RU2822193C2 - Mesothelinic car receptors and use thereof - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: there are presented: a polypeptide composition specifically interacting with human mesothelin, an immunoreactive cell for enhancing the immune response to cancer and a method for preparing the same, a pharmaceutical composition for enhancing the immune response to cancer, a nucleic acid composition for producing an immunoreactive cell and an expression vector. Also disclosed is a kit for enhancing the immune response to cancer, a method of treating neoplasia in a subject and treating a subject having a recurrent neoplasia, method of increasing or prolonging the survival rate of a subject having neoplasia, a method of reducing a tumour load in a subject, a method of increasing production of an immunoactivating cytokine in response to a cancer cell or a pathogen in a subject, a method of treating an inflammatory disease in a subject and a method for preventing transplant rejection in a subject.

EFFECT: invention is used to enhance the immune response to cancer, as well as to increase the production of an immunoactivating cytokine in response to a cancer cell or pathogen in a subject.

90 cl, 13 tbl, 94 dwg, 6 ex

Description

CROSS REFERENCE TO RELATED APPLICATION

This application claims benefit from 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 by each of which claims priority.

LIST OF SEQUENCES

This application contains a Sequence Listing that was submitted in ASCII format on the EFS-Web system and is hereby incorporated by reference in its entirety. The ASCII copy created on May 13, 2020 is named 0727341041SL_ST25.TXT and is 144318 bytes in size.

1. INTRODUCTION

The present invention relates to methods and compositions for enhancing the immune response against cancer and pathogens. It refers to chimeric antigen receptors (CARs) that specifically interact with human mesothelin, as well as immunoreactive cells that include such CAR receptors. The mesothelin-targeted CAR receptors disclosed herein have improved immunoactivating properties, including antitumor activity, while at the same time having properties to minimize CAR-induced toxicity and immunogenicity.

2. BACKGROUND OF THE ART

Cellular immunotherapy is a therapy with curative potential for the treatment of cancer. T cells and other immune cells can be modified to target tumor antigens by introducing genetic material encoding artificial or synthetic antigen receptors, called chimeric antigen receptors (CARs), specific for selected antigens. Targeted T-cell therapy using CAR receptors has recently demonstrated clinical success in the treatment of several hematological malignancies. However, translation of CAR-expressing T cell therapy to solid tumors poses several obstacles that must be overcome to achieve clinical benefit. Malignant cells adapt to create an immunosuppressive microenvironment to protect themselves from immune recognition and destruction. This tumor microenvironment poses a challenge to the application of treatments that involve stimulation of the immune response, such as targeted T cell therapy. Solid tumors may also be restricted to anatomical compartments that prevent efficient T cell migration, lack sufficient expression of agonistic costimulatory ligands, and/or express negative regulators of T cell function. Thus, successful eradication of solid tumors requires effective tumor infiltration and overcoming tumor-induced immunosuppression. In addition, solid tumors pose a problem for the selection of optimal immune targets - antigens, targeted interaction with which will allow active T cells to destroy the tumor with minimal or tolerable toxic effects on non-tumor tissues.

Thus, there is a need for new therapeutic strategies to engineer CAR receptors for the treatment of cancer, particularly solid tumors, where such strategies are capable of inducing active tumor killing with minimal toxicity and immunogenicity.

3. SUMMARY OF THE INVENTION

The present invention provides polypeptide compositions comprising: (a) a chimeric antigen receptor (CAR) that specifically interacts with mesothelin (eg, human mesothelin); and (b) a dominant negative form of programmed death protein 1 (PD-1 DN); immunoreactive cells including such polypeptide compositions, and the use of these polypeptide compositions and immunoreactive cells, for example, for the treatment of cancer.

The present invention provides polypeptide compositions. In some embodiments, the polypeptide composition includes: i) a chimeric antigen receptor (CAR) and ii) a dominant negative form of programmed death protein 1 (PD-1 DN), wherein the CAR includes: (a) an extracellular antigen binding domain and (b) an intracellular signaling domain a domain comprising a modified CD3ζ polypeptide comprising an ITAM2 variant and an ITAM3 variant, wherein the ITAM2 variant and ITAM3 variant each include two loss-of-function mutations.

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

In some embodiments, the PD-1 DN includes: (a) at least a portion of a programmed death protein 1 (PD-1) extracellular domain including a ligand binding region, and (b) a first transmembrane domain.

In some embodiments, the first transmembrane domain of the PD-1 DN includes 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- polypeptide 1, CTLA-4 polypeptide, CD27 polypeptide, CD40/My88 peptide, NKGD2 peptide, or a combination thereof. In some embodiments, the first transmembrane domain of the PD-1 DN includes a CD8 polypeptide. In some embodiments, the CD8 polypeptide contained in the first transmembrane domain of the PD-1 DN includes amino acids 137-207 of SEQ ID NO: 86. In some embodiments, the PD-1 DN does not have an intracellular domain. In some embodiments, the PD-1 DN includes amino acids 21-165 of SEQ ID NO: 48 and amino acids 137-207 of SEQ ID NO: 86.

In some embodiments, the extracellular antigen binding domain of the CAR specifically binds to human mesothelin with an EC50 value of from about 1 nM to about 25 nM. In some embodiments, the extracellular antigen binding domain of CAR specifically binds to human mesothelin with an EC50 value of approximately 20 nM.

In some embodiments, the extracellular antigen binding domain of the CAR includes a single chain variable fragment (scFv), a Fab that is optionally cross-linked, or an F(ab) ₂ . In some embodiments, the extracellular antigen binding domain of the CAR includes a human scFv. In some embodiments, the extracellular antigen binding domain of the CAR recognizes human mesothelin with a mesothelin expression level of approximately 1000 or more mesothelin binding sites/cell.

In some 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%, according to is at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 82. In some embodiments, the variable region heavy chain includes the amino acid sequence set forth in SEQ ID NO: 82.

In some 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%, according to is at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 83. In some embodiments, the variable region The light chain includes the amino acid sequence set forth in SEQ ID NO: 83.

In some 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%, according to is at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 82, and the light chain variable region includes 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 is at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 83. In some embodiments, the heavy chain variable region includes the amino acid sequence set forth in SEQ ID NO: 82, and the light chain variable region includes the amino acid sequence set forth in SEQ ID NO: 83.

In some embodiments, the extracellular antigen binding domain of the CAR includes a linker between a heavy chain variable region and a light chain variable region.

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

In some embodiments, each of the loss-of-function mutations in the modified CD3ζ CAR polypeptide is present at a position of a tyrosine amino acid residue. In some embodiments, the ITAM2 variant includes or consists of the amino acid sequence set forth in SEQ ID NO: 29. In some embodiments, the ITAM3 variant includes or consists of the amino acid sequence set forth in SEQ ID NO: 33. In some embodiments, the modified CD3ζ polypeptide includes native ITAM1. In some embodiments, native ITAM1 includes or consists of the amino acid sequence set forth in SEQ ID NO: 23. In some embodiments, the modified CD3ζ polypeptide includes or consists of the amino acid sequence set forth in SEQ ID NO: 35.

In some embodiments, the CAR includes or consists of the amino acid sequence set forth in SEQ ID NO: 56.

In some embodiments, the CAR further includes a second transmembrane domain. In some embodiments, the second transmembrane domain of the CAR includes 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, CTLA-4, CD27 polypeptide, CD40/My88 peptide, NKGD2 peptide, or a combination thereof. In some embodiments, the second transmembrane domain of the CAR includes a CD28 polypeptide

In some embodiments, the intracellular signaling domain of the CAR further includes a co-stimulatory signaling domain. In some embodiments, the costimulatory signaling region includes 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 combinations thereof. In some embodiments, the costimulatory signaling region includes a CD28 polypeptide.

The present invention provides immunoreactive cells comprising the polypeptide composition disclosed herein. In some embodiments, the PD-1 DN and/or CAR is expressed recombinantly. In some embodiments, the PD-1 DN and/or CAR is expressed from a vector. In some embodiments, the immunoreactive cell is selected from the group consisting of a T cell, a natural killer (NK) cell, or a pluripotent stem cell from which lymphoid cells can differentiate. In some embodiments, the pluripotent stem cell is an embryonic stem cell or induced pluripotent stem cells. In some embodiments, the immunoreactive cell is a T cell. In some embodiments, the T cell is selected from the group consisting of a cytotoxic T lymphocyte (CTL), a regulatory T cell, and a natural killer T (NKT) cell. In some embodiments, the immunoreactive cell is autologous. In some embodiments, the immunoreactive cell is allogeneic.

The present invention also provides compositions comprising an immunoreactive cell as disclosed herein. In some embodiments, the composition is a pharmaceutical composition, further comprising a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition includes from about 10 ⁴ to 10 ⁶ immunoreactive cells. In some embodiments, the pharmaceutical composition includes at least about 10 ⁵ immunoreactive cells. In some embodiments, the pharmaceutical composition includes approximately 10 ⁵ immunoreactive cells. In some embodiments, the pharmaceutical composition is for preventing and/or treating neoplasia in a subject, treating a subject having a recurrence of neoplasia, reducing tumor burden in a subject, increasing or prolonging survival of a subject having neoplasia, preventing and/or treating an inflammatory disease in a subject, and /or preventing graft rejection in a subject who is an organ transplant recipient.

In addition, the present invention provides nucleic acid compositions comprising a polynucleotide encoding a polypeptide composition disclosed herein. In some embodiments, the polynucleotide includes the nucleotide sequence set forth in SEQ ID NO: 123. In some embodiments, the polynucleotide includes the nucleotide sequence set forth in SEQ ID NO: 124. The present invention also provides vectors comprising the nucleic acid compositions disclosed herein. In some embodiments, the vector is a retroviral vector. In some embodiments, the retroviral vector is a γ-retroviral vector or a lentiviral vector.

The present invention provides methods for producing the immunoreactive cell disclosed herein. In some embodiments, the method includes introducing into an immunoreactive cell a polypeptide composition disclosed herein, a nucleic acid composition disclosed herein, or a vector disclosed herein.

The present invention provides kits comprising a polypeptide composition disclosed herein, a nucleic acid composition disclosed herein, a vector disclosed herein, an immunoreactive cell disclosed herein, or a pharmaceutical composition disclosed herein. In some embodiments, the kit further includes written instructions for treating and/or preventing the tumor.

In addition, the present invention provides various methods of using the immunoreactive cell described above. For example, the present invention provides methods for reducing tumor burden in a subject, wherein the method comprises administering to the subject an effective amount of immunoreactive cells or a pharmaceutical composition disclosed herein. In some embodiments, the method reduces the number of tumor cells, reduces tumor size, and/or eliminates tumors in a subject.

The present invention also provides methods for increasing or prolonging the survival of a subject having neoplasia, wherein the method comprises administering to the subject an effective amount of an immunoreactive cell disclosed herein or a pharmaceutical composition disclosed herein.

In some embodiments, the tumor or neoplasia is a solid tumor. In some embodiments, the solid tumor is selected from the group consisting of mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic carcinoma, carcinoma endometrial cancer, gastric cancer, cholangiocarcinoma, cervical cancer, salivary gland cancer and combinations thereof.

The present invention provides methods for treating a subject having a relapse of neoplasia, wherein the method comprises administering to the subject an effective amount of immunoreactive cells or a pharmaceutical composition disclosed herein. In some embodiments, the subject received immunotherapy prior to said administration of the immunoreactive cells or composition.

In addition, the present invention provides methods for increasing the production of an immune-activating cytokine in response to a cancer cell or pathogen in a subject. In some embodiments, the method includes administering to a subject an effective amount of immunoreactive cells or a pharmaceutical composition disclosed herein. In some 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.

In accordance with the invention disclosed herein, the various methods described above may involve administering at least one immunomodulatory agent. In some embodiments, the at least one immunomodulatory agent is selected from the group consisting of immunostimulants, immune checkpoint blockers, radiotherapy agents, chemotherapeutic agents, and combinations thereof. In some embodiments, the immunostimulatory agents are selected from the group consisting of IL-12, an agonistic co-stimulatory monoclonal antibody, and combinations thereof. In some 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 combinations thereof. In some embodiments, the agonistic costimulatory monoclonal antibody is an anti-4-1BB antibody. In some embodiments, the immune checkpoint blockers are selected from the group consisting of anti-PD-L1 antibodies, anti-CTLA-4 antibodies, anti-PD-1 antibodies, anti-LAG3 antibodies, anti-B7-H3 antibodies, anti-TIM3 antibodies, and combinations thereof. In some embodiments, the immune checkpoint blocker is an anti-PD-L1 antibody or an anti-PD-1 antibody. In some embodiments, the subject is a human.

In some embodiments, the immunoreactive cell is administered pleurally or intrapleurally to the subject.

The present invention also provides a method for preventing and/or treating an inflammatory disease in a subject. In some embodiments, the method includes administering to a subject an immunoreactive cell or pharmaceutical composition disclosed herein. In some embodiments, the immunoreactive cell is an immunoinhibitory cell. In some embodiments, the immunoinhibitory cell is a regulatory T cell. In some embodiments, the inflammatory disease is pancreatitis. In some embodiments, the subject is a human. In some embodiments, the subject is an organ transplant recipient. In some embodiments, the subject is a pancreas transplant recipient.

The present invention also provides a method for preventing graft rejection in a subject who is an organ transplant recipient. In some embodiments, the method includes administering to a subject an immunoreactive cell or pharmaceutical composition disclosed herein. In some embodiments, the immunoreactive cell is an immunoinhibitory cell. In some embodiments, the immunoinhibitory cell is a regulatory T cell. In some embodiments, the subject is a human. In some embodiments, the subject is a pancreas transplant recipient.

4. BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description, provided by way of example and not intended to limit the invention disclosed herein to the specific embodiments described, can be understood in conjunction with the accompanying drawings.

Figure 1 depicts a polypeptide composition in accordance with some embodiments of the invention disclosed herein. The polypeptide composition includes a CAR including an anti-mesothelin scFv (MSLN), a CD28 transmembrane domain, a CD28 cytoplasmic signaling domain, a CD3zeta signaling domain (eg, including an ITAM2 variant and an ITAM3 variant). CAR is fused to PD1DNR (and the PD-1 signaling domain) via the P2A cleavable peptide. SP: signal peptide; scFv: single chain variable fragment; TM: transmembrane domain; cyt: cytosolic domain; DNR: dominant negative receptor; LTR: long terminal repeat.

Figure 2 depicts various designs disclosed in Example 2.

Figures 3A-3D show virus production in the RD114 producer cell line. 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 an anti-Fab antibody. Blank RD114 served as a negative control. Figure 3A shows empty RD114 (as a negative control). Figure 3B shows the undiluted supernatant. Figure 3C shows the supernatant at a 1:2 dilution; and Figure 3D shows the supernatant at a 1:4 dilution.

Figures 4A-4E show transduction of human T cells M28z1XX-P2A-PD1DNR - donor H116-2. PHA-activated T cells were transduced with various concentrations of RD114 viral supernatant (Figure 4A shows a 1:2 dilution, Figure 4B shows a 1:5 dilution, Figure 4C shows a 1:7 dilution, Figure 4D shows a 1:15 dilution, and Figure 4E shows untransduced ("UT") cells and were stained for CAR expression with an anti-Fab antibody and for PD1DNR with an anti-PD1 antibody by flow cytometry.

Figures 5A-5E show transduction of human T cells M28z1XX-P2A-PD1DNR - donor H18. PHA-activated T cells were transduced with various concentrations of RD114 viral supernatant (Figure 5A shows a 1:2 dilution, Figure 5B shows a 1:5 dilution, Figure 5C shows a 1:10 dilution, Figure 5D shows a 1:15 dilution, and Figure 5E shows untransduced ("UT") cells and were stained for CAR expression with an anti-Fab antibody and for PD1DNR with an anti-PD1 antibody by flow cytometry.

Figures 6A-6F show transduction of human T cells M28z1XX-P2A-PD1DNR - donor H19. PHA-activated T cells were transduced with various concentrations of RD114 viral supernatant (Figure 6A shows a 1:2 dilution, Figure 6B shows a 1:5 dilution, Figure 6C shows a 1:7 dilution; Figure 6D shows a 1:10 dilution, Figure 6E shows a 1:15 dilution and Figure 6F shows untransduced ("UT") cells and were stained for CAR expression with an anti-Fab antibody and for PD1DNR with an anti-PD1 antibody by flow cytometry.

Figures 7A-7C show the correlation of vector copy number (VCN) with mean fluorescence intensity (MFI). PHA-activated T cells were transduced with various concentrations of RD114 viral supernatant and stained for CAR expression with anti-Fab antibody and flow cytometric analysis. Genomic DNA of transduced T cells was isolated and vector copy number was determined as VCN/μg DNA by qPCR. MFI of CAR-positive cells was correlated with VCN/μg DNA for three different donors. Figure 7A shows donor H19; Figure 7B shows donor H18 and Figure 7C shows donor H116-2.

Figure 8 shows cytotoxicity for transduced T cells from 3 different donors. MSLN-high target cells (MGM) were co-cultured with M28z1xx-PD1DNR CAR T cells from different donors at different E:T ratios using an electrical impedance-based assay. M28z1xx-PD1DNR CAR T cell mediated cytolysis of MGM cells at an E:T ratio of 1:1. M28z1xx-PD1DNR CAR T cells killed target cells with high levels of MSLN.

Figure 9 shows an example of electrical resistance-based cytotoxicity (eCTL) measurement.

Figure 10 shows the parameters for comparative analysis of different designs using eCTL.

Figures 11A-11E show the expression of MSLN and PD-L1 of the target cell lines. Mesothelioma cell lines (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)) assessed for MSLN and PD-L1 expression using flow cytometry. MGM, MGM-PDL1, and A549GM overexpressed MSLN; MGM-PDL1 cells further showed overexpression of PD-L1.

Figures 12A-12E show the expression of CAR and PD-1 in transduced T cells. Human T cells transduced with M28z (as shown in Figure 12A), M28z1xx (as shown in Figure 12B), M28z-PD1DNR (as shown in Figure 12C), and M28z1xx-PD1DNR (as shown in Figure 12D) were analyzed for CAR expression when stained with anti-myc antibody and for PD1/PD1DNR expression when stained with anti-PD1 antibody by flow cytometry. Figure 12E shows untransduced (“UT”) T cells.

Figures 13A-13C show a comparative analysis of the antitumor efficacy of CAR T cells bearing the 1XX domain and PD1DNR against MSLN-high (MGM) tumor cells. MSLN-high (MGM) target cells were cocultured with one of M28z, M28z1xx, M28z-PD1DNR, M28z1XX-PD1DNR, or nontransduced T cells at the indicated E:T ratios. Antitumor efficacy was assessed using an electrical impedance assay. Figure 13A shows an E:T ratio of approximately 3:1. Figure 13B shows an E:T ratio of approximately 1:1. Figure 13C shows an E:T ratio of approximately 0.33:1.

Figure 14 shows a comparative analysis of the cytotoxicity of CAR T cells bearing the 1xx domain and PD1DNR against MSLN-high (MGM) tumor cells. Chromium-51-labeled MSLN-high (MGM) target cells were cocultured with one of M28z, M28z1xx, M28z-PD1DNR, M28z1xx-PD1DNR, or non-transduced T cells at the indicated E:T ratio for 18 hours. Cytotoxicity was determined by the release of chromium-51 by CTL.

Figures 15A-15C show a comparative analysis of the antitumor efficacy of CAR T cells bearing the 1xx domain and PD1DNR against MSLN-negative tumor cells (MSTOG). MSLN-negative target cells (MSTOG) were cocultured with one of M28z, M28z1xx, M28z-PD1DNR, M28z1xx-PD1DNR, or non-transduced T cells at the indicated E:T ratios. Antitumor efficacy was assessed using an electrical impedance assay. Figure 15A shows an E:T ratio of approximately 3:1. Figure 15B shows an E:T ratio of approximately 1:1. Figure 15C shows an E:T ratio of approximately 0.33:1.

Figure 16 shows a comparative analysis of the cytotoxicity of CAR T cells bearing the 1XX domain and PD1DNR against MSLN-negative tumor cells (MSTOG). Chromium-51-labeled MSLN-negative target cells (MSTOG) were cocultured with one of M28z, M28z1XX, M28z-PD1DNR, M28z1XX-PD1DNR, or nontransduced T cells at the indicated E:T ratio for 18 hours. Cytotoxicity was determined by the release of chromium-51 by CTL.

Figures 17A-17C show a comparative analysis of the antitumor efficacy of CAR T cells bearing the 1XX domain and PD1DNR against tumor cells with high levels of MSLN and overexpression of PDL1. Target cells with high levels of MSLN and overexpression of PDL1 (MGM-PDL1) were cocultured with one of M28z, M28z1XX, M28z-PD1DNR, M28z1XX-PD1DNR, or non-transduced T cells at the indicated E:T ratios. Antitumor efficacy was assessed using an electrical impedance assay. Figure 17A shows an E:T ratio of approximately 3:1. Figure 17B shows an E:T ratio of approximately 1:1. Figure 17C shows an E:T ratio of approximately 0.33:1.

Figures 18A-18C show a comparative analysis of the antitumor efficacy of CAR T cells harboring the 1xx domain and PD1DNR against MSLN-high tumor cells (A549GM). High MSLN target cells (A549GM) were cocultured with one of M28z, M28z1xx, M28z-PD1DNR, M28z1xx-PD1DNR, or non-transduced T cells at the indicated E:T ratios. Antitumor efficacy was assessed using an electrical impedance assay. Figure 18A shows an E:T ratio of approximately 10:1. Figure 18B shows an E:T ratio of approximately 5:1. Figure 18C shows an E:T ratio of approximately 2:1.

Figures 19A-19C show a comparative analysis of the antitumor efficacy of CAR T cells bearing the 1xx domain and PD1DNR against low MSLN (A549G) tumor cells. Low MSLN target cells (A549G) were cocultured with one of M28z, M28z1xx, M28z-PD1DNR, M28z1xx-PD1DNR, or non-transduced T cells at the indicated E:T ratios. Antitumor efficacy was assessed using an electrical impedance 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.

Figures 20A-20D show the results of various treatments in an in vivo study. Figure 20A shows the comparative in vivo effectiveness of CAR T cells - M28z, M28z with PD-1 antibody and M28z with PD1DNR. Figure 20B shows the comparative in vivo efficacy of M28z and M28z1XX+PD1DNR CAR T cells. Figure 20C shows tumor burden imaging demonstrating systemic antitumor immunity following reintroduction of tumors. Figure 20D shows ex vivo immunofluorescence staining of orthotopic MPM tumors demonstrating CAR T cell infiltration.

Figure 21 shows the structure and components of the M28z1XXPD1DNR CAR. In contrast to M28z, M28z1XXPD1DNR CAR T cells carry a mutant CD3ζ signaling domain with one functional ITAM and coexpress PD1DNR, which consists of the transmembrane and hinge domains of CD8 and lacks the intracellular PD-1 signaling domain present in endogenous PD1.

Figure 22 shows the structure of CAR T cell vectors.

Figure 23 shows the expression of mesothelin (MSLN), PD-L1 and GFP on tumor cell lines. MGM, MGM-PDL1, and MSTOG tumor cells were examined for the expression of mesothelin (left panels), PD-L1 (middle panels), and GFP (right panels) by flow cytometry. Density plots showing the relative intensity of expression as a function of side scatter area (y-axis) are shown.

Figures 24A-24D show a mouse model of orthotopic MPM. Figure 24A shows a macroscopic view of human MPM (top left panel), reproduced in a mouse model of MPM (top right panel), with tumor involvement of the heart, lungs, and mediastinal structures, and tumor invading the chest wall (bottom panel). Figure 24B shows extensive tumor vascularization as demonstrated by CD34 immunofluorescence. Figure 24C shows that progression of tumor burden observed by BLI correlates with MRI tumor volume measurements at corresponding time points. Figure 24D shows the progression of tumor burden observed using sequential BLI and MRI.

Figures 25A-25C show the expression of mesothelin in human tissues by immunohistochemical analysis. Figure 25A shows the expression of mesothelin in the MPM compared to normal pleura and pericardium. Figure 25B shows the expression of mesothelin in lung adenocarcinoma compared to normal lung tissue. Figure 25C shows mesothelin expression in triple negative breast cancer compared to normal breast tissue.

Figure 26 shows that the expression of M28z1XXPD1DNR can be titrated using different dilutions of the viral supernatant. Human T cells were transduced with various dilutions of viral supernatant encoding M28z1XXPD1DNR (left panels) or mycM28z1XXPD1DNR (middle panels). CAR (y-axis) and PD1 (x-axis) expression of viable CD3-positive cells was assessed by flow cytometry. The results presented are from 1 donor, representative of 3 different donors.

Figure 27 shows that CAR expression, as measured by MFI, correlates with VCN. Human T cells obtained from 3 different donors were transduced with different dilutions of retroviral supernatant encoding either M28z1XXPD1DNR or mycM28z1XXPD1DNR. MFI values of CAR-positive T cells (determined by flow cytometry) were plotted against VCN (determined by qPCR). The R2 value was obtained from linear regression analysis (black line).

Figures 28A-28D show the expression of PD-1 and PD1DNR in mycM28z1XXPD1DNR and mycM28z CAR T cells. Figure 28A shows the percentage of CAR surface expression on mycM28z and mycM28z1XXPD1DNR CAR T cells. Figure 28B shows the percentage of CD3 positive cells positive for PD-1 surface expression. Figure 28C shows the MFI of PD-1 expression on the surface of CD3-positive cells. Figure 28D shows the relative expression of extracellular and intracellular domain mRNA of PD1, shown as fold change compared to expression of non-transduced T cells.

Figure 29 shows that M28z1XXPD1DNR-expressing T cells with or without the myc tag exhibited identical antitumor efficacy in vitro. Human T cells obtained from 3 different donors were transduced with either M28z1XXPD1DNR (red) or mycM28z1XXPD1DNR (green) (transduction range, 37%-63%) and cocultured with MGM cells (green; arrow indicates time of T cell addition ). The antitumor efficacy of both constructs was compared at the indicated E:T ratios using an electrical impedance-based cytotoxicity assay.

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 cocultured with one of the MGM, MGM-PDL1, or MSTOG tumor cells at the indicated E:T ratios. The cytotoxicity of CAR T cells was assessed using a 51Cr release assay after 18 h of co-culture. Untransduced (orange) T cells served as controls.

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 contacted with MGM or MGM-PDL1 target cells for 48 h at an E:T ratio of 1:1. CAR T cell accumulation was quantified by counting the absolute number of CAR T cells after each antigen stimulation.

Figure 32 shows that mycM28z1XXPD1DNR CAR T cells exhibited similar cytotoxicity as mycM28z CAR T cells when initially stimulated with antigen. Human T cells transduced with mycM28z1XXPD1DNR (blue) or mycM28z (red) were cocultured with 51Cr-labeled MGM or MGM-PDL1 target cells at the indicated E:T ratios. Cytotoxicity was assessed after 18 h using a 51Cr release assay. Untransduced T cells (orange) served as controls.

Figure 33 shows that mycM28z1XXPD1DNR CAR T cells maintained antitumor efficacy upon repeated antigen stimulation. Human T cells transduced with mycM28z1XXPD1DNR (blue) or mycM28z (red) were repeatedly exposed to MGM (left panels) or MGM-PDL1 (right panels) target cells for 48 h at an E:T ratio of 3:1 during 4 stimulations, followed by 2 additional stimulations at an E:T ratio of 1:1. Cytotoxicity of CAR T cells was assessed using a 51Cr release assay during the fourth and seventh antigen stimulation at the indicated E:T ratios, after 18 h of coculture.

Figure 34 shows that mycM28z1XXPD1DNR CAR T cells secreted effector cytokines when stimulated with antigen. Human T cells transduced with mycM28z1XXPD1DNR (blue) or mycM28z (red) were re-exposed to MGM (top row) or MGM-PDL1 (bottom row) target cells for 48 h at an E:T ratio of 1:1. Cell-free supernatant was collected 24 h after the first, third, and sixth antigen stimulation, and the secretion of effector cytokines was assessed using the Luminex assay.

Figure 35 shows that intrapleural administration of a single low dose of 3x10 ⁴ mycM28z1XXPD1DNR CAR T cells demonstrates antitumor efficacy in vivo. Female NSG mice bearing an orthotopic MGM tumor received 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 using BLI. The indicated time point represents day 15 after CAR T cell administration, when mice receiving P28z CAR T cells began to die. Statistical significance was determined using an unpaired Student's t test (2-tailed). ***p<0.001.

Figures 36A-36D show that intrapleurally administered mycM28z1XXPD1DNR CAR T cells exhibited antitumor efficacy in vivo and increased survival. Figure 3A shows series of BLI images of MGM-PDL1 tumors from tumor-bearing female NSG mice treated with a single dose of mycM28z (1×10 ⁵ ) or mycM28z1XXPD1DNR (1×10 ⁵ or 5×10 ⁴ ) CAR T cells (n=7-8) . Shown are 4 mice per treatment group in a ventral position. Figure 36B shows corresponding series of BLI tumors (average dorsal and ventral) showing the tumor burden of each treated mouse. Figure 36C shows the corresponding weights of the mice after treatment. Figure 36D shows a Kaplan-Meier survival analysis comparing the efficacy of mycM28z and mycM28z1XXPD1DNR CAR T cells in vivo. The survival curve was analyzed using the log-rank test. *p<0.05, **p<0.01.

Figure 37 shows the detection of mycM28z1XXPD1DNR CAR T cells in the primary tumor of mice after intrapleural treatment. Pleural tumor of MGM mice treated with ^5x105 untransduced T cells (left), mycM28z CAR T cells (middle), or mycM28z1XXPD1DNR CAR T cells (right). Tumor tissue was collected 3 days after intrapleural injection of T cells, fixed, and stained ex vivo for tumor mesothelin (green), human CD45-positive cells (red), and DAPI (cell nucleus, blue) by immunofluorescence.

Figures 38A and 38B show that mycM28z1XXPD1DNR CAR T cells prevented tumor re-engraftment when the tumor was reintroduced in vivo. Figure 38A shows a diagram illustrating a tumor reintroduction experiment: 68 days after intrapleural administration of mycM28z or mycM28z1XXPD1DNR CAR T cells (single dose of 1×10 ⁵ CAR T cells) and after pleural eradication of MGM-PDL1 tumor cells (inoculated dose 8×10 ⁵ ), mice were intraperitoneally reinfected 10 times with increasing doses (from 2×10 ⁶ to 11×10 ⁶ ) of MGM tumor cells every 4-8 days. Figure 38B shows a series of BLI images demonstrating tumor burden following a single intrapleural dose of mycM28z (2 mice, red lines) or mycM28z1XXPD1DNR (3 mice, black lines) CAR T cells and tumor re-infusion starting on treatment day 68. Black arrows indicate time points of repeated intraperitoneal infection with tumors with increasing doses.

Figures 39A-39C show that M28z1XXPD1DNR CAR T cells produced using clinical study vector stock have in vivo antitumor efficacy and prolong survival. Figure 39A shows series of BLI images of MGM tumors in tumor-bearing female NSG mice treated with 6x10 ⁴ (n=8) or 2x10 ⁵ (n=10) M28z1XXPD1DNR CAR T cells produced at CTCEF using viral supernatant for clinical research. Figure 39B shows the corresponding weight of the mice after treatment. Figure 39C shows Kaplan-Meier survival analysis.

Figure 40 shows the average body weights of intermediate sacrifices of male mice. Groups 1 (no tumor control), 3 (solvent control), and 5 (test drug) are shown.

Figure 41 shows the average body weights of intermediate sacrifices of female mice. Groups 2 (no tumor control), 4 (control) and 6 (study drug) are shown.

Figure 42 shows the average body weights at final sacrifice of male mice. Groups 7 (no tumor control), 9 (solvent control), and 11 (test drug) are shown.

Figure 43 shows the average body weights at final sacrifice of female mice. Groups 8 (no tumor control), 10 (solvent control), and 12 (test drug) are shown.

Figure 44 shows the identification of human T cells in tumors of mice treated with CAR T cells and treated with vehicle. Intrapleural tumor tissue cells obtained from CAR T cell-treated and vehicle-treated mice were stained with DAPI, anti-human CD45 AFC/CY7 conjugates, and anti-human CD3 PE/CY7 conjugates to detect viable human T cells by flow cytometry. Shown are density plots of human CD3 expression (x-axis) and human CD45 expression (y-axis) in DAPI-negative (live) single cells. Gate indicates cells staining positive for human CD45 and human CD3, representing human T cells.

Figure 45 shows the identification of human T cells in the spleen of CAR T cell-treated and vehicle-treated mice. Spleen tissue cells obtained from CAR T cell-treated and vehicle-treated mice were stained with DAPI, anti-human CD45 AFC/CY7 conjugates, and anti-human CD3 PE/CY7 conjugates to detect viable human T cells by flow cytometry. Density plots of human CD3 expression (X-Axis) and human CD45 expression (Y-Axis) in DAPI-negative (live) single cells are shown. Gate indicates cells staining positive for human CD45 and human CD3, representing human T cells.

Figure 46 shows the BLI of male mice.

Figure 47 shows the BLI of female mice.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention provides polypeptide compositions comprising a chimeric antigen receptor (CAR) targeting mesothelin and a dominant negative form of programmed death protein 1 (PD-1 DN), and immunoreactive cells (for example, T cells or NK cells) comprising polypeptide compositions. The present invention also provides methods of using such a polypeptide composition for inducing and/or enhancing the immune response of an immunoreactive cell to a target antigen, and/or treating and/or preventing neoplasia or other diseases/disorders where reduction in immune cell depletion is desired.

Constant exposure of antigen to T cells, such as in cancer, results in an altered state of T cell differentiation called exhaustion, which renders CAR T cells dysfunctional (Youngblood et al., Int Immunol.2010;22(10):797- 803; Wherry et al., Nat Rev Immunol.2015; 15(8): 486-499). Previous studies have shown that CAR activation potential is associated with three ITAMs (1-2-3) present in the cytoplasmic domain of CD3ζ (Acuto et al., Nat Rev Immunol. 2003; 3(12): 939-951; Love et al. , Cold Spring Harb Perspect Biol 2010; 2(6): a002485. Recent studies have shown that this CAR activation potential can be regulated by mutating ITAM motifs, thereby reducing their functionality. Importantly, it was shown that by introducing point mutations into the second and third positions of the ITAM motifs (1-X-X; herein referred to as “1XX”) of the CD3ζ domain, the direction of CAR T cell differentiation was changed from an exhaustive terminal state to a balanced state effector state and memory state in the presence of high antigenic stimulation (Feucht et al., Nat Med. 2019; 25(1): 82-88).

Another barrier that CAR T cells face in the solid tumor microenvironment is inhibition of their cytolytic activity mediated by PD1, an inhibitory receptor that is expressed upon antigen-mediated T cell activation. In addition, tumor cells upregulate the expression of coinhibitory ligands such as PD-L1 following exposure to T cell-secreted proapoptotic cytokines (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. 20(16): 4262-4273). To overcome this barrier, we combined mesothelin-specific CAR T cells with PD1 blocking antibodies to rescue exhausted CAR T cells, restoring the antitumor efficacy of CAR T cells in an orthotopic mouse model (Cherkassky et al., J Clin Invest. 2016 ; 126(8): 3130-3144; Cancer Cell 2019; To avoid repeated dosing of PD1 checkpoint blockers and their associated clinical side effects, we demonstrated that using a cell-natural PD1 checkpoint blockade strategy, in which PD1DNRs are cotransduced into the T cell along with a second generation CAR, ultimately does transduced cells are 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).

Therefore, to develop CAR T cells with an improved therapeutic profile, functional persistence, and resistance to tumor-mediated inhibition, we incorporated 1XX and PD1DNR components into a second-generation CAR vector design, allowing these cells to function effectively in the highly immunosuppressive microenvironment of solid tumors.

For purposes of clarity of disclosure, but not limitation, the detailed description is divided into the following subsections:

5.1. Definitions;

5.2. Polypeptide compositions;

5.2.1. PD-1 DN;

5.2.2. Mesothelin-targeting CAR receptors; And

5.2.3. Exemplary polypeptide compositions;

5.3. Immunoreactive cells;

5.4. Nucleic acid compositions and vectors;

5.5. Polypeptides and analogues;

5.6. Pharmaceutical compositions and administration;

5.7. Compositions;

5.8. Methods of treatment; And

5.9. Sets

5.1. Definitions

Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly known to one skilled in the art.

As used herein, the term "about" or "approximately" means within the acceptable range of error for a particular value, as determined by one skilled in the art, which will depend in part on how the value is measured or determined, i.e. from the limitations of the measurement system. For example, "about" may mean within 3 or greater than 3 standard deviations, in accordance with practice in the art. Alternatively, "about" may mean a range of up to 20%, such as up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly in relation to biological systems or processes, the term may mean within an order of magnitude, for example within 5 times or 2 times the value.

By "immunoreactive cell" is meant a cell that functions in an immune response, or a precursor or progeny thereof.

By “activates an immunoreactive cell” is meant the induction of signaling or changes in protein expression in a cell leading to the initiation of an immune response. For example, when CD3 chains cluster in response to ligand binding and immunoreceptor tyrosine inhibitory motifs (ITAMs), a signal transduction cascade is formed. In some embodiments, upon binding of a chimeric antigen receptor (CAR) to an antigen, formation of an immunological synapse occurs, which involves clustering of many molecules in the vicinity of the bound receptor (eg, CD4 or CD8, CD3γ/δ/ε/ζ, etc.). This clustering of membrane-bound signaling molecules allows ITAM motifs contained in CD3 chains to be phosphorylated. This phosphorylation, in turn, initiates the T cell activation pathway, ultimately activating transcription factors such as NF-κB and AP-1. These transcription factors induce global T cell gene expression with increased production of IL-2 for proliferation and expression of essential T cell regulatory proteins to initiate a T cell-mediated immune response.

By “stimulates an immunoreactive cell” is meant a signal that results in a robust and long-term immune response. In various embodiments, this occurs following activation of an immunoreactive cell (eg, a T cell) or is simultaneously mediated through receptors including, but not limited to, CD28, CD137 (4-1BB), OX40, CD40, and ICOS. Receiving multiple co-stimulatory signals may be important for generating a robust and long-term T cell-mediated immune response. T cells can quickly go into an inhibited state and stop responding to the antigen. Although the effects of these co-stimulatory signals may vary, they generally result in increased gene expression with the generation of long-lived, proliferative, and antiapoptotic T cells that respond effectively to the antigen until complete and sustained eradication.

As used herein, the term “antibody” refers not only to intact antibody molecules, but also to fragments of antibody molecules that retain the ability to bind an immunogen. Such fragments are also well known in the art and are routinely 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 F(ab') ₂ and Fab fragments. F(ab') ₂ and Fab fragments that lack the Fc portion of the intact antibody are cleared more quickly from the circulation and may have less nonspecific tissue binding than the intact antibody (Wahl et al., J. Nucl. Med 24: 316-325 (1983)). As used herein, antibodies include whole native antibodies, bispecific antibodies; chimeric antibodies; Fab, Fab', single chain fragment V region (scFv), fusion polypeptides and non-standard antibodies. In some embodiments, the antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains linked by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as _VH ) and a heavy chain constant region ( _CH ). The heavy chain constant region consists of three domains, CH1, CH2 and CH3. Each light chain consists of a light chain variable region (abbreviated herein as V _L ) and a light chain constant region C _L . The light chain constant region consists of a single domain, C _L . The V _H and V _L regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), which alternate with more conserved regions called framework regions (FR). Each V _H and V _L consists of three CDR regions and four FR regions, located from N-terminus to C-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The heavy and light chain variable regions contain a binding domain that interacts with the antigen. Antibody constant regions can mediate the binding of immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (C1q) of the classical complement system.

As used herein, "CDR regions" are defined as the amino acid sequences of the complementarity determining regions of an antibody, which are the hypervariable regions of the immunoglobulin heavy and light chains. See, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 4th U.S. Department of Health and Human Services, National Institutes of Health (1987). Typically, antibodies contain in the variable region three CDRs or CDR regions of the heavy chain and three CDRs or CDR regions of the light chain. CDR regions provide the majority of contact residues for binding of an antibody to an antigen or epitope. In some embodiments, the boundaries of the CDR regions are determined using the Kabat system (Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242) . In some embodiments, the CDR regions are identified in accordance with the Kabat system.

As used herein, the term “single chain variable fragment” or “scFv” is a fusion protein of the heavy ( _VH ) and light chain ( _VL ) variable regions of an immunoglobulin covalently linked to form a _VH :: _VL heterodimer. Either _VH and _VL are connected directly or are connected by a linker encoding a peptide (e.g., 10, 15, 20, 25 amino acids) that connects the N-terminus of _VH to the C-terminus of _VL or the C-terminus of _VH to N -end V _L . The linker is usually rich in glycine for flexibility, and serine or threonine for solubility. "Linker", as used herein, shall mean a functional group (eg, chemical or polypeptide) that covalently joins two or more polypeptides or nucleic acids, causing them to bind to each other. As used herein, the term “peptide linker” refers to one or more amino acids used to link two proteins to each other (eg, to link the V _H and V _L domains). In some embodiments, the linker contains or consists of the amino acid sequence set forth in SEQ ID NO: 66, which is presented below:

GGGGSGGGGSGGGGS [SEQ ID NO: 66].

An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 66 is provided in SEQ ID NO: 50, which is given below:

GGAGGTGGAGGCTCAGGAGGAGGAGGCAGTGGAGGTGGTGGGTCA [SEQ ID NO: 50].

An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 66 is provided in SEQ ID NO: 51, which is given below.

GGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCA [SEQ ID NO: 51]

Despite the removal of constant regions and the introduction of a linker, scFv proteins retain the specificity of the original immunoglobulin. Antibodies from a single chain Fv polypeptide can be expressed from a nucleic acid comprising the V _H and V _L coding sequences, as described in Huston, et al. (Proc. Nat. Acad. Sci. USA, 85: 5879-5883, 1988). See also US Patents 5,091,513, 5,132,405 and 4,956,778; and US patent publications 20050196754 and 20050196754. Antagonistic scFv fragments having inhibitory activity have been described (see, for example, 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; 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). see, for example, Peter et al., J Bioi Chern 2003 25278(38): 36740-7; Xie et al., Nat Biotech 1997 15(8): 768-71; 5-6): 427-55; Ho et al., BioChim Biophys Acta 2003 1638(3): 257-66).

As used herein, "F(ab)" refers to a fragment of an antibody structure that binds to an antigen but is monovalent and lacks an Fc portion, e.g., an antibody when digested by the enzyme papain produces two F(ab) fragments and an Fc portion. fragment (eg, heavy chain constant region (H); Fc region, does not bind antigen).

As used herein, "F(ab') ₂ "refers to an antibody fragment generated by pepsin digestion of whole IgG antibodies, such fragment containing two antigen binding (ab') (divalent) regions, where each (ab') region includes two separate amino acid chains, part of the H chain and the light (L) chain, connected by an SS bond, for antigen binding, and where the remaining parts of the H chains are connected to each other. The "F(ab')2" fragment can be divided into two separate Fab' fragments.

As used herein, the term “vector” refers to any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc., that is capable of replication when associated with the desired regulatory elements and that can carry gene sequences into cells. Thus, the term includes cloning and expression vehicles, as well as viral vectors and plasmid vectors.

As used herein, the term “expression vector” refers to a recombinant nucleic acid sequence, that is, a recombinant DNA molecule containing the desired coding sequence and the corresponding nucleic acid sequences necessary for expression of the operably linked coding sequence in a particular host organism. Nucleic acid sequences required for expression in prokaryotes typically include a promoter, an operator (optional), and a ribosome binding site, often together with other sequences. Eukaryotic cells are known to use promoters, enhancers, and termination and polyadenylation signals.

As used herein, the term "affinity" means the magnitude of binding strength. Affinity may depend on the closeness of the stereochemical correspondence between antibody paratopes and antigenic determinants, on the size of the contact area between them, and/or on the distribution of charged and hydrophobic groups. Methods for calculating the affinity of an antibody for an antigen are known in the art, including, but not limited to, various antigen binding experiments, such as functional assays (eg, flow cytometry analysis).

The term "chimeric antigen receptor" or "CAR" as used herein refers to a molecule comprising an extracellular antigen-binding domain that is fused to an intracellular signaling domain that is capable of activating or stimulating an immunoreactive cell, and a transmembrane domain. In some embodiments, the extracellular antigen binding domain of the CAR includes scFv. scFv can be obtained by merging the variable regions of the heavy and light chains of an antibody. Alternatively or additionally, the scFv can be derived from Fab fragments (rather than an antibody, e.g. derived from Fab libraries). In some embodiments, the scFv is fused to a transmembrane domain and then to an intracellular signaling domain. In some embodiments, the CAR is selected to have high binding affinity for an antigen.

As used herein, the term “nucleic acid molecules” includes any nucleic acid molecule encoding a polypeptide or fragment thereof of interest. Such nucleic acid molecules need not be 100% homologous or identical to the endogenous nucleic acid sequence, but may exhibit substantial identity.

By “substantially identical” or “substantially homologous” is meant an amino acid sequence or nucleic acid molecule exhibiting at least about 50% homology or identity to a reference amino acid sequence (e.g., any of the amino acid sequences described herein) or a reference nucleic acid sequence acids (eg, any of the nucleic acid sequences described herein). In some embodiments, such 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 or reference nucleic acid used for comparison.

Sequence identity can be measured using sequence analysis software (e.g., Sequence Analysis Software Package developed by Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP programs). PRETTYBOX). Such programs compare identical or similar sequences, assigning degrees of homology to various substitutions, deletions and/or other modifications. Conservative substitutions usually include substitutions in the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. An exemplary approach to determining the degree of identity may use the BLAST program, where a probability score ranging from e-3 to e-100 indicates a closely related sequence.

As used herein, the percentage of homology between two amino acid sequences is equivalent to the percentage of identity between the two sequences. The percent identity between two sequences is a function of the number of identical positions in the sequences (i.e., % homology=number of identical positions/total number of positions ×100), taking into account the number of gaps and the length of each gap that must be introduced to optimally align the two sequences. Comparing sequences and determining the percent identity between two sequences can be done using a mathematical algorithm.

The percentage of homology between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4: 11-17 (1988)), which was included in the program ALIGN (version 2.0), using the PAM120 residue substitution weight table, a gap length penalty of 12, and a gap penalty of 4. Additionally, the percentage of homology between two amino acid sequences can be determined using the Needleman and Wunsch algorithm and Wunsch, J. Mol. Biol. 48: 444-453 (1970)), which was included in the GAP program in the GCG software package (available at www.gcg.com), using either the Blossum 62 matrix or the PAM250 matrix. , and skip weights 16, 14, 12, 10, 8, 6 or 4 and length weights 1, 2, 3, 4, 5 or 6.

Additionally or alternatively, the amino acid sequences of the invention disclosed herein may further be used as a "query sequence" to perform searches in public databases, for example, to identify related sequences. Such a search can be performed using the XBLAST program (version 2.0) described in Altschul, et al. (1990) J. Mol. Biol. 215:403-10. A BLAST search for proteins can be performed using the XBLAST program with the parameters score=50, wordlength=3, obtaining amino acid sequences homologous to the specified sequences (for example, the heavy and light chain variable region sequences of scFv m903, m904, m905, m906 and m900), disclosed herein. Gapped BLAST can be used to obtain gap alignments for comparison purposes, as described in Altschul et al., (1997) Nucleic Acids Res. 25(17): 3389-3402. When using the BLAST and Gapped BLAST programs, you can use the default settings of the corresponding programs (for example, XBLAST and NBLAST). The term "constitutive expression" or "constitutively expressed" as used herein refers to expression or expression under any physiological conditions.

By “disease” is meant any condition, disease or disorder that damages or disrupts the normal function of a cell, tissue or organ, such as neoplasia and infection of a cell by a pathogen.

By "effective amount" is meant an amount sufficient to produce a therapeutic effect. In some embodiments, an "effective amount" is an amount sufficient to stop, improve, or inhibit ongoing proliferation, growth, or metastasis (eg, invasion or migration) of the neoplasia.

By "modulate" we mean a positive or negative change. Examples of modulation include about 1%, about 2%, about 5%, about 10%, about 25%, about 50%, about 75%, or about 100% change.

By "increase" is meant a positive change of at least approximately 5%. The change may be about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, about 100% or more.

By "decrease" is meant a negative change of at least about 5%. The change may be about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, or even about 100%.

By "isolated cell" is meant a cell separated from the molecular and/or cellular components that normally accompany the cell.

The terms "isolated", "purified" or "biologically pure" refer to a material that has been freed to varying degrees from the components that normally accompany it in its natural state. "Isolate" means the degree of separation from the source or environment. "Purify" means a higher degree of separation than isolation. A “purified” or “biologically pure” protein is sufficiently free from other materials such that any impurities do not significantly affect the biological properties of the protein or cause other adverse effects. In other words, a nucleic acid or peptide is purified if it is substantially free of cellular material, viral material or culture medium when produced using recombinant DNA techniques or chemical precursors or other chemical reagents in chemical synthesis. Purity and homogeneity are usually determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" may mean that the nucleic acid or protein produces essentially a single band on an electrophoretic gel. In the case of a protein that can be subject to modifications, such as phosphorylation or glycosylation, different modifications can result in different isolated proteins that can be purified separately.

By "neoplasia" is meant a disease characterized by abnormal proliferation of a cell or tissue and its subsequent migration or invasion into other tissues or organs. Neoplastic growth is usually uncontrolled and progressive and occurs under conditions that do not or can cause normal cells to stop multiplying. Neoplasia may affect various types of cells, tissues, or organs, including, but not limited to, an organ 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, ovaries, pancreas, prostate, skeletal muscle, skin, spinal cord, spleen, stomach, testicles, thymus, thyroid gland, trachea, genitourinary tract, ureter, urethra, uterus and vagina, or tissue or cell of this type. Neoplasias include malignant tumors such as sarcomas, carcinomas, or plasmacytomas (malignant tumors of plasma cells). In some embodiments, the neoplasia is a solid tumor. Neoplasia can be a primary tumor or a primary cancer. In addition, neoplasia may have a metastatic status.

As used herein, the term “conservative sequence modification” refers to an amino acid modification that does not significantly affect or alter the binding characteristics of a mesothelin-targeted CAR disclosed herein (eg, an extracellular antigen-binding domain CAR) comprising the amino acid sequence. Conservative modifications may include amino acid substitutions, additions and deletions. Modifications can be introduced into the extracellular antigen binding domain of the CAR disclosed herein using standard methods 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 substitutions in which an amino acid residue is replaced by an amino acid from the same group. For example, amino acids can be classified by charge: positively charged amino acids include lysine, arginine, histidine, negatively charged amino acids include aspartic acid, glutamic acid, 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 (acid polar), glutamic acid (acid polar), glutamine, histidine (basic polar), lysine (basic polar), serine, threonine and tyrosine; non-polar amino acids include alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan and valine. Thus, one or more amino acid residues in the CDR region can be replaced with other amino acid residues from the same group, and the modified antibody can be tested for retention of function (ie, the functions specified in paragraphs (c)-(l) above) using functional assays described herein. In some embodiments, no more than one, no more than two, no more than three, no more than four, no more than five residues in the specified sequence or CDR region are modified.

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 synthesized proteins that directs their entry into the secretory pathway. Examples of leader sequences include, but are not limited to, human IL-2 signal sequence (eg, MYRMQLLSCIALSLALVTNS [SEQ ID NO: 67]), mouse IL-2 signal sequence (eg, MYSMQLASCVTLTLVLLVNS [SEQ ID NO: 68]); a human kappa leader sequence (eg, METPAQLLFLLLLWLPDTTG [SEQ ID NO: 69]), a mouse kappa leader sequence (eg, METDTLLLWVLLLWVPGSTG [SEQ ID NO: 70]); a human CD8 leader sequence (eg, MALPVTALLLPLALLLHAARP [SEQ ID NO: 71]); truncated human CD8 signal peptide (eg, MALPVTALLLPLALLLHA [SEQ ID NO: 72]); human albumin signal sequence (eg, MKWVTFISLLFSSAYS [SEQ ID NO: 73]); and a human prolactin signal sequence (eg, MDSKGSSQKGSRLLLLLVVSNLLLCQGVVS [SEQ ID NO: 74]).

In some embodiments, the CAR includes a CD8 signal peptide at the N-terminus, for example, the signal peptide is coupled to the extracellular antigen binding domain of the CAR. In some embodiments, the CD8 signal peptide includes or consists of the amino acid sequence set forth in SEQ ID NO: 71.

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

ATGGCCCTGCCAGTAACGGCTCTGCTGCTGCCACTTGCTCTGCTCCTCCATGCAGCCAGGCCT [SEQ ID NO: 125]

The terms “includes” and “including” are intended to have the broad meaning assigned to them under US Patent Law and may mean “includes”, “comprising” and the like.

As used herein, “treatment” refers to a clinical intervention in an attempt to modify the course of a disease in the individual or cell being treated, and may be carried out either prophylactically or during clinical pathology. Therapeutic effects of treatment include, but are not limited to, prevention of onset or recurrence of disease, relief of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction in the rate of disease progression, improvement or alleviation of pathological condition and remission or improved prognosis. By preventing the progression of a disease or disorder, the treatment may prevent deterioration due to the disorder in an affected subject or a subject diagnosed with or suspected of having a disorder, but the treatment may also prevent the occurrence of a disorder or symptom of a disorder in a subject at risk of developing a disease or suspected of having a disorder. diseases.

An "individual" or "subject" as used herein is 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, sporting animals, rodents and pets. Non-limiting examples of non-human animals include rodents such as mice, rats, hamsters and guinea pigs; rabbits; dogs; cats; sheep; pigs; goats; cattle; horses; and non-human primates such as apes and monkeys. The term "immunodeficient" as used herein refers to a subject who has an immunodeficiency. The subject is highly vulnerable to opportunistic infections, infections caused by organisms that do not normally cause disease in a person with a healthy immune system, but may affect people with a poorly functioning or suppressed immune system.

Other aspects of the invention disclosed herein are described below and are within the scope of the invention disclosed herein.

5.2. Polypeptide compositions

The present invention provides polypeptide compositions comprising a mesothelin-targeted chimeric antigen receptor (CAR) and a dominant negative form of programmed death protein 1 (PD-1 DN).

5.2.1. Dominant negative form of programmed death protein 1 (PD-1 DN)

A dominant negative form of programmed death protein 1 (termed “PD-1 DN”) can enhance the therapeutic efficacy of a CAR immunoreactive cell. In some embodiments, the PD-1 DN includes: (a) at least a portion of a programmed death protein 1 (PD-1) extracellular domain including a ligand binding region, and (b) a transmembrane domain.

In some embodiments, an immunoreactive cell, such as a T cell or a progenitor cell thereof, is modified to express a dominant negative form (DN form) of PD-1.

Malignant cells adapt to generate an immunosuppressive microenvironment that protects cells from immune recognition and elimination (Sharpe et al., Dis. Model Mech. 2015; 8: 337-350). The immunosuppressive microenvironment imposes restrictions on immunotherapy methods. The invention disclosed herein solves this limitation by expressing in the immunoreactive cell or its progenitor cell a DN form of an inhibitor of the cell-mediated immune response. Details of the DN forms of cell-mediated immune response inhibitors are disclosed in WO2017/040945 and WO2017/100428, each of which is incorporated herein in its entirety.

Programmed cell death protein 1 (PD-1) is a negative immune regulator of activated T cells through interaction 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 members of the B7 family. The protein structure includes an extracellular IgV domain, followed by a transmembrane region and an intracellular tail. The intracellular tail contains two phosphorylation sites located at the immunoreceptor tyrosine inhibitory motif and the immunoreceptor tyrosine switching motif. PD-1 negatively regulates TCR signaling. Phosphatases SHP-1 and SHP-2 bind to the cytoplasmic tail of PD-1 upon ligand binding. PD-L1 upregulation is one of the mechanisms that tumor cells use to evade the host immune system. In preclinical and clinical trials, PD-1 blockade with antagonistic antibodies induced antitumor responses mediated by the host's endogenous immune system.

In some embodiments, the PD-1 polypeptide consists of the amino acid sequence GenBank accession number NP_005009.2 (SEQ ID NO: 48) or a fragment thereof. In some embodiments, amino acids 1-20 of SEQ ID NO: 48 are a PD-1 signal peptide (or peptide signal). In some embodiments, amino acids 21-170 of SEQ ID NO: 48 represent the extracellular domain of PD-1. In some embodiments, amino acids 171-191 of SEQ ID NO: 48 represent the transmembrane domain of PD-1. In some embodiments, amino acids 192-288 of SEQ ID NO: 48 represent the intracellular domain of PD-1. SEQ ID NO: 48 is given below:

MQIPQAPWPV VWAVLQLGWR PGWFLDSPDR PWNPPTFSPA LLVVTEGDNA TFTCSFSNTS ESFVLNWYRM SPSNQTDKLA AFPEDRSQPG QDCRFRVTQL PNGRDFHMSV VRARRNDSGT YLCGAISLAP KAQIKESLRA ELRVTERRAE VPTAHPSPSP RPAGQFQTLV VGVVGGLLGS LVLLVWV LAV ICSRAARGTI GARRTGQPLK EDPSAVPVFS VDYGELDFQW REKTPEPPVP CVPEQTEYAT IVFPSGMGTS SPARRGSADG PRSAQPLRPE DGHCSWPL [SEQ ID NO: 48]

In some embodiments, the extracellular domain of PD-1 includes a ligand binding domain (referred to as an “extracellular ligand binding domain”). In some embodiments, the extracellular ligand binding domain of PD-1 is fused to one or more heterologous polypeptide sequences, that is, 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 optionally a heterologous signal peptide including the various signal peptides described herein. In addition, the PD-1 DN may include a transmembrane domain, which is optionally a heterologous transmembrane domain including any of the various transmembrane domains described herein.

