WO2024226858A1

WO2024226858A1 - Methods for viral vector manufacturing

Info

Publication number: WO2024226858A1
Application number: PCT/US2024/026349
Authority: WO
Inventors: Julia Jaemi PROCTOR; Ashley BALTES; Dorothy Copeland MAXWELL; Benjamin John TILLOTSON; Roland Maxwell Houck ASHTON
Original assignee: Juno Therapeutics Inc
Current assignee: Juno Therapeutics Inc
Priority date: 2023-04-26
Filing date: 2024-04-25
Publication date: 2024-10-31
Anticipated expiration: 2025-10-26

Abstract

Provided herein are methods for developing a lentiviral vector manufacturing process by quality of design methods. The methods can be used select upstream process parameters that can be employed in conjunction with a downstream process to result in a process with desired vector performance and high efficiency recovery and purity of viral vector.

Description

METHODS FOR VIRAL VECTOR MANUFACTURING

Cross-Reference to Related Applications

[0001] This application claims priority to U.S. Provisional Patent Application No. 63/462,211, filed April 26, 2023, entitled “METHODS FOR VIRAL VECTOR MANUFACTURING,” which is herein incorporated by reference in its entirety for all purposes.

Reference to an Electronic Sequence Listing

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 73504-20274.40.XML created March 18, 2024 which is 85,872 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.

Field

[0003] The present disclosure provides methods for developing a lentiviral vector manufacturing process by quality of design methods. The methods can be used select upstream process parameters that can be employed in conjunction with a downstream process to result in a process with desired vector performance and high efficiency recovery and purity of viral vector.

Background

[0004] Lentiviral vectors (LVVs) are often used as the gene delivery tool for manufacturing CAR T cell therapies. There is a growing need for new LVV manufacturing platforms that can reduce cost of goods manufactured (COGm) and meet supply demands of commercial cell therapy products. Provided herein are embodiments that meet such needs.

Summary

[0005] Provided herein is a method for determining upstream lentiviral manufacturing conditions, the method comprising: (a) performing a plurality of transfection reactions by transfecting host cells with a plurality of transfection mixtures, each transfection mixture comprising (1) a mixture of plasmids for the production of a lentiviral vector, wherein the mixture of plasmids comprise at least two lentiviral helper plasmids and a transfer plasmid encoding a transgene, and (2) a transfection agent, wherein each of the plurality of transfection mixtures has a different mass fraction ratio of plasmids in the mixture of plasmids and the same total amount of DNA; (b) harvesting the culture supernatant from each transfection reaction, and optionally clarifying the supernatant of the harvested culture to produce a clarified harvest; (c) determining lentiviral vector performance from each of the harvested culture supernatants or the clarified harvest, wherein viral vector performance is determined from both an analytical measure of a cell-line titer and a primary cell titer; and (d) selecting a transfection mixture for an upstream processing method for producing a lentiviral vector based on the lentiviral vector performance that has been determined for the transfection mixture.

[0006] Also provided herein is a method for determining upstream lentiviral manufacturing conditions, the method comprising: (a) performing a plurality of transfection reactions by transfecting host cells with a plurality of transfection mixtures, each transfection mixture comprising (1) a mixture of plasmids for the production of a lentiviral vector, wherein the mixture of plasmids comprise at least one lentiviral packaging plasmid encoding a viral gene selected from rev, gag or pol, an envelope plasmid encoding VSV-G, and a transfer plasmid encoding a transgene, and (2) a transfection agent, wherein each of the plurality of transfection mixtures has a different mass fraction ratio of plasmids in the mixture of plasmids and the same total amount of DNA, wherein the mass fraction of the envelope plasmid is independently from 0.04 to 0.2; (b) harvesting the culture supernatant from each transfection reaction, and optionally clarifying the supernatant of the harvested culture to produce a clarified harvest; (c) determining lentiviral vector performance from each of the harvested culture supernatants or the clarified harvest; and (d) selecting a transfection mixture for an upstream processing method for producing a lentiviral vector based on the lentiviral vector performance that has been determined for the transfection mixture. In some embodiments, the lentiviral vector performance is determined from both an analytical measure of a cell line titer and a primary cell titer.

[0007] Provided herein is a method for determining upstream lentiviral manufacturing conditions, the method comprising: (a) performing a plurality of transfection reactions by transfecting host cells with a plurality of transfection mixtures, each transfection mixture comprising (1) a mixture of plasmids for the production of a lentiviral vector, wherein the mixture of plasmids comprise at least one lentiviral helper plasmid and a transfer plasmid encoding a transgene and (2) a transfection agent, wherein each of the plurality of transfection mixtures has a different mass fraction ratio of plasmids in the mixture of plasmids and the same total amount of DNA; (b) harvesting the culture supernatant from each transfection reaction, and optionally clarifying the supernatant of the harvested culture to produce a clarified harvest; (c) determining lentiviral vector performance from each of the harvested culture supernatants or the clarified harvest, wherein viral vector performance is determined from both an analytical measure of a cell line titer and a primary cell titer; (d) performing a downstream purification on the harvested culture supernatant or the clarified harvest from each of a subset of the transfection reactions; and (e) selecting a transfection mixture for an upstream processing method for producing a lentiviral vector based on the downstream purification performance that has been determined for the transfection mixture.

[0008] In some of any of such embodiments, the at least two helper plasmids encode at least one packaging viral gene and at least one envelope viral gene. In some embodiments, the envelope viral gene is VSV-G. In some embodiments, the packaging viral gene is rev, gag or pol or a combination of any of the foregoing. In some embodiments, the mixture of plasmids comprises a packaging plasmid encoding gag and pol (Gag-Pol plasmid), an envelope (Env) plasmid encoding VSV-G, and a transfer plasmid encoding the transgene. In some of any of such embodiments, the mixture of plasmids is a three-plasmid mixture, a four-plasmid mixture or a five-plasmid mixture.

[0009] In some of any of such embodiments, the mixture of plasmids is a three plasmid mixture composed of a packaging plasmid encoding gag and pol (Gag-Pol plasmid), an envelope (Env) plasmid encoding VSV-G, and a transfer plasmid encoding the transgene. In some of any of such embodiments, the mixture of plasmids is a four-plasmid mixture composed of a packaging plasmid encoding gag and pol (Gag-Pol plasmid), a packaging plasmid encoding rev (Rev plasmid), an envelope (Env) plasmid encoding VSV-G, and a transfer plasmid encoding the transgene.

[0010] In some of any of such embodiments, the subset of transfection reactions each have a different mass fraction ratio of the packaging plasmid encoding Gag-Pol or the envelope plasmid encoding VSV-G relative to the other plasmids in the mixture of plasmids. In some of any of such embodiments, the subset of transfection reactions each have a different mass fraction ratio of the packaging plasmid encoding Gag-Pol relative to the other plasmids in the mixture of plasmids. In some of any of such embodiments, the subset of transfection reactions each have a different mass ratios of the envelope plasmid encoding VSV-G relative to the other plasmids in the mixture of plasmids. In some of any of such embodiments, the subset of transfection reactions each have a different mass ratio of the packaging plasmid encoding Gag- Pol and a different mass ratio of the envelope plasmid encoding VSV-G, relative to the other plasmids in the mixture of plasmids. In some of any of such embodiments, the subset of transfection mixtures are candidate transfection mixtures that were identified to have the highest levels of viral vector performance from among the plurality of transfection mixtures in (c).

[0011] In some of any of such embodiments, the plurality of transfection reactions is a design of experiments (DOE).

[0012] In some of any of such embodiments, the mass fraction of the plasmid encoding VSV-G is varied in the plurality of transfection mixtures. In some of any of such embodiments, the mass fraction of the plasmid encoding VSV-G in each of the transfection mixtures is independently from 0.04 to 0.2. In some of any of such embodiments, the mass fraction of the plasmid encoding VSV-G among each of the transfection mixtures is independently from 0.046 to 0.15. In some of any of such embodiments, the mass fraction of each of one or more of the plasmids encoding rev, gag or pol is held constant among each of the transfection mixtures.

[0013] In some of any of such embodiments, the mass fraction of the plasmid encoding rev is held constant among each of the transfection mixtures. In some of any of such embodiments, the mass fraction of the plasmid encoding rev among each of the transfection mixtures is independently from 0.04 to 0.08, optionally from 0.04 to 0.06. In some of any of such embodiments, the mass fraction of the plasmid encoding rev among each of the transfection mixtures is held constant at about 0.049.

[0014] In some of any of such embodiments, the mass fraction of the plasmid encoding gag and/or pol, optionally gag and pol, is held constant among each of the transfection mixtures. In some of any of such embodiments, the mass fraction of the plasmid encoding gag and/or pol, optionally gag and pol, among each of the transfection mixtures is independently from 0.1 to 0.25, optionally from 0.15 and 0.2. In some of any of such embodiments, the mass fraction of the plasmid encoding gag and/or pol, optionally gag and pol, among each of the transfection mixtures is held constant at about 0.178.

[0015] In some of any of such embodiments, the mass fraction of the plasmid encoding the transgene is varied in the plurality of transfection mixtures. In some of any of such embodiments, the mass fraction of the plasmid encoding the transgene among each of the transfection mixtures is independently from 0.47 to 0.82, optionally from 0.58 to 0.74.

[0016] In some of any of such embodiments, the plurality of transfection mixtures is 3 to 50, optionally 3 to 30.

[0017] In some of any of such embodiments, the candidate transfection mixture in (d) is identified using multiple-response optimization. In some embodiments, the multiple-response optimization is characterized by a desirability function for each analytical measure of a cell line titer and a primary cell titer. In some embodiments, the desirability function for primary cell titer is to be maximized over the cell line titer. In some embodiments, the desirability function for primary cell titer and cell line titer are equal. In some of any of such embodiments, the desirability function for primary cell titer is about 1.0.

[0018] In some of any of such embodiments, the primary cell titer is a functional titer determined by a primary cell transduction assay. In some embodiments, the primary cell transduction assay comprises transducing target cells with the harvested culture supernatant or clarified harvest, incubating the transduced target cells under conditions for expression of the transgene, and analyzing expression of the transgene by the cells. In some embodiments, cell surface expression of the transgene is analyzed by flow cytometry. In some of any of such embodiments, the target cells are T cells and prior to the transducing the method comprises activating the T cells with a T cell stimulatory reagent, optionally wherein the T cell stimulatory reagent is an anti-CD3/anti-CD28 activation reagent. In some of any of such embodiments, the T cells are primary cells selected from a subject, optionally wherein the subject is a healthy subject. In some of any of such embodiments, the T cells are CD4+ T cells, CD8+ T cells or CD4+ and CD8+ T cells. In some of any of such embodiments, the target cells are T cells and incubating the transduced target cells under conditions for expression of the transgene comprises incubating the cells in the presence of one or more T cell stimulatory recombinant cytokines, optionally IL-2 IL-7, IL- 15 or IL-21 or a combination of any of the foregoing. In some embodiments, the incubating expands the T cells. In some embodiments, the incubating is for 2 to 10 days.

[0019] In some of any of such embodiments, the target cells are T cells and incubating the transduced target cells under conditions for expression of the transgene comprises incubating the cells in basal media without recombinant cytokines. In some embodiments, the incubating is for 12 hours to 48 hours. [0020] In some of any of such embodiments, the cell line titer is a functional titer or and/or an infectious titer. In some of any of such embodiments, the desirability function of the cell line titer is less than 1.0, optionally 0.5. In some of any of such embodiments, the desirability function of the cell line titer is about 1.0.

[0021] In some of any of such embodiments, the analytical measure of a cell line titer is an analytical measure in at least two cell line titer assays. In some embodiments, the at least two cell line titer assays are at least one infectious titer and at least one functional titer. In some of any of such embodiments, the desirability function of each of the at least two cell line titer is less than 1.0, optionally less than 0.5. In some of any of such embodiments, the desirability function of each of the at least two cell line titer combined is about 1.0.

[0022] In some of any of such embodiments, the infectious titer is determined by an endpoint dilution assay (TCID50) or a qPCR lentivirus titer assay. In some of any of such embodiments, the infectious titer is determined by a qPCR lentivirus titer assay. In some embodiments, the qPCR comprises primers and probes for amplification of a region of the LTR of the genomic RNA. In some of any of such embodiments, the functional titer is determined by a cell-based transduction assay. In some embodiments, transduction is determined by measuring transgene expression by flow cytometry. In some of any of such embodiments, the cell line titer assay is titer on an immortalized cell line. In some embodiments, the immortalized cell line is a Jurkat cell line.

[0023] In some of any of such embodiments, the downstream purification is by chromatography. In some of any of such embodiments, the downstream purification comprises chromatography and ultrafiltration/diafiltration (UF/DF). In some of any of such embodiments, the chromatography is by a method selected from the group consisting of heparin affinity, gel filtration and anion-exchange (AEX). In some of any of such embodiments, the chromatography is anion-exchange (AEX). In some of any of such embodiments, the downstream purification further comprises sterile filtration.

[0024] In some of any of such embodiments, the downstream purification performance for selecting the candidate transfection mixture in (f) is elution profile for vector performance, residual protein, residual BSA, residual plasmid or host-cell DNA or a combination of any of the foregoing. [0025] In some embodiments, the elution profile for vector performance comprises testing a plurality of elution fractions for infectious titer of the viral vector, optionally wherein infectious titer is assessed using a cell-based transduction assay.

[0026] In some of any of such embodiments, the method comprises optionally repeating steps (a)-(d) to identify different candidate transfection mixtures if the downstream purification performance is not acceptable.

[0027] In some of any of such embodiments, the method further comprises: (g) varying one or more parameters in a downstream process for producing the lentiviral vector comprising the downstream purification, wherein the downstream process is carried out from material harvested from an upstream process using the selected candidate transfection mixture.

[0028] In some embodiments, the upstream processing method comprises: (i) transfecting the host cells in a large-scale culture with the selected transfection mixture; (ii) harvesting the supernatant from the transfected culture; and (iii) clarifying the harvested culture supernatant by centrifugation or filtration.

[0029] In some of any of such embodiments, the host cells are adherent cells. In some of any of such embodiments, the host cells are suspension cells. In some of any of such embodiments, the host cells are HEK293T cells or a derivative thereof, optionally wherein the host cells are HEK 293T/17 cells.

[0030] In some of any of such embodiments, an endonuclease is added to the culture supernatant prior to harvesting the supernatant from the transfected culture. In some embodiments, the endonuclease is Benzonase.

[0031] In some of any of such embodiments, the transgene is a chimeric antigen receptor (CAR). In some embodiments, the chimeric antigen receptor is a monospecific CAR. In some embodiments, the chimeric antigen receptor is a bispecific CAR.

[0032] In some of any of such embodiments, the transfection agent is a cationic polymer. In some embodiments, the cationic polymer is polyethylenimine (PEI). In some embodiments, the mass ratio of the mixture of plasmid DNA to PEI is 5:1 to 1:5, optionally 3:1 to 1:3. In some of any of such embodiments, the mass ratio of the mixture of plasmid DNA to PEI is 1:1. In some of any of such embodiments, the mixture of plasmid DNA and PEI are provided as a transfection complex, optionally stabilized with fetal bovine serum (FBS) or human serum albumin (HSA).

[0033] Provided herein is a method manufacturing a lentiviral vector, the method comprising: (a) transiently transfecting host cells to produce a transfected culture with (i) a transfection mixture selected according to any of the provided methods, and (ii) a transfection agent to make a transfected culture, and optionally: (b) harvesting the supernatant from the transfected culture; (c) clarifying the harvested culture supernatant by filtration; (d) capturing and concentrating the lentiviral vector from the harvested and clarified supernatant by anion exchange chromatography (AEX); (e) ultrafiltering and diafiltering the lentiviral vector using Tangential Flow Filtration (TFF); (f) sterile filtering the lentiviral vector.

[0034] Provided herein is a method of manufacturing a lentiviral vector, the method comprising: (a) transiently transfecting host cells with a transfection mixture comprising (1) a mixture of plasmids for the production of a lentiviral vector, wherein the mixture of plasmids is a four-plasmid mixture comprising a lentiviral packaging plasmid encoding gag and pol at a mass fraction from 0.15 and 0.2, a lentiviral packaging plasmid encoding rev at a mass fraction from 0.04 to 0.06, an envelope plasmid encoding VSV-G at a mass fraction from 0.04 to 0.15, and a transfer plasmid encoding a chimeric antigen receptor at a mass fraction from 0.58 to 0.74, and (2) a transfection agent; (b) harvesting the supernatant from the transfected culture; (c) clarifying the harvested culture supernatant by filtration; (d) capturing and concentrating the lentiviral vector from the harvested and clarified supernatant by anion exchange chromatography (AEX); (e) ultrafiltering and diafiltering the lentiviral vector using Tangential Flow Filtration (TFF); (f) sterile filtering the lentiviral vector.

[0035] In some embodiments, the mass fraction of the packaging plasmid encoding gag and pol is about 0.178, the mass fraction of the lentiviral packaging plasmid encoding rev is about 0.049, the mass fraction of the envelope plasmid encoding VSV-G is about 0.046, and the mass fraction of the transfer plasmid encoding the CAR is about 0.727.

[0036] In some of any of such embodiments, the AEX chromatography includes a wash with about 300 mM NaCl and an elution with about 450 mM NaCl. In some of any of such embodiments, clarifying the harvested culture supernatant is by membrane filtration. In some embodiments, the membrane filtration is with a dual-layer filter. In some of any of such embodiments, sterile filtering is by membrane filtration. In some embodiments, sterile filtering is with a dual-layer filter.

[0037] In some of any of such embodiments, the host cells are HEK293 cells. In some embodiments, the HEK293 cells are HEK-293T/17 cells.

[0038] In some of any of such embodiments, the CAR is an anti-CD19 CAR. In some of any of such embodiments, the CAR contains in order from N-terminus to C-terminus: an extracellular antigen-binding domain that is the scFv set forth in SEQ ID NO: 43, the spacer set forth in SEQ ID NO:1, the transmembrane domain set forth in SEQ ID NO: 8, the 4- IBB costimulatory signaling domain set forth in SEQ ID NO: 12, and the signaling domain of a CD3- zeta (CD3Q chain set forth in SEQ ID NO: 13. In some of any of such embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO:57 or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 57. In some of any of such embodiments, the nucleotide sequence encoding the CAR comprises the sequence set forth in SEQ ID NO:58 or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 58.

[0039] In some of any of the provided embodiments, the volume of the transfection reaction is from 5 mL to 5000 mL or from about 5 mL to about 5000 mL. In some of any of the provided embodiments, the volume of the transfection reaction is from or from about 10 mL to 2000 mL. In some of any of the provided embodiments, the volume of the transfection reaction from or from about 500 mL to 2000 mL.

Brief Description of the Drawings

[0040] FIG. 1 shows a schematic of transfection of producer host cells with four plasmids for lentiviral vector production in an upstream process. The four plasmids are the transfer plasmid carrying the gene of interest (also called transgene plasmid), the Gag-Pol plasmid carrying capsid and enzymes for packaging, the Rev plasmid carrying the Rev gene for packaging and the VSV-G plasmid carrying the envelope protein.

[0041] FIG. 2 shows a subset of (six) plasmid ratio conditions tested from the plasmid ratio mixture DOE experiment performed to show the range of CAR expression (% CD3+CAR+ cells) achieved over the same four vector volumes tested in the DP process.

[0042] FIG. 3 shows varying correlations between cell line functional titer (Jurkat titer assay) and primary cell functional titer by CAR expression (%CD3+CAR+ T cells) based on amount of gag-pol plasmid used in the mixture DOE experiment studying plasmid ratios during transfection. Each dot represents a different plasmid ratio condition. The dots surrounded by the solid line have gag-pol plasmid ratios of about 0.00-0.10, the dots surrounded by the small circle dotted line have gag-pol plasmid ratios of about 0.15-0.25 and the dots surrounded by the large dash dot line have gag-pol plasmid ratios of about 0.30-0.40. [0043] FIG. 4 provides a broad schematic for the upstream and downstream processes in a lentiviral manufacturing process.

[0044] FIG. 5 depicts a JMP Prediction Profiler for cell line titer (e.g. Jurkat Titer, LVTA19 Titer), and a primary cell functional titer by CAR expression (CD3+CAR+; calculated lU/mL) as a function of plasmid ratios for gag-pol plasmid, rev plasmid, vsv-g plasmid and transgene plasmid used in the upstream process. The primary cell functional titer was an average of the CAR frequency (%CD3+CAR+) values * # of cells transduced / vector volume added for the two lowest vector volume conditions tested. For the JMP model, the positive linear lines in the far right Desirability graphs indicate desirability is set to maximize cell line titer (Jurkat titer and LVTA19 titer; importance =0.5 and primary cell functional titer (calculated CAR titer; importance = 1.0).

[0045] FIG. 6 shows a titration of functional performance (frequency of CD3+CAR+ T- cells) of lentiviral vector (increasing vector volume) produced from upstream processes using different selected plasmid ratios (0.046 VSV-G or 0.15 VSV-G). For comparison, also shown are CD3+ CAR+ frequency transduction vector performance for Optimum DSP Yield Ratio (“low VSV-G”) upstream process. A hashed line representing desired CAR expression also is shown.

[0046] FIG. 7 shows an overlay of cell line functional titer (Jurkat titer assay) and residual dsDNA of each elution fraction of lentiviral vector purified in the AEX chromatography step of the downstream process from material produced using plasmid ratio #1 (0.046 VSV-G) or plasmid ratio #3 (0.15 VSV-G) based on NaCl (mM) concentration used for elution. Also shown is cell line infectious titer and residual dsDNA of each elution fraction of lentiviral vector purified in the AEX chromatography step of the downstream process from material produced using the optimum DSP Yield Titer (“low VSV-G”).

Detailed Description

[0047] Provided herein are methods for the development of viral vectors, such as lentiviral vectors. The provided embodiments provide for improved methods for developing lentiviral vector (LVV) manufacturing methods by using a Quality by Design (QbD) approach. In particular, the QbD approach provided herein is particularly ideal for developing a lentiviral vector for use in a cellular drug product process in which the LVV is for use in transducing primary cells. [0048] A problem with existing methods of manufacture is that the processes for optimizing upstream and downstream processes are typically carried out independently or without considerations of factors that impact downstream process performance. For instance, typical LVV product development involves locking parameters in an upstream process without consideration of impact on a downstream process. The result is that when the process is applied to a downstream process an overall low yield may be achieved.

[0049] Balancing yield, impurity clearance, and vector quality in downstream processing is a major challenge faced when developing LVV processes. The provided embodiments are based on observations herein that high upstream physical and vector titer readouts may not always be predictive of the resulting overall process yield and the transduction frequency achieved in primary cells. The approaches to LVV process development disclosed herein indicate that assessing functionality/performance of the LVV (termed “primary cell titer” herein) is an important readout. It was also found herein that interactions between upstream parameters and downstream unit operations, such as anion-exchange chromatography, can impact the efficiency of a process, including overall purity and recovery of LVV. For instance, as shown herein, the plasmid ratio during the transient transfection step was identified as a parameter that impacted the relationship between upstream productivity, overall process yields through downstream unit operations, and drug product critical quality attributes (CQAs).

[0050] The above observations have resulted in the provided methods in which impact of upstream process parameters on downstream performance are considered to optimize overall recovery in a downstream process. For instance, provided methods include assessing functional titer early to elucidate the impact of upstream process parameters on transduction and transgene (e.g. CAR) expression in primary cells thereby ensuring upstream process parameters are chosen that are most likely to meet a target product profile (TPP). The provided methods also have established that plasmid ratios used in transfection of producer cells in an upstream process, and particularly of the plasmid encoding the envelope protein (e.g., VSV-G), is an important variable in achieving high yield in a downstream process. In some cases, the provided methods involve determining a VSV-G plasmid ratio or VSV-G plasmid ratio range that allows effective downstream purification of the LVV and using the determined VSV-G plasmid ratio or VSV-G plasmid ratio range for optimizing upstream performance parameters. In some cases, the provided methods comprise using lower overall VSV-G plasmid ratios relative to other plasmids (e.g., 0.150 or less mass fraction ratio), thereby providing desired upstream performance along with effective downstream purification to ensure high recovery. In some embodiments, provided methods also can include doing some downstream testing of harvested LVV from upstream processes prior to locking an upstream process, and if necessary, re-evaluating upstream material process to meet a target product profile. The provided methods minimize inefficiencies in a downstream process due to upstream harvest material being incompatible with a downstream platform. As a result, based on downstream performance, upstream process parameters, such as plasmid ratio, are chosen that meet the transgene (e.g. CAR) target and achieve target yield and impurity clearance in a downstream purification process (e.g., involving chromatography, TFF and sterile filtration).

[0051] The provided methods allow for design of LVV manufacturing methods that satisfy demands for quality LVV reagents used for producing engineered cell therapies. Notably, the provided methods ensure that LVV vectors used to produce engineered T cell therapy drug products, such as CAR-T cell therapies, are fully functional and meet the target product profile (TPP). In addition, the QbD approach can allow for selection of upstream and downstream processes to achieve this faster than existing methods by saving months (e.g., 3-4 months) of development, since the methods consider impacts of upstream process parameters on overall process yields and purity in downstream processes. The resulting methods thus enable scalability of downstream purification processes while achieving high recovery as a result of overall process yields in the downstream process. These methods thus address issues of vector supply by maximizing the number of patients that can be treated per vector manufacturing lot.

[0052] All publications, including patent documents, scientific articles and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference.

[0053] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. I. UPSTREAM PROCESS DEVELOPMENT AND DESIGN OF MIXTURE

METHODS

[0054] Provided herein is a method for determining upstream viral vector process conditions using a design of mixtures approach. In some embodiments, the provided embodiments are based on recognition that an upstream process for preparing viral vector is impacted not only by the ratio of plasmid mixtures in the transfection reaction, but also that this choice of parameters can impact efficiency and effectiveness (e.g., recovery and/or purity) of the downstream process. Moreover, although typical processes for upstream process development in lentiviral manufacturing assess performance in a cell line-based titer assay, it is found herein that this does not necessarily correlate to determination of optimal conditions for functional expression of a transgene in primary cells. Thus, provided methods herein also consider performance in a primary cell titer assay, such as functional titer by transgene expression in primary cells.

[0055] In some embodiments, the method includes (a) performing a plurality of transfection reactions by transfecting host cells with a plurality of transfection mixtures, each transfection mixture comprising (1) a mixture of plasmids for the production of a retroviral vector, such as a lentiviral vector, wherein the mixture of plasmids comprise at least one helper plasmid and a transfer plasmid encoding a transgene, and (2) a transfection agent, wherein each of the plurality of transfection mixtures has a different mass fraction ratio of plasmids in the mixture of plasmids and the same total amount of DNA; (b) harvesting the culture supernatant from each transfection reaction; (c) determining viral vector performance from each of the harvested culture supernatants, wherein viral vector performance is determined from both an analytical measure of a cell line titer and a primary cell titer; and (d) selecting a transfection mixture for an upstream processing method for producing a lentiviral vector based on the lentiviral vector performance that has been determined for the transfection mixture. In some embodiments, the culture supernatant from each transfection reaction can be clarified before determining virial vector performance. In such embodiments, viral vector performance is determined on the clarified harvest.

[0056] In some embodiments, the impact of parameter design in an upstream process are assessed for their impact on the downstream process for producing the viral vector, prior to selecting the transfection mixture for an upstream processing method. In some embodiments, the method includes (a) performing a plurality of transfection reactions by transfecting host cells with a plurality of transfection mixtures, each transfection mixture comprising (1) a mixture of plasmids for the production of a lentiviral vector, wherein the mixture of plasmids comprise at least one lentiviral helper plasmid and a transfer plasmid encoding a transgene and (2) a transfection agent, wherein each of the plurality of transfection mixtures has a different mass ratio of plasmids in the mixture of plasmids and the same total amount of DNA; (b) harvesting the culture supernatant from each transfection reaction; (c) determining lentiviral vector performance from each of the harvested culture supernatants, wherein viral vector performance is determined from both an analytical measure of a cell line titer and a primary cell titer; (d) performing one or more steps of a downstream process for viral vector production on the harvested culture supernatant or the clarified harvest from each of a subset of the transfection reactions; and (e) selecting a transfection mixture for an upstream processing method for producing a lentiviral vector based on the downstream purification performance that has been determined for the transfection mixture. In some embodiments, the culture supernatant from each transfection reaction can be clarified before determining virial vector performance. In such embodiments, viral vector performance is determined on the clarified harvest.

[0057] The one or more steps in the downstream process for manufacturing viral vector can be any as described in Section II.B. In some embodiments, the feed from harvests or clarified harvests of a subset of transfections of the design mixture are used to feed into the one or more steps of the downstream process. In some embodiments, the one or more steps of the downstream process at least includes a downstream purification step. In some embodiments, the downstream purification step is a capture chromatography, such as anion exchange chromatography (AEX). In some embodiments, the one or more downstream purification steps that are performed in the provided design method is a capture chromatography step such as AEX and Ultrafiltration/Diafiltration (UF/DF). In some embodiments, the one or more downstream purification steps that are performed in the provided design method is a capture chromatography step such as AEX and sterile filtration. In some embodiments, the one or more downstream purification steps that are performed in the provided design method is a capture chromatography step such as AEX, Ultrafiltration/Diafiltration (UF/DF), and sterile filtration.

[0058] In some embodiments, following the plurality of transfection reactions in the upstream process for producing the viral vector (e.g. lentiviral vector) in a design of experiment, the harvest of each of the plurality of transfection reactions is assessed. In some embodiments, the crude supernatant is assessed for titer. In some embodiments, the crude supernatant is clarified, such as by a filtration method. Exemplary methods of clarification are described in Section ILA. In some embodiments, titer is assessed using one or more cell line titer method(s) and a primary cell titer method.

[0059] In some embodiments, titer is the response in the design of experiments. Methods for assessing titer are described below. In some embodiments, the titer can be a cell line titer. In some embodiments, the titer can be a primary cell titer. In some embodiments, a multipleresponse optimization is carried out in which at least one cell line-based titer and at least one primary cell-based titer are assessed. In some aspects, a particular titer assay (e.g. cell line-based or primary cell-based) is weighted. In some embodiments, the weight determines how the desirability is distributed such that it determines the shape of the desirability function for each response. The weight can be used to translate the response scale to the zero-to-one desirability scale to determine the individual desirability of a response. In provided aspects, a weight is selected from 0.1 to 10 to emphasize or de-emphasize desirability of a response. A weight less than one places less emphasis on the response. A weight equal to one places equal important on the responds. A weight of greater than one (e.g. maximum 10) places more emphasis on the response.

[0060] In some embodiments, the weight of the cell line-based titer is equal to the weight of the primary cell line-based titer. In some embodiments, the weight of the cell-line based titer is 1.0 and the weight of the primary cell line-based titer is 1.0. In some embodiments, the weight of the primary cell titer assay is maximized over the cell line titer assay, for example the weight of the primary cell titer assay is 1.0 or greater and the weight of the cell line titer assay is less than 1.0. In some embodiments, the weight of the cell line titer assay is maximized over the primary cell titer assay, for example the weight of the cell line titer assay is 1.0 or greater and the weight of the primary cell titer assay is less than 1.0.

[0061] In some embodiments, the cell line-based titer assay is at least one cell line-based titer assay. In some embodiments, the at least one cell line-based titer assay is an infectious titer, a functional titer or both an infectious titer and a functional titer. In some embodiments, the at least one cell-line based titer is determined by at least two different cell line -based titer assays. In some embodiments, the at least one cell-line based titer is determined by an infectious titer and a functional titer. In some embodiments, the weight of the at least two different cell linebased titer assays is the same. In some embodiments, the weight of the at least two different cell line-based titer assays is different. In some embodiments, the at least one cell-line based titer is determined by an infectious titer and a functional titer, and the weight of the infectious titer assay and the functional titer assay is the same. In some embodiments, the weight of the infectious titer assay is 0.5 and the weight of the functional titer assay is 0.5. In some embodiments, the at least one cell line-based titer is determined by an infectious titer and a functional titer, and the weight of the infectious titer assay and the functional titer assay are different. In some embodiments, the weight of the functional titer assay is maximized over the infectious titer assay, for example the weight of the functional titer assay is 1.0 or greater and the weight of the infectious titer assay is less than 1.0. In some embodiments, the weight of the infectious titer assay is maximized over the functional titer assay, for example the weight of the infectious titer assay is 1.0 or greater and the weight of the functional titer assay is less than 1.0.

[0062] In some embodiments, the primary cell-based titer assay is at least one primary cellbased titer assay. In some embodiments, the at least one primary cell-based titer assay is an infectious titer, a functional titer or both an infectious titer and a functional titer. In some embodiments, the primary cell- based titer is determined by a functional titer.

[0063] In some embodiments, the at least one primary cell-based titer is determined by an infectious titer and a functional titer. In some embodiments, the weight of the at least two different primary cell-based titer assays is the same. In some embodiments, the weight of the at least two different primary cell-based titer assays is different. In some embodiments, the at least one primary cell-based titer is determined by an infectious titer and a functional titer, and the weight of the infectious titer assay and the functional titer assay is the same. In some embodiments, the weight of the infectious titer assay is 0.5 and the weight of the functional titer assay is 0.5. In some embodiments, the at least one primary cell-based titer is determined by an infectious titer and a functional titer, and the weight of the infectious titer assay and the functional titer assay are different. In some embodiments, the weight of the functional titer assay is maximized over the infectious titer assay, for example the weight of the functional titer assay is 1.0 or greater and the weight of the infectious titer assay is less than 1.0. In some embodiments, the weight of the infectious titer assay is maximized over the functional titer assay, for example the weight of the infectious titer assay is 1.0 or greater and the weight of the functional titer assay is less than 1.0.

[0064] In some embodiments of the provided methods, a multiple-response optimization is carried out in which titer responses include two cell-line based titers, generally a functional titer and an infectious titer, and a primary cell-based titer. In some embodiments, the primary cell- based titer is a functional titer based on transgene expression. In some embodiments, the weight of each cell-line based tier is 0.5 and the weight of the primary cell-based titer is 1.0.

[0065] Various software packages are known and available to a skilled artisan for design of experiments analysis. An exemplary package is JMP® software from SAS (e.g., available from jmp.com).

[0066] In some embodiments, candidate plasmid mass fraction ratios that achieve the highest titer response as a measure of viral vector performance are selected. In some embodiments, the candidate plasmid mass fraction ratios are determined from the harvest or clarified harvest of the upstream process.

[0067] In some embodiments, the method includes first identifying a subset of mass fraction ratios based on titer response as a measure of viral vector performance from the harvest or clarified harvest of the upstream process, and then performing a downstream purification on the harvest culture supernatant or the clarified harvest from each of the subset of the transfection reactions. The downstream purification harvest is then assessed for titer response. The titer response can be a cell line-based titer, a primary cell- based titer or can include both a cell linebased titer and a primary cell-based titer. The performance in the downstream process by titer can then be used to select a candidate transfection reaction for an upstream processing method (e.g. particular mass fraction ratio of plasmids for transfection).

