BR112018069075B1 - Methods and compositions for transducing lymphocytes and their regulated expansion. - Google Patents

Abstract

This disclosure provides methods for genetically modifying lymphocytes and methods for performing adoptive cell therapy that include transducing T cells and/or NK cells without prior ex vivo stimulation. The methods typically include genetically modified signaling polypeptides that may include a lymphoproliferative element and/or a chimeric antigen receptor (CAR), for example, microenvironment-restricted CAR. Further examples of such genetically modified signaling polypeptides are provided herein, as are vectors, such as retroviral vectors, packaging cell lines, and methods for producing them. Additionally, recombinant retroviruses and methods for producing them are provided. Numerous controls are provided, including riboswitches that are controlled, for example, in vivo, by nucleoside analogs.

Description

CROSS-REFERENCE TO RELATED REQUESTS

[0001] This application claims the benefit of Provisional Application No. 62/390,093, filed March 19, 2016; Provisional Application No. 62/360,041, filed July 8, 2016; and Provisional Application No. 62/467,039, filed March 3, 2017. These applications are incorporated by reference herein in their entirety.

SEQUENCE LISTING

[0002] This application incorporates by reference the material from the electronic Sequence Listing filed concurrently with this document. The material in the electronic Sequence Listing is submitted as a text file (.txt) entitled “F1_001_Sequence_listing.txt” created on March 16, 2017, which has a file size of 230 KB, and is incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0003] This disclosure relates to the field of immunology or, more specifically, to the genetic modification of T lymphocytes or other immune cells and methods for producing retroviruses and controlling gene expression.

BACKGROUND TO THE REVELATION

[0004] Lymphocytes isolated from a subject (e.g., patient) can be activated in vitro and genetically modified to express synthetic proteins that enable redirected interconnection to other cells and environments based on embedded genetic programs. An example of such a synthetic protein is a chimeric antigen receptor (CAR). A CAR that is currently used is a fusion of an extracellular recognition domain (e.g., an antigen-binding domain), a transmembrane domain, and one or more intracellular signaling domains encoded by a replicating incompetent retrovirus.

[0005] Although retroviruses have shown efficacy in infecting non-dividing cells, resting CD4 and CD8 lymphocytes are refractory to gene transduction by these vectors. To overcome this difficulty, these cells are typically activated in vitro using stimulation reagents before genetic modification with the CAR gene vector can occur. After stimulation and transduction, the genetically modified cells are expanded in vitro and subsequently reintroduced into a lymphodepleted patient. Through antigen interconnection in vivo, the intracellular signaling portion of CAR can initiate an activation-related response in an immune cell and the release of cytolytic molecules to induce tumor cell death.

[0006] Current methods require extensive manipulation and fabrication of proliferative T cells outside the body before their reinfusion into the patient, as well as lymphodepletion chemotherapy to release cytokines and deplete competing receptors to facilitate T cell engraftment. Such CAR therapies can no longer be controlled for in vivo spread rate once introduced into the body, nor can they be safely targeted to targets that are also expressed outside the tumor. As a result, CAR therapies today are typically infused from cells expanded ex vivo for 12 to 28 days using doses of 1 x 10⁵ to 1 x 10⁸ cells/kg and are targeted to targets, for example, tumor targets, for which on-target and off-tumor toxicity is generally acceptable. These relatively long ex vivo expansion times create problems of cell viability and sterility, as well as sample identity, in addition to scalability challenges. Therefore, there is a significant need for a more effective, scalable T-cell or NK-cell therapy.

SUMMARY OF THE INVENTION

[0007] In one aspect, a method is provided herein for genetically modifying and expanding lymphocytes of a subject comprising: A. placing resting T cells and/or NK cells of the subject ex vivo without requiring prior ex vivo stimulation into contact with recombinant retroviruses comprising: 1. a pseudotyping element on their surface that has the ability to bind to a T cell and/or NK cell and facilitate the fusion of the recombinant retrovirus membrane to the same; and ii. a polynucleotide comprising one or more transcriptional units functionally linked to an active promoter on T cells and/or NK cells, wherein one or more transcriptional units encode a first genetically modified signaling polypeptide regulated by an in vivo control element, wherein said first genetically modified signaling polypeptide comprises a lymphoproliferative element, wherein said contact facilitates the transduction of at least some of the resting T cells and/or NK cells by the recombinant retroviruses, thereby producing genetically modified T cells and/or NK cells; B. Introduce genetically modified T cells and/or NK cells into the subject; and expose genetically modified T cells and/or NK cells in vivo to a compound that binds to the control element in vivo to affect the expression of the first genetically modified signaling polypeptide and promote and/or enhance lymphocyte expansion, engraftment, and/or persistence in vivo, thus genetically modifying and expanding the subject's lymphocytes. In the illustrative embodiments, transduction is performed without ex vivo stimulation.

[0008] In the above aspect and in any of the method aspects for genetically modifying and expanding lymphocytes or for performing cell therapy in this document, if not mentioned in the broader aspect, in certain embodiments, the polynucleotide further comprises a transcriptional unit encoding a second genetically modified signaling polypeptide comprising a first chimeric antigen receptor comprising an antigen-specific targeting region (ASTR), a transmembrane domain and an intracellular activation domain.

[0009] In another aspect, a method for performing adoptive cell therapy in a subject is provided in this document, comprising: A. collecting blood from the subject; B. placing resting T cells and/or NK cells from the subject's blood ex vivo in contact with recombinant retroviruses, wherein the recombinant retroviruses comprise i. a pseudotyping element on their surface that has the ability to bind to a T cell and/or NK cell and facilitate membrane fusion of the recombinant retroviruses to them; and ii. a polynucleotide comprising one or more transcriptional units functionally linked to an active promoter on T cells and/or NK cells, wherein one or more transcriptional units encode a first genetically modified signaling polypeptide comprising a lymphoproliferative element whose expression is regulated by an in vivo control element and a second genetically modified signaling polypeptide comprising a chimeric antigen receptor comprising an antigen-specific targeting region (ASTR), a transmembrane domain and an intracellular activation domain, wherein said contact results in at least some of the resting T cells and/or NK cells becoming genetically modified; andC. Reintroducing genetically modified T cells and/or NK cells into the subject, wherein the expansion, engraftment, and/or persistence of the genetically modified T cells and/or NK cells occurs in vivo within the subject, and wherein the method between blood collection and reintroduction of the genetically modified T cells and/or NK cells is performed in no more than 24 hours, thus performing adoptive cell therapy in the subject.

[0010] A method for performing adoptive cell therapy in a subject is provided in another aspect of this document, comprising: A. collecting blood from a subject; B. isolating peripheral blood mononuclear cells (PBMCs) comprising resting T cells and/or resting NK cells; C. placing the subject's resting T cells and/or resting NK cells ex vivo in contact with recombinant retroviruses, wherein the recombinant retroviruses comprise a pseudotyping element on their surface that has the ability to bind to a resting T cell and/or NK cell and facilitate the fusion of the recombinant retrovirus membrane to the same, wherein said contact facilitates the transduction of the resting T cells and/or NK cells by the recombinant retroviruses, thereby producing genetically modified T cells and/or NK cells; and D. reintroducing the genetically modified cells into the subject within 24 hours of collecting blood from the subject, thereby performing adoptive cell therapy in the subject.

[0011] In another aspect of this document, a method for transducing resting lymphocytes from a subject is provided, comprising placing resting T cells and/or resting NK cells from a subject ex vivo in contact with recombinant retroviruses, wherein the recombinant retroviruses comprise a pseudotyping element on their surface that has the ability to bind to a resting T cell and/or resting NK cell and facilitate the fusion of the recombinant retrovirus membrane to the same, wherein said contact facilitates the transduction of the resting T cells and/or NK cells by the recombinant retroviruses, thus producing genetically modified T cells and/or NK cells. In illustrative embodiments of this aspect, at least 10, 20 or 25% of the resting T cells and/or NK cells or between 10% and 70% or 20% and 50% of the T cells and/or NK cells are transduced as a result of the process.

[0012] A method for transducing resting T cells and/or resting NK cells from isolated blood is provided in another aspect of this document, comprising: A. collecting blood from a subject; B. isolating peripheral blood mononuclear cells (PBMCs) comprising resting T cells and/or resting NK cells; C. placing the subject's resting T cells and/or resting NK cells ex vivo in contact with recombinant retroviruses, wherein the recombinant retroviruses comprise a pseudotyping element on their surface that has the ability to bind to a resting T cell and/or resting NK cell and facilitate the fusion of the recombinant retrovirus membrane to the same, wherein said contact facilitates the transduction of at least 5% of the resting T cells and/or resting NK cells by the recombinant retroviruses, thereby producing genetically modified T cells and/or NK cells, thereby transducing resting T cells and/or NK cells.

[0013] In one aspect, a recombinant retrovirus is provided in this document comprising: A. one or more pseudotyping elements capable of binding to a T cell and/or an NK cell and facilitating the fusion of the recombinant retrovirus membrane to them; B. a polynucleotide comprising one or more transcriptional units functionally linked to an active promoter on T cells and/or NK cells, wherein one or more transcriptional units encode a first genetically modified signaling polypeptide comprising a chimeric antigen receptor comprising an antigen-specific targeting region, a transmembrane domain and an intracellular activation domain and a second genetically modified signaling polypeptide comprising a lymphoproliferative element; wherein the expression of the first genetically modified signaling polypeptide and/or the second genetically modified signaling polypeptide is regulated by an in vivo control element; and C. an activation element on its surface, wherein the activation element has the ability to bind to a T cell and/or NK cell and is not encoded by a polynucleotide in the recombinant retrovirus.

[0014] In another aspect, a recombinant retrovirus is provided in this document comprising: A. a pseudotyping element on its surface that has the ability to bind to a T cell and/or NK cell and facilitate the fusion of the recombinant retrovirus membrane to the same, wherein said pseudotyping element comprises cytoplasmic domain deletion variants of a measles virus F polypeptide and/or a measles virus H polypeptide; B. a polynucleotide comprising one or more transcriptional units functionally linked to an active promoter on T cells and/or NK cells, wherein one or more transcriptional units encode a first genetically modified signaling polypeptide comprising a chimeric antigen receptor comprising an antigen-specific targeting region, a transmembrane domain and an intracellular activation domain and a second genetically modified signaling polypeptide comprising a constitutively active IL-7 receptor mutant; wherein the expression of the IL-7 receptor mutant is regulated by a riboswitch that binds to a nucleoside analog antiviral drug; and C. a polypeptide with the ability to bind to CD3 and a polypeptide with the ability to bind to CD28, wherein said polypeptides are expressed on the surface of a recombinant retrovirus; have the ability to bind to a T cell and/or NK cell; and are not encoded by a polynucleotide in the recombinant retrovirus. In the illustrative embodiments of this aspect, the binding of the nucleoside analog antiviral drug to the riboswitch increases the expression of the IL-7 receptor mutant.

[0015] In any of the method or composition aspects provided herein, unless already mentioned in the broader aspect, the recombinant retrovirus or recombinant retroviruses comprise or additionally comprise an activation element on their surface that has the capability to activate a resting T cell and/or a resting NK cell.

[0016] In any of the methods or compositions in this document that mention a T cell and/or an NK cell, or a resting T cell or a resting NK cell, in certain illustrative embodiments, the cell is a T cell.

[0017] Typically, the recombinant retrovirus in any of the methods and compositions provided in this document is replication-defective. That is, the virus cannot replicate. In the illustrative embodiments, the retrovirus is a lentivirus, such as a replication-defective HIV lentivirus.

[0018] In the illustrative embodiments of any of the aspects of the methods for genetically modifying and expanding lymphocytes or for performing adoptive cell therapy in this document, or similar methods, between 10% and 75%, or 10% and 70%, or 10% and 60%, or 10% and 50%, or 10% and 25%, or 20% and 75%, or 20% and 50%, or at least 10%, 20% or 25% of resting T cells are transduced and between 0% and 75% of NK cells are transduced. In other modalities, between 5% and 80%, or 10% and 80%, or 10% and 70%, or 10% and 60%, or 10% and 50%, or 10% and 25%, or 10% and 20%, or 20% and 50% of resting NK cells are transduced.

[0019] In the illustrative embodiments of any of the aspects of the methods for genetically modifying and expanding lymphocytes or for performing adoptive cell therapy in this document, or similar methods or any compositions provided in this document, unless explicitly mentioned in the broader aspect, the expression of said second genetically modified signaling polypeptide is regulated by the in vivo control element.

[0020] In the illustrative embodiments of any of the aspects of the methods for genetically modifying and expanding lymphocytes or for performing cell therapy in this document, or similar methods, if not explicitly mentioned in the broader aspect of the method, contact may be performed for between 15, 30 or 45 minutes or 1, 2, 3, 4, 5, 6, 7 or 8 hours at the lower end of the range, and between 6, 8, 10, 12, 18, 24, 36, 48 and 72 hours at the upper end of the range. For example, in the illustrative embodiments, contact is performed for between 2 and 24 hours, or between 4 and 12 hours, or between 4 and 8 hours.

[0021] In illustrative embodiments of any aspect of the methods for genetically modifying and expanding lymphocytes or for performing adoptive cell therapy in this document, or similar methods, if not explicitly mentioned in the broader aspect, the method may further comprise exposing genetically modified T cells and/or NK cells in vivo to a compound that binds to the control element in vivo to affect the expression of the first genetically modified signaling polypeptide and, optionally, the second genetically modified signaling polypeptide and to promote lymphocyte expansion, engraftment and/or persistence in vivo.

[0022] In the illustrative embodiments of any aspect of the methods for genetically modifying and expanding lymphocytes or for performing adoptive cell therapy in this document, or similar methods, if not explicitly mentioned in the broader aspect, the genetically modified T cells and/or NK cells undergo 8, 7, 6, 5, 4, 3 or fewer ex vivo cell divisions before being introduced or reintroduced into the subject.

[0023] In the illustrative embodiments of any aspect of the methods for genetically modifying and expanding lymphocytes or for performing cell therapy in this document, or similar methods, if not explicitly mentioned in the broader aspect, the expansion, engraftment and/or persistence of genetically modified T cells and/or NK cells in vivo depend on the presence or absence of the control element-binding compound in vivo and, in the illustrative embodiments, depend on the presence of the control element-binding compound in vivo.

[0024] In illustrative embodiments of any aspect of the methods for genetically modifying and expanding lymphocytes or for performing adoptive cell therapy in this document, or similar methods, if not explicitly mentioned in the broader aspect, the subject is not exposed to a lymphodepleting agent within 7, 14, or 21 days of making contact, during contact, and/or within 7, 14, or 21 days after the modified T cells and/or NK cells are introduced into the subject. In other embodiments, the subject is not exposed to a lymphodepleting agent during contact.

[0025] In the illustrative embodiments of any of the aspects of the methods for genetically modifying and expanding lymphocytes or for performing cell therapy in this document, or similar methods, if not explicitly mentioned in the broader aspect, resting T cells and/or resting NK cells are in contact with recombinant retroviruses for between 15 minutes and 12 hours.

[0026] In illustrative embodiments of any aspect of the methods for genetically modifying and expanding lymphocytes or for performing adoptive cell therapy in this document, or similar methods, if not explicitly mentioned in the broader aspect, the method additionally includes the step of separating recombinant retroviruses from T cells and/or NK cells after contact but before introduction. In illustrative embodiments of any aspect of the methods for genetically modifying and expanding lymphocytes or for performing cell therapy in this document, or similar methods, if not explicitly mentioned in the broader aspect, said exposure step comprises administering a dose of the compound to the subject before or during contact and/or after the genetically modified T cells and/or NK cells have been introduced into the subject.

[0027] In illustrative embodiments of any aspect of the methods for genetically modifying and expanding lymphocytes or for performing adoptive cell therapy in this document, or similar methods, if not explicitly mentioned in the broader aspect, the method comprises collecting blood comprising T cells and/or NK cells from the subject prior to ex vivo contact of the T cells and/or NK cells with recombinant retroviruses, and wherein introduction is reintroduction. For example, between 20 and 250 ml of blood are withdrawn from the subject.

[0028] In the illustrative embodiments of any of the aspects of the methods for genetically modifying and expanding lymphocytes or for performing cell therapy in this document, or similar methods, if not explicitly mentioned in the broader aspect, no more than 8, 12, 24 or 48 hours elapse between the time the blood is collected from the subject and the time the modified T cells and/or NK cells are reintroduced into the subject.

[0029] In the illustrative embodiments of any of the aspects of the methods for genetically modifying and expanding lymphocytes or for performing cell therapy in this document, or similar methods, if not explicitly mentioned in the broader aspect, between 4 or 8 hours at the lower end and 12, 24, 36, or 48 hours at the upper end of the range elapse between the time the blood is collected from the subject and the time the modified T cells and/or NK cells are reintroduced into the subject.

[0030] In the illustrative embodiments of any of the aspects of the methods for genetically modifying and expanding lymphocytes or for performing adoptive cell therapy in this document, or similar methods, if not explicitly mentioned in the broader aspect, all steps after the blood is collected and before the blood is reintroduced are performed in a closed system in which a person monitors the closed system throughout the processing. In another embodiment, all steps after the blood is collected and before the blood is reintroduced are performed in a closed system that remains in the same environment as the subject.

[0031] In illustrative embodiments of any of the methods and compositions provided herein that include one or more genetically modified signaling polypeptides, unless otherwise specified in the broader aspect, a genetically modified signaling polypeptide comprises or further comprises an antigen-specific targeting region (ASTR) and a transmembrane domain connecting the ASTR to the lymphoproliferative element. The ASTR of this genetically modified signaling polypeptide has the ability to bind to a first tumor antigen and, when present, the ASTR of the second genetically modified signaling polypeptide has the ability to bind to a second tumor antigen. In illustrative embodiments, the first genetically modified signaling polypeptide and/or the second genetically modified signaling polypeptide further comprise a co-stimulatory domain. Furthermore, the first genetically modified signaling polypeptide and/or the second genetically modified signaling polypeptide further comprise a stem. Furthermore, the first genetically modified signaling polypeptide further comprises an intracellular activation domain. The intracellular activation domain in the first genetically modified signaling polypeptide and/or the second genetically modified signaling polypeptide may be derived from CD3 zeta.

[0032] In illustrative embodiments of any of the methods and compositions provided herein that include a lymphoproliferative element, the lymphoproliferative element may comprise a T cell survival motif. The T cell survival motif may comprise all or a functional fragment of the IL-7 receptor, IL-15 receptor, or CD28. In other embodiments, the lymphoproliferative element may include a cytokine or cytokine receptor polypeptide or a fragment thereof comprising a signaling domain. For example, the lymphoproliferative element may comprise an interleukin polypeptide covalently linked to its cognate interleukin receptor polypeptide via a binder. Alternatively, the lymphoproliferative element may be an intracellular signaling domain of an IL-7 receptor, an intracellular signaling domain of an IL-12 receptor, an intracellular signaling domain of IL-23, an intracellular signaling domain of IL-27, an intracellular signaling domain of an IL-15 receptor, an intracellular signaling domain of an IL-21 receptor, or an intracellular signaling domain of a transforming growth factor β (TGFβ) decoy receptor. In other illustrative embodiments, the lymphoproliferative element is constitutively active. Furthermore, the lymphoproliferative element may include a mutated IL-7 receptor or a fragment thereof, which may additionally include a constitutively active mutated IL-7 receptor or a constitutively active fragment thereof.

[0033] In illustrative embodiments of any of the methods and compositions provided herein that include a recombinant retrovirus or recombinant retroviruses, unless explicitly cited in the broader aspect, the recombinant retroviruses may comprise on their surface an activation element comprising: A. a membrane-bound polypeptide capable of binding to CD3; and/or B. a membrane-bound polypeptide capable of binding to CD28.

[0034] Furthermore, the membrane-bound polypeptide capable of binding to CD3 is a polypeptide capable of binding to CD3 that can be fused to a heterologous GPI anchor-binding sequence, and the membrane-bound polypeptide capable of binding to CD28 can be a polypeptide capable of binding to CD28 that is fused to a heterologous GPI anchor-binding sequence. In some embodiments, the membrane-bound polypeptide capable of binding to CD28 is CD80, CD86, or a functional fragment thereof that has the ability to induce CD28-mediated activation of Akt, such as the extracellular domain of CD80.

[0035] In illustrative embodiments of any of the methods and compositions provided herein that include recombinant retroviruses, the membrane-bound polypeptide capable of binding to CD3 may be an anti-CD3 scFV linked to a CD14 GPI anchor binding sequence, and the membrane-bound polypeptide capable of binding to CD28 may be CD80, or the extracellular domain thereof, linked to a CD16B GPI anchor binding sequence. In illustrative embodiments of any of the methods and compositions provided herein that include recombinant retroviruses, the recombinant retroviruses may comprise, on their surface, an anti-CD3 scFV linked to a CD14 GPI anchor binding sequence, CD80, or the extracellular domain thereof, linked to a CD16B GPI anchor binding sequence and an IL-7 fusion polypeptide, or an active fragment thereof, and DAF comprising a GPI anchor binding sequence. In the illustrative embodiments of any of the methods and compositions provided herein that include recombinant retrovirus, IL-7, or an active fragment thereof, and DAF fusion, the anti-CD3 scFV and CD80, or extracellular domain thereof, each comprise a DAF signaling sequence.

[0036] In illustrative embodiments of any of the methods and compositions provided herein that include a recombinant retrovirus or recombinant retroviruses, unless explicitly cited in the broader aspect, the recombinant retroviruses may comprise on their surface a membrane-bound cytokine. The membrane-bound cytokine may be IL-7, IL-15, or an active fragment thereof. In other embodiments, the membrane-bound cytokine is a fusion polypeptide of IL-7, or an active fragment thereof, and DAF. For example, the fusion polypeptide may comprise the DAF signaling sequence (nucleotides 1 to 31 of SEQ ID NO:107), IL-7 without its signaling sequence (nucleotides 32 to 187 of SEQ ID NO:107), and a DAF fragment that includes its GPI anchor binding sequence (nucleotides 188 to 527 of SEQ ID NO:107).

[0037] In illustrative embodiments of any of the method and composition aspects provided herein, the pseudotyping element may comprise one or more heterologous envelope proteins. In other examples, the pseudotyping element may include one or more viral polypeptides recognized by T cells. The one or more pseudotyping elements may comprise a polypeptide of Measles Virus F, a polypeptide of Measles Virus H, and/or a fragment thereof. The one or more pseudotyping elements may be cytoplasmic domain deletion variants of a polypeptide of Measles Virus F and/or a polypeptide of Measles Virus H.

[0038] In illustrative embodiments of any of the methods and compositions provided herein that include the in vivo control element, the in vivo control element is the lymphoproliferative element, wherein the lymphoproliferative is inactive or less active in promoting the proliferation of T cells and/or NK cells in the absence of the compound, and wherein the compound is a molecular chaperone that binds to the lymphoproliferative element and induces the activity of the lymphoproliferative element.

[0039] In the illustrative embodiments of any of the methods and compositions provided herein that include the in vivo control element, the in vivo control element may be a polynucleotide comprising a riboswitch. The riboswitch may have the ability to bind to a nucleoside analog, and the compound that binds to the in vivo control element is the nucleoside analog. The nucleoside analog may be an antiviral agent. The antiviral agent may be acyclovir or penciclovir.

[0040] In illustrative embodiments of any of the methods and compositions provided herein that include a genetically modified signaling polypeptide, which includes an ASTR, the ASTR of either or both genetically modified signaling polypeptides may bind to a tumor-associated antigen. In some illustrative embodiments, the antigen-specific targeting region of the second genetically modified polypeptide is a microenvironment-restricted antigen-specific targeting region.

[0041] In illustrative embodiments of any of the methods and compositions provided herein that include a recombinant retrovirus or recombinant retroviruses, if not explicitly cited in the broader aspect, the recombinant retroviruses may encode a recognition domain for an approved monoclonal antibody biologic. In some embodiments, the recognition domain is expressed in the same transcript as the chimeric antigen receptor and in which the recognition domain is separated from the chimeric antigen receptor by a ribosome cleavage and/or jump signal. The ribosome cleavage and/or jump signal may be 2A-1. The recognition domain may include a polypeptide that is recognized by an EGFR-recognizing antibody or an epitope thereof. The recognition domain may be an EGFR mutant that is recognized by an EGFR antibody and expressed on the surface of transduced T cells and/or NK cells as another control mechanism provided herein. In related embodiments, the recognition domain may include a polypeptide that is recognized by an antibody that recognizes EGFR or an epitope thereof.

[0042] In any of the methods or compositions provided herein that include a lymphoproliferative element, the lymphoproliferative element may be a miRNA or shRNA that stimulates the STAT5 pathway or inhibits the SOCS pathway. For example, said miRNA or shRNA is a miRNA that binds to a nucleic acid encoding a protein selected from the group consisting of: ABCG1, SOCS, TGFbR2, SMAD2, cCBL, and PD1. In the illustrative embodiments for any of the recombinant retroviruses or transduced cells provided herein or methods that include them, such recombinant retroviruses or transduced cells may encode a miRNA or shRNA, for example, within an intron, in some embodiments, 1, 2, 3, or 4 embodiments that bind to nucleic acids encoding one or more of the following target genes expressed in endogenous T cells: PD-1; CTLA4; TCR alpha; TCR beta; CD3 zeta; SOCS; SMAD2; miR-155; IFN gamma; cCBL; TRAIL2; PP2A; or ABCG1. For example, in one embodiment, a combination of the following miRNAs can be included in a recombinant retrovirus or transduced cell genome: TCR alpha, CD3 zeta, IFN gamma, and PD-1; and, in another embodiment, SOCS 1, IFN gamma, TCR alpha, and CD3 zeta.

[0043] In the illustrative embodiments of any of the methods and compositions provided in this document, recombinant retroviruses, mammalian cells and/or packaging cells may comprise a Vpx polypeptide. The Vpx polypeptide may be, for example, a fusion polypeptide and, in some examples, especially in packaging cells, a membrane-bound Vpx polypeptide.

[0044] In any of the methods or compositions provided in this document, one or more pseudotyping elements may include an envelope protein from vesicular stomatitis virus (VSV-G), an envelope protein from feline endogenous virus (RD114), an oncoretroviral amphotopic envelope protein, or an oncoretroviral ecotropic envelope protein, or functional fragments thereof.

[0045] There are provided in this document in another aspect a genetically modified T cell and/or NK cell comprising: a. a first genetically modified signaling polypeptide comprising a lymphoproliferative element; and b. a second genetically modified signaling polypeptide comprising a chimeric antigen receptor comprising an antigen-specific targeting region (ASTR), a transmembrane domain and an intracellular activation domain.

[0046] In any of the methods provided herein that include a mammalian packaging cell, including an aspect of a recombinant retrovirus packaging system, or a method for producing a recombinant retrovirus, for example, the packageable RNA genome is encoded by a polynucleotide functionally linked to a promoter, wherein said promoter is constitutively active or inducible by the first transactivator or the second transactivator. The packageable RNA genome may be encoded by a polynucleotide functionally linked to a promoter, wherein said promoter is inducible by the second transactivator. A promoter used herein to trigger the expression of the first and/or second genetically modified signaling polypeptides is typically active in target cells, for example, lymphocytes, PBLs, T cells and/or NK cells, but, in the illustrative embodiments, is not active in the packaging cell line. The second transactivator may regulate the expression of an activation element with the ability to bind to and activate the target cell. In any of the methods provided in this document that involve a mammalian packaging cell, including an aspect of a recombinant retrovirus packaging system, or a method for producing a recombinant retrovirus, for example, a packageable RNA genome, in some embodiments, the expression of the packageable RNA genome can be regulated by the second transactivator.

[0047] Furthermore, the packageable RNA genome may comprise, from 5' to 3': 1.) a 5' long terminal repeat or an active fragment thereof; 2.) a nucleic acid sequence encoding a cis retroviral acting RNA packaging element; 3.) a nucleic acid sequence encoding a first target polypeptide; 4.) a promoter that is active in the target cell; and 5.) a 3' long terminal repeat or an active fragment thereof.

[0048] In some embodiments, the nucleic acid sequence encoding the first target polypeptide is in reverse orientation to an RNA encoding retroviral components for packaging and assembly.

[0049] In any of the methods provided herein that include a mammalian packaging cell, including an aspect of a recombinant retrovirus packaging system, or a method for producing a recombinant retrovirus, for example, the first target polypeptide comprises a first genetically modified signaling polypeptide, wherein said first genetically modified signaling polypeptide comprises a lymphoproliferative element. The packageable RNA genome may further comprise a nucleic acid sequence encoding a second target polypeptide. The second target polypeptide may comprise a second genetically modified signaling polypeptide including a chimeric antigen receptor comprising: 1.) a first antigen-specific targeting region; 2.) a first transmembrane domain; and 3.) a first intracellular activation domain.

[0050] In any of the methods provided in this document that include a mammalian packaging cell, including an aspect of recombinant retrovirus packaging, or a method for producing a recombinant retrovirus, for example, the mammalian cell, for example, the packaging cell may include a nucleic acid sequence encoding Vpx, for example, in the second or in an optional third transcriptional unit or in an additional transcriptional unit that is functionally linked to the first inducible promoter. The mammalian cell, which may be a packaging cell, may be a 293 cell.

[0051] In any of the methods provided in this document that involve a mammalian packaging cell, including an aspect of recombinant retrovirus packaging, or a method for producing a recombinant retrovirus, a first ligand may be rapamycin and a second ligand may be tetracycline or doxorubicin, or the first ligand may be tetracycline or doxorubicin and the second ligand may be rapamycin.

[0052] In some respects, a cell that has been transduced with any of the recombinant retroviruses provided herein is provided. The cell may be, for example, a lymphocyte, such as a T cell or NK cell. The cell, in the illustrative embodiments, is a human cell.

[0053] One aspect provided in this document is a method for expanding modified T cells and/or NK cells in a subject, wherein said method comprises: a.) placing isolated resting T cells and/or resting NK cells obtained from said subject in contact with recombinant retrovirus of any of the embodiments disclosed in this document; b.) introducing the genetically modified T cells and/or NK cells into the subject; and c.) providing an effective amount of acyclovir, an acyclovir prodrug, penciclovir or a penciclovir prodrug to said subject, wherein said modified T cells and/or NK cells proliferate in said subject upon administration of acyclovir, an acyclovir prodrug, penciclovir or a penciclovir prodrug, thereby expanding the modified T cells and/or NK cells in the subject.

[0054] In another aspect, a method is provided herein for interrupting the expansion, engraftment and/or persistence of modified T cells and/or NK cells in a subject, said method comprising: a.) placing isolated quiescent T cells and/or resting NK cells obtained from said subject in contact with recombinant retrovirus of any of the embodiments disclosed herein; b.) introducing the modified T cells and/or NK cells into the subject; c.) administering an effective amount of acyclovir, an acyclovir prodrug, penciclovir or a penciclovir prodrug to said subject to expand the modified T cells and/or NK cells in the subject, wherein said modified T cells and/or NK cells proliferate in said subject upon administration of acyclovir, an acyclovir prodrug, penciclovir or a penciclovir prodrug, thereby expanding the modified PBLs in the subject; ed.) to interrupt the administration of acyclovir, an acyclovir prodrug, penciclovir or a penciclovir prodrug, whereby said T cell and/or modified NK cells cease proliferation in said subject upon interruption of the administration of acyclovir, an acyclovir prodrug, penciclovir or a penciclovir prodrug, thereby controlling the expansion, engraftment and/or persistence of the T cell and/or modified NK cells in the subject.

[0055] In another aspect, a method for treating cancer in a subject is provided herein, wherein the method comprises: a. placing isolated quiescent T cells and/or resting NK cells obtained from said subject in contact with the recombinant vector of any of the embodiments disclosed herein; b. introducing the genetically modified T cells and/or NK cells into the subject; and c. administering an effective amount of acyclovir, an acyclovir prodrug, penciclovir or a penciclovir prodrug to said subject to expand the modified T cells and/or NK cells in the subject, wherein said modified T cells and/or NK cells proliferate in said subject upon administration of acyclovir, an acyclovir prodrug, penciclovir or a penciclovir prodrug, and wherein the chimeric antigen receptor on said modified T cells and/or NK cells binds to cancer cells in said subject, thereby treating the cancer in the subject.

[0056] In another aspect, there are provided in this document a transduced T cell and/or NK cell comprising a recombinant polynucleotide comprising one or more transcriptional units functionally linked to an active promoter in T cells and/or NK cells, wherein one or more transcriptional units encode a first genetically modified signaling polypeptide regulated by an in vivo control element, wherein said first genetically modified signaling polypeptide comprises a constitutively active IL-7 receptor mutant, and wherein the in vivo control element has the ability to bind and/or is designed and/or configured to bind to a compound in vivo.

[0057] In another aspect, a retroviral packaging system is provided in this document comprising: a mammalian cell comprising: A. a first transactivator expressed from a constitutive promoter and capable of binding to a first ligand and a first inducible promoter to affect the expression of a functionally linked nucleic acid sequence in the presence versus absence of the first ligand; B. a second transactivator capable of binding to a second ligand and a second inducible promoter and affecting the expression of a functionally linked nucleic acid sequence in the presence versus absence of the second ligand; and C. a packageable RNA genome for a retroviral particle, wherein the first transactivator regulates the expression of the second transactivator and a retroviral REV protein, wherein the second transactivator regulates the expression of a gag polypeptide, a pol polypeptide, and one or more pseudotyping elements with the ability to bind to a target cell and facilitate membrane fusion to it, and wherein the retroviral proteins are derived from a retrovirus. Embodiments of this aspect may include any of the embodiments provided herein for the elements mentioned in other aspects.

[0058] In another aspect, a method for producing a recombinant retrovirus is provided in this document comprising: A. cultivating a population of packaging cells to accumulate a first transactivator, wherein the packaging cells comprise the first transactivator expressed from a first constitutive promoter, wherein the first transactivator has the ability to bind to a first ligand and to an inducible first promoter to affect the expression of a nucleic acid sequence functionally linked thereto in the presence versus absence of the first ligand, and wherein the expression of a second transactivator and a retroviral REV protein is regulated by the first transactivator; B. incubating the population of packaging cells comprising the accumulated first transactivator in the presence of the first ligand to accumulate the second transactivator and the retroviral REV protein, wherein the second transactivator has the ability to bind to a second ligand and to an inducible second promoter to affect the expression of a nucleic acid sequence functionally linked thereto in the presence versus absence of the second ligand; eC. Incubate the population of packaging cells comprising the second transactivator and the accumulated retroviral REV protein in the presence of the second ligand, thereby inducing the expression of a gag polypeptide, a pol polypeptide, and one or more pseudotyping elements, thereby producing the recombinant retrovirus, in which a packable RNA genome is encoded by a polynucleotide functionally linked to a third promoter, in which said third promoter is constitutively active or inducible by the first transactivator or the second transactivator, and in which one or more pseudotyping elements have the ability to bind to a target cell and/or facilitate membrane fusion of the recombinant retrovirus to the same.

[0059] In some embodiments of aspects of the retroviral packaging system and method for producing a recombinant retrovirus provided in this document, the mammalian cell additionally comprises an activation element capable of binding to and activating a target cell, and the first transactivator regulates the expression of the activation element. The activation element is on the surface of the retrovirus and the activation element may include: a membrane-bound polypeptide capable of binding to CD3; and/or a membrane-bound polypeptide capable of binding to CD28. The membrane-bound polypeptide capable of binding to CD3 is a polypeptide capable of binding to CD3 that is fused to a heterologous GPI anchor binding sequence and the membrane-bound polypeptide capable of binding to CD28 is a polypeptide capable of binding to CD28 that is fused to a heterologous GPI anchor binding sequence. The membrane-bound polypeptide capable of binding to CD28, in some embodiments, comprises CD80, CD86, or a functional fragment thereof that has the ability to induce CD28-mediated activation of Akt, such as the extracellular domain of CD80. In other embodiments, the membrane-bound polypeptide capable of binding to CD3 is an anti-CD3 scFv linked to a CD14 GPI anchor binding sequence, and the membrane-bound polypeptide capable of binding to CD28 is CD80, or an extracellular fragment thereof, linked to a CD16B GPI anchor binding sequence.

[0060] In some embodiments of aspects of the retroviral packaging system and method for producing a recombinant retrovirus provided in this document, the mammalian cell additionally comprises a membrane-bound cytokine, and the first transactivator regulates the expression of the membrane-bound cytokine. The membrane-bound cytokine may be, for example, IL-7, IL-15, or an active fragment thereof. The membrane-bound cytokine in the embodiments may be a fusion polypeptide of IL-7, or an active fragment thereof, and DAF. For example, the fusion polypeptide may comprise the signaling sequence of DAF and IL-7 without its signaling sequence, followed by residues 36 to 525 of DAF.

[0061] In some embodiments of the aspects of the retroviral packaging system and method for producing a recombinant retrovirus provided in this document, the mammalian cell comprises, associated with its membrane, an activation element comprising an anti-CD3 scFV linked to a CD14 GPI anchor binding sequence and a CD80 linked, or an extracellular fragment thereof, to a CD16B GPI anchor binding sequence; and a membrane-bound cytokine comprising an IL-7 fusion polypeptide, or an active fragment thereof, and DAF comprising a GPI anchor binding sequence, wherein the first transactivator regulates the expression of each of the activation element and the membrane-bound cytokine. In some embodiments, the IL-7, or an active fragment thereof, and the DAF fusion, the anti-CD3 scFV and the CD80, or an extracellular fragment thereof, each comprise a DAF signaling sequence.

[0062] In some embodiments of the retroviral packaging system aspects and method for producing a recombinant retrovirus provided in this document, the mammalian cell additionally comprises a Vpx polypeptide. In these or other embodiments, one or more pseudotyping elements comprise one or more viral polypeptides recognized by T cells. One or more pseudotyping elements may comprise a Measles Virus F polypeptide, a Measles Virus H polypeptide, and/or a fragment thereof. In certain embodiments, one or more pseudotyping elements are cytoplasmic domain deletion variants of a Measles Virus F polypeptide and/or a Measles Virus H polypeptide.

[0063] In some embodiments of aspects of the retroviral packaging system and method for producing a recombinant retrovirus provided in this document, the packageable RNA genome is encoded by a polynucleotide functionally linked to a third promoter, wherein said third promoter is constitutively active or inducible by the first transactivator or the second transactivator. In the illustrative embodiments, the packageable RNA genome is encoded by a polynucleotide functionally linked to a third promoter, wherein said third promoter is inducible by the second transactivator.

[0064] In some embodiments of aspects of the retroviral packaging system and method for producing a recombinant retrovirus provided in this document, the packageable RNA genome further comprises, from 5' to 3': a) a 5' long terminal repeat or active fragment thereof; b) a nucleic acid sequence encoding a retroviral cis-acting RNA packaging element; c) a nucleic acid sequence encoding a first target polypeptide and an optional second target polypeptide; d) a fourth promoter functionally linked to the first target polypeptide and the optional second polypeptide, wherein said fourth promoter is active in the target cell but not active in the packaging cell line; and e) a 3' long terminal repeat or active fragment thereof.

[0065] In some embodiments of aspects of the retroviral packaging system and method for producing a recombinant retrovirus provided in this document, including the construct immediately above, the third promoter promotes transcription or expression in the opposite direction from transcription or expression promoted from the fourth promoter.

[0066] In some embodiments of aspects of the retroviral packaging system and method for producing a recombinant retrovirus provided in this document, the packageable RNA genome encodes the recombinant retrovirus of any embodiment disclosed in this disclosure, wherein the first target polypeptide and the second target polypeptide are the first genetically modified signaling polypeptide and the second genetically modified signaling polypeptide, respectively. In some embodiments, for example, the packageable RNA genome further comprises an in vivo control element functionally linked to the nucleic acid encoding the first genetically modified signaling polypeptide or the second genetically modified signaling polypeptide. The in vivo control element in the illustrative embodiments is a riboswitch. The riboswitch in the illustrative embodiments has the ability to bind to a compound, and the compound that binds to the in vivo control element is a nucleoside analog, and the nucleoside analog may be an antiviral drug, for example, acyclovir or penciclivr.

[0067] In some embodiments of aspects of the retroviral packaging system and method for producing a recombinant retrovirus provided in this document, the packageable RNA genome additionally comprises an intron comprising a polynucleotide encoding a miRNA or shRNA. The intron may be adjacent to and downstream of the fourth promoter.

[0068] In some embodiments of the retroviral packaging system aspects and method for producing a recombinant retrovirus provided in this document, the target cell may be a T cell and/or an NK cell.

[0069] In some embodiments of aspects of the retroviral packaging system and method for producing a recombinant retrovirus provided in this document, one or more pseudotyping elements comprise an envelope protein of vesicular stomatitis virus (VSV-G), an envelope protein of feline endogenous virus (RD114), an oncoretroviral amphotopic envelope protein or an oncoretroviral ecotropic envelope protein or functional fragments thereof.

[0070] In some embodiments of aspects of the retroviral packaging system and method for producing a recombinant retrovirus provided in this document, the packageable RNA genome is 11,000 KB or less or 10,000 KB or less in size. In some embodiments of aspects of the retroviral packaging system and method for producing a recombinant retrovirus provided in this document, the first target polypeptide comprises a first genetically modified signaling polypeptide, wherein said first genetically modified signaling polypeptide comprises a lymphoproliferative element, and the second target polypeptide comprises a second genetically modified signaling polypeptide including a CAR.

[0071] In one aspect, an isolated polynucleotide is provided in this document for regulating the expression of a target polynucleotide comprising: a polynucleotide encoding a target polynucleotide functionally linked to a promoter and a riboswitch, wherein the riboswitch comprises: a.) an aptamer domain capable of binding to a nucleoside analog antiviral drug and having reduced guanine or 2'-deoxyguanosine binding relative to the nucleoside analog antiviral drug; and b.) a function-switching domain capable of regulating the expression of the target polynucleotide, wherein the binding of the nucleoside analog by the aptamer domain induces or suppresses the expression-regulating activity of the function-switching domain, thereby regulating the expression of the target polynucleotide.

[0072] In the illustrative embodiments of any of the methods and compositions provided herein that include the in vivo control element, the control element may be a polynucleotide comprising a riboswitch. The riboswitch may have the ability to bind to a nucleoside analog, and the compound that binds to the in vivo control element is the nucleoside analog. The nucleoside analog may be an antiviral agent. The antiviral agent may be acyclovir or penciclovir. The riboswitch may preferably bind to acyclovir rather than penciclovir, or preferably it may bind to penciclovir rather than acyclovir. The riboswitch may have reduced binding to the nucleoside analog antiviral drug at temperatures above 37°C, 37.5°C, 38°C, 38.5°C or 39°C, for example, above 39°C. The riboswitch can be between 35, 40, 45, and 50 nucleotides long at the lower end of the band and 60, 65, 70, 75, 80, 85, 90, 95, and 100 nucleotides long at the upper end of the band, for example, between 45 and 80 nucleotides long. In the illustrative embodiments of any of the methods and compositions provided herein that include the riboswitch, the target polynucleotide that is regulated by the riboswitch may include a region encoding a miRNA, an shRNA, and/or a polypeptide. The target polynucleotide may encode a lymphoproliferative element. The target polynucleotide may be functionally linked to a promoter. The target polynucleotide may include a region encoding a polypeptide, and the polypeptide may include a chimeric antigen receptor comprising an antigen-specific targeting region, a transmembrane domain, and an intracellular activation domain. In illustrative embodiments of any of the methods and compositions provided herein that include the riboswitch, the function-switching domain may regulate an internal ribosome entry site, accessibility of pre-mRNA splicing donor in the viral gene construct, translation, transcription termination, transcript degradation, miRNA expression, or shRNA expression, thereby regulating the expression of the target polynucleotide. The riboswitch may include a ribozyme. In illustrative embodiments of any of the methods and compositions provided herein that include the riboswitch, the isolated polynucleotide may be a molecular cloning vector or an expression vector. In the illustrative embodiments of any of the methods and compositions provided in this document that include the riboswitch, the isolated polynucleotide can be integrated into a retroviral genome or a mammalian chromosome or fragment thereof.

[0073] Another aspect provided herein is a method for genetically modifying and expanding lymphocytes of a subject comprising: A. collecting blood from the subject; B. placing the subject's blood T cells and/or NK cells ex vivo in contact with recombinant retroviruses comprising: i. a pseudotyping element on its surface that has the ability to bind to a T cell and/or NK cell and facilitate the fusion of the recombinant retrovirus membrane to the same, wherein said pseudotyping element comprises cytoplasmic domain deletion variants of a measles virus F polypeptide and/or a measles virus H polypeptide; ii. a polypeptide with the ability to bind to CD3 and a polypeptide with the ability to bind to CD28, wherein said polypeptides are expressed on the surface of a recombinant retrovirus and have the ability to bind to a T cell and/or an NK cell and further wherein said polypeptides are not encoded by a polynucleotide in the recombinant retrovirus; (iii. a polynucleotide comprising one or more transcriptional units functionally linked to an active promoter on T cells and/or NK cells, wherein one or more transcriptional units encode a first genetically modified signaling polypeptide comprising a constitutively active IL-7 receptor mutant and a second genetically modified signaling polypeptide comprising a chimeric antigen receptor comprising an antigen-specific targeting region (ASTR), a transmembrane domain and an intracellular activation domain, wherein the expression of the IL-7 receptor mutant is regulated by a riboswitch that binds to a nucleoside analog antiviral drug, wherein the binding of the nucleoside analog antiviral drug to the riboswitch increases the expression of the IL-7 receptor mutant, and wherein said contact results in at least some of the resting T cells and/or NK cells becoming genetically modified; C. reintroduce the genetically modified T cells and/or NK cells into the subject; and D. To expose genetically modified T cells and/or NK cells in vivo to a nucleoside analog antiviral drug to promote the expansion of T cells and/or NK cells, wherein the method between blood collection and reintroduction of genetically modified T cells and/or NK cells is performed in no more than 24 hours and/or without requiring prior ex vivo stimulation, thereby genetically modifying and expanding the subject's lymphocytes.

[0074] In illustrative embodiments of this method aspect, the retrovirus is a lentivirus. In another illustrative embodiment, the recombinant retrovirus genetically modifies a T cell. In another illustrative embodiment, the polypeptide capable of binding to CD3 and the polypeptide capable of binding to CD28 are each fused to a heterologous GPI anchor binding sequence. In some cases, the polypeptide capable of binding to CD3 may be scFVFc anti-CD3 or scFV anti-CD3, and the polypeptide capable of binding to CD28 may be CD80. The scFVFc anti-CD3 or scFV anti-CD3 and CD80 may each be additionally fused to a DAF signaling sequence. In another illustrative embodiment, the recombinant retroviruses additionally comprise on their surface a fusion polypeptide comprising a cytokine covalently linked to DAF. In some cases, the cytokine may be IL-7 or IL-15, and the fusion polypeptide may comprise the DAF signaling sequence, IL-7 without its signaling sequence, and a DAF fragment comprising a GPI anchor-binding sequence.

[0075] In another illustrative embodiment of this method aspect immediately above, the riboswitch additionally controls the expression of the chimeric antigen receptor in a manner regulated by the binding of the riboswitch to the nucleoside analog antiviral drug which, in some cases, is acyclovir and/or penciclovir. In another embodiment, the constitutively active IL-7 can be replaced by a miRNA or shRNA. In some cases, the miRNA or shRNA can be encoded by nucleic acids within an intron.

[0076] Another aspect provided in this document is a recombinant retrovirus comprising: A. a pseudotyping element on its surface that has the ability to bind to a T cell and/or NK cell and facilitate the fusion of the recombinant retrovirus membrane to them, wherein said pseudotyping element comprises cytoplasmic domain deletion variants of a measles virus F polypeptide and/or a measles virus H polypeptide; B. a polynucleotide comprising one or more transcriptional units functionally linked to an active promoter on T cells and/or NK cells, wherein one or more transcriptional units encode a first genetically modified signaling polypeptide comprising a chimeric antigen receptor comprising an antigen-specific targeting region, a transmembrane domain and an intracellular activation domain and a second genetically modified signaling polypeptide comprising a constitutively active IL-7 receptor mutant; wherein the expression of the IL-7 receptor mutant is regulated by a riboswitch that binds to a nucleoside analog antiviral drug, wherein the binding of the nucleoside analog antiviral drug to the riboswitch increases the expression of the IL-7 receptor mutant; and C. a polypeptide with the ability to bind to CD3 and a polypeptide with the ability to bind to CD28, wherein said polypeptides are expressed on the surface of a recombinant retrovirus; have the ability to bind to a T cell and/or NK cell; and are not encoded by a polynucleotide in the recombinant retrovirus.

[0077] In the illustrative embodiments of the recombinant retrovirus aspect immediately above, the retrovirus is a lentivirus. In other illustrative embodiments of the method, the polypeptide capable of binding to CD3 and the polypeptide capable of binding to CD28 are each fused to a heterologous GPI anchor binding sequence. In some cases, the polypeptide capable of binding to CD3 may be scFVFc anti-CD3 or scFV anti-CD3, and the polypeptide capable of binding to CD28 may be CD80. The scFVFc anti-CD3 or scFV anti-CD3 and CD80 may each be additionally fused to a DAF signaling sequence. In another illustrative embodiment, the recombinant retroviruses additionally comprise on their surface a fusion polypeptide comprising a cytokine covalently linked to DAF. In some cases, the cytokine may be IL-7 or IL-15, and the fusion polypeptide may comprise the DAF signaling sequence, IL-7 without its signaling sequence, and a DAF fragment comprising a GPI anchor-binding sequence.

[0078] In another embodiment illustrating the recombinant retrovirus aspect immediately above, the riboswitch additionally controls the expression of the chimeric antigen receptor in a manner regulated by the binding of the riboswitch to the nucleoside analog antiviral drug which, in some cases, is acyclovir and/or penciclovir. In another embodiment, the constitutively active IL-7 can be replaced by a miRNA or shRNA. The miRNA or shRNA can be encoded by nucleic acids within an intron.

[0079] Another aspect provided in this document is a method for producing a recombinant retrovirus comprising: A. cultivating a population of packaging cells to accumulate a first transactivator, wherein the packaging cells comprise the first transactivator expressed from a constitutive promoter, wherein the first transactivator has the ability to bind to a first ligand and to an inducible first promoter to affect the expression of a nucleic acid sequence functionally linked thereto in the presence versus absence of the first ligand, and wherein the expression of a second transactivator and a retroviral REV protein is regulated by the first transactivator; B. incubate the population of packaging cells comprising the first transactivator accumulated in the presence of the first ligand to accumulate the second transactivator and the retroviral REV protein and an activation element typically on its surface, comprising a polypeptide with the ability to bind to CD3 and a polypeptide with the ability to bind to CD28, wherein the second transactivator has the ability to bind to a second ligand and a second inducible promoter to affect the expression of a nucleic acid sequence functionally linked to it in the presence versus absence of the second ligand; andC. Incubate the population of packaging cells comprising a second transactivator and accumulated retroviral REV protein in the presence of the second ligand, thereby inducing the expression of a gag polypeptide, a pol polypeptide, and a pseudotyping element capable of binding to a T cell and/or an NK cell and facilitating the fusion of the recombinant retrovirus membrane to them, wherein said pseudotyping element comprises cytoplasmic domain deletion variants of a measles virus F polypeptide and/or a measles virus H polypeptide, wherein a packageable RNA genome is encoded by a polynucleotide functionally linked to a third promoter, and wherein said promoter is inducible by the second transactivator, wherein the packageable RNA genome comprises, from 5' to 3': i. a 5' long terminal repeat or active fragment thereof; ii. a nucleic acid sequence encoding a cis-acting retroviral RNA packaging element; iii. a nucleic acid sequence encoding a first genetically modified signaling polypeptide comprising a chimeric antigen receptor and a second genetically modified signaling polypeptide comprising a constitutively active IL-7 receptor mutant separated by a cleavage signal; iv. a fourth promoter that is active on T cells and/or NK cells; and v. a long 3' terminal repeat or active fragment thereof, wherein the packageable RNA genome further comprises a riboswitch that binds to a nucleoside analog antiviral drug, wherein the binding of the riboswitch to the nucleoside analog antiviral drug increases the expression of the IL-7 receptor mutant, thereby producing the recombinant retrovirus.