In some embodiments, the PD-1 DN comprises an extracellular domain of a PD-1 polypeptide (e.g., amino acids 21-170 in SEQ ID NO: 48) or a ligand binding portion thereof (e.g., amino acids 21-165 in SEQ ID NO: 48). A cell expressing such a PD-1 DN may be unable or may have a reduced ability to signal the PD-1 immune checkpoint pathway. In some embodiments, PD-1 DN is a deletion mutant consisting of a deletion of the intracellular domain (eg, PD-1 DN lacks amino acids 192-288 in SEQ ID NO: 48), or a portion thereof. PD-1, consisting of a deletion of the intracellular domain, may have a reduced or inhibited PD-1-mediated immune checkpoint pathway.

In some embodiments, the PD-1 DN includes an extracellular PD-1 ligand binding domain. In some embodiments, the PD-1 DN includes an extracellular ligand binding domain of a PD-1 polypeptide and a transmembrane domain of a PD-1 polypeptide. In some embodiments, the PD-1 DN includes or consists of the amino acid sequence set forth in SEQ ID NO: 58 (or amino acids 21-165 in SEQ ID NO: 48). SEQ ID NO: 58 is given below.

PGWFLDSPDR PWNPPTFSPA LLVVTEGDNA TFTCSFSNTS ESFVLNWYRM SPSNQTDKLA AFPEDRSQPG QDCRFRVTQL PNGRDFHMSV VRARRNDSGT YLCGAISLAP KAQIKESLRA ELRVTERRAE VPTAHPSPSP RPAGQ [SEQ ID NO: 58]

An exemplary nucleotide sequence encoding SEQ ID NO: 58 (or amino acids 21-165 in SEQ ID NO: 48) is provided in SEQ ID NO: 59, which is listed below.

CCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAACGCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCTTTCCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACACAACTGCCC AACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGCGCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAAGGCGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGAAGGGCAGAAGTGCCCAGCCCACCCCAGCCCCTCACCCAGGCCAGCCGGCCAG [SEQ ID NO: 59]

In some embodiments, the PD-1 DN further includes a signal peptide, for example, the PD-1 DN includes an extracellular ligand binding domain of a PD-1 polypeptide, a transmembrane domain of a PD-1 polypeptide, and a signal peptide of a PD-1 polypeptide. In some embodiments, the signal peptide includes 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 provided in SEQ ID NO: 60, which is listed below.

ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAACTGGGCTGGCGG [SEQ ID NO: 60]

In some embodiments, the PD-1 DN includes or consists of amino acids 1-165 in SEQ ID NO: 48.

An exemplary nucleotide sequence encoding amino acids 1-165 in SEQ ID NO: 48 is provided in SEQ ID NO: 61, which is given below.

ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAACTGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAACGCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCG CTTTCCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACACAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGCGCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAAGGCGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAAGAGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGG CCAGCCGGCCAG [SEQ ID NO: 61]

In some embodiments, the PD-1 DN includes or consists of amino acids 21-151 of SEQ ID NO: 48. In some embodiments, the PD-1 DN includes or consists of amino acids 1-151 of SEQ ID NO: 48. In some embodiments PD-1 DN includes or consists of amino acids 21-151 in SEQ ID NO: 48. In some embodiments, PD-1 DN includes or consists of the amino acid sequence starting from amino acid 21 in SEQ ID NO: 48 to the amino acid between amino acids 151- 165 at SEQ ID NO: 48.

In some embodiments, the PD-1 DN further includes a CD8 polypeptide. In some embodiments, the PD-1 DN includes a PD-1 extracellular domain or a portion thereof (eg, an extracellular ligand binding domain) fused to a CD8 transmembrane domain and/or hinge domain. In some embodiments, the PD-1 DN includes a CD8 transmembrane domain (eg, amino acids 183-203 in SEQ ID NO: 86). Such embodiments are representative of a chimeric form of DN comprising a transmembrane domain from another (heterologous) polypeptide. As described above, a PD-1 DN comprising a heterologous domain, such as a transmembrane domain, may optionally include additional sequence from a heterologous polypeptide. In some embodiments, the PD-1 DN includes additional sequence from a heterologous polypeptide from the N-terminus of the transmembrane domain. In some embodiments, the PD-1 DN includes a CD8 hinge domain. In some embodiments, the heterologous sequence includes an additional N-terminal CD8 polypeptide sequence (eg, amino acids 137-182 (or optionally beginning with amino acids 138 or 139) of SEQ ID NO: 86). In some embodiments, the PD-1 DN includes additional sequence from a heterologous polypeptide from the C terminus of the CD8 transmembrane domain. In some embodiments, the additional C-terminal sequence is amino acids 204-209 of SEQ ID NO: 86.

In some embodiments, the PD-1 DN includes 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 an additional C -terminal sequence of the CD8 polypeptide (eg, amino acids 204-207 of SEQ ID NO: 86). In some embodiments, the PD-1 DN includes a CD8 polypeptide consisting of amino acids 137-207 of SEQ ID NO: 86.

An exemplary nucleotide sequence encoding amino acids 137-207 of SEQ ID NO: 86 is provided in SEQ ID NO: 62, which is given below:

CCCACCACGACGCCAGCGCCGCGACCACCAACCCCGGCGCCCACGATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCCTGGCCGGGACTTGTGGGGTCCTTCCTGTCACTGGTTATCACCCTTTACTGCAAC [SEQ ID NO: 62]

In some embodiments, the PD-1 DN includes 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 an additional C- the terminal sequence of the CD8 polypeptide (eg, amino acids 204-209 of SEQ ID NO: 86). In some embodiments, the PD-1 DN includes a CD8 polypeptide consisting of amino acids 137-209 of SEQ ID NO: 86.

An exemplary nucleotide sequence encoding amino acids 137-209 of SEQ ID NO: 86 is provided in SEQ ID NO: 63, which is given below:

CCCACCACGACGCCAGCGCCGCGACCACCAACCCCGGCGCCCACGATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCCTGGCCGGGACTTGTGGGGTCCTTCCTGTCACTGGTTATCACCCTTTACTGCAACCACAGG [SEQ ID NO: 63]

In some embodiments, the PD-1 DN includes the amino acid sequence set forth in SEQ ID NO: 49, which is listed below.

MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNA

TFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQL

PNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAE

VPTAHPSPSPRPAGQAAAPTTTPAPRPPTPAPTIASQPLSLRPEACRPAA

GGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCN [SEQ ID NO: 49]

An exemplary nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 49 is shown in SEQ ID NO: 64, which is given below:

ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAACTGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAACGCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCC GCTTTCCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACACAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGCGCAATGACAGCGGCACCTAC CTCTGTGGGGCCATCTCCCTGGCCCCCAAGGCGCAGATCAAAGAGAGCCTGCGGCAGAGCTCAGGGTGACAGAGAAGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAG CCGGCCAGGCGGCCGCACCCACCACGACGCCAGCGCCGACCACCAACCCCGGCGCCCACGATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCCTGGCCGGGACTTGTGGGGTCCTTCCTGTCACTGGTTATCACCCTTTACTGCAAC [SEQ ID NO: 6 4]

In some embodiments, the PD-1 DN includes the amino acid sequence set forth in SEQ ID NO: 118, which is listed below.

MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNA

TFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQL

PNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAE

VPTAHPSPSPRPAGQAAAPTTTPAPRPPTPAPTIASQPLSLRPEACRPAA

GGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHR [SEQ ID NO: 118]

An exemplary nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 118 is shown in SEQ ID NO: 119, which is given below:

ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAACTGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAACGCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCTTTCCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACACAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGCGCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAAGGCGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGAAGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCGGCCAGGCGGCCGCACCCACCACGACGCCAGCGCCGCGACCACCAACCCCGGCGCCCACGATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCCTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAACCACAGG [SEQ ID NO: 119]

In some embodiments, the PD-1 DN transmembrane domain includes a hydrophobic alpha helix that spans at least a portion of the membrane. Different transmembrane domains lead to different receptor stability. In accordance with the invention disclosed herein, the transmembrane domain of a PD-1 DN may include a native or modified transmembrane domain of any polypeptide disclosed herein, for example, any transmembrane domain that may be contained in a chimeric antigen receptor. In some embodiments, the transmembrane domain is a CD8 polypeptide, CD28 polypeptide, CD3ζ polypeptide, CD40 polypeptide, 4-1BB polypeptide, OX40 polypeptide, CD84 polypeptide, CD166 polypeptide, CD8a polypeptide, CD8b polypeptide, ICOS polypeptide, ICAM-1 polypeptide, CTLA polypeptide 4, a CD27 polypeptide, a CD40/My88 peptide, an NKGD2 peptide, a synthetic polypeptide (not based on an immune response associated protein), or a combination thereof. In some embodiments, the transmembrane domain is a CD8 polypeptide. The details of these transmembrane domains will be described in the section below.

5.2.2. Mesothelin-targeted chimeric antigen receptor (CAR)

The polypeptide composition disclosed herein includes a CAR that specifically interacts with mesothelin, for example, human mesothelin.

CAR receptors are engineered receptors that convey or impart specificity of interest to an immune effector cell. CAR receptors can be used to transfer the specificity of a monoclonal antibody to a T cell; with the transfer of their coding sequence mediated by retroviral vectors.

There are three generations of CAR receptors. “First generation” CAR receptors typically consist of an extracellular antigen-binding domain (eg, scFv) that is fused to a transmembrane domain that is fused to a cytoplasmic/intracellular signaling domain. “First-generation” CAR receptors can mediate de novo antigen recognition and induce activation of both CD4 ⁺ and CD8 ⁺ T cells through their CD3ζ chain signaling domain in a single fusion molecule independent of HLA-mediated antigen presentation. In “second generation” CAR receptors, the intracellular signaling domains of various co-stimulatory molecules (eg, CD28, 4-1BB, ICOS, OX40, CD27, CD40/My88, and NKGD2) are added to the cytoplasmic tail of the CAR, transducing additional signals to the T cell. “Second generation” CAR receptors include receptors that mediate both costimulation (eg, CD28 or 4-1BB) and activation (CD3ζ). “Third generation” CAR receptors include receptors that mediate multiple co-stimulation (eg, CD28 and 4-1BB) and activation (CD3ζ). In some embodiments, the CAR is a second generation CAR. In some embodiments, the CAR includes an extracellular antigen-binding domain that binds an antigen, a transmembrane domain, and an intracellular signaling domain, wherein the intracellular signaling domain includes a co-stimulatory signaling domain. In some embodiments, the CAR further includes a hinge/spacer region.

5.2.2.1. Extracellular antigen-binding domain of the CAR receptor

The extracellular antigen-binding domain of CAR specifically binds to mesothelin, such as human mesothelin. In some embodiments, the extracellular antigen binding domain is a scFv. In some embodiments, the scFv is a human scFv. In some embodiments, the scFv is a humanized scFv. In some embodiments, the scFv is a murine scFv. In some embodiments, the extracellular antigen binding domain of the CAR is a Fab that is optionally cross-linked. In some embodiments, the extracellular antigen binding domain of the CAR is F(ab) ₂ . In some embodiments, any of the preceding molecules may be contained in a heterologous sequence fusion protein to form an extracellular antigen binding domain. In some embodiments, the scFv is identified by screening a scFv phage library using an antigen-Fc fusion protein. The scFv can be derived from a mouse carrying the human V _L and/or V _H genes. scFv can also be replaced by a camelid heavy chain (eg VHH camel, llama, etc.) or a partial natural cell surface receptor ligand.

Mesothelin is an immunogenic cell surface antigen that is overexpressed in solid malignant tumors. Mesothelin is involved in proliferation, adhesion, invasion, cell signaling and metastasis. Studies have shown that serum-soluble mesothelin-related peptide secreted by mesothelin-expressing tumors can be measured in humans and mice and has been shown to correlate with response to therapy and prognosis. In normal tissues, mesothelin is expressed only in the pleura, pericardium and peritoneum at low levels. The recombinant anti-mesothelin immunotoxin SS1P showed specificity in vivo and significant antitumor activity in patients. In the pancreatic cancer vaccine trial, patients with a survival benefit had robust CD8 ⁺ T cell responses to mesothelin associated with a postvaccination delayed-type hypersensitivity reaction. Specific mesothelin-derived T cell epitopes have been shown to activate human T cells with efficient lysis of human tumors expressing mesothelin. In summary, there is strong evidence that adoptive immunotherapy targeting mesothelin can specifically target mesothelin-expressing tumors.

In some embodiments, the CAR binds to human mesothelin. In some embodiments, human mesothelin includes or consists of the amino acid sequence NCBI number: AAV87530.1 (SEQ ID NO: 75) or a fragment thereof.

SEQ ID NO: 75 is given below:

MALPTARPLL GSCGTPALGS LLFLLFSLGW VQPSRTLAGE TGQEAAPLDG VLANPPNISS LSPRQLLGFP CAEVSGLSTE RVRELAVALA QKNVKLSTEQ LRCLAHRLSE PPEDLDALPL DLLLFLNPDA FSGPQACTHF FSRITKANVD LLPRGAPERQ RLLPAALACW GVRGSLLSEA DVRALGGLAC DLPGRFVAES PRLVS CPGPLDQDQQ EAARAALQGG GPPYGPPSTW SVSTMDALRG LLPVLGQPII RSIPQGIVAA WRQRSSRDPS WRQPERTILR PRFRREVEKT ACPSGKKARE IDESLIFYKK WELEACVDAA LLATQMDRVN AIPFTYEQLD VLKHKLDELY PQGYPESVIQ HLGYLFLKMS PEDIRK WNVT SLETLKALLE VNKGHEMSPQ VATLIDRFVK GRGQLDKDTL DTLTAFYPGY LCSLSPEELS SVPPSSIWAV RPQDLDTCDP RQLDVLYPKA RLAFQNMNGS EYFVKIQSFL GGAPTEDLKA LSQQNVSMDL ATFMKLRTDA VLPLTVAEVQ KLLGPHVEGL KAEERHRPVR DWILRQRQDD LDTLGLGLQG GIPNGYLVLD LSVQEALSGT PCLLGPGPVL TVLALLLAST LA [SEQ ID NO: 75]

In some embodiments, the extracellular antigen binding domain of a CAR (embodied, for example, in a scFv or analog thereof) binds to human mesothelin with an EC50 value of from about 1 nM to about 25 nM, as measured by an enzyme-linked immunosorbent assay (ELISA). In some embodiments, the extracellular antigen binding domain of the CAR has an EC50 value of approximately 20 nM when measured by ELISA. In some embodiments, the extracellular antigen binding domain of CAR includes an anti-mesothelin antibody or antigen binding portion thereof described in US Pat. No. 8,357,783, which is incorporated herein by reference in its entirety. In some embodiments, the extracellular antigen binding domain of the CAR is derived from the heavy chain variable region and the light chain variable region of an antibody that binds to human mesothelin, for example, antibody m912, as 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 some embodiments, the extracellular antigen binding domain of the CAR is derived from a Fab (eg, a human or mouse Fab library).

Binding of the extracellular antigen binding domain (embodiment, e.g., scFv or analogue thereof) of a CAR receptor can be confirmed, e.g., by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS assay, bioassay (eg, growth inhibition), or Western -blotting. Each of these assays typically detects the presence of protein-antibody complexes of particular interest using a labeled reagent (eg, antibody or scFv) specific for the complex of interest. For example, scFv can be radiolabeled 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 included in this document by reference). The radioactive isotope can be detected using means such as a γ-ray counter or scintillation counter, or by autoradiography. In some embodiments, the mesothelin-targeted 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 some embodiments, the human mesothelin-targeted scFv is tagged with GFP.

In some embodiments, the extracellular antigen binding domain of the CAR binds to human mesothelin at a mesothelin level of approximately 1000 or more mesothelin binding sites/cell. In some embodiments, the extracellular antigen binding domain of the CAR binds to human mesothelin at a mesothelin level of from about 1,000 to about 50,000 mesothelin binding sites/cell. In some embodiments, the extracellular antigen binding domain of CAR does not bind to human mesothelin at mesothelin expression levels of less than 1000 mesothelin binding sites/cell, such as human mesothelin expressed in normal tissues, such as normal pleural, pericardial, and peritoneal tissues. In some embodiments, the extracellular antigen binding domain of the CAR does not bind human mesothelin at mesothelin expression levels greater than 50,000 mesothelin binding sites/cell. In some embodiments, the human scFV contained in the CAR binds to human mesothelin at a mesothelin expression level of from about 1,000 to about 50,000 mesothelin binding sites/cell. In some embodiments, the human scFV contained in the CAR does not bind to human mesothelin at a mesothelin expression level greater than 50,000 or less than 1,000 mesothelin binding sites/cell.

In some embodiments, the extracellular antigen binding domain of a CAR (e.g., scFv) includes a heavy chain variable region (V _H ) including a CDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 76, or a conservative modification thereof, CDR2, comprising or consisting of the amino acid sequence presented in SEQ ID NO: 77, or a conservative modification thereof, and CDR3, including or consisting of the amino acid sequence presented in SEQ ID NO: 78, or a conservative modification thereof. In some embodiments, V _H includes CDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 76, CDR2 including or consisting of the amino acid sequence set forth in SEQ ID NO: 77, and CDR3 including or consisting of the amino acid sequence , presented in SEQ ID NO: 78.

In some embodiments, the extracellular antigen-binding domain of a CAR (e.g., scFv) includes a light chain variable region (V _L ) comprising a CDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 79, or a conservative modification thereof, CDR2, comprising or consisting of the amino acid sequence presented in SEQ ID NO: 80, or a conservative modification thereof, and CDR3, including or consisting of the amino acid sequence presented in SEQ ID NO: 81, or a conservative modification thereof. In some embodiments, VL includes CDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 79, CDR2 including or consisting of the amino acid sequence set forth in SEQ ID NO: 80, and CDR3 including or consisting of the amino acid sequence set forth in SEQ ID NO: 80. presented in SEQ ID NO: 81.

In some embodiments, V _H includes CDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 76, or a conservative modification thereof, CDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 77, or a conservative modification thereof , and CDR3, including or consisting of the amino acid sequence presented in SEQ ID NO: 78, a conservative modification thereof; and V _L includes CDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 79 or a conservative modification thereof, CDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 80 or a conservative modification thereof, and CDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 81, or a conservative modification thereof. In some embodiments, V _H includes CDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 76, CDR2 including or consisting of the amino acid sequence set forth in SEQ ID NO: 77, and CDR3 including or consisting of the amino acid sequence presented in SEQ ID NO: 78; and V _L includes CDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 79, CDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 80, and CDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 81. In some embodiments, CDR regions are identified according to the Kabat numbering system.

In some embodiments, the heavy chain variable region (V _H ) includes the amino acid sequence set forth in SEQ ID NO: 82. In some embodiments, the light chain variable region (V L ) includes the amino acid sequence set forth in SEQ ID NO: 83. In some embodiments, the light chain variable region (V _L ) includes the amino acid sequence set forth in SEQ ID NO: 83. In embodiments, V _H includes the amino acid sequence set forth in SEQ ID NO: 82, and V _L includes the amino acid sequence set forth in SEQ ID NO: 83, optionally with (iii) a linker sequence, such as a linker peptide, between V _H and V _L . In some embodiments, the linker includes or consists of an amino acid sequence set forth in SEQ ID NO: 66. In some embodiments, V _H includes an amino acid sequence that is at least about 80% (e.g., at least about 85%, according to is at least about 90% or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO: 82. For example, V _H includes an amino acid sequence that is about 80%, about 81%, about 82%, approximately 83%, approximately 84%, approximately 85%, approximately 86%, approximately 87%, approximately 88%, approximately 89%, approximately 90%, approximately 91%, approximately 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. In some embodiments, V _H includes an amino acid sequence set forth in SEQ ID NO: 82. In some embodiments, V _L includes 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 includes an amino acid sequence that is about 80%, about 81% , approximately 82%, approximately 83%, approximately 84%, approximately 85%, approximately 86%, approximately 87%, approximately 88%, approximately 89%, approximately 90%, approximately 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. In some embodiments, V _L includes an amino acid sequence set forth in SEQ ID NO: 83. In some embodiments, V _H includes an amino acid sequence that is at least about 80% (e.g., at least is 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, and V _L includes 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. In some embodiments, V _H includes an amino acid the sequence presented in SEQ ID NO: 82, and V _L includes the amino acid sequence presented in SEQ ID NO: 83.

An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 82 is provided in SEQ ID NO: 52.

An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 83 is provided in SEQ ID NO: 53.

In some embodiments, the extracellular antigen binding domain of a CAR (e.g., scFv) includes 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%, approximately 89%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98% or approximately 99%) homologous or identical to the amino acid sequence set forth in SEQ ID NO: 84. In some embodiments, the extracellular antigen binding domain of a CAR (e.g., scFv) includes or consists of the amino acid sequence set forth in SEQ ID NO: 84. In some embodiments embodiment, the extracellular antigen binding domain of the CAR (eg, scFv) specifically binds to a human mesothelin polypeptide (eg, a human mesothelin polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 75).

An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 84 is provided in SEQ ID NO: 85.

In some embodiments, the scFv is a human scFv.

The sequences of SEQ ID NOs: 52, 53 and 76-85 are given below:

CAGGTTCAGCTTCAGGAGAGTGGCCCAGGCCTGGTGAAGCCAAGTGAGACTCTCAGCTTGACTTGCACAGTTTCTGGAGGCAGTGTCTCCTCAGGCAGCTATTATTGGTCCTGGATTCGGCAGCCCCCTGGGAAAGGCCTGGAGTGGATTGGGTACATATATTACAGTGGCAGCACAAATTACAATCCATCCCTGAAGTCTCGAGTAACTATCAGTGTGGACACAAGCAAGAATCAGTTTTC ACTCAAACTGTCTTCTGTGACTGCTGCTGACACTGCTGTTTATTATTGTGCCAGGGAGGGGAAAAATGGGGCATTTGATATTTGGGGTCAGGGCACAATGGTGACAGTCAGCTCT [SEQ ID NO: 52]

CGCCATCAGATGACTCAGTCCCCCTCCAGTCTTTCTGCCTCAGTTGGGGATAGAGTGACCATCACATGCAGAGCAAGTCAGAGCATATCATCCTACTGAACTGGTACCAGCAGAAGCCAGGGAAAGCCCCCAAATTGCTGATTTATGCAGCCTCAAGTCTCCAGAGTGGGGTGCCAAGCAGGTTCTCAGGCAGTGGCAGTGGGACAGATTTCACATTGACAATCAGCTCCCTCCAACCTGAAG ATTTTGCCACCTACTATTGCCAGCAATCCTACAGCACGCCCCTGACTTTTGGAGGTGGCACAAAGGTAGAGATCAAGAGGACT [SEQ ID NO: 53]

GGSVSSGSYY [SEQ ID NO: 76]

IYYSGST [SEQ ID NO: 77]

AREGKNGAFDIW [SEQ ID NO: 78]

QSISSY [SEQ ID NO: 79]

AASS [SEQ ID NO: 80]

QQSYSTPLTF [SEQ ID NO: 81]

[SEQ ID NO: 82]

RHQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIKRT [SEQ ID NO: 83]

QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGSYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREGKNGAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSRHQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQ SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIKRT [SEQ ID NO: 84]

CAGGTTCAGCTTCAGGAGAGTGGCCCAGGCCTGGTGAAGCCAAGTGAGACTCTCAGCTTGACTTGCACAGTTTCTGGAGGCAGTGTCTCCTCAGGCAGCTATTATTGGTCCTGGATTCGGCAGCCCCCTGGGAAAGGCCTGGAGTGGATTGGGTACATATATTACAGTGGCAGCACAAATTACAATCCATCCCTGAAGTCTCGAGTAACTATCAGTGTGGACACAAGCAAGAATCAGTTTTC ACTCAAACTGTCTTCTGTGACTGCTGCTGACACTGCTGTTTATTATTGTGCCAGGGAGGGGAAAAATGGGGCATTTGATATTTGGGGTCAGGGCACAATGGTGACAGTCAGCTCTGGAGGTGGAGGCTCAGGAGGAGGAGGCAGTGGAGGTGGTGGGTCACGCCATCAGATGACTCAGTCCCCCTCCAGTCTTTCTGCCTCAGTTGGGGATAGAGTGACCATCACATGCAGAGCAAGTCAGAGCATAT CATCCTATCTGAACTGGTACCAGCAGAAGCCAGGGAAAGCCCCCAAATTGCTGATTTATGCAGCCTCAAGTCTCCAGAGTGGGGTGCCAAGCAGGTTCTCAGGCAGTGGCAGTGGGACAGATTTCACATTGACAATCAGCTCCCTCCAACCTGAAGATTTTGCCACCTACTATTGCCAGCAATCCTACAGCACGCCCCTGACTTTTGGAGGTGGCACAAAGGTAGAGATCAAGAGGACT [SEQ ID NO: 85]

In some 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%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98% or approximately 99%) homologous or identical to the amino acid sequence presented in SEQ ID NO: 36, which is given below.

S SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTSGQAG [SEQ ID NO: 36]

An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 36 is provided in SEQ ID NO: 37, which appears below.

caggtgcagctgcaggagtccggcccaggactggtgaagccttcggagaccctgtccctc 60

acctgcactgtctctggtggctccgtcagcagtggtagttactactggagctggatccgg 120

cagcccccagggaagggactggagtggattgggtatatctattacagtgggagcaccaac 180

tacaacccctccctcaagagtcgagtcaccatatcagtagacacgtccaagaaccagttc 240

tccctgaagctgagctctgtgaccgctgcggacacggccgtgtattactgtgcgagagag 300

gggaagaatggggcttttgatatctggggccaagggacaatggtcaccgtctcttcagcc 360

tccaccaagggccatcggtcttccccctggcaccctcctccaagagcacctctgggggc 420

acagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtgg 480

aactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcagga 540

ctctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctac 600

atctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaa 660

tcttgtgacaaaactagtggccaggccggccac 693 [SEQ ID NO: 37]

In some 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 by 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% ) is homologous or identical to the amino acid sequence shown in SEQ ID NO: 38, which is given below.

DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSGFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC [SEQ ID NO: 38]

An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 38 is provided in SEQ ID NO: 39, which appears below.

gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcacc 60

atcacttgccgggcaagtcagagcattagcagctatttaaattggtatcagcagaaacca 120

gggaaagcccctaagctcctgatctatgctgcatccagtttgcaaagtggggtcccatca 180

gggttcagtggcagtggatctgggacagatttcactctcaccatcagcagtctgcaacct 240

gaagatttgcaacttactactgtcaacagagttacagtaccccgctcactttcggcgga 300

gggaccaaggtggagatcaaacgaactgtggctgcaccatctgtcttcatcttcccgcca 360

tctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctat 420

cccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccag 480

gagagtgtcacagagcaggacagcaaggacagcacctactgcctcagcagcaccctgacg 540

ctgagcaaagcagactacgagaaacacaaactctacgcctgcgaagtcacccatcagggc 600

ctgagctcgcccgtcacaaagagcttcaacaggggagagt [SEQ ID NO: 39]

In some 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%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98% or approximately 99%) homologous or identical to the amino acid sequence presented in SEQ ID NO: 40, which is given below.

RHQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSGFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC [SEQ ID NO: 40]

In some 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%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98% or approximately 99%) homologous or identical to the amino acid sequence presented in SEQ ID NO: 41, which is given below.

[SEQ ID NO: 41]

In some embodiments, the light chain variable region includes amino acids 1-107 of SEQ ID NO: 38. In some embodiments, the light chain variable region includes amino acids 1-107 of SEQ ID NO: 40.

In some embodiments, the extracellular antigen binding domain of a CAR (e.g., scFv) includes 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%, approximately 89%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98% or approximately 99%) homologous or identical to the amino acid sequence shown in SEQ ID NO: 42, which is listed below.

QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGSYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREGKNGAFDIWGQGTMVTVSSSGGGGSGGGGSGGGGSRHQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQ SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIKGQAGHHHHHHGDYKDDDDKG [SEQ ID NO: 42]

An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 42 is provided in SEQ ID NO: 45, which appears below.

atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccgcaggtgcagctgcaggagtccggcccaggactggtgaagccttcggagaccctgtccctcacctgcactgtctctggtggctccgtcagcagtggtagttactactggagctggatccggcagcccccagggaagggactggagtggatt gggtatatctattacagtgggagcaccaactacaacccctccctcaagagtcgagtcaccatatcagtagacacgtccaagaaccagttctccctgaagctgagctctg tgaccgctgcggacacggccgtgtattactgtgcgagagaggggaagaatggggcttttgatatctggggccaagggacaatggtcaccgtctcttcaggtggaggcgg ttcaggcggaggtggctctggcggtggcggatcacgacatcagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagtcagagcattagcagctatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcatccagtttgcaaagtggggtcccatca aggttcagtggcagtggatctgggacagatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttactactgtcaacagagttacagtaccccgctcactttcggcggagggaccaaggtggagatcaaacggactgcggccgca [SEQ ID NO: 45]

An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 42 is provided in SEQ ID NO: 46, which appears below. The nucleic acid sequence shown in SEQ ID NO: 46 is synthetically optimized for codon usage, which can enhance CAR expression.

ATGGCGCTGCCGGTGACCGCGCTGCTGCTGCCGCTGGCGCTGCTGCTGCATGCGGCGCGCCCGCAGGTGCAGCTGCAGGAAAGCGGCCCGGGCCTGGTGAAACCGAGCGAAACCCTGAGCCTGACCTGCACCGTGAGCGGCGGCAGCGTGAGCAGCGGCAGCTATTATTGGAGCTGGATTCGCCAGCCGCCGGGCAAAGGCCTGGAATGGATTGGCTATATTTATTATAGCGGCAGCACCAACTATA ACCCGAGCCTGAAAAGCCGCGTGACCATTAGCGTGGATACCAGCAAAAACCAGTTTAGCCTGAAACTGAGCAGCGTGACCGCGGCGGATACCGCGGTGTATTATTGCGCGCGCGAAGGCAAAAACGGCGCGTTTGATATTTGGGGCCAGGGCACCATGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCGCCATCAGATGACCCAGAGCCCGAGCAGCCTGA GCGCGAGCGTGGGCGATCGCGTGACCATTACCTGCCGCGCGAGCCAGAGCATTAGCAGCTATCTGAACTGGTATCAGCAGAAACCGGGCAAAGCGCCGAAACTGCTGATTTATGCGGCGAGCAGC CTGCAGAGCGGCGTGCCGAGCCGCTTTAGCGGCAGCGGCAGCGGCACCGATTTTACCCTGACCATTAGCAGCCTGCAGCCGGAAGATTTTGCGACCTATTATTGCCAGCAGCTATAGCACCCCG CTGACCTTTGGCGGCGGCACCAAAGTGGAAATTAAACGCACCGCGGCGGCG [SEQ ID NO: 46]

An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 42 is provided in SEQ ID NO: 47, which is given below. The nucleic acid sequence shown in SEQ ID NO: 47 is synthetically optimized for codon usage, which can enhance CAR expression.

atggccCTCCCGGTAACGGCTCTGCTGCTTCCACTCGCACTGCTCTTGCATGCTGCCAGACCACAGGTCCAGCTGCAGGAGAGTGGGCCTGGACTGGTTAAGCCGAGTGAGACACTTTCCTTGACGTGCACTGTGAGCGGGGGAAGTGTGTCCTCAGGTAGTTATTACTGGTCCTGGATTCGCCAGCCACCAGGAAAGGGACTGGAGTGGATAGGTTATATCTATTATTCTGGCAGCACTAA TTACAATCTTCTCTCAAAAGTAGGGTGACAATTTCAGTGGATACTTCCAAAAATCAGTTTAGTCTGAAGCTCAGCTCTGTGACAGCTGCTGATACTGCAGTTTACTACTGCGCCAGGGAGGGGAAGAATGGCGCCTTCGATATTTGGGGACAGGGCACTATGGTGACTGTATCAAGCGGAGGCGGTGGCAGCGGCGGGGGAGGGAGTGGAGGCGGCGGGTCTCGACATCAGATGACACAGAGCC CATCATCACTTAGCGCCAGCTTGGCGACCGGGTTACGATAACATGCAGGGCTTCCCAATCTATCAGTTCTTATCTGAACTGGTATCAGCAGAAACCAGGTAAGGCCCCAAGCTGCTCATC TACGCAGCCTCATCCCTGCAGAGCGGCGTCCCTAGTCGATTTTCCGGTAGTGGGTCAGGGACAGATTTTACCCTGACTATCAGTTCACTGCAGCCCGAGGACTTCGCGACATACTATTGCCA ACAGTCCTATAGTACACCCTTGACATTTGGCGGCGGACTAAAGTAGAAATTAAACGCACCgcggccgca [SEQ ID NO: 47]

V _H and/or V _L amino acid sequences consisting of at least about 80%, at least about 80%, at least about 85%, at least about 90%, or at least about by 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 a specific 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 ) may include substitutions (eg conservative substitutions), insertions or deletions from the specified sequence(s) while still retaining the ability to bind the target antigen (eg mesothelin). In some embodiments, a total of 1-10 amino acids are replaced, inserted, and/or deleted in a specific 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 some embodiments, substitutions, insertions, or deletions are present in regions outside the CDR regions (eg, FR regions) of the extracellular antigen-binding domain. In some embodiments, the extracellular antigen binding domain comprises a V _H and/or V _L sequence selected from the group consisting of SEQ ID NOs: 82 and 83, including post-translational modifications of such sequence (SEQ ID NOs: 82 and 83).

5.2.2.2. Transmembrane domain of CAR

In some embodiments, the CAR includes a transmembrane domain. In some embodiments, the transmembrane domain of the CAR includes a hydrophobic alpha helix that spans at least a portion of the membrane. Different transmembrane domains provide different receptor stability. Once the antigen is recognized, the receptors form a cluster and the signal is transmitted to the cell. In accordance with the invention disclosed herein, the transmembrane domain of a CAR may include a native 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 an immune response associated protein), or a combination thereof.

CD8

In some embodiments, the transmembrane domain includes a CD8 polypeptide (eg, a CD8 transmembrane domain or a portion thereof). In some embodiments, the transmembrane domain includes the human CD8 transmembrane domain or a portion thereof. In some embodiments, the CD8 polypeptide includes 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 NCBI sequence number: NP_001139345.1 (SEQ ID NO : 86), or a fragment thereof, and/or may optionally include up to one or two or three conservative amino acid substitutions. In some embodiments, the CD8 polypeptide includes or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 86 that has a length of at least about 20, or at least about 30, or at least about 40, or at least about 50, or at least about 60, or at least about 70, and up to about 235 amino acids. In some embodiments, the 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 some embodiments The CAR of the invention disclosed herein includes 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 some embodiments, the transmembrane domain of the CAR includes a CD8 polypeptide comprising or consisting of amino acids 137- 207 from SEQ ID NO: 86.

MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVELKCQVLLSNPTSGCSWLFQPRGAAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVC KCPRPVVKSGDKPSLSARYV [SEQ ID NO: 86]

In some embodiments, the transmembrane domain includes the mouse CD8 transmembrane domain or a portion thereof. In some embodiments, the CD8 polypeptide includes 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 NCBI sequence number: AAA92533.1 (SEQ ID NO : 87), or a fragment thereof, and/or may optionally include up to one or two or three conservative amino acid substitutions. In some embodiments, the CD8 polypeptide includes or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 87 that has a length of at least about 20, or at least about 30, or at least about 40, or at least about 50, or at least about 60, or at least about 70, or at least about 100, or at least about 200 and up to 247 amino acids. In some embodiments, the CD8 polypeptide comprises or consists of the 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 some embodiments embodiment, the transmembrane domain of the CAR includes a CD8 polypeptide that includes or consists of amino acids 151-219 of SEQ ID NO: 87.

1 MASPLTRFLS LNLLLLMGESI ILGSGEAKPQ APELRIFPKK MDAELGQKVD LVCEVLGSVS

61 QGCSWLFQNS SSKLPQPTFV VYMASSHNKI TWDEKLNSSK LFSAVRDTNN KYVLTLNKFS

121 KENEGYYFCS VISNSVMYFS SVVPVLQKVN STTTKPVLRT PSPVHPTGTS QPQRPEDCRP

181 RGSVKGTGLD FACDIYIWAP LAGICVAPLL SLIITLICYH RSRKRVCKCP RPLVRQEGKP

241 RPSEKIV [SEQ ID NO: 87]

In some embodiments, the CD8 polypeptide includes or consists of the amino acid sequence set forth in SEQ ID NO: 88, which is set forth below:

STTTKPVLRTPSPVHPTGTSQPQRPEDCRPRGSVKGTGLDFACDIYIWAPLAGICVALLLSLIITLICY [SEQ ID NO: 88]

An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 88 is provided in SEQ ID NO: 89, which is given below.

TCTACTACTACCAAGCCAGTGCTGCGAACTCCCTCACCTGTGCACCCTACCGGGACATCTCAGCCCCAGAGACCAGAAGATTGTCGGCCCCGTGGCTCAGTGAAGGGGACCGGATTGGACTTCGCCTGTGATATTTACATCTGGGCACCCTTGGCCGGAATCTGCGTGGCCCTTCTGCTGTCCTTGATCATCACTCTCATCTGCTAC [SEQ ID NO: 89]

CD28

In some embodiments, the CAR transmembrane domain includes a CD28 polypeptide (eg, the CD28 transmembrane domain or a portion thereof). In some embodiments, the transmembrane domain includes the human CD28 transmembrane domain or a portion thereof. In some embodiments, the CD28 polypeptide includes 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 NCBI sequence number: NP_006130 (SEQ ID NO: 90 ), or a fragment thereof, and/or may optionally include up to one or two or three conservative amino acid substitutions. In some embodiments, the CD28 polypeptide includes 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 in length , or at least about 50, and up to about 220 amino acids. In some embodiments, the CD28 polypeptide comprises or consists of the amino acid sequence of amino acids 1-220, 1-50, 50-100, 100-150, 114-220, 153-179, 150-200, or 200-220 from SEQ ID NO: 90 In some embodiments, the CAR transmembrane domain comprises a CD28 polypeptide comprising or consisting of SEQ ID NO: 92 (or amino acids 153-179 of SEQ ID NO: 90). An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 92 or amino acids 153-179 of SEQ ID NO: 90 is provided in SEQ ID NO: 93. In some embodiments, the CAR transmembrane domain includes a CD28 polypeptide comprising or consisting of the amino acid sequence amino acids 114-220 of SEQ ID NO: 90. An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 92 (or amino acids 153-179 of SEQ ID NO: 90) is provided in SEQ ID NO: 91. SEQ ID NO: 90 -93 are given below:

1 MLRLLLALNL FPSIQVTGNK ILVKQSPMLV AYDNAVNLSC KYSYNLFSRE FRASLHKGLD

61 SAVEVCVVYG NYSQQLQVYS KTGFNCDGKL GNESVTFYLQ NLYVNQTDIY FCKIEVMYPP

121 PYLDNEKSNG TIIHVKGKHL CPSPLFPGPS KPFWVLVVVG GVLACYSLLV TVAFIIFWVR

181 SKRSRLLHSD YMNMTPRRPG PTRKHYQPYA PPRDFAAYRS [SEQ ID NO: 90]

ttttgggtgctggtggtggttggtggagtcctggcttgctatagcttgctagtaacagtggcctttattattttctgggtg [SEQ ID NO: 91]

FWVLVVVGGV LACYSLLVTV AFIIFWV [SEQ ID NO: 92].

TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTG [SEQ ID NO: 93]

In some embodiments, the transmembrane domain includes the mouse CD28 transmembrane domain or a portion thereof. In some embodiments, the CD28 polypeptide includes 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 NCBI sequence number: NP_031668.3 (SEQ ID NO : 97) or a fragment thereof, and/or may optionally include up to one or two or three conservative amino acid substitutions. In some embodiments, the CD28 polypeptide includes or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 97 that has a length of 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 some 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 from SEQ ID NO: 97 In some embodiments, the CAR transmembrane domain comprises a CD28 polypeptide comprising or consisting of amino acids 151-177 of SEQ ID NO: 97.

SEQ ID NO: 97 is given below:

1 MTLRLLFLAL NFFSVQVTEN KILVKQSPLL VVDSNEVSLS CRYSYNLLAK EFRASLYKGV

61 NSDVEVCVGN GNFTYQPQFR SNAEFNCDGD FDNETVTFRL WNLHVNHTDI YFCKIEFMYP

121 PPYLDNERSN GTIIHIKEKH LCHTQSSPKL FWALVVVAGV LFCYGLLVTV ALCVIWTNSR

181 RNRLLQSDYM NMTPRRPGLT RKPYQPYAPA RDFAAYRP [SEQ ID NO: 97]

CD84

In some embodiments, the CAR transmembrane domain includes a native or modified CD84 polypeptide transmembrane domain 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 at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to NCBI sequence number: NP_001171808.1 (SEQ ID NO: 1) or a fragment thereof , and/or may optionally include up to one or two or up to three conservative amino acid substitutions. In some embodiments, the CD84 polypeptide includes or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 1 that is at least about 20 in length, or at least about 30 in length, or at least about 40 in length, or at least about 50 in length. and up to approximately 345 amino acids. In some 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 some embodiments, the CAR transmembrane domain includes or consists of a CD84 polypeptide comprising or consisting of amino acids 226-250 of SEQ ID NO: 1.

SEQ ID NO: 1 is given below:

1 maqhhlwill lclqtwpeaa gkdseiftvn gilgesvtfp vniqeprqvk iiawtsktsv

61 ayvtpgdset apvvtvthrn yyerihalgp nynlvisdlr medagdykad intqadpytt

121 tkrynlqiyr rlgkpkitqs lmasvnstcn vtltcsveke eknvtynwsp lgeegnvlqi

181 fqtpedqelt ytctaqnpvs nnsdsisarq lcadiamgfr thhtgllsvl amffllvlil

241 ssvflfrlfk rrqgrifpeg sclntftknp yaaskktiyt yimasrntqp aesriydeil

301 qskvlpskee pvntvysevq fadkmgkast qdskppgtss yeivi [SEQ ID NO: 1]

An exemplary nucleotide sequence encoding amino acids 226-250 of SEQ ID NO: 1 is provided in SEQ ID NO: 2, which appears below.

TTGCTGACGTGCTGGCTATGTTCTTTCTGCTTGTTCTCATTCTGTCTTCAGTGTTTTTGTTCCGTTTGTTCAAG [SEQ ID NO: 2]

CD166

In some embodiments, the CAR transmembrane domain includes a native or modified CD166 polypeptide transmembrane domain 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 at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to NCBI sequence number: NP_001618.2 (SEQ ID NO: 3) or a fragment thereof , and/or may optionally include up to one or two or up to three conservative amino acid substitutions. In some embodiments, the CD166 polypeptide includes or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 3 that has a length of 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 some embodiments, the CD166 polypeptide comprises or consists of the amino acid sequence of amino acids 1-583, 1-50, 50-100, 100-150, 150-200, 200-300, 300-400, 400-500, 528-549, or 500 -583 of SEQ ID NO: 3. In some embodiments, the CD166 polypeptide contained in the transmembrane domain of a CAR receptor disclosed herein includes or consists of the amino acid sequence of amino acids 528-553 of SEQ ID NO: 3. In some embodiments, the polypeptide CD166, contained in the transmembrane domain of CAR, includes or consists of the amino acid sequence of amino acids 528-549 of SEQ ID NO: 3.

SEQ ID NO: 3 is given below:

1 MESKGASSCR LLFCLLISAT VFRPGLGWYT VNSAYGDTII IPCRLDVPQN LMFGKWKYEK

61 PDGSPVFIAF RSSTKKSVQY DDVPEYKDRL NLSENYTLSI SNARISDEKR FVCMLVTEDN

121 VFEAPTIVKV FKQPSKPEIV SKALFLETEQ LKKLGDCISE DSYPDGNITW YRNGKVLHPL

181 EGAVVIIFKK EMDPVTQLYT MTSTLEYKTT KADIQMPFTC SVTYYGPSGQ KTIHSEQAVF

241 DIYYPTEQVT IQVLPPKNAI KEGDNITLKC LGNGNPPPEE FLFYLPGQPE GIRSSNTYTL

301 TDVRRNATGD YKCSLIDKKS MIASTAITVH YLDLSLNPSG EVTRQIGDAL PVSCTISASR

361 NATVVWMKDN IRLRSSPFS SLHYQDAGNY VCETALQEVE GLKKRESLTL IVEGKPQIKM

421 TKKTDPSGLS KTIICHVEGF PKPAIQWTIT GSGSVINQTE ESPYINGRYY SKIIISPEEN

481 VTLTCTAENQ LERTVNSLNV SAISIPEHDE ADEISDENRE KVNDQAKLIV GIVVGLLLAA

541 LVAGVVYWLY MKKSKTASKH VNKDLGNMEE NKKLEENNHK TEA [SEQ ID NO: 3]

An exemplary nucleotide sequence encoding amino acids 528-553 of SEQ ID NO: 3 is provided in SEQ ID NO: 4, which appears below.

CTAATTGTGGGAATCGTTGTTGGTCTCCTCCTTGCTGCCCTTGTTGCTGGTGTCGTCTACTGGCTGTACATGAAGAAG [SEQ ID NO: 4]

CD8a

In some embodiments, the CAR transmembrane domain includes a native or modified CD8a polypeptide transmembrane domain 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 is at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the sequence consisting of NCBI number: NP_001139345.1 (SEQ ID NO: 5) or fragments thereof, and/or may optionally include up to one or two or three conservative amino acid substitutions. In some embodiments, the CD8a polypeptide includes or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 5 that has a length of at least about 20, or at least about 30, or at least about 40, or at least about 50 and up to approximately 235 amino acids. In some 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 some embodiments embodiment, the CAR transmembrane domain comprises a CD8a polypeptide comprising or consisting of amino acids 183-207 of SEQ ID NO: 5. SEQ ID NO: 5 is given below:

1 MALPVTALLL PLALLLHAAR PSQFRVSPLD RTWNLGETVE LKCQVLLSNP TSGCSWLFQP

61 RGAAASPTFL LYLSQNKPKA AEGLDTQRFS GKRLGDTFVL TLSDFRRENE GYYFCSALSN

121 SIMYFSHFVP VFLPAKPTTT PAPRPPTPAP TIASQPLSLR PEACRPAAGG AVHTRGLDFA

181 CDIYIWAPLA GTCGVLLLSL VITLYCNHRN RRRVCKCPRP VVKSGDKPSL SARYV

[SEQ ID NO: 5]

An exemplary nucleotide sequence encoding amino acids 183-207 of SEQ ID NO: 5 is provided in SEQ ID NO: 6, which appears below.

atctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgcaac [SEQ ID NO: 6]

CD8b

In some embodiments, the CAR transmembrane domain includes a native or modified CD8b polypeptide transmembrane domain 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 is at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to sequence number NCBI: NP_742099.1 (SEQ ID NO: 7), or its fragments, and/or may optionally include up to one or two or three conservative amino acid substitutions. In some embodiments, the CD8b polypeptide includes or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 7 that has a length of at least about 20, or at least about 30, or at least about 40, or at least about 50 and up to approximately 221 amino acids. In some 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 some embodiments embodiment, the CAR transmembrane domain comprises a CD8b polypeptide comprising or consisting of amino acids 171-195 of SEQ ID NO: 7. SEQ ID NO: 7 is given below:

1 MRPRLWLLLA AQLTVLHGNS VLQQTPAYIK VQTNKMVMLS CEAKISLSNM RIYWLRQRQA

61 PSSDSHHEFL ALWDSAKGTI HGEEVEQEKI AVFRDASRFI LNLTSVKPED SGIYFCMIVG

121 SPELTFGKGT QLSVVDFLPT TAQPTKKSTL KKRVCRLPRP ETQKGPLCSP ITLGLLVAGV

181 LVLLVSLGVA IHLCCRRRRA RLRFMKQLRL HPLEKCSRMD Y [SEQ ID NO: 7]

An exemplary nucleotide sequence encoding amino acids 171-195 of SEQ ID NO: 7 is provided in SEQ ID NO: 8, which appears below.

ATCACCCTTGGCCTGCTGGTGGCTGGCGTCCTGGTTCTGCTGGTTTCCCTGGGAGTGGCCATCCACCTGTGCTGC [SEQ ID NO: 8]

ICOS

In some embodiments, the CAR transmembrane domain includes a native or modified ICOS polypeptide transmembrane domain or a portion thereof. An 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 at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to NCBI sequence number: NP_036224.1 (SEQ ID NO: 9) or a fragment thereof , and/or may optionally include up to one or two or up to three conservative amino acid substitutions. In some embodiments, the ICOS polypeptide includes or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 9 that has a length of at least about 20, or at least about 30, or at least about 40, or at least about 50 and up to approximately 199 amino acids. In some 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 some embodiments, the CAR transmembrane domain includes an ICOS polypeptide comprising or consisting of amino acids 141-165 of SEQ ID NO: 9. SEQ ID NO: 9 is given below:

1 MKSGLWYFFL FCLRIKVLTG EINGSANYEM FIFHNGGVQI LCKYPDIVQQ FKMQLLKGGQ

61 ILCDLTKTKG SGNTVSIKSL KFCHSQLSNN SVSFFLYNLD HSHANYYFCN LSIFDPPPFK

121 VTLTGGYLHI YESQLCCQLK FWLPIGCAAF VVVCILGCIL ICWLTKKKYS SSVHDPNGEY

181 MFMRAVNTAK KSRLTDVTL [SEQ ID NO: 9]

An exemplary nucleotide sequence encoding amino acids 141-165 of SEQ ID NO: 9 is provided in SEQ ID NO: 10, which appears below.

TTCTGGTTACCCATAGGATGTGCAGCCTTTGTTGTAGTCTGCATTTTGGGATGCATACTTATTTGTTGGCTTACA [SEQ ID NO: 10]

CTLA-4

In some embodiments, the CAR transmembrane domain includes a native or modified CTLA-4 polypeptide transmembrane domain or a portion thereof. A 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%, is at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to sequence number NCBI: NP_005205.2 (SEQ ID NO: 11) or fragment thereof, and/or may optionally include up to one or two or up to three conservative amino acid substitutions. In some embodiments, the CTLA-4 polypeptide includes or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 11 that is at least about 20 in length, or at least about 30 in length, or at least about 40 in length, or at least about 40 in length. approximately 50 to approximately 223 amino acids. In some 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 from SEQ ID NO: 11. B In some embodiments, the CAR transmembrane domain comprises a CTLA-4 polypeptide comprising or consisting of amino acids 162-186 of SEQ ID NO: 11. SEQ ID NO: 11 is set forth below:

1 MACLGFQRHK AQLNLATRTW PCTLLFFLLF IPVFCKAMHV AQPAVVLASS RGIASFVCEY

61 ASPGKATEVR VTVLRQADSQ VTEVCAATYM MGNELTFLDD SICTGTSSGN QVNLTIQGLR

121 AMDTGLYICK VELMYPPPYY LGIGNGTQIY VIDPEPCPDS DFLLWILAAV SSGLFFYSFL

181 LTAVSLSKML KKRSPLTTGV YVKMPPTEPE CEKQFQPYFI PIN [SEQ ID NO: 11]

An exemplary nucleotide sequence encoding amino acids 162-186 of SEQ ID NO: 11 is provided in SEQ ID NO: 12, which appears below.

TTCCTCCTCTGGATCCTTGCAGCAGTTAGTTCGGGGGTTGTTTTTTTATAGCTTTCTCCTCACAGCTGTTTCTTTG [SEQ ID NO: 12]

ICAM-1

In some embodiments, the CAR transmembrane domain includes a native or modified ICAM-1 polypeptide transmembrane domain 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%, is at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to sequence number NCBI: NP_000192.2 (SEQ ID NO: 13) or fragment thereof, and/or may optionally include up to one or two or up to three conservative amino acid substitutions. In some embodiments, the ICAM-1 polypeptide includes or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 13 that has a length of at least about 20, or at least about 30, or at least about 40, or at least from about 50 to about 220 amino acids. In some 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 some embodiments, the CAR transmembrane domain comprises an ICAM-1 polypeptide comprising or consisting of amino acids 481-507 of SEQ ID NO: 13. SEQ ID NO: 13 is set forth below:

1 MAPSSPRPAL PALLVLLGAL FPGPGNAQTS VSPSKVILPR GGSVLVTCST SCDQPKLLGI

61 ETPLPKKELL LPGNNRKVYE LSNVQEDSQP MCYSNCPDGQ STAKTFLTVY WTPERVELAP

121 LPSWQPVGKN LTLRCQVEGG APRANLTVVL LRGEKELKRE PAVGEPAEVT TTVLVRRDHH

181 GANFSCRTEL DLRPQGLELF ENTSAPYQLQ TFVLPATPPQ LVSPRVLEVD TQGTVVCSLD

241 GLFPVSEAQV HLALGDQRLN PTVTYGNDSF SAKASVSVTA EDEGTQRLTC AVILGNQSQE

301 TLQTVTIYSF PAPNVILTKP EVSEGTEVTV KCEAHPRAKV TLNGVPAQPL GPRAQLLLKA

361 TPEDNGRSFS CSATLEVAGQ LIHKNQTREL RVLYGPRLDE RDCPGNWTWP ENSQQTPMCQ

421 AWGNPLPELK CLKDGTFPLP IGESVTVTRD LEGTYLCRAR STQGEVTRKV TVNVLSPRYE

481 IVIITVVAAA VIMGTAGLST YLYNRQRKIK KYRLQQAQKG TPMKPNTQAT PP [SEQ ID NO: 13]

An exemplary nucleotide sequence encoding amino acids 481-507 of SEQ ID NO: 13 is provided in SEQ ID NO: 14, which appears below.

ATTGTCATCATCACTGTGGTAGCAGCCGCAGTCATAATGGGCACTGCAGGCCTCAGCACGTACCCTCTATAACCGCCAGCGG [SEQ ID NO: 14]

5.2.2.3. CAR hinge/spacer region

In some embodiments, the CAR includes a hinge/spacer region that connects the extracellular antigen-binding domain to the transmembrane domain. The hinge/spacer region may be flexible enough to allow the antigen binding domain to be oriented in different directions, facilitating antigen recognition. In some embodiments, the hinge/spacer region of a CAR may include a native or modified hinge region of a CD8 polypeptide, CD28 polypeptide, CD3ζ polypeptide, CD40 polypeptide, 4-1BB polypeptide, OX40 polypeptide, CD84 polypeptide, CD166 polypeptide, CD8a polypeptide, CD8b polypeptide, ICOS polypeptide , ICAM-1 polypeptide, CTLA-4 polypeptide, CD27 polypeptide, CD40/My88 peptide, NKGD2 peptide, synthetic polypeptide (not based on immune response associated protein), or a combination thereof. The hinge/spacer region may be an IgG1 hinge region or a CH ₂ CH ₃ immunoglobulin region and CD3 portions, a CD28 polypeptide portion (e.g., a portion of SEQ ID NO: 90), a CD8 polypeptide portion (e.g., a portion of SEQ ID NO: 86, or a portion of SEQ ID NO: 87), a variant of any of the foregoing, which 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 to them, or a synthetic spacer sequence.

CD28

In some embodiments, the CAR hinge/spacer region includes a native or modified CD28 polypeptide hinge region or a portion thereof, as described herein. In some embodiments, the CAR hinge/spacer region 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 given below.

IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP [SEQ ID NO: 15]

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 provided in SEQ ID NO: 54, which appears below.

attgaagttatgtatcctcctccttacctagacaatgagaagagcaatggaaccattatccatgtgaaagggaaacacctttgtccaagtcccctatttcccggaccttctaagccc [SEQ ID NO: 54]

CD84

In some embodiments, the CAR hinge/spacer region includes a native or modified CD84 polypeptide hinge region or a portion thereof, as described herein. In some embodiments, the CAR hinge/spacer region 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 provided in SEQ ID NO: 16, which is given below.

CAAGAGCTGACTTACACGTGTACAGCCCAGAACCCTGTCAGCAACAATTCTGACTCCATCTCTGCCCGGCAGCTCTGTGCAGACATCGCAATGGGCTTCCGTACTCACCACACCGGG [SEQ ID NO: 16]

CD166

In some embodiments, the CAR hinge/spacer region includes a native or modified CD166 polypeptide hinge region or a portion thereof, as described herein. In some embodiments, the CAR hinge/spacer region 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 provided in SEQ ID NO: 17, which is given below.

ACCAACTGGAGAGAACAGTAAACTCCTTGAATGTCTCTGCTATAAGTATTCCAGAACACGATGAGGCAGACGAGATAAGTGATGAAAACAGAGAAAAGGTGAATGACCAGGCAAAA [SEQ ID NO: 17]

In some embodiments, the CAR hinge/spacer region includes a CD166 polypeptide comprising or consisting of amino acids 484-527 of SEQ ID NO: 3. In some embodiments, the CAR hinge/spacer region includes a CD166 polypeptide comprising or consisting of amino acids 506-527 of SEQ ID NO: 3. In some embodiments, the CAR hinge/spacer region includes a CD166 polypeptide comprising or consisting of amino acids 517-527 of SEQ ID NO: 3. In some embodiments, the CAR hinge/spacer region includes a CD166 polypeptide comprising or consisting of from the amino acid sequence shown in SEQ ID NO: 109 or SEQ ID NO: 110. SEQ ID NO: 109 and 110 are given below.

