Chimeric antigen receptors with CD28 mutations and use thereof

Abstract

To provide chimeric antigen receptors with CD28 mutations and use thereof.SOLUTION: The present disclosure provides methods and compositions for enhancing the immune response toward cancers and pathogens. The present disclosure relates to chimeric antigen receptors (CARs) comprising a mutated CD28 intracellular motif, and cells comprising such CARs. The presently disclosed subject matter further relates to the use of the cells for treating diseases, e.g, for treating cancers. The subject of the present disclosure provides chimeric antigen receptors (CARs) comprising a mutated CD28 intracellular motif, namely a mutated YMNM motif.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 970,401, filed February 5, 2020, the contents of which are incorporated herein by reference in their entirety and which claims priority to this provisional patent application. Sequence Listing

[0002] This application contains a Sequence Listing that has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. The ASCII copy created on February 4, 2021 is named 072734.1189_ST25.txt and is 70,968 bytes in size.

[0003] 1.Technical Field The present disclosure provides methods and compositions for enhancing immune response against cancer and pathogens.The present disclosure relates to chimeric antigen receptors (CARs) that comprise mutant CD28 intracellular motifs, i.e. mutant YMNM motifs.The subject matter of the present disclosure also provides cells that comprise CARs and compositions that comprise cells, as well as the use of cells and compositions for treating disease, such as treating cancer. [Background technology]

[0004] 2. Background technology Cell-based immunotherapy is a therapy with therapeutic potential for the treatment of cancer. T cells and other immune cells can be modified to target tumor antigens by the introduction of genetic material that encodes a natural or modified T cell receptor (TCR) specific for a selected antigen, or a synthetic receptor for the antigen, called a chimeric antigen receptor (CAR). Patient engineered CAR T cells have demonstrated remarkable efficacy against a variety of liquid and solid malignancies.

[0005] CARs in clinical use and in preclinical development mainly use costimulatory signaling domains such as CD28 or 4-1BB. CD28 is a transmembrane protein that plays a key role in T cell activation through its role as a costimulatory molecule and is an essential part of CD28-based CAR constructs. Persistence, especially the functional persistence of these CARs, has been shown to be associated with better outcomes. There is an unmet need for improved CARs with enhanced proliferation and persistence and / or improved efficiency and activity compared to existing CARs. Summary of the Invention [Means for solving the problem]

[0006] 3. Summary of the Invention The subject matter of the present disclosure provides chimeric antigen receptors (CARs) that contain a mutated CD28 intracellular motif, i.e., a mutated YMNM motif.

[0007] The present disclosure provides a chimeric antigen receptor (CAR) comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising at least one costimulatory signaling domain comprising a CD28 polypeptide comprising a mutated YMNM motif.

[0008] In certain embodiments, the CD28 polypeptide has reduced recruitment of the p85 subunit of phosphoinositide 3-kinase (PI3K) compared to the CD28 molecule containing the native YMNM motif. In certain embodiments, the p85 subunit of PI3K does not bind to the mutant YMNM motif. In certain embodiments, the mutant YMNM motif consists of the amino acid sequence depicted in YxNx (SEQ ID NO: 21), where x is not methionine (M). In certain embodiments, the mutant YMNM motif consists of the amino acid sequence depicted in YENV (SEQ ID NO:22), YSNV (SEQ ID NO:23), YKNL (SEQ ID NO:24), YENQ (SEQ ID NO:25), YKNI (SEQ ID NO:26), YINQ (SEQ ID NO:27), YHNK (SEQ ID NO:28), YVNQ (SEQ ID NO:29), YLNP (SEQ ID NO:30), YLNT (SEQ ID NO:31), YDND (SEQ ID NO:66), YENI (SEQ ID NO:67), YENL (SEQ ID NO:68), YKNQ (SEQ ID NO:72), YKNV (SEQ ID NO:73), or YANG (SEQ ID NO:87). In certain embodiments, the mutant YMNM motif consists of the amino acid sequence depicted in YSNV (SEQ ID NO:23), YENV (SEQ ID NO:22), or YKNI (SEQ ID NO:26). In certain embodiments, the mutant YMNM motif consists of the amino acid sequence depicted in YSNV (SEQ ID NO:23). In certain embodiments, the mutated YMNM motif binds to growth factor receptor-bound receptor 2 (Grb2) and / or Grb2-associated adaptor downstream of Shc (GADS).

[0009] In certain embodiments, the mutant YMNM motif does not bind to Grb2 and / or GADS. In certain embodiments, the mutant YMNM motif consists of the amino acid sequence depicted in YMxM (SEQ ID NO: 20), where x is not aspartic acid (N). In certain embodiments, the mutant YMNM motif consists of the amino acid sequence depicted in YMDM (SEQ ID NO: 32), YMPM (SEQ ID NO: 79), YMRM (SEQ ID NO: 37), or YMSM (SEQ ID NO: 80). In certain embodiments, the mutant YMNM motif consists of the amino acid sequence depicted in YMDM (SEQ ID NO: 32). In certain embodiments, the mutant YMNM motif consists of the amino acid sequence depicted in YbxM (SEQ ID NO: 33), where x is not aspartic acid (N) and b is not methionine (M). In certain embodiments, the mutant YMNM motif consists of the amino acid sequence set forth in YTHM (SEQ ID NO: 34), YVLM (SEQ ID NO: 35), YIAM (SEQ ID NO: 36), YVEM (SEQ ID NO: 83), YVKM (SEQ ID NO: 85), or YVPM (SEQ ID NO: 86). In certain embodiments, the mutant YMNM motif consists of the amino acid sequence set forth in YMxb (SEQ ID NO: 65), where x is not aspartic acid (N) and b is not methionine (M). In certain embodiments, the mutant YMNM motif consists of the amino acid sequence set forth in YMAP (SEQ ID NO: 77). In certain embodiments, the p85 subunit of PI3K signaling binds to the mutant YMNM motif.

[0010] In certain embodiments, the mutant YMNM motif does not bind to Grb2 and / or GADS or the p85 subunit of PI3K. In certain embodiments, the mutant YMNM motif consists of the amino acid sequence depicted in Ybxb (SEQ ID NO: 43), where x is not aspartic acid (N) and b is not methionine (M). In certain embodiments, the mutant YMNM motif consists of the amino acid sequence depicted in YGGG (SEQ ID NO: 44), YAAA (SEQ ID NO: 45), YFFF (SEQ ID NO: 46), YETV (SEQ ID NO: 69), YQQQ (SEQ ID NO: 70), YHAE (SEQ ID NO: 71), YLDL (SEQ ID NO: 74), YLIP (SEQ ID NO: 75), YLRV (SEQ ID NO: 76), YTAV (SEQ ID NO: 82), or YVHV (SEQ ID NO: 84). In certain embodiments, the mutant YMNM motif consists of the amino acid sequence depicted in YGGG (SEQ ID NO: 44).

[0011] In certain embodiments, the mutant YMNM motif can regulate PI3K signaling by restricting the number of methionine residues that can bind to the p85 subunit of PI3K. In certain embodiments, the mutant YMNM motif consists of the amino acid sequence set forth in YMNx (SEQ ID NO: 38) or YxNM (SEQ ID NO: 39), where x is not methionine (M). In certain embodiments, the mutant YMNM motif consists of the amino acid sequence set forth in YMNV (SEQ ID NO: 40), YENM (SEQ ID NO: 41), and YMNQ (SEQ ID NO: 42), YMNL (SEQ ID NO: 78), or YSNM (SEQ ID NO: 81).

[0012] In certain embodiments, the extracellular antigen binding domain binds to an antigen. In certain embodiments, the antigen is a tumor antigen or a pathogen antigen. In certain embodiments, the antigen is a tumor antigen. In certain embodiments, the tumor antigen is CD19, mesothelin, AXL, TIM3, HVEM, MUC16, MUC1, CAIX, CEA, CD8, CD7, CD10, CD20, CD22, CD30, CLL1, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD70, CD74, CD99, CD123, CD133, CD138, EGP-2, EGP-40, EpCAM, Erb-B (e.g., Eerb-B2, Erb-B3, Erb-B4), FBP, fetal acetylcholine receptor, folate receptor-alpha, GD2, GD3, HER-2, hTER Selected from the group consisting of T, IL-13R-α2, κ-light chain, KDR, LeY, L1 cell adhesion molecule, MAGE-A1, ERBB2, MAGEA3, CT83 (also known as KK-LC-1), p53, MART1, GP100, proteinase 3 (PR1), tyrosinase, survivin, hTERT, EphA2, NKG2D ligand, NY-ESO-1, carcinoembryonic antigen (h5T4), PSCA, PSMA, ROR1, TAG-72, VEGF-R2, WT-1, BCMA, CD44V6, NKCS1, EGF1R, EGFR-VIII, ADGRE2, CCR1, LILRB2, PRAME, HPV E6 oncoprotein, and HPV E7 oncoprotein. In certain embodiments, the tumor antigen is CD19.

[0013] In certain embodiments, the mutated YMNM motif consists of the amino acid sequence set forth in YMDM (SEQ ID NO: 32). In certain embodiments, the extracellular antigen-binding domain binds to CD19. In certain embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 51.

[0014] In certain embodiments, the mutated YMNM motif consists of the amino acid sequence set forth in YKNI (SEQ ID NO: 26). In certain embodiments, the extracellular antigen-binding domain binds to CD19. In certain embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 55.

[0015] In certain embodiments, the mutated YMNM motif consists of the amino acid sequence set forth in YENV (SEQ ID NO: 22). In certain embodiments, the extracellular antigen-binding domain binds to CD19. In certain embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 53.

[0016] In certain embodiments, the mutated YMNM motif consists of the amino acid sequence set forth in YSNV (SEQ ID NO: 64). In certain embodiments, the extracellular antigen-binding domain binds to CD19. In certain embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 57.

[0017] In certain embodiments, the mutated YMNM motif consists of the amino acid sequence set forth in YGGG (SEQ ID NO: 63). In certain embodiments, the extracellular antigen-binding domain binds to CD19. In certain embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 61.

[0018] The subject matter of the present disclosure also provides a cell comprising a CAR as described herein. In certain embodiments, the cell is an immunoresponsive cell. In certain embodiments, the cell is a lymphoid cell or a myeloid cell. In certain embodiments, the cell is selected from the group consisting of T cells, natural killer (NK) cells, and stem cells from which lymphoid cells can be differentiated. In certain embodiments, the cell is a T cell. In certain embodiments, the T cell is selected from the group consisting of cytotoxic T lymphocytes (CTLs), gamma delta T cells, tumor-infiltrating lymphocytes (TILs), regulatory T cells, natural killer T (NKT) cells, and tumor-reactive lymphocytes.

[0019] Furthermore, the subject matter of the present disclosure provides a composition comprising the cell described herein.In certain embodiments, the composition is a pharmaceutical composition further comprising a pharma-ceutically acceptable excipient.In certain embodiments, the composition is for treating and / or preventing neoplasm and / or pathogen infection.

[0020] The subject matter of the present disclosure further provides a method for reducing tumor burden in a subject.In certain embodiments, the method comprises administering to a subject the cell described herein or the composition described herein.In certain embodiments, the method reduces the number of tumor cells, reduces tumor size, and / or eradicates tumor in a subject.

[0021] The subject matter of the present disclosure further provides a method for treating and / or preventing a neoplasm. In certain embodiments, the method comprises administering to a subject a cell as described herein or a composition as described herein.

[0022] The subject matter of the present disclosure further provides a method for prolonging survival of a subject having a neoplasm. In certain embodiments, the method comprises administering to the subject a cell described herein or a composition described herein.

[0023] In certain embodiments, the neoplasm and / or tumor is selected from the group consisting of B-cell leukemia, B-cell lymphoma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), non-Hodgkin's lymphoma, Burkitt's lymphoma, acute myeloid leukemia (AML), and mixed phenotype acute leukemia (MPAL).

[0024] The subject matter of the present disclosure further provides a method for producing antigen-specific cells.In certain embodiments, the method comprises introducing into cells the nucleic acid sequence encoding the CAR described herein.In certain embodiments, the nucleic acid sequence is present on a vector.In certain embodiments, the vector is a retroviral vector.

[0025] The subject matter of the present disclosure further provides a nucleic acid molecule encoding a CAR as described herein. In certain embodiments, the nucleic acid molecule comprises the nucleotide sequence set forth in SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, or SEQ ID NO:58. The subject matter of the present disclosure further provides a vector comprising the nucleic acid molecule as described herein. In certain embodiments, the vector is a gamma-retroviral vector (rector).

[0026] The subject matter of the present disclosure further provides a host cell expressing the nucleic acid molecules described herein. In certain embodiments, the host cell is a T cell.

[0027] Furthermore, the subject matter of the present disclosure provides a kit comprising a CAR described herein, a cell described herein, a composition described herein, a nucleic acid molecule described herein, or a vector described herein. In certain embodiments, the kit further comprises written instructions for treating and / or preventing a neoplasm and / or a pathogen infection. 4. Brief description of the drawings [Brief description of the drawings]

[0028] [Figure 1] FIG. 1 is a schematic diagram of physiological CD28 signaling.

[0029] [Diagram 2] FIG. 2 is a schematic diagram of CD28 modification to modulate PI3Kp85 binding to CD28.

[0030] [Diagram 3]Figure 3A-D show that CD28-YKNI mutant CAR T cells had a strong killing capacity in vitro, which was comparable to certain CAR T cells. Human CD19-targeted CAR T cells expressing a truncated EGFR domain (Etah19) were co-cultured with CD19+NALM6 cells expressing GFP-ff luciferase (NALM6gL) at different effector:tumor ratios. Tumor cell lysis (relative to non-signaling CAR T cells) was measured by bioluminescence after 24 hours. h28Z: CD28-based + CD3Z signaling domain; hBBZ: 4-1BB-based + CD3Z signaling domain; h28h1XX: CD28-based + CD3Z signaling domain with mutant ITAM2 and ITAM3; hYKNIZ: mutant CD28-based (YMNM->YKNI) + CD3Z signaling domain. pStimx# indicates post-stimulation, and the number indicates the number of previous stimulations. Figure 3A shows the results of tumor cell lysis after preparation. Figure 3B shows the results of tumor cell lysis after one stimulation. Figure 3C shows the results of tumor cell lysis after two stimulations. Figure 3D shows the results of tumor cell lysis after four stimulations.

[0031] [Figure 4-1] Figures 4A-N show that mutant CD28 CAR T cells demonstrated distinct pro-inflammatory cytokine secretion profiles. Human CD19-targeted CAR T cells were cultured alone and co-cultured with CD19+NALM6 cells at a 1:1 effector:tumor ratio. After 24 hours, supernatants were collected and cytokines were measured utilizing a bead-based multiplex assay. Figures 4A-G show the cytokine profile from donor V, and Figures 4H-N show the cytokine profile from donor IV. (Figures 4A, 4H) GMCSF; (Figures 4B, 4I) IFN-γ; (Figures 4C, 4J) IL-13; (Figures 4D-4K) IL-17; (Figures 4E-4L) IL-9; (Figures 4F-4M) IL-2; (Figures 4G-4N) TNF-α. [Figure 4-2] Same as above. [Figure 4-3] Same as above. [Figure 4-4] Same as above. [Figure 4-5] Same as above. [Figure 4-6] Same as above. [Diagram 4-7] Same as above.

[0032] [Diagram 5] Figure 5 shows that CD28-YKNI mutant CAR T cells did not have any quantitative difference in proliferation in response to repeated antigen exposure. Human CD19-targeted CAR T cells were co-cultured with NALM6 at an E:T ratio of 1:5 and a concentration of 50,000 CAR T cells / mL. Approximately every 5 days, CAR T cells were counted and characterized by flow cytometry, and the starting number of tumor cells was added back into the culture (indicated by the arrow).

[0033] [Figure 6] Figure 6 shows that CD28-YKNI mutant CAR T cells retained the memory phenotype in the context of repeated antigen encounters, compared to CD28 and CD28-1xx CAR T cells. Human CD19-targeted CAR T cells were co-cultured with NALM6 at an E:T ratio of 1:5 and a concentration of 50,000 CAR T cells / mL. Approximately every 5 days, CAR T cells were counted and characterized by flow cytometry for memory phenotype (CD62L+), and the starting number of NALM6 tumor cells was added back into the culture (indicated by the arrow).

[0034] [Figure 7] Figure 7 shows that CD28-YKNI mutant CAR T cells maintained a relatively balanced CD8:CD4 ratio in the context of repeated antigen encounters compared to CD28 and CD28-1xx CAR T cells. Human CD19-targeted CAR T cells were co-cultured with NALM6 at an E:T ratio of 1:5 and a concentration of 50,000 CAR T cells / mL. Approximately every 5 days, CAR T cells were counted and characterized by flow cytometry for CD4 / CD8 distribution, and the starting number of NALM6 tumor cells was added back into the culture (indicated by the arrow).

[0035] [Figure 8-1] Figure 8 shows that CD28-YKNI mutant CAR T cells demonstrated lower blastogenesis after single or multiple activation. CAR T cells were co-cultured with NALM6gL at an initial E:T of 1:5 (single stimulation, blue). In parallel, CAR T cells were repeatedly stimulated with the same amount of tumor for a total of 5 stimulations (single stimulation every 12 hours; red). Approximately 10 days after the start of the co-culture, size / blastogenesis (assessed by forward scatter) was assessed by flow cytometry. [Figure 8-2] Same as above.

