Chimeric antigen receptors having a CD28 mutation and uses thereof

Abstract

The present disclosure provides methods and compositions for enhancing immune responses to cancer and pathogens. The present disclosure relates to chimeric antigen receptors (CARs) comprising mutant CD28 intracellular motifs, and cells comprising such CARs. The subject matter of the present disclosure further relates to the use of the cells for treating diseases, for example, cancer. The subject matter of the present disclosure provides chimeric antigen receptors (CARs) comprising mutant CD28 intracellular motifs, i.e., mutant YMNM motifs.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 62 / 970,401, filed on February 5, 2020, the content of which is hereby incorporated by reference in its entirety, and claims priority based on this provisional patent application. Sequence Listing

[0002] This application includes a sequence listing submitted in ASCII format via EFS - Web, the content of which is hereby incorporated by reference in its entirety. A copy of the ASCII created on February 4, 2021, is named 072734.1189_ST25.txt and has a size of 70,968 bytes.

[0003] 1. Technical Field The present disclosure provides methods and compositions for enhancing the immune response against cancer and pathogens. The present disclosure relates to chimeric antigen receptors (CARs) comprising a mutant CD28 intracellular motif, namely a mutant YMNM motif. The subject matter of the present disclosure also provides cells comprising the CAR and compositions comprising the cells, as well as the use of the cells and compositions for treating diseases, for example, for treating cancer.

Background Art

[0004] 2. Background Art 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 introducing genetic material encoding 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). Engineered CAR T cells from patients have demonstrated significant efficacy against a variety of liquid and solid malignancies.

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

[0006] 3. Gist of the Invention The subject matter of the present disclosure provides a chimeric antigen receptor (CAR) comprising a mutant CD28 intracellular motif, i.e., a mutant 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 co-stimulatory signaling domain comprising a CD28 polypeptide comprising a mutant YMNM motif.

[0008] In certain embodiments, the CD28 polypeptide reduces the recruitment of the p85 subunit of phosphoinositide 3-kinase (PI3K) compared to a CD28 molecule comprising a 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 set forth 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 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). In certain embodiments, the mutant 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). In certain embodiments, the mutant YMNM motif consists of the amino acid sequence set forth in YSNV (SEQ ID NO: 23). In certain embodiments, the mutant YMNM motif binds to Grb2-related adaptor (GADS) downstream of growth factor receptor-bound receptor 2 (Grb2) and / or Shc.

[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 set forth in YMxM (SEQ ID NO: 20), where x is not asparagine (N). In certain embodiments, the mutant YMNM motif consists of the amino acid sequence set forth 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 set forth in YMDM (SEQ ID NO: 32). In certain embodiments, the mutant YMNM motif consists of the amino acid sequence set forth in YbxM (SEQ ID NO: 33), where x is not asparagine (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 asparagine (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 the 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 set forth in Ybxb (SEQ ID NO: 43), where x is not asparagine (N) and b is not methionine (M). In certain embodiments, the mutant 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 mutant YMNM motif consists of the amino acid sequence set forth in YGGG (SEQ ID NO: 44).

[0011] In certain embodiments, the mutant YMNM motif can regulate PI3K signaling by limiting 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 selected from the group consisting of 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-α, GD2, GD3, HER-2, hTERT, 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, cancer fetal antigen (h5T4), PSCA, PSMA, ROR1, TAG-72, VEGF-R2, WT-1, BCMA, CD44V6, NKCS1, EGF1R, EGFR-VIII, ADGRE2, CCR1, LILRB2, PRAME, HPV E6 cancer protein, and HPV E7 cancer protein. In certain embodiments, the tumor antigen is CD19.

[0013] In certain embodiments, the mutant 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 mutant 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 mutant 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 mutant 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 mutant 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 cells comprising the CARs described herein. In certain embodiments, the cells are immunoreactive cells. In certain embodiments, the cells are lymphoid or myeloid cells. In certain embodiments, the cells are selected from the group consisting of T cells, natural killer (NK) cells, and stem cells from which lymphoid cells can differentiate. In certain embodiments, the cells are T cells. In certain embodiments, the T cells are selected from the group consisting of cytotoxic T lymphocytes (CTLs), γδ 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 cells described herein. In certain embodiments, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable excipient. In certain embodiments, the composition is for treating and / or preventing neoplasms and / or pathogen infections.

[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 the subject the cells described herein or the composition described herein. In certain embodiments, the method reduces the number of tumor cells in the subject, reduces tumor size, and / or eradicates the tumor.

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

[0022] The subject matter of the present disclosure further provides a method for prolonging the survival of a subject having a neoplasm. In certain embodiments, the method comprises administering to the subject the cells described herein or the 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 lymphoma, Burkitt 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 the cells a 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] Furthermore, the subject matter of the present disclosure provides nucleic acid molecules encoding the CARs 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 vectors comprising the nucleic acid molecules described herein. In certain embodiments, the vector is a γ-retroviral vector.

[0026] The subject matter of the present disclosure further provides host cells that express 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 kits comprising the CARs described herein, the cells described herein, the compositions described herein, the nucleic acid molecules described herein, or the vectors described herein. In certain embodiments, the kit further comprises written instructions for treating and / or preventing a neoplasm and / or pathogen infection. 4. Brief Description of the Drawings

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[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 reduces the recruitment of the p85 subunit of phosphoinositide 3-kinase (PI3K) signaling as compared to a CD28 molecule comprising a native 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 Grb2-related adaptor downstream of growth factor receptor-bound receptor 2 (Grb2) and / or Shc (GADS) binds 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., immune-responsive cells, e.g., T cells or NK cells) comprising the CARs 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 an immune response against a target antigen and / or to treat and / or prevent a neoplasm or tumor and / or a pathogen infection. The subject matter of the present disclosure is at least partially based on the discovery that cells comprising a CAR comprising a mutant CD28 intracellular motif (i.e., a mutant YMNM motif) exhibit an enhanced anti-tumor effect as 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 by the present specification and the examples.

[0055] For the purpose of clarity, and not limitation, the detailed description is divided into the following subsections: 5.1. Definitions; 5.2. Chimeric Antigen Receptors (CARs); 5.3. Cells; 5.4. Compositions and Vectors; 5.5. Polypeptides; 5.6. Formulations and Administration; 5.7. Methods of Treatment; and 5.8. Kits 5.1. Definitions

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

[0057] As used herein, the terms “about” or “approximately” mean 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., the limitations of the measurement system. For example, “about” can mean within 3 or more standard deviations, per the convention in the art. Alternatively, “about” can mean within up to 20%, such as within up to 10%, up to 5%, or up to 1% of a given value. Alternatively, especially with respect to biological systems or processes, the term can mean within an order of magnitude, such as within 5-fold or 2-fold of a value.

[0058] “Immunoresponsive cell” means a cell that functions in an immune response, or a precursor or progeny thereof. In certain embodiments, the immunoresponsive cell is a lymphoid cell. Non-limiting examples of lymphoid cells include T cells, natural killer (NK) cells, B cells, and stem cells from which lymphoid cells can differentiate. In certain embodiments, the immunoresponsive cell is a myeloid cell.

[0059] "Activating an immunoreactive cell" means inducing signal transduction or a 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 an endogenous TCR or exogenous CAR binds to an antigen, formation of an immune synapse occurs, including 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 enables the ITAM motifs contained within the CD3 chains to be phosphorylated. This phosphorylation then initiates a T cell activation pathway that ultimately activates transcription factors such as NF-κB and AP-1. These transcription factors induce the overall gene expression of 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 an immunoreactive cell" means a signal that results in a robust and sustained immune response. In various embodiments, this occurs after activation of immune cells (e.g., T cells) or occurs simultaneously and is mediated by receptors including, but not limited to, CD28, CD137 (4-1BB), OX40, CD40, and ICOS. Receiving multiple stimulatory signals can be important for initiating a robust and long-term T cell-mediated immune response. T cells can become rapidly inhibited and non-responsive to antigens. The effects of these co-stimulatory signals can vary, but they generally result in increased gene expression to generate long-lived, proliferative, and anti-apoptotic T cells that respond robustly to antigens towards complete and sustained eradication.

[0061] As used herein, the term "antigen recognition receptor" refers to a receptor that can activate an immunoreactive cell (e.g., a T cell) in response to binding to an antigen.

[0062] As used herein, "CDR" is defined as the amino acid sequence of the complementarity determining regions of an antibody, which are the hypervariable regions of the immunoglobulin heavy and light chains. See, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 4th U.S. Department of Health and Human Services, National Institutes of Health (1987). Generally, an antibody contains three heavy chain and three light chain CDRs, i.e., CDR regions, in the variable region. The CDRs provide most of the contact residues for the binding of the antibody to an 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., U.S. Department 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 fusion protein of the variable regions of an immunoglobulin heavy chain (V H ::V L and light chain (V H ) covalently joined to form a heterodimer. V L and V H are either directly joined or joined by a linker (e.g., 10, 15, 20, 25 amino acids) encoding a peptide that connects the N-terminus of V L to the C-terminus of V H , or the C-terminus of V L to the N-terminus of V H , or the C-terminus of V L to the N-terminus of V

[0063]

[0063] As used herein, "linker" refers to a functional group (e.g., a chemical substance or polypeptide) that covalently binds two or more polypeptides or nucleic acids so as to connect them to each other. As used herein, "peptide linker" refers to one or more amino acids used to couple two proteins together (e.g., to couple V H and V L domains).

