Mesothelin CAR and its uses

Abstract

The subject matter of the present disclosure provides a polypeptide composition comprising a chimeric antigen receptor (CAR) targeting mesothelin; and a dominant negative form of programmed death 1 (PD-1DN). Also provided are immune response cells comprising such polypeptide compositions and the use of the polypeptide compositions and immune response cells for treatment, such as the treatment of solid tumors.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 62 / 848,983, filed May 16, 2019, and U.S. Provisional Application No. 62 / 975,966, filed February 13, 2020, the contents of each of which are incorporated by reference in their entirety, and claims the benefit of each claimed priority.

[0003] Sequence Listing

[0004] This application contains a sequence listing, which was submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. The ASCII copy was created on May 13, 2020, is named 0727341041SL_ST25.TXT, and is 144,318 bytes in size. Technical Field

[0005] The present disclosure provides methods and compositions for enhancing immune responses to cancer and pathogens. It relates to a chimeric antigen receptor (CAR) specifically targeting human mesothelin, and immune response cells comprising such CARs. The CAR targeting mesothelin disclosed herein has enhanced immune activation properties, including anti-tumor activity, while having the characteristics of minimizing CAR-induced toxicity and immunogenicity. Background Art

[0006] Cell-based immunotherapy is a treatment with the potential to cure cancer. T cells and other immune cells can be modified to target tumor antigens by introducing genetic material encoding artificial or synthetic antigen receptors (called chimeric antigen receptors (CARs)) specific for selected antigens. Targeted T cell therapies using CARs have recently achieved clinical success in the treatment of some hematological malignancies. However, there are several obstacles to the application of CAR-expressing T cell therapies to solid tumors that must be overcome to achieve clinical benefit. Malignant cells adapt to create an immunosuppressive microenvironment to protect themselves from immune recognition and clearance. This tumor microenvironment poses challenges to therapeutic approaches that involve stimulating an immune response, such as targeted T cell therapies. Solid tumors may also be confined to anatomical compartments that hinder effective T cell trafficking, lack expression of agonistic co-stimulatory ligands, and / or express negative regulators of T cell function. Therefore, successful elimination of solid tumors requires effective tumor penetration and overcoming tumor-induced immunosuppression. In addition, solid tumors pose challenges in selecting optimal immune target antigens that can eradicate the tumor through potent T cells while minimizing or tolerating toxicity to non-tumor tissues.

[0007] Therefore, new therapeutic strategies are needed to design CARs for the treatment of cancer, especially solid tumors, that can induce effective tumor eradication with minimal toxicity and immunogenicity. Summary of the Invention

[0008] The presently disclosed subject matter provides polypeptide compositions comprising (a) a chimeric antigen receptor (CAR) that specifically targets mesothelin (e.g., human mesothelin); (b) a dominant negative form of programmed death 1 (PD-1 DN); immune response cells comprising such polypeptide compositions, and uses of these polypeptide compositions and immune response cells, e.g., for treating cancer.

[0009] The subject matter of the present disclosure provides polypeptide compositions. In certain embodiments, the polypeptide compositions comprise: i) a chimeric antigen receptor (CAR) and ii) a dominant negative form of programmed death 1 (PD-1DN), wherein the CAR comprises (a) an extracellular antigen binding domain and (b) an intracellular signaling domain comprising a modified CD3ζ polypeptide, wherein the CD3ζ polypeptide comprises an ITAM2 variant and an ITAM3 variant, wherein each of the ITAM2 variant and the ITAM3 variant comprises two loss-of-function mutations.

[0010] In certain embodiments, the extracellular antigen-binding domain comprises: a heavy chain variable region comprising a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 76, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 77, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 78; and a light chain variable region comprising a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 79, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 80, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 81.

[0011] In certain embodiments, the PD-1 DN comprises: (a) at least a portion of the extracellular domain of programmed death 1 (PD-1) comprising a ligand binding region, and (b) a first transmembrane domain.

[0012] In certain embodiments, the first transmembrane domain of PD-1 DN comprises a CD8 polypeptide, a CD28 polypeptide, a CD3ζ polypeptide, a CD4 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, a CD166 polypeptide, a CD166 polypeptide, a CD8a polypeptide, a CD8b polypeptide, an ICOS polypeptide, an ICAM-1 polypeptide, a CTLA-4 polypeptide, a CD27 polypeptide, a CD40 / My88 peptide, an NKGD2 peptide, or a combination thereof. In certain embodiments, the first transmembrane domain of PD-1 DN comprises a CD8 polypeptide. In certain embodiments, the CD8 polypeptide contained in the first transmembrane domain of PD-1 DN comprises amino acids 137 to 207 of SEQ ID NO: 86. In certain embodiments, PD-1 DN lacks an intracellular domain. In certain embodiments, PD-1 DN comprises amino acids 21 to 165 of SEQ ID NO: 48 and amino acids 137 to 207 of SEQ ID NO: 86.

[0013] In certain embodiments, the extracellular antigen binding domain of CAR specifically binds to human mesothelin with an EC50 value of about 1 nM to about 25 nM. In certain embodiments, the extracellular antigen binding domain of CAR specifically binds to human mesothelin with an EC50 value of about 20 nM.

[0014] In certain embodiments, the extracellular antigen binding domain of CAR comprises a single-chain variable fragment (scFv), an optionally cross-linked Fab, or F(ab)2. In certain embodiments, the extracellular antigen binding domain of CAR comprises a human scFv. In certain embodiments, the extracellular antigen binding domain of CAR recognizes human mesothelin expressing a level of about 1000 or more mesothelin binding sites / cell.

[0015] In certain embodiments, the heavy chain variable region comprises an amino acid sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence of SEQ ID NO: 82. In certain embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 82.

[0016] In certain embodiments, the light chain variable region comprises an amino acid sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence of SEQ ID NO: 83. In certain embodiments, the light chain variable region comprises the amino acid sequence of SEQ ID NO: 83.

[0017] In certain embodiments, the heavy chain variable region comprises an amino acid sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence of SEQ ID NO: 82, and the light chain variable region comprises an amino acid sequence that is at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence of SEQ ID NO: NO: 83 has an amino acid sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homology or identity. In certain embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 82, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 83.

[0018] In certain embodiments, the extracellular antigen binding domain of the CAR comprises a linker between the heavy chain variable region and the light chain variable region.

[0019] In certain embodiments, the leader sequence is covalently linked to the N-terminus of the extracellular antigen-binding domain. In certain embodiments, the leader sequence comprises a CD8 polypeptide. In certain embodiments, the CD8 polypeptide consists of the amino acid sequence set forth in SEQ ID NO:71. In certain embodiments, at least a portion of the PD-1 extracellular domain comprises amino acids 21 to 165 of SEQ ID NO:48.

[0020] In certain embodiments, each loss-of-function mutation in the modified CD3ζ polypeptide of the CAR is located at a tyrosine amino acid residue. In certain embodiments, the ITAM2 variant comprises or consists of the amino acid sequence set forth in SEQ ID NO: 29. In certain embodiments, the ITAM3 variant comprises or consists of the amino acid sequence set forth in SEQ ID NO: 33. In certain embodiments, the modified CD3ζ polypeptide comprises native ITAM1. In certain embodiments, native ITAM1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23. In certain embodiments, the modified CD3ζ polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 35.

[0021] In certain embodiments, the CAR comprises or consists of the amino acid sequence shown in SEQ ID NO:56.

[0022] In certain embodiments, CAR also includes a second membrane spaning domain.In certain embodiments, the second membrane spaning domain of CAR includes CD8 polypeptides, CD28 polypeptides, CD3 ζ polypeptides, CD4 polypeptides, 4-1BB polypeptides, OX40 polypeptides, CD166 polypeptides, CD166 polypeptides, CD8a polypeptides, CD8b polypeptides, ICOS polypeptides, ICAM-1 polypeptides, CTLA-4 polypeptides, CD27 polypeptides, CD40 / My88 peptides, NKGD2 peptides or its combinations.In certain embodiments, the second membrane spaning domain of CAR includes CD28 polypeptides.

[0023] In certain embodiments, the intracellular signaling domain of CAR further comprises a costimulatory signaling domain. In certain embodiments, the costimulatory signaling region comprises a CD28 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a DAP-10 polypeptide, a CD27 polypeptide, a CD40 / My88 polypeptide, an NKGD2 polypeptide, or a combination thereof. In certain embodiments, the costimulatory signaling region comprises a CD28 polypeptide.

[0024] The subject matter of the present disclosure provides immune response cells comprising the polypeptide compositions disclosed herein. In certain embodiments, PD-1 DN and / or CAR are recombinantly expressed. In certain embodiments, PD-1 DN and / or CAR are expressed by a vector. In certain embodiments, the immune response cells are selected from T cells, natural killer (NK) cells, and pluripotent stem cells from which lymphocytes can be differentiated. In certain embodiments, the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells. In certain embodiments, the immune response cells are T cells. In certain embodiments, the T cells are selected from cytotoxic T lymphocytes (CTLs), regulatory T cells, and natural killer T (NKT) cells. In certain embodiments, the immune response cells are autologous. In certain embodiments, the immune response cells are allogeneic.

[0025] The presently disclosed subject matter also provides compositions comprising the immune response cells disclosed herein. In certain embodiments, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutical composition comprises about 10 4 to 10 6 In certain embodiments, the pharmaceutical composition comprises at least about 10 5 In certain embodiments, the pharmaceutical composition comprises about 10 5 In certain embodiments, the pharmaceutical composition is used to prevent and / or treat a tumor in a subject, treat a subject with tumor recurrence, reduce a subject's tumor burden, increase or prolong the survival of a subject with a tumor, prevent and / or treat an inflammatory disease in a subject, and / or prevent graft rejection in a subject receiving an organ transplant.

[0026] In addition, the presently disclosed subject matter provides nucleic acid compositions comprising polynucleotides encoding the polypeptide compositions disclosed herein. In certain embodiments, the polynucleotide comprises the nucleotide sequence set forth in SEQ ID NO: 123. In certain embodiments, the polynucleotide comprises the nucleotide sequence set forth in SEQ ID NO: 124. The presently disclosed subject matter also provides vectors comprising the nucleic acid compositions disclosed herein. In certain embodiments, the vector is a retroviral vector. In certain embodiments, the retroviral vector is a gamma-retroviral vector or a lentiviral vector.

[0027] The presently disclosed subject matter provides methods for producing the immune response cells disclosed herein. In certain embodiments, the methods comprise introducing a polypeptide composition disclosed herein, a nucleic acid composition disclosed herein, or a vector disclosed herein into an immune response cell.

[0028] The subject matter of the present disclosure provides a kit comprising a polypeptide composition of the present disclosure, a nucleic acid composition of the present disclosure, a vector of the present disclosure, an immune response cell of the present disclosure, or a pharmaceutical composition of the present disclosure. In certain embodiments, the kit further comprises written instructions for treating and / or preventing tumors.

[0029] In addition, the subject matter of the present disclosure provides various methods for using the above-mentioned immune response cells. For example, the subject matter of the present disclosure provides a method for reducing tumor burden in a subject, wherein the method comprises administering to the subject an effective amount of immune response cells or a pharmaceutical composition disclosed herein. In certain embodiments, the method reduces the number of tumor cells in the subject, reduces the size of the tumor, and / or eradicates the tumor.

[0030] The presently disclosed subject matter also provides methods of increasing or prolonging the survival of a subject having a tumor, wherein the method comprises administering to the subject an effective amount of the immune response cells of the present disclosure or the pharmaceutical composition of the present disclosure.

[0031] In certain embodiments, the tumor or tumor is a solid tumor. In certain embodiments, the solid tumor is selected from mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic carcinoma, endometrial cancer, gastric tumor, bile duct cancer, cervical cancer, salivary gland cancer, and combinations thereof.

[0032] The presently disclosed subject matter provides a method for treating a subject with tumor recurrence, the method comprising administering to the subject an effective amount of an immune response cell or pharmaceutical composition disclosed herein. In certain embodiments, the subject receives immunotherapy prior to administration of the immune response cell or composition.

[0033] In addition, the subject of the present disclosure provides a method for increasing the production of immune activation cytokines to respond to the cancer cells or pathogens of the subject. In certain embodiments, the method includes administering an effective amount of immune response cells or pharmaceutical compositions disclosed herein to the subject. In certain embodiments, the immune activation cytokine is selected from granulocyte macrophage colony stimulating factor (GM-CSF), IFN-α, IFN-β, IFN-γ, TNF-α, IL-2, IL-3, IL-6, IL-11, IL-7, IL-12, IL-15, IL-21, interferon regulatory factor 7 (IRF7) and combinations thereof.

[0034] According to the subject matter of the present disclosure, the above-mentioned various methods may include administering at least one immunomodulator. In certain embodiments, at least one immunomodulator is selected from immunostimulators, checkpoint immune blockers, radiotherapeutics, chemotherapeutics, and combinations thereof. In some embodiments, the immunostimulator is selected from IL-12, agonist co-stimulatory monoclonal antibodies, and combinations thereof. In certain embodiments, the immunostimulator is IL-12. In some embodiments, agonist co-stimulatory monoclonal antibodies are selected from anti-4-1BB antibodies, anti-OX40 antibodies, anti-ICOS antibodies, and combinations thereof. In certain embodiments, agonist co-stimulatory monoclonal antibodies are anti-4-1BB antibodies. In certain embodiments, checkpoint immune blockers are selected from anti-PD-L1 antibodies, anti-CTLA-4 antibodies, anti-PD-1 antibodies, anti-LAG3 antibodies, anti-B7-H3 antibodies, anti-TIM3 antibodies, and combinations thereof. In certain embodiments, the checkpoint immune blockers are anti-PD-L1 antibodies or anti-PD-1 antibodies. In certain embodiments, the subject is a human.

[0035] In certain embodiments, the immune response cells are administered to the subject transpleurally or intrapleurally.

[0036] The subject matter of the present disclosure also provides a method for preventing and / or treating an inflammatory disease in a subject. In certain embodiments, the method comprises administering to the subject an immune response cell or pharmaceutical composition of the present disclosure. In certain embodiments, the immune response cell is an immunosuppressive cell. In certain embodiments, the immunosuppressive cell is a regulatory T cell. In certain embodiments, the inflammatory disease is pancreatitis. In certain embodiments, the subject is a human. In certain embodiments, the subject is an organ transplant recipient. In certain embodiments, the subject is a pancreatic transplant recipient.

[0037] The disclosed subject matter further provides methods for preventing graft rejection in a subject receiving an organ transplant. In certain embodiments, the methods comprise administering to the subject an immune response cell or pharmaceutical composition of the present disclosure. In certain embodiments, the immune response cell is an immunosuppressive cell. In certain embodiments, the immunosuppressive cell is a regulatory T cell. In certain embodiments, the subject is a human. In certain embodiments, the subject is a recipient of a pancreatic transplant. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The following detailed description is given by way of example and is not intended to limit the subject matter of the present disclosure to the specific embodiments described, and can be understood in conjunction with the accompanying drawings.

[0039] Figure 1Describe a polypeptide composition according to certain embodiments of the present disclosure. The polypeptide composition comprises a CAR comprising an anti-mesothelin (MSLN) scFv, a CD28 transmembrane domain, a CD28 cytoplasmic signaling domain, and a CD3ζ signaling domain (e.g., comprising an ITAM2 variant and an ITAM3 variant). The CAR is fused to the PD1DNR (and PD1 signaling domain) via a cleavable P2A peptide. SP: signal peptide; scFv: single-chain variable fragment; TM: transmembrane domain; cyt: cytoplasmic domain; DNR: dominant negative receptor; LTR: long terminal repeat.

[0040] Figure 2 The various constructs disclosed in Example 2 are described.

[0041] Figures 3A-3D Virus production in the production cell line RD114 is described. RD114 cells were transduced with H29 viral supernatant at different dilutions (undiluted, 1:2, and 1:4) and stained for CAR expression by flow cytometry using an anti-Fab antibody. RD114 blank cells served as a negative control. Figure 3A RD114 null is shown (as a negative control). Figure 3B Shown undiluted; Figure 3C The supernatant was diluted 1:2; and Figure 3D A 1:4 dilution of the supernatant is shown.

[0042] Figures 4A-4E Describes transduction of human T cells with M28z1XX-P2A-PD1DNR-donor H116-2. PHA-activated T cells were transduced with different concentrations of RD114 viral supernatant ( Figure 4A Display 1:2, Figure 4B Display 1:5, Figure 4C Display 1:7, Figure 4D Display 1:15, Figure 4E Untransduced ("UT") is shown and stained for CAR expression by anti-Fab staining and PD1 DNR staining by anti-PD1 staining using flow cytometry.

[0043] Figures 5A-5E Describes transduction of human T cells with M28z1XX-P2A-PD1DNR-donor H18. PHA-activated T cells were transduced with different concentrations of RD114 viral supernatant ( Figure 5A Display 1:2, Figure 5B Display 1:5, Figure 5C Display 1:10, Figure 5D Display 1:15, Figure 5E Untransduced ("UT") is shown and stained for CAR expression by anti-Fab staining and PD1 DNR staining by anti-PD1 staining using flow cytometry.

[0044] Figures 6A-6F Describes transduction of human T cells with M28z1XX-P2A-PD1DNR-donor H19. PHA-activated T cells were transduced with different concentrations of RD114 viral supernatant ( Figure 6A Display 1:2, Figure 6B Display 1:5, Figure 6C Display 1:7; Figure 6D Display 1:10, Figure 6E Display 1:15, Figure 6F Untransduced ("UT") is shown and stained for CAR expression by anti-Fab staining and PD1 DNR staining by anti-PD1 staining using flow cytometry.

[0045] Figures 7A-7C The correlation between vector copy number (VCN) and mean fluorescence intensity (MFI) was described. PHA-activated T cells were transduced with different concentrations of RD114 viral supernatant and stained for CAR expression by anti-Fab staining and analyzed by flow cytometry. Genomic DNA from transduced T cells was isolated, and vector copy number (VCN / μg DNA) was determined using qPCR. The MFI of CAR-positive cells was correlated with VCN / μg DNA from three different donors. Figure 7A Donor H19 is shown; Figure 7B Donor H18 is shown. Figure 7C Donor H116-2 is shown.

[0046] Figure 8 Characterize the cytotoxicity of transduced T cells from three different donors. High MSLN target cells (MGM) were co-cultured with M28z1xx-PD1DNR CAR T cells from different donors at varying E:T ratios using an impedance-based assay. M28z1xx-PD1DNR CAR T cells mediated cytolysis of MGM cells at a 1:1 E:T ratio. M28z1xx-PD1DNR CAR T cells killed high MSLN target cells.

[0047] Figure 9 An example of impedance-based cytotoxicity measurement (eCTL) is described.

[0048] Figure 10 Describes the parameters for comparative analysis of various constructs using eCTL.

[0049] Figures 11A-11E Characterize the expression of MSLN and PD-L1 in target cell lines. Figure 11A As shown), MGM-PDL1 (as Figure 11B shown) and MSTOG (as Figure 11CAs shown) and lung cancer (A549GM (as shown) Figure 11D MSLN and PD-L1 expression in MGM, MGM-PDL1, and A549GM cell lines were analyzed. MGM, MGM-PDL1, and A549GM cells overexpressed MSLN; MGM-PDL1 cells additionally overexpressed PD-L1.

[0050] Figures 12A-12E Describe the expression of CAR and PD1 in T cells transduced with M28z (e.g. Figure 12A As shown), M28z1xx (as Figure 12B As shown), M28z-PD1DNR (as Figure 12C as shown) and M28z1xx-PD1DNR (as shown) Figure 12D Human T cells transduced with (as indicated) were analyzed by flow cytometry for CAR expression by anti-myc staining and for PD1 / PD1DNR expression by anti-PD1 staining. Figure 12E Untransduced ("UT") T cells are shown.

[0051] Figures 13A-13C The present study describes a comparative analysis of the antitumor efficacy of CAR T cells carrying 1XX domains and PD1DNR against MSLN-high tumor cells (MGM). MSLN-high target cells (MGM) were co-cultured with M28z, M28z1XX, M28z-PD1DNR, M28z1XX-PD1DNR, or untransduced T cells at the indicated E:T ratios. Antitumor efficacy was assessed using an impedance-based assay. Figure 13A Showing an E:T ratio of approximately 3:1. Figure 13B Shows an E:T ratio of approximately 1:1. Figure 13C Showing an E:T ratio of approximately 0.33:1.

[0052] Figure 14 Comparative analysis of the cytotoxicity of CAR T cells carrying 1xx constructs and PD1DNR against high MSLN tumor cells (MGM). High MSLN target cells (MGM) labeled with Chromium 51 were co-cultured with M28z, M28z1xx, M28z-PD1DNR, M28z1xx-PD1DNR, or untransduced T cells at the indicated E:T ratios for 18 hours. Cytotoxicity was determined by Chromium 51 CTL assay.

[0053] Figures 15A-15CThe present study describes a comparative analysis of the antitumor efficacy of CAR T cells carrying the 1xx domain and PD1DNR against MSLN-negative tumor cells (MSTOG). MSLN-negative target cells (MSTOG) were co-cultured with M28z, M28z1xx, M28z-PD1DNR, M28z1xx-PD1DNR, or untransduced T cells at the indicated E:T ratios. Antitumor efficacy was assessed using an impedance-based assay. Figure 15A Showing an E:T ratio of approximately 3:1. Figure 15B Shows an E:T ratio of approximately 1:1. Figure 15C Showing an E:T ratio of approximately 0.33:1.

[0054] Figure 16 The present invention describes a comparative analysis of the cytotoxicity of CAR T cells carrying the 1XX domain and PD1DNR against MSLN-negative tumor cells (MSTOG). MSLN-negative target cells (MSTOG) labeled with Chromium 51 were co-cultured with M28z, M28z1XX, M28z-PD1DNR, M28z1XX-PD1DNR, or untransduced T cells at the indicated E:T ratios for 18 hours. Cytotoxicity was determined by Chromium 51 CTL.

[0055] Figures 17A-17C This study describes a comparative analysis of the antitumor efficacy of CAR T cells carrying 1XX domains and PD1DNR against PDL1-overexpressing MSLN-high tumor cells. PDL1-overexpressing (MGM-PDL1)-high MSLN target cells were co-cultured with M28z, M28z1XX, M28z-PD1DNR, M28z1XX-PD1DNR, or untransduced T cells at the indicated E:T ratios. Antitumor efficacy was assessed using an impedance-based assay. Figure 17A Showing an E:T ratio of approximately 3:1. Figure 17B Shows an E:T ratio of approximately 1:1. Figure 17C Showing an E:T ratio of approximately 0.33:1.

[0056] Figures 18A-18C The present study describes a comparative analysis of the antitumor efficacy of CAR T cells carrying the 1xx domain and PD1DNR against MSLN-high tumor cells (A549GM). MSLN-high target cells (A549GM) were co-cultured with M28z, M28z1xx, M28z-PD1DNR, M28z1xx-PD1DNR, or untransduced T cells at the indicated E:T ratios. Antitumor efficacy was assessed using an impedance-based assay. Figure 18A Showing an E:T ratio of approximately 10:1. Figure 18B Showing an E:T ratio of approximately 5:1. Figure 18C Showing an E:T ratio of approximately 2:1.

[0057] Figures 19A-19C Comparative analysis of the antitumor efficacy of CAR T cells carrying 1xx domains and PD1DNR: low MSLN tumor cells (A549G). Low MSLN target cells (A549G) were co-cultured with M28z, M28z1xx, M28z-PD1DNR, M28z1xx-PD1DNR, or untransduced T cells at the indicated E:T ratios. Antitumor efficacy was assessed using an impedance-based assay. Figure 19A Showing an E:T ratio of approximately 10:1. Figure 19B Showing an E:T ratio of approximately 5:1. Figure 19C Showing an E:T ratio of approximately 2:1.

[0058] Figures 20A-20D Describe the results of in vivo studies of various treatments. Figure 20A Shown is a comparison of the in vivo efficacy of CAR T cells of M28z, M28z with PD1 antibody, and M28z with PD1DNR. Figure 20B Shown is the in vivo efficacy comparison of M28z and M28z1XX+PD1DNR CART cells. Figure 20C Shows tumor burden imaging, demonstrating systemic anti-tumor immunity after tumor re-challenge. Figure 20D Ex vivo immunofluorescence staining of orthotopic MPM tumors showing CAR T cell infiltration.

[0059] Figure 21 Describe the structure and components of the M28z1XXPD1DNR CAR. In contrast to M28z, M28z1XXPD1DNR CAR T cells have a mutated CD3ζ signaling domain, a single functional ITAM, and co-express PD1DNR, which consists of the CD8 transmembrane and hinge domains, and lack the intracellular PD1 signaling domain present in endogenous PD1.

[0060] Figure 22 Describe the structure of the CAR T cell vector.

[0061] Figure 23 Mesothelin (MSLN), PD-L1, and GFP expression in tumor cell lines. MGM, MGM-PDL1, and MSTOG tumor cells were analyzed by flow cytometry for mesothelin (left panel), PD-L1 (center panel), and GFP (right panel). Shown are density plots depicting relative expression intensity, plotted against side scatter area (Y-axis).

[0062] Figures 24A-24D Description of an orthotopic MPM mouse model. Figure 24A The gross appearance of human MPM (upper left panel) is recapitulated in the MPM mouse model (upper right panel), with tumor encapsulating cardiac, lung, and mediastinal structures, and tumor invading the chest wall (lower panel). Figure 24B The tumors showed extensive vascularity as demonstrated by CD34 immunofluorescence. Figure 24C Tumor burden progression monitored by BLI was shown to correlate with tumor volume measured by MRI at the respective time points. Figure 24D Shown is the progression of tumor burden monitored by serial BLI and MRI.

[0063] Figures 25A-25C The analysis of mesothelin expression in human tissues by immunohistochemistry is described. Figure 25A Showing the expression of mesothelin in MPM and normal pleura and pericardium. Figure 25B Showing the expression of mesothelin in lung adenocarcinoma and normal lung tissue. Figure 25C Showing the expression of mesothelin in triple-negative breast cancer and normal breast tissue.

[0064] Figure 26 The figure shows that M28z1XXPD1DNR expression can be titrated using different dilutions of viral supernatant. Human T cells were transduced with different dilutions of viral supernatant encoding M28z1XXPD1DNR (left panel) or mycM28z1XXPD1DNR (center panel). CAR (Y-axis) and PD1 (X-axis) expression on live CD3-positive cells was assessed by flow cytometry. The results depicted are representative of one of three different donors.

[0065] Figure 27 Figure 3 depicts CAR expression relative to VCN as measured by MFI. Human T cells from three different donors were transduced with different dilutions of retroviral supernatant encoding M28z1XXPD1DNR or mycM28z1XXPD1DNR. The MFI of CAR-positive T cells (determined by flow cytometry) is plotted against VCN (determined by qPCR). 2 Values ​​are from linear regression analysis (black line).

[0066] Figures 28A-28D Characterize the expression of PD1 and PD1DNR in mycM28z1XXPD1DNR and mycM28z CAR T cells. Figure 28A The percentage of CAR surface expression on mycM28z and mycM28z1XXPD1DNR CART cells is shown. Figure 28B The percentage of CD3-positive cells showing positive PD1 surface expression. Figure 28C Shown are the MFI of PD1 surface expression on CD3-positive cells. Figure 28D Shown are the fold changes in relative mRNA expression of the PD1 extracellular and PD1 intracellular domains compared to untransduced T cells.

[0067] Figure 29Figure 3: T cells expressing M28z1XXPD1DNR (with or without myc tag) exhibited equivalent antitumor efficacy in vitro. Human T cells from three different donors were transduced with M28z1XXPD1DNR (red) or mycM28z1XXPD1DNR (green) (transduction range, 37%-63%) and co-cultured with MGM cells (green; arrows indicate the time of T cell addition). The antitumor efficacy of the two constructs was compared at the indicated E:T ratios using an impedance-based cytotoxicity assay.

[0068] Figure 30 MycM28z1XXPD1DNR CAR T cells mediate antigen-specific, HLA-independent tumor lysis. Human T cells transduced with mycM28z1XXPD1DNR (blue) or mycM28z (red) were co-cultured with MGM, MGM-PDL1, or MSTOG tumor cells at the indicated E:T ratios. After 18 hours of co-culture, 51 Cr release assay to evaluate CAR T cell cytotoxicity. Untransduced T cells (orange) served as a control.

[0069] Figure 31 Figure 3. Depicts the accumulation of mycM28z1XXPD1DNR CAR-T cells after stimulation with mesothelin-expressing tumor cells. Human T cells transduced with mycM28z1XXPD1DNR (blue) or mycM28z (red) were repeatedly exposed to MGM or MGM-PDL1 target cells at a 1:1 E:T ratio for 48 hours. After each antigen stimulation, CAR T cell accumulation was quantified by absolute CAR T cell counts.

[0070] Figure 32 The results show that mycM28z1XXPD1DNR CAR-T cells exhibited similar cytotoxicity to mycM28zCAR-T cells under initial antigen stimulation. Human T cells transduced with mycM28z1XXPD1DNR (blue) or mycM28z (red) were compared with 51 Cr-labeled MGM or MGM-PDL1 target cells were co-cultured at the indicated E:T ratios and used 18 hours later. 51 Cytotoxicity was assessed by Cr release assay. Untransduced T cells (orange) served as a control.

[0071] Figure 33Figure 3: MycM28z1XXPD1DNR CAR T cells maintain anti-tumor efficacy under repeated antigen stimulation. Human T cells transduced with mycM28z1XXPD1DNR (blue) or mycM28z (red) were repeatedly exposed to MGM (left panel) or MGM-PDL1 (right panel) target cells at an E:T ratio of 3:1 for 48 hours for a total of 4 stimulations, followed by 2 additional stimulations at an E:T ratio of 1:1. After 18 hours of co-culture, the cells were stimulated with the fourth and seventh antigens at the indicated E:T ratios. 51 Cr release assay was used to evaluate the cytotoxicity of CAR T cells.

[0072] Figure 34 Figure 3. MycM28z1XXPD1DNR CAR T cells secrete effector cytokines upon antigen stimulation. Human T cells transduced with mycM28z1XXPD1DNR (blue) or mycM28z (red) were repeatedly exposed to MGM (top row) or MGM-PDL1 (bottom row) target cells at a 1:1 E:T ratio for 48 hours. Cell-free supernatants were collected 24 hours after the first, third, and sixth antigen exposures, and effector cytokine secretion was assessed by Luminex analysis.

[0073] Figure 35 Describe the use of a single low dose of 3 × 10 mycM28z1XXPD1DNR CAR T cells. 4 Intrapleural administration showed anti-tumor efficacy in vivo. Female NSG mice bearing orthotopic MGM tumors received a single intrapleural dose of P28z CART cells (n=6, red bars) or mycM28z1XXPD1DNR CAR T cells (n=10, blue bars). Tumor burden was determined by BLI. The time points shown represent day 15 after CAR T cell administration, when mice treated with P28z CAR T cells began to die. Statistical significance was determined using an unpaired Student's t-test (two-tailed). ***p<0.001.

[0074] Figures 36A-36D describe that intrapleurally administered mycM28z1XXPD1DNR CAR T cells exhibit antitumor efficacy and improve survival in vivo. Figure 3A A single dose of mycM28z (1×10 5 ) or mycM28z1XXPD1DNR (1×10 5 or 5×10 4 ) Serial tumor BLI of female NSG mice bearing MGM-PDL1 tumors treated with CAR T cells (n=7-8). Four mice per treatment group in ventral position are shown. Figure 36BCorresponding serial tumor BLI (average of dorsal and ventral sides) are shown, indicating the tumor burden for each treated mouse. Figure 36C The corresponding mouse body weights after treatment are shown. Figure 36D Shown is a Kaplan-Meier survival analysis comparing the in vivo efficacy of mycM28z and mycM28z1XXPD1DNR CAR T cells. Survival curves were analyzed using the log-rank test. *p < 0.05, **p < 0.01.

[0075] Figure 37 The mycM28z1XXPD1DNR CART cells were detected in primary tumors of mice treated intrapleurally. 5 Mice bearing pleural MGM tumors were treated with untransduced T cells (left), mycM28z CAR T cells (center), or mycM28z1XXPD1DNR CAR T cells (right). Tumor tissues were collected 3 days after intrapleural injection of T cells, fixed, and stained ex vivo for tumor mesothelin (green), human CD45-positive cells (red), and DAPI (nuclei, blue) by immunofluorescence.

