonal antibodies.

The epidermal growth factor receptor, also known as EGFR, ErbB1 and HER1, is a cell surface receptor for a member of the epidermal growth factor family of extracellular ligands. Alterations in EGFR activity have been implicated in certain cancers. In some embodiments, a gene encoding an EGFR polypeptide comprising a human epidermal growth factor receptor (EGFR) is constructed by removing a nucleic acid sequence encoding a polypeptide comprising a membrane-remote EGF binding domain and a cytoplasmic signaling tail but retaining an extracellular membrane-proximal antigenic determinant recognized by an anti-EGFR antibody. Preferably, the antibody is a known commercially available anti-EGFR monoclonal antibody, such as cetuximab, matuzumab, necitumumab or panitumumab.

Others have shown that biotinylated cetuximab combined with anti-biotin microbeads was used for immunomagnetic selection to successfully enrich T cells that had been lentivirally transduced with constructs containing EGFRt from as low as 2% of the population to greater than 90% purity without observable toxicity to the cell preparation. In addition, others have shown that constitutive expression of this inert EGFR molecule does not affect T cell phenotype or effector function such as that directed by the coordinately expressed chimeric antigen receptor (CAR), CD19R. In addition, others have shown that EGFR was successfully used as an in vivo tracking marker for T cell transplantation in mice via flow cytometry analysis. In addition, it has been shown that EGFR was successfully used as an in vivo tracking marker for T cell transplantation in mice via flow cytometry analysis. The antibody-dependent cellular cytotoxicity (ADCC) pathway demonstrated that EGFR has suicide gene potential. The inventors of the present invention have successfully expressed eTag in PBMC using lentiviral vectors, and have found that PBMC exposed to cetuximab express eTag in vitro, providing an effective elimination mechanism for PBMC. Therefore, EGFR can be used as a non-immune gene selection tool, tracking marker, and suicide gene for transduced T cells with immunotherapy potential. EGFR nucleic acid can also be detected by methods well known in the art.

In some embodiments provided herein, EGFR is expressed as part of a single polypeptide that also includes CAR or is expressed as part of a single polypeptide that includes a lymphoproliferative component lymphoproliferative component. In some embodiments, the amino acids encoding the EGFR recognition domain can be separated from the amino acids encoding the chimeric antigen receptor by a cleavage signal and/or a ribosome skipping sequence. The ribosome skipping and/or cleavage signal can be any ribosome skipping and/or cleavage signal known in the art. Without being limited by theory, the ribosome skipping sequence can be, for example, T2A (also referred to as 2A-1) having an amino acid sequence of GSGEGRGSLLTCGDVEENPGP (SEQ IDNO: 77). Without being limited by theory, other examples of cleavage signals and ribosome skipping sequences include FMDV 2A (F2A), horse type A rhinovirus 2A (abbreviated as E2A), porcine Teschenovirus 1 2A (P2A), and Thoseaasigna virus 2A (T2A). In some embodiments, the polynucleotide sequence encoding the recognition domain can be on the same transcript as the CAR or lymphoproliferative component, but separated from the polynucleotide sequence encoding the CAR or lymphoproliferative component by an internal ribosome entry site.

In other embodiments as exemplified herein, the recognition domain can be expressed as part of a fusion polypeptide fused to a lymphoproliferative component. Such constructs provide the advantage of taking up less genomic space on the RNA genome compared to individual polypeptides, especially in combination with other "space-saving" components provided herein. In an illustrative embodiment, the eTag is expressed as a fusion polypeptide fused to an IL7Rα mutant, as experimentally demonstrated herein.

Chimeric Antigen Receptor

In some aspects of the present invention, the engineered signaling polypeptide is a chimeric antigen receptor (CAR) or a polynucleotide encoding a CAR, which is referred to herein as "CAR" for simplicity. The CAR of the present invention includes: a) at least one antigen-specific targeting region (ASTR); b) a transmembrane domain; and c) an intracellular activation domain. In illustrative embodiments, the antigen-specific targeting region of CAR is the scFv portion of an antibody directed against a target antigen. In illustrative embodiments, the intracellular activation domain is from CD3Z, CD3D, CD3E, CD3G, CD79A, CD79B, DAP12, FCER1G, FCGR2A, FCGR2C, DAP10/CD28 or ZAP70, and in some other illustrative embodiments, from CD3z. In illustrative embodiments, CAR further includes a costimulatory domain, such as any one of the costimulatory domains provided above in the regulatory domain chapters, and in other illustrative embodiments, the costimulatory domain is 4-1BB (CD137), CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR and HVEM intracellular costimulatory domain. In certain embodiments, CAR includes any one of the transmembrane domains listed above in the transmembrane domain chapters.

The CAR of the present invention may be present in the plasma membrane of a eukaryotic cell (e.g., a mammalian cell), wherein suitable mammalian cells include, but are not limited to, cytotoxic cells, T lymphocytes, stem cells, progeny of stem cells, progenitor cells, progeny of progenitor cells, and NK cells, NK-T cells, and macrophages. When present in the plasma membrane of a eukaryotic cell, the CAR of the present invention is activated in the presence of one or more target antigens (under certain conditions, bound to ASTR). The target antigen is the second member of the specific binding pair. The target antigen of the specific binding pair may be a soluble (e.g., not bound to a cell) factor; a factor present on the surface of a cell such as a target cell; a factor present on the surface of an entity; a factor present on a lipid bilayer; and the like. When the ASTR is an antibody and the second member of the specific binding pair is an antigen, the antigen may be a soluble (e.g., not bound to a cell) antigen; an antigen present on the surface of a cell such as a target cell; an antigen present on the surface of an entity; an antigen present on a lipid bilayer; and the like.

In some cases, the CAR of the present invention increases the expression of at least one nucleic acid in the cell when present in the plasma membrane of a eukaryotic cell and activated by one or more target antigens. For example, in some cases, the CAR of the present invention increases the expression of at least one nucleic acid in the cell by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about 2 times, at least about 2.5 times, at least about 5 times, at least about 10 times, or more than 10 times when present in the plasma membrane of a eukaryotic cell and activated by one or more target antigens, compared to the transcription level of the nucleic acid in the absence of one or more target antigens.

As an example, the CAR of the present invention may include an intracellular signaling polypeptide containing an immunoreceptor tyrosine-based activation motif (ITAM).

In some cases, the CAR of the present invention, when present in the plasma membrane of a eukaryotic cell and activated by one or more target antigens, can increase the production of one or more cytokines by the cell. For example, when the CAR of the present invention is present in the plasma membrane of a eukaryotic cell and activated by one or more target antigens, the amount of cytokines produced by the cell in the absence of one or more target antigens can be compared to the amount of cytokines produced by the cell, which can increase the production of cytokines by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about 2 times, at least about 2.5 times, at least about 5 times, at least about 10 times, or more than 10 times. The cytokines whose production can be increased include, but are not limited to, interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-a), IL-2, IL-15, IL-12, IL-4, IL-5, IL-10; chemokines; growth factors; and the like.

In some cases, the CAR of the present invention, when present in the plasma membrane of a eukaryotic cell and activated by one or more target antigens, can result in both increased transcription of nucleic acids in the cell and increased production of cytokines by the cell.

In some cases, the CAR of the present invention, when present in the plasma membrane of a eukaryotic cell and activated by one or more target antigens, produces cytotoxic activity of the cell toward the target cell, the target cell expresses an antigen on its cell surface, and the antigen binds to the antigen binding domain of the first polypeptide of the CAR. For example, when the eukaryotic cell is a cytotoxic cell (e.g., NK cell or cytotoxic T lymphocyte), the CAR of the present invention, when present in the plasma membrane of a eukaryotic cell and activated by one or more target antigens, increases the cytotoxic activity of the cell toward the target cell, and the target cell expresses one or more target antigens on its cell surface. For example, when the eukaryotic cell is a NK cell or a T lymphocyte, the CAR of the present invention, when present in the plasma membrane of the eukaryotic cell and activated by one or more target antigens, increases the cytotoxic activity of the cell by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about 2 times, at least about 2.5 times, at least about 5 times, at least about 10 times, or more than 10 times, compared to the cytotoxic activity of the cell in the absence of one or more target antigens.

In some cases, the CARs of the invention, when present in the plasma membrane of a eukaryotic cell and activated by one or more target antigens, can produce events associated with other CAR activation, such as proliferation and expansion (due to enhanced cell division or anti-apoptotic responses).

In some cases, the CARs of the invention, when present in the plasma membrane of a eukaryotic cell and activated by one or more target antigens, can produce other CAR activation-related events, such as intracellular signaling regulation, cell differentiation, or cell death.

In some cases, the CAR of the present invention is microenvironmentally restricted. This property is generally a result of the microenvironmentally restricted nature of the ASTR domain of the CAR. Therefore, the CAR of the present invention may have a lower binding affinity or, in illustrative embodiments, may have a higher binding affinity for one or more target antigens under conditions of the microenvironment than under conditions of the normal physiological environment.

Lymphoproliferative component Lymphoproliferative component

Despite the continuous increase of cells, the number of peripheral T lymphocytes remains at a very stable level throughout adulthood, due to the proliferation of cells removed from the thymus and in response to antigen encounters, and due to the loss of cells that remove antigen-specific effects after antigen clearance (Marrak, P. et al. 2000. Nat Immunol 1: 107-111; Freitas, A.A. et al. 2000. Annu Rev Immunol 18: 83-111). The size of the peripheral T cell compartment is regulated by multiple factors that affect both proliferation and survival. However, in the context of lymphopenia, T lymphocytes divide independently of cognate antigens, due to the "acute constant proliferation" mechanism that maintains the size of the peripheral T cell compartment. The conditions of lymphopenia have been established in individuals or patients during adoptive cell therapy by in vivo proliferation of T cells and their introduction into individuals with lympho-consumption, resulting in the engraftment of transferred T cells and enhanced anti-tumor function. However, lymphodepletion of an individual is undesirable because it can cause serious side effects, including immune disturbances and death.

Studies have shown that lympholysis removes endogenous lymphocytes that serve as reservoirs for stabilizing cytokines, thereby releasing cytokines to induce survival and proliferation of adoptively transferred cells. Some cytokines such as IL-7 and IL-15 are known to mediate antigen-independent proliferation of T cells and are therefore able to induce stable proliferation in a non-lymphopenic environment. However, these cytokines and their receptors have inherent control mechanisms that prevent lymphoproliferative diseases in the steady state.

Many aspects provided herein include lymphoproliferative elements, lymphoproliferative elements, or nucleic acids encoding them, typically as part of an engineered signaling polypeptide. Therefore, in some aspects of the present invention, the engineered signaling polypeptide is a lymphoproliferative element, a lymphoproliferative element (LE), such as a chimeric lymphoproliferative element, a lymphoproliferative element (CLE). Typically, a LE comprises an extracellular domain, a transmembrane domain, and at least one intracellular signaling domain that drives hyperplasia, and in illustrative embodiments, comprises a second intracellular signaling domain. As illustrated herein (see, e.g., Examples 11 and 12), there are first and second intracellular signaling domains of a CLE, and the first intracellular signaling domain is positioned between a membrane-associated motif and a second intracellular domain. In illustrative embodiments, the intracellular domain of LE or the first intracellular domain with two or more intracellular domains in LE is not a functional intracellular activation domain from an ITAM intracellular domain, such as an intracellular domain from CD3Z, CD3D, CD3E, CD3G, CD79A, CD79B, DAP12, FCER1G, FCGR2A, FCGR2C, DAP10/CD28 or ZAP70, and in other illustrative sub-embodiments, the intracellular domain of CD3z. In illustrative embodiments, the second intracellular domain of LE is not a co-stimulatory domain of 4-1BB (CD137), CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR and HVEM. In illustrative embodiments, the LE extracellular domain does not include a single-chain variable fragment (scFv). In other illustrative embodiments, the LE extracellular domain that activates LE when combined with a binding partner does not include a single-chain variable fragment (scFv).

CLE does not include both ASTR and activation domains from: CD3Z, CD3D, CD3E, CD3G, CD79A, CD79B, DAP12, FCER1G, FCGR2A, FCGR2C, DAP10/CD28 or ZAP70. Without being limited by theory, it is believed that the extracellular domain and transmembrane domain play a supporting role in LE, ensuring that the intracellular signaling domain is in an effective conformation/orientation/positioning for driving proliferation. Therefore, it is believed that the ability of LE to drive proliferation is provided by the intracellular domain of LE, and it is believed that the extracellular domain and transmembrane domain play a secondary role relative to the intracellular domain. The lymphoproliferative component includes an intracellular domain that is a signaling polypeptide that is capable of driving proliferation of T cells or NK cells associated with the membrane via a membrane-associated motif (e.g., a transmembrane domain) and is oriented in an active conformation or capable of being oriented to an active conformation. In illustrative embodiments, the ASTR of the LE does not include a scFv. Provided herein are strategies for associating the intracellular domain with a membrane, such as by including a transmembrane domain, a GPI anchor, a myristoylated region, a palmitoylated region, and/or a prenylated region. In some embodiments, the lymphoproliferative component does not include an extracellular domain.

The extracellular domain, transmembrane domain, and intracellular domain of the LE can vary in their respective amino acid lengths. For example, for embodiments comprising replication-deficient recombinant retroviral particles, there are limitations on the length of polynucleotides that can be packaged into retroviral particles so that LEs with shorter amino acid sequences may be advantageous in certain illustrative embodiments. In some embodiments, the total length of the LE may be between 3 and 4000 amino acids, such as between 10 and 3000 amino acids, 10 and 2000 amino acids, 50 and 2000 amino acids, 250 and 2000 amino acids, and in illustrative embodiments, between 50 and 1000 amino acids, 100 and 1000 amino acids, or 250 and 1000 amino acids. When present to form an extracellular domain and a transmembrane domain, the extracellular domain may be between 1 and 1000 amino acids, and typically between 4 and 400 amino acids, between 4 and 200 amino acids, between 4 and 100 amino acids, between 4 and 50 amino acids, between 4 and 25 amino acids, or between 4 and 20 amino acids. In one embodiment, for the extracellular domain and transmembrane domain of this aspect of the invention, the extracellular region is GGGS. The transmembrane domain or the transmembrane region of the extracellular domain and transmembrane domain may be between 10 and 250 amino acids, and more typically at least 15 amino acids in length, and may be, for example, between 15 and 100 amino acids, 15 and 75 amino acids, 15 and 50 amino acids, 15 and 40 amino acids, 15 and 30 amino acids in length. The intracellular signaling domain may be, for example, between 10 and 1000 amino acids, 10 and 750 amino acids, 10 and 500 amino acids, 10 and 250 amino acids, or 10 and 100 amino acids. In illustrative embodiments, the intracellular signaling domain may be at least 30 amino acids, or between 30 and 500 amino acids, 30 and 250 amino acids, 30 and 150 amino acids, 30 and 100 amino acids, 50 and 500 amino acids, 50 and 250 amino acids, 50 and 150 amino acids, or 50 and 100 amino acids. In some embodiments, the intracellular signaling domain of a particular gene is at least 90%, 95%, 98%, 99%, 100% identical to at least 10, 25, 30, 40, or 50 amino acids from a sequence of that intracellular signaling domain, such as a sequence provided herein for that intracellular domain, up to the size of the entire intracellular domain sequence, and may include, for example, up to an additional 1, 2, 3, 4, 5, 10, 20, or 25 amino acids, with the proviso that such sequence is still capable of providing any of the LE properties disclosed herein.

In Examples 10, 11, and 12 herein, it was identified that CLE promotes proliferation of PBMC cell cultures transduced with lentiviral particles encoding CLE between day 7 and day 21, day 28, day 35, and/or day 42 after transduction. These examples provide tests and/or criteria that can be used to identify whether any test polypeptide, including LE or a test domain of LE, such as the first intracellular domain or the second intracellular domain or both the first and second intracellular domains, is indeed LE or an effective intracellular domain of LE, or a particularly effective LE or an intracellular domain of LE. Thus, in certain embodiments, any aspect or other embodiment of a polynucleotide or nucleic acid including LE or encoding LE provided herein may state that LE satisfies or provides the following properties or is capable of providing and/or possessing the following properties: the properties of any one or more of the identified tests or criteria for identifying LE provided herein, or cells genetically modified and/or transduced with retroviral particles (such as lentiviral particles encoding LE) are capable of providing, adapting to, possessing the following properties and/or being modified to achieve the results of one or more of the stated tests. In one embodiment, the LE provides, is capable of providing and/or possesses the following properties (or cells genetically modified and/or transduced with retroviral particles encoding the LE are capable of, suitable for, possess the following properties and/or are modified to provide): compared to control retroviral particles (e.g., lentiviral particles) under the same conditions, in the absence of exogenously added cytokines, between days 7 and 21, 28, 35 and/or 42 of in vivo culture after transduction, the lentivirus comprising a nucleic acid encoding the LE and an anti-CD19 CAR comprising a CD3ζ intracellular activation domain but not a co-stimulatory domain. In some embodiments, lymphoproliferative component testing for improved or enhanced survival, expansion and/or proliferation of cells transduced with retroviral particles (e.g., lentiviral particles) having a genome encoding a test construct encoding a putative LE (test cell) can be performed based on comparison with control cells, which can be, for example, untransduced cells, or cells transduced with control retroviral (e.g., lentiviral) particles identical to the lentiviral particles, comprising nucleic acid encoding a lymphoproliferative component, but lacking a lymphoproliferative component, or lacking one or more intracellular domains of a test polypeptide construct, but comprising the same extracellular domain (if present) and the same transmembrane region or membrane targeting region of the respective test polypeptide construct. In some embodiments, control cells are transduced with retroviral particles (e.g., lentiviral particles) having a genome encoding a lymphoproliferative component or an intracellular domain thereof identified as an exemplified lymphoproliferative component herein. In this embodiment, the test criteria may include at least sufficient enrichment, survival and/or expansion, or the absence of a statistical difference in enrichment, survival and/or expansion when tested using retroviral particles (e.g., lentiviral particles) having a genome encoding the test construct relative to that encoding a control lymphoproliferative component, typically by analyzing cells transduced therewith. In some embodiments, the exemplary or illustrative embodiments of lymphoproliferative components herein are illustrative embodiments of control lymphoproliferative components for such testing.

In some embodiments, this test for the improved properties of the presumed or tested lymphoproliferative component is performed by performing replication and/or performing statistical tests. The skilled person will recognize that many statistical tests can be used for this lymphoproliferative component test. It is envisioned that this test in these embodiments will be any such test known in the art. In some embodiments, the statistical test can be a T test or a Mann-Whitney-Wilcoxon test. In some embodiments, the standardized enrichment level of the test construct is significant at a p-value of less than 0.1 or less than 0.05 or less than 0.01.

In another embodiment, the LE provides, is capable of providing and/or possesses the following properties (or cells genetically modified and/or transduced with the LE are capable of providing, are suitable for, possess the following properties and/or are modified to provide): in the absence of exogenously added cytokines, between days 7 and 21, 28, 35 and/or 42 of in vivo culture, when transduced with an anti-CD19 CAR comprising a CD3ζ intracellular activation domain but not a co-stimulatory domain, pre-activated PBMCs transduced with a nucleic acid encoding the LE are expanded by at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold, or between 1.5-fold and 25-fold, or between 2-fold and 20-fold, or between 2-fold and 15-fold, or between 5-fold and 25-fold, or between 5-fold and 20-fold, or between 5-fold and 15-fold. In some embodiments, the test is performed in the presence of PBMCs, e.g., at a 1:1 ratio of transduced cells to PBMCs (which may, e.g., be from a matched donor), and in some embodiments, the test is performed in the absence of PBMCs. In some embodiments, the amplification analysis of any of these tests is performed as provided in Example 11 or Example 12. In some embodiments, the test may include other statistical tests and cutoffs, such as a P value below 0.1, 0.05, or 0.01, wherein the test polypeptide or nucleic acid encoding the test polypeptide is required to meet one or both thresholds (i.e., fold amplification and statistical cutoff).

For any one of the lymphoproliferative component tests provided herein, between the 7th day and the 14th day, the 21st day, the 28th day, the 35th day, the 42nd day or the 60th day after transduction, the number of test cells and the number of control cells are compared. In certain embodiments, the number of test and control cells can be measured by sequencing DNA and counting the identifiers present in each construct. In certain embodiments, the number of test and control cells can be directly counted, for example, with a hemocytometer or a cell counter. In certain embodiments, all test cells and control cells can be grown in the same container, hole or flask. In certain embodiments, test cells can be inoculated in one or more holes, flasks or containers, and control cells can be inoculated in one or more flasks or containers. In certain embodiments, test and control cells can be inoculated in holes or flasks separately, for example, one cell per hole. In certain embodiments, enrichment levels can be used to compare the number of test cells and control cells. In some embodiments, the enrichment level of a test or control construct can be calculated by dividing the number of cells at a later time point (day 14, day 21, day 28, day 35, or day 45) by the number of cells at day 7 for each construct. In some embodiments, the enrichment level of a test or control construct can be calculated by dividing the number of cells at a time point (day 14, day 21, day 28, day 35, or day 45) by the number of cells at that time point for cells that have not been transduced. In some embodiments, the enrichment level of each test construct can be normalized to the enrichment level of the respective control construct to produce a normalized enrichment level. In some embodiments, the LE encoded in the test construct provides (or cells genetically modified and/or transduced with retroviral particles (e.g., lentiviral particles) having a genome encoding the LE are capable of providing, suitable for, possess the following characteristics and/or are modified to provide) at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold normalized enrichment level, or between 1.5-fold and 25-fold normalized enrichment level, or between 3-fold and 20-fold normalized enrichment level, or between 5-fold and 25-fold normalized enrichment level, or between 5-fold and 20-fold normalized enrichment level, or between 5-fold and 15-fold normalized enrichment level. Enrichment can be measured, for example, by direct cell counting. The cutoff value can be based on a single test, or two, three, four or five replicates, or based on a number of replicates. The cutoff value can be met when the lymphoproliferative component meets one or more replicate tests, or meets or exceeds the cutoff value for all replicates. In some embodiments, enrichment is measured as log ₂ ((normalized count data on test day + 1)/(normalized count data on day 7 + 1)).

As demonstrated in Example 10, Example 11, and Example 12, CLE was identified from a construct library including constructs encoding a test chimeric polypeptide (designed to include an intracellular domain believed to induce proliferation and/or survival of lymphoid or myeloid cells) and an anti-CD19 CAR (including an intracellular activation domain but not a co-stimulatory domain). Pre-activation was performed in a pre-activation reaction mixture containing PBMC, a commercial medium for lymphocytes (complete OpTmizer ^™ CTS ^™ T-cell expansion SFM), recombinant human interleukin-2 (100 IU/ml), and anti-CD3 Ab (OKT3) (50 ng/ml), which was performed overnight at 37°C. After pre-activation, after adding the test and control lentiviral particles to the pre-activation reaction mixture, transduction was performed overnight at 37°C at a multiplicity of infection (MOI) of 5. Some control lentiviral particles contained constructs encoding polypeptides having an extracellular domain and a transmembrane domain but not having an intracellular domain. In contrast, the test lentiviral particles include constructs encoding polypeptides with an extracellular domain and a transmembrane domain and one or two intracellular domains. After transduction, complete OpTmizer ^TM CTS ^TM T-cell expansion SFM will be added to dilute the reaction mixture 5 to 20 times, and the cells will be cultured at 37°C for up to 45 days. After the 7th day after transduction, the culture is "fed" or not ("unfed") with additional PBMCs that are not matched to the transduced donor. After initially forming the transduction reaction mixture, no additional cytokines (e.g., IL-2, IL-7 or IL-15 and no other lymphoid mitogens) are added to these cultures that are not present in the commercial medium. Amplification is measured by analyzing the enrichment of the cell counts that are actually counted as the nucleic acid sequence counts of the unique identifiers of each construct in the mixed culture PBMC cell population, so that enrichment is positive, calculated as the logarithm of the ratio between the standardized count plus one on the last day of analysis and the count plus one on the 7th day with a base of 2. Additional details regarding the tests performed to identify LEs are provided in Examples 10, 11, and 12, including the experimental conditions.

As shown in Example 10, Example 11 and Example 12, constructs are identified as CLEs because CLEs induce proliferation/expansion in these fed or unfed cultures without the need to add cytokines (such as IL-2) between day 7 and day 21, day 28, day 35 and/or day 42. For example, Example 10 identifies effective CLEs by identifying test CLEs that provide increased expansion of these in vitro cultures between day 7 and day 21, day 28, day 35 and/or day 42 after transduction compared to control constructs that do not include any intracellular domains, regardless of whether non-transduced PBMCs are fed or not. Example 10 reveals that, in contrast to each control construct (not including an intracellular domain) present on day 7 after transduction, at least one and typically more than one test CLE that includes an intracellular domain from a test gene provides more expansion. Example 10 further provides a statistical method for identifying abnormally effective genes for the first intracellular domain and one or more exemplary intracellular domains from these genes. The method uses a Mann-Whitney-Wilcoxon test and a false discovery cutoff rate of less than 0.1 or less than 0.05. Example 12 identifies particularly effective genes for the first intracellular domain or the second intracellular domain, for example, by analyzing the gene score calculated as the combined score of all constructs with the gene. As shown in some of the tests performed in Example 12, such analysis can use the following cutoff values: greater than 1, or greater than a negative control construct and without any intracellular domain, or greater than 2.

As shown by the examples herein, in exemplary aspects and embodiments including LE (which typically includes CAR), such as the methods and uses thereof provided herein for genetically modifying and/or transducing, genetically modifying and/or transducing cells, the genetically modified cells are modified so as to possess new properties that the cells did not previously possess prior to genetic modification and/or transduction. This property can be provided by genetic modification using nucleic acids encoding CAR or LE (and in illustrative embodiments, both CAR and LE). For example, in certain embodiments, the genetically modified and/or transduced cells are capable of, adapted for, possess and/or modified to survive and/or proliferate in ex vivo culture for at least 7 days, 14 days, 21 days, 28 days, 35 days, 42 days or 60 days, or between day 7 and day 14, day 21, day 28, day 35, day 42 or day 60 after transduction, in the absence of added IL-2 or in the absence of added cytokines such as IL-2, IL-15 or IL-7, and in certain illustrative embodiments, in the presence of an antigen recognized by a CAR, wherein the method comprises genetically modifying and/or transducing with retroviral particles having a pseudotyping component and, optionally, an isolated or fused activation domain on its surface, and generally no pre-activation is required.

By being capable of enhancing survival and/or proliferation in certain embodiments, it is meant that the genetically modified and/or transduced cell exhibits, is capable of, is suitable for, possesses and is modified for improved survival or expansion in ex vivo or in vitro culture in a culture medium in the absence of one or more added cytokines (such as IL-2, IL-15 or IL-7) or added lymphocyte mitogens, as compared to a control cell that was genetically modified and/or transduced before it was genetically modified and/or transduced or a control cell that was transduced with a retroviral particle that is the same as the tested retroviral particle comprising LE or a putative LE but not having LE or an intracellular domain of LE, wherein said survival or proliferation of said control cell is promoted by said one or more cytokines (such as IL-2, IL-15 or IL-7) or said lymphocyte mitogens added to the culture medium. By added cytokines or lymphocyte mitogens, it is meant that the cytokines or lymphocyte mitogens are added to the culture medium from an exogenous source such that the concentration of the cytokines or lymphocyte mitogens is increased in the culture medium during the culture of the cells compared to the initial culture medium, and in some embodiments, the initial culture medium may not be present prior to the addition. By "addition" or "exogenous addition", it is meant that such cytokines or lymphocyte mitogens are added to the lymphocyte medium used to culture genetically modified and/or transduced cells after genetic modification and/or transduction, wherein the culture medium may or may not already possess the cytokines or lymphocyte mitogens. In the absence of exogenously added cytokines or lymphocyte mitogens, all or part of the medium comprising a mixture of multiple medium components is typically stored and, in illustrative embodiments, transported to the site where the culture occurs. In some embodiments, the lymphocyte medium is purchased from a supplier and a user, such as a technician who is not employed by the supplier and is not located within the supplier's facility, adds exogenously added cytokines or lymphocyte mitogens to the lymphocyte medium and then cultures the genetically modified and/or transduced cells in the presence or absence of such exogenously added cytokines or lymphocyte mitogens.

In some embodiments, improved or enhanced survival, expansion and/or proliferation can be demonstrated as an increase in the number of cells determined by sequencing DNA from cells transduced with retroviral particles (e.g., lentiviral particles) having a genome encoding a CLE and counting the occurrence of sequences present in the unique identifier from each CLE. In some embodiments, improved survival and/or improved expansion can be determined by directly counting cells with a hemocytometer or cell counter at each time point. In some embodiments, improved survival and/or improved expansion and/or enrichment can be calculated by dividing the number of cells at a later time point (day 21, day 28, day 35, and/or day 45) by the number of cells at day 7 for each construct. In some embodiments, cells can be counted by a hemocytometer or cell counter. In some embodiments, the enrichment level determined by nucleic acid counts or cell counts for each specific test construct can be normalized to the enrichment level of a respective control construct (i.e., a construct having the same extracellular domain and transmembrane domain but lacking the intracellular domain present in the test construct). In these embodiments, the LE encoded in the construct provides (or cells genetically modified and/or transduced with retroviral particles (e.g., lentiviral particles) having a genome encoding the LE are capable of, suitable for, possess the following properties and/or are modified to provide) at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold normalized enrichment level, or between 1.5-fold and 25-fold normalized enrichment level, or between 3-fold and 20-fold normalized enrichment level, or between 5-fold and 25-fold normalized enrichment level, or between 5-fold and 20-fold normalized enrichment level, or between 5-fold and 15-fold normalized enrichment level.

In the illustrative embodiments herein, one or more lymphoproliferative components are introduced into T cells and/or NK cells, typically by genetically modifying and/or transducing T cells and/or NK cells with replication-deficient recombinant retroviral particles, the genome of which encodes a lymphoproliferative component as part of an engineered signaling polypeptide. Lymphoproliferative component The lymphoproliferative component may include a signaling intracellular domain or may include more than one intracellular domain. Lymphoproliferative component In certain illustrative embodiments, the lymphoproliferative component includes two intracellular domains. Tables 8 to 25 provided herein and discussed in Examples 10, 11, and 12 provide an intracellular domain for CLE, and provide CLE with two intracellular domains identified using the experimental methods provided herein. For CLEs that include two intracellular signaling domains in these examples, the first intracellular signaling domain is between the transmembrane domain and the second intracellular signaling domain. The "intracellular signaling domain" of LE is sometimes referred to herein as the "intracellular domain" of LE. These examples confirm that the vast majority of the first intracellular domains tested (P3 portions) enhance the effect of proliferation/expansion of genetically modified PBMCs, and all of the second intracellular domains tested (P4 portions) were found to be found in at least one and generally more effective CLE construct (e.g., see Table 25). Some of these P4 portions were identified as being able to promote proliferation/expansion of genetically modified PBMCs without the P3 portion, and thus able to serve as the first intracellular signaling domain of LE. In addition, the candidate chimeric polypeptides tested included different combinations of extracellular and intracellular domains identified in Table 7 and shown to be present in at least one of the top 100 constructs (Table 25). In some embodiments, the intracellular domain of the lymphoproliferative component may include any of the intracellular portions (S036 to S216) listed in Table 7, including the P3 portion in Example 10, Example 11, or Example 12, or in the absence of the P3 portion, any P4 portion identified in Example 10, Example 11, or Example 12 as having activity inducing expansion between day 7 and day 21 or at a later time point. Without being limited by theory, in a CLE containing two intracellular signaling domains, the first intracellular signaling domain (P3 in Example 10, Example 11, or Example 12 and Figures 15 to 18) is selected to have a greater effect on the ability of CLE to induce proliferation of transduced cells and can be referred to as the core or primary signaling domain of CLE. The second intracellular signaling domain (P4 in Example 10, Example 11, and Example 12 and Figures 15 to 18) is selected to serve as a co-stimulatory domain of the first intracellular signaling domain. In addition, the extracellular domain and the transmembrane domain (P1/2 in Example 11 and Figures 16 to 17; and P1 and P2 in Examples 10 and 12 and Figures 15 to 18, respectively) are designed to play a role in directing the first intracellular signaling domain to an active conformation or orientation. In some embodiments, the intracellular signaling domain may have at least 80%, 90%, or 95%, or may have 100% sequence identity with any one of SEQ ID NOs: 404 to 509. In some embodiments, the intracellular signaling domain may have at least 80%, 90%, or 95%, or may have 100% sequence identity with an intracellular domain from BTLA, CD2, CD3D, CD3E, CD3G, CD3Z, CD4, CD8A, CD8B, CD27, mutant delta Lck CD28, CD28, CD40, CD79A, CD79B, CD137, CRLF2, CSF2RB, CSF2RA, CSF3R, DAP10/CD28, DAP12, EPOR, FCER1G, FCGR2A, FCGR2C, FCGRA2, GHR, ICOS, IFNAR1, IFNAR2, IFNGR1, IFNGR2, IFNLR1, IL1R1, IL1RAP, IL 1RL1, IL1RL2, IL2RA, IL2RB, IL2RG, IL3RA, IL4R, IL5RA, IL6R, IL6ST, IL7RA, IL9R, IL10RA, IL 10RB, IL11RA, IL12RB1, IL12RB2, IL13RA1, IL13RA2, IL15RA, IL17RA, IL17RB, IL17RC, IL17RD, IL17RE, IL18R1, IL18RAP, IL20RA, IL20RB, IL21R, IL22RA1, IL23R, IL27RA, IL31RA, LEPR, LIFR, LMP1, MPL, MYD88, OSMR, PRLR, TNFRSF4, TNFRSF8, TNFRSF9, TNFRSF14, TNFRSF18 or ZAP70, or variants or fragments thereof that promote signaling. In some embodiments, the intracellular domain may have at least 80%, 90%, or 95%, or may have 100% sequence identity to an intracellular domain from CD2, CD4, CD8A, CD8B, CD40, CD79B, CRLF2, CSF2RB, CSF2RA, CSF3R, EPOR, FCGR2C, FCGR2A, GHR, IFNAR1, IFNAR2, IFNGR1, IFNGR2, IFNLR1, IL1R1, IL1RAP, IL1RL1, IL1RL2, IL2RA, IL2RB, I L2RG, IL3RA, IL4R, IL5RA, IL6R, IL6ST, IL9R, IL10RA, IL10RB, IL11RA, IL13RA1, IL13RA2, IL17RA, IL17RB, IL17RC, IL17RD, IL17RE, IL18R1, IL18RAP, IL20RA, IL20RB, IL22RA1, IL31RA, LEPR, LIFR, LMP1, MPL, MYD88, OSMR and PRLR, or variants or fragments that promote signaling.

In some illustrative embodiments, the lymphoproliferative component lymphoproliferative component may be or may include a cytokine, or in other illustrative embodiments, a cytokine receptor, or a fragment including its signaling domain, which activates a STAT3 path, a STAT4 path, or even in other illustrative embodiments, a Jak/STAT5 path. Therefore, in a non-limiting example, the lymphoproliferative component lymphoproliferative component may be a cytokine receptor or an active fragment including its signaling domain, such as an interleukin receptor, or an active fragment including its signaling domain that activates STAT5. Therefore, the lymphoproliferative component lymphoproliferative component is a polypeptide that promotes hyperplasia and optionally survives (anti-apoptosis) and optionally provides a costimulatory signal that enhances the differentiation state of lymphocytes, hyperplastic potential, or resistance to cell death. In illustrative embodiments, the lymphoproliferative component lymphoproliferative component is a polypeptide that induces hyperplasia of T cells and/or NK cells. The illustrative lymphoproliferative component lymphoproliferative component induces hyperplasia by activating STAT5. Thus, fragments of this lymphoproliferative component lymphoproliferative component, in illustrative embodiments, retain the ability to induce proliferation of T cells and/or NK cells by activating STAT5.

In illustrative embodiments, the lymphoproliferative component, when present in genetically modified PBMCs, lymphocytes, or genetically modified T cells and/or NK cells, is capable of promoting lymphocyte proliferation/expansion and survives in culture, optionally ex vivo or in vitro, for 6, 7, 14, 21 or 35 days in culture, in the absence of exposure of the cells to cytokines (such as IL-15, IL-7, or, in illustrative embodiments, IL-2), in some illustrative embodiments, further in the absence of the target of the ASTR of the CAR expressed by the cells, or in certain illustrative embodiments, in the presence of an antigen recognized by the CAR (wherein the method comprises genetically and/or transducing using a retroviral particle having a pseudodifferentiation component and, optionally, a separate or fusion activation domain on its surface, and generally without the need for pre-activation).

In some of the methods and compositions presented herein, the lymphoproliferative component is used to promote the proliferation or expansion of genetically modified T cells in vivo without having to deplete individual lymph. Therefore, the non-limiting illustrative embodiments of the methods provided herein (including inserting the lymphoproliferative component into the resting T cells and/or NK cells of an individual, usually by transducing such T cells and/or NK cells) can be performed without depleting individual lymph before, during and/or after performing the method, or before, during and/or after collecting blood from an individual before performing this method, or before, during and/or after genetically modifying T cells and/or NK cells from an individual in vitro, and/or before, during and/or after reintroducing the genetically modified T cells and/or NK cells into an individual. Factors that promote the proliferation of T cells in vivo include cytokines and their receptors, wherein the receptors generally include a ligand binding domain and a signaling domain. In some embodiments, the lymphoproliferative element used in the methods and compositions disclosed herein is a cytokine and/or a cytokine receptor. The cytokine may be an interleukin, and the cytokine receptor may be an interleukin receptor. The lymphoproliferative element may be a functional fragment of a cytokine and/or a functional fragment of a cytokine receptor (such as a signaling domain thereof), wherein the fragment is capable of promoting the proliferation of T cells, for example, by activating STAT5.

In some embodiments, the cytokine lymphoproliferative component of the methods and compositions herein includes one or more of the following: interleukin-7 (IL-7) or its receptor (IL-7R), or its signaling domain; interleukin-12 (IL-12) or its receptor (IL-12R), or its signaling domain; interleukin-23 (IL-23) or its receptor composed of IL-12Rβ1 and IL-23R, or its signaling domain; interleukin-2 7 (IL-27) or its receptor (IL-27R), or its signaling domain; interleukin-15 (IL-15) or its receptor (IL-15R), or its signaling domain; interleukin-21 (IL-21) or its receptor (IL-21R), or its signaling domain; or transforming growth factor β (TGFβ) or its receptor (TGFβR) or its signaling domain; or TGFβ decoy receptor (TGF-β-dominant-negative receptor II (DNRII)). In some embodiments, the lymphoproliferative component lymphoproliferative component is IL-12R or TGFβ decoy receptor (TGF-β-dominant-negative receptor II (DNRII)).

IL-7 binds to the IL-7 receptor, a heterodimer consisting of IL-7Rα and a common γ chain. Binding generates a signaling cascade that is critical for T cell development in the thymus and survival in the periphery. Binding of IL-7 to the IL-7 receptor is known to activate the Jak/STAT5 pathway.

IL-12 is involved in the differentiation of naive T cells into Th1 cells (Hsieh CS et al. 1993. Science. 260 (5107): 547-9) and is called a T cell stimulator. IL-12 binds to the IL-12 receptor (which is a heterodimeric receptor formed by IL-12R-β1 and IL-12R-β2). IL12 can act by activating STAT4, but has been shown to also activate STAT5 in T cells (Ahn, H. et al. 1998. J. Immun. 161: 5893-5900). The IL-12 family consists of cytokines IL-12, IL-23, and IL-27. The receptor for IL-23 consists of IL-12Rβ1 and IL-23R. IL-27 is a heterodimeric cytokine composed of two different genes (Epstein-Barr virus-induced gene 3 (EBI3) and IL-27p28). IL-27 interacts with the IL-27 receptor.

IL-15 is a T cell and NK cell stimulator similar in structure and function to IL-2. Both cytokines induce T cell proliferation; and their shared function is thought to result from the use of two receptors, IL-2/IL-15Rβ and a common γ chain. The signaling pathway of IL-15 begins by binding to the IL-15Rα receptor and subsequent presentation to surrounding cells with IL-15Rβγc complexes on their cell surfaces. Upon binding to IL-15β, the subunit activates Janus kinase 1 (Jak1) and the γc subunit Janus kinase 3 (Jak3), which leads to phosphorylation and activation of STAT3 and STAT5.

IL-21 is expressed in activated human CD4+T cells and NK T cells, and IL-21 expression is upregulated in Th2 and Th17 subsets of T helper cells. IL-21 receptor (IL-21R) is expressed on the surface of T cells, B cells and NK cells and is structurally similar to the receptors of other I type cytokines like IL-2R or IL-15. IL-21R needs to be dimerized with common gamma chains (γc) in order to combine IL-21. When attached to IL-21, IL-21 receptor works via Jak/STAT path, thereby activating STAT1, STAT3 and STAT5.

The TGFβ decoy receptor (TGF-β—dominant-negative receptor II (DNRII)) blocks TGFβ signaling by competing with the native receptor for TGFβ binding. TGFβ-DNRII is a kinase-inactive, truncated form of RII that contains the extracellular TGFβ binding domain and transmembrane domain of RII. TGFβ-DNRII binds ligand but does not phosphorylate and activate RI, thereby reducing or eliminating Smad phosphorylation.

Mutations that increase the functionality of IL-7Rα have been identified in individuals with B-cell and T-cell acute lymphoblastic leukemia (B-ALL and T-ALL) (Zenatti PP et al. 2011. Nat Genet 43:932-939; Snochat, C. et al. 2011. J Exp Med 208:901-908; McElroy, C.A. et al. 2012. PNAS 109 (7): 2503-2508). Mutations include insertions and deletions in the N-terminal region of the IL-7Rα TMD, in which almost all sequences contain additional Cys residues, and S165 to C165 mutations. Cysteine leads to constitutive activation of the receptor. Some of the mutations in all T groups activate JAK1. These IL-7R mutants with increased functionality can be used as one of the lymphoproliferative components in any of the aspects provided herein.

Therefore, in some embodiments, the lymphoproliferative component lymphoproliferative component is a mutated IL-7 receptor. In other embodiments, the mutated IL-7 receptor is constitutively active, thereby activating the JAK-STAT5 pathway in the absence of cytokine ligands. In yet other embodiments, the mutated IL-7 receptor comprises 1 to 10 amino acid insertions at a position between 237 and 254, the insertion comprising a cysteine residue that includes the ability to constitutively activate the STAT5 pathway. In some embodiments, the mutated IL-7 receptor is IL-7Rα-insPPCL (represented by SEQ ID NO: 82). In addition, in some chimeric lymphoproliferative component lymphoproliferative component (CLE) embodiments provided herein, one or more (but not all) of the domains are from IL-7Rα-insPPCL.

In some embodiments, the lymphoproliferative component lymphoproliferative component is a chimeric cytokine receptor, such as, but not limited to, a cytokine connected to its receptor, which usually constitutively activates the same STAT pathway as the corresponding activated wild-type cytokine receptor (such as STAT3, STAT4, and in illustrative embodiments, STAT5). In some embodiments, the chimeric cytokine receptor is an interleukin or a fragment thereof, which is connected to or covalently connected to its cognate receptor via a connector. In some embodiments, the chimeric cytokine receptor is an IL-7 connected to IL-7Rα. In other embodiments, the chimeric cytokine receptor is an IL-7 connected to an IL-7Rα domain, such as an extracellular domain of IL-7Rα and/or a transmembrane domain of IL-7Rα. In some embodiments, the lymphoproliferative component lymphoproliferative component is a cytokine receptor that is not connected to a cytokine, and in fact in some embodiments, the lymphoproliferative component provided herein is a constitutively active cytokine receptor that is not connected to a cytokine. These chimeric IL-7 receptors typically constitutively activate STAT5 when expressed.

In the illustrative embodiments of any one of the methods and compositions comprising a lymphoproliferative component lymphoproliferative component provided herein, wherein the lymphoproliferative component lymphoproliferative component is a cytokine or cytokine receptor polypeptide or a fragment thereof comprising a signaling domain, the lymphoproliferative component lymphoproliferative component may include a portion covalently linked to its homologous interleukin receptor polypeptide via a connector. Typically, this portion of a homologous interleukin receptor includes a functional portion of an extracellular domain and a transmembrane domain capable of binding an interleukin cytokine. In some embodiments, the intracellular domain is an intracellular portion of a homologous interleukin receptor. In some embodiments, the intracellular domain is an intracellular portion of different cytokine receptors capable of promoting lymphocyte proliferation. In some embodiments, the lymphoproliferative component lymphoproliferative component is an interleukin polypeptide covalently linked to its full-length homologous interleukin receptor polypeptide via a connector.

In illustrative embodiments of any of the methods and compositions provided herein that include a lymphoproliferative component, the intracellular domain can be derived from the intracellular portion of the transmembrane protein CD40. The CD40 protein contains several binding sites for TRAF proteins. Without being limited by theory, the binding sites for TRAF1, TRAF2, and TRAF3 are located in the membrane distal domain of the intracellular portion of CD40 and include the amino acid sequence PXQXT (SEQ ID NO: 522), wherein each X can be any amino acid (corresponding to amino acids 35 to 39 of SEQ ID NO: 416) (Elgueta et al., Immunol Rev. 2009 May; 229(1): 152-72). TRAF2 has also been shown to bind to the consensus sequence SXXE (SEQ ID NO: 523), wherein each X can be any amino acid (corresponding to amino acids 57 to 60 of SEQ ID NO: 416) (Elgueta et al., Immunol Rev. 2009 May; 229(1): 152-72). The significant binding site for TRAF6 is located in the membrane proximal domain of the intracellular portion of CD40 and includes the consensus sequence QXPXEX (SEQ ID NO: 524), where each X can be any amino acid (corresponding to amino acids 16 to 21 of SEQ ID NO: 416) (Lu et al., J Biol Chem. 2003 Nov 14; 278(46): 45414-8). In an illustrative embodiment, the intracellular portion of the transmembrane protein CD40 may include all binding sites for TRAF proteins. TRAF binding sites are known in the art, and the skilled artisan will be able to identify corresponding TRAF binding sites in similar CD40 polypeptides. In some embodiments, suitable intracellular domains may include domains having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in SEQ ID NO: 416 or SEQ ID NO: 417. In some embodiments, the intracellular domain derived from CD40 has a length of about 30 amino acids (aa) to about 35 aa, about 35 aa to about 40 aa, about 40 aa to about 45 aa, about 45 aa to about 50 aa, about 50 aa to about 55 aa, about 55 aa to about 60 aa, or about 60 aa to about 65 aa. In illustrative embodiments, the intracellular domain derived from CD40 has a length of about 30 aa to about 66 aa, e.g., 30 aa to 65 aa or 50 aa to 66 aa. In illustrative embodiments of a lymphoproliferative element comprising a first intracellular domain derived from CD40, the second intracellular domain may not be an intracellular domain derived from MyD88, a CD28 family member (e.g., CD28, ICOS), a pattern recognition receptor, a C-reactive protein receptor (i.e., Nodi, Nod2, PtX3-R), a TNF receptor (i.e., CD40, RANK/TRANCE-R, OX40, 4-1BB), a HSP receptor (Lox-1 and CD91), or CD28. Pattern recognition receptors include, but are not limited to, endocytic pattern recognition receptors (i.e., mannose receptor, scavenger receptors (i.e., Mac-1, LRP, peptidoglycan, teichoic acid, toxins, CD1 1c/CR4)); external signal pattern recognition receptors (Toll-like receptors (TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10), peptidoglycan recognition protein (PGRP binds bacterial peptidoglycan and CD14)); external signal pattern recognition receptors (i.e., NOD receptors 1 and 2) and RIG1.

In the illustrative embodiments of any of the methods and compositions provided herein including a lymphoproliferative component, the intracellular domain may be derived from the intracellular portion of CD27. The serine at amino acid 219 of the full-length CD27 (corresponding to the serine at amino acid 6 of SEQ ID NO:413) is shown to be phosphorylated. In some embodiments, a suitable intracellular domain may include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to at least 10, 15, 20 or all of the amino acids in SEQ ID NO:413. In some embodiments, the intracellular domain derived from CD27 has a length of about 30 amino acids (aa) to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, or about 45aa to about 50aa.

In the illustrative embodiments of any of the methods and compositions provided herein including a lymphoproliferative component, the intracellular domain may be derived from the intracellular portion of CSF2RB. The full-length CSF2RB contains a Box 1 motif at amino acids 474 to 482 (corresponding to amino acids 14 to 22 of SEQ ID NO: 421). The tyrosine at amino acid 766 of the full-length CSF2RB (corresponding to the tyrosine at amino acid 306 of SEQ ID NO: 421) is shown to be phosphorylated. In some embodiments, a suitable intracellular domain may include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to at least 10, 15, 20 or all of the amino acids in SEQ ID NO: 421. In some embodiments, the intracellular domain derived from CSF2RB has a length of about 30 aa to about 35 aa, about 35 aa to about 40 aa, about 40 aa to about 45 aa, about 45 aa to about 50 aa, about 50 aa to about 55 aa, about 55 aa to about 60 aa, about 60 aa to about 65 aa, about 65 aa to about 70 aa, about 70 aa to about 100 aa, about 100 aa to about 125 aa, about 125 aa to 150 aa, about 150 aa to about 175 aa, about 175 aa to about 200 aa, about 200 aa to about 250 aa, about 250 aa to 300 aa, about 300 aa to 350 aa, about 350 aa to about 400 aa, or about 400 aa to about 450 aa.

In illustrative embodiments of any of the methods and compositions provided herein that include a lymphoproliferative component, the intracellular domain can be derived from the intracellular portion of IL2RB. Full-length IL2RB contains a Box 1 motif at amino acids 278 to 286 (corresponding to amino acids 13 to 21 of SEQ ID NO: 448). In some embodiments, a suitable intracellular domain can include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in SEQ ID NO: 448. In some embodiments, the intracellular domain derived from IL2RB has a length of about 30 aa to about 35 aa, about 35 aa to about 40 aa, about 40 aa to about 45 aa, about 45 aa to about 50 aa, about 50 aa to about 55 aa, about 55 aa to about 60 aa, about 60 aa to about 65 aa, about 65 aa to about 70 aa, about 70 aa to about 100 aa, about 100 aa to about 125 aa, about 125 aa to 150 aa, about 150 aa to about 175 aa, about 175 aa to about 200 aa, about 200 aa to about 250 aa, or about 250 aa to 300 aa.

In the illustrative embodiments of any of the methods and compositions provided herein including a lymphoproliferative component, the intracellular domain can be derived from the intracellular portion of IL6ST. Full-length IL6ST contains a Box 1 motif at amino acids 651 to 659 (corresponding to amino acids 10 to 18 of SEQ ID NO: 455). Serine at amino acids 661, 667, 782, 789, 829, and 839 of full-length IL6ST (corresponding to amino acids 20, 26, 141, 148, 188, and 198 of SEQ ID NO: 455, respectively) is shown to be phosphorylated. In some embodiments, a suitable intracellular domain may include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20 or all of the amino acids in SEQ ID NO:454 or SEQ ID NO:455. In some embodiments, the intracellular domain derived from IL6ST has a length of about 30 aa to about 35 aa, about 35 aa to about 40 aa, about 40 aa to about 45 aa, about 45 aa to about 50 aa, about 50 aa to about 55 aa, about 55 aa to about 60 aa, about 60 aa to about 65 aa, about 65 aa to about 70 aa, about 70 aa to about 100 aa, about 100 aa to about 125 aa, about 125 aa to 150 aa, about 150 aa to about 175 aa, about 175 aa to about 200 aa, about 200 aa to about 250 aa, or about 250 aa to 300 aa.

In illustrative embodiments of any of the methods and compositions provided herein that include a lymphoproliferative component, the intracellular domain may be derived from the intracellular portion of IL17RE. The full-length IL17RE contains a TIR domain at amino acids 372 to 495 (corresponding to amino acids 13 to 136 of SEQ ID NO: 473). In some embodiments, a suitable intracellular domain may include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to at least 10, 15, 20 or all of the amino acids in SEQ ID NO: 473. In some embodiments, the intracellular domain derived from IL17RE has a length of about 30 aa to about 35 aa, about 35 aa to about 40 aa, about 40 aa to about 45 aa, about 45 aa to about 50 aa, about 50 aa to about 55 aa, about 55 aa to about 60 aa, about 60 aa to about 65 aa, about 65 aa to about 70 aa, about 70 aa to about 100 aa, about 100 aa to about 125 aa, about 125 aa to 150 aa, about 150 aa to about 175 aa, or about 175 aa to about 200 aa.

In illustrative embodiments of any of the methods and compositions provided herein that include a lymphoproliferative component, the intracellular domain can be derived from the intracellular portion of IL2RG. Full-length IL2RG contains a box 1 motif at amino acids 286 to 294 (corresponding to amino acids 3 to 11 of SEQ ID NO: 449). In some embodiments, a suitable intracellular domain can include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in SEQ ID NO: 449. In some embodiments, the intracellular domain derived from IL2RG has a length of about 30 amino acids (aa) to about 35 aa, about 35 aa to about 40 aa, about 40 aa to about 45 aa, about 45 aa to about 50 aa, about 50 aa to about 55 aa, about 55 aa to about 60 aa, about 60 aa to about 65 aa, about 65 aa to about 70 aa, or about 70 aa to about 100 aa.

In illustrative embodiments of any of the methods and compositions provided herein that include a lymphoproliferative component, the intracellular domain may be derived from an intracellular portion of IL18R1. Full-length IL18R1 contains a TIR domain at amino acids 222 to 364 (corresponding to amino acids 28 to 170 of SEQ ID NO: 474). In some embodiments, a suitable intracellular domain may include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to at least 10, 15, 20 or all of the amino acids in SEQ ID NO: 474. In some embodiments, the intracellular domain derived from IL18R1 has a length of about 30 aa to about 35 aa, about 35 aa to about 40 aa, about 40 aa to about 45 aa, about 45 aa to about 50 aa, about 50 aa to about 55 aa, about 55 aa to about 60 aa, about 60 aa to about 65 aa, about 65 aa to about 70 aa, or about 70 aa to about 100 aa.

In illustrative embodiments of any of the methods and compositions provided herein that include a lymphoproliferative component, the intracellular domain can be derived from an intracellular portion of IL27RA. Full-length IL27RA contains a box 1 motif at amino acids 554 to 562 (corresponding to amino acids 17 to 25 of SEQ ID NO: 481). In some embodiments, a suitable intracellular domain can include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to at least 10, 15, 20, or all of the amino acids in SEQ ID NO: 481 or SEQ ID NO: 482. In some embodiments, the intracellular domain derived from IL27RA has a length of about 30 aa to about 35 aa, about 35 aa to about 40 aa, about 40 aa to about 45 aa, about 45 aa to about 50 aa, about 50 aa to about 55 aa, about 55 aa to about 60 aa, about 60 aa to about 65 aa, about 65 aa to about 70 aa, or about 70 aa to about 100 aa.

In illustrative embodiments of any of the methods and compositions provided herein that include a lymphoproliferative component, the intracellular domain may be derived from an intracellular portion of IFNGR2. Full-length IFNGR2 contains a dileucine internalization motif at amino acids 276 to 277 (corresponding to amino acids 8 to 9 of SEQ ID NO: 438). In some embodiments, a suitable intracellular domain may include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to at least 10, 15, 20 or all of the amino acids in SEQ ID NO: 438. In some embodiments, the intracellular domain derived from IFNGR21 has a length of about 30 aa to about 35 aa, about 35 aa to about 40 aa, about 40 aa to about 45 aa, about 45 aa to about 50 aa, about 50 aa to about 55 aa, about 55 aa to about 60 aa, about 60 aa to about 65 aa, or about 65 aa to about 70 aa.

In illustrative embodiments of any of the methods and compositions provided herein that include a lymphoproliferative component, the intracellular domain can be derived from the intracellular portion of MyD88. The MyD88 protein has an N-terminal death domain (corresponding to amino acids 29 to 106 of SEQ ID NO: 492) that mediates interaction with other death domain-containing proteins, an intermediate domain (corresponding to amino acids 107 to 156 of SEQ ID NO: 492) that interacts with IL-1R-related kinases, and a C-terminal TIR domain (corresponding to amino acids 160 to 304 of SEQ ID NO: 492) that associates with the TLR-TIR domain (Biol Res. 2007; 40(2): 97-112). MyD88 also has a canonical nuclear localization and export motif. Point mutations have been identified in MyD88 and include loss-of-function mutations L93P and R193C (corresponding to L93P and R196C in SEQ ID NO: 492) and gain-of-function mutation L265P (corresponding to L260P in SEQ ID NO: 492) (Deguine and Barton. F1000 Prime Rep. 2014 Nov 4;6:97). In some embodiments, the portion of protein MyD88 may include all of the domains disclosed herein. The domains, motifs, and point mutations of MyD88 are known in the art, and the skilled artisan will be able to identify corresponding domains, motifs, and point mutations in similar MyD88 polypeptides. In some embodiments, a suitable intracellular domain may include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20 or all of the amino acids in SEQ ID NOs:492 to 501. In some embodiments, the intracellular domain derived from MyD88 has a length of about 30 aa to about 35 aa, about 35 aa to about 40 aa, about 40 aa to about 45 aa, about 45 aa to about 50 aa, about 50 aa to about 55 aa, about 55 aa to about 60 aa, about 60 aa to about 65 aa, about 65 aa to about 70 aa, about 70 aa to about 100 aa, about 100 aa to about 125 aa, about 125 aa to 150 aa, about 150 aa to about 175 aa, about 175 aa to about 200 aa, about 200 aa to about 250 aa, about 250 aa to 300 aa, or about 300 aa to 350 aa. In illustrative embodiments, the intracellular domain derived from MyD88 has a length of about 30 aa to about 350 aa, e.g., 50 aa to 350 aa, or 100 aa to 350 aa, 100 aa to 304 aa, 100 aa to 296 aa, 100 aa to 251 aa, 100 aa to 191 aa, 100 aa to 172 aa, 100 aa to 146 aa, or 100 aa to 127 aa. In illustrative embodiments of a lymphoproliferative element comprising a first intracellular domain derived from MyD88, the second intracellular domain may not be an intracellular domain derived from a CD28 family member (e.g., CD28, ICOS), a pattern recognition receptor, a C-reactive protein receptor (i.e., Nodi, Nod2, PtX3-R), a TNF receptor (i.e., CD40, RANK/TRANCE-R, OX40, 4-1BB), a HSP receptor (Lox-1 and CD91), or CD28.

In illustrative embodiments of any of the methods and compositions provided herein that include a lymphoproliferative component, the intracellular domain can be derived from a portion of the transmembrane protein MPL. The transmembrane MPL protein contains a Box1 motif, PXXP (SEQ ID NO: 525), and a Box2 motif, which is a region with increased serine and glutamate content (corresponding to amino acids 46 to 64 in SEQ ID NO: 491), in which each X can be any amino acid (corresponding to amino acids 17 to 20 in SEQ ID NO: 491) (Drachman and Kaushansky. Proc Natl Acad Sci U S A. 1997 Mar 18; 94(6): 2350-5). Even though the presence of the Box2 motif is not always required for a proliferative signal, Box1 and Box2 motifs are also involved in binding to JAKs and signal transduction (Murakami et al., Proc Natl Acad Sci USA. 1991 Dec 15;88(24):11349-53; Fukunaga et al., EMBO J. 1991 Oct;10(10):2855-65; and O'Neal and Lee. Lymphokine Cytokine Res. 1993 Oct;12(5):309-12). Many cytokine receptors have hydrophobic residues at positions -1, -2, and -6 relative to the Box1 motif (corresponding to amino acids 16, 15, and 11, respectively, of SEQ ID NO: 491) that form a "switch motif" that requires cytokine-induced JAK2 activation but does not require JAK2 binding (Constantinescu et al., Mol Cell. 2001 Feb; 7(2):377-85; and Huang et al., Mol Cell. 2001 Dec; 8(6):1327-38). A deletion of the region encompassing amino acids 70 to 95 in SEQ ID NO: 491 was shown to support viral transformation in the context of v-mpl (Benit et al. J Virol. 1994 Aug; 68(8):5270-4), thus indicating that this region is not essential for the function of mpl in this context. Morello et al., Blood 1995 Jul;86(8):557-71, using the same deletion, showed that this region is not required for stimulating transcription of an erythropoietin receptor-responsive CAT receptor gene construct, and it was further seen that this deletion resulted in slightly enhanced transcription, which is expected to remove non-essential and negative components in this region as suggested by Drachman and Kaushansky. Thus, in some embodiments, the MPL intracellular signaling domain does not include the region comprising amino acids 70 to 95 in SEQ ID NO: 491. In full-length MPL, lysine K553 (corresponding to K40 of SEQ ID NO: 491) and K573 (corresponding to K60 of SEQ ID NO: 491) have been shown to be negative regulatory sites that act as part of a ubiquitination targeting motif (Saur et al., Blood 2010 Feb 11;115(6):1254-63). Thus, in some embodiments herein, the MPL intracellular signaling domain does not include these ubiquitination targeting motif residues. In full-length MPL, tyrosines Y521 (corresponding to Y8 of SEQ ID NO: 491), Y542 (corresponding to Y29 of SEQ ID NO: 491), Y591 (corresponding to Y78 of SEQ ID NO: 491), Y626 (corresponding to Y113 of SEQ ID NO: 491), and Y631 (corresponding to Y118 of SEQ ID NO: 491) were shown to be phosphorylated (Varghese et al., Front Endocrinol (Lausanne). 2017 Mar 31;8:59). Y521 and Y591 of full-length MPL are negative regulatory sites that act as a lysosomal targeting motif (Y521) or through interaction with the adaptor protein AP2 (Y591) (Drachman and Kaushansky, Proc Natl Acad Sci USA. 1997 Mar 18;94(6):2350-5; and Hitchcock et al., Blood. 2008 Sep 15;112(6):2222-31). Y626 and Y631 of full-length MPL are positive regulatory sites (Drachman and Kaushansky, Proc Natl Acad Sci USA. 1997 Mar 18;94(6):2350-5), and the murine homolog of Y626 is required for cellular differentiation and phosphorylation of Shc (Alexander et al., EMBO J. 1996 Dec 2;15(23):6531-40), and Y626 is also required for constitutive signaling in MPL with the W515A mutation described below (Pecquet et al., Blood. 2010 Feb 4;115(5):1037-48). MPL contains a binding motif NXXY (SEQ ID NO: 526) that binds Shc phosphotyrosine, where each X can be any amino acid (corresponding to amino acids 110 to 113 of SEQ ID NO: 491), and this tyrosine is phosphorylated and is essential for TPO-dependent phosphorylation of Shc, SHIP, and STAT3 (Laminet et al., J Biol Chem. 1996 Jan 5;271(1):264-9; and van der Geer et al., Proc Natl Acad Sci U S A. 1996 Feb 6;93(3):963-8). MPL also contains a STAT3 consensus binding sequence YXXQ (SEQ ID NO: 527), where each X can be any amino acid (corresponding to amino acids 118 to 121 of SEQ ID NO: 491) (Stahl et al., Science. 1995 Mar 3;267(5202):1349-53). The tyrosine of this sequence can be phosphorylated, and MPL is capable of partial STAT3 recruitment (Drachman and Kaushansky, Proc Natl Acad Sci USA. 1997 Mar 18;94(6):2350-5). MPL also contains the sequence YLPL (SEQ ID NO:528) (corresponding to amino acids 113 to 116 of SEQ ID NO:491), which is similar to the consensus binding site for STAT5 recruitment of pYLXL (SEQ ID NO:529), where pY is a phosphotyrosine and X can be any amino acid (May et al., FEBS Lett. 1996 Mar 30;394(2):221-6). Using computer simulations, Lee et al. found that clinically relevant mutations in the transmembrane domain of MPL should activate MPL in the following order of activation effect: W515K (corresponding to the amino acid substitution W2K of SEQ ID NO: 491) > S505A (corresponding to the amino acid substitution S14A of SEQ ID NO: 395) > W515I (corresponding to the amino acid substitution W2I of SEQ ID NO: 491) > S505N (corresponding to the amino acid substitution S14N of SEQ ID NO: 395, which was tested as T075 (SEQ ID NO: 396) in Example 12) (PLoS One. 2011; 6(8): e23396). It is predicted that simulations of these mutations may result in constitutive activation of JAK2, the kinase partner of MPL. In some embodiments, the intracellular portion of MPL may include all domains and motifs described herein and present in SEQ ID NO: 491. In some embodiments, the transmembrane portion of MPL may include all domains and motifs described herein and present in SEQ ID NO: 395. The domains, motifs and point mutations of MPL provided herein are known in the art, and the skilled artisan will recognize that the intracellular signaling domains of MPL herein will include, in illustrative embodiments, corresponding domains, motifs and point mutations that are shown to promote proliferative activity, and will not include those that are shown to inhibit MPL proliferative activity. In some embodiments, a suitable intracellular domain may include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20 or all of the amino acids in SEQ ID NO: 491. In some embodiments, the intracellular domain derived from MPL has about 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, about 45aa to about 50aa, about 50aa to about 55aa, about 55aa to about 60aa, about 60aa to about 65aa, about 65aa to about 70aa, about 70aa to about 100aa, about 100aa to about 125aa, about 125aa to 150aa, In an illustrative embodiment, the intracellular domain derived from MPL has a length of about 30aa to about 200aa, such as 30aa to 150aa, 30aa to 119aa, 30aa to 121aa, 30aa to 122aa, or 50aa to 125aa.

In illustrative embodiments of any of the methods and compositions provided herein that include a lymphoproliferative component, the intracellular domain may be derived from a portion of the transmembrane protein CD79B, also known as B29; IGB; AGM6. CD79B contains an ITAM motif at residues 193 to 212 (corresponding to amino acids 16 to 30 of SEQ ID NO: 419). CD79B has two tyrosines Y196 and Y207 known to be phosphorylated (corresponding to Y16 and Y27 of SEQ ID NO: 419). In some embodiments, the intracellular portion of the transmembrane protein CD79B may include the ITAM motifs and known phosphorylation sites described herein. The motifs and phosphorylatable tyrosines of CD79B are known in the art, and a skilled person will be able to identify corresponding motifs and phosphorylatable tyrosines in similar CD79B polypeptides. In some embodiments, suitable intracellular domains may include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to at least 10, 15, 20, or all of the amino acids in SEQ ID NO: 419. In some embodiments, the intracellular domain derived from CD79B has a length of about 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, or about 45aa to about 50aa. In illustrative embodiments, the intracellular domain derived from CD79B has a length of about 30aa to about 50aa. In some embodiments, a suitable CD79B intracellular activation domain may comprise a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a stretch of at least 10, 15, 20 or all amino acids of the following sequence: LDKDDSKAGMEEDHT[YEGLDIDQTATYEDI]VTLRTGEVKWSVGEHPGQE (SEQ ID NO:419), wherein the ITAM motif is described in brackets.

In illustrative embodiments of any of the methods and compositions provided herein that include a lymphoproliferative component, the intracellular domain may be derived from a portion of the transmembrane protein OSMR. OSMR contains a Box 1 motif at amino acids 771 to 779 of isoform 3 (corresponding to amino acids 16 to 30 of SEQ ID NO: 502). OSMR has two serines at amino acids 829 and 890 of isoform 3 known to be phosphorylated (serines at amino acids 65 and 128 of SEQ ID NO: 502). In some embodiments, the intracellular portion of protein OSMR may include the box 1 motif and known phosphorylation sites described herein. The motifs and phosphorylatable tyrosines of OSMR are known in the art, and a skilled person will be able to identify corresponding motifs and phosphorylatable serines in similar OSMR polypeptides. In some embodiments, a suitable intracellular domain may include a domain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a stretch of at least 10, 15, 20, or all of the amino acids in SEQ ID NO:502. In some embodiments, the intracellular domain derived from OSMR has a length of about 30 aa to about 35 aa, about 35 aa to about 40 aa, about 40 aa to about 45 aa, about 45 aa to about 50 aa, about 50 aa to about 55 aa, about 55 aa to about 60 aa, about 60 aa to about 65 aa, about 65 aa to about 70 aa, about 70 aa to about 100 aa, about 100 aa to about 125 aa, about 125 aa to 150 aa, about 150 aa to about 175 aa, about 175 aa to about 200 aa, or about 200 aa to about 250 aa.

In the illustrative embodiments of any one of the methods and compositions including lymphoproliferative components provided herein, the intracellular domain can be derived from the part of the transmembrane protein PRLR.PRLR contains the growth hormone receptor binding domain at the amino acid 185 to 261 (corresponding to the amino acid 28 to 104 of SEQID NO:503) of isoform 6.The growth hormone receptor binding domain of PRLR is known in the art, and the technician will be able to identify the corresponding domain in similar PRLR polypeptides.In certain embodiments, the intracellular domain suitable for can include at least 10, 15, 20 or all of the amino acids in SEQ ID NO:503 with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity. In some embodiments, the intracellular domain derived from PRLR has a length of about 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, about 45aa to about 50aa, about 50aa to about 55aa, about 55aa to about 60aa, about 60aa to about 65aa, about 65aa to about 70aa, about 70aa to about 100aa, about 100aa to about 125aa, about 125aa to 150aa, about 150aa to about 175aa, about 175aa to about 200aa, about 200aa to about 250aa, about 250aa to 300aa, about 300aa to 350aa, or about 350aa to about 400aa.

In illustrative embodiments of any of the methods, uses, and composition aspects provided herein that include a lymphoproliferative component, the intracellular domain can be, the lymphoproliferative component can include an intracellular domain or a fragment thereof that includes a signaling domain from: CSF2RB, CRLF2, CSF2RA, CSF3R, EPOR, GHR, IFNAR1, IFNAR2, IFNGR1, IFNGR2, IFNLR1, IL1R1, IL1RAP, IL1RL1, IL1RL2, IL2RA, IL2RB, IL2RG, IL3RA, IL5 RA, IL6R, IL6ST, IL7RA, IL9R, IL10RA, IL10RB, IL11RA, IL12RB1, IL12RB2, IL13RA1, IL13RA2, IL15RA, IL17RB, IL17RC, IL17RD, IL18R1, IL18RAP, IL20RA, IL20RB, IL21R, IL22RA1, IL23R, IL27RA, IL31RA, LEPR, LIFR, LMP1, MPL, MyD88, OSMR or PRLR. Each of these genes has an intracellular domain that is present at the P3 position on at least one construct in the top 100 hits of at least one of the copies of library 3.1. In some embodiments, the lymphoproliferative component may include an intracellular domain or a fragment thereof that includes a signaling domain from CSF2RB, CRLF2, CSF2RA, CSF3R, EPOR, GHR, IFNAR1, IFNAR2, IFNGR1, IFNGR2, IFNLR1, IL1R1, IL1RAP, IL1RL1, IL1RL2, IL2RA, IL2RB, IL2RG, IL3RA, IL5RA, IL6R, IL6ST, IL9R, IL10RA, IL10RB, IL11RA, IL13RA1, IL13RA2, IL17RB, IL17RC, IL17RD, IL18R1, IL18RAP, IL20RA, IL20RB, IL22RA1, IL31RA, LEPR, LIFR, LMP1, MPL, MyD88, OSMR, or PRLR. In some embodiments, the lymphoproliferative component may include an intracellular domain or fragment thereof that includes a signaling domain from CSF2RB, CSF2RA, CSF3R, EPOR, IFNGR1, IFNGR2, IL1R1, IL1RAP, IL1RL1, IL2RA, IL2RG, IL5RA, IL6R, IL9R, IL10RB, IL11RA, IL12RB1, IL12RB2, IL13RA2, IL15RA, IL17RD, IL21R, IL23R, IL27RA, IL31RA, LEPR, MPL, MyD88, or OSMR. Each of these genes has an intracellular domain that is present at the P3 position on at least one construct that does not have an intracellular domain in the P4 position and is in the top 100 hits of at least one of the replicates of library 3.1. In some embodiments, the lymphoproliferative component may include an intracellular domain or a fragment thereof that includes a signaling domain from CSF2RB, CSF2RA, CSF3R, EPOR, IFNGR1, IFNGR2, IL1R1, IL1RAP, IL1RL1, IL2RA, IL2RG, IL5RA, IL6R, IL9R, IL10RB, IL11RA, IL13RA2, IL17RD, IL31RA, LEPR, MPL, MyD88, or OSMR. In some embodiments, the lymphoproliferative component may include an intracellular domain or a fragment thereof that includes a signaling domain from CSF2RB, CSF3R, IFNAR1, IFNGR1, IL2RB, IL2RG, IL6ST, IL10RA, IL12RB2, IL17RC, IL17RE, IL18R1, IL22RA1, IL27RA, IL31RA, MPL, MyD88, OSMR, or PRLR (Table 1). Each of these genes has an intracellular domain that is significantly enriched at the P3 position across the repeats of library 3.1 (p<0.1). In some embodiments, the lymphoproliferative component may include an intracellular domain or a fragment thereof comprising a signaling domain from CSF2RB, CSF3R, IFNAR1, IFNGR1, IL2RB, IL2RG, IL6ST, IL10RA, IL17RC, IL17RE, IL18R1, IL22RA1, IL31RA, MPL, MyD88, OSMR, or PRLR. In some embodiments, the lymphoproliferative component may include an intracellular domain or a fragment thereof comprising a signaling domain from CSF2RB, CSF3R, IFNGR1, IL2RB, IL2RG, IL6ST, IL10RA, IL12RB2, IL17RE, IL18R1, IL22RA1, IL27RA, IL31RA, MPL, MyD88, OSMR, or PRLR (Table 1). Each of these genes has an intracellular domain that is significantly enriched at the P3 position across the repeats of library 3.1 (p<0.05). In some embodiments, the lymphoproliferative component may include an intracellular domain or a fragment thereof that includes a signaling domain from: CSF2RB, CSF3R, IFNGR1, IL2RB, IL2RG, IL6ST, IL10RA, IL17RE, IL18R1, IL22RA1, IL31RA, MPL, MyD88, OSMR, or PRLR. In some embodiments, the lymphoproliferative component may include an intracellular domain or a fragment thereof that includes a signaling domain from: CSF2RB, CSF3R, IL2RB, IL2RG, IL6ST, IL27RA, IL31RA, MPL, or MyD88. Each of these genes has an intracellular domain that is significantly enriched at the P3 position across at least 2 repeats of library 3.1 (p<0.05). In some embodiments, the lymphoproliferative component may include an intracellular domain or a fragment thereof that includes a signaling domain from: CSF2RB, CSF3R, IL2RB, IL2RG, IL6ST, IL31RA, MPL, or MyD88.

In some embodiments, the lymphoproliferative component may include the following sequence: S054, S057, S058, S059, S062, S063, S064, S069, S072, S077, S081, S082, S083, S084, S085, S086, S087, S098, S099, S100, S101, S102, S103, S104, S105, S106, S109, S115, S116, S117, S120, S121, S126, S129, S130, S135, S136, S137 7, S138, S141, S142, S143, S145, S147, S148, S154, S155, S156, S157, S158, S161, S165, S168, S169, S170, S171, S174, S175, S176, S177, S180, S183, S186, S189, S190, S191, S192, S193, S194, S195, S196, S197, S198, S199 or S202 (as shown in Table 7), or any fragment thereof that includes a signaling domain. Each of these portions was present at the P3 position on at least one construct in the top 100 hits of at least one of the replicates of library 3.1. In some embodiments, the lymphoproliferative component may include the sequence of S057, S058, S059, S064, S069, S072, S084, S085, S099, S100, S101, S102, S104, S106, S115, S116, S126, S130, S135, S137, S138, S142, S143, S148, S158, S165, S168, S169, S170, S171, S174, S175, S176, S177, S186, S190, S191, S192, S193, S197, S198, or S199 (as shown in Table 7), or any fragment thereof that includes a signaling domain. Each of these portions is present at the P3 position on at least one construct that does not have an intracellular domain in the P4 position and in the top 100 hits of at least one of the repeats of library 3.1. In some embodiments, the lymphoproliferative component may include the following sequences as shown in Table 7: S057, S062, S063, S064, S081, S084, S105, S106, S117, S129, S138, S149, S161, S168, S169, S170, S186, S190, S191, S192, S194, S196, S197, S198, S199 or S202 (Table 1), or any fragment thereof that includes a signaling domain. Each of these portions is significantly enriched at the P3 position across the repeats of library 3.1.

In illustrative embodiments of any of the methods, uses, and composition aspects provided herein that include a lymphoproliferative component, the intracellular domain may include an intracellular domain or fragment thereof that includes a signaling domain from CD3D, CD3E, CD3G, CD27, CD28, CD40, CD79A, CD79B, FCER1G, FCGR2C, FCGRA2, ICOS, TNFRSF4, TNFRSF8, TNFRSF9, TNFRSF14, or TNFRSF18. Each of these genes has an intracellular domain that is present at the P4 position on at least one construct in the top 100 hits of at least one of the repeats of library 3.1. In some embodiments, the lymphoproliferative component may include an intracellular domain or fragment thereof that includes a signaling domain from CD40, CD79B, FCGR2C, or FCGRA2. In some embodiments, the lymphoproliferative component may include an intracellular domain or fragment thereof including a signaling domain from: CD3D, CD3G, CD40, CD79A, ICOS, TNFRSF8, or TNFRSF9. Each of these genes has an intracellular domain that is present at the P4 position on at least one construct that does not have an intracellular domain in the P3 position and is in the top 100 hits of at least one of the repeats of library 3.1. In some embodiments, the lymphoproliferative component may include an intracellular domain or fragment thereof including a signaling domain from CD40. In some embodiments, the lymphoproliferative component may include an intracellular domain or fragment thereof including a signaling domain from a TNF receptor family member, and in illustrative embodiments as a second intracellular signaling domain, such as a TNF receptor family member shown in Table 2. In some embodiments, the lymphoproliferative component may include an intracellular domain or fragment thereof that includes a signaling domain from CD27, CD40, CD79B, TNFRSF4, TNFRSF8, TNFRSF9, or TNFRSF18 (Table 2). Each of these genes has an intracellular domain that is significantly enriched at the P4 position across the repeats of library 3.1 (p<0.1). In some embodiments, the lymphoproliferative component may include an intracellular domain or fragment thereof that includes a signaling domain from CD40 or CD79B. In some embodiments, the lymphoproliferative component may include the sequence of S037, S038, S039, S047, S048, S049, S050, S051, S052, S053, S074, S075, S076, S080, S211, S212, S213, S214, S215, or S216 (as shown in Table 7), or any fragment thereof including a signaling domain. Each of these portions is present at the P4 position on at least one construct in the top 100 hits of at least one of the repeats of library 3.1. In some embodiments, the lymphoproliferative component may include the sequence of S037, S039, S050, S051, S052, S080, S212, or S213 (as shown in Table 7), or any fragment thereof including a signaling domain. Each of these portions is present at the P4 position on at least one construct that does not have an intracellular domain in the P3 position and in the top 100 hits of at least one of the repeats of library 3.1. In some embodiments, the lymphoproliferative component may include the following sequences as shown in Table 7: S047, S050, S051, S053, S211, S212, S213, or S215 (Table 2), or any fragment thereof that includes a signaling domain. Each of these portions is significantly enriched at the P4 position across the repeats of library 3.1.

In some embodiments, the lymphoproliferative component may include a cytokine receptor or fragment comprising a signaling domain thereof. In some embodiments, the cytokine receptor may be CD27, CD40, CRLF2, CSF2RA, CSF2RB, CSF3R, EPOR, GHR, IFNAR1, IFNAR2, IFNGR1, IFNGR2, IFNLR1, IL1R1, IL1RAP, IL1RL1, IL1RL2, IL2R, IL2RA, IL2RB, IL2RG, IL3RA, IL4R, IL5RA, IL6R, IL6ST, IL7R, IL7RA, IL9R, IL10RA, IL10RB, IL11RA, IL12R B1, IL13R, IL13RA1, IL13RA2, IL15R, IL15RA, IL17RA, IL17RB, IL17RC, IL17RE, IL18R1, IL18RAP, IL20RA, IL20RB, IL21R, IL22RA1, IL23R, IL27R, IL27RA, IL31RA, LEPR, LIFR, MPL, OSMR, PRLR, TGFβR, TGFβ decoy receptor, TNFRSF4, TNFRSF8, TNFRSF9, TNFRSF14, or TNFRSF18. In some embodiments, the cytokine receptor can be CD27, CD40, CRLF2, CSF2RA, CSF2RB, CSF3R, EPOR, GHR, IFNAR1, IFNAR2, IFNGR1, IFNGR2, IFNLR1, IL1R1, IL1RAP, IL1RL1, IL1RL2, IL2RA, IL2RB, IL2RG, IL3RA, IL4R, IL5RA, IL6R, IL6ST, IL7RA, IL9R, IL10 RA, IL10RB, IL11RA, IL13RA1, IL13RA2, IL15RA, IL17RA, IL17RB, IL17RC, IL17RE, IL18R1, IL18RAP, IL20RA, IL20RB, IL22RA1, IL27RA, IL31RA, LEPR, LIFR, MPL, OSMR, PRLR, TNFRSF4, TNFRSF8, TNFRSF9, TNFRSF14 or TNFRSF18. Exemplary embodiments of chimeric cytokine receptors comprising functional intracellular domains of the cytokine receptors listed above were tested in Examples 11 and 12 to confirm that chimeric cytokine receptors comprising these functional intracellular domains can serve as lymphoproliferative components.

Table 1 provides the identifiers of the most effective first intracellular signaling domain (P3) portions and corresponding genes for the 8 pool 3.1 replicates when all constructs were analyzed independent of the P4 portion. In some embodiments, the lymphoproliferative component may include a first intracellular domain or a variant or fragment thereof that includes a signaling domain from the following genes or respective portions in parentheses that were most effective by this statistical analysis (P<0.1): CSF2RB (S057), CSF3R (S062, S063, and S064), IFNAR1 (S081), IFNGR1 (S084), IL2RB (S105), IL2RG (S106) 、IL6ST(S117), IL10RA(S129), IL12RB2(S138), IL17RE(S149), IL22RA1(S161), IL27RA(S168 and S169), IL31RA(S170), MPL(S186), MyD88(S190, S191, S192, S194, S196, S197, S198), OSMR(S199) and PRLR(S202). In some embodiments, the lymphoproliferative component may include a first intracellular domain or a variant or fragment thereof, which includes a signaling domain from the following genes or respective portions in parentheses, which are most effective by this statistical analysis of at least 2 libraries (P<0.1): CSF2RB (S057), CSF3R (S062, S063 and S064), IL2RB (S105), IL2RG (S106), IL6ST (S117), IL27RA (S168 and S169), IL31RA (S170), MPL (S186) and MyD88 (S190, S191, S192, S194, S196, S197, S198). In some embodiments, the lymphoproliferative component may include an intracellular domain or variant or fragment thereof that includes a signaling domain from the following genes or respective portions in parentheses: CSF2RB (S057), CSF3R (S062, S063, and S064), IL2RB (S105), IL6ST (S117), IL27RA (S168 and S169), IL31RA (S170), MPL (S186), and MyD88 (S190, S191, S192, S194, S196, S197, S198). Each of these genes is represented in a construct that is the most effective by this statistical analysis of at least 2 pools, even when the statistical cutoff of P is less than 0.05. In some embodiments, the lymphoproliferative component may include an intracellular domain or fragment thereof that includes a signaling domain from CSF3R, IL6ST, IL27RA, MPL, or MyD88. Each of these genes is represented in a construct that is most effective by this statistical analysis of at least 2 libraries of Example 10, even when the statistical cutoff of P is less than 0.05, wherein the libraries are not fed/unfed pairs from the same replicate. As mentioned in Table 1, some genes and portions at P3 are significant when the intracellular domain is present at P4, some genes and portions at P3 are significant when the stop codon is present at P4, and some genes and portions at P3 are significant when the intracellular domain or stop codon is present at P4. In some embodiments, the lymphoproliferative component may include a first intracellular domain or a variant or fragment thereof, which includes a signaling domain from the following genes or respective portions in parentheses, which is significant at a p-value of less than 0.1 when the P4 portion is the second intracellular domain: CSFS2RB (S057), CSF3R (S062 and S064), IL2RB (S105), IL2RG (S106), IL6ST (S117), IL10RA (S129), IL17RC, IL17RE (S149), IL27RA (S168 and S169), IL31RA (S170), MPL (S186), MyD88 (S190, S192, S194, S196 and S197), OSMR (S199) and PRLR (S202). In some embodiments, the lymphoproliferative component may include a first intracellular domain or a variant or fragment thereof, which includes a signaling domain from the following genes or respective portions in parentheses, which is significant at a p-value of less than 0.05 when the P4 portion is the second intracellular domain: CSFS2RB (S057), CSF3R (S062 and S064), IL2RB (S105), IL2RG (S106), IL6ST (S117), IL10RA (S129), IL17RE (S149), IL27RA (S168 and S169), IL31RA (S170), MPL (S186), MyD88 (S190, S194 and S197), OSMR (S199) and PRLR (S202). In some embodiments, the lymphoproliferative component may include a first intracellular domain or a variant or fragment thereof, which includes a signaling domain from the following genes or respective portions in parentheses, which are significant at a p-value of less than 0.1 when the P4 portion is the stop codon X002: CSF3R (S063), IFNAR1 (S081), IFNGR1 (S084), IL2RB (S105), IL2RG (S106), IL6ST (S117), IL12RB2 (S138), IL17RE (S149), IL18R1, IL22RA1 (S161), IL27RA (S169), IL31RA (S170), MyD88 (S191, S196 and S198), and PRLR (S202). In some embodiments, the lymphoproliferative component may include a first intracellular domain or a variant or fragment thereof, which includes a signaling domain from the following genes or respective portions in parentheses, which are significant at a p-value of less than 0.05 when the P4 portion is the stop codon X002: CSF3R (S063), IFNGR1 (S084), IL2RB (S105), IL2RG (S106), IL6ST (S117), IL12RB2 (S138), IL17RE (S149), IL18R1, IL22RA1 (S161), IL27RA (S169), IL31RA (S170), MyD88 (S191 and S198), and PRLR (S202).

Table 2 provides identifiers for the most potent second intracellular signaling domain (P4) portions and corresponding genes when all constructs were analyzed independently of the P3 portion for the 8 library 3.1 replicates in Example 10. The following genes are represented by the second intracellular signaling domain portion that was identified in the constructs shown to be most potent by this statistical analysis (P<0.1 or p<0.05), with the portion number in parentheses: CD27 (S047), CD40 (S050 and S051), CD79B (S053), TNFRSF4 (S211), TNFRSF8 (S212), TNFRSF9 (S213), and TNFRSF18 (S215). The following second intracellular domain genes are represented by the second intracellular signaling domain portion that was most effective by this statistical analysis of at least 2 libraries (P<0.1), with the portion number in parentheses: CD40 (S050 and S051), TNFRSF8 (S212), and TNFRSF18 (S215). The following second intracellular domain genes were represented by second intracellular signaling domain portions that were most effective by this statistical analysis of at least 2 libraries even when the statistical cutoff for P was less than 0.05: CSF2RB (S057), CSF3R (S062, S063, and S064), IL2RB (S105), IL6ST (S117), IL27RA (S168 and S169), IL31RA (S170), MPL (S186), and MyD88 (S190, S191, S192, S194, S196, S197, S198). As indicated in Table 2, some genes and portions at P4 were significant when the intracellular domain was present at P3, some genes and portions at P4 were significant when the linker X001 was present at P3, and some genes and portions at P4 were significant when the intracellular domain or linker was present at P3. In some embodiments, the lymphoproliferative component may include a second intracellular domain or a variant or fragment thereof, which includes a signaling domain from the following genes or respective portions in parentheses, which are significant at a p-value of less than 0.1 when the P3 portion is the first intracellular domain: CD27 (S047), CD40 (S050 and S051), CD79B (S053), TNFRSF4 (S211), TNFRSF8 (S212) and TNFRSF18 (S215). In some embodiments, the lymphoproliferative component may include a second intracellular domain or a variant or fragment thereof, which includes a signaling domain from the following genes or respective moieties in parentheses, which is significant at a p-value of less than 0.05 when the P3 moiety is the first intracellular domain: CD27 (S047), CD40 (S050 and S051), CD79B (S053), TNFRSF4 (S211), TNFRSF8 (S212), and TNFRSF18 (S215). In some embodiments, the lymphoproliferative component may include a second intracellular domain or a variant or fragment thereof, which includes a signaling domain from the following genes or respective moieties in parentheses, which is significant at a p-value of less than 0.1 when the P3 moiety is linker X001: CD27 (S047), CD40 (S050), TNFRSF9 (S213), and TNFRSF18. In some embodiments, the lymphoproliferative component may include a second intracellular domain or a variant or fragment thereof, which includes a signaling domain from the following genes or respective portions in parentheses, which are significant at a p-value less than 0.05 when the P3 portion is linker X001: CD27 (S047), CD40 (S050), TNFRSF9 (S213) and TNFRSF18.

In Example 10, constructs were statistically analyzed to determine which P4 portions were most effective for a particular P3 portion, and the results are listed in Table 3. As shown in Example 10, cells transduced with polynucleotides encoding lymphoproliferative components contained intracellular signaling domains derived from the following pairs of genes enriched at a p-value of less than 0.1: CSF2RA and TNFRSF4; CSF2RA and CD28; CSF2RA and TNFRSF8; CSF2RA and CD27; CSFR3 and CD79B; IFNAR2 and TNFRSF14; IL1RAP and CD79A; IL3RA and CD40; IL10RA and CD79B; IL11RA and F CGRA2; IL13RA2 and TNFRSF14; IL18RAP and CD3G; IL27RA and FCGRA2; LEPR and CD3G; LIFR and TNFRSF18; MPL and CD40; MPL and CD79B; MPL and TNFRSF4; MPL and CD79A; MPL and CD3G; MyD88 and CD79B; or MyD88 and CD3D, wherein the intracellular signaling domain from the first gene of each pair is the P3 portion and the intracellular signaling domain from the second gene of each pair is the P4 portion. Unless otherwise stated, or stated in the broadest aspect, in illustrative embodiments of any of the methods and compositions provided herein that include a lymphoproliferative component, the lymphoproliferative component comprises an intracellular signaling domain derived from the following pair of genes: CSF2RA and TNFRSF4; CSF2RA and CD28; CSF2RA and TNFRSF8; CSF2RA and CD27; CSFR3 and CD79B; IFNAR2 and TNFRSF14; IL1RAP and CD79A; IL3RA and CD40; IL10RA and CD79B; IL11RA and FCGRA2; IL13RA2 and TNFRSF14; IL18RAP and CD3G; IL27RA and FCGRA2; LEPR and CD3G; LIFR and TNFRSF18; MPL and CD40; MPL and CD79B; MPL and TNFRSF4; MPL and CD79A; MPL and CD3G; MyD88 and CD79B; or MyD88 and CD3D. Unless otherwise stated, or stated in the broadest aspect, in illustrative embodiments of any of the methods and compositions provided herein that include a polynucleotide encoding a lymphoproliferative element, the polynucleotide may encode an intracellular signaling domain derived from the following pair of genes: CSF2RA and TNFRSF4; CSF2RA and CD28; CSF2RA and TNFRSF8; CSF2RA and CD27; CSFR3 and CD79B; IFNAR2 and TNFRSF14; IL1RAP and CD79 A; IL3RA and CD40; IL10RA and CD79B; IL11RA and FCGRA2; IL13RA2 and TNFRSF14; IL18RAP and CD3G; IL27RA and FCGRA2; LEPR and CD3G; LIFR and TNFRSF18; MPL and CD40; MPL and CD79B; MPL and TNFRSF4; MPL and CD79A; MPL and CD3G; MyD88 and CD79B; or MyD88 and CD3D. As shown in Example 10, cells transduced with a polynucleotide encoding a lymphoproliferative component contained intracellular signaling domains derived from the following pairs of genes enriched at a p-value of less than 0.05: CSF2RA and TNFRSF4; CSF2RA and CD28; CSFR3 and CD79B; IFNAR2 and TNFRSF14; IL10RA and CD79B; IL11RA and FCGRA2; IL27RA and FCGRA2; LIFR and TNFRSF18; MPL and CD40; MPL and CD79B; MPL and TNFRSF4; MPL and CD79A; MPL and CD3G; and MyD88 and CD79B, wherein the intracellular signaling domain from the first gene of each pair is the P3 portion and the intracellular signaling domain from the second gene of each pair is the P4 portion. Unless stated otherwise, or stated in the broadest aspect, in illustrative embodiments of any of the methods and compositions provided herein that include a lymphoproliferative component, the lymphoproliferative component comprises an intracellular signaling domain derived from the following pair of genes: CSF2RA and TNFRSF4; CSF2RA and CD28; CSFR3 and CD79B; IFNAR2 and TNFRSF14; IL10RA and CD79B; IL11RA and FCGRA2; IL27RA and FCGRA2; LIFR and TNFRSF18; MPL and CD40; MPL and CD79B; MPL and TNFRSF4; MPL and CD79A; MPL and CD3G; and MyD88 and CD79B. Unless stated otherwise, or stated in the broadest aspect, in illustrative embodiments of any of the methods and compositions provided herein that include a polynucleotide encoding a lymphoproliferative element, the polynucleotide may encode an intracellular signaling domain derived from the following pair of genes: CSF2RA and TNFRSF4; CSF2RA and CD28; CSFR3 and CD79B; IFNAR2 and TNFRSF14; IL10RA and CD79B; IL11RA and FCGRA2; IL27RA and FCGRA2; LIFR and TNFRSF18; MPL and CD40; MPL and CD79B; MPL and TNFRSF4; MPL and CD79A; MPL and CD3G; and MyD88 and CD79B. As demonstrated in Example 10, cells transduced with a polynucleotide encoding a lymphoproliferative component contained the following pairs of intracellular signaling domains (the amino acid sequences of which are shown in Table 7) enriched at a p-value of less than 0.1: S058 and S211; S058 and S049; S059 and S212; S059 and S047; S064 and S053; S083 and S214; S101 and S052; S109 and S050; S129 and S053; S135 and S076; S142 and S214; S155 and S039; S169 and S076; S177 and S039; S180 and S216; S186 and S050; S186 and S051; S186 and S053; S186 and S211; S186 and S039; S192 and S053; S194 and S037; and S195 and S053. Unless otherwise stated, or stated in the broadest aspect, in illustrative embodiments of any of the methods and compositions provided herein that include a lymphoproliferative component, the lymphoproliferative component comprises the following pairs of intracellular signaling domains: S058 and S211; S058 and S049; S059 and S212; S059 and S047; S064 and S053; S083 and S214; S101 and S052; S109 and S212; 050; S129 and S053; S135 and S076; S142 and S214; S155 and S039; S169 and S076; S177 and S039; S180 and S216; S186 and S050; S186 and S051; S186 and S053; S186 and S211; S186 and S039; S192 and S053; S194 and S037; and S195 and S053. Unless otherwise stated, or stated in the broadest aspect, in illustrative embodiments of any of the methods and compositions provided herein that include a polynucleotide encoding a lymphoproliferative element, the polynucleotide comprises the following pair of intracellular signaling domains: S058 and S211; S058 and S049; S059 and S212; S059 and S047; S064 and S053; S083 and S214; S101 and S052; S109 and S050; S129 and S053; S135 and S076; S142 and S214; S155 and S039; S169 and S076; S177 and S039; S180 and S216; S186 and S050; S186 and S051; S186 and S053; S186 and S211; S186 and S039; S192 and S053; S194 and S037; and S195 and S053. As demonstrated in Example 10, cells transduced with a polynucleotide encoding a lymphoproliferative component contained the following pairs of intracellular signaling domains (the amino acid sequences of which are shown in Table 7) enriched at a p-value of less than 0.05: S058 and S211; S058 and S049; S064 and S053; S083 and S214; S129 and S053; S135 and S076; S169 and S076; S180 and S216; S186 and S050; S186 and S051; S186 and S053; S186 and S211; S186 and S039; and S195 and S053. Unless stated otherwise, or stated in the broadest aspect, in illustrative embodiments of any of the methods and compositions provided herein that include a lymphoproliferative component, the lymphoproliferative component comprises the following pairs of intracellular signaling domains: S058 and S211; S058 and S049; S064 and S053; S083 and S214; S129 and S053; S135 and S076; S169 and S076; S180 and S216; S186 and S050; S186 and S051; S186 and S053; S186 and S211; S186 and S039; and S195 and S053. Unless stated otherwise, or stated in the broadest aspect, in illustrative embodiments of any of the methods and compositions provided herein comprising a polynucleotide encoding a lymphoproliferative element, the polynucleotide comprises the following pair of intracellular signaling domains: S058 and S211; S058 and S049; S064 and S053; S083 and S214; S129 and S053; S135 and S076; S169 and S076; S180 and S216; S186 and S050; S186 and S051; S186 and S053; S186 and S211; S186 and S039; and S195 and S053.