[0068] In some embodiments, the provided methods for determining lentiviral manufacturing conditions includes (a) performing a plurality of transfection reactions by transfecting host cells with a plurality of transfection mixtures, each transfection mixture comprising (1) a mixture of plasmids for the production of a lentiviral vector, wherein the mixture of plasmids comprise at least one lentiviral helper plasmid and a transfer plasmid encoding a transgene and (2) a transfection agent, wherein each of the plurality of transfection mixtures has a different mass fraction ratio of plasmids in the mixture of plasmids and the same total amount of DNA; (b) harvesting the culture supernatant from each transfection reaction, and optionally clarifying the supernatant of the harvested culture to produce a clarified harvest; (c) determining lentiviral vector performance from each of the harvested culture supernatants or the clarified harvest, wherein viral vector performance is determined from both an analytical measure of a cell line titer and a primary cell titer; (d) performing a downstream purification on the harvested culture supernatant or the clarified harvest from each of a subset of the transfection reactions; and (e) selecting a transfection mixture for an upstream processing method for producing a lentiviral vector based on the downstream purification performance that has been determined for the transfection mixture.

[0069] Non-limiting descriptions of aspects of the provided methods are further described in the following subsections.

A. Design of Mixtures

[0070] In embodiments of the provided methods, the method uses not only the ratios of plasmids used in the transient transfection but also the titer information of the harvested material (e.g. crude or clarified) for optimizing the selection of the plasmids, and the ratios in which to mix them in order to achieve a final viral vector product with desired properties, such as desired transgene expression, purity and/or recovery. In some embodiments, the experiment design (DOE) methods assume a fixed set of plasmid materials and provide methods for the selection of optimal ratios for the plasmids in the mixture. As such, the methods allow selection of optimal transfection reactions for an upstream process method that depends on the relative proportions of the plasmids in the mixture and not on the amount of the mixture. In some cases, the plasmid ratio of the envelope plasmid (e.g., encoding VSV-G) is varied within a defined range of the experiment design. Additionally or alternatively, both the titer information of the harvested material (e.g. crude or clarified) is considered on cell lines and on primary cells in making a selection of the plasmid ratios.

[0071] In some embodiments, the transfection reaction is carried out by transient cotransfection of host cells with a retroviral (e.g. lentiviral) vector backbone plasmid carrying a transgene of interest with helper plasmids expressing viral packaging functions. The transient transfection can be carried out using methods as described in Section II.A in an upstream viral vector manufacturing process. In particular embodiments, the genes for helper packaging functions are split between separate plasmids along with the transgene plasmid and the plasmids are provided as a mixture in the transient transfection. In the provided methods, the mass fraction of one or more of the different plasmids is varied in the mixture design. In some embodiments, the transfection reaction is carried out in which a plurality of transfection reactions are performed, where each plasmid component of the mixture is an input variable and each of the plurality of reactions has a different mass fraction ratio of plasmids in the mixture and the same total amount of DNA.

[0072] In some embodiments, the mixture designs is a simplex lattice, simplex centroid, simplex axial or extreme vertex design. [0073] The different functions necessary for the production of a lentiviral vector can be provided to the cells by any number of plasmids. In particular, these functions may be provided by at least two, three, four, five or more plasmids. In some embodiments, at least two plasmids are helper plasmids that encode genes that are necessary for packaging of the viral vector. In some embodiments, the at least two helper plasmids encode at least one packaging viral gene and at least one envelope viral gene. The envelope viral gene can be any as described in Section ILA. In some embodiments, the envelope viral gene is for pseudotyping the viral vector (e.g. lentiviral vector) with a heterologous envelope protein. In some embodiments, the heterologous envelope protein is an envelope protein derived from a different virus or a chimeric envelope protein. In some embodiments, the envelope viral gene is VSV-G. In some embodiments, the mixture of plasmids includes an envelope plasmid encoding VSV-G. In some embodiments, the packaging viral gene is rev, gag or pol or a combination of any of the foregoing. In some embodiments, the at least one packaging plasmid encodes gag and pol (Gag-Pol plasmid). In some embodiments, there are at least two packaging plasmids in which one encodes gag and pol (Gag-Pol plasmid) and the other encodes rev (Rev plasmid). In some embodiments, at least one plasmid is a transgene plasmid that encodes the transgene of interest. In some embodiments, the transgene of interest is between a lentiviral 3'-LTR and a lentiviral 5'-LTR. Each function (or component) can be derived from any suitable lentivirus. In some embodiments, the gag, pol, rev and lentiviral genome (3'-LTR and a 5'-LTR) are derived from an HIV virus, in particular from HIV-1 or HIV-2. In a particular embodiment, the different mixture of plasmids for production of a lentiviral vector are provided to the host cell such as a mammalian cell, for example a 293T cell growing by transfection.

[0074] In some embodiments, the transgene of interest is a transgene encoding a heterologous protein for which it is desired to delivery to a target cell. In particular embodiments, the target cell is a primary cell. In some embodiments, the target cell is a lymphocyte. In some embodiments, the target cell is a T cell. In some embodiments, the transgene of interest is a chimeric antigen receptor (CAR). In some embodiments, the CAR can be any of the CAR sequence known to a skilled artisan. Exemplary CARs are described in Section III. In some embodiments, the transgene of interest is an anti-CD19 CAR.

[0075] In some embodiments, the mixture of plasmids is a three -plasmid mixture, a four- plasmid mixture or a five-plasmid mixture. In some embodiments, the mixture of plasmids includes a packaging plasmid encoding gag and pol (Gag-Pol plasmid), an envelope (Env) plasmid encoding VSV-G, and a transfer plasmid encoding the transgene. In some embodiments, the mixture of plasmids further includes a plasmid that encodes rev (Rev plasmid).

[0076] In some embodiments, the mixture of plasmids is a three plasmid mixture composed of a packaging plasmid encoding gag and pol (Gag-Pol plasmid), an envelope (Env) plasmid encoding VSV-G, and a transfer plasmid encoding the transgene. In some embodiments, the host cells are transfected with three plasmids adapted for producing lentiviral vectors, wherein one plasmid encodes lentiviral Gag and Pol, one plasmid encodes envelope proteins (Env plasmid), and one plasmid encodes a transgene of interest.

[0077] In some embodiments, the mixture of plasmids is a four-plasmid mixture composed of a packaging plasmid encoding gag and pol (Gag-Pol plasmid), a packaging plasmid encoding rev (Rev plasmid), an envelope (Env) plasmid encoding VSV-G, and a transfer plasmid encoding the transgene. In some embodiments, the host cells are transfected with four plasmids adapted for producing lentiviral vectors, wherein one plasmid encodes lentiviral Gag and Pol proteins (Gag-Pol plasmid), one plasmid encodes a lentiviral Rev protein (Rev plasmid), one plasmid encodes envelope proteins (Env plasmid), and one plasmid encodes a transgene of interest.

[0078] In some embodiments, the mixture of plasmids includes a first plasmid that is a packaging plasmid that encodes gag and pol (Gag-Pol plasmid), a second plasmid that is a packaging plasmid that encodes rev (Rev plasmid), a third plasmid that is an envelope (Env) plasmid that encodes VSV-G and a fourth plasmid that is a transfer plasmid that encodes a transgene of interest.

[0079] In some embodiments, the mass fraction of one or more plasmids in the mixture of plasmids is altered among each of the plurality of mixture of plasmids in the design of experiments. In some embodiments, the mass fraction of at least two plasmids in the mixture of plasmids is altered among each of the plurality of mixture of plasmids in the design of experiments. In some embodiments, the mass fraction of at least three plasmids in the mixture of plasmids is altered among each of the plurality of mixture of plasmids in the design of experiments. In some embodiments, the mass fraction of at least four plasmids in the mixture of plasmids is altered among each of the plurality of mixture of plasmids in the design of experiments. [0080] In some embodiments, one or more constraints on the mixture components can be implemented in the design. In some embodiments, the mass fraction ratio of at least one of the plasmids is held constant among the plurality of mixtures. In some embodiments, the mass fraction of one plasmid is altered while the others are kept constant. In some embodiments, the mass fraction of two plasmids are altered while the others are kept constant. In some embodiments, the mass fraction of only one plasmid is kept constant and the mass fraction of the other plasmids are altered.

[0081] In some embodiments, the mass fraction of at least one of the plasmids encoding rev, gag or pol is held constant among each of the different mixture of plasmids. In some embodiments, the mass fraction of the plasmid encoding rev is held constant among each of the different mixture of plasmids. In some embodiments, the mass fraction of the plasmid encoding gag and/or pol is held constant among each of the different mixture of plasmids. In some embodiments, the mass fraction of the plasmid encoding gag and pol is held constant among each of the different mixture of plasmids.

[0082] It is within the level of a skilled artisan to choose mass fractions for each of the plasmids in the mixture to generate a plurality of plasmid mixtures with different plasmid ratios in the design of experiments, in which the sum of the mass fractions is 1. For instance, mass fractions of a plasmid or plasmids in the mixture of plasmids can be chosen based on the scale of the experiment, constraints on the design, predefined mass fraction ranges of a particular plasmid (e.g. envelope plasmid encoding VSV-G), and other factors known to a skilled artisan. In some embodiments each of the different mixture of plasmids can be used to transfect host cells in a plurality of transfection reactions in an upstream process, and each can be assessed for titer response in accord with the provided methods.

[0083] In some embodiments, the mass fraction of the plasmid encoding gag and pol (Gag- Pol plasmid) in the mixture is 0.01 to 0.6, such as from 0.01 to 0.4, 0.01 to 0.2, 0.01 to 1.0, 0.01 to 0.08, 0.01 to 0.06, 0.01 to 0.04, 0.01 to 0.02, 0.02 to 0.6, 0.02 to 0.4, 0.02 to 0.2, 0.02 to 0.1, 0.02 to 0.08, 0.02 to 0.06, 0.02 to 0.04, 0.04 to 0.6, 0.04 to 0.6, 0.04 to 0.4, 0.04 to 0.2, 0.04 to 0.10, 0.04 to 0.08, 0.04 to 0.06, 0.06 to 0.6, 0.06 to 0.4, 0.06 to 0.2, 0.06 to 0.1, 0.06 to 0.08, 0.08 to 0.6, 0.08 to 0.4, 0.08 to 0.2, 0.08 to 0.1, 0.1 to 0.6, 0.1 to 0.4, 0.1 to 0.2, 0.2 to 0.6, 0.2 to 0.4 or 0.4 to 0.6. In some embodiments, the mass fraction of the plasmid encoding gag and pol (Gag-Pol plasmid) in the mixture is 0.1 to 0.25. In some embodiments, the mass fraction of the plasmid encoding rev in the mixture is 0.1, 0.15, 0.2, 0.25, or any value between any of the foregoing. In some embodiments, the mass fraction of the plasmid encoding gag and pol (Gag- Pol plasmid) in the mixture is 0.15 and 0.2. In some embodiments, the mass fraction of the plasmid encoding gag-pol is different among some of the plurality of plasmid mixtures used for the transfection reactions. In some embodiments, the mass fraction of the plasmid encoding gag- pol is different among each of the plurality of plasmid mixtures used for the transfection reactions. In some embodiments, the mass fraction of the plasmid encoding gag-pol is held constant among the plurality of plasmid mixtures used for the transfection reactions.

[0084] In some embodiments, the mass fraction of the plasmid encoding rev (Rev plasmid) in the mixture is 0.01 to 0.6, such as from 0.01 to 0.4, 0.01 to 0.2, 0.01 to 1.0, 0.01 to 0.08, 0.01 to 0.06, 0.01 to 0.04, 0.01 to 0.02, 0.02 to 0.6, 0.02 to 0.4, 0.02 to 0.2, 0.02 to 0.1, 0.02 to 0.08, 0.02 to 0.06, 0.02 to 0.04, 0.04 to 0.6, 0.04 to 0.4, 0.04 to 0.2, 0.04 to 0.10, 0.04 to 0.08, 0.04 to 0.06, 0.06 to 0.6, 0.06 to 0.4, 0.06 to 0.2, 0.06 to 0.1, 0.06 to 0.08, 0.08 to 0.6, 0.08 to 0.4, 0.08 to 0.2, 0.08 to 0.1, 0.1 to 0.6, 0.1 to 0.4, 0.1 to 0.2, 0.2 to 0.6, 0.2 to 0.4 or 0.4 to 0.6. In some embodiments, the mass fraction of the plasmid encoding rev (Rev plasmid) in the mixture is 0.04 to 0.08. In some embodiments, the mass fraction of the plasmid encoding rev in the mixture is 0.04, 0.05, 0.06, 0.07, 0.08 or any value between any of the foregoing. In some embodiments, the mass fraction of the plasmid encoding rev (Rev Plasmid) in the mixture is from 0.04 to 0.06. In some embodiments, the mass fraction of the plasmid encoding rev is different among some of the plurality of plasmid mixtures used for the transfection reactions. In some embodiments, the mass fraction of the plasmid encoding rev is different among each of the plurality of plasmid mixtures used for the transfection reactions. In some embodiments, the mass fraction of the plasmid encoding rev is held constant among the plurality of plasmid mixtures used for the transfection reactions.

[0085] In some embodiments, the mass fraction of the plasmid encoding VSV-G (Env plasmid) in the mixture is from 0.01 to 0.6, such as from 0.01 to 0.4, 0.01 to 0.2, 0.01 to 1.0, 0.01 to 0.08, 0.01 to 0.06, 0.01 to 0.04, 0.01 to 0.02, 0.02 to 0.6, 0.02 to 0.4, 0.02 to 0.2, 0.02 to 0.1, 0.02 to 0.08, 0.02 to 0.06, 0.02 to 0.04, 0.04 to 0.6, 0.04 to 0.4, 0.04 to 0.2, 0.04 to 0.10, 0.04 to 0.08, 0.04 to 0.06, 0.06 to 0.6, 0.06 to 0.4, 0.06 to 0.2, 0.06 to 0.1, 0.06 to 0.08, 0.08 to 0.6, 0.08 to 0.4, 0.08 to 0.2, 0.08 to 0.1, 0.1 to 0.6, 0.1 to 0.4, 0.1 to 0.2, 0.2 to 0.6, 0.2 to 0.4 or 0.4 to 0.6. In some embodiments, the mass fraction of the plasmid encoding VSV-G (Env plasmid) in the mixture is from 0.04 to 0.2. In some embodiments, the mass fraction of the plasmid encoding VSV-G (Env plasmid) in the mixture is 0.04, 0.06, 0.08, 0.1, 0.12, 0.14, 0.16, 0.18 or 0.2 or any value between any of the foregoing. In some embodiments, the mass fraction of the plasmid encoding VSV-G (Env plasmid) in the mixture is from 0.046 to 0.15. In some embodiments, the mass fraction of the plasmid encoding VSV-G is different among some of the plurality of plasmid mixtures used for the transfection reactions. In some embodiments, the mass fraction of the plasmid encoding VSV-G is different among each of the plurality of plasmid mixtures used for the transfection reactions. In some embodiments, the mass fraction of the plasmid encoding VSV-G is held constant among the plurality of plasmid mixtures used for the transfection reactions.

[0086] In some embodiments, the mass fraction of the plasmid encoding the transgene (transfer plasmid) in the mixture is from 0.47 to 0.82. In some embodiments, the mass fraction of the plasmid encoding the transgene (transfer plasmid) in the mixture is 0.48, 0.50, 0.52, 0.54, 0.56, 0.58, 0.60, 0.62, 0.64, 0.66, 0.68, 0.7, 0.72, 0.74, 0.76, 0.78, 0.8, 0.82 or any value between any of the foregoing. In some embodiments, the mass fraction of the plasmid encoding the transgene (transfer plasmid) in the mixture is from 0.58 to 0.74. In some embodiments, the mass fraction of the transfer plasmid is different among some of the plurality of plasmid mixtures used for the transfection reactions. In some embodiments, the mass fraction of the transfer plasmid is different among each of the plurality of plasmid mixtures used for the transfection reactions. In some embodiments, the mass fraction of the transfer plasmid is held constant among the plurality of plasmid mixtures used for the transfection reactions.

[0087] In some embodiments, the VSV-G plasmid mass fraction is constrained in a defined range in the defined experiments. In some embodiments, the defined range is a mass fraction of 0.04 to 0.2. In some embodiments, the method for determining upstream lentiviral manufacturing conditions includes (a) performing a plurality of transfection reactions by transfecting host cells with a plurality of transfection mixtures, each transfection mixture comprising (1) a mixture of plasmids for the production of a lentiviral vector, wherein the mixture of plasmids comprise at least one lentiviral packaging plasmid encoding a viral gene selected from rev, gag or pol, an envelope plasmid encoding VSV-G, and a transfer plasmid encoding a transgene, and (2) a transfection agent, wherein each of the plurality of transfection reactions has a different mass fraction ratio of plasmids in the mixture of plasmids and the same total amount of DNA, wherein the mass fraction of the envelope plasmid is independently from 0.04 to 0.2; (b) harvesting the culture supernatant from each transfection reaction, and optionally clarifying the supernatant of the harvested culture to produce a clarified harvest; (c) determining lentiviral vector performance from each of the harvested culture supernatants or the clarified harvest; and (d) selecting a transfection mixture for an upstream processing method for producing a lentiviral vector based on the lentiviral vector performance that has been determined for the transfection mixture.

[0088] In some embodiments, the plasmid ratio by mass fraction for each of the plurality of different plasmid mixtures is selected where the sum of mass fractions is 1.

[0089] The person skilled in the art can adapt the transfection method to the particular cell culture implemented. In particular, the amount of total DNA (comprising in particular the DNA from the mixture of plasmids required for production of a recombinant viral vector) can vary. In some embodiments, the total amount of DNA is 0.2 pg/106 cells to 12 pg/106 cells. In some embodiments, the total amount of DNA is 0.2 pg/106 cells, 0.4 pg/106 cells, 0.6 pg/106 cells, 0.8 pg/106 cells, 1 pg/106 cells, 2 pg/106 cells, 3 pg/106 cells, 4 pg/106 cells, 5 pg/106 cells, 6 pg/106 cells, 7 pg/106 cells, 8 pg/106 cells, 9 pg/106 cells, 10 pg/106 cells, 11 pg/106 cells or 12 pg/106 cells or any value between any of the foregoing. In some embodiments, the total amount of DNA is 0.3 pg/106 cells to 2 pg/106 cells. In some embodiments, the total amount of DNA is 0.5 pg/106 cells to 1.5 pg/106 cells.

[0090] In some embodiments, each of the different mixture of plasmids are provided to the host cell for a plurality of different transfection reactions. In some embodiments, the number of different transfection reactions is 3 to 100, such as 3 to 70, 3 to 50, 3 to 30, 3 to 15, 15 to 100, 15 to 70, 15 to 50, 15 to 30, 30 to 100, 30 to 70, 30 to 50, 50 to 100 or 50 to 70. In some embodiments, the number of different transfection reaction is at or about 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99 or any value between any of the foregoing. In some embodiments, the number of different transfection reactions is 3 to 50. In some embodiments, the number of different transfection reaction is 3 to 30.

B. Determination of Cell Line Titer

[0091] In some embodiments, the harvest or clarified harvest for each of the plurality of transfection reactions is assessed for titer in at least one cell line-based titer assay.

[0092] In some embodiments, the cell line titer is determined using a cell line that is a defined population of cells that can be maintained in culture for an extended period of time. In some embodiments, the cell line is a population of cells that retain stability of certain phenotypes and functions. In some embodiments, the cell line is clonal, such as a cell line wherein the entire population of cells originated from a single common ancestor cell. In some embodiments, the cell line is derived from a stem cell or population of stem cells.

[0093] In some embodiments, a cell line titer is assayed using a cell line that is immortalized. In some embodiments, the cell line is an immortalized cell line that is a population of cells that have evaded cellular senescence via mutation. In some embodiments, the immortalized cell line for use in determining a cell line titer can be grown for prolonged periods in vitro.

[0094] In some embodiments, the cell line is any permissive cells that can be transduced by the viral vector (e.g. lentiviral vector). In some embodiments, the cell line is chosen based on the transgene and its desired cell target. As an example, a T cell line is a target cell for a transgene encoding a chimeric antigen receptor (CAR) or a T cell receptor (TCR). It is within the level of a skilled artisan to choose an appropriate cell line for assessing titer.

[0095] In some embodiments, the cell line titer is assessed using a T cell line. In some embodiments, the T cell line is a Jurkat cell line or any derivative thereof. In some embodiments, the T cell line is selected from J.CaM1.6, CCRF-CEM, MJ [Gi l], SUP-T1 [VB], T ALL-104, Jurkat Clone E6-1, HH, MOLT-3, J45.01, CEM/C1, Loucy, I 9.2, CCRF-HSB-2, P116.C139, 12.1, C5/MJ, J.gammal.WT, Pl 16, H9/HTLV-IIIB, MOLT-4, CEM/C2, CEM-CM3, J.RT3-T3.5, JK28, or A3.

[0096] In some embodiments, the cell line titer is assessed using an epithelial cell line, such as HT1080-HEK293 cells. In some embodiments, the epithelial cell line is selected from HSAEC1-KT, hTERT-HMEl [ME16C], NuLi-1, HBEC3-KT, RPTEC/TERT1, hTERT EP156T, SV7tert PDGF tumor-1, hTERT RPE-1, hTERT-HPNE, CuFi-5, CuFi-4, CuFi-6, CuFi-1, CP-B (CP-52731), CP-C (CP-94251), CP-A (KR-42421). In some embodiments, the cell line is HT-1080 [HT1080], 293 [HEK-293], 293T, HEK-293.2sus, HEK293S GnTL.

[0097] In some embodiments, the cell line is a Jurkat cell.

[0098] Techniques for assessing titer of a lentiviral vector are known in the art. Non-limiting examples of available techniques for quantifying titer include determination by infectious or functional titer assays.

[0099] For example, the infectious titer (e.g. lU/mL) of a lentiviral vector can be assessed physically and/or molecularly, such as in qPCR or digital droplet PCR (ddPCR) as described in, e.g., M. Lock et al, Hu Gene Therapy Methods, Hum Gene Ther Methods 25(2): 115-25. 2014, which is incorporated herein by reference. In some embodiments, infectious titer is determined by serially diluting vector virus on cells, such as HEK cells or any permissible host cell line in the case of determination of cell line infectious titer. After incubation for a time period to achieve infection and transduction of the transgene via insertion in the lentiviral genome, cells are lysed and genomic nucleic acid is extracted. In some embodiments, the genomic nucleic acid is genomic RNA. In some embodiments, the genomic nucleic acid is genomic DNA. qPCR or ddPCR is performed, for example using probes against the LTRs in the lentivector genome to quantify vector titer (e.g., infectious titer) and against a cellular target to quantify the proportion of infected cells at a particular dilution. In some embodiments, qPCR (and/or ddPCR) is performed to amplify regions of the lentiviral vector LTR. In some embodiments, the qPCR comprises primers and probes for amplification of a region of the LTR. In some embodiments, the qPCR comprises primers and probes for amplification of a region of the LTR that is selected from is R, U5, U3, Psi, or PBS. In some embodiments, the region of the LTR is R-U5, U5-Psi, U5-U3, PBS-Psi. In some embodiments, the region of the LTR is AU3. In some embodiments, the primers and probes target integrated copies of the Rev response element (RRE). In some embodiments, qPCR is performed using primers and probes specific for the WPRE region.

[0100] In some of any of the provided embodiments, infectious titer is measured as Infectious Units per unit of volume, such as lU/mL. In some embodiments, the infectious titer is measured as plaque forming units (PEU) or foci forming units (LEU) per unit of volume, such as PFU/mL. In some embodiments, the infectious titer is measured as vector copy number (VCN), genome copy number (GCN), or vector genome (VG) per unit of volume, such as VG/mL.

[0101] In some embodiments, functional titer can be determined using a transduction assay in which titrations of the lentiviral vector comprising a transgene are introduced to permissive cells and the expression of said transgene is measured, such as in Transducing Units (TU) per unit of volume (e.g. TU/mL). In some embodiments, a transduction assay is performed by serially diluting vector virus on cells, such as Jurkat T cells or any permissible host cell line in the case of determination of cell line functional titer. After incubation for a time period to achieve infection and transduction of the transgene, such as between 24 and 72 hours, cells are incubated with a detectable label for the transgene (e.g., a fluorescently labeled antibody to a CAR) before counted via flow cytometry. Reagents for detecting surface expression of a CAR are well known to a skilled artisan. In some embodiments, the reagent is an anti-idiotypic antibody. Anti-idiotypic antibodies directed against CARs are known and can be chosen by a skilled artisan depending on the particular CAR. Exemplary anti-idiotypic antibodies directed against anti-CD19 CARs that contains an antigen-binding domain derived from FMC63 or SJ25C1 are known, see e.g., PCT publication No. WO2018/023100.

[0102] In some embodiments, the functional titer is measured as transducing or transduction units (TU) per unit of volume, such as TU/mL. In some embodiments, the functional titer is measured as a percent of transgene expressing cells detected, such as %CAR+.

[0103] In some embodiments, titer can be determined using an endpoint dilution (TCID50) method, which determines the dilution of virus at which 50% of the cell cultures are infected/transduced and hence, generally, can determine the titer within a certain range, such as one log.

C. Determination of Primary Cell Functional Titer

[0104] In some embodiments, the harvest or clarified harvest for each of the plurality of transfection reactions is assessed for titer in at least one primary cell titer assay.

[0105] In some embodiments, the primary cell is chosen based on the transgene and its desired cell target. As an example, a primary T cell is a target cell for a transgene encoding a chimeric antigen receptor (CAR) or a T cell receptor (TCR). It is within the level of a skilled artisan to choose an appropriate primary cell for assessing titer. In provided embodiments, the primary cells is obtained or isolated from a subject. In some embodiments, the subject can be a healthy or normal subject. In some embodiments, the subject is a subject that has a disease or conditions, such as a cancer or an autoimmune or inflammatory disease or condition. In some embodiments, a primary cell titer is assayed using a primary cells obtained from a patient sample, such as obtained from a blood sample or an apheresis or leukapheresis.

[0106] In some embodiments, the cell line titer and primary cell titer are assayed using the same method. In some embodiments, any of the methods described above but as determined in a primary cell can be assessed. Non-limiting examples of available techniques for quantifying titer include determination by infectious or functional titer assays.

[0107] For example, the infectious titer (e.g. lU/mL) of a lentiviral vector can be assessed physically and/or molecularly, such as in qPCR or digital droplet PCR (ddPCR) as described in, e.g., M. Lock et al, Hu Gene Therapy Methods, Hum Gene Ther Methods 25(2): 115-25. 2014, which is incorporated herein by reference. In some embodiments, infectious titer is determined by serially diluting vector virus on cells, such as HEK cells or any permissible host cell line in the case of determination of cell line infectious titer. After incubation for a time period to achieve infection and transduction of the transgene via insertion in the lentiviral genome, cells 1 are lysed and genomic DNA extracted. qPCR or ddPCR is performed, for example using probes against the LTRs in the lentivector genome to quantify vector titer (e.g., infectious titer) and against a cellular target to quantify the proportion of infected cells at a particular dilution.

[0108] In some of any of the provided embodiments, infectious titer is measured as Infectious Units per unit of volume, such as lU/mL. In some embodiments, the infectious titer is measured as plaque forming units (PFU) or foci forming units (FFU) per unit of volume, such as PFU/mL. In some embodiments, the infectious titer is measured as vector copy number (VCN), genome copy number (GCN), or vector genome (VG) per unit of volume, such as VG/mL.

[0109] In particular embodiments, the primary cell titer is determined in an assay for primary cell functional titer.

[0110] In some embodiments, the primary cell functional titer is assessed in primary cells after they have been following one or more steps of a manufacturing process as described herein. In some embodiments, the primary cell functional titer is assessed using a primary cells derived from a patient or subject sample.

[0111] In some embodiments, functional titer can be determined using a transduction assay in which titrations of the lentiviral vector comprising a transgene are introduced to cells and the expression of said transgene is measured, such as in Transducing Units (TU) per unit of volume (e.g. TU/mL). In some embodiments, a transduction assay is performed by serially diluting vector virus on primary cells. After incubation for a time period to achieve infection and transduction of the transgene, such as between 24 and 72 hours, cells are incubated with a detectable label for the transgene (e.g., a fluorescently labeled antibody to a CAR) before counted via flow cytometry. Reagents for detecting surface expression of a CAR are well known to a skilled artisan. In some embodiments, the reagent is an anti-idiotypic antibody. Anti- idiotypic antibodies directed against CARs are known and can be chosen by a skilled artisan depending on the particular CAR. Exemplary anti-idiotypic antibodies directed against antiCD 19 CARs that contains an antigen-binding domain derived from FMC63 or SJ25C1 are known, see e.g., PCT publication No. WO2018/023100.

[0112] In some embodiments, the functional titer is measured as transducing or transduction units (TU) per unit of volume, such as TU/mL. In some embodiments, the functional titer is measured as a percent of transgene expressing cells detected, such as %CAR+.

[0113] In some embodiments, the functional titer is determined on an output composition of primary cells produced by a cell therapy manufacturing methods. In some embodiments, the incubation is in under condition for cultivation of the primary cells such as in the presence of one or more recombinant cytokines. In some embodiments, the incubation is under conditions for expanding cells. Various methods of manufacturing cell therapy compositions are known, any of which can be used in according with determining a primary cell titer. In some embodiments, the harvested culture supernatant or clarified harvest is used to transduce (e.g. serial dilution) primary cells of an input composition of T cells, optionally after T cell stimulation, followed by incubation of the primary cells to produce an output composition.

[0114] In certain embodiments, the output composition contains cells that express the transgene, such as a recombinant receptor, e.g., a CAR, such as an anti-CD19 CAR. In particular embodiments, the cells of the output composition are produced under conditions that simulate methods use for producing cells that are suitable for administration to a subject as a therapy, e.g., an autologous cell therapy. In some embodiments, the generation of an output composition of engineered primary cells is carried out on a smaller scale than my traditionally be used for larger-scale cell therapy production. In some embodiments, the output composition is a composition of enriched CD4+ or CD8+ T cells.

[0115] In some embodiments, the process for generating or producing an output composition of engineered cells is by a process that includes some or all of the steps of: collecting or obtaining a biological sample; isolating, selecting, or enriching input cells from the biological sample; incubating the input cells under stimulating conditions; engineering the stimulated cells to express or contain a recombinant polynucleotide, e.g., a polynucleotide encoding a recombinant receptor such as a CAR; cultivating the engineered cells, e.g. to a threshold amount, density, or expansion; formulating the cultivated cells in an output composition. In some embodiments, prior to the stimulating the cells of the input composition, the method can include cryopreserving and storing the input cells and thawing the cells for subsequent stimulation step for transduction. In some embodiments, the methods include cultivating the engineered cells, e.g., under conditions that promote proliferation and/or expansion. In particular embodiments, the provided methods may be used in connection with harvesting, collecting, and/or formulating output compositions produced after the cells have been incubated, activated, stimulated, engineered, transduced, transfected, and/or cultivated. The cells of the output composition can then be assessed for functional titer in accord with provided methods.

[0116] In some embodiments, the engineered composition of output cells are T cells and are engineered from an input composition of primary T cells. In some embodiments, engineered cells, such as those that express an anti-CD19 CAR as described, used in accord with the provided methods and uses are produced or generated by exemplary processes as described in, for example, WO 2019/089855 and WO 2015/164675. Exemplary methods for producing an engineered composition of output cells are described below.

[0117] Input Composition

[0118] In some embodiments, the methods include isolating, selecting, or enriching input cells from a biological sample from a subject. Accordingly, the cells in some embodiments are primary cells, e.g., primary human cells.

[0119] The input composition of cells may be isolated from a sample, such as a biological sample, e.g., one obtained from or derived from a subject. In some embodiments, the subject is a healthy subject. In some embodiments, the subject from which the cell is isolated is one having the disease or condition or in need of a cell therapy or to which cell therapy will be administered. The subject in some embodiments is a human in need of a particular therapeutic intervention, such as the adoptive cell therapy for which cells are being isolated, processed, and/or engineered.

[0120] The samples include tissue, fluid, and other samples taken directly from the subject, as well as samples resulting from one or more processing steps, such as separation, centrifugation, washing, and/or incubation. The biological sample can be a sample obtained directly from a biological source or a sample that is processed. Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived therefrom.

[0121] In some aspects, the sample from which the cells are derived or isolated is blood or a blood-derived sample, or is or is derived from an apheresis or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsil, or other organ, and/or cells derived therefrom. Samples include, in the context of cell therapy, e.g., adoptive cell therapy, samples from autologous and allogeneic sources.

[0122] The cells generally are eukaryotic cells, such as mammalian cells, and typically are human cells. In some embodiments, the cells are derived from the blood, bone marrow, lymph, or lymphoid organs, are cells of the immune system, such as cells of the innate or adaptive immunity, e.g., myeloid or lymphoid cells, including lymphocytes, typically T cells and/or NK cells. Other exemplary cells include stem cells, such as multipotent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs). The cells typically are primary cells, such as those isolated directly from a subject and/or isolated from a subject and frozen. In some embodiments, the cells include one or more subsets of T cells or other cell types, such as whole T cell populations, CD4⁺ cells, CD8⁺ cells, and subpopulations thereof, such as those defined by function, activation state, maturity, potential for differentiation, expansion, recirculation, localization, and/or persistence capacities, antigen- specificity, type of antigen receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and/or degree of differentiation. With reference to the subject to be treated, the cells may be allogeneic and/or autologous. Among the methods include off-the-shelf methods. In some aspects, such as for off-the-shelf technologies, the cells are pluripotent and/or multipotent, such as stem cells, such as induced pluripotent stem cells (iPSCs). In some embodiments, the methods include isolating cells from the subject, preparing, processing, culturing, and/or engineering them, and reintroducing them into the same subject, before or after cryopreservation.

[0123] Among the sub-types and subpopulations of T cells and/or of CD4⁺ and/or of CD8⁺ T cells are naive T (TN) cells, effector T cells (TEFF), memory T cells and sub-types thereof, such as stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM), or terminally differentiated effector memory T cells, tumor-infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha/beta T cells, and delta/gamma T cells.

[0124] In some embodiments, the cells are natural killer (NK) cells. In some embodiments, the cells are monocytes or granulocytes, e.g., myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and/or basophils.

[0125] In some embodiments, isolation of the cells includes one or more preparation and/or non-affinity based cell separation steps. In some examples, cells are washed, centrifuged, and/or incubated in the presence of one or more reagents, for example, to remove unwanted components, enrich for desired components, lyse or remove cells sensitive to particular reagents. In some examples, cells are separated based on one or more property, such as density, adherent properties, size, sensitivity and/or resistance to particular components. [0126] In some examples, cells from the circulating blood of a subject are obtained, e.g., by apheresis or leukapheresis. The samples, in some aspects, contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and/or platelets, and in some aspects contains cells other than red blood cells and platelets.

[0127] In some embodiments, the blood cells collected from the subject are washed, e.g., to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments, the wash solution lacks calcium and/or magnesium and/or many or all divalent cations. In some aspects, a washing step is accomplished a semi-automated “flow-through” centrifuge (for example, the Cobe 2991 cell processor, Baxter) according to the manufacturer’s instructions. In some aspects, a washing step is accomplished by tangential flow filtration (TFF) according to the manufacturer’s instructions. In some embodiments, the cells are resuspended in a variety of biocompatible buffers after washing, such as, for example, Ca⁺⁺/Mg⁺⁺ free PBS. In certain embodiments, components of a blood cell sample are removed and the cells directly resuspended in culture media.

[0128] In some embodiments, the methods include density-based cell separation methods, such as the preparation of white blood cells from peripheral blood by lysing the red blood cells and centrifugation through a Percoll or Ficoll gradient.

