Mesothelin CAR and its uses

Abstract

The presently disclosed subject matter provides a polypeptide composition comprising a chimeric antigen receptor (CAR) that targets mesothelin; and a dominant-negative form of programmed cell death 1 (PD-1 DN). Also provided are immune response cells comprising such polypeptide compositions, and uses of the polypeptide compositions and immune response cells for treatment, e.g., treating solid tumors. The presently disclosed subject matter provides methods and compositions for enhancing immune responses against cancer and pathogens. The present invention relates to chimeric antigen receptors (CARs) that specifically target human mesothelin and immune response cells comprising such CARs. The mesothelin-targeting CARs of the present disclosure have enhanced immune activation properties, including antitumor activity, while minimizing CAR-induced toxicity and immunogenicity.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 848,983, filed May 16, 2019, and U.S. Provisional Patent Application No. 62 / 975,966, filed February 13, 2020, the contents of each of which are incorporated by reference in their entirety and to each of which priority is claimed.

[0002] Sequence Listing This application contains a Sequence Listing that was submitted in ASCII format via EFS-Web and is incorporated herein by reference in its entirety. The ASCII copy created on May 13, 2020 is named 0727341041SL_ST25.TXT and is 144,318 bytes in size.

[0003] 1. Introduction The subject matter of the present disclosure provides methods and compositions for enhancing immune responses against cancer and pathogens. The present invention relates to chimeric antigen receptors (CARs) that specifically target human mesothelin and immune response cells that contain such CARs. The mesothelin-targeting CARs of the present disclosure have enhanced immune activation properties, including antitumor activity, while having features that minimize CAR-induced toxicity and immunogenicity. [Background technology]

[0004] 2. Background of the invention Cell-based immunotherapy is a potentially curative therapy for the treatment of cancer. T cells and other immune cells can be modified to target tumor antigens through the introduction of genetic material that encodes an artificial or synthetic receptor for the antigen, termed a chimeric antigen receptor (CAR), specific for the selected antigen. Targeted T cell therapy using CARs has recently shown clinical success in the treatment of some hematological malignancies. However, the translation of CAR-expressing T cell therapy into solid tumors poses several barriers that must be overcome to achieve clinical benefit. Malignant cells adapt to generate an immunosuppressive microenvironment to protect themselves from immune recognition and elimination. This tumor microenvironment poses a challenge for methods of treatment that involve the stimulation of an immune response, such as targeted T cell therapy. Solid tumors may also be confined within anatomical compartments that impede efficient T cell trafficking, lack expression of agonistic costimulatory ligands, and / or express negative regulators of T cell function. Thus, successful elimination of solid tumors requires effective tumor infiltration and overcoming tumor-induced immune suppression. In addition, solid tumors pose challenges in selecting optimal immune targets-antigens whose targeting allows potent T cell-mediated eradication of the tumor with minimal or tolerable toxicity to non-tumor tissues.

[0005] Therefore, there is a need for novel therapeutic strategies to design CARs for treating cancer, especially solid tumors, that are capable of inducing potent tumor eradication with minimal toxicity and immunogenicity. Summary of the Invention [Means for solving the problem]

[0006] 3. Overview of the Invention The subject matter of the present disclosure provides polypeptide compositions comprising (a) a chimeric antigen receptor (CAR) that specifically targets mesothelin (e.g., human mesothelin) and (b) a dominant negative form of programmed cell death 1 (PD-1 DN), immune response cells comprising such polypeptide compositions, and uses of these polypeptide compositions and immune response cells, for example, to treat cancer.

[0007] The subject matter of the present disclosure provides a polypeptide composition. In certain embodiments, the polypeptide composition comprises: i) a chimeric antigen receptor (CAR), and ii) a dominant negative form of programmed cell death 1 (PD-1 DN), the CAR comprises (a) an extracellular antigen binding domain, and (b) an intracellular signaling domain comprising a modified CD3ζ polypeptide comprising an ITAM2 variant and an ITAM3 variant, each of which comprises two loss-of-function mutations.

[0008] In certain embodiments, the extracellular antigen binding domain comprises: a heavy chain variable region comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 76, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 77, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 78; and a light chain variable region comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 79, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 80, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 81.

[0009] In certain embodiments, the PD-1 DN comprises: (a) at least a portion of the extracellular domain of programmed cell death 1 (PD-1) that includes the ligand-binding region, and (b) the first transmembrane domain.

[0010] 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 comprised in the first transmembrane domain of PD-1 DN comprises amino acids 137-207 of SEQ ID NO:86. In certain embodiments, the PD-1 DN lacks an intracellular domain. In certain embodiments, the PD-1 DN comprises amino acids 21-165 of SEQ ID NO:48 and amino acids 137-207 of SEQ ID NO:86.

[0011] In certain embodiments, the extracellular antigen-binding domain of the 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 the CAR specifically binds to human mesothelin with an EC50 value of about 20 nM.

[0012] In certain embodiments, the extracellular antigen-binding domain of the CAR is a single chain variable fragment (scFv), optionally cross-linked Fab, or F(ab) 2 In certain embodiments, the extracellular antigen binding domain of the CAR comprises a human scFv. In certain embodiments, the extracellular antigen binding domain of the CAR recognizes human mesothelin at about 1,000 or more mesothelin binding sites / cellular mesothelin expression level.

[0013] 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 to identical to the amino acid sequence set forth in SEQ ID NO: 82. In certain embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:82.

[0014] 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 to identical to the amino acid sequence set forth in SEQ ID NO: 83. In certain embodiments, the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:83.

[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 to identical to the amino acid sequence set forth in SEQ ID NO:82. and 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 to identical to the amino acid sequence set forth in SEQ ID NO: 83. In certain embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 82 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:83.

[0016] 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.

[0017] In certain embodiments, the leader is covalently linked to the N-terminus of the extracellular antigen-binding domain. In certain embodiments, the leader 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 extracellular domain of PD-1 comprises amino acids 21-165 of SEQ ID NO: 48.

[0018] In certain embodiments, each of the loss-of-function mutations in the modified CD3ζ polypeptide of the CAR is in 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 a native ITAM1. In certain embodiments, the 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.

[0019] In certain embodiments, the CAR comprises or consists of the amino acid sequence set forth in SEQ ID NO:56.

[0020] In certain embodiments, the CAR further comprises a second transmembrane domain.In certain embodiments, the second transmembrane domain of the CAR 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 second transmembrane domain of the CAR comprises a CD28 polypeptide.

[0021] 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.

[0022] The subject matter of the present disclosure provides an immune response cell comprising the polypeptide composition disclosed herein. In certain embodiments, the PD-1 DN and / or CAR are recombinantly expressed. In certain embodiments, the PD-1 DN and / or CAR are expressed from a vector. In certain embodiments, the immune response cell is selected from the group consisting of T cells, natural killer (NK) cells, and pluripotent stem cells from which lymphoid cells 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 cell is a T cell. In certain embodiments, the T cell is selected from the group consisting of cytotoxic T lymphocytes (CTLs), regulatory T cells, and natural killer T (NKT) cells. In certain embodiments, the immune response cell is autologous. In certain embodiments, the immune response cell is allogeneic.

[0023] The subject matter of the present disclosure further provides a composition comprising the immune response cells disclosed herein. In certain embodiments, the composition is a pharmaceutical composition further comprising a pharma- ceutically acceptable excipient. In certain embodiments, the pharmaceutical composition comprises about 10 4 ~10 pieces 6 In certain embodiments, the pharmaceutical composition comprises between about 10 immune response cells. 5 In certain embodiments, the pharmaceutical composition comprises about 10 immune response cells. 5 In certain embodiments, the pharmaceutical composition is for preventing and / or treating a neoplasm in a subject, for treating a subject with a recurrence of a neoplasm, for reducing tumor burden in a subject, for increasing or prolonging survival of a subject with a neoplasm, for preventing and / or treating an inflammatory disease in a subject, and / or for preventing graft rejection in a subject who is a recipient of an organ transplant.

[0024] In addition, the subject of the present disclosure provides a nucleic acid composition comprising a polynucleotide encoding the polypeptide composition 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 subject of the present disclosure further provides a vector comprising the nucleic acid composition of the present disclosure. In certain embodiments, the vector is a retroviral vector. In certain embodiments, the retroviral vector is a gamma-retroviral vector or a lentiviral vector.

[0025] The subject matter of the present disclosure provides a method of producing an immune response cell as disclosed herein. In certain embodiments, the method comprises introducing a polypeptide composition of the present disclosure, a nucleic acid composition of the present disclosure, or a vector of the present disclosure into an immune response cell.

[0026] 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 use in treating and / or preventing a neoplasm.

[0027] Furthermore, the subject matter of the present disclosure provides various methods of using the immune response cells described above.For example, the subject matter of the present disclosure provides a method of reducing tumor burden in a subject, comprising administering to the subject an effective amount of the immune response cells or pharmaceutical composition of the present disclosure.In certain embodiments, the method reduces the number of tumor cells in the subject, reduces tumor size, and / or eradicates tumor.

[0028] The presently disclosed subject matter also provides a method of increasing or prolonging survival of a subject having a neoplasm, the method comprising administering to the subject an effective amount of an immune response cell of the present disclosure or a pharmaceutical composition of the present disclosure.

[0029] In certain embodiments, the tumor or neoplasm is a solid tumor. In certain embodiments, the solid tumor is selected from the group consisting of mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colorectal cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic carcinoma, endometrial cancer, stomach cancer, cholangiocarcinoma, cervical cancer, salivary gland cancer, and combinations thereof.

[0030] The presently disclosed subject matter provides a method of treating a subject having a recurrence of a neoplasm, comprising administering to the subject an effective amount of an immune response cell or pharmaceutical composition disclosed herein, in certain embodiments, the subject has undergone immunotherapy prior to said administration of the immune response cell or composition.

[0031] In addition, the subject matter of the present disclosure provides a method for increasing immune-activating cytokine production in response to cancer cells or pathogens in a subject. In certain embodiments, the method comprises administering to the subject an effective amount of an immune response cell or pharmaceutical composition disclosed herein. In certain embodiments, the immune-activating cytokine is selected from the group consisting of 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.

[0032] In accordance with the subject matter of the present disclosure, the above various methods may include administering at least one immunomodulatory agent. In certain embodiments, the at least one immunomodulatory agent is selected from the group consisting of an immunostimulatory agent, a checkpoint immune blockade agent, a radiotherapy agent, a chemotherapy agent, and a combination thereof. In some embodiments, the immunostimulatory agent is selected from the group consisting of IL-12, an agonistic costimulatory monoclonal antibody, and a combination thereof. In certain embodiments, the immunostimulatory agent is IL-12. In some embodiments, the agonistic costimulatory monoclonal antibody is selected from the group consisting of an anti-4-1BB antibody, an anti-OX40 antibody, an anti-ICOS antibody, and a combination thereof. In certain embodiments, the agonistic costimulatory monoclonal antibody is an anti-4-1BB antibody. In certain embodiments, the checkpoint immune blockade agent is selected from the group consisting of an anti-PD-L1 antibody, an anti-CTLA-4 antibody, an anti-PD-1 antibody, an anti-LAG3 antibody, an anti-B7-H3 antibody, an anti-TIM3 antibody, and a combination thereof. In certain embodiments, the checkpoint immune blockade agent is an anti-PD-L1 antibody or an anti-PD-1 antibody. In certain embodiments, the subject is a human.

[0033] In certain embodiments, the immune response cells are administered pleurally or intrapleurally to the subject.

[0034] The subject matter of the present disclosure further includes a method for preventing and / or treating inflammatory disease in a subject. In certain embodiments, the method includes administering to a subject the immune response cell or pharmaceutical composition of the present disclosure. In certain embodiments, the immune response cell is an immune inhibitory cell. In certain embodiments, the immune inhibitory 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.

[0035] The subject matter of the present disclosure further provides a method for preventing graft rejection in a subject who is a recipient of an organ transplant.In certain embodiments, the method comprises administering to a subject the immune response cell or pharmaceutical composition of the present disclosure.In certain embodiments, the immune response cell is an immune inhibitory cell.In certain embodiments, the immune inhibitory 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.

[0036] 4. Brief description of the drawings The following detailed description is provided by way of example and is not intended to limit the subject matter of the present disclosure to the particular embodiments described and may be understood in conjunction with the accompanying drawings. [Brief description of the drawings]

[0037] [Figure 1] Figure 1 represents a polypeptide composition according to a certain embodiment of the subject matter 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, a CD3 zeta signaling domain (including, for example, ITAM2 variants and ITAM3 variants). The CAR is fused to PD1DNR (and PD1 signaling domain) via a cleavable P2A peptide. SP: signaling peptide; scFv: single chain variable fragment; TM: transmembrane domain; cyt: cytoplasmic domain; DNR: dominant negative receptor; LTR: long terminal repeat. [Diagram 2] FIG. 2 shows the various constructs disclosed in Example 2. [Diagram 3] Figures 3A-3D show virus production in the producer cell line RD114. RD114 cells were transduced with various dilutions of H29 virus supernatant (undiluted, 1:2, and 1:4) and stained for CAR expression by flow cytometry using an anti-Fab antibody. RD114 empty served as a negative control. Figure 3A shows RD114 empty (as a negative control). Figure 3B shows undiluted, Figure 3C shows a dilution of the supernatant 1:2, and Figure 3D shows a dilution of the supernatant 1:4. [Figure 4A] Figures 4A-4E show transduction of human T cell-donor H116-2 with M28z1XX-P2A-PD1DNR. PHA-activated T cells were transduced with various concentrations of RD114 viral supernatant (Figure 4A shows 1:2, Figure 4B shows 1:5, Figure 4C shows 1:7, Figure 4D shows 1:15, and Figure 4E shows untransduced ("UT")) and stained for CAR expression by anti-Fab staining and for PD1DNR by anti-PD1 staining using flow cytometry. [Figure 4B] Same as above. [Figure 4C] Same as above. [Figure 4D] Same as above. [Figure 4E] Same as above. [Figure 5A] Figures 5A-5E show transduction of human T cells-donor H18 with M28z1XX-P2A-PD1DNR. PHA-activated T cells were transduced with various concentrations of RD114 viral supernatant (Figure 5A shows 1:2, Figure 5B shows 1:5, Figure 5C shows 1:10, Figure 5D shows 1:15, and Figure 5E shows untransduced ("UT")) and stained for CAR expression by anti-Fab staining and for PD1DNR by anti-PD1 staining using flow cytometry. [Figure 5B] Same as above. [Figure 5C] Same as above. [Figure 5D] Same as above. [Figure 5E] Same as above. [Figure 6A] Figures 6A-6F show transduction of human T cells-donor H19 with M28z1XX-P2A-PD1DNR. PHA-activated T cells were transduced with various concentrations of RD114 viral supernatant (Figure 6A shows 1:2, Figure 6B shows 1:5, Figure 6C shows 1:7, Figure 6D shows 1:10, Figure 6E shows 1:15, and Figure 6F shows untransduced ("UT")) and stained for CAR expression by anti-Fab staining and for PD1DNR by anti-PD1 staining using flow cytometry. [Figure 6B] Same as above. [Figure 6C] Same as above. [Figure 6D] Same as above. [Figure 6E] Same as above. [Figure 6F] Same as above. [Figure 7-1] Figures 7A-7C show the correlation of vector copy number (VCN) with median fluorescence intensity (MFI). PHA-activated T cells were transduced with various concentrations of RD114 viral supernatant and stained for CAR expression by anti-Fab staining and flow cytometry analysis. Genomic DNA of transduced T cells was isolated and vector copy number was determined as VCN per µg of DNA using qPCR. MFI of CAR-positive cells was correlated with VCN per µg of DNA for three different donors. Figure 7A shows donor H19, Figure 7B shows donor H18, and Figure 7C shows donor H116-2. [Figure 7-2] Same as above. [Figure 8] Figure 8 shows cytotoxicity for transduced T cells from three different donors. Using an impedance-based assay, MSLN high target cells (MGM) were co-cultured with M28z1xx-PD1DNR CAR T cells from different donors at various E:T ratios. M28z1xx-PD1DNR CAR T cells mediated cytolysis of MGM cells at an E:T ratio of 1:1. M28z1xx-PD1DNR CAR T cells killed MSLN high target cells. [Figure 9] FIG. 9 shows an example of impedance-based cytotoxicity assay (eCTL). [Figure 10] FIG. 10 shows parameters of a comparative analysis of the various constructs using eCTL. [Figure 11A]Figures 11A-11E show MSLN and PD-L1 expression in target cell lines. Mesothelioma (MGM (shown in Figure 11A), MGM-PDL1 (shown in Figure 11B) and MSTOG (shown in Figure 11C)) and lung cancer (A549GM (shown in Figure 11D) and A549G (shown in Figure 11e)) cell lines were assessed for MSLN and PD-L1 expression by flow cytometry. MGM, MGM-PDL1 and A549GM overexpressed MSLN, MGM-PDL1 cells further overexpressed PD-L1. [Figure 11B] Same as above. [Figure 11C] Same as above. [Figure 11D] Same as above. [Figure 11E] Same as above. [Figure 12A] Figures 12A-12E show CAR and PD1 expression of transduced T cells. Human T cells transduced with M28z (shown in Figure 12A), M28z1xx (shown in Figure 12B), M28z-PD1DNR (shown in Figure 12C) and M28z1xx-PD1DNR (shown in Figure 12D) were analyzed for CAR expression by anti-myc staining and for PD1 / PD1DNR expression by anti-PD1 staining using flow cytometry. Figure 12E shows untransduced ("UT") T cells. [Figure 12B] Same as above. [Figure 12C] Same as above. [Figure 12D] Same as above. [Figure 12E] Same as above. [Figure 13A] Figures 13A-13C show comparative analysis of anti-tumor efficiency of CAR T cells carrying 1XX domain and PD1DNR on MSLN-high tumor cells (MGM). MSLN-high target cells (MGM) were co-cultured with either M28z, M28z1xx, M28z-PD1DNR, M28z1XX-PD1DNR or untransduced T cells at the indicated E:T ratios. Anti-tumor efficiency was evaluated using an impedance-based assay. Figure 13A shows an E:T ratio of about 3:1. Figure 13B shows an E:T ratio of about 1:1. Figure 13C shows an E:T ratio of about 0.33:1. [Figure 13B] Same as above. [Figure 13C] Same as above. [Figure 14] Figure 14 shows a comparative analysis of the cytotoxicity of CAR T cells carrying the 1xx domain and PD1DNR on MSLN-high tumor cells (MGM). Chromium-51-labeled MSLN-high target cells (MGM) were co-cultured with either M28z, M28z1xx, M28z-PD1DNR, M28z1xx-PD1DNR or untransduced T cells at the indicated E:T ratio for 18 hours. Cytotoxicity was determined by Chromium-51 CTL. [Figure 15A] Figures 15A-15C show comparative analysis of anti-tumor efficiency of CAR T cells carrying 1xx domain and PD1DNR on MSLN-negative tumor cells (MSTOG). MSLN-negative target cells (MSTOG) were co-cultured with either M28z, M28z1xx, M28z-PD1DNR, M28z1xx-PD1DNR or untransduced T cells at the indicated E:T ratios. Anti-tumor efficiency was evaluated using an impedance-based assay. Figure 15A shows an E:T ratio of about 3:1. Figure 15B shows an E:T ratio of about 1:1. Figure 15C shows an E:T ratio of about 0.33:1. [Figure 15B] Same as above. [Figure 15C] Same as above. [Figure 16] Figure 16 shows a comparative analysis of the cytotoxicity of CAR T cells carrying 1XX domain and PD1DNR on MSLN-negative tumor cells (MSTOG). Chromium-51-labeled MSLN-negative target cells (MSTOG) were co-cultured with either M28z, M28z1XX, M28z-PD1DNR, M28z1XX-PD1DNR or untransduced T cells at the indicated E:T ratio for 18 hours. Cytotoxicity was determined by Chromium-51 CTL. [Figure 17A]Figures 17A-17C show comparative analysis of antitumor efficiency of CAR T cells carrying 1XX domain and PD1DNR on MSLN-high tumor cells overexpressing PDL1. MSLN-high target cells overexpressing PDL1 (MGM-PDL1) were co-cultured with either M28z, M28z1XX, M28z-PD1DNR, M28z1XX-PD1DNR or untransduced T cells at the indicated E:T ratios. Antitumor efficiency was evaluated using an impedance-based assay. Figure 17A shows an E:T ratio of about 3:1. Figure 17B shows an E:T ratio of about 1:1. Figure 17C shows an E:T ratio of about 0.33:1. [Figure 17B] Same as above. [Figure 17C] Same as above. [Figure 18A] Figures 18A-18C show comparative analysis of anti-tumor efficiency of CAR T cells carrying 1xx domain and PD1DNR on MSLN-high tumor cells (A549GM). MSLN-high target cells (A549GM) were co-cultured with either M28z, M28z1xx, M28z-PD1DNR, M28z1xx-PD1DNR or untransduced T cells at the indicated E:T ratios. Anti-tumor efficiency was evaluated using an impedance-based assay. Figure 18A shows an E:T ratio of about 10:1. Figure 18B shows an E:T ratio of about 5:1. Figure 18C shows an E:T ratio of about 2:1. [Figure 18B] Same as above. [Figure 18C] Same as above. [Figure 19A] Figures 19A-19C show comparative analysis of anti-tumor efficiency of CAR T cells carrying 1xx domains and PD1DNR:MSLN low tumor cells (A549G). MSLN low target cells (A549G) were co-cultured with either M28z, M28z1xx, M28z-PD1DNR, M28z1xx-PD1DNR or untransduced T cells at the indicated E:T ratios. Anti-tumor efficiency was assessed using an impedance-based assay. Figure 19A shows an E:T ratio of approximately 10:1. Figure 19B shows an E:T ratio of approximately 5:1. Figure 19C shows an E:T ratio of approximately 2:1. [Figure 19B] Same as above. [Figure 19C] Same as above. [Figure 20-1] Figures 20A-20D show the results of in vivo studies of various treatments. Figure 20A shows the relative efficacy in vivo of CAR T cells-M28z, M28z with PD1 antibody, and M28z with PD1DNR. Figure 20B shows the relative efficacy in vivo of M28z and M28z1XX+PD1DNR CAR T cells. Figure 20C shows tumor burden imaging showing systemic anti-tumor immunity after tumor rechallenge. Figure 20D shows ex vivo immunofluorescence staining of orthotopic MPM tumors showing CAR T cell infiltration. [Figure 20-2] Same as above. [Figure 21] Figure 21 shows the structure and components of the M28z1XXPD1DNR CAR. In contrast to M28z, M28z1XXPD1DNR CAR T cells possess a mutated CD3ζ signaling domain with a single functional ITAM, co-express PD1DNR, which consists of the CD8 transmembrane and hinge domains, and lack the intracellular PD1 signaling domain present in endogenous PD1. [Figure 22-1] Figure 22 shows the structure of the CAR T cell vector. [Figure 22-2] Same as above. [Figure 23-1] Figure 23 shows the expression of mesothelin (MSLN), PD-L1, and GFP in tumor cell lines. MGM, MGM-PDL1, and MSTOG tumor cells were analyzed for their expression of mesothelin (left panel), PD-L1 (middle panel), and GFP (right panel) by flow cytometry. Density plots showing relative expression intensity plotted against side scatter area (Y axis) are shown. [Figure 23-2] Same as above. [Figure 24]Figures 24A-24D show an orthotopic MPM mouse model. Figure 24A shows that the macroscopic findings of human MPM (top left panel) are reproduced in a mouse model of MPM with tumor-engulfed heart, lung, and mediastinal structures (top right panel) as well as tumors invading the chest wall (bottom panel). Figure 24B shows that the expanding vascularity of the tumor is demonstrated by CD34 immunofluorescence. Figure 24C shows that tumor burden progression monitored by BLI correlates with tumor volume measurements by MRI at each time point. Figure 24D shows tumor burden progression monitored by serial BLI and MRI. [Figure 25A] Figures 25A-25C show the expression of mesothelin in human tissues by immunohistochemistry. Figure 25A shows the expression of mesothelin in MPM versus normal pleura and pericardium. Figure 25B shows the expression of mesothelin in lung adenocarcinoma versus normal lung tissue. Figure 25C shows the expression of mesothelin in triple-negative breast cancer versus normal breast tissue. [Figure 25B] Same as above. [Figure 25C] Same as above. [Figure 26-1] Figure 26 shows that expression of M28z1XXPD1DNR can be titrated using various dilutions of viral supernatant. Human T cells were transduced with various dilutions of viral supernatant encoding M28z1XXPD1DNR (left panel) or mycM28z1XXPD1DNR (middle panel). CAR (Y axis) and PD1 (X axis) expression of viable CD3 positive cells was assessed by flow cytometry. Results shown are from a representative example of one of three different donors. [Figure 26-2] Same as above. [Figure 27]Figure 27 shows that CAR expression measured by MFI correlates with VCN. Human T cells from three different donors were transduced with various dilutions of retroviral supernatants encoding either M28z1XXPD1DNR or mycM28z1XXPD1DNR. The MFI of CAR-positive T cells (determined by flow cytometry) was plotted against VCN (determined by qPCR). R2 values ​​were derived from linear regression analysis (black line). [Figure 28] Figures 28A-28D show expression of PD1 and PD1DNR in mycM28z1XXPD1DNR and mycM28z CAR T cells. Figure 28A shows the percentage of CAR surface expression of mycM28z and mycM28z1XXPD1DNR CAR T cells. Figure 28B shows the percentage of CD3 positive cells relative to PD1 surface expression. Figure 28C shows the MFI of PD1 surface expression of CD3 positive cells. Figure 28D shows the relative mRNA expression of PD1 extracellular domain and PD1 intracellular domain shown as fold change compared to that of untransduced T cells. [Figure 29-1] Figure 29 shows that M28z1XXPD1DNR expressing T cells with or without myc tag exhibit identical antitumor efficiency in vitro. Human T cells from three different donors were transduced with either M28z1XXPD1DNR (red) or mycM28z1XXPD1DNR (green) (transduction range, 37%-63%) and co-cultured with MGM cells (green; arrows indicate time of T cell addition). Antitumor efficiency of both constructs was compared at the indicated E:T ratios using an impedance-based cytotoxicity assay. [Figure 29-2] Same as above. [Diagram 30]Figure 30 shows that 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 either MGM, MGM-PDL1, or MSTOG tumor cells at the indicated E:T ratios. CAR T cell cytotoxicity was assessed by 51Cr release assay after 18 hours of co-culture. Untransduced T cells (orange) served as a control. [Diagram 31] Figure 31 shows the accumulation of mycM28z1XXPD1DNR CAR T cells upon 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 an E:T ratio of 1:1 for 48 hours. CAR T cell accumulation was quantified by absolute CAR T cell counts after each antigen stimulation. [Diagram 32] Figure 32 shows that mycM28z1XXPD1DNR CAR T cells exhibited similar cytotoxicity as mycM28z CAR T cells upon initial antigen stimulation. Human T cells transduced with mycM28z1XXPD1DNR (blue) or mycM28z (red) were co-cultured with 51Cr-labeled MGM or MGM-PDL1 target cells at the indicated E:T ratios. Cytotoxicity was assessed after 18 hours using a 51Cr release assay. Untransduced T cells (orange) served as a control. [Diagram 33] Figure 33 shows that mycM28z1XXPD1DNR CAR T cells retained antitumor efficacy upon 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 4 stimulations for 48 hours, followed by an E:T ratio of 1:1 for two additional stimulations. CAR T cell cytotoxicity was assessed by 51Cr release assay at the indicated E:T ratios and upon the 4th and 7th antigen stimulations after 18 hours of co-culture. [Diagram 34] Figure 34 shows that mycM28z1XXPD1DNR CAR T cells secreted effector cytokines upon antigen stimulation. Human T cells transduced with mycM28z1XXPD1DNR (blue) or mycM28z (red) were repeatedly exposed to MGM (top) or MGM-PDL1 (bottom) target cells at an E:T ratio of 1:1 for 48 hours. Cell-free supernatants were collected 24 hours after the first, third, and sixth antigen exposures, and effector cytokine secretion was evaluated by Luminex assay. [Diagram 35] Figure 35 shows that a single low dose of 3x104 intrapleural administration of mycM28z1XXPD1DNR CAR T cells demonstrates antitumor efficacy in vivo. Female NSG mice bearing orthotopic MGM tumors were treated with a single intrapleural dose of either P28z CAR T cells (n=6, red bar) or mycM28z1XXPD1DNR CAR T cells (n=10, blue bar). Tumor burden was measured by BLI. The time point shown represents day 15 after CAR T cell administration, the time point when mice treated with P28z CAR T cells began to become moribund. Statistical significance was determined using an unpaired Student's t-test (two-tailed). ***p<0.001. [Figure 36-1] Figures 36A-36D show that intrapleurally administered mycM28z1XXPD1DNR CAR T cells exhibit in vivo antitumor efficiency and increase survival. Figure 3A shows sequential tumor BLI of female NSG mice bearing MGM-PDL1 tumors treated with a single dose of mycM28z (1x105) or mycM28z1XXPD1DNR (1x105 or 5x104) CAR T cells (n=7-8). Four mice per treatment group are shown in prone position. Figure 36B shows the corresponding sequential BLI (average of dorsal and ventral) showing the tumor burden of each treated mouse. Figure 36C shows the corresponding mouse body weights after treatment. Figure 36D shows 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. [Figure 36-2] Same as above. [Figure 37] Figure 37 shows detection of mycM28z1XXPD1DNR CAR T cells in primary tumors of mice treated intrapleurally. Pleural MGM tumors from mice were treated with 5x105 untransduced T cells (left), mycM28z CAR T cells (middle), or mycM28z1XXPD1DNR CAR T cells (right). Three days after intrapleural T cell injection, tumor tissues were harvested, fixed, and stained ex vivo for tumor mesothelin (green), human CD45 positive cells (red), and DAPI (cell nuclei, blue) by immunofluorescence. [Figure 38] Figures 38A and 38B show that mycM28z1XXPD1DNR CAR T cells resisted tumor reconstitution upon repeated tumor challenge in vivo. Figure 38A shows a schematic diagram illustrating the following tumor rechallenge experiment: 68 days after intrapleural administration of mycM28z or mycM28z1XXPD1DNR CAR T cells (single dose of 1 x 105 CAR T cells) and eradication from the pleura of MGM-PDL1 tumor cells (inoculation dose of 8 x 105), mice were intraperitoneally rechallenged 10 times with increasing doses (2 x 106 to 11 x 106) of MGM tumor cells every 4 to 8 days. Figure 38B shows sequential BLI showing tumor challenge 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 of increasing doses. [Figure 39-1] Figures 39A-39C show that M28z1XXPD1DNR CAR T cells produced using clinical trial vector stock have anti-tumor efficiency in vivo and prolong survival. Figure 39A shows sequential tumor BLI of female NSG mice bearing MGM tumors treated with 6x104 (n=8) or 2x105 (n=10) M28z1XXPD1DNR CAR T cells produced by CTCEF using clinical trial viral supernatant. Figure 39B shows the corresponding mouse body weights after treatment. Figure 39C shows Kaplan-Meier survival analysis. [Figure 39-2] Same as above. [Diagram 40] Figure 40 shows the mean body weights at interim sacrifice for male mice. Groups 1 (non-tumor control), 3 (vehicle control), and 5 (test article) are shown. [Diagram 41] Figure 41 shows the mean body weights at interim sacrifice for female mice. Groups 2 (non-tumor control), 4 (control), and 6 (test article) are shown. [Diagram 42] Figure 42 shows the mean body weights at terminal sacrifice for male mice. Groups 7 (non-tumor control), 9 (vehicle control), and 11 (test article) are shown. [Diagram 43] Figure 43 shows the mean body weights at terminal sacrifice for female mice. Groups 8 (non-tumor control), 10 (vehicle control), and 12 (test article) are shown. [Figure 44-1] Figure 44 shows the identification of human T cells in tumors of 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 to detect viable human T cells by flow cytometry. Density plots of human CD3 expression (X-axis) and human CD45 expression (Y-axis) of DAPI-negative (live) single cells are shown. Gates indicate cells stained positive for human CD45 and human CD3, representing human T cells. [Figure 44-2] Same as above. [Figure 44-3] Same as above. [Figure 44-4] Same as above. [Figure 45-1]Figure 45 shows the identification of human T cells in the spleens of mice treated with CAR T cells and mice treated with vehicle. Spleen tissue cells from mice treated with CAR T cells and mice treated with vehicle were stained with DAPI, anti-human CD45 APC / CY7, and anti-human CD3 PE / CY7 antibodies to detect live human T cells by flow cytometry. Density plots of human CD3 expression (X-axis) and human CD45 expression (Y-axis) of DAPI-negative (live) single cells are shown. Gates indicate cells stained positive for human CD45 and human CD3, which represent human T cells. [Figure 45-2] Same as above. [Figure 45-3] Same as above. [Figure 45-4] Same as above. [Figure 46] FIG. 46 shows BLI of male mice. [Figure 47] FIG. 47 shows BLI of female mice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0038] 5. Detailed Description of the Invention The presently disclosed subject matter provides polypeptide compositions comprising a chimeric antigen receptor (CAR) that targets mesothelin and a dominant negative form of programmed cell death 1 (PD-1 DN), as well as immune response cells (e.g., T cells or NK cells) comprising the polypeptide compositions. The presently disclosed subject matter also provides methods of using such polypeptide compositions to induce and / or enhance the immune response of immune response cells to a target antigen and / or to treat and / or prevent neoplasms or other diseases / disorders where reduced immune cell wasting is desirable.