[0064] Despite the removal of the constant region and the introduction of a linker, the scFv protein retains the specificity of the original immunoglobulin. Single-chain Fv polypeptide antibodies are described by Huston et al., Proc Nat Acad Sci USA (1988); 85: 5879-5883,; U.S. Pat. Nos. 5,091,513, 5,132,405 and 4,956,778; and U.S. Patent Application Publication Nos. 20050196754 and 20050196754, such as V H and V LIt can be expressed from a nucleic acid containing a coding array. Agonistic scFvs having inhibitory activity have been described (Zhao et al., Hybridoma (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 having 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" means a measure of binding strength. Affinity can depend on the closeness of the stereochemical fit between the binding site of the antibody and the epitope, the size of the contact area between them, and / or the distribution of charged and hydrophobic groups. Methods for calculating the affinity of an antibody for an antigen include, but are not limited to, various antigen-binding experiments, such as functional assays (e.g., flow cytometry assays), and are known in the art.

[0066] As used herein, the term "chimeric antigen receptor" or "CAR" refers to a molecule comprising an extracellular antigen-binding domain fused to an intracellular signaling domain capable of activating an immune-responsive cell, and a transmembrane domain. In certain embodiments, the extracellular antigen-binding domain of the CAR comprises 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 (obtained instead from an antibody, for example, from a Fab library). In certain embodiments, the scFv is fused to the transmembrane domain and then to the intracellular signaling domain.

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

[0068] "Substantially identical" or "substantially homologous" means 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 arrays is a function of the number of identical positions shared by the arrays (i.e., % homology = number of identical positions / total number of positions × 100), taking into account the number of gaps that need to be introduced for optimal alignment of the two arrays and the length of each gap. Comparison of arrays and determination of the percent identity between two arrays can be achieved using mathematical algorithms.

[0070] Array identity can be measured using array analysis software (e.g., the Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or the PILEUP / PRETTYBOX programs). Such software matches identical or similar arrays by assigning a degree 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 for determining the degree of identity, the BLAST program can be used, and a probability score between e-3 and e-100 indicates closely related arrays.

[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)), incorporated into 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 incorporated into 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" for performing a search against a public database, for example, to identify related sequences. Such a search can be performed using the XBLAST program (version 2.0) of Altschul et al. (1990) J. Mol. Biol. 215:403-10. By performing a BLAST protein search using the XBLAST program with a score = 50 and a word length = 3, amino acid sequences homologous to the specified sequences disclosed herein (e.g., the heavy chain variable region sequences and light chain variable region sequences of scFv m903, m904, m905, m906, and m900) can be obtained. To obtain a gapped alignment for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing the BLAST and Gapped BLAST programs, the default parameters of each program (e.g., XBLAST and NBLAST) can be used.

[0073] "Effective amount" means an amount sufficient to affect a beneficial or desired clinical result upon treatment. The effective amount can be administered to a subject in one or more than one dose. In certain embodiments, the effective amount can be an amount sufficient to alleviate, ameliorate, stabilize, reverse, or delay the progression of a disease or to reduce the pathological severity of a disease. The effective amount can be determined by a physician for each case and is within the skill of those in the art. When determining an appropriate dosage to achieve an effective amount, several factors are typically considered. These factors include the age, sex, and weight of the subject, the condition being treated, the severity of the condition, and the form and effective concentration of the cells being administered.

[0074] "Modulate" means to change 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] "Increase" means to change positively by at least about 5%. The change can be about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, about 100% or more.

[0076] "Reduce" means to change negatively by at least about 5%. The change 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 substance in which the components normally associated with it as found in its natural state are absent to varying degrees. "Isolating" refers to the degree of separation from the original source or surroundings. "Purifying" refers to a higher degree of separation than isolation. A "purified" or "biologically pure" protein does not contain other materials to such an extent that any impurities substantially affect the biological properties of the protein or cause other adverse results. That is, a nucleic acid or peptide is purified if, when produced by recombinant DNA technology, it substantially does not contain cellular material, viral material, or culture medium, or if chemically synthesized, it substantially does not contain chemical precursors or other chemicals. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" can indicate that a nucleic acid or protein yields essentially one band on an electrophoretic gel. In the case of a protein that can be subjected to modifications such as phosphorylation or glycosylation, different modifications can result in different isolated proteins that can be separately purified.

[0078] "Isolated cell" means a cell that has been separated from the molecules and / or components of the cell that are naturally associated with it.

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

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

[0081] "Signal sequence" or "leader sequence" means a peptide sequence (e.g., 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 the United States Patent Law and can mean "includes", "including", etc.

[0083] As used herein, "treatment" refers to a clinical intervention intended to alter the course of a disease in an individual or cell being treated, and can be performed either for prophylaxis or during the course of clinical pathology. The therapeutic effects of treatment include, but are not limited to, prevention of the occurrence or recurrence of disease, alleviation of symptoms, reduction of the direct or indirect pathological severity of the disease, prevention of metastasis, reduction of the rate of disease progression, amelioration or palliation of the disease state, and remission or improvement of the prognosis. By preventing the progression of a disease or disorder, treatment can prevent exacerbation due to the disorder in an affected or diagnosed subject or a subject suspected of having the disorder, but treatment can also prevent the onset of the disorder or the symptoms of the disorder in a subject at risk of or suspected of having the disorder.

[0084] "Individual" or "subject" as used herein refers to a vertebrate, e.g., a human or 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 state" as used herein refers to a subject having immunodeficiency. A subject is highly vulnerable to infections caused by organisms that do not cause disease in a person with a normal, healthy immune system but can affect a person with an insufficiently functioning or suppressed immune system.

[0085] Other aspects of the subject matter of this disclosure are described in the following disclosure and are within the scope of the subject matter of this disclosure. 5.2. Chimeric Antigen Receptor (CAR)

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

[0087] A CAR is an engineered receptor that grafts or confers specificity for a target onto an immune effector cell. CARs can be used to graft the specificity of monoclonal antibodies onto T cells; this involves transfer of their coding sequences facilitated by retroviral vectors.

[0088] There are three generations of CARs. A "first-generation" CAR typically consists of an extracellular antigen-binding domain (e.g., scFv) fused to a transmembrane domain fused to a cytoplasmic / intracellular signaling domain. A "first-generation" CAR can provide de novo antigen recognition and, independent of HLA-mediated antigen presentation, CD4 + T cells and CD8 +It can cause the activation of both T cells. "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. "Second-generation" CARs include those that provide both costimulation (e.g., CD28 or 4-1BB) and activation (CD3ζ). "Third-generation" CARs include those that provide both multiple costimulations (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 includes an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, and the intracellular signaling domain includes a costimulatory signaling domain. In certain embodiments, the CAR further includes a hinge / spacer region. In certain embodiments, the antigen recognition receptor is a third-generation CAR that includes multiple costimulatory signaling domains.

[0089] In certain embodiments, the CAR can include an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, and the extracellular antigen-binding domain specifically binds to an antigen that 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. In the tumor-related embodiments described herein, any tumor antigen (e.g., an antigenic peptide) can be used. Sources of antigens include, but are not limited to, cancer proteins. The antigen can be expressed as a peptide or as an intact protein or a portion thereof. The intact protein or a portion thereof can be wild-type or mutagenized. In certain embodiments, the antigen is 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-α, GD2, GD3, HER-2, hTERT, 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, cancer fetal antigen (h5T4), PSCA, PSMA, ROR1, TAG-72, VEGF-R2, WT-1, BCMA, CD44V6, NKCS1, EGF1R, EGFR-VIII, ADGRE2, CCR1, LILRB2, PRAME, HPV E6 cancer protein, and HPV E7 cancer protein. 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.

Chemical formula

[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 treating and / or preventing pathogen infection or other infectious diseases. 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 virus, 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, bunyavirus, phlebovirus and Nairobi virus); Arenaviridae (Arena viridae) (hemorrhagic fever virus); Reoviridae (e.g., reovirus, orbivirus and rotavirus); Birnaviridae; Hepadnaviridae (hepatitis B virus); Parvoviridae (Parvovirida) (parvovirus); Papovaviridae (papillomavirus, polyomavirus); Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus (HSV) 1 and 2, varicella-zoster virus, cytomegalovirus (CMV), herpesvirus); Poxviridae (e.g., smallpox virus, vaccinia virus, poxvirus); and Iridoviridae (e.g., African swine fever virus);and 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 = parenteral transmission, class 2 = enteric transmission (i.e., hepatitis C) agents; Norwalk and related viruses, and astroviruses), human papillomavirus (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), Epstein-Barr virus (EBV), human immunodeficiency virus (HIV), or influenza virus. 5.2.2. Extracellular antigen-binding domain of the CAR

[0098] In certain embodiments, the extracellular antigen-binding domain comprises an scFv. In certain embodiments, the scFv is a human scFv. In certain embodiments, the scFv is a humanized scFv. In certain embodiments, the scFv is a mouse scFv. In certain embodiments, the scFv is identified by screening an scFv phage library with an 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 an F(ab)2. Any of the aforementioned molecules may be included in a fusion protein with a heterologous sequence to form the extracellular antigen-binding domain.

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

[0101] 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 specific protein-antibody complex by using a labeled reagent (e.g., an antibody, or scFv) specific for the complex of interest. For example, an scFv can be radiolabeled and used in a radioimmunoassay (RIA) (see, e.g., Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March 1986, incorporated herein by reference). The radioisotope 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 of the CAR (e.g., scFv) comprises or consists of the amino acid sequence set forth in SEQ ID NO: 2 and specifically binds to the 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 a V that has 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%) identical or the same as the amino acid sequence set forth in SEQ ID NO: 3 H comprises. For example, the extracellular antigen-binding domain of the CAR (e.g., scFv) comprises a V that has 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% identical or the same as the amino acid sequence set forth in SEQ ID NO: 3 H comprises. In certain embodiments, the extracellular antigen-binding domain comprises a V that comprises the amino acid sequence set forth in SEQ ID NO: 3 H comprises. 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 a V that has 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%) identical or the same as the amino acid sequence set forth in SEQ ID NO: 4 LIt includes. For example, the extracellular antigen-binding domain of CAR (e.g., scFv) has 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 and includes V L It includes. In certain embodiments, the extracellular antigen-binding domain includes V that includes the amino acid sequence set forth in SEQ ID NO: 4 H It includes. SEQ ID NO: 4 is provided in Table 1 below.