[0036] [Figure 9] Figures 9A-B show metabolic profiles measured in CAR T cells 9 days after single or multiple stimulations in donors A and B. Oxygen consumption rate (OCR) (Figure 9A) and extracellular acidification rate (ECAR) (Figure 9B) were measured in stimulated CAR T cells.

[0037] [Figure 10-1] Figures 10A-B show that CD28-YKNI mutant CAR T cells expressed lower levels of co-inhibitory molecules in the context of single or multiple stimulations. Expression of LAG3 and PD1 (Figure 10A) and TIM-3 and PD1 (Figure 10B) was measured in CD28-YKNI mutant CAR T cells (ah19hYKNIhZ) and wild-type CAR T cells (ah19h28hZ) under single or multiple stimulations. [Figure 10-2] Same as above. [Figure 10-3] Same as above. [Figure 10-4] Same as above.

[0038] [Figure 11]Figure 11 shows that CD28-YKNI mutant CD19-targeted CAR T cells outperformed standard CD28-based CAR T cells in vivo. NCG mice were inoculated with 106 NALM6gfp+ffLUC+ tumor cells and treated with CAR T cells 4 days later. Survival rates were plotted. CAR T cells were derived from two different healthy donors.

[0039] [Figure 12] FIG. 12 is a schematic diagram of exemplary CD28 mutants with altered PI3Kp85 and Grb2 / GADS binding ability relative to CD28.

[0040] [Figure 13] Figure 13 shows that CD28-YKNI mutant CAR T cells demonstrated comparable killing capacity in a 24-h killing assay. Human CD19-targeted CAR T cells expressing a truncated EGFR domain (Etah19) were co-cultured with CD19+NALM6 cells expressing GFP-ff luciferase (NALM6gL) at different effector:tumor ratios, and tumor cell lysis (vs. non-signaling CAR T cells) was measured by bioluminescence after 24 h.

[0041] [Figure 14] Figure 14 shows that CD28-Yxxx mutant CD19-targeted CAR T cells (YKNI, YENV, and YMDM) outperformed standard CD28-based CAR T cells in vitro. Human CD19-targeted CAR T cells were co-cultured with NALM6 at an E:T ratio of 1:5 and a starting concentration of 25,000 CAR T cells / mL. CAR+ and NALM6 concentrations were measured daily and plotted over 6 days.

[0042] [Figure 15-1]Figure 15 shows that CD28 mutants demonstrated a favorable exhaustion immune phenotype. CAR T cells were co-cultured with NALM6gL at an initial E:T of 1:5 (single stimulation). In parallel, CAR T cells were repeatedly stimulated with the same amount of tumor for a total of five stimulations (one stimulation every 12 hours). Approximately 10 days after the start of the co-culture, exhaustion markers (TIM3 and PD1) were evaluated by flow cytometry. [Figure 15-2] Same as above.

[0043] [Figure 16] Figure 16 shows the survival curves of NCG mice inoculated with 1x106 NALM6gfp+ffLUC+ tumor cells and treated with different CAR T cells.

[0044] [Figure 17] Figure 17 shows the survival curves of NCG mice inoculated with 1x106 NALM6gfp+ffLUC+ tumor cells and treated with different CAR T cells.

[0045] [Figure 18-1] Figure 18 shows bioluminescence images of NCG mice inoculated with 1x106 NALM6gfp+ffLUC+ tumor cells and treated with different CAR T cells. Bioluminescence was measured weekly. [Figure 18-2] Same as above. [Figure 18-3] Same as above. [Figure 18-4] Same as above. [Figure 18-5] Same as above.

[0046] [Figure 19-1] Figure 19 shows that CD28-Yxxx mutant CD19-targeted CAR T cells demonstrated potent long-term cytotoxicity in vitro. Human CD19-targeted CAR T cells (line with diamonds) were co-cultured with NALM6gL (line with circles) at an E:T ratio of 1:15. CAR+T cell and NALM6 concentrations were measured daily and plotted in cells / mL over 7 days. [Figure 19-2] Same as above.

[0047] [Figure 20] Figure 20 shows that CD28-Yxxx mutant CD19-targeted CAR T cells displayed a favorable exhaustion immune phenotype. CAR T cells were co-cultured with NALM6gL at an E:T ratio of 1:15 or 1:30. After 5 days, the expression of exhaustion markers, including LAG3, TIM3 and PD1, in CAR T cells was evaluated by flow cytometry.

[0048] [Figure 21] Figure 21 shows the survival curve of NCG mice receiving CD28-Yxxx mutant CD19-targeting CAR T cells. NCG mice were inoculated with 1x106 NALM6gfp+ffLUC+ tumor cells and treated with 500,000 CAR T cells 4 days later. CAR T cells were derived from two different healthy donors.

[0049] [Figure 22] Figure 22 shows the survival curve of NCG mice receiving CD28-Yxxx mutant CD19-targeting CAR T cells. NCG mice were inoculated with 1x106 NALM6gfp+ffLUC+ tumor cells and treated with 200,000 CAR T cells 4 days later. CAR T cells were derived from a single healthy donor.

[0050] [Diagram 23] Figure 23 shows that CD28-Yxxx mutant CD19-targeted CAR T cells exhibited enhanced proliferation in vitro, regardless of antigen density. Human CD19-targeted CD28-Yxxx mutant CAR T cells were co-cultured with NALM6gL at either high or low CD19 antigen density, at an E:T ratio of 1:1. Every 6 days, CAR+T cells were counted and restimulated with NALM6gL for a total of three stimulations.

[0051] [Figure 24-1]Figures 24A-C show that CD28-Yxxx mutant CD19-targeted CAR T cells demonstrated a unique cytokine secretion profile upon exposure to antigen. Human CD19-targeted CD28-Yxxx mutant CAR T cells were co-cultured with NALM6gL. After 24 hours, supernatants were collected and cytokines including interleukin-2 (Figure 24A), TNF-α (Figure 24A), GM-CSF (Figure 24B), interferon-γ (Figure 24B), IL-9 (Figure 24C), and IL-17 (Figure 24C) were measured by Luminex bead-based multiplex assay. [Figure 24-2] Same as above. [Figure 24-3] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0052] 5. Detailed Description The subject matter of the present disclosure provides a chimeric antigen receptor (CAR) comprising at least one costimulatory signaling domain comprising a CD28 polypeptide comprising a mutant YMNM motif. The CD28 polypeptide has reduced recruitment of the p85 subunit of phosphoinositide 3-kinase (PI3K) signaling compared to a CD28 molecule comprising a natural YMNM motif. In certain embodiments, the p85 subunit of PI3K signaling does not bind to the mutant YMNM motif. In certain embodiments, the p85 subunit of PI3K signaling does not bind to the mutant YMNM motif, and growth factor receptor-bound receptor 2 (Grb2) and / or downstream Grb2-associated adaptor (GADS) of Shc bind to the mutant YMNM motif. In certain embodiments, Grb2 and / or GADS do not bind to the mutant YMNM motif. In certain embodiments, Grb2 and / or GADS do not bind to the mutant YMNM motif, and the p85 subunit of PI3K signaling binds to the mutant YMNM motif.

[0053] The subject matter of the present disclosure also provides cells (e.g., immunoresponsive cells, e.g., T cells or NK cells) comprising the CAR of the present disclosure. The subject matter of the present disclosure further provides methods of using the cells of the present disclosure to induce and / or enhance immune responses to target antigens and / or to treat and / or prevent neoplasms or tumors, and / or pathogen infections. The subject matter of the present disclosure is based, at least in part, on the discovery that cells comprising a CAR comprising a mutant CD28 intracellular motif (i.e., a mutant YMNM motif) exhibit enhanced anti-tumor effects compared to cells comprising a CAR comprising a native CD28 intracellular motif (i.e., a native YMNM motif).

[0054] Non-limiting embodiments of the present disclosure are illustrated in the specification and by way of examples.

[0055] For purposes of clarity of disclosure, and not by way of limitation, the detailed description is divided into the following subsections: 5.1. Definition; 5.2. Chimeric antigen receptor (CAR); 5.3.Cell; 5.4. Compositions and Vectors; 5.5. Polypeptides; 5.6. Formulation and Administration; 5.7. Method of treatment; and 5.8.Kit 5.1.Definition

[0056] Unless otherwise defined, all technical and scientific terms used herein have the meanings that are commonly understood by those skilled in the art.The following references provide those skilled in the art with the general definitions of many of the terms used in the subject matter of this disclosure: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd Edition, 1994); The Cambridge Dictionary of Science and Technology (Walker, ed., 1988); The Glossary of Genetics, 5th Edition, R.Rieger et al. (ed.), Springer Verlag (1991); and Hale&Marham, The Harper Collins Dictionary of Biology (1991).

[0057] As used herein, the term "about" or "approximately" means within an acceptable error range of a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., on the limitations of the measurement system. For example, "about" can mean within 3 or more than 3 standard deviations, according to the practice in the art. Alternatively, "about" can mean within a range of up to 20%, e.g., up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, e.g., within 5-fold or 2-fold of a value.

[0058] "Immunoresponsive cell" refers to a cell that functions in immune response, or its precursor or progeny. In certain embodiments, the immunoresponsive cell is a cell of lymphoid system. Non-limiting examples of lymphoid system cells include T cells, natural killer (NK) cells, B cells, and stem cells from which lymphoid cells can be differentiated. In certain embodiments, the immunoresponsive cell is a cell of myeloid system.

[0059] "Activating an immunoresponsive cell" refers to the induction of signal transduction or change in protein expression in a cell that results in the initiation of an immune response. For example, when CD3 chains cluster in response to ligand binding and immunoreceptor tyrosine-based inhibitory motifs (ITAMs), a signal transduction cascade is produced. In certain embodiments, when endogenous TCR or exogenous CAR binds to an antigen, the formation of an immune synapse occurs, which includes the clustering of many molecules (e.g., CD4 or CD8, CD3γ / δ / ε / ζ, etc.) near the bound receptor. This clustering of membrane-bound signaling molecules allows the ITAM motifs contained within the CD3 chains to be phosphorylated. This phosphorylation then initiates the T cell activation pathway that ultimately activates transcription factors such as NF-κB and AP-1. These transcription factors induce global gene expression in T cells to increase IL-2 production for proliferation and expression of master regulator T cell proteins to initiate a T cell-mediated immune response.

[0060] "Stimulating immune responsive cells" refers to signals that result in robust and sustained immune responses. In various embodiments, this occurs after activation of immune cells (e.g., T cells) or occurs simultaneously, mediated by receptors including, but not limited to, CD28, CD137 (4-1BB), OX40, CD40 and ICOS. Receiving multiple stimulatory signals may be important to initiate robust and long-lasting T cell-mediated immune responses. T cells can quickly become inhibited and unresponsive to antigens. The effects of these costimulatory signals may vary, but they generally result in increased gene expression to generate long-lived, proliferative, and anti-apoptotic T cells that robustly respond to antigens, toward complete and sustained eradication.

[0061] As used herein, the term "antigen-recognizing receptor" refers to a receptor that can activate an immunoresponsive cell (eg, a T cell) in response to binding to an antigen.

[0062] As used herein, "CDR" is 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, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 4th USD Department of Health and Human Services, National Institutes of Health (1987). Generally, antibodies contain three heavy chain and three light chain CDRs, i.e., CDR regions, in the variable region. The CDRs provide the majority of contact residues for the binding of the antibody to the antigen or epitope. In certain embodiments, the CDR regions are described using the Kabat system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., USDepartment of Health and Human Services (1991); NIH Publication No. 91-3242). In certain embodiments, the CDRs are identified according to the IMGT numbering system. As used herein, the term "single chain variable fragment" or "scFv" refers to a V H ::V L Covalently linked immunoglobulin heavy chains (V H ) and light chain (V L ) is a fusion protein of the variable region of H and V L are either directly bonded or V H N-terminus of V L and the C-terminus of V H The C-terminus of V L The linker is usually glycine-rich for flexibility and serine or threonine-rich for solubility. The linker is usually glycine-rich for flexibility and serine or threonine-rich for solubility.

[0063] "Linker," as used herein, is intended to mean a functional group (e.g., a chemical or polypeptide) that covalently bonds two or more polypeptides or nucleic acids so that they are connected to one another. As used herein, a "peptide linker" refers to a peptide linker that is used to couple two proteins together (e.g., V H and V L "Coupling" refers to one or more amino acids used to couple a domain together.

[0064] Despite the removal of the constant region and the introduction of the linker, the scFv protein retains the specificity of the original immunoglobulin. Single chain Fv polypeptide antibodies can be prepared by the VFv synthesis method as described by Huston et al., Proc Nat Acad Sci USA (1988); 85:5879-5883; U.S. Patent Nos. 5,091,513, 5,132,405 and 4,956,778; and U.S. Patent Application Publication Nos. 20050196754 and 20050196754. H and V LIt can be expressed from a nucleic acid containing the coding sequence. Agonistic scFvs with inhibitory activity have been described (Zhao et al., Hyrbidoma (Larchmt) 2008;27(6):455-51; Peter et al., J Cachexia Sarcopenia Muscle (2013);4(1):79-86; Shieh et al., J Imunol (2009);183(4):2277-85; Giomarelli et al., Thromb Haemost (2007);97(6):955-63; Fife et al., JCI (2006);116(8):2252-61; Brocks et al., Immunotechnology, (1997);3(3):173-84; Moosmayer et al., Ther Immunol (1995);2(10):31-40). Agonistic scFvs with stimulatory activity have been described (Peter et al., J Biol Chem (2003); 25278(38):36740-7; Xie et al., Nat Biotech (1997); 15(8):768-71; Ledbetter et al., Crit Rev Immunol (1997); 17(5-6):427-55; Ho et al., BioChem Biophys Acta (2003); 1638(3):257-66).

[0065] As used herein, the term "affinity" refers to a measure of binding strength. Affinity may depend on the closeness of the stereochemical fit between the antibody binding site and the antigenic determinant, the size of the contact area between them, and / or the distribution of charged and hydrophobic groups. Methods for calculating the affinity of an antibody to an antigen are known in the art, including but not limited to various antigen binding experiments, such as functional assays (e.g., flow cytometry assays).

[0066] The term "chimeric antigen receptor" or "CAR" as used herein refers to a molecule that includes an extracellular antigen-binding domain fused to an intracellular signaling domain that can activate an immunoresponsive cell, and a transmembrane domain. In certain embodiments, the extracellular antigen-binding domain of the CAR includes an scFv. The scFv can be derived from the fusion of the variable heavy and light regions of an antibody. Alternatively or additionally, the scFv can be derived from Fab's (e.g., obtained from a Fab library instead of from an antibody). In certain embodiments, the scFv is fused to a transmembrane domain and then to an intracellular signaling domain.

[0067] As used herein, the term "nucleic acid molecule" includes any nucleic acid molecule that encodes a polypeptide of interest. Such nucleic acid molecules need not be 100% homologous or identical to an endogenous nucleic acid sequence, but may exhibit substantial identity.

[0068] "Substantially identical" or "substantially homologous" refers to a polypeptide or nucleic acid molecule that exhibits at least about 50% identity or homology to a reference amino acid sequence (e.g., any one of the amino acid sequences described herein) or a reference nucleic acid sequence (e.g., any one of the nucleic acid sequences described herein). In certain embodiments, such a sequence is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical or homologous to the amino acid sequence or nucleic acid sequence used for comparison.

[0069] The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = number of identical positions / total number of positions x 100), taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of the percent identity between two sequences can be accomplished using a mathematical algorithm.

[0070] Sequence identity can be determined using sequence analysis software (e.g., Sequence Analysis The degree of identity can be measured using the BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs (Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determine the degree of identity, the BLAST program can be used, and a probability score between e-3 and e-100 indicates closely related sequences.

[0071] The percent homology or identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) as implemented in the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Additionally, the percent homology or identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) algorithm as implemented in the GAP program in the GCG software package (available at www.gcg.com) using either a Blossum62 matrix or a PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0072] Additionally or alternatively, the amino acid sequences of the subject matter of the present disclosure can be further used as a "query sequence" to perform searches against public databases, for example to identify related sequences. Such searches can be performed using the XBLAST program (version 2.0) of Altschul et al. (1990) J. Mol. Biol. 215:403-10. BLAST protein searches can be performed using the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the specified sequences disclosed herein (e.g., heavy and light chain variable region sequences of scFv m903, m904, m905, m906, and m900). To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (eg, XBLAST and NBLAST) can be used.

[0073] An "effective amount" is an amount sufficient to affect beneficial or desired clinical results upon treatment. An effective amount can be administered to a subject in one or more doses. In certain embodiments, an effective amount can be an amount sufficient to palliate, improve, stabilize, reverse or delay the progression of a disease, or reduce the pathological severity of a disease. An effective amount can be determined by a physician on a case-by-case basis, and is within the skill of a person of ordinary skill in the art. When determining the appropriate dosage to achieve an effective amount, several factors are usually taken into consideration. These 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 cells being administered.

[0074] By "modulate" is meant to alter, either positively or negatively. Exemplary modulations include changes of about 1%, about 2%, about 5%, about 10%, about 25%, about 50%, about 75%, or about 100%.

[0075] By "increase" is meant a positive alteration of at least about 5%. The alteration can be about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, about 100% or more.

[0076] By "reduce" is meant to negatively alter by at least about 5%. The alteration can be about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, or even about 100%.

