阅读说明：本技术 嵌合抗原受体细胞的制备及其用途 (Preparation of chimeric antigen receptor cells and uses thereof ) 是由 肖磊 蒲程飞 曹志远 吴昭 于 2018-05-30 设计创作，主要内容包括：本文描述的实施方案涉及包含基因修饰的CAR细胞的组合物及其用于治疗癌症的用途。本公开的一些实施方案涉及用于T细胞应答增强和/或CAR细胞制备的组合物和方法。例如,方法可包括获得包含CAR的细胞,并在所述CAR的细胞外结构域识别的试剂存在下培养所述细胞。(Embodiments described herein relate to compositions comprising genetically modified CAR cells and their use for treating cancer. Some embodiments of the present disclosure relate to compositions and methods for T cell response enhancement and/or CAR cell preparation. For example, a method can include obtaining a cell comprising a CAR and culturing the cell in the presence of an agent recognized by the extracellular domain of the CAR.)

1. A method, comprising:

providing a cell comprising a Chimeric Antigen Receptor (CAR); and

culturing the cell in the presence of an agent that binds to the extracellular domain of the CAR to obtain a CAR cell.

2. The method of claim 1, further comprising:

introducing into the cell a nucleic acid sequence encoding a CAR and a nucleic acid sequence encoding hTERT or SV40LT or a combination thereof.

3. The method of claim 1 or 2, wherein the agent binds the extracellular domain of the CAR and mediates a response of the cell comprising the CAR or the agent is an extracellular domain of an antigen to which the extracellular domain of the CAR binds.

4. The method of claim 3, wherein the antigen is Epidermal Growth Factor Receptor (EGFR), variant III of epidermal growth factor receptor (EGFRvIII), human epidermal growth factor receptor 2(HER2), Mesothelin (MSLN), prostate-specific membrane antigen (PSMA), carcinoembryonic antigen (CEA), bisialoganglioside 2(GD2), interleukin-13 Ra2(IL13R α 2), glypican-3 (GPC3), Carbonic Anhydrase IX (CAIX), L1 cell adhesion molecule (L1-CAM), cancer antigen 125(CA125), cluster of differentiation 133(CD133), Fibroblast Activation Protein (FAP), cancer/testis antigen 1B (CTAG1B), mucin 1(MUC1), folate receptor α (FR- α), CD19, FZD10, HR TSLR, PRLR, Muc17, GUCY2C, CD207, CD3, CD5, CD B Cell Maturation Antigen (BCMA) or CD 4.

5. The method of claim 1 or 2, wherein the ratio of the amount of the agent to the number of the CAR cells after culturing with the agent is 1:5000 to 1:5(μ g/10)⁴Individual cells).

6. The method of claim 1 or 2, wherein the concentration of the agent in the culture medium is from 2 to 10⁴ng/ml。

7. The method of claim 1 or 2, wherein providing the T cell comprising the CAR comprises culturing the T cell without the agent for at least 8 days after introducing the vector comprising the nucleic acid sequence encoding the CAR into the T cell, and culturing the T cell in the presence of the agent comprises culturing the T cell after the at least 8 days.

8. The method of claim 1 or 2, wherein the ratio of the number of the CAR cells expressing the CAR to the cells not expressing the CAR is greater than the ratio when the cells are cultured without the agent.

9. The method of claim 1 or 2, wherein integration of the nucleic acid sequence encoding hTERT, nucleic acid encoding SV40LT, or a combination thereof comprises genomic integration of the nucleic acid sequence encoding hTERT, nucleic acid encoding SV40LT, or a combination thereof and constitutive expression of hTERT, SV40 LT-encoding nucleic acid, or a combination thereof.

10. The method of claim 1 or 2, wherein expression of the nucleic acid sequence encoding hTERT or SV40LT, or a combination thereof, is modulated by an inducible expression system.

11. The method of claim 1 or 2, further comprising:

introducing a nucleic acid sequence encoding a suicide gene into the cell, and culturing the cell with a nucleoside analog in a manner that allows expression of the suicide gene, such that the nucleoside analog is cytotoxic to the CAR cell.

12. The method of claim 1 or 2, wherein the CAR comprises an extracellular domain, a spacer domain, a transmembrane domain, and an intracellular domain.

13. The method of claim 12, wherein the spacer domain of the CAR comprises the amino acid sequence of SEQ ID No.:68 or 69, or the transmembrane domain of the CAR comprises the amino acid sequence of SEQ ID No.:72 or 75 and the spacer domain of the CAR comprises the amino acid sequence of SEQ ID No.: 68.

14. The method of claim 1 or 2, wherein the agent comprises the extracellular domain of at least one of CD19, FZD10, TSHR, PRLR, Muc17, GUCY2C, CD207, CD3, CD5, or CD4, or at least one of amino acids SEQ ID: 18-27.

15. The method of claim 1 or 2, wherein the agent comprises at least one of the extracellular domains of GCC, B7-H4, Prostate Specific Membrane Antigen (PSMA), carcinoembryonic antigen (CEA), IL13R α, her-2, CD19, CD20, CD22, CD123, NY-ES0-1, HIV-I Gag, Lewis Y, Mart-I, gplOO, tyrosinase, WT-I, H TERI, MUC16, mesothelin, MIC-A, MIC-B, estrogen, progesterone, RON, or one or more members of the ULBP/RAETl family.

16. The method of any one of claims 1-15, wherein the cell is a T cell or a Natural Killer (NK) cell.

17. A pharmaceutical composition comprising the CAR cell obtained by the method of any one of claims 1-16.

18.A modified T cell comprising a nucleic acid sequence encoding hTERT or a nucleic acid encoding SV40LT or a combination thereof, wherein the nucleic acid sequence encoding hTERT or a nucleic acid encoding SV40LT or a combination thereof is integrated into the genome of the modified T cell and the modified T cell constitutively expresses hTERT, SV40LT or a combination thereof.

19. The modified T cell of claim 18, wherein:

the modified T cell further comprises a nucleic acid sequence encoding a CAR;

the modified T cell is capable of inhibiting a cell expressing an antigen to which the CAR binds; and is

The CAR and the nucleic acid sequence encoding hTERT or SV40LT, or a combination thereof, are expressed as gene products as separate polypeptides.

20. The modified T cell of claim 18, wherein expression of the nucleic acid sequence encoding hTERT, the nucleic acid encoding SV40LT, or a combination thereof is modulated by an inducible expression system, and/or the modified T cell comprises a nucleic acid sequence encoding a suicide gene.

Technical Field

The present disclosure relates to modified cells, in particular, to compositions comprising modified cells and their use for treating diseases and conditions.

Background

Over 25 years ago, scientists developed Chimeric Antigen Receptors (CARs) for expression on T cells. Chimeric Antigen Receptor (CAR) technology binds the antigen recognition domain of a particular antibody to the intracellular domain of a T Cell Receptor (TCR). T cells genetically modified to target certain malignancies have shown good clinical results. During CAR T cell therapy, the physician will draw blood from the patient and harvest its cytotoxic T cells. The cells were then re-engineered in the laboratory so they could know how to attack each patient's specific cancer. Patients are usually chemotherapy prior to or during CAR T cell therapy to eliminate some of their existing immune cells. However, chemotherapy can result in a significant reduction in T cells in patients. Although most patients will recover within 9 months and their immune cells will reach pre-chemotherapy levels, some patients may not be able to generate sufficient T cells for continued CAR T cell therapy. This puts the life of these patients at risk. Furthermore, for CAR T therapy, long-term maintenance of CAR T cells in patients is important for patient prognosis in tumor treatment. For example, if the long-term presence of CAR T cells can be maintained, the technique can be effective in reducing tumor recurrence.

Disclosure of Invention

Embodiments described herein relate to compositions comprising genetically modified CAR cells and their use for treating diseases and conditions.

Some embodiments of the present disclosure relate to a method comprising: providing a cell; introducing into a cell a nucleic acid sequence encoding a CAR and a nucleic acid sequence encoding hTERT, SV40LT, or a combination thereof; and culturing the cell in the presence of an agent recognized by the extracellular domain of the CAR, thereby producing the modified CAR cell.

In some embodiments, the integration of the nucleic acid sequence encoding hTERT, the nucleic acid encoding SV40LT, or a combination thereof comprises genomic integration of the nucleic acid sequence encoding hTERT, the nucleic acid encoding SV40LT, or a combination thereof and constitutive expression of hTERT, SV40LT, or a combination thereof. In some embodiments, the expression of hTERT, SV40LT, or a combination thereof is modulated by an inducible expression system. In some embodiments, the method can further comprise introducing a nucleic acid sequence encoding a suicide gene into the cell, and culturing the CAR cell comprising the suicide gene and the nucleic acid encoding the CAR with a nucleoside analog in a manner that allows expression of the suicide gene such that the nucleoside analog is cytotoxic. In some embodiments, the cell is a T cell or a Natural Killer (NK) cell.

In some embodiments, the CAR comprises an extracellular domain, a transmembrane domain, and an intracellular domain. In some embodiments, the intracellular domain comprises a costimulatory signaling domain including an intracellular domain of a costimulatory molecule selected from the group consisting of: CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and any combination thereof.

In some embodiments, the antigen is Epidermal Growth Factor Receptor (EGFR), variant iii of epidermal growth factor receptor (egfrviii), human epidermal growth factor receptor 2(HER2), Mesothelin (MSLN), prostate-specific membrane antigen (PSMA), carcinoembryonic antigen (CEA), bis-sialylganglioside 2(GD2), interleukin-13 Ra2(IL13R α), glypican-3 (GPC3), carbonic anhydrase ix (ix cai), L1 cell adhesion molecule (L1-CAM), cancer antigen 125(CA125), cluster of differentiation 133(CD133), Fibroblast Activation Protein (FAP), cancer/testis antigen 1B (CTAG1B), mucin 1 (mulr B), MUC1 c1, tslr 3984, CD 9617, CD 9636, CD 369685, CD 369617, CD 3619, CD 369625, CD 369617, CD 3643, CD 369685, CD.

In some embodiments, the agent is an antibody that binds the extracellular domain of the CAR. In some embodiments, the antibody is a human IgG antibody and/or a Fab fragment that binds human IgG. In some embodiments, the modulatory compound comprises an extracellular domain of at least one of CD19, FZD10, TSHR, PRLR, Muc17, GUCY2C, CD207, CD3, CD5, or CD 4. In some embodiments, the modulating compound comprises the amino acid sequence: 41-47 of at least one of SEQ ID. In some embodiments, the modulating compound binds to the amino acid sequence: 21 and at least one of SEQ ID Nos. 48 to 53. In some embodiments, the CAR cell comprises at least one of SEQ ID Nos. 38, 35, 39, and 40.

In some embodiments, the CAR cells cultured in the presence of the agent exhibit an increase in cell growth of about 1.5 to 2-fold compared to CAR cells cultured in the absence of the agent. In some embodiments, the CAR cells cultured in the presence of the agent exhibit an increase in cell growth of about 1.5 to 3-fold compared to CAR cells cultured in the absence of the agent. In some embodiments, the CAR cells cultured in the presence of the agent exhibit about a 2-fold increase in cell growth compared to CAR cells cultured in the absence of the agent. In some embodiments, the cell density of the CAR cells in the culture medium is at least about 25 x 10⁴Individual cells/mL cell culture medium. In some embodiments, the cell density of the CAR cells in the culture medium is less than about 200 x 10⁴Individual cells/mL cell culture medium. In some embodiments, the cell density of the CAR cells in the culture medium is at about 50 x 10⁴To about 200X 10⁴Between cells/mL cell culture medium. In some embodiments, the cell density of the CAR cells in the culture medium is at about 50 x 10⁴To about 100X 10⁴Individual cell/mL cellBetween the culture media.

In some embodiments, the CAR cell is sensitive to tetracycline from the cell culture medium. In some embodiments, the CAR cell comprises a third nucleic acid sequence encoding an inverse tetracycline transactivator (rtTA). In some embodiments, the expression of hTERT or SV40LT is regulated by rtTA such that hTERT or SV40LT is expressed in the presence of tetracycline. In some embodiments, the concentration of tetracycline in the cell culture medium is not less than about 2 μ g/ml. In some embodiments, the tetracycline is selected from the group consisting of: tetracycline, demeclocycline, meclocycline, doxycycline, lymecycline, methacycline, minocycline, oxytetracycline, rolicycline, and chlortetracycline. In some embodiments, the tetracycline is doxycycline.