NQLERTVNSLNVPAISIPEHDEADEISDENREKVNDQAK [SEQ ID NO: 109]

AAANQLERTVNSLNVSAISIPEHDEADEISDENREKVNDQAK [SEQ ID NO: 110]

In some embodiments, the CD166 polypeptide contained in the hinge/spacer region and transmembrane domain of the CAR includes 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 NO: 111-117 are given below.

PEHDEADEISDENREKVNDQAKLIVGIVVGLLLAALVAGVVYWLYMKK [SEQ ID NO: 111]

ENREKVNDQAKLIVGIVVGLLLAALVAGVVYWLYMKK [SEQ ID NO: 112]

NQLERTVNSLNVPAISIPEHDEADEISDENREKVNDQAKLIVGIVVGLLLAALVAGVVYWLYMKK [SEQ ID NO: 113]

TCTAENQLERTVNSLNVSAISIPEHDEADEISDENREKVNDQAKLIVGIVVGLLLAALVAGVVYWL [SEQ ID NO: 114]

PEHDEADEISDENREKVNDQAKLIVGIVVGLLLAALVAGVVYWL [SEQ ID NO: 115]

NQLERTVNSLNVSAISIPEHDEADEISDENREKVNDQAKLIVGIVVGLLLAALVAGVVYWL [SEQ ID NO: 116]

AAANQLERTVNSLNVSAISIPEHDEADEISDENREKVNDQAKLIVGIVVGLLLAALVAGVVYWLYMKK [SEQ ID NO: 117]

CD8a

In some embodiments, the CAR hinge/spacer region includes a native or modified CD8a polypeptide hinge region or a portion thereof, as described herein. In some embodiments, the CAR hinge/spacer region 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 provided in SEQ ID NO: 18, which is given below.

cccaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgaggggctggacttcgcctgtgat [SEQ ID NO: 18]

CD8b

In some embodiments, the hinge/spacer region of the CAR includes a native or modified hinge region of a CD8b polypeptide, as described herein. In some embodiments, the CD8b polypeptide contained in the hinge/spacer region of the CAR includes 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 provided in SEQ ID NO: 7. NO: 19, which is given below.

CTGAGTGTGGTTGATTTCCTTCCCACCACTGCCCAGCCCACCAAGAAGTCCACCCTCAAGAAGAGTGTGCCGGTTACCCAGGCCAGAGACCCAGAAGGGCCCACTTTGTAGCCCC [SEQ ID NO: 19]

ICOS

In some embodiments, the CAR hinge/spacer region includes a native or modified ICOS polypeptide hinge region or a portion thereof, as described herein. In some embodiments, the CAR hinge/spacer region 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 provided in SEQ ID NO: 20, which is given below.

tctcatgccaactattacttctgcaacctatcaatttttgatcctcctccttttaaagtaactcttacaggaggatatttgcatatttatgaatcacaactttgttgccagctgaag [SEQ ID NO: 20]

CTLA-4

In some embodiments, the CAR hinge/spacer region includes a native or modified CTLA-4 polypeptide hinge region or a portion thereof, as described herein. In some embodiments, the CAR hinge/spacer region 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 provided in SEQ ID NO: 11. NO: 21, which is given below.

GACACGGGACTCTACATCTGCAAGGTGGAGCTCATGTACCCACCGCCATACTACCTGGGCATAGGCAACGGAACCCAGATTTATGTAATTGATCCAGAACCGTGCCCAGATTCTGAC [SEQ ID NO: 21]

ICAM-1

In some embodiments, the CAR hinge/spacer region includes a native or modified ICAM-1 polypeptide hinge region or a portion thereof, as described herein. In some embodiments, the CAR hinge/spacer region 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 provided in SEQ ID NO: 13. NO: 22, which is given below.

GGGGAATCAGTGACTGTCACTCGAGATCTTGAGGGCACCTACCTCTGTCGGGCCAGGAGCACTCAAGGGGAGGTCACCCGCAAGGTGACCGTGAATGTGCTCTCCCCCCGGTATGAG [SEQ ID NO: 22]

In some embodiments, the mesothelin-targeted CAR includes a hinge/spacer region. In some embodiments, the hinge/spacer region is located between the extracellular antigen binding domain and the transmembrane domain. In some embodiments, the hinge/spacer region includes a CD8 polypeptide, CD28 polypeptide, CD3ζ polypeptide, CD4 polypeptide, 4-1BB polypeptide, OX40 polypeptide, CD166 polypeptide, CD8a polypeptide, CD8b polypeptide, ICOS polypeptide, ICAM-1 polypeptide, CTLA-4 polypeptide , CD27 polypeptide, CD40/My88 peptide, NKGD2 peptide, synthetic polypeptide (not based on immune response associated protein), or a combination thereof. In some embodiments, the transmembrane domain includes a CD8 polypeptide, CD28 polypeptide, CD3ζ polypeptide, CD4 polypeptide, 4-1BB polypeptide, OX40 polypeptide, CD166 polypeptide, CD8a polypeptide, CD8b polypeptide, ICOS polypeptide, ICAM-1 polypeptide, CTLA-4 polypeptide, polypeptide CD27, CD40/My88 peptide, NKGD2 peptide, synthetic polypeptide (not based on immune response associated protein), or a combination thereof.

In some embodiments, the transmembrane domain and the hinge/spacer region are derived from the same molecule. In some embodiments, the transmembrane domain and the hinge/spacer region are derived from different molecules. In some embodiments, the CAR hinge/spacer region includes a CD28 polypeptide, and the CAR transmembrane domain includes a CD28 polypeptide. In some embodiments, the CAR hinge/spacer region includes a CD28 polypeptide, and the CAR transmembrane domain includes a CD28 polypeptide. In some embodiments, the CAR hinge/spacer region includes a CD84 polypeptide, and the CAR transmembrane domain includes a CD84 polypeptide. In some embodiments, the CAR hinge/spacer region includes a CD166 polypeptide, and the CAR transmembrane domain includes a CD166 polypeptide. In some embodiments, the CAR hinge/spacer region includes a CD8a polypeptide, and the CAR transmembrane domain includes a CD8a polypeptide. In some embodiments, the CAR hinge/spacer region includes a CD8b polypeptide, and the CAR transmembrane domain includes a CD8b polypeptide. In some embodiments, the CAR hinge/spacer region includes a CD28 polypeptide and the CAR transmembrane domain includes an ICOS polypeptide.

5.2.2.4. Intracellular signaling domain of CAR

A. CD3ζ

In some embodiments, the CAR receptor includes an intracellular signaling domain. In some embodiments, the CAR intracellular signaling domain includes a CD3ζ polypeptide that can activate or stimulate a cell (eg, a lymphoid lineage cell, eg, a T cell). Wild-type (“native”) CD3ζ includes three immunoreceptor tyrosine activating motifs (“ITAMs”) (e.g., ITAM1, ITAM2, and ITAM3), three basic residue-rich region (BRS) regions (BRS1, BRS2, and BRS3), and transmits signal activation into a cell (eg, a lymphoid lineage cell, eg, a T cell) upon antigen binding. The intracellular signaling domain of the native CD3ζ chain is the main transmitter of signals from endogenous TCR receptors.

In some embodiments, the intracellular signaling domain of the CAR includes a native CD3ζ polypeptide. In some embodiments, the native CD3ζ polypeptide includes 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 is 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 NCBI sequence number: NP_932170 (SEQ ID NO: 94) or its fragment. In some embodiments, the native CD3ζ polypeptide includes or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 94 that has a length of at least about 20, or at least about 30, or at least about 40, or at least about 50, or at least about 100, or at least about 110 and up to about 164 amino acids. In some embodiments, the native CD3ζ polypeptide includes or consists of the amino acid sequence of amino acids 1-50, 50-100, 100-150, 50-164, 55-164, or 150-164 of SEQ ID NO: 94. In some embodiments, the native polypeptide CD3ζ comprises or consists of the amino acid sequence of amino acids 52-164 of SEQ ID NO: 94.

SEQ ID NO: 94 is given below:

1 MKWKALFTAA ILQAQLPITE AQSFGLLDPK LCYLLDGILF IYGVILTALF LRVKFSRSAD

61 APAYQQGQNQ LYNELNLGRR EEYDVLDKRR GRDPEMGGKP QRRKNPQEGL YNELQKDKMA

121 EAYSEIGMKG ERRRGKGHDG LYQGLSTATK DTYDALHMQA LPPR [SEQ ID NO: 94]

In some embodiments, the CD3ζ polypeptide includes 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 is 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 95, or fragment thereof, and/or may optionally include up to one or two or up to three conservative amino acid substitutions. SEQ ID NO: 95 is given below:

RVKFSRSADA PAYQQGQNQL YNELNLGRRE EYDVLDKRRG RDPEMGGKPR RKNPQEGLYN

ELQKDKMAEA YSEIGMKGER RRGKGHDGLY QGLSTATKDT YDALHMQALP PR [SEQ ID NO: 95]

An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 95 is provided in SEQ ID NO: 96, which is given below.

AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCG CCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC [SEQ ID NO: 96]

In some embodiments, the CAR intracellular signaling domain includes a modified CD3ζ polypeptide. In some embodiments, the CAR intracellular signaling domain includes a modified human CD3ζ polypeptide. In some embodiments, the modified CD3ζ polypeptide includes 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 is at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 35, or a fragment thereof, and/or may optionally include up to one or two or up to three conservative amino acid substitutions. SEQ ID NO: 35 is given below:

RVKFSRSADA PAYQQGQNQL YNELNLGRRE EYDVLDKRRG RDPEMGGKPR RKNPQEGLFN

ELQKDKMAEA FSEIGMKGER RRGKGHDGLF QGLSTATKDT FDALHMQALP PR [SEQ ID NO: 35]

An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 35 is provided in SEQ ID NO: 55, which is given below.

agagtgaagttcagcaggagcgcagacgcccccgcgtaccagcaggggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtTcaatgaactgcagaaagataagatggcggaggcct TcagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttTccaggggctcagtacagccaccaaggacacctTcgacgcccttcacatgcaggccctgccccctcgc [SEQ ID NO: 55]

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

QNQLYNELNLGRREEYDVLDKR [SEQ ID NO: 23]

An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 23 is provided in SEQ ID NO: 24, which appears below.

cagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagaga [SEQ ID NO: 24]

In some embodiments, the modified CD3ζ polypeptide includes an ITAM1 variant including one or more loss-of-function mutations. In some embodiments, the ITAM1 variant includes or consists of two loss-of-function mutations. In some embodiments, each of the one or more (eg, two) loss-of-function mutations includes or consists of a mutation of a tyrosine residue in ITAM1. In some embodiments, an ITAM1 variant (eg, a variant consisting of two loss-of-function mutations) includes or consists of the amino acid sequence set forth in SEQ ID NO: 25, which is set forth below.

QNQLFNELNLGRREEFDVLDKR [SEQ ID NO: 25]

An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 25 is provided in SEQ ID NO: 26, which appears below.

cagaaccagctctTtaacgagctcaatctaggacgaagagaggagtTcgatgttttggacaagaga [SEQ ID NO: 26]

In some embodiments, the modified CD3ζ polypeptide includes native ITAM2. In some embodiments, native ITAM2 includes or consists of the amino acid sequence set forth in SEQ ID NO: 27, which is listed below.

QEGLYNELQKDKMAEAYSEIGMK [SEQ ID NO: 27]

An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 27 is provided in SEQ ID NO: 28, which appears below.

caggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaa [SEQ ID NO: 28]

In some embodiments, the modified CD3ζ polypeptide includes an ITAM2 variant including one or more loss-of-function mutations. In some embodiments, the ITAM2 variant includes or consists of two loss-of-function mutations. In some embodiments, each of the one or more (eg, two) loss-of-function mutations includes or consists of a mutation of a tyrosine residue in ITAM2. In some embodiments, an ITAM2 variant (eg, a variant consisting of two loss-of-function mutations) includes or consists of the amino acid sequence set forth in SEQ ID NO: 29, which is set forth below.

QEGLFNELQKDKMAEAFSEIGMK [SEQ ID NO: 29]

An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 29 is provided in SEQ ID NO: 30, which is given below.

caggaaggcctgtTcaatgaactgcagaaagataagatggcggaggcctTcagtgagattgggatgaaa [SEQ ID NO: 30]

In some embodiments, the modified CD3ζ polypeptide includes native ITAM3. In some embodiments, native ITAM3 includes or consists of the amino acid sequence set forth in SEQ ID NO: 31, which is listed below.

HDGLYQGLSTATKDTYDALHMQ [SEQ ID NO: 31]

An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 31 is provided in SEQ ID NO: 32, which appears below.

cacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcag [SEQ ID NO: 32]

In some embodiments, the modified CD3ζ polypeptide includes an ITAM3 variant including one or more loss-of-function mutations. In some embodiments, the ITAM3 variant includes or consists of two loss-of-function mutations. In some embodiments, each of the one or more (eg, two) loss-of-function mutations includes or consists of a mutation of a tyrosine residue in ITAM3. In some embodiments, an ITAM3 variant (eg, a variant consisting of two loss-of-function mutations) includes or consists of the amino acid sequence set forth in SEQ ID NO: 33, which is set forth below.

HDGLFQGLSTATKDTFDALHMQ [SEQ ID NO: 33]

An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 33 is provided in SEQ ID NO: 34, which appears below.

cacgatggcctttTccaggggctcagtacagccaccaaggacacctTcgacgcccttcacatgcag [SEQ ID NO: 34]

In some embodiments, the CAR intracellular signaling domain comprises a modified CD3ζ polypeptide comprising an ITAM1 variant including 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, including or consisting of one or more loss-of-function mutations, or a combination thereof.

In some embodiments, the CAR intracellular signaling domain includes a modified CD3ζ polypeptide including an ITAM2 variant including or consisting of one or more (e.g., two) loss-of-function mutations, and an ITAM3 variant including or consisting of one or more (e.g., two) loss-of-function mutations. In some embodiments, the intracellular signaling domain of CAR includes a modified CD3ζ polypeptide comprising 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 some embodiments, the CAR intracellular signaling domain comprises a modified CD3ζ polypeptide comprising 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 presented in SEQ ID NO: 33 (for example, in the construct designated "1XX"). In some embodiments, the modified CD3ζ polypeptide includes or consists of the amino acid sequence set forth in SEQ ID NO: 35.

In some embodiments, the CAR intracellular signaling domain includes a modified CD3ζ polypeptide including an ITAM1 variant including or consisting of one or more (e.g., two) loss-of-function mutations, and an ITAM3 variant including or consisting of one or more (e.g., two) loss-of-function mutations. In some embodiments, the CAR intracellular signaling domain comprises a modified CD3ζ polypeptide comprising an ITAM1 variant comprising or consisting of two loss-of-function mutations, native ITAM2 and an ITAM3 variant comprising or consisting of two loss-of-function mutations. In some embodiments, the CAR intracellular signaling domain 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 presented in SEQ ID NO: 33 (for example, in the construct designated "X2X").

In some embodiments, the CAR intracellular signaling domain includes a modified CD3ζ polypeptide including an ITAM1 variant including or consisting of one or more (e.g., two) loss-of-function mutations, and an ITAM2 variant including or consisting of one or more (e.g., two) loss-of-function mutations. In some embodiments, the CAR intracellular signaling domain includes a modified CD3ζ polypeptide comprising or consisting of an ITAM1 variant comprising two loss-of-function mutations, an ITAM2 variant comprising or consisting of two loss-of-function mutations, and native ITAM3. In some embodiments, the CAR intracellular signaling domain comprises a 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 native ITAM3, consisting of the amino acid sequence presented in SEQ ID NO: 31 (for example, in the construct designated "XX3").

In some embodiments, the CAR intracellular signaling domain comprises a modified CD3ζ polypeptide comprising an ITAM1 variant comprising one or more (eg, two) loss-of-function mutations. In some embodiments, the CAR intracellular signaling domain includes a modified CD3ζ polypeptide comprising an ITAM1 variant comprising or consisting of two loss-of-function mutations, native ITAM2 and native ITAM3. In some embodiments, the CAR intracellular signaling domain comprises a modified CD3ζ polypeptide comprising a variant ITAM1 consisting of the amino acid sequence set forth in SEQ ID NO: 25, native ITAM2 consisting of the amino acid sequence set forth in SEQ ID NO: 27, and native ITAM3. consisting of the amino acid sequence presented in SEQ ID NO: 31 (for example, in the construct designated "X23").

In some embodiments, the CAR intracellular signaling domain includes a modified CD3ζ polypeptide including native ITAM1, native ITAM2, and an ITAM3 variant including one or more (eg, two) loss-of-function mutations. In some embodiments, the CAR intracellular signaling domain includes a modified CD3ζ polypeptide including native ITAM1, native ITAM2, and an ITAM1 variant comprising or consisting of two loss-of-function mutations. In some embodiments, the CAR intracellular signaling domain comprises a modified CD3ζ polypeptide comprising native ITAM1 consisting of the amino acid sequence set forth in SEQ ID NO: 23, 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 presented in SEQ ID NO: 33 (for example, in the construct designated "12X").

In some embodiments, the CAR intracellular signaling domain includes a modified CD3ζ polypeptide including native ITAM1, an ITAM2 variant including one or more (eg, two) loss-of-function mutations, and native ITAM3. In some embodiments, the CAR intracellular signaling domain includes a modified CD3ζ polypeptide including native ITAM1, an ITAM2 variant comprising or consisting of two loss-of-function mutations, and native ITAM3. In some embodiments, the CAR intracellular signaling domain comprises a modified CD3ζ polypeptide comprising 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 native ITAM3 variant , consisting of the amino acid sequence presented in SEQ ID NO: 31 (for example, in the construct designated “1X3”).

In some embodiments, the CAR intracellular signaling domain includes a modified CD3ζ polypeptide including a deletion of one or two ITAMs. In some embodiments, the modified CD3ζ polypeptide includes or consists of a deletion of ITAM1 and ITAM2, e.g., the modified CD3ζ polypeptide includes native ITAM3 or a variant of ITAM3, and does not include ITAM1 or ITAM2. In some embodiments, the modified CD3ζ polypeptide includes native ITAM3 consisting of the amino acid sequence set forth in SEQ ID NO: 31 and does not include ITAM1 (native or modified) or ITAM2 (native or modified) (for example, in the construct designated "D12" ).

In some embodiments, the modified CD3ζ polypeptide includes or consists of a deletion of ITAM2 and ITAM3, e.g., the modified CD3ζ polypeptide includes native ITAM1 or a variant of ITAM1, and does not include ITAM2 or ITAM3. In some embodiments, the modified CD3ζ polypeptide includes native ITAM1 consisting of the amino acid sequence set forth in SEQ ID NO: 23 and does not include ITAM2 (native or modified) or ITAM3 (native or modified) (e.g., in a construct designated "D23" ).

In some embodiments, the modified CD3ζ polypeptide includes or consists of a deletion of ITAM1 and ITAM3, e.g., the modified CD3ζ polypeptide includes native ITAM2 or a variant of ITAM2, and does not include ITAM1 or ITAM3. In some embodiments, the modified CD3ζ polypeptide includes native ITAM2 consisting of the amino acid sequence set forth in SEQ ID NO: 27 and does not include ITAM1 (native or modified) or ITAM3 (native or modified) (for example, in the construct designated "D13" ).

In some embodiments, the modified CD3ζ polypeptide includes or consists of a deletion of ITAM1, e.g., the modified CD3ζ polypeptide includes native ITAM2 or an ITAM2 variant, and native ITAM3 or an ITAM3 variant, and does not include ITAM1 (native or modified).

In some embodiments, the modified CD3ζ polypeptide includes or consists of a deletion of ITAM2, for example, the modified CD3ζ polypeptide includes native ITAM1 or an ITAM1 variant, and native ITAM3 or an ITAM3 variant, and does not include ITAM2 (native or modified).

In some embodiments, the modified CD3ζ polypeptide includes or consists of a deletion of ITAM3, e.g., the modified CD3ζ polypeptide includes native ITAM1 or an ITAM1 variant, and native ITAM2 or an ITAM2 variant, and does not include ITAM3 (native or modified).

B. Costimulatory signaling region

In some embodiments, the intracellular signaling domain of the CAR further includes at least a co-stimulatory signaling region. In some embodiments, the costimulatory signaling region includes at least a portion of a costimulatory molecule that can provide optimal activation of lymphocytes.

As used herein, “costimulatory molecules” refer to cell surface molecules other than antigen receptors or their ligands that are required for an effective lymphocyte response to an antigen. Non-limiting examples of co-stimulatory 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 surface of cells that, upon binding to its receptor, produces a costimulatory response, i.e. an intracellular response that causes stimulation when an antigen binds to its CAR molecule. Costimulatory ligands include, but are not limited to, CD80, CD86, CD70, OX40L and 4-1BBL. As one example, the 4-1BB ligand (i.e., 4-1BBL) can bind to 4-1BB (also known as “CD137”), providing an intracellular signal that, in combination with the CAR signal, induces CAR ⁺ T- effector function cells. CAR receptors including an intracellular signaling domain including a co-stimulatory signaling region including 4-1BB, ICOS or DAP-10 are disclosed in US 7,446,190, which is incorporated herein by reference in its entirety.

In some embodiments, the CAR intracellular signaling domain includes a co-stimulatory signaling region including a CD28 polypeptide (eg, the CD28 intracellular domain or a portion thereof). In some embodiments, the co-stimulatory signaling region includes the intracellular domain of human CD28 or a portion thereof. In some embodiments, the co-stimulatory signaling region includes a CD28 polypeptide comprising or consisting of amino acids 180-220 of SEQ ID NO: 90.

In some embodiments, the co-stimulatory signaling region comprises a CD28 polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 101 (or amino acids 180-220 of SEQ ID NO: 90). SEQ ID NO: 101 is given below.

RSKRSRLLHS DYMNMTPRRP GPTRKHYQPY APPRDFAAYR S [SEQ ID NO: 101]

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 provided in SEQ ID NO: 102, which appears below.

AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC [SEQ ID NO: 102]

In some embodiments, the costimulatory signaling region comprises a CD28 polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 108 (or amino acids 180-219 of SEQ ID NO: 90). SEQ ID NO: 108 is given below.

RSKRSRLLHS DYMNMTPRRP GPTRKHYQPY APPRDFAAYR K [SEQ ID NO: 108]

In some embodiments, the co-stimulatory signaling region includes the intracellular domain of mouse CD28 or a portion thereof. In some embodiments, the co-stimulatory signaling region includes or consists of amino acids 178-218 of SEQ ID NO: 97.

An exemplary nucleotide sequence encoding amino acids 178-218 of SEQ ID NO: 97 is provided in SEQ ID NO: 98, which appears below.

aat agtagaagga acagactcct tcaaagtgac tacatgaaca tgactccccg gaggcctggg ctcactcgaa agccttacca gccctacgcc cctgccagag actttgcagc gtaccgcccc [SEQ ID NO: 98]

In some embodiments, the co-stimulatory signaling region includes 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 set forth below:

NSRRNRLLQS DYMNMTPRRP GLTRKPYQPY APARDFAAYR P [SEQ ID NO: 99].

An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 99 is provided in SEQ ID NO: 100, which is given below.

AATAGTAGAAGGAACAGACTCCTTTCAAAGTGACTACATGAACATGACTCCCCGGAGGCCTGGGCTCACTCGAAAGCCTTACCAGCCCTACGCCCCTGCCAGAGACTTTGCAGCGTACCGCCCC [SEQ ID NO: 100]

In some embodiments, the costimulatory signaling region includes a portion of a first costimulatory molecule and a portion of a second costimulatory molecule, for example, a CD28 intracellular domain and a 4-1BB intracellular domain or a CD28 intracellular domain and an OX40 intracellular domain.

In some embodiments, the co-stimulatory signaling region includes a 4-1BB polypeptide (eg, the 4-1BB intracellular domain or a portion thereof). In some embodiments, the co-stimulatory signaling region includes the human intracellular 4-1BB domain or a portion thereof. 4-1BB may act as a tumor necrosis factor (TNF) ligand and have stimulatory activity. In some embodiments, the 4-1BB polypeptide includes 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 is 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 NCBI sequence number: NP_001552.2 (SEQ ID NO: 103) or a fragment thereof, and/or may optionally include up to one or two or up to three conservative amino acid substitutions. In some embodiments, the 4-1BB polypeptide includes or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 103 that has a length of at least about 20, at least about 25, or at least about 30, or at least about 40, or at least about 50 and up to about 255 amino acids. In some embodiments, the 4-1BB polypeptide comprises or consists of the amino acid sequence of amino acids 1-255, 1-50, 50-100, 100-150, 150-200, 214-255, or 200-255 from SEQ ID NO: 103. B In some embodiments, the co-stimulatory signaling region comprises a 4-1BB polypeptide comprising or consisting of SEQ ID NO: 104 (or amino acids 214-255 of SEQ ID NO: 103). SEQ ID NO: 103 and 104 are given below:

1 MGNSCYNIVA TLLLVLNFER TRSLQDPCSN CPAGTFCDNN RNQICSPCPP NSFSSAGGQR

61 TCDICRQCKG VFRTRKECSS TSNAECDCTP GFHCLGAGCS MCEQDCKQGQ ELTKKGCKDC

121 CFGTFNDQKR GICRPWTNCS LDGKSVLVNG TKERDVVCGP SPADLSPGAS SVTPPAPARE

181 PGHSPQIISF FLALTSTALL FLLFFLTLRF SVVKRGRKKL LYIFKQPFMR PVQTTQEEDG

241 CSCRFPEEEE GGCEL [SEQ ID NO: 103]

KRGRKKLLYI FKQPFMRPVQ TTQEEDGCSC RFPEEEEGGC EL [SEQ ID NO: 104].

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 provided in SEQ ID NO: 105, which appears below.

AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG [SEQ ID NO: 105]

In some embodiments, the co-stimulatory signaling region includes an OX40 polypeptide (eg, an OX40 intracellular domain or a portion thereof). In some embodiments, the co-stimulatory signaling region includes the intracellular domain of human OX40 or a portion thereof. In some embodiments, the OX40 polypeptide includes 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 NCBI sequence number: NP_003318.1 (SEQ ID NO : 106) or a fragment thereof, and/or may optionally include up to one or two or three conservative amino acid substitutions. In some embodiments, the OX40 polypeptide includes 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 in length , or at least about 50 and up to about 277 amino acids. In some 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 set forth below .

1 MCVGARRLGR GPCAALLLLG LGLSTVTGLH CVGDTYPSND RCCHECRPGN GMVSRCSRSQ

61 NTVCRPCGPG FYNDVVSSKP CKPCTWCNLR SGSERKQLCT ATQDTVCRCR AGTQPLDSYK

121 PGVDCAPCPP GHFSPGDNQA CKPWTNCTLA GKHTLQPASN SSDAICEDRD PPATQPQETQ

181 GPPARPITVQ PTEAWPRTSQ GPSTRPVEVP GGRAVAAILG LGLVLGLLGP LAILLALYLL

241 RRDQRLPPDA HKPPGGGSFR TPIQEEQADA HSTLAKI [SEQ ID NO: 106]

In some embodiments, the co-stimulatory signaling region includes an ICOS polypeptide (eg, the ICOS intracellular domain or a portion thereof). In some embodiments, the co-stimulatory signaling region includes the human ICOS intracellular domain or a portion thereof. In some embodiments, the ICOS polypeptide includes 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 NCBI sequence number: NP_036224 (SEQ ID NO: 65 ) or a fragment thereof, and/or may optionally include up to one or two or three conservative amino acid substitutions. In some embodiments, the ICOS polypeptide includes 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 in length , or at least about 50 and up to about 199 amino acids. In some 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 set forth below.

1 MKSGLWYFFL FCLRIKVLTG EINGSANYEM FIFHNGGVQI LCKYPDIVQQ FKMQLLKGGQ

61 ILCDLTKTKG SGNTVSIKSL KFCHSQLSNN SVSFFLYNLD HSHANYYFCN LSIFDPPPFK

121 VTLTGGYLHI YESQLCCQLK FWLPIGCAAF VVVCILGCIL ICWLTKKKYS SSVHDPNGEY

181 MFMRAVNTAK KSRLTDVTL [SEQ ID NO: 65]

In some embodiments, a mesothelin-directed CAR disclosed herein further includes an inducible promoter for expression of nucleic acid sequences in human cells. Promoters for use in CAR gene expression may be a constitutive promoter such as the ubiquitin C (UbiC) promoter.

In some embodiments, mutation sites and/or connections between domains/motifs/regions of CARs derived from different proteins are deimmunized. The immunogenicity of connections between different CAR fragments can be predicted using the NetMHC 4.0 server. For each peptide containing at least one amino acid from the following fragment, the binding affinity for HLA A, B and C can be predicted for all alleles. The immunogenicity index of each peptide can be set for each peptide. The immunogenicity index can be calculated using the following formula: Immunogenicity index=[(50-binding affinity)*HLA frequency] _n . n is the prediction number for each peptide.

5.2.2.5. Exemplary CAR Receptors

In some embodiments, the mesothelin-targeted CAR includes:

(a) an extracellular antigen binding domain comprising a V _H including a CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 76, a CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 77, and a CDR3 consisting of the amino acid sequence, presented in SEQ ID NO: 78; and V _L 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) a transmembrane domain comprising a CD28 polypeptide (eg, a human CD28 transmembrane domain or a portion thereof);

(c) a CD28 hinge/spacer region (eg, a human CD28 hinge/spacer region or a portion thereof); And

(d) an intracellular signaling domain comprising: (i) a modified CD3ζ polypeptide (e.g., a modified human CD3ζ polypeptide) including native ITAM1, an ITAM2 variant consisting of two loss-of-function mutations, and an ITAM3 variant consisting of two loss-of-function mutations , and (ii) a co-stimulatory signaling region comprising a CD28 polypeptide (eg, a human CD28 polypeptide, eg, a human CD28 intracellular domain or a portion thereof).

In some embodiments, the transmembrane domain includes 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).

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

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

In some embodiments, the co-stimulatory signaling region includes 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).

In some embodiments, the CAR includes 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%, is at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 56. In some embodiments, CAR includes the amino acid sequence set forth in SEQ ID NO: 56. SEQ ID NO: 56 is set forth below.

QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGSYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREGKNGAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSRHQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSG VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIKRTAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEY DVLDKRRGRDPEMGGKPRRKNPQEGLFNELQKDKMAEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTFDALHMQALPPR [SEQ ID NO: 56]

An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 56 is provided in SEQ ID NO: 57, which is given below.

CAGGTTCAGCTTCAGGAGAGTGGCCCAGGCCTGGTGAAGCCAAGTGAGACTCTCAGCTTGACTTGCACAGTTTCTGGAGGCAGTGTCTCCTCAGGCAGCTATTATTGGTCCTGGATTCGGCAGCCCCCTGGGAAAGGCCTGGAGTGGATTGGGTACATATATTACAGTGGCAGCACAAATTACAATCCATCCCTGAAGTCTCGAGTAACTATCAGTGTGGACACAAGCAAGAATCAGTTTTCACTCAAACTGTCTTCTGTGACTGCTGCTGACACTGCTGTTTATTATTGTGCCAGGGAGGGGAAAAATGGGGCATTTGATATTTGGGGTCAGGGCACAATGGTGACAGTCAGCTCTGGAGGTGGAGGCTCAGGAGGAGGAGGCAGTGGAGGTGGTGGGTCACGCCATCAGATGACTCAGTCCCCCTCCAGTCTTTCTGCCTCAGTTGGGGATAGAGTGACCATCACATGCAGAGCAAGTCAGAGCATATCATCCTATCTGAACTGGTACCAGCAGAAGCCAGGGAAAGCCCCCAAATTGCTGATTTATGCAGCCTCAAGTCTCCAGAGTGGGGTGCCAAGCAGGTTCTCAGGCAGTGGCAGTGGGACAGATTTCACATTGACAATCAGCTCCCTCCAACCTGAAGATTTTGCCACCTACTATTGCCAGCAATCCTACAGCACGCCCCTGACTTTTGGAGGTGGCACAAAGGTAGAGATCAAGAGGACTGCGGCCGCAATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTTCAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTTCAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTTCCAGGGGCTCAGTACAGCCACCAAGGACACCTTCGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC [SEQ ID NO: 57]

In some embodiments, the CAR further includes a CD8 leader sequence. In some embodiments, the CD8 leader sequence includes or consists of the amino acid sequence set forth in SEQ ID NO: 71.

An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 71 is provided in SEQ ID NO: 120, which is given below.

ATGGCCCTGCCAGTAACGGCTCTGCTGCTGCCACTTGCTCTGCTCCTCCATGCAGCCAGGCCT [SEQ ID NO: 120]

In some embodiments, the CAR includes 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%, is at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 43, which is listed below. In some embodiments, the CAR includes or consists of the amino acid sequence set forth in SEQ ID NO: 43. SEQ ID NO: 43 includes a CD8 leader sequence that consists of the amino acid sequence set forth in SEQ ID NO: 71. SEQ ID NO: 43 is provided below:

MALPVTALLLPLALLLHAARPQVQLQESGPGLVKPSETLSLTCTVSGGSVSSGSYYWSWI

RQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR

EGKNGAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSRHQMTQSPSSLSASVGDRVTITCRA

SQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFAT

YYCQQSYSTPLTFGGGTKVEIKRTAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLF

PGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHY

QPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMG

GKPRRKNPQEGLFNELQKDKMAEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTFDALHM

QALPPR [SEQ ID NO: 43]

An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 43 is provided in SEQ ID NO: 44, which is given below.

ATGGCCCTGCCAGTAACGGCTCTGCTGCTGCCACTTGCTCTGCTCCTCCATGCAGCCAGGCCTCAGGTTCAGCTTCAGGAGAGTGGCCCAGGCCTGGTGAAGCCAAGTGAGACTCTCAGCTTGACTTGCACAGTTTCTGGAGGCAGTGTCTCCTCAGGCAGCTATTATTGGTCCTGGATTCGGCAGCCCCCTGGGAAAGGCCTGGAGTGGATTGGGTACATATATTACAGTGGCAGCACAAA TTACAATCCATCCCTGAAGTCTCGAGTAACTATCAGTGTGGACACAAGCAAGAATCAGTTTTCACTCAAACTGTCTTCTGTGACTGCTGCTGACACTGCTGTTTATTATTGTGCCAGGGAGG GGAAAAATGGGGCATTTGATATTTGGGGTCAGGGCACAATGGTGACAGTCAGCTCTGGAGGTGGAGGCTCAGGAGGAGGAGGCAGTGGAGGTGGTGGGTCACGCCATCAGATGACTCAGTCCC CCTCCAGTCTTTCTGCCTCAGTTGGGGATAGAGTGACCATCACATGCAGAGCAAGTCAGAGCATATCATCCTACTGAACTGGTACCAGCAGAAGCCAGGGAAAGCCCCCAAATTGCTGATTTATGCAGCCTCAAGTCTCCAGAGTGGGGTGCCAAGCAGGTTCTCAGGCAGTGGCAGTGGGACAGATTTCACATTGACAATCAGCTCCCTCCAACCTGAAGATTTTGCCACCTACTATTGCCA GCAATCCTACAGCACGCCCCTGACTTTTGGAGGTGGCACAAAGGTAGAGATCAAGAGGACTGCGGCCGCAATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAA CCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAG TGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACC ACGCGACTTCGCAGCCTATCGCTCCAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACG [SEQ ID NO: 44]

5.2.3. Exemplary Polypeptide Compositions

In some embodiments, the polypeptide composition includes a mesothelin-targeted 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.

In some embodiments, the polypeptide composition includes a mesothelin-targeted 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.

In some embodiments, the polypeptide composition includes a mesothelin-targeted 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.

In some embodiments, the polypeptide composition includes a mesothelin-targeted 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.

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

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

In some embodiments, the polypeptide composition includes a mesothelin-targeted CAR comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 56, and a CD8 leader sequence comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 71, and PD- 1 DN comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 49.

In some embodiments, the polypeptide composition includes a mesothelin-targeted CAR comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 56, and a CD8 leader sequence comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 71, and PD- 1 DN comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 118.

5.3. Immunoreactive cells

The present invention provides immunoreactive cells comprising the polypeptide composition disclosed herein. In some embodiments, the CAR is capable of activating an immunoreactive cell. In some embodiments, the polypeptide composition is capable of promoting an antitumor effect of an immunoreactive cell. Immunoreactive cells can be transduced with a polypeptide composition, and such cells will co-express CAR and PD-1 DN.

The immunoreactive cells of the invention disclosed herein may be cells of the lymphoid lineage. The lymphoid lineage, including B, T and natural killer (NK) cells, ensures the production of antibodies, regulation of the cellular immune system, detection of foreign agents in the blood, detection of cells foreign to the host, etc. Non-limiting examples of immunoreactive cells of the lymphoid lineage include T cells, natural killer (NK) cells, embryonic stem cells, and pluripotent stem cells (eg, cells from which lymphoid cells can differentiate). T cells can be lymphocytes that mature in the thymus and are primarily responsible for cellular immunity. T cells are part of the adaptive immune system. The T cells of the invention disclosed herein may be any type of T cell, including, but not limited to, T helper cells, cytotoxic T cells, memory T cells (including central memory T cells, like T stem cells memory cells (or stem-like memory T cells), and two types of effector memory T cells: for example, TEM cells and TEMRA 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 (CTL or killer T cells) are a subset of T lymphocytes that can cause the death of infected somatic or tumor cells. modified to target specific antigens through the introduction of an antigen recognition receptor, such as a CAR or TCR. In some embodiments, the immunoresponsive cell is a T cell. The T cell may be a CD4 ⁺ T cell or a CD8 ⁺ T cell. In some embodiments, the T cell is a CD4 ⁺ T cell. In some embodiments, the T cell is a CD8 ⁺ T cell.

Natural killer (NK) cells can be lymphocytes that are part of cellular immunity and participate in the innate immune response. NK cells do not require prior activation to exert their cytotoxic effects on target cells.

Types of human lymphocytes according to the invention disclosed herein include, but are not limited to, peripheral donor lymphocytes, for example, those disclosed in Sadelain, M., et al. 2003 Nat Rev Cancer 3: 35-45 (disclosure of peripheral donor lymphocytes genetically modified to express CAR receptors), in Morgan, R.A., et al. 2006 Science 314: 126-129 (which discloses peripheral donor lymphocytes genetically modified to express a T-cell receptor complex that recognizes full-length tumor antigens and includes an α and β heterodimer), in Panelli, M.C., et al. 2000 J Immunol 164:495-504; Panelli, M.C., et al. 2000 J Immunol 164: 4382-4392 (which discloses cultures of lymphocytes derived from tumor infiltrating lymphocytes (TILs) in tumor biopsy specimens), and Dupont, J., et al. 2005 Cancer Res 65: 5417-5427; Papanicolaou, G. A., et al. 2003 Blood 102: 2498-2505 (which discloses antigen-specific peripheral blood leukocytes selectively expanded in vitro using artificial antigen-presenting cells (iAPCs) or activated dendritic cells). Immunoreactive cells (eg, T cells) may be autologous, non-autologous (eg, allogeneic), or may be derived in vitro from modified progenitor or stem cells.

In some embodiments, an immunoreactive cell disclosed herein includes a mesothelin-targeted 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.

In some embodiments, immunoreactive cells disclosed herein comprising one or more CAR and/or PD-1/DN polypeptides according to the invention disclosed herein are allogeneic or autologous EBV-sensitized cytotoxic T lymphocytes (CTLs). For example, obtaining EBV-responsive cytotoxic T cells may involve isolating PBMCs from a EBV-seropositive autologous or allogeneic donor and enriching them for T cells by eliminating monocytes and NK cells. EBV-sensitized cytotoxic T cells can also be generated by contacting donor PBMCs or purified donor T cells with “stimulator” cells that express one or more EBV antigens and present the EBV antigen(s) to unstimulated T cells, resulting in stimulation and expansion of EBV-sensitized CTLs. Specifically, in some embodiments, such methods include obtaining a sample of cells (eg, PBMCs) from a subject comprising CD3 ⁺ cells, and contacting said CD3 ⁺ cells with antigen and/or antigen-presenting stimulatory cells. In some embodiments, CD3 ⁺ T cells are isolated from the sample prior to exposure to the antigen using a method known in the art (eg, positive selection of CD3 ⁺ cells from the sample and/or negative selection by eliminating unwanted cells or components from the sample). In some embodiments, such methods include selection by fluorescence-activated cell sorting (FACS), using anti-CD3 beads (e.g., magnetic beads), plastic adhesion, NK cell elimination using anti-CD56 antibodies, elutriation, and/or combinations this. EBV antigens include, for example, latent membrane protein (LMP) and EBV nuclear antigen (EBNA) proteins such as LMP-1, LMP-2A and LMP-2B, and EBNA-1, EBNA-2, EBNA-3A, EBNA- 3B, EBNA-3C and EBNA-LP. Cytotoxic T cells that include T cell receptor(s) that recognize one or more EBV-specific antigens are considered to be “sensitized” by such EBV antigen(s) and are therefore referred to herein as “EBV-sensitized cytotoxic T cells.” . Known methods for generating populations of allogeneic or autologous EBV-specific cytotoxic T cells, which may include one or more of the CAR polypeptides disclosed herein, 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 herein by reference. Likewise, cytotoxic T cells can be “sensitized” to other viral antigens, including cytomegalovirus (CMV), papillomavirus (eg, HPV), adenovirus, polyomavirus (eg, BKV, JCV, and Merkel cell virus), retrovirus (eg, HTLV- I, also including lentivirus such as HIV), picornavirus (eg hepatitis A virus), hepadnavirus (eg hepatitis B virus), hepacivirus (eg hepatitis C virus), deltavirus (eg hepatitis D virus), hepevirus (eg , hepatitis E virus), etc. In some embodiments, the target antigen is derived from an oncovirus. In some embodiments, the T cells used to generate the CAR-T cells disclosed herein are multifunctional T cells, such as those T cells that are capable of inducing multiple immune effector functions that provide a more effective immune response against a pathogen than cells that produce, for example, only one immune effector (eg, one biomarker such as a cytokine or CD107a). Less polyfunctional, monofunctional, or even “exhausted” T cells may dominate immune responses during chronic infections, thereby negatively impacting protection against virus-associated complications. In some embodiments, the CAR-T cells disclosed herein are multifunctional. In some embodiments, at least about 50% of the T cells used to generate the CAR-T cells disclosed herein are CD4 ⁺ T cells. In some such embodiments, the T cells are less than about 50% CD4 ⁺ T cells. In some embodiments, the T cells are predominantly CD4 ⁺ T cells.

In some embodiments, at least about 50% of the T cells used to generate the CAR-T cells disclosed herein are CD8 ⁺ T cells. In some such embodiments, the T cells are less than about 50% CD8 ⁺ T cells. In some embodiments, the T cells are predominantly CD8 ⁺ T cells. In some embodiments, T cells (eg, sensitized T cells and/or CAR-T cells described herein) are stored in a cell library or bank prior to administration to a subject.

An immunoreactive cell disclosed herein may further include at least one exogenous costimulatory ligand, wherein the immunoreactive cell coexpresses or is induced to coexpress a mesothelin-specific CAR and at least one exogenous costimulatory ligand. The interaction between a mesothelin-specific CAR and at least one costimulatory ligand provides a non-antigen-specific signal important for full activation of an immunoreactive cell (eg, a T cell). Costimulatory ligands include, but are not limited to, members of the tumor necrosis factor (TNF) superfamily and ligands of the immunoglobulin (Ig) superfamily. TNF is a cytokine involved in systemic inflammation and which stimulates the acute phase response. Its main role is to regulate immune cells. Members of the TNF superfamily share a number of common characteristics. Most members of the TNF superfamily are synthesized as type II transmembrane proteins (with an extracellular C terminus) containing a short cytoplasmic segment and a relatively long extracellular region. Members of the TNF superfamily include, but are not limited to, nerve growth factor (NGF), CD40L (CD40L)/CD154, CD137L/4-1BBL, TNF-α, CD134L/OX40L/CD252, CD27L/CD70, Fas ligand (FasL), CD30L /CD153, tumor necrosis factor beta (TNFβ)/lymphotoxin alpha (LTα), lymphotoxin beta (LTβ), CD257/B cell activating factor (BAFF)/Blys/THANK/Tall-1, glucocorticoid-inducible 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 that are involved in cell recognition, binding, or adhesion. These proteins have similar structural features to immunoglobulins - they contain an immunoglobulin domain (fold). Immunoglobulin superfamily ligands include, but are not limited to, CD80 and CD86, both CD28 ligands, PD-L1/(B7-H1), which are PD-1 ligands.

In some 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 some embodiments, the costimulatory ligand is 4-1BBL. 4-1BBL can be covalently linked to the 5' end of the extracellular antigen binding domain of a mesothelin-targeted CAR. Alternatively, 4-1BBL can be covalently linked to the 3' end of the intracellular signaling domain of a mesothelin-targeted CAR.

In addition, an immunoreactive cell disclosed herein may further include at least one exogenous cytokine, wherein the immunoreactive cell coexpresses or is induced to coexpress a mesothelin-specific CAR and at least one exogenous cytokine. In some 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-17. 21. In some embodiments, the at least one exogenous cytokine includes IL-12. In some embodiments, the immunoreactive cell coexpresses a mesothelin-targeted CAR and exogenous IL-12. IL-12 can be covalently linked to the 3' end of the intracellular signaling domain of the mesothelin-targeted CAR.

In addition, the immunoreactive cells may express a second CAR that binds to a second antigen that is mesothelin or an antigen other than mesothelin. CARs that can be used as a second CAR in combination with a mesothelin-specific CAR in the invention disclosed herein include the 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. 7,446,190, which are incorporated herein by reference in their entirety, e.g., CD19-targeted CARs (see U.S. 7,446,190; U.S. 2013/0071414), HER2-targeted CARs (see Ahmed, et al., Clin Cancer Res., 2010), MUC16-targeted CAR receptors (see Chekmasova, et al., 2011), prostate-specific membrane antigen (PSMA)-targeted CAR receptors (e.g., Zhong, et al., Molecular Therapy, 18(2): 413-420 (2010), which are incorporated herein by reference in their entirety. Immunoreactive cells expressing two or more antigen recognition receptors (eg, CAR receptors) are described in WO 2014/055668. , which is incorporated herein by reference in its entirety.

The second antigen may 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. Sources of tumor antigen include, but are not limited to, cancer proteins. The second antigen may be expressed as a peptide or an intact protein or a portion thereof. The intact protein or part thereof may be native or mutant. 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 may be carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), CD5, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD123, CD133, CD138, cytomegalovirus (CMV)-infected cell antigen (eg, cell surface antigen), epithelial glycoprotein 2 (EGP 2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyrosine- protein kinases ErbB2, Erb-B3, Erb-B4, folate binding protein (FBP), fetal 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 alpha-2 subunit (IL-13Rα2), κ-light chain, insertion kinase domain receptor (KDR), Lewis A (CA19.9), Lewis Y (LeY ), cell adhesion molecule L1 (L1CAM), melanoma antigen family A, 1 (MAGE-AI), Mucin 16 (Muc-16), Mucin 1 (Muc-1), NKG2D ligands, cancer-testis antigen NY-ESO-1 , ofetal antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), tumor glycoprotein 72 (TAG-72), vascular endothelial growth factor R2 (VEGF-R2), Wilms tumor protein (WT- 1), transmembrane receptor tyrosine protein kinase type 1 (ROR1), or a combination thereof.

Suitable pathogen antigens for use in treating an infection caused by a pathogen or other infectious disease, such as in an immunocompromised subject, include, but are not limited to, viral antigens present in cytomegalovirus (CMV), Epstein-Barr virus (EBV), human immunodeficiency virus ( HIV) and influenza virus. Immunoreactive cells that include a second CAR that specifically interacts with a viral antigen can be used to treat viral diseases. In some embodiments, a mesothelin-targeting CAR and a second CAR that binds to a CMV antigen are coexpressed on immunoreactive cells (eg, cytotoxic T lymphocytes) and can be used to treat CMV.

Mesothelin-specific or mesothelin-directed human lymphocytes that can be used in the methods of the invention disclosed herein include, but are not limited to, peripheral donor lymphocytes, such as those described in Sadelain, M., et al. 2003 Nat Rev Cancer 3: 35-45 (disclosure of peripheral donor lymphocytes genetically modified to express CAR receptors), in Morgan, R.A., et al. 2006 Science 314: 126-129 (which discloses peripheral donor lymphocytes genetically modified to express a T-cell receptor complex that recognizes full-length tumor antigens and includes an α and β heterodimer), in Panelli, M.C., et al. 2000 J Immunol 164:495-504; Panelli, M.C., et al. 2000 J Immunol 164: 4382-4392 (which discloses cultures of lymphocytes derived from tumor infiltrating lymphocytes (TILs) in tumor biopsy specimens), and Dupont, J., et al. 2005 Cancer Res 65: 5417-5427; Papanicolaou, G. A., et al. 2003 Blood 102: 2498-2505 (which discloses antigen-specific peripheral blood leukocytes selectively expanded in vitro using artificial antigen-presenting cells (iAPCs) or activated dendritic cells). Immunoreactive cells (eg, T cells) may be autologous, non-autologous (eg, allogeneic), or may be derived in vitro from modified progenitor or stem cells.

Assays can be used to compare the effect of costimulatory signaling on enhancing the proliferation of effector function and accumulation of T cells transduced with a mesothelin-targeted CAR upon repeated (weekly) antigen stimulation. Peripheral blood lymphocytes (PBL) can be obtained from healthy volunteers according to an IRB-approved protocol and transduced. The efficiency of gene transfer can be monitored using FACS analysis to quantify the proportion of GFP ⁺ (transduced) T cells and/or using quantitative PCR. Using a well established co-culture system (Gade, TP, et al. Cancer Res. 65, 9080-9088 (2005); Gong, MC, et al. Neoplasia. 1, 123-127 (1999); Latouche, J. B. & Sadelain, M. Nat. Biotechnol. 18, 405-409 (2000)) can determine whether fibroblastic AAPCs expressing mesothelin (compared to mesothelin controls) can direct the release of cytokines from transduced T cells (LUMINEX cell supernatant assay 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 impact of CD80 and/or 4-1BBL on T cell survival, proliferation and efficacy can be assessed. T cells can be restimulated with mesothelin ⁺ target cells (MSLN ⁺ ) and determined whether T cell proliferation and cytokine response remain similar or decrease with restimulation. Cytotoxicity assays with different E:T ratios can be performed using chromium release assays. Statistical analysis may optionally be performed using two-way analysis of variance followed by pairwise multiple comparison procedures, in which data may be expressed as mean ±SEM. CD4 and CD8 T cell subtypes (activated effector, central memory, effector memory) can be identified to determine which conditions are favorable for the maintenance or expansion of the central memory phenotype.

In some embodiments, an immunoreactive cell (e.g., a T cell) disclosed herein expresses 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 copies of the mesothelin-directed CAR vector/cell. For example, an immunoreactive cell (eg, a T cell) disclosed herein expresses about 1, about 2, about 3, or about 4 copies of a mesothelin-targeting CAR vector/cell. In some embodiments, an immunoreactive cell (eg, a T cell) disclosed herein expresses from about 3 to about 4 copies of the mesothelin-targeting CAR vector/cell. In some embodiments, the cytotoxicity and cytokine production of the immunoreactive cell (eg, T cell) is proportional to the level of mesothelin-specific CAR expression in the cell. For example, the higher the level of CAR expression in an immunoreactive cell, the higher the cytotoxicity and cytokine production that the immunoreactive cell exhibits. An immunoreactive cell (eg, a T cell) having a high level of mesothelin CAR receptor expression can cause antigen-specific cytokine production or secretion and/or exhibit cytotoxicity to a tissue or cell having a low level of mesothelin expression, for example, about 2000 or less than, about 1000 or less, about 900 or less, about 800 or less, about 700 or less, about 600 or less, about 500 or less, about 400 or less, about 300 or less, about 200 or less, about 100 or fewer mesothelin/cell binding sites. Additionally or alternatively, the cytotoxicity and cytokine production of an immunoreactive cell (eg, a T cell) disclosed herein is proportional to the level of expression of human mesothelin in the target tissue or cell. For example, the higher the level of expression of human mesothelin in the target, the higher the cytotoxicity and cytokine production that the immunoreactive cell exhibits.

In some embodiments, the target cells are heterogeneous MSLN-expressing cells, which are a population of cells including cells with low MSLN expression and cells with high MSLN expression. An immunoreactive cell disclosed herein may exhibit increased cytotoxicity and antitumor activity against cells with low MSLN expression (e.g., about 2000 or less, about 1000 or less, about 900 or less, about 800 or less, about 700 or less, about 600 or less, about 500 or less, about 400 or less, about 300 or less, about 200 or less, or about 100 or less MSLN binding sites/cell) in the presence of cells with high MSLN expression. In some embodiments, even in the presence of cells with high MSLN expression, the immunoreactive cell does not exhibit increased cytotoxicity or nonspecific killing against MSLN-negative cells. Thus, an immunoreactive cell can exhibit enhanced cytotoxicity and antitumor activity against low MSLN expressing cells in the presence of high MSLN expressing cells while maintaining safety against MSLN negative cells.

In some embodiments, the immunoreactive cell may express one or more adhesion molecules that can increase the avidity of the MSLN-specific CAR, particularly if the CAR is a low affinity CAR. Non-limiting examples of adhesion molecules include CD2 and VLA-4. CD2 expressed on an immunoreactive cell can bind to CD58 expressed on a target cell (eg, a cancer cell). VLA-4 expressed on an immunoreactive cell can bind to VCAM-1 on a target cell (eg, a cancer cell).

The crude source of CTL may be any known in the art, such as fetal, neonatal or adult bone marrow or other hematopoietic cell source such as fetal liver, peripheral blood or umbilical cord blood. Various methods can be used to separate cells. For example, negative selection methods allow cells other than CTLs to be initially removed. MAbs are particularly useful for identifying markers associated with specific cell lineages and/or differentiation stages for both positive and negative selection.

Most terminally differentiated cells can be initially removed by relatively rough separation. For example, magnetic bead separation may be used initially to remove large numbers of irrelevant cells. In some embodiments, at least about 80%, typically at least 70%, of the total hematopoietic cells will be removed prior to cell isolation.

Separation procedures include, but are not limited to, density gradient centrifugation; return to original state; binding to particles that change cell density; separation in a magnetic field using magnetic beads coated with antibodies; affinity chromatography; cytotoxic agents combined or used in conjunction with mAbs, including, but not limited to, complement and cytotoxins; as well as panning with the antibody attached to a solid matrix, such as a plate, chip, elutriation or any other convenient technique.

Separation and analysis methods include, but are not limited to, flow cytometry, which may have varying degrees of complexity, such as multiple color channels, small-angle and obtuse-angle light scattering detection channels, and resistance channels.

Cells can be selected from dead cells by using dead cell-binding dyes such as propidium iodide (PI). In some embodiments, cells are collected in a medium containing 2% fetal calf serum (FCS) or 0.2% bovine serum albumin (BSA), or any other suitable, such as sterile isotonic, medium.

5.4. Nucleic acid compositions and vectors

The present invention provides nucleic acid compositions encoding the polypeptide compositions disclosed herein (eg, those disclosed in Section 5.2). In some embodiments, the nucleic acid composition includes a polynucleotide encoding a polypeptide composition disclosed herein (eg, disclosed in Section 5.2). Vectors including such nucleic acid compositions and cells including such nucleic acid compositions or vectors are also provided.

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

The nucleic acid compositions can be administered to subjects and/or delivered into cells using known methods or as described herein.

Genetic modification of an immunoreactive cell (eg, T cell or NK cell) can be accomplished by transducing a substantially homogeneous composition of cells with a recombinant DNA construct. In some embodiments, a retroviral vector (gamma-retroviral or lentiviral) is used to introduce a DNA construct into a cell. For example, a polynucleotide encoding an antigen recognition receptor can be cloned into a retroviral vector and expression can be directed from its endogenous promoter, from a retroviral long terminal repeat, or from a promoter specific to the type of target cell of interest. Non-viral vectors can also be used.

To initially genetically modify an immunoreactive cell to introduce an antigen recognition receptor (eg, CAR or TCR), a retroviral vector is typically used for transduction, but any other suitable viral vector or non-viral delivery system may be used. The CAR and PD-1 DN can be constructed in a single multicistronic expression cassette, in multiple expression cassettes of a single vector, or in multiple vectors. Examples of elements that create a polycistronic expression cassette 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 virus IRES, hepatitis C virus IRES, pestivirus IRES, aphthovirus IRES, picornavirus IRES, poliovirus IRES, and encephalomyocarditis virus IRES), as well as cleavable linkers (e.g., 2A peptides, e.g., P2A, T2A, E2A and F2A peptides). In some embodiments, the P2A peptide includes or consists of the amino acid sequence set forth in SEQ ID NO: 107, which is listed below:

GSGATNFSLLKQAGDVEENPGPM [SEQ ID NO: 107]

In some embodiments, the P2A peptide includes or consists of the amino acid sequence set forth in SEQ ID NO: 121, which is listed below:

ATNFSLLKQAGDVEENPGP [SEQ ID NO: 121]

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

GCAACAAACTTCTCACTACTCAAACAAGCAGGTGACGTGGAGGAGAATCCCGGCCCA [SEQ ID NO: 122]

Combinations of a retroviral vector and an appropriate packaging line are also suitable if the capsid proteins will be functional for infecting human cells. Various cell lines are known to produce amphotropic viruses, 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). Also suitable are non-amphotropic particles, for example particles pseudotyped with the VSVG, RD114 or GALV envelope glycoproteins, as well as any other particles known in the art.