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

Chemical formula

[0107] In certain embodiments, the extracellular antigen-binding domain of CAR (e.g., scFv) includes V that includes the amino acid sequence set forth in SEQ ID NO: 6 or a conservative modification thereof H V that includes CDR1, the amino acid sequence set forth in SEQ ID NO: 7 or a conservative modification thereof H CDR2, and V that includes the amino acid sequence set forth in SEQ ID NO: 8 or a conservative modification thereof H CDR3. SEQ ID NOs: 6-8 are provided in Table 1.

[0108] In certain embodiments, the extracellular antigen-binding domain of CAR (e.g., scFv) includes V that includes the amino acid sequence set forth in SEQ ID NO: 9 or a conservative modification thereof L CDR1, V that includes the amino acid sequence set forth in SEQ ID NO: 10 or a conservative modification thereofL A V containing CDR2 and the amino acid sequence set forth in SEQ ID NO: 11 or a conservative modification thereof L contains CDR3. SEQ ID NOs: 9-11 are provided in Table 1.

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

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

Table 1

[0111] As used herein, the term "conservative array modification" refers to amino acid modifications that do not significantly affect or alter the binding properties of the CARs of the present disclosure (e.g., the extracellular antigen-binding domain of the CAR) that include amino acid sequences. Conservative modifications can include amino acid substitutions, additions, and deletions. The modifications can be introduced into the human scFv of the CARs of the present disclosure by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis. Amino acids can be grouped by their physicochemical properties such as charge and polarity. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid within the same group. For example, amino acids can be classified by charge: positively charged amino acids include lysine, arginine, and histidine; negatively charged amino acids include aspartic acid and glutamic acid; and neutral-charged amino acids include alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. Additionally, amino acids can be classified by polarity: polar amino acids include arginine (basic polar), asparagine, aspartic acid (acidic polar), glutamic acid (acidic polar), glutamine, histidine (basic polar), lysine (basic polar), serine, threonine, and tyrosine; nonpolar amino acids include alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine. Thus, one or more amino acid residues within the CDR regions can be replaced with other amino acid residues from the same group, and the modified 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, one or fewer, two or fewer, three or fewer, four or fewer, five or fewer residues within the specified sequence or CDR region are modified.

[0112] For a specified array (e.g., array numbers 3 and 4), V having at least about 80%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% (e.g., about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) homology or identity H and / or V L amino acid sequences may include substitutions (e.g., conservative substitutions), insertions, or deletions compared to a specific sequence(s), but retain the ability to bind to a target antigen (e.g., CD19). In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in a specific sequence (e.g., array numbers 3 and 4). In certain embodiments, the substitution, insertion, or deletion occurs in a region outside of the CDR (e.g., FR) of the extracellular antigen-binding domain. In certain embodiments, the extracellular antigen-binding domain is V selected from the group consisting of array numbers 3 and 4, including post-translational modifications of the sequence (array numbers 3 and 4). H and / or V L sequence. 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 extends across at least a portion of the membrane. Different transmembrane domains result in different receptor stabilities. After antigen recognition, receptor clustering and signals are transmitted to the cell. In certain embodiments, the transmembrane domain of the CAR comprises the native or modified transmembrane domain of CD8, CD28, CD3ζ, CD4, 4-1BB, OX40, ICOS, CD84, CD166, CD8a, CD8b, ICAM-1, CTLA-4, CD27, CD40, NKGD2, or a combination thereof.

[0114] In certain embodiments, the transmembrane domain of the CAR comprises a CD28 polypeptide (e.g., the transmembrane domain of CD28 or a portion thereof). In certain embodiments, the transmembrane domain of the CAR comprises the transmembrane domain of human CD28 or a portion thereof. In certain embodiments, the CD28 polypeptide comprises 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 amino acid sequence having NCBI Reference No.: NP_006130 (SEQ ID NO: 12) or a fragment thereof, and / or may contain up to one or up to two or up to three conservative amino acid substitutions as needed. 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 acid positions 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 the amino acid sequence of amino acid positions 153-179 of SEQ ID NO: 12. SEQ ID NO: 12 is provided below.

Chemical formula

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

Chemical formula

[0116] In certain embodiments, the transmembrane domain of the CAR comprises the transmembrane domain of murine CD28 or a portion thereof. In certain embodiments, the CD28 polypeptide comprises an amino acid sequence or a fragment thereof 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 No.: NP_031668.3 (SEQ ID NO: 14), and / or may optionally include 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 contiguous 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 at positions 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 that comprises or consists of the amino acids at positions 151-177 of SEQ ID NO: 14. SEQ ID NO: 14 is provided below.

Chemical formula

[0117] In certain embodiments, the transmembrane domain of the CAR comprises a CD8 polypeptide (e.g., the transmembrane domain of CD8 or a portion thereof). In certain embodiments, the transmembrane domain of the CAR comprises the transmembrane domain of human CD8 or a portion thereof. In certain embodiments, the CD8 polypeptide comprises an amino acid sequence or a fragment thereof 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 No.: NP_001139345.1 (SEQ ID NO: 15), 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: 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 acid positions 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 acid positions 137-209 of SEQ ID NO: 15. SEQ ID NO: 15 is provided below. [Chemical formula]

[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 or a fragment thereof 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 No.: AAA92533.1 (SEQ ID NO: 16), and / or may optionally include up to one or up to two or up to three conservative amino acid substitutions. In certain embodiments, the CD8 polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 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 acid positions 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. [Chemical formula]

[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 flexible enough to allow the antigen-binding domain to be oriented in different directions while maintaining the activation activity of the CAR to facilitate antigen recognition.

[0120] In certain embodiments, the hinge / spacer region of the CAR comprises a native or modified hinge region of CD8, CD28, CD3ζ, 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 can be a hinge region derived from IgG1, or a CH2CH3 region of an immunoglobulin and 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 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 the 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 a cell (e.g., a lymphoid cell, e.g., a T cell). Wild-type ( "native") CD3ζ comprises three functional immunoreceptor activation tyrosine motifs (ITAMs) and three functional basic rich stretch (BRS) regions (BRS1, BRS2, and BRS3). CD3ζ transmits an activation signal to a cell (e.g., a lymphoid cell, e.g., a T cell) after antigen binding. The intracellular signaling domain of the CD3ζ chain is the primary transducer of signals from the endogenous TCR.

[0122] In certain embodiments, the intracellular signaling domain of the CAR comprises native CD3ζ. In certain embodiments, the CD3ζ polypeptide comprises an amino acid sequence or a fragment thereof 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), 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 acid positions 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 the CAR comprises a CD3ζ polypeptide comprising or consisting of the amino acid sequence of amino acid positions 52-164 of SEQ ID NO: 17. SEQ ID NO: 17 is provided below. [Chemical formula]

[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. [Chemical formula]

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

[0125] As used herein, "co-stimulatory molecule" refers to a cell surface molecule other than an antigen receptor or its ligand that can provide an efficient response of lymphocytes to an antigen. In certain embodiments, the co-stimulatory molecule can result in optimal lymphocyte activation. Non-limiting examples of co-stimulatory 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. A co-stimulatory molecule can bind to a co-stimulatory ligand, which is a protein expressed on the cell surface that, when bound to its receptor, results in a co-stimulatory response (i.e., an intracellular response that affects the stimulation provided when the CAR binds to its target antigen).

[0126] In certain embodiments, the at least one co-stimulatory signaling domain comprises a CD28 polypeptide comprising a mutant 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 cluster of differentiation 28, Tp44, and the CD28 molecule. CD28 possesses an intracellular domain that contains an intracellular motif important for effective signaling of CD28. In certain embodiments, the CD28 intracellular domain contains an intracellular subdomain (also known as an “intracellular motif”) that regulates the signaling pathway 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 adapter molecules that interact with these motifs via 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-related adapter protein 2 (GADS). Grb2 can bind to Gab1 and Gab2 and can then mobilize the p85 subunit of PI3K.

[0129] In certain embodiments, the native YMNM motif consists of the amino acid sequence set forth in YMNM (SEQ ID NO: 19). In certain embodiments, the native 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 native 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, a CD28 polypeptide comprising a variant YMNM motif of the present disclosure reduces the recruitment of the p85 subunit of PI3K as compared to a CD28 molecule comprising a native YMNM motif.

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

[0132] In certain embodiments, the variant YMNM binds to the p85 subunit of PI3K but does not bind to Grb2 and / or GADS. Since the binding of PI3K p85 is retained, downstream signaling of PI3K is retained intact. Since the binding of Grb2 / GADS is blocked, the recruitment of the p85 subunit of PI3K caused by the binding of Grb2 to Gab1 and Gab2 is reduced or blocked. Furthermore, downstream signaling of Grb2 / GADS is blocked. Such variant YMNM motifs are referred to as "PI3K-permissive variants."

[0133] In certain embodiments, the variant YMNM does not bind to the p85 subunit of PI3K and does not bind to Grb2 and / or GADS. Such variant YMNM motifs are referred to as "non-functional variants." Non-functional variants do not result in the binding of PI3K, Grb2, or GADS to CD28 at the YMNM motif, but do not preclude the binding of these signaling molecules to other locations on the CD28 molecule.