[0129] In some embodiments, at least a portion of the selection step includes incubation of cells with a selection reagent. The incubation with a selection reagent or reagents, e.g., as part of selection methods which may be performed using one or more selection reagents for selection of one or more different cell types based on the expression or presence in or on the cell of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acid. In some embodiments, any known method using a selection reagent or reagents for separation based on such markers may be used. In some embodiments, the selection reagent or reagents result in a separation that is affinity- or immunoaffinity-based separation. For example, the selection in some aspects includes incubation with a reagent or reagents for separation of cells and cell populations based on the cells’ expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, followed generally by washing steps and separation of cells having bound the antibody or binding partner, from those cells having not bound to the antibody or binding partner. [0130] In some aspects of such processes, a volume of cells is mixed with an amount of a desired affinity-based selection reagent. The immunoaffinity-based selection can be carried out using any system or method that results in a favorable energetic interaction between the cells being separated and the molecule specifically binding to the marker on the cell, e.g., the antibody or other binding partner on the solid surface, e.g., particle. In some embodiments, methods are carried out using particles such as beads, e.g. magnetic beads, that are coated with a selection agent (e.g. antibody) specific to the marker of the cells. The particles (e.g. beads) can be incubated or mixed with cells in a container, such as a tube or bag, while shaking or mixing, with a constant cell density-to-particle (e.g., bead) ratio to aid in promoting energetically favored interactions. In other cases, the methods include selection of cells in which all or a portion of the selection is carried out in the internal cavity of a centrifugal chamber, for example, under centrifugal rotation. In some embodiments, incubation of cells with selection reagents, such as immunoaffinity-based selection reagents, is performed in a centrifugal chamber. In certain embodiments, the isolation or separation is carried out using a system, device, or apparatus described in International Patent Application, Publication Number W02009/072003, or US 20110003380 Al. In one example, the system is a system as described in International Publication Number W02016/073602.

[0131] In some embodiments, the isolation methods include the separation of different cell types based on the expression or presence in the cell of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acid. In some embodiments, any known method for separation based on such markers may be used. In some embodiments, the separation is affinity- or immunoaffinity-based separation. For example, the isolation in some aspects includes separation of cells and cell populations based on the cells’ expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, followed generally by washing steps and separation of cells having bound the antibody or binding partner, from those cells having not bound to the antibody or binding partner.

[0132] Such separation steps can be based on positive selection, in which the cells having bound the reagents are retained for further use, and/or negative selection, in which the cells having not bound to the antibody or binding partner are retained. In some examples, both fractions are retained for further use. In some aspects, negative selection can be particularly useful where no antibody is available that specifically identifies a cell type in a heterogeneous population, such that separation is best carried out based on markers expressed by cells other than the desired population.

[0133] The separation need not result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection of or enrichment for cells of a particular type, such as those expressing a marker, refers to increasing the number or percentage of such cells, but need not result in a complete absence of cells not expressing the marker. Likewise, negative selection, removal, or depletion of cells of a particular type, such as those expressing a marker, refers to decreasing the number or percentage of such cells, but need not result in a complete removal of all such cells.

[0134] In some examples, multiple rounds of separation steps are carried out, where the positively or negatively selected fraction from one step is subjected to another separation step, such as a subsequent positive or negative selection. In some examples, a single separation step can deplete cells expressing multiple markers simultaneously, such as by incubating cells with a plurality of antibodies or binding partners, each specific for a marker targeted for negative selection. Likewise, multiple cell types can simultaneously be positively selected by incubating cells with a plurality of antibodies or binding partners expressed on the various cell types.

[0135] For example, in some aspects, specific subpopulations of T cells, such as cells positive or expressing high levels of one or more surface markers, e.g., CD28⁺, CD62L⁺, CCR7⁺, CD27⁺, CD127⁺, CD4⁺, CD8⁺, CD45RA⁺, and/or CD45RO⁺ T cells, are isolated by positive or negative selection techniques.

[0136] For example, CD3⁺, CD28⁺ T cells can be positively selected using anti-CD3/anti- CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3/CD28 T Cell Expander).

[0137] In some embodiments, isolation is carried out by enrichment for a particular cell population by positive selection, or depletion of a particular cell population, by negative selection. In some embodiments, positive or negative selection is accomplished by incubating cells with one or more antibodies or other binding agent that specifically bind to one or more surface markers expressed or expressed (marker⁺) at a relatively higher level (marker¹¹¹⁸¹¹) on the positively or negatively selected cells, respectively.

[0138] In particular embodiments, a biological sample, e.g., a sample of PBMCs or other white blood cells, are subjected to selection of CD4+ T cells, where both the negative and positive fractions are retained. In certain embodiments, CD8+ T cells are selected from the negative fraction. In some embodiments, a biological sample is subjected to selection of CD8+ T cells, where both the negative and positive fractions are retained. In certain embodiments, CD4+ T cells are selected from the negative fraction.

[0139] In some embodiments, T cells are separated from a PBMC sample by negative selection of markers expressed on non-T cells, such as B cells, monocytes, or other white blood cells, such as CD 14. In some aspects, a CD4⁺ or CD8⁺ selection step is used to separate CD4⁺ helper and CD8⁺ cytotoxic T cells. Such CD4⁺ and CD8⁺ populations can be further sorted into sub-populations by positive or negative selection for markers expressed or expressed to a relatively higher degree on one or more naive, memory, and/or effector T cell subpopulations.

[0140] In some embodiments, CD8⁺ cells are further enriched for or depleted of naive, central memory, effector memory, and/or central memory stem cells, such as by positive or negative selection based on surface antigens associated with the respective subpopulation. In some embodiments, enrichment for central memory T (TCM) cells is carried out to increase efficacy, such as to improve long-term survival, expansion, and/or engraftment following administration, which in some aspects is particularly robust in such sub-populations. See Terakura et al. (2012) Blood.1:72-82; Wang et al. (2012) J Immunother. 35(9):689-701. In some embodiments, combining TcM-enriched CD8⁺ T cells and CD4⁺T cells further enhances efficacy.

[0141] In embodiments, memory T cells are present in both CD62L⁺ and CD62L" subsets of CD8⁺ peripheral blood lymphocytes. PBMC can be enriched for or depleted of CD62L'CD8⁺ and/or CD62L⁺CD8⁺ fractions, such as using anti-CD8 and anti-CD62L antibodies.

[0142] In some embodiments, the enrichment for central memory T (TCM) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and/or CD 127; in some aspects, it is based on negative selection for cells expressing or highly expressing CD45RA and/or granzyme B. In some aspects, isolation of a CD8⁺ population enriched for TCM cells is carried out by depletion of cells expressing CD4, CD 14, CD45RA, and positive selection or enrichment for cells expressing CD62L. In one aspect, enrichment for central memory T (TCM) cells is carried out starting with a negative fraction of cells selected based on CD4 expression, which is subjected to a negative selection based on expression of CD 14 and CD45RA, and a positive selection based on CD62L. Such selections in some aspects are carried out simultaneously and in other aspects are carried out sequentially, in either order. In some aspects, the same CD4 expression-based selection step used in preparing the CD8⁺ cell population or subpopulation, also is used to generate the CD4⁺ cell population or sub- population, such that both the positive and negative fractions from the CD4-based separation are retained and used in subsequent steps of the methods, optionally following one or more further positive or negative selection steps.

[0143] In a particular example, a sample of PBMCs or other white blood cell sample is subjected to selection of CD4⁺ cells, where both the negative and positive fractions are retained. The negative fraction then is subjected to negative selection based on expression of CD 14 and CD45RA or CD 19, and positive selection based on a marker characteristic of central memory T cells, such as CD62L or CCR7, where the positive and negative selections are carried out in either order.

[0144] CD4⁺ T helper cells are sorted into naive, central memory, and effector cells by identifying cell populations that have cell surface antigens. CD4⁺ lymphocytes can be obtained by standard methods. In some embodiments, naive CD4⁺ T lymphocytes are CD45RO", CD45RA⁺, CD62L⁺, CD4⁺ T cells. In some embodiments, central memory CD4⁺ cells are CD62L⁺ and CD45RO⁺. In some embodiments, effector CD4⁺ cells are CD62L" and CD45RO".

[0145] In one example, to enrich for CD4⁺ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CDl lb, CD16, HLA-DR, and CD8. In some embodiments, the antibody or binding partner is bound to a solid support or matrix, such as a magnetic bead or paramagnetic bead, to allow for separation of cells for positive and/or negative selection. For example, in some embodiments, the cells and cell populations are separated or isolated using immunomagnetic (or affinity magnetic) separation techniques (reviewed in Methods in Molecular Medicine, vol. 58: Metastasis Research Protocols, Vol. 2: Cell Behavior In Vitro and In Vivo, p 17-25 Edited by: S. A. Brooks and U. Schumacher © Humana Press Inc., Totowa, NJ).

[0146] In some aspects, the sample or composition of cells to be separated is incubated with small, magnetizable or magnetically responsive material, such as magnetically responsive particles or microparticles, such as paramagnetic beads (e.g., such as Dynabeads or MACS beads). The magnetically responsive material, e.g., particle, generally is directly or indirectly attached to a binding partner, e.g., an antibody, that specifically binds to a molecule, e.g., surface marker, present on the cell, cells, or population of cells that it is desired to separate, e.g., that it is desired to negatively or positively select.

[0147] In some embodiments, the magnetic particle or bead comprises a magnetically responsive material bound to a specific binding member, such as an antibody or other binding partner. There are many well-known magnetically responsive materials used in magnetic separation methods. Suitable magnetic particles include those described in Molday, U.S. Pat. No. 4,452,773, and in European Patent Specification EP 452342 B, which are hereby incorporated by reference. Colloidal sized particles, such as those described in Owen U.S. Pat. No. 4,795,698, and Liberti et al., U.S. Pat. No. 5,200,084 are other examples.

[0148] The incubation generally is carried out under conditions whereby the antibodies or binding partners, or molecules, such as secondary antibodies or other reagents, which specifically bind to such antibodies or binding partners, which are attached to the magnetic particle or bead, specifically bind to cell surface molecules if present on cells within the sample.

[0149] In some aspects, the sample is placed in a magnetic field, and those cells having magnetically responsive or magnetizable particles attached thereto will be attracted to the magnet and separated from the unlabeled cells. For positive selection, cells that are attracted to the magnet are retained; for negative selection, cells that are not attracted (unlabeled cells) are retained. In some aspects, a combination of positive and negative selection is performed during the same selection step, where the positive and negative fractions are retained and further processed or subject to further separation steps.

[0150] In certain embodiments, the magnetically responsive particles are coated in primary antibodies or other binding partners, secondary antibodies, lectins, enzymes, or streptavidin. In certain embodiments, the magnetic particles are attached to cells via a coating of primary antibodies specific for one or more markers. In certain embodiments, the cells, rather than the beads, are labeled with a primary antibody or binding partner, and then cell-type specific secondary antibody- or other binding partner (e.g., streptavidin)-coated magnetic particles, are added. In certain embodiments, streptavidin-coated magnetic particles are used in conjunction with biotinylated primary or secondary antibodies.

[0151] In some embodiments, the magnetically responsive particles are left attached to the cells that are to be subsequently incubated, cultured and/or engineered; in some aspects, the particles are left attached to the cells for administration to a patient. In some embodiments, the magnetizable or magnetically responsive particles are removed from the cells. Methods for removing magnetizable particles from cells are known and include, e.g., the use of competing non-labeled antibodies, and magnetizable particles or antibodies conjugated to cleavable linkers. In some embodiments, the magnetizable particles are biodegradable. [0152] In some embodiments, the affinity-based selection is via magnetic-activated cell sorting (MACS) (Miltenyi Biotec, Auburn, CA). Magnetic Activated Cell Sorting (MACS) systems are capable of high-purity selection of cells having magnetized particles attached thereto. In certain embodiments, MACS operates in a mode wherein the non-target and target species are sequentially eluted after the application of the external magnetic field. That is, the cells attached to magnetized particles are held in place while the unattached species are eluted. Then, after this first elution step is completed, the species that were trapped in the magnetic field and were prevented from being eluted are freed in some manner such that they can be eluted and recovered. In certain embodiments, the non-target cells are labelled and depleted from the heterogeneous population of cells.

[0153] In certain embodiments, the isolation or separation is carried out using a system, device, or apparatus that carries out one or more of the isolation, cell preparation, separation, processing, incubation, culture, and/or formulation steps of the methods. In some aspects, the system is used to carry out each of these steps in a closed or sterile environment, for example, to minimize error, user handling and/or contamination. In one example, the system is a system as described in International Patent Application, Publication Number W02009/072003, or US 20110003380 Al.

[0154] In some embodiments, the system or apparatus carries out one or more, e.g., all, of the isolation, processing, engineering, and formulation steps in an integrated or self-contained system, and/or in an automated or programmable fashion. In some aspects, the system or apparatus includes a computer and/or computer program in communication with the system or apparatus, which allows a user to program, control, assess the outcome of, and/or adjust various aspects of the processing, isolation, engineering, and formulation steps.

[0155] In some aspects, the separation and/or other steps is carried out using CliniMACS system (Miltenyi Biotec), for example, for automated separation of cells on a clinical-scale level in a closed and sterile system. Components can include an integrated microcomputer, magnetic separation unit, peristaltic pump, and various pinch valves. The integrated computer in some aspects controls all components of the instrument and directs the system to perform repeated procedures in a standardized sequence. The magnetic separation unit in some aspects includes a movable permanent magnet and a holder for the selection column. The peristaltic pump controls the flow rate throughout the tubing set and, together with the pinch valves, ensures the controlled flow of buffer through the system and continual suspension of cells. [0156] The CliniMACS system in some aspects uses antibody-coupled magnetizable particles that are supplied in a sterile, non-pyrogenic solution. In some embodiments, after labelling of cells with magnetic particles the cells are washed to remove excess particles. A cell preparation bag is then connected to the tubing set, which in turn is connected to a bag containing buffer and a cell collection bag. The tubing set consists of pre-assembled sterile tubing, including a pre-column and a separation column, and are for single use only. After initiation of the separation program, the system automatically applies the cell sample onto the separation column. Labelled cells are retained within the column, while unlabeled cells are removed by a series of washing steps. In some embodiments, the cell populations for use with the methods described herein are unlabeled and are not retained in the column. In some embodiments, the cell populations for use with the methods described herein are labeled and are retained in the column. In some embodiments, the cell populations for use with the methods described herein are eluted from the column after removal of the magnetic field, and are collected within the cell collection bag.

[0157] In certain embodiments, separation and/or other steps are carried out using the CliniMACS Prodigy system (Miltenyi Biotec). The CliniMACS Prodigy system in some aspects is equipped with a cell processing unity that permits automated washing and fractionation of cells by centrifugation. The CliniMACS Prodigy system can also include an onboard camera and image recognition software that determines the optimal cell fractionation endpoint by discerning the macroscopic layers of the source cell product. For example, peripheral blood is automatically separated into erythrocytes, white blood cells and plasma layers. The CliniMACS Prodigy system can also include an integrated cell cultivation chamber which accomplishes cell culture protocols such as, e.g., cell differentiation and expansion, antigen loading, and long-term cell culture. Input ports can allow for the sterile removal and replenishment of media and cells can be monitored using an integrated microscope. See, e.g., Klebanoff et al. (2012) J Immunother. 35(9): 651-660, Terakura et al. (2012) Blood.1:72-82, and Wang et al. (2012) J Immunother. 35(9):689-701.

[0158] In some embodiments, a cell population described herein is collected and enriched (or depleted) via flow cytometry, in which cells stained for multiple cell surface markers are carried in a fluidic stream. In some embodiments, a cell population described herein is collected and enriched (or depleted) via preparative scale (FACS)-sorting. In certain embodiments, a cell population described herein is collected and enriched (or depleted) by use of microelectromechanical systems (MEMS) chips in combination with a FACS-based detection system (see, e.g., WO 2010/033140, Cho et al. (2010) Lab Chip 10, 1567-1573; and Godin et al. (2008) J Biophoton. l(5):355-376. In both cases, cells can be labeled with multiple markers, allowing for the isolation of well-defined T cell subsets at high purity.

[0159] In some embodiments, the antibodies or binding partners are labeled with one or more detectable marker, to facilitate separation for positive and/or negative selection. For example, separation may be based on binding to fluorescently labeled antibodies. In some examples, separation of cells based on binding of antibodies or other binding partners specific for one or more cell surface markers are carried in a fluidic stream, such as by fluorescence- activated cell sorting (FACS), including preparative scale (FACS) and/or microelectromechanical systems (MEMS) chips, e.g., in combination with a flow-cytometric detection system. Such methods allow for positive and negative selection based on multiple markers simultaneously.

[0160] In some embodiments, the preparation methods include steps for freezing, e.g., cryopreserving, the cells, either before or after isolation, incubation, and/or engineering. In some embodiments, the freeze and subsequent thaw step removes granulocytes and, to some extent, monocytes in the cell population. In some embodiments, the cells are suspended in a freezing solution, e.g., following a washing step to remove plasma and platelets. Any of a variety of known freezing solutions and parameters in some aspects may be used. One example involves using PBS containing 20% DMSO and 8% human serum albumin (HSA), or other suitable cell freezing media. This is then diluted 1 : 1 with media so that the final concentration of DMSO and HSA are 10% and 4%, respectively. In some embodiments, the cell compositions are stored in a formulation containing at or about 5%, 6%, 7%, 7.5%, 8%, 9% or 10% dimethylsulfoxide, or a range defined by any of the foregoing, such as at or about 7.5% DMSO. In some aspects, the compositions are stored in a formulation containing at or about 0.5%, 1%, 2% or 2.5% (v/v) of 25% human albumin, or a range defined by any of the foregoing, such as at or about 1% (v/v) 25% human albumin. The cells are generally then frozen to -80° C. at a rate of 1° per minute and stored in the vapor phase of a liquid nitrogen storage tank.

[0161] In some embodiments, the isolation and/or selection results in one or more input compositions of enriched T cells, e.g., CD3+ T cells, CD4+ T cells, and/or CD8+ T cells. In some embodiments, two or more separate input composition are isolated, selected, enriched, or obtained from a single biological sample. In some embodiments, separate input compositions are isolated, selected, enriched, and/or obtained from separate biological samples collected, taken, and/or obtained from the same subject.

[0162] In certain embodiments, the one or more input compositions is or includes a composition of enriched T cells that includes at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or at or at about 100% CD3+ T cells. In particular embodiment, the input composition of enriched T cells consists essentially of CD3+ T cells.

[0163] In some embodiments, the input composition is a composition of enriched T cells, enriched CD4+ T cells, and/or enriched CD8+ T cells (herein after also referred to as compositions of enriched T cells, compositions of enriched CD4+ T cells, and compositions of enriched CD8+ T cells, respectively).

[0164] In some embodiments, a composition enriched in CD4+ T cells contains at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 99.9% CD4+ T cells. In particular embodiments, the composition of enriched CD4+ T cells contains 100% CD4+ T cells contains about 100% CD4+ T cells. In certain embodiments, the composition of enriched T cells includes or contains less than 20%, less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% CD8+ T cells, and/or contains no CD8+ T cells, and/or is free or substantially free of CD8+ T cells. In certain embodiments, the input composition of CD4+ T cells includes less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% CD8+ T cells, and/or contains no CD8+ T cells, and/or is free or substantially free of CD8+ T cells. In some embodiments, the composition of enriched T cells consists essentially of CD4+ T cells.

[0165] In certain embodiments, the one or more compositions is or includes a composition of CD8+ T cells that is or includes at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or at or at about 100% CD8+ T cells. In certain embodiments, the composition of CD8+ T cells contains less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% CD4+ T cells, and/or contains no CD4+ T cells, and/or is free of or substantially free of CD4+ T cells. In some embodiments, the composition of enriched T cells consists essentially of CD8+ T cells.

[0166] In certain embodiments, the process is performed with two or more input compositions of enriched T cells, such as a separate CD4+ composition and a separate CD8+ composition, that are separately processed and engineered from the same starting or initial biological sample and re-infused back into the subject at a defined ratio, e.g. 1:1 ratio of CD4+ to CD8+ T cells.

J. A div at ion and Stimulation

[0167] In certain embodiments, the process for producing an output composition of engineered cells further can include one or more of: activating and/or stimulating a cells, e.g., cells of an input composition. In some embodiments, gene transfer is accomplished by first stimulating the cell, such as by combining cells with a stimulus that induces a response such as proliferation, survival, and/or activation, e.g., as measured by expression of a cytokine or activation marker. In certain embodiments, the gene transfer is accomplished by first incubating the cells under stimulating conditions, such as by any of the methods described.

[0168] In some embodiments, the cells are incubated and/or cultured prior to transduction. The conditions for incubation or culture can include one or more of particular media, temperature, oxygen content, carbon dioxide content, time, agents, e.g., nutrients, amino acids, antibiotics, ions, and/or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate the cells.

[0169] In some embodiments, the stimulating conditions or agents include one or more agent, e.g., ligand, which is capable of stimulating or activating an intracellular signaling domain of a TCR complex. In some aspects, the agent turns on or initiates TCR/CD3 intracellular signaling cascade in a T cell. Such agents can include antibodies, such as those specific for a TCR, e.g. anti-CD3. In some embodiments, the stimulating conditions include one or more agent, e.g. ligand, which is capable of stimulating a costimulatory receptor, e.g., anti- CD28. In some embodiments, such agents and/or ligands may be, bound to solid support such as a bead, and/or one or more cytokines. Optionally, the expansion method may further comprise the step of adding anti-CD3 and/or anti-CD28 antibody to the culture medium (e.g., at a concentration of at least about 0.5 ng/ml). In some embodiments, the stimulating agents include IL-2, IL- 15 and/or IL-7. In some aspects, the IL-2 concentration is at least about 10 units/mL. The cytokines may also include any described in section I.C.4.

[0170] In some embodiments, the stimulatory reagent contains a bead and one or more agents that directly interact with a macromolecule on the surface of a cell. In certain embodiments, the bead (e.g., a paramagnetic bead) interacts with a cell via one or more agents (e.g., an antibody) specific for one or more macromolecules on the cell (e.g., one or more cell surface proteins). In certain embodiments, the bead (e.g., a paramagnetic bead) is labeled with a first agent described herein, such as a primary antibody (e.g., an anti-biotin antibody) or other biomolecule, and then a second agent, such as a secondary antibody (e.g., a biotinylated anti- CD3 antibody) or other second biomolecule (e.g., streptavidin), is added, whereby the secondary antibody or other second biomolecule specifically binds to such primary antibodies or other biomolecule on the particle.

[0171] In some embodiments, the stimulatory reagent comprises a bead that contains a metal oxide core (e.g., an iron oxide core) and a coat, wherein the metal oxide core comprises at least one polysaccharide (e.g., dextran), and wherein the coat comprises at least one polysaccharide (e.g., amino dextran), at least one polymer (e.g., polyurethane) and silica. In some embodiments the metal oxide core is a colloidal iron oxide core. In certain embodiments, the one or more agents include an antibody or antigen-binding fragment thereof. In particular embodiments, the one or more agents include an anti-CD3 antibody and an anti-CD28 antibody. In some embodiments, the stimulatory reagent comprises an anti-CD3 antibody, anti-CD28 antibody, and an anti-biotin antibody. In some embodiments, the stimulatory reagent comprises an anti-biotin antibody. In some embodiments, the bead has a diameter of about 3 pm to about 10 pm. In some embodiments, the bead has a diameter of about 3 pm to about 5 pm. In certain embodiments, the bead has a diameter of about 3.5 pm.

[0172] In some embodiments, the stimulatory reagent comprises one or more agents that are attached to a bead comprising a metal oxide core (e.g., an iron oxide inner core) and a coat (e.g., a protective coat), wherein the coat comprises polystyrene. In certain embodiments, the beads are monodispersed, paramagnetic (e.g., superparamagnetic) beads comprising a paramagnetic (e.g., superparamagnetic) iron core, e.g., a core comprising magnetite (Fe3O4) and/or maghemite (yFe2O3) c and a polystyrene coat or coating. In some embodiments, the bead is non-porous. In some embodiments, the beads contain a functionalized surface to which the one or more agents are attached. In certain embodiments, the one or more agents are covalently bound to the beads at the surface. In some embodiments, the one or more agents include an antibody or antigen-binding fragment thereof. In some embodiments, the one or more agents include an anti-CD3 antibody and an anti-CD28 antibody. In some embodiments, the one or more agents include an anti-CD3 antibody and/or an anti-CD28 antibody, and an antibody or antigen fragment thereof capable of binding to a labeled antibody (e.g., biotinylated antibody), such as a labeled anti-CD3 or anti-CD28 antibody. In certain embodiments, the beads have a density of about 1.5 g/cm3 and a surface area of about 1 m2/g to about 4 m2/g. In particular embodiments, the beads are monodisperse superparamagnetic beads that have a diameter of about 4.5 pm and a density of about 1.5 g/cm3. In some embodiments, the beads the beads are monodisperse superparamagnetic beads that have a mean diameter of about 2.8 pm and a density of about 1.3 g/cm3.

[0173] In some embodiments, the population of enriched T cells is incubated with stimulatory reagent a ratio of beads to cells at or at about 3:1, 2.5:1, 2:1, 1.5:1, 1.25:1, 1.2:1, 1.1:1, 1:1, 0.9:1, 0.8:1, 0.75:1, 0.67:1, 0.5:1, 0.3:1, or 0.2:1. In particular embodiments, the ratio of beads to cells is between 2.5:1 and 0.2:1, between 2:1 and 0.5:1, between 1.5:1 and 0.75:1, between 1.25:1 and 0.8:1, between 1.1:1 and 0.9:1. In particular embodiments, the ratio of beads to cells is about 1:1 or is 1:1.

[0174] [0769] In particular embodiments, the stimulatory reagent contains an oligomeric reagent, e.g., a streptavidin mutein reagent, that is conjugated, linked, or attached to one or more agent, e.g., ligand, which is capable of activating an intracellular signaling domain of a TCR complex. In some embodiments, the oligomeric stimulatory reagent is or includes a reversible system in which at least one agent (e.g., an agent that is capable of producing a signal in a cell such as a T cell) is associated, e.g., reversibly associated, with the oligomeric reagent. Nonlimiting examples of oligomeric stimulatory reagents may be found, for example, in International published PCT Appl. No. WO 2018/197949, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the reagent contains a plurality of binding sites capable of binding, e.g., reversibly binding, to the agent.

[0175] In particular embodiments, the oligomeric particle reagent stimulatory agent is composed of and/or contains a plurality of streptavidin or streptavidin mutein tetramers. In some embodiments, the oligomeric particle has a radius, e.g., an average radius, of between 70 nm and 125 nm, inclusive; a molecular weight of between 1 x 107 g/mol and 1 x 109 g/mol, inclusive; and/or between 1,000 and 5,000 streptavidin or streptavidin mutein tetramers, inclusive. In some embodiments, the oligomeric particle reagent is bound, e.g., reversibly bound, to an anti-CD3 and/or an anti-CD28 antibody or antigen binding fragment thereof, via a binding partner, e.g., a streptavidin binding peptide, e.g. Strep-tag® II. In particular embodiments, the one or more agents is an anti-CD3 and/or an anti CD28 Fab containing a binding partner, e.g., a streptavidin binding peptide, e.g. Strep-tag® II. In some embodiments, the cells are stimulated in the presence of, of about, or of at least 0.01 pg, 0.02 pg, 0.03 pg, 0.04 pg, 0.05 pg, 0.1 pg, 0.2 pg, 0.3 pg, 0.4 pg, 0.5 pg, 0.75 pg, 1 pg, 2 pg, 3 pg, 4 pg, 5 pg, 6 pg, 7 pg, 8 pg, 9 pg, or 10 pg of the oligomeric stimulatory reagent per 106 cells.

[0176] In some aspects, incubation is carried out in accordance with techniques such as those described in US Patent No. 6,040,177 to Riddell et al., Klebanoff et al. (2012) J Immunother. 35(9): 651-660, Terakura et al. (2012) Blood.1:72-82, and/or Wang et al. (2012) J Immunother. 35(9):689-701.

[0177] In some embodiments, the T cells are expanded by adding to a culture-initiating composition feeder cells, such as non-dividing peripheral blood mononuclear cells (PBMC), (e.g., such that the resulting population of cells contains at least about 5, 10, 20, or 40 or more PBMC feeder cells for each T lymphocyte in the initial population to be expanded); and incubating the culture (e.g. for a time sufficient to expand the numbers of T cells). In some aspects, the non-dividing feeder cells can comprise gamma-irradiated PBMC feeder cells. In some embodiments, the PBMC are irradiated with gamma rays in the range of about 3000 to 3600 rads to prevent cell division. In some aspects, the feeder cells are added to culture medium prior to the addition of the populations of T cells.

[0178] In some embodiments, the stimulating conditions include temperature suitable for the growth of human T lymphocytes, for example, at least about 25 degrees Celsius, generally at least about 30 degrees, and generally at or about 37 degrees Celsius. Optionally, the incubation may further comprise adding non-dividing EBV-transformed lymphoblastoid cells (LCL) as feeder cells. LCL can be irradiated with gamma rays in the range of about 6000 to 10,000 rads. The LCL feeder cells in some aspects is provided in any suitable amount, such as a ratio of LCL feeder cells to initial T lymphocytes of at least about 10:1.

[0179] In embodiments, antigen-specific T cells, such as antigen- specific CD4+ and/or CD8+ T cells, are obtained by stimulating naive or antigen specific T lymphocytes with antigen. For example, antigen- specific T cell lines or clones can be generated to cytomegalovirus antigens by isolating T cells from infected subjects and stimulating the cells in vitro with the same antigen.

[0180] In some embodiments, the total duration of the incubation, e.g. with the stimulating agent, is between or between about 1 hour and 96 hours, 1 hour and 72 hours, 1 hour and 48 hours, 4 hours and 36 hours, 8 hours and 30 hours or 12 hours and 24 hours, such as at least or at least about 6 hours, 12 hours, 18 hours, 24 hours, 36 hours or 72 hours. In some embodiments, the further incubation is for a time between or about between 1 hour and 48 hours, 4 hours and 36 hours, 8 hours and 30 hours or 12 hours and 24 hours, inclusive.

[0181] In particular embodiments, the stimulating conditions include incubating, culturing, and/or cultivating a composition of enriched T cells with and/or in the presence of one or more cytokines. In particular embodiments, the one or more cytokines are recombinant cytokines. In some embodiments, the one or more cytokines are human recombinant cytokines. In certain embodiments, the one or more cytokines bind to and/or are capable of binding to receptors that are expressed by and/or are endogenous to T cells. In particular embodiments, the one or more cytokines is or includes a member of the 4-alpha-helix bundle family of cytokines. In some embodiments, members of the 4-alpha-helix bundle family of cytokines include, but are not limited to, interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-7 (IL-7), interleukin-9 (IL-9), interleukin 12 (IL-12), interleukin 15 (IL-15), granulocyte colony- stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF).

[0182] In some embodiments, the stimulation results in activation and/or proliferation of the cells, for example, prior to transduction.

2. Transduction

[0183] In some embodiments, the provided methods involve methods of transducing cells by contacting, e.g., incubating, a cell composition comprising a plurality of cells with a viral vector. In some embodiments, the cells to be transduced are or comprise primary cells obtained from a subject, such as cells enriched and/or selected from a subject.

[0184] In some embodiments, methods for genetic engineering by transduction are carried out by contacting one or more cells of a composition with viral vector (e.g. lentiviral vector) in accord with the provided methods. In some embodiments, the contacting can be effected with centrifugation, such as spinoculation (e.g. centrifugal inoculation). Such methods include any of those as described in International Publication Number WO2016/073602. Exemplary centrifugal chambers include those produced and sold by Biosafe SA, including those for use with the Sepax® and Sepax® 2 system, including an A-200/F and A-200 centrifugal chambers and various kits for use with such systems. Exemplary chambers, systems, and processing instrumentation and cabinets are described, for example, in US Patent No. 6,123,655, US Patent No. 6,733,433 and Published U.S. Patent Application, Publication No.: US 2008/0171951, and published international patent application, publication no. WO 00/38762, the contents of each of which are incorporated herein by reference in their entirety. Exemplary kits for use with such systems include, but are not limited to, single-use kits sold by BioSafe SA under product names CS-430.1, CS-490.1, CS-600.1 or CS-900.2.

[0185] In some embodiments, the contacting can be effected with centrifugation, such as spinoculation (e.g., centrifugal inoculation). In some embodiments, the composition containing cells, the vector, e.g., viral particles and reagent can be rotated, generally at relatively low force or speed, such as speed lower than that used to pellet the cells, such as from or from about 600 rpm to 1700 rpm (e.g., at or about or at least 600 rpm, 1000 rpm, or 1500 rpm or 1700 rpm). In some embodiments, the rotation is carried at a force, e.g., a relative centrifugal force, of from or from about 100 g to 3200 g (e.g., at or about or at least at or about 100 g, 200 g, 300 g, 400 g, 500 g, 1000 g, 1500 g, 2000 g, 2500 g, 3000 g or 3200 g), as measured for example at an internal or external wall of the chamber or cavity. The term “relative centrifugal force” or RCF is generally understood to be the effective force imparted on an object or substance (such as a cell, sample, or pellet and/or a point in the chamber or other container being rotated), relative to the earth’s gravitational force, at a particular point in space as compared to the axis of rotation. The value may be determined using well-known formulas, taking into account the gravitational force, rotation speed and the radius of rotation (distance from the axis of rotation and the object, substance, or particle at which RCF is being measured).

[0186] In some embodiments, the viral vector is provided by serial dilution of harvested culture supernatant or clarified supernatant.

[0187] In some embodiments, the method involves contacting or incubating, the cells with the viral vectors or supernatant containing same. In some embodiments, the contacting is for 30 minutes to 72 hours, such as 30 minute to 48 hours, 30 minutes to 24 hours or 1 hour to 24 hours, such as at least or at least about 30 minutes, 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 36 hours or more.

[0188] In some embodiments, contacting is performed in solution. In some embodiments, the cells and viral particles are contacted in a volume of from or from about 0.5 mL to 500 mL, such as from or from about 0.5 mL to 200 mL, 0.5 mL to 100 mL, 0.5 mL to 50 mL, 0.5 mL to 10 mL, 0.5 mL to 5 mL, 5 mL to 500 mL, 5 mL to 200 mL, 5 mL to 100 mL, 5 mL to 50 mL, 5 mL to 10 mL, 10 mL to 500 mL, 10 mL to 200 mL, 10 mL to 100 mL, 10 mL to 50 mL, 50 mL to 500 mL, 50 mL to 200 mL, 50 mL to 100 mL, 100 mL to 500 mL, 100 mL to 200 mL or 200 mL to 500 mL. [0189] In certain embodiments, the input cells are treated, incubated, or contacted with particles that comprise binding molecules that bind to or recognize the recombinant receptor that is encoded by the viral DNA.

3. Cultivation and Expansion

[0190] In some embodiments, methods of producing an output composition of engineered primary cells include one or more steps for cultivating cells, e.g., cultivating cells under conditions that promote proliferation and/or expansion. In some embodiments, cells are cultivated under conditions that promote proliferation and/or expansion subsequent to a step of transduction. In particular embodiments, the cells are cultivated after the cells have been incubated under stimulating conditions and transduced or transfected with a recombinant polynucleotide, e.g., a polynucleotide encoding a recombinant receptor. Thus, in some embodiments, a composition of CAR-positive T cells that has been engineered by transduction is cultivated under conditions that promote proliferation and/or expansion.