In some embodiments, the CAR cell comprises a fourth nucleic acid sequence encoding a suicide gene such that the nucleoside analog is cytotoxic to the CAR cell when the CAR cell is cultured in the presence of the nucleoside analog in a manner that allows expression of the suicide gene, hi some embodiments, the suicide gene is selected from the group consisting of thymidine kinase of herpes simplex virus, thymidine kinase of varicella zoster virus, and bacterial cytosine deaminase. in some embodiments, the suicide gene is thymidine kinase of herpes simplex virus. in some embodiments, the nucleoside analog is selected from the group consisting of ganciclovir, acyclovir, penciclovir, valacyclovir, trifluorothymidine, 1- [ 2-deoxy, 2-fluoro, β -D-arabinofuranosyl ] -5-iodouracil, arabinoadenosine (ara-A), arabinothymidine (araT)1- β -D-arabinofuranosyl thymine, 5-ethyl-2 '-amino-5' -diamino-5 '-amino-5' -iodouridine, arabinoside, and in some embodiments, the nucleoside analog is AZaD-uridine.

Some embodiments relate to an isolated cell obtained using the methods described herein. Some embodiments relate to a composition comprising an isolated population of cells. Some embodiments relate to a method of enhancing a T cell response in a subject and/or treating a tumor in a subject, the method comprising: administering an effective amount of a composition described herein.

Some embodiments relate to a modified cell comprising a nucleic acid sequence encoding hTERT, a nucleic acid encoding SV40LT, or a combination thereof, wherein integration of the nucleic acid sequence encoding hTERT, the nucleic acid encoding SV40LT, or the combination thereof comprises genomic integration of the nucleic acid sequence encoding hTERT, the nucleic acid encoding SV40LT, or the combination thereof and constitutive expression of hTERT, SV40LT, or the combination thereof.

In some embodiments, the modified cell is a T cell and further comprises a nucleic acid sequence encoding a CAR, and the modified cell is capable of inhibiting the cell from expressing an antigen recognized by the CAR. In some embodiments, the nucleic acid encoding the CAR and the nucleic acid encoding hTERT, the nucleic acid encoding SV40LT, or a combination thereof are expressed as gene products of separate polypeptides.

In some embodiments, expression of a nucleic acid sequence encoding hTERT, a nucleic acid encoding SV40LT, or a combination thereof is modulated by an inducible expression system in some embodiments, the inducible expression system is a rtTA-TRE system that increases or activates expression of an SV40LT gene or an hTERT gene or a combination thereof in some embodiments, the modified cell comprises a nucleic acid sequence encoding a suicide gene in some embodiments, the modified cell is a T cell or an NK cell in some embodiments, the suicide gene is an HSV-TK system in some embodiments, the modified cell is a proliferative T cell in some embodiments, the TCR comprises an extracellular domain, a transmembrane domain, and an intracellular domain, and the extracellular domain binds to a tumor antigen in some embodiments, the tumor antigen comprises HER LT, CD LT, a kappa, or LIGHT chain, CD LT, CD 36123, CD LT, CD 364, CD5, CD-TCR 14, CD5, CD-5, CD-TCR 72, CD-14, CD-5, CD-hcg, CD-14, CD-hcp, CD-hcg, CD-hcp, a, hcp.

Some embodiments relate to a method of generating a CAR T cell, the method comprising: propagating the T cell by transferring one or more nucleic acid sequences to the T cell to obtain a proliferative T cell; and introducing a nucleic acid sequence encoding a CAR into the proliferating T cell to obtain a CAR T cell, the CAR comprising an extracellular domain, a transmembrane domain, and an intracellular domain.

In some embodiments, the expanded T cell is any modified T cell described herein. In some embodiments, the one or more nucleic acid sequences comprise the Tet-inducible HPV16-E6/E7 expression system. In some embodiments, the T cell is a primary T cell extracted from the subject. In some embodiments, the T cell is a T cell with reduced immunogenicity as compared to a corresponding wild-type T cell in response to T cell infusion. Some embodiments relate to a method of treating a disease or condition, the method comprising: administering to a human patient a pharmaceutical composition comprising the modified cells described herein. In some embodiments, the disease or condition is AIDS, and the pharmaceutical composition comprises a cell comprising a CAR having an antigen binding domain that binds a molecule on the surface of HIV. In some embodiments, the disease or condition is cancer, and the pharmaceutical composition comprises a modified cell comprising a CAR, wherein the antigen binding domain of the CAR binds to a molecule on the cancer cell, and the number of endogenous TCRs on the cell is reduced. In some embodiments, the nucleic acid encoding the CAR is integrated into the genome of the T cell.

Some embodiments relate to a CAR T cell comprising: a nucleic acid sequence encoding a CAR comprising an extracellular domain, a transmembrane domain and an intracellular domain comprising a CD 3-zeta signaling domain and a signaling domain of a costimulatory molecule, wherein the TCR gene of the T cell is disrupted such that expression of the TCR is reduced or eliminated. In some embodiments, the CAR T cell comprises a modified T cell described herein.

Some embodiments relate to a CAR T cell comprising: a nucleic acid sequence encoding a CAR comprising an extracellular domain, a transmembrane domain, and an intracellular domain comprising a CD 3-zeta signaling domain and a signaling domain of a costimulatory molecule, wherein the CD4 gene of the T cell is disrupted such that expression of endogenous CD4 is reduced. In some embodiments, the antigen binding domain of the CAR binds to a molecule on the surface of HIV. In some embodiments, the CAR-T cell comprises a modified T cell described herein.

Some embodiments relate to a method of producing a conditionally proliferative T cell, the method comprising: transferring one or more nucleic acid sequences to a T cell to obtain a proliferative T cell, wherein the one or more nucleic acid sequences encode a peptide such that expression of the peptide causes the T cell to become a proliferative T cell, and the peptide is modulated by an inducible expression system, an inducible suicide system, or a combination thereof. In some embodiments, the peptide is hTERT, SV40LT, or a combination thereof. In some embodiments, the inducible expression system is a rtTA-TRE system. In some embodiments, the inducible suicide system is an HSV-TK system or an inducible caspase-9 system.

Some embodiments relate to a method of treating a disease or condition, the method comprising: preparing conditionally proliferative T cells using the methods described herein; culturing the conditionally proliferating T cells in a medium comprising tetracycline or doxycycline; culturing the conditionally proliferating T cells in a medium that does not contain any tetracycline or doxycycline; obtaining a T cell with reduced expression of its SV40LT gene or hTERT gene; and administering to a subject in need thereof a pharmaceutical composition comprising the T cell.

Some embodiments relate to a pharmaceutical composition comprising proliferative T cells obtained using the methods described herein for treating a disease or condition, the method for treating a disease or condition comprising: preparing conditionally proliferative T cells using the methods described herein; culturing the conditionally proliferating T cells in a medium comprising tetracycline or doxycycline; culturing the conditionally proliferating T cells in a medium that does not contain tetracycline or doxycycline; obtaining a T cell with reduced expression of its SV40LT gene or hTERT gene; and administering to the subject a pharmaceutical composition comprising the T cell. In some embodiments, the method may further comprise administering ganciclovir to a subject responsive to certain predetermined conditions.

Some embodiments relate to a population of T cells comprising modified cells in which an endogenous gene associated with a biosynthetic or trafficking pathway of a TCR gene of the modified cells is disrupted such that expression of the endogenous TCR is reduced.

Some embodiments relate to a population of T cells comprising modified cells wherein an endogenous gene associated with a biosynthetic or trafficking pathway of a PD-1 gene of the modified cells is disrupted such that expression of the PD-1 gene is reduced. In some embodiments, the modified cell comprises a nucleic acid sequence encoding a truncated PD-1, which truncated PD-1 reduces the inhibitory effect of apoptosis ligand 1(PD-L1) on human T cells.

Some embodiments relate to a method comprising: providing a cell comprising a CAR, and culturing the cell in the presence of an agent recognized by the extracellular domain of the CAR to obtain the CAR cell.

Some embodiments relate to a method for in vitro CAR cell preparation, the method comprising: providing a cell; introducing a nucleic acid sequence encoding a CAR into a cell to obtain a CAR cell; and culturing the CAR cell in the presence of an agent recognized by the extracellular domain of the CAR to obtain the CAR cell.

Some embodiments relate to a method for enriching a CAR-expressing cell, the method comprising: providing a cell; introducing a nucleic acid sequence encoding a CAR into a cell to obtain a cell that expresses the CAR (CAR cell) and a cell that does not express the CAR; and culturing the CAR cell in the presence of an agent that binds the extracellular domain of the CAR to enrich the CAR-expressing cell.

Some embodiments relate to a method for in vitro CAR cell preparation, the method comprising the following steps in the following order: (a) introducing a nucleic acid sequence encoding a CAR into a cell to obtain a CAR cell; (b) culturing the CAR cells using a first culture medium for a predetermined time; and (c) culturing the CAR cell using a second culture medium, wherein the first culture medium does not comprise an agent, the second culture medium comprises an agent, and the agent binds to the extracellular domain of the CAR.

In some embodiments, the agent is a compound that binds to the extracellular domain of a CAR and mediates a cellular response, in some embodiments, the modulating compound is a ligand of the extracellular domain of a CAR or an antigen to which the extracellular domain of a CAR binds, in some embodiments, the agent is an extracellular domain of an antigen to which the extracellular domain of a CAR binds, in some embodiments, the antigen is an Epidermal Growth Factor Receptor (EGFR), variant III of the epidermal growth factor receptor (EGFRvIII), human epidermal growth factor receptor 2(HER2), Mesothelin (MSLN), Prostate Specific Membrane Antigen (PSMA), carcinoembryonic antigen (CEA), bissialoganglioside 2(GD2), interleukin-13 Ra2(IL13R α), glypican-3 (GPC R α), Carbonic Anhydrase (CAIX), L R α cell adhesion molecule (L R α -CAM), cancer antigen 125(CA125), Clusterin (CD), reticulocyte activating protein (GPC 36133), oncomelan-R α), CD R α, CD-III, CD-13, CD R α, CD-III, CD-III.

In some embodiments, the co-stimulatory molecule of the CAR comprises at least one of CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-L ICOS, lymphocyte function-associated antigen-I (LFA-1), CD2, CD7, LIGHT, NKG2C, or B7-H3. In some embodiments, the CAR comprises an extracellular domain, a transmembrane domain, and an intracellular domain comprising a CD 3-zeta signaling domain and a signaling domain of a costimulatory molecule. In some embodiments, the cell is an NK cell or a T cell, or a combination thereof. In some embodiments, the modulatory compound is a soluble antigen produced by a eukaryotic or bacterial expression system.

In some embodiments, the ratio of the amount of the agent to the number of CAR cells after culturing the CAR cells with the agent is 1:50 to 1:5(μ g/10)⁴Individual cell), 1:500 to 1:5(μ g/10)⁴Individual cell), or 1:5000 to 10:5(μ g/10)⁴Individual cells). In some embodiments, culturing the cells comprises culturing the cells using a medium comprising at least one of anti-CD 3 beads, anti-CD 28 beads, and IL 2. In some embodiments, the ratio of the amount of the agent to the number of CAR cells after culturing the CAR cells with the agent is 1:50 to 1:5(μ g/10)⁴Individual cells). In some embodiments, of the CAR on CAR cells cultured in the presence of an agentThe copy number is greater than when the CAR cells are cultured without the agent. In some embodiments, the ratio of the number of cells expressing the CAR to the number of cells not expressing the CAR when cultured in the presence of the agent is greater than when the cells are cultured without the agent. In some embodiments, culturing the CAR cell in the presence of the agent comprises: culturing the CAR cell in the presence of the agent for a predetermined period of time, or culturing the CAR cell in the presence of the agent for at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days. In some embodiments, the predetermined period of time is from 7 to 100 days. In some embodiments, the number of CAR cells that produce a memory T cell phenotype when cultured in the presence of the agent is greater than when the CAR cells are cultured without the agent. In some embodiments, the amount of cytokine produced by the CAR cell when cultured in the presence of the agent is greater than the amount of cytokine produced by the CAR cell when the CAR cell is cultured without the agent.

In some embodiments, the CAR cells are derived from a healthy donor and have reduced expression of endogenous TCR genes and/or HLA I. In some embodiments, the CAR cells are derived from a healthy donor and do not elicit a Graft Versus Host Disease (GVHD) response or elicit a reduced GVHD response in a human recipient compared to the GVHD response elicited by primary human T cells isolated from the same human donor, and the expression of endogenous TCR genes and/or HLA I is not reduced, or the expression of endogenous TCR genes and/or HLAI is not disrupted, and endogenous TCR genes and/or HLA I is normally expressed. In some embodiments, the CAR T cell is a T cell comprising a nucleic acid sequence encoding hTERT, a nucleic acid encoding SV40LT, or a combination thereof.

In some embodiments, the CAR cell comprises a nucleic acid sequence encoding hTERT and a nucleic acid encoding SV40 LT. In some embodiments, the expression of hTERT is modulated by an inducible expression system. In some embodiments, the expression of the SV40LT gene is modulated by an inducible expression system. In some embodiments, the inducible expression system is a rtTA TRE that increases or activates expression of the SV40LT gene, the hTERT gene, or a combination thereof. In some embodiments, the CAR cell comprises a nucleic acid sequence encoding a suicide gene. In some embodiments, the suicide gene is the HSV-TK system.