In certain illustrative embodiments, the lymphoproliferative element comprises a cytokine receptor or a fragment thereof comprising a signaling domain that activates the Jak/STAT5 pathway. For example, this lymphoproliferative element may include the intracellular domains of IL21R, IL27R, IL31RA, LIFR, and OSMR. As shown in the experiments of Example 11 and Example 12 and Tables 8 to 18, chimeric lymphoproliferative elements comprising the intracellular domains of these genes were found in candidate chimeric polypeptides that induced the highest degree of proliferation in PBMCs cultured in the absence of exogenous cytokines such as IL-2.

In some embodiments, the lymphoproliferative component The lymphoproliferative component may comprise an intracellular domain that is an interleukin or an interleukin receptor and is part of an optimal construct identified in libraries 1A, 1.1A, 2B, 2.1B, 3A, 3.1A, 3B, 3.1B, 4B, and/or 4.1B in Examples 11 and 12 (Tables 8 to 18). In some embodiments, the lymphoproliferative component The lymphoproliferative component may comprise an intracellular domain that is a cytokine receptor and is part of an optimal construct identified in libraries 1A, 1.1A, 2B, 2.1B, 3A, 3.1A, 3B, 3.1B, 4B, and/or 4.1B in Examples 11 and 12 (Tables 8 to 18). In some embodiments, the lymphoproliferative component The lymphoproliferative component may comprise an intracellular domain that includes at least one ITAM motif and is part of the optimal construct identified in libraries 1A, 1.1A, 2B, 2.1B, 3A, 3.1A, 3B, 3.1B, 4B and/or 4.1B of Example 11 and Example 12 (Tables 8 to 18). In some embodiments, the lymphoproliferative component may comprise one of the following intracellular domains that is part of the top construct in at least one of libraries 3A, 3.1A, 3B, 3.1B, 4B or 4.1B.

In illustrative embodiments, the lymphoproliferative element The lymphoproliferative element may comprise an intracellular domain from IL7R, IL12RB1, IL15RA or IL27RA, which is present in the constructs that showed particularly noteworthy enrichment (i.e., caused the highest degree of proliferation) in the initial screen and the repeated screen as detailed in Examples 11 and 12 (Tables 20-24).

In illustrative embodiments, the lymphoproliferative component may comprise an intracellular domain from the following cytokine receptors: CD27, CD40, CRLF2, CSF2RA, CSF3R, EPOR, GHR, IFNAR1, IFNAR2, IFNGR2, IL1R1, IL1RL1, IL2RA, IL2RG, IL3RA, IL5RA, IL6R, IL7R, IL9R, IL10RB, IL11RA, IL12RB1, IL13RA 1, IL13RA2, IL15RA, IL17RB, IL18R1, IL18RAP, IL20RB, IL22RA1, IL27RA, IL31RA, LEPR, MPL, OSMR, PRLR, TNFRSF4, TNFRSF8, TNFRSF9, TNFRSF14 or TNFRSF18, which are present in the constructs that showed particularly noteworthy enrichment in the initial screen and the repeated screen as detailed in Examples 11 and 12 (Tables 20 to 24).

In illustrative embodiments, the lymphoproliferative element The lymphoproliferative element may comprise an intracellular domain from CD3D, CD3E, CD3G, CD79A, CD79B, FCER1G, FCGR2A or FCGR2C that includes at least one ITAM motif and is present in constructs that showed particularly noteworthy enrichment in the initial screen and repeated screens as detailed in Examples 11 and 12 (Tables 20 to 24).

In some embodiments, the lymphoproliferative component of this paragraph (which is shown in Examples 11 and 12 to be active in constructs having only a single intracellular domain) can be an intracellular domain of a lymphoproliferative component having two or more intracellular domains, or in illustrative embodiments, a lymphoproliferative component having a single intracellular domain (i.e., the lymphoproliferative component does not comprise two or more intracellular domains). In illustrative embodiments, the intracellular domain in the lymphoproliferative component comprises a domain from CD40, CRLF2, CSF2RA, CSF3R, EPOR, FCGR2A, IFNAR2, IFNGR2, IL1R1, IL3RA, IL7R, IL10RB, IL11RA, IL12RB1, IL13RA2, IL18RAP, IL31RA, MPL, MYD88, TNFRSF14, or TNFRSF18 that is present in constructs that showed particularly noteworthy enrichment in the initial screen and repeat screen as detailed in Example 12 (Tables 23 and 24) as single intracellular signaling domains. In illustrative embodiments, the intracellular domain in the lymphoproliferative element comprises a domain from IL7R or IL12RB1, which is present in constructs that showed particularly noteworthy enrichment in the initial screen and the repeated screen as detailed in Example 12 (Tables 23 and 24). In some embodiments, the intracellular domain in the lymphoproliferative element having a single intracellular domain can be a cytokine receptor. In illustrative embodiments, a lymphoproliferative component having a single intracellular domain The cytokine receptor in the lymphoproliferative component comprises a domain from CD40, CRLF2, CSF2RA, CSF3R, EPOR, IFNAR2, IFNGR2, IL1R1, IL3RA, IL7R, IL10RB, IL11RA, IL12RB1, IL13RA2, IL18RAP, IL31RA, MPL, TNFRSF14, or TNFRSF18, which is present in constructs that showed particularly noteworthy enrichment in the initial and repeat screens as detailed in Example 12 (Tables 23 and 24). In some embodiments, the intracellular domain in the lymphoproliferative component of the lymphoproliferative component may include at least one ITAM motif. In an illustrative embodiment, a lymphoproliferative component comprising at least one ITAM motif The intracellular domain in the lymphoproliferative component comprises a domain from FCGR2A, which is present in constructs that showed particularly noteworthy enrichment in the initial screen and in the repeated screen as detailed in Example 12 (Table 23).

In illustrative embodiments, lymphoproliferative components The lymphoproliferative components may include costimulatory domains from CD27, CD28, OX40 (also known as TNFRSF4), GITR (also known as TNFRSF18) or HVEM (also known as TNFRSF14), which are present in constructs that show particularly noteworthy enrichment in initial screening and repeated screening as described in detail in Examples 11 and 12 (Tables 20 to 24). In some embodiments, the lymphoproliferative component comprising a costimulatory domain from OX40 does not include an intracellular domain from CD3Z, CD28, 4-1BB, ICOS, CD27, BTLA, CD30, GITR or HVEM. In some embodiments, the lymphoproliferative component comprising a costimulatory domain from GITR does not include an intracellular domain from CD3Z, CD28, 4-1BB, ICOS, CD27, BTLA, CD30 or HVEM. In some embodiments, the lymphoproliferative component comprising a costimulatory domain from CD28 does not include an intracellular domain from CD3Z, 4-1BB, ICOS, CD27, BTLA, CD30 or HVEM. In some embodiments, the lymphoproliferative component comprising a costimulatory domain from OX40, CD3Z, CD28, 4-1BB, ICOS, CD27, BTLA, CD30, GITR or HVEM does not include a coiled coil spacer domain of a transmembrane domain. In some embodiments, the lymphoproliferative component comprising a costimulatory domain from GITR does not include an intracellular domain from CD3Z, which is the N-terminal of the costimulatory domain of GITR.

In some embodiments, the lymphoproliferative component can be one of the following constructs: M024-S190-S047, M025-S050-S197, M036-S170-S047, M012-S045-S048, M049-S194-S064, M025-S190-S050, M025-S190-S05, E013-T041 -S186-S051, E013-T028-S186-S051, E014-T015-S186-S051, E011-T016-S186-S050, E011-T073-S186-S050 or E013-T011-S186-S211, all of which stimulate proliferation of resting lymphocytes after transduction as shown in Example 13 (Figures 19 to 20). In certain embodiments, the lymphoproliferative component comprises an extracellular domain from CSF3R, IL3RA, ICOS, CRLF, CSF2RA, LIFR or CD40; a first intracellular domain from MyD88, CD40 or MPL, and/or a second intracellular domain from CD27 or MyD88.

In some embodiments, the lymphoproliferative element The lymphoproliferative element can be the construct E013-T041-S186-S051, which stimulates proliferation of resting lymphocytes after administration of replication-defective recombinant retroviral particles displaying UCHT1 scFvFc-GPI as shown and analyzed in Example 16 (FIG. 22A and 22B). In other embodiments, the lymphoproliferative element The lymphoproliferative element is IL7-IL7RA-IL2RB, as shown and analyzed in Example 16.

As detailed in Example 11, for libraries 1A and 1.1A, constructs with domains from cytokine receptors CD27, IL1RL1, IL6R, IL31RA, TNFRSF4, or TNFRSF18 showed particularly noteworthy enrichment in both libraries 1A and 1.1A (Table 20).

As detailed in Example 11, for libraries 2B and 2.1B, constructs with domains from cytokine receptors CD40, IFNGR2, GHR, IL10RB, IL11RA, IL13RA2, IL17RB, IL22RA1, TNFRSF14, or TNFRSF9 showed particularly noteworthy enrichment in both libraries 2B and 2.1B (Table 21).

As detailed in Example 12, for libraries 3A and 3.1A, constructs with domains from cytokine receptors CD27, CD40, CSF2RA, MPL, OSMR, TNFRSF4, or TNFRSF18 showed particularly noteworthy enrichment in both libraries 3A and 3.1A (Table 22).

As described in detail in Example 12, for libraries 3B and 3.1B, constructs with domains from cytokine receptors CD27, CD40, CRLF2, CSF2RA, CSF3R, EPOR, IFNAR1, IFNAR2, IFNGR2, IL1RL1, IL2RG, IL3RA, IL5RA, IL6R, IL7R, IL9R, IL10RB, IL11RA, IL12RB1, IL13RA1, IL13RA2, IL15RA, IL18RAP, IL20RB, IL27RA, IL31RA, LEPR, MPL, OSMR, PRLR, TNFRSF4, TNFRSF8, TNFRSF9, TNFRSF14, or TNFRSF18 showed particularly noteworthy enrichment in both libraries 3B and 3.1B (Table 23).

As described in detail in Example 12, for libraries 4B and 4.1B, constructs with domains from cytokine receptors CD27, CD40, CRLF2, CSF2RA, CSF3R, EPOR, IFNAR2, IFNGR2, IL1R1, IL2RA, IL3RA, IL2RG, IL6R, IL7R, IL10RB, IL11RA, IL31RA, IL13RA1, IL13RA2, IL18R1, MPL, OSMR, TNFRSF4, TNFRSF9, or TNFRSF18 showed particularly noteworthy enrichment in both libraries 4B and 4.1B (Table 24).

In certain illustrative embodiments, the lymphoproliferative element The lymphoproliferative element comprises the intracellular domains of CD40, MPL, and IL2Rb, which in the examples herein have been demonstrated to promote PBMC proliferation.

In some embodiments, the lymphoproliferative element lymphoproliferative element may not be a cytokine receptor. In some embodiments, a lymphoproliferative element other than a cytokine receptor lymphoproliferative element may include an intracellular signaling domain from: CD2, CD3D, CD3G, CD3Z, CD4, CD8RA, CD8RB, CD28, CD79A, CD79B, FCER1G, FCGR2A, FCGR2C, or ICOS. Exemplary embodiments of chimeric lymphoproliferative elements lymphoproliferative elements including these recited genes are provided in Examples 11 and 12 and the tables cited therein.

In some embodiments, the CLE is not IL-15 linked to an IL-2/IL-15 receptor.

In some of the methods and compositions disclosed herein, the expression of the lymphoproliferative component is induced and may even be dependent on the combination of a compound and a control component (as discussed in WO2017/165245A2, WO2018/009923A1, and WO2018/161064A1), which is a riboswitch in a non-limiting embodiment. In some embodiments, the lymphoproliferative component is expressed by a promoter active in T cells and/or NK cells. For the methods and compositions provided herein, a skilled artisan will recognize that the promoter is active in T cells and/or NK cells and can be used to express a first engineered signaling polypeptide or a second engineered signaling polypeptide, or any component thereof. In illustrative embodiments, this promoter is inactive in a packaging cell line (such as a packaging strain disclosed herein). In some embodiments, the promoter is the EF1α promoter or the murine stem cell virus (MSCV) promoter (Jones et al., Human Gene Therapy (2009) 20: 630-40). In an illustrative embodiment, the promoter is the T cell-specific CD3ζ promoter.

In some embodiments, the lymphoproliferative element is microenvironmentally restricted. For example, the lymphoproliferative element can be a mutated receptor that differentially binds its respective cytokine under abnormal conditions relative to physiological conditions. For example, an IL-7R can be used that binds IL7 more strongly in a tumor environment than in a normal physiological environment.

In certain embodiments, the lymphoproliferative component lymphoproliferative component is fused to a recognition domain or an elimination domain. Such recognition domains or elimination domains are disclosed in more detail herein. This fusion provides, especially when using a truncated or otherwise mutated lymphoproliferative component lymphoproliferative component, the advantage of requiring fewer polynucleotides in the retroviral genome. This is important in the illustrative embodiments provided herein, because it helps to allow more nucleic acids encoding functional components to be included in the retroviral genome and because it adds a mechanism, if its proliferation is no longer needed or its proliferation is harmful to the organism, the cell expressing the lymphoproliferative component lymphoproliferative component can be killed by this mechanism.

In some embodiments, lymphoproliferative element lymphoproliferative element (including CLE) comprises an intracellular activation domain as disclosed above. In some illustrative embodiments, lymphoproliferative element lymphoproliferative element is a CLE comprising an intracellular activation domain, and the intracellular activation domain comprises an ITAM-containing domain. Therefore, CLE may comprise an intracellular activation domain, and the intracellular activation domain has at least 80%, 90%, 95%, 98% or 100% sequence identity with CD3Z, CD3D, CD3E, CD3G, CD79A, CD79B, DAP12, FCER1G, FCGR2A, FCGR2C, DAP10/CD28 or ZAP70 domains provided herein, wherein CLE does not comprise ASTR. In certain illustrative embodiments, the intracellular activation domain is an ITAM-containing domain from the following: CD3D, CD3G, CD3Z, CD79A, CD79B, FCER1G, FCGR2A or FCGR2C. CLE comprising an intracellular activation domain is effective in promoting proliferation of PBMCs in vitro in culture in the absence of exogenous cytokines (such as exogenous IL-2) as demonstrated herein in Examples 11 and 12 and associated Tables 19 to 24. In some embodiments, provided herein is a CLE comprising an intracellular domain from: CD3D, CD3Z, CD3Z, CD79A, FCER1G.

In some embodiments, one or more domains of lymphoproliferative component lymphoproliferative component are fused to the regulatory domain (such as costimulatory domain) and/or intracellular activation domain of CAR. In some embodiments, one or more intracellular domains of lymphoproliferative component lymphoproliferative component may be part of the same polypeptide as CAR or may be fused or functionally connected to some components of CAR as appropriate. In other embodiments, engineered signaling polypeptides may include ASTR, intracellular activation domain (such as CD3 ζ signaling domain), costimulatory domain and lymphoproliferative domain. Other details about costimulatory domain, intracellular activation domain, ASTR and other CAR domains are disclosed elsewhere herein.

In the illustrative embodiments herein, the T cell and/or NK cell survival component is introduced into the resting T cells and/or resting NK cells, usually by transducing the resting T cells and/or resting NK cells with replication-deficient recombinant retroviral particles, the genome of which encodes a T cell and/or NK cell survival component as part of an engineered signaling polypeptide. In some embodiments, the lymphoproliferative component lymphoproliferative component is also a T cell and/or NK cell survival component. As discussed above, some of the lymphoproliferative components of the lymphoproliferative component not only promote hyperplasia, but also promote cell survival. In some embodiments, the T cell and/or NK cell survival cell primitive is not a lymphoproliferative component lymphoproliferative component. In some embodiments, the T cell and/or NK cell survival primitive may be a CD28 T cell survival primitive or a CD137 cell survival primitive. These T cell survival primitives may be found in engineered signaling polypeptides including ASTRs (such as scFv). In an illustrative embodiment, the T cell survival motif is a CD28 T cell survival motif or a CD137 motif connected to a scFv via a CD8a transmembrane domain or a CD28 transmembrane domain. In certain embodiments, the intracellular signaling domain comprises a polypeptide sequence comprising an immunoreceptor tyrosine-based activation motif (ITAM). In a certain embodiment, the polypeptide sequence is a CD3 zeta signaling domain.

In some embodiments, the lymphoproliferative component lymphoproliferative component is not a polypeptide, but instead comprises inhibitory RNA. In some embodiments, the method, use, composition and product of the process according to any aspect herein include a lymphoproliferative component lymphoproliferative component comprising inhibitory RNA and a lymphoproliferative component lymphoproliferative component for an engineered signaling polypeptide. In the embodiment where the lymphoproliferative component lymphoproliferative component is an inhibitory RNA, the inhibitory RNA may be a miRNA that stimulates the STAT5 path by generally utilizing the negative regulator in the SOCS path to degrade or downregulate and enhance the activation of STAT5. In some embodiments, miRNA refers to mRNA encoding a protein that affects hyperplasia, such as but not limited to ABCG1, SOCS1, TGFbR2, SMAD2, cCBL and PD1. In illustrative embodiments, as exemplified herein, this inhibitory RNA (e.g., miRNA) may be located in an intron in a packaging cell and/or a replication-defective recombinant retroviral particle genome and/or a retroviral vector, and is generally expressed by driving a promoter active in T cells and/or NK cells. Without being limited by theory, it is believed that inclusion of introns in the transcription unit results in higher expression and/or stability of the transcript. Therefore, the ability to place miRNAs in introns of retroviral genomes adds to the teachings of the present invention, which overcomes the challenges of prior art attempts to maximize activity to the size limitations of retroviruses (such as lentiviral genomes). In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 miRNAs (in illustrative embodiments, 2 to 5 (e.g., 4) miRNAs (one or more of which each bind to nucleic acids encoding one or more of ABCG1, SOCS1, TGFbR2, SMAD2, cCBL and PD1)) can be included in the recombinant retroviral genome and delivered to target cells, such as T cells and/or NK cells, using the methods provided herein. In fact, as provided herein, 1, 2, 3 or 4 miRNAs can be delivered in a single intron (such as the EF1a intron).

ABCG1 is an ATP-binding cassette transporter that negatively regulates the proliferation of thymocytes and peripheral lymphocytes (Armstrong et al. 2010. J Immunol 184(1):173-183).

SOCS1 is a member of the SOCS (suppressor of cytokine signaling) family of negative regulators of cytokine signaling that inhibit the Jak/Stat pathway (such as STAT5). SOCS1 is also known as JAB (Janus kinase binding protein), SSI-1 (Stat-induced Stat inhibitor-1), and TIP3 (Tec interacting protein).

TGFbR2 is a member of the serine/threonine protein kinase family that binds TGF-β, forming a complex that phosphorylates the protein, subsequently enters the nucleus and regulates the transcription of genes associated with proliferation.

SMAD2 mediates the signaling of transforming growth factor (TGF)-β and regulates multiple cellular processes, such as cell proliferation, apoptosis, and differentiation.

cCBL is an E3 ubiquitin ligase that inhibits TCR signaling by dephosphorylating and inactivating ZAP-70 and through internalization of the TCR.

PD1 (CD279) is a cell surface receptor expressed on T cells and ProB cells. PD-1 binds to two ligands, PD-L1 and PD-L2. Signal transduction via PD-1 plays a role in preventing cell activation.

In some embodiments, the lymphoproliferative component herein is a polypeptide comprising an intracellular region of any one of the genes in Table 19. In some embodiments, the lymphoproliferative component lymphoproliferative component comprises or is an intracellular domain identified in a chimeric polypeptide of Table 19. In these embodiments, the lymphoproliferative component lymphoproliferative component may be a polypeptide that is a chimeric polypeptide (see CLE below), or is not a CLE, but comprises or is an intracellular domain of a gene identified in the P3 (first intracellular domain) position of Table 19. Table 19 identifies CLE constructs that promote proliferation of PBMCs between day 7 and the last day when the second intracellular domain is not present on the construct. In some sub-embodiments of this embodiment, the lymphoproliferative component lymphoproliferative component is a chimeric polypeptide (i.e., CLE, see below) comprising a combination of a transmembrane domain and an intracellular domain with or without an extracellular domain comprising a dimerization motif.

In some embodiments, the lymphoproliferative component comprises MPL or is MPL or a variant or fragment thereof, including at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the intracellular domain of MPL, with or without the transmembrane domain and/or extracellular domain of MPL, and/or a variant or fragment having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the intracellular domain of MPL, with or without the transmembrane domain and/or extracellular domain of MPL, wherein the variant and/or fragment retains the ability to promote cellular proliferation of PBMCs (and in some embodiments, T cells). In illustrative embodiments, the lymphoproliferative component comprises the intracellular domain of MPL, or a variant or fragment thereof, which variant or fragment comprises at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the intracellular domain of MPL, and the lymphoproliferative component does not comprise the transmembrane domain of MPL. In some embodiments, the lymphoproliferative component comprises the intracellular domain of MPL, or a variant or fragment thereof, which variant or fragment comprises at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the intracellular domain of MPL, and the lymphoproliferative component comprises the transmembrane domain of MPL. In some embodiments, the MPL fragments included in the compositions and methods herein have and/or retain a JAK-2 binding domain. In some embodiments, the MPL fragments included herein have or retain the ability to activate STAT. The whole intracellular domain of MPL is SEQ ID NO: 491 (part S186 in Tables 8 to 19). MPL is a receptor for thrombopoietin. Several cytokines such as thrombopoietin and EPO are referred to in the literature and herein as hormones or cytokines.

In some embodiments (which provide a separate aspect of the present invention), provided herein are chimeric polypeptides of chimeric lymphoproliferative elements lymphoproliferative elements (CLE) and isolated polynucleotides and nucleic acid sequences encoding them. CLE may include any of the domains and/or domains derived from the specific genes discussed in this section. Similarly, the isolated polynucleotides and nucleic acid sequences encoding CLE may encode any of the domains and/or domains derived from the specific genes discussed in this section as part of CLE. Exemplary CLE is illustrated in Figures 15 to 18. The CLE herein promotes the proliferation of T cells and/or NK cells and may also promote the survival of T cells and/or NK cells as appropriate. Some CLEs promote proliferation and may also promote the survival of other types of PBMCs (e.g., B cells) as appropriate. The embodiments provided herein including nucleic acid sequences encoding CLE and CAR are referred to herein as CLE CAR polynucleotide embodiments for ease of design. In some embodiments, CLE may include a transmembrane domain and a first intracellular domain. Furthermore, in illustrative embodiments, as shown in Figures 15-18, the CLE includes an extracellular domain and/or a second intracellular domain (and in further embodiments, a third intracellular domain, a fourth intracellular domain, etc.). The chimeric polypeptides herein are chimeric in that at least one domain is from a polypeptide that is different from at least one of the other domains and such chimeras cannot be naturally found in an organism without human intervention. Some CLEs include ligands for receptors and some CLEs do not include ligands for receptors.

Without being limited by theory, these CLEs are designed to promote the proliferation and, optionally, cell survival of B cells, NK cells, and/or T cells in a constitutive manner (i.e., without the need for ligand binding for activation). None of the CLEs are found in nature, and many CLEs have components that are not usually expressed in vivo in B cells, T cells, and/or NK cells, and/or some candidate CLEs are not usually known to specifically promote cell proliferation and/or cell survival signaling of B cells, T cells, and/or NK cells. For example, MPL is not usually expressed in B cells, T cells, and/or NK cells. Unexpectedly, the new CLEs identified by screening a large number of candidate chimeric polypeptides as described in Examples 11 and 12 promote PBMC cell proliferation. These CLEs help meet the long-term needs of identifying mechanisms that stimulate the proliferation and, optionally, survival of B cells, T cells, and/or NK cells, such as will be beneficial for important clinically relevant technologies, such as CAR-T and T cells that are genetically engineered to express defined TCRs. Therefore, it is believed that some CLEs will provide T cells and/or NK cells with the ability to expand in vivo without requiring lymphodepletion of the host.

The chimeric lymphoproliferative component provided herein, the lymphoproliferative component and the isolated polynucleotides and nucleic acids encoding them may be included in any aspect provided herein including the lymphoproliferative component lymphoproliferative component. For example, the first engineered signaling polypeptide may be or may include a CLE in the aspect provided herein, the CLE including one or more transcription units encoding the first engineered signaling polypeptide regulated by the control component. In addition, a separate aspect of the present invention is provided, which specifically includes a CLE. For example, these aspects include isolated chimeric lymphoproliferative polypeptides, isolated polynucleotides and nucleic acids encoding them, and vectors, including plasmids, viruses and retroviral vectors (including these nucleic acid sequences or isolated polynucleotides). These aspects further include methods for transducing or transfecting PBMCs (such as B cells, or specifically T cells and NK cells) with isolated polynucleotides and vectors containing them. These cells may be isolated unaltered cells, or they may be modified cells, such as cells genetically modified such as to express defined TCRs or CAR-T cells.

The lymphoproliferative modules provided herein typically include a transmembrane domain. For example, the transmembrane domain can have 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity to any of the transmembrane domains from the following genes and representative sequences: CD8β (SEQ ID NO:47), CD4 (SEQ ID NO:48), CD3ζ (SEQ ID NO:49), CD28 (SEQ ID NO:50), CD134 (SEQ ID NO:51), CD7 (SEQ ID NO:51), CD2 (SEQ ID NO:322), CD3D (SEQ ID NO:323), CD3E (SEQ ID NO:324), CD3G (SEQ ID NO:325), CD3Z (SEQ ID NO:326), CD4 (SEQ ID NO:327), CD8A (SEQ ID NO:328), CD8B (SEQ ID NO:329), CD27 (SEQ ID NO:330), CD28 (SEQ ID NO:331), CD40 (SEQ ID NO:332), ID NO:332), CD79A (SEQ ID NO:333), CD79B (SEQ ID NO:334), CRLF2 (SEQ ID NO:335), CRLF2 (SEQ ID NO:336), CSF2RA (SEQ ID NO:337), CSF2RB (SEQ ID NO:339), CSF3R (SEQ ID NO:340), CSF 3R (SEQ ID NO:341), EPOR (SEQ ID NO:342), EPOR (SEQ ID NO:343), FCER1G (SEQ ID NO:344), FCGR2C (SEQ ID NO:345), FCGRA2 (SEQ ID NO:346), GHR (SEQ ID NO:347), GHR (SEQ ID NO:348), ICOS (SEQ ID NO:349), IF NAR (SEQ ID NO:350), IFNAR2 (SEQ ID NO:351), IFNGR1 (SEQ ID NO:352), IFNGR2 (SEQ ID NO:353), IFNLR1 (SEQ ID NO:354), IL1R1 (SEQ ID NO:355), IL1RAP (SEQ ID NO:356), IL1RL1 (SEQ ID NO:357), IL1RL2 (SEQ ID NO:358), IL2RA (SEQ ID NO:35 9), IL2RB (SEQ ID NO:360), IL2RG (SEQ ID NO:361), IL3RA (SEQ ID NO:362), IL4R (SEQ ID NO:363), IL5RA (SEQ ID NO:364), IL6R (SEQ ID NO:365), IL6ST (SEQ ID NO:366), IL7RA (SEQ ID NO:367), IL7RA (SEQ ID NO: 368), IL9R (SEQ ID NO:369), IL10RA (SEQ ID NO:370), IL10RB (SEQ ID NO:371), IL11RA (SEQ ID NO:372), IL12RB1 (SEQ ID NO:373), IL12RB2 (SEQ ID NO:374), IL13RA1 (SEQ ID NO:375), IL13RA2 (SEQ ID NO:376), IL15RA (SEQ ID NO:377), IL17RA (SEQ ID NO:378), IL17RB (SEQ ID NO:379), IL17RC (SEQ ID NO:380), IL17RD (SEQ ID NO:381), IL17RE (SEQ ID NO:382), IL18R1 (SEQ ID NO:383), IL18RAP (SEQ ID NO:384), IL20RA (SEQ ID NO:385), IL20RB (SEQ ID NO:378) :386), IL21R (SEQ ID NO:387), IL22RA1 (SEQ ID NO:388), IL23R (SEQ ID NO:389), IL27RA (SEQ ID NO:390), IL27RA (SEQ ID NO:391), IL31RA (SEQ ID NO:392), LEPR (SEQ ID NO:393), LIFR (SEQ ID NO:394), MPL (SEQ ID NO:395), MPL (SEQ ID NO:396), OSMR (S EQ ID NO:397), PRLR (SEQ ID NO:398), TNFRSF4 (SEQ ID NO:399), TNFRSF8 (SEQ ID NO:400), TNFRSF9 (SEQ ID NO:401), TNFRSF14 (SEQ ID NO:402) and TNFRSF18 (SEQ ID NO:403). TM domains suitable for use in any engineered signaling polypeptide include, but are not limited to, constitutively active cytokine receptors, TM domains from LMP1, and TM domains from type 1 TM proteins comprising a dimerization motif, as discussed in more detail herein. In any of the aspects disclosed herein containing a transmembrane domain from a type I transmembrane protein, the transmembrane domain may be a type I growth factor receptor, a hormone receptor, a T cell receptor, or a TNF family receptor.

In some embodiments, CLE includes both the extracellular portion and the transmembrane portion from the same part (in illustrative embodiments, the same receptor), one of which is a mutant in illustrative embodiments, thus forming an extracellular domain and a transmembrane domain. These domains may be from a cytokine receptor or a mutant thereof, or a hormone receptor or a mutant thereof, and in some embodiments, when expressed in at least some cell types, it is reported to be constitutively active. In illustrative embodiments, this extracellular domain and transmembrane domain do not include a ligand binding region. It is believed that these domains do not bind ligands when present in CLE and expressed in B cells, T cells and/or NK cells. Mutations in these receptor mutants may occur in the transmembrane region or in the extracellular juxtamembrane region. Without being limited by theory, mutations in at least some of the extracellular-transmembrane domains of CLE provided herein are responsible for the signal transduction of CLE in the absence of a ligand by bringing together activation chains that are not usually together or by changing the confirmation of connecting transmembrane domains and/or intracellular domains.

One aspect of the transmembrane domains utilizing receptor mutants provided herein is an isolated polynucleotide comprising one or more nucleic acid sequences, wherein:

A first nucleic acid sequence of the one or more nucleic acid sequences encodes a chimeric polypeptide comprising, in an amino to carboxyl orientation,

(a) an extracellular domain and a transmembrane domain from a cytokine receptor or a hormone receptor, wherein at least one of the extracellular domain and the transmembrane domain comprises a mutation found in a constitutively active mutant of a cytokine receptor, and wherein the extracellular sequence does not bind a ligand of a cytokine receptor; and

(b) a first intracellular domain selected from the intracellular domains of a gene having a first intracellular domain and optionally a second intracellular domain of a selected polypeptide identified in Tables 8 to 12, wherein the chimeric polypeptide promotes cell proliferation of B cells, T cells and/or NK cells.

The extracellular region of this extracellular domain and transmembrane domain in these embodiments is generally long enough to form a connector, in illustrative embodiments, a flexible connector between the transmembrane domain and another functional peptide region, such as a gap domain, which in some embodiments is connected to the amino terminus of the extracellular region. Therefore, the extracellular region, when present to form an extracellular domain and a transmembrane domain, can be between 1 amino acid and 1000 amino acids in length, and is generally between 4 amino acids and 400 amino acids, between 4 amino acids and 200 amino acids, between 4 amino acids and 100 amino acids, between 4 amino acids and 50 amino acids, between 4 amino acids and 25 amino acids, or between 4 amino acids and 20 amino acids. In one embodiment, the extracellular region is GGGS for the extracellular domain and transmembrane domain of this aspect of the invention.

As discussed in more detail below, whether as part of an embodiment comprising an extracellular domain and a transmembrane domain (as one shown, for example, as libraries 1A, 1.1A, 1.1B, 2B, and 2.1B of Example 11) or as part of an embodiment comprising an extracellular dimerization motif (as shown, for example, as libraries 3A, 3B, 3.1A, 3.1B, 4B, and 4.1B of Example 12), the transmembrane domain is typically at least long enough to cross the plasma membrane. Thus, the transmembrane domain or the transmembrane region of the extracellular domain and the transmembrane domain may be between 10 and 250 amino acids, and more typically is at least 15 amino acids in length, and may be, for example, between 15 and 100 amino acids, between 15 and 75 amino acids, between 15 and 50 amino acids, between 15 and 40 amino acids, or between 15 and 30 amino acids.

Exemplary extracellular domains and transmembrane domains of CLEs of embodiments comprising these domains (in illustrative embodiments, extracellular domains) are generally less than 30 amino acids from the membrane proximal extracellular domains of mutant receptors reported to be constitutive together with the transmembrane domain, which do not require ligand binding for activation of the associated intracellular domain. In illustrative embodiments, these extracellular domains and transmembrane domains include IL7RA Ins PPCL, CRLF2 F232C, CSF2RB V449E, CSF3R T640N, EPOR L251C I252C, GHRE260C I270C, IL27RA F523C, and MPL S505N. Additional non-limiting examples of such extracellular domains and transmembrane domains are provided in Table 7 and exemplified in Example 11 and corresponding tables. In some embodiments, the extracellular domain and the transmembrane domain do not comprise more than 10, 20, 25, 30, or 50 constituent amino acids in sequence that are identical to a portion of the extracellular domain and/or transmembrane domain of IL7RA or a mutant thereof. In some embodiments, the extracellular domain and the transmembrane domain are not IL7RA Ins PPCL. In some embodiments, the extracellular domain and the transmembrane domain do not comprise more than 10, 20, 25, 30, or 50 constituent amino acids in sequence that are identical to a portion of the extracellular domain and/or transmembrane domain of IL15R.

Additional exemplary transmembrane domains are provided in Example 12. These transmembrane domains in the illustrative embodiments are from type I transmembrane proteins.

Thus, in one aspect, provided herein are isolated polynucleotides comprising one or more nucleic acid sequences, wherein:

A first nucleic acid sequence of the one or more nucleic acid sequences encodes a chimeric polypeptide comprising, in an amino to carboxyl orientation,

a) a transmembrane domain from a type I transmembrane protein; and

b) a first intracellular domain selected from the intracellular domains of a gene having a first intracellular domain of a selected polypeptide identified in Tables 13 to 18, wherein the chimeric polypeptide promotes cell proliferation of B cells, T cells and/or NK cells.

In illustrative embodiments, the chimeric polypeptide promotes cell proliferation of PBMCs (e.g., B cells and/or NK cells, and/or in illustrative embodiments, T cells). As shown in Example 12, these chimeric polypeptides are capable of promoting cell proliferation of PBMCs in the absence of exposure of PBMCs to exogenous cytokines (such as IL-2, IL-15 or IL-7) during culture (e.g., in the absence of adding IL-2 to the culture medium of PBMCs (e.g., B cells, T cells or NK cells) expressing nucleic acids encoding the chimeric polypeptides). As illustrated in Example 12, for these embodiments, IL-2 may be added during transduction of PBMCs, but not in subsequent culture. For example, the chimeric polypeptides disclosed herein are lymphoproliferative components lymphoproliferative components due to their ability to promote cell proliferation and, optionally, cell survival of PBMCs (and in illustrative embodiments, T cells) without the need to add IL-2 during culture of PBMCs (i.e., T cells) after culture day 1, day 2, day 3, day 4, day 5, or day 7 after the PBMCs are transduced with nucleic acids encoding chimeric lymphoproliferative components lymphoproliferative components, as appropriate. Example 13 provides another example of a method that can be used to identify and analyze lymphoproliferative components lymphoproliferative components and describe the properties or capabilities of lymphoproliferative components. Such analysis can include, for example, measuring the relevant lymphoproliferative activity of such lymphoproliferative components lymphoproliferative components, such as by analyzing the magnitude of enrichment provided by genetically modified lymphocytes expressing such lymphoproliferative components lymphoproliferative components compared to control lymphocytes that do not express such lymphoproliferative components lymphoproliferative components lymphoproliferative components.

In one embodiment of this aspect, the first nucleic acid sequence further encodes an extracellular domain, and in illustrative embodiments, the extracellular domain comprises a dimerization motif. In illustrative embodiments of this aspect, the extracellular domain comprises a leucine zipper. In some embodiments, the leucine zipper is from a jun polypeptide, such as c-jun. In certain embodiments, the c-jun polypeptide is the c-jun polypeptide region of ECD-11.

In embodiments of any of these aspects where the transmembrane domain is a type I transmembrane protein, the transmembrane domain may be a type I growth factor receptor, a hormone receptor, a T cell receptor, or a TNF family receptor. In an embodiment of any of the aspects and embodiments where the chimeric polypeptide comprises an extracellular domain and wherein the extracellular domain comprises a dimerization motif, the transmembrane domain may be a type I cytokine receptor, a hormone receptor, a T cell receptor, or a TNF family receptor.

Exemplary transmembrane domains include any transmembrane domain used in Example 12. In some embodiments, the transmembrane domain is from CD4, CD8RB, CD40, CRLF2, CSF2RA, CSF3R, EPOR, FCGR2C, GHR, ICOS, IFNAR1, IFNGR1, IFNGR2, IL1R1, IL1RAP, IL2RG, IL3RA, IL5RA, IL6ST, IL7RA, IL10RB, IL11RA, IL13RA2, IL17RA, IL17RB, IL17RC, IL17RE, IL18R1, IL18RAP, IL20RA, IL22RA1, IL31RA, LEPR, PRLR, and TNFRSF8, or mutants thereof that are known to promote signaling activity in certain cell types when these mutants are present in the constructs provided in Example 12. In some embodiments, the transmembrane domain is from CD40, ICOS, FCGR2C, PRLR, IL3RA, or IL6ST. In some embodiments, the transmembrane domain is a specific transmembrane domain provided for these genes in the constructs for any one or more of Tables 13 to 18 or a transmembrane domain from any of the constructs in the first 50, 25, or 10 listed constructs in those tables. In illustrative embodiments, the transmembrane domain is from CD40 or ICOS (as non-limiting examples, the transmembrane domains for these genes are provided in Table 7) or a fragment thereof that retains the ability to cross the membrane, and/or a mutant thereof that is known to promote constitutive signaling activity in certain cell types.

Exemplary transmembrane domains from this aspect are shown as P2 in Tables 13 to 18. For library 3A, when found at the transmembrane domain position (P2) of the candidate chimeric polypeptide components herein, transmembrane domains (P2) from CD40, CD8B, CRLF2, CSF2RA, GCGR2C, ICOS, IFNAR1, IFNGR1, IL10RB, IL18R1, IL18RAP, IL3RA, LEPR, and PRLR, or mutants thereof known to promote constitutive signaling activity in certain cell types when these mutants are present in the constructs provided in Example 12, promote proliferation of PBMCs between the 7th day and the last day when the data of all constructs with transmembrane domains derived from the gene are considered in combination. In the screening provided in Examples 11 and 12, cells were transduced with replication-defective recombinant retroviral particles containing the library and then fed into PBMCs or not, as discussed in Examples 11 and 12. Screens where cells were fed to PBMCs were identified with an "A" after the library number, e.g., library 1A, and screens where cells were not fed to PBMCs were identified with a "B" after the library number, e.g., library 1.1B. In addition, screens of libraries identified as containing a ".1" (e.g., library 1.1A) were performed in the same manner to screen corresponding libraries that did not have a ".1", e.g., library 1.1A was a repeated screen of library 1A. For library 3B, transmembrane domains from CD40, ICOS, CSF2RA, IL18R1, IL3RA, and TNFRSF14, or mutants thereof known to promote constitutive signaling activity in certain cell types when these mutants are present in the constructs provided in Example 12, were most able to promote cell proliferation. For library 4B, transmembrane domains from IL3RA, CSFR2RA, IL18R1, CD8B, CD40, ICOS, or mutants thereof known to promote constitutive signaling activity in certain cell types when these mutants are present in the constructs provided in Example 12 are most capable of promoting cell proliferation. In illustrative embodiments, the transmembrane domains in the CLE provided herein are transmembrane domains from CD40 and ICOS. Examples of transmembrane domains or portions and/or mutants thereof present in constructs that promote cell proliferation for libraries 3A, 3B, 3.1A, 3.1B, 4B, and 4.1B are provided in the table provided in Example 12.

In some embodiments of any of the aspects herein, the chimeric polypeptide comprises a transmembrane domain identified in any one of the constructs shown in Tables 13-18 except for the transmembrane domain mutant V449E of CSF2RB.

In some embodiments, the extracellular domain and the transmembrane domain are the viral protein LMP1 or mutants and/or fragments thereof. LMP1 is a multi-span transmembrane protein known to activate cell signaling independently of ligands when targeting lipid rafts or when fused to CD40 (Kaykas et al., EMBO J. 20: 2641 (2001)). Fragments of LMP1 are usually long enough to span the plasma membrane and activate the attached intracellular domain. For example, LMP1 can be between 15 amino acids and 386 amino acids, between 15 amino acids and 200 amino acids, between 15 amino acids and 150 amino acids, between 15 amino acids and 100 amino acids, between 18 amino acids and 50 amino acids, between 18 amino acids and 30 amino acids, between 20 amino acids and 200 amino acids, between 20 amino acids and 150 amino acids, between 20 amino acids and 50 amino acids, between 20 amino acids and 30 amino acids, between 20 amino acids and 100 amino acids, between 20 amino acids and 40 amino acids, or between 20 amino acids and 25 amino acids. Mutants and/or fragments of LMP1 retain their ability to activate the intracellular domain when included in the CLE provided herein. In addition, if present, the extracellular domain includes at least 1, but typically at least 4 amino acids and is typically connected to another functional polypeptide, such as an interstitial domain, such as an eTag. In some embodiments, the lymphoproliferative component comprises an LMP1 transmembrane domain. In illustrative embodiments, the lymphoproliferative element comprises an LMP1 transmembrane domain, and the one or more intracellular domains do not comprise an intracellular domain from a TNFRSF protein (i.e., CD40, 4-IBB, RANK, TACI, OX40, CD27, GITR, LTR, and BAFFR), TLR1-TLR13, integrin, FcγRIII, Dectin1, Dectin2, NOD1, NOD2, CD16, IL-2R, type I and II interferon receptors, such as C Chemokine receptors for CR5 and CCR7, G protein-coupled receptors, TREM1, CD79A, CD79B, Ig-α, IPS-1, MyD88, RIG-1, MDA5, CD3Z, MyD88ΔTIR, TRIF, TRAM, TIRAP, MAL, BTK, RTK, RAC1, SYK, NALP3 (NLRP3), NALP3ΔLRR, NALP1, CARD9, DAI, IPAG, STING, Zap70 or LAT.

In other embodiments of CLE provided herein, the extracellular domain includes a dimerization portion. Many different dimerization portions disclosed herein can be used in these embodiments. In illustrative embodiments, the dimerization portion is capable of homodimerization. Without being limited by theory, the dimerization portion can provide an activation function to the intracellular domain connected to it via the transmembrane domain. This activation can be provided, for example, after dimerization of the dimerization portion, which can change the orientation of the intracellular domain connected to it via the transmembrane domain, or it can bring the intracellular domain into proximity. The extracellular domain with the dimerization portion can also serve to connect the identification marker to the cell expressing the CLE. In some embodiments, the dimerization agent can be located intracellularly rather than extracellularly. In some embodiments, more than one or more dimerization domains can be used.

The extracellular domain of the embodiment in which the extracellular domain has a dimerization motif is long enough to form a dimer, such as a leucine zipper dimer. Therefore, the extracellular domain including the dimerization portion can be 15 amino acids to 100 amino acids, 20 amino acids to 50 amino acids, 30 amino acids to 45 amino acids or 35 amino acids to 40 amino acids, in an illustrative embodiment, the c-Jun portion of the c-Jun extracellular domain provided in Table 7. The extracellular domain of the polypeptide including the dimerization portion may not retain other functionalities. For example, for leucine zipper dimers, these leucine zippers are able to form dimers because they retain the motif of leucine separated by 7 residues along α. However, the leucine zipper portion of certain embodiments of CLE provided herein may or may not retain its DNA binding function.

An exemplary extracellular domain of this type is a leucine zipper domain from a Jun portion such as c-Jun. For example, the extracellular domain may be a variant of c-Jun found in NM_002228_3 (Table 7). This extracellular domain is used in the constructs discussed in Example 18.

A spacer between 1 and 4 alanines may be included in the CLE between the extracellular domain and the transmembrane domain having the dimerization portion. Without being limited by theory, it is believed that the alanine spacer affects signal transduction of the intracellular domain connected to the leucine zipper extracellular region via the transmembrane domain by changing the orientation of the intracellular domain.

The first intracellular domain and the second intracellular domain of CLE provided herein are intracellular signaling domains of genes known in at least some cell types to promote proliferation, survival (anti-apoptosis) and/or provide costimulatory signals that enhance differentiation state, proliferative potential or resistance to cell death. Therefore, these intracellular domains can be intracellular domains from lymphoproliferative components and costimulatory domains provided herein. Some of the intracellular domains of known candidate chimeric polypeptides can activate JAK1/JAK2 signaling, JAK3, STAT1, STAT2, STAT3, STAT4, STAT5 and STAT6. Intracellular domains from IFNAR1, IFNGR1, IFNLR1, IL2RB, IL4R, IL5RB, IL6R, IL6ST, IL7RA, IL9R, IL10RA, IL21R, IL27R, IL31RA, LIFR and OSMR are known in the art for activating JAK1 signaling. Intracellular domains from CRLF2, CSF2RA, CSF2RB, CSF3R, EPOR, GHR, IFNGR2, IL3RA, IL5RA, IL6ST, IL20RA, IL20RB, IL23R, IL27R, LEPR, MPL and PRLR are known in the art for activating JAK2. Intracellular domains from IL2RG are known in the art for activating JAK3. Intracellular domains from GHR, IFNAR1, IFNAR2, IFNGR1, IFNGR2, IL2RB, IL2RG, IL4R, IL5RA, IL5RB, IL7RA, IL9R, IL21R, IL22RA1, IL31RA, LIFR, MPL and OSMR are known in the art for activating STAT1. Intracellular domains from IFNAR1 and IFNAR2 are known in the art for activating STAT2. Intracellular domains from GHR, IL2RB, IL2RG, IL6R, IL7RA, IL9R, IL10RA, IL10RB, IL21R, IL22RA1, IL23R, IL27R, IL31RA, LEPR, LIFR, MPL, and OSMR are known in the art for activating STAT3. Intracellular domains from IL12RB1 are known in the art for activating STAT4. Intracellular domains from CSF2RA, CSF2RB, CSF3R, EPOR, GHR, IL2RB, IL2RG, IL3RA, IL4R, IL5RA, IL5RB, IL7RA, IL9R, IL15RA, IL20RA, IL20RB, IL21R, IL22RA1, IL31RA, LIFR, MPL, OSMR, and PRLR are known in the art for activating STAT5. Intracellular domains from IL4R and OSMR are known in the art for activating STAT6. The gene found in the first intracellular domain and its intracellular domain are identical to the second intracellular domain, except that if the first and second intracellular domains are identical, at least one, and typically both, the transmembrane domain and the extracellular domain are not from the same gene.

Exemplary intracellular domains include those from the constructs listed in Tables 8 to 12. Exemplary intracellular domains from these genes empirically determined to be active in the experiments of Example 11 are provided in the first intracellular domain (P3) and second intracellular domain (P4) positions of the table provided in Example 11 and as further listed in this section below. Illustrative intracellular domains identified in the top hits screened for Example 11 include CD40, CSF2RA, IFNAR1, IL1RAP, IL4R, IL6ST, IL11RA, IL12RB2, IL17RA, IL17RD, IL17RE, IL18R1, IL21R, IL23R, MPL, and MyD88. Illustrative intracellular domains identified in the top hits screened for Example 12 include CD40, LEPR, MyD88, IFNAR2, MPL, IL18R1, IL13RA2, IL10RB, IL23R, or CSF2RA. In certain illustrative embodiments, the first intracellular domain is MPL, LEPR, MyD88, or IFNAR2. For these certain illustrative embodiments, non-limiting exemplary second intracellular domains (P4) domains are those linked to genes corresponding to MPL, LEPR, MyD88, IFNAR2, CD40, CD79B, or CD27 first intracellular domains (P3) (provided in Tables 13 to 18), including, in some non-limiting examples, the first intracellular domains and/or second intracellular domains of those genes provided in these tables. For these certain illustrative embodiments, other non-limiting exemplary second intracellular domain (P4) domains are linked to the corresponding MPL, LEPR, MYD88, IFNAR2, CD40, CD79B or CD27 first intracellular domain in the table provided in Example 12, including, as non-limiting examples, the first intracellular domain and/or second intracellular domain of those genes provided in these tables. In some embodiments, the chimeric lymphoproliferative component lymphoproliferative component can be any of the polypeptides in Tables 19 to 23 of Example 11 and Example 12.

In certain illustrative embodiments, the second intracellular domain is from CD3D, CD3ζ, CD27, CD40, CD79A, CD79B, FCER1G, FCGRA2, ICOS, TNFRSF4 and TNFRSF8, or mutants thereof known to promote signaling activity in certain cell types when these mutants are present in the constructs provided in Example 12. In certain illustrative embodiments, the second intracellular domain is from CD40, CD79B, TNFRSF4, TNFRSF9, TNFRSF14, FCGRA2, CD3ζ or CD27, including the intracellular domains of CD40, CD79B and CD27 in illustrative examples. For these certain illustrative embodiments, non-limiting exemplary first intracellular domains (P3) are those linked to the genes of CD40, CD79B, TNFRSF4, TNFRSF9, TNFRSF14, FCGRA2, CD3G, or CD27, including, in illustrative examples, the intracellular domains of CD40, CD79B, or CD27 in Tables 13 to 18, including, as non-limiting examples, the first intracellular domains and/or second intracellular domains of those genes provided in these tables. For these certain illustrative embodiments, other non-limiting exemplary first intracellular (P3) domains are those linked to the genes of CD40, CD79B, TNFRSF4, TNFRSF9, TNFRSF14, FCGRA2, CD3G, or CD27, including, in illustrative examples, the intracellular domains of the CD40, CD79B, and CD27 second intracellular domains in the table of these genes as P3 provided in Example 12, including, as non-limiting examples, the first intracellular domains and/or second intracellular domains of those genes provided in these tables.

In illustrative embodiments, the first intracellular domain is from MPL and the second intracellular domain is from OX40, CD40, CD79A or CD79B. As shown in Example 10, at a false discovery rate of 0.05, when included as a second intracellular domain of a chimeric lymphoproliferative component containing MPL as the first intracellular domain, the intracellular domains from these genes were shown to significantly increase proliferation in at least one of library 3.1 or library 4.1. In other illustrative embodiments, the first intracellular domain is from MPL and the second intracellular domain is from OX40, CD40 or CD79B. In some embodiments, the first intracellular domain contains a sequence from S186 (SEQ ID NO: 491) or a variant or fragment thereof that promotes signal transduction, and the second intracellular domain contains one of the following sequences: S050, S051, S052, S053 or S211 (SEQ ID NOs: 416 to 419 and 504, respectively), or a variant or fragment thereof that promotes signal transduction. In illustrative embodiments, the first intracellular domain is from CSF2RA and the second intracellular domain is from OX40, CD27, CD28 or CD30. As shown in Example 10, at a false discovery rate of 0.1, when included as a second intracellular domain of a chimeric lymphoproliferative component containing CSF2RA as the first intracellular domain, the intracellular domains from these genes were shown to significantly increase the proliferation of at least one of library 3.1 or library 4.1. In other illustrative embodiments, the first intracellular domain is from CSF2RA and the second intracellular domain is from OX40 or CD28. As shown in Example 10, at a false discovery rate of 0.05, when included as a second intracellular domain of a chimeric lymphoproliferative component containing CSF2RA as the first intracellular domain, the intracellular domains from these genes were shown to significantly increase the proliferation of at least one of library 3.1 or library 4.1. In some embodiments, the first intracellular domain contains a sequence from S059 (SEQ ID NO: 423) or a variant or fragment thereof that promotes signaling, and the second intracellular domain contains one of the sequences from S047 (SEQ ID NO: 413) or S212 (SEQ ID NO: 505), or a variant or fragment thereof that promotes signaling. In some embodiments, the first intracellular domain contains a sequence from S058 (SEQ ID NO: 422) or a variant or fragment thereof that promotes signaling, and the second intracellular domain contains a sequence from S211 (SEQ ID NO: 504), or a variant or fragment thereof that promotes signaling. In illustrative embodiments, the first intracellular domain is from CSF3R, and the second intracellular domain is from CD79B. As shown in Example 10, at a false discovery rate of 0.05, when included as a second intracellular domain of a chimeric lymphoproliferative component containing CSF3R as the first intracellular domain, the intracellular domain from CD79B was shown to significantly increase the proliferation of at least one of library 3.1 or library 4.1. In some embodiments, the first intracellular domain contains a sequence from S064 (SEQ ID NO: 426) or a variant or fragment thereof that promotes signaling, and the second intracellular domain contains a sequence from S053 (SEQ ID NO: 419), or a variant or fragment thereof that promotes signaling. In an illustrative embodiment, the first intracellular domain is from IL11RA, and the second intracellular domain is from FCGRA2. As shown in Example 10, at a false discovery rate of 0.05, when included as a second intracellular domain of a chimeric lymphoproliferative component containing IL11RA as the first intracellular domain, the intracellular domain from FCGRA2 was shown to significantly increase the proliferation of at least one of library 3.1 or library 4.1. In some embodiments, the first intracellular domain contains a sequence from S135 (SEQ ID NO: 461) or a variant or fragment thereof that promotes signaling, and the second intracellular domain contains a sequence from S076 (SEQ ID NO: 431), or a variant or fragment thereof that promotes signaling. In illustrative embodiments, the first intracellular domain is from LIFR and the second intracellular domain is from GITR. As shown in Example 10, at a false discovery rate of 0.05, when included as a second intracellular domain of a chimeric lymphoproliferative component containing LIFR as the first intracellular domain, the intracellular domain from GITR was shown to significantly increase the proliferation of at least one of library 3.1 or library 4.1. In some embodiments, the first intracellular domain contains a sequence from S180 (SEQ ID NO: 489) or a variant or fragment thereof that promotes signaling, and the second intracellular domain contains a sequence from S216 (SEQ ID NO: 509), or a variant or fragment thereof that promotes signaling. In illustrative embodiments, the first intracellular domain is from MyD88 and the second intracellular domain is from CD3D or CD79B. As shown in Example 10, at a false discovery rate of 0.1, when included as a second intracellular domain of a chimeric lymphoproliferative component containing MyD88 as the first intracellular domain, the intracellular domains from these genes were shown to significantly increase the proliferation of at least one of library 3.1 or library 4.1. In some embodiments, the first intracellular domain contains a sequence from S192 (SEQ ID NO: 495) or a variant or fragment thereof that promotes signaling, and the second intracellular domain contains a sequence from S053 (SEQ ID NO: 419), or a variant or fragment thereof that promotes signaling. In some embodiments, the first intracellular domain contains a sequence from S194 (SEQ ID NO: 497) or a variant or fragment thereof that promotes signaling, and the second intracellular domain contains a sequence from S037 (SEQ ID NO: 405), or a variant or fragment thereof that promotes signaling. In some embodiments, the first intracellular domain contains a sequence from S195 (SEQ ID NO: 498), or a variant or fragment thereof that promotes signaling, and the second intracellular domain contains a sequence from S053 (SEQ ID NO: 419), or a variant or fragment thereof that promotes signaling.

Detailed information (including sequence information) about exemplary intracellular domains of the above genes identified in Examples 11 and 12 in constructs that promote cell survival and proliferation is provided in Table 7. The intracellular domains that may be included in the CLE embodiments provided herein include mutants and/or fragments of the intracellular domains of the stated genes, with the proviso that such mutants and/or fragments maintain the ability to promote proliferation, survival (anti-apoptosis) and/or provide costimulatory signals that enhance differentiation state, proliferative potential or resistance to cell death. Thus, the intracellular domain may be, for example, between 10 and 1000 amino acids, 10 and 750 amino acids, 10 and 500 amino acids, 10 and 250 amino acids, or 10 and 100 amino acids. In illustrative embodiments, the intracellular domain may be at least 30 amino acids, or between 30 and 500 amino acids, 30 and 250 amino acids, 50 and 500 amino acids, 50 and 250 amino acids. In illustrative embodiments, the first intracellular domain is from IFNAR2 and the second intracellular domain is from HVEM. As shown in Example 10, at a false discovery rate of 0.05, when included as a second intracellular domain of a chimeric lymphoproliferative component containing IFNAR2 as the first intracellular domain, the intracellular domain from HVEM was shown to significantly increase the proliferation of at least one of library 3.1 or library 4.1. In some embodiments, the first intracellular domain contains a sequence from S083 (SEQ ID NO: 436) or a variant or fragment thereof that promotes signal transduction, and the second intracellular domain contains a sequence from S214 (SEQ ID NO: 507), or a variant or fragment thereof that promotes signal transduction. In illustrative embodiments, the first intracellular domain is from IL1RAP, and the second intracellular domain is from CD79A. As shown in Example 10, at a false discovery rate of 0.1, when included as a second intracellular domain of a chimeric lymphoproliferative component containing IL1RAP as the first intracellular domain, an intracellular domain from CD79A was shown to significantly increase the proliferation of at least one of library 3.1 or library 4.1. In some embodiments, the first intracellular domain contains a sequence from S101 (SEQ ID NO: 444) or a variant or fragment thereof that promotes signaling, and the second intracellular domain contains a sequence from S052 (SEQ ID NO: 418), or a variant or fragment thereof that promotes signaling. In an illustrative embodiment, the first intracellular domain is from IL3RA and the second intracellular domain is from CD40. As shown in Example 10, at a false discovery rate of 0.1, when included as a second intracellular domain of a chimeric lymphoproliferative component containing IL3RA as the first intracellular domain, an intracellular domain from CD40 was shown to significantly increase the proliferation of at least one of library 3.1 or library 4.1. In some embodiments, the first intracellular domain contains a sequence from S109 (SEQ ID NO: 450) or a variant or fragment thereof that promotes signaling, and the second intracellular domain contains a sequence from S050 (SEQ ID NO: 416), or a variant or fragment thereof that promotes signaling. In illustrative embodiments, the first intracellular domain is from IL10RA and the second intracellular domain is from CD79B. As shown in Example 10, at a false discovery rate of 0.05, when included as a second intracellular domain of a chimeric lymphoproliferative component containing IL10RA as the first intracellular domain, the intracellular domain from CD79B was shown to significantly increase the proliferation of at least one of library 3.1 or library 4.1. In some embodiments, the first intracellular domain contains a sequence from S129 (SEQ ID NO: 450) or a variant or fragment thereof that promotes signaling, and the second intracellular domain contains a sequence from S050 (SEQ ID NO: 416), or a variant or fragment thereof that promotes signaling. In illustrative embodiments, the first intracellular domain is from IL13RA2 and the second intracellular domain is from HVEM. As shown in Example 10, at a false discovery rate of 0.1, the intracellular domain from HVEM was shown to significantly increase proliferation of at least one of Library 3.1 or Library 4.1 when included as a second intracellular domain of a chimeric lymphoproliferative component containing IL13RA2 as the first intracellular domain. In some embodiments, the first intracellular domain contains a sequence from S142 (SEQ ID NO: 466), or a variant or fragment thereof that promotes signaling, and the second intracellular domain contains a sequence from S214 (SEQ ID NO: 507), or a variant or fragment thereof that promotes signaling. In illustrative embodiments, the first intracellular domain is from IL18RAP, and the second intracellular domain is from CD3ζ. As shown in Example 10, at a false discovery rate of 0.1, when included as a second intracellular domain of a chimeric lymphoproliferative component containing IL18RAP as the first intracellular domain, the intracellular domain from CD3ζ was shown to significantly increase the proliferation of at least one of library 3.1 or library 4.1. In some embodiments, the first intracellular domain contains a sequence from S155 (SEQ ID NO: 475) or a variant or fragment thereof that promotes signaling, and the second intracellular domain contains a sequence from S039 (SEQ ID NO: 407), or a variant or fragment thereof that promotes signaling. In an illustrative embodiment, the first intracellular domain is from IL27RA and the second intracellular domain is from FCGRA2. As shown in Example 10, at a false discovery rate of 0.05, when included as a second intracellular domain of a chimeric lymphoproliferative component containing IL27RA as the first intracellular domain, the intracellular domain from FCGRA2 was shown to significantly increase the proliferation of at least one of library 3.1 or library 4.1. In some embodiments, the first intracellular domain contains a sequence from S169 (SEQ ID NO: 482) or a variant or fragment thereof that promotes signaling, and the second intracellular domain contains a sequence from S076 (SEQ ID NO: 431), or a variant or fragment thereof that promotes signaling. In illustrative embodiments, the first intracellular domain is from LEPR, and the second intracellular domain is from CD3G. As shown in Example 10, at a false discovery rate of 0.1, when included as a second intracellular domain of a chimeric lymphoproliferative component containing LEPR as the first intracellular domain, the intracellular domain from CD3G was shown to significantly increase the proliferation of at least one of library 3.1 or library 4.1. In some embodiments, the first intracellular domain contains a sequence from S177 (SEQ ID NO: 488) or a variant or fragment thereof that promotes signaling, and the second intracellular domain contains a sequence from S039 (SEQ ID NO: 407), or a variant or fragment thereof that promotes signaling.

In some embodiments, all domains of CLE are not IL-7 receptor or a mutant thereof, and/or a fragment thereof having at least 10, 15, 20 or 25 consecutive amino acids of IL-7 receptor, or are not IL-15 receptor or a mutant thereof, and/or a fragment thereof having at least 10, 15, 20 or 25 consecutive amino acids of IL-15 receptor. In some embodiments, CLE does not comprise a combination of the first intracellular domain and the second intracellular domain of CD40 and MyD88.

In illustrative embodiments, CLE includes a recognition domain and/or an elimination domain. Details about the recognition domain and/or elimination domain are provided in other sections herein. Any of the recognition domains and/or elimination domains provided herein may be part of a CLE. Typically, the recognition domain is connected to the N-terminus of the extracellular domain. Without being limited by theory, in some embodiments, the extracellular domain includes the function of providing a connector (in illustrative embodiments, a flexible connector) to connect the recognition domain to a cell expressing the CLE.

In illustrative embodiments, as illustrated in Figures 15 and 16, a CLE provided herein is co-expressed with a CAR that can be designed according to any of the CAR embodiments provided herein or otherwise known in the art.

Some embodiments provided herein are isolated polynucleotides and nucleic acid sequences encoding any one of the CLEs provided herein, as illustrated in Figures 15 to 18. These isolated polynucleotides may include any components known in the art for providing expression of transcripts (such as transcripts encoding CLE). For example, isolated polynucleotides may include promoter components active in B cells, T cells, and/or NK cells. These promoters suitable for these embodiments are known in the art, some of which are identified in other sections of the present invention. For example, a skilled technician will understand how to involve isolated polynucleotides and vectors containing them, as discussed in more detail in Examples 11 and 12, for expressing any one of the CLE embodiments provided herein. For example, in some embodiments, a Kozak sequence is provided within 10 nucleotides upstream of the ATG start site, which encodes the amino terminal amino acid of CLE or CAR upstream of CLE on the same transcription unit. In some embodiments, the polynucleotide may be a translation initiation sequence. In some embodiments, the polynucleotide may be a translation initiation sequence, which may be AGAGGATCCATG (SEQ ID NO: 533). In some embodiments, the translation initiation sequence may be a Kozak-type sequence, also referred to herein as Kozak. In some embodiments, the Kozak-type sequence may be CCACCAT/UG(G) (SEQ ID NO: 515), CCGCCAT/UG(G) (SEQ ID NO: 516), GCCGCCGCCAT/UG(G) (SEQ ID NO: 517), or GCCGCCGCCAT/U(G) (SEQ ID NO: 531). In any of the embodiments disclosed herein, an internal ribosome binding site may be used instead of a Kozak-type sequence. Thus, in polynucleotide embodiments comprising a nucleic acid encoding a CLE provided herein, the polynucleotide may further comprise a Kozak-related sequence, a WPRE element, and one or more of a plurality of termination sequences, such as a double termination sequence or a triple termination sequence. In some embodiments, the triple termination sequence of the nucleic acid encoding CLE may be upstream of the promoter of CLE. In illustrative embodiments, the triple termination sequence may be included upstream of the 5' end of the promoter within 10, 25, 50 or 100 nucleotides. In some embodiments, the triple termination sequence in the nucleic acid encoding CLE may be included after the promoter and before the associated Kozak-related sequence. In illustrative embodiments, the triple termination sequence may be included downstream of the 3' end of the promoter within 10, 25, 50 or 100 nucleotides, and/or upstream of the 5' end of the Kozak-related sequence within 10, 25, 50 or 100 nucleotides. In some embodiments, the triple termination sequence of the nucleic acid encoding CLE may be included after the coding sequence of CLE. In illustrative embodiments, the triple termination sequence may be included downstream of the 3' end of the stop codon of CLE within 10, 25, 50 or 100 nucleotides. In some embodiments, the triple termination sequence may be at the 3' end of the coding sequence of CLE. The nucleic acid encoding CLE may include one, two, three or more triple termination sequences. In an illustrative embodiment, the nucleic acid encoding CLE may include a triple termination sequence upstream of the promoter, a triple termination sequence downstream of the promoter and upstream of the Kozak-related sequence, and a triple termination sequence downstream of the coding sequence of CLE. In any of the embodiments disclosed herein, the triple termination sequence may include three termination codons in the same reading frame. In an illustrative embodiment, the triple termination sequence may be SEQ ID NO: 520. When using multiple termination sequences with multiple termination codons in the same reading frame, the skilled person will understand that the multiple termination sequences should be in the same reading frame as the coding sequence of CLE and/or CAR. In an illustrative embodiment of any of the embodiments disclosed herein, the triple termination sequence may include three termination codons in different reading frames. In an illustrative embodiment, the triple termination sequence may be SEQ ID NO: 534.

In addition, the polynucleotides including the nucleic acid sequences encoding the CLE provided herein also generally include a signal sequence to directly express the plasma membrane. Exemplary signal sequences are generally provided herein and provided in other sections. Components may be provided on the transcript so that both CAR and CLE are expressed from the same transcript. This construct is advantageous in that less nucleic acid is required than when these components are expressed from different transcripts, which is an important feature that enables these constructs to be present in a vector (such as a retroviral genome), for example, for transfection or transduction of B cells, T cells, and/or NK cells.

Numerous chimeric polypeptide candidates are involved and identified as lymphoproliferative components in Examples 11 and 12 herein. For library 1A (which includes constructs encoding chimeric polypeptides and CARs), 172 top candidate chimeric polypeptides were identified (see Table 8), and when cultured in the absence of IL-2, PBMC proliferation was promoted between the 7th day and the last day. For library 2B (which includes constructs encoding chimeric polypeptides but not including CARs), 167 top candidate chimeric polypeptides were identified (see Table 9), and when cultured in the absence of IL-2, PBMC proliferation was promoted between the 7th day and the last day. Certain illustrative CLEs and polynucleotides and nucleic acid sequences encoding them, and methods including any of these include intracellular domains from genes with matched domains (e.g., transmembrane genes and first intracellular genes and optionally second intracellular genes) on the constructs provided in Tables 8 to 12, and (in non-limiting examples) specific matched domains provided in these tables, some of which are described in the illustrative embodiments section herein. In an illustrative embodiment, intracellular domains from CLEs with matched domains (e.g., a transmembrane gene and a first and optionally a second intracellular gene) on constructs with particularly noteworthy enrichment in the first and repeated screens with the same P1 to P2, P3, and P4 domains (e.g., libraries 1A and 1.1A) can be used as shown in Tables 8 to 12. For libraries 1A and 1.1A, constructs M001-S116-S044, M024-S192-S045, M001-S047-S102, M048-S195-S043, M012-S216-S211, and M030-S170-S194 had particularly noteworthy enrichment in both screens.

Additional information about the first and second intracellular domains in constructs with particularly noteworthy enrichment in the screening of both library 1A and library 1.1A is provided in Table 20, including the genes from which the first and second intracellular domains are derived, whether the first and/or second intracellular domains are cytokines receptors, and whether the first and/or second intracellular domains have at least one ITAM motif. As shown in Example 17, constructs with intracellular domains from IL6R, MYD88, CD27, MYD88, TNFRSF18, or IL31RA (when present in the first intracellular domain (P3)) and constructs with intracellular domains from CD8B, IL1RL1, CD8A, TNFRSF4, or MYD88 (when present in the second intracellular domain (P4)) show particularly noteworthy enrichment in both library 1A and 1.1A (Table 20). Constructs with domains from cytokine receptors IL6R, CD27, TNFRSF18, or IL31RA when present in the first intracellular domain (P3), and constructs with domains from cytokine receptors IL1RL1 or TNFRSF4 when present in the second intracellular domain (P4), showed particularly noteworthy enrichment in both pools 1A and 1.1A (Table 20).

For pools 2B and 2.1B, constructs M007-S049-S051, M007-S050-S039, M012-S050-S043, M012-S161-S213, M030-S142-S049, M001-S145-S130, M018-S085-S039, M018-S075-S053, M012-S135-S074, and M007-S214-S077 had particularly noteworthy enrichments in both screens. For the purposes of the replicate screens, constructs with particularly noteworthy enrichments were those with _log2 ((normalized count data on the last day + 1)/(normalized count data on day 7 + 1)) values greater than 2.

Additional information about the first and second intracellular domains in constructs with particularly noteworthy enrichment in the screen for both pool 2B and pool 2.1B is provided in Table 21, including the genes from which the first and second intracellular domains are derived, whether the first and/or second intracellular domains are cytokine receptors, and whether the first and/or second intracellular domains have at least one ITAM motif. Constructs with intracellular domains from CD28, CD40, IL22RA1, IL13RA2, IL17RB, IFNGR2, FCGR2C, IL11RA, or TNFRSF14 were present in the first intracellular domain (P3), and constructs with intracellular domains from CD40, CD3ζ, CD8A, TNFRSF9, CD28, IL10RB, CD79B, FCER1G, or GHR were present in the second intracellular domain (P4), showing particularly noteworthy enrichment in both pools 2B and 2.1B (Table 21). In some embodiments provided herein, the intracellular domain of the lymphoproliferative component is from CD40, IL22RA1, IL13RA2, IL17RA, IL17RB, IFNGR2, and FCGR2C. Constructs with domains from cytokine receptors CD40, IL22RA1, IL13RA2, IL17RB, IFNGR2, IL11RA, or TNFRSF14 were present in the first intracellular domain (P3), and constructs with domains from cytokine receptors TNFRSF9, IL10RB, GHR, or CD40 were present in the second intracellular domain (P4), showing particularly noteworthy enrichment in both pools 2B and 2.1B (Table 21). Constructs with a domain containing the ITAM motif of FCGR2C present in the first intracellular domain (P3) and constructs with a domain containing the ITAM motif of CD3G, CD79B or FCER1G present in the second intracellular domain (P4) showed particularly noteworthy enrichment in both pools 2B and 2.1B (Table 21).

In an initial interim analysis, for library 3A (which includes constructs encoding chimeric polypeptides and CARs), 126 top candidate chimeric polypeptides were identified (see Table 13) that promoted PBMC proliferation between day 7 and the last day of transduced PBMCs stimulated with untransduced PBMCs as described in Example 12 when cultured in the absence of IL-2. For an initial interim analysis of library 3B (which includes constructs encoding chimeric polypeptides and CARs), 127 top candidate chimeric polypeptides were identified (see Table 14) that promoted PBMC proliferation between day 7 and the last day of transduced PBMCs not stimulated with untransduced PBMCs as described in Example 12 when cultured in the absence of IL-2. For library 4B (which included constructs encoding chimeric polypeptides instead of CARs), 154 top candidate chimeric polypeptides were identified (see Table 17) that promoted PBMC proliferation between day 7 and the last day of transduced PBMCs stimulated with non-transduced PBMCs when cultured in the absence of IL-2 as described in Example 12.

After further decoding (which provides deep decoding), in libraries 3A and 3B, which include constructs encoding chimeric polypeptides and CARs and transduced PBMCs supplemented with (library 3A) or not supplemented with (library 3B) freshly transduced PBMCs, 134 top candidates were identified for library 3A on day 21, 124 top candidates were identified for library 3A on day 35, and 131 top candidates were identified for library 3B on day 21 (see Tables 13 and 14, respectively) when cultured in the absence of IL-2 (i.e., IL-2 was not added to the culture medium during the culture period after the initial transduction), IL-7 or any other exogenous cytokine, promoting PBMC proliferation between day 7 and the last day.

Certain illustrative CLEs and polynucleotides and nucleic acid sequences encoding them and methods comprising any of these include intracellular domains from genes with matched domains (e.g., transmembrane genes and first intracellular genes and optionally second intracellular genes) on constructs provided in Tables 13 to 18 and (in non-limiting examples) specific matched domains provided on these tables, some of which are described in the illustrative embodiments section herein. In illustrative CLE embodiments, it is contemplated that the intracellular domains identified in the data provided in Examples 11 and 12 from the first intracellular domain or second intracellular domain position can be used interchangeably as the first intracellular domain or the second intracellular domain. In illustrative embodiments, intracellular domains from genes with matched domains (e.g., transmembrane genes and first intracellular genes and optionally second intracellular genes) on constructs with particularly noteworthy enrichments in the first and repeated screenings of the same P1, P2, P3 and P4 domains (e.g., libraries 3A and 3.1A) can be used. For libraries 3A and 3.1A, constructs E008/E013-T041-S186-S050, E006/E011-T077-S186-S211, E007/E012-T021-S186-S051, E009/E014-T041-S186-S053, E007/E012-T0 73-S186-S053, E006/E011-T017-S186-S051, E006/E011-T031-S186-S211, E006/E011-T011-S186-S050, E006/E011-T011-S186-S047, E007/E012 -T00 1-S186-S050, E006/E011-T041-S186-S051, E008/E013-T028-S186-S076, E009/E014-T029-S199-S053, E009/E014-T062-S186-S216, E007/E012-T006-S058-S051, E009/E014-T076-S186-S211 and E007/E012-T001-S186-S047 had particularly noteworthy enrichments in both screens, where the P1 portions separated by slashes here include different tags for libraries 3A and 3.1A as shown in Tables 7, 13 and 15. For the purpose of replicate screening, constructs with particularly noteworthy enrichments were those with _log2 ((normalized count data on the last day + 1)/(normalized count data on day 7 + 1)) values greater than 2.

Additional information about the first and second intracellular domains in constructs with particularly noteworthy enrichment in the screens for both library 3A and library 3.1A is provided in Table 22, including the genes from which the first and second intracellular domains are derived, whether the first and/or second intracellular domains are cytokine receptors, and whether the first and/or second intracellular domains have at least one ITAM motif. Constructs with intracellular domains from MPL, OSMR, or CSF2RA were present in the first intracellular domain (P3), and constructs with intracellular domains from CD40, TNFRSF4, CD79B, CD27, FCGR2A, or TNFRSF18 were present in the second intracellular domain (P4), showing particularly noteworthy enrichment in both libraries 3A and 3.1A (Table 22). Constructs with domains from cytokine receptors MPL, OSMR or CSF2RA were present in the first intracellular domain (P3), and constructs with domains from cytokine receptors CD40, TNFRSF4, CD27 or TNFRSF18 were present in the second intracellular domain (P4), showing particularly noteworthy enrichment in both pools 3A and 3.1A (Table 22). Constructs with domains containing ITAM motifs of MPL or OSMR were present in the second intracellular domain (P4), showing particularly noteworthy enrichment in both pools 3A and 3.1A (Table 22).

For 3B and 3.1B, constructs E007/E012-T017-S186-S051, E007/E012-T073-S186-S053, E008/E013-T028-S186-S047, E006/E011-T011-S186-S047, E007/E012-T082-S176-S214, E006/E011-T046-S047 S186-S052, E008/E013-T029-S186-S052, E009/E014-T011-S186-S053, E008/E013-T032-S186-S039, E007/E012-T034-S186-S051, E007/E012-T0 41-S192-S213, E006/E011-T014-S06 9-S213, E006/E011-T022-S186-S053, E006/E011-T023-S115-S075, E006/E011-T029-S106-S213, E006/E011-T032-S155-S080, E006/E011-T041- S186-S216, E006/E011-T057-S135-S 080, E006/E011-T072-S191-X002, E006/E011-T077-S186-S216, E006/E011-T080-S141-S080, E007/E012-T001-X001-S214, E007/E012-T007-S0 59-S211、E007/E012-T016-S186-S052、 E007/E012-T031-S186-S053, E007/E012-T044-S102-S052, E007/E012-T044-S142-X002, E007/E012-T055-S069-S053, E007/E012-T063-S176-S2 16. E007/E012-T065-S157-S075, E00 8/E013-T008-S085-X002, E008/E013-T011-S085-S048, E008/E013-T021-S109-X002, E008/E013-T021-S168-S211, E008/E013-T032-S064-S214, E008/E013-T037-S170-S215, E008/E 013-T038-S176-S048, E008/E013-T039-S137-S216, E008/E013-T041-S141-S053, E008/E013-T045-S177-S048, E008/E013-T048-S109-S074, E0 08/E013-T073-S199-S075, E009/E014 -T001-S157-S074, E009/E014-T005-S196-S049, E009/E014-T011-S130-X002, E009/E014-T013-S155-X002, E009/E014-T017-S186-S076, E009/E 014-T021-S142-S080, E009/E014-T02 3-S082-S076, E009/E014-T038-S196-S037, E009/E014-T055-S186-S052, E009/E014-T060-S175-S053, E009/E014-T070-S085-S212, E008/E013- T026-S054-S213, E009/E014-T007-S 120-S053, E007/E012-T045-S186-S211, E008/E013-T073-S186-X002, E008/E013-T074-S186-X002, E007/E012-T055-S186-S053, E008/E013-T0 36-S186-S053, E007/E012-T017-S058 -S053, E008/E013-T030-S189-S080, E006/E011-T029-S081-S047, E009/E014-T044-S194-S050, E006/E011-T028-S121-X002, E008/E013-T028-S1 86-S053, E009/E014-T078-S142-S2 13. E009/E014-T041-S186-S051, E008/E013-T006-S186-S050, E006/E011-T028-S186-S075, E006/E011-T040-S120-S038, E007/E012-T044-S11 5-S211, E009/E014-T039-S176-S075, E 007/E012-T028-S186-S050, E008/E013-T031-S202-S050, E007/E012-T072-S192-S053, E006/E011-T065-X001-S051, E007/E012-T030-S062-X0 02. E007/E012-T073-S186-X002, E009 /E014-T056-S186-S053, E008/E013-T046-S137-X002, E006/E011-T016-S136-S076, E007/E012-T032-S142-S037, E007/E012-T065-S120-S215, E0 09/E014-T077-S186-S047, E009/E0 14-T001-S126-S051, E006/E011-T030-S121-S039, E008/E013-T006-S176-S213, E009/E014-T032-S130-S215, E008/E013-T041-S186-S039, E00 9/E014-T021-S186-S047, E008/E013- T026-S137-S214, E007/E012-T029-S116-S075, E008/E013-T026-S106-S049, and E007/E012-T032-S168-S075 had particularly notable enrichments in both screens, where the P1 portion separated by slashes here included different markers of libraries 3B and 3.1B, as shown in Tables 7, 14, and 16. For the purposes of the replicate screens, constructs with particularly notable enrichments were those with _log2 ((normalized count data on the last day + 1)/(normalized count data on day 7 + 1)) values greater than 2.

Additional information about the first and second intracellular domains in constructs with particularly noteworthy enrichment in the screens for both library 3B and library 3.1B is provided in Table 23, including the genes from which the first and second intracellular domains are derived, whether the first and/or second intracellular domains are cytokine receptors, and whether the first and/or second intracellular domains have at least one ITAM motif. The first and/or second intracellular domains have at least one ITAM motif from MPL, LEPR, MYD88, EPOR, IL5RA, IL2RG, IL18RAP, IL11RA, IL13RA1, CSF2RA, IL1RL1, IL13RA2, IL20RB, IFNGR2, IL3RA, IL27RA, CSF3R, IL31RA, IL12RB1, OSMR, IL10RB, IFNAR2, CRLF2, IL7R, IFNAR1, PRLR, IL9R, IL6R, or IL15RA When constructs with an intracellular domain from CD40, CD79B, CD27, TNFRSF14, CD79A, CD3G, TNFRSF9, FCGR2C, ICOS, TNFRSF18, TNFRSF4, CD28, FCER1G, FCGR2A, CD3D, TNFRSF8, or CD3E were present in the second intracellular domain (P4), they showed particularly noteworthy enrichment in both pools 3B and 3.1B (Table 23). Constructs with an intracellular domain from MPL, LEPR, EPOR, IL5RA, IL2RG, IL18RAP, IL11RA, IL13RA1, CSF2RA, IL1RL1, IL13RA2, IL20RB, IFNGR2, IL3RA, IL27RA, CSF3R, IL31RA, IL12RB1, OSMR, IL10RB, IFNAR2, CRLF2, IL7R, IFNAR1, PRLR, IL9R, IL6R, or IL15RA when present in the first intracellular domain (P3) and constructs with an intracellular domain from CD40, CD27, TNFRSF14, TNFRSF9, TNFRSF18, TNFRSF4, or TNFRSF8 when present in the second intracellular domain (P4) showed particularly noteworthy enrichment in both pools 3B and 3.1B (Table 23). Constructs with domains containing the ITAM motif of CD79B, CD79A, CD3G, FCGR2C, FCER1G, FCGR2A, CD3D, or CD3E showed particularly noteworthy enrichment in both pools 3B and 3.1B when present in the second intracellular domain (P4) (Table 23).

For libraries 4B and 4.1B, constructs E007/E012-T078-S154-S047, E008/E013-T062-S186-X002, E008/E013-T055-S186-S050, E009/E014-T057-S186-S050, E007/E012-T077-S054-S053, E007/E01 2-T034-S135-S211, E009/E014-T071-X001-S216, E009/E014-T011-S141-S037, E008/E013-T041-S186-S037, E006/E011-T038-S106-S039, E006 /E011-T011-S121-X002、E007/ E012-T007-S085-S215, E006/E011-T041-S186-S050, E007/E012-T008-S064-S051, E006/E011-T041-S186-S047, E008/E013-T045-S186-S051, E0 08/E013-T003-S104-S216, E0 06/E011-T019-S186-S053, E008/E013-T071-S064-S080, E006/E011-T021-S054-X002, E006/E011-T003-S135-X002, E009/E014-T020-S199-S21 3. E008/E013-T027-S121-S211, E009/E014-T032-S195-X002, E009/E014-T050-S171-X002, E008/E013-T069-S083-X002, E008/E013-T026-S116-S038, E009/E014-T072-S195-X0 02. E007/E012-T047-S058-S2 11. E008/E013-T046-S142-S080, E006/E011-T065-S186-S076, E006/E011-T062-S069-X002, E007/E012-T047-S098-X002, E009/E014-T069-S099 -S048, E008/E013-T039-S141 -S050, E006/E011-T052-S130-S052, E008/E013-T041-S186-X002, E007/E012-T019-S120-X002, E008/E013-T045-S186-S053, E006/E011-T003-S 170-S039, E007/E012-T047-S 058-S051, E007/E012-T069-S109-X002, E009/E014-T019-S130-S075, and E006/E011-T047-S054-S053 had particularly notable enrichments in both screens, where the P1 portion separated by slashes here included different markers of libraries 4B and 4.1B, as shown in Tables 7, 17, and 18. For the purposes of the replicate screens, constructs with particularly notable enrichments were those with _log2 ((normalized count data on the last day + 1)/(normalized count data on day 7 + 1)) values greater than 2. Additional information about the first and second intracellular domains in constructs with particularly noteworthy enrichment in the screens for both library 4B and library 4.1B is provided in Table 24, including the genes from which the first and second intracellular domains are derived, whether the first and/or second intracellular domains are cytokine receptors, and whether the first and/or second intracellular domains have at least one ITAM motif. Constructs with an intracellular domain from IL18R1, MPL, CRLF2, IL11RA, IL13RA1, IL2RG, IL7R, IFNGR2, CSF3R, IL2RA, OSMR, MYD88, IL31RA, IFNAR2, IL6R, CSF2RA, IL13RA2, EPOR, IL1R1, IL10RB, or IL3RA when present in the first intracellular domain (P3) and constructs with an intracellular domain from CD27, CD40, CD79B, TNFRSF4, TNFRSF18, CD3D, CD3G, CD27, ICOS, TNFRSF9, CD3E, FCGR2A, CD28, CD79A, or FCGR2C when present in the second intracellular domain (P4) showed particularly noteworthy enrichment in both pools 4B and 4.1B (Table 24). Constructs with an intracellular domain from IL18R1, MPL, CRLF2, IL11RA, IL13RA1, IL2RG, IL7R, IFNGR2, CSF3R, IL2RA, OSMR, IL31RA, IFNAR2, IL6R, CSF2RA, IL13RA2, EPOR, IL1R1, IL10RB, or IL3RA when present in the first intracellular domain (P3) and constructs with an intracellular domain from CD27, CD40, TNFRSF4, TNFRSF18, or TNFRSF9 when present in the second intracellular domain (P4) showed particularly noteworthy enrichment in both pools 4B and 4.1B (Table 24). Constructs with domains containing the ITAM motif of CD79B, CD3D, CD3G, CD3E, FCGR2A, CD79A or FCGR2C showed particularly noteworthy enrichment in both pools 4B and 4.1B when present in the second intracellular domain (P4) (Table 24).

In illustrative embodiments, the CLE comprises an intracellular domain from any one of the constructs in Tables 19 to 23. In some illustrative embodiments, the CLE comprises an intracellular domain from any one of the constructs in Tables 19 to 23 having only a single intracellular domain (the other P3 or P4 is a connector or stop).

Information about various exemplary CLE domains provided herein is found in Table 7. For example, the first CLE identified in Table 8 is M008-S212-S075. For this CLE, the extracellular domain and transmembrane domain (P1-2) are M008, the first intracellular domain (P3) is S212, and the second intracellular domain (P4) is S075. When considering a nucleic acid encoding it, such as M008, detailed information about the identifiers of these domains and modules is found in Table 7. Table 7 reveals that M008 is ECDTM-8, and more specifically an interleukin 7 receptor alpha (IL7RA) mutant with a PPCL insertion and the construct includes an eTag.

For Pool 1A, when found at the first intracellular domain position (P3) of the candidate chimeric polypeptide, the intracellular domains from CD3D, CD3E, CD8A, CD27, CD40, CD79B, IFNAR1, IL2RA, IL3RA, IL13RA2, TNFRSF8, and TNFRSF9, or mutants thereof having signaling activity, promoted proliferation of PBMCs between day 7 and the last day when the data for all constructs having the first intracellular domain (P3) derived from the gene were considered in combination. Thus, exemplary embodiments of CLE provided herein have intracellular domains from CD3D, CD3E, CD8A, CD27, CD40, CD79B, IFNAR1, IL2RA, IL3RA, IL13RA2, TNFRSF8, and TNFRSF9, including mutants thereof that retain signaling activity as the second intracellular domain (or, in illustrative embodiments, the first intracellular domain).

For Pool 1A, when found at the second intracellular domain position (P4) of the candidate chimeric polypeptide, the intracellular domains from CD3D, CD3ζ, CD8A, CD8B, CD27, CD40, CD79B, CRLF2, FCGR2C, ICOS, IL2RA, IL13RA1, IL13RA2, IL15RA, TNFRSF9, and TNFRSF18, or mutants thereof known to have signaling activity, promoted proliferation of PBMCs between day 7 and the last day when the data for all constructs having a second intracellular domain (P4) derived from that gene were considered in combination. Examples of second intracellular domains, or portions thereof and/or mutants present in constructs that promote cell proliferation are provided in the table in Example 11. Thus, exemplary embodiments of CLE provided herein have intracellular domains from CD3D, CD3G, CD8A, CD8B, CD27, CD40, CD79B, CRLF2, FCGR2C, ICOS, IL2RA, IL13RA1, IL13RA2, IL15RA, TNFRSF9, and TNFRSF18, including mutants thereof that retain signaling activity as the first intracellular domain (or in illustrative embodiments, the second intracellular domain).

For Pool 3A, the first intracellular (P3) domains from CSF2RA, IFNAR1, IL1RAP, IL4R, IL6ST, IL11RA, IL12RB2, IL17RA, IL17RD, IL17RE, IL18R1, IL21R, IL23R, MPL, and MyD88, or mutants thereof known to promote signaling activity in certain cell types when these mutants are present in the constructs provided in Example 12, when found at the first intracellular domain (P3) of the candidate chimeric polypeptide components herein, promoted proliferation of PBMCs between day 7 and the last day or between day 7 and day 21 when the data for all constructs having a first intracellular domain derived from the gene were considered in combination. This conclusion is based on the enrichment of sequence counts of constructs in mixed cultured PBMC cell populations, such that when the results for all constructs for a gene are combined, for pool 3A, the enrichment calculated as the base 2 logarithm of the ratio between the normalized counts plus one on the last day indicated on the table and the normalized counts plus one on day 7 is at least 2. For pool 3B, when analyzed in this manner, the first intracellular domains from CSF2RB, IL4R, and MPL, or mutants thereof known to promote signaling activity in certain cell types when these mutants are present in the constructs provided in Example 12, performed best. Examples of first intracellular domains or portions thereof and/or mutants present in constructs that promote cell proliferation for pools 3A, 3B, 3.1A, and 3.1B are provided in Tables 13 to 16.

For library 3A, when found at the second intracellular domain (P4) of the candidate chimeric polypeptide components herein, the second intracellular (P4) domains from CD3D, CD3G, CD27, CD40, CD79A, CD79B, FCER1G, FCGRA2, ICOS, TNFRSF4, and TNFRSF8, or mutants thereof known to promote signaling activity in certain cell types when these mutants are present in the constructs provided in Example 12, promote proliferation of PBMCs between days 7 and 21 or between days 7 and the last day indicated on the table when the data for all constructs having a second intracellular domain derived from the gene are considered in combination. This conclusion is based on the enrichment of the sequence counts of the constructs in the mixed cultured PBMC cell population, such that when the results for all constructs for one gene are combined, for library 3A, the enrichment calculated as the logarithm to the base 2 of the ratio between the normalized counts plus one on the last day indicated on the table and the normalized counts plus one on day 7 is at least 2. Examples of second intracellular domains or portions and/or mutants thereof present in constructs that promote cell proliferation for libraries 3A and 3B are provided in Tables 13-18.

After an initial interim decoding analysis, for library 3A, when found at the first intracellular domain (P3) of the candidate chimeric polypeptide components herein, the first intracellular (P3) domains from IL17RD, IL17RE, IL1RAP, IL23R and MPL, or mutants thereof known to promote signaling activity in certain cell types, promoted the proliferation of PBMCs between days 7 and 21 when the data of all constructs having the first intracellular domain derived from the gene were considered in combination. For library 3B, when analyzed in this manner, the first intracellular domains from IL21R, MPL or OSMR, or mutants thereof known to promote signaling activity in certain cell types, performed best. Examples of first intracellular domains or portions thereof and/or mutants present in constructs that promote cell proliferation for libraries 3A and 3B are provided in the table in Example 12. Thus, exemplary embodiments of CLE provided herein have intracellular domains from IL17RD, IL17RE, IL1RAP, IL23R, and MPL F9, including mutants thereof that retain signaling activity as the second intracellular domain (or in illustrative embodiments, the first intracellular domain).

After an initial decoding analysis, for library 3A, when found at the second intracellular domain (P4) of the candidate chimeric polypeptide components herein, the second intracellular (P4) domains from CD27, CD3ζ, CD40, and CE79B, or mutants thereof known to promote signaling activity in certain cell types, promoted proliferation of PBMCs between the 7th day and the last day indicated on the table when the data of all constructs having a second intracellular domain derived from the gene were considered in a combined manner. For library 3B, when analyzed in this manner, the second intracellular domain from CD40, or mutants thereof known to promote signaling activity in certain cell types, performed best. Thus, exemplary embodiments of CLE provided herein have intracellular domains from CD27, CD3ζ, CD40, and CE79B, including mutants thereof that retain signaling activity as the first intracellular domain (or in illustrative embodiments, the second intracellular domain).