[0191] In some embodiments, cultivation is carried out under conditions that promote proliferation and/or expansion. In some embodiments, such conditions may be designed to induce proliferation, expansion, activation, and/or survival of cells in the population. In particular embodiments, the stimulating conditions can include one or more of particular media, temperature, oxygen content, carbon dioxide content, time, agents, e.g., nutrients, amino acids, antibiotics, ions, and/or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to promote growth, division, and/or expansion of the cells.

[0192] In particular embodiments, the cells are cultivated in the presence of one or more cytokines. In particular embodiments, the one or more cytokines are recombinant cytokines. In some embodiments, the one or more cytokines are human recombinant cytokines. In certain embodiments, the one or more cytokines bind to and/or are capable of binding to receptors that are expressed by and/or are endogenous to T cells. In particular embodiments, the one or more cytokines, e.g. a recombinant cytokine, is or includes a member of the 4-alpha-helix bundle family of cytokines. In some embodiments, members of the 4-alpha-helix bundle family of cytokines include, but are not limited to, interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-7 (IL-7), interleukin-9 (IL-9), interleukin 12 (IL-12), interleukin 15 (IL-15), granulocyte colonystimulating factor (G-CSF), and granulocyte-macrophage colony- stimulating factor (GM-CSF). In some embodiments, the one or more recombinant cytokine includes IL-2, IL-7 and/or IL- 15. In some embodiments, the cells, e.g., engineered cells, are cultivated in the presence of a cytokine, e.g., a recombinant human cytokine, at a concentration of between 1 lU/mL and 2,000 lU/mL, between 10 lU/mL and 100 lU/mL, between 50 lU/mL and 200 lU/mL, between 100 lU/mL and 500 lU/mL, between 100 lU/mL and 1,000 lU/mL, between 500 lU/mL and 2,000 lU/mL, or between 100 lU/mL and 1,500 lU/mL.

[0193] In some embodiments, cells are incubated, such as for cultivation or expansion, in the presence of recombinant IL-2, e.g., human recombinant IL-2, at a concentration between 1 lU/mL and 500 lU/mL, between 10 lU/mL and 250 lU/mL, between 50 lU/mL and 200 lU/mL, between 50 lU/mL and 150 lU/mL, between 75 lU/mL and 125 lU/mL, between 100 lU/mL and 200 lU/mL, or between 10 lU/mL and 100 lU/mL. In particular embodiments, cells are incubated, such as for cultivation or expansion, in the presence of recombinant IL-2 at a concentration at or at about 50 lU/mL, 60 lU/mL, 70 lU/mL, 80 lU/mL, 90 lU/mL, 100 lU/mL, 110 lU/mL, 120 lU/mL, 130 lU/mL, 140 lU/mL, 150 lU/mL, 160 lU/mL, 170 lU/mL, 180 lU/mL, 190 lU/mL, or 100 lU/mL. In some embodiments, the cells are incubated, such as for cultivation or expansion, in the presence of or of about 100 lU/mL of recombinant IL-2, e.g., human recombinant IL-2.

[0194] In some embodiments, the cells are incubated, such as for cultivation or expansion, in the presence of recombinant IL-7, e.g., human recombinant IL-7, at a concentration between 100 lU/mL and 2,000 lU/mL, between 500 lU/mL and 1,000 lU/mL, between 100 lU/mL and 500 lU/mL, between 500 lU/mL and 750 lU/mL, between 750 lU/mL and 1,000 lU/mL, or between 550 lU/mL and 650 lU/mL. In particular embodiments, the cells are incubated, such as for cultivation or expansion, in the presence of IL-7 at a concentration at or at about 50 IU/mL,100 lU/mL, 150 lU/mL, 200 lU/mL, 250 lU/mL, 300 lU/mL, 350 lU/mL, 400 lU/mL, 450 lU/mL, 500 lU/mL, 550 lU/mL, 600 lU/mL, 650 lU/mL, 700 lU/mL, 750 lU/mL, 800 lU/mL, 750 lU/mL, 750 lU/mL, 750 lU/mL, or 1,000 lU/mL. In particular embodiments, the cells are incubated, such as for cultivation or expansion, in the presence of or of about 600 lU/mL of recombinant IL-7, e.g., human recombinant IL-7.

[0195] In some embodiments, the cells are incubated, such as for cultivation or expansion, in the presence of recombinant IL- 15, e.g., human recombinant IL- 15, at a concentration between 1 lU/mL and 500 lU/mL, between 10 lU/mL and 250 lU/mL, between 50 lU/mL and 200 lU/mL, between 50 lU/mL and 150 lU/mL, between 75 lU/mL and 125 lU/mL, between 100 lU/mL and 200 lU/mL, or between 10 lU/mL and 100 lU/mL. In particular embodiments, cell are incubated, such as for cultivation or expansion, in the presence of recombinant IL- 15 at a concentration at or at about 50 lU/mL, 60 lU/mL, 70 lU/mL, 80 lU/mL, 90 lU/mL, 100 lU/mL, 110 lU/mL, 120 lU/mL, 130 lU/mL, 140 lU/mL, 150 lU/mL, 160 lU/mL, 170 lU/mL, 180 lU/mL, 190 lU/mL, or 200 lU/mL. In some embodiments, the cell are incubated, such as for cultivation or expansion, in the presence of or of about 100 lU/mL of recombinant IL-15, e.g., human recombinant IL- 15.

[0196] In particular embodiments, the cells are incubated, such as for cultivation or expansion, in the presence of IL-2, IL-7, and/or IL-15. In some embodiments, the IL-2, IL-7, and/or IL-15 are recombinant. In certain embodiments, the IL-2, IL-7, and/or IL-15 are human. In particular embodiments, the one or more cytokines are or include human recombinant IL-2, IL-7, and/or IL-15. In certain embodiments, the cells are stimulated or subjected to stimulation under stimulating conditions in the presence of recombinant IL-2, IL-7, and IL- 15. In certain embodiments, the cells are incubated, such as for cultivation or expansion, in the presence of recombinant IL-2 of or of about 100 lU/mL, recombinant IL-7 of or of about 600 lU/mL, and recombinant IL- 15 of or of about 100 lU/mL.

[0197] In some embodiments, the cultivation is performed under conditions that generally include a temperature suitable for the growth of primary immune cells, such as human T lymphocytes, for example, at least about 25 degrees Celsius, generally at least about 30 degrees, and generally at or about 37 degrees Celsius. In some embodiments, the composition of enriched T cells is incubated at a temperature of 25 to 38°C, such as 30 to 37°C, for example at or about 37 °C ± 2 °C. In some embodiments, the incubation is carried out for a time period until the culture, e.g. cultivation or expansion, results in a desired or threshold density, number or dose of cells. In some embodiments, the incubation is greater than or greater than about or is for about or 24 hours, 48 hours, 72 hours, 96 hours, 5 days, 6 days, 7 days, 8 days, 9 days or more.

[0198] In particular embodiments, the cultivation is performed in a closed system. In certain embodiments, the cultivation is performed in a closed system under sterile conditions. In particular embodiments, the cultivation is performed in the same closed system as one or more steps of the provided systems. In some embodiments the composition of enriched T cells is removed from a closed system and placed in and/or connected to a bioreactor for the cultivation. Examples of suitable bioreactors for the cultivation include, but are not limited to, GE Xuri W25, GE Xuri W5, Sartorius BioSTAT RM 20 | 50, Finesse SmartRocker Bioreactor Systems, and Pall XRS Bioreactor Systems. In some embodiments, the bioreactor is used to perfuse and/or mix the cells during at least a portion of the cultivation step.

[0199] In some embodiments, the mixing is or includes rocking and/or motioning. In some cases, the bioreactor can be subject to motioning or rocking, which, in some aspects, can increase oxygen transfer. Motioning the bioreactor may include, but is not limited to rotating along a horizontal axis, rotating along a vertical axis, a rocking motion along a tilted or inclined horizontal axis of the bioreactor or any combination thereof. In some embodiments, at least a portion of the incubation is carried out with rocking. The rocking speed and rocking angle may be adjusted to achieve a desired agitation. In some embodiments the rock angle is 20°, 19°, 18°, 17°, 16°, 15°, 14°, 13°, 12°, 11°, 10°, 9°, 8°, 7°, 6°, 5°, 4°, 3°, 2° or 1°. In certain embodiments, the rock angle is between 6-16°. In other embodiments, the rock angle is between 7-16°. In other embodiments, the rock angle is between 8-12°. In some embodiments, the rock rate is 1, 2, 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, 40 rpm. In some embodiments, the rock rate is between 4 and 12 rpm, such as between 4 and 6 rpm, inclusive.

[0200] In some embodiments, the bioreactor maintains the temperature at or near 37°C and CO2 levels at or near 5% with a steady air flow at, at about, or at least 0.01 L/min, 0.05 L/min, 0.1 L/min, 0.2 L/min, 0.3 L/min, 0.4 L/min, 0.5 L/min, 1.0 L/min, 1.5 L/min, or 2.0 L/min or greater than 2.0 L/min. In certain embodiments, at least a portion of the cultivation is performed with perfusion, such as with a rate of 290 ml/day, 580 ml/day, and/or 1160 ml/day, e.g., depending on the timing in relation to the start of the cultivation and/or density of the cultivated cells. In some embodiments, at least a portion of the cell culture expansion is performed with a rocking motion, such as at an angle of between 5° and 10°, such as 6°, at a constant rocking speed, such as a speed of between 5 and 15 RPM, such as 6 RMP or 10 RPM.

II. METHODS OF MANUFACTURING VIRAL VECTORS

[0201] Provided herein are methods of manufacturing viral vectors. In some embodiments, a polynucleotide encoding a transgene, such as a recombinant receptor and/or additional polypeptide is contained in such vectors. In some embodiments, the one or more vector(s) can be used to transform a host cell, e.g., a cell for engineering. Exemplary vectors include vectors include viral vectors, such as lentiviral vectors.

[0202] Viral vectors, including retroviral vectors, have become the dominant method for the introduction of genes into mammalian, e.g., human cells. The provided viral vector particles contain a genome derived from a retroviral genome based vector, such as derived from a gammaretroviral or lentiviral genome based vector. Any of a large number of such suitable vector genomes are known ((see, e.g., Koste et al. (2014) Gene Therapy 2014 Apr 3. doi: 10.1038/gt.2014.25; Carlens et al. (2000) Exp Hematol 28(10): 1137-46; Alonso-Camino et al. (2013) Mol Ther Nucl Acids 2, e93; Park et al., Trends Biotechnol. 2011 November 29(11): 550-557; Pfeifer and Verma (2001) Annu. Rev. Genomics Hum. Genet., 2:177-211). In some embodiments, the viral vector particles contain a genome derived from a retroviral genome based vector, such as derived from a lentiviral genome based vector. In some embodiments, the viral vector particle is a lentiviral vector particle.

[0203] In some aspects of the provided viral vectors, a recombinant nucleic acid (e.g., transgene) encoding a recombinant protein, such as an antigen receptor, such as a chimeric antigen receptor (CAR) or a transgenic T cell receptor (TCR), is contained and/or located between the 5' LTR and 3' LTR sequences of the vector genome. In some embodiments, the recombinant protein is an antigen receptor. In some embodiments, the recombinant protein is a T cell receptor (TCR). In some embodiments, the recombinant protein is a chimeric antigen receptor (CAR). In some embodiments, recombinant nucleic acids are transferred into cells using retroviral vectors, such as lentiviral vectors or gamma-retroviral vectors (see, e.g., Koste et al. (2014) Gene Therapy 2014 Apr. 3. doi: 10.1038/gt.2014.25; Carlens et al. (2000) Exp Hematol 28(10): 1137-46; Alonso-Camino et al. (2013) Mol Ther Nucl Acids 2, e93; Park et al., Trends Biotechnol. 2011 Nov. 29(11): 550-557. Retroviruses are useful as delivery vectors because of their ability to integrate their genes into the host genome, transferring a large amount of foreign genetic material, infecting a broad spectrum of species and cell types and of being packaged in special cell lines (Miller, 1992). Lentiviruses, in contrast to other retroviruses, in some contexts may be used for transducing certain non-dividing cells.

[0204] Non-limiting examples of lentiviral vectors include those derived from a lentivirus, such as Human Immunodeficiency Vims 1 (HIV-1), HIV-2, an Simian Immunodeficiency Vims (SIV), Human T-lympho tropic vims 1 (HTLV-1), HTLV-2 or equine infection anemia vims (E1AV). In some embodiments, the viral vector genome is a lentivirus genome, such as an HIV- 1 genome or an SIV genome. For example, lentiviral vectors have been generated by multiply attenuating virulence genes, for example, the genes env, vif, vpu and nef can be deleted, making the vector safer for therapeutic purposes. Lentiviral vectors are known. See Naldini et al., (1996 and 1998); Zufferey et al., (1997); Dull et al., 1998, U.S. Pat. Nos. 6,013,516; and 5,994,136). In some embodiments, these viral vectors are plasmid-based or virus-based, and are configured to carry the essential sequences for incorporating foreign nucleic acid, for selection, and for transfer of the nucleic acid into a host cell. Known lentiviruses can be readily obtained from depositories or collections such as the American Type Culture Collection (“ATCC”; 10801 University Blvd., Manassas, Va. 20110-2209), or isolated from known sources using commonly available techniques.

[0205] In some embodiments, two components are involved in making a virus-based gene delivery system: first, packaging plasmids, encompassing the structural proteins as well as the enzymes necessary to generate a viral vector particle, and second, the transfer plasmid, i.e., the genetic material to be transferred. Biosafety safeguards can be introduced in the design of one or both of these components. In some embodiments, the packaging plasmid can contain all HIV-1 proteins other than envelope proteins (Naldini et ah, 1998). In some embodiments, viral vectors can lack additional viral genes, such as those that are associated with virulence, e.g. vpr, vif, vpu and nef, and/or Tat, a primary transactivator of HIV. In some embodiments, packaging systems for lentiviral vectors, such as HIV-based lentiviral vectors, include separate packaging plasmids that together comprise only three genes of the parental virus: gag, pol and rev, which reduces or eliminates the possibility of reconstitution of a wild-type virus through recombination.

[0206] In some embodiments, the viral genome vector can contain sequences of the 5' and 3' LTRs of a retrovirus, such as a lentivirus. In some aspects of the provided viral vectors, the recombinant nucleic acid encoding a recombinant protein, such as provided as part of an expression cassette containing the transgene under the control of a promoter, is contained and/or located between the 5' LTR and 3' LTR sequences of the vector genome, including wildtype LTRs or portions or chimeric portions thereof. In some aspects, the viral genome construct may contain sequences from the 5' and 3' LTRs of a lentivirus, and in particular can contain the R and U5 sequences from the 5' LTR of a lentivirus and an inactivated or self-inactivating 3' LTR from a lentivirus. The LTR sequences can be LTR sequences from any lentivirus from any species. For example, they may be LTR sequences from HIV, SIV, FIV or BIV. Typically, the LTR sequences are HIV LTR sequences.

[0207] In some embodiments, the viral vector, such as an HIV viral vector, lacks additional transcriptional units. In some embodiments, the vector genome can contain an inactivated or self-inactivating 3' LTR (Zufferey et al. J Virol 72: 9873, 1998; Miyoshi et al., J Virol 72:8150, 1998). For example, the vector genome can contain deletion in the U3 region of the 3' LTR of the DNA used to produce the viral vector RNA, which can generate a self-inactivating (SIN) vector. This deletion can then be transferred to the 5' LTR of the proviral DNA during reverse transcription. In some embodiments, the 3' LTR is deleted for the promoter and the enhancer of U3. In some embodiments enough sequence can be eliminated, including the removal of a TATA box, to abolish the transcriptional activity of the LTR. This can prevent production of full-length vector RNA in transduced cells. Thus, some embodiments include a deletion in the U3 region of the 3' LTR of the DNA. In some embodiments, this does not affect vector titers or the in vitro or in vivo properties of the vector.

[0208] Optionally, the U3 sequence from the lentiviral 5' LTR can be replaced with a promoter sequence in the viral construct, such as a heterologous promoter sequence. This can increase the titer of virus recovered from the packaging cell line. An enhancer sequence can also be included. Any enhancer/promoter combination that increases expression of the viral RNA genome in the packaging cell line may be used. In one example, the CMV enhancer/promoter sequence is used (U.S. Pat. No. 5,385,839 and U.S. Pat. No. 5,168,062).

[0209] In some embodiments, the viral vector genome may also contain additional genetic elements. The types of elements that can be included in the constructs are not limited in any way and can be chosen by one with skill in the art. In some embodiments, the vector genome contains sequences derived from a viral genome (e.g. lentiviral genome) that are non-coding regions of the genome that facilitate or provide recognition signals for DNA or RNA synthesis and processing. In some embodiments, such sequences can include cis-acting sequences that can be involved in packaging or encapsidation, reverse transcription and transcription and/or gene transfer or integration. In some embodiments, cis-activating sequences provided as part of the viral vector are derived from the same lentivirus or retrovirus -like organism.

[0210] In some embodiments, a signal that facilitates nuclear entry of the viral genome in the target cell may be included. An example of such a signal is the Flap sequence (also called a DNA Flap sequence) formed from the cPPT and CTS components that are part of the pol gene of a viral vector genome, such as a lentiviral vector genome. In some embodiments, a Flap sequence includes a portion of viral nucleic acid that contains a cPPT and/or a CTS region, but in which is deleted 5' and 3' portions of the pol gene that are not necessary for Flap function. In some cases, the viral vector does not contain a functional Flap region. As discussed below, in some embodiments a viral vector contains viral nucleic acid containing a variant Flap that lacks all or a portion of one or both of the cPPT and CTS region. [0211] In some embodiments, the lentiviral vector genome can contain elements selected among a splice donor site (SD), a splice acceptor site (SA) and/or a Rev-responsive element (RRE). In some embodiments, RRE is provided to allow export of viral messenger RNA from the nucleus to the cytosol after binding of the Rev protein provided as part of a helper plasmid during viral packaging. In some embodiments, the vector genome can contain the psi (w) packaging signal, which, in some cases, can be derived from the N-terminal fragment of the gag ORF. In some embodiments, the psi packaging signal sequence can be modified by frameshift mutation(s) in order to prevent any interference of a possible transcription/translation of gag peptide, with that of the transgene.

[0212] In some embodiments, provided is a viral vector, such as a lentiviral vector, that contains a recombinant genome containing in order between the 5' and 3' LTR sequences of the

[0213] vector genome: an RRE; a polynucleotide containing viral nucleic acid comprising a functional DNA Flap containing a cPPT and CTS that is inserted upstream of a promoter controlling expression of a polynucleotide encoding a recombinant protein; a transgene containing a promoter controlling expression of a polynucleotide encoding the recombinant protein and the polynucleotide encoding the recombinant protein, such as an antigen receptor (e.g. a CAR); and a polynucleotide containing a modified PRE operably linked to the nucleic acid encoding the recombinant protein. In some embodiments, the recombinant genome comprises the sequence 5' LTR-RRE-cPPT-CTS-transgene(s)-modified PRE-3' LTR. In some embodiments, the modified PRE in the viral vector, such as lentiviral vector is as described in WO2016115177. In some embodiments, the lentiviral vector is an HIV-1 derived lentiviral vector.

[0214] In some embodiments, among the provided polynucleotides, including viral vectors, are those containing variations in viral Flap sequences (deemed “variant Flap” polynucleotides or sequences). Such polynucleotides include those containing one or more modifications, e.g., deletion(s), within a viral Flap sequence within the polynucleotide. The variations can include complete deletion of a Flap sequence, or sub-part thereof, within a viral sequence of the polynucleotide. Such polynucleotides include viral vectors, such as a lentiviral vector, containing such variant Flap sequences. In some embodiments, the modified Flap in the viral vector, such as lentiviral vector is as described in WO2016115177.

[0215] In certain embodiments, the risk of insertional mutagenesis can be minimized by constructing the retroviral vector genome, such as lentiviral vector genome, to be integration defective. A variety of approaches can be pursued to produce a non-integrating vector genome. In some embodiments, a mutation(s) can be engineered into the integrase enzyme component of the pol gene, such that it encodes a protein with an inactive integrase. In some embodiments, the vector genome itself can be modified to prevent integration by, for example, mutating or deleting one or both attachment sites, or making the 3' LTR-proximal polypurine tract (PPT) non-functional through deletion or modification. In some embodiments, non-genetic approaches are available; these include pharmacological agents that inhibit one or more functions of integrase. The approaches are not mutually exclusive; that is, more than one of them can be used at a time. For example, both the integrase and attachment sites can be non-functional, or the integrase and PPT site can be non-functional, or the attachment sites and PPT site can be nonfunctional, or all of them can be non-functional. Such methods and viral vector genomes are known and available (see Philpott and Thrasher, Human Gene Therapy 18:483, 2007; Engelman et al. J Virol 69:2729, 1995; Brown et al J Virol 73:9011 (1999); WO 2009/076524;

McWilliams et al., J Virol 77:11150, 2003; Powell and Levin J Virol 70:5288, 1996).

[0216] In some embodiments, the vector contains sequences for propagation in a host cell, such as a prokaryotic host cell. In some embodiments, the nucleic acid of the viral vector contains one or more origins of replication for propagation in a prokaryotic cell, such as a bacterial cell. In some embodiments, vectors that include a prokaryotic origin of replication also may contain a gene whose expression confers a detectable or selectable marker such as drug resistance.

[0217] Viral vector production processes include upstream and downstream processes. The upstream process involves transfecting a particular cell type with a plurality of plasmids including packaging plasmids coding for certain viral genes and a transfer plasmid containing the recombinant nucleic acid that code for a gene of interest (also called a “transgene”) that, when expressed in the particular cell type, ultimately produce the desired viral particles which can then be harvested for use in clinical and/or research settings. In some embodiments, the recombinant nucleic acid, e.g., one encoding the desired sequence, such as an expression cassette, is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication defective. In order to produce virions, packaging plasmids encoding Gag, Pol, Rev, and optionally the Tat genes is introduced into a packaging cell line with a transfer plasmid that contains the recombinant nucleic acid between the LTRs ad a psi packaging signal. An envelope protein, such as VSV-G, is encoded by a third separate plasmid. When the plasmids are introduced into a host cell, the packaging sequence may permit the RNA transcript of the transfer plasmid to be packaged into viral particles, which then may be secreted into the culture media. The media containing the recombinant retroviruses in some embodiments is then collected, and then in downstream processes is concentrated and purified, and used for gene transfer.

A. Upstream Process

[0218] In some embodiments, a host cell for production of a viral vector is transfected with one or more plasmid vectors containing the components necessary to generate the particles. The host cells can express or be made to express essential lentiviral (e.g. HIV-1) genes to allow the generation of lentiviral particles. These genes can be expressed by several plasmids. In some aspects, in order to prevent replication of the genome in the target cell, however, endogenous viral genes required for replication are removed and provided separately in the host cell line. In some embodiments, multiple vectors are utilized to separate the various genetic components that generate the retroviral vector particles. In some such embodiments, providing separate vectors to the host cell reduces the chance of recombination events that might otherwise generate replication competent viruses.

[0219] In some embodiments, a host cell line is transfected with a lentiviral expression plasmid (also called “transfer plasmid”) containing a cis-acting psi (Y) packaging sequence and the transgene gene inserted between the lentiviral LTRs to allow target cell integration along with several helper plasmids encoding the virus enzymatic and/or structural components, such as Env, Gag, pol and/or rev. The helper plasmids provide the helper functions as well as structural and replication proteins in trans required to produce the lentivirus. In some embodiments, the packaging plasmid(s) can contain all retroviral, such as HIV-1, proteins other than envelope proteins (Naldini et al., 1998). In other embodiments, viral vectors can lack additional viral genes, such as those that are associated with virulence, e.g. vpr, vif, vpu and nef, and/or Tat, a primary transactivator of HIV.

[0220] In some embodiments, lentiviral vectors, such as HIV-based lentiviral vectors, comprise only three genes of the parental virus: gag, pol and rev, which reduces or eliminates the possibility of reconstitution of a wild-type virus through recombination. In some embodiments, a GagPol packaging plasmid containing the gag and pol genes encoding for structural and enzymatic components and a Rev plasmid containing the rev gene encoding for Rev regulatory protein are separately introduced into a packaging cell line. In some embodiments, a single plasmid vector having all of the retroviral components can be used. In some embodiments, an envelope plasmid encoding an env gene also can be introduced, which, in some cases, can result in viral particles pseudotyped with alternative Env proteins. In some embodiments, the retroviral vector particle, such as lentiviral vector particle, is pseudotyped to increase the transduction efficiency of host cells. For example, a retroviral vector particle, such as a lentiviral vector particle, is pseudotyped with a VSV-G glycoprotein, which provides a broad cell host range extending the cell types that can be transduced. The envelope plasmid may also be called a pseudotyping plasmid.

[0221] The env gene can be derived from any appropriate virus, such as a retrovirus. In some embodiments, the env is an amphotropic envelope protein which allows transduction of cells of human and other species. Some embodiments use retroviral-derived env genes, including, but not limited to: Moloney murine leukemia virus (MoMuLV or MMLV), Harvey murine sarcoma vims (HaMuSV or HSV), murine mammary tumor virus (MuMTV or MMTV), gibbon ape leukemia vims (GaLV or GALV), human immunodeficiency vims (HIV) and Rous sarcoma vims (RSV). In some embodiments, other env genes such as Vesicular stomatitis vims (VSV) protein G (VSVG), that of hepatitis viruses, and of influenza also can be used.

[0222] In some embodiments, the transfer plasmid comprises an HIV lentiviral vector backbone comprising a packaging sequence and a recombinant nucleic acid encoding a human therapeutic transgene. In particular embodiments, the recombinant nucleic acid encodes a recombinant receptor, such as a CAR.

[0223] In some embodiments, a host cell can be transfected with a transfer plasmid containing a cis-acting psi (Y) packaging sequence and the transgene gene inserted between the lentiviral LTRs to allow target cell integration; a packaging plasmid or plasmids encoding the pol, gag, rev and/or tat viral genes and, in some cases, containing the rev-response element (RRE) and an envelope plasmid, such as a plasmid encoding an envelope protein, such as the G protein of the Vesicular Stomatitis Vims (VSV-G) envelope gene.

[0224] In some embodiments, the plasmid gene expression is under the control of operably linked regulatory sequences, e.g., a promoter or enhancer. The regulatory sequence in some embodiments can be any eukaryotic promoter or enhancer, including for example, EFla, PGK, the Moloney murine leukemia vims promoter-enhancer element, the human cytomegalovirus enhancer, the vaccinia P7.5 promoter or the like. In some embodiments, the regulatory sequence is one which is not endogenous to the lentivirus from which the vector is being constructed. Thus, if the vector is being made from SIV, the SIV regulatory sequence found in the SIV LTR may be replaced by a regulatory element which does not originate from SIV.

[0225] In some embodiments, a host cell can be transiently transfected with the one or more helper plasmids encoding one or more viral proteins, including at least one packaging plasmid and an envelope plasmid, and a transfer plasmid containing the recombinant nucleic acid encoding the transgene (e.g. CAR). The host cells are thus cells or cell-lines that can produce or release viral vector particles carrying the gene of interest. In some embodiments, the host cell provides components or is made to provide components that are required in trans for the packaging of the viral genomic RNA into lentiviral vector particles, including viral regulatory and structural proteins. In some embodiments, the host cells may be any cell line that is capable of expressing lentiviral proteins and producing functional lentiviral vector particles. In some aspects, suitable host cells include 293 (ATCC CCL X), 293T, HeLA (ATCC CCL 2), D17 (ATCC CCL 183), MDCK (ATCC CCL 34), BHK (ATCC CCL- 10) and Cf2Th (ATCC CRL 1430) cells.

[0226] In some embodiments, these cells can further be anchorage dependent, which means that these cells will grow, survive, or maintain function optimally when attached to a surface such as glass or plastic. In some embodiments, these cells can be suspension-adapted such that these cells do not require attachment to a surface. In some embodiments, the host cells may be neoplastically transformed cells. In some embodiments, host cells for transfection with the lentiviral vector and packaging plasmids include, for example, mammalian primary cells; established mammalian cell lines, such as COS, CHO, HeLa, NIH3T3, 293T, 293F, LV293, HEK 293, and PC 12 cells; amphibian cells, such as Xenopus embryos and oocytes; other vertebrate cells; insect cells (for example, Drosophila), yeast cells (for example, S. cerevisiae, S. pombe, or Pichia pastoris) and prokaryotic cells (for example, E. coli).

[0227] In an embodiment of the method, the host cells may comprise mammalian cells, such as HEK 293 suspension cells. The host cells may be another mammalian cell type including, but not limited to, HeLa, U20S, A549, HT1080, CAD, P19, NIH 3T3, L929, N2a, recombinant Chinese hamster ovary (CHO), MCF-7, Y79, SO-Rb50, Hep G2, DUKX-X1 1, J558L, and/or baby hamster kidney (BHK) cells.

[0228] In some embodiments, the host cells are adherent cells and an adherent cell culture is transfected. [0229] In some embodiments, the host cells are suspension cells and a suspension cell culture is transfected. In some embodiments, the host cells are selected from the group consisting of HEK293 cells, HEK293S cells, HEK293T cells adapted for suspension culture (HEK293Ts), HEK293F cells, HEK293FT cells, HEK293FTM cells, and HEK293E cells. In some embodiments, the host cells are HEK293Ts cells. In some embodiments, the host cells are HEK 293T/17 cells.

[0230] In some embodiments, a retroviral vector, such as a lentiviral vector, can be produced in a host cell, such as an exemplary HEK 293T cell line, by introduction of plasmids to allow generation of lentiviral particles. In some embodiments, a host cell is transfected and/or contains a polynucleotide encoding gag and pol, and a polynucleotide encoding a recombinant receptor, such as an antigen receptor, for example, a CAR. In some embodiments, the host cell is optionally and/or additionally transfected with and/or contains a polynucleotide encoding a rev protein. In some embodiments, the host cell is optionally and/or additionally transfected with and/or contains a polynucleotide encoding a non-native envelope glycoprotein, such as VSV-G. In some such embodiments, approximately one to three days, such as two days, after transfection of cells, e.g. HEK 293T cells, the cell supernatant contains recombinant lentiviral vectors, which can be recovered and titered.

[0231] In some embodiments, the plasmids are introduced via transfection or infection into the host cells. Methods for transfection or infection are well known. Non-limiting examples include calcium phosphate, DEAE-dextran and lipofection methods, electroporation and microinjection. In some embodiments, a transfection reagent is added to facilitate transfection. In some embodiments, a transfection reagent is a molecule that increases the transfection of DNA into the host cell. Illustrative examples of transfection reagents suitable for use in particular embodiments contemplated herein include but are not limited to calcium phosphate, cationic lipids, and cationic polymers.

[0232] In some embodiments, the transfection reagent is a calcium phosphate solution.

[0233] Illustrative examples of cationic lipids suitable for use in particular embodiments contemplated herein include but are not limited to N-[l-(2,3-dioleoyloxy)propel]-N,N,N- trimethylammonium (DOTMA); 2,3-dioleyloxy-N-[2-spermine carboxamide] ethyl-N,N- dimethyl-1 -propanammonium trifluoroacetate (DOSPA, Lipofectamine); l,2-dioleoyl-3- trimethylammonium-propane (DOTAP); N-[l-(2,3-dimyristyloxy) propyl] -N,N-dimethyl- N-(2- hydroxy ethyl) ammonium bromide (DMRIE), 3-P-|N-(N.N -dimethylaminoethane) carbamoyl] cholesterol (DC-Chol); dioctadecyl amidoglyceryl spermine (DOGS, Transfectam); and imethyldioctadecylammonium bromide (DDAB).

[0234] In some embodiments, the transfection reagent is a cationic polymer, such as polyethylenimine (PEI).

[0235] In some embodiments, an upstream process for producing a retroviral vector, such as a lentiviral vector (LVV), involves inoculating a culture with host cells, transiently transfecting the host cells in the culture with a plasmid mixture comprising one or more helper plasmids, a transfer plasmid, and a transfection agent. In some embodiments, the one or more helper plasmids includes at least one packaging plasmid encoding gag, pol and rev and an envelope plasmid. In some embodiments, the one or more helper plasmids includes a gag-pol packaging plasmid, a rev packaging plasmid and an envelope plasmid. In some embodiments, the envelope plasmid encodes an envelope glycoprotein including but not limited to an envelope glycoprotein from a Vesicular stomatitis virus (VSV) envelope protein or variant thereof (e.g., VSV-G), a Cocal virus (COCV) envelope protein or variant thereof, a Maraba virus (MARAV) envelope protein or variant thereof, a Piry virus (PIRYV) envelope protein or variant thereof, a Nipah vims (NiV) envelope protein or variant thereof, a Sendai vims (SeV) envelope protein or variant thereof, a Morbillivirus envelope protein or variant thereof, a Canine distemper (CDV) envelope protein or variant thereof, a Measles vims (MV) envelope protein or variant thereof, a Sindbis vims (SINV) envelope protein or variant thereof, a Gibbon ape leukemia vims (GALV) envelope protein or variant thereof, a Feline endogenous retrovirus (RD114) envelope protein or variant thereof, a Feline leukemia vims (FeEV) envelope protein or variant thereof, a Baboon endogenous retrovirus (BaEV) envelope protein or variant thereof, a Hepatitis B (HBV) envelope protein or variant thereof, a Hepatitis C (HCV) envelope protein or variant thereof, and a Rabis vims (RABV) envelope protein or variant thereof. In some embodiments, the envelope plasmid encodes VSV-G.

[0236] In some embodiments, an upstream process for producing lentiviral vector (EVV) involves inoculating a culture with host cells, transiently transfecting the host cells in the culture with a plasmid mixture comprising a gag-pol packaging plasmid, a rev packaging plasmid, an envelope plasmid encoding VSV-G, a transfer plasmid encoding a transgene (e.g. a CAR), and a transfection reagent. In some embodiments, the transfer plasmid encodes a recombinant receptor, such as a CAR. In some embodiments, the mixture of plasmids is a four-plasmid mixture comprising a lentiviral packaging plasmid encoding gag and pol at a mass fraction ratio of between 0.15 and 0.2, a lentiviral packaging plasmid encoding rev at a mass fraction ratio of 0.04 to 0.06, an envelope plasmid encoding VSV-G at a mass fraction ratio of 0.04 to 0.15, and a transfer plasmid encoding a transgene at a mass fraction ratio of 0.58 to 0.74. In some embodiments, the mass fraction ratio of the packaging plasmid encoding gag and pol is about 0.178, the mass fraction ratio of the lentiviral packaging plasmid encoding rev is about 0.049, the mass ratio of the envelope plasmid encoding VSV-G is about 0.046, and the mass fraction ratio of the transfer plasmid encoding the transgene is about 0.727. In some embodiments, the transgene is a chimeric antigen receptor. In some embodiments, the chimeric antigen receptor is an anti-CD19 CAR. A skilled artisan is familiar with anti-CD19 CAR sequences and exemplary anti-CD19 Cars are described in Section III.

[0237] In some embodiments, the transfection reagent is a cationic polymer (e.g., polyethylenimine, also called PEI) for cationic polymer-mediated transient transfection. Cationic polymer based reagents can be particularly helpful in transfecting cells that exhibit low efficiency when transfected using lipid based reagents. When used at optimal concentrations, these reagents exhibit low toxicity. Polymer based transfection reagents can be used to transfect suspension cultures, primary cells, a variety of eukaryotic cells lines, adherent cells, etc. Many natural and synthetic cationic polymer based transfection reagents are currently available. The cationic polymer may in some examples comprise PEI, but in other embodiments, the cationic polymer may be different. In some embodiments, PEI Pro is the cationic polymer. Exemplary cationic polymers that can be used include but are not limited to histones, poly-L-lysine, polyamidoamine dendrimers, protamine and/or any combination thereof, and the like. Exemplary methods of transfection of packaging cell lines using a cationic polymer such as PEI are described in PCT publ. No. WO2021222133).