Some embodiments relate to an isolated cell obtained by the above method.

Some embodiments relate to a pharmaceutical composition comprising an isolated cell obtained by the above method.

Some embodiments relate to a method for stimulating an anti-tumor immune response in a subject, the method comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition. Some embodiments relate to a pharmaceutical composition for use in treating cancer, the method for treating cancer comprising administering an effective amount of the pharmaceutical composition to a subject in need thereof. In some embodiments, the spacer domain of the CAR comprises the amino acid sequence of SEQ ID No. 68 or 69. In some embodiments, the transmembrane domain of the CAR comprises the amino acid sequence of SEQ ID No. 72 or 75 and the spacer domain of the CAR comprises the amino acid sequence of SEQ ID No. 68.

Some embodiments relate to a method comprising: administering to a subject in need thereof an effective amount of a T cell comprising a CAR to provide a T cell response; and administering an effective amount of a presenting cell expressing a soluble agent recognizable by the extracellular domain of the CAR.

Some embodiments relate to a method of enhancing a T cell response in a subject, the method comprising: administering to the subject an effective amount of a T cell comprising a CAR to provide a T cell response; and administering an effective amount of a presenting cell expressing a soluble agent recognizable by the extracellular domain of the CAR to enhance a T cell response in the subject. In some embodiments, enhancing a T cell response in a subject comprises selectively enhancing proliferation of a T cell comprising a CAR.

Some embodiments relate to a method of enhancing treatment of a condition in a subject using a CAR cell. In some embodiments, the method comprises administering to the subject a population of cells expressing the agent and a population of CAR cells. In other embodiments, the method comprises administering to the subject a vaccine derived from the agent and a population of CAR cells. The CAR cell comprises a nucleic acid sequence encoding the CAR, and the extracellular domain of the CAR recognizes the agent.

Some embodiments relate to a method of enhancing proliferation of CAR cells in a subject having a disease. The method comprises the following steps: preparing a cell comprising a CAR; administering to the subject an effective amount of a CAR cell; introducing into a cell a nucleic acid sequence encoding an agent recognizable by the extracellular domain of the CAR to obtain a modified cell, and administering to the subject an effective amount of the modified cell.

In some embodiments, the agent is a ligand of the extracellular domain of the CAR the agent is an antigen to which the extracellular domain of the CAR binds, and the agent comprises at least one of the extracellular domains of Epidermal Growth Factor Receptor (EGFR), variant III of epidermal growth factor receptor (EGFRvIII), human epidermal growth factor receptor 2 (HER), Mesothelin (MSLN), prostate-specific membrane antigen (PSMA), carcinoembryonic antigen (CEA), bis-sialylganglioside 2 (GD), interleukin-13 Ra (IL 13), glypican-3 (GPC), carbonic anhydrase IX, L cell adhesion molecule (L-CAM), cancer antigen 125(CA125), cluster of differentiation 133(CD133), Fibroblast Activation Protein (FAP), cancer/testis antigen 1B (CTAG 1), mucin 1 (MUC), folate receptor (FR-), CD, FZD, TSHR, MUC, CD17, CAR, GULR 2, CD4, CD-1B (CTAG 1), CD-NO-III), and optionally a polypeptide or a polypeptide, wherein the agent is expressed by a CD-III domain, and/CD-III, or a CD-III, and/CD-III, or a polypeptide, and a polypeptide, wherein the agent is a polypeptide, or a polypeptide, and wherein the agent is a polypeptide, or a protein.

Some embodiments relate to an isolated nucleic acid sequence encoding a CAR comprising an extracellular domain, a spacer domain, a transmembrane domain, and an intracellular domain. The extracellular domain of the CAR binds a tumor antigen, and the spacer domain comprises the amino acid sequence of SEQ ID No. 67 or 68.

Some embodiments relate to an isolated nucleic acid sequence encoding a CAR comprising an extracellular domain, a spacer domain, a transmembrane domain, and an intracellular domain. The extracellular domain of the CAR binds a tumor antigen; the spacer domain comprises the amino acid sequence of seq id No. 69; and the transmembrane domain comprises the amino acid sequence of SEQ ID No. 73 or 74.

In some embodiments, the antigen-binding fragment comprises a Fab or scfv, in some embodiments, a tumor antigen comprises HER2, CD19, CD20, CD22, kappa or LIGHT chain, CD30, CD33, CD123, CD38, ROR1, ErbB3/4, EGFR, EGFRvIII, EphA2, FAP, carcinoembryonic antigen, EGP2, EGP40, mesothelin, TAG 40, PSMA, NKG2 40 ligand, B40-H40, IL-13 receptor 402, IL-11 receptor 40, MUC 40, CA 40, GD 40, HMW-MAA, CD171, Lewis Y, G250/CAIX, HLA-AI a 40, NY-MAA-NY-40, PSC 40, GD 40, CD-MAA, CD 72, CD-x, CD40, CD-T40, CD-receptor, CD40, CD-receptor.

Some embodiments relate to a vector comprising the above-described isolated nucleic acid sequence.

Some embodiments relate to a cell comprising the above-described isolated nucleic acid sequence.

Some embodiments relate to a composition comprising a T cell population comprising the above-described isolated nucleic acid sequence.

Some embodiments relate to a method for stimulating an anti-tumor immune response or treating a condition in a subject. The method comprises administering to a subject an effective amount of a pharmaceutical composition comprising a population of human T cells comprising the above-described isolated nucleic acid sequence.

Some embodiments relate to a method comprising: providing a cell comprising the isolated nucleic acid sequence described above, and culturing the cell in the presence of an agent recognized by the extracellular domain of the CAR.

Some embodiments relate to a method for in vitro CAR cell preparation. The method comprises the following steps: providing a cell; introducing any of the above isolated nucleic acid sequences into a cell to obtain a CAR cell; and culturing the CAR cell in the presence of an agent recognized by the extracellular domain of the CAR.

Some embodiments relate to a method for enriching a CAR-expressing cell. The method comprises the following steps: providing a cell; introducing any of the above isolated nucleic acid sequences into a cell to obtain a cell that expresses a CAR (CAR cell) and a cell that does not express a CAR; and culturing the CAR cell in the presence of an agent that binds the extracellular domain of the CAR to enrich the CAR-expressing cell.

In some embodiments, the agent is a ligand for the extracellular domain of a CAR in some embodiments, the agent is an antigen to which the extracellular domain of a CAR binds in some embodiments, the agent is an extracellular domain of an antigen in some embodiments, the antigen is Epidermal Growth Factor Receptor (EGFR), variant III of epidermal growth factor receptor (EGFRvIII), human epidermal growth factor receptor 2(HER2), Mesothelin (MSLN), Prostate Specific Membrane Antigen (PSMA), carcinoembryonic antigen (CEA), bissialoglioside 2(GD2), interleukin-13 Ra2(IL13R α), glypican-3 (GPC3), Carbonic Anhydrase IX (CAIX), L1 cell adhesion molecule (L1-CAM), cancer antigen 125(CA125), cluster of differentiation 133(CD133), fibroblast activation protein (GPC), cancer/testis antigen 1B (CTAG1B), mucin 1(MUC1), cancer antigen 125(CA125), CD 35133), CD 5842, CD 465, CD27, CD 465, CD27, CD 53, CD 465, CD5, CD 465, CD5, CD 465, CD5, CD 465, CD5, CD 465, or at least one or a combination of an antibody which causes a cell to stimulate a folate in some embodiments, or a cell response in a cell.

This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.

Drawings

The embodiments are described with reference to the accompanying drawings. The use of the same reference symbols in different drawings indicates similar or identical items.

Figure 1 shows a schematic diagram illustrating T cell culture with or without antigen and shows a histogram showing the results of cell expansion of non-transduced T cells and CAR T cells using media without reagents according to embodiments of the present disclosure.

Figure 2 shows a table with various parameters for comparing T cells cultured with or without antigen.

Figure 3 shows the results of flow cytometric analysis demonstrating that CD19 maintains CAR T19 cell activity.

Figure 4 shows the results of flow cytometric analysis demonstrating that CD19 stimulates and/or induces CAR T cells to produce a memory cell phenotype.

Figure 5 shows the results of flow cytometric analysis demonstrating that CD19 stimulates and/or induces CAR T cells to produce a memory cell phenotype. The analysis results show that CD19 can stabilize the state of the cells. 402 and 406 of fig. 4 indicate the cell debris levels of cells cultured with and without CD19, respectively. 404 and 408 of fig. 4 indicate the proportion of cell debris for cells cultured with and without CD19, respectively.

Figure 6 shows a histogram demonstrating the ability of CD19 to enhance the release of IFN γ.

Figure 7 shows the results of flow cytometric analysis demonstrating that CD19 maintained the presence of CAR T cells.

Figure 8 shows the results of flow cytometric analysis demonstrating that TSHR maintains CAR T-TSHR cellular activity.

FIG. 9 shows Δ MFI (mean fluorescence intensity) for CART-TSHR cells. MFI refers to the mean fluorescence of a population of cells and is calculated as a numerical value.

Figure 10 shows the results of additional flow cytometric analyses demonstrating that TSHR maintains CAR T-TSHR cellular activity.

Figure 11 shows the cell morphology of CAR T-TSHR cells cultured with and without TSHR.

Figure 12 shows a schematic of the structure of an exemplary CAR molecule and a portion of the cell membrane.

Figure 13 shows the results of expansion of various constructs of CARs and T cells with CARs. T cells with various constructs of the CAR were cultured for predetermined times, respectively. Flow cytometric analysis of cultured T cells on day 1 and day 15; the cell expansion rate was measured. The histograms show the fold expansion of CAR T cells cultured with or without the CD19 extracellular domain.

Figure 14 shows flow cytometric analysis of CAR T cell expansion in four groups as shown in figure 13. The CAR T cells were cultured without the CD19 extracellular domain for 15 days.

Figure 15 shows flow cytometric analysis of CAR T cell expansion in four groups as shown in figure 13. The CAR T cells were cultured with CD19 extracellular domain for 15 days.

Figure 16 shows flow cytometric analysis of CAR expression levels on CAR T cells in four groups as shown in figure 13. The CAR T cells were cultured without the CD19 extracellular domain for 15 days.

Figure 17 shows flow cytometric analysis of CAR expression levels on CAR T cells in four groups as shown in figure 13. The CAR T cells were cultured with CD19 extracellular domain for 20 days.

Figure 18 shows flow cytometric analysis of CD4/CD8 phenotypic changes in CAR T cells.

FIG. 19 shows flow cytometric analysis of the phenotypic change of CD4/CD8 in CAR T cells.

Figure 20 shows flow cytometric analysis of CAR expression levels of CAR T cells in two groups as shown in figure 13. The CAR T cells were cultured with CD19 extracellular domain for 17 days.

Figure 21 shows flow cytometric analysis of killing assay of CAR T cells.

Figure 22 shows flow cytometric analysis of IFN-g release from CAR T cells.

FIG. 23 shows a schematic of a plurality of DNA constructs.

Fig. 24 shows a fluorescent photograph of T cell killing.

Fig. 25 shows a fluorescent photograph of T cell killing.

Fig. 26 shows a fluorescent photograph of T cell killing.

Figure 27 shows a graph of a multiple immortalized single cell sequencing assay.

Figure 28 shows a graph of a plurality of immortalized single cell sequencing assays.

Figure 29 shows a graph of a plurality of immortalized single cell sequencing assays.

Figure 30 shows a graph of a plurality of immortalized single cell sequencing assays.

Figure 31 shows a graph of a multiple immortalized single cell sequencing assay.

Figure 32 shows flow cytometric results of CAR expression in immortalized T cells. The two ordinates in the upper graph represent the expression of CAR. The abscissa is the expression of CD279(PD 1). The isotype control is on the left, and CAR antibody is on the right. T cells were infected with a vector comprising ef1a-TK-IRES-rtTa-TRE-hTERT DNA and a vector comprising hCD19 CAR-encoding DNA. During culture, CD19 peptide was added to stimulate the growth of T cells. Cellular CAR expression can be seen to be 82.87%. Qualitative + quantitative results. The following table shows copy number experiments for CARs. It can be seen that 224,1151 copies of CAR were present per 1 μ g of gDNA in CAR T cells, which induced expression of hCD19 in the expression system, which is a quantitative result. "dt mix" refers to a control group comprising cells transduced with ef1a-TK-IRES-rtTA-TRE-hTERT only, without DNA encoding anti-CD 19 CAR.