[0076] Figure 38A and 38B We show that mycM28z1XXPD1DNR CAR T cells resist tumor reestablishment after repeated tumor challenge in vivo. Figure 38A The scheme of tumor rechallenge experiments is shown: mycM28z or mycM28z1XXPD1DNRCAR T cells were administered intrapleurally (single dose 1×10 5 CAR-T cells) and intrapleural eradication of MGM-PDL1 tumor cells (8×10 5 After the vaccination dose), every 4-8 days, the patient was given an increasing dose (2×10 6 to 11×10 6 ) were intraperitoneally challenged with MGM tumor cells 10 times. Figure 38B Serial BLIs are shown, indicating tumor burden after a single intrapleural dose of mycM28z (2 mice, red line) or mycM28z1XXPD1DNR (3 mice, black line) CAR T cells and tumor rechallenge starting on treatment day 68. Black arrows indicate the time points of intraperitoneal tumor rechallenge with increasing doses.

[0077] Figures 39A-39C Describes that M28z1XXPD1DNRCAR T cells generated using a vector library for clinical trials have anti-tumor efficacy and prolonged survival in vivo. Figure 39A 6×10 cells were produced by CTCEF using viral supernatant used in clinical trials. 4(n=8) or 2×10 5 Serial tumor BLI of MGM tumor-bearing female NSG mice treated with M28z1XXPD1DNR CAR T cells (n=10). Figure 39B The corresponding mouse body weights after treatment are shown. Figure 39C Kaplan-Meier survival analysis is shown.

[0078] Figure 40 Depicts the mean body weight of male mice at mid-term sacrifice. Shown are Group 1 (non-tumor control), Group 3 (control), and Group 5 (test article).

[0079] Figure 41 Depicts the mean body weight of female mice at mid-term sacrifice. Shown are Group 2 (non-tumor control), Group 4 (control), and Group 6 (test article).

[0080] Figure 42 Depicts the mean body weight of male mice at final sacrifice. Shown are Group 7 (non-tumor control), Group 9 (control), and Group 11 (test article).

[0081] Figure 43 Depicts the mean body weight of female mice at final sacrifice. Shown are Groups 8 (non-tumor control), 10 (control), and 12 (test article).

[0082] Figure 44 Figure 3. Identification of human T cells in tumors from CAR T cell-treated and vehicle-treated mice. Intrapleural tumor tissue cells from CAR T cell-treated and vehicle-treated mice were stained with DAPI, anti-human CD45 APC / CY7, and anti-human CD3 PE / CY7 antibodies, and live human T cells were detected by flow cytometry. Shown are density plots of human CD3 expression (X-axis) and human CD45 expression (Y-axis) on DAPI-negative (live) single cells. The gate shows cells that stain positive for human CD45 and human CD3, representing human T cells.

[0083] Figure 45 Figure 3. Identification of human T cells in the spleens of CAR T cell-treated and vehicle-treated mice. Splenic tissue from CAR T cell-treated and vehicle-treated mice was stained with DAPI, anti-human CD45 APC / CY7, and anti-human CD3 PE / CY7 antibodies, and live human T cells were detected by flow cytometry. Shown are density plots of human CD3 expression (X-axis) and human CD45 expression (Y-axis) on DAPI-negative (live) single cells. The gate shows cells that stain positive for both human CD45 and human CD3, representing human T cells.

[0084] Figure 46 Describes BLI in male mice.

[0085] Figure 47 Describes BLI of female mice. DETAILED DESCRIPTION

[0086] The subject matter disclosed in the present invention provides a peptide composition comprising a dominant negative form of a chimeric antigen receptor (CAR) targeting mesothelin and programmed death 1 (PD-1DN), and an immune response cell (e.g., T cell or NK cell) comprising the peptide composition. The disclosed subject matter also provides a method for inducing and / or enhancing the immune response of immune response cells to target antigens and / or treating and / or preventing tumors or other diseases / disorders that require reducing immune cell exhaustion using the peptide composition.

[0087] Continuous antigen exposure of T cells, such as in cancer, leads to changes in the differentiation state of T cells, called exhaustion, which makes CART cells dysfunctional (Youngblood et al., Int Immunol. 2010; 22 (10): 797-803; Wherry et al., Nat Rev Immunol. 2015; 15 (8): 486-499). Previous studies have shown that CAR activation potential is associated with three ITAMs (1-2-3) present in the CD3ζ cytoplasmic domain (Acuto et al., Nat Rev Immunol. 2003; 3 (12): 939-951; Love et al., Cold Spring Harb Perspect Biol. 2010; 2 (6): a002485). Recent studies have shown that this CAR activation potential can be calibrated by mutating ITAMs, thereby reducing its function. Importantly, studies have shown that by introducing point mutations in the second and third ITAMs (1-XX; designated herein as “1XX”) of the CD3ζ domain, the fate of CAR T cells is altered from an exhausted state to a balanced effector and memory state in the presence of high antigen exposure (Feucht et al., Nat Med. 2019; 25(1): 82-88).

[0088] Another obstacle encountered by CAR T cells in the solid tumor microenvironment is the inhibition of cytolytic activity mediated by PD1, an inhibitory receptor expressed after antigen-mediated T cell activation. In addition, after exposure to pro-apoptotic cytokines secreted by T cells, tumor cells enhance the expression of co-inhibitory ligands such as PD-L1 (McGray et al., Mol Ther. 2014; 22(1): 206-218; Spranger et al., Sci Transl Med. 2013; 5(200): 200ra116; Moon et al., Clin Cancer Res. 2014; 20(16): 4262-4273). To overcome this obstacle, our research group combined mesothelin-targeting CAR T cells with PD1 blocking antibodies to rescue exhausted CAR T cells and restore the anti-tumor efficacy of CAR T cells in our orthotopic mouse model (Cherkassky et al., J Clin Invest. 2016; 126(8): 3130-3144; Grosser et al., Cancer Cell. 2019; 36(5): 471-482). To avoid the repeated use of PD1 checkpoint blockade and the associated clinical adverse reactions, our research group has demonstrated that the use of a cell-intrinsic PD1 checkpoint blockade strategy, in which PD1DNR is co-transduced into T cells with second-generation CAR, ultimately renders the transduced cells resistant to tumor PD-L1-mediated inhibition in the solid tumor microenvironment (Cherkassky et al., J Clin Invest. 2016; 126(8): 3130-3144; Grosser et al., Cancer Cell. 2019; 36(5): 471-482).

[0089] Therefore, to develop CART cells with enhanced therapeutic characteristics, functional persistence, and resistance to tumor-mediated suppression, the inventors incorporated 1XX and PD1DNR components into the second-generation CAR vector design, which enabled these cells to function effectively in the microenvironment of highly immunosuppressive solid tumors.

[0090] For the purpose of clarity of disclosure and not limitation, the detailed description is divided into the following subsections:

[0091] 5.1. Definitions;

[0092] 5.2. Peptide compositions;

[0093] 5.2.1.PD-1 DN;

[0094] 5.2.2. CAR targeting mesothelin; and

[0095] 5.2.3. Exemplary polypeptide compositions;

[0096] 5.3. Immune response cells;

[0097] 5.4. Nucleic acid compositions and vectors;

[0098] 5.5. Peptides and analogs;

[0099] 5.6. Pharmaceutical Compositions and Administration;

[0100] 5.7. Preparations;

[0101] 5.8. Treatment methods; and

[0102] 5.9. Kit

[0103] 5.1. Definitions

[0104] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0105] As used herein, the term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, according to practice in the art, "about" can mean within 3 or more standard deviations. Alternatively, "about" can mean a range of up to 20%, e.g., up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, e.g., within 5-fold or 2-fold a value.

[0106] "Immune response cell" refers to a cell that plays a role in an immune response, or a progenitor cell or progeny cell thereof.

[0107] "Activating immune response cells" refers to signal transduction or changes in inducing intracellular protein expression, thereby inducing an immune response. For example, when CD3 chains aggregate in response to ligand binding and immunoreceptor tyrosine-based inhibitory motifs (ITAMs), signal transduction cascades are generated. In certain embodiments, when chimeric antigen receptors (CARs) are bound to antigens, immune synapses are formed, which include aggregation of many molecules (e.g., CD4 or CD8, CD3γ / δ / ε / ζ, etc.) near the binding receptor. This aggregation of membrane-bound signaling molecules allows phosphorylation of the ITAM motifs contained in the CD3 chains. This phosphorylation, in turn, initiates the T cell activation pathway, ultimately activating transcription factors such as NF-κB and AP-1. These transcription factors induce the global gene expression of T cells to increase the production of IL-2, promote proliferation and the expression of master regulator T cell proteins, thereby initiating T cell-mediated immune responses.

[0108] "Stimulating immune response cells" refers to signals that produce a strong and sustained immune response. In various embodiments, this occurs after activation of immune response cells (e.g., T cells), or simultaneously through receptors including but not limited to CD28, CD137 (4-1BB), OX40, CD40, and ICOS. Receiving a variety of co-stimulatory signals is important for establishing a strong, long-term T cell-mediated immune response. T cells are quickly suppressed and become unresponsive to antigens. Although the effects of these co-stimulatory signals may be different, they typically result in increased gene expression, resulting in long-lived, proliferating, and anti-apoptotic T cells that react strongly to antigens, thereby achieving thorough and sustained eradication.

[0109] As used herein, the term "antibody" refers not only to complete antibody molecules, but also to antibody molecule fragments that retain immunogen binding ability. Such fragments are also well known in the art and are frequently used both in vitro and in vivo. Therefore, as used herein, the term "antibody" refers not only to complete immunoglobulin molecules, but also to the well-known active fragments F(ab')2 and Fab. F(ab')2 and Fab fragments that lack the Fc fragment of a complete antibody are cleared from the circulation faster and have less non-specific tissue binding than complete antibodies (Wahl et al., J.Nucl.Med.24:316-325 (1983). As used herein, antibodies include all-natural antibodies, bispecific antibodies, chimeric antibodies, Fab, Fab', single-chain V region fragments (scFv), fusion polypeptides and unconventional antibodies. In certain embodiments, an antibody is a glycoprotein chain comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as V H ) and a heavy chain constant region (C H The heavy chain constant region is composed of three domains: CH1, CH2 and CH3. Each light chain consists of a light chain variable region (abbreviated as V L ) and a light chain constant region C L The light chain constant region consists of a domain C L Composition. H and V L They can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). H and V LIt consists of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with the antigen. The constant region of an antibody mediates the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and the first component (C1q) of the classical complement system.

[0110] As used herein, "CDR" is defined as the complementary determining region amino acid sequence of an antibody, which is the hypervariable region of the immunoglobulin heavy and light chains. See, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, U.S. Department of Health and Human Services, National Institutes of Health (1987). Typically, an antibody comprises three heavy chain and three light chain CDRs or CDR regions in the variable region. CDRs provide most of the contact residues for the binding of an antibody to an antigen or epitope. In certain embodiments, the Kabat system is used to describe CDR regions (Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, 5th edition, U.S. Department of Health and Human Services, National Institutes of Health Publishing No. 91-3242). In certain embodiments, CDRs are identified according to the Kabat system.

[0111] As used herein, the term "single-chain variable fragment" or "scFv" is an immunoglobulin heavy chain variable region (V H ) and light chain variable region (V L ) covalently linked to form V H ::V L Heterodimer fusion protein. H and V L Directly linked or linked by a peptide-encoded linker (e.g., 10, 15, 20, 25 amino acids), connecting V H The N-terminal and V L C-terminus, or V H The C-terminus and V L The N-terminus of the peptide is the N-terminus of the peptide. Linkers are typically rich in glycine for flexibility and serine or threonine for solubility. "Linker" as used herein shall refer to a functional group (e.g., a chemical or polypeptide) that covalently links two or more polypeptides or nucleic acids to connect them to each other. As used herein, "peptide linker" refers to one or more amino acids (e.g., a linker V) used to link two proteins together. H and V LIn certain embodiments, the linker comprises or consists of the amino acid sequence shown in SEQ ID NO: 66, which is provided below:

[0112]

[0113] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO:66 is set forth in SEQ ID NO:50, which is provided below:

[0114]

[0115] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO:66 is set forth in SEQ ID NO:51, which is provided below:

[0116]

[0117] Despite the removal of the constant region and the introduction of a linker, scFv retains the specificity of the original immunoglobulin. Single-chain Fv polypeptide antibodies can be prepared from V-terminal fragments including those described by Huston et al. H and V LThe invention relates to expressing a nucleic acid encoding a gene. (Proc. Nat. Acad. Sci. USA, 85: 5879-5883 1988). See also U.S. Patent Nos. 5,091,513, 5,132,405 and 4,956,778; and U.S. Patent Publication Nos. 20050196754 and 20050196754. Antagonistic scFvs with inhibitory activity have been described (see, e.g., Zhao et al., Hyrbidoma (Larchmt) 2008 27(6):455-51; Peter et al., J Cachexia Sarcopenia Muscle 2012 August 12; Shieh et al., J Imunol 2009 183(4):2277-85; Giomarelli et al., Thromb Haemost 2007 97(6):955-63; Fife et al., J Clin Invst 2006 116(8):2252-61; Brocks et al., Immunotechnology 1997 3(3):173-84; Moosmayer et al., Ther Immunol 1995 2(10:31-40). Agonistic scFvs with stimulatory activity have been described (see, e.g., Peter et al., J Biol Chern 2003 25278(38):36740-7; Xie et al., Nat Biotech 1997 15(8):768-71; Ledberter et al., Crit Rev Immunol 1997 17(5-6):427-55; Ho et al., BioChim Biophys Acta 2003 1638(3):257-66).

[0118] As used herein, "F(ab)" refers to a fragment of an antibody structure that binds to an antigen but is monovalent and does not have an Fc portion, e.g., papain digestion of an antibody produces two F(ab) fragments and an Fc fragment (e.g., a heavy (H) chain constant region; an Fc region that does not bind to an antigen).

[0119] As used herein, "F(ab')2" refers to an antibody fragment produced by pepsin digestion of whole IgG antibodies, wherein the fragment has two antigen-binding (ab') (divalent) regions, wherein each (ab') region comprises two separate amino acid chains, a portion of an H chain and a light chain (L) connected by an SS bond for antigen binding, with the remaining H chain portions linked together. The "F(ab')2" fragment can be split into two separate Fab' fragments.

[0120] As used herein, the term "vector" refers to any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc., that is capable of replication and transfer of gene sequences into cells when combined with appropriate control elements. Thus, the term includes cloning and expression vectors, as well as viral vectors and plasmid vectors.

[0121] As used herein, the term "expression vector" refers to a recombinant nucleic acid sequence, i.e., a recombinant DNA molecule, which contains the desired coding sequence and appropriate nucleic acid sequences required for expression of an operably linked coding sequence in a particular host organism. The nucleic acid sequences necessary for expression in prokaryotes typically include a promoter, an operator (optional), and a ribosome binding site, often along with other sequences. Eukaryotic cells are known to utilize promoters, enhancers, termination, and polyadenylation signals.

[0122] As used herein, the term "affinity" refers to a measure of binding strength. Affinity may depend on the closeness of the stereochemical match between the antibody binding site and the antigenic determinant, the size of the contact area between them, and / or the distribution of charged and hydrophobic groups. Methods for calculating the affinity of an antibody for an antigen are known in the art, including but not limited to various antigen binding experiments, such as functional assays (e.g., flow cytometric analysis).

[0123] The term "chimeric antigen receptor" or "CAR" as used herein refers to a molecule comprising an extracellular antigen binding domain and a transmembrane domain fused to an intracellular signaling domain capable of activating or stimulating an immune response cell. In certain embodiments, the extracellular antigen binding domain of CAR comprises scFv. scFv can be obtained by fusing the variable heavy and light regions of an antibody. Alternatively or additionally, scFv can be derived from Fab (rather than from an antibody, e.g., from a Fab library). In certain embodiments, scFv is fused to a transmembrane domain and then to an intracellular signaling domain. In certain embodiments, a CAR is selected that has a high binding affinity to the antigen.

[0124] As used herein, the term "nucleic acid molecule" includes any nucleic acid molecule encoding a desired polypeptide or fragment thereof. Such a nucleic acid molecule need not be 100% homologous or similar to an endogenous nucleic acid sequence, but may exhibit substantial identity.

[0125] "Substantial identity" or "substantial homology" refers to an amino acid sequence or nucleic acid molecule that has at least about 50% homology or identity to a reference amino acid sequence (e.g., any amino acid sequence described herein) or a reference nucleic acid sequence (e.g., any nucleic acid sequence described herein). In certain embodiments, the sequence has at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or at least about 100% homology or identity to the sequence of the reference amino acid or reference nucleic acid used for comparison.

[0126] Sequence identity can be measured by using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wisconsin 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning homology to various substitutions, deletions, and / or other modifications. Conservative substitutions generally include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine, lysine, arginine; and phenylalanine, tyrosine. In an exemplary method for determining the degree of identity, the BLAST program can be used, and probability scores between e-3 and e-100 indicate closely related sequences.

[0127] As used herein, the percent homology between two amino acid sequences is equivalent to the percent identity between the two sequences. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = number of identical positions / total number of positions × 100), taking into account the number of gaps and the length of each gap, which need to be introduced to achieve optimal alignment of the two sequences. The comparison of sequences and the determination of the percent identity between the two sequences can be accomplished using a mathematical algorithm.

[0128] The percent homology between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4: 11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using the PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent homology between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. 48: 444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (visit www.gcg.com), using either the Blossum 62 matrix or the PAM250 matrix, a gap weight of 16, 14, 12, 10, 8, 6, or 4, and a length weight of 1, 2, 3, 4, 5, or 6.

[0129] Additionally or alternatively, the amino acid sequences of the disclosed subject matter can also be used as a "query sequence" to perform searches against public databases, e.g., to identify related sequences. Such searches can be performed using the XBLAST program (version 2.0) of Altschul et al. (1990) J. Mol. Biol. 215:403-10. BLAST protein searches can be performed using the XBLAST program with a score of 50 and a word length of 3 to obtain amino acid sequences homologous to the specific sequences disclosed herein (e.g., the heavy and light chain variable region sequences of scFvm903, m904, m905, m906, and m900). To obtain gapped alignments for comparison, Gapped BLAST can be used, as described in Altschul et al. (1997) Nucleic Acid Res. 25(17):3389-3402. When using BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. As used herein, the term "constitutive expression" or "constitutive expression" refers to expression or expressed under all physiological conditions.

[0130] "Disease" refers to any condition, illness, or disorder that damages or interferes with the normal function of cells, tissues, or organs, such as tumors and infection with cellular pathogens.

[0131] "Effective amount" refers to an amount sufficient to produce a therapeutic effect. In certain embodiments, an "effective amount" is an amount sufficient to prevent, improve or inhibit the continued proliferation, growth or metastasis (eg, invasion or migration) of a tumor.

[0132] "Modulation" refers to a positive or negative change. Exemplary modulations include changes of about 1%, about 2%, about 5%, about 10%, about 25%, about 50%, about 75%, or about 100%.

[0133] "Increase" means a positive change of at least 5%. The change may be about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, about 100% or more.

[0134] "Decrease" refers to a negative change of at least 5%. The change may be about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, or even about 100%.

[0135] An "isolated cell" is a cell that has been separated from the molecular and / or cellular components with which it naturally accompanies the cell.

[0136] The terms "isolated," "purified," or "biologically pure" refer to substances that are free to varying degrees from components that normally accompany them in their native state. "Isolated" refers to a degree of separation from an original source or environment. "Purified" means a higher degree of separation than isolated. A "pure" or "biologically pure" protein is sufficiently free of other substances such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. In other words, a nucleic acid or polypeptide is "pure" if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "pure" can mean that a nucleic acid or protein forms essentially a single band in an electrophoretic gel. For proteins that can be modified (e.g., phosphorylated or glycosylated), different modifications can produce different isolated proteins, which can be purified separately.

[0137] " tumor " refers to a kind of disease characterized by the pathological proliferation of cells or tissues and its subsequent migration or invasion to other tissues or organs. Tumor growth is usually uncontrolled and progressive, and occurs under the conditions that can not induce or cause normal cell proliferation to stop. Tumor can affect multiple cell types, tissues or organs, including but not limited to being selected from the following organs: bladder, bone, brain, breast, cartilage, glial cells, esophagus, fallopian tube, gall bladder, heart, intestine, kidney, liver, lung, lymph node, nervous tissue, ovary, pancreas, prostate, skeletal muscle, skin, spinal cord, spleen, stomach, testicle, thymus, thyroid, trachea, genitourinary tract, ureter, urethra, uterus and vagina, or its tissue or cell type. Tumor includes cancer, such as sarcoma, malignant epithelial tumor or plasmacytoma (malignant tumor of plasma cells). In some embodiments, tumor is a solid tumor. Tumor can be a primary tumor or primary cancer. In addition, tumor may be in a metastatic state.

[0138] As used herein, the term "conservative sequence modification" refers to amino acid modifications that do not significantly affect or change the binding characteristics of the CAR targeting mesothelin (e.g., the extracellular antigen-binding domain of CAR) comprising an amino acid sequence disclosed herein. Conservative modifications may include amino acid replacements, additions, and deletions. Modifications may be introduced into the extracellular antigen-binding domain of the CAR disclosed herein by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Amino acids can be grouped according to their physicochemical properties (such as charge and polarity). Conservative amino acid replacement refers to replacing amino acid residues with amino acids of the same group. For example, amino acids can be classified by charge: positively charged amino acids include lysine, arginine, histidine, negatively charged amino acids include aspartic acid, glutamic acid, and neutrally charged amino acids include alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In addition, amino acids can be grouped by polarity: polar amino acids include arginine (basic polarity), asparagine, aspartic acid (acidic polarity), glutamic acid (acidic polarity), glutamine, histidine (basic polarity), lysine (basic polarity), serine, threonine, and tyrosine; non-polar amino acids include alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine. Thus, one or more amino acid residues within a CDR region can be replaced with other amino acid residues from the same group, and the altered antibody can be tested for retained function (i.e., the functions described in (c) to (l) above) using the functional assays described herein. In certain embodiments, no more than one, no more than two, no more than three, no more than four, no more than five residues within a specific sequence or CDR region are altered.

[0139] A "signal sequence" or "leader sequence" refers to a peptide sequence (eg, 5, 10, 15, 20, 25, or 30 amino acids) present at the N-terminus of a newly synthesized protein that directs the protein into the secretory pathway. Exemplary leader sequences include, but are not limited to, human IL-2 signal sequence (e.g., MYRMQLLSCIALSLALVTNS [SEQ ID NO: 67]), mouse IL-2 signal sequence (e.g., MYSMQLASCVTLTLVLLVNS [SEQ ID NO: 68]); human kappa leader sequence (e.g., METPAQLLFLLLLWLPDTTG [SEQ ID NO: 69]), mouse kappa leader sequence (e.g., METDTLLLWVLLLWVPGSTG [SEQ ID NO: 70]); human CD8 leader sequence (e.g., MALPVTALLLPLALLLHAARP [SEQ ID NO: 71]); truncated human CD8 signal peptide (e.g., MALPVTALLLPLALLLHA [SEQ ID NO: 72]); human albumin signal sequence (e.g., MKWVTFISLLFSSAYS [SEQ ID NO: 73]); and human prolactin signal sequence (e.g., MDSKGSSQKGSRLLLLLVVSNLLLCQGVVS [SEQ ID NO: IDNO:74]).

[0140] In some embodiments, CAR comprises a CD8 signal peptide at the N-terminus, for example, a signal peptide is connected to the extracellular antigen binding domain of CAR. In some embodiments, the CD8 signal peptide comprises or consists of the amino acid sequence shown in SEQ ID NO:71.

[0141] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 71 is set forth in SEQ ID NO: 125. SEQ ID NO: 125 is provided below.

[0142]

[0143] The terms "comprising" and "including" have the broadest meanings given to them in U.S. patent law and may mean "including," "comprising," and the like.

[0144] As used herein, "treatment" refers to clinical intervention that attempts to alter the course of disease in the individual or cell being treated, and can be performed for prevention or during clinical pathology. Therapeutic benefits include, but are not limited to, preventing the onset or recurrence of a disease, alleviating symptoms, alleviating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating the disease state, and alleviating or improving prognosis. By preventing the progression of a disease or condition, treatment can prevent the condition from worsening in an affected or diagnosed subject or a subject suspected of having the disease, but treatment can also prevent the onset of the disease or symptoms of the disease in a subject at risk of or suspected of having the disease.

[0145] As used herein, an "individual" or "subject" is a vertebrate, such as a human or a non-human animal (e.g., a mammal). Mammals include, but are not limited to, humans, primates, livestock, sports animals, rodents, and pets. Non-limiting examples of non-human animal subjects include rodents, such as mice, rats, hamsters, and guinea pigs; rabbits; dogs; cats; sheep; pigs; goats; cattle; horses; and non-human primates, such as apes and monkeys. As used herein, the term "immunocompromised" refers to a subject that has an immune deficiency. A subject is highly susceptible to opportunistic infections, i.e., infections caused by organisms that would not normally cause illness in a person with a healthy immune system, but that can affect a person with a poorly functioning or suppressed immune system.

[0146] Other aspects of the presently disclosed subject matter are described in the following disclosure and are within the scope of the presently disclosed subject matter.

[0147] 5.2. Peptide Compositions

[0148] The presently disclosed subject matter provides polypeptide compositions comprising a chimeric antigen receptor (CAR) targeting mesothelin and a dominant negative form of programmed death 1 (PD-1 DN).

[0149] 5.2.1. Dominant negative form of programmed death 1 (PD-1 DN)

[0150] A dominant negative form of programmed death 1 (referred to as "PD-1 DN") can enhance the therapeutic effect of immune response cells containing CAR. In certain embodiments, the PD-1 DN comprises (a) at least a portion of the extracellular domain of programmed death 1 (PD-1) comprising a ligand binding region, and (b) a transmembrane domain.

[0151] In certain embodiments, immune response cells (eg, T cells or precursors thereof) are engineered to express a dominant negative form (DN form) of PD-1.

[0152] Malignant cells adapt to produce an immunosuppressive microenvironment that protects cells from immune recognition and elimination (Sharpe et al., Dis. Model Mech. 2015; 8: 337-350). The immunosuppressive microenvironment limits immunotherapy methods. The present disclosure addresses this limitation by expressing a DN form of a cell-mediated immune response inhibitor in an immune response cell or its precursor cell. Detailed information on the DN form of a cell-mediated immune response inhibitor is disclosed in WO2017 / 040945 and WO2017 / 100428 (the contents of each of which are incorporated herein in their entirety).

[0153] Programmed cell death protein 1 (PD-1) is a negative immune regulator when activated T cells bind to their corresponding ligands (PD-L1 and PD-L2, expressed endogenously on macrophages and dendritic cells). PD-1 is a type I membrane protein composed of 268 amino acids. PD-1 has two ligands, PD-L1 and PD-L2, which are members of the B7 family. The protein's structure consists of an extracellular IgV domain, followed by a transmembrane region and an intracellular tail. The intracellular tail contains two phosphorylation sites located within an immunoreceptor tyrosine-based inhibitory motif and an immunoreceptor tyrosine-based switch motif. PD-1 negatively regulates TCR signaling. Phosphatases SHP-1 and SHP-2 bind to the cytoplasmic tail of PD-1 through ligand binding. Upregulation of PD-L1 is one of the mechanisms by which tumor cells evade the host immune system. In preclinical and clinical trials, blocking PD-1 with antagonistic antibodies has induced anti-tumor responses mediated by the host's endogenous immune system.

[0154] In certain embodiments, the PD-1 polypeptide consists of amino acids having GenBank No. NP_005009.2 (SEQ ID No: 48) or a fragment thereof. In certain embodiments, amino acids 1 to 20 of SEQ ID NO: 48 are the signal peptide (or peptide signal) of PD-1. In certain embodiments, amino acids 21 to 170 of SEQ ID NO: 48 are the extracellular domain of PD-1. In certain embodiments, amino acids 171 to 191 of SEQ ID NO: 48 are the transmembrane domain of PD-1. In certain embodiments, amino acids 192 to 288 of SEQ ID NO: 48 are the intracellular domain of PD-1. SEQ ID NO: 48 is provided below:

[0155]

[0156] In certain embodiments, the extracellular domain of PD-1 comprises a ligand-binding domain (referred to as an "extracellular ligand-binding domain"). In certain embodiments, the extracellular ligand-binding domain of PD-1 is fused to one or more heterologous polypeptide sequences, i.e., the PD-1 DN is a chimeric sequence. For example, the extracellular ligand-binding domain of PD-1 may be fused at its N-terminus to a signal peptide that may be a heterologous signal peptide (including the various signal peptides described herein). In addition, the PD-1 DN may comprise a transmembrane domain that may be a heterologous transmembrane domain, including any of the various transmembrane domains described herein.

[0157] In certain embodiments, the PD-1 DN comprises the extracellular domain of the PD-1 polypeptide (e.g., amino acids 21 to 170 of SEQ ID NO: 48) or a ligand-binding portion thereof (e.g., amino acids 21 to 165 of SEQ ID NO: 48). Cells expressing such PD-1 DN may lack or have reduced ability to signal in the PD-1 immune checkpoint pathway. In certain embodiments, the PD-1 DN is a deletion mutant consisting of a deletion of the intracellular domain (e.g., the PD-1 DN lacks amino acids 192 to 288 of SEQ ID NO: 48) or a portion thereof. PD-1 consisting of a deletion of the intracellular domain may reduce or inhibit the PD-1-mediated immune checkpoint pathway.

[0158] In certain embodiments, the PD-1 DN comprises the extracellular ligand-binding domain of PD-1. In certain embodiments, the PD-1 DN comprises the extracellular ligand-binding domain of a PD-1 polypeptide and the transmembrane domain of a PD-1 polypeptide. In certain embodiments, the PD-1 DN comprises or consists of the amino acid sequence of SEQ ID NO:58 (or amino acids 21 to 165 of SEQ ID NO:48). SEQ ID NO:58 is provided below.

[0159]

[0160] An exemplary nucleotide sequence encoding SEQ ID NO:58 (or amino acids 21 to 165 of SEQ ID NO:48) is set forth in SEQ ID NO:59 provided below.

[0161]

[0162] In certain embodiments, the PD-1 DN further comprises a signal peptide. For example, the PD-1 DN comprises the extracellular ligand-binding domain of the PD-1 polypeptide, the transmembrane domain of the PD-1 polypeptide, and the signal peptide of the PD-1 polypeptide. In certain embodiments, the signal peptide comprises or consists of amino acids 1-20 of SEQ ID NO:48. An exemplary nucleotide sequence encoding amino acids 1-20 of SEQ ID NO:48 is set forth in SEQ ID NO:60, provided below.

[0163]

[0164] In certain embodiments, the PD-1 DN comprises or consists of amino acids 1 to 165 of SEQ ID NO:48.

[0165] An exemplary nucleotide sequence encoding amino acids 1-165 of SEQ ID NO:48 is set forth in SEQ ID NO:61 provided below.

[0166]

[0167] In certain embodiments, the PD-1 DN comprises or consists of amino acids 21 to 151 of SEQ ID NO: 48. In certain embodiments, the PD-1 DN comprises or consists of amino acids 1 to 151 of SEQ ID NO: 48. In certain embodiments, the PD-1 DN comprises or consists of amino acids 21 to 151 of SEQ ID NO: 48. In certain embodiments, the PD-1 DN comprises or consists of the amino acid sequence starting from amino acid 21 of SEQ ID NO: 48 to amino acids 151 to 165 of SEQ ID NO: 48.

[0168] In certain embodiments, the PD-1 DN further comprises a CD8 polypeptide. In certain embodiments, the PD-1 DN comprises the extracellular domain of PD-1 or a portion thereof (e.g., an extracellular ligand binding domain) fused to the transmembrane domain and / or hinge domain of CD8. In certain embodiments, the PD-1 DN comprises the transmembrane domain of CD8 (e.g., amino acids 183 to 203 of SEQ ID NO: 86). Such embodiments represent chimeric DN forms comprising transmembrane domains from different (heterologous) polypeptides. As described above, the PD-1 DN comprising a heterologous domain (e.g., a transmembrane domain) may optionally include additional sequences from a heterologous polypeptide. In certain embodiments, the PD-1 DN comprises additional sequences from the N-terminus of a heterologous polypeptide from the transmembrane domain. In certain embodiments, the PD-1 DN comprises the hinge domain of CD8. In certain embodiments, the heterologous sequence comprises an additional N-terminal sequence of a CD8 polypeptide (e.g., amino acids 137 to 182 (or optionally starting at amino acids 138 or 139) of SEQ ID NO: 86). In certain embodiments, the PD-1 DN comprises an additional sequence from the C-terminus of a heterologous polypeptide from the CD8 transmembrane domain. In certain embodiments, the additional C-terminal sequence is amino acids 204 to 209 of SEQ ID NO: 86.