[0039] For example, persistent antigen exposure of T cells in cancer leads to an alteration in the T cell differentiation state, termed exhaustion, that renders CAR T cells functionally impaired (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 capacity 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 demonstrated that this CAR activation capacity can be modulated by mutating the ITAMs, thereby reducing their functionality. Importantly, the introduction of point mutations in the second and third ITAMs (1-XX; herein designated "1XX") of the CD3 ζ domain has been shown to shift the fate of CAR T cells 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).

[0040] Another obstacle that CAR T cells encounter in the solid tumor microenvironment is the inhibition of their cytolytic activity mediated through PD1, an inhibitory receptor expressed upon antigen-mediated T cell activation. In addition, tumor cells enhance the expression of co-inhibitory ligands such as PD-L1 after exposure to proapoptotic cytokines secreted by T cells (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 group combined mesothelin-targeted CAR T cells with a PD1-blocking antibody 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 repeated administration of PD1 checkpoint blockade and associated clinical side effects, our group has demonstrated that using a cell-specific PD1 checkpoint inhibition strategy in which PD1DNR is co-transduced with a second-generation CAR into T cells 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).

[0041] Therefore, to develop CAR T cells with enhanced therapeutic profile, functional persistence, and resistance to tumor-mediated inhibition, we incorporated 1XX and PD1DNR components into a second-generation CAR vector design, enabling these cells to perform efficiently in the highly immunosuppressive microenvironment of solid tumors.

[0042] For purposes of clarity of disclosure, and not by way of limitation, the detailed description is divided into the following subsections: 5.1. Definition; 5.2. Polypeptide Compositions; 5.2.1.PD-1 DN; 5.2.2. Mesothelin-targeted CAR; and 5.2.3. Exemplary Polypeptide Compositions; 5.3. Immune response cells; 5.4. Nucleic Acid Compositions and Vectors; 5.5. Polypeptides and analogs; 5.6. Pharmaceutical Compositions and Administration; 5.7. Preparations; 5.8. Treatment methods; and 5.9.Kit

[0043] 5.1.Definition Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art.

[0044] As used herein, the term "about" or "approximately" means within an acceptable error range of a particular value as determined by a person skilled in the art, which depends in part on the method of measuring or determining the value, i.e., the limitations of the measurement system. For example, "about" can mean within 3 standard deviations or within 3 or more standard deviations with respect to the practice in the art. Alternatively, "about" can mean within a range of up to 20%, such as 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 of a value, such as within 5-fold or within 2-fold.

[0045] By "immune response cell" is meant a cell that functions in the immune response, or a precursor or progeny thereof.

[0046] "Activating immune response cells" refers to the induction of signal transduction or change in protein expression in cells that results in the initiation of an immune response. For example, when CD3 chains form clusters in response to binding of a ligand and immunoreceptor tyrosine-based inhibitory motifs (ITAMs), a signal transduction cascade is generated. In certain embodiments, when a chimeric antigen receptor (CAR) binds to an antigen, the formation of an immune synapse occurs, which includes the clustering of many molecules (e.g., CD4 or CD8, CD3γ / δ / ε / ζ, etc.) in the vicinity of the bound receptor. This clustering of membrane-bound signal transduction molecules allows the ITAM motifs contained within the CD3 chains to become phosphorylated. This phosphorylation then 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 IL-2 production for proliferation, and the expression of master regulatory T cell proteins to initiate T cell-mediated immune responses.

[0047] "Stimulating immune response cells" refers to signals that result in a robust and sustained immune response. In various embodiments, this occurs after activation of immune response cells (e.g., T cells) or is mediated simultaneously through receptors including, but not limited to, CD28, CD137 (4-1BB), OX40, CD40, and ICOS. Receiving multiple costimulatory signals can be important to initiate a robust and long-lasting T cell-mediated immune response. T cells can be quickly inhibited and become unresponsive to antigens. The effects of these costimulatory signals can be diverse, but they generally result in increased gene expression to generate long-lived, proliferative, anti-apoptotic T cells that robustly respond to antigens for complete and sustained eradication.

[0048] As used herein, the term "antibody" refers not only to intact antibody molecules, but also to fragments of antibody molecules that retain immunogen-binding ability. Such fragments are also well known in the art and are commonly used both in vitro and in vivo. Thus, as used herein, the term "antibody" refers not only to intact immunoglobulin molecules, but also to the well-known active fragments F(ab') 2 and Fab. F(ab') which lacks the Fe fragment of an intact antibody. 2 and Fab fragments are cleared more rapidly from the circulation and may have less non-specific tissue binding of intact antibodies (Wahl et al., J. Nucl. Med. 24:316-325 (1983)). As used herein, antibodies include whole natural antibodies, bispecific antibodies, chimeric antibodies, Fab, Fab', single chain V region fragments (scFv), fusion polypeptides, and atypical antibodies. In certain embodiments, antibodies are glycoproteins comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (herein referred to as V H ) and heavy chain constant (C H The heavy chain constant region is composed of three domains, CH1, CH2, and CH3. Each light chain comprises a light chain variable region (herein referred to as V L ) and light chain constant C L The light chain constant region consists of one domain, C L It consists of V H and V L The regions can be further subdivided into highly variable regions called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). H and V Lis composed of three CDRs and four FRs arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of complement (C1q) of the classical complement system.

[0049] As used herein, "CDR" is defined as the amino acid sequence of the complementarity determining region of an antibody, which is the hypervariable region of the immunoglobulin heavy and light chains. See, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 4th US Department of Health and Human Services, National Institutes of Health (1987). Generally, an antibody comprises three heavy and three light chain CDRs or CDR residues in the variable region. CDRs provide the majority of contact residues for antibody binding to antigen or epitope. In certain embodiments, CDR regions are represented using Kabat's system (Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242). In certain embodiments, CDRs are identified according to Kabat's system.

[0050] As used herein, the term "single-chain variable fragment" or "scFv" refers to a fragment of an immunoglobulin heavy chain (V H ) and light chain (V L ) are covalently linked to the variable region of V H ::V L It is a fusion protein that forms a heterodimer. Hand V L is directly bonded or V H N-terminus of V L or V H The C-terminus of V L The nucleic acids are linked by a peptide-encoded linker (e.g., 10, 15, 20, 25 amino acids) that connects to the N-terminus of the V. The linker is usually glycine-rich for flexibility and serine- or threonine-rich for solubility. "Linker" as used herein means a functional group (e.g., a chemical group or a polypeptide) that covalently bonds two or more polypeptides or nucleic acids, thereby connecting them to each other. As used herein, "peptide linker" refers to a peptide that is used to link two proteins together (e.g., V H and V L Linker refers to one or more amino acids used to link (connect) domains. In certain embodiments, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 66, provided below: [ka] .

[0051] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO:66 is set forth in SEQ ID NO:50, provided below: [ka] .

[0052] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO:66 is set forth in SEQ ID NO:51, provided below: [ka]

[0053] Despite the removal of the constant region and the introduction of a linker, the scFv protein retains the specificity of the original immunoglobulin. Single-chain Fv polypeptide antibodies are derived from the VFv polypeptide described in Huston, et al. (Proc. Nat. Acad. Sci. USA, 85:5879-5883, 1988). H and V L It can be expressed from a nucleic acid containing the coding sequence. See also U.S. Patent Nos. 5,091,513, 5,132,405, and 4,956,778; and U.S. Patent Application 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 Imunol2009 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 Bioi Chern 2003 25278(38):36740-7; Xie et al., Nat Biotech 1997 15(8):768-71; Ledbetter et al., Crit Rev Immunol1997 17(5-6):427-55; Ho et al., BioChim Biophys Acta 2003 1638(3):257-66).

[0054] As used herein, "F(ab)" refers to the fragment of an antibody structure that binds to an antigen but is monovalent and does not have the Fc portion; for example, digestion of an antibody with the enzyme papain produces two F(ab) fragments and an Fc fragment (e.g., heavy (H) chain constant region; the Fc region that does not bind to antigen).

[0055] As used herein, "F(ab') 2 " refers to an antibody fragment produced by pepsin digestion of a whole IgG antibody, which fragment has two antigen-binding (ab') (bivalent) regions, each (ab') region containing two separate amino acid chains, a portion of a H chain and a light (L) chain linked by an S-S bond for binding to the antigen, with the remaining portions of the H chains linked together. The "F(ab')2" fragment can be split into two separate Fab' fragments.

[0056] As used herein, the term "vector" refers to any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc., which is capable of replication and transfer of genetic sequences into a cell when associated with the proper control elements. Thus, the term includes cloning and expression vehicles as well as viral vectors and plasmid vectors.

[0057] As used herein, the term "expression vector" refers to a recombinant nucleic acid sequence, i.e., a recombinant DNA molecule that contains a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host organism. Nucleic acid sequences necessary for expression in prokaryotic cells usually include a promoter, an operator (optionally), and a ribosome binding site, often along with other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals.

[0058] As used herein, the term "affinity" refers to the measurement of binding strength. Affinity may depend on the closeness of the stereochemical fit 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 to an antigen are known in the art and include, but are not limited to, various antigen binding experiments, such as functional assays (e.g., flow cytometry assays).

[0059] The term "chimeric antigen receptor" or "CAR" as used herein refers to a molecule that comprises an intracellular signaling domain capable of activating or stimulating immune response cells, and an extracellular antigen binding domain fused to a transmembrane domain. In certain embodiments, the extracellular antigen binding domain of the CAR comprises an scFv. The scFv can be derived from fusing the variable heavy and light chain regions of an antibody. Alternatively or additionally, the scFv can be derived from a Fab' (e.g., rather than an antibody from a Fab library). In certain embodiments, the scFv is fused to a transmembrane domain, which is then fused to an intracellular signaling domain. In certain embodiments, the CAR is selected to have a high binding affinity to the antigen.

[0060] As used herein, the term "nucleic acid molecule" includes any nucleic acid molecule or fragment thereof that encodes a polypeptide of interest. Such nucleic acid molecules do not necessarily have to be 100% homologous or identical to an endogenous nucleic acid sequence, but may exhibit substantial identity.

[0061] "Substantially identical" or "substantially homologous" refers to an amino acid sequence or nucleic acid molecule that exhibits at least about 50% homology or identity to a reference amino acid sequence (e.g., any one of the amino acid sequences described herein) or a reference nucleic acid sequence (e.g., any one of the nucleic acid sequences described herein). In certain embodiments, such a sequence is 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% homologous or identical to the sequence of the reference amino acid sequence or reference nucleic acid sequence used for comparison.

[0062] Sequence identity can be measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically 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 approach to determine the degree of identity, the BLAST program may be used with a probability score between e-3 and e-100, which indicates closely related sequences.

[0063] As used herein, the percent homology between two amino acid sequences is equivalent to the percent identity between two sequences.The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap (i.e., % homology=number of identical positions / total number of positions×100).Comparing sequences and determining the percent identity between two sequences can be achieved using mathematical algorithms.

[0064] 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)) incorporated into the ALIGN program (version 2.0), using a PAM120 weighted residue table, a gap extension penalty of 12, and a gap penalty of 4. Additionally, the percent homology between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) incorporated into the GAP program in the GCG software package (available at www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0065] Additionally or alternatively, the amino acid sequence of the subject matter of the present disclosure can be further used as a "query sequence" to perform searches against public databases, for example 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 with the XBLAST program, score=50, wordlength=3, to obtain amino acid sequences homologous to the specified sequences disclosed herein (e.g., heavy and light chain variable region sequences of scFv m903, m904, m905, m906, and m900). To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids 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. The term "constitutive expression" or "constitutively expressed" as used herein refers to expression or being expressed under all physiological conditions.

[0066] By "disease" is meant any condition, disease, or disorder that damages or interferes with the normal function of a cell, tissue, or organ, such as neoplasms and pathogenic infection of a cell.

[0067] "Effective amount" means an amount sufficient to have a therapeutic effect. In certain embodiments, an "effective amount" is an amount sufficient to stop, ameliorate, or inhibit the continued proliferation, growth, or metastasis (e.g., invasion or migration) of a neoplasm.

[0068] By "modulate" is meant to alter, either positively or negatively. Exemplary modulations include changes of about 1%, about 2%, about 5%, about 10%, about 25%, about 50%, about 75%, or about 100%.

[0069] By "increase" is meant a positive alteration of at least about 5%. The alteration can be about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, about 100%, or more.

[0070] By "reduce" is meant to negatively alter by at least about 5%. The alteration can be about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, or even about 100%.

[0071] By "isolated cell" is meant a cell that has been separated from molecules and / or cellular components that naturally accompany the cell.

[0072] The terms "isolated," "purified," or "biologically pure" refer to material that is free to various degrees from components that normally accompany it when found in its native state. "Isolate" refers to a degree of separation from the original source or surroundings. "Purify" refers to a degree of separation that is higher than isolation. A "purified" or "biologically pure" protein is substantially free of other materials such that no impurities substantially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide is purified if it is substantially free of cellular material, viral material, or culture medium if produced by recombinant DNA technology, or chemical precursors or other chemicals if chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" may indicate that the nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. In the case of proteins that can be subject to modifications, such as phosphorylation or glycosylation, different modifications can result in different isolated proteins that can be individually purified.

[0073] "Neoplasm" refers to a disease characterized by pathological proliferation of cells or tissues and subsequent migration or invasion into other tissues or organs. Neoplasmic growth is typically uncontrolled and progressive, occurring under conditions that do not induce replication or cause replication arrest in normal cells. Neoplasms can affect a variety of cell types, tissues, or organs, including, but not limited to, organs selected from the group consisting of bladder, bone, brain, breast, cartilage, glia, esophagus, fallopian tube, gallbladder, heart, intestine, kidney, liver, lung, lymph node, nervous tissue, ovary, pancreas, prostate, skeletal muscle, skin, spinal cord, spleen, stomach, testes, thymus, thyroid, trachea, urogenital tract, ureter, urethra, uterus, and vagina, or tissues or cell types thereof. Neoplasms include cancers, such as sarcomas, carcinomas, or plasmacytomas (malignant tumors of plasma cells). In certain embodiments, the neoplasm is a solid tumor. The neoplasm can be a primary tumor or a primary cancer. In addition, the neoplasm can be in a metastatic state.

[0074] As used herein, the term "conservative sequence modification" refers to an amino acid modification that does not significantly affect or significantly change the binding characteristics of the mesothelin-targeting CAR (e.g., the extracellular antigen-binding domain of the CAR) of the present disclosure, including the amino acid sequence. Conservative modifications can include amino acid substitutions, additions, and deletions. Modifications can be introduced into the extracellular antigen-binding domain of the CAR of the present disclosure by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Amino acids can be classified into groups according to their physicochemical properties, such as charge and polarity. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid in the same group. For example, amino acids can be classified by charge: positively charged amino acids include lysine, arginine, and histidine, negatively charged amino acids include aspartic acid and glutamic acid, and neutrally charged amino acids include alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. Additionally, amino acids can be classified by polarity: polar amino acids include arginine (basic polar), asparagine, aspartic acid (acidic polar), glutamic acid (acidic polar), glutamine, histidine (basic polar), lysine (basic polar), serine, threonine, and tyrosine; nonpolar amino acids include alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine. Thus, one or more amino acid residues in the CDR regions can be exchanged for other amino acid residues in the same group, and the altered antibodies can be tested for retention of function (i.e., the functions described in (c) through (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 in a specified sequence or CDR region are altered.

[0075] By "signal sequence" or "leader sequence" is meant 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 its entry into the secretory pathway. Exemplary leader sequences include, but are not limited to, the 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: 74]).

[0076] In certain embodiments, the CAR comprises a CD8 signal peptide at the N-terminus, e.g., the signal peptide is connected to the extracellular antigen-binding domain of the CAR. In certain embodiments, the CD8 signal peptide comprises or consists of the amino acid sequence set forth in SEQ ID NO:71.

[0077] An exemplary nucleotide sequence that encodes the amino acid sequence of SEQ ID NO:71 is set forth in SEQ ID NO:125. SEQ ID NO:125 is provided below. [ka]

[0078] The terms "comprises" and "comprising" are intended to have the broad meaning ascribed to them in U.S. patent law and can mean "includes," "including," etc.

[0079] As used herein, "treatment" refers to clinical intervention in an attempt to change the course of disease in the treated individual or cell, and can be performed either prophylactically or during the course of clinical pathology. The therapeutic effect of treatment includes, but is not limited to, prevention of disease onset or recurrence, alleviation of symptoms, reduction of any direct or indirect pathological consequences of disease, prevention of metastasis, reduction of disease progression rate, improvement or mitigation of disease state, and remission or improvement of prognosis. By preventing the progression of disease or disorder, treatment can prevent the deterioration of the disorder in subjects who are affected or diagnosed, or subjects suspected of having the disorder, but treatment can also prevent the onset of the disorder or symptoms of the disorder in subjects at risk of the disorder or subjects suspected of having the disorder.

[0080] An "individual" or "subject" as used herein refers to a vertebrate, such as a human or a non-human animal, such as a mammal. Mammals include, but are not limited to, humans, primates, farm animals, sport 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; cows; horses; and non-human primates, such as apes and monkeys. The term "immunocompromised" as used herein refers to a subject with an immune deficiency. The subject is highly susceptible to opportunistic infections, which are infections caused by organisms that do not usually cause disease in people with healthy immune systems, but can affect people with poorly functioning or suppressed immune systems.

[0081] Other aspects of the presently disclosed subject matter are described in the disclosure that follows and are within the scope of the presently disclosed subject matter.

[0082] 5.2. Polypeptide Compositions The subject matter of the present disclosure provides polypeptide compositions comprising a mesothelin-targeting chimeric antigen receptor (CAR) and a dominant negative form of programmed cell death 1 (PD-1 DN).

[0083] 5.2.1. Programmed cell death 1 dominant negative (PD-1 DN) A dominant negative form of programmed cell death 1 (referred to as "PD-1 DN") can enhance the therapeutic efficacy of immune response cells, including CARs. In certain embodiments, PD-1 DN comprises (a) at least a portion of the extracellular domain of programmed cell death 1 (PD-1), including the ligand-binding region, and (b) the transmembrane domain.

[0084] In certain embodiments, immune response cells, such as T cells or their precursor cells, are engineered to express a dominant negative (DN) form of PD-1.

[0085] Malignant cells adapt to generate an immunosuppressive microenvironment that protects them from immune recognition and elimination (Sharpe et al., Dis. Model Mech. 2015;8:337-350). The immunosuppressive microenvironment is a limitation for immunotherapy methods. The subject matter of the present disclosure addresses this limitation by expressing a DN type of inhibitor of cellular immune response in immune response cells or their precursor cells. Details of the DN type of inhibitor of cellular immune response are disclosed in WO2017 / 040945 and WO2017 / 100428, the contents of each of which are incorporated herein by reference in their entirety.

[0086] Programmed cell death protein 1 (PD-1) is a negative immune regulator of activated T cells through engagement with its corresponding ligands, PD-L1 and PD-L2, expressed on endogenous macrophages and dendritic cells. PD-1 is a type I membrane protein of 268 amino acids. PD-1 has two ligands, PD-L1 and PD-L2, which are B7 family members. The structure of the protein includes an extracellular IgV domain followed by a transmembrane region and an intracellular tail. The intracellular tail contains two phosphorylation sites located at an immunoreceptor tyrosine-based inhibitory motif and an immunoreceptor tyrosine-based switch motif. PD-1 negatively regulates TCR signals. SHP-1 and SHP-2 phosphatases bind to the cytoplasmic tail of PD-1 upon ligand binding. Upregulation of PD-L1 is one mechanism used by tumor cells to evade the host immune system. In preclinical and clinical trials, blockade of PD-1 with antagonistic antibodies induced antitumor responses mediated through the host's endogenous immune system.

[0087] In certain embodiments, the PD-1 polypeptide consists of the amino acid sequence having GenBank No. NP_005009.2 (SEQ ID NO:48) or a fragment thereof. In certain embodiments, amino acids 1-20 of SEQ ID NO:48 are the signal peptide (or peptide signal) of PD-1. In certain embodiments, amino acids 21-170 of SEQ ID NO:48 are the extracellular domain of PD-1. In certain embodiments, amino acids 171-191 of SEQ ID NO:48 are the transmembrane domain of PD-1. In certain embodiments, amino acids 192-288 of SEQ ID NO:48 are the intracellular domain of PD-1. SEQ ID NO:48 is provided below: [ka]

[0088] In certain embodiments, the extracellular domain of PD-1 comprises a ligand-binding domain (referred to as the "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 can be fused at its N-terminus to a signal peptide, which is a heterologous signal peptide, optionally including various signal peptides described herein. In addition, the PD-1 DN can comprise a transmembrane domain, which is a heterologous transmembrane domain, optionally including any of the various transmembrane domains described herein.

[0089] In certain embodiments, the PD-1 DN comprises the extracellular domain of a PD-1 polypeptide (e.g., amino acids 21-170 of SEQ ID NO: 48) or a ligand-binding portion thereof (e.g., amino acids 21-165 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 or a portion thereof (e.g., the PD-1 DN lacks amino acids 192-288 of SEQ ID NO: 48). A PD-1 consisting of a deletion of the intracellular domain may reduce or inhibit the immune checkpoint pathway mediated by PD-1.