[0077] The terms "isolated," "purified," or "biologically pure" refer to a material that is free to various degrees of components that normally accompany it as found in its native state. "Isolate" refers to a degree of separation from the original source or surroundings. "Purify" refers to a degree of separation greater than isolation. A "purified" or "biologically pure" protein is sufficiently free of other materials such that any impurities do not substantially affect the biological properties of the protein or cause other deleterious consequences. That is, a nucleic acid or peptide is purified if it is substantially free of cellular material, viral material, or culture medium if produced by recombinant DNA technology, or substantially free of chemical precursors or other chemicals if chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" may refer to a nucleic acid or protein that gives rise to essentially one band in an electrophoretic gel. In the case of proteins that can be subject to modifications, such as phosphorylation or glycosylation, different modifications may result in different isolated proteins that can be purified separately.

[0078] By "isolated cell" is meant a cell that has been separated from molecules and / or cellular components that naturally accompany the cell.

[0079] As used herein, the term "antigen-binding domain" refers to a domain capable of specifically binding to a particular antigenic determinant or set of antigenic determinants present on a cell.

[0080] "Neoplasm" refers to a disease characterized by pathological proliferation of cells or tissues and subsequent migration to or invasion of other tissues or organs. Neoplastic growth is typically uncontrolled and progressive, occurring under conditions that would not induce or cause the cessation of normal cell proliferation. Neoplasms can affect a variety of cell types, tissues, or organs, including but not limited to organs selected from the group consisting of bladder, bone, brain, breast, cartilage, glia, esophagus, fallopian tube, gallbladder, heart, intestine, kidney, liver, lung, lymph nodes, nervous tissue, ovaries, pancreas, prostate, skeletal muscle, skin, spinal cord, spleen, stomach, testes, thymus, thyroid, trachea, urogenital tract, ureter, urethra, uterus, and vagina, or tissues or cell types thereof. Neoplasia includes cancers such as sarcoma, carcinoma, or plasmacytoma (malignant tumor of plasma cells).

[0081] By "signal sequence" or "leader sequence" is meant a peptide sequence (eg, 5, 10, 15, 20, 25 or 30 amino acids) present at the N-terminus of a newly synthesized protein that directs entry into the secretory pathway.

[0082] The terms "comprises" and "comprising" are intended to have the broad meaning ascribed to them in U.S. patent law and can mean "includes," "including," etc.

[0083] As used herein, "treatment" refers to clinical intervention that seeks to change the disease course of the individual or cell being treated, and can be performed either for prevention or during the course of clinical pathology.The therapeutic effect of treatment includes, but is not limited to, prevention of disease occurrence or recurrence, alleviation of symptoms, reduction of the direct or indirect pathological severity of disease, prevention of metastasis, slowing the rate of disease progression, amelioration or alleviation of disease state, and remission or improvement of prognosis.By preventing the progression of disease or disorder, treatment can prevent the deterioration of the disorder in subjects who have been affected or diagnosed or suspected of having the disorder, but treatment can also prevent the onset of the disorder or the symptoms of the disorder in subjects who are at risk of the disorder or suspected of having the disorder.

[0084] An "individual" or "subject" herein is a vertebrate, e.g., a human or a non-human animal, e.g., a mammal. Mammals include, but are not limited to, humans, primates, livestock, sport animals, rodents and pets. Non-limiting examples of non-human animal subjects include rodents, such as mice, rats, hamsters and guinea pigs; rabbits; dogs; cats; sheep; pigs; goats; cows; horses; and non-human primates, such as apes and monkeys. The term "immunocompromised" as used herein refers to a subject with an immune deficiency. The subject is highly vulnerable to opportunistic infections, infections caused by organisms that do not normally cause disease in people with healthy immune systems, but can affect people with poorly functioning or suppressed immune systems.

[0085] Other aspects of the presently disclosed subject matter are described in the disclosure that follows and are within the scope of the presently disclosed subject matter. 5.2. Chimeric Antigen Receptors (CARs)

[0086] In certain embodiments, the present disclosure provides a chimeric antigen receptor (CAR) comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising at least one costimulatory signaling domain comprising a CD28 polypeptide comprising a mutated CD28 intracellular motif, i.e., a mutated YMNM motif.

[0087] CAR is an engineered receptor that grafts or confers a specificity of interest to immune effector cells. CAR can be used to graft the specificity of monoclonal antibodies into T cells; with the transfer of their coding sequences facilitated by retroviral vectors.

[0088] There are three generations of CARs. "First generation" CARs are typically composed of an extracellular antigen binding domain (e.g., scFv) fused to a transmembrane domain fused to a cytoplasmic / intracellular signaling domain. "First generation" CARs can provide de novo antigen recognition and bind to CD4 through their CD3 ζ chain signaling domain in a single fusion molecule, independent of HLA-mediated antigen presentation. + T cells and CD8 +The "second generation" CARs can cause both activation of T cells and activation of T cells. The "second generation" CARs add intracellular signaling domains from various costimulatory molecules (e.g., CD28, 4-1BB, ICOS, OX40) to the cytoplasmic tail of the CAR to provide additional signals to T cells. The "second generation" CARs include those that provide both costimulation (e.g., CD28 or 4-1BB) and activation (CD3ζ). The "third generation" CARs include those that provide multiple costimulation (e.g., CD28 and 4-1BB) and activation (CD3ζ). In certain embodiments, the antigen recognition receptor is a second generation CAR. In certain embodiments, the CAR comprises an extracellular antigen binding domain that binds to an antigen, a transmembrane domain, and an intracellular signaling domain, and the intracellular signaling domain comprises a costimulatory signaling domain. In certain embodiments, the CAR further comprises a hinge / spacer region. In certain embodiments, the antigen recognition receptor is a third generation CAR that comprises multiple costimulatory signaling domains.

[0089] In certain embodiments, a CAR can comprise an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, where the extracellular antigen-binding domain specifically binds to an antigen, which can be a tumor antigen or a pathogen antigen. 5.2.1. Antigen

[0090] In certain embodiments, the CAR binds to a tumor antigen or a pathogen antigen.

[0091] In certain embodiments, the CAR binds to a tumor antigen. Any tumor antigen (e.g., an antigenic peptide) can be used in the tumor-associated embodiments described herein. Sources of antigens include, but are not limited to, cancer proteins. Antigens can be expressed as peptides or as intact proteins or portions thereof. Intact proteins or portions thereof can be native or mutagenized. In certain embodiments, antigens are expressed in tumor tissue. Non-limiting examples of tumor antigens include mesothelin, AXL, TIM3, HVEM, CD19, MUC16, MUC1, CAIX, CEA, CD8, CD7, CD10, CD20, CD22, CD30, CLL1, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD70, CD74, CD99, CD123, CD133, CD138, EGP-2, EGP-40, EpCAM, Erb-B (e.g., Erb-B2, Erb-B3, Erb-B4), FBP, fetal acetylcholine receptor, folate receptor-alpha, GD2, GD3, HER-2, hT These include ERT, IL-13R-α2, κ-light chain, KDR, LeY, L1 cell adhesion molecule, MAGE-A1, MAGEA3, CT83 (also known as KK-LC-1), p53, MART1, GP100, proteinase 3 (PR1), tyrosinase, survivin, hTERT, EphA2, NKG2D ligand, NY-ESO-1, carcinoembryonic antigen (h5T4), PSCA, PSMA, ROR1, TAG-72, VEGF-R2, WT-1, BCMA, CD44V6, NKCS1, EGF1R, EGFR-VIII, ADGRE2, CCR1, LILRB2, PRAME, HPV E6 oncoprotein, and HPV E7 oncoprotein. In certain embodiments, the tumor antigen is CD19.

[0092] In certain embodiments, the CAR binds to a CD19 polypeptide. In certain embodiments, the CAR binds to a human CD19 polypeptide. In certain embodiments, the human CD19 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1 or a portion thereof. SEQ ID NO: 1 is provided below. [ka]

[0093] In certain embodiments, the CAR binds to the extracellular domain of CD19 (e.g., human CD19).

[0094] In certain embodiments, the CAR binds to a pathogen antigen, for example, for use in the treatment and / or prevention of pathogen infection or other infectious disease. Non-limiting examples of pathogens include viruses, bacteria, fungi, parasites and protozoa that can cause disease.

[0095] Non-limiting examples of viruses include Retroviridae (e.g., human immunodeficiency viruses, e.g., HIV-1 (HDTV-III, LAVE or HTLV-III / LAV, or HIV-III; and other isolates such as HIV-LP); Picornaviridae (e.g., poliovirus, hepatitis A virus; enterovirus, human coxsackievirus, rhinovirus, echovirus); Caliciviridae (Calciviridae) (e.g., strains causing gastroenteritis); Togaviridae (e.g., equine encephalitis virus, rubella virus); Flaviviridae (Flaviridae) (e.g., dengue virus, encephalitis virus, yellow fever virus);Coronaviridae (Coronoviridae) (e.g. coronavirus);Rhabdoviridae (e.g. vesicular stomatitis virus, rabies virus);Filoviridae (e.g. Ebola virus);Paramyxoviridae (e.g. parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus);Orthomyxoviridae (e.g. influenza virus);Bunyaviridae (Bungaviridae) (e.g. hantavirus, bungavirus, phlebovirus and naira virus);Arenaviridae (Arena viridae) (hemorrhagic fever viruses); Reoviridae (e.g., reovirus, orbivirus, and rotavirus); Birnaviridae; Hepadnaviridae (hepatitis B viruses); Parvoviridae (Parvovirida) (parvoviruses); Papovaviridae (papillomaviruses, polyomaviruses); Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex viruses (HSV) 1 and 2, varicella-zoster virus, cytomegalovirus (CMV), herpes viruses; Poxviridae (e.g., smallpox virus, vaccinia virus, poxvirus); and Iridoviridae (e.g., African swine fever virus);as well as unclassified viruses (e.g., agents of delta hepatitis (thought to be a defective satellite of hepatitis B virus), non-A, non-B hepatitis (class 1 = internally transmitted, class 2 = agents of parenteral transmission (i.e., hepatitis C); Norwalk and related viruses, and astroviruses), human papillomaviruses (i.e., HPV), JC virus, Epstein-Barr virus, Merkel cell polyomavirus;

[0096] Non-limiting examples of bacteria include Pasteurella, Staphylococci, Streptococcus, Escherichia coli, Pseudomonas species, and Salmonella species. Specific examples of infectious bacteria include Helicobacter pyloris, Borelia burgdorferi, Legionella pneumophilia, Mycobacteria species (e.g., M. tuberculosis, M. avium, M. intracellulare, M. kansaii, M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae (Group B Streptococcus), Streptococcus (viridans group), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic species), Streptococcus pneumoniae, pathogenic Campylobacter species, Enterococcus species, Haemophilus influenzae, Bacillus antracis, corynebacterium diphtheriae, corynebacterium species, Erysipelothrix rhusiopathiae, Clostridium perfringers, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturella multocida, Bacteroides species, Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponema pertenue, Leptospira, Rickettsia, clostridium difficile, and Actinomyces israelli.

[0097] In certain embodiments, the pathogen antigen is a viral antigen present in cytomegalovirus (CMV), a viral antigen present in Epstein-Barr virus (EBV), a viral antigen present in human immunodeficiency virus (HIV), or a viral antigen present in influenza virus. 5.2.2. Extracellular Antigen-Binding Domain of CAR

[0098] In certain embodiments, the extracellular antigen-binding domain comprises scFv. In certain embodiments, the scFv is human scFv. In certain embodiments, the scFv is humanized scFv. In certain embodiments, the scFv is mouse scFv. In certain embodiments, the scFv is identified by screening an scFv phage library with antigen-Fc fusion protein.

[0099] In certain embodiments, the extracellular antigen-binding domain comprises a Fab. In certain embodiments, the Fab is cross-linked. In certain embodiments, the extracellular antigen-binding domain comprises a F(ab) 2 Any of the foregoing molecules may be included in a fusion protein with a heterologous sequence to form an extracellular antigen-binding domain.

[0100] In certain embodiments, the extracellular antigen binding domain of the CAR (e.g., scFv) is about 1×10 -6 M or less dissociation constant (K d In certain embodiments, K d is about 1 x 10 -6 M or less, approximately 1 x 10 -7 M or less, approximately 1 x 10 -8 M or less, or about 1 x 10 -9 In certain non-limiting embodiments, K d is about 1 x 10 -8 M or less. In certain non-limiting embodiments, K d is about 1×10- 9 M or less.

[0101] The binding of the extracellular antigen-binding domain of the CAR can be confirmed, for example, by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growth inhibition), or Western blot assay. Each of these assays generally detects the presence of a particular protein-antibody complex of interest by using a labeled reagent (e.g., antibody, or scFv) specific for the complex of interest. For example, scFv can be radioactively labeled and used in radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March 1986, incorporated herein by reference). Radioisotopes can be detected by means such as a gamma counter or scintillation counter or by autoradiography. In certain embodiments, the 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 one embodiment, the human scFv is labeled with GFP.

[0102] In certain embodiments, the CDRs are identified according to the IMGT numbering system.

[0103] In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) of the CAR comprises or consists of the amino acid sequence set forth in SEQ ID NO:2 and specifically binds to a CD19 polypeptide (e.g., a human CD19 polypeptide, e.g., a human CD19 polypeptide having the amino acid sequence SEQ ID NO:1, or a portion thereof).

[0104] In certain embodiments, the extracellular antigen binding domain of the CAR (e.g., scFv) comprises an amino acid sequence that is at least about 80% (e.g., at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:3. H For example, the extracellular antigen binding domain of a CAR (e.g., scFv) can comprise an VFv that comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO:3. H In certain embodiments, the extracellular antigen-binding domain comprises the V H SEQ ID NO:3 is provided in Table 1 below.

[0105] In certain embodiments, the extracellular antigen binding domain of the CAR (e.g., scFv) comprises an amino acid sequence that is at least about 80% (e.g., at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:4. LFor example, the extracellular antigen binding domain of the CAR (e.g., scFv) can comprise a VFv that comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO:4. L In certain embodiments, the extracellular antigen-binding domain comprises the V H SEQ ID NO:4 is provided in Table 1 below.

[0106] In certain embodiments, the extracellular antigen-binding domain of the CAR comprises the amino acid sequence set forth in SEQ ID NO:3. H and V comprising the amino acid sequence set forth in SEQ ID NO:4 L In certain embodiments, V H and V L are linked via a linker. In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO: 5. SEQ ID NO: 5 is provided below. [ka]

[0107] In certain embodiments, the extracellular antigen binding domain (e.g., scFv) of the CAR comprises a VF comprising the amino acid sequence set forth in SEQ ID NO:6, or a conservative modification thereof. H CDR1, a V comprising the amino acid sequence set forth in SEQ ID NO:7 or a conservative modification thereof H CDR2 and V comprising the amino acid sequence set forth in SEQ ID NO:8 or a conservative modification thereof H SEQ ID NOs: 6 to 8 are provided in Table 1.

[0108] In certain embodiments, the extracellular antigen binding domain of the CAR (e.g., scFv) comprises a VFV comprising the amino acid sequence set forth in SEQ ID NO: 9, or a conservative modification thereof. L CDR1, a V comprising the amino acid sequence set forth in SEQ ID NO: 10 or a conservative modification thereofL CDR2, and a V domain having the amino acid sequence set forth in SEQ ID NO:11 or a conservative modification thereof L SEQ ID NOs: 9 to 11 are provided in Table 1.

[0109] In certain embodiments, the extracellular antigen binding domain (e.g., scFv) of the CAR comprises a VF comprising the amino acid sequence set forth in SEQ ID NO:6, or a conservative modification thereof. H CDR1, a V comprising the amino acid sequence set forth in SEQ ID NO:7 or a conservative modification thereof H CDR2, a V comprising the amino acid sequence set forth in SEQ ID NO:8 or a conservative modification thereof H CDR3, a V comprising the amino acid sequence set forth in SEQ ID NO:9 or a conservative modification thereof L CDR1, V with the amino acid sequence set forth in SEQ ID NO: 10 or conservative modifications L CDR2, and a V domain having the amino acid sequence set forth in SEQ ID NO:11 or a conservative modification thereof L Includes CDR3.

[0110] In certain embodiments, the extracellular antigen binding domain of the CAR (e.g., scFv) comprises the amino acid sequence set forth in SEQ ID NO:6. H CDR1, V comprising the amino acid sequence set forth in SEQ ID NO:7 H CDR2, comprising the amino acid sequence set forth in SEQ ID NO:8 H CDR3, V comprising the amino acid sequence set forth in SEQ ID NO:9 L CDR1, V comprising the amino acid sequence set forth in SEQ ID NO:10 L CDR2, and V comprising the amino acid sequence set forth in SEQ ID NO:11 L Includes CDR3. Table 1 [Table 1]

[0111] As used herein, the term "conservative sequence modification" refers to an amino acid modification that does not significantly affect or alter the binding properties of a CAR of the present disclosure (e.g., the extracellular antigen-binding domain of a CAR) that contains the amino acid sequence. Conservative modifications can include amino acid substitutions, additions, and deletions. Modifications can be introduced into the human scFv of a CAR of the present disclosure by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Amino acids can be classified into groups according to physicochemical properties, such as charge and polarity. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid within the same group. For example, amino acids can be classified according to charge: positively charged amino acids include lysine, arginine, histidine, negatively charged amino acids include aspartic acid, glutamic acid, and neutrally charged amino acids include alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. Furthermore, amino acids can be classified according to polarity: polar amino acids include arginine (basic polar), asparagine, aspartic acid (acidic polar), glutamic acid (acidic polar), glutamine, histidine (basic polar), lysine (basic polar), serine, threonine, and tyrosine; non-polar amino acids include alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine. Thus, one or more amino acid residues in a CDR region can be replaced with other amino acid residues from the same group, and the altered antibody can be tested for retained function (i.e., the functions described in (c) to (l) above) using the functional assays described herein. In certain embodiments, no more than one, no more than two, no more than three, no more than four, no more than five residues in a given sequence or CDR region are altered.