[0169] In certain embodiments, the PD-1 DN comprises the transmembrane domain of a CD8 polypeptide (e.g., amino acids 183 to 203 of SEQ ID NO: 86), the hinge domain of a CD8 polypeptide (e.g., amino acids 137 to 182 of SEQ ID NO: 86), and an additional C-terminal sequence of a CD8 polypeptide (e.g., amino acids 204 to 207 of SEQ ID NO: 86). In certain embodiments, the PD-1 DN comprises a CD8 polypeptide consisting of amino acids 137 to 207 of SEQ ID NO: 86.

[0170] An exemplary nucleotide sequence encoding amino acids 137 to 207 of SEQ ID NO:86 is set forth in SEQ ID NO:62, which is provided below:

[0171]

[0172] In certain embodiments, the PD-1 DN comprises the transmembrane domain of a CD8 polypeptide (e.g., amino acids 183 to 203 of SEQ ID NO: 86), the hinge domain of a CD8 polypeptide (e.g., amino acids 137 to 182 of SEQ ID NO: 86), and an additional C-terminal sequence of a CD8 polypeptide (e.g., amino acids 204 to 209 of SEQ ID NO: 86). In certain embodiments, the PD-1 DN comprises a CD8 polypeptide consisting of amino acids 137 to 209 of SEQ ID NO: 86.

[0173] An exemplary nucleotide sequence encoding amino acids 137 to 209 of SEQ ID NO:86 is set forth in SEQ ID NO:63 provided below:

[0174]

[0175] In certain embodiments, the PD-1 DN comprises the amino acid sequence set forth in SEQ ID NO:49 provided below.

[0176]

[0177] An exemplary nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO:49 is set forth in SEQ ID NO:64, which is provided below:

[0178]

[0179] In certain embodiments, the PD-1 DN comprises the amino acid sequence set forth in SEQ ID NO: 118 provided below.

[0180]

[0181] An exemplary nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 118 is set forth in SEQ ID NO: 119, which is provided below:

[0182]

[0183] In certain embodiments, the transmembrane domain of PD-1 DN comprises a hydrophobic alpha helix across at least a portion of the membrane. Different transmembrane domains result in different receptor stability. According to the present disclosure, the transmembrane domain of PD-1 DN may include the natural or modified transmembrane domain of any polypeptide disclosed herein, for example, any transmembrane domain that may be included in a chimeric antigen receptor. In certain embodiments, the transmembrane domain is a CD8 polypeptide, a CD28 polypeptide, a CD3 ζ polypeptide, a CD40 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, a CD84 polypeptide, a CD166 polypeptide, a CD8a polypeptide, a CD8b polypeptide, an ICOS polypeptide, an ICAM-1 polypeptide, a CTLA-4 polypeptide, a CD27 polypeptide, a CD40 / My88 peptide, a NKGD2 peptide, a synthetic polypeptide (not based on a protein associated with an immune response) or a combination thereof. In certain embodiments, the transmembrane domain is a CD8 polypeptide. The details of these transmembrane domains will be described in the following sections.

[0184] 5.2.2. Chimeric Antigen Receptor (CAR) Targeting Mesothelin

[0185] The polypeptide compositions disclosed herein comprise CARs that specifically target mesothelin (eg, human mesothelin).

[0186] CAR is an engineered receptor that can be implanted or confer specificity to immune effector cells. CAR can be used to transfer the specificity of a monoclonal antibody to a T cell; transfer of its coding sequence is facilitated by retroviral vectors.

[0187] There are three generations of CARs. “First generation” CARs typically consist of an extracellular antigen binding domain (e.g., scFv) fused to a transmembrane domain, which is fused to a cytoplasmic / intracellular signaling domain. “First generation” CARs can provide de novo antigen recognition and activate CD4 through the CD3ζ chain signaling domain in a single fusion molecule. + and CD8 +T cells are independent of HLA-mediated antigen presentation. "Second generation" CAR adds intracellular signaling domains (e.g., CD28, 4-1BB, ICOS, OX40, CD27, CD40 / My88, and NKGD2) from various costimulatory molecules to the cytoplasmic tail of CAR to provide additional signals to T cells. "Second generation" CAR includes CAR that provides costimulation (e.g., CD28 or 4-1BB) and activation (CD3ζ) simultaneously. "Third generation" CAR includes CAR that provides multiple costimulations (such as CD28 and 4-1BB) and activation (CD3ζ). In certain embodiments, CAR is a second generation CAR. In certain embodiments, CAR includes an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain bound to an antigen, wherein the intracellular signaling domain includes a costimulatory signaling domain. In certain embodiments, CAR also includes a hinge / spacer.

[0188] 5.2.2.1. Extracellular Antigen Binding Domain of CAR

[0189] The extracellular antigen binding domain of CAR specifically binds to mesothelin, such as human mesothelin. In certain embodiments, the extracellular antigen binding domain is scFv. In certain embodiments, scFv is human scFv. In certain embodiments, scFv is humanized scFv. In certain embodiments, scFv is mouse scFv. In certain embodiments, the extracellular antigen binding domain of CAR is Fab, which is optionally cross-linked. In certain embodiments, the extracellular antigen binding domain of CAR is F(ab)2. In certain embodiments, any of the above molecules may be included in a fusion protein with a heterologous sequence to form an extracellular antigen binding domain. In certain embodiments, scFv is identified by screening a scFv phage library using an antigen Fc fusion protein. scFv can be obtained from a phage library carrying human V L and / or V H The scFv can also be replaced by a camelid heavy chain (e.g., VHH from Camelidae, Alpaca, etc.) or a portion of a natural ligand for a cell surface receptor.

[0190] Mesothelin is an immunogenic cell surface antigen that is highly expressed in solid cancers. Mesothelin is involved in cell proliferation, adhesion, invasion, cell signaling, and metastasis. Studies have shown that serum soluble mesothelin-related peptides secreted by mesothelin-expressing tumors can be detected in humans and mice and have been shown to be associated with treatment response and prognosis. In normal tissues, mesothelin is only expressed at low levels in the pleura, pericardium, and peritoneum. The anti-mesothelin recombinant immunotoxin SS1P showed specific and significant anti-tumor activity in patients. In a pancreatic cancer vaccine trial, patients with a survival advantage had consistent CD8 +T cell responses to mesothelin are associated with vaccine-induced delayed-type hypersensitivity reactions. Specific T cell epitopes derived from mesothelin have been found to activate human T cells to effectively lyse mesothelin-expressing human tumors. Therefore, there is strong supportive evidence that adoptive immunotherapy targeting mesothelin can be effective against mesothelin-expressing tumors.

[0191] In certain embodiments, the CAR binds to human mesothelin. In certain embodiments, the human mesothelin comprises or consists of the amino acid sequence of NCBI reference number AAV87530.1 (SEQ ID No: 75) or a fragment thereof.

[0192] SEQ ID NO:75 is shown below:

[0193]

[0194] In certain embodiments, the extracellular antigen binding domain of CAR (e.g., comprising scFv or its analogs) binds to human mesothelin with an EC50 value of about 1 nM to about 25 nM as measured by enzyme-linked immunosorbent assay (ELISA). In certain embodiments, the extracellular antigen binding domain of CAR has an EC50 value of about 20 nM as measured by ELISA. In certain embodiments, the extracellular antigen binding domain of CAR comprises an anti-mesothelin antibody or antigen-binding portion thereof described in U.S. Patent No. 8,357,783, the entire contents of which are incorporated herein by reference. In certain embodiments, the extracellular antigen binding domain of CAR is derived from the heavy chain variable region and light chain variable region of an antibody that binds to human mesothelin, for example, Feng et al. in Mol. Cancer Therapy (2009); 8 (5): antibody m912 disclosed in 1113-1118, the entire contents of which are incorporated herein by reference. Antibody m912 was isolated from a human Fab library by screening for recombinant mesothelin. In certain embodiments, the extracellular antigen binding domain of the CAR is derived from a Fab library (e.g., from a human or mouse Fab library).

[0195] The binding of the extracellular antigen binding domain of CAR (embodiments, for example, in scFv or its analogs) can be confirmed by, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, in vivo detection (such as growth inhibition), or Western Blot method. Each of these analyses is typically performed by using a labeling reagent (e.g., antibody or scFv) specific to the target complex to detect the presence of a protein-antibody complex of a specific target. For example, scFv can be radiolabeled and used for radioimmunoassay (RIA) (e.g., see Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986, incorporated herein by reference). Radioisotopes can be detected by using methods such as a gamma counter or a scintillation counter or autoradiography. In certain embodiments, the extracellular antigen binding domain targeting mesothelin is labeled with a fluorescent marker. Non-limiting examples of fluorescent labels include green fluorescent protein (GFP), blue fluorescent proteins (e.g., EBFP, EBFP2, Azurite, and mKalama1), cyan fluorescent proteins (e.g., ECFP, Cerulean, and CyPet), and yellow fluorescent proteins (e.g., YFP, Citrine, Venus, and YPet). In certain embodiments, the human scFv targeting mesothelin is labeled with GFP.

[0196] In certain embodiments, the extracellular antigen binding domain of CAR binds to human mesothelin at a mesothelin level of about 1000 or more mesothelin binding sites / cell. In certain embodiments, the extracellular antigen binding domain of CAR binds to human mesothelin at a mesothelin level of about 1000 to about 50,000 mesothelin binding sites / cell. In certain embodiments, the extracellular antigen binding domain of CAR does not bind to human mesothelin with a mesothelin expression level of less than 1000 mesothelin binding sites / cell, such as human mesothelin expressed in normal tissues, such as normal pleural, pericardial, and peritoneal tissues. In certain embodiments, the extracellular antigen binding domain of CAR does not bind to human mesothelin with a mesothelin expression level of more than 50,000 mesothelin binding sites / cell. In certain embodiments, the human scFv contained in CAR binds to human mesothelin at a mesothelin expression level of about 1000 to about 50,000 mesothelin binding sites / cell. In certain embodiments, the human scFv contained in the CAR does not bind to mesothelin in humans expressing mesothelin at a level greater than 50,000 or less than 1,000 mesothelin binding sites per cell.

[0197] In certain embodiments, the extracellular antigen binding domain of the CAR (e.g., scFv) comprises a heavy chain variable region (V H ), the heavy chain variable region comprises: a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 76 or a conservative modification thereof, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 77 or a conservative modification thereof, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 78 or a conservative modification thereof. In certain embodiments, V H The invention comprises: a CDR1 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 76, a CDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 77, and a CDR3 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 78.

[0198] In certain embodiments, the extracellular antigen binding domain of the CAR (e.g., scFv) comprises a light chain variable region (V L ), the light chain variable region comprises: a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 79 or a conservative modification thereof, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 80 or a conservative modification thereof, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 81 or a conservative modification thereof. In certain embodiments, V L The invention comprises: a CDR1 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 79, a CDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 80, and a CDR3 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 81.

[0199] In certain embodiments, V H comprising: a CDR1 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 76 or a conservative modification thereof, a CDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 77 or a conservative modification thereof, and a CDR3 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 78 or a conservative modification thereof; and the V L The invention comprises: a CDR1 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 79 or a conservative modification thereof, a CDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 80 or a conservative modification thereof, and a CDR3 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 81 or a conservative modification thereof. In certain embodiments, V Hcomprising: a CDR1 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 76, a CDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 77, and a CDR3 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 78; the V L The CDRs are comprised of a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 79, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 80, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 81. In certain embodiments, the CDRs are identified according to the Kabat numbering system.

[0200] In certain embodiments, the heavy chain variable region (V H ) comprises the amino acid sequence shown in SEQ ID NO: 82. In certain embodiments, the light chain variable region (V L ) comprises the amino acid sequence shown in SEQ ID NO: 83. In certain embodiments, the V H Comprising the amino acid sequence shown in SEQ ID NO: 82, the V L comprising the amino acid sequence shown in SEQ ID NO: 83, optionally in V H and V L There is (iii) a linker sequence, such as a linker peptide. In certain embodiments, the linker comprises or consists of the amino acid sequence shown in SEQ ID NO: 66. In certain embodiments, V H comprising an amino acid sequence having at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homology or identity to the amino acid sequence set forth in SEQ ID NO: 82. For example, V H comprising an amino acid sequence having about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homology or identity to the amino acid sequence of SEQ ID NO: 82. In certain embodiments, V H Contains the amino acid sequence shown in SEQ ID NO: 82. In certain embodiments, V L comprising an amino acid sequence having at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homology or identity to the amino acid sequence set forth in SEQ ID NO: 83. For example, V Lcomprising an amino acid sequence having about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homology or identity to the amino acid sequence of SEQ ID NO: 83. In certain embodiments, V L Contains the amino acid sequence shown in SEQ ID NO: 83. In certain embodiments, V H comprising an amino acid sequence having at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homology or identity to the amino acid sequence of SEQ ID NO: 82, and V L comprising an amino acid sequence having at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homology or identity to the amino acid sequence set forth in SEQ ID NO: 83. In certain embodiments, V H Comprising the amino acid sequence shown in SEQ ID NO: 82, V L Comprising the amino acid sequence shown in SEQ ID NO:83.

[0201] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:82 is shown in SEQ ID NO:52.

[0202] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:83 is shown in SEQ ID NO:53.

[0203] In certain embodiments, the extracellular antigen binding domain of CAR (e.g., scFv) comprises an amino acid sequence having at least about 80%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% (e.g., about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) homology or identity to the amino acid sequence shown in SEQ ID NO: 84. In certain embodiments, the extracellular antigen binding domain of CAR (e.g., scFv) comprises or consists of the amino acid sequence shown in SEQ ID NO: 84. In certain embodiments, the extracellular antigen binding domain of the CAR (e.g., scFv) specifically binds to a human mesothelin polypeptide (e.g., a human mesothelin polypeptide comprising the amino acid sequence shown in SEQ ID NO: 75).

[0204] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO:84 is shown in SEQ ID NO:85.

[0205] In certain embodiments, the scFv is a human scFv.

[0206] SEQ ID NOs: 52, 53, and 76-85 are shown below:

[0207]

[0208]

[0209] In certain embodiments, the heavy chain variable region comprises an amino acid sequence that is at least about 80%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% (e.g., about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) homologous or identical to the amino acid sequence of SEQ ID NO:36 provided below.

[0210]

[0211] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 36 is set forth in SEQ ID NO: 37 provided below.

[0212]

[0213] In certain embodiments, the light chain variable region comprises an amino acid sequence that is at least about 80%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% (e.g., about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) homologous or identical to the amino acid sequence of SEQ ID NO:38 provided below.

[0214]

[0215] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 38 is set forth in SEQ ID NO: 39 provided below.

[0216]

[0217] In certain embodiments, the light chain variable region comprises an amino acid sequence that is at least about 80%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% (e.g., about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) homologous or identical to the amino acid sequence of SEQ ID NO:40 provided below.

[0218]

[0219] In certain embodiments, the heavy chain variable region comprises an amino acid sequence that is at least about 80%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% (e.g., about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) homologous or identical to the amino acid sequence of SEQ ID NO:41 provided below.

[0220]

[0221] In certain embodiments, the light chain variable region comprises amino acids 1-107 of SEQ ID NO: 38. In certain embodiments, the light chain variable region comprises amino acids 1-107 of SEQ ID NO: 40.

[0222] In certain embodiments, the extracellular antigen binding domain of the CAR (e.g., scFv) comprises an amino acid sequence that is at least about 80%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% (e.g., about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) homologous or identical to the amino acid sequence of SEQ ID NO: 42 provided below.

[0223]

[0224] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:42 is set forth in SEQ ID NO:45 provided below.

[0225]

[0226]

[0227] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 42 is set forth as SEQ ID NO: 46 provided below. The nucleic acid sequence set forth in SEQ ID NO: 46 is comprehensively optimized for codon usage, which can increase expression of CAR.

[0228]

[0229] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 42 is set forth in SEQ ID NO: 47 provided below. The nucleic acid sequence shown in SEQ ID NO: 47 is comprehensively optimized for codon usage, which can increase expression of CAR.

[0230]

[0231] A V consisting of a V having at least about 80%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% (e.g., about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) homology or sequence identity to a specified sequence (e.g., SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:41, or SEQ ID NO:42). H and / or V L The amino acid sequence may comprise substitutions (e.g., conservative substitutions), insertions, or deletions relative to the specified sequence, but retains the ability to bind to the target antigen (e.g., mesothelin). In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in the specified sequence (e.g., SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 41, or SEQ ID NO: 42). In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs of the extracellular antigen-binding domain (e.g., in the FRs). In certain embodiments, the extracellular antigen-binding domain comprises a V sequence selected from SEQ ID NOs: 82 and 83. H and / or V L Sequences, including post-translational modifications of the sequences (SEQ ID NOs: 82 and 83).

[0232] CAR transmembrane domain

[0233] In certain embodiments, CAR includes a transmembrane domain. In certain embodiments, the transmembrane domain of CAR includes a hydrophobic alpha helix across at least a portion of the membrane. Different transmembrane domains lead to different receptor stability. After antigen recognition, receptors aggregate and send signals to cells. According to the present disclosure, the transmembrane domain of CAR may include CD8 polypeptides, CD28 polypeptides, CD3 ζ polypeptides, CD40 polypeptides, 4-1BB polypeptides, OX40 polypeptides, CD84 polypeptides, CD166 polypeptides, CD8a polypeptides CD8b polypeptides, ICOS polypeptides, ICAM-1 polypeptides, CTLA-4 polypeptides, CD27 polypeptides, CD40 / My88 peptides, NKGD2 peptides, synthetic polypeptides (not based on proteins associated with immune responses) or a combination thereof of natural or modified transmembrane domains.

[0234] CD8

[0235] In certain embodiments, the transmembrane domain comprises a CD8 polypeptide (e.g., a transmembrane domain of CD8 or a portion thereof). In certain embodiments, the transmembrane domain comprises a transmembrane domain of human CD8 or a portion thereof. In certain embodiments, the CD8 polypeptide comprises or consists of an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or at least about 100% homology or identity to a sequence or fragment thereof with NCBI reference number NP_001139345.1 (SEQ ID No: 86) and / or optionally comprising at most one, at most two, or at most three conservative amino acid substitutions. In certain embodiments, the CD8 polypeptide comprises or consists of an amino acid sequence of a contiguous portion of SEQ ID NO: 86 having a length of at least about 20, or at least about 30, or at least about 40, or at least about 50, or at least about 60, or at least about 70, and at most about 235 amino acids. In certain embodiments, the CD8 polypeptide comprises or consists of the amino acid sequence of amino acids 1 to 235, 1 to 50, 50 to 100, 100 to 150, 150 to 200, 137 to 209, or 200 to 235 of SEQ ID NO: 86. In certain embodiments, the CAR of the presently disclosed subject matter comprises a transmembrane domain comprising a CD8 polypeptide comprising or consisting of the amino acid sequence of amino acids 137 to 209 of SEQ ID NO: 86. In certain embodiments, the transmembrane domain of the CAR comprises a CD8 polypeptide comprising or consisting of amino acids 137 to 207 of SEQ ID NO: 86.

[0236]

[0237] In certain embodiments, the transmembrane domain comprises the transmembrane domain of mouse CD8 or a portion thereof. In certain embodiments, the CD8 polypeptide comprises or consists of an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homology or identity to the sequence of NCBI reference number AAA92533.1 (SEQ ID NO: 87) or a fragment thereof and / or optionally comprising at most one, at most two, or at most three conservative amino acid substitutions. In certain embodiments, the CD8 polypeptide comprises or consists of an amino acid sequence of a contiguous portion of SEQ ID NO: 87, having a length of at least about 20, or at least about 30, or at least about 40, or at least about 50, or at least about 60, or at least about 70, or at least about 100, or at least about 200, and at most 247 amino acids. In certain embodiments, the CD8 polypeptide comprises or consists of an amino acid sequence of amino acids 1 to 247, 1 to 50, 50 to 100, 100 to 150, 150 to 200, 151 to 219, or 200 to 247 of SEQ ID NO: 87. In certain embodiments, the transmembrane domain of the CAR comprises a CD8 polypeptide comprising or consisting of amino acids 151 to 219 of SEQ ID NO: 87.

[0238]

[0239] In certain embodiments, the CD8 polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO:88, which is provided below:

[0240]

[0241] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO:88 is set forth in SEQ ID NO:89, which is provided below.

[0242]

[0243] CD28

[0244] In certain embodiments, the transmembrane domain of CAR comprises a CD28 polypeptide (e.g., a transmembrane domain of CD28 or a portion thereof). In certain embodiments, the transmembrane domain comprises a transmembrane domain of human CD28 or a portion thereof. In certain embodiments, the CD28 polypeptide comprises or consists of a sequence with NCBI reference number NP_006130 (SEQ ID NO: 90) or a fragment thereof having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or at least about 100% homology or identity and / or may optionally comprise an amino acid sequence of up to one or up to two or up to three conservative amino acid substitutions. In certain embodiments, the CD28 polypeptide comprises or consists of an amino acid sequence of a continuous portion of SEQ ID NO: 90, having a length of at least about 20, at least about 25, or at least about 30, or at least about 40, or at least about 50, and a maximum of about 220 amino acids. In certain embodiments, the CD28 polypeptide comprises or consists of an amino acid sequence of amino acids 1 to 220, 1 to 50, 50 to 100, 100 to 150, 114 to 220, 153 to 179, 150 to 200, or 200 to 220 of SEQ ID NO: 90. In certain embodiments, the transmembrane domain of the CAR comprises a CD28 polypeptide comprising or consisting of SEQ ID NO: 92 (or amino acids 153 to 179 of SEQ ID NO: 90). An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 92 or amino acids 153 to 179 of SEQ ID NO: 90 is set forth in SEQ ID NO: 93. In certain embodiments, the transmembrane domain of the CAR comprises a CD28 polypeptide comprising or consisting of an amino acid sequence of amino acids 114 to 220 of SEQ ID NO: 90. An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 92 (or amino acids 153 to 179 of SEQ ID NO: 90) is shown in SEQ ID NO: 91. SEQ ID NOs: 90-93 are shown below:

[0245]

[0246] In certain embodiments, the transmembrane domain comprises the transmembrane domain of mouse CD28, or a portion thereof. In certain embodiments, the CD28 polypeptide comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the sequence of NCBI Reference No. NP_031668.3 (SEQ ID NO: 97), or a fragment thereof, and / or optionally comprises up to one, two, or three conservative amino acid substitutions. In certain embodiments, the CD28 polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 97 and is at least about 20, at least about 30, at least about 40, or at least about 50, and up to 218 amino acids in length. In certain embodiments, the CD28 polypeptide comprises or consists of an amino acid sequence of amino acids 1 to 218, 1 to 50, 50 to 100, 100 to 150, 114 to 220, 150 to 200, 151 to 177, or 200 to 218 of SEQ ID NO: 97. In certain embodiments, the transmembrane domain of the CAR comprises a CD28 polypeptide comprising or consisting of amino acids 151 to 177 of SEQ ID NO: 97.

[0247] SEQ ID NO:97 is shown below:

[0248]

[0249] CD84

[0250] In certain embodiments, the transmembrane domain of the CAR comprises a natural or modified transmembrane domain of a CD84 polypeptide or a portion thereof. The CD84 polypeptide may have an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or at least about 100% homology or identity to the sequence of NCBI reference number NP_001171808.1 (SEQ ID No: 1) or a fragment thereof, and / or may optionally comprise at most one, at most two, or at most three conservative amino acid substitutions. In certain embodiments, the CD84 polypeptide comprises or consists of an amino acid sequence of a contiguous portion of SEQ ID NO: 1, having a length of at least about 20, or at least about 30, or at least about 40, or at least about 50, and at most about 345 amino acids. In certain embodiments, the CD84 polypeptide comprises or consists of an amino acid sequence of amino acids 1 to 345, 1 to 50, 50 to 100, 100 to 150, 150 to 200, 226 to 250, 250 to 300, or 300 to 345 of SEQ ID NO: 1. In certain embodiments, the transmembrane domain of the CAR comprises or consists of a CD84 polypeptide comprising or consisting of amino acids 226 to 250 of SEQ ID NO: 1.

[0251] The SEQ ID NO: 1 is shown below:

[0252]

[0253] An exemplary nucleotide sequence encoding amino acids 226 to 250 of SEQ ID NO: 1 is set forth in SEQ ID NO: 2 provided below.

[0254]

[0255] CD166

[0256] In certain embodiments, the membrane spaning domain of CAR includes a natural or modified membrane spaning domain of CD166 polypeptides or a part thereof.CD166 polypeptides may have a sequence or fragment thereof with NCBI reference number NP_001618.2 (SEQ ID NO:3) with at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or at least about 100% homology or identity and / or may optionally include up to one or up to two or up to three conservative amino acid replacement amino acid sequences. In certain embodiments, CD166 polypeptides include or are composed of SEQ ID NO:3 continuous portion amino acid sequence, its length is at least about 20, or at least about 30, or at least about 40, or at least about 50, at least about 100, and up to about 583 amino acids. In certain embodiments, the CD166 polypeptide comprises or is comprised of SEQ ID NO:3 amino acid 1 to 583, 1 to 50, 50 to 100, 100 to 150, 150 to 200, 200 to 300, 300 to 400, 400 to 500, 528 to 549 or 500 to 583 amino acid sequence composition. In certain embodiments, the CD166 polypeptide comprised in the transmembrane domain of CAR of the present disclosure comprises or is comprised of SEQ ID NO:3 amino acid 528 to 553 amino acid sequence composition. In certain embodiments, the CD166 polypeptide comprised in the transmembrane domain of CAR comprises or is comprised of SEQ ID NO:3 amino acid 528 to 549 amino acid sequence composition.

[0257] SEQ ID NO: 3 is shown below:

[0258]

[0259] An exemplary nucleotide sequence encoding amino acids 528 to 553 of SEQ ID NO: 3 is provided as SEQ ID NO: 4 below.

[0260]

[0261] CD8a

[0262] In certain embodiments, the transmembrane domain of the CAR includes a natural or modified transmembrane domain of a CD8a polypeptide or a portion thereof. The CD8a polypeptide may have an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or at least about 100% homology or identity to a sequence consisting of NCBI reference number NP_001139345.1 (SEQ ID No: 5) or a fragment thereof, and / or may optionally contain at most one, at most two, or at most three conservative amino acid substitutions. In certain embodiments, the CD8a polypeptide comprises or consists of an amino acid sequence of a continuous portion of SEQ ID NO: 5 having a length of at least about 20, or at least about 30, or at least about 40, or at least about 50, and at most about 235 amino acids. In certain embodiments, the CD8a polypeptide comprises or consists of an amino acid sequence of amino acids 1 to 235, 1 to 50, 50 to 100, 100 to 150, 183 to 207, 150 to 200, or 200 to 235 of SEQ ID NO: 5. In certain embodiments, the transmembrane domain of the CAR comprises a CD8a polypeptide comprising or consisting of amino acids 183 to 207 of SEQ ID NO: 5. SEQ ID NO: 5 is provided below:

[0263]

[0264] An exemplary nucleotide sequence encoding amino acids 183 to 207 of SEQ ID NO: 5 is set forth in SEQ ID NO: 6 provided below.

[0265]

[0266] In certain embodiments, the transmembrane domain of the CAR comprises a natural or modified transmembrane domain of a CD8b polypeptide or a portion thereof. The CD8b polypeptide may have an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or at least about 100% homology or identity to a sequence or fragment thereof with NCBI reference number NP_742099.1 (SEQ ID No: 7), and / or may optionally comprise at most one, at most two, or at most three conservative amino acid substitutions. In certain embodiments, the CD8b polypeptide comprises or consists of an amino acid sequence of a continuous portion of SEQ ID NO: 7 having a length of at least about 20, or at least about 30, or at least about 40, or at least about 50 and at most about 221 amino acids. In certain embodiments, the CD8b polypeptide comprises or consists of an amino acid sequence of amino acids 1 to 221, 1 to 50, 50 to 100, 100 to 150, 171 to 195, 150 to 200, or 200 to 221 of SEQ ID NO: 7. In certain embodiments, the transmembrane domain of the CAR comprises a CD8b polypeptide comprising or consisting of amino acids 171 to 195 of SEQ ID NO: 7. SEQ ID NO: 7 is provided below:

[0267]

[0268] An exemplary nucleotide sequence encoding amino acids 171 to 195 of SEQ ID NO: 7 is set forth in SEQ ID NO: 8 provided below.

[0269]

[0270] ICOS

[0271] In certain embodiments, the transmembrane domain of the CAR comprises a native or modified transmembrane domain of an ICOS polypeptide or a portion thereof. The ICOS polypeptide may have an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or at least about 100% homology or identity to the sequence of NCBI reference number NP_036224.1 (SEQ ID No: 9) or a fragment thereof, and / or may optionally comprise up to one, up to two, or up to three conservative amino acid substitutions. In certain embodiments, the ICOS polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 9, having a length of at least about 20, or at least about 30, or at least about 40, or at least about 50, or up to about 199 amino acids. In certain embodiments, the ICOS polypeptide comprises or consists of amino acids 1 to 199, 1 to 50, 50 to 100, 100 to 150, 141 to 165, or 150 to 199 of SEQ ID NO: 9. In certain embodiments, the transmembrane domain of the CAR comprises an ICOS polypeptide comprising or consisting of amino acids 141 to 165 of SEQ ID NO: 9. SEQ ID NO: 9 is shown below:

[0272]

[0273] An exemplary nucleotide sequence encoding amino acids 141 to 165 of SEQ ID NO: 9 is set forth in SEQ ID NO: 10 provided below.

[0274]

[0275] In certain embodiments, the transmembrane domain of the CAR comprises a native or modified transmembrane domain of a CTLA-4 polypeptide or a portion thereof. The CTLA-4 polypeptide may have an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homology or identity to the sequence of NCBI Reference No. NP_005205.2 (SEQ ID No: 11) or a fragment thereof, and / or may optionally contain up to one, two, or three conservative amino acid substitutions. In certain embodiments, the CTLA-4 polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 11 and is at least about 20, or at least about 30, or at least about 40, or at least about 50, and up to about 223 amino acids in length. In certain embodiments, the CTLA-4 polypeptide comprises or consists of an amino acid sequence of amino acids 1 to 223, 1 to 50, 50 to 100, 100 to 150, 162 to 186, 150 to 200, or 200 to 223 of SEQ ID NO: 11. In certain embodiments, the transmembrane domain of the CAR comprises a CTLA-4 polypeptide comprising or consisting of amino acids 162 to 186 of SEQ ID NO: 11. SEQ ID NO: 11 is shown below:

[0276]

[0277] An exemplary nucleotide sequence encoding amino acids 162 to 186 of SEQ ID NO: 11 is set forth in SEQ ID NO: 12 provided below.

[0278]

[0279] ICAM-1

[0280] In certain embodiments, the transmembrane domain of the CAR comprises a native or modified transmembrane domain of an ICAM-1 polypeptide or a portion thereof. The ICAM-1 polypeptide may have an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the sequence of NCBI Reference No. NP_000192.2 (SEQ ID NO: 13) or a fragment thereof, and / or may optionally comprise up to one, two, or three conservative amino acid substitutions. In certain embodiments, the ICAM-1 polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 13 and is at least about 20, or at least about 30, or at least about 40, or at least about 50, and up to about 220 amino acids in length. In certain embodiments, the ICAM-1 polypeptide comprises or consists of amino acids 1 to 532, 1 to 50, 50 to 100, 100 to 150, 150 to 200, 200 to 300, 300 to 400, 481 to 507, 400 to 500, or 500 to 532 of SEQ ID NO: 13. In certain embodiments, the transmembrane domain of the CAR comprises an ICAM-1 polypeptide comprising or consisting of amino acids 481 to 507 of SEQ ID NO: 13. SEQ ID NO: 13 is shown below:

[0281]

[0282] An exemplary nucleotide sequence encoding amino acids 481 to 507 of SEQ ID NO: 13 is set forth in SEQ ID NO: 14 provided below.

[0283]

[0284] 5.2.2.3. CAR Hinge / Spacer

[0285] In some embodiments, CAR includes hinge / spacer for connecting extracellular antigen-binding domain to membrane spaning domain.Hinge / spacer can be flexible enough to allow antigen-binding domain to be oriented in different directions to promote antigen recognition.In some embodiments, hinge / spacer of CAR can include CD8 polypeptides, CD28 polypeptides, CD3 ζ polypeptides, CD40 polypeptides, 4-1BB polypeptides, OX40 polypeptides, CD84 polypeptides, CD166 polypeptides, CD8a polypeptides, CD8b polypeptides, ICOS polypeptides, ICAM-1 polypeptides, CTLA-4 polypeptides, CD27 polypeptides, CD40 / My88 peptides, NKGD2 peptides, synthetic polypeptides (not based on proteins associated with immune response) or a combination thereof Natural or modified hinge region. The hinge / spacer can be a hinge region from IgG1, or a portion of the CH2CH3 region and CD3 of an immunoglobulin, a portion of a CD28 polypeptide (e.g., a portion of SEQ ID NO:90), a portion of a CD8 polypeptide (e.g., a portion of SEQ ID NO:86 or a portion of SEQ ID NO:87), a variant having at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% homology or identity to any of the foregoing, or a synthetic spacer sequence.