[0080] In an illustrative embodiment of the method, the riboswitch additionally controls the expression of the chimeric antigen receptor in a manner regulated by the binding of the riboswitch to the nucleoside analog antiviral drug. In another illustrative embodiment, the nucleoside analog antiviral drug is acyclovir and/or penciclovir. In another illustrative embodiment, the packageable RNA genome additionally comprises a recognition domain, wherein the recognition domain comprises a polypeptide that is recognized by an antibody that recognizes EGFR or an epitope thereof. In another illustrative embodiment, the first ligand is rapamycin and the second ligand is tetracycline or doxorubicin, or the first ligand is tetracycline or doxorubicin and the second ligand is rapamycin. In another illustrative embodiment, the packaging cell additionally comprises a nucleic acid sequence encoding Vpx in the second or third optional transcriptional unit or in an additional transcriptional unit that is functionally linked to the first inducible promoter. In another illustrative embodiment, the polypeptide capable of binding to CD3 and the polypeptide capable of binding to CD28 are each fused to a heterologous GPI anchor binding sequence. In some cases, the polypeptide capable of binding to CD3 may be anti-CD3 scFVFc or anti-CD3 scFV, or anti-CD2 scFv, and the polypeptide capable of binding to CD28 may be CD80. The anti-CD3 scFVFc or anti-CD3 scFV and CD80 may each be additionally fused to a DAF signaling sequence. In another illustrative embodiment, the expression of a fusion polypeptide comprising a DAF-covalently linked cytokine is also induced. In some cases, the cytokine may be IL-7 or IL-15, and the fusion polypeptide may comprise the DAF signaling sequence, IL-7 without its signaling sequence, and a DAF fragment comprising a GPI anchor binding sequence. In another illustrative embodiment, the riboswitch additionally controls the expression of the chimeric antigen receptor in a manner regulated by the binding of the riboswitch to the nucleoside analog antiviral drug, which in some cases is acyclovir and/or penciclovir. In another embodiment, the constitutively active IL-7 may be replaced by a miRNA or shRNA. The miRNA or shRNA may be encoded by nucleic acids within an intron. In an illustrative embodiment, the retrovirus that is produced is a lentivirus.

[0081] A genetically modified lymphocyte comprising: A. a first genetically modified signaling polypeptide comprising a constitutively active IL-7 receptor mutant; and B. a second genetically modified signaling polypeptide comprising a chimeric antigen receptor comprising an antigen-specific targeting region (ASTR), a transmembrane domain and an intracellular activation domain.

[0082] In the illustrative embodiments of the genetically modified lymphocyte aspect above, the genetically modified lymphocyte is a T cell and/or an NK cell. In certain embodiments, the lymphocyte is a T cell. In another illustrative embodiment, the expression of said first genetically modified signaling polypeptide and/or said second genetically modified signaling polypeptide is regulated by a riboswitch that binds to a nucleoside analog antiviral drug, wherein the binding of the nucleoside analog antiviral drug to the riboswitch increases the expression of the IL-7 receptor mutant. In another embodiment, the constitutively active IL-7 receptor can be replaced by a miRNA or an shRNA. The miRNA or shRNA can be additionally encoded by nucleic acids within an intron.

[0083] A genetically modified T cell and/or NK cell comprising: a. a first genetically modified signaling polypeptide comprising a lymphoproliferative element; and b. a second genetically modified signaling polypeptide comprising a chimeric antigen receptor comprising an antigen-specific targeting region (ASTR), a transmembrane domain and an intracellular activation domain.

[0084] In illustrative embodiments of the appearance of genetically modified T cells and/or NK cells, the lymphoproliferative element is constitutively active and, in some cases, is a constitutively active mutated IL-7 receptor or a fragment thereof. In another illustrative embodiment, the expression of the first genetically modified signaling polypeptide and/or the second genetically modified signaling polypeptide is regulated by an in vivo control element. In some cases, the in vivo control element is a polynucleotide comprising a riboswitch. In some cases, the riboswitch has the ability to bind to a nucleoside analog, and when the nucleoside analog is present, the first genetically modified signaling polypeptide and/or the second genetically modified polypeptide are expressed. In other illustrative embodiments, the genetically modified T cells and/or NK cells have, on their surface, an activation element, a pseudotyping element, and/or a membrane-bound cytokine. In some cases, the activation element comprises a membrane-bound polypeptide capable of binding to CD3; and/or a membrane-bound polypeptide capable of binding to CD28. In one embodiment, the activation element comprises scFV anti-CD3 fused to a heterologous GPI anchor-binding sequence and/or CD80 fused to a heterologous GPI anchor-binding sequence. In an illustrative embodiment, the pseudotyping element comprises a Measles Virus F polypeptide, a Measles Virus H polypeptide, and/or cytoplasmic domain deletion variants of a Measles Virus F polypeptide and/or a Measles Virus H polypeptide. In other embodiments, the membrane-bound cytokine is a fusion polypeptide comprising IL-7, or a fragment thereof, fused to DAF, or a fragment thereof comprising a GPI anchor-binding sequence.

[0085] In one aspect, a method is provided herein for genetically modifying and expanding lymphocytes of a subject comprising: A. placing resting T cells and/or NK cells of the subject ex vivo, typically without requiring prior ex vivo stimulation, into contact with recombinant retroviruses comprising: i. a pseudotyping element on their surface that has the ability to bind to a T cell and/or NK cell and facilitate the fusion of the recombinant retrovirus membrane to them; and ii. A. a polynucleotide comprising one or more transcriptional units functionally linked to an active promoter on T cells and/or NK cells, wherein one or more transcriptional units encode a first genetically modified signaling polypeptide regulated by an in vivo control element, wherein said first genetically modified signaling polypeptide comprises a lymphoproliferative element, and optionally encode a second genetically modified signaling polypeptide optionally regulated by an in vivo control element, wherein the second genetically modified signaling polypeptide comprises an intracellular activation domain and optionally other components of a CAR, wherein said contact facilitates the transduction of at least some of the resting T cells and/or NK cells by recombinant retroviruses, thereby producing genetically modified T cells and/or NK cells; B. introduce the genetically modified T cells and/or NK cells into the subject; to expose genetically modified T cells and/or NK cells in vivo to a compound that acts as the in vivo control element to affect the expression of the first genetically modified signaling polypeptide and promote the expansion, engraftment, and/or persistence of lymphocytes in vivo, thereby genetically modifying and expanding the subject's lymphocytes.

[0086] In the illustrative embodiments, transduction is performed without ex vivo stimulation. In the illustrative embodiments, the compound is a molecular chaperone, such as a small molecular chaperone. In the illustrative embodiments, the binding of the molecular chaperone to the lymphoproliferative element increases the proliferative activity of the lymphoproliferative element. The molecular chaperone can be administered to the subject before blood is collected, during contact, and/or after T cells and/or NK cells are introduced into the subject. It will be understood by this aspect, in which the compound is the in vivo control element, that the compound typically has the ability to bind to a lymphoproliferative element and/or a component of a CAR and does not bind to such lymphoproliferative element or CAR component during the performance of the method. Other embodiments and teachings related to the methods provided in this document that include transfecting a T cell and/or an NK cell with a recombinant retrovirus apply to this aspect, including a molecular chaperone embodiment as well.

[0087] In another aspect, a method is provided in this document for selecting a microenvironment-restricted antigen-specific targeting region comprising selecting from a polypeptide presentation library by: a. subjecting polypeptides from the polypeptide presentation library to a binding assay under a normal physiological condition and a binding assay under an abnormal condition; and b. selecting a polypeptide that exhibits an increase in binding activity under the abnormal condition compared to the physiological condition, thereby selecting the microenvironment-restricted antigen-specific targeting region.

[0088] In another aspect, a method is provided in this document for isolating a microenvironment-restricted antigen-specific targeting region comprising: selection of a polypeptide library: a) placing the polypeptide library under abnormal conditions in contact with a target antigen bound to a solid support, wherein clones expressing polypeptides that bind to the target antigen remain bound to the solid support through the target antigen; b) incubating the solid supports with bound polypeptides under physiological conditions; and c) collecting clones that elute from the solid support under physiological conditions, thereby isolating the microenvironment-restricted antigen-specific targeting region.

[0089] In another aspect, a chimeric antigen receptor for binding to a target antigen is provided in this document comprising: a) at least one microenvironment-restricted antigen-specific targeting region selected by selection from a polypeptide library and having increased activity in a binding assay under an abnormal condition compared to a normal physiological condition; b) a transmembrane domain; and c) an intracellular activation domain.

[0090] In another aspect, a chimeric antigen receptor for binding to a target antigen is provided in this document comprising: a) a microenvironmentally restricted antigen-specific targeting region that exhibits increased binding to the target antigen under an abnormal condition compared to a normal physiological environment, wherein the antigen-specific targeting region binds to the target; b) a transmembrane domain; and c) an intracellular activation domain.

[0091] In illustrative embodiments of any of the methods and compositions provided herein that include the microenvironment-restricted antigen-specific targeting region (ASTR), the ASTR may have an increase of at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% in antigen-binding affinity in the assay under abnormal conditions compared to normal conditions. Abnormal conditions may include hypoxia, an acidic pH, a higher concentration of lactic acid, a higher concentration of hyaluronan, a higher concentration of albumin, a higher concentration of adenosine, a higher concentration of R-2-hydroxyglutarate, a higher concentration of PAD enzymes, higher pressure, higher oxidation, and lower nutrient availability. Microenvironment-restricted ASTR can exhibit increased antigen binding at pH 6.7 compared to pH 7.4. Microenvironment-restricted ASTR can exhibit increased antigen binding in a tumor environment and/or in vitro tumor surrogate assay conditions, relative to a corresponding physiological condition. The target may be 4-1BB, ST4, adenocarcinoma antigen, alpha-fetoprotein, AXL, BAFF, B-lymphoma cell, C242 antigen, CA-125, carbonic anhydrase 9 (CA-IX), C-MET, CCR4, CD152, CD19, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44 v6, CD51, CD52, CD56, CD74, CD80, CEA, CNT0888, CTLA-4, DRS, EGFR, EpCAM, CD3, FAP, fibronectin extra domain B, folate receptor 1, GD2, ganglioside GD3, glycoprotein 75, GPNMB, HER2/neu, HGF, receptor kinase of human scatter factor, IGF-1 receptor, IGF-I, IgG1, IL-CAM, IL-13, IL-6, insulin-like growth factor receptor I, nSP1 integrin, nvP3 integrin, MORAb-009, MS4A1, MUC1, CanAg mucin, N-glycolylneuraminic acid, NPC-1C, PDGF-R α, PDL192, phosphatidylserine, prostate carcinoma cells, RANKL, RON, ROR1, ROR2 SCH 900105, SDC1, SLAMF7, TAG-72, tenascin C, TGF beta 2, TGF-P, TRAIL-R1, TRAIL-R2, CTAA16 tumor antigen, VEGF-A, VEGFR-1, VEGFR2, and vimentin. An ASTR can be an antibody, an antigen, a ligand, a receptor-binding domain of a ligand, a receptor, a receptor-binding domain, or an affibody. An ASTR can be a full-length antibody, a single-chain antibody, a Fab fragment, a Fab' fragment, a (Fab')2 fragment, an Fv fragment, a divalent single-chain antibody, or a diabody. An ASTR can include both a heavy chain and a light chain of an antibody. The antibody can be a variable single-chain fragment. In some embodiments, the heavy and light chains can be separated by a binder, wherein the binder is between 6 and 100 amino acids in length. In some embodiments, the heavy chain can be in an N-terminal position relative to the light chain in the chimeric antigen receptor, and in some embodiments, the light chain can be in an N-terminal position relative to the heavy chain in the chimeric antigen receptor.

[0092] In illustrative embodiments of any of the methods that include a polypeptide presentation library, the polypeptide presentation library may be a phage presentation library or a yeast presentation library. The polypeptide presentation library may be an antibody presentation library. The antibody presentation library may be a human or humanized antibody presentation library. The antibody presentation library may be a virgin library. The methods may include infecting bacterial cells with the collected phage to generate a refined phage presentation library and repeating the contact, incubation, and collection for 1 to 1,000 cycles, using the refined phage presentation library generated from a previous cycle.

[0093] In illustrative embodiments of any of the methods provided in this document that involve isolating or selecting a microenvironmentally restricted ASTR, the method may include determining the nucleotide sequence of a polynucleotide encoding the microenvironmentally restricted antigen-specific targeting region, thereby determining the polypeptide sequence of the microenvironmentally restricted ASTR. The methods may include producing a microenvironmentally restricted biological chimeric antigen receptor by generating a polynucleotide encoding a polypeptide comprising the microenvironmentally restricted ASTR, a transmembrane domain, and an intracellular activation domain. The library may be a single-chain antibody library.

[0094] Methods for isolating a microenvironment-restricted ASTR may include repeated selection between 1 and 1,000 times. Methods for isolating a microenvironment-restricted ASTR may be performed without mutating polynucleotides encoding the microenvironment-restricted antigen-specific targeting region isolated between selection cycles. Methods for isolating a microenvironment-restricted ASTR may be performed by culturing, high-fidelity amplification, and/or dilution of polynucleotides encoding antigen-specific targeting regions or host organisms including the same, between selection cycles. Methods may include, prior to repetition, mutagenizing the selected and/or isolated microenvironment-restricted antigen-specific targeting region. Methods may include determining the sequence of the selected and/or isolated microenvironment-restricted antigen-specific targeting region, and/or a polynucleotide encoding the same, after one or more selection cycles by long-read DNA sequencing. Methods may include determining the sequence before and after expansion of the isolated microenvironment-restricted ASTR. Methods for isolating a microenvironment-restricted ASTR can be performed without repeat selection. Methods for isolating a microenvironment-restricted ASTR can be performed without mutating a polynucleotide encoding the isolated microenvironment-restricted ASTR after the microenvironment-restricted ASTR is isolated.

[0095] In illustrative embodiments of any of the compositions provided herein that include a chimeric antigen receptor with a microenvironment-restricted ASTR, the microenvironment-restricted ASTR can be identified by selection from an antibody library. In some embodiments, the microenvironment-restricted ASTR is identified by selection from a phage-presentation or yeast-presentation library. In some embodiments, the chimeric antigen receptor comprises a bispecific ASTR.

[0096] In other respects, a transduced T cell and/or NK cell comprising a recombinant polynucleotide comprising one or more transcriptional units functionally linked to an active promoter in T cells and/or NK cells is provided herein, wherein one or more transcriptional units encode a first genetically modified signaling polypeptide regulated by an in vivo control element, wherein said first genetically modified signaling polypeptide comprises a constitutively active IL-7 receptor mutant, and wherein the in vivo control element has the ability to bind to a compound in vivo or is configured to bind to a compound in vivo.

[0097] In another aspect, a recombinant retrovirus comprising a recombinant polynucleotide comprising one or more transcriptional units functionally linked to an active promoter on T cells and/or NK cells is provided herein, wherein one or more transcriptional units encode a first genetically modified signaling polypeptide regulated by an in vivo control element, wherein said first genetically modified signaling polypeptide comprises a constitutively active IL-7 receptor mutant, and wherein the in vivo control element has the ability to bind to a compound in vivo or is configured to bind to a compound in vivo.

[0098] In another aspect, a method for transducing a T cell and/or NK cell is provided herein comprising placing a T cell and/or NK cell in contact with a recombinant retrovirus comprising a recombinant polynucleotide comprising one or more transcriptional units functionally linked to an active promoter in T cells and/or NK cells, wherein one or more transcriptional units encode a first genetically modified signaling polypeptide regulated by an in vivo control element, wherein said first genetically modified signaling polypeptide comprises a constitutively active IL-7 receptor mutant, and wherein the in vivo control element has the ability to bind to a compound in vivo under transduction conditions, thereby transducing the T cell and/or NK cell.

[0099] In the illustrative embodiments of the T cell and/or NK cell aspects, the recombinant retrovirus aspects, and the method aspects provided in the preceding paragraphs, the recombinant polynucleotide further comprises a transcriptional unit encoding a second genetically modified signaling polypeptide comprising a first chimeric antigen receptor comprising an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain. In other illustrative embodiments, the lymphoproliferative element comprises a mutated IL-7 receptor or a fragment thereof. In other illustrative embodiments, the in vivo control element is a polynucleotide comprising a riboswitch. In some cases, the riboswitch has the ability to bind to a nucleoside analog, and the compound that binds to the in vivo control element is the nucleoside analog. In some cases, the nucleoside analog is an antiviral agent, such as, for example, acyclovir or penciclovir. In certain embodiments, the antiviral agent is acyclovir. In other illustrative embodiments, the constitutively active IL-17 receptor mutant is fused to EGFR or an epitope thereof. In other illustrative embodiments, the constitutively active IL-7 receptor mutant comprises an eTag. In other illustrative embodiments, the constitutively active IL-7 receptor mutant comprises a PPCL insertion. In other illustrative embodiments, the constitutively active IL-7 receptor mutant comprises a PPCL insertion at a position equivalent to position 243 in a wild-type human IL-8 receptor. In other illustrative embodiments, the transduced T cell or NK cell is a transduced T cell.

BRIEF DESCRIPTION OF THE DRAWINGS

[0100] Figure 1 shows a schematic drawing of illustrative compositions including a packaging cell (100) and a recombinant retrovirus (200) produced by the packaging cell (100). In Figure 1, several vectors (referred to as recombinant polynucleotides (110)) with the capacity to encode aspects of the invention are packaged into a recombinant retrovirus (200) that includes in its genome a first genetically modified signaling polypeptide that includes a lymphoproliferative element and, in some embodiments, a second genetically modified signaling polypeptide that is a chimeric antigen receptor or a CAR. The recombinant retrovirus expresses on its membrane a pseudotyping element (in a non-limiting embodiment, a Measles Virus hemagglutinin polypeptide (H) and a Measles Virus fusion polypeptide (F) or cytoplasmic domain deletion variants thereof) (240) that allows the retrovirus to bind and fuse to a target cell; an activation element (in non-limiting embodiments, an activation element that has a polypeptide with the ability to bind to CD28 and a polypeptide with the ability to bind to CD3) (210 and 220, respectively) that has the ability to bind to and activate a resting T cell; and a membrane-bound cytokine (in a non-limiting embodiment and IL-7 DAF fusion polypeptide) (230). The parts identified as (250), (260), (270), (280) and (290) are Src-FLAG-Vpx, HIV gag matrix, HIV gag capsid, HIV RNA and HIV pol, respectively.

[0101] Figure 2 shows a schematic drawing of illustrative compositions including a recombinant retrovirus produced by a packaging cell (200) and a resting T cell (300) transfected by the recombinant retrovirus (200). Elements on the surface of the retrovirus bind to receptors and/or ligands on the surface of a resting T cell. The pseudotyping element may include a binding polypeptide and a fusogenic polypeptide (in non-limiting embodiments, a Measles Virus hemagglutinin polypeptide (H) and a Measles Virus fusion polypeptide (F)) or cytoplasmic domain deletion variants thereof) that facilitate the binding and fusion of the retrovirus to the T cell. In non-limiting embodiments, the retrovirus includes on its surface an activation element (in non-limiting embodiments, an activation element that has a polypeptide capable of binding to CD28 and a polypeptide capable of binding to CD3) that has the ability to activate the resting T cell by interconnecting the T cell receptor complex and, optionally, a co-receptor (320). Furthermore, membrane-bound cytokines (in non-limiting embodiments, an IL-7 DAF fusion polypeptide) present on the retrovirus surface bind to IL-7Rα (310) on the surface of the resting T cell. The retrovirus fuses with the T cell, and the polynucleotides encoding the first genetically modified signaling polypeptide that includes the lymphoproliferative element (in the illustrative embodiments, a constitutively active IL-7Rα) (370) are reverse transcribed in the cytosol before migrating to the nucleus to be incorporated into the DNA of the activated T cell. In some embodiments, virus-packaged Src-FLAG-Vpx (250) enters the cytosol of resting T cells and promotes the degradation of SAMHD1 (350), resulting in an increased pooling of cytoplasmic dNTPs available for reverse transcription. In some embodiments, the polynucleotides may also encode a second genetically modified signaling polypeptide that includes a CAR (360). In some embodiments, the lymphoproliferative element is expressed when a compound binds to an in vivo control element that regulates its expression (in a non-limiting example, the in vivo control element is a riboswitch that binds to a nucleoside analog). In some embodiments, the expression of the CAR is also regulated by the in vivo control element. The (330) portion is SLAM and CD46. The (340) portion is CD3.

[0102] Figures 3A to 3E show schematic drawings of vector systems. Figure 3A shows a construct containing a polynucleotide sequence encoding an FRB domain fused to the NFKB p65 activator domain (p65 AD) and a ZFHD1 DNA-binding domain fused to three FKBP repeats that is constitutively expressed. The construct in Figure 3A also includes HIV1 REV and Vpx as an SrcFlagVpx fusion under the rapamycin-inducible ZFHD1/p65 AD promoter. Figure 3B shows a construct containing a polynucleotide encoding an rtTA sequence under the control of the ZFHD1/p65 AD promoter. Figure 3C shows a construct containing a polynucleotide encoding a puromycin resistance gene flanked by loxP sites and the extracellular MYC tag flanked by lox2272 sites. Both selectable markers are under the control of a BiTRE promoter, which is flanked by FRT sites. Figure 3D shows a construct containing a polynucleotide encoding RFP flanked by loxP sites that is under the control of a TRE promoter and a single FRT site between the TRE promoter and the 5' loxP site of RFP. Figure 3E shows a construct containing a polynucleotide encoding GFP flanked by loxP sites that is under the control of the TRE promoter and a single FRT site between the TRE promoter and the 5' loxP site of GFP. The constructs in Figures 3C to 3E function as storage blocks for other polynucleotide sequences for insertion into the genome of the packaging cell line.

[0103] Figures 4A to 4C show schematic drawings of constructs. Figure 4A shows a construct containing a tricistronic polynucleotide encoding scFvFc anti-CD3 (clone UCHT1) with a CD14 GPI anchor binding site, CD80 extracellular domain (ECD) capable of binding to CD28 with a CD16B GPI anchor binding site, and IL-7 fused to decay-accelerating factor (DAF) with transposon sequences flanking the polynucleotide region for integration into the HEK293S genome. Figure 4B shows a construct containing a polynucleotide with a BiTRE promoter and a polynucleotide region encoding gag and pol polypeptides in one direction and a polynucleotide region encoding the FΔx and HΔy proteins of the measles virus in the other direction. Figure 4C shows a construct containing a polynucleotide sequence encoding a CAR and the lymphoproliferative element IL7Rα-insPPCL under the control of a CD3Z promoter that is not active in HEK293S cells, wherein the CAR and IL7Rα-insPPCL are separated by a polynucleotide sequence encoding a T2A ribosome jump sequence, and the IL7Rα-insPPCL has a ribozyme controlled by an acyclovir riboswitch. The CAR-containing construct additionally includes cPPT/CTS, an RRE sequence, and a polynucleotide sequence encoding HIV-1 Psi (^). The entire polynucleotide sequence in the CAR-containing construct to be integrated into the genome is flanked by FRT sites.

[0104] Figures 5A to 5C show molecular structures of acyclovir (Figure 5A), penciclovir (Figure 5B) and 2‘-deoxyguanosine (Figure 5C) as representative nucleoside analogs for selective riboswitch control.

[0105] Figure 6 represents the Mesoplasma florum type I-A deoxyguanosine riboswitch regulatory region and associated gene product. The sequence is the reverse complement of the M. florum L1 genomic DNA (AE017263.1) nt624396 to nt625670 which is identical to the M. florum W37 genomic DNA (CP006778.1) nt636277 to nt637550. The deoxyguanosine-binding aptamer sequence used for initial screening is indicated in bold and underlined. The downstream gene product (class Ib (aerobic) ribonucleotide reductase, beta subunit) is indicated in uppercase letters.

[0106] Figure 7 represents the type I-A deoxyguanosine riboswitch aptamer regions of M. florum targeted for directed evolution strategy. Nucleotides within empty ovals were targeted for randomization. Nucleotides within striped ovals were targeted for insertion/deletion and randomization.

[0107] Figures 8A and 8B represent the type I-A deoxyguanosine riboswitch aptamer screening library of M. florum. In Figure 8A, nucleotides within boxes with solid lines are sequence regions targeted for randomization, and nucleotides within boxes with dashed lines are sequence regions targeted for insertion/deletion and randomization. Figure 8B shows the possible sequences generated through mutation (random nucleotides ("N")) and deletion/insertion.

[0108] Figure 9 depicts the M. florum type I-A deoxyguanosine riboswitch aptamer oligo library synthesized as a reverse complement with additional base pairs added to enable PCR amplification and T7 promoter addition for in vitro transcription for library screening. The corresponding T7 promoter amplification primer and reverse amplification primer are also shown.

[0109] Figure 10 represents the guanosine xpt riboswitch regulatory region of Bacillus subtilis and the associated gene product. The sequence is the reverse complement of the genomic DNA of B. subtilis subsp. subtilis 6051-HGW (CP003329.1) nt2319439 to nt2320353. The guanosine-binding aptamer sequence used for initial screening is indicated in bold and underlined. The downstream gene product (xanthine phosphoribosyltransferase xpt) is indicated in uppercase letters.

[0110] Figure 11 represents the guanosine xpt riboswitch aptamer regions of B. subtilis targeted for directed evolution strategy. Nucleotides within empty ovals were targeted for randomization. Nucleotides within striped ovals were targeted for insertion/deletion and randomization.

[0111] Figures 12A and 12B represent the B. subtilis guanosine xpt riboswitch aptamer screening library. In Figure 12A, nucleotides within boxes with solid lines are sequence regions targeted for randomization, and nucleotides within boxes with dashed lines are sequence regions targeted for insertion/deletion and randomization. Figure 12B shows the possible sequences generated through mutation (random nucleotides ("N")) and deletion/insertion.

[0112] Figure 13 depicts the B. subtilis guanosine xpt riboswitch aptamer oligo library synthesized as a reverse complement with additional base pairs added to enable PCR amplification and T7 promoter addition for in vitro transcription for library screening. The corresponding T7 promoter amplification primer and reverse amplification primer are also shown.

[0113] Figure 14 shows the construction of the selection library. The library was built based on known guanosine and deoxyguanosine binding RNA (Pikovskaya, 2013).

[0114] Figure 15 shows an illustration of graphene oxide (GrO) aptamer selection. In step (1), RNA was transcribed and purified. In step (2), the purified RNA was eluted. In step (3), aptamers were incubated with counter-targets and buffer. In step (4), sequences bound to counter-targets or buffer components were removed with graphene oxide. In step (5), centrifugation separated non-specifically responsive species within the supernatant, which is then discarded. Two additional 5-minute washes removed most residual buffer-binding and counter-target-binding sequences. In step (6), a 1X acyclovir-in-selection buffer solution was added to the GrO-bound library for positive selection so that potential aptamer sequences desorb from GrO through interaction with the positive target. In step (7), a final centrifugation step separates the target-binding sequences in the supernatant from the non-responsive sequences still adsorbed to GrO. In step (8), the selected sequences were reverse transcribed, then the library was amplified by PCR, then transcribed to generate the library for the next selection cycle.

[0115] Figure 16 shows an illustration of parallel evaluation of graphene oxide. Enriched libraries undergoing parallel evaluation were divided into four equal portions. Library samples were then added to graphene oxide and incubated to load the library onto the graphene oxide. Two 5-minute washes were used to remove non-binding material. For the positive sample (acyclovir) and special target (penciclovir), each target was prepared separately in 1X selection buffer at 1 μM; the counter-target replaced the positive target with 10 μM of each counter-target in solution; the negative sample replaced the positive target with an equal volume of nuclease-free water. The samples were then combined with their respective graphene oxide preparations and incubated. After incubation, the samples were centrifuged to recover their supernatants, and library recovery was determined by reading a NanoDrop-1000 spectrophotometer (Thermo Fisher Scientific; Wilmington, DE). The remaining library sample was analyzed on denaturing PAGE. Images of the gels were taken after staining/decolorization with Gel-Star. Bands corresponding to the expected library size were recovered for a follow-up parallel evaluation cycle, with the acyclovir positive target replacing counter-targets for the pre-loading incubation of the negative, counter, and special target samples. The material recovered from the second parallel evaluation was used for sequencing and analysis.

[0116] Figure 17 shows seven aptamer candidates against acyclovir. The free energy for each aptamer was computed at 37 °C and 1 M Na+ by Quikfold 3.0 (Zuker 2003). The sequences were identified using proprietary algorithms. The underlined regions in each sequence are the PCR primer hybridization regions.

[0117] Figure 18 shows seven aptamer candidates against penciclovir. The free energy for each aptamer was computed at 37 °C and 1 M Na+ by Quikfold 3.0 (Zuker 2003). The sequences were identified using proprietary algorithms. The underlined regions in each sequence are the PCR primer hybridization regions.

[0118] Figure 19A provides a schematic drawing of IL7Rα variants tested for lymphoproliferative/survival activity when expressed in PBMCs. Figure 19B provides a bar graph showing the percentage of PBMC viability in the presence and absence of IL-2.

[0119] Figure 20 shows a plot of transduction efficacy against MOI for negatively selected and unstimulated T cells transduced using lentiviruses pseudotyped with VSV-G or truncated versions of MV(Ed) F and H polypeptides. The symbols are misaligned for enhanced clarity.

[0120] Figure 21 is a graph showing that miRNAs targeting CD3zeta located in the EF-1alpha promoter intron have the ability to knock down CD3 complex expression.

DEFINITIONS

[0121] As used in this document, the term "chimeric antigen receptor" or "CAR" or "CARs" refers to genetically modified receptors that graft antigen specificity onto cells, for example, T cells, NK cells, macrophages, and stem cells. The CARs of the invention include at least one antigen-specific targeting region (ASTR) and an intracellular activation domain (IAD) and may include a stem, a transmembrane (TM) domain, and one or more co-stimulatory domains (CSDs). In another embodiment, the CAR is a bispecific CAR, which is specific for two different antigens or epitopes. After the ASTR specifically binds to a target antigen, the IAD activates intracellular signaling. For example, the IAD can redirect T cell reactivity and specificity to a selected target in a non-MHC restricted manner by exploiting the antigen-binding properties of antibodies. Non-MHC restricted antigen recognition provides CAR-expressing T cells with the ability to recognize an antigen independently of antigen processing, thus bypassing a major tumor escape mechanism. Furthermore, when expressed on T cells, CARs advantageously do not dimerize with endogenous T cell receptor (TCR) alpha and beta chains.

[0122] As used in this document, the term "microenvironment" means any portion or region of a tissue or body that has constant or temporal physical or chemical differences from other tissue regions or body regions. For example, a "tumor microenvironment," as used in this document, refers to the environment in which a tumor exists, which is the non-cellular area within the tumor and the area directly outside the tumor tissue but not belonging to the intracellular compartment of the cancer cell itself. The tumor microenvironment may refer to any and all conditions of the tumor environment, including conditions that create a structural and/or functional environment for the malignant process to survive and/or expand and/or spread. For example, the tumor microenvironment may include alterations in conditions such as, but not limited to, pressure, temperature, pH, ionic strength, osmotic pressure, osmolality, oxidative stress, concentration of one or more solutes, electrolyte concentration, glucose concentration, hyaluronan concentration, lactic acid or lactate concentration, albumin concentration, adenosine levels, R-2-hydroxyglutarate levels, pyruvate concentration, oxygen concentration and/or the presence of oxidants, reductants or cofactors, as well as other conditions that a person skilled in the art will understand.

[0123] As used interchangeably in this document, the terms "polynucleotide" and "nucleic acid" refer to a polymeric form of nucleotides of any length, or ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-stranded, double-stranded or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derived nucleotide bases.

[0124] As used in this document, the term "antibody" includes polyclonal and monoclonal antibodies, including intact antibodies and antibody fragments that retain specific antigen binding. Antibody fragments may be, but are not limited to, antigen-binding fragments (Fab), Fab' fragments, F(ab')2 fragments, Fv fragments, Fab'-SH fragments, (Fab')2 Fv fragments, Fd fragments, recombinant IgG (rIgG) fragments, single-chain antibody fragments, including single-chain variable fragments (scFv), divalent scFvs, trivalent scFvs, and single-domain antibody fragments (e.g., sdAb, sdFv, nanobody). The term includes genetically modified and/or otherwise modified forms of immunoglobulins, such as intracorporates, pepticobodies, chimeric antibodies, single-chain antibodies, fully human antibodies, humanized antibodies, fusion proteins including an antigen-specific targeting region of an antibody and a non-antibody protein, heteroconjugated antibodies, multispecific antibodies, for example, bispecific, diabodies, triabodies and tetrabodies, tandem di-scFvs and tandem tri-scFvs. Unless otherwise specified, the term “antibody” should be understood as including functional antibody fragments thereof. The term also includes intact or full-length antibodies, including antibodies of any class or subclass, including IgG and its subclasses, IgM, IgE, IgA and IgD.

[0125] As used in this document, the term "antibody fragment" includes a portion of an intact antibody, for example, the antigen-binding or variable region of an intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng. 8(10): 1057 to 1062 (1995)); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with one antigen-binding site, and a residual "Fc" fragment, a designation that reflects the ability to crystallize easily. Pepsin treatment produces an F(ab')2 fragment that has two antigen-binding sites and also has the ability to crosslink the antigen.

[0126] As used interchangeably in this document, the terms antibody fragments “single-chain Fv”, “scFv” or “sFv” include the antibody VH and VL domains, wherein these domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide additionally includes a polypeptide spacer or binder between the VH and VL domains that enables the scFv to form the desired structure for antigen binding. For an analysis of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, volume 113, Rosenburg and Moore eds., Springer-Verlag, New York, pages 269 to 315 (1994).

[0127] As used herein, "naturally occurring" VH and VL domains refer to VH and VL domains that have been isolated from a host without further molecular evolution to alter their affinities when generated in an scFv format under specific conditions such as those disclosed in US Patent No. 8709755 B2 and Application WO/2016/033331A1.

[0128] As used in this document, the term "affinity" refers to the equilibrium constant for the reversible binding of two agents and is expressed as a dissociation constant (Kd). Affinity may be at least 1 time greater, at least 2 times greater, at least 3 times greater, at least 4 times greater, at least 5 times greater, at least 6 times greater, at least 7 times greater, at least 8 times greater, at least 9 times greater, at least 10 times greater, at least 20 times greater, at least 30 times greater, at least 40 times greater, at least 50 times greater, at least 60 times greater, at least 70 times greater, at least 80 times greater, at least 90 times greater, at least 100 times greater, or at least 1,000 times greater, or more, than the affinity of an antibody for unrelated amino acid sequences. The affinity of an antibody to a target protein can be, for example, from about 100 nanomolar (nM) to about 0.1 nM, from about 100 nM to about 1 picomolar (pM), or from about 100 nM to about 1 femtomolar (fM) or more. As used in this document, the term “avidity” refers to the resistance of a complex of two or more agents to dissociation after dilution. The terms “immunoreactive” and “preferentially binds” are used interchangeably in this document in relation to antibodies and/or antigen-binding fragments.

[0129] As used in this document, the term “bond” refers to a direct association between two molecules due to, for example, covalent, electrostatic, hydrophobic and ionic interactions and/or hydrogen bonding, including interactions such as salt bridges and water bridges. Non-specific bonding could refer to bonding with an affinity less than about 10-7 M, for example, bonding with an affinity of 10-6 M, 10-5 M, 10-4 M, etc.

[0130] As used in this document, reference to a "cell surface expression system" or "cell surface presentation system" refers to the presentation or expression of a protein or portion thereof on the surface of a cell. Typically, a cell is generated that expresses proteins of interest fused to a cell surface protein. For example, a protein is expressed as a fusion protein with a transmembrane domain.

[0131] As used in this document, the term "element" includes polypeptides, including polypeptide fusions, polypeptide regions and mutants or functional fragments thereof, and polynucleotides, including microRNAs and shRNAs and mutants or functional fragments thereof.

[0132] As used in this document, the term "region" is any segment of a polypeptide or polynucleotide.

[0133] As used in this document, a "domain" is a region of a polypeptide or polynucleotide with a functional and/or structural property.

[0134] As used in this document, the terms "stem" or "stem domain" refer to a flexible polypeptide connecting region that provides structural flexibility and spacing to flanking polypeptide regions and may consist of natural or synthetic polypeptides. A stem may be derived from a hinge or hinge region of an immunoglobulin (e.g., IgG1) that is generally defined as extending from Glu216 to Pro230 of human IgG1 (Burton (1985) Molec. Immunol., 22:161–206). The hinge regions of other IgG isotypes may be aligned with the IgG1 sequence by placing the first and last cysteine residues that form disulfide bonds between heavy chains (S-S) in the same positions. The stem may be naturally occurring or non-naturally occurring, including, but not limited to, an altered hinge region, as disclosed in U.S. Patent No. 5,677,425. The stem may include a complete hinge region derived from an antibody of any class or subclass. The stem may also include regions derived from CD8, CD28, or other receptors that provide a similar function in providing flexibility and spacing to flanking regions.

[0135] The term "isolate," as used in this document, means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the materials coexisting in the natural system, is isolated. Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition and could still be isolated so that such vector or composition is not part of its natural environment.

[0136] As used in this document, a “polypeptide” is a single chain of amino acid residues linked by peptide bonds. A polypeptide does not fold into a fixed structure nor does it undergo any post-translational modification. A “protein” is a polypeptide that folds into a fixed structure. “Polypeptides” and “proteins” are used interchangeably in this document.

[0137] . As used in this document, a polypeptide may be “purified” to remove contaminating components from a natural environment of the polypeptide, for example, materials that would interfere with diagnostic or therapeutic uses for the polypeptide, such as, for example, enzymes, hormones, and other proteaceous or non-proteaceous solutes. A polypeptide may be purified (1) to more than 90%, more than 95%, or more than 98% by weight of the antibody as determined by the Lowry method, for example, more than 99% by weight, (2) to a degree sufficient to obtain at least 15 N-terminal or internal amino acid sequence residues using a spin cup sequencer, or (3) to homogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or non-reducing conditions using silver stain or Coomassie blue.

[0138] As used in this document, the term "immune cells" generally includes erythrocytes (white blood cells) that are derived from hematopoietic stem cells (HSCs) produced in the bone marrow. "Immune cells" include, for example, lymphocytes (T cells, B cells, natural killer (NK) cells) and myeloid-derived cells (neutrophils, eosinophils, basophils, monocytes, macrophages, dendritic cells).

[0139] As used in this document, "T cell" includes all types of immune cells that express CD3, including helper T cells (CD4+ cells), cytotoxic T cells (CD8+ cells), regulatory T cells (Tregs), and gamma-delta T cells.

[0140] As used in this document, a "cytotoxic cell" includes CD8+ T cells, natural killer (NK) cells, NK-T cells, Yδ T cells, a subpopulation of CD4+ cells, and neutrophils that are cells with the ability to mediate cytotoxicity responses.

[0141] As used in this document, the term "stem cells" generally includes pluripotent or multipotent stem cells. "Stem cells" include, for example, embryonic stem cells (ES); mesenchymal stem cells (MSCs); induced pluripotent stem cells (iPSCs); and committed progenitor cells (hematopoietic stem cells (HSCs); bone marrow-derived cells, etc.).

[0142] As used in this document, the terms "treatment," "to treat," and similar terms refer to achieving a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of partially or completely curing a disease and/or adverse effect attributable to the disease. "Treatment," as used in this document, includes any treatment of a disease in a mammal, for example, in a human being, and includes: (a) preventing the disease from occurring in an individual who may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, that is, stopping its development; and (c) attenuating the disease, that is, causing regression of the disease.

[0143] As used interchangeably in this document, the terms "individual," "subject," "host," and "patient" refer to a mammal, including, but not limited to, humans, murines (e.g., rats, mice), lagomorphs (e.g., rabbits), non-human primates, humans, canines, felines, ungulates (e.g., equines, bovines, ovines, porcines, caprines), etc.

[0144] As used in this document, the terms "therapeutically effective amount" or "efficient amount" refer to the amount of an agent or combined amounts of two agents that, when administered to a mammal or other subject to treat a disease, is sufficient to effect such treatment for the disease. The "therapeutically effective amount" will vary depending on the agent (or agents), the disease and its severity, and the age, weight, etc., of the individual being treated.

[0145] As used in this document, the term "evolution" or "evolve" refers to the use of one or more mutagenesis methods to generate a different polynucleotide encoding a different polypeptide, which is an enhanced biological molecule and/or contributes to the generation of another enhanced biological molecule. "Physiological" or "normal" or "normal physiological" conditions are conditions such as, but not limited to, pressure, temperature, pH, ionic strength, osmotic pressure, osmolality, oxidative stress, concentration of one or more solutes, concentration of electrolytes, concentration of glucose, concentration of hyaluronan, concentration of lactic acid or lactate, concentration of albumin, adenosine levels, R-2-hydroxyglutarate levels, concentration of pyruvate, concentration of oxygen and/or presence of oxidants, reductants or cofactors, as well as other conditions, which are considered to be within a normal range at the site of administration, or in the tissue or organ at the site of action, in a subject.

[0146] As used in this document, a “genetically modified cell” includes cells that contain exogenous nucleic acids whether or not the exogenous nucleic acids are integrated into the cell's genome.

[0147] A “polypeptide”, as used herein, may include part of or an entire protein molecule as well as any post-translational or other modifications.

[0148] A pseudotyping element, as used herein, may include a "binding polypeptide" comprising one or more polypeptides, typically glycoproteins, that identify and bind to the host target cell, and one or more "fusogenic polypeptides" that mediate the fusion of the host target cell and retroviral membranes, thereby enabling a retroviral genome to enter the host target cell. The "binding polypeptide," as used herein, may also be referred to as a "T cell and/or NK cell binding polypeptide" or a "target interconnection element," and the "fusogenic polypeptide" may also be referred to as a "fusogenic element."

[0149] A “resting” lymphocyte, such as a resting T cell, is a lymphocyte in the G0 stage of the cell cycle that does not express activation markers, such as Ki-67. Resting lymphocytes may include naive T cells that have never encountered a specific antigen and memory T cells that have been altered by a previous encounter with an antigen. A “resting” lymphocyte may also be referred to as a “quiescent” lymphocyte.

[0150] As used in this document, “lymphoid depletion” involves methods that reduce the number of lymphocytes in a subject, for example, by administering a lymphoid depleting agent. Lymphoid depletion can also be achieved by fractionated whole-body or partial-body radiotherapy. A lymphoid depleting agent may be a chemical compound or composition with the ability to decrease the number of functional lymphocytes in a mammal when administered to the mammal. An example of such an agent is one or more chemotherapeutic agents. Such agents and dosages are known and may be selected by a treating physician depending on the subject being treated. Examples of lymphoid depleting agents include, but are not limited to, fludarabine, cyclophosphamide, cladribine, denileukin diftitox, or combinations thereof.

[0151] It should be understood that the present disclosure and the aspects and embodiments provided herein are not limited to the particular examples disclosed, and as such may vary. It should also be understood that the terminology used herein is for the purpose of disclosing only particular examples and embodiments, and is not intended to be limiting, as the scope of the present disclosure will be limited only by the appended claims.

[0152] When a range of values is given, it is understood that each intervening value, up to the tenth of a unit of the lower limit, unless the context clearly indicates otherwise, between the upper and lower limits of that range and any other value mentioned or intervening in that mentioned range is considered part of the disclosure. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges and are also part of the invention, subject to any limit specifically excluded in the mentioned range. When the indicated range includes one or both limits, ranges that exclude one or both of these included limits are also included in the invention. When multiple low values and multiple high values for ranges are given, a person skilled in the art will recognize that a selected range will include a low value that is less than the high value. All headings in this descriptive report are for the convenience of the reader and are not limiting.

[0153] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by an individual of ordinary skill in the art to which this invention pertains. Although any methods and materials similar or equivalent to those described herein may also be used in the practice of the present invention, preferred methods and materials will now be described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in conjunction with which the publications are cited.

[0154] It should be noted that as used in this document and the accompanying claims, the singular forms “a”, “an”, “the” and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, the reference to “a chimeric antigen receptor” includes a plurality of such chimeric antigen receptors and equivalents thereof known to those skilled in the art, and so forth. It is further noted that claims may be drafted to exclude any optional element. As such, this statement is intended to serve as a background for the use of such exclusive terminology as “only”, “just” and the like in connection with the citation of elements of the claim or the use of a “negative” limitation.

[0155] It is understood that certain features of the invention, which are, for clarity, described in the context of separate embodiments, can also be provided in combination in a single embodiment. Conversely, several features of the invention, which are, for brevity, described in the context of a single embodiment, can also be provided separately or in any suitable subcombination. All combinations of the embodiments belonging to the invention are specifically covered by the present invention and are disclosed herein as if each and every combination were individually and explicitly disclosed. Additionally, all subcombinations of the various embodiments and elements thereof are also specifically covered by the present invention and are disclosed herein as if each and every said subcombination were individually and explicitly disclosed herein.

DETAILED DESCRIPTION

[0156] The present disclosure overcomes these challenges of the prior art by providing methods and compositions for genetically modifying lymphocytes and methods for performing adoptive cell therapy that include transducing T cells and/or NK cells that require much less ex vivo time, for example, 24, 12 or 8 hours or less and, in some modalities, without prior ex vivo stimulation. These methods are well suited for closed-system ex vivo processing of a subject's blood and can be performed with the subject present in the same environment as and/or, in some modalities, within their line of sight of their blood or isolated blood cells at all times during the performance of the method. More specifically, the aspects and modalities of the disclosure in this document overcome the problems associated with current adoptive cell therapies by providing methods for transducing resting T cells and/or resting NK cells that typically utilize a pseudotyping element that facilitates the binding and fusion of a recombinant retrovirus to a resting T cell and/or resting NK cell, to facilitate the genetic modification of resting T cells and/or NK cells by recombinant retroviruses. Furthermore, the methods provided in this document overcome the problems of the technique by using, in the illustrative embodiments, a chimeric antigen receptor and a lymphoproliferative element whose expression is under the control of an in vivo control element, such that exposure of the subject to a compound that binds to the in vivo control element, or termination of such exposure, promotes the expansion of genetically modified T cells and/or NK cells in vivo.

[0157] As a result of these and other improvements disclosed in detail in this document, in one aspect, a method is provided in this document for modifying resting T cells and/or resting NK cells of a subject, such as a patient who has a disease or disorder, wherein the subject's blood is collected; the resting T cells and/or NK cells are genetically modified by contacting them with a recombinant retrovirus; and the genetically modified cells are reintroduced into the subject typically within a shorter time period than previous methods, for example, within 24 hours and, in some non-limiting embodiments, within 12 hours and/or without further expansion of the genetically modified T cell and/or NK cell population ex vivo, for example, such that the genetically modified resting T cells and/or NK cells do not undergo more than 4 cell divisions ex vivo. Thus, the methods provided in this document can be performed in significantly less time than current CAR therapies, thereby providing processes by which a subject can remain in a clinic for the entire duration of the ex vivo steps. This facilitates the performance of the ex vivo steps in a closed system, reducing the chances of contamination and mixing of patient samples, and can be more readily performed by clinical laboratories.

[0158] Consequently, Figures 1 and 2 provide schematic diagrams of the illustrative compositions used in the methods provided in this document. Figure 1 provides a diagram of a packaging cell (100) and a recombinant retrovirus produced by such packaging cell (200). The packaging cell (100) includes recombinant polynucleotides (110) incorporated into its genome which include recombinant transcriptional elements expressing retroviral proteins and several different membrane-bound polypeptides under the control of inducible promoters that are regulated by transactivators, which bind to and are activated by ligands. These transactivators, inducible promoters and ligands are used to induce the sequential expression and accumulation of cell membrane-bound polypeptides that will be incorporated into the recombinant retrovirus membrane as well as retroviral components necessary for retrovirus packaging and assembly.

[0159] As a result of the sequential induced expression of the various polynucleotides, as discussed in detail in the present document below, the illustrative packaging cell (100) illustrated in Figure 1 is produced and can be used in the illustrative methods to produce recombinant retroviruses used in the methods for transfecting T cells and/or resting NK cells ((300) in Figure 2) provided in the present document. The packaging cell (100), in non-limiting illustrative embodiments, includes in its genome nucleic acids encoding a packageable retroviral RNA genome that includes at least some of the elements of a retroviral genome required for retrovirus packaging and assembly (as non-limiting illustrative examples, a retroviral psi element, a retroviral gag polypeptide and a retroviral pol polypeptide).

[0160] Some membrane-bound polypeptides incorporated into or associated with the cell membrane of the packaging cell will become incorporated into or associated with the retrovirus, but are not encoded by the retroviral genome. For example, the packaging cell and the recombinant retrovirus formed from it may include a retroviral Vpx polypeptide (250), which, in non-limiting illustrative examples, may be expressed as a membrane-associated fusion protein, for example, an Src-Flag-Vpx polypeptide; a pseudotyping element that may include a linking polypeptide and a fusogenic polypeptide (240), which, in a non-limiting embodiment, includes a Measles Virus hemagglutinin polypeptide (H) and a Measles Virus fusion polypeptide (F), or cytoplasmic domain deletion variants thereof; Optionally, one or more activating elements (210, 220), which, in a non-limiting embodiment, include a membrane-bound polypeptide capable of binding to CD3 and a membrane-bound polypeptide capable of binding to CD28; and/or, optionally, a membrane-bound cytokine (230), a non-limiting embodiment of which is a fusion polypeptide that includes IL-7 fused to DAF, or a fragment thereof. Several other specific types of these membrane-bound polypeptides are provided in this document.

[0161] As a result of the sequential expression of transcriptional elements by the packaging cell, a recombinant retrovirus is produced. The retroviral RNA genome within and typically integrated into the genome of the packaging cell that becomes the genome of the recombinant retrovirus includes retroviral components (as illustrative, non-limiting examples, retroviral Gag and Pol polynucleotides) that are necessary for retroviral production, infection, and integration into the genome of a host cell, which is typically a resting T cell and/or NK cell. Furthermore, the retroviral genome additionally includes polynucleotides encoding one or typically two genetically modified signaling polypeptides provided herein. One of the genetically modified signaling polypeptides typically encodes a lymphoproliferative element (in non-limiting examples, a constitutive interleukin 7 receptor mutant) and the other genetically modified signaling polypeptide typically encodes a chimeric antigen receptor.

[0162] The recombinant retrovirus (200) is then used to transduce a resting T cell and/or resting NK cell (300) in the methods provided in this document. As shown in Figure 2, after the resting T cell and/or resting NK cell (300) are brought into contact with the recombinant retrovirus (200), the membrane polypeptides discussed above on the surface of the retrovirus bind to receptors and/or ligands on the surface of the resting T cell and/or resting NK cell (300). For example, the pseudotyping element, which, as indicated above, may include a binding polypeptide that binds to molecules on the surface of resting T cells and/or resting NK cells and a fusogenic polypeptide, facilitates the binding and fusion of the retrovirus (200) to the T cell and/or NK cell membrane. The activation element (or elements) (210, 220) activates the resting T cell and/or NK cell (300) by interconnecting the T cell receptor complex, a process that occurs over the course of time following contact or subsequent incubation. Furthermore, membrane-bound cytokines (230) may be present on the retrovirus surface and may bind to cytokine receptors (310) on the surface of the resting T cell and/or NK cell (300), thus further promoting binding and activation. Therefore, without delving into theory, in the illustrative embodiments provided herein, as a result of one or more of these recombinant retrovirus components (200), ex vivo stimulation or activation may not be required by an element that is no longer within or on the retrovirus (200). This, in turn, helps to reduce the ex vivo time required for completion of the methods in these illustrative methods provided herein.