[0090] 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 set forth in SEQ ID NO:58 (or amino acids 21-165 of SEQ ID NO:48). SEQ ID NO:58 is provided below. [ka]

[0091] An exemplary nucleotide sequence encoding SEQ ID NO:58 (or amino acids 21-165 of SEQ ID NO:48) is set forth in SEQ ID NO:59, provided below. [ka]

[0092] 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 a PD-1 polypeptide, the transmembrane domain of a PD-1 polypeptide, and the signal peptide of a 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. [ka]

[0093] In certain embodiments, the PD-1 DN comprises or consists of amino acids 1-165 of SEQ ID NO:48.

[0094] An exemplary nucleotide sequence encoding amino acids 1-165 of SEQ ID NO:48 is set forth in SEQ ID NO:61, provided below. [ka]

[0095] 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 with amino acid 21 of SEQ ID NO: 48 through between amino acids 151 to 165 of SEQ ID NO: 48.

[0096] 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., the 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 183-203 of SEQ ID NO: 86). Such embodiments represent chimeric DN types that comprise transmembrane domains from different (heterologous) polypeptides. As described above, PD-1 DNs that comprise a heterologous domain, such as a transmembrane domain, may optionally comprise additional sequences from a heterologous polypeptide. In certain embodiments, the PD-1 DN comprises additional sequences from a heterologous polypeptide at the N-terminus of the transmembrane domain. In certain embodiments, the PD-1 DN comprises the hinge domain of CD8. In certain embodiments, the heterologous sequence comprises additional N-terminal sequences of a CD8 polypeptide (e.g., amino acids 137-182 (or optionally starting at amino acid 138 or 139) of SEQ ID NO: 86). In certain embodiments, the PD-1 DN comprises additional sequence from a heterologous polypeptide at the C-terminus of the transmembrane domain of CD8. In certain embodiments, the additional C-terminal sequence is amino acids 204-209 of SEQ ID NO:86.

[0097] In certain embodiments, the PD-1 DN comprises the transmembrane domain of a CD8 polypeptide (e.g., amino acids 183-203 of SEQ ID NO: 86), the hinge domain of a CD8 polypeptide (e.g., amino acids 137-182 of SEQ ID NO: 86), and additional C-terminal sequences of a CD8 polypeptide (e.g., amino acids 204-207 of SEQ ID NO: 86). In certain embodiments, the PD-1 DN comprises a CD8 polypeptide consisting of amino acids 137-207 of SEQ ID NO: 86.

[0098] An exemplary nucleotide sequence encoding amino acids 137-207 of SEQ ID NO:86 is set forth in SEQ ID NO:62, provided below: [ka]

[0099] In certain embodiments, the PD-1 DN comprises a transmembrane domain of a CD8 polypeptide (e.g., amino acids 183-203 of SEQ ID NO: 86), a hinge domain of a CD8 polypeptide (e.g., amino acids 137-182 of SEQ ID NO: 86), and additional C-terminal sequences of a CD8 polypeptide (e.g., amino acids 204-209 of SEQ ID NO: 86). In certain embodiments, the PD-1 DN comprises a CD8 polypeptide consisting of amino acids 137-209 of SEQ ID NO: 86.

[0100] An exemplary nucleotide sequence encoding amino acids 137-209 of SEQ ID NO:86 is set forth in SEQ ID NO:63, provided below: [ka]

[0101] In one specific embodiment, the PD-1 DN comprises the amino acid sequence set forth in SEQ ID NO:49, provided below. [ka]

[0102] An exemplary nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO:49 is set forth in SEQ ID NO:64, provided below: [ka] [ka]

[0103] In certain embodiments, the PD-1 DN comprises the amino acid sequence set forth in SEQ ID NO: 118, provided below. [ka]

[0104] An exemplary nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO:118 is set forth in SEQ ID NO:119, provided below: [ka]

[0105] In certain embodiments, the transmembrane domain of PD-1 DN comprises a hydrophobic alpha helix that spans at least a portion of the membrane. Different transmembrane domains result in different receptor stabilities. In accordance with the subject matter of the present disclosure, the transmembrane domain of PD-1 DN may comprise any transmembrane domain that may be comprised in a natural or modified transmembrane domain of any polypeptide disclosed herein, such as a chimeric antigen receptor. In certain embodiments, the transmembrane domain is a CD8 polypeptide, a CD28 polypeptide, a CD3ζ 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, an 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. Details of these transmembrane domains are described in the following sections.

[0106] 5.2.2. Mesothelin-targeted chimeric antigen receptors (CARs) Polypeptide compositions of the disclosure include a CAR that specifically targets mesothelin, e.g., human mesothelin.

[0107] CAR is an engineered receptor that grafts or confers a specificity of interest to immune effector cells. CAR can be used to graft the specificity of monoclonal antibodies into T cells, and the transfer of its coding sequence is facilitated by retroviral vectors.

[0108] There are three generations of CARs. "First generation" CARs are typically composed of an extracellular antigen binding domain (e.g., scFv) fused to a transmembrane domain fused to a cytoplasmic / intracellular signaling domain. "First generation" CARs can provide de novo antigen recognition and bind to CD4 through its CD3 ζ chain signaling domain in a single fusion molecule, independent of HLA-mediated antigen presentation.+ and CD8 + "Second generation" CARs add intracellular signaling domains from various costimulatory molecules (e.g., CD28, 4-1BB, ICOS, OX40, CD27, CD40 / My88, and NKGD2) to the cytoplasmic tail of the CAR to provide additional signals to T cells. "Second generation" CARs include CARs that provide both costimulation (e.g., CD28 or 4-1BB) and activation (CD3ζ). "Third generation" CARs include CARs that provide multiple costimulation (e.g., CD28 and 4-1BB) and activation (CD3ζ). In certain embodiments, the CAR is a second generation CAR. In certain embodiments, the CAR comprises an extracellular antigen binding domain that binds to an antigen, a transmembrane domain, and an intracellular signaling domain, and the intracellular signaling domain comprises a costimulatory signaling domain. In certain embodiments, the CAR further comprises a hinge / spacer region.

[0109] 5.2.2.1. Extracellular Antigen-Binding Domain of the CAR The extracellular antigen-binding domain of the CAR specifically binds to mesothelin, for example human mesothelin. In certain embodiments, the extracellular antigen-binding domain is an scFv. In certain embodiments, the scFv is a human scFv. In certain embodiments, the scFv is a humanized scFv. In certain embodiments, the scFv is a mouse scFv. In certain embodiments, the extracellular antigen-binding domain of the CAR is an optionally cross-linked Fab. In certain embodiments, the extracellular antigen-binding domain of the CAR is an F(ab) 2 In certain embodiments, any of the above molecules are included in a fusion protein with a heterologous sequence to form the extracellular antigen-binding domain. In certain embodiments, the scFv is identified by screening an scFv phage library with an antigen-Fc fusion protein. The scFv is a human V L and / or V HThe scFv can also be replaced by camelid heavy chains (e.g. VHH from camel, llama, etc.) or partial natural ligands of cell surface receptors.

[0110] Mesothelin is an immunogenic cell surface antigen highly expressed in solid cancers. Mesothelin is involved in cell proliferation, adhesion, invasion, cell signaling, and metastasis. Studies have demonstrated that serum soluble mesothelin-related peptides secreted by mesothelin-expressing tumors can be measured in both humans and mice, and they have been shown to correlate with treatment response and prognosis. In normal tissues, mesothelin is expressed at low levels only in the pleura, pericardium, and peritoneum. Anti-mesothelin recombinant immunotoxin SS1P shows in vivo specificity and significant antitumor activity in patients. In clinical trials of pancreatic cancer vaccines, patients with a survival advantage showed consistent CD8 expression against mesothelin, which is associated with vaccine-induced delayed-type hypersensitivity responses. + The T cell response was observed in mesothelin-derived human tumors. Specific T cell epitopes derived from mesothelin were shown to activate human T cells to efficiently lyse mesothelin-expressing human tumors. Thus, there is strong evidence that adoptive immunotherapy targeting mesothelin can target mesothelin-expressing tumors.

[0111] In certain embodiments, the CAR binds to human mesothelin. In certain embodiments, the human mesothelin comprises or consists of an amino acid sequence having NCBI reference number: AAV87530.1 (SEQ ID NO: 75), or a fragment thereof.

[0112] SEQ ID NO:75 is provided below: [ka]

[0113] In certain embodiments, the extracellular antigen-binding domain of the CAR (e.g., embodied in an scFv or analog thereof) 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 the CAR has an EC50 value of about 20 nM as measured by ELISA. In certain embodiments, the extracellular antigen-binding domain of the CAR comprises an anti-mesothelin antibody or antigen-binding portion thereof described in U.S. Pat. No. 8,357,783, which is incorporated herein by reference in its entirety. In certain embodiments, the extracellular antigen-binding domain of the CAR is derived from an antibody that binds to human mesothelin, such as the heavy and light chain variable regions of antibody m912 disclosed in Feng et al., Mol. Cancer Therapy (2009);8(5):1113-1118, which is incorporated herein by reference in its entirety. Antibody m912 was isolated from a human Fab library by panning against recombinant mesothelin. In certain embodiments, the extracellular antigen-binding domain of the CAR is derived from a Fab' (e.g., from a human or mouse Fab library).

[0114] The binding of the extracellular antigen-binding domain of the CAR (in embodiments, e.g., scFv or analog thereof) can be confirmed, for example, by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growth inhibition), or Western blot assay. Each of these assays generally detects the presence of a particular protein-antibody complex of interest by using a labeling reagent (e.g., antibody or scFv) specific for the complex of interest. For example, scFv can be radioactively labeled and used in a radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986, which is incorporated herein by reference). Radioactive isotopes can be detected by means such as the use of a gamma counter or scintillation counter, or autoradiography. In certain embodiments, the mesothelin-targeting extracellular antigen-binding domain is labeled with a fluorescent marker. Non-limiting examples of fluorescent markers include green fluorescent protein (GFP), blue fluorescent protein (e.g., EBFP, EBFP2, Azurite, and mKalama1), cyan fluorescent protein (e.g., ECFP, Cerulean, and CyPet), and yellow fluorescent protein (e.g., YFP, Citrine, Venus, and YPet). In certain embodiments, the mesothelin-targeting human scFv is labeled with GFP.

[0115] In certain embodiments, the extracellular antigen binding domain of the CAR binds to human mesothelin at about 1,000 or more mesothelin binding sites / cell mesothelin levels. In certain embodiments, the extracellular antigen binding domain of the CAR binds to human mesothelin at about 1,000 to about 50,000 mesothelin binding sites / cell mesothelin levels. In certain embodiments, the extracellular antigen binding domain of the CAR does not bind to human mesothelin at less than 1,000 mesothelin binding sites / cell mesothelin expression levels, 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 the CAR does not bind to human mesothelin at more than 50,000 mesothelin binding sites / cell mesothelin expression levels. In certain embodiments, the human scFV contained in the CAR binds to human mesothelin at about 1,000 to about 50,000 mesothelin binding sites / cellular mesothelin expression levels. In certain embodiments, the human scFV contained in the CAR does not bind to human mesothelin at more than 50,000 or less than 1,000 mesothelin binding sites / cellular mesothelin expression levels.

[0116] In certain embodiments, the extracellular antigen binding domain of the CAR (e.g., scFv) comprises a heavy chain variable region (VCH) comprising a CDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 76 or a conservative modification thereof, a CDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 77 or a conservative modification thereof, and a CDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 78 or a conservative modification thereof. H In certain embodiments, V H comprises a CDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 76, a CDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 77, and a CDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 78.

[0117] In certain embodiments, the extracellular antigen binding domain of the CAR (e.g., scFv) comprises a light chain variable region (VCH) comprising a CDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 79 or a conservative modification thereof, a CDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 80 or a conservative modification thereof, and a CDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 81 or a conservative modification thereof. L In certain embodiments, V L comprises a CDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:79, a CDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:80, and a CDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:81.

[0118] In certain embodiments, V H comprises a CDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 76 or a conservative modification thereof, a CDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 77 or a conservative modification thereof, and a CDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 78 or a conservative modification thereof; and V L comprises a CDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 79 or a conservative modification thereof, a CDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 80 or a conservative modification thereof, and a CDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 81 or a conservative modification thereof. H comprises a CDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 76, a CDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 77, and a CDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 78, and L comprises a CDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 79, a CDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 80, and a CDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 81. In certain embodiments, the CDRs are identified according to the Kabat numbering system.

[0119] In certain embodiments, the heavy chain variable region (V H ) comprises the amino acid sequence set forth in SEQ ID NO: 82. In certain embodiments, the light chain variable region (V L ) comprises the amino acid sequence set forth in SEQ ID NO: 83. In certain embodiments, V H comprises the amino acid sequence set forth in SEQ ID NO: 82, L comprises the amino acid sequence set forth in SEQ ID NO: 83, and optionally (iii) V H and V L In certain embodiments, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 66. H comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO: 82. H comprises an amino acid sequence that is 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% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 82. H comprises the amino acid sequence set forth in SEQ ID NO: 82. L comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO: 83. For example, V L comprises an amino acid sequence that is 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% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 83. Lcomprises the amino acid sequence set forth in SEQ ID NO: 83. In certain embodiments, V H comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:82, L comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO: 83. H comprises the amino acid sequence set forth in SEQ ID NO: 82, L comprises the amino acid sequence set forth in SEQ ID NO:83.

[0120] An exemplary nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO:82 is set forth in SEQ ID NO:52.

[0121] An exemplary nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO:83 is set forth in SEQ ID NO:53.

[0122] 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 set forth in SEQ ID NO: 84. In certain embodiments, the extracellular antigen binding domain of the CAR (e.g., scFv) comprises or consists of the amino acid sequence set forth 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 set forth in SEQ ID NO: 75).

[0123] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO:84 is set forth in SEQ ID NO:85.

[0124] In certain embodiments, the scFv is a human scFv.

[0125] SEQ ID NOs: 52, 53, and 76-85 are provided below: [ka] [ka]

[0126] 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 set forth in SEQ ID NO:36, provided below. [ka]

[0127] An exemplary nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO:36 is set forth in SEQ ID NO:37, provided below. [ka]

[0128] 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 set forth in SEQ ID NO:38, provided below. [ka]

[0129] An exemplary nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO:38 is set forth in SEQ ID NO:39, provided below. [ka]

[0130] 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 set forth in SEQ ID NO:40, provided below. [ka]

[0131] 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 set forth in SEQ ID NO:41, provided below. [ka]

[0132] 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.

[0133] 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 set forth in SEQ ID NO:42, provided below. [ka]

[0134] An exemplary nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO:42 is set forth in SEQ ID NO:45, provided below. [ka] [ka]

[0135] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 42 is set forth below in SEQ ID NO: 46. The nucleic acid sequence set forth in SEQ ID NO: 46 is synthetically optimized with respect to codon usage, which can increase expression of the CAR. [ka]

[0136] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 42 is set forth below in SEQ ID NO: 47. The nucleic acid sequence set forth in SEQ ID NO: 47 is synthetically optimized with respect to codon usage, which can increase expression of the CAR. [ka]

[0137] A V 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 sequence identical to a particular 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 contain substitutions (e.g., conservative substitutions), insertions, or deletions compared to a designated sequence, but may retain the ability to bind to a target antigen (e.g., mesothelin). In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in a particular 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 (e.g., FRs) of the extracellular antigen-binding domain. In certain embodiments, the extracellular antigen-binding domain comprises a V-terminal amino acid sequence selected from the group consisting of SEQ ID NOs: 82 and 83, including post-translational modifications of the sequences (SEQ ID NOs: 82 and 83). H and / or V L Contains arrays.

[0138] 5.2.2.2.Transmembrane domain of CAR In certain embodiments, the CAR comprises a transmembrane domain. In certain embodiments, the transmembrane domain of the CAR comprises a hydrophobic alpha helix that spans at least a portion of the membrane. Different transmembrane domains result in different receptor stabilities. After antigen recognition, the receptors form clusters and transmit signals to the cell. In accordance with the subject matter of the present disclosure, the transmembrane domain of the CAR can comprise a natural or modified transmembrane domain of a CD8 polypeptide, a CD28 polypeptide, a CD3ζ 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, an NKGD2 peptide, a synthetic polypeptide (not based on a protein associated with an immune response), or a combination thereof.

[0139] CD8 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 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 a sequence having NCBI reference number: NP_001139345.1 (SEQ ID NO: 86), or a fragment thereof, and / or may optionally include up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, the CD8 polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 86 that is 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 up to about 235 amino acids in length. In certain embodiments, a CD8 polypeptide comprises or consists of the amino acid sequence of amino acids 1-235, 1-50, 50-100, 100-150, 150-200, 137-209, or 200-235 of SEQ ID NO: 86. In certain embodiments, a 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 137-209 of SEQ ID NO: 86. In certain embodiments, the transmembrane domain of the CAR comprises a CD8 polypeptide comprising or consisting of amino acids 137-207 of SEQ ID NO: 86. [ka]

[0140] 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 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 a sequence having NCBI reference number: AAA92533.1 (SEQ ID NO: 87), or a fragment thereof, and / or may optionally contain up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, the CD8 polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 87 that is 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 up to 247 amino acids in length. In certain embodiments, a CD8 polypeptide comprises or consists of an amino acid sequence of amino acids 1-247, 1-50, 50-100, 100-150, 150-200, 151-219, or 200-247 of SEQ ID NO: 87. In certain embodiments, the transmembrane domain of a CAR comprises a CD8 polypeptide comprising or consisting of amino acids 151-219 of SEQ ID NO:87. [ka]

[0141] In certain embodiments, the CD8 polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO:88, provided below: [ka]

[0142] An exemplary nucleotide sequence that encodes the amino acid sequence of SEQ ID NO:88 is set forth in SEQ ID NO:89, provided below. [ka]

[0143] CD28 In certain embodiments, the transmembrane domain of the 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 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 a sequence having NCBI reference number: NP_006130 (SEQ ID NO: 90), or a fragment thereof, and / or may optionally include 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 that is a contiguous portion of SEQ ID NO: 90 that 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 220 amino acids in length. In certain embodiments, the CD28 polypeptide comprises or consists of the 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 the amino acid sequence of 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 the 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 set forth in SEQ ID NO: 91. SEQ ID NOs: 90 to 93 are provided below: [ka] [ka]

[0144] 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 a sequence having NCBI reference number: NP_031668.3 (SEQ ID NO: 97), or a fragment thereof, and / or may optionally contain 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 that is a contiguous portion of SEQ ID NO: 97 that is at least about 20, or at least about 30, or 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 the amino acid sequence of amino acids 1-218, 1-50, 50-100, 100-150, 114-220, 150-200, 151-177, or 200-218 of SEQ ID NO: 97. In certain embodiments, the transmembrane domain of the CAR comprises a CD28 polypeptide comprising or consisting of amino acids 151-177 of SEQ ID NO:97.

[0145] SEQ ID NO:97 is provided below: [ka]

[0146] CD84 In certain embodiments, the transmembrane domain of the CAR comprises a native 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% homologous or identical to a sequence having NCBI reference number: NP_001171808.1 (SEQ ID NO: 1), or a fragment thereof, and / or may optionally include up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, the CD84 polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 1 that is at least about 20, or at least about 30, or at least about 40, or at least about 50, and up to about 345 amino acids in length. In certain embodiments, the CD84 polypeptide comprises or consists of the amino acid sequence of amino acids 1-345, 1-50, 50-100, 100-150, 150-200, 226-250, 250-300, or 300-345 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 226-250 of SEQ ID NO:1.

[0147] SEQ ID NO:1 is provided below: [ka]

[0148] An exemplary nucleotide sequence encoding amino acids 226-250 of SEQ ID NO:1 is set forth in SEQ ID NO:2, provided below. [ka]

[0149] CD166 In certain embodiments, the transmembrane domain of the CAR comprises a native or modified transmembrane domain of a CD166 polypeptide or a portion thereof. The CD166 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 a sequence having NCBI reference number: NP_001618.2 (SEQ ID NO: 3), or a fragment thereof, and / or may optionally include up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, the CD166 polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 3 that 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 length. In certain embodiments, the CD166 polypeptide comprises or consists of the amino acid sequence of amino acids 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 of SEQ ID NO: 3. In certain embodiments, the CD166 polypeptide comprised in the transmembrane domain of a CAR of the present disclosure comprises or consists of the amino acid sequence of amino acids 528 to 553 of SEQ ID NO: 3. In certain embodiments, the CD166 polypeptide comprised in the transmembrane domain of a CAR comprises or consists of the amino acid sequence of amino acids 528 to 549 of SEQ ID NO: 3.

[0150] SEQ ID NO:3 is provided below: [ka]

[0151] An exemplary nucleotide sequence encoding amino acids 528-553 of SEQ ID NO:3 is set forth in SEQ ID NO:4, provided below. [ka]

[0152] CD8a In certain embodiments, the transmembrane domain of the CAR comprises a native or modified transmembrane domain of a CD8a polypeptide or a portion thereof. The CD8a 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 a sequence consisting of NCBI Reference Number: NP_001139345.1 (SEQ ID NO: 5), or a fragment thereof, and / or may optionally contain up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, the CD8a polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 5 that is at least about 20, or at least about 30, or at least about 40, or at least about 50, and up to about 235 amino acids in length. In certain embodiments, the CD8a polypeptide comprises or consists of the amino acid sequence of amino acids 1-235, 1-50, 50-100, 100-150, 183-207, 150-200, or 200-235 of SEQ ID NO: 5. In certain embodiments, the transmembrane domain of the CAR comprises a CD8a polypeptide comprising or consisting of amino acids 183-207 of SEQ ID NO: 5. SEQ ID NO: 5 is provided below: [ka]

[0153] An exemplary nucleotide sequence encoding amino acids 183-207 of SEQ ID NO:5 is set forth in SEQ ID NO:6, provided below. [ka]

[0154] CD8b In certain embodiments, the transmembrane domain of the CAR comprises a native or modified transmembrane domain of a CD8b polypeptide or a portion thereof. The CD8b 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 a sequence having NCBI reference number: NP_742099.1 (SEQ ID NO: 7), or a fragment thereof, and / or may optionally include up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, the CD8b polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 7 that is at least about 20, or at least about 30, or at least about 40, or at least about 50, and up to about 221 amino acids in length. In certain embodiments, the CD8b polypeptide comprises or consists of the amino acid sequence of amino acids 1-221, 1-50, 50-100, 100-150, 171-195, 150-200, or 200-221 of SEQ ID NO: 7. In certain embodiments, the transmembrane domain of the CAR comprises a CD8b polypeptide comprising or consisting of amino acids 171-195 of SEQ ID NO: 7. SEQ ID NO: 7 is provided below: [ka]

[0155] An exemplary nucleotide sequence encoding amino acids 171-195 of SEQ ID NO:7 is set forth in SEQ ID NO:8, provided below. [ka]

[0156] ICOS In certain embodiments, the transmembrane domain of CAR comprises a natural or modified transmembrane domain of ICOS polypeptide or a portion thereof. 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% homologous or identical to a sequence having NCBI reference number: NP_036224.1 (SEQ ID NO: 9) or a fragment thereof, and / or may optionally include up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, ICOS polypeptide comprises or consists of an amino acid sequence that is a continuous portion of SEQ ID NO: 9 that is at least about 20, 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 1-199, 1-50, 50-100, 100-150, 141-165, or 150-199 of SEQ ID NO: 9. In certain embodiments, the transmembrane domain of the CAR comprises an ICOS polypeptide comprising or consisting of amino acids 141-165 of SEQ ID NO: 9. SEQ ID NO: 9 is provided below: [ka]

[0157] An exemplary nucleotide sequence encoding amino acids 141-165 of SEQ ID NO:9 is set forth in SEQ ID NO:10, provided below. [ka]

[0158] CTLA-4 In certain embodiments, the transmembrane domain of the CAR comprises a natural 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% homologous or identical to the sequence having NCBI reference number: NP_005205.2 (SEQ ID NO: 11), or a fragment thereof, and / or may optionally include up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, the CTLA-4 polypeptide comprises or consists of an amino acid sequence that is a continuous portion of SEQ ID NO: 11 that 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 the amino acid sequence of amino acids 1-223, 1-50, 50-100, 100-150, 162-186, 150-200, or 200-223 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 162-186 of SEQ ID NO: 11. SEQ ID NO: 11 is provided below: [ka]

[0159] An exemplary nucleotide sequence encoding amino acids 162-186 of SEQ ID NO:11 is set forth in SEQ ID NO:12, provided below. [ka]

[0160] ICAM-1 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 a sequence having NCBI reference number: NP_000192.2 (SEQ ID NO: 13) or a fragment thereof, and / or may optionally include up to one, or up to two, or up to 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 that 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 the amino acid sequence of amino acids 1-532, 1-50, 50-100, 100-150, 150-200, 200-300, 300-400, 481-507, 400-500, or 500-532 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 481-507 of SEQ ID NO: 13. SEQ ID NO: 13 is provided below: [ka]

[0161] An exemplary nucleotide sequence encoding amino acids 481-507 of SEQ ID NO:13 is set forth in SEQ ID NO:14, provided below. [ka]

[0162] Hinge / spacer region of CAR In certain embodiments, the CAR comprises a hinge / spacer region that links the extracellular antigen-binding domain to the transmembrane domain. The hinge / spacer region may be sufficiently flexible to allow the antigen-binding domain to face different directions to facilitate antigen recognition. In certain embodiments, the hinge / spacer region of the CAR may comprise a natural or modified hinge region of a CD8 polypeptide, a CD28 polypeptide, a CD3ζ 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, an NKGD2 peptide, a synthetic polypeptide (not based on a protein related to immune response), or a combination thereof. The hinge / spacer region may be a hinge region of IgG1, or a CH of an immunoglobulin. 2 CH 3 The spacer sequence may be a region, and a portion of CD3, 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), or a variation of any of the foregoing that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% homologous or identical thereto, or a synthetic spacer sequence.