[0134] In certain embodiments, the variant YMNM retains only one of the two methionine residues present in the YMNM motif (i.e., YMxx or YxxM). These motifs may regulate signaling via PI3K by limiting the number of methionine residues that can bind to the p85 subunit of PI3K. Such variant YMNM motifs are referred to as "hybrid "HEMI" variants." 5.2.4.1. GADS / Grb-2 Permissive Variants

[0135] In certain embodiments, the variant YMNM motif is a GADS / Grb-2 permissive variant. In certain embodiments, the variant 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 variant 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). In certain embodiments, the variant YMNM motif consists of the amino acid sequence set forth in YSNV (SEQ ID NO: 23). In certain embodiments, the variant YMNM motif consists of the amino acid sequence set forth in YKNI (SEQ ID NO: 26). In certain embodiments, the variant YMNM motif consists of the amino acid sequence set forth in YENV (SEQ ID NO: 22). In certain embodiments, the variant YMNM motif consists of the amino acid sequence set forth in YKNL (SEQ ID NO: 24). 5.2.4.2. PI3K Permissive Variants

[0136] In certain embodiments, the mutant YMNM motif is a PI3K permissive variant. In certain embodiments, the mutant YMNM motif consists of the amino acid sequence set forth in YMxM (SEQ ID NO: 20), where x is not asparagine (N). 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, the mutant YMNM motif consists of the amino acid sequence set forth 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 set forth in YMDM (SEQ ID NO: 32).

[0137] In certain embodiments, the mutant YMNM motif consists of the amino acid sequence set forth in YbxM (SEQ ID NO: 33), where x is not asparagine (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 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).

[0138] In certain embodiments, the variant YMNM motif consists of the amino acid sequence set forth in YMxb (SEQ ID NO: 65), where x is not asparagine (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 variant YMNM motif consists of the amino acid sequence set forth in YMAP (SEQ ID NO: 77).

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

[0140] In certain embodiments, the variant YMNM motif is a hybrid "HEMI" variant. In certain embodiments, the variant 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 variant 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). 5.2.4.4. Non - functional variants

[0141] In certain embodiments, the mutant YMNM motif is a non-functional variant. In certain embodiments, the mutant YMNM motif consists of the amino acid sequence Ybxb (SEQ ID NO: 43), where x is not asparagine (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 mutant 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 mutant 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 co-stimulatory signaling domain comprising a CD28 polypeptide comprising a mutant YMNM motif consisting of the amino acid sequence set forth in YENV (SEQ ID NO: 22), and the CD28 polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 47. SEQ ID NO: 47 is provided below.

Chemical formula

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

Chemical formula

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

Chemical formula

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

Chemical formula

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

Chemical formula

[0147] In certain embodiments, the intracellular signaling domain of the CAR comprises a first co-stimulatory signaling domain comprising a CD28 polypeptide comprising a mutant YMNM motif (disclosed herein), and a second co-stimulatory signaling domain comprising the intracellular domain of a co-stimulatory molecule. In certain embodiments, the co-stimulatory 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 co-stimulatory 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), and / or may optionally contain 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 acid positions 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.

Chemical formula

[0149] In certain embodiments, the CAR is a CD19-targeted CAR. In certain embodiments, the CAR comprises (a) an extracellular antigen-binding domain that binds to human CD19 and comprises a V H CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 6, a V H CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7, a V H CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8, a V L CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 9, a V L CDR2, and a V L CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 10; (b) a transmembrane domain comprising the transmembrane domain of CD28 or a portion thereof, and (c) an intracellular signaling domain comprising a co-stimulatory signaling domain comprising (i) a CD3ζ polypeptide, and (ii) a CD28 polypeptide comprising a mutant YMNM motif. In certain embodiments, the mutant 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). In certain embodiments, V 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, the exemplary CD19-targeted CAR comprises a mutant YMNM motif consisting of the amino acid sequence set forth in YMDM (SEQ ID NO: 32). In certain embodiments, the exemplary CD19-targeted CAR consists of the amino acid sequence set forth in SEQ ID NO: 51 provided below.

Chemical formula

[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.

Chem.

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

Chem.

[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.

Chem.

[0154] In certain embodiments, an exemplary CD19-targeted CAR comprises a mutant 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.

Chem.

Chem.

[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.

Chem.

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

Chemical Structure

[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.

Chemical Structure

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

Chemical Structure

[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.

Chemical Structure

Chemical Structure

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

[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. [Chemistry] [Chemistry] 5.3. Cells

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

[0163] In certain embodiments, the cells are lymphoid cells. Lymphoid cells can provide functions such as antibody production, regulation of the cellular immune system, detection of foreign substances in the blood, and detection of foreign cells in the host. Non-limiting examples of lymphoid cells include T cells, natural killer (NK) cells, B cells, dendritic cells, and stem cells from which lymphoid cells can differentiate. In certain embodiments, the stem cells are pluripotent stem cells (e.g., embryonic stem cells).

[0164] In certain embodiments, the cell is a T cell. T cells mature in the thymus and can be lymphocytes that are mainly responsible for cellular 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: for example, TEM cells and TEMRA cells, regulatory T cells (also known as suppressor T cells), tumor-reactive lymphocytes, tumor-infiltrating lymphocytes (TIL), natural killer T cells, mucosa-associated invariant T cells, and γδ T cells. Cytotoxic T cells (CTLs or killer T cells) are a subset of T lymphocytes that can induce the death of infected somatic cells or tumor cells. The patient's own T cells may be genetically modified to target specific antigens by the introduction of a CAR. In certain embodiments, the immunoreactive cell is a T cell. T cells can be CD4 + T cells or CD8 + T cells. In certain embodiments, the T cell is a CD4 + T cell. In certain embodiments, the T cell is a CD8 + T cell.

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

[0166] The types of human lymphocytes of the subject matter of the present disclosure include, but are not limited to, peripheral donor lymphocytes. For example, Sadelaín et al., Nat Rev Cancer (2003);3:35-45 (disclosing peripheral donor lymphocytes genetically modified to express a chimeric antigen receptor (CAR)), Morgan, R.A. et al., 2006 Science 314:126-129 (disclosing peripheral donor lymphocytes genetically modified to express a full-length tumor antigen recognition T cell receptor complex 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 (AAPCs) or pulsed dendritic cells).

[0167] Cells (e.g., T cells) can be derived in vitro from autologous, allogeneic (e.g., allogeneic), or engineered precursors or stem cells.

[0168] The cells of the subject matter of the present disclosure can be myeloid cells. Non-limiting examples of myeloid cells include monocytes, macrophages, neutrophils, dendritic cells, basophils, eosinophils, megakaryocytes, mast cells, erythrocytes, platelets, and stem cells from which myeloid cells can differentiate. In certain embodiments, the stem cells are pluripotent stem cells (e.g., embryonic stem cells or induced pluripotent stem cells).

[0169] In certain embodiments, the cells of the present disclosure can modulate the tumor microenvironment. Tumors have a microenvironment that opposes the host immune response, including a series of mechanisms by malignant cells to protect themselves from immune recognition and elimination. This "hostile tumor microenvironment" includes infiltrating regulatory CD4 +It includes various immunosuppressive factors, including expression of ligands targeting T cells (Tregs), myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), immunosuppressive cytokines including TGF-β, and immunosuppressive 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 impede an effective antitumor immune response. 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 the CARs of the present 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 a change in protein expression in cells (e.g., activated immunoreactive cells) that results in a decrease in the immune response. Polypeptides known to suppress or decrease the immune response 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 against neoplastic cells. In various embodiments, inhibiting, blocking, or antagonizing the interaction of immunosuppressive polypeptides and / or their ligands enhances the immune response of immunoreactive cells.

[0172] In certain embodiments, immune-stimulatory activity refers to the induction of signaling or changes in protein expression in cells that result in an increase in the immune response (e.g., activated immunoreactive cells). Immune-stimulatory activity can include pro-inflammatory activity. Polypeptides known to stimulate or increase the immune response 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 the immune response against neoplastic cells. In various embodiments, promoting, stimulating, or activating pro-inflammatory polypeptides and / or their ligands enhances the immune response of immunoreactive cells.

[0173] Cells comprising a CAR and a soluble scFv that binds to a polypeptide having immunosuppressive or immune-stimulatory activity are disclosed in International Publication No. WO 2014 / 134165, which is incorporated herein by reference in its entirety.

[0174] In certain embodiments, the cell further comprises exogenous CD40L. Cells comprising a CAR and exogenous CD40L are disclosed in International Publication No. WO 2014 / 134165.

[0175] Further, in certain embodiments, the cell is engineered to express IL-18. In certain embodiments, the cell further comprises an exogenous IL-18 polypeptide or a fragment thereof. In certain embodiments, the cell further comprises 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, e.g., 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, which is incorporated herein by reference in its entirety.

[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 that contain a CAR and are engineered to express IL-33, e.g., that contain 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, which is incorporated herein by reference in its entirety.

[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 that contain a CAR and are engineered to express IL-36, e.g., that contain 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, which is incorporated herein by reference in its entirety. 5.4. Compositions and Vectors

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

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

[0180] Furthermore, the subject matter of the present disclosure provides a nucleic acid composition comprising a polynucleotide encoding a CAR of the present disclosure (e.g., as 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 present 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 transcriptional 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 and / or into 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 gammaretroviral vector or a lentiviral vector) is used to introduce the DNA construct into the cells. For example, a polynucleotide encoding an antigen recognition receptor can be cloned into a retroviral vector and expression can be driven from its endogenous promoter, the long terminal repeat of the retrovirus, or a promoter specific for the target cell type of interest. Non-viral vectors can similarly be used.