[0112] V has at least about 80%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% (e.g., about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) homology or identity to a specified sequence (e.g., SEQ ID NOs: 3 and 4). H and / or V L The amino acid sequence may contain substitutions (e.g., conservative substitutions), insertions, or deletions compared to the specified sequence(s), but retain the ability to bind to the target antigen (e.g., CD19). In certain embodiments, a total of 1-10 amino acids are substituted, inserted, and / or deleted in the specified sequence (e.g., SEQ ID NOs: 3 and 4). In certain embodiments, the substitutions, insertions, or deletions occur in the regions outside the CDRs (e.g., FRs) of the extracellular antigen-binding domain. In certain embodiments, the extracellular antigen-binding domain is a V-seq sequence selected from the group consisting of SEQ ID NOs: 3 and 4, including post-translational modifications of the sequences (SEQ ID NOs: 3 and 4). H and / or V L Contains arrays. 5.2.3.Transmembrane domain of CAR

[0113] In certain non-limiting embodiments, the transmembrane domain of the CAR comprises a hydrophobic alpha helix that spans at least a portion of the membrane. Different transmembrane domains result in different receptor stabilities. After antigen recognition, receptor clusters and signals are transmitted to cells. In certain embodiments, the transmembrane domain of the CAR comprises the native or modified transmembrane domain of CD8, CD28, CD3zeta, CD4, 4-1BB, OX40, ICOS, CD84, CD166, CD8a, CD8b, ICAM-1, CTLA-4, CD27, CD40, NKGD2, or combinations thereof.

[0114] In certain embodiments, the transmembrane domain of the CAR comprises a CD28 polypeptide (e.g., a transmembrane domain of CD28 or a portion thereof). In certain embodiments, the transmembrane domain of the CAR comprises a transmembrane domain of human CD28 or a portion thereof. In certain embodiments, the CD28 polypeptide comprises or consists of an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% homologous or identical to an amino acid sequence having NCBI reference number: NP_006130 (SEQ ID NO: 12), or a fragment thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. In certain embodiments, the CD28 polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 12 and is at least 20, or at least 30, or at least 40, or at least 50, and up to 220 amino acids in length. In certain embodiments, the CD28 polypeptide comprises or consists of the amino acid sequence of amino acids 1-220, 1-50, 50-100, 100-150, 150-200, 153-179, or 200-220 of SEQ ID NO: 12. In certain embodiments, the transmembrane domain of the CAR comprises a CD28 polypeptide comprising or consisting of amino acids 153-179 of SEQ ID NO: 12. SEQ ID NO: 12 is provided below. [ka]

[0115] An exemplary nucleotide sequence encoding amino acids 153-179 of SEQ ID NO:12 is set forth in SEQ ID NO:13, provided below. [ka]

[0116] In certain embodiments, the transmembrane domain of the CAR comprises the transmembrane domain of mouse CD28 or a portion thereof. In certain embodiments, the CD28 polypeptide comprises or consists of an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% homologous or identical to the sequence having NCBI reference number: NP_031668.3 (SEQ ID NO: 14), or a fragment thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. In certain non-limiting embodiments, the CD28 polypeptide comprises or consists of an amino acid sequence that is a continuous portion of SEQ ID NO: 14, and is at least 20, or at least 30, or at least 40, or at least 50, and up to 218 amino acids in length. In certain embodiments, the CD28 polypeptide comprises or consists of the amino acid sequence of amino acids 1-220, 1-50, 50-100, 100-150, 150-200, 151-177, or 200-218 of SEQ ID NO: 14. In certain embodiments, the transmembrane domain of the CAR comprises a CD28 polypeptide comprising or consisting of amino acids 151-177 of SEQ ID NO: 14. SEQ ID NO: 14 is provided below. [ka]

[0117] In certain embodiments, the transmembrane domain of the CAR comprises a CD8 polypeptide (e.g., a transmembrane domain of CD8 or a portion thereof). In certain embodiments, the transmembrane domain of the CAR comprises a transmembrane domain of human CD8 or a portion thereof. In certain embodiments, the CD8 polypeptide comprises or consists of an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous or identical to a sequence having NCBI reference number: NP_001139345.1 (SEQ ID NO: 15), or a fragment thereof, and / or optionally comprises up to one or up to two or up to three conservative amino acid substitutions. In certain embodiments, the CD8 polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 15 and is at least 20, or at least 30, or at least 40, or at least 50, and up to 235 amino acids in length. In certain embodiments, the CD8 polypeptide comprises or has 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: 15. In certain embodiments, the transmembrane domain of the CAR comprises a CD8 polypeptide comprising or consisting of amino acids 137-209 of SEQ ID NO: 15. SEQ ID NO: 15 is provided below. [ka]

[0118] In certain embodiments, the transmembrane domain of the CAR comprises the transmembrane domain of mouse CD8 or a portion thereof. In certain embodiments, the CD8 polypeptide comprises or consists of an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous or identical to the sequence having NCBI reference number: AAA92533.1 (SEQ ID NO: 16), or a fragment thereof, and / or may optionally contain up to one or up to two or up to three conservative amino acid substitutions. In certain embodiments, the CD8 polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 16, and is at least about 20, or at least about 30, or at least about 40, or at least about 50, or at least about 60, or at least about 70, or at least about 100, or at least about 200, and up to 247 amino acids in length. In certain embodiments, the CD8 polypeptide comprises or has 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: 16. In certain embodiments, the transmembrane domain of the CAR comprises a CD8 polypeptide comprising or consisting of amino acids 151-219 of SEQ ID NO: 16. SEQ ID NO: 16 is provided below. [ka]

[0119] In certain embodiments, the CAR further comprises a spacer region that links the extracellular antigen-binding domain to the transmembrane domain. The spacer region can be sufficiently flexible to allow the antigen-binding domain to be oriented in different directions to facilitate antigen recognition while maintaining the activation activity of the CAR.

[0120] In certain embodiments, the hinge / spacer region of the CAR comprises a native or modified hinge region of CD8, CD28, CD3zeta, CD40, 4-1BB, OX40, CD84, CD166, CD8a, CD8b, ICOS, ICAM-1, CTLA-4, CD27, CD40, NKGD2, a synthetic polypeptide (not based on a protein associated with an immune response), or a combination thereof. The hinge / spacer region may be a hinge region from IgG1, or a CH of an immunoglobulin. 2 CH 3 The region may be a portion of CD3, a portion of a CD28 polypeptide (e.g., a portion of SEQ ID NO: 12 or 14), a portion of a CD8 polypeptide (e.g., a portion of SEQ ID NO: 15 or 16), a variant of any of the foregoing that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% homologous or identical to any of the foregoing, or a synthetic spacer sequence. 5.2.4. Intracellular signaling domain of CAR

[0121] In certain embodiments, the CAR comprises an intracellular signaling domain. In certain embodiments, the intracellular signaling domain of the CAR comprises a CD3ζ polypeptide. CD3ζ can activate or stimulate cells (e.g., lymphoid cells, e.g., T cells). Wild-type ("natural") CD3ζ comprises three functional immunoreceptor tyrosine-based activation motifs (ITAMs), three functional basic-rich stretch (BRS) regions (BRS1, BRS2 and BRS3). CD3ζ transmits activation signals to cells (e.g., lymphoid cells, e.g., T cells) after antigen binding. The intracellular signaling domain of the CD3ζ chain is the primary transmitter of signals from endogenous TCR.

[0122] In certain embodiments, the intracellular signaling domain of the CAR comprises native CD3ζ. In certain embodiments, the CD3ζ polypeptide comprises or consists of an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous or identical to the sequence having NCBI reference number: NP_932170 (SEQ ID NO: 17), or a fragment thereof, and / or may optionally contain up to one or up to two or up to three conservative amino acid substitutions. In certain embodiments, the CD3ζ polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 17 and is at least 20, or at least 30, or at least 40, or at least 50, and up to 164 amino acids in length. In certain embodiments, the CD3ζ polypeptide comprises or consists of the amino acid sequence of amino acids 1-164, 1-50, 50-100, 52-164, 100-150, or 150-164 of SEQ ID NO: 17. In certain embodiments, the intracellular signaling domain of a CAR comprises a CD3ζ polypeptide comprising or consisting of amino acids 52-164 of SEQ ID NO: 17. SEQ ID NO: 17 is provided below. [ka]

[0123] In certain embodiments, the intracellular signaling domain of the CAR comprises a CD3ζ polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 18. SEQ ID NO: 18 is provided below. [ka]

[0124] In certain embodiments, the intracellular signaling domain of CAR further comprises at least one costimulatory signaling domain.In certain embodiments, the at least one costimulatory signaling domain comprises at least one costimulatory molecule or a portion thereof.In certain embodiments, the at least one costimulatory signaling domain comprises at least one intracellular domain of costimulatory molecule or a portion thereof.

[0125] As used herein, "costimulatory molecule" refers to a cell surface molecule other than antigen receptor or its ligand that can provide an efficient response of lymphocytes to antigen. In certain embodiments, costimulatory molecules can provide optimal lymphocyte activation. Non-limiting examples of costimulatory molecules include CD28, 4-1BB, OX40, ICOS, DAP-10, CD27, CD40, NKGD2, CD2, FN14, HVEM, LTBR, ​​CD28H, TNFR1, TNFR2, BAFF-R, BCMA, TACI, TROY, RANK, CD40, CD27, CD30, EDAR, XEDAR, GITR, DR6, and NGFR, and combinations thereof. Costimulatory molecules can bind to costimulatory ligands, which are proteins expressed on the cell surface that, when bound to their receptors, generate a costimulatory response (i.e., an intracellular response that affects the stimulation that is provided when the CAR binds to its target antigen).

[0126] In certain embodiments, at least one costimulatory signaling domain comprises a CD28 polypeptide comprising a mutated YMNM motif.

[0127] CD28 is a transmembrane protein that plays an important role in T cell activation through its function as a costimulatory molecule.CD28 is also known as differentiation cluster 28, Tp44, and CD28 molecule.CD28 possesses an intracellular domain that contains intracellular motifs that are important for effective signaling of CD28.In certain embodiments, CD28 intracellular domain contains intracellular subdomains (also known as "intracellular motifs") that regulate signaling pathways after TCR stimulation.

[0128] CD28 contains three intracellular motifs: the YMNM motif and two proline-rich motifs: the PRRP motif and the PYAP motif. The CD28 intracellular motifs can serve as docking sites for several adaptor molecules that interact with these motifs through their SH2 or SH3 domains. Such interactions transmit downstream signals that terminate in transcription factors that regulate gene expression. For example, the native YMNM motif binds to the p85 subunit of phosphoinositide 3-kinase (PI3K). The native YMNM motif also binds to growth factor receptor-bound protein 2 (Grb2) and / or Grb2-associated adaptor protein 2 (GADS). Grb2 can bind to Gab1 and Gab2, which in turn recruits the p85 subunit of PI3K.

[0129] In certain embodiments, the naturally occurring YMNM motif consists of the amino acid sequence set forth in YMNM (SEQ ID NO: 19). In certain embodiments, the naturally occurring YMNM motif binds to the p85 subunit of PI3K via the consensus sequence YMxM (SEQ ID NO: 20), where x is not aspartic acid (N). In certain embodiments, the naturally occurring YMNM motif binds to Grb2 and / or GADs via the consensus sequence YxNx (SEQ ID NO: 21), where x is not methionine (M).

[0130] In certain embodiments, CD28 polypeptides containing a mutant YMNM motif of the present disclosure have reduced recruitment of the p85 subunit of PI3K compared to CD28 molecules containing a native YMNM motif.

[0131] In certain embodiments, the p85 subunit of PI3K does not bind to the mutant YMNM motif, thereby reducing the recruitment of the p85 subunit of PI3K to the CD28 polypeptide. The mutant YMNM motif that blocks the binding of the p85 subunit of PI3K retains its binding to Grb2 and / or GADS. Thus, the downstream signaling of Grb2 / GADS remains intact, for example, the downstream signaling leading to IL-2 secretion remains intact. Such mutant YMNM motifs are called "GADS / Grb2 permissive mutants".

[0132] In certain embodiments, mutant YMNM binds to p85 subunit of PI3K but not to Grb2 and / or GADS. Since PI3K p85 binding is preserved, downstream signaling of PI3K is kept intact. Since Grb2 / GADS binding is blocked, recruitment of PI3K p85 subunit caused by Grb2 binding to Gab1 and Gab2 is reduced or blocked. Furthermore, downstream signaling of Grb2 / GADS is blocked. Such mutant YMNM motif is called "PI3K permissive mutant".

[0133] In certain embodiments, mutant YMNM does not bind to the p85 subunit of PI3K and does not bind to Grb2 and / or GADS. Such mutant YMNM motifs are called "non-functional mutants". Non-functional mutants do not cause PI3K, Grb2, or GADS to bind to CD28 at the YMNM motif, but do not exclude these signaling molecules from binding to other sites on the CD28 molecule.

[0134] In certain embodiments, mutant YMNM retains only one of the two methionine residues present in the YMNM motif (i.e., YMxx or YxxM). These motifs may regulate signaling through PI3K by limiting the number of methionine residues that can bind to the p85 subunit of PI3K. Such mutant YMNM motifs are referred to as "hybrid "HEMI" mutants." GADS / Grb-2 permissive mutants

[0135] In certain embodiments, the mutant YMNM motif is a GADS / Grb-2 permissive mutant. In certain embodiments, the mutant YMNM motif consists of the amino acid sequence set forth in YxNx (SEQ ID NO: 21), where x is not methionine (M). In certain embodiments, x is selected from the group consisting of the amino acids A, R, N, D, C, E, Q, G, H, I, K, F, P, S, T, W, Y, V, and L. In certain embodiments, the mutant YMNM motif consists of the amino acid sequence depicted in YENV (SEQ ID NO:22), YSNV (SEQ ID NO:23), YKNL (SEQ ID NO:24), YENQ (SEQ ID NO:25), YKNI (SEQ ID NO:26), YINQ (SEQ ID NO:27), YHNK (SEQ ID NO:28), YVNQ (SEQ ID NO:29), YLNP (SEQ ID NO:30), YLNT (SEQ ID NO:31), YDND (SEQ ID NO:66), YENI (SEQ ID NO:67), YENL (SEQ ID NO:68), YKNQ (SEQ ID NO:72), YKNV (SEQ ID NO:73), or YANG (SEQ ID NO:87). In certain embodiments, the mutant YMNM motif consists of the amino acid sequence depicted in YSNV (SEQ ID NO:23). In certain embodiments, the mutant YMNM motif consists of the amino acid sequence depicted in YKNI (SEQ ID NO:26). In certain embodiments, the mutant YMNM motif consists of the amino acid sequence depicted in YENV (SEQ ID NO:22). In one particular embodiment, the mutated YMNM motif consists of the amino acid sequence set forth in YKNL (SEQ ID NO:24). PI3K permissive mutants

[0136] In certain embodiments, the mutant YMNM motif is a PI3K-permissive mutant. In certain embodiments, the mutant YMNM motif consists of the amino acid sequence depicted in YMxM (SEQ ID NO: 20), where x is not aspartic acid (N). In certain embodiments, x is selected from the group consisting of amino acids A, R, D, C, E, Q, G, H, I, K, M, F, P, S, T, W, Y, V, and L. In certain embodiments, the mutant YMNM motif consists of the amino acid sequence depicted in YMDM (SEQ ID NO: 32), YMPM (SEQ ID NO: 79), YMRM (SEQ ID NO: 37), or YMSM (SEQ ID NO: 80). In certain embodiments, the mutant YMNM motif consists of the amino acid sequence depicted in YMDM (SEQ ID NO: 32).

[0137] In certain embodiments, the mutated YMNM motif consists of the amino acid sequence set forth in YbxM (SEQ ID NO: 33), where x is not aspartic acid (N) and b is not methionine (M). In certain embodiments, x is selected from the group consisting of the amino acids A, R, D, C, E, Q, G, H, I, K, M, F, P, S, T, W, Y, V, and L. In certain embodiments, b is selected from the group consisting of the amino acids A, R, N, C, E, Q, G, H, I, K, N, F, P, S, T, W, Y, V, and L. In certain embodiments, the mutated YMNM motif consists of the amino acid sequence set forth in YTHM (SEQ ID NO: 34), YVLM (SEQ ID NO: 35), YIAM (SEQ ID NO: 36), YVEM (SEQ ID NO: 83), YVKM (SEQ ID NO: 85), or YVPM (SEQ ID NO: 86).