FIG. 33 shows a graph indicating that the cells are double-switch T cells, CD8+ monoclonal cells (Dox concentration 2. mu.g/mL). rTetR was used to construct anti-CD 19CAR proliferative T cells. Thus, when doxycycline (Dox) was added to the culture, hTERT was expressed. When Dox was not added, hTERT was not expressed and the cells gradually started to die.

FIG. 34 shows a graph indicating double safety T/CAR T cells 1+/-TK (ganciclovir).

FIG. 35 shows the results of flow cytometric analysis indicating double safety T/CAR T cells 1+/-TK (ganciclovir).

FIG. 36 shows the results of flow cytometric analysis indicating double safety T/CAR T cells 1+/-TK (ganciclovir). Experimental cells: CD8+ CZY-1SDS-T and NT cells; TK usage: the injection is injected every 24 hours, and the concentration in an adult is 71.45ng/ml/5000 ng/kg. In vitro assay concentration gradient: 357.2ng/ml, 142.9ng/ml, 71.45ng/ml, 35.725ng/ml, 14.28ng/ml, 3.6 ng/ml. As an example, FIG. 36 shows the use of 35.725ng/ml in controls and experiments. The starting cells were cultured at a cell density of 50w/ml of 200 w.

Figure 37 shows concentration gradient culture functional tests showing CD8+ dual switch CAR T cells.

Fig. 38 shows the optimal culture concentrations: 50w/ml to 100w/ml, and low or high concentrations can inhibit the growth of double-switch T cells. W/ml means 1 ten thousand per ml.

Fig. 39 shows the results of a two-switch CART cell killing assay.

Fig. 40 shows the results of the cell killing analysis.

Fig. 41 shows the results of the cell killing analysis. Fig. 26 and 27 are the results of killing analysis (primary t cell cd3 knockdown). After CAR and hTERT were knocked out and transfected, universal CART was made. The flow chart is as follows: the 32.17% on the left are cd3 knock-out cd3 cells, as are the 79.16% on the left. The sequencing peak pattern can be seen from a distinct set of peaks to demonstrate that the knock-out has occurred.

Fig. 42 shows CD3 negative cells obtained using ZFNs and purified with CD3 microbeads. After purification, CD3 negative cells were inoculated with APC-CD3 antibody and the results of flow cytometry indicated that the knockdown was successful, with 99.7% of the cells being CD 3-. These CD3 negative cells were then transfected into the genome of these cells with a lentiviral copy of dual switch hTERT and CAR.

Figure 43 shows survival and growth of various CAR T cells. In group 1 (hTert CD19 CAR), primary T cells obtained from healthy donors were transduced with a nucleic acid sequence encoding CD19CAR and a nucleic acid sequence encoding hTert. In group 2(CD 19 CARs), primary T cells were transduced with a nucleic acid sequence encoding a CD19 CAR. CAR T cells comprising nucleic acid sequences encoding hTERT showed long-term survival. Of these CAR T cells, cells cultured with cell culture medium containing CD19ECD had a higher cell growth rate than cells cultured with cell culture medium not containing CD19 ECD. CAR T cells that do not comprise a nucleic acid sequence encoding hTERT begin to die after about 20 days after the cells are transduced with the nucleic acid sequence encoding CAR.

Figure 44 shows cell growth of various sets of CAR T cells under different conditions. A: group 1 (hTERT + DOX + CD 19): proliferative CD19CAR T cells (hTERT) were cultured in media containing ECD CD19 and Dox. Group 2 (hTERT + DOX): proliferative CD19CAR T cells (hTERT) were cultured in medium containing Dox but without ECD CD 19. Group 3 (no hTERT CD19 CAR-T): CD19 CART cells were cultured in medium without Dox and ECD CD 19. B: group 1: CD19CAR T cells (h19CAR) were cultured in media without ECD CD19 and Dox. Group 2: proliferative CD19CAR T cells with dual switches (dual switch h19CAR + Dox) were cultured in medium containing Dox but without ECD CD 19. Group 3: proliferative CD19CAR T cells with dual switches (dual switch h19CAR + Dox + CD19) were cultured in medium containing Dox and ECD CD 19. These results indicate that the agents and/or proliferative modifications are useful for long-term maintenance of CART cells in vitro.

Figure 45 shows flow cytometric analysis (gated by single live cells) indicating expression of anti-TSHR CAR molecules on T cells. anti-TSHR CAR T cells were constructed and expression of CAR molecules was detected by flow cytometry. Expression of the CAR molecule was observed compared to non-transduced T cells.

Figure 46 shows flow cytometric analysis (gated by single viable cells) indicating overexpression of TSHR on T cells. Lentiviral vectors are used to construct antigen over-expressed T cells (TSHR). Expression of TSHR molecules on the surface of T cells was observed (IgG on the left, anti-TSHR FITC on the right).

FIG. 47 shows cytokine release (IL-2) in peripheral blood of mice.

FIG. 48 shows cytokine release (IFN-. gamma.) in peripheral blood of mice.

FIG. 49 shows cytokine release (IL-4) in peripheral blood of mice.

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods and materials are described. For the purposes of this disclosure, the following terms are defined below.

The article "a/an" is used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. For example, "an element" means one element or more than one element.

By "about" is meant an amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by as much as 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from a reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

As used herein, the term "activate" refers to a cellular state that has been sufficiently stimulated to induce detectable cellular proliferation. Activation may also be associated with induced cytokine production and detectable effector function. The term "activated T cell" especially refers to a T cell undergoing cell division.

The term "antibody" is used in its broadest sense and refers to monoclonal antibodies (includingFull length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity or function. The antibodies of the present disclosure may exist in various forms, including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F (ab)₂And single chain and humanized Antibodies (Harlow et al, 1999, In: Using Antibodies: antibody Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al, 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al, 1988, Proc. Natl. Acad. Sci. USA 85: 5879-.

The term "antibody fragment" refers to a portion of a full-length antibody, e.g., the antigen-binding or variable region of an antibody. Other examples of antibody fragments include Fab, Fab ', F (ab')₂And Fv fragments; a diabody; a linear antibody; a single chain antibody molecule; and multispecific antibodies formed from antibody fragments.

The term "Fv" refers to the smallest antibody fragment that contains the entire antigen recognition and antigen binding site. The fragment consists of a dimer of one heavy and one light chain variable region domain in close, non-covalent association. By folding of these two domains, six hypervariable loops (each 3 loops from the H and L chains, respectively) are created, which contribute amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three Complementarity Determining Regions (CDRs) specific for an antigen) has the ability to recognize and bind antigen, although with lower affinity than the entire binding site (dimer).

As used herein, "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in all antibody molecules in their naturally occurring configuration. As used herein, "antibody light chain" refers to the smaller of the two types of polypeptide chains present in all antibody molecules in their naturally occurring configuration. The kappa and lambda light chains refer to the two major antibody light chain isotypes.

The term "synthetic antibody" refers to an antibody produced using recombinant DNA techniques, such as, for example, an antibody expressed by a bacteriophage. The term also includes antibodies generated by synthesizing DNA molecules encoding the antibodies and expressing the DNA molecules to obtain the antibodies or to obtain the amino acids encoding the antibodies. Synthetic DNA is obtained using techniques available and well known in the art.

The term "antigen" refers to a molecule that elicits an immune response, which may involve antibody production or activation of specific immunocompetent cells, or both. Antigens include any macromolecule, including all proteins or peptides, or molecules derived from recombinant or genomic DNA. For example, a DNA comprising a nucleotide sequence or partial nucleotide sequence encoding a protein or peptide that elicits an immune response, thus encoding the term "antigen" as used herein. An antigen need not be encoded by only the full-length nucleotide sequence of a gene. The antigen may be generated, synthesized or derived from a biological sample, including a tissue sample, a tumor sample, a cell, or a biological fluid.

As used herein, the term "anti-tumor effect" refers to a biological effect associated with decreased tumor volume, decreased tumor cell number, decreased metastasis number, decreased tumor cell proliferation, decreased tumor cell survival, increased life expectancy of a subject having tumor cells, or improvement of various physiological symptoms associated with a cancerous condition. First, an "anti-tumor effect" can also be exhibited by the ability of peptides, polynucleotides, cells and antibodies to prevent tumorigenesis.

The term "autoantigen" refers to an antigen that is mistaken by the immune system as foreign. Autoantigens include cellular proteins, phosphoproteins, cell surface proteins, cellular lipids, nucleic acids, glycoproteins, including cell surface receptors.

The term "autologous" is used to describe material derived from a subject, which is subsequently reintroduced into the same subject.

The term "allogeneic" is used to describe grafts derived from different subjects of the same species. As an example, the donor subject may be related or unrelated or the recipient subject, but the donor subject has similar immune system markers as the recipient subject.

The term "xenogeneic" is used to describe grafts derived from subjects of different species. As an example, the donor subject and the recipient subject are from different species, and the donor subject and the recipient subject may be genetically and immunologically incompatible.

The term "cancer" is used to refer to a disease characterized by rapid and uncontrolled growth of abnormal cells. Cancer cells can spread locally, or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers include breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, and the like.

Throughout this specification, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.

The phrase "consisting of" is intended to include and be limited to anything following the phrase "consisting of. Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and that no other elements may be present.

The phrase "consisting essentially of means includes any elements listed after the phrase, and may include other elements that do not interfere with or contribute to the activities or actions specified in the present disclosure for the listed elements. Thus, the phrase "consisting essentially of.

The terms "complementary" and "complementary" refer to polynucleotides (i.e., nucleotide sequences) that are related together by the base pairing rules. For example, the sequence "A-G-T" is complementary to the sequence "T-C-A". Complementarity may be "partial," in which only some of the nucleic acids' bases are matched according to the base pairing rules. Alternatively, there may be "complete" or "total" complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands.

The term "corresponds to" or "corresponds to" refers to (a) a polynucleotide having a nucleotide sequence that is substantially identical or complementary to all or a portion of a reference polynucleotide sequence or that encodes an amino acid sequence that is identical to an amino acid sequence in a peptide or protein; or (b) a peptide or polypeptide having an amino acid sequence substantially identical to an amino acid sequence in a reference peptide or protein.

The term "co-stimulatory ligand" refers to a molecule on an antigen presenting cell (e.g., APC, dendritic cell, B cell, etc.) that specifically binds to a cognate co-stimulatory molecule on T cells, thereby providing a signal in addition to the primary signal provided by, for example, TCR/CD3 complex binding to peptide-loaded MHC molecules that mediates T cell responses, including at least one of proliferation, activation, differentiation, and other cellular responses the co-stimulatory ligand may include B7-1(CD80), B7-2(CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible co-stimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, HLA-G, MICA a, MICB, HVEM, lymphotoxin 40 receptor, 3/TR 40, ILT 40, hvt 40, CD40, agonist of CD40, agonist binding to HLA-G, MICA, MICB, HVEM, lymphotoxin 40 receptor binding to 3/TR 40, ligand specific binding to CD40, and agonist binding to CD40, particularly CD40, and agonist antibodies such as antibodies to CD40, CD-1, CD40, and antibodies specific binding to antibodies, such as CD 36x-1, CD40, and CD40, and antibodies that bind to cell specific ligands.

The term "co-stimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a co-stimulatory ligand, thereby mediating a co-stimulatory response, such as proliferation, of the T cell. Costimulatory molecules include MHC class I molecules, BTLA, and Toll-like receptors.

The term "co-stimulatory signal" refers to a signal that, in combination with a primary signal (such as TCR/CD3 linkage), results in up-or down-regulation of T cell proliferation and/or key molecules. The terms "disease" and "condition" may be used interchangeably, or may be different, in that a particular disease or condition may not have a known causative agent (and therefore the etiology has not been solved), and thus it is not yet a recognized disease, but is merely an undesirable condition or syndrome in which a clinician has identified a more or less set of particular symptoms. The term "disease" is a health state of a subject, wherein the subject is unable to maintain homeostasis, and wherein the health of the subject continues to deteriorate if the disease is not improved. In contrast, a "disorder" in a subject is a state of health in which the animal is able to maintain homeostasis, but in which the state of health of the animal is less favorable than in the absence of the disorder. If not treated in time, the condition does not necessarily lead to a further reduction in the health status of the animal.

The term "effective" means sufficient to achieve a desired, expected, or intended result. For example, an "effective amount" in the context of treatment may be an amount of a compound sufficient to produce a therapeutic or prophylactic benefit.

The term "encoding" refers to the inherent property of a particular nucleotide sequence in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes having defined nucleotide sequences (i.e., rRNA, tRNA, and mRNA) or defined amino acid sequences and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to the gene produces the protein in a cell or other biological system. The nucleotide sequence of which is identical to the mRNA sequence (except for the substitution of "T" with "U") and is typically provided in the sequence listing, the coding strand, as well as the non-coding strand that serves as the template for transcription of the gene or cDNA, may be referred to as the protein or other product encoding the gene or cDNA.