[0184] To initially genetically modify cells to contain the CARs of the present disclosure, retroviral vectors can be used for transduction, although any other suitable viral vector or non-viral delivery system can be used. The antigen recognition receptor can be constructed with a single multicistronic expression cassette, multiple expression cassettes of a single vector, or multiple vectors. Examples of elements for creating multicistronic expression cassettes include various viral and non-viral internal ribosome entry sites (IRESs, e.g., FGF-1 IRES, FGF-2 IRES, VEGF IRES, IGF-II IRES, NF-κB IRES, RUNX1 IRES, p53 IRES, hepatitis A IRES, hepatitis C IRES, pestivirus IRES, aphthovirus IRES, picornavirus IRES, poliovirus IRES, and encephalomyocarditis virus IRES) and cleavable linkers (e.g., 2A peptides, e.g., P2A, T2A, E2A, and F2A peptides), but are not limited thereto. Combinations of retroviral vectors with suitable packaging strains are also suitable, and the capsid protein will be functional for infecting human cells. Various 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, e.g., particles pseudotyped with VSVG, RD114, or GALV envelope 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 virus supernatant alone or concentrated vector stock, 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] Cells can be modified using other transduction viral vectors. In certain embodiments, the selected vector exhibits high infection efficiency and stable integration and expression (see, e.g., 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. U.S.A. 94:10319, 1997). Other viral vectors that can be used include, for example, adenovirus, lentivirus, and adeno-associated viral vectors, vaccinia virus, bovine papillomavirus, or herpes virus, 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 for vectors).Retroviral vectors have been particularly well developed and are used in clinical settings (Rosenberg et al., N Engl J Med (1990); 323:370, 1990; Anderson et al., U.S. Patent No. 5,399,346).

[0187] Non-viral approaches can also be used for genetic modification of cells. For example, nucleic acids can be introduced into cells by administering them in the presence of lipofection (Feigner et al., Proc Natl Acad Sci U.S.A. (1987); 84:7413; Ono et al., Neurosci Lett (1990); 17:259; Brigham et al., Am J Med 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), or by microinjection under surgical conditions (Wolff et al., Science (1990); 247:1465). Other non-viral means for gene transfer include transfection in vitro using calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. Liposomes can also potentially be useful for delivery of DNA to cells. Transplantation of normal genes into diseased tissues of a subject can also be achieved by introducing the normal nucleic acid into a cell type that can be cultured 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 recognition receptors of the present disclosure to cells or a subject. In certain embodiments, the CRISPR system is used to deliver the antigen recognition receptors of the present disclosure disclosed herein. In certain embodiments, zinc finger nucleases are used to deliver the antigen recognition receptors. In certain embodiments, the TALEN system is used to deliver the antigen recognition receptors of the present disclosure.

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

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

[0191] Transcription activator-like effector nuclease (TALEN) is a restriction enzyme that can be engineered to cleave specific sequences of DNA. The TALEN system operates on substantially the same principle as ZFN. 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 33 - 34 amino acid repeat motifs with two variable positions that have strong recognition for specific nucleotides. By assembling arrays 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 within the genome. cDNA expression for use in polynucleotide therapies is driven from any suitable promoter (e.g., human cytomegalovirus (CMV), simian virus 40 (SV40), or metallothionein promoter) and can be regulated by any suitable mammalian regulatory element or intron (e.g., elongation factor 1a enhancer / promoter / intron construct). For example, if desired, the expression of the nucleic acid can be directed using enhancers known to preferentially direct gene expression in specific cell types. The enhancers used can include, but are not limited to, those characterized as tissue or cell-specific enhancers. Alternatively, when genomic clones are used as therapeutic constructs, regulation can be mediated by cognate regulatory sequences or, if desired, by regulatory sequences derived from a heterologous source that includes any of the above promoters or regulatory elements.

[0192] Methods for delivering genome editing agents / systems can vary as needed. In certain embodiments, the components of a 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, hydrodynamic pressure, continuous infusion, 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). 5.5. Polypeptide

[0193] The subject matter of the present disclosure provides methods for optimizing amino acid or nucleic acid sequences by effecting changes in the sequences. Such changes can 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 CD3ζ). The analogs can differ from the naturally occurring polypeptides disclosed herein by differences in amino acid sequence, post-translational modifications, or both. The analogs can 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 sequences of the subject matter of the present disclosure. The length of the sequence comparison is at least 5, 10, 15 or 20 amino acid residues, e.g., at least 25, 50, or 75 amino acid residues, or more than 100 amino acid residues. Again, in an exemplary approach for determining the degree of identity, the BLAST program can be used, e -3 and e -100The probability scores between it and [object] indicate arrays that are closely related. Modifications include the chemical derivatization of polypeptides in vivo and in vitro, such as acetylation, carboxylation, phosphorylation, or glycosylation. Such modifications can occur during the synthesis or processing of the polypeptide or in subsequent treatments with isolated modifying enzymes. Analogs can also differ from naturally occurring polypeptides by changes in the primary sequence. These include both genetic variants of natural and induced types (e.g., Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual (2nd Edition), CSH Press, 1989, or Ausubel et al., as described above, resulting from random mutagenesis by irradiation or exposure to ethyl methyl sulfate or site-directed mutagenesis). Also included are cyclic peptides, molecules and analogs containing residues other than L-amino acids (e.g., D-amino acids or unnatural or synthetic amino acids, such as β or γ amino acids).

[0194] In addition to full-length polypeptides, the subject matter of the present disclosure also provides any fragment of the polypeptides disclosed herein. As used herein, the term "fragment" means at least 5, 10, 13, or 15 amino acids. In certain embodiments, the fragment comprises at least 20 contiguous amino acids, at least 30 contiguous amino acids, or at least 50 contiguous amino acids. In certain embodiments, the fragment comprises at least 60 - 80, 100, 200, 300 or more contiguous amino acids. Fragments can be generated by methods known to those skilled in the art or can result from normal protein processing (e.g., removal of amino acids from nascent polypeptides not required for biological activity, or removal of amino acids by alternative mRNA splicing or alternative protein processing events). 5.6. Formulations 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, such as an isotonic aqueous solution, suspension, emulsion, dispersion, or viscous composition, and can be buffered to a selected pH. Liquid preparations are generally easier to prepare than gels, other viscous compositions, and solid compositions. Further, liquid compositions are somewhat advantageous, particularly for administration 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 contain a carrier, for example, a solvent or dispersion medium containing water, physiological saline, phosphate buffered saline, polyols (such as glycerol, propylene glycol, liquid polyethylene glycol, etc.) and suitable mixtures thereof.

[0196] A sterile injectable solution can be prepared by incorporating the genetically modified cells into a suitable solvent in the required amounts, optionally together with various amounts of other components. Such compositions may be admixed with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, etc. The composition can also be lyophilized. The composition can contain auxiliary substances such as wetting agents, dispersing agents, or emulsifying agents (such as methylcellulose), pH buffering agents, gelling or thickening additives, preservatives, flavoring agents, coloring agents, etc., depending on the route of administration and the desired preparation. Appropriate preparations can be prepared without undue experimentation, referring 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 antibacterial preservatives, antioxidants, chelating agents, and buffering agents. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, etc. Prolonged absorption in injectable pharmaceutical forms can be achieved by using 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 the genetically modified cells.

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

[0199] If desired, the viscosity of the composition can be maintained at a selected level using pharmaceutically acceptable thickening agents. For example, methylcellulose is readily and economically available and easy to handle. Other suitable thickening agents include, for example, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, carbomer, etc. The concentration of the thickening substance can depend on the selected agent. The important point is to use an amount that will achieve the selected viscosity. Clearly, the selection of the appropriate carrier and other additives will depend on the exact route of administration and the nature of the particular dosage form, e.g., whether the composition is formulated into a liquid dosage form (e.g., the composition is a solution, suspension, gel or other liquid form, e.g., a time-release form or a liquid-filled form).

[0200] Compositions containing the cells of the present disclosure can be administered systemically or directly to a subject to induce and / or enhance an immune response against an antigen and / or to treat and / or prevent neoplasia. In certain embodiments, the cells of the present disclosure or a composition containing them are injected directly into the organ of interest (e.g., the organ affected by neoplasia). Alternatively, the cells of the present disclosure or a composition containing them can be indirectly supplied to the organ of interest, for example, by administration to the circulatory system (e.g., the tumor vasculature). Proliferating agents and differentiating agents can be supplied before, during, or after administration of the cells or 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 usually be administered intravascularly in any physiologically acceptable vehicle, but can also be introduced into bone or other convenient sites where the cells can find an appropriate 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, between about 10 4 and about 10 10 cells, between about 10 4 and about 10 7 cells, between about 10 5 and about 10 7 cells, between about 10 5 and about 10 9 cells, between about 10 5 and about 10 10 cells, between about 10 6 and about 10 8 cells of the present disclosure are administered to the subject. More effective cells may be administered in even fewer numbers. Usually, at least about 1×10 5 cells are administered, ultimately reaching about 1×10 10 cells or more. In certain embodiments, at least about 1×10 5 cells, about 2×10 5 cells, about 5×10 5 cells, about 1×10 6cells, about 5×10 6 cells, about 1×10 7 cells, about 5×10 7 cells, about 1×10 8 cells, about 5×10 8 cells, about 1×10 9 cells, or about 5×10 9 cells of the present disclosure are administered to a subject. In certain embodiments, about 10 5 cells and about 10 6 cells of the present disclosure between are administered to a subject. In certain embodiments, about 1×10 5 cells of the present disclosure are administered to a subject. In certain embodiments, about 2×10 5 cells of the present disclosure are administered to a subject. In certain embodiments, about 5×10 5 cells of the present disclosure are administered to a subject. In certain embodiments, about 1×10 6 cells of the present disclosure are administered to a subject. Although an exact determination of what may be considered an effective dose can be based on individual factors for each subject, including the size, age, sex, weight, and condition of the particular subject, the dosage can be readily ascertained by one of ordinary skill in the art from the present disclosure and knowledge of the art.