[0163] By binding to the T cell and/or NK cell (200), the retrovirus then fuses with the T cell and/or NK cell (300), and the polypeptides and nucleic acids in the retrovirus enter the T cell and/or NK cell (300). As indicated above, one of these polypeptides in the retrovirus is the Vpx polypeptide (250). The Vpx polypeptide (250) binds to and induces the degradation of the restriction factor SAMHD1 (350), which degrades free dNTPs in the cytoplasm. Thus, the concentration of free dNTPs in the cytoplasm increases as Vpx degrades SAMHD1, and reverse transcription activity is increased, thus facilitating reverse transcription of the retroviral genome and integration into the T cell and/or NK cell genome.

[0164] After integration of the retroviral genome into the T cell and/or NK cell (200), the T cell and/or NK cell genome includes nucleic acids encoding the signaling polypeptide that encodes the lymphoproliferative element (370) and, optionally, the signaling polypeptide that encodes the CAR (360). The expression of the lymphoproliferative element and, optionally, the CAR is under the control of a control element in vivo. Exposure to a compound that binds to the control element in vivo, which occurs in vivo by administering it to a subject whose T cell and/or NK cell (300) have been transduced, promotes the proliferation of the T cell and/or NK cell (300) in vivo by expressing the lymphoproliferative element and, optionally, as a result of CAR expression and CAR binding to its target cell. Thus, the T cells and/or NK cells that are transduced with recombinant retroviruses in this document have one or more signals that trigger proliferation and/or inhibit cell death, which, in turn, in the illustrative embodiments, avoids the requirements of previous methods to lymphodeplete a host before returning the transduced T cells and/or NK cells to the subject. This, in turn, in the illustrative embodiments, further reduces the processing time requirement before the transduced T cells and/or NK cells are reintroduced into a subject. Thus, in the illustrative embodiments, no more than 36 hours, 24 hours, 12 hours, or in some cases even 8 hours of time are required from blood collection from the subject to blood reintroduction to the subject, which fundamentally alters the CAR-T process of previous methods. Furthermore, the in vivo control element provides one of the safety mechanisms also provided in this document. For example, ceasing administration of the compound may decrement or even terminate the expression of the lymphoproliferative element and, optionally, the CAR, thus ending a proliferation and/or survival signal for the transduced T cell and/or NK cell and their progeny.

METHODS FOR PERFORMING ADOPTIVE CELL THERAPY

[0165] In certain respects, methods for performing adoptive cell therapy in a subject are provided in this document, as an illustrative example, the method may include the following: A. collecting blood from a subject; B. isolating peripheral blood mononuclear cells (PBMCs) comprising resting T cells and/or resting NK cells; C. placing the subject's resting T cells and/or resting NK cells ex vivo in contact with recombinant retroviruses, wherein the recombinant retroviruses comprise a pseudotyping element on their surface that has the ability to bind to a resting T cell and/or NK cell and facilitate the fusion of the recombinant retrovirus membrane to the same, wherein said contact facilitates the transduction of the resting T cells and/or NK cells by the recombinant retroviruses, thus producing genetically modified T cells and/or NK cells; and D. reintroduce the genetically modified cells into the subject within 36, 24, 12, or even 8 hours of collecting the subject's blood, thus performing adoptive cell therapy on the subject.

[0166] In some respects provided in this document, methods with similar steps are referred to as methods for genetically modifying and expanding a subject's lymphocytes. A person skilled in the art will understand that the discussion in this document, insofar as it applies to methods and compositions for performing adoptive cell therapy, also applies to methods for genetically modifying and expanding a subject's lymphocytes.

[0167] Typically, the adoptive cell therapy methods of the present disclosure are performed by autologous transfer, wherein cells are isolated and/or otherwise prepared from the subject who is to receive the cell therapy or from a sample derived from such subject. Thus, in some respects, cells are derived from a subject, for example, a patient, who needs treatment, and the cells, after isolation and processing, are administered to the same subject. In some embodiments of the methods and compositions disclosed herein, a subject who has a disease or disorder enters a medical facility where the subject's blood is drained using known methods, such as venipuncture. In certain embodiments, the volume of blood drained from a subject is between 10, 15, 20, 25, 30, 35, 40, 50, 75, or 100 ml at the lower end of the range and 200, 250, 300, 350, 400, 500, 750, 1,000, 2,000, or 2,500 ml at the upper end of the range. In some embodiments, between 10 and 400 ml are drained from the subject. In some embodiments, between 20 and 250 ml of blood are drained from the subject. In some embodiments, the blood is fresh when it is processed. In any of the embodiments disclosed herein, fresh blood may be blood that has been withdrawn from a subject less than 15, 30, 45, 60, 90, 120, 150, or 180 minutes prior. In some modalities, the blood is processed using the methods described in this document without storage.

[0168] Contact between T cells and/or NK cells and recombinant retroviruses typically facilitates the transduction of T cells and/or NK cells by the recombinant retrovirus. For all of this disclosure, a transduced T cell and/or NK cell includes the progeny of ex vivo transduced cells that retain at least some of the nucleic acids or polynucleotides that are incorporated into the cell during ex vivo transduction. In the methods in this document that refer to the “reintroduction” of a transduced cell, it will be understood that such a cell is not typically in a transduced state when it is collected from the blood of a subject. A subject in any of the aspects disclosed in this document may be, for example, an animal, a mammal, and, in the illustrative embodiments, a human being.

[0169] Without adhering to theory, in non-limiting illustrative methods, the delivery of a polynucleotide encoding a lymphoproliferative element, such as a constitutively active IL7 mutant, to a resting T cell and/or NK cell ex vivo, which can integrate into the T cell or NK cell genome, provides that cell with a trigger for in vivo expansion without the need to lymphodeplete the host. Thus, in the illustrative embodiments, the subject is not exposed to a lymphodepleting agent within 1, 2, 3, 4, 5, 6, 7, 10, 14, 21, or 28 days or within 1 month, 2 months, 3 months, or 6 months of contact, during contact, and/or within 1, 2, 3, 4, 5, 6, 7, 10, 14, 21, or 28 days or within 1 month, 2 months, 3 months, or 6 months after the modified T cells and/or NK cells are reintroduced to the subject. Furthermore, in non-limiting illustrative embodiments, the methods provided herein may be performed without exposing the subject to a lymphodepleting agent during the step in which a recombinant retrovirus is in contact with the subject's resting T cells and/or resting NK cells and/or during the entire ex vivo method.

[0170] Therefore, methods for expanding genetically modified T cells and/or NK cells in a subject in vivo are a feature of some embodiments of the present disclosure. In the illustrative embodiments, such methods are free from ex vivo propagation or substantially free from propagation.

[0171] This entire method/process of draining blood from a subject to reintroducing the blood back into the subject after ex vivo transduction of T cells and/or NK cells, in the illustrative embodiments not limiting in this document, may occur over a period of time less than 48 hours, less than 36 hours, less than 24 hours, less than 12 hours, less than 11 hours, less than 10 hours, less than 9 hours, less than 8 hours, less than 7 hours, less than 6 hours, less than 5 hours, less than 4 hours, less than 3 hours or less than 2 hours. In other embodiments, the entire method/process of draining/collecting blood from a subject for reintroduction of the blood back into the subject after ex vivo transduction of T cells and/or NK cells, in the illustrative embodiments not limiting in this document, occurs over a period of time between 1 hour and 12 hours, or between 2 hours and 8 hours, or between 4 hours and 12 hours, or between 4 hours and 24 hours, or between 8 hours and 24 hours, or between 8 hours and 36 hours, or between 8 hours and 48 hours, or between 12 hours and 24 hours, or between 12 hours and 36 hours, or between 12 hours and 48 hours, or over a period of time between 15, 30, 60, 90, 120, 180 and 240 minutes at the lower end of the range and 120, 180, and 240, 300, 360, 420, and 480 minutes at the upper end of the range. In other embodiments, the entire method/process of draining/collecting blood from a subject for reintroduction of the blood back into the subject after ex vivo transduction of T cells and/or NK cells occurs over a time period between 1, 2, 3, 4, 6, 8, 10, and 12 hours at the lower end of the range, and 8, 9, 10, 11, 12, 18, 24, 36, or 48 hours at the upper end of the range. In some embodiments, the genetically modified T cells and/or NK cells are separated from the recombinant retroviruses after the time period in which contact occurs.

[0172] Because the methods provided in this document for adoptive cell therapy and related methods for modifying resting T cells and/or resting NK cells ex vivo before expanding them in vivo can be performed in significantly less time than previous methods, fundamental improvements in safety and patient care as well as product manufacturability are made possible. Therefore, it is expected that such processes will be favorable in the view of regulatory agencies responsible for approving such processes when performed in vivo for therapeutic purposes. For example, the subject in non-limiting examples can remain in the same building (e.g., infusion clinic) or environment as the instrument processing their blood or sample for the entire time the sample is being processed before the modified T cells and/or NK cells are reintroduced into the patient. In illustrative, non-limiting embodiments, a subject remains within the site line and/or within 30, 48, 15, 24, 7.62, or 3.66 meters (100, 50, 25, or 12 feet) or an arm's length of their blood or cells being processed, throughout the entire method/process of draining/collecting the subject's blood for reintroduction of the blood to the subject after ex vivo transduction of T cells and/or NK cells. In other illustrative, non-limiting embodiments, a subject remains awake and/or at least one person may continue to monitor the subject's blood or cells being processed, completely and/or continuously throughout the entire method/process of draining/collecting the subject's blood for reintroduction of the blood to the subject after ex vivo transduction of T cells and/or NK cells. Due to the embodiments provided in this document, the entire method/process for adoptive cell therapy and/or for transducing T cells and/or NK cells at rest, from draining/collecting blood from the subject to reintroducing the blood to the subject after ex vivo transduction of T cells and/or NK cells, can be performed with continuous monitoring by a human being. In other illustrative, non-limiting embodiments, at no point in the entire method/process from draining/collecting blood from the subject to reintroducing the blood to the subject after ex vivo transduction of T cells and/or NK cells are blood cells incubated in an environment where no person is present. In other illustrative, non-limiting embodiments, the entire method/process from draining/collecting blood from the subject to reintroducing the blood to the subject after ex vivo transduction of T cells and/or NK cells is performed near the subject and/or in the same environment as the subject and/or near the subject's bed or chair. Thus, sample identity mix-ups can be avoided, as well as lengthy and costly incubations over periods of days or weeks. This is further facilitated by the fact that the methods provided in this document are readily adaptable to closed and automated blood processing systems, where a blood sample and its components to be reintroduced into the subject only come into contact with disposable, single-use components.

[0173] The methods for performing adoptive cell therapy provided in this document typically include methods for transducing T cells and/or resting NK cells, which are distinct aspects of the present disclosure. A person skilled in the art will recognize that the details provided in this document for transducing T cells and/or NK cells can apply to any aspect that includes such a step(s). Consequently, a method for transducing a T cell and/or an NK cell, typically a resting T cell and/or a resting NK cell, is provided in certain aspects hereof, which includes placing the resting T cell and/or resting NK cell in contact with a recombinant retrovirus, wherein the recombinant retrovirus typically comprises a pseudotyping element on its surface that has the ability to bind to the resting T cell and/or NK cell and facilitate the fusion of the recombinant retrovirus membrane to the same, wherein said contact (and incubation under contact conditions) facilitates the transduction of the resting T cell and/or NK cell by the recombinant retroviruses, thus producing the genetically modified T cell and/or NK cell. Further embodiments of such a method may include any of the retrovirus embodiments, lymphoproliferative elements, CARs, pseudotyping elements, riboswitches, activation elements, membrane-bound cytokines, miRNAs and/or other elements disclosed herein. This method for transducing a T cell and/or NK cell can be performed in vitro or ex vivo.

[0174] In adoptive cell therapy methods and any method provided herein that includes transducing resting T cells and/or resting NK cells ex vivo, typically, neutrophils/granulocytes are separated from blood cells before the cells are contacted with the recombinant retrovirus. In some embodiments, peripheral blood mononuclear cells (PBMCs), including peripheral blood lymphocytes (PBLs) such as T cells and/or NK cells, are isolated from other components of a blood sample using, for example, apheresis and/or density gradient centrifugation. In some embodiments, neutrophils are removed before PBMCs and/or T cells and/or NK cells are processed, contacted with a recombinant retrovirus, transduced, or transfected. With reference to the subject to be treated, the cells may be allogeneic and/or autologous.

[0175] As non-limiting examples, in some embodiments, PBMCs are isolated using a Sepax or Sepax 2 cell processing system (BioSafe). In some embodiments, PBMCs are isolated using a CliniMACS Prodigy cell processor (Miltenyi Biotec). In some embodiments, an automated apheresis separator is used that draws blood from the subject, passes the blood through a device that selects a particular cell type (such as, for example, PBMCs) and returns the remainder to the subject. Density gradient centrifugation may be performed after apheresis. In some embodiments, PBMCs are isolated using a leukoreduction filter device. In some embodiments, magnetic microsphere-activated cell sorting is then used to purify a specific population of PBMCs, such as PBLs or a subset thereof, according to a cell phenotype (i.e., positive selection). Other purification methods can also be used, such as substrate adhesion, which uses a substrate that mimics the environment in which a T cell is found during recruitment, enabling them to adhere and migrate, or negative selection, in which unwanted cells are targeted for removal with antibody complexes that target unwanted cells. In some embodiments, erythrocyte rosette formation can be used to purify cells.

[0176] In some illustrative embodiments of any of the relevant aspects in this document, PBLs include T cells and/or NK cells. The T cells and/or NK cells that are brought into contact with recombinant retroviruses of the present disclosure during certain embodiments in this document, for example, in methods for modifying lymphocytes and methods for performing adoptive cell therapy, are primarily resting T cells. In some embodiments, the T cells and/or NK cells consist of between 95 and 100% resting cells (Ki-67-). In some embodiments, the T cells and/or NK cells that are brought into contact with recombinant retroviruses include between 90, 91, 92, 93, 94 and 95% resting cells at the lower end of the range and 96, 97, 98, 99 or 100% resting cells at the upper end of the range. In some embodiments, the T cells and/or NK cells include naive cells.

[0177] In some embodiments of the methods and compositions disclosed in this document, T cells and/or NK cells are brought into ex vivo contact with recombinant retroviruses to genetically modify the T cells and/or NK cells to elicit a targeted immune response in the subject when reintroduced into the subject. During the contact period, the recombinant retroviruses identify and bind to T cells and/or NK cells, at which point the host and retroviral cell membranes begin to fuse. Then, through the process of transduction, the genetic material of the recombinant retroviruses enters the T cells and/or NK cells and is incorporated into the host cell DNA. Lentiviral transduction methods are known. Exemplary methods are described in, for example, Wang et al. (2012) J. Immunother. 35(9): 689 to 701; Cooper et al. (2003) Blood. 101:1637 to 1644; Verhoeyen et al. (2009) Methods Mol Biol. 506: 97 to 114; and Cavalieri et al. (2003) Blood. 102(2): 497 to 505.

[0178] Many of the methods provided in this document involve the transduction of T cells and/or NK cells. The methods are known in the art for transducing T cells and/or NK cells ex vivo with retroviruses, such as lentiviruses. The methods provided in this document, in the illustrative embodiments, do not require ex vivo activation or stimulation. Thus, this step common in previous methods can be avoided in the present method, although an ex vivo stimulating molecule (or molecules), such as anti-CD3 and/or anti-CD28 microspheres, may be present during transduction. However, with the illustrative methods provided in this document, ex vivo stimulation is not required. In certain exemplary methods, between 3 and 10 multiplicities of infection (MOI) and, in some embodiments, between 5 and 10 MOI units of the retrovirus, for example, lentivirus, may be used.

[0179] The transduction reaction can be performed in a closed system, such as a Sepax system, as discussed in this document, where the transduction reaction can be performed in disposable bags loaded into the system. Blood cells, such as PBMCs, from the blood sample collected from the subject can be brought into contact with the recombinant retroviruses disclosed in this document, in a bag as soon as these blood cells are separated, isolated and/or purified away from granulocytes, including neutrophils, which are not typically present during the contact step (i.e., the transduction reaction).

[0180] The retrovirus can be introduced into the bag containing the isolated PBMCs, thus making contact with the PBMCs. The time from the subject's blood collection to the time when blood cells, such as PBMCs, are added to the transduction reaction bag can be between 30 minutes and 4 hours, between 30 minutes and 2 hours, or about 1 hour in some examples. Additives such as media, human serum albumin, human AB+ serum, and/or subject-derived serum can be added to the transduction reaction mixture. Media is typically present, such as those known in the art for ex vivo processes (as non-limiting examples, X-VIVO 15 medium (Lonza) or CTS (Thermo Fisher Scientific)). Supportive cytokines can be added to the transduction reaction mixture, such as IL2, IL7, or IL15, or those found in HSA.

[0181] The transduction reaction mixture can be incubated between 23 and 39 °C and, in some illustrative embodiments, at 37 °C. In certain embodiments, the transduction reaction can be performed at 37 to 39 °C for faster fusion/transduction. dGTP can be added to the transduction reaction. The transduction reaction mixture can be incubated for 1 to 12 hours and, in some embodiments, 6 to 12 hours. After transduction, before the transduced T cells and/or NK cells are infused back into the subject, the cells are removed by washing the transduction reaction mixture. For example, the system, such as a Sepax instrument, can be used to wash the cells, for example, with 10 to 50 ml of washing solution, before the transduced cells are infused back into the subject. In some modalities, neutrophils are removed before PBMCs and/or T cells and/or NK cells are processed, placed in contact with a recombinant retrovirus, transduced, or transfected.

[0182] In an illustrative embodiment for performing adoptive cell therapy, blood is collected from a subject in a blood bag, and the blood bag is attached to a cell processing system, such as a Sepax cell processing system. PBMCs isolated using the cell processing system are collected in a bag, brought into contact with recombinant retrovirus under conditions sufficient to transduce T cells and/or NK cells, and incubated. After incubation, the bag containing the mixture of PBMCs and recombinant retrovirus is attached to a cell processing system, and the PBMCs are washed. The washed PBMCs are collected in a bag and reinfused into the subject. In some modalities, the entire method, from blood collection to reinfusion of transduced T and/or NK cells, is performed within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 18, or 24 hours. In the illustrative modalities, the entire method is performed within 12 hours.

[0183] In some embodiments, the target cells for recombinant retroviruses are PBLs. In some embodiments, the target cells are T cells and/or NK cells. In some embodiments, the T cells are helper T cells and/or killer T cells.

[0184] In some embodiments, the recombinant retroviruses provided in this document have pseudotyping elements on their surface that have the ability to bind to T cells and/or NK cells and facilitate membrane fusion of the recombinant retroviruses to them. In other embodiments, the recombinant retroviruses have activation elements on their surface that have the ability to bind to resting T cells and/or NK cells. In still other embodiments, the recombinant retroviruses have membrane-bound cytokines on their surface. In some embodiments, the recombinant retroviruses include a polynucleotide that has one or more transcriptional units encoding one or more genetically modified signaling polypeptides, one or more of which includes a lymphoproliferative element. In other embodiments, when two signaling polypeptides are used, one includes a lymphoproliferative element and the other is typically a chimeric antigen receptor (CAR) that includes an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain. As indicated in this document, an activation element (or elements) that is typically associated with the surface of a recombinant retrovirus provided in this document has the ability to, and as a result of contact with resting T cells and/or NK cells for a sufficient period of time and under appropriate conditions, activate the resting T cells and/or NK cells. It will be understood that such activation occurs over time during a contact step of the methods in this document. Furthermore, it will be understood that, in some embodiments where a pseudotyping element is found on the surface of a recombinant retrovirus that binds to a T cell and/or an NK cell, in the methods in this document, activation may be induced by the binding of the pseudotyping element. An activation element is optional in these embodiments.

[0185] Further details regarding a pseudotyping element, an activation element, a membrane-bound cytokine, a genetically modified signaling polypeptide, a lymphoproliferative element, and a CAR are provided in other sections of this document.

[0186] In some embodiments of the methods and compositions disclosed in this document, between 5% and 90% of the total lymphocytes collected from the blood are transduced. In some embodiments, the percentage of lymphocytes that are transduced is between 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 and 60% at the lower end of the range and 50, 55, 60, 65, 70, 75, 80, 85 and 90% at the upper end of the range. In some modalities, the percentage of lymphocytes that are transduced is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, or at least 60%.

[0187] In some embodiments of the methods and compositions disclosed herein, the genetically modified T cells and/or NK cells are reintroduced, reintroduced, or reinfused into the subject without further ex vivo manipulation, such as stimulation and/or activation of T cells and/or NK cells. In prior art methods, ex vivo manipulation is used for stimulation/activation of T cells and/or NK cells and for expansion of genetically modified T cells and/or NK cells prior to introducing the genetically modified T cells and/or NK cells into the subject. In prior art methods, this typically takes days or weeks and requires a subject to return to a clinic for a blood infusion days or weeks after an initial blood drain. In some embodiments of the methods and compositions disclosed herein, T cells and/or NK cells are not stimulated ex vivo by exposure to anti-CD3/anti-CD28 solid supports, such as, for example, anti-CD3/anti-CD28 coated microspheres, prior to contact of the T cells and/or NK cells with the recombinant retroviruses. As such, an ex vivo propagation-free method is provided herein. In other embodiments, the genetically modified T cells and/or NK cells are not expanded ex vivo or are only expanded by a small number of cell divisions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cell division cycles), but are instead expanded, or predominantly expanded, in vivo, i.e., within the subject. In some embodiments, no additional medium is added to allow for further cell expansion. In some embodiments, no cellular fabrication of PBLs occurs while the PBLs are in contact with the recombinant retrovirus. In illustrative embodiments, no cellular fabrication of PBLs occurs while the PBLs are ex vivo. In previous adoptive cell therapy methods, subjects were lymphodepleted prior to reinfusion with genetically modified T cells and/or NK cells. In some embodiments, patients or subjects are not lymphodepleted prior to blood withdrawal. In some embodiments, patients or subjects are not lymphodepleted prior to reinfusion with genetically modified T cells and/or NK cells.

[0188] In any of the modalities disclosed in this document, the number of T cells and/or NK cells to be reinfused into a subject may be between 1 x 10⁻⁶, 2.5 x 10⁻⁶, 5 x 10⁻⁶, 1 x 10⁻⁶, 2.5 x 10⁻⁶, 5 x 10⁻⁶, 1 x 10⁻⁶, 2.5 x 10⁻⁵, 5 x 10⁻⁵, 1 x 10⁻⁶, 2.5 x 10⁻⁵, 5 x 10⁻⁵, 1 x 10⁻⁶, 2.5 x 10⁻⁶, 5 x 10⁻⁶ and 1 x 10⁻⁷ cells/kg at the extreme end. 105 1 106 2 5 106 5 106 lower end of the range and 5 x 10, 1 x 10, 2.5 x 10, 5 x 10, 1 x 10, 2.5 x 10, 5 x 10, 1 x 107, 2.5 x 107, 5 x 107 and 1 x 108 cells/kg at the upper end of the range. In the illustrative embodiments, the number of T cells and/or NK cells to be reinfused into a subject may be between 1 x 10⁴, 2.5 x 10⁴, 5 x 10⁴ and 1 x 10⁵ cells/kg at the lower end of the range and 2.5 x 10⁴, 5 x 10⁴, 1 x 10⁵, 2.5 x 10⁵, 5 x 10⁵ and 1 x 10⁶ cells/kg at the upper end of the range. In some modalities, the number of PBLs to be reinfused into a subject may be less than 5 x 10⁵, 1 x 10⁶, 2 x 10⁶, 5 x 10⁶, 1 x 10⁷, 2 x 10⁷, 5 x 10⁷, and 1 x 10⁸ cells at the lower end of the range and 2.5 x 10⁶, 5 x 10⁶, 1 x 10⁷, 2.5 x 10⁷, 5 x 10⁷, 1 x 10⁸, 2.5 x 10⁸, 5 x 10⁸, and 1 x 10⁹ cells at the upper end of the range. In some modalities, the number of T cells and/or NK cells available for reinfusion in a 70 kg subject or patient is between 7 x 10⁵ and 2.5 x 10⁸ cells. In other modalities, the number of T cells and/or NK cells available for transduction is approximately 7 x 10⁶ plus or minus 10%.

[0189] In the methods disclosed in this document, the entire adoptive cell therapy procedure, from blood withdrawal to reinfusion of genetically modified T cells and/or NK cells, can advantageously be performed in a shorter time than previous methods. In some embodiments, the entire adoptive cell therapy procedure can be performed in less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 18 or 24 hours. In the illustrative embodiments, the entire adoptive cell therapy procedure can be performed in less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 hours. In some modalities, the entire adoptive cell therapy procedure can be performed between 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 15 hours at the lower end of the range and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 18, or 24 hours at the upper end of the range.

[0190] In some embodiments provided herein, the steps of withdrawing a blood sample from a subject, contacting T cells and/or NK cells with recombinant retroviruses, and/or introducing genetically modified T cells and/or NK cells into the subject occur in a closed system. A closed system is a culture process that is generally closed or completely closed to contamination. An advantage of the present invention is that methods for performing CAR therapy in a closed system are provided herein. One of the greatest risks to safety and regulatory control in cell processing procedures is the risk of contamination through frequent exposure to the environment as found in traditional open cell culture systems. To mitigate this risk, particularly in the absence of antibiotics, some commercial processes have been developed that focus on the use of disposable (single-use) equipment. However, even with their use under aseptic conditions, there is always a risk of contamination from the openings of the bottles for sampling or adding additional growth medium. To overcome this problem, a closed-system process is provided in this document, a process that is designed and can be operated in such a way that the product is not exposed to the external environment. This is important due to the fact that the external environment is not typically sterile. Material transfer occurs through sterile connections or pipe welding. Air for gas exchange occurs through a gas-permeable membrane or other similar additions, via a 0.2 μm filter to prevent environmental exposure.

[0191] In some embodiments, the closed system includes an ex vivo circulation system connected to the subject’s in vivo circulatory system such that blood is drained and then circulated to the ex vivo circulatory system before being reintroduced to the subject. In some embodiments, the ex vivo circulatory system includes a system or apparatus for isolating PBLs and/or a system or apparatus for isolating T cells and/or NK cells, in combination with the system or apparatus for exposing the cells to the recombinant retrovirus. In some embodiments, the closed system does not allow the T cells and/or NK cells to be exposed to air.

[0192] Such closed-system methods can be performed with commercially available devices. For example, the method can be performed on devices adapted for closed-system T-cell production. Such devices include a G-Rex™, a WAVE Bioreactor™, an OriGen PermaLife™ bag, and a VueLife® bag.

[0193] In some embodiments of the methods and compositions disclosed herein, genetically modified T cells and/or NK cells within a subject are exposed to a compound that binds to an in vivo control element present on them, wherein the in vivo control element is a part of the genetic material introduced by recombinant retroviruses. In some embodiments, the in vivo control element may be a riboswitch and the compound may bind to the aptamer domain of the riboswitch. In some embodiments, the in vivo control element may be a molecular chaperone. In any of the embodiments disclosed herein, the compound may be a nucleoside analog. In some embodiments, the nucleoside analog may be a nucleoside analog antiviral drug, wherein an antiviral drug is a compound approved by the Food and Drug Administration for antiviral treatment or a compound in an antiviral clinical trial in the United States. In illustrative embodiments, the compound may be acyclovir or penciclovir. In some embodiments, the compound may be famciclovir, the oral prodrug of penciclovir, or valacyclovir, the oral prodrug of acyclovir. The binding of the compound to the control element in vivo affects the expression of the introduced genetic material and, therefore, the propagation of genetically modified T cells and/or NK cells.

[0194] In some embodiments, the nucleoside analog antiviral drug or prodrug, for example, acyclovir, valacyclovir, penciclovir, or famciclovir, is administered to the subject before, concomitantly with, and/or after PBLs are isolated from the subject's blood and before T cells and/or NK cells are brought into contact with a recombinant retrovirus. In some embodiments, the prodrug or nucleoside analog antiviral drug is administered to the subject between 5, 10, 15, 30, and 60 minutes at the lower end of the range and 1, 5, 2, 3, 4, 5, 6, 8, 12, or 24 hours at the upper end of the range before PBLs are isolated from the blood or before T cells and/or NK cells are brought into contact with a recombinant retrovirus. In other embodiments, the prodrug or nucleoside analog antiviral drug is administered to the subject for between 1.5, 2, 3, 4, 5, 6, 8, 12, or 24 hours at the lower end of the range and 1, 2, 3, 4, 5, 6, 7, 10, 14, 21, or 28 days at the upper end of the range, after PBLs are isolated from the blood and T cells and/or NK cells are brought into contact with a recombinant retrovirus using the methods provided in this document. In some embodiments, the prodrug or nucleoside analog antiviral drug is administered to the subject for at least 1.5, 2, 3, 4, 5, 6, 8, 12, or 24 hours, or at least 2, 3, 4, 5, 6, 7, 10, 14, 21, or 28 days after PBLs are isolated from the blood and T cells and/or NK cells are brought into contact with a recombinant retrovirus using the methods provided in this document. In some embodiments, the prodrug or nucleoside analog antiviral drug is administered to the subject for at least 1, 2, 3, 4, 5, 7, 10, 14, 21, 28, 30, 60, 90, or 120 days, or 5, 6, 9, 12, 24, 36, 48, 60, 72, 84, 96, 120 months, or indefinitely after the PBLs have been reinfused into the subject. In any of the embodiments disclosed herein, the prodrug or nucleoside analog antiviral drug may be administered before and/or during the reinfusion of the PBLs and/or after the PBLs have been reinfused.

[0195] In some embodiments, the compound that binds to the control element in vivo is administered once, twice, three times, or four times daily to the subject. In some embodiments, daily doses of the compound are given for 1 week, 2 weeks, 4 weeks, 3 months, 6 months, 1 year, until a subject is free of a disease, such as cancer-free, or indefinitely. The drug, in the illustrative embodiments, is a nucleoside analog antiviral drug that binds to a nucleoside analog, such as a riboswitch, as disclosed in more detail herein.

[0196] The methods are known in the art for delivering drugs, or small molecules or biological products, and can be used in the methods provided in this document. Any such methods can be used to deliver candidate drugs or compounds or antibodies for use in the methods of the present invention. For example, common routes of administration include non-invasive peroral (through the mouth), topical (skin), transmucosal (nasal, buccal/sublingual, vaginal, ocular and rectal) and inhalation routes. Many protein and peptide drugs, such as monoclonal antibodies, have to be delivered by injection or a nanoneedle arrangement. For example, many immunizations rely on the delivery of protein drugs and are often done by injection.

GENETICALLY MODIFIED SIGNALING POLYPEPTIDE (OR POLYPEPTIDES)

[0197] In some embodiments, recombinant retroviruses used to contact T cells and/or NK cells have a polynucleotide that has one or more transcriptional units encoding one or more genetically modified signaling polypeptides, one or more of which include a lymphoproliferative element. In some embodiments, a signaling polypeptide includes any combination of the following: an extracellular antigen-binding domain (or antigen-specific targeting region or ASTR), a stem, a transmembrane domain, an intracellular activation domain, a lymphoproliferative element, a modulator domain (such as a costimulatory domain), and a T cell survival motif. In the illustrative embodiments, at least one, two, or all of the genetically modified signaling polypeptides are a CAR. In some embodiments, when two signaling polypeptides are used, one encodes a lymphoproliferative element and the other encodes a chimeric antigen receptor (CAR) that includes an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activation domain. In other embodiments, a CAR may include a lymphoproliferative element fused to an antigen-specific targeting region. In other embodiments, when the lymphoproliferative element is a constitutively active interleukin receptor, such as a known variant of IL-7Rα, no antigen-specific targeting region is required because binding is not ligand-dependent. A person of ordinary skill in the art would be able to reconfigure the system to place the lymphoproliferative element and the CAR on distinct polynucleotides with similar or dissimilar control elements for the methods and compositions disclosed herein. A person skilled in the art will recognize that such genetically modified polypeptides may also be referred to as recombinant polypeptides.

SPECIFIC TARGETING REGION FOR ANTIGEN

[0198] In some embodiments, a genetically modified signaling polypeptide includes a specific linking pair member, which is typically an ASTR, sometimes referred to as an antigen-binding domain in this document. Specific linking pairs include, but are not limited to, antigen-antibody linking pairs; ligand-receptor linking pairs; and the like. Thus, a specific linking pair member suitable for use in a genetically modified signaling polypeptide of the present disclosure includes an ASTR that is an antibody, an antigen, a ligand, a receptor-binding domain of a ligand, a receptor, a ligand-binding domain of a receptor, and an affibody.

[0199] An ASTR suitable for use in a genetically modified signaling polypeptide of the present disclosure may be any antigen-binding polypeptide. In certain embodiments, the ASTR is an antibody, such as a full-length antibody, a single-chain antibody, a Fab fragment, a Fab' fragment, a (Fab')2 fragment, an Fv fragment, and a divalent single-chain antibody or a diabody.

[0200] In some embodiments, the ASTR is a single-stranded Fv (scFv). In some embodiments, the heavy chain is at the N-terminal position of the light chain in the genetically modified signaling polypeptide. In other embodiments, the light chain is at the N-terminal position of the heavy chain in the genetically modified signaling polypeptide. In any of the disclosed embodiments, the heavy and light chains may be separated by a binder as discussed in more detail in this document. In any of the disclosed embodiments, the heavy or light chain may be at the N-terminus of the genetically modified signaling polypeptide and is typically C-terminal to another domain, such as a signaling sequence or peptide.

[0201] Other antibody-based recognition domains (cAb VHH (camellid antibody variable domains) and humanized versions, IgNAR VH (shark antibody variable domains) and humanized versions, sdAb VH (single-domain antibody variable domains) and "camelized" antibody variable domains) are suitable for use with the genetically modified signaling polypeptides and methods for using the genetically modified signaling polypeptides of the present disclosure. In some cases, T cell receptor (TCR)-based recognition domains, such as single-chain TCRs (scTv, two single-chain domain TCRs containing VαVβ), are also suitable for use.

[0202] In some embodiments, ASTRs can be multispecific antibodies, for example, bispecific antibodies. Multispecific antibodies have binding specificities for at least two different sites. In certain embodiments, one of the binding specificities is for one target antigen and the other is for another target antigen. In certain embodiments, bispecific antibodies can bind to two different epitopes of a target antigen. Bispecific antibodies can also be used to target cytotoxic agents to cells expressing a target antigen. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0203] An ASTR suitable for use in a genetically modified signaling polypeptide of the present disclosure may have a variety of antigen-binding specificities. In some cases, the antigen-binding domain is specific for an epitope present on an antigen that is expressed (synthesized) by a target cell. In one example, the target cell is a cancer cell-associated antigen. The antigen associated with a cancer cell can be an antigen associated with, for example, a breast cancer cell, a B-cell lymphoma, a Hodgkin lymphoma cell, an ovarian cancer cell, a prostate cancer cell, a mesothelioma, a lung cancer cell (e.g., a small cell lung cancer cell), a non-Hodgkin B-cell lymphoma (B-NHL) cell, an ovarian cancer cell, a prostate cancer cell, a mesothelioma cell, a lung cancer cell (e.g., a small cell lung cancer cell), a melanoma cell, a chronic lymphocytic leukemia cell, an acute lymphocytic leukemia cell, a neuroblastoma cell, a glioma, a glioblastoma, a medulloblastoma, a colorectal cancer cell, etc. A cancer cell-associated antigen can also be expressed by a non-cancerous cell.

[0204] Non-limiting examples of antigens to which an ASTR of a genetically modified signaling polypeptide can bind include, for example, CD19, CD20, CD38, CD30, ERBB2, CA125, MUC-1, prostate-specific membrane antigen (PSMA), CD44 surface adhesion molecule, mesothelin, carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR), EGFRvIII, vascular endothelial growth factor receptor-2 (VEGFR2), high molecular weight melanoma-associated antigen (HMW-MAA), MAGE-Al, IL-13R-α2, GD2, Axl, Ror2 and the like.

[0205] In some cases, a member of a specific linking pair suitable for use in a genetically modified signaling polypeptide is an ASTR that is a ligand for a receptor. Ligands include, but are not limited to, cytokines (e.g., IL-13, etc.); growth factors (e.g., heregulin; vascular endothelial growth factor (VEGF); and the like); an integrin-binding peptide (e.g., a peptide comprising the sequence Arg-Gly-Asp); and the like.

[0206] When the member of a specific linking pair in a genetically modified signaling polypeptide is a ligand, the genetically modified signaling polypeptide can be activated in the presence of a second specific linking pair member, wherein the second specific linking pair member is a receptor for the ligand. For example, where the ligand is VEGF, the second specific linking pair member can be a VEGF receptor, including a soluble VEGF receptor.

[0207] As noted above, in some cases, the member of a specific linking pair that is included in a genetically modified signaling polypeptide is an ASTR that is a receptor, for example, a receptor for a ligand, a co-receptor, etc. The receptor may be a ligand-binding fragment of a receptor. Suitable receptors include, but are not limited to, a growth factor receptor (e.g., a VEGF receptor); a killer cell lecithin-like receptor (NKG2D) subfamily K member polypeptide (receptor for MICA, MICB, and ULB6); a cytokine receptor (e.g., an IL-13 receptor; an IL-2 receptor; etc.); CD27; a natural cytotoxicity receptor (NCR) (e.g., NKP30 polypeptide (NCR3/CD337) (receptor for HLA-B-associated transcript 3 (BAT3) and B7-H6); etc.); etc.

STEM

[0208] In some embodiments, the genetically modified signaling polypeptide includes a stem that is located in the portion of the genetically modified signaling polypeptide that lies outside the cell and is interposed between the ASTR and the transmembrane domain. In some cases, the stem has at least 85, 90, 95, 96, 97, 98, 99, or 100% identity with a wild-type CD8 stem region (TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGG AVHTRGLDFA (SEQ ID NO:79)), has at least 85, 90, 95, 96, 97, 98, 99, or 100% identity with a wild-type CD28 stem region (FCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO:80)), or has at least 85, 90, 95, 96, 97, 98, 99, or 100% identity with a wild-type immunoglobulin heavy chain stem region. In a genetically modified signaling polypeptide, the employed stem allows the... A specific antigen-targeting region, and typically the entire genetically modified signaling polypeptide, retains increased binding to a target antigen.

[0209] The stem region can have a length of about 4 amino acids to about 50 amino acids, for example, from about 4 aa to about 10 aa, from about 10 aa to about 15 aa, from about 15 aa to about 20 aa, from about 20 aa to about 25 aa, from about 25 aa to about 30 aa, from about 30 aa to about 40 aa or from about 40 aa to about 50 aa.

[0210] In some cases, the stem of a genetically modified signaling polypeptide includes at least one cysteine. For example, in some cases, the stem may include the sequence Cys-Pro-Pro-Cys (SEQ ID NO:62). If present, a cysteine in the stem of a first genetically modified signaling polypeptide may be available to form a disulfide bond with a stem in a second genetically modified signaling polypeptide.

[0211] The stems may include amino acid sequences of immunoglobulin hinge regions that are known in the art; see, for example, Tan et al. (1990) Proc. Natl. Acad. Sci. USA 87:162; and Huck et al. (1986) Nucl. Acids Res. 14:1779. As non-limiting examples, an immunoglobulin hinge region may include a domain with at least 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 or 100% sequence identity with an extension of at least 10, 15, 20 or all amino acids from any of the following amino acid sequences: DKTHT (SEQ ID NO:63); CPPC (SEQ ID NO:62); CPEPKSCDTPPPCPR (SEQ ID NO:64) (see, for example, Glaser et al. (2005) J. Biol. Chem. 280:41.494); ELKTPLGDTTHT (SEQ ID NO:65); KSCDKTHTCP (SEQ ID NO:66); KCCVDCP (SEQ ID NO:67); KYGPPPC (SEQ ID NO:68); EPKSCDKTHTCPPCP (SEQ ID NO:69) (human IgG1 hinge); ERKCCVECPPCP (SEQ ID NO:70) (human IgG2 hinge); ELKTPLGDTTHTCPRCP (SEQ ID NO:71) (human IgG3 hinge); SPNMVPHAHHAQ (SEQ ID NO:72) (human IgG4 hinge); and similar. The stem may include a hinge region with an amino acid sequence from a human IgG1, IgG2, IgG3, or IgG4 hinge region. The stem may include one or more amino acid substitutions and/or insertions and/or deletions compared to a wild-type (naturally occurring) hinge region. For example, His229 of the human IgG1 hinge may be replaced by Tyr, so that the stem includes the sequence EPKSCDKTYTCPPCP (see, for example, Yan et al. (2012) J. Biol. Chem. 287:5.891). The stem may include an amino acid sequence derived from human CD8; for example, the stem may include the amino acid sequence: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO:73) or a variant thereof.

TRANSMEMBRANE DOMAIN

[0212] A genetically modified signaling polypeptide of the present disclosure may include transmembrane domains for insertion into a eukaryotic cell membrane. The transmembrane domain may be interposed between the ASTR and the costimulatory domain. The transmembrane domain may be interposed between the stem and the costimulatory domain, such that the chimeric antigen receptor includes, in order from amino terminus (N-terminal) to carboxyl terminus (C-terminal): an ASTR; a stem; a transmembrane domain; and an activation domain.

[0213] Any transmembrane (TM) domain that provides insertion of a polypeptide into the cell membrane of a eukaryotic cell (e.g., mammalian) is suitable for use in the aspects and embodiments disclosed herein. Non-limiting examples of suitable TM domains for any of the aspects or embodiments provided herein include a domain with at least 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% sequence identity with an extension of at least 10, 15, 20, or all amino acids from any of the following TM domains: a) CD* alpha (IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO:46)); b) CD8 beta (LGLLVAGVLVLLVSLGVAIHLCC (SEQ ID NO:47)); c) CD4 (ALIVLGGVAGLLLFIGLGIFFCVRC (SEQ ID NO:48)); d) CD3Z (LCYLLDGILFIYGVILTALFLRV (SEQ ID NO:49); e) CD28 (FWVLVVVGGVLACYSLLVTVAFIFIFWV (SEQ ID NO:50)); f) CD134 (OX40): (VAAILGLGLVLGLLGPLAILLALYLL (SEQ ID NO:51)); g) CD7(ALPAALAVISFLLGLGLGVACVLA (SEQ ID NO:52)), h) CD8TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAG TCGVLLLSLVITLYC (SEQ ID NO:75), and i) CD28 IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACY SLLVTVAFIIFWV (SEQ ID NO:76).

[0214] By way of non-limiting examples, a transmembrane domain of an aspect of the invention may have at least 80, 90 or 95% sequence identity with the transmembrane domain of SEQ ID NO:46, the transmembrane domain of CD8 beta, the transmembrane domain of CD4, the transmembrane domain of CD3 zeta, the transmembrane domain of CD28, the transmembrane domain of CD134 or the transmembrane domain of CD7.

INTRACELLULAR ACTIVATION DOMAIN

[0215] The intracellular activation domains suitable for use in a genetically modified signaling polypeptide of the present disclosure, when activated, typically induce the production of one or more cytokines; increased cell death and/or increased proliferation of CD8+ T cells, CD4+ T cells, natural killer T cells, Yδ T cells and/or neutrophils. The activation domains may also be referred to as activation domains in this document.

[0216] In some embodiments, the intracellular activation domain includes at least one (e.g., one, two, three, four, five, six, etc.) ITAM motif as described below. In some embodiments, the intracellular activation domain includes DAP10/CD28 type signaling chains. In some embodiments, the intracellular activation domain is not covalently linked to the membrane-bound genetically modified signaling polypeptide but is instead diffused into the cytoplasm. As non-limiting examples, an intracellular activation domain of an aspect of the invention may have at least 80%, 90%, or 95% sequence identity with the CD3Z, CD3D, CD3E, CD3G, CD79A, DAP12, FCERlG, DAP10/CD28, or ZAP70 domains as described above.

[0217] Intracellular activation domains suitable for use in a genetically modified signaling polypeptide of the present disclosure include intracellular signaling polypeptides containing a tyrosine-based immunoreceptor activation motif (ITAM). An ITAM motif is YX1X2L/I, wherein X1 and X2 are independently any amino acid. In some cases, the intracellular activation domain of a genetically modified signaling polypeptide includes 1, 2, 3, 4, or 5 ITAM motifs. In some cases, an ITAM motif is repeated twice in an intracellular activation domain, wherein the first and second occurrences of the ITAM motif are separated from each other by 6 to 8 amino acids, for example, (YX1X2L/I)(X3)n(YX1X2L/I), wherein n is an integer from 6 to 8, and each of the 6 to 8 X3 can be any amino acid. In some cases, the intracellular activation domain of a genetically modified signaling polypeptide includes 3 ITAM motifs.

[0218] A suitable intracellular activation domain may be a portion containing an ITAM motif that is derived from a polypeptide containing an ITAM motif. For example, a suitable intracellular activation domain may be a domain containing an ITAM motif from any protein containing an ITAM motif. Thus, a suitable intracellular activation domain need not contain the entire sequence of the entire protein from which it is derived. Examples of suitable ITAM motif-containing polypeptides include, but are not limited to: CD3Z (CD3 zeta); CD3D (CD3 delta); CD3E (CD3 epsilon); CD3G (CD3 gamma); CD79A (alpha chain of antigen-receptor complex-associated protein); DAP12; and FCERlG (gamma chain of Fc I epsilon receptor).

[0219] In some cases, the intracellular activation domain is derived from the CD3 zeta chain of T cell surface glycoprotein (also known as CD3Z, T cell T3 receptor zeta chain, CD247, CD3-ZETA, CD3H, CD3Q, T3Z, TCRZ, etc.). For example, a suitable intracellular activation domain may include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with an extension of at least 10, 15, 20, or all amino acids in the following sequences, or with a contiguous extension of about 100 amino acids to about 110 amino acids (aa), from about 110 aa to about 115 aa, from about 115 aa to about 120 aa, from about 120 aa to about 130 aa, from about 130 aa to about 140 aa, from about 140 aa to about 150 aa, or from about 150 aa to about 160 aa of any of the following amino acid sequences (2 isoforms): HDGLYQGLSTATKDTYDALH M QALPPR (SEQ ID NO:11) orMKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFS RSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNP QEG LYNELQ KD KMAEAYSEIG MKGERRRGKGHDG LYQGLSTATKDTYDALH MQALPPR (SEQ ID NO:12), where the ITAM reasons are in bold and underlined.

[0220] Likewise, a suitable intracellular polypeptide activation domain may include a portion containing an ITAM motif of the full-length CD3 zeta amino acid sequence. Thus, a suitable intracellular activation domain may include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with an extension of at least 10, 15, 20, or all amino acids in the following sequences, or with a contiguous extension of about 100 amino acids to about 110 amino acids (aa), from about 110 aa to about 115 aa, from about 115 aa to about 120 aa, from about 120 aa to about 130 aa, from about 130 aa to about 140 aa, from about 140 aa to about 150 aa, or from about 150 aa to about 160 aa of any of the following amino acid sequences: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTY DALHMQALPPR (SEQ ID NO:13);RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQ RRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT YDALHMQALPPR (SEQ ID NO:81); NQLYNELNLGRREEYDVLDKR SEQ ID NO:14); EGLYNELQKDKMAEAYSEIGMK (SEQ ID NO:15); or DGLYQGLSTATKDTYDALHMQ (SEQ ID NO:16), where the ITAM reasons are in bold and underlined.

[0221] In some cases, the intracellular activation domain is derived from the CD3 delta chain of T cell surface glycoprotein (also known as CD3D; CD3-DELTA; T3D; CD3 antigen, delta subunit; CD3 delta; CD3d antigen, delta polypeptide (TiT3 complex); OKT3, delta chain; T cell T3 receptor delta chain; CD3 delta chain and T cell surface glycoprotein; etc.). Thus, a suitable intracellular activation domain may include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with an extension of at least 10, 15, 20, or all amino acids in the following sequences, or with a contiguous extension of about 100 amino acids to about 110 amino acids (aa), from about 110 aa to about 115 aa, from about 115 aa to about 120 aa, from about 120 aa to about 130 aa, from about 130 aa to about 140 aa, from about 140 aa to about 150 aa, or from about 150 aa to about 160 aa of any of the following amino acid sequences: MEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDI TRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVAGIIV TDVIATLLLALGVFCFAGHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYS HLGGNWARNK (SEQ ID NO:17) or MEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDI TRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRTADTQALLRNDQVYQPL RDRDDAQYSHLGGNWARNK (SEQ ID NO:18), wherein the ITAM motifs are in bold and underlined.

[0222] Likewise, a suitable intracellular activation domain polypeptide may comprise a portion containing an ITAM motif of the full-length CD3 delta amino acid sequence. Thus, a suitable intracellular activation domain may include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with an extension of at least 10, 15, 20 or all amino acids in the following sequence: DQVYQPLRDRDDAQYSHLGGN (SEQ ID NO:19), wherein the ITAM motifs are in bold and underlined.

[0223] In some cases, the intracellular activation domain is derived from the CD3 epsilon chain of T cell surface glycoprotein (also known as CD3e, T cell surface antigen T3/Leu-4 epsilon chain, CD3 epsilon chain of T cell surface glycoprotein, AI504783, CD3, CD3 epsilon, T3e, etc.). Thus, a suitable intracellular activation domain may include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with an extension of at least 10, 15, 20, or all amino acids in the following sequences, or with a contiguous extension of about 100 amino acids to about 110 amino acids (aa), from about 110 aa to about 115 aa, from about 115 aa to about 120 aa, from about 120 aa to about 130 aa, from about 130 aa to about 140 aa, from about 140 aa to about 150 aa, or from about 150 aa to about 160 aa of the following amino acid sequence:MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQY PGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSK PEDANFYLYLRARVCENCMEMDMSVATIVIVDICITGGLLLLVYYWSKNRKAKA KPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI (SEQ ID NO:20), wherein the ITAM motifs are in bold and underlined.

[0224] Likewise, a suitable intracellular activation domain polypeptide may comprise a portion containing an ITAM motif of the full-length CD3 epsilon amino acid sequence. Thus, a suitable intracellular activation domain may include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with an extension of at least 10, 15, 20 or all amino acids in the following sequence: NPDYEPIRKGQRDLYSGLNQR (SEQ ID NO:21), wherein the ITAM motifs are in bold and underlined.

[0225] In some cases, the intracellular activation domain is derived from the CD3 gamma chain of T cell surface glycoprotein (also known as CD3G, T cell T3 receptor gamma chain, CD3-GAMMA, T3G, gamma polypeptide (TiT3 complex), etc.). Thus, a suitable intracellular activation domain may include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with an extension of at least 10, 15, 20, or all amino acids in the following sequences, or with a contiguous extension of about 100 amino acids to about 110 amino acids (aa), from about 110 aa to about 115 aa, from about 115 aa to about 120 aa, from about 120 aa to about 130 aa, from about 130 aa to about 140 aa, from about 140 aa to about 150 aa, or from about 150 aa to about 160 aa of the following amino acid sequence: MEQGKGLAVLILAIILLQGTLAQSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITW FKDGKMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQ NCIELNAATISGFLFAEIVSIFVLAVGVYFIAGQDGVRQSRASDKQTLLPNDQLY QPLKDREDDQYSHLQGNQLRRN (SEQ ID NO:22), where the ITAM motifs are in bold and underlined.

[0226] Likewise, a suitable intracellular activation domain polypeptide may comprise a portion containing an ITAM motif of the full-length CD3 gamma amino acid sequence. Thus, a suitable intracellular activation domain may include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with an extension of at least 10, 15, 20 or all amino acids in the following sequence: DQLYQPLKDREDDQYSHLQGN (SEQ ID NO:23), wherein the ITAM motifs are in bold and underlined.