[0138] In certain embodiments, the mutated YMNM motif consists of the amino acid sequence set forth in YMxb (SEQ ID NO: 65), where x is not aspartic acid (N) and b is not methionine (M). In certain embodiments, x is selected from the group consisting of the amino acids A, R, D, C, E, Q, G, H, I, K, M, F, P, S, T, W, Y, V, and L. In certain embodiments, b is selected from the group consisting of the amino acids A, R, N, C, E, Q, G, H, I, K, N, F, P, S, T, W, Y, V, and L. In certain embodiments, the mutated YMNM motif consists of the amino acid sequence set forth in YMAP (SEQ ID NO: 77).

[0139] Certain mutant YMNM motifs are described in Mol Cell Proteomics. 2010 November;9(11):2391-404; Virology. 2015 May;0:568-577, both of which are incorporated by reference in their entireties. 5.2.4.3. Hybrid "HEMI" variants

[0140] In certain embodiments, the mutant YMNM motif is a hybrid "HEMI" mutant. In certain embodiments, the mutant YMNM motif consists of the amino acid sequence set forth in YMNx (SEQ ID NO: 38) or YxNM (SEQ ID NO: 39), where x is not methionine (M). In certain embodiments, x is selected from the group consisting of the amino acids A, R, N, C, E, Q, G, H, I, K, N, F, P, S, T, W, Y, V, and L. In certain embodiments, the mutant YMNM motif consists of the amino acid sequence set forth in YMNV (SEQ ID NO: 40), YENM (SEQ ID NO: 41), YMNQ (SEQ ID NO: 42), YMNL (SEQ ID NO: 78), or YSNM (SEQ ID NO: 81). Non-functional variants

[0141] In certain embodiments, the mutant YMNM motif is a non-functional mutant. In certain embodiments, the mutant YMNM motif is comprised of the amino acid sequence Ybxb (SEQ ID NO: 43), where x is not aspartic acid (N) and b is not methionine (M). In certain embodiments, x is selected from the group consisting of A, R, D, C, E, Q, G, H, I, K, M, F, P, S, T, W, Y, V, and L. In certain embodiments, b is selected from the group consisting of A, R, N, D, C, E, Q, G, H, I, K, F, P, S, T, W, Y, V, and L. In certain embodiments, the mutated YMNM motif consists of the amino acid sequence set forth in YGGG (SEQ ID NO: 44), YAAA (SEQ ID NO: 45), YFFF (SEQ ID NO: 46), YETV (SEQ ID NO: 69), YQQQ (SEQ ID NO: 70), YHAE (SEQ ID NO: 71), YLDL (SEQ ID NO: 74), YLIP (SEQ ID NO: 75), YLRV (SEQ ID NO: 76), YTAV (SEQ ID NO: 82), or YVHV (SEQ ID NO: 84). In certain embodiments, the mutated YMNM motif consists of the amino acid sequence set forth in YGGG (SEQ ID NO: 44).

[0142] In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling domain comprising a CD28 polypeptide comprising a mutated YMNM motif consisting of the amino acid sequence set forth in YENV (SEQ ID NO: 22), wherein the CD28 polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 47. SEQ ID NO: 47 is provided below. [ka]

[0143] In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling domain comprising a CD28 polypeptide comprising a mutated YMNM motif consisting of the amino acid sequence set forth in YKNI (SEQ ID NO: 26), wherein the CD28 polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 48. SEQ ID NO: 48 is provided below. [ka]

[0144] In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling domain comprising a CD28 polypeptide comprising a mutated YMNM motif consisting of the amino acid sequence set forth in YMDM (SEQ ID NO: 32), wherein the CD28 polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 49. SEQ ID NO: 49 is provided below. [ka]

[0145] In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling domain comprising a CD28 polypeptide comprising a mutated YMNM motif consisting of the amino acid sequence set forth in YGGG (SEQ ID NO: 44), wherein the CD28 polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 63. SEQ ID NO: 63 is provided below. [ka]

[0146] In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling domain comprising a CD28 polypeptide comprising a mutated YMNM motif consisting of the amino acid sequence set forth in YSNV (SEQ ID NO: 23), wherein the CD28 polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 64. SEQ ID NO: 64 is provided below. [ka]

[0147] In certain embodiments, the intracellular signaling domain of the CAR comprises a first costimulatory signaling domain comprising a CD28 polypeptide comprising a mutated YMNM motif (disclosed herein), and a second costimulatory signaling domain comprising an intracellular domain of a costimulatory molecule. In certain embodiments, the costimulatory molecule is selected from the group consisting of 4-1BB, OX40, ICOS, DAP-10, CD30, CD271, BAFFR, BCMA, DR3, FN14, HVEM, LTBR, ​​RANK, TACI, TNFR1, TNFR2, TROY, EPOR, IL1RAcP, IL18R1, IL18RAP, ST2, and combinations thereof.

[0148] In certain embodiments, the second costimulatory signaling domain comprises a 4-1BB polypeptide (e.g., the intracellular domain of 4-1BB or a portion thereof). In certain embodiments, the 4-1BB polypeptide comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to the amino acid sequence having NCBI Ref. No.: NP_001552 (SEQ ID NO: 50), or a fragment thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. In certain embodiments, the 4-1BB polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 50, and is at least 20, or at least 30, or at least 40, or at least 50, or at least 100, or at least 150, or at least 150, and up to 255 amino acids in length. In certain 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 of SEQ ID NO: 50. SEQ ID NO: 50 is provided below. [ka] 5.2.5. Example CAR

[0149] In certain embodiments, the CAR is a CD19-targeting CAR. In certain embodiments, the CAR (a) binds to human CD19 and comprises the amino acid sequence set forth in SEQ ID NO:6. H CDR1, V comprising the amino acid sequence set forth in SEQ ID NO:7 H CDR2, comprising the amino acid sequence set forth in SEQ ID NO:8 H CDR3, V comprising the amino acid sequence set forth in SEQ ID NO:9 L CDR1, V comprising the amino acid sequence set forth in SEQ ID NO:10 L CDR2, and V comprising the amino acid sequence set forth in SEQ ID NO:11 L (b) an extracellular antigen-binding domain comprising a CDR3; (b) a transmembrane domain comprising the transmembrane domain of CD28 or a portion thereof; and (c) an intracellular signaling domain comprising (i) a CD3ζ polypeptide, and (ii) a costimulatory signaling domain comprising a CD28 polypeptide comprising a mutated YMNM motif. In certain embodiments, the mutated YMNM motif consists of the amino acid sequence set forth in YMDM (SEQ ID NO: 32), YKNI (SEQ ID NO: 26), YENV (SEQ ID NO: 22), YSNV (SEQ ID NO: 23), YKNL (SEQ ID NO: 24), or YGGG (SEQ ID NO: 44). ... H and V L are linked via a linker having the amino acid sequence set forth in SEQ ID NO:5.

[0150] In certain embodiments, an exemplary CD19-targeting CAR comprises a mutated YMNM motif consisting of the amino acid sequence set forth in YMDM (SEQ ID NO: 32). In certain embodiments, an exemplary CD19-targeting CAR consists of the amino acid sequence set forth in SEQ ID NO: 51, provided below. [ka]

[0151] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO:51 is set forth in SEQ ID NO:52, provided below. [ka]

[0152] In certain embodiments, an exemplary CD19-targeting CAR comprises a mutated YMNM motif consisting of the amino acid sequence set forth in YENV (SEQ ID NO: 22). In certain embodiments, an exemplary CD19-targeting CAR consists of the amino acid sequence set forth in SEQ ID NO: 53, provided below. [ka]

[0153] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO:53 is set forth in SEQ ID NO:54, provided below. [ka]

[0154] In certain embodiments, an exemplary CD19-targeted CAR comprises a mutated YMNM motif consisting of the amino acid sequence YKNI (SEQ ID NO: 26). In certain embodiments, an exemplary CD19-targeted CAR consists of the amino acid sequence set forth in SEQ ID NO: 55, provided below. [ka] [ka]

[0155] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO:55 is set forth in SEQ ID NO:56, provided below. [ka]

[0156] In certain embodiments, an exemplary CD19-targeting CAR comprises a mutated YMNM motif consisting of the amino acid sequence YSNV (SEQ ID NO: 23). In certain embodiments, an exemplary CD19-targeting CAR consists of the amino acid sequence set forth in SEQ ID NO: 57, provided below. [ka]

[0157] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO:57 is set forth in SEQ ID NO:58, provided below. [ka]

[0158] In certain embodiments, an exemplary CD19-targeting CAR comprises a mutated YMNM motif consisting of the amino acid sequence YKNL (SEQ ID NO: 24). In certain embodiments, an exemplary CD19-targeting CAR consists of the amino acid sequence set forth in SEQ ID NO: 59, provided below. [ka]

[0159] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO:59 is set forth in SEQ ID NO:60, provided below. [ka] [ka]

[0160] In certain embodiments, an exemplary CD19-targeting CAR comprises a mutated YMNM motif consisting of the amino acid sequence YGGG (SEQ ID NO: 44). In certain embodiments, an exemplary CD19-targeting CAR consists of the amino acid sequence set forth in SEQ ID NO: 61, provided below. [ka]

[0161] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO:61 is set forth in SEQ ID NO:62, provided below. [ka] [ka] 5.3.Cells

[0162] The subject matter of the present disclosure provides a cell comprising the CAR of the present disclosure (e.g., as disclosed in section 5.2).In certain embodiments, the cell is selected from the group consisting of lymphoid cells and myeloid cells.In certain embodiments, the cell is an immunoresponsive cell.In certain embodiments, the immunoresponsive cell is a lymphoid cell.

[0163] In certain embodiments, the cell is a lymphoid cell. The lymphoid cell can provide antibody production, regulation of cellular immune system, detection of foreign substances in blood, detection of foreign cells to host, etc. Non-limiting examples of lymphoid cell include T cell, natural killer (NK) cell, B cell, dendritic cell, and stem cell from which lymphoid cell can be differentiated. In certain embodiments, the stem cell is a pluripotent stem cell (e.g., embryonic stem cell).

[0164] In certain embodiments, the cells are T cells. T cells can be lymphocytes that mature in the thymus and are primarily responsible for cell-mediated immunity. T cells are involved in the adaptive immune system. The T cells of the subject matter of the present disclosure can be any type of T cell, including, but not limited to, helper T cells, cytotoxic T cells, memory T cells (central memory T cells, stem cell-like memory T cells (or stem-like memory T cells), and two types of effector memory T cells: e.g., TEM cells and TEMRA cells, regulatory T cells (also known as suppressor T cells), tumor-reactive lymphocytes, tumor-infiltrating lymphocytes (TILs), natural killer T cells, mucosal-associated invariant T cells, and gamma delta T cells. Cytotoxic T cells (CTLs or killer T cells) are a subset of T lymphocytes that can induce the death of infected somatic or tumor cells. The patient's own T cells may be genetically modified to target specific antigens by the introduction of a CAR. In certain embodiments, the immunoresponsive cells are T cells. T cells are CD4 + T cells or CD8 + In certain embodiments, the T cells are CD4 + In certain embodiments, the T cells are CD8 + T cells.

[0165] In certain embodiments, the cell is a NK cell. Natural killer (NK) cells are part of cellular immunity and can be lymphocytes that act during natural immune response. NK cells do not require prior activation to perform cytotoxic effects on target cells.

[0166] The types of human lymphocytes of the subject matter of this disclosure include, but are not limited to, peripheral donor lymphocytes. See, for example, Sadelain et al., Nat Rev Cancer (2003); 3: 35-45 (disclosing peripheral donor lymphocytes genetically modified to express CARs), Morgan, RA et al., 2006 Science 314: 126-129 (disclosing peripheral donor lymphocytes genetically modified to express full-length tumor antigen-recognizing T cell receptor complexes including α and β heterodimers), Panelli et al., J Immunol (2000); 164: 495-504; Panelli et al., J Immunol (2000); 164: 4382-4392 (disclosing lymphocyte cultures derived from tumor-infiltrating lymphocytes (TILs) in tumor biopsies), and Dupont et al., Cancer Res (2005); 65: 5417-5427; Papanicolaou et al., Blood (2003); 102: 2498-2505 (disclosing antigen-specific peripheral blood leukocytes selectively expanded in vitro using artificial antigen presenting cells (AAPC) or pulsed dendritic cells).

[0167] The cells (eg, T cells) can be derived in vitro from autologous, non-autologous (eg, allogeneic), or engineered precursor or stem cells.

[0168] The subject cell of the present disclosure can be myeloid cell.Non-limiting examples of myeloid cell include monocyte, macrophage, neutrophil, dendritic cell, basophil, neutrophil, eosinophil, megakaryocyte, mast cell, erythrocyte, platelet, and stem cell that myeloid cell can differentiate.In certain embodiments, stem cell is pluripotent stem cell (for example, embryonic stem cell or induced pluripotent stem cell).

[0169] In certain embodiments, the cells of the present disclosure can regulate the tumor microenvironment. Tumors have a microenvironment hostile to the host immune response that involves a series of mechanisms by malignant cells to protect themselves from immune recognition and elimination. This "hostile tumor microenvironment" is characterized by the infiltrating regulatory CD4 +The tumor microenvironment includes a variety of immunosuppressive factors, including T cells (Tregs), myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), immunosuppressive cytokines including TGF-β, and expression of ligands that target immunoinhibitory receptors (CTLA-4 and PD-1) expressed by activated T cells. These mechanisms of immunosuppression play a role in maintaining tolerance and suppressing inappropriate immune responses, but within the tumor microenvironment, these mechanisms prevent effective anti-tumor immune responses. Collectively, these immunosuppressive factors can induce either significant anergy or apoptosis of adoptively transferred CAR-modified T cells upon encounter with target tumor cells.

[0170] In certain embodiments, cells can be transduced with a CAR of the disclosure such that the cells express the CAR.

[0171] In certain embodiments, the cells further comprise a soluble single-chain variable fragment (scFv) that binds to a polypeptide having immunosuppressive or immunostimulatory activity. In certain embodiments, immunosuppressive activity refers to the induction of signaling or changes in protein expression in cells (e.g., activated immunoresponsive cells) that result in a decrease in immune response. Polypeptides known to suppress or decrease immune responses through their binding include CD47, PD-1, CTLA-4, and their corresponding ligands including SIRPa, PD-L1, PD-L2, B7-1, and B7-2. Such polypeptides are present in the tumor microenvironment and inhibit the immune response to neoplastic cells. In various embodiments, inhibiting, blocking, or antagonizing the interaction of immunosuppressive polypeptides and / or their ligands enhances the immune response of immunoresponsive cells.

[0172] In certain embodiments, immune stimulatory activity refers to the induction of signaling or changes in protein expression in cells (e.g., activated immunoresponsive cells) that result in an increased immune response. Immune stimulatory activity can include pro-inflammatory activity. Polypeptides known to stimulate or increase immune responses through their binding include CD28, OX-40, 4-1BB, and their corresponding ligands including B7-1, B7-2, OX-40L, and 4-1BBL. Such polypeptides are present in the tumor microenvironment and activate immune responses against neoplastic cells. In various embodiments, promoting, stimulating, or agonizing pro-inflammatory polypeptides and / or their ligands enhances the immune response of immunoresponsive cells.

[0173] Cells comprising a CAR and a soluble scFv that binds a polypeptide having immunosuppressive or immunostimulatory activity are disclosed in WO 2014 / 134165, which is incorporated by reference in its entirety.

[0174] In certain embodiments, the cells further comprise exogenous CD40L. Cells comprising a CAR and exogenous CD40L are disclosed in WO 2014 / 134165.

[0175] Furthermore, in certain embodiments, the cells are engineered to express IL-18. In certain embodiments, the cells further comprise an exogenous IL-18 polypeptide or a fragment thereof. In certain embodiments, the cells further comprise a modified promoter / enhancer at the IL-18 locus, which can increase IL-18 gene expression (e.g., a constitutive or inducible promoter arranged to drive IL-18 gene expression). Cells comprising a CAR and engineered to express IL-18, for example, comprising an exogenous IL-18 polypeptide or a fragment thereof or a modified promoter / enhancer at the IL-18 locus, are disclosed in International Publication No. WO 2018 / 027155, the entirety of which is incorporated by reference.

[0176] Additionally or alternatively, the cells are engineered to express IL-33. In certain embodiments, the cells further comprise an exogenous IL-33 polypeptide or a fragment thereof. In certain embodiments, the cells further comprise a modified promoter / enhancer at the IL-33 locus, which can increase IL-33 gene expression (e.g., a constitutive or inducible promoter arranged to drive IL-33 gene expression). Cells comprising a CAR and engineered to express IL-33, for example, comprising an exogenous IL-33 polypeptide or a fragment thereof or a modified promoter / enhancer at the IL-33 locus, are disclosed in International Publication No. WO 2019 / 099479, the entirety of which is incorporated by reference.

[0177] Additionally or alternatively, the cells are engineered to express IL-36. In certain embodiments, the cells further comprise an exogenous IL-36 polypeptide or a fragment thereof. In certain embodiments, the cells further comprise a modified promoter / enhancer at the IL-36 locus, which can increase IL-36 gene expression (e.g., a constitutive or inducible promoter arranged to drive IL-36 gene expression). Cells comprising a CAR and engineered to express IL-36, for example, comprising an exogenous IL-36 polypeptide or a fragment thereof or a modified promoter / enhancer at the IL-36 locus, are disclosed in International Publication No. WO 2019 / 099483, the entirety of which is incorporated by reference. 5.4. Compositions and Vectors

[0178] The presently disclosed subject matter provides compositions comprising a CAR of the present disclosure (e.g., those disclosed in Section 5.2). Cells comprising such compositions are also provided.