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

[0204] One skilled in the art can readily determine the amounts of cells, as well as any additives, vehicles, and / or carriers, administered in the composition and the method. Typically, any additives (in addition to the active cell(s) and / or agent(s)) are present in an amount of a 0.001-50% (by weight) solution in phosphate buffered saline, and the active ingredient is present in an order of micrograms to milligrams, for example, 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 an animal or human, the following can be determined: toxicity, such as by determining the lethal dose (LD) and LD50 in a suitable animal model, such as rodents like mice; the dosage of the composition(s) that induces a suitable response, the concentration of the components in the composition(s), and the timing of administration of the composition(s). Such determinations do not require undue experimentation from the knowledge of one skilled in the art, the present disclosure, and the literature cited herein. Also, the duration of continuous administration can be confirmed without undue experimentation.

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

[0206] Administration of the composition can be autologous or allogeneic. For example, the cells can be obtained from one subject and administered to the same subject or a different compatible subject. Cells derived from peripheral blood or its progeny (e.g., in vivo, ex vivo, or in vitro derived) can be administered. When administering the composition of the present disclosure (e.g., a pharmaceutical composition comprising the cells of the present disclosure), it can be formulated in a unit dosage injectable form (solution, suspension, emulsion).

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

[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 and compositions containing them of the present disclosure can be used for treatment or medicine. The subject matter of the present disclosure provides various methods of using cells (e.g., T cells, such as CD4 + T cells or CD8 + T cells) or compositions containing them. For example, the cells and compositions containing them of the present disclosure can be used to reduce the tumor burden in a subject. The cells of the present disclosure can reduce the number of tumor cells in a subject, reduce the tumor size, and / or eradicate the tumor. The cells and compositions containing them of the present disclosure can be used to treat and / or prevent neoplasms or tumors in a subject. The cells and compositions containing them of the present disclosure can be used to extend the survival of a subject suffering from a neoplasm or tumor. The cells and compositions containing them of the present disclosure can be used to treat and / or prevent pathogen infections in a subject. Such methods include administering a cell or a composition containing it (e.g., a pharmaceutical composition) of the present disclosure to achieve a desired effect, such as alleviating existing symptoms or preventing recurrence. For treatment, the amount administered is an amount effective to produce the desired effect. The effective amount can be supplied in a single or a series of administrations. The effective amount can be supplied 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 containing them. For example, the subject matter of the present disclosure provides a method for reducing the tumor burden in a subject. In certain embodiments, the method for reducing the tumor burden includes administering a cell or a composition containing it of the present disclosure to a subject. The cells of the present disclosure can reduce the number of tumor cells in a subject, reduce the tumor size, and / or eradicate the tumor.

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

[0211] The subject matter of the present disclosure also provides a method of increasing or prolonging the survival of a subject having a neoplasm. In certain embodiments, the method of increasing or prolonging the survival of a subject having 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 the tumor burden in the subject. Further, the subject matter of the present disclosure provides a method for increasing an 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 a neoplasm or tumor include B-cell leukemia, B-cell lymphoma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), multiple myeloma, lymphoma (Hodgkin lymphoma, non-Hodgkin lymphoma), glioblastoma, myelodysplastic syndrome (MDS), and chronic myelogenous leukemia (CML), bone cancer, intestinal cancer, liver cancer, skin cancer, head and neck cancer, melanoma (cutaneous or intraocular malignant melanoma), renal cancer (e.g., clear cell carcinoma), pharyngeal cancer, prostate cancer (e.g., hormone-resistant prostate adenocarcinoma), blood cancer (e.g., leukemia, lymphoma, and myeloma), uterine cancer, rectal cancer, cancer of the anal region, bladder cancer, brain cancer, stomach cancer, testicular cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, 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 myelogenous leukemia, polycythemia vera, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, pediatric solid tumor, lymphocytic lymphoma, bladder cancer, kidney or ureteral cancer, renal pelvis carcinoma, central nervous system (CNS) neoplasm, primary CNS lymphoma, tumor angiogenesis, spinal cord axis tumor, brainstem glioma, pituitary adenoma, Kaposi sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancer including those induced by asbestos, Waldenström macroglobulinemia, heavy chain disease, and solid tumors such as sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing tumor, leiomyosarcoma, rhabdomyosarcoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms tumor, cervical cancer, salivary gland cancer, uterine cancer, testicular cancer, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, epithelioma, 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 lymphoma, Burkitt 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 of increasing or prolonging the survival of a subject having a pathogen infection. In certain embodiments, the method comprises administering to the subject an 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 goals of treatment may include reducing or reversing disease progression and / or ameliorating side effects. The subject may have a history of symptoms that have already been treated, in which case the goals of treatment typically include reducing or delaying the risk of recurrence.

[0216] To avoid or minimize the risk of immunological complications (known as "malignant T cell transformation"), such as graft-versus-host disease (GvHD), or when healthy tissue expresses the same target antigen as tumor cells, leading to a similar outcome as GvHD, further modifications can be introduced into the cells (e.g., T cells) of the present disclosure. 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 the EGFRt polypeptide. The EGFRt polypeptide can enable T cell elimination by administering an anti-EGFR monoclonal antibody (e.g., cetuximab). EGFRt can be covalently linked upstream of the CAR. The suicide gene can be included within a vector containing the nucleic acid encoding the CAR of the present disclosure. Thus, administration of a prodrug (e.g., AP1903, which can activate iCasp-9) designed to activate the suicide gene during malignant T cell transformation (e.g., GVHD) causes apoptosis in suicide gene-activated cells expressing the CAR. The incorporation of a suicide gene, for example, into the CAR of the present disclosure provides an additional level of safety, with the ability to eliminate most receptor-expressing cells within a very short period. The cells (e.g., T cells) of the present disclosure with the incorporated suicide gene can be preferentially eliminated at a given time point after cell injection or eradicated at the earliest signs of toxicity. 5.8. Kit

[0217] The subject matter of the present disclosure provides a kit for inducing and / or enhancing an immune response in a subject, treating and / or preventing a neoplasm or tumor in a subject, reducing the tumor burden in a subject, increasing or prolonging the survival of a subject having a neoplasm, and / or treating and / or preventing a pathogen infection. In certain embodiments, the kit comprises a cell or a composition comprising the cell of the present disclosure. 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 forms known in the art. Such a container can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding a medicament. In certain non-limiting embodiments, the kit comprises a nucleic acid molecule encoding the CAR of the present disclosure.

[0218] If desired, the cell and / or nucleic acid molecule are provided with instructions for administering the cell or nucleic acid molecule to a subject having or at risk of developing neoplasia. The instructions generally include information regarding the use of the composition for treating and / or preventing neoplasia. In certain embodiments, the instructions include at least one of the following: a description of the therapeutic agent; a dosage schedule and administration for treating or preventing neoplasia; cautions; warnings; directions; counter-indications; overdose information; adverse reactions; animal pharmacology; clinical studies; and / or references. The instructions may be printed directly on the container (if present), or as a label affixed to the container, or printed as a separate sheet, pamphlet, card, or folder supplied within or with the container.

Examples

[0219] 6. Examples The subject matter of the present disclosure will be better understood by reference to the following examples, which are provided by way of illustration of the subject matter of the present disclosure and not by way of limitation. Example 1: In Vitro and In Vivo Characterization of CD28 Mutant CAR T Cells

[0220] CD28 binds directly to the PI3K p85 subunit (through the presence of the YMxM consensus in YMNM in YMNM), and Grb2 (which binds to the YxNx consensus motif) binds to Gab1 and Gab2, which in turn can recruit the PI3K p85 subunit and initiate downstream signaling, so PI3K signaling appears to be redundant.

[0221] Given the diversity of adapter molecules that can adhere to the CD28 molecule, the functional fate of T cells (i.e., effector cell cytotoxicity, cytokine secretion, activation, survival, memory formation, and exhaustion) is most likely the result of the sum of the downstream signaling cascades derived from the binding of these adapter molecules to CD28. Therefore, modification of these CD28 motifs can alter signaling and allow or restrict the binding of various adapter molecules that can result in enhanced effector function and / or reduced dysfunction. Considering the involvement of PI3K signaling in the final differentiation of T cells, the redundancy of this signaling pathway may be harmful to effector function, and regulating it may be beneficial (Figure 1).

[0222] Considering the ability of the YMNM motif of CD28 to determine binding partners (adapter molecules), and the ability of those partners to determine the fate of T cells through several signaling cascades derived from PI3K, Grb2, and GADS, several CD28 mutations were created that allow or exclude the binding of these adapter molecules to CD28 (Figure 2, Figure 12). Characterization of CD28-YKNI mutant CAR T cells

[0223] CD28-YKNI mutant CAR T cells were generated. Various human CD19-targeted CAR T cells expressing a truncated EGFR domain (Etah19) were co-cultured with CD19+ NALM6 cells (NALM6gL) expressing GFP-ff luciferase at various effector:tumor ratios. Lysis of tumor cells (relative to non-signaling CAR T cells) was measured by bioluminescence after 24 hours. CD28-YKNI mutant CAR T cells were found to have potent killing ability in vitro (Figures 3A-3D).

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

[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, the 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 to the cultures at different time points. CD28-YKNI mutant CAR T cells also had potent killing and proliferation ability in vitro (Figure 5). CD28-YKNI mutant CAR T cells retained a memory phenotype under conditions of repeated antigen encounter compared to CD28 and CD28-1xx CAR T cells (Figure 6). CD28-YKNI mutant CAR T cells retained a relatively balanced CD8:CD4 ratio under conditions of repeated antigen encounter compared to CD28 and CD28-1xx CAR T cells (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 restimulated with the same amount of tumor for a total of 5 stimulations (1 stimulation every 12 hours). Approximately 10 days after the start of co-culture, size / immaturity (evaluated by forward scatter) was evaluated by flow cytometry. CD28-YKNI mutant CAR T cells demonstrated lower immaturity 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 rate (OCR) was measured in Etah19h28Z and Etah19h28Zp33 after 5 stimulations with the leukemia antigen, but this difference was not present after only 1 stimulation. The increase in cellular basal oxygen consumption suggested a preferential dependence on oxidative phosphorylation as the main energy generation mechanism and explained the metabolic requirements for enhanced CAR T cell proliferation. This was further confirmed by the increase in basal OCR after further stimulation with tumor antigen (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 for lactate production during glycolysis. The increase in basal ECAR in stimulated CAR T cells suggested an increase in glycolytic activity, which is typically measured in T cells activated with antigen. An increase in ECAR was observed in Etah19hMUThZ, but it was minimal, suggesting that T cells with this modification did not experience significant stimulation.