[0238] In some embodiments, the transfection process includes combining the plurality of plasmids with a cationic polymer (e.g., PEI), thereby condensing the DNA plasmids into positively charged particles that bind to anionic cell surfaces. Once bound, the transfection complex (e.g., PEI-DNA transfection complex) may be endocytosed by the cells, and the DNA (e.g., plurality of transfer plasmids) may be released into the cytoplasm of the cells. In some embodiments, a solution of the plasmid DNA is mixed with the cationic polymer (e.g. polyethylenimine or PEI) prior to contacted with the packaging cells. In some embodiments, the plasmid DNA is mixed with PEI prior to the contacting at a particular mass ratio amount to produce a complex of DNA:PEI. In some embodiments, the mass ratio of DNA to PEI is 5: 1 to 1:5, such as 3:1 to 1:3. In some embodiments, the mass ratio of DNA to pEI is about 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5 or any value between any of the foregoing. In some embodiments, the DNA plasmid solution and the PEI solution are mixed at a 1:1 DNA:PEI mass ratio. In some embodiments, the mixed DNA and PEI can then be contacted with a polyplex stabilizing agent (e.g., FBS or HSA).

[0239] In some embodiments, transfection is with a PEI/DN A complex of a certain size. In some aspects, transfection efficacy, and in turn, expression of desired genes, may in some examples be adversely impacted by a non-optimal size (e.g., too large or too small) of a transfection complex that includes a cationic polymer and DNA (e.g., a DNA plasmid or a plurality of DNA plasmids). Stabilizing a size of a polyplex in order to provide a transfection complex of an optimal size for transfection can improve downstream gene expression stemming from the transfection procedure. The predetermined size range may comprise a range in which transfection of a population of cells with the polymer-DNA transfection complex is more efficient and/or effective than if the polymer-DNA transfection complex size were outside of the predetermined size range. As an example, transfection of a population of cells with such a polymer-DNA transfection complex having dimensions within the predetermined size range increases viral vector titer (e.g., LVV titer) at harvest as compared with a population of cells transfected with the polymer-DNA transfection complex having dimensions outside of the predetermined size range. In examples, the predetermined size range is from 200-1400 nm in diameter. For example, between 400-1000 nm in diameter. Specifically, between 400-450 nm in diameter, or between 450-500 nm in diameter, or between 500-550 nm in diameter, or between 550-600 nm in diameter, or between 600-650 nm in diameter, or between 650-700 nm in diameter, or between 700-750 nm in diameter, or between 750-800 nm in diameter, or between 800-850 nm in diameter, or between 850-900 nm in diameter, or between 900-950 nm in diameter, or between 950-1000 nm in diameter. In some embodiments, a method for stabilizing a size of a polyplex comprises mixing together a first solution comprising deoxyribonucleic acid (DNA) with a second solution comprising a cationic polymer (e.g. PEI) to obtain a polyplex solution, and at a predetermined time subsequent to mixing together the first solution and the second solution, adding the polyplex stabilizing agent to the polyplex solution to stabilize the size of the polyplex.

[0240] In an embodiments, the polyplex stabilizing agent can be an albumin. The albumin can be a bovine serum albumin (BSA) or a human serum albumin (HSA). In some embodiments the transfection is carried out in the presence of fetal bovine serum (FBS) which contains BSA. In an embodiment, the PEI-DNA transfection complex stabilizing agent may be nonrecombinant HSA, recombinant HSA or a combination thereof. For example, the recombinant HSA may in some examples be purified Pichia pastoris, although other organisms may be used without departing from the scope of this disclosure. Examples include but are not limited to Saccharomyces cerevisiae, Hansenula polymorpha, Kluyveromyces lactis, etc. In some embodiments, the organism is P. pastoris, or S. cerevisiae.

[0241] In embodiments of an upstream process, the host cells are thawed and seeded for culture, seeded host cells are cultured to a desired density, the medium exchanged, and the cells transfected with a mixture comprising a transfection reagent, and the plurality of plasmids including the packaging plasmids encoding viral accessory genes and an envelope plasmid encoding an env gene (e.g. VSV-G) and a transfer plasmid encoding a packageable viral vector genome comprising a therapeutic transgene, such as a CAR. After a sufficient time for transfection, another medium exchange is performed and the transfected host cells are cultured to produce viral vector for about one to about three days.

[0242] In some embodiments, an upstream manufacturing process for a retroviral vector, e.g., lentiviral vector, involves culturing and expanding host cells to a volume until a sufficient amount of host cells to seed for culture is obtained, seeding a container (e.g. flask or bioreactor) with host packaging cells and culturing the cells for transfection, exchanging the culture medium, transfecting the host cells with a mixture that includes a transfection reagent and a mixture of a plurality of plasmids including helper plasmids encoding viral accessory genes (e.g., gag, pol and rev) and an envelope gene (e.g. VSV-G) and a transfer plasmid encoding a recombinant nucleic acid transgene, culturing the transfected cells to complete the transfection, exchanging the culture medium in the container, and culturing the cells for about one to about three days to produce the viral vector.

[0243] In some embodiments, the upstream process can be carried out on a small scale. In some embodiments, a small scale process for transfecting cells can be useful for upstream development procedures, such as in accord with the methods described in Section I. In some embodiments, the culture has a volume of 10 mL to 1000 mL, such as about 10 mL, about 15 mL, about 20 mL, about 30 mL, about 40 mL, about 50 mL, about 75 mL, about 100 mL, about 250 mL, about 500 mL, about 750 mL or about 1000 mL or any value between any of the foregoing. In some embodiments, the harvest of an upstream process is about 15 mL to about 20 mL. In some embodiments, the culture is inoculated with about 2 x 105 to about 15 x 105 viable host cells. In some embodiments, the host cells are cultured in plate or a flask.

[0244] In some embodiments, the upstream process can be carried out using a large-scale culture. In some embodiments, the culture has a volume of 5 L to 2000 L. In some embodiments, the culture has a volume of 5L, 10 L, 30 L, 50 L, 100 L, 200 L, 500 L, 1000 L, 2000 L, or any value between any of the foregoing. In some embodiments, the culture is inoculated with about 10.0 x 108 to about 150.0 x 108 viable host cells. In some embodiment, the host cells are cultured in a bioreactor. In some embodiments, a large scale upstream process includes thawing and culturing and expanding the host cells in progressively larger volumes until a sufficient amount of host cells to seed a working volume (e.g. 200 L) for large scale culture, such as in a bioreactor, is achieved.

[0245] In some embodiments, the transfection mixture (a mixture containing (1) a mixture of plasmids for the production of a lentiviral vector, wherein the mixture of plasmids comprise at least one lentiviral helper plasmid and a transfer plasmid encoding a transgene and (2) a transfection reagent) has a volume of from or from about 0.5 mL to 500 mL, such as from or from about 0.5 mL to 200 mL, 0.5 mL to 100 mL, 0.5 mL to 50 mL, 0.5 mL to 10 mL, 0.5 mL to 5 mL, 5 mL to 500 mL, 5 mL to 200 mL, 5 mL to 100 mL, 5 mL to 50 mL, 5 mL to 10 mL, 10 mL to 500 mL, 10 mL to 200 mL, 10 mL to 100 mL, 10 mL to 50 mL, 50 mL to 500 mL, 50 mL to 200 mL, 50 mL to 100 mL, 100 mL to 500 mL, 100 mL to 200 mL or 200 mL to 500 mL. In some embodiments, the transfection mixture has a volume of 5 mL In some embodiments, the transfection mixture has a volume of 50 mL. In some embodiments, the transfection mixture has a volume of 500 mL. In some embodiments, the transfection mixture has a volume of 1000 mL. In some embodiments, the transfection mixture has a volume of 2000 mL.

[0246] In some embodiments, the transfection mixture (a mixture containing (1) a mixture of plasmids for the production of a lentiviral vector, wherein the mixture of plasmids comprise at least one lentiviral helper plasmid and a transfer plasmid encoding a transgene, and (2) a transfection reagent) has a volume of from or from about 5 mL to 5000 mL, such as from or from about 5 mL to 2000 mL, 5 mL to 1000 mL, 5 mL to 500 mL, 5 mL to 100 mL, 5 mL to 50 mL, 50 mL to 5000 mL, 50 mL to 200 mL, 50 mL to 1000 mL, 50 mL to 500 mL, 50 mL to 100 mL, 100 mL to 5000 mL, 100 mL to 2000 mL, 100 mL to 1000 mL, 100 mL to 500 mL, 500 mL to 5000 mL, 500 mL to 2000 mL, 500 mL to 1000 mL, 1000 mL to 5000 mL, 1000 mL to 2000 mL or 2000 mL to 5000 mL. [0247] In some embodiments, the transfection reaction is in a volume of from or from about 5 mL to 5000 mL, such as from or from about 5 mL to 2000 mL, 5 mL to 1000 mL, 5 mL to 500 mL, 5 mL to 100 mL, 5 mL to 50 mL, 50 mL to 5000 mL, 50 mL to 200 mL, 50 mL to 1000 mL, 50 mL to 500 mL, 50 mL to 100 mL, 100 mL to 5000 mL, 100 mL to 2000 mL, 100 mL to 1000 mL, 100 mL to 500 mL, 500 mL to 5000 mL, 500 mL to 2000 mL, 500 mL to 1000 mL, 1000 mL to 5000 mL, 1000 mL to 2000 mL or 2000 mL to 5000 mL. In some embodiments, the transfection reaction is in a volume of 5 mL. In some embodiments, the transfection reaction is in a volume of 50 mL. In some embodiments, the transfection reaction is in a volume of 500 mL. In some embodiments, the transfection reaction is in a volume of 1000 mL. In some embodiments, the transfection reaction is in a volume of 2000 mL.

[0248] In some embodiments, the transfection reaction is in a volume of from or from about 0.5 mL to 500 mL, such as from or from about 0.5 mL to 200 mL, 0.5 mL to 100 mL, 0.5 mL to 50 mL, 0.5 mL to 10 mL, 0.5 mL to 5 mL, 5 mL to 500 mL, 5 mL to 200 mL, 5 mL to 100 mL, 5 mL to 50 mL, 5 mL to 10 mL, 10 mL to 500 mL, 10 mL to 200 mL, 10 mL to 100 mL, 10 mL to 50 mL, 50 mL to 500 mL, 50 mL to 200 mL, 50 mL to 100 mL, 100 mL to 500 mL, 100 mL to 200 mL or 200 mL to 500 mL.

[0249] In particular embodiments, the host cells are transfected for about 12 hours to about 72 hours. In some embodiments, the host cells are transfected for 12 hours, 24 hours, 36 hours, 48 hours, 60 hour, 72 hours, or any value between any of the foregoing. In some embodiments, media is exchanged at least one time after adding the transfection mixture to host cells. In some embodiments, supernatant media from transfected cultures is collected 1 to 3 days after transfection.

[0250] Culture of the host cells after transfection produces viral vector particles that contain the viral vector genome. When a recombinant plasmid and the retroviral LTR and packaging sequences are introduced into a special cell line, the packaging sequences may permit the RNA transcript of the recombinant plasmid to be packaged into viral particles, which then may be secreted into the culture media.

[0251] The media containing the recombinant retroviruses in some embodiments is then collected, optionally clarified. The harvested material can then be titered by methods as described or can be fed into a downstream process, such as described below. For example, in some aspects, after cotransfection of the packaging plasmids and the transfer vector to the host cell, the viral vector particles are recovered from the culture media and titered by standard methods used by those of skill in the art.

[0252] In some embodiments, the crude harvest material is collected. In some embodiments, the crude harvest material may be further clarified to remove cell debris and other biological impurities. In some embodiments, clarification can be carried out by centrifugation. In some embodiments, clarification is by counterflow centrifugation (CFC). In some embodiments, clarification can be carried out by filtration. Various filtration systems for clarification are known to a skilled artisan. In some embodiments, the clarification is by depth filtration. In some embodiments, the clarification is by membrane filtration. In some embodiments, the clarification is first by depth filtration and then by membrane filtration.

[0253] A depth filter does not have a defined pore size or structure. Depth filters comprise gradient density structures specifically designed to retain particles of a defined size. The particles are retained within the whole depth of the filter media. Depth filter media may comprise cellulose, polyethersulfone (PES), diatomaceous earth, or other materials suitable to retain contaminants of a particular size. Membrane filters, in contrast, retain particles of a particular size excluded by the pore size of the membrane at the membrane surface. In particular embodiments, membrane filters have a prefiltration membrane and a filtration membrane. In particular embodiments, the prefiltration membranes have larger pore sizes than filtration membranes and function to reduce clogging or fouling of the filtration membrane. Multiple formats of depth filters and membrane filters are commercially available.

[0254] In some embodiments, clarification is by membrane filtration. In some embodiments, a skilled artisan can readily choose an appropriate size based on the size of a lentivirus vector, which typically has a size of 80-100 nm. In some embodiments, the filter has a pore size between 0.2 pM and 0.6 pM, such as an 0.22 pm or 0.45 pm. In some embodiments, a 0.45 pm filter pore size is used. In particular embodiments, the membrane filtration is dual-layer filtration. In particular embodiments, the dual-layer filter comprising a prefilter membrane and a filtration membrane. In particular embodiments, the dual-layer filter comprises a prefilter membrane comprising a prefilter pore size of about 0.45 pm to about 0.8 pm and a final filtration membrane comprising a final filter pore size of about 0.22 pm to about 0.45 pm. In particular embodiments, the dual-layer filter comprises a prefilter membrane comprising a prefilter pore size of about 0.45 pm to about and a final filtration membrane comprising a final filter pore size of about 0.22 pm. In particular embodiments, the dual-layer filter comprises a prefilter membrane comprising a prefilter pore size of about 0.8 pm and a final filtration membrane comprising a final filter pore size of about 0.45 pm. In some embodiments, a membrane filter is used, such as one with an 0.8+0.45 pm pore size.

[0255] In some embodiments, the filtrate flux rate (the flow rate at which a sample passes through the membrane per unit area per unit time) can be chosen depending on the scale of the upstream process or other factors known to a skilled artisan. In some embodiments, flow conditions for filtration can be carried out at a particular liters/m2/hour or LMH. In some embodiments, the filtrate flux rate is between 100 and 300 LMH, such as 125 LMH, 150 LMH, 175 LMH, 200 LMH, 225 LMH, 250 LMH or 275 LMH, or any value between the foregoing. In some embodiments, the filtrate flux rate is between 125 and 175 LMH. In some embodiments, the filtrate flux rate is between 200 and 275 LMH, such as about 250 LMH.

[0256] In some embodiments, the clarified harvested material is further treated with a DNA endonuclease. In provided embodiments, an endonuclease to the culture supernatant of the harvested culture or the clarified harvest at the end of the viral vector production period. The viral vector supernatant may comprise residual nucleic acids including, but not limited to RNA, plasmid DNA from host cell transfection and genomic DNA from lysis of host cells during viral vector production. Such residual nucleic acids are potentially toxic and decrease the efficacy of any viral vector produced from the manufacturing processes contemplated herein. In some embodiments, a nuclease digestion step reduces the amount of these residual nucleic acids in the viral vector production supernatant.

[0257] In some embodiments, the nuclease is an endonuclease. In some embodiments, the endonuclease is a DNA/RNA endonuclease (an endonuclease that cleaves both DNA and RNA). Illustrative examples of endonucleases suitable for use in particular embodiments include, but are not limited to Benzonase® endonuclease (EMD Millipore), Denarase® endonuclease (c- LEcta GmbH), Decontaminase™ endonuclease (AG Scientific), and recombinant NucA protein from Serratia marcescens. In particular embodiments, a Benzonase® endonuclease is added to the viral vector production supernatant at the conclusion of the viral vector production process. In some embodiments, magnesium also is added, which can be necessary for nuclease activity. IN some embodiments, 1-2 mM Mg2+ is added for activity of Benzonase®. In some embodiments, the endonuclease digestion is performed at a concentration of about 20 U/ml to about 70 U/ml. In some embodiments, the endonuclease digestion is performed at a concentration of about 50 U/ml to about 70 U/ml. In some embodiments, the endonuclease digestion is performed at a concentration of about 55 U/ml to about 65 U/ml, such as about 60 U/mL.

[0258] In particular embodiments, the nuclease digestion step is performed at a suitable temperature and for a time sufficient to digest contaminating nucleic acids present in the viral vector production supernatant. In particular embodiments, nuclease digestion is performed from about 2°C to about 8 °C overnight. In particular embodiments, nuclease digestion is performed from about 36°C to about 38°C for about one, about two, or about three hours. In some embodiments, endonuclease digestion is performed at about 36°C, about 37°C, or about 38°C for about one, about two, or about three hours. In more preferred embodiments, Benzonase® endonuclease digestion is performed at about 37 °C for about 1 to 2 hours.

[0259] In some embodiments, the nuclease treatment is performed on harvested material before clarification. In such an example, after the nuclease has sufficiently digested the extracellular nucleic acids present in the viral vector production supernatant, the supernatant is clarified and filtered. In other embodiments, the nuclease treatment is performed on the clarified harvested material.

[0260] After the viral vector production supernatant has been harvested and clarified, it may optionally be stored at a suitable temperature, e.g., at about 4°C to -80°C. In some embodiments, after harvesting and clarification, and optional storage, the viral vector is purified and concentrated by a downstream process. In some embodiments, after harvesting and clarification, and optional storage, the viral vector is subjected to analytical testing, such as for determination of infectious titer and functional titer as described in Section I.

[0261]

B. Downstream Process

[0262] In some embodiments, harvested materials, more typically clarified harvested material, from an upstream process is subjected to a downstream process for concentration and purification of the lentiviral vector. In some embodiments, the downstream process involves capturing and concentrating the viral vector in the resultant clarified filtrate using chromatography, such as affinity chromatography or cation exchange chromatography; ultrafiltering and diafiltering the viral vector using tangential flow filtration (TFF); and filtering the purified and concentrated material by sterile filtration. In some embodiments, the viral vector can be formulated for fill. In some embodiments, the viral vector can be frozen. [0263] Downstream viral vector manufacturing processes include a downstream purification step involving chromatography. Chromatography is usually performed on a column packed with a resin or bead designed to capture the viral vector from the harvested and clarified viral vector production supernatant and to allow the undesired impurities in the harvested and clarified viral vector production supernatant to pass through the column. Captured viral vector is then displaced or eluted from the column using desorption agents. In particular embodiments, the chromatography is ion-exchange chromatography, size-exclusion chromatography, affinity chromatography, or multi-modal chromatography.

[0264] Ion exchange chromatography (IEX) involves the separation of ionizable molecules based on their total charge. IEX includes both anion exchange chromatography and cation exchange chromatography. Anion exchange chromatography (AEX) exploits the negatively charged surface of viral vector particles for purification purposes. In particular embodiments, the viral vector from the harvested and clarified viral vector production supernatant is captured and concentrated using anion exchange chromatography.

[0265] In some embodiments, chromatography is AEX. In some embodiments, AEX reduces the level of residual impurities (residual host cell protein (HCP), bovine serum albumin (BSA), host cell DNA (dsDNA), and plasmid) in the lentiviral vector clarified harvest and also can reduce the feed stream volume for subsequent processing, such as by the Ultrafiltration/Diafiltration (UF/DF) process. In some embodiments, the column contains a resin that acts as a strong anion exchanger with a positively charged quaternary amine group. AEX chromatography thus separates molecules present in the feed stream based on their total charge. AEX resins are created by covalently linking positively charged functional groups to a solid matrix. In some embodiments, a clarified harvest material, optionally after treatment with an endonuclease, is loaded onto the AEX column at low ionic strength (i.e. low salt concentration) and then washed with buffers with increasing ionic strength (i.e. increasing salt concentration) to remove undesired impurities. The vector is then eluted using a buffer of defined ionic strength (i.e. defined salt concentration). Elution by differences in ionic strength (i.e. differences in salt concentration) relies on charged salt ions competing with bound molecules for binding sites on the charged resin. Molecules with fewer charged groups tend to elute at lower salt concentrations, and those with more charged groups elute at higher salt concentrations. Differences in charge between desired product and impurities can be exploited to achieve separation between vector and residual HCP, BSA, dsDNA, and plasmid. [0266] In some embodiments, the column is washed one or more times with the wash buffer and then eluted. In some embodiments, AEX chromatography includes a wash with 200 to 350 mM NaCl, such as a wash with 200 nM NaCl, 250 nM NaCl, 300 nM NaCl or 350 nM NaCl, or a value between any of the foregoing. In some embodiments, the AEX chromatography includes a wash with about 300 mM NaCl. In some embodiments, the vector is then eluted at a higher salt concentration from the wash. In some embodiments, the vector eluted with 350 nM to 500 nM NaCl, such as eluted with 350 nM, 400 nM, 450 nM or 500 nM NaCl, or a value between any of the foregoing. In some embodiments, the AEX chromatography includes an elution with about 450 mM NaCl. In some embodiments, the AEX chromatography includes a wash with about 300 mM NaCl and an elution with about 450 mM NaCl.

[0267] Other chromatography methods also can be used. Cation exchange chromatography is another form of ion exchange chromatography (IEX). Cation exchange chromatography uses a negatively charged ion exchange resin with an affinity for molecules having net positive surface charges. Here, the pH of a lentiviral supernatant can be adjusted below the LVV iso electric point to give the LVV an overall positive net surface charge which binds it to the negatively charged resin beads.

[0268] Accordingly, in various embodiments, the LVV supernatant is pumped over an ionexchange chromatography column. In some embodiments, the LVV supernatant is pumped over a cation exchange chromatography column. In a particular embodiment, the LVV supernatant is pumped over a sulfate cation exchange chromatography (e.g., Toyopearl™ Sulfate-650F). In some embodiments, the sulfate cation exchange chromatography comprises a column having bead size of about 45 pm and/or a mean pore size of about 100 nm.

[0269] Size-exclusion chromatography (SEC) separates molecules based on their sizes using a resin that comprises beads with a defined pore size. Molecules elute from SEC resins in order of size: large molecules that are not trapped in bead pores travel a shorter distance and elute first and small molecules that are slowed by the bead pores elute last. Beads of different pore sizes can be purchased to achieve the desired resolution. SEC has been used to purify wild-type retroviruses and retroviral vectors. Retroviral vectors are excluded from the bead pores due to their large size and elute in the void volume of the column while lower molecular weight contaminants are retarded by the column and elute in later fractions. In particular embodiments, the viral vector from the harvested and clarified viral vector production supernatant is captured and concentrated using size-exclusion chromatography. [0270] Affinity chromatography (AC) separates molecules based on their highly selective affinity for particular chromatographic adsorbents. Unfortunately, little is known about the composition of the viral membrane, which complicates the selection of suitable adsorbents. Viral vectors have been engineered to express affinity tags on their surface to facilitate purification, e.g., MoMLV modified to express hexahistidine affinity tags purified by immobilized metal affinity chromatography (IMAC). MoMLV viral vectors have also been purified by exploiting the interaction between streptavidin and biotin. Heparin affinity chromatography has been used to purify viral vectors that use heparan sulfate as cell surface receptor, including pseudotyped retroviral vectors, e.g. , VSV-G pseudotyped lentiviral vectors. In particular embodiments, the viral vector from the harvested and clarified viral vector production supernatant is captured and concentrated using affinity chromatography.

[0271] Multimodal or mixed-mode chromatography (MMC) incorporates multiple modes of chromatography in a single resin. MMC enhances the selectivity of the resin because molecules can be separated based on several of their characteristics, rather than just a single one.

[0272] Downstream viral vector manufacturing processes contemplated in particular embodiments further comprise an ultrafiltration step to further purify and concentrate the viral vector and a diafiltration step to exchange the buffer of the concentrated and filtered viral vector buffer to diafiltration buffer.

[0273] In particular embodiments, viral vectors are filtered and concentrated and subsequently diafiltered using tangential flow filtration. In preferred embodiments, viral vectors are filtered and concentrated and subsequently diafiltered using tangential flow filtration. Hollow fiber TFF modules or filters have been used to simultaneously concentrate and remove impurities to yield highly active retroviral vectors. Hollow fiber TFF modules or filters have also been used as a convenient tool for diafiltering viral vectors into buffers suitable for bulk viral vector formulation.

[0274] In particular embodiments, the TFF systems comprise pumping the viral vector containing feed solution into the hollow fiber TFF module. The pore size of the TFF module is selected such that the viral vector does not pass through the pores and is concentrated in the retentate, the solution retained in the TFF module; whereas the permeate containing impurities passes through the pores.

[0275] In particular embodiments, the TFF systems are used to perform diafiltration or buffer exchange of a viral vector containing solution. TFF systems are an effective way to remove, modify, and/or exchange change ion concentration, pH, salts, sugars, non-aqueous solvents, separate unbound molecules, and remove low molecular weight contaminants. In particular embodiments, a hollow fiber TFF module or filter is used to perform diafiltration and/or ultrafiltration to further purify, concentrate, and perform a buffer exchange. Suitable TFF systems for use in particular embodiments contemplated herein are commercially available, e.g., from EMD Millipore, Sigma, GE Healthcare, Sartorius, and Repligen.

[0276] In particular embodiments, hollow fiber TFF modules or filters comprise a pore size of about 100 kD to about 500 kDa and a surface area of about 0.5 m2 to about 20 m2. In some embodiments, the pore size is about 100 kDa, about 200 kDa, about 300 kDa, about 400 kDa or about 500 kDa, or any value between any of the foregoing. In some embodiments, the surface area is about 1.00 m2, about 1.50 m2, about 2.00 m2, or about 2.50 m2, or any value between any of the foregoing. In some embodiments, the surface area is about 1.25 m2, about 1.30 m2, about 1.35 m2, about 1.40 m2, about 1.45 m2, about 1.50 m2, or any value between any of the foregoing.

[0277] In particular embodiments, downstream viral vector manufacturing processes comprise an ultrafiltration step performed using a hollow fiber TFF module, and further comprise a diafiltration step performed using the hollow fiber TFF module to exchange the buffer containing the viral vector to a diafiltration buffer in preparation for formulation. The buffer can be any suitable buffer and/or pharmaceutically acceptable medium. The viral vector is formulated to stabilize the vector and to retain vector activity through freeze/thaw cycles.

[0278] The resulting eluate can be further processed after the chromatography step or after chromatography and filtration steps using a filter to further remove impurities. In particular embodiments, the downstream process includes anion exchange chromatography (AEX) and ultrafiltration and diafiltration (UF/DF), followed by sterile filtration. In particular embodiments, sterile filtration as a last step in the process is necessary where the chromatography (e.g. AEX) cannot be performed aseptically at scale. The filtration step further removes impurities from the viral vector before formulation and fill. In particular embodiments, the filtering step comprises filtering the concentrated viral vector through a dual-layer filter comprising a pre-filter pore size of about 0.45 pm to about 0.8 pm and a final filter pore size of about 0.20 pm to about 0.45 pm. In particular embodiments, the filtering step comprises filtering the concentrated viral vector through a dual-layer filter comprising a pre-filter pore size of about 0.45 pm and a final filter pore size of about 0.20 pm. [0279] In some embodiments, the filtrate flux rate (the flow rate at which a sample passes through the membrane per unit area per unit time) can be chosen depending on the various factors known to a skilled artisan, such as titer recovery, BSA clearance, dsDNA clearance, or turbidity reduction. In some embodiments, flow conditions for filtration can be carried out at a particular liters/m2/hour or LMH. In some embodiments, the filtrate flux rate is between 100 and 300 LMH, such as 125 LMH, 150 LMH, 175 LMH, 200 LMH, 225 LMH, 250 LMH or 275 LMH, or any value between the foregoing. In some embodiments, the filtrate flux rate is between 120 and 200 LMH. In some embodiments, the filtrate flux rate is between 170 and 190 LMH, such as about 181 LMH.

[0280] In particular embodiments, after the formulated viral vector is passed through the filter and recovered, a final fill finish is performed on the filtered formulated viral vector and subsequently cryopreserved. The fill process can involve filling containers, e.g., vials, ampules, etc., with formulated viral vector and finishing the process of packaging the viral vector for distribution. After the fill finish, the cryopreservation of the formulated viral vector is performed such that stability and biological activity of the vector is substantially maintained, and/or such that loss of viral vector stability and biological activity is minimized. In some embodiments, the filtered formulated viral vector is cryopreserved or frozen at a temperature less than about - 65°C, less than about -70°C, less than about - 75°C, or less than about -80°C. In particular embodiments, the cooling rate is 1° to 3° C/minute.

[0281] Recovered and/or produced retroviral vector particles, such as lentiviral vectors, can be used to transduce target cells using the methods as described. Once in the target cells, the viral RNA is reverse-transcribed, imported into the nucleus and stably integrated into the host genome. One or two days after the integration of the viral RNA, the expression of the recombinant protein, e.g. antigen receptor, such as CAR, can be detected.

III. NUCLEUS ACID ENCODING A HETEROLOGOUS PROTEIN

[0282] In some embodiments, the viral vector contains a nucleic acid (e.g., polynucleotide) that encodes a heterologous recombinant protein. In some embodiments, the heterologous recombinant protein or molecule is or includes a recombinant receptor, e.g., an antigen receptor, SB-transposons, e.g., for gene silencing, capsid-enclosed transposons, homologous double stranded nucleic acid, e.g., for genomic recombination or reporter genes (e.g., fluorescent proteins, such as GFP) or luciferase). [0283] In some embodiments, the viral vector contains a nucleic acid (e.g., polynucleotide) that encodes a recombinant receptor and/or chimeric receptor, such as a heterologous receptor protein. The recombinant receptor, such as heterologous receptor, may include antigen receptors, such as functional non-TCR antigen receptors, including chimeric antigen receptors (CARs), and other antigen-binding receptors such as transgenic T cell receptors (TCRs). The receptors may also include other receptors, such as other chimeric receptors, such as receptors that bind to particular ligands and having transmembrane and/or intracellular signaling domains similar to those present in a CAR.

[0284] In any of such examples, the nucleic acid (e.g., polynucleotide) is inserted or located in a region of the viral vector, such as generally in a non-essential region of the viral genome. In some embodiments, the nucleic acid (e.g., polynucleotide) is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication defective.

[0285] In some embodiments, the encoded recombinant antigen receptor, e.g., CAR, is one that is capable of specifically binding to one or more ligand on a cell or disease to be targeted, such as a cancer, infectious disease, inflammatory or autoimmune disease, or other disease or condition, including those described herein for targeting with the provided methods and compositions.

[0286] In certain embodiments, an exemplary antigen is or includes avP6 integrin (avb6 integrin), B cell maturation antigen (BCMA), B7-H3, B7-H6, carbonic anhydrase 9 (CA9, also known as CAIX or G250), a cancer-testis antigen, cancer/testis antigen IB (CTAG, also known as NY-ESO-1 and LAGE-2), carcinoembryonic antigen (CEA), a cyclin, cyclin A2, C-C Motif Chemokine Ligand 1 (CCL-1), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7/8, CD123, CD138, CD171, epidermal growth factor protein (EGFR), truncated epidermal growth factor protein (tEGFR), type III epidermal growth factor receptor mutation (EGFR vIII), epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrinB2, ephrine receptor A2 (EPHa2), estrogen receptor, Fc receptor like 5 (FCRL5; also known as Fc receptor homolog 5 or FCRH5), fetal acetylcholine receptor (fetal AchR), a folate binding protein (FBP), folate receptor alpha, ganglioside GD2, O-acetylated GD2 (OGD2), ganglioside GD3, glycoprotein 100 (gplOO), G Protein Coupled Receptor 5D (GPCR5D), Her2/neu (receptor tyrosine kinase erb-B2), Her3 (erb-B3), Her4 (erb-B4), erbB dimers, Human high molecular weight-melanoma-associated antigen (HMW-MAA), hepatitis B surface antigen, Human leukocyte antigen Al (HLA-A1), Human leukocyte antigen A2 (HLA-A2), IL- 22 receptor alpha(IL-22Ra), IL-13 receptor alpha 2 (IL-13Ra2), kinase insert domain receptor (kdr), kappa light chain, LI cell adhesion molecule (Ll-CAM), CE7 epitope of Ll-CAM, Leucine Rich Repeat Containing 8 Family Member A (LRRC8A), Lewis Y, Melanoma- associated antigen (MAGE)-Al, MAGE-A3, MAGE-A6, mesothelin, c-Met, murine cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, natural killer group 2 member D (NKG2D) ligands, melan A (MART-1), neural cell adhesion molecule (NCAM), oncofetal antigen, Preferentially expressed antigen of melanoma (PRAME), progesterone receptor, a prostate specific antigen, prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), Receptor Tyrosine Kinase Like Orphan Receptor 1 (R0R1), survivin, Trophoblast glycoprotein (TPBG also known as 5T4), tumor-associated glycoprotein 72 (TAG72), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor 2 (VEGFR2), Wilms Tumor 1 (WT-1), a pathogen- specific antigen, or an antigen associated with a universal tag, and/or biotinylated molecules, and/or molecules expressed by HIV, HCV, HBV or other pathogens. Antigens targeted by the receptors in some embodiments include antigens associated with a B cell malignancy, such as any of a number of known B cell marker. In some embodiments, the antigen is or includes CD20, CD19, CD22, R0R1, CD45, CD21, CD5, CD33, Igkappa, Iglambda, CD79a, CD79b, or CD30.

[0287] In some embodiments, the exemplary antigens are orphan tyrosine kinase receptor ROR1, tEGFR, Her2, Ll-CAM, CD 19, CD20, CD22, mesothelin, CEA, and hepatitis B surface antigen, anti-folate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, EGP-2, EGP-4, 0EPHa2, ErbB2, 3, or 4, FBP, fetal acetylcholine receptor, GD2, GD3, HMW-MAA, IL-22R- alpha, IL-13R-alpha2, kdr, kappa light chain, Lewis Y, LI -cell adhesion molecule, MAGE-A1, mesothelin, MUC1, MUC16, PSCA, NKG2D Ligands, NY-ESO-1, MART-1, gplOO, oncofetal antigen, ROR1, TAG72, VEGF-R2, carcinoembryonic antigen (CEA), prostate specific antigen, PSMA, Her2/neu, estrogen receptor, progesterone receptor, ephrinB2, CD123, CS-1, c-Met, GD-2, and MAGE A3, CE7, Wilms Tumor 1 (WT-1), a cyclin, such as cyclin Al (CCNA1), and/or biotinylated molecules, and/or molecules expressed by and/or characteristic of or specific for HIV, HCV, HBV, HPV, and/or other pathogens and/or oncogenic versions thereof.

[0288] In some embodiments, the antigen is or includes a pathogen- specific or pathogen- expressed antigen. In some embodiments, the antigen is a viral antigen (such as a viral antigen from HIV, HCV, HBV, etc.), bacterial antigens, and/or parasitic antigens. [0289] Antigen receptors, including CARs and recombinant TCRs, and production and introduction thereof, in some embodiments include those described, for example, in international patent application publication numbers W0200014257, WO2013126726, WO2012/129514, WO2014031687, WO2013/ 166321, W02013/071154, WO2013/ 123061, U.S. patent application publication numbers US2002131960, US2013287748, US20130149337, U.S. Patent Nos.: 6,451,995, 7,446,190, 8,252,592, , 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and 8,479,118, and European patent application number EP2537416, and/or those described by Sadelain et al., Cancer Discov. 2013 April; 3(4): 388-398; Davila et al. (2013) PLoS ONE 8(4): e61338; Turtle et al., Curr. Opin. Immunol., 2012 October; 24(5): 633-39; Wu et al., Cancer, 2012 March 18(2): 160-75.