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

Other transducing viral vectors can be used to modify an immunoreactive cell. In some embodiments, the selected vector exhibits 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., 263-267, 1996; 10319, 1997). Other viral vectors that may be used include, for example, adenoviral, lentiviral and adeno-associated virus vectors, vaccinia virus, bovine papillomavirus, or herpes virus such as Epstein-Barr virus (also see, for example, vectors in Miller, Human Gene Therapy 15-14, 1990; Friedman, Science 244: 1275-1281, 1989; Eglitis et al., BioTechniques 6: 608-614, 1988; , 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; : 407-416, 1991; Miller et al., Biotechnology 7: 980-990, 1989; LeGal La Salle et al., Science 259: 988-990, 1993; and Johnson, Chest 107: 77S-83S, 1995). Retroviral vectors are particularly well established and are used in clinical settings (Rosenberg et al., N. Engl. J. Med 323: 370, 1990; Anderson et al., US Patent 5,399,346).

In some embodiments, the vector encoding a polypeptide composition disclosed herein is a retroviral vector, for example, an SGF γ-retroviral vector, which may be a Moloney murine leukemia virus-based retroviral vector. In some embodiments, the vector includes or consists of the nucleotide sequence set forth in SEQ ID NO: 123, which is listed below:

ATGGCCCTGCCAGTAACGGCTCTGCTGCTGCCACTTGCTCTGCTCCTCCATGCAGCCAGGCCTCAGGTTCAGCTTCAGGAGAGTGGCCCAGGCCTGGTGAAGCCAAGTGAGACTCTCAGCTTGACTTGCACAGTTTCTGGAGGCAGTGTCTCCTCAGGCAGCTATTATTGGTCCTGGATTCGGCAGCCCCCTGGGAAAGGCCTGGAGTGGATTGGGTACATATATTACAGTGGCAGCACAAATTACAATCCATCCCTGAAGTCTCGAGTAACTATCAGTGTGGACACAAGCAAGAATCAGTTTTCACTCAAACTGTCTTCTGTGACTGCTGCTGACACTGCTGTTTATTATTGTGCCAGGGAGGGGAAAAATGGGGCATTTGATATTTGGGGTCAGGGCACAATGGTGACAGTCAGCTCTGGAGGTGGAGGCTCAGGAGGAGGAGGCAGTGGAGGTGGTGGGTCACGCCATCAGATGACTCAGTCCCCCTCCAGTCTTTCTGCCTCAGTTGGGGATAGAGTGACCATCACATGCAGAGCAAGTCAGAGCATATCATCCTATCTGAACTGGTACCAGCAGAAGCCAGGGAAAGCCCCCAAATTGCTGATTTATGCAGCCTCAAGTCTCCAGAGTGGGGTGCCAAGCAGGTTCTCAGGCAGTGGCAGTGGGACAGATTTCACATTGACAATCAGCTCCCTCCAACCTGAAGATTTTGCCACCTACTATTGCCAGCAATCCTACAGCACGCCCCTGACTTTTGGAGGTGGCACAAAGGTAGAGATCAAGAGGACTgcggccgcaattgaagttatgtatcctcctccttacctagacaatgagaagagcaatggaaccattatccatgtgaaagggaaacacctttgtccaagtcccctatttcccggaccttctaagcccttttgggtgctggtggtggttggtggagtcctggcttgctatagcttgctagtaacagtggcctttattattttctgggtgaggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgccgccccgggcccacccgcaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctccagagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtTcaatgaactgcagaaagataagatggcggaggcctTcagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttTccaggggctcagtacagccaccaaggacacctTcgacgcccttcacatgcaggccctgccccctcgcGGATCTGGAGCAACAAACTTCTCACTACTCAAACAAGCAGGTGACGTGGAGGAGAATCCCGGCCCAATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAACTGGGCTGGCGGCCAGGATGGTTCTTAGACTCCCCAGACAGGCCCTGGAACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAACGCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCTTTCCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACACAACTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGCGCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAAGGCGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGAAGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCCAGCCGGCCAGGCGGCCGCACCCACCACGACGCCAGCGCCGCGACCACCAACCCCGGCGCCCACGATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCCTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAACCACAGGCGGATCCAATAAcagccactcgaggatccGGATTAGTCCAATTTGTTAAAGACAGGATATCAGTGGTCCAGGCTCTAGTTTTGACTCAACAATATCACCAGCTGAAGCCTATAGAGTACGAGCCATAGATAAAATAAAAGATTTTATTTAGTCTCCAGAAAAAGGGGGGAATGAAAGACCCCACCTGTAGGTTTGGCAAGCTAGCTTAAGTAACGCCATTTTGCAAGGCATGGAAAAATACATAACTGAGAATAGAGAAGTTCAGATCAAGGTCAGGAACAGATGGAACAGCTGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGAACAGCTGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGTCCCCAGATGCGGTCCAGCCCTCAGCAGTTTCTAGAGAACCATCAGATGTTTCCAGGGTGCCCCAAGGACCTGAAATGACCCTGTGCCTTATTTGAACTAACCAATCAGTTCGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTCCCCGAGCTCAATAAAAGAGCCCACAACCCCTCACTCGGGGCGCCAGTCCTCCGATTGACTGAGTCGCCCGGGTACCCGTGTATCCAATAAACCCTCTTGCAGTTGCATCCGACTTGTGGTCTCGCTGTTCCTTGGGAGGGTCTCCTCTGAGTGATTGACTACCCGTCAGCGGGGGTCTTTCACACATGCAGCATGTATCAAAATTAATTTGGTTTTTTTTCTTAAGTATTTACATTAAATGGCCATAGTACTTAAAGTTACATTGGCTTCCTTGAAATAAACATGGAGTATTCAGAATGTGTCATAAATATTTCTAATTTTAAGATAGTATCTCCATTGGCTTTCTACTTTTTCTTTTATTTTTTTTTGTCCTCTGTCTTCCATTTGTTGTTGTTGTTGTTTGTTTGTTTGTTTGTTGGTTGGTTGGTTAATTTTTTTTTAAAGATCCTACACTATAGTTCAAGCTAGACTATTAGCTACTCTGTAACCCAGGGTGACCTTGAAGTCATGGGTAGCCTGCTGTTTTAGCCTTCCCACATCTAAGATTACAGGTATGAGCTATCATTTTTGGTATATTGATTGATTGATTGATTGATGTGTGTGTGTGTGATTGTGTTTGTGTGTGTGACTGTGAAAATGTGTGTATGGGTGTGTGTGAATGTGTGTATGTATGTGTGTGTGTGAGTGTGTGTGTGTGTGTGTGCATGTGTGTGTGTGTGACTGTGTCTATGTGTATGACTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTTGTGAAAAAATATTCTATGGTAGTGAGAGCCAACGCTCCGGCTCAGGTGTCAGGTTGGTTTTTGAGACAGAGTCTTTCACTTAGCTTGGAATTCactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgatgacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagcattgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccAAGCTTTGCTCTTAGGAGTTTCCTAATACATCCCAAACTCAAATATATAAAGCATTTGACTTGTTCTATGCCCTAGGGGGCGGGGGGAAGCTAAGCCAGCTTTTTTTAACATTTAAAATGTTAATTCCATTTTAAATGCACAGATGTTTTTATTTCATAAGGGTTTCAATGTGCATGAATGCTGCAATATTCCTGTTACCAAAGCTAGTATAAATAAAAATAGATAAACGTGGAAATTACTTAGAGTTTCTGTCATTAACGTTTCCTTCCTCAGTTGACAACATAAATGCGCTGCTGAGCAAGCCAGTTTGCATCTGTCAGGATCAATTTCCCATTATGCCAGTCATATTAATTACTAGTCAATTAGTTGATTTTTATTTTTGACATATACATGTGAATGAAAGACCCCACCTGTAGGTTTGGCAAGCTAGCTTAAGTAACGCCATTTTGCAAGGCATGGAAAAATACATAACTGAGAATAGAAAAGTTCAGATCAAGGTCAGGAACAGATGGAACAGCTGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGAACAGCTGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGTCCCCAGATGCGGTCCAGCCCTCAGCAGTTTCTAGAGAACCATCAGATGTTTCCAGGGTGCCCCAAGGACCTGAAATGACCCTGTGCCTTATTTGAACTAACCAATCAGTTCGCTTCTCGCTTCTGTTCGCGCGCTTATGCTCCCCGAGCTCAATAAAAGAGCCCACAACCCCTCACTCGGGGCGCCAGTCCTCCGATTGACTGAGTCGCCCGGGTACCCGTGTATCCAATAAACCCTCTTGCAGTTGCATCCGACTTGTGGTCTCGCTGTTCCTTGGGAGGGTCTCCTCTGAGTGATTGACTACCCGTCAGCGGGGGTCTTTCATTTGGGGGCTCGTCCGGGATCGGGAGACCCCTGCCCAGGGACCACCGACCCACCACCGGGAGGTAAGCTGGCCAGCAACTTATCTGTGTCTGTCCGATTGTCTAGTGTCTATGACTGATTTTATGCGCCTGCGTCGGTACTAGTTAGCTAACTAGCTCTGTATCTGGCGGACCCGTGGTGGAACTGACGAGTTCGGAACACCCGGCCGCAACCCTGGGAGACGTCCCAGGGACTTCGGGGGCCGTTTTTGTGGCCCGACCTGAGTCCTAAAATCCCGATCGTTTAGGACTCTTTGGTGCACCCCCCTTAGAGGAGGGATATGTGGTTCTGGTAGGAGACGAGAACCTAAAACAGTTCCCGCCTCCGTCTGAATTTTTGCTTTCGGTTTGGGACCGAAGCCGCGCCGCGCGTCTTGTCTGCTGCAGCATCGTTCTGTGTTGTCTCTGTCTGACTGTGTTTCTGTATTTGTCTGAAAATATGGGCCCGGGCTAGcctgttaccactCCCTTAAGTTTGACCTTAGGTCACTGGAAAGATGTCGAGCGGATCGCTCACAACCAGTCGGTAGATGTCAAGAAGAGACGTTGGGTTACCTTCTGCTCTGCAGAATGGCCAACCTTTAACGTCGGATGGCCGCGAGACGGCACCTTTAACCGAGACCTCATCACCCAGGTTAAGATCAAGGTCTTTTCACCTGGCCCGCATGGACACCCAGACCAGGTccccTACATCGTGACCTGGGAAGCCTTGGCTTTTGACCCCCCTCCCTGGGTCAAGCCCTTTGTACACCCTAAGCCTCCGCCTCCTCTTCCTCCATCCGCCCCGTCTCTCCCCCTTGAACCTCCTCGTTCGACCCCGCCTCGATCCTCCCTTTATCCAGCCCTCACTCCTTCTCTAGGCGCCCCCATATGGCCATATGAGATCTTATATGGGGCACCCCCGCCCCTTGTAAACTTCCCTGACCCTGACATGACAAGAGTTACTAACAGCCCCTCTCTCCAAGCTCACTTACAGGCTCTCTACTTAGTCCAGCACGAAGTCTGGAGACCTCTGGCGGCAGCCTACCAAGAACAACTGGACCGACCGGTGGTACCTCACCCTTACCGAGTCGGCGACACAGTGTGGGTCCGCCGACACCAGACTAAGAACCTAGAACCTCGCTGGAAAGGACCTTACACAGTCCTGCTGACCACCCCCACCGCCCTCAAAGTAGACGGCATCGCAGCTTGGATACACGCCGCCCACGTGAAGGCTGCCGACCCCGGGGGTGGACCATCCTCTAGACTGCC [SEQ ID NO: 123]

In some embodiments, the vector includes or consists of the nucleotide sequence set forth in SEQ ID NO: 124, which is set forth below:

ATGGCCCTGCCAGTAACGGCTCTGCTGCTGCCACTTGCTCTGCTCCTCCATGCAGCCAGGCCTCAGGTTCAGCTTCAGGAGAGTGGCCCAGGCCTGGTGAAGCCAAGTGAGACTCTCAGCTTGACTTGCACAGTTTCTGGAGGCAGTGTCTCCTCAGGCAGCTATTATTGGTCCTGGATTCGGCAGCCCCCTGGGAAAGGCCTGGAGTGGATTGGGTACATATATTACAGTGGCAGCACAAA TTACAATCCATCCCTGAAGTCTCGAGTAACTATCAGTGTGGACACAAGCAAGAATCAGTTTTCACTCAAACTGTCTTCTGTGACTGCTGCTGACACTGCTGTTTATTATTGTGCCAGGGAGGGGAAAAATGGGGCATTTGATATTTGGGTCAGGGCACAATGGTGACAGTCAGCTCTGGAGGTGGAGGCTCAGGAGGAGGAGGCAGTGGAGGTGGTGGGTCACGCCATCAGATGACTCAGTC CCCCTCCAGTCTTTCTGCCTCAGTTGGGGATAGAGTGACCATCACATGCAGAGCAAGTCAGAGCATATCATCCTACTGAACTGGTACCAGCAGAAGCCAGGGAAAGCCCCCAAATTGCTGATTTATGCAGCCTCAAGTCTCCAGAGTGGGGTGCCAAGCAGGTTCTCAGGCAGTGGCAGTGGGACAGATTTCACATTGACAATCAGCTCCCTCCAACCTGAAGATTTTGCCACCTACTATTG CCAGCAATCCTACAGCACGCCCCTGACTTTTGGAGGTGGCACAAAGGTAGAGATCAAGAGGACTgcggccgcaattgaagttatgtatcctcctccttacctagacaatgagaagagcaatggaaccattatccatgtgaaagggaaacacctttgtccaagtcccctatttcccggaccttctaagcccttttgggtgctggtggtggttgg tggagtcctggcttgctatagcttgctagtaacagtggcctttattattttctgggtgaggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgccgccccgggcccacccgcaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctccagagtgaagttcagcaggagcgcagacg cccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtTcaatgaactgcagaaagataagatggcggaggcctTcagtgagattgggatgaaaggcgagcg ccggaggggcaaggggcacgatggcctttTccaggggctcagtacagccaccaaggacacctTcgacgcccttcacatgcaggccctgccccctcgcGGATCTGGAGCAACAAA CTTCTCACTACTCAAACAAGCAGGTGACGTGGAGGAGAATCCCGGCCCAATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAACTGGGCTGGCGGCCAGG ATGGTTCTTAGACTCCCCAGACAGGCCCTGGAACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAACGCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCTTTCCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACACAACTGCCCAACGGG CGTGACTTCCACATGAGCGTGGTCAGGGCCCGGCGCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGGCCCCCAAGGCGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGG TGACAGAGAGAAGGGCAGAAGTGCCCAGCCCACCCCAGCCCCTCACCCAGGCCAGCCGGCCAGGCGGCCGCACCCACCACGACGCCAGCGCCGCGACCACCAACCCCGGCGCCCACGATC GCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCCTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAACCACAGGCGGATCCAATAAcagccactcgaggatccGGATTAGTCCAATTTGTTAAAGACAGGATATCAGTGGTCCAGG CTCTAGTTTTGACTCAACAATATCACCAGCTGAAGCCTATAGAGTACGAGCCATAGATAAAATAAAAGATTTTATTTAGTCTCCAGAAAAAGGGGGGAATGAAAGACCCCACCTGTAGGTTTGGCAAGCTAGCTTAAGTAACGCCATTTTGCAAGGCATGGAAAAATACATAACTGAGAATAGAGAAGTTCAGATCAAGGTCAGGAACAGATGGAACAGCTGAATATGGGCCAAACAGGATATCTGTGGTA AGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGAACAGCTGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGTCCCCAGATGCGGTCCAGCCCTCAGCAGTTTCTAGAGAACCATCAGATGTTTCCAGGGTGCCCCAAGGACCTGAAATGACCCTGTGCCTTATTTGAACTAACCAATCAGTTCGCTTCTCGCTTCTGTTC GCGCGCTTCTGCTCCCCGAGCTCAATAAAAGAGCCCACAACCCCACTCGGGGCGCCAGTCCTCCGATTGACTGAGTCGCCCGGGTACCCGTGTATCCAATAAACCCTCTTGCAGTTGCATCCGACTTGTGGTCTCGCTGTTCCTTGGGAGGGTCTCCTCTGAGTGATTGACTACCCGTCAGCGGGGGTCTTTCACATGCAGCATGTATCAAAATTAATTTGGTTTTTTTTCTTAAGTATT TACATTAAAATGGCCATAGTACTTAAAGTTACATTGGCTTCCTTGAAATAAACATGGAGTATTCAGAATGTGTCATAAATATTTCTAATTTTAAGATAGTATCTCCATTGGCTTTCTACTTTTTCTTTTATTTTTTTTTGTCCTCTGTCTTCCATTTGTTGTTGTTGTTGTTTGTTTGTTTGTTTGTTGGTTGGTTGGTTAATTTTTTTTTAAAGATCCTACACTATAGTTCAAGCTAGACTAT TAGCTACTCTGTAACCCAGGGTGACCTTGAAGTCATGGGTAGCCTGCTGTTTTAGCCTTCCCACATCTAAGATTACAGGTATGAGCTATCATTTTTGGTATATTGATTGATTGATTGATTGATGTGTGTGTGTGTGATTGTGTTTGTGTGTGTGAtTGTGtATATGTGTGTATGGtTGTGTGTGAtTGTGTGTATGTATGTtTGTGTGTGAtTGTGTGTGTGatTGTGCATGTG TGTGTGTGTGAtTGTGTtTATGTGTATGAtTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTTGTGtAtAtATATTTATGGTAGTGAGAGgCAACGCT CCGGCTCAGGTGTCAGGTTGGTTTTTGAGACAGAGTCTTTCACTTAGCTTGGAATTCactggccgtcgttttacaacgtcgtgactgggaaaccctggcgttacccaactta atcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatatggtgcactctcagtacaatctgctctgatgccgcatagtta agccagccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagct gcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtt tcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcatttgcctt cctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcactttttaaagttctgctatgtggcgcggtattatcccgtattga cgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggat catgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctgg ctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacga cggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagttactcatatatactt tagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaa accaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtTcttcta gtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgt gtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagcTAtgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcgga acaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatt tttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctca catalog ttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccAAGCTTTGCTCTTAGGAGTTTCCTAATACATCCCAAACTCAAATATA TAAAGCATTTGACTTGTTCTATGCCCTAGGGGGCGGGGGGAAGCTAAGCCAGCTTTTTTTAACATTTAAAATGTTAATTCCATTTTTAAATGCACAGATGTTTTTATTTCATAAGGGTTTCAATGTGCATGAATGCTGCAATATTCCTGTTACCAAAGCTAGTATAAATAAAAATAGATAAACGTGGAAATTACTTAGAGTTTCTGTCATTAACGTTTCCTTCCTCAGTTGACAACATAAATGCGCT GCTGAGAAGCCAGTTTGCATCTGTCAGGATCAATTTCCCATTATGCCAGTCATATTAATTACTAGTCAATTAGTTGATTTTTATTTTTGACATATACATGTGAAAGACCCCACCTGTAGGTTTGGCAAGCTAGCTTAAGTAACGCCATTTTGCAAGGCATGGAAAAATACATAACTGAGAATAGAAAAGTTCAGATCAAGGTCAGGAACAGATGGAACAGCTGAATATGGGCCAAACAGGATATCTGTGG TAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGAACAGCTGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGTCCCCAG ATGCGGTCCAGCCCTCAGCAGTTTCTAGAGAACCATCAGATGTTTCCAGGGTGCCCCAAGGACCTGAAATGACCCTGTGCCTTATTTGAACTAACCAATCAGTTCGCTTCTCGCTTCTGTTCG CGCGCTTcTGCTCCCCGAGCTCAATAAAAGAGCCCACAACCCCTCACTCGGcGCGCCAGTCCTCCGATTGACTGAGTCGCCCGGGTACCCGTGTATCCAATAAACCCTCTTGCAGTTGCATCCGACTTGTGGTCTCGCTGTTCCTTGGGAGGGTCTCCTCTGAGTGATTGACTACCCGTCAGCGGGGGTCTTTCATTTGGGGGCTCGTCCGGGATCGGGAGACCCCTGCCCAGGGA CCACCGACCCACCACCGGGAGGTAAGCTGGCCAGCAACTTATCTGTGTCTGTCCGATTGTCTAGTGTCTATGACTGATTTTATGCGCCTGCGTCGGTACTAGTTAGCTAACTAGCTCTGTATCTGGCGGACCCGTGGTGGAACTGACGAGTTCGGAACACCCGGCCGCAACCCTGGGAGACGTCCCAGGGACTTCGGGGGCCGTTTTTGTGGCCCGACCTGAGTCCTAAAATCCCGATCG TTTAGGACTCTTTGGTGCACCCCCCTTAGAGGAGGGATATGTGGTTCTGGTAGGAGACGAGAACCTAAAACAGTTCCCGCCTCCGTCTGAATTTTTGCTTTCGGTTTGGGACCGAAGCCGCGCCGCGCGTCTTGTCTGCTGCAGCATCGTTCTGTGTTGTCTCTGTCTGACTGTGTTTCTGTATTTGTCTGAAAATATGGGCCCGGGCTAGcctgttaccactCCCTTAAGTTTGACCTTA GGTCACTGGAAAGATGTCGAGCGGATCGCTCACAACCAGTCGGTAGATGTCAAGAAGAGACGTTGGGTTACCTTCTGCTCTGCAGAATGGCCAACCTTTAACGTCGGATGGCCGCGAGACGGCACCTTTAACCGAGACCTCATCACCCAGGTTAAGATCAAGGTCTTTTCACCTGGCCCGCATGGACACCCAGACCAGGTccccTACATCGTGACCTGGGAAGCCTTGGCTTTTGACCCCCC TCCCTGGGTCAAGCCCTTTGTACACCCTAAGCCTCCGCCTCCTCTTCCTCCATCCGCCCCGTCTCTCCCCCTTGAACCTCCTCGTTCGACCCCGCCTCGATCCTCCCTTTATCCAGCCCTCACTC CTTCTCTAGGCGCCCCCATATGGCCATATGAGATCTTATATGGGGCACCCCCGCCCCTTGTAAACTTCCCTGACCCTGACATGACAAGAGTTACTAACAGCCCCTCTCCAAGCTCACTTACAG GCTCTCTACTTAGTCCAGCACGAAGTCTGGAGACCTCTGGCGGCAGCCTACCAAGAACAACTGGACCGACCGGTGGTACCTCACCCTTACCGAGTCGGCGACACAGTGTGGGTCCGCCGACACCAGACTAAGAACCTAGAACCTCGCTGGAAAGGACCTTACACAGTCCTGCTGACCACCCCCACCGCCCTCAAAGTAGACGGCATCGCAGCTTGGATACACGCCGCCCACGTGAAGGCTGCCG ACCCCGGGGGTGGACCATCCTCTAGACTGCC [SEQ ID NO: 124]

Non-viral approaches can also be used to genetically modify an immunoreactive cell. For example, a nucleic acid molecule can be introduced into an immunoreactive cell by introducing the nucleic acid in the presence of lipofection (Feigner et al., Proc. Natl. Acad. Sci. U.S.A. 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) 263: 14621, 1988; Wu et al., Journal of Biological Chemistry 264: 16985, 1989) or by microinjection in a surgical setting (Wolff et al., Science 247: 1465, 1990). Other nonviral gene transfer methods include in vitro transfection using calcium phosphate, DEAE-dextran, electroporation, and protoplast fusion. Liposomes can also potentially be used to deliver DNA into cells. Transplantation of normal genes into diseased tissues of a subject can also be achieved by transfer of normal nucleic acid into cells of a cultured type ex vivo (for example, into an autologous or heterologous primary cell or its progeny), after which the cell (or its progeny) is introduced into the target tissue or injected systematically. Recombinant receptors can also be created or produced using transposases or targeting nucleases (eg, zinc finger nucleases, meganucleases or TALE nucleases, CRISPR). Transient expression can be achieved by electroporation of RNA.

Any methods of targeted genome editing can be used to express a polypeptide composition. In some embodiments, the CRISPR system is used to express a polypeptide composition disclosed herein. In some embodiments, zinc finger nucleases are used to express a polypeptide composition disclosed herein. In some embodiments, the TALEN system is used to express a polypeptide composition disclosed herein.

The Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) system is a genome editing tool discovered in prokaryotic cells. When used for genome editing, such a system includes Cas9 (a protein capable of modifying DNA using crRNA as a guide), CRISPR RNA (crRNA, contains RNA used by the Cas9 nuclease to guide it to the correct site in the host DNA, along with a region that binds to tracrRNA (usually in the form of a hairpin), forming an active complex with Cas9), transactivating crRNA (tracrRNA, binds to crRNA and forms an active complex with Cas9) and an optional DNA repair template site (DNA that directs the cellular repair process, ensuring the insertion of a specific DNA sequence ). CRISPR/Cas9 often uses a plasmid to transfect target cells. CrRNA must be designed for each specific application because it is the sequence that Cas9 uses to identify and directly bind to target DNA in the cell. The repair template carrying the CAR expression cassette must also be designed for each specific application, as it must overlap with the sequences on either side of the cut and encode the insertion sequence. Multiple crRNAs and tracrRNAs can be packaged together to form a single guide RNA (sgRNA). Such an sgRNA can be coupled to the Cas9 gene and converted into a plasmid for transfection into cells.

Zinc finger nuclease (ZFN) is an artificial restriction enzyme created by combining the DNA binding domain of a zinc finger with a DNA cleavage domain. The zinc finger domain can be designed to target specific DNA sequences, allowing zinc finger nuclease to target desired sequences in genomes. The DNA-binding domains of individual ZFNs typically contain many distinct zinc finger repeats, and each can recognize multiple base pairs. The most common method for creating a new zinc finger domain is to combine smaller zinc finger “modules” that have known specificity. The most common cleavage domain in ZFN is the nonspecific cleavage domain from the type II restriction endonuclease FokI. By using the endogenous homologous recombination (HR) machinery and a homologous DNA template carrying a CAR expression cassette, ZFN can be used to insert a CAR expression cassette into the genome. When the target sequence is cleaved by ZFN nucleases, the HR apparatus searches for homology between the damaged chromosome and the homologous DNA template, and then copies the template sequence between the two broken ends of the chromosome, as a result of which the homologous DNA template is integrated into the genome.

Transcription activator-like effector nucleases (TALENs) are restriction enzymes that can be engineered to cut specific DNA sequences. The TALEN system works on almost the same principle as the ZFN. They are created by combining the DNA-binding domain of transcription activator-like effectors with a DNA cleavage domain. Transcription activator-like effectors (TALEs) consist of 33–34 amino acid-long repeat motifs with two variable positions that are capable of reliably recognizing specific nucleotides. By assembling sets of such TALE effectors, the TALE DNA-binding domain can be designed to bind the desired DNA sequence and thus direct the nuclease to cut at specific locations in the genome. Expression of cDNA for use in polynucleotide therapy methods may be directed from any suitable promoter (eg, human cytomegalovirus (HCMV), simian virus 40 (SV40) or metallothioneion promoters) and may be regulated by any suitable mammalian regulatory element or intron (eg, enhancer structure /promoter/intron of elongation factor 1α). For example, enhancers that are known to preferentially direct gene expression in specific cell types by regulating nucleic acid expression can be used if desired. Enhancers used may include, without limitation, enhancers that are characterized as tissue-specific or cell-specific enhancers. Alternatively, if a genomic clone is used as a therapeutic construct, regulation may be mediated by cognate regulatory sequences or, optionally, regulatory sequences derived from a heterologous source, including any of the promoters or regulatory elements described above.

Methods of delivery of genome editing tools/systems may vary depending on the need. In some embodiments, components of the selected genome editing method are delivered as DNA constructs in one or more plasmids. In some embodiments, the components are delivered using viral vectors. Common delivery methods include, but are not limited to, electroporation, microinjection, gene gun, impalefection, hydrostatic pressure, continuous infusion, sonication, magnetofection, adeno-associated viruses, envelope protein pseudotyping of viral vectors, replication competent vectors, cis- and trans-acting elements, herpes simplex virus and chemical carriers (eg, oligonucleotides, lipoplexes, polymersomes, polyplexes, dendrimers, inorganic nanoparticles and cell penetrating peptides).

5.5. Polypeptides and analogues

Also disclosed herein, the invention includes a polypeptide disclosed herein (e.g., mesothelin, CD28, CD8, CD3ζ and PD-1 DN, etc.), or fragments thereof, that are modified in ways that improve their antitumor activity in expression in an immunoreactive cell. The present invention provides methods for optimizing an amino acid sequence or a nucleic acid sequence by changing the sequence. Such changes may include certain mutations, deletions, insertions or post-translational modifications. The present invention further includes analogs of any polypeptide disclosed herein (including, but not limited to, mesothelin, CD28, CD8, CD3ζ and PD-1 DN). Analogues may differ from the polypeptide disclosed herein by differences in amino acid sequence and/or post-translational modifications. Analogs may 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 greater homology to all or part of the amino acid sequence according to the invention disclosed herein. The length of the sequence comparison is at least 5, 10, 15 or 20 amino acid residues, such as at least 25, 50 or 75 amino acid residues, or greater than 100 amino acid residues. Again, an exemplary approach to determining the degree of identity may use the BLAST program with a probability score of e ^-3 to e ^-100 indicating a closely related sequence. Modifications include chemical derivatization of polypeptides in vivo and in vitro, such as acetylation, carboxylation, phosphorylation or glycosylation; such modifications may occur during synthesis or processing of the polypeptide or after treatment with isolated modifying enzymes. Analogues may also differ from polypeptides by changes in the primary sequence. Such changes include genetic variants, natural and induced (for example, those resulting from random mutagenesis by radiation or ethane methyl sulfonate, or from 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 that contain residues other than L-amino acids, such as D-amino acids or unnatural or synthetic amino acids, such as β- or γ-amino acids.

In addition to full-length polypeptides, the invention disclosed herein also provides fragments of any 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 some embodiments, the fragment includes at least 20 consecutive amino acids, at least 30 consecutive amino acids, or at least 50 consecutive amino acids. In some embodiments, the fragment includes at least 60-80, 100, 200, 300, or more consecutive amino acids. The fragments can be produced by methods known to those skilled in the art, or can be formed as a result of normal protein processing (for example, the removal of amino acids from a synthesized polypeptide that are not required for biological activity, or the removal of amino acids as a result of alternative splicing events of mRNA or alternative protein processing) .

Non-protein analogues have a chemical structure that serves to mimic the functional activity of the protein disclosed herein. Such analogues may exhibit the physiological activity of the parent polypeptide. Methods for constructing analogs are well known in the art, and synthesis of analogs can be accomplished according to such methods by modifying the chemical structures such that the resulting analogs have greater antineoplastic activity than the parent polypeptide when expressed in an immunoreactive cell. Such chemical modifications include, but are not limited to, substitution of alternative R groups and changes in the degree of saturation of specific carbon atoms compared to the original polypeptide. In some embodiments, the protein analogues are relatively resistant to degradation in vivo, allowing for a longer lasting therapeutic effect when administered. Assays for measuring functional activity include, without limitation, those described in the Examples below.

In accordance with the invention disclosed herein, polynucleotides encoding an extracellular antigen binding domain that specifically binds to human mesothelin (eg, scFv, Fab or (Fab) ₂ ), CD3ζ, CD8, CD28, 4-1BB, 4-1BBL and IL- 12 can be modified by codon optimization. Codon optimization can modify both natural and recombinant gene sequences to achieve the highest possible levels of productivity in any given expression system. Factors that influence the various stages of protein expression include codon adaptability, mRNA structure, and various cis elements in transcription and translation. Any suitable codon optimization methods or technologies that are known to those skilled in the art can be used to modify the polynucleotides of the invention disclosed herein, including, but not limited to, OptimumGene™, Encor and Blue Heron optimization.

Codon optimization can be performed based on four different algorithms (eg, Blue Heron and Encore algorithms). The codon optimized sequences resulting from all four algorithms are mixed and removed all CPGs and BamH1 for optimal cloning. In some embodiments, the codon-optimized nucleotide sequence is approximately 70% homologous to the original sequence before codon optimization. To obtain efficient expression in an immunoreactive cell (eg, primary human T cells), the codon-optimized nucleotide sequence is ligated to a CD8 leader sequence, for example, a polynucleotide encoding SEQ ID NO: 71. The CD8 leader sequence ensures optimal cleavage of the signal sequence preceding the severe scFv chains (QVQL). Codon optimization optimizes expression of a mesothelin-specific CAR in an immunoreactive cell, such as primary human T cells from multiple donors, with good transduction efficiency. Multiple copies of CAR vectors in T cells from multiple donors are tested for functional potency, specificity, and sensitivity against a variety of hematological and solid tumor cells with varying mesothelin expression. A codon-optimized mesothelin-targeted CAR with a vector copy number of 1-4 (more specifically, approximately 3-4) provides very effective cytotoxicity against targets with high mesothelin expression, but with minimal reactivity against targets with low mesothelin expression, i.e. e. normal tissue. The above-described genetic engineering means of creating a mesothelin-specific CAR receptor that is reactive against cancer cells expressing high levels of mesothelin while sparing normal tissue expressing low levels of mesothelin is optimal for use as a clinical vector for cancer therapy with a guarantee of safety .

5.6. Pharmaceutical compositions and administration

The invention disclosed herein provides compositions comprising cells disclosed herein (eg, as disclosed in Section 5.3). The number of cells contained in the compositions may vary depending on the purpose of use of the composition, and/or the size, age, sex, weight and condition of the subject who receives the composition. In some embodiments, the composition includes from about 10 ⁴ to about 10 ¹⁰ , from about 10 ⁴ to about 10 ⁶ , from about 10 ⁵ to about 10 ⁶ , from about 10 ⁵ to about 10 ⁷ , from about 10 ⁵ to about 10 ⁹ or from about 10 ⁶ to about 10 ⁸ immunoreactive cells disclosed herein. In some embodiments, the composition includes at least about 1×10 ⁵ , at least about 5×10 ⁵ , at least about 1×10 ⁶ , at least about 1×10 ⁷ , at least about 1×10 ⁸ immunoreactive cells disclosed herein. In some embodiments, the composition includes approximately 1×10 ⁵ cells disclosed herein.

Compositions comprising the immunoreactive cells disclosed herein may be provided systemically or directly to a subject to induce and/or enhance an immune response to an antigen, and/or treat and/or prevent a tumor, pathogenic infection or infectious disease, inflammatory disease, or transplant rejection. In some embodiments, immunoreactive cells disclosed herein or compositions comprising them are directly administered to an organ of interest (eg, an organ affected by neoplasia). Also disclosed herein, immunoreactive cells or compositions comprising them are provided indirectly to an organ of interest, for example, when administered to the cardiovascular system (eg, tumor vasculature). Expansion and differentiation agents can be provided before, during or after administration of cells or compositions to increase the production of T cells, NK cells or CTL cells in vitro or in vivo.

The immunoreactive cells disclosed herein can be administered in any physiologically acceptable environment, typically by the intravascular route, although they can also be administered into bone or other convenient site where the cells can find a suitable site for regeneration and differentiation (eg, the thymus). Typically, at least about 1x10 ⁵ cells will be administered, ultimately reaching about 1x10 ¹⁰ or more. The immunoreactive cells disclosed herein may include a purified population of cells. Those skilled in the art will readily be able to determine the percentage of immunoreactive cells disclosed herein in a population using various known techniques such as fluorescence activated cell sorting (FACS). Suitable purity range boundaries in populations comprising the immunoreactive cells disclosed herein are from about 50% to about 55%, from about 5% to about 60%, and from about 65% to about 70%. In some embodiments, the purity is from about 70% to about 75%, from about 75% to about 80%, or from about 80% to about 85%. In some embodiments, the purity is from about 85% to about 90%, from about 90% to about 95%, and from about 95% to about 100%. Doses can be easily adjusted by specialists (for example, a decrease in purity may require an increase in dose). The cells may be administered by injection, catheter, or the like.

The compositions disclosed herein may be pharmaceutical compositions comprising the immunoreactive cells or precursors thereof disclosed herein and a pharmaceutically acceptable carrier. Administration can be autologous or heterologous. For example, immunoreactive cells or progenitors can be obtained from one subject and administered to the same subject or another compatible subject. Peripheral blood-derived immunoreactive cells or their progeny (eg, obtained in vivo, ex vivo, or in vitro) can be administered by localized injection, including catheter administration, systemic injection, localized injection, intravenous injection, or parenteral administration. When a therapeutic composition of the present invention is administered (eg, a pharmaceutical composition comprising an immunoreactive cell disclosed herein), it may be formulated in a standard injectable form (solution, suspension, emulsion).

5.7. Compositions

Compositions comprising the immunoreactive cells disclosed herein may conveniently be presented as sterile liquid preparations, for example, isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may be buffered to a selected pH. Liquid preparations are generally easier to prepare than gels, other viscous compositions, and solid compositions. In addition, liquid compositions are slightly more convenient to administer, particularly by injection. On the other hand, viscous compositions can be formulated in an appropriate viscosity range to provide longer periods of contact with specific tissues. Liquid and viscous compositions may include carriers, which may be a solvent or dispersion medium containing water, saline, phosphate-buffered saline, polyhydric alcohol (eg, glycerol, propylene glycol, liquid polyethylene glycol, etc.) and suitable mixtures thereof.

Sterile injection solutions can be prepared by incorporating genetically modified immunoreactive cells in the required amount of a suitable solvent, with varying amounts of other ingredients as necessary. Such compositions may be present in admixture with a suitable carrier, diluent or excipient such as sterile water, saline, glucose, dextrose and the like. The compositions may also be lyophilized. The compositions may contain excipients such as wetting, dispersing or emulsifying agents (eg methylcellulose), pH buffering agents, gelling or viscosity enhancing agents, preservatives, flavoring agents, coloring agents, etc., depending on the desired route of administration and formulation. . Standard texts such as REMINGTON'S PHARMACEUTICAL SCIENCE, 17th edition, 1985, incorporated herein by reference, provide information on preparing suitable preparations without undue experimentation.

Various additives may be added to enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents and buffering agents. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, etc. Prolonged absorption of the injectable pharmaceutical form can be achieved by using substances that delay absorption, for example, aluminum monostearate and gelatin. However, according to the invention disclosed herein, any medium, diluent or additive used must be compatible with the genetically modified immunoreactive cells or their precursors.

The compositions may be isotonic, i.e. they can have the same osmotic pressure as blood and tear fluid. The desired isotonicity of the compositions can be achieved by using sodium chloride or other pharmaceutically acceptable substances such as dextrose, boric acid, sodium tartrate, propylene glycol or other inorganic or organic solutes. Sodium chloride is particularly preferred in the case of buffers containing sodium ions.

The viscosity of the composition can, if necessary, be maintained at a selected level by using a pharmaceutically acceptable thickener. For example, methylcellulose is readily, economically available and easy to work with. Other suitable thickeners include, for example, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, carbomer and the like. The concentration of the thickener may depend on the product chosen. The important point is to use an amount that will achieve the selected viscosity. Obviously, the selection of suitable carriers and other additives will depend on the precise route of administration and the nature of the particular dosage form, such as a liquid dosage form (e.g., whether the composition is to be formulated as a solution, suspension, gel, or other liquid form, such as sustained release form or liquid-filled form).

The number of cells administered will vary depending on the subject being treated. In some embodiments, a human subject is administered from about 10 ⁴ to about 10 ¹⁰ , from about 10 ⁵ to about 10 ⁹ , from about 10 ⁴ to about 10 ⁶ , from about 10 ⁵ to about 10 ⁶ , from about 10 ⁵ to about 10 ⁷ or from about 10 ⁶ to about 10 ⁸ immunoreactive cells disclosed herein. More effective cells can be administered in even smaller quantities. In some embodiments, a human subject is administered at least about 1×10 ⁵ , at least about 1×10 ⁶ , at least about 1×10 ⁷ , 1×10 ⁸ , at least about 2×10 ⁸ , at least about 3×10 ⁸ , at least about 4×10 ⁸ , or at least about 5×10 ⁸ immunoreactive cells disclosed herein. The precise determination of what is considered an effective dose may be based on factors individual to each subject, including the size, age, sex, weight and condition of the particular subject. Dosages can be readily determined by those skilled in the art from the present disclosure and available knowledge. In some embodiments, the subject is administered approximately 1×10 ⁵ cells disclosed herein.

One skilled in the art will readily be able to determine the number of cells and optional additives, excipients and/or carrier in the compositions and methods administered. Typically, any additives (in addition to the active cell(s) and/or agent(s)) are present in an amount of from 0.001 to 50% (by weight) solution in phosphate-buffered saline, and the active ingredient is present in an amount on the order of micrograms or milligrams, such as from about 0.0001 to about 5 weight. %, preferably from about 0.0001 to about 1 wt. %, even more preferably from about 0.0001 to about 0.05 wt. % or from about 0.001 to about 20 wt. %, from about 0.01 to about 10 wt. % or from about 0.05 to about 5 wt. %. For any composition administered to an animal or human, the following may be determined: toxicity, for example by determining the lethal dose (LD) and LD50 in a suitable animal model, eg rodents such as mice; and the dose of the composition(s), the concentration of the components therein, and the time of administration of the composition(s) that produce the appropriate response. Such determinations do not require undue experimentation based on the knowledge available to one skilled in the art given herein as well as the documents cited therein. In addition, the timing of successive administrations can be established without unnecessary experimentation.

5.8. Treatment methods

Immunoreactive cells and compositions comprising them according to the invention disclosed herein can be used to treat and/or prevent a tumor caused by a pathogen infection, infectious disease, inflammatory disease or transplant rejection. Such immunoreactive cells may be administered to a subject (eg, a human subject) in need of treatment or prevention of a solid tumor (eg, mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer , gastric cancer, synovial sarcoma, thymic carcinoma, endometrial carcinoma, gastric cancer and/or cholangiocarcinoma). In some embodiments, the immunoreactive cell is a T cell. The T cell may be a CD4 ⁺ T cell or a CD8 ⁺ T cell. In some embodiments, the T cell is a CD4 ⁺ T cell.

The present invention provides methods for inducing and/or enhancing an immune response in a subject in need thereof. The immunoreactive cells disclosed herein and compositions comprising them can be used to treat and/or prevent neoplasia in a subject. The immunoreactive cells disclosed herein and compositions incorporating them can be used to prolong the survival of a subject suffering from neoplasia. The immunoreactive cells disclosed herein and compositions incorporating them may also be used to treat and/or prevent pathogen-induced infection or other infectious disease in a subject, such as an immunocompromised human subject. Such methods include administering the immunoreactive cells disclosed herein or a composition comprising them (eg, a pharmaceutical composition) in an amount effective to achieve the desired effect, whether alleviation of an existing condition or prevention of relapse. In the case of treatment, the amount administered is the amount effective to achieve the desired effect. An effective amount may be provided in one or a series of administrations. An effective amount may be provided as a bolus or continuous perfusion.

An "effective amount" (or "therapeutically effective amount") is an amount sufficient to produce a beneficial or desired clinical result in a treatment. An effective amount may be administered to a subject in one or more doses. With respect to treatment, an effective amount is an amount that is sufficient to alleviate, attenuate, stabilize, reverse, or slow the progression of a disease, or otherwise reduce the pathological consequences of the disease. The effective amount is usually determined on an individual basis by the attending physician and is within the skill of the art. Typically, when determining the appropriate dose, several factors are taken into account in order to achieve an effective amount. Such factors include the age, sex and weight of the subject, the condition being treated, the severity of the condition, and the form and effective concentration of the immunoreactive cells administered.

In the case of adoptive immunotherapy using antigen-specific T cells, cell doses typically are infused in the range of about 10 ⁶ to about 10 ¹⁰ (eg, 10 ⁹ ). However, due to the high potency of the polypeptide compositions disclosed herein, fewer cells as disclosed herein are required to achieve the desired effects. For example, approximately 1×10 ⁵ cells disclosed herein are sufficient to achieve the desired effects.

Following administration of the immunoreactive cells to a subject and subsequent differentiation, immunoreactive cells are induced that are specifically directed against a particular antigen (eg, human mesothelin). "Induction" of T cells may involve inactivation of antigen-specific T cells, for example, by ablation or anergy. Inactivation is particularly useful for establishing and restoring tolerance, for example in autoimmune disorders. The immunoreactive cells of the invention disclosed herein can be administered by any route 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. into the thymus. In some embodiments, the immunoreactive cells and/or compositions comprising them are administered pleurally to a subject in need. In some embodiments, the immunoreactive cells and/or compositions comprising them are administered intrapleurally to a subject in need.

The present invention provides various methods of using immunoreactive cells (eg, T cells). For example, the present invention provides methods for reducing the tumor burden in a subject. In some embodiments, a method of reducing tumor burden comprises administering an effective amount of immunoreactive cells disclosed herein or a composition comprising them to a subject. An immunoreactive cell disclosed herein can reduce the number of tumor cells, reduce the size of a tumor, and/or eliminate a tumor in a subject. The tumor may be a solid tumor. Non-limiting examples of solid tumor include mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic carcinoma, endometrial carcinoma, gastric cancer and cholangiocarcinoma.

The present invention also provides methods for increasing or prolonging the survival of a subject having neoplasia. In some embodiments, a method of increasing or prolonging the survival of a subject having neoplasia comprises administering an effective amount of immunoreactive cells disclosed herein or a composition comprising them to the subject. The method may cause a reduction or elimination of a tumor burden in a subject. In addition, the present invention provides methods for enhancing an immune response in a subject, comprising administering an immunoreactive cell disclosed herein or a composition comprising the same to the subject. The present invention also provides methods for treating and/or preventing a tumor in a subject, comprising administering an immunoreactive cell disclosed herein or a composition comprising the same to the subject.

In some embodiments, the neoplasia is a solid tumor. Neoplasia can be a primary tumor or a primary cancer. In addition, neoplasia may have a metastatic status.

Malignant tumors whose growth can be inhibited using immunoreactive cells according to the invention disclosed herein include malignant tumors typically responsive to immunotherapy. Non-limiting examples of malignancies for treatment include mesothelioma, lung cancer (eg, non-small cell lung cancer), pancreatic cancer, ovarian cancer, breast cancer (eg, metastatic breast cancer, metastatic triple negative breast cancer), colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic carcinoma, endometrial carcinoma, gastric cancer, cholangiocarcinoma, cervical cancer and salivary gland cancer. In addition, the invention disclosed herein includes refractory or relapsed malignant tumors, the growth of which can be slowed by the use of immunoreactive cells according to the invention disclosed herein.

Examples of other neoplasias or malignancies that may be treated using the methods of the invention disclosed herein include bone cancer, colon cancer, liver cancer, skin cancer, head or neck cancer, melanoma (cutaneous or intraocular malignant melanoma), kidney cancer, (eg, clear cell carcinoma), throat cancer, prostate cancer (eg, hormone-resistant prostate adenocarcinoma), hematologic malignancies (eg, leukemia, lymphoma, and myeloma), uterine cancer, rectal cancer, anal cancer, bladder cancer, brain cancer brain, stomach cancer, testicular cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, leukemia (eg, acute leukemia, acute lymphocytic leukemia, acute myelocytic 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 disease, non-Hodgkin's lymphoma), small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer , soft tissue sarcoma, urethral cancer, penile cancer, solid tumors in children, lymphocytic lymphoma, bladder cancer, kidney or ureteral cancer, renal pelvis cancer, central nervous system (CNS) neoplasia, primary CNS lymphoma, tumor angiogenesis, axis tumor spine, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, cancers caused by environmental factors, including those caused by asbestos, including Waldenström's macroglobulinemia, heavy chain disease and solid tumors, such as sarcomas and carcinomas (for example, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, squamous cell carcinoma, cell carcinoma, adenocarcinoma, carcinoma sweat glands, sebaceous carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms tumor, cervical cancer, salivary gland cancer, uterine cancer, testicular cancer , bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodenroglioma, schwannoma, meningioma, melanoma, neuroblastoma and retinoblastoma).

In addition, the present invention provides methods for increasing the production of an immune-activating cytokine in response to a cancer cell or pathogen in a subject. In some embodiments, the method includes administering an immunoreactive cell disclosed herein or a composition comprising the same to a subject. 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 some embodiments, the immunoreactive cells increase the production of GM-CSF, IFN-γ, and/or TNF-α.

The invention disclosed herein provides therapies that are particularly useful for treating solid tumors (eg, mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, cancer stomach, synovial sarcoma, thymic carcinoma, endometrial carcinoma, gastric cancer and cholangiocarcinoma). Solid tumors can be primary tumors or tumors in a metastatic state. Some solid tumors are heterogeneous MSLN-expressing tumors, such as breast cancer (eg, TNBC), lung cancer, ovarian cancer, pancreatic cancer, esophageal cancer, colon cancer, gastric cancer, and malignant pleural mesothelioma (MPM). Heterogeneous MSLN-expressing cells (eg, tumor cells) are a population of cells including cells with low levels of MSLN expression and cells with high levels of MSLN expression. An immunoreactive cell disclosed herein may exhibit increased cytotoxicity and antitumor activity against cells with low MSLN expression (e.g., about 2000 or less, about 1000 or less, about 900 or less, about 800 or less, about 700 or less, about 600 or less, about 500 or less, about 400 or less, about 300 or less, about 200 or less, or about 100 or less MSLN binding sites/cell) in the presence of cells with high levels of MSLN expression. In some embodiments, even in the presence of cells with high MSLN expression, immunoreactive cells do not exhibit increased cytotoxicity or nonspecific cytolysis of MSLN-negative cells. Thus, an immunoreactive cell may exhibit enhanced cytotoxicity and antitumor activity against low MSLN expressing cells in the presence of high MSLN expressing cells while remaining safe for MSLN negative cells.

In addition, the invention disclosed herein provides methods for treating subjects with an infection caused by a pathogen (eg, a viral infection, a bacterial infection, a fungal infection, a parasitic infection, or a protozoal infection). The invention disclosed herein is particularly useful for enhancing the immune response in a subject with an immunodeficiency. Examples of viral infections susceptible to treatment using the method of the invention include, but are not limited to, infections caused by cytomegalovirus (CMV), Epstein-Barr virus (EBV), human immunodeficiency virus (HIV), and influenza virus. Thus, the present invention provides a method of treating or preventing infection caused by a pathogen in a subject, comprising administering an effective amount of immunoreactive cells disclosed herein or a composition comprising them.

In accordance with the invention disclosed herein, the various methods described above may involve administering at least one immunomodulatory agent. Non-limiting examples of immunomodulatory agents include immunostimulants, immune checkpoint blockers, radiotherapy agents, and chemotherapeutic agents. In some embodiments, the immunomodulatory agent is an immunostimulant agent. Non-limiting examples of immunostimulatory agents include IL-12 and agonistic costimulatory monoclonal antibodies. In some embodiments, the immunostimulatory agent is IL-12. In some embodiments, immunoreactive cells disclosed herein or a composition comprising them in combination with an anti-IL-12 antibody 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 some embodiments, the agonistic co-stimulatory monoclonal antibody is an anti-4-1BB antibody.

In some embodiments, immunoreactive cells disclosed herein or a composition comprising them not only can demonstrate tumor-targeted adoptive T cell therapy, but can also enhance T cell function through the generation of improved antigen receptors and through interfering with the host microenvironment through immunomodulation using IL- 12. Of all immunotherapeutic approaches, IL-12, a multifunctional cytokine, is considered one of the most promising approaches for the treatment of breast cancer (Boggio, K., et al., Cancer Res 60, 359-364 (2000); Czerniecki, B. J. et al., Cancer Res 67, 1842-1852 (2007); Nanni, P. et al., J Exp Med 194, 1195-1205 (2001)). IL-12 is considered a master regulator of type 1 adaptive cellular immunity, a critical pathway involved in antitumor responses (Del Vecchio, M., et al., Clin Cancer Res 13, 4677-4685 (2007)). IL-12 modulates antitumor responses at various levels, including polarization of CD4 T cells toward a Th1 phenotype (Wesa, et al., J Immunother 30, 75-82 (2007)), enhancement of T and NK cell effector functions (Curtsinger et al., 2007). al., J Exp Med 197, 1141-1151 (2003)), remodeling of the innate immune response (Chmielewski et al., Cancer Res 71, 5697-5706 (2011)) and regulation of tumor angiogenesis (Voest et al., J Natl Cancer Inst 87, 581-586 (1995). The immunomodulatory and antiangiogenic functions of IL-12 have provided the basis for the use of this cytokine in combination with an immunoreactive cell of the invention disclosed herein for the treatment of cancer, such as breast cancer (eg, TNBC). In 148 clinical trials involving the administration of IL-12 to patients with cancer (36 of which were recently reported), successful phase II studies were administered intraperitoneally (Lenzi et al. Clin.Cancer Res. 8, 3686-3695 (2002)) or subcutaneous ( Mahviet et al. Cancer Gene Ther. 14, 717-723 (2007); Kang et al., Hum. Gene Ther. 12, 671-684 (2001). administration of IL-12 showed that paracrine secretion of IL-12 generated by gene transfer could induce immunity against tumor locally and at a distant site. Although several studies have documented the antitumor efficacy of IL-12 in preclinical models of breast cancer (BC) (Boggio et al. Cancer Res 60, 359-364 (2000); Nanniet al. J Exp Med 194, 1195-1205 (2001)) , the significant toxicity resulting from administration of recombinant human IL-12 observed in several clinical studies in advanced malignancies precludes its clinical use. To overcome this limitation, several groups have demonstrated that intratumoral delivery of IL-12 using adenoviral vectors induces tumor regression and T cell activation in preclinical models of breast cancer (Gyorffy et al. J Immunol 166, 6212-6217 (2001); Bramson et al ., Hum Gene Ther 7, 1995-2002 (1996)). Later, Sabel et al. used polylactic acid microspheres to release IL-12 into the tumor and found that the antitumor response was mediated primarily by NK cells (Sabel et al. Breast Cancer Res Treat 122, 325-336 (2010)). Other studies have used mesenchymal stromal cells to locally deliver IL-12 to breast cancer in mice (Eliopoulos et al. Cancer Res 68, 4810-4818 (2008)). A phase I study 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 cytokine secretion by NK cells (Bekaii-Saabet al. Cancer Therapeutics 8, 2983 -2991 (2009)). Therefore, IL-12 may have great promise as an antitumor agent, and its use as a costimulator in an adoptive T-cell therapy approach is well justified.

In some embodiments, the immunomodulatory agent is an immune checkpoint blocker. Non-limiting examples of immune checkpoint blockers include anti-PD-L1 antibodies, anti-CTLA-4 antibodies, anti-PD-1 antibodies, anti-LAG3 antibodies, anti-B7-H3 antibodies, and anti-TIM3 antibodies. In some embodiments, the immune checkpoint blocker is an anti-PD-L1 antibody. In some embodiments, an immunoreactive cell according to the invention disclosed herein, or a composition comprising it, in combination with an anti-PD-L1 antibody, can be used to treat breast cancer (BC), such as TNBC.

Programmed cell death protein ligand 1 (PD-L1/B7-H4/CD274) is an inhibitory signal typically expressed in actively inflamed tissues that serves as a negative feedback loop to limit T cell activation. PD-L1 expression is generally absent in non-inflamed healthy tissues (including breast (Dong et al. Nature Medicine 8, 793-800 (2002))) but is instead most abundant in cancerous tissues, especially in cancerous tissues with an inflammatory infiltrate (Spranger et al. Science translational medicine 5, 200ra116 (2013)). This association with inflammation is likely due to upregulation of PD-L1 when tumor cells are exposed to T cell-secreted cytokines produced upon T cell activation. This expression profile is observed in breast cancer, with 50-75% of breast cancer specimens staining positive for PD-L1, and expression is significantly associated with a heavy lymphocytic infiltrate (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., BCM Cancer 8, 57 (2008)). Breast cancer may also intrinsically express PD-L1, secondary to oncogenic signaling. Activation of the PI(3)K pathway leads to upregulation of PD-L1 protein in breast cancer cells, and activation of PI(3)K in patient tumors is significantly correlated with PD-L1 expression (Crane et al. Oncogen 28, 306-312 ( 2009)). Expression of PD-1 by activated T cells spatially and temporally couples the ligand to receptor expression in the immunosuppressive tumor microenvironment (TME). Expression of PD-L1 in breast cancer tissues suggests that it is an immunotherapeutic target in these patients. The effectiveness of PD-L1/PD-1 blockade in various preclinical cancer models (including breast (Ge et al. Cancer letter 336, 253-259 (2013))) has paved the way for phase I studies using PD-L1- or PD- 1-specific antibodies in patients with advanced cancer. A phase I study (using an anti-PD-1 antibody) demonstrated efficacy only in PD-L1+ patients (Topalian et al., New England Journal of Medicine 366, 2443-2454 (2012)). Genetically engineered T cells provide unique advantages to overcome coinhibitory checkpoints and the typical lack of costimulation found in the TME. CAR-expressing T cells are essentially engineered to optimize their co-stimulatory requirements to support T cell expansion, survival, and function.

In some embodiments, the immunomodulatory agent is a radiotherapy agent. The localized, radiation-induced immunological milieu may not only provide the preconditions for enhancing the engraftment of targeted T cells in the tumor (thus eliminating the need for systemic lymphodepletion regimens), but also the immunological responses resulting from the combination of radiotherapy and adaptive T -cell therapy also increases abscopal antitumor efficacy. In radiation-resistant tumors, 4-1BB co-stimulatory signaling in CAR T cells can overcome immunoinhibition. In some embodiments, the immunomodulatory agent is a chemotherapeutic agent, including, but not limited to, cisplatin. Cisplatin-induced secretion of chemokines and cytokines may promote MSLN-directed and endogenous T cell responses.