[0163] CD28 In certain embodiments, the hinge / spacer region of the CAR comprises a native or modified hinge region or portion thereof of a CD28 polypeptide described herein. In certain embodiments, the hinge / spacer region of the CAR comprises a CD28 polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 15 (or amino acids 114-152 of SEQ ID NO: 90). SEQ ID NO: 15 is provided below. [ka]

[0164] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO:15 (or amino acids 114-152 of SEQ ID NO:90) is set forth in SEQ ID NO:54, provided below. [ka]

[0165] CD84 In certain embodiments, the hinge / spacer region of the CAR comprises a native or modified hinge region or portion thereof of a CD84 polypeptide described herein. In certain embodiments, the hinge / spacer region of the CAR comprises a CD84 polypeptide comprising or consisting of amino acids 187-225 of SEQ ID NO: 1. An exemplary nucleotide sequence encoding amino acids 187-225 of SEQ ID NO: 1 is set forth in SEQ ID NO: 16, provided below. [ka]

[0166] CD166 In certain embodiments, the hinge / spacer region of the CAR comprises a native or modified hinge region or portion thereof of a CD166 polypeptide described herein. In certain embodiments, the hinge / spacer region of the CAR comprises a CD166 polypeptide comprising or consisting of amino acids 489-527 of SEQ ID NO: 3. An exemplary nucleotide sequence encoding amino acids 489-527 of SEQ ID NO: 3 is set forth in SEQ ID NO: 17, provided below. [ka]

[0167] In certain embodiments, the hinge / spacer region of the CAR comprises a CD166 polypeptide comprising or consisting of amino acids 484-527 of SEQ ID NO:3. In certain embodiments, the hinge / spacer region of the CAR comprises a CD166 polypeptide comprising or consisting of amino acids 506-527 of SEQ ID NO:3. In certain embodiments, the hinge / spacer region of the CAR comprises a CD166 polypeptide comprising or consisting of amino acids 517-527 of SEQ ID NO:3. In certain embodiments, the hinge / spacer region of the CAR comprises a CD166 polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO:109 or SEQ ID NO:110. SEQ ID NOs:109 and 110 are provided below. [ka]

[0168] In certain embodiments, the CD166 polypeptide contained in the hinge / spacer region and transmembrane domain of the CAR comprises or consists of the amino acid sequence set forth 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 NOs: 111-117 are provided below. [ka] [ka]

[0169] CD8a In certain embodiments, the hinge / spacer region of the CAR comprises a native or modified hinge region or portion thereof of a CD8a polypeptide described herein. In certain embodiments, the hinge / spacer region of the CAR comprises a CD8a polypeptide comprising or consisting of amino acids 137-182 of SEQ ID NO: 5. An exemplary nucleotide sequence encoding amino acids 137-182 of SEQ ID NO: 5 is set forth in SEQ ID NO: 18, provided below. [ka]

[0170] CD8b In certain embodiments, the hinge / spacer region of the CAR comprises a native or modified hinge region of a CD8b polypeptide described herein. In certain embodiments, the CD8b polypeptide comprised in the hinge / spacer region of the CAR comprises or consists of amino acids 132-170 of SEQ ID NO:7. An exemplary nucleotide sequence encoding amino acids 132-170 of SEQ ID NO:7 is set forth in SEQ ID NO:19, provided below. [ka]

[0171] ICOS In certain embodiments, the hinge / spacer region of the CAR comprises a native or modified hinge region or portion thereof of an ICOS polypeptide described herein. In certain embodiments, the hinge / spacer region of the CAR comprises an ICOS polypeptide comprising or consisting of amino acids 102-140 of SEQ ID NO: 9. An exemplary nucleotide sequence encoding amino acids 102-140 of SEQ ID NO: 9 is set forth in SEQ ID NO: 20, provided below. [ka]

[0172] CTLA-4 In certain embodiments, the hinge / spacer region of the CAR comprises a native or modified hinge region or portion thereof of a CTLA-4 polypeptide described herein. In certain embodiments, the hinge / spacer region of the CAR comprises a CTLA-4 polypeptide comprising or consisting of amino acids 123-161 of SEQ ID NO: 11. An exemplary nucleotide sequence encoding amino acids 123-161 of SEQ ID NO: 11 is set forth in SEQ ID NO: 21, provided below. [ka]

[0173] ICAM-1 In certain embodiments, the hinge / spacer region of the CAR comprises a native or modified hinge region or portion thereof of an ICAM-1 polypeptide described herein. In certain embodiments, the hinge / spacer region of the CAR comprises an ICAM-1 polypeptide comprising or consisting of amino acids 442-480 of SEQ ID NO: 13. An exemplary nucleotide sequence encoding amino acids 442-480 of SEQ ID NO: 13 is set forth in SEQ ID NO: 22, provided below. [ka]

[0174] In certain embodiments, the mesothelin-targeting CAR comprises a hinge / spacer region. In certain embodiments, the hinge / spacer region is located between the extracellular antigen-binding domain and the transmembrane domain. In certain embodiments, the hinge / spacer region 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 related to immune response), or a combination thereof. In certain embodiments, the transmembrane domain comprises a CD8 polypeptide, a CD28 polypeptide, a CD3zeta 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.

[0175] In certain embodiments, the transmembrane domain and the hinge / spacer region are derived from the same molecule. In certain embodiments, the transmembrane domain and the hinge / spacer region are derived from different molecules. In certain embodiments, the hinge / spacer region of the CAR comprises a CD28 polypeptide, and the transmembrane domain of the CAR comprises a CD28 polypeptide. In certain embodiments, the hinge / spacer region of the CAR comprises a CD28 polypeptide, and the transmembrane domain of the CAR comprises a CD28 polypeptide. In certain embodiments, the hinge / spacer region of the CAR comprises a CD84 polypeptide, and the transmembrane domain of the CAR comprises a CD84 polypeptide. In certain embodiments, the hinge / spacer region of the CAR comprises a CD166 polypeptide, and the transmembrane domain of the CAR comprises a CD166 polypeptide. In certain embodiments, the hinge / spacer region of the CAR comprises a CD8a polypeptide, and the transmembrane domain of the CAR comprises a CD8a polypeptide. In certain embodiments, the hinge / spacer region of the CAR comprises a CD8b polypeptide, and the transmembrane domain of the CAR comprises a CD8b polypeptide. In certain embodiments, the hinge / spacer region of the CAR comprises a CD28 polypeptide and the transmembrane domain of the CAR comprises an ICOS polypeptide.

[0176] 5.2.2.4. CAR Intracellular Signaling Domain A.CD3ζ In certain embodiments, the CAR comprises an intracellular signaling domain. In certain embodiments, the intracellular signaling domain of the CAR comprises a CD3ζ polypeptide, which can activate or stimulate cells (e.g., lymphoid lineage cells, e.g., T cells). Wild-type ("natural") CD3ζ comprises three immunoreceptor tyrosine-based activation motifs ("ITAMs") (e.g., ITAM1, ITAM2, and ITAM3), three base-rich stretch (BRS) regions (BRS1, BRS2, ​​and BRS3), and sends activation signals to cells (e.g., lymphoid lineage cells, e.g., T cells) after antigen binding. The intracellular signaling domain of the natural CD3ζ chain is the primary transmitter of endogenous TCR signals.

[0177] In certain embodiments, the intracellular signaling domain of the CAR comprises a native CD3ζ polypeptide. In certain embodiments, the native 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%, at least about 100% homologous or identical to the sequence having NCBI reference number: NP_932170 (SEQ ID NO: 94) or a fragment thereof. In certain embodiments, the native CD3ζ polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 94 that is 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 up to about 164 amino acids in length. In certain embodiments, a native CD3 ζ 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, a native CD3 ζ polypeptide comprises or consists of the amino acid sequence of amino acids 52 to 164 of SEQ ID NO: 94.

[0178] SEQ ID NO:94 is provided below: [ka]

[0179] 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%, at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO:95, or a fragment thereof, and / or may optionally include up to one, or up to two, or up to three conservative amino acid substitutions. SEQ ID NO:95 is provided below: [ka]

[0180] An exemplary nucleotide sequence that encodes the amino acid sequence of SEQ ID NO:95 is set forth in SEQ ID NO:96, provided below. [ka]

[0181] In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ polypeptide. In certain embodiments, the intracellular signaling domain of the CAR comprises a modified human CD3ζ polypeptide. In certain embodiments, the modified 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%, at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 35, or a fragment thereof, and / or may optionally include up to one, or up to two, or up to three conservative amino acid substitutions. SEQ ID NO: 35 is provided below: [ka] [ka]

[0182] An exemplary nucleotide sequence that encodes the amino acid sequence of SEQ ID NO:35 is set forth in SEQ ID NO:55, provided below. [ka]

[0183] In certain embodiments, the modified CD3ζ polypeptide comprises one, two or three ITAM variants. In certain embodiments, the modified CD3ζ polypeptide comprises a native ITAM1. In certain embodiments, the native ITAM1 comprises or consists of the amino acid sequence set forth in SEQ ID NO:23. [ka]

[0184] An exemplary nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO:23 is set forth in SEQ ID NO:24, provided below. [ka]

[0185] 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 the 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 set forth in SEQ ID NO: 25, provided below. [ka]

[0186] An exemplary nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO:25 is set forth in SEQ ID NO:26, which is provided below. [ka]

[0187] In certain embodiments, the modified CD3ζ polypeptide comprises a native ITAM2. In certain embodiments, the native ITAM2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 27, provided below. [ka]

[0188] An exemplary nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO:27 is set forth in SEQ ID NO:28, provided below. [ka]

[0189] 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 of 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 set forth in SEQ ID NO: 29, as provided below. [ka]

[0190] An exemplary nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO:29 is set forth in SEQ ID NO:30, provided below. [ka]

[0191] 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 set forth in SEQ ID NO:31, provided below. [ka]

[0192] An exemplary nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO:31 is set forth in SEQ ID NO:32, provided below. [ka]

[0193] 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 of a tyrosine residue of 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 set forth in SEQ ID NO: 33, as provided below. [ka]

[0194] An exemplary nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO:33 is set forth in SEQ ID NO:34, provided below. [ka]

[0195] In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ 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.

[0196] In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ 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 CD3ζ 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 CD3ζ polypeptide comprising a native ITAM1 consisting of the amino acid sequence set forth in SEQ ID NO:23, an ITAM2 variant consisting of the amino acid sequence set forth in SEQ ID NO:29, and an ITAM3 variant consisting of the amino acid sequence set forth in SEQ ID NO:33 (e.g., a construct designated "1XX"). In certain embodiments, the modified CD3ζ polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO:35.

[0197] In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ 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 CD3ζ 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 CD3ζ polypeptide comprising an ITAM1 variant consisting of the amino acid sequence set forth in SEQ ID NO:25, 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 "X2X").

[0198] In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ 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 an modified CD3ζ polypeptide comprising an ITAM1 variant comprising or consisting of two loss-of-function mutations, 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 an modified CD3ζ polypeptide comprising an ITAM1 variant consisting of the amino acid sequence set forth in SEQ ID NO:25, an ITAM2 variant consisting of the amino acid sequence set forth in SEQ ID NO:29, and a native ITAM3 consisting of the amino acid sequence set forth in SEQ ID NO:31 (e.g., a construct designated "XX3").

[0199] In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ 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 CD3ζ 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 CD3ζ polypeptide comprising an ITAM1 variant consisting of the amino acid sequence set forth in SEQ ID NO:25, a native ITAM2 consisting of the amino acid sequence set forth in SEQ ID NO:27, and a native ITAM3 consisting of the amino acid sequence set forth in SEQ ID NO:31 (e.g., a construct designated "X23").

[0200] In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ 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 CD3ζ 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 CD3ζ 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 "12X").

[0201] In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ 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 CD3ζ 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 CD3ζ polypeptide comprising a native ITAM1 consisting of the amino acid sequence set forth in SEQ ID NO:23, an ITAM2 variant consisting of the amino acid sequence set forth in SEQ ID NO:29, and a native ITAM3 variant consisting of the amino acid sequence set forth in SEQ ID NO:31 (e.g., a construct designated "1X3").

[0202] In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ polypeptide comprising one or two ITAM deletions. In certain embodiments, the modified CD3ζ polypeptide comprises or consists of deletions of ITAM1 and ITAM2, for example, the modified CD3ζ polypeptide comprises a natural ITAM3 or an ITAM3 variant, and does not comprise ITAM1 or ITAM2. In certain embodiments, the modified CD3ζ polypeptide comprises a natural ITAM3 consisting of the amino acid sequence set forth in SEQ ID NO:31, and does not comprise ITAM1 (natural or modified) or ITAM2 (natural or modified) (e.g., the construct designated "D12").

[0203] In certain embodiments, the modified CD3ζ polypeptide comprises or consists of a deletion of ITAM2 and ITAM3, e.g., the modified CD3ζ polypeptide comprises a native ITAM1 or ITAM1 variant, and does not comprise ITAM2 or ITAM3. In certain embodiments, the modified CD3ζ polypeptide comprises a 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., the construct designated "D23").

[0204] In certain embodiments, the modified CD3ζ polypeptide comprises or consists of a deletion of ITAM1 and ITAM3, e.g., the modified CD3ζ polypeptide comprises a native ITAM2 or ITAM2 variant, and does not comprise ITAM1 or ITAM3. In certain embodiments, the modified CD3ζ polypeptide comprises a 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").

[0205] In certain embodiments, the modified CD3ζ polypeptide comprises or consists of a deletion of ITAM1, for example, the modified CD3ζ polypeptide comprises a native ITAM2 or ITAM2 variant, and a native ITAM3 or ITAM3 variant, and does not comprise ITAM1 (native or modified).

[0206] In certain embodiments, the modified CD3ζ polypeptide comprises or consists of a deletion of ITAM2, for example, the modified CD3ζ polypeptide comprises a native ITAM1 or ITAM1 variant, and a native ITAM3 or ITAM3 variant, and does not comprise ITAM2 (native or modified).

[0207] In certain embodiments, the modified CD3ζ polypeptide comprises or consists of a deletion of ITAM3, for example, the modified CD3ζ polypeptide comprises a native ITAM1 or ITAM1 variant, and a native ITAM2 or ITAM2 variant, and does not comprise ITAM3 (native or modified).

[0208] B. Costimulatory Signaling Regions In certain embodiments, the intracellular signaling domain of the CAR further comprises at least a costimulatory signaling region. In certain embodiments, the costimulatory signaling region comprises at least a portion of a costimulatory molecule, which can provide optimal lymphocyte activation.

[0209] As used herein, "costimulatory molecule" refers to a cell surface molecule other than an antigen receptor or its ligand, which is necessary for an efficient response of lymphocytes to antigen. Non-limiting examples of costimulatory molecules include CD28, 4-1BB, OX40, ICOS, DAP-10, CD27, CD40, and NKGD2. A costimulatory molecule can bind to a costimulatory ligand, which is a protein expressed on the cell surface that, when bound to its receptor, produces a costimulatory response, i.e., an intracellular response that stimulates only if the antigen binds to its CAR molecule. Costimulatory ligands include, but are not limited to, CD80, CD86, CD70, OX40L, and 4-1BBL. As an example, 4-1BB ligand (i.e., 4-1BBL) is a CAR signaling ligand that, in combination with the CAR signaling, stimulates the CAR molecule. + It can bind to 4-1BB (also known as "CD137") to provide an intracellular signal that induces effector cell function of T cells. CARs that contain intracellular signaling domains that include costimulatory signaling regions that include 4-1BB, ICOS, or DAP-10 are disclosed in U.S. Patent No. 7,446,190, the entirety of which is incorporated herein by reference.

[0210] In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region that comprises a CD28 polypeptide (e.g., the intracellular domain of CD28 or a portion thereof). In certain embodiments, the costimulatory signaling region comprises the intracellular domain of human CD28 or a portion thereof. In certain embodiments, the costimulatory signaling region comprises a CD28 polypeptide that comprises or consists of amino acids 180-220 of SEQ ID NO:90.

[0211] In certain embodiments, a costimulatory signaling region comprises a CD28 polypeptide comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 101 (or amino acids 180-220 of SEQ ID NO: 90). SEQ ID NO: 101 is provided below. [ka]

[0212] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO:101 (or amino acids 180-220 of SEQ ID NO:90) is set forth in SEQ ID NO:102, provided below. [ka]

[0213] In certain embodiments, a costimulatory signaling region comprises a CD28 polypeptide comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 108 (or amino acids 180-219 of SEQ ID NO: 90). SEQ ID NO: 108 is provided below. [ka]

[0214] 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 178-218 of SEQ ID NO:97.

[0215] An exemplary nucleotide sequence encoding amino acids 178-218 of SEQ ID NO:97 is set forth in SEQ ID NO:98, provided below. [ka]

[0216] In certain embodiments, the costimulatory signaling region comprises or consists of a CD28 polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 99. SEQ ID NO: 99 is provided below: [ka] .

[0217] An exemplary nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO:99 is set forth in SEQ ID NO:100, provided below. [ka]

[0218] In certain embodiments, the costimulatory signaling region comprises a portion of a first costimulatory molecule and a portion of a second costimulatory molecule, for example, the intracellular domain of CD28 and the intracellular domain of 4-1BB or the intracellular domain of CD28 and the intracellular domain of OX40.

[0219] In certain embodiments, the costimulatory signaling region comprises a 4-1BB polypeptide (e.g., the intracellular domain of 4-1BB or a portion thereof). In certain embodiments, the costimulatory signaling region comprises the intracellular domain of human 4-1BB or a portion thereof. 4-1BB can act as a tumor necrosis factor (TNF) ligand and can have stimulatory activity. In certain embodiments, the 4-1BB 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 a sequence having NCBI reference number: NP_001552.2 (SEQ ID NO: 103) or a fragment thereof, and / or may optionally include up to one, or up to two, or up to 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 that 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 255 amino acids in length. In certain embodiments, the 4-1BB polypeptide comprises or consists of an amino acid sequence of amino acids 1-255, 1-50, 50-100, 100-150, 150-200, 214-255, or 200-255 of SEQ ID NO: 103. In certain embodiments, the costimulatory signaling region comprises a 4-1BB polypeptide that comprises or consists of SEQ ID NO: 104 (or amino acids 214-255 of SEQ ID NO: 103). SEQ ID NOs: 103 and 104 are provided below: [ka] .

[0220] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO:104 (or amino acids 214-255 of SEQ ID NO:103) is set forth in SEQ ID NO:105, provided below. [ka]

[0221] 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 a sequence having NCBI reference number: NP_003318.1 (SEQ ID NO: 106) or a fragment thereof, and / or may optionally comprise up to one, or up to two, or up to 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 that 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 277 amino acids in length. In certain embodiments, the OX40 polypeptide comprises or consists of the amino acid sequence of amino acids 1-277, 1-50, 50-100, 100-150, 150-200, or 200-277 of SEQ ID NO: 106. SEQ ID NO: 106 is provided below. [ka]

[0222] 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 an intracellular domain of human ICOS 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 a sequence having NCBI reference number: NP_036224 (SEQ ID NO: 65), or a fragment thereof, and / or may optionally include up to one, or 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 that 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 1-199, 1-50, 50-100, 100-150, or 150-199 of SEQ ID NO: 65. SEQ ID NO: 65 is provided below. [ka]

[0223] In certain embodiments, the mesothelin-targeting CAR of the present disclosure further comprises an inducible promoter to express the nucleic acid sequence in human cells. The promoter used to express the CAR gene can be a constitutive promoter, such as the ubiquitin C (UbiC) promoter.

[0224] In certain embodiments, the mutation sites and / or junctions between domains / motifs / regions of CARs derived from different proteins are deimmunized. The immunogenicity of the junctions between different CAR parts can be predicted using the NetMHC 4.0 server. For each peptide containing at least one amino acid from the following parts, the binding affinity to HLA A, B, and C for all alleles can be predicted. A score of the immunogenicity of each peptide can be assigned to each peptide. The immunogenicity score is calculated according to the following formula: immunogenicity score = [(50 - binding affinity) * HLA frequency] n where n is the expected number of each peptide.

[0225] 5.2.2.5. Illustrated CAR In certain embodiments, a mesothelin-targeting CAR is: (a) a V comprising a CDR1 having the amino acid sequence set forth in SEQ ID NO: 76, a CDR2 having the amino acid sequence set forth in SEQ ID NO: 77, and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 78; H and a V comprising a CDR1 consisting of the amino acid sequence set forth in SEQ ID NO:79, a CDR2 consisting of the amino acid sequence set forth in SEQ ID NO:80, and a CDR3 consisting of the amino acid sequence set forth in SEQ ID NO:81. L an extracellular antigen-binding domain comprising; (b) a transmembrane domain containing CD28 polypeptide (e.g., the transmembrane domain of human CD28 or a portion thereof); (c) a CD28 hinge / spacer region (e.g., the hinge / spacer region of human CD28 or a portion thereof); and (d) (i) a modified CD3ζ polypeptide (e.g., a modified human CD3ζ polypeptide) comprising a native ITAM1, an ITAM2 variant comprising two loss-of-function mutations, and an ITAM3 variant comprising two loss-of-function mutations; and (ii) an intracellular signaling domain comprising a costimulatory signaling region comprising a CD28 polypeptide (e.g., a human CD28 polypeptide, e.g., the intracellular domain of human CD28 or a portion thereof). Includes.

[0226] In certain embodiments, the transmembrane domain comprises a CD28 polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 92 (or amino acids 153-179 of SEQ ID NO: 90).

[0227] 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).

[0228] In certain embodiments, the modified CD3ζ polypeptide consists of the amino acid sequence set forth in SEQ ID NO:35.

[0229] In certain embodiments, the costimulatory signaling region comprises a CD28 polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 101 (or amino acids 180-220 of SEQ ID NO: 90).

[0230] In certain embodiments, a 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%, at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 56. In certain embodiments, a CAR comprises the amino acid sequence set forth in SEQ ID NO: 56. SEQ ID NO: 56 is provided below. [ka]

[0231] An exemplary nucleotide sequence that encodes the amino acid sequence of SEQ ID NO:56 is set forth in SEQ ID NO:57, provided below. [ka] [ka]

[0232] In certain embodiments, the CAR further comprises a CD8 leader. In certain embodiments, the CD8 leader comprises or consists of the amino acid sequence set forth in SEQ ID NO:71.

[0233] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO:71 is set forth in SEQ ID NO:120, provided below. [ka]

[0234] 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%, at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 43, provided below. In certain embodiments, the CAR comprises or consists of the amino acid sequence set forth in SEQ ID NO: 43. SEQ ID NO: 43 comprises a CD8 leader consisting of the amino acid sequence set forth in SEQ ID NO: 71. SEQ ID NO: 43 is provided below: [ka]

[0235] An exemplary nucleotide sequence that encodes the amino acid sequence of SEQ ID NO:43 is set forth in SEQ ID NO:44, provided below. [ka] [ka]

[0236] 5.2.3. EXEMPLARY POLYPEPTIDE COMPOSITIONS In certain embodiments, the polypeptide composition comprises a mesothelin-targeting CAR comprising, or consisting of, the amino acid sequence set forth in SEQ ID NO:56, and a PD-1 DN comprising, or consisting of, amino acids 21-165 of SEQ ID NO:48.

[0237] In certain embodiments, the polypeptide composition comprises a mesothelin-targeting CAR comprising, or consisting of, the amino acid sequence set forth in SEQ ID NO:56, and a PD-1 DN comprising, or consisting of, amino acids 1-165 of SEQ ID NO:48.

[0238] In certain embodiments, the polypeptide composition comprises a mesothelin-targeting CAR comprising, or consisting of, the amino acid sequence set forth in SEQ ID NO:56, and a PD-1 DN comprising, or consisting of, the amino acid sequence set forth in SEQ ID NO:49.

[0239] In certain embodiments, the polypeptide composition comprises a mesothelin-targeting CAR comprising, or consisting of, the amino acid sequence set forth in SEQ ID NO:56, and a PD-1 DN comprising, or consisting of, the amino acid sequence set forth in SEQ ID NO:118.

[0240] In certain embodiments, the polypeptide composition comprises a mesothelin-targeting CAR comprising, or consisting of, the amino acid sequence set forth in SEQ ID NO:56, and a CD8 leader comprising, or consisting of, the amino acid sequence set forth in SEQ ID NO:71, and a PD-1 DN comprising, or consisting of, amino acids 21-165 of SEQ ID NO:48.

[0241] In certain embodiments, the polypeptide composition comprises a mesothelin-targeting CAR comprising, or consisting of, the amino acid sequence set forth in SEQ ID NO:56, and a CD8 leader comprising, or consisting of, the amino acid sequence set forth in SEQ ID NO:71, and a PD-1 DN comprising, or consisting of, amino acids 1-165 of SEQ ID NO:48.

[0242] In certain embodiments, the polypeptide composition comprises a mesothelin-targeting CAR comprising, or consisting of, the amino acid sequence set forth in SEQ ID NO:56, and a CD8 leader comprising, or consisting of, the amino acid sequence set forth in SEQ ID NO:71, and a PD-1 DN comprising, or consisting of, the amino acid sequence set forth in SEQ ID NO:49.

[0243] In certain embodiments, the polypeptide composition comprises a mesothelin-targeting CAR comprising, or consisting of, the amino acid sequence set forth in SEQ ID NO:56, and a CD8 leader comprising, or consisting of, the amino acid sequence set forth in SEQ ID NO:71, and a PD-1 DN comprising, or consisting of, the amino acid sequence set forth in SEQ ID NO:118.

[0244] 5.3. Immune response cells The subject matter of the present disclosure provides an immune response cell comprising the polypeptide composition disclosed herein.In certain embodiments, the CAR can activate the immune response cell.In certain embodiments, the polypeptide composition can promote the anti-tumor effect of the immune response cell.The polypeptide composition can be transduced into the immune response cell so that the cell co-expresses the CAR and PD-1 DN.

[0245] The immune response cells of the subject matter of the present disclosure can be cells of the lymphoid lineage. The lymphoid lineage, including B, T, and natural killer (NK) cells, provide antibody production, regulation of the cell-mediated immune system, detection of foreign substances in the blood, detection of cells foreign to the host, and the like. Non-limiting examples of immune response cells of the lymphoid lineage include T cells, natural killer (NK) cells, embryonic stem cells, and pluripotent stem cells (e.g., cells from which cells of the lymphoid lineage can differentiate). T cells mature in the thymus and can be lymphocytes that are primarily responsible for cell-mediated immunity. T cells participate in the adaptive immune system. The T cells of the subject matter of the present disclosure can be helper T cells, cytotoxic T cells, memory T cells (central memory T cells, stem cell-like memory T cells (or stem cell-like memory T cells), and two types of effector memory T cells: e.g., T EM Cells and T EMRAThe immune response cells may be any type of T cell, including, but not limited to, CD4 T cells, regulatory T cells (also known as suppressor T cells), natural killer T cells, mucosal-associated invariant T cells, and γδ T cells. Cytotoxic T cells (CTLs or killer T cells) are a subset of T lymphocytes capable of inducing the death of infected somatic or tumor cells. The patient's own T cells may be genetically modified to target specific antigens through the introduction of antigen recognition receptors, such as CARs or TCRs. In certain embodiments, the immune response cells are T cells. T cells are CD4 T cells. + T cells or CD8 + In certain embodiments, the T cells are CD4 + In certain embodiments, the T cells are CD8 + T cells.

[0246] Natural killer (NK) cells can be lymphocytes that are part of the cell-mediated immunity and act during the innate immune response. NK cells do not require prior activation to exert their cytotoxic effect on target cells.

[0247] Types of human lymphocytes of the presently disclosed subject matter include peripheral blood donor lymphocytes, e.g., Sadelain, M., et al. 2003 Nat Rev Cancer 3:35-45 (disclosing peripheral blood donor lymphocytes genetically modified to express CARs), Morgan, RA, et al. 2006 Science 314:126-129 (disclosing peripheral blood donor lymphocytes genetically modified to express full-length tumor antigen-recognizing T cell receptor complexes containing 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 antigen-specific peripheral blood leukocytes selectively expanded in vitro using artificial antigen presenting cells (AAPC) or pulsed dendritic cells). Immune response cells (e.g., T cells) can be autologous, non-autologous (e.g., allogeneic), or derived from in vitro engineered progenitor or stem cells.

[0248] In certain embodiments, an immune response cell of the disclosure comprises a mesothelin-targeting CAR comprising, or consisting of, the amino acid sequence set forth in SEQ ID NO:56, and a PD-1 DN comprising, or consisting of, amino acids 1-165 of SEQ ID NO:48.