[0229] 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 5 stimulations (once every 12 hours). Approximately 10 days after the start of co - culture, exhaustion markers (LAG3 and PD1) were evaluated by flow cytometry. CD28 - YKNI mutant CAR T cells expressed lower levels of co - inhibitory molecules (LAG3 and PD1, TIM - 3 and PD1) in both single - or multiple - stimulation settings (Figures 10A - 10B)

[0230] The in - vivo anti - tumor effect of mutant - based CAR T cells was measured. NCG mice were inoculated with 10 6 NALM6gfp + ffLUC + tumor cells 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] Various human CD19 - targeted CD28 mutant CAR T cells (CD28 - YKNI, CD28 - YMDM, CD28 - YGGG, CD28 - YENV, CD28 - YKNL, and CD28 - YSNV) expressing a truncated EGFR domain (Etah19) were co - cultured with CD19+NALM6 cells (NALM6gL) expressing GFP - ff luciferase at various effector:tumor ratios, and lysis of tumor cells (relative to non - signaling CAR T cells) was measured by bioluminescence after 24 hours. CD28 - YKNI mutant CAR T cells showed equivalent killing ability in the 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 an initial concentration of 25,000 CAR T cells / mL. The concentrations of CAR+ and NALM6 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). The CD28 mutants exhibited potent long-term cytotoxic ability in vitro.

[0233] CAR T cells were co-cultured with NALM6gL at an initial E:T (single stimulation) of 1:5. In parallel, CAR T cells were restimulated with the same amount of tumor for a total of 5 stimulations (1 stimulation every 12 hours). Approximately 10 days after the start of co-culture, exhaustion markers (TIM3 and PD1) were evaluated by flow cytometry. The CD28 mutants demonstrated a favorable exhausted 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). NCG mice were inoculated with 1e6 NALM6gfp + ffLUC + tumor cells and treated with CAR T cells 4 days later. Survival rates were graphed. Bioluminescence was measured weekly. The CAR T cells were derived from a single healthy donor. Example 2: Characterization of CD28 Mutant CAR T Cells

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

[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 cytotoxic ability 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. Five days later, exhausted marker (LAG3, TIM3, and PD1) expression CAR T cells were evaluated by flow cytometry. CD28-Yxxx mutant CD19-targeted CAR T cells had a favorable exhausted immune phenotype (Figure 20).

[0238] To show the in vivo antitumor effect of CD28 mutant CAR T cells, NCG mice were inoculated with 1×10 6 cells of NALM6gfp + ffLUC + tumor cells 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 and outperformed standard CD28-based CAR T cells (Figures 21 and 22).

[0239] CD28-Yxxx mutant CD19-targeted CAR T cells exhibited 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 in either high or low CD19 antigen density. Every 6 days, CAR+ T cells were counted and restimulated with NALM6gL for a total of 3 stimulations. CD28-Yxxx mutant CD19-targeted CAR T cells showed enhanced proliferation in vitro compared to standard CD28-based CAR T cells under both high and low antigen density CD19 tumor cell conditions (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, the supernatant was collected and cytokines including interleukin-2, TNF-α, GM-CSF, interferon-γ, IL-9, and IL-17 were measured by a Luminex bead-based multiplex assay. CD28-Yxxx mutant CD19-targeted CAR T cells demonstrated a unique cytokine secretion profile upon antigen exposure (Figures 24A - 24C).

[0241] Although some of the subject matter and its advantages of the present disclosure have been described in detail, it is to be understood that various changes, substitutions, and alterations can be made herein without departing from the spirit and scope of the present disclosure. Further, the scope of the present application is not intended to be limited to the specific embodiments of the processes, machines, manufactures, and compositions of matter, and methods described herein. As will be readily understood by those skilled in the art from the disclosure of the present subject matter, processes, machines, manufactures, compositions of matter, or methods that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein, whether currently existing or hereafter developed in accordance with the present subject matter, may be utilized. Accordingly, the appended claims are intended to include such processes, machines, manufactures, compositions of matter, or methods within their scope.

[0242] A variety of patents, patent applications, publications, product descriptions, protocols, and sequence accession numbers are cited throughout this application, and the disclosures thereof are hereby incorporated by reference in their entirety for all purposes. The present invention provides, for example, the following items. (Item 1) A chimeric antigen receptor (CAR) comprising at least one costimulatory signaling domain comprising a CD28 polypeptide comprising an extracellular antigen-binding domain, a transmembrane domain, and a mutant YMNM motif, and an intracellular signaling domain. (Item 2) The CAR according to Item 1, wherein the CD28 polypeptide reduces the recruitment of the p85 subunit of phosphoinositide 3-kinase (PI3K) as compared to a CD28 molecule comprising a native YMNM motif. (Item 3) The CAR according to Item 1 or 2, wherein the p85 subunit of PI3K does not bind to the mutant YMNM motif. (Item 4) The CAR according to Item 3, wherein the mutant YMNM motif consists of the amino acid sequence set forth in YxNx (SEQ ID NO: 21), and x is not methionine (M). (Item 5) The CAR according to Item 3 or 4, wherein the mutant 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) The CAR according to any one of Items 3 to 5, wherein the mutant 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) The CAR according to any one of items 3 to 6, wherein the mutant YMNM motif consists of the amino acid sequence set forth in YSNV (SEQ ID NO: 23). (Item 8) The CAR according to any one of items 3 to 7, wherein the mutant YMNM motif binds to growth factor receptor-bound receptor 2 (Grb2) and / or Grb2-related adapter downstream of Shc (GADS). (Item 9) The CAR according to item 1 or 2, wherein the mutant YMNM motif does not bind to Grb2 and / or GADS. (Item 10) The CAR according to item 9, wherein the mutant YMNM motif consists of the amino acid sequence set forth in YMxM (SEQ ID NO: 20), and x is not asparagine (N). (Item 11) The CAR according to item 9 or 10, wherein the mutant YMNM motif consists of the amino acid sequence set forth in YMDM (SEQ ID NO: 32), YMPM (SEQ ID NO: 79), YMRM (SEQ ID NO: 37), or YMSM (SEQ ID NO: 80). (Item 12) The CAR according to any one of items 9 to 11, wherein the mutant YMNM motif consists of the amino acid sequence set forth in YMDM (SEQ ID NO: 32). (Item 13) The CAR according to item 9, wherein the mutant YMNM motif consists of the amino acid sequence set forth in YbxM (SEQ ID NO: 33), x is not asparagine (N), and b is not methionine (M). (Item 14) The CAR according to item 13, wherein 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). (Item 15) The CAR according to item 9, wherein the mutant YMNM motif consists of the amino acid sequence set forth in YMxb (SEQ ID NO: 65), x is not asparagine (N), and b is not methionine (M). (Item 16) The CAR according to item 15, wherein the mutant YMNM motif consists of the amino acid sequence set forth in YMAP (SEQ ID NO: 77). (Item 17) The CAR according to any one of items 9 to 16, wherein the p85 subunit of PI3K signaling binds to the mutant YMNM motif. (Item 18) The CAR according to item 1 or 2, wherein the mutant YMNM motif does not bind to Grb2 and / or GADS or the p85 subunit of PI3K. (Item 19) The CAR according to item 18, wherein the mutant YMNM motif consists of the amino acid sequence set forth in Ybxb (SEQ ID NO: 43), where x is not aspartic acid (N) and b is not methionine (M). (Item 20) The CAR according to item 19, wherein the mutant 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) The CAR according to any one of items 18 to 20, wherein the mutant YMNM motif consists of the amino acid sequence set forth in YGGG (SEQ ID NO: 44). (Item 22) The CAR according to 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) The CAR according to item 22, wherein 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). (Item 24) The CAR according to item 22 or 23, wherein 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). (Item 25) The CAR according to any one of items 1 to 24, wherein the extracellular antigen-binding domain binds to an antigen. (Item 26) The CAR according to item 25, wherein the antigen is a tumor antigen or a pathogen antigen. (Item 27) The CAR according to item 25 or 26, wherein the antigen is a tumor antigen. (Item 28) The CAR according to item 27, wherein the tumor antigen is selected from the group consisting of 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-α, GD2, GD3, HER-2, hTERT, 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, cancer fetal antigen (h5T4), PSCA, PSMA, ROR1, TAG-72, VEGF-R2, WT-1, BCMA, CD44V6, NKCS1, EGF1R, EGFR-VIII, ADGRE2, CCR1, LILRB2, PRAME, HPV E6 cancer protein, and HPV E7 cancer protein. (Item 29) The CAR according to item 28, wherein the tumor antigen is CD19. (Item 30) The CAR according to any one of items 1 to 29, wherein the mutant YMNM motif consists of the amino acid sequence set forth in YMDM (SEQ ID NO: 32). (Item 31) The CAR according to item 30, wherein the extracellular antigen-binding domain binds to CD19. (Item 32) The CAR according to item 31, comprising the amino acid sequence set forth in SEQ ID NO: 51. (Item 33) The CAR according to any one of items 1 to 29, wherein the mutant YMNM motif consists of the amino acid sequence set forth in YKNI (SEQ ID NO: 26). (Item 34) The CAR according to item 33, wherein the extracellular antigen-binding domain binds to CD19. (Item 35) The CAR according to item 34, comprising the amino acid sequence set forth in SEQ ID NO: 55. (Item 36) The CAR according to any one of items 1 to 29, wherein the mutant YMNM motif consists of the amino acid sequence set forth in YENV (SEQ ID NO: 22). (Item 37) The CAR according to item 36, wherein the extracellular antigen-binding domain binds to CD19. (Item 38) The CAR according to item 37, comprising the amino acid sequence set forth in SEQ ID NO: 53. (Item 39) The CAR according to any one of items 1 to 29, wherein the mutant YMNM motif consists of the amino acid sequence set forth in YSNV (SEQ ID NO: 64). (Item 40) The CAR according to item 39, wherein the extracellular antigen-binding domain binds to CD19. (Item 41) The CAR according to item 40, comprising the amino acid sequence set forth in SEQ ID NO: 57. (Item 42) The CAR according to any one of items 1 to 29, wherein the mutant YMNM motif consists of the amino acid sequence set forth in YGGG (SEQ ID NO: 63). (Item 43) The CAR according to item 42, wherein the extracellular antigen-binding domain binds to CD19. (Item 44) The CAR according to item 43, comprising the amino acid sequence set forth in SEQ ID NO: 61. (Item 45) A cell comprising the CAR according to any one of items 1 to 44. (Item 46) The cell according to item 45, which is an immunoreactive cell. (Item 47) The cell according to item 45 or 46, which is a lymphoid cell or a myeloid cell. (Item 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 differentiate. (Item 49) The cell according to any one of items 45 to 48, which is a T cell. (Item 50) The cell according to item 48 or 49, wherein the T cell is selected from the group consisting of cytotoxic T lymphocytes (CTLs), γδ T cells, tumor-reactive lymphocytes, tumor-infiltrating lymphocytes (TILs), regulatory T cells, and natural killer T (NKT) cells. (Item 51) A composition comprising the cell according to any one of items 45 to 50. (Item 52) The composition according to item 51, which is a pharmaceutical composition further comprising a pharmaceutically acceptable excipient. (Item 53) The composition according to item 51 or 52, which is for treating and / or preventing neoplasms or tumors, and / or pathogen infections. (Item 54) A method for reducing the tumor burden of a subject, the method comprising administering to the subject the cell according to any one of items 45 to 50 or the composition according to any one of items 51 to 53. (Item 55) The method according to item 54, which reduces the number of tumor cells, reduces the tumor size, and / or eradicates the tumor in the subject. (Item 56) A method for treating and / or preventing a neoplasm or tumor, 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 57) A method for prolonging the survival of a subject having a neoplasm or tumor, 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 58) The method according to 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 lymphoma, Burkitt lymphoma, acute myeloid leukemia (AML), and mixed phenotype acute leukemia (MPAL). (Item 59) A method for producing antigen-specific cells, comprising introducing into a cell a nucleic acid molecule encoding a CAR according to any one of items 1 to 44. (Item 60) The method according to item 59, wherein the nucleic acid molecule is present on a vector. (Item 61) The method according to item 60, wherein the vector is a retroviral vector. (Item 62) A nucleic acid molecule encoding a CAR according to any one of items 1 to 44. (Item 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) A vector comprising the nucleic acid molecule according to item 62 or 63. (Item 65) The vector according to item 64, wherein the vector is a γ-retroviral vector. (Item 66) A host cell expressing the nucleic acid molecule according to item 64 or 65. (Item 67) The host cell according to item 66, which is a T cell. (Item 68) A kit comprising a CAR according to any one of items 1 to 44, a cell according to any one of items 35 to 40, a composition according to any one of items 51 to 53, a nucleic acid molecule according to item 62 or 63, or a vector according to item 64 or 65. (Item 69) The kit according to item 68, further comprising a written instruction for treating and / or preventing a neoplasm, tumor, and / or pathogen infection.