[0227] In some cases, the intracellular activation domain is derived from CD79A (also known as B cell antigen receptor complex-associated protein alpha chain; CD79a antigen (immunoglobulin-associated alpha); MB-1 membrane glycoprotein; Ig-alpha; membrane-bound immunoglobulin-associated protein; surface IgM-associated protein; etc.). Thus, a suitable intracellular activation domain may include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with an extension of at least 10, 15, 20, or all amino acids in the following sequences, or with a contiguous extension of about 100 amino acids to about 110 amino acids (aa), from about 110 aa to about 115 aa, from about 115 aa to about 120 aa, from about 120 aa to about 130 aa, from about 130 aa to about 140 aa, from about 140 aa to about 150 aa, or from about 150 aa to about 160 aa of any of the following amino acid sequences: MPGGPGVLQALPATIFLLFLLSAVYLGPGCQALWMHKVPASLMVSLGEDAHF QCPHNSSNNANVTWWRVLHGNYTWPPEFLGPGEDPNGTLIIQNVNKSHGGI YVCRVQEGNESYQQSCGTYLRVRQPPPRPFLDMGEGTKNRIITAEGIILLFCA VVPGTLLLFRKRWQNEKLGLDAGDEYEDENLYEGLNLDDCSMYEDISRGLQG TYQDVGSLNIGDVQLEKP (SEQ ID NO:24) or MPGGPGVLQALPATIFLLFLLSAVYLGPGCQALWMHKVPASLMVSLGEDAHF QCPHNSSNNANVTWWRVLHGNYTWPPEFLGPGEDPNEPPPRPFLDMGEGT KNRIITAEGIILLFCAVVPGTLLLFRKRWQNEKLGLDAGDEYEDENLYEGLNLD DCSMYEDISRGLQGTYQDVGSLNIGDVQLEKP (SEQ ID NO:25), where the ITAM reasons are in bold and underlined.

[0228] Likewise, a suitable intracellular activation domain polypeptide may comprise a portion containing an ITAM motif of the full-length CD79A amino acid sequence. Thus, a suitable intracellular activation domain may include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with an extension of at least 10, 15, 20 or all amino acids in the following sequence: ENLYEGLNLDDCSMYEDISRG (SEQ ID NO:26), wherein the ITAM motifs are in bold and underlined.

[0229] In some cases, the intracellular activation domain is derived from DAP12 (also known as TYROBP; TYRO tyrosine kinase-binding protein; KARAP; PLOSL; DNAX-activating protein 12; KAR-associated protein; TYRO tyrosine kinase-binding protein; killer cell activating receptor-associated protein; killer cell activating receptor-associated protein; etc.). For example, a suitable intracellular activation domain may include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with an extension of at least 10, 15, 20, or all amino acids in the following sequences, or with a contiguous extension of about 100 amino acids to about 110 amino acids (aa), from about 110 aa to about 115 aa, from about 115 aa to about 120 aa, from about 120 aa to about 130 aa, from about 130 aa to about 140 aa, from about 140 aa to about 150 aa, or from about 150 aa to about 160 aa of any of the following amino acid sequences (4 isoforms): NO:27) RLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYK (SEQ ID NO:29), orMGGLEPCSRLLLLPLLLAVSDCSCSTVSPGVLAGIVMGDLVLTVLIALAVYFLG RLVPRGRGAAEATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYK (SEQ ID NO:30), where the ITAM reasons are in bold and underlined.

[0230] Likewise, a suitable intracellular activation domain polypeptide may comprise a portion containing an ITAM motif of the full-length DAP12 amino acid sequence. Thus, a suitable intracellular activation domain may include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with an extent of at least 10, 15, 20 or all amino acids in the following sequence: ESPYQELQGQRSDVYSDLNTQ (SEQ ID NO:31), wherein the ITAM motifs are in bold and underlined.

[0231] In some cases, the intracellular activation domain is derived from FCERlG (also known as FCRG; Fc receptor I epsilon gamma chain; Fc receptor gamma chain; fc-epsilon RI-gamma; fcRgamma; fceRI gamma; high-affinity immunoglobulin receptor epsilon gamma subunit; high-affinity immunoglobulin E receptor gamma chain; etc.). For example, a suitable intracellular activation domain may include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with an extension of at least 10, 15, 20, or all amino acids in the following sequences, or with a contiguous extension of about 50 amino acids to about 60 amino acids (aa), about 60 aa to about 70 aa, about 70 aa to about 80 aa, or about 80 aa to about 88 aa of the following amino acid sequence: MIPAVVLLLLLLVEQAAALGEPQLCYILDAILFLYGIVLTLLYCRLKIQVRKAAITS YEKSDGVYTGLSTRNQETYETLKHEKPPQ (SEQ ID NO:32), where the ITAM reasons are in bold and underlined.

[0232] Likewise, a suitable intracellular activation domain polypeptide may comprise a portion containing an ITAM motif of the full-length FCER1G amino acid sequence. Thus, a suitable intracellular activation domain may include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with an extension of at least 10, 15, 20 or all amino acids in the following sequence: DGVYTGLSTRNQETYETLKHE (SEQ ID NO:33), wherein the ITAM motifs are in bold and underlined.

[0233] Suitable intracellular activation domains for use in a genetically modified signaling polypeptide of the present disclosure include a DAP10/CD28 type signaling chain. An example of a DAP10 signaling chain is the amino acid sequence that is: RPRRSPAQDGKVYINMPGRG (SEQ ID NO:34). In some embodiments, a suitable intracellular activation domain includes a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with an extension of at least 10, 15, 20 or all amino acids in the following sequence: RPRRSPAQDGKVYINMPGRG (SEQ ID NO:34).

[0234] An example of a CD28 signaling chain is the amino acid sequence that is FWVLVWGGVLACYSLLVTVAFHFWVRSKRSRLLHSDYMNMTPRRPGPTRKH YQPYAPPRDFAAYRS (SEQ ID NO:35). In some embodiments, a suitable intracellular domain includes a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with an extension of at least 10, 15, 20 or all amino acids in the following sequence: FWVLVWGGVLACYSLLVTVAFHFWVRSKRSRLLHSDYMNMTPRRPGPTRKH YQPYAPPRDFAAYRS (SEQ ID NO:35).

[0235] Suitable intracellular activation domains for use in a genetically modified signaling polypeptide of the present disclosure include a ZAP70 polypeptide, for example, a suitable intracellular activation domain may include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with an extension of at least 10, 15, 20, or all amino acids in the following sequences or with a contiguous extension of about 300 amino acids to about 400 amino acids, from about 400 amino acids to about 500 amino acids, or from about 500 amino acids to 619 amino acids, of the following amino acid sequence: MPDPAAHLPFFYGSISRAEAEEHLKLAGMADGLFLLRQCLRSLGGYVLSLVHD VRFHHFPIERQLNGTYAIAGGKAHCGPAELCEFYSRDPDGLPCNLRKPCNRP SGLEPQPGVFDCLRDAMVRDYVRQTWKLEGEALEQAIISQAPQVEKLIATTAH ERMPWYHSSLTREEAERKLYSGAQTDGKFLLRPRKEQGTYALSLIYGKTVYH YLISQDKAGKYCIPEGTKFDTLWQLVEYLKLKADGLIYCLKEACPNSSASNASG AAAPTLPAHPSTLTHPQRRIDTLNSDGYTPEPARITSPDKPRPMPMDTSVYES PYSDPEELKDKKLFLKRDNLLIADIELGCGNFGSVRQGVYRMRKKQIDVAIKVL KQGTEKADTEEMMREAQIMHQLDNPYIVRLIGVCQAEALMLVMEMAGGGPLH KFLVGKREEIPVSNVAELLHQVSMGMKYLEEKNFVHRDLAARNVLLVNRHYAK ISDFGLSKALGADDSYYTARSAGKWPLKWYAPECINFRKFSSRSDVWSYGVT MWEALSYGQKPYKKMKGPEVMAFIEQGKRMECPPECPPELYALMSDCWIYK WEDRPDFLTVEQRMRACYYSLASKVEGPPGSTQKAEAACA (SEQ ID NO:36).

LYMPHOPROLIFERATIVE ELEMENTS

[0236] Peripheral T lymphocyte numbers are maintained at remarkably stable levels throughout adulthood, despite the continuous addition of cells due to thymic emigration and proliferation in response to antigen encounter, and cell loss due to removal of antigen-specific effectors after antigen release (Marrak, P. et al. 2000. Nat Immunol 1:107-111; Freitas, A.A. et al. 2000. Annu Rev Immunol 18:83-111). The size of the peripheral T cell compartment is regulated by multiple factors that influence both proliferation and survival. However, in a lymphopenic environment, T lymphocytes divide independently of the cognate antigen due to mechanisms of "acute homeostatic proliferation" that maintain the size of the peripheral T cell compartment. The conditions for lymphopenia were established in subjects or patients during adoptive cell therapy by proliferating T cells in vitro and introducing them into lymphodepleted subjects, resulting in improved graft and antitumor function of the transferred T cells. However, lymphodepletion of a subject is undesirable because it can cause serious side effects, including immune dysfunction and death.

[0237] Studies have shown that lymphodepletion removes endogenous lymphocytes that function as cellular scavengers for homeostatic cytokines, thereby releasing cytokines to induce the survival and proliferation of adoptively transferred cells. Some cytokines, such as IL-7 and IL-15, are known to mediate antigen-independent T-cell proliferation and thus have the capacity to stimulate homeostatic proliferation in non-lymphopenic environments. However, these cytokines and their receptors have intrinsic control mechanisms that prevent lymphoproliferative disorders in homeostasis.

[0238] Many of the aspects provided in this document include a lymphoproliferative element or a nucleic acid encoding the same, typically as part of a genetically modified signaling polypeptide. In the illustrative embodiments in this document, a lymphoproliferative element is introduced into a resting T cell and/or resting NK cell, typically by transducing the resting T cell and/or resting NK cell with a retrovirus whose genome encodes the lymphoproliferative element as part of a genetically modified signaling polypeptide. The lymphoproliferative element may be a cytokine or, in further illustrative embodiments, a cytokine receptor, or a fragment that includes a signaling domain thereof, which activates a STAT3 pathway, a STAT4 pathway, or, in still other illustrative embodiments, a Jak/STAT5 pathway. As such, a lymphoproliferative element can be, in a non-limiting example, a cytokine receptor, or an active fragment that includes a signaling domain thereof, such as an interleukin receptor, or an active fragment that includes a signaling domain thereof, which activates STAT5. Thus, a lymphoproliferative element is a polypeptide that induces the proliferation of a T cell and/or NK cell. The illustrative lymphoproliferative elements induce proliferation by activating STAT5. Thus, fragments of such lymphoproliferative elements retain the ability to induce the proliferation of T cells and/or NK cells, in the illustrative embodiments, by activating STAT5.

[0239] In some of the methods and compositions presented in this document, a lymphoproliferative element is used to promote the proliferation or expansion of genetically modified T cells in vivo without having to lymphodeplete the subjects. As such, illustrative, non-limiting embodiments of the methods provided in this document that include inserting a lymphoproliferative element into a resting T cell and/or NK cell of a subject, typically transducing such T cell and/or NK cell, can be performed without lymphodepleting the subject before, during and/or after performing the method or without lymphodepleting the subject before, during and/or after collecting blood from a subject before performing such method or without lymphodepleting the subject before, during and/or after genetically modifying the subject's T cells or NK cells ex vivo and/or before, during or after reintroducing the genetically modified T cells and/or NK cells into the subject. Factors that promote T cell proliferation in vivo include cytokines and their receptors, wherein a receptor typically includes a ligand-binding domain and a signaling domain. In some embodiments, the lymphoproliferative element used in the methods and compositions disclosed herein is a cytokine and/or a cytokine receptor. The cytokine may be an interleukin, and the cytokine receptor may be an interleukin receptor. The lymphoproliferative element may be a functional fragment of a cytokine and/or a functional fragment of a cytokine receptor, such as a signaling domain thereof, wherein the fragment has the ability to promote T cell proliferation, for example, by activating STAT5.

[0240] In some embodiments, the lymphoproliferative cytokine element in the methods and compositions in this document includes one or more of the following: Interleukin-7 (IL-7) or its receptor (IL-7R) or a signaling domain thereof; Interleukin-12 (IL-12) or its receptor (IL-12R) or a signaling domain thereof; Interleukin-23 (IL-23) or its receptor composed of IL-12R β1 and IL-23R or a signaling domain thereof; Interleukin-27 (IL-27) or its receptor (IL-27R) or a signaling domain thereof; Interleukin-15 (IL-15) or its receptor (IL-15R) or a signaling domain thereof; Interleukin-21 (IL-21) or its receptor (IL-21R) or a signaling domain thereof; or transforming growth factor β (TGFβ) or its receptor (TGFβR) or a signaling domain thereof; or the TGFβ decoy receptor (TGF-β-dominant negative receptor II (DNRII)). In some embodiments, the lymphoproliferative element is IL-12R or the TGFβ decoy receptor (TGF-β-dominant negative receptor II (DNRII)).

[0241] IL-7 binds to the IL-7 receptor, a heterodimer consisting of IL-7R alpha and a common gamma chain receptor. Binding results in a cascade of signals important for T cell development within the thymus and for survival within the periphery. IL-7 binding to the IL-7 receptor is known to activate the Jak/STAT5 pathway.

[0242] IL-12 is involved in the differentiation of naive T cells into Th1 cells (Hsieh CS et al. 1993. Science. 260(5107):547-549) and is known as a T cell stimulating factor. IL-12 binds to the IL-12 receptor, which is a heterodimeric receptor formed by IL-12R-β1 and IL-12R-β2. IL-12 can act by activating STAT4, but it has also been shown to activate STAT5 in T cells (Ahn, H., et al. 1998. J. Immun. 161:5893-5900). The IL-12 family is composed of the cytokines IL-12, IL-23, and IL-27. The receptor for IL-23 is composed of IL-12R-β1 and IL-23R. IL-27 is a heterodimeric cytokine composed of two distinct genes, the Epstein-Barr virus-induced gene 3 (EBI3) and IL-27p28. IL-27 interacts with the IL-27 receptor.

[0243] IL-15 is a T-cell and NK-cell stimulating factor that is similar in structure and function to IL-2. Both cytokines induce T-cell proliferation; and their shared functions are believed to result from both receptors using the common Y chains and IL-2/IL-15Rβ. The IL-15 signaling pathway begins with binding to the IL-15Rα receptor, with subsequent presentation to surrounding cells carrying the IL-15Rβyc complex on their cell surface. Upon binding, the IL-15β subunit activates Janus kinase 1 (Jak1) and Janus kinase 3 (Jak3) of the yc subunit, leading to phosphorylation and activation of STAT3 and STAT5.

[0244] IL-21 is expressed on human CD4+ T cells and NK T cells, and IL-21 expression is upregulated in Th2 and Th17 subsets of helper T cells. The IL-21 receptor (IL-21R) is expressed on the surface of T, B, and NK cells and is similar in structure to receptors for other type I cytokines such as IL-2R or IL-15. IL-21R requires dimerization with the common gamma chain (YC) in order to bind to IL-21. When bound to IL-21, the IL-21 receptor acts via the Jak/STAT pathway, activating STAT1, STAT3, and STAT5.

[0245] TGFβ decoy receptors (TGF-β—dominant negative receptor II (DNRII)) block TGFβ signaling by competing with natural receptors for TGFβ binding. TGFβ-DNRII is a truncated form without kinase activity of RII that contains the extracellular TGFβ-binding domain and the transmembrane domain of RII. TGFβ-DNRII binds to the ligand but does not phosphorylate and activate RII, thus decreasing or eliminating Smad phosphorylation.

[0246] Gain-of-function mutations in IL-7Rα have been identified in subjects with B-cell and T-cell acute lymphoblastic leukemias (B-ALL and T-ALL) (Zenatti PP, et al. 2011. Nat Genet 43:932-939; Snochat, C. et al. 2011. J Exp Med 208:901-908; McElroy, C.A. et al. 2012. PNAS 109(7):2503-2508). The mutations included insertions and deletions in the N-terminal region of IL-7Rα TMD, with almost all sequences containing an extra Cys residue and an S165 to C165 mutation. Cysteine resulted in constitutive receptor activation. Some of the mutations in the T-all group activated JAK1. These gain-of-function IL-7R mutants can be used in any of the aspects provided in this document as a lymphoproliferative element (or elements).

[0247] Consequently, in some embodiments, the lymphoproliferative element is a mutated IL-7 receptor. In other embodiments, the mutated IL-7 receptor is constitutively active, activating the JAK-STAT5 pathway in the absence of the cytokine ligand. In still other embodiments, the mutated IL-7 receptor comprises a 1 to 10 amino acid insertion at a position between 237 and 254 that includes a cysteine residue that includes the ability to constitutively activate the STAT5 pathway. In some embodiments, the mutated IL-7 receptor is IL-7Rα-insPPCL (represented by SEQ ID NO:82).

[0248] In some embodiments, the lymphoproliferative element is a chimeric cytokine receptor, such as, but not limited to, a cytokine bound to its receptor that typically and constitutively activates the same STAT pathway as a corresponding activated wild-type cytokine receptor, such as STAT3, STAT4, and, in illustrative embodiments, STAT5. In some embodiments, the chimeric cytokine receptor is an interleukin, or a fragment thereof, bound or covalently linked to its cognate receptor, or a fragment thereof, by means of a binder. In some embodiments, the chimeric cytokine receptor is IL-7 bound to IL-7Rα. In other embodiments, the chimeric cytokine receptor is IL-7 bound to a domain of IL-7Rα, such as, for example, the extracellular domain of IL-7Rα and/or the transmembrane domain of IL-7Rα. In some embodiments, the lymphoproliferative element is a cytokine receptor that is not agglutinated to a cytokine and, in fact, in the illustrative embodiments provided herein, a lymphoproliferative element is a constitutively active cytokine receptor that is not agglutinated to a cytokine. These chimeric IL-7 receptors typically and constitutively activate STAT5 when expressed.

[0249] In some embodiments, the lymphoproliferative element is not a cytokine or a cytokine receptor, but a miRNA that stimulates the STAT5 pathway, typically potentiating STAT5 activation by degrading a negative regulator in the SOCS pathway. In some embodiments, the miRNA is for proteins that affect proliferation, such as, but not limited to, ABCG1, SOCS1, TGFbR2, SMAD2, cCBL, and PD1. In the illustrative embodiments, as exemplified in this document, such miRNAs may be localized to introns in a packaging cell and/or a recombinant retrovirus genome, typically with expression triggered by a promoter that is active in a T cell and/or NK cell. Without adhering to theory, it is believed that the inclusion of introns in transcription units results in higher expression and/or stability of transcripts. As such, the ability to place miRNAs within introns of a retroviral genome complements the teachings of the present disclosure, overcoming the challenges in the prior art of attempting to obtain maximum activities within the size constraints of a retroviral, such as a lentivirus genome. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 miRNAs, in illustrative embodiments between 2 and 5, for example, 4 miRNAs, one or more of which each binds to nucleic acids encoding one or more of ABCG1, SOCS1, TGFbR2, SMAD2, cCBL, and PD1, can be incorporated into the recombinant retroviral genome and delivered to a target cell, for example, T cells and/or NK cells, using the methods provided in this document. In fact, as provided in this document, 1, 2, 3, or 4 miRNAs can be delivered on a single intron, such as the EF1a intron.

[0250] ABCG1 is an ATP-binding cassette transporter that negatively regulates the proliferation of peripheral lymphocytes and thymocytes (Armstrong et al. 2010. J Immunol 184(1):173 to 183).

[0251] SOCS1 is a member of the SOCS (Suppressor of Cytokine Signaling) family of negative regulators of cytokine signal transduction that inhibit the Jak/Stat pathway, like STAT5. SOCS1 is also known as JAB (Janus Kinase Binding Protein), SSI-1 (Stat-Induced State Inhibitor-1), and TIP3 (Tec-Interacting Protein).

[0252] TGFbR2 is a member of the serine/threonine kinase protein family that binds to TGF-β, forming a complex that phosphorylates proteins that then enter the nucleus and regulate the transcription of genes related to proliferation.

[0253] SMAD2 mediates the transforming growth factor (TGF)-β signal and regulates multiple cellular processes, such as proliferation, apoptosis, and differentiation.

[0254] cCBL is an E3 ubiquitin ligase that inhibits TCR signaling by dephosphorylation and inactivation of ZAP-70 and through TCR internalization.

[0255] PD1 (CD279) is a cell surface receptor expressed on T cells and ProB cells. PD-1 binds to two ligands, PD-L1 and PD-L2. Signaling via PD-1 functions to prevent cell activation.

[0256] In some of the methods and compositions disclosed herein, the expression of the lymphoproliferative element is induced and may even be dependent on the binding of a compound to an in vivo control element (as discussed elsewhere herein) which, in non-limiting embodiments, is a ribowsitch. In some embodiments, the lymphoproliferative element is expressed from an active promoter in a T cell and/or an NK cell. For the methods and compositions provided herein, a person skilled in the art will recognize that the promoters are known to be active in T cells and/or NK cells and can be used to express a first genetically modified signaling polypeptide or a second genetically modified signaling polypeptide or any component thereof. In the illustrative embodiments, such a promoter is not active in a packaging cell line, such as the packaging cell lines disclosed herein. In some embodiments, the promoter is an EF1a promoter or the murine stem cell virus (MSCV) promoter (Jones et al., Human Gene Therapy (2009) 20: 630-640). In the illustrative embodiments, the promoter is the T-cell specific CD3 zeta promoter.

[0257] In some modalities, the lymphoproliferative element is restricted to the microenvironment. For example, the lymphoproliferative element may be a mutated receptor that binds to its respective cytokine differentially under abnormal versus physiological conditions. For example, an IL-7R that can bind to IL7 more strongly in a tumor environment than in a normal physiological environment may be used.

[0258] In some embodiments, the lymphoproliferative element is fused to a recognition or scavenging domain. Such recognition or scavenging domains are disclosed in more detail in this document. Such fusion provides the advantage, especially when a truncated or mutated lymphoproliferative element is used, of requiring fewer polynucleotides in the retroviral genome. This is important in the illustrative embodiments provided in this document because it helps to allow more nucleic acids encoding functional elements to be included in the retroviral genome. In other embodiments, the lymphoproliferative element is fused to a co-stimulatory domain and/or an intracellular activation domain. A lymphoproliferative element, as disclosed in this document, is not a chimeric antigen receptor (CAR) or an intracellular activation domain or co-stimulatory domain thereof. However, in some embodiments, a lymphoproliferative element may be fused to an antigen-specific targeting region (ASTR) and activated by the binding of the ASTR to its antigen. In still other embodiments, a genetically modified signaling polypeptide may include an ASTR, an intracellular activation domain (such as a CD3 zeta signaling domain), a co-stimulatory domain, and a lymphoproliferative domain. Further details relating to co-stimulatory domains, intracellular activation domains, ASTRs, and other CAR domains are disclosed elsewhere in this document.

[0259] In the illustrative embodiments in this document, a T cell and/or NK cell survival element is introduced into a resting T cell and/or resting NK cell, typically by transducing the resting T cell and/or resting NK cell with a retrovirus whose genome encodes the T cell and/or NK cell survival element as part of a genetically modified signaling polypeptide. In some embodiments, a lymphoproliferative element is also a T cell and/or NK cell survival element. As discussed above, some of the lymphoproliferative elements not only promote proliferation but also promote cell survival. In some embodiments, the T cell and/or NK cell survival motif is not a lymphoproliferative element. For example, the T cell and/or NK cell survival motif may be a CD28 T cell survival motif or a CD137 cell survival motif. Such T cell survival motifs can be found in genetically modified signaling polypeptides that include an ASTR, such as an scFV. In one illustrative embodiment, the T cell survival motif is a CD28 T cell survival motif or a CD137 motif connected to an scFv via a CD8a transmembrane domain or a CD28 transmembrane domain. In certain embodiments, said intracellular signaling domain comprises a polypeptide sequence comprising a tyrosine-based immunoreceptor activating motif (ITAM). In one particular embodiment, said polypeptide sequence is a CD3Z signaling domain.

MODULATING DOMAINS

[0260] Modulatory domains can alter the effect of the intracellular activation domain in the genetically modified signaling polypeptide, including enhancing or suppressing downstream effects of the activation domain or altering the nature of the response. Modulatory domains suitable for use in a genetically modified signaling polypeptide of the present disclosure include co-stimulatory domains. A suitable modulator domain for inclusion in the genetically modified signaling polypeptide may have a length of approximately 30 to approximately 70 amino acids (aa), for example, a modulator domain may have a length of approximately 30 aa to approximately 35 aa, approximately 35 aa to approximately 40 aa, approximately 40 aa to approximately 45 aa, approximately 45 aa to approximately 50 aa, approximately 50 aa to approximately 55 aa, approximately 55 aa to approximately 60 aa, approximately 60 aa to approximately 65 aa, or approximately 65 aa to approximately 70 aa. In other cases, the modulating domain may have a length of about 70 aa to about 100 aa, from about 100 aa to about 200 aa, or greater than 200 aa.

[0261] Co-stimulatory domains typically enhance and/or alter the nature of the response to an activating domain. Suitable co-stimulatory domains for use in a genetically modified signaling polypeptide of the present disclosure are generally receptor-derived polypeptides. In some embodiments, the co-stimulatory domains homodimerize. A subject co-stimulatory domain may be an intracellular portion of a transmembrane protein (i.e., the co-stimulatory domain may be derived from a transmembrane protein). Non-limiting examples of suitable co-stimulatory polypeptides include, but are not limited to, 4-IBB (CD137), CD27, CD28, Lck-binding deleted CD28 (ICΔ), ICOS, OX40, BTLA, CD27, CD30, GITR, and HVEM. For example, a co-stimulatory domain of an aspect of the invention may have at least 80%, 90%, or 95% sequence identity with the co-stimulatory domain of 4-IBB (CD137), CD27, CD28, CD28 deleted for Lck binding (ICΔ), ICOS, OX40, BTLA, CD27, CD30, GITR, or HVEM. For example, a co-stimulatory domain of an aspect of the invention may have at least 80%, 90%, or 95% sequence identity with the co-stimulatory domain of non-limiting examples of suitable co-stimulatory polypeptides that include, but are not limited to, 4-IBB (CD137), CD27, CD28, CD28 deleted for Lck binding (ICΔ), ICOS, OX40, BTLA, CD27, CD30, GITR, or HVEM. For example, a co-stimulatory domain of an aspect of the invention may have at least 80%, 90%, or 95% sequence identity with the co-stimulatory domain of 4-IBB (CD137), CD27, CD28, CD28 deleted for Lck binding (ICΔ), ICOS, OX40, BTLA, CD27, CD30, GITR, or HVEM.

[0262] A co-stimulatory domain suitable for inclusion in a genetically modified signaling polypeptide may have a length of about 30 amino acids to about 70 amino acids (aa), for example, a co-stimulatory domain may have a length of about 30 aa to about 35 aa, about 35 aa to about 40 aa, about 40 aa to about 45 aa, about 45 aa to about 50 aa, about 50 aa to about 55 aa, about 55 aa to about 60 aa, about 60 aa to about 65 aa or about 65 aa to about 70 aa. In other cases, the co-stimulatory domain may have a length of about 70 aa to about 100 aa, from about 100 aa to about 200 aa, or greater than 200 aa.

[0263] In some cases, the co-stimulatory domain is derived from an intracellular portion of the CD137 transmembrane protein (also known as TNFRSF9; CD137; 4-lBB; CDwl37; ILA; etc.). For example, a suitable co-stimulatory domain may include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with an extension of at least 10, 15, 20, or all amino acids in the following amino acid sequence: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO:1). In some of these modalities, the co-stimulatory domain has a length of approximately 30 aa to approximately 35 aa, approximately 35 aa to approximately 40 aa, approximately 40 aa to approximately 45 aa, approximately 45 aa to approximately 50 aa, approximately 50 aa to approximately 55 aa, approximately 55 aa to approximately 60 aa, approximately 60 aa to approximately 65 aa, or approximately 65 aa to approximately 70 aa.

[0264] In some cases, the costimulatory domain is derived from an intracellular portion of the CD28 transmembrane protein (also known as Tp44). For example, a suitable costimulatory domain may include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with an extension of at least 10, 15, 20, or all amino acids in the following amino acid sequence: RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO:2). In some of these modalities, the co-stimulatory domain has a length of approximately 30 aa to approximately 35 aa, approximately 35 aa to approximately 40 aa, approximately 40 aa to approximately 45 aa, approximately 45 aa to approximately 50 aa, approximately 50 aa to approximately 55 aa, approximately 55 aa to approximately 60 aa, approximately 60 aa to approximately 65 aa, or approximately 65 aa to approximately 70 aa.

[0265] In some cases, the costimulatory domain is derived from an intracellular portion of the CD28 transmembrane protein deleted for Lck binding (ICΔ). For example, a suitable costimulatory domain may include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with an extension of at least 10, 15, 20, or all amino acids in the following amino acid sequence: RSKRSRLLHSDYMNMTPRRPGPTRKHYQAYAAARDFAAYRS (SEQ ID NO:3). In some of these modalities, the co-stimulatory domain has a length of approximately 30 aa to approximately 35 aa, approximately 35 aa to approximately 40 aa, approximately 40 aa to approximately 45 aa, approximately 45 aa to approximately 50 aa, approximately 50 aa to approximately 55 aa, approximately 55 aa to approximately 60 aa, approximately 60 aa to approximately 65 aa, or approximately 65 aa to approximately 70 aa.

[0266] In some cases, the co-stimulatory domain is derived from an intracellular portion of the transmembrane protein ICOS (also known as AILIM, CD278, and CVID1). For example, a suitable co-stimulatory domain may include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with an extension of at least 10, 15, 20, or all amino acids in the following amino acid sequence: TKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTL (SEQ ID NO:4). In some of these modalities, the co-stimulatory domain has a length of approximately 30 aa to approximately 35 aa, approximately 35 aa to approximately 40 aa, approximately 40 aa to approximately 45 aa, approximately 45 aa to approximately 50 aa, approximately 50 aa to approximately 55 aa, approximately 55 aa to approximately 60 aa, approximately 60 aa to approximately 65 aa, or approximately 65 aa to approximately 70 aa.

[0267] In some cases, the co-stimulatory domain is derived from an intracellular portion of the OX40 transmembrane protein (also known as TTNFRSF4, RP5-902P8.3, ACT35, CD134, OX-40, TXGPlL). For example, a suitable co-stimulatory domain may include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with an extension of at least 10, 15, 20, or all amino acids in the following amino acid sequence: RRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI (SEQ ID NO:5). In some of these modalities, the co-stimulatory domain has a length of approximately 30 aa to approximately 35 aa, approximately 35 aa to approximately 40 aa, approximately 40 aa to approximately 45 aa, approximately 45 aa to approximately 50 aa, approximately 50 aa to approximately 55 aa, approximately 55 aa to approximately 60 aa, approximately 60 aa to approximately 65 aa, or approximately 65 aa to approximately 70 aa.

[0268] In some cases, the costimulatory domain is derived from an intracellular portion of the CD27 transmembrane protein (also known as S 152, T 14, TNFRSF7, and Tp55). For example, a suitable costimulatory domain may include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with an extension of at least 10, 15, 20, or all amino acids in the following amino acid sequence: HQRRKYRSNKGESPVEPAEPCRYSCPREEEGSTIPIQEDYRKPEPACSP (SEQ ID NO:6). In some of these modalities, the co-stimulatory domain has a length of approximately 30 aa to approximately 35 aa, approximately 35 aa to approximately 40 aa, approximately 40 aa to approximately 45 aa, and approximately 45 aa to approximately 50 aa.

[0269] In some cases, the co-stimulatory domain is derived from an intracellular portion of the transmembrane protein BTLA (also known as BTLAl and CD272). For example, a suitable co-stimulatory domain may include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with an extension of at least 10, 15, 20, or all amino acids in the following amino acid sequence:CCLRRHQGKQNELSDTAGREINLVDAHLKSEQTEASTRQNSQVLLSETGIYDN DPDLCFRMQEGSEVYSNPCLEENKPGIVYASLNHSVIGPNSRLARNVKEAPTE YASICVRS (SEQ ID NO:7).

[0270] In some cases, the costimulatory domain is derived from an intracellular portion of the CD30 transmembrane protein (also known as TNFRSF8, DlS166E, and Ki-1). For example, a suitable co-stimulatory domain may include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a length of about 100 amino acids to about 110 amino acids (aa), about 110 aa to about 115 aa, about 115 aa to about 120 aa, about 120 aa to about 130 aa, about 130 aa to about 140 aa, about 140 aa to about 150 aa, about 150 aa to about 160 aa, or about 160 aa to about 185 aa of the following sequence of amino acids: RRACRKRIRQKLHLCYPVQTSQPKLELVDSRPRRSSTQLRSGASVTEPVAEE RGLMSQPLMETCHSVGAAYLESLPLQDASPAGGPSSPRDLPEPRVSTEHTNN KIEKIYIMKADTVIVGTVKAELPEGRGLAGPAEPELEEELEADHTPHYPEQETE PPLGSCSDVMLSVEEEGKEDPLPTAASGK (SEQ ID NO:8).

[0271] In some cases, the co-stimulatory domain is derived from an intracellular portion of the transmembrane protein GITR (also known as TNFRSF18, RP5-902P8.2, AITR, CD357, and GITR-D). For example, a suitable co-stimulatory domain may include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with an extension of at least 10, 15, 20, or all amino acids in the following amino acid sequence: HIWQLRSQCMWPRETQLLLEVPPSTEDARSCQFPEEERGERSAEEKGRLGD LWV (SEQ ID NO:9). In some of these modalities, the co-stimulatory domain has a length of approximately 30 aa to approximately 35 aa, approximately 35 aa to approximately 40 aa, approximately 40 aa to approximately 45 aa, approximately 45 aa to approximately 50 aa, approximately 50 aa to approximately 55 aa, approximately 55 aa to approximately 60 aa, approximately 60 aa to approximately 65 aa, or approximately 65 aa to approximately 70 aa.

[0272] In some cases, the co-stimulatory domain is derived from an intracellular portion of the HVEM transmembrane protein (also known as TNFRSF14, RP3-395M20.6, ATAR, CD270, HVEA, HVEM, LIGHTR, and TR2). For example, a suitable co-stimulatory domain may include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with an extension of at least 10, 15, 20, or all amino acids in the following amino acid sequence: CVKRRKPRGDVVKVIVSVQRKRQEAEGEATVIEALQAPPDVTTVAVEETIPSFT GRSPNH (SEQ ID NO:10). In some of these embodiments, the co-stimulatory domain of both the first and second polypeptides has a length of approximately 30 aa to approximately 35 aa, approximately 35 aa to approximately 40 aa, approximately 40 aa to approximately 45 aa, approximately 45 aa to approximately 50 aa, approximately 50 aa to approximately 55 aa, approximately 55 aa to approximately 60 aa, approximately 60 aa to approximately 65 aa, or approximately 65 aa to approximately 70 aa.

BINDER

[0273] In some cases, the genetically modified signaling polypeptide includes a binder between any two adjacent domains. For example, a binder may be between the transmembrane domain and the first costimulatory domain. As another example, the ASTR may be an antibody and a binder may be between the heavy chain and the light chain. As another example, a binder may be between the ASTR and the transmembrane domain and a costimulatory domain. As another example, a binder may be between the costimulatory domain and the intracellular activation domain of the second polypeptide. As another example, the binder may be between the ASTR and the intracellular signaling domain.

[0274] The binding peptide can have any of a variety of amino acid sequences. Proteins can be joined by a spacer peptide, usually of a flexible nature, although other chemical linkages are not excluded. A binder can be a peptide between about 1 and about 100 amino acids in length or between about 1 and about 25 amino acids in length. These binders can be produced using synthetic binder-coding oligonucleotides to couple the proteins. Peptide binders with a degree of flexibility can be used. The binding peptides can have virtually any amino acid sequence, bearing in mind that suitable binders will have a sequence that results in a generally flexible peptide. Small amino acids, such as glycine and alanine, are used in creating a flexible peptide. Creating such sequences is routine for those skilled in the art.

[0275] Suitable binders can be readily selected and can be any suitable one from different lengths, such as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids or 7 amino acids to 8 amino acids and can have 1, 2, 3, 4, 5, 6 or 7 amino acids.

[0276] Exemplary flexible binders include glycine (G)n polymers, glycine-serine polymers (including, for example, (GS)n, GSGGSn, GGGSn and GGGGSn where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers and other flexible binders known in the art. Glycine and glycine-serine polymers are of interest since both amino acids are relatively unstructured and therefore can serve as a neutral link between components. Glycine polymers are of particular interest since glycine accesses significantly more phi-psi space even than alanine and is much less constrained than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173 to 11142 (1992)). Exemplary flexible binders include, but are not limited to, GGGGSGGGGSGGGGS (SEQ ID NO:53), GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO:54), GGGGSGGGSGGGGS (SEQ ID NO:55), GGSG (SEQ ID NO:56), GGSGG (SEQ ID NO:57), GSGSG (SEQ ID NO:58), GSGGG (SEQ ID NO:59), GGGSG (SEQ ID NO:60), GSSSG (SEQ ID NO:61), and the like. A person of ordinary skill in the art will recognize that the design of a peptide conjugated to any of the elements described above may include binders that are wholly or partially flexible, such that the binder may include a flexible binder as well as one or more portions that confer a less flexible structure.

CHIMERIC ANTIGEN RECEPTOR

[0277] In some aspects of the present invention, a genetically modified signaling polypeptide is a chimeric antigen receptor (CAR) or a polynucleotide encoding a CAR, which, for simplicity, is referred to herein as "CAR". In some embodiments, a CAR of the present disclosure includes: a) at least one antigen-specific targeting region (ASTR); b) a transmembrane domain; and c) an intracellular activation domain. In illustrative embodiments, the antigen-specific targeting region of the CAR is an scFv portion of an antibody to the target antigen.

[0278] A CAR of the present disclosure may be present in the plasma membrane of a eukaryotic cell, for example, a mammalian cell, wherein suitable mammalian cells include, but are not limited to, a cytotoxic cell, a T lymphocyte, a stem cell, a progeny of a stem cell, a progenitor cell, a progeny of a progenitor cell and an NK cell, an NK-T cell and a macrophage. When present in the plasma membrane of a eukaryotic cell, a CAR of the present disclosure is active in the presence of one or more target antigens that, under certain conditions, bind to the ASTR. The target antigen is the second member of the specific binding pair. The target antigen of the specific binding pair may be a soluble factor (e.g., not bound to a cell); a factor present on the surface of a cell, such as a target cell; a factor presented on a solid surface; a factor present in a lipid bilayer; and the like. When the ASTR is an antibody, and the second member of the specific binding pair is an antigen, the antigen can be a soluble antigen (e.g., not bound to a cell); an antigen present on the surface of a cell, such as a target cell; an antigen presented on a solid surface; an antigen present in a lipid bilayer; and the like.

[0279] In some cases, a CAR of the present disclosure, when present in the plasma membrane of a eukaryotic cell and when activated by one or more target antigens, increases the expression of at least one nucleic acid in the cell. For example, in some cases, a CAR of the present disclosure, when present in the plasma membrane of a eukaryotic cell and when activated by one or more target antigens, increases the expression of at least one nucleic acid in the cell by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about 2 times, at least about 2.5 times, at least about 5 times, at least about 10 times or more than 10 times, compared to the level of nucleic acid transcription in the absence of one or more target antigens.

[0280] As an example, the CAR of the present disclosure may include an intracellular signaling polypeptide containing a tyrosine-based immunoreceptor activation motif (ITAM).

[0281] A CAR of the present disclosure, when present in the plasma membrane of a eukaryotic cell and when activated by one or more target antigens, may, in some cases, result in increased production of one or more cytokines by the cell. For example, a CAR of the present disclosure, when present in the plasma membrane of a eukaryotic cell and when activated by one or more target antigens, may increase the production of a cytokine by the cell by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about 2 times, at least about 2.5 times, at least about 5 times, at least about 10 times or more than 10 times, compared to the amount of cytokine produced by the cell in the absence of one or more target antigens. Cytokines whose production can be increased include, but are not limited to, interferon gamma (IFN-Y), tumor necrosis factor-alpha (TNF-α), IL-2, IL-15, IL-12, IL-4, IL-5, IL-10; a chemokine; a growth factor; and similar substances.

[0282] In some cases, a CAR of the present disclosure, when present in the plasma membrane of a eukaryotic cell and when activated by one or more target antigens, can result in either an increase in the transcription of a nucleic acid in the cell or an increase in the production of a cytokine by the cell.

[0283] In some cases, a CAR of the present disclosure, when present in the plasma membrane of a eukaryotic cell and when activated by one or more target antigens, results in cytotoxic activity by the cell towards a target cell that expresses on its cell surface an antigen to which the antigen-binding domain of the first polypeptide of the CAR binds. For example, when the eukaryotic cell is a cytotoxic cell (e.g., an NK cell or a cytotoxic T lymphocyte), a CAR of the present disclosure, when present in the cell's plasma membrane and when activated by one or more target antigens, increases the cytotoxic activity of the cell towards a target cell that expresses on its cell surface one or more target antigens. For example, when the eukaryotic cell is an NK cell or a T lymphocyte, a CAR of the present revelation, when present in the cell's plasma membrane and when activated by one or more target antigens, increases the cytotoxic activity of the cell by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about 2 times, at least about 2.5 times, at least about 5 times, at least about 10 times, or more than 10 times, compared to the cytotoxic activity of the cell in the absence of one or more target antigens.

[0284] In some cases, a CAR of the present disclosure, when present in the plasma membrane of a eukaryotic cell and when activated by one or more target antigens, may result in other events related to CAR activation, such as proliferation and expansion (either due to increased cell division or anti-apoptotic responses).

[0285] In some cases, a CAR of the present disclosure, when present in the plasma membrane of a eukaryotic cell and when activated by one or more target antigens, may result in other events related to CAR activation, such as modulation of intracellular signaling, cell differentiation or cell death.

[0286] A CAR of the present disclosure may be present in a eukaryotic cell membrane, wherein the first and second polypeptides of the CAR are not covalently linked to each other. A CAR of the present disclosure may be present in a eukaryotic cell membrane as a single heterodimer that is not covalently linked to any other polypeptide in the membrane. Alternatively, a first CAR of the present disclosure may be present in a eukaryotic cell membrane as a heterodimer that is covalently or non-covalently linked to a second CAR of the present disclosure. In some cases, the first and second CARs are covalently linked via a disulfide bond formed between the cysteines present in a stem present in both the first polypeptide of the first CAR and the first polypeptide of the second CAR.

[0287] In some cases, a CAR of the present disclosure may be present in a eukaryotic cell membrane, wherein the first polypeptides of the CAR include an antibody fragment and the second polypeptides of the CAR include a signal transduction domain derived from a cytokine receptor, such that, upon dimerization, the CAR may represent a heterodimeric signalbody CAR, for example, a signalbody composed of at least two independent polypeptides. A "signalbody," as it is known in the art, is a unique chimeric macromolecule composed of an antibody fragment and a signal transduction domain derived from a cytokine receptor. In certain cases, a heterodimeric signalbody CAR of the present disclosure, when present in the cell membrane of a eukaryotic cell, dimerized by a dimerizer and activated by an antigen, for example, an oligomerized antigen, may induce oligomerization of the heterodimeric signalbody CAR. Such ligand-induced oligomerization of a heterodimeric signaling body CAR can activate, for example, enhance or perpetuate, for example, maintain, signal transduction; for example, ligand-induced oligomerization of a heterodimeric signaling body CAR can transmit a signal that stimulates a cellular response. In some cases, a plurality of heterodimeric signaling body CARs can be used combinatorially to stimulate a desired cellular response.

[0288] In some embodiments, the CARs of the present disclosure are microenvironmentally restricted. This property is typically the result of the microenvironmentally restricted nature of the CAR's ASTR domain. Thus, the CARs of the present disclosure may have a lower binding affinity or, in illustrative embodiments, may have a higher binding affinity to one or more target antigens under a condition (or conditions) in a microenvironment than under a condition in a normal physiological environment.

SEQUENCE RECOMBINATION

[0289] In certain cases, the sequences of genetically modified signaling polypeptides, which may be referred to herein as recombinant polypeptides, can be reassigned or deleted in a cell through the use of site-specific recombinant technology. In certain embodiments, the cell activation-related response to a particular genetically modified signaling polypeptide can be altered by site-specific recombination; for example, a first intracellular activation domain of a genetically modified signaling polypeptide that elicits a first activation-related response can be exchanged for a second intracellular activation domain that elicits a second activation-related response. As will become clear to a person skilled in the art, site-specific recombination can be used in a cell to exchange any domain or sequence of a genetically modified signaling polypeptide for any other domain or sequence as disclosed herein. As will also become clear to a person skilled in the art, site-specific recombination can be used in a cell to delete any domain or sequence of a genetically modified signaling polypeptide. Such sequence and domain exchange or excision is known in the art; see, for example, domain switching in signal bodies as described in Tone et al. (2013) Biotechnology and Bioengineering, 3219–3226, the disclosure of which is disclosed herein by reference. The mechanisms and requirements for performing site-specific recombination in vivo are well known in the art; see, for example, Grindley et al. (2006) Annual Review of Biochemistry, 567–605 and Tropp (2012) Molecular Biology (Jones & Bartlett Publishers, Sudbury, MA), the disclosures of which are incorporated herein by reference.

[0290] In some embodiments, the genetically modified signaling polypeptides are generated by fusing all the different domains discussed above together to form a fusion protein. The genetically modified signaling polypeptide is typically generated by a transcriptional unit comprising polynucleotide sequences that encode the different domains of the genetically modified signaling polypeptides as discussed herein. In some embodiments, the ASTR of the present invention, which functions to recognize and bind to an antigen on target cells, is restricted to the microenvironment.

[0291] The wild-type or native protein that is suitable for use wholly or in part for at least its antigen-binding domain as an ASTR in the present invention can be revealed by generating a protein library and screening the library for a protein with a desired antigen-binding affinity. The wild-type protein can be revealed by screening a cDNA library. A cDNA library is a combination of cloned cDNA fragments (complementary DNA) inserted into a collection of host cells, which together constitute some portion of the organism's transcriptome. cDNA is produced from the complementarily transcribed mRNA and therefore contains the coding sequence for an organism's expressed proteins. Information in cDNA libraries is a powerful and useful tool for revealing proteins with desired properties by screening libraries for proteins with the desired binding affinity to the target antigen. COMBINATIONS In some embodiments, a polynucleotide provided by recombinant retroviruses has one or more transcriptional units encoding specific combinations of one or more genetically modified signaling polypeptides. In some methods and compositions provided in this document, genetically modified T cells include combinations of one or more genetically modified signaling polypeptides after T cell transduction by recombinant retroviruses. It will be understood that the reference to a first polypeptide, a second polypeptide, a third polypeptide, etc. is for convenience, and elements in a “first polypeptide” and those in a “second polypeptide” mean that the elements are in different polypeptides that are referred to as first or second for reference and convention only, typically in elements or steps further away from that specific polypeptide.

[0292] In some embodiments, the first genetically modified signaling polypeptide includes an extracellular antigen-binding domain, which has the ability to bind to an antigen, and an intracellular signaling domain. In other embodiments, the first genetically modified signaling polypeptide also includes a T cell survival motif and/or a transmembrane domain. In some embodiments, the first genetically modified signaling polypeptide does not include a co-stimulatory domain, while in other embodiments, the first genetically modified signaling polypeptide does not include a co-stimulatory domain.

[0293] In some embodiments, a second genetically modified signaling polypeptide includes a lymphoproliferative gene product and, optionally, an extracellular antigen-binding domain. In some embodiments, the second genetically modified signaling polypeptide also includes one or more of the following: a T-cell survival motif, an intracellular signaling domain, and one or more costimulatory domains. In other embodiments, when two genetically modified signaling polypeptides are used, at least one is a CAR.

[0294] In one embodiment, one or more genetically modified signaling polypeptides are expressed under a T cell-specific promoter or a general promoter under the same transcript wherein, in the transcript, nucleic acids encoding the genetically modified signaling polypeptides are separated by nucleic acids encoding one or more internal ribosomal entry sites (IREs) or one or more protease cleavage peptides.

[0295] In certain embodiments, the polynucleotide encodes two genetically modified signaling polypeptides wherein the first genetically modified signaling polypeptide includes a first extracellular antigen-binding domain, which has the ability to bind to a first antigen, and a first intracellular signaling domain, but not a co-stimulatory domain, and the second polypeptide includes a second extracellular antigen-binding domain, which has the ability to bind to VEGF, and a second intracellular signaling domain, such as, for example, the signaling domain of a co-stimulatory molecule. In a certain embodiment, the first antigen is PSCA, PSMA, or BCMA. In a certain embodiment, the first extracellular antigen-binding domain comprises an antibody or fragment thereof (e.g., scFv), for example, an antibody or fragment thereof specific for PSCA, PSMA, or BCMA. In a certain embodiment, the second extracellular antigen-binding domain that binds to VEGF is a receptor for VEGF, i.e., VEGFR. In certain disciplines, VEGFR is VEGFR1, VEGFR2, or VEGFR3. In one particular discipline, VEGFR is VEGFR2.

[0296] In certain embodiments, the polynucleotide encodes two genetically modified signaling polypeptides wherein the first genetically modified signaling polypeptide includes an extracellular tumor antigen-binding domain and a CD3Z signaling domain, and the second genetically modified signaling polypeptide includes an antigen-binding domain, wherein the antigen is an angiogenic or vasculogenic factor, and one or more co-stimulatory molecule signaling domains. The angiogenic factor may be, for example, VEGF. The one or more co-stimulatory molecule signaling motifs may comprise, for example, co-stimulatory signaling domains of each of CD27, CD28, OX40, ICOS, and 4-1BB.

[0297] In certain embodiments, the polynucleotide encodes two genetically modified signaling polypeptides wherein the first genetically modified signaling polypeptide includes an extracellular tumor antigen-binding domain and the CD3Z signaling domain, the second polypeptide comprises an antigen-binding domain, which has the ability to bind to VEGF, and co-stimulatory signaling domains of each of CD27, CD28, OX40, ICOS and 4-1BB. In an additional embodiment, the first signaling polypeptide or the second signaling polypeptide also has a T cell survival motif. In some embodiments, the T cell survival motif is, or is derived from, an intracellular signaling domain of the IL-7 receptor (IL-7R), an intracellular signaling domain of the IL-12 receptor, an intracellular signaling domain of the IL-15 receptor, an intracellular signaling domain of the IL-21 receptor, or an intracellular signaling domain of the transforming growth factor β receptor (TGFβ) or the TGFβ decoy receptor (TGF-β—dominant negative receptor II (DNRII)).

[0298] In certain embodiments, the polynucleotide encodes two genetically modified signaling polypeptides wherein the first genetically modified signaling polypeptide includes an extracellular tumor antigen-binding domain and a CD3Z signaling domain, and the second genetically modified signaling polypeptide includes an antigen-binding domain, which has the ability to bind to VEGF, an intracellular T cell survival motif of IL-7 receptor and co-stimulatory signaling domains of each of CD27, CD28, OX40, ICOS and 4-1BB.

[0299] In some embodiments, more than two signaling polypeptides are encoded by the polynucleotide. In certain embodiments, only one of the genetically modified signaling polypeptides includes an antigen-binding domain that binds to a tumor-associated antigen or a tumor-specific antigen; each of the genetically modified signaling polypeptides comprises an antigen-binding domain that binds to an antigen that is not a tumor-associated antigen or a tumor-specific antigen. In other embodiments, two or more of the genetically modified signaling polypeptides include antigen-binding domains that bind to one or more tumor-associated antigens or tumor-specific antigens, wherein at least one of the genetically modified signaling polypeptides comprises an antigen-binding domain that does not bind to a tumor-associated antigen or a tumor-specific antigen.