A. Chimeric Antigen Receptor

[0290] In some embodiments, the nucleic acid (e.g., polynucleotide) contained in a genome of the viral vector encodes a chimeric antigen receptor (CAR). The CAR is generally a genetically engineered receptor with an extracellular ligand binding domain, such as an extracellular portion containing an antibody or fragment thereof, linked to one or more intracellular signaling components. In some embodiments, the chimeric antigen receptor includes a transmembrane domain and/or intracellular domain linking the extracellular domain and the intracellular signaling domain. Such molecules typically mimic or approximate a signal through a natural antigen receptor and/or signal through such a receptor in combination with a costimulatory receptor.

[0291] In some embodiments, CARs are constructed with a specificity for a particular marker, such as a marker expressed in a particular cell type to be targeted by adoptive therapy, e.g., a cancer marker and/or any of the antigens described. Thus, the CAR typically includes one or more antigen-binding fragment, domain, or portion of an antibody, or one or more antibody variable domains, and/or antibody molecules. In some embodiments, the CAR includes an antigen-binding portion or portions of an antibody molecule, such as a variable heavy chain (VH) or antigen-binding portion thereof, or a single-chain antibody fragment (scFv) derived from the variable heavy (VH) and variable light (VL) chains of a monoclonal antibody (mAb).

[0292] In some embodiments, engineered cells, such as T cells, are provided that express a CAR with specificity for a particular antigen (or marker or ligand), such as an antigen expressed on the surface of a particular cell type. In some embodiments, the antigen is a polypeptide. In some embodiments, it is a carbohydrate or other molecule. In some embodiments, the antigen is selectively expressed or overexpressed on cells of the disease or condition, e.g., the tumor or pathogenic cells, as compared to normal or non-targeted cells or tissues. In other embodiments, the antigen is expressed on normal cells and/or is expressed on the engineered cells.

[0293] In particular embodiments, the recombinant receptor, such as chimeric receptor, contains an intracellular signaling region, which includes a cytoplasmic signaling domain or region (also interchangeably called an intracellular signaling domain or region), such as a cytoplasmic (intracellular) region capable of inducing a primary activation signal in a T cell, for example, a cytoplasmic signaling domain or region of a T cell receptor (TCR) component (e.g., a cytoplasmic signaling domain or region of a zeta chain of a CD3-zeta (CD3Q chain or a functional variant or signaling portion thereof) and/or that comprises an immunoreceptor tyrosine-based activation motif (IT AM). In some embodiments, the CAR comprises an extracellular antigen-recognition domain that specifically binds to a target antigen and an intracellular signaling domain comprising an ITAM. In some embodiments, the intracellular signaling domain comprises an intracellular domain of a CD3-zeta (CD3Q chain.

[0294] In some embodiments, the chimeric receptor further contains an extracellular ligandbinding domain that specifically binds to a ligand (e.g., antigen) antigen. In some embodiments, the chimeric receptor is a CAR that contains an extracellular antigen-recognition domain that specifically binds to an antigen. In some embodiments, the ligand, such as an antigen, is a protein expressed on the surface of cells. In some embodiments, the CAR is a TCR-like CAR and the antigen is a processed peptide antigen, such as a peptide antigen of an intracellular protein, which, like a TCR, is recognized on the cell surface in the context of a major histocompatibility complex (MHC) molecule.

[0295] Exemplary antigen receptors, including CARs, and methods for engineering and introducing such receptors into cells, include those described, for example, in international patent application publication numbers W0200014257, WO2013126726, WO2012/129514, WO2014031687, WO2013/ 166321, W02013/071154, WO2013/ 123061, U.S. patent application publication numbers US2002131960, US2013287748, US20130149337, U.S. Patent Nos.: 6,451,995, 7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and 8,479,118, and European patent application number EP2537416, and/or those described by Sadelain et al., Cancer Discov. 2013 April; 3(4): 388-398; Davila et al. (2013) PLoS ONE 8(4): e61338; Turtle et al., Curr. Opin. Immunol., 2012 October; 24(5): 633-39; Wu et al., Cancer, 2012 March 18(2): 160-75. In some aspects, the antigen receptors include a CAR as described in U.S. Patent No. 7,446,190, and those described in International Patent Application Publication No. WO/2014055668 Al. Examples of the CARs include CARs as disclosed in any of the aforementioned publications, such as WO2014031687, US 8,339,645, US 7,446,179, US 2013/0149337, U.S. Patent No. 7,446,190, US Patent No. 8,389,282, Kochenderfer et al., 2013, Nature Reviews Clinical Oncology, 10, 267-276 (2013); Wang et al. (2012) J. Immunother. 35(9): 689-701; and Brentjens et al., Sci Transl Med. 2013 5(177). See also WO2014031687, US 8,339,645, US 7,446,179, US 2013/0149337, U.S. Patent No. 7,446,190, and US Patent No. 8,389,282.

[0296] In some embodiments, the CAR is constructed with a specificity for a particular antigen (or marker or ligand), such as an antigen expressed in a particular cell type to be targeted by adoptive therapy, e.g., a cancer marker, and/or an antigen intended to induce a dampening response, such as an antigen expressed on a normal or non-diseased cell type. Thus, the CAR typically includes in its extracellular portion one or more antigen binding molecules, such as one or more antigen-binding fragment, domain, or portion, or one or more antibody variable domains, and/or antibody molecules. In some embodiments, the CAR includes an antigenbinding portion or portions of an antibody molecule, such as a single-chain antibody fragment (scFv) derived from the variable heavy (VH) and variable light (VL) chains of a monoclonal antibody (mAb).

[0297] In some embodiments, the antibody or antigen-binding portion thereof is expressed on cells as part of a recombinant receptor, such as an antigen receptor. Among the antigen receptors are functional non-TCR antigen receptors, such as chimeric antigen receptors (CARs). Generally, a CAR containing an antibody or antigen-binding fragment that exhibits TCR-like specificity directed against peptide-MHC complexes also may be referred to as a TCR-like CAR. In some embodiments, the extracellular antigen binding domain specific for an MHC- peptide complex of a TCR-like CAR is linked to one or more intracellular signaling components, in some aspects via linkers and/or transmembrane domain(s). In some embodiments, such molecules can typically mimic or approximate a signal through a natural antigen receptor, such as a TCR, and, optionally, a signal through such a receptor in combination with a co stimulatory receptor.

[0298] In some embodiments, the recombinant receptor, such as a chimeric receptor (e.g., CAR), includes a ligand-binding domain that binds, such as specifically binds, to an antigen (or a ligand). Among the antigens targeted by the chimeric receptors are those expressed in the context of a disease, condition, or cell type to be targeted via the adoptive cell therapy. Among the diseases and conditions are proliferative, neoplastic, and malignant diseases and disorders, including cancers and tumors, including hematologic cancers, cancers of the immune system, such as lymphomas, leukemias, and/or myelomas, such as B, T, and myeloid leukemias, lymphomas, and multiple myelomas.

[0299] In some embodiments, the antigen (or a ligand) is a polypeptide. In some embodiments, it is a carbohydrate or other molecule. In some embodiments, the antigen (or a ligand) is selectively expressed or overexpressed on cells of the disease or condition, e.g., the tumor or pathogenic cells, as compared to normal or non-targeted cells or tissues. In other embodiments, the antigen is expressed on normal cells and/or is expressed on the engineered cells. In some embodiments, the antigen is associated with a disease or condition, such as cancer, an autoimmune disease or disorder, or an infectious disease. In some embodiments, the antigen receptor, e.g., CAR, specifically binds to a universal tag.

[0300] In some embodiments, the CAR contains an antibody or an antigen-binding fragment e.g., scFv) that specifically recognizes an antigen, such as an intact antigen, expressed on the surface of a cell.

[0301] In some embodiments, the antigen (or a ligand) is a tumor antigen or cancer marker. In some embodiments, the antigen (or a ligand) the antigen is or includes avP6 integrin (avb6 integrin), B cell maturation antigen (BCMA), B7-H3, B7-H6, carbonic anhydrase 9 (CA9, also known as CAIX or G250), a cancer-testis antigen, cancer/testis antigen IB (CTAG, also known as NY-ESO-1 and LAGE-2), carcinoembryonic antigen (CEA), a cyclin, cyclin A2, C-C Motif Chemokine Ligand 1 (CCL-1), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7/8, CD123, CD138, CD171, epidermal growth factor protein (EGFR), truncated epidermal growth factor protein (tEGFR), type III epidermal growth factor receptor mutation (EGFR vIII), epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrinB2, ephrine receptor A2 (EPHa2), estrogen receptor, Fc receptor like 5 (FCRL5; also known as Fc receptor homolog 5 or FCRH5), fetal acetylcholine receptor (fetal AchR), a folate binding protein (FBP), folate receptor alpha, ganglioside GD2, O-acetylated GD2 (OGD2), ganglioside GD3, glycoprotein 100 (gplOO), G Protein Coupled Receptor 5D (GPCR5D), Her2/neu (receptor tyrosine kinase erb-B2), Her3 (erb-B3), Her4 (erb-B4), erbB dimers, Human high molecular weight-melanoma-associated antigen (HMW-MAA), hepatitis B surface antigen, Human leukocyte antigen Al (HLA-A1), Human leukocyte antigen A2 (HLA-A2), IL- 22 receptor alpha(IL-22Ra), IL-13 receptor alpha 2 (IL-13Ra2), kinase insert domain receptor (kdr), kappa light chain, LI cell adhesion molecule (Ll-CAM), CE7 epitope of Ll-CAM, Leucine Rich Repeat Containing 8 Family Member A (LRRC8A), Lewis Y, Melanoma- associated antigen (MAGE)-Al, MAGE-A3, MAGE-A6, mesothelin, c-Met, murine cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, natural killer group 2 member D (NKG2D) ligands, melan A (MART-1), neural cell adhesion molecule (NCAM), oncofetal antigen, Preferentially expressed antigen of melanoma (PRAME), progesterone receptor, a prostate specific antigen, prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), Receptor Tyrosine Kinase Like Orphan Receptor 1 (R0R1), survivin, Trophoblast glycoprotein (TPBG also known as 5T4), tumor-associated glycoprotein 72 (TAG72), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor 2 (VEGFR2), Wilms Tumor 1 (WT-1), a pathogen- specific antigen, or an antigen associated with a universal tag, and/or biotinylated molecules, and/or molecules expressed by HIV, HCV, HBV or other pathogens. Antigens targeted by the receptors in some embodiments include antigens associated with a B cell malignancy, such as any of a number of known B cell marker. In some embodiments, the antigen is or includes CD20, CD19, CD22, R0R1, CD45, CD21, CD5, CD33, Igkappa, Iglambda, CD79a, CD79b, or CD30.

[0302] The chimeric receptors, such as CARs, generally include an extracellular antigen binding domain that is an antigen-binding portion or portions of an antibody molecule. In some embodiments, the antigen-binding domain is a portion of an antibody molecule, generally a variable heavy (Vn) chain region and/or variable light (VL) chain region of the antibody, e.g., an scFv antibody fragment. In some embodiments, the CAR includes an antigen-binding portion or portions of an antibody molecule, such as a single-chain antibody fragment (scFv) derived from the variable heavy (Vn) and variable light (VL) chains of a monoclonal antibody (mAb). In some embodiments, the antigen-binding domain is a single domain antibody (sdAb), such as sdFv, nanobody, VHH and VNAR. In some embodiments, an antigen-binding fragment comprises antibody variable regions joined by a flexible linker.

[0303] In some embodiments, the antibody or an antigen-binding fragment (e.g. scFv or VH domain) specifically recognizes an antigen, such as CD 19. In some embodiments, the antibody or antigen-binding fragment is derived from, or is a variant of, antibodies or antigen-binding fragment that specifically binds to CD 19. In some embodiments, the antigen is CD 19. In some embodiments, the antibody or an antigen-binding fragment (e.g. scFv) contains a variable heavy chain and a variable light chain with six CDRs, CDRH1-3 and CDRL1-3, that confer binding to CD19.

[0304] In some embodiments, the scFv contains a VH and a VL derived from an antibody or an antibody fragment specific to CD19. In some embodiments, the extracellular binding domain of the CD 19 CAR is derived from an antibody specific to CD 19, including, for example, SJ25C1 (Bejcek et al., Cancer Res. 55:2346-2351 (1995)), HD37 (Pezutto et al., J. Immunol. 138(9):2793-2799 (1987)), 4G7 (Meeker et al., Hybridoma 3:305-320 (1984)), B43 (Bejcek (1995)), BLY3 (Bejcek (1995)), B4 (Freedman et al., 70:418-427 (1987)), B4 HB12b (Kansas & Tedder, J. Immunol. 147:4094-4102 (1991); Yazawa et al., Proc. Natl. Acad. Sci. USA 102:15178-15183 (2005); Herbst et al., J. Pharmacol. Exp. Ther. 335:213-222 (2010)), BU12 (Callard et al., J. Immunology, 148(10): 2983-2987 (1992)), and CLB-CD19 (De Rie Cell. Immunol. 118:368-381(1989)). In any of these embodiments, the extracellular binding domain of the CD 19 CAR can comprise or consist of the VH, the VL, and/or one or more CDRs of any of the antibodies. In some embodiments, the antibody or antibody fragment that binds CD 19 is a mouse derived antibody such as FMC63 and SJ25C1. In some embodiments, the antibody or antibody fragment is a human antibody, e.g., as described in U.S. Patent Publication No. US 2016/0152723.

[0305] In some embodiments the antigen-binding domain includes a VH and/or VL derived from FMC63, which, in some aspects, can be an scFv. FMC63 generally refers to a mouse monoclonal IgGl antibody raised against Nalm-1 and -16 cells expressing CD19 of human origin (Ling, N. R., et al. (1987). Leucocyte typing 111. 302). In some embodiments, the FMC63 antibody comprises CDR-H1 and CDR-H2 set forth in SEQ ID NO: 38 and 39, respectively, and CDR-H3 set forth in SEQ ID NO: 40 or 54 and CDR-L1 set forth in SEQ ID NO: 35 and CDR- L2 set forth in SEQ ID NO: 36 or 55 and CDR-L3 sequences set forth in SEQ ID NO: 37 or 56. In some embodiments, the FMC63 antibody comprises the heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 41 and the light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 42.

[0306] In some embodiments, the scFv comprises a variable light chain containing the CDR- -L1 sequence of SEQ ID NO:35, a CDR-L2 sequence of SEQ ID NO:36, and a CDR-L3 sequence of SEQ ID NO: 37 and/or a variable heavy chain containing a CDR-H1 sequence of SEQ ID NO:38, a CDR-H2 sequence of SEQ ID NO:39, and a CDR-H3 sequence of SEQ ID NO:40, or a variant of any of the foregoing having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto. In some embodiments, the scFv comprises a variable heavy chain region of FMC63 set forth in SEQ ID NO:41 and a variable light chain region of FMC63 set forth in SEQ ID NO:42, or a variant of any of the foregoing having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.

[0307] In some embodiments, the FMC63 antibody comprises CDR-H1 and CDR-H2 set forth in SEQ ID NO: 38 and 39, respectively, and CDR-H3 set forth in SEQ ID NO: 40 or 54 and CDR-L1 set forth in SEQ ID NO: 35 and CDR-L2 set forth in SEQ ID NO: 36 or 55 and CDR-L3 sequences set forth in SEQ ID NO: 37 or 56. In some embodiments, the FMC63 antibody comprises the heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 41 and the light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 42. In some embodiments, the scFv comprises a variable light chain containing the CDR— LI sequence of SEQ ID NO:35, a CDR-L2 sequence of SEQ ID NO:36, and a CDR- L3 sequence of SEQ ID NO:37 and/or a variable heavy chain containing a CDR-H1 sequence of SEQ ID NO:38, a CDR-H2 sequence of SEQ ID NO:39, and a CDR-H3 sequence of SEQ ID NO:40, or a variant of any of the foregoing having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto. In some embodiments, the scFv comprises a variable heavy chain region of FMC63 set forth in SEQ ID NO:41 and a variable light chain region of FMC63 set forth in SEQ ID NO:42, or a variant of any of the foregoing having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.

[0308] In some embodiments, the variable heavy and variable light chains are connected by a linker. In some embodiments, the linker is set forth in SEQ ID NO:24. In some embodiments, the scFv comprises, in order, a VH, a linker, and a VL. In some embodiments, the scFv comprises, in order, a VL, a linker, and a VH. In some embodiments, the scFv is encoded by a sequence of nucleotides set forth in SEQ ID NO:25 or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:25. In some embodiments, the scFv comprises the sequence of amino acids set forth in SEQ ID NO:43 or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:43. [0309] In some embodiments the antigen-binding domain includes a VH and/or VL derived from SJ25C1, which, in some aspects, can be an scFv. SJ25C1 is a mouse monoclonal IgGl antibody raised against Nalm-1 and -16 cells expressing CD19 of human origin (Ling, N. R., et al. (1987). Leucocyte typing 111. 302). In some embodiments, the SJ25C1 antibody comprises CDR-H1, CDR-H2 and CDR-H3 set forth in SEQ ID NOS: 47-49, respectively, and CDR-L1, CDR-L2 and CDR-L3 sequences set forth in SEQ ID NOS: 44-46, respectively. In some embodiments, the SJ25C1 antibody comprises the heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 50 and the light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 51. In some embodiments, the scFv comprises a variable light chain containing a CDR-L1 sequence of SEQ ID NO:44, a CDR-L2 sequence of SEQ ID NO: 45, and a CDR-L3 sequence of SEQ ID NO:46 and/or a variable heavy chain containing a CDR-H1 sequence of SEQ ID NO:47, a CDR-H2 sequence of SEQ ID NO:48, and a CDR-H3 sequence of SEQ ID NO:49,, or a variant of any of the foregoing having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto. In some embodiments, the scFv comprises a variable heavy chain region of SJ25C1 set forth in SEQ ID NO:50 and a variable light chain region of SJ25C1 set forth in SEQ ID NO:51, or a variant of any of the foregoing having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto. In some embodiments, the variable heavy and variable light chains are connected by a linker. In some embodiments, the linker is set forth in SEQ ID NO:52. In some embodiments, the scFv comprises, in order, a VH, a linker, and a VL. In some embodiments, the scFv comprises, in order, a VL, a linker, and a VH. In some embodiments, the scFv comprises the sequence of amino acids set forth in SEQ ID NO:53 or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:53.

[0310] Exemplary antigen receptors, e.g., CARs, also include the CARs of FDA-approved products BREYANZI® (lisocabtagene maraleucel), TECARTUS™ (brexucabtagene autoleucel), KYMRIAH™ (tisagenlecleucel), and YESCARTA™ (axicabtagene ciloleucel). In some of any of the provided embodiments, the CAR is the CAR of BREYANZI® (lisocabtagene maraleucel), TECARTUS™ (brexucabtagene autoleucel), KYMRIAH™ (tisagenlecleucel), YESCARTA™ (axicabtagene ciloleucel). In some of any of the provided embodiments, the CAR is the CAR of BREYANZI® (lisocabtagene maraleucel, see Sehgal et al., 2020, Journal of Clinical Oncology 38:15_suppl, 8040; Teoh et al., 2019, Blood 134(Supplement_l):593; and Abramson et al., 2020, The Lancet 396(10254): 839-852). In some of any of the provided embodiments, the CAR is the CAR of TECARTUS™ (brexucabtagene autoleucel, see Mian and Hill, 2021, Expert Opin Biol Ther; 21(4):435-441; and Wang et al., 2021, Blood 138(Supplement 1):744). In some of any of the provided embodiments, the CAR is the CAR of KYMRIAH™ (tisagenlecleucel, see Bishop et al., 2022, N Engl J Med 386:629:639; Schuster et al., 2019, N Engl J Med 380:45-56; Halford et al., 2021, Ann Pharmacother 55(4):466-479; Mueller et al., 2021, Blood Adv. 5(23):4980-4991; and Fowler et al., 2022, Nature Medicine 28:325-332). In some of any of the provided embodiments, the CAR is the CAR of YESCARTA™ (axicabtagene ciloleucel, see Neelapu et al., 2017, N Engl J Med 377(26):2531- 2544; Jacobson et al., 2021, The Lancet 23(l):P91-103; and Locke et al., 2022, N Engl J Med 386:640-654).

[0311] In some aspects, the recombinant receptor, e.g., a chimeric antigen receptor, includes the extracellular portion containing one or more antigen binding domains, such as an antibody or fragment thereof, and one or more intracellular signaling region or domain (also interchangeably called a cytoplasmic signaling domain or region). In some aspects, the recombinant receptor, e.g., CAR, further includes a spacer and/or a transmembrane domain or portion. In some aspects, the spacer and/or transmembrane domain can link the extracellular portion containing the antigen- binding domain and the intracellular signaling region(s) or domain(s).

[0312] In some embodiments, the recombinant receptor such as the CAR, further includes a spacer, which may include a hinge domain. In some embodiments, the spacer in a CD8a hinge domain, for example, a human CD8a hinge domain. In some embodiments, the CD8a hinge domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:59. In some embodiments, the hinge domain comprises a CD28 hinge domain, for example, a human CD28 hinge domain. In some embodiments, the CD28 hinge domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:60. In some embodiments, the CD28 hinge domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:61. In some embodiments, the hinge domain has a sequence of amino acids that has at least 80% sequence identity, such as at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any of the foregoing.

[0313] In some embodiments, the spacer may be or include at least a portion of an immunoglobulin constant region or variant or modified version thereof, such as a hinge region, e.g., an IgG4 hinge region, and/or a CH1/CL and/or Fc region. In some embodiments, the recombinant receptor further comprises a spacer and/or a hinge region. In some embodiments, the constant region or portion is of a human IgG, such as IgG4 or IgGl. In some aspects, the portion of the constant region serves as a spacer region between the antigen-recognition component, e.g., scFv, and transmembrane domain. The spacer can be of a length that provides for increased responsiveness of the cell following antigen binding, as compared to in the absence of the spacer. In some examples, the spacer is at or about 12 amino acids in length or is no more than 12 amino acids in length. Exemplary spacers include those having at least about 10 to 229 amino acids, about 10 to 200 amino acids, about 10 to 175 amino acids, about 10 to 150 amino acids, about 10 to 125 amino acids, about 10 to 100 amino acids, about 10 to 75 amino acids, about 10 to 50 amino acids, about 10 to 40 amino acids, about 10 to 30 amino acids, about 10 to 20 amino acids, or about 10 to 15 amino acids, and including any integer between the endpoints of any of the listed ranges. In some embodiments, a spacer region has about 12 amino acids or less, about 119 amino acids or less, or about 229 amino acids or less. Exemplary spacers include IgG4 hinge alone, IgG4 hinge linked to CH2 and CH3 domains, or IgG4 hinge linked to the CH3 domain. Exemplary spacers include, but are not limited to, those described in Hudecek et al. (2013) Clin. Cancer Res., 19:3153, Hudecek et al. (2015) Cancer Immunol Res. 3(2): 125- 135 or international patent application publication number WO2014031687.

[0314] In some embodiments, the spacer contains only a hinge region of an IgG, such as only a hinge of IgG4 or IgGl, such as the hinge only spacer set forth in SEQ ID NO: 1, and encoded by the sequence set forth in SEQ ID NO: 2. In some embodiments, the spacer is an Ig hinge, e.g., and IgG4 hinge, linked to a CH2 and/or CH3 domains. In some embodiments, the spacer is an Ig hinge, e.g., an IgG4 hinge, linked to CH2 and CH3 domains, such as set forth in SEQ ID NO: 4. In some embodiments, the spacer the spacer is an Ig hinge, e.g., an IgG4 hinge, linked to a CH3 domain only, such as set forth in SEQ ID NO: 3. In some embodiments, the spacer is or comprises a glycine-serine rich sequence or other flexible linker such as known flexible linkers. In some embodiments, the constant region or portion is of IgD. In some embodiments, the spacer has the sequence set forth in SEQ ID NO: 5. In some embodiments, the spacer has a sequence of amino acids that exhibits at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOS: 1, 3, 4 and 5. [0315] In some aspects, the spacer is a polypeptide spacer that (a) comprises or consists of all or a portion of an immunoglobulin hinge or a modified version thereof or comprises about 15 amino acids or less, and does not comprise a CD28 extracellular region or a CD8 extracellular region, (b) comprises or consists of all or a portion of an immunoglobulin hinge, optionally an IgG4 hinge, or a modified version thereof and/or comprises about 15 amino acids or less, and does not comprise a CD28 extracellular region or a CD8 extracellular region, or (c) is at or about 12 amino acids in length and/or comprises or consists of all or a portion of an immunoglobulin hinge, optionally an IgG4, or a modified version thereof; or (d) consists or comprises the sequence of amino acids set forth in SEQ ID NOS: 1, 3-5, 27-34 or 24, or a variant of any of the foregoing having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto, or (e) comprises or consists of the formula X1PPX2P, where Xi is glycine, cysteine or arginine and X2 is cysteine or threonine.

[0316] In some embodiments, the antigen receptor comprises an intracellular domain linked directly or indirectly to the extracellular domain. In some embodiments, the chimeric antigen receptor includes a transmembrane domain linking the extracellular domain and the intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises an IT AM. For example, in some aspects, the antigen recognition domain (e.g. extracellular domain) generally is linked to one or more intracellular signaling components, such as signaling components that mimic activation through an antigen receptor complex, such as a TCR complex, in the case of a CAR, and/or signal via another cell surface receptor. In some embodiments, the chimeric receptor comprises a transmembrane domain linked or fused between the extracellular domain (e.g. scFv) and intracellular signaling domain. Thus, in some embodiments, the antigenbinding component (e.g., antibody) is linked to one or more transmembrane and intracellular signaling domains.

[0317] In one embodiment, a transmembrane domain that naturally is associated with one of the domains in the receptor, e.g., CAR, is used. In some instances, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.

[0318] The transmembrane domain in some embodiments is derived either from a natural or from a synthetic source. Where the source is natural, the domain in some aspects is derived from any membrane-bound or transmembrane protein. Transmembrane regions include those derived from (z.e. comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 (4-1BB), or CD154. Alternatively the transmembrane domain in some embodiments is synthetic. In some aspects, the synthetic transmembrane domain comprises predominantly hydrophobic residues such as leucine and valine. In some aspects, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain. In some embodiments, the linkage is by linkers, spacers, and/or transmembrane domain(s). In some aspects, the transmembrane domain contains a transmembrane portion of CD28 or a variant thereof. The extracellular domain and transmembrane can be linked directly or indirectly. In some embodiments, the extracellular domain and transmembrane are linked by a spacer, such as any described herein.

[0319] In some embodiments, the transmembrane domain is a transmembrane domain of human CD28 or variant thereof, e.g., a 27-amino acid transmembrane domain of a human CD28 (Accession No.: P10747.1). In some embodiments, the transmembrane domain is a transmembrane domain that comprises the sequence of amino acids set forth in SEQ ID NO: 8 or a sequence of amino acids that exhibits at least or at least about85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:8. In some embodiments, the transmembrane-domain containing portion of the recombinant receptor comprises the sequence of amino acids set forth in SEQ ID NO: 9 or a sequence of amino acids having at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.

[0320] In some embodiments, the transmembrane domain of the is a transmembrane domain of a human CD8a. In some embodiments, the transmembrane domain is a transmembrane domain that comprises the sequence of amino acids set forth in SEQ ID NO: 62 or a sequence of amino acids that exhibits at least or at least about85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:62.

[0321] In some embodiments, the recombinant receptor, e.g., CAR, includes at least one intracellular signaling component or components, such as an intracellular signaling region or domain. T cell activation is in some aspects described as being mediated by two classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences), and those that act in an antigenindependent manner to provide a secondary or co- stimulatory signal (secondary cytoplasmic signaling sequences). In some aspects, the CAR includes one or both of such signaling components. Among the intracellular signaling region are those that mimic or approximate a signal through a natural antigen receptor, a signal through such a receptor in combination with a costimulatory receptor, and/or a signal through a costimulatory receptor alone. In some embodiments, a short oligo- or polypeptide linker, for example, a linker of between 2 and 10 amino acids in length, such as one containing glycines and serines, e.g., glycine-serine doublet, is present and forms a linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR.

[0322] In some embodiments, upon ligation of the CAR, the cytoplasmic domain or intracellular signaling region of the CAR activates at least one of the normal effector functions or responses of the immune cell, e.g., T cell engineered to express the CAR. For example, in some contexts, the CAR induces a function of a T cell such as cytolytic activity or T-helper activity, such as secretion of cytokines or other factors. In some embodiments, a truncated portion of an intracellular signaling region of an antigen receptor component or costimulatory molecule is used in place of an intact immuno stimulatory chain, for example, if it transduces the effector function signal. In some embodiments, the intracellular signaling regions, e.g., comprising intracellular domain or domains, include the cytoplasmic sequences of the T cell receptor (TCR), and in some aspects also those of co-receptors that in the natural context act in concert with such receptor to initiate signal transduction following antigen receptor engagement, and/or any derivative or variant of such molecules, and/or any synthetic sequence that has the same functional capability. In some embodiments, the intracellular signaling regions, e.g., comprising intracellular domain or domains, include the cytoplasmic sequences of a region or domain that is involved in providing costimulatory signal.

[0323] In some aspects, the CAR includes a primary cytoplasmic signaling sequence that regulates primary activation of the TCR complex. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or IT AMs. Examples of ITAM containing primary cytoplasmic signaling sequences include those derived from CD3 zeta chain, FcR gamma, CD3 gamma, CD3 delta and CD3 epsilon. In some embodiments, cytoplasmic signaling molecule(s) in the CAR contain(s) a cytoplasmic signaling domain, portion thereof, or sequence derived from CD3 zeta.

[0324] In some embodiments, the receptor includes an intracellular component of a TCR complex, such as a TCR CD3 chain that mediates T-cell activation and cytotoxicity, e.g., CD3 zeta chain. Thus, in some aspects, the antigen-binding portion is linked to one or more cell signaling modules. In some embodiments, cell signaling modules include CD3 transmembrane domain, CD3 intracellular signaling domains, and/or other CD transmembrane domains. In some embodiments, the receptor, e.g., CAR, further includes a portion of one or more additional molecules such as Fc receptor y, CD8alpha, CD8beta, CD4, CD25, or CD16. For example, in some aspects, the CAR or other chimeric receptor includes a chimeric molecule between CD3- zeta (CD3-Q or Fc receptor y and CD8alpha, CD8beta, CD4, CD25 or CD16.

[0325] In some embodiments, the intracellular (or cytoplasmic) signaling region comprises a human CD3 chain, optionally a CD3 zeta stimulatory signaling domain or functional variant thereof, such as a 112 AA cytoplasmic domain of isoform 3 of human CD3^ (Accession No.: P20963.2) or a CD3 zeta signaling domain as described in U.S. Patent No.: 7,446,190 or U.S. Patent No. 8,911,993. In some embodiments, the intracellular signaling region comprises the sequence of amino acids set forth in SEQ ID NO: 13, 14 or 15 or a sequence of amino acids that exhibits at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 13, 14 or 15. In some embodiments, the CD3(^ signaling domain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the CD3(^ signaling domain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the CD3(^ signaling domain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 15.

[0326] In the context of a natural TCR, full activation generally requires not only signaling through the TCR, but also a costimulatory signal. Thus, in some embodiments, to promote full activation, a component for generating secondary or co-stimulatory signal is also included in the CAR. In other embodiments, the CAR does not include a component for generating a costimulatory signal. In some aspects, an additional CAR is expressed in the same cell and provides the component for generating the secondary or costimulatory signal.

[0327] In some embodiments, the chimeric antigen receptor contains an intracellular domain of a T cell costimulatory molecule. In some embodiments, the CAR includes a signaling domain and/or transmembrane portion of a costimulatory receptor, such as CD28, 4- IBB, 0X40 (CD134), CD27, DAP10, DAP12, ICOS and/or other costimulatory receptors. In some embodiments, the CAR includes a costimulatory region or domain of CD28 or 4- IBB, such as of human CD28 or human 4- IBB. [0328] In some embodiments, the intracellular signaling region or domain comprises an intracellular costimulatory signaling domain of human CD28 or functional variant or portion thereof, such as a 41 amino acid domain thereof and/or such a domain with an LL to GG substitution at positions 186-187 of a native CD28 protein. In some embodiments, the intracellular signaling domain can comprise the sequence of amino acids set forth in SEQ ID NO: 10 or 11 or a sequence of amino acids that exhibits at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 10 or 11. In some embodiments, the intracellular region comprises an intracellular costimulatory signaling domain of 4- IBB or functional variant or portion thereof, such as a 42-amino acid cytoplasmic domain of a human 4-1BB (Accession No. Q07011.1) or functional variant or portion thereof, such as the sequence of amino acids set forth in SEQ ID NO: 12 or a sequence of amino acids that exhibits at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 12.

[0329] In some aspects, the same CAR includes both the primary (or activating) cytoplasmic signaling regions and costimulatory signaling components.

[0330] In some embodiments, the activating domain is included within one CAR, whereas the costimulatory component is provided by another CAR recognizing another antigen. In some embodiments, the CARs include activating or stimulatory CARs, costimulatory CARs, both expressed on the same cell (see WO2014/055668). In some aspects, the cells include one or more stimulatory or activating CAR and/or a costimulatory CAR. In some embodiments, the cells further include inhibitory CARs (iCARs, see Fedorov et al., Sci. Transl. Medicine, 5(215) (December, 2013), such as a CAR recognizing an antigen other than the one associated with and/or specific for the disease or condition whereby an activating signal delivered through the disease-targeting CAR is diminished or inhibited by binding of the inhibitory CAR to its ligand, e.g., to reduce off-target effects.

[0331] In some embodiments, the two receptors induce, respectively, an activating and an inhibitory signal to the cell, such that ligation of one of the receptor to its antigen activates the cell or induces a response, but ligation of the second inhibitory receptor to its antigen induces a signal that suppresses or dampens that response. Examples are combinations of activating CARs and inhibitory CARs (iCARs). Such a strategy may be used, for example, to reduce the likelihood of off-target effects in the context in which the activating CAR binds an antigen expressed in a disease or condition but which is also expressed on normal cells, and the inhibitory receptor binds to a separate antigen which is expressed on the normal cells but not cells of the disease or condition.

[0332] In some aspects, the chimeric receptor is or includes an inhibitory CAR (e.g. iCAR) and includes intracellular components that dampen or suppress an immune response, such as an IT AM- and/or co stimulatory-promoted response in the cell. Exemplary of such intracellular signaling components are those found on immune checkpoint molecules, including PD-1, CTLA4, LAG3, BTLA, OX2R, TIM-3, TIGIT, LAIR-1, PGE2 receptors, EP2/4 Adenosine receptors including A2AR. In some aspects, the engineered cell includes an inhibitory CAR including a signaling domain of or derived from such an inhibitory molecule, such that it serves to dampen the response of the cell, for example, that induced by an activating and/or costimulatory CAR.

[0333] In some cases, CARs are referred to as first, second, and/or third generation CARs. In some aspects, a first generation CAR is one that solely provides a CD3-chain induced signal upon antigen binding; in some aspects, a second-generation CARs is one that provides such a signal and costimulatory signal, such as one including an intracellular signaling domain from a costimulatory receptor such as CD28 or CD137; in some aspects, a third generation CAR in some aspects is one that includes multiple costimulatory domains of different costimulatory receptors.