[0286] CD28

[0287] In certain embodiments, as described herein, the hinge / spacer region of CAR comprises a natural or modified hinge region of a CD28 polypeptide or a portion thereof. In certain embodiments, the hinge / spacer region of CAR comprises a CD28 polypeptide comprising or consisting of an amino acid sequence shown in SEQ ID NO: 15 (or amino acids 114 to 152 in SEQ ID NO: 90). SEQ ID NO: 15 is provided below.

[0288]

[0289] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 15 (or amino acids 114 to 152 of SEQ ID NO: 90) is set forth in SEQ ID NO: 54 provided below.

[0290]

[0291] CD84

[0292] In certain embodiments, the hinge / spacer region of the CAR comprises a native or modified hinge region of a CD84 polypeptide or a portion thereof, as described herein. In certain embodiments, the hinge / spacer region of the CAR comprises a CD84 polypeptide comprising or consisting of amino acids 187 to 225 of SEQ ID NO: 1. An exemplary nucleotide sequence encoding amino acids 187 to 225 of SEQ ID NO: 1 is set forth in SEQ ID NO: 16 provided below.

[0293]

[0294] CD166

[0295] In certain embodiments, as described herein, the hinge / spacer of CAR includes the natural or modified hinge region of CD166 polypeptide or a part thereof. In certain embodiments, the hinge / spacer of CAR includes CD166 polypeptide, and the polypeptide includes or is made up of SEQ ID NO:3 amino acid 489 to 527. Encoding SEQ ID NO:3 amino acid 489 to 527 exemplary nucleic acid sequences are listed in SEQ ID NO provided below: 17.

[0296]

[0297] In certain embodiments, the hinge / spacer region of CAR includes a CD166 polypeptide comprising or consisting of SEQ ID NO:3 amino acids 484 to 527. In certain embodiments, the hinge / spacer region of CAR includes a CD166 polypeptide comprising or consisting of SEQ ID NO:3 amino acids 506 to 527. In certain embodiments, the hinge / spacer region of CAR includes a CD166 polypeptide comprising or consisting of SEQ ID NO:3 amino acids 517 to 527. In certain embodiments, the hinge / spacer region of CAR includes a CD166 polypeptide comprising or consisting of SEQ ID NO:109 or SEQ ID NO:110 amino acid sequence. SEQ ID NOs:109 and 110 are shown below.

[0298]

[0299] In certain embodiments, the CD166 polypeptide included in the hinge / spacer and transmembrane domain of CAR comprises or consists of an amino acid sequence shown in SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116 or SEQ ID NO: 117. SEQ ID NO: 111-117 are as follows.

[0300]

[0301] CD8a

[0302] In certain embodiments, as described herein, the hinge / spacer region of the CAR comprises a native or modified hinge region of a CD8a polypeptide or a portion thereof. In certain embodiments, the hinge / spacer region of the CAR comprises a CD8a polypeptide comprising or consisting of amino acids 137 to 182 of SEQ ID NO: 5. An exemplary nucleotide sequence encoding amino acids 137 to 182 of SEQ ID NO: 5 is set forth in SEQ ID NO: 18 provided below.

[0303]

[0304] CD8b

[0305] In certain embodiments, the hinge / spacer region of the CAR comprises a native or modified hinge region of a CD8b polypeptide as described herein. In certain embodiments, the CD8b polypeptide contained in the hinge / spacer region of the CAR comprises or consists of amino acids 132 to 170 of SEQ ID NO: 7. An exemplary nucleotide sequence encoding amino acids 132 to 170 of SEQ ID NO: 7 is set forth in SEQ ID NO: 19 provided below.

[0306]

[0307] ICOS

[0308] In certain embodiments, the hinge / spacer region of the CAR comprises a native or modified hinge region of an ICOS polypeptide or portion thereof, as described herein. In certain embodiments, the hinge / spacer region of the CAR comprises an ICOS polypeptide comprising or consisting of amino acids 102 to 140 of SEQ ID NO: 9. An exemplary nucleotide sequence encoding amino acids 102 to 140 of SEQ ID NO: 9 is set forth in SEQ ID NO: 20 provided below.

[0309]

[0310] CTLA-4

[0311] In certain embodiments, the hinge / spacer region of the CAR comprises a native or modified hinge region of a CTLA-4 polypeptide or a portion thereof, as described herein. In certain embodiments, the hinge / spacer region of the CAR comprises a CTLA-4 polypeptide comprising or consisting of amino acids 123 to 161 of SEQ ID NO: 11. An exemplary nucleotide sequence encoding amino acids 123 to 161 of SEQ ID NO: 11 is set forth in SEQ ID NO: 21 provided below.

[0312]

[0313] ICAM-1

[0314] In certain embodiments, the hinge / spacer region of the CAR comprises a native or modified hinge region of an ICAM-1 polypeptide or a portion thereof, as described herein. In certain embodiments, the hinge / spacer region of the CAR comprises an ICAM-1 polypeptide comprising or consisting of amino acids 442 to 480 of SEQ ID NO: 13. An exemplary nucleotide sequence encoding amino acids 442 to 480 of SEQ ID NO: 13 is set forth in SEQ ID NO: 22 provided below.

[0315]

[0316] In certain embodiments, the CAR targeting mesothelin includes hinge / spacer. In certain embodiments, hinge / spacer is located between the extracellular antigen binding domain and the transmembrane domain. In certain embodiments, hinge / spacer includes CD8 polypeptides, CD28 polypeptides, CD3ζ polypeptides, CD4 polypeptides, 4-1BB polypeptides, OX40 polypeptides, CD166 polypeptides, CD8a polypeptides, CD8b polypeptides, ICOS polypeptides, ICAM-1 polypeptides, CTLA-4 polypeptides, CD27 polypeptides, CD40 / My88 peptides, NKGD2 peptides, synthetic polypeptides (not based on proteins associated with immune response), or combinations thereof. In certain embodiments, the transmembrane domain comprises a CD8 polypeptide, a CD28 polypeptide, a CD3ζ polypeptide, a CD4 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, a CD166 polypeptide, a CD8a polypeptide, a CD8b polypeptide, an ICOS polypeptide, an ICAM-1 polypeptide, a CTLA-4 polypeptide, a CD27 polypeptide, a CD40 / My88 peptide, an NKGD2 peptide, a synthetic polypeptide (not based on a protein associated with an immune response), or a combination thereof.

[0317] In certain embodiments, the transmembrane domain and hinge / spacer are derived from the same molecule. In certain embodiments, the transmembrane domain and hinge / spacer are derived from different molecules. In certain embodiments, the hinge / spacer of CAR comprises a CD28 polypeptide and the transmembrane domain of CAR comprises a CD28 polypeptide. In certain embodiments, the hinge / spacer of CAR comprises a CD28 polypeptide and the transmembrane domain of CAR comprises a CD28 polypeptide. In certain embodiments, the hinge / spacer of CAR comprises a CD84 polypeptide and the transmembrane domain of CAR comprises a CD84 polypeptide. In certain embodiments, the hinge / spacer of CAR comprises a CD166 polypeptide and the transmembrane domain of CAR comprises a CD166 polypeptide. In certain embodiments, the hinge / spacer of CAR comprises a CD8a polypeptide and the transmembrane domain of CAR comprises a CD8a polypeptide. In certain embodiments, the hinge / spacer of CAR comprises a CD8b polypeptide and the transmembrane domain of CAR comprises a CD8b polypeptide. In certain embodiments, the hinge / spacer of CAR comprises a CD28 polypeptide and the transmembrane domain of CAR comprises an ICOS polypeptide.

[0318] 5.2.2.4. Intracellular Signaling Domain of CAR

[0319] A.CD3ζ

[0320] In certain embodiments, CAR comprises an intracellular signaling domain. In certain embodiments, the intracellular signaling domain of CAR comprises a CD3ζ polypeptide, which can activate or stimulate cells (e.g., lymphoid cells, such as T cells). Wild-type ("natural") CD3ζ comprises three immunoreceptor tyrosine-based activation motifs ("ITAM") (e.g., ITAM1, ITAM2, and ITAM3), three alkaline-rich stretches (BRS) regions (BRS1, BRS2, ​​and BRS3), and transmits activation signals to cells (e.g., lymphoid cells, such as T cells) after antigen binding. The intracellular signaling domain of the natural CD3ζ chain is the main transmitter of endogenous TCR signals.

[0321] In certain embodiments, the intracellular signaling domain of the CAR comprises a native CD3 zeta polypeptide. In certain embodiments, the native CD3 zeta polypeptide comprises or consists of an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or at least about 100% homology or identity to the sequence of NCBI reference number NP_932170 (SEQ ID No: 94) or a fragment thereof. In certain embodiments, the native CD3 zeta polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 94 and has a length of at least about 20, or at least about 30, or at least about 40, or at least about 50, or at least about 100, or at least about 110, and a maximum of about 164 amino acids. In certain embodiments, the native CD3 zeta polypeptide comprises or consists of the amino acid sequence of amino acids 1 to 50, 50 to 100, 100 to 150, 50 to 164, 55 to 164, or 150 to 164 of SEQ ID NO: 94. In certain embodiments, the native CD3 zeta polypeptide comprises or consists of the amino acid sequence of amino acids 52 to 164 of SEQ ID NO: 94.

[0322] SEQ ID NO:94 is provided below:

[0323]

[0324] In certain embodiments, the CD3ζ polypeptide comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or at least about 100% homologous or identical to the amino acid sequence of SEQ ID NO: 95, or a fragment thereof, and / or may optionally comprise up to one, two, or three conservative amino acid substitutions. SEQ ID NO: 95 is shown below:

[0325]

[0326] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO:95 is set forth in SEQ ID NO:96 provided below.

[0327]

[0328] In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3 zeta polypeptide. In certain embodiments, the intracellular signaling domain of the CAR comprises a modified human CD3 zeta polypeptide. In certain embodiments, the modified CD3 zeta polypeptide comprises or consists of an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homology or identity to the amino acid sequence shown in SEQ ID NO: 35 or a fragment thereof, and / or an amino acid sequence that optionally comprises at most one, at most two, or at most three conservative amino acid substitutions. SEQ ID NO: 35 is provided below:

[0329]

[0330] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO:35 is set forth in SEQ ID NO:55 provided below.

[0331]

[0332] In certain embodiments, the modified CD3ζ polypeptide comprises one, two, or three ITAM variants. In certain embodiments, the modified CD3ζ polypeptide comprises native ITAM1. In certain embodiments, native ITAM1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23.

[0333]

[0334] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:23 is set forth in SEQ ID NO:24 provided below.

[0335]

[0336] In certain embodiments, the modified CD3ζ polypeptide comprises an ITAM1 variant comprising one or more loss-of-function mutations. In certain embodiments, the ITAM1 variant comprises or consists of two loss-of-function mutations. In certain embodiments, each of one or more (e.g., two) loss-of-function mutations comprises or consists of a mutation of a tyrosine residue in ITAM1. In certain embodiments, the ITAM1 variant (e.g., a variant consisting of two loss-of-function mutations) comprises or consists of the amino acid sequence shown in SEQ ID NO: 25 provided below.

[0337]

[0338] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:25 is set forth in SEQ ID NO:26 provided below.

[0339]

[0340] In certain embodiments, the modified CD3ζ polypeptide comprises native ITAM2. In certain embodiments, native ITAM2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 27 provided below.

[0341]

[0342] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:27 is set forth in SEQ ID NO:28, as shown below.

[0343]

[0344] In certain embodiments, the modified CD3ζ polypeptide comprises an ITAM2 variant comprising one or more loss-of-function mutations. In certain embodiments, the ITAM2 variant comprises or consists of two loss-of-function mutations. In certain embodiments, each of the one or more (e.g., two) loss-of-function mutations comprises or consists of a mutation in a tyrosine residue in ITAM2. In certain embodiments, the ITAM2 variant (e.g., a variant consisting of two loss-of-function mutations) comprises or consists of the amino acid sequence shown in SEQ ID NO: 29 provided below.

[0345]

[0346] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:29 is set forth in SEQ ID NO:30 provided below.

[0347]

[0348] In certain embodiments, the modified CD3ζ polypeptide comprises a native ITAM3. In certain embodiments, the native ITAM3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 31 provided below.

[0349]

[0350] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:31 is set forth in SEQ ID NO:32 provided below.

[0351]

[0352] In certain embodiments, the modified CD3ζ polypeptide comprises an ITAM3 variant comprising one or more loss-of-function mutations. In certain embodiments, the ITAM3 variant comprises or consists of two loss-of-function mutations. In certain embodiments, each of the one or more (e.g., two) loss-of-function mutations comprises or consists of a mutation in a tyrosine residue in ITAM3. In certain embodiments, the ITAM3 variant (e.g., a variant consisting of two loss-of-function mutations) comprises or consists of the amino acid sequence shown in SEQ ID NO: 33 provided below.

[0353]

[0354] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:33 is set forth in SEQ ID NO:34, as shown below.

[0355]

[0356] In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3 zeta polypeptide comprising: an ITAM1 variant comprising or consisting of one or more loss-of-function mutations, an ITAM2 variant comprising or consisting of one or more loss-of-function mutations, and / or an ITAM3 variant comprising or consisting of one or more loss-of-function mutations, or a combination thereof.

[0357] In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3 zeta polypeptide comprising: an ITAM2 variant comprising or consisting of one or more (e.g., two) loss-of-function mutations, and an ITAM3 variant comprising or consisting of one or more (e.g., two) loss-of-function mutations. In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3 zeta polypeptide comprising: a native ITAM1, an ITAM2 variant comprising or consisting of two loss-of-function mutations, and an ITAM3 variant comprising or consisting of two loss-of-function mutations. In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3 zeta polypeptide comprising: a native ITAM1 consisting of the amino acid sequence of SEQ ID NO: 23, an ITAM2 variant consisting of the amino acid sequence of SEQ ID NO: 29, and an ITAM3 variant consisting of the amino acid sequence of SEQ ID NO: 33 (e.g., a construct designated as "1XX"). In certain embodiments, the modified CD3 zeta polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 35.

[0358] In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3 zeta polypeptide comprising an ITAM1 variant comprising or consisting of one or more (e.g., two) loss-of-function mutations, and an ITAM3 variant comprising or consisting of one or more (e.g., two) loss-of-function mutations. In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3 zeta polypeptide comprising an ITAM1 variant comprising or consisting of two loss-of-function mutations, a native ITAM2, and an ITAM3 variant comprising or consisting of two loss-of-function mutations. In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3 zeta polypeptide comprising an ITAM1 variant consisting of the amino acid sequence of SEQ ID NO: 25, a native ITAM2 consisting of the amino acid sequence of SEQ ID NO: 27, and an ITAM3 variant consisting of the amino acid sequence of SEQ ID NO: 33 (e.g., a construct designated "X2X").

[0359] In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3 zeta polypeptide comprising: an ITAM1 variant comprising or consisting of one or more (e.g., two) loss-of-function mutations and an ITAM2 variant comprising or consisting of one or more (e.g., two) loss-of-function mutations. In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3 zeta polypeptide comprising or consisting of an ITAM1 variant comprising two loss-of-function mutations, an ITAM2 variant comprising or consisting of two loss-of-function mutations, and native ITAM3. In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3 zeta polypeptide comprising an ITAM1 variant consisting of the amino acid sequence of SEQ ID NO: 25, an ITAM2 variant consisting of the amino acid sequence of SEQ ID NO: 29, and a native ITAM3 consisting of the amino acid sequence of SEQ ID NO: 31 (e.g., a construct designated "XX3").

[0360] In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3 zeta polypeptide comprising an ITAM1 variant comprising one or more (e.g., two) loss-of-function mutations. In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3 zeta polypeptide comprising an ITAM1 variant comprising or consisting of two loss-of-function mutations, a native ITAM2, and a native ITAM3. In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3 zeta polypeptide comprising an ITAM1 variant consisting of the amino acid sequence of SEQ ID NO: 25, a native ITAM2 consisting of the amino acid sequence of SEQ ID NO: 27, and a native ITAM3 consisting of the amino acid sequence of SEQ ID NO: 31 (e.g., a construct designated "X23").

[0361] In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3 zeta polypeptide comprising a native ITAM1, a native ITAM2, and an ITAM3 variant comprising one or more (e.g., two) loss-of-function mutations. In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3 zeta polypeptide comprising a native ITAM1, a native ITAM2, and an ITAM1 variant comprising or consisting of two loss-of-function mutations. In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3 zeta polypeptide comprising a native ITAM1 consisting of the amino acid sequence set forth in SEQ ID NO: 23, a native ITAM2 consisting of the amino acid sequence set forth in SEQ ID NO: 27, and an ITAM3 variant consisting of the amino acid sequence set forth in SEQ ID NO: 33 (e.g., a construct designated as "12X").

[0362] In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3 zeta polypeptide comprising a native ITAM1, an ITAM2 variant comprising one or more (e.g., two) loss-of-function mutations, and a native ITAM3. In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3 zeta polypeptide comprising a native ITAM1, an ITAM2 variant comprising or consisting of two loss-of-function mutations, and a native ITAM3. In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3 zeta polypeptide comprising a native ITAM1 consisting of the amino acid sequence of SEQ ID NO: 23, an ITAM2 variant consisting of the amino acid sequence of SEQ ID NO: 29, and a native ITAM3 variant consisting of the amino acid sequence of SEQ ID NO: 31 (e.g., a construct designated as "1X3").

[0363] In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3 zeta polypeptide comprising a deletion of one or two ITAMs. In certain embodiments, the modified CD3 zeta polypeptide comprises or consists of a deletion of ITAM1 and ITAM2, for example, the modified CD3 zeta polypeptide comprises a natural ITAM3 or ITAM3 variant and does not comprise ITAM1 or ITAM2. In certain embodiments, the modified CD3 zeta polypeptide comprises a natural ITAM3 consisting of the amino acid sequence shown in SEQ ID NO: 31 and does not comprise ITAM1 (natural or modified) or ITAM2 (natural or modified) (e.g., a construct designated as "D12").

[0364] In certain embodiments, the modified CD3 zeta polypeptide comprises or consists of a deletion of ITAM2 and ITAM3, for example, the modified CD3 zeta polypeptide comprises native ITAM1 or an ITAM1 variant, and does not comprise ITAM2 or ITAM3. In certain embodiments, the modified CD3 zeta polypeptide comprises native ITAM1 consisting of the amino acid sequence set forth in SEQ ID NO: 23, and does not comprise ITAM2 (native or modified) or ITAM3 (native or modified) (e.g., a construct designated "D23").

[0365] In certain embodiments, the modified CD3 zeta polypeptide comprises or consists of a deletion of ITAM1 and ITAM3, for example, the modified CD3 zeta polypeptide comprises native ITAM2 or an ITAM2 variant and does not comprise ITAM1 or ITAM3. In certain embodiments, the modified CD3 zeta polypeptide comprises native ITAM2 consisting of the amino acid sequence set forth in SEQ ID NO: 27 and does not comprise ITAM1 (native or modified) or ITAM3 (native or modified) (e.g., a construct designated "D13").

[0366] In certain embodiments, the modified CD3ζ polypeptide comprises or consists of a deletion of ITAM1, for example, the modified CD3ζ polypeptide comprises native ITAM2 or an ITAM2 variant, and native ITAM3 or an ITAM3 variant, and does not comprise ITAM1 (native or modified).

[0367] In certain embodiments, the modified CD3ζ polypeptide comprises or consists of an ITAM2 deletion, for example, the modified CD3ζ polypeptide comprises native ITAM1 or an ITAM1 variant, and native ITAM3 or an ITAM3 variant, and does not comprise ITAM2 (native or modified).

[0368] In certain embodiments, the modified CD3ζ polypeptide comprises or consists of a deletion of ITAM3, for example, the modified CD3ζ polypeptide comprises native ITAM1 or an ITAM1 variant, and native ITAM2 or an ITAM2 variant, and does not comprise ITAM3 (native or modified).

[0369] B. Costimulatory signal transduction region

[0370] In certain embodiments, the intracellular signaling domain of the CAR further comprises at least one costimulatory signaling region. In certain embodiments, the costimulatory signaling region comprises at least a portion of a costimulatory molecule that can provide optimal lymphocyte activation.

[0371] As used herein, " co-stimulatory molecules " refer to cell surface molecules other than antigen receptors or their ligands required for the effective response of lymphocytes to antigens. Non-limiting examples of co-stimulatory molecules include CD28, 4-1BB, OX40, ICOS, DAP-10, CD27, CD40 and NKGD2. Co-stimulatory molecules can be combined with co-stimulatory ligands, and the ligand is a protein expressed on the cell surface, which produces a co-stimulatory response when bound to a receptor, i.e., affects the intracellular response of the provided stimulation when the antigen is bound to its CAR molecule. Co-stimulatory ligands include but are not limited to CD80, CD86, CD70, OX40L and 4-1BBL. As an example, 4-1BB ligands (i.e., 4-1BBL) can be bound to 4-1BB (also referred to as "CD137") to provide intracellular signals, which are combined with CAR signals to induce CAR + Effector cell function of T cells. CARs comprising an intracellular signaling domain containing a costimulatory signaling region (including 4-1BB, ICOS or DAP-10) are disclosed in US 7,446,190, which is incorporated herein by reference in its entirety.

[0372] In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region comprising a CD28 polypeptide (e.g., an intracellular domain of CD28 or a portion thereof). In certain embodiments, the costimulatory signaling region comprises an intracellular domain of human CD28 or a portion thereof. In certain embodiments, the costimulatory signaling region comprises a CD28 polypeptide comprising or consisting of amino acids 180 to 220 of SEQ ID NO: 90.

[0373] In certain embodiments, the costimulatory signaling region comprises a CD28 polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 101 (or amino acids 180 to 220 of SEQ ID NO: 90). SEQ ID NO: 101 is provided below.

[0374]

[0375] An exemplary nucleotide sequence encoding SEQ ID NO: 101 (or the amino acid sequence of amino acids 180 to 220 of SEQ ID NO: 90) is set forth in SEQ ID NO: 102 provided below.

[0376]

[0377] In certain embodiments, the costimulatory signaling region comprises a CD28 polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 108 (or amino acids 180 to 219 of SEQ ID NO: 90). SEQ ID NO: 108 is provided below.

[0378]

[0379] In certain embodiments, the costimulatory signaling region comprises the intracellular domain of mouse CD28 or a portion thereof. In certain embodiments, the costimulatory signaling region comprises or consists of amino acids 178 to 218 of SEQ ID NO: 97.

[0380] An exemplary nucleotide sequence encoding amino acids 178 to 218 of SEQ ID NO: 97 is set forth in SEQ ID NO: 98 provided below.

[0381]

[0382] In certain embodiments, the costimulatory signaling region comprises or consists of a CD28 polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 99. SEQ ID NO: 99 is provided below:

[0383]

[0384] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:99 is set forth in SEQ ID NO:100 provided below.

[0385]

[0386] In certain embodiments, the costimulatory signaling region comprises a portion of a first costimulatory molecule and a portion of a second costimulatory molecule, e.g., the intracellular domain of CD28 and the intracellular domain of 4-1BB or the intracellular domain of CD28 and the intracellular domain of OX40.

[0387] In certain embodiments, the costimulatory signaling region comprises a 4-1BB polypeptide (e.g., an intracellular domain of 4-1BB or a portion thereof). In certain embodiments, the costimulatory signaling region comprises an intracellular domain of human 4-1BB or a portion thereof. 4-1BB can act as a tumor necrosis factor (TNF) ligand and has stimulatory activity. In certain embodiments, the 4-1BB polypeptide comprises or consists of a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homology or identity to a sequence or fragment thereof with NCBI reference number NP_001552.2 (SEQ ID NO: 103), and / or may optionally comprise an amino acid sequence having at most one, at most two, or at most three conservative amino acid substitutions. In certain embodiments, the 4-1BB polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 103, having a length of at least about 20, at least about 25, or at least about 30, or at least about 40, or at least about 50, and up to about 255 amino acids. In certain embodiments, the 4-1BB polypeptide comprises or consists of an amino acid sequence of amino acids 1 to 255, 1 to 50, 50 to 100, 100 to 150, 150 to 200, 214 to 255, or 200 to 255 of SEQ ID NO: 103. In certain embodiments, the costimulatory signaling region comprises a 4-1BB polypeptide comprising or consisting of SEQ ID NO: 104 (or amino acids 214 to 255 of SEQ ID NO: 103). SEQ ID NOs: 103 and 104 are shown below:

[0388]

[0389] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 104 (or amino acids 214 to 255 of SEQ ID NO: 103) is set forth in SEQ ID NO: 105 provided below.

[0390]

[0391] In certain embodiments, the costimulatory signaling region comprises an OX40 polypeptide (e.g., an intracellular domain of OX40 or a portion thereof). In certain embodiments, the costimulatory signaling region comprises an intracellular domain of human OX40 or a portion thereof. In certain embodiments, the OX40 polypeptide comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to the sequence of NCBI Reference No. NP_003318.1 (SEQ ID No: 106) or a fragment thereof, and / or optionally comprises at most one, at most two, or at most three conservative amino acid substitutions. In certain embodiments, the OX40 polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 106 and is at least about 20, at least about 25, or at least about 30, or at least about 40, or at least about 50, and at most about 277 amino acids in length. In certain embodiments, the OX40 polypeptide comprises or consists of the amino acid sequence of amino acids 1 to 277, 1 to 50, 50 to 100, 100 to 150, 150 to 200, or 200 to 277 of SEQ ID NO: 106. SEQ ID NO: 106 is provided below.

[0392]

[0393] In certain embodiments, the costimulatory signaling region comprises an ICOS polypeptide (e.g., an intracellular domain of ICOS or a portion thereof). In certain embodiments, the costimulatory signaling region comprises the intracellular domain of human ICO or a portion thereof. In certain embodiments, the ICOS polypeptide comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to the sequence of NCBI Reference No. NP_036224 (SEQ ID NO: 65) or a fragment thereof, and / or may optionally contain up to one, up to two, or up to three conservative amino acid substitutions. In certain embodiments, the ICOS polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 65 and is at least about 20, at least about 25, or at least about 30, or at least about 40, or at least about 50, and up to about 199 amino acids in length. In certain embodiments, the ICOS polypeptide comprises or consists of the amino acid sequence of amino acids 1 to 199, 1 to 50, 50 to 100, 100 to 150, or 150 to 199 of SEQ ID NO: 65. SEQ ID NO: 65 is provided below.

[0394]

[0395] In certain embodiments, the CAR targeting mesothelin disclosed in the present invention further comprises an inducible promoter for expressing the nucleic acid sequence in human cells. The promoter for expressing the CAR gene can be a constitutive promoter, such as ubiquitin C (UbiC) promoter.

[0396] In certain embodiments, mutation sites and / or CAR domains / motifs / regions from different proteins are deimmunized. The immunogenicity of the connections between different CAR parts can be predicted using the NetMHC 4.0 server. For each peptide containing at least one amino acid from the next part, the binding affinity of all alleles to HLA A, B, and C can be predicted. An immunogenicity score for each peptide can be assigned to each peptide. The immunogenicity score can be calculated using the formula: Immunogenicity score = [(50-binding affinity) * HLA frequency] n . n is the number of predictions per peptide.

[0397] 5.2.2.5. Exemplary CAR

[0398] In certain embodiments, the CAR targeting mesothelin comprises:

[0399] (a) an extracellular antigen-binding domain comprising V H and V L , the V H comprising a CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 76, a CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 77, and a CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 78; the V L comprising a CDR1 consisting of the amino acid sequence of SEQ ID NO: 79, a CDR2 consisting of the amino acid sequence of SEQ ID NO: 80, and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 81;

[0400] (b) a transmembrane domain comprising a CD28 polypeptide (e.g., a transmembrane domain of human CD28 or a portion thereof);

[0401] (c) a CD28 hinge / spacer region (e.g., a hinge / spacer region of human CD28 or a portion thereof); and

[0402] (d) an intracellular signaling domain comprising (i) a modified CD3 zeta polypeptide (e.g., a modified human CD3 zeta polypeptide) comprising a native ITAM1, an ITAM2 variant consisting of two loss-of-function mutations, and an ITAM3 variant consisting of two loss-of-function mutations, and (ii) a costimulatory signaling region comprising a CD28 polypeptide (e.g., a human CD28 polypeptide, e.g., an intracellular domain of human CD28 or a portion thereof).

[0403] In certain embodiments, the transmembrane domain comprises a CD28 polypeptide consisting of the amino acid sequence of SEQ ID NO:92 (or amino acids 153 to 179 of SEQ ID NO:90).

[0404] In certain embodiments, the CD28 hinge / spacer region consists of the amino acid sequence set forth in SEQ ID NO: 15 (or amino acids 114 to 152 of SEQ ID NO: 90).

[0405] In certain embodiments, the modified CD3ζ polypeptide consists of the amino acid sequence shown in SEQ ID NO:35.

[0406] In certain embodiments, the costimulatory signaling region comprises a CD28 polypeptide consisting of the amino acid sequence of SEQ ID NO: 101 (or amino acids 180 to 220 of SEQ ID NO: 90).

[0407] In certain embodiments, the CAR comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or at least about 100% homologous or identical to the amino acid sequence of SEQ ID NO: 56. In certain embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 56. SEQ ID NO: 56 is provided below.

[0408]

[0409] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO:56 is set forth in SEQ ID NO:57 provided below.

[0410]

[0411] In certain embodiments, the CAR further comprises a CD8 leader. In certain embodiments, the CD8 leader comprises or consists of the amino acid sequence shown in SEQ ID NO: 71.

[0412] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO:71 is set forth in SEQ ID NO:120 provided below.

[0413]

[0414] In certain embodiments, the CAR comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homology or identity to the amino acid sequence of SEQ ID NO: 43, provided below. In certain embodiments, the CAR comprises or consists of the amino acid sequence of SEQ ID NO: 43. SEQ ID NO: 43 includes a CD8 leader consisting of the amino acid sequence of SEQ ID NO: 71. SEQ ID NO: 43 is provided below:

[0415]

[0416] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO:43 is set forth in SEQ ID NO:44 provided below.

[0417]

[0418] 5.2.3. Exemplary Polypeptide Compositions

[0419] In certain embodiments, the polypeptide composition comprises: a mesothelin-targeting CAR comprising or consisting of the amino acid sequence shown in SEQ ID NO:56, and a PD-1 DN comprising or consisting of amino acids 21 to 165 of SEQ ID NO:48.

[0420] In certain embodiments, the polypeptide composition comprises: a mesothelin-targeting CAR comprising or consisting of the amino acid sequence shown in SEQ ID NO:56, and a PD-1 DN comprising or consisting of amino acids 1 to 165 of SEQ ID NO:48.

[0421] In certain embodiments, the polypeptide composition comprises: a mesothelin-targeting CAR comprising or consisting of the amino acid sequence shown in SEQ ID NO:56, and a PD-1 DN comprising or consisting of the amino acid sequence shown in SEQ ID NO:49.

[0422] In certain embodiments, the polypeptide composition comprises: a mesothelin-targeting CAR comprising or consisting of the amino acid sequence shown in SEQ ID NO: 56, and a PD-1 DN comprising or consisting of the amino acid sequence shown in SEQ ID NO: 118.

[0423] In certain embodiments, the polypeptide composition comprises: a mesothelin-targeting CAR comprising or consisting of the amino acid sequence shown in SEQ ID NO: 56, a CD8 leader sequence comprising or consisting of the amino acid sequence shown in SEQ ID NO: 71, and a PD-1 DN comprising or consisting of amino acids 21 to 165 of SEQ ID NO: 48.

[0424] In certain embodiments, the polypeptide composition comprises: a mesothelin-targeting CAR comprising or consisting of the amino acid sequence shown in SEQ ID NO: 56, a CD8 leader sequence comprising or consisting of the amino acid sequence shown in SEQ ID NO: 71, and a PD-1 DN comprising or consisting of amino acids 1 to 165 of SEQ ID NO: 48.

[0425] In certain embodiments, the polypeptide composition comprises: a CAR targeting mesothelin comprising or consisting of the amino acid sequence shown in SEQ ID NO: 56, a CD8 leader sequence comprising or consisting of the amino acid sequence shown in SEQ ID NO: 71, and a PD-1 DN comprising or consisting of the amino acid sequence shown in SEQ ID NO: 49.

[0426] In certain embodiments, the polypeptide composition comprises: a mesothelin-targeting CAR comprising or consisting of the amino acid sequence shown in SEQ ID NO: 56, a CD8 leader sequence comprising or consisting of the amino acid sequence shown in SEQ ID NO: 71, and a PD-1 DN comprising or consisting of the amino acid sequence shown in SEQ ID NO: 118.