The term "exogenous" refers to a molecule that does not naturally occur in a wild-type cell or organism, but is typically introduced into the cell by molecular biological techniques. Examples of exogenous polynucleotides include vectors, plasmids, and/or artificial nucleic acid constructs encoding the desired proteins. With respect to polynucleotides and proteins, the term "endogenous" or "native" refers to a naturally occurring polynucleotide or amino acid sequence that may be found in a given wild-type cell or organism. Likewise, a particular polynucleotide sequence isolated from a first organism and transferred to a second organism by molecular biological techniques is generally considered to be an "exogenous" polynucleotide or amino acid sequence relative to the second organism. In particular embodiments, a polynucleotide sequence may be "introduced" into a microorganism already containing such polynucleotide sequence by molecular biological techniques, e.g., to produce one or more additional copies of the originally naturally occurring polynucleotide sequence, thereby facilitating overexpression of the encoded polypeptide.

The term "expression" refers to the transcription and/or translation of a particular nucleotide sequence driven by its promoter.

The term "expression vector" refers to a vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. The expression vector includes sufficient cis-acting elements for expression; other elements for expression may be provided by the host cell or in an in vitro expression system. Expression vectors include all vectors known in the art that incorporate recombinant polynucleotides, such as cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).

The term "homologous" refers to sequence similarity or sequence identity between two polypeptides or between two polynucleotides when a position in two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, the molecules are homologous at that position. The percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared x 100. For example, two sequences are 60% homologous if 6 of 10 positions in the two sequences are matching or homologous. For example, the DNA sequences ATTGCC and TATGGC share 50% homology. When aligning the two sequences, a comparison is made to obtain maximum homology.

The term "immunoglobulin" or "Ig" refers to a class of proteins used as antibodies. The five members included in such proteins are IgA, IgG, IgM, IgD and IgE. IgA is a primary antibody found in body secretions such as saliva, tears, breast milk, gastrointestinal secretions, and mucous secretions of the respiratory and genitourinary tracts. IgG is the most common circulating antibody. In most subjects, IgM is the primary immunoglobulin produced in the primary immune response. It is the most effective immunoglobulin in agglutination, complement fixation and other antibody responses, and is important for defense against bacteria and viruses. IgD is an immunoglobulin that does not have known antibody function but can act as an antigen receptor. IgE is an immunoglobulin that mediates immediate hypersensitivity by causing mast cells and basophils to release mediators upon exposure to allergen.

The term "isolated" refers to a material that is substantially or essentially free of components that normally accompany it in its natural state. The material may be a cell or a macromolecule, such as a protein or nucleic acid. For example, an "isolated polynucleotide" as used herein refers to a polynucleotide that has been purified from the sequences that flank it in a naturally occurring state, e.g., a DNA fragment that has been removed from the sequences that are normally adjacent to the fragment. Alternatively, "isolated peptide" or "isolated polypeptide" and the like as used herein refers to the in vitro isolation and/or purification of a peptide or polypeptide molecule from its native cellular environment as well as from its association with other components of a cell.

The term "substantially purified" refers to a material that is substantially free of components with which it is normally associated in its natural state. For example, a substantially purified cell refers to a cell that has been isolated from other cell types that are normally associated in their naturally occurring or native state. In some cases, a substantially purified cell population refers to a homogenous cell population. In other instances, the term simply refers to a cell that has been isolated from a cell with which it is naturally associated in its native state. In some embodiments, the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro.

In the context of the present disclosure, the following abbreviations for commonly occurring nucleic acid bases are used. "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

Unless otherwise indicated, "nucleotide sequences encoding amino acid sequences" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence encoding a protein or RNA may also include introns to the extent that the nucleotide sequence encoding the protein may contain one or more introns in some forms.

The term "lentivirus" refers to a genus of the family retroviridae. Lentiviruses are unique among retroviruses, which are capable of infecting non-dividing cells; they can deliver large amounts of genetic information into the DNA of host cells, and therefore they are one of the most efficient methods of gene delivery vectors. HIV, SIV and FIV are examples of lentiviruses. Vectors derived from lentiviruses provide a means to achieve significant levels of gene transfer in vivo.

The term "modulate" refers to mediating a detectable increase or decrease in the level of a response in a subject as compared to the level of a response in a subject in the absence of a treatment or compound, and/or as compared to the level of a response in an otherwise identical but untreated subject. The term encompasses interfering with and/or affecting a natural signal or response, thereby mediating a beneficial therapeutic response in a subject, preferably a human.

A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.

The term "under transcriptional control" means that a promoter is operably linked to a polynucleotide and is in the correct position and orientation relative to the polynucleotide to control the initiation of transcription by RNA polymerase and expression of the polynucleotide.

The term "overexpressed" tumor antigen or "overexpression" of a tumor antigen is intended to indicate an abnormal expression level of a tumor antigen in cells from a diseased region (e.g., a solid tumor) in a particular tissue or organ of a patient relative to the expression level in normal cells from that tissue or organ. Patients with solid tumors or hematologic malignancies characterized by overexpression of tumor antigens can be identified by standard assays known in the art.

"parenteral" administration of immunogenic compositions includes, for example, subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), intrasternal injection, or infusion techniques.

The terms "patient," "subject," and "individual" and the like are used interchangeably herein and refer to any human, animal, or living organism suitable for use in the methods described herein. In certain non-limiting embodiments, the patient, subject, or individual is a human or an animal. In some embodiments, the term "subject" is intended to include living organisms (e.g., mammals) in which an immune response can be elicited. Examples of subjects include humans and animals, such as dogs, cats, mice, rats, and transgenic species thereof.

A subject in need of treatment or in need thereof includes a subject having a disease, condition, or disorder in need of treatment. Wherein a subject in need thereof further comprises a subject in need of treatment to prevent a disease, condition, or disorder.

The term "polynucleotide" or "nucleic acid" refers to mRNA, RNA, cRNA, rRNA, cDNA, or DNA. The term generally refers to any type of nucleotide, either in polymeric form, ribonucleotides or deoxyribonucleotides or in modified form, that is at least 10 bases in length. The term includes all forms of nucleic acid, including single-stranded and double-stranded forms of nucleic acid.

The terms "polynucleotide variant" and "variant" and the like refer to a polynucleotide that exhibits substantial sequence identity to a reference polynucleotide sequence or a polynucleotide that hybridizes to a reference sequence under stringent conditions as defined below. These terms also encompass polynucleotides that are distinguished from a reference polynucleotide by the addition, deletion or substitution of at least one nucleotide. Thus, the terms "polynucleotide variant" and "variant" include polynucleotides in which one or more nucleotides have been added or deleted or replaced by a different nucleotide. In this regard, it is well known in the art that certain alterations, including mutations, additions, deletions and substitutions, may be made to a reference polynucleotide such that the altered polynucleotide retains a biological function or activity of the reference polynucleotide or has increased activity (i.e., is optimized) relative to the reference polynucleotide. Polynucleotide variants include, for example, polynucleotides having at least 50% (and at least 51% to at least 99% and all integer percentages therebetween, e.g., 90%, 95%, or 98%) sequence identity to a reference polynucleotide sequence described herein. The terms "polynucleotide variant" and "variant" also include naturally occurring allelic variants and orthologs.

"polypeptide," "polypeptide fragment," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acid residues and variants and synthetic analogs thereof. Thus, these terms apply to amino acid polymers in which one or more amino acid residues is a synthetic non-naturally occurring amino acid, such as a chemical analog of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. In some embodiments, a polypeptide may include an enzymatic polypeptide or "enzyme" that generally catalyzes (i.e., increases the rate of) various chemical reactions.

The term "polypeptide variant" refers to a polypeptide that is distinguished from a reference polypeptide sequence by the addition, deletion, or substitution of at least one amino acid residue. In certain embodiments, a polypeptide variant is distinguished from a reference polypeptide by one or more substitutions, which may be conservative or non-conservative. In certain embodiments, polypeptide variants comprise conservative substitutions, and in this regard, it is well known in the art that some amino acids may be changed to other amino acids with substantially similar properties without changing the nature of the polypeptide activity. Polypeptide variants also encompass polypeptides in which one or more amino acids have been added or deleted or replaced by a different amino acid residue.

The term "promoter" refers to a DNA sequence that is recognized by or introduced into the synthetic machinery of a cell to initiate specific transcription of a polynucleotide sequence. The term "expression control sequence" refers to a DNA sequence necessary for the expression of an operably linked coding sequence in a particular host organism. Suitable control sequences for prokaryotes include, for example, promoters, optionally operator sequences and ribosome binding sites. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

The terms "bind", "binding" or "interact with" refer to a molecule that recognizes and adheres to a specific second molecule in a sample or organism, but does not substantially recognize or adhere to other structurally unrelated molecules in the sample. The term "specifically binds," as used herein with respect to an antibody, refers to an antibody that recognizes a specific antigen but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to an antigen from one or more species. However, this cross-species reactivity does not change the specific classification of the antibody itself. In another example, an antibody that specifically binds to an antigen can also bind to different allelic forms of the antigen. However, this cross-reactivity does not change the specific classification of the antibody itself. In some cases, the term "specific binding" or "specific binding" may be used to refer to the interaction of an antibody, protein or peptide with a second chemical species, meaning that the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, antibodies recognize and bind to a specific protein structure, rather than to any protein. If the antibody is specific for epitope "A", the presence of a molecule comprising epitope A (or free, unlabeled A) in a reaction comprising label "A" and the antibody will reduce the amount of label A bound to the antibody.

By "statistically significant" is meant that the results are unlikely to occur by chance. Statistical significance can be determined by any method known in the art. Common significance metrics include the p-value, which is the frequency or probability of an event occurrence observed when a ghost is set to true. If the obtained p-value is less than the significance level, the null hypothesis is rejected. In a simple case, the significance level is defined as a p-value of 0.05 or less. An amount that is "reduced" or "minor" is typically a "statistically significant" or physiologically significant amount, and can include about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8,9, 10, 15, 20, 30, 40, or 50 or more times (e.g., 100, 500, 1000 times) (including all integers and decimal points between 1 and greater than 1, such as 1.5, 1.6, 1.7, 1.8, etc.) the amount or level of reduction described herein.

The term "stimulation" refers to a primary response induced by the binding of a stimulating molecule (e.g., the TCR/CD3 complex) to its cognate ligand, thereby mediating a signaling event, such as signaling via the TCR/CD3 complex.

The term "stimulatory molecule" refers to a molecule on a T cell that specifically binds to a cognate stimulatory ligand present on an antigen presenting cell. For example, a functional signaling domain derived from a stimulatory molecule is the zeta chain associated with the T cell receptor complex.

The term "stimulatory ligand" refers to a ligand that, when present on an antigen presenting cell (e.g., APC, dendritic cell, B cell, etc.), can specifically bind to a cognate binding partner (referred to herein as a "stimulatory molecule") on a cell, e.g., a T cell, thereby mediating a primary response of the T cell, including activation, initiation of an immune response, proliferation, and similar processes. Stimulatory ligands are well known in the art and encompass, inter alia, peptide-loaded MHC class I molecules, anti-CD 3 antibodies, superagonist anti-CD 28 antibodies, and superagonist anti-CD 2 antibodies.

The term "therapeutic" refers to treatment and/or prevention. Therapeutic effects can be obtained by inhibiting, alleviating or eradicating the disease state or alleviating the symptoms of the disease state.

The term "therapeutically effective amount" refers to an amount of a compound of the invention that will elicit the biological or medical response of a tissue, system or subject that is being sought by the researcher, veterinarian, medical doctor or other clinician. The term "therapeutically effective amount" includes an amount of a compound that, when administered, is sufficient to prevent the development of, or alleviate to some extent, one or more signs or symptoms of the condition or disease being treated. The therapeutically effective amount will vary depending on the compound, the disease and its severity and the age, weight, etc., of the subject to be treated.

The term "treating a disease" refers to reducing the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.

The term "transfected" or "transformed" or "transduced" refers to the process of transferring or introducing an exogenous nucleic acid into a host cell. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed or transduced with an exogenous nucleic acid. The cells include primary subject cells and progeny thereof.

A "vector" is a polynucleotide that comprises an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the interior of a cell. Many vectors are known in the art, including linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes an autonomously replicating plasmid or virus. The term also includes non-plasmid and non-viral compounds that facilitate transfer of nucleic acids into cells, such as polylysine compounds, liposomes, and the like. Examples of the viral vector include an adenovirus vector, an adeno-associated virus vector, a retrovirus vector and the like. For example, lentiviruses are complex retroviruses which, in addition to the common retroviral genes gag, pol and env, also contain other genes with regulatory or structural functions. Lentiviral vectors are well known in the art. Some examples of lentiviruses include human immunodeficiency virus: HIV-1, HIV-2 and simian immunodeficiency virus: and (6) SIV. Lentiviral vectors are generated by multiple attenuation of HIV virulence genes, e.g., deletion of genes env, vif, vpr, vpu, and nef, rendering the vector biologically safe.