[0334] In some embodiments, the CAR encompasses one or more, e.g., two or more, costimulatory domains and an activation domain, e.g., primary activation domain, in the cytoplasmic portion. Exemplary CARs include intracellular components of CD3-zeta, CD28, and 4- IBB.

[0335] In some embodiments, the antigen receptor further includes a marker and/or cells expressing the CAR or other antigen receptor further includes a surrogate marker, such as a cell surface marker, which may be used to confirm transduction or engineering of the cell to express the receptor. In some aspects, the marker includes all or part (e.g., truncated form) of CD34, a NGFR, or epidermal growth factor receptor, such as truncated version of such a cell surface receptor (e.g., tEGFR). In some embodiments, the nucleic acid encoding the marker is operably linked to a polynucleotide encoding for a linker sequence, such as a cleavable linker sequence, e.g., T2A. For example, a marker, and optionally a linker sequence, can be any as disclosed in published patent application No. WO2014031687. For example, the marker can be a truncated EGFR (tEGFR) that is, optionally, linked to a linker sequence, such as a T2A cleavable linker sequence.

[0336] An exemplary polypeptide for a truncated EGFR (e.g. tEGFR) comprises the sequence of amino acids set forth in SEQ ID NO: 7 or 16 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 7 or 16. An exemplary T2A linker sequence comprises the sequence of amino acids set forth in SEQ ID NO: 6 or 17 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 6 or 17.

[0337] In some embodiments, the marker is a molecule, e.g., cell surface protein, not naturally found on T cells or not naturally found on the surface of T cells, or a portion thereof. In some embodiments, the molecule is a non-self molecule, e.g., non-self protein, i.e., one that is not recognized as “self’ by the immune system of the host into which the cells will be adoptively transferred.

[0338] In some embodiments, the marker serves no therapeutic function and/or produces no effect other than to be used as a marker for genetic engineering, e.g., for selecting cells successfully engineered. In other embodiments, the marker may be a therapeutic molecule or molecule otherwise exerting some desired effect, such as a ligand for a cell to be encountered in vivo, such as a costimulatory or immune checkpoint molecule to enhance and/or dampen responses of the cells upon adoptive transfer and encounter with ligand.

[0339] In some embodiments, the chimeric antigen receptor includes an extracellular portion containing the antibody or fragment described herein. In some aspects, the chimeric antigen receptor includes an extracellular portion containing the antibody or fragment described herein and an intracellular signaling domain. In some embodiments, the antibody or fragment includes an scFv or a single-domain VH antibody and the intracellular domain contains an ITAM. In some aspects, the intracellular signaling domain includes a signaling domain of a zeta chain of a CD3-zeta (CD3Q chain. In some embodiments, the CD3-zeta chain is a human CD3-zeta chain. In some embodiments, the intracellular signaling region further comprises a CD28 and CD 137 (4- IBB, TNFRSF9) co- stimulatory domains, linked to a CD3 zeta intracellular domain. In some embodiments, the CD28 is a human CD28. In some embodiments, the 4-1BB is a human 4-1BB. In some embodiments, the chimeric antigen receptor includes a transmembrane domain disposed between the extracellular domain and the intracellular signaling region. In some aspects, the transmembrane domain contains a transmembrane portion of CD28. The extracellular domain and transmembrane can be linked directly or indirectly. In some embodiments, the extracellular domain and transmembrane are linked by a spacer, such as any described herein.

[0340] In some embodiments, the CAR contains an antibody, e.g., an antibody fragment, a transmembrane domain that is or contains a transmembrane portion of CD28 or a functional variant thereof, and an intracellular signaling domain containing a signaling portion of CD28 or functional variant thereof and a signaling portion of CD3 zeta or functional variant thereof. For example, in some embodiments, the CAR includes an antibody such as an antibody fragment, including scFvs, e.g. specific for CD19 such as any described above, a spacer, such as a spacer containing a portion of an immunoglobulin molecule, such as a hinge region and/or one or more constant regions of a heavy chain molecule, such as an Ig-hinge containing spacer, a transmembrane domain containing all or a portion of a CD28-derived transmembrane domain, a CD28-derived intracellular signaling domain, and a CD3 zeta signaling domain.

[0341] In some embodiments, the CAR contains an antibody, e.g., antibody fragment, a transmembrane domain that is or contains a transmembrane portion of CD28 or a functional variant thereof, and an intracellular signaling domain containing a signaling portion of a 4- IBB or functional variant thereof and a signaling portion of CD3 zeta or functional variant thereof. In some such embodiments, the receptor further includes a spacer containing a portion of an Ig molecule, such as a human Ig molecule, such as an Ig hinge, e.g. an IgG4 hinge, such as a hinge- only spacer. In some embodiments, the CAR includes an antibody or fragment, such as scFv, e.g. specific for CD 19 such as any described above, a spacer such as any of the Ig-hinge containing spacers, a CD28-derived transmembrane domain, a 4-lBB-derived intracellular signaling domain, and a CD3 zeta-derived signaling domain.

[0342] In particular embodiments, the CAR is a CD19-directed CAR containing an scFv antigen-binding domain from FMC63; an immunoglobulin hinge spacer, a transmembrane domain, and an intracellular signaling domain containing a costimulatory signaling region that is a signaling domain of 4- IBB and a signaling domain of a CD3-zeta (CD3Q chain. In some embodiments, the scFv contains the sequence set forth in SEQ ID NO:43. In some embodiments, the scFv ha a VL having CDRs having an amino acid sequences RASQDISKYLN (SEQ ID NO: 35), an amino acid sequence of SRLHSGV (SEQ ID NO: 36), and an amino acid sequence of GNTLPYTFG (SEQ ID NO: 37); and a VH with CDRs having an amino acid sequence of DYGVS (SEQ ID NO: 38), an amino acid sequence of VIWGSETTYYNSALKS (SEQ ID NO: 39) and YAMDYWG (SEQ ID NO: 40)). In some embodiments, the transmembrane domain has the sequence set forth in SEQ ID NO:8. In some embodiments, the transmembrane domain has a sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:8. In some embodiments, the 4-1BB costimulatory signaling domain has the sequence set forth in SEQ ID NO: 12. In some embodiments, the 4- IBB costimulatory signaling domain has a sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 12. In some embodiments, the CD3-zeta domain has the sequence set forth in SEQ ID NO: 13. In some embodiments, the CD3zeta signaling domain has a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.

[0343] In some embodiments, the CAR contains in order from N-terminus to C-terminus: an extracellular antigen-binding domain that is an scFv comprising a variable heavy chain region of FMC63 set forth in SEQ ID NO:41 and a variable light chain region of FMC63 set forth in SEQ ID NO:42, such as the scFv set forth in SEQ ID NO: 43, the CD8a hinge domain of SEQ ID NO:59, the CD8a transmembrane domain of SEQ ID NO:62, the 4- IBB costimulatory domain of SEQ ID NO: 12, the CD3(^ signaling domain of SEQ ID NO: 13. In some embodiments, the CAR has a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of the foregoing sequences. In some embodiments, the CAR has a sequence of amino acids having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 63. In some embodiments, the CAR has the sequence set forth in SEQ ID NO: 63.

[0344] In some embodiments, the CAR contains in order from N-terminus to C-terminus: an extracellular antigen-binding domain that is an scFv comprising a variable heavy chain region of FMC63 set forth in SEQ ID NO:41 and a variable light chain region of FMC63 set forth in SEQ ID NO:42, such as the scFv set forth in SEQ ID NO: 43, the CD28 hinge domain of SEQ ID NO:60, the CD28 transmembrane domain of SEQ ID NO:8 or 9, the CD28 costimulatory domain of SEQ ID NO: 10, the CD3(^ signaling domain of SEQ ID NO: 13. In some embodiments, the CAR has a sequence of amino acids having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 64. In some embodiments, the CAR has the sequence set forth in SEQ ID NO: 64. [0345] In some embodiments, the CD19-directed CAR binds to CD 19 and mediates cytokine production and/or cytotoxic activity against CD 19+ target cells when expressed in a T cell and stimulated via the CAR, such as by binding to CD 19.

[0346] In particular embodiments of any of the provided methods, the CAR contains in order from N-terminus to C-terminus: an extracellular antigen-binding domain that is the scFv set forth in SEQ ID NO: 43, the spacer set forth in SEQ ID NO:1, the transmembrane domain set forth in SEQ ID NO:8, the 4-1BB costimulatory signaling domain set forth in SEQ ID NO: 12, and the signaling domain of a CD3-zeta (CD3Q chain set forth in SEQ ID NO: 13.

[0347] In some embodiments, the CAR has a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:57. In some embodiments, the CAR comprises the sequence set forth in SEQ ID NO:68. In some embodiments, the CAR is set forth in SEQ ID NO:57. In some embodiments, the CAR is encoded by a sequence of nucleotides set forth in SEQ ID NO:69 or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:58. In some embodiments, the CAR is encoded by a sequence of nucleotides set forth in SEQ ID NO:58

[0348] In some embodiments, nucleic acid molecules encoding such CAR constructs further includes a sequence encoding a T2A ribosomal skip element and/or a tEGFR sequence, e.g., downstream of the sequence encoding the CAR. In some embodiments, the sequence encodes a T2A ribosomal skip element set forth in SEQ ID NO: 6 or 17, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 6 or 17. In some embodiments, T cells expressing an antigen receptor (e.g. CAR) can also be generated to express a truncated EGFR (EGFRt) as a non-immunogenic selection epitope (e.g. by introduction of a construct encoding the CAR and EGFRt separated by a T2A ribosome switch to express two proteins from the same construct), which then can be used as a marker to detect such cells (see e.g. U.S. Patent No. 8,802,374). In some embodiments, the sequence encodes an tEGFR sequence set forth in SEQ ID NO: 7 or 16, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 7 or 16. In some cases, the peptide, such as T2A, can cause the ribosome to skip (ribosome skipping) synthesis of a peptide bond at the C-terminus of a 2A element, leading to separation between the end of the 2A sequence and the next peptide downstream (see, for example, de Felipe. Genetic Vaccines and Ther. 2:13 (2004) and deFelipe et al. Traffic 5:616-626 (2004)). Many 2A elements are known. Examples of 2A sequences that can be used in the methods and nucleic acids disclosed herein, without limitation, 2A sequences from the foot-and-mouth disease virus (F2A, e.g., SEQ ID NO: 21), equine rhinitis A virus (E2A, e.g., SEQ ID NO: 20), Thosea asigna virus (T2A, e.g., SEQ ID NO: 6 or 17), and porcine teschovirus- 1 (P2A, e.g., SEQ ID NO: 18 or 19) as described in U.S. Patent Publication No. 20070116690.

[0349] The recombinant receptors, such as CARs, expressed by the cells administered to the subject generally recognize or specifically bind to a molecule that is expressed in, associated with, and/or specific for the disease or condition or cells thereof being treated. Upon specific binding to the molecule, e.g., antigen, the receptor generally delivers an immuno stimulatory signal, such as an IT AM-transduced signal, into the cell, thereby promoting an immune response targeted to the disease or condition. For example, in some embodiments, the cells express a CAR that specifically binds to an antigen expressed by a cell or tissue of the disease or condition or associated with the disease or condition.

IV. DEFINITIONS

[0350] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[0351] The term “about” as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se.

[0352] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. For example, “a” or “an” means “at least one” or “one or more.”

[0353] Throughout this disclosure, various aspects of the claimed subject matter are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the claimed subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the claimed subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the claimed subject matter. This applies regardless of the breadth of the range.

[0354] The terms “polypeptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues and are not limited to a minimum length. Polypeptides, including the provided receptors and other polypeptides, e.g., linkers or peptides, may include amino acid residues including natural and/or non-natural amino acid residues. The terms also include postexpression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, and phosphorylation. In some aspects, the polypeptides may contain modifications with respect to a native or natural sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.

[0355] As used herein, “percent (%) amino acid sequence identity” and “percent identity” when used with respect to an amino acid sequence (reference polypeptide sequence) is defined as the percentage of amino acid residues in a candidate sequence (e.g., the subject antibody or fragment) that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various known ways, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Appropriate parameters for aligning sequences can be determined, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0356] An amino acid substitution may include replacement of one amino acid in a polypeptide with another amino acid. The substitution may be a conservative amino acid substitution or a non-conservative amino acid substitution. Amino acid substitutions may be introduced into a binding molecule, e.g., antibody, of interest and the products screened for a desired activity, e.g., retained/improved antigen binding, decreased immunogenicity, or improved ADCC or CDC.

[0357] Amino acids generally can be grouped according to the following common sidechain properties:

[0358] (1) hydrophobic: Norleucine, Met, Ala, Vai, Leu, He;

[0359] (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;

[0360] (3) acidic: Asp, Glu;

[0361] (4) basic: His, Lys, Arg;

[0362] (5) residues that influence chain orientation: Gly, Pro;

[0363] (6) aromatic: Trp, Tyr, Phe.

[0364] In some embodiments, conservative substitutions can involve the exchange of a member of one of these classes for another member of the same class. In some embodiments, non-conservative amino acid substitutions can involve exchanging a member of one of these classes for another class.

[0365] As used herein, a composition refers to any mixture of two or more products, substances, or compounds, including cells. It may be a solution, a suspension, liquid, powder, a paste, aqueous, non-aqueous or any combination thereof.

[0366] As used herein, “enriching” when referring to one or more particular cell type or cell population, refers to increasing the number or percentage of the cell type or population, e.g., compared to the total number of cells in or volume of the composition, or relative to other cell types, such as by positive selection based on markers expressed by the population or cell, or by negative selection based on a marker not present on the cell population or cell to be depleted. The term does not require complete removal of other cells, cell type, or populations from the composition and does not require that the cells so enriched be present at or even near 100% in the enriched composition.

[0367] As used herein, a statement that a cell or population of cells is “positive” for a particular marker refers to the detectable presence on or in the cell of a particular marker, typically a surface marker. When referring to a surface marker, the term refers to the presence of surface expression as detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting said antibody, wherein the staining is detectable by flow cytometry at a level substantially above the staining detected carrying out the same procedure with an isotype-matched control or fluorescence minus one (FMO) gating control under otherwise identical conditions and/or at a level substantially similar to that for cell known to be positive for the marker, and/or at a level substantially higher than that for a cell known to be negative for the marker.

[0368] As used herein, a statement that a cell or population of cells is “negative” for a particular marker refers to the absence of substantial detectable presence on or in the cell of a particular marker, typically a surface marker. When referring to a surface marker, the term refers to the absence of surface expression as detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting said antibody, wherein the staining is not detected by flow cytometry at a level substantially above the staining detected carrying out the same procedure with an isotype-matched control or fluorescence minus one (FMO) gating control under otherwise identical conditions, and/or at a level substantially lower than that for cell known to be positive for the marker, and/or at a level substantially similar as compared to that for a cell known to be negative for the marker.

[0369] The term “vector” is used herein to refer to a nucleic acid molecule, microorganism, or virus capable of transferring or transporting another nucleic acid molecule to a cell or genome. Illustrative examples of vectors include, for example, plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, bacteria, and viral vectors.

[0370] The term “viral vector” refers to a nucleic acid molecule that includes virus-derived nucleic acid elements that typically facilitate transfer of the nucleic acid molecule or integration into a cell and/or genome. The term “viral vector” includes a modified virus or viral particle capable of transferring a nucleic acid into a cell and/or genome. Viral vectors may contain structural and/or functional genetic elements that are primarily derived from a virus. Viral vectors suitable for use in preferred embodiments, include but are not limited to retroviral vectors and lentiviral vectors. In particular embodiments, a viral vector comprises a 5' LTR, a packaging signal, a cPPT/FLAP element, a RNA export element, a transgene, and a 3' LTR. Viral vectors may optionally comprise post-transcriptional regulatory elements and poly adenylation signals/sequences .

[0371] As used herein, the term “retrovirus” or “retroviral vector” refers to a viral vector that reverse transcribes its genomic RNA into a linear double-stranded DNA copy and subsequently covalently integrates its genomic DNA into a host genome. Illustrative retroviral vectors suitable for use in particular embodiments, include, but are not limited to those derived from Moloney murine leukemia vims (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma vims (HaMuSV), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Friend murine leukemia vims, Murine Stem Cell Virus (MSCV) and Rous Sarcoma Virus (RSV)) and lentivirus.

[0372] As used herein, the term “lentivirus” with reference to a lentiviral vector refers to a group (or species) of complex retroviruses. Illustrative lentiviral vectors suitable for use in particular embodiments contemplated herein include, but are not limited to those derived from HIV (human immunodeficiency vims; including HIV type 1, and HIV type 2); visna-maedi virus (VMV); the caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immune deficiency virus (BIV); and simian immunodeficiency virus (SIV).

[0373] The term “Self-inactivating” (SIN) viral vectors refers to replication-defective vectors, e.g., retroviral or lentiviral vectors, in which the right (3') LTR enhancer-promoter region, known as the U3 region, has been modified (e.g., by deletion or substitution) to prevent viral transcription beyond the first round of viral replication. This is because the right (3') LTR U3 region is used as a template for the left (5') LTR U3 region during viral replication and, thus, the viral transcript cannot be made without the U3 enhancer- promoter. Self-inactivation is typically achieved through the introduction of a deletion in the U3 region of the 3' LTR of the vector DNA, i. e., the DNA used to produce the vector RNA. Thus, during reverse transcription, this deletion is transferred to the 5' LTR of the proviral DNA. In the case of lentiviral vectors, it has been discovered that such vectors tolerate significant U3 deletions, including the removal of the LTR TATA box (e.g., deletions from -418 to -18), without significant reductions in vector titers.

[0374] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein. [0375] As used herein, a “subject” is a mammal, such as a human or other animal, and typically is human. In some embodiments, the subject, e.g., patient, to whom the agent or agents, cells, cell populations, or compositions are administered, is a mammal, typically a primate, such as a human. In some embodiments, the primate is a monkey or an ape. The subject can be male or female and can be any suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects. In some embodiments, the subject is a non-primate mammal, such as a rodent.

V. EXEMPLARY EMBODIMENTS

[0376] Among the provided embodiments are:

1. A method for determining upstream lentiviral manufacturing conditions, the method comprising:

(a) performing a plurality of transfection reactions by transfecting host cells with a plurality of transfection mixtures, each transfection mixture comprising (1) a mixture of plasmids for the production of a lentiviral vector, wherein the mixture of plasmids comprise at least two lentiviral helper plasmids and a transfer plasmid encoding a transgene, and (2) a transfection agent, wherein each of the plurality of transfection mixtures has a different mass fraction ratio of plasmids in the mixture of plasmids and the same total amount of DNA;

(b) harvesting the culture supernatant from each transfection reaction, and optionally clarifying the supernatant of the harvested culture to produce a clarified harvest;

(c) determining lentiviral vector performance from each of the harvested culture supernatants or the clarified harvest, wherein viral vector performance is determined from both an analytical measure of a cell-line titer and a primary cell titer; and

(d) selecting a transfection mixture for an upstream processing method for producing a lentiviral vector based on the lentiviral vector performance that has been determined for the transfection mixture.

2. A method for determining upstream lentiviral manufacturing conditions, the method comprising:

(a) performing a plurality of transfection reactions by transfecting host cells with a plurality of transfection mixtures, each transfection mixture comprising (1) a mixture of plasmids for the production of a lentiviral vector, wherein the mixture of plasmids comprise at least one lentiviral packaging plasmid encoding a viral gene selected from rev, gag or pol, an envelope plasmid encoding VSV-G, and a transfer plasmid encoding a transgene, and (2) a transfection agent, wherein each of the plurality of transfection mixtures has a different mass fraction ratio of plasmids in the mixture of plasmids and the same total amount of DNA, wherein the mass fraction of the envelope plasmid is independently from 0.04 to 0.2;

(c) determining lentiviral vector performance from each of the harvested culture supernatants or the clarified harvest; and

3. The method of embodiment 2, wherein the lentiviral vector performance is determined from both an analytical measure of a cell line titer and a primary cell titer.

4. A method for determining upstream lentiviral manufacturing conditions, the method comprising:

(a) performing a plurality of transfection reactions by transfecting host cells with a plurality of transfection mixtures, each transfection mixture comprising (1) a mixture of plasmids for the production of a lentiviral vector, wherein the mixture of plasmids comprise at least one lentiviral helper plasmid and a transfer plasmid encoding a transgene and (2) a transfection agent, wherein each of the plurality of transfection mixtures has a different mass fraction ratio of plasmids in the mixture of plasmids and the same total amount of DNA;

(c) determining lentiviral vector performance from each of the harvested culture supernatants or the clarified harvest, wherein viral vector performance is determined from both an analytical measure of a cell line titer and a primary cell titer;

(d) performing a downstream purification on the harvested culture supernatant or the clarified harvest from each of a subset of the transfection reactions; and

(e) selecting a transfection mixture for an upstream processing method for producing a lentiviral vector based on the downstream purification performance that has been determined for the transfection mixture. 5. The method of embodiment 1 and 4, wherein the at least two helper plasmids encode at least one packaging viral gene and at least one envelope viral gene.

6. The method of embodiment 5, wherein the envelope viral gene is VSV-G.

7. The method of embodiment 5, wherein the packaging viral gene is rev, gag or pol or a combination of any of the foregoing.

8. The method of embodiment 7, wherein the mixture of plasmids comprises a packaging plasmid encoding gag and pol (Gag-Pol plasmid), an envelope (Env) plasmid encoding VSV-G, and a transfer plasmid encoding the transgene.

9. The method of any of embodiments 1-8, wherein the mixture of plasmids is a three-plasmid mixture, a four-plasmid mixture or a five-plasmid mixture.

10. The method of any of embodiments 1-9, wherein the mixture of plasmids is a three plasmid mixture composed of a packaging plasmid encoding gag and pol (Gag-Pol plasmid), an envelope (Env) plasmid encoding VSV-G, and a transfer plasmid encoding the transgene.

11. The method of any of embodiment 1-9, wherein the mixture of plasmids is a four- plasmid mixture composed of a packaging plasmid encoding gag and pol (Gag-Pol plasmid), a packaging plasmid encoding rev (Rev plasmid), an envelope (Env) plasmid encoding VSV-G, and a transfer plasmid encoding the transgene.

12. The method of any of embodiments 8-11, wherein the subset of transfection reactions each have a different mass fraction ratio of the packaging plasmid encoding Gag-Pol or the envelope plasmid encoding VSV-G relative to the other plasmids in the mixture of plasmids.

13. The method of any of embodiments 8-12, wherein the subset of transfection reactions each have a different mass fraction ratio of the packaging plasmid encoding Gag-Pol relative to the other plasmids in the mixture of plasmids.

14. The method of any of embodiments 8-13, wherein the subset of transfection reactions each have a different mass ratios of the envelope plasmid encoding VSV-G relative to the other plasmids in the mixture of plasmids.

15. The method of any of embodiments 8-14, wherein the subset of transfection reactions each have a different mass ratio of the packaging plasmid encoding Gag-Pol and a different mass ratio of the envelope plasmid encoding VSV-G, relative to the other plasmids in the mixture of plasmids. 16. The method of any of embodiments 4-15, wherein the subset of transfection mixtures are candidate transfection mixtures that were identified to have the highest levels of viral vector performance from among the plurality of transfection mixtures in (c).

17. The method of any of embodiments 1-16, wherein the plurality of transfection reactions is a design of experiments (DOE).

18. The method of any of embodiments 4, 7 and 9, wherein the mass fraction of the plasmid encoding VSV-G is varied in the plurality of transfection mixtures.

19. The method of any of embodiments 6, and 8-18 , wherein the mass fraction of the plasmid encoding VSV-G in each of the transfection mixtures is independently from 0.04 to 0.2.

20. The method of any of embodiments 6 and 8-19, wherein the mass fraction of the plasmid encoding VSV-G among each of the transfection mixtures is independently from 0.046 to 0.15.

21. The method of any of embodiments 7-20, wherein the mass fraction of each of one or more of the plasmids encoding rev, gag or pol is held constant among each of the transfection mixtures.

22. The method of any of embodiments 7-21, wherein the mass fraction of the plasmid encoding rev is held constant among each of the transfection mixtures.

23. The method of any of embodiments 7-22, wherein the mass fraction of the plasmid encoding rev among each of the transfection mixtures is independently from 0.04 to 0.08, optionally from 0.04 to 0.06.

24. The method of any of embodiments 7-23, wherein the mass fraction of the plasmid encoding rev among each of the transfection mixtures is held constant at about 0.049.

25. The method of any of embodiments 7-24, wherein the mass fraction of the plasmid encoding gag and/or pol, optionally gag and pol, is held constant among each of the transfection mixtures.

26. The method of any of embodiments 7-25, wherein the mass fraction of the plasmid encoding gag and/or pol, optionally gag and pol, among each of the transfection mixtures is independently from 0.1 to 0.25, optionally from 0.15 and 0.2.

27. The method of any of embodiments 7-26, wherein the mass fraction of the plasmid encoding gag and/or pol, optionally gag and pol, among each of the transfection mixtures is held constant at about 0.178. 28. The method of any of embodiments 1-27, wherein the mass fraction of the plasmid encoding the transgene is varied in the plurality of transfection mixtures.

29. The method of any of embodiments 1-28, wherein the mass fraction of the plasmid encoding the transgene among each of the transfection mixtures is independently from 0.47 to 0.82, optionally from 0.58 to 0.74.

30. The method of any of embodiments 1-29, wherein the plurality of transfection mixtures is 3 to 50, optionally 3 to 30.

31. The method of any of embodiments 1-30, wherein the candidate transfection mixture in (d) is identified using multiple-response optimization.

32. The method of embodiment 31, wherein the multiple-response optimization is characterized by a desirability function for each analytical measure of a cell line titer and a primary cell titer.

33. The method of embodiment 32, wherein the desirability function for primary cell titer is to be maximized over the cell line titer.

34. The method of embodiment 32, wherein the desirability function for primary cell titer and cell line titer are equal.

35. The method of embodiment 33 or embodiment 34, wherein the desirability function for primary cell titer is about 1.0.

36. The method of any of embodiments 1-35, wherein the primary cell titer is a functional titer determined by a primary cell transduction assay.

37. The method of embodiment 36, wherein the primary cell transduction assay comprises transducing target cells with the harvested culture supernatant or clarified harvest, incubating the transduced target cells under conditions for expression of the transgene, and analyzing expression of the transgene by the cells.

38. The method of embodiment 37, wherein cell surface expression of the transgene is analyzed by flow cytometry.

39. The method of embodiment 37 or embodiment 38, wherein the target cells are T cells and prior to the transducing the method comprises activating the T cells with a T cell stimulatory reagent, optionally wherein the T cell stimulatory reagent is an anti-CD3/anti-CD28 activation reagent.

40. The method of embodiment 39, wherein the T cells are primary cells selected from a subject, optionally wherein the subject is a healthy subject. 41. The method of embodiment 39 or embodiment 40, wherein the T cells are CD4+

T cells, CD8+ T cells or CD4+ and CD8+ T cells.

42. The method of any of embodiments 37-41, wherein the target cells are T cells and incubating the transduced target cells under conditions for expression of the transgene comprises incubating the cells in the presence of one or more T cell stimulatory recombinant cytokines, optionally IL-2 IL-7, IL- 15 or IL-21 or a combination of any of the foregoing.

43. The method of embodiment 42, wherein the incubating expands the T cells.

44. The method embodiment 42 or embodiment 43, wherein the incubating is for 2 to 10 days.

45. The method of any of embodiments 37-41, wherein the target cells are T cells and incubating the transduced target cells under conditions for expression of the transgene comprises incubating the cells in basal media without recombinant cytokines.

46. The method of embodiment 45, wherein the incubating is for 12 hours to 48 hours.

47. The method of any of embodiments 1-46, wherein the cell line titer is a functional titer or and/or an infectious titer.

48. The method of any of embodiments 32-47, wherein the desirability function of the cell line titer is less than 1.0, optionally 0.5.

49. The method of any of embodiments 32-47, wherein the desirability function of the cell line titer is about 1.0.

50. The method of any of embodiments 1-49, wherein the analytical measure of a cell line titer is an analytical measure in at least two cell line titer assays.

51. The method of embodiment 50, wherein the at least two cell line titer assays are at least one infectious titer and at least one functional titer.

52. The method of embodiment 50 or embodiment 51, wherein the desirability function of each of the at least two cell line titer is less than 1.0, optionally less than 0.5.

53. The method of embodiment 50 or embodiment 51, wherein the desirability function of each of the at least two cell line titer combined is about 1.0.

54. The method of any of embodiments 47-53, wherein the infectious titer is determined by an endpoint dilution assay (TCID50) or a qPCR lentivirus titer assay.

55. The method of any of embodiments 47-54, wherein the infectious titer is determined by a qPCR lentivirus titer assay. 56. The method of embodiment 55, wherein the qPCR comprises primers and probes for amplification of a region of the LTR of the genomic RNA.

57. The method of any of embodiment 47-53, wherein the functional titer is determined by a cell-based transduction assay.

58. The method of any of embodiments 57, wherein transduction is determined by measuring transgene expression by flow cytometry.

59. The method of any of embodiments 1-58, wherein the cell line titer assay is titer on an immortalized cell line.

60. The method of embodiment 59, wherein the immortalized cell line is a Jurkat cell line.

61. The method of any of embodiments 1-60, wherein the downstream purification is by chromatography.

62. The method of any of embodiments 1-61, wherein the downstream purification comprises chromatography and ultrafiltration/diafiltration (UF/DF).

63. The method of embodiment 61 and embodiment 62, wherein the chromatography is by a method selected from the group consisting of heparin affinity, gel filtration and anion- exchange (AEX).

64. The method of any of embodiments 61-63, wherein the chromatography is anion- exchange (AEX).

65. The method of any of embodiments 61-64, wherein the downstream purification further comprises sterile filtration.

66. The method of any of any of embodiments 1-65, wherein the downstream purification performance for selecting the candidate transfection mixture in (f) is elution profile for vector performance, residual protein, residual BSA, residual plasmid or host-cell DNA or a combination of any of the foregoing.

67. The method of embodiment 66, wherein elution profile for vector performance comprises testing a plurality of elution fractions for infectious titer of the viral vector, optionally wherein infectious titer is assessed using a cell-based transduction assay.

68. The method of any of embodiments 1-67, comprising optionally repeating steps (a)-(d) to identify different candidate transfection mixtures if the downstream purification performance is not acceptable.

69. The method of any of embodiments 1-68, wherein the method further comprises: (g) varying one or more parameters in a downstream process for producing the lentiviral vector comprising the downstream purification, wherein the downstream process is carried out from material harvested from an upstream process using the selected candidate transfection mixture.

70. The method of embodiment 69, wherein the upstream processing method comprises:

(i) transfecting the host cells in a large-scale culture with the selected transfection mixture;

(ii) harvesting the supernatant from the transfected culture; and

(iii) clarifying the harvested culture supernatant by centrifugation or filtration.

71. The method of any of embodiments 1-70, wherein the host cells are adherent cells.

72. The method of any of embodiments 1-70, wherein the host cells are suspension cells.

73. The method of any of embodiments 1-72, wherein the host cells are HEK293T cells or a derivative thereof, optionally wherein the host cells are HEK 293T/17 cells.

74. The method of any of embodiments 1-73, wherein the endonuclease is added to the culture supernatant prior to harvesting the supernatant from the transfected culture.

75. The method of embodiment 74, wherein the endonuclease is Benzonase.

76. The method of any of embodiments 1-75, wherein the transgene is a chimeric antigen receptor (CAR).

77. The method of embodiment 76, wherein the chimeric antigen receptor is a monospecific CAR.

78. The method of embodiment 77, wherein the chimeric antigen receptor is a bispecific CAR.

79. The method of any of embodiments 1-78, wherein the transfection agent is a cationic polymer.

80. The method of embodiment 79, wherein the cationic polymer is polyethylenimine (PEI).

81. The method of embodiment 80, wherein the mass ratio of the mixture of plasmid DNA to PEI is 5:1 to 1:5, optionally 3:1 to 1:3. 82. The method of embodiment 80 or embodiment 81, wherein the mass ratio of the mixture of plasmid DNA to PEI is 1:1.

83. The method of any of embodiments 80-82, wherein the mixture of plasmid DNA and PEI are provided as a transfection complex , optionally stabilized with fetal bovine serum (FBS) or human serum albumin (HSA).

84. A method of manufacturing a lentiviral vector, the method comprising:

(a) transiently transfecting host cells to produce a transfected culture with (i) a transfection mixture selected according to the method of any one of embodiments 1-83 and (ii) a transfection agent to make a transfected culture, and optionally:

(b) harvesting the supernatant from the transfected culture;

(c) clarifying the harvested culture supernatant by filtration;

(d) capturing and concentrating the lentiviral vector from the harvested and clarified supernatant by anion exchange chromatography (AEX);

(e) ultrafiltering and diafiltering the lentiviral vector using Tangential Flow Filtration (TFF);

(f) sterile filtering the lentiviral vector.

85. A method of manufacturing a lentiviral vector, the method comprising:

(a) transiently transfecting host cells to produce a transfected culture with a transfection mixture comprising (1) a mixture of plasmids for the production of a lentiviral vector, wherein the mixture of plasmids is a four-plasmid mixture comprising a lentiviral packaging plasmid encoding gag and pol at a mass fraction from 0.15 and 0.2, a lentiviral packaging plasmid encoding rev at a mass fraction from 0.04 to 0.06, an envelope plasmid encoding VSV-G at a mass fraction from 0.04 to 0.15, and a transfer plasmid encoding a chimeric antigen receptor at a mass fraction from 0.58 to 0.74, and (2) a transfection agent;

(b) harvesting the supernatant from the transfected culture;

(c) clarifying the harvested culture supernatant by filtration;

(f) sterile filtering the lentiviral vector. 86. The method of embodiment 85, wherein the mass fraction of the packaging plasmid encoding gag and pol is about 0.178, the mass fraction of the lentiviral packaging plasmid encoding rev is about 0.049, the mass fraction of the envelope plasmid encoding VSV- G is about 0.046, and the mass fraction of the transfer plasmid encoding the CAR is about 0.727.

87. The method of embodiment 85 or embodiment 86, wherein the AEX chromatography includes a wash with about 300 mM NaCl and an elution with about 450 mM NaCl.

88. The method of any of embodiments 84-86, wherein the transfection agent is a cationic polymer, optionally wherein the cationic polymer is polyethylenimine (PEI).

89. The method of any of embodiments 84-88, wherein clarifying the harvested culture supernatant is by membrane filtration.

90. The method of embodiment 89, wherein the membrane filtration is with a duallayer filter.

91. The method of any of embodiments 84-90, wherein sterile filtering is by membrane filtration.

92. The method of embodiment 91, wherein sterile filtering is with a dual-layer filter.

93. The method of any of embodiments 84-92, wherein the host cells are HEK293 cells.

94. The method of embodiment 93, wherein the HEK293 cells are HEK-293T/17 cells.

95. The method of any of embodiments 84-94, wherein the CAR is an anti-CD19 CAR.

96. The method of any of embodiments 84-95, wherein the CAR contains in order from N-terminus to C-terminus: an extracellular antigen-binding domain that is the scFv set forth in SEQ ID NO: 43, the spacer set forth in SEQ ID NO:1, the transmembrane domain set forth in SEQ ID NO:8, the 4-1BB costimulatory signaling domain set forth in SEQ ID NO: 12, and the signaling domain of a CD3-zeta (CD3Q chain set forth in SEQ ID NO: 13.