[0249] In certain embodiments, the immune response cells of the present disclosure comprising one or more of the 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 can involve isolating PBMCs from an EBV-seropositive autologous or allogeneic donor and enriching them for T cells by depletion of monocytes and NK cells. EBV-sensitized cytotoxic T cells can also be produced by contacting donor PBMCs or purified donor T cells with "stimulator" cells that express one or more EBV antigens and present EBV antigens to unstimulated T cells, thereby resulting in the stimulation and expansion of EBV-sensitized CTLs. In particular, in certain embodiments, such methods involve the use of CD3 + obtaining a cell (e.g., PBMC) sample from a subject comprising said CD3 + In certain embodiments, the method further comprises contacting the cell with an antigen and / or an antigen-presenting stimulatory cell. + After isolation of T cells from a sample, the cells are contacted with an antigen by methods known in the art (e.g., CD3 +Positive selection of cells and / or negative selection by depleting undesirable cells or components from the sample). In certain embodiments, such methods include selection using fluorescence-activated cell sorting (FACS) with anti-CD3 beads (e.g., magnetic beads), plastic adherence, depletion of NK cells using anti-CD56, elutriation, and / or a combination thereof. EBV antigens include, for example, latent membrane proteins (LMPs) and EBV nuclear antigen (EBNA) proteins, such as LMP-1, LMP-2A, and LMP-2B, as well as EBNA-1, EBNA-2, EBNA-3A, EBNA-3B, EBNA-3C, and EBNA-LP. Cytotoxic T cells that contain a T cell receptor that recognizes one or more EBV-specific antigens are considered to be "sensitized" to those EBV antigens and are therefore referred to herein as "EBV-sensitized cytotoxic T cells." Known methods for generating allogeneic or autologous EBV-specific cytotoxic T cell populations that may comprise one or more of the 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 for their teachings. Similarly, cytotoxic T cells can be "sensitized" to other viral antigens, including cytomegalovirus (CMV), papillomavirus (e.g., HPV), adenovirus, polyomavirus (e.g., BKV, JCV, and Merkel cell virus), retrovirus (including lentiviruses such as HTLV-I, HIV), picornavirus (e.g., Hepatitis A virus), hepadnavirus (e.g., Hepatitis B virus), hepacivirus (e.g., Hepatitis C virus), deltavirus (e.g., Hepatitis D virus), hepevirus (e.g., Hepatitis E virus), and the like. In certain embodiments, the target antigen is derived from an oncogenic virus.In certain embodiments, the T cells used to generate the CAR-T cells of the present disclosure are multifunctional T cells, e.g., T cells capable of inducing multiple immune effector functions that provide a more effective immune response against pathogens than cells that only produce a single immune effector (e.g., a single biomarker such as a cytokine or CD107a). Less multifunctional, monofunctional, or even "exhausted" T cells may dominate the immune response during chronic infections, thus negatively impacting protection against 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 are less than about 50% CD4 + In certain embodiments, the T cells are primarily CD4 + T cells.

[0250] 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 certain embodiments, the T cells are primarily CD8 + T cells. In certain embodiments, T cells (e.g., primed T cells and / or CAR-T cells as described herein) are stored in a cell library or bank and then administered to a subject.

[0251] The immune response cell of the present disclosure may further comprise at least one exogenous costimulatory ligand, so that the immune response cell is co-expressed or induced to co-express the mesothelin-specific CAR and at least one exogenous costimulatory ligand. The interaction between the mesothelin-specific CAR and at least one costimulatory ligand provides a non-antigen-specific signal that is important for the complete activation of the immune response cell (e.g., T cell). Costimulatory ligands include, but are not limited to, members of the tumor necrosis factor (TNF) superfamily and members of the immunoglobulin (Ig) superfamily ligands. TNF is a cytokine involved in systemic inflammation and stimulates the acute phase response. Its main role is to regulate immune cells. TNF superfamily members share several common features. The majority of TNF superfamily members are synthesized as type II transmembrane proteins (extracellular C-terminus) that contain a short cytoplasmic segment and a relatively long extracellular region. TNF superfamily members 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 beta (TNFβ) / lymphotoxin-alpha (LTα), lymphotoxin-beta (LTβ), CD257 / B cell activating factor (BAFF) / Blys / THANK / Tall-1, glucocorticoid-induced TNF receptor ligand (GITRL), and TNF-related apoptosis-inducing ligand (TRAIL), LIGHT (TNFSF14). The immunoglobulin (Ig) superfamily is a large group of cell surface and soluble proteins involved in cellular recognition, binding, or adhesion processes. These proteins share structural features with immunoglobulins, that is, they have immunoglobulin domains (folds). Immunoglobulin superfamily ligands include, but are not limited to, CD80 and CD86, both of which are ligands for CD28, and PD-L1 / (B7-H1), which is a ligand for PD-1.

[0252] In certain embodiments, the at least one costimulatory ligand is selected from the group consisting of 4-1BBL, CD80, CD86, CD70, OX40L, CD48, TNFRSF14, PD-L1, and combinations thereof. In certain embodiments, the costimulatory ligand is 4-1BBL. 4-1BBL can be covalently linked to the 5' end of the extracellular antigen binding domain of the mesothelin-targeting CAR. Alternatively, 4-1BBL can be covalently linked to the 3' end of the intracellular signaling domain of the mesothelin-targeting CAR.

[0253] Furthermore, the immune response cells of the present disclosure may further comprise at least one exogenous cytokine, such that the immune response cells co-express or are induced to co-express the mesothelin-targeted CAR and at least one exogenous cytokine. In certain embodiments, the at least one exogenous cytokine is selected from the group consisting of IL-2, IL-3, IL-6, IL-7, IL-11, IL-12, IL-15, IL-17, and IL-21. In certain embodiments, the at least one exogenous cytokine comprises IL-12. In certain embodiments, the immune response cells co-express the mesothelin-targeted CAR and exogenous IL-12. The IL-12 can be covalently bound to the 3' end of the intracellular signaling domain of the mesothelin-targeted CAR.

[0254] In addition, the immune response cell can express a second CAR that binds to a second antigen, which is mesothelin or an antigen other than mesothelin. CARs that can be used as a second CAR in combination with the mesothelin-specific CAR of the presently disclosed subject matter include those CARs 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 U.S. Pat. No. 7,446,190, which are incorporated by reference herein in their entireties, such as CD19-targeted CARs (see U.S. Pat. No. 7,446,190; U.S. Patent Application Publication No. 2013 / 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 (see, e.g., Zhong, et al., Molecular Therapy, 18(2):413-420 (2010), all of which are incorporated herein by reference in their entirety. Immune response cells expressing two or more antigen recognition receptors (e.g., CARs) are described in WO2014 / 055668, which is incorporated herein by reference in its entirety.

[0255] 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 tumor antigen includes, but is not limited to, cancer protein. The second antigen can be expressed as a peptide or as an intact protein or a part thereof. The intact protein or a part 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 is 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, an antigen of a cytomegalovirus (CMV) infected cell (e.g., a cell surface antigen), 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), death acetylcholine receptor (AChR), folate receptor-a, ganglioside G2 (GD2), ganglioside G3 (GD3), human epidermal growth factor receptor 2 (HER -2), human telomerase reverse transcriptase (hTERT), interleukin-13 receptor subunit alpha-2 (IL-13Rα2), kappa-light chain, kinase inactivation domain receptor (KDR), LewisA (CA19.9), LewisY (LeY), L1 cell adhesion molecule (LlCAM), melanoma antigen family A1 (MAGE-AI), mucin 16 (Muc-16), mucin 1 (Muc-1), NKG2D ligand, cancer-testis antigen NY-ESO-1, carcinoembryonic 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), Wilms tumor protein (WT-1), tyrosine protein kinase transmembrane receptor type 1 (ROR1), or a combination thereof.

[0256] Suitable pathogen antigens for use in the treatment of pathogen infections or other infectious diseases, for example in immunocompromised subjects, include, but are not limited to, viral antigens present in cytomegalovirus (CMV), Epstein-Barr virus (EBV), human immunodeficiency virus (HIV), and influenza virus. Immune response cells comprising a second CAR that targets viral antigens can be used to treat viral diseases. In certain embodiments, mesothelin-targeting CAR and a second CAR that binds to CMV antigens can be co-expressed in immune response cells (e.g., cytotoxic T lymphocytes) and used to treat CMV.

[0257] Mesothelin-specific or mesothelin-targeted human lymphocytes that can be used in the methods of the presently disclosed subject matter include peripheral blood donor lymphocytes, such as those described in Sadelain, M., et al. 2003 Nat Rev Cancer 3:35-45 (disclosing peripheral blood donor lymphocytes genetically modified to express CARs), Morgan, RA, et al. 2006 Science 314:126-129 (disclosing peripheral blood donor lymphocytes genetically modified to express a full-length tumor antigen-recognizing T cell receptor complex containing 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 antigen-specific peripheral blood leukocytes selectively expanded in vitro using artificial antigen presenting cells (AAPC) or pulsed dendritic cells). Immune response cells (e.g., T cells) can be autologous, non-autologous (e.g., allogeneic), or derived from in vitro engineered progenitor or stem cells.

[0258] The assay may be used to compare the effect of costimulatory signaling on the proliferation, effector function, and enhanced accumulation of mesothelin-targeted CAR-transduced T cells with repeated (weekly) antigen stimulation. Peripheral blood lymphocytes (PBLs) may be collected from healthy volunteers and transduced according to an IRB-approved protocol. Gene transfer efficiency was monitored by FACS analysis to detect GFP. +The (transduced) T cell fraction may be quantified and / or monitored by quantitative PCR. Using well-established co-culture systems (Gade, TP, et al. Cancer Res. 65, 9080-9088 (2005); Gong, MC, et al. Neoplasia. 1, 123-127 (1999); Latouche, JB & Sadelain, M. Nat.Biotechnol. 18, 405-409 (2000)), it may be determined whether mesothelin-expressing fibroblast AAPC (compared to mesothelin controls) direct cytokine release from transduced T cells (cell supernatant LUMINEX assays for 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 effect of CD80 and / or 4-1BBL on T cell survival, proliferation, and efficacy may be assessed. T cells may be stimulated with mesothelin + (MSLN + ) may be exposed to repeated stimulation with target cells to determine whether T cell proliferation and cytokine responses remain similar or are impaired by repeated stimulation. Cytotoxicity assays with multiple E:T ratios may be performed using chromium release assays. Statistical analysis may be performed by two-way ANNOVA followed by paired multiple comparison procedures as appropriate, in which case data may be expressed as mean ± SEM. CD4 and CD8 T cell subtypes (activated effector, central memory, effector memory) may be identified to determine which conditions favor the maintenance or expansion of the central memory phenotype.

[0259] In certain embodiments, 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 per cell of a mesothelin-targeted CAR. For example, immune response cells (e.g., T cells) of the present disclosure express about 1, about 2, about 3, or about 4 vector copies per cell of a mesothelin-targeted CAR. In certain embodiments, immune response cells (e.g., T cells) of the present disclosure express about 3 to about 4 vector copies per cell of a mesothelin-targeted CAR. In certain embodiments, the cytotoxicity and cytokine production of immune response cells (e.g., T cells) are proportional to the expression level of a mesothelin-specific CAR in the cell. For example, the higher the expression level of CAR in an immune response cell, the higher 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 antigen-specific cytokine production or secretion and / or exhibit cytotoxicity against tissues or cells with low levels of mesothelin expression, e.g., about 2,000 or less, about 1,000 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 / cells. Additionally or alternatively, the cytotoxicity and cytokine production of immune response cells (e.g., T cells) of the present disclosure is proportional to the expression level of human mesothelin in the target tissue or cell. For example, the higher the expression level of human mesothelin in the target, the higher the cytotoxicity and cytokine production of the immune response cells.

[0260] In certain embodiments, the target cell is a heterogeneous MSLN-expressing cell, which is a cell population that includes MSLN low-expressing cells and MSLN high-expressing cells.In the presence of MSLN high-expressing cells, the immune response cells of the present disclosure can show increased cytotoxicity and antitumor activity against MSLN low-expressing cells (e.g., about 2,000 or less, about 1,000 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 MSLN high-expressing cells, the immune response cells do not show increased cytotoxicity or non-specific killing against MSLN negative cells. Thus, immune response cells can exhibit increased cytotoxicity and antitumor activity against MSLN low expressing cells in the presence of MSLN high expressing cells, but retain safety against MSLN negative cells.

[0261] In certain embodiments, immune response cells can express one or more adhesion molecules that can increase the avidity of MSLN-specific CAR, especially when the 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).

[0262] Non-purified CTL origins, such as bone marrow, fetal, neonatal, or adult origins, or other hematopoietic cell origins, such as fetal liver, peripheral blood, or umbilical cord blood, can be known in the art.Various techniques can be used to separate cells.For example, negative selection methods can first remove non-CTL.mAbs are particularly useful for identifying markers associated with specific cell lineages and / or differentiation stages for both positive and negative selection.

[0263] First, the majority of terminally differentiated cells can be removed by a relatively crude separation. For example, magnetic bead separation can be used first to remove many unrelated cells. In certain embodiments, at least about 80%, usually at least 70%, of the total hematopoietic cells are removed prior to cell isolation.

[0264] Separation procedures include, but are not limited to, density gradient centrifugation; resedimentation; coupling to particles that modify cell density; magnetic separation with antibody-coated magnetic beads; affinity chromatography; cytotoxic agents bound to or used in conjunction with the mAb, including, but not limited to, complement and cytotoxins; and panning with antibodies bound to a solid matrix, e.g., a plate, chip, elutriation, or any other convenient technique.

[0265] Techniques for separation and analysis include, but are not limited to, flow cytometry, which can have various degrees of sophistication, such as multiple color channels, small and obtuse angle light scatter detection channels, impedance channels.

[0266] Cells can be selected for dead cells by using a dye that associates with dead cells, such as propidium iodide (PI). In certain embodiments, cells are harvested in medium containing 2% fetal calf serum (FCS) or 0.2% bovine serum albumin (BSA), or any other suitable, e.g., sterile, isotonic medium.

[0267] 5.4. Nucleic Acid Compositions and Vectors The subject matter of the present disclosure provides nucleic acid compositions encoding the polypeptide compositions disclosed herein (e.g., as disclosed in Section 5.2). In certain embodiments, the nucleic acid compositions comprise polynucleotides encoding the polypeptide compositions disclosed herein (e.g., as disclosed in Section 5.2). Also provided are vectors comprising such nucleic acid compositions and cells comprising such nucleic acid compositions or vectors.

[0268] In certain embodiments, the nucleic acid composition further comprises a promoter operably linked to the polypeptide composition.In certain embodiments, the promoter is endogenous or exogenous.In certain embodiments, the exogenous promoter is selected from the elongation factor (EF)-1 promoter, the CMV promoter, the SV40 promoter, the PGK promoter, and the metallothionein promoter.In certain embodiments, the promoter is an inducible promoter.In certain embodiments, the inducible promoter is selected from the NFAT transcription response element (TRE) promoter, the CD69 promoter, the CD25 promoter, and the IL-2 promoter.

[0269] The nucleic acid compositions can be administered to a subject and / or delivered to a cell by methods known in the art or described herein.

[0270] Genetic modification of immune response cells (e.g., T cells or NK cells) can be achieved by transducing recombinant DNA constructs into substantially homogeneous cell compositions. In certain embodiments, retroviral vectors (gammaretroviruses or lentiviruses) are used to introduce DNA constructs into cells. For example, a polynucleotide encoding an antigen-recognizing receptor can be cloned into a retroviral vector, and expression can be driven from its endogenous promoter, from a retroviral long terminal repeat sequence, or from a promoter specific to the target cell type of interest. Non-viral vectors can also be used.

[0271] When immune response cells are first genetically modified to contain an antigen-recognizing receptor (e.g., CAR or TCR), a retroviral vector is typically used for transduction, although any other suitable viral vector or non-viral delivery system can be used. The CAR and PD-1 DN can be constructed in a single polycistronic expression cassette, in multiple expression cassettes in a single vector, or in multiple vectors. Exemplary elements for creating polycistronic expression cassettes include, but are not limited to, various viral and non-viral internal ribosome entry sites (IRES, e.g., FGF-1 IRES, FGF-2 IRES, VEGF IRES, IGF-II IRES, NF-κB IRES, RUNX1 IRES, p53 IRES, Hepatitis A IRES, Hepatitis C IRES, Pestivirus IRES, Aphthovirus 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, provided below: [ka]

[0272] In certain embodiments, the P2A peptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 121, provided below: [ka]

[0273] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO:121 is set forth in SEQ ID NO:122, provided below: [ka]

[0274] If the capsid protein is functional to infect human cells, the combination of retroviral vector with a suitable packaging cell line is also suitable.Various amphotropic virus producing 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 particles, such as those pseudotyped with VSVG, RD114, or GALV envelopes, and any other particles known in the art, are also suitable.

[0275] Possible transduction methods also include direct co-culture of the cells with producer cells, e.g., by the method of Bregni, et al. (1992) Blood 80:1418-1422, or culture with viral supernatant alone or concentrated vector stocks in the presence or absence of appropriate growth factors and polycations, e.g., by the method of Xu, et al. (1994) Exp. Hemat. 22:223-230; and Hughes, et al. (1992) J. Clin. Invest. 89:1817.

[0276] 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, for example, Cayouette et al., Human Gene Therapy 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; and Miyoshi et al., Proc. Natl. Acad. Sci. USA 94:10319, 1997).Other viral vectors that can be used include, for example, adenovirus, lentivirus, and adeno-associated viral vectors, vaccinia virus, bovine papilloma virus, or herpes viruses, such as Epstein-Barr virus (see, 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; Moen, 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 have been particularly well developed and have been used in clinical settings (Rosenberg et al., N. Engl. J. Med 323:370, 1990; Anderson et al., U.S. Patent No. 5,399,346).

[0277] In certain embodiments, the vector encoding the polypeptide composition of the present disclosure is a retroviral vector, such as a Moloney murine leukemia-based retroviral vector, SGFγ-retroviral vector. In certain embodiments, the vector comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 123, provided below: [ka] [ka] [ka]

[0278] In certain embodiments, the vector comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 124, provided below: [ka] [ka] [ka]

[0279] Non-viral approaches can also be used for the genetic modification of immune response cells. For example, lipofection (Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413, 1987; Ono et al., Neuroscience Letters 17:259, 1990; Brigham et al., Am. J. Med. Sci. 298:278, 1989; Staubinger et al., Methods in Enzymology 101:512, 1983), asialoorosomucoid-polylysine conjugation (Wu et al., Journal of Biological Chemistry 263:14621, 1988; Wu et al., Journal of Biological Chemistry 264:16985, 1989), or in the surgical setting by microinjection (Wolff et al., Science 247:1465, Nucleic acid molecules can be introduced into immune response cells by administering the nucleic acid via the ELISA kit (1990). Other non-viral means for gene transfer include in vitro transfection using calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. Liposomes may possibly be beneficial for delivering DNA to cells. Transplanting normal genes into diseased tissues of subjects can also be achieved by transferring normal nucleic acids ex vivo into culturable cell types (e.g., autologous or heterologous primary cells or their progeny) and then injecting the cells (or their progeny) into target tissues or systemically. Recombinant receptors can also be induced or obtained using transposases or targeted nucleases (e.g., zinc finger nucleases, meganucleases, or TALE nucleases, CRISPR). Transient expression can be obtained by RNA electroporation.

[0280] 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 nuclease is used to express the polypeptide composition disclosed herein.In certain embodiments, the TALEN system is used to express the polypeptide composition disclosed herein.

[0281] 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 its guide), CRISPR RNA (crRNA, containing the RNA used by Cas9 to guide it to a precise section of the host DNA along with a region that binds to tracrRNA (usually in the form of a hairpin loop) that forms an active complex with Cas9), transactivating crRNA (tracrRNA, which binds to crRNA to form an active complex with Cas9), and an optional section of DNA repair template (DNA that guides the cell repair process to allow the insertion of a specific DNA sequence). CRISPR / Cas9 is often transfected into target cells using a plasmid. Since the crRNA is the sequence that Cas9 uses to identify and directly bind to the target DNA in the cell, the crRNA must be designed for each application. The repair template with the CAR expression cassette must also be designed for each application, since it must overlap the sequences on either side of the cut and code for the insertion sequence. Multiple crRNAs and tracrRNAs can be packaged together to form a single guide RNA (sgRNA), which can be combined with a Cas9 gene and made into a plasmid to be transfected into cells.

[0282] Zinc finger nucleases (ZFNs) are artificial restriction enzymes that are generated by combining a zinc finger DNA binding domain with a DNA cleavage domain. The zinc finger domain can be engineered to target specific DNA sequences that allow the zinc finger nuclease to target a desired sequence in the genome. The DNA binding domain of an individual ZFN typically contains multiple individual zinc finger repeats, each capable of recognizing multiple base pairs. The most common method for generating novel zinc finger domains is to combine smaller zinc finger "modules" of known specificity. The most common cleavage domain in ZFNs is the non-specific cleavage domain from the type II restriction endonuclease FokI. Using the endogenous homologous recombination (HR) machinery and a homologous DNA template bearing the CAR expression cassette, ZFNs can be used to insert a CAR expression cassette into the genome. Once the targeting sequence is cleaved by the ZFN, the HR machinery searches for homology between the damaged chromosome and the homologous DNA template, and then copies the sequence of the template between the two cut ends of the chromosome, thereby integrating the homologous DNA template into the genome.

[0283] Transcription activator-like effector nucleases (TALENs) are restriction enzymes that can be engineered to cleave specific sequences in DNA. TALEN systems operate on roughly the same principle as ZFNs. They are generated by combining a transcription activator-like effector DNA binding domain with a DNA cleavage domain. Transcription activator-like effectors (TALEs) are composed of a repeating motif of 33-34 amino acids with two variable positions that strongly recognize specific nucleotides. By assembling an array of these TALEs, the TALE DNA binding domain can be engineered to bind to a desired DNA sequence, thereby guiding the nuclease to cleave at a specific location in the genome. cDNA expression for use in polynucleotide therapy can be directed 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, the expression of nucleic acid can be directed using enhancers known to preferentially direct gene expression in specific cell types.The enhancers used include, but are not limited to, those characterized as tissue- or cell-specific enhancers.Alternatively, when genomic clones are used as therapeutic constructs, regulation can be mediated by homologous regulatory sequences, including any of the promoters or regulatory elements described above, or by regulatory sequences from heterologous sources if desired.

[0284] The method of delivery of genome editing agent / system can vary according to need.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 vector.Common delivery methods include but are not limited to electroporation, microinjection, gene gun, impalefection, hydrostatic pressure, continuous infusion, sonication, magnetofection, adeno-associated virus, envelope protein pseudotyping of viral vector, replication-competent vector cis and trans-acting elements, herpes simplex virus, chemical vehicle (e.g., oligonucleotide, lipoplex, polymersome, polyplex, dendrimer, inorganic nanoparticle, and cell-penetrating peptide).

[0285] 5.5. Polypeptides and Analogs The subject matter of the present disclosure also includes polypeptides disclosed herein (such as mesothelin, CD28, CD8, CD3ζ, and PD-1 DN) or fragments thereof that have been modified to enhance their antineoplastic activity when expressed in immune response cells. The subject matter of the present disclosure provides methods for optimizing amino acid or nucleic acid sequences by making alterations to the sequence. Such alterations may include certain mutations, deletions, insertions, or post-translational modifications. The subject matter of the present disclosure further includes analogs of any of the polypeptides disclosed herein (including, but not limited to, mesothelin, CD28, CD8, CD3ζ, and PD-1 DN). Analogs differ from the polypeptides disclosed herein by differences in amino acid sequence, by post-translational modifications, or both. Analogs exhibit 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 a portion of the amino acid sequence of the subject 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. Again, in an exemplary approach to determining the degree of identity, the BLAST program uses a sequence comparison program to determine the degree of identity of closely related sequences. -3 From e -100A probability score between the . Modifications include in vivo and in vitro chemical derivatization of the polypeptide, such as acetylation, carboxylation, phosphorylation, or glycosylation, which may occur during polypeptide synthesis or processing, or after treatment with an isolated modifying enzyme. Analogs may also differ from the polypeptide by changes in the primary sequence. These include both natural and induced genetic variants (e.g., resulting from random mutagenesis by exposure to radiation or ethanemethylsulfamic acid, or by site-directed mutagenesis 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, such as D-amino acids or non-naturally occurring or synthetic amino acids, such as β- or γ-amino acids.

[0286] In addition to full-length polypeptides, the subject matter of the present disclosure also provides fragments of any one of the polypeptides or peptide domains disclosed herein. As used herein, the term "fragment" means at least 5, 10, 13, or 15 amino acids. In certain embodiments, the fragment comprises at least 20 contiguous amino acids, at least 30 contiguous amino acids, or at least 50 contiguous amino acids. In certain embodiments, the fragment comprises at least 60-80, 100, 200, 300, or more contiguous amino acids. Fragments can be generated by methods known to those of skill in the art or can result from normal protein processing (e.g., removal of amino acids from a nascent polypeptide that are not required for biological activity, or removal of amino acids by alternative mRNA splicing or alternative protein processing events).

[0287] Non-protein analogs have chemical structures designed to mimic the functional activity of the proteins disclosed herein. Such analogs may exceed the biological activity of the original polypeptide. Methods for designing analogs are well known in the art, and the synthesis of analogs can be carried out according to such methods by modifying the chemical structure so that the resulting analog increases the antineoplastic activity of the original polypeptide when expressed in immune response cells. These chemical modifications include, but are not limited to, the substitution of alternative R groups and changing the degree of saturation at certain carbon atoms of the reference polypeptide. In certain embodiments, protein analogs are relatively resistant to in vivo degradation, thereby resulting in a more sustained therapeutic effect upon administration. Assays for measuring functional activity include, but are not limited to, the assays described in the examples below.

[0288] In accordance with the presently disclosed subject matter, human mesothelin (e.g., scFv, Fab, or (Fab) 2 Polynucleotides encoding extracellular antigen-binding domains that specifically bind to CD3ζ, CD8, CD28, 4-1BB, 4-1BBL, and IL-12 can be modified by codon optimization. Codon optimization can modify both naturally occurring and recombinant gene sequences to achieve the highest possible level of productivity in any given expression system. Factors involved in different stages of protein expression include codon compatibility, mRNA structure, and various cis elements in transcription and translation. Any suitable codon optimization method or technique known in the art, including but not limited to OptimumGene™, Encor optimization, and Blue Heron, can be used to modify the polynucleotides of the subject matter of the present disclosure.

[0289] Codon optimization can be performed based on four different algorithms (e.g., Blue Heron and Encore algorithms). The codon-optimized sequences from all four algorithms are blended, and all CPGs and BAM-H1 are removed for optimal cloning. In certain embodiments, the codon-optimized nucleotide sequence is about 70% homologous to the original sequence before codon optimization. To obtain efficient expression in immune response cells (e.g., human primary T cells), the codon-optimized nucleotide sequence is ligated to a polynucleotide encoding a CD8 leader, e.g., SEQ ID NO: 71. The CD8 leader provides optimal signal cleavage before the scFv heavy chain (QVQL). Codon optimization optimizes mesothelin CAR expression in immune response cells, e.g., multiple human donor primary T cells, with good transduction efficiency. Multiple CAR vector copy numbers in multiple donor T cells are tested for functional efficiency, specificity, and sensitivity against multiple hematological and solid cancer cells with various mesothelin expression. Codon-optimized mesothelin-targeting CARs with vector copy numbers of 1-4 (more specifically, about 3-4) provide highly efficient cytotoxicity against high mesothelin-expressing targets, but show minimal reactivity against low mesothelin-expressing targets, i.e., normal tissues. The above genetic manipulations in generating specific mesothelin CARs that are reactive against high mesothelin-expressing cancer cells but have no effect on low mesothelin-expressing normal tissues are optimal for use as vectors in the clinic for cancer therapy while ensuring safety.