As shown in Tables 13 to 18, when found at the first intracellular domain (P3) of the candidate chimeric polypeptides herein, the first intracellular (P3) domain from MPL, LEPR, MYD88, IFNAR2, or mutants thereof that retain signaling activity in some cases, when considering the first intracellular domain that most frequently appears in the top candidate constructs, promotes proliferation of PBMCs between day 7 and the end of the experiment. Examples of first intracellular domains or portions thereof and/or mutants present in constructs that promote cell proliferation for libraries 3A, 3B, 3.1A, 3.1B, 4B, and 4.1B are provided in the table in Example 12. Thus, exemplary embodiments of CLE provided herein have intracellular domains from MPL, LEPR, MYD88, IFNAR2, including mutants thereof that retain signaling activity as the second intracellular domain (or, in illustrative embodiments, the first intracellular domain).

As shown in Tables 13 to 18, when found at the second intracellular domain (P4) of the candidate chimeric polypeptides herein, the second intracellular (P4) domain from CD40, CD79B, CD27, or in some cases, mutants thereof that retain signaling activity, when considering the first intracellular domain that most frequently appears in the top candidate constructs, promotes proliferation of PBMCs between day 7 and the last day indicated on the table. Examples of first intracellular domains or portions thereof and/or mutants present in constructs that promote cell proliferation for libraries 3A, 3B, 3.1A, 3.1B, 4B, and 4.1B are provided in the table in Example 12. Thus, exemplary embodiments of CLE provided herein have intracellular domains from CD40, CD79B, CD27, including mutants thereof that retain signaling activity as first intracellular domains (or in illustrative embodiments, second intracellular domains).

In illustrative embodiments, the construct of CLE encodes the intracellular domain of MPL at P3 and the intracellular domain of OX40, CD40, CD79A or CD79B at P4. As shown in Example 10, the intracellular domains from these genes showed that when included at P4 of a chimeric lymphoproliferative component containing MPL at P3, at least one of library 3.1 or library 4.1 significantly increased proliferation at a false discovery rate of 0.05. In illustrative embodiments, the construct encodes the intracellular domain of MPL at P3 and the intracellular domain of OX40, CD40 or CD79B at P4. In some embodiments, the construct encodes the sequence of S186 (SEQ ID NO: 491) at P3, or a variant or fragment thereof that promotes signaling, and the construct encodes the sequence of S050, S051, S052, S053, or S211 (SEQ ID NOs: 416 to 419 and 504, respectively), or a variant or fragment thereof that promotes signaling at P4. In illustrative embodiments, the construct of CLE encodes the intracellular domain of CSF2RA at P3 and the intracellular domain of OX40, CD27, or CD30 at P4. As shown in Example 10, the intracellular domains from these genes showed that when included at P4 in a chimeric lymphoproliferative component containing CSF2RA at P3, proliferation was significantly increased in at least one of library 3.1 or library 4.1 at a false discovery rate of 0.1. In other illustrative embodiments, the construct encodes the intracellular domain of CSF2RA at P3 and the intracellular domain of OX40 at P4. As shown in Example 10, the intracellular domain from OX40 exhibits a significant increase in proliferation at a false discovery rate of 0.05 in at least one of library 3.1 or library 4.1 when included at P4 of a chimeric lymphoproliferative component containing CSF2RA at P3. In some embodiments, the construct encodes a sequence of S059 (SEQ ID NO: 423) at P3, or a variant or fragment thereof that promotes signaling, and the construct encodes a sequence of S047 (SEQ ID NO: 413) or S212 (SEQ ID NO: 505) at P4, or a variant or fragment thereof that promotes signaling. In some embodiments, the construct encodes a sequence of S058 (SEQ ID NO: 422) at P3, or a variant or fragment thereof that promotes signaling, and the construct encodes a sequence of S211 (SEQ ID NO: 504) at P4, or a variant or fragment thereof that promotes signaling. In illustrative embodiments, the construct of CLE encodes the intracellular domain of CSF3R at P3 and the intracellular domain of CD79B at P4. As shown in Example 10, the intracellular domain from CD79B showed that when included at P4 of the chimeric lymphoproliferative component containing CSF3R at P3, it significantly increased proliferation at a false discovery rate of 0.05 in at least one of library 3.1 or library 4.1. In some embodiments, the construct encodes the sequence of S064 (SEQ ID NO: 426) at P3 or a variant or fragment thereof that promotes signaling, and the construct encodes the sequence of S053 (SEQ ID NO: 419) at P4, or a variant or fragment thereof that promotes signaling. In illustrative embodiments, the construct of CLE encodes the intracellular domain of IL1RL1 at P3 and the intracellular domain of GITR at P4. As shown in Example 10, the intracellular domain from GITR was shown to significantly increase proliferation at a false discovery rate of 0.1 in at least one of library 3.1 or library 4.1 when included at P4 of a chimeric lymphoproliferative component containing IL1RL1 at P3. In some embodiments, the construct encodes the sequence of S102 (SEQ ID NO: 445) at P3, or a variant or fragment thereof that promotes signaling, and the construct encodes the sequence of S216 (SEQ ID NO: 509) at P4, or a variant or fragment thereof that promotes signaling. In an illustrative embodiment, the construct of CLE encodes the intracellular domain from IL5RA at P3 and the intracellular domain from deltaLck CD28 at P4. As shown in Example 10, the intracellular domain from mutant delta Lck CD28 was shown to significantly increase proliferation at a false discovery rate of 0.05 in at least one of library 3.1 or library 4.1 when included at P4 of a chimeric lymphoproliferative component containing IL5RA at P3. In some embodiments, the construct encodes the sequence of S115 (SEQ ID NO: 453) at P3, or a variant or fragment thereof that promotes signaling, and the construct encodes the sequence of S048 (SEQ ID NO: 414) at P4, or a variant or fragment thereof that promotes signaling. In an illustrative embodiment, the construct of CLE encodes the intracellular domain from IL11RA at P3 and the intracellular domain from FCGRA2 at P4. As shown in Example 10, the intracellular domain from FCGRA2 showed that when included at P4 of a chimeric lymphoproliferative component containing IL11RA at P3, it significantly increased proliferation at a false discovery rate of 0.05 in at least one of library 3.1 or library 4.1. In some embodiments, the construct encodes the sequence of S135 (SEQ ID NO: 461) at P3 or a variant or fragment thereof that promotes signaling, and the construct encodes the sequence of S076 (SEQ ID NO: 431) at P4 or a variant or fragment thereof that promotes signaling. In an illustrative embodiment, the construct of CLE encodes the intracellular domain from LIFR at P3 and the intracellular domain from GITR at P4. As shown in Example 10, the intracellular domain from GITR showed that when included at P4 of a chimeric lymphoproliferative component containing LIFR at P3, it significantly increased proliferation at a false discovery rate of 0.05 in at least one of library 3.1 or library 4.1. In some embodiments, the construct encodes the sequence of S180 (SEQ ID NO: 489) at P3 or a variant or fragment thereof that promotes signaling, and the construct encodes the sequence of S216 (SEQ ID NO: 509) at P4 or a variant or fragment thereof that promotes signaling. In illustrative embodiments, the construct of CLE encodes the intracellular domain of MyD88 from P3 and the intracellular domain of CD3D from P4. As shown in Example 10, the intracellular domain from CD3D showed that when included at P4 of the chimeric lymphoproliferative component containing MyD88 at P3, at least one of library 3.1 or library 4.1 significantly increased proliferation at a false discovery rate of 0.1. In some embodiments, the construct encodes the sequence of S194 (SEQ ID NO: 497) at P3 or a variant or fragment thereof that promotes signaling, and the construct encodes the sequence of S037 (SEQ ID NO: 405) at P4 or a variant or fragment thereof that promotes signaling.

In illustrative embodiments, the construct of CLE encodes the intracellular domains of two genes from the following gene pairs, or variants or fragments thereof: CSF2RA and TNFRSF4; CSF2RA and CD28; CSF2RA and TNFRSF8; CSF2RA and CD27; CSFR3 and CD79B; IFNAR2 and TNFRSF14; IL1RAP and CD79A; IL3RA and CD40; IL10RA and CD79B; IL11RA and FCGRA2; IL13RA2 and TNFRSF14; IL18RAP and CD3G; IL27RA and FCGRA2; LEPR and CD3G; LIFR and TNFRSF18; MPL and CD40; MPL and CD79B; MPL and TNFRSF4; MPL and CD3G; MyD88 and CD79B; and MyD88 and CD3D. At a p of less than 0.1, portions encoding the intracellular domains from these pairs of genes were the most effective in Example 10 (Table 3). In some embodiments, the construct of CLE encodes the intracellular domains or variants or fragments thereof from two of the following pairs of genes: CSF2RA and TNFRSF4; CSF2RA and CD28; CSFR3 and CD79B; IFNAR2 and TNFRSF14; IL10RA and CD79B; IL11RA and FCGRA2; IL27RA and FCGRA2; LIFR and TNFRSF18; MPL and CD40; MPL and CD40; MPL and CD79B; MPL and TNFRSF4; MPL and CD3G; and MyD88 and CD79B. At a p of less than 0.05, portions encoding the intracellular domains from these pairs of genes were the most effective pairs in Example 10 (Table 3). In some embodiments, the construct of CLE encodes sequences from two of the following pairs of portions (as shown in Table 7) or variants or fragments thereof: S058 and S211; S058 and S049; S059 and S212; S059 and S047; S064 and S053; S083 and S214; S101 and S052; S109 and S050; S129 and S053; S 135 and S076; S142 and S214; S155 and S039; S169 and S076; S177 and S039; S180 and S216; S186 and S050; S186 and S051; S186 and S053; S186 and S211; S186 and S039; S192 and S053; S194 and S037; and S195 and S053. At p less than 0.1, these pairs of moieties were the most effective moieties in Example 10 (Table 3). In some embodiments, the construct of CLE encodes sequences from two of the following pairs of moieties (as shown in Table 7) or variants or fragments thereof: S058 and S211; S058 and S049; S064 and S053; S083 and S214; S129 and S053; S135 and S076; S169 and S076; S180 and S216; S186 and S050; S186 and S051; S186 and S053; S186 and S211; S186 and S039; and S195 and S053. At p less than 0.05, these pairs of moieties were the most effective moieties in Example 10 (Table 3).

In the notable embodiments of any of the isolated polypeptides or the vector aspects and embodiments provided herein comprising a first nucleic acid sequence encoding a chimeric polypeptide that promotes cell proliferation of PBMC, B cells, T cells and/or NK cells (i.e., CLE) and a second nucleic acid sequence encoding a chimeric antigen receptor (CAR) comprising an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain, the first nucleic acid sequence and the second nucleic acid sequence may be separated by a ribosomal skipping sequence. In some embodiments, the ribosomal skipping sequence is F2A, E2A, P2A, or T2A.

As disclosed herein, a lymphoproliferative component (as exemplified herein with CLE) may include a dimerization motif in some illustrative embodiments to help enhance and/or modulate its activity, for example to affect a signal in a cell, such as a signal that affects protein activity or gene expression. This dimerization motif is typically part or all of the extracellular domain thereof. In some embodiments, the dimerization moiety may be linked to the recognition and interstitial sequences of the extracellular domain of CLE herein. Indeed, in some embodiments, a lymphoproliferative component may include the entire lymphoproliferative component from a protein, except that the extracellular domain includes a dimerization moiety.

In some embodiments of lymphoproliferative components and CLEs including dimerizing agents and polynucleotides and nucleic acids encoding them, the dimerization motif may include an amino acid sequence from a transmembrane homodimeric polypeptide that naturally exists as a homodimer. For example, the dimerization motif may be a leucine zipper polypeptide, such as a Jun polypeptide, as exemplified in Example 12 herein. In some embodiments, these transmembrane homodimeric polypeptides may include early activation antigen CD69 (CD69), transfer receptor protein 1 (CD71), B cell differentiation antigen (CD72), T cell surface protein touch (CD96), endoglin (Cd105), killer cell lectin-like receptor subfamily B member 1 (Cd161), P-selectin glycoprotein ligand 1 (Cd162), glutamyl aminopeptidase (Cd249), tumor necrosis factor receptor superfamily member 16 (CD271), cadherin-1 (E-cadherin) (Cd324) or an active fragment thereof. In some embodiments, the dimerization motif may include an amino acid sequence from a transmembrane protein that dimerizes after binding of a ligand (also referred to herein as a dimer or dimerizer). In some embodiments, the dimerization motif and dimer may include (wherein the dimer is in parentheses after the dimer binding pair): FKBP and FKBP (rapamycin); GyrB and GyrB (coumermycin); DHFR and DHFR (methotrexate); or DmrB and DmrB (AP20187). As mentioned above, rapamycin may be used as a dimer. Alternatively, a rapamycin derivative or analog may be used (see, e.g., WO96/41865, WO 99/36553, WO 01/14387; and Ye et al. (1999) Science 283: 88-91). For example, analogs, homologues, derivatives and other compounds structurally related to rapamycin ("rapamycin analogs") include, among other things, variants of rapamycin with one or more of the following modifications related to rapamycin: demethylation, elimination or replacement of the methoxy group at C7, C42 and/or C29; elimination, derivatization or replacement of the hydroxyl group at C13, C43 and/or C28; reduction, elimination or derivatization of the ketone at C14, C24 and/or C30; replacement of the 6-membered methylpiperidine ring with a 5-membered prolyl ring; and substitutional substitution of the cyclohexyl ring or replacement of the cyclohexyl ring with a substituted cyclopentyl ring. Additional information is presented in, for example, U.S. Pat. Nos. 5,525,610, 5,310,903, 5,362,718 and 5,527,907. Selective epimerization of the C-28 hydroxyl group is described (see, for example, WO 01/14387). Additional synthetic dimerizing agents suitable for use as substitutes for rapamycin include those described in U.S. Patent Publication No. 2012/0130076. As mentioned above, coumermycin can be used as a dimerizing agent. Alternatively, coumermycin analogs (see, e.g., Farrar et al. (1996) Nature 383: 178-181; and U.S. Patent No. 6,916,846) can be used. As mentioned above, in some cases, the dimerizing agent is methotrexate, such as non-cytotoxic, homologous bifunctional methotrexate dimers (see, e.g., U.S. Patent No. 8,236,925).

In some embodiments, a lymphoproliferative assembly (in an illustrative embodiment, CLE) comprising a dimerization motif may be active in the absence of a dimerizing agent when present in the plasma membrane of a eukaryotic cell. For example, a lymphoproliferative assembly comprising a dimerization motif comprising a leucine zipper or comprising a dimerization motif from a transmembrane homodimeric polypeptide may be active in the absence of a dimerizing agent, wherein the transmembrane homodimeric polypeptide includes CD69, CD71, CD72, CD96, Cd105, Cd161, Cd162, Cd249, CD271, Cd324, active mutants thereof, and/or active fragments thereof. In some embodiments, a lymphoproliferative assembly comprising a dimerization motif may be active in the presence of a dimerizing agent when present in the plasma membrane of a eukaryotic cell. For example, a lymphoproliferative component including a dimerization motif from FKBP, GyrB, DHFR, or DmrB can be active in the presence of a respective dimerizing agent or analog thereof (eg, rapamycin, coumermycin, methotrexate, and AP20187), respectively.

In the method embodiments including a lymphoproliferative element (e.g., CLE) including a dimer for dimerizing a polypeptide comprising a dimerization motif, the dimer can be administered to the host (subject) using convenient means capable of producing the desired effect. Thus, the dimer can be incorporated into a variety of formulations for administration. More specifically, the dimer can be formulated into a pharmaceutical composition by combining with a suitable pharmaceutically acceptable carrier or diluent, and can be formulated into preparations in solid, semisolid, liquid or gaseous form, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants and aerosols.

A skilled artisan will readily appreciate that dosage levels may vary depending on the particular dimer, the severity of the symptoms, and the individual's sensitivity to side effects. The optimal dosage for a given compound can be readily determined by a skilled artisan using a variety of means.

The dimer is administered to a subject using any available method and route suitable for drug delivery, including in vivo or ex vivo methods as well as systemic and topical routes of administration.

In any aspect or embodiment in which the extracellular domain of CLE comprises a dimerization motif, the dimerization motif may be selected from the group consisting of: polypeptides containing a leucine zipper motif, CD69, CD71, CD72, CD96, Cd105, Cd161, Cd162, Cd249, CD271, and Cd324, and mutants and/or active fragments thereof that retain dimerization ability. In any of the aspects or embodiments in which the extracellular domain of CLE comprises a dimerization motif, the dimerization motif may require a dimerizer, and the dimerization motif and the related dimerizer may be selected from the group consisting of: FKBP and rapamycin or an analog thereof, GyrB and coumermycin or an analog thereof, DHFR and methotrexate or an analog thereof, or DmrB and AP20187 or an analog thereof, and mutants and/or active fragments of the dimerization proteins that retain dimerization ability.

In illustrative embodiments of any aspect or embodiment in which the extracellular domain of CLE comprises a dimerization motif, the extracellular domain may comprise a leucine zipper motif. In some embodiments, the leucine zipper motif is from a jun polypeptide, such as c-jun. In certain embodiments, the c-jun polypeptide is a c-jun polypeptide region of ECD-11.

In any of the aspects or embodiments provided herein that include a lymphoproliferative component, the lymphoproliferative component can be a polypeptide comprising a membrane targeting region, a dimerization (multimerization) domain, a first intracellular domain, and an optional second intracellular domain, and an optional third intracellular domain, and an optional fourth intracellular domain, wherein the first intracellular domain and the optional second intracellular domain, the third intracellular domain, and the fourth intracellular domain are from any gene having an intracellular domain identified in Tables 8 to 18, or are selected from any of the first intracellular domain and the second intracellular domain identified in Tables 8 to 18, and wherein the internal dimerization and/or multimerization lymphoproliferative component promotes cell proliferation of PBMCs (and in illustrative embodiments, B cells, T cells, and/or NK cells). This polypeptide may be referred to herein as an internal dimerization and/or multimerization lymphoproliferative component. In certain embodiments, a connector may be added between any of the domains. In certain embodiments, the first intracellular domain and the second intracellular domain are MyD88 and CD40. In certain embodiments, the first intracellular domain and the second intracellular domain are not MyD88 and CD40. In some embodiments, the intracellular domain is MPL or an intracellular fragment thereof that retains the ability to promote PBMC cell proliferation. In some embodiments, the intracellular domain is not MPL or an intracellular fragment thereof that retains the ability to promote PBMC cell proliferation. In some embodiments, the intracellular domain is IL2Rb, or an intracellular fragment thereof that retains the ability to promote PBMC cell proliferation. In some embodiments, the intracellular domain is not IL2Rb, or an intracellular fragment thereof that retains the ability to promote PBMC cell proliferation. In some embodiments, the intracellular domain is IL2Ra, or an intracellular fragment thereof that retains the ability to promote PBMC cell proliferation. In some embodiments, the intracellular domain is not IL2Ra, or an intracellular fragment thereof that retains the ability to promote PBMC cell proliferation. In some embodiments, the intracellular domain is MyD88, or an intracellular fragment thereof that retains the ability to promote PBMC cell proliferation. In some embodiments, the intracellular domain is not MyD88 or an intracellular fragment thereof that retains the ability to promote PBMC cell proliferation.

The dimerization domain of this internal dimerization and/or multimerization polypeptide lymphoproliferative component in the illustrative embodiment can be located between the membrane targeting region and the first intracellular domain or after the intracellular domain (see, e.g., Spencer et al. J Clin Invest. 2011 April; 121(4): 1524-34) or between intracellular domains. In certain embodiments, the dimerization (or multimerization) domain comprises a multimerization or dimerization ligand binding site, such as a FKBP region, e.g., FKBP12. In some embodiments, the polypeptide may comprise an extracellular domain, such as any of the extracellular domains provided herein, e.g., in Tables 8 to 12 of Example 11 or in Tables 13 to 18 of Example 12, with or without a recognition domain and a gap domain. In certain embodiments, the membrane targeting region is selected from the group consisting of a myristoylated region, a palmitoylated region, a prenylated region, and a transmembrane sequence of a receptor. In certain embodiments, the membrane targeting region is a myristoylated region. The internal dimeric and/or multimeric lymphoproliferative components are typically attached to the plasma membrane via a membrane targeting region, for example, with an N-terminal myristate. In illustrative embodiments, the FKBP region binds FK506.

The internal dimerization and/or multimerization lymphoproliferative component in one embodiment is an integral part of a system using an analog of the lipid permeable dimeric immunosuppressant drug FK506, which loses its normal biological activity while gaining the ability to crosslink molecules genetically fused to the FK506 binding protein FKBP12. By fusing one or more FKBPs and myristoylation sequences to the cytoplasmic signaling domain of the target receptor, we can stimulate signaling in a dimer drug-dependent but ligand and ectodomain-independent manner. This provides temporal control, reversibility using monomeric drug analogs, and enhanced specificity to the system. The high affinity of the third generation AP20187/AP1903 dimer drug for its binding domain FKBP12 allows for specific activation of the recombinant receptor in vivo without inducing nonspecific side effects via endogenous FKBP12. FKBP12 variants with amino acid substitutions and deletions (such as FKBP12V36) that bind to the dimer drug can also be used. Additionally, the synthetic ligands are resistant to protease degradation, making them more effective than most delivered protein agents in activating the receptor in vivo.

Pseudotyped components

Many of the methods and compositions provided herein include pseudotype components. Pseudotyping of replication-deficient recombinant retroviral particles with heterologous envelope glycoproteins generally changes the tropism of the virus and facilitates transduction of host cells. As used herein, pseudotype components may include: "binding polypeptides", which include one or more polypeptides (usually glycoproteins) that recognize and bind to target host cells; and one or more "fusion-promoting polypeptides", which mediate fusion of the retrovirus with the target host cell membrane, thereby allowing the retroviral genome to enter the target host cell. In some embodiments provided herein, the pseudotype component is provided as a polypeptide/protein, or is provided as a nucleic acid sequence encoding a polypeptide/protein.

In some embodiments, the pseudotyping component is a feline endogenous virus (RD114) envelope protein, an amphotropic envelope protein, an ecotropic envelope protein, a herpes oral virus envelope protein (VSV-G), a baboon reverse transcriptase envelope glycoprotein (BaEV), a murine leukemia virus envelope protein (MuLV), and/or paramyxovirus measles envelope proteins H and F.

In some embodiments, the pseudotyped component may be wild-type BaEV. Without being limited by theory, BaEV contains an R peptide that is shown to inhibit transduction. In some embodiments, BaEV may contain a deletion of the R peptide. In some embodiments, BaEV may contain a deletion of the inhibitory R peptide after the nucleotide encoding the amino acid sequence HA (referred to herein as BaEVΔR (HA)). In some embodiments, BaEV may contain a deletion of the inhibitory R peptide after the nucleotide encoding the amino acid sequence HAM (referred to herein as BaEVΔR (HAM)).

In some embodiments, the pseudotyping component includes a binding polypeptide and a fusion polypeptide derived from different proteins. For example, the replication-defective recombinant retroviral particles in the methods and compositions disclosed herein can be pseudotyped by the fusion (F) polypeptide and the hemagglutinin (H) polypeptide of the measles virus (MV), as non-limiting examples, a clinical wild-type strain of MV, and a vaccine strain including the Edmonston strain (MV-Edm) or a fragment thereof. Without being limited by theory, it is believed that both the hemagglutinin (H) and fusion (F) polypeptides can play a role in entry into host cells, wherein the H protein binds MV to receptors CD46, SLAM, and Nectin-4 on the target cell, and F mediates fusion of the retroviral and host cell membranes. In an illustrative embodiment, particularly when the target cell is a T cell and/or a NK cell, the binding polypeptide is a measles virus H polypeptide, and the fusion polypeptide is a measles virus F polypeptide.

In some studies, lentiviral particles pseudotyped with truncated F and H polypeptides showed significant increases in titer and transduction efficiency (Funke et al. 2008. Molecular Therapy. 16(8):1427-1436), (Frecha et al. 2008. Blood. 112(13):4843-4852). The highest titer was obtained when the F cytoplasmic tail was truncated by 30 residues (also known as MV(Ed)-FΔ30 (SEQ ID NO:105)). For the H variant, the best truncation occurred when 18 or 19 residues were deleted (MV(Ed)-HΔ18 (SEQ ID NO: 106) or (MV(Ed)-HΔ19)), but variants with a 24-residue truncation also produced the best titers with and without alanine substitution of the deleted residue (MV(Ed)-HΔ24 (SEQ ID NO: 235) and MV(Ed)-HΔ24+A).

In some embodiments, including those directed to transduction of T cells and/or NK cells, the replication-defective recombinant retroviral particles in the methods and compositions disclosed herein are pseudotyped with mutant or variant versions of the measles virus fusion (F) polypeptide and hemagglutinin (H) polypeptide (in illustrative examples, cytoplasmic domain deletion variants of the measles virus F and H polypeptides). In some embodiments, the mutated F and H polypeptides are "truncated H" or "truncated F" polypeptides, the cytoplasmic portion of which has been truncated, i.e., amino acid residues (or encoding nucleic acids of the corresponding nucleic acid molecules encoding the protein) have been deleted. "HΔY" and "FΔX" represent such truncated H and F polypeptides, respectively, wherein "Y" refers to 1 to 34 residues that have been deleted from the amino terminus, and "X" refers to 1 to 35 residues that have been deleted from the carboxyl terminus of the cytoplasmic domain. In another embodiment, the "truncated F polypeptide" is FΔ24 or FΔ30 and/or the "truncated H protein" is selected from the group consisting of: HΔ14, HΔ15, HΔ16, HΔ17, HΔ18, HΔ19, HΔ20, HΔ21+A, HΔ24 and HΔ24+4A, more preferably HΔ18 or HΔ24. In an illustrative embodiment, the truncated F polypeptide is MV(Ed)-FΔ30, and the truncated H polypeptide is MV(Ed)-HΔ18.

In some embodiments, the fusion polypeptide includes multiple components expressed as a polypeptide. In some embodiments, the binding polypeptide and the fusion polypeptide are translated from the same transcript but from a separate ribosome binding site; in other embodiments, the binding polypeptide and the fusion polypeptide are separated by a cleavage peptide site (which is not limited by theory, cleaved after translation, as is common in the literature) or a ribosome skipping sequence. In some embodiments, the translation of the binding polypeptide and the fusion polypeptide from the separated ribosome binding site produces a higher amount of fusion polypeptide than the binding polypeptide. In some embodiments, the ratio of fusion polypeptide to binding polypeptide is at least 2: 1, at least 3: 1, at least 4: 1, at least 5: 1, at least 6: 1, at least 7: 1 or at least 8: 1. In some embodiments, the ratio of fusion polypeptide to binding polypeptide is between the low end of the range of 1.5: 1, 2: 1 or 3: 1 and the high end of the range of 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1 or 10: 1.

Activation components

Many of the methods and composition aspects of the present invention include an activation component (also referred to herein as a T cell activation component), or a nucleic acid encoding an activation component. Restrictions associated with lentiviral (LV) transduction into resting T cells are attributed to a series of pre- and post-entry barriers and cell restriction factors (Strebel et al. 2009. BMC Medicine 7: 48). One limitation is that envelope pseudotyped LV particles cannot recognize potential receptors and mediate fusion with cell membranes. However, under certain conditions, transduction of resting T cells by HIV-1-based lentiviral vectors is most likely after T cell receptor (TCR) CD3 complex and CD28 co-stimulation (Korin and Zack. 1998. Journal of Virology. 72: 3161-8, Maurice et al. 2002. Blood 99: 2342-50), and via exposure to cytokines (Cavalieri et al. 2003).

The cells of the immune system (such as T lymphocytes) recognize and interact with specific antigens via receptors or receptor complexes, and when recognizing or interacting with such antigens, the cells are activated and expanded in vivo. An example of this receptor is an antigen-specific T lymphocyte receptor complex (TCR/CD3). T cell receptors (TCR) are expressed on the surface of T lymphocytes. A component (CD3) is responsible for intracellular signaling after TCR is occupied by ligands. The T lymphocyte receptor for antigen CD3 complex (TCR/CD3) recognizes antigenic peptides presented to it by proteins of major tissue compatibility complex (MHC). The complex of MHC and peptide is expressed on the surface of antigen presenting cells and other T lymphocyte targets. The stimulation of TCR/CD3 complexes leads to the activation of T lymphocytes and subsequent antigen-specific immune responses. TCR/CD3 complexes play an important role in the effector function and regulation of the immune system. Therefore, the activation assembly provided herein is combined with one or more components of the T cell receptor-related complex, for example, by combining with CD3 to activate T cells. In some cases, the activation assembly can be activated alone. In other cases, activation requires activation via the TCR receptor complex in order to further activate the cell.

T lymphocytes also require a second, co-stimulatory signal to become fully active in vivo. Without this signal, T lymphocytes are unresponsive to antigens bound to the TCR, or become anergic.

However, the transduction and amplification of T cells do not require a second co-stimulatory signal. This co-stimulatory signal is provided, for example, by CD28, a T lymphocyte protein that interacts with CD80 and CD86 on antigen-producing cells. As used herein, the functional extracellular fragment of CD80 retains its ability to interact with CD28. OX40, 4-1BB and ICOS (inducible co-stimulators) (other T lymphocyte proteins) provide co-stimulatory signals when bound to their corresponding ligands OX40L, 4-1BBL and ICOSLG.

Activation of the T cell receptor (TCR) CD3 complex and co-stimulation through CD28 can occur by ex vivo exposure to a solid surface (e.g., beads) coated with anti-CD3 and anti-CD28. In some embodiments of the methods and compositions disclosed herein, resting T cells are activated by exposure to a solid surface coated with anti-CD3 and anti-CD28 ex vivo. In other embodiments, resting T cells or NK cells (T cells in illustrative embodiments) are activated by exposure to soluble anti-CD3 antibodies (e.g., at 50 ng/ml to 150 ng/ml, or 75 ng/ml to 125 ng/ml, or 100 ng/ml). In this embodiment, it can be part of a method for genetically modifying or transducing (without prior activation in illustrative embodiments), and such activation and/or contacting can be performed for, for example, 8 hours or less, 4 hours or less, or between 2 hours and 8 hours, between 2 hours and 4 hours, or between 2 hours and 3 hours.

In certain illustrative embodiments of the methods and compositions provided herein, polypeptides capable of binding CD3 and/or CD28 are presented as "activation components" on the surface of replication-deficient recombinant retroviral particles in the methods and compositions disclosed herein, which are also aspects of the present invention. In some embodiments, the activation components on the surface of replication-deficient recombinant retroviral particles may include one or more polypeptides capable of binding OX40, 4-1BB or ICOS. In some embodiments, the activation component may be a T cell surface protein agonist. The activation component may include a co-stimulatory polypeptide that serves as a ligand for a T cell surface protein. In some embodiments, the co-stimulatory polypeptide may include one or more of OX40L, 4-1BBL or ICOSLG. In some embodiments, one or more copies of these activation components may be expressed on the surface of replication-deficient recombinant retroviral particles as polypeptides separated and different from the pseudotyped components. In some embodiments, the activation component may be expressed as a fusion polypeptide on replication-deficient recombinant retroviral particles. In illustrative embodiments, the fusion polypeptide includes one or more activation components and one or more pseudotyped components. In other illustrative embodiments, the fusion polypeptide includes anti-CD3 and viral envelope proteins, such as OKT-3scFv fused to the amino terminus of MuLV envelope protein, as shown in Maurice et al. (2002). Because of its ability to activate resting T cells, polypeptides that bind CD3, CD28, OX40, 4-1BB or ICOS are referred to as activation components. In certain embodiments, nucleic acids encoding this activation component are found in the genome of replication-defective recombinant retroviral particles containing activation components on their surface. In other embodiments, nucleic acids encoding activation components are not found in replication-defective recombinant retroviral particle genomes. In yet other embodiments, nucleic acids encoding activation components are found in the genome of viral packaging cells.

In some embodiments, the activation component is a polypeptide capable of binding to CD3. In some embodiments, the polypeptide capable of binding to CD3 is an anti-CD3 antibody, or a fragment thereof that retains the ability to bind to CD3. In an illustrative embodiment, the anti-CD3 antibody or its fragment is a single-chain anti-CD3 antibody, such as but not limited to an anti-CD3 scFv. In another illustrative embodiment, the polypeptide capable of binding to CD3 is an anti-CD3 scFv Fc.

A large number of anti-human CD3 monoclonal antibodies and antibody fragments thereof are available and can be used in the present invention, including but not limited to UCHT1, OKT-3, HIT3A, TRX4, X35-3, VIT3, BMA030 (BW264/56), CLB-T3/3, CRIS7, YTH12.5, F111409, CLB-T3.4.2, TR-66, WT31, WT32, SPv-T3b, 11D8, XIII-141, XIII46, XIII-87, 12F6, T3/RW2-8C8, T3/RW24B6, OKT3D, M-T301, SMC2, and F101.01.

In some embodiments, the activation component is a polypeptide capable of binding to CD28. In some embodiments, the polypeptide capable of binding to CD28 is an anti-CD28 antibody, or a fragment thereof that retains the ability to bind to CD28. In other embodiments, the polypeptide capable of binding to CD28 is CD80, CD86, or a fragment thereof that can bind to CD28 and induce CD28-mediated activation of Akt, such as an external fragment of CD80. In some aspects herein, an external fragment of CD80 means a fragment that is usually present outside the cell in the standard cell location of CD80 that retains the ability to bind to CD28. In an illustrative embodiment, an anti-CD28 antibody or a fragment thereof is a single-chain anti-CD28 antibody, such as, but not limited to, an anti-CD28 scFv. In another illustrative embodiment, the polypeptide capable of binding to CD28 is CD80, or a fragment of CD80, such as an external fragment of CD80.

Anti-CD28 antibodies are known in the art and may include, as non-limiting examples, monoclonal antibody 9.3, an IgG2a antibody (Dr. Jeffery Ledbetter, Bristol Myers Squibb Corporation, Seattle, Wash.), monoclonal antibody KOLT-2 (IgG1 antibody), 15E8 (IgG1 antibody), 248.23.2 (IgM antibody), and EX5.3D10 (IgG2a antibody).

In one illustrative embodiment, the activation component includes two polypeptides, a polypeptide capable of binding to CD3 and a polypeptide capable of binding to CD28.

In certain embodiments, the polypeptide capable of binding to CD3 or CD28 is an antibody (single-chain monoclonal antibody) or an antibody fragment (e.g., a single-chain antibody fragment). Thus, the antibody fragment may be, for example, a single-chain fragment variable region (scFv), an antibody binding (Fab) fragment of an antibody, a single-chain antigen binding fragment (scFab), a single-chain antigen binding fragment without cysteine (scFabΔC), a fragment variable region (Fv), a structure specific for an adjacent antigenic determinant of an antigen (CRAb), or a single domain antibody (VH or VL).

In any of the embodiments disclosed herein, the activation component or the nucleic acid encoding it may include a dimerization or higher polymerizing motif. Dimerization or polymerizing motifs are known in the art and those skilled in the art will understand how to incorporate them into polypeptides for effective dimerization or multimerization. For example, in some embodiments, the activation component including a dimerization motif may be one or more polypeptides capable of binding to CD3 and/or CD28. In some embodiments, the polypeptide capable of binding to CD3 is an anti-CD3 antibody or a fragment thereof that retains the ability to bind to CD3. In an illustrative embodiment, the anti-CD3 antibody or its fragment is a single-chain anti-CD3 antibody, such as, but not limited to, an anti-CD3 scFv. In another illustrative embodiment, the polypeptide capable of binding to CD3 is an anti-CD3 scFv Fc, which in some embodiments is considered to have a dimerization motif but does not have any additional dimerization motifs of anti-CD3, because it is known that the anti-CD3 scFv Fc construct is capable of dimerization without the need for a separate dimerization motif.

In some embodiments, the dimerization or multimerization motif or the nucleic acid sequence encoding it may be an amino acid sequence from a transmembrane polypeptide naturally present as a homodimer or multimer. In some embodiments, the dimerization or multimerization motif or the nucleic acid sequence encoding it may be an amino acid sequence from a fragment of a natural protein or an engineered protein. In one embodiment, the homodimeric polypeptide is a polypeptide containing a leucine zipper motif (leucine zipper polypeptide). For example, the leucine zipper is derived from c-JUN, a non-limiting example of which is disclosed as being related to the chimeric lymphoproliferative element (CLE) herein.

In some embodiments, these transmembrane homodimeric polypeptides may include early activation antigen CD69 (CD69), transfer receptor protein 1 (CD71), B cell differentiation antigen (CD72), T cell surface protein touch (CD96), endoglin (Cd105), killer cell lectin-like receptor subfamily B member 1 (Cd161), P-selectin glycoprotein ligand 1 (Cd162), glutamyl aminopeptidase (Cd249), tumor necrosis factor receptor superfamily member 16 (CD271), cadherin-1 (E-cadherin) (Cd324) or active fragments thereof. In some embodiments, the dimerization motif and the nucleic acid encoding it may include an amino acid sequence from a transmembrane protein that dimerizes after binding of a ligand (also referred to herein as a dimer or dimerizer). In some embodiments, the dimerization motif and dimer may include (wherein the dimer is in parentheses after the dimer binding pair): FKBP and FKBP (rapamycin); GyrB and GyrB (coumermycin); DHFR and DHFR (methotrexate); or DmrB and DmrB (AP20187). As mentioned above, rapamycin can be used as a dimer. Alternatively, a rapamycin derivative or analog can be used (see, e.g., WO96/41865, WO 99/36553, WO 01/14387; and Ye et al. (1999) Science 283: 88-91). For example, analogs, homologues, derivatives and other compounds structurally related to rapamycin ("rapamycin analogs") include, among other things, variants of rapamycin with one or more of the following modifications related to rapamycin: demethylation, elimination or replacement of the methoxy group at C7, C42 and/or C29; elimination, derivatization or replacement of the hydroxyl group at C13, C43 and/or C28; reduction, elimination or derivatization of the ketone at C14, C24 and/or C30; replacement of the 6-membered methylpiperidine ring with a 5-membered prolyl ring; and substitutional substitution of the cyclohexyl ring or replacement of the cyclohexyl ring with a substituted cyclopentyl ring. Additional information is presented in, for example, U.S. Pat. Nos. 5,525,610, 5,310,903, 5,362,718 and 5,527,907. Selective epimerization of the C-28 hydroxyl group is described (see, for example, WO 01/14387). Additional synthetic dimerizing agents suitable for use as substitutes for rapamycin include those described in U.S. Patent Publication No. 2012/0130076. As mentioned above, coumermycin can be used as a dimerizing agent. Alternatively, coumermycin analogs (see, e.g., Farrar et al. (1996) Nature 383: 178-181; and U.S. Patent No. 6,916,846) can be used. As mentioned above, in some cases, the dimerizing agent is methotrexate, such as non-cytotoxic, homologous bifunctional methotrexate dimers (see, e.g., U.S. Patent No. 8,236,925).

In some embodiments, when present on the surface of a replication-deficient recombinant retroviral particle, an activation assembly comprising a dimerization motif may be active in the absence of a dimerizing agent. For example, an activation assembly comprising a dimerization motif from a transmembrane homodimeric polypeptide may be active in the absence of a dimerizing agent, wherein the transmembrane homodimeric polypeptide includes CD69, CD71, CD72, CD96, Cd105, Cd161, Cd162, Cd249, CD271, Cd324, active mutants thereof, and/or active fragments thereof. In some embodiments, the activation assembly may be an anti-CD3 single-chain fragment and include a dimerization motif selected from the group consisting of CD69, CD71, CD72, CD96, Cd105, Cd161, Cd162, Cd249, CD271, Cd324, active mutants thereof, and/or active fragments thereof.

In some embodiments, when present on the surface of a replication-defective recombinant retroviral particle, an activation component comprising a dimerization motif may be active in the presence of a dimerizing agent. For example, an activation component comprising a dimerization motif from FKBP, GyrB, DHFR, or DmrB may be active in the presence of a respective dimerizing agent or an analog thereof (e.g., rapamycin, coumermycin, methotrexate, and AP20187), respectively. In some embodiments, the activation component may be a single-chain antibody fragment against anti-CD3 or anti-CD28 or another molecule that binds CD3 or CD28, and the dimerization motif and the dimerizing agent may be selected from the group consisting of FKBP and rapamycin or an analog thereof, GyrB and coumermycin or an analog thereof, DHFR and methotrexate or an analog thereof, or DmrB and AP20187 or an analog thereof.

In some embodiments, the activation assembly is fused to a heterologous signal sequence and/or a heterologous membrane adhesion sequence, both of which help guide the activation assembly to the membrane. The heterologous signal sequence targets the activation assembly to the endoplasmic reticulum, wherein the heterologous membrane adhesion sequence is covalently linked to one or more fatty acids (also referred to as post-translational lipid modification) such that the activation assembly fused to the heterologous membrane adhesion sequence is anchored in the lipid rafts of the plasma membrane. In some embodiments, post-translational lipid modification can occur via myristoylation, palmitoylation or GPI anchoring. Myristoylation is a post-translational protein modification corresponding to the covalent attachment of a 14-carbon saturated fatty acid (myristic acid) to the N-terminal glycine of a eukaryotic or viral protein. Palmitoylation is a post-translational protein modification corresponding to a C16 acyl chain and cysteine, and less to the covalent attachment of serine and threonine residues of a protein. GPI anchoring refers to the connection of glycosylphosphatidylinositol or GPI to the C-terminal of a protein during post-translational modification.

In some embodiments, the heterologous membrane linking sequence is a GPI anchor linking sequence. The heterologous GPI anchor linking sequence can be derived from any known GPI-anchored protein (reviewed in Ferguson MAJ, Kinoshita T, Hart GW. Glycosylphosphatidylinositol Anchors. In: Varki A, Cummings RD, Esko JD et al., ed. Essentials of Glycobiology. 2nd edition. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 2009. Chapter 11). In some embodiments, the heterologous GPI anchor linking sequence is a GPI anchor linking sequence from CD14, CD16, CD48, CD55 (DAF), CD59, CD80 and CD87. In some embodiments, the heterologous GPI anchor linking sequence is derived from CD16. In illustrative embodiments, the heterologous GPI anchor sequence is derived from the Fc receptor FcγRIIIb (CD16b) or decay accelerating factor (DAF), otherwise known as complement decay accelerating factor or CD55.

In some embodiments, one or both of the activation components include a heterologous signal sequence that helps direct the expression of the activation component to the cell membrane. Any signal sequence that is active in the packaging cell line can be used. In some embodiments, the signal sequence is a DAF signal sequence. In illustrative embodiments, the activation component is fused to a DAF cycle sequence at its N-terminus and a GPI anchor linker sequence at its C-terminus.

In an illustrative embodiment, the activation component includes an anti-CD3 scFvFc fused to a GPI anchoring sequence derived from CD14, and CD80 fused to a GPI anchoring sequence derived from CD16b; and both are expressed on the surface of a replication-deficient recombinant retroviral particle provided herein. In some embodiments, the anti-CD3 scFvFc is fused to a DAF signal sequence at its N-terminus and a GPI anchoring sequence derived from CD14 at its C-terminus, and CD80 is fused to a DAF signal sequence at its N-terminus and a GPI anchoring sequence derived from CD16b at its C-terminus; and both are expressed on the surface of a replication-deficient recombinant retroviral particle provided herein. In some embodiments, the DAF signal sequence includes amino acid residues 1-30 of the DAF protein.

Membrane-bound cytokines

Some embodiments of the methods and compositions provided herein include membrane-bound cytokines, or polynucleotides encoding membrane-bound cytokines. Cytokines are usually, but not always, secretory proteins. Naturally secreted cytokines can be engineered into membrane-bound fusion proteins. Membrane-bound cytokines fusion polypeptides are included in the methods and compositions disclosed herein, and are also aspects of the present invention. In some embodiments, replication-deficient recombinant retroviral particles have membrane-bound cytokines fusion polypeptides on their surfaces that can bind to T cells and/or NK cells and promote their proliferation and/survival. Typically, membrane-bound polypeptides are incorporated into the membrane of replication-deficient recombinant retroviral particles, and when cells are transduced by replication-deficient recombinant retroviral particles, the fusion of the retroviral and host cell membranes produces polypeptides that bind to the membrane of the transduced cells.

In some embodiments, the cytokine fusion polypeptide includes IL-2, IL-7, IL-15 or an activated fragment thereof. The membrane-bound cytokine fusion polypeptide is generally a cytokine fused to a heterologous signal sequence and/or a heterologous membrane linking sequence. In some embodiments, the heterologous membrane linking sequence is a GPI anchor linking sequence. The heterologous GPI anchor linking sequence can be derived from any known GPI-anchored protein (reviewed in Ferguson MAJ, Kinoshita T, Hart GW. Glycosylphosphatidylinositol Anchors. In: Varki A, Cummings RD, Esko JD et al., ed. Essentials of Glycobiology. 2nd edition. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 2009. Chapter 11). In some embodiments, the heterologous GPI anchor linking sequence is a GPI anchor linking sequence from CD14, CD16, CD48, CD55 (DAF), CD59, CD80 and CD87. In some embodiments, the heterologous GPI anchoring sequence is derived from CD 16. In some embodiments, the heterologous GPI anchoring sequence is derived from Fc receptor FcγRIIIb (CD16b). In some embodiments, the GPI anchor is the GPI anchor of DAF.

In an illustrative embodiment, the membrane-bound cytokine is a fusion polypeptide of the cytokine fused to DAF. DAF is known to accumulate in lipid rafts incorporated into the membranes of replication-defective recombinant retroviral particles budding from packaging cells. Thus, without being limited by theory, it is believed that the DAF fusion protein is preferentially targeted to the portion of the membrane of the packaging cell that will become part of the recombinant retroviral membrane.

In a non-limiting illustrative embodiment, the interleukin fusion polypeptide is IL-7, or an active fragment thereof fused to DAF. In a specific non-limiting illustrative embodiment, the fusion interleukin polypeptide comprises, in order: a DAF signal sequence (residues 1-31 of DAF), IL-7 without its signal sequence, and residues 36-525 of DAF.

Packaging cell lines/methods for producing recombinant retroviral particles

The present invention provides mammalian packaging cells and packaging cell lines that produce replication-deficient recombinant retroviral particles. The cell lines that produce replication-deficient recombinant retroviral particles are also referred to herein as packaging cell lines. A non-limiting example of this method is provided in Example 1 herein. The cells of the packaging cell line may be adherent cells or suspension cells. In illustrative embodiments, the packaging cell line may be a suspension cell line, i.e., a cell line that does not adhere to a surface during growth. The cells may be grown in a chemically defined medium and/or a serum-free medium. In some embodiments, the packaging cell line may be a suspension cell line derived from an adherent cell line, such as HEK293, which may be grown under conditions that produce a HEK293 cell line adapted to suspension according to methods known in the art. The packaging cell line is typically grown in a chemically defined medium. In some embodiments, the packaging cell line medium may include serum. In some embodiments, the packaging cell line medium may include a serum substitute, as known in the art. In illustrative embodiments, the packaging cell line medium may be a serum-free medium. This medium can be a chemically defined serum-free formulation manufactured in accordance with the current Good Manufacturing Practice (CGMP) regulations of the US Food and Drug Administration (FDA). The packaging cell line medium can be xeno-free and intact. In some embodiments, the packaging cell line medium is cleared by a regulatory agency for use in ex vivo cell processing, such as an FDA 510(k) cleared device. It should be understood herein (where it is stated that the medium includes a composition of a base medium and a medium supplement including a catalog number of a medium supplement such as A1048501 or A1048503) that the composition is intended to mean a composition of the medium and the added supplement. Typically, the manufacturing medium and supplements provide instructions regarding the volume of the supplement added.

Therefore, in one aspect, the present invention provides a method for producing replication-defective recombinant retroviral particles, comprising: A. culturing packaging cells in a serum-free medium containing a suspension, wherein the packaging cells contain a nucleic acid sequence encoding a packageable RNA genome, REV protein, gag polypeptide, pol polypeptide and pseudotyping components of replication-defective recombinant retroviral particles; and B. collecting the replication-defective recombinant retroviral particles from the serum-free medium. In another aspect, the present invention provides a method for transducing lymphocytes with replication-defective recombinant retroviral particles, comprising: A. culturing packaging cells in a serum-free medium containing a suspension, wherein the packaging cells comprise nucleic acid sequences encoding a packageable RNA genome, REV protein, gag polypeptide, pol polypeptide and pseudotyping components of replication-defective recombinant retroviral particles; B. collecting the replication-defective recombinant retroviral particles from the serum-free medium; and C. contacting the lymphocytes with the replication-defective recombinant retroviral particles, wherein the contacting is performed for less than 24 hours, 20 hours, 18 hours, 12 hours, 8 hours, 4 hours or 2 hours (or between 1 hour, 2 hours, 3 hours or 4 hours at the low end of the range and 4 hours, 6 hours, 8 hours, 12 hours, 18 hours, 20 hours or 24 hours at the high end of the range), thereby transducing the lymphocytes.

In another aspect, provided herein is a retroviral packaging system comprising: a mammalian cell comprising: a) a first transactivator expressed from a constitutive promoter and capable of binding a first ligand and a first inducible promoter to affect expression of a nucleic acid sequence operably linked thereto in the presence and absence of the first ligand; b) a second transactivator capable of binding a second ligand and a second inducible promoter and affecting expression of a nucleic acid sequence operably linked thereto in the presence and absence of the second ligand; and c) a packageable RNA genome of a retroviral particle, wherein the first transactivator regulates expression of the second transactivator, and wherein the second transactivator regulates expression of a retroviral polypeptide contained in the viral package, such as a gag polypeptide, a pol polypeptide and/or a pseudotyping component and, optionally, other polypeptides to be incorporated into or onto the replication-defective recombinant retroviral particle and believed to be toxic to the packaging cell line, e.g., HEK-293. In some aspects, the second transactivator itself is cytotoxic to the packaging cell line. The pseudotyped component is generally capable of binding to the cell membrane of the target cell and facilitating its fusion, as discussed in detail herein. Therefore, without being limited by theory, the system provides the ability to accumulate certain polypeptides/proteins (e.g., non-toxic proteins) that cannot inhibit or cannot substantially inhibit or are considered to be unable to inhibit the proliferation or survival of mammalian cells, while culturing mammalian cell populations for several days or indefinitely, and controlling the induction of polypeptides (e.g., toxic polypeptides) that are expected to be used for retroviral products but have inhibitory or can have inhibitory or have been reported to have inhibitory survival and/or proliferation of mammalian cells, until the later time when replication-defective recombinant retroviral particles are close to being generated and collected. The packaged RNA genome is generally encoded by a polynucleotide operably connected to a promoter (sometimes referred to herein as a third promoter for convenience), wherein the third promoter is generally induced by the first transactivator or the second transactivator. In illustrative embodiments, the packaged RNA genome is encoded by a polynucleotide operably connected to a third promoter, wherein the third promoter is induced by the second transactivator. Thus, the packageable RNA genome can be produced at a later time point close to when replication-defective recombinant retroviral particles will be harvested.

Those skilled in the art will appreciate that many different transactivators, ligands, and inducible promoters can be used in retroviral packaging systems. Such inducible promoters can be isolated and derived from many organisms, such as eukaryotes and prokaryotes. Modification of an inducible promoter derived from a first organism for use in a second organism (e.g., a first prokaryote and a second eukaryote, a first eukaryote and a second prokaryote, etc.) is known in the art. Such inducible promoters, and systems based on such inducible promoters but also including other control proteins include, but are not limited to, alcohol-regulated promoters (e.g., alcohol dehydrogenase I (alcA) gene promoter, promoters responsive to alcohol transactivator proteins (AlcR), etc.), tetracycline-regulated promoters (e.g., promoter systems including TetActivators, TetON, TetOFF, etc.), steroid-regulated promoters (e.g., rat glucocorticoid receptor promoter system, human estrogen receptor promoter, etc.), and promoters responsive to alcohol transactivator proteins (e.g., TetON, TetOFF, etc.). Systems, retinoid promoter systems, thyroid promoter systems, ecdysone promoter systems, mifepristone promoter systems, etc.), metal-regulated promoters (e.g., metallothionein promoter systems, etc.), promoters regulated by related pathogens (e.g., salicylic acid-regulated promoters, ethylene-regulated promoters, benzothiadiazole-regulated promoters, etc.), temperature-regulated promoters (e.g., heat shock-inducible promoters (e.g., HSP-70, HSP-90, soybean heat shock promoters, etc.)), light-regulated promoters, synthetic inducible promoters, and the like. In some embodiments, a mifepristone-regulated system may be used. In some embodiments, a mifepristone-inducible system with an autoregulatory feedback loop may be used. In some embodiments, the GAL4-regulated fusion protein is expressed from a construct that also contains a transposon end repeat site and a lox site and a FRT site. In some embodiments, the GAL4-regulated fusion protein controls the expression of a reverse tet transactivator (rtTA) and a BiTRE. In some embodiments, another construct with lox sites and FRT sites contains a GAL4 upstream activating sequence (UAS) and an E1b TATA box promoter driving a reporter such as mCherry. In some embodiments, the GAL4 regulatory fusion protein binds to the GAL4 upstream activating sequence (UAS) on both the promoter that controls the expression of the GAL4 regulatory fusion protein and the promoter that controls the expression of the target polynucleotide. In some embodiments, mifepristone, doxycycline, and puromycin will be used for induction and selection of packaging cell lines.

In some embodiments, either or both of the transactivators can be divided into two or more polypeptides. In some embodiments, the two or more polypeptides may include a DNA binding domain and an activation domain capable of stimulating transcription on a separate polypeptide. This "activation domain" is not confused with an "activation component", such as a polypeptide that binds to CD3, which is capable of activating T cells and/or NK cells, and is usually activated when in contact with this T cell and/or NK cell, as discussed in detail herein. The separated polypeptide may further include a fusion with a polypeptide that can dimerize by adding a ligand. In some embodiments, the activation domain may be a p65 activation domain or a functional fragment thereof. In an illustrative embodiment of the packaging system herein, the DNA binding domain may be a DNA binding domain from ZFHD1 or a functional fragment thereof. In some embodiments, one polypeptide may be a fusion with FKBP or its functional mutant and/or its fragment or multiple FKBPs, and another polypeptide may be a fusion with the FRB domain of mTOR or its functional mutant and/or its fragment, and the ligand may be rapamycin or a functional rapamycin analog. In some embodiments, FRB contains mutants K2095P, T2098L and/or W2101F. In some embodiments, the isolated polypeptide may be FKBP or a functional fragment thereof, and CalcineurinA or a functional fragment thereof, and the dimer may be FK506. In some embodiments, the isolated polypeptide may be FKBP or a functional fragment thereof, and CyP-Fas or a functional fragment thereof, and the dimer may be FKCsA. In some embodiments, the isolated polypeptide may be GAI or a functional fragment thereof, and GID1 or a functional fragment thereof, and the dimer may be gibberellin. In some embodiments, the isolated polypeptide may be Snap-tag and HaloTag or a functional fragment thereof and the dimer may be HaXS. In some embodiments, the isolated polypeptide may include the same polypeptide. For example, the DNA binding domain and the activation domain may be expressed as a fusion protein with FKBP or GyrB, and the dimer may be FK1012 or coumermycin, respectively. In some embodiments, the inducible promoter may be a DNA sequence to which the DNA binding domain normally binds. In some embodiments, the inducible promoter may be different from the DNA sequence to which the DNA binding domain normally binds. In some embodiments, the transactivator may be rtTA, the ligand may be tetracycline or doxycycline, and the inducible promoter may be a TRE. In illustrative embodiments, the first transactivator is the p65 activation domain fused to FRB and the ZFHD1 DNA binding domain fused to three FKBP polypeptides, and the first ligand is rapamycin. In other illustrative embodiments, the second transactivator may be rtTA, the second ligand may be tetracycline or doxycycline, and the inducible promoter may be a TRE.

In some embodiments, the first transactivator may regulate expression of a component to control nuclear export of a transcript containing a consensus sequence, such as HIV Rev, and the consensus sequence may be a Rev response component. In illustrative embodiments, the target cell is a T cell.

In some embodiments, the pseudotyped component is a retroviral envelope polypeptide. The pseudotyped component generally includes a binding polypeptide and a fusion polypeptide for binding the target cell to the viral membrane and promoting membrane fusion of the target cell and the viral membrane, as discussed in more detail herein. In some embodiments, the pseudotyped component is a feline endogenous virus (RD114) envelope protein, a tumor retrovirus double-tropic envelope protein, a tumor retrovirus tropic envelope protein, and/or a vesicular stomatitis virus envelope protein (VSV-G). In illustrative embodiments, the pseudotyped component includes a binding polypeptide and a fusion polypeptide derived from different proteins, as discussed in further detail herein. For example, in illustrative embodiments, especially when the target cell is a T cell and/or a NK cell, the binding polypeptide is a hemagglutinin (H) polypeptide of a measles virus (e.g., an Edmonston strain of a measles virus) or a cytoplasmic domain deletion variant thereof, and the other fusion polypeptide is a fusion (F) polypeptide of a measles virus (e.g., an Edmonston strain of a measles virus) or a cytoplasmic domain deletion variant thereof. In some embodiments, the fusion polypeptide may include multiple components expressed as one polypeptide. In some embodiments, the binding polypeptide and the fusion polypeptide may be translated from the same transcript, but from different ribosomal binding sites, or the polypeptide may be cleaved using a peptide cleavage signal or a ribosomal skipping sequence after translation, as disclosed elsewhere herein, to produce the binding polypeptide and the fusion polypeptide. In some embodiments, when the binding polypeptide is a measles virus H polypeptide or a cytoplasmic domain deletion thereof, and the fusion polypeptide is a measles virus F polypeptide or a cytoplasmic domain deletion thereof, the translation of the F polypeptide and the H polypeptide from separate ribosomal binding sites produces a higher amount of F polypeptide than the H polypeptide. In some embodiments, the ratio of the F polypeptide (or its cytoplasmic domain deletion) to the H polypeptide (or its cytoplasmic domain deletion) is at least 2:1, at least 3:1, at least 4:1, at least 5:1, at least 6:1, at least 7:1, or at least 8:1.

In some embodiments, the first transactivator can regulate the expression of an activation component capable of binding to and activating a target cell, such as a T cell or a NK cell. Any activation component disclosed herein can be expressed. For example, in these embodiments, the activation component may include: a.) a membrane-bound polypeptide capable of binding to and activating CD3; and/or b.) a membrane-bound polypeptide capable of binding to and activating CD28. In some embodiments, the membrane-bound polypeptide capable of binding to and activating CD3 may be an anti-CD3 antibody. In other embodiments, the anti-CD3 antibody may be an anti-CD3 scFvFc. In some embodiments, the membrane-bound polypeptide capable of binding to and activating CD28 is CD80, CD86, or a functional fragment thereof, such as the extracellular domain of CD80.

In some embodiments, the second transactivator can regulate the expression of a packageable RNA genome, which can include RNA encoding one or more target polypeptides, including as non-limiting examples any of the engineered signaling polypeptides disclosed herein and/or one or more (e.g., two or more) inhibitory RNA molecules. It should be noted that it is foreseeable that the retroviral packaging system aspects and the method aspects of preparing replication-defective recombinant retroviral particles are not limited to preparing replication-defective recombinant retroviral particles for transducing T cells and/or NK cells, but are also applicable to any cell type that can be transduced by replication-defective recombinant retroviral particles. In some illustrative embodiments, the packageable RNA genome is designed to express one or more target polypeptides, including as non-limiting examples any of the engineered signaling polypeptides disclosed herein and/or one or more (e.g., two or more) inhibitory RNA molecules that are oriented oppositely (e.g., encoded on opposite strands and in opposite directions) to retroviral components such as gag and pol. For example, the packageable RNA genome includes 5' to 3': 5' long terminal repeats or active truncations thereof; nucleic acid sequences encoding retroviral cis-acting RNA packaging components; nucleic acid sequences encoding a first target polypeptide and optionally a second target polypeptide, the target polypeptide being, for example, but not limited to, an engineered signaling polypeptide in an opposite orientation, which can drive a promoter in this opposite direction relative to the 5' long terminal repeats and the cis-acting RNA packaging components, which in some embodiments is referred to as a "fourth" promoter for convenience only (and sometimes referred to herein as a promoter active in T cells and/or NK cells), which is active in target cells such as T cells and/or NK cells, but in illustrative examples, is not active in packaging cells, or is only inducible or minimally active in packaging cells; and 3' long terminal repeats or active truncations thereof. In some embodiments, the packageable RNA genome may include a central polypurine region (cPPT)/central termination sequence (CTS) component. In some embodiments, the retroviral cis-acting RNA packaging component may be HIV Psi. In some embodiments, the retroviral cis-acting RNA packaging component may be a Rev response component. In exemplary embodiments, the engineered signaling polypeptide driven by a promoter oriented opposite to the 5' long terminal repeat is one or more engineered signaling polypeptides disclosed herein, and may optionally express one or more inhibitory RNA molecules as disclosed herein and in more detail in WO2017/165245A2, WO2018/009923A1, and WO2018/161064A1.

It should be understood that promoter numbering such as first promoter, second promoter, third promoter, fourth promoter, etc. is for convenience only. A promoter referred to as a "fourth" promoter should not be considered to imply the presence of any other promoters, such as first promoter, second promoter, or third promoter, unless other promoters are explicitly recited. It should be noted that each of the promoters is capable of driving transcripts to be expressed in an appropriate cell type, and such transcripts form a transcription unit.

In some embodiments, the engineered signaling polypeptide may include a first lymphoproliferative component. Suitable lymphoproliferative components are disclosed in other parts herein. As a non-limiting example, the lymphoproliferative component may be expressed as a fusion with a recognition domain, such as an eTag, as disclosed herein. In some embodiments, the packaged RNA genome may further include a nucleic acid sequence encoding a second engineered polypeptide, including a chimeric antigen receptor encoding any CAR embodiment provided herein. For example, the second engineered polypeptide may include a first antigen-specific targeting region, a first transmembrane domain, and a first intracellular activation domain. Examples of antigen-specific targeting regions, transmembrane domains, and intracellular activation domains are disclosed elsewhere herein. In some embodiments where the target cell is a T cell, a promoter active in the target cell is active in the T cell, as disclosed elsewhere herein.

In some embodiments, the engineered signaling polypeptide may include a CAR, and the nucleic acid sequence may encode any CAR embodiment provided herein. For example, the engineered polypeptide may include a first antigen-specific targeting region, a first transmembrane domain, and a first intracellular activation domain. Examples of antigen-specific targeting regions, transmembrane domains, and intracellular activation domains are disclosed elsewhere herein. In some embodiments, the packaged RNA genome may further include a nucleic acid sequence encoding a second engineered polypeptide. In some embodiments, the second engineered polypeptide may be a lymphoproliferative component. In some embodiments in which the target cell is a T cell or a NK cell, a promoter active in the target cell is active in a T cell or a NK cell, as disclosed elsewhere herein.

In some embodiments, the packaged RNA genome may further include a riboswitch, as discussed in WO2017/165245A2, WO2018/009923A1, and WO2018/161064A1. In some embodiments, the nucleic acid sequence encoding the engineered signaling polypeptide may be reversely oriented relative to the 5' to 3' orientation established by the 5'LTR and the 3'LTR. In other embodiments, the packaged RNA genome may further include a riboswitch, and the riboswitch may be reversely oriented as appropriate. In any of the embodiments disclosed herein, a polynucleotide comprising any of the components may include a primer binding site. In illustrative embodiments, a transcription blocker or a polyA sequence may be placed near a gene to prevent or reduce unregulated transcription. In any of the embodiments disclosed herein, the nucleic acid sequence encoding Vpx may be located on a second transcription unit or on a third transcription unit that is optionally present, or on an additional transcription unit that is operably linked to a first inducible promoter.

In another aspect herein, a mammalian packaging cell line is provided that comprises a packaging RNA genome of a replication-defective retroviral particle, wherein the packaging RNA genome comprises:

a. 5' long terminal repeat or its activated fragment;

b. a nucleic acid sequence encoding a retroviral cis-acting RNA packaging component;

c. a polynucleotide comprising one or more nucleic acid sequences operably linked to a promoter active in T cells and/or NK cells, wherein a first nucleic acid sequence in the one or more nucleic acids encodes one or more (e.g., two or more) inhibitory RNA molecules against one or more RNA targets, and a second nucleic acid sequence in the one or more nucleic acids encodes a chimeric antigen receptor (CAR) comprising an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain; and

d. 3’ long terminal repeat or its activated fragment.

The inhibitory RNA molecules in the above aspects may include any of the inhibitory RNA molecules provided in other parts of the disclosure, as non-limiting examples, shRNA or miRNA.

In some embodiments of the mammalian packaging cell line aspect, the polynucleotide of (c) may be in the opposite orientation to the nucleic acid sequence encoding the retroviral cis-acting RNA packaging component (b), the 5' long terminal repeat (a) and/or the 3' long terminal repeat (d), for example relative to the orientation established by the 5' LTR and the 3' LTR.

In some embodiments of the mammalian packaging cell line aspect, expression of the packageable RNA genome is driven by an inducible promoter active in the mammalian packaging cell line.

In illustrative embodiments, the promoter that is active in T cells and/or NK cells that drive the expression of the inducible RNA and CAR in the above-provided aspects is not active or has only minimal activity or inducible activity in the packaging cell line. In illustrative embodiments, this promoter that is active in T cells and/or NK cells is located on the packageable RNA genome between the nucleic acids encoding one (e.g., two) or more inducible RNAs and CARs and 3'LTRs.

In any of the aspects involving packaged cells or cell lines encoding one or more inhibitory RNA molecules for one or more RNA targets, at least one inhibitory RNA molecule and in some embodiments, all inhibitory RNA molecules may include a 5' strand and a 3' strand that are partially or completely complementary to each other, wherein the 5' strand and the 3' strand are capable of forming an 18 to 25 nucleotide RNA duplex. In some embodiments, the 5' strand length may be 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides, and the 3' strand length may be 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides. In some embodiments, the 5' strand and the 3' strand length may be the same or different. In some embodiments, the RNA duplex may include one or more mismatches. In alternative embodiments, the RNA duplex has no mismatches.

In any of the aspects provided above for the packaged cells or cell lines encoding inhibitory RNA molecules for one or more RNA targets herein, the inhibitory RNA molecule may be a miRNA or shRNA. In some embodiments, the inhibitory molecule may be a precursor of a miRNA, such as a Pri-miRNA or a Pre-miRNA, or a precursor of an shRNA. In some embodiments, one or more inhibitory RNA molecules may be artificially derived miRNAs or shRNAs. In other embodiments, the inhibitory RNA molecule may be a dsRNA (transcribed or artificially introduced) processed into a siRNA or the siRNA itself. In some embodiments, the inhibitory RNA molecule may be a miRNA or shRNA having a sequence not found in nature, or having at least one functional segment not found in nature, or having a combination of functional segments not found in nature. In illustrative embodiments, at least one or all of the inhibitory RNA molecules are miR-155.

In any of the aspects provided above for the packaged cells or cell lines encoding inhibitory RNA molecules for one or more RNA targets herein, the one or more inhibitory RNA molecules may, in some embodiments, comprise a 5' to 3' orientation: a 5' arm, a 5' stem, a loop, a 3' stem that is partially or completely complementary to the 5' stem, and a 3' arm. In some embodiments, at least one of the two or more inhibitory RNA molecules has this arrangement. In other embodiments, all of the two or more inhibitory RNA molecules have this arrangement. In some embodiments, the 5' stem may be 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In some embodiments, the 3' stem may be 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In some embodiments, the loop length may be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides. In some embodiments, the 5' arm, the 3' arm, or both are derived from a naturally occurring miRNA. In some embodiments, the 5' arm, the 3' arm, or both are derived from a naturally occurring miRNA selected from the group consisting of miR-155, miR-30, miR-17-92, miR-122, and miR-21. In illustrative embodiments, the 5' arm, the 3' arm, or both are derived from miR-155. In some embodiments, the 5' arm, the 3' arm, or both are derived from Mus musculus miR-155 or Homo sapiens miR-155. In some embodiments, the 5' arm has a sequence set forth in SEQ ID NO: 256 or a functional variant thereof, such as the same length as SEQ ID NO: 256, or 95%, 90%, 85%, 80%, 75%, or 50% of the length of SEQ ID NO: 256, or 100 nucleotides or less, 95 nucleotides or less, 90 nucleotides or less, 85 nucleotides or less, 80 nucleotides or less, 75 nucleotides or less, 70 nucleotides or less, 65 nucleotides or less, 60 nucleotides or less, 55 nucleotides or less, 50 nucleotides or less, 45 nucleotides or less, 40 nucleotides or less, 35 nucleotides or less, 30 nucleotides or less, or 25 nucleotides or less; and a sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to SEQ ID NO: 256. In some embodiments, the 3' arm has a sequence set forth in SEQ ID NO: 260 or a functional variant thereof, such as a length identical to SEQ ID NO: 260, or 95%, 90%, 85%, 80%, 75%, or 50% of the length of SEQ ID NO: 206, or 100 nucleotides or less, 95 nucleotides or less, 90 nucleotides or less, 85 nucleotides or less, 80 nucleotides or less, 75 nucleotides or less, 70 nucleotides or less, 65 nucleotides or less, 60 nucleotides or less, 55 nucleotides or less, 50 nucleotides or less, 45 nucleotides or less, 40 nucleotides or less, 35 nucleotides or less, 30 nucleotides or less, or 25 nucleotides or less; and a sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to SEQ ID NO: 260. In some embodiments, the 3' arm comprises nucleotides 221 to 283 of the Mus musculus BIC.

In another aspect, provided herein is a method for preparing replication-defective recombinant retroviral particles, comprising: culturing a population of packaging cells to accumulate a first transactivator, wherein the packaging cells comprise the first transactivator expressed by a constitutive promoter, wherein the first transactivator is capable of binding a first ligand and a first inducible promoter to affect expression of a nucleic acid sequence operably linked thereto in the presence and absence of the first ligand, and wherein expression of a second transactivator is regulated by the first transactivator; culturing the population of packaging cells comprising the accumulated first transactivator in the presence of the first ligand to accumulate the second transactivator , wherein the second transactivator is capable of binding to a second ligand and a second inducible promoter to affect the expression of a nucleic acid sequence operably linked thereto in the presence and absence of the second ligand; and the packaging cell population comprising the accumulated second transactivator is cultured in the presence of the second ligand, thereby inducing the expression of retroviral polypeptides involved in viral packaging, such as gag polypeptides, pol polypeptides and/or pseudotyping components, and optionally other polypeptides believed to inhibit mammalian cell proliferation or survival, which will be incorporated into or onto the replication-defective recombinant retrovirus, thereby producing replication-defective recombinant retroviral particles. In illustrative embodiments, the packageable RNA genome is encoded by a polynucleotide operably linked to a promoter (sometimes referred to as a "third" promoter for convenience), wherein the third promoter is constitutively active or inducible by the first transactivator or, in illustrative embodiments, by the second transactivator, thereby producing replication-defective recombinant retroviral particles. The pseudotyping component is generally capable of binding to the cell membrane of the target cell and promoting fusion of the target cell membrane with the membrane of the replication-defective recombinant retroviral particle. The pseudotyping component may be any envelope protein known in the art. In some embodiments, the envelope protein may be a vesicular stomatitis virus envelope protein (VSV-G), a feline endogenous virus (RD114) envelope protein, an oncomplex retrovirus amphotropic envelope protein, and/or an oncomplex retrovirus tropic envelope protein. One skilled in the art will appreciate that a variety of different transactivators, ligands, and inducible promoters may be used in methods for preparing replication-defective recombinant retroviral particles. Suitable transactivators, ligands, and inducible promoters are disclosed elsewhere herein, including above. A skilled artisan will further appreciate that the above teachings relating to the retroviral packaging system aspects provided herein are also applicable to methods for preparing replication-defective recombinant retroviral particle aspects, and vice versa.

In some embodiments, the expression of the first transactivator can regulate the nuclear export of the transcript containing the consensus sequence, such as HIV Rev, and the consensus sequence can be a Rev response component (RRE). In illustrative embodiments, the target cell is generally a T cell. In some embodiments, HIV RRE and the polynucleotide region encoding HIV Rev can be replaced by HIV-2RRE and the polynucleotide region encoding HIV-2Rev, respectively. In some embodiments, HIV RRE and the polynucleotide region encoding HIV Rev can be replaced by SIV RRE and the polynucleotide region encoding SIV Rev, respectively. In some embodiments, HIV RRE and the polynucleotide region encoding HIV Rev can be replaced by RemRE and the polynucleotide region encoding beta retrovirus Rem, respectively. In some embodiments, HIV RRE and the polynucleotide region encoding HIV Rev can be replaced by delta retrovirus RexRRE and the polynucleotide region encoding delta retrovirus Rex, respectively. In some embodiments, Rev class proteins are not required and PRE can be replaced by cis-acting RNA components such as constitutive transport components (CTE).

In some embodiments, the pseudotyped component is a viral envelope protein. The pseudotyped analysis component generally includes a binding polypeptide and a fusion polypeptide for binding the virus to the target cell membrane and promoting membrane fusion of the virus and the target cell membrane. In some embodiments, the pseudotyped component may be a feline endogenous virus (RD114) envelope protein, a tumor retrovirus double-tropic envelope protein, a tumor retrovirus tropic envelope protein and/or a vesicular stomatitis virus envelope protein (VSV-G). In illustrative embodiments, the pseudotyped component includes a binding polypeptide and a fusion polypeptide derived from different proteins, as further discussed in detail herein. For example, in an illustrative embodiment, especially when the target cell is a T cell and/or a NK cell, the binding polypeptide may be a cytoplasmic domain deletion variant of the measles virus H polypeptide and the fusion polypeptide may be a cytoplasmic domain deletion variant of the measles virus F polypeptide. In some embodiments, a fusion polypeptide may include multiple components expressed as one polypeptide. In some embodiments, the binding polypeptide and the fusion polypeptide can be translated from the same transcript and from different ribosomal binding sites, or the polypeptide can be cleaved post-translationally using a peptide cleavage signal or ribosomal skipping sequence, as disclosed elsewhere herein, to produce a binding polypeptide and a fusion polypeptide. In some embodiments, translation of the binding polypeptide and the fusion polypeptide from separate ribosomal binding sites produces a higher amount of fusion polypeptide than the binding polypeptide. In some embodiments, the ratio of fusion polypeptide to binding polypeptide is at least 2:1, at least 3:1, at least 4:1, at least 5:1, at least 6:1, at least 7:1, or at least 8:1.

In some embodiments, the first transactivator can regulate the expression of an activation component that can bind to and activate a target cell such as a T cell. In these embodiments, the activation component can include: a.) a membrane-bound polypeptide that can bind to and activate CD3; and/or b.) a membrane-bound polypeptide that can bind to and activate CD28. In some embodiments, the membrane-bound polypeptide that can bind to and activate CD28 is CD80, CD86, or a functional fragment thereof. In some embodiments, the replication-defective recombinant retroviral particle can include an activation component on the retroviral membrane and a retroviral RNA in the nucleocapsid, thereby producing a replication-defective recombinant retroviral particle.

In some embodiments, the second transactivator can regulate the expression of an RNA including from 5' to 3': a 5' long terminal repeat or an active truncated fragment thereof; a nucleic acid sequence encoding a retroviral cis-acting RNA packaging component; a nucleic acid sequence encoding a first target polypeptide and, optionally, a second target polypeptide (as a non-limiting example, one or two engineered signaling polypeptides); a promoter active in a target cell; and a 3' long terminal repeat or an active truncated fragment thereof. In some embodiments, the RNA may include a cPPT/CTS component. In some embodiments, the RNA may include a primer binding site. In some embodiments, the retroviral cis-acting RNA packaging component may be HIV Psi. In some embodiments, the retroviral cis-acting RNA packaging component may be a Rev response component. In any of the embodiments disclosed herein, the retroviral components (including RRE and Psi) on the RNA may be located at any position, as will be understood by those skilled in the art. In illustrative embodiments, the engineered signaling polypeptide is one or more of the engineered signaling polypeptides disclosed herein.

In some embodiments, the engineered signaling polypeptide may include a first lymphoproliferative component. Suitable lymphoproliferative components are disclosed in other parts herein. In some illustrative embodiments, the lymphoproliferative component is an IL-7 receptor mutant fused to a recognition domain, such as an eTag. In some embodiments, the packaged RNA genome may further include a nucleic acid sequence encoding a second engineered polypeptide, including a chimeric antigen receptor encoding any CAR embodiment provided herein. For example, the second engineered polypeptide may include a first antigen-specific targeting region, a first transmembrane domain, and a first intracellular activation domain. Examples of antigen-specific targeting regions, transmembrane domains, and intracellular activation domains are disclosed elsewhere herein. In some embodiments where the target cell is a T cell, a promoter active in the target cell is active in the T cell, as disclosed elsewhere herein.

In some embodiments, the packaged RNA genome may further include a riboswitch, as discussed in WO2017/165245A2, WO2018/009923A1, and WO2018/161064A1. In some embodiments, the nucleic acid sequence encoding the engineered signaling polypeptide may be in reverse orientation. In other embodiments, the packaged RNA genome may further include a riboswitch, and the riboswitch may be in reverse orientation as appropriate. In any of the embodiments disclosed herein, a polynucleotide comprising any of the components may include a primer binding site. In illustrative embodiments, a transcription blocker or polyA sequence may be placed near the gene to prevent or reduce unregulated transcription. In any of the embodiments disclosed herein, the nucleic acid sequence encoding Vpx may be located on the second transcription unit or on the third transcription unit, which may be present, or on an additional transcription unit operably linked to the first inducible promoter.

In some embodiments of packaging systems or methods for preparing replication-deficient recombinant retroviral particle aspects, the encoded RNA may include introns, which may, for example, be transcribed from the same promoter used to express the target polypeptide. In certain illustrative embodiments, such introns may encode 1, 2, 3, or 4 miRNAs. In these and other embodiments of packaging systems or methods for preparing replication-deficient recombinant retroviral particle aspects, the packaged RNA genome size is 11,000 KB or less, and in some instances 10,000 KB or less.

In some embodiments, the first transactivator may affect the expression of one or more non-toxic polypeptides. In some embodiments, the second transactivator may affect the expression of one or more toxic polypeptides. For example, the first transactivator may induce the expression of retroviral proteins Rev and Vpx in addition to polypeptides that are transported to the cell membrane of the packaging cell, and the second transactivator may induce the expression of retroviral proteins GAG, POL, MV (Ed) -FΔ30 and MV (Ed) -HΔ18 or MV (Ed) -HΔ24 and the expression of the lentiviral genome. In some embodiments, the first transactivator may affect the expression of one or more toxic polypeptides and/or the second transactivator may affect the expression of one or more non-toxic polypeptides.

In another aspect, provided herein is a mammalian packaging cell comprising: a) a first transcription unit in the genome of the mammalian packaging cell, comprising a nucleic acid sequence encoding a first transactivator, wherein the first transcription unit is operably linked to a constitutive promoter, and wherein the transactivator is capable of binding to a first inducible promoter and affecting expression of a nucleic acid sequence operably linked thereto in the presence and absence of a first ligand, and wherein the first transactivator is capable of binding to the first ligand; b) a second transcription unit and optionally a third transcription unit in the genome of the mammalian packaging cell, comprising a nucleic acid sequence encoding a retroviral REV protein and a nucleic acid sequence encoding a second transactivator, wherein the second transactivator is capable of binding to a second inducible promoter and affecting expression of a nucleic acid sequence operably linked thereto in the presence and absence of a second ligand, wherein the second transactivator is capable of binding to the second ligand, and wherein the second transcription unit and optionally a third transcription unit c) a fourth transcription unit in the genome of a mammalian packaging cell and, if applicable, a fifth transcription unit, which comprises a nucleic acid sequence encoding a retroviral gag polypeptide and a retroviral pol polypeptide, and a binding polypeptide and a fusion polypeptide capable of binding to and promoting fusion of a target cell membrane with a retroviral membrane, wherein the fourth transcription unit and, if applicable, the fifth transcription unit are operably linked to the second inducible promoter; and d) a sixth transcription unit in the genome of a mammalian packaging cell, which comprises, from 5' to 3', a 5' LTR or an active truncated fragment thereof, a nucleic acid sequence encoding a retroviral cis-acting RNA packaging component, a cPPT/CTS component, the reverse complement of a nucleic acid sequence encoding an engineered signaling polypeptide, an intron, a promoter active in a target cell, and a 3' LTR or an active truncated fragment thereof, wherein the sixth transcription unit is operably linked to the second inducible promoter.

In another aspect, provided herein is a method for preparing replication-defective recombinant retroviral particles, comprising: 1.) culturing a population of packaging cells to accumulate a first transactivator, wherein the packaging cells comprise: a) a first transcription unit in the genome of a mammalian packaging cell, comprising a nucleic acid sequence encoding the first transactivator, wherein the first transcription unit is operably linked to a constitutive promoter, and wherein the transactivator is capable of binding to a first inducible promoter and affecting expression of a nucleic acid sequence operably linked thereto in the presence and absence of a first ligand, and wherein the first transactivator is capable of binding to the first ligand; b) a second transcription unit and optionally a third transcription unit in the genome of a mammalian packaging cell, comprising a nucleic acid encoding a retroviral REV protein. The invention relates to a method for preparing a mammalian packaging cell comprising: a first transcription unit comprising a nucleic acid sequence encoding a retroviral gag polypeptide and a retroviral pol polypeptide, and a nucleic acid sequence encoding a second transactivator capable of binding to a second inducible promoter and affecting the expression of a nucleic acid sequence operably linked thereto in the presence and absence of a second ligand, wherein the second transactivator is capable of binding to the second ligand, and wherein the second transcription unit and optionally the third transcription unit are operably linked to the first inducible promoter; c) a fourth transcription unit and optionally the fifth transcription unit in the genome of a mammalian packaging cell, which comprises a nucleic acid sequence encoding a retroviral gag polypeptide and a retroviral pol polypeptide, and a binding polypeptide and a fusion polypeptide capable of binding to and promoting fusion of a retroviral membrane with a target cell membrane, wherein the fourth transcription unit and optionally the fifth transcription unit are operably linked to the a second inducible promoter; and d) a sixth transcription unit in the genome of a mammalian packaging cell, which comprises, from 5' to 3', a 5' LTR or an active truncated fragment thereof, a primer binding site (PBS), a nucleic acid sequence encoding a retroviral cis-acting RNA packaging component, a cPPT/CTS component, a reverse complement of a nucleic acid sequence encoding an engineered signaling polypeptide, an intron, a target cell promoter active in a target cell, and a 3' LTR or an active truncated fragment thereof, wherein the fifth transcription unit is operably linked to the second inducible promoter; and 2) culturing a population of packaging cells comprising the first transactivator in the presence of the first ligand to accumulate the second transactivator and the retroviral REV protein; and 3) culturing a population of packaging cells comprising the second transactivator in the presence of the second ligand The invention relates to a packaging cell group containing a retroviral REV protein, a retroviral gag polypeptide, a retroviral pol polypeptide, a binding polypeptide, a fusion polypeptide and a retroviral RNA (a retroviral RNA including a 5'LTR or an activation fragment thereof from 5' to 3', a PBS, a retroviral cis-acting RNA packaging component, a reverse complement of a nucleic acid sequence encoding an engineered signaling polypeptide, a target cell promoter, and a 3'LTR or an active truncated fragment thereof), wherein replication-defective recombinant retroviral particles are formed and released by the packaging cells, and wherein the replication-defective recombinant retroviral particles include the binding polypeptide and/or the fusion polypeptide on the retroviral membrane and the retroviral RNA in the nucleocapsid, thereby producing replication-defective recombinant retroviral particles.

In one aspect provided herein, a retroviral packaging system can include a mammalian cell comprising: 1.) a first transactivator expressed from a constitutive promoter and capable of binding a first ligand and a first inducible promoter to affect expression of a nucleic acid sequence operably linked thereto in the presence and absence of the first ligand; 2.) a second transactivator capable of binding a second ligand and a second inducible promoter and affecting expression of a nucleic acid sequence operably linked thereto in the presence and absence of the second ligand; and 3.) a packageable RNA genome of a reverse transcriptase particle, wherein the first transactivator regulates expression of the second transactivator, HIV REV, IL7 GPI DAF and activation component, and wherein the second transactivator regulates expression of a gag polypeptide, a pol polypeptide, a retroviral cis-acting RNA packaging component, and one or more envelope polypeptides. In illustrative embodiments, the first transactivator may be an FRB domain fused to a p65 activation domain and one or more FKBP domains fused to a ZFHD1 DNA binding domain, the first ligand may be rapamycin, and the first inducible promoter may be one or more ZFHD1 binding sites. In illustrative embodiments, the second transactivator may be an rtTA protein, the second ligand may be tetracycline or doxycycline, and the second inducible promoter may be a TRE promoter or a bidirectional TRE promoter. In illustrative embodiments, the retroviral cis-acting RNA packaging component may be HIV Psi. In illustrative embodiments, one or more envelope proteins include a cytoplasmic domain deletion variant of the F polypeptide and H polypeptide of the measles virus. In illustrative embodiments, a transcription blocker or polyA sequence may be placed near the gene to prevent or reduce unregulated transcription. In some embodiments, a rapamycin-doxycycline inducible lentiviral genome with a riboswitch (SEQ ID NO: 83) may be used. In some embodiments, rapamycin-doxycycline can be used to induce GAG POL ENV (SEQ ID NO: 84). In some embodiments, rapamycin can be used to induce TET activator (SEQ ID NO: 85). In some embodiments, rapamycin inducer can be used to induce REV srcVpx (SEQ ID NO: 86).

Some aspects of the present invention include cells or cells, which are mammalian cells used as packaging cells to prepare replication-defective recombinant retroviral particles in illustrative examples, such as lentiviral particles for transducing T cells and/or NK cells. Any of a wide variety of cells can be selected to produce viruses or viral particles in vitro, such as re-directed recombinant retroviral particles according to the present invention. Eukaryotic cells, especially mammalian cells, are generally used, including human cells, ape cells, canine cells, cat cells, equine cells and rodent cells. In illustrative examples, the cells are human cells. In other illustrative embodiments, the cells proliferate indefinitely and are therefore immortal. Examples of cells that can be advantageously used in the present invention include NIH3T3 cells, COS cells, Madin-Darby canine kidney cells, human embryonic 293T cells and any cells derived from such cells, such as gpnlslacZ Cells derived from 293T cells. Highly transfectable cells, such as human embryonic kidney 293T cells, can be used. "Highly transfectable" means that at least about 50%, preferably at least about 70%, and most preferably at least about 80% of the cells can express the genes of the introduced DNA.

Suitable mammalian cells include primary cells and immortalized cell lines. Suitable mammalian cell lines include human cell lines, non-human primate cell lines, rodent (e.g., mouse, rat) cell lines, and the like. Suitable mammalian cell lines include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC No. CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RAT1 cells, mouse L cells (ATCC No. CCL1.3), human embryonic kidney (HEK) cells (ATCC No. CRL1573), HLHepG2 cells, Hut-78, Jurkat, HL-60, NK cell lines (e.g., NKL, NK92, and YTS), and the like.

In any of the embodiments disclosed herein, the method of preparing a replication-defective recombinant retroviral particle may comprise growing mammalian packaging cells to 50%, 60%, 70%, 80%, 90% or 95% confluence or confluence, or to 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of peak cell density or peak cell density, and then splitting or diluting the cells. In some embodiments, a stirred tank reactor may be used to grow the cells. In some embodiments, the cells may be split at least about 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:12, 1:15, or 1:20 using methods that will be understood by a skilled artisan. In some embodiments, the cells may be diluted to 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of peak cell density. In some embodiments, after splitting or diluting the cells, the cells can be grown for 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 10 hours, or 16 hours, or 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days before adding the first ligand. In some embodiments, the cells are grown for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 21 days, or 28 days in the presence of the first ligand, which in illustrative embodiments can be rapamycin or a rapamycin analog. In some embodiments, a second ligand may be added and the cells grown for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 21 days, or 28 days. In illustrative embodiments, the second ligand may be tetracycline or doxycycline. The culture conditions will depend on the cells and ligand used, and methods are known in the art. Specific examples of conditions for culturing and inducing HEK293S cells are shown in Example 2.

As disclosed herein, replication-deficient recombinant retroviral particles are a common tool for gene delivery (Miller, Nature (1992) 357: 455-460). The ability of replication-deficient recombinant retroviral particles to deliver unaligned nucleic acid sequences to a wide range of rodents, primates, and human somatic cells makes replication-deficient recombinant retroviral particles more suitable for gene transfer to cells. In some embodiments, replication-deficient recombinant retroviral particles can be derived from alpha retrovirus, beta retrovirus, gamma retrovirus, delta retrovirus, epsilon retrovirus, lentivirus, or foamy virus. There are many types of retroviruses suitable for the methods disclosed herein. For example, murine leukemia virus (MLV), human immunodeficiency virus (HIV), equine infectious anemia virus (EIAV), mouse mammary tumor virus (MMTV), Rous sarcoma virus (RSV), Fuchsina sarcoma virus (FuSV), Moloney murine leukemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus (A-MLV), avian myelocytopathic virus-29 (MC29), and avian erythrocytic virus (AEV) can be used. A detailed list of retroviruses can be found in Coffin et al. ("Retroviruses", 1977, Cold Spring Harbor Laboratory Press edition: J M Coffin, S M Hughes, H E Varmus, pages 758-763). Details on the genomic structure of some retroviruses can be found in the art. For example, details about HIV can be found in NCBI Genbank (ie Genome Accession No. AF033819).

In illustrative embodiments, the replication-deficient recombinant retroviral particles may be derived from a lentivirus. In some embodiments, the replication-deficient recombinant retroviral particles may be derived from HIV, SIV or FIV. In other illustrative embodiments, the replication-deficient recombinant retroviral particles may be derived from human immunodeficiency virus (HIV) in the lentivirus genus. Lentiviruses are complex retroviruses that contain other genes with regulatory or structural functions in addition to the common retroviral genes gag, pol and env. The higher complexity enables the lentivirus to regulate its life cycle during latent infection. A typical lentivirus is human immunodeficiency virus (HIV), the pathogen of AIDS. In vivo, HIV can infect terminally differentiated cells that rarely divide, such as lymphocytes and macrophages.

In illustrative embodiments, the replication-deficient recombinant retroviral particles provided herein contain a Vpx polypeptide. The Vpx polypeptide can be expressed in a packaging cell line after the Vpx encoding nucleic acid is integrated into its genome, for example as a cell membrane-bound protein incorporated into the retroviral membrane (Durand et al., J. Virol. (2013) 87: 234-242). Retroviral membrane-bound Vpx can be constructed sequentially by treatment with viral proteases so that free Vpx is released once incorporated into the viral particle. This example of a Vpx fusion with this function is Src-Flag-Vpx, which includes the membrane targeting domain (MGSSKSKPKDP) (SEQ ID NO: 227) of the first 11 amino acids of c-Src, followed by the viral protease cleavage domain KARVLAEA (SEQ ID NO: 228), followed by Flag-tagged Vpx.

Without being limited by theory, the Vpx polypeptide helps the transduction of resting cells by stimulating the efficiency of the reverse transcription process by degrading the limiting factor SAMHD1. Therefore, it is believed that in the methods provided herein, wherein Vpx is present in replication-defective recombinant retroviral particles used to transduce T cells and/or NK cells, Vpx is released into the cytoplasm of resting T cells or resting NK cells after transduction of cells by replication-defective recombinant retroviral particles containing Vpx. Vpx then degrades SAMHD1, which increases free dNTPs, which in turn stimulates reverse transcription of the retroviral genome.

In some embodiments, the replication-deficient recombinant retroviral particles provided herein include and/or contain Vpu polypeptides. Vpu polypeptides can be expressed from plasmids or packaging cell lines as viral membrane proteins, for example, incorporated into retroviral membranes, after Vpu encoding nucleic acids are integrated into their genomes. Recombinations formed by Vpu and its sequence codons optimized for expression in humans can be constructed and expressed so that free Vpu can be incorporated into viral particles. Vpu is an auxiliary protein found in HIV-1 and some HIV-1-related simian immunodeficiency viruses (SIV) isolates (such as SIVcpz, SIVgsn, and SIVmon) but not in HIV-2 or most SIV isolates. Vpu protein is involved in the downregulation of CD4 and the antagonism of binding proteins for particle release. More importantly, Vpu shares structural similarities with viroporins and, in particular, viroporin M2 from influenza virus A (IAV), and therefore, Vpu is shown to form cation-selective ion channels and permeate membranes in a variety of models. It has been shown that IAV-M2 helps acidify particles and thus favors early release from the endosomal pathway for entry, thereby reducing the amount of terminally transported products.

Example 4 herein shows that transduction of resting PBMC by retroviral particles is enhanced by the presence of Vpu in the retroviral particles. Without being limited by theory, the Vpu polypeptide assists the transduction of resting cells by accelerating the acidification of the lentiviral pseudotype, thereby allowing more capsids to reach the cytoplasm faster. Acidification of the virus interior leads to weakening of the electrostatic interactions between the viral structural proteins, and thus helps to decompose the capsid and ribonucleic acid protein (RNP) complex. Therefore, it is believed that in the method provided herein (wherein Vpu is present in the replication-deficient recombinant retroviral particle membrane for transducing T cells and/or NK cells), after transducing cells by replication-deficient recombinant retroviral particles containing Vpu, Vpu accelerates the acidification of the lentiviral particles in the endosomal pathway, and is conducive to the release of the capsid into the cytoplasm of resting T cells and/or NK cells. Therefore, in some embodiments, a Vpu polypeptide is included, which contains or is a fragment of Vpu, and the Vpu fragment retains the ability to promote the transduction of resting PBMCs (and in some embodiments, resting T cells). The fragment may comprise a fragment of Vpu that is at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to wild-type Vpu. In some embodiments, the Vpu polypeptide includes a Vpu fragment that retains the ability to promote acidification of lentiviral particles in the endosomal pathway.

