Chimeric antigen receptors targeting mesothelin and use thereof

Abstract

The presently disclosed subject matter provides methods for treating neoplasia using cells comprising an antigen-recognizing receptor (e.g., a chimeric antigen receptor (CAR)) that specifically targets mesothelin.

Description

[0001] CHIMERIC ANTIGEN RECEPTORS TARGETING MESOTHELIN AND USE THEREOF

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 63 / 657,491, filed June 7, 2024, the content of which is incorporated by reference in its entirety.

[0004] SEQUENCE LISTING

[0005] A Sequence Listing conforming to the rules of WIPO Standard ST.26 is hereby incorporated by reference. Said Sequence Listing has been filed as an electronic document via PatentCenter encoded as XML in UTF-8 text. The electronic document, created on June 4, 2025, is entitled “0727341769_SL” and is 90,307 bytes in size.

[0006] TECHNICAL FIELD

[0007] The presently disclosed subject matter provides methods for treating neoplasia (e.g., cancer) using cells comprising an antigen-recognizing receptor (e.g., a chimeric antigen receptor (CAR)) that specifically targets mesothelin.

[0008] BACKGROUND

[0009] Cell-based immunotherapy is a therapy with curative potential for the treatment of cancer. T cells and other immune cells may be modified to target tumor antigens through the introduction of genetic material coding for artificial or synthetic receptors for antigen, termed Chimeric Antigen Receptors (CARs), specific to selected antigens. Targeted T cell therapy using CARs has shown recent clinical success in treating some hematologic malignancies. However, translating CAR-expressing T cell therapy to solid tumors poses several obstacles that must be overcome to achieve clinical benefit. Malignant cells adapt to generate an immunosuppressive microenvironment to protect themselves from immune recognition and elimination. This tumor microenvironment poses a challenge to methods of treatment involving stimulation of an immune response, such as targeted T cell therapies. Solid tumors may also be restricted within anatomical compartments that impede efficient T cell trafficking, lack expression of agonistic costimulatory ligands and / or express negative regulators of T cell function. The successful elimination of solid tumors thus requires effective tumor infiltration and overcoming tumor-induced immunosuppression. In addition, solid tumors pose a challenge for selecting optimal immune targets - antigens whose targeting would enable tumor eradication by potent T cells, with minimal or tolerable toxicity to non-tumor tissues. Accordingly, there are needs for novel therapeutic strategies to design CARs for treating cancers, particularly, solid tumors, which strategies capable of inducing potent tumor eradication with minimal toxicity and immunogenicity.

[0010] SUMMARY OF THE INVENTION

[0011] The presently disclosed subject matter provides polypeptide compositions comprising (a) a chimeric antigen receptor (CAR) that specifically targets mesothelin (e.g., human mesothelin).

[0012] In certain non-limiting embodiments, the presently disclosed subject matter provides an antigen-recognizing receptor comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen-binding domain specifically binds to mesothelin and comprises a heavy chain variable region comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 6 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8 or a conservative modification thereof; and a light chain variable region comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 9 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11 or a conservative modification thereof. In certain embodiments, the heavy chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 6, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8; and the light chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 9, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11.

[0013] In certain embodiments, the extracellular antigen-binding domain is a single-chain variable fragment (scFv), a Fab, which is optionally crosslinked, or a F(ab)2. In certain embodiments, the extracellular antigen-binding domain is a humanized scFv. In certain embodiments, one or more of the scFv, Fab and F(ab)2 are comprised in a fusion protein with a heterologous sequence to form the extracellular antigen-binding domain.

[0014] In certain embodiments, the heavy chain variable region comprises an amino acid sequence that is at least 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% or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 12. In certain embodiments, the light chain variable region comprises an amino acid sequence that is at least 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% or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 13. In certain embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 12, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 13.

[0015] In certain embodiments, the extracellular antigen-binding domain comprises a linker between the heavy chain variable region and the light chain variable region of the extracellular antigen-binding domain. In certain embodiments, the linker consists of the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. In certain embodiments, the linker consists of the amino acid sequence set forth in SEQ ID NO: 1.

[0016] In certain embodiments, a signal peptide is covalently joined to the 5’ terminus of the extracellular antigen-binding domain. In certain embodiments, the heavy chain variable region and the light chain variable region are positioned from the N- to the C-terminus: VH-VL. In certain embodiments, the heavy chain variable region and the light chain variable region are positioned from the N- to the C-terminus: VL-VH.