Studies have shown that patients with lung adenocarcinoma (LAC) and malignant pleural mesothelioma (MPM), who have high levels of cytotoxic tumor-infiltrating lymphocytes (cTILs) and low levels of regulatory T cells (Tregs), have a better prognosis and longer survival-free survival. progression (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 CAR can be used to stimulate cTILs in LAC and MPM. Servais (2012) and Kachala (2013) reported that MSLN is overexpressed and contributes to the aggressiveness of LAC and MPM, justifying the selection of MSLN as a target of CAR T-cell therapy. Higher percentages of TILs after cisplatin therapy and radiotherapy were associated with improved treatment outcomes in both mouse models and patients.

Cisplatin and radiation therapy-induced tumor and abscopal immunomodulation may provide the preconditioning necessary for better engraftment of adoptively transferred T cells; T cell costimulation strategies to harness tumor and stromal immunomodulation may enhance the antitumor efficacy of both endogenous and adoptively transferred T cells.

In addition, various methods described above for using immunoreactive cells (eg, T cells) expressing a mesothelin-specific CAR, for example, to treat cancer in a subject or to reduce tumor burden in a subject, can be combined with antigen modulation of cancer cells. Immunoreactive cells (eg, T cells) expressing a mesothelin-specific CAR can target and destroy MSLN expressed on the membrane (termed “cell membrane MSLN”) of a tumor or cancer cell, but not cytoplasmic MSLN. Some tumors or cancers (eg, lung cancer and mesothelioma) have low cell membrane MSLN content but high cytoplasmic MSLN content. Modulation of a cancer cell antigen may increase cell membrane MSLN expression in a tumor or cancer cell, which may make the tumor or cancer cell more likely to be targeted by a CAR-expressing immunoreactive cell, and thus more susceptible to cytolysis caused by the immunoreactive cell. In some embodiments, the cancer cell antigen modulation is radiation.

Other modifications may be made to immunoreactive cells (eg, T cells) to prevent or minimize the risks of immunological complications (known as "malignant transformation of T cells"), such as graft-versus-host disease (GVHD), or cases where healthy tissue express the same target antigens as tumor cells, leading to GVHD-like findings. A possible solution to this problem is to introduce a suicide gene into CAR-expressing T cells. Suitable suicide genes include, but are not limited to, herpes simplex virus thymidine kinase (hsv-tk), inducible suicide gene Caspase 9 (iCasp-9), and human epidermal growth factor receptor truncated polypeptide (EGFRt). In some embodiments, the suicide gene is an EGFRt polypeptide. The EGFRt polypeptide can mediate T cell clearance upon administration of an anti-EGFR monoclonal antibody (eg, cetuximab). EGFRt can be covalently attached to the 3' end of the intracellular signaling domain of a mesothelin-targeted CAR. The suicide gene can be introduced into a vector containing nucleic acids encoding the mesothelin-specific CAR receptors disclosed herein. In this case, administration of a prodrug designed to activate a suicide gene (eg, a prodrug (eg, AP1903) that can activate iCasp-9) during malignant transformation of T cells (eg, GVHD) initiates apoptosis in suicide gene-activated CAR-expressing CARs. T cells.

In addition, the present invention provides a method for preventing and/or treating an inflammatory disease in a subject. In some embodiments, the method includes administering an immunoreactive cell disclosed herein or a composition comprising the same to a subject. In some embodiments, the immunoreactive cell is an immunoinhibitory cell. In some embodiments, the immunoinhibitory cell is a regulatory T cell. In some embodiments, the inflammatory disease is pancreatitis. In some embodiments, the subject is a human. In some embodiments, the subject is an organ transplant recipient, such as a pancreas transplant recipient.

In addition, the present invention provides a method for preventing graft rejection in a subject who is an organ transplant recipient. In some embodiments, the method includes administering an immunoreactive cell disclosed herein or a composition comprising the same to a subject. In some embodiments, the immunoreactive cell is an immunoinhibitory cell. In some embodiments, the immunoinhibitory cell is a regulatory T cell. In some embodiments, the subject is a human. In another embodiment, the subject is a pancreas transplant recipient.

A mesothelin-directed CAR disclosed herein can be transduced into an immunoinhibitory cell, such as a regulatory T cell. The transduced immunoinhibitory cell can be administered to a subject (eg, a human) having inflammatory pathologies or an inflammatory disease. In some embodiments, the inflammatory site or site of inflammatory disease has a high level of expression of mesothelin recognized by the MSLN-CAR disclosed herein. The inflammatory disease can be severe, such as acute pancreatitis. In addition, the transduced immunoinhibitory cell can be administered to a subject who is an organ transplant recipient.

In addition, a mesothelin-targeting CAR disclosed herein, as well as a second CAR directed against an MHC antigen, can be co-transduced into an immunoinhibitory cell (e.g., a regulatory T cell), whereby the immunoinhibitory cell can specifically assemble at the pancreas transplant site glands. In some embodiments, an MHC class I subject receives a pancreas transplant from an MHC class II donor; the recipient's regulatory T cells are transduced with the MSLN-specific CAR disclosed herein and a second CAR directed against an MHC class II antigen, and thus the transduced recipient regulatory T cells are collected/aggregated at the pancreas transplant site and avoid graft or organ rejection .

Eligible human subjects for therapy generally comprise two treatment groups, which can be differentiated by clinical criteria. Subjects with “advanced disease” or “high tumor burden” are subjects who carry a clinically measurable tumor. A clinically measurable tumor is one that can be detected by tumor mass (eg, by palpation, computed tomography, sonogram, mammogram, or x-ray; positive biochemical or histopathological markers alone are not sufficient to identify this population). The pharmaceutical composition is administered to such subjects to induce an antitumor effect in order to alleviate their condition. Ideally, this results in a reduction in tumor mass, but any clinical improvement is considered effective. Clinical improvement includes a reduction in the risk or rate of progression, or a reduction in the pathological consequences of the tumor.

The second group of eligible subjects is known in the art as the "adjuvant group." These are individuals who have a history of neoplasia but have demonstrated a response to another treatment regimen. Previous therapy may include, but is not limited to, surgical resection, radiation therapy, and conventional chemotherapy. As a result, such individuals have no clinically measurable tumor. However, it is suspected that they are at risk of disease progression either close to the original tumor site or due to metastases. This group can be divided into high- and low-risk individuals. This division is made on the basis of signs observed before or after the initial treatment. These features are known in the medical field and are defined accordingly for each specific neoplasia. Features typical of high-risk subgroups include patients whose tumor has spread to nearby tissue or who have lymph node involvement.

Another group has a genetic predisposition to neoplasia but has not yet demonstrated clinical signs of neoplasia. For example, women who test positive for a genetic mutation associated with breast cancer but are still of childbearing age may wish to receive one or more of the immunoreactive cells described herein for prophylactic treatment to prevent the occurrence of neoplasia until they will not be suitable for preventive surgery.

Due to the surface expression of the CAR receptor against mesothelin and PD-1 DN, which enhances the antitumor effect of the immunoreactive cell, adoptively transferred T or NK cells have increased and selective cytolytic activity at the tumor site. In addition, after their localization in a tumor or viral infection and their proliferation, T cells transform the tumor or viral infection into a highly permeable environment for a wide range of immune cells involved in the physiological antitumor or antiviral response (tumor-infiltrating lymphocytes, NK-, NKT -cells, dendritic cells and macrophages).

In addition, the present invention provides methods for treating and/or preventing a pathogen-induced infection (eg, 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 immunoreactive cells disclosed herein or a composition comprising them to a subject having an infection caused by a pathogen. Examples of viral infections susceptible to treatment include, but are not limited to, infections caused by cytomegalovirus (CMV), Epstein-Barr virus (EBV), human immunodeficiency virus (HIV), and influenza virus.

Other modifications may be made to immunoreactive cells (eg, T cells) to prevent or minimize the risks of immunological complications (known as "malignant transformation of T cells"), such as graft-versus-host disease (GVHD), or cases where healthy tissue express the same target antigens as tumor cells, leading to GVHD-like findings. A possible solution to this problem is to introduce a suicide gene into CAR-expressing T cells. Suitable suicide genes include, but are not limited to, herpes simplex virus thymidine kinase (hsv-tk), inducible suicide gene Caspase 9 (iCasp-9), and human epidermal growth factor receptor truncated polypeptide (EGFRt). In some embodiments, the suicide gene is an EGFRt polypeptide. The EGFRt polypeptide can mediate T cell clearance upon administration of an anti-EGFR monoclonal antibody (eg, cetuximab). EGFRt can be covalently attached upstream of the antigen recognition receptor of a CAR receptor disclosed herein. The suicide gene can be introduced into a vector comprising nucleic acids encoding the CAR receptor disclosed herein. In this case, administration of a prodrug designed to activate a suicide gene (eg, a prodrug (eg, AP1903) that can activate iCasp-9) during malignant transformation of T cells (eg, GVHD) initiates apoptosis in suicide gene-activated CAR-expressing CARs. T cells. Incorporation of a suicide gene into the CAR disclosed herein improves safety due to the ability to eliminate a large portion of CAR T cells in a very short time. An immunoreactive cell (eg, a T cell) disclosed herein that includes a suicide gene may be advantageously eliminated immediately following infusion of CAR T cells or eliminated upon early signs of toxicity.

5.9. Sets

The invention disclosed herein provides kits for inducing and/or enhancing an immune response and/or treating and/or preventing a tumor or pathogen-induced infection in a subject. In some embodiments, the kit includes an effective amount of immunoreactive cells disclosed herein or a pharmaceutical composition including them. In some embodiments, the kit includes a sterile container; such containers may be boxes, ampoules, bottles, vials, tubes, bags, sachets, blister packs, or other suitable container forms known in the art. Such containers may be made of plastic, glass, laminated paper, metal foil, or other materials suitable for storing medications. In some embodiments, the kit includes an isolated nucleic acid molecule encoding an anti-mesothelin CAR and an isolated nucleic acid molecule encoding PD-1 DN in an expressed form, which may optionally be contained in the same or different vectors.

Optionally, the immunoreactive cells and/or nucleic acid molecules are provided with instructions for administering the cells or nucleic acid molecules to a subject who has or is at risk of developing neoplasia or a pathogen-associated infection or immune disorder. The instructions generally contain information about the use of the composition for the treatment and/or prevention of neoplasia or infection caused by a pathogen. In some embodiments, the instructions include at least one of the following: a description of the therapeutic agent; a regimen for the treatment or prevention of neoplasia caused by a pathogen infection or immune disorder, or symptoms thereof; precautionary measures; warnings; indications; contraindications; information about overdose; unwanted reactions; pharmacology in animals; clinical studies and/or references. Instructions may be printed directly on the container (if available), or provided as a label on the container, or as a separate sheet, brochure, card, or folder provided in or with the container.

6. EXAMPLES

The practice of the present invention uses, unless otherwise indicated, standard techniques of molecular biology (including recombination techniques), microbiology, cell biology, biochemistry and immunology, which are within the competence of one skilled in the art. Such methods are fully described in the literature, for example, in "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 methods may be used to prepare the polynucleotides and polypeptides disclosed herein and, as such, may be considered in the making and practice of the invention disclosed herein. Particularly useful techniques for specific embodiments will be discussed in the sections that follow.

The following examples are used to provide those skilled in the art with a complete disclosure and description of how to make and use the cells and compositions disclosed herein, and are not intended to limit the scope of what the inventors believe to be their invention.

Example 1

The polypeptide composition disclosed herein was created. The polypeptide composition includes: (i) a CAR that binds to human mesothelin, and (ii) a dominant negative form of programmed death protein 1 (PD-1 DN), as shown in Figure 1. The mesothelin-targeted CAR includes: (a) a signaling a CD8 peptide (e.g., a CD8 signal peptide consisting of the amino acid sequence set forth in SEQ ID NO: 71), (b) an extracellular antigen-binding domain that is a scFv comprising a V _H including a CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 76, CDR2, consisting of the amino acid sequence presented in SEQ ID NO: 77, and CDR3, having the amino acid sequence presented in SEQ ID NO: 78; and V _L 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 , (c) a transmembrane domain that includes a CD28 polypeptide (e.g., a CD28 polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 92 (or amino acids 153-179 of SEQ ID NO: 90)), (d) a hinge/spacer the CD28 region (e.g., a CD28 polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 15 (or amino acids 114-152 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 co-stimulatory 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 180-220 of SEQ ID NO: 90) ). The PD-1 DN includes a PD-1 signal peptide consisting of amino acids 1-20 of SEQ ID NO: 48, a PD-1 extracellular domain consisting of amino acids 21-165 of SEQ ID NO: 48, and a CD8 polypeptide consisting of amino acids 137-207 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 CAR and PD-1 DN, as shown in Figure 1. The polypeptide composition is designated "M28z1XXPD1DNR".

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 provided in SEQ ID NO: 123. Another exemplary nucleotide sequence encoding the polypeptide composition is provided in SEQ ID NO: 124.

Example 2

The activities of M28z1XX-P2A-PD1DNR, having the structure of the polypeptide composition as described in Example 1, were studied. The structures of the alternative and control constructs were compared with M28z1XX-P2A-PD1DNR, as shown in Figure 2.

Viral vectors incorporating CAR constructs were produced in the RD114 producer cell line 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 an anti-Fab antibody. Blank RD114 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 with anti-Fab antibody and PD1DNR with anti-PD1 antibody staining by flow cytometry. The correlation between vector copy number (VCN) and mean fluorescence intensity (MFI) was examined. 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/μg DNA by qPCR. As shown in Figures 7A-7C, MFI of CAR-positive cells correlated with VCN/μg DNA for all three donors tested. The transduction ratios of human CD4 ⁺ and CD8 ⁺ T cells are shown in Table 1.

The cytolytic effects of M28z1xx-PD1DNR CAR T cells were then examined. MSLN-high (MGM) target cells were co-cultured with M28z1XX-PD1DNR CAR T cells from different donors at different E:T ratios using impedance-based assays. The results are shown in Figure 8. As shown in Figure 8, T cells transduced with M28z1XX-PD1DNR CAR demonstrated effective cytotoxicity to all three different donors tested. Effector cytotoxicity was present at several E:T ratios (data not shown).

Conclusions:

M28z1xx-PD1DNR vectors were successfully produced in RD114 cells. Stable producer cell lines were successfully obtained for all constructs. Viral vectors were titrated to yield transduction rates of ~40–60% in T cells from multiple donors. CD4 and CD8 T cells were successfully transduced to express CAR and PD1DNR. A correlation was observed between vector copy number and transduction.

Example 3

This example describes a comparative analysis of various designs, including the M28z1XX-PD1DNR. Cytotoxicity was measured using an impedance assay. The principle of electrical resistance cytotoxicity (eCTL) measurement is shown in Figure 9. Comparative analysis parameters are shown in Figure 10, including CAR constructs, donors, CAR targets, and E:T ratios. The expression of MSLN and PD-L1 in target cell lines was measured. Mesothelioma (MGM, MGM-PDL1, and MSTOG) and lung cancer (A549GM and A549G) cell lines were assessed for MSLN and PD-L1 expression by flow cytometry. The results are shown in Figures 11A-11E. MGM, MGM-PDL1 and A549GM overexpress MSLN. MGM-PDL1 cells further expressed PD-L1.

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

A comparative analysis of the antitumor efficacy of CAR T cells expressing M28z, M28z1xx, M28z-PD1DNR, or M28z1xx-PD1DNR against MSLN-high tumor cells (MGM) was performed. MSLN target cells (MGM) were cocultured with either M28z, M28z1xx, M28z-PD1DNR, M28z1XX-PD1DNR, or non-transduced T cells at different E:T ratios. Antitumor efficacy was assessed using an resistance-based assay. The results are shown in Figures 13A-13C. In addition, chromium-51-labeled MSLN-rich target cells (MGM) were co-cultured with M28z, M28z1xx, M28z-PD1DNR, M28z1xx-PD1DNR or non-transduced T cells at different E:T ratios for 18 hours. Cytotoxicity was determined by the release of chromium-51 by CTL. The results are shown in Figure 14.

A comparative analysis was then performed measuring the antitumor efficacy of CAR T cells expressing M28z, M28z1xx, M28z-PD1DNR, or M28z1xx-PD1DNR against MSLN-negative tumor cells (MSTOG). MSLN-negative target cells (MSTOG) were cocultured with M28z, M28z1xx, M28z-PD1DNR, M28z1xx-PD1DNR, or nontransduced T cells at the indicated E:T ratios. Antitumor efficacy was assessed using an resistance-based assay. The results are shown in Figures 15A-15C. In addition, MSLN-negative target cells (MSTOG) labeled with chromium-51 were co-cultured with M28z, M28z1XX, M28z-PD1DNR, M28z1XX-PD1DNR or non-transduced T cells at different E:T ratios for 18 hours. Cytotoxicity was determined by the release of chromium-51 by CTL. The results are shown in Figure 16.

In addition, a comparative analysis of the antitumor efficacy of CAR T cells expressing M28z, M28z1xx, M28z-PD1DNR, or M28z1xx-PD1DNRag was performed compared with high-MSLN tumor cells overexpressing PDL1. Target cells with high levels of MSLN and overexpression of PDL1 (MGM-PDL1) were co-cultured with M28z, M28z1XX, M28z-PD1DNR, M28z1XX-PD1DNR or non-transduced T cells at different E:T ratios. Antitumor efficacy was assessed using an resistance-based assay. The results are shown in Figures 19A-19C. Similarly, the comparative analysis of the antitumor efficacy of CAR T cells expressing M28z, M28z1xx, M28z-PD1DNR or M28z1xx-PD1DNRag against MSLN-high tumor cells (A549GM) was carried out, the results are shown in Figures 18A-18C; and also comparatively analyzed the antitumor efficacy of CAR T cells expressing M28z, M28z1xx, M28z-PD1DNR or M28z1xx-PD1DNR against tumor cells with low MSLN (A549G), the results are shown in Figures 19A-19C.

Conclusions:

M28z1xx-PD1DNR constructs caused lysis of MSLN ⁺ target cells in an E:T ratio-dependent manner, with results reproducible with different T cell donors in different cancer forms (lung cancer and mesothelioma cell lines). The specific cytotoxic effect correlated with MSLN expression levels, and was effective against MSLN ⁺ target cells with high PD-L1 expression.

Example 4 - Regional Delivery of Mesothelin-Targeted CAR T Cells for Clinical Use with Self-Checkpoint PD-1 Cell Blockade: Advancement to Phase I Study

Summary: This case study provides evidence of the preclinical safety and improved antitumor efficacy of M28z1XXPD1DNR CAR T cells for clinical use.

Methods: Comparative cytotoxicity, proliferation, and cytokine secretion of human T cells modified to express M28z or M28z1XXPD1DNR CARs were assessed using chromium release, accumulation, and Luminex assays, respectively. The antitumor efficacy of a single dose (1x10 ⁵ CAR T cells; E:T 1:1000) of intrapleurally administered M28z or M28z1XXPD1DNR CAR T cells was studied in NSG mice with orthotopic pleural mesothelioma using serial bioluminescence imaging and survival comparison. After tumor elimination, functional persistence of CAR T cells was tested upon re-infection with the tumor (increasing doses from 2x10 ⁶ to 11x10 ⁶ tumor cells).

Results: In vitro, both M28z and M28z1XXPD1DNR CAR T cells exhibited antigen-specific cytotoxicity, accumulation, and secretion of effector cytokines (see Table 2). In vivo, a single dose of M28z1XXPD1DNR CAR T cells resulted in tumor clearance, increased survival, and resistance to tumor re-engraftment after 10 tumor re-challenges (see Table 2) compared with a single dose of M28z CAR T cells.

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

Table 2 - Comparison of M28z and M28z1XXPD1DNR CAR T-cell designs M28z M28z1XXPD1DNR Target Mesothelin Mesothelin Costimulatory domain CD28 CD28 CD3z No mutations 2 mutant ITAMs (1XX) Proprietary T cell checkpoint blockade (PD1DNR) No Yes In vitro results Human T cell transduction range 25%-82% 30%-87% Expression of PD-1 extracellular domain mRNA compared to non-transduced domain, fold 4 157 Cytotoxicity range E:T 10:1 35%-45% 25%-51% E:T 5:1 28%-44% 20%-38% E:T 2:1 17%-32% 14%-24% Accumulation, range, multiplicity 110-390 53-622 Effector cytokines (E:T 1:1, 24 h), range IL-2 18-23 ng/ml 9-19 ng/ml TNF-α 545-977 pg/ml 380-852 pg/ml IFN-γ 8-11 ng/ml 6-15 ng/ml In vivo results Removal of the tumor 26 days 19 days Median survival 56 days Not achieved Tumor progression as measured by bioluminescence imaging after reinfection Re-infection 3 times in 15 days +1log +0.2 log Re-infection 10 times in 52 days +1-2log +0.2 log Current state In progress to Phase II clinical trial • Obtained vector and viral supernatant for clinical use

Example 5 - Next generation CAR T cell with blockade of the cell's own PD-1: Clinical rationale, development of a preclinical and clinical trial protocol

Malignant pleural mesothelioma (MPM) is a cancer with low mutational load and low PDL1 expression with disappointing responses to anti-PD1 antibodies. An ongoing phase I/II study (NCT02414269, n=41) established the safety and antitumor efficacy of intrapleural mesothelin-targeted chimeric antigen receptor (M28z CAR) T cells followed by PD-1 antibody. CAR T cells using an antibody blockade strategy of the cell's own PD1 are safe and can provide antitumor efficacy against both low and high PDL1 tumors without the need for repeated administration of anti-PD1 antibody.

Summary: This case study provides evidence of the preclinical safety and enhanced antitumor efficacy of M28z1XXPD1DNR CAR T cells for clinical use.

Methods: M28z and M28z1XXPD1DNR (modified CD3z domain dominant negative PD-1 receptor) CARs for clinical use were transduced into multiple donor T cells as effectors, and low and high PDL1 MPM cells were used as targets. At different E:T ratios, comparative in vitro and in vivo antitumor efficacy was assessed in mice with orthotopic MPM. Systemic antitumor immunity was tested by repeated tumor challenge at a distant site.

Results: In vitro, no significant differences were noted between M28z and M28z1XXPD1DNR CAR (antigen-specific cytotoxicity, accumulation and secretion of effector cytokines). In vivo, a single dose (1x10 ⁵ CAR T cells) of intrapleurally administered M28z CAR T cells, either with a boost of anti-PD1 antibody or the cell's own PD1DNR, resulted in comparable tumor clearance, increased survival, and weight gain. See Figure 20A and Table 3. In mice with orthotopic MPM, a single low dose (1 x 10 ⁵ CAR T cells) of M28z1XXPD1DNR CAR T cells eliminated pleural tumors, demonstrating increased systemic immunity compared to M28z CAR T cells due resistance to repeated tumor infections at a distant site in the abdominal cavity without any toxic effects (PD1DNR binds to mouse PDL1/2). See Figures 20B and 20C. The collected tumors showed increased and deeper infiltration of CAR T cells compared to non-transduced T cells. See Figure 20D.

Conclusions: Single intrapleural administration of low doses of M28z1XXPD1DNR CAR T cells demonstrates feasibility, safety, tumor clearance, functional persistence, and systemic antitumor immunity.

Table 3 - Comparison of characteristics of PD-1 DNR CAR T-cell therapy and checkpoint blocker CAR T-cell therapy Characteristic PD-1 DNR CAR T cells Checkpoint blockers with CAR T cells Mechanism
Potential doses
Targeted therapy Internal cellular
One-time
Yes External cellular
Multiple
No Toxic effect Limited by tumor Systemic Limits of tumor penetration Unlikely Limits of Antibody Penetration Patient selection
Sensitivity to antigens Not required
Plural Tumors with high PD-1L
Plural

Example 6

1. Brief description

Malignant pleural mesothelioma (MPM) is a rare and fatal malignancy associated with asbestos exposure. MPM is a regionally aggressive primary pleural malignancy characterized by invasion of vital organs or the chest wall (Carbone et al., CA Cancer J Clin. 2019; 69(5): 402-429). Most patients (60-70%) have locoregionally advanced disease and are unresectable at presentation (Nelson et al., J Clin Oncol. 2017; 35(29): 3354-3362; Flores et al., J Thorac Oncol. 2007; 2 (10): 957-965). Even with successful completion of a combination of chemotherapy, aggressive surgical resection and radiation therapy, the average survival of patients after treatment is only 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 first-line systemic treatment for patients with MPM is cisplatin with pemetrexed, based on a median overall survival of 12.1 months compared with 9.3 months for cisplatin alone (Vogelzang et al., J Clin Oncol. 2003;21(14) ): 2636-2644). Because MPM patients have low tumor mutational burden and low programmed death ligand 1 (PD-L1) expression, their response to immune checkpoint inhibitors is limited and there remains a huge unmet need (Yarchoan et al., JCI Insight. 2019; 4 (6); Forde et al., Curr Treat Options Oncol.2019; The local nature, potential accessibility, and relative absence of metastases make MPM a suitable candidate for regionally targeted therapy (Nelson et al., J Clin Oncol. 2017; 35(29): 3354-3362).

This case study describes preclinical studies conducted to support the clinical use of intrapleural dose of chimeric antigen receptor (CAR) T cells M28z1XXPD1DNR, a new investigational drug for the treatment of patients diagnosed (histologically or cytologically documented) with MPM who have received at least one chemotherapy regimen , and in whom the presence of a tumor is documented.

This is a single-center, phase I study with a maximum number of participants of 36, designed to evaluate the safety, dose requirement, and efficacy of targeting genetically targeted autologous M28z1XXPD1DNR CAR T cells after pretreatment with cyclophosphamide. In this study, 5 dose levels were planned: 1 x 10 ⁶ , 3 x 10 ⁶ , 6 x 10 ⁶ , 1 x 10 ⁷ and 3 x 10 ⁷ M28z1XXPD1DNR CAR T cells/kg, assuming no dose-limiting toxicities. M28z1XXPD1DNR CAR T cells are administered by infusion through an indwelling pleural catheter. Before treatment, patients are screened for mesothelin expression by immunohistochemical analysis of tumor biopsy samples and/or blood levels of soluble mesothelin-related peptides.

M28z1XXPD1DNR CAR T cells are autologous T cells transduced ex vivo with gammaretroviral vector stock supernatant obtained from the master vector-producing cell bank, 293VEC GALV SFG M28z1XXPD1DNR. The main CAR components encoded in the vector are as follows:

1) human scFv against mesothelin for targeted interaction with tumors expressing mesothelin,

2) Human CD28 co-stimulatory domain for signaling T cell survival and proliferation,

3) Point mutant human CD3ζ with one functional immunoreceptor tyrosine activating motif (ITAM) for calibrated T cell activation, and

4) Dominant negative programmed cell death protein receptor 1 (PD1) (PD1DNR) to protect T cells from entering a state of dysfunction or exhaustion when exposed to antigen (see Figure 21).

Mesothelin is a cancer cell surface antigen that is overexpressed in most forms of MPM, lung cancer, triple negative breast cancer, pancreatic cancer and ovarian cancer, as well as some forms of esophageal cancer (Pastan et al., Cancer Res. 2014; 74 (11): 2907-2912; Kachala et al., Clin Cancer Res. 20(4): 1020-1028; Tang et al., Anticancer Agents Med Chem. 13(2): 276-280; et al., Clin Cancer Res. 2012; Mol Cancer Ther. 11(3): 517-525; Breast Cancer Res Treat 2012; ). We have previously demonstrated that mesothelin overexpression promotes the aggressiveness of lung adenocarcinomas (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. 11(3): 517-525), and triple-negative breast cancer (n=250) (Tozbikian et al., PLoS One.2014 ; 9(12): e114900). In addition to the fact that mesothelin expression in tumors is relatively high compared to normal tissues, it is also expressed at very low levels on normal mesothelial surfaces of the 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 against solid tumors. The biological function of mesothelin is not well understood and is being studied.

Previously, mesothelin-targeted CAR T cells were administered intravenously to humans (3 x 10 ⁸ cells/m ² or 4.8 x 10 ⁷ cells/dose) in a clinical study conducted at the University of Pennsylvania (NCT01355965), where the CAR included a murine scFv. The anaphylactic reaction observed in 1 patient was reported to be caused by anti-mouse antibody responses that were raised against the humanized portion of the mouse scFv in the CAR construct used in this study (Beatty et al., Cancer Immunol Res. 2014). At the same time, in a recent phase I study (NCT02414269) conducted in the authors' laboratory, mesothelin-targeted CAR T cells (up to 6x10 ⁷ CAR T cells/kg), which consisted of fully human scFv, were intrapleurally administered. derived from a human Fab library (Feng et al., Mol Cancer Ther. 2009). To date, 40 patients have been treated, with no dose-limiting toxic effects observed. Preliminary efficacy in this study was observed in a cohort of patients (n=18) who received CAR T-cell therapy followed by a minimum of 3 doses of pembrolizumab (an anti-PD1 antibody) with an additional three-month follow-up period. Importantly, 83% of patients in this cohort did not require new or additional treatment at 6 months, with half of the patients receiving no additional treatment at 18 months. In addition, in most patients, CAR T cells were detected in the peripheral blood >100 days after intrapleural administration, indicating persistence of these cells in the patient.

In support of the first human clinical study, the pharmacology program for M28z1XXPD1DNR CAR T cells consists of a series of orthogonal in vitro specificity, cytotoxicity, cytokine accumulation and secretion studies, as well as in vivo tumor efficacy and survival studies in mice, results that suggest an effective dose for translation into clinical use. Table 4 provides general information about the nonclinical pharmacological and toxicological studies performed and their main results.

Table 4. General information on pharmacology and toxicology studies and their main results Experiment Target Main results Expression of mesothelin during human tumors Determination of cell surface distribution of mesothelin in human tumors - Mesothelin is expressed in solid tumors
- Lack of mesothelin expression in normal lung and breast tissue Transduction Determination of transduction efficiency with viral supernatant - The concentration of the viral supernatant is directly proportional to the transduction efficiency of CAR and PD1DNR Vector copy number (VCN) Determination of the number of vector insertions in the T-cell genome - Positive correlation between CAR and VCN expressions
- CAR transduction optimized to 35%-70% to maintain VCN≤5 Quantification of PD1DNR Accurate quantification of endogenous PD-1 and PD1DNR levels - PD-1 extracellular domain is upregulated at the protein level (>2-fold) and mRNA level (>100-fold) for mycM28z1XXPD1DNR CAR T cells Myc-tag interaction Study of the influence of the myc tag on CAR function - M28z1XXPD1DNR CAR T cells with and without myc tag demonstrate identical antitumor efficacy in vitro Cytotoxicity Determination of antigen-specific cytotoxicity - mycM28z1XXPD1DNR CAR T cells mediate antigen-specific HLA-independent tumor lysis Accumulation CAR T cell accumulation study - mycM28z1XXPD1DNR CAR T cells were found to proliferate and accumulate up to 622 times more after six antigen stimulations Repeated antigenic stimulation Cytotoxicity study with continuous contact with antigen - Initial antigen stimulation: Similar cytotoxicity of mycM28z1XXPD1DNR and mycM28z CAR T cells
- Repeated antigen stimulation: Cytotoxicity was maintained for mycM28z1XXPD1DNR but decreased for mycM28z CAR T cells Cytokine secretion Quantitative determination of cytokine secretion upon antigen stimulation - mycM28z1XXPD1DNR CAR T cells secrete effector cytokines (IL-2, IFN-γ, TNF-α) when stimulated by antigen
- Secretion of effector cytokines decreased with repeated antigen stimulation Antitumor efficacy in vivo In vivo antitumor efficacy and survival study - Efficacy and tumor clearance observed with a single intrapleural dose of 3x10 ⁴ mycM28z1XXPD1DNR CAR T cells/mouse
- Mice treated with mycM28z1XXPD1DNR (dose of 5 x 10 ⁴ and 1 x 10 ⁵ ) CAR T cells remained tumor-free until the end of the study (day 68; median survival not reached compared to 50 days for mice treated with 1 x 10 ⁵ mycM28z CAR T cells)
- Weight gain and lack of toxicity in mice treated with mycM28z1XXPD1DNR CAR T cells Immunofluorescence staining of tumor ex vivo Detection of CAR T cells in primary tumors in mice after intrapleural treatment - Applied mycM28z1XXPD1DNR CAR T lymphocytes in situ found tumor penetrating and enriched in peritumoral areas Repeated antigen stimulation in vivo Study of functional persistence and long-term antitumor efficacy - A single intrapleural dose of 1x10 ⁵ mycM28z1XXPD1DNR CAR T cells demonstrated excellent functional persistence and improved long-term antitumor activity by resisting intraperitoneal tumor engraftment upon re-challenge
- mycM28z CAR T cells became dysfunctional upon repeated antigen stimulation Antitumor efficacy of CAR T cells for clinical use in vivo Confirmation of the antitumor efficacy of cryopreserved T cells transduced with stock vector solution for a clinical study - Cryopreserved M28z1XXPD1DNR CAR T cells for clinical study demonstrated high viability after thawing, exhibited antitumor efficacy and prolonged survival in vivo
- No toxic effects were observed Toxic effect In vivo toxicity study - No mortality or pathology
- Absence of significant clinical signs
- No significant difference in body weight compared to controls without tumor
- Histopathology: No microscopic findings associated with acute or delayed toxicity
- Hematology: Female mice sacrificed on study day 15 had a high mean monocyte %; lack of correlation with the results of microscopic studies.
- Clinical Biochemistry: Male mice sacrificed on study day 14 had a low mean total protein value; lack of correlation with the results of microscopic studies.
- Single orthotopic administration of 1x10 ⁵ mycM28z1XXPD1DNR CAR T cells in a xenograft model of mesothelioma was well tolerated.

To facilitate the discovery of CAR T cells in preclinical studies, CAR T cells containing a myc tag at the N terminus of a mesothelin-specific scFv (mycM28z1XXPD1DNR) were generated. To assess the concordance between mycM28z1XXPD1DNR and M28z1XXPD1DNR CAR T cells, the transduction efficiency of viral supernatants was compared and consistent, concentration-dependent expression of vector components was observed between the two constructs. In addition, CAR and PD1DNR were expressed proportionally in each transduced cell due to the presence of the self-cleaving P2A peptide, which efficiently mediates the expression of the bicistronic transgene. When comparing the percentage of transduced cells expressing CAR with the number of vector copy numbers inserted into the T cell genome, a positive linear relationship was observed between the dilution of the viral supernatant used for transduction and the resulting vector copy number (VCN). Based on these observations, it was determined that a range of 35-70% CAR-expressing T cells provides optimal transduction efficiency that: (1) maintains a low VCN:cell ratio and (2) reduces the risk of insertional mutagenesis, which typically increases with high VCN:cell ratio.

To confirm the expression of PD1DNR and its differentiation from its endogenous counterpart, flow cytometry and qPCR analyzes were performed to measure and compare cell surface protein expression and intracellular PD1 mRNA expression, respectively, in T cells transduced with mycM28z1XXPD1DNR and mycM28z. At the cell surface protein level, compared with mycM28z CAR T cells, mycM28z1XXPD1DNR CAR T cells exhibited a 2-fold increase in the percentage of cells staining positive for PD1, and a 3-fold increase in the mean fluorescence intensity (MFI) exhibited by PD1-positive cells . At the mRNA level, compared to non-transduced T cells, mycM28z CAR T cells showed a 4-fold increase in both extracellular and intracellular PD1 domains, whereas mycM28z1XXPD1DNR CAR T cells showed a 157-fold increase in the extracellular PD1 domain and only a 2-fold increase. fold increase in the intracellular domain of PD1. This expression of the extracellular domain of PD1, increased by orders of magnitude, indicates high expression of PD1DNR, which serves to combat checkpoint inhibition.

Finally, to rule out any possible interaction of the myc tag (used in preclinical studies) with impaired CAR function, the antitumor efficacy of mycM28z1XXPD1DNR and M28z1XXPD1DNR CAR T cells was compared using an electrical impedance-based cytotoxicity assay, which revealed no differences in the kinetics and overall cytolysis of mesothelin -positive tumor cells from 3 different donors, confirming that the myc tag does not affect CAR function.

The specificity, cytotoxicity, accumulation, and cytokine secretion of mycM28z1XXPD1DNR CAR T cells were then examined. In a ⁵¹ Cr release cytotoxicity assay, mycM28z1XXPD1DNR CAR T cells demonstrated antigen-specific and human leukocyte antigen (HLA)-independent cytotoxicity against mesothelin-positive tumor cells with both constitutive expression and overexpression of PD-L1. No nonspecific cytotoxicity was observed against mesothelin-negative tumor cells. mycM28z1XXPD1DNR CAR T cells did not exhibit any cytotoxicity against PD-L1 overexpressing targets in the absence of mesothelin antigen expression. In an antigen challenge assay (antigen stress test), both mycM28z1XXPD1DNR and mycM28z CAR T cells expanded up to 622 times over a period of 6 antigen challenges. At the time points analyzed, the constructs exhibited similar cytotoxicity after the first antigen challenge and maintained similar cytotoxicity until the fourth antigen challenge. When the effector-to-target (E:T) ratio was further decreased to increase antigen stress, mycM28z1XXPD1DNR CAR T cells maintained cytotoxicity better than mycM28z CAR T cells upon the seventh antigen stimulation. However, over the course of the assay, secretion of effector cytokines (IL-2, IFN-γ, and TNF-α) was progressively decreased in both mycM28z1XXPD1DNR and mycM28z CAR T cells, indicating the safety profile of both constructs.

MPM tumor cells cotransduced with firefly luciferase (ffLuc) were injected intrapleurally to engraft tumors representing the MPM orthotopic cancer model. To monitor tumor growth non-invasively, tumor-bearing mice were intraperitoneally injected with luciferin at a dose of 150 mg/kg and imaged on an IVIS Spectrum imaging system (PerkinElmer, Waltham, MA) after 15 minutes using a protocol optimized for quantitative monitoring of pleural tumor regression or progression, which was previously published by the authors' laboratory (Servais et al., Curr Protoc Pharmacol. 2011; Chapter 14: Unit 14 21).

In an initial experiment conducted to investigate the antitumor activity of CAR T cells in vivo, NSG mice with orthotopic tumors were administered a single intrapleural dose of ^3x104 mycM28z1XXPD1DNR CAR T cells and compared with mice receiving a single intrapleural dose of control CAR T cells specific to prostate-specific membrane antigen (P28z). After fifteen days, mice treated with mycM28z1XXPD1DNR CAR T cells showed a significant reduction in tumor burden (p=0.0002), while mice treated with P28z CAR T cells developed agony due to high tumor burden.

In the second in vivo study, mice with orthotopic tumors were divided into 3 groups, each of which received a single intrapleural dose of ^1x105 or ^5x104 mycM28z1XXPD1DNR CAR T cells or ^1x105 mycM28z CAR T cells. Serial tumor imaging showed a reduction in tumor burden as early as 5 days after CAR T cell administration, with complete tumor clearance, determined by a decrease in bioluminescence imaging (BLI) signal to baseline, around day 19 in mice treated with ^1x105 mycM28z1XXPD1DNR CAR T -cells, and at approximately day 26 in mice treated with 1x10 ⁵ mycM28z CAR T cells. Mice treated with either dose of mycM28z1XXPD1DNR CAR T cells remained tumor-free until the end of the study (68 days). Median survival was 50 days in mice treated with 1x10 ⁵ mycM28z CAR T cells; median survival was not achieved in mice treated with any dose of mycM28z1XXPD1DNR CAR T cells (>68 days; p=0.0085-0.0427). Ex vivo immunofluorescence imaging of pleural tumors showed that mycM28z1XXPD1DNR CAR T cells surrounded the tumor periphery at high density and infiltrated the tumor parenchyma 3 days after regional delivery. These results were reproduced using T cells from multiple donors with varying percentages of CD4 and CD8 T cells transduced with the mycM28z1XXPD1DNR CAR.

To investigate the functional persistence of mycM28z1XXPD1DNR CAR T cells, mice with orthotopic MPM that received a single intrapleural dose of 1×10 ⁵ mycM28z1XXPD1DNR or mycM28z CAR T cells were reinfected with increasing doses (2×10 ⁶ to 11×10 ⁶ cells/dose ) mesothelin-positive tumor cells injected intraperitoneally (it is more appropriate to re-introduce cells into the abdominal cavity than into the pleural cavity) every 4-8 days, up to 10 times. The BLI signal in mice treated with mycM28z1XXPD1DNR CAR T cells peaked in intensity shortly after each tumor rechallenge and returned to baseline levels at all time points after rechallenge. In contrast, mice treated with mycM28z CAR T cells showed a similar trend up to 5 tumor reinfections, but failed to control tumor recovery after administration of higher tumor doses at later time points in tumor reinfection (6 to 10). which led to tumor recurrence and agonal state. mycM28z1XXPD1DNR CAR T cells prevented intraperitoneal tumor formation over 10 repeat infections, even >126 days after a single intrapleural dose of 1x10 ⁵ CAR T cells, without any obvious signs of toxicity. Under conditions of high antigenic stress, mycM28z1XXPD1DNR CAR T cells exhibited superior functional persistence and increased antitumor efficacy compared to mycM28z CAR T cells in vivo. To confirm that this increased efficacy was not due to graft-versus-host disease, which is typically observed in NSG mice treated with CAR T cells, non-antigen-expressing targets were administered at this time point, resulting in increased BLI signal of tumors without an antitumor response. confirming that the observed antitumor efficacy was antigen-specific.

To confirm the antitumor efficacy of cryopreserved T cells transduced with M28z1XXPD1DNR CAR-encoding viral supernatant obtained for use in clinical trials, M28z1XXPD1DNR CAR T cells generated in the Laboratory of Cell Therapy and Cell Engineering (CTCEF) of the Memorial Cancer Center. Sloan-Kettering (MSK), thawed (viability: 88% after thawing) and administered intrapleurally to mice with orthotopic MPM at a dose of 6x10 ⁴ and 2x10 ⁵ CAR T cells/mouse. Tumor regression and resolution was observed after both doses, with 100% of mice surviving to the end of the observation period (day 70), whereas tumors in untreated mice progressed to death by day 19. Cryopreserved CAR T cells showed high viability after thawing and were effective without any signs of toxicity.

During the above-described efficacy experiments, no toxic effects were observed in mice, and the weight of the animals remained stable throughout the study.

Section 3 of this Example (entitled “Preclinical Toxicology”) describes a study conducted in mice to specifically evaluate the potential toxicity of mycM28z1XXPD1DNR CAR T cells in a mouse model of orthotopic MPM. Mortality, pathological severity, weight, clinical signs, complete blood count and clinical chemistry, gross autopsy and histopathological examination were assessed in 96 (48 male and 48 female) NSG mice bearing 8-day-old orthotopic mesothelioma that were randomly assigned to the group control and treatment. A dose of 1x10 ⁵ CAR T cells/mouse or control medium (5x10 ⁶ CAR T cells/kg) was administered as a single dose via orthotopic injection. On days 2 and 14 after administration of CAR T cells or medium (intermediate and final sacrifice, respectively), mice were anesthetized for necropsy and evaluation of CBC and clinical blood chemistry parameters. Day 14 was chosen as the time point for sacrifice because the tumor had either significantly regressed or had been eliminated by this time point (as confirmed by BLI or autopsy data from previous experiments). Performing sacrifice and necropsy at this time point allows for investigation of any targeted, non-tumor effects (scFv used in the present CAR reacts with mouse mesothelin) (Feng et al., Mol Cancer Ther. 2009) on normal tissues, particularly pleura, abdominal cavity and pericardium, with low levels of mesothelin expression following peak CAR T cell expansion in the absence of tumor burden with high antigen expression.

No mortality or severe morbidity was observed in the animals in this study, with the exception of 2 animals in the vehicle control group, which were separately sacrificed due to severe condition and difficulty breathing 20-22 days after tumor injection. Previous work in our laboratory has shown that vehicle-treated control animals can succumb to tumor burden approximately 20 to 22 days after tumor injection (Servais et al., Clin Cancer Res. 2012; Servais et al., Curr. Protoc Pharmacol. 2011; Unit14 21; Sci Transl Med. 2014; 261ra151; J Clin Invest. 126(8): 3130-3144; et al., PLoS One. 2011; 6(10): e26722). Therefore, the killings of these animals were unplanned, but not unexpected. Mortality or pathology was not associated with CAR T cells. Animals that received the control medium showed a progressive decrease in body weight over the study period and a significant difference in weight compared to control mice without tumors and mice that received mycM28z1XXPD1DNR CAR T cells. This was associated with an increase in tumor burden in control animals receiving medium. No significant clinical signs were observed in mice treated with mycM28z1XXPD1DNR CAR T cells. Mild skin peeling was observed in one mouse receiving the study drug, which was attributed to irritation caused by the surgical clamps, as no other animals were affected and the animals' activity was normal. The mice appeared normal throughout the observation period.

Female mice treated with mycM28z1XXPD1DNR CAR T cells that were sacrificed 14 days after CAR T cell administration had a high mean percentage of monocytes (mean, 18.44%, n=5) (p≤0.0001) compared with control tumor-bearing mice treated with medium (mean, 3.34%, n=5). The reference range established for the percentage of monocytes is 0.9-18%. However, this did not coincide with the microscopic examination data. No other significant or abnormal results were observed for the hematological parameters assessed. Any differences between the study drug groups and the matched vehicle groups were within normal reference ranges or were not biologically significant or statistically significant.

Male mice treated with mycM28z1XXPD1DNR CAR T cells that were sacrificed 14 days after CAR T cell administration had a low mean total protein (mean 3.83 g/dL, n=5) (p=0.0022) compared to control tumor-bearing mice that received medium (mean 4.68 g/dL, n=4). The reference range established for total protein is 4.1-6.4 g/dL. However, this did not coincide with the microscopic examination data. No other adverse effects regarding clinical biochemistry parameters were observed with the administration of the study drug. Any differences between the study drug groups and the corresponding vehicle groups were within normal reference ranges or were not biologically significant or statistically significant.

A review of histopathological findings revealed that there were no microscopic findings associated with acute or delayed toxicity from mycM28z1XXPD1DNR CAR T cell administration at the interim and final sacrifice days. Microscopic findings from animals in the intermediate-kill CAR-T cell groups included the presence of mixed cellular infiltration in tumor xenografts. This was considered to be related to the administration of the study drug but not to any toxic effects of the study drug. Any other findings observed were determined to have occurred sporadically, at the same frequency as in control animals, or to have been common among the species/strains used.

mycM28z1XXPD1DNR CAR T cells were detected in the tumor and spleen 8 days after intrapleural administration, with BLI showing that after 2 weeks, tumor burden was significantly reduced in mice treated with CAR T cells, confirming not only successful administration but also pharmacological activity of the study drug. Mouse plasma cytokine levels obtained at the same time point showed slightly higher levels of IL-4 in mice treated with CAR T cells than in mice treated with control media. Levels of IL-10, IL-6, KC/GRO, and TNF-α were generally low and did not differ significantly between mice receiving CAR T cells and mice receiving control media. IFN-γ, IL-12p70, IL-1β, IL-2 and IL-5 were not detected (below the limit of quantitation).

In conclusion, the data show that M28z1XXPD1DNR CAR T cells are well tolerated. The dose of 1x10 ⁵ cells/mouse is 5 times the initial patient dose (1x10 ⁶ cells/kg based on body weight), corresponding to 5x10 ⁶ cells/kg. M28z1XXPD1DNR CAR T cells contain the same antigen targeting moiety as M28z CAR T cells for which safety data in patients is already available (n=50). In the present clinical trial (IND16354), the authors did not observe any dose-limiting toxicity and extratumoral toxicity when up to 6 x 10 ⁷ M28z CAR T cells/kg was administered intrapleurally in combination with a PD1 checkpoint blocking antibody administered 3 weeks after administration of CAR T cells. Four patients received a second dose of intrapleural M28z CAR T cells after multiple doses of anti-PD1 antibody (4-week washout period) without observed toxicity. Taken together, all available preclinical and clinical data reasonably indicate that the proposed starting dose and route of administration of M28z1XXPD1DNR CAR T cells does not pose an unacceptable risk to our patients.

2. Preclinical pharmacology

A. Methods

CAR vectors. Vectors used in preclinical studies are presented in Table 5.

Table 5. Brief description of vectors used in preclinical studies Vector Protein expression mycM28z Mesotlin-specific scFv (myc-tag), CD28, CD3ζ mycM28z1XXPD1DNR Mesotlin-specific scFv (myc-tag), CD28, CD3ζ (1XX), P2A, PD1DNR M28z1XXPD1DNR (for research) Mesotlin-specific scFv, CD28, CD3ζ (1XX), P2A, PD1DNR M28z1XXPD1DNR (for clinical use*) Mesotlin-specific scFv, CD28, CD3ζ (1XX), P2A, PD1DNR GFP-ffLuc (transduced into tumor cells) GFP, firefly luciferase P28z (negative control) Prostate specific membrane antigen (PSMA) scFv, CD28, CD3ζ *For clinical use: CAR T cells produced in the MSK Cell Therapy and Cell Engineering Laboratory using viral supernatant prepared for clinical use.

Mesothelin-targeting CAR constructs contain a mesothelin-specific scFv (clone m912) (Feng et al., Mol Cancer Ther. 2009) fused to the CD28 co-stimulatory domain and the CD3ζ signaling domain (M28z). The CD3ζ chain was mutated in two of its three ITAM motifs, leaving one functional ITAM (designated 1XX) (Feucht et al., Nat Med. 2019; 25(1): 82-88). CAR was fused to PD1DNR via the P2A site from porcine teschovirus 1. PD1DNR consists of the PD1 signal 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 does not contain the PD1 signaling domain, which provides T cell intrinsic checkpoint blockade. To facilitate CAR detection, a myc tag (with the amino acid sequence EQKLISEEDL ×2) was fused to the N terminus of the scFv in the mycM28z and mycM28z1XXPD1DNR constructs. To eliminate any potential risk of immunogenicity in humans, the M28z1XXPD1DNR design for clinical use does not contain a myc tag. In addition, protein expression is codon optimized to avoid any immunogenicity in the CAR and PD1DNR construct. The detailed structure of the constructs used in preclinical studies is presented in Figure 22.

Expression of CAR constructs is under the control of the Moloney murine leukemia virus long terminal repeat (LTR) from the retroviral SFG vector (Riviere et al., Proc Natl Acad Sci US A. 1995; 92(15): 6733-6737). Expression of CAR and PD1DNR is directed by retroviral LTR.

All CAR vectors were transfected into 293T H29 packaging cell lines, and the viral supernatant produced by these cells was used for transduction and generation of stable 293T RD114 cell lines.

CAR T cells. Primary human T lymphocytes were isolated from the blood of healthy volunteer donors in accordance with a protocol approved by the center's institutional review board. 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 supernatant containing the mycM28z, mycM28z1XXPD1DNR, or M28z1XXPD1DNR vectors by spinoculation at 1800 g for 60 minutes at 24°C in 6-well culture plates coated with 15 μg/ml RetroNectin (Takara, Shiga, Japan). After spinoculation, transduced PBMCs were maintained in RPMI-1640 supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 20 U/ml IL-2. Transduction efficiency was determined by flow cytometric analysis of myc-tag expression on scFv-tagged CARs or by staining with an anti-human IgG antibody specific for the F(ab') ₂ fragment to detect scFv expression of untagged CAR from M28z1XXPD1DNR. CAR T cells were tested for >70% viability, >95% T cell purity by anti-human CD3 staining, 35-70% transduction efficiency by flow cytometry, and CD4/CD8 expression. Characteristics of T cells used in preclinical studies are presented in Table 6.

Table 6. Characteristics of T cells used in preclinical studies MCPC donor % CD3 % CD3+ CAR+ % CD3+ CAR CD4+ % CD3+ CAR CD8+ H16 97-98 43-62 48-57 33-45 H18 97-99 38-57 42-45 45-52 H19 98 37-64 41-46 47-51 C 97-98 65-66 37-38 55-57 C1 97 64-70 46 48-49 H1 97 56-60 38 Not determined

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

Table 7. Brief description of tumor cells used in preclinical studies Tumor cell Protein expression MSTOG GFP, ffLuc MGM GFP, ffLuc, mesothelin MGM-PDL1 GFP, ffLuc, mesothelin, PD-L1

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

Flow cytometry. Flow cytometry was performed using an Attune NxT flow cytometer (ThermoScientific, Waltham, MA) or a BD LSRFortessa (BD Biosciences, San Jose, CA). Expression of human mesothelin on the surface of tumor cells was detected using phycoerythrin-conjugated rat anti-human mesothelin IgG2a antibody (R&D Systems, Minneapolis, MN). Expression of human PD-L1 on the surface of tumor cells was detected using phycoerythrin cyanine 7-conjugated mouse anti-human PD-L1 IgG1 (BD Biosciences). Human T cells were analyzed for their cell surface expression of human CD3 using allophycocyanin-cyanin 7-conjugated mouse anti-human CD3 IgG2a or phycoerythrin-cyanin 7-conjugated mouse anti-human CD3 IgG1 antibody (BioLegend, San Diego, CA) and Human CD4 or human CD8 using fluorescein isothiocyanate-conjugated mouse anti-human CD4 IgG1 antibody (BioLegend) or Alexa Fluor 488-conjugated mouse anti-human CD8 IgG1 antibody (BioLegend), respectively. Cell surface expression of CAR was quantified using phycoerythrin-conjugated anti-myc tag antibody (Cell Signaling Technology, Danvers, MA) or Alexa Fluor 647 F(ab') ₂ -conjugated specific goat anti-human F(ab') antibody. ₂ (Jackson ImmunoResearch, West Grove, PA). Cell surface expression of PD1 on CAR T cells was analyzed using Brilliant Violet 711-conjugated mouse anti-human PD1 IgG1 antibody (BioLegend). For ex vivo detection of human T cells, treated mouse tissue was stained with phycoerythrin-cyanine 7-conjugated mouse anti-human CD3 IgG1 antibody and allophycocyanine-cyanine 7-conjugated mouse anti-human CD45 IgG1 antibody (BioLegend). Differentiation of live cells and 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). Table 8 shows the antibodies used for flow cytometry.

Table 8. Antibodies for flow cytometry in nonclinical pharmacology studies Marker Clone(s) View Antibody format Fluorophores Manufacturer CD3 HIT3a Mouse IgG2a AFC-CY7 BioLegend UCHT1 Mouse IgG1 FE-CY7 BioLegend CD45 2D1 Mouse IgG1 AFC-CY7 BioLegend CD4 RPA-T4 Mouse IgG1 FITC BioLegend CD8 RPA-T8 Mouse IgG1 AF700 BioLegend PD1 EH12.2H7 Mouse IgG1 BV711 BioLegend Myc-tag 1B11 Mouse IgG2a FE Cell Signaling F(ab') ₂ Polyclonal Goat F(ab') ₂ AF647 Jackson ImmunoResearch Mesothelin 420411 Rat IgG2a FE R&D Systems PD-L1 MIH1 Mouse IgG1 FE-CY7 BD Biosciences

Definition of VCN. Total genomic DNA from CAR T cells was isolated using a Miniprep Kit (Qiagen, Hilden, Germany). PCR primers and TaqMan probes were used to detect SFG and the albumin housekeeping gene (ALB). Probe and primer sequences for human SFG:

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]

Probe and primer sequences for human ALB:

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

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

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

The amplification reaction (25 µl) contained 5 µl (150 µg) genomic DNA and 12.5 µl TaqMan Fast Advanced Master Mix (ThermoFisher Scientific), 0.8 µl primers (forward and reverse), 0.2 µl TaqMan probe and 5 .7 µl distilled water. qPCR conditions were as follows: 50°C (2 min), 95°C (20 min), then 42 cycles at 95°C (15 sec) and 60°C (1 min), using a QuantStudio 7 real-time PCR system -Flex (ThermoFisher Scientific). All PCR measurements were performed in triplicate. The ratio of VCN per cell was calculated as (average number of SFG/average number of ALB)*2. Average amounts were extrapolated from SFG and ALB standard curves.

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

Primer sequences for GAPDH:

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

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

Primer sequences for the extracellular domain of PD-1 (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]

Primer sequences for the extracellular domain of PD-1 (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]

The cDNA was diluted 5-fold for subsequent qPCR analysis. The amplification reaction (20 μl) contained cDNA from 200 ng of total RNA and 10 μl of QuantiTect SYBR Green PCR Mix (Qiagen), 4 μl of primers (forward and reverse, 200 nM each) and distilled water. qPCR conditions were as follows: 95°C (15 min), 95°C (20 min), then 45 cycles of 94°C (15 sec), 60°C (30 sec), 72°C (30 sec), and 50 °C (20 seconds for data acquisition) using the QuantStudio 7 Flex Real-Time PCR System (ThermoFisher Scientific). All PCR measurements were performed in triplicate. All primers were synthesized at Integrated DNA Technologies (Coralville, IA), and amplification efficiency (E) values were calculated. Relative expression of target genes was normalized to the reference group as a ratio according to the Pfaffl formula (Pfaffl, Nucleic Acids Res. 2001; 29(9): e45):

Relative coefficient = (E _target ) ^{ΔCttarget (control-sample)} / (E _ref ) ^{ΔCtref (control-sample)}

Target=extracellular/intracellular domain of PD1; Ref=GAPDH; control=untransduced T cells.