The range is as follows: throughout this disclosure, various aspects of the present disclosure may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have explicitly disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual values within the stated range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

The present disclosure relates to isolated nucleic acid sequences, vectors comprising the isolated nucleic acid sequences, modified cells, and methods of using the cells to treat cancer.

Some aspects of the disclosure relate to the surprising discovery that culturing CAR cells in vitro with an agent can enhance the efficacy of the CAR cells and/or the efficiency of CAR cell production, achieve long-term in vitro maintenance of the CAR cells, and/or induce the CAR T cells to develop a memory T cell phenotype. In these cases, the CAR expressed by the CAR cell recognizes and/or binds the agent. In some embodiments, the agent is a regulatory compound that binds to an extracellular component of the CAR and/or activates a signaling pathway of the CAR, thereby stimulating the T cell expressing the CAR. For example, a regulatory compound can bind to a CAR of a T cell and mediate a T cell response, including activation, initiation of an immune response, and/or proliferation.

Some aspects of the disclosure relate to modified T cell/CAR T cells (i.e., proliferative cells or long-lived cells) that can be grown multiple times. Such proliferating cells maintain the functions of normal T cells/CAR T cells, such as cell therapy functions. In some embodiments, a dual switch may be designed to modulate the growth of proliferative T cell/CAR T cells. Embodiments herein contemplate a mechanism that includes one or two control switches. The first switch comprises rtTA-TRE-hTERT/SV40 LT. rtTA-TRE is eukaryotic cell induced regulatory gene expression. By adding tetracycline to induce expression of hTERT (human telomerase reverse transcriptase) or SV40LT (SV40 large T antigen), an immortalized phenotype can be produced. The second regulating switch is EF1 a-TK. The TK gene is a suicide gene. In the case of ganciclovir addition, the agent will allow the suicide gene to function to regulate cell death itself. In some embodiments, CAR T cells with one or two control switches can allow the CAR T cells to survive longer and retain the associated biological functions while remaining effective and safe. Furthermore, in addition to lentiviruses, various other methods included in the present invention can be used to generate T cells, such as knock-in methods for inserting a genome into another genome and the use of other vectors (e.g., retroviral vectors).

Embodiments of the present disclosure relate to compositions and methods for treating conditions using Chimeric Antigen Receptor (CAR) cells. The term "chimeric antigen receptor" or alternatively "CAR" refers to a recombinant polypeptide construct comprising at least an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain (e.g., a cytoplasmic domain). In some embodiments, the domains in the CAR polypeptide construct are in the same polypeptide chain (e.g., comprise a chimeric fusion protein) or are discontinuous from one another (e.g., in different polypeptide chains).

In some embodiments, the intracellular signaling domain may comprise a functional signaling domain derived from a stimulatory molecule and/or a co-stimulatory molecule as described above. In certain embodiments, the intracellular signaling domain comprises a functional signaling domain derived from a primary signaling domain (e.g., the primary signaling domain of CD 3-zeta). In other embodiments, the intracellular signaling domain further comprises one or more functional signaling domains derived from at least one co-stimulatory molecule. A costimulatory signaling region refers to a portion of a CAR that includes the intracellular domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are necessary for an effective response of lymphocytes to an antigen.

Between the extracellular domain and the transmembrane domain of the CAR, a spacer domain (i.e., a hinge domain) can be incorporated. As used herein, the term "spacer domain" refers to any oligopeptide or polypeptide that functions to connect a transmembrane domain to an extracellular domain or a cytoplasmic domain in a polypeptide chain. The spacer domain may comprise up to 300 amino acids, preferably 10 to 100 amino acids, and most preferably 25 to 50 amino acids.

The extracellular domain of a CAR may include an antigen binding domain (e.g., scFv, single domain antibody or TCR (e.g., TCR α binding domain or TCR β binding domain)) that targets a particular tumor marker (e.g., tumor antigen), tumor antigen is a protein that elicits an immune response produced by tumor cells, particularly a T cell-mediated immune response tumor antigen is well known in the art and includes, for example, glioma-associated antigen, carcinoembryonic antigen (CEA), β -human chorionic gonadotropin, Alpha Fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2(AS), enterocarboxylesterase, mut hsp70-2, M-CSF, prostatase (prostatase), Prostate Specific Antigen (PSA), NY-ESO-1, LAGE-1a, p53, prostate specific protein (prostein), PSMA, herhercar, psm 2/ne, IGF-B-84, and, if CD cell receptor antigen is CD 6346, CD 9-IGF-CD 9, CD 9-IGF-B, CD 9-IGF-c, CD-B-IGF-B-IGF-9, and CD-IGF-B-c-B-f-B-c-B-c (CD-B).

In some embodiments, the extracellular ligand-binding domain comprises a scFv comprising a light chain Variable (VL) region and a heavy chain Variable (VH) region of a target antigen-specific monoclonal antibody connected by a flexible linker. Single chain variable region fragments were prepared by linking the light chain variable region and/or the heavy chain variable region using short linking peptides (Bird et al, Science 242:423-426, 1988). An example of a linker peptide is a peptide having the amino acid sequence (GGGGS)₃(SEQ ID:76) which bridges about 3.5nm between the carboxy terminus of one variable region and the amino terminus of the other variable region. Has designed and used itThe linker of his sequence (Bird et al, 1988, supra). In general, the linker may be a short flexible polypeptide and preferably comprises about 20 amino acid residues or less. In turn, the linker can be modified to perform additional functions, such as drug attachment or attachment of a solid support. Single-stranded variants can be produced recombinantly or synthetically. For synthetic production of scFv, an automated synthesizer can be used. For recombinant production of scFv, a suitable plasmid containing a polynucleotide encoding a scFv can be introduced into a suitable host cell, which can be a eukaryote, such as a yeast, plant, insect, or mammalian cell, or can be a prokaryote, such as e. Polynucleotides encoding the scFv of interest can be prepared by conventional procedures such as ligation of the polynucleotides. The resulting scFv can be isolated using standard protein purification techniques known in the art.

In some embodiments, the tumor antigen comprises a HER, CD, kappa, or light chain, CD123, CD, ROR, ErbB/4, EGFR, EGFRvIII, EphA, FAP, carcinoembryonic antigen, EGP, mesothelin, TAG, PSMA, NKG2 ligand, B-H, IL-13 receptor 2, IL-11 receptor, MUC, CA, GD, HMW-MAA, CD171, Lewis Y, G250/CAIX, HLA-AI MAGE A, HLA-A2-ESO-1, PSC, folate receptor-, CD44 v/8, 8H, NCAM, VEGF receptor, 5T, fetal AchR, NKG2 ligand, CD44v, TEM, or a virus-associated antigen expressed by the tumor.

In some embodiments, a genetically modified cell is contemplated. In some embodiments, the modified cell can comprise a nucleic acid sequence encoding hTERT, a nucleic acid encoding SV40LT, or a combination thereof. In certain embodiments, the modified cell can comprise a first nucleic acid sequence encoding hTERT and/or a second nucleic acid sequence encoding SV40 LT. For example, the nucleic acid sequence encoding hTERT has the sequence of SEQ ID No. 6 and the nucleic acid sequence encoding SV40LT has the sequence of SEQ ID No. 7.

In some embodiments, the modified cell is a T cell or an NK cell. In certain embodiments, the modified cell is a proliferative T cell. A proliferating cell refers to a genetically modified cell that has a higher proliferative capacity than a wild-type cell. Several techniques can be implemented to obtain proliferative cells. For example, hTERT, SV40LT, and/or other genes can be transferred to cells to obtain proliferative cells. In some embodiments, mRNA encoding constructs (e.g., hTERT and/or SV40LT) can be injected into cells to achieve transient gene expression in these cells. In other embodiments, a vector encoding a construct (e.g., hTERT and/or SV40LT) can be introduced into a cell to obtain a proliferative cell. For example, at least a portion of the vector may be integrated into the genome of the cell. In these cases, the integration of the nucleic acid sequence encoding hTERT, the nucleic acid encoding SV40LT, or a combination thereof can include genomic integration of the nucleic acid sequence encoding hTERT, the nucleic acid encoding SV40LT, or a combination thereof and constitutive expression of hTERT, SV40LT, or a combination thereof.

As described above, some embodiments relate to a multistep control of proliferative capacity.A eukaryotic cell-induced expression system can be used to modulate the proliferative capacity of T cells, hTERT and/or SV40LT can be expressed by the continued addition of "tetracycline" to these cells, however, if the provision of tetracycline is terminated, hTERT and/or SV40LT may not be expressed.

In some embodiments, the expression of the nucleic acid sequence encoding hTERT, the nucleic acid encoding SV40LT, or a combination thereof is modulated by an inducible expression system. For example, the inducible expression system is an rTTA TRE that increases or activates expression of the SV40LT gene, hTERT gene, or a combination thereof. Inducible expression systems allow for the temporally and spatially controlled activation and/or expression of genes. For example, tetracycline-controlled transcriptional activation is a method of inducible gene expression in which transcription is reversibly turned on or off in the presence of the antibiotic tetracycline or one of its derivatives (e.g., doxycycline). For example, inducible suicide gene expression systems allow for the temporally and spatially controlled activation and/or expression of suicide genes, which results in cells killing themselves through apoptosis.

In some embodiments, the modified cell may comprise a nucleic acid sequence encoding a suicide gene. For example, the suicide gene is the HSV-TK system.

In some embodiments, the tumor antigen comprises a HER2, CD19, CD20, CD22, kappa or LIGHT chain, CD30, CD33, CD123, CD38, ROR1, ErbB3/4, EGFR, EGFRvIII, EphA2, FAP, carcinoembryonic antigen, EGP2, mesothelin, TAG 2, PSMA, NKzeta G2 2 ligand, B2-H2, IL-13 receptor 22, IL-11 receptor 2, MUC 2, CA 2, GD2, HMW-MAA, CD171, Lewis Y, G250/CAIX, CAR-72, MUC 2, CD-PSC 72, CD-2, CD-MAG 72, CD-1-PSC 2, CD-PSC 72, CD-5, CD-14, CD-5, CD-CD 2, CD-related signal domain, CD-binding, VEGF-related, CD-binding, VEGF-binding, VEGF-related, CD-binding, or a signaling domains, wherein the combination thereof may comprise a nucleic acid, which, as a, or a signal transduction, which, in a molecule, which, may comprise a, as a stimulation in a combination, a stimulating, a stimulating, or in a combination, a cell signaling molecule, which, may comprise a co-2, a stimulating, a stimulating, a co-2.

In some embodiments, the TCR gene of the T cell is disrupted such that expression of the endogenous TCR is reduced. In certain embodiments, the targeting vector associated with the TCR gene is integrated into the genome of the T cell such that expression of the endogenous TCR is eliminated.

In some embodiments, the CD4 gene of the T cell is disrupted such that expression of endogenous CD4 is reduced. In certain embodiments, the antigen binding domain of the CAR binds to a molecule on the surface of HIV.

Some embodiments relate to a method for making a modified cell having a CAR (CAR cell). In some embodiments, the method may comprise providing a cell; and introducing into the cell a nucleic acid sequence encoding a CAR and a nucleic acid sequence encoding hTERT, SV40LT, or a combination thereof. In some embodiments, the integration of the nucleic acid sequence encoding hTERT, the nucleic acid encoding SV40LT, or a combination thereof comprises genomic integration of the nucleic acid sequence encoding hTERT, the nucleic acid encoding SV40LT, or a combination thereof and constitutive expression of hTERT, SV40LT, or a combination thereof. In some embodiments, the expression of the nucleic acid sequence encoding hTERT, SV40LT, or a combination thereof is modulated by an inducible expression system. In some embodiments, the method can further comprise culturing the CAR cell in the presence of an agent recognized by the extracellular domain of the CAR.

In some embodiments, the method may further comprise introducing into the cell a nucleic acid sequence encoding a suicide gene, in some embodiments, the agent is a regulatory compound that binds to an extracellular component of the CAR and mediates a cellular response, for example, the regulatory compound is a ligand of the extracellular domain of the CAR or an antigen to which the extracellular domain of the CAR binds, in some embodiments, the agent is an extracellular domain of an antigen to which the extracellular domain of the CAR binds, for example, the antigen is Epidermal Growth Factor Receptor (EGFR), variant iii of epidermal growth factor receptor (egfrviii), human epidermal growth factor receptor 2(HER2), Mesothelin (MSLN), Prostate Specific Membrane Antigen (PSMA), carcinoembryonic antigen (CEA), bis-ganglioside 2(GD2), interleukin-13 Ra2(IL13R α), glypican-3 (GPC3), carbonic anhydrase ix (caix), L2 cell adhesion molecule (L8-1), cancer antigen (CA125), interleukin-13 Ra2(IL13R α), glypican-3 (GPC3), carbonic anhydrase ix (caix), L2 cell adhesion molecule (L2) cell adhesion molecule (L8-1-CD 6319), CD 465, CD 75, CD 35, CD 75, CD5, CD 75, CD8, CD 75, CD8, CD 9, CD5, CD 9, CD5, CD 9, CD8, CD 9, CD8, CD 9.