Retroviral genome size

In the methods and compositions provided herein, the recombinant retroviral genome (in non-limiting illustrative examples, a lentiviral genome) has a limit on the number of polynucleotides that can be packaged into viral particles. In some embodiments provided herein, the polypeptide encoded by the polynucleotide coding region may be a truncation or other deletion that retains functional activity, so that the polynucleotide coding region is encoded by fewer nucleotides than the polynucleotide coding region of the wild-type polypeptide. In some embodiments, the polypeptide encoded by the polynucleotide coding region may be a fusion polypeptide that can be expressed by a promoter. In some embodiments, the fusion polypeptide may have a cleavage signal to produce two or more functional polypeptides by a fusion polypeptide and a promoter. In addition, some functions that are not needed after the initial ex vivo transduction are not included in the retroviral genome, but are present on the surface of the replication-deficient recombinant retroviral particles via the packaging cell membrane. These different strategies are used herein to maximize the functional components packaged in the replication-deficient recombinant retroviral particles.

In some embodiments, the recombinant retroviral genome to be packaged may be between 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, and 8,000 nucleotides at the low end of the range and 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, and 11,000 nucleotides at the high end of the range. The retroviral genome to be packaged includes one or more polynucleotide regions encoding the first and second engineered signaling polypeptides as disclosed in detail herein. In some embodiments, the retroviral genome to be packaged may be less than 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, or 11,000 nucleotides. Packageable functions discussed elsewhere herein include retroviral sequences required for retroviral assembly and packaging, such as retroviral rev, gag, and pol coding regions, as well as 5'LTR and 3'LTR or active truncated fragments thereof, nucleic acid sequences encoding retroviral cis-acting RNA packaging components, and cPPT/CTS components. In addition, in illustrative embodiments, the replication-defective recombinant retroviral particles herein may include any one or more or all of the following (in some embodiments in the opposite orientation relative to the 5' to 3' orientation established by the retroviral 5'LTR and 3'LTR (as shown in FIG. 4C as a non-limiting example)): one or more polynucleotide regions encoding first and second engineered signaling polypeptides, at least one of which includes at least one lymphoproliferative component; a second engineered signaling polypeptide that may include a chimeric antigen receptor; a miRNA (control component), such as a riboswitch, which generally regulates expression of the first and/or second engineered signaling polypeptide; a recognition domain, an intron, a promoter active in a target cell, such as a T cell, a 2A cleavage signal, and/or an IRES.

Recombinant Retroviral Particles

Recombinant retroviral particles are disclosed in the methods and compositions provided herein, for example, for transducing T cells and/or NK cells to prepare genetically modified T cells and/or NK cells. The recombinant retroviral particles themselves are aspects of the present invention. Typically, the recombinant retroviral particles included in the aspects provided herein are replication-defective, meaning that the recombinant retroviral particles are unable to replicate when they leave the packaging cell. In illustrative embodiments, the recombinant retroviral particles are lentiviral particles.

In some aspects, a replication-deficient recombinant retroviral particle is provided herein for transducing cells, usually lymphocytes and in illustrative embodiments, T cells and/or NK cells. Replication-deficient recombinant retroviral particles may include any of the pseudotyped components discussed elsewhere herein. In some embodiments, replication-deficient recombinant retroviral particles may include any of the activation components discussed elsewhere herein. In one aspect, replication-deficient recombinant retroviral particles including polynucleotides are provided herein, the polynucleotides including: A. One or more transcription units operably linked to a promoter active in T cells and/or NK cells, wherein the one or more transcription units encode a chimeric antigen receptor (CAR); and B. pseudotyped components and T cell activation components on its surface, wherein the T cell activation component is not encoded by a polynucleotide in a replication-deficient recombinant retroviral particle. In some embodiments, the T cell activation component may be any of the activation components discussed elsewhere herein. In illustrative embodiments, the T cell activation component may be an anti-CD3 scFvFc. In another aspect, a replication-deficient recombinant retroviral particle is provided herein, comprising a polynucleotide comprising one or more transcription units operably linked to a promoter active in T cells and/or NK cells, wherein the one or more transcription units encode a first polypeptide comprising a chimeric antigen receptor (CAR) and a second polypeptide comprising a lymphoproliferative component. In some embodiments, the lymphoproliferative component may be a chimeric lymphoproliferative component. In illustrative embodiments, the lymphoproliferative component does not include IL-7 linked to an IL-7 receptor alpha chain or a fragment thereof. In some embodiments, the lymphoproliferative component does not include IL-15 linked to an IL-2/IL-15 receptor beta chain.

In some aspects, provided herein is a replication-deficient recombinant retroviral particle comprising a polynucleotide comprising one or more transcription units operably linked to a promoter active in T cells and/or NK cells, wherein the one or more transcription units encode a first polypeptide comprising a chimeric antigen receptor (CAR) and a second polypeptide comprising a chimeric lymphoproliferative component (e.g., a constitutively active chimeric lymphoproliferative component). In illustrative embodiments, the chimeric lymphoproliferative component does not comprise a cytokine linked to its cognate receptor or to a fragment of its cognate receptor.

In some aspects, a recombinant retroviral particle is provided herein, comprising (i) a pseudotyped component that is capable of binding to T cells and/or NK cells and promoting membrane fusion of the recombinant retroviral particle; (ii) a polynucleotide having one or more transcription units operably linked to a promoter active in T cells and/or NK cells, wherein the one or more transcription units encode a first engineered signaling polypeptide having a chimeric antigen receptor (including an antigen-specific targeting region, a transmembrane domain, and an intracellular activation domain), and a second engineered signaling polypeptide comprising at least one lymphoproliferative component; wherein the expression of the first engineered signaling polypeptide and/or the second engineered signaling polypeptide is regulated by an in vivo control component; and (iii) an activation component on its surface, wherein the activation component is capable of binding to T cells and/or NK cells and is not encoded by a polynucleotide in the recombinant retroviral particle. In some embodiments, the promoter active in T cells and/or NK cells is not active in the packaging cell line, or is only active in the packaging cell line in an inducible manner. In any of the embodiments disclosed herein, one of the first and second engineered signaling polypeptides can have a chimeric antigen receptor and the other engineered signaling polypeptide can have at least one lymphoproliferative component.

The present invention provides various components and combinations of components included in replication-defective recombinant retroviral particles, such as pseudotyped components, activation components, and membrane-bound cytokines, and nucleic acid sequences included in the genome of replication-defective recombinant retroviral particles, such as but not limited to nucleic acids encoding CAR; nucleic acids encoding lymphoproliferative components; nucleic acids encoding control components (such as riboswitches); promoters, especially constitutively active or inducible promoters in T cells; and nucleic acids encoding inhibitory RNA molecules. In addition, various aspects provided herein (such as methods for preparing recombinant retroviral particles, methods for performing adoptive cell therapy, and methods for transducing T cells) produce and/or include replication-defective recombinant retroviral particles. The replication-defective recombinant retrovirus produced and/or included in such methods itself forms a replication-defective recombinant retroviral particle composition as an independent aspect of the present invention, and the composition may be in a separated form. Such compositions may be in a dried (e.g., lyophilized) form, or may be in a suitable solution or medium form known in the art for storage and use of retroviral particles.

Therefore, as a non-limiting example, in another aspect, there is provided herein a replication-deficient recombinant retroviral particle having a polynucleotide in its genome, the polynucleotide having one or more nucleic acid sequences operably connected to a promoter active in T cells and/or NK cells, in some cases, the nucleic acid sequence includes a first nucleic acid sequence encoding one or more (e.g., two or more) inhibitory RNA molecules for one or more RNA targets and a second nucleic acid sequence encoding a chimeric antigen receptor or CAR, as described herein. In other embodiments, there is a third nucleic acid sequence encoding at least one lymphoproliferative component that is not an inhibitory RNA molecule previously described herein. In certain embodiments, the polynucleotide includes one or more riboswitches as mentioned herein, the riboswitch being operably connected to the first nucleic acid sequence, the second nucleic acid sequence, and/or the third nucleic acid sequence (if present). In this construct, the expression of one or more inhibitory RNAs, CARs, and/or one or more lymphoproliferative components that are not inhibitory RNAs is controlled by a riboswitch. In some embodiments, two to 10 inhibitory RNA molecules are encoded by a first nucleic acid sequence. In other embodiments, two to six inhibitory RNA molecules are encoded by the first nucleic acid sequence. In illustrative embodiments, four inhibitory RNA molecules are encoded by the first nucleic acid sequence. In some embodiments, the first nucleic acid sequence encodes one or more inhibitory RNA molecules and is located in an intron. In certain embodiments, the intron includes all or part of a promoter. The promoter may be a Pol I, Pol II, or Pol III promoter. In some illustrative embodiments, the promoter is a Pol II promoter. In some embodiments, the intron is adjacent to a promoter active in T cells and/or NK cells and is located downstream of the promoter. In some embodiments, the intron is EF1-α intron A.

The recombinant retroviral particle embodiments herein include those in which the retroviral particles include a genome including one or more nucleic acids encoding one or more inhibitory RNA molecules. Various alternative embodiments of such nucleic acids encoding inhibitory RNA molecules that may be included in the genome of the retroviral particles (including such nucleic acids and combinations of other nucleic acids encoding CAR or lymphoproliferative components other than inhibitory RNA molecules) are included in, for example, the inhibitory RNA portion provided herein and in various other paragraphs of the combination of these embodiments. In addition, various alternatives to such replication-deficient recombinant retroviruses can be identified by the exemplary nucleic acids disclosed in the packaging cell line state disclosed herein. Skilled industry insiders will recognize that the disclosure of this part of the recombinant retroviral particles including the genome encoding one or more (e.g., two or more) inhibitory RNA molecules can be combined with various alternatives to such nucleic acids encoding inhibitory RNA molecules provided in other parts of this article. In addition, skilled industry insiders will recognize that such nucleic acids encoding one or more inhibitory RNA molecules can be combined with various other functional nucleic acid components provided herein, such as (e.g.,) disclosed herein focusing on inhibitory RNA molecules and nucleic acids encoding these molecules. In addition, the various examples of specific inhibitory RNA molecules provided elsewhere herein can be used in the recombinant retroviral particle aspects of the present invention.

The essential elements of a recombinant retroviral vector such as a lentiviral vector are known in the art. These elements are included in the packaging cell line section and in the details provided in the examples section for preparing recombinant retroviral particles. For example, lentiviral particles typically include packaging components REV, GAG, and POL, which can be delivered to a packaging cell line via one or more packaging plasmids; pseudotyping components, various examples provided herein, which can be delivered to a packaging cell line via a pseudotyped plasmid; and a genome, which is produced by a polynucleotide delivered to a host cell via a transfer plasmid. This polynucleotide typically includes a viral LTR and a psi packaging signal. The 5'LTR may be a chimeric 5'LTR fused to a heterologous promoter, including a 5''LTR that is independent of Tat transactivation. The transfer plasmid can be self-inactivated, for example by removing the U3 region of the 3'LTR. In some non-limiting embodiments, for any composition or method aspects and embodiments provided herein including reverse transcription particles, Vpu (such as a polypeptide comprising Vpu (sometimes referred to herein as a "Vpu polypeptide")) including (but not limited to) Src-FLAG-Vpu is packaged in a retroviral particle. In some non-limiting embodiments, Vpx (such as Src-FLAG-Vpx) is packaged in a retroviral particle. Without being limited by theory, after transduction of T cells, Vpx enters the cytosol of the cell and promotes the degradation of SAMHD1, thereby increasing the cytoplasmic dNTP pool available for reverse transcription. In some non-limiting embodiments, for any composition or method aspects and embodiments including reverse transcription particles provided herein, Vpu and Vpx are packaged in a retroviral particle.

The retroviral particles (e.g., lentiviral particles) included in the various aspects of the present invention are non-replicating in illustrative embodiments, particularly for safety reasons for embodiments including introducing cells transduced with such retroviral particles into individuals. When replication-defective retroviral particles are used to transduce cells, the retroviral particles are not produced by the transduced cells. Modification of the retroviral genome is known in the art to ensure that the retroviral particles comprising the genome are replication-defective. However, it should be understood that in some embodiments, for any of the aspects provided herein, replication-competent recombinant retroviral particles can be used.

Skilled practitioners will recognize that different types of vectors, such as expression vectors, may be used to deliver the functional components discussed herein to packaging cells and/or to T cells. Illustrative aspects of the invention utilize retroviral vectors, and (in some specific illustrative embodiments) lentiviral vectors. Other suitable expression vectors may be used to implement certain embodiments herein. Such expression vectors include, but are not limited to, viral vectors (e.g., those based on vaccinia virus; poliovirus; adenovirus (see, e.g., Li et al., Invest Opthalmol Vis Sci 35:2543-2549, 1994; Borras et al., Gene Ther 6:515-524, 1999; Li and Davidson, PNAS 92:7700-7704, 1995; Sakamoto et al., H Gene Ther 5:1088-1097, 1999; WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655); adeno-associated virus (see, e.g., Ali et al., Hum Gene Ther 9:81 86, 1998, Flannery et al., PNAS 94:6916 6921, 1997; Bennett et al., Invest Opthalmol Vis Sci 38:2857 2863, 1997; Jomary et al., Gene Ther 4:683 690, 1997, Rolling et al., Hum Gene Ther 10:641 648, 1999; Ali et al., Hum Mol Genet 5:591 594, 1996; WO Srivastava, Samulski et al., J. Vir. (1989) 63:3822-3828, 93/09239; Mendelson et al., Virol. (1988) 166:154-165; and Flotte et al., PNAS (1993) 90:10613-10617); SV40; herpes simplex virus; or a retroviral vector (e.g., mouse leukemia virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus), such as a gamma retrovirus; or human immunodeficiency virus (see, e.g., Miyoshi et al., PNAS 94:10319-23, 1997; Takahashi et al., J Virol 73:7812-7813, 1997; 7816,1999); and similar.

In illustrative embodiments, the retroviral particle is a lentiviral particle.Such retroviral particles typically include a retroviral genome within a capsid within a viral envelope.

In some embodiments, a DNA-containing viral particle is used instead of a recombinant retroviral particle. Such viral particles may be adenovirus, adeno-associated virus, herpes virus, cytomegalovirus, poxvirus, vaccinia virus, influenza virus, vesicular stomatitis virus (VSV), or Sindbis virus. A skilled artisan will understand how to modify the methods disclosed herein for different viruses and retroviruses or retroviral particles. When using viral particles comprising a DNA genome, a skilled artisan will understand that these genomes may include functional units to induce integration of all or part of the DNA genome of the viral particle into the genome of a T cell transduced with such a virus.

In some embodiments, HIV RRE and the polynucleotide region encoding HIV Rev can be replaced by the N-terminal RGG box RNA binding motif and the polynucleotide region encoding ICP27. In some embodiments, the polynucleotide region encoding HIV Rev can be replaced by one or more polynucleotide regions encoding adenovirus E1B 55-kDa and E4 Orf6.

In one aspect, the present invention provides a container (such as a commercial container or packaging) or a kit comprising it, comprising a separated replication-defective recombinant retroviral particle according to any one of the replication-defective recombinant retroviral particle aspects provided herein. In addition, in another aspect, a container (such as a commercial container or packaging) or a kit comprising it is provided herein, comprising a separated packaging cell according to any one of the packaging cell and/or packaging cell line aspects provided herein, in an illustrative embodiment, a separated packaging cell from a packaging cell line. In some embodiments, the kit includes an additional container, which includes additional reagents, such as a buffer or reagent for use in the method provided herein. In addition, in certain aspects, any replication-defective recombinant retroviral particle provided herein in any aspect is provided herein for use in the manufacture of a kit for genetically modifying a T cell or NK according to any aspect provided herein. In addition, in certain aspects, any packaging cell and/or packaging cell line provided herein in any aspect is provided herein for use in the manufacture of replication-defective recombinant retroviral particles according to any aspect provided herein.

In one aspect, provided herein are commercial containers containing replication-deficient recombinant retroviral particles and instructions for use to treat tumor growth in an individual, wherein the replication-deficient recombinant retroviral particles include a polynucleotide in their genome, the polynucleotide comprising one or more nucleic acid sequences operably linked to a promoter active in T cells and/or NK cells. In some embodiments, a nucleic acid sequence of one or more nucleic acid sequences may encode a chimeric antigen receptor (CAR), the chimeric antigen receptor comprising an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain. In some embodiments, a nucleic acid sequence of one or more nucleic acid sequences may encode one or more inhibitory RNA molecules for one or more RNA targets.

The container containing the recombinant retroviral particles can be a tube, a vial, a well plate, or other container for storing the recombinant retroviral particles. The test kit can include two or more containers, wherein the second or other containers can include, for example, a solution or medium for transducing T cells and/or NK cells, and/or the second or other containers can include a pH regulating pharmaceutical agent. Any of these containers can have industrial strength and grade. The replication defective recombinant retroviral particles in such an aspect including a test kit and a nucleic acid encoding an inhibitory RNA molecule can be any one of the embodiments of such replication defective recombinant retroviral particles provided herein, including any one of the embodiments of the inhibitory RNA provided herein.

In another aspect, a replication-deficient recombinant retroviral particle is provided herein for use in the manufacture of a kit for genetically modifying T cells and/or NK cells, wherein the use of the kit includes: contacting T cells or NK cells with replication-deficient recombinant retroviral particles in vitro, wherein the replication-deficient recombinant retroviral particles include a pseudotyped component on the surface and a T cell activation component on the surface, wherein the contact helps to transduce T cells or NK cells by replication-deficient recombinant retroviral particles, thereby producing genetically modified T cells or NK cells. In some embodiments, T cells or NK cells may be from an individual. In some embodiments, the T cell activation component may be membrane-bound. In some embodiments, the contact may be performed at a low end of 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours or 8 hours and at a high end of 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 10 hours, 12 hours, 15 hours, 18 hours, 21 hours and 24 hours, for example, between 1 hour and 12 hours. The replication-defective recombinant retroviral particles used to make the kit may include any of the aspects, embodiments, or subembodiments discussed elsewhere herein.

In another aspect, provided herein is a pharmaceutical composition for treating or preventing cancer or tumor growth comprising a replication-defective recombinant retroviral particle as an active ingredient. In another aspect, provided herein is an infusion composition for treating or preventing cancer or tumor growth comprising a replication-defective recombinant retroviral particle. The replication-defective recombinant retroviral particle of the pharmaceutical composition or infusion composition may include any of the aspects, embodiments, or sub-embodiments discussed above or elsewhere herein.

Genetically modified T cells and NK cells

In embodiments of the methods and compositions herein, a genetically modified lymphocyte that is a separate aspect of the present invention is produced. Such genetically modified lymphocytes may be transduced lymphocytes. In one aspect, genetically modified T cells or NK cells are provided herein, wherein the T cells or NK cells are genetically modified to express a first engineered signaling polypeptide. In some embodiments, the first engineered signaling polypeptide may be a lymphoproliferative component or a CAR including an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain. In some embodiments, the T cell or NK cell may further include a second engineered signaling polypeptide that may be a CAR or a lymphoproliferative component. In some embodiments, the lymphoproliferative component may be a chimeric lymphoproliferative component. In some embodiments, the T cell or NK cell may further include a pseudotyped component on the surface. In some embodiments, the T cell or NK cell may further include an activation component on the surface. The CAR, lymphoproliferative component, pseudotyped component, and activation component of the genetically modified T cell or NK cell may include any of the aspects, embodiments, or sub-embodiments disclosed herein. In an illustrative embodiment, the activating component may be an anti-CD3 scFvFc.

Some aspects provided herein include genetically modified T cells or NK cells made by transducing resting T cells and/or resting NK cells according to a method, the method comprising contacting the resting T cells and/or resting NK cells with replication-defective recombinant retroviral particles ex vivo, wherein the replication-defective recombinant retroviral particles comprise a pseudotyping component on their surface and a membrane-bound T cell activation component on their surface, wherein the contacting facilitates transduction of the resting T cells and/or NK cells by the replication-defective recombinant retroviral particles, thereby producing genetically modified T cells and/or NK cells, and wherein the contacting is performed between a low end of a range of 1 hour, 2 hours, 3 hours, 4 hours or 6 hours and a high end of a range of 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 18 hours, 20 hours or 24 hours, for example, between 1 hour and 12 hours.

In some embodiments, the genetically modified lymphocytes are lymphocytes, such as T cells or NK cells, that have been genetically modified to express a first engineered signaling polypeptide comprising at least one lymphoproliferative component and/or a second engineered signaling polypeptide comprising a chimeric antigen receptor, the chimeric antigen receptor comprising an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain. In some embodiments of any of the aspects herein, NK cells are NKT cells. NKT cells are a subset that expresses CD3 and typically co-expresses αβT cell receptors and also expresses multiple molecular markers (NK1.1 or CD56) that are typically associated with NK cells.

The genetically modified lymphocytes of the present invention have heterologous nucleic acid sequences that have been introduced into the lymphocytes by recombinant DNA methods. For example, the heterologous sequences in the illustrative embodiments are inserted into the lymphocytes during the method for transducing the lymphocytes provided herein. Heterologous nucleic acids are found in the lymphocytes and in some embodiments are integrated or not integrated into the genome of the genetically modified lymphocytes.

In illustrative embodiments, heterologous nucleic acids are integrated into the genome of genetically modified lymphocytes. In illustrative embodiments, such lymphocytes are produced using the methods provided herein for transducing lymphocytes using recombinant retroviral particles. Such recombinant retroviral particles may include polynucleotides encoding chimeric antigen receptors, which typically include at least one antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain. In other parts of the present invention, various embodiments of replication-deficient recombinant retroviral particles and polynucleotides encoded in the genome of replication-deficient retroviral particles are provided herein, and the replication-deficient retroviral particles can be used to produce genetically modified lymphocytes that themselves form another aspect of the present invention.

The genetically modified lymphocytes of the present invention can be separated in vitro. For example, such lymphocytes can be found in media and other solutions for ex vivo transduction as provided herein. Lymphocytes can be present in the blood collected from individuals using the method provided herein in a form that is not genetically modified, and then genetically modified during the transduction method. Genetically modified lymphocytes can be found inside individuals after they are genetically modified, after they are introduced or reintroduced into individuals. Genetically modified lymphocytes can be resting T cells or resting NK cells, or genetically modified T cells or NK cells can actively divide, especially after they are expressed in some of the functional components provided in the nucleic acid inserted into T cells or NK cells after transduction as disclosed herein.

In one aspect, provided herein is a transduced and/or genetically modified T cell or NK cell, comprising a recombinant polynucleotide, the recombinant polynucleotide comprising one or more transcription units in its genome operably connected to a promoter active in T cells and/or NK cells, the transcription unit expressing one or more of the functional components provided in any one of the aspects and embodiments of the present invention. For example, one or more transcripts may express CAR, which may include any one of the CAR components provided herein, such as ASTR (as a non-limiting example, MBR-ASTR), a transmembrane domain, and an intracellular signaling domain, and may further include a regulatory domain as a non-limiting example. In addition, provided herein is one or more functional components expressed in transduced and/or genetically modified T cells or NK cells, one or more of the lymphoproliferative components, such as constitutively active IL-7 receptor mutants or other lymphoproliferative components that are not inhibitory RNA molecules (such as miRNA or shRNA), recognition domains, and/or elimination domains.

In one aspect, provided herein is a genetically modified T cell or NK cell comprising:

a. one or more (eg, two or more) inhibitory RNA molecules directed against one or more RNA targets; and

b. a chimeric antigen receptor (CAR) comprising an antigen-specific targeting region (ASTR), a transmembrane domain and an intracellular activation domain,

And/or nucleic acids encoding inhibitory RNA molecules and CARs against one or more RNA targets, wherein the one (e.g., two) or more inhibitory RNA molecules and CARs or nucleic acids encoding them are encoded by or are genetically modified nucleic acid sequences of T cells and/or NK cells.

The genetically modified T cells or NK cells may be a genetically modified T cell population and/or NK cell population comprising one (e.g., two) or more inhibitory RNA molecules against one or more RNA targets; and CAR.

In some embodiments of the above T cells or NK cells comprising one or more (e.g., two or more) inhibitory RNA molecules and CARs or nucleic acids encoding the same, the components that may be included as part of the CAR or may be expressed together with the lymphoproliferative component or used to control the lymphoproliferative component may include any of the specific embodiments provided herein.

In some embodiments of the state of the T cell or NK cell comprising one or more (e.g., two or more) inhibitory RNA molecules and CAR or nucleic acids encoding them immediately above, CAR is a microenvironment restricted organism (MRB)-CAR and/or a genetically modified T cell or NK cell may further include at least one lymphoproliferative component that is not an inhibitory RNA molecule and/or a nucleic acid encoding a lymphoproliferative component. In such embodiments, the lymphoproliferative component is encoded by a nucleic acid sequence modified to the gene of the T cell and/or NK cell. In such embodiments, any one of the lymphoproliferative components disclosed herein may be used and/or encoded. For example, at least one lymphoproliferative component may be a constitutively active IL-7 receptor.

In some embodiments of the state that T cells or NK cells include one or more (e.g., two or more) inhibitory RNA molecules and CAR or nucleic acids encoding them immediately above, the inhibitory RNA molecule is a precursor of miRNA or shRNA. In some embodiments of this state, the one (e.g., two) or more inhibitory RNA molecules are polycistronic. In some embodiments of this state, the one (e.g., two) or more inhibitory RNA molecules are directed to the same or (in illustrative embodiments) different RNA targets. In some embodiments of this state, most or all of the one (e.g., two) or more inhibitory RNA molecules reduce the expression of endogenous TCR.

In some embodiments of the aspect that the T cell or NK cell comprises one or more (e.g., two or more) inhibitory RNA molecules and CAR or nucleic acid encoding the same, the RNA target is an mRNA transcribed from a gene selected from the group consisting of: PD-1, CTLA4, TCRα, TCRβ, CD3ζ, SOCS, SMAD2, miR-155 target, IFNγ, cCBL, TRAIL2, PP2A, and ABCG1. In some embodiments of this aspect, at least one of the one (e.g., two) or more inhibitory RNA molecules is miR-155.

In some embodiments of the state that the T cell or NK cell comprises one or more (e.g., two or more) inhibitory RNA molecules and CAR or nucleic acids encoding the same, the ASTR of CAR is MRB ASTR and/or the ASTR of CAR binds to a tumor-associated antigen. In addition, in some embodiments of the above state, the first nucleic acid sequence is operably connected to a riboswitch, which is, for example, capable of binding to a nucleoside analog, and in an illustrative embodiment is an antiviral drug, such as acyclovir.

In the methods and compositions disclosed herein, expression of the engineered signaling polypeptide is regulated by a control component, and in some embodiments, the control component is a polynucleotide comprising a riboswitch. In certain embodiments, the riboswitch is capable of binding a nucleoside analog, and when the nucleoside analog is present, one or both of the engineered signaling polypeptides are expressed.

The genetically modified lymphocytes disclosed herein may also have polypeptides on their surface that are remnants of fusion of replication-defective recombinant retroviral particles during the transduction methods provided herein. Such polypeptides may include activation components, pseudotype components, and/or one or more fusion polypeptides including cytokines.

In one aspect, provided herein is a genetically modified T cell and/or NK cell expressing one or more (e.g., two or more) inhibitory RNA molecules and chimeric antigen receptors or CARs for one or more RNA targets, as disclosed herein. In some embodiments, genetically modified T cells and/or NK cells further express at least one lymphoproliferative component disclosed herein that is not an inhibitory RNA molecule. In certain embodiments, genetically modified T cells and/or NK cells also express one or more riboswitches that control the expression of one or more inhibitory RNA molecules, CARs, and/or at least one lymphoproliferative component that is not an inhibitory RNA molecule. In some embodiments, genetically modified T cells and/or NK cells express two to 10 inhibitory RNA molecules. In other embodiments, genetically modified T cells and/or NK cells express two to six inhibitory RNA molecules. In illustrative embodiments, genetically modified T cells and/or NK cells express four inhibitory RNA molecules.

Nucleic Acids

The present invention provides nucleic acids encoding polypeptides of the present invention. In some embodiments, the nucleic acid will be DNA, including, for example, recombinant expression vectors. In some embodiments, the nucleic acid will be RNA, such as RNA synthesized in vivo.

In some cases, the nucleic acid provides, for example, production of a polypeptide of the invention in a mammalian cell. In other cases, the individual nucleic acid provides amplification of a nucleic acid encoding a polypeptide of the invention.

The nucleotide sequence encoding the polypeptide of the present invention may be operably linked to transcription control elements, such as a promoter and an enhancer.

Suitable promoters and enhancer elements are known in the art. For expression in bacterial cells, suitable promoters include, but are not limited to, lacI, lacZ, T3, T7, gpt, λP, and trc. For expression in eukaryotic cells, suitable promoters include, but are not limited to, light chain and/or heavy chain immunoglobulin gene promoters and enhancer elements; cytomegalovirus immediate early promoter; herpes simplex virus thymidine kinase promoter; early and late SV40 promoters; promoters present in the long terminal repeats of retroviruses; mouse metallothionein-I promoter; and various tissue-specific promoters known in the art.

Suitable reversible promoters (including reversible inducible promoters) are known in the art. Such reversible promoters can be isolated and derived from many organisms, such as eukaryotes and prokaryotes. Modification of a reversible promoter derived from a first organism (e.g., a first prokaryote and a second eukaryote, a first eukaryote and a second prokaryote, etc.) for use in a second organism is known in the art. Such reversible promoters, and systems based on such reversible promoters but also including other control proteins include, but are not limited to, alcohol-regulated promoters (e.g., alcohol dehydrogenase I (alcA) gene promoter, promoters responsive to alcohol transactivator proteins (AlcR), etc.), tetracycline-regulated promoters (e.g., promoter systems including TetActivators, TetON, TetOFF, etc.), steroid-regulated promoters (e.g., rat glucocorticoid receptor promoter system, human estrogen receptor promoter system , retinoid promoter system, thyroid promoter system, ecdysone promoter system, mifepristone promoter system, etc.), metal-regulated promoters (e.g., metallothionein promoter system, etc.), related pathogen-regulated promoters (e.g., salicylic acid-regulated promoters, ethylene-regulated promoters, benzothiadiazole-regulated promoters, etc.), temperature-regulated promoters (e.g., heat shock-inducible promoters (e.g., HSP-70, HSP-90, soybean heat shock promoter, etc.)), light-regulated promoters, synthetic inducible promoters, and the like.

In some cases, a locus or construct or transgene containing a suitable promoter is irreversibly converted via induction of an inducible system. Suitable systems for inducing irreversible conversion are well known in the art, for example, induction of irreversible conversion can utilize Cre-lox mediated recombination (see, for example, Fuhrmann-Benzakein et al., PNAS (2000) 28: e99, the disclosure of which is incorporated herein by reference). Any suitable combination of recombinases, endonucleases, ligases, recombination sites, etc. known in the art can be used to generate irreversibly converted promoters. The methods, mechanisms, and requirements for performing site-specific recombination described elsewhere herein are used to generate irreversibly converted promoters and are well known in the art, see, for example, Grindley et al. (2006) Annual Review of Biochemistry, 567-605 and Tropp (2012) Molecular Biology (Jones & Bartlett Publishers, Sudbury, MA), the disclosure of which is incorporated herein by reference.

In some cases, the promoter is a CD8 cell-specific promoter, a CD4 cell-specific promoter, a neutrophil-specific promoter, or a NK-specific promoter. For example, the CD4 gene promoter can be used, see, for example, Salmon et al. (1993) Proc. Natl. Acad. Sci. USA 90: 7739 and Marodon et al. (2003) Blood 101: 3416. As another example, the CD8 gene promoter can be used. NK cell-specific expression can be achieved by using the Neri (page 46) promoter; see, for example, Eckelhart et al. (2011) Blood 117: 1565.

In some embodiments, for example, for expression in yeast cells, suitable promoters are constitutive promoters, such as ADH1 promoter, PGK1 promoter, ENO promoter, PYK1 promoter, and the like; or regulatable promoters, such as GALI promoter, GAL10 promoter, ADH2 promoter, PH05 promoter, CUP1 promoter, GAL7 promoter, MET25 promoter, MET3 promoter, CYC1 promoter, HIS3 promoter, ADH1 promoter, PGK promoter, GAPDH promoter, ADC1 promoter, TRP1 promoter, URA3 promoter, LEU2 promoter, ENO promoter, TP1 promoter, and AOX1 (e.g., for use in Pichia pastoris). The selection of suitable vectors and promoters is within the level of those skilled in the art.

Suitable promoters for use in prokaryotic host cells include, but are not limited to, bacteriophage T7 RNA polymerase promoter, trp promoter, lac operator promoter, hybrid promoters (e.g., lac/tac hybrid promoter, tac/trc hybrid promoter, trp/lac promoter, T7/lac promoter); trc promoter; tac promoter and the like; araBAD promoter; promoters regulated in vivo, such as ssaG promoter or related promoters (see, e.g., U.S. Patent Publication No. 20040131637), pagC promoter (Pulkkinen and Miller, J. Bacterial., 1991: 173(1): 86-93 ; Alpuche-Aranda et al., PNAS, 1992; 89(21):10079-83), nirB promoter (Harborne et al. (1992) Mal. Micro. 6:2805-2813), and the like (see, e.g., Dunstan et al. (1999) Infect. Immun. 67:5133-5141; McKelvie et al. (2004) Vaccine 22:3243-3255; and Chatfield et al. (1992) Biotechnol. 10:888-892); σ70 promoter, e.g., consensus σ70 promoter (see, e.g., Genome Accession Nos. AX798980, AX798961, and AX798183); stationary phase promoters, such as dps promoter, spv promoter, and the like; promoters derived from pathogenicity island SPI-2 (see, e.g., WO 96/17951); actA promoter (see, e.g., Shetron-Rama et al. (2002) Infect. Immun. 70:1087-1096); rpsM promoter (see, e.g., Valdivia and Falkow (1996). Mal. Microbial. 22:367); tet promoter (see, e.g., Hillen, W. and Wissmann, A. (1989) In Saenger, W. and Heinemann, U. (eds.), Topics in Molecular and Structural Biology, Protein-Nucleic Acids Interaction. Macmillan, London, UK, Vol. 10, pp. 143-162); SP6 promoter (see, e.g., Melton et al. (1984) Nucl. Acids Res. 12:7035); and the like. Suitable strong promoters for prokaryotes such as E. coli include, but are not limited to, Trc, Tac, T5, T7, and Pλ. Non-limiting examples of operons for bacterial host cells include the lactose promoter operator (when in contact with lactose, the LacI repressor protein changes conformation, thereby preventing the LacI repressor protein from binding to the operator), the tryptophan promoter operator (when complexed with tryptophan, the TrpR repressor protein has a conformation that binds the operator; in the absence of tryptophan, the TrpR repressor protein has a conformation that does not bind the operator) and the tac promoter operator (see, e.g., deBoer et al. (1983) Proc. Natl. Acad. Sci. U.S.A. 80:21-25).

The nucleotide sequence encoding the polypeptide of the present invention may be present in an expression vector and/or a cloning vector. The nucleotide sequences encoding two separate polypeptides may be cloned in the same or different vectors. The expression vector may include a selective marker, a replication origin, and other components that provide for replication and/or maintenance of the vector. Suitable expression vectors include, for example, plasmids, viral vectors, and the like.

A large number of suitable vectors and promoters are known to those skilled in the art; many are commercially available for the production of individual recombinant constructs. The following bacterial vectors are provided by way of example: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, CA, USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden). The following eukaryotic vectors are provided by way of example: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene) pSVK3, pBPV, pMSG, and pSVL (Pharmacia).

The expression vectors usually have convenient restriction sites located near the promoter sequence to provide for the insertion of the nucleic acid sequence encoding the heterologous protein. A selectable marker that operates in the expression host may be present.

As described above, in some embodiments, the nucleic acid encoding the polypeptide of the present invention will be RNA, such as RNA synthesized in vitro. Methods for synthesizing RNA in vitro are known in the art; any known method can be used to synthesize RNA comprising a nucleotide sequence encoding a polypeptide of the present invention. Methods for introducing RNA into host cells are known in the art. See, for example, Zhao et al. (2010) Cancer Res. 15: 9053. Introducing RNA comprising a nucleotide sequence encoding a polypeptide of the present invention into a host cell can be performed in vitro, ex vivo, or in vivo. For example, host cells (e.g., NK cells, cytotoxic T lymphocytes, etc.) can be electroporated in vitro or ex vivo by RNA comprising a nucleotide sequence encoding a polypeptide of the present invention.

Various aspects and embodiments comprising polynucleotides, nucleic acid sequences and/or transcription units and/or vectors comprising the same further comprise one or more of a Kozak type sequence (also referred to herein as a Kozak-related sequence), a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and a double stop codon or a triple stop codon, wherein one or more stop codons of the double stop codon or the triple stop codon define the end of reading from at least one of the one or more transcription units. In certain embodiments, the polynucleotides, nucleic acid sequences and/or transcription units and/or vectors comprising the same further comprise a Kozak type sequence having a 5' nucleotide within 10 nucleotides upstream of the start codon of at least one of the one or more transcription units. The Kozak consensus sequence of Kozak assay 699 vertebrate mRNAs (GCC)GCCRCCATG (SEQ ID NO: 530), wherein R is a purine (A or G) (Kozak. Nucleic Acids Res. 1987 Oct 26; 15(20): 8125-48). In one embodiment, the Kozak type sequence is or includes CCACCAT/UG(G) (SEQ ID NO:515), CCGCCAT/UG(G) (SEQ ID NO:516), GCCGCCGCCAT/UG(G) (SEQ ID NO:517), or GCCGCCACCAT/UG(G) (SEQ ID NO:532) (wherein the nucleotides in parentheses represent optional nucleotides and the nucleotides separated by slashes indicate different possible nucleotides at that position, e.g., depending on whether the nucleic acid is DNA or RNA). In these embodiments including an AU/TG start codon, A can be considered as position 0. In certain illustrative embodiments, the nucleotides at -3 and +4 are the same, e.g., -3 and +4 can be G. In another embodiment, the Kozak type sequence includes an A or G in the 3rd position upstream of ATG, wherein ATG is the start codon. In another embodiment, the Kozak type sequence includes an A or G in the 3rd position upstream of AUG, wherein AUG is the start codon. In an illustrative embodiment, the Kozak-type sequence is (GCC)GCCRCCATG (SEQ ID NO: 530), wherein R is a purine (A or G). In an illustrative embodiment, the Kozak-type sequence is GCCGCCACCAUG (SEQ ID NO: 518). In another embodiment, which can be combined with the preceding embodiments comprising a Kozak-type sequence and/or the following embodiments comprising a triple codon, the polynucleotide comprises a WPRE element. The WPRE is characterized in the art (see, e.g., (Higashimoto et al., Gene Ther. 2007; 14: 1298)) and exemplified in Examples 11 and 12 herein. In some embodiments, the WPRE element is located 3' to the stop codon of one or more transcription units and 5' to the 3' LTR of the polynucleotide. In another embodiment, which can be combined with one or both of the preceding embodiments (i.e., an embodiment in which the polynucleotide comprises a Kozak-type sequence and/or an embodiment in which the polynucleotide comprises a WPRE), one or more transcription units are terminated by one or more stop codons of a double stop codon or a triple stop codon, wherein the double stop codon comprises a first stop codon in a first reading frame and a second stop codon in a second reading frame or a first stop codon in a frame with a second stop codon, and wherein the triple stop codon comprises a first stop codon in a first reading frame, a second stop codon in a second reading frame, and a third stop codon in a third reading frame or a first stop codon in a frame with a second stop codon or a third stop codon.

A triple stop codon herein includes three stop codons, one in each reading frame, within 10 nucleotides of each other, and preferably with overlapping sequences, or three stop codons in the same reading frame, preferably at consecutive codons. A double stop codon means two stop codons, each in a different reading frame, within 10 nucleotides of each other, and preferably with overlapping sequences, or two stop codons in the same reading frame, preferably at consecutive codons.

In some of the methods and compositions disclosed herein, the introduction of DNA into PBMCs, B cells, T cells and/or NK cells and, optionally, the incorporation of DNA into the host cell genome is performed using methods that do not utilize replication-defective recombinant retroviral particles. For example, other viral vectors such as those derived from adenovirus, adenovirus-associated virus, or herpes simplex virus type 1 may be utilized, as non-limiting examples.

In some embodiments, the methods provided herein may include transfecting and/or transducing target cells with non-viral vectors. In one of the embodiments disclosed herein that utilize non-viral vectors to transfect target cells, non-viral vectors (including naked DNA) may be introduced into target cells (such as PBMCs, B cells, T cells, and/or NK cells) using methods including electroporation, nucleofection, liposome formulations, lipids, dendrimers, cationic polymers (such as poly(ethyleneimine) (PEI) and poly(l-lysine) (PLL)), nanoparticles, cell penetrating peptides, microinjection, and/or non-integrated lentiviral vectors. In some embodiments, DNA may be introduced into target cells (such as PBMCs, B cells, T cells, and/or NK cells) in a complex with liposomes and protamine. Other methods for ex vivo transfection and/or transduction of T cells and/or NK cells that can be used in the embodiments of the methods provided herein are known in the art (see, e.g., Morgan and Boyerinas, Biomedicines. 2016 Apr 20;4(2).pii:E9, which is incorporated herein by reference in its entirety).

In some embodiments of the methods provided herein, the DNA can be integrated into the genome using a transposon-based vector system by co-transfecting, co-nucleofecting, or co-electroporating the target DNA into a plasmid containing transposon ITR fragments at the 5' and 3' ends of the gene of interest or co-transfecting, co-nucleofecting, or co-electroporating the transposase vector system into DNA or mRNA or protein or site-specific serine recombinase (such as phiC31 that integrates the gene of interest into the pseudo-attP site in the human genome), in which case the DNA vector may contain a 34 bp to 40 bp attB site, which is a recognition sequence for the recombinase (Bhaskar Thyagarajan et al. Site-Specific Genomic Integration in Mammalian Cells Mediated by Phage Integrase, Mol Cell Biol. 2001 June; 21(12): 3926–3934) and co-transfected with a recombinase. For T cells and/or NK cells, transposon-based systems useful in certain methods provided herein utilize the Sleeping Beauty DNA vector system (see, e.g., U.S. Pat. No. 6,489,458 and U.S. Patent Application No. 15/434,595, which are incorporated herein by reference in their entirety), the PiggyBac DNA vector system (see, e.g., Manuri et al., Hum Gene Ther. 2010 April; 21(4): 427-37, which are incorporated herein by reference in their entirety), or the Tol2 transposon system (see, e.g., Tsukahara et al., Gene Ther. 2015 February; 22(2): 209–215, which are incorporated herein by reference in their entirety) in the form of DNA, mRNA or protein. In some embodiments, the transposon and/or transposase of the transposon-based vector system can be produced as a minicircle DNA vector before introduction into T cells and/or NK cells (see, e.g., Hudecek et al., Recent Results Cancer Res. 2016; 209: 37-50 and Monjezi et al., Leukemia. 2017 January; 31(1): 186-194, which are incorporated herein by reference in their entirety). CAR or lymphoproliferative components can also be integrated into the genome by adding 50 bp to 1000 bp homology arms homologous to the integration 5' and 3' of the target site using CRISPR or TALEN-mediated integration to define and specific sites in the genome (Jae Seong Lee et al. Scientific Reports 5, Article No.: 8572 (2015), site-specific integration in CHO cells mediated by CRISPR/Cas9 and homology-directed DNA repair pathways). CRISPR or TALEN provides specific and gene-targeted cleavage and the construct will be integrated via homology-mediated end joining (Yao X et al. Cell Res. 2017 June; 27(6):801-814. doi:10.1038/cr.2017.76. 2017 May 19 Electronic Version). CRISPR or TALEN can be transfected as DNA, mRNA or protein via target plasmid.

Packaging cells

The present invention provides packaging cells and mammalian cell lines that are packaging cell lines, which produce replication-defective recombinant retroviral particles that genetically modify target mammalian cells and their mammalian cell lines. In illustrative embodiments, the packaging cells contain nucleic acid sequences encoding a packageable RNA genome, REV protein, gag polypeptide, pol polypeptide, and pseudotyping components of replication-defective recombinant retroviral particles.

Suitable mammalian cells include primary cells and immortalized cell lines. Suitable mammalian cell lines include human cell lines, non-human primate cell lines, rodent (e.g., mouse, rat) cell lines, and the like. Suitable mammalian cell lines include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC No. CRL9618, No. CCL61, No. CRL9096), HEK293 cells (e.g., ATCC No. CRL-1573), suspension-adapted HEK293 cells, Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RAT1 cells, mouse L cells (ATCC No. CCL1.3), human embryonic kidney (HEK) cells (ATCC No. CRL1573), HLHepG2 cells, Hut-78, Jurkat, HL-60, NK cell lines (e.g., NKL, NK92 and YTS), and the like.

In some cases, the cell is not an immortalized cell line, but a cell or ex vivo cell (e.g., a primary cell) obtained from an individual. For example, in some cases, the cell is an immune cell obtained from an individual. As another example, the cell is a stem cell or progenitor cell obtained from an individual. In any one of the embodiments and aspects provided herein including B cells, T cells or NK cells, the cell may be an autologous cell or it may be an allogeneic cell (also referred to herein as an allogeneic cell). In some embodiments, the allogeneic cell may be a genetically engineered allogeneic cell. Allogeneic cells (such as allogeneic T cells) and methods for genetically engineering allogeneic cells are known in the art. In some embodiments, the allogeneic cell may be an immortalized cell. In some embodiments in which the allogeneic cell is a T cell, the T cell is genetically engineered so that at least one of its T cell receptor chains does not work or is at least partially missing. In some embodiments, the allogeneic cell may be modified so that it lacks all or part of the B2 microglobulin. In some embodiments where allogeneic cells are used in the methods herein, the lymphoproliferative element and/or CLE may function to reduce the number of cells necessary to practice the invention and may facilitate cell production of T cells, B cells, or NK cells from the individual.

Methods of activating immune cells

The present invention provides a method for activating immune cells (lymphocytes in illustrative embodiments) in vitro, in vivo or in vitro. The method generally involves contacting immune cells (in vitro, in vivo or in vitro) with activation components or with one or more target antigens, wherein the immune cells are genetically modified to produce the CAR of the present invention (which is MRB-CAR in some embodiments). In the presence of one or more target antigens, CAR activates immune cells to produce activated immune cells. Immune cells include, for example, cytotoxic T lymphocytes, NK cells, CD4 ⁺ T cells, T regulatory (Treg) cells, γδT cells, NK-T cells, neutrophils, etc. In other embodiments, T cells are contacted with T cell activators to activate T cells. These methods are provided herein and discussed in the activation component section of this article.

Contacting genetically modified immune cells (e.g., T lymphocytes, NK cells) with one or more target antigens can increase the amount of cytokines produced by the immune cells by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about 2 times, at least about 2.5 times, at least about 5 times, at least about 10 times, or more than 10 times compared to the amount of cytokines produced by the immune cells in the absence of the one or more target antigens. The cytokines whose production can be increased include, but are not limited to, IL-2 and IFN-γ.

Contacting a genetically modified immune cell (e.g., a cytotoxic T lymphocyte) with an AAR can increase the cytotoxic activity of the cytotoxic cell by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, or more than 10-fold compared to the cytotoxic activity of the cytotoxic cell in the absence of one or more target antigens.

Contacting the genetically modified immune cells (e.g., cytotoxic T lymphocytes) with one or more target antigens can increase the cytotoxic activity of the cytotoxic cells by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, or more than 10-fold compared to the cytotoxic activity of the cytotoxic cells in the absence of the one or more target antigens.

In other embodiments, contacting a genetically modified host cell with an antigen can increase or decrease cell proliferation, cell survival, cell death, and the like, for example, depending on the host immune cell.

Inhibitory RNA molecules

In certain embodiments, the methods of the invention provided herein include inhibiting the expression of one or more endogenous genes expressed in T cells and/or NK cells. The methods provided herein illustrate the ability to prepare recombinant retroviral particles that express one or more (and in illustrative embodiments, two or more) inhibitory RNA molecules, such as miRNA or shRNA that can be used in such methods. In fact, the methods provided herein illustrate that such inhibitory RNA molecules can be encoded in introns including, for example, the Ef1a intron. This method teaches the use of this method to maximize the functional components that can be included in the packaged retroviral genome to overcome the shortcomings of previous teachings and maximize the effectiveness of such recombinant retroviral particles in adoptive T cell therapy.

In some embodiments, the inhibitory RNA molecule includes a 5' strand and a 3' strand (in some examples, a sense strand and an antisense strand) that are partially or completely complementary to each other, such that the two strands are able to form an RNA duplex of 18 to 25 nucleotides within a cellular environment. The 5' strand may be 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, and the 3' strand may be 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. The 5' strand and the 3' strand may be the same or different lengths, and the RNA duplex may include one or more mismatches. Alternatively, the RNA duplex has no mismatches.

The inhibitory RNA molecules included in the compositions and methods provided herein are not present in and/or are not naturally expressed in the T cells into which they are inserted into their genomes in certain illustrative embodiments. In some embodiments, the inhibitory RNA molecules are miRNA or shRNA. In some embodiments, when a nucleic acid encoding siRNA is mentioned herein or in a priority application, especially in the context of a nucleic acid being part of a genome, it will be understood that such nucleic acids are capable of forming siRNA precursors such as miRNA or shRNA in cells treated by DICER to form double-stranded RNA that generally interacts with or becomes part of a RISK complex. In some embodiments, the inhibitory molecule in one embodiment of the present invention may be a precursor of a miRNA (such as a Pri-miRNA or Pre-miRNA), or a precursor of an shRNA. In some embodiments, miRNA or shRNA is artificially derived (i.e., an artificial miRNA or siRNA). In other embodiments, the inhibitory RNA molecule is a dsRNA (transcribed or artificially introduced) or siRNA itself that is processed into a siRNA. In some embodiments, the miRNA or shRNA has a sequence that is not found in nature, or has at least one functional segment that is not found in nature, or has a combination of functional segments that are not found in nature.

In some embodiments, the inhibitory RNA molecule is placed in a first nucleic acid molecule in a series or multiple arrangements so that multiple miRNA sequences are simultaneously expressed from a single polycistronic miRNA transcript. In some embodiments, the inhibitory RNA molecule can be directly or indirectly connected to each other using a non-functional connector sequence. In some embodiments, the connector sequence length can be between 5 nucleotides and 120 nucleotides, and in some embodiments can be between 10 nucleotides and 40 nucleotides, as a non-limiting example. In illustrative embodiments, the first nucleic acid sequence encoding one or more (e.g., two or more) inhibitory RNAs and the second nucleic acid sequence encoding CAR (e.g., MRB-CAR) are operably connected to a promoter having constitutive activity or inducible in T cells or NK cells. Therefore, the inhibitory RNA molecule (e.g., miRNA) and CAR are expressed in a polycistronic manner. In addition, a functional sequence can be expressed from the same transcript. For example, any of the lymphoproliferative components that are not inhibitory RNA molecules provided herein can be expressed from the transcript identical to CAR and one or more (e.g., two or more) inhibitory RNA molecules.

In some embodiments, the inhibitory RNA molecule is a naturally occurring miRNA, such as but not limited to miR-155. Alternatively, an artificial miRNA can be produced in which a hybridization/complementary stem structure can be formed and a sequence for a target RNA is placed in a miRNA framework including a microRNA flanking sequence and a loop for microRNA processing, and can be optionally derived from a naturally occurring miRNA identical to the flanking sequence between the stem sequence. Therefore, in some embodiments, the inhibitory RNA molecule is oriented from 5' to 3' to include: a 5' microRNA flanking sequence, a 5' stem, a loop, a 3' stem partially or completely complementary to the 5' stem, and a 3' microRNA flanking sequence. In some embodiments, the 5' stem (also referred to herein as the 5' arm) may be 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In some embodiments, the 3' stem (also referred to herein as the 3' arm) may be 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In some embodiments, the length of the loop is 3 to 40, 10 to 40, 20 to 40, or 20 to 30 nucleotides, and in illustrative embodiments, the length of the loop may be 18, 19, 20, 21, or 22 nucleotides. In some embodiments, one stem is two nucleotides longer than the other stem. The longer stem may be the 5' stem or the 3' stem.

In some embodiments, the 5' microRNA flanking sequence, the 3' microRNA flanking sequence, or both are derived from naturally occurring miRNAs, such as, but not limited to, miR-155, miR-30, miR-17-92, miR-122, and miR-21. In certain embodiments, the 5' microRNA flanking sequence, the 3' microRNA flanking sequence, or both are derived from miR-155, such as miR-155 from Mus musculus or Homo sapiens. Inserting a synthetic miRNA stem-loop into the miR-155 framework (i.e., the 5' microRNA flanking sequence, the 3' microRNA flanking sequence, and the loop between the miRNA 5' stem and the 3' stem) is known to those of ordinary skill in the art (Chung, K. et al. 2006. Nucleic Acids Research. 34(7): e53; US 7,387,896). SIBR (synthetic inhibitory BIC-derived RNA) sequences (Chung et al. 2006, supra) for example have a 5' microRNA flanking sequence consisting of nucleotides 134 to 161 (SEQ ID NO: 256) of the Mus musculus BIC non-coding mRNA (Genbank ID AY096003.1) and a 3' microRNA flanking sequence consisting of nucleotides 223 to 283 of the Mus musculus BIC non-coding mRNA (Genbank ID AY096003.1). In one study, the SIBR sequence was modified (eSIBR) to enhance the expression of miRNA (Fowler, D.K. et al. 2015. Nucleic acids Research 44(5): e48). In some embodiments of the present invention, the miRNA may be placed in a SIBR or eSIBR miR-155 framework. In the illustrative embodiments herein, the miRNA is placed in a miR-155 framework, which includes the 5' microRNA flanking sequence of miR-155 shown by SEQ ID NO: 256, the 3' microRNA flanking sequence shown by SEQ ID NO: 260 (nucleotides 221 to 265 of the Mus musculus BIC non-coding mRNA); and a modified miR-155 loop (SEQ ID NO: 258). Thus, in some embodiments, the 5' microRNA flanking sequence of miR-155 is SEQ ID NO: 256 or a functional variant thereof, such as the same length as SEQ ID NO: 256, or 95%, 90%, 85%, 80%, 75% or 50% of the length of SEQ ID NO: 256, or 100 nucleotides or less, 95 nucleotides or less, 90 nucleotides or less, 85 nucleotides or less, 80 nucleotides or less, 75 nucleotides or less, 70 nucleotides or less, 65 nucleotides or less, 60 nucleotides or less, 55 nucleotides or less, 50 nucleotides or less, 45 nucleotides or less, 40 nucleotides or less, 35 nucleotides or less, 30 nucleotides or less, or 25 nucleotides or less in length; and is identical to SEQ ID NO: 256. NO:256 Sequences that are at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical. In some embodiments, the 3' microRNA flanking sequence of miR-155 is SEQ ID NO: 260 or a functional variant thereof, such as the same length as SEQ ID NO: 260, or 95%, 90%, 85%, 80%, 75%, or 50% of the length of SEQ ID NO: 260, or a length of 100 nucleotides or less, 95 nucleotides or less, 90 nucleotides or less, 85 nucleotides or less, 80 nucleotides or less, 75 nucleotides or less, 70 nucleotides or less, 65 nucleotides or less, 60 nucleotides or less, 55 nucleotides or less, 50 nucleotides or less, 45 nucleotides or less, 40 nucleotides or less, 35 nucleotides or less, 30 nucleotides or less, or 25 nucleotides or less; and a sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to SEQ ID NO: 260. However, any known microRNA framework that functions to provide appropriate processing within the cell of the miRNA into which it is inserted to form a mature miRNA capable of inhibiting the expression of its bound target mRNA is encompassed by the present invention.

In some embodiments, at least one, at least two, at least three, or at least four of the inhibitory RNA molecules encoded by the nucleic acid sequence in the polynucleotide of the replication-defective recombinant retroviral particle have the following arrangement in a 5' to 3' orientation: a 5' microRNA flanking sequence, a 5' stem, a loop, a 3' stem partially or completely complementary to the 5' stem, and a 3' microRNA flanking sequence. In some embodiments, all inhibitory RNA molecules have the following arrangement in a 5' to 3' orientation: a 5' microRNA flanking sequence, a 5' stem, a loop, a 3' stem partially or completely complementary to the 5' stem, and a 3' microRNA flanking sequence. As disclosed herein, the inhibitory RNA molecules may be separated by one or more connector sequences, which in some embodiments have no function other than as a spacer between the inhibitory RNA molecules.

In some embodiments, when two or more inhibitory RNA molecules are included (in some instances, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 inhibitory RNA molecules are included), these inhibitory RNA molecules are directed against the same or different RNA targets (such as mRNA transcribed from related genes). In illustrative embodiments, the first nucleic acid sequence includes between 2 and 10, between 2 and 8, between 2 and 6, between 2 and 5, between 3 and 5, or between 3 and 6 inhibitory RNA molecules. In an illustrative embodiment, the first nucleic acid sequence includes four inhibitory RNA molecules.

In some embodiments, the RNA target is an mRNA transcribed from a gene expressed by a free T cell, such as, but not limited to, PD-1 (prevents inactivation); CTLA4 (prevents inactivation); TCRα (safely prevents autoimmunity); TCRb (safety-prevents autoimmunity); CD3Z (safety-prevents autoimmunity); SOCS1 (prevents inactivation); SMAD2 (prevents inactivation); miR-155 target (promotes activation); IFNγ (reduces CRS); cCBL (prolongs signal transduction); TRAIL2 (prevents death); PP2A (prolongs signal transduction); ABCG1 (increases cholesterol microcontent by limiting the clearance of cholesterol). In an illustrative example, the miRNA inserted into the genome of a T cell in the method provided herein is directed to a target, such that the proliferation of the T cell is induced and/or enhanced and/or apoptosis is inhibited.

In some embodiments, the RNA target includes an mRNA encoding a component of a T cell receptor (TCR) complex. Such components may include components for producing and/or forming a T cell receptor complex and/or components for the proper function of a T cell receptor complex. Therefore, in one embodiment, at least one of the two or more inhibitory RNA molecules reduces the formation and/or function of a TCR complex (in an illustrative embodiment, one or more endogenous TCR complexes of a T cell). The T cell receptor complex includes TCR alpha, TCR b, CD3 d, CD3 e, CD3 g, and CD3 z. These components are known to have complex interdependencies, such that a reduction in the expression of any one subunit will result in reduced expression and function of the complex. Therefore, in one embodiment, the RNA target is an mRNA expressed in one or more of TCR alpha, TCR b, CD3 d, CD3 e, CD3 g, and CD3 z that are endogenous to a transduced T cell. In certain embodiments, the RNA target is an mRNA transcribed from an endogenous TCR alpha or TCR beta gene of a T cell, the genome of which comprises a first nucleic acid sequence encoding one or more miRNAs. In illustrative embodiments, the RNA target is an mRNA transcribed from a TCRα gene. In certain embodiments, inhibitory RNA molecules directed against mRNA transcribed from a target gene with similar expected utility can be combined. In other embodiments, inhibitory RNA molecules directed against target mRNA transcribed from a target gene with complementary utility can be combined. In certain embodiments, two or more inhibitory RNA molecules are directed against mRNA transcribed from target genes CD3Z, PD1, SOCS1, and/or IFNγ.

In some embodiments provided herein, two or more inhibitory RNA molecules can be delivered in a single intron, such as, but not limited to, EF1-αa intron A. Intron sequences that can be used to carry the miRNA of the present invention include any introns processed in T cells. As shown herein, one advantage of such an arrangement is that this helps to maximize the ability of miRNA sequences to be included in the size of the retroviral genome for delivering such sequences to T cells using the methods provided herein. This is especially true when the intron of the first nucleic acid sequence includes all or part of the promoter sequence (such as lymphoproliferative components that are not inhibitory RNA molecules) for expressing introns, CAR sequences, and other functional sequences provided herein in a polycistronic manner. The sequence requirements of introns are known in the art. In some embodiments, such intron processing is operably connected to a riboswitch, such as any riboswitch disclosed herein. Therefore, a riboswitch can provide a regulatory component for controlling the expression of one or more miRNA sequences on a first nucleic acid sequence. Thus, illustrative embodiments provided herein are combinations of miRNAs directed against endogenous T cell receptor subunits, wherein expression of the miRNAs is regulated by a riboswitch, which can be any of the riboswitches discussed herein.

In some embodiments, inhibitory RNA molecules may be provided on multiple nucleic acid sequences that may be included on the same or different transcription units. For example, a first nucleic acid sequence may encode one or more inhibitory RNA molecules and be expressed from a first promoter, and a second nucleic acid sequence may encode one or more inhibitory RNA molecules and be expressed from a second promoter. In illustrative embodiments, two or more inhibitory RNA molecules are located on a first nucleic acid sequence expressed from a single promoter. The promoter used to express such miRNAs is typically a promoter that is inactive in a packaging cell used to express a retroviral particle that delivers the miRNA in its genome to a target T cell, but such promoters are constitutively active or in an inducible manner in T cells. The promoter may be a Pol I, Pol II, or Pol III promoter. In some illustrative embodiments, the promoter is a Pol II promoter.

Characteristics and commercial production methods

The present invention provides a variety of methods and compositions, which can be used as research reagents in scientific experiments and for commercial production. This scientific experiment may include a method for characterizing lymphocytes (such as NK cells, and in illustrative embodiments, T cells) using a method for genetically modifying (e.g., transducing) lymphocytes provided herein. These methods can be used, for example, to study the activation of lymphocytes and the detailed molecular mechanisms by which these cells can be transduced through their activation. In addition, provided herein will be used, for example, as a research tool to better understand the factors that affect T cell proliferation and survival through genetically modified lymphocytes. These genetically modified lymphocytes (such as NK cells, and in illustrative embodiments, T cells) can be used for commercial production in addition, for example, for producing certain factors that can be collected or tested or used to produce commercial products, such as growth factors and immunomodulators.

The scientific experiments and/or features of lymphocytes may include any of the aspects, embodiments, or sub-embodiments provided herein for analyzing or comparing lymphocytes. In some embodiments, T cells and/or NK cells may be transduced with replication-defective recombinant retroviral particles provided herein including polynucleotides. In some embodiments, transduced T cells and/or NK cells may include polynucleotides including polynucleotides encoding polypeptides of the present invention, such as CAR, lymphoproliferative components, and/or activation components. In some embodiments, polynucleotides may include inhibitory RNA molecules as discussed elsewhere herein. In some embodiments, the lymphoproliferative component may be a chimeric lymphoproliferative component.

treatment method

The present invention provides various treatment methods using CAR. The CAR of the present invention can mediate cytotoxicity to target cells when present in T lymphocytes or NK cells. The CAR of the present invention binds to antigens present on target cells, thereby mediating the killing of target cells by T lymphocytes or NK cells that are genetically modified to produce CAR. The ASTR of CAR binds to antigens present on the surface of target cells.

The present invention provides a method for killing or inhibiting the growth of target cells, which method comprises contacting a cytotoxic immune effector cell (e.g., a cytotoxic T cell or NK cell) that has been genetically modified to produce an individual CAR, such that the T lymphocyte or NK cell recognizes an antigen present on the surface of the target cell and mediates the killing of the target cell.

The present invention provides a method for treating a disease or disorder in an individual suffering from the disease or disorder, the method comprising: a. introducing an expression vector comprising a polynucleotide sequence encoding a CAR into peripheral blood cells obtained from the individual to produce genetically engineered cytotoxic cells; and b. administering the genetically engineered cytotoxic cells to the individual.

In the methods provided herein comprising administering genetically modified T cells and/or NK cells to an individual (particularly an individual suffering from or suspected of having cancer), the method may further comprise delivering an effective dose of an immune checkpoint inhibitor to the individual. This checkpoint inhibitor delivery may occur before, after, or simultaneously with the administration of the genetically modified T cells and/or NK cells. Immune checkpoint inhibitors are known and various compounds are approved and in clinical studies. In the case of some immune checkpoint inhibitors, checkpoint molecules (many of which are targets of checkpoint inhibitor compounds) include the following:

CD27. This molecule supports antigen-specific expansion of naive T cells and is essential for the generation of T cell memory. Celldex Therapeutics is studying CDX-1127, an agonistic anti-CD27 pure antibody.

CD28. This molecule is constitutively expressed on nearly all human CD4+ T cells and on approximately half of all CD8 T cells. CD28 is the target of the TGN1412 "superagonist".

CD40. This molecule (found on various immune system cells including antigen presenting cells) has CD40L, otherwise known as CD154, as its ligand and is transiently expressed on the surface of activated CD4+ T cells. Anti-CD40 agonist simplex antibodies are licensed to Roche.

CD122. This molecule, which is the interleukin-2 receptor beta subunit, is known to increase the proliferation of CD8+ effector T cells. Nektar Therapeutics has developed NKTR-214, an immunostimulatory cytokine that is biased by CD122.

CD137. When this molecule (also called 4-1BB) is bound by the CD137 ligand, the result is T-cell proliferation. Pieris Pharmaceuticals has developed an engineered lipocalin with bispecificity for CD137 and HER2.

OX40. This molecule (also known as CD134) has OX40L or CD252 as its ligand. Like CD27, OX40 promotes the expansion of effector and memory T cells, however, it is also known for its ability to inhibit the differentiation and activity of T regulatory cells and to regulate cytokine production. AstraZeneca has three drugs in development targeting OX40: MEDI0562, a humanized OX40 agonist, MEDI6469, a murine OX4 agonist; and MEDI6383 (OX40 agonist).

GITR is an abbreviation for glucocorticoid-induced TNFR family-related gene, which promotes T cell expansion, including Treg expansion. TG Therapeutics is studying anti-GITR antibodies.

ICOS. This molecule is short for inducible T-cell co-stimulator and is also known as CD278, which is expressed on activated T cells. Jounce Therapeutics is developing ICOS agonists.

A2AR. The adenosine A2A receptor is considered an important checkpoint in cancer therapy because adenosine in the immune microenvironment leading to activation of the A2a receptor is a negative immune feedback loop and the tumor microenvironment has a relatively high concentration of adenosine.

B7-H3 (also known as CD276) was originally understood to be a co-stimulatory molecule but is now considered co-inhibitory. MacroGenics is developing MGA271, an Fc-optimized, pure-line antibody targeting B7-H3.

B7-H4 (also known as VTCN1) is expressed by tumor cells and tumor-associated macrophages and plays a role in tumor escape.

BTLA. This molecule is an abbreviation for B and T lymphocyte attenuator and is also known as CD272, which has HVEM (herpes virus entry mediator) as its ligand.

CTLA-4 is an abbreviation for cytokine T-lymphocyte-associated protein 4 and is also known as CD152, which is the target of Bristol-Myers Squibb's compound ipilimumab (Yervoy).

IDO is the abbreviation for indoleamine 2,3-dioxygenase, which is a tryptophan degrading enzyme with immunosuppressive properties. Another important molecule is TDO, tryptophan 2,3-dioxygenase. IDO. Newlink Genetics and Incyte have identified IDO pathway inhibitors.

KIR is the abbreviation for killer cell immunoglobulin-like receptor, which is a receptor for MHC class I molecules on natural killer cells. Bristol-Myers Squibb has developed Lirilumab, a simple antibody to KIR.

LAG3, which is short for lymphocyte activation gene-3, acts to suppress immune responses by acting on Tregs, and CD8+ T cells have a direct effect. Bristol-Myers Squibb has developed an anti-LAG3 pure antibody called BMS-986016.

PD-1 is the abbreviation of programmed death 1 (PD-1) receptor, which has two ligands, PD-L1 and PD-L2. This checkpoint is the target of Merck & Co.'s melanoma drug Keytruda.

TIM-3 is the abbreviation for T cell immunoglobulin domain and leukocyte domain 3, which is expressed on activated human CD4+ T cells and regulates Th1 and Th17 cytokines.

VISTA (protein) is the abbreviation for V-domain Ig inhibitory factor of T cell activation. VISTA is initially expressed on hematopoietic cells.

Individuals suitable for treatment

A variety of subjects are suitable for treatment by the methods and compositions of the present invention. Suitable subjects include any subject, such as a human or non-human animal, who has a disease or condition, has been diagnosed with a disease or condition, is at risk of having a disease or condition, has had a disease or condition and is at risk of recurring the disease or condition, has been treated with a drug for the disease or condition and has failed to respond to such treatment, or has been treated with a drug for the disease or condition but has relapsed after an initial response to such treatment.

Individuals suitable for treatment by immunomodulatory approaches include individuals with autoimmune diseases; individuals who are organ or tissue transplant recipients and the like; immunocompromised individuals; and individuals infected with pathogens.

Illustrative Embodiments

This exemplary embodiment section provides the exemplary aspects and embodiments provided herein and is further discussed throughout this specification. For the sake of brevity and convenience, all disclosed aspects and embodiments and all possible combinations of disclosed aspects and embodiments are not listed in this section. It should be understood that the embodiments provided are specific embodiments for many aspects, as discussed in this entire disclosure. It is intended that in view of the complete disclosure herein, any individual embodiment described below or in this complete disclosure may be combined with any aspect described below or in this complete disclosure, wherein it is an additional element that can be added to the aspect or because it is a narrower element for an element that has been presented in the aspect. This combination is specifically discussed in other sections of this detailed description.

In one aspect, provided herein is a replication-defective recombinant retroviral particle comprising a polynucleotide comprising:

A. one or more transcription units operably linked to a promoter active in T cells and/or NK cells, wherein the one or more transcription units encode a chimeric antigen receptor (CAR); and

B. A pseudotyping component and an activation component (e.g., a NK cell activation component, or in illustrative embodiments, a T cell activation component) on its surface, wherein the activation component is not encoded by a polynucleotide in the replication-defective recombinant retroviral particle.

In another aspect, a replication-deficient recombinant retroviral particle is provided herein, comprising a polynucleotide comprising one or more transcription units operably linked to a promoter active in T cells and/or NK cells, wherein the one or more transcription units encode a first polypeptide comprising a chimeric antigen receptor (CAR) and a second polypeptide comprising a lymphoproliferative component. In this section and in this disclosure, various embodiments of these particles such as, but not limited to, lymphoproliferative components, activation components, pseudotype components, control components, CARs, and other components are provided herein.

In another aspect, provided herein are replication-deficient recombinant retroviral particles comprising polynucleotides, the polynucleotides comprising one or more transcription units operably linked to a promoter active in T cells and/or NK cells, wherein the one or more transcription units encode a first polypeptide comprising a chimeric antigen receptor (CAR) and a second polypeptide comprising a chimeric lymphoproliferative component (e.g., a constitutively active chimeric lymphoproliferative component). In illustrative embodiments, the chimeric lymphoproliferative component does not include a cytokine linked to its cognate receptor or to a fragment of its cognate receptor. In this section and in this disclosure, various embodiments of these particles such as, but not limited to, lymphoproliferative components, activation components, pseudotyped components, control components, CARs, and other components are provided herein.

In another aspect, the present invention provides a replication-deficient recombinant retroviral particle comprising:

A. a polynucleotide comprising one or more transcription units operably linked to a promoter active in T cells and/or NK cells, wherein the one or more transcription units encode a chimeric antigen receptor (CAR); and

B. A pseudotyping component and a T cell activation component on its surface, wherein the T cell activation component is not encoded by a polynucleotide in the replication-defective recombinant retroviral particle, and wherein the T cell activation component is an anti-CD3 scFvFc antibody. In this section and throughout this disclosure, various embodiments of these particles such as, but not limited to, lymphoproliferative components, activation components, pseudotyping components, control components, CARs, and other components are provided herein.

In another aspect, the present invention provides a replication-deficient recombinant retroviral particle comprising:

A. one or more pseudotyping components that are capable of binding to T cells and/or NK cells and facilitating membrane fusion of the replication-defective recombinant retroviral particles therewith;

B. A polynucleotide comprising one or more transcription units operably linked to a promoter active in T cells and/or NK cells, wherein the one or more transcription units encode a first engineered signaling polypeptide comprising a chimeric antigen receptor (comprising an antigen-specific targeting region, a transmembrane domain, and an intracellular activation domain) and a second engineered signaling polypeptide comprising at least one lymphoproliferative component; wherein expression of the first engineered signaling polypeptide and/or the second engineered signaling polypeptide is regulated by a control component; and

C. An activation component on its surface, wherein the activation component is capable of binding to T cells and/or NK cells and is not encoded by a polynucleotide in the replication-defective recombinant retroviral particle. In this section and in this disclosure, various embodiments of these particles such as (but not limited to) lymphoproliferative components, activation components, pseudotyped components, control components, CARs and other components are provided herein.

In some aspects, provided herein are genetically modified lymphocytes (such as T cells or NK cells) made by ex vivo transduction of resting T cells and/or resting NK cells with any of the replication-deficient recombinant retroviral particles provided herein, wherein the contact facilitates transduction of resting T cells and/or NK cells by replication-deficient recombinant retroviral particles, thereby producing genetically modified T cells and/or NK cells. In some embodiments, the replication-deficient recombinant retroviral particles comprise a pseudotyped component on its surface and a membrane-bound T cell activation component on its surface. In this section and in this disclosure, various embodiments of this cell are provided herein, such as, but not limited to, contact time, lymphoproliferative component, activation component, pseudotyped component, control component, and other components.

In another aspect, the use of any replication-deficient recombinant retroviral particles in the manufacture of a kit for genetically modifying T cells and/or NK cells is provided herein, wherein the use of the kit includes the steps of any one of the method aspects and embodiments provided herein. For example, in another aspect, a replication-deficient recombinant retroviral particle is provided herein for the manufacture of a kit for genetically modifying T cells or NK cells, wherein the use of the kit includes: contacting T cells or NK cells with replication-deficient recombinant retroviral particles in vitro, wherein the replication-deficient recombinant retroviral particles include pseudotyped components on the surface and T cell activation components generally on the surface, wherein the contact helps to transduce T cells or NK cells by replication-deficient recombinant retroviral particles, thereby producing genetically modified T cells or NK cells. In some embodiments, the T cell activation component can be membrane-bound. In illustrative embodiments, the T cell activation component activates T cells via a T cell receptor-associated receptor complex. In some embodiments, the contacting can be performed between a low end of 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours or 8 hours and a high end of 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 10 hours, 12 hours, 15 hours, 18 hours, 21 hours and 24 hours, such as between 1 hour and 12 hours or between 1 hour and 5 hours. In this use aspect and other use aspects herein, the replication-defective recombinant retroviral particles used to make the kit may include any of the replication-defective recombinant retroviral particle aspects and embodiments provided herein. Various other embodiments of these methods such as, but not limited to, other contact times, lymphoproliferative components, activation components, pseudotype components, percentages of cells transfected/genetically modified, control components, and other components are provided herein.

In another aspect, provided herein are transduced and/or genetically modified lymphocytes, typically T cells and/or NK cells, and typically resting T cells and/or resting NK cells, comprising contacting lymphocytes with replication-deficient recombinant retroviral particles, wherein the replication-deficient recombinant retroviral particles typically include pseudotyped components on their surface, wherein the contact (which can also be considered as incubation under contact conditions) facilitates transduction of resting T cells and/or NK cells by replication-deficient recombinant retroviral particles, thereby producing genetically modified T cells and/or NK cells. The pseudotyped components are typically capable of binding to resting T cells and/or NK cells and typically facilitate their own membrane fusion or binding to other proteins of replication-deficient recombinant retroviral particles. In some embodiments of the method, the replication-deficient recombinant retroviral particles include an activation component, such as a T cell activation component that activates T cells via a T cell receptor-associated complex. This activation component can be an anti-CD3 antibody, such as an anti-CD3 scFv or an anti-CD3 scFvFc. Various exemplary and illustrative contact times are provided herein. Various other embodiments of these methods are provided herein such as, but not limited to, contact time, lymphoproliferative components, activation components, pseudotyping components, percentage of transfected/genetically modified cells, control components, Kozak-type sequences, WPRE components, triple termination sequences, and other components.

In another aspect, provided herein is a method for genetically modifying and/or transducing lymphocytes, the method comprising contacting lymphocytes with replication-deficient recombinant retroviral particles ex vivo, wherein the replication-deficient recombinant retroviral particles comprise a pseudotyping component on their surface and a membrane-bound T cell activation component on their surface, wherein the contact facilitates transduction of lymphocytes by the replication-deficient recombinant retroviral particles, thereby producing genetically modified lymphocytes. In some embodiments, the membrane-bound T cell activation component is an anti-CD3 antibody, such as an anti-CD3 scFv or an anti-CD3 scFvFc. Various other embodiments of these methods are provided herein, such as, but not limited to, contact time, lymphoproliferative component, activation component, pseudotyping component, percentage of cells transfected/genetically modified, control component, Kozak-like sequence, WPRE, triple termination, and other components.

In another aspect, provided herein is a method of transducing resting lymphocytes of an individual, the method comprising contacting resting T cells and/or resting NK cells with replication-defective recombinant retroviral particles ex vivo, wherein the replication-defective recombinant retroviral particles comprise a pseudotyping component on their surface that is capable of binding to resting T cells and/or resting NK cells and facilitating membrane fusion of the replication-defective recombinant retroviral particles therewith, wherein the contacting facilitates transduction of resting T cells and/or NK cells by the replication-defective recombinant retroviral particles, thereby producing genetically modified T cells and/or NK cells. In illustrative embodiments of this aspect, at least 10%, 20%, or 25% of resting T cells and/or NK cells, or between 10% and 70%, between 10% and 50%, or between 20% and 50% of T cells and/or NK cells are transduced as a result of the method. Various other embodiments of these methods are provided herein such as, but not limited to, contact time, lymphoproliferative components, activation components, pseudotyping components, percentage of transfected/genetically modified cells, control components, and other components.

In another aspect, provided herein is a method for transducing resting T cells and/or resting NK cells from isolated cells, comprising:

A. Collect blood from individuals;

B. Isolating peripheral blood mononuclear cells (PBMCs) containing resting T cells and/or resting NK cells;

C. contacting resting T cells and/or resting NK cells of an individual with replication-defective recombinant retroviral particles ex vivo, wherein the replication-defective recombinant retroviral particles comprise a pseudotyping component on their surface that is capable of binding to resting T cells and/or resting NK cells and facilitates binding of the replication-defective recombinant retroviral particles to their membranes, wherein the contacting facilitates transduction of at least 5% of resting T cells and/or NK cells by the replication-defective recombinant retroviral particles, thereby generating genetically modified T cells and/or NK cells, thereby transducing resting T cells and/or NK cells. Various other embodiments of these methods are provided herein, such as, but not limited to, contact time, lymphoproliferative component, activation component, pseudotyping component, percentage of transfected/genetically modified cells, control component, and other components.

Typically, the packaging cell line is used to produce replication-deficient recombinant retroviral particles discussed elsewhere herein. In some embodiments, the packaging cell line may be a suspension cell line. In illustrative embodiments, the packaging cell line may be grown in a serum-free medium. In some embodiments, the lymphocytes may be from an individual. In illustrative embodiments, the lymphocytes may be from the blood of an individual. Various other embodiments of these methods such as, but not limited to, contact time, lymphoproliferative components, activation components, pseudotyping components, percentage of cells transfected/genetically modified, control components, and other components are provided herein.

In another aspect, provided herein is a method for transducing lymphocytes with a replication-defective recombinant retroviral particle, comprising:

A. transfecting packaging cells with a set of vectors comprising components of replication-defective recombinant retroviral particles, wherein the packaging cells are grown in a serum-free medium containing a suspension;

B. collecting the replication-defective recombinant retroviral particles from serum-free medium; and

C. Contacting lymphocytes with replication-defective recombinant retroviral particles. Various embodiments of these methods are provided herein such as, but not limited to, contact time, lymphoproliferative components, activation components, pseudotyping components, percentage of transfected/genetically modified cells, control components, and other components.

In another aspect, provided herein is a method for transducing lymphocytes with replication-defective recombinant retroviral particles, comprising:

A. culturing packaging cells in a suspension in a serum-free medium, wherein the packaging cells contain nucleic acid sequences encoding the packaging RNA genome, REV protein, gag polypeptide, pol polypeptide and pseudotyping components of the replication-defective retroviral particles;

B. collecting the replication-defective recombinant retroviral particles from serum-free medium; and

C. contacting the lymphocyte with the replication-defective recombinant retroviral particle, wherein the contacting is performed for less than 12 hours, thereby transducing the lymphocyte. Various other embodiments of these methods are provided herein, such as, but not limited to, other contact times, lymphoproliferative components, activation components, pseudotyping components, percentages of transfected/genetically modified cells, control components, and other components.

Other embodiments of methods for genetically modifying and/or transducing PBMCs, lymphocytes, NK cells, and/or in the illustrative embodiments, T cells, or related methods, uses, or products of the method aspects herein are provided in other sections of the disclosure within and outside this illustrative embodiments section, for example, in a section entitled "METHODS FOR TRANSDUCING AND/OR GENETICALLY MODIFYING LYMPHOCYTES". For example, unless incompatible with an aspect or stated in an aspect, other embodiments of any of the above methods or related uses or products of method aspects for genetically modifying and/or transducing may include any of the embodiments of replication-defective recombinant retroviral particles, lymphoproliferative components, CARs, pseudotyping components, riboswitches, activation components, membrane-bound cytokines, miRNAs, Kozak-like sequences, WPREs, triple stop codons, and/or other components disclosed herein. In certain illustrative embodiments, the retroviral particle is a lentiviral particle.

Unless incompatible with or stated in an aspect, in illustrative embodiments, the packaging cell is an immortalized cell comprising a DNA nucleic acid stably integrated therein encoding a packageable RNA genome. In some embodiments, the gag polypeptide and the pol polypeptide are expressed from one or more inducible promoters, wherein the method further comprises: during the culture period, adding a transactivator to induce expression of the gag polypeptide and the pol polypeptide from the one or more inducible promoters. Various embodiments of these methods such as, but not limited to, contact time, lymphoproliferative components, activation components, control components, and other components are provided herein.

Unless incompatible with an aspect or stated in an aspect, in illustrative embodiments of any of the method aspects for genetically modifying and/or transducing lymphocytes (T lymphocytes), for genetically modifying and expanding lymphocytes (e.g., T lymphocytes), or performing cell therapy or the like herein, the contacting may be performed for a low end of a range of between 5 seconds, 10 seconds, 15 seconds, 30 seconds, or 45 seconds, or 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes, or 45 minutes, or 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, or 8 hours, and a high end of a range of between 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours, and 72 hours. For example, in illustrative embodiments, the contacting is carried out for between 2 hours and 24 hours, between 2 hours and 20 hours, between 2 hours and 6 hours, between 1 hour and 20 hours, between 1 hour and 12 hours, between 1 hour and 4 hours, between 4 hours and 12 hours, or between 4 hours and 8 hours. In some embodiments, the replication-defective recombinant retroviral particles may be washed immediately after adding the cells to be genetically modified and/or transduced, such that the contacting time is carried out for the length of time it takes to wash the replication-defective recombinant retroviral particles. Thus, typically, the contacting includes at least an initial contacting step in which the retroviral particles are contacted with the cells in suspension in the transduction reaction mixture. In some embodiments, unless stated otherwise, or otherwise stated in the broadest aspects provided herein, in any of the methods, methods for producing a product, or uses thereof, contacting of PBMCs, NK cells, and in illustrative embodiments, T cells with replication-defective recombinant retroviral particles may be performed for between 1 minute and 12 hours, between 5 minutes and 12 hours, between 10 minutes and 12 hours, between 15 minutes and 12 hours, between 30 minutes and 12 hours, or between 1 hour and 24 hours, such as between 1 hour and 12 hours or between 1 hour and 6 hours. In some embodiments, contacting may be performed for less than 24 hours, such as less than 12 hours, less than 8 hours, less than 4 hours, less than 3 hours, less than 2 hours, less than 1 hour, less than 30 minutes, less than 15 minutes, less than 10 minutes, less than 5 minutes, less than 4 minutes, less than 3 minutes, less than 2 minutes, or less than 1 minute. In illustrative embodiments, contacting is performed for between 1 minute and 12 hours, between 5 minutes and 12 hours, between 15 minutes and 12 hours, between 30 minutes and 12 hours, between 1 hour and 14 hours, between 1 hour and 12 hours, between 1 hour and 6 hours, between 1 hour and 4 hours, or between 2 hours and 14 hours. Such methods may be performed without prior activation.

Unless incompatible with an aspect, or already stated in an aspect, in the illustrative embodiments for genetically modifying and/or transducing lymphocytes (e.g., T lymphocytes), for genetically modifying and expanding lymphocytes (e.g., T lymphocytes), or for performing any of the method aspects of cell therapy herein or similar methods, other embodiments of the time range of methods of genetically modifying and/or transducing or related uses or products of the method aspects herein are contemplated to be provided in other sections of the present disclosure within and outside of this illustrative embodiments section, for example, in the section entitled “(Methods for Transducing and/or Genetically Modifying Lymphocytes) METHODS FOR TRANSDUCING AND/OR GENETICALLY MODIFYING LYMPHOCYTES”.

Unless incompatible with an aspect, or stated in an aspect, in illustrative embodiments for genetically modifying and/or transducing lymphocytes (e.g., T lymphocytes), for genetically modifying and expanding lymphocytes (e.g., T lymphocytes), or for performing any of the method aspects of cell therapy herein or similar methods, the cells are activated during contacting and are not activated at all or for more than 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, or 8 hours prior to contacting. In certain illustrative embodiments, activation by a component that is not present on the surface of the retroviral particle does not require genetic modification and/or transduction of the cell. Thus, no such activation or stimulating component is required, except on the retroviral particle, before, during, or after contacting.

Unless incompatible with an aspect or stated in an aspect, in an illustrative embodiment of any of the method aspects for genetically modifying and/or transducing lymphocytes (e.g., T lymphocytes), for genetically modifying and expanding lymphocytes, or for performing cell therapy herein, or a similar method, wherein the replication-defective recombinant retroviral particle comprises a polynucleotide comprising one or more transcription units operably linked to a promoter active in T cells and/or NK cells, wherein the one or more transcription units encode a first polypeptide comprising a chimeric antigen receptor (CAR), and in the description In some embodiments, the second polypeptide comprises a chimeric lymphoproliferative component, and the genetically modified T cell or NK cell is capable of, suitable for, possesses, and/or is modified to survive and/or proliferate in ex vivo culture for at least 7 days, 14 days, 21 days, 28 days, 35 days, 42 days, or 60 days, or between 7 days and 14 days, 21 days, 28 days, 35 days, 42 days, or 60 days after transduction in the absence of added IL-2 or in the absence of added cytokines (such as IL-2, IL-15, or IL-7) and in the presence of an antigen recognized by the CAR.

Unless incompatible with an aspect or stated in an aspect, in the methods, kits, uses or compositions (e.g., polynucleotides, packaging cells or replication-defective recombinant retroviral particles) provided herein, the replication-defective recombinant retroviral particles contain or further contain an activation component on their surface that is capable of activating T cells and/or NK cells. Typically, an activation component such as a T cell activation component is not encoded by a polynucleotide in the replication-defective recombinant retroviral particle.

Unless incompatible with the aspect or stated in the aspect, in the methods, kits, uses or compositions (e.g., polynucleotides, packaging cells or replication-defective recombinant retroviral particles) provided herein describing T cells and/or NK cells or resting T cells and/or resting NK cells, in certain illustrative embodiments, the cell is a T cell.

Unless incompatible with the aspect or stated in the aspect, generally, the recombinant retroviral particles in any of the methods, kits, uses or compositions (e.g., polynucleotides, packaging cells or replication-defective recombinant retroviral particles) provided herein are replication-defective, that is, non-replicable. In illustrative embodiments, the retrovirus is a lentivirus, such as a replication or replication-defective HIV lentivirus. In illustrative embodiments, the retrovirus is a lentivirus, such as a replication-defective or replication-defective HIV lentiviral particle. Therefore, in the illustrative embodiments of any of the methods, kits, uses or compositions (e.g., polynucleotides, packaging cells or replication-defective recombinant retroviral particles) provided herein, if not described or otherwise described, the replication-defective recombinant retroviral particles are lentiviral particles and/or the genetically modified cells are genetically modified T cells.

Unless incompatible with the aspect or stated in the aspect, in the illustrative embodiments of any one of the aspects provided herein, the lymphocytes may be T cells and/or NK cells. In further embodiments, the T cells and/or NK cells may be resting T cells and/or resting NK cells.

Unless incompatible with an aspect or stated in an aspect, in illustrative embodiments of any of the method aspects for transducing, genetically modifying and expanding lymphocytes, or for performing adaptive cell therapy or the like as described herein, between 10% and 75%, or between 10% and 70%, or between 10% and 60%, or between 10% and 50%, or between 10% and 25%, or between 20% and 75%, or between 20% and 50%, or at least 10%, 20% or 25% of resting T cells are transduced or between 0% and 75% of NK cells are transduced. In other aspects, between 5% and 80%, or between 10% and 80%, or between 10% and 70%, or between 10% and 60%, or between 10% and 50%, or between 10% and 25%, or between 10% and 20%, or between 20% and 50% of resting NK cells are transduced.

Unless incompatible with an aspect or stated in an aspect, in illustrative embodiments of any of the method aspects for genetically modifying and expanding lymphocytes or for performing adaptive cell therapy or similar therapies described herein or any composition provided herein, expression of the second engineered signaling polypeptide is regulated by a control element.

Unless incompatible with the aspect or stated in the aspect, in any of the methods, uses, process products or compositions herein including genetically modified T cells or NK cells or replication-deficient recombinant retroviral particles, this genetically modified T cell or NK cell or replication-deficient recombinant retroviral particle may include a polynucleotide comprising one or more transcription units operably linked to a promoter active in T cells and/or NK cells, wherein the one or more transcription units encode a first polypeptide comprising a chimeric antigen receptor (CAR) and/or a second polypeptide comprising a T cell lymphoproliferative component. In some embodiments, the expression of CAR and/or lymphoproliferative components is controlled by a control component or a different control component for each. In some embodiments, CAR and/or lymphoproliferative components may be expressed on the same polypeptide, and in illustrative embodiments, CAR and/or lymphoproliferative components are expressed as independent polypeptides. In some embodiments, CAR is an MRB CAR. In some embodiments, the chimeric lymphoproliferative component is constitutively active, such as a constitutively active chimeric cytokine receptor. In this embodiment, the constitutively active chimeric cytokine receptor can be controlled by a control component.

Unless incompatible with an aspect or stated in an aspect, in any of the aspects comprising a polynucleotide herein, the polynucleotide comprises one or more transcription units operably linked to a promoter active in T cells and/or NK cells, wherein the one or more transcription units encode a first polypeptide comprising a chimeric antigen receptor (CAR) and/or a second polypeptide comprising a T cell lymphoproliferative component, the polynucleotide may further comprise one or more of a Kozak-related sequence, a WPRE element, and multiple termination sequences (such as a double stop codon or a triple stop codon), wherein one or more of the double stop codon or the triple stop codon defines the termination of reading from at least one of the one or more transcription units. In certain embodiments, the polynucleotide comprises a Kozak-type sequence selected from the group consisting of CCACCAT/UG(G) (SEQ ID NO:515), CCGCCAT/UG(G) (SEQ ID NO:516), GCCGCCGCCAT/UG(G) (SEQ ID NO:517), or GCCGCCACCAT/UG(G) (SEQ ID NO:532). In certain embodiments, the -3 and +4 nucleotides relative to the start codon of the first nucleic acid sequence both comprise G. In another embodiment that may be combined with the preceding embodiment comprising a Kozak-type sequence and/or the following embodiment comprising a triple stop codon, the polynucleotide comprises a WPRE element. In some embodiments, the WPRE element is located 3' to the stop codon of one or more transcription units and 5' to the 3' LTR of the polynucleotide. In another embodiment that may be combined with any one or both of the preceding embodiments (i.e., an embodiment in which the polynucleotide comprises a Kozak-type sequence and/or an embodiment in which the polynucleotide comprises a WPRE), the one or more transcription units are terminated by one or more stop codons of a double stop codon or a triple stop codon.

Unless incompatible with an aspect or stated in an aspect, in the illustrative embodiments of any of the methods, uses and compositions provided herein that include a lymphoproliferative component, the lymphoproliferative component may be a chimeric lymphoproliferative component. In any of the embodiments herein that include a chimeric lymphoproliferative component, the chimeric lymphoproliferative component may be a chimeric cytokine receptor. In any of the embodiments herein that include a chimeric lymphoproliferative component, the chimeric lymphoproliferative component may be IL-7 connected to an IL-7 receptor alpha, or IL-7 that is not connected to an IL-7 receptor alpha. In some embodiments, a chimeric cytokine receptor comprises an IL-7 connected to an IL-7 receptor alpha, or a fragment thereof that retains the ability to promote the proliferation of T cells and/or NK cells, and wherein the chimeric cytokine receptor is constitutively active. In some embodiments, the chimeric cytokine receptor comprises IL-7 covalently linked to a functional extracellular fragment of an IL-7 receptor capable of binding IL-7, an IL-7 receptor transmembrane domain, and an IL-7 receptor signaling domain.

Unless incompatible with an aspect or stated in an aspect, in any of the aspects or embodiments herein comprising a chimeric lymphoproliferative component, the chimeric lymphoproliferative component may not be a chimeric cytokine receptor. In illustrative embodiments of any of the methods and compositions provided herein comprising a lymphoproliferative component, the chimeric lymphoproliferative component may be a chimeric cytokine receptor. In illustrative embodiments of any of the methods and compositions provided herein comprising a chimeric cytokine receptor, the chimeric cytokine receptor comprises an intracellular signaling domain of an IL-7 receptor, an intracellular signaling domain of an IL-12 receptor, an intracellular signaling domain of an IL-15/IL-2 receptor (such as an IL-15/IL-2β receptor), an intracellular signaling domain of an IL-21 receptor, an intracellular signaling domain of an IL-23 receptor, an intracellular signaling domain of an IL-27 receptor, an intracellular signaling domain of a transforming growth factor β (TGFβ) decoy receptor, or CD28. In some embodiments, the chimeric cytokine receptor comprises IL-7 fused to an IL-7 receptor.