[0179] In certain embodiments, the CAR of the present disclosure is encoded by a nucleic acid molecule operably linked to a promoter.

[0180] Additionally, the presently disclosed subject matter provides nucleic acid compositions comprising a polynucleotide encoding a CAR of the present disclosure (e.g., those disclosed in Section 5.2). Cells comprising such nucleic acid compositions are also provided.

[0181] In certain embodiments, the nucleic acid composition further comprises a promoter operably linked to the polynucleotide encoding the CAR of the disclosure.

[0182] In certain embodiments, the promoter is endogenous or exogenous.In certain embodiments, the exogenous promoter is selected from the elongation factor (EF)-1 promoter, the cytomegalovirus immediate early promoter (CMV) promoter, the simian virus 40 early promoter (SV40) promoter, the phosphoglycerate kinase (PGK) promoter, and the metallothionein promoter.In certain embodiments, the promoter is an inducible promoter.In certain embodiments, the inducible promoter is selected from the NFAT transcription response element (TRE) promoter, the CD69 promoter, the CD25 promoter, and the IL-2 promoter.

[0183] The compositions and nucleic acid compositions can be administered to a subject or delivered to cells by methods known in the art or as described herein. Genetic modification of cells (e.g., T cells or NK cells) can be achieved by transducing a substantially homogeneous cell composition with a recombinant DNA construct. In certain embodiments, a retroviral vector (e.g., a gamma retroviral vector or a lentiviral vector) is used to introduce a DNA construct into cells. For example, a polynucleotide encoding an antigen-recognizing receptor can be cloned into a retroviral vector and expression can be driven from its endogenous promoter, the retroviral long terminal repeat, or a promoter specific for the target cell type of interest. Non-viral vectors can be used as well.

[0184] To initially genetically modify cells to contain the CAR of the present disclosure, retroviral vectors can be used for transduction, but any other suitable viral vectors or non-viral delivery systems can be used. Antigen recognition receptors can be constructed in a single multicistronic expression cassette, multiple expression cassettes in a single vector, or multiple vectors. Examples of elements that create polycistronic expression cassettes include, but are not limited to, various viral and non-viral internal ribosome entry sites (IRES, such as FGF-1 IRES, FGF-2 IRES, VEGF IRES, IGF-II IRES, NF-κB IRES, RUNX1 IRES, p53 IRES, Hepatitis A IRES, Hepatitis C IRES, Pestivirus IRES, Aphthovirus IRES, Picornavirus IRES, Poliovirus IRES and Encephalomyocarditis virus IRES) and cleavable linkers (e.g., 2A peptides, such as P2A, T2A, E2A and F2A peptides). A combination of a retroviral vector with an appropriate packaging line will also be suitable, and the capsid protein will be functional to infect human cells. A variety of amphotropic virus-producing cell lines are known, including, but not limited to, PA12 (Miller et al., (1985) Mol Cell Biol (1985); 5:431-437); PA317 (Miller et al., Mol Cell Biol (1986); 6:2895-2902); and CRIP (Danos et al., Proc Natl Acad Sci USA (1988); 85:6460-6464). Non-amphotropic particles, such as particles pseudotyped with VSVG, RD114 or GALV envelopes and any others known in the art, are also suitable.

[0185] Possible methods of transduction also include direct co-culture of cells with producer cells (Bregni et al., Blood (1992); 80:1418-1422), or culture with viral supernatant alone or concentrated vector stocks, with or without appropriate growth factors and polycations (Xu et al., Exp Hemat (1994); 22:223-230; and Hughes et al., J Clin Invest (1992); 89:1817).

[0186] Other transduction virus vectors can be used to modify cells.In certain embodiments, the vector selected 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., Science, 272:263-267, 1996; and Miyoshi et al., Proc.Natl.Acad.Sci.USA 94:10319, 1997). Other viral vectors that can be used include, for example, adenovirus, lentivirus, and adeno-associated viral vectors, vaccinia virus, bovine papilloma virus, or herpes viruses, such as Epstein-Barr virus (see, e.g., Miller, Human Gene Thera (1990); 15-14; Friedman, Science 244:1275-1281, 1989; Eglitis et al., BioTechniques (1988); 6:608-614; Tolstoshev et al., Cur Opin Biotechnol (1990); 1:55-61; Sharp, The Lancet (1991); 337:1277-78; Cornetta et al., Nucleic Acid Research and Molecular Biology 36:311-22, 1987; Anderson, Science (1984); 226:401-409; Moen, Blood Cells 17:407-16, 1991; Miller et al., Biotechnol (1989); 7:980-90; LeGal La Salle et al., Science (1993); 259:988-90; and Johnson, Chest (1995) 107:77S-83S (see also vectors).Retroviral vectors have been particularly well developed and have been used in clinical settings (Rosenberg et al., N Engl J Med (1990);323:370,1990; Anderson et al., US Pat. No. 5,399,346).

[0187] Non-viral approaches can also be used for the genetic modification of cells. For example, by administering nucleic acids in the presence of lipofection (Feigner et al., Proc Natl. Acad Sci USA(1987);84:7413;Ono et al., Neurosci Lett(1990);17:259;Brigham et al., Am J Med Nucleic acid molecules can be introduced into cells by microinjection (Wolff et al., Science (1990); 247: 1465), or under surgical conditions (Sci (1989); 298: 278; Staubinger et al., Methods in Enzymol (1983); 101: 512; Wu et al., J Biol Chem (1988); 263: 14621; Wu et al., J Biol Chem (1989); 264: 16985). Other non-viral means for gene transfer include in vitro transfection using calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. Liposomes can also be potentially useful for the delivery of DNA into cells. The transplantation of normal genes into the diseased tissue of a subject can also be achieved by transferring normal nucleic acids into a culturable cell type ex vivo (e.g., autologous or heterologous primary cells or their progeny), and then injecting the cells (or their derivatives) into the target tissue or by systemic injection. Recombinant receptors can also be induced or obtained using transposases or targeted nucleases (e.g., zinc finger nucleases, meganucleases, or TALE nucleases, CRISPR). Transient expression can also be obtained by RNA electroporation.

[0188] Any targeted genome editing method can also be used to deliver the antigen-recognizing receptor of the present disclosure to cell or subject.In certain embodiments, the CRISPR system is used to deliver the antigen-recognizing receptor of the present disclosure disclosed herein.In certain embodiments, the zinc finger nuclease is used to deliver the antigen-recognizing receptor.In certain embodiments, the TALEN system is used to deliver the antigen-recognizing receptor of the present disclosure.

[0189] The clustered regularly interspaced short palindromic repeats (CRISPR) system is a genome editing tool found in prokaryotic cells. When utilized for genome editing, the system includes Cas9 (a protein that can modify DNA using crRNA as its guide), CRISPR RNA (crRNA, an RNA used by Cas9 to guide it to the correct section of host DNA, along with a region that binds to tracrRNA (generally in a hairpin loop form) and forms an active complex with Cas9), transactivating crRNA (tracrRNA, binds to crRNA and forms an active complex with Cas9), and any section of DNA repair template (DNA that guides the cell repair process allowing for the insertion of a specific DNA sequence). CRISPR / Cas9 often uses a plasmid to transfect target cells. The crRNA must be designed for each application, as it is the sequence that Cas9 uses to identify the target DNA in the cell and bind directly to the target DNA. The repair template with the CAR expression cassette must also be designed for each application, as it must overlap with sequences on either side of the cut and code for the insertion sequence. Multiple crRNAs and tracrRNAs can be packaged together to form a single guide RNA (sgRNA), which can be spliced ​​together with the Cas9 gene and made into a plasmid for transfection into cells.

[0190] Zinc finger nucleases (ZFNs) are artificial restriction enzymes generated by combining a zinc finger DNA binding domain with a DNA cleavage domain. The zinc finger domain can be engineered to target specific DNA sequences allowing the zinc finger nuclease to target desired sequences within the genome. The DNA binding domain of an individual ZFN typically contains multiple individual zinc finger repeats, each capable of recognizing multiple base pairs. The most common method of generating new zinc finger domains is to combine smaller zinc finger "modules" of known specificity. The most common cleavage domain in ZFNs is the non-specific cleavage domain derived from the type II restriction endonuclease FokI. Using a homologous DNA template with endogenous homologous recombination (HR) machinery and a CAR expression cassette, ZFNs can be used to insert the CAR expression cassette into the genome. Once the target sequence is cleaved by the ZFN, the HR machinery searches for homology between the damaged chromosome and a homologous DNA template and then copies the sequence of the template between the two cleaved ends of the chromosome, thereby integrating the homologous DNA template into the genome.

[0191] Transcription activator-like effector nucleases (TALENs) are restriction enzymes that can be engineered to cleave specific sequences of DNA. TALEN systems work on roughly the same principle as ZFNs. They are generated by combining a transcription activator-like effector DNA binding domain with a DNA cleavage domain. Transcription activator-like effectors (TALEs) are composed of a 33-34 amino acid repeat motif with two variable positions that have strong recognition for specific nucleotides. By assembling an array of these TALEs, the TALE DNA binding domain can be engineered to bind to a desired DNA sequence, thereby guiding the nuclease to cleave at a specific location in the genome. cDNA expression for use in polynucleotide therapy can be directed from any suitable promoter (e.g., human cytomegalovirus (CMV), simian virus 40 (SV40), or metallothionein promoters) and regulated by any suitable mammalian regulatory element or intron (e.g., elongation factor 1a enhancer / promoter / intron structure). For example, if desired, enhancers known to preferentially direct gene expression in specific cell types can be used to direct the expression of nucleic acid.The enhancers used can include, but are not limited to, those characterized as tissue- or cell-specific enhancers.Alternatively, if a genomic clone is used as a therapeutic construct, regulation can be mediated by homologous regulatory sequences, or, if desired, by regulatory sequences derived from heterologous sources, including any of the promoters or regulatory elements described above.

[0192] The method for delivering genome editing agent / system may vary as required.In certain embodiments, the components of the selected genome editing method are delivered as DNA constructs in one or more plasmids.In certain embodiments, the components are delivered via viral vectors.Common delivery methods include, but are not limited to, electroporation, microinjection, gene gun, impalefection, hydrostatic pressure, continuous injection, sonication, magnetofection, adeno-associated virus, envelope protein pseudotyping of viral vectors, cis and trans-acting elements of replication-competent vectors, herpes simplex virus, and chemical vehicles (e.g., oligonucleotides, lipoplexes, polymersomes, polyplexes, dendrimers, inorganic nanoparticles, and cell membrane-permeable peptides). Polypeptides

[0193] The subject matter of the present disclosure provides a method for optimizing an amino acid sequence or a nucleic acid sequence by generating sequence changes. Such changes may include certain mutations, deletions, insertions, or post-translational modifications. The subject matter of the present disclosure further includes analogs of any naturally occurring polypeptide disclosed herein, including but not limited to CD19, CD8, CD28, 4-1BB, and CD3zeta. Analogs may differ from naturally occurring polypeptides disclosed herein by differences in amino acid sequence, post-translational modifications, or both. 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 more homology to all or a portion of the naturally occurring amino acid sequence of the subject matter of the present disclosure. The length of sequence comparison is at least 5, 10, 15 or 20 amino acid residues, for example at least 25, 50 or 75 amino acid residues, or more than 100 amino acid residues. Again, an exemplary approach for determining the degree of identity can use the BLAST program, e.g. -3 and e -100A probability score between indicates a closely related sequence. Modifications include in vivo and in vitro chemical derivatization of polypeptides, such as acetylation, carboxylation, phosphorylation, or glycosylation. Such modifications may occur during synthesis or processing of the polypeptide or in subsequent treatment with isolated modifying enzymes. Analogs may also differ from naturally occurring polypeptides by changes in the primary sequence. These include natural and derived forms (see, e.g., Sambrook, Fritsch and Maniatis, Molecular Included are genetic variants, both from random mutagenesis by exposure to irradiation or ethanemethyl sulfate or site-directed mutagenesis, as described in Cloning: A Laboratory Manual (2nd 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 (e.g., D-amino acids or unnatural or synthetic amino acids, e.g., β or γ amino acids).

[0194] In addition to full-length polypeptides, the subject matter of the present disclosure also provides fragments of any of the polypeptides disclosed herein. As used herein, the term "fragment" refers to at least 5, 10, 13, or 15 amino acids. In certain embodiments, a fragment comprises at least 20 contiguous amino acids, at least 30 contiguous amino acids, or at least 50 contiguous amino acids. In certain embodiments, a fragment comprises at least 60-80, 100, 200, 300, or more contiguous amino acids. Fragments can be generated by methods known to those of skill in the art or can result from normal protein processing (e.g., removal of amino acids from a nascent polypeptide that are not required for biological activity, or removal of amino acids by alternative mRNA splicing or alternative protein processing events). 5.6. Formulation and Administration

[0195] The subject matter of the present disclosure also provides a composition comprising the cells of the present disclosure. The composition comprising the cells of the present disclosure can be conveniently prepared as a sterile liquid preparation, for example, an isotonic aqueous solution, a suspension, an emulsion, a dispersion, or a viscous composition, and can be buffered to a selected pH. Liquid preparations are usually easier to prepare than gels, other viscous compositions, and solid compositions. In addition, liquid compositions are somewhat more convenient to administer, particularly by injection. On the other hand, viscous compositions can be formulated within an appropriate viscosity range to achieve a longer contact period with a particular tissue. The liquid or viscous composition can include a carrier, and can be, for example, a solvent or dispersion medium containing water, saline, phosphate buffered saline, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), and suitable mixtures thereof.

[0196] Sterile injectable solutions can be prepared by incorporating the genetically modified cells in the required amount of a suitable solvent with various amounts of other ingredients as desired. Such compositions may be mixed with suitable carriers, diluents, or excipients, such as sterile water, saline, glucose, dextrose, etc. The compositions may also be lyophilized. Depending on the route of administration and the preparation desired, the compositions may contain auxiliary substances such as wetting agents, dispersing agents, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or thickening additives, preservatives, flavoring agents, coloring agents, etc. Suitable preparations can be prepared without undue experimentation by reference to standard texts such as "REMINGTON'S PHARMACEUTICAL SCIENCE", 17th Edition, 1985, which is incorporated herein by reference.

[0197] Various additives can be added to enhance the stability and sterility of the composition, including antimicrobial preservatives, antioxidants, chelating agents, and buffers. Prevention of microbial action can be ensured by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. Prolonged absorption of injectable pharmaceutical forms can be achieved by the use of agents that delay absorption, such as aluminum monostearate and gelatin. However, according to the subject matter of the present disclosure, any vehicle, diluent, or additive used must be compatible with genetically modified cells.

[0198] The composition can be isotonic, i.e., they can have the same osmotic pressure as blood and tear fluid.The desired isotonicity of the composition can be achieved using sodium chloride, or other pharma- ceutically acceptable agents, such as dextrose, boric acid, sodium tartrate, propylene glycol, or other inorganic or organic solutes.Sodium chloride can be used especially for buffers containing sodium ions.

[0199] The viscosity of the composition can be maintained at a selected level, if desired, by using a pharma- ceutically acceptable thickening agent. For example, methylcellulose is readily and economically available and easy to handle. Other suitable thickening agents include, for example, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, carbomer, and the like. The concentration of the thickening agent can depend on the selected drug. 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 exact route of administration and the nature of the particular dosage form, for example, a liquid dosage form (for example, whether the composition is formulated into a solution, suspension, gel, or other liquid form, for example, a time-release form or a liquid-filled form).

[0200] The composition comprising the cells of the present disclosure can be systemically or directly provided to a subject to induce and / or enhance immune response to an antigen and / or treat and / or prevent neoplasia.In certain embodiments, the cells of the present disclosure or the composition comprising the same are directly injected into the organ of interest (e.g., the organ affected by neoplasia).Alternatively, the cells of the present disclosure or the composition comprising the same are indirectly provided to the organ of interest, for example, by administration to the circulatory system (e.g., tumor vasculature).Proliferative and differentiation agents can be provided before, during, or after administration of the cells or the composition to increase the production of cells (e.g., T cells or NK cells) in vitro or in vivo.

[0201] The cells of the present disclosure can be administered in any physiologically acceptable vehicle, usually intravascularly, but can also be introduced into bone or other convenient sites where the cells can find a suitable site for regeneration and differentiation (e.g., the thymus).

[0202] The number of cells administered can vary depending on the subject being treated. In certain embodiments, about 10 4 pcs and about 10 10 Between pieces, about 10 4 pcs and about 10 7 Between pieces, about 10 5 pcs and about 10 7 Between pieces, about 10 5 pcs and about 10 9 Between pieces, about 10 5 pcs and about 10 10 Between or about 10 6 pcs and about 10 8 Between about 1×10 cells of the present disclosure are administered to a subject. More effective cells may be administered in even smaller numbers. Typically, at least about 1×10 5 The final number of cells administered was approximately 1 × 10 10 In certain embodiments, the number of mAbs reaches at least about 1×10 or more. 5 pieces, approximately 2×10 5 pieces, about 5×10 5 pieces, about 1×10 6pieces, about 5×10 6 pieces, about 1×10 7 pieces, about 5×10 7 pieces, about 1×10 8 pieces, about 5×10 8 pieces, about 1×10 9 Pieces, or about 5 x 10 9 In certain embodiments, about 10 cells of the present disclosure are administered to a subject. 5 pcs and about 10 6 Between about 1×10 cells of the present disclosure are administered to a subject. In certain embodiments, between about 1×10 5 The cells of the present disclosure are administered to a subject. In certain embodiments, about 2×10 5 The cells of the present disclosure are administered to a subject. In certain embodiments, about 5×10 5 The cells of the present disclosure are administered to a subject. In certain embodiments, about 1×10 6 The cells of the present disclosure are administered to the subject.The exact determination of what can be considered as an effective dose can be based on factors specific to each subject, including the size, age, sex, weight and symptoms of the particular subject.The dosage can be easily ascertained by those skilled in the art from the present disclosure and knowledge in the art.