[0017] In certain non-limiting embodiments, the presently disclosed subject matter provides an antigen-recognizing receptor, comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen-binding domain specifically binds to mesothelin and comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDR1, a CDR2, and a CDR3 of a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 12, and the light chain variable region comprises a CDR1, a CDR2, and a CDR3 of a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 13. In certain non-limiting embodiments, the presently disclosed subject matter provides an antigen-recognizing receptor, comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen-binding domain specifically binds to mesothelin and comprises a single chain a single chain variable region (scFv) comprising the amino acid sequence set forth in SEQ ID NO: 16. In certain embodiments, the transmembrane domain comprises a CD8 polypeptide, a CD28 polypeptide, a CD3(^ polypeptide, a CD4 polypeptide, a 4-1BB polypeptide, an 0X40 polypeptide, an ICOS polypeptide, a CTLA-4 polypeptide, a PD-1 polypeptide, a LAG-3 polypeptide, a 2B4 polypeptide, a BTLA polypeptide, or a combination thereof. In certain embodiments, the intracellular signaling domain comprises a CD3(^ polypeptide. In certain embodiments, the intracellular signaling domain further comprises at least one co-stimulatory signaling region. In certain embodiments, the at least one co-stimulatory signaling region comprises a CD28 polypeptide, a 4-1BB polypeptide, an 0X40 polypeptide, an ICOS polypeptide, a DAP- 10 polypeptide, or a combination thereof. In certain embodiments, the antigen-recognizing receptor is a chimeric antigen receptor (CAR). In certain embodiments, the antigen-recognizing receptor is recombinantly expressed or expressed from a vector.

[0018] In certain non-limiting embodiments, the presently disclosed subject matter provides a cell comprising the antigen-recognizing receptor disclosed herein. In certain embodiments, the antigen-recognizing receptor is constitutively expressed on the surface of the cell. In certain embodiments, the cell is an immunoresponsive cell. In certain embodiments, the cell is a cell of the lymphoid lineage or a cell of the myeloid lineage. In certain embodiments, the cell is selected from the group consisting of a T cell, a Natural Killer (NK) cell, and a stem cell from which a lymphoid cell may be differentiated. In certain embodiments, the cell is a T cell. In certain embodiments, the T cell is a cytotoxic T lymphocyte (CTL) or a regulatory T cell. In certain embodiments, the stem cell is a pluripotent stem cell. In certain embodiments, the pluripotent stem cell is an embryoid stem cell or an induced pluripotent stem cell.

[0019] In certain non-limiting embodiments, the presently disclosed subject matter provides a nucleic acid molecule encoding the antigen-recognizing receptor disclosed herein. In certain non-limiting embodiments, the presently disclosed subject matter provides a vector comprising the nucleic acid molecule disclosed herein. In certain embodiments, the vector is a retroviral vector. In certain embodiments, the retroviral vector is a y-retroviral vector or a lentiviral vector. In certain non-limiting embodiments, the presently disclosed subject matter provides a lipid nanoparticle comprising the nucleic acid disclosed herein.

[0020] In certain non-limiting embodiments, the presently disclosed subject matter provides a composition comprising the cell, the nucleic acid molecule, the vector, or the lipid nanoparticle disclosed herein. In certain embodiments, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

[0021] In certain non-limiting embodiments, the presently disclosed subject matter provides a method of treating and / or preventing a tumor in a subject in need thereof and / or increasing or lengthening survival of a subject having a tumor, the method comprising administering to the subject an effective amount of the cell or the composition disclosed herein. In certain embodiments, the method reduces the number of tumor cells, reduces tumor size, and / or eradicates the tumor in the subject. In certain embodiments, the tumor is associated with mesothelin. In certain embodiments, the tumor is a solid tumor. In certain embodiments, the solid tumor is selected from mesothelioma, renal cancer, bladder cancer, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic carcinoma, endometrial carcinoma, stomach cancer, or cholangiocarcinoma.