Unless incompatible with an aspect or stated in an aspect, in the illustrative embodiments of any one of the methods and compositions provided herein including a lymphoproliferative component, the lymphoproliferative component may include a T cell survival primitive. The T cell survival primitive may include all or functional fragments of an IL-7 receptor, an IL-15 receptor, or CD28. In other embodiments, the lymphoproliferative component may include a cytokine or a cytokine receptor polypeptide, or a fragment thereof comprising a signaling domain. For example, the lymphoproliferative component may include an interleukin polypeptide and the lymphoproliferative component may further include a portion of its homologous interleukin receptor polypeptide covalently linked via a connector. Typically, this portion of a homologous interleukin receptor includes an extracellular domain capable of binding to an interleukin cytokine and a functional portion of a transmembrane domain. In some embodiments, the intracellular domain is an intracellular portion of a homologous interleukin receptor. In some embodiments, the intracellular domain is an intracellular portion of a different interleukin receptor that is capable of promoting lymphocyte proliferation and optionally survives in vitro or in vitro in culture for 6 days, 7 days, 14 days, 21 days, or 35 days or more during culture in the absence of exposing the lymphocytes to exogenous cytokines (such as IL-15, IL-7, and in illustrative embodiments, IL-2) and optionally in the absence of a target for an ASTR of the CAR expressed by the lymphocytes, or in certain illustrative embodiments, in the presence of an antigen recognized by the CAR, wherein the method comprises genetically modifying and/or transducing with a retroviral particle having a pseudotyping component and, optionally, an isolated or fused activation domain on its surface and typically without the need for pre-activation.

Unless incompatible with an aspect or stated in an aspect, in some embodiments, the lymphoproliferative element is an interleukin polypeptide covalently linked to its full-length cognate interleukin receptor polypeptide via a linker. Alternatively, the lymphoproliferative element can be the intracellular signaling domain of the IL-7 receptor, the intracellular signaling domain of the IL-12 receptor, the intracellular signaling domain of the IL-23 receptor, the intracellular signaling domain of the IL-27 receptor, the intracellular signaling domain of the IL-15 receptor, the intracellular signaling domain of the IL-21 receptor, or the intracellular signaling domain of the transforming growth factor β (TGFβ) decoy receptor.

Unless incompatible with an aspect or stated in an aspect, in some illustrative embodiments, the lymphoproliferative component is constitutively active. In addition, the lymphoproliferative component may include a mutant IL-7 receptor or a fragment thereof, which may further include a constitutively active mutant IL-7 receptor or a constitutively active fragment thereof. In some embodiments, the lymphoproliferative component or chimeric cytokine receptor comprises an IL7 InsPPCL mutant. Unless incompatible with an aspect or stated in an aspect, in any one of the aspects including the lymphoproliferative component, the lymphoproliferative component may be any CLE listed in the lymphoproliferative component section herein. For example, in illustrative embodiments, CLE comprises a domain from a gene gene particularly referred to as particularly noteworthy and/or the optimal construct (Top Constructs) relative to the examples herein.

Unless incompatible with an aspect or stated in an aspect, in any of the aspects and embodiments disclosed herein including a lymphoproliferative component, such as the products of the method, use, composition and process aspects, in illustrative embodiments, the genetically modified PBMCs, lymphocytes or genetically modified T cells and/or NK cells are able to survive and/or proliferate/expand in culture ex vivo or in vitro for at least 6 days, 7 days, 14 days, 21 days or 35 days or more during culture in the absence of exposure of the cells to cytokines (such as IL-15, IL-7, and in illustrative embodiments, IL-2) and optionally an ASTR to the CAR expressed by the cell, or in certain illustrative embodiments, in the presence of an antigen recognized by the CAR (particularly for embodiments in which the method comprises genetically modifying and/or transducing with retroviral particles having a pseudotyped component and optionally an isolated or fused activation domain on its surface and pre-activation is generally not required). In any of the embodiments disclosed herein comprising a CAR and a lymphoproliferative element, the genetically modified T cells and/or NK cells can be capable of being implanted into a mouse without being exposed to the target recognized by the CAR.

Unless incompatible with an aspect or stated in an aspect, in the illustrative embodiments of any of the methods and compositions provided herein that include replication-deficient recombinant retroviral particles, the replication-deficient recombinant retroviral particles may include an activation component on its surface. In illustrative embodiments, the activation component activates T cells via a T cell receptor-associated complex. In some cases, the activation component may only activate T cells. In other cases, the activation component requires activation via a TCR receptor complex to further activate T cells. Therefore, in some embodiments, the activation component comprises:

A. a membrane-bound polypeptide capable of binding to CD3; and/or

B. Membrane-bound polypeptides capable of binding to CD28.

In addition, the membrane-bound polypeptide capable of binding to CD3 is a polypeptide capable of binding to CD3 fused to a heterologous GPI anchoring linking sequence, and the membrane-bound polypeptide capable of binding to CD28 may be a polypeptide capable of binding to CD28 fused to a heterologous GPI anchoring linking sequence. In some embodiments, the membrane-bound polypeptide capable of binding to CD28 is CD80, CD86, or a functional fragment thereof capable of inducing CD28-mediated activation of Akt (such as the extracellular domain of CD80).

Unless incompatible with an aspect or stated in an aspect, in the illustrative embodiments of any of the methods and compositions provided herein that are replication-deficient recombinant retroviral particles or that include replication-deficient recombinant retroviral particles, the activation component can be a membrane-bound polypeptide capable of binding to CD3, such as anti-CD3 scFv or anti-CD3 scFvFc. In the illustrative embodiments of any of the methods and compositions provided herein that include replication-deficient recombinant retroviral particles and anti-CD3, anti-CD3 can be anti-CD3 scFvFc fused to a heterologous GPI anchoring linker sequence. In the illustrative embodiments of any of the methods and compositions provided herein that include replication-deficient recombinant retroviral particles, the membrane-bound polypeptide capable of binding to CD3 can be an anti-CD3 scFv bound to a CD14 GPI anchoring linker sequence, and the membrane-bound polypeptide capable of binding to CD28 can be CD80 or its extracellular domain bound to a CD16B GPI anchoring linker sequence. In illustrative embodiments of any of the methods and compositions provided herein that include replication-deficient recombinant retroviral particles, the replication-deficient recombinant retroviral particles may include on their surface an anti-CD3 scFv that binds to a CD14 GPI anchoring sequence, or CD80 or its extracellular domain that binds to a CD16B GPI anchoring sequence, and a fusion polypeptide of IL-7 or its activation fragment, and, and DAF that includes a GPI anchoring sequence. In illustrative embodiments of any of the methods and compositions provided herein that include replication-deficient recombinant retroviral particles, IL-7 or its activation fragment, and DAF fusion, anti-CD3 scFv, and CD80 and its extracellular domain each include a DAF signal sequence.

Unless otherwise stated, or stated in a broad aspect, in the illustrative embodiments of any of the methods and compositions provided herein that include replication-deficient recombinant retroviral particles, the replication-deficient recombinant retroviral particles may include a membrane-bound cytokine on its surface. The membrane-bound cytokine may be IL-7, IL-15, or an activated fragment thereof. In other embodiments, the membrane-bound cytokine is a fusion polypeptide of IL-7 or an activated fragment thereof and DAF. For example, the fusion polypeptide may include a DAF signal sequence (nucleotides 1 to 34 of SEQ ID NO: 286), IL-7 without its signal sequence (nucleotides 35 to 186 of SEQ ID NO: 286), and a DAF fragment including its GPI anchor linker sequence (nucleotides 187 to 532 of SEQ ID NO: 286).

Unless incompatible with an aspect or stated in an aspect, in illustrative embodiments of any of the methods and composition aspects provided herein, the pseudotyped component may comprise one or more heterologous envelope proteins. In other examples, the pseudotyped component may comprise one or more viral polypeptides recognized by T cells. The one or more pseudotyped components may comprise a measles virus F polypeptide, a measles virus H polypeptide, and/or a fragment thereof. The one or more pseudotyped components may be a cytoplasmic domain deletion variant of a measles virus F polypeptide and/or a measles virus H polypeptide.

Unless incompatible with an aspect or stated in an aspect, in illustrative embodiments of any of the methods and compositions provided herein that include a control component, the control component is a control component that modulates a lymphoproliferative component, wherein the lymphoproliferative component is inactive or has less activity in promoting proliferation of T cells and/or NK cells in the absence of the compound, and wherein the compound is a molecular chaperone that binds to the lymphoproliferative component and induces activity of the lymphoproliferative component.

Unless incompatible with an aspect or stated in an aspect, in illustrative embodiments of any of the methods and compositions provided herein that include a control component, the control component can be a polynucleotide comprising a riboswitch. The riboswitch can be capable of binding a nucleoside analog and the compound that binds the control component is a nucleoside analog. The nucleoside analog can be an antiviral agent. The antiviral agent can be acyclovir or penciclovir.

Unless incompatible with an aspect or stated in an aspect, in the illustrative embodiments of any of the methods and compositions provided herein that include engineered signaling polypeptides (which include ASTRs), the ASTRs of one or both of the engineered signal overlapping polypeptides may bind to tumor-associated antigens. In some illustrative embodiments, the antigen-specific targeting region of the second engineered polypeptide is a microenvironment-restricted antigen-specific targeting region (MRB-ASTR). In some embodiments, the microenvironment may be an in vivo microenvironment, such as a tumor, tissue, non-tumor tissue, normal tissue, or tissue that has undergone a transient change in pH. For example, tissues that typically undergo transient changes in pH include muscle tissue under anaerobic conditions or muscle tissue that has undergone exercise or inflamed tissue or tissue that is undergoing inflammation. In some embodiments that include a target mammalian cell, the target mammalian cell may be a tumor cell or a non-tumor cell or a normal cell.

Unless incompatible with an aspect or stated in an aspect, in the illustrative embodiments of any one of the methods and compositions provided herein including one or more replication-deficient recombinant retroviral particles, if not explicitly stated in the broadest aspect, the replication-deficient recombinant retroviral particles may encode the recognition domain of a biologically validated monoclonal antibody. In some embodiments, the recognition domain is expressed on the same transcript as the chimeric antigen receptor, and wherein the recognition domain is separated from the chimeric antigen receptor by ribosome skipping and/or cleavage signals. The ribosome skipping and/or cleavage signal may be 2A-1. The recognition domain may include a polypeptide or an antigenic determinant thereof recognized by an antibody that recognizes EGFR. The recognition domain may be an EGFR mutant recognized by an EGFR antibody and expressed on the surface of a transduced T cell and/or NK cell as another control mechanism provided herein. In a related embodiment, the recognition domain may include a polypeptide or an antigenic determinant thereof recognized by an antibody that recognizes EGFR.

Unless incompatible with any one of the aspects, methods, uses, process products or stated in one aspect, the method may further include collecting blood from the individual. The method may further include reintroducing genetically modified T cells and/or NK cells into the individual after ex vivo contact with the individual's resting T cells and/or resting NK cells. In certain embodiments, the resting T cells and/or resting NK cells contacted are from the individual's blood, and the expansion of the genetically modified T cells and/or NK cells occurs in the individual. In certain embodiments, the step between collecting blood and reintroducing genetically modified T cells and/or NK cells is performed within no more than 24 hours, 18 hours, 12 hours, 8 hours, 6 hours, or 4 hours. Other times are provided herein. In certain embodiments, the individual is not exposed to a lympho-depleting agent within 7 days of contact, during contact, and/or within 7 days after the genetically modified T cells and/or NK cells are reintroduced into the individual.

In one aspect, provided herein is a method of transducing and/or genetically modifying lymphocytes (e.g., T cells and/or NK cells) of an individual, in illustrative embodiments, resting lymphocytes (resting T cells and/or NK cells), the method comprising contacting resting T cells and/or resting NK cells of an individual ex vivo with replication-defective recombinant retroviral particles, wherein the replication-defective recombinant retroviral particles comprise a pseudotyping component on their surface and a membrane-bound anti-CD3 scFvFc antibody on their surface that is capable of binding to resting T cells and/or resting NK cells and facilitates fusion of the replication-defective recombinant retroviral particles therewith, wherein the contacting facilitates transduction of resting T cells and/or NK cells with the replication-defective recombinant retroviral particles, thereby generating genetically modified T cells and/or NK cells.

In one aspect, provided herein is a method for transducing and/or genetically modifying resting T cells and/or resting NK cells from isolated blood, the method comprising: collecting blood from a subject; isolating peripheral blood mononuclear cells (PBMCs) comprising resting T cells and/or resting NK cells; and contacting the resting T cells and/or resting NK cells of the subject ex vivo with replication-defective recombinant retroviral particles for an effective time, wherein the replication-defective recombinant retroviral particles comprise a pseudotyping component on their surface and a membrane-bound anti-CD3 scFvFc antibody on their surface, thereby producing genetically modified T cells and/or NK cells, thereby transducing resting T cells and/or NK cells.

In the present invention, in the embodiment of transducing and/or genetically modifying T lymphocytes (e.g., T cells and/or NK cells) comprising membrane-bound anti-CD3 scFvFc antibodies, the pseudotyped component is in certain embodiments a herpes oral virus envelope protein (VSV-G). In some embodiments, the replication-deficient recombinant retroviral particle further comprises a membrane-bound polypeptide capable of binding to CD28, which may include, for example, the extracellular domain of CD80, CD86, or a functional fragment thereof that retains the ability to bind to CD28. In some embodiments, the anti-CD3 scFvFc antibody is fused to a heterologous GPI anchoring linking sequence. In some embodiments, the anti-CD3 scFvFc antibody is not encoded by a polynucleotide in the replication-deficient recombinant retroviral particle.

In the embodiment of transducing and/or genetically modifying T lymphocytes (e.g., T cells and/or NK cells) comprising membrane-bound anti-CD3 scFvFc antibodies herein, the recombinant retroviral particle may further include a polynucleotide comprising one or more transcription units operably linked to a promoter active in T cells and/or NK cells, wherein the one or more transcription units encode a chimeric antigen receptor. In some embodiments, the membrane-bound polypeptide capable of binding to CD3 is not encoded by a polynucleotide in the replication-deficient recombinant retroviral particle. In some embodiments, the anti-CD3 scFvFc antibody is not encoded by a polynucleotide in the replication-deficient recombinant retroviral particle.

In another aspect, provided herein is a method for transducing and/or genetically modifying resting lymphocytes of an individual, the method comprising contacting resting T cells and/or resting NK cells of the individual with replication-defective recombinant retroviral particles ex vivo, wherein the replication-defective recombinant retroviral particles comprise a pseudotyping component on their surface and a membrane-bound polypeptide capable of binding to CD3 on their surface, but not a membrane-bound polypeptide capable of binding to CD28 and activating CD28 on their surface, wherein the contact facilitates transduction of resting T cells and/or NK cells using the replication-defective recombinant retroviral particles, thereby producing genetically modified T cells and/or NK cells.

In another aspect, provided herein is a method for transducing and/or genetically modifying resting T cells and/or resting NK cells from isolated blood, the method comprising: collecting blood from an individual; isolating peripheral blood mononuclear cells (PBMCs) containing resting T cells and/or resting NK cells; and contacting the individual's resting T cells and/or resting NK cells ex vivo with replication-deficient recombinant retroviral particles for an effective time, wherein the replication-deficient recombinant retroviral particles comprise a pseudotyping component on their surface and a membrane-bound polypeptide capable of binding to CD3 on their surface, but not a membrane-bound polypeptide capable of binding to CD28 and activating CD28 on their surface, thereby producing genetically modified T cells and/or NK cells, thereby transducing resting T cells and/or NK cells.

In the aspect of transducing and/or genetically modifying resting T lymphocytes herein, the resting T lymphocytes include a pseudotyped component on its surface and a membrane-bound polypeptide capable of binding to CD3 on its surface, but not a membrane-bound polypeptide capable of binding to CD28 and activating CD28 on its surface, the pseudotyped component can be, for example, herpes oral virus envelope protein (VSV-G). In illustrative embodiments, the membrane-bound polypeptide capable of binding to CD3 is an anti-CD3 scFvFc antibody, which in some embodiments is fused to a heterologous GPI anchor linker sequence. In some embodiments of this aspect, the contact is performed for at least 2 hours, or between 2 hours and 24 hours, or between 2 hours and 6 hours. In some embodiments, the detectable marker is encoded by the genome of the replication-deficient recombinant retroviral particle and is detected in T cells and/or NK cells after transduction. In some embodiments, the membrane-bound polypeptide capable of binding to CD3 is not encoded by a polynucleotide in the replication-deficient recombinant retroviral particle. In some embodiments, the detectable marker is encoded by the genome of the replication-defective recombinant retroviral particle and is detected in T cells and/or NK cells following transduction.

In another aspect, provided herein is a replication-deficient recombinant retroviral particle comprising: one or more pseudotyping components; a polynucleotide comprising one or more transcription units operably linked to a promoter active in T cells and/or NK cells, wherein the one or more transcription units encode a chimeric antigen receptor; and a pseudotyping component on its surface and an activation component on its surface, wherein the activation component is capable of binding to T cells and/or NK cells and is not encoded by the polynucleotide in the replication-deficient recombinant retroviral particle, and wherein the activation component is an anti-CD3 scFvFc antibody.

In another aspect, provided herein is a replication-deficient recombinant retroviral particle comprising: one or more pseudotyping components capable of binding to T cells and/or NK cells and facilitating fusion of the replication-deficient recombinant retroviral particle to the membrane thereon;

A polynucleotide comprising one or more transcription units operably linked to a promoter active in T cells and/or NK cells, wherein the one or more transcription units encode a chimeric antigen receptor; and a pseudotyped component on its surface and an activation component on its surface, wherein the activation component is capable of binding to T cells and/or NK cells and is not encoded by a polynucleotide in a replication-defective recombinant retroviral particle, and wherein the activation component is a membrane-bound polypeptide capable of binding to CD3 on its surface, but is not a membrane-bound polypeptide capable of binding to CD28 on its surface and activating CD28.

In some embodiments of the replication-deficient recombinant retroviral particle aspects herein, the recombinant retroviral particle further comprises a polynucleotide comprising one or more transcription units operably linked to a promoter active in T cells and/or NK cells, wherein the one or more transcription units encode a chimeric antigen receptor. In some embodiments of these aspects, the membrane-bound polypeptide capable of binding to CD3 is not encoded by the polynucleotide in the replication-deficient recombinant retroviral particle. In some embodiments of these aspects, the anti-CD3 scFvFc antibody is not encoded by the polynucleotide in the replication-deficient recombinant retroviral particle.

In any of the aspects and embodiments herein, such as those comprising a polypeptide or a nucleic acid encoding the same, including, for example, an activation component present on the surface of a replication-deficient recombinant retroviral particle, the activation component may further include, for example, a fusion polypeptide with a polypeptide capable of binding to CD28 or, in an illustrative embodiment, CD3, a dimerization motif that is active in the absence of a dimerizing agent. In some embodiments, the activation component may be an anti-CD3 single-chain antibody and the dimerization motif may be selected from the group consisting of: CD69, CD71, CD72, CD96, Cd105, Cd161, Cd162, Cd249, CD271, and Cd324, and mutants and/or active fragments thereof that retain dimerization ability.

In any of the aspects and embodiments herein, such as those comprising a polypeptide or a nucleic acid encoding the same, including, for example, an activation component present on the surface of a replication-defective recombinant retroviral particle, the activation component may further include, for example, a fusion polypeptide with a polypeptide capable of binding to CD28 or, in illustrative embodiments, CD3, a dimerization motif that is active in the absence of a dimerizing agent. In some embodiments, the activation component may be an anti-CD3 single-chain antibody and the dimerization motif may be selected from the group consisting of: FKBP and rapamycin or an analog thereof, GyrB and coumermycin or an analog thereof, DHFR and methotrexate or an analog thereof, or DmrB and AP20187 or an analog thereof, and mutants and/or active fragments of the dimerization proteins that retain the dimerization ability.

In any of the aspects and embodiments herein, such as those comprising a polypeptide or a nucleic acid encoding the same, comprising, for example, an activation component present on the surface of a replication-defective recombinant retroviral particle, the activation component may further comprise an anti-CD3-scFvFc-GPI portion (UCHT1) and, optionally, VSV-G.

In one embodiment of the above aspect, the polynucleotide further comprises a Kozak type sequence, a WPRE component, and one or more of a double stop codon or a triple stop codon, wherein one or more of the double stop codon or the triple stop codon defines the termination of reading from at least one of the one or more transcription units. In certain embodiments, the polynucleotide comprises a Kozak type sequence selected from the following: CCACCAT/UG(G) (SEQ ID NO:515), CCGCCAT/UG(G) (SEQ ID NO:516), GCCGCCGCCAT/UG(G) (SEQ ID NO:517), or GCCGCCACCAT/UG(G) (SEQ ID NO:532). In certain embodiments, the -3 and +4 nucleotides relative to the start codon of the first nucleic acid sequence both comprise G. In another embodiment that may be combined with the foregoing embodiment comprising a Kozak type sequence and/or the following embodiment comprising a triple stop codon, the polynucleotide comprises a WPRE component. In some embodiments, the WPRE component is located 3' to the stop codon of the one or more transcription units and 5' to the 3' LTR of the polynucleotide. In another embodiment that may be combined with any or both of the preceding embodiments (i.e., embodiments wherein the polynucleotide comprises a Kozak-type sequence and/or embodiments wherein the polynucleotide comprises a WPRE), one or more transcription units are terminated by one or more of a double stop codon or a triple stop codon.

In some embodiments of any of the aspects provided herein that include an ASTR, the ASTR can be an anti-tag ASTR. This method can further include, for example, a tag-bound antibody that binds to a target molecule, such as a target protein on a target cell.

In any of the embodiments and aspects provided herein comprising B cells, T cells, or NK cells, the cell can be an allogeneic cell, or it can be non-allogeneic.

In any of the aspects and embodiments disclosed herein, DNA is transduced into PBMC, B cells, T cells and/or NK cells and optionally DNA is incorporated into the host cell genome. Methods that do not utilize replication-defective recombinant retroviral particles can be used to perform. For example, other viral vectors, such as those derived from adenovirus, adenovirus-associated virus, or herpes simplex virus type 1, can be used as non-limiting examples. In other embodiments, these aspects and embodiments may include transfection and/or transduction of target cells with non-viral vectors. In any of these embodiments of transfecting target cells with non-viral vectors disclosed herein, non-viral vectors including naked DNA can be introduced into target cells (such as PBMC, B cells, T cells and/or NK cells) using methods including: electroporation, nuclear transfection, liposome preparations, lipids, dendrimers, cationic polymers such as poly (ethyleneimine) (PEI) and poly (l-lysine) (PLL), nanoparticles, cell penetrating peptides, microinjection and/or non-integrating lentiviral vectors. In some embodiments of the methods and compositions provided herein, the DNA can be integrated into the genome by co-transfection, co-nucleofection or co-electroporation of target DNA using a transposon-based vector system as a plasmid containing transposon ITR fragments at the 5' and 3' ends of the gene of interest and a transposase vector system. The transposase or other enzymes used in these methods (e.g., CRISPR or TALEN) can be co-transfected with the target plasmid as DNA, mRNA or protein.

In another aspect, the present invention provides a replication-defective recombinant retroviral particle, each comprising:

A. a pseudotyping component on its surface, which is capable of binding to T cells and/or NK cells and facilitating the fusion of the replication-defective recombinant retroviral particles with their membranes, wherein the pseudotyping component comprises a cytoplasmic domain deletion variant of a measles virus F polypeptide and/or a measles virus H polypeptide;

B. A polynucleotide comprising one or more transcription units operably linked to a promoter active in T cells and/or NK cells, wherein the one or more transcription units encode a first engineered signaling polypeptide comprising a chimeric antigen receptor (which comprises an antigen-specific targeting region, a transmembrane domain, and an intracellular activation domain) and a second engineered signaling polypeptide comprising a constitutively active IL-7 receptor mutant; wherein expression of the IL-7 receptor mutant is regulated by a riboswitch that binds a nucleoside analog antiviral drug; and

C. A polypeptide capable of binding to CD3 and a polypeptide capable of binding to CD28, wherein the polypeptide is expressed on the surface of a replication-deficient recombinant retroviral particle; is capable of binding to T cells and/or NK cells; and is not encoded by a polynucleotide in the replication-deficient recombinant retroviral particle. In an illustrative embodiment of this aspect, the binding of a nucleoside analog antiviral drug to a riboswitch increases the expression of an IL-7 receptor mutant.

In one aspect, provided herein is a method for genetically modifying and expanding lymphocytes of a subject, the method comprising:

A. contacting a subject's resting T cells and/or NK cells ex vivo without prior ex vivo stimulation with a replication-defective recombinant retroviral particle comprising:

i. a pseudotyping component on its surface, which is capable of binding to T cells and/or NK cells and facilitating membrane fusion of the replication-defective recombinant retroviral particles therewith; and

ii. a polynucleotide comprising one or more transcription units operably linked to a promoter active in T cells and/or NK cells, wherein the one or more transcription units encode a first engineered signaling polypeptide regulated by a control component, wherein the first engineered signaling polypeptide comprises at least one lymphoproliferative component,

wherein the contacting facilitates transduction of at least some of the resting T cells and/or NK cells with the replication-defective recombinant retroviral particles, thereby generating genetically modified T cells and/or NK cells;

B. introducing genetically modified T cells and/or NK cells into the individual; and

C. Exposing genetically modified T cells and/or NK cells in vivo to a compound that binds a control component to affect the expression of the first engineered signaling polypeptide and promote and/or enhance the expansion, engraftment and/or persistence of lymphocytes in vivo, thereby genetically modifying and expanding lymphocytes of an individual. In an illustrative embodiment, transduction is performed without the need for in vitro stimulation.

In any of the above aspects and method aspects for genetically modifying and expanding lymphocytes or for performing cell therapy herein, if not recited in the broadest aspect, in certain embodiments, the polynucleotide further comprises a transcription unit encoding a second engineered signaling polypeptide comprising a first chimeric antigen receptor comprising an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain.

In another aspect, provided herein is a method for performing adoptive cell therapy on a subject, the method comprising:

A. Collect blood from individuals;

B. contacting resting T cells and/or NK cells from the blood of an individual ex vivo with replication-defective recombinant retroviral particles, wherein the replication-defective recombinant retroviral particles comprise

i. a pseudotyping component on its surface, which is capable of binding to T cells and/or NK cells and facilitating membrane fusion of the replication-defective recombinant retroviral particles thereon; and

ii. a polynucleotide comprising one or more transcription units operably linked to a promoter active in T cells and/or NK cells, wherein the one or more transcription units encode a first engineered signaling polypeptide comprising at least one lymphoproliferative component, the expression of which is regulated by a control component, and a second engineered signaling polypeptide comprising a chimeric antigen receptor comprising an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain,

wherein the contacting produces at least some of the resting T cells and/or NK cells that become genetically modified; and

C. Reintroducing the genetically modified T cells and/or NK cells into the individual, wherein expansion, engraftment and/or persistence of the genetically modified T cells and/or NK cells occurs in the individual, and wherein the method between collecting blood and reintroducing the genetically modified T cells and/or NK cells is performed within no more than 24 hours, thereby performing adoptive cell therapy on the individual.

Another aspect of the present invention provides a method for performing adoptive cell therapy on an individual, the method comprising:

A. Collect blood from individuals;

B. Isolating peripheral blood mononuclear cells (PBMCs) containing resting T cells and/or resting NK cells;

C. contacting the individual's resting T cells and/or resting NK cells ex vivo with replication-defective recombinant retroviral particles, wherein the replication-defective recombinant retroviral particles comprise a pseudotyping component on their surface that is capable of binding to resting T cells and/or NK cells and facilitating membrane fusion of the replication-defective recombinant retroviral particles thereto, wherein the contacting facilitates transduction of resting T cells and/or NK cells with the replication-defective recombinant retroviral particles, thereby generating genetically modified T cells and/or NK cells; and

D. Reintroducing the genetically modified cells into the individual within 24 hours of collecting blood from the individual, thereby performing adoptive cell therapy in the individual.

In illustrative embodiments of any of the method aspects for genetically modifying and expanding lymphocytes or for performing adoptive cell therapy or similar methods described herein, if not explicitly stated in the broadest aspect, the method may further comprise exposing the genetically modified T cells and/or NK cells in vivo to a compound that binds a control component to affect the expression of the first engineered signaling polypeptide and, if applicable, the second engineered signaling polypeptide, and promoting the expansion, engraftment, and/or persistence of the lymphocytes in vivo.

In illustrative embodiments of any of the method aspects for genetically modifying and expanding lymphocytes or for performing adoptive cell therapy or similar methods described herein, if not explicitly stated in the broadest aspect, the genetically modified T cells and/or NK cells undergo 8, 7, 6, 5, 4, 3 or fewer cell divisions ex vivo prior to being introduced or reintroduced into an individual.

In illustrative embodiments of any of the method aspects for genetically modifying and expanding lymphocytes or for performing adoptive cell therapy or similar methods described herein, if not explicitly stated in the broadest aspect, the expansion, engraftment and/or persistence of the genetically modified T cells and/or NK cells in vivo depends on the presence or absence of a compound that binds a control component, and in illustrative embodiments depends on the presence of a compound that binds a control component.

In illustrative embodiments of any of the method aspects for genetically modifying and expanding lymphocytes or for performing adoptive cell therapy or similar methods described herein, if not explicitly stated in the broadest aspect, the individual is not exposed to a lymphodepleting agent within 7 days, 14 days, or 21 days of performing the contacting or during the contacting, and/or within 7 days, 14 days, or 21 days after the modified T cells and/or NK cells are introduced into the individual. In other embodiments, the individual is not exposed to a lymphodepleting agent during the contacting.

In the illustrative embodiments of any of the method aspects for genetically modifying and expanding lymphocytes or for performing adoptive cell therapy or similar methods described herein, if not explicitly stated in the broadest aspect, the T cell and/or NK cell conditions used in the contacting step may be any of the conditions, such as contact time, provided in other paragraphs in this illustrative embodiments section and other sections herein for methods for genetically modifying and/or transducing (including those methods that do not include an explicit in vivo step).

In an illustrative embodiment for genetically modifying and amplifying lymphocytes or for performing any of the adoptive cell therapy or similar methods herein, if not explicitly described in the broadest aspect, the method further includes a step of separating replication-defective recombinant retroviral particles from T cells and/or NK cells after contact. In methods including introduction or reintroduction, separation is performed before introduction. In an illustrative embodiment for genetically modifying and amplifying lymphocytes or for performing any of the adoptive cell therapy or similar methods herein, if not explicitly described in the broadest aspect, the exposure step includes administering a dose of compound to an individual before or during contact, and/or after genetically modified T cells and/or NK cells have been introduced into the individual.

In an illustrative embodiment of any of the method aspects for genetically modifying and expanding lymphocytes or for performing adoptive cell therapy or similar methods herein, if not explicitly stated in the broadest aspect, the method comprises collecting blood containing T cells and/or NK cells from an individual before contacting the T cells and/or NK cells ex vivo with replication-deficient recombinant retroviral particles, and wherein the introduction is reintroduction. For example, between 20 ml and 250 ml of blood is drawn from the individual.

In illustrative embodiments of any of the method aspects for genetically modifying and expanding lymphocytes or for performing adoptive cell therapy or similar methods described herein, if not explicitly stated in the broadest aspect, no more than 8 hours, 12 hours, 24 hours, or 48 hours elapse between the time blood is collected from the individual and the time the modified T cells and/or NK cells are reintroduced into the individual.

In illustrative embodiments of any of the method aspects for genetically modifying and expanding lymphocytes or for performing adoptive cell therapy or similar methods described herein, if not explicitly stated in the broadest aspect, the range between the time of collection of blood from the individual and the time of reintroduction of the modified T cells and/or NK cells into the individual is between 4 hours or 8 hours on the low end and 12 hours, 24 hours, 36 hours, or 48 hours on the high end.

In the aspects and embodiments provided herein comprising administering genetically modified T cells and/or NK cells to a subject, particularly when the subject has or is suspected of having cancer, the method may further comprise delivering to the subject an effective amount of an immune checkpoint inhibitor.

In an illustrative embodiment of any of the method aspects for genetically modifying and expanding lymphocytes or for performing adoptive cell therapy or similar methods herein, if not explicitly stated in the broadest aspect, all steps after blood collection and before reintroduction of blood are performed in a closed system in which a person monitors the closed system throughout the process. In another embodiment, after blood collection and before reintroduction of blood are performed in a closed system maintained in the same room as the individual.

In the illustrative embodiments of any of the methods and compositions provided herein including one or more transcription units or one or more engineered signaling polypeptides, if not explicitly described in the broadest aspect, one of the transcription units or one of the engineered signaling polypeptides may encode or include or further include an antigen-specific targeting region (ASTR) and a transmembrane domain that connects the ASTR to a lymphoproliferative component. The ASTR of this engineered signaling polypeptide is capable of binding to a first tumor antigen, and when present, the ASTR of the second engineered signaling polypeptide is capable of binding to a second tumor antigen. In illustrative embodiments, the first engineered signaling polypeptide and/or the second engineered signaling polypeptide further include a co-stimulatory domain. In addition, the first engineered signaling polypeptide and/or the second engineered signaling polypeptide further include a handle. In addition, the first engineered signaling polypeptide further includes an intracellular activation domain. The intracellular activation domain on the first engineered signaling polypeptide and/or the second engineered signaling polypeptide may be derived from CD3ζ. In illustrative embodiments of any of the methods and compositions provided herein that include a lymphoproliferative component, the lymphoproliferative component can comprise MPL or can be MPL or a variant and/or fragment thereof, including at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the intracellular domain of MPL, with or without the transmembrane domain and/or the extracellular domain of MPL; and/or variants and/or fragments having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the intracellular domain of MPL, with or without the transmembrane domain and/or the extracellular domain of MPL, wherein the variant and/or fragment retains the ability to promote cellular proliferation of PBMCs (in some embodiments, T cells).

In any of the methods or compositions provided herein including a lymphoproliferative component, the method or composition may further include an inhibitory RNA molecule (such as miRNA or shRNA) that stimulates the STAT5 pathway or inhibits the SOCS pathway, or the lymphoproliferative component may be replaced by the inhibitory RNA molecule. For example, the inhibitory RNA molecule may bind to a nucleic acid encoding a protein selected from the group consisting of ABCG1, SOCS, TGFbR2, SMAD2, cCBL, and PD1. In illustrative embodiments of any of the replication-deficient recombinant retroviral particles or transduced cells provided herein or methods comprising the same, such replication-deficient recombinant retroviral particles or transduced cells may encode two or more inhibitory RNA molecules, such as miRNA or shRNA in an intron, in some embodiments 1, 2, 3 or 4 inhibitory RNA molecules, which bind to nucleic acids encoding one or more of the following target endogenous T cell-expressed genes: PD-1, CTLA4, TCRα, TCRβ, CD3ζ, SOCS, SMAD2, miR-155, IFNγ, cCBL, TRAIL2, PP2A or ABCG1. For example, in one embodiment, a combination of miRNAs targeting any of the following may be included in the genome of the replication-deficient recombinant retroviral particles or transduced cells: TCRα, CD3ζ, IFNγ, and PD-1; and in another embodiment SOCS 1, IFNγ, TCRα and CD3ζ.

In illustrative embodiments of any of the methods and compositions provided herein, the replication-defective recombinant retroviral particles, mammalian cells, and/or packaging cells may comprise a Vpu polypeptide. The Vpu polypeptide may be, for example, a fusion polypeptide, and in some instances, particularly in packaging cells, a membrane-bound Vpu polypeptide. In illustrative embodiments of any of the methods and compositions provided herein, the replication-defective recombinant retroviral particles, mammalian cells, and/or packaging cells may comprise both a Vpu polypeptide and a Vpx polypeptide.

In illustrative embodiments of any of the methods and compositions provided herein, replication-defective recombinant retroviral particles, mammalian cells and/or packaging cells can comprise a Vpx polypeptide. The Vpx polypeptide can be, for example, a fusion polypeptide, and in some instances, particularly in packaging cells, is a membrane-bound Vpx polypeptide.

In any of the methods or compositions provided herein, one or more pseudotyping components may include herpes oral virus envelope protein (VSV-G), feline endogenous virus (RD114) envelope protein, oncorretroviral amphotropic envelope protein, or oncorretroviral ecotropic envelope protein, or a functional fragment thereof.

In one aspect, provided herein are genetically modified T cells and/or NK cells, wherein the T cells and/or NK cells have been genetically modified to express a first engineered signaling polypeptide comprising a lymphoproliferative component and a second engineered signaling polypeptide comprising a CAR, wherein the CAR comprises an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain.

In another aspect, provided herein are genetically modified T cells and/or NK cells comprising: a pseudotyping component on the surface of the T cell or NK cell and an activation component on the surface of the T cell or NK cell, wherein the T cell or NK cell has been genetically modified to express a first engineered signaling polypeptide comprising a lymphoproliferative component and a second engineered signaling polypeptide comprising a chimeric antigen receptor comprising an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain.

In another aspect, provided herein are genetically modified T cells and/or NK cells comprising:

A. a first engineered signaling polypeptide comprising at least one lymphoproliferative component; and

B. A second engineered signaling polypeptide comprising a chimeric antigen receptor comprising an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain.

In some embodiments, in the absence of a target target that exposes the cell to a cytokine (such as IL-15, IL-7, and in illustrative embodiments, IL-2) and an ASTR for a CAR expressed by the cell, or in certain illustrative embodiments in the presence of an antigen recognized by the CAR (particularly wherein the genetically modified T cells or NK cells are genetically modified and/or transfected with retroviral particles having a pseudotyped component and an optionally isolated or fused activation domain on its surface and generally without the need for pre-activation), the genetically modified T cells and/or NK cells are able to survive and/or proliferate and/or expand in culture in vitro or in vitro for 6 days, 7 days, 14 days, 21 days, or 35 days, 42 days, or more during culture. In some embodiments, the genetically modified T cells and/or NK cells comprise a chimeric cytokine receptor. In some embodiments, the genetically modified T cells and/or NK cells do not comprise a chimeric cytokine receptor.

In any of the methods provided herein including mammalian packaging cells (including replication-deficient recombinant retroviral particle packaging system aspects, or methods for preparing replication-deficient recombinant retroviral particles), for example, the packaged RNA genome is encoded by a polynucleotide operably linked to a promoter, wherein the promoter has constitutive activity or can be induced by a first transactivator or a second transactivator. The packaged RNA genome can be encoded by a polynucleotide operably linked to a promoter, wherein the promoter can be induced by a second transactivator. The promoter used herein to drive the expression of the first and/or second engineered signaling polypeptide is typically active in target cells (e.g., lymphocytes, PBLs, T cells, and/or NK cells), but in illustrative embodiments, is not active in packaging cell lines. The second transactivator can regulate the expression of an activation component that can bind to and activate target cells. In any of the methods provided herein that include mammalian packaging cells (including replication-defective recombinant retroviral particle packaging system aspects, or methods for preparing replication-defective recombinant retroviral particles (e.g., packageable RNA genomes in some embodiments)), expression of the packageable RNA genome can be regulated by a second transactivator.

In addition, the RNA genome can be packaged to include from 5' to 3':

1.) 5' long terminal repeat or its activation fragment;

2.) a nucleic acid sequence encoding a retroviral cis-acting RNA packaging component;

3.) a nucleic acid sequence encoding a first target polypeptide and/or a nucleic acid sequence encoding one or more (e.g., two or more) inhibitory RNA molecules;

4.) a promoter that is active in the target cell; and

5.) 3’ long terminal repeat or its activation fragment.

In some embodiments, the nucleic acid sequence encoding the first target polypeptide is in the opposite orientation to the RNA encoding the retroviral components for packaging and assembly and the 5'LTR.

In any of the methods provided herein including mammalian packaging cells (including replication-defective recombinant retroviral particle packaging system aspects, or methods for preparing replication-defective recombinant retroviral particles), for example, the first target polypeptide comprises a first engineered signaling polypeptide and wherein the first engineered signaling polypeptide comprises at least one lymphoproliferative component. The packageable RNA genome may further comprise a nucleic acid sequence encoding a second target polypeptide. The second target polypeptide may comprise a second engineered signaling polypeptide comprising a chimeric antigen receptor, the chimeric antigen receptor comprising:

1.) a first antigen-specific targeting region;

2.) the first transmembrane domain; and

3.) The first intracellular activation domain.

In any of the methods provided herein that include mammalian packaging cells (including replication-defective recombinant retroviral particle packaging system aspects, or methods for preparing replication-defective recombinant retroviral particles (e.g., mammalian cells)), for example, the packaging cells may include, for example, a nucleic acid sequence encoding a Vpu polypeptide on the second transcription unit or, optionally, a third transcription unit, or on other transcription units operably linked to a first inducible promoter. In any of the methods provided herein that include mammalian packaging cells (including replication-defective recombinant retroviral particle packaging system aspects, or methods for preparing replication-defective recombinant retroviral particles (e.g., mammalian cells), for example, the packaging cells may include, for example, a nucleic acid sequence encoding both a Vpx polypeptide and a Vpu polypeptide on the second transcription unit or, optionally, a third transcription unit, or on other transcription units operably linked to a first inducible promoter. The mammalian cell that can be a packaging cell can be a 293 cell.

In any of the methods provided herein that include mammalian packaging cells (including replication-defective recombinant retroviral particle packaging system aspects, or methods for preparing replication-defective recombinant retroviral particles (e.g., mammalian cells)), for example, the packaging cell may include, for example, a nucleic acid sequence encoding Vpx on the second transcription unit or, optionally, on the third transcription unit, or on other transcription units operably linked to the first inducible promoter. The mammalian cell that can be a packaging cell can be a 293 cell.

In any of the methods provided herein comprising mammalian packaging cells (including replication-defective recombinant retroviral particle packaging system aspects, or methods for preparing replication-defective recombinant retroviral particles), the first ligand can be rapamycin and the second ligand can be tetracycline or doxorubicin, or the first ligand can be tetracycline or doxorubicin and the second ligand can be rapamycin.

In some aspects, provided herein is a cell transduced by any one of the replication-deficient recombinant retroviral particles provided herein. The cell can be, for example, a lymphocyte, such as a T cell or a NK cell. In illustrative embodiments, the cell is a human cell.

In one aspect, provided herein is a method for expanding modified T cells and/or NK cells in an individual, the method comprising:

A. contacting isolated resting T cells and/or resting NK cells obtained from the individual with a replication-defective recombinant retroviral particle of any one of the embodiments disclosed herein;

B. introducing genetically modified T cells and/or NK cells into the individual; and

C. providing an effective amount of acyclovir, acyclovir prodrug, penciclovir or penciclovir prodrug to the individual, wherein the modified T cells and/or NK cells proliferate in the individual after administration of acyclovir, acyclovir prodrug, penciclovir or penciclovir prodrug, thereby expanding the modified T cells and/or NK cells in the individual.

In another aspect, provided herein is a method for stopping expansion, engraftment, and/or persistence of modified T cells and/or NK cells in an individual, the method comprising:

A. contacting isolated resting T cells and/or NK cells obtained from the individual with a replication-defective recombinant retroviral particle of any one of the embodiments disclosed herein;

B. introducing the modified T cells and/or NK cells into the individual;

C. administering to the subject an effective amount of acyclovir, acyclovir prodrug, penciclovir or penciclovir prodrug to expand the modified T cells and/or NK cells in the subject, wherein the modified T cells and/or NK cells proliferate in the subject after administration of acyclovir, acyclovir prodrug, penciclovir or penciclovir prodrug, thereby expanding the modified PBL in the subject; and

D. Ceasing administration of acyclovir, acyclovir prodrug, penciclovir or penciclovir prodrug, wherein the modified T cells and/or NK cells cease to proliferate in the individual after ceasing administration of acyclovir, acyclovir prodrug, penciclovir or penciclovir prodrug, thereby controlling the expansion, engraftment and/or persistence of the modified T cells and/or NK cells in the individual.

In another aspect, provided herein is a method of treating cancer in an individual, the method comprising:

A. contacting isolated resting T cells and/or NK cells obtained from the individual with a replication-defective recombinant retroviral particle according to any one of the embodiments disclosed herein;

B. introducing genetically modified T cells and/or NK cells into the individual; and

C. administering an effective amount of acyclovir, acyclovir prodrug, penciclovir or penciclovir prodrug to the individual to expand the modified T cells and/or NK cells in the individual, wherein the modified T cells and/or NK cells proliferate in the individual after administration of acyclovir, acyclovir prodrug, penciclovir or penciclovir prodrug, and wherein the chimeric antigen receptor in the modified T cells and/or NK cells binds to the cancer cells in the individual, thereby treating the cancer in the individual.

In another aspect, provided herein is a transduced T cell and/or NK cell comprising a recombinant polynucleotide comprising one or more transcription units operably linked to a promoter active in T cells and/or NK cells, wherein the one or more transcription units encode a first engineered signaling polypeptide regulated by a control component, wherein the first engineered signaling polypeptide comprises a constitutively active IL-7 receptor mutant, and wherein the control component is capable of binding to a compound in vivo and/or is designed and/or configured to bind to a compound.

In another aspect, a retroviral packaging system is provided herein, comprising:

A mammalian cell comprising:

A. a first transactivator, which is expressed from a constitutive promoter and is capable of binding a first ligand and a first inducible promoter for affecting expression of a nucleic acid sequence operably linked thereto in the presence versus absence of the first ligand;

B. a second transactivator capable of binding a second ligand and a second inducible promoter and affecting expression of a nucleic acid sequence operably linked thereto in the presence versus absence of the second ligand; and

C. RNA genome packaged into retroviral particles,

Wherein the first transactivator regulates the expression of the second transactivator and the retroviral REV protein, wherein the second transactivator regulates the expression of the gag polypeptide, the pol polypeptide and one or more pseudotyped components capable of binding to a target cell and facilitating fusion with its membrane, and wherein the retroviral protein is derived from a retrovirus. Embodiments of this aspect may include any of the embodiments provided herein for the embodiments in other aspects.

In another aspect, provided herein is a method for producing a replication-defective recombinant retroviral particle, comprising:

A. culturing a population of packaging cells to accumulate a first transactivator, wherein the packaging cells comprise the first transactivator expressed by a first constitutive promoter, wherein the first transactivator is capable of binding a first ligand and a first inducible promoter for affecting expression of a nucleic acid sequence operably linked thereto in the presence of the first ligand as opposed to in the absence of the first ligand, and wherein expression of a second transactivator and a retroviral REV protein is regulated by the first transactivator;

B. culturing a population of packaging cells comprising the accumulated first transactivator in the presence of a first ligand to accumulate a second transactivator and a retroviral REV protein, wherein the second transactivator is capable of binding a second ligand and a second inducible promoter for affecting expression of a nucleic acid sequence operably linked thereto in the presence of the second ligand as opposed to in its absence; and

C. culturing a population of packaging cells containing the accumulated second transactivator and the retroviral REV protein in the presence of a second ligand, thereby inducing the expression of the gag polypeptide, the pol polypeptide and one or more pseudotyping components, thereby producing replication-defective recombinant retroviral particles,

Wherein the packageable RNA genome is encoded by a polynucleotide operably linked to a third promoter, wherein the third promoter is constitutively active or inducible by the first transactivator or the second transactivator, and wherein one or more pseudotyping components are capable of binding to target cells and/or facilitating membrane fusion with the replication-defective recombinant retroviral particle.

In some embodiments of the retroviral packaging system and the method for manufacturing replication-deficient recombinant retroviral particles provided herein, the mammalian cell further comprises an activation component capable of binding to a target cell (usually a lymphocyte, such as a NK cell or a T cell in an illustrative embodiment) and activating the target cell, and the first transactivator regulates the expression of the activation component. The activation component is on the surface of the replication-deficient recombinant retroviral particle and wherein the activation component may include: a membrane-bound polypeptide capable of binding to CD3; and/or a membrane-bound polypeptide capable of binding to CD28. The membrane-bound polypeptide capable of binding to CD3 is a polypeptide capable of binding to CD3 fused to a heterologous GPI anchor linking sequence, and the membrane-bound polypeptide capable of binding to CD28 is a polypeptide capable of binding to CD28 fused to a heterologous GPI anchor linking sequence. The membrane-bound polypeptide capable of binding to CD28 comprises CD80, CD86, or a functional fragment thereof (such as the extracellular domain of CD80) capable of inducing activation of Akt mediated by CD28 in some embodiments. In other embodiments, the membrane-bound polypeptide capable of binding to CD3 is an anti-CD3 scFv or anti-CD3 scFvFc bound to a CD14 GPI anchor linker sequence, and the membrane-bound polypeptide capable of binding to CD28 is CD80, or an extracellular fragment thereof bound to a CD16B GPI anchor linker sequence.

In some embodiments of the retroviral packaging system and the method for manufacturing replication-deficient recombinant retroviral particle aspects provided herein, the mammalian cell further comprises a membrane-bound cytokine, and the first transactivator regulates the expression of the membrane-bound cytokine. The membrane-bound cytokine can be, for example, IL-7, IL-15, or an activated fragment thereof. The membrane-bound cytokine in the embodiment can be a fusion polypeptide of IL-7, or an activated fragment thereof, and DAF. For example, the fusion polypeptide can comprise a DAF signal sequence and IL-7 without its signal sequence, followed by residues 36 to 525 of DAF.

In some embodiments of the retroviral packaging system and methods for producing replication-defective recombinant retroviral particles provided herein, a mammalian cell comprises associated with its membrane: an activation component comprising an anti-CD3 scFv or anti-CD3 scFvFc bound to a CD14 GPI anchoring linker sequence, or CD80 or an extracellular fragment thereof bound to a CD16B GPI anchoring linker sequence; and a membrane-bound cytokine comprising a fusion polypeptide of IL-7 or its activation fragment and DAF comprising a GPI anchoring linker sequence, and wherein the first transactivator regulates the expression of each of the activation component and the membrane-bound cytokine. In some embodiments, IL-7 or its activation fragment and DAF fusion, anti-CD3 scFv or anti-CD3 scFvFc and CD80 or an extracellular fragment thereof each comprise a DAF signal sequence.

In some embodiments of the retroviral packaging system and the method for manufacturing a replication-deficient recombinant retroviral particle aspect provided herein, the mammalian cell further comprises a Vpu polypeptide. In some embodiments of the retroviral packaging system and the method for manufacturing a replication-deficient recombinant retroviral particle aspect provided herein, the mammalian cell further comprises a Vpu polypeptide and a Vpx polypeptide. In some embodiments of the retroviral packaging system and the method for manufacturing a replication-deficient recombinant retroviral particle aspect provided herein, the mammalian cell further comprises a Vpx polypeptide. In these or other embodiments, one or more pseudotyped components comprise one or more viral polypeptides recognized by T cells. One or more pseudotyped components may comprise a measles virus F polypeptide, a measles virus H polypeptide and/or a fragment thereof. In certain illustrative embodiments, one or more pseudotyped components are cytoplasmic domain deletion variants of a measles virus F polypeptide and/or a measles virus H polypeptide.

In some embodiments of the retroviral packaging system and methods for producing replication-defective recombinant retroviral particle aspects provided herein, the packaged RNA genome is encoded by a polynucleotide operably linked to a third promoter, wherein the third promoter is constitutively active or inducible by a first transactivator or a second transactivator. In illustrative embodiments, the packaged RNA genome is encoded by a polynucleotide operably linked to a third promoter, wherein the third promoter is inducible by a second transactivator.

In some embodiments of the retroviral packaging systems and methods for producing replication-defective recombinant retroviral particles provided herein, the packageable RNA genome further comprises from 5' to 3':

a) 5' long terminal repeat or its activation fragment;

b) a nucleic acid sequence encoding a retroviral cis-acting RNA packaging component;

c) a nucleic acid sequence encoding a first target polypeptide and, optionally, a second target polypeptide;

d) a fourth promoter operably linked to the first target polypeptide and optionally the second target polypeptide, wherein the fourth promoter is active in the target cell but inactive in the packaging cell line; and

e) 3' long terminal repeat or an activated fragment thereof.

In some embodiments of the retroviral packaging systems and methods for making replication-defective recombinant retroviral particle aspects provided herein that include the constructs immediately above, the third promoter promotes transcription or expression in the opposite direction to that promoted by the fourth promoter.

In some embodiments of the retroviral packaging system and the method for manufacturing a replication-deficient recombinant retroviral particle aspect provided herein, the packaged RNA genome encodes the replication-deficient recombinant retroviral particle of any embodiment disclosed in the present invention, wherein the first target polypeptide and the second target polypeptide are the first engineered signaling polypeptide and the second engineered signaling polypeptide, respectively. In some embodiments, for example, the packaged RNA genome further comprises a control component operably linked to a nucleic acid encoding the first engineered signaling polypeptide or the second engineered signaling polypeptide. The control component is a riboswitch in an illustrative embodiment. The riboswitch is capable of binding a compound in an illustrative embodiment and the compound that binds the control component is a nucleoside analog, and the nucleoside analog can be an antiviral drug, such as acyclovir or penciclovir.

In some embodiments of the retroviral packaging system and the method for manufacturing replication-defective recombinant retroviral particle aspects provided herein, the packaged RNA genome further comprises an intron, which comprises a polynucleotide encoding an inhibitory RNA molecule (such as miRNA or shRNA). The intron may be adjacent to the fourth promoter or downstream of the fourth promoter.

In some embodiments of the retroviral packaging systems and methods for producing replication-defective recombinant retroviral particle aspects provided herein, the target cells can be T cells and/or NK cells.

In some embodiments of the retroviral packaging systems and methods for producing replication-defective recombinant retroviral particles provided herein, one or more pseudotyping components comprise vesicular stomatitis virus envelope protein (VSV-G), feline endogenous virus (RD114) envelope protein, oncorretroviral amphotropic envelope protein, or oncorretroviral ecotropic envelope protein, or a functional fragment thereof.

In some embodiments of the retroviral packaging systems and methods for producing replication-defective recombinant retroviral particles provided herein, the packaged RNA genome size is 11,000 KB or less, or 10,000 KB or less. In some embodiments of the retroviral packaging systems and methods for producing replication-defective recombinant retroviral particles provided herein, the first target polypeptide comprises a first engineered signaling polypeptide and wherein the first engineered signaling polypeptide comprises at least one lymphoproliferative component, and the second target polypeptide comprises a second engineered signaling polypeptide comprising a CAR.

In an illustrative embodiment of any of the methods and compositions provided herein including a control component, a polynucleotide comprising a riboswitch may be provided. The riboswitch may be capable of binding to a nucleoside analog and the compound binding to the control component is a nucleoside analog. The nucleoside analog may be an antiviral agent. The antiviral agent may be acyclovir or penciclovir. The riboswitch may preferably bind to acyclovir via penciclovir or preferably bind to penciclovir via acyclovir. The riboswitch may reduce binding to a nucleoside analog antiviral drug at a temperature higher than 37°C, 37.5°C, 38°C, 38.5°C or 39°C (e.g., higher than 39°C). The riboswitch may be between the low end of a range of 35, 40, 45 and 50 nucleotides in length and the high end of a range of 60, 65, 70, 75, 80, 85, 90, 95 and 100 nucleotides in length, for example, between 45 and 80 nucleotides in length. In the illustrative embodiments of any one of the methods and compositions provided herein including riboswitches, the polynucleotide target polynucleotide regulated by the riboswitch may include a region encoding miRNA, shRNA and/or polypeptide. The polynucleotide target polynucleotide may encode a lymphoproliferative component. The polynucleotide target polynucleotide may be operably linked to a promoter. The polynucleotide target polynucleotide may include a region encoding a polypeptide, and the polypeptide may include a chimeric antigen receptor, which includes an antigen-specific targeting region, a transmembrane domain and an intracellular activation domain. In the illustrative embodiments of any one of the methods and compositions provided herein including riboswitches, the functional switching domain may regulate the accessibility of pre-mRNA splicing donors in the internal ribosome entry site, viral gene constructs, translation, transcription termination, transcription degradation, miRNA expression or shRNA expression, thereby regulating the expression of the polynucleotide target polynucleotide. The riboswitch may include a ribonuclease. In the illustrative embodiments of any one of the methods and compositions including riboswitches provided herein, the isolated polynucleotide may be a molecular cloning vector or an expression vector. In the illustrative embodiments of any of the methods and compositions provided herein including riboswitches, the isolated polynucleotides may be integrated into a retroviral genome or into a mammalian chromosome or fragment thereof. In the illustrative embodiments of any of the aspects and embodiments herein including riboswitches, the riboswitch regulates pre-mRNA splicing. For example, the riboswitch may include a branch point sequence between two exons on a polynucleotide and/or transcription unit generally herein. Therefore, the binding of a nucleoside-like antiviral compound or drug to a riboswitch regulates exon splicing. Such embodiments may include a polynucleotide comprising a first exon, a second exon, and a riboswitch between the first exon and the second exon, wherein the riboswitch comprises a branch point sequence, and wherein the binding of a nucleoside-like antiviral compound or drug (e.g., acyclovir) to the riboswitch regulates exon splicing of the first and second exons. In the illustrative embodiments of any of the aspects and embodiments herein including riboswitches, the riboswitch may control gene expression by regulating pre-mRNA trans-splicing. In such embodiments, the riboswitch is located within a trans-splicing intron. For example, one aspect can include a polynucleotide comprising a first exon, a second exon, and a riboswitch between the first exon and the second exon, wherein the riboswitch comprises a branch point sequence, and wherein binding of acyclovir to the riboswitch modulates exonic splicing of the first and second exons.

In an illustrative embodiment of any replication-deficient recombinant retroviral particle, the retroviral particle is a lentiviral particle. In other illustrative embodiments of the method, a polypeptide capable of binding to CD3 and a polypeptide capable of binding to CD28 are each fused to a heterologous GPI anchoring connection sequence. In some cases, the polypeptide capable of binding to CD3 may be an anti-CD3 scFvFc or an anti-CD3 scFv, and the polypeptide capable of binding to CD28 may be CD80. Anti-CD3 scFvFc or anti-CD3scFv and CD80 may each be further fused to a DAF signal sequence. In another illustrative embodiment, the replication-deficient recombinant retroviral particle further comprises a fusion polypeptide on its surface, the fusion polypeptide comprising a cytokine covalently linked to DAF. In some cases, the cytokine may be IL-7 or IL-15, and the fusion polypeptide may comprise a fragment of a DAF signal sequence, IL-7 without its signal sequence, and a DAF comprising a GPI anchoring connection sequence.

In another illustrative embodiment of any replication-deficient recombinant retroviral particle aspect herein, the riboswitch further controls the expression of the chimeric antigen receptor in a manner regulated by the binding of the riboswitch to a nucleoside analog antiviral drug, which in some cases is acyclovir and/or penciclovir. In another embodiment, the constitutively active IL-7 can be replaced by a miRNA or shRNA or a nucleic acid encoding a miRNA or shRNA, and IL-7 can be present. The miRNA or shRNA can be encoded by a nucleic acid within an intron.

Another aspect provided herein is a method for producing a replication-defective recombinant retroviral particle comprising:

A. culturing a population of packaging cells to accumulate a first transactivator, wherein the packaging cells comprise the first transactivator expressed by a constitutive promoter, wherein the first transactivator is capable of binding a first ligand and a first inducible promoter for affecting expression of a nucleic acid sequence operably linked thereto in the presence of the first ligand as opposed to in the absence of the first ligand, and wherein expression of a second transactivator and a retroviral REV protein is regulated by the first transactivator;

B. culturing a population of packaging cells comprising the accumulated first transactivator in the presence of a first ligand to accumulate a second transactivator and a retroviral REV protein and an activation module generally on its surface, the activation module comprising a polypeptide capable of binding to CD3 and comprising a polypeptide capable of binding to CD28, wherein the second transactivator is capable of binding to a second ligand and a second inducible promoter for affecting expression of a nucleic acid sequence operably linked thereto in the presence of the second ligand as opposed to in its absence; and

C. Culturing a population of packaging cells containing accumulated second transactivators and retroviral REV proteins in the presence of a second ligand, thereby inducing the expression of gag polypeptide, pol polypeptide, and a pseudotyping component capable of binding to T cells and/or NK cells and facilitating membrane fusion with replication-defective recombinant retroviral particles, wherein the pseudotyping component comprises a cytoplasmic domain deletion variant of a measles virus F polypeptide and/or a measles virus H polypeptide,

wherein the packageable RNA genome is encoded by a polynucleotide operably linked to a third promoter, and wherein the promoter is inducible by a second transactivator,

The packaging RNA genome includes from 5' to 3':

i. 5' long terminal repeat or its activation fragment;

ii. a nucleic acid sequence encoding a retroviral cis-acting RNA packaging component;

iii. a nucleic acid sequence encoding a first engineered signaling polypeptide comprising a chimeric antigen receptor and a second engineered signaling polypeptide comprising a constitutively active IL-7 receptor mutant separated by a cleavage signal;

iv. a fourth promoter that is active in T cells and/or NK cells; and

v.3' long terminal repeat or its activation fragment,

Replication-defective recombinant retroviral particles are thereby produced.

In an illustrative embodiment of the method, the packageable RNA genome further comprises a riboswitch that binds a nucleoside analog antiviral drug, wherein binding the riboswitch to the nucleoside analog antiviral drug increases the expression of the IL-7 receptor mutant. In some embodiments, the riboswitch further controls the expression of the chimeric antigen receptor in a manner regulated by binding of the riboswitch to the nucleoside analog antiviral drug. In another illustrative embodiment, the nucleoside analog antiviral drug is acyclovir and/or penciclovir. In another illustrative embodiment, the packageable RNA genome further comprises a recognition domain, wherein the recognition domain comprises a polypeptide recognized by an antibody that recognizes EGFR or an antigenic determinant thereof. In another illustrative embodiment, the first ligand is rapamycin and the second ligand is tetracycline or doxorubicin; or the first ligand is tetracycline or doxorubicin, and the second ligand is rapamycin. In another illustrative embodiment, the packaging cell further comprises a nucleic acid sequence encoding Vpu on the second or optionally selected third transcription unit or on other transcription units operably linked to the first inducible promoter. In another illustrative embodiment, the packaging cell further comprises a nucleic acid sequence encoding a Vpu polypeptide and a Vpx polypeptide on a second or optionally a third transcription unit or on other transcription units operably linked to a first inducible promoter. In another illustrative embodiment, the packaging cell further comprises a nucleic acid sequence encoding a Vpx on a second or optionally a third transcription unit or on other transcription units operably linked to a first inducible promoter. In another illustrative embodiment, the polypeptide capable of binding to CD3 and the polypeptide capable of binding to CD28 are each fused to a heterologous GPI anchor linking sequence. In some cases, the polypeptide capable of binding to CD3 may be an anti-CD3 scFvFc or an anti-CD3 scFv, and the polypeptide capable of binding to CD28 may be CD80. The anti-CD3 scFvFc or the anti-CD3 scFv and CD80 may each be further fused to a DAF signal sequence. In another illustrative embodiment, the expression of a fusion polypeptide comprising a cytokine covalently linked to DAF is also included. In some cases, the cytokine may be IL-7 or IL-15, and the fusion polypeptide may comprise a fragment of a DAF signal sequence, IL-7 without its signal sequence, and DAF comprising a GPI anchor linker sequence. In another illustrative embodiment, the riboswitch further controls the expression of the chimeric antigen receptor in a manner regulated by the binding of the riboswitch to a nucleoside analog antiviral drug, which in some cases is acyclovir and/or penciclovir. In another embodiment, the constitutively active IL-7 may be replaced by miRNA or shRNA or a nucleic acid encoding miRNA or shRNA, and IL-7 may be present. The miRNA or shRNA may be encoded by a nucleic acid within an intron. In an illustrative embodiment, the retroviral particle is a lentiviral particle.

In another aspect herein, a genetically modified lymphocyte is provided, comprising:

A. a first engineered signaling polypeptide comprising a constitutively active IL-7 receptor mutant; and

B. A second engineered signaling polypeptide comprising a chimeric antigen receptor comprising an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain.

In the illustrative embodiments of the above genetically modified lymphocytes, the genetically modified lymphocytes are T cells and/or NK cells. In certain embodiments, the lymphocytes are T cells. In another illustrative embodiment, the expression of the first engineered signaling polypeptide and/or the second engineered signaling polypeptide is regulated by a riboswitch that binds a nucleoside analog antiviral drug, wherein the binding of the nucleoside analog antiviral drug to the riboswitch increases the expression of the IL-7 receptor mutant. In another embodiment, the genetically modified lymphocytes express at least one (e.g., two) inhibitory RNA molecules, such as miRNA or shRNA. The inhibitory RNA molecules may be further encoded by nucleic acids within introns.

Another aspect herein provides a genetically modified T cell and/or NK cell comprising:

a. a first engineered signaling polypeptide comprising at least one lymphoproliferative component; and

b. A second engineered signaling polypeptide comprising a chimeric antigen receptor comprising an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain.

In illustrative embodiments of genetically modified T cells and/or NK cell profiles, the lymphoproliferative component is constitutively active, and in some cases is a constitutively active mutant IL-7 receptor or fragment thereof. In another illustrative embodiment, the expression of the first engineered signaling polypeptide and/or the second engineered signaling polypeptide is regulated by a control component. In some cases, the control component is a polynucleotide comprising a riboswitch. In some cases, the riboswitch is capable of binding to a nucleoside analog and when the nucleoside analog is present, the first engineered signaling polypeptide and/or the second engineered signaling polypeptide are expressed. In other illustrative embodiments, genetically modified T cells and/or NK cells have an activation component, a pseudotype component, and/or a membrane-bound cytokine on their surface. In some cases, the activation component comprises a membrane-bound polypeptide capable of binding to CD3; and/or a membrane-bound polypeptide capable of binding to CD28. In certain embodiments, the activation component comprises an anti-CD3 scFv or anti-CD3 scFvFc fused to a heterologous GPI anchoring sequence and/or CD80 fused to a heterologous GPI anchoring sequence. In an illustrative embodiment, the pseudotyping component comprises a measles virus F polypeptide, a measles virus H polypeptide, and/or a cytoplasmic domain deletion variant of a measles virus F polypeptide and/or a measles virus H polypeptide. In other embodiments, the membrane-bound cytokine is a fusion polypeptide comprising IL-7 or a fragment thereof fused to DAF or comprising a GPI anchoring sequence.

In one aspect, provided herein is a method for genetically modifying and expanding lymphocytes of a subject, comprising:

A. contacting a subject's resting T cells and/or NK cells ex vivo, usually without prior ex vivo stimulation, with replication-defective recombinant retroviral particles comprising:

i. a pseudotyping component on its surface that is capable of binding to T cells and/or NK cells and facilitating membrane fusion with the replication-defective recombinant retroviral particle; and

ii. a polynucleotide comprising one or more transcription units operably linked to a promoter active in T cells and/or NK cells, wherein the one or more transcription units: encode a first engineered signaling polypeptide regulated by a control component, wherein the first engineered signaling polypeptide comprises at least one lymphoproliferative component; and optionally encode a second engineered signaling polypeptide optionally regulated by a control component, wherein the second engineered signaling polypeptide comprises an intracellular activation domain and optionally other components of a CAR, wherein the contacting facilitates transduction of at least some of the resting T cells and/or NK cells using replication-defective recombinant retroviral particles, thereby generating genetically modified T cells and/or NK cells;

B. introducing genetically modified T cells and/or NK cells into the individual; and

The genetically modified T cells and/or NK cells are exposed in vivo to a compound that acts as a control component to affect the expression of the first engineered signaling polypeptide and promote the expansion, engraftment and/or persistence of the lymphocytes in vivo, thereby genetically modifying and expanding the lymphocytes of the individual.

In illustrative embodiments, transduction is performed without in vitro stimulation. In illustrative embodiments, the compound is a chaperone, such as a small molecule chaperone. In illustrative embodiments, the combination of a chaperone with a lymphoproliferative component increases the proliferative activity of the lymphoproliferative component. The chaperone can be administered to an individual before blood is collected, during contact, and/or after T cells and/or NK cells are introduced into an individual. It will be understood that the compound is usually able to bind to a component of a lymphoproliferative component and/or CAR, and to bind to this lymphoproliferative component or CAR component during the execution of the method. Other embodiments and teachings related to the method provided herein including transfection of T cells and/or NK cells with replication-deficient recombinant retroviral particles are applied to this aspect, and also include chaperone embodiments.

In another aspect, provided herein is a chimeric antigen receptor for binding to a target antigen, the chimeric antigen receptor comprising:

a) an antigen-specific targeting region restricted by a microenvironment, which exhibits increased binding to a target antigen under abnormal conditions compared to normal physiological conditions, wherein the antigen-specific targeting region binds to the target;

b) a transmembrane domain; and

c) Intracellular activation domain.

In an illustrative embodiment of any of the methods and compositions provided herein comprising an antigen-specific targeting region (ASTR) restricted by a microenvironment, the ASTR may increase binding affinity for the target antigen by at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% in an analysis under abnormal conditions compared to normal conditions. Abnormal conditions may be hypoxia, acidic pH, higher concentrations of lactic acid, higher concentrations of hyaluronic acid, higher concentrations of albumin, higher concentrations of adenosine, higher concentrations of R-2-hydroxyglutaric acid, higher concentrations of PAD enzymes, higher pressure, higher oxidation reactions, and lower nutrient availability. For example, an ASTR restricted by a microenvironment may exhibit an increase in antigen binding at a pH of 6.7 compared to a pH of 7.4. An ASTR restricted by a microenvironment may exhibit an increase in antigen binding in a tumor environment and/or in vivo tumor environment analysis conditions associated with corresponding physiological conditions. The target can be 4-1BB, ST4, adenocarcinoma antigen, α-fetoprotein, AXL, BAFF, B-lymphoma cells, C242 antigen, CA-125, carbonic anhydrase-9 (CA-IX), C-MET, CCR4, CD 152, CD 19, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44 v6, CD51, CD52, CD56, CD74, CD80, CEA, CNT0888, CTLA-4, DRS, EGFR, EpCAM, CD3, FAP, fibronectin ectodomain-B, folate receptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HER2/neu, HGF, human scatter factor receptor kinase, IGF-1 receptor, IGF-I, IgG1, Ll-CAM, IL-13, IL-6, insulin-like growth factor I receptor, integrin nSP1, integrin nvP3, MORAb-009, MS4A1, MUC1, mucin CanAg, N-glucosylneuramine, NPC-1C, PDGF-R a, PDL192, phosphatidylserine, prostate cancer cells, RANKL, RON, ROR1, ROR2 SCH900105, SDC1, SLAMF7, TAG-72, tenascin C, TGFβ2, TGF-β, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2 and vimentin. ASTRs may be antibodies, antigens, ligands, receptor binding domains of ligands, receptors, ligand binding domains of receptors, or affinity bodies. ASTRs may be full-length antibodies, single-chain antibodies, Fab fragments, Fab' fragments, (Fab') ₂ fragments, Fv fragments, and bivalent single-chain antibodies or bifunctional antibodies. ASTRs may include heavy chains and light chains from antibodies. The antibody may be a single-chain variable fragment. In some embodiments, the heavy chain and the light chain may be separated by a connector, wherein the connector is between 6 and 100 amino acids in length. In some embodiments, the heavy chain may be located at the N-terminus of the light chain on the chimeric antigen receptor, and in some embodiments the light chain may be located at the N-terminus of the heavy chain on the chimeric antigen receptor.

In the illustrative embodiments of any of the methods provided herein including a polypeptide display library, the polypeptide display library may be a phage display library or a yeast display library. The polypeptide display library may be an antibody display library. The antibody display library may be a human or humanized antibody display library. The antibody display library may be a native library. The method may include infecting bacterial cells with collected phages to produce a refined phage display library, and repeating contact, cultivation and collection for 1 to 1000 cycles, using the refined phage display library produced by the aforementioned cycles.

In another aspect, provided herein is a transduced T cell and/or NK cell comprising a recombinant polynucleotide comprising one or more transcription units operably linked to a promoter active in T cells and/or NK cells, wherein the one or more transcription units encode a first engineered signaling polypeptide regulated by a control component, wherein the first engineered signaling polypeptide comprises a constitutively active IL-7 receptor mutant, and wherein the control component is capable of binding to a compound in vitro or in vivo, or is configured to bind to a compound in vivo.

In another aspect, provided herein is a replication-defective recombinant retroviral particle comprising a recombinant polynucleotide comprising one or more transcription units operably linked to a promoter active in T cells and/or NK cells, wherein the one or more transcription units encode a first engineered signaling polypeptide regulated by a control component (which may be an in vivo control component), wherein the first engineered signaling polypeptide comprises a constitutive IL-7 receptor mutant, and wherein the control component is capable of binding to a compound in vivo, or is configured to bind to a compound in vivo.

In another aspect, provided herein is a method for transducing T cells and/or NK cells, the method comprising: contacting T cells and/or NK cells with a replication-defective recombinant retroviral particle comprising a recombinant polynucleotide, the recombinant polynucleotide comprising one or more transcription units operably linked to a promoter active in T cells and/or NK cells, wherein the one or more transcription units encode a first engineered signaling polypeptide regulated by a control component, wherein the first engineered signaling polypeptide comprises a constitutive IL-7 receptor mutant, and wherein the in vivo control component is capable of binding to a compound in vivo or in vitro under transduction conditions, thereby transducing T cells and/or NK cells.

In the illustrative embodiments of the transduced T cell and/or NK cell aspects, replication-deficient recombinant retroviral particle aspects, and method aspects provided in the preceding paragraphs, the recombinant polynucleotide further comprises a transcription unit encoding a second engineered signaling polypeptide comprising a first chimeric antigen receptor, the first chimeric antigen receptor comprising an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain. In other illustrative embodiments, the lymphoproliferative component comprises a mutated IL-7 receptor or a fragment thereof. In other illustrative embodiments, the control component is a polynucleotide comprising a riboswitch. In some cases, the riboswitch is capable of binding to a nucleoside analog and the compound that binds the control component is a nucleoside analog. In some cases, the nucleoside analog is an antiviral agent, such as acyclovir or penciclovir. In certain embodiments, the antiviral agent is acyclovir. In other illustrative embodiments, a constitutively active IL-7 receptor mutant is fused to EGFR or its antigenic determinant. In other illustrative embodiments, a constitutively active IL-7 receptor mutant comprises an eTag. In other illustrative embodiments, the constitutively active IL-7 receptor mutant comprises a PPCL insertion. In other illustrative embodiments, the constitutively active IL-7 receptor mutant comprises a PPCL insertion at a position equivalent to position 243 in the wild-type human IL-8 receptor. In other illustrative embodiments, the transduced T cells and/or NK cells are transduced T cells.

In one aspect, provided herein is a replication-defective recombinant retroviral particle comprising a polynucleotide in its genome, the polynucleotide comprising one or more nucleic acid sequences operably linked to a promoter active in T cells and/or NK cells, wherein:

a. a first nucleic acid sequence of the one or more nucleic acid sequences encodes one or more (e.g., two or more) inhibitory RNA molecules against one or more RNA targets, and

b. The second nucleic acid sequence of the one or more nucleic acid sequences encodes a chimeric antigen receptor (CAR), which comprises an antigen-specific targeting region (ASTR), a transmembrane domain and an intracellular activation domain.

In another aspect herein, a mammalian packaging cell line is provided that comprises a packaging RNA genome of a replication-defective recombinant retroviral particle, wherein the packaging RNA genome comprises:

a. 5' long terminal repeat or its activated fragment;

b. a nucleic acid sequence encoding a retroviral cis-acting RNA packaging component;

c. a polynucleotide comprising one or more nucleic acid sequences operably linked to a promoter active in T cells and/or NK cells, wherein a first nucleic acid sequence of the one or more nucleic acids encodes one or more (e.g., two or more) inhibitory RNA molecules against one or more RNA targets, and a second nucleic acid sequence of the one or more nucleic acid sequences encodes a chimeric antigen receptor (CAR) comprising an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain; and

d. 3’ long terminal repeat or its activated fragment.

In some embodiments of the mammalian packaging cell line aspect, the polynucleotide of (c) can be in the opposite orientation to the nucleic acid sequence encoding the retroviral cis-acting RNA packaging component (b), the 5' long terminal repeat (a) and/or the 3' long terminal repeat (d).

In some embodiments of the mammalian packaging cell line aspect, expression of the packageable RNA genome is driven by an inducible promoter active in the mammalian packaging cell line.

In some embodiments of the mammalian packaging cell line aspect, the retroviral cis-acting RNA packaging element can comprise a central polypurine tract (cPPT)/central termination sequence, HIV Psi, or a combination thereof.

Another aspect of the present invention provides a retroviral vector comprising a packageable RNA genome for replication-defective recombinant retroviral particles, wherein the packageable RNA genome comprises:

a. 5' long terminal repeat or its activated fragment;

b. a nucleic acid sequence encoding a retroviral cis-acting RNA packaging component;

c. a polynucleotide comprising one or more nucleic acid sequences operably linked to a promoter active in T cells and/or NK cells, wherein a first nucleic acid sequence of the one or more nucleic acids encodes one or more (e.g., two or more) inhibitory RNA molecules against one or more RNA targets, and a second nucleic acid sequence of the one or more nucleic acid sequences encodes a chimeric antigen receptor (CAR) comprising an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain; and

d. 3’ long terminal repeat or its activated fragment.

In some embodiments of the retroviral vector aspect, the polynucleotide of (c) may be in the opposite orientation to the nucleic acid sequence encoding the retroviral cis-acting RNA packaging component (b), the 5' long terminal repeat (a) and/or the 3' long terminal repeat (d).

In some embodiments of the retroviral vector aspect, expression of the packageable RNA genome is driven by an inducible promoter active in a mammalian packaging cell line.

In some embodiments of the retroviral vector aspect, the retroviral cis-acting RNA packaging component may include a central polypurine tract (cPPT)/central termination sequence, HIV Psi, or a combination thereof. The retroviral vector may optionally include an antibiotic resistance gene and/or a detectable marker.

In another aspect, provided herein is a method for genetically modifying or transducing lymphocytes (e.g., T cells and/or NK cells) or a population thereof of an individual, the method comprising contacting lymphocytes (e.g., T cells and/or NK cells) or a population thereof of an individual in vitro with a replication-defective recombinant retroviral particle, the replication-defective recombinant retroviral particle comprising a polynucleotide in its genome, the polynucleotide comprising one or more nucleic acid sequences operably linked to a promoter active in lymphocytes (e.g., T cells and/or NK cells), wherein a first nucleic acid sequence of the one or more nucleic acid sequences encodes a gene targeting one or more One or more (e.g., two or more) inhibitory RNA molecules targeting multiple RNA targets, and a second nucleic acid sequence of the one or more nucleic acid sequences encodes a chimeric antigen receptor (CAR), which comprises an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain, wherein the contact facilitates genetic modification and/or transduction of lymphocytes (e.g., T cells and/or NK cells) or at least some of the lymphocytes (e.g., T cells and/or NK cells) using replication-defective recombinant retroviral particles, thereby producing genetically modified and/or transduced lymphocytes (e.g., T cells and/or NK cells).

In some embodiments of the method provided immediately above, lymphocytes (e.g., T cells and/or NK cells) or a colony thereof modified and/or transduced by genetic means are introduced into an individual. In some embodiments, lymphocytes (e.g., T cells and/or NK cells) or a colony thereof modified and/or transduced by genetic means undergo 4 or fewer cell divisions in vitro before being introduced or reintroduced into an individual. In some embodiments, lymphocytes are resting T cells and/or resting NK cells between 1 hour and 12 hours of contact with replication-deficient recombinant retroviral particles. In some embodiments, the time of collecting blood from an individual and the time of reintroducing genetically modified T cells and/or NK cells into an individual are no more than 8 hours. In some embodiments, all steps after collecting blood and before reintroducing blood are performed in a closed system, and people monitor the closed system during the entire treatment period.

In another aspect, provided herein is a genetically modified T cell and/or NK cell comprising:

a. one or more (eg, two or more) inhibitory RNA molecules directed against one or more RNA targets; and

b. A chimeric antigen receptor (CAR) comprising an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain, wherein the one or more (e.g., two or more) inhibitory RNA molecules and the CAR are encoded by genetically modified nucleic acid sequences of T cells and/or NK cells.

In some embodiments of genetically modified T cells and/or NK cell aspects, genetically modified T cells and/or NK cells also include at least one lymphoproliferative component that is not an inhibitory RNA molecule, wherein the lymphoproliferative component is encoded by a genetically modified nucleic acid of T cells and/or NK cells. In some embodiments, the inhibitory RNA molecule, CAR and/or at least one lymphoproliferative component are expressed as a polycistronic substance. In illustrative embodiments, the inhibitory RNA molecule is expressed by a single polycistronic transcript.

In another aspect, a replication-deficient recombinant retroviral particle is provided herein for use in a method for genetically modifying lymphocytes of an individual for treating tumor growth, wherein the replication-deficient recombinant retroviral particle comprises a polynucleotide in its genome, the polynucleotide comprising one or more nucleic acid sequences operably linked to a promoter active in T cells and/or NK cells, wherein the first nucleic acid sequence of the one or more nucleic acid sequences encodes a chimeric antigen receptor (CAR), the chimeric antigen receptor comprising an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain, and the second nucleic acid sequence encodes a lymphoproliferative component, wherein the method comprises contacting an individual's T cells and/or NK cells in vitro, and the contact helps to transduce at least some of the resting T cells and/or NK cells using replication-deficient recombinant retroviral particles, thereby producing genetically modified T cells and/or NK cells. In some embodiments, CAR is MRB-CAR. In any one of the aspects including MRB-CAR provided herein, MRB-CAR may have reduced binding to its cognate antigen at one pH value than at different pH values. In some embodiments, MRB-CAR may have a reduced binding to its cognate antigen at a pH higher than 7.0, 7.1, 7.2, 7.3, 7.4 or 7.5 than at a pH lower than 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 or 7.0. In other embodiments, MRB-CAR may have a reduced binding at a higher pH than at a lower pH. For example, MRB-CAR may have a reduced binding to its cognate antigen at a pH lower than 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 or 7.0 than at a pH higher than 7.0, 7.1, 7.2, 7.3, 7.4 or 7.5. In other embodiments, MRB-CAR presents an increased binding in the pH of the tumor compared to the pH of the blood. In some embodiments, the agent is used in a method, which further includes introducing genetically engineered T cells and/or NK cells into an individual. In some embodiments, the agent may be sodium bicarbonate, tris-hydroxymethylaminomethane, an equimolar hypertonic solution of sodium bicarbonate and sodium carbonate, or a proton pump inhibitor such as esomeprazole, esomeprazole and naproxen, lansoprazole, omeprazole, and rabeprazole.

In another aspect, provided herein is a replication-defective recombinant retroviral particle for use in a method for genetically modifying T cells and/or NK cells of a subject for treating tumor growth, wherein the method comprises:

a) contacting the individual's T cells and/or NK cells in vitro with a replication-defective recombinant retroviral particle comprising a polynucleotide in its genome, the polynucleotide comprising one or more nucleic acid sequences operably linked to a promoter active in T cells and/or NK cells, wherein a first nucleic acid sequence of the one or more nucleic acid sequences encodes one or more (e.g., two or more) inhibitory RNA molecules against one or more RNA targets, and a second nucleic acid sequence of the one or more nucleic acid sequences encodes a chimeric antigen receptor (CAR) comprising an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain, wherein the contact facilitates transduction of at least some of the resting T cells and/or NK cells with the replication-defective recombinant retroviral particle, thereby generating genetically modified T cells and/or NK cells; and

b) introducing the genetically modified T cells and/or NK cells into an individual, thereby genetically modifying the T cells and/or NK cells of the individual.

In the aspects provided immediately above, in some embodiments, a population of T cells and/or NK cells is contacted in the contacting step and introduced into the individual in the introducing step.

In another aspect, provided herein is the use of a replication-defective recombinant retroviral particle in the manufacture of a kit for genetically modifying T cells and/or NK cells of an individual, wherein the use of the kit comprises:

A. contacting the individual's T cells and/or NK cells in vitro with a replication-defective recombinant retroviral particle comprising a polynucleotide in its genome, the polynucleotide comprising one or more nucleic acid sequences operably linked to a promoter active in T cells and/or NK cells, wherein a first nucleic acid sequence of the one or more nucleic acid sequences encodes one or more (e.g., two or more) inhibitory RNA molecules against one or more targets, and a second nucleic acid sequence of the one or more nucleic acid sequences encodes a chimeric antigen receptor (CAR) comprising an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain, wherein the contact facilitates transduction of at least some of the resting T cells and/or NK cells with the replication-defective recombinant retroviral particle, thereby generating genetically modified T cells and/or NK cells; and

B. Introducing genetically modified T cells and/or NK cells into an individual, thereby genetically modifying the individual's T cells and/or NK cells.

In another aspect, provided herein is a use of a replication-deficient recombinant retroviral particle in the manufacture of a medicament for genetically modifying T cells and/or NK cells of an individual, wherein the use of the medicament comprises:

A) contacting the individual's T cells and/or NK cells in vitro with a replication-defective recombinant retroviral particle comprising a polynucleotide in its genome, the polynucleotide comprising one or more nucleic acid sequences operably linked to a promoter active in T cells and/or NK cells, wherein a first nucleic acid sequence of the one or more nucleic acid sequences encodes one or more (e.g., two or more) inhibitory RNA molecules against one or more targets, and a second nucleic acid sequence of the one or more nucleic acid sequences encodes a chimeric antigen receptor (CAR) comprising an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain, wherein the contacting facilitates transduction of at least some of the resting T cells and/or NK cells with the replication-defective recombinant retroviral particle, thereby generating genetically modified T cells and/or NK cells; and

B) introducing the genetically modified T cells and/or NK cells into the individual, thereby genetically modifying the T cells and/or NK cells of the individual.

In another aspect, provided herein is a commercial container containing a replication-defective recombinant retroviral particle and instructions for use thereof for treating tumor growth in an individual, wherein the replication-defective recombinant retroviral particle comprises a polynucleotide in its genome, the polynucleotide comprising one or more nucleic acid sequences operably linked to a promoter active in T cells and/or NK cells, wherein a first nucleic acid sequence of the one or more nucleic acid sequences encodes one or more (e.g., two or more) inhibitory RNA molecules against one or more RNA targets, and a second nucleic acid sequence of the one or more nucleic acid sequences encodes a chimeric antigen receptor (CAR) comprising an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain.

In some embodiments, in the form of a commercial container, the instructions direct the user to contact the individual's T cells and/or NK cells in vitro to facilitate transduction of at least one resting T cell and/or NK cell of the individual using replication-defective recombinant retroviral particles, thereby producing genetically modified T cells and/or NK cells.

In any of the aspects provided herein including a polynucleotide comprising one or more nucleic acid sequences operably linked to a promoter active in T cells and/or NK cells, wherein the first nucleic acid sequence of the one or more nucleic acid sequences encodes one or more (e.g., two or more) inhibitory RNA molecules for one or more RNA targets, and the second nucleic acid sequence of the one or more nucleic acid sequences encodes a chimeric antigen receptor (CAR) comprising an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain, the polynucleotide may further include a third nucleic acid sequence encoding at least one lymphoproliferative component that is not an inhibitory RNA molecule. In some embodiments, the lymphoproliferative component may be a cytokine or a cytokine receptor polypeptide, or a fragment thereof comprising a signaling domain. In some embodiments, the lymphoproliferative component is constitutively active. In certain embodiments, the lymphoproliferative component may be an IL-7 receptor or a fragment thereof. In illustrative embodiments, the lymphoproliferative component may be a constitutively active IL-7 receptor or a constitutively active fragment thereof.

In any one of the aspects including polynucleotides provided herein (including one or more nucleic acid sequences operably linked to a promoter active in T cells and/or NK cells), wherein the first nucleic acid sequence of the one or more nucleic acid sequences encodes one or more (e.g., two or more) inhibitory RNA molecules for one or more RNA targets, the inhibitory RNA molecules may include 5' chains and 3' chains partially or completely complementary to each other in some embodiments, wherein the 5' chain and the 3' chain can form 18 to 25 nucleotide RNA double helices. In some embodiments, the 5' chain length may be 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides, and the 3' chain length may be 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides. In some embodiments, the 5' chain and the 3' chain length may be the same or different. In some embodiments, the RNA double helix may include one or more mismatches. In alternative embodiments, the RNA double helix does not have a mismatch.

In any of the aspects provided herein including polynucleotides (comprising one or more nucleic acid sequences operably linked to a promoter active in T cells and/or NK cells), the first nucleic acid sequence of the one or more nucleic acid sequences may encode one or more (e.g., two or more) inhibitory RNA molecules for one or more RNA targets, and the inhibitory RNA molecules may be miRNA or shRNA. In some embodiments, the inhibitory molecule may be a precursor of miRNA, such as Pri-miRNA or pre-miRNA, or a precursor of shRNA. In some embodiments, the inhibitory molecule may be an artificially derived miRNA or shRNA. In other embodiments, the inhibitory RNA molecule may be a dsRNA (transcribed or artificially introduced) or siRNA itself that is processed into siRNA. In some embodiments, the inhibitory RNA molecule may be a miRNA or shRNA having a sequence not found in nature, or having at least one functional segment not found in nature, or having a combination of functional segments not found in nature. In illustrative embodiments, at least one or all of the inhibitory RNA molecules are miR-155.

In any of the aspects provided herein including polynucleotides (comprising one or more nucleic acid sequences operably linked to a promoter active in T cells and/or NK cells), the first nucleic acid sequence of the one or more nucleic acid sequences may encode one or more (e.g., two or more) inhibitory RNA molecules for one or more RNA targets, and in some embodiments, the inhibitory RNA molecules may be oriented from 5' to 3' to include: 5' arms, 5' stems, loops, 3' stems partially or completely complementary to the 5' stems, and 3' arms. In some embodiments, at least one of the two or more inhibitory RNA molecules has this arrangement. In other embodiments, all of the two or more inhibitory RNA molecules have this arrangement. In some embodiments, the 5' stem length may be 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides. In some embodiments, the 3' stem length may be 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides. In some embodiments, the loop length may be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides. In some embodiments, the 5' arm, the 3' arm, or both are derived from a naturally occurring miRNA. In some embodiments, the 5' arm, the 3' arm, or both are derived from a naturally occurring miRNA selected from the group consisting of miR-155, miR-30, miR-17-92, miR-122, and miR-21. In illustrative embodiments, the 5' arm, the 3' arm, or both are derived from miR-155. In some embodiments, the 5' arm, the 3' arm, or both are derived from Mus musculus miR-155 or Homo sapiens miR-155. In some embodiments, the 5' arm has a sequence set forth in SEQ ID NO: 256 or a functional variant thereof, such as a sequence that is identical in length to SEQ ID NO: 256, or 95%, 90%, 85%, 80%, 75% or 50% of the length of SEQ ID NO: 256, or 100 nucleotides or less, 95 nucleotides or less, 90 nucleotides or less, 85 nucleotides or less, 80 nucleotides or less, 75 nucleotides or less, 70 nucleotides or less, 65 nucleotides or less, 60 nucleotides or less, 55 nucleotides or less, 50 nucleotides or less, 45 nucleotides or less, 40 nucleotides or less, 35 nucleotides or less, 30 nucleotides or less, or 25 nucleotides or less; and at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to SEQ ID NO: 256. In some embodiments, the 3' arm has a sequence set forth in SEQ ID NO: 260 or a functional variant thereof, such as a sequence that is identical in length to SEQ ID NO: 260, or 95%, 90%, 85%, 80%, 75% or 50% of the length of SEQ ID NO: 260, or 100 nucleotides or less, 95 nucleotides or less, 90 nucleotides or less, 85 nucleotides or less, 80 nucleotides or less, 75 nucleotides or less, 70 nucleotides or less, 65 nucleotides or less, 60 nucleotides or less, 55 nucleotides or less, 50 nucleotides or less, 45 nucleotides or less, 40 nucleotides or less, 35 nucleotides or less, 30 nucleotides or less, or 25 nucleotides or less; and at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to SEQ ID NO: 260. In some embodiments, the 3' arm comprises nucleotides 221 to 283 of the Mus musculus BIC.

In any of the aspects provided herein including polynucleotides (comprising one or more nucleic acid sequences operably linked to a promoter active in T cells and/or NK cells), the first nucleic acid sequence of the one or more nucleic acid sequences may encode two or more inhibitory RNA molecules for one or more RNA targets, and in some embodiments, the two or more inhibitory RNA molecules may be located continuously in the first nucleic acid sequence. In some embodiments, the inhibitory RNA molecules may be directly or indirectly adjacent to each other using non-functional connector sequences. In some embodiments, the connector sequence length may be between 5 and 120 nucleotides, or between 10 and 40 nucleotides in length.

In any of the aspects provided herein including polynucleotides (comprising one or more nucleic acid sequences operably linked to a promoter active in T cells and/or NK cells), the first nucleic acid sequence of the one or more nucleic acid sequences may encode two or more inhibitory RNA molecules for one or more RNA targets, and in some embodiments, the first nucleic acid sequence encodes two to four inhibitory RNA molecules. In illustrative embodiments, the first nucleic acid sequence includes between 2 and 10, between 2 and 8, between 2 and 6, between 2 and 5, between 2 and 4, between 3 and 5, or between 3 and 6 inhibitory RNA molecules. In an illustrative embodiment, the first nucleic acid sequence includes four inhibitory RNA molecules.

In any of the aspects provided herein including a polynucleotide comprising one or more nucleic acid sequences operably linked to a promoter active in T cells and/or NK cells, the first nucleic acid sequence of the one or more nucleic acid sequences may encode one or more (e.g., two or more) inhibitory RNA molecules for one or more RNA targets, and the one or more (e.g., two or more) inhibitory RNA molecules may be in an intron. In some embodiments, the intron is in the promoter. In illustrative embodiments, the intron is EF-1α intron A. In some embodiments, the intron is adjacent to the promoter and downstream of the promoter, and in illustrative embodiments, the intron is inactive in packaging cells for producing replication-defective recombinant retroviral particles.

In any of the aspects provided herein including a polynucleotide (comprising one or more nucleic acid sequences operably linked to a promoter active in T cells and/or NK cells), wherein the first nucleic acid sequence of the one or more nucleic acid sequences encodes two or more inhibitory RNA molecules directed against one or more RNA targets, in some embodiments, the two or more inhibitory RNA molecules may be directed against different targets. In an alternative embodiment, the two or more inhibitory RNA molecules are directed against the same target. In some embodiments, the RNA target is an mRNA transcribed from a gene expressed by a free T cell, such as, but not limited to: PD-1 (prevents inactivation); CTLA4 (prevents inactivation); TCRα (safely prevents autoimmunity); TCRb (safe-prevents autoimmunity); CD3Z (safe-prevents autoimmunity); SOCS1 (prevents inactivation); SMAD2 (prevents inactivation); miR-155 target (promotes activation); IFNγ (reduces CRS); cCBL (prolongs signaling); TRAIL2 (prevents death); PP2A (prolongs signaling); ABCG1 (increases cholesterol microcontent by limiting cholesterol clearance). In some embodiments, the RNA target is an mRNA transcribed from a gene encoding a component of the T cell receptor (TCR) complex. In some embodiments, at least one of the two or more inhibitory RNA molecules can reduce the expression of a T cell receptor (in illustrative embodiments, one or more endogenous T cell receptors of a T cell). In certain embodiments, the RNA target may be mRNA transcribed from an endogenous TCRα or TCRβ gene of a T cell whose genome comprises a first nucleic acid sequence encoding one or more miRNAs. In an illustrative embodiment, the RNA target is mRNA transcribed from a TCRα gene.