[0203] The cells of the present disclosure may include a population of purified cells. One of skill in the art can easily determine the percentage of cells of the present disclosure in a population using various well-known methods, such as fluorescence-activated cell sorting (FACS). Suitable ranges of purity in a population containing immunoresponsive cells of the present disclosure are about 50% to about 55%, about 5% to about 60%, and about 65% to about 70%. In certain embodiments, the purity is about 70% to about 75%, about 75% to about 80%, or about 80% to about 85%. In certain embodiments, the purity is about 85% to about 90%, about 90% to about 95%, and about 95% to about 100%. The dosage can be easily adjusted by one of skill in the art (e.g., a decrease in purity may require an increase in dosage). The cells can be introduced by injection, catheter, etc.

[0204] One of skill in the art can readily determine the amount of cells and any additives, vehicles, and / or carriers in the compositions and administered in the methods. Typically, any additives (in addition to the active cell(s) and / or drug(s)) are present in an amount of 0.001-50% (by weight) solution in phosphate buffered saline, and the active ingredient is present in the order of micrograms to milligrams, e.g., about 0.0001-about 5% by weight, about 0.0001-about 1% by weight, about 0.0001-about 0.05% by weight, or about 0.001-about 20% by weight, about 0.01-about 10% by weight, or about 0.05-about 5% by weight. For any composition administered to animals or humans, the following can be determined: toxicity, such as by determining the lethal dose (LD) and LD50 in an appropriate animal model, e.g., rodents such as mice; the dosage of the composition(s) that elicits an appropriate response, the concentrations of components in the composition(s) and the timing of administering the composition(s). Such determinations do not require undue experimentation from the knowledge of one of ordinary skill in the art, this disclosure, and the documents cited herein, and the time of sequential administration can be ascertained without undue experimentation.

[0205] In certain embodiments, the composition is a pharmaceutical composition comprising the cells of the present disclosure and a pharma- ceutically acceptable carrier.

[0206] The administration of the composition can be autologous or xenogeneic.For example, cells can be obtained from one subject and administered to the same subject or different compatible subjects.Peripheral blood-derived cells or their progeny (e.g., in vivo, ex vivo or in vitro derived) can be administered.When administering the composition of the present disclosure (e.g., the pharmaceutical composition comprising the cells of the present disclosure), it can be formulated in a unit dose injectable form (solution, suspension, emulsion).

[0207] The cells and compositions of the disclosure can be administered by any method known in the art, including, but not limited to, oral administration, intravenous administration, subcutaneous administration, intranodal administration, intratumoral administration, intrathecal administration, intrapleural administration, intraosseous administration, intraperitoneal administration, pleural administration, and direct administration to a subject. 5.7. Treatment Method

[0208] The subject matter of the present disclosure provides a method for inducing and / or increasing an immune response in a subject in need thereof. The cells of the present disclosure and compositions comprising the same can be used in therapy or medicine. The subject matter of the present disclosure can be used to inducing and / or increasing an immune response in a subject in need thereof. The cells (e.g., T cells, e.g., CD4 + T cells or CD8 + Various methods of using the cells of the present disclosure or compositions comprising the same (e.g., T cells) or compositions comprising the same are provided. For example, the cells of the present disclosure and compositions comprising the same can be used to reduce tumor burden in a subject. The cells of the present disclosure can reduce the number of tumor cells, reduce tumor size, and / or eradicate tumors in a subject. The cells of the present disclosure and compositions comprising the same can be used to treat and / or prevent neoplasms or tumors in a subject. The cells of the present disclosure and compositions comprising the same can be used to prolong the survival of a subject suffering from a neoplasm or tumor. The cells of the present disclosure and compositions comprising the same can be used to treat and / or prevent pathogen infection in a subject. Such methods include administering the cells of the present disclosure or compositions comprising the same (e.g., pharmaceutical compositions) to achieve a desired effect, e.g., alleviation of existing symptoms or prevention of recurrence. For treatment, the amount administered is an amount effective to produce the desired effect. An effective amount can be provided in one or a series of administrations. An effective amount can be provided by bolus or continuous perfusion.

[0209] The subject matter of the present disclosure provides various methods of using cells (e.g., T cells) or compositions comprising the same.For example, the subject matter of the present disclosure provides a method of reducing tumor burden in a subject.In certain embodiments, the method of reducing tumor burden comprises administering to a subject the cells of the present disclosure or compositions comprising the same.The cells of the present disclosure can reduce the number of tumor cells, reduce tumor size, and / or eradicate tumor in a subject.

[0210] The tumor may be a solid tumor. Non-limiting examples of solid tumors include mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colorectal cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic carcinoma, endometrial carcinoma, stomach cancer, melanoma, hepatocellular carcinoma, renal cell carcinoma, soft tissue sarcoma, and bile duct carcinoma.

[0211] The subject matter of the present disclosure also provides a method for increasing or prolonging the survival of a subject with a neoplasm. In certain embodiments, the method for increasing or prolonging the survival of a subject with a neoplasm comprises administering to the subject an immunoresponsive cell of the present disclosure or a composition comprising the same. The method can reduce or eradicate tumor burden in the subject. Furthermore, the subject matter of the present disclosure provides a method for increasing immune response in a subject, comprising administering to the subject a cell of the present disclosure or a composition comprising the same. The subject matter of the present disclosure further provides a method for treating and / or preventing a neoplasm in a subject, comprising administering to the subject a cell of the present disclosure or a composition comprising the same.

[0212] Non-limiting examples of neoplasms or tumors include B-cell leukemia, B-cell lymphoma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), multiple myeloma, lymphoma (Hodgkin's lymphoma, non-Hodgkin's lymphoma), glioblastoma, myelodysplastic syndrome (MDS), and chronic myeloid leukemia (CML), bone cancer, intestinal cancer, liver cancer, skin cancer, head and neck cancer, melanoma (cutaneous or intraocular malignant melanoma), kidney cancer (e.g., clear cell carcinoma), pharyngeal cancer, prostate cancer (e.g., hormone refractory prostate adenocarcinoma), blood cancer (e.g., leukemia, lymphoma, and myeloma), uterine cancer, rectal cancer, anal region cancer, bladder cancer, brain cancer, stomach cancer (stomach cancer), and the like. cancer), testicular cancer, carcinoma of the fallopian tube, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, leukemia (e.g., acute leukemia, acute lymphocytic leukemia, acute myeloid leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myeloid leukemia, polycythemia vera, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of childhood, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brain Solid tumors such as stem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, heavy chain diseases including Waldenstrom's macroglobulinemia, and sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelioma, lymphangiosarcoma, lymphangioendothelioma, synovium, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchial carcinoma, hepatocellular carcinoma, bile duct (nile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, salivary gland carcinoma, uterine cancer, testicular cancer, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, meningioma, melanoma, neuroblastoma, and retinoblastoma).

[0213] In certain embodiments, the tumor or neoplasm is selected from the group consisting of B-cell leukemia, B-cell lymphoma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), non-Hodgkin's lymphoma, Burkitt's lymphoma, acute myeloid leukemia (AML), and mixed phenotype acute leukemia (MPAL). In certain embodiments, the CAR binds to CD19.

[0214] The subject matter of the present disclosure also provides a method for increasing or prolonging the survival of a subject with pathogen infection.In certain embodiments, the method comprises administering to a subject the immunoresponsive cell of the present disclosure or a composition comprising the same.Non-limiting pathogen infections include HIV and fungal infections.

[0215] The subject may have an advanced form of the disease, in which case the goal of treatment may include reducing or reversing disease progression and / or ameliorating side effects. The subject may have a history of a condition that has already been treated, in which case the goal of treatment typically includes reducing or delaying the risk of recurrence.

[0216] Further modifications may be introduced into the cells (e.g., T cells) of the present disclosure to avoid or minimize the risk of immunological complications (known as "malignant T cell transformation"), such as graft-versus-host disease (GvHD), or where healthy tissue expresses the same target antigen as tumor cells, resulting in a similar outcome to GvHD. A potential solution to this problem is to engineer a suicide gene into the cells of the present disclosure. Suitable suicide genes include, but are not limited to, herpes simplex virus thymidine kinase (hsv-tk), inducible caspase 9 suicide gene (iCasp-9), and truncated human epidermal growth factor receptor (EGFRt) polypeptide. In certain embodiments, the suicide gene is an EGFRt polypeptide. The EGFRt polypeptide can enable T cell elimination by administering an anti-EGFR monoclonal antibody (e.g., cetuximab). The EGFRt can be covalently linked upstream of the CAR. The suicide gene can be included in a vector that includes a nucleic acid encoding the CAR of the present disclosure. Thus, administration of a prodrug designed to activate a suicide gene (e.g., a prodrug (e.g., AP1903 that can activate iCasp-9) during malignant T cell transformation (e.g., GVHD) causes apoptosis in suicide gene-activated cells expressing a CAR. The incorporation of a suicide gene, for example, into a CAR of the present disclosure, confers an additional level of safety with the ability to eliminate most receptor-expressing cells within a very short period of time. Cells (e.g., T cells) of the present disclosure with an incorporated suicide gene can be preemptively eliminated at a given time point after cell infusion or eradicated at the earliest sign of toxicity. 5.8.Kit

[0217] The subject matter of the present disclosure provides a kit for inducing and / or enhancing immune response in a subject, treating and / or preventing neoplasm or tumor in a subject, reducing tumor burden in a subject, increasing or prolonging the survival of a subject with a neoplasm in a subject, and / or treating and / or preventing pathogen infection.In certain embodiments, the kit comprises a cell of the present disclosure or a composition comprising the same.In certain embodiments, the kit comprises a sterile container; such a container can be a box, an ampoule, a bottle, a vial, a tube, a bag, a pouch, a blister pack, or other suitable container form known in the art.Such a container can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicines.In certain non-limiting embodiments, the kit comprises a nucleic acid molecule encoding a CAR of the present disclosure.

[0218] If desired, the 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. The instructions generally include information regarding the use of the composition for the treatment and / or prevention of neoplasia. In certain embodiments, the instructions include at least one of the following: description of the therapeutic agent; dosage schedule and administration for the treatment or prevention of neoplasia; cautions; warnings; instructions; counter-indications; overdosage information; adverse reactions; animal pharmacology; clinical studies; and / or references. The instructions may be printed directly on the container (if present), or may be printed as a label attached to the container, or as a separate sheet, pamphlet, card, or folder that is provided in or with the container. EXAMPLES

[0219] 6. Working Example The presently disclosed subject matter will be better understood by reference to the following examples, which are provided by way of illustration of the presently disclosed subject matter, and not by way of limitation. Example 1: In vitro and in vivo characterization of CD28 mutant CAR T cells

[0220] PI3K signaling appears to be redundant, as not only does CD28 bind directly to the PI3K p85 subunit (through the presence of the YMxM consensus in YMNM), but Grb2 (which binds to a YxNx consensus motif) binds Gab1 and Gab2, which in turn can recruit the PI3K p85 subunit and initiate downstream signaling.

[0221] Given the diverse number of adaptor molecules that can attach to the CD28 molecule, the functional outcomes of T cells (i.e., effector cytotoxicity, cytokine secretion, activation, survival, memory formation and exhaustion) are most likely the result of the sum of downstream signaling cascades derived from the binding of these adaptor molecules to CD28. Thus, modification of these CD28 motifs may allow or restrict the binding of various adaptor molecules that may alter signaling and result in enhanced effector function and / or reduced dysfunction. Given the involvement of PI3K signaling in the terminal differentiation of T cells, redundancy in this signaling pathway may be detrimental to effector function, and modulating it may be beneficial (Figure 1).

[0222] Given the ability of the YMNM motif of CD28 to determine its binding partners (adapter molecules) and that these partners determine T cell fate through several signaling cascades derived from PI3K, Grb2, and GADS, we generated several CD28 mutations that either allow or exclude the binding of these adaptor molecules to CD28 (Figure 2, Figure S12). Characterization of CD28-YKNI mutant CAR T cells

[0223] CD28-YKNI mutant CAR T cells were generated. Different human CD19-targeted CAR T cells expressing a truncated EGFR domain (Etah19) were co-cultured with CD19+NALM6 cells expressing GFP-ff luciferase (NALM6gL) at different effector:tumor ratios. Tumor cell lysis (for non-signaling CAR T cells) was measured by bioluminescence after 24 hours. CD28-YKNI mutant CAR T cells were found to have potent killing capacity in vitro (Figure 3A-D).

[0224] Human CD19-targeted CAR T cells were cultured alone or co-cultured with CD19+NALM6 cells at a 1:1 effector:tumor ratio. After 24 hours, supernatants were collected and cytokines were measured using a bead-based multiplex assay. CD28-YKNI mutant CAR T cells had a unique cytokine secretion profile (Figure 4A-N).

[0225] Various human CD19-targeted CAR T cells were co-cultured with NALM6 at an E:T ratio of 1:5 and a concentration of 50,000 CAR T cells / mL. Approximately every 5 days, CAR T cells were counted and characterized by flow cytometry, memory phenotype (CD62L+) and CD4 / CD8 distribution. Starting numbers of tumor cells were added back into the culture at different time points. CD28-YKNI mutant CAR T cells also had potent killing and proliferation capacity in vitro (Figure 5). CD28-YKNI mutant CAR T cells were characterized by CD28 and CD28-1xx Compared with CAR T cells, CD28-YKNI mutant CAR T cells retained their memory phenotype in the context of repeated antigen encounters (Figure 6). Compared with CAR T cells, they maintained a relatively balanced CD8:CD4 ratio in the setting of repeated antigen encounters (Figure 7).

[0226] CD28-YKNI mutant CAR T cells demonstrated a restricted activation profile after single and multiple stimulations. CAR T cells were co-cultured with NALM6gL at an initial E:T of 1:5 (single stimulation). In parallel, CAR T cells were repeatedly stimulated with the same amount of tumor for a total of five stimulations (one stimulation every 12 hours). Approximately 10 days after the start of the co-culture, size / blastogenesis (assessed by forward scatter) was assessed by flow cytometry. CD28-YKNI mutant CAR T cells demonstrated lower blastogenesis after single or multiple activation (Figure 8).

[0227] The metabolic profile of CD28-YKNI mutant CAR T cells was measured 9 days after single or multiple stimulations. CD28-YKNI mutant CAR T cells demonstrated significantly lower basal respiration after single or multiple stimulations (Figure 9A). Significantly higher basal oxygen consumption rates (OCR) were measured in Etah19h28Z and Etah19h28Zp33 after 5 stimulations with leukemia antigens, but this difference was not present after only 1 stimulation. The increase in basal oxygen consumption of the cells suggested a preferential reliance on oxidative phosphorylation as the main energy generating mechanism, explaining the metabolic demands required for enhanced CAR T cell proliferation. This was further confirmed by the increase in basal OCR after further stimulations with tumor antigens (e.g., 5 stimulations compared to 1 stimulation).

[0228] CD28-YKNI mutant CAR T cells also demonstrated significantly lower lactate production after single or multiple stimulations (Figure 9B). Extracellular acidification rate (ECAR) is a measurable surrogate of lactate production during glycolysis. An increase in basal ECAR in stimulated CAR T cells suggests increased glycolytic activity, which is typically measured in T cells activated with antigen. An increase in ECAR in Etah19hMUThZ was observed, but was minimal, suggesting that T cells with this modification did not experience significant stimulation.

[0229] CAR T cells were co-cultured with NALM6gL in an initial E:T of 1:5 (single stimulation). In parallel, CAR T cells were repeatedly stimulated with the same amount of tumor for a total of five stimulations (single stimulation every 12 hours). Approximately 10 days after the start of the co-culture, exhaustion markers (LAG3 and PD1) were assessed by flow cytometry. CD28-YKNI mutant CAR T cells expressed lower levels of co-inhibitory molecules (LAG3 and PD1, TIM-3 and PD1) in the context of single or multiple stimulations (Figures 10A-B).

[0230] The in vivo antitumor efficacy of mutation-based CAR T cells was measured. 6 NALM6gfp + ffLUC + Tumor cells were inoculated and treated with CAR T cells 4 days later. CAR T cells were derived from two different healthy donors. CD28-YKNI mutant CD19-targeted CAR T cells outperformed standard CD28-based CAR T cells in vivo (Figure 11). Characterization of other CD28 mutant CAR T cells

[0231] Different human CD19-targeting CD28 mutant CAR T cells expressing a truncated EGFR domain (Etah19) (CD28-YKNI, CD28-YMDM, CD28-YGGG, CD28-YENV, CD28-YKNL, and CD28-YSNV) CD28 were co-cultured with CD19+ NALM6 cells expressing GFP-ff luciferase (NALM6gL) at different effector:tumor ratios, and tumor cell lysis (relative to non-signaling CAR T cells) was measured by bioluminescence after 24 hours. The T cells exhibited comparable killing capacity in a 24-hour killing assay (Figure 13).