[0427] 5.3. Immune Response Cells

[0428] The present disclosure provides immune response cells comprising the polypeptide compositions disclosed herein. In certain embodiments, CAR can activate immune response cells. In certain embodiments, the polypeptide compositions can promote the anti-tumor efficacy of the immune response cells. Immune response cells can be transduced with the polypeptide compositions so that the cells co-express CAR and PD-1 DN.

[0429] The immune response cells of the present disclosure can be lymphoid cells. The lymphoid system including B cells, T cells and natural killer (NK) cells provides the production of antibodies, the regulation of the cellular immune system, the detection of foreign bodies in the blood, the detection of foreign cells in the host, etc. Non-limiting examples of lymphoid immune response cells include T cells, natural killer (NK) cells, embryonic stem cells, and pluripotent stem cells (e.g., cells that can differentiate into lymphocytes). T cells can be mature lymphocytes in the thymus, which are mainly responsible for cell-mediated immunity. T cells participate in the adaptive immune system. The T cells of the present disclosure can be any type of T cells, including but not limited to helper T cells, cytotoxic T cells, memory T cells (including central memory T cells, stem cell-like memory T cells (or stem-like memory T cells) and two effector memory T cells: for example, T EM cells and T EMRA Cells, regulatory T cells (also called suppressor T cells), natural killer T cells, mucosal-associated constant T cells and gamma delta T cells. Cytotoxic T cells (CTL or killer T cells) are a subset of T lymphocytes that can induce the death of infected somatic cells or tumor cells. The patient's own T cells can be genetically modified to target specific antigens by introducing antigen recognition receptors (such as CAR or TCR). In certain embodiments, the immune response cells are T cells. T cells can be CD4 + T cells or CD8 + In some embodiments, the T cells are CD4 + In some embodiments, the T cells are CD8 + T cells.

[0430] Natural killer (NK) cells are lymphocytes that are part of cell-mediated immunity and play a role in the innate immune response. NK cells do not require prior activation to exert cytotoxic effects on target cells.

[0431] Types of human lymphocytes of the presently disclosed subject matter include, but are not limited to, peripheral donor lymphocytes, such as those disclosed in Sadelain, M. et al. 2003 Nat Rev Cancer 3:35-45 (disclosing peripheral donor lymphocytes genetically engineered to express CAR), Morgan, RA et al. 2006 Science 314:126-129 (disclosing peripheral donor lymphocytes genetically engineered to express the full-length tumor antigen-recognizing T cell receptor complex (including α and β heterodimers)), Panelli, MC et al. 2000 J Immunol 164:495-504; Panelli, MC et al. 2000 J Immunol 164:4382-4392 (disclosing cultures of lymphocytes derived from tumor infiltrating lymphocytes (TILs) in tumor biopsies), and Dupont, J. et al. 2005 Cancer Res 65:5417-5427; Papanicolaou, GA et al. 2003 Blood 102:2498-2505 (disclosing selective in vitro expansion of antigen-specific peripheral blood leukocytes using artificial antigen-presenting cells (AAPCs) or pulsed dendritic cells). Immune response cells (e.g., T cells) can be autologous, non-autologous (e.g., allogeneic), or obtained in vitro from engineered progenitor cells or stem cells.

[0432] In certain embodiments, the immune response cell disclosed herein comprises a mesothelin-targeting CAR comprising or consisting of the amino acid sequence shown in SEQ ID NO: 56, and a PD-1 DN comprising or consisting of amino acids 1 to 165 of SEQ ID NO: 48.

[0433] In certain embodiments, the immune response cells of the present disclosure comprising one or more CAR and / or PD-1 / DN polypeptides of the present disclosure are allogeneic or autologous EBV-sensitized cytotoxic T lymphocytes (CTLs). For example, the generation of EBV-sensitized cytotoxic T cells may involve isolating PBMCs from an autologous or allogeneic donor that is positive for EBV serotype, and enriching T cells by removing monocytes and NK cells. EBV-sensitized cytotoxic T cells can also be produced by contacting donor PBMCs or purified donor T cells with "stimulation" cells that express one or more EBV antigens and present EBV antigens to unstimulated T cells, thereby causing stimulation and amplification of EBV-sensitized CTLs. It is noteworthy that in certain embodiments, such methods are directed to cells comprising CD3 + A cell sample (eg, PBMC) is obtained from a subject with the cells, and the CD3 +The cells are contacted with antigens and / or antigen presenting stimulatory cells. In certain embodiments, prior to contact with the antigen, the cells are positively selected for CD3 + cells and / or negatively select by removing unwanted cells or components from the sample) to isolate CD3 +T cell.In certain embodiments, such method comprises using fluorescence activated cell sorting (FACS), anti-CD3 beads (such as magnetic beads), plastic adhesion, using anti-CD56 to exhaust NK cells, elutriation and / or its combination to select.EBV antigen includes, for example, latent membrane protein (LMP) and EBV nuclear antigen (EBNA) protein, such as LMP-1, LMP-2A, LMP-2B and EBNA-1, EBNA-2, EBNA-3A, EBNA-3B, EBNA-3C and EBNA-LP. The cytotoxic T cells comprising the T cell receptors for identifying one or more EBV specific antigens are considered to be "sensitive" to these EBV antigens, and are therefore referred to herein as "EBV sensitive cytotoxic T cells". Methods for generating allogeneic or autologous EBV-specific cytotoxic T cell populations that may contain one or more CAR polypeptides of the present disclosure are described, for example, in Barker et al., Blood 116(23):5045-49 (2010); Doubrovina et al., Blood 119(11):2644-56 (2012); Koehne et al., Blood 99(5):1730-40 (2002); and Smith et al., Cancer Res. 72(5):1116-25 (2012), which are incorporated by reference. Similarly, cytotoxic T cells can be "sensitive" to other viral antigens, including cytomegalovirus (CMV), papillomavirus (e.g., HPV), adenovirus, polyomavirus (e.g., BKV, JCV, and Merkel cell virus), retrovirus (e.g., HTLV-I, also including slow viruses, such as HIV), picornavirus (e.g., hepatitis A virus), hepadnavirus (e.g., hepatitis B virus), hepatitis C virus (e.g., hepatitis C virus), hepatitis D virus (e.g., hepatitis D virus), hepatitis E virus (e.g., hepatitis E virus), etc. In certain embodiments, the target antigen is from a tumor virus. In certain embodiments, the T cells used to produce the CAR T cells of the present disclosure are multifunctional T cells, e.g., those capable of inducing multiple immune effector functions, which provide a more effective immune response to pathogens than cells that only produce, for example, a single immune effector (e.g., a single biomarker, such as a cytokine or CD107a). During chronic infection, T cells with low function, single function, or even "exhausted" may dominate the immune response, thereby negatively impacting the prevention of virus-related complications. In certain embodiments, the CAR T cells of the present disclosure are multifunctional. In certain embodiments, at least about 50% of the T cells used to generate the CAR T cells of the present disclosure are CD4 + In certain such embodiments, the T cells have less than about 50% CD4 +In some embodiments, the T cells are primarily CD4 + T cells.

[0434] In certain embodiments, at least about 50% of the T cells used to generate the CAR T cells of the present disclosure are CD8 + In certain such embodiments, the T cells are less than about 50% CD8 + In some embodiments, the T cells are primarily CD8 + T cells. In certain embodiments, T cells (e.g., sensitized T cells and / or CAR T cells described herein) are stored in a cell bank or reservoir prior to administration to a subject.

[0435] The immune response cells disclosed in the present invention may also include at least one exogenous costimulatory ligand so that the immune response cells co-express or are induced to co-express mesothelin-specific CAR and at least one exogenous costimulatory ligand. The interaction between mesothelin-specific CAR and at least one costimulatory ligand provides a non-antigen-specific signal that is very important for the complete activation of immune response cells (e.g., T cells). Costimulatory ligands include but are not limited to tumor necrosis factor (TNF) superfamily members and immunoglobulin (Ig) superfamily ligands. TNF is a cytokine that participates in systemic inflammation and stimulates acute phase reactions. Its main function is to regulate immune cells. TNF superfamily members have many common features. Most TNF superfamily members are synthesized as type I transmembrane proteins (extracellular C-terminus) comprising a short cytoplasmic fragment and a relatively long extracellular region. Members of the TNF superfamily include, but are not limited to, nerve growth factor (NGF), CD40L (CD40L) / CD154, CD137L / 4-1BBL, TNF-α, CD134L / OX40L / CD252, CD27L / CD70, Fas ligand (FasL), CD30L / CD153, tumor necrosis factor β (TNFβ) / lymphotoxin α (LTα), lymphotoxin β (LTβ), CD257 / B cell activating factor (BAFF) / Blys / THANK / Tall-1, glucocorticoid-induced TNF receptor ligand (GITRL), TNF-related apoptosis-inducing ligand (TRAIL), and LIGHT (TNFSF14). The immunoglobulin (Ig) superfamily is a large class of cell surface and soluble proteins involved in cell recognition, binding, or adhesion. These proteins share structural features with immunoglobulins—they possess an immunoglobulin domain (fold). Immunoglobulin superfamily ligands include, but are not limited to, CD28's two ligands CD80 and CD86, and PD-1's ligand PD-L1 / (B7-H1).

[0436] In certain embodiments, at least one co-stimulatory ligand is selected from 4-1BBL, CD80, CD86, CD70, OX40L, CD48, TNFRSF14, PD-L1, and combinations thereof. In certain embodiments, the co-stimulatory ligand is 4-1BBL. 4-1BBL can be covalently linked to the 5' end of the extracellular antigen binding domain of a CAR targeting mesothelin. Alternatively, 4-1BBL can be covalently linked to the 3' end of the intracellular signaling domain of a CAR targeting mesothelin.

[0437] In addition, the immune response cells disclosed in the present invention may further comprise at least one exogenous cytokine, so that the immune response cells co-express or are induced to co-express mesothelin-specific CAR and at least one exogenous cytokine. In certain embodiments, at least one exogenous cytokine is selected from IL-2, IL-3, IL-6, IL-7, IL-11, IL-12, IL-15, IL-17 and IL-21. In certain embodiments, at least one exogenous cytokine comprises IL-12. In certain embodiments, the immune response cells co-express CAR targeting mesothelin and exogenous IL-12. IL-12 can be covalently linked to the 3' end of the intracellular signaling domain of the CAR targeting mesothelin.

[0438] Additionally, the immune response cell may express a second CAR that binds to a second antigen (mesothelin or an antigen other than mesothelin). In the presently disclosed subject matter, CARs that can be used as a second CAR in conjunction with a mesothelin-specific CAR include those described in Sadelain et al., "The Basic Principles of Chimeric Antigen Receptor Design" Cancer Discovery, OF1-11, (2013), Chicaybam et al., (2011), Brentjens et al., Nature Medicine 9:279-286 (2003), and US7,446,190, which are incorporated herein by reference in their entireties, e.g., CD19-targeted CARs (see US7,446,190; US2013 / 0071414), HER2-targeted CARs (see Ahmed et al., Clin Cancer Res., 2010), MUC16-targeted CARs (see Chekmasova et al., 2011), prostate-specific membrane antigen (PSMA)-targeted CARs (e.g., Zhong et al., Molecular Biology, 2012). The CARs described in WO2014 / 055668, the entirety of which is incorporated herein by reference.

[0439] The second antigen can be a tumor antigen or a pathogen antigen. Any suitable tumor antigen (antigenic peptide) is suitable for use in the tumor-related embodiments described herein. The source of the tumor antigen includes, but is not limited to, cancer proteins. The second antigen can be expressed as a peptide or a complete protein or a portion thereof. The complete protein or a portion thereof can be natural or mutated. Suitable second antigens include, but are not limited to, prostate-specific membrane antigen (PSMA) and prostate stem cell antigen (PCSA). In some embodiments, the tumor antigen can be carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), CD5, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD123, CD133, CD138, antigens of cytomegalovirus (CMV) infected cells (e.g., cell surface antigens), epithelial glycoprotein 2 (EGP2), epithelial glycoprotein 40 (EGP-40), Epithelial cell adhesion molecule (EpCAM), receptor tyrosine protein kinases Erb-B2, Erb-B3, Erb-B4, folate binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor α, ganglioside G2 (GD2), ganglioside G3 (GD3), human epidermal growth factor receptor 2 (HER-2), human telomerase reverse transcriptase (hTERT), interleukin-13 receptor subunit α-2 (IL-13Rα2), kappa light chain, kinase insert domain receptor (KDR), Lewis A (CA19.9), Lewis Y (LeY), L1 cell adhesion molecule (LlCAM), melanoma antigen family A, 1 (MAGE-AI), mucin 16 (Muc-16), mucin 1 (Muc-1), NKG2D ligand, cancer testis antigen NY-ESO-1, tumor-fetal antigen (h5T4), prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), tumor-associated glycoprotein 72 (TAG-72), vascular endothelial growth factor R2 (VEGF-R2), Wilm's tumor protein (WT-1), tyrosine-protein kinase transmembrane receptor type 1 (ROR1), or a combination thereof.

[0440] Suitable pathogenic antigens for treating pathogen infection or other infectious diseases, for example, in immunocompromised subjects, including but not limited to viral antigens present in cytomegalovirus (CMV), Epstein-Barr virus (EBV), human immunodeficiency virus (HIV) and influenza virus. The immune response cells including the second CAR targeting viral antigens can be used to treat viral diseases. In certain embodiments, the CAR targeting mesothelin and the second CAR combining CMV antigens are co-expressed in immune response cells (e.g., cytotoxic T lymphocytes) and can be used to treat CMV.

[0441] Mesothelin-specific or mesothelin-targeted human lymphocytes that can be used in the methods of the presently disclosed subject matter include, but are not limited to, peripheral donor lymphocytes, e.g., Sadelain, M., et al. 2003 Nat Rev Cancer 3:35-45 (disclosing peripheral donor lymphocytes genetically modified to express CAR), Morgan, RA et al. 2006 Science 314:126-129 (disclosing peripheral donor lymphocytes genetically engineered to express the full-length tumor antigen-recognizing T cell receptor complex (including α and β heterodimers)), Panelli, MC et al. 2000 J Immunol 164:495-504; Panelli, MC et al. 2000 J Immunol 164:4382-4392 (disclosing lymphocyte cultures derived from tumor infiltrating lymphocytes (TILs) in tumor biopsies), and Dupont, J. et al. 2005 Cancer Res 65:5417-5427; Papanicolaou, GA et al. 2003 Blood 102:2498-2505 (disclosing the use of artificial antigen presenting cells (AAPCs) or pulsed dendritic cells to selectively expand antigen-specific peripheral blood leukocytes in vitro). Immune response cells (such as T cells) can be autologous, non-autologous (such as allogeneic), or derived in vitro from engineered progenitor cells or stem cells.

[0442] The assay can be used to compare the effects of costimulatory signals on the proliferation, effector function, and accumulation of T cells transduced with a CAR targeting mesothelin after repeated (weekly) antigen stimulation. Peripheral blood lymphocytes (PBL) can be harvested from healthy volunteers and transduced according to an IRB-approved protocol. Gene transfer efficiency can be monitored by FACS analysis to quantify GFP expression. +The ratio of (transduced) T cells and / or monitored by quantitative PCR. Using a mature co-culture system (Gade, TP et al., Cancer Res. 65 9080-9088 (2005); Gong, MC et al., Neoplasia. 1123-127 (1999); Latouche, JB and Sadelain, M. Nat. Biotechnol. 18405-409 (2000)), it can be determined whether fibroblasts AAPC expressing mesothelin (vs. mesothelin control group) directly release cytokines from transduced T cells (cell supernatant LUMINEX analysis of IL-2, IL-4, IL-10, IFN-γ, TNF-α and GM-CSF), T cell proliferation (by CFSE labeling) and T cell survival (by annexin V staining). The effects of CD80 and / or 4-1BBL on T cell survival, proliferation and efficacy can be assessed. T cells may be exposed to mesothelin under repeated stimulation. + (MSLN + ) target cells and determine whether T cell proliferation and cytokine responses remain similar or diminish after repeated stimulation. Cytotoxicity assays with multiple E:T ratios can be performed using a chromium release assay. Statistical analysis can be performed using a two-way ANNOVA followed by pairwise multiple comparisons, with data presented as mean ± SEM. CD4 and CD8 T cell subtypes (activated effector, central memory, effector memory) can be determined to determine which conditions favor the maintenance or expansion of the central memory phenotype.

[0443] In certain embodiments, the immune response cells (e.g., T cells) of the present disclosure express about 1 to about 4, about 2 to about 4, about 3 to about 4, about 1 to about 2, about 1 to about 3, or about 2 to about 3 vector copies / cell of the CAR targeting mesothelin. For example, the immune response cells (e.g., T cells) of the present disclosure express about 1, about 2, about 3, or about 4 vector copies / cell of the CAR targeting mesothelin. In certain embodiments, the immune response cells (e.g., T cells) of the present disclosure express about 3 to about 4 vector copies / cell of the CAR targeting mesothelin. In certain embodiments, the cytotoxicity and cytokine production of immune response cells (e.g., T cells) are proportional to the expression level of mesothelin-specific CAR in the cell. For example, the higher the CAR expression level in the immune response cell, the greater the cytotoxicity and cytokine production exhibited by the immune response cell. Immune response cells (e.g., T cells) with high mesothelin CAR expression levels can induce the production or secretion of antigen-specific cytokines and / or exhibit cytotoxicity to tissues or cells with low mesothelin expression levels, e.g., about 2000 or less, about 1000 or less, about 900 or less, about 800 or less, about 700 or less, about 600 or less, about 500 or less, about 400 or less, about 300 or less, about 200 or less, about 100 or less mesothelin binding sites / cell. Additionally or alternatively, the cytotoxicity and cytokine production of the immune response cells (e.g., T cells) of the present disclosure are proportional to the expression level of human mesothelin in the target tissue or target cell. For example, the higher the expression level of human mesothelin in the target cell, the greater the cytotoxicity and cytokine production exhibited by the immune response cells.

[0444] In certain embodiments, the target cells are heterogeneous MSLN-expressing cells, which are cell populations comprising low MSLN-expressing cells and high MSLN-expressing cells. The immune response cells disclosed herein can exhibit enhanced cytotoxicity and anti-tumor activity against low MSLN-expressing cells (e.g., about 2000 or less, about 1000 or less, about 900 or less, about 800 or less, about 700 or less, about 600 or less, about 500 or less, about 400 or less, about 300 or less, about 200 or less, or about 100 or less MSLN binding sites / cell) in the presence of high MSLN-expressing cells. In certain embodiments, even in the presence of high MSLN-expressing cells, the cytotoxicity or nonspecific killing of the immune response cells against MSLN-negative cells does not increase. Therefore, the immune response cells exhibit increased cytotoxicity and anti-tumor activity against low MSLN-expressing cells in the presence of high MSLN-expressing cells, while maintaining safety against MSLN-negative cells.

[0445] In certain embodiments, immune response cells may express one or more adhesion molecules that increase the affinity of MSLN-specific CAR, especially when CAR is a low-affinity CAR. Non-limiting examples of adhesion molecules include CD2 and VLA-4. CD2 expressed on immune response cells can bind to CD58 expressed on target cells (e.g., cancer cells). VLA-4 expressed on immune response cells can bind to VCAM-1 on target cells (e.g., cancer cells).

[0446] The unpurified source of CTL can be any source known in the art, such as bone marrow, fetus, newborn or adult or other hematopoietic cell sources, such as fetal liver, peripheral blood or umbilical cord blood. Various techniques can be used to separate cells. For example, negative selection methods can be used to initially remove non-CTL. Monoclonal antibodies (mAbs) are particularly useful when identifying markers associated with specific cell lineages and / or differentiation stages of positive and negative selection.

[0447] Most of the terminally differentiated cells can be initially removed by relatively coarse separation. For example, magnetic bead separation can be used to initially remove a large number of irrelevant cells. In certain embodiments, at least about 80%, and typically at least 70%, of the total hematopoietic cells will be removed prior to cell separation.

[0448] Separation procedures include, but are not limited to, density gradient centrifugation; repositioning; coupling to particles that alter cell density; magnetic separation using antibody-coated magnetic beads; affinity chromatography; cytotoxic agents conjugated to or used in conjunction with mAbs, including but not limited to complement and cytotoxins; and panning, elutriation, or any other convenient technique using antibodies attached to a solid matrix (e.g., plate, chip).

[0449] Techniques used for separation and analysis include, but are not limited to, flow cytometry, which can have varying degrees of complexity, eg, multiple color channels, low-angle and obtuse-angle light scatter detection channels, impedance channels.

[0450] Cells can be selected for dead cells by using a dye associated with dead cells, such as propidium iodide (PI).In certain embodiments, cells are collected in culture medium containing 2% fetal calf serum (FCS) or 0.2% bovine serum albumin (BSA) or any other suitable, e.g., sterile, isotonic, culture medium.

[0451] 5.4. Nucleic Acid Compositions and Vectors

[0452] The presently disclosed subject matter provides nucleic acid compositions encoding the polypeptide compositions disclosed herein (e.g., disclosed in Section 5.2). In certain embodiments, the nucleic acid compositions comprise polynucleotides encoding the polypeptide compositions disclosed herein (e.g., one disclosed in Section 5.2). Also provided are vectors comprising such nucleic acid compositions, and cells comprising such nucleic acid compositions or vectors.

[0453] In some embodiments, the nucleic acid composition further comprises a promoter operably coupled to the polypeptide composition. In some embodiments, the promoter is endogenous or exogenous. In some embodiments, the exogenous promoter is selected from elongation factor (EF)-1 promoter, CMV promoter, SV40 promoter, PGK promoter, and metallothionein promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the inducible promoter is selected from NFAT transcriptional response element (TRE) promoter, CD69 promoter, CD25 promoter, and IL-2 promoter.

[0454] The nucleic acid composition can be administered to a subject and / or delivered into a cell by methods known in the art or as described herein.

[0455] Genetic modification of immune response cells (e.g., T cells or NK cells) can be accomplished by transducing a substantially homogeneous cell composition with a recombinant DNA construct. In certain embodiments, a retroviral vector (gamma retrovirus or slow virus) is used to introduce a DNA construct into a cell. For example, the polynucleotide encoding an antigen recognition receptor can be cloned into a retroviral vector and can be driven by its endogenous promoter, a retroviral long terminal repeat sequence, or a promoter specific to the target cell type. Non-viral vectors can also be used.

[0456] For the initial genetic modification of immune response cells to include antigen recognition receptors (e.g., CAR or TCR), retroviral vectors are typically used for transduction, but any other suitable viral vector or non-viral delivery system may also be used. CAR and PD-1 DN can be constructed in multiple expression cassettes on a single vector or in single or multicistronic expression cassettes on multiple vectors. Examples of elements for constructing multicistronic expression cassettes include, but are not limited to, various viral and non-viral internal ribosome entry sites (IRES, such as FGF-1 IRES, FGF-2 IRES, VEGF IRES, IGF-II IRES, NF-κB IRES, RUNX1 IRES, p53 IRES, Hepatitis A IRES, Hepatitis C IRES, Plague virus IRES, Oral virus IRES, Picornavirus IRES, Poliovirus IRES, and Encephalomyocarditis virus IRES) and cleavable linkers (e.g., 2A peptides, such as P2A, T2A, E2A, and F2A peptides). In certain embodiments, the P2A peptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 107, which is provided below:

[0457]

[0458] In certain embodiments, the P2A peptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 121, which is provided below:

[0459]

[0460] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 121 is shown in SEQ ID NO: 122, which is provided below:

[0461]

[0462] Combinations of retroviral vectors and appropriate packaging lines are also suitable, wherein the capsid protein is effective for infecting human cells. Various amphotropic virus production cell lines are known, including but not limited to PA12 (Miller et al. (1985) Mol. Cell. Biol. 5: 431-437); PA317 (Miller et al. (1986) Mol. Cell. Biol. 6: 2895-2902) and CRIP (Danos et al. (1988) Proc. Natl. Acad. Sci. USA 85: 6460-6464). Non-amphotropic (viral) particles are also suitable, for example, pseudotyped (viral) particles packaged using VSVG, RD114 or GALV, and any other pseudotyped (viral) particles known in the art.

[0463] Possible transduction methods also include direct co-cultivation of cells with producer cells, for example, by the method of Bregni et al. (1992) Blood 80:1418-1422, or cultivation using viral supernatant alone or using concentrated carriers with or without appropriate growth factors and polycations, for example, by the method of Xu et al. (1994) Exp. Hemat. 22:223-230; and Hughes et al. (1992) J. Clin. Invest. 89:1817.

[0464] Other transduction virus vectors can be used to modify immune response cells. In certain embodiments, the selected vector shows high infection efficiency and stable integration and expression (see, e.g., Cayouette et al., Human Gene Therpy 8:423-430, 1997; Kido et al., Current Eye Research 15:833-844, 1996; Bloomer et al., Journal of Virology 71:6641-6649, 1997; Naldini et al., Science 272:263-267, 1996; Miyoshi et al., Proc. Natl. Acad. Sci. USA 94:10319, 1997). Other viral vectors that can be used include, for example, adenovirus, lentivirus, and adenylate-associated virus vectors, vaccinia virus, bovine papilloma virus, or herpes virus, such as Epstein-Barr virus (see also vectors in, e.g., Miller, Human Gene Therapy 15-14, 1990; Friedman, Science 244:1275-1281, 1989; Eglitis et al., BioTechniques 6:608-614, 1988; Tolstoshev et al., Current Opinion in Biotechnology 1:55-61, 1990; Sharp, The Lancet 337:1277-1278, 1991; Cornetta et al., Nucleic Acid Research and Molecular Biology 36:311-322, 1987; Anderson, Science 226:401-409, 1984; Mone, Blood Cells 17:407-416, 1991; Miller et al., Biotechnology 7:980-990, 1989; LeGal La Salle et al., Science 259:988-990, 1993; and Johnson, Chest 107:77S-83S, 1995). Retroviral vectors are particularly well developed and have been used clinically (Rosenberg et al., N. Engl. J. Med 323:370, 1990; Anderson et al., U.S. Pat. No. 5,399,346).

[0465] In certain embodiments, the vector encoding the polypeptide composition of the present disclosure is a retroviral vector, such as an SGFγ-retroviral vector, which can be a Moloney mouse leukemia retroviral vector. In certain embodiments, the vector comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 123, which is provided below:

[0466]

[0467]

[0468]

[0469] In certain embodiments, the vector comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 124, which is provided below:

[0470]

[0471]

[0472]

[0473] Non-viral methods can also be used for genetic modification of immune response cells. For example, nucleic acid molecules can be introduced into immune response cells by administering the nucleic acid in the presence of lipofectamine (Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413, 1987; Ono et al., Neurosicience Letters 17:259, 1990; Brigham et al., Am. J. Med. Sci. 298:278, 1989; Staubinger et al., Methods inenzymology 101:512, 1983), by asialopolylysine complexation (Wu et al., Journal of Biological Chemistry 263:14621, 1988; Wu et al., Journal of Biological Chemistry 264:16985, 1989), or by microinjection under surgical conditions (Wolff et al., Science 247:1465, 1990). Other non-viral gene transfer methods include in vitro transfection using calcium phosphate, DEAE-dextran, electroporation, and protoplast fusion. Liposomes may also be useful for delivering DNA to cells. Normal genes can be transplanted into the affected tissues of the subject by transplanting normal nucleic acids into a cell type cultured in vitro (e.g., autologous or heterologous primary cells or their offspring), and then the cells (or their offspring) are injected into the target tissue or injected systemically. Recombinant receptors can also be derived or obtained using transposases or targeted nucleases (e.g., zinc finger nucleases, homing endonucleases, or TALE nucleases, CRISPR). Transient expression can be obtained by RNA electroporation.

[0474] Any targeted genome editing method can be used to express the polypeptide composition. In certain embodiments, the CRISPR system is used to express the polypeptide composition disclosed herein. In certain embodiments, zinc finger nucleases are used to express the polypeptide composition disclosed herein. In certain embodiments, the TALEN system is used to express the polypeptide composition disclosed herein.

[0475] The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system is a genome editing tool found in prokaryotic cells. When used for genome editing, the system includes Cas9 (a protein that can modify DNA using crRNA as a guide), CRISPR RNA (crRNA, which contains RNA used to guide Cas9 to the correct part of the host DNA and a region (usually in the form of a hairpin loop) that binds to tracrRNA, which forms an active complex with Cas9), a transactivating crRNA (tracrRNA, which binds to the crRNA and forms an active complex with Cas9), and an optional portion of the DNA repair template (DNA that guides the cell's repair process, allowing the insertion of a specific DNA sequence). CRISPR / Cas9 is typically used to transfect target cells using a plasmid. The crRNA needs to be designed for each application because this is the sequence that Cas9 uses to recognize and directly bind to the target DNA in the cell. A repair template carrying the CAR expression cassette also needs to be designed for each application because it must overlap with the sequences on either side of the cut and encode the insertion sequence. Multiple crRNAs and tracrRNAs can be packaged together to form a single guide RNA (sgRNA). The sgRNA can be combined with the Cas9 gene and made into a plasmid for transfection into cells.

[0476] Zinc finger nucleases (ZFNs) are artificial restriction endonucleases composed of a zinc finger DNA-binding domain and a DNA-cleavage domain. The zinc finger domain can be designed to target specific DNA sequences, enabling the zinc finger nuclease to target desired sequences within the genome. The DNA-binding domain of a single ZFN typically contains multiple individual zinc finger repeats, each of which can recognize multiple base pairs. The most common method for generating new zinc finger domains is to combine smaller zinc finger "modules" with known specificities. The most common cleavage domain in ZFNs is the nonspecific cleavage domain from the type IIs restriction endonuclease Fok I. ZFNs can be used to insert CAR expression cassettes into the genome using the endogenous homologous recombination (HR) machinery and a homologous DNA template carrying a CAR expression cassette. When the target sequence is cleaved by the ZFN, the HR machinery searches for homology between the damaged chromosome and the homologous DNA template, then replicates the template sequence between the two broken ends of the chromosome, resulting in the integration of the homologous DNA template into the genome.

[0477] Transcription activator-like effector nucleases (TALENs) are restriction endonucleases that can be engineered to cut specific DNA sequences. The TALEN system works in much the same way as ZFNs. They are generated by combining a transcription activator-like effector DNA binding domain and a DNA cleavage domain. Transcription activator-like effectors (TALEs) consist of a 33-34 amino acid repeat motif with two variable positions (with strong recognition for specific nucleotides). By assembling arrays of these TALEs, the TALE DNA binding domain can be engineered to bind to the desired DNA sequence, thereby guiding the nuclease to cut at a specific location in the genome. cDNA expression for polynucleotide therapy methods can be controlled by any suitable promoter (e.g., human cytomegalovirus (CMV), simian virus 40 (SV40), or metallothionein promoters) and regulated by any suitable mammalian regulatory element or intron (e.g., elongation factor 1a enhancer / promoter / intron structure). For example, if desired, enhancers known to preferentially control gene expression in specific cell types can be used to control the expression of nucleic acids. The enhancers used may include, but are not limited to, those with tissue or cell specificity. Alternatively, if the genomic clone is used as a therapeutic construct, regulation may be mediated by homologous regulatory sequences or, if desired, by regulatory sequences of heterologous origin, including any of the promoters or regulatory elements described above.

[0478] The method of delivering genome editing agents / systems can vary as needed. In certain embodiments, the components of the selected genome editing method are delivered as DNA constructs in one or more plasmids. In certain embodiments, the components are delivered via viral vectors. Common delivery methods include, but are not limited to, electroporation, microinjection, gene guns, puncture transfection, hydrostatic pressure, continuous infusion, ultrasound, magnetic infection, adeno-associated viruses, pseudotyped envelope proteins of viral vectors, replication-active vector cis- and trans-acting elements, herpes simplex viruses, and chemical carriers (e.g., oligonucleotides, lipoplexes, polymer vesicles, polymers, dendrimers, inorganic nanoparticles, and cell-penetrating peptides).