Claims

**Claim 1** A chimeric antigen receptor (CAR) comprising at least one costimulatory signaling domain comprising a CD28 polypeptide containing an extracellular antigen-binding domain, a transmembrane domain, and a mutant YMNM motif, and an intracellular signaling domain, wherein the mutant YMNM motif consists of the amino acid sequence set forth in YKN I (SEQ ID NO: 26), YENV (SEQ ID NO: 22), or YSNV (SEQ ID NO: 23), the CAR. **Claim 2** a) The CD28 polypeptide reduces the recruitment of the p85 subunit of phosphoinositide 3-kinase (PI3K) as compared to a CD28 molecule containing a native YMNM motif; and / or b) The p85 subunit of PI3K does not bind to the mutant YMNM motif, the CAR according to claim 1. **Claim 3** The CAR according to any one of claims 1 to 2, wherein the mutant YMNM motif binds to Grb2-related adaptor (GADS) downstream of growth factor receptor-bound receptor 2 (Grb2) and / or Shc. **Claim 4** The CAR according to any one of claims 1 to 3, wherein the mutant YMNM motif can regulate PI3K signaling by limiting the number of methionine residues capable of binding to the p85 subunit of PI3K. **Claim 5** The CAR according to any one of claims 1 to 4, wherein the extracellular antigen-binding domain binds to an antigen. **Claim 6** The CAR according to claim 5, wherein the antigen is a tumor antigen or a pathogen antigen. **Claim 7** The CAR according to claim 5 or 6, wherein the antigen is a tumor antigen. **Claim 8** The CAR according to claim 7, wherein the tumor antigen is selected from the group consisting of 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-α, GD2, GD3, HER-2, hTERT, 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, cancer fetal antigen (h5T4), PSCA, PSMA, ROR1, TAG-72, VEGFR-2, WT-1, BCMA, CD44V6, NKCS1, EGFR1, EGFR-VIII, ADGRE2, CCR1, LILRB2, PRAME, HPV E6 cancer protein, and HPV E7 cancer protein.

9. The CAR according to claim 8, wherein the tumor antigen is CD19.

10. The CAR according to any one of claims 1 to 9, wherein the mutant YMNM motif consists of the amino acid sequence set forth in YKNI (SEQ ID NO: 26).

11. The CAR according to claim 10, wherein the extracellular antigen-binding domain binds to CD19.

12. The CAR according to claim 11, comprising the amino acid sequence set forth in SEQ ID NO:

55.

13. The CAR according to any one of claims 1 to 9, wherein the mutant YMNM motif consists of the amino acid sequence set forth in YENV (SEQ ID NO: 22).

14. The CAR according to claim 13, wherein the extracellular antigen-binding domain binds to CD19.

15. The CAR according to claim 14, comprising the amino acid sequence set forth in SEQ ID NO:

53.

16. The CAR according to any one of claims 1 to 9, wherein the mutant YMNM motif consists of the amino acid sequence set forth in YSNV (SEQ ID NO: 23).

17. The CAR according to claim 16, wherein the extracellular antigen-binding domain binds to CD19.

18. The CAR according to claim 17, comprising the amino acid sequence set forth in SEQ ID NO:

57.

19. A cell comprising the CAR according to any one of claims 1 to 18.

20. The cell according to claim 19, which is an immunoreactive cell.

21. The cell according to claim 19 or 20, which is a lymphoid cell or a myeloid cell.

22. The cell according to any one of claims 19 to 21, which is selected from the group consisting of a T cell, a natural killer (NK) cell, and a stem cell from which a lymphoid cell can be differentiated.

23. The cell according to any one of claims 19 to 22, which is a T cell.

24. The cell according to claim 22 or 23, wherein the T cell is selected from the group consisting of a cytotoxic T lymphocyte (CTL), a γδ T cell, a tumor-reactive lymphocyte, a tumor-infiltrating lymphocyte (TIL), a regulatory T cell, and a natural killer T (NKT) cell.

25. A composition comprising the cell according to any one of claims 19 to 24.

26. The composition according to claim 25, which is a pharmaceutical composition further comprising a pharmaceutically acceptable excipient.

27. The composition according to claim 25 or 26, which is for treating and / or preventing a neoplasm or tumor, and / or a pathogen infection.

28. The composition according to any one of claims 25 to 27, for reducing the tumor burden of a subject.

29. The composition according to claim 28, wherein the number of tumor cells in the subject is decreased, the tumor size is decreased, and / or the tumor is eradicated.

30. The composition according to any one of claims 25 to 27, for treating and / or preventing a neoplasm or tumor.

31. The composition according to any one of claims 25 to 27, for prolonging the survival of a subject having a neoplasm or tumor.

32. The composition according to any one of claims 28 to 31, 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 lymphoma, Burkitt lymphoma, acute myeloid leukemia (AML), and mixed phenotype acute leukemia (MPAL).

33. A method for producing antigen-specific cells, the method comprising introducing a nucleic acid molecule encoding a CAR according to any one of claims 1 to 18 into a cell.

34. The method according to claim 33, wherein the nucleic acid molecule is present on a vector.

35. The method according to claim 34, wherein the vector is a retroviral vector.

36. A nucleic acid molecule encoding a CAR according to any one of claims 1 to 18.

37. The nucleic acid molecule according to claim 36, comprising the nucleotide sequence set forth in SEQ ID NO: 54, SEQ ID NO: 56, or SEQ ID NO:

58.

38. A vector comprising the nucleic acid molecule according to claim 36 or 37.

39. The vector according to claim 38, wherein the vector is a gamma-retroviral vector.

40. A host cell expressing the nucleic acid molecule according to claim 38 or 39.

41. The host cell according to claim 40, which is a T cell.

42. A kit comprising a CAR according to any one of claims 1 to 18, a cell according to any one of claims 19 to 24, a composition according to any one of claims 25 to 32, a nucleic acid molecule according to claim 36 or 37, or a vector according to claim 38 or 39.

43. The kit according to claim 42, further comprising written instructions for treating and / or preventing a neoplasm, tumor and / or pathogen infection.

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