⁵¹ Cr cytotoxicity assay. Cytotoxicity of mycM28z1XXPD1DNR CAR T cells compared to mycM28z T cells was determined using a standard 51Cr release assay. In 96-well round-bottom plates, 5 x 10 ⁵ to 1 x 10 ⁶ total T cells in 200 µl RPMI with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin were serially diluted 1: 2 in 100 µl of medium. Target cells were incubated with 75 µCi ⁵¹ Cr per 1 x 10 ⁶ cells for 2 hours and resuspended to a final concentration of 5 x 10 ³ cells/100 µl. After 3 washes with media, 100 μl of target cells were added to T cells in triplicate and incubated for 4-18 h in a humidity-controlled incubator with 5% CO ₂ at 37°C. Supernatants were collected, seeded into 96-well Lumina plates (PerkinElmer) and measured on a PerkinElmer TopCount. Spontaneous release of ⁵¹ Cr was assessed in target cells incubated with media alone, and maximum release of ⁵¹ Cr was determined with target cells incubated in 100 μl of 0.2% Triton X-100. The percentage of specific lysis was calculated as follows: [(experimental counts per minute (cpm)-spontaneous cpm release)/(total cpm-spontaneous cpm release)]+/-standard error of the mean, and analyzed using Microsoft Excel (Microsoft, Redmond, WA) or GraphPad Prism (GraphPad Software, La Jolla, CA).

Electrical resistance analysis. CAR T cell-induced lysis of target cells in vitro was assessed in real time using the xCELLigence Real Time Cell Analysis (ACEA Biosciences, San Diego, CA). First, 50 μL of RPMI with 10% FBS, 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin as culture medium for target and effector cells was added to a 96-well microtiter plate coated with gold microelectrodes (ACEA Biosciences) to measure background resistance. Then, 10,000 target cells were seeded in 100 μl of medium per well and target cell adhesion was monitored for 24–34 h before adding CAR T cells in 50 μl of medium in triplicate at an E:T ratio of 1:1–1:3. To assess changes in resistance resulting from CAR T cell-induced lysis and detachment of target cells, data were recorded every 15 min in a humidity-controlled incubator with 5% CO ₂ at 37°C for up to 4 days after addition of effector cells.

Repeated antigenic stimulation. To investigate the antitumor efficacy of CAR T cells upon antigen re-stimulation in vitro, 3.3 x 10 ⁵ -1 x 10 ⁶ T cells transduced with mycM28z1XXPD1DNR or mycM28z as a control were co-cultured with 3.3 x 10 ⁵ irradiated cells - targets (E:T ratio 1:1 to 3:1) in 1 ml RPMI with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin in 24-well cell culture plates. After 48 h of coculture, T cells were pooled, enumerated, analyzed for CAR expression by flow cytometry, and replated at the same E:T ratio with irradiated target cells for 6 cycles of antigen reexposure. After 1, 3, and 6 cycles of antigen exposure, CAR T cell cytotoxicity was assessed using ^51Cr release and resistance assays.

Accumulation. Accumulation was assessed by co-cultivating 3.3 x 10 ⁵ T cells transduced with mycM28z1XXPD1DNR or mycM28z control with 3.3 x 10 ⁵ irradiated target cells (E:T ratio, 1:1) in 1 ml RPMI with 10 % FBS, 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin in 24-well cell culture plates. After 48 h of coculture, T cells were pooled, counted, analyzed for their CAR expression by flow cytometry, and replated at the same E:T ratio with irradiated target cells for up to 6 cycles of antigen reexposure. The number of CAR T cells after each cycle of antigen stimulation was used to determine the accumulation of CAR T cells over time by the absolute number of T cells.

Quantitative determination of cytokines. Cytokine release assays were performed by co-cultivating 3.3 x 10 ⁵ T cells transduced with mycM28z1XXPD1DNR or mycM28z control with 3.3 x 10 ³ target cells (E:T ratio, 1:1) in RPMI with 10% FBS, 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin in 24-well cell culture plates. After 48 h of coculture, T cells were pooled, enumerated, analyzed for their CAR expression by flow cytometry, and replated at the same E:T ratio with irradiated target cells for up to 6 cycles of antigen reexposure. For cytokine quantification, supernatants were collected 24 hours after coculture for repeated stimulation with antigens 1, 3, and 6 and centrifuged at 800 g for 10 min at room temperature to remove cells and debris. Cytokine levels were determined in duplicate using the Human Cytokine Magnetic 30-plex Panel (Invitrogen, Carlsbad, CA) and the MAGPIX system (Luminex, Austin, TX) according to the manufacturers' instructions.

Orthotopic mouse model. Orthotopic tumor models are considered more clinically relevant and better suited for predicting drug efficacy than standard subcutaneous tumor models. Due to the fact that tumor cells are implanted directly into the organ of origin, these tumors reflect the initial situation (eg, microenvironment) much better than traditional subcutaneous xenograft tumor models. The combination of tumor cells transfected with the luciferase gene, together with orthotopic implantation of these cells, allows non-invasive imaging of tumor growth, tumor distribution, and metastatic growth.

Female and male NOD/SCID gamma mice, 6–10 weeks old (The Jackson Laboratory, Bar Harbor, ME), were used to create the orthotopic model. All procedures were performed in accordance with the approved protocols of the center's Institutional Animal Care and Use Committee (IACUC). The mice were anesthetized by inhalation of isoflurane and oxygen. To engraft orthotopic MPM tumors, direct intrapleural injection of mesothelin-expressing cells (8×10 ⁵ tumor cells) in 200 μl of serum-free medium was performed through the right thoracic incision. Tumor engraftment occurred in >95% of mice after inoculation by 8–12 days postinjection. If moribund, mice were sacrificed according to IACUC guidelines.

This model reproduces the human tumor microenvironment as it reflects the overall appearance and histopathological profile of the MPM (see Figure 24A). In addition, the extensive lymphovascularization of MPM tumors in the present mouse model (see Figure 24B) is characteristic of human MPM.

BLI is a sensitive in vivo method that can detect as few as 1×10 ³ tumor cells in the pleural space. Standardization and sensitivity are based on the inventors' own experiments (Kachala et al., Clin Cancer Res. 2014; 20(4): 1020-1028; Servais et al., Clin Cancer Res. 2012; Servais et al., Curr Protoc Pharmacol. 2011; Chapter 14: Servais et al., PLoS One.2011; 6(10): e26722; J Mol Med (Berl 89(8): 753-769). A strong relationship was observed between tumor BLI signal and pleural tumor volume determined by magnetic resonance imaging (MRI), the gold standard for tumor volume assessment (r=0.86, p<0.0001, corrected for internal clustering in mice; Figures 24C and 24D). The quantitative BLI results obtained are explained by the fact that the tumor grows along the chest wall as a thickening of the pleural lining, minimizing the depth of the tumor (Figure 24A). Thus, BLI in an orthotopic pleural cancer model can provide an accurate quantification of tumor burden, thereby providing a comparable standard for non-invasive serial assessment of biomarker performance in living mice. Using immunohistochemical analysis and flow cytometry, mesothelin expression was observed to be maintained in the orthotopic model of MPM even in late stages of the disease (data not shown).

Mouse tissue processing. Mice were sacrificed with CO ₂ , and pleural tumors and spleens were collected in a 50-mL ice-cold RPMI-1640 conical tube. For processing, the tissue was ground through a 40-μm cell sieve and centrifuged at 450 g for 5 min at 4°C. If the cell pellet appeared bloody, it was resuspended in 2 ml ACK lysis buffer (Lonza, Basel, Switzerland) and incubated for 5 minutes at room temperature. After an additional centrifugation step at 450 g for 5 minutes at 4°C, the cell pellet was resuspended in PBS with 5% bovine serum albumin for washing and antibody staining for direct use in flow cytometry.

Immunofluorescence. Female NSG mice implanted with MGM pleural tumors were injected with ^5x105 mycM28z1XXPD1DNR CAR T cells, mycM28z CAR T cells, or untransduced T cells. Three days after injection, mice were sacrificed, pleural tumors were isolated, fixed in 4% paraformaldehyde overnight at room temperature, and processed for paraffin embedding using a Leica ASP6025 histology processor (Leica Biosystems, Wetzlar, Germany). Freshly cut 5 μm paraffin sections were stained for sequential double immunofluorescence on a Leica Bond RX (Leica Biosystems) using 1.25 μg/ml mouse monoclonal anti-CD45 antibody clone 2B11+PD7/26 (Dako) for 1 hour with Alexa tyramide detection for 10 minutes. Fluor 594 1:200 (Life Technologies, Carlsbad, CA) on Leica Bond using method F, then 0.03 µg/ml rabbit monoclonal clone D9R5G to mesothelin (Cell Signaling) for 1 hour with detection for 10 minutes with Alexa Fluor 488 tyramide 1 :200 (Life Technologies) on Leica Bond using Method F. Sections were pretreated with Leica Bond ER2 buffer (Leica Biosystems) for 20 min at 100°C before each staining. After staining, sections were mounted in Mowiol medium for digital scanning on a Vectra 3.0 multispectral microscope (Perkin Elmer) using a 20× objective.

Histological examination of tumors and immunostaining. Histopathological evaluation of tumors was performed after hematoxylin and eosin staining of tissue samples embedded in paraffin and fixed with 4% paraformaldehyde. Immunohistochemical analysis for human mesothelin was performed with mouse anti-human mesothelin IgG antibody (1:100; Vector Labs, Burlingame, CA) using the Ventana platform. Mesothelin was scored by a laboratory technician blinded to the clinical data as follows: 0 (no staining), 1 (weak expression), 2 (moderate expression), and 3 (strong expression). The distribution of mesothelin-positive tumor cells compared to all tumor cells detected in a single sample was scored as 0 (absent), 1 (1–50%), and 2 (51–100%).

MRI. MRI was performed using a Bruker 4.7T USR scanner (Bruker Biospin, Ettlingen, Germany) equipped with a 400 mT/m gradient coil and a custom-made birdcage resonator with an internal diameter of 32 mm. Axial chest MRI images were acquired using a rapid spin-echo RARE sequence [repetition time (TR)=1.7 sec, echo time (TE)=40 msec and 12 mean values] triggered by the animal's respiration to reduce motion artifacts caused by breathing. The slice thickness was 0.7 mm, and the in-plane image resolution was 117 × 156 mm. Tumor volumes (mm ³ ) were measured by tracing the tumor boundaries in each section using Bruker ParaVision Xtip software (Bruker Biospin) and then calculated from the areas of tumor regions in each section.

BLI. BLI was used for noninvasive imaging of tumor burden. Mice were intraperitoneally injected with D-luciferin at a dose of 150 mg/kg. Tumor bioluminescence was measured after 15 min in mice in dorsal and ventral positions using an IVIS Spectrum in vivo imaging system (PerkinElmer). The average total flux at the dorsal and ventral positions was presented as the BLI signal in photons per second.

Quantitative determination of cytokines in mouse blood plasma samples. Plasma cytokine levels in a subset of mice from the toxicology study were quantified using the V-Plex Mouse Proinflamasted Panel 1 10-plex assay (Meso Scale Diagnostics, Rockville, MD). The following cytokines were analyzed: IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, KC/GRO and TNF-α. When possible, 60 μl of plasma was diluted 2-fold and applied to duplicate wells. Results (n=2 per sample) are presented in picograms per milliliter of plasma. Cytokine quantification was performed in the Immune Monitoring Laboratory at MSK.

B. Human tissue mesothelin and in vitro CAR T cell studies

B.1. Expression of mesothelin in tumor and normal tissues

Published studies have observed very low expression of mesothelin in normal pleura, peritoneal cavity and pericardium. Extensive studies of human MPM, lung adenocarcinoma, and triple-negative breast cancer tissues along with normal control tissues have been performed using mesothelin immunohistochemistry (Kachala et al., Clin Cancer Res. 2014; 20(4): 1020-1028; Servais et al. , Clin Cancer Res.2012; Rizket al., Cancer Epidemiol Biomarkers Prev. 21(3): 482-486. As shown in Figures 25A-25C, mesothelin is overexpressed in human MPM and lung carcinoma compared to normal tissues (see Figure 25A). Mesothelin was overexpressed in 69% of lung adenocarcinomas with no expression in normal lungs (see Figure 25B). Likewise, mesothelin is not expressed in normal breast tissue, but is overexpressed in triple negative breast cancer (see Figure 25C).

B2. Transduction of human T cells using M28z1XXPD1DNR

Viral supernatant encoding M28z1XXPD1DNR or mycM28z1XXPD1DNR obtained from stable 293T RD114 cell lines was titrated to assess human T cell transduction efficiency by flow cytometry. T cells successfully transduced CAR and PD1DNR with donor-dependent surface expression levels ranging from 32% to 73% CAR and 22% to 64% PD1 (including PD1DNR and endogenous PD1 expression) for viral supernatant dilutions tested (see Figure 26 ). CAR and PD1DNR were expressed proportionally due to bicistronic transgene expression mediated by self-cleaving P2A peptide.

AT 3. Vector copy number (VCN)

After successful transduction of human T cells using M28z1XXPD1DNR, the next step was to examine the relationship between CAR expression and the integration of vector copies into the T cell genome. A close relationship between MFI of CAR-positive cells and VCN was found in several donors and dilutions of viral supernatant encoding M28z1XXPD1DNR or mycM28z1XXPD1DNR, yielding R ² values ranging from 0.89 to 0.99 (see Figure 27). Transduction efficiency and VCN were found to be donor dependent, but high VCN (>5/cell) were observed particularly with transduction efficiency >70% and low VCN (<1/cell) were observed particularly with transduction efficiency <35% (data not shown). To avoid the high risk of insertional mutagenesis and ensure efficient T cell transduction, viral supernatants were titrated for all constructs to obtain transduction efficiencies in the range of 35–70% for all subsequent in vitro and in vivo experiments.

AT 4. M28z1XXPD1DNR CAR T cells overexpress the extracellular domain of PD1

Human T cells transduced with M28z1XXPD1DNR express PD1DNR, a decoy receptor that does not contain the intracellular signaling domain of PD1. Relative surface expression of PD1 protein and mRNA expression in mycM28z1XXPD1DNR CAR T cells and mycM28z CAR T cells were examined by flow cytometry and qPCR, respectively. With comparable levels of CAR surface expression (64-70%, see Figure 28A), cell surface PD1 staining resulted in the detection of higher levels of PD1 in both percentage of positive cells (2-fold) and intensity (3-fold). 4 times) on mycM28z1XXPD1DNR CAR T cells compared to mycM28z CAR T cells (see Figures 28B and 28C). Detection of PD1 cell surface expression was limited to the extracellular domain that was present in PD1DNR as well as endogenous PD1. Therefore, although higher surface expression of the extracellular domain of PD1 was induced and indicative of PD1DNR expression, differentiating PD1DNR and endogenous PD1 at the cell surface level was not possible.

To further investigate the differences in expression of PD1DNR and endogenous PD1, relative mRNA expression was examined by qPCR. To distinguish between the expression of PD1DNR and endogenous PD1, primers specific for the extracellular and intracellular domains of PD1 were selected, and the mRNA expression of the extracellular and intracellular PD1 domains of mycM28z1XXPD1DNR and mycM28z CAR T cells was measured compared with non-transduced T cells. mycM28z CAR T cells do not express PD1DNR and therefore express only endogenous PD1 at an equimolar ratio of extracellular to intracellular domain. While expression of the intracellular domain was exclusive only in the case of endogenous PD1, the extracellular domain was expressed by both PD1DNR and endogenous PD1. mycM28z CAR T cells were found to express 4-fold higher levels of extracellular and intracellular domains of PD1 compared to non-transduced T cells. However, mycM28z1XXPD1DNR CAR T cells exhibited 157-fold upregulation of the extracellular domain of PD1; non-transduced T cells showed only a two-fold upregulation of the PD1 intracellular domain (see Figure 28D).

Thus, these data confirm that PD1DNR is overexpressed at both the mRNA and protein levels compared to endogenous PD1 in mycM28z1XXPD1DNR CAR T cells. This finding provides the basis for intrinsic T cell checkpoint blockade.

AT 5. Functional comparison of M28z1XXPD1DNR CAR T cells with and without myc tag

To facilitate detection of CAR expression, M28z1XXPD1DNR CAR T cells were generated with a myc tag fused to the N terminus of an anti-mesothelin scFv, resulting in mycM28z1XXPD1DNR. To rule out any possible interaction of the myc tag with CAR function, a direct comparison of the antitumor efficacy of M28z1XXPD1DNR (no tag) and mycM28z1XXPD1DNR CAR T cells was performed. Human T cells obtained from 3 independent donors transduced with both M28z1XXPD1DNR and mycM28z1XXPD1DNR, at similar levels of transduction (37-63%, as determined by flow cytometry with cells stained with goat F(ab') ₂ fragment specific against human F(ab') ₂ ) showed no differences in the kinetics and overall lysis of MGM target cells at different E:T ratios (see Figure 29).

AT 6. mycM28z1XXPD1DNR CAR T cells mediate antigen-specific, HLA-independent tumor lysis

The cytotoxic activity of mycM28z1XXPD1DNR in comparison with mycM28z CAR T cells was determined against a panel of tumor cell lines, including human mesothelioma cells (MSTO-211H) with (GM) and without (G) mesothelin expression, as well as constitutive PD-L1 expression, using ⁵¹ Cr release analysis. mycM28z1XXPD1DNR CAR T cells efficiently lysed MGM and MGM-PDL1 targets, as did mycM28z CAR T cells, when cocultured for 18 hours with mesothelin-expressing tumor cells at various E:T ratios, as shown in the cytotoxicity assay results below ( see Figure 30). Nonspecific lysis by either mycM28z1XXPD1DNR or mycM28z CAR T cells was not observed against mesothelin-negative MSTOG tumor cells. Untransduced T cells did not show killing of either target. These results confirm that M28z1XXPD1DNR CAR T cells kill target cells in a mesothelin-specific and HLA-independent manner.

AT 7. mycM28z1XXPD1DNR CAR T cells accumulate

To investigate whether mycM28z1XXPD1DNR CAR T cells support T cell accumulation upon repeated antigen stimulation with mesothelin-expressing tumor cells with inducible PD-L1 expression (MGM) or constitutive PD-L1 expression (MGM-PDL1), mycM28z1XXPD1DNR CAR T cell expansion quantified and compared with mycM28z CAR T cell expansion. Over the course of 6 repeated stimulations with the mycM28z1XXPD1DNR antigen, CAR T cells expanded up to 622-fold, similar to mycM28z CAR T cells (see Figure 31).

AT 8. Antitumor efficacy of mycM28z1XXPD1DNR CAR T cells upon repeated antigen stimulation

Upon first exposure to antigen, mycM28z1XXPD1DNR CAR T cells exhibited E:T-dependent cytotoxicity against MGM and MGM-PDL1 target cells, but showed no difference in cytotoxicity compared to mycM28z CAR T cells (see Figure 32). To examine the cytotoxic capacity of mycM28z1XXPD1DNR and mycM28z CAR T cells during chronic antigen exposure (or high antigen stress), CAR T cells were repeatedly cocultured with irradiated MGM or MGM-PDL1 target cells every 48 h at an E:T ratio of 3 :1 for 4 cycles. At the end of the third cycle, the CAR T cell sample was subjected to a fourth antigen stimulation in the ⁵¹ Cr cytotoxicity assay. mycM28z1XXPD1DNR and mycM28z CAR T cells showed comparable cytotoxicity during the fourth antigen stimulation (see Figure 33).

Then, to further enhance antigenic stress, mimicking the high tumor burden in solid tumors, CAR T cells were cocultured with target cells at an E:T ratio of 1:1 in the fifth and sixth cycles of antigenic stimulation. At the end of the sixth cycle, another sample of CAR T cells was collected and subjected to the seventh cycle of antigen stimulation in the ⁵¹ Cr cytotoxicity assay. Seventh antigen challenge cytotoxicity of mycM28z CAR T cells against both target cell lines was significantly reduced. In contrast, mycM28z1XXPD1DNR CAR T cells maintained cytotoxicity toward target cells. mycM28z1XXPD1DNR CAR T cells compared to mycM28z CAR T cells demonstrated superior tumor cell killing with target cells with high constitutive expression of PD-L1 (see Figure 33), although cytotoxicity was reduced compared to the first and fourth antigen stimulations. Taken together, these data indicate that mycM28z CAR T cells reach a state of exhaustion with prolonged antigen exposure, whereas mycM28z1XXPD1DNR CAR T cells are able to maintain antitumor activity even under conditions of high antigen stress. The observed effects depended on biological parameters such as donor, E:T ratio, and co-culture time between tumor and CAR T cells.

AT 9. Target-stimulated cytokine release by mycM28z1XXPD1DNR CAR T cells

The secretion of effector cytokines mycM28z1XXPD1DNR and mycM28z CAR T cells upon repeated antigen stimulation was assessed using the Luminex assay. Both mycM28z1XXPD1DNR and mycM28z CAR T cells secreted high levels of IL-2, IFN-γ, and TNF-α after the first antigen stimulation. However, secretion of effector cytokines by both CAR T cells was reduced upon repeated antigen stimulation (see Figure 34).

C. In vivo studies

S.1. Antitumor efficacy of mycM28z1XXPD1DNR CAR T cells visualized by ffLuc tumor cells

Using an orthotopic model, a series of in vivo mouse experiments were performed to study the specificity and efficacy of mycM28z1XXPD1DNR CAR T cells by measuring tumor burden and animal survival. Serial BLI was used to confirm tumor engraftment, to equalize tumor burden between intervention groups before initiation of T-cell therapy, and then to measure response to therapy.

To investigate the antitumor efficacy of mycM28z1XXPD1DNR CAR T cells, a single low dose of 3x10 ⁴ CAR T cells was administered intrapleurally to female NSG mice 13 days after orthotopic MGM tumor inoculation. The low dose was specifically chosen to mimic the high tumor antigenic load experienced by CAR T cells. Compared to control mice treated with P28z CAR T cells, mice treated with a single dose of mycM28z1XXPD1DNR CAR T cells showed a significant reduction in tumor burden 15 days after T cell administration (p=0.0002) (see Figure 35) . At day 15 post-treatment, mice injected with P28z CAR T cells began to succumb as expected due to high tumor burden, while mice injected with mycM28z1XXPD1DNR CAR T cells showed no signs of toxicity.

Observing the efficacy and tumor eradication with a single dose of 3x10 ⁴ mycM28z1XXPD1DNR CAR T cells/mouse (corresponding to 1.2x10 ⁶ -1.5x10 ⁶ cells/kg; with a mouse weight of 20-25 g), the authors The inventors decided to increase the dose level by 3-4 times to study the toxic effect in mice (described in Section 3 of this Example (under the heading "Preclinical Toxicology")).

In a subsequent experiment, female NSG mice bearing an established MGM-PDL1 pleural tumor were given a single intrapleural injection of 1 x 10 ⁵ or 5 x 10 ⁴ mycM28z1XXPD1DNR CAR T cells per mouse 11 days after tumor inoculation (E:T ratio, 1:1000-1 :2000; from 2.5x10 ⁶ to 5x10 ⁶ cells/kg; average mouse weight 20 g) or 1x10 ⁵ mycM28z CAR T cells/mouse as control. E:T ratios were calculated based on tumor burden quantification as described previously (Servais et al., Curr Protoc Pharmacol. 2011; Chapter 14: Unit14 21; Servais et al., PLoS One. 2011; 6(10): e26722) .

Serial tumor imaging with D-luciferin (luciferin for ffluc) showed a reduction in tumor burden according to BLI as early as 5 days after CAR T cell administration, with complete tumor clearance (BLI background signal) around day 19 in mice treated with ^1x105 mycM28z1XXPD1DNR CAR T cells, and at approximately day 26 in mice treated with 1x10 ⁵ mycM28z CAR T cells (see Figures 36A and 36B). Untreated mice died 9-12 days after the start of treatment (see Figure 36B). Tumor eradication was maintained with mycM28z1XXPD1DNR CAR T cells (dose of 1 x 10 ⁵ and 5 x 10 ⁴ ) until the end of the study on day 68. However, mice treated with mycM28z CAR T cells were sacrificed before day 68 due to a state of agony and weight loss , an autopsy revealed the presence of a tumor. Mice receiving either dose of mycM28z1XXPD1DNR CAR T cells gained weight linearly throughout the study, while mice receiving mycM28z CAR T cells lost weight by the end of the study (see Figure 36C). Median survival was 12 days in untreated mice and 50 days in mice treated with 1x10 ⁵ mycM28z CAR T cells; median survival was not achieved in mice treated with any dose of mycM28z1XXPD1DNR CAR T cells (see Figure 36D). Survival was significantly increased in mice receiving mycM28z1XXPD1DNR CAR T cells compared to mice receiving mycM28z CAR T cells (p=0.0085 for ^5x104 mycM28z1XXPD1DNR and p=0.0427 for ^1x105 mycM28z1XXPD1DNR by compared to 1x10 ⁵ mycM28 CAR T cells). No overt clinical signs of toxicity were observed in mice treated with mycM28z1XXPD1DNR CAR T cells.

C.2. Detection of mycM28z1XXPD1DNR CAR T cells in the primary tumor

In a subset of mice, following intrapleural injection of 5x10 ⁵ mycM28z1XXPD1DNR CAR T cells, mycM28z CAR T cells, or untransduced T cells into NSG mice bearing an MGM pleural tumor, the pleural tumor was removed within 3 days of injection and examined for infiltration by human T cells. cells when stained for human CD45 and human tumor mesothelin using immunofluorescence.

In mice treated with non-transduced T cells, only a few non-transduced T cells were found in the tumor periphery and parenchyma, whereas mycM28z1XXPD1DNR CAR T cells and mycM28z CAR T cells were found in higher densities in the peritumoral region and at the tumor interface and peritumoral region (see Figure 37). These data suggest that regionally (intrapleurally) administered mesothelin-targeted T cells are concentrated in the peritumoral region and infiltrate from the tumor periphery into the tumor.

C.3. Antitumor activity of mycM28z1XXPD1DNR CAR T cells during tumor re-challenge in vivo

To study the functional persistence of mycM28z1XXPD1DNR CAR T cells, MGM-PDL1 pleural tumor-bearing mice (n=5) treated with a single intrapleural dose of 1x10 ⁵ mycM28z1XXPD1DNR or mycM28z CAR T cells were reinfected intraperitoneally with increasing doses of MGM tumor cells. Re-challenge was initiated 68 days after intrapleural T cell administration and after pleural tumor clearance with an initial boost dose of 2 x 10 ⁶ tumor cells. The tumor dose was increased to 11×10 ⁶ tumor cells for 10 repeat infections every 4-8 days (see Figure 38A). Tumor reinfection was associated with an increase in BLI signal shortly after each tumor injection, followed by a decrease in BLI, indicating T cell-mediated tumor regression (see Figure 38B). Peak BLI signals increased following subsequent increases in tumor cell doses, with mice receiving mycM28z CAR T cells exhibiting a significantly higher increase in BLI signal compared to mice receiving the same dose of mycM28z1XXPD1DNR CAR T cells. Specifically, the BLI signal in mice treated with mycM28z1XXPD1DNR CAR T cells peaked shortly after each tumor rechallenge, but returned to baseline BLI signal even at the highest rechallenge dose (see Figure 38B). In contrast, mice treated with mycM28z CAR T cells showed a decrease in BLI signal after an initial increase in BLI up to 5 tumor re-challenges, however, they eventually lost the ability to control tumor re-engraftment after higher tumor doses, resulting in a persistent increase in signal BLI and tumor burden and agonal state. mycM28z1XXPD1DNR CAR T cells prevented intraperitoneal tumor engraftment over 10 repeat infections, even >126 days after a single intrapleural dose of 1x10 ⁵ CAR T cells, without any obvious clinical signs of toxicity. These data indicate that mycM28z1XXPD1DNR CAR T cells are capable of tumor control not only locally in the pleural space, but also in distant parts of the body. In vivo, mycM28z1XXPD1DNR CAR T cells remained functional upon prolonged exposure to antigen upon repeated antigen stimulation and persisted for a long time, whereas mycM28z CAR T cells became dysfunctional upon repeated antigen stimulation, indicating superior functional persistence and enhanced long-term antitumor activity. mycM28z1XXPD1DNR CAR T cell activity. This increased efficacy was confirmed to be not due to graft-versus-host disease. Non-antigen-expressing targets were administered and tumor BLI enhancement without an antitumor response was noted, confirming that antitumor efficacy is antigen-specific.

C.4. Antitumor efficacy of M28z1XXPD1DNR T cells for clinical use

The purpose of this study was to confirm the antitumor efficacy of cryopreserved M28z1XXPD1DNR CAR T cells that were generated at MSK's Cell Therapy and Cell Engineering Facility (CTCEF). Before intrapleural injection, aliquots of cryopreserved M28z1XXPD1DNR CAR T cells were thawed in RPMI-1640 with 10% FBS, washed two times in RPMI-1640 without FBS, and resuspended in RPMI-1640 without FBS as injection medium. CAR T cell viability after thawing and before injection was determined to be 88% using acridine orange/propidium iodide staining. CAR T cells were administered intrapleurally to female NSG mice bearing MGM pleural tumors at a dose of 6x10 ⁴ or 2x10 ⁵ viable CAR T cells/mouse (n=8-10). Serial BLI of tumors revealed tumor clearance at day 16 in mice treated with 2x10 ⁵ CAR T cells (see Figure 39A). Mice injected with 6x10 ⁴ CAR T cells showed tumor regression and clearance at day 29 (see Figure 39A). No weight loss was observed in any of the treatment groups, whereas untreated tumor-bearing mice lost weight and died within 19 days of treatment (see Figures 39B and 39C). No toxic effects were observed in mice treated with CAR T cells, and 100% of treated mice remained alive at the end of the observation period (day 70, see Figure 39C). This study confirmed that cryopreserved M28z1XXPD1DNR CAR T cells generated using parental vectors obtained for a prospective clinical trial exhibited high viability after thawing, exhibited antitumor efficacy in vivo, and were well tolerated in mice.

3. Preclinical toxicology

A. Summary of Nonclinical Safety Studies

Binding and activity of M28z1XXPD1DNR CAR T cells are specific to human mesothelin, so there is no ideal pharmacologically relevant species in which to conduct preclinical safety studies. In addition, variability in the expression profiles of target antigens and differences in clearance mechanisms and immunogenicity of human polypeptides such as CARs in immunocompetent mice hinder the use of animals to predict CAR T cell toxicity in humans. Because M28z1XXPD1DNR CAR T cells have relevant pharmacodynamic effects (cytokines, accumulation, tumor regression) in an orthotopic immunodeficient mouse model expressing human mesothelin, a safety study was conducted in this xenogeneic model. This was a Good Laboratory Practice (GLP) study conducted at MSK in the Antitumor Activity Core Laboratory. This section discusses the design, methods, and results of this study.

The selected dose of CAR T cells for this study was 1x10 ⁵ mycM28z1XXPD1DNR CAR T cells. This dose was chosen because it is 3-4 times higher than the minimal effective dose tested ( ^3x104 mycM28z1XXPD1DNR CAR T cells) for the treatment of orthotopic mesothelioma tumors in our preclinical mouse model. It is important to note that the selected dose of CAR T cells (1x10 ⁵ cells/mouse or 5x10 ⁶ cells/kg) is 5 times higher than the starting dose proposed in the current study (1x10 ⁶ cells/kg). In our published studies, mesothelin-targeted CAR with PD1DNR (Cherkassky et al., J Clin Invest. 2016; 126(8): 3130-3144) at a dose of 40,000 to 50,000 CAR T cells was shown to eliminate high pleural tumor burden load in mice with orthotopic pleural mesothelioma.

CAR T cells were delivered as a single dose via intrapleural injection to NSG mice bearing mesothelioma xenografts in the pleural cavity. Unlike other agents, such as antibodies, for which intrapleural administration is initially limited to the pleural cavity, in mice, as in humans, intrapleurally administered CAR T cells have been shown to circulate systemically within one or two days and are not limited to the pleural cavity (Adusumilli et al., Sci Transl Med. 2014; 6(261): 261ra151; Cherkassky et al., J Clin Invest. 2016; 126(8): 3130-3144; Adusumilli et al., Cancer Res. 2019; 79 (13)). In particular, the inventors observed that in mice, as in humans, intrapleurally administered CAR T cells are activated by antigen and proliferate 5-10 times higher than the initial dose administered within a short period of time (Adusumilli et al., Sci Transl Med. 2014; 6(261): 261ra151; J Clin Invest. 2016; 126(8): 3130-3144), so the actual number of CAR T cells tested in this study is 5-10. times the initially administered dose for a short period of time.

When assessing the safety of M28z1XXPD1DNR CAR T cells for medical use, the following factors are important.

1. Previous experience with the medical use of CAR T cells shows that exceeding the pharmacodynamic effect is the main cause of adverse events, which can be controlled and contained. In the inventors' Phase I clinical trial, there were no reports of adverse events associated with non-tumor toxicity of mesothelin-targeted CAR T cells (Adusumilli et al., Cancer Res.2019; 79(13)).

2. In in vitro studies, the pharmacological activity of mycM28z1XXPD1DNR CAR T cells was found to correlate with the expression of mesothelin on the target cell surface.

3. In our orthotopic MPM model, specific cytotoxicity and increased survival were observed following a single intrapleural injection of mycM28z1XXPD1DNR CAR T cells into mice bearing MPM tumors. Without treatment with mycM28z1XXPD1DNR CAR T cells, these mice would have died within 20-22 days of tumor implantation. Within 7 days, the tumor burden was significantly reduced, and in animals recovery continued after 68 days; In contrast, untreated control animals had a median survival of 12 days, and mycM28z-treated control animals had a median survival of 50 days.

Mortality and disease severity, weight, clinical signs, hematology and clinical biochemistry, gross necropsy findings, and histopathological findings were assessed in 96 (48 male and 48 female) NSG mice (Jackson Laboratory) bearing 8-day-old orthotopic MGM tumors randomly assigned to groups. control and treatment. A dose of 1 x 10 ⁵ cells/mouse (approximately 5 x 10 ⁶ cells/kg) in RPMI-1640 was selected, which corresponds to at least approximately 3-4 times the minimum effective dose determined in in vitro and in vivo control experiments on this model (3×10 ⁴ cells/mouse). The dosage and dissection regimen were chosen rationally: (a) although a higher initial test dose may allow investigation of any immediate adverse effects from dosing (such effects were not observed in current clinical studies), the biology and pharmacology do not reflect the functional persistence and proliferation of mycM28z1XXPD1DNR CAR T -cells (i.e., in the solid tumor microenvironment, it is important to ensure that relatively higher numbers of CAR T cells are present in the tumor/body for an extended period of time than with short-term, high-dose-induced tumor eradication kinetics), and (b) day 14 was chosen because the tumor had either largely regressed or been eliminated at this time point (as confirmed by BLI or autopsy data from previous experiments) (Adusumilli et al., Sci Transl Med. 2014; 6(261): 261ra151; Cherkassky et al. J Clin Invest 2016; 126(8): 3130-3144). Killing and dissection at this time point allows for investigation of any targeted non-tumor effects (scFv used in the present CAR reacts with mouse mesothelin) (Feng et al., Mol Cancer Ther. 2009) on normal tissues, particularly the pleura, peritoneum and pericardium, with low levels of mesothelin expression after peak expansion of CAR T cells in the absence of tumor burden with high mesothelin expression.

mycM28z1XXPD1DNR CAR T cells or control medium were administered in a single dose by orthotopic injection on study day 1 (male mice) or study day 2 (female mice). All animals were monitored for mortality and disease severity twice a week until study day -8, followed by daily monitoring (working days) until study day 1. After dosing, animals were observed daily until the end of the study (day 15 of the study). Body weight was recorded twice a week until study day -8, followed by daily monitoring (working days) until study day 1. After dosing, body weight was recorded daily until the end of the study (day 15 of the study). Clinical signs were recorded twice a week until study day 1, followed by daily monitoring from study days 1 to 15. On study day 2 (intermediate male mouse sacrifice), study day 3 (interim female mouse sacrifice), study day 14 (final male mouse sacrifice), and study day 15 (final female mouse sacrifice), mice were anesthetized with isoflurane and blood was collected for hematology studies. and clinical biochemistry. For macroscopic and complete dissection, tissues were collected and fixed in formalin. No tissue was discarded during dissection. During necropsy, macroscopic examination of each animal was performed by members of the Central Laboratory for the Evaluation of Antitumor Activity. Any macroscopic lesions or other abnormalities were recorded using standard terminology and provided to the technologist for comparison with microscopic findings. For histopathological analysis, all tissues from dissected animals were preserved in formalin. After at least 24 hours in fixative, the tissues were processed and embedded in paraffin. The paraffin blocks were then cut into 4-μm-thick sections. The resulting unstained slides were then stained with hematoxylin and eosin. The slides were then sent to HSRL for analysis by a board-certified laboratory technician. Lesions were recorded using morphological diagnoses according to standard nomenclature. On study days 9 (male mice) and 10 (female mice), plasma was collected from a subset of mice and cytokine assessments were performed at the Central Immune Monitoring Laboratory at MSK (not GLP). In addition, spleens and tumors were harvested from a subset of mice to identify mycM28z1XXPD1DNR CAR T cells by flow cytometry (non-GLP). On study days -1/1, 7/8 and 14/15, a subset of animals were imaged using luciferin (dorsal/ventral) to assess tumor burden and study drug efficacy.

No mortality or disease severity was observed in the animals in this study, with the exception of 2 control animals in the imaging cohort that were selectively sacrificed due to severe condition and difficulty breathing on study days 12 and 14 (days 20–22 post-tumor injection). Previous work in our laboratory has shown that control animals can succumb to tumor burden approximately 20-22 days after tumor injection (Servais et al., Clin Cancer Res. 2012; Servais et al., Curr Protoc Pharmacol. 2011; Chapter 14: Unit14 21; Sci Transl Med.2014; 6(261): 261ra151; J Clin Invest 2016(8): 3130-3144; One. 2011; 6(10): e26722). Therefore, these killings were unplanned, but not unexpected. Mortality or disease severity during study drug administration was not monitored.

Animals treated with control media showed a progressive decrease in body weight during the study and a significant difference in weight compared to tumor-free control mice and mice treated with mycM28z1XXPD1DNR CAR T cells. This was associated with an increase in tumor burden in control animals.

No significant clinical signs were observed in mice treated with mycM28z1XXPD1DNR CAR T cells. Mild desquamation was observed in one mouse treated with the study drug, which was attributed to irritation caused by surgical clamps, as no other animals were harmed and the animals' activity was normal. The mice appeared normal throughout the observation period.

Female mice treated with mycM28z1XXPD1DNR CAR T cells in group 12 (final sacrifice on day 15 of the study) had a high mean percentage of monocytes (mean, 18.44%, n=5) (p≤0.0001) compared with group 10 (tumor with environmental control) (mean, 3.34%, n=5). The reference range established for the percentage of monocytes is 0.9-18%. However, this did not correlate with microscopic findings. No other significant or abnormal results were observed when assessing the complete blood count parameters. Any differences between the study drug groups and the matched vehicle groups were within normal reference ranges or were not biologically relevant or statistically significant.

Male mice treated with mycM28z1XXPD1DNR CAR T cells in group 11 (final sacrifice on day 14 of the study) had a low mean total protein value (mean, 3.83 g/dL, n=5) (p=0.0022) compared with group 9 (tumor with environmental control) (mean, 4.68 g/dL, n=4). The reference range established for total protein is 4.1–6.4 g/dL. However, this did not correlate with microscopic findings. No other adverse effects in terms of clinical biochemistry parameters were observed with the administration of the study drug. Any differences between the study drug groups and the matched vehicle groups were within normal reference ranges or were not biologically relevant or statistically significant.

Tumor tissue was not collected at the time of necropsy, but was observed in many animals at interim sacrifice, as expected. At the time of final sacrifice, tumor burden was noticeable in control animals receiving medium alone, but in most cases little or no tumor lesions were observed in animals receiving study drug. Macroscopic observations in individual animal necropsies included a mottled liver, a reduced spleen, a blue seminal vesicle, a dark green gall bladder, a blue gall bladder, an oily kidney, a liver bleb, and a white spot on the spleen. None of these results correlated with microscopic findings. A small spleen is expected in NSG mice due to lymphocyte depletion.

Review of the histopathological findings showed no microscopic findings at interim (study day 2/3) or final (study day 14/15) killing days associated with acute or delayed toxicity resulting from study drug administration. Microscopic findings from animals in groups 5 and 6 (test sample; interim sacrifice) included the presence of mixed cellular infiltration in xenograft tumors in the lungs. This was thought to be related to study drug administration but was not due to any study drug toxicity. This result was confirmed in a separate study using immunofluorescence staining of mycM28z1XXPD1DNR CAR T cells in the primary tumor of mice treated with intrapleural therapy (see Section 2.C.2 of this Example). Any other results observed were determined to occur sporadically, at the same frequency as controls, or to be common for the species/strains used.

In vivo, BLI on day -1/1 of the study showed successful tumor introduction in all animals in each group. Following administration of control medium, tumor burden in the male and female mice groups increased at each time point measured, with two animals requiring early imaging due to severe condition. After administration of the study drug, the tumor burden in animals in the groups of male and female mice increased 1 week after injection; however, after 2 weeks the load decreased significantly. Human T cells were detected in spleen and tumor tissue 8 days after intrapleural injection in mice treated with mycM28z1XXPD1DNR CAR T cells, but not in mice treated with medium. Mouse plasma cytokine levels obtained at the same time point showed slightly increased levels of IL-4 in CAR T cell-treated mice compared with control-treated mice. Levels of IL-10, IL-6, KC/GRO, and TNF-α were generally low and did not show significant differences between mice receiving CAR T cells and mice receiving control media. IFN-γ, IL-12p70, IL-1β, IL-2 and IL-5 were not detected (below the limit of quantitation).

The purpose of this study was to evaluate the acute and delayed toxicity of mycM28z1XXPD1DNR CAR T cells in NSG mice after a single orthotopic injection. During the study, data on body weight, clinical signs, complete blood count, clinical biochemistry and histopathological data were collected and analyzed. The results of this study indicate that a single orthotopic administration of 1×10 ⁵ mycM28z1XXPD1DNR CAR T cells in a mesothelioma xenograft model is well tolerated.

B. Methods

Testing system. Experiments were performed on 6-8 week old male and female NSG mice from the Jackson Laboratory. Mice in the tumor groups received 800,000 MGM cells/mouse via intrapleural injection on study day 1 (male mice) and study day 2 (female mice). These mice were expected to develop symptoms of pleural disease approximately 5 to 20 days after injection.

Study drug. The study drug (mycM28z1XXPD1DNR CAR T cells) was prepared by the inventors at MSK. PBMCs from a healthy donor were thawed on 05/11/2019 and transduced on 07/11/2019 with retroviral particles encoding mycM28z1XXPD1DNR. Before injection, transduced PBMCs were maintained in RPMI-1640 medium with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 20 U/ml IL-2. On injection days 11/13/2019 (male mice) and 11/14/2019 (female mice), transduced cells were pooled, analyzed by flow cytometry for CD3 and CAR expression, washed and resuspended in media (RPMI-1640 without FBS and without phenol red ) and kept on ice until injection.

mycM28z1XXPD1DNRCAR T cells were resuspended in vehicle to a final concentration of 5×10 ⁵ viable CAR T cells/ml. Cell viability was measured. The prepared solution was then transferred to the vivarium on ice for immediate use. The study drug, ready for use and stored on ice, was considered stable under these conditions throughout the study period. The preparation of the CAR T-cell preparation was carried out in the laboratory of the authors of the invention in a laminar flow hood at room temperature under aseptic conditions. CAR viability and expression were determined pre- and post-dose.

Wednesday. The media used in the preparation of the CAR T cell formulations and for administration to the control group was RPMI-1640 Sterile Medium without FBS and without Phenol Red (GIBCO, Cat. No. 32404, Lot No. 2099376). The medium was transferred to the vivarium on ice for immediate use. The media, ready for use and stored on ice, was considered stable under these conditions throughout the study period, as confirmed by the manufacturer's certificate of analysis. The preparation of the medium was carried out in the laboratory of the authors of the invention in a laminar flow hood at room temperature under aseptic conditions.

Study design. All cohorts were randomized to study arms prior to Day 1 of the study. Upon arrival, animals were randomly removed from their transport cages and assigned to their respective treatment groups as shown in Table 9 below. Mice were identified using an ear punch.

Mice in the tumor groups received 8×10 ⁵ MGM cells via orthotopic pleural injection 8 days before study drug administration. mycM28z1XXPD1DNR CAR T cells or control medium were administered similarly via orthotopic injection once on study day 1 (male mice) or study day 2 (female mice). Tumor cells, medium and the test drug were administered in an injection volume of 200 μl/mouse.

Table 9. Distribution into groups Group* Mice/
Group Floor Mouse ID# Blood/tissue collection Opening Intermediate killing (24 h) Day 2/3 1. No tumor - Control medium 3 Male 1-3 CBC & Chem General 2. No tumor - Control medium 3 Female 4-6 CBC & Chem General 3. Tumor - Control medium 10 Male 7-16 CBC & Chem Complete 4. Tumor - Control medium 10 Female 17-26 CBC & Chem Complete 5. Tumor - Investigational drug 10 Male 27-36 CBC & Chem Complete 6. Tumor - Investigational drug 10 Female 37-46 CBC & Chem Complete Final killing Day 14/15 7. No tumor - Control medium 3 Male 47-49 CBC & Chem General 8. No tumor - Control medium 3 Female 50-52 CBC & Chem General 9. Tumor - Control medium 5 Male 53-57 CBC & Chem Complete 10. Tumor - Control medium 5 Female 58-62 CBC & Chem Complete 11. Tumor - Investigational drug 5 Male 63-67 CBC & Chem Complete 12. Tumor - Investigational drug 5 Female 68-72 CBC & Chem Complete TK/cytokines 13. Tumor - Control medium 3 Male 73-75 Tumor, spleen, 200 µl EDTA No 14. Tumor - Control medium 3 Female 76-78 Tumor, spleen, 200 µl EDTA No 15. Tumor - Investigational drug 3 Male 79-81 Tumor, spleen, 200 µl EDTA No 16. Tumor - Investigational drug 3 Female 82-84 Tumor, spleen, 200 µl EDTA No Visualization 17. Tumor - Control medium 3 Male 85-87 No No 18. Tumor - Control medium 3 Female 88-90 No No 19. Tumor - Investigational drug 3 Male 91-93 No No 20. Tumor - Investigational drug 3 Female 94-96 No No *Decoding of groups: No tumor - Control medium: No tumor mice + RPMI1640
Tumor - Control medium: Mice with tumor MGM+RPMI1640
Tumor - Investigational drug: Tumor-bearing mice MGM+1×10 ⁵ mycM28z1XXPD1DNR CAR T cells

Clinical signs and body weight measurements were recorded throughout the study to assess pathology, and acute and delayed toxicity were assessed at 1 and 14 days post-dose, respectively. Complete blood count, clinical chemistry, and histopathology data were collected and assessed at early and late time points to determine study drug tolerability.

Estimated parameters.

Mortality and severity of the disease. All animals were monitored for mortality and disease severity twice a week until study day -8, followed by daily observation (on weekdays) until study day 1. After dosing, animals were observed daily until the end of the study (day 15 of the study).

Body mass. Body weight was recorded twice a week before study day -8, followed by daily monitoring (working days) until study day 1. After dosing, body weight was recorded daily until the end of the study (study day 15).

Clinical signs. Clinical signs were recorded twice a week before study day 1, followed by daily monitoring from study days 1 to 15.

General blood analysis. On day 2 of the study (male groups 1, 3, 5), day 3 of the study (female groups 2, 4, 6), day 14 of the study (male groups 7, 9, 11) and day 15 of the study (female groups 8, 10, 12) Mice were anesthetized with isoflurane and whole blood was collected in an EDTA tube for the following measurements, as shown in Table 10:

Table 10. General blood test indicators Neutrophils Leukocytes Lymphocytes Red blood cells Monocytes Hemoglobin Eosinophils Hematocrit Basophils Average red blood cell volume % Neutrophils Average erythrocyte hemoglobin % Lymphocytes Average hemoglobin concentration in erythrocytes % Monocytes Distribution of red blood cells by volume % Eosinophils Platelet count % Basophils Average platelet volume

Clinical biochemistry. On day 2 of the study (male groups 1, 3, 5), day 3 of the study (female groups 2, 4, 6), day 14 of the study (male groups 7, 9, 11) and day 15 of the study (female groups 8, 10, 12) mice were anesthetized with isoflurane. Whole blood was collected into a serum separation tube. The serum was separated and tested by the following measurements as shown in Table 11:

Table 11. Clinical biochemistry indicators Blood urea nitrogen Cholesterol Creatinine Alanine aminotransferase Phosphorus Aspartate aminotransferase Calcium Alkaline phosphatase Total protein Total bilirubin Albumen Sodium Globulin Potassium Albumin/globulin ratio Chloride Glucose Sodium/potassium ratio

Opening. On day 2 of the study (male groups 1, 3, 5), day 3 of the study (female groups 2, 4, 6), day 14 of the study (male groups 7, 9, 11) and day 15 of the study (female groups 8, 10, 12) mice were euthanized by CO ₂ inhalation. A general autopsy of animals was performed in groups 1, 2, 7 and 8, and a complete autopsy of animals in groups 3-6 and 9-12. Tissues were collected and fixed in formalin. No tissue was discarded during dissection.

Observations of general pathology. During necropsy, macroscopic examinations of each animal were performed by members of the Central Antitumor Activity Evaluation Laboratory. Any macroscopic lesions or other abnormal findings were recorded using standard terminology and provided to the technologist for comparison with the microscopic findings.

Histopathology. All tissues of dissected animals were preserved in formaldehyde. After at least 24 hours in fixative, the tissues listed in Table 12 were processed and embedded in paraffin (tissues indicated with an asterisk were first decalcified before embedding). The paraffin blocks were then cut into 4-μm thick sections. The resulting unstained slides were then stained with hematoxylin and eosin. The slides were then sent to HSRL for review by a board-certified laboratory technician. Lesions were recorded using morphological diagnoses according to standardized nomenclature.

Table 12. Tissues examined microscopically Adrenal glands Pancreas Bone (bone marrow)* Rectum Brain (brain stem, cerebellum and cerebrum) Salivary glands Esophagus Leather Eyes Spleen Gallbladder Sternum* Heart Knee-joint* Inguinal lymph node Stomach Intestine (duodenum, jejunum, ileum, cecum, colon) Testicles, epididymis, seminal vesicles and prostate (males) Kidneys Thymus Liver Thyroid Lungs Language Mesenteric lymph node Trachea Oral-nasal cavity (nasopharynx)* Bladder Ovaries, horns, uterus, cervix and vagina (females) Spinal column*

Cytokine (non-GLP) assay. On study days 9 (male mice) and 10 (female mice), blood was collected from mice in groups 13, 15 (male), 14, and 16 (female) into EDTA tubes. The plasma was separated and frozen. Cytokine assessments were performed at the Central Immune Monitoring Laboratory at MSK (not GLP).

Toxicokinetic studies: Identification of CAR T cells using flow cytometry (non-GLP). On study days 9 (male mice) and 10 (female mice), blood was collected from mice in groups 13, 15 (male), 14, and 16 (female). A gross necropsy was performed on all animals, with tumor and spleen tissue placed in RPMI medium. Samples were immediately provided to the inventors for identification of mycM28z1XXPD1DNR CAR T cells using flow cytometry (non-GLP).

Bioluminescence imaging. On study days -1/1, 7/8 and 14/15, groups of 17-20 animals were imaged using luciferin (dorsal/ventral) to assess tumor burden and study drug efficacy.

Statistical methods. Group means and standard deviations were calculated for body weight, complete blood count, and clinical chemistry. For complete blood count and clinical chemistry, the percentage difference between the mean of the study drug and corresponding vehicle groups was calculated at each time point. The statistical significance of these differences was analyzed using an unpaired t test and was considered statistically significant at p < 0.05. Statistical analyzes were performed using Ascentos preclinical laboratory information systems software version 1.3.4 developed by PDS Life Sciences (Mt. Arlington, NJ). A review of clinical data was performed by assessing individual potential outlier values using the 1.5×IQR criterion and laboratory-established reference parameter ranges. Any values identified as outliers were removed from statistical analysis.

C. Results

S.1. Mortality and disease severity

Previous work by the inventors has shown that control animals can succumb to tumor burden approximately 20-22 days after tumor injection. Two control animals in the imaging cohort (86 [group 17] and 88 [group 19]) were selectively sacrificed on study days 12 and 14 due to pathology and difficulty breathing. Macroscopic necropsy of each animal after sacrifice showed a significant tumor burden in the chest cavity surrounding the lungs and heart. Both animals were given control media and showed pathology within the expected window. Thus, although these killings were unplanned, they were not unexpected. No other mortality or pathology was observed in the animals in this study.

C.2. Body mass

A summary of the average body weight in each group is shown in Figures 40-43. Animals in group 9 (environment control) showed a progressive decrease in body weight during the study and a significant difference in weight compared to control animals without tumors (group 7) and animals in the study drug group (group 11) on day 14 of the study. This is explained by the increase in tumor burden in control animals (see Figure 42). Similarly, animals in group 10 (control environment) showed a significant difference in body weight compared to group 8 (control environment without tumor) and group 12 (test drug), from day 13 of the study until the end of the study (see Figure 43 ). It is also associated with tumor burden. All animals showed a decrease in body weight the day after surgery (day 2 for male mice and day 3 for female mice), which was attributed to surgery. Tumor-free control mice and mice treated with mycM28z1XXPD1DNR CAR T cells regained their original pre-surgery weight within 2-3 days after surgery and gradually gained weight throughout the remainder of the study, whereas mice treated with medium regained body weight there was little or no tumor after surgery, and the animals gradually lost weight while their tumor burden increased (see Figures 42 and 43).

C.3. Clinical signs

Animals in group 17 (control environment) showed signs of hair loss (85, 87) and flaking (87), which was associated with an aggressive caged animal. Once the animals were separated, the signs decreased, indicating that the observations were related to the animals fighting. Animal 86 exhibited difficulty breathing and decreased activity on day 12 of the study, which was associated with tumor burden and led to selective sacrifice. Animal 93 (group 19, study drug) exhibited mild desquamation; however, this was due to irritation caused by the surgical clamps as no other animal in the cage was harmed and the animals' activity was normal. Animal 88 (group 18, control environment) had red eyes 2 days before selective killing. This clinical sign could be an early sign of the animal's discomfort before selective killing due to tumor burden.

No other significant clinical signs were observed during this study; the mice appeared normal throughout the observation period.

C.4. General blood analysis

Group 12 (female mice, study drug) had a high mean percentage of monocytes (mean, 18.44%, n=5) (p=≤0.0001) compared to group 10 (tumor with control medium) (mean, 3.34%, n=5) . The reference range established for the percentage of monocytes is 0.9-18%. However, this did not correlate with microscopic findings.

No other significant or abnormal results were observed for the CBC parameters assessed. Any differences between the study drug-treated groups and the corresponding vehicle-treated groups were within normal reference ranges or were not biologically relevant or statistically significant.

C.5. Clinical biochemistry

Group 11 (male mice, study drug) had a low mean total protein (mean, 3.83 g/dL, n=5) (p=0.0022) compared to group 9 (control tumors) (mean, 4.68 g/dl, n=4). The reference range established for total protein is 4.1–6.4 g/dL. However, this did not correlate with microscopic findings.

No other adverse effects in terms of clinical biochemistry parameters were observed with the administration of the study drug. Any differences between the study drug groups and the matched vehicle groups were within normal reference ranges or were not biologically relevant or statistically significant.

S.6. Observations of macroscopic pathology

Tumor tissue was not collected at the time of necropsy, but was observed in many animals at interim sacrifice, as expected. At the time of final sacrifice, tumor burden was visible in animals receiving control media, but in most cases little or no tumor lesions were observed in animals receiving study drug. Additional findings obtained during the autopsy are detailed in Table 13 below.

Table 13. Macroscopic observations with correlation with microscopic results General observation Observed animal(s)
(group number) Result correlation Related to the study drug? Liver spots 11 (G3 - Control environment)
32 (G5 - Investigational drug)
59 (G10 - Control environment)
60 (G10 - Control environment)
71 (G12 - Investigational drug) Did not correlate with microscopic examination results No Reduced spleen 11 (G3 - Control environment)
16 (G3 - Control environment)
25 (G4 - Control environment)
30 (G5 - Investigational drug)
34 (G5 - Investigational drug)
40 (G6 - Investigational drug)
45 (G6 - Investigational drug)
56 (G9 - Control environment)
58 (G10 - Control environment)
59 (G10 - Control environment)
60 (G10 - Control environment)
65 (G11 - Investigational drug)
71 (G12 - Investigational drug) Lymphopenia is an expected component of the NSG mouse phenotype No Blue seminal vesicle 57 (G9 - Control environment) Did not correlate with microscopic examination results No Dark green gallbladder 107 (G9 - Control environment) Did not correlate with microscopic examination results No Blue gallbladder 62 (G10 - Control environment) Did not correlate with microscopic examination results No Oily bud, with spots 21 (G4 - Control environment)
71 (G12 - Investigational drug) Did not correlate with microscopic examination results No Blister in the liver 40 (G6 - Investigational drug) Did not correlate with microscopic examination results No White spot on the spleen 69 (G12 - Investigational drug) Moderate metaplasia - considered a spontaneous result No

No other macroscopic observations were noted at necropsy.

C.7. Histopathology

A review of the histopathological findings revealed that at interim (day 2) and final (day 14) sacrifice there were no microscopic findings associated with early or late toxicity resulting from study drug administration.