[0479] 5.5. Peptides and analogs

[0480] The present disclosure also includes polypeptides disclosed herein (e.g., mesothelin, CD28, CD8, CD3ζ, and PD-1 DN, etc.) or fragments thereof, which have been modified to enhance their anti-tumor activity when expressed in immune response cells. The present disclosure provides methods for optimizing amino acid sequences or nucleic acid sequences by changing the sequence. Such changes may include certain mutations, deletions, insertions, or post-translational modifications. The present disclosure also includes analogs of any polypeptide disclosed herein (including but not limited to mesothelin, CD28, CD8, CD3ζ, and PD-1 DN). Analogs may differ from the polypeptides disclosed herein by amino acid sequence differences, post-translational modifications, or both. Analogs may show at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more homology with all or part of the amino acid sequence of the present disclosure. The length of the sequence comparison is at least 5, 10, 15 or 20 amino acid residues, for example, at least 25, 50 or 75 amino acid residues, or more than 100 amino acid residues. Similarly, in an exemplary method of determining similarity, the BLAST program can be used, and the probability score is in e -3 and e -100 ] between closely related sequences. Modifications include in vivo and in vitro chemical derivatization of polypeptides, such as acetylation, carboxylation, phosphorylation, or glycosylation; such modifications may occur during polypeptide synthesis or processing, or after treatment with isolated modifying enzymes. Analogs may also differ from polypeptides produced by altering the original sequence. These include genetic variations, including natural variations and induced variations (e.g., random mutations by irradiation or exposure to ethylamine methylsulfate or site-specific mutations as described in Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual (2d ed.), CSH Press, 1989, or Ausubel et al., supra). Also included are cyclized peptides, molecules, and analogs containing residues other than L-amino acids, for example, D-amino acids or non-naturally occurring or synthetic amino acids, such as β or γ amino acids.

[0481] In addition to full-length polypeptides, the disclosed subject matter also provides a fragment of any polypeptide or peptide domain disclosed herein. As used herein, the term "fragment" refers to at least 5, 10, 13 or 15 amino acids. In certain embodiments, the fragment comprises at least 20 continuous amino acids, at least 30 continuous amino acids or at least 50 continuous amino acids. In certain embodiments, the fragment comprises at least 60 to 80, 100, 200, 300 or more continuous amino acids. The fragment can be produced by methods known to those skilled in the art, or can be produced by normal protein processing (for example, removing amino acids that are not needed for biological activity from nascent polypeptides, or removing amino acids by selective mRNA splicing or selective protein processing events).

[0482] Non-protein analogs have chemical structures designed to mimic the functional activities of the proteins disclosed herein. Such analogs may exceed the physiological activity of the original polypeptide. Methods for mimetic design are well known in the art, and analogs can be synthesized by modifying the chemical structure according to these methods so that the resulting analogs increase the anti-tumor activity of the original polypeptide when expressed in immune response cells. These chemical modifications include, but are not limited to, replacing alternative R groups and changing the saturation of specific carbon atoms of the reference polypeptide. In certain embodiments, protein analogs are relatively resistant to degradation in vivo, thereby producing a more lasting therapeutic effect upon administration. Assays for measuring functional activity include, but are not limited to, the assays described in the following examples.

[0483] According to the present disclosure, polynucleotides encoding extracellular antigen binding domains that specifically bind to human mesothelin (e.g., scFv, Fab, or (Fab)2), CD3ζ, CD8, CD28, 4-1BB, 4-1BBL, and IL-12 can be modified by codon optimization. Codon optimization can alter naturally occurring and recombinant gene sequences to achieve the highest productivity levels in any given expression system. Factors involved in different stages of protein expression include codon adaptability, mRNA structure, and various cis elements in transcription and translation. Any suitable codon optimization method or technique known to those skilled in the art can be used to modify the polynucleotides of the present disclosure, including but not limited to OptimumGene. TM , Encor Optimization and BlueHeron.

[0484] Codon optimization can be performed based on four different algorithms (e.g., Blue Heron and Encore algorithms). The codon optimized sequences obtained from all four algorithms are mixed, and all CPGs and BAM-H1 are removed to obtain the best clone. In certain embodiments, the codon optimized nucleic acid sequence is about 70% homologous to the original sequence before codon optimization. In order to obtain efficient expression in immune response cells (e.g., primary human T cells), the codon optimized nucleic acid sequence is connected to a CD8 leader sequence, such as a polynucleotide encoding SEQ ID NO: 71. The CD8 leader sequence provides optimal signal cleavage before the scFv heavy chain (QVQL). Codon optimization optimizes the expression of mesothelin CAR in immune response cells (e.g., multiple human donor primary T cells) with good transduction efficiency. Functional efficiency, specificity, and sensitivity tests are performed on multiple CAR vector copy numbers in multiple donor T cells for a variety of hematologic cancer cells and solid cancer cells expressing different mesothelins. A codon-optimized mesothelin-targeting CAR with a vector copy number of 1-4 (more specifically, approximately 3-4) provides highly effective cytotoxicity against mesothelin-highly expressed targets, but has minimal reactivity against mesothelin-lowly expressed targets (i.e., normal tissues). The above genetic engineering produces a specific mesothelin CAR that is responsive to mesothelin-highly expressed cancer cells while sparing normal tissues with low mesothelin expression. This CAR is optimal for use as a clinical vector for cancer treatment while ensuring safety.

[0485] 5.6. Pharmaceutical Composition and Administration

[0486] The presently disclosed subject matter provides compositions comprising the cells disclosed herein (e.g., as disclosed in Section 5.3). The number of cells included in the composition can vary depending on the intended use of the composition and / or the size, age, sex, weight, and condition of the subject receiving the composition. In certain embodiments, the composition comprises about 10 4 to about 10 10 , about 10 4 to about 10 6 , about 10 5 to about 10 6 , about 10 5 to about 10 7 , about 10 5 to about 10 9 or about 10 6 to about 10 8 In certain embodiments, the composition comprises at least about 1×10 5 , at least about 5×10 5 , at least about 1×10 6 , at least about 1×10 7 , at least about 1×108 In certain embodiments, the composition comprises about 1×10 5 cells of the present disclosure.

[0487] Compositions comprising immune response cells of the present disclosure can be provided to subjects systematically or directly for inducing and / or enhancing immune responses to antigens and / or treating and / or preventing tumors, pathogen infections or infectious diseases, inflammatory diseases or transplant rejection. In certain embodiments, immune response cells of the present disclosure or compositions comprising them are directly injected into target organs (e.g., organs affected by tumors). Alternatively, immune response cells of the present disclosure or compositions comprising them are provided to target organs indirectly, for example, by administration into the circulatory system (e.g., tumor vasculature). Amplifiers and differentiation agents can be provided before, during, or after administration of cells or compositions to increase the production of T cells, NK cells, or CTL cells in vitro or in vivo.

[0488] The immune response cells of the present disclosure may be administered in any physiologically acceptable excipient, typically intravascularly, but they may also be introduced into the bone or other convenient site where the cells can find a suitable location for regeneration and differentiation (e.g., the thymus). Typically, at least about 1×10 5 cells, eventually reaching about l×l0 10 Or more. Immune response cells of the present disclosure may comprise purified cell populations. Those skilled in the art can use various well-known methods, such as fluorescence activated cell sorting (FACS), to easily determine the percentage of immune response cells of the present disclosure in a colony. Suitable purity ranges in the colony comprising immune response cells of the present disclosure are about 50% to about 55%, about 5% to about 60%, and about 65% to about 70%. In certain embodiments, purity is about 70% to about 75%, about 75% to about 80%, or about 80% to about 85%. In certain embodiments, purity is about 85% to about 90%, about 90% to about 95%, and about 95% to about 100%. Those skilled in the art can easily adjust dosage (for example, purity reduction may require increased dosage). Cells can be introduced by injection, catheter or similar means.

[0489] The composition of the present disclosure can be a pharmaceutical composition comprising the immune response cells of the present disclosure or its progenitor cells and a pharmaceutically acceptable carrier. Administration can be autologous or heterologous. For example, the immune response cells or progenitor cells can be obtained from a subject and administered to the same subject or different compatible subjects. The immune response cells derived from peripheral blood or their progeny (e.g., in vivo, in vitro or in vitro derived) can be administered by local injection, including catheter administration, systemic injection, local injection, intravenous injection or parenteral administration. When administering the therapeutic composition of the present disclosure (e.g., a pharmaceutical composition comprising the immune response cells of the present disclosure), it can be formulated into an injectable form (solution, suspension, emulsion) of a unit dose.

[0490] 5.7. Formulation

[0491] Compositions comprising immune response cells of the present disclosure can be conveniently provided as sterile liquid preparations, such as isotonic aqueous solutions, suspensions, emulsions, dispersions or viscous compositions that can be buffered to a selected pH value. Liquid preparations are generally easier to prepare than gels, other viscous compositions and solid compositions. In addition, liquid compositions are more convenient to administer, especially by injection. On the other hand, viscous compositions can be formulated within a suitable viscosity range to provide a longer contact time with a specific tissue. Liquid or viscous compositions can include carriers, which can be excipients or dispersion media, including, for example, water, saline, phosphate buffered saline, polyols (such as glycerol, propylene glycol, liquid polyethylene glycol, etc.) and suitable mixtures thereof.

[0492] Sterile injection solution can be prepared by adding genetically modified immune response cells to the appropriate excipient of the desired amount and adding different amounts of other ingredients as needed. Such compositions can be mixed with suitable carriers, diluents or excipients (such as sterile water, normal saline, glucose, dextrose, etc.). The compositions can also be lyophilized. The compositions can include auxiliary substances, such as wetting agents, dispersants or emulsifiers (such as methylcellulose), pH buffers, gels or viscosity-increasing additives, preservatives, flavorings, coloring agents, etc., depending on route of administration and required preparation method. Standard texts, such as the 17th edition of "REMINGTON'S PHARMACEUTICAL SCIENCE" in 1985, are incorporated herein by reference and can be used to prepare suitable preparations without excessive experiments.

[0493] Various additives that enhance the stability and sterility of the composition can be added, including antimicrobial preservatives, antioxidants, chelating agents, and buffers. Prevention of microbial activity can be ensured by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, etc.). The absorption of the injectable pharmaceutical form can be prolonged by using agents that delay absorption, such as aluminum monostearate and gelatin. However, according to the subject matter of the present disclosure, any carrier, diluent, or additive used must be compatible with the genetically modified immune response cells or their progenitor cells.

[0494] The compositions can be isotonic, i.e., they can have the same osmotic pressure as blood and tears. The desired isotonicity of the composition can be achieved using sodium chloride or other pharmaceutically acceptable agents such as dextrose, boric acid, sodium tartrate, propylene glycol, or other inorganic or organic solutes. Sodium chloride is particularly suitable for buffers containing sodium ions.

[0495] If necessary, pharmaceutically acceptable thickening agents can be used to maintain the viscosity of the composition at a selected level. For example, methylcellulose is easily available and economical, easy to use. Other suitable thickening agents include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer etc. The concentration of the thickening agent depends on selected medicament. The important point is to use the amount that can reach selected viscosity. Obviously, the selection of suitable carriers and other additives will depend on the character of specific route of administration and specific dosage form, for example liquid dosage form (for example, whether the composition is formulated as solution, suspension, gel or other liquid forms (for example time-release form or liquid-filled form)).

[0496] The number of cells to be administered will vary depending on the subject being treated. In certain embodiments, approximately 10 4 With about 10 10 Between, about 10 5 With about 10 9 Between, about 10 4 With about 10 6 Between, about 10 5 With about 10 6 Between, about 10 5 With about 10 7 Between or about 10 6 With about 10 8 In some embodiments, at least about 1×10 5 , at least about 1×10 6 , at least about 1×10 7 , 1×10 8 , at least about 2×10 8 , at least about 3×10 8 , at least about 4×108 , or at least about 5×10 8 The immune response cells of the present disclosure are administered to a human subject. The precise determination of an effective amount may be based on individual factors of each subject, including their size, age, sex, weight, and the condition of the particular subject. A person skilled in the art can readily determine the dosage based on the present disclosure and knowledge in the art. In certain embodiments, about 1×10 5 The cells disclosed herein are administered to a subject.

[0497] Those skilled in the art can easily determine the amount of cells and optional additives, excipients and / or carriers in the composition and the method of administration. Typically, any additive (except active cells and / or reagents) is present in the amount of a solution of 0.001 to 50% (weight) in phosphate buffered saline, and the active ingredient is present in the order of micrograms to milligrams, such as about 0.0001 to about 5wt%, about 0.0001 to about 1wt%, about 0.0001 to about 0.05wt% or about 0.001 to about 20wt%, about 0.01 to about 10wt% or about 0.05 to about 5wt%. For any component administered to an animal or human, the following can be determined: toxicity, for example, by measuring lethal dose (LD) and LD50 in a suitable animal model (such as rodents, such as mice); the dosage of the composition that causes an appropriate reaction, the concentration of the components therein and the time of administering the composition. Such determination does not require unnecessary experiments based on the knowledge of those skilled in the art, the present disclosure and the documents cited herein. Moreover, the time of continuous administration can be determined without unnecessary experiments.

[0498] 5.8. Treatment

[0499] The immune response cells of the present disclosure and compositions comprising the same can be used to treat and / or prevent tumors, pathogen infections, infectious diseases, inflammatory diseases or transplant rejection. Such immune response cells can be administered to subjects (e.g., human subjects) who need them to treat or prevent solid tumors (e.g., mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumors, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic carcinoma, endometrial cancer, gastric tumors and / or bile duct cancer). In certain embodiments, the immune response cells are T cells. The T cells can be CD4 + T cells or CD8 + In some embodiments, the T cells are CD4 + T cells.

[0500] The present disclosure provides methods for inducing and / or increasing an immune response in a subject in need thereof. The immune response cells disclosed in the present invention and compositions comprising the same can be used to treat and / or prevent tumors in a subject. The immune response cells disclosed in the present invention and compositions comprising the same can be used to prolong the survival of a subject suffering from a tumor. The immune response cells disclosed in the present invention and compositions comprising the same can also be used to treat and / or prevent pathogen infection or other infectious diseases in a subject, for example, a human subject with a low immune function. Such methods include administering the immune response cells disclosed in the present invention or a composition comprising the same (e.g., a pharmaceutical composition) in an effective amount to achieve the desired effect, whether to alleviate an existing condition or to prevent recurrence. For treatment, the dosage is an effective amount that produces the desired effect. The effective amount can be provided in one or a series of administrations. The effective amount can be provided by pill or continuous infusion.

[0501] An "effective amount" (or "therapeutically effective amount") refers to an amount sufficient to produce a beneficial or desired clinical result through treatment. An effective amount can be administered to a subject once or multiple times. In terms of treatment, an effective amount is an amount sufficient to alleviate, improve, stabilize, reverse or slow the progression of a disease, or otherwise reduce the pathological consequences of the disease. The effective amount is usually determined by a physician based on the specific circumstances and is within the skill of those skilled in the art. Several factors are generally considered when determining the appropriate dosage to achieve an effective amount. These factors include the age, sex and weight of the subject, the disease being treated, the severity of the disease, and the form and effective concentration of the administered immune response cells.

[0502] For adoptive immunotherapy using antigen-specific T cells, approximately 10 6 to about 10 10 (For example, about 10 9 However, due to the high efficiency of the polypeptide composition of the present invention, a smaller number of cells of the present invention is required to achieve the desired effect. For example, about 1×10 5 A single cell of the present disclosure is sufficient to achieve the desired effect.

[0503] After the immune response cells are administered into the subject and subsequently differentiated, the immune response cells are induced, specifically targeting a specific antigen (e.g., human mesothelin). "Inducing" T cells may include inactivation of antigen-specific T cells, such as by deletion or anergy. Inactivation is particularly useful for establishing or rebuilding tolerance, such as in autoimmune diseases. The immune response cells of the present disclosure can be administered by any method known in the art, including but not limited to pleural administration, intravenous administration, subcutaneous administration, intranodal administration, intratumoral administration, intrathecal administration, intrapleural administration, intraperitoneal administration, and direct administration to the thymus. In certain embodiments, immune response cells and / or compositions comprising the same are administered to the pleura of a subject in need. In certain embodiments, immune response cells and / or compositions comprising the same are administered intrapleurally to a subject in need.

[0504] The present disclosure provides various methods for using immune response cells (e.g., T cells). For example, the present disclosure provides methods for reducing tumor burden in a subject. In certain embodiments, the method for reducing tumor burden comprises administering to a subject an effective amount of immune response cells of the present invention or a composition comprising the same. The immune response cells disclosed herein can reduce the number of tumor cells in a subject, reduce tumor size, and / or eradicate the tumor. The tumor can be a solid tumor. Non-limiting examples of solid tumors include mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumors, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic carcinoma, endometrial cancer, gastric tumors, and bile duct cancer.

[0505] The presently disclosed subject matter also provides methods for increasing or extending the survival of a subject with a tumor. In certain embodiments, the method for increasing or extending the survival of a subject with a neoplastic tumor comprises administering to the subject an effective amount of the immune response cells disclosed herein or a composition comprising the same. The method can reduce or eliminate the tumor burden in the subject. In addition, the presently disclosed subject matter provides methods for increasing the immune response in a subject, comprising administering to the subject the immune response cells disclosed herein or a composition comprising the same. The presently disclosed subject matter also provides methods for treating and / or preventing tumors in a subject, comprising administering to the subject the immune response cells disclosed herein or a composition comprising the same.

[0506] In certain embodiments, the tumor is a solid tumor. The tumor can be a primary tumor or a primary cancer. Additionally, the tumor can be in a metastatic state.

[0507] The cancers whose growth can be suppressed using the immune response cells of the present disclosure include cancers that are generally responsive to immunotherapy. Non-limiting examples of cancer treatment include mesothelioma, lung cancer (e.g., non-small cell lung cancer), pancreatic cancer, ovarian cancer, breast cancer (e.g., metastatic breast cancer, metastatic triple-negative breast cancer), colon cancer, pleural tumors, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic carcinoma, endometrial cancer, gastric tumor, bile duct cancer, cervical cancer, and salivary gland cancer. In addition, the present disclosure includes refractory or recurrent malignancies whose growth can be suppressed using the immune response cells of the present disclosure.

[0508] Examples of other tumors or cancers that can be treated using the methods of the presently disclosed subject matter include bone cancer, intestinal cancer, liver cancer, skin cancer, head and neck cancer, melanoma (malignant melanoma of the skin or intraocular), kidney cancer (e.g., clear cell carcinoma), laryngeal cancer, prostate cancer (e.g., hormone-refractory prostate cancer), blood cancers (e.g., leukemias, lymphomas, and myeloma), uterine cancer, rectal cancer, cancer of the anal region, bladder cancer, brain cancer, stomach cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, leukemias (e.g., acute leukemias, acute lymphoblastic leukemias, acute myeloid leukemias, Leukemia, acute myeloid leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma (Hodgkin's disease, non-Hodgkin's disease), small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethra cancer, penile cancer, solid tumors in children, lymphocytic lymphoma, bladder cancer, kidney cancer or ureter cancer, renal pelvis cancer, central nervous system tumors (CNS), Primary central nervous system lymphoma, tumor angiogenesis, spinal axis tumors, brain stem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers, including asbestos-induced cancers including Waldenstrom macroglobulinemia, heavy chain disease, and solid tumors such as sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, Myosoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, hepatocarcinoma, Nile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, salivary gland cancer, uterine cancer, testicular cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligoglioma, schwannoma, meningioma, melanoma, neuroblastoma, and retinoblastoma).

[0509] In addition, the subject disclosed in the present invention provides a method for increasing immune activation cytokine production to respond to cancer cells or pathogens in a subject. In some embodiments, the method includes administering to the subject an immune response cell of the present disclosure or a composition comprising the same. The immune activation cytokine can be granulocyte macrophage colony stimulating factor (GM-CSF), IFN-α, IFN-β, IFN-γ, TNF-α, IL-2, IL-3, IL-6, IL-11, IL-7, IL-12, IL-15, IL-21, interferon regulatory factor 7 (IRF7) and combinations thereof. In some embodiments, immune response cells increase the production of GM-CSF, IFN-γ and / or TNF-α.

[0510] The presently disclosed subject matter provides therapies that are particularly useful for treating solid tumors (solid tumors are, for example, mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumors, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic carcinoma, endometrial cancer, gastric tumors, and bile duct cancer). Solid tumors can be primary tumors or metastatic tumors. Certain solid tumors are heterogeneous MSLN-expressing tumors, such as breast cancer (e.g., TNBC), lung cancer, ovarian cancer, pancreatic cancer, esophageal cancer, colon cancer, gastric cancer, and malignant pleural mesothelioma (MPM). Heterogeneous MSLN-expressing cells (e.g., tumor cells) are cell populations comprising low MSLN-expressing cells and high MSLN-expressing cells. In the presence of high MSLN expressing cells, the immune response cells disclosed herein can exhibit enhanced cytotoxicity and anti-tumor activity against low MSLN expressing cells (e.g., about 2000 or less, about 1000 or less, about 900 or less, about 800 or less, about 700 or less, about 600 or less, about 500 or less, about 400 or less, about 300 or less, about 200 or less, or about 100 or less MSLN binding sites / cell). In certain embodiments, even in the presence of high MSLN expressing cells, the immune response cells do not exhibit increased cytotoxicity or nonspecific killing against MSLN-negative cells. Thus, the immune response cells can exhibit increased cytotoxicity and anti-tumor activity against low MSLN expressing cells in the presence of high MSLN expressing cells while retaining safety against MSLN-negative cells.

[0511] In addition, the subject matter of the present disclosure provides a method for treating a subject suffering from a pathogen infection (e.g., a viral infection, a bacterial infection, a fungal infection, a parasitic infection, or a protozoan infection). The subject matter of the present disclosure is particularly suitable for enhancing the immune response of a subject with a low immune function. Exemplary viral infections susceptible to treatment using the methods of the present invention include, but are not limited to, cytomegalovirus (CMV), Epstein-Barr virus (EBV), human immunodeficiency virus (HIV), and influenza virus infection. Therefore, the subject matter of the present disclosure provides a method for treating or preventing a pathogen infection in a subject, the method comprising administering an effective amount of an immune response cell of the present disclosure or a composition comprising the same.

[0512] According to the present disclosure, the various methods described above may include administering at least one immunomodulator. Non-limiting examples of immunomodulators include immunostimulators, checkpoint immune blockers, radiotherapeutics, and chemotherapeutics. In certain embodiments, the immunomodulator is an immunostimulator. Non-limiting examples of immunostimulators include IL-12 and agonist costimulatory monoclonal antibodies. In certain embodiments, the immunostimulator is IL-12. In certain embodiments, the immune response cells of the present disclosure or compositions comprising the same can be used to treat breast cancer (BC) in combination with anti-IL-12 antibodies, such as metastatic triple-negative breast cancer (TNBC). Non-limiting examples of agonist costimulatory monoclonal antibodies include anti-4-1BB antibodies, anti-OX40 antibodies, and anti-ICOS antibodies. In certain embodiments, agonist costimulatory monoclonal antibodies are anti-4-1BB antibodies.

[0513] In certain embodiments, the immune response cells of the present disclosure or compositions comprising the same can not only demonstrate tumor-targeted adoptive T cell therapy, but also can enhance T cell function by designing improved antigen receptors and intervening in the host microenvironment through immunomodulation using IL-12. Among all immunotherapy methods, a multifunctional cytokine IL-12 is considered to be one of the most promising methods for treating BC (Boggio, K. et al., Cancer Res 60, 359-364 (2000); Czerniecki, BJ et al., Cancer Res 67, 1842-1852 (2007); Nanni, P. et al., J Exp Med 194, 1195-1205 (2001)). IL-12 is considered to be the main regulator of adaptive type 1 cell-mediated immunity, which is a key pathway involved in anti-tumor response (Del Vecchio, M. et al., Clin Cancer Res 13, 4677-4685 (2007)). IL-12 regulates anti-tumor responses at different levels, including differentiation of CD4 T cells toward a Th1 phenotype (Wesa et al., J Immunother 30, 75-82 (2007)), enhancing T cell and NK effector function (Curtsinger et al., J Exp Med 197, 1141-1151 (2003)), remodeling innate immune responses (Chmilewski et al., Cancer Res 71, 5697-5706 (2011)), and regulating tumor angiogenesis (Voest et al., J Natl Cancer Inst 87, 581-586 (1995)). The immunomodulatory and anti-angiogenic functions of IL-12 provide a rationale for combining this cytokine with the immune response cells of the present disclosure for treating cancer, such as BC (e.g., TNBC). In 148 clinical trials, including the administration of IL-12 to cancer patients (36 of which were recently reported), phase II studies were successfully conducted by intraperitoneal injection (Lenzi et al., Clin. Cancer Res. 8, 3686-3695 (2002)) or subcutaneous injection (Mahvi et al., Cancer Gene Ther. 14, 717-723 (2007); Kang et al., Hum. Gene Ther. 12, 671-684 (2001)). IL-12 has been shown to induce immunity against local and distant tumors through paracrine secretion of IL-12 produced by gene transfer.Although some studies have demonstrated the anticancer effect of IL-12 in preclinical models of breast cancer (BC) (Boggio et al., Cancer Res 60, 359-364 (2000); Nanni et al., J Exp Med 194, 1195-1205 (2001)), the significant toxicity caused by the administration of recombinant human IL-12 observed in some late-stage cancer clinical trials has hindered its clinical application. To overcome this limitation, many groups have demonstrated that in clinical models of BC, intratumoral delivery of IL-12 using adenoviral vectors can induce tumor regression and T cell activation (Gyorffy et al., J Immunol 166, 6212-6217 (2001); Bramson et al., Hum Gene Ther 7, 1995-2002 (1996)). Recently, Sabel et al. used polylactic acid microspheres to release IL-12 into tumors and found that the anti-tumor response was mainly mediated by NK cells (Sabel et al., Brest Cancer Res 122, 325-336 (2010)). Other researchers used mesenchymal stromal cells to locally deliver IL-12 to mouse BC (Eliopoulos et al., Cancer Res 68, 4810-4818 (2008)). A phase I trial of paclitaxel and trastuzumab combined with IL-12 in patients with HER2 / neu-expressed malignancies showed that IL-12 and trastuzumab had significant synergistic effects in stimulating NK cell cytokine secretion (Bekaii-Saab et al., Molecular cancer therapeutics 8, 2983-2991 (2009)). Therefore, IL-12 has considerable prospects as an anticancer agent, and it is reasonable to use it as a costimulator in adoptive T cell therapy.

[0514] In certain embodiments, the immunomodulator is a checkpoint immune blocker. Non-limiting examples of checkpoint immune blockers include anti-PD-L1 antibodies, anti-CTLA-4 antibodies, anti-PD-1 antibodies, anti-LAG3 antibodies, anti-B7-H3 antibodies, and anti-TIM3 antibodies. In certain embodiments, the checkpoint immune blocker is an anti-PD-L1 antibody. In certain embodiments, the immune response cells of the present disclosure or compositions comprising the same can be used in combination with anti-PD-L1 antibodies to treat breast cancer (BC), such as TNBC.

[0515] Programmed cell death ligand 1 (PD-L1 / B7-H4 / CD274) is an inhibitory signal that is usually expressed in active inflammatory tissues and used as a negative feedback loop to limit T cell activation. PD-L1 expression is usually absent in non-inflammatory normal tissues (including breast (Dong et al., Nature medicine 8, 793-800 (2002))). On the contrary, PD-L1 expression is most common in cancer tissues, especially in tissues with inflammatory infiltration (Spranger et al., Science translational medicine 5, 200ra116 (2013)). This association with inflammation may be due to PD-L1 upregulation when tumor cells are exposed to cytokines secreted by T cells produced by T cell activation. This expression pattern is demonstrated in BC, with 50%-75% of BC samples staining positive for PD-L1, and PD-L1 expression is closely associated with severe lymphocytic infiltration (Brown et al., Journal of immunology 170, 1257-1266 (2003); Ghebeh et al., Neoplasia 8, 190-198 (2006); Ghebeh et al., BMC cancer 8, 57 (2008)). BC-infiltrating T cells also express PD-L1 in 54% of patients (Ghebeh et al., BMC cancer 8, 57 (2008)). Some BC may also naturally express PD-L1, secondary to oncogenic signals. Activation of the PI(3)K pathway leads to upregulation of PD-L1 protein in BC cells, and PI(3)K activation in patient tumors is significantly correlated with PD-L1 expression (Crane et al., Oncogene 28, 306-312 (2009)). Activated T cells express PD-1, which spatially and temporally links ligands to receptor expression in immunosuppressive TME. The expression of PD-L1 in BC tissues indicates that it is an immunotherapy target for these patients. The efficacy of PD-L1 / PD-1 blockers in a variety of preclinical cancer models (including breast cancer (Ge et al., Cancer letters 336, 253-259 (2013))) paves the way for Phase I trials using PD-L1 or PD-1 targeted antibodies to treat advanced cancer patients. A Phase I study (using PD-1 antibodies) proved to be effective only for PD-L1+ patients (Topalian et al., The New England journal of medicine 366, 2443-2454 (2012)). Genetically engineered T cells provide unique advantages for overcoming the co-inhibitory checkpoints and typical costimulation lack within TME. CAR-expressing T cells are indeed engineered to optimize their costimulation requirements to support the expansion, survival, and function of T cells.

[0516] In some embodiments, the immunomodulator is a radiotherapeutic agent. The local, radiation-induced immune environment can not only provide a prerequisite for enhancing the implantation of targeted T cells in tumors (thereby eliminating the need for systemic lymphodepletion therapy), but also the immune response produced by the combination of radiotherapy and adoptive T cell therapy also enhances the anti-tumor efficacy of distant sites. In radiation-resistant tumors, 4-1BB co-stimulatory signaling in CART cells can overcome immunosuppression. In some embodiments, the immunomodulator is a chemotherapeutic agent, including but not limited to cisplatin. Cisplatin-induced chemokine and cytokine secretion can promote MSLN targeting and endogenous T cell responses.

[0517] Studies have shown that patients with lung adenocarcinoma (LAC) and malignant pleural mesothelioma (MPM) who have high levels of cytotoxic tumor-infiltrating lymphocytes (cTILs) and low levels of regulatory T cells (Tregs) have better prognosis and longer progression-free survival (Servais et al., Clin Cancer Res (May 1, 2012); 18: 2478-2489; Kachala et al., Clin Cancer Res (2013); 20(4); 1020–8). Adoptive T cell therapy using CARs targeting MSLN can be used to promote cTILs in LAC and MPM. Servais (2012) and Kachala (2013) reported that MSLNs are overexpressed in LAC and MPMs and promote aggressiveness, justifying the selection of MSLNs as a target for CAR T cell therapy. Increased TIL proportions after cisplatin and radiotherapy are associated with improved prognosis in mouse models and patients.

[0518] Tumor radiotherapy-cisplatin treatment-induced tumor and remote immune regulation can provide the required preconditioning for better engraftment of adoptively transferred T cells; T cell co-stimulation strategies utilizing tumor and stromal immune regulation can enhance the anti-tumor efficacy of endogenous and adoptively transferred T cells.

[0519] In addition, the different methods described above using immune response cells (e.g., T cells) expressing mesothelin-specific CARs, such as for treating cancer in a subject, or for reducing a subject's tumor burden, can be combined with cancer cell antigen regulation. Immune response cells (e.g., T cells) expressing mesothelin-specific CARs can target and kill MSLN (referred to as "cell membrane MSLN") expressed on the membrane of tumors or cancer cells, but cannot kill cytoplasmic MSLN. Certain tumors or cancers (such as lung cancer and mesothelioma) have lower cell membrane MSLN but higher cytoplasmic MSLN. Cancer cell antigen regulation can increase the expression of cell membrane MSLN in tumors or cancer cells, which can make tumors or cancer cells more likely to be targeted by immune response cells expressing CAR, thereby making them more likely to be killed by immune response cells. In certain embodiments, cancer cell antigen regulation is radiation.

[0520] Further modifications can be introduced into immune response cells (e.g., T cells) to avoid or minimize the risk of immune complications (called "malignant T cell transformation"), such as graft-versus-host disease (GvHD), or when healthy tissue expresses the same target antigen as tumor cells, resulting in results similar to GvHD. A potential solution to this problem is to engineer suicide genes into T cells expressing CARs. Suitable suicide genes include, but are not limited to, herpes simplex virus thymidine kinase (hsv-tk), inducible Caspase 9 suicide gene (iCasp-9), and truncated human epidermal growth factor receptor (EGFRt) polypeptides. In certain embodiments, the suicide gene is an EGFRt polypeptide. The EGFRt polypeptide can eliminate T cells by administering an anti-EGFR monoclonal antibody (e.g., cetuximab). EGFRt can be covalently linked to the 3' end of the intracellular signaling domain of a CAR targeting mesothelin. The suicide gene can be contained in a vector comprising a nucleic acid encoding a mesothelin-specific CAR of the present disclosure. In this way, administration of a prodrug designed to activate a suicide gene (e.g., a prodrug such as AP1903, which activates iCasp-9) during malignant T cell transformation (e.g., GVHD) triggers apoptosis in suicide gene-activated CAR-expressing T cells.