[0300] In some modalities, the tumor-associated antigen or tumor-specific antigen is Her2, prostate stem cell antigen (PSCA), PSMA (prostate-specific membrane antigen), B-cell maturation antigen (BCMA), alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen-125 (CA-125), CA19-9, calretinin, MUC-1, epithelial membrane protein (EMA), epithelial tumor antigen (ETA), tyrosinase, melanoma-associated antigen (MAGE), CD34, CD45, CD99, CD117, chromogranin, cytokeratin, desmin, glial fibrillary acidic protein (GFAP), macroscopic cystic disease fluid protein (GCDFP-15), HMB-45 antigen, melan-A protein (melanoma antigen recognized by T lymphocytes; MART-1), myo-D1, muscle-specific actin (MSA), neurofilament, neuron-specific enolase (NSE), placental alkaline phosphatase, synaptophysin, thyroglobulin, thyroid transcription factor-1, dimeric form of pyruvate kinase type M2 isoenzyme (tumor M2-PK), CD19, CD22, CD27, CD30, CD70, GD2 (ganglioside G2), EphA2, CSPG4, CD138, FAP (Fibroblast Activating Protein), CD171, kappa, lambda, 5T4, integrin αvβ6, integrin αvβ3 (CD61), galactin, K-Ras (V-Ki-ras2 Kirsten mouse sarcoma viral oncogene), Ral-B, B7-H3, B7-H6, CAIX, CD20, CD33, CD44, CD44v6, CD44v7/8, CD123, EGFR, EGP2, EGP40, EpCAM, fetal AchR, FRα, GD3, HLA-A1+MAGE1, HLA-A1+NY-ESO-1, IL-11Rα, IL-13Rα2, Lewis-Y, Muc16, NCAM, NKG2D ligands, NY-ESO-1, PRAME, ROR1, Survivin, TAG72, TEMs, VEGFR2, EGFRvIII (epidermal growth factor variant III), sperm protein 17 (Sp17), mesothelin, PAP (prostatic acid phosphatase), prosteine, TARP (T-cell receptor gamma alternative reading frame protein), Trp-p8, STEAP1 (prostate epithelial antigen with transmembrane salts 1), an abnormal ras protein or a protein p53 abnormal.

[0301] In some embodiments, the first genetically modified signaling polypeptide includes a first extracellular antigen-binding domain that binds to a first antigen, and a first intracellular signaling domain; and a second genetically modified signaling polypeptide includes a second extracellular antigen-binding domain that binds to a second antigen, or a receptor that binds to the second antigen; and a second intracellular signaling domain, wherein the second genetically modified signaling polypeptide does not comprise a co-stimulatory domain. In a particular embodiment, the first antigen-binding domain and the second antigen-binding domain are independently an antigen-binding portion of a receptor or an antigen-binding portion of an antibody. In a particular embodiment, each or both the first antigen-binding domain and the second antigen-binding domain are scFv antibody fragments. In certain embodiments, the first genetically modified signaling polypeptide and/or the second genetically modified signaling polypeptide additionally comprise a transmembrane domain. In a particular embodiment, the first genetically modified signaling polypeptide or the second genetically modified signaling polypeptide comprises a T cell survival motif, for example, any of the T cell survival motifs described herein.

[0302] In another embodiment, the first genetically modified signaling polypeptide includes a first extracellular antigen-binding domain that binds to HER2 and the second genetically modified signaling polypeptide includes a second extracellular antigen-binding domain that binds to MUC-1.

[0303] In another embodiment, the second extracellular antigen-binding domain of the second genetically modified signaling polypeptide binds to an interleukin.

[0304] In another embodiment, the second extracellular antigen-binding domain of the second genetically modified signaling polypeptide binds to a damage-associated molecular pattern (DAMP; also known as an alarmin). In other embodiments, a DAMP is a heat shock protein, high-mobility group box 1 chromatin-associated protein (HMGB1), S100A8 (also known as MRP8 or calgranulin A), S100A9 (also known as MRP14 or calgranulin B), serum amyloid A (SAA), deoxyribonucleic acid, adenosine triphosphate, uric acid, or heparin sulfate.

[0305] In certain embodiments, the said second antigen is an antigen in an antibody that binds to an antigen presented by a tumor cell.

[0306] In some modalities, the activation of signal transduction via the second genetically modified signaling polypeptide is non-antigenic, but is associated with hypoxia. In certain modalities, hypoxia is induced by activation of hypoxia-inducible factor-1α (HIF-1α), HIF-1β, HIF-2α, HIF-2β, HIF-3α or HIF-3β.

[0307] In some embodiments, the expression of one or more genetically modified signaling polypeptides is regulated by an in vivo control element, which is disclosed in more detail in this document.

ADDITIONAL SEQUENCES

[0308] Genetically modified signaling polypeptides, such as CARs, may additionally include one or more additional polypeptide domains, wherein such domains include, but are not limited to, a signaling sequence; an epitope tag; an affinity domain; and a polypeptide that produces a detectable signal. Non-limiting examples of additional domains for any of the aspects or embodiments provided herein include a domain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any of the following sequences as described below: a signaling sequence, an epitope tag, an affinity domain, or a polypeptide that produces a detectable signal.

[0309] Signaling sequences that are suitable for use in a subject CAR, for example, in the first polypeptide of a subject CAR, include any eukaryotic signaling sequence, including a naturally occurring signaling sequence, a synthetic (e.g., artificial) signaling sequence, etc. In some embodiments, for example, the signaling sequence may be the CD8 signaling sequence MALPVTALLLPLALLLHAARP (SEQ ID NO:74).

[0310] Suitable epitope labels include, but are not limited to, hemagglutinin (HA; e.g., YPYDVPDYA; SEQ ID NO:37); FLAG (e.g., DYKDDDDK; SEQ ID NO:38); c-myc (e.g., EQKLISEEDL; SEQ ID NO:39) and the like.

[0311] Affinity domains include peptide sequences that can interact with a binding partner, for example, one immobilized on a solid support, useful for identification or purification. DNA sequences encoding multiple consecutive unique amino acids, such as histidine, when fused to the expressed protein, can be used for one-step purification of the recombinant protein by high-affinity binding to a resin column, such as nickel sepharose. Exemplary affinity domains include His5 (HHHHH; SEQ ID NO:40), HisX6 (HHHHHH; SEQ ID NO:41), c-myc (EQKLISEEDL; SEQ ID NO:39), Flag (DYKDDDDK; SEQ ID NO:38), Strep Tag (WSHPQFEK; SEQ ID NO:42), hemagglutinin, e.g., HA Tag (YPYDVPDYA; SEQ ID NO:37), GST, thioredoxin, cellulose-binding domain, RYIRS (SEQ ID NO:43), Phe-His-His-Thr (SEQ ID NO:44), chitin-binding domain, S-peptide, T7 peptide, SH2 domain, C-end RNA tag, WEAAAREACCRECCARA (SEQ ID NO:45), metal-binding domains, e.g., zinc-binding domains or binding domains to calcium, such as those from calcium-binding proteins, for example, calmodulin, troponin C, calcineurin B, myosin light chain, recoverin, S-modulin, visinin, VILIP, neurocalcin, hypocalcin, frequenin, caltractin, calpain large subunit, S100 proteins, parvalbumin, calbindin D9K, calbindin D28K and calretinin, inteins, biotin, streptavidin, MyoD, Id, leucine zipper sequences and maltose-binding protein.

[0312] Suitable signal-producing proteins include, for example, fluorescent proteins; enzymes that catalyze a reaction that generates a detectable signal as a product; and the like.

[0313] Some suitable fluorescent proteins include, but are not limited to, green fluorescent protein (GFP) or variants thereof, blue fluorescent variant of GFP (BFP), cyan fluorescent variant of GFP (CFP), yellow fluorescent variant of GFP (YFP), enhanced GFP (EGFP), enhanced CFP (ECFP), enhanced YFP (EYFP), GFPS65T, Emerald, Topaz (TYFP), Venus, Citrine, mCitrine, GFPuv, destabilized EGFP (dEGFP), destabilized ECFP (dECFP), destabilized EYFP (dEYFP), mCFPm, Cerulean, T-Sapphire, CyPet, YPet, mKO, HcRed, t-HcRed, DsRed, DsRed2, DsRed monomer, J-Red, dimer2, t-dimer2(12), mRFPl, pocilloporin, Renilla GFP, Monster GFP, paGFP, Kaede protein and kindling protein, phycobiliproteins and phycobiliprotein conjugates including β-F-coerythrin, R-F-coerythrin and allophycocyanin. Other examples of fluorescent proteins include mHoneydew, mBanana, mOrange, dTomato, tdTomato, mTangerine, mStrawberry, mCherry, mGrapel, mRaspberry, mGrape2, mPlum (Shaner et al. (2005) Nat. Methods 2:905 to 909) and the like. Any of a variety of fluorescent and colored proteins from the Anthozoan species, as described in, for example, Matz et al. (1999) Nature Biotechnol. 17:969 to 973, is suitable for use.

[0314] Suitable enzymes include, but are not limited to, horseradish peroxidase (HRP), alkaline phosphatase (AP), beta-galactosidase (GAL), glucose-6-phosphate dehydrogenase, beta-N-acetylglucosaminidase, β-glucuronidase, invertase, xanthine oxidase, firefly luciferase, glucose oxidase (GO) and the like.

DOMAIN OF RECOGNITION AND/OR ELIMINATION

[0315] Any of the recombinant retroviruses provided herein may include nucleic acids encoding a recognition or deletion domain as part of, or separate from, the nucleic acids encoding any of the genetically modified signaling polypeptides provided herein. Thus, any of the genetically modified signaling polypeptides provided herein may include a recognition or deletion domain. For example, any of the CARs disclosed herein may include a recognition or deletion domain. Furthermore, a recognition or deletion domain may be expressed together with, or even fused to, any of the lymphoproliferative elements disclosed herein. Recognition or deletion domains are expressed on T cells and/or NK cells, but are not expressed on the retrovirus.

[0316] In some embodiments, the recognition or scavenging domain may be derived from the herpes simplex virus (HSV-tk)-derived thymidine kinase enzyme or inducible caspase-9. In some embodiments, the recognition or scavenging domain may include a modified endogenous cell surface molecule, for example, as disclosed in U.S. Patent No. 8,802,374. The modified endogenous cell surface molecule may be any cell surface-related receptor, ligand, glycoprotein, adhesion molecule, antigen, integrin, or differentiation cluster (CD) that is modified. In some embodiments, the modified endogenous cell surface molecule is a truncated tyrosine kinase receptor. In one aspect, the truncated tyrosine kinase receptor is a member of the epidermal growth factor receptor (EGFR) family (e.g., ErbB1, ErbB2, ErbB3, ErbB4). In some embodiments, the recognition domain may be a polypeptide that is recognized by an antibody that recognizes the extracellular domain of an EGFR member. In some embodiments, the recognition domain may be at least 20 contiguous amino acids of an EGFR family member or, for example, between 20 and 50 contiguous amino acids of an EGFR family member. For example, SEQ ID NO:78 is an exemplary polypeptide that is recognized and, under appropriate conditions, bound by an antibody that recognizes the extracellular domain of an EGFR member. Such extracellular EGFR epitopes are sometimes referred to herein as eTags. In the illustrative embodiments, such epitopes are recognized by commercially available anti-EGFR monoclonal antibodies.

[0317] The epidermal growth factor receptor, also known as EGFR, ErbB1, and HER1, is a cell surface receptor for members of the epidermal growth factor family of extracellular ligands. Alterations in EGFR activity have been implicated in certain cancers. In some embodiments, a gene encoding an EGFR polypeptide including human epidermal growth factor receptor (EGFR) is constructed by removing nucleic acid sequences encoding polypeptides including the distal membrane EGF-binding domain and the cytoplasmic signaling tail, but retaining the proximal extracellular membrane epitope recognized by an anti-EGFR antibody. Preferably, the antibody is a commercially available anti-EGFR monoclonal antibody known, such as cetuximab, matuzumab, necitumumab, or panitumumab.

[0318] Others have shown that the application of biotinylated cetuximab to immunomagnetic selection in combination with antibiotic microspheres successfully enriches lentivirally transduced T cells with EGFRt-containing constructs from as low as 2% of the population to more than 90% purity without observable toxicity to the cell preparation. Furthermore, others have shown that the constitutive expression of this inert EGFR molecule does not affect T cell phenotype or effector function as directed by the coordinately expressed chimeric antigen receptor (CAR), CD19R. Additionally, others have shown that, through flow cytometric analysis, EGFR was successfully used as an in vivo tracking marker for T cell engraftment in mice. Moreover, EGFR has demonstrated suicide gene potential through antibody-dependent cell cytotoxicity (ADCC) pathways mediated by Erbitux®. The inventors of the present disclosure successfully expressed eTag in PBMCs using lentiviral vectors and found that in vitro eTag expression by PBMCs exposed to cetuximab provided an effective scavenging mechanism for PBMCs. Thus, EGFR can be used as a non-immunogenic screening tool, tracking marker, and suicide gene for transduced T cells that have immunotherapeutic potential. EGFR nucleic acid can also be detected by well-known means in the art.

[0319] In some embodiments provided in this document, EGFR is expressed as part of a single polypeptide that also includes the CAR or as part of a single polypeptide that includes the lymphoproliferative element. In some embodiments, the amino acid sequence encoding the EGFR recognition domain can be separated from the amino acid sequence encoding the chimeric antigen receptor by a cleavage signal and/or a ribosome jump sequence. The ribosome jump and/or cleavage signal can be any ribosome jump and/or cleavage signal known in the art. Without adhering to theory, the ribosome jump sequence could be, for example, 2A-1 with the amino acid sequence GSGEGRGSLLTCGDVEENPGP (SEQ ID NO:77). Without adhering to theory, other examples of cleavage signals and ribosome jump sequences include FMDV 2A (F2A); Equine rhinitis virus A 2A (abbreviated as E2A); porcine teschovirus-1 2A (P2A); and Thoseaasigna virus 2A (T2A). In some embodiments, the polynucleotide sequence encoding the recognition domain may be in the same transcript as the CAR or lymphoproliferative element, but separated from the polynucleotide sequence encoding the CAR or lymphoproliferative element by an internal ribosome entry site.

[0320] In other embodiments, as empirically exemplified herein, a recognition domain can be expressed as part of a fusion polypeptide, fused to a lymphoproliferative element. Such constructs provide the advantage, especially in combination with other “space-saving” elements provided herein, of occupying less genomic space in an RNA genome compared to separate polypeptides. In an illustrative embodiment, an eTag is expressed as a fusion polypeptide, fused to an IL7Ra mutant, as experimentally demonstrated herein.

PSEUDOTYPING ELEMENTS

[0321] Many of the methods and compositions provided in this document include pseudotyping elements. Retrovirus pseudotyping with heterologous envelope glycoproteins typically alters a virus's tropism and facilitates transduction into host cells. A pseudotyping element, as used in this document, may include a "binding polypeptide" comprising one or more polypeptides, typically glycoproteins, that identify and bind to the target host cell, and one or more "fusogenic polypeptides" that mediate the fusion of the target host and retroviral cell membranes, thus enabling a retroviral genome to enter the target host cell. In some embodiments provided in this document, the pseudotyping elements are provided as a polypeptide (or polypeptides)/protein (or proteins) or as nucleic acid sequences encoding the polypeptide (or polypeptides)/protein (or proteins).

[0322] In some embodiments, the pseudotyping element is the envelope protein of the feline endogenous virus (RD114), the oncoretroviral amphotopic envelope protein, the oncoretroviral ecotropic envelope protein, the envelope protein of the vesicular stomatitis virus (VSV-G) and/or the envelope proteins of the measles paramyxovirus H and F.

[0323] In some embodiments, the pseudotyping elements include a linking polypeptide and a fusogenic polypeptide derived from different proteins. For example, recombinant retroviruses of the methods and compositions disclosed herein can be pseudotyped with the fusion (F) and hemagglutinin (H) polypeptides of measles virus (MV), as non-limiting examples, clinical wild-type strains of MV and vaccine strains including the Edmonston strain (MV-Edm) or fragments thereof. Without adhering to theory, it is believed that both hemagglutinin (H) and fusion (F) polypeptides play a role in entry into host cells wherein the H protein binds MV to CD46, SLAM, and Nectin-4 receptors on target cells and F mediates the fusion of retroviral and host cell membranes. In an illustrative embodiment, especially when the target cell is a T cell and/or NK cell, the binding polypeptide is a Measles Virus H polypeptide and the fusogenic polypeptide is a Measles Virus F polypeptide.

[0324] In some studies, lentiviral particles pseudotyped with truncated F and H polypeptides showed a significant increase in titers and transduction efficacy (Funke et al. 2008. Molecular Therapy. 16(8):1427 to 1436), (Frecha et al. 2008. Blood. 112(13):4843 to 4852). The highest titers were obtained when the F cytoplasmic tail was truncated by 30 residues (designated MV(Ed)-FΔ30 (SEQ ID NO:105)). For the H variants, optimal truncation occurred when 18 or 19 residues were deleted (MV(Ed)-HΔ18 (SEQ ID NO:106) or MV(Ed)-HΔ19), although variants with a truncation of 24 residues with and without the replacement of deleted residues by alanine (MV(Ed)-HΔ24 (SEQ ID NO:235) and MV(Ed)-HΔ24+A) also resulted in optimal titrations.

[0325] In some embodiments, including those targeting T cell and/or NK cell transduction, the recombinant retroviruses of the methods and compositions disclosed herein are pseudotyped with mutated versions or variants of the measles virus fusion (F) and hemagglutinin (H) polypeptides, in the illustrative examples, cytoplasmic domain deletion variants of the measles virus F and H polypeptides. In some embodiments, the mutated F and H polypeptides are "truncated H" or "truncated F" polypeptides, whose cytoplasmic portion has been truncated, i.e., amino acid residues (or nucleic acids encoding the corresponding nucleic acid molecule that encodes the protein) have been deleted. "HΔY" and "FΔX" designate such truncated H and F polypeptides, respectively, wherein "Y" refers to 1 to 34 residues that have been deleted from the amino terminuses, and "X" refers to 1 to 35 residues that have been deleted from the carboxy terminuses of the cytoplasmic domains. In a further embodiment, the "truncated F polypeptide" is FΔ24 or FΔ30 and/or the "truncated H protein" is selected from the group consisting of HΔ14, HΔ15, HΔ16, HΔ17, HΔ18, HΔ19, HΔ20, HΔ21+A, HΔ24 and HΔ24+4A, more preferably HΔ18 or HΔ24. In an illustrative embodiment, the truncated F polypeptide is MV(Ed)-FΔ30 and the truncated H polypeptide is MV(Ed)-HΔ18.

[0326] In some embodiments, the fusogenic polypeptide includes multiple elements expressed as a single polypeptide. In some embodiments, the binding polypeptide and fusogenic polypeptide are translated from the same transcript but from separate ribosome-binding sites; in other embodiments, the binding polypeptide and fusogenic polypeptide are separated by a cleavage peptide site, which, without adhering to theory, is cleaved after translation, as is common in the literature, or a ribosome jump sequence. In some embodiments, translation of the binding polypeptide and fusogenic polypeptide from separate ribosome-binding sites results in a higher amount of fusogenic polypeptide compared to the binding polypeptide. In some embodiments, the ratio between fusogenic polypeptide and binding polypeptide is at least 2:1, at least 3:1, at least 4:1, at least 5:1, at least 6:1, at least 7:1, or at least 8:1. In some embodiments, the ratio between the fusogenic polypeptide and the linking polypeptide is between 1.5:1, 2:1, or 3:1 at the lower end of the range and 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1 at the upper end of the range.

ACTIVATION ELEMENTS

[0327] Many aspects of the methods and composition of the present disclosure include an activation element or a nucleic acid encoding an activation element. Restrictions associated with lentiviral (LV) transduction in resting T cells are attributed to a number of pre-entry and post-entry barriers as well as cellular restrictive factors (Strebel et al 2009. BMC Medicine 7:48). One restriction is the inability of envelope pseudotyped LV particles to recognize potential receptors and mediate fusion to the cell membrane. However, under certain conditions, transduction of resting T cells with HIV-1-based lentiviral vectors is possible primarily through T cell receptor (TCR) CD3 complex and CD28 co-stimulation (Korin & Zack. 1998. Journal of Virology. 72:3.161 to 3.168, Maurice et al. 2002. Blood 99:2.342 to 2.350), as well as through exposure to cytokines (Cavalieri et al 2003).

[0328] Cells of the immune system, such as T lymphocytes, recognize and interact with specific antigens through receptors or receptor complexes that, upon recognition or interaction with such antigens, cause cell activation and expansion throughout the body. An example of such a receptor is the antigen-specific T lymphocyte receptor complex (TCR/CD3). The T cell receptor (TCR) is expressed on the surface of T lymphocytes. One component, CD3, is responsible for intracellular signaling after the TCR is occupied by the ligand. The T lymphocyte receptor for the antigen-CD3 complex (TCR/CD3) recognizes antigenic peptides that are presented to it by proteins of the major histocompatibility complex (MHC). MHC and peptide complexes are expressed on the surface of antigen-presenting cells and other T lymphocyte targets. Stimulation of the TCR/CD3 complex results in T lymphocyte activation and a subsequent antigen-specific immune response. The TCR/CD3 complex plays a central role in the effector function and regulation of the immune system.

[0329] T lymphocytes also require a second co-stimulatory signal to become fully active. Without such a signal, T lymphocytes are either unresponsive to antigen binding to the TCR or become anergic. Such a co-stimulatory signal, for example, is provided by CD28, a T lymphocyte protein, which interacts with CD80 and CD86 on antigen-producing cells. ICOS (Inducible Co-stimulatory), another T lymphocyte protein, provides a co-stimulatory signal when bound to the ICOS ligand.

[0330] Activation of the T cell receptor CD3 complex (TCR) and co-stimulation with CD28 can occur by ex vivo exposure to solid surfaces (e.g., microspheres) coated with anti-CD3 and anti-CD28. In some embodiments of the methods and compositions disclosed herein, resting T cells are activated by ex vivo exposure to solid surfaces coated with anti-CD3 and anti-CD28.

[0331] In certain illustrative embodiments of the methods and compositions provided herein, polypeptides that have the ability to bind to CD3 and/or CD28 are presented as "activation elements" on the surface of recombinant retroviruses of the methods and compositions disclosed herein, which are also aspects of the invention. The polypeptides that bind to CD3 and/or CD28 are referred to as "activation elements" because of their ability to activate resting T cells.

[0332] In some embodiments, the activating element is a polypeptide capable of binding to CD3. In some embodiments, the polypeptide capable of binding to CD3 is an anti-CD3 antibody or a fragment thereof that retains the ability to bind to CD3. In the illustrative embodiments, the anti-CD3 antibody or fragment thereof is a single-chain anti-CD3 antibody, such as, but not limited to, an anti-CD3 scFV. In another illustrative embodiment, the polypeptide capable of binding to CD3 is anti-CD3scFvFc.

[0333] Various anti-human CD3 monoclonal antibodies and antibody fragments thereof are available and can be used in the present invention, including but not limited to UCHT1, OKT-3, HIT3A, TRX4, X35-3, VIT3, BMA030 (BW264/56), CLB-T3/3, CRIS7, YTH12.5, F111409, CLB-T3.4.2, TR-66, WT31, WT32, SPv-T3b, 11D8, XIII-141, XIII46, XIII-87, 12F6, T3/RW2-8C8, T3/RW24B6, OKT3D, M-T301, SMC2 and F101.01.

[0334] In some embodiments, the activating element is a polypeptide capable of binding to CD28. In some embodiments, the polypeptide capable of binding to CD28 is an anti-CD28 antibody or a fragment thereof that retains the ability to bind to CD28. In other embodiments, the polypeptide capable of binding to CD28 is CD80, CD86, or a functional fragment thereof that has the ability to bind to CD28 and induce CD28-mediated activation of Akt, such as an external fragment of CD80. In illustrative embodiments, the anti-CD28 antibody or fragment thereof is a single-chain anti-CD28 antibody, such as, but not limited to, an anti-CD28 scFV. In another illustrative embodiment, the polypeptide capable of binding to CD28 is CD80 or a fragment of CD80, such as an external fragment of CD80.

[0335] Anti-CD28 antibodies are known in the art and may include, as non-limiting examples, monoclonal antibody 9.3, an IgG2a antibody (Dr. Jeffery Ledbetter, Bristol Myers Squibb Corporation, Seattle, Wash.), monoclonal antibody KOLT-2, an IgG1 antibody, 15E8, an IgG1 antibody, 248.23.2, an IgM antibody, and EX5.3D10, an IgG2a antibody.

[0336] In an illustrative embodiment, an activation element includes two polypeptides, one polypeptide with the ability to bind to CD3 and one polypeptide with the ability to bind to CD28.

[0337] In certain embodiments, the polypeptide with the ability to bind to CD3 or CD28 is an antibody, a single-chain monoclonal antibody, or an antibody fragment, for example, a single-chain antibody fragment. Consequently, the antibody fragment may be, for example, a single-chain fragment variable region (scFv), an antibody-binding fragment (Fab) of an antibody, a single-chain antigen-binding fragment (scFab), a single-chain antigen-binding fragment without cysteines (scFabΔC), a fragment variable region (Fv), a construct specific for adjacent epitopes of an antigen (CRAb), or a single-domain antibody (VH or VL).

[0338] In some embodiments, an activation element is fused to a heterologous signaling sequence and/or a heterologous membrane-binding sequence, both of which help direct the activation element to the membrane. The heterologous signaling sequence directs the activation element to the endoplasmic reticulum, where the heterologous membrane-binding sequence covalently binds to one or more fatty acids (also known as post-translational lipid modification) so that the activation elements fused to the heterologous membrane-binding sequence are anchored to the lipid rafts of the plasma membrane. In some embodiments, post-translational lipid modification can occur via myristoylation, palmitoylation, or GPI docking. Myristoylation is a post-translational protein modification that corresponds to the covalent bonding of a 14-carbon saturated fatty acid, myristic acid, to the N-terminal glycine of a eukaryotic or viral protein. Palmitoylation is a post-translational protein modification that corresponds to the covalent attachment of a C16 acyl chain to cysteines, and less frequently to serine and threonine residues, of proteins. GPI docking refers to the attachment of glycosylphosphatidylinositol, or GPI, to the C-terminus of a protein during post-translational modification.

[0339] In some embodiments, the heterologous membrane-binding sequence is a GPI anchor-binding sequence. The heterologous GPI anchor-binding sequence may be derived from any known GPI-anchored protein (reviewed in Ferguson MAJ, Kinoshita T, Hart GW. Glycosylphosphatidylinositol Anchors. In: Varki A, Cummings RD, Esko JD, et al., editors. Essentials of Glycobiology. 2nd edition. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 2009. Chapter 11). In some embodiments, the heterologous GPI anchor-binding sequence is the GPI anchor-binding sequence of CD14, CD16, CD48, CD55 (DAF), CD59, CD80, and CD87. In some embodiments, the heterologous GPI anchor-binding sequence is derived from CD16. In the illustrative embodiments, the heterologous GPI anchor binding sequence is derived from the Fc FcYRIIIb (CD16b) receptor or decay-accelerating factor (DAF), otherwise known as complement decay-accelerating factor or CD55.

[0340] In some embodiments, one or both activation elements include a heterologous signaling sequence to help direct the expression of the activation element to the cell membrane. Any signaling sequence that is active in the packaging cell lineage can be used. In some embodiments, the signaling sequence is a DAF signaling sequence. In the illustrative embodiments, an activation element is fused to a DAF signaling sequence at its N-terminus and a GPI anchor-binding sequence at its C-terminus.

[0341] In an illustrative embodiment, the activation element includes scFVFc anti-CD3 fused to a CD14-derived GPI anchor binding sequence and CD80 fused to a CD16b-derived GPI anchor binding sequence; and both are expressed on the surface of a recombinant retrovirus provided in this document. In some embodiments, scFVFc anti-CD3 is fused to a DAF signaling sequence at its N-terminus and a CD14-derived GPI anchor binding sequence at its C-terminus, and CD80 is fused to a DAF signaling sequence at its N-terminus and a CD16b-derived GPI anchor binding sequence at its C-terminus; and both are expressed on the surface of a recombinant retrovirus provided in this document. In some embodiments, the DAF signaling sequence includes amino acid residues 1 to 30 of the DAF protein.

Membrane-bound cytokines

[0342] Some embodiments of the method and composition aspects disclosed herein include a membrane-bound cytokine or polynucleotides encoding a membrane-bound cytokine. Cytokines are typically, but not always, secreted proteins. Cytokines that are naturally secreted can be genetically modified as fusion proteins to be membrane-bound. Membrane-bound cytokine fusion polypeptides are included in the methods and compositions disclosed herein and are also an aspect of the invention. In some embodiments, recombinant retroviruses have a membrane-bound cytokine fusion polypeptide on their surface that has the ability to bind to a T cell and/or NK cell and promote their proliferation and/or survival. Typically, membrane-bound polypeptides are incorporated into the membranes of recombinant retroviruses, and when a cell is transduced by the recombinant retrovirus, the fusion of the retroviral and host cell membranes results in the polypeptide being bound to the membrane of the transduced cell.

[0343] In some embodiments, the cytokine fusion polypeptide includes IL-7, IL-15, or an active fragment thereof. Membrane-bound cytokine fusion polypeptides are typically a cytokine fused to a heterologous signaling sequence and/or a heterologous membrane-binding sequence. In some embodiments, the heterologous membrane-binding sequence is a GPI anchor-binding sequence. The heterologous GPI anchor-binding sequence may be derived from any known GPI-anchored protein (reviewed in Ferguson MAJ, Kinoshita T, Hart GW. Glycosylphosphatidylinositol Anchors. In: Varki A, Cummings RD, Esko JD, et al., editors. Essentials of Glycobiology. 2nd edition. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 2009. Chapter 11). In some embodiments, the heterologous GPI anchor binding sequence is the GPI anchor binding sequence of CD14, CD16, CD48, CD55 (DAF), CD59, CD80, and CD87. In some embodiments, the heterologous GPI anchor binding sequence is derived from CD16. In one illustrative embodiment, the heterologous GPI anchor binding sequence is derived from the Fc FcYRIIIb receptor (CD16b). In some embodiments, the GPI anchor is the DAF GPI anchor.

[0344] In the illustrative embodiments, the membrane-bound cytokine is a fusion polypeptide of a cytokine fused to DAF. DAF is known to accumulate in lipid rafts that are incorporated into the membranes of retroviruses that germinate from packaging cells. Consequently, without adhering to theory, it is believed that DAF fusion proteins are preferentially targeted to portions of packaging cell membranes that will become part of a recombinant retroviral membrane.

[0345] In non-limiting illustrative embodiments, the cytokine fusion polypeptide is IL-7, or an active fragment thereof, fused to DAF. In a specific non-limiting illustrative embodiment, the cytokine fusion polypeptide includes, in order: the DAF signaling sequence (DAF residues 1 to 31), IL-7 without its signaling sequence, and DAF residues 36 to 525.

IN VIVO CONTROL ELEMENT RIBOSWITCHES

[0346] Some of the compositions and methods provided in this document include one or more riboswitches or polynucleotides containing one or more riboswitches, which form distinct aspects of the present disclosure. Riboswitches are a common feature in bacteria for regulating gene expression and are a means of achieving RNA control of biological functions. Riboswitches are polynucleotides that may be present in the 5' untranslated region of mRNAs and enable regulatory control over gene expression through the binding of a small molecule ligand that induces or suppresses riboswitch activity. Typically, the riboswitch controls a gene product involved in the generation of the small molecule ligand, thus forming a feedback loop. Riboswitches typically act in a cis manner, although riboswitches have been identified that act in a trans manner. Natural riboswitches consist of two domains: an aptamer domain that binds to the ligand through a three-dimensional folded RNA structure and a function-switching domain that induces or suppresses activity in the riboswitch based on the absence or presence of the ligand. Thus, there are two ligand-sensitive conformations achieved by the riboswitch, representing activated and deactivated states (Garst et al., 2011). The function-switching domain can affect the expression of a polynucleotide by regulating: an internal ribosome entry site, accessibility of pre-mRNA splicing donors in the retroviral gene construct, translation, transcription termination, transcript degradation, miRNA expression, or shRNA expression (Dambach and Winkler 2009). Function switching domains and aptamers can be used as modular components that enable synthetic RNA devices to control gene expression, or as native aptamers, mutated/evolved native aptamers, or fully synthetic aptamers that are identified from screening random RNA libraries (McKeague et al 2016).

[0347] The purine riboswitch family represents one of the largest families with over 500 sequences found (Mandal et al 2003; US20080269258; and WO2006055351). Purine riboswitches share a similar structure consisting of three conserved helical elements/rod structures (P1, P2, P3) with intervening junction/loop elements (J1-2, L2, J2-3, L3, J3-1). The aptamer domains of the purine family of riboswitches naturally vary in their affinity/regulation for various purine compounds, such as adenine, guanine, adenosine, guanosine, deoxyadenosine, deoxyguanosine (Figure 5), etc. due to sequence variation (Kim et al. 2007).

[0348] In one aspect, an isolated polynucleotide is provided in this document for regulating the expression of a target polynucleotide including: a polynucleotide encoding the target polynucleotide functionally linked to a promoter and a riboswitch, wherein the riboswitch includes: a.) an aptamer domain capable of binding to a nucleoside analog antiviral drug and having reduced guanine or 2'-deoxyguanosine binding relative to the nucleoside analog antiviral drug; and b.) a function-switching domain capable of regulating the expression of the target polynucleotide, wherein the binding of the nucleoside analog by the aptamer domain induces or suppresses the expression-regulating activity of the function-switching domain, thereby regulating the expression of the target gene. In some embodiments, the target polynucleotide may be a polypeptide coding region, a miRNA, or an shRNA. In a non-limiting example, the riboswitch is functionally linked to a nucleic acid encoding a polypeptide, miRNA, or shRNA with in vivo activity, for example, that is effective in treating a disease. For example, in such a non-limiting example, the riboswitch is functionally linked to a nucleic acid encoding a chimeric antigen receptor. In the non-limiting illustrative examples provided herein, the target polynucleotide encodes one or more genetically modified signaling polypeptides included in various other aspects of the present disclosure. In these non-limiting illustrative examples, the riboswitch and the target polynucleotide encoding one or more genetically modified signaling polypeptides may be found in the genome of a packaging cell, a recombinant retrovirus, a T cell, and/or an NK cell.

[0349] In some embodiments, the aptamer domain may be between 30, 35, 40, 45, 50, 55, 60, 65, and 70 nucleotides long at the lower end of the band and 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, and 100 nucleotides long at the upper end of the band, for example, between 45 and 80 nucleotides long, between 45 and 60 nucleotides long, or between 45 and 58 nucleotides long. In the illustrative embodiments, the nucleoside analog antiviral drug may be the pharmaceutical ligand acyclovir (also known as acyclovir and acycloguanosine) or penciclovir (Figure 5). In some embodiments, the aptamer domain may have a binding affinity to the nucleoside analog antiviral drug that is greater than, for example, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 times greater than the binding affinity to the nucleoside or nucleotide.

[0350] The in vivo control element promotes the expansion of transduced T cells in vivo. In some modes, expansion depends on the presence of the control element. However, in other modes, the expansion of transduced T cells may be at least partially triggered by other factors, such as the presence of interleukins within the subject and the binding of the ASTR of a CAR on the recombinant T cell to its ligand.

[0351] In some embodiments, a nucleoside analog antiviral drug, for example, acyclovir or penciclovir, is administered to a subject before, during, and/or after PBLs are isolated from the blood and before T cells and/or NK cells are brought into contact with a recombinant retrovirus that includes an in vivo control element, which, in non-limiting illustrative examples, is a riboswitch, that binds to the nucleoside analog antiviral drug and regulates the expression of one or more target polynucleotides. The one or more target polynucleotides may encode one or more polypeptides which, in non-limiting illustrative examples, are one or more genetically modified signaling polypeptides, at least one of which encodes a lymphoproliferative element. In some embodiments, the nucleoside analog antiviral drug, for example, acyclovir or penciclovir, is administered to the subject for between 5, 10, 15, 30, and 60 minutes at the lower end of the range and 1, 5, 2, 3, 4, 5, 6, 8, 12, 24, 48, or 72 hours at the upper end of the range, before PBLs are isolated from the blood or before T cells and/or NK cells are brought into contact with a recombinant retrovirus. In some embodiments, the nucleoside analog antiviral drug, for example, acyclovir or penciclovir, is administered to the subject for between 1.5, 2, 3, 4, 5, 6, 8, 12 or 24 hours at the lower end of the range, 1, 2, 3, 4, 5, 6, 7, 10, 14, 21 or 28 days at the upper end of the range, after PBLs are isolated from the blood or after T cells and/or NK cells are brought into contact with a recombinant retrovirus in the methods provided in this document. In some embodiments, the nucleoside analog antiviral drug, for example, acyclovir or penciclovir, is administered to the subject for at least 1.5, 2, 3, 4, 5, 6, 8, 12 or 24 hours or at least 2, 3, 4, 5, 6, 7, 10, 14, 21 or 28 days after PBLs are isolated from the blood or after T cells and/or NK cells are brought into contact with a recombinant retrovirus in the methods provided in this document. In some embodiments, the nucleoside analog antiviral drug, for example, acyclovir or penciclovir, is administered to the subject for at least 1, 2, 3, 4, 5, 7, 10, 14, 21, 28, 30, 60, 90, or 120 days, or 5, 6, 9, 12, 24, 36, 48, 60, 72, 84, 96, 120 months, or indefinitely after the PBLs have been reinfused into the subject. In any of the embodiments disclosed herein, the nucleoside analog antiviral drug may be administered before and/or during the reinfusion of the PBLs and/or after the PBLs have been reinfused. In some modalities, the nucleoside analog antiviral drug is administered until a subject no longer experiences symptoms of, or is afflicted by, a disease to which the target polynucleotide is related.

[0352] In some embodiments, the aptamer domain may preferentially bind to penciclovir rather than acyclovir or, alternatively, to another antiviral agent, so that concomitant antiviral therapy may be used without affecting the riboswitch. In some embodiments, the aptamer domain may bind to penciclovir with a binding affinity greater than, for example, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 times greater than the aptamer domain that binds to acyclovir or another antiviral agent. In some embodiments, the aptamer domain may preferentially bind to acyclovir rather than penciclovir or, alternatively, to another antiviral agent, so that concomitant antiviral therapy can be used without affecting the riboswitch. In some embodiments, the aptamer domain may bind to acyclovir with a binding affinity greater than, for example, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 times greater than the aptamer domain that binds to penciclovir or another antiviral agent. In some embodiments, oral prodrugs of penciclovir (famciclovir) and acyclovir (valacyclovir) may be given to a subject.

[0353] In some embodiments, the aptamer domain of an isolated polynucleotide may share at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or be identical to any of the sequences of SEQ IDs 87 to 93 and may retain the ability to bind to acyclovir and a reduced ability to bind to guanine or 2'-deoxyguanosine relative to the nucleoside analog antiviral drug, and in which the aptamer domain retains the ability to induce or suppress the expression regulation activity of the function-switching domain when bound by acyclovir. In some embodiments, the aptamer domain of an isolated polynucleotide may share at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or be identical to the aptamer domain of SEQ IDs 94 to 100 and may retain the ability to bind to penciclovir and a reduced ability to bind to guanine or 2'-deoxyguanosine relative to the nucleoside analog antiviral drug, and in which the aptamer domain retains the ability to induce or suppress the expression regulation activity of the function-switching domain when bound by penciclovir. In some embodiments, a region of an isolated polynucleotide or a region of a riboswitch may share at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or be identical to any of the sequences in SEQ ID Nos: 87 to 100.

[0354] In some embodiments, a DNA sequence containing a region of an aptamer domain of an isolated polynucleotide may share at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or be identical to any of the sequences in SEQ ID Nos: 108 to 221. In some embodiments, a region of an isolated polynucleotide may share at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or be identical to any of the sequences in SEQ ID Nos: 108 to 221.

[0355] In some embodiments, a DNA sequence containing a region of an aptamer domain of an isolated polynucleotide may share at least 80%, 85%, 90%, 91%, 91.84%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or be identical to SEQ ID NO:108. In some embodiments, a DNA sequence containing a region of an aptamer domain of an isolated polynucleotide may share at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 95.83%, 96%, 97%, 98%, or 99% sequence identity or be identical to SEQ ID NO:147. In some embodiments, a DNA sequence containing a region of an aptamer domain of an isolated polynucleotide may share at least 80%, 85%, 90%, 91%, 92%, 93%, 93.88%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or be identical to SEQ ID NO:164. In some embodiments, a DNA sequence containing a region of an aptamer domain of an isolated polynucleotide may share at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 95.83%, 96%, 97%, 98%, or 99% sequence identity or be identical to SEQ ID NO:183. In some embodiments, a DNA sequence containing a region of an aptamer domain of an isolated polynucleotide may share at least 80%, 85%, 90%, 91%, 91.84%, 92%, 93%, 94%, 95%, 95.83%, 96%, 97%, 98%, or 99% sequence identity or be identical to SEQ ID NO:198.

[0356] In some embodiments, a region of an isolated polynucleotide may include any of the consensus sequences of SEQ IDs:222 to 226. In some embodiments, a region of an isolated polynucleotide may share at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 95.83%, 96%, 97%, 98% or 99% sequence identity or be identical to any of the sequences of SEQ IDs:222 to 226.

[0357] In any of the embodiments disclosed in this document, the isolated polynucleotide may retain the ability to bind to acyclovir and/or penciclovir. In any of the embodiments disclosed herein, an isolated polynucleotide may be the reverse complement of any of the sequences in SEQ ID Nos: 87 to 100 or SEQ ID Nos: 108 to 221. In any of the embodiments disclosed herein, an isolated polynucleotide may be an RNA transcript or version of the DNA sequences in SEQ ID Nos: 108 to 221 or of the DNA sequences complementary to SEQ ID Nos: 108 to 221. In any of the embodiments disclosed herein, an isolated polynucleotide may be a reverse transcript or DNA version of any of the RNA sequences in SEQ ID Nos: 87 to 100 or the DNA strand complementary to a reverse transcript of any of the RNA sequences in SEQ ID Nos: 87 to 100.

[0358] In some embodiments provided in this document, riboswitch scaffolds can be used for mutational analysis or molecular evolution. The riboswitches selected for mutational or molecular evolution can be from any known organism, for example, bacteria. In some embodiments, the type IA deoxyguanosine riboswitch from Mesoplasma florum can be used for molecular evolution. In some embodiments, the derived aptamer domain can be at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% identical to the aptamer domain of the type I-A deoxyguanosine riboswitch from Mesoplasma florum (SEQ ID NO:237). In other embodiments, the xpt riboswitch from Bacillus subtilis can be used. In some embodiments, the derived aptamer domain may be at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% identical to the aptamer domain of the xpt riboswitch from Bacillus subtilis (SEQ ID NO:243).

[0359] Aptamer domains can be used as modular components and combined with any of the function-switching domains to affect the RNA transcript. In any of the embodiments disclosed herein, the riboswitch can affect the RNA transcript by regulating any of the following activities: internal ribosomal entry site (IRES), accessibility of pre-mRNA splicing donor, translation, transcription termination, transcript degradation, miRNA expression, or shRNA expression. In some embodiments, the function-switching domain can control the binding of an anti-IRES to an IRES (see, for example, Ogawa, RNA (2011), 17:478-488, the disclosure of which is incorporated by reference herein in its entirety). In any of the embodiments disclosed herein, the presence or absence of the small molecule ligand can cause the riboswitch to affect the RNA transcript. In some embodiments, the riboswitch may include a ribozyme. Riboswitches with ribozymes can inhibit or enhance transcript degradation of target polynucleotides in the presence of the small molecule ligand. In some embodiments, the ribozyme may be a gun class ribozyme, a hammerhead class ribozyme, a twist class ribozyme, an axe class ribozyme, or the HDV (hepatitis delta virus) ribozyme.

[0360] In any of the embodiments disclosed herein, the riboswitch may be located at various positions relative to the target polynucleotide, as is generally known for riboswitches. In some embodiments, the riboswitch may regulate the accessibility of pre-mRNA splicing donor and may be located before the target polynucleotide. In some embodiments, the riboswitch may regulate the inclusion of a poly(A) tail and be located after the target polynucleotide. In some embodiments, the riboswitch may regulate an anti-IRES and be located upstream of an IRES. In non-limiting illustrative embodiments, a riboswitch provided herein may be located at any of these positions relative to a nucleic acid encoding one or more genetically modified signaling polypeptides provided herein.

[0361] In some embodiments, the riboswitch can be destabilized at temperatures above 37.5 °C, 38 °C, 38.5 °C, 39 °C, 39.5 °C, or 40 °C so that the riboswitch is no longer responsive to the ligand. In some embodiments, molecular evolution can be used to select riboswitches that are destabilized at temperatures above 37.5 °C, 38 °C, 38.5 °C, 39 °C, 39.5 °C, or 40 °C.

[0362] In some embodiments, the target polynucleotide may encode a miRNA, shRNA, and/or a polypeptide, wherein the target polynucleotide is functionally linked to a promoter. In some embodiments, the target polynucleotide may encode a lymphoproliferative element. In some embodiments, the target polynucleotide may be a miRNA or shRNA. In some embodiments, the miRNA or shRNA may enhance the STAT5 pathway or inhibit the SOCS pathway. In some embodiments, the miRNA or shRNA may target transcripts of SOCS1, SMAD2, TGFβ, or PD-1. In some embodiments, the miRNA is miR-155. In some embodiments, the target polynucleotide encodes a polypeptide, and the polypeptide may include a CAR that includes an antigen-specific targeting region, a transmembrane domain, and an intracellular activation domain.

[0363] In another aspect, an isolated polynucleotide is provided in this document for regulating the expression of a target polynucleotide including: a polynucleotide encoding the target polynucleotide functionally linked to a promoter and a riboswitch, wherein the riboswitch includes: a.) an aptamer domain capable of binding to a nucleoside analog antiviral drug with a binding affinity at least twice that with which the aptamer domain binds to guanine or 2'-deoxyguanosine; and b.) a function-switching domain capable of regulating the expression of the target polynucleotide, wherein the binding of the nucleoside analog by the aptamer domain induces or suppresses the expression-regulating activity of the function-switching domain. In some embodiments, the aptamer domain can bind to the nucleoside analog antiviral drug with a binding affinity at least 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 times greater than the aptamer domain binds to guanine or 2'-deoxyguanosine. In some embodiments, the aptamer domain may be between 30, 35, 40, 45, 50, 55, 60, 65, and 70 nucleotides long at the lower end of the band and 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, and 100 nucleotides long at the upper end of the band, for example, between 45 and 80 nucleotides long or between 45 and 58 nucleotides long. In the illustrative embodiments, the nucleoside analog antiviral drug may be the pharmaceutical ligand acyclovir (also known as acyclovir and acycloguanosine) or penciclovir. In some embodiments, the aptamer domain may have a binding affinity to the nucleoside analog antiviral drug that is greater than, for example, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 times greater than the binding affinity to the nucleoside or nucleotide. In some embodiments, the binding of the nucleoside analog by the aptamer domain may induce activity in the riboswitch.

[0364] In some embodiments, the aptamer domain may be specific for penciclovir and have no reactivity to acyclovir or, alternatively, to another antiviral agent, so that concomitant antiviral therapy may be used without affecting the riboswitch. In some embodiments, the aptamer domain may bind to penciclovir with a binding affinity at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 times greater than the aptamer domain that binds to acyclovir or another antiviral agent. In some embodiments, the aptamer domain may be specific for acyclovir and have no reactivity to penciclovir or, alternatively, to another antiviral agent, so that concomitant antiviral therapy may be used without affecting the riboswitch. In some embodiments, the aptamer domain can bind to acyclovir with a binding affinity at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times greater than the aptamer domain that binds to acyclovir or another antiviral agent. In some embodiments, the oral prodrugs of penciclovir (famciclovir) and acyclovir (valacyclovir) can be given to a subject. In some embodiments, the derived aptamer domain can be at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% identical to the aptamer domain of the type I-A deoxyguanosine riboswitch from Mesoplasma florum. In some embodiments, the derived aptamer domain may be at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% identical to the aptamer domain of the Bacillus subtilis xpt riboswitch. In any of the embodiments disclosed herein, the riboswitch may affect the RNA transcript by regulating any of the following activities: internal ribosomal entry site, accessibility of pre-mRNA splicing donor in the retroviral gene construct, translation, transcription termination, transcript degradation, miRNA expression, or shRNA expression. In some embodiments, the switching domain may control the binding of an anti-IRES to an IRES. In any of the embodiments disclosed herein, the presence or absence of the small molecule ligand may cause the riboswitch to affect the RNA transcript. In some embodiments, the riboswitch may include a ribozyme. Riboswitches with ribozymes can inhibit or enhance transcript degradation of genes of interest in the presence of the small molecule ligand. In some embodiments, the ribozyme may be a gun class ribozyme, a hammerhead class ribozyme, a twist class ribozyme, an axe class ribozyme, or the HDV (hepatitis delta virus) ribozyme. In some embodiments, the riboswitch may be destabilized at temperatures above 37.5 °C, 38 °C, 38.5 °C, 39 °C, 39.5 °C, or 40 °C so that the riboswitch is no longer responsive to the ligand. In some embodiments, molecular evolution can be used to select riboswitches that are destabilized at temperatures above 37.5 °C, 38 °C, 38.5 °C, 39 °C, 39.5 °C, or 40 °C. In some embodiments, the target polynucleotide may encode a miRNA, shRNA, and/or a polypeptide, wherein the target polynucleotide is functionally linked to a promoter. In some embodiments, the target polynucleotide may encode a lymphoproliferative element. In some embodiments, the target polynucleotide may be a miRNA, and optionally, the miRNA may stimulate the STAT5 pathway or inhibit the SOCS pathway. In some embodiments, the miRNA may target transcripts of SOCS1, SHP, SMAD2, TGFβ, or PD-1. In these embodiments, the miRNA can be miR-155. In some embodiments, the target polynucleotide encodes a polypeptide, and the polypeptide may include a CAR that includes an antigen-specific targeting region, a transmembrane domain, and an intracellular activation domain. Additional CAR embodiments are disclosed elsewhere in this document.

[0365] In some embodiments, aptamer evolution can be performed by selecting aptamers from randomized native purine or guanine aptamer libraries using SELEX (Systematic Evolution of Ligands by Exponential Enrichment) methods, including, but not limited to, those methods employing graphene oxide in the selection and screening process. In other embodiments, random mutagenesis methodology, such as error-prone PCR, can be used to evolve aptamer constructs or riboswitch constructs where the aptamer is incorporated in the context of any of the riboswitch activities described herein by screening in vitro or in mammalian cells. In other embodiments, random nucleotide libraries can be used in riboswitch evolution. In any of the embodiments disclosed herein, riboswitches can be identified from screening such libraries in vitro or in mammalian cells.

[0366] In some embodiments, the evolved or derived aptamer domain may have increased binding to analogs of the native ligand and decreased binding to the native ligand. In some embodiments, the aptamer domain may be configured to have increased binding to analogs of the native ligand and decreased binding to the native ligand. In some embodiments, the aptamer domain may be derived from the purine riboswitch family. In some embodiments, the native ligand may be a nucleoside or nucleotide, and the analog may be a nucleoside analog or a nucleotide analog. In some embodiments, the nucleoside analog is an antiviral drug. In illustrative embodiments, the aptamer domains may be derived from 2'-deoxyguanosine and guanine riboswitch scaffolds, and the derived aptamer domains may show reduced binding to 2'-deoxyguanosine and guanine relative to the wild-type riboswitch.