Microscopic findings in animals in groups 5 and 6 (test drug) included the presence of a mixed cellular infiltrate in tumor xenografts in the lungs. This was considered to be related to the administration of the study drug but was not due to any toxicity of the study drug. This result was confirmed in a separate study using immunofluorescent staining of mycM28z1XXPD1DNR CAR T cells in the primary tumor of intrapleurally treated mice (see Section 2.C.2 of this Example).

Any other results observed were determined to occur sporadically, at the same frequency as controls, or to be common for the species/strains used.

P.8. Cytokine analysis

Cytokine levels in plasma collected from groups 13-16 (8 days after study drug/vehicle administration) were measured to examine the toxicity of mycM28z1XXPD1DNR CAR T cells compared to vehicle controls on peripheral cytokine expression in treated mice. The following cytokines were analyzed: IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, KC/GRO and TNF-α.

The results of this assay demonstrated undetectable levels of IFN-γ, IL-12p70, IL-1β, IL-2, and IL-5 (below the limit of quantitation) in all animals, regardless of whether they received CAR T cells or control medium. IL-4 was detected at low levels and was the only cytokine that showed slightly higher levels in mice treated with CAR T cells than in mice treated with control media. Levels of all other cytokines that were detected above the limit of quantitation, such as IL-10, IL-6, KC/GRO, and TNF-α, were generally low and did not differ significantly between mice treated with CAR T cells and mice treated with CAR T cells. received control medium.

P.9. Identification of mycM28z1XXPD1DNR CAR T cells in tumor and spleen

To confirm mycM28z1XXPD1DNR CAR T cell injection, groups 13-16 were sacrificed 8 days after test drug/medium administration. The spleen and tumor were isolated, processed, and stained for human T cells (CD45+ CD3+) by flow cytometry. Human T cells were detected in tumor and spleen tissue from all mice treated with mycM28z1XXPD1DNR CAR T cells, but not in mice treated with medium (see Figures 44 and 45).

P.10. Bioluminescence Imaging

Whole body optical BLI (IVIS) was performed in mice in groups 17–20 to provide a qualitative assessment of MGM tumor burden. Images obtained on day -1/1 of the study showed successful tumor administration in all animals in each group. After administration of control medium, the tumor burden of animals in groups 17 (male mice) and 18 (female mice) increased at each measurement time point, with two animals requiring early imaging due to severe condition. After administration of the study drug, the tumor burden in animals in groups 19 (male mice) and 20 (female mice) increased 1 week after injection; however, after 2 weeks the load was significantly reduced. The imaging results of male and female mice are shown in Figures 46 and 47, respectively.

D. Discussion

Extensive pharmacological studies were performed to determine the antitumor efficacy of M28z1XXPD1DNR CAR T cells in vitro and in vivo. mycM28z1XXPD1DNR CAR T cells have been shown to kill target cells in a mesothelin-dependent and HLA-independent manner and accumulate and secrete effector cytokines upon antigen stimulation. A sophisticated antigen stress test using repeated antigen stimulation showed that mycM28z1XXPD1DNR CAR T cells were able to maintain cytotoxicity longer than mycM28z CAR T cells in the assay. In vivo, antigen rechallenge demonstrated greater functional persistence of mycM28z1XXPD1DNR CAR T cells compared with mycM28z CAR T cells, resulting in consistent tumor regression over a series of 10 tumor rechallenges with escalating tumor doses in mycM28z1XXPD1DNR CAR T-treated mice -cells, but ultimately the tumors progressed and recurred in mice treated with mycM28z CAR T cells.

Pharmacological studies in animals are more relevant to study the antitumor efficacy of CAR T cells because they cover important aspects of pharmacology and pharmacokinetics that cannot be studied in in vitro assays. Factors such as route of administration, migration to the tumor site, homing to lymphoid organs, systemic circulation, persistence in vivo and, despite the limitations associated with the use of an immunodeficient mouse strain, interaction with the tumor immune microenvironment ultimately modulate antitumor efficacy. . We have developed a translationally relevant orthotopic mouse model of pleural mesothelioma that resembles the human disease (Servais et al., Clin Cancer Res. 2012). We have shown that regional (intrapleural) administration of T cells significantly suppresses not only pleural disease but also disseminated tumor sites (Adusumilli et al., Sci Transl Med. 2014; 6(261): 261ra151). More importantly, the results observed in the ongoing clinical trial (IND 16354) reflect the authors' initial observations in an orthotopic mouse model of MPM, supporting the validity of the model in terms of clinical relevance (Adusumilli et al., Cancer Res. 2019;79(13) )). In this study, complete tumor eradication was observed with a single low dose of 3 x 10 ⁴ mycM28z1XXPD1DNR CAR T cells delivered regionally, and no evidence of targeted non-tumor toxic effects was observed. A toxicity study of a single orthotopic dose of 1x10 ⁵ mycM28z1XXPD1DNR CAR T cells in a xenograft mouse model of mesothelioma with a 2-week recovery period confirmed that mycM28z1XXPD1DNR CAR T cells were well tolerated. There were no significant results for mortality, clinical follow-up, changes in body weight or macroscopic examination. Microscopic examination revealed a mixed cellular infiltrate in tumor xenografts that was not observed in media-treated mice but is indicative of mycM28z1XXPD1DNR CAR T cell pharmacology. Treated male mice had a low mean total protein and treated female mice showed a high mean monocyte percentage at the 2 week time point, but neither of these values correlated with any microscopic findings.

CAR T cells, including mesothelin-targeted CAR T cells, have been evaluated in humans and were reported to be safe, with certain manageable side effects such as fever, chills, myalgias, hypersensitivity, and anaphylaxis from -for inflammation caused by T cells. The risk of replication-competent retroviruses will be minimized through extensive product testing. In addition, the patient's T cells will be tested every 12 weeks for up to 2 years or until the disease progresses. Patients will be monitored for survival for 15 years after receiving CAR T cells.

A combined assessment of these data was used to establish a safe starting dose for medical use and to support a dose escalation regimen.

Embodiments of the invention disclosed herein

It will be apparent from the above description that the invention disclosed herein may be subject to changes and modifications to adapt it to various applications and conditions. Such embodiments are also included within the scope of the following claims.

The listing of a list of elements in any variable definition herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The description of an embodiment herein includes this embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

All patents and publications referenced in this specification are incorporated herein by reference to the extent that each independent patent and publication were specifically and individually stated to be incorporated by reference.

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LIST OF SEQUENCES

<110> MEMORIAL SLOAN-KETTERING CANCER CENTER

<120> MESOTHELIN CAR RECEPTORS AND THEIR APPLICATION

<130> 072734.1041

<150> US 62/848,983

<151> 2019-05-16

<150>US 62/975,966

<151> 2020-02-13

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<213> Homo sapiens

<400> 7

Met Arg Pro Arg Leu Trp Leu Leu Leu Ala Ala Gln Leu Thr Val Leu

1 5 10 15

His Gly Asn Ser Val Leu Gln Gln Thr Pro Ala Tyr Ile Lys Val Gln

20 25 30

Thr Asn Lys Met Val Met Leu Ser Cys Glu Ala Lys Ile Ser Leu Ser

35 40 45

Asn Met Arg Ile Tyr Trp Leu Arg Gln Arg Gln Ala Pro Ser Ser Asp

50 55 60

Ser His His Glu Phe Leu Ala Leu Trp Asp Ser Ala Lys Gly Thr Ile

65 70 75 80

His Gly Glu Glu Val Glu Gln Glu Lys Ile Ala Val Phe Arg Asp Ala

85 90 95

Ser Arg Phe Ile Leu Asn Leu Thr Ser Val Lys Pro Glu Asp Ser Gly

100 105 110

Ile Tyr Phe Cys Met Ile Val Gly Ser Pro Glu Leu Thr Phe Gly Lys

115 120 125

Gly Thr Gln Leu Ser Val Val Asp Phe Leu Pro Thr Thr Ala Gln Pro

130 135 140

Thr Lys Lys Ser Thr Leu Lys Lys Arg Val Cys Arg Leu Pro Arg Pro

145 150 155 160

Glu Thr Gln Lys Gly Pro Leu Cys Ser Pro Ile Thr Leu Gly Leu Leu

165 170 175

Val Ala Gly Val Leu Val Leu Leu Val Ser Leu Gly Val Ala Ile His

180 185 190

Leu Cys Cys Arg Arg Arg Arg Ala Arg Leu Arg Phe Met Lys Gln Leu

195 200 205

Arg Leu His Pro Leu Glu Lys Cys Ser Arg Met Asp Tyr

210 215 220

<210> 8

<211> 75

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 8

atcacccttg gcctgctggt ggctggcgtc ctggttctgc tggtttccct gggagtggcc 60

atccacctgt gctgc 75

<210> 9

<211> 199

<212> PROTEIN

<213> Homo sapiens

<400> 9

Met Lys Ser Gly Leu Trp Tyr Phe Phe Leu Phe Cys Leu Arg Ile Lys

1 5 10 15

Val Leu Thr Gly Glu Ile Asn Gly Ser Ala Asn Tyr Glu Met Phe Ile

20 25 30

Phe His Asn Gly Gly Val Gln Ile Leu Cys Lys Tyr Pro Asp Ile Val

35 40 45

Gln Gln Phe Lys Met Gln Leu Leu Lys Gly Gly Gln Ile Leu Cys Asp

50 55 60

Leu Thr Lys Thr Lys Gly Ser Gly Asn Thr Val Ser Ile Lys Ser Leu

65 70 75 80

Lys Phe Cys His Ser Gln Leu Ser Asn Asn Ser Val Ser Phe Phe Leu

85 90 95

Tyr Asn Leu Asp His Ser His Ala Asn Tyr Tyr Phe Cys Asn Leu Ser

100 105 110

Ile Phe Asp Pro Pro Pro Phe Lys Val Thr Leu Thr Gly Gly Tyr Leu

115 120 125

His Ile Tyr Glu Ser Gln Leu Cys Cys Gln Leu Lys Phe Trp Leu Pro

130 135 140

Ile Gly Cys Ala Ala Phe Val Val Val Cys Ile Leu Gly Cys Ile Leu

145 150 155 160

Ile Cys Trp Leu Thr Lys Lys Lys Tyr Ser Ser Ser Val His Asp Pro

165 170 175

Asn Gly Glu Tyr Met Phe Met Arg Ala Val Asn Thr Ala Lys Lys Ser

180 185 190

Arg Leu Thr Asp Val Thr Leu

195

<210> 10

<211> 75

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 10

ttctggttac ccataggatg tgcagccttt gttgtagtct gcattttggg atgcatactt 60

atttgttggc ttaca 75

<210> 11

<211> 223

<212> PROTEIN

<213> Homo sapiens

<400> 11

Met Ala Cys Leu Gly Phe Gln Arg His Lys Ala Gln Leu Asn Leu Ala

1 5 10 15

Thr Arg Thr Trp Pro Cys Thr Leu Leu Phe Phe Leu Leu Phe Ile Pro

20 25 30

Val Phe Cys Lys Ala Met His Val Ala Gln Pro Ala Val Val Leu Ala

35 40 45

Ser Ser Arg Gly Ile Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly

50 55 60

Lys Ala Thr Glu Val Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln

65 70 75 80

Val Thr Glu Val Cys Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr

85 90 95

Phe Leu Asp Asp Ser Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val

100 105 110

Asn Leu Thr Ile Gln Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile

115 120 125

Cys Lys Val Glu Leu Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly

130 135 140

Asn Gly Thr Gln Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser

145 150 155 160

Asp Phe Leu Leu Trp Ile Leu Ala Ala Val Ser Ser Gly Leu Phe Phe

165 170 175

Tyr Ser Phe Leu Leu Thr Ala Val Ser Leu Ser Lys Met Leu Lys Lys

180 185 190

Arg Ser Pro Leu Thr Thr Gly Val Tyr Val Lys Met Pro Pro Thr Glu

195 200 205

Pro Glu Cys Glu Lys Gln Phe Gln Pro Tyr Phe Ile Pro Ile Asn

210 215 220

<210> 12

<211> 75

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 12

ttcctcctct ggatccttgc agcagttagt tcggggttgt ttttttatag ctttctcctc 60

acagctgttt ctttg 75

<210> 13

<211> 532

<212> PROTEIN

<213> Homo sapiens

<400> 13

Met Ala Pro Ser Ser Pro Arg Pro Ala Leu Pro Ala Leu Leu Val Leu

1 5 10 15

Leu Gly Ala Leu Phe Pro Gly Pro Gly Asn Ala Gln Thr Ser Val Ser

20 25 30

Pro Ser Lys Val Ile Leu Pro Arg Gly Gly Ser Val Leu Val Thr Cys

35 40 45

Ser Thr Ser Cys Asp Gln Pro Lys Leu Leu Gly Ile Glu Thr Pro Leu

50 55 60

Pro Lys Lys Glu Leu Leu Leu Pro Gly Asn Asn Arg Lys Val Tyr Glu

65 70 75 80

Leu Ser Asn Val Gln Glu Asp Ser Gln Pro Met Cys Tyr Ser Asn Cys

85 90 95

Pro Asp Gly Gln Ser Thr Ala Lys Thr Phe Leu Thr Val Tyr Trp Thr

100 105 110

Pro Glu Arg Val Glu Leu Ala Pro Leu Pro Ser Trp Gln Pro Val Gly

115 120 125

Lys Asn Leu Thr Leu Arg Cys Gln Val Glu Gly Gly Ala Pro Arg Ala

130 135 140

Asn Leu Thr Val Val Leu Leu Arg Gly Glu Lys Glu Leu Lys Arg Glu

145 150 155 160

Pro Ala Val Gly Glu Pro Ala Glu Val Thr Thr Thr Val Leu Val Arg

165 170 175

Arg Asp His His Gly Ala Asn Phe Ser Cys Arg Thr Glu Leu Asp Leu

180 185 190

Arg Pro Gln Gly Leu Glu Leu Phe Glu Asn Thr Ser Ala Pro Tyr Gln

195 200 205

Leu Gln Thr Phe Val Leu Pro Ala Thr Pro Pro Gln Leu Val Ser Pro

210 215 220

Arg Val Leu Glu Val Asp Thr Gln Gly Thr Val Val Cys Ser Leu Asp

225 230 235 240

Gly Leu Phe Pro Val Ser Glu Ala Gln Val His Leu Ala Leu Gly Asp

245 250 255

Gln Arg Leu Asn Pro Thr Val Thr Tyr Gly Asn Asp Ser Phe Ser Ala

260 265 270

Lys Ala Ser Val Ser Val Thr Ala Glu Asp Glu Gly Thr Gln Arg Leu

275 280 285

Thr Cys Ala Val Ile Leu Gly Asn Gln Ser Gln Glu Thr Leu Gln Thr

290 295 300

Val Thr Ile Tyr Ser Phe Pro Ala Pro Asn Val Ile Leu Thr Lys Pro

305 310 315 320

Glu Val Ser Glu Gly Thr Glu Val Thr Val Lys Cys Glu Ala His Pro

325 330 335

Arg Ala Lys Val Thr Leu Asn Gly Val Pro Ala Gln Pro Leu Gly Pro

340 345 350

Arg Ala Gln Leu Leu Leu Lys Ala Thr Pro Glu Asp Asn Gly Arg Ser

355 360 365

Phe Ser Cys Ser Ala Thr Leu Glu Val Ala Gly Gln Leu Ile His Lys

370 375 380

Asn Gln Thr Arg Glu Leu Arg Val Leu Tyr Gly Pro Arg Leu Asp Glu

385 390 395 400

Arg Asp Cys Pro Gly Asn Trp Thr Trp Pro Glu Asn Ser Gln Gln Thr

405 410 415

Pro Met Cys Gln Ala Trp Gly Asn Pro Leu Pro Glu Leu Lys Cys Leu

420 425 430

Lys Asp Gly Thr Phe Pro Leu Pro Ile Gly Glu Ser Val Thr Val Thr

435 440 445

Arg Asp Leu Glu Gly Thr Tyr Leu Cys Arg Ala Arg Ser Thr Gln Gly

450 455 460

Glu Val Thr Arg Lys Val Thr Val Asn Val Leu Ser Pro Arg Tyr Glu

465 470 475 480

Ile Val Ile Ile Thr Val Val Ala Ala Ala Val Ile Met Gly Thr Ala

485 490 495

Gly Leu Ser Thr Tyr Leu Tyr Asn Arg Gln Arg Lys Ile Lys Lys Tyr

500 505 510

Arg Leu Gln Gln Ala Gln Lys Gly Thr Pro Met Lys Pro Asn Thr Gln

515 520 525

Ala Thr Pro Pro

530

<210> 14

<211> 81

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 14

attgtcatca tcactgtggt agcagccgca gtcataatgg gcactgcagg cctcagcacg 60

tacctctata accgccagcg g 81

<210> 15

<211> 39

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 15

Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn

1 5 10 15

Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu

20 25 30

Phe Pro Gly Pro Ser Lys Pro

35

<210> 16

<211> 117

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 16

caagagctga cttacacgtg tacagcccag aaccctgtca gcaacaattc tgactccatc 60

tctgcccggc agctctgtgc agacatcgca atgggcttcc gtactcacca caccggg 117

<210> 17

<211> 116

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 17

accaactgga gagaacagta aactccttga atgtctctgc tataagtatt ccagaacacg 60

atgaggcaga cgagataagt gatgaaaaca gagaaaaggt gaatgaccag gcaaaa 116

<210> 18

<211> 138

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 18

cccaccacga cgccagcgcc gcgaccacca acaccggcgc cccacatcgc gtcgcagccc 60

ctgtccctgc gcccagaggc gtgccggcca gcggcggggg gcgcagtgca cacgagggg 120

ctggacttcg cctgtgat 138

<210> 19

<211> 117

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 19

ctgagtgtgg ttgatttcct tcccaccact gcccagccca ccaagaagtc caccctcaag 60

aagagagtgt gccggttacc caggccagag acccagaagg gcccactttg tagcccc 117

<210> 20

<211> 117

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 20

tctcatgcca actattactt ctgcaaccta tcaatttttg atcctcctcc ttttaaagta 60

actcttacag gaggatattt gcatatttat gaatcacaac tttgttgcca gctgaag 117

<210> 21

<211> 117

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 21

gacacgggac tctacatctg caaggtggag ctcatgtacc caccgccata ctacctgggc 60

ataggcaacg gaacccagat ttatgtaatt gatccagaac cgtgcccaga ttctgac 117

<210> 22

<211> 117

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 22

ggggaatcag tgactgtcac tcgagatctt gagggcacct acctctgtcg ggccaggagc 60

actcaagggg aggtcacccg caaggtgacc gtgaatgtgc tctccccccg gtatgag 117

<210> 23

<211> 22

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 23

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

1 5 10 15

Asp Val Leu Asp Lys Arg

20

<210> 24

<211> 66

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 24

cagaaccagc tctataacga gctcaatcta ggacgaagag aggagtacga tgttttggac 60

aagaga 66

<210> 25

<211> 22

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 25

Gln Asn Gln Leu Phe Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Phe

1 5 10 15

Asp Val Leu Asp Lys Arg

20

<210> 26

<211> 66

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 26

cagaaccagc tctttaacga gctcaatcta ggacgaagag aggagttcga tgttttggac 60

aagaga 66

<210> 27

<211> 23

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 27

Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala

1 5 10 15

Tyr Ser Glu Ile Gly Met Lys

20

<210> 28

<211> 69

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 28

caggaaggcc tgtacaatga actgcagaaa gataagatgg cggaggccta cagtgagatt 60

gggatgaaa 69

<210> 29

<211> 23

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 29

Gln Glu Gly Leu Phe Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala

1 5 10 15

Phe Ser Glu Ile Gly Met Lys

20

<210> 30

<211> 69

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 30

caggaaggcc tgttcaatga actgcagaaa gataagatgg cggaggcctt cagtgagatt 60

gggatgaaa 69

<210> 31

<211> 22

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 31

His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr

1 5 10 15

Asp Ala Leu His Met Gln

20

<210> 32

<211> 66

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 32

cacgatggcc tttaccaggg tctcagtaca gccaccaagg acacctacga cgcccttcac 60

atgcag 66

<210> 33

<211> 22

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 33

His Asp Gly Leu Phe Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Phe

1 5 10 15

Asp Ala Leu His Met Gln

20

<210> 34

<211> 66

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 34

cacgatggcc ttttccaggg gctcagtaca gccaccaagg acaccttcga cgcccttcac 60

atgcag 66

<210> 35

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 35

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Phe Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Phe Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Phe Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Phe Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 36

<211> 230

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 36

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Val Ser Ser Gly

20 25 30

Ser Tyr Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Glu Gly Lys Asn Gly Ala Phe Asp Ile Trp Gly Gln Gly

100 105 110

Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr Ser Gly Gln Ala Gly

225 230

<210> 37

<211> 693

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 37

caggtgcagc tgcaggagtc cggcccagga ctggtgaagc cttcggagac cctgtccctc 60

acctgcactg tctctggtgg ctccgtcagc agtggtagtt actactggag ctggatccgg 120

cagcccccag ggaagggact ggagtggatt gggtatatct attacagtgg gagcaccaac 180

tacaacccct ccctcaagag tcgagtcacc atatcagtag acacgtccaa gaaccagttc 240

tccctgaagc tgagctctgt gaccgctgcg gacacggccg tgtattactg tgcgagagag 300

gggaagaatg gggcttttga tatctggggc caagggacaa tggtcaccgt ctcttcagcc 360

tccaccaagg gcccatcggt cttccccctg gcaccctcct ccaagagcac ctctgggggc 420

acagcggccc tgggctgcct ggtcaaggac tacttccccg aaccggtgac ggtgtcgtgg 480

aactcaggcg ccctgaccag cggcgtgcac accttcccgg ctgtcctaca gtcctcagga 540

ctctactccc tcagcagcgt ggtgaccgtg ccctccagca gcttgggcac ccagacctac 600

atctgcaacg tgaatcacaa gcccagcaac accaaggtgg acaagaaagt tgagcccaaa 660

tcttgtgaca aaactagtgg ccaggccggc cac 693

<210> 38

<211> 214

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 38

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Gly Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 39

<211> 640

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 39

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

gggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240

gaagattttg caacttacta ctgtcaacag agttacagta ccccgctcac tttcggcgga 300

gggaccaagg tggagatcaa acgaactgtg gctgcaccat ctgtcttcat cttcccgcca 360

tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 420

cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 480

gagagtgtca cagagcagga cagcaaggac agcacctact gcctcagcag caccctgacg 540

ctgagcaaag cagactacga gaaacacaaa ctctacgcct gcgaagtcac ccatcagggc 600

ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt 640

<210> 40

<211> 214

<212> PROTEIN

<213> Homo sapiens

<400> 40

Arg His Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Gly Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 41

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 41

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Val Ser Ser Gly

20 25 30

Ser Tyr Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Glu Gly Lys Asn Gly Ala Phe Asp Ile Trp Gly Gln Gly

100 105 110

Thr Met Val Thr Val Ser Ser Ser

115 120

<210> 42

<211> 262

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 42

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Val Ser Ser Gly

20 25 30

Ser Tyr Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Glu Gly Lys Asn Gly Ala Phe Asp Ile Trp Gly Gln Gly

100 105 110

Thr Met Val Thr Val Ser Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Arg His Gln Met Thr Gln Ser Pro Ser

130 135 140

Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala

145 150 155 160

Ser Gln Ser Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly

165 170 175

Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly

180 185 190

Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu

195 200 205

Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln

210 215 220

Gln Ser Tyr Ser Thr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu

225 230 235 240

Ile Lys Gly Gln Ala Gly His His His His His Gly Asp Tyr Lys

245 250 255

Asp Asp Asp Asp Lys Gly

260

<210> 43

<211> 486

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 43

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu

20 25 30

Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly

35 40 45

Ser Val Ser Ser Gly Ser Tyr Tyr Trp Ser Trp Ile Arg Gln Pro Pro

50 55 60

Gly Lys Gly Leu Glu Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr

65 70 75 80

Asn Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr

85 90 95

Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp

100 105 110

Thr Ala Val Tyr Tyr Cys Ala Arg Glu Gly Lys Asn Gly Ala Phe Asp

115 120 125

Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Gly Gly Gly Gly

130 135 140

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg His Gln Met Thr

145 150 155 160

Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile

165 170 175

Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn Trp Tyr Gln

180 185 190

Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser

195 200 205

Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr

210 215 220

Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr

225 230 235 240

Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu Thr Phe Gly Gly Gly

245 250 255

Thr Lys Val Glu Ile Lys Arg Thr Ala Ala Ala Ile Glu Val Met Tyr

260 265 270

Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn Gly Thr Ile Ile His

275 280 285

Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu Phe Pro Gly Pro Ser

290 295 300

Lys Pro Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr

305 310 315 320

Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys

325 330 335

Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg

340 345 350

Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp

355 360 365

Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala

370 375 380

Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu

385 390 395 400

Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp

405 410 415

Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu

420 425 430

Phe Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Phe Ser Glu Ile

435 440 445

Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Phe

450 455 460

Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Phe Asp Ala Leu His Met

465 470 475 480

Gln Ala Leu Pro Pro Arg

485

<210> 44

<211> 1458

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 44

atggccctgc cagtaacggc tctgctgctg ccacttgctc tgctcctcca tgcagccagg 60

cctcaggttc agcttcagga gagtggccca ggcctggtga agccaagtga gactctcagc 120

ttgacttgca cagtttctgg aggcagtgtc tcctcaggca gctattattg gtcctggatt 180

cggcagcccc ctgggaaagg cctggagtgg attgggtaca tatattacag tggcagcaca 240

aattacaatc catccctgaa gtctcgagta actatcagtg tggacacaag caagaatcag 300

ttttcactca aactgtcttc tgtgactgct gctgacactg ctgtttatta ttgtgccagg 360

gaggggaaaa atggggcatt tgatatttgg ggtcagggca caatggtgac agtcagctct 420

ggaggtggag gctcaggagg aggaggcagt ggaggtggtg ggtcacgcca tcagatgact 480

cagtccccct ccagtctttc tgcctcagtt ggggatagag tgaccatcac atgcagagca 540

agtcagagca tatcatccta tctgaactgg taccagcaga agccagggaa agcccccaaa 600

ttgctgattt atgcagcctc aagtctccag agtggggtgc caagcaggtt ctcaggcagt 660

ggcagtggga cagatttcac attgacaatc agctccctcc aacctgaaga ttttgccacc 720

tactattgcc agcaatccta cagcacgccc ctgacttttg gaggtggcac aaaggtagag 780

atcaagagga ctgcggccgc aattgaagtt atgtatcctc ctccttacct agacaatgag 840

aagagcaatg gaaccattat ccatgtgaaa gggaaacacc tttgtccaag tcccctattt 900

cccggacctt ctaagccctt ttgggtgctg gtggtggttg gtggagtcct ggcttgctat 960

agcttgctag taacagtggc ctttattatt ttctgggtga ggagtaagag gagcaggctc 1020

ctgcacagtg actacatgaa catgactccc cgccgccccg ggcccacccg caagcattac 1080

cagccctatg ccccaccacg cgacttcgca gcctatcgct ccagagtgaa gttcagcagg 1140

agcgcagacg cccccgcgta ccagcagggc cagaaccagc tctataacga gctcaatcta 1200

ggacgaagag aggagtacga tgttttggac aagagacgtg gccgggaccc tgagatgggg 1260

ggaaagccga gaaggaagaa ccctcaggaa ggcctgttca atgaactgca gaaagataag 1320

atggcggagg ccttcagtga gattgggatg aaaggcgagc gccggagggg caaggggcac 1380

gatggccttt tccaggggct cagtacagcc accaaggaca ccttcgacgc ccttcacatg 1440

caggccctgc cccctcgc 1458

<210> 45

<211> 801

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 45

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccgcaggtgc agctgcagga gtccggccca ggactggtga agccttcgga gaccctgtcc 120

ctcacctgca ctgtctctgg tggctccgtc agcagtggta gttactactg gagctggatc 180

cggcagcccc cagggaaggg actggagtgg attgggtata tctattacag tgggagcacc 240

aactacaacc cctccctcaa gagtcgagtc accatatcag tagacacgtc caagaaccag 300

ttctccctga agctgagctc tgtgaccgct gcggacacgg ccgtgtatta ctgtgcgaga 360

gaggggaaga atggggcttt tgatatctgg ggccaaggga caatggtcac cgtctcttca 420

ggtggaggcg gttcaggcgg aggtggctct ggcggtggcg gatcacgaca tcagatgacc 480

cagtctccat cctccctgtc tgcatctgta ggagacagag tcaccatcac ttgccgggca 540

agtcagagca ttagcagcta tttaaattgg tatcagcaga aaccagggaa agcccctaag 600

ctcctgatct atgctgcatc cagtttgcaa agtggggtcc catcaaggtt cagtggcagt 660

ggatctggga cagatttcac tctcaccatc agcagtctgc aacctgaaga ttttgcaact 720

tactactgtc aacagagtta cagtaccccg ctcactttcg gcggagggac caaggtggag 780

atcaaacgga ctgcggccgc a 801

<210> 46

<211> 801

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 46

atggcgctgc cggtgaccgc gctgctgctg ccgctggcgc tgctgctgca tgcggcgcgc 60

ccgcaggtgc agctgcagga aagcggcccg ggcctggtga aaccgagcga aaccctgagc 120

ctgacctgca ccgtgagcgg cggcagcgtg agcagcggca gctattattg gagctggatt 180

cgccagccgc cgggcaaagg cctggaatgg attggctata tttattatag cggcagcacc 240

aactataacc cgagcctgaa aagccgcgtg accattagcg tggataccag caaaaaccag 300

tttagcctga aactgagcag cgtgaccgcg gcggataccg cggtgtatta ttgcgcgcgc 360

gaaggcaaaa acggcgcgtt tgatatttgg ggccagggca ccatggtgac cgtgagcagc 420

ggcggcggcg gcagcggcgg cggcggcagc ggcggcggcg gcagccgcca tcagatgacc 480

cagagcccga gcagcctgag cgcgagcgtg ggcgatcgcg tgaccattac ctgccgcgcg 540

agccagagca ttagcagcta tctgaactgg tatcagcaga aaccgggcaa agcgccgaaa 600

ctgctgattt atgcggcgag cagcctgcag agcggcgtgc cgagccgctt tagcggcagc 660

ggcagcggca ccgattttac cctgaccatt agcagcctgc agccggaaga ttttgcgacc 720

tattattgcc agcagagcta tagcaccccg ctgacctttg gcggcggcac caaagtggaa 780

attaaacgca ccgcggcggc g 801

<210> 47

<211> 801

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 47

atggccctcc cggtaacggc tctgctgctt ccactcgcac tgctcttgca tgctgccaga 60

ccacaggtcc agctgcagga gagtgggcct ggactggtta agccgagtga gacactttcc 120

ttgacgtgca ctgtgagcgg gggaagtgtg tcctcaggta gttattactg gtcctggatt 180

cgccagccac caggaaaggg actggagtgg ataggttata tctattattc tggcagcact 240

aattacaatc cttctctcaa aagtagggtg acaatttcag tggatacttc caaaaatcag 300

tttagtctga agctcagctc tgtgacagct gctgatactg cagtttacta ctgcgccagg 360

gaggggaaga atggcgcctt cgatatttgg ggacagggca ctatggtgac tgtatcaagc 420

ggaggcggtg gcagcggcgg gggagggagt ggaggcggcg ggtctcgaca tcagatgaca 480

cagagcccat catcacttag cgccagcgtt ggcgaccggg ttacgataac atgcagggct 540

tcccaatcta tcagttctta tctgaactgg tatcagcaga aaccaggtaa ggcccccaag 600

ctgctcatct acgcagcctc atccctgcag agcggcgtcc ctagtcgatt ttccggtagt 660

gggtcaggga cagattttac cctgactatc agttcactgc agcccgagga cttcgcgaca 720

tactattgcc aacagtccta tagtacaccc ttgacatttg gcggcgggac taaagtagaa 780

attaaacgca ccgcggccgc a 801

<210> 48

<211> 288

<212> PROTEIN

<213> Homo sapiens

<400> 48

Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln

1 5 10 15

Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp

20 25 30

Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp

35 40 45

Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val

50 55 60

Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala

65 70 75 80

Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg

85 90 95

Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg

100 105 110

Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu

115 120 125

Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val

130 135 140

Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro

145 150 155 160

Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly

165 170 175

Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Cys

180 185 190

Ser Arg Ala Ala Arg Gly Thr Ile Gly Ala Arg Arg Thr Gly Gln Pro

195 200 205

Leu Lys Glu Asp Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr Gly

210 215 220

Glu Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val Pro

225 230 235 240

Cys Val Pro Glu Gln Thr Glu Tyr Ala Thr Ile Val Phe Pro Ser Gly

245 250 255

Met Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro Arg

260 265 270

Ser Ala Gln Pro Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro Leu

275 280 285

<210> 49

<211> 239

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 49

Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln

1 5 10 15

Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp

20 25 30

Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp

35 40 45

Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val

50 55 60

Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala

65 70 75 80

Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg

85 90 95

Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg

100 105 110

Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu

115 120 125

Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val

130 135 140

Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro

145 150 155 160

Arg Pro Ala Gly Gln Ala Ala Ala Pro Thr Thr Thr Pro Ala Pro Arg

165 170 175

Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg

180 185 190

Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly

195 200 205

Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr

210 215 220

Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Asn

225 230 235

<210> 50

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 50

ggaggtggag gctcaggagg aggaggcagt ggaggtggtg ggtca 45

<210> 51

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 51

ggtggaggcg gttcaggcgg aggtggctct ggcggtggcg gatca 45

<210> 52

<211> 357

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 52

Cys Ala Gly Gly Thr Thr Cys Ala Gly Cys Thr Thr Cys Ala Gly Gly

1 5 10 15

Ala Gly Ala Gly Thr Gly Gly Cys Cys Cys Ala Gly Gly Cys Cys Thr

20 25 30

Gly Gly Thr Gly Ala Ala Gly Cys Cys Ala Ala Gly Thr Gly Ala Gly

35 40 45

Ala Cys Thr Cys Thr Cys Ala Gly Cys Thr Thr Gly Ala Cys Thr Thr

50 55 60

Gly Cys Ala Cys Ala Gly Thr Thr Thr Cys Thr Gly Gly Ala Gly Gly

65 70 75 80

Cys Ala Gly Thr Gly Thr Cys Thr Cys Cys Thr Cys Ala Gly Gly Cys

85 90 95

Ala Gly Cys Thr Ala Thr Thr Ala Thr Thr Gly Gly Thr Cys Cys Thr

100 105 110

Gly Gly Ala Thr Thr Cys Gly Gly Cys Ala Gly Cys Cys Cys Cys Cys

115 120 125

Thr Gly Gly Gly Ala Ala Ala Gly Gly Cys Cys Thr Gly Gly Ala Gly

130 135 140

Thr Gly Gly Ala Thr Thr Gly Gly Gly Thr Ala Cys Ala Thr Ala Thr

145 150 155 160

Ala Thr Thr Ala Cys Ala Gly Thr Gly Gly Cys Ala Gly Cys Ala Cys

165 170 175

Ala Ala Ala Thr Thr Ala Cys Ala Ala Thr Cys Cys Ala Thr Cys Cys

180 185 190

Cys Thr Gly Ala Ala Gly Thr Cys Thr Cys Gly Ala Gly Thr Ala Ala

195 200 205

Cys Thr Ala Thr Cys Ala Gly Thr Gly Thr Gly Gly Ala Cys Ala Cys

210 215 220

Ala Ala Gly Cys Ala Ala Gly Ala Ala Thr Cys Ala Gly Thr Thr Thr

225 230 235 240

Thr Cys Ala Cys Thr Cys Ala Ala Ala Cys Thr Gly Thr Cys Thr Thr

245 250 255

Cys Thr Gly Thr Gly Ala Cys Thr Gly Cys Thr Gly Cys Thr Gly Ala

260 265 270

Cys Ala Cys Thr Gly Cys Thr Gly Thr Thr Thr Ala Thr Thr Ala Thr

275 280 285

Thr Gly Thr Gly Cys Cys Ala Gly Gly Gly Ala Gly Gly Gly Gly Ala

290 295 300

Ala Ala Ala Ala Thr Gly Gly Gly Gly Cys Ala Thr Thr Thr Gly Ala

305 310 315 320

Thr Ala Thr Thr Thr Gly Gly Gly Gly Thr Cys Ala Gly Gly Gly Cys

325 330 335

Ala Cys Ala Ala Thr Gly Gly Thr Gly Ala Cys Ala Gly Thr Cys Ala

340 345 350

Gly Cys Thr Cys Thr

355

<210> 53

<211> 327

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 53

cgccatcaga tgactcagtc cccctccagt ctttctgcct cagttgggga tagagtgacc 60

atcacatgca gagcaagtca gagcatatca tcctatctga actggtacca gcagaagcca 120

gggaaagccc ccaaattgct gatttatgca gcctcaagtc tccagagtgg ggtgccaagc 180

aggttctcag gcagtggcag tgggacagat ttcacattga caatcagctc cctccaacct 240

gaagattttg ccacctacta ttgccagcaa tcctacagca cgcccctgac ttttggaggt 300

ggcacaaagg tagagatcaa gaggact 327

<210> 54

<211> 117

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 54

attgaagtta tgtatcctcc tccttaccta gacaatgaga agagcaatgg aaccattatc 60

catgtgaaag ggaaacacct ttgtccaagt cccctatttc ccggaccttc taagccc 117

<210> 55

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 55

agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 60

tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120

cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgttcaat 180

gaactgcaga aagataagat ggcggaggcc ttcagtgaga ttgggatgaa aggcgagcgc 240

cggaggggca aggggcacga tggccttttc caggggctca gtacagccac caaggacacc 300

ttcgacgccc ttcacatgca ggccctgccc cctcgc 336

<210> 56

<211> 465

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 56

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Val Ser Ser Gly

20 25 30

Ser Tyr Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Glu Gly Lys Asn Gly Ala Phe Asp Ile Trp Gly Gln Gly

100 105 110

Thr Met Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

Ser Gly Gly Gly Gly Ser Arg His Gln Met Thr Gln Ser Pro Ser Ser

130 135 140

Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser

145 150 155 160

Gln Ser Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys

165 170 175

Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val

180 185 190

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

195 200 205

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

210 215 220

Ser Tyr Ser Thr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

225 230 235 240

Lys Arg Thr Ala Ala Ala Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu

245 250 255

Asp Asn Glu Lys Ser Asn Gly Thr Ile Ile His Val Lys Gly Lys His

260 265 270

Leu Cys Pro Ser Pro Leu Phe Pro Gly Pro Ser Lys Pro Phe Trp Val

275 280 285

Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr

290 295 300

Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu

305 310 315 320

His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg

325 330 335

Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg

340 345 350

Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln

355 360 365

Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu

370 375 380

Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly

385 390 395 400

Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Phe Asn Glu Leu Gln

405 410 415

Lys Asp Lys Met Ala Glu Ala Phe Ser Glu Ile Gly Met Lys Gly Glu

420 425 430

Arg Arg Arg Gly Lys Gly His Asp Gly Leu Phe Gln Gly Leu Ser Thr

435 440 445

Ala Thr Lys Asp Thr Phe Asp Ala Leu His Met Gln Ala Leu Pro Pro

450 455 460

Arg

465

<210> 57

<211> 1395

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 57

caggttcagc ttcaggagag tggcccaggc ctggtgaagc caagtgagac tctcagcttg 60

acttgcacag tttctggagg cagtgtctcc tcaggcagct attattggtc ctggattcgg 120

cagccccctg ggaaaggcct ggagtggatt gggtacatat attacagtgg cagcacaaat 180

tacaatccat ccctgaagtc tcgagtaact atcagtgtgg acacaagcaa gaatcagttt 240

tcactcaaac tgtcttctgt gactgctgct gacactgctg tttattattg tgccagggag 300

gggaaaaatg gggcatttga tatttggggt cagggcacaa tggtgacagt cagctctgga 360

ggtggaggct caggaggagg aggcagtgga ggtggtggggt cacgccatca gatgactcag 420

tccccctcca gtctttctgc ctcagttggg gatagagtga ccatcacatg cagagcaagt 480

cagagcatat catcctatct gaactggtac cagcagaagc cagggaaagc ccccaaattg 540

ctgatttatg cagcctcaag tctccagagt ggggtgccaa gcaggttctc aggcagtggc 600

agtgggacag atttcacatt gacaatcagc tccctccaac ctgaagattt tgccacctac 660

tattgccagc aatcctacag cacgcccctg acttttggag gtggcacaaa ggtagagatc 720

aagaggactg cggccgcaat tgaagttatg tatcctcctc cttacctaga caatgagaag 780

agcaatggaa ccattatcca tgtgaaaggg aaacaccttt gtccaagtcc cctatttccc 840

ggaccttcta agcccttttg ggtgctggtg gtggttggtg gagtcctggc ttgctatagc 900

ttgctagtaa cagtggcctt tattattttc tgggtgagga gtaagaggag caggctcctg 960

cacagtgact acatgaacat gactccccgc cgccccgggc ccacccgcaa gcattaccag 1020

ccctatgccc caccacgcga cttcgcagcc tatcgctcca gagtgaagtt cagcaggagc 1080

gcagacgccc ccgcgtacca gcagggccag aaccagctct ataacgagct caatctagga 1140

cgaagagagg agtacgatgt tttggacaag agacgtggcc gggaccctga gatgggggga 1200

aagccgagaa ggaagaaccc tcaggaaggc ctgttcaatg aactgcagaa agataagatg 1260

gcggaggcct tcagtgagat tgggatgaaa ggcgagcgcc ggaggggcaa ggggcacgat 1320

ggccttttcc aggggctcag tacagccacc aaggacacct tcgacgccct tcacatgcag 1380

gccctgcccc ctcgc 1395

<210> 58

<211> 145

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 58

Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr

1 5 10 15

Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn Ala Thr Phe

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln

145

<210> 59

<211> 435

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 59

ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt ctccccagcc 60

ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct gcagcttctc caacacatcg 120

gagagcttcg tgctaaactg gtaccgcatg agccccagca acgacgga caagctggcc 180

gctttccccg aggaccgcag ccagcccggc caggactgcc gcttccgtgt cacacaactg 240

cccaacgggc gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc 300

tacctctgtg gggccatctc cctggccccc aaggcgcaga tcaaagagag cctgcgggca 360

gagctcaggg tgacagagag aagggcagaa gtgcccacag cccaccccag cccctcaccc 420

aggccagccg gccag 435

<210> 60

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 60

atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact gggctggcgg 60

<210> 61

<211> 495

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 61

atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact gggctggcgg 60

ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt ctccccagcc 120

ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct gcagcttctc caacacatcg 180

gagagcttcg tgctaaactg gtaccgcatg agccccagca acgacgga caagctggcc 240

gctttccccg aggaccgcag ccagcccggc caggactgcc gcttccgtgt cacacaactg 300

cccaacgggc gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc 360

tacctctgtg gggccatctc cctggccccc aaggcgcaga tcaaagagag cctgcgggca 420

gagctcaggg tgacagagag aagggcagaa gtgcccacag cccaccccag cccctcaccc 480

aggccagccg gccag 495

<210> 62

<211> 213

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 62

cccaccacga cgccagcgcc gcgaccacca accccggcgc ccacgatcgc gtcgcagccc 60

ctgtccctgc gcccagaggc gtgccggcca gcggcggggg gcgcagtgca cacgagggg 120

ctggacttcg cctgtgatat ctacatctgg gcgcccctgg ccgggacttg tggggtcctt 180

ctcctgtcac tggttatcac cctttactgc aac 213

<210> 63

<211> 219

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 63

cccaccacga cgccagcgcc gcgaccacca accccggcgc ccacgatcgc gtcgcagccc 60

ctgtccctgc gcccagaggc gtgccggcca gcggcggggg gcgcagtgca cacgagggg 120

ctggacttcg cctgtgatat ctacatctgg gcgcccctgg ccgggacttg tggggtcctt 180

ctcctgtcac tggttatcac cctttactgc aaccacagg 219

<210> 64

<211> 717

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 64

atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact gggctggcgg 60

ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt ctccccagcc 120

ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct gcagcttctc caacacatcg 180

gagagcttcg tgctaaactg gtaccgcatg agccccagca acgacgga caagctggcc 240

gctttccccg aggaccgcag ccagcccggc caggactgcc gcttccgtgt cacacaactg 300

cccaacgggc gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc 360

tacctctgtg gggccatctc cctggccccc aaggcgcaga tcaaagagag cctgcgggca 420

gagctcaggg tgacagagag aagggcagaa gtgcccacag cccaccccag cccctcaccc 480

aggccagccg gccaggcggc cgcacccacc acgacgccag cgccgcgacc accaaccccg 540

gcgcccacga tcgcgtcgca gcccctgtcc ctgcgcccag aggcgtgccg gccagcggcg 600

ggggggcgcag tgcacacgag ggggctggac ttcgcctgtg atatctacat ctgggcgccc 660

ctggccggga cttgtggggt ccttctcctg tcactggtta tcacccttta ctgcaac 717

<210> 65

<211> 199

<212> PROTEIN

<213> Homo sapiens

<400> 65

Met Lys Ser Gly Leu Trp Tyr Phe Phe Leu Phe Cys Leu Arg Ile Lys

1 5 10 15

Val Leu Thr Gly Glu Ile Asn Gly Ser Ala Asn Tyr Glu Met Phe Ile

20 25 30

Phe His Asn Gly Gly Val Gln Ile Leu Cys Lys Tyr Pro Asp Ile Val

35 40 45

Gln Gln Phe Lys Met Gln Leu Leu Lys Gly Gly Gln Ile Leu Cys Asp

50 55 60

Leu Thr Lys Thr Lys Gly Ser Gly Asn Thr Val Ser Ile Lys Ser Leu

65 70 75 80

Lys Phe Cys His Ser Gln Leu Ser Asn Asn Ser Val Ser Phe Phe Leu

85 90 95

Tyr Asn Leu Asp His Ser His Ala Asn Tyr Tyr Phe Cys Asn Leu Ser

100 105 110

Ile Phe Asp Pro Pro Pro Phe Lys Val Thr Leu Thr Gly Gly Tyr Leu

115 120 125

His Ile Tyr Glu Ser Gln Leu Cys Cys Gln Leu Lys Phe Trp Leu Pro

130 135 140

Ile Gly Cys Ala Ala Phe Val Val Val Cys Ile Leu Gly Cys Ile Leu

145 150 155 160

Ile Cys Trp Leu Thr Lys Lys Lys Tyr Ser Ser Ser Val His Asp Pro

165 170 175

Asn Gly Glu Tyr Met Phe Met Arg Ala Val Asn Thr Ala Lys Lys Ser

180 185 190

Arg Leu Thr Asp Val Thr Leu

195

<210> 66

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 66

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 67

<211> 20

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 67

Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu

1 5 10 15

Val Thr Asn Ser

20

<210> 68

<211> 20

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 68

Met Tyr Ser Met Gln Leu Ala Ser Cys Val Thr Leu Thr Leu Val Leu

1 5 10 15

Leu Val Asn Ser

20

<210> 69

<211> 20

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 69

Met Glu Thr Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro

1 5 10 15

Asp Thr Thr Gly

20

<210> 70

<211> 20

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 70

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly

20

<210> 71

<211> 21

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 71

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro

20

<210> 72

<211> 18

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 72

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala

<210> 73

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 73

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Ser Ser Ala Tyr Ser

1 5 10 15

<210> 74

<211> 30

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 74

Met Asp Ser Lys Gly Ser Ser Gln Lys Gly Ser Arg Leu Leu Leu Leu

1 5 10 15

Leu Val Val Ser Asn Leu Leu Leu Cys Gln Gly Val Val Ser

20 25 30

<210> 75

<211> 622

<212> PROTEIN

<213> Homo sapiens

<400> 75

Met Ala Leu Pro Thr Ala Arg Pro Leu Leu Gly Ser Cys Gly Thr Pro

1 5 10 15

Ala Leu Gly Ser Leu Leu Phe Leu Leu Phe Ser Leu Gly Trp Val Gln

20 25 30

Pro Ser Arg Thr Leu Ala Gly Glu Thr Gly Gln Glu Ala Ala Pro Leu

35 40 45

Asp Gly Val Leu Ala Asn Pro Pro Asn Ile Ser Ser Leu Ser Pro Arg

50 55 60

Gln Leu Leu Gly Phe Pro Cys Ala Glu Val Ser Gly Leu Ser Thr Glu

65 70 75 80

Arg Val Arg Glu Leu Ala Val Ala Leu Ala Gln Lys Asn Val Lys Leu

85 90 95

Ser Thr Glu Gln Leu Arg Cys Leu Ala His Arg Leu Ser Glu Pro Pro

100 105 110

Glu Asp Leu Asp Ala Leu Pro Leu Asp Leu Leu Leu Phe Leu Asn Pro

115 120 125

Asp Ala Phe Ser Gly Pro Gln Ala Cys Thr His Phe Phe Ser Arg Ile

130 135 140

Thr Lys Ala Asn Val Asp Leu Leu Pro Arg Gly Ala Pro Glu Arg Gln

145 150 155 160

Arg Leu Leu Pro Ala Ala Leu Ala Cys Trp Gly Val Arg Gly Ser Leu

165 170 175

Leu Ser Glu Ala Asp Val Arg Ala Leu Gly Gly Leu Ala Cys Asp Leu

180 185 190

Pro Gly Arg Phe Val Ala Glu Ser Ala Glu Val Leu Leu Pro Arg Leu

195 200 205

Val Ser Cys Pro Gly Pro Leu Asp Gln Asp Gln Gln Glu Ala Ala Arg

210 215 220

Ala Ala Leu Gln Gly Gly Gly Pro Pro Tyr Gly Pro Pro Ser Thr Trp

225 230 235 240

Ser Val Ser Thr Met Asp Ala Leu Arg Gly Leu Leu Pro Val Leu Gly

245 250 255

Gln Pro Ile Ile Arg Ser Ile Pro Gln Gly Ile Val Ala Ala Trp Arg

260 265 270

Gln Arg Ser Ser Arg Asp Pro Ser Trp Arg Gln Pro Glu Arg Thr Ile

275 280 285

Leu Arg Pro Arg Phe Arg Arg Glu Val Glu Lys Thr Ala Cys Pro Ser

290 295 300

Gly Lys Lys Ala Arg Glu Ile Asp Glu Ser Leu Ile Phe Tyr Lys Lys

305 310 315 320

Trp Glu Leu Glu Ala Cys Val Asp Ala Ala Leu Leu Ala Thr Gln Met

325 330 335

Asp Arg Val Asn Ala Ile Pro Phe Thr Tyr Glu Gln Leu Asp Val Leu

340 345 350

Lys His Lys Leu Asp Glu Leu Tyr Pro Gln Gly Tyr Pro Glu Ser Val

355 360 365

Ile Gln His Leu Gly Tyr Leu Phe Leu Lys Met Ser Pro Glu Asp Ile

370 375 380

Arg Lys Trp Asn Val Thr Ser Leu Glu Thr Leu Lys Ala Leu Leu Glu

385 390 395 400

Val Asn Lys Gly His Glu Met Ser Pro Gln Val Ala Thr Leu Ile Asp

405 410 415

Arg Phe Val Lys Gly Arg Gly Gln Leu Asp Lys Asp Thr Leu Asp Thr

420 425 430

Leu Thr Ala Phe Tyr Pro Gly Tyr Leu Cys Ser Leu Ser Pro Glu Glu

435 440 445

Leu Ser Ser Val Pro Pro Ser Ser Ile Trp Ala Val Arg Pro Gln Asp

450 455 460

Leu Asp Thr Cys Asp Pro Arg Gln Leu Asp Val Leu Tyr Pro Lys Ala

465 470 475 480

Arg Leu Ala Phe Gln Asn Met Asn Gly Ser Glu Tyr Phe Val Lys Ile

485 490 495

Gln Ser Phe Leu Gly Gly Ala Pro Thr Glu Asp Leu Lys Ala Leu Ser

500 505 510

Gln Gln Asn Val Ser Met Asp Leu Ala Thr Phe Met Lys Leu Arg Thr

515 520 525

Asp Ala Val Leu Pro Leu Thr Val Ala Glu Val Gln Lys Leu Leu Gly

530 535 540

Pro His Val Glu Gly Leu Lys Ala Glu Glu Arg His Arg Pro Val Arg

545 550 555 560

Asp Trp Ile Leu Arg Gln Arg Gln Asp Asp Leu Asp Thr Leu Gly Leu

565 570 575

Gly Leu Gln Gly Gly Ile Pro Asn Gly Tyr Leu Val Leu Asp Leu Ser

580 585 590

Val Gln Glu Ala Leu Ser Gly Thr Pro Cys Leu Leu Gly Pro Gly Pro

595 600 605

Val Leu Thr Val Leu Ala Leu Leu Leu Ala Ser Thr Leu Ala

610 615 620

<210> 76

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 76

Gly Gly Ser Val Ser Ser Gly Ser Tyr Tyr

1 5 10

<210> 77

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 77

Ile Tyr Tyr Ser Gly Ser Thr

15

<210> 78

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 78

Ala Arg Glu Gly Lys Asn Gly Ala Phe Asp Ile Trp

1 5 10

<210> 79

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 79

Gln Ser Ile Ser Ser Tyr

15

<210> 80

<211> 4

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 80

Ala Ala Ser Ser

1

<210> 81

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 81

Gln Gln Ser Tyr Ser Thr Pro Leu Thr Phe

1 5 10

<210> 82

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 82

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Val Ser Ser Gly

20 25 30

Ser Tyr Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Glu Gly Lys Asn Gly Ala Phe Asp Ile Trp Gly Gln Gly

100 105 110

Thr Met Val Thr Val Ser Ser

115

<210> 83

<211> 109

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 83

Arg His Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr

100 105

<210> 84

<211> 243

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 84

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Val Ser Ser Gly

20 25 30

Ser Tyr Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Glu Gly Lys Asn Gly Ala Phe Asp Ile Trp Gly Gln Gly

100 105 110

Thr Met Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

Ser Gly Gly Gly Gly Ser Arg His Gln Met Thr Gln Ser Pro Ser Ser

130 135 140

Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser

145 150 155 160

Gln Ser Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys

165 170 175

Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val

180 185 190

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

195 200 205

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

210 215 220

Ser Tyr Ser Thr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

225 230 235 240

Lys Arg Thr

<210> 85

<211> 729

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 85

caggttcagc ttcaggagag tggcccaggc ctggtgaagc caagtgagac tctcagcttg 60

acttgcacag tttctggagg cagtgtctcc tcaggcagct attattggtc ctggattcgg 120

cagccccctg ggaaaggcct ggagtggatt gggtacatat attacagtgg cagcacaaat 180

tacaatccat ccctgaagtc tcgagtaact atcagtgtgg acacaagcaa gaatcagttt 240

tcactcaaac tgtcttctgt gactgctgct gacactgctg tttattattg tgccagggag 300

gggaaaaatg gggcatttga tatttggggt cagggcacaa tggtgacagt cagctctgga 360

ggtggaggct caggaggagg aggcagtgga ggtggtggggt cacgccatca gatgactcag 420

tccccctcca gtctttctgc ctcagttggg gatagagtga ccatcacatg cagagcaagt 480

cagagcatat catcctatct gaactggtac cagcagaagc cagggaaagc ccccaaattg 540

ctgatttatg cagcctcaag tctccagagt ggggtgccaa gcaggttctc aggcagtggc 600

agtgggacag atttcacatt gacaatcagc tccctccaac ctgaagattt tgccacctac 660

tattgccagc aatcctacag cacgcccctg acttttggag gtggcacaaa ggtagagatc 720

aagaggact 729

<210> 86

<211> 235

<212> PROTEIN

<213> Homo sapiens

<400> 86

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Ser Gln Phe Arg Val Ser Pro Leu Asp Arg Thr