In any of the aspects provided herein including a polynucleotide (comprising one or more nucleic acid sequences operably linked to a promoter active in T cells and/or NK cells), a first nucleic acid sequence of one or more nucleic acid sequences may encode one or more (e.g., two or more) inhibitory RNA molecules for one or more RNA targets, and a second nucleic acid sequence of one or more nucleic acid sequences encodes a chimeric antigen receptor (CAR) comprising an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain, in some embodiments, CAR is a microenvironmentally restricted organism (MRB)-CAR. In other embodiments, the ASTR of CAR binds to a tumor-associated antigen. In other embodiments, the ASTR of CAR is a microenvironmentally restricted organism (MRB)-ASTR.

In any of the aspects provided herein including polynucleotides (comprising one or more nucleic acid sequences operably linked to a promoter active in T cells and/or NK cells), the first nucleic acid sequence of the one or more nucleic acid sequences may encode one or more (e.g., two or more) inhibitory RNA molecules for one or more RNA targets, and the second nucleic acid sequence of the one or more nucleic acid sequences encodes a chimeric antigen receptor (CAR), the chimeric antigen receptor comprising an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain, and in some cases, the third nucleic acid sequence of the one or more nucleic acid sequences encoding at least one lymphoproliferative component is not an inhibitory RNA molecule, in some embodiments, any one or all of the first nucleic acid sequence, the second nucleic acid sequence, and the third nucleic acid sequence may be operably linked to a riboswitch. In some embodiments, the riboswitch is capable of binding nucleoside analogs. In some embodiments, the nucleoside analogs are antiviral drugs.

In any of the aspects provided herein including replication-deficient recombinant retroviral particles, in some embodiments, the replication-deficient recombinant retroviral particles include a pseudotyped component on their surface that is capable of binding to T cells and/or NK cells and facilitates membrane fusion with the replication-deficient recombinant retroviral particles. In some embodiments, the pseudotyped component may be a measles virus F polypeptide, a measles virus H polypeptide, a VSV-G polypeptide, or any fragment thereof that retains the ability to bind to resting T cells and/or resting NK cells. In illustrative embodiments, the pseudotyped component is VSV-G.

In any of the aspects provided herein including replication-deficient recombinant retroviral particles, in some embodiments, the replication-deficient recombinant retroviral particles include an activation component on their surface, the activation component comprising a membrane-bound polypeptide capable of binding to CD3, and/or a membrane-bound polypeptide capable of binding to CD28. In some embodiments, the membrane-bound polypeptide capable of binding to CD3 is fused to a heterologous GPI anchoring linking sequence, and/or the membrane-bound polypeptide capable of binding to CD28 is fused to a heterologous GPI anchoring linking sequence. In some embodiments, the membrane-bound polypeptide capable of binding to CD3 is an anti-CD3 scFv or an anti-CD3 scFvFc. In illustrative embodiments, the membrane-bound polypeptide capable of binding to CD3 is an anti-CD3 scFvFc. In illustrative embodiments, the membrane-bound polypeptide capable of binding to CD28 is CD80, or its extracellular domain bound to the CD16B GPI anchoring linking sequence.

In any of the aspects provided herein comprising a replication-defective recombinant retroviral particle, in some embodiments, the replication-defective recombinant retroviral particle comprises a nucleic acid on its surface encoding a domain recognized by a biologically validated monoclonal antibody.

In the aspect of the chimeric polypeptide provided below that promotes the cell proliferation of PBMC (e.g., B cells, T cells and/or NK cells), the chimeric polypeptide may be referred to herein as a chimeric lymphoproliferative element (CLE). The embodiments provided below that include nucleic acid sequences encoding CLE and CAR are referred to herein as CLE CAR polynucleotide embodiments.

In embodiments and subembodiments of any of the aspects and embodiments provided herein (including those in the above paragraphs of this section), the lymphoproliferative component can be any of the chimeric lymphoproliferative components provided herein, including below in this section. In other embodiments and subembodiments of any of the aspects and embodiments of CAR herein, the lymphoproliferative component is, in illustrative embodiments, any of the chimeric lymphoproliferative components encoded by the nucleic acids in the CLECAR polynucleotide (and related polypeptide) embodiments provided herein, e.g., described below in this section.

Unless incompatible with an aspect, or stated in an aspect, in certain illustrative embodiments, any aspect or other embodiment provided herein comprising a lymphoproliferative element (LE) or a nucleic acid encoding a lymphoproliferative element may state that the lymphoproliferative element satisfies, is suitable for satisfying, possesses, has the ability to, or possesses the properties of any of the identified tests for identifying the LE provided herein, or that cells genetically modified and/or transduced by a retroviral particle (e.g., a lentiviral particle) having a genome encoding the LE are capable of, suitable for, possess, and/or modified to achieve the results of any one or more of the following tests:

a) improving the expansion of pre-activated PBMCs transduced with retroviral particles comprising a nucleic acid encoding a lymphoproliferative component between day 7 and day 21, day 28, day 35 and/or day 42 of in vitro culture in the absence of exogenously added cytokines, when transduced with a nucleic acid encoding an anti-CD19 CAR comprising a CD3ζ intracellular activation domain but without a co-stimulatory domain, compared to a control construct under the same conditions; and/or

b) when transduced with a nucleic acid encoding an anti-CD19 CAR comprising a CD3ζ intracellular activation domain but without a co-stimulatory domain, in the absence of exogenously added cytokines, pre-activated PBMCs transduced with retroviral particles comprising a nucleic acid encoding a lymphoproliferative component are expanded by at least 2-fold, 3-fold, 4-fold, 6-fold, 7-fold or 8-fold, or between 3-fold and 25-fold, between 5-fold and 25-fold, between 5-fold and 20-fold, between 5-fold and 15-fold, between 7-fold and 15-fold or between 7-fold and 12-fold between days 7 and 21, 28, 35 and/or 42 of in vitro culture.

In some embodiments, a statistical test is used to demonstrate improvement in amplification according to element a) in the previous paragraph. The statistical test can use a cutoff value of, for example, a p-value equal to or less than 0.10, 0.05, or 0.01. For example, the statistical test can be a T test. The control construct can be a control construct of the lymphoproliferative component provided herein, or a control construct that does not include LE, or in illustrative embodiments, the control construct is the same as the nucleic acid construct that includes the lymphoproliferative component, but does not have the lymphoproliferative component or the intracellular domain of the lymphoproliferative component.

In one aspect, provided herein are isolated polynucleotides comprising one or more nucleic acid sequences, wherein:

A first nucleic acid sequence of the one or more nucleic acid sequences encodes a chimeric polypeptide comprising, in an amino to carboxyl orientation,

a) an extracellular domain and a transmembrane domain from a cytokine receptor or a hormone receptor, wherein at least one of the extracellular domain and the transmembrane domain comprises

i. a mutation found in a constitutively active mutant of a cytokine receptor, wherein the extracellular sequence does not bind a ligand of the cytokine receptor, or

ii. the extracellular domain and transmembrane domain from LMP1; and

b) a first intracellular domain selected from the intracellular domain of a gene having a first intracellular domain of a selected polypeptide identified in Tables 8 to 11, wherein the chimeric polypeptide promotes cellular proliferation of B cells, T cells and/or NK cells.

In an embodiment of the aspect immediately following the above paragraph, the first nucleic acid sequence further encodes a second intracellular domain, wherein the second intracellular domain may be upstream (i.e., 5') of the first intracellular domain, or in an illustrative embodiment, the second intracellular domain is downstream of the first intracellular domain. In a sub-embodiment of this embodiment, the first intracellular domain and the second intracellular domain combination comprise the first intracellular domain and the second intracellular domain combination of the selected polypeptide. Therefore, for clarity, in non-limiting exemplary embodiments, the selected polypeptide is M008-S212-S075. In this embodiment, the first intracellular domain of the chimeric polypeptide comprises or is TNFRSF8 transcript variant 1 (NM_001243_4) (S212) and the second polypeptide comprises or is FCGR2C (NM_201563_5) (S075).

In one embodiment of this aspect, the chimeric polypeptide comprises a transmembrane domain, a first intracellular domain, and a second intracellular domain of a selected polypeptide. In one embodiment of this aspect, the chimeric polypeptide comprises an extracellular domain and a transmembrane domain, a first intracellular domain, and a second intracellular domain of a selected polypeptide. Therefore, for the sake of clarity, in non-limiting exemplary embodiments, the selected polypeptide is M008-S212-S075. In this embodiment, the extracellular domain and transmembrane domain of the chimeric polypeptide comprise or are Myc LMP1 (NC_007605_1) (M008), the first intracellular domain of the chimeric polypeptide comprises or is TNFRSF8 transcript variant 1 (NM_001243_4) (S212), and the second intracellular domain of the chimeric polypeptide comprises or is FCGR2C (NM_201563_5) (S075). In a related sub-embodiment, the chimeric polypeptide comprises the extracellular and transmembrane domains of the selected polypeptide, the first intracellular domain, and the second intracellular domain, except that the extracellular domain and the transmembrane domain optionally comprise a recognition domain or an elimination domain, or do not comprise a recognition domain or an elimination domain. In a related sub-embodiment, the chimeric polypeptide comprises the extracellular and transmembrane domains of the selected polypeptide, the first intracellular domain, and the second intracellular domain, except that the extracellular and transmembrane domains comprise a recognition or elimination domain of the selected polypeptide, or another clearance tag. In another embodiment of the aspect provided in the above paragraph, the first intracellular domain and the second intracellular domain combination comprise the first intracellular domain of the selected polypeptide and any intracellular domain of any gene identified in the second intracellular domain of any candidate polypeptide in Tables 8 to 11.

In another embodiment of this aspect, the polynucleotide further comprises a second nucleic acid sequence encoding a chimeric antigen receptor (CAR), the chimeric antigen receptor comprising an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain. In a sub-embodiment of this aspect, the first intracellular domain and the second intracellular domain are combined as the first intracellular domain and the second intracellular domain of the selected polypeptide, and the selected polypeptide is identified in Tables 8 to 11. In another sub-embodiment of this embodiment, the chimeric polypeptide comprises an extracellular domain and a transmembrane domain, a first intracellular domain, and a second intracellular domain of the selected polypeptide, and the selected polypeptide is identified in Tables 8 to 11. In a related sub-embodiment, the chimeric polypeptide comprises an extracellular domain and a transmembrane domain, a first intracellular domain, and a second intracellular domain of the selected polypeptide, except that the extracellular domain and the transmembrane domain optionally comprise a recognition or elimination domain, and wherein the recognition or elimination domain is a recognition or elimination domain of the extracellular domain of the selected polypeptide, or other recognition or elimination domains, and wherein the selected polypeptide is identified in Tables 8 to 11.

In another aspect, provided herein are isolated polynucleotides comprising one or more nucleic acid sequences, wherein:

A first nucleic acid sequence of the one or more nucleic acid sequences encodes a chimeric polypeptide comprising, in an amino to carboxyl orientation,

a) an extracellular domain and a transmembrane domain from a cytokine receptor or a hormone, wherein at least one of the extracellular domain and the transmembrane domain comprises

i. a mutation found in a constitutively active mutant of a cytokine receptor, wherein the extracellular sequence does not bind a ligand of the cytokine receptor, or

ii. the extracellular domain and transmembrane domain from LMP1; and

b) a first intracellular domain selected from the intracellular domain of CD3D, CD3E, CD8A, CD27, CD40, CD79B, IFNAR1, IL2RA, IL3RA, IL13RA2, TNFRSF8, TNFRSF9, IL31RA or MyD88, wherein the chimeric polypeptide promotes cell proliferation of PBMCs, in illustrative embodiments, B cells, T cells and/or NK cells.

In an embodiment of the aspect immediately following the above paragraph, the first nucleic acid sequence further encodes a second intracellular domain, wherein the second intracellular domain may be upstream (i.e., 5') of the first intracellular domain, or in an illustrative embodiment, the second intracellular domain is downstream of the first intracellular domain. In a sub-embodiment of this embodiment, the second intracellular domain is from a gene of a second intracellular domain in a selected polypeptide identified in any table of Example 11 as producing an enrichment higher than 0, 1, or 2 on the 35th day versus the 7th day, wherein the first intracellular domain is from a gene of a first intracellular domain of the selected polypeptide. In another sub-embodiment of this sub-embodiment or embodiment, the first intracellular domain and the second intracellular domain are combined as a first intracellular domain and a second intracellular domain from a selected polypeptide identified in any table of Example 11 as producing an enrichment higher than 0, 1, or 2 on the 35th day versus the 7th day. In another embodiment of the embodiment or sub-embodiment in this paragraph, the first intracellular domain is from a first gene of a selected first intracellular domain of a selected polypeptide selected in Tables 8 to 11 and the second intracellular domain is from a second gene of a selected second intracellular domain of a selected polypeptide. In another embodiment of the embodiments or subembodiments in this paragraph, the first intracellular domain is a polypeptide selected from Tables 8-11 and the second intracellular domain is a polypeptide selected from Tables 8-11.

In another sub-embodiment of the above embodiment in which the first nucleic acid sequence further encodes a second intracellular domain, the second intracellular domain is selected from a second intracellular domain identified in any table in Example 11 as producing an enrichment greater than 0, 1, or 2 on day 35 versus day 7 for the corresponding first intracellular domain. In sub-embodiments of these embodiments, the first intracellular domain is selected from an intracellular domain of CD27, CD40, or CD79B. In other sub-embodiments of this embodiment, the first intracellular domain is selected from an intracellular domain of CD3D, CD3E, CD8A, CD27, CD40, CD79B, IFNAR1, IL2RA, IL3RA, IL13RA2, TNFRSF8, TNFRSF9, IL31RA, or MyD88 identified in any table in Example 11 as producing an enrichment greater than 0, 1, or 2 on day 35 versus day 7. In another embodiment of the embodiment or sub-embodiment in this paragraph, the first intracellular domain is from a first gene of a chimeric polypeptide selected in Tables 8 to 11 and the second intracellular domain is from a second gene of a chimeric polypeptide selected in Tables 8 to 11. In another embodiment of the embodiments or subembodiments in this paragraph, the first intracellular domain is from the first gene of the chimeric polypeptide selected in Tables 8 to 11 and the second intracellular domain is from the intracellular domain of CD3D, CD3ζ, CD8A, CD8B, CD27, CD40, CD79B, CRLF2, FCGR2C, ICOS, IL2RA, IL13RA1, IL13RA2, IL15RA, TNFRSF9 and TNFRSF18. In another embodiment of the embodiments or subembodiments in this paragraph, the first intracellular domain is from the selected polypeptide identified in Tables 8 to 11 and the second intracellular domain is from the selected polypeptide identified in Tables 8 to 11. In a related subembodiment, the chimeric polypeptide comprises the extracellular domain and the transmembrane domain of the selected polypeptide, the first intracellular domain and the second intracellular domain, except that the extracellular domain and the transmembrane domain optionally comprise a clearance tag, or do not comprise a clearance tag, and wherein the selected polypeptide is identified in Tables 8 to 11. In a related sub-embodiment, the chimeric polypeptide comprises the extracellular domain and the transmembrane domain of the selected polypeptide, the first intracellular domain and the second intracellular domain, except that the extracellular domain and the transmembrane domain comprise the recognition or elimination domain of the selected polypeptide, or other recognition or elimination domains, and wherein the selected polypeptide is recognized in Tables 8 to 11.

In another embodiment of this aspect, the first intracellular domain selects the intracellular domain of CD3D, CD3E, CD8A, CD27, CD40, CD79B, IFNAR1, IL2RA, IL3RA, IL13RA2, TNFRSF8 or TNFRSF9, and the polynucleotide further comprises a second nucleic acid sequence encoding a chimeric antigen receptor (CAR), the chimeric antigen receptor comprising an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain. In a sub-embodiment of this aspect, the first intracellular domain is from the gene of the first intracellular domain of the selected polypeptide and the second intracellular domain is from the gene of the second intracellular domain of the selected polypeptide, and the selected polypeptide is identified in Tables 8 to 11. In a sub-embodiment of this aspect, the first intracellular domain and the second intracellular domain are combined as the first intracellular domain and the second intracellular domain of the selected polypeptide, and the selected polypeptide is identified in Tables 8 to 11. In a sub-embodiment of this embodiment, the chimeric polypeptide comprises the extracellular domain and the transmembrane domain, the first intracellular domain and the second intracellular domain of the selected polypeptide, and the selected polypeptide is identified in Tables 8 to 11. In a related sub-embodiment, the chimeric polypeptide comprises the extracellular and transmembrane domains, the first intracellular domain and the second intracellular domain of the selected polypeptide, except that the extracellular domain and the transmembrane domain optionally comprise a recognition or elimination domain, and wherein the recognition or elimination domain is the recognition or elimination domain of the extracellular domain of the selected polypeptide, or other recognition or elimination domain, and wherein the selected polypeptide is identified in Tables 8 to 11.

In another aspect, provided herein are isolated polynucleotides comprising one or more nucleic acid sequences, wherein:

A first nucleic acid sequence of the one or more nucleic acid sequences encodes a chimeric polypeptide comprising, in an amino to carboxyl orientation,

(a) an extracellular domain and a transmembrane domain from a cytokine receptor or a hormone, wherein at least one of the extracellular domain and the transmembrane domain comprises

i. a mutation found in a constitutively active mutant of a cytokine receptor, wherein the extracellular sequence does not bind a ligand of the cytokine receptor, or

ii. the extracellular domain and transmembrane domain from LMP1; and

(b) a first intracellular domain selected from the group consisting of an intracellular domain of CD3D, CD3Z, CD8A, CD8B, CD27, CD40, CD79B, CRLF2, FCGR2C, ICOS, IL2RA, IL13RA1, IL13RA2, IL15RA, TNFRSF9, and TNFRSF18, wherein the chimeric polypeptide promotes cell proliferation of PBMCs, and in illustrative embodiments, B cells, T cells, and/or NK cells.

In Example 11, the intracellular domain described in the immediately above aspect is identified as the notable second intracellular domain. However, it should be understood that in some embodiments, the intracellular domain identified as the notable second intracellular domain is the first intracellular domain of the chimeric polypeptide of these embodiments.

In another aspect, provided herein are isolated polynucleotides comprising one or more nucleic acid sequences, wherein:

A first nucleic acid sequence of the one or more nucleic acid sequences encodes a chimeric polypeptide comprising, in an amino to carboxyl orientation,

a) an extracellular domain and a transmembrane domain from a cytokine receptor or a hormone, wherein at least one of the extracellular domain and the transmembrane domain comprises i. a mutation found in a constitutively active mutant of a cytokine receptor, and wherein the extracellular sequence does not bind a ligand of a cytokine receptor, or

ii. the extracellular domain and transmembrane domain from LMP1; and

b) a first intracellular domain selected from the group consisting of the intracellular domains of CD3D, CD3E, CD8A, CD27, CD40, CD79B, IFNAR1, IL2RA, IL3RA, IL13RA2, TNFRSF8, TNFRSF9, IL31RA, or MyD88,

wherein the chimeric polypeptide promotes cell proliferation of PBMCs, and in illustrative embodiments, B cells, T cells and/or NK cells, and wherein the extracellular domain and the transmembrane domain are selected from the extracellular domain and the transmembrane domain of IL7RA Ins PPCL, LMP1, CRLF2 F232C, CSF2RB V449E, CSF3R T640N, EPOR L251C I252C, GHR E260C I270C, IL27RA F523C and MPL S505N.

In certain embodiments of any of the above aspects of polynucleotides encoding chimeric polypeptides, wherein the chimeric polypeptide promotes cell proliferation of PBMCs, and in illustrative embodiments, B cells, T cells and/or NK cells, the extracellular domain and the transmembrane domain are selected from CSF3RT640N and MPL S505N. In illustrative embodiments, the extracellular domain and the transmembrane domain are from CSF3RT640N and in certain embodiments, the isolated polynucleotide does not comprise a CAR. In certain sub-embodiments, the extracellular domain and the transmembrane domain are CSF3RT640N provided in Table 7, but the recognition domain and the elimination domain are optional. In certain embodiments, the first intracellular domain and the second intracellular domain are from any of the polypeptides provided in Example 11 and the table including the intracellular domain and the extracellular domain and the first intracellular domain herein. In other sub-embodiments, the extracellular domain and the intracellular domain are from MPL S505N, and in certain embodiments, the extracellular domain and the transmembrane domain of MPL S505N provided in Table 7 except the recognition domain and the elimination domain are optional.

In another aspect, provided herein are isolated polynucleotides comprising one or more nucleic acid sequences, wherein:

A first nucleic acid sequence of the one or more nucleic acid sequences encodes a chimeric polypeptide comprising, in an amino to carboxyl orientation,

a) a transmembrane domain from a type I transmembrane protein; and

b) a first intracellular domain selected from the intracellular domain of a gene having a first intracellular domain of a selected polypeptide identified in Tables 12 to 17, wherein the chimeric polypeptide promotes cell proliferation and or survival of B cells, T cells and/or NK cells.

As shown in the additional Example 12, such chimeric polypeptides are capable of promoting cell survival and/or proliferation of PBMCs in vivo or in vitro or ex vivo for 6 days, 7 days, 14 days, 21 days, 35 days, 42 days or more during culture in the absence of exposure of PBMCs, T cells, B cells or NK cells to cytokines (such as IL-15, IL-7, and in illustrative embodiments, IL-2) and, optionally, in the absence of the target of the ASTR of the CAR expressed by the cells.

In one embodiment of the aspect in the paragraph immediately above and in any embodiment of this aspect in the following section, the first intracellular domain is an intracellular domain of a gene having a first intracellular domain in Tables 12 to 17, and in other illustrative embodiments, one of the first 50, 25 or 10 constructs is listed in Tables 12 to 17, and in other illustrative embodiments, one of the first 50, 25 or 10 constructs is listed in two, three, four or five of the six tables in Tables 12 to 17, or all of the tables. In one embodiment, the first intracellular domain is an intracellular domain identified in Table 18. Table 18 identifies constructs that promote the proliferation of PBMCs between day 7 and day 21 or day 35, wherein the second intracellular domain is not present in the construct. In some embodiments of this embodiment, the chimeric polypeptide comprises a combination of a transmembrane domain and an intracellular domain, with or without an extracellular domain comprising a dimerization motif of Table 18.

In one embodiment of the aspect in the immediately preceding paragraph and in any embodiment of this aspect in the following section, the first nucleic acid sequence further encodes an extracellular domain, and in illustrative embodiments, the extracellular domain comprises a dimerization motif. In any aspect or embodiment in which the extracellular domain of CLE comprises a dimerization motif, the dimerization motif can be selected from the group consisting of a polypeptide containing a leucine zipper motif, CD69, CD71, CD72, CD96, Cd105, Cd161, Cd162, Cd249, CD271, and Cd324, and mutants and/or active fragments thereof that retain dimerization ability. In any of the aspects or embodiments in which the extracellular domain of CLE comprises a dimerization motif, the dimerization motif may require a dimerizer, and the dimerization motif and the relevant dimerizer may be selected from the group consisting of: FKBP and rapamycin or an analog thereof, GyrB and coumermycin or an analog thereof, DHFR and methotrexate or an analog thereof, or DmrB and AP20187 or an analog thereof, and mutants and/or active fragments of the dimeric proteins that retain the dimerization ability.

In illustrative embodiments of any aspect or embodiment in which the extracellular domain of CLE comprises a dimerization motif, the extracellular domain may comprise a leucine zipper motif. In some embodiments, the leucine zipper motif is from a jun polypeptide, such as c-jun. In certain embodiments, the c-jun polypeptide is a c-jun polypeptide region of ECD-11.

In one embodiment of this aspect, the first nucleic acid sequence further encodes a second intracellular domain, wherein the second intracellular domain may be upstream (i.e., 5') of the first intracellular domain, or in illustrative embodiments, the second intracellular domain is downstream of the first intracellular domain. In a sub-embodiment of this embodiment, the first intracellular domain and the second intracellular domain combination comprise the first intracellular domain of the selected polypeptide and any intracellular domain of any gene identified in the second intracellular domain of any candidate polypeptide in Tables 12 to 17, and in illustrative embodiments in Tables 12 to 17, and in other embodiments, one of the first 50, 25, or 10 constructs is listed in Tables 12 to 17, and in other illustrative embodiments, one of the first 50, 25, or 10 constructs is listed in two, three, four, or five, or all of the tables in Tables 12 to 17. In a sub-embodiment of this embodiment, the first intracellular domain and the second intracellular domain combination comprise the first intracellular domain and the second intracellular domain of the selected polypeptide.

In one embodiment of this aspect, the chimeric polypeptide comprises a transmembrane domain, a first intracellular domain, and a second intracellular domain of a selected polypeptide. In one embodiment of this aspect, the chimeric polypeptide comprises an extracellular and transmembrane domain, a first intracellular domain, and a second intracellular domain of a selected polypeptide, wherein the extracellular domain optionally comprises a recognition domain or an elimination domain.

In a related sub-embodiment, the chimeric polypeptide comprises the extracellular and transmembrane domains, the first intracellular domain, and the second intracellular domain of the selected polypeptide, except that the extracellular and transmembrane domains optionally comprise a recognition domain or an elimination domain or do not comprise a recognition domain or an elimination domain. In a related sub-embodiment, the chimeric polypeptide comprises the extracellular and transmembrane domains, the first intracellular domain, and the second intracellular domain of the selected polypeptide, except that the extracellular and transmembrane domains comprise a recognition domain or an elimination domain of the selected polypeptide, or another recognition domain or an elimination domain.

In another embodiment of this aspect, the polynucleotide further comprises a second nucleic acid sequence encoding a chimeric antigen receptor (CAR), the chimeric antigen receptor comprising an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain. In a sub-embodiment of this aspect, the first intracellular domain and the second intracellular domain are combined to form a first intracellular domain and a second intracellular domain of a selected polypeptide, and the selected polypeptide is identified in Tables 12 to 17, and in other embodiments, one of the first 50, 25, or 10 constructs is listed in Tables 12 to 17, and in other embodiments, one of the first 50, 25, or 10 constructs is listed in two, three, four, or five, or all of the tables in Tables 12 to 17. In another embodiment of this embodiment, the chimeric polypeptide comprises the extracellular domain and transmembrane domain, the first intracellular domain and the second intracellular domain of the selected polypeptide, and the selected polypeptide recognizes in Tables 12 to 17, or in illustrative embodiments, one of the first 50, 25 or 10 constructs is listed in Tables 12 to 17, in other embodiments, one of the first 50, 25 or 10 constructs is listed in two, three, four or five, or all of the tables in Tables 12 to 17. In a related sub-embodiment, the chimeric polypeptide comprises the extracellular domain and transmembrane domain, the first intracellular domain and the second intracellular domain of the selected polypeptide, except that the extracellular domain and transmembrane domain optionally comprise a recognition domain or elimination domain, and wherein the recognition domain or elimination domain is the recognition domain or elimination domain of the extracellular domain of the selected polypeptide, or another recognition domain or elimination domain, and wherein the selected polypeptide recognizes in Tables 12 to 17.

In another aspect, provided herein are isolated polynucleotides comprising one or more nucleic acid sequences, wherein:

A first nucleic acid sequence of the one or more nucleic acid sequences encodes a chimeric polypeptide comprising, in an amino to carboxyl orientation,

a) a transmembrane domain from a type I transmembrane protein; and

b) a first intracellular domain selected from the group consisting of the intracellular domains of LEPR, MYD88, IFNAR2, MPL, IL18R1, IL13RA2, IL10RB, IL23R or CSF2RA,

Wherein the chimeric polypeptide promotes cell proliferation of PBMCs, and in illustrative embodiments, B cells, T cells and/or NK cells.

In an embodiment of the aspect immediately preceding the paragraph and in any embodiment of this aspect in the following section, the first nucleic acid sequence further encodes an extracellular domain, and in illustrative embodiments, the extracellular domain comprises a dimerization motif. In illustrative embodiments of this aspect, the extracellular domain comprises a leucine zipper. In some embodiments, the leucine zipper is from a jun polypeptide, such as c-jun. In certain embodiments, the c-jun polypeptide is a c-jun polypeptide region of ECD-11.

In another embodiment of this aspect in which the first intracellular domain is selected from the intracellular domain of LEPR, MYD88, IFNAR2, MPL, IL18R1, IL13RA2, IL10RB, IL23R or CSF2RA, the first nucleic acid sequence further encodes a second intracellular domain, wherein the second intracellular domain may be upstream (i.e., 5') of the first intracellular domain, or in illustrative embodiments, the second intracellular domain is downstream of the first intracellular domain. In a sub-embodiment of this embodiment, the second intracellular domain is from a gene of a second intracellular domain in a selected polypeptide identified in any table of Example 12 as producing an enrichment of greater than 0, 1 or 2 at day 21 versus day 7, wherein the first intracellular domain is from a gene of a first intracellular domain of a selected polypeptide. In another sub-embodiment of this sub-embodiment or embodiment, the first intracellular domain and the second intracellular domain are combined as a first intracellular domain and a second intracellular domain from a selected polypeptide identified in any table of Example 12 as producing an enrichment of greater than 0, 1 or 2 at day 21 versus day 7. In another embodiment of the embodiments or sub-embodiments in this paragraph, the first intracellular domain is from a first gene of a selected first intracellular domain of a selected polypeptide in Tables 12 to 17 and the second intracellular domain is from a second gene of a selected second intracellular domain of a selected polypeptide. In another embodiment of the embodiments or sub-embodiments in this paragraph, the first intracellular domain is from a polypeptide selected in Tables 12 to 17 and the second intracellular domain is from a selected polypeptide. In another embodiment of this aspect or any embodiment herein, the polynucleotide further comprises a second nucleic acid sequence encoding a chimeric antigen receptor (CAR) comprising an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain.

In one embodiment of this aspect or in a sub-embodiment or other sub-embodiment of the embodiment and sub-embodiment comprising the second intracellular domain in the preceding paragraph, the first intracellular domain is the intracellular domain of LEPR, MYD88, IFNAR2 and MPL. In another embodiment, the first intracellular domain is the intracellular domain of LEPR, IFNAR2 and MPL. In another embodiment, the first intracellular domain is not MyD88. In another embodiment, the first intracellular domain is not MyD88 and the second intracellular domain is not CD40. In a sub-embodiment of this embodiment in this paragraph, the polynucleotide further comprises a second nucleic acid sequence encoding a chimeric antigen receptor (CAR), which comprises an antigen-specific targeting region (ASTR), a transmembrane domain and an intracellular activation domain.

In one embodiment of this aspect of the invention, MPL is the first intracellular domain and the second intracellular domain is from the gene for the second intracellular domain in the selected polypeptides identified in Tables 12 to 17. In another sub-embodiment of this embodiment, the first intracellular domain and the second intracellular domain combination comprises the first intracellular domain and the second intracellular domain from the selected chimeric polypeptides identified in Tables 12 to 17. In some sub-embodiments of these embodiments, the orientation of the first intracellular domain and the second intracellular domain is opposite so that the carboxyl terminal residue of the second domain is bound to the first domain or to the spacer between the second domain and the first domain.

In one embodiment of this aspect of the invention, LEPR is the first intracellular domain and the second intracellular domain is from the gene for the second intracellular domain in the selected polypeptides identified in Tables 12 to 17. In other sub-embodiments of this embodiment, the first intracellular domain and the second intracellular domain combination comprises the first intracellular domain and the second intracellular domain from the selected chimeric polypeptides identified in Tables 12 to 17. In some sub-embodiments of these embodiments, the orientation of the first intracellular domain and the second intracellular domain is opposite, such that the carboxyl terminal residue of the second domain is bound to the first domain or to the spacer between the second domain and the first domain.

In one embodiment of this aspect of the invention, IFNAR2 is the first intracellular domain and the second intracellular domain is from the gene for the second intracellular domain in a selected polypeptide identified in Tables 12 to 17. In other sub-embodiments of this embodiment, the first intracellular domain and the second intracellular domain combination comprises the first intracellular domain and the second intracellular domain from a selected chimeric polypeptide identified in Tables 12 to 17. In some sub-embodiments of these embodiments, the orientation of the first intracellular domain and the second intracellular domain is opposite, such that the carboxyl terminal residue of the second domain is bound to the first domain or to the spacer between the second domain and the first domain.

In one embodiment of this aspect of the invention, MyD88 is the first intracellular domain and the second intracellular domain is from the gene for the second intracellular domain in a selected polypeptide identified in Tables 12 to 17. In another sub-embodiment of this embodiment, the first intracellular domain and the second intracellular domain combination comprises the first intracellular domain and the second intracellular domain from a selected chimeric polypeptide identified in Tables 12 to 17. In some sub-embodiments of these embodiments, the orientation of the first intracellular domain and the second intracellular domain is opposite, such that the carboxyl terminal residue of the second domain is bound to the first domain or to the spacer between the second domain and the first domain.

In some embodiments of the isolated polynucleotide aspects of the intracellular domain of the above wherein the first intracellular domain is selected from LEPR, MYD88, IFNAR2, IL17RD, IL17RE, IL1RAP, IL23R and MPL, the transmembrane domain is selected from the transmembrane domain of CD40, CD8B, CRLF2, CSF2RA, GCGR2C, ICOS, IFNAR1, IFNGR1, IL10RB, IL18R1, IL18RAP, IL3RA and PRLR. In some illustrative embodiments, the transmembrane domain is the transmembrane domain of CD40 or ICOS. In some sub-embodiments, the first intracellular domain is from LEPR, MYD88, IFNAR2 and MPL. For example, in some embodiments, the transmembrane domain is the transmembrane domain from CD40 and the first intracellular domain is the intracellular domain from MPL.

In some embodiments of the isolated polynucleotide aspects above wherein the first intracellular domain is selected from the intracellular domains of LEPR, MYD88, IFNAR2, IL17RD, IL17RE, IL1RAP, IL23R and MPL, the second intracellular domain is from CD40, CD79B or CD27. In some subembodiments, the first intracellular domain is from LEPR, MYD88, IFNAR2 and MPL. For example, in some embodiments, the first intracellular domain is MPL and the second intracellular domain is CD40. In all embodiments in this paragraph, the order of the first intracellular domain and the second intracellular domain on the polypeptide can be reversed.

In another aspect, provided herein are isolated polynucleotides comprising one or more nucleic acid sequences, wherein:

A first nucleic acid sequence of the one or more nucleic acid sequences encodes a chimeric polypeptide comprising, in an amino to carboxyl orientation,

a) a transmembrane domain from a type I transmembrane protein; and

b) a first intracellular domain selected from the intracellular domain of a gene having a first intracellular domain of a selected polypeptide identified in Tables 12 to 17, wherein the chimeric polypeptide promotes cell proliferation of B cells, T cells and/or NK cells

Wherein the chimeric polypeptide promotes cell proliferation of PBMCs, and in illustrative embodiments, B cells, T cells and/or NK cells. In some embodiments, the NK cells are NKT cells.

In one embodiment of the aspect in the immediately preceding paragraph and in any embodiment of this aspect in the following section, the first nucleic acid sequence further encodes an extracellular domain, and in illustrative embodiments, the extracellular domain comprises a dimerization motif. In any aspect or embodiment in which the extracellular domain of CLE comprises a dimerization motif, the dimerization motif can be selected from the group consisting of a polypeptide containing a leucine zipper motif, CD69, CD71, CD72, CD96, Cd105, Cd161, Cd162, Cd249, CD271, and Cd324, and mutants and/or active fragments thereof that retain dimerization ability. In any aspect or embodiment in which the extracellular domain of CLE herein comprises a dimerization motif, the dimerization motif may require a dimerizer, and the dimerization motif and the relevant dimerizer may be selected from the group consisting of: FKBP and rapamycin or an analog thereof, GyrB and coumermycin or an analog thereof, DHFR and methotrexate or an analog thereof, or DmrB and AP20187 or an analog thereof, and mutants and/or active fragments of the dimeric proteins that retain the dimerization ability.

In illustrative embodiments of any aspect or embodiment of the extracellular domain of CLE herein comprising a dimerization motif, the extracellular domain may comprise a leucine zipper motif. In some embodiments, the leucine zipper motif is from a jun polypeptide, such as c-jun. In certain embodiments, the c-jun polypeptide is a c-jun polypeptide region of ECD-11.

In one embodiment of this aspect, the first nucleic acid sequence further encodes a second intracellular domain, wherein the second intracellular domain may be upstream (i.e., 5') of the first intracellular domain, or in illustrative embodiments, the second intracellular domain is downstream of the first intracellular domain. In a sub-embodiment of this embodiment, the first intracellular domain and the second intracellular domain combination comprise the first intracellular domain of the selected polypeptide and any intracellular domain of any gene identified in the second intracellular domain of any candidate polypeptide in Tables 12 to 17. In a sub-embodiment of this embodiment, the first intracellular domain and the second intracellular domain combination comprise the first intracellular domain and the second intracellular domain of the selected polypeptide.

In one embodiment of this aspect, the chimeric polypeptide comprises a transmembrane domain, a first intracellular domain, and a second intracellular domain of a selected polypeptide. In one embodiment of this aspect, the chimeric polypeptide comprises an extracellular and transmembrane domain, a first intracellular domain, and a second intracellular domain of a selected polypeptide, wherein the extracellular domain optionally comprises a recognition domain or an elimination domain.

In a related sub-embodiment, the chimeric polypeptide comprises the extracellular and transmembrane domains of the selected polypeptide, the first intracellular domain and the second intracellular domain, except that the extracellular domain and the transmembrane domain optionally comprise a recognition domain or an elimination domain, or do not comprise a recognition domain or an elimination domain. In a related sub-embodiment, the chimeric polypeptide comprises the extracellular and transmembrane domains of the selected polypeptide, the first intracellular domain and the second intracellular domain, except that the extracellular domain and the transmembrane domain optionally comprise a recognition domain or an elimination domain of the selected polypeptide, or another recognition domain or an elimination domain.

In another embodiment of this aspect, the polynucleotide further comprises a second nucleic acid sequence encoding a chimeric antigen receptor (CAR), the chimeric antigen receptor comprising an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain. In a sub-embodiment of this aspect, the first intracellular domain and the second intracellular domain are combined as the first intracellular domain and the second intracellular domain of the selected polypeptide, and the selected polypeptide is identified in Tables 12 to 17. In another embodiment of this embodiment, the chimeric polypeptide comprises an extracellular domain and a transmembrane domain, a first intracellular domain, and a second intracellular domain of the selected polypeptide, and the selected polypeptide is identified in Tables 12 to 17. In a related sub-embodiment, the chimeric polypeptide comprises an extracellular domain and a transmembrane domain, a first intracellular domain, and a second intracellular domain of the selected polypeptide, except that the extracellular domain and the transmembrane domain optionally comprise a recognition domain or an elimination domain, and wherein the recognition domain or the elimination domain is a recognition domain or an elimination domain of the extracellular domain of the selected polypeptide, or another recognition domain or an elimination domain, and wherein the selected polypeptide is identified in Tables 12 to 17.

In another aspect, provided herein are isolated polynucleotides comprising one or more nucleic acid sequences, wherein:

A first nucleic acid sequence of the one or more nucleic acid sequences encodes a chimeric polypeptide comprising, in an amino to carboxyl orientation,

a) a transmembrane domain from a type I transmembrane protein; and

b) a first intracellular domain selected from the group consisting of the intracellular domains of LEPR, MYD88, IFNAR2, IL17RD, IL17RE, IL1RAP, IL23R and MPL,

Wherein the chimeric polypeptide promotes cell proliferation of PBMCs, and in illustrative embodiments, B cells, T cells and/or NK cells.

In an embodiment of the aspect immediately preceding the paragraph and in any embodiment of this aspect in the following section, the first nucleic acid sequence further encodes an extracellular domain, and in illustrative embodiments, the extracellular domain comprises a dimerization motif. In illustrative embodiments of this aspect, the extracellular domain comprises a leucine zipper. In some embodiments, the leucine zipper is from a jun polypeptide, such as c-jun. In certain embodiments, the c-jun polypeptide is a c-jun polypeptide region of ECD-11.

In another embodiment of this aspect in which the first intracellular domain is selected from the intracellular domains of LEPR, MYD88, IFNAR2, IL17RD, IL17RE, IL1RAP, IL23R and MPL, the first nucleic acid sequence further encodes a second intracellular domain, wherein the second intracellular domain may be upstream (i.e., 5') of the first intracellular domain, or in illustrative embodiments, the second intracellular domain is downstream of the first intracellular domain. In a sub-embodiment of this embodiment, the second intracellular domain is from a gene of a second intracellular domain in a selected polypeptide identified in any table of Example 12 as producing an enrichment of greater than 0, 1 or 2 on day 21 versus day 7, wherein the first intracellular domain is from a gene of a first intracellular domain of a selected polypeptide. In another sub-embodiment of this sub-embodiment or embodiment, the first intracellular domain and the second intracellular domain are combined as a first intracellular domain and a second intracellular domain from a selected polypeptide identified in any table of Example 12 as producing an enrichment of greater than 0, 1 or 2 on day 21 versus day 7. In another embodiment of the embodiments or sub-embodiments in this paragraph, the first intracellular domain is from a first gene of a selected first intracellular domain of a selected polypeptide in Tables 12 to 17 and the second intracellular domain is from a second gene of a selected second intracellular domain of a selected polypeptide. In another embodiment of the embodiments or sub-embodiments in this paragraph, the first intracellular domain is from a polypeptide selected in Tables 12 to 17 and the second intracellular domain is from a selected polypeptide. In another embodiment of this aspect or any embodiment herein, the polynucleotide further comprises a second nucleic acid sequence encoding a chimeric antigen receptor (CAR) comprising an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain.

In certain embodiments of any of the methods or isolated polynucleotide embodiments herein comprising a chimeric polypeptide, wherein the chimeric polypeptide promotes cell proliferation of PBMCs, and in illustrative embodiments, B cells, T cells and/or NK cells, the first and/or second intracellular domains (in illustrative embodiments, the first intracellular domain) are intracellular domains from LEPR, MYD88, IFNAR2, MPL, IL18R1, IL13RA2, IL10RB, IL23R or CSF2RA. In some embodiments, the first intracellular domain is from MPL. In exemplary subembodiments, the chimeric polypeptide comprises an extracellular domain comprising a dimerization motif provided herein.

In certain embodiments of any of the methods or isolated polynucleotide embodiments of the present invention, the transmembrane domain is from CD40, ICOS, FCGR2C, PRLR, IL3RA or IL6ST, wherein the chimeric polypeptide promotes cell proliferation of PBMC, and in illustrative embodiments, promotes cell proliferation of B cells, T cells and/or NK cells. In some illustrative embodiments, the transmembrane domain is from CD40 or ICOS. In exemplary sub-embodiments, the chimeric polypeptide comprises an extracellular domain comprising a dimerization motif provided herein.

In certain embodiments of any of the methods or isolated polynucleotide embodiments of the present invention, the first and/or second intracellular domains (in illustrative embodiments, the second intracellular domains) are intracellular domains from CD40, CD79B, TNFRSF4, TNFRSF9, TNFRSF14, FCGRA2, CD3G or CD27, wherein the chimeric polypeptide promotes cell proliferation of PBMC, and in illustrative embodiments, promotes cell proliferation of B cells, T cells and/or NK cells. In some illustrative embodiments, the second intracellular domain is from CD40, CD79B or CD27. In exemplary sub-embodiments, the chimeric polypeptide comprises an extracellular domain comprising a dimerization motif provided herein.

In certain embodiments of any of the methods or isolated polynucleotide embodiments of the present invention comprising a chimeric polypeptide, the first and/or second intracellular domain is an intracellular domain (P3 or P4 component) of any of the chimeric polypeptides in Tables 12 to 17, wherein the chimeric polypeptide promotes cell proliferation of PBMCs, and in illustrative embodiments, promotes cell proliferation of B cells, T cells and/or NK cells. In some embodiments, the chimeric polypeptide comprises any P2, P3 and P4 combination of any of the chimeric polypeptides in Tables 12 to 17.

In embodiments where the transmembrane domain is a type I transmembrane protein, the transmembrane domain may be a type I cytokine receptor, a hormone receptor, a T cell receptor, or a TNF family receptor. In embodiments where the chimeric polypeptide comprises a transmembrane domain and where the transmembrane domain comprises a dimerization motif, the transmembrane domain may be a type I cytokine receptor, a hormone receptor, a T cell receptor, or a TNF family receptor. In illustrative embodiments of any of these embodiments in this paragraph, the first intracellular domain is selected from the intracellular domains of LEPR, MYD88, IFNAR2, IL17RD, IL17RE, IL1RAP, IL23R, and MPL.

In embodiments of any of the aspects and embodiments of a polynucleotide comprising a nucleic acid sequence encoding a chimeric polypeptide, the chimeric polypeptide comprises an extracellular domain, which may comprise 1, 2, 3 or 4 amino acid spacers within 20 amino acids of the carboxyl terminus of the extracellular domain (and in illustrative embodiments, at the carboxyl terminus of the extracellular domain) such that the spacer separates the extracellular domain from the transmembrane domain. In some embodiments, 1, 2, 3 or 4 amino acid spacers comprise alanine residues, and in some cases, only alanine residues. In illustrative sub-embodiments of embodiments comprising such spacers, the extracellular domain comprises a dimerization motif, such as a leucine zipper motif.

It should be understood that the first intracellular domain and the second intracellular domain of the chimeric polypeptides provided herein comprising the aspects and embodiments of the cell proliferation of PBMC, B cell, T cell and/or NK cell can include mutants and/or fragments of the gene, and the limiting condition is that the chimeric polypeptide retains its ability to promote the cell proliferation of PBMC, B cell, T cell and/or NK cell. These mutants and/or fragments usually retain signal transduction activity so that the chimeric polypeptide retains its promotion of survival and proliferation activity. It should be understood that the first intracellular domain and the second intracellular domain can be opposite so as to be used in any aspect and embodiment of the chimeric polypeptides comprising the cell proliferation of PBMC, B cell, T cell and/or NK cell provided herein. Therefore, it should be understood in these embodiments that the first intracellular domain is the second intracellular domain and vice versa.

In any of the aspects and embodiments comprising polynucleotides provided herein, the polynucleotides include a nucleic acid sequence encoding a chimeric polypeptide that promotes cell proliferation of PBMCs, B cells, T cells and/or NK cells, which may further encode a recognition domain or an elimination domain. In some embodiments, the recognition domain or the elimination domain is a recognition domain of a biologically approved monoclonal antibody. In some embodiments, the recognition domain or the elimination domain comprises a polypeptide recognized by an antibody that recognizes EGFR, or an antigenic determinant thereof. In some embodiments, the recognition domain or the elimination domain comprises a polypeptide recognized by an antibody that recognizes MYC, or an antigenic determinant thereof.

In any of the aspects and embodiments comprising polynucleotides provided herein, the polynucleotides include a nucleic acid sequence encoding a chimeric polypeptide that promotes cell proliferation of PBMCs, B cells, T cells, and/or NK cells, the nucleic acid sequence and/or the second nucleic acid sequence are operably linked to a first promoter active in B cells, T cells, and/or NK cells. In some illustrative embodiments, the first promoter is active in T cells and/or NK cells.

In any of the isolated polynucleotide aspects and embodiments provided herein having a nucleic acid sequence encoding a chimeric polypeptide that promotes cell proliferation of PBMCs, B cells, T cells and/or NK cells, for polynucleotide embodiments comprising a second intracellular domain, a connector may be present between the transmembrane domain and the first intracellular domain and/or between the first intracellular domain and the second intracellular domain. In some embodiments, for embodiments comprising an extracellular domain, a connector between 1 and 4 alanines is present between the extracellular domain and the transmembrane domain.

In the embodiments provided herein comprising a first nucleic acid sequence encoding a chimeric polypeptide that promotes cell proliferation of PBMC, B cells, T cells and/or NK cells and a second nucleic acid sequence encoding a chimeric antigen receptor (CAR) through separation of polynucleotides or vectors and any one of the embodiments, the chimeric antigen receptor comprises an antigen-specific targeting region (ASTR), a transmembrane domain and an intracellular activation domain, and the first nucleic acid sequence and the second nucleic acid sequence can be separated by a ribosomal skipping sequence. In some embodiments, the ribosomal skipping sequence is F2A, E2A, P2A or T2A.

In embodiments of any of the isolated polynucleotide aspects and embodiments of chimeric polypeptides that promote cell proliferation of PBMCs, B cells, T cells, and/or NK cells provided herein, the polynucleotide further comprises a Kozak-type sequence, a WPRE component, and one or more of a double stop codon or a triple stop codon, wherein one or more of the double stop codon or the triple stop codon defines the termination of reading from at least one of the one or more transcription units. In certain embodiments, the polynucleotide further comprises a Kozak-type sequence selected from the following: CCACCAT/UG(G) (SEQ ID NO: 515), CCGCCAT/UG(G) (SEQ ID NO: 516), GCCGCCGCCAT/UG(G) (SEQ ID NO: 517), or GCCGCCGCCAT/UG. In certain embodiments, the -3 and +4 nucleotides relative to the start codon of the first nucleic acid sequence both comprise G. In another embodiment that can be combined with the foregoing embodiment comprising a Kozak-type sequence and/or the following embodiment comprising a triple stop codon, the polynucleotide comprises a WPRE component. In some embodiments, the WPRE element is located 3' to the stop codon of one or more transcription units and 5' to the 3' LTR of the polynucleotide. In another embodiment that may be combined with any or both of the preceding embodiments (i.e., an embodiment in which the polynucleotide comprises a Kozak-type sequence and/or an embodiment in which the polynucleotide comprises a WPRE), one or more transcription units terminate with one or more stop codons in a double stop codon or a triple stop codon.

In one aspect, provided herein is a nucleic acid vector comprising

Embodiments of any of the isolated polynucleotide aspects and embodiments provided herein encoding a chimeric polypeptide that promotes cell proliferation of PBMCs, B cells, T cells and/or NK cells,

a. Copy source;

b. antibiotic resistance genes; and/or

c. One or more viral and/or retroviral sequences selected from retroviral long terminal repeats (LTRs) or active fragments thereof, retroviral cis-acting RNA packaging components, and/or retroviral central polypurine tract/central termination sequence (CTS) components.

In certain embodiments of this aspect, the vector is a plasmid. In other embodiments, the vector is a replication-defective viral vector, such as a replication-defective retroviral vector or particle. In these embodiments, the replication-defective reverse transcription vector or particle comprises a 5' retroviral LTR or an active fragment thereof, a retroviral cis-acting RNA packaging component, and a 3' retroviral LTR or an active fragment thereof, wherein the 5' retroviral LTR and the 3' retroviral LTR are flanked by isolated polynucleotides. In some embodiments, the isolated polynucleotide is in the opposite orientation to the nucleic acid sequence encoding the retroviral cis-acting RNA packaging component, the 5' long terminal repeat sequence and/or the 3' long terminal repeat sequence.

In some aspects, provided herein is a replication-defective recombinant retroviral particle comprising a retroviral genome and further comprising an envelope and a capsid, the retroviral genome comprising any of the aspects and embodiments comprising isolated polynucleotides herein, the isolated polynucleotides comprising a first nucleic acid sequence encoding a chimeric polypeptide that promotes cell proliferation of PBMC, B cells, T cells and/or NK cells. The retroviral particle may additionally include any of the retroviral components described in the present disclosure. In one embodiment, the retroviral genome further comprises a second nucleic acid sequence encoding a chimeric antigen receptor (CAR), the chimeric antigen receptor comprising an antigen-specific targeting region (ASTR), a transmembrane domain and an intracellular activation domain. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence may be spaced apart by a ribosomal skipping sequence. In some embodiments, the ribosomal skipping sequence is F2A, E2A, P2A or T2A. In these embodiments, the first nucleic acid sequence and the second nucleic acid sequence are generally on the same transcription unit.

In another embodiment, a mammalian cell is provided herein, wherein the mammalian cell (eg, human cell) is a B cell, T cell or NK cell comprising an isolated polynucleotide, according to any one of the aspects and embodiments comprising an isolated polynucleotide herein, the isolated polynucleotide may be integrated into the genome of the cell as appropriate, and the isolated polynucleotide comprises a first nucleic acid sequence encoding a chimeric polypeptide that promotes cell proliferation of PBMC, B cells, T cells and/or NK cells. In one embodiment, the isolated polynucleotide in the mammalian cell further comprises a second nucleic acid sequence encoding a chimeric antigen receptor (CAR), the chimeric antigen receptor comprising an antigen-specific targeting region (ASTR), a transmembrane domain and an intracellular activation domain. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence may be spaced apart by a ribosomal skipping sequence. In some embodiments, the ribosomal skipping sequence is F2A, E2A, P2A or T2A. In these embodiments, the first nucleic acid sequence and the second nucleic acid sequence are generally on the same transcription unit. In some embodiments, the cell is a T cell or a NK cell, and in certain illustrative embodiments, the cell is a human T cell.

In another aspect, there is provided herein a packaging component comprising an isolated polynucleotide according to any one of the aspects and embodiments comprising isolated polynucleotides herein, the isolated polynucleotides can be integrated into the genome of the cell, comprising a first nucleic acid sequence encoding a chimeric polypeptide that promotes cell proliferation of PBMC, B cells, T cells and/or NK cells. In some embodiments, the isolated polynucleotide in the cell further comprises a second nucleic acid sequence encoding a chimeric antigen receptor (CAR), the chimeric antigen receptor comprising an antigen-specific targeting region (ASTR), a transmembrane domain and an intracellular activation domain. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence can be spaced apart by a ribosome skipping sequence. In some embodiments, the ribosome skipping sequence is F2A, E2A, P2A or T2A. In these embodiments, the first nucleic acid sequence and the second nucleic acid sequence are generally on the same transcription unit. In some embodiments, the cell is provided herein for producing a retroviral particle.

In one aspect, a method for promoting cell proliferation of PBMC (e.g., B cells, T cells and/or NK cells) is provided herein, the method comprising transducing and/or genetically modifying cells with a vector encoding a lymphoproliferative component (and in illustrative embodiments, a chimeric polypeptide according to any one of the embodiments provided herein), wherein the chimeric polypeptide promotes PBMC proliferation. The method may include culturing cells for at least 7 days, 14 days, 21 days, 28 days, 35 days, 42 days or 60 days. Typically, this culture is performed in the presence of IL-2, for example, after transduction with a medium that can induce IL-2 in some embodiments.

In one aspect, a method for transfecting, transducing and/or genetically modifying B cells, T cells and/or NK cells (in some non-limiting embodiments, allogeneic cells, autologous cells or non-allogeneic cells) is provided herein, the method comprising contacting the cell with an isolated polynucleotide according to any one of the aspects and embodiments comprising isolated polynucleotides herein, the isolated polynucleotides comprising a first nucleic acid sequence encoding a chimeric polypeptide that promotes cell proliferation of PBMC, B cells, T cells and/or NK cells (CLE aspects and embodiments). In some embodiments of this method aspect, the cell is a T cell and the isolated polynucleotide further comprises a second nucleic acid sequence encoding a chimeric antigen receptor (CAR), the chimeric antigen receptor comprising an antigen-specific targeting region (ASTR), a transmembrane domain and an intracellular activation domain. In other words, the method for transfecting, transducing and/or genetically modifying B cells, T cells and/or NK cells may include contacting the cell with an isolated polynucleotide according to any one of the CLE or CLE CAR embodiments herein. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence can be separated by a ribosomal skipping sequence. In some embodiments, the ribosomal skipping sequence is F2A, E2A, P2A or T2A. In these embodiments, the first nucleic acid sequence and the second nucleic acid sequence are generally on the same transcription unit. In some embodiments, the cell is a T cell or a NK cell, and in some illustrative embodiments, the cell is a human T cell. The contact is carried out under effective conditions for transfection, transduction and/or genetically modifying cells. In some embodiments, the isolated polynucleotides at the time of contact are in a carrier according to any one of the vector aspects and embodiments provided herein. In some embodiments, the isolated polynucleotides are in non-replicating retroviral particles at the time of contact.

In some embodiments of any of the aspects herein, the NK cells are NKT cells.

The following non-limiting examples are provided only by way of illustrating illustrative embodiments, and in no way limit the scope and spirit of the present invention. In addition, it should be understood that any invention disclosed or claimed herein encompasses all variations, combinations and arrangements of any one or more features described herein. Any one or more features may be explicitly excluded from the claims, even if a specific exclusion is not explicitly set forth herein. It should also be understood that, unless generally skilled in the art will understand, otherwise, according to the particular method disclosed herein or other methods known in the art, the disclosure of the reagent used in the method is intended to be synonymous with (and provide support for) the method involving the use of the reagent. In addition, unless generally skilled in the art will understand, otherwise the specification and/or claims disclose a method, any one or more of the reagents disclosed herein can be used for the method.

Examples

Example 1. Engineering of a retroviral packaging and transduction system to target resting T cells for selection of T cell integration and expression by PBMCs.

Although it is possible to produce high-titer lentiviral vectors using transient transfection, this method carries the risk of producing replication-deficient recombinant retroviruses (RCRs) and is not amplifiable for clinical applications. Here, multiple constructs encoding inducible promoters and their regulators are synchronously introduced into HEK293 suspension adaptive cells (HEK293S) to stably produce viral components, CAR genes and their regulatory components to produce stable retroviral packaging cell lines. Two different inducible systems can be used to temporarily control gene expression. One system is based on the dimerization induced by rapamycin- or rapamycin analogs of two transcription factors. One transcription factor consists of three copies of the FKPB protein fused to the ZFHD1 DNA binding domain and the other transcription factor consists of the FRB protein fused to the p65 activation domain. Rapamycin or rapamycin analogs dimerize transcription factors to form ZFHD1/p65 AD and can activate gene transcription at the 12xZFHD1 binding site.

A series of vectors as shown in Figures 3A to 3E are generated by flanking transposon sequences for integration into the HEK293S genome. Once integrated into the genome of the cell, these sequences act as regulatory components and lox sites and/or FRT sites (referred to herein as landing sites) for subsequent integration using Cre and/or flp recombinases. The initial 5 constructs contain polynucleotide sequences encoding puromycin resistance, GFP, RFP, and targeting cell membranes via N-terminal PLss (bovine prolactin signal peptide) and anchored to the cell membrane via the C-terminal transmembrane anchoring domain of platelet-derived growth factor receptor (PDGFR) extracellular MYC markers. The initial 5 constructs may also include constitutive minimal CMV promoters and minimal IL-2 promoters, promoters based on ZFHD1 regulated by rapamycin, tetracycline responsive element (TRE) promoters, or bidirectional TRE (BiTRE) promoters. The construct in FIG. 3A contains polynucleotide sequences encoding the FRB domain fused to the NFκB p65 activation subdomain (p65 AD) and the ZFHD1 DNA binding domain fused to three constitutively expressed FKBP repeats. The construct in FIG. 3A also includes HIV1 REV and HSV VP65 domains SrcFlagVpx under the rapamycin-inducible ZFHD1/p65 AD promoter. The construct in FIG. 3B includes polynucleotides encoding the rtTA sequence under the control of the ZFHD1/p65 AD promoter. The construct in FIG. 3C includes polynucleotides encoding a puromycin resistance gene flanked by loxP sites and an extracellular MYC marker flanked by lox2272 sites. Both of these selectable markers are under the control of the BiTRE promoter, which is flanked by FRT sites. The construct in FIG. 3D includes polynucleotides encoding GFP flanked by loxP sites under the control of the TRE promoter. The construct in FIG. 3D also includes a single FRT site between the TRE promoter and the 5'loxP site of GFP. The construct in FIG. 3E includes a polynucleotide encoding RFP flanked by loxP sites under the control of the ZFHD1/p65AD promoter. The construct in FIG. 3E also includes a single FRT site between the ZFHD1/p65AD promoter and the 5'loxP site of RFP. The constructs in FIG. 3C to FIG. 3E serve as landing points for other polynucleotide sequences to be inserted into the genome of the packaging cell line. The polynucleotide sequence to be inserted can be flanked by lox sites and inserted into the genome using Cre recombinase and loxP sites. This results in the insertion and simultaneous removal of genomic regions encoding puromycin resistance, extracellular MYC markers, GFP, and RFP. Alternatively, the polynucleotide sequence can be flanked by FRT sites and inserted into the genome using flp recombinase and FRT sites, followed by the removal of the polynucleotide sequences encoding puromycin resistance, extracellular MYC markers, GFP, and RFP using Cre recombinase.

To generate packaging cell lines with landing sites integrated into the genome, HEK293S cells were co-transfected with equimolar concentrations of the five plasmids (FIG. 3A-3E) plus 5 μg of in vitro transcribed piggybac transposase mRNA or 5 μg of a plasmid with a promoter for expressing piggybac transposase in the presence of PEI at a ratio of 2:1 or 3:1 PEI to DNA (w/w) or 2 μg to 5 μg piggybac transposase protein using a cationic peptide mixture. Transfected cells were selected with puromycin in the presence of 100 nm rapamycin and 1 μg/mL doxycycline for 2 to 5 days, followed by fluorescence activated cell sorting to collect cells expressing GFP and RFP. Sorted cells were grown in the absence of puromycin, rapamycin and doxycycline for 5 days, and cells expressing GFP and RFP were removed, and myc-positive cells were also removed with myc beads. Individual clones from negatively sorted cells were then screened for induction of GFP and RFP using rapamycin and doxycycline as well as cloned single cells. DNA from the clones was harvested and sequenced for integration analysis. Clones that were positive for robust induced expression of GFP and RFP in the presence of rapamycin and doxycycline, and limited background expression in the absence of rapamycin and doxycycline were amplified and stacked.

HEK293S cells with constructs from Figures 3A to 3E integrated into the genome were then transfected with constructs containing tricistronic polynucleotides encoding DAF signal sequence/anti-CD3 scFvFc (UCHT1)/CD14 GPI anchoring site (SEQ ID NO: 287), DAF signal sequence/CD80 extracellular domain capable of binding CD28/CD16B GPI anchoring site (SEQ ID NO: 286) and DAF signal sequence/IL-7/DAF (SEQ ID NO: 286) and transposon sequences flanking the polynucleotide region for integration into the HEK293S genome (Figure 4). After transfection, cells were expanded for 2 days in the absence of rapamycin and doxycycline and clones that were constitutively red were selected. Positive clones were then transiently transfected with constructs for expressing Cre recombinase to remove remaining genomic DNA and the RFP coding region. Simultaneously transfected another construct containing a polynucleotide with a BiTRE promoter, a polynucleotide region encoding a gag polypeptide and a pol polypeptide in one direction, and a polynucleotide region encoding a measles virus F protein and a H protein in the other direction (FIG. 4B). The Cre recombinase integrates the construct into the genome to produce the integrated sequence shown in FIG. 4B. The resulting pure lines are evaluated for protein expression in the presence of doxycycline and rapamycin and analyzed by deep sequencing for genomic integration. The remaining TRE-responsive GFP site is retained for insertion into the lentiviral genome.

Example 2. Generation of lentiviral vectors and retroviral packaging.

The retroviral packaging stable cell line produced in Example 1 is transfected with a construct (Fig. 4C) for expressing Flp recombinase and a construct containing a polynucleotide sequence, which encodes CAR and the lymphoproliferative component IL7Rα-insPPCL under the control of CD3Z promoter (which is inactive in HEK293S cells), wherein CAR and IL7Rα-insPPCL are separated by a polynucleotide sequence encoding T2A ribosomal skipping sequence, and IL7Rα-insPPCL has a ribonuclease controlled by acyclovir riboswitch. The construct containing CAR further includes cPPT/CTS and RRE sequences and a polynucleotide sequence encoding HIV-1Psi. The entire polynucleotide sequence on the construct containing CAR to be integrated into the genome is flanked by FRT sites. The successful integration of the construct containing CAR results in the constitutive expression of GFP removed by transient transfection with a construct for expressing Cre recombinase. The CAR-containing construct in the retroviral genome can be in the reverse orientation relative to the 5' to 3' direction established by the 5' long terminal repeat and the 3' long terminal repeat. The HEK293S strain is grown in serum-free medium. After growing to peak cell density in a stirred tank reactor, the cells were diluted to 70% peak cell density and treated with 100nM rapamycin for 2 days to induce the expression of early genes REV, Vpx and aCD3 scFv CD16B GPI, aCD28 scFvCD16B GPI and IL-7SD GPI DAF, followed by the addition of 1μg/mL doxycycline to the medium to induce the expression of structural components of the lentiviral genome such as GagPol, MV (Ed) -FΔ30, MV (Ed) -HΔ18 and therapeutic targets. The level of viral yield is checked by qPCR and p24 ELISA of the packaging sequence. Viruses are collected by deep filtration of cells and concentration/diafiltration using TFF boxes, followed by rapid freezing for vials.

Example 3. Efficiency of transduction of freshly isolated and unstimulated human T cells by retroviral particles pseudotyped with VSV-G and expressing anti-CD3 scFvFc on their surface.

Recombinant lentiviral particles were produced by transiently transfecting 293T cells (Lenti-X ^™ 293T, Clontech) with separate lentiviral packaging plasmids encoding gag/pol and rev and a pseudotyped plasmid encoding VSV-G. A third generation lentiviral expression vector encoding GFP, anti-CD19 chimeric antigen receptor, and eTAG referred to herein as F1-0-03 (FIG. 5) (containing a deletion in the 3'LTR resulting in self-inactivation) was co-transfected with the packaging plasmids. Cells were adapted to suspension culture by continuous growth in Freestyle ^™ 293 expression medium (ThermoFisher Scientific). Cells in suspension were inoculated at 1×10 ⁶ cells/mL (30 mL) in a 125 mL Erlenmeyer flask and immediately transfected using polyethyleneimine (PEI) (Polysciences) dissolved in weak acid.

Plasmid DNA was diluted in 1.5 ml Gibco ^™ Opti-MEM ^™ medium for 30 mL of cells. To obtain lentiviral particles pseudotyped with VSV-G, the total DNA used (1 μg/mL of culture volume) was a mixture of 4 plasmids with the following molar ratios: 2× genomic plasmid (F1-0-03), 1× Rev-containing plasmid, 1× VSV-G-containing plasmid, and 1× gag/pol-containing plasmid. To obtain lentiviral particles pseudotyped with VSV-G and expressing anti-CD3-scFvFc-GPI on their surface, the total DNA used (1 μg/mL of culture volume) was a mixture of 5 plasmids with the following molar ratios: 2× genomic plasmid (F1-0-03), 1× Rev-containing plasmid, 1× VSV-G-containing plasmid, 1× anti-CD3-scFvFc-GPI-containing plasmid, and 1× gag/pol-containing plasmid. To obtain lentiviral particles pseudotyped with VSV-G and expressing anti-CD3-scFvFc-GPI and CD80-GPI on their surface, the total DNA used (1 μg/mL culture volume) was a mixture of 6 plasmids with the following molar ratios: 2× genomic plasmid (F1-0-03), 1× Rev-containing plasmid, 1× VSV-G-containing plasmid, 1× anti-CD3-scFvFc-containing plasmid, 1× CD80-containing plasmid, and 1× gag/pol-containing plasmid. Separately, PEI was diluted to 2 μg/mL (culture volume, 2:1 ratio to DNA) in 1.5 ml Gibco ^™ Opti-MEM ^™ . After 5 minutes of room temperature incubation, the two solutions were thoroughly mixed together and incubated at room temperature for 20 minutes. The final volume (3 ml) was added to the cells. The cells were then incubated at 37°C for 72 hours with rotation at 125 rpm and 8% _CO2 . Plasmids containing anti-CD3-scFvFc-GPI include scFv derived from OKT3 or UCHT1 and a GPI anchor linker sequence. The UCHT1 scFvFc-GPI vector encodes a peptide (SEQ ID NO: 278) including a human Igκ signal peptide (NCBI GI No. CAA45494.1 amino acids 1 to 22) fused to a UCHT1 scFv (NCBI GI No. CAH69219.1 amino acids 21 to 264) fused to a human IgG1 Fc having an A at position 115 substituted with T (NCBI GI No. AEV43323.1 amino acids 1 to 231) fused to a human DAF GPI anchor linker sequence (NCBI GI No. NP_000565 amino acids 345 to 381). The OKT3scFvFc-GPI vector encodes a peptide (SEQ ID NO: 279) comprising a human Igκ signal peptide (amino acid 1 to amino acid 22 of NCBI GI No. CAA45494.1) fused to an OKT3 scFv (SEQ ID NO: 285) fused to a human IgG1 Fc (amino acid 1 to amino acid 231 of NCBI GI No. AEV43323.1) fused to a human DAF GPI anchor linker sequence (amino acid 345 to amino acid 381 of NCBI GI No. NP_000565). The CD80-GPI vector encodes a peptide (SEQ ID NO: 280) comprising the human CD80 signal peptide and the extracellular domain (amino acid 1 to amino acid 242 of NCBI GI No. NP_005182) fused to the human CD16b GPI anchor linker sequence (amino acid 196 to amino acid 233 of NCBI GI No. NP_000561).

After 72 hours, the supernatant was collected and clarified by centrifugation at 1,200 g for 10 minutes. The clarified supernatant was poured into a new tube. The lentiviral particles were precipitated by centrifugation at 3,300 g overnight at 4°C. The supernatant was discarded and the lentiviral particle pellet was resuspended in 1:100 of the original volume of X-Vivo ^™ 15 medium (Lonza). The lentiviral particles were titrated using serial dilutions and flow cytometric analysis of GFP expression in 293T and Jurkat cells 72 hours after transduction.

Peripheral blood mononuclear cells (PBMCs) were first isolated from fresh blood in ACD (acid citrate) tubes from donors 12F and 12M or from buffy coat from donor 13F, collected and distributed by San Diego Blood Bank, CA. Ficoll-Paque 2000 based on SepMate ^™ 50 (Stemcell ^™ ) was performed according to the manufacturer's instructions. Gradient density separation of PBMC on (GE Healthcare Life Sciences). 30mL of blood or blood clot yellow layer diluted in PBS-2% HIFCS (heat-inactivated fetal bovine serum) was layered according to each SepMate ^TM test tube. After centrifugation at 1,200g for 20 minutes at room temperature, the PBMC layer was collected, pooled and washed three times with 45mL of PBS-2% HIFCS, and centrifuged at 400g for 10 minutes at room temperature. Then the pellet was cultivated in 10mL of RBC lysis buffer (Alfa Aesar) at room temperature for 10 minutes, and washed an additional two times with 45mL of PBS-2% HIFCS, and centrifuged at 400g for 10 minutes at room temperature. The final wash was performed in the following transduction and culture media: X-Vivo ^TM 15 of donor 13F or RPMI-1640+10% HIFCS of donor 12F and donor 12M. No additional steps were taken to remove monocytes. After isolation, fresh and unstimulated PBMCs were resuspended in the respective media to a final concentration of 1×10 ⁶ /mL and transduced with the previously disclosed lentiviral particles (in duplicate or triplicate). Transduction was performed for 14 hours at 37°C, 5% CO ₂ in X-Vivo ^™ 15 medium for donor 13F or in RPMI-1640 + 10% HIFCS for donor 12F and donor 12M. Transduction was typically performed at MOI 1 in a 12-well plate format at 1 ml/well. For kinetic experiments, 0.5×10 ⁶ PBMCs/mL were transduced in 7 mL of final solution at MOI 1, with rotation at 125 rpm and 8% CO ₂ , incubated in a 125 mL shake flask at 37°C for 2 to 20 hours. After incubation with lentivirus for the selected time, wash three times with X-Vivo ^™ 15 medium for donor 13F or PBS + 2% HIFCS for donor 12F and donor 12M, and finally incubate at a cell density of 1×10 ₆ /mL in X-Vivo ^™ 15 medium for donor 13F or RPMI-1640 + 10% HIFCS for donor 12F and donor ^12M at 37°C, 5% CO 2. Starting on day 3 after transduction, and every 2-3 days thereafter, the medium volume was doubled and supplemented with IL-2 to a final concentration of 100U/mL. Samples were collected on different days after transduction (day 3 to day 17) to evaluate the transduction efficiency of each type of lentivirus produced using GFP expression levels.

On different days after transduction, 100 μL of cells were collected and analyzed for the expression of GFP in the CD3+ cell population by flow cytometry for lentiviral particles pseudotyped with VSV-G, lentiviral particles pseudotyped with VSV-G and displaying anti-CD3 scFvFc-GPI on their surface, or lentiviral particles pseudotyped with VSV-G and displaying anti-CD3 scFvFc-GPI and CD80-GPI on their surface with or without OKT3 antibody (Biolegend) at 1 μg/ml.

Figures 6A and 6B show histograms of the percentage (%) of CD3+GFP+ cells in the total CD3+ population and the absolute number of cells per well of the CD3+GFP+ population, respectively, 14 hours after transduction of freshly isolated and unstimulated PBMCs from donor 12M with the indicated lentiviral particles on days 3, 6, 9, 13, and 17. Each bar represents the mean +/- SD of replicates. Figures 6A and 6B show that freshly isolated and unstimulated PBMCs were effectively transduced with VSV-G pseudotyped lentiviral particles and anti-CD3-scFvFc displayed on the surface of the lentiviral particles. Anti-CD3 scFv's derived from OKT3 or UCHT1 were effective when in the form of scFvFc-GPI.

Figures 7A and 7B show histograms of the percentage (%) of CD3+GFP+ cells in the total CD3+ population and the absolute number of cells per well of the CD3+GFP+ population, respectively, 14 hours after transduction of freshly isolated and unstimulated PBMCs from donor 13F with the indicated lentiviral particles on days 3 and 6. Please note that "A" is the result using lentiviral particles pseudotyped with VSV-G (three replicates); "B" is the result using lentiviral particles pseudotyped with VSV-G in the presence of OKT3 Ab (1 μg/mL) added to the transduction medium (two replicates); "C" is the result using lentiviral particles pseudotyped with VSV-G (three replicates) displaying GPI-anchored UCHT1scFvFc on its surface; and "D" is the result using lentiviral particles pseudotyped with VSV-G (two replicates) displaying GPI-anchored UCHT1scFvFc and GPI-anchored CD80 on its surface. Each bar represents the mean +/- SD of two or three replicates, as indicated in Figure 7A. Figures 7A and 7B show that freshly isolated and unstimulated PBMs were also efficiently transduced with lentivirus pseudotyped with VSV-G and displaying anti-CD3-scFvFc-GPI and CD80-GPI on their surface when transduction was performed for 14 hours.

FIG8A and FIG8B show a histogram of the percentage (%) of CD3+GFP+ cells in the total CD3+ population and a histogram of the absolute number of cells per well of the CD3+GFP+ population on days 3, 6, and 9 after the indicated exposure (2 to 20 hours) of freshly isolated and unstimulated PBMCs from donor 12M transduced with the indicated lentiviral particles, respectively. Transduction was performed in a dish or shaker flask as indicated. Each bar represents the mean +/-SD of two replicates of lentiviral particles pseudotyped with VSV-G ("[VSV-G]"); other experiments do not have replicates. FIG8A and FIG8B show that freshly isolated and unstimulated PBMCs can be effectively transduced in as little as 2 hours with lentiviral particles pseudotyped with VSV-G and displaying anti-CD3 scFvFc and CD80 on their surface.

Of note, subsequent experiments were performed using lentiviral particles generated by transducing freshly isolated resting PBMCs for 2.5 hours in the presence of soluble anti-CD3 antibody (100 ng/ml) using only Rev, VSV-G, gag/pol, and F1-0-03 (but not vectors encoding anti-CD3 or CD80). Transduction was achieved using 3 different soluble anti-CD3 antibody clones. The transduction efficiencies measured by CD3+GFP+ cells on day 6 were 4.31%, 3.27%, and 0.52% for the CD3 antibody clones HIT3A, OKT3, and UCHT1, respectively. The background transduction efficiency in the absence of soluble antibody was 0.06%.

Example 4. Transduction efficiency of freshly isolated and unstimulated human T cells by retroviral particles containing a VSV-G pseudotyping component and a membrane-bound activation component and optionally the Vpu protein.

Recombinant lentiviral particles were produced by transiently transfecting 293T cells (Lenti-X ^™ 293T, Clontech) with separate lentiviral packaging plasmids encoding gag/pol and rev and a pseudotyped plasmid encoding VSV-G. A third generation lentiviral expression vector encoding GFP, anti-CD19 chimeric antigen receptor, and eTAG (FIG. 5), referred to herein as F1-0-03, was co-transfected with the packaging plasmid. Cells were adapted to suspension culture by continuous growth in Freestyle ^™ 293 expression medium (ThermoFisher Scientific). Cells in suspension were inoculated at 1×10 ⁶ cells/ml (30 mL) in a 125 mL Erlenmeyer bottle and immediately transfected with polyethyleneimine (PEI) (Polysciences) dissolved in a weak acid.

Plasmid DNA was diluted in 1.5 ml Gibco ^™ Opti-MEM ^™ medium for 30 mL of cells. To obtain lentiviral particles pseudotyped with VSV-G [VSV-G], the total DNA used (1 μg/mL of culture volume) was a mixture of 4 plasmids with the following molar ratios: 2× genomic plasmid (F1-0-03), 1× Rev-containing plasmid, 1× VSV-G-containing plasmid, and 1× gag/pol-containing plasmid. To obtain lentiviral particles pseudotyped with VSV-G and displaying anti-CD3-scFvFc-GPI on their surface [VSV-G+UCHT1], the total DNA used (1 μg/mL of culture volume) was a mixture of 5 plasmids with the following molar ratios: 2× genomic plasmid (F1-0-03), 1× Rev-containing plasmid, 1× VSV-G-containing plasmid, 1× anti-CD3-scFvFc-GPI-containing plasmid and 1× gag/pol-containing plasmid. To obtain lentiviral particles pseudotyped with VSV-G, displaying anti-CD3-scFvFc-GPI and containing the auxiliary protein Vpu [VSV-G+UCHT1+VpuCH], the total DNA used (1 μg/mL of culture volume) was a mixture of 6 plasmids with the following molar ratios: 2× genomic plasmid (F1-0-03), 1× Rev-containing plasmid, 1× VSV-G-containing plasmid, 1× anti-CD3-scFvFc-GPI-containing plasmid, 1× Vpu-CH-containing plasmid and 1× gag/pol-containing plasmid. Separately, PEI was diluted to 2 μg/mL (culture volume, 2:1 ratio to DNA) in 1.5 ml Gibco ^™ Opti-MEM ^™ . After 5 minutes of incubation at room temperature, the two solutions were thoroughly mixed together and incubated at room temperature for 20 minutes. The final volume (3 ml) was added to the cells. The cells were then incubated at 37°C with rotation at 125 rpm and 8% _CO2 for 72 hours. The anti-CD3-scFvFc-GPI containing plasmid includes scFv derived from UCHT1 and a GPI anchor linker sequence derived from DAF (CD55). The UCHT1 scFvFc-GPI vector encodes a peptide (SEQ ID NO: 278) including a human Igκ signal peptide (amino acids 1 to 22 of NCBI GI No. CAA45494.1) fused to UCHT1 scFv (amino acids 21 to 264 of NCBI GI No. CAH69219.1), to human IgG1 Fc (amino acids 1 to 231 of NCBI GI No. AEV43323.1), to a human DAF GPI anchor linker sequence (amino acids 345 to 381 of NCBI GI No. NP_000565). The plasmid containing Vpu-CH encodes a peptide (MFDFIARVDYRVGVVALVVALIIAIIVWSIVYIEYRKLLKQRKIDWLIKRIRERAEDSGNESDGEIEELSTMVDMEHLRLLDDL*) comprising the Vpu sequence from HIV-1M CH167-CC isolate (amino acids 1-84, GeneBank: AGF30946.1).

After 72 hours, the supernatant was collected and clarified by centrifugation at 1,200 g for 10 minutes. The clarified supernatant was poured into a new tube. The lentiviral particles were precipitated by overnight centrifugation at 3,300 g at 4°C. The supernatant was discarded and the lentiviral particle pellet was resuspended in 1:100 of the original volume of X-Vivo ^™ 15 medium (Lonza). The lentiviral particles were titrated by serial dilution and analysis of GFP expression by flow cytometry in 293T and Jurkat cells 72 hours after transduction.

Peripheral blood mononuclear cells (PBMC) were separated from the buffy coat and collected and distributed by San Diego Blood Bank, CA. Ficoll-Paque 2000 based on SepMate ^™ 50 (Stemcell ^™ ) was performed according to the manufacturer's instructions. Gradient density separation of PBMCs on Sigma-Aldrich (GE Healthcare Life Sciences). 30 mL of the buffy coat diluted in X-Vivo ^™ 15 medium was layered according to each SepMate ^™ tube. After centrifugation at 1,200 g for 20 minutes at room temperature, the PBMC layers were collected, pooled and washed three times with 45 mL of X-Vivo ^™ 15 medium and centrifuged at 400 g for 10 min at room temperature. The pellets were then incubated in 10 mL of RBC lysis buffer (Alfa Aesar) for 10 minutes at room temperature and washed an additional two times with 45 mL of X-Vivo ^™ 15 medium and centrifuged at 400 g for 10 min at room temperature. No additional steps were taken to remove monocytes. After separation, fresh and unstimulated PBMCs were resuspended in X-Vivo ^™ 15 to a final concentration of 1×10 ⁶ /mL and transduced with the previously disclosed lentiviral particles (two replicates). Transduction was performed for 3 days at 37°C, 5% _CO2 in X-Vivo ^™ 15 medium. Transduction was typically performed in 1 ml/well in a 12-well plate format at an MOI of 10 for [VSV-g] and at an MOI of 1 for [VSV-G+UCHT1] and [VSV-G+UCHT1+Vpu-CH] (in duplicate). Three days after transduction, samples were collected and analyzed by FACS for absolute cell counts and GFP expression in the CD3+ population.

Figures 9A and 9B show a histogram of the percentage (%) of CD3+GFP+ cells in the total CD3+ population 3 days after transduction of freshly isolated and unstimulated PBMCs with the indicated lentiviral particles (Figure 9A) and a histogram of the absolute cell counts per well of the live CD3+ population (Figure 9B), respectively. Each bar represents the mean +/-SD of two replicates. Figure 9A shows that the addition of the anti-CD3-scFvFc-GPI portion (UCHT1) to the VSV-G pseudotyped lentiviral particles resulted in higher transduction efficiency for freshly isolated and unstimulated PBMCs compared to [VSV-G] alone. Figure 9A also shows that the addition of Vpu-CH can further improve the transduction efficiency of the double pseudotyped vector [VSV-G+UCHT1]. Figure 9B shows that the UCHT1 portion enhances cell stimulation in absolute cell counts per microliter compared to VSV-G alone. Addition of Vpu and UCHT1 similarly enhanced cell stimulation in absolute cell counts compared to VSV-G alone.

Example 5. Transduction of unstimulated human PBMCs by a 4-hour exposure to retroviral particles pseudotyped with VSV-G and expressing anti-CD3 scFvFc on their surface is inhibited by antiviral drugs.

In this example, unstimulated human PBMCs were efficiently transduced by a brief 4-hour exposure to retroviral particles pseudotyped with VSV-G and not displaying UCHT1 scFvFc-GPI on their surface, either alone or in combination with CD80-GPI. The presence of a combination of the antiviral drugs dapline (a reverse transcriptase inhibitor) and dolutegravir (an integrase inhibitor) was added to one sample to inhibit transduction.

Recombinant lentiviral particles were produced in 293T cells (Lenti-X ^™ 293T, Clontech) adapted to suspension culture in Freestyle ^™ 293 expression medium (Thermo Fisher Scientific). Cells were transiently transfected using PEI with a genomic plasmid and separate packaging plasmids encoding gag/pol, rev, and a pseudotyped plasmid encoding VSV-G as described in Example 3. For some samples, the transfection reaction mixture also included a plasmid encoding UCHT1 scFvFc-GPI alone or together with another plasmid encoding CD80-GPI as further described in Example 3. The genomic plasmid was a third generation lentiviral expression vector encoding GFP, anti-CD19 chimeric antigen receptor, and eTAG referred to herein as F1-0-03 ( FIG. 5 ). F1-0-03 did not include a lymphoproliferative component.

PBMCs were prepared from the buffy coat as described in Example 3 without any additional steps to remove monocytes. After separation, 1 ml of X-Vivo 15 containing 1×10 ⁶ unstimulated PBMCs was inoculated into each well of a 96-well plate. Viral particles were added at an MOI of 1 and the plates were incubated for 4 hours at 37° C. and 5% CO _2. After 4 hours of transduction, the cells in each well were washed 3 times in DPBS + 2% HSA before being resuspended in 1 ml of X-Vivo 15 and incubated at 37° C. and 5% CO _2. For samples treated with antiviral drugs, dapline and dolutegravir were added to a final concentration of 10 μM during transduction and subsequent incubation. Exogenous cytokines were not added to the samples at any time. Samples were collected on day 6 to determine the transduction efficiency based on GFP expression, as determined by FAC analysis using lymphocyte gates based on forward and side scatter.

Figures 10A and 10B show histograms of the percentage (%) of GFP+ cells (Figure 10A) and the absolute number of GFP+ cells per well (Figure 10B) on day 6 after transduction of unstimulated PBMCs. These histograms show that unstimulated human PBMCs can be transduced by a 4-hour exposure to VSV-G pseudotyped lentiviral particles encoding F1-0-03 and not displaying UCHT1 scFvFc-GPI alone or in combination with CD80-GPI. In addition, as shown for VSV-G pseudotyped lentiviral particles displaying UCHT1 scFvFc-GPI, the transduction efficiency of PBMCs by these lentiviral particles is significantly reduced by the presence of a drug combination that inhibits reverse transcription and integration. This example shows that after a brief 4-hour exposure to these retroviral particles, the genetic modification and transgene expression of these PBMCs is not a pseudotransduction, but rather a result of transduction, where the viral transgene RNA is reverse transcribed, integrated into the genome of the PBMCs, and expressed.

Example 6. Transduction efficiency of unstimulated PBMCs incubated for 4 hours with retroviral particles pseudotyped with envelopes derived from VSV-G, BaEV or MuLV and optionally expressing anti-CD3 scFvFc on their surface.

In this example, lentiviral particles pseudotyped with various envelope proteins were incubated with unstimulated human PBMCs for 4 hours and transduction efficiency was assessed.

Recombinant lentiviral particles were produced in 293T cells (Lenti-X ^™ 293T, Clontech) adapted to suspension culture in Freestyle ^™ 293 expression medium (Thermo Fisher Scientific). Cells were transiently transfected using PEI with genomic plasmids and separate packaging plasmids encoding gag/pol, rev, and pseudotyped plasmids. For some samples, the transduction reaction mixture also included a plasmid encoding UCHT1 scFvFc-GPI. The genomic plasmid used for the samples in this example was F1-3-219 or F1-3-451 described in other examples herein. The pseudotyped plasmids used for the samples in this example encoded envelope proteins from: VSV-G (SEQ ID NO:548), BaEVwt (SEQ ID NO:535), MuLV (SEQ ID NO:538) (sometimes referred to as MLV), a modification of the BaEV envelope in which the fusion inhibitory R peptide was removed by truncation of BaEVwt before methionine residue 546 (SEQ ID NO:536) or valine residue 547 (SEQ ID NO:537), or U-MuLV (SEQ ID NO:539) in which the anti-CD3 scFv from UCHT1 was fused to the amino terminus of the MuLV envelope.

Experiments using lentiviral particles encoding F1-3-219 and F1-3-451 were performed on different days, and PBMCs from different donors were used, but the same protocol was followed. On day 0, PBMCs were prepared from the yellow layer of blood clots as described in Example 3 without any additional steps to remove monocytes. After separation, 1 ml of X-Vivo15 containing 1×10 ⁶ unstimulated PBMCs was inoculated into each well of a 96-well plate. Unless otherwise indicated, viral particles were added at an MOI of 1 and the plates were incubated at 37° C. and 5% CO ₂ for 4 hours. After 4 hours of transduction, cells in each well were washed 3 times in DPBS + 2% HSA before being resuspended in 1 ml of X-Vivo15, and incubated at 37° C. and 5% CO _2. Exogenous cytokines were not added to the sample at any time. Samples were collected on day 3 or day 6 to determine transduction efficiency based on GFP expression as determined by FAC analysis using a lymphocyte gate based on forward and side scatter.

For the first transduction testing VSV-G and baboon envelope proteins, the transduction efficiency of the lentiviral particles encoding F1-3-219 was measured on day 6. FIG. 11A shows the FLAG+% and FIG. 11B shows the number of FLAG+ cells per microliter of lentivirus pseudotyped as indicated. Each of the lentiviral particles, including those with VSV-G or baboon envelope proteins, was able to promote transduction compared to non-transduced cells. The higher transduction rate by lentiviral particles pseudotyped with VSV-G in this example relative to that in Example 5 is likely a result of the chimeric lymphoproliferative component encoded by F1-3-219. Viral particles pseudotyped with VSV-G and expressing UCHT1 scFvFc-GPI and viral particles pseudotyped with BaEVΔR(HA) showed the highest transduction efficiency in this experiment.

For the first transduction testing VSV-G and baboon envelope proteins, transduction efficiency of lentiviral particles encoding F1-3-451 was measured on day 6. Figure 12A shows CD3+FLAG+% and Figure 12B shows viable CD3+FLAG+ cells% of lentivirus pseudotyped as indicated. Each of the lentiviral particles, including those with VSV-G or murine leukemia virus envelope proteins, was able to promote transduction compared to non-transduced cells. Viral particles pseudotyped with MuLV and not displaying UCHT1 ScFv as either a single envelope protein and UCHT1 scFvFv-GPI [MuLV+U] or as an envelope fusion protein [U-MuLV] showed the highest transduction efficiency in this experiment.

Example 7. Time course of retroviral transduction of unstimulated PBMCs by exposure for 4 hours or less than 1 minute.

In this experiment, recombinant lentiviral particles were incubated with unstimulated PBMCs for between 4 hours and less than 1 minute and examined for their ability to transduce PBMCs and promote their survival and/or proliferation in vitro in the absence of any exogenous cytokines.

method

Recombinant lentiviral particles were produced in 293T cells (Lenti-X ^TM 293T, Clontech) adapted to suspension culture in Freestyle ^TM 293 expression medium (Thermo Fisher Scientific). Cells were transiently transfected using PEI with a genomic plasmid and separate packaging plasmids encoding gag/pol, rev, and a pseudotyped plasmid encoding VSV-G as described in Example 3. For some samples, the transduction reaction mixture also included a plasmid encoding UCHT1 scFvFc-GPI as further described in Example 3. Two genomic plasmids were used in this example. The first plasmid included a Kozak sequence, a CD8a signal peptide, a FLAG tag, and an anti-CD19:CD8:CD3z CAR, followed by a triple termination sequence (F1-3-253). The second plasmid included the Kozak sequence, CD8a signal peptide, FLAG tag and anti-CD19:CD8:CD3z CAR, T2A and CLE DL3A-4 (E013-T041-S186-S051), followed by a triple termination sequence (F1-3-451).

On day 0, Ficoll-Paque (GE Healthcare Life Sciences) and SepMate ^TM -50 (Stemcell ^TM Technologies) enrich PBMCs from buffy coats (San Diego Blood Bank) from 2 donors with density gradient concentration. No additional steps were taken to remove monocytes. After separation, PBMCs were diluted to 1×10 ⁶ PBMCs per 1 ml of X-Vivo 15 (LONZA), and 1 ml was inoculated into each well of a 96-well plate. Cells from each donor were also left for expression analysis by FACS. Anti-CD3, anti-CD28, IL-2, IL-7 or other exogenous cytokines were not added before transduction to activate or otherwise stimulate lymphocytes. Lentiviral particles were directly added to unstimulated PBMCs at an MOI of 1. Before washing the cells of the transduction mixture 3 times with DPBS + 2% HSA, the transductions were incubated at 37°C and 5% _CO2 for 4 hours, 2 hours, 30 minutes, 15 minutes, 7.5 minutes, 5 minutes, 2.5 minutes, or not at all. Then, the cells in each well were resuspended in 1 ml X-Vivo15 and incubated at 37°C and 5% _CO2 . For samples treated with antiviral drugs, during transduction, dapline or dolutegravir was added to a final concentration of 10 μM, and the transduction reaction was incubated for 4 hours at 37°C and 5% _CO2 . After three washes, the drugs were supplemented at the same concentration in the recovery medium. No exogenous cytokines were added to the samples at any time. Samples were collected on day 6, and the transduction efficiency based on FLAG expression was determined by FACS analysis using lymphocyte gates based on forward and side scatter.

result

In this example, it was found that an incubation period of less than 1 minute was as effective as a 4-hour incubation period in promoting the transduction of unstimulated PBMCs by recombinant lentiviral particles. Figure 13 shows the CD3+FLAG+ absolute cell counts (per microliter) on the 6th day after the indicated period of unstimulated PBMCs from 1 donor were transduced by different recombinant lentiviral particles. The ability of each of the recombinant lentiviral particles to transduce PBMCs was similar in all incubation periods. This is particularly evident for lentiviral particles expressing anti-CD3scFvFc-GPI, and has a higher transduction efficiency than its non-anti-CD3scFvFc-GPI expressing counterpart. For all incubation times examined, the total number of transduced PBMCs was greater in those samples transduced by [F1-3-451GU] than in those samples transduced by [F1-3-253GU] indicating that DL3A CLE encoded in F1-3-451 promotes the survival and/or proliferation of these cells. Inhibition of transduction by dapline (reverse transcriptase) and dolutegravir (integrase inhibitor) as shown in Figure 13 demonstrated that the genetic modification and transgene expression of these PBMCs was not a pseudo-transduction, but rather a result of transduction in which viral transgene RNA was reverse transcribed, integrated into the genome of the PBMCs, and expressed. Similar results were observed using PBMCs from a second donor.

Example 8. Construction of the IL-7 receptor lymphoproliferative module.