[0367] In some embodiments, the riboswitch can regulate donor accessibility of pre-mRNA splicing in the retroviral gene construct, wherein the retroviral construct triggers CAR genes or other genes of interest from the reverse strand under a general promoter or a T cell-specific promoter. In other embodiments, the riboswitch can regulate an IRES in the retroviral gene construct, wherein the retroviral construct triggers the translation of CAR genes or other genes of interest. In other embodiments, the riboswitch can control the transcription terminator of RNA, miRNA, or gene transcripts or can control transcript translation. In other embodiments, the nucleoside analog riboswitch can be integrated into a ribozyme to inhibit or enhance transcript degradation of CAR genes or other genes of interest in the presence of the nucleoside analog.

[0368] In some embodiments, the isolated polynucleotide for regulating the expression of a target polynucleotide, which includes a polynucleotide encoding the target polynucleotide functionally linked to a promoter and a riboswitch that binds to a nucleoside analog antiviral drug, is a molecular cloning vector. The molecular cloning vector can be any type of molecular cloning vector known in the art. As non-limiting examples, the vector can be a plasmid, a virus, or a retrovirus, any of which can be an expression vector. Such an expression vector can encode any of the target polynucleotides previously provided in this document. One or more multiple cloning and/or restriction sites can be included in a 5’ or 3’ molecular cloning vector to a riboswitch provided in this document so that the riboswitch is functionally linked to a target polynucleotide inserted into the multiple cloning and/or restriction site.

MOLECULAR CHAPERONES

[0369] In one aspect, a method is provided herein for genetically modifying and expanding lymphocytes of a subject comprising: A. placing resting T cells and/or NK cells of the subject ex vivo, typically without requiring prior ex vivo stimulation, into contact with recombinant retroviruses comprising: i. a pseudotyping element on their surface that has the ability to bind to a T cell and/or NK cell and facilitate the fusion of the recombinant retrovirus membrane to them; and ii. A. A polynucleotide comprising one or more transcriptional units functionally linked to an active promoter on T cells and/or NK cells, wherein one or more transcriptional units encode a first genetically modified signaling polypeptide regulated by an in vivo control element, wherein said first genetically modified signaling polypeptide comprises a lymphoproliferative element and/or a chimeric antigen receptor, wherein said contact facilitates the transduction of at least some of the resting T cells and/or NK cells by recombinant retroviruses, thereby producing genetically modified T cells and/or NK cells; B. Introduce the genetically modified T cells and/or NK cells into the subject; and C. Expose the genetically modified T cells and/or NK cells in vivo to a compound that acts as the in vivo control element to affect the expression of the first genetically modified signaling polypeptide and promote lymphocyte expansion in vivo, thereby genetically modifying and expanding the subject's lymphocytes.

[0370] In the illustrative embodiments, transduction is performed without ex vivo stimulation. In the illustrative embodiments, the compound is a molecular chaperone, such as a small molecule molecular chaperone. In the illustrative embodiments, the binding of the molecular chaperone to the lymphoproliferative element and/or CAR component increases the proliferative activity of the lymphoproliferative element and/or CAR. The molecular chaperone can be administered to the subject before blood is collected, during contact, and/or after T cells and/or NK cells are introduced into the subject. Some embodiments of this aspect involve collecting blood from the subject. In these embodiments, the introduction is a reintroduction of the cells that were collected and genetically modified prior to reintroduction. The entire process, in the illustrative embodiments, is a shorter process than the methods of the prior art, as for other aspects in this document. For example, the entire process can be completed in less than 48 hours, less than 24 hours, or less than 12 hours. The entire process, in other modalities, can be completed in 2, 4, 6, or 8 hours at the lower end of the range and 12, 24, 36, or 48 hours at the upper end of the range.

[0371] Consequently, in some embodiments of the methods and compositions provided herein, the in vivo control element is a molecular chaperone. Compared to other embodiments herein with other in vivo control elements, such as riboswitches that typically bind to a compound to affect the expression of a lymphoproliferative element or other component of a first or second genetically modified signaling polypeptide herein, molecular chaperones are compounds that are the in vivo control elements and, as such, directly affect the activity of, typically by binding to, a lymphoproliferative element or other component of a first or second genetically modified signaling polypeptide herein. In illustrative examples of such embodiments of the methods herein that involve the administration of molecular chaperones, a lymphoproliferative element, membrane-bound cytokine, and/or CAR component may be an inactive or less active lymphoproliferative element, membrane-bound cytokine, and/or CAR component that is bound by the molecular chaperone to enhance its activity. Thus, the target binding site for a molecular chaperone is typically a target polypeptide. In some embodiments, as indicated, the polypeptide may be a first and/or second genetically modified signaling polypeptide, or a polypeptide component thereof, whose activity is affected by binding to the molecular chaperone, which, in the illustrative embodiments, is a small-molecule molecular chaperone. In some embodiments, the polypeptide may include a lymphoproliferative element whose activity is regulated, in the illustrative embodiments, increasingly regulated by a molecular chaperone, preferably a small-molecule molecular chaperone. The molecular chaperone in the methods provided herein may be a compound that binds to the mutant lymphoproliferative element and/or inactive CAR component, thereby making them active.

[0372] In other embodiments, a lymphoproliferative element or other signaling domain has been mutated to allow transit to the plasma membrane only in the presence of a small molecule synthetic chaperone. In other embodiments, the chaperone promotes the stability of the lymphoproliferative element or other signaling domain or protein and the half-life as an enhancer.

[0373] It will be understood that aspects and embodiments of the present invention include many of the same steps and compositions provided in detail herein. Consequently, it will be understood that the teachings throughout this descriptive report that refer to these common elements apply to aspects and embodiments that utilize a molecular chaperone as the in vivo control element, which typically binds to a lymphoproliferative element or other target molecule directly, in addition to or instead of other in vivo control elements provided herein, such as riboswitches, which typically utilize a molecule, such as a drug, that binds to the riboswitch.

[0374] In some embodiments, a molecular chaperone is a compound that can regulate the subcellular localization of a target, for example, the appropriate folding and transit of a target protein, such as a lymphoproliferative element and/or a component of a CAR, from the endoplasmic reticulum to the plasma membrane or its half-life on the surface. In other embodiments, a molecular chaperone can promote the functional conformation of a dysfunctional target, thus acting as an enhancer. Examples of molecules that act as chaperones or enhancers for naturally mutated proteins include lumacaftor and ivacaftor. These proteins act on mutant CFTR chloride channel variants, such as G551D or F508del. Ivacaftor enhances the activity of the G551D or F508del mutated ion channel, while lumacaftor promotes the stabilization of mutant chloride channels and subsequent potentiation by ivacaftor. Such chaperone-dependent proteins can be generated from naturally functional proteins and screened for functional activity only in the presence of molecular chaperones. Thus, such proteins are only active when the chaperone is present. Examples of such molecules that can be screened for specific chaperone activity include small-molecule antivirals or anti-infectives that show no activity against normal human proteins. Consequently, in one embodiment, the molecular chaperone used in the methods in this document is a small-molecule antiviral or anti-infective compound that shows no activity against normal human proteins.

[0375] In some embodiments, genetically modified lymphocytes may be exposed and/or a subject may receive the molecular chaperone. In some embodiments, the compound is administered to the subject before, during, and/or after PBLs are isolated from the blood and before T cells and/or NK cells are brought into contact with a recombinant retrovirus. The recombinant retrovirus in such embodiments includes a lymphoproliferative element and/or inactive or less active CAR component that binds to or is regulated by the molecular chaperone compound.

[0376] For any of the modalities provided in this document for modifying and expanding lymphocytes, which may be part of adoptive cell therapy methods, the compound may be administered to the subject for between 5, 10, 15, 30 and 60 minutes at the lower end of the range and 1, 5, 2, 3, 4, 5, 6, 8, 12 or 24 hours at the upper end of the range, before PBLs are isolated from the blood or before T cells and/or NK cells are brought into contact with a recombinant retrovirus. In some embodiments, the compound is administered to the subject for between 1.5, 2, 3, 4, 5, 6, 8, 12 or 24 hours at the lower end of the range, %, 1, 2, 3, 4, 5, 6, 7, 10, 14, 21 or 28 days at the upper end of the range, after PBLs are isolated from the blood or after T cells and/or NK cells are brought into contact with a recombinant retrovirus in the methods provided in this document. In some embodiments, the compound is administered to the subject for at least 1.5, 2, 3, 4, 5, 6, 8, 12, or 24 hours, or at least 2, 3, 4, 5, 6, 7, 10, 14, 21, or 28 days after PBLs are isolated from the blood or after T cells and/or NK cells are brought into contact with a recombinant retrovirus using the methods provided in this document. In some embodiments, the compound is administered to the subject for at least 1, 2, 3, 4, 5, 7, 10, 14, 21, 28, 30, 60, 90, or 120 days, or 5, 6, 9, 12, 24, 36, 48, 60, 72, 84, 96, 120 months, or indefinitely after the PBLs have been reinfused into the subject. In any of the embodiments disclosed herein, the compound may be administered before and/or during the reinfusion of the PBLs and/or after the PBLs have been reinfused.

[0377] For any of the embodiments in this document, the molecular chaperones are not the in vivo control elements that are linked by compounds that regulate and/or activate them. The molecular chaperones are compounds, preferably small molecule compounds, that are the in vivo control elements and regulate the activity of lymphoproliferative elements and/or functional components of CARs.

Packaging cell lines/methods for producing recombinant retroviruses

[0378] In one aspect, a retroviral packaging system is provided in the present document comprising: a mammalian cell comprising: a) a first transactivator expressed from a constitutive promoter and capable of binding to a first ligand and a first inducible promoter to affect the expression of a functionally linked nucleic acid sequence in the presence versus absence of the first ligand; a) a second transactivator capable of binding to a second ligand and a second inducible promoter and affecting the expression of a functionally linked nucleic acid sequence in the presence versus absence of a second ligand; (c) a packageable RNA genome for a retroviral particle, wherein the first transactivator regulates the expression of the second transactivator, and wherein the second transactivator regulates the expression of retroviral polypeptides involved in viral packaging, such as, for example, a gag polypeptide, a pol polypeptide and/or a pseudotyping element and, optionally, other polypeptides that will become incorporated within or on the recombinant retrovirus and that are believed to be toxic to packaging cell lines, such as, for example, HEK-293. In certain respects, the second transactivator itself is cytotoxic to packaging cell lines. Pseudotyping elements typically have the ability to bind to a cell membrane of a target cell and facilitate fusion to it, as discussed in detail in this document. Thus, without adhering to theory, the system provides the ability to accumulate certain polypeptides/proteins that do not inhibit, or do not substantially inhibit, or are not believed to inhibit the proliferation or survival of mammalian cells, for example, non-toxic proteins, while cultivating a population of mammalian cells for days or indefinitely and controlling the induction of polypeptides that are desired for the retroviral product but that are inhibitors, or may be inhibitors, or have been reported to be inhibitors of mammalian cell survival and/or proliferation, for example, toxic polypeptides, until a later time closer to when the retroviruses will be produced and collected. The packageable RNA genome is typically encoded by a polynucleotide functionally linked to a promoter, sometimes referred to herein as a third promoter for convenience, wherein said third promoter is typically inducible by either the first transactivator or the second transactivator. In the illustrative embodiments, the packageable RNA genome is encoded by a polynucleotide functionally linked to a third promoter, wherein said third promoter is inducible by the second transactivator. As such, the packageable RNA genome can be produced at a later time point, closer to when the retrovirus will be collected.

[0379] A person skilled in the art will understand that many different transactivators, ligands, and inducible promoters can be used in the retroviral packaging system. Such inducible promoters can be isolated and derived from many organisms, for example, eukaryotes and prokaryotes. The modification of inducible promoters derived from a first organism for use in a second organism, for example, a first prokaryote and a second eukaryote, a first eukaryote and a second prokaryote, etc., is well known in the art. Such inducible promoters and systems based on such inducible promoters, but also including additional control proteins, include, but are not limited to, alcohol-regulated promoters (e.g., alcohol dehydrogenase I gene promoter (alcA), alcohol transactivator protein responsive promoters (AlcR), etc.), tetracycline-regulated promoters (e.g., promoter systems including TetActivators, TetON, TetOFF, etc.), steroid-regulated promoters (e.g., rat glucocorticoid receptor promoter systems, human estrogen receptor promoter systems, retinoid promoter systems, thyroid promoter systems, ecdysone promoter systems, mifepristone promoter systems, etc.), metal-regulated promoters (e.g., metallothionein promoter systems, etc.), pathogenesis-related regulated promoters (e.g., salicylic acid-regulated promoters, ethylene-regulated promoters, etc.). benzothiadiazole, etc.), temperature-regulated promoters (e.g., heat shock inducible promoters (e.g., HSP-70, HSP-90, soybean heat shock promoter, etc.), light-regulated promoters, synthetic inducible promoters, and the like. In some embodiments, a mifepristone-regulated system may be used. In some embodiments, a mifepristone-inducible system with an autoregulatory feedback loop may be used. In some embodiments, a regulatory fusion protein GAL4 is expressed from a construct that also contains the terminal transposon repeats and lox and FRT sites. In some embodiments, the regulatory fusion protein GAL4 counter-expresses a reverse tet transactivator (rtTA) and BiTRE. In some embodiments, another construct with lox and FRT sites contains an upstream activation sequence (UAS) of GAL4 and an E1b TATA box promoter that triggers a reporter such as mCherry. In some embodiments, a GAL4 regulatory fusion protein binds to GAL4 upstream activation sequences (UAS) in both the promoter that controls the expression of the GAL4 regulatory fusion protein and the promoter that controls the expression of a target polynucleotide. In some embodiments, mifepristone, doxycycline, and puromycin will be used for induction and selection of the packaging cell lineage.

[0380] In some embodiments, each or both transactivators may be split into two or more polypeptides. In some embodiments, the two or more polypeptides may include a DNA-binding domain and an activation domain with the ability to stimulate transcription into separate polypeptides. This “activation domain” should not be confused with an “activation element,” such as a CD3-binding polypeptide that has the ability to activate a T cell and/or NK cell and does not typically activate such T cell and/or NK cell when placed in contact with them, as discussed in detail in this document. The separate polypeptides may additionally include fusions with polypeptides capable of dimerization through the addition of a ligand. In some embodiments, the activation domain may be the p65 activation domain or a functional fragment thereof. In the illustrative embodiments of the packaging systems in this document, the DNA-binding domain may be the ZFHD1 DNA-binding domain or a functional fragment thereof. In some embodiments, one polypeptide may be a fusion with FKBP, or mutants and/or functional fragments thereof, or multiple FKBPs, and another polypeptide may be a fusion with the mTOR FRB domain, or mutants and/or functional fragments thereof, and the ligand may be rapamycin or a functional rapalog. In some embodiments, the FRB contains the K2095P, T2098L, and/or W2101F mutations. In some embodiments, the detached polypeptides may be FKBP, or functional fragments thereof, and Calcineurin A, or functional fragments thereof, and the dimerizing agent may be FK506. In some embodiments, the detached polypeptides may be FKBP, or functional fragments thereof, and CyP-Fas, or functional fragments thereof, and the dimerizing agent may be FKCsA. In some embodiments, the detached polypeptides may be GAI, or functional fragments thereof, and GID1, or functional fragments thereof, and the dimerizing agent may be gibberellin. In some embodiments, the detached polypeptides may be Snap-tag and HaloTag, or functional fragments thereof, and the dimerizing agent may be HaXS. In some embodiments, the detached polypeptides may include the same polypeptide. For example, the DNA-binding domain and the activation domain may be expressed as fusion proteins with FKBP or GyrB, the dimerizing agent may be FK1012 or coumermycin, respectively. In some embodiments, the inducible promoter may be the DNA sequence to which the DNA-binding domain typically binds. In some embodiments, the inducible promoter may vary from the DNA sequence to which the DNA-binding domain typically binds. In some embodiments, the transactivator may be an rtTA, the ligand may be tetracycline or doxycycline, and the inducible promoter may be TRE. In the illustrative embodiments, the first transactivator is the p65 activation domain fused to FRB and the ZFHD1 DNA-binding domain fused to three FKBP polypeptides, and the first ligand is rapamycin. In further illustrative embodiments, the second transactivator may be an rtTA, the second ligand may be tetracycline or doxycycline, and the inducible promoter may be TRE.

[0381] In some embodiments, the first transactivator can regulate the expression of an element to control the nuclear export of transcripts containing a consensus sequence, such as an HIV Rev, and the consensus sequence can be the Rev response element. In the illustrative embodiments, the target cell is a T cell.

[0382] In some embodiments, the pseudotyping element is a retroviral envelope polypeptide. The pseudotyping element typically includes a binding polypeptide and a fusogenic polypeptide to bind to and facilitate fusion of target cell membranes and viral membranes, as discussed in more detail in this document. In some embodiments, the pseudotyping element is the envelope protein of feline endogenous virus (RD114), the oncoretroviral amphotopic envelope protein, the oncoretroviral ecotropic envelope protein, and/or the envelope protein of vesicular stomatitis virus (VSV-G). In illustrative embodiments, the pseudotyping element includes a binding polypeptide and a fusogenic polypeptide derived from different proteins, as discussed in more detail in this document. For example, in an illustrative embodiment, especially when the target cell is a T cell and/or NK cell, the binding polypeptide is a hemagglutinin (H) polypeptide from a Measles Virus (such as the Edmonston strain of Measles Virus), or a cytoplasmic domain deletion variant thereof, and the fusogenic polypeptide is a fusion (F) polypeptide from a Measles Virus (such as the Edmonston strain of Measles Virus), or a cytoplasmic domain deletion variant thereof. In some embodiments, the fusogenic polypeptide may include multiple elements expressed as a single polypeptide. In some embodiments, the linking polypeptide and the fusogenic polypeptide can be translated from the same transcript but from separate ribosome binding sites, or the polypeptide is cleaved after translation using a peptide cleavage signal or a ribosome jump sequence, as disclosed elsewhere in this document, to generate the linking polypeptide and the fusogenic polypeptide. In some embodiments, when the binding polypeptide is a Measles Virus H polypeptide, or a cytoplasmic domain deletion thereof, and the fusogenic polypeptide is a Measles Virus F polypeptide, or a cytoplasmic domain deletion thereof, translation of the F and H polypeptides from separate ribosome binding sites results in a higher amount of the F polypeptide compared to the H polypeptide. In some embodiments, the ratio between the F polypeptides (or cytoplasmic domain deletions thereof) and H polypeptides (or cytoplasmic domain deletions thereof) is at least 2:1, at least 3:1, at least 4:1, at least 5:1, at least 6:1, at least 7:1, or at least 8:1.

[0383] In some embodiments, the first transactivator can regulate the expression of an activation element capable of binding to and activating a target cell, such as a T cell. Any of the activation elements disclosed herein can be expressed. For example, in these embodiments, the activation element may include: a.) a membrane-bound polypeptide capable of binding to and activating CD3; and/or b.) a membrane-bound polypeptide capable of binding to and activating CD28. In some embodiments, the membrane-bound polypeptide capable of binding to and activating CD28 is CD80, CD86, or functional fragments thereof, such as the extracellular domain of CD80.

[0384] In some embodiments, the second transactivator can regulate the expression of an RNA encoding one or more target polypeptides, including, as a non-limiting example, any of the genetically modified signaling polypeptides disclosed herein. It should be noted that it is contemplated that the aspect of the retroviral packaging system and the method for producing an aspect of the recombinant retrovirus are not limited to the production of recombinant retroviruses for T cell and/or NK cell transduction, but rather for any cell type that can be transduced by recombinant retroviruses. The RNA, in certain illustrative embodiments, includes, in opposite orientation (e.g., encoding on the opposite strand and in opposite orientation), retroviral components such as gag and pol. For example, the RNA may include from 5' to 3': a 5' long terminal repeat, or an active truncated fragment thereof; a nucleic acid sequence encoding a retroviral cis-acting RNA packaging element; A nucleic acid sequence encoding a first and, optionally, a second target polypeptide, such as, but not limited to, a genetically modified signaling polypeptide (or polypeptides) that may be expelled from a promoter, which, in some embodiments, is called a “fourth” promoter for convenience only; a promoter that is active in a target cell; and an active 3’ long terminal repeat, or truncated fragment thereof. In some embodiments, the RNA may include a central polypurine tract (cPPT)/central termination sequence element (CTS). In some embodiments, the retroviral cis-acting RNA packaging element may be HIV Psi. In some embodiments, the retroviral cis-acting RNA packaging element may be the Rev Response Element. The genetically modified signaling polypeptide in the illustrative embodiments is one or more of the genetically modified signaling polypeptides disclosed herein.

[0385] It will be understood that the number of promoters, such as a first, second, third, fourth, etc. promoter, is for convenience only. A promoter who is called a “fourth” promoter should not be taken to imply that there are any additional promoters, such as first, second, or third promoters, unless such other promoters are explicitly mentioned.

[0386] In some embodiments, the genetically modified signaling polypeptide may include a first lymphoproliferative element. Suitable lymphoproliferative elements are disclosed in other sections of this document. As a non-limiting example, the lymphoproliferative element may be expressed as a fusion with a recognition domain, such as an eTag, as disclosed in this document. In some embodiments, the packageable RNA genome may additionally include a nucleic acid sequence encoding a second genetically modified polypeptide including a chimeric antigen receptor encoding any CAR embodiment provided in this document. For example, the second genetically modified polypeptide may include a first antigen-specific targeting region, a first transmembrane domain, and a first intracellular activation domain. Examples of antigen-specific targeting regions, transmembrane domains, and intracellular activation domains are disclosed elsewhere in this document. In some embodiments where the target cell is a T cell, the promoter that is active in a target cell is active in a T cell, as disclosed elsewhere in this document.

[0387] In some embodiments, the packageable RNA genome may additionally include a riboswitch, as discussed in other sections of this document. In some embodiments, the nucleic acid sequence encoding the genetically modified signaling polypeptide may be reverse-oriented. In additional embodiments, the packageable RNA genome may additionally include a riboswitch, and optionally, the riboswitch may be reverse-oriented. In any of the embodiments disclosed herein, a polynucleotide including any of the elements may include a primer-binding site. In the illustrative embodiments, transcription blockers or polyA sequences may be placed close to the genes to prevent or reduce unregulated transcription. In any of the embodiments disclosed herein, a nucleic acid sequence encoding Vpx in the second or third optional transcriptional unit or in an additional transcriptional unit that is functionally linked to the first inducible promoter.

[0388] In another aspect, a method for producing a recombinant retrovirus is provided in this document, including: cultivating a population of packaging cells to accumulate a first transactivator, wherein the packaging cells include the first transactivator expressed from a constitutive promoter, wherein the first transactivator has the ability to bind to a first ligand and to a first inducible promoter to affect the expression of a nucleic acid sequence functionally linked to it in the presence versus absence of the first ligand, and wherein the expression of a second transactivator is regulated by the first transactivator; incubating the population of packaging cells including the first transactivator accumulated in the presence of the first ligand to accumulate the second transactivator, wherein the second transactivator has the ability to bind to a second ligand and to a second inducible promoter to affect the expression of a nucleic acid sequence functionally linked to it in the presence versus absence of the second ligand; and incubate the population of packaging cells including the accumulated second transactivator in the presence of the second ligand, thereby inducing the expression of retroviral polypeptides involved in viral packaging, such as, for example, a gag polypeptide, a pol polypeptide and/or a pseudotyping element and, optionally, other polypeptides believed to inhibit the proliferation or survival of mammalian cells that will become incorporated into or on the recombinant retrovirus, thus producing the recombinant retrovirus. In the illustrative embodiments, a packageable RNA genome is encoded by a polynucleotide functionally linked to a promoter, sometimes conveniently referred to as a “third” promoter, wherein said third promoter is either constitutively active or inducible by either the first transactivator or, in the illustrative embodiments, the second transactivator, thus producing the recombinant retrovirus. Pseudotyping elements typically have the ability to bind to a target cell membrane and facilitate the fusion of the target cell membrane to the recombinant retrovirus membrane. Pseudotyping elements can be any envelope proteins known in the art. In some embodiments, the envelope protein may be vesicular stomatitis virus (VSV-G) envelope protein, feline endogenous virus (RD114) envelope protein, oncoretroviral amphotopic envelope protein, and/or oncoretroviral ecotropic envelope protein. A person skilled in the art will understand that many different transactivators, ligands, and inducible promoters can be used in the method to produce a recombinant retrovirus. Suitable transactivators, ligands, and inducible promoters are disclosed elsewhere in this document, including above. A person skilled in the art will further understand that the teachings previously presented in this document relating to an aspect of the retroviral packaging system provided herein also apply to aspects of the method for producing recombinant retroviruses, and vice versa.

[0389] In some embodiments, the first transactivator can regulate the expression of an element to control the nuclear export of transcripts containing a consensus sequence, such as an HIV Rev, and the consensus sequence can be the Rev Response Element (RRE). In illustrative embodiments, the target cell is typically a T cell. In some embodiments, HIV RREs and the polynucleotide region encoding HIV Rev can be replaced by HIV-2 RREs and a polynucleotide region encoding HIV-2 Rev, respectively. In some embodiments, HIV RREs and the polynucleotide region encoding HIV Rev can be replaced by SIV RREs and a polynucleotide region encoding SIV Rev, respectively. In some embodiments, HIV RREs and the polynucleotide region encoding HIV Rev can be replaced by RemREs and a polynucleotide region encoding a Rem betaretrovirus, respectively. In some embodiments, HIV RREs and the HIV Rev-encoding polynucleotide region can be replaced by a RexRRE deltaretrovirus and a Rex deltaretrovirus-encoding polynucleotide region, respectively. In some embodiments, a Rev-type protein is not required, and RREs can be replaced by acting RNA elements, such as the constitutive transport element (CTE).

[0390] In some embodiments, the pseudotyping element is a viral envelope protein. The pseudotyping element typically includes a binding polypeptide and a fusogenic polypeptide to bind to and facilitate membrane fusion of target cell and viral membranes. In some embodiments, the pseudotyping element may be the envelope protein of feline endogenous virus (RD114), the oncoretroviral amphotopic envelope protein, the oncoretroviral ecotropic envelope protein, and/or the envelope protein of vesicular stomatitis virus (VSV-G). In illustrative embodiments, the pseudotyping element includes a binding polypeptide and a fusogenic polypeptide derived from different proteins, as discussed in more detail in this document. For example, in an illustrative embodiment, especially when the target cell is a T cell and/or NK cell, the binding polypeptide may be a cytoplasmic domain deletion variant of a Measles Virus H polypeptide and the fusogenic polypeptide may be the cytoplasmic domain deletion variant of a Measles Virus F polypeptide. In some embodiments, the fusogenic polypeptide may include multiple elements expressed as a polypeptide. In some embodiments, the binding polypeptide and the fusogenic polypeptide may be translated from the same transcript and translated from separate ribosome binding sites, or the polypeptide may be cleaved post-translation using a peptide cleavage signal or a ribosome jump sequence, as disclosed elsewhere in this document, to generate the binding polypeptide and the fusogenic polypeptide. In some embodiments, translation of the binding polypeptide and fusogenic polypeptide from separate ribosome-binding sites results in a higher amount of fusogenic polypeptide compared to binding polypeptide. In some embodiments, the ratio between fusogenic polypeptide and binding polypeptide is at least 2:1, at least 3:1, at least 4:1, at least 5:1, at least 6:1, at least 7:1, or at least 8:1.

[0391] In some embodiments, the first transactivator can regulate the expression of an activation element capable of binding to and activating a target cell, such as a T cell. In these embodiments, the activation element may include: a.) a membrane-bound polypeptide capable of binding to and activating CD3; and/or b.) a membrane-bound polypeptide capable of binding to and activating CD28. In some embodiments, the membrane-bound polypeptide capable of binding to and activating CD28 is CD80, CD86, or functional fragments thereof. In some embodiments, the recombinant retrovirus may include the activation element in a retroviral membrane and the retroviral RNA within a nucleocapsid, thus producing a recombinant retrovirus.

[0392] In some embodiments, the second transactivator may regulate the expression of an RNA including from 5' to 3': a 5' long terminal repeat, or an active truncated fragment thereof; a nucleic acid sequence encoding a retroviral cis-acting RNA packaging element; a nucleic acid sequence encoding a first target polypeptide and an optional second target polypeptide, as a non-limiting example, one or two genetically modified signaling polypeptides; a promoter that is active in a target cell; and a 3' long terminal repeat, or an active truncated fragment thereof. In some embodiments, the RNA may include a cPPT/CTS element. In some embodiments, the RNA may include a primer binding site. In some embodiments, the retroviral cis-acting RNA packaging element may be HIV Psi. In some embodiments, the retroviral cis-acting RNA packaging element may be the Rev Response Element. In any of the embodiments disclosed herein, the retroviral components in the RNA, including RRE and Psi, may be located in any position, as a person skilled in the art will understand. The genetically modified signaling polypeptide in the illustrative embodiments is one or more of the genetically modified signaling polypeptides disclosed herein.

[0393] In some embodiments, the genetically modified signaling polypeptide may include a first lymphoproliferative element. Suitable lymphoproliferative elements are disclosed in other sections of this document. In some embodiments, the lymphoproliferative element is an IL-7 receptor mutant fused to a recognition domain, such as an eTag. In some embodiments, the packageable RNA genome may additionally include a nucleic acid sequence encoding a second genetically modified polypeptide including a chimeric antigen receptor encoding any CAR embodiment provided in this document. For example, the second genetically modified polypeptide may include a first antigen-specific targeting region, a first transmembrane domain, and a first intracellular activation domain. Examples of antigen-specific targeting regions, transmembrane domains, and intracellular activation domains are disclosed elsewhere in this document. In some embodiments where the target cell is a T cell, the promoter that is active in a target cell is active in a T cell, as disclosed elsewhere in this document.

[0394] In some embodiments, the packageable RNA genome may additionally include a riboswitch, as discussed in other sections of this document. In some embodiments, the nucleic acid sequence encoding the genetically modified signaling polypeptide may be reverse-oriented. In additional embodiments, the packageable RNA genome may additionally include a riboswitch, and optionally, the riboswitch may be reverse-oriented. In any of the embodiments disclosed herein, a polynucleotide including any of the elements may include a primer-binding site. In the illustrative embodiments, transcription blockers or polyA sequences may be placed close to the genes to prevent or reduce unregulated transcription. In any of the embodiments disclosed herein, a nucleic acid sequence encoding Vpx in the second or third optional transcriptional unit or in an additional transcriptional unit that is functionally linked to the first inducible promoter.

[0395] In some embodiments of packaging system aspects or methods for producing retroviruses, the encoded RNA may include an intron, which may be transcribed, for example, from the same promoter to express the target polypeptide (or target polypeptides). Such an intron may encode 1, 2, 3, or 4 miRNAs, in certain illustrative embodiments. In these and other embodiments of packaging system aspects or methods for producing retroviruses, the packageable RNA genome is 11,000 KB or less and, in some cases, 10,000 KB or less in size.

[0396] In some embodiments, the first transactivator may affect the expression of one or more polypeptides that are non-toxic. In some embodiments, the second transactivator may affect the expression of one or more polypeptides that are toxic. For example, the first transactivator may induce the expression of the retroviral proteins Rev and Vpx in addition to the polypeptides that will be transported to the cell membrane of the packaging cell, and the second transactivator may induce the expression of the retroviral proteins GAG, POL, MV(Ed)-FΔ30, and MV(Ed)-HΔ18 or MV(Ed)-HΔ24 and the expression of the lentiviral genome. In some embodiments, the first transactivator may affect the expression of one or more polypeptides that are toxic and/or the second transactivator may affect the expression of one or more polypeptides that are non-toxic.

[0397] In another aspect, a mammalian packaging cell is provided in this document, including: a.) a first transcriptional unit in the genome of the mammalian packaging cell, including a nucleic acid sequence encoding a first transactivator, wherein said first transcriptional unit is functionally linked to a constitutive promoter and wherein said transactivator has the ability to bind to an inducible first promoter and affect the expression of a nucleic acid sequence functionally linked thereto in the presence versus absence of a first linker, and wherein said first transactivator has the ability to bind to said first linker; b.) a second transcriptional unit and an optional third transcriptional unit in the genome of the mammalian packaging cell, including a nucleic acid sequence encoding a retroviral REV protein and a nucleic acid sequence encoding a second transactivator with the ability to bind to a second inducible promoter and affect the expression of a functionally linked nucleic acid sequence in the presence versus absence of a second ligand, wherein the second transactivator has the ability to bind to the second ligand, and wherein the second transcriptional unit and the optional third transcriptional unit are functionally linked to the first inducible promoter; (c.) a fourth transcriptional unit and an optional fifth transcriptional unit in the genome of the mammalian packaging cell, including a nucleic acid sequence encoding a retroviral gag polypeptide and a retroviral pol polypeptide, and a binding polypeptide and a fusogenic polypeptide that have the ability to bind and facilitate the fusion of a target cell membrane and the retroviral membrane, wherein the fourth transcriptional unit and the optional fifth transcriptional unit are functionally linked to the second inducible promoter; and d) a sixth transcriptional unit in the mammalian packaging cell genome, including, from 5' to 3', a 5' LTR, or active truncated fragment thereof, a nucleic acid sequence encoding a cis retroviral acting RNA packaging element, a cPPT/CTS element, a reverse complement of a nucleic acid sequence encoding a genetically modified signaling polypeptide, an intron, a promoter that is active in a target cell, and a 3' LTR, or active truncated fragment thereof, wherein the sixth transcriptional unit is functionally linked to the second inducible promoter.

[0398] In another aspect, a method for producing a recombinant retrovirus is provided in this document, which includes: 1.) cultivating a population of packaging cells to accumulate a first transactivator, wherein the packaging cells include: a.) a first transcriptional unit in the mammalian packaging cell genome, including a nucleic acid sequence encoding a first transactivator, wherein said first transcriptional unit is functionally linked to a constitutive promoter and wherein said transactivator has the ability to bind to an inducible first promoter and affect the expression of a nucleic acid sequence functionally linked thereto in the presence versus absence of a first linker, and wherein said first transactivator has the ability to bind to said first linker; b.) a second transcriptional unit and an optional third transcriptional unit in the genome of the mammalian packaging cell, including a nucleic acid sequence encoding a retroviral REV protein and a nucleic acid sequence encoding a second transactivator with the ability to bind to a second inducible promoter and affect the expression of a functionally linked nucleic acid sequence in the presence versus absence of a second ligand, wherein the second transactivator has the ability to bind to the second ligand, and wherein the second transcriptional unit and the optional third transcriptional unit are functionally linked to the first inducible promoter; (c.) a fourth transcriptional unit and an optional fifth transcriptional unit in the genome of the mammalian packaging cell, including a nucleic acid sequence encoding a retroviral gag polypeptide and a retroviral pol polypeptide, and a binding polypeptide and a fusogenic polypeptide that have the ability to bind and facilitate the fusion of the retroviral membrane with a target cell membrane, wherein the fourth polypeptide and the optional fifth transcriptional unit are functionally linked to the second inducible promoter; and d.) a sixth transcriptional unit in the mammalian packaging cell genome, including a 5' to 3' 5' LTR, or active truncated fragment thereof, a primer-binding site (PBS), a nucleic acid sequence encoding a retroviral cis-acting RNA packaging element, a cPPT/CTS element, a reverse complement of a nucleic acid sequence encoding a genetically modified signaling polypeptide, an intron, a target cell promoter that is active in a target cell, a 3' LTR, or active truncated fragment thereof, wherein the fifth transcriptional unit is functionally linked to the second inducible promoter; and 2.) incubate the packaging cell population including the first transactivator in the presence of the first ligand to accumulate the second transactivator and the retroviral REV protein; and 3.) incubate the population of packaging cells including the second transactivator and the retroviral REV protein in the presence of the second ligand, thereby inducing the expression of the retroviral gag polypeptide, the retroviral pol polypeptide, the linking polypeptide, the fusogenic polypeptide, and a retroviral RNA including 5' to 3', a 5' LTR, or an active fragment thereof, PBS, the retroviral cis-acting RNA packaging element, the reverse complement of the nucleic acid sequence encoding the genetically modified signaling polypeptide, the target cell promoter, and a 3' LTR, or an active truncated fragment thereof, wherein recombinant retroviruses are formed and released from the packaging cells, and wherein the recombinant retroviruses include the linking polypeptide and/or the fusogenic polypeptide in a retroviral membrane and the retroviral RNA within a nucleocapsid, thus producing recombinant retroviruses.

[0399] In one aspect provided in this document, the retroviral packaging system may include a mammalian cell comprising: 1.) a first transactivator expressed from a constitutive promoter and with the ability to bind to a first ligand and to a first inducible promoter to affect the expression of a nucleic acid sequence functionally linked to it in the presence versus absence of the first ligand; 2.) a second transactivator with the ability to bind to a second ligand and to a second inducible promoter and affect the expression of a nucleic acid sequence functionally linked to it in the presence versus absence of a second ligand; and 3.) a packageable RNA genome for a retroviral particle, wherein the first transactivator regulates the expression of the second transactivator, HIV Rev, an IL7 GPI DAF, and an activation element, and wherein the second transactivator regulates the expression of a gag polypeptide, a pol polypeptide, a retroviral cis-acting RNA packaging element, and one or more envelope polypeptides. In the illustrative embodiments, the first transactivator may be an FRB domain fused to a p65 activation domain and one or more FKBP domains fused to a ZFHD1 DNA-binding domain, the first linker may be rapamycin, and the first inducible promoter may be one or more ZFHD1 binding sites. In the illustrative embodiments, the second transactivator may be an rtTA protein, the second linker may be tetracycline or doxycycline, and the second inducible promoter may be a TRE promoter or a bidirectional TRE promoter. In the illustrative embodiments, the retroviral cis-acting RNA packaging element may be HIV Psi. In the illustrative embodiments, one or more envelope proteins include cytoplasmic domain deletion variants of F and H polypeptides from a Measles Virus. In the illustrative embodiments, transcription blockers or polyA sequences may be placed near genes to prevent or reduce unregulated transcription. In some embodiments, a rapamycin-doxycycline-inducible lentiviral genome with a riboswitch may be used (SEQ ID NO:83). In some embodiments, a rapamycin-doxycycline-inducible GAG POL ENV may be used (SEQ ID NO:84). In some embodiments, a rapamycin-doxycycline-inducible TET activator may be used (SEQ ID NO:85). In some embodiments, a rapamycin-inducible REV srcVpx may be used (SEQ ID NO:86).

[0400] Some aspects of the present disclosure include or are cells, in the illustrative examples, mammalian cells, which are used as packaging cells to produce retroviruses, such as lentiviruses, for transduction of T cells and/or NK cells. Any of a wide variety of cells can be selected for the in vitro production of a virus, such as a redirected retrovirus, according to the invention. Eukaryotic cells are typically used, particularly mammalian cells including cells from humans, apes, canines, felines, equines, and rodents. In the illustrative examples, the cells are human cells. In further illustrative embodiments, the cells reproduce indefinitely and are therefore immortal. Examples of cells that can be advantageously used in the present invention include NIH 3T3 cells, COS cells, Madin-Darby canine kidney cells, human embryonic 293T cells, and any cells derived from such cells, such as gpnlslacZ ΦNX cells, which are derived from 293T cells. Highly transfectable cells, such as human embryonic 293T kidney cells, can be used. By "highly transfectable" it is understood that at least about 50%, more preferably at least about 70%, and most preferably at least about 80% of the cells can express the genes of the introduced DNA.

[0401] Suitable mammalian cells include primary cells and immortalized cell lines. Suitable mammalian cell lines include human cell lines, non-human primate cell lines, rodent cell lines (e.g., mouse, rat) and the like. Suitable mammalian cell lines include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) at CCL-2), CHO cells (e.g., ATCC at CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC at CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC at CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC at CCL10), PC12 cells (ATCC at CRL1721), COS cells, COS-7 cells (ATCC at CRL1651), RAT1 cells, mouse L cells (ATCC at CCL113), human embryonic kidney (HEK) cells (ATCC at CRL1573), HLHepG2, Hut-78, Jurkat, HL-60 cells, NK cell lines (e.g., NKL, NK92 and YTS) and similar models.

[0402] In any of the embodiments disclosed herein, the methods for producing a recombinant retrovirus may include culturing a mammalian packing cell at 50%, 60%, 70%, 80%, 90%, or 95% confluency or at 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% peak cell density and then splitting and diluting the cells. In some embodiments, a reactor with a stirring tank may be used to culture the cells. In some embodiments, the cells may be split at least about 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:12, 1:15, or 1:20 using methods that one skilled in the art will understand. In some embodiments, cells can be diluted to 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% peak cell density. In some embodiments, after cell division or dilution, cells can be cultured for 1, 2, 3, 4, 5, 6, 7, 8, 10, or 16 hours or 1, 2, 3, 4, 5, 6, or 7 days before the addition of the first ligand. In some embodiments, cells are cultured in the presence of the first ligand for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, or 28 days in the presence of the first ligand, which, in the illustrative embodiments, can be rapamycin or a rapalog. In some embodiments, a second ligand can be added and the cells can be cultured for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, or 28 days, which, in the illustrative embodiments, may be tetracycline or doxycycline. The culture conditions will depend on the cells and ligands used, and the methods are known in the art. A specific example of the conditions for culturing and inducing HEK293S cells is shown in Example 8.

[0403] As disclosed in this document, recombinant retroviruses are a common tool for gene delivery (Miller, Nature (1992) 357:455-460). The ability of recombinant retroviruses to deliver an un-rearranged nucleic acid sequence into a wide range of somatic cells of rodents, primates, and humans makes recombinant retroviruses well-suited for transferring genes into a cell. In some embodiments, the recombinant retrovirus may be derived from the genus Alpharetrovirus, the genus Betaretrovirus, the genus Gammaretrovirus, the genus Deltaretrovirus, the genus Epsilonretrovirus, the genus Lentivirus, or the genus Spumavirus. There are many retroviruses suitable for use in the methods disclosed in this document. For example, murine leukemia virus (MLV), human immunodeficiency virus (HIV), equine infectious anemia virus (EIAV), mouse mammary tumor virus (MMTV), Rous sarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murine leukemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus (A-MLV), avian myelocytoma virus-29 (MC29), and avian erythroblastosis virus (AEV) can be used. A detailed list of retroviruses can be found in Coffin et al. (“Retroviruses” 1997 Cold Spring Harbor Laboratory Press Eds: J M Coffin, S M Hughes, H E Varmus pages 758 to 763). Details about the genomic structure of some retroviruses can be found in the technique. As an example, details about HIV can be found from NCBI Genbank (i.e., Genome Accession Number AF033819).

[0404] In illustrative embodiments, the recombinant retrovirus may be derived from the genus Lentivirus. In some embodiments, the recombinant retrovirus may be derived from HIV, SIV, or FIV. In further illustrative embodiments, the recombinant retrovirus may be derived from the human immunodeficiency virus (HIV) in the genus Lentivirus. Lentiviruses are complex retroviruses that, in addition to the common retroviral genes gag, pol, and env, contain other genes with regulatory or structural functions. The higher complexity allows the lentivirus to modulate its own life cycle, such as during the course of latent infection. A typical lentivirus is the human immunodeficiency virus (HIV), the etiological agent of AIDS. In vivo, HIV can infect terminally differentiated cells that rarely divide, such as lymphocytes and macrophages.

[0405] In the illustrative embodiments, the recombinant retroviruses provided in this document contain the Vpx polypeptide. The Vpx polypeptide can be expressed in a packaging cell line, after integration of a Vpx-encoding nucleic acid into its genome, for example, as a cell membrane-bound protein that is incorporated into a retroviral membrane (Durand et al., J. Virol. (2013) 87: 234-242). A retroviral membrane-bound Vpx can be constructed with a processing sequence for a viral protease so that free Vpx is released once incorporated into a viral particle. One example of a Vpx fusion with this functionality is Src-Flag-Vpx, which includes a membrane targeting domain (MGSSKSKPKDP) (SEQ ID NO:227) from the first 11 amino acids of c-Src followed by a viral protease cleavage domain KARVLAEA (SEQ ID NO:228) followed by Flag-tagged Vpx.

[0406] Without adhering to theory, the Vpx polypeptide assists in the transduction of resting cells by stimulating the efficiency of the reverse transcription process by degrading the restriction factor SAMHD1. Consequently, it is believed that, in the methods provided in this document where Vpx is present in a recombinant retrovirus used to transduce T cells and/or NK cells, Vpx is released into the cytoplasm of a resting T cell or a resting NK cell upon transduction of the cell by a recombinant retrovirus containing Vpx. Vpx then degrades SAMHD1, which causes an increase in free dNTPs, which, in turn, stimulate reverse transcription of the retroviral genome.

RETROVIRAL GENOME SIZE

[0407] In the methods and compositions provided in this document, recombinant retroviral genomes, in the non-limiting illustrative examples, lentiviral genomes, have a limitation on the number of polynucleotides that can be packaged into the viral particle. In some embodiments provided in this document, the polypeptides encoded by the polynucleotide coding region may be truncations or other deletions that retain functional activity so that the polynucleotide coding region is encoded by fewer nucleotides than the polynucleotide coding region for the wild-type polypeptide. In some embodiments, the polypeptides encoded by the polynucleotide coding region may be fusion polypeptides that can be expressed from a promoter. In some embodiments, the fusion polypeptide may have a cleavage signal to generate two or more functional polypeptides from a fusion polypeptide and a promoter. Furthermore, some functions that are not required after initial ex vivo transduction are not included in the retroviral genome, but instead are present on the surface of the virus or retrovirus via cell membrane packaging. These various strategies are used in this paper to maximize the functional elements that are packaged within the retrovirus.

[0408] In some embodiments, the recombinant retroviral genome to be packaged may have between 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000 and 8,000 nucleotides at the lower end of the band and 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000 and 11,000 nucleotides at the upper end of the band. The retroviral genome to be packaged includes one or more polynucleotide regions encoding a first and a second gene-modification signaling polypeptide as disclosed in detail herein. In some embodiments, the recombinant retroviral genome to be packaged may be smaller than 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, or 11,000 nucleotides. The functions discussed elsewhere in this document that may be packaged include retroviral sequences required for retroviral assembly and packaging, such as retroviral rev, gag, and pol coding regions, as well as a 5' LTR and a 3' LTR, or an active truncated fragment thereof, a nucleic acid sequence encoding a retroviral cis-acting RNA packaging element, and a cPPT/CTS element. Furthermore, in the illustrative embodiments, a recombinant virus or retrovirus in this document may include any one or more or all of the following, in some embodiments, in reverse orientation of these retroviral functional regions: one or more polynucleotide regions encoding a first and a second genetic modification signaling polypeptide, at least one of which includes a lymphoproliferative element and may additionally include an ASTR; a second genetically modified signaling polypeptide that may include a chimeric antigen receptor; an in vivo control element, such as a riboswitch, that typically regulates the expression of the first and/or second genetic modification signaling polypeptides; a recognition domain, an intron, a promoter that is active in a target cell, such as a T cell, a 2A cleavage signal, and/or an IRES.

Recombinant retroviruses

[0409] Recombinant retroviruses are disclosed in the methods and compositions provided herein, for example, to transduce T cells and/or NK cells to produce genetically modified T cells and/or NK cells. The recombinant retroviruses themselves are aspects of the present invention. In some embodiments, the recombinant retroviruses are replication incompetent, meaning that a retrovirus cannot replicate once it leaves the packaging cell. In some embodiments, the recombinant retroviruses may be adenoviruses, adeno-associated viruses, herpesviruses, cytomegaloviruses, poxviruses, avipoxviruses, influenza viruses, vesicular stomatitis viruses (VSV), or Sindbis viruses. A person skilled in the art will understand how to modify the methods disclosed herein for use with different retroviruses. For example, in some embodiments, the HIV RREs and the HIV Rev-encoding polynucleotide region can be replaced by N-terminal RGG box RNA-binding motifs and a polynucleotide region encoding ICP27. In some embodiments, the HIV Rev-encoding polynucleotide region can be replaced by one or more polynucleotide regions encoding E1B 55-kDa and E4 Orf6 adenoviruses.

[0410] Consequently, a recombinant retrovirus is provided in some embodiments herein that includes (i) a pseudotyping element capable of binding to a T cell and/or NK cell and facilitating the fusion of the recombinant retrovirus membrane to them; (ii) a polynucleotide having one or more transcriptional units functionally linked to an active promoter on T cells and/or NK cells, wherein one or more transcriptional units encode a first genetically modified signaling polypeptide having a chimeric antigen receptor that includes an antigen-specific targeting region, a transmembrane domain and an intracellular activation domain and a second genetically modified signaling polypeptide that includes a lymphoproliferative element; wherein the expression of the first genetically modified signaling polypeptide and/or the second genetically modified signaling polypeptide is regulated by an in vivo control element; (iii) an activation element on its surface, wherein the activation element has the ability to bind to a T cell and/or NK cell and is not encoded by a polynucleotide in the recombinant retrovirus. In some embodiments, the active principle in T cells and/or NK cells is not active in the packaging cell line. In any of the embodiments disclosed herein, either of the first and second genetically modified signaling polypeptides may have a chimeric antigen receptor and the other genetically modified signaling polypeptide may have a lymphoproliferative element.

Genetically modified T cells and NK cells

[0411] In the embodiments of the methods and compositions in this document, genetically modified lymphocytes are produced, which are a separate aspect of the invention. In some embodiments, genetically modified lymphocytes are lymphocytes, such as T cells and/or NK cells, that have been genetically modified to express a first genetically modified signaling polypeptide comprising a lymphoproliferative element and/or a second genetically modified signaling polypeptide comprising a chimeric antigen receptor, which includes an antigen-specific targeting region (ASTR), a transmembrane domain and an intracellular activation domain.

[0412] In the methods and compositions disclosed herein, the expression of one or both genetically modified signaling polypeptides is typically regulated by an in vivo control element, and, in some embodiments, the in vivo control element is a polynucleotide comprising a riboswitch. In certain embodiments, the riboswitch has the ability to bind to a nucleoside analog and, when the nucleoside analog is present, one or both genetically modified signaling polypeptides are expressed.

[0413] The genetically modified lymphocytes disclosed herein may also have polypeptides expressed on their surface, such as one or more polypeptides that function as an activation element, one or more polypeptides that function as a pseudotyping element, and/or one or more fusion polypeptides that include a cytokine. In some embodiments, the genetically modified lymphocytes have an activation element on their surface. The activation element may have a membrane-bound polypeptide with the ability to bind to CD3; and/or a membrane-bound polypeptide with the ability to bind to CD28. In some embodiments, the activation element is scFvFc anti-CD3 fused to a heterologous GPI anchor binding sequence and/or CD80 fused to a heterologous GPI anchor binding sequence. In some embodiments, the genetically modified lymphocytes have a pseudotyping element on their surface. In some embodiments, the genetically modified lymphocytes have a fusion polypeptide on their surface where the fusion polypeptide is a cytokine covalently linked to DAF. In some embodiments, the cytokine is IL-7 or IL-15. In the illustrative embodiments, the cytokine is IL-7. In some embodiments, the cytokine is missing its signaling sequence. In the illustrative embodiments, the cytokine is inserted into DAF behind its signaling sequence.

NUCLEIC ACIDS

[0414] The present disclosure provides the nucleic acid coding polypeptides of the present disclosure. A nucleic acid will, in some embodiments, be DNA, including, for example, a recombinant expression vector. A nucleic acid will, in some embodiments, be RNA, for example, RNA synthesized in vitro.

[0415] In some cases, a nucleic acid provides the production of a present revelation polypeptide, for example, in a mammalian cell. In other cases, a nucleic acid subject provides the amplification of the nucleic acid encoding a present revelation polypeptide.

[0416] A nucleotide sequence encoding a polypeptide of the present disclosure can be functionally linked to a transcriptional control element, for example, a promoter and enhancer, etc.

[0417] The promoter and enhancer elements are known in the art. For expression in a bacterial cell, suitable promoters include, but are not limited to, lacl, lacZ, T3, T7, gpt, lambda P, and trc. For expression in a eukaryotic cell, suitable promoters include, but are not limited to, enhancer elements and promoters of light and/or heavy chain immunoglobulin genes; immediate early cytomegalovirus promoter; herpes simplex virus thymidine kinase promoter; early and late SV40 promoters; promoter present in long terminal repeats of a retrovirus; mouse metallothionein-I promoter; and various tissue-specific promoters known in the art.