[0290] 5.6. Pharmaceutical Compositions and Administration The subject matter of the present disclosure provides compositions (e.g., as disclosed in Section 5.3) comprising the cells of the present disclosure. The amount of cells contained in the composition may vary depending on the intended use of the composition and / or the size, age, sex, weight, and condition of the subject to which the composition is administered. In certain embodiments, the composition comprises about 10 4 ~about 10 10 Between pieces, about 10 4 ~about 10 6 Between pieces, about 10 5 ~about 10 6Between pieces, about 10 5 ~about 10 7 Between pieces, about 10 5 ~about 10 9 Between pieces, or about 10 6 ~about 10 8 In certain embodiments, the composition comprises between about 1×10 5 Pieces, at least about 5 x 10 5 At least about 1 x 10 pieces 6 At least about 1 x 10 pieces 7 At least about 1 x 10 pieces 8 In certain embodiments, the composition comprises about 1×10 immune response cells of the present disclosure. 5 The cell of the present disclosure.

[0291] The composition comprising the immune response cells of the present disclosure can be provided systemically or directly to a subject to induce and / or enhance immune responses to antigens and / or treat and / or prevent neoplasms, pathogen infections or infectious diseases, inflammatory diseases, or transplant rejection. In certain embodiments, the immune response cells of the present disclosure or a composition comprising the same are directly injected into an organ of interest (e.g., an organ affected by a neoplasm). Alternatively, the immune response cells of the present disclosure or a composition comprising the same are provided indirectly to an organ of interest, for example, by administration into the circulatory system (e.g., the vasculature of a tumor). Expansion and differentiation agents can be provided before, during, or after administration of the cells or compositions to increase the production of T cells, NK cells, or CTL cells in vitro or in vivo.

[0292] The immune response cells of the present disclosure can be administered in any physiologically acceptable vehicle, usually intravenously, but they may also be introduced into bone or other convenient sites (e.g., the thymus) where the cells may find a site for appropriate regeneration and differentiation. Typically, at least about 1×10 cells are administered. 5 The final dose is approximately l x l0 10or more. The immune response cells of the present disclosure may comprise a purified cell population. One of skill in the art can easily determine the percentage of immune response cells of the present disclosure in a population using various well-known methods, such as fluorescence-activated cell sorting (FACS). Suitable ranges of purity in a population containing 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, the purity is about 70% to about 75%, about 75% to about 80%, or about 80% to about 85%. In certain embodiments, the purity is about 85% to about 90%, about 90% to about 95%, and about 95% to about 100%. The dosage can be easily adjusted by one of skill in the art (e.g., a decrease in purity may require an increase in dosage). The cells can be introduced by injection, catheter, etc.

[0293] The composition of the present disclosure may be a pharmaceutical composition comprising the immune response cells of the present disclosure or their progenitors and a pharma- ceutically acceptable carrier. Administration may be autologous or xenogeneic. For example, immune response cells or progenitors may be obtained from one subject and administered to the same subject or to a different compatible subject. Peripheral blood derived immune response cells or their progeny (e.g., derived in vivo, ex vivo, or in vitro) may be administered via catheter administration, systemic injection, local injection, intravenous injection, or local injection, including parenteral administration. When administering the therapeutic composition of the subject of the present disclosure (e.g., a pharmaceutical composition comprising the immune response cells of the present disclosure), it may be formulated into an injectable unit dosage form (solution, suspension, emulsion).

[0294] 5.7. Preparations The compositions comprising the immune response cells of the present disclosure can be conveniently provided as a sterile liquid preparation, such as an isotonic aqueous solution, suspension, emulsion, dispersion, or viscous composition, which can be buffered to a selected pH. Liquid preparations are usually easier to prepare than gels, other viscous compositions, and solid compositions. In addition, liquid compositions are somewhat more convenient to administer, especially by injection. On the other hand, viscous compositions can be formulated within an appropriate viscosity range to provide a longer contact period with a particular tissue. Liquid or viscous compositions can include a carrier, which can be a solvent or dispersion medium, including, for example, water, saline, phosphate buffered saline, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), and suitable mixtures thereof.

[0295] Sterile injectable solutions can be prepared by incorporating the genetically modified immune response cells in the required amount of an appropriate solvent with various amounts of other ingredients, if desired. Such compositions can be in admixture with suitable carriers, diluents, or excipients, such as sterile water, saline, glucose, dextrose, and the like. The compositions can also be lyophilized. Depending on the route of administration and the preparation desired, the compositions can contain auxiliary substances, such as wetting agents, dispersing agents, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling agents, or thickening additives, preservatives, flavoring agents, coloring agents, and the like. Standard texts, such as "REMINGTON'S PHARMACEUTICAL SCIENCE", 17th edition, 1985, incorporated herein by reference, may be consulted for preparing suitable preparations without undue experimentation.

[0296] Various additives can be added to enhance the stability and sterility of the composition, including antimicrobial preservatives, antioxidants, chelating agents, and buffers. Prevention of microbial action can be ensured by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. The sustained absorption of injectable pharmaceutical forms can be achieved by the use of agents that delay absorption, such as aluminum monostearate and gelatin. However, according to the subject matter of the present disclosure, any vehicle, diluent, or additive used must be compatible with genetically modified immune response cells or their precursor cells.

[0297] The composition 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 pharma- ceutically acceptable agents, such as dextrose, boric acid, sodium tartrate, propylene glycol, or other inorganic or organic solutes.Sodium chloride can be especially for buffers containing sodium ions.

[0298] The viscosity of the composition can be maintained at a selected level using a pharma- ceutically acceptable thickening agent if desired. For example, methylcellulose is readily available, inexpensive, and easy to handle. Other suitable thickening agents include, for example, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, carbomer, and the like. The concentration of the thickening agent can depend on the drug selected. The important point is to use an amount that achieves the selected viscosity. Obviously, the selection of suitable carriers and other additives depends on the exact route of administration and the nature of a particular dosage form, for example, a liquid dosage form (for example, whether the composition is formulated into a solution, suspension, gel, or another liquid dosage form, such as a sustained release dosage form or a liquid fill dosage form).

[0299] The amount of cells administered will vary with respect to the subject being treated. In certain embodiments, about 10 4 ~about 10 10 Between pieces, about 10 5 ~about 10 9 Between pieces, about 104 ~about 10 6 Between pieces, about 10 5 ~about 10 6 Between pieces, about 10 5 ~about 10 7 Between pieces, or about 10 6 ~about 10 8 Between about 1×10 immune response cells of the present disclosure are administered to a human subject. More effective cells may be administered in smaller numbers. In certain embodiments, at least about 1×10 5 At least about 1 x 10 pieces 6 At least about 1 x 10 pieces 7 pieces, 1×10 8 At least about 2 x 10 pieces 8 At least about 3 x 10 pieces 8 Pieces, at least about 4 x 10 8 pieces, or at least about 5 × 10 8 The immune response cells of the present disclosure are administered to a human subject. The exact amount that will be an effective dose can be determined based on individual factors for each subject, including the size, age, sex, weight, and condition of the particular subject. The dosage can be easily ascertained by those skilled in the art from the present disclosure and knowledge in the art. In certain embodiments, about 1×10 5 The cells of the present disclosure are administered to a subject.

[0300] One of skill in the art can readily determine the amount of cells and optional additives, vehicles, and / or carriers to be administered in the compositions and methods. Typically, any additives (in addition to the active cells and / or agents) are present in an amount of 0.001 to 50% by weight solution in phosphate buffered saline, and the active ingredient is present in the order of micrograms to milligrams, e.g., about 0.0001 to about 5% by weight, about 0.0001 to about 1% by weight, about 0.0001 to about 0.05% by weight, or about 0.001 to about 20% by weight, about 0.01 to about 10% by weight, or about 0.05 to about 5% by weight. For any composition administered to animals or humans, the following can be determined: toxicity, e.g., by determining the lethal dose (LD) and LD50 in a suitable animal model, e.g., a rodent such as a mouse; the dosage of the composition that elicits an appropriate response, the concentration of the components therein, and the time at which the composition is administered. Such determinations do not require undue experimentation from the knowledge of one of ordinary skill in the art, this disclosure, and the documents cited herein, and the time of sequential administration can be ascertained without undue experimentation.

[0301] 5.8. Treatment Method The immune response cells of the subject matter of the present disclosure and compositions comprising same can be used to treat and / or prevent neoplasms, pathogen infections, infectious diseases, inflammatory diseases, or transplant rejection. Such immune response cells can be administered to a subject (e.g., a human subject) in need thereof for the treatment or prevention of 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 cancer, endometrial cancer, stomach cancer, and / or cholangiocarcinoma). In certain embodiments, the immune response cells are T cells. T cells are CD4 + T cells or CD8 + In certain embodiments, the T cells are CD4 + T cells.

[0302] The subject matter of the present disclosure provides a method of inducing and / or increasing an immune response in a subject in need thereof. The immune response cells of the present disclosure and compositions comprising the same can be used to treat and / or prevent a neoplasm in a subject. The immune response cells of the present disclosure and compositions comprising the same can be used to prolong the survival of a subject suffering from a neoplasm. The immune response cells of the present disclosure and compositions comprising the same can also be used to treat and / or prevent a pathogen infection or other infectious disease in a subject, such as an immunocompromised human subject. Such methods include administering an effective amount of the immune response cells of the present disclosure or a composition comprising the same (e.g., a pharmaceutical composition) to achieve a desired effect, whether it is mitigation of an existing condition or prevention of recurrence. For treatment, the amount administered is an amount effective to produce the desired effect. An effective amount can be provided in one or a series of administrations. An effective amount can be provided in a bolus or continuous perfusion.

[0303] An "effective amount" (or "therapeutically effective amount") is an amount sufficient to produce beneficial or desired clinical results upon treatment. An effective amount can be administered to a subject in one or more doses. In terms of treatment, an effective amount is an amount that is sufficient to palliate, improve, stabilize, reverse, or delay the progression of a disease, or otherwise reduce the pathological consequences of a disease. An effective amount is generally determined by a physician on a case-by-case basis and is within the skill of the art. When determining the appropriate dosage to achieve an effective amount, several factors are typically considered. These factors include the age, sex, and weight of the subject, the condition being treated, the severity of the condition, and the dosage form and effective concentration of the immune response cells being administered.

[0304] In adoptive immunotherapy using antigen-specific T cells, approximately 10 6 ~about 10 10 range (e.g., about 10 9 A cell dose of about 1×10 cells is typically injected. However, due to the high efficiency of the polypeptide compositions of the present disclosure, a smaller amount of the cells of the present disclosure is required to achieve the desired effect. For example, about 1×10 cells are injected.5 A single cell of the present disclosure is sufficient to achieve the desired effect.

[0305] The immune response cells are administered to a subject and then differentiated to induce immune response cells that are specific for one particular antigen (e.g., human mesothelin). "Induction" of T cells can include inactivation of antigen-specific T cells, such as by deletion or anergy. Inactivation is particularly useful for establishing or re-establishing tolerance, such as in the case of autoimmune disorders. The immune response cells of the subject matter 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, the immune response cells and / or compositions comprising the same are administered pleurally to a subject in need thereof. In certain embodiments, the immune response cells and / or compositions comprising the same are administered intrapleurally to a subject in need thereof.

[0306] The subject matter of the present disclosure provides various methods of using immune response cells (e.g., T cells). For example, the subject matter of the present disclosure provides a method of reducing tumor burden in a subject. In certain embodiments, the method of reducing tumor burden comprises administering to a subject an effective amount of the immune response cells of the present disclosure or a composition comprising the same. The immune response cells of the present disclosure can reduce the number of tumor cells, reduce tumor size, and / or eradicate tumors in a subject. 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 tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic cancer, endometrial cancer, stomach cancer, and cholangiocarcinoma.

[0307] The subject matter of the present disclosure also provides a method for increasing or prolonging the survival of a subject with a neoplasm. In certain embodiments, the method for increasing or prolonging the survival of a subject with a neoplastic neoplasm comprises administering to the subject an effective amount of an immune response cell of the present disclosure or a composition comprising the same. The method can reduce or eradicate tumor burden in the subject. Thus, the subject matter of the present disclosure provides a method for increasing the immune response in a subject, comprising administering to the subject an immune response cell of the present disclosure or a composition comprising the same. The subject matter of the present disclosure further provides a method for treating and / or preventing a neoplasm in a subject, comprising administering to the subject an immune response cell of the present disclosure or a composition comprising the same.

[0308] In certain embodiments, the neoplasm is a solid tumor. The neoplasm may be a primary tumor or a primary cancer. In addition, the neoplasm may be in a metastatic state.

[0309] Cancers whose growth can be inhibited using the immune response cells of the subject of the present disclosure include cancers that typically respond to immunotherapy.Non-limiting examples of cancers for 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 cancer, endometrial cancer, stomach cancer, bile duct cancer, cervical cancer, and salivary gland cancer.In addition, the subject of the present disclosure includes refractory or recurrent malignant tumors whose growth can be inhibited using the immune response cells of the subject of the present disclosure.

[0310] Examples of other neoplasms or cancers that may 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 (cutaneous or intraocular malignant melanoma), kidney cancer (e.g., clear cell carcinoma), pharyngeal cancer, prostate cancer (e.g., hormone-refractory prostate adenocarcinoma), blood cancer (e.g., leukemia, lymphoma, 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, leukemia (e.g. acute leukemia, acute lymphocytic leukemia, acute myeloid leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma (Hodgkin's lymphoma, non-Hodgkin's lymphoma), small intestine cancer, endocrine system cancer, thyroid cancer , parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) neoplasms, primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers including asbestos-induced cancer, Waldenstrom Ström's macroglobulinemia, heavy chain disease, and solid tumors such as sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovium, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, hepatoma, bile duct (nile) carcinoma, duct cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, salivary gland cancer, uterine cancer, testicular cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, meningioma, melanoma, neuroblastoma, and retinoblastoma).

[0311] In addition, the subject matter of the present disclosure provides a method for increasing immune-activating cytokine production in response to cancer cells or pathogens in a subject. In certain embodiments, the method includes administering to a subject an immune response cell of the present disclosure or a composition comprising the same. The immune-activating 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 certain embodiments, the immune response cell increases the production of GM-CSF, IFN-γ, and / or TNF-α.

[0312] The subject matter of the present disclosure provides a particularly useful therapy for treating solid tumors (e.g., mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic cancer, endometrial cancer, stomach cancer, and cholangiocarcinoma). The solid tumor may be a primary tumor or a tumor in a metastatic state. 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 that include MSLN-low expressing cells and MSLN-high expressing cells. The immune response cells of the present disclosure can exhibit increased cytotoxicity and antitumor activity against MSLN low-expressing cells (e.g., about 2,000 or less, about 1,000 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 MSLN high-expressing cells. In certain embodiments, even in the presence of MSLN high-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 antitumor activity against MSLN low-expressing cells in the presence of MSLN high-expressing cells, but retain safety against MSLN negative cells.

[0313] Furthermore, the subject matter of the present disclosure provides a method for treating a subject with 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 useful for enhancing immune response in immunocompromised subjects. Exemplary viral infections that are susceptible to treatment using the method of the present disclosure include, but are not limited to, cytomegalovirus (CMV), Epstein-Barr virus (EBV), human immunodeficiency virus (HIV), and influenza virus infection. Thus, the subject matter of the present disclosure provides a method for treating or preventing a pathogen infection in a subject, comprising administering an effective amount of the immune response cell of the present disclosure or a composition comprising the same.

[0314] In accordance with the subject matter of the present disclosure, the above various methods may include administering at least one immunomodulatory agent. Non-limiting examples of immunomodulatory agents include immunostimulatory agents, checkpoint immune blockade agents, radiotherapy agents, and chemotherapeutic agents. In certain embodiments, the immunomodulatory agent is an immunostimulatory agent. Non-limiting examples of immunostimulatory agents include IL-12, and agonistic costimulatory monoclonal antibodies. In certain embodiments, the immunostimulatory agent is IL-12. In certain embodiments, the immune response cells of the present disclosure in combination with anti-IL-12 antibodies or compositions comprising the same can be used to treat breast cancer (BC), such as metastatic triple-negative breast cancer (TNBC). Non-limiting examples of agonistic costimulatory monoclonal antibodies include anti-4-1BB antibodies, anti-OX40 antibodies, and anti-ICOS antibodies. In certain embodiments, the agonistic costimulatory monoclonal antibody is anti-4-1BB antibodies.

[0315] In certain embodiments, the immune response cells of the present disclosure or compositions comprising the same can not only exhibit tumor-targeting adoptive T cell therapy, but also enhance T cell function through improved antigen receptor design and through intervention in the host microenvironment by immune modulation using IL-12. Among all immunotherapy approaches, IL-12, a multifunctional cytokine, is considered to be one of the most promising approaches 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 thought to be a master regulator of adaptive type 1 cell-mediated immunity, a crucial pathway involved in anti-tumor responses (Del Vecchio, M., et al., Clin Cancer Res 13, 4677-4685 (2007)). IL-12 modulates anti-tumor responses at various levels, including polarization of CD4 T cells toward a Th1 phenotype (Wesa, et al., J Immunother 30, 75-82 (2007)), boosting T cell and NK effector function (Curtsinger et al., J Exp Med 197, 1141-1151 (2003)), remodeling the innate immune response (Chmielewski 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 the rationale for using this cytokine in combination with the immune response cells of the subject matter of the present disclosure to treat cancer, such as BC (e.g., TNBC).Of 148 clinical trials involving administration of IL-12 to patients with cancer (36 of which have been reported recently), successful Phase II clinical trials were by intraperitoneal administration (Lenzi et al. Clin.Cancer Res. 8, 3686-3695 (2002)) or subcutaneous administration (Mahvi et al. Cancer Gene Ther. 14, 717-723 (2007); Kang et al. Hum.Gene Ther. 12, 671-684 (2001)). IL-12 shows that paracrine secretion of IL-12 produced by gene transfer can induce immunity against tumors at local and distant sites. Although several studies have demonstrated the anticancer efficacy 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)), significant toxicity due to the administration of recombinant human IL-12 has been observed in several clinical trials in advanced cancer, precluding its clinical use. To overcome this limitation, many research groups have demonstrated that intratumoral delivery of IL-12 using adenoviral vectors induces tumor regression and T cell activation in preclinical models of BC (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 antitumor response was primarily mediated by NK cells (Sabel et al. Breast Cancer Res Treat 122, 325-336 (2010)). Others used mesenchymal stromal cells to locally deliver IL-12 to mouse BC (Eliopoulos et al. Cancer Res 68, 4810-4818 (2008)).A phase I clinical trial of paclitaxel and trastuzumab in combination with IL-12 in patients with HER2 / neu expressing malignancies showed impressive synergy between IL-12 and trastuzumab in stimulating NK cell cytokine secretion (Bekaii-Saab et al. Molecular cancer therapeutics 8, 2983-2991 (2009)). Thus, IL-12 shows considerable promise as an anticancer agent, and its use as a costimulator in adoptive T cell therapy approaches is well justified.

[0316] In certain embodiments, the immunomodulatory agent is a checkpoint immune blockade agent. Non-limiting examples of checkpoint immune blockade agents include anti-PD-L1 antibody, anti-CTLA-4 antibody, anti-PD-1 antibody, anti-LAG3 antibody, anti-B7-H3 antibody, and anti-TIM3 antibody. In certain embodiments, the checkpoint immune blockade agent is an anti-PD-L1 antibody. In certain embodiments, the subject immune response cell of the present disclosure or a composition comprising the same can be used in combination with anti-PD-L1 antibody to treat breast cancer (BC), such as TNBC.

[0317] Programmed cell death ligand 1 (PD-L1 / B7-H4 / CD274) is an inhibitory signal typically expressed in active inflamed tissues and acts as a negative feedback loop to limit T cell activation. PD-L1 expression is typically absent in non-inflamed normal tissues, including breast (Dong et al. Nature medicine 8, 793-800 (2002)), and instead is most abundant in cancer tissues, especially those with inflammatory infiltrates (Spranger et al. Science translational medicine 5, 200ra116 (2013)). This association with inflammation is likely due to upregulation of PD-L1 upon exposure of tumor cells to T cell-secreted cytokines that are produced during T cell activation. This expression pattern is demonstrated by BC, where 50%-75% of BC specimens stain positive for PD-L1 and expression is strongly associated with heavy lymphocytic infiltrates (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 expressed PD-L1 in 54% of patients (Ghebeh et al. BMC cancer 8, 57 (2008)). BC may also express PD-L1 innately and secondarily to oncogenic signaling. 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 correlates significantly with PD-L1 expression (Crane et al. Oncogene 28, 306-312 (2009)). Expression of PD-1 by activated T cells spatially and temporally links the ligand to receptor expression within the immunosuppressive TME. Expression of PD-L1 in BC tissues suggests that it is a target for immunotherapy for these patients.The efficacy of PD-L1 / PD-1 blockade in multiple preclinical cancer models, including breast (Ge et al. Cancer letters 336, 253-259 (2013)), paved the way for Phase I clinical trials using PD-L1- or PD-1-targeting antibodies for patients with advanced cancer. Phase I clinical trials (using PD-1 antibodies) demonstrated efficacy exclusively in PD-L1+ patients (Topalian et al. The New England journal of medicine 366, 2443-2454 (2012)). Engineered T cells offer a unique advantage to overcome coinhibitory checkpoints and typically lack the costimulation found within the TME. CAR-expressing T cells have indeed been engineered to optimize their costimulatory requirements to support T cell expansion, survival, and function.

[0318] In some embodiments, the immunomodulatory agent is a radiotherapy agent. Not only can the local radiation-induced immunological environment provide the prerequisites for enhancing the engraftment of targeted T cells in tumors (thereby eliminating the need for systemic lymphodepletion regimens), but the immune response resulting from the combination of radiotherapy and adoptive T cell therapy also enhances long-range antitumor efficacy. In radioresistant tumors, 4-1BB costimulatory signaling in CAR-T cells can overcome immune inhibition. In some embodiments, the immunomodulatory agent is a chemotherapeutic agent, including but not limited to cisplatin. Cisplatin-induced secretion of chemokines and cytokines can promote MSLN-targeted T cell responses and endogenous T cell responses.

[0319] Studies have shown that patients with lung adenocarcinoma (LAC) and malignant pleural mesothelioma (MPM) who exhibit high levels of cytotoxic tumor-infiltrating lymphocytes (cTIL) and low levels of regulatory T cells (Treg) have a 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 MSLN-targeted CARs can be used to promote cTIL in LAC and MPM. Servais (2012) and Kachala (2013) reported that MSLN is overexpressed in LAC and MPM and promotes aggressiveness, justifying the selection of MSLN as a target for CAR T cell therapy. A higher proportion of TILs after cisplatin and radiation therapy is associated with improved outcomes in both mouse models and patients.

[0320] Tumor and long-range immunomodulation induced by tumor radiotherapy and cisplatin therapy may provide the necessary prerequisites for better engraftment of adoptively transferred T cells; T cell costimulatory strategies to exploit tumor and stromal immunomodulation could enhance the antitumor efficacy of both endogenous and adoptively transferred T cells.

[0321] In addition, the above-mentioned various methods of using immune response cells (e.g., T cells) expressing mesothelin-specific CARs can be combined with modulation of cancer cell antigens, for example, to treat cancer in a subject or to reduce tumor burden in a subject. Immune response cells (e.g., T cells) expressing mesothelin-specific CARs can target and kill MSLN expressed on the membrane of tumor or cancerous cells (referred to as "cell membrane MSLN"), but cannot target and kill cytoplasmic MSLN. Certain tumors or cancers (e.g., lung cancer and mesothelioma) have low cell membrane MSLN but high cytoplasmic MSLN. Modulation of cancer cell antigens can increase the expression of cell membrane MSLN in tumor or cancerous cells, which makes the tumor or cancerous cells more likely to be targeted by CAR-expressing immune response cells, and thus more susceptible to killing by immune response cells. In certain embodiments, the modulation of cancer cell antigens is radiation.

[0322] To avoid or minimize the risk of immunological complications (known as "malignant T-cell transformation"), such as graft-versus-host disease (GvHD), or in cases where healthy tissue expresses the same target antigen as tumor cells, resulting in a similar outcome to GvHD, further modifications can be introduced into immune response cells (e.g., T cells). A potential solution to this problem is to engineer a suicide gene into the CAR-expressing T cells. Suitable suicide genes include, but are not limited to, herpes simplex thymidine kinase (hsv-tk), inducible caspase 9 suicide gene (iCasp-9), and truncated human epidermal growth factor receptor (EGFRt) polypeptide. In certain embodiments, the suicide gene is an EGFRt polypeptide. The EGFRt polypeptide can allow for the elimination of T cells by administering an anti-EGFR monoclonal antibody (e.g., cetuximab). The EGFRt can be covalently linked to the 3' end of the intracellular signaling domain of the mesothelin-targeting CAR. The suicide gene can be included in a vector that includes 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 upon malignant T cell transformation (e.g., GVHD) (e.g., a prodrug (e.g., AP1903 that can activate iCasp-9)) induces apoptosis in suicide gene-activated CAR-expressing T cells.

[0323] In addition, the subject of the present disclosure provides a method for preventing and / or treating inflammatory disease in a subject.In certain embodiments, the method comprises administering to a subject the immune response cell of the present disclosure or a composition comprising the same.In certain embodiments, the immune response cell is an immune inhibitory cell.In certain embodiments, the immune inhibitory 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, for example a pancreas transplant recipient.

[0324] Furthermore, the subject of the present disclosure provides a method for preventing graft rejection in a subject who is a recipient of an organ transplant.In certain embodiments, the method comprises administering to a subject the immune response cell of the present disclosure or a composition comprising the same.In certain embodiments, the immune response cell is an immune inhibitory cell.In certain embodiments, the immune inhibitory cell is a regulatory T cell.In certain embodiments, the subject is a human.In further embodiments, the subject is a recipient of a pancreatic transplant.

[0325] The mesothelin-targeting CAR of the present disclosure can be transduced into immunoinhibitory cells, such as regulatory T cells. The transduced immunoinhibitory cells can be administered to a subject (e.g., a human) with an inflammatory condition or disease. In some embodiments, the site of inflammation or disease has high-level expression of mesothelin, which is recognized by the MSLN-CAR of the present disclosure. The inflammatory condition can be extreme, for example, severe pancreatitis. In addition, the transduced immunoinhibitory cells can be administered to a subject who is a recipient of an organ transplant.