[0521] In addition, the presently disclosed subject matter provides a method for preventing and / or treating an inflammatory disease in a subject. In certain embodiments, the method comprises administering to the subject an immune response cell of the present disclosure or a composition comprising the same. In certain embodiments, the immune response cell is an immunosuppressive cell. In certain embodiments, the immunosuppressive cell is a regulatory T cell. In certain embodiments, the inflammatory disease is pancreatitis. In certain embodiments, the subject is a human. In certain embodiments, the subject is an organ transplant recipient, such as a pancreas transplant recipient.

[0522] In addition, the presently disclosed subject matter provides a method for preventing graft rejection in a subject who is an organ transplant recipient. In certain embodiments, the method comprises administering to the subject an immune response cell of the present disclosure or a composition comprising the same. In certain embodiments, the immune response cell is an immunosuppressive cell. In certain embodiments, the immunosuppressive cell is a regulatory T cell. In certain embodiments, the subject is a human. In another embodiment, the subject is a pancreas transplant recipient.

[0523] The CAR targeting mesothelin disclosed herein can be transduced into immunosuppressive cells, such as regulatory T cells. The transduced immunosuppressive cells can be administered to subjects (e.g., people) with inflammatory conditions or inflammatory diseases. In some embodiments, the inflammatory site or inflammatory disease site has a high expression level of mesothelin, which is recognized by the MSLN-CAR disclosed herein. Inflammatory conditions may be very serious, such as severe pancreatitis. In addition, the transduced immunosuppressive cells can be administered to subjects receiving organ transplants.

[0524] In addition, the CAR targeting mesothelin disclosed herein and the second CAR targeting an MHC antigen can be co-transduced into immunosuppressive cells (e.g., regulatory T cells) so that the immunosuppressive cells can specifically aggregate at the site of the transplanted pancreas. In certain embodiments, an MHC class I subject receives a pancreas transplant from an MHC class II donor; the recipient's regulatory T cells are transduced with the MSLN-specific CAR disclosed herein and a second CAR targeting an MHC class II antigen, so that the recipient's transduced regulatory T cells aggregate / pool at the site of the transplanted pancreas to avoid graft or organ rejection.

[0525] Human subjects suitable for treatment generally include two treatment groups that can be distinguished by clinical criteria. Subjects with "advanced disease" or "high tumor burden" are those with clinically measurable tumors. A clinically measurable tumor is one that can be detected based on tumor mass (e.g., by palpation, CAT scan, sonogram, mammography, or X-ray; positive biochemical or histopathological markers alone are not sufficient to identify this population). These subjects are administered a pharmaceutical composition to elicit an anti-tumor response with the goal of alleviating their condition. Ideally, the tumor mass will be reduced as a result, and any clinical improvement is beneficial. Clinical improvement includes a reduction in the risk or rate of progression of the tumor or a reduction in pathological consequences.

[0526] The second group of suitable subjects is referred to in the art as the "adjuvant group." These individuals have a history of tumors that have responded to another form of treatment. Previous treatments may include, but are not limited to, surgical resection, radiation therapy, and traditional chemotherapy. Therefore, these individuals do not have clinically measurable tumors. However, they are suspected to be at risk for disease progression, either near the primary tumor site or as metastases. This group can be further subdivided into high-risk and low-risk individuals. The subdivisions are based on characteristics observed before and after initial treatment. These characteristics are known in the clinical art and are applicable to each different tumor. Typical characteristics of the high-risk subgroup are tumors that invade adjacent tissues or show lymph node involvement.

[0527] Another group has a genetic predisposition to tumors but has not yet demonstrated clinical symptoms of tumors. For example, a woman who tests positive for a genetic mutation associated with breast cancer but is still of childbearing age may wish to receive prophylactic treatment with one or more immune response cells as described herein to prevent the development of tumors until she is eligible for preventive surgery.

[0528] As a result of the surface expression of anti-mesothelin CAR and PD-1 DN, which enhance the anti-tumor efficacy of immune response cells, adoptively transferred T or NK cells have enhanced selective cytolytic activity at the tumor site. In addition, after localizing to the tumor or viral infection and proliferating, T cells transform the tumor or viral infection site into a highly conductive environment for a variety of immune cells to participate in physiological anti-tumor or anti-viral responses (tumor-infiltrating lymphocytes, NK cells, NKT cells, dendritic cells and macrophages).

[0529] In addition, the subject matter of the present disclosure provides a method for treating and / or preventing a pathogen infection (e.g., a viral infection, a bacterial infection, a fungal infection, a parasitic infection, or a protozoan infection) in a subject (e.g., an immunocompromised subject). The method may comprise administering an effective amount of an immune response cell of the present disclosure or a composition comprising the same to a subject suffering from a pathogen infection. Exemplary viral infections susceptible to treatment include, but are not limited to, cytomegalovirus (CMV), Epstein-Barr virus (EBV), human immunodeficiency virus (HIV), and influenza virus infection.

[0530] Further modifications may be introduced into the immune response cells (such as T cells) of the present disclosure to avoid or minimize the risk of immune complications (called "malignant T cell transformation"), such as graft-versus-host disease (GvHD), or when healthy tissue expresses the same target antigen as tumor cells, resulting in GvHD-like results. A potential solution to this problem is to engineer suicide genes into the immune response cells of the present disclosure. Suitable suicide genes include, but are not limited to, herpes simplex virus thymidine kinase (hsv-tk), inducible Caspase 9 suicide gene (iCasp-9), and truncated human epidermal growth factor receptor (EGFRt) polypeptides. In certain embodiments, the suicide gene is an EGFRt polypeptide. The EGFRt polypeptide can eliminate T cells by administering anti-EGFR monoclonal antibodies (e.g., cetuximab). EGFRt can be covalently linked to the upstream of the antigen recognition receptor of the CAR of the present disclosure. The suicide gene can be contained in a vector comprising a nucleic acid encoding the CAR of the present disclosure. In this way, administration of a prodrug intended to activate the suicide gene (e.g., a prodrug (e.g., AP1903, which activates iCasp-9)) during malignant T cell transformation (e.g., GVHD) triggers apoptosis of CAR-expressing T cells activated by the suicide gene. Incorporating the suicide gene into the CAR of the present disclosure can eliminate most of the CAR T cells in a very short period of time, thereby improving safety. The immune response cells (e.g., T cells) of the present disclosure that are combined with the suicide gene can be pre-cleared at a specific time point after CAR T cell infusion, or eradicated at early signs of toxicity.

[0531] 5.9. Kit

[0532] The present disclosure provides kits for inducing and / or enhancing an immune response and / or treating and / or preventing a tumor or pathogen infection in a subject. In certain embodiments, the kit comprises an effective amount of immune response cells of the present disclosure or a pharmaceutical composition comprising the same. In certain embodiments, the kit comprises a sterile container; these containers can be boxes, ampoules, bottles, vials, tubes, bags, pouches, blister packs or other suitable container forms known in the art. These containers can be made of plastic, glass, laminated paper, metal foil or other materials suitable for holding drugs. In certain embodiments, the kit includes an isolated nucleic acid molecule encoding an anti-mesothelin CAR and an isolated nucleic acid molecule encoding an expressible form of PD-1 DN, which may optionally be contained in the same or different vectors.

[0533] If necessary, immune response cells and / or nucleic acid molecules are provided, along with instructions for administering these cells or nucleic acid molecules to subjects suffering from tumors or pathogens or immune disorders or at risk of developing them. The instructions typically include information about using the composition to treat and / or prevent tumors or pathogen infections. In certain embodiments, the instructions include at least one of the following: a description of the therapeutic agent; a dosage schedule and administration for treating or preventing tumors, pathogen infections or immune disorders or their symptoms; precautions; warnings; indications; contraindications; overdose information; adverse reactions; animal pharmacology; clinical studies; and / or reference materials. The instructions can be printed directly on the container (if any), or affixed to the container as a label, or as a separate sheet of paper, brochure, card, or folder within or provided with the container.

[0534] 6. Examples

[0535] Unless otherwise indicated, the practice of the present invention employs conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are well within the capabilities of those skilled in the art. These techniques are fully explained in the literature, for example, in Molecular Cloning: A Laboratory Manual, Second Edition (Sambrook, 1989); Oligonucleotide Synthesis (Gait, 1984); Animal Cell Culture (Freshney, 1987); Enzymology Methods, Handbook of Experimental Immunology (Weir, 1996); Gene Transfer Vectors for Mammalian Cells (Miller and Calos, 1987); Current Protocols in Molecular Biology (Ausubel, 1987); PCR: Polymerase Chain Reaction (Mullis, 1994); Current Protocols in Immunology (Coligan, 1991). These techniques are applicable to the production of polynucleotides and polypeptides disclosed herein and, therefore, may be considered in making and implementing the disclosed subject matter. Particularly useful techniques for specific embodiments will be discussed in the following sections.

[0536] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the cells and compositions of the present disclosure and are not intended to limit the scope of what the inventors regard as their invention.

[0537] Example 1

[0538] The polypeptide composition of the present disclosure is prepared. The polypeptide composition comprises: (i) a CAR that binds to human mesothelin and (ii) a dominant negative form of programmed cell death 1 (PD-1 DN), such as Figure 1The CAR targeting mesothelin comprises (a) a CD8 signal peptide (e.g., a CD8 signal peptide consisting of an amino acid sequence as shown in SEQ ID NO: 71), (b) an extracellular antigen binding domain, which is a scFv comprising a V H , the V H comprising a CDR1 consisting of the amino acid sequence of SEQ ID NO: 76, a CDR2 consisting of the amino acid sequence of SEQ ID NO: 77, and a CDR3 having the amino acid sequence of SEQ ID NO: 78; and V L , the V L comprising a CDR1 consisting of the amino acid sequence of SEQ ID NO:79, a CDR2 consisting of the amino acid sequence of SEQ ID NO:80, and a CDR3 consisting of the amino acid sequence of SEQ ID NO:81, (c) a transmembrane domain comprising a CD28 polypeptide (e.g., a CD28 polypeptide consisting of the amino acid sequence of SEQ ID NO:92 (or amino acids 153 to 179 of SEQ ID NO:90)), (d) a CD28 hinge / spacer region (e.g., a CD28 polypeptide consisting of the amino acid sequence of SEQ ID NO:15 (or amino acids 114 to 152 of SEQ ID NO:90)), and (e) an intracellular signaling domain comprising a modified CD3 zeta polypeptide consisting of the amino acid sequence of SEQ ID NO:35 and a costimulatory signaling region comprising a CD28 polypeptide (e.g., a CD28 polypeptide consisting of the amino acid sequence of SEQ ID NO:101 (or amino acids 180 to 220 of SEQ ID NO:90)). The PD-1 DN comprises a PD-1 signal peptide consisting of amino acids 1 to 20 of SEQ ID NO: 48, a PD-1 extracellular domain consisting of amino acids 21 to 165 of SEQ ID NO: 48, and a CD8 polypeptide consisting of amino acids 137 to 207 of SEQ ID NO: 86. The polypeptide composition further comprises a P2A peptide having an amino acid sequence as shown in SEQ ID NO: 121, which is located between the CAR and the PD-1 DN, as shown in FIG. Figure 1 The peptide composition is designed as "M28z1XXPD1DNR".

[0539] The CAR contained in the polypeptide structure has an amino acid sequence as shown in SEQ ID NO: 56. An exemplary nucleic acid sequence encoding the polypeptide structure is shown in SEQ ID NO: 123. Another exemplary nucleic acid sequence encoding the polypeptide composition is shown in SEQ ID NO: 124.

[0540] Example 2

[0541] The present invention studies the activity of M28z1XX-P2A-PD1DNR having the structure of the polypeptide composition described in Example 1. Figure 2 As shown, the structures of the alternative and control constructs were compared to M28z1XX-P2A-PD1DNR.

[0542] Viral vectors containing CAR constructs were generated in the production cell line RD114. Figures 3A-3D As shown. RD114 cells were transduced with different dilutions of H29 virus supernatant (undiluted, 1:2 and 1:4) and CAR expression was stained by flow cytometry using anti-Fab antibody. RD114 blank was used as a negative control. Figures 4A-4E , 5A-5E and 6A-6F, human T cells were successfully transduced with M28z1XX-P2A-PD1DNR. PHA-activated T cells were transduced with different concentrations of RD114 viral supernatant, and CAR expression was stained by anti-Fab staining and PD1DNR was stained by anti-PD1 staining by flow cytometry. The present invention studies whether the vector copy number (VCN) is related to the mean fluorescence intensity (MFI). PHA-activated T cells were transduced with different concentrations of RD114 viral supernatant, and CAR expression was stained by anti-Fab staining and flow cytometry analysis. The genomic DNA of the transduced T cells was isolated, and the vector copy number was determined as VCN / μg DNA using qPCR. As Figures 7A-7C As shown, the MFI of CAR-positive cells correlated with VCN / μg DNA for all three donors tested. Human CD4 + and CD8 + The transduction rates of T cells are shown in Table 1 .

[0543] Table 1

[0544]

[0545] Next, the present invention studied the cytolytic effect of M28z1XX-PD1DNR CAR T cells. Using an impedance-based assay, high MSLN target cells (MGM) were co-cultured with M28z1XX-PD1DNR CAR T cells from different donors at different E:T ratios. The results are shown in Figure 2. Figure 8 As shown. Figure 8 As shown, T cells transduced with the M28z1XX-PD1DNR CAR exhibited potent cytotoxicity against all three different donors tested, with effector cytotoxicity spanning multiple E:T ratios (data not shown).

[0546] in conclusion:

[0547] The M28z1XX-PD1DNR vector was successfully produced in RD114 cells. All constructs were successfully established as stable producer cell lines. The viral vector was titrated to produce approximately 40-60% transduction in multiple donor T cells. Both CD4 and CD8 T cells were successfully transduced to express the CAR and PD1DNR. A correlation between vector copy number and transduction was observed.

[0548] Example 3

[0549] This example describes a comparative analysis of various constructs, including M28z1XX-PD1DNR. Cytotoxicity was determined using an impedance-based assay. The principle of impedance-based cytotoxicity measurement (eCTL) is as follows: Figure 9 The parameters for comparative analysis are shown in Figure 10 As shown in Figure 2, including CAR construct, donor, CAR target and E:T ratio. The expression of MSLN and PD-L1 in target cell lines was detected. The expression of MSLN and PD-L1 in mesothelioma (MGM, MGM-PDL1 and MSTOG) and lung cancer (A549GM and A549G) cell lines was detected by flow cytometry. The results are shown in Figure 2. Figures 11A-11E As shown. MGM, MGM-PDL1, and A549GM overexpress MSLN. MGM-PDL1 cells additionally overexpress PD-L1.

[0550] Simultaneous detection of CAR and PD1 expression in transduced T cells. Using flow cytometry, CAR expression in human T cells transduced with M28z, M28z1xx, M28z-PD1DNR, or M28z1XX-PD1DNR was analyzed by anti-myc staining, and PD1 / PD1DNR expression was analyzed by anti-PD1 staining. The results are shown in Figure 2. Figures 12A-12E shown.

[0551] The antitumor efficacy of CAR T cells expressing M28z, M28z1XX, M28z-PD1DNR, or M28z1XX-PD1DNR against high MSLN tumor cells (MGM) was compared. High MSLN target cells (MGM) were co-cultured with M28z, M28z1XX, M28z-PD1DNR, M28z1XX-PD1DNR, or untransduced T cells at different E:T ratios. Antitumor efficacy was assessed using an impedance-based assay. Figures 13A-13C In addition, high MSLN target cells (MGM) labeled with Cr-51 were co-cultured with M28z, M28z1XX, M28z-PD1DNR, M28z1XX-PD1DNR, or untransduced T cells at different E:T ratios for 18 hours. Cytotoxicity was determined by Cr-51 CTL. The results are shown in Figure 14 shown.

[0552] Next, we compared the antitumor efficacy of CAR T cells expressing M28z, M28z1XX, M28z-PD1DNR, or M28z1XX-PD1DNR against MSLN-negative tumor cells (MSTOG). MSLN-negative target cells (MSTOG) were co-cultured with M28z, M28z1XX, M28z-PD1DNR, M28z1XX-PD1DNR, or untransduced T cells at the specified E:T ratio. Antitumor efficacy was assessed using an impedance-based assay. Figures 15A-15C In addition, MSLN-negative target cells (MSTOG) labeled with Cr-51 were co-cultured with M28z, M28z1XX, M28z-PD1DNR, M28z1XX-PD1DNR, or untransduced T cells at different E:T ratios for 18 h. Cytotoxicity was determined by Cr-51 CTL assay. The results are shown in Figure 16 shown.

[0553] In addition, the antitumor efficacy of CAR T cells expressing M28z, M28z1XX, M28z-PD1DNR, or M28z1XX-PD1DNR against high MSLN tumor cells overexpressing PDL1 was compared and analyzed. High MSLN target cells overexpressing PDL1 (MGM-PDL1) were co-cultured with M28z, M28z1XX, M28z-PD1DNR, M28z1XX-PD1DNR, or untransduced T cells at different E:T ratios. Antitumor efficacy was evaluated using an impedance-based assay. The results are shown in Figure 3. Figures 19A-19C Similarly, the comparative analysis of the antitumor efficacy of CAR T cells expressing M28z, M28z1XX, M28z-PD1DNR or M28z1XX-PD1DNR against high MSLN tumor cells (A549GM) was determined, and the results are shown in Figure 2. Figures 18A-18C and comparative analysis of the anti-tumor efficacy of CAR T cells expressing M28z, M28z1XX, M28z-PD1DNR or M28z1XX-PD1DNR against low MSLN tumor cells (A549G), as shown in FIG. Figures 19A-19C shown.

[0554] in conclusion:

[0555] The M28z1XX-PD1DNR construct kills MSLNs in an E:T ratio-dependent manner + The results were reproduced in different T cell donors with different cancers (lung cancer and mesothelioma cell lines). Targeted killing was associated with the expression level of MSLN, and the PD-L1 high expression of MSLN + Target cells are effective.

[0556] Example 4 - Regional infusion of clinical-grade mesothelin-targeted CAR T cells with intracellular PD-1 checkpoint blockade Send: Converted to Phase I trial

[0557] Summarize: This example provides evidence of preclinical safety and enhanced anti-tumor efficacy of clinical-grade M28z1XXPD1DNR CAR T cells.

[0558] method: Comparative cytotoxicity, proliferation, and cytokine secretion of human T cells engineered to express M28z or M28z1XXPD1DNRCAR were assessed by chromium release, accumulation, and Luminex analysis, respectively. A single dose (1×10 5 Antitumor efficacy of intrapleural administration of M28z or M28z1XXPD1DNR CAR T cells (CAR T cells; E:T 1:1000) in NSG mice bearing orthotopic pleural mesothelioma. After tumor eradication, repeated tumor challenge (2×10 6 to 11×10 6 The functional persistence of CAR T cells was tested with increasing doses of tumor cells.

[0559] result: In vitro, both M28z and M28z1XXPD1DNR CAR T cells exhibited antigen-specific cytotoxicity, accumulation, and effector cytokine secretion (Table 2). In vivo, a single dose of M28z1XXPD1DNR CAR T cells resulted in tumor eradication, improved survival, and resistance to tumor reconstitution after 10 tumor rechallenges (Table 2), compared with a single dose of M28z CAR T cells.

[0560] in conclusion: Data on the safety, tumor eradication, and functional persistence of CAR T cells without anti-PD-1 antibodies support the initiation of a phase I clinical trial of intrapleurally administered M28z1XXPD1DNR CAR T cells in patients with pleural mesothelioma.

[0561] Table 2 Comparison of M28z and M28z1XXPD1DNR CAR T cell constructs

[0562]

[0563] Example 5 - Next-Generation CAR T Cells with Cell-Intrinsic PD-1 Blockade: Clinical Rationale, Preclinical, and Clinical Trial Protocol Development

[0564] Malignant pleural mesothelioma (MPM) is a cancer with a low mutational burden and low PDL1 expression, which inhibits the response to anti-PD-1 antibodies. In an ongoing phase I / II trial (NCT02414269, n=41), the safety and antitumor efficacy of intrapleural administration of mesothelin-targeted chimeric antigen receptor (M28z CAR) T cells followed by PD-1 antibodies have been demonstrated. CART cells with a cell-intrinsic anti-PD-1 strategy are safe and can provide antitumor efficacy against both low and high PDL1 tumors without the need for repeated administration of anti-PD-1 antibodies.

[0565] Summarize: This example provides evidence of preclinical safety and enhanced anti-tumor efficacy of clinical-grade M28z1XXPD1DNR CAR T cells.

[0566] method Clinical-grade M28z and M28z1XXPD1DNR (modified CD3z domain with PD-1 dominant negative receptor) CARs were transduced into multiple donor T cells as effectors, and MPM cells with low and high PDL1 were used as targets. Comparative in vitro and in vivo antitumor efficacy was evaluated in mice with orthotopic MPM at different E:T ratios. Systemic antitumor immunity was tested by repeated tumor challenge at distant sites.

[0567] result: In vitro, no significant differences were found between M28z and M28z1XXPD1DNR CARs (antigen-specific cytotoxicity, accumulation, and effector cytokine secretion). 5 Intrapleural administration of M28z CAR T cells, or repeated administration of anti-PD1 antibodies or cell-intrinsic PD1 DNR, resulted in similar tumor eradication, improved survival, and weight gain. Figure 20A and Table 3. In orthotopic MPM mice, a single low dose (1×10 5 CAR T cells) eradicated pleural tumors and demonstrated enhanced systemic immunity by protecting against tumor rechallenge at distant peritoneal sites without any toxicity compared to M28z CAR T cells (PD1DNR binds to mouse PDL1 / 2). Figure 20B and Figure 20C The obtained tumors showed higher and deeper CAR T cell penetration compared to non-transduced T cells. Figure 20D .

[0568] in conclusion:A single, low-dose intrapleural administration of M28z1XXPD1DNR CAR T cells demonstrated feasibility, safety, tumor eradication, functional persistence, and systemic antitumor immunity.

[0569] Table 3 Comparison of therapeutic characteristics of PD-1 DNR CAR T cells and CAR T cell checkpoint blockade

[0570]

[0571]

[0572] Example 6

[0573] 1. Summary

[0574] Malignant pleural mesothelioma (MPM) is a rare and fatal malignant tumor associated with asbestos exposure. MPM is a regionally invasive primary pleural malignancy characterized by invasion of vital organs or the chest wall (Carbone et al., CA Cancer J Clin. 2019; 69(5): 402-429). Most patients (60%-70%) report having locally advanced disease and are unresectable (Nelson et al., J Clin Oncol. 2017; 35(29): 3354-3362; Flores et al., J Thorac Oncol. 2007; 2(10): 957-965). Even with successful completion of a combination of chemotherapy, aggressive surgical resection, and radiation therapy, the median survival of treated patients is only 9-17 months (Flores et al., J Thorac Oncol. 2007; 2(10): 957-965). Since 2003, there have been no new FDA-approved therapies for MPM (Tsao et al., J Thorac Oncol. 2018; 13(11): 1655-1667). The current standard of care for first-line systemic treatment of MPM patients is cisplatin plus pemetrexed, with a median overall survival of 12.1 months, compared to 9.3 months with cisplatin alone (Vogelzang et al., J Clin Oncol. 2003; 21(14): 2636-2644). Because MPM patients have a lower tumor mutation burden and lower programmed death ligand 1 (PD-L1) expression, their response to immune checkpoint inhibitors is limited, and a large unmet need remains (Yarchoan et al., JCI Insight. 2019; 4(6); Forde et al., Curr Treat Options Oncol. 2019; 20(2): 18). The limited scope, potential accessibility, and relative lack of metastasis when present make MPMs suitable candidates for regionally targeted therapies (Nelson et al., J Clin Oncol. 2017; 35(29): 3354-3362).

[0575] This example describes a nonclinical study conducted to support the clinical use of intrapleural dosing of M28z1XXPD1DNR chimeric antigen receptor (CAR) T cells, an investigational new drug for the treatment of patients diagnosed with (histologically or cytologically documented) MPM who have received at least one chemotherapy regimen and have documented tumors.

[0576] This is a single-center, phase I study with up to 36 participants designed to evaluate the safety, dose requirements, and targeting efficiency of genetically directed autologous M28z1XXPD1DNR CAR T cells after cyclophosphamide pretreatment. There are five planned dose levels in this study: 1×10 6, 3×10 6 , 6×10 6 , 1×10 7 and 3×10 7 M28z1XXPD1DNR CAR T cells / kg, provided there are no dose-limiting toxicities. M28z1XXPD1DNR CAR T cells are infused via an indwelling pleural catheter. Before treatment, patients are screened for mesothelin expression by immunohistochemical analysis of biopsied tumors and / or blood levels of soluble mesothelin-related peptide.

[0577] M28z1XXPD1DNR CAR T cells are autologous T cells transduced in vitro with a γ-retroviral vector stock supernatant generated from the vector production master cell bank 293VEC-GALV-SFG-M28z1XXPD1DNR. The main components of the CAR encoded in the vector are:

[0578] 1) Human anti-mesothelin scFv for targeting mesothelin-expressing tumors,

[0579] 2) Human CD28 co-stimulatory domain for transmitting T cell survival and proliferation signals,

[0580] 3) human CD3ζ with a single functional immunoreceptor tyrosine-based activation motif (ITAM) for point mutations for calibrating T cell activation, and

[0581] 4) Programmed cell death protein 1 (PD1) dominant negative receptor (PD1DNR), which protects T cells from entering a state of dysfunction or exhaustion after antigen exposure (see Figure 21 ).

[0582] Mesothelin is a cancer cell surface antigen that is overexpressed in most MPMs, lung cancer, triple-negative breast cancer, pancreatic and ovarian cancers, and some esophageal cancers (Pastan et al., Cancer Res. 2014; 74(11):2907-2912; Kachala et al., Clin Cancer Res. 2014; 20(4):1020-1028; Tang et al., Anticancer Agents Med Chem. 2013; 13(2):276-280; Servais et al., Clin Cancer Res. 2012; Kelly et al., Mol Cancer Ther. 2012; 11(3):517-525; Tchou et al., Breast Cancer Res Treat. 2012; 133(2):799-804). The inventors have previously demonstrated that mesothelin overexpression promotes the aggressiveness of lung adenocarcinoma (n=1200) (Kachala et al., Clin Cancer Res. 2014; 20(4): 1020-1028), MPM (n=250) (Servais et al., Clin Cancer Res. 2012; Kelly et al., Mol Cancer Ther. 2012; 11(3): 517-525) and triple-negative breast cancer (n=250) (Tozbikian et al., PLoS One. 2014; 9(12): e114900). In addition to the relatively high expression of mesothelin in tumors, the expression level of mesothelin on the surface of normal peritoneal, pleural and pericardial mesothelial cells is also very low compared with normal tissues (Villena-Vargas et al., Ann Cardiothorac Surg. 2012; 1(4): 466-471), making it an ideal target for CAR T cell therapy of solid tumors. The biological function of mesothelin is still unclear and under investigation.

[0583] Previously, in a clinical study conducted at the University of Pennsylvania (NCT01355965), CAR T cells targeting mesothelin were intravenously injected into humans (3×10 8 cells / m 2 or 4.8×10 7cells / dose), where the CAR contained a murine scFv. It was reported that the allergic reaction seen in one patient was caused by an anti-mouse antibody response developed against the humanized mouse scFv portion of the CAR construct used in that study (Beatty et al., Cancer Immunol Res. 2014). In contrast, in a recent Phase I study conducted by the inventors’ laboratory (NCT02414269), CAR T cells targeting mesothelin (up to 6×10 7 CAR T cells / kg) are administered intrapleurally and consist of fully human scFvs from a human Fab library (Feng et al., Mol Cancer Ther. 2009). To date, 40 patients have been treated without observed dose-limiting toxicities. This study observed preliminary efficacy in a group of patients (n=18) who received CAR T cell therapy followed by at least 3 doses of pembrolizumab (anti-PD1) and were followed up for an additional 3 months. Importantly, 83% of patients in this group did not require new or additional treatment at 6 months, and half of the patients did not receive additional treatment within 18 months. In addition, in most patients, CAR T cells were detected in peripheral blood >100 days after intrapleural administration, indicating that these cells persisted in the patients.

[0584] To support the first-in-human clinical trial, the pharmacology program for the M28z1XXPD1DNR CAR T cells consisted of a series of orthogonal in vitro studies of specificity, cytotoxicity, accumulation, and cytokine secretion, as well as in vivo studies of tumor efficacy and survival in mice. The results were translated into a recommended effective dose for clinical application. Table 4 provides a comprehensive summary of the nonclinical pharmacology and toxicology analyses and their key results.

[0585] Table 4 Comprehensive summary of pharmacological and toxicological analyses and their main results

[0586]

[0587]

[0588]

[0589] To facilitate testing of CAR T cells in preclinical studies, CAR T cells containing a myc tag at the N-terminus of a mesothelin-specific scFv (mycM28z1XXPD1DNR) were generated. To assess the consistency of mycM28z1XXPD1DNR and M28z1XXPD1DNR CAR T cells, the transduction efficiency of viral supernatants was compared and consistent concentration-dependent expression of vector components was observed between the two constructs. Furthermore, CAR and PD1DNR were expressed proportionally within each transduced cell due to the presence of the P2A self-cleaving peptide, which effectively mediates bicistronic transgene expression. When the percentage of transduced cells expressing CAR was compared with the number of vector copies inserted into the T cell genome, a positive linear correlation was observed between the dilution of viral supernatant used for transduction and the resulting vector copy number (VCN). From these observations, it was determined that a range of 35%–70% of CAR-expressing T cells ensured optimal transduction efficiency, which (1) maintained a low VCN:cell ratio and (2) reduced the risk of insertional mutagenesis, which is typically associated with higher VCN:cell ratios.

[0590] To confirm the expression of PD1DNR and distinguish it from its endogenous counterpart, flow cytometry and qPCR analysis were used to measure and compare the mRNA expression of cell surface protein and intracellular PD1 in mycM28z1XXPD1DNR- and mycM28z-transduced T cells, respectively. At the cell surface protein level, mycM28z1XXPD1DNRCAR T cells showed a 2-fold increase in the percentage of cells staining positive for PD1, and a 3-fold increase in the mean fluorescence intensity (MFI) of PD1-positive cells compared to mycM28z CAR T cells. At the mRNA level, mycM28z CAR T cells showed a 4-fold increase in both the extracellular and intracellular domains of PD1 relative to untransduced T cells, while mycM28z1XXPD1DNR CAR T cells showed a 157-fold increase in the extracellular domain of PD1 and only a 2-fold increase in the intracellular domain of PD1. The magnitude of the higher expression of the PD1 extracellular domain indicates high expression of PD1DNR, which is used to resist checkpoint inhibition.

[0591] Finally, to exclude any potential interference of the myc tag (used in preclinical studies) with CAR function, the antitumor efficacy of mycM28z1XXPD1DNR and M28z1XXPD1DNR CAR T cells was compared using an impedance-based cytotoxicity assay, which showed no differences in the kinetics and overall killing of mesothelin-positive tumor cells from three different donors, confirming that the myc tag did not interfere with CAR function.

[0592] Next, the specificity, cytotoxicity, accumulation, and cytokine secretion of mycM28z1XXPD1DNR CAR T cells were analyzed. 51 In the Cr cytotoxicity test, mycM28z1XXPD1DNR CAR T cells exhibited antigen-specific and human leukocyte antigen (HLA)-independent cytotoxicity against mesothelin-positive tumor cells, with constitutive expression and overexpression of PD-L1. No nonspecific cytotoxicity was observed against mesothelin-negative tumor cells. In the absence of mesothelin antigen expression, mycM28z1XXPD1DNR CAR T cells did not express any cytotoxicity against target cells overexpressing PD-L1. After repeated antigen stimulation tests (antigen stress tests), it was found that the amplification multiples of mycM28z1XXPD1DNR and mycM28z CART cells were as high as 622 times during 6 antigen stimulations. At the time points tested, the constructs exhibited similar cytotoxicity after the first antigen stimulation and maintained similar cytotoxicity until the fourth antigen stimulation. When the effector-to-target (E:T) ratio was further reduced to increase antigenic stress, mycM28z1XXPD1DNR CAR T cells retained better cytotoxicity than mycM28z CAR T cells at the seventh antigen stimulation. However, the secretion of effector cytokines (IL-2, IFN-γ, and TNF-α) by mycM28z1XXPD1DNR and mycM28z CAR T cells gradually decreased over the course of the trial, indicating the safety of both constructs.

[0593] MPM tumor cells co-transduced with firefly luciferase (ffLuc) were administered intrapleurally to establish tumors representing an MPM carcinoma in situ model. To non-invasively monitor tumor growth, tumor-bearing mice were injected intraperitoneally with 150 mg / kg of luciferin using an optimized protocol previously published by our laboratory for quantitative monitoring of pleural tumor regression or progression, and visualized 15 minutes later on an IVIS Spectrum Imaging System (PerkinElmer, Waltham, MA) (Servais et al., Curr Protoc Pharmacol. 2011; Chapter 14: Unit 14 21).