[0232] Various human CD19-targeted CAR T cells were co-cultured with NALM6 at an E:T ratio of 1:5 and a starting concentration of 25,000 CAR T cells / mL. CAR+ and NALM6 concentrations were measured daily and plotted over 6 days. CD28-Yxxx mutant CD19-targeted CAR T cells (YKNI, YENV, and YMDM) outperformed standard CD28-based CAR T cells in vitro (Figure 14). CD28 mutants displayed potent long-term cytotoxicity capacity in vitro.

[0233] CAR T cells were co-cultured with NALM6gL at an initial E:T of 1:5 (single stimulation). In parallel, CAR T cells were repeatedly stimulated with the same amount of tumor for a total of five stimulations (one stimulation every 12 hours). Approximately 10 days after the start of the co-culture, exhaustion markers (TIM3 and PD1) were assessed by flow cytometry. CD28 mutants demonstrated a favorable exhaustion immune phenotype (Figure 15).

[0234] CD28-Yxxx mutant CD19-targeted CAR T cells (YKNI, YENV, and YMDM) outperformed standard CD28-based CAR T cells in vitro (Figures 16-18). + ffLUC + Tumor cells were inoculated and treated with CAR T cells 4 days later. Survival was graphed. Bioluminescence was measured weekly. CAR T cells were derived from a single healthy donor. Example 2: Characterization of CD28 mutant CAR T cells

[0235] We generated CD28-Yxxx mutant CD19-targeted CAR T cells, including YENV, YKNI, YGGG, YMDM and YSNV CD19-targeted CAR T cells. We characterized the in vitro and in vivo properties of these CD28 mutant CAR T cells.

[0236] CD28-Yxxx mutant CD19-targeted CAR T cells were co-cultured with NALM6gL at an E:T ratio of 1:15. The concentration of NALM6 was measured daily and plotted over 7 days, plotted in cells / mL. It was observed that CD28-Yxxx mutant CD19-targeted CAR T cells (CD28-YENV, CD28-YKNI, CD28-YGGG, CD28-YMDM and CD28-YSNV) outperformed standard CD28-based CAR T cells and demonstrated potent long-term cytotoxicity capacity in vitro (Figure 19).

[0237] CD28-Yxxx mutant CD19-targeted CAR T cells were co-cultured with NALM6gL at E:T ratios of 1:15 and 1:30. After 5 days, exhaustion marker (LAG3, TIM3 and PD1)-expressing CAR T cells were assessed by flow cytometry. CD28-Yxxx mutant CD19-targeted CAR T cells had a favorable exhaustion immunophenotype (Figure 20).

[0238] To demonstrate the in vivo antitumor efficacy of CD28 mutant CAR T cells, 1 × 10 6 NALM6gfp + ffLUC + Tumor cells were inoculated and treated with 500,000 or 200,000 CAR T cells 4 days later. CD28-Yxxx mutant CD19-targeted CAR T cells demonstrated enhanced tumor control in vivo, outperforming standard CD28-based CAR T cells (Figure 21 and Figure 22).

[0239] CD28-Yxxx mutant CD19-targeted CAR T cells displayed enhanced proliferation in vitro, independent of antigen density. Human CD19-targeted CD28-Yxxx mutant CAR T cells were co-cultured with NALM6gL at a 1:1 E:T ratio, either at high or low CD19 antigen density. Every 6 days, CAR+ T cells were counted and restimulated with NALM6gL for a total of three stimulations. CD28-Yxxx mutant CD19-targeted CAR T cells displayed enhanced proliferation in vitro relative to standard CD28-based CAR T cells in the context of both high and low antigen density CD19 tumor cells (Figure 23).

[0240] The cytokine secretion profile of CD28-Yxxx mutant CD19-targeted CAR T cells was measured. Human CD19-targeted CD28-Yxxx mutant CAR T cells were co-cultured with NALM6gL. After 24 hours, supernatants were collected and cytokines including interleukin-2, TNF-α, GM-CSF, interferon-γ, IL-9, and IL-17 were measured by Luminex bead-based multiplex assay. CD28-Yxxx mutant CD19-targeted CAR T cells demonstrated a unique cytokine secretion profile upon exposure to antigen (Figure 24A-C).

[0241] Although the subject matter of the present disclosure and some of its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the present disclosure. Moreover, the scope of the present application is not intended to be limited to the specific embodiments of the process, machine, manufacture, and composition of matter, and method described herein. As one skilled in the art would readily appreciate from the disclosure of the subject matter of the present disclosure, in accordance with the subject matter of the present disclosure, any currently existing or hereafter developed process, machine, manufacture, composition of matter, or method may be utilized that performs substantially the same function or achieves substantially the same result as the corresponding embodiment described herein. Accordingly, it is intended that the appended claims include within their scope such processes, machines, manufacture, compositions of matter, or methods.

[0242] Various patents, patent applications, publications, product descriptions, protocols, and sequence accession numbers are cited throughout this application, the disclosures of which are incorporated herein by reference in their entireties for all purposes. The present invention provides, for example, the following items. (Item 1) A chimeric antigen receptor (CAR) comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising at least one costimulatory signaling domain comprising a CD28 polypeptide containing a mutated YMNM motif. (Item 2) 2. The CAR of item 1, wherein the CD28 polypeptide has reduced recruitment of the p85 subunit of phosphoinositide 3-kinase (PI3K) compared to a CD28 molecule containing a native YMNM motif. (Item 3) 3. The CAR of item 1 or 2, wherein the p85 subunit of PI3K does not bind to the mutant YMNM motif. (Item 4) The CAR of item 3, wherein the mutant YMNM motif consists of an amino acid sequence set forth in YxNx (SEQ ID NO: 21), where x is not methionine (M). (Item 5) The CAR of item 3 or 4, wherein the mutated YMNM motif consists of the amino acid sequence set forth in YENV (SEQ ID NO: 22), YSNV (SEQ ID NO: 23), YKNL (SEQ ID NO: 24), YENQ (SEQ ID NO: 25), YKNI (SEQ ID NO: 26), YINQ (SEQ ID NO: 27), YHNK (SEQ ID NO: 28), YVNQ (SEQ ID NO: 29), YLNP (SEQ ID NO: 30), YLNT (SEQ ID NO: 31), YDND (SEQ ID NO: 66), YENI (SEQ ID NO: 67), YENL (SEQ ID NO: 68), YKNQ (SEQ ID NO: 72), YKNV (SEQ ID NO: 73), or YANG (SEQ ID NO: 87). (Item 6) 6. The CAR of any one of items 3 to 5, wherein the mutated YMNM motif consists of the amino acid sequence set forth in YSNV (SEQ ID NO: 23), YENV (SEQ ID NO: 22), or YKNI (SEQ ID NO: 26). (Item 7) 7. The CAR of any one of items 3 to 6, wherein the mutated YMNM motif consists of the amino acid sequence set forth in YSNV (SEQ ID NO: 23). (Item 8) 8. The CAR of any one of items 3 to 7, wherein the mutated YMNM motif binds to growth factor receptor-bound receptor 2 (Grb2) and / or Grb2-associated adaptor downstream of Shc (GADS). (Item 9) 3. The CAR of item 1 or 2, wherein the mutated YMNM motif does not bind to Grb2 and / or GADS. (Item 10) The CAR of item 9, wherein the mutant YMNM motif consists of an amino acid sequence set forth in YMxM (SEQ ID NO: 20), wherein x is not aspartic acid (N). (Item 11) The CAR of item 9 or 10, wherein the mutated YMNM motif consists of the amino acid sequence set forth in YMDM (sequence number 32), YMPM (sequence number 79), YMRM (sequence number 37), or YMSM (sequence number 80). (Item 12) 12. The CAR of any one of items 9 to 11, wherein the mutated YMNM motif consists of the amino acid sequence set forth in YMDM (SEQ ID NO: 32). (Item 13) Item 9. The mutant YMNM motif comprises an amino acid sequence represented by YbxM (SEQ ID NO: 33), where x is not aspartic acid (N) and b is not methionine (M). CAR as stated. (Item 14) The CAR of item 13, wherein the mutated YMNM motif consists of the amino acid sequence set forth in YTHM (sequence number 34), YVLM (sequence number 35), YIAM (sequence number 36), YVEM (sequence number 83), YVKM (sequence number 85), or YVPM (sequence number 86). (Item 15) 10. The CAR of item 9, wherein the mutant YMNM motif consists of the amino acid sequence set forth in YMxb (SEQ ID NO: 65), wherein x is not aspartic acid (N) and b is not methionine (M). (Item 16) The CAR of item 15, wherein the mutated YMNM motif consists of the amino acid sequence set forth in YMAP (sequence number 77). (Item 17) 17. The CAR of any one of items 9 to 16, wherein the p85 subunit of PI3K signaling binds to a mutant YMNM motif. (Item 18) 3. The CAR of item 1 or 2, wherein the mutated YMNM motif does not bind to Grb2 and / or GADS or the p85 subunit of PI3K. (Item 19) 19. The CAR of item 18, wherein the mutant YMNM motif consists of the amino acid sequence set forth in Ybxb (SEQ ID NO: 43), wherein x is not aspartic acid (N) and b is not methionine (M). (Item 20) 20. The CAR of item 19, wherein the mutated YMNM motif consists of the amino acid sequence set forth in YGGG (SEQ ID NO: 44), YAAA (SEQ ID NO: 45), YFFF (SEQ ID NO: 46), YETV (SEQ ID NO: 69), YQQQ (SEQ ID NO: 70), YHAE (SEQ ID NO: 71), YLDL (SEQ ID NO: 74), YLIP (SEQ ID NO: 75), YLRV (SEQ ID NO: 76), YTAV (SEQ ID NO: 82), or YVHV (SEQ ID NO: 84). (Item 21) 21. The CAR of any one of items 18 to 20, wherein the mutated YMNM motif consists of the amino acid sequence set forth in YGGG (SEQ ID NO: 44). (Item 22) 3. The CAR of item 1 or 2, wherein the mutant YMNM motif can regulate PI3K signaling by limiting the number of methionine residues that can bind to the p85 subunit of PI3K. (Item 23) 23. The CAR of item 22, wherein the mutant YMNM motif consists of the amino acid sequence set forth in YMNx (sequence number 38) or YxNM (sequence number 39), wherein x is not methionine (M). (Item 24) The CAR of item 22 or 23, wherein the mutated YMNM motif consists of the amino acid sequence set forth in YMNV (SEQ ID NO: 40), YENM (SEQ ID NO: 41), and YMNQ (SEQ ID NO: 42), YMNL (SEQ ID NO: 78), or YSNM (SEQ ID NO: 81). (Item 25) 25. The CAR of any one of items 1 to 24, wherein the extracellular antigen binding domain binds to an antigen. (Item 26) 26. The CAR of claim 25, wherein the antigen is a tumor antigen or a pathogen antigen. (Item 27) 27. The CAR of item 25 or 26, wherein the antigen is a tumor antigen. (Item 28) The tumor antigen is CD19, mesothelin, AXL, TIM3, HVEM, MUC16, MUC1, CA1X, CEA, CD8, CD7, CD10, CD20, CD22, CD30, CLL1, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD70, CD74, CD99, CD123, CD133, CD138, EGP-2, EGP-40, EpCAM, Erb-B, FBP, fetal acetylcholine receptor, folate receptor-alpha, GD2, GD3, HER-2, hTERT, IL-13R-alpha2, kappa-light receptor, 28. The CAR of item 27, wherein the CAR is selected from the group consisting of: IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37, IL-38, IL-39, IL-40, IL-41, IL-42, IL-43, IL-44, IL-45, IL-46, IL-47, IL-48, IL-49, IL-59, IL-59, IL-59, IL-59, IL-59, IL-59, IL-59, IL-59, IL-60, IL-61, IL-62, IL-63, IL-64, IL-65, IL-76, IL-87, IL-88, IL-89 ... (Item 29) 29. The CAR of item 28, wherein the tumor antigen is CD19. (Item 30) 30. The CAR of any one of items 1 to 29, wherein the mutated YMNM motif consists of the amino acid sequence set forth in YMDM (SEQ ID NO: 32). (Item 31) 31. The CAR of paragraph 30, wherein the extracellular antigen binding domain binds to CD19. (Item 32) 32. The CAR of item 31, comprising the amino acid sequence set forth in SEQ ID NO:51. (Item 33) 30. The CAR of any one of items 1 to 29, wherein the mutated YMNM motif consists of the amino acid sequence set forth in YKNI (SEQ ID NO: 26). (Item 34) 34. The CAR of claim 33, wherein the extracellular antigen binding domain binds to CD19. (Item 35) The CAR of item 34, comprising the amino acid sequence set forth in SEQ ID NO: 55. (Item 36) 30. The CAR of any one of items 1 to 29, wherein the mutated YMNM motif consists of the amino acid sequence set forth in YENV (SEQ ID NO: 22). (Item 37) 37. The CAR of paragraph 36, wherein the extracellular antigen binding domain binds to CD19. (Item 38) 38. The CAR of item 37, comprising the amino acid sequence set forth in SEQ ID NO: 53. (Item 39) 30. The CAR of any one of items 1 to 29, wherein the mutated YMNM motif consists of the amino acid sequence set forth in YSNV (SEQ ID NO: 64). (Item 40) 40. The CAR of paragraph 39, wherein the extracellular antigen binding domain binds to CD19. (Item 41) 41. The CAR of item 40, comprising the amino acid sequence set forth in SEQ ID NO:57. (Item 42) 30. The CAR of any one of items 1 to 29, wherein the mutated YMNM motif consists of the amino acid sequence set forth in YGGG (SEQ ID NO: 63). (Item 43) 43. The CAR of claim 42, wherein the extracellular antigen binding domain binds to CD19. (Item 44) 44. The CAR of item 43, comprising the amino acid sequence set forth in SEQ ID NO: 61. (Item 45) A cell comprising the CAR of any one of items 1 to 44. (Item 46) 46. ​​The cell of item 45, which is an immunoresponsive cell. (Item 47) 47. The cell according to item 45 or 46, which is a lymphoid or myeloid cell. (Item 48) 48. The cell according to any one of items 45 to 47, which is selected from the group consisting of T cells, natural killer (NK) cells, and stem cells from which lymphoid cells can be differentiated. (Item 49) 49. The cell of any one of items 45 to 48, which is a T cell. (Item 50) 50. The cell of item 48 or 49, wherein the T cell is selected from the group consisting of cytotoxic T lymphocytes (CTLs), gamma delta T cells, tumor-reactive lymphocytes, tumor-infiltrating lymphocytes (TILs), regulatory T cells, and natural killer T (NKT) cells. (Item 51) 51. A composition comprising the cells of any one of items 45 to 50. (Item 52) 52. The composition according to item 51, which is a pharmaceutical composition further comprising a pharma- ceutically acceptable excipient. (Item 53) 53. The composition according to item 51 or 52, for treating and / or preventing a neoplasm or tumor, and / or a pathogen infection. (Item 54) 52. A method for reducing tumor burden in a subject, comprising administering to the subject a cell according to any one of items 45 to 50 or a composition according to any one of items 51 to 53. (Item 55) 55. The method of claim 54, wherein the number of tumor cells is reduced, tumor size is reduced, and / or the tumor is eradicated in the subject. (Item 56) A method for treating and / or preventing a neoplasm or tumor, comprising administering to said subject a cell according to any one of items 45 to 50 or a composition according to any one of items 51 to 53. (Item 57) A method for prolonging survival of a subject having a neoplasm or tumor, comprising administering to said subject a cell according to any one of items 45 to 50 or a composition according to any one of items 51 to 53. (Item 58) 58. The method of any one of items 54 to 57, wherein the neoplasm and / or tumor is selected from the group consisting of B-cell leukemia, B-cell lymphoma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), non-Hodgkin's lymphoma, Burkitt's lymphoma, acute myeloid leukemia (AML) and mixed phenotype acute leukemia (MPAL). (Item 59) 45. A method for producing an antigen-specific cell, comprising introducing into a cell a nucleic acid molecule encoding a CAR described in any one of items 1 to 44. (Item 60) 60. The method of item 59, wherein the nucleic acid molecule is present on a vector. (Item 61) 61. The method of item 60, wherein the vector is a retroviral vector. (Item 62) 45. A nucleic acid molecule encoding the CAR of any one of items 1 to 44. (Item 63) 63. The nucleic acid molecule according to item 62, comprising the nucleotide sequence set forth in SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, or SEQ ID NO:58. (Item 64) 64. A vector comprising the nucleic acid molecule of item 62 or 63. (Item 65) 65. The vector according to item 64, wherein the vector is a gamma-retroviral vector. (Item 66) 66. A host cell expressing the nucleic acid molecule of item 64 or 65. (Item 67) 67. The host cell of paragraph 66, which is a T cell. (Item 68) A kit comprising the CAR of any one of items 1 to 44, the cell of any one of items 35 to 40, the composition of any one of items 51 to 53, the nucleic acid molecule of item 62 or 63, or the vector of item 64 or 65. (Item 69) 70. The kit of item 68, further comprising written instructions for treating and / or preventing a neoplasm, tumor, and / or pathogen infection.

Claims

[Claim 1] The invention described in the specification.

Images