97. The method of any of embodiments 84-96, wherein the CAR comprises the amino acid sequence set forth in SEQ ID NO:57 or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 57.

I l l 98. The method of any of embodiments 84-97, wherein the nucleotide sequence encoding the CAR comprises the sequence set forth in SEQ ID NO:58 or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 58.

99. The method of any of embodiments 4-98, wherein the volume of the transfection reaction is from 5 mL to 5000 mL or from about 5 mL to about 5000 mL

100. The method of any of embodiments 4-99, wherein the volume of the transfection reaction is from or from about 10 mL to 2000 mL.

101. The method of any of embodiments 4-100, wherein the volume of the transfection reaction from or from about 500 mL to 2000 mL.

VI. EXAMPLES

[0377] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

Example 1: Assessment of Upstream Process Features in a Lentiviral Vector Manufacturing Process on Viral Vector Performance

[0378] A mixture design of experiments (DOE) was carried out to study the relationship between several factors in an upstream lentiviral manufacturing process on the performance of a lentiviral vector carrying an exemplary anti-CD19 chimeric antigen receptor (CAR) transgene. In the studies, the relative levels (mass fractions) of the four plasmids used in the transfection step were analyzed while holding the overall amount of DNA added constant (FIG. 1).

[0379] A mixture DOE was used to interrogate the four plasmids used during transfection. HEK-293T/17 cells were transfected with different ratios of four plasmids necessary for lentiviral vector production, transgene plasmid, Rev plasmid, VSV-G plasmid, and Gag-Pol plasmid. The mixture DOE was generated with a random block (two blocks containing 13 conditions each). In each mixture DOE, each of the four plasmids were a proportion of the final mixture and, therefore, the proportions of each of the four plasmids added together equal 1.0 (see Table El). [0380] Table El: Mass Fraction of Plasmid Added to Each TFXN (transfection) Complex per Condition.

A. Transfection and Harvest

[0381] HEK-293T/17 cells were thawed and expanded for several passages prior to seeding the cells for vector production. At seed for production, 26 x T-75 vessels were seeded at 2.0 x 10⁴ viable cells per centimeter square (vc/cm²) and were expanded for about 3 days before transfection in a 5% CO2 humidified incubator maintained at 37 °C. [0382] Before transfection, the DNA plasmid solutions were prepared in a 50 mL conical tube for each T-75 vessel. The plasmids were added at amounts in Table El to about 5.4 mL of a pH neutral media (e.g. BalanCD® media; Irvine Scientific, Santa Ana, CA, PN 91165), and then were swirled to mix. The total plasmid DNA concentration was about 0.241 pg/cm2. A master mix was prepared by mixing equal volume the above DNA plasmid solution and a BalanCD®- polyethyleneimine (PEI) solution together. Each master mix that was prepared contained a different molar ratio of transgene:rev:env:gag/pol plasmid and a 1:1 DNA:PEI mass ratio.

[0383] The combined master mix containing transfection (TFXN) complexes of DNA:PEI were diluted directly into the pre-TFXN media (DMEM + 9.1% FBS) to form the TFXN media. The media used for transfection also included fetal bovine serum which contains bovine serum albumin (BSA); however human serum albumin (HSA) can be added to the mix instead. The spent growth media was removed from each T-75 flask and replaced with about 16.25 mL of the appropriate TFXN media. Each T-75 flask was returned to the controlled 37 °C, 5% CO2 humidified incubator and media was exchanged about 22 hours after transfection with fresh TFXN media. About 30 hours after media exchange, the media was harvested from each flask into 50 mL conical tubes and subjected to a clarifying spin (200 x g, 5 minutes). About 13.0 mL of each harvest sample was then treated with 60 U/mL of Benzonase and 1.2 mM MgSO4 and incubated at 37 °C for about 1 hour. Once this incubation was complete, the Benzonase- treated harvest samples were sampled and stored at -80 °C for analytical testing.

B. Analytical Testing

[0384] Crude lentiviral vector harvests described above were assessed for vector cell line titer and primary cell functional titer by monitoring CAR expression in primary cells.

[0385] Cell line titer was determined using by a functional titer transduction assay using a Jurkat titer assay (TU/mL) or by an infectious titer assay using a LVTA19 (lU/mL) titer assay. In the Jurkat titer assay, a Jurkat cell line was transduced in dilution series with lentiviral vector, and 24-72 hours post-transduction cells having the CAR protein on their surface were detected with fluorescently labeled antibody (e.g. anti-idiotypic antibody directed against CAR) via flow cytometry. For the LVTA19 titer assay, HEK293 cells were transduced in dilution series with lentiviral vector, 24 hours post-transduction the cells were lysed and the genomic DNA was extracted, and qPCR was then used to assay the extracted DNA using probes against the LTRs in the lentivector genome. [0386] For assessing the primary cell functional titer based on CAR expression in primary cells, primary T cells were isolated by immunoaffinity-based enrichment from healthy donor and activated with an anti-CD3/anti-CD28 bead activation reagent. The activated cells were transduced with clarified viral vector harvest material, cultivated with cytokines under conditions for expansion, and harvested engineered T cells were monitored for CAR expression using an anti-idiotypic antibody directed against the CAR.

[0387] In addition to cell line titer and primary cell functional titer by CAR expression, other analytical measurements that were assessed included physical titer assays by p24 ELISA (total p24 assay), genomes titer by qPCR (RNA genomes assay) and residual host cell proteins.

[0388] Based on the CAR+ expression as an analytical measure, the results showed that different plasmid ratios in the upstream process for transfecting producer cells impact the CAR+ transduction plateau (FIG. 2). A comparison of cell line titer to CAR expression is shown in FIG. 3 for different plasmid mass fraction preparations as a function of the gag-pol plasmid amount. The results show that cell line titer alone does not predict viral vector performance in the CAR-T cell manufacturing process, since higher titers of vector preparations did not consistently correlate to the degree of CAR expression. The slope of correlation between cell line titer and CAR expression was dependent on the amount of gag-pol plasmid used in the plasmid ratio.

Example 2: Impact of VSV-G Plasmid Amount in Upstream and Downstream Development on Recovery and Purity of Lenti viral Vector

[0389] Crude harvested material from an upstream process substantially as described in Example 1 was purified in a downstream process (FIG. 4). As described in Example 1 above, the VSV-G plasmid amount impacts viral vector performance of crude harvested vector in the downstream process. In particular, a high amount of VSV-G plasmid was associated with high titer and CAR-T cell expression analytical measurements of viral vector performance in the upstream process. In the downstream process, crude harvested material generated from either an upstream process using plasmid amounts from an exemplary condition with “low VSV-G’ plasmid amount of 0.046 and a “high VSV-G” plasmid amount of 0.235 were evaluated in a downstream process to further identify parameters that impacted the downstream process. Table E2 sets forth plasmid ratios used for these studies.

Table E2

[0390] Upstream material was generated substantially as described in Example 1 using the plasmid ratios in Table E2 for transfection of HEK-293T/17 cells. From upstream material, the downstream purification process involved sequential process steps beginning with clarification, purification using anion-exchange chromatography (AEX), and concentration and buffer exchange by ultrafiltration and diafiltration, sterile filtration and then fill and finish. For AEX, clarified harvested material was loaded onto an anion exchange column with a positively charged quaternary amine group ligand at a low ionic strength (i.e. low salt concentrations), washed with buffers of increasing ionic strength (i.e. increasing salt concentrations) and eluted with salt (NaCl). The AEX was used to reduce the level of residual impurities (residual host cell protein (HCP), bovine serum albumin (BSA), host cell DNA (hcDNA), residual double stranded DNA (dsDNA) and plasmid DNA in the lentiviral vector clarified harvest and to reduce the feed stream volume for subsequent processing by the Ultrafiltration/Diafiltration (UF/DF).

[0391] These studies found that material produced using the “high VSV-G” plasmid ratio did not perform as well in the AEX chromatography step as the “low VSV-G” ratio. The “High VSV-G” material eluted earlier in the gradient (-400 mM NaCl) than the “Low VSV-G” material (-640 mM NaCl). Specifically, the poor resolution in the AEX profiles of infectious titer, residual BSA and residual HCP did not allow for a wash step, which is necessary to removing residual BSA and residual HCP. The “Low VSV-G” condition resulted in higher percent recovery across sterile filtration. Additionally, the overall process recovery with the “high VSV-G” plasmid ratio was 9.20% compared with the overall process recovery with the “low VSV-G” plasmid ratio was 14.6%.

[0392] These results are consistent with a finding that the amount of VSV-G plasmid added during the transfection step in the upstream process directly impacts the performance of the downstream purification process (resolution on the AEX step for residual protein/DNA removal and overall downstream titer yield). The results indicate that downstream performance is derived largely by VSV-G (surface protein) amount.

[0393] These results further indicate that identifying a plasmid amount in an upstream analysis as described in Example 1 versus by a downstream analysis as described in this example can result in identification of different optimal plasmid ratios. For instance, analysis of upstream process performance would indicate a higher VSV-G plasmid amount is desirable, whereas analysis of downstream performance indicates a lower VSV-G plasmid amount is more ideal.

Example 3: Method to Identify Plasmid Ratios to Maximize Performance in Upstream and Downstream Process

[0394] The observations described in Examples 1 and 2 demonstrated that when optimizing plasmid ratios to produce lentiviral vector, the outcome (optimal) ratio depends on the design goal (e.g. highest infectious titer, lowest residual impurities, highest CAR+ frequency, or highest purity after AEX).

[0395] As a model for identifying plasmid ratios, the JMP model described in Example 1 was used to identify plasmid ratios to maximize vector performance in the upstream process, in which the desirability function for the model was based on maximizing cell line titer (e.g. LVTA19 and Jurkat assays) at an importance level of 0.5 and maximizing primary cell functional titer by CAR expression at an importance level 1.0. Results in Example 1 support that using both a cell based titer method and primary cell functional titer method (e.g. primary cell transduction performance) is necessary since cell line titer alone is not a good predictor of T-cell transduction performance. Since the lentiviral vector is used in processes for producing CAR- expressing cells, CAR expression by primary cells was deemed to be the more important analytical measurement int this model.

[0396] As shown in FIG. 5, maximizing desirability based on cell line titer and primary cell functional titer (transduction of primary T cells) without any constraints on the model , resulted in a proposed VSV-G mass fraction of 0.235 an amount shown to be too high for optimal downstream purification performance as described in Example 2.

[0397] Since results in Example 2 predicted that the amount of VSV-G plasmid added during the transfection step directly impacts the performance of the downstream purification process, the JMP model was modified to maximize cell line titer and primary cell functional titer (primary T cell transduction) performance while also holding VSV-G at a level known to be compatible with downstream purification. In the updated model, the VSV-G mass fraction was set to a constant value of 0.046, which matched the “low VSV-G” plasmid amount assessed in Example 1 (also referred to as “Optimum DSP Yield Ratio”). Using the importance level criteria above (infectious titer 0.5 and functional titer 1.0), the JMP model was used to predict the optimal levels of the remaining three plasmids (gag-pol, rev, and transgene) in an upstream process. Additional levels of VSV-G were also fixed in the model (0.10 and 0.15) and then JMP was used to predict the optimal mass fractions of the other three plasmids. After performing several of these predictions, it was observed that the optimized Gag-Pol mass fractions were all relatively similar. Based on these results, the Gag-Pol mass fraction was chosen as 0.178 by averaging the identified values. The chosen Rev plasmid amount was 0.049, which was the lowest mass fraction tested, as minimizing Rev consistently gave the highest cell line titer and highest maximum primary cell functional titer (transduction frequency) values.

[0398] After several iterations of this modeling process, the fixed values for gag-pol and rev plasmid were determined and plasmid ratios set for each of the three conditions as shown in Table E3. Table E3 also depicts the previously selected “Low VSV-G” plasmid ratio (“Reference”). A similar process could be followed where the levels of the remaining three plasmids are allowed to vary independently for each VSV-G mass fraction to be tested.

Table E3.

Example 4. Assessment of Downstream Process Performance Using Plasmid Amounts from JMP Model

[0399] Upstream process harvest material was generated using the three plasmid ratios identified in Example 3 (Table E3). The harvest material generated was sampled and then forward processed for clarification and then used in downstream processing.

[0400] HEK-293T/17 cells were thawed and expanded for several passages, seeded for production at 3.5 x 104 vc/cm2, and expanded for about 3 days before transfection in a 37 °C, 5% CO2 humidified incubator. The DNA plasmid solutions were generated by adding the different plasmids amounts in Table E3 to a pH neutral media (e.g. BalanCD® media; Irvine Scientific, Santa Ana, CA, PN 91165), and then were swirled to mix. A master mix was prepared by mixing equal volume of the above DNA plasmid solution and a BalanCD®- polyethyleneimine (PEI) solution together at a 1:1 DNA:PEI mass ratio, which was diluted into pre-TFXN media (DMEM + 9.1% FBS) to form the TFXN media. Spent growth media was removed from cells and replaced with TFXN media and incubated at 37 °C, 5% CO2 incubator until media exchange about 20-24 hours after transfection with fresh TFXN media. Crude virus was harvested 28 - 32 hours after media exchange. All harvest samples were centrifuged at 200 x g for 5 minutes and processed for clarification by filtration.

[0401] Harvest and clarified harvest material from each of the three plasmid ratios were measured in the Jurkat Titer Assay and in the primary functional titer assay (primary T cell CAR expression assay) substantially as described in Example 1. As shown in FIG. 6, the maximum CD3+CAR+ frequency achieved by the crude viral vector produced in the upstream process using conditions with plasmid ratio #1 (0.046 VSV-G) and plasmid ratio #3 (0.15 VSV-G) were significantly higher (nearly 70%) than the previous Optimum DSP Yield “low VSV-G” ratio (less than 30%) conditions described in Examples 1 and 2.

[0402] The analytical measurements of vector performance shown above indicate that any of the three plasmid ratios tested would improve the vector performance for the process as compared to the “low VSV-G” plasmid ratio conditions described in Examples 1 and 2. However, as described in the Example 2 above, the plasmid ratio used in the transfection step can have an impact on downstream processing performance. To assess the impact on downstream performance, the clarified harvested material generated in upstream processes using the three new plasmid ratios was forward processed through downstream AEX chromatography to observe their effect on the elution profile for vector yield as well as residual protein (HCP,BSA), and residual DNA (dsDNA, plasmid) clearance. The NaCl concentration for elution was increased stepwise from 150 - 750 mM NaCl in 50 mM NaCl increments every 3 column volumes (CVs). Each 3 CV elution fraction (FXN) was collected separately and immediately diluted 5X into 25 mM Tris, pH 7.5. The flow through (FT) and elution FXNs were analyzed for Jurkat titer and residuals.

[0403] For all three plasmid ratios, the elution trends were similar for host cell proteins (HCP), with the majority of residual HCP eluting in the FXNs at or before 300 mM NaCl. This suggests the varying plasmid ratios in the conditions tested do not affect the elution profile for residual HCP.

[0404] FIG. 7 shows an overlay of Jurkat titer and residual dsDNA (Quant-iT™ PicoGreen™ dsDNA Assay Kit; ThermoFisher Scientific, Cat. No. P7589) for each vector elution fraction for upstream transfection Plasmid Ratio #1 (0.046 VSV-G) and Plasmid Ratio #3 (0.15 VSV-G). The Plasmid Ratio #2 performed similarly to Plasmid Ratio #1 (data not shown). The maximum Jurkat titer was observed in the 350 mM and 400 mM NaCl fraction for each plasmid ratio, suggesting the varying ratios do not lead to a shift in the vector elution profile. Plasmid Ratio #3 (0.15 VSV-G amount) showed the highest Jurkat titer out of the three proposed plasmid ratios. The other two plasmid ratios showed similar Jurkat titers to each other. For dsDNA elution, the run with Plasmid Ratio #3 (0.15 VSV-G) showed a peak between 350 mM - 450 mM NaCl, which was sharper than the run with Plasmid Ratio #1. The runs with Plasmid Ratio #1 (0.046 VSV-G) and Plasmid Ratio #2 (0.1 VSV-G) showed a similar trend, although a peak in dsDNA concentration at 550 mM NaCl was observed for Plasmid Ratio #2 (0.1 VSV-G; data not shown). Each run showed a peak co-eluting with the vector in the 400 and 450 mM NaCl fractions, however the run with Plasmid Ratio #3 (0.15 VSV-G) showed a much higher concentration of dsDNA in those fractions. This plasmid ratio contained the highest level of VSV-G (0.15 by mass fraction) of the three new plasmid ratios.

[0405] Due to high co-elution of dsDNA with the vector for Plasmid Ratio #3 (0.15 VSV- G), this ratio was eliminated. Clarified Harvest material prepared using Plasmid Ratio #1 (0.046 VSV-G) and Plasmid Ratio #2 (0.10 VSV-G) were each forward processed through AEX Chromatography step to confirm the wash and elution conditions chosen. The Plasmid Ratio #2 (0.10 VSV-G) eluate material had higher dsDNA per infectious titer unit compared with the Plasmid Ratio #1 (0.046 VSV-G) eluate material in this experiment. Due to this, Plasmid Ratio #2 (0.10 VSV-G) ratio was eliminated. The Plasmid Ratio #1 (0.046 VSV-G) AEX eluate material was forward processed through an ultrafiltration and diafiltration (UF/DF) step and a sterile filtration step. The overall process recovery achieved using Plasmid Ratio #1 (0.046 VSV-G) was 30%.

[0406] Without wishing to be bound by theory, it is thought that the dsDNA is bound to the vector in higher amounts when more VSVG plasmid is used for transfection since VSV-G is a “sticky” protein. Together, based on the titer data, CD3+CAR+ frequency data, and downstream performance, the results indicated that plasmid ratio #1 (0.046 VSV-G; Table E4) maintained downstream purification performance (through the AEX chromatography unit operation) best in these studies.

Table E4

Example 5: Transfection Reaction Scaling

[0407] A plasmid ratio identified by the mixture design of experiments (DOE) described in Example 1 was used to assess the improvement on transduction using different processes, such as due to differences in volumes of the transfection reaction. In this experiment, an exemplary plasmid ratio identified in Example 1 was used to assess the relationship between transfection volume in an upstream lentiviral manufacturing process on the harvest and performance of a lentiviral vector carrying an exemplary anti-CD19 chimeric antigen receptor (CAR) transgene. In the studies, the mass fractions and concentration of the four plasmids used in the transfection step were held constant while volume (see volume of transfection mixture as provided in Table E5) was varied. The plasmid ratio selected from the initial mixture DOE experiment was tested in multiple increasing scales.

[0408] HEK-293T/17 cells were transfected with an exemplary candidate plasmid ratio of four plasmids necessary for lentiviral vector production: transgene plasmid, Rev plasmid, VSV- G plasmid, and Gag-Pol plasmid. Cells were transfected at any of a small scale (IX), medium scale (4.6X) and large scale (32X) as shown in the Table E5 below.

Table E5: Volume of Each TFXN (transfection) Complex per Condition.

[0409] HEK-293T/17 cells were thawed and expanded for several passages prior to seeding the cells for vector production. At seed for production, vessels were seeded with viable HEK- 293T/17 cells and expanded for about 3 days before transfection in a5% CO2 humidified incubator maintained at 37 °C. Before transfection, the DNA plasmid solutions were prepared in an appropriate sized container. The plasmids were added at a specific exemplary plasmid ratio (relative level of each plasmid as a predetermined mass fraction) to a pH neutral medium, and then were swirled to mix. A master mix was prepared by mixing together (i) the DNA plasmid solution and (ii) solution of pH neutral medium containing polyethyleneimine (PEI). The combined master mix containing transfection (TFXN) complexes of DNA:PEI were diluted directly into the pre-TFXN media (DMEM + 9.1% FBS) to form the TFXN media scaled for volume as described in Table E5.

[0410] The spent growth media was removed from each culture vessel (e.g., cell culture flask or cell culture vessel such as a bioreactor) and replaced with the TFXN media. Each cell culture vessel was maintained at 37 °C and media was exchanged at a set time after transfection with fresh TFXN media. After a set production duration following the media exchange, the media was harvested from each culture vessel.

[0411] Further assessment of the selected plasmid ratio at each of the three volumetric scales tested demonstrated consistent analytical results (including titer and p24; data not shown). Moreover, further testing revealed successful recoveries at larger scales and across sites. These results demonstrate the scalability of the manufacturing process and its successful use at larger scales. The results also support the robustness of method used to select the plasmid ratio and the selected plasmid ratio for use in the manufacturing process. [0412] The present invention is not intended to be limited in scope to the particular disclosed embodiments, which are provided, for example, to illustrate various aspects of the invention. Various modifications to the compositions and methods described will become apparent from the description and teachings herein. Such variations may be practiced without departing from the true scope and spirit of the disclosure and are intended to fall within the scope of the present disclosure.

VII. SEQUENCES

Claims

CLAIMS WHAT IS CLAIMED:

(b) harvesting the culture supernatant from each transfection reaction, and optionally clarifying the supernatant of the harvested culture to produce a clarified harvest; (c) determining lentiviral vector performance from each of the harvested culture supernatants or the clarified harvest; and

3. The method of claim 2, wherein the lentiviral vector performance is determined from both an analytical measure of a cell line titer and a primary cell titer.

(e) selecting a transfection mixture for an upstream processing method for producing a lentiviral vector based on the downstream purification performance that has been determined for the transfection mixture.

5. The method of claim 1 and 4, wherein the at least two helper plasmids encode at least one packaging viral gene and at least one envelope viral gene.

6. The method of claim 5, wherein the envelope viral gene is VSV-G.

7. The method of claim 5, wherein the packaging viral gene is rev, gag or pol or a combination of any of the foregoing.

8. The method of claim 7, wherein the mixture of plasmids comprises a packaging plasmid encoding gag and pol (Gag-Pol plasmid), an envelope (Env) plasmid encoding VSV-G, and a transfer plasmid encoding the transgene.

9. The method of any of claims 1-8, wherein the mixture of plasmids is a three- plasmid mixture, a four-plasmid mixture or a five-plasmid mixture.

10. The method of any of claims 1-9, wherein the mixture of plasmids is a three plasmid mixture composed of a packaging plasmid encoding gag and pol (Gag-Pol plasmid), an envelope (Env) plasmid encoding VSV-G, and a transfer plasmid encoding the transgene.

11. The method of any of claim 1-9, wherein the mixture of plasmids is a four- plasmid mixture composed of a packaging plasmid encoding gag and pol (Gag-Pol plasmid), a packaging plasmid encoding rev (Rev plasmid), an envelope (Env) plasmid encoding VSV-G, and a transfer plasmid encoding the transgene.

12. The method of any of claims 8-11, wherein the subset of transfection reactions each have a different mass fraction ratio of the packaging plasmid encoding Gag-Pol or the envelope plasmid encoding VSV-G relative to the other plasmids in the mixture of plasmids.

13. The method of any of claims 8-12, wherein the subset of transfection reactions each have a different mass fraction ratio of the packaging plasmid encoding Gag-Pol relative to the other plasmids in the mixture of plasmids.

14. The method of any of claims 8-13, wherein the subset of transfection reactions each have a different mass ratios of the envelope plasmid encoding VSV-G relative to the other plasmids in the mixture of plasmids.

15. The method of any of claims 8-14, wherein the subset of transfection reactions each have a different mass ratio of the packaging plasmid encoding Gag-Pol and a different mass ratio of the envelope plasmid encoding VSV-G, relative to the other plasmids in the mixture of plasmids.

16. The method of any of claims 4-15, wherein the subset of transfection mixtures are candidate transfection mixtures that were identified to have the highest levels of viral vector performance from among the plurality of transfection mixtures in (c).

17. The method of any of claims 1-16, wherein the plurality of transfection reactions is a design of experiments (DOE).

18. The method of any of claims 4, 7 and 9, wherein the mass fraction of the plasmid encoding VSV-G is varied in the plurality of transfection mixtures.

19. The method of any of claims 6, and 8-18 , wherein the mass fraction of the plasmid encoding VSV-G in each of the transfection mixtures is independently from 0.04 to 0.2.

20. The method of any of claims 6 and 8-19, wherein the mass fraction of the plasmid encoding VSV-G among each of the transfection mixtures is independently from 0.046 to 0.15.

21. The method of any of claims 7-20, wherein the mass fraction of each of one or more of the plasmids encoding rev, gag or pol is held constant among each of the transfection mixtures.

22. The method of any of claims 7-21, wherein the mass fraction of the plasmid encoding rev is held constant among each of the transfection mixtures.

23. The method of any of claims 7-22, wherein the mass fraction of the plasmid encoding rev among each of the transfection mixtures is independently from 0.04 to 0.08, optionally from 0.04 to 0.06.

24. The method of any of claims 7-23, wherein the mass fraction of the plasmid encoding rev among each of the transfection mixtures is held constant at about 0.049.

25. The method of any of claims 7-24, wherein the mass fraction of the plasmid encoding gag and/or pol, optionally gag and pol, is held constant among each of the transfection mixtures.

26. The method of any of claims 7-25, wherein the mass fraction of the plasmid encoding gag and/or pol, optionally gag and pol, among each of the transfection mixtures is independently from 0.1 to 0.25, optionally from 0.15 and 0.2.

27. The method of any of claims 7-26, wherein the mass fraction of the plasmid encoding gag and/or pol, optionally gag and pol, among each of the transfection mixtures is held constant at about 0.178.

28. The method of any of claims 1-27, wherein the mass fraction of the plasmid encoding the transgene is varied in the plurality of transfection mixtures.

29. The method of any of claims 1-28, wherein the mass fraction of the plasmid encoding the transgene among each of the transfection mixtures is independently from 0.47 to 0.82, optionally from 0.58 to 0.74.

30. The method of any of claims 1-29, wherein the plurality of transfection mixtures is 3 to 50, optionally 3 to 30.

31. The method of any of claims 1-30, wherein the candidate transfection mixture in (d) is identified using multiple-response optimization.

32. The method of claim 31, wherein the multiple-response optimization is characterized by a desirability function for each analytical measure of a cell line titer and a primary cell titer.

33. The method of claim 32, wherein the desirability function for primary cell titer is to be maximized over the cell line titer.

34. The method of claim 32, wherein the desirability function for primary cell titer and cell line titer are equal.

35. The method of claim 33 or claim 34, wherein the desirability function for primary cell titer is about 1.0.

36. The method of any of claims 1-35, wherein the primary cell titer is a functional titer determined by a primary cell transduction assay.

37. The method of claim 36, wherein the primary cell transduction assay comprises transducing target cells with the harvested culture supernatant or clarified harvest, incubating the transduced target cells under conditions for expression of the transgene, and analyzing expression of the transgene by the cells.

38. The method of claim 37, wherein cell surface expression of the transgene is analyzed by flow cytometry.

39. The method of claim 37 or claim 38, wherein the target cells are T cells and prior to the transducing the method comprises activating the T cells with a T cell stimulatory reagent, optionally wherein the T cell stimulatory reagent is an anti-CD3/anti-CD28 activation reagent.

40. The method of claim 39, wherein the T cells are primary cells selected from a subject, optionally wherein the subject is a healthy subject.

41. The method of claim 39 or claim 40, wherein the T cells are CD4+ T cells, CD8+

T cells or CD4+ and CD8+ T cells.

42. The method of any of claims 37-41, wherein the target cells are T cells and incubating the transduced target cells under conditions for expression of the transgene comprises incubating the cells in the presence of one or more T cell stimulatory recombinant cytokines, optionally IL-2 IL-7, IL- 15 or IL-21 or a combination of any of the foregoing.

43. The method of claim 42, wherein the incubating expands the T cells.

44. The method claim 42 or claim 43, wherein the incubating is for 2 to 10 days.

45. The method of any of claims 37-41, wherein the target cells are T cells and incubating the transduced target cells under conditions for expression of the transgene comprises incubating the cells in basal media without recombinant cytokines.

46. The method of claim 45, wherein the incubating is for 12 hours to 48 hours.

47. The method of any of claims 1-46, wherein the cell line titer is a functional titer or and/or an infectious titer.

48. The method of any of claims 32-47, wherein the desirability function of the cell line titer is less than 1.0, optionally 0.5.

49. The method of any of claims 32-47, wherein the desirability function of the cell line titer is about 1.0.

50. The method of any of claims 1-49, wherein the analytical measure of a cell line titer is an analytical measure in at least two cell line titer assays.

51. The method of claim 50, wherein the at least two cell line titer assays are at least one infectious titer and at least one functional titer.

52. The method of claim 50 or claim 51, wherein the desirability function of each of the at least two cell line titer is less than 1.0, optionally less than 0.5.

53. The method of claim 50 or claim 51, wherein the desirability function of each of the at least two cell line titer combined is about 1.0.

54. The method of any of claims 47-53, wherein the infectious titer is determined by an endpoint dilution assay (TCID50) or a qPCR lentivirus titer assay.

55. The method of any of claims 47-54, wherein the infectious titer is determined by a qPCR lentivirus titer assay.

56. The method of claim 55, wherein the qPCR comprises primers and probes for amplification of a region of the LTR of the genomic RNA.

57. The method of any of claim 47-53, wherein the functional titer is determined by a cell-based transduction assay.

58. The method of any of claims 57, wherein transduction is determined by measuring transgene expression by flow cytometry.

59. The method of any of claims 1-58, wherein the cell line titer assay is titer on an immortalized cell line.

60. The method of claim 59, wherein the immortalized cell line is a Jurkat cell line.

61. The method of any of claims 1-60, wherein the downstream purification is by chromatography .

62. The method of any of claims 1-61, wherein the downstream purification comprises chromatography and ultrafiltration/diafiltration (UF/DF).

63. The method of claim 61 and claim 62, wherein the chromatography is by a method selected from the group consisting of heparin affinity, gel filtration and anion-exchange (AEX).

64. The method of any of claims 61-63, wherein the chromatography is anion- exchange (AEX).

65. The method of any of claims 61-64, wherein the downstream purification further comprises sterile filtration.

66. The method of any of any of claims 1-65, wherein the downstream purification performance for selecting the candidate transfection mixture in (f) is elution profile for vector performance, residual protein, residual BSA, residual plasmid or host-cell DNA or a combination of any of the foregoing.

67. The method of claim 66, wherein elution profile for vector performance comprises testing a plurality of elution fractions for infectious titer of the viral vector, optionally wherein infectious titer is assessed using a cell-based transduction assay.

68. The method of any of claims 1-67, comprising optionally repeating steps (a)-(d) to identify different candidate transfection mixtures if the downstream purification performance is not acceptable.

69. The method of any of claims 1-68, wherein the method further comprises:

(g) varying one or more parameters in a downstream process for producing the lentiviral vector comprising the downstream purification, wherein the downstream process is carried out from material harvested from an upstream process using the selected candidate transfection mixture.

70. The method of claim 69, wherein the upstream processing method comprises:

(i) transfecting the host cells in a large-scale culture with the selected transfection mixture; (ii) harvesting the supernatant from the transfected culture; and

71. The method of any of claims 1-70, wherein the host cells are adherent cells.

72. The method of any of claims 1-70, wherein the host cells are suspension cells.

73. The method of any of claims 1-72, wherein the host cells are HEK293T cells or a derivative thereof, optionally wherein the host cells are HEK 293T/17 cells.

74. The method of any of claims 1-73, wherein an endonuclease is added to the culture supernatant prior to harvesting the supernatant from the transfected culture.

75. The method of claim 74, wherein the endonuclease is Benzonase.

76. The method of any of claims 1-75, wherein the transgene is a chimeric antigen receptor (CAR).

77. The method of claim 76, wherein the chimeric antigen receptor is a monospecific CAR.

78. The method of claim 77, wherein the chimeric antigen receptor is a bispecific CAR.

79. The method of any of claims 1-78, wherein the transfection agent is a cationic polymer.

80. The method of claim 79, wherein the cationic polymer is polyethylenimine (PEI).

81. The method of claim 80, wherein the mass ratio of the mixture of plasmid DNA to PEI is 5:1 to 1:5, optionally 3:1 to 1:3.

82. The method of claim 80 or claim 81, wherein the mass ratio of the mixture of plasmid DNA to PEI is 1:1.

83. The method of any of claims 80-82, wherein the mixture of plasmid DNA and PEI are provided as a transfection complex , optionally stabilized with fetal bovine serum (FBS) or human serum albumin (HSA).

84. A method of manufacturing a lentiviral vector, the method comprising:

(a) transiently transfecting host cells to produce a transfected culture with (i) a transfection mixture selected according to the method of any one of claims 1-83 and (ii) a transfection agent to make a transfected culture, and optionally:

(b) harvesting the supernatant from the transfected culture;

(c) clarifying the harvested culture supernatant by filtration;

(f) sterile filtering the lentiviral vector.

85. A method of manufacturing a lentiviral vector, the method comprising:

(b) harvesting the supernatant from the transfected culture;

(c) clarifying the harvested culture supernatant by filtration;

(d) capturing and concentrating the lentiviral vector from the harvested and clarified supernatant by anion exchange chromatography (AEX); (e) ultrafiltering and diafiltering the lentiviral vector using Tangential Flow Filtration

(TFF);

(f) sterile filtering the lentiviral vector.

86. The method of claim 85, wherein the mass fraction of the packaging plasmid encoding gag and pol is about 0.178, the mass fraction of the lentiviral packaging plasmid encoding rev is about 0.049, the mass fraction of the envelope plasmid encoding VSV-G is about 0.046, and the mass fraction of the transfer plasmid encoding the CAR is about 0.727.

87. The method of claim 85 or claim 86, wherein the AEX chromatography includes a wash with about 300 mM NaCl and an elution with about 450 mM NaCl.

88. The method of any of claims 84-86, wherein the transfection agent is a cationic polymer, optionally wherein the cationic polymer is polyethylenimine (PEI).

89. The method of any of claims 84-88, wherein clarifying the harvested culture supernatant is by membrane filtration.

90. The method of claim 89, wherein the membrane filtration is with a dual-layer filter.

91. The method of any of claims 84-90, wherein sterile filtering is by membrane filtration.

92. The method of claim 91, wherein sterile filtering is with a dual-layer filter.

93. The method of any of claims 84-92, wherein the host cells are HEK293 cells.

94. The method of claim 93, wherein the HEK293 cells are HEK-293T/17 cells.

95. The method of any of claims 84-94, wherein the CAR is an anti-CD19 CAR.

96. The method of any of claims 84-95, wherein the CAR contains in order from N- terminus to C-terminus: an extracellular antigen-binding domain that is the scFv set forth in SEQ ID NO: 43, the spacer set forth in SEQ ID NO:1, the transmembrane domain set forth in SEQ ID NO:8, the 4- IBB costimulatory signaling domain set forth in SEQ ID NO: 12, and the signaling domain of a CD3-zeta (CD3Q chain set forth in SEQ ID NO: 13.

97. The method of any of claims 84-96, wherein the CAR comprises the amino acid sequence set forth in SEQ ID NO:57 or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 57.

98. The method of any of claims 84-97, wherein the nucleotide sequence encoding the CAR comprises the sequence set forth in SEQ ID NO:58 or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 58.

99. The method of any of claims 4-98, wherein the volume of the transfection reaction is from 5 mL to 5000 mL or from about 5 mL to about 5000 mL

100. The method of any of claims 4-99, wherein the volume of the transfection reaction is from or from about 10 mL to 2000 mL.

101. The method of any of claims 4-100, wherein the volume of the transfection reaction from or from about 500 mL to 2000 mL.