[0418] Suitable reversible promoters, including reversible inducible promoters, are known in the art. Such reversible promoters can be isolated and derived from many organisms, for example, eukaryotes and prokaryotes. The modification of reversible promoters derived from a first organism for use in a second organism, for example, a first prokaryote and a second eukaryote, a first eukaryote and a second prokaryote, etc., is well known in the art. Such reversible promoters and systems based on such reversible promoters, but also comprising additional control proteins, include, but are not limited to, alcohol-regulated promoters (e.g., alcohol dehydrogenase I gene promoter (alcA), alcohol transactivator protein responsive promoters (AlcR), etc.), tetracycline-regulated promoters (e.g., promoter systems including TetActivators, TetON, TetOFF, etc.), steroid-regulated promoters (e.g., rat glucocorticoid receptor promoter systems, human estrogen receptor promoter systems, retinoid promoter systems, thyroid promoter systems, ecdysone promoter systems, mifepristone promoter systems, etc.), metal-regulated promoters (e.g., metallothionein promoter systems, etc.), pathogenesis-related regulated promoters (e.g., salicylic acid-regulated promoters, ethylene-regulated promoters, etc.). benzothiadiazole, etc.), temperature-regulated promoters (e.g., heat shock inducible promoters (e.g., HSP-70, HSP-90, soybean heat shock promoter, etc.), light-regulated promoters, synthetic inducible promoters, and the like.

[0419] In some cases, the locus or construct or trans gene containing the appropriate promoter is irreversibly switched through the induction of an inducible system. Suitable systems for inducing an irreversible switch are well known in the art, for example, the induction of an irreversible switch can make use of Crelox-mediated recombination (see, for example, Fuhrmann-Benzakein, et al., PNAS (2000) 28:e99, the disclosure of which is incorporated herein by reference). Any suitable combination of recombinase, endonuclease, ligase, recombination sites, etc., known in the art can be used in the generation of an irreversibly switchable promoter. Methods, mechanisms, and requirements for performing site-specific recombination, described elsewhere in this document, find use in the generation of irreversibly switched promoters and are well known in the art, see, for example, Grindley et al. (2006) Annual Review of Biochemistry, 567 to 605 and Tropp (2012) Molecular Biology (Jones & Bartlett Publishers, Sudbury, MA), whose findings are incorporated into this document by way of reference.

[0420] In some cases, the promoter is a CD8 cell-specific promoter, a CD4 cell-specific promoter, a neutrophil-specific promoter, or an NK cell-specific promoter. For example, a CD4 gene promoter may be used; see, for example, Salmon et al. (1993) Proc. Natl. Acad. Sci. USA 90:7739; and Marodon et al. (2003) Blood 101:3416. As another example, a CD8 gene promoter may be used. NK cell-specific expression may be achieved by using a Neri (p46) promoter; see, for example, Eckelhart et al. (2011) Blood 117:1565.

[0421] In some embodiments, for example, for expression in a yeast cell, a suitable promoter is a constitutive promoter, such as an ADH1 promoter, a PGK1 promoter, an ENO promoter, a PYK1 promoter and the like; or a regulable promoter, such as a GALI promoter, a GAL1O promoter, an ADH2 promoter, a PH05 promoter, a CUP1 promoter, a GAL7 promoter, a MET25 promoter, a MET3 promoter, a CYCl promoter, a HIS3 promoter, an ADH1 promoter, a PGK promoter, a GAPDH promoter, an ADCl promoter, a TRP1 promoter, a URA3 promoter, a LEU2 promoter, an ENO promoter, a TP1 promoter and AOX1 (for example, for use in Pichia). The selection of the appropriate vector and promoter is well within the level of the average person skilled in the art.

[0422] Promoters suitable for use in prokaryotic host cells include, but are not limited to, a T7 bacteriophage RNA polymerase promoter; a trp promoter; a lac operon promoter; a hybrid promoter, for example, a lac/tac hybrid promoter, a tac/trc hybrid promoter, a trp/lac promoter, a T7/lac promoter; a trc promoter; a tac promoter and the like; an araBAD promoter; regulated promoters in vivo, such as an ssaG promoter or a related promoter (see, for example, U.S. Patent Publication 20040131637), a pagC promoter (Pulkkinen and Miller, J. Bacterial., 1991; 173(1): 86 to 93; Alpuche-Aranda et al., PNAS, 1992; 89(21): 10,079 to 10,083), a nirB promoter (Harborne et al. (1992) Mal. Micro. 6:2,805 to 2,813) and the like (see, for example, Dunstan et al. (1999) Infect. Immun. 67:5,133 to 5,141; McKelvie et al. (2004) Vaccine 22:3,243 to 3,255; and Chatfield et al. (1992) Biotechnol. 10:888 to 892); a sigma70 promoter, for example, a consensus sigma70 promoter (see, for example, GenBank Accession Numbers AX798980, AX798961 and AX798183); a stationary phase promoter, for example, a dps promoter, an spv promoter and the like; a promoter derived from the SPI-2 pathogenicity island (see, for example, W096/17951); an actA promoter (see, for example, Shetron-Rama et al. (2002) Infect. Immun. 70:1087 to 1096); an rpsM promoter (see, for example, Valdivia and Falkow (1996). Mal. Microbial. 22:367); a tet promoter (see, for example, Hillen, W. and Wissmann, A. (1989) in Saenger, W. and Heinemann, U. (eds), Topics in Molecular and Structural Biology, Protein-Nucleic Acid Interaction. Macmillan, London, UK, Volume 10, pages 143 to 162); an SP6 promoter (see, for example, Melton et al. (1984) Nucl. Acids Res. 12:7035); and similar promoters. Strong promoters suitable for use in prokaryotes, such as Escherichia coli, include, but are not limited to, Trc, Tac, T5, T7, and PLambda. Non-limiting examples of operators for use in bacterial host cells include a lactose promoter operator (Laci repressor protein changes conformation when placed in contact with lactose, thus preventing Laci repressor protein from binding to the operator), a tryptophan promoter operator (when complexed with tryptophan, the TrpR repressor protein has a conformation that binds to the operator; in the absence of tryptophan, the TrpR repressor protein has a conformation that does not bind to the operator), and a tac promoter operator (see, for example, deBoer et al. (1983) Proc. Natl. Acad. Sci. USA 80:21-25).

[0423] The nucleotide sequence encoding a revelation polypeptide may be present in an expression vector and/or a cloning vector. Nucleotide sequences encoding two separate polypeptides may be cloned into the same vectors or into separate vectors. An expression vector may include a selectable marker, an origin of replication, and other features that provide vector replication and/or maintenance. Suitable expression vectors include, for example, plasmids, viral vectors, and the like.

[0424] Large numbers of suitable vectors and promoters are known to those skilled in the art; many are commercially available for generating a recombinant subject construct. The following bacterial vectors are provided by way of example: pBs, phagescript, PsiXl74, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, CA, USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden). The following eukaryotic vectors are provided by way of example: pWLneo, pSV2cat, pOG44, PXRl, pSG (Stratagene) pSVK3, pBPV, pMSG, and pSVL (Pharmacia).

[0425] Expression vectors generally have convenient restriction sites close to the promoter sequence to provide insertion of nucleic acid sequences encoding heterologous proteins. An operational selectable marker in the expression host may be present. Suitable expression vectors include, but are not limited to, viral vectors (e.g., viral vectors based on vaccinia virus; poliovirus; adenovirus (see, for example, Li et al., Invest Ophthalmol Vis Sci 35:2543 to 2549, 1994; Borras et al., Gene Ther 6:515 to 524, 1999; Li and Davidson, PNAS 92:7700 to 7704, 1995; Sakamoto et al., H Gene Ther 5:1088 to 1097, 1999; documents in WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655); adeno-associated virus ... example, Ali et al., Hum Gene Ther 9:81 to 86, 1998, Flannery et al., PNAS 94:6916 to 6921, 1997; Bennett et al., Invest Opthalmol Vis Sci 38:2857 to 2863, 1997; Jomary et al., Gene Ther 4:683 to 690, 1997, Rolling et al., Hum Gene Ther 10:641 to 648, 1999; Ali et al., Hum Mol Genet 5:591 to 594, 1996; Srivastava in WO 93/09239, Samulski et al., J. Vir. (1989) 63:3822 to 3828; Mendelson et al., Virol. (1988) 166:154 to 165; and Flotte et al., PNAS (1993) 90: 10,613 to 10,617); SV40; herpes simplex virus; gamma retrovirus; human immunodeficiency virus (see, for example, Miyoshi et al., PNAS 94:10,319 to 10,323, 1997; Takahashi et al., J Virol 73:7,812 to 7,816, 1999); a retroviral vector (e.g., Murine Leukemia Virus, splenic necrosis virus and retrovirus-derived vectors such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus and mammary tumor virus); and the like.

[0426] As noted above, in some embodiments, a nucleic acid encoding a polypeptide of the present disclosure will, in some embodiments, be RNA, for example, RNA synthesized in vitro. Methods for the in vitro synthesis of RNA are known in the art; any known method can be used to synthesize RNA including a nucleotide sequence encoding a polypeptide of the present disclosure. Methods for introducing RNA into a host cell are known in the art. See, for example, Zhao et al. (2010) Cancer Res. 15:9053. The introduction of RNA including a nucleotide sequence encoding a polypeptide of the present disclosure into a host cell can be performed in vitro or ex vivo. For example, a host cell (e.g., an NK cell, a cytotoxic T lymphocyte, etc.) can be electroporated in vitro or ex vivo with RNA comprising a nucleotide sequence encoding a polypeptide of the present disclosure.

CELLS

[0427] The present disclosure provides mammalian cell lines that produce recombinant retroviruses that genetically modify mammalian target cells and the mammalian target cells themselves.

[0428] Suitable mammalian cells include primary cells and immortalized cell lines. Suitable mammalian cell lines include human cell lines, non-human primate cell lines, rodent cell lines (e.g., mouse, rat), and the like. Suitable mammalian cell lines include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) at CCL-2), CHO cells (e.g., ATCC at CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC at CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC at CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC at CCL10), PC12 cells (ATCC at CRL1721), COS cells, COS-7 cells (ATCC at CRL1651), RAT1 cells, mouse L cells (ATCC at CCL113), human embryonic kidney (HEK) cells (ATCC at CRL1573), HLHepG2, Hut-78, Jurkat, HL-60 cells, NK cell lines (e.g., NKL, NK92 and YTS) and similar models.

[0429] In some cases, the cell is not an immortalized cell line, but instead a cell (e.g., a primary cell) obtained from an individual or an ex vivo cell. For example, in some cases, the cell is an immune cell obtained from an individual. As another example, the cell is a stem cell or progenitor cell obtained from an individual.

METHODS FOR ACTIVATING AN IMMUNE CELL

[0430] The present disclosure provides methods for activating an immune cell in vitro, in vivo, or ex vivo. The methods generally involve placing an immune cell (in vitro, in vivo, or ex vivo) in contact with one or more target antigens, wherein the immune cell has been genetically modified to produce a CAR restricted to the microenvironment of the present disclosure. In the presence of one or more target antigens, the microenvironment-restricted CAR activates the immune cell, thereby producing an activated immune cell. Immune cells include, for example, a cytotoxic T lymphocyte, an NK cell, a CD4+ T cell, a regulatory T cell (Treg), a Yδ T cell, an NK-T cell, neutrophils, etc.

[0431] Contact of a genetically modified immune cell (e.g., a T lymphocyte, an NK cell) with one or more target antigens can increase the production of a cytokine by the immune cell by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about 2 times, at least about 2.5 times, at least about 5 times, at least about 10 times, or more than 10 times, compared to the amount of cytokine produced by the immune cell in the absence of one or more target antigens. Cytokines whose production can be increased include, but are not limited to, IL-2 and IFN-γ.

[0432] Contact of a genetically modified cytotoxic cell (e.g., cytotoxic T lymphocyte) with AAR can increase the cytotoxic activity of the cytotoxic cell by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about 2 times, at least about 2.5 times, at least about 5 times, at least about 10 times, or more than 10 times, compared to the cytotoxic activity of the cytotoxic cell in the absence of one or more target antigens.

[0433] Contact of a genetically modified cytotoxic cell (e.g., cytotoxic T lymphocyte) with one or more target antigens can increase the cytotoxic activity of the cytotoxic cell by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about 2 times, at least about 2.5 times, at least about 5 times, at least about 10 times, or more than 10 times, compared to the cytotoxic activity of the cytotoxic cell in the absence of one or more target antigens.

[0434] In other modalities, for example, depending on the host immune cell, contact of a genetically modified host cell with an antigen can increase or decrease cell proliferation, cell survival, cell death and the like.

METHODS FOR PRODUCING AN ANTIGEN-SPECIFIC TARGETING REGION RESTRICTED TO THE MICROENVIRONMENT

[0435] In some embodiments, the antigen-binding domains (also referred to herein as "antigen-specific target regions" or "ASTRs") of CARs bind constitutively to their cognate antigens. In other embodiments, ASTRs may be microenvironmentally restricted, preferentially binding, or only binding to their cognate antigen under certain abnormal conditions, such as those existing in the tumor microenvironment, as disclosed in more detail herein. Microenvironmentally restricted ASTRs that bind preferentially or exclusively under abnormal conditions of a tumor microenvironment may provide a reduction in non-tumor target effects insofar as antigen binding under normal physiological conditions is reduced, in some situations, to levels below detection by immunoassays. In certain aspects, the CARs provided herein include a microenvironmentally restricted ASTR that specifically binds to a target protein, wherein the ASTR is an scFv fragment that includes a variable heavy chain region and a variable light chain region.

[0436] Certain illustrative embodiments of the aspects disclosed in this document, for example, the methods, cells, cell lines, retroviruses, polynucleotides or vectors disclosed in this document include CARs that include antigen-specific targeting regions restricted to the microenvironment.

[0437] Consequently, in one aspect, a chimeric antigen receptor for binding to a target antigen is provided in the present document which includes: a) a microenvironmentally restricted antigen-specific targeting region that exhibits increased binding to the target antigen in an abnormal condition compared to a normal physiological environment, where the antigen-specific targeting region binds to the target; b) a transmembrane domain; and c) an intracellular activation domain.

[0438] In another aspect, a chimeric antigen receptor for binding to a target antigen is provided in this document which includes: a) at least one microenvironment-restricted antigen-specific targeting region selected by selection from a polypeptide library and which has an increase in activity in a target antigen binding assay under an abnormal condition compared to a normal physiological condition; b) a transmembrane domain; and c) an intracellular activation domain.

[0439] In some embodiments of any aspect disclosed herein, any of the chimeric antigen receptors may be microenvironmentally restricted such that they exhibit increased binding activity in an abnormal condition compared to a normal physiological condition. In some illustrative embodiments of any aspect disclosed herein, the microenvironmentally restricted ASTR is identified from an initial polypeptide library without mutation/evolutionary library members prior to screening/evolution and/or without mutation during or between optional repeated screening cycles. Exemplary transmembrane domains and intracellular activation domains may be any of those disclosed herein for CARs.

[0440] In one aspect, a method for selecting a microenvironmentally restricted ASTR is provided in this document comprising selecting from a polypeptide presentation library: a. subjecting polypeptides from the polypeptide presentation library to a target antigen binding assay under a normal physiological condition and a target antigen binding assay under an abnormal condition; and b. selecting a polypeptide that exhibits an increase in target antigen binding activity in the abnormal condition compared to the physiological condition, thereby selecting the microenvironmentally restricted antigen-specific targeting region.

[0441] In another aspect, a method for isolating a microenvironmentally restricted ASTR is provided in this document, which includes selection of a polypeptide library: placing the polypeptide library under abnormal conditions in contact with a target antigen bound to a solid support, wherein clones expressing polypeptides that bind to the target antigen remain bound to the solid support through the target antigen; incubating the solid supports with bound polypeptides under physiological conditions; and collecting clones that elute from the solid support under physiological conditions, thereby isolating the microenvironmentally restricted antigen-specific targeting region.

[0442] In some illustrative embodiments of any aspect disclosed herein, the microenvironment-restricted antigen-specific targeting region is identified from an initial polypeptide library screening with no mutation/evolution members of the library prior to screening and/or no mutation/evolution during or between optional repeated screening or selection cycles.

[0443] Normal physiological conditions may include those of temperature, pH, osmotic pressure, osmolality, oxidative stress, and electrolyte concentration that would be considered within a normal range at the site of administration, or in the tissue or organ at the site of action, to a subject. An abnormal condition is one that deviates from the normally acceptable range for that condition. In one aspect, a microenvironment-restricted antigen-specific targeting region (i.e., polypeptide) is virtually inactive under normal conditions, but is active under non-normal conditions to a level that is equal to or better than under normal conditions. For example, in one aspect, the microenvironment-restricted antigen-specific targeting region is virtually inactive at body temperature, but is active at lower temperatures. In another aspect, the microenvironment-restricted antigen-specific targeting region is reversibly or irreversibly inactivated under normal conditions. In a further aspect, the microenvironment-restricted antigen-specific targeting region is a therapeutic protein. In another aspect, the microenvironment-restricted antigen-specific targeting region is used as a drug or therapeutic agent. In yet another aspect, the microenvironment-restricted antigen-specific targeting region is more or less active in highly oxygenated blood, such as, for example, after passage through the lung or in the lower pH environments found in the kidney.

[0444] In some embodiments, a single screening cycle is performed to obtain the antigen-specific targeting region restricted to the microenvironment. In certain embodiments, the screening or selection method is repeated after identifying free polypeptides that bind to antigen under abnormal conditions and do not bind under physiological conditions, or cells expressing a test polypeptide that had these properties, or phage coated with a test polypeptide that has such properties in an initial or previous cycle. In some methods, the phages that are collected are used to infect cells, which may be infected with helper phage as well, in order to amplify the collected phage. In other methods where the polypeptides on the cell surface are tested, the collected cells may be cultured to "amplify" the polypeptides expressed by the cells by amplifying the polynucleotides in the cells that encode the polypeptides. In some methods, amplification is performed by culturing cells that express the identified polypeptides without performing a process to mutate the polynucleotides that encode the identified polypeptides between cycles. Thus, the polypeptides that were collected in a previous cycle are enriched by amplifying cells that contain polynucleotides encoding those collected polypeptides.

[0445] The screening or selection method can be performed once or repeated 1 to 1,000 times. In the illustrative examples, the selection is repeated 1 to 20 times or 2 to 10 times or 2 to 5 times.

[0446] In other methods, microenvironment-restricted ASTRs against an antigen of interest (i.e., target antigen) are performed using one or more mutation/evolution cycles between selection cycles. In one method, a wild-type protein is identified, for example, by generating a polypeptide or protein library and screening the polypeptide or protein library for a polypeptide or protein with a desired binding affinity to a target antigen. In some embodiments where wild-type proteins are antibodies, wild-type antibodies can be revealed by generating and screening polyclonal or monoclonal antibody libraries, including phage-presenting antibody libraries, for example, humanized phage-presenting antibody libraries.

[0447] Evolved ASTRs can be generated by subjecting wild-type protein, or a nucleic acid sequence encoding wild-type protein, to a mutagenesis process to produce a population of mutant polypeptides that can be screened to identify a mutant ASTR with increased activity (e.g., heightened binding affinity to the target antigen) in a tumor environment and/or in a tumor surrogate assay condition in vitro, compared to a normal physiological environment. Examples of such methods are provided in document no. WO2016033331 ("CONDITIONALLY ACTIVE CHIMERIC ANTIGEN RECEPTORS FOR MODIFIED T CELLS") or in U.S. Patent no. 8,709,755, both incorporated herein by reference in their entirety. This method for generating a microenvironmentally restricted antibody is incorporated herein by reference in its entirety.

[0448] In other embodiments, microenvironment-restricted antigen-specific polypeptides (i.e., targeting regions, e.g., antibodies) can be identified by screening an initial polypeptide library under abnormal versus physiological conditions and identifying a test polypeptide from the initial polypeptide library that binds preferentially or exclusively under abnormal versus physiological conditions. In some examples, the microenvironment-restricted antigen-specific polypeptides identified and isolated (i.e., targeting regions, e.g., antibodies) identified from an initial polypeptide library in an initial polypeptide library screening bind to their cognate antigen preferentially or exclusively under abnormal versus physiological conditions. In such cases, no mutation/evolution cycle is performed. Consequently, the method in the illustrative embodiments is performed without mutated polynucleotides encoding the microenvironment-restricted antigen-specific targeting region isolated between screening cycles (e.g., selection cycles) or is performed only for a single binding assay under abnormal versus physiological conditions to isolate and identify the microenvironment-restricted antigen-specific polypeptide (i.e., targeting region, e.g., antibody). The method can be performed by culturing, amplifying with high fidelity, and/or diluting polynucleotides encoding antigen-specific targeting regions, or host organisms including them, between screening and/or selection cycles, without any mutation/evolution. Furthermore, the method can be performed without repeating the screening and/or selection and can be performed without mutation/evolution of a polynucleotide encoding the microenvironment-restricted antigen-specific targeting region isolated, after the microenvironment-restricted antigen-specific polypeptide (i.e., target region, e.g., antibody) is isolated.

[0449] Assays for use in the methods provided herein for detecting the binding of a polypeptide to a cognate linking partner include cell-based assays and, in particular, assays performed using cell surface presentation systems, such as mammalian cell surface presentation systems. In an exemplary method, nucleic acids encoding a polypeptide or a library of variant polypeptides, including a library of modified polypeptides, can be introduced into a vector suitable for expression in cells, such as mammalian cells. The cells are then transfected with the vector, and the polypeptide (or polypeptides) is expressed by the cells. The cell library containing surface-expressed polypeptides can be placed in contact with a solution containing a surface-bound or soluble cognate linking partner. Binding activity can be detected using any assay that can detect binding to the cell surface. Activity can also be assessed by evaluating a functional activity of the polypeptide or polypeptides. Any cell-based assay known to those skilled in the art is contemplated for use in the methods provided herein, including cell proliferation assays, cell death assays, flow cytometry, cell separation techniques, fluorescence-activated cell sorting (FACS), phase microscopy, fluorescence microscopy, receptor binding assays, cell signaling assays, immunocytochemistry, and reporter gene assays. In some examples, the assays are fluorescence-activated cell sorting (FACS) assays.

[0450] Polypeptides or proteins can be expressed by mammalian cells as secreted soluble molecules, cell surface molecules, or intracellular antibodies. In one exemplary method, cells can be transfected with a protein library under conditions whereby most or all cells have a member of the protein library anchored to the cell surface. Optionally, an expression system can be used in which most mammalian cell transfectants have only one plasmid integrated into their genome. Therefore, most (i.e., at least about 70% or about 80% or about 90%) of the transfectants express one or more molecules of a polypeptide. This can be verified, for example, by isolating and culturing individual transfectants; and amplifying and sequencing the expressed sequences to determine if they have a single sequence.

[0451] In some examples of the methods provided in this document, the polypeptides are antibodies presented on the surface of mammalian cells. Any antibody described in this document can be expressed on the surface of mammalian cells, including full-length bivalent functional antibodies, such as IgG antibodies. The antibody can be a fragment, for example, Fab fragments or scFv fragments. Antibodies may include an Fc region, such as an scFv-Fc, or a full-length antibody comprising two heavy and two light chains. A person skilled in the art can select a suitable antibody fragment. For example, ScFv-Fcs and full-length antibodies produced in mammalian cells may have several advantages over scFv or Fab fragments.

[0452] Solid supports that can be used in the binding assays provided in this document include any carrier that has the ability to be attached to a polypeptide binding partner, such as a ligand, receptor, or antigen. Typically, to facilitate high-throughput screening, a cognate binding partner is attached to the solid support. Examples of carriers for use as solid supports in the methods provided in this document include, but are not limited to, glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amyloses, natural and modified celluloses, polyacrylamides, agaroses, and magnetic solid supports, such as solid supports that include magnetite. The solid support may be one or more spheres or particles, microspheres, a tube or plate surface, a filter membrane, and other solid supports known in the art. Exemplary solid support systems include, but are not limited to, a flat surface constructed, for example, of glass, silicon, metal, nylon, cellulose, plastic, or a composite, including membranes or multi-cavity plates; or they may be in the form of a microsphere, such as a silica gel, a controlled-pore glass, a magnetic or cellulose microsphere. Furthermore, such methods can be adapted for use in suspension or in column form. In some embodiments, the microenvironment-restricted antigen-specific polypeptide (i.e., the target region, e.g., antibody) is identified and isolated by bioselection from a phage-presenting or yeast surface-presenting antibody library (e.g., humanized antibody) (Colby et al., "Engineering Antibody Affinity by Yeast Surface Display", Meth. Enzym. 388, 26 (2004)) with an immobilized target antigen. For example, either a naive humanized antibody library or a synthetic humanized antibody library can be selected using the phage presentation or yeast surface presentation methods in this document. In some embodiments, in an initial phage presentation process, phage clones can be transferred to a mammalian vector and used for a mammalian cell surface screening method (see, for example, Yoon et al., BMC Biotechnology 12:62; 1472–6750 (2012)). An exemplary method for performing phage presentation to isolate a microenvironment-restricted antigen-specific target region is provided in Example 2.

[0453] A microenvironmentally restricted ASTR identified using methods provided in this document may be an antibody, an antigen, a ligand, a receptor-binding domain of a ligand, a receptor, a ligand-binding domain of a receptor, or an affibody. In embodiments where the microenvironmentally restricted ASTR is an antibody, it may be a full-length antibody, a single-chain antibody, a Fab fragment, a Fab' fragment, a (Fab')2 fragment, an Fv fragment, and a divalent single-chain antibody or a diabody, wherein the antigen-specific targeting region comprises a heavy chain and a light chain of an antibody. In some embodiments, the microenvironmentally restricted ASTR is a variable single-chain fragment. Such a variable single-chain fragment may have heavy and light chains separated by a binder, wherein the binder is between 6 and 100 amino acids in length. In some forms, the heavy chain is in an N-terminal position relative to the light chain in the chimeric antigen receptor. In other forms, the light chain is in an N-terminal position relative to the heavy chain. The microenvironment-restricted ASTR may be a bispecific ASTR.

[0454] Microenvironment-restricted ASTRs identified using methods provided herein are typically polypeptides, and more specifically polypeptide antibodies, and in the illustrative embodiments, single-chain antibodies. These polypeptides may bind to their cognate antigens with higher or lower affinity under abnormal versus normal conditions, but in the illustrative embodiments, they bind with higher affinity under abnormal conditions than under normal conditions. In some embodiments, these polypeptides may bind to their cognate antigen with an affinity 10%, 20%, 25%, 50%, 75%, 90%, 95%, or 99% higher under abnormal conditions than under physiological (i.e., normal) conditions. In some embodiments, the ASTRs identified using the methods provided herein do not bind to their cognate antigens under normal physiological conditions at any detectable level above the background levels obtained using negative controls, such as negative control antibodies.

[0455] The nucleotide sequence encoding a microenvironment-restricted ASTR isolated by the method provided in this document can be determined by sequencing the nucleotides of the collected cell expressing microenvironment-restricted antigen-specific targeting. This nucleotide sequence information can then be used to produce a microenvironment-restricted biological chimeric antigen receptor (MRB-CAR) by generating a polynucleotide encoding a polypeptide comprising the microenvironment-restricted antigen-specific targeting region, a transmembrane domain, and an intracellular activation domain. The microenvironment-restricted antigen-specific targeting regions can be cloned into a CAR construct expression system, which can be used to generate recombinant lentiviruses that include CAR in their genome, and then the recombinant lentiviruses can be used to transduce T cells to test CAR-mediated tumor antigen-expressing target cell killing in a tumor-selective environment compared to physiological conditions.

CONDITIONS FOR CONDITIONAL ACTIVITY

[0456] In the methods provided in this document, the activity of one or more polypeptides, such as, for example, single-chain antibodies, is screened or tested under two different sets of conditions that simulate a condition or conditions in two different physiological environments, such as, for example, a diseased microenvironment and the normal physiological condition of a non-diseased microenvironment. Typically, the conditions are conditions that can be simulated or replicated in vitro. A set of conditions may include one or more conditions to simulate a microenvironment associated with a disease. The disease may alter intracellular and extracellular homeostasis. For example, the diseased microenvironment may simulate one or more conditions in a tumor microenvironment or a cancer microenvironment. Typically, the difference or differences in activity under the two sets of conditions may result in the conditional activity of the molecule. Thus, a molecule that exhibits greater activity under the first set of conditions (e.g., simulating conditions in a tumor microenvironment) compared to the second set of conditions (e.g., simulating conditions in a normal or non-diseased environment) is identified as a candidate molecule that is restricted to the microenvironment.

[0457] The two sets of conditions may be selected to vary by one or more parameters that differ in two physiological environments, as described herein or known to one skilled in the art, including, but not limited to, chemical conditions, biological conditions, or physical conditions. The parameters that may be varied between the two sets may include one or more conditions selected from pressure, temperature, pH, ionic strength, osmotic pressure, osmolality, oxidative stress, turbidity, light exposure (including UV, infrared, or visible light), concentration of one or more solutes, such as electrolytes, glucose concentration, hyaluronan concentration, lactic acid or lactate concentration, albumin concentration, adenosine levels, R-2-hydroxyglutarate levels, pyruvate concentration, oxygen concentration, and/or the presence of oxidants, reductants, or cofactors. By varying the buffer and electrolyte systems in the calibration solutions, physiological conditions such as pH, buffering capacity, ionic environment, temperature, glucose concentration, and ionic strength can be adjusted to those of the biological environment to be simulated. The set of conditions simulating a normal physiological environment can be selected to be different from the set of conditions simulating a disease microenvironment, such as a tumor microenvironment, by one or more of the conditions described in this document.

[0458] For example, as discussed below, several parameters of the tumor microenvironment differ compared to a non-tumor microenvironment, including, but not limited to, oxygen concentration, pressure, presence of cofactors, pH, hyaluronan concentration, lactate concentration, albumin concentration, adenosine levels, R-2-hydroxyglutarate levels, and pyruvate concentration. Any of these parameters can be replicated in vitro to simulate one or more conditions that exist in a tumor or cancer environment compared to conditions that exist in a non-tumor or normal environment. Normal physiological conditions that can be simulated include environments found in healthy or non-diseased tissue in any location in the body, such as in the GI tract, skin, vasculature, blood, and extracellular matrix. Typically, in the assays in this document, physiological conditions can be similar in vitro by the choice of buffer used to assess protein activity. For example, any one or more conditions of a diseased microenvironment (such as a tumor microenvironment) and a non-diseased environment can be simulated by differences in the assay buffer used to assess activity in the assay. Therefore, in the methods in this document for identifying an environment-restricted polypeptide, a component or components or feature or features of an assay buffer are altered or made different in a first assay to test activity under a first condition and in a second assay to test activity under a second condition. For example, as discussed in this document, several parameters of the tumor microenvironment differ compared to a non-tumor environment, including, but not limited to, oxygen, pressure, presence of cofactors, pH, hyaluronan concentration (such as increased or decreased hyaluronan concentration), lactate concentration (such as increased or decreased lactate concentration), albumin concentration (such as increased or decreased albumin concentration), adenosine levels (such as increased or decreased adenosine levels), R-2-hydroxyglutarate levels (such as increased or decreased R-2-hydroxyglutarate levels), and pyruvate concentration (including increased or decreased pyruvate concentration). More specifically, conditions in a tumor microenvironment may include lower pH, higher concentrations of hyaluronan, higher concentrations of lactate and pyruvate, higher concentrations of albumin, increased adenosine levels, increased levels of R-2-hydroxyglutarate, hypoxia, lower glucose concentration, and slightly higher temperature compared to the non-tumor microenvironment. For example, a microenvironment-restricted ASTR is virtually inactive at normal body temperature but is active at a higher temperature in a tumor microenvironment. Furthermore, the microenvironment-restricted antibody is less active in normal oxygenated blood but more active under the less oxygenated environment found in a tumor. In yet another aspect, the microenvironment-restricted antibody is less active at a normal physiological pH of 7.2 to 7.8 but more active under the acidic pH of 5.8 to 7.0 or 6.0 to 6.8 found in a tumor microenvironment. For example, the antibody restricted to the microenvironment is more active at a pH of 6.7 than at a pH of 7.4. There are other conditions in the tumor microenvironment known to a person skilled in the art that can also be used as the condition in the present invention under which conditionally active ASTRs have different binding affinities. The in vitro assay conditions that mimic these in vivo tumor conditions are referred to herein as in vitro tumor surrogate assay conditions.

[0459] Any one or more of these conditions can be simulated in vitro by choosing a particular assay buffer. The composition of the assay buffer simulating a disease microenvironment can be selected to be identical to the composition of the assay buffer simulating a normal environment, with the exception of one or more known or described conditions in this document that are altered in the disease microenvironment. Furthermore, in screening or identifying the activity of one or more polypeptides under two different sets of conditions, generally the only conditions that are varied in the assay refer to the buffer conditions simulating the in vivo microenvironment. The other assay conditions, such as time, temperature, and incubation conditions, can be the same for both sets of conditions. Typically, the same buffer base is used in both disease-simulating and normal-simulating microenvironment conditions, but the buffer composition design can be made to differ in one or more parameters, such as pH, oxygen, pressure, presence of cofactors, pH, hyaluronan concentration (such as increased or decreased hyaluronan concentration), lactate concentration (such as increased or decreased lactate concentration), albumin concentration (such as increased or decreased albumin concentration), and/or pyruvate concentration (including increased or decreased pyruvate concentration). In both disease-simulating and normal-simulating microenvironment conditions, any buffer base known to one skilled in the art may be used.

METHODS FOR GENERATING A MICROENVIRONMENT-RESTRICTED CELL

[0460] The present disclosure provides a method for generating a microenvironmentally restricted cell. The method generally involves modifying a mammalian cell with an expression vector (e.g., a plasmid or a retrovirus) or RNA (e.g., RNA transcribed in vitro), including nucleotide sequences encoding the microenvironmentally restricted CARs of the present disclosure. The genetically modified cell is microenvironmentally restricted in the presence of one or more target antigens. The genetic modification can be performed in vivo, in vitro, or ex vivo. The cell can be an immune cell (e.g., a T lymphocyte, a T helper cell, or an NK cell), a stem cell, a progenitor cell, etc.

[0461] In some cases, genetic modification is performed ex vivo. For example, a T lymphocyte, a stem cell, a T helper cell, or an NK cell is obtained from an individual; and the cell from the individual is genetically modified to express a CAR of the present disclosure. The genetically modified cell is restrictable to the microenvironment in the presence of one or more target antigens. In some cases, the genetically modified cell is activated ex vivo. In other cases, the genetically modified cell is introduced into an individual (e.g., the individual from whom the cell was obtained); and the genetically modified cell is activated in vivo. For example, when one or more target antigens are present on the surface of a cell in the individual, there is no need to administer the antigen. The genetically modified cell comes into contact with the antigen present on the surface of a cell in the individual, and the genetically modified cell is activated. For example, when the genetically modified cell is a T lymphocyte, the genetically modified cell can exhibit cytotoxicity toward a cell that expresses one or more target antigens on its surface to which the CAR binds.

METHODS FOR TEMPORARY REDUCTION OF CAR-T TARGET BINDING SENSITIVE TO TUMOR MICROENVIRONMENT

[0462] A method for the temporary reduction of tumor microenvironment-sensitive CAR-T target binding through pharmacological modification of vascular and tissue pH is provided in this document. Microenvironmentally controlled scFvs in CAR-T cells provide an additional level of protection against non-tumor target toxicity, requiring local tumor environmental conditions to enable T cell interconnection. While attractive for some monoclonal antibody therapies, adoptive cell therapy can create local environments that are temporarily permissive for their CAR-T targets. For example, CAR-T cells activated in local tissues can further reduce local pH, depending on the cytoplasmic domains present in the CAR construct. In other cases, cytokine release syndrome and other morbidity associated with adoptive cell therapy can result in loss of blood bicarbonate buffering capacity, leading to lactic acidosis. It is established that adoptive cell therapies administered by intravenous infusion result in temporary pulmonary trapping. For some cell therapies, the infusion rate requires constant monitoring of dissolved oxygen (Fischer et al. Stem Cells Dev. June 2009; 18(5): 683-691). The extent of lung trapping depends on cell size, activation state, cell dose, and infusion rate. Cruz et al (Cytotherapy. October 2010; 12(6): 743-749) report adverse findings from over 300 T-cell infusions that low doses and slow infusion can reduce lung trapping. However, with certain high-potency CAR-T cells, targets present even at low levels in the lung endothelium, such as Her2 (Morgan et al. Mol Ther. April 2010; 18(4): 843-851), can result in immediate toxicity that cannot be controlled and leads to rapid patient deterioration due to the high CAR-T cell concentration in the lung after infusion and the presence of the T-cell target in these tissues. In other cases, the presence of T-cell targets in other non-target tissues, such as the bile duct, can create non-target toxicities outside the tumor that cannot be controlled (Lamers Mol Ther. April 2013; 21(4): 904-912) and result in severe organ toxicity before other agents, such as steroids or cell-killing epitopes, can be used. Although venous and arterial plasma have a strong buffering capacity against acidosis, conditions such as respiratory acidosis, shock, metabolic acidosis, and ischemic acidosis can occur in cancer patients treated with adoptive cell therapy.

[0463] In some embodiments, methods for temporarily increasing vascular pH under specific conditions to reduce the affinity of microenvironmentally controlled scFvs for their antigens are provided. A deviation of 0.4 U in blood pH can reduce the affinity of certain scFvs by more than 10-fold. In some embodiments, therapeutic pH control can be achieved via IV or oral routes of administration. In some embodiments, inactivation of binding affinity can be achieved with bicarbonate. In other embodiments, Tris-hydroxymethyl aminomethane (also known as tromethamine, trometamol, and THAM) and Carbicarb™ (and equimolar hypertonic solution of sodium bicarbonate and sodium carbonate) are used to increase blood pH by a sufficient amount to alleviate non-tumor target toxicities. In still other modalities, small molecule proton pump inhibitors can also be used to increase blood pH and/or tissue pH by a sufficient amount to alleviate non-tumor target toxicities. Proton pump inhibitors include, but are not limited to, esomeprazole (Nexium), esomeprazole and naproxen (Vimovo), lansoprazole (Prevacid), omeprazole (Prilosec and Zegerid), and rabeprazole (Aciphex). Administration of proton pump inhibitors can be used effectively over longer periods of time to modulate the binding affinity of the antigen-binding domain to its cognate antigen for days, weeks, months, or years. In other modalities, the affinity of the antigen-binding domain to its cognate antigen can also be modulated by altering blood pH and/or tissue pH by controlling transcription, translation, membrane expression, and the stability of transporters and pumps. Examples of such transporters and pumps for modulating pH include, but are not limited to, proton pumps, members of the sodium proton exchanger (NHE) family, the bicarbonate transporter (BCT) family, and the monocarboxylate transporter family. In certain modalities, bicarbonate, THAM, or Caricarb™ can be administered prior to or concomitantly with CAR-T cell infusion in patients expressing pH-controlled scFvs. Such treatment will alleviate the immediate cytotoxicity that is otherwise associated with the temporary pulmonary entrapment of CAR-T cell infusions.

ADDITIONAL MODALITIES

[0464] In certain embodiments, the methods provided herein for the present disclosure include inhibiting the expression of one or more endogenous genes expressed in T cells and/or NK cells. The methods provided herein illustrate the ability to produce recombinant retroviruses expressing miRNA or shRNA, for example, which can be used for such methods. In fact, the methods provided herein illustrate that such miRNA or shRNA can be encoded within introns, including, for example, an Ef1a intron. This leverages the present teachings of the methods to maximize the functional elements that can be included in a packageable retroviral genome to overcome the disadvantages of previous teachings and maximize the effectiveness of such recombinant retroviruses in adoptive T cell therapy.

[0465] In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 miRNAs, in the illustrative embodiments between 2 and 5, for example, 4 miRNAs, which bind to nucleic acids encoding one or more of the following endogenous T cell-expressed target genes, can be included in the recombinant retroviral genome and delivered to T cells and/or NK cells using the methods provided herein. In fact, as provided herein, 1, 2, 3 or 4 miRNAs can be delivered to a single intron, such as the EF1a intron. The endogenous T cell-expressed target genes may include the following, with a non-limiting expected benefit of such inactivation in parentheses: PD-1 (prevent inactivation); CTLA4 (prevent inactivation); TCRa (safety - prevent autoimmunity); TCRb (safety - prevent autoimmunity); CD3Z (safety - prevent autoimmunity); SOCS (prevent inactivation); SMAD2 (prevent inactivation); miR-155 (promote activation); IFN gamma (reduce CRS); cCBL (prolong signaling); TRAIL2 (prevent death); PP2A (prolong signaling); ABCG1 (increase cholesterol microdomain content by limiting cholesterol release).

[0466] In certain embodiments, miRNAs targeting genes with similar expected utilities can be combined. In other embodiments, miRNAs targeting genes with complementary utilities can be combined. In some embodiments, the combinations may include CD3Z, PD1, SOCS1 and/or IFN gamma.

TREATMENT METHODS

[0467] The present disclosure provides several methods of treatment using a CAR. A CAR of the present disclosure, when present on a T lymphocyte or an NK cell, can mediate cytotoxicity towards a target cell. A CAR of the present disclosure binds to an antigen present on a target cell, thus mediating the elimination of a target cell by a T lymphocyte or an NK cell genetically modified to produce the CAR. The ASTR of the CAR binds to an antigen present on the surface of a target cell.

[0468] The present disclosure provides methods for exterminating or inhibiting the proliferation of a target cell, wherein the method involves contacting a cytotoxic immune effector cell (e.g., a cytotoxic T cell or an NK cell) that is genetically modified to produce a subject CAR, such that the T lymphocyte or NK cell recognizes an antigen present on the surface of a target cell and mediates the extermination of the target cell.

[0469] The present disclosure provides a method for treating a disease or disorder in an individual who has the disease or disorder, wherein the method includes: a. introducing an expression vector including a polynucleotide sequence encoding a CAR into peripheral blood cells obtained from the subject to produce a genetically modified cytotoxic cell; and b. administering the genetically modified cytotoxic cell to the subject.

SUITABLE SUBJECTS FOR TREATMENT

[0470] A variety of subjects are suitable for treatment with the methods and compositions presented in this document. Suitable subjects include any individual, for example, a human or non-human animal that has a disease or disorder, that has been diagnosed with a disease or disorder, that is at risk of developing a disease or disorder, that has had a disease or disorder and is at risk of recurrence of the disease or disorder, that has been treated with an agent for the disease or disorder and has failed to respond to such treatment, or that has been treated with an agent for the disease or disorder but has relapsed after an initial response to such treatment.

[0471] Suitable subjects for treatment with an immunomodulatory method include individuals who have an autoimmune disorder; individuals who are organ or tissue transplant recipients; and similar individuals who are immunocompromised; and individuals who are infected with a pathogen.

[0472] The following non-limiting examples are provided only by way of illustration of exemplary embodiments and in no way limit the scope and spirit of the present disclosure. Furthermore, it should be understood that any inventions disclosed or claimed herein encompass all variations, combinations, and permutations of any one or more features described herein. Any one or more features may be explicitly excluded from the claims even if the specific exclusion is not explicitly stated herein. It should also be understood that the disclosure of a reagent for use in a method is intended to be synonymous with (and provide support for) that method involving the use of that reagent, according to the specific methods disclosed herein or other methods known in the art, unless a person of ordinary skill in the art would understand otherwise. Additionally, where the descriptive report and/or claims disclose a method, any one or more of the reagents disclosed herein may be used in the method, unless a person of ordinary skill in the art would understand otherwise.

EXAMPLES EXAMPLE 1. GENERATION OF RIBOSWITCHES THAT RESPOND SPECIFICALLY TO NUCLEOSIDE ANALOG ANTIVIRAL DRUGS.

[0473] This example provides a method for screening libraries based on natural structural riboswitches that bind to guanosine and deoxyguanosine. These riboswitches were used as scaffolds to develop induced libraries for the selection of aptamers that specifically bind to a nucleoside analog ligand. Previously, isothermal titration calorimetry was used to show that these natural riboswitches bind to their native ligands. Further tests showed that a deoxyguanosine switch also interacted weakly with the nucleoside analogs acyclovir and penciclovir, leading to the redesign of this sequence into a new library. Single-stranded riboswitch regions that were targeted for mutation variant sequences that specifically respond to acyclovir or penciclovir were selected.

MATERIALS

[0474] The selection components guanine, guanosine, deoxyguanosine, acyclovir, and penciclovir were ordered from Sigma-Aldrich (St. Louis, MO). Acyclovir was the initial target, while penciclovir was an analyte of special interest used in later cycles, and guanine, guanosine, and deoxyguanosine were used as counter-targets. Graphene oxide (GrO), to be used as the partitioning medium, was purchased from Angstron Materials (Dayton, OH). HEPES (pH 7.3) and MgCl2 were purchased from Amersco LLC (Solon, OH). KCl was purchased from Teknova (Hollister, CA). The selection buffer was prepared at 5X (1X as HEPES 50 mM, KCl 100 mM, MgCl2 20 mM, pH 7.3). Targets, counter-targets, and oligos were reconstituted in nuclease-free water for preliminary analysis and aptamer screening. Aliquots were prepared for all targets and stored at -20 °C to maximize shelf life.

APTAMER LIBRARY GENERATION

[0475] The initial aptamer library template was synthesized by IBA GmbH (Gottingen, Germany) as the reverse complement of the sequences in Figure 14. In Figure 14, the nucleotides in boxes are single-stranded in the known sequences, with "mutations" introduced during synthesis to allow better binding to analogs of the original targets. For nucleotides within boxes outlined with solid lines, substitution mutations were allowed; for nucleotides within boxes outlined with dashed lines, substitution mutations as well as insertions or deletions were allowed. The primers were synthesized by IDT (Coralville, IA) as single-stranded DNA. The T7 primer (SEQ ID NO:240) was combined with library template sequences for primer extension with Titanium Taq DNA polymerase (Clontech; Mountain View, CA). The primer-extended material was transcribed using the T7 High Yield Transcription Kit (Epicentre; Madison, WI) and then purified by 10% denaturing polyacrylamide gel electrophoresis (PAGE) with 8 M urea before use in the selection process. During selection, the library was reverse transcribed using SuperScript IV Reverse Transcriptase (Invitrogen; Carlsbad, CA) with the reverse primer (SEQ ID NO:241) and amplified using Titanium Taq DNA polymerase (Clontech; Mountain View, CA). The aptamer with SEQ ID NO:248 had a J2-3 loop variance of -3 to -1 and a diversity of ~2.25x10¹⁰. The aptamer with SEQ ID NO:250 had a J2-3 loop variation from 0 (native) to +5 and a diversity of ~9.38x1014. The two oligonucleotides (SEQ ID NOs:249 and 250) were mixed at a ratio of 1:4,160 to produce equimolar diversity in the combined library cluster, with a total diversity of ~9.38x1013.

LIBRARY SCREENING

[0476] Library screening was conducted using a Systematic Evolution of Ligands by Exponential Enrichment with graphene oxide (GO-SELEX) approach (Figure 15) (Park et al., 2012), taking advantage of the π-π interaction that gives graphene oxide a high affinity for single-stranded nucleic acids (Zeng et al., 2015). The aim was to select sequences that did not interact with the 1X selection buffer or with the counter-targets (guanine, guanosine and deoxyguanosine), but did not bind to the positive target acyclovir.

[0477] For each cycle, a given amount of library was first re-folded in 1X selection buffer (denaturation for 5 minutes at 90 °C, 5 minutes at 4 °C, then room temperature). Counter-targets were then added to the folded libraries and incubated for 30 minutes at 37 °C. The exceptions to this were cycles 1 and 2, where counter-targets were only briefly (< 1 minute) included to help load the library into GrO. After allowing the library to interact with the counter-targets and buffer components, the unbound library was loaded into GrO (mass equal to 100 times the mass of the library at the start of the cycle) over the course of a 10-minute incubation at 37 °C. The solution was then centrifuged at 7,000 x g to pellet the GrO. The supernatant, which contained sequences bound to counter-targets and/or buffer, was removed. The pellet was then washed twice with 200 μl of 1X selection buffer, centrifuging at 7,000 x g and removing the supernatant after each wash. A solution containing a positive target was then added and allowed to elute the GrO library under the conditions indicated in Table 1 for up to 60 minutes at 37 °C, essentially allowing the target to compete with graphene oxide for library binding. Sequences that bind most strongly to the target would desorb from the graphene oxide and remain bound to the target at the end of the incubation. A final centrifugation step separated the released material, located in the supernatant, from the unresponsive library that remained bound to the graphene oxide.

[0478] After positive selection, the recovered RNA purified using 10% denaturing PAGE with 8 M Urea was then quantified using a spectrophotometer reading (Table 1), reverse transcribed with SuperScript IV and amplified using PCR with Titanium Taq DNA polymerase. The amplification products were transcribed into RNA for the next selection cycle.

[0479] Three levels of stringency were implemented throughout the selection process (Table 1). The first two selection cycles did not include screening against counter-targets to maximize library loading on GrO. Additionally, a large excess of acyclovir was used in positive incubations to maximize library recovery, hence the low stringency designation. Counter-target incubations were introduced after library recovery was achieved, as intermediate stringency conditions. The acyclovir-to-library ratio was also reduced during these three cycles to increase library competition for target binding. Once more than 10% recovery was achieved, the final high-stringency selection cycles were implemented. The counter-target/library ratio remained high, and the positive target/library ratio was set to 1:1 while the positive incubation time was reduced to select faster binding sequences. Since library recovery was shown to remain above 10% after more than two cycles of high stringency conditions, parallel evaluations were conducted. TABLE 1. SELECTION AND EVALUATION CONDITIONS. CONDITIONS USED FOR EACH SELECTION OR INCUBATION CYCLE, WITH RECOVERY AS THE RATIO BETWEEN THE RECOVERED SAMPLE AND THE INPUT LIBRARY FOR EACH CYCLE. LIBRARY ENRICHMENT WAS MONITORED THROUGHOUT THE SELECTION COURSE. *COUNTER-TARGETS USED FOR LOADING, NON-EXTENDED INCUBATION. t PRE-LOADING INCUBATION CONDUCTED WITH GROUPED COUNTER-TARGETS. $ PRE-LOADING INCUBATION CONDUCTED WITH THE ACYCLOVIR POSITIVE TARGET. This was done to minimize the recovery of cross-reactive species. The following abbreviations are used in this table: "X targets" are counter-targets; "target (+)" is acyclovir or pencyclovir; "(+) incubation time (min)" is the time the solution from "Library: Target (+)" was incubated in GrO. G0 is generation 0 and so on; R1 is cycle 1 and so on. FOR THE PARALLEL EVALUATION (PARALLEL 1 AND PARALLEL 2), INCUBATIONS WERE PERFORMED WITH: (-) 1X SELECTION BUFFER ONLY, (X) COUNTER-TARGETS IN 1X SELECTION BUFFER, (+) ACYCLOVIR IN 1X SELECTION BUFFER AND (P) PENCICLOVIR IN 1X SELECTION BUFFER.