[0326] In addition, the mesothelin-targeting CAR of the present disclosure and a second CAR targeting an MHC antigen can be co-transduced into immunoinhibitory cells (e.g., regulatory T cells), thereby allowing the immunoinhibitory cells to be specifically collected at the site of the transplanted pancreas. In certain embodiments, an MHC class I subject is transplanted with a pancreatic graft from an MHC class II donor, and the recipient's regulatory T cells are transduced with the MSLN-specific CAR of the present disclosure and a second CAR targeting an MHC class II antigen, such that the recipient's transduced regulatory T cells can be collected / pooled at the site of the transplanted pancreas to avoid graft or organ rejection.

[0327] Human subjects suitable for treatment typically 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. Clinically measurable tumors are tumors that can be detected based on tumor mass (e.g., palpation, CAT scan, sonogram, mammogram, or X-ray; positive biochemical or histopathological markers alone are insufficient to identify this population). Pharmaceutical compositions are administered to these subjects to induce an anti-tumor response with the aim of alleviating the condition. Ideally, a reduction in tumor mass occurs as a result, but any clinical improvement is beneficial. Clinical improvement includes a reduction in the risk or rate of progression, or a reduction in the pathological consequences of the tumor.

[0328] The second group of suitable subjects is known in the art as the "adjuvant group". These are individuals who have a history of neoplasms but have responded to other treatment modalities. Previous treatments may include, but are not limited to, surgical resection, radiation therapy, and conventional chemotherapy. As a result, these individuals do not have clinically measurable tumors. However, they are suspected to be at risk for disease progression, either near the original tumor site or by metastasis. This group can be further subdivided into high-risk and low-risk individuals. Subdivision is based on features observed before or after the initial treatment. These features are known in the clinical arts and are appropriately defined for each different neoplasm. Typical features for the high-risk subgroup are those whose tumor invades adjacent tissues or those who show lymph node metastasis.

[0329] Another group has a genetic predisposition to neoplasia, but has not yet shown evidence of clinical signs of neoplasia. For example, a woman who tests positive for a genetic mutation associated with breast cancer, but is still of childbearing age, may wish to administer one or more of the immune response cells described herein in a treatment to prophylactically prevent the development of neoplasia until it is appropriate to perform prophylactic surgery.

[0330] As a result of surface expression of the anti-mesothelin CAR and PD-1 DN, which enhance the antitumor effects of immune response cells, adoptively transferred T or NK cells are endowed with enhanced selective cytolytic activity at the tumor site. Furthermore, following their localization to the tumor or viral infection and their proliferation, the T cells then render the tumor or viral infection site a highly conducive environment for a wide range of immune cells (tumor-infiltrating lymphocytes, NK, NKT cells, dendritic cells, and macrophages) involved in the physiological antitumor or antiviral response.

[0331] 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 protozoal infection) in a subject, such as an immunocompromised subject. The method may include administering an effective amount of the immune response cell of the present disclosure or a composition comprising the same to a subject with 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.

[0332] To avoid or minimize the risk of immunological complications (known as "malignant T cell transformation"), such as graft-versus-host disease (GvHD), or where healthy tissue expresses the same target antigen as tumor cells resulting in a similar outcome to GvHD, further modifications can be introduced into the immune response cells (e.g., T cells) of the present disclosure. A potential solution to this problem is to engineer a suicide gene into the immune response cells of the present disclosure. Suitable suicide genes include, but are not limited to, herpes simplex thymidine kinase (hsv-tk), inducible caspase 9 suicide gene (iCasp-9), and truncated human epidermal growth factor receptor (EGFRt) polypeptide. In certain embodiments, the suicide gene is an EGFRt polypeptide. The EGFRt polypeptide can allow for the elimination of T cells by administering an anti-EGFR monoclonal antibody (e.g., cetuximab). The EGFRt can be covalently linked upstream of the antigen recognition receptor of the CAR of the present disclosure. The suicide gene can be included in a vector that includes a nucleic acid encoding the CAR of the present disclosure. In this way, administration of a prodrug (e.g., a prodrug (e.g., AP1903 that can activate iCasp-9) designed to activate a suicide gene upon malignant T cell transformation (e.g., GVHD) induces apoptosis in suicide gene-activated CAR-expressing T cells. Incorporation of a suicide gene into the CAR of the present disclosure provides an additional level of safety by allowing a large proportion of the CAR T cells to be eliminated in a very short period of time. Immune response cells (e.g., T cells) of the present disclosure incorporating a suicide gene can be pre-eliminated at a predetermined time point after CAR T cell infusion or eradicated at the earliest sign of toxicity.

[0333] 5.9.Kit The subject matter of the present disclosure provides a kit for inducing and / or enhancing immune response in a subject and / or treating and / or preventing neoplasms or pathogen infections.In certain embodiments, the kit comprises an effective amount of the immune response cell of the present disclosure or a pharmaceutical composition comprising the same.In certain embodiments, the kit comprises a sterile container; such container may be a box, an ampoule, a bottle, a vial, a tube, a bag, a pouch, a blister pack, or other suitable container known in the art.Such container may be made of plastic, glass, laminated paper, metal foil, or other material suitable for holding a drug.In certain embodiments, the kit comprises 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 be included in the same or different vectors as needed.

[0334] If desired, the immune response cells and / or nucleic acid molecules are provided with instructions for administering the cells or nucleic acid molecules to a subject having or at risk of developing a neoplasm or pathogen, or immune disorder. The instructions generally include information about the use of the composition for the treatment and / or prevention of a neoplasm or pathogen infection. In certain embodiments, the instructions include at least one of the following: description of the therapeutic agent; dosing schedule and administration for the treatment or prevention of a neoplasm, pathogen infection, or immune disorder or symptoms thereof; warnings; cautions; indications; contraindications; overdose information; adverse reactions; animal pharmacology; clinical trials; and / or references. The instructions may be printed directly on the container (if present), or may be printed as a label attached to the container, or as a separate sheet, pamphlet, or folder provided in or with the container. EXAMPLES

[0335] 6. Working Example The practice of the present disclosure employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are fully explained in such publications as "Molecular Cloning: A Laboratory Manual", second edition (Sambrook, 1989); "Oligonucleotide Synthesis" (Gait, 1984); "Animal Cell Culture" (Freshney, 1987); "Methods in Enzymology" "Handbook of Experimental Immunology" (Weir, 1996); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Current Protocols in Molecular Biology" (Ausubel, 1987); "PCR: The Polymerase Chain Reaction", (Mullis, 1994); "Current Protocols in Immunology" (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides disclosed herein and therefore may be considered in making and practicing the presently disclosed subject matter. Particularly useful techniques of certain embodiments are discussed in the following sections.

[0336] The following examples are put forth to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the disclosed cells and compositions and are not intended to limit the scope of what the inventors regard as the invention.

[0337] Example 1 A polypeptide composition of the present disclosure was generated. The polypeptide composition includes: (i) a CAR that binds to human mesothelin, and (ii) a dominant negative form of programmed cell death 1 (PD-1 DN), as shown in Figure 1. The mesothelin-targeting CAR includes (a) a CD8 signal peptide (e.g., a CD8 signal peptide consisting of the amino acid sequence set forth in SEQ ID NO: 71), (b) a VDR1 having an amino acid sequence set forth in SEQ ID NO: 76, a CDR2 having an amino acid sequence set forth in SEQ ID NO: 77, and a CDR3 having an amino acid sequence set forth in SEQ ID NO: 78. H and a V comprising a CDR1 consisting of the amino acid sequence set forth in SEQ ID NO:79, a CDR2 consisting of the amino acid sequence set forth in SEQ ID NO:80, and a CDR3 consisting of the amino acid sequence set forth in SEQ ID NO:81. L (c) an extracellular antigen-binding domain which is an scFv comprising: (a) a transmembrane domain comprising a CD28 polypeptide (e.g., a CD28 polypeptide consisting of the amino acid sequence set forth in 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 set forth in SEQ ID NO: 15 (or amino acids 114 to 152 of SEQ ID NO: 90)); and (e) an intracellular signaling domain comprising a modified CD3ζ polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 35, and a costimulatory signaling region comprising a CD28 polypeptide (e.g., a CD28 polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 101 (or amino acids 180 to 220 of SEQ ID NO: 90)). 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 also includes a P2A peptide having the amino acid sequence set forth in SEQ ID NO: 121, and located between the CAR and PD-1 DN as shown in Figure 1. The polypeptide composition is designated "M28z1XXPD1DNR."

[0338] The CAR contained in the polypeptide construct has the amino acid sequence set forth in SEQ ID NO: 56. An exemplary nucleotide sequence encoding the polypeptide construct is set forth in SEQ ID NO: 123. Another exemplary nucleotide sequence encoding the polypeptide composition is set forth in SEQ ID NO: 124.

[0339] Example 2 We investigated the activity of M28z1XX-P2A-PD1DNR, which has the structure of the polypeptide composition described in Example 1. The structures of alternative and control constructs were compared to M28z1XX-P2A-PD1DNR, as shown in FIG.

[0340] Viral vectors containing the CAR construct were generated in the producer cell line RD114 as shown in Figures 3A-3D. RD114 cells were transduced with various dilutions of H29 viral supernatant (undiluted, 1:2, and 1:4) and stained for CAR expression by flow cytometry using anti-Fab antibodies. RD114 empty served as a negative control. Human T cells were successfully transduced with M28z1XX-P2A-PD1DNR as shown in Figures 4A-4E, 5A-5E, and 6A-6F. PHA-activated T cells were transduced with various concentrations of RD114 viral supernatant and stained for CAR expression by anti-Fab staining and for PD1DNR by anti-PD1 staining using flow cytometry. We studied whether vector copy number (VCN) correlated with median fluorescence intensity (MFI). PHA-activated T cells were transduced with various concentrations of RD114 viral supernatant and stained for CAR expression by anti-Fab staining and flow cytometry analysis. Genomic DNA of transduced T cells was isolated and vector copy number was determined as VCN per μg of DNA using qPCR. As shown in Figures 7A-7C, the MFI of CAR-positive cells correlated with VCN per μg of DNA for all three tested donors. Human CD4 + and CD8 + The T cell transduction ratios are shown in Table 1. [Table 1]

[0341] Next, the cytolytic effect of M28z1xx-PD1DNR CAR T cells was studied. Using an impedance-based assay, MSLN-rich target cells (MGM) from different donors were co-cultured with M28z1XX-PD1DNR CAR T cells at various E:T ratios. The results are shown in Figure 8. As shown in Figure 8, M28z1XX-PD1DNR CAR-transduced T cells demonstrated effective cytotoxicity against all three different donors tested. Effector cytotoxicity spanned multiple E:T ratios (data not shown). Conclusion:

[0342] M28z1xx-PD1DNR vectors were successfully generated in RD114 cells. Stable producer cell lines were successfully established for all constructs. Viral vectors were titrated to obtain approximately 40-60% transduction of multiple donor T cells. CD4 and CD8 T cells were successfully transduced to express CAR and PD1DNR. A correlation between vector copy number and transduction was observed.

[0343] Example 3 This example describes the comparative analysis of various constructs, including M28z1XX-PD1DNR. Cytotoxicity was measured by using impedance assay. The principle of impedance-based cytotoxicity measurement (eCTL) is shown in Figure 9. The parameters of the comparative analysis, including CAR construct, donor, CAR target and E:T ratio, are shown in Figure 10. MSLN and PD-L1 expression in target cell lines was measured. Mesothelioma (MGM, MGM-PDL1 and MSTOG) and lung cancer (A549GM and A549G) cell lines were evaluated for MSLN and PD-L1 expression by flow cytometry. The results are shown in Figures 11A-11E. MGM, MGM-PDL1 and A549GM overexpressed MSLN. MGM-PDL1 cells further overexpressed PD-L1.

[0344] The CAR and PD1 expression of transduced T cells was also measured. Human T cells transduced with M28z, M28z1xx, M28z-PD1DNR or M28z1xx-PD1DNR were analyzed for CAR expression by anti-myc staining and for PD1 / PD1DNR expression by anti-PD1 staining using flow cytometry. The results are shown in Figures 12A-12E.

[0345] A comparative analysis of the antitumor efficiency of CAR T cells expressing M28z, M28z1xx, M28z-PD1DNR or M28z1xx-PD1DNR against MSLN-high tumor cells (MGM) was performed. MSLN-high target cells (MGM) were co-cultured with either M28z, M28z1xx, M28z-PD1DNR, M28z1XX-PD1DNR or untransduced T cells at various E:T ratios. Antitumor efficiency was evaluated using an impedance-based assay. The results are shown in Figures 13A-13C. Furthermore, Chromium-51-labeled MSLN-high target cells (MGM) were co-cultured with either M28z, M28z1xx, M28z-PD1DNR, M28z1xx-PD1DNR or untransduced T cells at various E:T ratios for 18 h. Cytotoxicity was determined by Chromium-51 CTL. The results are shown in Figure 14.

[0346] Next, comparative analysis of the antitumor efficiency of CAR T cells expressing M28z, M28z1xx, M28z-PD1DNR or M28z1xx-PD1DNR against MSLN-negative tumor cells (MSTOG) was measured. MSLN-negative target cells (MSTOG) were co-cultured with either M28z, M28z1xx, M28z-PD1DNR, M28z1xx-PD1DNR or untransduced T cells at the indicated E:T ratios. Antitumor efficiency was evaluated using an impedance-based assay. The results are shown in Figures 15A-15C. Furthermore, Chromium-51-labeled MSLN-negative target cells (MSTOG) were co-cultured with either M28z, M28z1XX, M28z-PD1DNR, M28z1XX-PD1DNR or untransduced T cells at various E:T ratios for 18 h. Cytotoxicity was determined by Chromium-51 CTL. The results are shown in Figure 16.

[0347] Furthermore, a comparative analysis of the antitumor efficiency of CAR T cells expressing M28z, M28z1xx, M28z-PD1DNR or M28z1xx-PD1DNR against MSLN high tumor cells overexpressing PDL1 was measured. MSLN high target cells overexpressing PDL1 (MGM-PDL1) were co-cultured with either M28z, M28z1XX, M28z-PD1DNR, M28z1XX-PD1DNR or untransduced T cells at various E:T ratios. Antitumor efficiency was evaluated using an impedance-based assay. The results are shown in Figures 19A-19C. Similarly, a comparative analysis of the anti-tumor efficiency of CAR T cells expressing M28z, M28z1xx, M28z-PD1DNR or M28z1xx-PD1DNR against MSLN-high tumor cells (A549GM) was measured, and the results are shown in Figures 18A-18C; a comparative analysis of the anti-tumor efficiency of CAR T cells expressing M28z, M28z1xx, M28z-PD1DNR or M28z1xx-PD1DNR against MSLN-low tumor cells (A549G) was measured, and the results are shown in Figures 19A-19C. Conclusion:

[0348] The M28z1xx-PD1DNR construct was expressed in the MSLN depending on the E:T ratio. + Targeted cell killing was observed, and the results were replicated here for different T cell donors with different cancers (lung cancer and mesothelioma cell lines). Targeted killing correlated with the level of MSLN expression, with MSLNs with high expression of PD-L1 + It was effective against the target cells.

[0349] Example 4 Localized delivery of clinical-grade mesothelin-targeted CAR T cells with cell-intrinsic PD-1 checkpoint blockade: A bridge to phase I trials Summary: This example provides evidence of the preclinical safety and enhanced antitumor efficacy of clinical-grade M28z1XXPD1DNR CAR T cells.

[0350] Methods: The relative cytotoxicity, proliferation, and cytokine secretion of human T cells engineered to express M28z or M28z1XXPD1DNR CAR were assessed by chromium release, accumulation, and Luminex assays, respectively. A single dose (1 × 10 5 The antitumor efficacy of 10 CAR T cells (E:T 1:1000) was investigated in NSG mice bearing orthotopic pleural mesothelioma by serial bioluminescence imaging and comparison of survival. After tumor eradication, the functional persistence of CAR T cells was evaluated against tumor burden (2 × 10 6 pieces~11×10 6 The tumor cells were then cultured in a 3x1000 swelled 4 ... with increasing doses of 1000 tumour cells each.

[0351] Results: In vitro, both M28z and M28z1XXPD1DNR CAR T cells exhibited antigen-specific cytotoxicity, accumulation, and effector cell secretion (see Table 2). In vivo, a single dose of M28z1XXPD1DNR CAR T cells conferred tumor eradication, enhanced survival, and resistance to tumor re-formation upon 10 tumor rechallenges relative to a single dose of M28z CAR T cells (see Table 2).

[0352] Conclusions: Data on safety, tumor eradication, and functional durability of CAR T cells, without the use of anti-PD1 antibodies, support the initiation of a phase I clinical trial of intrapleural administration of M28z1XXPD1DNR CAR T cells in patients with pleural mesothelioma. [Table 2]

[0353] Example 5 Next-generation CAR T cells with cell-intrinsic PD-1 blockade: Clinical rationale, preclinical and clinical trial protocol development Malignant pleural mesothelioma (MPM) is a cancer with low mutational burden and low PDL1 expression that prevents response to anti-PD1 antibodies. In an ongoing Phase I / II study (NCT02414269, n=41), we established the safety and antitumor efficiency of intrapleurally administered mesothelin-targeted chimeric antigen receptor (M28z CAR) T cells followed by PD-1 antibodies. CAR T cells with a cell-intrinsic anti-PD1 strategy are safe and can provide antitumor efficiency against both low and high PDL1 tumors without the need for repeated administration of anti-PD1 antibodies.

[0354] Summary: This example provides evidence of the preclinical safety and enhanced antitumor efficacy of clinical-grade M28z1XXPD1DNR CAR T cells.

[0355] Methods: Clinical grade M28z and M28z1XXPD1DNR (modified CD3z domain containing PD-1 dominant negative receptor) CARs were transduced in multiple donor T cells as effectors and used as targets MPM cells with low and high PDL1. Relative antitumor efficacies in vitro and in vivo at various E:T ratios were evaluated in orthotopic MPM-bearing mice. Systemic antitumor immunity was tested by repeated tumor challenge at distant sites.

[0356] Results: In vitro, no significant differences were observed between M28z and M28z1XXPD1DNR CARs (antigen-specific cytotoxicity, accumulation, and effector cytokine secretion). In vivo, a single dose (1 × 10) administered intrapleurally with either repeated doses of anti-PD1 antibody or cell-specific PD1DNR 5 CAR T cells) resulted in comparable tumor eradication and enhanced survival with weight gain. See Figure 20A and Table 3. In mice with orthotopic MPM, a single, low dose (1 x 10 5M28z1XXPD1DNR CAR T cells (1 CAR T cell) demonstrated enhanced systemic immunity compared to M28z CAR T cells by eradicating pleural tumors and resistant to tumor rechallenge at a distant peritoneal site without any toxicity (PD1DNR binds to mouse PDL1 / 2). See Figures 20B and 20C. Recovered tumors demonstrate higher and deeper CAR T cell infiltration compared to untransduced T cells. See Figure 20D.

[0357] Conclusions: A single, low-dose intrapleural administration of M28z1XXPD1DNR CAR T cells demonstrates feasibility, safety, tumor eradication, durable function, and systemic antitumor immunity. [Table 3]

[0358] Example 6 1. Overview Malignant pleural mesothelioma (MPM) is a rare, fatal malignancy associated with asbestos exposure. MPM is a locally invasive primary malignancy of the pleura characteristically with involvement of vital organs or the chest wall (Carbone et al., CA Cancer J Clin. 2019;69(5):402-429). The majority of patients (60%-70%) at presentation have locoregionally 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 combined chemotherapy, aggressive surgical resection, and radiation therapy, treated patients only have a median survival of 9-17 months (Flores et al., J Thorac Oncol. 2007;2(10):957-965). There have been no new FDA-approved treatments for MPM since 2003 (Tsao et al., J Thorac Oncol. 2018;13(11):1655-1667). The current standard of care for first-line systemic treatment of patients with MPM is cisplatin and pemetrexed, based on a median overall survival of 12.1 months compared to 9.3 months for cisplatin alone (Vogelzang et al., J Clin Oncol. 2003;21(14):2636-2644). Patients with MPM have low tumor mutational burden and low programmed death-ligand 1 (PD-L1) expression, limiting their response to immune checkpoint inhibitors and persisting a huge unmet need (Yarchoan et al., JCI Insight. 2019;4(6);Forde et al., Curr Treat Options Oncol. 2019;20(2):18). The localized nature of MPM, its potential accessibility, and the relative lack of metastases at presentation make it an excellent candidate for locally targeted therapy (Nelson et al., J Clin Oncol. 2017;35(29):3354-3362).

[0359] This example describes a non-clinical study conducted to support the clinical use of an intrapleural dose of a new investigational agent, M28z1XXPD1DNR chimeric antigen receptor (CAR) T cells, for the treatment of patients with a diagnosis of MPM (documented histologically or cytologically) who have received at least one chemotherapy regimen and have a documented tumor.

[0360] This is a single-center Phase I study of up to 36 participants diagnosed to evaluate the safety, dosing requirements, and targeting efficiency of genetically derived autologous M28z1XXPD1DNR CAR T cells after conditioning with cyclophosphamide. There are five planned dose levels in this study: 1 × 10 per kg. 6 , 3×10 6 , 6×10 6 , 1×10 7 , and 3 × 10 7 M28z1XXPD1DNR CAR T cells, provided there is no dose-limiting toxicity. M28z1XXPD1DNR CAR T cells are infused via an indwelling pleural catheter. Patients are screened for mesothelin expression by immunohistochemical analysis of pre-treatment biopsy tumor and / or blood levels of soluble mesothelin-related peptide.

[0361] M28z1XXPD1DNR CAR T cells are autologous T cells transduced ex vivo with the supernatant of gammaretroviral vector stock generated from the vector-producing master cell bank 293VEC-GALV-SFG-M28z1XXPD1DNR. The main components of the vector-encoded CAR are: 1) a human anti-mesothelin scFv to target tumors that express mesothelin; 2) the human CD28 costimulatory domain to signal for T cell survival and proliferation; 3) a point-mutated human CD3ζ with a single functional immunoreceptor tyrosine-based activation motif (ITAM) for calibrated T cell activation; and 4) Programmed cell death protein 1 (PD1) dominant negative receptor (PD1DNR) to prevent T cells from becoming dysfunctional or exhausted upon exposure to antigens. (See FIG. 21).

[0362] Mesothelin is a cancer cell surface antigen that is overexpressed in the majority of MPM, lung, triple-negative breast, pancreatic, and ovarian cancers, and in 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). We previously demonstrated that mesothelin overexpression promotes invasion in 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 mesothelin expression in tumors compared to normal tissues, it is also expressed at very low levels on the mesothelial surfaces of normal peritoneum, pleura, and pericardium (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 functions of mesothelin are poorly understood and are under investigation.

[0363] Previously, in a clinical study conducted at the University of Pennsylvania (NCT01355965), mesothelin-targeting CAR T cells, in which the CAR contained a mouse scFv, were administered intravenously to humans (1m 2 Winning 3 x 10 8 cells or 4.8 x 10 per dose 7 cells per kg). An anaphylactic reaction noted in one patient was reported to be caused by an anti-mouse antibody response generated against the humanized mouse scFv portion of the CAR construct used in that study (Beatty et al., Cancer Immunol Res. 2014). In contrast, a recent Phase I study (NCT02414269) from our laboratory used mesothelin-targeting CAR T cells (up to 6 × 10 per kg) composed of fully human scFvs derived from a human Fab library. 7 100 CAR T cells) were administered intrapleurally (Feng et al., Mol Cancer Ther. 2009). To date, 40 patients have been treated without observing dose-limiting toxicity. Preliminary efficacy was observed in this study in a cohort of patients (n=18) who received CAR T cell therapy followed by three doses of pembrolizumab (anti-PD1) and an additional 3 months of follow-up. Importantly, 83% of patients in this cohort did not require new or additional treatment at 6 months, and half of the patients did not receive additional treatment for 18 months. Furthermore, in the majority of patients, CAR T cells were detected in the peripheral blood more than 100 days after intrapleural administration, indicating that these cells persist in the patient's body.

[0364] In support of first-in-human clinical trials, the pharmacology program for M28z1XXPD1DNR CAR T cells will consist of a series of orthogonal in vitro specificity, cytotoxicity, accumulation, and cytokine secretion studies in mice and in vivo tumor efficacy and viability studies, the results of which will suggest effective doses for transitioning to clinical use. Table 4 provides a comprehensive overview of the nonclinical pharmacology and toxicology assays that were performed, along with their key findings. [Table 4-1] [Table 4-2] [Table 4-3]

[0365] To facilitate detection of CAR T cells in preclinical studies, we generated CAR T cells containing a myc tag at the N-terminus of the mesothelin-specific scFv (mycM28z1XXPD1DNR). To assess the consistency between mycM28z1XXPD1DNR and M28z1XXPD1DNR CAR T cells, we compared the transduction efficiency of viral supernatants and observed consistent concentration-dependent expression of vector components between the two constructs. Furthermore, both CAR and PD1DNR were proportionally expressed in cells transduced with each other due to the presence of the P2A self-cleaving peptide, which efficiently mediates the expression of the bicistronic transgene. When comparing the percentage of transduced cells expressing CAR to the number of vector copy inserts in the T cell genome, we observed a positive linear association between the dilution of viral supernatant used for transduction and the resulting vector copy number (VCN). From these observations, it was determined that T cells expressing a CAR in the range of 35%–70% ensure optimal transduction efficiency, which (1) maintains a low VCN:cell ratio and (2) reduces the risk of insertional mutagenesis, which is usually higher at higher VCN:cell ratios.

[0366] To confirm the expression of PD1DNR and distinguish it from its endogenous counterpart, flow cytometry and qPCR assays were performed to measure and compare cell surface protein and intracellular mRNA expression for PD1 in mycM28z1XXPD1DNR and mycM28z transduced T cells, respectively. At the cell surface protein level, mycM28z1XXPD1DNR CAR T cells exhibited a 2-fold increase in the percentage of cells stained positive for PD1 and a 3-fold increase in the median fluorescence intensity (MFI) exhibited by PD-1 positive cells compared to mycM28z CAR T cells. At the mRNA level, mycM28z CAR T cells exhibited a 4-fold increase in both PD1 extracellular and intracellular domains compared to untransduced T cells, whereas mycM28z1XXPD1DNR CAR T cells exhibited a 157-fold increase in PD1 extracellular domain and only a 2-fold increase in PD1 intracellular domain. An order of magnitude higher expression of the PD1 extracellular domain indicates higher expression of PD1DNR, which plays an antagonistic role in checkpoint inhibition.

[0367] Finally, to rule out any potential interference of the myc tag (used in preclinical studies) with CAR function, an impedance-based cytotoxicity assay was used to compare the antitumor efficiency of mycM28z1XXPD1DNR CAR T cells and M28z1XXPD1DNR CAR T cells and revealed no differences in the kinetics and overall killing of mesothelin-positive tumor cells across three different donors, confirming that the myc tag does not interfere with CAR function.