20 25 30

Trp Asn Leu Gly Glu Thr Val Glu Leu Lys Cys Gln Val Leu Leu Ser

35 40 45

Asn Pro Thr Ser Gly Cys Ser Trp Leu Phe Gln Pro Arg Gly Ala Ala

50 55 60

Ala Ser Pro Thr Phe Leu Leu Tyr Leu Ser Gln Asn Lys Pro Lys Ala

65 70 75 80

Ala Glu Gly Leu Asp Thr Gln Arg Phe Ser Gly Lys Arg Leu Gly Asp

85 90 95

Thr Phe Val Leu Thr Leu Ser Asp Phe Arg Arg Glu Asn Glu Gly Tyr

100 105 110

Tyr Phe Cys Ser Ala Leu Ser Asn Ser Ile Met Tyr Phe Ser His Phe

115 120 125

Val Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg

130 135 140

Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg

145 150 155 160

Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly

165 170 175

Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr

180 185 190

Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Asn His

195 200 205

Arg Asn Arg Arg Arg Val Cys Lys Cys Pro Arg Pro Val Val Lys Ser

210 215 220

Gly Asp Lys Pro Ser Leu Ser Ala Arg Tyr Val

225 230 235

<210> 87

<211> 247

<212> PROTEIN

<213> Mus musculus

<400> 87

Met Ala Ser Pro Leu Thr Arg Phe Leu Ser Leu Asn Leu Leu Leu Met

1 5 10 15

Gly Glu Ser Ile Ile Leu Gly Ser Gly Glu Ala Lys Pro Gln Ala Pro

20 25 30

Glu Leu Arg Ile Phe Pro Lys Lys Met Asp Ala Glu Leu Gly Gln Lys

35 40 45

Val Asp Leu Val Cys Glu Val Leu Gly Ser Val Ser Gln Gly Cys Ser

50 55 60

Trp Leu Phe Gln Asn Ser Ser Ser Lys Leu Pro Gln Pro Thr Phe Val

65 70 75 80

Val Tyr Met Ala Ser Ser His Asn Lys Ile Thr Trp Asp Glu Lys Leu

85 90 95

Asn Ser Ser Lys Leu Phe Ser Ala Val Arg Asp Thr Asn Asn Lys Tyr

100 105 110

Val Leu Thr Leu Asn Lys Phe Ser Lys Glu Asn Glu Gly Tyr Tyr Phe

115 120 125

Cys Ser Val Ile Ser Asn Ser Val Met Tyr Phe Ser Ser Val Val Pro

130 135 140

Val Leu Gln Lys Val Asn Ser Thr Thr Thr Lys Pro Val Leu Arg Thr

145 150 155 160

Pro Ser Pro Val His Pro Thr Gly Thr Ser Gln Pro Gln Arg Pro Glu

165 170 175

Asp Cys Arg Pro Arg Gly Ser Val Lys Gly Thr Gly Leu Asp Phe Ala

180 185 190

Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Ile Cys Val Ala Pro

195 200 205

Leu Leu Ser Leu Ile Ile Thr Leu Ile Cys Tyr His Arg Ser Arg Lys

210 215 220

Arg Val Cys Lys Cys Pro Arg Pro Leu Val Arg Gln Glu Gly Lys Pro

225 230 235 240

Arg Pro Ser Glu Lys Ile Val

245

<210> 88

<211> 69

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 88

Ser Thr Thr Thr Lys Pro Val Leu Arg Thr Pro Ser Pro Val His Pro

1 5 10 15

Thr Gly Thr Ser Gln Pro Gln Arg Pro Glu Asp Cys Arg Pro Arg Gly

20 25 30

Ser Val Lys Gly Thr Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp

35 40 45

Ala Pro Leu Ala Gly Ile Cys Val Ala Leu Leu Leu Ser Leu Ile Ile

50 55 60

Thr Leu Ile Cys Tyr

65

<210> 89

<211> 207

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 89

tctactacta ccaagccagt gctgcgaact ccctcacctg tgcaccctac cgggacatct 60

cagccccaga gaccagaaga ttgtcggccc cgtggctcag tgaaggggac cggattggac 120

ttcgcctgtg atatttacat ctgggcaccc ttggccggaa tctgcgtggc ccttctgctg 180

tccttgatca tcactctcat ctgctac 207

<210> 90

<211> 220

<212> PROTEIN

<213> Homo sapiens

<400> 90

Met Leu Arg Leu Leu Leu Ala Leu Asn Leu Phe Pro Ser Ile Gln Val

1 5 10 15

Thr Gly Asn Lys Ile Leu Val Lys Gln Ser Pro Met Leu Val Ala Tyr

20 25 30

Asp Asn Ala Val Asn Leu Ser Cys Lys Tyr Ser Tyr Asn Leu Phe Ser

35 40 45

Arg Glu Phe Arg Ala Ser Leu His Lys Gly Leu Asp Ser Ala Val Glu

50 55 60

Val Cys Val Val Tyr Gly Asn Tyr Ser Gln Gln Leu Gln Val Tyr Ser

65 70 75 80

Lys Thr Gly Phe Asn Cys Asp Gly Lys Leu Gly Asn Glu Ser Val Thr

85 90 95

Phe Tyr Leu Gln Asn Leu Tyr Val Asn Gln Thr Asp Ile Tyr Phe Cys

100 105 110

Lys Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser

115 120 125

Asn Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro

130 135 140

Leu Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly

145 150 155 160

Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile

165 170 175

Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met

180 185 190

Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro

195 200 205

Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser

210 215 220

<210> 91

<211> 81

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 91

ttttgggtgc tggtggtggt tggtggagtc ctggcttgct atagcttgct agtaacagtg 60

gcctttatta ttttctgggt g 81

<210> 92

<211> 27

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 92

Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu

1 5 10 15

Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val

20 25

<210> 93

<211> 81

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 93

ttttgggtgc tggtggtggt tggtggagtc ctggcttgct atagcttgct agtaacagtg 60

gcctttatta ttttctgggt g 81

<210> 94

<211> 164

<212> PROTEIN

<213> Homo sapiens

<400> 94

Met Lys Trp Lys Ala Leu Phe Thr Ala Ala Ile Leu Gln Ala Gln Leu

1 5 10 15

Pro Ile Thr Glu Ala Gln Ser Phe Gly Leu Leu Asp Pro Lys Leu Cys

20 25 30

Tyr Leu Leu Asp Gly Ile Leu Phe Ile Tyr Gly Val Ile Leu Thr Ala

35 40 45

Leu Phe Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr

50 55 60

Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg

65 70 75 80

Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met

85 90 95

Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn

100 105 110

Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met

115 120 125

Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly

130 135 140

Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala

145 150 155 160

Leu Pro Pro Arg

<210> 95

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 95

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 96

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 96

agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 60

tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120

cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180

gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240

cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300

tacgacgccc ttcacatgca ggccctgccc cctcgc 336

<210> 97

<211> 218

<212> PROTEIN

<213> Mus musculus

<400> 97

Met Thr Leu Arg Leu Leu Phe Leu Ala Leu Asn Phe Phe Ser Val Gln

1 5 10 15

Val Thr Glu Asn Lys Ile Leu Val Lys Gln Ser Pro Leu Leu Val Val

20 25 30

Asp Ser Asn Glu Val Ser Leu Ser Cys Arg Tyr Ser Tyr Asn Leu Leu

35 40 45

Ala Lys Glu Phe Arg Ala Ser Leu Tyr Lys Gly Val Asn Ser Asp Val

50 55 60

Glu Val Cys Val Gly Asn Gly Asn Phe Thr Tyr Gln Pro Gln Phe Arg

65 70 75 80

Ser Asn Ala Glu Phe Asn Cys Asp Gly Asp Phe Asp Asn Glu Thr Val

85 90 95

Thr Phe Arg Leu Trp Asn Leu His Val Asn His Thr Asp Ile Tyr Phe

100 105 110

Cys Lys Ile Glu Phe Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Arg

115 120 125

Ser Asn Gly Thr Ile Ile His Ile Lys Glu Lys His Leu Cys His Thr

130 135 140

Gln Ser Ser Pro Lys Leu Phe Trp Ala Leu Val Val Val Ala Gly Val

145 150 155 160

Leu Phe Cys Tyr Gly Leu Leu Val Thr Val Ala Leu Cys Val Ile Trp

165 170 175

Thr Asn Ser Arg Arg Asn Arg Leu Leu Gln Ser Asp Tyr Met Asn Met

180 185 190

Thr Pro Arg Arg Pro Gly Leu Thr Arg Lys Pro Tyr Gln Pro Tyr Ala

195 200 205

Pro Ala Arg Asp Phe Ala Ala Tyr Arg Pro

210 215

<210> 98

<211> 123

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 98

aatagtagaa ggaacagact ccttcaaagt gactacatga acatgactcc ccggaggcct 60

gggctcactc gaaagcctta ccagccctac gcccctgcca gagactttgc agcgtaccgc 120

ccc 123

<210> 99

<211> 41

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 99

Asn Ser Arg Arg Asn Arg Leu Leu Gln Ser Asp Tyr Met Asn Met Thr

1 5 10 15

Pro Arg Arg Pro Gly Leu Thr Arg Lys Pro Tyr Gln Pro Tyr Ala Pro

20 25 30

Ala Arg Asp Phe Ala Ala Tyr Arg Pro

35 40

<210> 100

<211> 123

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 100

aatagtagaa ggaacagact ccttcaaagt gactacatga acatgactcc ccggaggcct 60

gggctcactc gaaagcctta ccagccctac gcccctgcca gagactttgc agcgtaccgc 120

ccc 123

<210> 101

<211> 41

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 101

Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr

1 5 10 15

Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro

20 25 30

Pro Arg Asp Phe Ala Ala Tyr Arg Ser

35 40

<210> 102

<211> 123

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 102

aggagtaaga ggagcaggct cctgcacagt gactacatga acatgactcc ccgccgcccc 60

gggcccaccc gcaagcatta ccagccctat gccccaccac gcgacttcgc agcctatcgc 120

tcc 123

<210> 103

<211> 255

<212> PROTEIN

<213> Homo sapiens

<400> 103

Met Gly Asn Ser Cys Tyr Asn Ile Val Ala Thr Leu Leu Leu Val Leu

1 5 10 15

Asn Phe Glu Arg Thr Arg Ser Leu Gln Asp Pro Cys Ser Asn Cys Pro

20 25 30

Ala Gly Thr Phe Cys Asp Asn Asn Arg Asn Gln Ile Cys Ser Pro Cys

35 40 45

Pro Pro Asn Ser Phe Ser Ser Ala Gly Gly Gln Arg Thr Cys Asp Ile

50 55 60

Cys Arg Gln Cys Lys Gly Val Phe Arg Thr Arg Lys Glu Cys Ser Ser

65 70 75 80

Thr Ser Asn Ala Glu Cys Asp Cys Thr Pro Gly Phe His Cys Leu Gly

85 90 95

Ala Gly Cys Ser Met Cys Glu Gln Asp Cys Lys Gln Gly Gln Glu Leu

100 105 110

Thr Lys Lys Gly Cys Lys Asp Cys Cys Phe Gly Thr Phe Asn Asp Gln

115 120 125

Lys Arg Gly Ile Cys Arg Pro Trp Thr Asn Cys Ser Leu Asp Gly Lys

130 135 140

Ser Val Leu Val Asn Gly Thr Lys Glu Arg Asp Val Val Cys Gly Pro

145 150 155 160

Ser Pro Ala Asp Leu Ser Pro Gly Ala Ser Ser Val Thr Pro Pro Ala

165 170 175

Pro Ala Arg Glu Pro Gly His Ser Pro Gln Ile Ile Ser Phe Phe Leu

180 185 190

Ala Leu Thr Ser Thr Ala Leu Leu Phe Leu Leu Phe Phe Leu Thr Leu

195 200 205

Arg Phe Ser Val Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe

210 215 220

Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly

225 230 235 240

Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

245 250 255

<210> 104

<211> 42

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 104

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 105

<211> 126

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 105

aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60

actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120

gaactg 126

<210> 106

<211> 277

<212> PROTEIN

<213> Homo sapiens

<400> 106

Met Cys Val Gly Ala Arg Arg Leu Gly Arg Gly Pro Cys Ala Ala Leu

1 5 10 15

Leu Leu Leu Gly Leu Gly Leu Ser Thr Val Thr Gly Leu His Cys Val

20 25 30

Gly Asp Thr Tyr Pro Ser Asn Asp Arg Cys Cys His Glu Cys Arg Pro

35 40 45

Gly Asn Gly Met Val Ser Arg Cys Ser Arg Ser Gln Asn Thr Val Cys

50 55 60

Arg Pro Cys Gly Pro Gly Phe Tyr Asn Asp Val Val Ser Ser Lys Pro

65 70 75 80

Cys Lys Pro Cys Thr Trp Cys Asn Leu Arg Ser Gly Ser Glu Arg Lys

85 90 95

Gln Leu Cys Thr Ala Thr Gln Asp Thr Val Cys Arg Cys Arg Ala Gly

100 105 110

Thr Gln Pro Leu Asp Ser Tyr Lys Pro Gly Val Asp Cys Ala Pro Cys

115 120 125

Pro Pro Gly His Phe Ser Pro Gly Asp Asn Gln Ala Cys Lys Pro Trp

130 135 140

Thr Asn Cys Thr Leu Ala Gly Lys His Thr Leu Gln Pro Ala Ser Asn

145 150 155 160

Ser Ser Asp Ala Ile Cys Glu Asp Arg Asp Pro Pro Ala Thr Gln Pro

165 170 175

Gln Glu Thr Gln Gly Pro Pro Ala Arg Pro Ile Thr Val Gln Pro Thr

180 185 190

Glu Ala Trp Pro Arg Thr Ser Gln Gly Pro Ser Thr Arg Pro Val Glu

195 200 205

Val Pro Gly Gly Arg Ala Val Ala Ala Ile Leu Gly Leu Gly Leu Val

210 215 220

Leu Gly Leu Leu Gly Pro Leu Ala Ile Leu Leu Ala Leu Tyr Leu Leu

225 230 235 240

Arg Arg Asp Gln Arg Leu Pro Pro Asp Ala His Lys Pro Pro Gly Gly

245 250 255

Gly Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp Ala His Ser

260 265 270

Thr Leu Ala Lys Ile

275

<210> 107

<211> 23

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 107

Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val

1 5 10 15

Glu Glu Asn Pro Gly Pro Met

20

<210> 108

<211> 41

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 108

Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr

1 5 10 15

Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro

20 25 30

Pro Arg Asp Phe Ala Ala Tyr Arg Lys

35 40

<210> 109

<211> 39

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 109

Asn Gln Leu Glu Arg Thr Val Asn Ser Leu Asn Val Pro Ala Ile Ser

1 5 10 15

Ile Pro Glu His Asp Glu Ala Asp Glu Ile Ser Asp Glu Asn Arg Glu

20 25 30

Lys Val Asn Asp Gln Ala Lys

35

<210> 110

<211> 42

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 110

Ala Ala Ala Asn Gln Leu Glu Arg Thr Val Asn Ser Leu Asn Val Ser

1 5 10 15

Ala Ile Ser Ile Pro Glu His Asp Glu Ala Asp Glu Ile Ser Asp Glu

20 25 30

Asn Arg Glu Lys Val Asn Asp Gln Ala Lys

35 40

<210> 111

<211> 48

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 111

Pro Glu His Asp Glu Ala Asp Glu Ile Ser Asp Glu Asn Arg Glu Lys

1 5 10 15

Val Asn Asp Gln Ala Lys Leu Ile Val Gly Ile Val Val Gly Leu Leu

20 25 30

Leu Ala Ala Leu Val Ala Gly Val Val Tyr Trp Leu Tyr Met Lys Lys

35 40 45

<210> 112

<211> 37

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 112

Glu Asn Arg Glu Lys Val Asn Asp Gln Ala Lys Leu Ile Val Gly Ile

1 5 10 15

Val Val Gly Leu Leu Leu Ala Ala Leu Val Ala Gly Val Val Tyr Trp

20 25 30

Leu Tyr Met Lys Lys

35

<210> 113

<211> 65

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 113

Asn Gln Leu Glu Arg Thr Val Asn Ser Leu Asn Val Pro Ala Ile Ser

1 5 10 15

Ile Pro Glu His Asp Glu Ala Asp Glu Ile Ser Asp Glu Asn Arg Glu

20 25 30

Lys Val Asn Asp Gln Ala Lys Leu Ile Val Gly Ile Val Val Gly Leu

35 40 45

Leu Leu Ala Ala Leu Val Ala Gly Val Val Tyr Trp Leu Tyr Met Lys

50 55 60

Lys

65

<210> 114

<211> 66

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 114

Thr Cys Thr Ala Glu Asn Gln Leu Glu Arg Thr Val Asn Ser Leu Asn

1 5 10 15

Val Ser Ala Ile Ser Ile Pro Glu His Asp Glu Ala Asp Glu Ile Ser

20 25 30

Asp Glu Asn Arg Glu Lys Val Asn Asp Gln Ala Lys Leu Ile Val Gly

35 40 45

Ile Val Val Gly Leu Leu Leu Ala Ala Leu Val Ala Gly Val Val Tyr

50 55 60

Trp Leu

65

<210> 115

<211> 44

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 115

Pro Glu His Asp Glu Ala Asp Glu Ile Ser Asp Glu Asn Arg Glu Lys

1 5 10 15

Val Asn Asp Gln Ala Lys Leu Ile Val Gly Ile Val Val Gly Leu Leu

20 25 30

Leu Ala Ala Leu Val Ala Gly Val Val Tyr Trp Leu

35 40

<210> 116

<211> 61

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 116

Asn Gln Leu Glu Arg Thr Val Asn Ser Leu Asn Val Ser Ala Ile Ser

1 5 10 15

Ile Pro Glu His Asp Glu Ala Asp Glu Ile Ser Asp Glu Asn Arg Glu

20 25 30

Lys Val Asn Asp Gln Ala Lys Leu Ile Val Gly Ile Val Val Gly Leu

35 40 45

Leu Leu Ala Ala Leu Val Ala Gly Val Val Tyr Trp Leu

50 55 60

<210> 117

<211> 68

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 117

Ala Ala Ala Asn Gln Leu Glu Arg Thr Val Asn Ser Leu Asn Val Ser

1 5 10 15

Ala Ile Ser Ile Pro Glu His Asp Glu Ala Asp Glu Ile Ser Asp Glu

20 25 30

Asn Arg Glu Lys Val Asn Asp Gln Ala Lys Leu Ile Val Gly Ile Val

35 40 45

Val Gly Leu Leu Leu Ala Ala Leu Val Ala Gly Val Val Tyr Trp Leu

50 55 60

Tyr Met Lys Lys

65

<210> 118

<211> 241

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 118

Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln

1 5 10 15

Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp

20 25 30

Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp

35 40 45

Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val

50 55 60

Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala

65 70 75 80

Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg

85 90 95

Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg

100 105 110

Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu

115 120 125

Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val

130 135 140

Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro

145 150 155 160

Arg Pro Ala Gly Gln Ala Ala Ala Pro Thr Thr Thr Pro Ala Pro Arg

165 170 175

Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg

180 185 190

Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly

195 200 205

Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr

210 215 220

Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Asn His

225 230 235 240

Arg

<210> 119

<211> 723

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 119

atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact gggctggcgg 60

ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt ctccccagcc 120

ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct gcagcttctc caacacatcg 180

gagagcttcg tgctaaactg gtaccgcatg agccccagca acgacgga caagctggcc 240

gctttccccg aggaccgcag ccagcccggc caggactgcc gcttccgtgt cacacaactg 300

cccaacgggc gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc 360

tacctctgtg gggccatctc cctggccccc aaggcgcaga tcaaagagag cctgcgggca 420

gagctcaggg tgacagagag aagggcagaa gtgcccacag cccaccccag cccctcaccc 480

aggccagccg gccaggcggc cgcacccacc acgacgccag cgccgcgacc accaaccccg 540

gcgcccacga tcgcgtcgca gcccctgtcc ctgcgcccag aggcgtgccg gccagcggcg 600

ggggggcgcag tgcacacgag ggggctggac ttcgcctgtg atatctacat ctgggcgccc 660

ctggccggga cttgtggggt ccttctcctg tcactggtta tcacccttta ctgcaaccac 720

agg 723

<210> 120

<211> 63

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 120

atggccctgc cagtaacggc tctgctgctg ccacttgctc tgctcctcca tgcagccagg 60

cct 63

<210> 121

<211> 19

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 121

Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn

1 5 10 15

Pro Gly Pro

<210> 122

<211> 57

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 122

gcaacaaact tctcactact caaacaagca ggtgacgtgg aggagaatcc cggccca 57

<210> 123

<211> 8613

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 123

atggccctgc cagtaacggc tctgctgctg ccacttgctc tgctcctcca tgcagccagg 60

cctcaggttc agcttcagga gagtggccca ggcctggtga agccaagtga gactctcagc 120

ttgacttgca cagtttctgg aggcagtgtc tcctcaggca gctattattg gtcctggatt 180

cggcagcccc ctgggaaagg cctggagtgg attgggtaca tatattacag tggcagcaca 240

aattacaatc catccctgaa gtctcgagta actatcagtg tggacacaag caagaatcag 300

ttttcactca aactgtcttc tgtgactgct gctgacactg ctgtttatta ttgtgccagg 360

gaggggaaaa atggggcatt tgatatttgg ggtcagggca caatggtgac agtcagctct 420

ggaggtggag gctcaggagg aggaggcagt ggaggtggtg ggtcacgcca tcagatgact 480

cagtccccct ccagtctttc tgcctcagtt ggggatagag tgaccatcac atgcagagca 540

agtcagagca tatcatccta tctgaactgg taccagcaga agccagggaa agcccccaaa 600

ttgctgattt atgcagcctc aagtctccag agtggggtgc caagcaggtt ctcaggcagt 660

ggcagtggga cagatttcac attgacaatc agctccctcc aacctgaaga ttttgccacc 720

tactattgcc agcaatccta cagcacgccc ctgacttttg gaggtggcac aaaggtagag 780

atcaagagga ctgcggccgc aattgaagtt atgtatcctc ctccttacct agacaatgag 840

aagagcaatg gaaccattat ccatgtgaaa gggaaacacc tttgtccaag tcccctattt 900

cccggacctt ctaagccctt ttgggtgctg gtggtggttg gtggagtcct ggcttgctat 960

agcttgctag taacagtggc ctttattatt ttctgggtga ggagtaagag gagcaggctc 1020

ctgcacagtg actacatgaa catgactccc cgccgccccg ggcccacccg caagcattac 1080

cagccctatg ccccaccacg cgacttcgca gcctatcgct ccagagtgaa gttcagcagg 1140

agcgcagacg cccccgcgta ccagcagggc cagaaccagc tctataacga gctcaatcta 1200

ggacgaagag aggagtacga tgttttggac aagagacgtg gccgggaccc tgagatgggg 1260

ggaaagccga gaaggaagaa ccctcaggaa ggcctgttca atgaactgca gaaagataag 1320

atggcggagg ccttcagtga gattgggatg aaaggcgagc gccggagggg caaggggcac 1380

gatggccttt tccaggggct cagtacagcc accaaggaca ccttcgacgc ccttcacatg 1440

caggccctgc cccctcgcgg atctggagca acaaacttct cactactcaa acaagcaggt 1500

gacgtggagg agaatcccgg cccaatgcag atcccacagg cgccctggcc agtcgtctgg 1560

gcggtgctac aactgggctg gcggccagga tggttcttag actccccaga caggccctgg 1620

aaccccccca ccttctcccc agccctgctc gtggtgaccg aaggggacaa cgccaccttc 1680

acctgcagct tctccaacac atcggagagc ttcgtgctaa actggtaccg catgagcccc 1740

agcaaccaga cggacaagct ggccgctttc cccgaggacc gcagccagcc cggccaggac 1800

tgccgcttcc gtgtcacaca actgcccaac gggcgtgact tccacatgag cgtggtcagg 1860

gcccggcgca atgacagcgg cacctacctc tgtggggcca tctccctggc ccccaaggcg 1920

cagatcaaag agagcctgcg ggcagagctc agggtgacag agagaagggc agaagtgccc 1980

acagcccacc ccagcccctc acccaggcca gccggccagg cggccgcacc caccacgacg 2040

ccagcgccgc gaccaccaac cccggcgccc acgatcgcgt cgcagcccct gtccctgcgc 2100

ccagaggcgt gccggccagc ggcggggggc gcagtgcaca cgagggggct ggacttcgcc 2160

tgtgatatct acatctgggc gcccctggcc gggacttgtg gggtccttct cctgtcactg 2220

gttatcaccc tttactgcaa ccacaggcgg atccaataac agccactcga ggatccggat 2280

tagtccaatt tgttaaagac aggatatcag tggtccaggc tctagttttg actcaacaat 2340

atcaccagct gaagcctata gagtacgagc catagataaa ataaaagatt ttatttagtc 2400

tccagaaaaa ggggggaatg aaagacccca cctgtaggtt tggcaagcta gcttaagtaa 2460

cgccattttg caaggcatgg aaaaatacat aactgagaat agagaagttc agatcaaggt 2520

caggaacaga tggaacagct gaatatgggc caaacaggat atctgtggta agcagttcct 2580

gccccggctc agggccaaga acagatggaa cagctgaata tgggccaaac aggatatctg 2640

tggtaagcag ttcctgcccc ggctcagggc caagaacaga tggtccccag atgcggtcca 2700

gccctcagca gtttctagag aaccatcaga tgtttccagg gtgccccaag gacctgaaat 2760

gaccctgtgc cttatttgaa ctaaccaatc agttcgcttc tcgcttctgt tcgcgcgctt 2820

ctgctccccg agctcaataa aagagcccac aacccctcac tcggggcgcc agtcctccga 2880

ttgactgagt cgcccgggta cccgtgtatc caataaaccc tcttgcagtt gcatccgact 2940

tgtggtctcg ctgttccttg ggagggtctc ctctgagtga ttgactaccc gtcagcgggg 3000

gtctttcaca catgcagcat gtatcaaaat taatttggtt ttttttctta agtatttaca 3060

ttaaatggcc atagtactta aagttacatt ggcttccttg aaataaacat ggagtattca 3120

gaatgtgtca taaatatttc taattttaag atagtatctc cattggcttt ctactttttc 3180

ttttattttt ttttgtcctc tgtcttccat ttgttgttgt tgttgtttgt ttgtttgttt 3240

gttggttggt tggttaattt ttttttaaag atcctacact atagttcaag ctagactatt 3300

agctactctg taacccaggg tgaccttgaa gtcatgggta gcctgctgtt ttagccttcc 3360

cacatctaag attacaggta tgagctatca tttttggtat attgattgat tgattgattg 3420

atgtgtgtgt gtgtgattgt gtttgtgtgt gtgactgtga aaatgtgtgt atgggtgtgt 3480

gtgaatgtgt gtatgtatgt gtgtgtgtga gtgtgtgtgt gtgtgtgtgc atgtgtgtgt 3540

gtgtgactgt gtctatgtgt atgactgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt 3600

gtgtgtgtgt gttgtgaaaa aatattctat ggtagtgaga gccaacgctc cggctcaggt 3660

gtcaggttgg tttttgagac agagtctttc acttagcttg gaattcactg gccgtcgttt 3720

tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc 3780

cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt 3840

tgcgcagcct gaatggcgaa tggcgcctga tgcggtattt tctccttacg catctgtgcg 3900

gtatttcaca ccgcatatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa 3960

gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg 4020

catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac 4080

cgtcatcacc gaaacgcgcg atgacgaaag ggcctcgtga tacgcctatt tttataggtt 4140

aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc 4200

ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa 4260

taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc 4320

cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa 4380

acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa 4440

ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg 4500

atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa 4560

gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc 4620

acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc 4680

atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta 4740

accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag 4800

ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca 4860

acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata 4920

gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc 4980

tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca 5040

ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca 5100

actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg 5160

taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa 5220

tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt 5280

gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat 5340

cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg 5400

gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga 5460

gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac 5520

tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt 5580

ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag 5640

cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc 5700

gaactgagat acctacagcg tgagcattga gaaagcgcca cgcttcccga agggaaaag 5760

gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca 5820

gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt 5880

cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc 5940

tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc 6000

cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc 6060

cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa 6120

ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac 6180

tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc 6240

caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa 6300

tttcacacag gaaacagcta tgaccatgat tacgccaagc tttgctctta ggagtttcct 6360

aatacatccc aaactcaaat atataaagca tttgacttgt tctatgccct agggggcggg 6420

gggaagctaa gccagctttt tttaacattt aaaatgttaa ttccatttta aatgcacaga 6480

tgtttttatt tcataagggt ttcaatgtgc atgaatgctg caatattcct gttaccaaag 6540

ctagtataaa taaaaataga taaacgtgga aattacttag agtttctgtc attaacgttt 6600

ccttcctcag ttgacaacat aaatgcgctg ctgagcaagc cagtttgcat ctgtcaggat 6660

caatttccca ttatgccagt catattaatt actagtcaat tagttgattt ttatttttga 6720

catatacatg tgaatgaaag accccacctg taggtttggc aagctagctt aagtaacgcc 6780

attttgcaag gcatggaaaa atacataact gagaatagaa aagttcagat caaggtcagg 6840

aacagatgga acagctgaat atgggccaaa caggatatct gtggtaagca gttcctgccc 6900

cggctcaggg ccaagaacag atggaacagc tgaatatggg ccaaacagga tatctgtggt 6960

aagcagttcc tgccccggct cagggccaag aacagatggt ccccagatgc ggtccagccc 7020

tcagcagttt ctagagaacc atcagatgtt tccagggtgc cccaaggacc tgaaatgacc 7080

ctgtgcctta tttgaactaa ccaatcagtt cgcttctcgc ttctgttcgc gcgcttatgc 7140

tccccgagct caataaaaga gcccacaacc cctcactcgg ggcgccagtc ctccgattga 7200

ctgagtcgcc cgggtacccg tgtatccaat aaaccctctt gcagttgcat ccgacttgtg 7260

gtctcgctgt tccttgggag ggtctcctct gagtgattga ctacccgtca gcggggggtct 7320

ttcatttggg ggctcgtccg ggatcgggag acccctgccc agggaccacc gacccaccac 7380

cgggaggtaa gctggccagc aacttatctg tgtctgtccg attgtctagt gtctatgact 7440

gattttatgc gcctgcgtcg gtactagtta gctaactagc tctgtatctg gcggacccgt 7500

ggtggaactg acgagttcgg aacacccggc cgcaaccctg ggagacgtcc cagggacttc 7560

gggggccgtt tttgtggccc gacctgagtc ctaaaatccc gatcgtttag gactctttgg 7620

tgcacccccc ttagaggagg gatatgtggt tctggtagga gacgagaacc taaaacagtt 7680

cccgcctccg tctgaatttt tgctttcggt ttgggaccga agccgcgccg cgcgtcttgt 7740

ctgctgcagc atcgttctgt gttgtctctg tctgactgtg tttctgtatt tgtctgaaaa 7800

tatgggcccg ggctagcctg ttaccactcc cttaagtttg accttaggtc actggaaaga 7860

tgtcgagcgg atcgctcaca accagtcggt agatgtcaag aagagacgtt gggttacctt 7920

ctgctctgca gaatggccaa cctttaacgt cggatggccg cgagacggca cctttaaccg 7980

agacctcatc acccaggtta agatcaaggt cttttcacct ggcccgcatg gacacccaga 8040

ccaggtcccc tacatcgtga cctgggaagc cttggctttt gacccccctc cctgggtcaa 8100

gccctttgta caccctaagc ctccgcctcc tcttcctcca tccgccccgt ctctccccct 8160

tgaacctcct cgttcgaccc cgcctcgatc ctccctttat ccagccctca ctccttctct 8220

aggcgccccc atatggccat atgagatctt atatggggca cccccgcccc ttgtaaactt 8280

ccctgaccct gacatgacaa gagttactaa cagcccctct ctccaagctc acttacaggc 8340

tctctactta gtccagcacg aagtctggag acctctggcg gcagcctacc aagaacaact 8400

ggaccgaccg gtggtacctc acccttaccg agtcggcgac acagtgtggg tccgccgaca 8460

ccagactaag aacctagaac ctcgctggaa aggaccttac acagtcctgc tgaccacccc 8520

caccgccctc aaagtagacg gcatcgcagc ttggatacac gccgcccacg tgaaggctgc 8580

cgaccccggg ggtggaccat cctctagact gcc 8613

<210> 124

<211> 8604

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 124

atggccctgc cagtaacggc tctgctgctg ccacttgctc tgctcctcca tgcagccagg 60

cctcaggttc agcttcagga gagtggccca ggcctggtga agccaagtga gactctcagc 120

ttgacttgca cagtttctgg aggcagtgtc tcctcaggca gctattattg gtcctggatt 180

cggcagcccc ctgggaaagg cctggagtgg attgggtaca tatattacag tggcagcaca 240

aattacaatc catccctgaa gtctcgagta actatcagtg tggacacaag caagaatcag 300

ttttcactca aactgtcttc tgtgactgct gctgacactg ctgtttatta ttgtgccagg 360

gaggggaaaa atggggcatt tgatatttgg ggtcagggca caatggtgac agtcagctct 420

ggaggtggag gctcaggagg aggaggcagt ggaggtggtg ggtcacgcca tcagatgact 480

cagtccccct ccagtctttc tgcctcagtt ggggatagag tgaccatcac atgcagagca 540

agtcagagca tatcatccta tctgaactgg taccagcaga agccagggaa agcccccaaa 600

ttgctgattt atgcagcctc aagtctccag agtggggtgc caagcaggtt ctcaggcagt 660

ggcagtggga cagatttcac attgacaatc agctccctcc aacctgaaga ttttgccacc 720

tactattgcc agcaatccta cagcacgccc ctgacttttg gaggtggcac aaaggtagag 780

atcaagagga ctgcggccgc aattgaagtt atgtatcctc ctccttacct agacaatgag 840

aagagcaatg gaaccattat ccatgtgaaa gggaaacacc tttgtccaag tcccctattt 900

cccggacctt ctaagccctt ttgggtgctg gtggtggttg gtggagtcct ggcttgctat 960

agcttgctag taacagtggc ctttattatt ttctgggtga ggagtaagag gagcaggctc 1020

ctgcacagtg actacatgaa catgactccc cgccgccccg ggcccacccg caagcattac 1080

cagccctatg ccccaccacg cgacttcgca gcctatcgct ccagagtgaa gttcagcagg 1140

agcgcagacg cccccgcgta ccagcagggc cagaaccagc tctataacga gctcaatcta 1200

ggacgaagag aggagtacga tgttttggac aagagacgtg gccgggaccc tgagatgggg 1260

ggaaagccga gaaggaagaa ccctcaggaa ggcctgttca atgaactgca gaaagataag 1320

atggcggagg ccttcagtga gattgggatg aaaggcgagc gccggagggg caaggggcac 1380

gatggccttt tccaggggct cagtacagcc accaaggaca ccttcgacgc ccttcacatg 1440

caggccctgc cccctcgcgg atctggagca acaaacttct cactactcaa acaagcaggt 1500

gacgtggagg agaatcccgg cccaatgcag atcccacagg cgccctggcc agtcgtctgg 1560

gcggtgctac aactgggctg gcggccagga tggttcttag actccccaga caggccctgg 1620

aaccccccca ccttctcccc agccctgctc gtggtgaccg aaggggacaa cgccaccttc 1680

acctgcagct tctccaacac atcggagagc ttcgtgctaa actggtaccg catgagcccc 1740

agcaaccaga cggacaagct ggccgctttc cccgaggacc gcagccagcc cggccaggac 1800

tgccgcttcc gtgtcacaca actgcccaac gggcgtgact tccacatgag cgtggtcagg 1860

gcccggcgca atgacagcgg cacctacctc tgtggggcca tctccctggc ccccaaggcg 1920

cagatcaaag agagcctgcg ggcagagctc agggtgacag agagaagggc agaagtgccc 1980

acagcccacc ccagcccctc acccaggcca gccggccagg cggccgcacc caccacgacg 2040

ccagcgccgc gaccaccaac cccggcgccc acgatcgcgt cgcagcccct gtccctgcgc 2100

ccagaggcgt gccggccagc ggcggggggc gcagtgcaca cgagggggct ggacttcgcc 2160

tgtgatatct acatctgggc gcccctggcc gggacttgtg gggtccttct cctgtcactg 2220

gttatcaccc tttactgcaa ccacaggcgg atccaataac agccactcga ggatccggat 2280

tagtccaatt tgttaaagac aggatatcag tggtccaggc tctagttttg actcaacaat 2340

atcaccagct gaagcctata gagtacgagc catagataaa ataaaagatt ttatttagtc 2400

tccagaaaaa ggggggaatg aaagacccca cctgtaggtt tggcaagcta gcttaagtaa 2460

cgccattttg caaggcatgg aaaaatacat aactgagaat agagaagttc agatcaaggt 2520

caggaacaga tggaacagct gaatatgggc caaacaggat atctgtggta agcagttcct 2580

gccccggctc agggccaaga acagatggaa cagctgaata tgggccaaac aggatatctg 2640

tggtaagcag ttcctgcccc ggctcagggc caagaacaga tggtccccag atgcggtcca 2700

gccctcagca gtttctagag aaccatcaga tgtttccagg gtgccccaag gacctgaaat 2760

gaccctgtgc cttatttgaa ctaaccaatc agttcgcttc tcgcttctgt tcgcgcgctt 2820

ctgctccccg agctcaataa aagagcccac aacccctcac tcggggcgcc agtcctccga 2880

ttgactgagt cgcccgggta cccgtgtatc caataaaccc tcttgcagtt gcatccgact 2940

tgtggtctcg ctgttccttg ggagggtctc ctctgagtga ttgactaccc gtcagcgggg 3000

gtctttcaca tgcagcatgt atcaaaatta atttggtttt ttttcttaag tatttacatt 3060

aaatggccat agtacttaaa gttacattgg cttccttgaa ataaacatgg agtattcaga 3120

atgtgtcata aatatttcta attttaagat agtatctcca ttggctttct actttttctt 3180

ttattttttt ttgtcctctg tcttccatt gttgttgttg ttgtttgttt gtttgtttgt 3240

tggttggttg gttaattttt ttttaaagat cctacactat agttcaagct agactattag 3300

ctactctgta acccaggtg accttgaagt catgggtagc ctgctgtttt agccttccca 3360

catctaagat tacaggtatg agctatcatt tttggtatat tgattgattg attgattgat 3420

gtgtgtgtgt gtgattgtgt ttgtgtgtgt gattgtgtat atgtgtgtat ggttgtgtgt 3480

gattgtgtgt atgtatgttt gtgtgtgatt gtgtgtgtgt gattgtgcat gtgtgtgtgt 3540

gtgattgtgt ttatgtgtat gattgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt 3600

gtgtgtgtgt tgtgtatata tatttatggt agtgagaggc aacgctccgg ctcaggtgtc 3660

aggttggttt ttgagacaga gtctttcact tagcttggaa ttcactggcc gtcgttttac 3720

aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc 3780

ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc 3840

gcagcctgaa tggcgaatgg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta 3900

tttcacaccg catatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc 3960

agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat 4020

ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt 4080

catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg 4140

tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa 4200

cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac 4260

cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg 4320

tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc 4380

tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg 4440

atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga 4500

gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc 4560

aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag 4620

aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga 4680

gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg 4740

cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga 4800

atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt 4860

tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact 4920

ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt 4980

ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg 5040

ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta 5100

tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac 5160

tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta 5220

aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt 5280

tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt 5340

tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt 5400

gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc 5460

agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg 5520

tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg 5580

ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt 5640

cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac 5700

tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg 5760

acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg 5820

gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat 5880

ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt 5940

tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg 6000

attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa 6060

cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc 6120

ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga 6180

aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg 6240

ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc 6300

acacaggaaa cagctatgac catgattacg ccaagctttg ctcttaggag tttcctaata 6360

catcccaaac tcaaatatat aaagcatttg acttgttcta tgccctaggg ggcgggggga 6420

agctaagcca gcttttttta acatttaaaa tgttaattcc attttaaatg cacagatgtt 6480

tttatttcat aagggtttca atgtgcatga atgctgcaat attcctgtta ccaaagctag 6540

tataaataaa aatagataaa cgtggaaatt acttagagtt tctgtcatta acgtttcctt 6600

cctcagttga caacataaat gcgctgctga gaagccagtt tgcatctgtc aggatcaatt 6660

tcccattatg ccagtcatat taattactag tcaattagtt gatttttatt tttgacatat 6720

acatgtgaaa gaccccacct gtaggtttgg caagctagct taagtaacgc cattttgcaa 6780

ggcatggaaa aatacataac tgagaataga aaagttcaga tcaaggtcag gaacagatgg 6840

aacagctgaa tatgggccaa acaggatatc tgtggtaagc agttcctgcc ccggctcagg 6900

gccaagaaca gatggaacag ctgaatatgg gccaaacagg atatctgtgg taagcagttc 6960

ctgccccggc tcaggggccaa gaacagatgg tccccagatg cggtccagcc ctcagcagtt 7020

tctagagaac catcagatgt ttccagggtg ccccaaggac ctgaaatgac cctgtgcctt 7080

atttgaacta accaatcagt tcgcttctcg cttctgttcg cgcgcttctg ctccccgagc 7140

tcaataaaag agcccacaac ccctcactcg gcgcgccagt cctccgattg actgagtcgc 7200

ccgggtaccc gtgtatccaa taaaccctct tgcagttgca tccgacttgt ggtctcgctg 7260

ttccttggga gggtctcctc tgagtgattg actacccgtc agcgggggtc tttcatttgg 7320

gggctcgtcc gggatcggga gacccctgcc cagggaccac cgacccacca ccgggaggta 7380

agctggccag caacttatct gtgtctgtcc gattgtctag tgtctatgac tgattttatg 7440

cgcctgcgtc ggtactagtt agctaactag ctctgtatct ggcggacccg tggtggaact 7500

gacgagttcg gaacacccgg ccgcaaccct gggagacgtc ccaggactt cgggggccgt 7560

ttttgtggcc cgacctgagt cctaaaatcc cgatcgttta ggactctttg gtgcaccccc 7620

cttagaggag ggatatgtgg ttctggtagg agacgagaac ctaaaacagt tcccgcctcc 7680

gtctgaattt ttgctttcgg tttgggaccg aagccgcgcc gcgcgtcttg tctgctgcag 7740

catcgttctg tgttgtctct gtctgactgt gtttctgtat ttgtctgaaa atatgggccc 7800

gggctagcct gttaccactc ccttaagttt gaccttaggt cactggaaag atgtcgagcg 7860

gatcgctcac aaccagtcgg tagatgtcaa gaagagacgt tgggttacct tctgctctgc 7920

agaatggcca acctttaacg tcggatggcc gcgagacggc acctttaacc gagacctcat 7980

cacccaggtt aagatcaagg tcttttcacc tggcccgcat ggacacccag accaggtccc 8040

ctacatcgtg acctgggaag ccttggcttt tgacccccct ccctgggtca agccctttgt 8100

acaccctaag cctccgcctc ctcttcctcc atccgccccg tctctccccc ttgaacctcc 8160

tcgttcgacc ccgcctcgat cctcccttta tccagccctc actccttctc taggcgcccc 8220

catatggcca tatgagatct tatatggggc acccccgccc cttgtaaact tccctgaccc 8280

tgacatgaca agagttacta acagcccctc tctccaagct cacttacagg ctctctactt 8340

agtccagcac gaagtctgga gacctctggc ggcagcctac caagaacaac tggaccgacc 8400

ggtggtacct cacccttacc gagtcggcga cacagtgtgg gtccgccgac accagactaa 8460

gaacctagaa cctcgctgga aaggacctta cacagtcctg ctgaccaccc ccaccgccct 8520

caaagtagac ggcatcgcag cttggataca cgccgcccac gtgaaggctg ccgaccccgg 8580

gggtggacca tcctctagac tgcc 8604

<210> 125

<211> 63

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 125

atggccctgc cagtaacggc tctgctgctg ccacttgctc tgctcctcca tgcagccagg 60

cct 63

<210> 126

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 126

aggaccttac acagtcctgc tgac 24

<210> 127

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 127

agaacctaga acctcgctgg a 21

<210> 128

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 128

ctgcgatgcc gtctactttg 20

<210> 129

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 129

tgctgaaaca ttcaccttcc atgcaga 27

<210> 130

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 130

tgaaacatac gttcccaaag agttt 25

<210> 131

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 131

ctctccttct cagaaagtgt gcatat 26

<210> 132

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 132

gaaggtgaag gtcggagt 18

<210> 133

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 133

catgggtgga atcatattgg aa 22

<210> 134

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 134

ccaggatggt tcttagactc cc 22

<210> 135

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 135

tttagcacga agctctccga t 21

<210> 136

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 136

acgagggaca ataggagcca 20

<210> 137

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic polynucleotide

<400> 137

ggcatactcc gtctgctcag 20

<---

Claims (102)

1. A polypeptide composition that specifically interacts with human mesothelin, including: i) chimeric antigen receptor (CAR), comprising: (a) an extracellular antigen binding domain comprising: a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence set forth in SEQ ID NO:76, a CDR2 consisting of the amino acid sequence set forth in SEQ ID NO:77, and a CDR3 consisting of the amino acid sequence presented 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 represented by SEQ ID NO: 29 and an ITAM3 variant comprising the amino acid sequence represented by SEQ ID NO: 33; And ii) a dominant negative form of programmed death protein 1 (PD-1 DN), including: (a) at least a portion of an extracellular domain of programmed death protein 1 (PD-1) including a ligand binding region, and (b) first transmembrane domain. 2. The polypeptide composition of claim 1, wherein the extracellular antigen binding domain of CAR specifically binds to human mesothelin with an EC ₅₀ value of from about 1 nM to about 25 nM. 3. The polypeptide composition of claim 1 or 2, wherein the extracellular antigen binding domain of the CAR comprises a single chain variable fragment (scFv), a Fab that is optionally cross-linked, or an F(ab) ₂ . 4. The polypeptide composition according to claim 3, wherein the extracellular antigen binding domain of the CAR includes a human scFv. 5. The polypeptide composition according to any one of claims 1 to 4, wherein the extracellular antigen binding domain of the CAR recognizes human mesothelin at a mesothelin expression level of approximately 1000 or more mesothelin binding sites/cell. 6. The polypeptide composition according to any one of claims 1 to 5, wherein the heavy chain variable region comprises an amino acid sequence that is at least about 80% homologous or identical to the amino acid sequence set forth in SEQ ID NO:82. 7. The polypeptide composition according to any one of claims 1 to 6, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:82. 8. The polypeptide composition according to any one of claims 1 to 7, wherein the light chain variable region comprising an amino acid sequence that is at least about 80% homologous or identical to the amino acid sequence set forth in SEQ ID NO:83. 9. The polypeptide composition according to any one of claims 1 to 8, wherein the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 83. 10. The polypeptide composition according to any one of claims 1 to 9, wherein the heavy chain variable region includes an amino acid sequence that is at least about 80% homologous or identical to the amino acid sequence presented in SEQ ID NO:82, and the light chain variable region includes an amino acid sequence that is at least about 80% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 83. 11. The polypeptide composition according to any one of claims 1 to 10, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:82 and the light chain variable region includes the amino acid sequence set forth in SEQ ID NO: 83. 12. The polypeptide composition according to any one of claims 1 to 11, wherein the extracellular antigen binding domain of the CAR includes a linker between a heavy chain variable region and a light chain variable region. 13. The polypeptide composition according to any one of claims 1 to 12, wherein the leader sequence is covalently linked to the N-terminus of the extracellular antigen-binding domain. 14. The polypeptide composition according to claim 13, wherein the leader sequence includes a CD8 polypeptide. 15. The polypeptide composition according to claim 14, wherein the CD8 polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 71. 16. The polypeptide composition according to any one of claims 1 to 15, wherein at least a portion of the extracellular domain of PD-1 includes amino acids 21-165 of SEQ ID NO: 48. 17. The polypeptide composition according to any one of claims 1 to 16, wherein the first transmembrane domain of the PD-1 DN includes 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 , CD8b polypeptide, ICOS polypeptide, ICAM-1 polypeptide, CTLA-4 polypeptide, CD27 polypeptide, CD40/My88 peptide, NKGD2 peptide, or a combination thereof. 18. The polypeptide composition according to claim 17, wherein the first transmembrane domain of the PD-1 DN includes a CD8 polypeptide. 19. The polypeptide composition of claim 18, wherein the CD8 polypeptide contained in the first transmembrane domain of the PD-1 DN comprises amino acids 137-207 of SEQ ID NO: 86. 20. The polypeptide composition according to any one of claims 1 to 19, 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. 21. The polypeptide composition according to any one of claims 1 to 20, wherein each of the two loss-of-function mutations occurs at a tyrosine amino acid residue. 22. The polypeptide composition according to any one of claims 1 to 21, wherein the ITAM2 variant includes or consists of the amino acid sequence presented in SEQ ID NO: 29. 23. The polypeptide composition according to any one of claims 1 to 22, wherein the ITAM3 variant includes or consists of the amino acid sequence set forth in SEQ ID NO: 33. 24. The polypeptide composition according to any one of claims 1 to 23, wherein the modified CD3ζ polypeptide includes native ITAM1. 25. The polypeptide composition of claim 24, wherein native ITAM1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23. 26. The polypeptide composition according to any one of claims 1 to 25, wherein the modified CD3ζ polypeptide includes or consists of the amino acid sequence set forth in SEQ ID NO: 35. 27. The polypeptide composition according to any one of claims 1 to 26, wherein PD-1 DN does not contain an intracellular domain. 28. The polypeptide composition according to any one of claims 1 to 27, wherein the CAR further includes a second transmembrane domain. 29. The polypeptide composition of claim 28, wherein 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, ICAM-1 polypeptide, CTLA-4 polypeptide, CD27 polypeptide, CD40/My88 peptide, NKGD2 peptide, or a combination thereof. 30. The polypeptide composition of claim 28 or 29, wherein the second transmembrane domain of the CAR comprises a CD28 polypeptide. 31. The polypeptide composition according to any one of claims 1 to 30, wherein the CAR intracellular signaling domain further includes a co-stimulatory signaling region. 32. The polypeptide composition of claim 31, wherein the co-stimulatory signaling region includes 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 combinations thereof. 33. The polypeptide composition of claim 31 or 32, wherein the co-stimulatory signaling region comprises a CD28 polypeptide. 34. The polypeptide composition according to any one of claims 1 to 33, wherein the CAR comprises the amino acid sequence set forth in SEQ ID NO: 56. 35. An immunoreactive cell for enhancing the immune response against cancer, comprising the polypeptide composition according to any one of claims 1 to 34, wherein the immunoreactive cell is selected from a T cell, a natural killer (NK) cell or a pluripotent stem cell from which a lymphoid cell can differentiate . 36. The immunoreactive cell according to claim 35, wherein PD-1 DN and/or CAR is expressed recombinantly. 37. The immunoreactive cell according to claim 35 or 36, wherein PD-1 DN and/or CAR is expressed from the vector. 38. The immunoreactive cell according to any one of claims 35 to 37, wherein the immunoreactive cell is selected from the group consisting of a T cell, a natural killer (NK) cell, and a pluripotent stem cell from which lymphoid cells can differentiate. 39. The immunoreactive cell according to claim 38, wherein the immunoreactive cell is a T cell. 40. The immunoreactive cell of claim 39, wherein the T cell is selected from the group consisting of a cytotoxic T lymphocyte (CTL), a regulatory T cell, and a natural killer T (NKT) cell. 41. The immunoreactive cell according to claim 38, wherein the pluripotent stem cell is an embryonic stem cell or an induced pluripotent stem cell. 42. An immunoreactive cell according to any one of claims 35-41, where the immunoreactive cell is autologous. 43. An immunoreactive cell according to any one of claims 35-41, where the immunoreactive cell is allogeneic. 44. A pharmaceutical composition for enhancing the immune response against cancer, comprising an effective amount of an immunoreactive cell according to any one of claims 35 to 43 and a pharmaceutically acceptable excipient. 45. The pharmaceutical composition according to claim 44, comprising from about 10 ⁴ to 10 ⁶ immunoreactive cells. 46. The pharmaceutical composition according to claim 44 or 45, comprising at least about 10 ⁵ immunoreactive cells. 47. The pharmaceutical composition according to any one of claims 44-46, comprising approximately 10 ⁵ immunoreactive cells. 48. The pharmaceutical composition according to any one of claims 44 to 47, which is intended for treating neoplasia in a subject, treating a subject having a recurrence of neoplasia, reducing tumor burden in a subject, increasing or prolonging survival of a subject having neoplasia, treating an inflammatory disease in a subject, and /or preventing graft rejection in a subject who is an organ transplant recipient. 49. A nucleic acid composition for producing an immunoreactive cell, including a polynucleotide encoding a polypeptide containing i) chimeric antigen receptor (CAR), comprising: a) an extracellular antigen binding domain comprising: a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence set forth in SEQ ID NO:76, a CDR2 consisting of the amino acid sequence set forth in SEQ ID NO:77, and a CDR3 consisting of the amino acid the sequence shown 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 represented by SEQ ID NO: 29 and an ITAM3 variant comprising the amino acid sequence represented by SEQ ID NO: 33; And ii) a dominant negative form of programmed death protein 1 (PD-1 DN), including: (a) at least a portion of an extracellular domain of programmed death protein 1 (PD-1) including a ligand binding region, and (b) first transmembrane domain. 50. The nucleic acid composition of claim 49, wherein the polynucleotide comprises the nucleotide sequence set forth in SEQ ID NO: 123. 51. The nucleic acid composition of claim 49, wherein the polynucleotide comprises the nucleotide sequence set forth in SEQ ID NO: 124. 52. An expression vector for producing an immunoreactive cell, comprising a nucleic acid composition according to any one of claims 49-51. 53. The vector according to claim 52, which is a retroviral vector. 54. The vector of claim 53, wherein the retroviral vector is a γ-retroviral vector or a lentiviral vector. 55. A method for producing an immunoreactive cell, comprising introducing into the immunoreactive cell a polypeptide composition according to any one of claims 1-34, a nucleic acid composition according to any one of claims 49-51 or a vector according to any one of claims 52-54, where the immunoreactive cell is selected from A T cell, natural killer (NK) cell, or pluripotent stem cell from which a lymphoid cell can differentiate. 56. A kit for enhancing the immune response against cancer, comprising a polypeptide composition according to any one of claims 1 to 34, a nucleic acid composition according to any one of claims 49 to 51, a vector according to any one of claims 52 to 54, an immunoreactive cell according to any one of claims 35-43 or a pharmaceutical composition according to any one of claims 44-48 and written instructions. 57. The kit of claim 56, wherein the written instructions include written instructions for treating neoplasia in a subject, treating a subject having a recurrence of neoplasia, reducing tumor burden in a subject, increasing or prolonging survival of a subject having neoplasia, treating an inflammatory disease in a subject, and/ or preventing graft rejection in a subject who is an organ transplant recipient. 58. A method of treating neoplasia in a subject, comprising administering to the subject an effective amount of immunoreactive cells according to any one of claims 35-43 or a pharmaceutical composition according to any one of claims 44-48. 59. A method of reducing tumor burden in a subject, comprising administering to the subject an effective amount of immunoreactive cells according to any one of claims 35-43 or a pharmaceutical composition according to any one of claims 44-48. 60. The method of claim 59, wherein the method reduces the number of tumor cells, reduces the size of the tumor, and/or eliminates the tumor in the subject. 61. The method according to any one of claims 58-60, where the neoplasia and/or tumor is a solid tumor. 62. The method according to claim 61, where the solid tumor is selected from the group consisting of mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma , thymic carcinoma, endometrial carcinoma, gastric cancer, cholangiocarcinoma and combinations thereof. 63. A method of treating a subject having a relapse of neoplasia, comprising administering to the subject an effective amount of immunoreactive cells according to any one of claims 35-43 or a pharmaceutical composition according to any one of claims 44-48. 64. The method of claim 63, wherein the subject has received immunotherapy prior to said administration of the immunoreactive cells or composition. 65. A method of increasing or prolonging the survival of a subject having neoplasia, comprising administering to the subject an effective amount of immunoreactive cells according to any one of claims 35-43 or a pharmaceutical composition according to any one of claims 44-48. 66. A method of increasing the production of an immune-activating cytokine in response to a cancer cell or pathogen in a subject, wherein the method comprises administering to the subject an effective amount of an immunoreactive cell according to any one of claims 35-43 or a pharmaceutical composition according to any one of claims 44-48. 67. The method according to claim 66, where 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. 68. The method according to any one of claims 58-67, further comprising administering at least one immunomodulatory agent. 69. The method of claim 68, wherein the at least one immunomodulatory agent is selected from the group consisting of immunostimulants, immune checkpoint blockers, radiotherapy agents, chemotherapeutic agents, and combinations thereof. 70. The method of claim 69, wherein the immunostimulant agent is selected from the group consisting of IL-12, an agonistic costimulatory monoclonal antibody, and combinations thereof. 71. The method according to claim 70, wherein the immunostimulating agent is IL-12. 72. The method of claim 69, wherein the agonistic co-stimulatory 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. 73. The method of claim 72, wherein the agonistic co-stimulatory monoclonal antibody is an anti-4-1BB antibody. 74. The method of claim 69, wherein the immune checkpoint blockers are selected from the group consisting of anti-PD-L1 antibodies, anti-CTLA-4 antibodies, anti-PD-1 antibodies, anti-LAG3 antibodies, anti-B7-H3 antibodies, anti-TIM3 antibodies and their combinations. 75. The method of claim 74, wherein the immune checkpoint blocker is an anti-PD-L1 antibody or an anti-PD-1 antibody. 76. The method according to any one of claims 58-75, where the subject is a person. 77. The method according to any one of claims 58-75, wherein the immunoreactive cell is pleurally or intrapleurally administered to the subject. 78. A method of treating an inflammatory disease in a subject, comprising administering to the subject immunoreactive cells according to any one of claims 35-43 or a pharmaceutical composition according to any one of claims 44-48. 79. The method of claim 78, wherein the immunoreactive cell is an immunoinhibitory cell. 80. The method of claim 79, wherein the immunoinhibitory cell is a regulatory T cell. 81. The method according to any one of claims 78-80, where the inflammatory disease is pancreatitis. 82. The method according to any one of claims 78-81, where the subject is a person. 83. The method according to any one of claims 78 to 82, wherein the subject is an organ transplant recipient. 84. The method according to any one of claims 78 to 83, wherein the subject is a pancreas transplant recipient. 85. A method for preventing transplant rejection in a subject who is an organ transplant recipient, comprising administering to the subject the immunoreactive cells of any one of claims 35 to 43 or a pharmaceutical composition of any of claims 44 to 48. 86. The method of claim 85, wherein the immunoreactive cell is an immunoinhibitory cell. 87. The method of claim 86, wherein the immunoinhibitory cell is a regulatory T cell. 88. The method according to any one of claims 85-87, where the subject is a person. 89. The method according to any one of claims 85-88, wherein the subject is a pancreas transplant recipient. 90. Immunoreactive cells according to any one of claims 35-43 or a pharmaceutical composition according to any one of claims 44-48 for use in treating neoplasia in a subject, treating a subject having a recurrence of neoplasia, reducing tumor burden in a subject, increasing or prolonging survival of a subject, having neoplasia, treating an inflammatory disease in a subject, and/or preventing graft rejection in a subject who is an organ transplant recipient.

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