A series of constitutively active IL7 receptor (IL7R) transmembrane mutants from T-cell lymphoblastic leukemia (243InsPPCL (SEQ ID NO:82); 246InsKCH (SEQ ID NO:101); 241InsFSCGP (SEQ ID NO:102); 244InsCHL (SEQ ID NO:103); and 244InsPPVCSVT (SEQ ID NO:104); all from Shochat et al. 2011, J. Exp. Med. Vol. 208 No. 5 901-908) were synthesized by overlapping oligonucleotide synthesis (DNA2.0, Newark, California). The synthetic constitutively active IL7R transmembrane mutant was inserted into the constitutively expressing lentiviral vector backbone immediately after the 2A ribosomal skipping sequence, followed by an anti-CD19 CD3ζ expression cassette including a CD8A handle (SEQ ID NO: 79) and a leader peptide (SEQ ID NO: 74). HEK293 packaging cells were transfected with IL7R transmembrane mutant lentiviral vector and lentiviral packaging constructs, grown, and viral supernatants were collected using methods known in the art. CD3/CD28 stimulated T cells were transduced with viral supernatants and grown in IL2-deficient AIM V, CTS OpTmizer T cell expansion SFM or X-VIVO 15 medium for 4 weeks, supplemented with frozen PBMCs from the same donor once a week. The resulting amplified transduced T cells expressing IL7R variants were cloned using FACS sorting and the sequence of the IL7R construct was identified by sequencing RT-PCR products. The 243InsPPCL variant (PPCL) (SEQ ID NO: 82) was selected for further evolution to generate a conditionally active CAR.

Example 9. Testing the activity of the IL-7 receptor lymphoproliferative/survival component in PBMCs.

To test the IL-7Rα variants for their ability to mediate cytokine-independent survival of T cells, thirty milliliters of human blood was drawn into blood collection tubes using acid citrate (ACD) as an anticoagulant. Whole blood was processed using density gradient concentration by Ficoll-Pacque ^™ (General Electric) according to the manufacturer's instructions to obtain peripheral blood mononuclear cells (PBMCs). Aliquots of PBMCs were aseptically transferred to the wells of a 12-well tissue culture plate with X-Vivo ^™ 15 culture medium (Lonza) to a final concentration in a final volume of 1 mL containing 500,000 viable cells/ml. Recombinant human interleukin-2 (IL-2) (Novoprotein) was also added to some samples at a concentration of 100 IU/ml. Activated anti-CD3 Ab (OKT3, Novoprotein) was added at a concentration of 50 ng/ml to activate PBMCs for viral transduction. The plates were cultured overnight in a standard humidified tissue culture incubator at 37°C and 5% carbon dioxide. After overnight incubation, lentiviral particle preparations containing the desired test construct ( FIG. 14A ) were added to individual wells at a multiplicity of infection (MOI) of 5. The plates were incubated overnight at 37° C. and 5% carbon dioxide in a standard humidified tissue culture incubator. After overnight incubation, the contents of each of the wells of the 12-well plate were collected and centrifuged to obtain a pellet. The samples were washed once with D-PBS + 2% human serum albumin (HSA), resuspended in X-Vivo 15 ^TM medium, and transferred to 6-well gas permeable cell culture device (Wilson Wolf). Additional X-Vivo ^™ 15 medium was added to bring the final volume of each well to 30 ml. A matched control sample of each of the constructs was transferred to The cells were incubated in a standard humidified tissue culture incubator at 37°C and 5% carbon dioxide. Device for 7 days. During every 2 to 3 days of culture, fresh IL-2 was added to the control samples containing IL-2. Matched test samples without IL-2 were not supplemented. Samples were removed on day 7 to track cell number and viability during expansion (Countess, ThermoFisher).

Figure 14A provides a schematic diagram of the tested IL7Rα constructs. These constructs were inserted into the recombinant lentiviral particle genome. Replication-deficient recombinant retroviral particles were used to transduce PBMCs. Figure 14A shows a schematic diagram of wild-type IL7Rα (SEQ ID NO: 229) consisting of a signal sequence (SS), an extracellular domain (ECD), a transmembrane (TM), and an intracellular domain (ICD). "1" indicates the site of the fibronectin type III domain; "2" indicates the site of the WSXWS motif; "3" indicates the Box 1 site; "4" indicates the site of the protein kinase C (PKC) phosphorylation site, and "5" indicates the Box 2 site.

Variant "A" is an IL-7Rα with InsPPCL (Shochat et al. 2011, J. Exp. Med. Vol. 208 No. 5901-908) at position 243 but without the S185C mutation, which is expressed on a transcript with a GFP polypeptide, a GSG linker, and a P2A ribosomal skipping sequence fused to its N-terminus. Variant "B" is an IL-7RαInsPPCL with a GFP polypeptide, a GSG linker, and a P2A ribosomal skipping sequence fused to its N-terminus and a Myc tag between the signal sequence and the extracellular domain. Variant "C" is similar to variant "B" except that its intracellular domain is truncated at position 292. Variant "D" is similar to variant "A" except that its intracellular domain is truncated at position 292. Variant "E" is an IL-7RαInsPPCL variant truncated at its N-terminus so that the signal sequence and most of the extracellular domain (residues 1 to 228) are absent; variant "E" also has a GFP polypeptide, a GSG linker, a P2A ribosomal skipping sequence, and an eTag fused to the N-terminus in the order according to the amino terminus. Numbering of amino acid residues is based on IL7Rα (NCBI GI No. 002176.2). T cells containing each of the variants were tested for viability using the Trypan Blue exclusion method in the presence or absence of IL-2.

As shown in Figure 14B, PBMC requires IL-2 for in vitro survival. As illustrated in Figure 14B, on the 7th day after transduction, untransfected PBMCs had about 80% viability in the presence of IL-2, and 0% viability in the absence of IL-2. On the 7th day after transduction, in the absence of IL-2, PBMCs with a full-length version of IL-7RαInsPPCL (IL-7Rα variants A and B in Figure 14A) had more than 20% viability, indicating that the expression of constitutively active IL-7RαInsPPCL receptors had survival activity in these cells. In addition, compared to wild-type IL-7 receptors, on the 7th day after transduction, T cells expressing IL-7RαInsPPCL variants (IL-7Rα variants C and D in Figure 14A) with truncated intracellular domains (ICD) had increased viability. Finally, the N-terminal IL-7 receptor mutant (IL-7Rα variant E in Figure 14A) had survival activity in these cells as shown in Figure 14B. Thus, this example demonstrates that certain IL-7 receptor mutants have survival activity when expressed in PBMCs.

Example 10. Analysis of intracellular signaling domains of lymphoproliferative components and whole chimeric constructs from iterative chimeric library screening.

This example confirms that the intracellular domains from the vast majority of candidate proliferation and/or survival genes, which have been reported to promote lymphoid or myeloid proliferation and/or survival under certain conditions, are effective in promoting proliferation and/or survival of PBMCs when cultured between day 7 and at least day 21 after transduction in the absence of exogenously added cytokines (such as IL-2). In addition, intracellular domains and corresponding genes that surprisingly promote particularly noteworthy amplification were identified at time points on day 21 and beyond.

Materials and Methods

Data preparation

The data used for this analysis is a subset of constructs in a collection of 8 replicate pools, 4 of which were prepared according to Pool 3.1A ("fed" with PBMC) (3.1.1a to 3.1.4a) and Pool 3.1B (not "fed" with PBMC) (3.1.1b to 3.1.4b) of Example 12 herein. Differential enrichment between day 7 and day 21 was analyzed for all 8 pools, and additional time points were analyzed as follows: Pool 3.1.1a - day 28 and day 35; Pool 3.1.1b - day 35; Pool 3.1.4a - day 28, day 35, and day 42; and Pool 3.1.4b - day 28. All constructs with either one (first intracellular domain tested only) or two intracellular domains (tested intracellular domain plus any second intracellular domain in the library) were analyzed at each time point compared to no intracellular domain (linker and stop) for the same extracellular and transmembrane domains.

Functional Analysis

A Mann-Whitney-Wilcoxon test was performed for differential enrichment of constructs containing a specific intracellular domain portion compared to constructs containing any other intracellular domain portion, where differential enrichment was defined as the difference between the enrichment of a construct with an intracellular domain and its counterpart with the same extracellular and transmembrane domains and no intracellular domain (log2 conversion ratio between normalized counts at day 21 and normalized counts at day 7, each with an increase of 1 pseudocount). P values can be adjusted for the false discovery rate (Benjamini-Hochberg) for each set tested (when considering one set tested per library), and the false discovery rate was set to 0.1 (Sober et al. Sci Rep. 2016 Dec 8; 6: 38439; and Biostatistics. 2017 Apr 1; 18(2): 275-294).

result

We sought to confirm that the intracellular signaling domains of various candidate genes reported to promote cell proliferation and/or survival of lymphoid or myeloid cells under certain conditions could provide effective intracellular signaling domains for lymphoproliferative elements included in the compositions and methods provided herein. Thus, chimeric lymphoproliferative elements were constructed that combined a first intracellular signaling domain with various extracellular domains and transmembrane domains (and in most cases, a second intracellular domain). These chimeric lymphoproliferative element (CLE) constructs include, from amino to carboxyl terminus, an extracellular domain (P1), a transmembrane domain (P2), and one or both of the two, a first intracellular signaling domain (P3), and a second intracellular signaling domain (P4) as schematically shown in FIG. 15 for nucleic acid constructs encoding these CLEs. The first intracellular signaling domain (P3) from the following genes (with identifiers of the portions provided in parentheses and sequences of the portions provided in Table 7) were tested: CRLF2 (S054), CSF2RB (S057), CSF2RA (S058 and S059), CSF3R (S062), CSF3R (S063), CSF3R (S064), EPOR (S069 and S072), GHR (S077), IFNAR1 (S081), IFNAR2 (S082 and S083), IFNGR1 (S084), IFNGR2 (S085), IFNLR 1 (S086), IL1R1 (S098 and S099), IL1RAP (S100 and S101), IL1RL1 (S102), IL1RL2 (S103), IL2RA (S104), IL2RB (S105), IL2RG (S106), IL3RA (S109), IL5R (S115), IL6R (S116), IL6ST (S117), IL7RA (S120), IL7RA (S121), IL9R (S126), IL10RA (S129), IL10RB (S130), IL11RA (S131), 35), IL12RB1(S136), IL12RB1(S137), IL12RB2(S138), IL13RA1(S141), IL13RA2(S142), IL15RA(S143), IL17RB(S145), IL17RC(S147), IL17RD(S148), IL18R1(S154), IL 18RAP(S155), IL20RA(S156), IL20RB(S157), IL21R(S158), IL22RA1(S161), IL23R(S165), IL27 RA (S168 and S169), IL31RA (S170 and S171), LEPR (S174, S175, S176 and S177), LIFR (S180), LMP1 (S183), MPL (S186), MyD88 (S189, S190, S191, S192, S193, S194, S195, S196, S197, and S198), OSMR (S199), PRLR (S202), IL17RA (S144), IL17RC (S146), IL17RE (S149) and IFNLR1 (S087). In addition, a connector was included as a P3 portion (X001) in the screen to generate a lymphoproliferative element lacking the first intracellular signaling domain.

In the 3.1 library, the "second" intracellular domain (P4) is intended to serve as a regulatory domain, although several of these P4 intracellular domains are from genes that have also been reported to promote proliferation and/or survival of lymphoid or myeloid cells under certain conditions, and thus may provide potent proliferation and/or survival signals in the absence of the "first" intracellular signaling domain (P3) on the lymphoproliferative element. The second intracellular signaling domain (P4) from the following genes (with partial identifiers provided in parentheses and partial sequences provided in Table 7) was tested: CD3D (S037), CD3E (S038), CD3G (S039), CD27 (S047), mutant delta LckCD28 (S048), CD28 (S049), CD40 (S050 and S051), CD79A (S052), CD79B (S053), FCER1G (S074), FCGR2C (S075), FCGRA2 (S076), ICOS (S080), TNFRSF4 (S211), TNFRSF8 (S212), TNFRSF9 (S213), TNFRSF14 (S214), and TNFRSF18 (S215 and S216). Additionally, stop codons were included in some candidate lymphoproliferative elements within the P4 domain to generate constructs with only a single intracellular domain.

As discussed in Example 12 for library 3.1, extracellular domains containing variants of c-Jun including a leucine zipper motif were used with eTAG and linkers containing 0, 1, 2, 3, or 4 alanines (E006, E007, E008, E009, and E010, respectively). Transmembrane domains or combined stalk and TM domains from the following genes (with partial identifiers provided in parentheses and partial sequences provided in Table 7) were tested: CD2 (T001), CD3D (T002), CD3E (T003), CD3G (T004), CD3Z (T005), CD4 (T006), CD8A (T007), CD8B (T008), CD27 (T009), CD28 (T010), CD40 (T011), CD79A (T012), CD79B (T013), CRLF2 (T014 and T015), CSF2RA (T016), CSF2RB (T017 and T018), CSF3R (T019 and T020), T020), EPOR (T021 and T022), FCER1G (T023), FCGR2C (T024), FCGRA2 (T025), GHR (T026 and T027), ICOS (T028), IFNAR1 (T029), IFNAR2 (T030), IFNGR1 (T031), IFNGR2 (T032), IFN LR1(T033), IL1R1(T034), IL1RAP(T035), IL1RL1(T036), IL1RL2(T037), IL2RA(T038), IL2RB(T039), IL2RG(T040), IL3RA(T041), IL4R (T042), IL5RA (T043), IL6R (T044), IL6ST (T045), IL7RA (T046 and T047), IL9R (T048), IL10RA (T049), IL10RB (T050), IL11RA (T051), IL12RB1 (T052), IL12RB2 (T053), IL13RA1 (T054), IL13RA2 (T055), IL15RA (T056), IL17RA (T057), IL17RB (T058), IL17RC (T059), IL17RD (T060), IL17RE (T061), IL18R1 (T062 ), IL18RAP(T063), IL20RA(T064), IL20RB(T065), IL21R(T066), IL22RA1(T067), IL23R(T068), IL27RA(T069), IL27RA(T070), IL31RA(T071), LEPR(T072), LIFR(T073), MP L(T074), MPL(T075), OSMR(T076), PRLR(T077), TNFRSF4(T078), TNFRSF8(T079), TNFRSF9(T080), TNFRSF14(T081) and TNFRSF18(T082).

In addition to the repetition of library 3.1 discussed above and further analyzed in this example, a total of twenty library screenings have been performed to confirm that various candidate intracellular domains can effectively promote T cell in vitro expansion in the absence of any exogenous T cell growth stimulator (e.g., exogenous IL-2) of the culture medium. Under various conditions similar to those provided in Examples 11 and 12 and using PBMCs from different donors, the construct includes one or two intracellular domains and a transmembrane domain and an extracellular domain as provided in Table 7. For libraries that do not include CAR in the tested constructs, less amplification is generally observed on the 21st day and the 7th day, and the transduction of many constructs encoding both CAR and CLE provides enrichment beyond the enrichment provided by the construct including only CAR between the 21st day and the 7th day. These general observations confirm that the tested lymphoproliferative component enhances the primary proliferation and/or amplification signal provided by anti-CD19 CAR in the presence of PBMCs, at least some of which are believed to express CD-19.

To confirm that many different intracellular domains that drive proliferation and/or survival of myeloid or lymphocytes under certain conditions can serve as the first intracellular signaling domain in the lymphoproliferative component provided herein to promote the expansion of transduced PBMCs in the absence of any exogenously added cytokines or exogenously added lymphocyte mitogens (e.g., anti-CD3 or anti-CD28) during culture, eight pool screens were performed for intracellular P3 and P4 domains according to the method provided for pool 3.1 according to Example 12, including 4 replicate pools according to 3.1A ("fed" with PBMCs) and 4 replicate pools according to 3.1B (not "fed" with PBMCs). Cells were cultured for 21 days after transduction in the absence of exogenous cytokines or other T cell stimulants, and the results were analyzed. Amplification at day 21 and later time points was determined by measuring enrichment, which was the ratio between the normalized counts (per million counts) at day 21 or later and the normalized counts at day 7 for that construct. Cells were analyzed for expansion at day 21, day 28, day 35, and day 42 for each available time point within the pool replicates compared to day 7. Constructs were ranked with respect to the level of expansion obtained at the time points tested compared to day 7.

Table 25 provides constructs that ranked in the top 100 constructs in terms of amplification at a time point relative to day 7 in any of these eight replicate pool screens at at least one time point. Cellular amplification quantification showed that each extracellular domain (P1 portion) and transmembrane domain (P2 portion) and all second intracellular domains (P4 portion) were represented in at least one top 100 construct in at least one of these eight pool screens. Of the 79 first intracellular signaling domains (P3 portions) tested in the 49 genes in these screens, only 6 intracellular signaling domain portions were not present in the top 100 constructs in any of the eight replicate pool screens, and different intracellular domain fragments from two of the genes were found in the top 100 constructs. Thus, intracellular signaling domains from only 3 of the tested genes were not represented in the top 100 constructs. No constructs without an intracellular signaling domain (i.e., constructs in which P3 is a linker (X001) and P4 is a stop codon (X002)) were found in the top 100 fractions. In fact, none of the constructs without an intracellular signaling domain were ranked in the top 350 in any of the eight replicate pool screens (the top ranked being 353 in pool 3.1.1b), validating the effectiveness of these lymphoproliferative component (i.e., driver) screens in identifying effective lymphoproliferative component constructs, and in particular, intracellular signaling domains that promote expansion of these cultures ex vivo or in vitro under these conditions.

These data support that a relatively large number of intracellular domains from genes reported to promote cell proliferation or survival in lymphoid or myeloid cells (including those cells successfully tested in these eight library screens) under certain conditions can be used as the first signaling intracellular domain of the lymphoproliferative component in the methods and compositions provided herein. The five genes (six intracellular domain P3 portions) that were not identified as providing top 100 enrichment in any of these screens were IL4R (portions S110 and S113), IL17RA (portion S144), IL17RC (portion S146), IL17RE (portion S149), and IFNLR1 (portion S087). It is possible that these P3 portions have technical issues that were not identified in the top 100 in any of the replicates, either in the gene construct or in the method used for detection. Therefore, it cannot be concluded from this data that these intracellular activation domains cannot form effective intracellular first signaling molecules. It is also noteworthy that IL17RC portion S146 was not found in any of the top 100 pools in these pool 3.1 replicates, but IL17RC intracellular domain portion S147 was found in the top 100 repertoires.

Without being limited by theory, the results that all extracellular domains (P1) and all transmembrane domains (P2) were present in at least one of the top 100 constructs from at least one library screen are consistent with the hypothesis that the extracellular and transmembrane domains play a supporting role in directing one or both intracellular signaling domains in these lymphoproliferative component constructs. Since the ECDs tested in the screen are known to form dimers, and the transmembrane domains are known to act as transmembrane polypeptides that can direct the intracellular signaling domain in an active conformation, it is expected that all ECDs and TMs tested in the screen will be effective at least some of the time when paired with an effective intracellular signaling domain.

It is believed that in complex lymphoproliferative component screens such as those analyzed in this example, as exemplified in more detail in Examples 11 and 12 separately herein, the most potent intracellular domains and complete chimeric constructs will appear in at least one screen out of the eight screens as top 100 hits out of the following identified numbers of constructs in each library: 3.1.1a 73,556; 3.1.1b 93,768; 3.1.2a 91,572; 3.1.2b 115,860; 3.1.3a 91,918; 3.1.3b 99,772; 3.1.4a 31,177; 3.1.4b 66,873. However, many potent intracellular domains do not appear as top 100 hits in all or even most screens. Without being limited by theory, it is believed that there are many factors that contribute to whether a lymphoproliferative component is relatively effective in a given experiment, in which cultures are generated from PBMCs transduced with a large, complex mixture of lentiviruses encoding candidate lymphoproliferative components, which compete with each other for amplification, and in which the transduced PBMCs are obtained from different donor individuals in each "feed-in" experiment and each "no-feed-in" experiment.

Portions containing the following genes were present in the top 100 constructs at P3 on the construct containing X002 at P4 and at least one of the pool 3.1 repeats (Table 25): CSF2RB, CSF2RA, CSF3R, EPOR, IFNGR1, IFNGR2, IL1R1, IL1RAP, IL1RL1, IL2RA, IL2RG, IL5RA, IL6R, IL9R, IL10RB, IL11RA, IL12RB1, IL12RB2, IL13RA2, IL15RA, IL17RD, IL21R, IL23R, IL27RA, IL31RA, LEPR, MPL, MyD88, or OSMR. The following parts were present in the first 100 constructs at P3 on constructs containing X002 at P4 and at least one of the library 3.1 repeats (Table 25): S057, S058, S059, S064, S069, S072, S084, S085, S099, S100, S101, S102, S104, S106, S115, S116, S117, S118, S119, S200, S201, S202, S203, S204, S205 16, S126, S130, S135, S137, S138, S142, S143, S148, S158, S165, S168, S169, S170, S171, S174, S175, S176, S177, S186, S190, S191, S192, S193, S197, S198 or S199.

The following gene containing portions were present in the first 100 constructs at P4 on the construct containing X001 at P3 and at least one of the library 3.1 repeats (Table 25): CD40, CD79B, FCGR2C, or FCGRA2. The following portions were present in the first 100 constructs at P4 on the construct containing X001 at P3 and at least one of the library 3.1 repeats (Table 25): S037, S039, S050, S051, S052, S080, S212, or S213.

Next, data from the 8 pool 3.1 replicate pool screening experiments were statistically analyzed to identify particularly potent intracellular P3 and P4 domains. Time points starting at day 21 and later were analyzed relative to day 7 cell counts (i.e., DNA barcode counts assuming equal cell counts), as discussed above for the top 100 pool analysis.

We use statistical analysis to identify especially effective intracellular domains (that is, P3 parts and P4 parts) independent of their intracellular partners. The difference enrichment value is calculated as the enrichment of the construct containing the intracellular part of interest and the enrichment of the construct with the same extracellular part and transmembrane part but without the intracellular domain, wherein the enrichment is expressed as the logarithm of the ratio between the normalized count value (per million counts) at a specific time point and the normalized count value (per million counts) at the 7th day with a false count of 1. Statistical analysis is performed by performing the Mann-Whitney-Wilcoxon test on each first intracellular domain (P3) compared to all other possible P3 parts. This test calculates the sum of the rankings for the construct containing the specific P3 part and all other constructs in the data set, and performs a chi-square test to determine whether the ranking distribution is significantly different. Apply Benjamini-Hochberg correction to obtain the p value adjusted for the false discovery rate of each individual library. First, all constructs unrelated to the P4 part are analyzed. When several parts represent variants of the same gene, the parts grouped by gene were also used for separate analysis. The same analysis was then performed only for the construct with a stop codon at P4 (X002 part).

Table 1 provides the identifiers of the most effective first intracellular signaling domain (P3) genes and portions using statistical analysis for the 8 library 3.1 replicates. An asterisk after the day number in Table 1 indicates that the P3 gene or portion was significant for the indicated level when analyzed only with termination at P4, and a cross after the day number indicates that the P3 gene or portion was significant for the indicated P level when analyzed with any P4 for a given P3. The absence of an asterisk or cross after the day number indicates that the significance and significant level for the indicated day was obtained for both the P3 portion or gene analyzed with all P4 portions of a given P3 and for the analysis of termination at P4 for a given P3. The following genes are represented by the portion of the first intracellular signaling domain identified in the constructs shown to be most effective by this statistical analysis (P<0.1), with the portion number in parentheses, or by the gene shown to be most effective by the gene analysis: CSF2RB (S057), CSF3R (S062, S063 and S064), IFNAR1 (S081), IFNGR1 (S084), IL2RB (S105), IL2RG (S106), IL6ST (S117 ), IL10RA (S129), IL12RB2 (S138), IL17RC (gene analysis only), IL17RE (S149), IL18R1 (gene analysis only), IL22RA1 (S161), IL27RA (S168 and S169), IL31RA (S170), MPL (S186), MyD88 (S190, S191, S192, S194, S196, S197, S198), OSMR (S199), and PRLR (S202). Of note, IL17RE (S149) was identified by this statistical analysis but was not shown on any of the top 100 lists for these libraries (see Table 25). It is believed that the lower representation of this portion of this library produces a false positive statistical result.

The following first intracellular domain genes are represented by the first intracellular signaling domain portion that was most effective by this statistical analysis of at least 2 libraries (P<0.1), with the portion number in parentheses: CSF2RB (S057), CSF3R (S062, S063, and S064), IL2RB (S105), IL2RG (S106), IL6ST (S117), IL27RA (S168 and S169), IL31RA (S170), MPL (S186), and MyD88 (S190, S191, S192, S194, S196, S197, S198).

The following first intracellular domain genes were represented by first intracellular signaling domain portions that were most effective by this statistical analysis of at least 2 pools even when the statistical cutoff for P was less than 0.05: CSF2RB (S057), CSF3R (S062, S063, and S064), IL2RB (S105), IL6ST (S117), IL27RA (S168 and S169), IL31RA (S170), MPL (S186), and MyD88 (S190, S191, S192, S194, S196, S197, S198). Finally, the following first intracellular domain genes were most effective by this statistical analysis of at least 2 pools even when the statistical cutoff for P was less than 0.05, where the pools were not fed/unfed pairs from the same replicate: CSF3R, IL6ST, IL27RA, MPL, and MyD88.

Table 1. Most effective P3 segments and genes from replicate screening of library 3.1.

Analysis of the P4 domain

Statistical analysis of P4 domains was performed as indicated above for P3 domains, except that any P3 domains or no P3 domains (linker (X001) at partial P3) were used to analyze the tested P4 domains. Table 2 provides the identifiers of the most effective second intracellular signaling domain (P4) genes and portions using statistical analysis for the 8 library 3.1 replicates. When analyzed only with the linker at P3, the asterisk after the day number in Table 2 indicates that the P4 gene or portion is significant for the indicated level, and when analyzed with any P3 for a given P4, the pound sign after the day number indicates that the P4 gene or portion is significant for the indicated P level. The absence of an asterisk or pound sign after the day number indicates that the significance and significant level of the indicated day are obtained for the P4 portion or gene for both the analysis of all P3 portions of a given P4 and the analysis of the linker at P3 of a given P4. The following genes with part numbers in parentheses are represented by the second intracellular signaling domain portion that was identified in the constructs shown to be most effective by this statistical analysis (P<0.1): CD27 (S047), CD40 (S050 and S051), CD79B (S053), TNFRSF4 (S211), TNFRSF8 (S212), TNFRSF9 (S213), and TNFRSF18 (S215). The following genes are represented by the second intracellular signaling domain portion that was most effective by this statistical analysis of at least 2 pools: CD40 (S050 and S051) and TNFRSF8 (S212). The following genes with partial numbers in parentheses are represented by the second intracellular signaling domain portion that was identified in the constructs shown to be most effective by this statistical analysis (P<0.05): CD27 (S047), CD40 (S050 and S051), TNFRSF4 (S211), TNFRSF8 (S212), TNFRSF9 (S213), and TNFRSF18 (S215). Of note, all of the most statistically significant second intracellular signaling domains were derived from TNF receptor family members.

Table 2. Most effective P4 parts and genes from replicate screening of library 3.1.

Further analysis was performed to identify the most effective second intracellular signaling domain (P4) partner for each first intracellular signaling domain. For this analysis, constructs with two intracellular domains were compared with their equivalents with only one intracellular domain (same extracellular domain, transmembrane domain and first intracellular domain). Differential enrichment was calculated as the difference between the enrichment values between the two constructs, where enrichment was expressed as the logarithm of the ratio between the normalized counts (per million counts) at the time point of interest and the normalized counts (per million counts) at day 7 (each increased by 1 pseudo count) with a base of 2. For each representative combination of the extracellular part, the transmembrane part and the first intracellular part, the differential expression values of the constructs with a specific second intracellular domain compared to other second intracellular domains were tested by Mann-Whitney-Wilcoxon to identify the parts associated with significant changes in the ranking distribution. The Benjamini-Hochberg method was used to adjust the P value for each test set, and the false discovery rate was set to 0.1 or 0.05.

Table 3 provides identifiers of the most potent second intracellular signaling domain (P4) partners for the first intracellular signaling domain (P3) portion and the corresponding gene using this statistical analysis for the 8 replicates of pool 3.1 and pool 3.0 (Example 12). The following pairs of genes with the gene from the P3 portion first and the gene from the P4 portion second represented by the first and second intracellular signaling domain portions identified as being on the same construct most potent by this statistical analysis (P<0.1) are: CSF2RA and TNFRSF4; CSF2RA and CD28; CSF2RA and TNFRSF8; CSF2RA and CD27; CSFR3 and CD79B; IFNAR2 and TNFRSF14; IL1 RAP and CD79A; IL3RA and CD40; IL10RA and CD79B; IL11RA and FCGRA2; IL13RA2 and TNFRSF14; IL18RAP and CD3G; IL27RA and FCGRA2; LEPR and CD3G; LIFR and TNFRSF18; MPL and CD40; MPL and CD79B; MPL and TNFRSF4; MPL and CD3G; MyD88 and CD79B; and MyD88 and CD3D. The following pairs of genes with genes first from the P3 portion and second from the P4 portion are represented by the first and second intracellular signaling domain portions, which were identified as being most effective on the same construct at a strict statistical level (P<0.05): CSF2RA and TNFRSF4; CSF2RA and CD28; CSFR3 and CD79B; IFNAR2 and TNFRSF14; IL10RA and CD79B; IL11RA and FCGRA2; IL27RA and FCGRA2; LIFR and TNFRSF18; MPL and CD40; MPL and CD40; MPL and CD79B; MPL and TNFRSF4; MPL and CD3G; and MyD88 and CD79B. The following pairs of genes with the P3 portion first and the P4 portion second were identified on the same construct as being most effective by this statistical analysis (P<0.1): S058 and S211; S058 and S049; S059 and S212; S059 and S047; S064 and S053; S083 and S214; S101 and S052; S109 and S050; S129 and S053; S 135 and S076; S142 and S214; S155 and S039; S169 and S076; S177 and S039; S180 and S216; S186 and S050; S186 and S051; S186 and S053; S186 and S211; S186 and S039; S192 and S053; S194 and S037; and S195 and S053. The following gene pairs, first with the P3 portion and secondly with the P4 portion, were identified on the same construct as being most effective by this statistical analysis (P<0.05): S058 and S211; S058 and S049; S064 and S053; S083 and S214; S129 and S053; S135 and S076; S169 and S076; S180 and S216; S186 and S050; S186 and S051; S186 and S053; S186 and S211; S186 and S039; and S195 and S053.

Thus, the intracellular signaling domains from the listed P4 genes appear to be particularly effective in costimulating the proliferative signals provided by the intracellular signaling domains of the listed P3 partner genes.

Table 3. P4 section of improved P3 section

This example confirms that the vast majority of the first intracellular domains tested are effective in driving expansion of transduced PBMCs between days 7 and 21 after transduction in the absence of exogenous cytokines when cultured ex vivo. In addition, various intracellular domains and first and second intracellular domain combinations were identified as being particularly effective in driving expansion under these conditions up to 21 days and later time points. Example 11. Identification of candidate chimeric polypeptide lymphoproliferative components.

In this example, two chimeric polypeptide pools (pool 1 and pool 2) of candidate (putative) chimeric lymphoproliferative components were assembled into viral vectors from a pool of extracellular-transmembrane blocking sequences, a pool of intracellular blocking sequences, and a pool of barcodes according to constructs encoding chimeric polypeptides provided in Figures 16-17. Proliferation of pool-transduced PBMCs was performed over time: genomic DNA was extracted from PBMCs at weekly intervals and the barcode region was amplified for DNA sequencing to estimate the number of cells in these mixed PBMC cultures containing each of the test candidate chimeric lymphoproliferative components. Thus, the test candidate chimeric lymphoproliferative components were screened for their ability to promote PBMC cell proliferation in the absence of IL-2 or any other exogenous cytokines.

Two libraries were prepared and analyzed in this study: the constructs in library 1 (which were used in the screening identified as libraries 1A, 1.1A, and 1.1B) were flanked by CAR at the 5' end of the candidate chimeric polypeptide, while the constructs in library 2 (which were used in the screening identified as libraries 2B and 2.1B) did not include CAR (Figures 16 and 17). Figure 16 provides a schematic diagram of a non-limiting exemplary transgenic expression cassette containing a polynucleotide sequence encoding a CAR of library 1 driven by an EF-1α promoter and a Kozak type sequence (GCCGCCACC (SEQ ID NO: 519)) in the backbone of a lentiviral vector and a candidate chimeric lymphoproliferative element (CLE). The CAR is FLAG-tagged and contains an ASTR for CD19, a stem and transmembrane portion of CD8, and an intracellular activation domain from CD3z. Each candidate lymphoproliferative component of library 1 includes 3 modules: 1 extracellular/transmembrane module (P1-2) and 2 intracellular modules (P3 and P4). The P1-2 modules also encode a recognition and/or elimination domain (TAG). A triple termination sequence (TAATAGTGA (SEQ ID NO: 520)) separates P4 from a DNA barcode (P5). WPRE (GTCCTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTG (SEQ ID NO:521)) is present between the last stop codon (starting 4 bp after the last nucleotide of the last stop codon) and the 3'LTR (which starts 83 nucleotides after the last nucleotide of the WPRE).

The CAR of pool 1 and P1-2 are separated by a polynucleotide sequence encoding a T2A ribosomal skipping sequence.

FIG17 provides a schematic diagram of a non-limiting exemplary transgenic expression cassette containing a polynucleotide sequence encoding a candidate CLE of library 2 driven by the EF-1α promoter and a Kozak-type sequence (GCCGCCACC (SEQ ID NO: 519)) in a lentiviral vector backbone. Each candidate lymphoproliferative element of library 2 includes three modules: an extracellular/transmembrane module (P1-2) and two intracellular modules (P3 and P4). The P1-2 modules also encode a recognition and/or elimination domain (TAG). A triple termination sequence (TAATAGTGA (SEQ ID NO: 520)) separates P4 from a DNA barcode (P5). WPRE (GTCCTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTG (SEQ ID NO:521)) is present between the last stop codon (starting 4 bp after the last nucleotide of the last stop codon) and the 3'LTR (which starts 83 nucleotides after the last nucleotide of the WPRE).

The assembled candidate chimeric polypeptide portions of the two libraries follow an identical design with 3 positions (1 extracellular/transmembrane module (P1-2) and 2 intracellular modules (P3 and P4), one of which may be an early termination signal). A GGS connector encoded by a nucleic acid sequence between each of the domains is included as a construct component product. Nucleic acids encoding Myc or eTAG (SEQ ID NO: 284) recognition and/or elimination domains (referred to as "eTAG" or "Myc Tag" in Table 7) in their entirety or in variant form are inserted into the 5' of the extracellular domain of each truncated cytokine receptor (or select LMP1 construct) as part of the P1-2 module so that the encoded candidate chimeric polypeptide includes a recognition and/or elimination domain in a frame with an extracellular domain. The signal sequence is inserted at the 5' end of the CAR encoding sequence in library 1 and at the 5' end of the encoding recognition and/or elimination domain sequence in library 2. A Kozak-type sequence (GCCGCCACC (SEQ ID NO: 519), which is also an enhanced Kozak-type sequence, is inserted within 10 nucleotides before the ATG start codon, which is at the 5' end of the signal sequence coding sequence in library 1 and at the 5' end of the coding recognition and/or elimination domain sequence (or selection LMP1 construct) in library 2. The fourth module contains random barcodes (~100 million combinations) generated by a random PCR assembly of ~10,000×~10,000 sub-barcodes. The coding sequence of the CAR of the library 1 construct encodes the signal sequence, FLAG tag, anti-CD19 ASTR scFv, CD8a stem, CD8a transmembrane domain, and CD3ζ intracellular activation domain. The expression of CAR (library 1 only) and CLE (library 1 and library 2) is driven by the EF1α promoter. In the construct of library 1, the T2A site is located between the CAR and the chimeric polypeptide coding sequence. In pools 1 and 2, a triple stop codon is present at the 3' end of P4.

Synthesis of viral vectors

Individual parts of the library assembly were designed and synthesized as DNA blocks with compatible type IIs enzyme (AarI) overhangs and linkers encoding glycine-glycine-serine sequences on the 5' and 3' ends of each part. The parts were then cloned individually into a universal plasmid vector. The assembly of each part was completed as a tube assembly, where 0.04 pmol of the part, 0.04 pmol of the barcode mix, and 0.04 pmol of the plasmid vector backbone were added to a 10 ul reaction, followed by the addition of 1 unit of AarI enzyme, 10 units of T4 DNA ligase, and 1X ligase buffer (50 mM Tris-HCl, 10 mM MgCl ₂ , 1 mM ATP, 10 mM DTT) for optimal ligase activity. Dissociation and ligation cycles were performed using the following thermal cycles: 2 min at 37°C and 5 min at 16°C; incubation at 50°C for 5 min for final dissociation; incubation at 80°C for 10 min to inactivate AarI, and a final hold at 4°C for 55 cycles. The assembled vector was then purified by conventional ethanol precipitation. The resulting purified vector was electroporated (Electro Micropulser, Bio-rad) into Top10 electrocompetent cells (Thermo Fisher Scientific) and directly recovered in liquid culture containing confocal antibiotics for selection and amplification. The culture was cultivated at 37°C for 10 to 12 hours and then collected, and the plasmid was purified using the ZymoPURE-EndoZero ^™ Plasmid Gigaprep Kit.

Lentiviral particle production

Recombinant lentiviral particles were produced by transiently transfecting 293T cells (Lenti-X ^™ 293T, Clontech) with separate lentiviral packaging plasmids encoding gag/pol and rev and a pseudotyped plasmid encoding VSV-G. Synthetic viral vectors encoding candidate chimeric polypeptides with or without co-expressed chimeric antigen receptors were co-transfected with the packaging plasmids. Cells were adapted to suspension culture by continuous growth in Freestyle ^™ 293 Expression Medium (ThermoFisher Scientific). Cells in suspension were inoculated at 1×10 ⁶ cells/mL (200 mL) in a 1L Erlenmeyer flask and immediately transfected using polyethyleneimine (PEI) (Polysciences) dissolved in weak acid.

Plasmid DNA was diluted in 10 ml ^Gibco Opti-MEM ^TM medium for 200 ml cells. To obtain lentiviral particles pseudotyped with VSV-G, the total DNA used (1 μg/mL of culture volume) was a mixture of 4 plasmids with the following molar ratios: 2× combined viral vector, 1× Rev-containing plasmid, 1× VSV-G-containing plasmid, and 1× gag/pol-containing plasmid. Separately, PEI was diluted to 2 μg/mL (culture volume, 2:1 ratio to DNA) in 10 ml ^Gibco Opti-MEM ^TM . After 5 minutes of room temperature incubation, the two solutions were thoroughly mixed together and incubated at room temperature for 20 minutes. The final volume (20 ml) was added to the cells. The cells were then incubated at 37° C. for 48 hours with rotation at 125 rpm and 8% CO ₂ .

After 48 hours, the supernatant was collected and clarified by centrifugation at 1,000g for 10 minutes. The viral supernatant was then filtered through a low protein binding Millex-HV 0.45μm PVDF filter (Millipore, catalog number SLHVR25LS). The clarified supernatant was poured into a new tube, 1 volume of Lenti-X PEG (Takara, 4×) was mixed with 3 volumes of the clarified supernatant, and incubated overnight at 4°C. The lentiviral particles were then precipitated by centrifugation at 1,500g and 4°C for 90 minutes. The supernatant was discarded, and the lentiviral particle pellet was resuspended in a 1:100 initial volume of complete PBMC growth medium without IL-2. The lentiviral particles were titrated using a p24 assay ELISA kit from Takara (#632200).

Transduction and culture of PBMCs

Whole human blood (101 ml) from healthy donors was collected into 200 ml blood bags containing CDP anticoagulant (Nanger; S-200). Blood was processed using density gradient centrifugation with Ficoll-Pacque ^™ (General Electric) using Sepax 2 S-100 devices (Biosafe; 14000) following the manufacturer's instructions and kit CS900.2 (BioSafe; 1008) to obtain peripheral blood mononuclear cells (PBMC). The yield of viable PBMC was 7.3×10 ⁷ cells. 1.5×10 ⁷ PBMCs were added to each of two G-Rex 100M cell culture devices (Wilson Wolf, 81100S) to reach a final concentration of 0.5×10 6 viable cells/ml in a final volume of 30 ml of complete OpTmizer ^™ CTS ^™ T Cell Expansion SFM (OpTmizer ^™ CTS ^™ T Cell Expansion Basal Medium 1 L (Thermo Fisher, A10221) supplemented with 26 ml OpTmizer ^™ CTS ^™ T Cell Expansion Supplement (Thermo Fisher, A10484-02), 25 ml CTS ^™ Immune Cell SR (Thermo Fisher, A2596101) and ¹⁰ ml CTS ^™ GlutaMAX ^™ -I Supplement (Thermo Fisher, A1286001) according to the manufacturer's instructions). Recombinant human interleukin-2 (IL-2) (Novoprotein) at a concentration of 100 IU/ml was also added along with an activating anti-CD3 Ab (OKT3, Novoprotein) at a concentration of 50 ng/ml to activate PBMCs for viral transduction. The G-Rex devices were incubated overnight at 37°C and 5% _CO2 in a standard humidified tissue culture incubator. After overnight incubation, a lentiviral particle preparation containing the construct encoding the chimeric polypeptide to be tested (pool 1 or pool 2) was added to individual G-Rex devices at a multiplicity of infection (MOI) of 5. The G-Rex devices (pool 1 and pool 2) were incubated overnight at 37°C and 5% _CO2 in a standard humidified tissue culture incubator. After overnight incubation, additional complete OpTmizer ^™ CTS ^™ T cell expansion SFM was added to bring the final volume of each G-Rex device to 500 ml. No additional IL-2 or other exogenous cytokines were added in this or the following cell culture steps. After PBMC isolation, Devices were incubated for 7 days at 37°C and 5% _CO2 in a standard humidified tissue culture incubator. On day 7, cells were collected from two G-Rex devices transduced with either pool 1 or pool 2 by centrifugation and resuspended in fresh complete OpTmizer ^™ CTS ^™ T cell expansion SFM without IL-2. 1.25×10 ⁷ pool 1 cells and 2.54×10 ⁷ pool 2 cells were placed on a new G-Rex 100M device containing 500 ml of complete OpTmizer ^™ CTS ^™ T cell expansion SFM without IL-2. A vial of thawed day 0 frozen donor-matched PBMCs (1.46×10 ⁷ PBMCs) was added to the G-Rex device containing cells transduced with pool 1 to provide CD19+ B cell activation to CD19 ASTR. In these screens, cells fed with PBMCs are designated by A after the pool number, e.g., screens performed using cells transduced with Pool 1 and fed with PBMCs are referred to herein as Pool 1A. Pool 2 did not receive Day 0 PBMCs. In these screens, cells not fed with PBMCs are designated by B after the pool number, e.g., screens performed using cells transduced with Pool 2 and not fed with PBMCs are referred to herein as Pool 2B. The G-Rex devices were incubated overnight at 37°C and 5% _CO2 in a standard humidified tissue culture incubator. On Day 14, cells were collected from both G-Rex devices (Pool 1A and Pool 2B) by centrifugation and resuspended in fresh complete OpTmizer ^™ CTS ^™ T Cell Expansion SFM without IL-2. Each of these cells was placed in an individual well of a 6-well G-rex dish (Wilson-Wolf; 80240M) containing 30 ml of complete OpTmizer ^™ CTS ^™ T cell expansion SFM without IL-2. A vial of thawed day 0 frozen donor-matched PBMCs was added to the well containing the pool 1A cells to provide CD19+ B cell activation for CD19 ASTR. The G-Rex device was incubated overnight at 37°C and 5% _CO2 in a standard humidified tissue culture incubator. On day 21, cells were collected from each well of the 6-well G-rex dish, and the treatment performed on day 14 was repeated. Samples (1×10 ⁵ to ~4-5×10 ⁶ cells) were collected on different days after transduction (day 7, day 21, day 28, and/or day 35), and genomic DNA was purified using GenElute ^™ mammalian genomic DNA microprep according to the kit protocol. In some cases, the sample is purified using density gradient centrifugation with Ficoll-Pacque ^TM (General Electric) to remove dead cells before genomic DNA extraction. The purified genomic DNA is sequenced using Illumina HiSeq to produce paired end 150bp readings. Typically, a subset of 10 million readings is extracted from each indexed fastq text file and analyzed using barcode_reader, which is a custom R script for extracting barcode sequences based on the presence of constant regions. The purified genomic DNA is also sequenced on the PacBio sequencing system to obtain a longer read length to associate the barcode with the construct. These screens are repeated and the library is designated as 1.1A, 1.1B, and 2.1B, where A and B indicate whether the cells as discussed above are fed with PBMC.

ANALYSE information

Once HiSeq data for all time points were obtained, the barcode count tables were merged based on the barcode ID. The counts were normalized to "counts per million" using the following formula: 1×10 ⁶ * raw counts / sum (raw counts of all barcodes in the sample), which is called the prevalence value. Barcodes with an average prevalence of 0.33 cpm or less per time point were discarded. Full-length construct-barcode associations obtained at selected time points by SMRT sequencing (Pacific Biosciences) were used to identify components of interest. Non-competent constructs that did not have 3 parts or ambiguous constructs with more than one construct cell were filtered out. The enrichment value of each component was calculated as (normalized counts at the final time point + 1) / (normalized counts at the initial time point + 1). Candidate chimeric polypeptide genes were identified as constructs with an enrichment above the threshold specific to each library.

result

The chimeric polypeptide candidate is indicated to have 3 test domains, including an extracellular and transmembrane domain (P1-2), a first intracellular domain (P3) and a second intracellular domain (P4) (Figures 16 and 17). In addition, the construct includes a DNA barcode to help analyze and identify the construct using next-generation sequencing. In addition, the most constructs shown in Table 7 include nucleic acid sequences encoding recognition and/or elimination domains in a frame with an extracellular domain and a transmembrane domain. However, the recognition and/or elimination domains of those constructs encoding the extracellular or transmembrane fragments of LMP1 are within the cells of those constructs containing such recognition and/or elimination domains. Library 1 construct encodes the CAR upstream of the chimeric polypeptide candidate. A total of 226,840 possible conceptual combinations were designed based on 20 different extracellular-transmembrane domains, 106 possible P3 domains and 107 possible P4 domains, one of which is a stop codon. After 7 days of viral vector assembly, lentiviral particle production, transduction of PBMCs and culture, not all conceptual constructs were detected. For library 1A, after transduction of PBMC and cultivation for 7 days, there are 57,815 constructs. For library 2B, after transduction of PBMC and cultivation for 7 days, there are 69,578 constructs. Detailed information on the candidates analyzed and detailed results are provided in Tables 8 to 12. Detailed information on each candidate part is provided in Table 7. The titles of Tables 8 to 12 are as follows: BlockSequence is the password of the P1-P2, P3 and P4 domains from Table 7 used in each construct; Norm_D7 is the standardized count on the 7th day; Enrich_DXX is the enrichment on the XXth day compared to the 7th day, where XX is the day indicated in the table, for example, Enrich_D21 is the enrichment on the 21st day compared to the 7th day and Enrich_D35 is the enrichment on the 35th day compared to the 7th day. The data of the construct are displayed immediately on the right row of the construct. The data of the two constructs are displayed in each column. For example, the first candidate identified in Table 8 is M008-S212-S075. For this candidate, the extracellular and transmembrane domains (P1-2) are M008, the first intracellular domain (P3) is S212, and the second intracellular domain (P4) is S075. Detailed information about the identity of these modules (e.g., M008) is found in Table 7. Table 7 reveals that M008 is LMP1 and that the construct includes a Myc tag. The amino acid sequences of the candidate domains are provided in Table 7. For clarity, the GenBank identifiers in Table 7 provide nucleic acid sequences encoding polypeptide domains. In some cases, the nucleic acid sequences are modified to aid cloning, or for codon optimization, but encode polypeptides identical to the GenBank sequences. In some cases, as apparent from the amino acid sequences provided in Table 7, portions of the polypeptides encoded by the GenBank sequences are used.

Candidate extracellular and transmembrane domains are selected from known mutant receptors, such as cytokines or hormone receptors that have been reported to be constitutively active when expressed in at least some cell types. Ligand binding domains are not included in candidate extracellular and transmembrane domains. Mutations in such receptor mutants occur in the transmembrane region or the proximal membrane region. Exemplary candidate extracellular and transmembrane domains include IL7RAIns PPCL, LMP1, CRLF2 F232C, CSF2RB V449E, CSF3R T640N, EPOR L251C I252C, GHR E260CI270C, IL27RA F523C and MPL S505N. Candidate extracellular and transmembrane domains include wild-type viral protein LMP1, which is a multi-transmembrane protein known to activate ligand-independent cell signaling when targeting lipid rafts or when fused with CD40 (Kaykas et al. EMBO J. 20: 2641 (2001)).

The identity of the potential polypeptides that select to occupy the first and second intracellular domains of the candidate chimeric polypeptide is consistent. These candidate intracellular domains are known to promote proliferation, survival (anti-apoptosis) and/or provide intracellular signaling domains of genes that provide costimulatory signals in at least some cell types, and the costimulatory signals enhance differentiation state, proliferation potential or resistance to cell death. Some of the intracellular domains of the known candidate chimeric polypeptides can activate JAK1/JAK2 signaling and STAT5. Intracellular domains from some of the genes listed in Table 7 are included in the library. Exemplary intracellular domains from these genes are provided in P3 and P4 positions in Tables 8 to 12. Detailed information about these P3 and P4 domains is provided in Table 7.

For library 1A including constructs encoding chimeric polypeptides and CARs, 172 optimal candidate chimeric polypeptides that promote PBMC proliferation to the maximum extent (and possibly survival) between day 7 and the last day indicated in the table were identified when cultured in the absence of IL-2 (see Table 8, wherein enrichment is calculated as Log2 (standardized counts on the last day indicated in the table + 1) - log2 (standardized counts on day 7 + 1)). It is worth noting that this screening experiment or all screening experiments of Example 11 and Example 12 are competitive experiments. Therefore, a negative result does not mean that the construct does not promote the proliferation of T cells in the absence of growth factors such as IL-2. It only means that relative to the other constructs tested, it is expressed better than other constructs in terms of proliferation.

For library 1A, for each extracellular and transmembrane mutant domain tested at the P1-2 position, some chimeric polypeptide constructs promoted cell proliferation between day 7 and the last day indicated on the table. When all the results of all constructs derived from the same position P1-2 mutants were combined, all tested extracellular and transmembrane domains produced a cell enrichment greater than 1 between day 7 and the last day indicated on the table (i.e., promoted cell proliferation). Examples of extracellular and/or transmembrane domains or portions and/or mutants present in constructs that promote cell proliferation are provided in Table 8. Examples of extracellular and/or transmembrane domains or portions and/or mutants present in constructs that promote cell proliferation in a repeated screening called library 1.1A are provided in Table 10. Examples of extracellular and/or transmembrane domains or portions and/or mutants present in constructs that promote cell proliferation in a repeated screening called library 1.1B (which includes cells that were not fed into PBMCs transduced with library 1) are provided in Table 11.

For Pool 1A, intracellular domains from CD3D, CD3E, CD8A, CD27, CD40, CD79B, IFNAR1, IL2RA, IL3RA, IL13RA2, TNFRSF8, and TNFRSF9, or mutants thereof, known to have signaling activity (if such mutants are present in the constructs provided in Table 8), when found to be in the first intracellular domain position (P3) of the candidate chimeric polypeptide, promoted PBMC proliferation between day 7 and the last day indicated on the table, when the data for all constructs having the first intracellular domain (P3) from that gene were considered in a combined manner. This conclusion is based on the enrichment of sequence counts of constructs in mixed cultured PBMC cell populations, when the results for all constructs of a gene are combined, such that for Pool 1A, the counts of this domain present on the last day indicated in the table are at least 3 times greater than on day 7. Examples of first intracellular domains, or portions thereof, and/or mutants present in constructs that promote the highest degree of cell proliferation are provided in Table 8. Examples of first intracellular domains or portions and/or mutants thereof present in constructs that promoted the highest degree of cell proliferation in the replicate screen designated as Pool 1.1A are provided in Table 10. Examples of first intracellular domains or portions and/or mutants thereof present in constructs that promoted cell proliferation in the replicate screen designated as Pool 1.1B (which included transduction of cells not fed PBMCs with Pool 1) are provided in Table 11.

For pool 1A, intracellular domains from CD3D, CD3Z, CD8A, CD8B, CD27, CD40, CD79B, CRLF2, FCGR2C, ICOS, IL2RA, IL13RA1, IL13RA2, IL15RA, TNFRSF9, and TNFRSF18, or mutants thereof, known to have signaling activity (if such mutants are present in the table), when found in the second intracellular domain position (P4) of the candidate chimeric polypeptide, promoted PBMC proliferation between day 7 and the last day indicated on the table, when the data for all constructs with the second intracellular domain (P4) from that gene were considered in combination. This conclusion is based on the enrichment of sequence counts of constructs in mixed cultured PBMC cell populations, when the results for all constructs of a gene are combined, such that for pool 1A, this domain count was present at least 2.9 times more on day 35 than on day 7. Examples of second intracellular domains, or portions thereof, and/or mutants present in constructs that promote the highest degree of cell proliferation are provided in Table 8. Examples of second intracellular domains or portions and/or mutants thereof present in constructs that promote cell proliferation in a replicate screen designated as Pool 1.1A are provided in Table 10. Examples of second intracellular domains or portions and/or mutants thereof present in constructs that promote cell proliferation in a replicate screen designated as Pool 1.1B (which included transduction of cells not fed PBMCs with Pool 1) are provided in Table 11.

More specifically, for library 1A, of the 5,996 constructs with MPL S505N (MPL proto-oncogene, thrombopoietin receptor with serine 505 mutated to asparagine) as the P1-P2 module, 165 were positive, defined as constructs with an enrichment greater than two for library 1A. Of the 436 constructs with CD3D (CD3d molecule) as the P3 module, 24 were positive. Of the 528 constructs with CD3E (CD3e molecule) as the P3 module, 17 were positive. Of the 261 constructs with CD8A (CD8a molecule) as the P3 module, 14 were positive. Of the 1,022 constructs with CD40 (CD40 molecule) as the P3 module, 50 were positive. Of the 568 constructs with IL3RA (interleukin 3 receptor subunit alpha) as the P3 module, 26 were positive. Of the 556 constructs with CD79B (CD79b molecule) as the P3 module, 27 were positive. Of the 131 constructs with IFNAR1 (interferon alpha and beta receptor subunit 1) as the P3 module, 7 were positive. Of the 519 constructs with IL13RA2 (interleukin 13 receptor subunit alpha 2) as the P3 module, 33 were positive. Of the 571 constructs with IL2RA (interleukin 2 receptor subunit alpha) as the P3 module, 28 were positive. Of the 618 constructs with CD27 (CD27 molecule) as the P3 module, 30 were positive. Of the 573 constructs with TNFRSF8 (TNF receptor superfamily member 8) as the P3 module, 15 were positive. Of the 573 constructs with TNFRSF9 (TNF receptor superfamily member 9) as the P3 module, 25 were positive. Of the 360 constructs with IL13RA2 (interleukin 13 receptor subunit alpha 2) as the P4 module, 25 were positive. Of the 490 constructs with IL2RA (interleukin 2 receptor subunit alpha) as the P4 module, 28 were positive. Of the 489 constructs with IL15RA (interleukin 15 receptor subunit alpha) as the P4 module, 22 were positive. Of the 590 constructs with CD8A (CD8a molecule) as the P4 module, 25 were positive. Of the 619 constructs with IL13RA1 (interleukin 13 receptor subunit alpha 1) as the P4 module, 25 were positive. Of the 637 constructs with CD3G (CD3g molecule) as the P4 module, 30 were positive. Of the 618 constructs with CD3D (CD3d molecule) as the P4 module, 37 were positive. Of the 673 constructs with CD27 (CD27 molecule) as the P4 module, 29 were positive. Of the 654 constructs with CD79B (CD79b molecule) as the P4 module, 33 were positive. Of the 728 constructs with TNFRSF9 (TNF receptor superfamily member 9) as the P4 module, 30 were positive. Of the 1,169 constructs with CD40 (CD40 molecule) as the P4 module, 53 were positive. Of the 1,324 constructs with TNFRSF18 (TNF receptor superfamily member 18) as the P4 module, 54 were positive. Of the 1,739 constructs with CD8B as the P4 module, 73 were positive. Of the 286 constructs with CRLF2 as the P4 module, 8 were positive. Of the 725 constructs with FCGR2C as the P4 module, 29 were positive. Of the 602 constructs with ICOS as the P4 module, 24 were positive.

For the purposes of the replicate screens, constructs with particularly noteworthy enrichments were those with _log2 ((normalized count data for the last day + 1)/(normalized count data for day 7 + 1)) values greater than 2 for both pools of the replicate pool. Constructs meeting this cutoff for Pools 1 and 2 are listed in Tables 20 and 21. For Pools 1A and 1.1A, constructs M001-S116-S044, M024-S192-S045, M001-S047-S102, M048-S195-S043, M012-S216-S211, and M030-S170-S194 had particularly noteworthy enrichments in both screens.

Additional information about the first and second intracellular domains in constructs with particularly noteworthy enrichment in the screens for both library 1A and library 1.1A is provided in Table 20, including the genes from which the first and second intracellular domains are derived, whether the first and/or second intracellular domains are cytokine receptors, and whether the first and/or second intracellular domains have at least one ITAM motif. Constructs with intracellular domains from IL6R, MYD88, CD27, TNFRSF18, or IL31RA were present in the first intracellular domain (P3), and constructs with intracellular domains from CD8B, IL1RL1, CD8A, TNFRSF4, or MYD88 were present in the second intracellular domain (P4), showing particularly noteworthy enrichment in both library 1A and 1.1A (Table 20). Constructs with domains from cytokine receptors IL6R, CD27, TNFRSF18, or IL31RA when present in the first intracellular domain (P3), and constructs with domains from cytokine receptors IL1RL1 or TNFRSF4 when present in the second intracellular domain (P4), showed particularly noteworthy enrichment in both pools 1A and 1.1A (Table 20).

In library 2B, which includes constructs encoding chimeric polypeptides and not encoding CARs, 167 optimal candidates that promoted PBMC proliferation between day 7 and the last day indicated on the table were identified when cultured in the absence of IL-2 (see Table 9, where enrichment was calculated as Log2 (normalized counts on the last day indicated on the table + 1) - log2 (normalized counts on day 7 + 1)). It is noteworthy that the general enrichment was less than that found for library 1A constructs (including CAR).

For Pool 2B, CSF3R T640N in the P1-2 position promoted cell proliferation better than other tested extracellular and transmembrane (P1-2) domains between day 7 and the last day indicated on the table, yielding an enrichment factor greater than 2.5 when all results from all constructs of extracellular and transmembrane domains were combined. Examples of extracellular and/or transmembrane domains or portions and/or mutants thereof present in constructs that promoted the highest degree of cell proliferation are provided in Table 9. Examples of extracellular and/or transmembrane domains or portions and/or mutants thereof present in constructs that promoted the highest degree of cell proliferation in the replicate screen designated Pool 2.1B are provided in Table 12.

For pool 2B, intracellular domains from CD8A, CD40, IFNAR1, IL31RA, and MyD88, or mutants thereof, known to have signaling activity (if such mutants are present in the constructs provided in the table), when found in the first intracellular domain position (P3) of the candidate chimeric polypeptide, promoted PBMC proliferation between day 7 and the last day indicated on the table, when the data of all constructs having the first intracellular domain (P3) from that gene were considered in combination. This conclusion is based on the enrichment of sequence counts of constructs in mixed cultured PBMC cell populations, when the results of all constructs for a gene are combined, such that for pool 2B, there are at least 2 times more counts of this domain present at day 28 than at day 7. Examples of first intracellular domains, or portions thereof, and/or mutants present in constructs that promote the greatest degree of cell proliferation are provided in Table 9. Examples of first intracellular domains, or portions thereof, and/or mutants present in constructs that promote the highest degree of cell proliferation in the repeated screens referred to as pool 2.1B are provided in Table 12.

For library 2B, the intracellular domain from CD27 or its mutants, which are known to have signaling activity (if such mutants are present in the constructs provided in the table), when found in the second intracellular domain position (P4) of the chimeric polypeptide candidate, promotes PBMC proliferation between day 7 and the last day indicated on the table, when the data of all constructs with the second intracellular domain (P4) from that gene are considered in combination. This conclusion is based on the enrichment of sequence counts of constructs in mixed cultured PBMC cell populations, when the results of all constructs of a gene are combined, so that for library 2B, the count of this domain present on day 28 is at least 11 times more than on day 7. Examples of second intracellular domains or portions thereof and/or mutants present in constructs that promote the greatest degree of cell proliferation are provided in Table 9. Examples of second intracellular domains or portions thereof and/or mutants present in constructs that promote the greatest degree of cell proliferation in the repeated screening referred to as library 2.1B are provided in Table 12.

More specifically, for library 2B, of the 6,909 constructs with CSF3R T640N (colony stimulating factor 3 receptor with threonine 640 mutated to asparagine) as the P1/P2 module of library 2B, 59 were positive, defined as constructs with an enrichment greater than two for library 2B. Of the 184 constructs with IFNAR1 (interferon alpha and beta receptor subunit 1) as the P3 module of library 2B, 3 were positive. Of the 1,258 constructs with CD40 (CD40 molecule) as the P3 module of library 2B, 17 were positive. Of the 851 constructs with CD27 (CD27 molecule) as the P4 module of library 2B, 11 were positive. Of the 418 constructs with CD8A as the P3 module of library 2B, 10 were positive. Of the 1,385 constructs with IL31RA as the P3 module of pool 2B, 12 were positive. Of the 5,757 constructs with MyD88 as the P3 module of pool 2B, 56 were positive.

For pools 2B and 2.1B, constructs M007-S049-S051, M007-S050-S039, M012-S050-S043, M012-S161-S213, M030-S142-S049, M001-S145-S130, M018-S085-S039, M018-S075-S053, M012-S135-S074, and M007-S214-S077 had particularly noteworthy enrichments in both screens. For the purposes of the replicate screens, constructs with particularly noteworthy enrichments were those with _log2 ((normalized count data on the last day + 1)/(normalized count data on day 7 + 1)) values greater than 2.

Additional information about the first and second intracellular domains in constructs with particularly noteworthy enrichment in the screen for both pool 2B and pool 2.1B is provided in Table 21, including the genes from which the first and second intracellular domains are derived, whether the first and/or second intracellular domains are cytokine receptors, and whether the first and/or second intracellular domains have at least one ITAM motif. Constructs with intracellular domains from CD28, CD40, IL22RA1, IL13RA2, IL17RB, IFNGR2, FCGR2C, IL11RA, or TNFRSF14 were present in the first intracellular domain (P3), and constructs with intracellular domains from CD40, CD3ζ, CD8A, TNFRSF9, CD28, IL10RB, CD79B, FCER1G, or GHR were present in the second intracellular domain (P4), showing particularly noteworthy enrichment in both pools 2B and 2.1B (Table 21). When constructs with domains from cytokine receptors CD40, IL22RA1, IL13RA2, IL17RB, IFNGR2, IL11RA, or TNFRSF14 were present in the first intracellular domain (P3), and when constructs with domains from cytokine receptors TNFRSF9, IL10RB, GHR, or CD40 were present in the second intracellular domain (P4), they showed particularly noteworthy enrichment in both pools 2B and 2.1B (Table 21). When constructs with domains containing ITAM motifs of FCGR2C were present in the first intracellular domain (P3), and when constructs with domains containing ITAM motifs of CD3G, CD79B, or FCER1G were present in the second intracellular domain (P4), they showed particularly noteworthy enrichment in both pools 2B and 2.1B (Table 21).

Example 12. Identification of Candidate Chimeric Polypeptide Lymphoproliferative Components.

In this example, two chimeric polypeptide pools (pool 3 and pool 4) of candidate (putative) chimeric lymphoproliferative components were assembled into viral vectors from an extracellular-transmembrane blocking sequence pool, an intracellular blocking sequence pool, and a barcode pool of constructs encoding chimeric polypeptides according to the provided in Figures 15 and 18. Proliferation of pool-transduced PBMCs was performed over time: genomic DNA was extracted from PBMCs at weekly intervals and the barcode region was amplified for DNA sequencing to estimate the number of cells in these mixed PBMC cultures containing each of the test candidate chimeric polypeptides. Thus, the test candidate chimeric polypeptides were screened for their ability to promote PBMC cell proliferation in the absence of exogenously added IL-2 or any other exogenous cytokines.

Two libraries were prepared and analyzed in this study: library 3 (which was used in the screening identified as libraries 3A, 3B, 3.1A and 3.1B) was flanked by CAR at the 5' end of the chimeric polypeptide, while library 4 (which was used in the screening identified as libraries 4B and 4.1B) did not include CAR. Figure 15 provides a schematic diagram of a non-limiting exemplary transgenic expression cassette containing a polynucleotide sequence encoding a CAR and candidate CLE of library 3 driven by an EF-1α promoter and a Kozak type sequence (GCCGCCACC (SEQ ID NO: 519)) in the backbone of a lentiviral vector. CAR is FLAG-tagged and contains an ASTR for CD19, a stem and transmembrane portion of CD8, and an intracellular activation domain from CD3z. Each candidate lymphoproliferative component of library 3 includes 4 modules: an extracellular module (P1), a transmembrane module (P2), and 2 intracellular modules (P3 and P4). The P1 module also encodes a Myc recognition and/or elimination domain. The triple stop sequence (TAATAGTGA (SEQ ID NO: 520) separated P4 from the DNA barcode (P5). The WPRE (GTCCTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTG (SEQ ID NO: 521)) was present between the last stop codon (starting 4 bp after the last nucleotide of the last stop codon) and the 3' LTR (which starts 83 nucleotides after the last nucleotide of the WPRE).

CAR and P1 of library 3 are separated by a polynucleotide sequence encoding a T2A ribosomal skipping sequence.

Figure 18 provides a schematic diagram of a non-limiting exemplary transgenic expression cassette containing a polynucleotide sequence encoding a candidate CLE of library 4 driven by the EF-1α promoter and a Kozak type sequence (GCCGCCACC (SEQ ID NO: 519)) in a lentiviral vector backbone. Each candidate lymphoproliferative element of library 4 includes four modules: an extracellular module (P1), a transmembrane module (P2), and two intracellular modules (P3 and P4). The P1 module also encodes a Myc recognition and/or elimination domain. A triple termination sequence (TAATAGTGA (SEQ ID NO: 520)) separates P4 from a DNA barcode (P5). WPRE (GTCCTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTG (SEQ ID NO:521)) is present between the last stop codon (starting 4 bp after the last nucleotide of the last stop codon) and the 3'LTR (which starts 83 nucleotides after the last nucleotide of the WPRE).

The assembled candidate chimeric polypeptide portions of the two libraries follow exactly the same design with 4 positions (1 extracellular module (P1), 1 transmembrane module (P2), and 2 intracellular modules (P3 and P4), one of which may be an early termination signal) (Figures 15 and 18). Nucleic acids encoding Myc tags or eTAG recognition and/or elimination domains (referred to as "eTAG" or "Myc Tag" in Table 7) in their entirety or in variant form are inserted at the 5' end of the CAR encoding sequence in library 3 and at the 5' end of the c-Jun domain in P1 in library 4. The signal sequence is inserted at the 5' end of the CAR encoding sequence in library 3 and at the 5' end of the recognition and/or elimination domain encoding sequence in library 4. The general design and construction of the libraries (including barcodes) are as disclosed in Example 11 herein, except for the P1 and P2 domains as described in more detail later in this Example. In addition, the clearance domain was on some constructs as indicated in Table 7 and when present was either an eTag variant or a Myc tag.

Viral vector synthesis and lentiviral production

The vectors were synthesized and lentiviral particles of each pool were produced as disclosed in Example 11.

Transduction and culture of PBMCs

For library 3A, all human blood (89.1 ml) from healthy donors was collected into 200 ml blood bags containing CDP anticoagulant (Nanger; S-200). The manufacturer's instructions and kit CS900.2 (BioSafe; 1008) were followed using Sepax 2S-100 devices (Biosafe; 14000) to process blood using density gradient centrifugation with Ficoll-Pacque ^™ (General Electric) to obtain peripheral blood mononuclear cells (PBMC). The yield of live PBMC was 8.4 × 10 ⁷ cells. Ten vials of 5 × 10 ⁶ PBMC cells per bottle were frozen for later use in CD19+B cells fed into the library 3A sample containing CD19-CAR. 3×10 ⁷ PBMCs were added to a G-Rex 100M cell culture device (Wilson Wolf, 81100S) to reach a final concentration of 0.5×10 6 viable cells/ml in a final volume of 60 ml complete OpTmizer ^™ CTS ^™ T Cell Expansion SFM (OpTmizer ^™ CTS ^™ T Cell Expansion Basal Medium 1 L (Thermo Fisher, A10221) supplemented with 26 ml OpTmizer ^™ CTS ^™ T Cell Expansion Supplement (Thermo Fisher, A10484-02), 25 ml CTS ^™ Immune Cell SR (Thermo Fisher, A2596101) and ¹⁰ ml CTS ^™ GlutaMAX ^™ -I Supplement (Thermo Fisher, A1286001) according to the manufacturer's instructions). Recombinant human interleukin-2 (IL-2) (Novoprotein) at a concentration of 100 IU/ml was also added along with an activating anti-CD3 Ab (OKT3, Novoprotein) at a concentration of 50 ng/ml to activate PBMCs for viral transduction. The G-Rex devices were incubated overnight at 37°C and 5% _CO2 in a standard humidified tissue culture incubator. After overnight incubation, a lentiviral particle preparation containing the construct encoding the chimeric polypeptide to be tested (library 3) was added to the G-Rex device at a multiplicity of infection (MOI) of 5. The G-Rex devices were incubated overnight at 37°C and 5% _CO2 in a standard humidified tissue culture incubator. After overnight incubation, additional complete OpTmizer ^™ CTS ^™ T cell expansion SFM was added to bring the final volume to 500 ml. No additional IL-2 or other exogenous cytokines were added in this or the following cell culture steps. After isolation from the PBMCs, Devices were incubated for 7 days at 37°C and 5% _CO2 in a standard humidified tissue culture incubator. On day 7, cells were collected from G-Rex devices transduced with Pool 3 by centrifugation and resuspended in fresh complete OpTmizer ^™ CTS ^™ T Cell Expansion SFM without IL-2. 5× ¹⁰⁷ Pool 3 cells were plated on a new G-Rex 100M device containing 500 ml of complete OpTmizer ^™ CTS ^™ T Cell Expansion SFM without IL-2 and labeled Pool 3A. 1.1× ¹⁰⁸ Pool 3 cells were plated on a new G-Rex 100M device containing 500 ml of complete OpTmizer ^™ CTS ^™ T Cell Expansion SFM without IL-2 and labeled Pool 3B. In these screens, cells that were not fed with PBMCs are designated by A after the library number, for example, screens performed using cells transduced with library 3 and fed with PBMCs are referred to herein as library 3A. A vial of thawed day 0 frozen donor-matched PBMCs (2×10 ⁷ PBMCs) was added to a G-Rex device containing cells transduced with library 3A to provide CD19+ B cell activation for CD19 ASTR. Library 3B does not receive day 0 PBMCs. In these screens, cells that were not fed with PBMCs are designated by B after the library number. For example, screens performed using cells transduced with library 3 and not fed with PBMCs are referred to herein as library 3B. The G-Rex device was incubated at 37°C and 5% CO ₂ in a standard humidified tissue culture incubator. On day 14, cells were collected from two G-Rex devices (bank 3A, bank 3B) by centrifugation and resuspended in fresh complete OpTmizer ^™ CTS ^™ T cell expansion SFM without IL-2. Each of these cells was placed in an individual well of a 6-well G-Rex plate (Wilson-Wolf; 80240M) containing 30 ml of complete OpTmizer ^™ CTS ^™ T cell expansion SFM without IL-2. A vial of thawed day 0 frozen donor-matched PBMCs (5×10 ⁶ PBMCs) was added to the G-Rex containing bank 3A to provide CD19+ B cell activation for CD19 ASTR. The G-Rex device was incubated at 37°C and 5% CO ₂ in a standard humidified tissue culture incubator. This process was repeated on days 21, 28, 35, and 42, except that two vials of frozen day 0 PBMCs were added to pool 3A on day 21 and one vial was added on days 28, 35, and 42. Pool 3B was not cultured for the past 21 days. These screens were repeated except that the Myc tag on the P1 portion was replaced with an eTag (as shown in Tables 7, 15, and 16) and designated as pools 3.1A and 3.1B, where the A and B indicated whether the cells were fed with PBMCs as discussed above.

Cells from pool 3A were prepared for analysis by flow cytometry (400 g, 5 minutes) at day 49. Cells were collected by centrifugation and layered on Ficoll (Ficoll-Paque Premium (GE, cat# 17-5442-02)) according to the manufacturer's instructions. 1.7 x 10 ⁶ cells were resuspended in 450 μl FACs staining buffer (554656, BD). The resuspended cells were aliquoted into 9 eppendorf tubes, each containing 50 μl or 1.8×10 ⁵ cells. 2.5 μl of human Fc block (564220, BD) was added to each tube and incubated at room temperature for 10 minutes. The following antibody formula was added to 50 μl sample to stain CD3, CD4, CD8, CD56, CD19, FLAG Tag (CAR+): 2.5 μl anti-CD3-BV421 antibody (clone OKT3, 317344, Biolegend), 2.5 μl anti-CD8-BV510 antibody (clone RPA-T8 (301048, Biolegend)), 2.5 μl anti-CD4-PE-Cy7 antibody (clone OKT4 (317412, Biolegend)), 2.5 μl anti-CD56-BV785 (362550, Biolegend) and 0.5 μl PE anti-FLAG Tag (anti-DYKDDDDK, Clone L5, 637310, Biolegend) for 5 color staining samples. 2.5 μl BB515 CD19 antibody (cloned HIB19, 564456, BD) and 0.5 ul PE anti-FLAG Tag antibody were added for 2 color staining samples. Single staining samples of CD3, CD4, CD8, CD56 or FLAGTag were also prepared using the above antibodies and volumes. 2.5 μl BV421 IgG (mouse IgG2a, 400259, Biolegend), 2.5 μl PE/CY5 IgG (mouse IgG2b, 400317, Biolegend), 2.5 μl BV510 IgG (mouse IgG1, 400171, Biolegend), 2.5 μl BV785 IgG (mouse IgG1, 400169, Biolegend) and 10 μl PE IgG (mouse IgG1, 555749, BD) were added for IgG control staining samples. No antibody was added for unstained controls. The sample was incubated on ice for 30 minutes. The sample was centrifuged (400g, 5 minutes) and the supernatant was removed. The sample was washed twice with 0.5ml FACS staining buffer. The stained cell aggregate was resuspended in 125μl FACS staining buffer and then fixed by adding 125ul BD fixation buffer (554655BD). The sample was cultivated for 15 minutes with fixation buffer at 4°C. The sample was washed twice with 500μl FACS staining buffer and resuspended with 0.4ml FACS staining buffer. FAC analysis of the sample was performed on a Novocyte flow cytometer (ACEA Biosciences) and lymphocyte gate analysis was used based on forward scatter and side scatter.

For Pool 4B, whole human blood (70 ml) from healthy donors was collected into 200 ml blood bags containing CDP anticoagulant (Nanger; S-200). Blood was processed using density gradient centrifugation with Ficoll-Pacque ^™ (General Electric) using Sepax 2 S-100 devices (Biosafe; 14000) following the manufacturer's instructions and kit CS900.2 (BioSafe; 1008) to obtain peripheral blood mononuclear cells (PBMCs). The yield of viable PBMCs was 4.9×10 ⁷ cells. 4.9×10 ⁷ PBMCs were added to a G-Rex 100M cell culture device (Wilson Wolf, 81100S) to reach a final concentration of 0.5×10 ⁶ viable cells/ml in a final volume of 98 ml of complete OpTmizer ^™ CTS ^™ T cell expansion SFM. Recombinant human interleukin-2 (IL-2) (Novoprotein) at a concentration of 100 IU/ml was also added along with an activating anti-CD3 Ab (OKT3, Novoprotein) at a concentration of 50 ng/ml to activate PBMCs for viral transduction. The G-Rex devices were incubated overnight at 37°C and 5% _CO2 in a standard humidified tissue culture incubator. After overnight incubation, a lentiviral particle preparation containing the construct encoding the chimeric polypeptide to be tested (Pool 4) was added to the G-Rex device at a multiplicity of infection (MOI) of 5. The G-Rex devices were incubated overnight at 37°C and 5% _CO2 in a standard humidified tissue culture incubator. After overnight incubation, additional complete OpTmizer ^™ CTS ^™ T Cell Expansion SFM was added to bring the final volume to 500 ml. No additional IL-2 was added in this or the following cell culture steps. After isolation from the PBMCs, Devices were incubated for 7 days at 37°C and 5% _CO2 in a standard humidified tissue culture incubator. On day 7, cells were collected from G-Rex devices transduced with pool 4 by centrifugation and resuspended in fresh complete OpTmizer ^™ CTS ^™ T Cell Expansion SFM without IL-2. 1.63× ¹⁰⁸ pool 4 cells were placed on a new G-Rex 100M device containing 500 ml of complete OpTmizer ^™ CTS ^™ T Cell Expansion SFM without IL-2 and labeled as pool 4. Pool 4 did not receive day 0 PBMCs. Therefore, in these screens, the pool was designated with B after the pool number, e.g., screens performed using cells transduced with pool 4 and not fed with PBMCs are referred to herein as pool 4B. G-Rex devices were incubated at 37°C and 5% _CO2 in a standard humidified tissue culture incubator. On day 14, cells were collected from the G-Rex device (Pool 4) by centrifugation and resuspended in fresh complete OpTmizer ^™ CTS ^™ T cell expansion SFM without IL-2. Each of these cells was placed in an individual well of a 6-well G-rex dish (Wilson-Wolf; 80240M) containing 30 ml of complete OpTmizer ^™ CTS ^™ T cell expansion SFM without IL-2. The G-Rex device was incubated at 37°C and 5% _CO2 in a standard humidified tissue culture incubator. Pool 4 samples were cultured for the past 21 days. These screens were repeated, except that the Myc tag on the P1 portion was replaced with an eTag (as shown in Tables 7 and 17) and designated as Pool 4.1B, where the B indicates that the cells were not fed with PBMCs as discussed above.

Samples (7×10 ⁴ to ～4-8×10 ⁶ cells) were collected on different days after transduction (day 7 and day 21), and genomic DNA was purified using GenElute ^TM mammalian genomic DNA micropreparation according to the kit protocol. In some cases, density gradient centrifugation using Ficoll-Pacque ^TM (General Electric) was used to purify the sample to remove dead cells before genomic DNA extraction. Purified genomic DNA was sequenced using Illumina HiSeq to generate paired-end 150bp reads. Typically, a subset of 10 million reads was extracted from each indexed fastq file and analyzed using barcode_reader, which is a custom R script for extracting barcode sequences based on the presence of constant regions. Purified genomic DNA was also sequenced on the PacBio sequencing system to associate the barcode with the construct.

ANALYSE information

Data analysis was performed as disclosed in Example 11 except for the different time points.

result

In this experiment, the chimeric polypeptide candidates were designed to have 4 test domains, including an extracellular domain (P1), a transmembrane domain (P2), a first intracellular domain (P3), and a second intracellular domain (P4) (Figures 15 and 18). As explained in Example 11, the constructs include DNA barcodes to help analyze and identify the constructs using next-generation sequencing. In addition, all constructs include nucleic acid sequences encoding recognition and/or elimination domains in a frame with an extracellular domain. The constructs in Library 3, but not Library 4, encode CARs upstream of the chimeric polypeptide candidates, which are structurally consistent with the CARs used in Library 1 (Example 11).

The extracellular domain used for library 3 and library 4 is a c-Jun variant including a leucine zipper motif (NM_002228_3) known as a dimerization motif (see, e.g., Chinenov and Kerppola, Oncogene. 2001 Apr 30; 20(19): 2438-52). Candidate chimeric polypeptides having a spacer between 0 and 4 alanine residues are included between the c-Jun extracellular domain and the transmembrane domain. Without being limited by theory, it is believed that the alanine spacer can affect signal transduction of the intracellular domain connected to the leucine extracellular domain via the transmembrane domain by changing the orientation of the connected transmembrane domain and/or intracellular domain.

Candidate transmembrane domains are selected from single transmembrane domains of known wild-type and mutant type I receptors, such as cytokines, hormones, co-stimulatory or activating receptors that have been reported to be constitutively active when expressed in at least some cell types. The mutations in such receptor mutants selected from libraries 3 and 4 occur in the transmembrane region. Exemplary transmembrane domains of libraries 3A, 3B, 3.1A, 3.1B, 4B and 4.1B can be identified in Tables 13 to 18. The titles of Tables 13 to 18 are as follows: BlockSequence is the code for the P1, P2, P3 and P4 domains from Table 7 used in each construct; Norm_D7 is the standardized count on the 7th day; Enrich_DXX is the enrichment of the XXth day versus the 7th day, where XX is the number of days indicated in the table, for example, Enrich_D21 is the enrichment of the 21st day versus the 7th day and Enrich_D35 is the enrichment of the 35th day versus the 7th day. The data of the construct is displayed immediately on the right line of the construct. Data for two constructs are shown in each column. For example, the first construct identified in Table 13 is E013-T047-S158-S080, which is described as P1-P2-P3-P4. In that construct, the transmembrane domain (P2) is T047, which is the transmembrane domain of IL7RA InsPPCL (interleukin 7 receptor), as provided in Table 7, and the amino acid sequence of this domain is included in this construct. The same first intracellular domain (P3) and second intracellular domain (P4) included in Example 11 are included in the library in this example, but one of the P3 portions is a spacer (see below).

A total of 5 possible conceptual combinations were designed based on the Jun leucine zipper extracellular domain and 0-4 threonine spacers (one of which is a 23 amino acid spacer) separated from 82 different transmembrane domains, 81 potential P3 domains, and 21 potential P4 domains (one of which is a stop codon). Not all conceptual constructs were detected after viral vector assembly, lentiviral particle production, transduction of PBMCs and culture for 7 days. For library 3A and library 3B, there were 68,951 constructs after transduction of PBMCs and culture for 7 days. For library 4B, there were 50,652 constructs after transduction of PBMCs and culture for 7 days. Detailed information about the candidates for analysis can be determined from Tables 7 and 13 to 18. The coding system of the construct is the same as that explained in Example 11, as described in the preceding paragraph.

For libraries 3A, 3B, 3.1A, 3.1B, 4B, and 4.1B, constructs that promoted PBMC cell proliferation were identified between day 7 and the last day indicated on the table. Certain polypeptides in a particular module of CLEs were among the top hits in libraries 3A, 3B, and 4. These CLEs promoted proliferation to the greatest extent. For example, exemplary constructs containing CD40 or ICOS at the P2 position; MPL, MyD88, LEPR, or IFNAR2 at the P3 position; and/or CD40, CD79B, or CD27 at the P4 position were the top 5 most common chimeric polypeptides in at least 2 of the 3 libraries 3A, 3B, and 4B (see Tables 13, 14, and 17). Clearly, MPL is the most common chimeric polypeptide at the P3 large interval for all 3 libraries. Note that the top hits from P3 (MP, MyD88, LEPR, and IFNAR2) are not included in the P4 position of these constructs.

In the constructs including encoding chimeric polypeptides and CARs and the transduced PBMCs supplemented with (library 3A) or without (library 3B) fresh untransduced PBMCs, 126 and 127 optimal candidates (see Tables 13 and 14) were identified respectively when cultured in the absence of IL-2 to promote PBMC proliferation between the 7th day and the last day indicated on the table. The optimal candidate lists of Tables 13 and 14 are generated by combining the top 100 most popular candidates on the 21st day based on the initial interim data analysis with the top 100 most enriched candidate constructs between the 21st day and the 7th day based on the initial interim data analysis. The initial interim data are generated by decoding the part of the encoded library, so that 66 of the top 100 constructs and 538 of the top 1000 constructs have been decoded for library 3A; and 77 of the top 100 constructs have been decoded for library 3B, and 464 of the top 1000 constructs.

For pool 3A, the transmembrane domain (P2) or mutants thereof from CD40, CD8B, CRLF2, CSF2RA, FCGR2C, ICOS, IFNAR1, IFNGR1, IL10RB, IL18R1, IL18RAP, IL3RA, LEPR, and PRLR are known to promote signaling activity in certain cell types (if such mutants are present in the constructs provided in the table), promoting proliferation of PBMCs between days 7 and 21, when data from all constructs with transmembrane domains from that gene are considered in combination (data not shown). This conclusion is based on the enrichment of sequence counts of constructs in mixed cultured PBMC cell populations, when the results for all constructs for a gene are combined, such that for pool 3A, the enrichment calculated as the base 2 logarithm of the ratio between the normalized counts plus one on day 21 and the normalized counts plus one on day 7 is at least 2. For Pool 3B, transmembrane domains from CD40, ICOS, CSF2RA, IL18R1, IL3RA, and TNFRSF14 were the best expressed when analyzed in this manner. Examples of transmembrane domains, or portions thereof, and/or mutants present in constructs that promote cell proliferation are provided in Tables 13 and 14 for Pools 3A and 3B at Day 35 and Day 21, respectively. Examples of transmembrane domains, or portions thereof, and/or mutants present in constructs that promote cell proliferation in the replicate screens designated Pools 3.1A and 3.1B are provided in Tables 15 and 16.

For library 3A, when promoting PBMC proliferation was found at the first intracellular domain position (P3) of the candidate chimeric polypeptide assembly herein between days 7 and 21, when the data of all constructs with the first intracellular domain from that gene were considered in a combined manner (data not shown), the first intracellular domain (P3) or mutants thereof from IL17RD, IL17RE, IL1RAP, IL23R and MPL were known to promote signaling activity in certain cell types (if such mutants were present in the constructs provided in the table). This conclusion is based on the enrichment of the sequence counts of the constructs in the mixed cultured PBMC cell population, when the results of all constructs of a gene were combined, so that for library 3A, the enrichment calculated by the logarithm of the ratio between the normalized count plus one on day 21 and the normalized count plus one on day 7 was at least 2. For library 3B, the first intracellular domains or mutants thereof from IL21R, MPL and IL2RB, which are known to promote signaling activity in certain cell types (if such mutants are present in the table), were the best expressed when analyzed in this manner. Examples of first intracellular domains or portions thereof and/or mutants present in constructs that promoted maximal cell proliferation for Pools 3A and 3B at Day 35 and Day 21, respectively, are provided in Tables 13 and 14. Examples of first intracellular domains or portions thereof and/or mutants present in constructs that promoted cell proliferation in the replicate screens designated Pools 3.1A and 3.1B are provided in Tables 15 and 16.

For library 3A, when the second intracellular domain position (P4) of the candidate chimeric polypeptide assembly herein was found to promote PBMC proliferation between days 7 and 21, when the data of all constructs with a second intracellular domain from that gene were considered in a combined manner (data not shown), the second intracellular domain (P4) or mutants thereof from CD27, CD3G, CD40, and CD79B were known to promote signaling activity in certain cell types (if such mutants were present in the table). This conclusion was based on the enrichment of the sequence counts of the constructs in the mixed cultured PBMC cell population, when the results of all constructs of a gene were combined, such that for library 3A, the enrichment calculated as the logarithm of the ratio between the normalized count plus one on day 21 and the normalized count plus one on day 7 was at least 2. For library 3B, the second intracellular domain from CD40 or mutants thereof (if such mutants were present in the table) that are known to promote signaling activity in certain cell types were the best expressed when analyzed in this manner. Examples of second intracellular domains, or portions thereof, and/or mutants present in constructs that promoted maximal cell proliferation for Pools 3A and 3B at Day 35 and Day 21, respectively, are provided in Tables 13 and 14. Examples of second intracellular domains, or portions thereof, and/or mutants present in constructs that promoted maximal cell proliferation in the replicate screens designated Pools 3.1A and 3.1B are provided in Tables 15 and 16.

Regarding specific genes, of the 798 constructs with CD8B (CD8b molecule) as the P2 portion, 111 were positive, defined as constructs with an enrichment greater than two (log2) for pool 3A or 3B on day 21. Of the 1,032 constructs with CD40 (CD40 molecule) as the P2 portion, 159 were positive. Of the 1,339 constructs with CRLF2 (cytokine receptor-like factor 2) as the P2 portion, 175 were positive. Of the 593 constructs with CSF2RA (colony stimulating factor 2 receptor alpha subunit) as the P2 portion, 108 were positive. Of the 714 constructs with FCGR2C (Fc fragment (gene/pseudogene) of IgG receptor IIc) as the P2 portion, 88 were positive. Of the 788 constructs with ICOS (inducible T cell co-stimulator) as the P2 portion, 108 were positive. Of the 882 constructs with IFNAR1 (interferon alpha and beta receptor subunit 1) as the P2 module, 106 were positive. Of the 806 constructs with IFNGR1 (interferon gamma receptor 1) as the P2 module, 104 were positive. Of the 938 constructs with IL3RA (interleukin 3 receptor subunit alpha) as the P2 module, 132 were positive. Of the 746 constructs with IL10RB (interleukin 10 receptor subunit alpha) as the P2 module, 99 were positive. Of the 814 constructs with IL18R1 (interleukin 18 receptor 1) as the P2 module, 131 were positive. Of the 552 constructs with IL18RAP (interleukin 18 receptor accessory protein) as the P2 module, 83 were positive. Of the 1,390 constructs with LEPR (leptin receptor) as the P2 module, 184 were positive. Of the 795 constructs with PRLR (prolactin receptor) as the P2 module, 123 were positive.

Of the 1,259 constructs with IL1RAP (interleukin 1 receptor accessory protein) as the P3 portion, 158 were positive, defined as constructs with an enrichment greater than two for pools 3A or 3B on day 21. Of the 200 constructs with IL17RD (interleukin 17 receptor D) as the P3 portion, 31 were positive. Of the 11 constructs with IL17RE (interleukin 17 receptor E) as the P3 module, 3 were positive. Of the 538 constructs with IL23R (interleukin 23 receptor) as the P3 module, 86 were positive. Of the 1,531 constructs with MPL (MPL proto-oncogene, thrombopoietin receptor) as the P3 module, 509 were positive.

Of the 3,124 constructs with CD3G (CD3g molecule) as the P4 portion, 458 were positive, defined as constructs with an enrichment greater than two for pools 3A or 3B on day 21. Of the 3,293 constructs with CD27 (CD27 molecule) as the P4 portion, 443 were positive. Of the 5,163 constructs with CD40 (CD40 molecule) as the P4 module, 724 were positive. Of the 4,486 constructs with CD79B (CD79b molecule) as the P4 module, 663 were positive. It is worth noting that for any construct, since this is a competitive assay, "negative" means that the construct does not compete in promoting proliferation, and non-constructs are not able to promote proliferation.

For the purpose of replicate screening, constructs with particularly noteworthy enrichments were those with _log2 ((normalized count data for the last day + 1)/(normalized count data for day 7 + 1)) values greater than 2 in both replicate screens, as listed in Tables 21 to 24 for Pool 3 and the 4 replicate pools. For Pools 3A and 3.1A, constructs E008/E013-T041-S186-S050, E006/E011-T077-S186-S211, E007/E012-T021-S186-S051, E009/E014-T041-S186-S053, E007/E012-T041-S186-S054, 73-S186-S053, E006/E011-T017-S186-S051, E006/E011-T031-S186-S211, E006/E011-T011-S186-S050, E006/E011-T011-S186-S047, E007/E012 -T00 1-S186-S050, E006/E011-T041-S186-S051, E008/E013-T028-S186-S076, E009/E014-T029-S199-S053, E009/E014-T062-S186-S216, E007/E012-T006-S058-S051, E009/E014-T076-S186-S211 and E007/E012-T001-S186-S047 had particularly noteworthy enrichments in both screens, where the P1 portions separated by slashes here include different tags for libraries 3A and 3.1A as shown in Tables 7, 13 and 15.

Additional information about the first and second intracellular domains in constructs with particularly noteworthy enrichment in the screens for both library 3A and library 3.1A is provided in Table 22, including the genes from which the first and second intracellular domains are derived, whether the first and/or second intracellular domains are cytokine receptors, and whether the first and/or second intracellular domains have at least one ITAM motif. Constructs with intracellular domains from MPL, OSMR, or CSF2RA were present in the first intracellular domain (P3), and constructs with intracellular domains from CD40, TNFRSF4, CD79B, CD27, FCGR2A, or TNFRSF18 were present in the second intracellular domain (P4), showing particularly noteworthy enrichment in both libraries 3A and 3.1A (Table 22). Constructs with domains from cytokine receptors MPL, OSMR or CSF2RA were present in the first intracellular domain (P3), and constructs with domains from cytokine receptors CD40, TNFRSF4, CD27 or TNFRSF18 were present in the second intracellular domain (P4), showing particularly noteworthy enrichment in both pools 3A and 3.1A (Table 22). Constructs with domains containing ITAM motifs of MPL or OSMR were present in the second intracellular domain (P4), showing particularly noteworthy enrichment in both pools 3A and 3.1A (Table 22).