[0022] In certain non-limiting embodiments, the presently disclosed subject matter provides a method for producing a cell comprising an antigen-recognizing receptor disclosed herein, comprising introducing into the cell a nucleic acid molecule that encodes the antigenrecognizing receptor. In certain non-limiting embodiments, the presently disclosed subject matter provides a kit for reducing tumor burden in a subject, treating and / or preventing a tumor in a subject, and / or increasing or lengthening survival of a subject having a tumor, comprising the cell disclosed herein. In certain embodiments, the kit further comprises written instructions for using the cell for reducing tumor burden in a subject, treating and / or preventing a tumor or neoplasm in a subject, and / or increasing or lengthening survival of a subject having a tumor.

[0023] BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The following Detailed Description, given by way of example, but not intended to limit the presently disclosed subject matter to specific embodiments described, may be understood in conjunction with the accompanying drawings.

[0025] Figure 1 shows activity of the R6P1-B1 clone against human mesothelin expressing cells detected by flow cytometry.

[0026] Figures 2A-2E illustrate the structure and interaction of the m912 scFv and R6P1-B1 scFv. Figure 2A shows B-factor view of the m912 scFv and R6P1-B1 scFv. Figure 2B shows the distribution of electrostatic forces of the m912 scFv and R6P1-B1 scFv. Figure 2C shows the lipophilicity of the m912 scFv and R6P1-B1 scFv. Figure 2D shows a summary of the properties of the m912 scFv and R6P1-B1 scFv and the predicted binding pocket. Figure 2E shows the interaction of m912 scFv and R6P1-B1 scFv with human mesothelin.

[0027] Figures 3A-3F depict various exemplary expression constructs encompassed by the presently disclosed subject matter. Figure 3 A illustrates a CAR construct comprising a mesothelin mM scFv, a CD28 costimulatory domain, and a CD3(^ intracellular domain. Figure 3B shows the expression of a CAR construct detected by flow cytometry. Figure 3C illustrates CAR constructs comprising two different mesothelin scFvs, a CD28 costimulatory domain, and a CD3(^ intracellular domain. Figure 3D illustrates different constructs disclosed herein. In MZ-KITv, the construct encodes an MZ CAR and a KITv polypeptide. In mM28Z and mMBBZ, the constructs encode CARs comprising the presently disclosed scFv and including a CD28 costimulatory domain or a 4- IBB costimulatory domain, respectively. Figure 3E illustrates a construct for the constitutive expression of (a) a CAR including a M912 scFv and a CD3(^ intracellular domain, and (b) a KITv polypeptide, and inducible expression of a CAR including a mM scFv and a CD3(^ intracellular domain. Figure 3F illustrates a construct for the constitutive expression of (a) a CAR including a mM scFv and a CD3(^ intracellular domain, and (b) a KITv polypeptide, and inducible expression of (c) a CAR including a M912 scFv, a CD28 costimulatory domain, and a CD3(^ intracellular domain, and (b) a PD-1 DNR polypeptide.

[0028] Figures 4A-4C depict mesothelin (MSLN)-targeted CAR transduction and cytotoxicity against human and mouse MSLN. Figure 4A shows transduction of T cells with the presently disclosed antigen-recognizing receptor. Figure 4B shows cytotoxic activity against MGM mesothelioma cells. Figure 4C shows cytotoxic activity against mouse AB 12 cells expressing mesothelin.

[0029] Figure 5 depicts flow cytometry analysis showing CAR transduction efficiency in T cells derived from nine independent human donors. T cells were transduced with the codon- optimized mM28z CAR construct. Transduction levels ranged from 30% to 73% across donors, demonstrating consistent and robust expression.

[0030] Figure 6 shows cytotoxic activity of M28z and mM28z CAR T cells against human mesothelioma MSTO-GM cells. CAR T cells were co-cultured with MSTO-GM cells at different effector-to-target (E:T) ratios for 48 hours. Cell viability was assessed postincubation. Controls include MSTO-GM cells alone (MGM) and untransduced T cells (UT).

[0031] Figure 7 shows cytotoxic activity of M28z and mM28z CAR T cells against murine mesothelioma AB 12 cells. CAR T cells were co-cultured with AB 12 cells at indicated E:T ratios for up to 48 hours. Cell viability was measured to assess cytotoxicity. Controls include AB 12 cells alone (AB12-mMSLN) and untransduced T cells (UT).