[0594] In initial experiments to investigate the in vivo antitumor activity of CAR T cells, NSG mice bearing orthotopic tumors received a single intrapleural administration of 3 × 10 4The researchers compared mice treated with mycM28z1XXPD1DNR CAR T cells with mice treated with a single intrapleural dose of control CAR T cells against prostate-specific membrane antigen (P28z). After 15 days, mice treated with mycM28z1XXPD1DNR CAR T cells showed a significant reduction in tumor burden (p = 0.0002), while mice treated with P28z CAR T cells began to die due to high tumor burden.

[0595] In the second in vivo study, mice bearing orthotopic tumors were divided into three groups, each receiving 1 × 10 5 or 5×10 4 mycM28z1XXPD1DNR CAR T cells or 1×10 5 A single intrapleural dose of mycM28z CAR T cells was administered. Serial tumor imaging showed a reduction in tumor burden as early as 5 days after CAR T cell administration, with complete tumor eradication determined by a decrease in bioluminescence imaging (BLI) signal to baseline levels. 5 The tumors of mice treated with mycM28z1XXPD1DNR CAR T cells were completely eradicated around day 19. 5 Mice treated with mycM28z CAR T cells had complete tumor eradication around day 26. Mice treated with either dose of mycM28z1XXPD1DNR CAR T cells remained tumor-free until the end of the study (day 68). 5 The median survival of mice treated with mycM28z CAR T cells was 50 days; mice treated with either dose of mycM28z1XXPD1DNR CAR T cells did not reach the median survival rate (>68 days; p=0.0085-0.0427). Immunofluorescence imaging of pleural tumors in vitro showed that 3 days after local delivery, mycM28z1XXPD1DNR CAR T cells surrounded the tumor at a high density and invaded the tumor parenchyma. These results were reproduced using T cells from multiple donors with different percentages of CD4 and CD8 T cells transduced with mycM28z1XXPD1DNR CAR.

[0596] To investigate the functional persistence of mycM28z1XXPD1DNR CAR T cells, patients who received a single intrapleural dose of 1×10 5 Orthotopic MPM mice were treated with mycM28z1XXPD1DNR or mycM28z CAR T cells by intraperitoneal administration every 4–8 days up to 10 times (repeated administration of cells in the peritoneal cavity is more feasible than in the pleural cavity). 6 to 11×10 6cells / dose) mesothelin-positive tumor cells. Shortly after each tumor re-challenge, the BLI signal of mice treated with mycM28z1XXPD1DNRCAR T cells peaked and returned to baseline levels at all re-challenge time points. In contrast, mice treated with mycM28z CAR T cells showed the same trend for up to 5 tumor re-challenges, but failed to control tumor reestablishment after higher tumor doses were given at later tumor re-challenge time points (6 to 10), resulting in tumor recurrence and moribund state. mycM28z1XXPD1DNR CAR T cells resisted intraperitoneal tumor establishment with 10 repeated challenges, even after a single intrapleural dose of 1×10 5 CAR T cells were administered for >126 days without any overt signs of toxicity. In a high antigen stress environment, mycM28z1XXPD1DNR CAR T cells demonstrated superior functional persistence and enhanced anti-tumor efficacy compared to mycM28z CAR T cells in vivo. To confirm that this enhanced efficacy was not due to graft-versus-host disease (GVHD), which is common in NSG mice treated with CAR T cells at this time point, non-antigen-expressing targets were administered, resulting in increased tumor BLI without anti-tumor responses, confirming that the observed anti-tumor efficacy was antigen-specific.

[0597] To verify the anti-tumor efficacy of cryopreserved T cells transduced with viral supernatant encoding M28z1XXPD1DNR CAR for clinical trials, M28z1XXPD1DNR CAR T cells generated by MSK Cell Therapy and Cell Engineering Facility (CTCEF) were introduced, and the M28z1XXPD1DNR CAR T cells were thawed (post-thaw survival rate: 88%) and cultured at 6×10 4 and 2×10 5 CAR T cells / mouse were injected intrapleurally into mice with orthotopic MPM. Tumor regression and eradication were observed at both doses, with 100% of mice surviving until the end of the observation period (day 70), while tumors in untreated mice progressed, leading to death on day 19. Cryopreserved CAR T cells showed high viability and efficacy after thawing, without any signs of toxicity.

[0598] During the above-mentioned efficacy experiments, no toxicity was observed in the mice and their body weight remained stable.

[0599] Section 3 of this Example (entitled "Nonclinical Toxicology") describes a study conducted in mice to specifically evaluate the potential toxicity of mycM28z1XXPD1DNR CAR T cells in an orthotopic mouse model of MPM. Mortality, morbidity, body weight, clinical signs, hematology and clinical chemistry, gross necropsy, and histopathology data were evaluated in 96 (48 male and 48 female) NSG mice bearing 8-day-old orthotopic mesothelioma tumors that were randomized into control and treatment groups. A single dose of 1 × 10 5 CAR T cells / mouse or vehicle control (5×10 6 CAR T cells / kg). On the 2nd and 14th day after administration of CAR T cells or vehicle (mid-term and final sacrifice, respectively), mice were sedated for autopsy and assessment of hematological and clinical chemistry parameters. Day 14 was selected as the time point for final sacrifice because the tumor either significantly regressed or was eradicated at this time point (as shown by BLI or autopsy in previous experiments). Sacrifice and autopsy at this time point can examine normal tissues with low expression levels of mesothelin, particularly any targeting, non-tumor effects of the pleura, peritoneum, and pericardium (the scFv used in our CAR responds to mouse mesothelin) (Feng et al., Mol Cancer Ther. 2009), in the absence of a tumor load with high antigen expression, accompanied by a peak in CAR T cell amplification.

[0600] No death or morbidity was observed in the animals in this study, except for two animals in the control vehicle-treated group, which underwent selective sacrifice 20-22 days after tumor administration due to morbidity and dyspnea. Previous work in our laboratory has shown that control vehicle-treated animals may become moribund due to tumor burden approximately 20-22 days after tumor administration (Servais et al., Clin Cancer Res. 2012; Servais et al., Curr Protoc Pharmacol. 2011; Chapter 14: Unit 1421; Adusumilli et al., Sci Transl Med. 2014; 6(261): 261ra151; Cherkassky et al., J Clin Invest. 2016; 126(8): 3130-3144; Servais et al., PLoS One. 2011; 6(10): e26722). Therefore, these sacrifices were unplanned, but not unexpected. No death or morbidity was traced back to CAR T cells. Animals receiving the vehicle control gradually lost weight during the study, with significant differences in body weight compared to non-tumor controls and mice treated with mycM28z1XXPD1DNR CAR T cells. This was attributed to increased tumor burden in the control vehicle-treated animals. No overt clinical signs were observed in mice treated with mycM28z1XXPD1DNR CAR T cells. Mild scabs were observed in one test article-treated mouse, which was attributed to irritation from the surgical clips; no other animals were affected and the animal's activity was normal. The mice appeared normal throughout the monitoring period.

[0601] The mean monocyte percentage in female mice treated with mycM28z1XXPD1DNR CAR T cells, which were ultimately sacrificed 14 days after CAR T cell administration, was higher (mean 18.44%, n=5) compared to tumor control vehicle mice (mean 3.34%, n=5) (p<0.0001). The established reference range for monocyte percentage is 0.9%-18%. However, this did not correlate with any microscopic observations. No other significant or abnormal results were observed for the hematological parameters evaluated. Any differences between the test article-treated groups and the corresponding vehicle-treated groups were within the normal reference range, or were biologically irrelevant or not statistically significant.

[0602] Male mice treated with mycM28z1XXPD1DNR CAR T cells were finally sacrificed 14 days after CAR T cell administration and had lower mean total protein values ​​(mean 3.83 g / dL, n=5) compared to tumor control vehicle mice (mean 4.68 g / dL, n=4) (p=0.0022). The established reference range for total protein was 4.1-6.4 g / dL. However, this did not correlate with any microscopic observations. No other adverse effects on clinical chemistry parameters were observed after administration of the test article. Any differences between the test article-treated groups and the corresponding vehicle-treated groups were within the normal reference range, or were biologically irrelevant or not statistically significant.

[0603] Histopathological examination revealed no microscopic findings related to acute or delayed toxicity associated with mycM28z1XXPD1DNR CAR T cell administration at the interim and final sacrifice dates. Microscopic findings in animals in the interim sacrifice group included the presence of mixed cellular infiltrates within the xenograft tumors. This was considered related to test article administration but not to any test article toxicity. Any other observed findings that were similar in incidence to the control group or were common to the species / strain used were determined to be incidental.

[0604] Eight days after intrapleural administration, mycM28z1XXPD1DNR CAR T cells were found in tumors and spleens, and BLI showed that at the 2-week time point, the tumor burden in CAR T cell-treated mice was significantly reduced, confirming the successful administration and the pharmacological activity of the test article. Plasma cytokine levels of mice obtained at the same time point showed that IL-4 levels were slightly higher in mice treated with CAR T cells than in mice receiving vehicle controls. IL-10, IL-6, KC / GRO, and TNF-α levels were generally low, with no significant differences between mice receiving CAR T cells and mice receiving vehicle controls. IFN-γ, IL-12p70, IL-1β, IL-2, and IL-5 were not detected (below the limit of quantification).

[0605] In summary, the data indicate that M28z1XXPD1DNR CAR T cells are well tolerated. 5 The dose of cells / mouse was higher than the starting dose for patients (1×10 6 cells / kg body weight) is 5 times higher, equivalent to 5×10 6 M28z1XXPD1DNR CAR T cells have the same antigen targeting moiety as M28z CAR T cells, and patient safety data (n=50) are available. In our clinical trial (IND16354), up to 6×10 7We did not observe any dose-limiting toxicities or on-target, non-tumor toxicities when we administered M28zCAR T cells / kg in combination with anti-PD1 checkpoint blockade antibodies 3 weeks after CAR T cells were administered. Four patients received a second dose of intrapleural M28zCAR T cells after multiple doses of anti-PD1 agents (with a 4-week washout period) without observed toxicities. In summary, all available preclinical and clinical data reasonably suggest that the proposed starting dose and route of administration of M28z1XXPD1DNR CAR T cells will not pose an unacceptable risk to our patients.

[0606] 2. Nonclinical pharmacology

[0607] A. Method

[0608] CAR Vectors Table 5 summarizes the vectors used in nonclinical studies.

[0609] Table 5 Overview of vectors used in nonclinical studies

[0610]

[0611] *Clinical-grade: CAR T cells manufactured by the MSK Cell Therapy and Cell Engineering Facility using viral supernatant produced for clinical trials.

[0612] The CAR construct targeting mesothelin comprises a mesothelin-specific scFv (clone m912) (Feng et al., Mol Cancer Ther. 2009) fused to a CD28 co-stimulatory domain and a CD3ζ signaling domain (M28z). The CD3ζ chain mutates in two of its three ITAMs to form a single functional ITAM (called 1XX) (Feucht et al., Nat Med. 2019; 25(1): 82-88). CAR is fused to PD1DNR via the P2A site derived from porcine Teschovirus-1. PD1DNR is composed of a PD1 signal peptide and a PD1 extracellular domain fused to a CD8 transmembrane and hinge domain (Cherkassky et al., J Clin Invest. 2016; 126(8): 3130-3144). This decoy receptor depletes the PD1 signaling domain, thereby providing T cell intrinsic checkpoint blockade. To facilitate detection of CAR, a myc tag (amino acid sequence EQKLISEEDL×2) was fused to the N-terminus of the scFv in the constructs mycM28z and mycM28z1XXPD1DNR. To avoid any potential risk of immunogenicity in humans, the clinical-grade construct M28z1XXPD1DNR does not contain a myc tag. In addition, codon optimization was performed for protein expression to avoid any immunogenicity when constructing CAR and PD1DNR. The detailed structure of the constructs used in nonclinical studies is shown in Figure 2. Figure 22 shown.

[0613] The expression of the CAR construct is controlled by the Moloney murine leukemia virus long terminal repeat (LTR) of the retroviral SFG vector (Riviere et al., Proc Natl Acad Sci US A. 1995; 92(15): 6733-6737). The expression of both CAR and PD1DNR is driven by the retroviral LTR.

[0614] All CAR vectors were transfected into the 293T H29 packaging cell line, and the viral supernatant produced by these cells was used to transduce and generate the stable 293T RD114 cell line.

[0615] CAR T cells: Human naive T lymphocytes were isolated from the blood of healthy volunteer donors according to a protocol approved by the Institutional Review Board. Peripheral blood mononuclear cells (PBMCs) activated by phytohemagglutinin were isolated by low-density centrifugation on lymphocyte separation medium (Corning, New York, NY). Two days after isolation, PBMCs were transduced with viral supernatants containing mycM28z, mycM28z1XXPD1DNR, or M28z1XXPD1DNR vectors by spinoculation at 1800 g for 60 minutes at 24°C on 6-well culture plates coated with 15 μg / mL RetroNectin (Takara, Shiga, Japan). After spinoculation, transduced PBMCs were stored in RPMI-1640 supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 units / mL penicillin, 100 μg / mL streptomycin, and 20 units / mL IL-2. Transduction efficiency was determined by flow cytometry analysis of the expression of the myc tag on the tagged CAR scFv or by staining with an anti-human IgG antibody specific for the F(ab')2 fragment for the untagged CAR scFv of M28z1XXPD1DNR. CAR T cells demonstrated >70% viability as determined by anti-human CD3 staining, >95% T cell purity, 35%-70% transduction efficiency by flow cytometry, and CD4 / CD8 expression. Table 6 summarizes the characteristics of the T cells used in the nonclinical studies.

[0616] Table 6 T cell characteristics used in nonclinical studies

[0617]

[0618] Tumor Cells Cells from the MSTO-211H human pleural mesothelioma cell line (ATCC CRL-2081) were genetically modified and used for in vitro and in vivo studies (Table 7).

[0619] Table 7 Overview of tumor cells used in nonclinical studies

[0620] tumor cells Protein expression MSTOG GFP, ffLuc MGM GFP, ffLuc, mesothelin MGM-PGL1 GFP, ffLuc, mesothelin, PD-L1

[0621] MSTO-211H is a biphasic mesothelin (MPM) cancer cell line that lacks expression of endogenous CD80 / 86 co-stimulatory ligands. MSTO-211H cells were retrovirally transduced to express GFP and ffLuc proteins, termed MSTOG, allowing for noninvasive in vivo BLI using the SFG retroviral vector constructed at MSK. Medium containing filtered virus was added to cells permeabilized with 8 μg / mL polybrene (Sigma-Aldrich, St. Louis, MO). After 24 hours, cells were re-infected with freshly collected virus. These cells were transduced with human mesothelin variant 1 (isolated from the human ovarian cancer cell line [OVCAR-3]) subcloned into the SFG retroviral vector to generate mesothelin+ MSTO-211H cells, termed MGM. Similarly, MGM cells were transduced with PD-L1 (OriGene cDNA subcloned into the SFG vector) to generate MGM-PDL1. Tumor cells were maintained in RPMI-1640 medium containing 10% FBS, 2 mM L-glutamine, 100 units / mL penicillin, and 100 μg / mL streptomycin in a 5% CO2 humidified incubator at 37°C. A linear correlation was observed between the number of luciferase-expressing tumor cells and in vitro BLI photon counts (Pearson r = 0.999, p < 0.0001, data not shown). The relative expression levels of the transduced proteins were expressed as shown in Figure 5. Figure 23 shown.

[0622] Flow cytometry was performed using an Attune NxT flow cytometer (ThermoScientific, Waltham, MA) or a BD LSR Fortessa (BD Biosciences, San Jose, CA). Human mesothelin cell surface expression on tumor cells was detected using phycoerythrin-conjugated anti-human mesothelin rat IgG2a (R&D Systems, Minneapolis, MN). Human PD-L1 cell surface expression on tumor cells was detected using phycoerythrin-cyanine 7-conjugated anti-human PD-L1 mouse IgG1 (BD Biosciences). The cell surface expression of human CD3 on human T cells was analyzed using allophycocyanin-cyanidin 7-conjugated anti-human CD3 mouse IgG2α or phycoerythrin-cyanidin 7-conjugated anti-human CD3 mouse IgG1 antibodies (BioLegend, San Diego, CA), and human CD4 or human CD8 were analyzed using fluorescein isothiocyanate-conjugated anti-human CD4 mouse IgG1 (BioLegend) or Alexa Fluor 488-conjugated anti-human CD8 mouse IgG1 (BioLegend). Phycoerythrin-conjugated anti-myc tag antibodies (Cell Signaling Technology, Danvers, MA) or Alexa Fluor 647-conjugated F(ab')2 fragment-specific goat anti-human F(ab')2 fragments (Jackson ImmunoResearch, West Grove, PA) were used to quantify the cell surface expression of CAR. Brilliant Violet 711-conjugated anti-human PD1 mouse IgG1 (BioLegend) was used to analyze the cell surface expression of PD1 on CAR T cells. For in vitro detection of human T cells, treated mouse tissues were stained with phycoerythrin cyanidin 7-conjugated anti-human CD3 mouse IgG1 antibody and allophycocyanin cyanidin 7-conjugated anti-human CD45 mouse IgG1 antibody (BioLegend). Cells were stained with 4'6-diamidino-2-phenylindole (DAPI, ThermoFisher Scientific, Waltham, MA) or eFluor 506 (ThermoFisher Scientific) to distinguish live and dead cells. Data analysis was performed using FCS Express (De Novo Software, Pasadena, CA) and FlowJo (BD Biosciences) software. Table 8 summarizes the antibodies used for flow cytometry.

[0623] Table 8 Flow cytometry antibodies used in nonclinical pharmacology studies

[0624]

[0625]

[0626] VCN was determined using the Miniprep Kit (Qiagen, Hilden, Germany) to isolate total genomic DNA from CAR T cells. TaqMan PCR primers and probes were used to detect SFG and the housekeeping gene albumin (ALB). Human SFG probe and primer sequences:

[0627] Probe sequence: 5'-VIC-AGGACCTTACACAGTCCTGCTGAC-TAMRA-3' [SEQ ID NO: 126]

[0628] Forward primer sequence: 5'-AGAACCTAGAACCTCGCTGGA-3' [SEQ ID NO: 127]

[0629] Reverse primer sequence: 5'-CTGCGATGCCGTCTACTTTG-3' [SEQ ID NO: 128]

[0630] Human ALB probe and primer sequences:

[0631] Probe sequence:

[0632] 5'-VIC-TGCTGAAACATTCACCTTCCATGCAGA-TAMRA-3'[SEQ ID NO:129]

[0633] Forward primer sequence: 5'-TGAAACATACGTTCCCAAAGAGTTT-3' [SEQ ID NO: 130]

[0634] Reverse primer sequence: 5'-CTCTCCTTCTCAGAAAGTGTGCATAT-3' [SEQ ID NO: 131]

[0635] Amplification reactions (25 μL) contained 5 μL (150 μg) of genomic DNA and 12.5 μL of TaqMan Fast Advanced Master Mix (ThermoFisher Scientific), 0.8 μL of primers (forward and reverse), 0.2 μL of TaqMan probe, and 5.7 μL of distilled water. qPCR conditions were as follows: 50°C (2 minutes), 95°C (20 minutes), followed by 42 cycles of 95°C (15 seconds) and 60°C (1 minute) using the QuantStudio 7-Flex Real-Time PCR System (ThermoFisher Scientific). All PCR assays were performed in triplicate. VCN per cell was calculated as the ratio (average number of SFG / average number of ALB) x 2. Average amounts were extrapolated from SFG and ALB standard curves.

[0636] Determination of PD1 mRNA expression Total RNA was isolated from CART cells using the Miniprep Kit (Qiagen), and reverse transcription was performed using the High-Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific). The SYBR Green assay was used to detect the extracellular and intracellular domains of human PDCD1. Human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used for normalization. The following primers were used.

[0637] GAPDH primer sequences:

[0638] Forward primer sequence: 5'-GAAGGTGAAGGTCGGAGT-3' [SEQ ID NO: 132]

[0639] Reverse primer sequence: 5'-CATGGGTGGAATCATATTGGAA-3' [SEQ ID NO: 133]

[0640] PD1 extracellular domain primer sequences (Yoon et al., Science. 2015; 349(6247): 1261669):

[0641] Forward primer sequence: 5'-CCAGGATGGTTCTTAGACTCCC-3' [SEQ ID NO: 134]

[0642] Reverse primer sequence: 5'-TTTAGCACGAAGCTCTCCGAT-3' [SEQ ID NO: 135]

[0643] PD1 intracellular domain primer sequences (Hsu et al., J Immunol. 2016; 197(5): 1884-1892):

[0644] Forward primer sequence: 5'-ACGAGGGACAATAGGAGCCA-3' [SEQ ID NO: 136]

[0645] Reverse primer sequence: 5'-GGCATACTCCGTCTGCTCAG-3' [SEQ ID NO: 137]

[0646] cDNA was diluted 5-fold for subsequent qPCR experiments. Amplification reactions (20 μL) contained cDNA generated from 200 ng of total RNA and 10 μL QuantiTect SYBR Green PCR Mix (Qiagen), 4 μL primers (forward and reverse, 200 nM each), and distilled water. qPCR conditions were as follows: 95°C (15 min), 95°C (20 min), followed by 45 cycles of 94°C (15 s), 60°C (30 s), 72°C (30 s), and 50°C (data collection for 20 s) using the QuantStudio 7Flex Real-Time PCR System (ThermoFisher Scientific). All PCR assays were performed in triplicate. All primers were synthesized by Integrated DNA Technologies (Coralville, IA), and amplification efficiency (E) values ​​were calculated. The relative expression of the target gene was normalized to the internal reference group according to the Pfaff formula (Pfaff, Nucleic Acids Res. 2001; 29(9):e45).

[0647] Relative ratio = (E 目的基因 ) ΔCt目的基因(对照-样品) / (E 内参基因 ) ΔCt内参基因(对照-样品)

[0648] Target gene = PD1 extracellular / intracellular domain; Reference gene = GAPDH; Control = untransduced T cells.

[0649] 51 Cr cytotoxicity assay The cytotoxicity of mycM28z1XXPD1DNR CAR T cells and mycM28z T cells was determined and compared by a standard 51Cr release assay. In a 96-well round-bottom plate, 5×10 cells were cultured in 200 μL RPMI containing 10% FBS, 2 mM L-glutamine, 100 units / mL penicillin, and 100 μg / mL streptomycin. 5 to 1×10 6Total T cells were serially diluted 1:2 in 100 μL culture medium. Target cells were mixed with 1×10 6 75 μCi for cells 51 Cr was incubated for 2 h and 5×10 3 After washing three times with culture medium, 100 μL of target cells were added to T cells in triplicate and incubated at 37°C in a 5% CO2 humidified incubator for 4-18 hours. The supernatant was collected, plated on a 96-well Lumina plate (PerkinElmer), and measured on a PerkinElmer TopCount. Spontaneous 51 was assessed in target cells cultured with culture medium alone. Cr Release was measured in target cells cultured in 100 μL 0.2% Triton X-100. Cr Release. The percentage of specific lysis was calculated as follows: [(experimental counts per minute (cpm) - spontaneous release cpm) / (total cpm - spontaneous release cpm)] × 100. Data are reported as the mean of three measurements + / - standard error of the mean and analyzed using Microsoft Excel (Microsoft, Redmond, WA) or GraphPad Prism (GraphPad Software, La Jolla, CA).

[0650] The impedance test uses the xCELLigence real-time cell analysis instrument (ACEA Biosciences, San Diego, CA) to evaluate in vitro CAR T cell-induced target cell killing in real time. First, 50 μL RPMI containing 10% FBS, 2mM L-glutamine, 100 units / mL penicillin and 100 μg / mL streptomycin as a culture medium for target cells and effector cells was added to a 96-well microtiter plate coated with a gold microelectrode (ACEA Biosciences) to measure background impedance. Secondly, 10,000 target cells were inoculated in 100 μL culture medium per well, and target cell adhesion was monitored for 24-34 hours before CAR T cells were added to 50 μL culture medium in triplicate at an E:T ratio of 1:1 to 1:3. In order to evaluate the impedance changes caused by target cell killing and detachment induced by CAR T cells, data were recorded every 15 minutes in a 5% CO2 humidified incubator at 37 ° C for 4 days after the effector cells were added.

[0651] Repeated antigen stimulation To study the anti-tumor efficacy of CAR T cells under repeated antigen stimulation in vitro, 3.3×10 5 to 1×10 6T cells with 3.3×10 5 Irradiated target cells (E:T ratio of 1:1 to 3:1) were co-cultured in 1 mL RPMI containing 10% FBS, 2 mM L-glutamine, 100 units / mL penicillin, and 100 μg / mL streptomycin in 24-well cell culture plates. After 48 hours of co-culture, T cells were aggregated, counted, analyzed for CAR expression by flow cytometry, and re-transplanted with irradiated target cells at the same E:T ratio for up to 6 rounds of repeated antigen exposure. After 1, 3, and 6 rounds of antigen exposure, the cells were treated with 51 Chromium release and impedance-based assays were used to assess the cytotoxicity of CART cells.

[0652] The accumulation of 3.3 × 10 5 T cells with 3.3×10 5 Irradiated target cells (E:T ratio of 1:1) were co-cultured in 1 mL RPMI containing 10% FBS, 2 mM L-glutamine, 100 units / mL penicillin, and 100 μg / mL streptomycin in 24-well cell culture plates to assess accumulation. After 48 hours of co-culture, T cells were aggregated, counted, analyzed for CAR expression by flow cytometry, and re-transplanted with irradiated target cells at the same E:T ratio for up to 6 rounds of repeated antigen exposure. The number of CAR T cells after each cycle of antigen stimulation was used to determine the accumulation of CAR T cells over time by absolute T cell counts.

[0653] Cytokine quantification was performed by transducing 3.3 × 10 cells with mycM28z1XXPD1DNR or mycM28z as a control. 5 T cells with 3.3×10 3 Irradiated target cells (E: T ratio of 1: 1) were co-cultured in 1 mL RPMI containing 10% FBS, 2 mM L-glutamine, 100 units / mL penicillin and 100 μg / mL streptomycin in 24-well cell culture plates for cytokine release assays. After 48 hours of co-culture, T cells were collected and counted by flow cytometry, their CAR expression was analyzed, and they were re-transplanted with irradiated target cells at the same E: T ratio for up to 6 rounds of repeated antigen exposure. For cytokine quantification, supernatants were collected after 24 hours of co-culture to repeat antigen stimulation 1, 3 and 6 and centrifuged at 800 g for 10 minutes at room temperature to remove cells and debris. Cytokine levels were determined in duplicate using the Human Cytokine Magnetic 30-plex panel (Invitrogen, Carlsbad, CA) and MAGPI...

Claims

1. A T cell comprising a polypeptide composition, wherein the polypeptide composition include: i) a chimeric antigen receptor (CAR) comprising the amino acid sequence shown in SEQ ID NO: 56; and ii) a dominant negative form of programmed death 1 (PD-1 DN), which comprises amino acids 21 to 165 of SEQ ID NO:48 and amino acids 137 to 207 of SEQ ID NO:

86.

2. The T cell according to claim 1, wherein the PD-1 DN and / or the CAR are recombinantly expressed.

3. The T cell according to claim 1, wherein the PD-1 DN and / or the CAR are expressed by a vector.

4. The T cell according to claim 1, wherein the T cell is selected from the group consisting of cytotoxic T lymphocytes (CTLs), regulatory T cells, and natural killer T (NKT) cells.

5. The T cell of any one of claims 1-4, wherein the cell is autologous.

6. The T cell of any one of claims 1-4, wherein the cell is allogeneic.

7. A pharmaceutical composition comprising an effective amount of the T cell according to any one of claims 1 to 6 and a pharmaceutically acceptable excipient.

8. The pharmaceutical composition according to claim 7, comprising 10 4 Up to 10 6 T cells.

9. The pharmaceutical composition according to claim 7, comprising at least 10 5 T cells.

10. The pharmaceutical composition according to any one of claims 7 to 9, comprising 10 5 T cells.

11. A pharmaceutical composition according to any one of claims 7 to 9, for preventing and / or treating a tumor in a subject, treating a subject with tumor recurrence, reducing a subject's tumor burden, increasing or prolonging the survival of a subject with a tumor, preventing and / or treating an inflammatory disease in a subject, and / or preventing graft rejection in a subject receiving an organ transplant.

12. A nucleic acid comprising the nucleotide sequence shown in SEQ ID NO: 123 or SEQ ID NO:

124. A vector comprising the nucleic acid according to claim 12. The vector according to claim 13 , which is a retroviral vector. The vector according to claim 14 , wherein the retroviral vector is a γ-retroviral vector or a lentiviral vector.

16. A method for producing T cells, the method comprising introducing the polypeptide composition mentioned in any one of claims 1 to 6, the nucleic acid of claim 12, or the vector of any one of claims 13 to 15 into T cells.

17. A kit comprising the T cell of any one of claims 1 to 6, the nucleic acid of claim 12, the vector of any one of claims 13 to 15, or the pharmaceutical composition of any one of claims 7 to 11.

18. The kit of claim 17, wherein the kit further comprises a method for treating and / or preventing a tumor in a subject, treating a subject with tumor recurrence, reducing a tumor burden in a subject, increasing or prolonging the survival of a subject with a tumor, wherein the tumor or tumor expresses mesothelin.

19. Use of an effective amount of the T cell of any one of claims 1-6, or the pharmaceutical composition of any one of claims 7-11, in the preparation of a medicament for preventing and / or treating a tumor in a subject, wherein the tumor is selected from mesothelioma, lung cancer, and a combination thereof.

20. Use of an effective amount of the T cell of any one of claims 1-6, or the pharmaceutical composition of any one of claims 7-11, in the preparation of a medicament for reducing the tumor burden in a subject, wherein the tumor is selected from mesothelioma, lung cancer, and a combination thereof.

21. The use according to claim 20, which reduces the number of tumor cells, reduces the size of a tumor, and / or eradicates a tumor in a subject.

22. Use of an effective amount of the T cell of any one of claims 1-6, or the pharmaceutical composition of any one of claims 7-11, in the preparation of a medicament for treating a subject with tumor recurrence, wherein the tumor is selected from mesothelioma, lung cancer, and a combination thereof.

23. The use according to claim 22, wherein the subject receives immunotherapy prior to the administration of the T cells or the composition.

24. Use of an effective amount of the T cell of any one of claims 1-6, or the pharmaceutical composition of any one of claims 7-11, in the preparation of a medicament for increasing or prolonging the survival of a subject suffering from a tumor, wherein the tumor is selected from mesothelioma, lung cancer, and a combination thereof.

25. Use of an effective amount of the T cell of any one of claims 1-6, or the pharmaceutical composition of any one of claims 7-11, in the preparation of a medicament for increasing the production of immune-activating cytokines in response to cancer cells in a subject, wherein the cancer cells are selected from mesothelioma, lung cancer, and a combination thereof.

26. The use according to claim 25, wherein the immune activation cytokine is selected from granulocyte macrophage colony stimulating factor (GM-CSF), IFN-α, IFN-β, IFN-γ, TNF-α, IL-2, IL-3, IL-6, IL-11, IL-7, IL-12, IL-15, IL-21, interferon regulatory factor 7 (IRF7), and combinations thereof.

27. The use according to any one of claims 19-26, further comprising administering at least one immunomodulator.

28. The use according to claim 27, wherein the at least one immunomodulatory agent is selected from the group consisting of an immunostimulant, a checkpoint immune blocker, a radiotherapeutic agent, a chemotherapeutic agent, and combinations thereof.

29. The use according to claim 28, wherein the immunostimulatory agent is selected from IL-12, agonist co-stimulatory monoclonal antibodies, and combinations thereof.

30. The use according to claim 29, wherein the immunostimulant is IL-12.

31. The use according to claim 28, wherein the agonist co-stimulatory monoclonal antibody is selected from anti-4-1BB antibody, anti-OX40 antibody, anti-ICOS antibody, and a combination thereof.

32. The use according to claim 31, wherein the agonist co-stimulatory monoclonal antibody is an anti-4-1BB antibody.

33. The use according to claim 28, wherein the checkpoint immune blocker is selected from anti-PD-L1 antibodies, anti-CTLA-4 antibodies, anti-PD-1 antibodies, anti-LAG3 antibodies, anti-B7-H3 antibodies, anti-TIM3 antibodies, and combinations thereof.

34. The use according to claim 33, wherein the checkpoint immune blocker is an anti-PD-L1 antibody or an anti-PD-1 antibody.

35. The use according to claim 27, wherein the subject is a human.

36. The use according to claim 27, wherein the T cells are administered to the subject transpleurally or intrapleurally.

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