For libraries 3B and 3.1B, constructs E007/E012-T017-S186-S051, E007/E012-T073-S186-S053, E008/E013-T028-S186-S047, E006/E011-T011-S186-S047, E007/E012-T082-S176-S214, E006/E011-T046 -S186-S052, E008/E013-T029-S186-S052, E009/E014-T011-S186-S053, E008/E013-T032-S186-S039, E007/E012-T034-S186-S051, E007/E012-T0 41-S192-S213, E006/E011-T014-S0 69-S213, E006/E011-T022-S186-S053, E006/E011-T023-S115-S075, E006/E011-T029-S106-S213, E006/E011-T032-S155-S080, E006/E011-T04 1-S186-S216, E006/E011-T057-S135- S080, E006/E011-T072-S191-X002, E006/E011-T077-S186-S216, E006/E011-T080-S141-S080, E007/E012-T001-X001-S214, E007/E012-T007-S0 59-S211, E007/E012-T016-S186-S052 . 216. E007/E012-T065-S157-S075, E0 08/E013-T008-S085-X002, E008/E013-T011-S085-S048, E008/E013-T021-S109-X002, E008/E013-T021-S168-S211, E008/E013-T032-S064-S214 , E008/E013-T037-S170-S215, E008/E 013-T038-S176-S048, E008/E013-T039-S137-S216, E008/E013-T041-S141-S053, E008/E013-T045-S177-S048, E008/E013-T048-S109-S074, E0 08/E013-T073-S199-S075, E009/E014 -T001-S157-S074, E009/E014-T005-S196-S049, E009/E014-T011-S130-X002, E009/E014-T013-S155-X002, E009/E014-T017-S186-S076, E009/E 014-T021-S142-S080, E009/E014-T02 3-S082-S076, E009/E014-T038-S196-S037, E009/E014-T055-S186-S052, E009/E014-T060-S175-S053, E009/E014-T070-S085-S212, E008/E013- T026-S054-S213, E009/E014-T007-S 120-S053, E007/E012-T045-S186-S211, E008/E013-T073-S186-X002, E008/E013-T074-S186-X002, E007/E012-T055-S186-S053, E008/E013-T0 36-S186-S053, E007/E012-T017-S058 -S053, E008/E013-T030-S189-S080, E006/E011-T029-S081-S047, E009/E014-T044-S194-S050, E006/E011-T028-S121-X002, E008/E013-T028-S1 86-S053, E009/E014-T078-S142-S2 13. E009/E014-T041-S186-S051, E008/E013-T006-S186-S050, E006/E011-T028-S186-S075, E006/E011-T040-S120-S038, E007/E012-T044-S11 5-S211, E009/E014-T039-S176-S075, E 007/E012-T028-S186-S050, E008/E013-T031-S202-S050, E007/E012-T072-S192-S053, E006/E011-T065-X001-S051, E007/E012-T030-S062-X0 02. E007/E012-T073-S186-X002, E009 /E014-T056-S186-S053、E008/E013-T046-S137-X002、E006/E011-T016-S136-S076、E007/E012-T032-S142-S037、E007/E012-T065-S120-S215、E 009/E014-T077-S186-S047, E009/E01 4-T001-S126-S051, E006/E011-T030-S121-S039, E008/E013-T006-S176-S213, E009/E014-T032-S130-S215, E008/E013-T041-S186-S039, E009/ E014-T021-S186-S047, E008/E013-T 026-S137-S214, E007/E012-T029-S116-S075, E008/E013-T026-S106-S049, and E007/E012-T032-S168-S075 had particularly noteworthy enrichments in both screens, wherein the P1 portion separated by slashes herein includes different tags of libraries 3B and 3.1B as shown in Tables 7, 14, and 16. For the purpose of replicate screens, constructs with particularly noteworthy enrichments were those with log ₂ ((normalized count data at the last day + 1)/(normalized count data at day 7 + 1))

Additional information about the first and second intracellular domains in constructs with particularly noteworthy enrichment in the screens for both library 3B and library 3.1B is provided in Table 23, including the genes from which the first and second intracellular domains are derived, whether the first and/or second intracellular domains are cytokine receptors, and whether the first and/or second intracellular domains have at least one ITAM motif. The first and/or second intracellular domains have at least one ITAM motif from MPL, LEPR, MYD88, EPOR, IL5RA, IL2RG, IL18RAP, IL11RA, IL13RA1, CSF2RA, IL1RL1, IL13RA2, IL20RB, IFNGR2, IL3RA, IL27RA, CSF3R, IL31RA, IL12RB1, OSMR, IL10RB, IFNAR2, CRLF2, IL7R, IFNAR1, PRLR, IL9R, IL6R, or IL15RA When constructs with an intracellular domain from CD40, CD79B, CD27, TNFRSF14, CD79A, CD3G, TNFRSF9, FCGR2C, ICOS, TNFRSF18, TNFRSF4, CD28, FCER1G, FCGR2A, CD3D, TNFRSF8, or CD3E were present in the second intracellular domain (P4), they showed particularly noteworthy enrichment in both pools 3B and 3.1B (Table 23). Constructs with domains from cytokine receptors MPL, LEPR, EPOR, IL5RA, IL2RG, IL18RAP, IL11RA, IL13RA1, CSF2RA, IL1RL1, IL13RA2, IL20RB, IFNGR2, IL3RA, IL27RA, CSF3R, IL31RA, IL12RB1, OSMR, IL10RB, IFNAR2, CRLF2, IL7R, IFNAR1, PRLR, IL9R, IL6R, or IL15RA when present in the first intracellular domain (P3) and constructs with domains from cytokine receptors CD40, CD27, TNFRSF14, TNFRSF9, TNFRSF18, TNFRSF4, or TNFRSF8 when present in the second intracellular domain (P4) showed particularly noteworthy enrichment in both pools 3B and 3.1B (Table 23). Constructs with domains containing the ITAM motif of CD79B, CD79A, CD3G, FCGR2C, FCER1G, FCGR2A, CD3D, or CD3E showed particularly noteworthy enrichment in both pools 3B and 3.1B when present in the second intracellular domain (P4) (Table 23).

FACs analysis showed that the expanded cells of pool 3A on day 49 were mainly CD3+, CD8+, CD56+ and FLAG-Tag+. The table immediately below shows the percentage of lymphocytes that stained positive for the specified markers and marker combinations. This data shows that the drivers identified from this pool screening mainly expanded the CD3+ T cell component.

Table 4. Percentages of lymphocytes staining positive for the indicated markers and marker combinations.

For pool 4B, which included a construct encoding a chimeric polypeptide without a CAR and transduced PBMCs that were not supplemented with fresh untransduced PBMCs, 154 top candidates were identified that promoted maximal PBMC proliferation between day 7 and the last day indicated in the table when cultured in the absence of IL-2 (see Table 17).

For pool 4B, when the transmembrane domain position (P2) of the candidate chimeric polypeptide assembly herein was found to promote cell proliferation of PBMCs between day 7 and the last day indicated on the table, the transmembrane domain (P2) or mutants thereof from CD40, CD8B, CSF2RA, IL18R1 and IL3RA are known to promote constitutive signaling activity in certain cell types (if such mutants are present in the constructs provided in the table) when the data of all constructs with transmembrane domains from that gene are considered in a combined manner. This conclusion is based on the enrichment of the sequence counts of the constructs in the mixed cultured PBMC cell population, such that when the results of all constructs for one gene are combined, for pool 4B, the enrichment calculated as the logarithm of the ratio between the normalized counts plus one on the last day indicated on the table and the normalized counts plus one on day 7 is at least 2. Examples of transmembrane domains or portions thereof and/or mutants present in constructs that promote cell proliferation in pool 4B are provided in Table 17. Examples of transmembrane domains or portions thereof and/or mutants present in constructs that promoted cell proliferation in an iterative screen designated library 4.1B are provided in Table 18.

For library 4B, when the first intracellular domain position (P3) of the candidate chimeric polypeptide assembly herein was found to promote cell proliferation of PBMCs between day 7 and the last day indicated on the table, the first intracellular domain (P3) or mutants thereof from CRLF2, CSF2RA, IFNGR2, IL4R, MPL and OSMR are known to promote signaling activity in certain cell types (if such mutants are present in the constructs provided in the table) when the data of all constructs having the first intracellular domain from that gene are considered in combination. This conclusion is based on the enrichment of the sequence counts of the constructs in the mixed cultured PBMC cell population, when the results of all constructs of a gene are combined, so that for library 4B, the enrichment calculated by the logarithm of the ratio between the normalized counts plus one on the last day indicated on the table and the normalized counts plus one on day 7 is greater than 0. Examples of first intracellular domains or portions and/or mutants thereof present in constructs that promote cell proliferation in library 4B are provided in Table 17. Examples of first intracellular domains or portions and/or mutants thereof present in constructs that promoted cell proliferation in an iterative screen designated Library 4.1B are provided in Table 18.

For library 4, when the second intracellular domain position (P4) of the candidate chimeric polypeptide assembly herein was found to promote the proliferation of PBMCs between day 7 and the last day indicated on the table, the second intracellular domain (P4) or mutants thereof from CD27, CD40 and CD79B were known to promote signal transduction activity in certain cell types (if such mutants are present in the constructs provided in the table) when the data of all constructs with the second intracellular domain from that gene were considered in a combined manner. This conclusion is based on the enrichment of sequence counts of constructs in mixed cultured PBMC cell populations, when the results of all constructs of a gene are combined, so that for library 4B, the count of this domain present on the last day is more than on day 7. Examples of second intracellular domains or portions and/or mutants present in constructs that promote cell proliferation in library 4B are provided in Table 17. Examples of second intracellular domains or portions and/or mutants present in constructs that promote cell proliferation in a repeated screen called library 4.1B are provided in Table 18.

For libraries 4B and 4.1B, constructs E007/E012-T078-S154-S047, E008/E013-T062-S186-X002, E008/E013-T055-S186-S050, E009/E014-T057-S186-S050, E007/E012-T077-S054-S053, E007/E01 2-T034-S135-S211, E009/E014-T071-X001-S216, E009/E014-T011-S141-S037, E008/E013-T041-S186-S037, E006/E011-T038-S106-S039, E006 /E011-T011-S121-X002、E007/E 012-T007-S085-S215, E006/E011-T041-S186-S050, E007/E012-T008-S064-S051, E006/E011-T041-S186-S047, E008/E013-T045-S186-S051, E00 8/E013-T003-S104-S216, E00 6/E011-T019-S186-S053, E008/E013-T071-S064-S080, E006/E011-T021-S054-X002, E006/E011-T003-S135-X002, E009/E014-T020-S199-S213 ,E008/E013-T027-S121-S211,E 009/E014-T032-S195-X002, E009/E014-T050-S171-X002, E008/E013-T069-S083-X002, E008/E013-T026-S116-S038, E009/E014-T072-S195-X0 02. E007/E012-T047-S058-S21 1. E008/E013-T046-S142-S080, E006/E011-T065-S186-S076, E006/E011-T062-S069-X002, E007/E012-T047-S098-X002, E009/E014-T069-S099 -S048, E008/E013-T039-S141-S 050, E006/E011-T052-S130-S052, E008/E013-T041-S186-X002, E007/E012-T019-S120-X002, E008/E013-T045-S186-S053, E006/E011-T003-S17 0-S039, E007/E012-T047-S05 8-S051, E007/E012-T069-S109-X002, E009/E014-T019-S130-S075, and E006/E011-T047-S054-S053 had particularly noteworthy enrichments in both screens, wherein the P1 portion separated by slashes herein included different tags of libraries 34B and 4.1B as shown in Tables 7, 17, and 18. For the purpose of the replicate screens, constructs with particularly noteworthy enrichments were those with _log2 ((normalized count data on the last day + 1)/(normalized count data on day 7 + 1)) values greater than 2.

Additional information about the first and second intracellular domains in constructs with particularly noteworthy enrichment in the screens for both library 4B and library 4.1B is provided in Table 24, including the genes from which the first and second intracellular domains are derived, whether the first and/or second intracellular domains are cytokine receptors, and whether the first and/or second intracellular domains have at least one ITAM motif. Constructs with an intracellular domain from IL18R1, MPL, CRLF2, IL11RA, IL13RA1, IL2RG, IL7R, IFNGR2, CSF3R, IL2RA, OSMR, MYD88, IL31RA, IFNAR2, IL6R, CSF2RA, IL13RA2, EPOR, IL1R1, IL10RB, or IL3RA when present in the first intracellular domain (P3) and constructs with an intracellular domain from CD27, CD40, CD79B, TNFRSF4, TNFRSF18, CD3D, CD3G, CD27, ICOS, TNFRSF9, CD3E, FCGR2A, CD28, CD79A, or FCGR2C when present in the second intracellular domain (P4) showed particularly noteworthy enrichment in both pools 4B and 4.1B (Table 24). Constructs with an intracellular domain from IL18R1, MPL, CRLF2, IL11RA, IL13RA1, IL2RG, IL7R, IFNGR2, CSF3R, IL2RA, OSMR, IL31RA, IFNAR2, IL6R, CSF2RA, IL13RA2, EPOR, IL1R1, IL10RB, or IL3RA when present in the first intracellular domain (P3) and constructs with an intracellular domain from CD27, CD40, TNFRSF4, TNFRSF18, or TNFRSF9 when present in the second intracellular domain (P4) showed particularly noteworthy enrichment in both pools 4B and 4.1B (Table 24). Constructs with domains containing the ITAM motif of CD79B, CD3D, CD3G, CD3E, FCGR2A, CD79A or FCGR2C showed particularly noteworthy enrichment in both pools 4B and 4.1B when present in the second intracellular domain (P4) (Table 24).

For library 4B, of the 572 constructs with CD8B (CD8b molecule) as the P2 portion, 21 were positive, defined as constructs with an enrichment greater than two for library 4B. Of the 897 constructs with CD40 (CD40 molecule) as the P2 module, 50 were positive. Of the 551 constructs with CSF2RA (colony stimulating factor 2 receptor alpha subunit) as the P2 module, 25 were positive. Of the 732 constructs with IL3RA (interleukin 3 receptor subunit alpha) as the P2 module, 47 were positive. Of the 738 constructs with IL18R1 (interleukin 18 receptor 1) as the P2 module, 44 were positive. Of the 1,683 constructs with MPL (MPL proto-oncogene, thrombopoietin receptor) as the P3 module, 305 were positive.

In the initial interim analysis of library 3A on the 21st day, 66 of the first 100 hits and 538 of the first 1000 hits have been decoded. Further decoding provides depth decoding and identification of 99 of the first 100 hits and 992 of the first 1000 hits of library 3A and collects data on the 35th day and updates analysis on the 21st day. In the initial analysis of library 3B on the 21st day, 77 of the first 100 hits and 464 of the first 1000 hits have been decoded in the initial interim analysis. Further decoding provides depth decoding and identification of 100 of the first 100 hits and 708 of the first 1000 hits of library 3B. The optimal constructs of library 3A and 3B and the 35th day data of library 3A and the 21st day data of library 3B are respectively displayed in Tables 13 and 14.

In pools 3A and 3B comprising constructs encoding chimeric polypeptides and CARs and transduced PBMCs supplemented with (pool 3A) or without (pool 3B) fresh untransduced PBMCs, 124 top candidates for pool 3A on day 35 that promoted maximal PBMC proliferation between day 7 and the last day indicated on the table were identified when cultured in the absence of IL-2 (i.e., IL-2 was not added to the culture medium during culture after the initial transduction), and 131 top candidates for pool 3B on day 21 were identified (see Tables 13 and 14, respectively). The optimal candidate lists of Table 13 (Pool 3A at day 35) and Table 14 (Pool 3B at day 21) were generated by combining the top 100 most popular candidates at day 21 (Pool 3B) or day 21 or 35 (Pool 3A) with the top 100 most enriched candidate constructs between day 21 and day 7 (Pool 3B) or between day 21 and day 7 or between day 35 and day 7 (Pool 3A) after deep decoding.

From the top hits table using the deep decoding results, certain polypeptides in a particular module of CLE are among the top hits in at least one of library 3A and library 3B and at least one of the time points of day 21 and day 35 (library 3A) or day 21 (library 3B). For example, exemplary constructs containing the following are among the top 5 most common chimeric polypeptides (see Tables 13 and 14): in the P2 position, CD40 (specifically, for example, a transmembrane domain with codon T011), ICOS (specifically, for example, a transmembrane domain with codon T028), FCGR2C (specifically, for example, a transmembrane domain with codon T024), PRLR (specifically, for example, a transmembrane domain with codon T077), IL3RA (specifically, for example, a transmembrane domain with codon T041), IL6ST (specifically, for example, a transmembrane domain with codon T0 in the P2 position, IL18R1 (specifically, for example, a transmembrane domain with codon T062); in the P3 position, MPL (specifically, for example, an intracellular domain with codon S186), IL18R1 (specifically, for example, an intracellular domain with codon S154), IL13RA2 (specifically, for example, an intracellular domain with codon S142), IL10RB (specifically, for example, an intracellular domain with codon S130), LEPR (specifically, for example, an intracellular domain with codon S176), IFNAR2 (specifically, for example, an intracellular domain with codon S177), S083), IL23R (specifically, for example, an intracellular domain with codon S165), MyD88 (specifically, for example, an intracellular domain with codon S194, and CSF2RA (specifically, for example, an intracellular domain with codon S058); and/or in position P4, CD40 (specifically, for example, an intracellular domain with codon S050 or S051), CD79B (specifically, for example, an intracellular domain with codon S053), TNFRSF4 (specifically, for example, an intracellular domain with codon S211), TNFRSF9 (specifically, for example, an intracellular domain with codon S214), The present invention discloses a chimeric polypeptide comprising a polypeptide having a P4 position in the P3 region of the constructs of the invention. The present invention discloses a chimeric polypeptide comprising a polypeptide having a P4 position in the P3 region of the constructs of the invention. The present invention discloses a chimeric polypeptide comprising a polypeptide having a P4 position in the P3 region of the constructs of the invention. The present invention discloses a chimeric polypeptide comprising a polypeptide having a P4 position in the P3 region of the constructs of the invention. The present invention discloses a chimeric polypeptide comprising a polypeptide having a P4 position in the P3 region of the constructs of the invention. The present invention discloses a chimeric polypeptide comprising a polypeptide having a P4 position in the P3 region of the constructs of the invention.

The following additional observations are noteworthy: P2_CD40, P2_ICOS, P3_MPL, P4_CD40, and P4_CD79B appear as the most common fraction among the top hits in each of Tables 13 and 14. P4_CD27 appears as the most common fraction in Tables 13 and 14. CSF2RB appears 4 times in the top 123 most enriched and is most repeated at P2, but mutation V449E does not appear in these top hits (Table 13). Therefore, in some embodiments herein, the chimeric polypeptide comprises a transmembrane domain identified in any of the constructs shown in Tables 13 and 14, except for the transmembrane domain mutation V449E of CSF2RB. Eight MyD88 variants appear at least once at the P3 position in the top 123 of Library 3A (Table 13). Two variants of CD40 tested in the library appear at least once at the P4 position in the top 123 list of Library 3A (Table 13). The combination of MPL at P3 and CD40 at P4 was represented in 26 constructs in the list of the top 123 constructs of library 3A (Table 13).

For genetic analysis of genes, for Pool 3A, the transmembrane domains from CD4, CD8B, CD40, CRLF2, CSF2RA, CSF3R, EPOR, FCGR2C, GHR, ICOS, IFNAR1, IFNGR1, IFNGR2, IL1R1, IL1RAP, IL2RG, IL3RA, IL5RA, IL6ST, IL7RA, IL10RB, IL11RA, IL13RA2, IL17RA, IL17RB, IL17RC, IL17RE, IL18R1, IL18RAP, IL20RA, IL22RA1, IL31RA, LEPR, PRLR, and TNFRSF8, which are known to promote signaling activity in certain cell types (P2) or mutants thereof (if such mutants are present in the constructs provided in the table), promoted proliferation of PBMCs between day 7 and the last day indicated when the data for all constructs with a transmembrane domain from that gene were considered in combination (Table 13). This conclusion is based on the enrichment of sequence counts of constructs in mixed cultured PBMC cell populations, such that when the results for all constructs for one gene are combined, for library 3A, the enrichment calculated as the base 2 logarithm of the ratio between the normalized counts plus one on the last day indicated on the table and the normalized counts plus one on day 7 is at least 2. For library 3B (Table 14), transmembrane domains from CD40, ICOS, and IL18R1, or mutants thereof (if such mutants are present in the table), which are known to promote signaling activity in certain cell types, are the best expressed when analyzed in this manner. For libraries 3A and 3B, examples of transmembrane domains or portions thereof and/or mutants present in constructs that promote the greatest degree of cell proliferation are provided in Tables 13 and 14. Examples of transmembrane domains or portions thereof and/or mutants present in constructs that promote cell proliferation in the replicate screens referred to as libraries 3.1A and 3.1B are provided in Tables 15 and 16.

For library 3A, the first intracellular domain (P3) or mutants thereof (if such mutants are present in the constructs provided in the table) from CSF2RA, IFNAR1, IL1RAP, IL4R, IL6ST, IL11RA, IL12RB2, IL17RA, IL17RD, IL17RE, IL18R1, IL21R, IL23R, MPL and MyD88, which are known to promote signaling activity in certain cell types, when found in the first intracellular domain position (P3) of the candidate chimeric polypeptide components herein, promoted cell proliferation of PBMCs between day 7 and the last day indicated on the table, when the data of all constructs having the first intracellular domain from that gene were considered in a combined manner. This conclusion is based on the enrichment of the sequence counts of the constructs in the mixed cultured PBMC cell population, such that when the results of all constructs for one gene are combined, for library 3A, the enrichment calculated as the logarithm of the ratio between the normalized counts plus one on the last day indicated on the table and the normalized counts plus one on day 7 is at least 2. For pool 3B, first intracellular domains from CSF2RB, IL4R, and MPL, or mutants thereof, known to promote signaling activity in certain cell types, if such mutants are present in the table, were the best expressed when analyzed in this manner. For pools 3A and 3B, examples of first intracellular domains, or portions thereof, and/or mutants present in constructs that promote the greatest degree of cell proliferation are provided in Tables 13 and 14. Examples of first intracellular domains, or portions thereof, and/or mutants present in constructs that promote cell proliferation in replicate screens designated as pools 3.1A and 3.1B are provided in Tables 15 and 16.

For library 3A, the second intracellular domain (P4) or mutants thereof from CD3D, CD3G, CD27, CD40, CD79A, CD79B, FCER1G, FCGRA2, ICOS, TNFRSF4 and TNFRSF8, which are known to promote signaling activity in certain cell types, if such mutants are present in the constructs provided in the table, are found to be in the second intracellular domain position (P4) of the candidate chimeric polypeptide assembly herein, and promote the proliferation of PBMCs between days 7 and 21 or between days 7 and the last day indicated on the table, when the data of all constructs having a second intracellular domain from that gene are considered in combination. This conclusion is based on the enrichment of the sequence counts of the constructs in the mixed cultured PBMC cell population, such that when the results of all constructs are combined for one gene, for library 3A, the enrichment calculated by the logarithm of the ratio between the normalized counts plus one on the last day indicated on the table and the normalized counts plus one on day 7 is at least 2. For Pool 3B, a second intracellular domain from CD40 or a mutant thereof, if such a mutant is present in the table, known to promote signaling activity in certain cell types, was the best expressed when analyzed in this manner, although a 2-fold log2 enrichment was not obtained. For Pools 3A and 3B, examples of second intracellular domains, or portions thereof and/or mutants, present in constructs that promoted the greatest degree of cell proliferation are provided in Tables 13 and 14. Examples of second intracellular domains, or portions thereof and/or mutants, present in constructs that promoted cell proliferation in replicate screens designated Pools 3.1A and 3.1B are provided in Tables 15 and 16.

Example 13. Characterization of individual lymphoproliferative components

In this example, the selected chimeric lymphoproliferative element (CLE) identified in the library screening described in Example 11 and Example 12 is evaluated individually. To further analyze the CLE identified in the library 2B screening, activated PBMCs are transduced overnight with lentiviral particles encoding a single CLE and cultured in the absence of exogenous cytokines. To further analyze the CLE identified in the library 3A screening, activated PBMCs are transduced overnight with lentiviral particles encoding CLE flanked with anti-CD19 CAR at the 5' end, and are cultured in the presence of donor-matched CD19+B cells added to the culture every 7 days, but in the absence of exogenous cytokines. The cells are cultured for up to 35 days to assess the ability of CLE to promote PBMC proliferation. This example further provides a method for characterizing any individual putative lymphoproliferative element and further confirms the identity of several highly active CLEs.

Recombinant retroviral particles were produced in 293T cells (Lenti-X ^™ 293T, Clontech) adapted for suspension culture in Freestyle ^™ 293 expression medium (ThermoFisher Scientific) serum-free chemically defined medium. The cells were transfected with PEI with genomic plasmid and 3 lentiviral packaging plasmids encoding gag/pol, rev and pseudotyped plasmids encoding as explained in Example 4. The selected CLEs identified in the library screening described in Examples 11 and 12 were regenerated from their portions (P1-2, P3 and P4 or P1, P2, P3 and P4) respectively and inserted into the same transgenic expression cassettes as the library from which they were first identified. The modular portions of each of the selected CLEs are shown in Figures 19 and 20 and the gene names and amino acid sequences are shown in Table 7. Figures 17 and 15 show the cassettes for library 2 and library 3, respectively. Cells transduced with lentiviral particles containing constructs identified by library screening are referred to as "DL" followed by the library number. For example, cells transduced with lentiviral particles containing constructs from library 2 are referred to as DL2. Thus, CLEs with the prefixes "DL2" and "DL3" were constructed in the cassettes shown in Figures 17 and 15, respectively. Similarly, "DC1-5" and "DC1-6" comprise CLEs inserted into the transgene expression cassette used for library 1 as shown in Figure 16. The chimeric lymphoproliferative element of DC1-5 encodes, from amino to carboxy terminus: residues 21-458 of human IL-7 (SEQ ID NO: 511), a flexible linker (SEQ ID NO: 53), and human IL-7Rα (SEQ ID NO: 229), which is the full-length IL-7Rα without its signal peptide. The CLE of DC1-6 encodes from amino to carboxyl terminus: the extracellular and transmembrane portions of IL-7 (SEQ ID NO: 511), a flexible linker (SEQ ID NO: 53), IL-7Rα (CD127) (SEQ ID NO: 513), and the intracellular domain of IL-2Rβ (CD122) (SEQ ID NO: 514). Chimeric proteins IL-7–IL-7Rα and IL-7–IL-7Rα–IL-2Rβ have been described previously (Hunter et al. Molecular Immunology 56 (2013): 1-11). IL-7 "DC2-5" and "DC2-6" comprise the CLE inserted into the transgenic expression cassette used in library 2 as shown in FIG. 17. The CLE in DC2-5 and DC2-6 is the same CLE as DC1-5 and DC1-6, respectively. Non-transduced PBMCs (NT), PBMCs transduced with a vector encoding eTag instead of CLE (F1-0-01), and PBMCs transduced with a vector encoding anti-CD19 CAR and eTag instead of CLE (F1-3-23) were included as negative controls.

The constructs from library 2B used in this example were DL2-1 (M024-S190-S047), DL2-2 (M025-S050-S197), DL2-3 (M036-S170-S047), DL2-4 (M012-S045-S048), DL2-5 (M049-S194-S064), DL2-6 (M025-S190-S050), and DL2-7 (M025-S190-S051).

The constructs from library 3A used in this example were DL3A-1 (E013-T047-S158-S080), DL3A-2 (E011-T024-S194-S039), DL3A-3 (E014-T040-S135-S076), DL3A-4 (E013-T041-S186-S051), DL3A-5 (E013-T064-S05 8-S212), DL3A-6 (E013-T028-S186-S051), DL3A-7 (E014-T015-S186-S051), DL3A-8 (E011-T016-S186-S050), DL3A-9 (E011-T073-S186-S050) and DL3A-10 (E013-T011-S186-S211).

On day 0, the cells were separated by Ficoll-Paque PBMCs were enriched from the buffy coat (San Diego Blood Bank) by density gradient centrifugation followed by lysis of erythrocytes (GE Healthcare Life Sciences). 1.5×10 ⁶ PBMCs were seeded in each well of a G-Rex 6-well plate (WilsonWolf, 80240M) in 3 ml complete OpTmizer ^™ CTS ^™ T cell expansion SFM supplemented with 100 IU/ml (IL-2) and 50 ng/ml anti-CD3 antibody (317326, Biolegend) to activate PBMCs for viral transduction. After overnight transduction at 37° C. and 5% CO ₂ , the lentiviral particles encoding the constructs described above were added directly to the activated PBMCs at an MOI of 5 and incubated overnight at 37° C. and 5% CO _2. The next day, the medium volume in each well was brought to 30 ml with complete OpTmizer ^™ CTS ^™ T cell expansion SFM and the plate was returned to the incubator. No IL-2, IL-7 or other exogenous cytokines were added during this or the following cell culture steps. Cells from each well were collected on day 7 to determine cell number, percent viability and percent transduced cells, which were defined as the percentage of FLAG-Tag+ or E-Tag+ cells by FACS analysis. The cells were then centrifuged, resuspended in fresh complete OpTmizer ^™ CTS ^™ T Cell Expansion SFM, and 0.5×10 ⁶ cells from each sample were re-seeded into the wells of a G-Rex 6-well plate in 30 ml complete OpTmizer ^™ CTS ^™ T Cell Expansion SFM. Previously frozen day 0 donor-matched PBMCs containing 0.5×10 ⁶ CD19+ B cells (as determined by FACS on day 0) were added to the "DL3A-" culture to provide CD19+ B cell activation to CD19 ASTR. This process was repeated on days 14, 20, and 28 before harvesting cells on day 35.

result

PBMCs transduced with lentiviral particles encoding CLE were cultured for 35 days in the absence of exogenous cytokines. Proliferation was expressed as fold expansion, which was calculated by dividing the total number of cells by the number of cells seeded at that time point. PBMCs transduced with each of the individually selected CLEs shown in FIG19 proliferated more than negative controls, which included untransduced PBMCs and PBMCs transduced with vectors encoding eTag instead of CLE ( FIG19 ). In addition, the following CLEs exhibited fold expansions greater than 23 on days 14, 21, 28, and 35, while the fold expansions of the negative controls were almost 0 for these time points: DL2-7, DC2-6, DL2-6, DL2-2, DL2-1, and DC2-5. PBMCs cultured in the presence of fresh donor-matched PBMCs added every 7 days, but in the absence of exogenous cytokines, were transduced with constructs encoding anti-CD19 CAR and different CLEs (shown in FIG20 ) and proliferated more than negative controls, including untransduced PBMCs and PBMCs transduced with vectors encoding anti-CD19 CAR and eTag instead of CLEs. All CLEs shown in FIG20 exhibited a fold expansion of greater than 23 on day 35, while the fold expansion of the negative control was almost 0 on day 35. The proliferation of PBMCs transduced with lentiviral particles containing polynucleotides encoding DL3A-1 (E013-T047-S158-S080), DL3A-2 (E011-T024-S194-S039), DL3A-3 (E014-T040-S135-S076), and DL3A-5 (E013-T064-S058-S212) was comparable to the negative control and is not shown in Figure 20. The expression of these CLEs in cells transduced with retroviral particles whose genomes encode these CLEs was not analyzed to confirm that they were indeed expressed.

Example 14. Expression of CLE that promotes survival and/or expansion of PBMCs.

In this example, when cultured in vitro for 21 days in the absence of exogenously added cytokines, 14 unique recombinant lentiviral particles exposed to unstimulated human PBMCs for 4 hours were assessed individually for their ability to promote transduction and subsequent survival and/or proliferation of PBMCs. The retroviral particles in this example were pseudotyped with VSV-G, expressed anti-CD3scFvFc-GPI on their surface, and contained transgenic expression cassettes encoding anti-CD19 CAR and 1 of 14 unique CLEs. Donors of matching PBMCs were added to the culture to provide CD19+B cell activation of CD19 CAR. No exogenous cytokines were added to the sample at any point in this example.

method

Recombinant lentiviral particles were produced in 293T cells (Lenti-X ^™ 293T, Clontech) adapted to suspension culture in Freestyle ^™ 293 expression medium (Thermo Fisher Scientific) serum-free chemically defined medium. Cells were transiently transfected using PEI with a genomic plasmid and separate packaging plasmids encoding gag/pol, rev, a pseudotyped plasmid encoding VSV-G, and a plasmid encoding UCHT1 scFvFc-GPI as described in Example 4. The genomic plasmid included a Kozak sequence, a CD8a signal peptide, a FLAG tag, and anti-CD19:CD8:CD3z CAR, T2A and CLE, followed by a triple termination sequence, as shown in FIG15 . The anti-CD19 CAR lacks the co-stimulatory domain. Thirteen different CLEs were identified as being more highly enriched or more ubiquitous in library 3.1.1A at day 35, and 1 CLE from library 3A was identified to be regenerated solely from the following parts thereof (P1, P2, P3, and P4): DL3.1A-1 (E006-T074-S194-S211), DL3.1A-2 (E010-T073-S186-S211), DL3.1A-3 (E009-T049-S106-S037), DL3.1A-4 (E009-T073-S190-S215), DL3.1A-5 (E009-T009-S165-S052), DL3.1A-6 (E009-T066-S167-S211). 8-S053), DL3.1A-7(E010-T024-S197-S214), DL3.1A-8(E009-T072-S186-S039), DL3.1A-9(E009-T038-S105-S050), DL3.1A-10(E006-T057-S105-S0 39), DL3.1A-11 (E006-T077-S069-S050), DL3.1A-12 (E009-T071-S186-S211), DL3.1A-13 (E009-T032-S105-S048) and DL3A-4 (E013-T041-S186-S051). Another genomic plasmid including the Kozak sequence, CD8a signal peptide, FLAG tag, anti-CD19:CD8:CD3z CAR, T2A and triple terminator sequence but lacking CLE (F1-3-253) was included as a control. Lentiviral particles encoding a genomic vector in which CLE is DL3A-4 but does not express UCHT1 scFvFc-GPI on its surface were also used as a control in this experiment.

On day 0, the cells were separated by Ficoll-Paque (GE Healthcare Life Sciences) Density gradient centrifugation followed by lysis of red blood cells enriched PBMCs from a leukocyte reduction system (LRS-WBC) (San Diego Blood Bank). . No additional steps were taken to remove monocytes. A portion of the cells from each donor was frozen at 2×10 ⁷ PBMCs per bottle for later use in feeding CD19-CAR expressing cells. Cells from each donor were also left for expression analysis by FAC. The remaining PBMCs were diluted to 1.0×10 ⁶ viable cells/ml in complete OpTmizerTM CTSTM T cell expansion SFM, and 5 ml was added to a 50 ml conical tube for each sample. Anti-CD3, anti-CD28, IL-2, IL-7 or other exogenous cytokines were not added before transduction to activate or otherwise stimulate lymphocytes. Lentiviral particles were added directly to unstimulated PBMCs in conical tubes at an MOI of 1 and incubated for 4 hours at 37°C and 5% CO ₂ . Cells were then washed 3 times in DPBS + 2% HSA before resuspending in 30 ml complete OpTmizer™ CTS™ T Cell Expansion SFM and transferred to 6-well gas permeable cell culture device (Wilson Wolf). Cells from each well were collected on day 7 to determine cell number, percent viability, and percent transduced cells, which was defined as the percentage of FLAG-Tag+ cells by FACS analysis using a lymphocyte gate. Half of the cells from each transduction were plated 6-well plates. Pre-refrigerated day 0 donor-matched PBMCs were added to each sample at a ratio of 1 transduced cell to 1 day 0 donor-matched CD19+B cell (the percentage of CD19+B cells in PBMCs was determined by FACs on day 0). Complete OpTmizer™ CTS™ T cell expansion SFM was then added to each well to bring the volume to 30 ml, and the transduced cells were cultured at 37°C and 5% _CO2 . On day 14, 15 ml of spent culture medium was replaced with fresh culture medium, and additional day 0 PBMCs were added at the same 1:1 ratio. Cells were assayed again on days 14 and 21 to determine cell number, percent viability, and expression of surface markers by FACS.

result

The lentiviral particles in this example are pseudotyped with VSV-G, express anti-CD3scFvFc-GPI on their surface, and contain transgenic expression cassettes encoding anti-CD19 CAR and 1 of 14 unique CLEs. Before washing the cells 3×, the lentivirus was exposed to unstimulated and presumably resting PBMCs for 4 hours and cultured in the absence of any exogenously added cytokines. Analysis of PBMCs on day 7 confirmed that each of the lentiviral particles including CLE and expressing anti-CD3scFvFc-GPI was effective in transducing PBMCs. In addition, analysis of the total number of CART cells on days 7, 14, and 21 showed that PBMCs transduced with constructs encoding anti-CD19CAR and CLE were able to better promote in vitro survival and/or expansion of CAR+ cells compared to control constructs encoding anti-CD19 CAR but not CLE (F1-3-253). The following table (Table 5) shows the total number of CAR+ cells transduced by the indicated constructs normalized to the total number of CAR+ cells transduced by F1-3-253 on days 7, 14, and 21. The following table also shows the percentage increase in the number of CAR+ cells transduced for each construct between days 7 and 14 and between days 7 and 21.

Table 5. Cell count analysis.

As supported by the data shown in Table 5, transduction of PBMCs with anti-CD19 CAR and the tested CLE provided improved expansion and/or survival at days 7, 14, and 21 compared to untransduced control cells or control cells transduced with lentiviral particles without CLE.

CLEs of particular note in this example for their ability to promote survival and proliferation of transduced PBMCs were DL3.1A-2 (E010-T073-S186-S211), DL3.1A-7 (E010-T024-S197-S214), DL3.1A-8 (E009-T072-S186-S039), DL3.1A-12 (E009-T071-S186-S211), and DL3A-4 (E013-T041-S186-S051).

Example 15. Peripheral blood mononuclear cell (PBMC) isolation, transduction and expansion.

The following examples illustrate the purposes of the closed system for ex vivo processing PBMC before in vivo expansion. As an example, 30ml to 200ml of human blood is extracted from an individual in a blood collection bag using acid citrate dextrose solution (ACD) as an anticoagulant. Alternatively, blood is drawn into a blood collection tube, a syringe or an equivalent and transferred to an empty blood collection bag or an IV bag. According to the manufacturer's instructions, a pure cell kit (catalog number #CS-900.2, Omniamed) is used on Sepax 2 cell processing systems (BioSafe) to process whole blood. Peripheral blood mononuclear cells (PBMC) are collected in a culture bag, or alternatively in a syringe. Aseptically, aliquots are collected for cell counting to determine the number of viable cells. PBMCs at a final concentration of 0.1×10 6 to 1.0×10 6 viable cells/ml were transferred to a G-Rex100 MCS gas permeable cell culture system device (Wilson Wolf) in X-VIVO 15 (Cat. No. 08-879H, Lonza) or CTS OpTmizer Cell Expansion SFM (Cat. No. A1048501, Thermo Fisher Scientific) medium with ¹⁰ IU/ml to 300 IU/ml IL-2 (Cat. No. 202-IL-010, R&D Systems) in a final volume of up to ²⁰⁰ ml. In addition to IL-2, CTS Immune Cell SR (Cat. No. A2596101, Thermo Fisher Scientific) can be added to the culture medium. Closed G-Rex gas permeable cell culture system devices can be pre-coated with fibronectin (Retronectin) (Catalog #CH-296, Takara) or a similar fibronectin-derived equivalent according to the manufacturer's instructions.

PBMCs isolated from peripheral blood were loaded onto PALL PBMC filters, washed once with 10 ml AIM V (Thermo Fisher Scientific) or X-VIVO 15 medium through the filter, and then perfused with 10 ml to 60 ml lentiviral stock (as prepared in Example 2) at 5 ml/hour at 37°C. The PBMCs were then washed again with AIM V, CTS OpTmizer T cell expansion SFM or X-VIVO 15 medium containing recombinant human DNase (Pulmozyme, Genentech), followed by washing with DNase-free Ringer's lactate (Lactated Ringers) (Catalog # L7500, Braun). The PBMCs were then perfused back into the syringe through the filter. The cells (target level of cells was 5×10 ⁵ to 1×10 ⁶ cells/kg) were then re-infused into the individual via intravenous infusion.

Depending on the riboswitch contained in the retroviral genome, a respective nucleoside analog antiviral drug or nucleoside analog antiviral prodrug (acyclovir, valacyclovir, penciclovir and famciclovir) is administered to the individual. Any therapeutically effective dose (such as 500 mg of nucleoside analog antiviral drug or prodrug) can be administered to the individual orally three times a day. Treatment with nucleoside analog antiviral drugs or prodrugs is preferably started before reinfusion (such as 2 hours before), and can also be started at the time of reinfusion or after some time of reinfusion. Treatment can continue for at least 1, 2, 3, 4, 5, 7, 10, 14, 21, 28, 30, 60, 90, 120 days or 5, 6, 9, 12, 24, 36 or 48 months or longer. Treatment may include administering nucleoside analog antiviral drugs or prodrugs once, twice, three times or four times a day. After reinfusion and treatment initiation, the number of infected cells is determined by blood counts on days 2, 5, 7, 11, 13, 18, 28, and 56 after reinfusion using qPCR to quantify the amount of viral genomes. Individuals experiencing fever or interleukin release syndrome can reduce or stop the dose or frequency of nucleoside analog antiviral drugs or prodrugs. If the infected T cells fail to expand 10,000 to 100,000 times on day 18, the dose or frequency of nucleoside analog antiviral drugs or prodrugs can be increased. The clinical response of an individual can be measured via FDG PET imaging and serial CT scans. The oral dose of nucleoside analog antiviral drugs or prodrugs can be reduced or stopped after an extended remission period or when excess T cell propagation exceeds 30% of the total peripheral T cell count.

Example 16. Proof of concept for various illustrative methods provided herein, including in vivo expansion of genetically modified lymphocytes.

This example provides illustrative methods for ex vivo transduction of PBMCs (including T cells) and in vivo expansion of these transduced PBMCs (including T cells). Such methods include illustrative 4-hour transduction methods with illustrative recombinant lentiviral particles expressing certain illustrative chimeric lymphoproliferative components. In addition, such methods provide additional illustrative methods for transducing PBMCs (which are typically T cells in illustrative embodiments) for 4 hours with replication-defective recombinant retroviral particles, wherein the replication-defective recombinant retroviral particles are produced by transfecting packaging cells with vectors encoding various components of the replication-defective recombinant retroviral particles in suspension in serum-free chemically defined medium.

Materials and Methods

Recombinant retroviral particles were produced in 293T cells (Lenti-X ^™ 293T, Clontech) adapted for suspension culture in Freestyle ^™ 293 Expression Medium (ThermoFisher Scientific), a serum-free chemically defined medium. Cells were transiently transfected using PEI with 3 genomic plasmids (described in detail below) and 1 of 3 separate packaging plasmids encoding gag/pol, rev and a pseudotyped plasmid encoding VSV-G as explained in Example 4. To produce lentiviral particles that also exhibit a membrane-bound activation component, a fourth packaging plasmid encoding a membrane-bound polypeptide capable of binding to CD3 (UCHT1 scFvFc-GPI) was co-transfected as described in Example 4. The genomic plasmid is a third generation lentiviral expression vector containing a deletion in the 3'LTR that results in self-inactivation, wherein the plasmid encodes the following:

(1) Anti-ROR2 MRB-CAR:T2A:eTag (F1-1-27): This genomic plasmid is identical to that shown in FIG5 , except that the sequence encoding the anti-CD19 CAR is replaced by an MRB-ASTR having scFvs that recognize human ROR2, CD8 stem and transmembrane sequence (SEQ ID NO: 75), CD137 (SEQ ID NO: 1), and CD3z (SEQ ID NO: 13). The recombinant lentiviral particle encoding this construct is called F1-1-27.

(2) Flag-anti-ROR2 MRB-CAR: T2A: CLE (F1-1-228 and F1-1-228U): This genomic plasmid is identical to the one shown in FIG5 , except that the sequence encoding anti-CD19 is replaced by a Flag tag covalently linked to the anti-ROR2 MRB-CAR described in (1) above, and GMCSFR ss: eTag is replaced by CLE. The CLE is a type I transmembrane protein comprising an extracellular dimerization module (P1), a transmembrane module (P2), and two intracellular modules (P3 and P4). The gene in position P1-P2-P3-P4 is MycTag 2A Jun–IL13RA–MPL–CD40. The codes for these modules are E013-T041-S186-S051 (see Table 7). This CLE was first identified in pools 3A, 3B, and 4B and was the 6th most enriched CLE in pool 3A between days 7 and 35. The recombinant retroviral particle encoding this construct was designated F1-1-228 and the recombinant retroviral particle encoding this construct and displaying UCHT1 scFvFc-GPI was designated F1-1-228U; or

(3) Flag-anti-CD19 CAR: T2A: CLE (F1-3-219 and F1-3-219U): The genomic plasmid is consistent with the anti-human CD19 CAR shown in Figure 5, except that the Flag tag is inserted between CD8ss and CAR, and the sequence encoding GMCSFRss: eTag is replaced by CLE. The CLE is a type I transmembrane protein that includes an interleukin polypeptide covalently linked to the extracellular and transmembrane module domains of its cognate interleukin receptor via a flexible linker and covalently linked to the intracellular domain of a different cytokine receptor. In particular, from the amino terminus to the carboxyl terminus, the plasmid encodes: IL-7 (SEQ ID NO: 511), a flexible linker (SEQ ID NO: 53), the extracellular and transmembrane portions of IL-7Rα (CD127) (SEQ ID NO: 513), and the intracellular domain of IL-2Rβ (CD122) (SEQ ID NO: 514). The recombinant retroviral particle encoding this construct was designated F1-3-219 and the recombinant retroviral particle encoding this construct and displaying UCHT1 scFvFc-GPI was designated F1-3-219U.

PBMC isolation, overnight transduction of PBMCs after ex vivo stimulation, followed by 15-day ex vivo expansion of engineered lymphocytes

On day 0, PBMCs were isolated from ACD peripheral blood of healthy volunteers with informed consent by density gradient centrifugation using Ficoll-Pacque ^™ (General Electric) using the CS-900.2 kit (BioSafe; 1008) on a Sepax 2 S-100 device (Biosafe; 14000) according to the manufacturer's instructions. 3.0×10 ⁷ live PBMCs were plated on 1 L G-Rex (Wilson-Wolf) and the volume was brought to 60 ml using complete OpTmizer™ CTS™ T cell expansion SFM supplemented with 100 IU/ml IL-2 (Novoprotein, GMP-CD66), 10 ng/ml IL-7 (Novoprotein, GMP-CD47) and 50 ng/ml anti ^{- CD3} antibody (OKT3, Novoprotein) to activate the PBMCs (which included T cells and NK cells) for viral transduction. After overnight incubation at 37°C and 5% _CO2 , lentiviral particles encoding anti-ROR2 MRB-CAR, F1-1-27 were added directly to activated PBMCs at an MOI of 5 (440ul) and incubated overnight at 37°C and 5% _CO2 . After overnight incubation, cells were fed by bringing the total volume of medium in G-Rex to 100ml using complete OpTmizer ^™ CTS ^™ T cell expansion SFM supplemented with NAC (Sigma) to increase the final concentration by 10mM and 100IU/ml recombinant human IL-2 and 10ng/ml recombinant human IL-7. G-Rex devices were incubated in a standard humidified tissue culture incubator at 37°C and 5% _CO2 with the addition of 100IU/ml recombinant human IL-2 and 10ng/ml recombinant human IL-7 solutions every 48 hours. Cells were expanded on day 15 before harvesting. The transduced cells were washed in freezing medium (70% RPMI 1640, 20% heat-inactivated FBS, 10% DMSO) and stored frozen in 1 ml aliquots at 5.0 x ¹⁰⁷ cells/ml for later use. Two days before use in Experiment A in the Examples below, eight vials (4.0×10 ⁸ ) of these cryopreserved F1-1-27-transduced PBMCs were thawed and cultured in 1 L of OpTmizer ^™ CTS ^™ T Cell Expansion Basal Medium (Thermo Fisher, A10484-02), 25 ml of CTS ^™ Immune Cell SR (Thermo Fisher, A2596101), and 10 ml of CTS ^™ GlutaMAX ^™ -I Supplement (Thermo Fisher, A1286001) containing 377 ml of complete OpTmizer ^™ CTS ^™ T Cell Expansion SFM supplemented with 100 IU/ml IL-2 (Novoprotein), 10 ng/ml IL-7 ⁽ Novoprotein), and sufficient NAC according to the manufacturer's ^instructions . Fisher, A10221))) in G-Rex100M for 2 days to increase the final concentration by 10 mM. Advantageous rapid transduction of PBMC isolation and resting lymphocytes without prior ex vivo stimulation and without ex vivo cell expansion

Whole human blood from two healthy volunteers with informed consent was collected into multiple 100 mm Vacutainer tubes (Becton Dickenson; 364606) containing 1.5 ml of citrate dextrose solution A anticoagulant (ACD peripheral blood). For each volunteer, blood from the Vacutainer tubes was pooled (185.2 ml for Group B and 182.5 ml for Group C) and distributed into two standard 500 ml blood collection bags. The following steps for PBMC enrichment via transduction were performed in a closed system.

Blood from 2 blood bags from each volunteer was processed sequentially using density gradient centrifugation with Ficoll-Paque ^™ (General Electric) using the CS-900.2 kit (BioSafe; 1008) on a Sepax 2S-100 device (Biosafe; 14000), using 2 wash cycles according to the manufacturer's instructions to obtain 45 ml of isolated PBMCs per run. The wash solution used in the Sepax 2 treatment was standard saline (Chenixin Pharm) + 2% human serum albumin (HSA) (Sichuan Yuanda Shuyang Pharmaceutical). The final cell resuspension solution was 45 ml complete OpTmizer ^™ CTS ^™ T Cell Expansion SFM (1 L of OpTmizer ^™ CTS ^™ T Cell Expansion Basal Medium (Thermo Fisher, A10221-03) supplemented with 26 ml OpTmizer ^™ CTS ^™ T Cell Expansion Supplement (Thermo Fisher, A10484-02), 25 ml CTS ^™ Immune Cell SR (Thermo Fisher, A2596101) and 10 ml CTS ^™ GlutaMAX ^™ -I Supplement (Thermo Fisher, A1286001)). Each treatment step on the Sepax 2 machine was approximately 1 hour and 12 minutes. The enriched PBMCs obtained from the two treatment rounds were pooled for Group B and Group C, respectively, and the cells were counted.

5.5×10 ⁷ freshly enriched live PBMCs were seeded into each of 4 standard blood collection bags for Group B and the volume was brought to 55 ml using complete OpTmizer ^™ CTS ^™ T Cell Expansion SFM to a cell density of 1.0×10 ⁶ /ml. 1.12×10 ⁸ freshly enriched live PBMCs were seeded into each of 2 standard blood collection bags for Group C and the volume was brought to 110 ml using complete OpTmizer ^™ CTS ^™ T Cell Expansion SFM to a cell density of 1.0×10 ⁶ /ml. No anti-CD3, anti-CD28, IL-2, IL-7 or other exogenous cytokines were added to activate or otherwise stimulate lymphocytes ex vivo prior to transduction. Lentiviral particles were added directly to unstimulated PBMCs in blood collection bags at an MOI of 1 as follows: 0.779 ml of F1-1-228 was added to one bag of Group B PBMCs and 3.11 ml of F1-1-228U was added to another bag of Group B PBMCs; 0.362 ml of F1-3-219 was added to one bag of Group C PBMCs and 3.52 ml of F1-3-219U was added to another bag of Group C PBMCs. The transduction reaction mixture was gently massaged to mix the contents and then incubated in the blood collection bag for four (4) hours at 37°C and 5% _CO2 in a standard humidified tissue culture incubator. PBMCs from each bag were then translated into 50 ml Conical tubes (to remove cells from the closed system in this proof-of-concept experiment) and washed 3 times in DPBS + 2% HSA before being resuspended in 5 ml DPBS + 2% HSA and counted. The following table shows the duration of each step in the process and the total time taken. No other processing was performed on these PBMCs before they were used in the experiments in this example.

Table 6. Elapsed time for each step after blood thawing and pooling.

In vitro transduction efficiency and cytokine-independent survival/proliferation of PBMCs transduced by the above method

2.0 x 10 ⁶ PBMCs that induced T cells and NK cells and were exposed to retrovirus for 4 hours as disclosed immediately below were inoculated in duplicate or triplicate into each well of a 6-well tissue culture dish of each sample. The dish was centrifuged and each sample was resuspended in 2 ml of complete OpTmizer ^TM CTS ^TM T cell expansion SFM. No cytokines were added. The dish was cultured in a standard humidified tissue culture incubator at 37°C and 5% CO ₂ for 6 days. Half (1 ml) of the cell suspension of each well was removed on the 3rd day and the remaining cells were removed on the 6th day to determine the number of cells, the percentage of survival and the percentage of transduced cells, which was defined as the percentage of FLAG-Tag+ cells by FACS analysis. The total cell count on the 6th day was doubled to account for the removal of half of the cells on the 3rd day.

The above method leads to the in vivo proliferation/survival and targeted killing of tumors by effector PBMCs

The xenograft model using NSG or NOD Scid Gamma mice was selected to detect the ability of human PBMCs transduced with F1-1-27, F1-1-228, F1-1-228U, F1-3-219 and F1-3-219U to survive, proliferate and kill homologous antigen-expressing tumors in vivo. NSG is a mouse strain lacking mature T cells, NK cells and B cells and is one of the most immune-deficient mouse strains described so far. These cellular components of the immune system are usually removed to enable human PBMCs to be transplanted in the case of innate, humoral or adaptive immune responses of the host. Due to the absence of a common γ chain outside mouse cells, the concentration of stable cytokines can usually be achieved only after radiation or lymphoid depletion chemotherapy in humans, which enables human cells transferred through acceptance to accept such cytokines. At the same time, these animals can also be used to transplant tumor xenograft targets to detect the efficacy of CAR killing targeted expression tumors. Although the presence of xenoreactive T-cell receptor antigens in effector cell products will ultimately lead to graft-versus-host disease, these models allow for short-term assessment of animal pharmacology and acute tolerance.

Raji cells (ATCC, Manassas, VA) expressing endogenous human CD19 and CHO cells (ATCC, Manassas, VA) transfected to stably express human ROR2 (CHO-ROR2) were used to provide antigens to stimulate CAR effector cells and generate consistent target tumors to determine the efficacy of CAR effector cells in killing homologous antigen-expressing tumors. Raji cells and transgenic CHO variants grew rapidly and developed disseminated malignancies after subcutaneous administration into NSG mice in combination with Matrigel artificial substrate.

Mice were treated according to protocols approved by the Institutional Animal Care and Use Committee of the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. Subcutaneous (sc) tumor xenografts were established in the posterior flank of female NOD-Prkdc ^scid Il2rg ^tm1 / Begen (B-NSG) mice (Beijing Biocytogen Co. Ltd.). In brief, cultured Raji cells and cultured CHO-ROR2 cells were washed separately in DPBS (Thermo Fisher), counted, resuspended in cold DPBS and mixed on ice with an appropriate volume of Matrigel ECM (Corning; final concentration 5 mg/mL) at a concentration of 0.5×10 ⁶ cells/200 microliters. Animals were prepared for injection using standard approved anesthesia and depilation (Nair) before injection. 200 μl of ECM containing cell suspension of Raji and CHO-ROR2 cells were subcutaneously injected into the posterior flank of 9-week-old or 10-week-old mice, respectively.

Five days after tumor inoculation, mice bearing CHO-ROR2 tumors with an average volume of ⁷⁷ mm3 were dosed intravenously (IV) by tail vein injection as follows: NSG mice in group A received 200 μl DPBS containing 1× ¹⁰⁷ PBMCs transduced with F1-1-27 lentiviral particles (n=4), or 200 μl DPBS alone (n=2); NSG mice in group B received 200 μl DPBS containing 0.85× ¹⁰⁷ PBMCs transduced with F1-1-228 lentiviral particles (n=2), 200 μl DPBS containing 0.85× ¹⁰⁷ PBMCs transduced with F1-1-228U lentiviral particles (n=2), or 200 μl DPBS alone (n=2). Similarly, mice in Group C bearing Raji tumors (average volume of 76 mm 3) were dosed intravenously (IV) 5 days after tumor inoculation by tail vein injection of 1×10 ⁷ PBMCs transduced with 200 μl DPBS alone (n=4) or with 200 μl DPBS containing F1-3-219 lentiviral particles (n=3) or 200 μl DPBS containing F1-3-219U lentiviral particles (n= ³ ). It should be noted that PBMCs with F1-1-228, F1-1-228U, F1-3-219, and F1-3-219U were transduced for dosing these mice using a protocol that favors rapid transduction of resting lymphocytes prior to ex vivo activation and without ex vivo cell expansion. The total time from total human blood collection to IV dosing of mice with transduced PBMCs was 14.5 hours for F1-1-228 and F1-1-228U and 11.5 hours for F1-3-219 and F1-3-219U.

Tumors were measured twice a week using calipers and tumor volume was calculated using the following equation: (longest diameter x shortest diameter ² )/2. Approximately 100 μl of blood was collected from each mouse on days 7, 14, and 21 for FACS and qPCR analysis. Blood, spleen, and tumors were also collected when mice were euthanized, consistent with autopsy indicators of tumor burden.

Flow Cytometry

For cells harvested from in vitro cultures - the cells were centrifuged and resuspended in 0.5 ml FACS staining buffer (554656, BD). 2.5 μl human Fc block (BD, 564220) was added to each sample and incubated at room temperature for 10 minutes. The cells were stained with 0.5 μl anti-FLAG Tag PE ((anti-DYKDDDDK) 637310, Biolegend)) and 0.5 μl Live/DeadFixable Green Dead cell stain (L34970, Thermo Fisher) for 30 min on ice. The cells were washed twice with FACS buffer, fixed in a 1:1 mixture of FACS buffer and BD Cytofix (554655, BD), treated with Novocyte (ACEA), and the data were analyzed using live valves based on forward and side scatter and live dead staining with NovoExpress software (ACEA). Transduced lymphocytes were measured as FLAG Tag+ cells.

For cells obtained from blood--red blood cells in freshly collected blood were lysed using lysis buffer (555899, BD) and the remaining cells were resuspended in 100 μl of FACS staining buffer. 2.5 μl of human Fc block (BD, 564220) was added to each sample and incubated at room temperature for 10 minutes. Cells were stained with biotinylated cetuximab on ice for 30 min. The stained cells were washed with FACS buffer and further stained with 5 μl of anti-human CD45-PE-Cy7 and 0.5 μl of anti-mouse CD45-FITC. 0.4 μl SA-PE was added to the samples of mice from these groups that were administered with cells transduced with F1-1-27, F1-1-228, F1-1-228U and PBS control. 1 μl of anti-FLAG Tag PE ((anti-DYKDDDDK) 637310, Biolegend) was added to samples from mice dosed with cells transduced with F1-3-219 and F1-3-219U and PBS control from this group. Cells were incubated on ice for 30 min, washed twice in FACS buffer, and resuspended in 100 μl FACS staining buffer with 1 μl 7-AAD (420404, Biolegend). Freshly stained samples were processed with Novocyte (ACEA) and the resulting data were analyzed using forward and side scatter based live valves, 7-AAD based live valves for human CD45+ using NovoExpress software (ACEA) and the expression of FLAG or eTag was detected.

qPCR

The presence of transduced lymphocytes was assessed by bioanalytical qPCR from genomic DNA (gDNA) isolated from blood samples. Genomic DNA was isolated from 50 μl blood samples using the QIAamp DNA Blood Mini Kit (Qiagen 51106) and further cleaned using the QIAamp DNA Micro Kit (56304). The isolated genomic DNA was subjected to a TaqMan assay (Thermo Fisher) using primers and probe sets specific for the 5'LTR of the lentivirus to detect transduced cells.

result

Lentiviral particles pseudotyped with VSV-G and encoding CAR and lymphoproliferative components were used in this experiment. Lentiviral particles F1-1-228 and F1-1-228U encode MRB-CAR as ROR2 and CLE including MycTag and 2A Jun dimerization domains, IL13RA transmembrane domains, and MPL and CD40 intracellular domains, and lentiviral particles F1-2-219 and F1-3-219U encode CAR as CD19 and CLE, which encode IL7 covalently linked to the extracellular and transmembrane portions of IL-7Rα (CD127) (SEQ ID NO: 513) and the intracellular domain of IL-2R (CD122). Lentiviral particles F1-1-228U and F1-3-219U also present UCHT1scFvFc-GPI on their surface. PBMCs were isolated from human blood by centrifugation with Ficoll-Paque ^TM density gradients. Before cell ex vivo activation, fresh PBMCs were transduced with lentiviral particles in standard blood bags. After 4 hours of transduction, the cells were washed and used in the following experiments. It will be appreciated by skilled artisans that the entire process from blood collection to washing cells can be performed in a closed system. As a control, PBMCs were activated overnight, transduced with lentiviral particles encoding CAR without encoding lymphoproliferative components or UCHT1scFvFc-GPI (F1-1-27), and cultured in vitro for 15 days (F1-1-27).

The transduced PBMCs were cultured in vitro in the absence of cytokines. Figure 21 shows the transduction efficiency at day 6. UCHT1scFvFc-GPI increases the transduction efficiency of F1-1-228 and F1-3-219. The transduction efficiency of resting PBMCs when exposed to F1-1-228U or F1-3-219U for 4 hours was 34% and 73%, respectively. Figure 22 shows the total number of viable cells in these cultures between day 3 and day 6. Resting PBMCs transduced with F1-1-228U or F1-3-219U survived and even proliferated between day 3 and day 6 in the absence of exogenous cytokines, while PBMCs transduced with F1-1-228 or F1-3-219 did not survive and even proliferated. These results demonstrate that lentiviral particles presenting the activation component and encoding the lymphoproliferative component can transduce quiescent PBMCs within 4 hours and that these transduced PBMCs can proliferate and survive in vitro when cultured for 6 days in the absence of exogenous cytokines.

Immunodeficient mice carrying ROR2 or CD19 tumors were intravenously administered 1×10 ⁷ PBMCs expressing CAR to ROR2 or CD19, respectively. The ability of transduced PBMCs to survive and proliferate in vivo was detected over time. Figures 23A to 23C show the lentiviral copies per microgram of genomic DNA isolated from the blood of mice that administered PBMCs expressing CAR by qPCR as a readout of transduced cells. Figure 23A shows the lentiviral copies per microgram of genomic DNA for F1-1-27, which does not encode a lymphoproliferative component, from an average of 884 on day 7 to less than the lower limit of quantification (LLOQ) on day 21. Figure 23B shows that the lentiviral copies for F1-1-228U were lower than the LLOQ on days 7 and 14, but the increase on day 21 was greater than the LLOQ and increased to an average of 1,939 on day 25. Figure 23C shows that when mice were euthanized, the lentiviral copies for F1-3-219U increased from below the LLOQ on day 7 to 20,430 on day 14. The lentiviral copies in control samples F1-1-228 and F1-3-219 were still below the LLOQ. Figure 24 shows the number of transduced cells per 200ul of blood as determined by FACS analysis of the eTag or FLAG-Tag of the CAR construct when mice were euthanized (the last time point is shown in Figure 23). A significant number of CAR+ cells were detected in the blood of mice dosed with cells transduced with F1-1-228U (5,857) and F1-3-219U (121,324).

The anti-tumor activity of PMBCs modified by gene mode to express lymphoproliferative components and anti-CD19 CAR or anti-ROR2 CAR is analyzed. Mice carrying CHO-ROR2 tumors or CD-19 expressing tumors are produced as provided above. Lymphocytes transduced with F1-1-228U or F1-3-219U kill tumors expressing their target antigens in vivo (Figure 25A and Figure 25B). In both cases, lymphocytes reduce tumor volume in a delayed manner. Not limited by theory, but this delay is consistent with the demand for injection cell amplification, and it takes time as observed by qPCR. Later time points cannot be studied in this experiment, because mice carrying ROR2 tumors have reached the euthanasia pointer for tumor load, and mice carrying Raji tumors have produced a large number of graft-versus-host disease caused by transduced and amplified lymphocytes in mice.

These data are shown together, and the retroviral particles presenting activation components and encoding lymphoproliferative components can transduce resting PBMC within 4 hours, and these transduced PBMCs can proliferate and survive in vivo. The two lymphoproliferative components MycTag 2A Jun-IL13Ra-MPL-CD40 and IL-7-IL-7Rα-IL-2Rβ tested in this experiment present the ability to promote survival and proliferation in vivo. In addition, these lymphocytes expressing MRB-CAR (F1-1-228U) or traditional CAR (F1-3-219U) transduced in this way can recognize and kill tumor cells in vivo.

Example 17. Functionality of miRNA inserted into the intron of the EF-1α promoter.

Design four separate Gene fragments, each containing a miR-155 framework including a miR-155 5' flanking sequence or "5'arm" (SEQ ID NO: 256) and a miR-155 3' flanking sequence or "3'arm" (SEQ ID NO: 260). The only miRNA fragment that targeted the CD3zeta mRNA transcript was used to replace the miR-155 stem-loop precursor. Contains a Assembled as a single strand into a 40 bp overlapping sequence in the EF-1α promoter intron. Assemble ultra commercial kit (NEBuilder, New England Biolabs, Inc.) to assemble

The synthetic EF-1α promoter and intron A containing miRNA (in sequence SEQ ID NO: 255) are part of the transgene expression block that drives the expression of GFP and eTag contained in the lentiviral vector backbone (the lentiviral vector backbone with GFP recognized by cetuximab and an exemplary eTag is referred to herein as F1-0-02; FIG. 26A and FIG. 26B ). Each of the SEQ ID NO: 255 indicated in Table 26 and the nucleotide positions of its individual components are the positions of the various "signatures" in Figure 26B. Proper assembly of the four miRNAs into the lentiviral vector backbone was confirmed by comprehensive sequencing of the modified EF-1α promoter and intronic regions.

Table 26. Nucleotide positions of features in SEQ ID NO:255.

Replication-defective lentiviral particles containing nucleic acids encoding four miRNAs for CD3ζ in their genome were produced by transiently co-transfecting the following four plasmids into suspended HEK293 cells: a plasmid containing nucleic acids encoding F1-0-02 modified to include four miRNAs targeting CD3zeta mRNA transcripts, a plasmid encoding VSV-G, a plasmid encoding REV, and a plasmid encoding GAG-POL. The lentiviral particle supernatant was collected after 48 hours and PEG precipitated for 24 hours. The supernatant was centrifuged and the pelleted lentiviral particles were resuspended in complete PBMC growth medium without IL-2. The 48-hour transduction of Jurkat cells was used to calculate the lentiviral particle titer.

For transduction, PBMCs were thawed on day 0 and incubated with 100 U/mL of hrIL-2 for 24 hours. On day 1, PBMCs were activated via CD3/CD28-bound beads. On day 2, activated PBMCs were transduced with lentiviral particles containing a genome with a nucleic acid sequence encoding miRNA at 10 MOI. Cells were expanded until day 11, with fresh hrIL-2 added every two days. On days 7, 9, and 11, 1 million cells were harvested for FACS analysis.

Cells were stained with PE-conjugated OKT-3 antibody (Biolegend) for CD3 Epsilon surface expression. Expression levels were determined using the mean fluorescence intensity (MF) of PE in the GFP-positive population (transduced cells). Expression levels in transduced cells were compared between reverse lentiviral particles derived from F1-0-02 and reverse lentiviral particles derived from F1-0-02, in which the nucleic acid sequence encoding the continuously localized CD3z miRNA was inserted into the EF-1α promoter and intron A.

The results are shown in Figure 27. This data shows that sequential miRNA targeting CD3zeta, encoded by a nucleic acid sequence within intron A of the EF-1 alpha promoter, is effective in knocking down the expression of the CD3 complex.

Example 18. Position dependence of continuous inhibitory RNA inserted into the intron of the EF-1α promoter.

clone

Four miRNA-expressing lentiviral vector constructs were designed to test the processing of individual miRNA precursors in a structure comprising four consecutive miRNA precursors. Table 27 shows the names of the individual constructs and the location of miR-TCRα in each construct.

Construct Position 1 Position 2 Location 3 Position 4 TCRa-P1 miR-TCRa miR-155 miR-PD-1 miR-CTLA-4 TCRa-P2 miR-155 miR-TCRa miR-PD-1 miR-CTLA-4 TCRa-P3 miR-155 miR-PD-1 miR-TCRa miR-CTLA-4 TCRa-P4 miR-155 miR-CTLA-4 miR-PD-1 miR-TCRa

Table 27. Constructs containing polycistronic miRNAs.

Each miRNA contained a specific order of the miR-155 framework used in Example 17, e.g., miR-155 5' arm (SEQ ID NO: 256), miR-155 3' arm (SEQ ID NO: 260), loop (SEQ ID NO: 258), and stem sequence as shown in Table 28. A Type II assembly approach was used to achieve assembly of the four miRNA fragments into their appropriate positions within the EF-1α intron of a lentiviral vector construct (F1-0-02; provided in Example 17 and shown in Figures 26A and 26B).

Table 28: Sequences in miRNA constructs

,in:

SEQ ID NO:267=TCRamiRNA stem 1; ATATGTACTTGGCTGGACAGC

SEQ ID NO:268=TCRamiRNA stem 2;GCTGTCCACAAGTACATAT

SEQ ID NO:269=Linker 1;CACATTTGGTGCCGGATGAAGCTCTTATGTTGCCGGTCAT

SEQ ID NO: 270=mir-155 stem 1; CTGTTAATGCTAATCGTGATA

SEQ ID NO: 271=mir-155 stem 2; TATCACGATTATTAACAG

SEQ ID NO:272 = Linker 2; GTTGCCGGAGTCTTGGCAGCGAGAGATCACTATCAACTAA

SEQ ID NO:273=PD-1miRNA stem 1; TACCAGTTTAGCACGAAGCTC

SEQ ID NO:274=PD-1miRNA stem 2;GAGCTTCGCTAAACTGGTA

SEQ ID NO:275 = Linker 3; GTGTTAATTGTCCATGTAGCGAGGCATCCTTATGGCGTGG

SEQ ID NO:276=CTLA-4miRNA stem 1; TGCCGCTGAAATCCAAGGCAA

SEQ ID NO: 277=CTLA-4miRNA stem 2; TTGCCTTGTTTTCAGCGGCA

Lentiviral particle production

The four constructs and control F1-0-02 (which does not include nucleic acid sequences encoding miRNA) are used to produce lentiviral particles in 30 mL suspension cultures of 293T cells. The lentiviral particles were collected and concentrated using PEG precipitation. Functional lentiviral particle titers were obtained by transducing Jurkat cells at multiple dilutions (1:1000, 1:10000, 1:100000), incubating lentiviral particles and cells at 37°C for 2 days, washing cells 2× with FACS buffer, and analyzing GFP using flow cytometry. Additional details on the production of lentiviral particles are provided in Example 17 herein.

divert

For transduction, PBMCs were thawed and recovered overnight in complete medium containing 100 U/mL hrIL-2. 1×10 ⁵ PBMCs were activated by exposure to CD3/CD28-bound beads for 24 hours. Cells were transduced in duplicate wells at an MOI of 10 with each of the four miRNA constructs or with control retroviral particles F1-0-02. Cells were supplied with 100 U/mL hrIL-2 every 3 days and expanded until day 10.

FACS

Cells were pooled for FACS analysis, which confirmed cells transduced with replication-defective lentiviral vectors. The results show that approximately equal amounts of miRNA-containing viruses were delivered to each well in the experiment.

Cell-to-ct miRNA RT-qPCR and analysis

RT-qPCR analysis was designed to detect expression and processing of miRNA precursors into mature processed miRs. The analysis was performed by first normalizing all miR-TCRa ct values to the RNU48 internal control to generate ΔCt values. Next, the ΔCt value of each transduced sample was subtracted from the ΔCt of the untransduced control to generate ΔΔCt. This value represents the amount of processed miR-TCRa miRNA in each transduced sample relative to the untransduced control.

As shown in Figure 28, RT-qPCR analysis successfully detected processed miR-TCRα in samples transduced with replication-defective lentiviral particles containing miR-TCRα. In addition, the results clearly indicated that there was no significant effect in miRNA TCRα processing at any of the four positions tested.

Example 19. miRNA expression increases in vivo survival and/or proliferation of transduced cells expressing CAR.

method

Library preparation

108 The gene fragments were used to generate a library of constructs each containing four miRNA precursors at consecutive positions 1 (P1), 2 (P2), 3 (P3) and 4 (P4). A miR-155 framework specific for P1, P2, P3 or P4 and comprising a 5' arm and a 3' arm as described in Example 17, wherein a unique miRNA framework targeting mRNA transcripts corresponding to 1 to 27 different genes is used to replace the miR-155 stem-loop precursor. For clarity, the sequence of the miRNA fragment differs for each position P1 to P4, even among miRNA fragments targeting mRNA transcripts corresponding to the same gene. Contains unique 40 bp overlapping sequences, and the Type II assembly method is used to assemble the four By these methods, a total diversity of 531,441 unique constructs (27 miRNAs at P1 x 27 miRNAs at P2 x 27 miRNAs at P3 x 27 miRNAs at P4) was possible.

The pool of miRNA constructs was cloned individually into the EF-1α intron A of F1-1-315 and F1-2-314 to generate pool 315 and pool 314, respectively. In addition to the EF-1α promoter, F1-1-315 also includes a CD8a signal peptide, anti-ROR2:CD28:CD3z CAR, T2A and eTag. Similarly, in addition to the EF-1α promoter, F1-2-314 includes a CD8a signal peptide, anti-Axl:CD8:CD3z CAR, T2A and eTag. Figures 26A and 26B show similar lentiviral vectors with an EF-1α promoter (including intron A with 4 miRNA precursors) that drive the expression of GFP instead of CAR.

cCBL and CD3z are among the 27 transcripts targeted in this example. The 4 miRNA sequences for cCBL are: SEQ ID NO: 540 for P1; SEQ ID NO: 541 for P2; SEQ ID NO: 542 for P3; and SEQ ID NO: 543 for P4. The 4 miRNA sequences for CD3z are: SEQ ID NO: 544 for P1; SEQ ID NO: 545 for P2; SEQ ID NO: 546 for P3; and SEQ ID NO: 547 for P4.

Note that due to its specific sequence (miRNA for CD3z) designed, it will only target RNA encoding endogenous CD3z and will not target RNA encoding CD3z domain within the CAR transgenic. This is achieved by designing the miRNA to target sequences within the 3'UTR of endogenous CD3z that are not present in the CAR transgenic. Alternatively, the miRNA can be targeted to the mRNA sequence encoding the CD3z intracellular domain of both endogenous CD3z and the CAR transgenic, with the restriction that the codon usage for the CAR transgenic is sufficiently different from that of the endogenous CD3z, so that the endogenous CD3z is targeted instead of the CAR transgenic mRNA for cleavage.

Lentiviral particle production

Pool 315 and pool 314 were used alone to produce lentiviral particles in 30 ml suspension cultures of 293T cells. The lentiviral particles were collected and concentrated using PEG precipitation. Additional details on the production of lentiviral particles are provided in Example 17 herein.

divert

On day 0, PBMCs were isolated from ACD peripheral blood and 5.0×10 7 viable PBMCs were seeded in 100 ml into each of ^{two 1L} G-Rex devices with complete OpTmizer ^™ CTS ^™ T cell expansion SFM supplemented with 100 IU/ml IL-2 (Novoprotein, GMP-CD66), 10 ng/ml IL-7 (Novoprotein, GMP-CD47) and 50 ng/ml anti-CD3 antibody (Novoprotein, GMP-A018) to activate PBMCs including T cells and NK cells for viral transduction. Lentiviral particles were added directly to the activated PBMCs in 1G-Rex of pool 315 and other G-Rex of pool 314 at an MOI of 5 and cultured overnight. G-Rex devices were incubated in a standard humidified tissue culture incubator at 37°C and 5% _CO2 , with 100 IU/ml recombinant human IL-2 and 10 ng/ml recombinant human IL-7 solutions added every 48 hours, and the cultures were expanded until day 12, at which time the cells were primarily T cells. Additional details on PBMC isolation, transduction, and ex vivo expansion are provided in Example 16 herein.

Tumor Cultivation and Administration of Transduced Cells.

The xenograft model using NOD Scid Gamma (NSG) mice was selected to detect the ability of human PBMCs transduced with lentiviral particles of library 315 or library 314 to survive and/or proliferate in vivo, wherein the tumor expresses or does not express the antigen recognized by the CAR encoded in the genome of these lentiviral particles. Mice were treated according to the protocol approved by the Institutional Animal Care and Use Committee of the Institute of Biochemistry and Cell of the Chinese Academy of Sciences. Subcutaneous (sc) tumor xenografts were established in the flank of 12-week-old female NOD-Prkdc ^scid Il2rg ^tm1 / Begen (B-NSG) mice (Beijing Biocytogen Co. Ltd.). Briefly, cultured CHO cells transfected to stably express human ROR2 (CHO-ROR2) or human AXL (CHO-AXL), cultured CHO cells were washed individually in DPBS (Thermo Fisher), counted, resuspended in cold DPBS, and mixed with an appropriate volume of Matrigel ECM (Corning; final concentration 5 mg/mL) at a concentration of 0.47×10 ⁶ cells/200 μl on ice. Animals were prepared for injection using standard approved anesthesia, with hair removed prior to injection (Nair). 200 μl of either cell suspension in ECM was injected subcutaneously into the posterior flank of CHO cells (n=2), CHO-ROR2 cells (n=1), and CHO-AXL cells (n=1), respectively.

Five days after tumor inoculation, one mouse bearing a CHO tumor and one mouse bearing a CHO-ROR2 tumor were intravenously (IV) administered with 200 μl DPBS containing 1×10 ⁷ PBMCs transduced with lentiviral particles from pool 315 by tail vein injection. Five days after tumor inoculation, one mouse bearing a CHO tumor and one mouse bearing a CHO-Axl tumor were intravenously (IV) administered with 200 μl DPBS containing 1×10 ⁷ PBMCs transduced with lentiviral particles from pool 314 by tail vein injection.

Tumor collection and DNA sequencing

The tumor was excised on the 20th day after administration of transduced PBMC. DNA was extracted from half of each tumor, and 4ug from each tumor was used as a template in a PCR reaction for 25 cycles to amplify the EF-1α intron. The amplicon was cloned into a sequencing vector, converted into bacteria and streaked onto a plate. 18 total colonies (about 5 per mouse) were selected, and DNA was prepared and analyzed using Sanger sequencing to determine the sequence of the sample of the miRNA construct present in the tumor.

result

Mouse xenograft model is used to determine whether the miRNA targeting cCBL or CD3z increases the in vivo hyperplasia and/or survival of transduced PBMC expressing CAR, wherein the xenograft is a tumor with or without the expression of the targeted antigen of CAR. For this analysis, a library of miRNA constructs consisting of miRNAs for cCBL, endogenous CD3z or 25 other target targets is produced. The analyzed miRNA construct contains 4 positions of 4 separate miRNAs, as shown in Figure 26B and Examples 17 and 18. Tumor DNA is analyzed by sequencing the EF-1α introns to identify which miRNA constructs are present 20 days after injection of transduced PBMCs, and therefore which miRNA constructs increase hyperplasia and/or survival.

531,441 different combinations of 4 consecutive miRNAs are possible. Among the 18 EF-1α introns sequenced, 13 contain miRNA constructs, wherein all 4 miRNAs in the construct are directed to one target, although the target is different among the constructs. It is worth noting that 2 EF-1α introns contain miRNA constructs with all 4 miRNAs for cCBL (SEQ ID NO: 540 to 543), and 1 EF-1α intron contains miRNA constructs with all 4 miRNAs for CD3z (SEQ ID NO: 544 to 547). 4 consecutive miRNAs for cCBL were found in CHO-ROR2 tumors from mice receiving PBMCs transduced with library 315, and in CHO tumors from mice receiving PBMCs transduced with library 314. 4 consecutive miRNAs for CD3z were found in CHO tumors from mice receiving PBMCs transduced with library 315. This indicates that knockout of each of cCBL and CD3z promotes survival and/or proliferation of T cells in the tumor microenvironment. In addition, this data indicates that there is a dosage effect, such that 4 species of miRNAs for cCBL and CD3z produce greater knockout of transcripts encoding these genes than 1, 2 or 3 species, and this increased knockout confers a survival and/or proliferation advantage.

The disclosed embodiments, examples, and experiments are not intended to limit the scope of the invention or to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to the numbers used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be accounted for. It should be understood that variations can be made to the methods as described without changing the basic aspects of the experiments intended to illustrate.

Those skilled in the art may design many modifications and other embodiments within the scope and spirit of the present invention. In fact, the materials, methods, diagrams, experiments, examples and embodiments described may be varied by those skilled in the art without changing the basic aspects of the present invention. Any of the disclosed embodiments may be used in conjunction with other disclosed embodiments.

In some cases, some concepts are described with reference to specific embodiments. However, it is understood by those skilled in the art that various modifications and changes may be made without departing from the scope of the invention as set forth in the following claims. Therefore, the description and drawings should be regarded in an illustrative rather than a restrictive sense, and all such modifications are intended to be included within the scope of the invention.

Table 7. Codes, names and amino acid sequences of parts P1-P2, P1, P2, P3 and P4

Table 8. Library 1A optimal constructs.

Table 9. Pool 2B optimal constructs.

Table 10. Library 1.1A optimal constructs.

Table 11. Library 1.1B optimal constructs.

Table 12. Library 2.1B optimal constructs.

Table 13. Pool 3A optimal constructs.

Table 14. Pool 3B optimal constructs.

Table 15. Library 3.1A optimal constructs.

Table 16. Library 3.1B optimal constructs.

Table 17. Pool 4B optimal constructs.

Table 18. Library 4.1B optimal constructs.

Table 19. Lib3 P4_STOP.

Table 20. Constructs with particularly noteworthy enrichments in libraries 1A and 1.1A.

Table 21. Constructs with particularly noteworthy enrichments in libraries 2B and 2.1B.

Table 22. Constructs with particularly noteworthy enrichments in libraries 3A and 3.1A.

Table 23. Constructs with particularly noteworthy enrichments in libraries 3B and 3.1B.

Table 24. Constructs with particularly noteworthy enrichments in libraries 4B and 4.1B.

Table 25. Constructs of the top 100 hits present in at least one replicate of library 3.1A or 3.1B.

Claims (32)

1. A replication-defective recombinant retroviral particle comprising:

A. One or more viral envelope proteins on the surface of the replication defective recombinant retroviral particle;

B. A membrane-bound T cell activating module on the surface of the replication-defective retroviral particle, wherein the membrane-bound T cell activating module is fused to a heterologous membrane adhesion sequence, and wherein the membrane-bound T cell activating module is a polypeptide capable of binding to CD3 on the surface of a T cell; and

C. A polynucleotide comprising one or more transcriptional units operably linked to a promoter active in a T cell, wherein the one or more transcriptional units encode

I. A first polypeptide comprising a lymphoproliferative component (LE), wherein the LE comprises an intracellular signaling domain ：CD40,CD79B,CRLF2,CSF2RA,CSF3R,EPOR,FCGR2A,FCGR2C,GHR,IFNAR1,IFNAR2,IFNGR2,IL1R1,IL1RL1,IL2RA,IL3RA,IL5RA,IL6R,IL9R,IL10RB,IL11RA,IL13RA1,IL13RA2,IL17RB,IL18R1,IL18RAP,IL20RB,IL22RA1,IL27RA,IL31RA,LEPR,MPL,MYD88,OSMR or PRLR from the following, and wherein the LE is capable of promoting survival and/or cell proliferation of T cells during culture without the need for exogenous cytokines, and

A second polypeptide comprising an antigen specific targeting region, a transmembrane domain, and an intracellular activation domain.

2. Use of a replication defective recombinant retroviral particle in the preparation of a kit for genetically modifying T cells of an individual wherein the use of the kit comprises:

Contacting the T cell ex vivo with the replication defective recombinant retroviral particle, wherein the replication defective recombinant retroviral particle comprises:

A. one or more viral envelope proteins on the surface of the replication defective retroviral particle,

B. A membrane-bound T cell activating component on the surface of the replication-defective retroviral particle, wherein the membrane-bound T cell activating component is a polypeptide capable of binding to CD3 on the surface of a T cell; and

C. a polynucleotide comprising one or more transcriptional units operably linked to a promoter active in a T cell, wherein the one or more transcriptional units encode a first polypeptide comprising a lymphoproliferative component (LE) or a first polypeptide comprising an LE and a second polypeptide comprising an antigen specific targeting region, a transmembrane domain, and an intracellular activation domain, wherein the LE comprises intracellular signaling domain ：CD40,CD79B,CRLF2,CSF2RA,CSF3R,EPOR,FCGR2A,FCGR2C,GHR,IFNAR1,IFNAR2,IFNGR2,IL1R1,IL1RL1,IL2RA,IL3RA,IL5RA,IL6R,IL9R,IL10RB,IL11RA,IL13RA1,IL13RA2,IL17RB,IL18R1,IL18RAP,IL20RB,IL22RA1,IL27RA,IL31RA,LEPR,MPL,MYD88,OSMR or PRLR from the following, thereby producing a genetically modified T cell,

Wherein the contacting is performed for less than 12 hours,

Wherein the use is performed without prior ex vivo stimulation of T cells, and

Wherein said contacting facilitates membrane fusion of said T cell with said replication defective recombinant retroviral particle, thereby producing a genetically modified T cell.

3. A genetically modified T cell comprising:

A. one or more viral envelope proteins on the surface of the T cell;

B. A membrane-bound T cell activating module on the surface of the T cell, wherein the membrane-bound T cell activating module is fused to a heterologous membrane adhesion sequence, and wherein the membrane-bound T cell activating module is a polypeptide capable of binding to CD3 on the surface of the T cell; and

C. A polynucleotide comprising one or more transcriptional units operably linked to a promoter active in a T cell, wherein the one or more transcriptional units encode

I. A first polypeptide comprising a lymphoproliferative component (LE), wherein the LE comprises an intracellular signaling domain ：CD40,CD79B,CRLF2,CSF2RA,CSF3R,EPOR,FCGR2A,FCGR2C,GHR,IFNAR1,IFNAR2,IFNGR2,IL1R1,IL1RL1,IL2RA,IL3RA,IL5RA,IL6R,IL9R,IL10RB,IL11RA,IL13RA1,IL13RA2,IL17RB,IL18R1,IL18RAP,IL20RB,IL22RA1,IL27RA,IL31RA,LEPR,MPL,MYD88,OSMR or PRLR from the following, and wherein the LE is capable of promoting survival and/or cell proliferation of T cells during culture without the need for exogenous cytokines, and

A second polypeptide comprising an antigen-specific targeting region, a transmembrane domain, and an intracellular activation domain.

4. A method for genetically modifying and/or transducing a T cell comprising contacting the T cell ex vivo with a replication defective recombinant retroviral particle comprising

A. one or more viral envelope proteins on the surface of the replication defective retroviral particle,

B. a membrane-bound T cell activating assembly on a surface thereof, wherein the membrane-bound T cell activating assembly is capable of binding to CD 3; and

C. A polynucleotide comprising one or more transcriptional units operably linked to a promoter active in a T cell, wherein the one or more transcriptional units encode a first polypeptide comprising a lymphoproliferative component (LE), or a first polypeptide comprising an LE and a second polypeptide comprising an antigen specific targeting region, a transmembrane domain, and an intracellular activation domain, wherein the LE comprises an intracellular signaling domain ：CD40,CD79B,CRLF2,CSF2RA,CSF3R,EPOR,FCGR2A,FCGR2C,GHR,IFNAR1,IFNAR2,IFNGR2,IL1R1,IL1RL1,IL2RA,IL3RA,IL5RA,IL6R,IL9R,IL10RB,IL11RA,IL13RA1,IL13RA2,IL17RB,IL18R1,IL18RAP,IL20RB,IL22RA1,IL27RA,IL31RA,LEPR,MPL,MYD88,OSMR or PRLR from the following, and

Wherein the contacting is performed for less than 12 hours,

Wherein the method is performed without prior ex vivo stimulation of T cells, and

Wherein said contacting facilitates membrane fusion of said T cells by said replication defective recombinant retroviral particle, thereby producing genetically modified T cells.

5. The use according to claim 2, the method for genetically modifying and/or transducing T cells according to claim 4, wherein the use or the method is performed without prior ex vivo stimulation of T cells.

6. The use according to claim 2, the method for genetically modifying and/or transducing T cells according to claim 4, wherein the T cells are resting T cells.

7. The use according to claim 2, the method for genetically modifying and/or transducing T cells according to claim 4, wherein the membrane bound T cell activating module is fused to a heterologous membrane adhesion sequence.

8. The use of claim 7, method for genetically modifying and/or transducing T cells, wherein the membrane-bound T cell activating component is an anti-CD 3 scFvFc antibody.

9. The use of claim 8, method for genetically modifying and/or transducing T cells, wherein the heterologous membrane adhesion sequence is a GPI anchor adhesion sequence.

10. The replication defective recombinant retroviral particle of claim 1, the genetically modified T cell of claim 3, wherein the membrane bound T cell activating component is an anti-CD 3 scFvFc antibody, and wherein the heterologous membrane adhesive sequence is a GPI anchor adhesive sequence.

11. The replication-defective recombinant retroviral particle of claim 1, the use of claim 2, the genetically modified T cell of claim 3, or the method for genetically modifying and/or transducing a T cell of claim 4, wherein the fusion polypeptide on the surface of the replication-defective recombinant retroviral particle comprises one or more viral envelope proteins and a membrane-bound T cell activating module, and wherein the membrane-bound T cell activating module comprises an anti-CD 3 antibody.

12. The replication-defective recombinant retroviral particle, use, genetically modified T cell, or method for genetically modifying and/or transducing a T cell according to claim 11, wherein one of the one or more viral envelope proteins is MuLV.

13. The replication defective recombinant retroviral particle, use, genetically modified T cell, or method for genetically modifying and/or transducing a T cell according to claim 12, wherein the anti-CD 3 antibody is UCHT1.

14. The replication-defective recombinant retroviral particle of claim 1, the use of claim 2, the genetically modified T cell of claim 3, or the method for genetically modifying and/or transducing a T cell of claim 4, wherein one of the one or more viral envelope proteins is MuLV, wherein the membrane bound T cell activating component comprises an anti-CD 3 antibody.

15. The replication-defective recombinant retroviral particle, use, genetically modified T cell, or method for genetically modifying and/or transducing a T cell according to claim 14, wherein MuLV is fused to an anti-CD 3 antibody.

16. The replication defective recombinant retroviral particle of claim 14, wherein the anti-CD 3 antibody is UCHT1.

17. The use of claim 2, the genetically modified T cell of claim 3, the method for genetically modifying and/or transducing a T cell of claim 4, wherein the one or more transcriptional units encode the first polypeptide comprising the LE and the second polypeptide comprising the antigen-specific targeting region, transmembrane domain, and intracellular activation domain.

18. The use of claim 2, the method of claim 4, wherein the use or the method further comprises:

collecting blood comprising T cells from an individual prior to contacting the T cells ex vivo with the replication defective recombinant retrovirus; and

Introducing the genetically modified T cell into the individual.

19. The use or method of claim 18, wherein the genetically modified T cells are not expanded ex vivo after the contacting and prior to being introduced into the individual.

20. The replication defective recombinant retroviral particle of claim 1, the use of claim 2, the genetically modified T cell of claim 3, the method for genetically modifying and/or transducing a T cell of claim 4, wherein the membrane bound T cell activating assembly comprises an anti-CD 3 antibody.

21. The replication defective recombinant retroviral particle of claim 1, the use of claim 2, the method for genetically modifying and/or transducing T cells of claim 4, wherein the replication defective recombinant retroviral particle is a lentiviral particle.

22. The replication defective recombinant retroviral particle of claim 1, the use of claim 2, the genetically modified T cell of claim 3, the method for genetically modifying and/or transducing a T cell of claim 4, wherein the LE is a chimeric LE comprising two or more intracellular signaling domains each from a different gene.

23. The replication defective recombinant retroviral particle of claim 1, the use of claim 2, the genetically modified T cell of claim 3, the method for genetically modifying and/or transducing a T cell of claim 4, wherein the LE is a chimeric LE comprising an intracellular signaling domain, a transmembrane domain and an extracellular domain.

24. The use, genetically modified T cell, method for genetically modifying and/or transducing a T cell, replication defective recombinant retroviral particle according to claim 23, wherein the extracellular domain of the chimeric LE comprises a dimeric motif or a multimerization.

25. The use, genetically modified T cell, method for genetically modifying and/or transducing a T cell, replication defective recombinant retroviral particle according to claim 23, wherein the extracellular domain of the chimeric LE does not bind to a ligand of an interleukin receptor.

26. The replication defective recombinant retroviral particle of claim 1, the use of claim 2, the genetically modified T cell of claim 3, the method for genetically modifying and/or transducing a T cell of claim 4, wherein the LE is constitutively active.

27. The replication defective recombinant retroviral particle of claim 1, the use of claim 2, the genetically modified T cell of claim 3, the method for genetically modifying and/or transducing a T cell of claim 4, wherein the LE possesses the following properties:

a) Under the same conditions, the amplification of pre-activated PBMCs transduced with retroviral particles comprising a nucleic acid construct encoding the LE is improved when transduced with a nucleic acid encoding an anti-CD 19 antigen specific targeting region comprising a cd3ζ intracellular activation domain but no co-stimulatory domain between day 7 and day 21 of in vitro culture in the absence of exogenously added cytokines compared to a control construct identical to the nucleic acid construct comprising the LE but without the LE; and/or

B) When transduced with a nucleic acid encoding an anti-CD 19 antigen-specific targeting region comprising a cd3ζ intracellular activating domain but no co-stimulatory domain, preactivated PBMCs transduced with a nucleic acid construct encoding the LE will be amplified at least 2-fold between day 7 and day 21 of in vitro culture in the absence of exogenously added cytokines.

28. The replication defective recombinant retroviral particle, use, genetically modified T cell, method for genetically modifying and/or transducing T cell according to claim 27, wherein the improvement is determined using a statistical method and a cut-off p value equal to or less than 0.1.

29. The replication defective recombinant retroviral particle of claim 1, the use of claim 2, the genetically modified T cell of claim 3, the method for genetically modifying and/or transducing a T cell of claim 4, wherein the LE comprises a first intracellular signaling domain and a second intracellular signaling domain, respectively, selected from the group consisting of: MPL and CD40; MPL and CD79B; MPL and TNFRSF4; MPL and CD79A; MPL and CD3G; CSF2RA and TNFRSF4; CSF2RA and CD28; CSF2RA and TNFRSF8; CSF2RA and CD27; CSFR3 and CD79B; IFNAR2 and TNFRSF14; IL3RA and CD40; IL10RA and CD79B; IL11RA and FCGR2A; IL13RA2 and TNFRSF14; IL18RAP and CD3G; IL27RA and FCGR2A; LEPR and CD3G; myD88 and CD79B; or MyD88 and CD3D.

30. The replication defective recombinant retroviral particle of claim 1, the use of claim 2, the genetically modified T cell of claim 3, the method for genetically modifying and/or transducing a T cell of claim 4, wherein the LE comprises an intracellular signaling domain of MPL.

31. The use, genetically modified T cell, method for genetically modifying and/or transducing a T cell, replication defective recombinant retroviral particle according to claim 30, wherein the LE further comprises an intracellular signaling domain of CD 40.

32. The replication defective recombinant retroviral particle of claim 20, wherein the replication defective recombinant retroviral particle further comprises a membrane-bound cytokine on the surface of the replication defective recombinant retroviral particle, wherein the membrane-bound cytokine comprises a fusion polypeptide of IL-7 and DAF, and wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 286.