[0032] Figures 8A-8D depict activation marker expression in mM28z CAR T cells upon exposure to soluble mesothelin (MSLN). Data demonstrate antigen-specific activation of mM28z CAR T cells. Figure 8 A shows CD69 expression. Figure 8B shows CD 107a expression. Figure 8C shows PD-1 expression. Figure 8D shows CD25 expression. Figures 9A and 9B depict the effect of soluble mesothelin on mM28z CAR T cell cytotoxicity. Figure 9A shows binding of mM28z CAR T cells to soluble mesothelin. Figure 9B shows cytotoxicity measured using a chromium-51 release assay following pre-incubation with mesothelin-containing supernatant. Results indicate no off-target cytotoxicity due to soluble antigen exposure.

[0033] Figure 10 shows dose-dependent binding of mM28z CAR T cells to recombinant soluble mesothelin (rMSLN). Flow cytometry analysis shows increased binding with increasing concentrations of rMSLN, confirming antigen specificity and sensitivity.

[0034] Figure 11 shows transduction efficiency of mM28z CAR T cells at decreasing vector concentrations. T cells were transduced using serial dilutions of the mM28z vector (1 : 10 to 1 :30). Flow cytometry analysis revealed sustained transduction levels (up to 46%) even at the lowest vector concentration tested.

[0035] DETAILED DESCRIPTION

[0036] The present disclosure provides uses of cells comprising an antigen-recognizing receptor (e.g., a chimeric antigen receptor (CAR)) that specifically targets mesothelin for treatments, e.g., for treating neoplasia. The cells can be immunoresponsive cells, e.g., genetically modified immunoresponsive cells e.g., T-cells or NK cells). The present disclosure is based, in part, on unexpected findings demonstrating that the antigen-recognizing receptors disclosed herein exhibit significantly higher expression levels in cells (e.g., T cells) compared to other known anti-mesothelin antigen-recognizing receptors. Without being bound by theory, the inventors believe that this enhanced expression is attributable to the unique antigen-binding domain disclosed herein.

[0037] Non-limiting embodiments of the present disclosure are described by the present specification and Examples.

[0038] For purposes of clarity of disclosure and not by way of limitation, the detailed description is divided into the following subsections:

[0039] 1. Definitions;

[0040] 2. Mesothelin;

[0041] 3. Antigen-Recognizing Receptors;

[0042] 4. Cells;

[0043] 5. Compositions and Vectors;

[0044] 6. Polypeptides;

[0045] 7. Formulations and Administration; 8. Methods of Treatment; and

[0046] 9. Exemplary Embodiments.

[0047] / . Definitions

[0048] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: 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). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.

[0049] As used herein, the term “about” or “approximately” means within an acceptable error range for the 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 than 3 standard deviations, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2- fold, of a value.

[0050] By “immunoresponsive cell” is meant a cell that functions in an immune response or a progenitor, or progeny thereof. In certain embodiments, the immunoresponsive cell is a cell of lymphoid lineage. Non-limiting examples of cells of lymphoid lineage include T-cells, Natural Killer (NK) cells, B cells, and stem cells from which lymphoid cells may be differentiated. In certain embodiments, the immunoresponsive cell is a cell of myeloid lineage.

[0051] By “activates an immunoresponsive cell” is meant induction of signal transduction or changes in protein expression in the cell resulting in initiation of an immune response. For example, when CD3 Chains cluster in response to ligand binding and immunoreceptor tyrosinebased inhibition motifs (ITAMs) a signal transduction cascade is produced. In certain embodiments, when an endogenous TCR or an exogenous CAR binds to an antigen, a formation of an immunological synapse occurs that includes clustering of many molecules near the bound receptor (e.g. CD4 or CD8, CD3(75 / s, etc.). This clustering of membrane bound signaling molecules allows for ITAM motifs contained within the CD3 chains to become phosphorylated. This phosphorylation in turn initiates a T-cell activation pathway ultimately activating transcription factors, such as NF-KB and AP-1. These transcription factors induce global gene expression of the T-cell to increase IL-2 production for proliferation and expression of master regulator T-cell proteins in order to initiate a T-cell mediated immune response.