[0480] For the two parallel evaluations, the library to be evaluated was divided into four equal quantities for preparation and re-folding as above (Figure 16). For each condition, 50 pmol of library were combined with 1X selection buffer, re-folded (90 °C for 5 minutes, 4 °C for 5 minutes), and then incubated with 200 μl of 10 μM combined counter-targets in 1X selection buffer for 30 minutes at 37 °C. These samples were then loaded into an amount of graphene oxide equal to 100 times the mass of the library in the sample and incubated for 10 minutes at 37 °C, and then washed twice with 200 μl of 1X selection buffer as before. The loaded graphene oxide samples were then incubated in parallel with 200 μl of the appropriate evaluation condition (1X selection buffer only, 10 μM pooled counter-targets, 1 μM penciclovir, 1 μM acyclovir for the first parallel evaluation or 0.5 μM acyclovir for the second parallel evaluation; in Table 1, these conditions are shown as: (-); (X); (P); (+); and (+), respectively) in 1X selection buffer for 30 minutes at 37 °C. A final centrifugation step separated the desorbed responsive library from the non-responsive graphene oxide-bound library. The responsive libraries were quantified using spectrophotometric reading (Table 1), verified using 10% denaturing PAGE with 8 M urea, and prepared for a second parallel evaluation. This follow-up assessment continued to use counter-targets for the pre-loading incubation of the positive sample, but used the positive target acyclovir for each other pre-incubation of the samples. This was done to minimize the representation of cross-reactive sequences in a given sample (i.e., responsive to counter-targets in the positive sample, responsive to acyclovir in the negative samples, to counter-targets, or to penciclovir). The material recovered from the second parallel assessment was quantified using spectrophotometric reading (Table 1), verified using 10% denaturing PAGE with 8 M urea, and prepared for reverse transcription sequencing and PCR to generate single-stranded DNA.

SEQUENCING

[0481] The initial library was subjected to more than 10 selection cycles based on GrO and parallel evaluation (Table 1). The GO-SELEX process is designed to enrich sequences across multiple selection cycles that bind to specific targets of interest and remove sequences that bind to buffer components or non-target compounds. As a result, the populations to be sequenced are expected to contain multiple copies of potential aptamer candidates.

[0482] The Illumina MiSeq system (San Diego, CA) was deployed to sequence aptamer libraries after parallel evaluation using a one-end read technique. Subsequent data analysis and deep sequencing reduces the large number of screening cycles that traditional SELEX requires, which can introduce error and bias due to the screening process (Schütze et al., 2011). Five samples were sequenced: the final generation library that responded to acyclovir, the final generation library that responded to counter-targets, the final generation library that responded to the 1X selection buffer (negative condition), the penultimate generation library that responded to acyclovir, and the final generation library that responded to the additional target of interest, penciclovir. From these datasets, sequence families were constructed at 95% homology (sequence similarity considering mutations, deletions, and insertions) for aptamer candidate identification. There were 1,711,535 raw sequences (124,600 unique sequences) from the library that responded to acyclovir and 2,074,832 raw sequences (110,149 unique sequences) from the library that responded to penciclovir.

SELECTION OF APTAMER CANDIDATE

[0483] Sequence family construction focused primarily on the similarity between sequences. This means that the frequency of a sequence in the positive target population was taken into account, but greater emphasis was placed on the degree of variation between similar sequences, with 95% homology being the minimum requirement (100% matching across the entire sequence is not necessary to join a family; up to 2 bases may be mismatched, inserted, or deleted).5 Families with the highest number of members would therefore be expected to rank highly as aptamer candidates. After families are constructed, consideration can be given to the relative presence of a family in a given population – families that occur frequently in both negative target and counter-target populations are considered weaker candidates10 insofar as they demonstrate a degree of non-specific interaction in binding to buffer or counter-target components. Additionally, families that demonstrate a high enrichment rate (i.e., a large ratio between the final positive population and the penultimate positive population) improve their candidacy, as the enrichment rate was linked to a candidate's binding affinity relative to the rest of the population (Levay et al., 2015; Wang et al., 2014). Under these conditions, several candidate families appear to be strong candidates for acyclovir (Table 2) and penciclovir binding. TABLE 2. DNA SEQUENCES CORRESPONDING TO THE NON-STEM REGION OF ACYCLOVIR-BINDING RNA RIBOSWITCHES. SEVEN FAMILIES WERE IDENTIFIED IN THE SCREENING: 582, 769, 795, 935, 946, 961, AND 996 WITH BETWEEN 1 AND 39 SEQUENCES IN EACH FAMILY. THE PERCENTAGE IDENTITY FOR EACH SEQUENCE IN THE FAMILY WAS COMPARED TO THE MOST PREDOMINANT SEQUENCE WITHIN EACH FAMILY (582-1, 769-1, 795-1, 935-1, 946-1, 961-1, AND 996-1). The percentage identity for each sequence in the family was also compared to the wild-type sequence.

[0484] The acyclovir-positive target produced seven strong candidates (SEQ ID NOs:87 to 93; RNA sequences including stem regions) corresponding to 5821 (SEQ ID NO:108), 769-1 (SEQ ID NO:147), 795-1 (SEQ ID NO:164), 935-1 (SEQ ID NO:183), 946-1 (SEQ ID NO:198), 961-1 (SEQ ID NO:220) and 996-1 (SEQ ID NO:221), each designated F1A (Figure 17). These sequences were the most prevalent sequences in each family (the DNA sequences of all members of each family were: 582 (SEQ ID NOs: 108 to 146); 769 (SEQ ID NOs: 147 to 163); 795 (SEQ ID NOs: 164 to 182); 935 (SEQ ID NOs: 183 to 197); 946 (SEQ ID NOs: 198 to 219); 961 (SEQ ID NO: 220); and 996 (SEQ ID NO: 221)). The consensus sequences show all possible substitutions or gaps at each nucleotide position for each family (SEQ ID NOs: 222 to 226). Since the goal was to identify aptamers from a library based on RNA known to bind to deoxyguanosine, strong candidates needed to have a minimal presence in the counter-target population. Candidates F1A-795, F1A-935, and F1A-946 met this criterion very well, as they were not detected in the counter-target population. F1A-996 and F1A-961 are considered the next best candidates in this respect, although they appear to a lesser degree in the counter-target population. Additionally, the candidates must appear minimally in the negative population, as those sequences desorbed from GrO without the influence of acyclovir and could represent false positives. F1A-935 and F1A-946 also performed ideally under this criterion, as they are not found in the negative population. Candidate F1A-769 was minimally detected in the negative population, with candidates F1A-961, F1A-795, and F1A-996 performing worse. The enrichment rate was the final condition to be considered, with F1A-935, F1A-946, and F1A-769 performing adequately. Candidate F1A-582 was included because it exhibited the highest enrichment rate, although it did not perform well under the other criteria. The remaining candidates did not perform well in relation to these four, but exhibited acceptable characteristics.

[0485] The additional target penciclovir yielded seven strong candidates (SEQ ID NOs: 94 to 100), each designated F1P (Figure 18). As before, the aim was to identify aptamers from a library based on RNA known to bind to deoxyguanosine, diverging from libraries enriched to bind to acyclovir (acyclovir) after Cycle 10. Strong candidates needed to have minimal presence in both counter-target and acyclovir populations to minimize cross-reactivity. Candidate F1P-923 met the first criterion, candidate F1P-710 met the second criterion, and candidate F1P-584 met both criteria to a degree. Candidate F1P-584 also demonstrated moderate favorability for penciclovir relative to the negative condition, as well as moderate enrichment relative to the previous generation response to acyclovir. The remaining candidates demonstrated either minimal favorability of penciclovir over acyclovir or minimal favorability of penciclovir over the counter-targets (F1P-837 and F1P-932; F1P-991 and F1P-718, respectively). These four candidates demonstrated some favorability for penciclovir over the negative condition that minimizes the chance of a false positive, although this criterion is not as significant if a candidate does not demonstrate selectivity for penciclovir over its analogs. The enrichment rate was the final condition to be considered, with F1P-923, F1P-932, and F1P-584 performing adequately.

[0486] Quantitative PAGE evaluation of selected aptamers was performed. Individually synthesized and transcribed aptamers were subjected to selection on graphene oxide (GrO) under physiological Mg++ (0.5 mM) and elution with acyclovir (+) or counter-targets (x). Aptamer fractions specifically diluted for each sample were subjected to PAGE for analysis.

[0487] 100 pmol of each aptamer candidate (porteste/strip) were resuspended in modified 1X selection buffer (50 mM HEPES, 100 mM KCl, 0.5 mM MgCl2, pH 7.3) and re-folded (90 °C for 5 min, then 4 °C for 5 min), then incubated at 37 °C for 30 min with 200 pmol (each) of pooled counter-targets or target. The final library concentration was 0.5 μM, the target/counter-target concentration was 1 μM (the incubation volume was 200 μl).

[0488] After target/counter-target incubation, 250 μg of GrO (Angstron Materials (Dayton, OH)) were added to adsorb the unbound candidate (10-minute incubation at 37 °C).

[0489] Samples were centrifuged for 5 minutes at 7,000 x g. The supernatant was recovered, denatured using 2X Formamide with 40 mM EDTA, and passed through a 10% denaturing PAGE solution with 8 M urea (supplier: American Bioanalytical; catalog number AB13021-01000, AB13022-01000). Continuous buffer was 1X TBE (supplier: Amresco/VWR; catalog number 0658-20L, diluted using DI water). DNA ladder was a 20/100 DNA ladder (IDT). Gels were stained with Gel Star (Lonza, 50535) and imaged on a blue light transilluminator.

[0490] Candidates F1A-769, F1A-795, F1A-946 and F1A-996 appear to exhibit a selective positive response in this quantitative PAGE assessment (good elution of the GrO aptamer with acyclovir targeting and relatively lower or minimal elution with counter-targets).

CONCLUSION

[0491] Strong candidates for acyclovir were identified after twelve cycles of iterative screening and parallel evaluation; reasonable candidates for penciclovir were identified after two cycles of screening and parallel evaluation.

EXAMPLE 2: ISOLATION OF CONDITIONAL scFvs.

[0492] Potential splicing site disadvantages are removed, and tumor antigen-specific scFvs are synthesized by overlapping oligo synthesis and cloned into the CAR construct containing the acyclovir responsive element and the primate CD3Z promoter. As an initial prototype, anti-ECD EPCAM or ERBB2scFv with a CD8-alpha signaling peptide, stem, and transmembrane domain is used. Solid tumor microenvironment-restricted CAR products are generated either using the methods described in US Patent No. 8,709,755 and PCT Publication WO/2016/033331A1, or by direct selection from human phage libraries under permissive or non-permissive conditions. Briefly, a human VH x VL library from Creative Biolabs (Shirley, NY) is selected under the following tumor-permissive conditions: 100 μg/ml hyaluronan, 100 kDa fraction (Lifecore Biomedical, Chaska, MN), 20 mg/ml recombinant HSA (Cyagen, Santa Clara, CA), 200 ng/ml recombinant human VEGF in 25 mM sodium bicarbonate buffer, 2 μM adenosine, 10 mM sodium lactate pH 6.7, after release with streptavidin magnetic microspheres (ThermoFisher, Carlsbad, CA) bound to biotinylated human IgG. Binding to the biotinylated target receptor ECD of EPCAM- and ERBB2-conjugated microspheres at 37 °C is performed under permissive conditions followed by serial washes under permissive conditions. Phages are released under physiological conditions (1 μg/ml hyaluronan, 20 mg/ml HSA, 25 mM bicarbonate, 1 mM sodium lactate, pH 7.2) followed by elution of limited variants with acid elution and rapid neutralization with 1 M Tris. Phages are expanded and genomic DNA is split for in-depth analysis of VH x VL strand sequences using long read sequencing (PacBio, Menlo Park, CA). Selection can be repeated for enrichment. VH x VL sequences showing preferential amplification of reads during the phage culture process compared to target enrichment are excluded for further analysis. Target-selectively bound phages that are enriched under permissive tumor conditions but released under physiological conditions are chosen for further characterization by cloning into the CAR construct expression system, lentivirus generation, and transduction into T cells to test CAR-mediated tumor antigen-expressing target cell killing in a tumor-selective environment compared to physiological conditions.

EXAMPLE 3: GENERATION OF MRB-CARs USING ScFvs RESTRICTED TO THE MICROENVIRONMENT.

[0493] Microenvironment-restricted ASTRs were obtained that were produced by subjecting VH and VL sequences with low selectivity for the tumor microenvironment to evolution as described in WO/2016/033331A1. Chimeric antigen receptors (CARs) for binding two tumor antigens, Axl or Ror2, with increased activity at the reduced pH of a tumor environment compared to normal tissue (such microenvironment-restricted biology is sometimes referred to in this document as MRB-CARs) were produced by incorporating the heavy and light chains of microenvironment-restricted single-chain antibodies into lentiviral expression vectors along with other CAR domains to generate MRB-CARs. These CARs included various combinations of amino-to-carboxy termination modules, which included a CD8 (P1) signaling peptide (SEQ ID NO:74); microenvironmentally restricted VH and VL combinations of anti-Ror2 and anti-Axl; a CD8 (SEQ ID NO:75) or CD28 (SEQ ID NO:76) transmembrane and stem domain (P5); a CD 137 (SEQ ID NO:1) or ICΔ (SEQ ID NO:3) co-stimulatory domain (P6); a CD3Z (SEQ ID NO:13) activation domain (P7); a 2A-1 ribosome jump sequence (SEQ ID NO:77) (P8); and an exemplary eTAG (SEQ ID NO:78) (P9).

[0494] Pan T cells were transduced with recombinant lentiviral particles expressing candidate CARs and the percentage of transfected cells was determined by determining the percentage of cells expressing eTag using FACS. Pan T cells were successfully transduced with recombinant lentiviral particles encoding candidate CARs and exhibited conditional activity in these transduced T cell assays at pH 6.7 versus pH 7.4.

[0495] The cytotoxic activity of candidate CARs against target cells expressing Axl or Ror2 was analyzed at a pH of 7.4 (physiological pH) or a pH of 6.7 (pH surrogate tumor assay condition). Many of the candidate CARs were more effective at lysing target cells at a pH of 6.7 than at a pH of 7.4.

EXAMPLE 4. CONSTRUCTION OF LIGAND-INDUCTIBLE RIBOSWITCHES.

[0496] The deoxyguanosine riboswitch aptamer and guanine riboswitch aptamers (Pikovskaya, 2014; Kim, 2007) or other purine riboswitch aptamers are synthesized as oligonucleotides. In one example, the deoxyguanosine riboswitch IA from Mesoplasma florum (underlined and bold in Figure 6; Figure 7) is selected for evolution to generate an acyclovir-responsive riboswitch. In another example, the guanine xpt riboswitch from Bacillus subtilis (underlined and bold in Figure 10; Figure 11) is selected for evolution to generate an acyclovir-responsive riboswitch. For each of these two examples, a random RNA library is generated with alternating nucleotides at targeted sequence positions in segments P2, P3, J1-2, and J2-3 (Figures 7 and 11). Each segment allows for 3 alternating nucleic acids at each targeted sequence position or, alternatively, deletion and insertion of 4 nucleotide bases at the +1 site at each targeted sequence position for saturation mutagenesis as indicated in Figures 8A, 8B, and 9 (IA of M. florum) and Figures 12A, 12B, and 13 (expt of B. subtilis). Primer extension and reagent preparation are followed by RNA transcription. The resulting RNA library is negatively selected on graphene oxide in the presence of guanine, guanosine, and deoxyguanosine followed by positive selection with acyclovir or penciclovir. During the positive and negative selection processes, physiological human cell magnesium levels (0.5 mM to 1.2 mM) are used, and the temperature is maintained at 37 °C. The recovered aptamers are reverse transcribed and amplified by PCR followed by transcription and subsequent screening for at least 8 successive selection cycles. In a parallel approach, the aptamers are screened with an additional negative screening at 40 °C. The resulting positive clusters are examined by sequencing and NextGen analysis. Individual aptamers are synthesized and examined for affinity by isothermal calorimetry at 35 to 40 °C at physiological human cell magnesium levels. After selection of acyclovir- and penciclovir-positive specific aptamers, the aptamers are integrated into hammerhead ribozymes and gun ribozymes. Acyclovir-positive selective aptamers are combined with gun ribozymes to identify acyclovir-regulated ribozymes. (Harris KA RNA. November 2015;21(11):1852-1858. doi: 10.1261/rna). The variants are subjected to gel displacement-based PAGE purification in the presence of acyclovir and absence of penciclovir. Additionally, the acycloguanosine-selective riboswitch is placed immediately 3' into a loop to a splicing acceptor upstream of the CAR/IL-7 construct. In the absence of acyclovir, the splicing site position is bound in the riboswitch complex, but in the presence of acyclovir it becomes accessible, generating a functional CAR transcript.

EXAMPLE 5. CONSTRUCTION OF IN VIVO PROPAGATION DOMAINS.

[0497] A series of constitutively active IL7 receptor transmembrane mutants (IL7R) of T-cell lymphoblastic leukemias (243 InsPPCL (SEQ ID NO:82); 246 InsKCH (SEQ ID NO:101); 241 InsFSCGP (SEQ ID NO:102); 244 InsCHL (SEQ ID NO:103); and 244 InsPPVCSVT (SEQ ID NO:104); all from Shochat et al 2011, J. Exp. Med. Volume 208 no 5 901-908) is synthesized by overlapping oligonucleotide synthesis (DNA2.0, Newark, California). The synthesized constitutively active IL7R transmembrane mutants are inserted into a constitutive expression lentiviral vector main chain behind a 2A ribosome jump sequence followed by an anti-CD19 CD3Z expression cassette, which includes a CD8A stem (SEQ ID NO:79) and an initiation peptide (SEQ ID NO:74). HEK293 packaging cells are transfected with the IL7R transmembrane mutant lentiviral vectors and lentiviral packaging constructs, and the cultured and viral supernatants are collected using methods known in the art. CD3/CD28-stimulated T cells are transduced with the viral supernatants and cultured in IL2-deficient AIM V, CTS OpTmizer Cell T Expansion SFM, or X-VIVO 15 media for 4 weeks, supplemented weekly with frozen PBMCs from the same donor. IL7R variants expressing expanded transduced T cells are cloned by FACS sorting, and the IL7R construct sequences are identified by RT-PCR product sequencing. The 243 InsPPCL (PPCL) variant (SEQ ID NO:82) is selected for further evolution to generate a conditionally active CAR.

EXAMPLE 6. SCREENING OF ACCESSORY COMPONENTS FOR CAR-T ACTIVATION AND PROPAGATION.

[0498] A series of protein-coding domains (ABCG1, SOCS1, SMAD2, TGFBR2, cCBL, and PD1) and miRNA sequences is constructed for incorporation into a synthetic intron on the reverse strand of a CD3 promoter-driven CAR cassette. Each construct containing the CD3 promoter-driven CAR cassette and a protein-coding domain or miRNA sequence includes a unique barcode for deep sequencing and is assembled using Gibson assembly followed by library transformation and expansion in E. coli. Viral stocks are produced and used to transduce CD3/CD28-stimulated T cells in AIM V, CTS OpTmizer T Cell Expansion SFM, or X-VIVO 15 IL2-free media and allowed to proliferate for 4 weeks in culture with serial DNA sampling for amplification and deep sequencing for barcode identification. The library is also subjected to PACBio full-length sequencing to determine library diversity and to decode barcode components. miRNA sequences and protein-coding domains are tested for synergistic activation of CAR CD3Z domains.

EXAMPLE 7. GENETIC MODIFICATION OF A RETROVIRAL TRANSDUCTION AND PACKAGING SYSTEM TO TARGET RESTING T CELLS FOR SELECTIVE T CELL INTEGRATION AND PBMC EXPRESSION.

[0499] Although the production of high-titer lentiviral vectors by temporary transfection is possible, this method carries the risk of generating non-replicating competent retroviruses (RCRs) and is not scalable for clinical applications. In the present paper, a stable retroviral packaging cell line is generated by the simultaneous introduction of multiple constructs encoding inducible promoters and their regulators into HEK293 suspension-adapted (HEK293S) cells to stably produce viral components, CAR genes, and their regulatory components. Two distinct inducible systems can be used to temporarily control gene expression. One system is based on rapamycin- or rapalog-induced dimerization of two transcription factors. One transcription factor consists of three copies of the FKPB protein fused to a ZFHD1 DNA-binding domain, and the other transcription factor consists of an FRB protein fused to a p65 activation domain. Rapamycin or rapalog dimerizes transcription factors to form ZFHD1/p65 AD and can activate gene transcription at 12xZFHD1 binding sites.

[0500] A series of vectors as shown in Figures 3A to 3E is generated with flanking transposon sequences for integration into the HEK293S genome. Once integrated into a cell's genome, these sequences function as regulatory components and lox and/or FRT sites for subsequent integration using Cre and/or flp recombinases, herein referred to as depot blocks. The initial 5 constructs contain polynucleotide sequences encoding puromycin resistance, GFP, RFP, and an extracellular MYC tag that is targeted to the cell membrane via an N-terminal PLss (bovine prolactin signaling peptide) and anchored to the cell membrane via a C-terminal transmembrane anchoring domain of platelet-derived growth factor receptor (PDGFR). The initial 5 constructs may also include constitutive minimal CMV and minimal IL-2 promoters, a rapamycin-regulated ZFHD1-based promoter, a tetracycline-responsive element (TRE) promoter, or a bidirectional TRE promoter (BiTRE). The construct in Figure 3A contains a polynucleotide sequence encoding the FRB domain fused to the NFKB p65 activator domain (p65 AD) and the ZFHD1 DNA-binding domain fused to three FKBP repeats that is constitutively expressed. The construct in Figure 3A also includes HIV1 REV and HSV VP65 domain SrcFlagVpx under the rapamycin-inducible ZFHD1/p65 AD promoter. The construct in Figure 3B includes a polynucleotide encoding an rtTA sequence under the control of the ZFHD1/p65 AD promoter. The construct in Figure 3C includes a polynucleotide encoding a puromycin resistance gene flanked by loxP sites and the extracellular MYC tag flanked by lox2272 sites. Both of these selectable markers are under the control of a BiTRE promoter, which is flanked by FRT sites. The construct in Figure 3D includes a polynucleotide encoding GFP flanked by loxP sites that is under the control of a TRE promoter. The construct in Figure 3D also includes a single FRT site between the TRE promoter and the 5' loxP site of GFP. The construct in Figure 3E includes a polynucleotide encoding RFP flanked by loxP sites that is under the control of the ZFHD1/p65 AD promoter. The construct in Figure 3E also includes a single FRT site between the ZFHD1/p65 AD promoter and the 5' loxP site of RFP. The constructs in Figures 3C to 3E function as storage blocks for other polynucleotide sequences for insertion into the genome of the packaging cell line. The polynucleotide sequences to be inserted can be flanked by lox sites and inserted into the genome using Cre recombinase and loxP sites. This results in the simultaneous insertion and removal of genomic regions encoding puromycin resistance, the extracellular MYC tag, GFP, and RFP. Alternatively, the polynucleotide sequences can be flanked by FRT sites and inserted into the genome using flp recombinase and FRT sites followed by the removal of polynucleotide sequences encoding puromycin resistance, the extracellular MYC tag, GFP, and RFP using Cre recombinase.

[0501] To generate the packaging cell line with genome-integrated storage blocks, HEK293S cells are co-transfected with equimolar concentrations of the 5 plasmids (Figures 3A to 3E) plus 5 μg of in vitro transcribed piggybac transposase mRNA or 5 μg of a plasmid with a promoter to express piggybac transposase in the presence of PEI at a 2:1 or 3:1 ratio between PEI and DNA (w/w) or 2 to 5 μg of piggybac transposase protein using a cationic peptide mixture. Transfected cells are screened with puromycin in the presence of 100 nm rapamycin and 1 µg/ml doxycycline for 2 to 5 days followed by fluorescence-activated cell sorting to collect cells expressing GFP and RFP. The sorted cells are cultured for 5 days in the absence of puromycin, rapamycin, and doxycycline, and cells expressing GFP and RFP are removed. Cells positive for myc are also removed with myc microspheres. Individual clones of negatively sorted cells are then screened for GFP and RFP induction by rapamycin and doxycycline, and single-cell cloning occurs. DNA from clones is collected and sequenced for integration analysis. Clones positive for strong inducible expression of GFP and RFP in the presence of rapamycin and doxycycline, but with limited background expression in the absence of rapamycin and doxycycline, are expanded and deposited.

[0502] HEK293S cells with the constructs from Figures 3A to 3E integrated into the genome are then transfected with a construct containing a tricistronic polynucleotide encoding a DAF/scFVFc anti-CD3 (UCHT1)/CD14 GPI anchor binding site signaling sequence (SEQ ID NO:252), a DAF/CD80 extracellular domain signaling sequence capable of binding to CD28/CD16B GPI anchor binding site (SEQ ID NO:253), and a DAF/IL-7/DAF signaling sequence (SEQ ID NO:107) and transposon sequences flanking the polynucleotide region for integration into the HEK293S genome (Figure 4A). Following transfection, cells are expanded for 2 days in the absence of rapamycin and doxycycline, and colonies that are constitutively red are selected. Positive colonies are then transiently transfected with a construct to express Cre recombinase to remove the remaining genomic DNA and the RFP coding region. Another construct (Figure 4B) containing a polynucleotide with a BiTRE promoter and a polynucleotide region encoding gag and pol polypeptides in one direction and a polynucleotide region encoding the F and H proteins of the measles virus in the other direction is transfected simultaneously. Cre recombinase integrates the construct into the genome to generate the integrated sequence shown in Figure 4B. The resulting colonies are assessed for protein expression in the presence of doxycycline and rapamycin and analyzed by deep sequencing for genomic integration. The remaining TRE-responsive GFP site is retained for lentiviral genome insertion.

EXAMPLE 8. LENTIVIRUS VECTOR GENERATION AND RETROVIRAL PACKAGING.

[0503] The stable retroviral packaging cell line generated in Example 7 is transfected with a construct (Figure 4C) to express Flp recombinase and a construct containing a polynucleotide sequence encoding a CAR and the lymphoproliferative element IL7Rα-insPPCL under the control of a CD3Z promoter that is not active in HEK293S cells, wherein the CAR and IL7Rα-insPPCL are separated by a polynucleotide sequence encoding a T2A ribosome jump sequence and the IL7Rα-insPPCL has an acyclovir riboswitch-controlled ribozyme. The CAR-containing construct additionally includes cPPT/CTS and RRE sequences and a polynucleotide sequence encoding HIV-1 Psi. The entire polynucleotide sequence in the CAR-containing construct to be integrated into the genome is flanked by FRT sites. Successful integration of the CAR-containing construct causes constitutive GFP expression, which is subsequently removed by temporary transfection with a construct to express Cre recombinase. The HEK293S cell line is cultured in serum-free medium. After growth to peak cell density in a stirred tank reactor, cells are diluted to a peak cell density of 70% and treated with 100 nM rapamycin for 2 days to induce expression of early genes REV, Vpx, and aCD3 scFv CD16B GPI, aCD28 scFv CD16B GPI, and IL-7 SD GPI DAF, followed by the addition of 1 µg/ml doxycycline to the medium to induce expression of structural elements such as Gag Pol, MV(Ed)-FΔ30, MV(Ed)-HΔ18, and the lentiviral genome, including the therapeutic target. Virus production levels are examined by qPCR of the packaging sequence and p24 ELISA. The virus is collected by deep filtration of cells and concentration/diafiltration using a TFF cartridge followed by rapid freezing for vial placement.

EXAMPLE 9. ISOLATION, TRANSDUCTION AND EXPANSION OF PERIPHERAL BLOOD MONONUCLEAR CELLS (PBMCs).

[0504] The following example illustrates the use of a closed system for ex vivo processing of PBMCs prior to in vivo expansion. As an example, 30 to 200 ml of human blood is drained from a subject with Acid Dextrose Citrate Solution (ACD) as an anticoagulant into a blood collection bag. Alternatively, the blood is drained into Vacutainer tubes, a syringe, or equivalent and transferred to an empty IV or blood collection bag. The whole blood is processed using a Neat Cell kit (catalog number CS-900.2, Omniamed) in a Sepax 2 (BioSafe) cell processing system according to the manufacturer's instructions. Peripheral blood mononuclear cells (PBMCs) are collected either into a culture bag or, alternatively, a syringe. An aliquot is aseptically withdrawn for cell counting to determine the number of viable cells. PBMCs are transferred to a G-Rex100MCS Gas Permeable Cell Culture System (Wilson Wolf) at a final concentration of 0.1 to 1.0 x 10⁶ viable cells/ml in X-VIVO 15 media (catalog number 08-879H, Lonza) or CTS Optimizer Cell Expansion SFM media (catalog number A1048501, Thermo Fisher Scientific) with 10 to 300 IU/ml of IL-2 (catalog number 202-IL-010, R&D Systems) to a final volume of up to 200 ml. In addition to IL-2, CTS Immune Cell SR (catalog number A2596101, Thermo Fisher Scientific) can be added to the medium. The closed G-Rex Gas Permeable Cell Culture System device can be pre-coated with Retronectin (catalog number CH-296, Takara) or a similar fibronectin-derived equivalent, according to the manufacturer's instructions.

[0505] PBMCs isolated from peripheral blood are loaded onto a PBMC PALL filter, washed once through the filter with 10 ml of AIM V media (Thermo Fisher Scientific) or X-VIVO 15 followed by infusion with 10 to 60 ml of lentivirus stock (as prepared in Example 8) at 37 °C at 5 ml/h. The PBMCs are then washed again with AIM V media, CTS Optimizer T Cell Expansion SFM, or X-VIVO 15 containing recombinant human DNase (Pulmozyme, Genentech) followed by a wash with DNase-free Lactated Ringers (catalog number L7500, Braun). The PBMCs are then reverse perfused through the filter into a syringe. The cells (target cell levels are 5 x 10⁵ to 1 x 10⁶ cells/kg) are then reinfused into the subject via intravenous infusion.

[0506] Depending on the riboswitch contained within the retroviral genome, the subject receives the respective nucleoside analog antiviral drug or nucleoside analog antiviral prodrug (acyclovir, valacyclovir, penciclovir, or famciclovir). Subjects may receive any therapeutically effective dose, such as 500 mg of the nucleoside analog antiviral drug or prodrug orally three times daily. Treatment with the nucleoside analog antiviral drug or prodrug preferably begins before reinfusion, such as 2 hours before, and may also begin at the time of reinfusion or at some time after reinfusion. Treatment may continue for at least 1, 2, 3, 4, 5, 7, 10, 14, 21, 28, 30, 60, 90, 120 days or 5, 6, 9, 12, 24, 36, or 48 months or more. Treatment may include administration of the nucleoside analog antiviral drug or prodrug once, twice, three, or four times daily. After reinfusion and treatment are initiated, the number of infected cells is determined by blood counts on days 2, 5, 7, 11, 13, 18, 28, and 56 post-reinfusion using qPCR to quantify the amount of viral genome. A subject who experiences fever or cytokine release syndrome may have the dose or frequency of the nucleoside analog antiviral drug or prodrug reduced or discontinued. If infected T cells fail to amplify 10,000 to 100,000 times by day 18, the dose or frequency of the nucleoside analog antiviral drug or prodrug may be increased. The subject's clinical response can be measured using serial FDG PET imaging and CT scans. The oral dosage of the nucleoside analog antiviral drug or prodrug may be reduced or discontinued after prolonged remission or in the case of excessive T cell spread beyond 30% of total peripheral T cell counts.

EXAMPLE 10: THERAPEUTIC INTERVENTION TO RAISE VASCULAR OR TISSUE PH.

[0507] To reduce the binding of an antigen-binding domain to its cognate antigen, NaHCO3 is administered as an IV bolus or by IV infusion. The standard dosage is 1 mg/kg of body weight as the initial dose followed by 0.5 mg/kg every 10 minutes. A 50 milliliter bolus of NaHCO3 will raise the serum pH by approximately 0.1 pH unit. If the pH is 7.0, it requires four 50 mEq ampoules of NaHCO3 to correct the pH to 7.40.

EXAMPLE 11: ACTIVITY TEST OF IL-7 RECEPTOR SURVIVAL/LYMPHOPROLIFERATIVE ELEMENTS IN PBMCs

[0508] To test IL-7Rα variants for their ability to mediate antigen-independent T-cell survival, thirty milliliters of human blood were drained with acid dextrose citrate (ACD) as an anticoagulant in Vacutainer tubes. Whole blood was processed using density gradient centrifugation with Ficoll-Pacque™ (General Electric) following the manufacturer's instructions to obtain peripheral blood mononuclear cells (PBMCs). Aliquots of PBMCs were aseptically transferred to wells of a 12-well tissue culture plate, along with X-Vivo™ 15 medium (Lonza) at a final concentration of 0.5 million viable cells/ml in a final volume of 1 ml. Recombinant human interleukin-2 (IL-2) (Novoprotein) was also added at a concentration of 100 IU/ml in some samples. Anti-CD3 activating antibody (OKT3, Novoprotein) was added at a concentration of 50 ng/ml to activate PBMCs for viral transduction. Plates were incubated overnight in a standard humidified tissue culture incubator at 37°C and 5% carbon dioxide. After overnight incubation, lentivirus particle preparations containing the desired test constructs (Figure 19A) were added to individual wells at a multiplicity of infection (MOI) of 5. The plate was incubated overnight in a standard humidified tissue culture incubator at 37°C and 5% carbon dioxide. After overnight incubation, the contents of each well of the 12-well plate were collected and centrifuged to obtain a pellet. The samples were washed once with D-PBS + 2% Human Serum Albumin (HSA), resuspended in X-Vivo15™ medium, and transferred to wells of a 6-well gas-permeable G-Rex® cell culture device (Wilson Wolf). More X-Vivo™ 15 medium was added to bring the final volume of each well to 30 ml. Corresponding control samples for each of the constructs were transferred to wells of a 6-well gas-permeable G-Rex® cell culture device (Wilson Wolf), and more medium was added to bring the final volume to 30 ml with 100 IU/ml of IL-2 for some control samples. The G-Rex® device was incubated in a standard humidified tissue culture incubator at 37°C and 5% Carbon Dioxide for 7 days. Fresh IL-2 was added to control samples containing IL-2 during culture every 2 to 3 days. The correlated test samples without IL-2 were not supplemented. Samples were removed to track cell numbers and viability during expansion (Countess, Thermo Fisher) on day 7.

[0509] Figure 19A provides a schematic drawing of the IL7Rα constructs that were tested. These constructs were inserted into a recombinant lentiviral genome. Recombinant retroviruses were used to transduce PBMCs. Figure 19A shows a schematic drawing of wild-type IL7Rα (SEQ ID NO:229), which consists of a signaling sequence (SS), an extracellular domain (ECD), a transmembrane (TM), and an intracellular domain (ICD). “1” indicates the site of a fibronectin type III domain; “2” indicates the site of a WSXWS motif; “3” indicates a Box 1 site, “4” indicates the site of a protein kinase C (PKC) phosphorylation site, and “5” indicates a Box 2 site.

[0510] Variant “A” is IL-7Rα with an InsPPCL at position 243 (Shochat et al 2011, J. Exp. Med. Volume 208 no 5 901 to 908), but without the S185C mutation, expressed in a transcript with a GFP polypeptide, a GSG binder, and a P2A ribosome jump sequence fused to its N-terminus. Variant “B” is IL-7Rα InsPPCL with a GFP polypeptide, a GSG binder, and a P2A ribosome jump sequence fused to its N-terminus, as well as a Myc tag between the signaling sequence and the extracellular domain. Variant “C” is similar to variant “B” except that its intracellular domain is truncated at position 292. Variant “D” is similar to variant “A” except that its intracellular domain is truncated at position 292. Variant “E” is the IL-7Rα InsPPCL variant truncated at its N-terminus so that the signaling sequence and most of the extracellular domain (residues 1 to 228) are not present; variant “E” also has a GFP polypeptide, a GSG binder, a P2A ribosome jump sequence, and an eTag fused to the N-terminus, in that order from the amino terminus. The amino acid residue numbering is based on IL7Rα (NCBI GI No. 002176.2). T cells containing each of the variants were tested for viability in the presence or absence of IL-2 using Trypan Blue exclusion.

[0511] As shown in Figure 19B, PBMCs require IL-2 for in vitro survival. As illustrated in Figure 19B, non-transfected PBMCs have approximately 80% viability in the presence of IL-2 and 0% viability in the absence of IL-2. PBMCs that have the full-length versions of IL-7Rα InsPPCL (IL-7Rα variants A and B in Figure 19A) had more than 20% viability in the absence of IL-2, indicating that the expression of the constitutively active IL-7Rα InsPPCL receptor has survival activity in these cells. Furthermore, T cells expressing IL-7Rα InsPPCL variants with a truncated intracellular domain (ICD) (IL-7Rα variants C and D in Figure 19A) had increased viability compared to the wild-type IL-7 receptor. Finally, the N-terminal IL-7 receptor mutant (IL-7Rα variant E in Figure 19A) as shown in Figure 19B had survival activity in these cells. Consequently, this example illustrates that the IL-7 receptor had survival activity when expressed in PBMCs.

EXAMPLE 12. TRANSDUCTION EFFICACY OF RECENTLY ISOLATED UNSTIMULATED HUMAN T CELLS BY MeVpp

[0512] Lentiviruses were produced by temporary transfection of 293T cells (Lenti-X 293T, Clontech) with lentiviral expression vectors. The cells were adapted to suspension culture by serial proliferation in Freestyle 293 Expression Medium (ThermoFisher Scientific). The cells in suspension were inoculated at 1 x 10⁶ cells/ml (30 ml) into a 125 ml Erlenmeyer flask and immediately transfected using PEI (Polysciences) dissolved in weak acid.

[0513] Plasmid DNA was diluted in 1.5 ml of Optimem medium per 30 ml of cells. For VSV-G pseudoparticles, the total DNA (1 μg/ml culture volume) was a mixture of 4 plasmids with the following molar ratios: genomic plasmid 2x, Rev-containing plasmid 1x, VSVg-containing plasmid 1x, and Gagpol-containing plasmid 1x. For the MV(Ed)-FΔ30/HΔ18 pseudoparticles, the total DNA (1 μg/ml of culture volume) was a mixture of 5 plasmids with the following molar ratios: genomic plasmid 2x, Rev-containing plasmid 1x, (2/3, two-thirds) x MV(Ed)-FΔ30-containing plasmid, (1/3, one-third) x MV(Ed)-HΔ18-containing plasmid and Gagpol-containing 1x plasmid. For the MV(Ed)-FΔ30/HΔ24 pseudoparticles, the total DNA (1 μg/ml culture volume) was a mixture of 5 plasmids with the following molar ratios: genomic plasmid 2x, Rev-containing plasmid 1x, (2/3, two-thirds) x MV(Ed)-FΔ30-containing plasmid, (1/3, one-third) x MV(Ed)-HΔ24-containing plasmid, and Gagpol-containing plasmid 1x. Separately, the PEI was diluted in 1.5 ml of Optimem at 2 μg/ml (culture volume, 2:1 ratio for DNA). After a 5-minute incubation at room temperature, the two solutions were thoroughly mixed and incubated at room temperature for a further 20 minutes. The final volume (3 ml) was added to the cells. The cells were then incubated at 37 °C for 48 hours with rotation at 120 rpm and with 5 to 8% CO2.

[0514] After 48 hours, the supernatants were collected by centrifugation at 1,000 g for 10 minutes. The supernatants were decanted into a fresh tube and % of the supernatant volume in PEG solution (PEG-IT, System Biosciences) was added. Lentiviral pseudotypes were precipitated by overnight incubation at 4 °C followed by centrifugation at 1,500 g for 20 minutes at 4 °C. The supernatant was removed, and the virus was resuspended in a 1:100 volume of PBS. The viruses were titrated by serial dilution and GFP expression in Raji cells, which express both CD46 and SLAM, 48 hours post-transduction, by flow cytometry.

[0515] Enriched peripheral blood T cells were first isolated from a fresh leukocyte cream of blood collected and distributed by the San Diego Blood Bank, CA. Briefly, density gradient separation based on SepMate™ (Stemcell™) of PBMCs on Ficoll-Paque PLUS® (GE Healthcare Life Sciences) was performed according to the manufacturer's instructions. Untouched T cells were then further enriched by negative selection of freshly isolated total PBMCs using the Dynabeads® Untouched T Cell Kit (Invitrogen) and manufacturer's instructions. After isolation, 2,6 E5 unstimulated and freshly isolated enriched peripheral blood T lymphocytes were transduced, in duplicate, with different vectors, at various multiplicities of infection (MOI). Transductions were conducted for 14 h at 37 °C in 100 µl of final 2% RPMI-HIFCS in a 96-well plate. After incubation with the vectors for 14 h, the cells were washed three times with 2% PBS-HIFCS and finally incubated at a cell density of 0.5 E6/ml in 10% RPMI-HIFCS at 37 °C until day 3.

[0516] Three days after transduction with VSV-Gpp or MeVpp, 1E5 cells were collected and analyzed by flow cytometry for GFP expression in the CD3+ cell population. Figure 20 shows a plot of transduction efficacy against MOI for negatively selected and unstimulated T cells. The symbols are misaligned for enhanced clarity. Unstimulated T cells were more effectively transduced using MV(Ed)-FΔ30/MV(Ed)-HΔ18 pseudoparticles (~70 to 80%) compared to VSV-Gpp (~0 to 5%) at an MOI of 1. Unstimulated T cells were also more effectively transduced using MV(Ed)-FΔ30/MV(Ed)-HΔ24 pseudotyping particles at an MOI of 5 (~70 to 80%) compared to VSV-Gpp at an MOI of 6 (~5 to 10%). The transduction efficiency of newly isolated pseudotyped lentivolver unstimulated human T cells is much higher when MeV envelope polypeptides are used for pseudotyping than when VSV-G is used.

EXAMPLE 13. DEMONSTRATION OF THE FUNCTIONALITY OF miRNAs INSERTED INTO THE EF-1ALFA PROMOTER INTRON

[0517] Four separate gBlocks® Gene Fragments were designed, each containing the miR-155 framework. For each gBlock®, a unique miRNA targeting the CD3zeta mRNA transcript was used to replace the miR-155 target sequence. Each gBlock® contained a 40 base pair overlap sequence designed to facilitate the assembly of all four gBlocks® as a single strand at the EF-1alpha promoter intron. The gBlocks® were assembled using a commercial Gibson® ultra assembly kit (NEBuilder, New England Biolabs, Inc.).

[0518] The EF-1alpha promoter and intron A (SEQ ID NO:255) were part of a transgene expression cassette that triggers the expression of GFP and eTag contained in a lentivirus vector backbone (the lentivirus backbone with the exemplary GFP and eTag recognized by cetuximab is referred to herein as F02). The nucleotide positions of each gBlock® and its respective components in SEQ ID NO:255 are denoted in Table 3. The appropriate assembly of four miRNAs in the lentivirus backbone was confirmed by comprehensive sequencing of the EF-1alpha promoter. TABLE 3. NUCLEOTIDE POSITIONS OF RESOURCES IN SEQ ID NO:255

[0519] Lentiviruses containing the four miRNAs targeting CD3zeta were produced by temporary cotransfection of four plasmids into HEK293 cells in suspension: the plasmid containing the four miRNAs targeting the CD3zeta mRNA transcript, a plasmid containing VSV-G, a plasmid containing REV, and a plasmid containing GAG-POL. The viral supernatant was collected after 48 hours and precipitated with PEG for 24 hours. The supernatants were centrifuged, and the pelleted virus was resuspended in IL-2-free complete PBMC growth medium. Viral titers were calculated by 48-hour transduction of Jurkat cells.

[0520] For transduction, PBMCs were thawed on Day 0 and incubated for 24 hours without 100 U/ml of hrIL-2. On Day 1, PBMCs were activated using CD3/CD28 conjugated microspheres. On Day 2, activated PBMCs were transduced with lentivirus containing miRNAs at an MOI of 10. Cells were expanded until Day 11, with fresh hrIL-2 added every two days. On Days 7, 9, and 11, 1 million cells were collected for FACS analysis.

[0521] Cells were stained for CD3 Epsilon surface expression using PE-conjugated OKT-3 antibody (Biolegend). Expression levels were determined by the mean fluorescence intensity (MF) of PE in the GFP-positive population (transduced cells). Expression levels in transduced cells were compared between wild-type virus (F02) and F02 virus containing CD3z miRNAs. Figure 21 shows that miRNAs targeting CD3zeta located in the EF-1alpha promoter intron have the ability to knock down CD3 complex expression.

[0522] The methods, examples, and experiments disclosed are not intended to limit the scope of the disclosure or to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to the numbers used (e.g., quantities, temperature, etc.), but some experimental errors and deviations should be taken into account. It should be understood that variations on the methods as described may be made without altering the fundamental aspects they are intended to illustrate.

[0523] Those skilled in the art may devise many modifications and other modalities within the scope and spirit of the present revelation. Indeed, variations in the materials, methods, designs, experiments, examples, and modalities described may be made by those skilled in the art without altering the fundamental aspects of the present revelation. Any of the modalities revealed may be used in combination with any other modality revealed.

[0524] In some cases, certain concepts have been described with reference to specific embodiments. However, an individual of ordinary skill in the art will realize that various modifications and alterations can be made without departing from the scope of the invention as presented in the claims below. Correspondingly, the descriptive report and figures should be considered in an illustrative rather than restrictive sense, and all such modifications are conceived as being included within the scope of the invention.

Claims (24)

1. Replication-incompetent recombinant lentivirus, CHARACTERIZED in that it comprises: A. a viral envelope protein on its surface; B. an anti-CD3 antibody on its surface, wherein the anti-CD3 antibody is not encoded by a polynucleotide in the recombinant lentivirus; and C. a polynucleotide encoding: i. a first genetically modified signaling polypeptide comprising a cytokine receptor polypeptide, wherein the cytokine receptor polypeptide is an intracellular signaling domain of an IL-7 receptor, an intracellular signaling domain of an IL-12 receptor, an intracellular signaling domain of an IL-15 receptor, or an intracellular signaling domain of an IL-21 receptor; and ii. a second genetically modified signaling polypeptide comprising a chimeric antigen receptor comprising an antigen-specific targeting region, a transmembrane domain, and an intracellular activation domain, wherein the intracellular activation domain comprises a CD3Z intracellular activation domain. 2. Replication-incompetent recombinant lentivirus, according to claim 1, CHARACTERIZED in that the first genetically modified signaling polypeptide is encoded in a reverse orientation with respect to a viral LTR, or an active fragment thereof, in the genome of the recombinant lentivirus. 3. Replication-incompetent recombinant lentivirus, according to any one of claims 1 to 2, CHARACTERIZED in that at least one of the promoters is not active in a packaging strain used to produce the replication-incompetent recombinant lentivirus. 4. Replication-incompetent recombinant lentivirus, according to any one of claims 1 to 3, CHARACTERIZED in that the lentivirus comprises a promoter, wherein the promoter is the EF1a promoter, the MSCV promoter, or the CD3z promoter. 5. Replication-incompetent recombinant lentivirus, according to any one of claims 1 to 4, CHARACTERIZED in that the polynucleotide further comprises an intron. 6. Replication-incompetent recombinant lentivirus according to claim 5, CHARACTERIZED in that the intron encodes an shRNA or one or more miRNAs. 7. Replication-incompetent recombinant lentivirus, according to any one of claims 1 to 5, CHARACTERIZED in that the polynucleotide further encodes one or more inhibitory RNA molecules directed against one or more RNA targets, wherein one or more RNA targets are mRNA transcripts of a gene selected from the group consisting of: PD-1, CTLA4, SOCS, SMAD2, a miR-155 target, IFN gamma, cCBL, TRAIL2, PP2A, ABCG1 and a component of the TCR complex. 8. Replication-incompetent recombinant lentivirus, according to any one of claims 1 to 5, CHARACTERIZED in that the polynucleotide further encodes two or more inhibitory RNA molecules directed against two or more RNA targets, wherein the two or more RNA targets comprise IFN-gamma transcribed mRNA and a component of the TCR complex. 9. Replication-incompetent recombinant lentivirus, according to any one of claims 1 to 8, CHARACTERIZED in that the first genetically modified signaling polypeptide is capable of activating a Jak pathway. 10. Replication-incompetent recombinant lentivirus, according to any one of claims 1 to 9, CHARACTERIZED in that the first genetically modified signaling polypeptide is capable of activating a STAT3 pathway, a STAT4 pathway, or a STAT5 pathway. 11. Replication-incompetent recombinant lentivirus, according to any one of claims 1 to 10, CHARACTERIZED in that the second genetically modified signaling polypeptide further comprises a costimulatory domain, wherein the costimulatory domain is selected from the intracellular portion of 4-IBB (CD137), CD27, CD28, CD28 deleted for binding to Lck (ICΔ), CD30, ICOS, OX40, BTLA, GITR and HVEM. 12. Replication-incompetent recombinant lentivirus, according to any one of claims 1 to 11, CHARACTERIZED in that the signaling domain of the cytokine receptor polypeptide is the intracellular signaling domain of an IL-15 receptor, wherein the IL-15 receptor is IL-2/IL-15Rβ. 13. Replication-incompetent recombinant lentivirus, according to any one of claims 1 to 11, CHARACTERIZED in that the signaling domain of the cytokine receptor polypeptide is the intracellular signaling domain of an IL-15 receptor, wherein the IL-15 receptor is the common gamma (Y) chain. 14. Replication-incompetent recombinant lentivirus, according to any one of claims 1 to 11, CHARACTERIZED in that the signaling domain of the cytokine receptor polypeptide is the intracellular signaling domain of an IL-7 receptor, wherein the IL-7 receptor is IL-7Rα. 15. Replication-incompetent recombinant lentivirus, according to any one of claims 1 to 14, CHARACTERIZED in that the first genetically modified signaling polypeptide is constitutively active. 16. Replication-incompetent recombinant lentivirus, according to claim 15, CHARACTERIZED in that the first constitutively active genetically modified signaling polypeptide is an IL-7Rα mutant, or a functional fragment thereof. 17. Replication-incompetent recombinant lentivirus, according to claim 16, CHARACTERIZED in that the constitutively active lymphoproliferative element is a functional fragment of an IL-7Rα mutant comprising amino acids 229 to 292 of SEQ ID NO:229 with an insertion of the amino acids PPCL at position 243. 18. Replication-incompetent recombinant lentivirus, according to any one of claims 1 to 17, CHARACTERIZED in that the expression of the lymphoproliferative element is regulated by a control element in vivo. 19. Replication-incompetent recombinant lentivirus, according to any one of claims 1 to 18, CHARACTERIZED in that it further comprises a polypeptide capable of binding to CD28 selected from an anti-CD28, CD80, CD86 antibody or a fragment thereof that retains the ability to bind to CD28. 20. Replication-incompetent recombinant lentivirus, according to any one of claims 1 to 19, CHARACTERIZED in that the anti-CD3 antibody is an anti-CD3 scFv. 21. Replication-incompetent recombinant lentivirus, according to any one of claims 1 to 20, CHARACTERIZED in that the anti-CD3 antibody is a GPI-anchored anti-CD3scFvFc. 22. Replication-incompetent recombinant lentivirus, according to any one of claims 1 to 21, CHARACTERIZED in that the viral envelope protein comprises an envelope protein of vesicular stomatitis virus (VSV-G), an envelope protein of feline endogenous virus (RD114), a polypeptide of Measles Virus F, a polypeptide of Measles Virus H, or a functional fragment thereof. 23. Replication-incompetent recombinant lentivirus, according to any one of claims 1 to 22, CHARACTERIZED in that at least one of the viral envelope proteins comprises anti-CD3 antibody. 24. Method for genetically modifying T cells, CHARACTERIZED in that it comprises: placing peripheral blood mononuclear cells (PBMCs) comprising T cells ex vivo in contact with recombinant lentiviruses that are incompetent in replication to form a transduction reaction mixture, wherein the recombinant lentiviruses that are incompetent in replication comprise: A. a viral envelope protein on their surface; B. an anti-CD3 antibody on their surface; and C. a polynucleotide encoding a first genetically modified signaling polypeptide comprising a cytokine receptor polypeptide, wherein the contact occurs for between 15 minutes and 14 hours before the T cells are removed by washing from the transduction reaction mixture, and wherein said contact facilitates the fusion of the recombinant lentiviruses that are incompetent in replication to the T cells to produce genetically modified T cells.