In some embodiments, the CAR cell exhibits an increase in cell growth of about 1.5 to 2-fold compared to a CAR cell cultured without the agent. In certain embodiments, the CAR cell exhibits an increase in cell growth of about 1.5 to 3 fold compared to a CAR cell cultured without the agent. In certain embodiments, the CAR cell exhibits an about 2-fold increase in cell growth compared to a CAR cell cultured without the agent.

In some embodiments, the cell density of the CAR cells in the culture medium is at least 25 ten thousand cells per ml of cell culture medium. In certain embodiments, the cell density of the CAR cells is less than 200 x 10⁴Individual cells/ml cell culture medium. In certain embodiments, the cell density of the CAR cells is at 50 x 10⁴To 200X 10⁴Between cells/ml cell culture medium. In certain embodiments, the cell density of the CAR cells is at 50 x 10⁴To 100X 10⁴Between cells/ml cell culture medium.

In some embodiments, the CAR cell is sensitive to tetracycline in the cell culture medium. For example, the CAR cell comprises a third nucleic acid sequence encoding an inverse tetracycline transactivator (rtTA). In certain embodiments, the expression of hTERT, SV40LT is regulated by rtTA such that hTERT, SV40LT is expressed in the presence of tetracycline. For example, the tetracycline is selected from the group of: tetracycline, demeclocycline, meclocycline, doxycycline, lymecycline, methacycline, minocycline, oxytetracycline, rolicycline, and chlortetracycline. In a specific embodiment, the tetracycline is doxycycline. In certain embodiments, the concentration of tetracycline in the cell culture medium is not less than 2 μ g/ml.

In some embodiments, the CAR cells may comprise a fourth nucleic acid sequence encoding a suicide gene such that the CAR cells are cultured with a nucleoside analog in a manner that allows expression of the suicide gene such that the nucleoside analog is cytotoxic, for example, the suicide gene is selected from the group consisting of thymidine kinase of herpes simplex virus, thymidine kinase of varicella zoster virus, and bacterial cytosine deaminase.

Some embodiments relate to an isolated cell obtained using the above method. In some embodiments, a composition comprising an isolated population of cells is provided. In some embodiments, a method of enhancing a T cell response in a subject and/or treating a tumor in a subject can comprise administering an effective amount of a composition.

In some embodiments, a method of generating a CAR T cell is contemplated. The method may comprise: propagating the T cell by transferring one or more nucleic acid sequences to the T cell to obtain a proliferative T cell; and introducing a nucleic acid sequence encoding a CAR into the proliferating T cell to obtain a CAR T cell, wherein the CAR comprises an extracellular domain, a transmembrane domain, and an intracellular domain. For example, the one or more nucleic acid sequences comprise the Tet-inducible HPV16-E6/E7 expression system.

In some embodiments, the T cell is a primary T cell extracted from the subject. In some embodiments, the T cell is a T cell with reduced immunogenicity as compared to a corresponding wild-type T cell in response to T cell infusion.

Some embodiments relate to a method of treating a disease or condition. The methods can include administering a pharmaceutical composition described herein (e.g., a modified T cell population) to a human patient. In certain embodiments, the disease or condition is AIDS, and the antigen binding domain of the CAR binds to a molecule on the surface of HIV. In certain embodiments, the disease or condition is cancer, and the antigen binding domain of the CAR binds to a molecule on the cancer cell and the endogenous TCR number is reduced.

Some embodiments relate to a CAR T cell comprising a nucleic acid sequence encoding a CAR, the CAR comprising an extracellular domain, a transmembrane domain, and an intracellular domain comprising a CD 3-zeta signaling domain and a signaling domain of a costimulatory molecule, wherein a TCR gene of the T cell is disrupted such that expression of the TCR is eliminated.

Some embodiments relate to a CAR T cell comprising a nucleic acid sequence encoding a CAR, the CAR comprising an extracellular domain, a transmembrane domain, and an intracellular domain comprising a CD 3-zeta signaling domain and a signaling domain of a co-stimulatory molecule, wherein the CD4 gene of the T cell is disrupted such that expression of endogenous CD4 is reduced. For example, the antigen binding domain of the CAR binds to a molecule on the surface of HIV and/or tumor cells.

Some embodiments relate to a method of producing conditionally proliferative T cells. The method can include transferring one or more nucleic acid sequences to a T cell to obtain a proliferative T cell, wherein the one or more nucleic acid sequences encode a peptide such that expression of the peptide causes the T cell to become a proliferative T cell, and the peptide is modulated by an inducible expression system, an inducible suicide system, or a combination thereof. In some embodiments, the peptide is hTERT, SV40LT, or a combination thereof. In certain embodiments, the inducible expression system is a rtTA TRE. In certain embodiments, the inducible suicide system is the HSV-TK system or the inducible caspase-9 system.

Some embodiments relate to a method of treating a disease or condition. The method comprises preparing conditionally proliferative T cells using the methods described herein; culturing the conditionally proliferating T cells in a medium comprising tetracycline or doxycycline; culturing the conditionally proliferating T cells in a medium that does not contain any tetracycline or doxycycline to obtain T cells with reduced expression of their SV40LT gene or hTERT gene; and administering to the subject a pharmaceutical composition comprising the obtained T cells.

Some embodiments relate to a pharmaceutical composition obtained using a method described herein for treating a disease or condition, the method for treating a disease or condition comprising preparing conditionally proliferative T cells using the method; culturing the conditionally proliferating T cells in a medium comprising tetracycline or doxycycline; culturing the conditionally proliferating T cells with a medium that does not contain tetracycline or doxycycline to obtain T cells with reduced expression of their SV40LT gene or hTERT gene; and administering to the subject a pharmaceutical composition comprising the obtained T cells. In certain embodiments, the method may further comprise administering ganciclovir to the subject in response to certain predetermined conditions.

In some embodiments, an endogenous gene associated with the biosynthetic or trafficking pathway of the TCR gene of the modified cell is disrupted such that expression of the endogenous TCR is reduced.

Some embodiments relate to a population of T cells comprising the modified cells described herein. In some embodiments, an endogenous gene associated with the biosynthetic or trafficking pathway of the PD-1 gene of the modified cell is disrupted, such that expression of endogenous PD-1 is reduced. In certain embodiments, the modified cell comprises a nucleic acid sequence encoding a truncated PD-1, which truncated PD-1 reduces the inhibitory effect of apoptosis ligand 1(PD-L1) on human T cells.

In some embodiments, a method for making a modified cell is provided. In some embodiments, the method can include obtaining a cell comprising a Chimeric Antigen Receptor (CAR); and culturing the cell in the presence of an agent recognized by the extracellular domain of the CAR. In some embodiments, the methods can be performed for in vitro CAR cell preparation. The method may comprise providing a cell; introducing a nucleic acid sequence encoding a CAR into a cell to obtain a CAR cell; and culturing the CAR cell in the presence of an agent recognized by the extracellular domain of the CAR. In some embodiments, the method can be performed to enrich for cells expressing the CAR. The method may comprise providing a cell; introducing a nucleic acid sequence encoding a CAR into a cell to obtain a cell that expresses the CAR (CAR cell) and a cell that does not express the CAR; and culturing the CAR cell in the presence of an agent that binds the extracellular domain of the CAR to enrich the CAR-expressing cell. In some embodiments, the methods can be performed for in vitro CAR cell preparation. The method may comprise the following steps in the following order: (a) introducing a nucleic acid sequence encoding a CAR into a cell to obtain a CAR cell; (b) culturing the CAR cells using a first culture medium for a predetermined time; and (c) culturing the CAR cell using a second medium, wherein the first medium does not comprise an agent; the second medium comprises an agent, and the agent binds to the extracellular domain of the CAR.

Some embodiments relate to isolated cells obtained by the above methods and pharmaceutical compositions comprising the isolated cells. Some embodiments relate to a method for stimulating an anti-tumor immune response in a subject. The method comprises administering to the subject an effective amount of the pharmaceutical composition. Some embodiments relate to a pharmaceutical composition for use in treating cancer, the method for treating cancer comprising administering to a subject an effective amount of the pharmaceutical composition.

In some embodiments, the modulating compound may comprise at least one of the amino acid sequences of CD19, FZD10, TSHR, PRLR, Muc17, GUCY2C, CD207, CD3, CD5, or CD 4. in some embodiments, the modulating compound comprises at least one of the amino acid sequences of SEQ ID:41-47 and 61-63. in some embodiments, the modulating compound binds the amino acid sequence of SEQ ID:55, 21, 48, 49, estrogen 40, 51-53, and 56-60. in some embodiments, at least one of the amino acid sequences of SEQ ID:41-47, and 56-60. in some embodiments, the modulating compound binds the amino acid sequence of SEQ ID:55, 21, 48, 49, 40, 51-53, and 56-60, in some embodiments, at least one of the group consisting of the soluble protein of the CD-2, the CD-WO 2, CD-9, the CD-123, the CD-2, the CD-123, the CD-2, the CD-III-2, the CD-III-IV, the CD-III-IV-II-IV, the CD-IV-III-IV, the CD-III-IV-III-IV, the CD-III-IV, the CD-IV-II, the CD-II-IV, the CD-III, the CD-.

In some embodiments, a "soluble antigen" is a polypeptide that does not bind to a cell membrane. Soluble antigens are most often ligand-binding polypeptides (e.g., receptors) that lack a transmembrane domain and a cytoplasmic domain. Soluble antigens may include additional amino acid residues, such as affinity tags that provide purification of the polypeptide or provide a site for attachment of the polypeptide to a substrate or immunoglobulin constant region sequence. When the soluble antigenic polypeptide lacks sufficient portions of the transmembrane and intracellular polypeptide segments to provide membrane anchoring or signal transduction, respectively, the soluble antigenic polypeptide is said to be substantially free of these segments. For example, many cell surface receptors occur naturally, while soluble counterparts are produced by proteolysis.

In certain embodiments, the antigen is Epidermal Growth Factor Receptor (EGFR), variant iii of epidermal growth factor receptor (egfrviii), human epidermal growth factor receptor 2(HER2), Mesothelin (MSLN), prostate-specific membrane antigen (PSMA), carcinoembryonic antigen (CEA), disialoganglioside 2(GD2), interleukin-13 Ra2(IL13R α), glypican-3 (GPC3), carbonic anhydrase ix (caix), L1 cell adhesion molecule (L1-CAM), cancer antigen 125(CA125), cluster of differentiation 133(CD133), Fibroblast Activation Protein (FAP), cancer/testis antigen 1B (CTAG1B), mucin 1(MUC1), receptor α (FR- α), CD19, FZD 36, hr, pr8917 lr, tslr 17, gcy 2B 207, CD 9634, CD 369685, CD 369638, CD 3638 mature cell antigen (CD 3985).

In some embodiments, the CAR comprises an extracellular domain, a transmembrane domain, and an intracellular domain comprising a CD 3-zeta signaling domain and a signaling domain of a costimulatory molecule. In certain embodiments, the co-stimulatory molecule of the CAR comprises at least one of CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-L ICOS, lymphocyte function-associated antigen-I (LFA-1), CD2, CD7, LIGHT, NKG2C, or B7-H3.

In some embodiments, the cell is an NK cell, a T cell, or a combination thereof. For example, the cells are T cells derived from primary T cells obtained from a healthy donor or subject.

In some embodiments, the ratio of the amount of the agent to the number of CAR cells after culturing the CAR cells with the agent is 1:50 to 1:5(μ g/10)⁴Individual cell), 1:500 to 1:5(μ g/10)⁴Individual cell), or 1:5000 to 1:5(μ g/10)⁴Individual cells). In certain embodiments, the ratio of the amount of agent to the number of CAR cells is 1:50 to 1:5(μ g/10)⁴Individual cells).

In some embodiments, the culture medium comprises at least one of anti-CD 3 beads, anti-CD 28 beads, and IL 2.

In some embodiments, the copy number of the CAR on the CAR cell is greater than when the CAR cell is cultured without the agent. In certain embodiments, the ratio of the number of cells expressing the CAR to cells not expressing the CAR is greater than when the cells are cultured without the agent.