[0052] By “stimulates an immunoresponsive cell” is meant a signal that results in a robust and sustained immune response. In various embodiments, this occurs after immune cell (e.g., T- cell) activation or concomitantly mediated through receptors including, but not limited to, CD28, CD137 (4-1BB), 0X40, CD40 and ICOS. Receiving multiple stimulatory signals can be important to mount a robust and long-term T-cell mediated immune response. T-cells can quickly become inhibited and unresponsive to antigen. While the effects of these costimulatory signals may vary, they generally result in increased gene expression in order to generate long lived, proliferative, and anti-apoptotic T-cells that robustly respond to antigen for complete and sustained eradication.

[0053] The term “antigen-recognizing receptor” as used herein refers to a receptor that is capable of recognizing a target antigen (e.g., mesothelin). In certain embodiments, the antigenrecognizing receptor is capable of activating an immune or immunoresponsive cell (e.g., a T- cell) upon its binding to the target antigen.

[0054] As used herein, “complementarity determining regions” or “CDRs” are defined as the complementarity determining region amino acid sequences of an antibody which are the hypervariable regions of immunoglobulin heavy and light chains. See, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 4th U. S. Department of Health and Human Services, National Institutes of Health (1987), or IMGT numbering system (Lefranc, The Immunologist (1999);7: 132-136; Lefranc et al., Dev. Comp. Immunol. (2003);27:55-77). Generally, antibodies comprise three heavy chain and three light chain CDRs or CDR regions in the variable region. CDRs provide the majority of contact residues for the binding of the antibody to the antigen or epitope. In certain embodiments, the CDRs regions are delineated using the Kabat numbering system.

[0055] As used herein, the term “single-chain variable fragment” or “scFv” is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of an immunoglobulin (e.g., mouse or human) covalently linked to form a VH::VL heterodimer. The heavy (VH) and light chains (VL) are either joined directly or joined by a peptide-encoding linker (e.g., 10, 15, 20, 25 amino acids), which connects the N-terminus of the VH with the C-terminus of the VL, or the C-terminus of the VH with the N-terminus of the VL. The linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility. The linker can link the heavy chain variable region and the light chain variable region of the extracellular antigen-binding domain. Non-limiting examples of linkers are disclosed in Shen et al., Anal. Chem. 80(6): 1910- 1917 (2008) and WO 2014 / 087010, the contents of which are hereby incorporated by reference in their entireties. In certain embodiments, the linker is a G4S linker.

[0056] In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO: 1, which is provided below: GGGGSGGGGSGGGGS [ SEQ ID NO : 1 ]

[0057] In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO: 2, which is provided below:

[0058] GGGGSGGGGSGGGSGGGGS [ SEQ ID NO : 2 ]

[0059] In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO: 3, which is provided below: GGGGSGGGGSGGGGSGGGSGGGGS [ SEQ ID NO : 3 ]

[0060] In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO: 4, which is provided below: GGGGSGGGGSGGGGSGGGGSGGGSGGGGS [ SEQ ID NO : 4 ]

[0061] Despite removal of the constant regions and the introduction of a linker, scFv proteins retain the specificity of the original immunoglobulin. Single chain Fv polypeptide antibodies can be expressed from a nucleic acid comprising VH - and VL encoding sequences as described by Huston, et al. Proc. Nat. Acad. Sci. USA, (1988);85:5879-5883; U.S. Patent Nos. 5,091,513, 5,132,405 and 4,956,778; and U.S. Patent Publication Nos. 20050196754 and 20050196754. Antagonistic scFvs having inhibitory activity have been described (see, e.g., Zhao et al., Hyrbidoma (Larchmt) (2008);27(6):455-51; Peter et al., J Cachexia Sarcopenia Muscle (2012);August 12; Shieh et al., J Imunol (2009); 183(4):2277-85; Giomarelli et al., Thromb Haemost (2007);97(6):955-63; Fife eta., J Clin Invst (2006);l 16(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 Chern (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., BioChim Biophys Acta (2003); 1638(3):257-66).

[0062] The term “chimeric antigen receptor” or “CAR” as used herein refers to a molecule comprising an extracellular antigen-binding domain that is fused to an intracellular signaling domain that is capable of activating or stimulating an immunoresponsive cell, and a transmembrane domain. In certain embodiments, the extracellular antigen-binding domain of a CAR comprises an scFv. The scFv can be derived from fusing the variable heavy and light regions of an antibody. Alternatively or additionally, the scFv may be derived from Fab’s (instead of from an antibody, e.g., obtained from Fab libraries). In certain embodiments, the scFv is fused to the transmembrane domain, which is fused to the intracellular signaling domain.