[0368] We next analyzed the specificity, cytotoxicity, accumulation, and cytokine secretion of mycM28z1XXPD1DNR CAR T cells. 51In Cr cytotoxicity assays, mycM28z1XXPD1DNR CAR T cells exhibited antigen-specific and human leukocyte antigen (HLA)-independent cytotoxicity against mesothelin-positive tumor cells with both constitutive and overexpression of PD-L1. No nonspecific cytotoxicity against mesothelin-negative tumor cells was observed. mycM28z1XXPD1DNR CAR T cells did not express any cytotoxicity against targets overexpressing PD-L1 in the absence of mesothelin antigen expression. When performing repeated antigen stimulation assays (antigen stress test), a maximum of 622-fold expansion was recognized in both mycM28z1XXPD1DNR CAR T cells and mycM28z CAR T cells over a period of six antigen stimulations. At the time points tested, the constructs demonstrated similar cytotoxicity after the first antigen stimulation and retained similar cytotoxicity up to the fourth antigen stimulation. When the effector-to-target (E:T) ratio was further reduced to increase antigen stress, mycM28z1XXPD1DNR CAR T cells retained cytotoxicity better than mycM28z CAR T cells upon the seventh round of antigen stimulation. However, secretion of effector cytokines (IL-2, IFN-γ, and TNF-α) gradually decreased in both mycM28z1XXPD1DNR and mycM28z CAR T cells over the course of the assay, indicating the safety profile of both constructs.

[0369] MPM tumor cells co-transduced with firefly luciferase (ffLuc) were administered intrapleurally to establish tumors that represent an orthotopic cancer model of MPM. To noninvasively monitor tumor growth, tumor-bearing mice were injected intraperitoneally with a 150 mg / kg dose of luciferin using a protocol optimized for quantitatively monitoring pleural tumor regression or progression previously published by our laboratory, and visualized 15 min later on an IVIS Spectrum imaging system (PerkinElmer, Waltham, MA).

[0370] In the first experiments conducted to study the antitumor activity of CAR T cells in vivo, orthotopic tumor-bearing NSG mice were treated with a single intrapleural dose of 3 × 10 4 Mice were treated with mycM28z1XXPD1DNR CAR T cells and compared to mice treated with a single intrapleural dose of control CAR T cells specific for 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), whereas mice treated with P28z CAR T cells began to become moribund due to high tumor burden.

[0371] In the second in vivo study, orthotopic tumor-bearing mice were distributed into three groups, each of which received a single intrapleural dose of 1 × 10 5 pcs or 5×10 4 mycM28z1XXPD1DNR CAR T cells or 1 x 10 5 MycM28z CAR T cells were administered to each patient. Serial tumor imaging was performed as early as 5 days after CAR T cell administration, i.e., 1 × 10 5 At approximately day 19, mice treated with mycM28z1XXPD1DNR CAR T cells had 1 × 10 5 Mice treated with mycM28z CAR T cells showed a reduction in tumor burden, including complete tumor eradication, at approximately day 26, as determined by a decrease in bioluminescence imaging (BLI) signal relative to baseline levels. Mice treated with either dose of mycM28z1XXPD1DNR CAR T cells remained tumor-free until the end of the study (68 days). Median survival was 1 × 10 5Median survival was not reached in mice treated with either dose of mycM28z1XXPD1DNR CAR T cells (>68 days; p=0.0085-0.0427) compared with 50 days in mice treated with mycM28z1XXPD1DNR CAR T cells. Ex vivo immunofluorescence imaging of pleural tumors revealed that mycM28z1XXPD1DNR CAR T cells densely surrounded the tumor periphery and invaded the tumor parenchyma by 3 days after local delivery. These results were replicated by the use of T cells derived from multiple donors with various percentages of CD4 and CD8 T cells transduced with the mycM28z1XXPD1DNR CAR.

[0372] To investigate the functional persistence of mycM28z1XXPD1DNR CAR T cells, 1 × 10 5 Mice with orthotopic MPM received a single intrapleural dose of either mycM28z1XXPD1DNR CAR T cells or mycM28z CAR T cells every 4–8 days for up to 10 doses in escalating doses (2 × 10 per dose). 6 ~11×10 6 cells) were reloaded with mesothelin-positive tumor cells administered intraperitoneally (repeated administration of cells is more feasible in the peritoneal cavity than in the pleural cavity). The BLI signal for mice treated with mycM28z1XXPD1DNR CAR T cells peaked immediately after each tumor reload and returned to baseline levels at all reload time points. In contrast, mice treated with mycM28z CAR T cells showed the same trend up to 5 tumor reloads, but were unable to control tumor re-establishment after administration of higher tumor doses at later tumor reload time points (6–10 times), resulting in tumor recurrence and moribundity. mycM28z1XXPD1DNR CAR T cells were reloaded with 1 × 10 CAR T cells without any obvious signs of toxicity. 5More than 126 days after a single intrapleural administration of 10 CAR T cells, the mycM28z1XXPD1DNR CAR T cells resisted intraperitoneal tumor establishment against 10 repeated challenges. Under high antigen stress conditions, mycM28z1XXPD1DNR CAR T cells exhibited superior functional persistence and enhanced antitumor efficacy compared to mycM28z CAR T cells in vivo. To confirm that this enhanced efficacy was not due to graft-versus-host disease, which is common in CAR T cell-treated NSG mice at this time point, targets that do not express the antigen were administered, resulting in increased tumor BLI without an antitumor response, confirming that the observed antitumor efficacy was antigen-specific.

[0373] To evaluate the antitumor efficiency of cryopreserved T cells transduced with viral supernatant encoding the M28z1XXPD1DNR CAR produced for use in clinical trials, M28z1XXPD1DNR CAR T cells generated by the MSK Cell Therapy and Cell Engineering Facility (CTCEF) were thawed (viability: 88% after thawing) and injected intrapleurally into mice with orthotopic MPM at 6 × 10 per mouse. 4 pcs and 2×10 5 Cryopreserved CAR T cells were injected at a dose of 100%. Tumor regression and eradication was observed at both doses with 100% of mice surviving until the end of the observation period (day 70), whereas untreated mice experienced tumor progression and died by day 19. Cryopreserved CAR T cells demonstrated high survival rates after thawing and were effective without any signs of toxicity.

[0374] No toxicity was observed in the mice when conducting the efficacy study and body weights remained stable throughout.

[0375] Section 3 of this Example (titled "Non-clinical Toxicology") describes studies 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 histopathological evaluations were evaluated in 96 (48 males and 48 females) NSG mice bearing 8-day-old orthotopic mesothelioma randomly assigned to control and treatment groups. 1×10 per mouse 5 Dose of CAR T cells or control vehicle (5 × 10 per kg) 6 CAR T cells) were administered once by orthotopic injection. Mice were sedated for necropsy and evaluation of hematology and clinical chemistry parameters on days 2 and 14 after CAR T cell or vehicle administration (intermediate and terminal sacrifice, respectively). Day 14 was chosen as the time point for terminal sacrifice because tumors had significantly regressed or eradicated at this time point (proven by BLI or necropsy from previous experiments). Performing sacrifice and necropsy at this time allows for examination of any on-target, off-tumor effects in normal tissues with low levels of mesothelin expression - specifically pleura, peritoneum, and pericardium - after the peak of CAR T cell expansion in the absence of tumor burden with high antigen expression (the scFv used in our CAR reacts with mesothelin in mice) (Feng et al., Mol Cancer Ther. 2009).

[0376] No mortality or morbidity was observed in the animals in this study, except for two animals from the control vehicle-treated group that underwent selective sacrifice due to mortality and forced respiration 20–22 days after tumor challenge. Previous work in our laboratory has shown that animals treated with control vehicle can become moribund due to tumor burden approximately 20-22 days after tumor challenge (Servais et al., Clin Cancer Res. 2012; Servais et al., Curr Protoc Pharmacol. 2011; Chapter 14:Unit14 21; 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). Thus, these sacrifices were unplanned but not unexpected. Neither mortality nor morbidity was influenced by the CAR T cells. Animals receiving the control vehicle showed a progressive loss of body weight and a significant difference in weight over the study period compared to non-tumor controls and mice treated with mycM28z1XXPD1DNR CAR T cells. This was attributed to an increase in tumor burden in animals treated with the control vehicle. No significant clinical signs were observed in mice treated with mycM28z1XXPD1DNR CAR T cells. One test article-treated mouse was observed to have slight crust formation, which was attributed to irritation caused by the hemostat, as other animals were unaffected and animal activity was normal. Mice appeared normal throughout the monitoring period.

[0377] Female mice treated with mycM28z1XXPD1DNR CAR T cells, which underwent terminal sacrifice 14 days after CAR T cell administration, had higher mean percent monocyte values ​​(mean, 18.44%, n=5) (p≦0.0001) compared to tumor control vehicle mice (mean, 3.34%, n=5). The established reference range for monocyte percentage is 0.9%-18%. However, this did not correlate with any of the microscopic findings. No other significant or abnormal results were observed for the hematological parameters evaluated. Any differences between the test article-treated and corresponding vehicle-treated groups were within the normal reference range or were not biologically relevant or statistically significant.

[0378] Male mice treated with mycM28z1XXPD1DNR CAR T cells, which underwent terminal sacrifice 14 days after CAR T cell administration, had lower mean total protein values ​​(mean, 3.83 g / dL, n=5) (p=0.0022) compared to tumor control vehicle mice (mean, 4.68 g / dL, n=4). The established reference range for total protein is 4.1-6.4 g / dL. However, this did not correlate with any microscopic findings. No other adverse effects on clinical chemistry parameters were observed with test article administration. Any differences between test article-treated and corresponding vehicle-treated groups were within the normal reference range or were not biologically relevant or statistically significant.

[0379] Histopathological examination showed the absence of any microscopic findings at the interim and terminal sacrifice dates related to acute or delayed toxicity resulting from mycM28z1XXPD1DNR CAR T cell administration. Microscopic findings for animals in the CAR T cell interim sacrifice group included the presence of mixed cellular infiltrates within the xenograft tumors, which were considered related to test article administration but not to any test article toxicity. Any other observed findings were determined to occur sporadically, with incidences similar to those in controls, or were common in the species / strain utilized.

[0380] mycM28z1XXPD1DNR CAR T cells were found in the tumor and spleen 8 days after intrapleural administration, and BLI revealed a significant reduction in tumor burden in mice treated with CAR T cells at 2 weeks, confirming both successful administration and pharmacological activity of the test article. Plasma cytokine levels in mice obtained at the same time points showed slightly higher IL-4 levels in mice treated with CAR T cells than in mice treated with vehicle control. Levels of IL-10, IL-6, KC / GRO, and TNF-α were generally low and not significantly different between mice receiving CAR T cells and vehicle control. IFN-γ, IL-12p70, IL-1β, IL-2, and IL-5 were undetectable (below limit of quantification).

[0381] In conclusion, the data show that M28z1XXPD1DNR CAR T cells are well tolerated. 5 The dose of cells was determined based on the patient's starting dose (1 × 10 cells per kg of body weight). 6 cells per kg), which is 5 × 10 6 This corresponds to 10 cells. M28z1XXPD1DNR CAR T cells have the same antigen targeting moiety as M28z CAR T cells, for which patient safety data (n=50) are already available. In our clinical trial (IND16354), up to 6×10 per kg administered intrapleurally in combination with an anti-PD1 checkpoint blockade antibody given 3 weeks after CAR T cell administration. 7 For M28z CAR T cells, we did not observe any dose-limiting toxicity and observed on-target, off-tumor toxicity. Four patients received a second dose of intrapleural M28z CAR T cells followed by multiple doses of anti-PD1 agents (with a 4-week washout period) without any observed toxicity. Taken together, all of the available preclinical and clinical data reasonably indicate that the proposed starting dose and route of administration of M28z1XXPD1DNR CAR T cells does not pose unacceptable risks to our patients.

[0382] 2. Nonclinical Pharmacology A. Method CAR Vectors. The vectors used in nonclinical studies are summarized in Table 5. [Table 5]

[0383] The mesothelin-targeting CAR construct contains a mesothelin-specific scFv (clone m912) (Feng et al., Mol Cancer Ther. 2009) fused to the CD28 costimulatory domain and the CD3 ζ signaling domain (M28z). The CD3 ζ chain was mutated at two of its three ITAMs, resulting in a single functional ITAM (designated 1XX) (Feucht et al., Nat Med. 2019;25(1):82-88). The CAR is fused to PD1DNR via the P2A site derived from porcine teschovirus-1. PD1DNR is composed of the PD1 signaling peptide and the PD1 extracellular domain fused to the CD8 transmembrane and hinge domains (Cherkassky et al., J Clin Invest. 2016;126(8):3130-3144). This decoy receptor is depleted of PD1 signaling domain, thereby resulting in 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 scFv in constructs mycM28z and mycM28z1XXPD1DNR. To avoid any potential risk of immunogenicity in humans, clinical grade construct M28z1XXPD1DNR does not contain a myc tag. Furthermore, protein expression is codon-optimized to avoid any immunogenicity in the construction of CAR and PD1DNR. The detailed structure of the constructs used in non-clinical studies is shown in Figure 22.

[0384] Expression of the CAR construct is under the control of 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). Expression of both CAR and PD1DNR is driven by the retroviral LTR.

[0385] All CAR vectors were transfected into the 293T H29 packaging cell line, and viral supernatants produced by these cells were used to transduce and generate stable 293T RD114 cell lines.

[0386] CAR T cells. Human primary T lymphocytes were isolated from the blood of healthy volunteer donors under an Institutional Review Board-approved protocol. Phytohemagglutinin-activated peripheral blood mononuclear cells (PBMCs) were isolated by low-density centrifugation in 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 min at 24 °C in 6-well culture plates coated with 15 μg / mL RetroNectin (Takara, Shiga, Japan). The day after spinoculation, transduced PBMCs were maintained 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. Expression of myc-tags in the scFv of tagged CARs or F(ab') 2Transduction efficiency was determined by staining with a fragment-specific anti-human IgG antibody to detect expression of the M28z1XXPD1DNR untagged CAR scFv. CAR T cells were tested for viability >70%, T cell purity >95% by anti-human CD3 staining, transduction efficiency of 35%-70% by flow cytometry, and CD4 / CD8 expression. Characteristics of T cells used in nonclinical studies are summarized in Table 6. [Table 6]

[0387] Tumor cells. Cells derived 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). [Table 7]

[0388] MSTO-211H is a biphasic MPM cancer cell line that lacks expression of endogenous CD80 / 86 costimulatory ligands. MSTO-211H cells were retrovirally transduced to express GFP and ffLuc proteins, designated MSTOG, to allow non-invasive in vivo BLI using the SFG retroviral vector constructed at MSK. Media containing filtered virus was added to cells permeabilized with 8ug / mL Polybrene (Sigma-Aldrich, St. Louis, MO). Cells were reinfected with freshly harvested virus 24 hours later. These cells were transduced with human mesothelin variant 1 (isolated from a human ovarian cancer cell line [OVCAR-3]) subcloned into the SFG retroviral vector to generate mesothelin+ MSTO-211H cells, designated MGM. Similarly, MGM cells were transduced with PD-L1 (OriGene cDNA subcloned into SFG vector) to obtain MGM-PDL1. 2Tumor cells were maintained in RPMI-1640 medium containing 10% FBS, 2 mM L-glutamine, 100 units / mL penicillin, and 100ug / mL streptomycin in a humidified incubator. A linear correlation was observed between tumor cells expressing luciferase and BLI photon counts in vitro (Pearson r=0.999, p<0.0001, data not shown). The relative expression levels of transduced proteins are shown in Figure 23.

[0389] Flow cytometry. Flow cytometry was performed using an Attune NxT Flow Cytometer (ThermoScientific, Waltham, MA) or BD LSRFortessa (BD Biosciences, San Jose, CA). Phycoerythrin-conjugated anti-human mesothelin rat IgG2a (R&D Systems, Minneapolis, MN) was used to detect human mesothelin cell surface expression on tumor cells. Phycoerythrin-cyanine 7-conjugated anti-human PD-L1 mouse IgG1 (BD Biosciences) was used to detect human PD-L1 cell surface expression on tumor cells. Human T cells were analyzed for cell surface expression of human CD3 using allophycocyanin-cyanin 7-conjugated anti-human CD3 mouse IgG2a or phycoerythrin-cyanin 7-conjugated anti-human CD3 mouse IgG1 antibodies (BioLegend, San Diego, CA), and human CD4 or human CD8 using fluorescein isothiocyanate-conjugated anti-human CD4 mouse IgG1 (BioLegend) or Alexa Fluor 488-conjugated anti-human CD8 mouse IgG1 (BioLegend), respectively. Phycoerythrin-conjugated anti-myc tag antibody (Cell Signaling Technology, Danvers, MA) or Alexa Fluor 647-conjugated F(ab') 2 Fragment-specific goat anti-human F(ab') 2Fragment (Jackson ImmunoResearch, West Grove, PA) was used to quantify cell surface expression of CAR. Cell surface expression of PD1 on CAR T cells was analyzed using Brilliant Violet 711-conjugated anti-human PD1 mouse IgG1 (BioLegend). For ex vivo detection of human T cells, processed mouse tissues were stained with phycoerythrin-cyanine 7-conjugated anti-human CD3 mouse IgG1 antibody and allophycocyanin-cyanine 7-conjugated anti-human CD45 mouse IgG1 antibody (BioLegend). Discrimination of live from dead cells was performed by staining cells with either 4',6-diamidino-2-phenylindole (DAPI, ThermoFisher Scientific, Waltham, MA) or eFluor 506 (ThermoFisher Scientific). 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. [Table 8]

[0390] Determination of VCN. Total genomic DNA from CAR T cells was isolated using a Miniprep Kit (Qiagen, Hilden, Germany). TaqMan PCR primers and probes were used to detect SFG and the housekeeping gene albumin (ALB). Human SFG probe and primer sequences: Probe sequence: 5'-VIC-AGGACCTTACACAGTCCTGCTGAC-TAMRA-3' [SEQ ID NO: 126] Forward primer sequence: 5'-AGAACCTAGAACCTCGCTGGA-3' [SEQ ID NO: 127] Reverse primer sequence: 5'-CTGCGATGCCGTCTACTTTG-3' [SEQ ID NO: 128] Human ALB probe and primer sequences: Probe sequence: 5'-VIC-TGCTGAAACATTCACCTTCCATGCAGA-TAMRA-3' [SEQ ID NO: 129] Forward primer sequence: 5'-TGAAACATACGTTCCCAAAGAGTTT-3' [SEQ ID NO: 130] Reverse primer sequence: 5'-CTCTCCTTCTCAGAAAGTGTGCATAT-3' [SEQ ID NO: 131]

[0391] 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 uL of distilled water. qPCR conditions were as follows: 50°C (2 min), 95°C (20 min), followed by 42 cycles of 95°C (15 s) and 60°C (1 min) using a QuantStudio 7-Flex Real-Time PCR System (ThermoFisher Scientific). All PCR measurements were performed in triplicate. VCN per cell is (mean amount of SFG / mean amount of ALB) * The mean amounts were calculated as the ratio of 2. The mean amounts were extrapolated from the SFG and ALB standard curves.

[0392] Determination of PD1 mRNA expression. Total RNA from CAR T cells was isolated using a Miniprep Kit (Qiagen) and subjected to reverse transcription using a High-Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific). 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: GAPDH primer sequence: Forward primer sequence: 5'-GAAGGTGAAGGTCGGAGT-3' [SEQ ID NO: 132] Reverse primer sequence: 5'-CATGGGTGGAATCATATTGGAA-3' [SEQ ID NO: 133] PD1 extracellular domain primer sequence (Yoon et al., Science. 2015;349(6247):1261669): Forward primer sequence: 5'-CCAGGATGGTTCTTAGACTCCC-3' [SEQ ID NO: 134] Reverse primer sequence: 5'-TTTAGCACGAAGCTCTCCGAT-3' [SEQ ID NO: 135] PD1 intracellular domain primer sequence (Hsu et al., J Immunol. 2016;197(5):1884-1892.): Forward primer sequence: 5'-ACGAGGGACAATAGGAGCCA-3' [SEQ ID NO: 136] Reverse primer sequence: 5'-GGCATACTCCGTCTGCTCAG-3' [SEQ ID NO: 137]

[0393] The cDNA was diluted 5-fold for the following qPCR assay. The amplification reaction (20 μL) contained cDNA derived from 200 ng of total RNA as well as 10 μL of QuantiTect SYBR Green PCR Mix (Qiagen), 4 μL of primers (forward and reverse, 200 nM each), and distilled water. The 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 (20 s for data collection) using a QuantStudio 7 Flex Real-Time PCR system (ThermoFisher Scientific). All PCR measurements 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 target genes was normalized to the reference group as a ratio according to the following Pfaffl formula (Pfaff, Nucleic Acids Res. 2001;29(9):e45): Relative ratio = (E 標的 ) ΔCt標的(対照-試料) / (E 参照 ) ΔCt参照(対照-試料) Target = ...

Claims

1. i) (a) an extracellular antigen-binding domain comprising a heavy chain variable region comprising CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 76, CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 77, and CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 78; and a light chain variable region comprising CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 79, CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 80, and CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 81; (b) an intracellular signaling domain comprising a modified CD3ζ polypeptide comprising an ITAM2 variant comprising the amino acid sequence set forth in SEQ ID NO:29 and an ITAM3 variant comprising the amino acid sequence set forth in SEQ ID NO:33; a chimeric antigen receptor (CAR) that binds to human mesothelin, comprising: ii) (a) at least a portion of the extracellular domain of Programmed Cell Death 1 (PD-1) comprising amino acids 21-165 of SEQ ID NO: 48; and (b) the first transmembrane domain A dominant negative form of programmed cell death 1 (PD-1 DN) containing wherein the CAR is fused to the PD-1 DN by a cleavable peptide.

2. i) the extracellular antigen-binding domain of the CAR specifically binds to human mesothelin with an EC50 value of 1 nM to 25 nM; ii) the extracellular antigen-binding domain of the CAR recognizes human mesothelin at a mesothelin expression level of 1,000 or more mesothelin binding sites / cell; iii) the extracellular antigen-binding domain of the CAR is a single chain variable fragment (scFv), Fab, or F(ab) ２ Including, iv) the extracellular antigen-binding domain of the CAR comprises a linker between the heavy chain variable region and the light chain variable region; and / or v) the extracellular antigen binding domain of the CAR comprises a leader covalently linked to the N-terminus of the extracellular antigen binding domain. The polypeptide of claim 1.

3. i) the heavy chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 82, and the light chain variable region comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 83; or ii) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 82, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 83; A polypeptide according to claim 1 or 2.

4. 4. The polypeptide of any one of claims 1-3, wherein the first transmembrane domain of the PD-1 DNA 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.

5. 5. The polypeptide of claim 4, wherein the first transmembrane domain of the PD-1 DNA comprises a CD8 polypeptide comprising amino acids 137-207 of SEQ ID NO:

86.

6. 6. The polypeptide of any one of claims 1-5, wherein the PD-1 DN comprises amino acids 21-165 of SEQ ID NO:48 and amino acids 137-207 of SEQ ID NO:

86.

7. The polypeptide according to any one of claims 1 to 6, wherein the modified CD3ζ polypeptide comprises a native ITAM1.

8. The polypeptide according to any one of claims 1 to 7, wherein the modified CD3ζ polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO:

35.

9. The polypeptide of any one of claims 1 to 8, wherein the PD-1 DN lacks an intracellular domain.

10. i) the CAR further comprises a second transmembrane domain; and / or ii) the intracellular signaling domain of the CAR further comprises a costimulatory signaling region. A polypeptide according to any one of claims 1 to 9.

11. 11. The polypeptide of claim 10, wherein the second transmembrane domain of the CAR comprises a CD28 polypeptide and / or the costimulatory signaling region comprises a CD28 polypeptide.

12. The polypeptide of any one of claims 1 to 11, wherein the CAR comprises the amino acid sequence set forth in SEQ ID NO:

56.

13. An immune response cell comprising a polypeptide according to any one of claims 1 to 12.

14. i) said PD-1 DN and said CAR are recombinantly expressed; and / or ii) the PD-1 DN and the CAR are expressed from a vector; The immune response cell of claim 13.

15. i) the immune response cells are selected from the group consisting of T cells, natural killer (NK) cells, and lymphoid cells differentiated from pluripotent stem cells; ii) the immune response cell is a T cell; iii) the immune response cell is a T cell selected from the group consisting of a cytotoxic T lymphocyte (CTL), a regulatory T cell, and a natural killer T (NKT) cell; iv) the immune response cells are lymphoid cells differentiated from embryonic stem cells or induced pluripotent stem cells; An immune response cell according to claim 13 or 14.

16. A pharmaceutical composition comprising an effective amount of the immune response cells according to any one of claims 13 to 15 and a pharma- ceutically acceptable excipient.

17. 10 ４ ~10 ６ between at least 10 ５ or 10 of said immune response cells. ５ The pharmaceutical composition of claim 16 comprising said immune response cells.

18. A nucleic acid comprising a polynucleotide encoding the polypeptide of any one of claims 1 to 12.

19. 19. The nucleic acid of claim 18, wherein the polynucleotide comprises the nucleotide sequence set forth in SEQ ID NO:123 or SEQ ID NO:

124.

20. A vector comprising the nucleic acid of claim 18 or 19.

21. The vector of claim 20, which is a retroviral vector, a gamma-retroviral vector or a lentiviral vector.

22. A method for producing an immune response cell, the method comprising introducing into an immune response cell a polypeptide according to any one of claims 1 to 12, a nucleic acid according to claim 18 or 19, or a vector according to claim 20 or 21.

23. A kit comprising a polypeptide according to any one of claims 1 to 12, a nucleic acid according to claim 18 or 19, a vector according to claim 20 or 21, an immune response cell according to any one of claims 13 to 15, or a pharmaceutical composition according to claim 16 or 17.

24. A composition comprising an immune response cell according to any one of claims 13 to 15 or a pharmaceutical composition according to claim 16 or 17 for the treatment and / or prevention of a neoplasm in a subject, for treating a subject with recurrence of a neoplasm, for reducing tumor burden in a subject, for increasing or prolonging survival of a subject with a neoplasm, for preventing and / or treating an inflammatory disease in a subject and / or for preventing graft rejection in a subject who is the recipient of an organ transplant.

25. 25. The composition of claim 24, which reduces the number of tumor cells, reduces tumor size, and / or eradicates the tumor in the subject.

26. The composition of claim 25, wherein the neoplasm and / or the tumor is a solid tumor.

27. 27. The composition of claim 26, wherein the solid tumor is selected from the group consisting of mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colorectal cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic carcinoma, endometrial carcinoma, gastric cancer, cholangiocarcinoma, and combinations thereof.

28. The composition of any one of claims 24 to 27, wherein the subject has undergone immunotherapy prior to administration of the composition.

29. 30. The composition of claim 28, wherein the subject is receiving or has received at least one agent selected from the group consisting of an immunostimulatory agent, a checkpoint immune blockade agent, and combinations thereof.

30. i) the immune stimulatory agent is selected from the group consisting of IL-12, agonistic costimulatory monoclonal antibodies, and combinations thereof; ii) the agonistic costimulatory monoclonal antibody is selected from the group consisting of an anti-4-1BB antibody, an anti-OX40 antibody, an anti-ICOS antibody, and combinations thereof; iii) the checkpoint immune blockade agent is selected from the group consisting of an anti-PD-L1 antibody, an anti-CTLA-4 antibody, an anti-PD-1 antibody, an anti-LAG3 antibody, an anti-B7-H3 antibody, an anti-TIM3 antibody, and combinations thereof; 30. The composition of claim 29.

31. The composition of any one of claims 24 to 30, wherein the immune response cells are administered pleurally or intrapleurally to the subject.

32. The composition of claim 31 , wherein the inflammatory disease is pancreatitis.

33. 32. The composition of claim 31 , wherein the subject is an organ transplant recipient.

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