In some embodiments, the CAR cell can be cultured in the presence of the agent for a predetermined period of time, or the CAR cell can be cultured in the presence of the agent for at least 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days. For example, the predetermined period of time is 7-100 days. In other embodiments, the CAR cell can be cultured without the agent for at least 8,9, 10, 11, 12, or 13 days after the vector comprising the nucleic acid sequence encoding the CAR is introduced into the cell, and then cultured with the agent. In particular embodiments, the CAR cell can be cultured without the agent for about 10 days after the vector comprising the nucleic acid sequence encoding the CAR is introduced into the cell, and then cultured with the agent. In certain embodiments, culturing the T cell in the presence of the agent comprises culturing the T cell with or without the agent for at least 8 days after introducing the vector comprising the nucleic acid sequence encoding the CAR into the T cell, and then culturing the T cell with the agent after the at least 8 days. In certain embodiments, culturing the T cell in the presence of the agent comprises culturing the T cell with or without the agent for at least 10 days after introducing the vector comprising the nucleic acid sequence encoding the CAR into the T cell, and then culturing the T cell with the agent after the at least 10 days.

In some embodiments, the number of CAR cells that produce a memory T cell phenotype when cultured in the presence of the agent is greater than when the CAR cells are cultured without the agent.

In some embodiments, when the CAR cell is cultured without the agent, the amount of cytokine produced by the CAR cell is greater than the amount of cytokine produced by the CAR cell.

In some embodiments, the CAR cells are derived from a healthy donor and have reduced expression of endogenous TCR genes and/or HLA I. In certain embodiments, the CAR cells are derived from a healthy donor and do not elicit a Graft Versus Host Disease (GVHD) response or elicit a reduced GVHD response in a human recipient compared to the GVHD response elicited by primary human T cells isolated from the same human donor, and the expression of endogenous TCR genes and/or HLA I is not reduced, or the expression of endogenous TCR genes and/or HLAI is not disrupted, and endogenous TCR genes and/or HLA I is normally expressed.

In some embodiments, the CAR T cell is a T cell comprising a nucleic acid sequence encoding hTERT, a nucleic acid encoding SV40LT, or a combination thereof. In certain embodiments, the CAR T cell can comprise a nucleic acid sequence encoding hTERT and a nucleic acid encoding SV40 LT. In certain embodiments, the expression of hTERT is modulated by an inducible expression system. In certain embodiments, the expression of the SV40LT gene is modulated by an inducible expression system. In certain embodiments, the inducible expression system is rtTATRE, which increases or activates expression of the SV40LT gene, hTERT gene, or a combination thereof.

In some embodiments, the CAR cell can comprise a nucleic acid sequence encoding a suicide gene. In certain embodiments, the suicide gene is the HSV-TK system.

Some embodiments relate to a method of in vivo cell expansion. In some embodiments, the method comprises administering to the subject an effective amount of a T cell comprising a CAR to provide a T cell response; and administering an effective amount of a presenting cell expressing a soluble agent recognizable by the extracellular domain of the CAR. In some embodiments, the method may be practiced to enhance a T cell response in a subject. The method can include administering to the subject an effective amount of a T cell comprising the CAR to provide a T cell response, and administering an effective amount of a presenting cell expressing a soluble agent recognizable by the extracellular domain of the CAR to enhance the T cell response in the subject. In certain embodiments, the presenting cell is a T cell, a dendritic cell, and/or an antigen presenting cell. In certain embodiments, enhancing a T cell response in a subject comprises selectively enhancing proliferation of a T cell comprising a CAR. In some embodiments, the methods can be used to enhance treatment of a condition in a subject using CAR cells. The method may comprise administering a population of cells expressing the agent or an agent formulated as a vaccine. In these cases, the CAR cell can comprise a nucleic acid sequence encoding the CAR, and the extracellular domain of the CAR can recognize the agent. In some embodiments, the method can be practiced to enhance proliferation of CAR cells in a subject having a disease. The method can include preparing a cell comprising a CAR; administering to the subject an effective amount of a CAR cell; introducing into a cell a nucleic acid sequence encoding an agent recognizable by the extracellular domain of the CAR, and administering to the subject an effective amount of the cell.

T cell responses in a subject refer to cell-mediated immunity associated with helper, killer, regulatory and other types of T cells. For example, T cell responses may include activities such as assisting other leukocytes in the immune process and identifying and destroying virus-infected cells and tumor cells. T cell responses in a subject can be measured by various indicators, such as many virus-infected cells and/or tumor cells that are killed by T cells, the amount of cytokines released by T cells when co-cultured with virus-infected cells and/or tumor cells, the level of proliferation of T cells in a subject, phenotypic changes in T cells (e.g., memory T cell changes), and the level of longevity or longevity of T cells in a subject.

In some embodiments, an in vitro killing assay can be performed by measuring the killing efficacy of CAR T cells by co-culturing the CAR T cells with antigen positive cells by showing a reduction in the number of corresponding antigen positive cells co-cultured with CAR T cells and an increase in the release of IFN γ, TNF α, etc., as compared to control cells that do not express the corresponding antigen, CAR T cells can be considered to have a killing effect on the corresponding antigen positive cells.

In some embodiments, the agent is a ligand for the extracellular domain of the CAR the agent is an antigen to which the extracellular domain of the CAR binds in certain embodiments, the agent includes at least one of the extracellular domains of Epidermal Growth Factor Receptor (EGFR), variant III of epidermal growth factor receptor (EGFRvIII), human epidermal growth factor receptor 2(HER2), Mesothelin (MSLN), Prostate Specific Membrane Antigen (PSMA), carcinoembryonic antigen (CEA), bis-sialoganglioside 2(GD2), interleukin-13 Ra2(IL13R α), glypican-3 (GPC3), Carbonic Anhydrase IX (CAIX), L1 cell adhesion molecule (L1-CAM), cancer antigen 125(CA125), cluster of differentiation 133(CD133), Fibroblast Activation Protein (FAP), cancer/testis antigen 1B (CTAG 5), mucin 1(MUC1), receptor α (NYLR α -24), CD α (CD133), CD 52), CD 8652, CD52, III-III, CD-13, CD 8653, CD-III-27, FAP-III.

In some embodiments, the CAR comprises an extracellular domain, a transmembrane domain, and an intracellular domain comprising a CD 3-zeta signaling domain and a signaling domain of a costimulatory molecule. In certain embodiments, the co-stimulatory molecule of the CAR comprises at least one of CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-L ICOS, lymphocyte function-associated antigen-I (LFA-1), CD2, CD7, LIGHT, NKG2C, or B7-H3.

In some embodiments, the cell or isolated cell is an NK cell, a T cell, or a combination thereof. In certain embodiments, the cell is attenuated as viable and replication deficient. In certain embodiments, the cells are attenuated to be viable and replication defective by gamma irradiation or chemical inactivation. In certain embodiments, the cells or isolated modified cells are obtained from Peripheral Blood Mononuclear Cells (PBMCs) of the subject. In certain embodiments, the cell is a T cell of a subject or a healthy donor. In certain embodiments, the cell is a T cell formulated as a vaccine. In certain embodiments, the cell is an attenuated tumor cell. In certain embodiments, the cell is a modified cell having reduced immunogenicity to allogeneic CAR therapy as compared to a wild-type cell.

In some embodiments, the agent is expressed by a cell, and expression of the agent is modulated by an inducible expression system. In certain embodiments, the agent is expressed by a cell, and expression of the agent is modulated by an inducible suicide gene expression system. In certain embodiments, the agent is a soluble antigen such that the antigen is released by the cell.

Some embodiments relate to an isolated nucleic acid sequence encoding a CAR having a spacer domain. In some embodiments, the isolated nucleic acid sequence can encode a CAR having an extracellular domain, a spacer domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain binds a tumor antigen and the spacer domain comprises the amino acid sequence of SEQ ID No. 68 or 69. In some embodiments, the isolated nucleic acid sequence can encode a CAR having an extracellular domain that binds a tumor antigen, a spacer domain comprising the amino acid sequence of SEQ ID No. 68, a transmembrane domain comprising the amino acid sequence of SEQ ID No. 72 or 75, and an intracellular domain.

Some embodiments relate to a vector comprising an isolated nucleic acid sequence, and to a cell comprising the isolated nucleic acid sequence. For example, the cell may be an NK cell, a T cell, or a combination thereof. Some embodiments relate to a composition comprising a population of T cells having an isolated nucleic acid sequence.

Some embodiments relate to a method for making a cell having a CAR and uses thereof. In some embodiments, the methods may be practiced for stimulating an anti-tumor immune response or treating a condition in a subject. The method can include administering to a subject an effective amount of a pharmaceutical composition comprising a population of human T cells comprising an isolated nucleic acid sequence. In some embodiments, the method can include obtaining a cell comprising an isolated nucleic acid sequence; and culturing the cell in the presence of an agent recognized by the extracellular domain of the CAR. In some embodiments, the methods can be performed for in vitro CAR cell preparation. The method may comprise providing a cell; introducing the isolated nucleic acid sequence into a cell to obtain a CAR cell; and culturing the CAR cell in the presence of an agent recognized by the extracellular domain of the CAR. In some embodiments, the method can be performed for enriching for cells expressing a CAR. The method may comprise providing a cell; introducing the isolated nucleic acid sequence into a cell to obtain a cell that expresses the CAR (CAR cell) and a cell that does not express the CAR; and culturing the CAR cell in the presence of an agent that binds the extracellular domain of the CAR to enrich the CAR-expressing cell.

In certain embodiments, the tumor antigen comprises HER, CD, kappa, or LIGHT chain, CD123, CD, ROR, ErbB/4, EGFR, EGFRvIII, EphA, FAP, carcinoembryonic antigen, EGP, mesothelin, TAG, PSMA, NKG2 ligand, B-H, IL-13 receptor 2, IL-11 receptor, MUC, CA, GD, HMW-MAA, CD171, Lewis Y, G250/CAIX, HLA-AI MAGE A, HLA-A2-ESO-1, PSC, folate receptor-, CD44 v/8, 8H, NCAM, VEGF receptor, 5T, fetal AchR, CD44, LFv, TEM or TEE 8. in certain embodiments, the cell-signaling domain comprises a stimulation domain, CD-4-signaling domain, CD-4 domain, CD-OS-signaling domain, CD-4 domain, CD-4 domain, CD-OS-CD-4 domain, CD-CD.

In some embodiments, the agent is a ligand for the extracellular domain of a CAR, in some embodiments, the agent is an antigen to which the extracellular domain of a CAR binds, in some embodiments, the agent is the extracellular domain of an antigen, in some embodiments, the antigen is Epidermal Growth Factor Receptor (EGFR), variant iii of epidermal growth factor receptor (egfrviii), human epidermal growth factor receptor 2(HER2), Mesothelin (MSLN), Prostate Specific Membrane Antigen (PSMA), carcinoembryonic antigen (CEA), bissialylganglioside 2(GD2), interleukin-13 Ra2(IL13R α), glypican-3 (GPC3), carbonic anhydrase ix caix), L1 cell adhesion molecule (L1-CAM), cancer antigen 125(CA125), cluster of differentiation 133(CD133), fibroblast activation protein (GPC), cancer/testis antigen 1B (CTAG 16), mucin 1(MUC1), cancer antigen 125(CA125), cluster of differentiation 133), CD5, CD 465, CD 4624, a cell stimulating antibody comprising at least one of the antibody that binds to a human folate, a cell, CD 465, CD5, CD 465, a human antibody, a cell stimulating antibody, a human antibody, a cell receptor, a binding antibody, a human antibody, a binding domain, a binding antibody, a CD 465, a binding antibody, and a binding antibody that causes a binding in some embodiments, in certain embodiments, CD 598, CD 465, CD5, CD 465, or a human extracellular domain, and at least one antibody that causes a human extracellular domain.

Nucleic acid sequences encoding the desired molecules can be obtained using recombinant methods known in the art, such as, for example, by screening libraries from cells expressing the genes, by deriving the genes from vectors known to contain them, or by isolating them directly from cells and tissues containing them using standard techniques. Alternatively, the gene of interest may be produced synthetically, rather than cloned.

Embodiments of the present disclosure also relate to a vector into which the DNA of the present disclosure is inserted. Vectors derived from retroviruses (such as lentiviruses) are suitable tools for achieving long-term gene transfer, as they allow long-term stable integration of transgenes and their propagation in progeny cells. Lentiviral vectors have a further advantage over vectors derived from oncogenic retroviruses such as murine leukemia virus, because they can transduce non-proliferating cells such as hepatocytes. They also have the additional advantage of low immunogenicity.

Expression of a CAR-encoding natural or synthetic nucleic acid is typically achieved by operably linking the nucleic acid encoding the CAR polypeptide or portion thereof to one or more promoters and incorporating the construct into an expression vector. The vectors may be suitable for replication and integration into eukaryotes. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters for regulating the expression of the desired nucleic acid sequence.

Additional information related to expression of synthetic nucleic acids encoding CARs and gene transfer into mammalian cells is provided in U.S. patent No. US8,906,682, which is incorporated by reference in its entirety.