[0063] By “substantially identical” or “substantially homologous” is meant a polypeptide or nucleic acid molecule exhibiting at least about 50% homologous or identical to a reference amino acid sequence (for example, any of the amino acid sequences described herein) or a reference nucleotide sequence (for example, any of the nucleotide sequences described herein). In certain embodiments, such a sequence is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence or the nucleotide sequence used for comparison.

[0064] Sequence identity can be measured by using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e-3 and e-100 indicating a closely related sequence.

[0065] The percent homology between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4: 11-17 (1988)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent homology between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0066] Additionally or alternatively, the amino acids sequences of the presently disclosed subject matter can further be used as a “query sequence” to perform a search against public databases to, for example, identify related sequences. Such searches can be performed using the XBLAST program (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to the specified sequences (e.g., heavy and light chain variable region sequences of scFv703) disclosed herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs e.g., XBLAST and NBLAST) can be used.

[0067] An “effective amount” is an amount sufficient to affect a beneficial or desired clinical result upon treatment. An effective amount can be administered to a subject in one or more doses. In certain embodiments, an effective amount can be an amount that is sufficient to palliate, ameliorate, stabilize, reverse or slow the progression of the disease, or otherwise reduce the pathological consequences of the disease. The effective amount can be determined by a physician on a case-by-case basis and is within the skill of one in the art. Several factors are typically taken into account when determining an appropriate dosage to achieve an effective amount. These factors include 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 administered.

[0068] By “modulate” is meant positively or negatively alter. Exemplary modulations include a about 1%, about 2%, about 5%, about 10%, about 25%, about 50%, about 75%, or about 100% change.

[0069] By “increase” is meant to alter positively by at least about 5%. An alteration may be by about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, about 100% or more.

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

[0071] The terms “isolated,” “purified,” or “biologically pure” refer to material that is free to varying degrees from components which normally accompany it as found in its native state. “Isolate” denotes a degree of separation from original source or surroundings. “Purify” denotes a degree of separation that is higher than isolation. A “purified” or “biologically pure” protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high-performance liquid chromatography. The term “purified” can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.

[0072] By “isolated cell” is meant a cell that is separated from the molecular and / or cellular components that naturally accompany the cell.

[0073] The term “antigen-binding domain” as used herein refers to a domain capable of specifically binding a particular antigenic determinant or set of antigenic determinants present on a cell.

[0074] By “neoplasia” is meant a disease characterized by the pathological proliferation of a cell or tissue and its subsequent migration to or invasion of other tissues or organs. Neoplasia growth is typically uncontrolled and progressive, and occurs under conditions that would not elicit, or would cause cessation of, multiplication of normal cells. Neoplasia can affect a variety of cell types, tissues, or organs, including but not limited to an organ selected from the group consisting of bladder, bone, brain, breast, cartilage, glia, esophagus, fallopian tube, gallbladder, heart, intestines, kidney, liver, lung, lymph node, nervous tissue, ovaries, pancreas, prostate, skeletal muscle, skin, spinal cord, spleen, stomach, testes, thymus, thyroid, trachea, urogenital tract, ureter, urethra, uterus, and vagina, or a tissue or cell type thereof. Neoplasia include cancers, such as sarcomas, carcinomas, or plasmacytomas (malignant tumor of the plasma cells). The neoplasia can a primary tumor or primary cancer. In addition, the neoplasia can be in metastatic status.

[0075] By “receptor” is meant a polypeptide, or portion thereof, present on a cell membrane that selectively binds one or more ligand.

[0076] By “recognize” is meant selectively binds to a target. A T-cell that recognizes a tumor can expresses a receptor (e.g., a TCR or CAR) that binds to a tumor antigen.

[0077] By “reference” or “control” is meant a standard of comparison. For example, the level of scFv-antigen binding by a cell expressing a CAR and an scFv may be compared to the level of scFv-antigen binding in a corresponding cell expressing CAR alone.

[0078] By “signal sequence” or “leader sequence” is meant a peptide sequence (e.g., 5, 10, 15, 20, 25 or 30 amino acids) present at the N-terminus of newly synthesized proteins that directs their entry to the secretory pathway. Exemplary leader sequences include, but is not limited to, a human IL-2 signal sequence (e.g., a human IL-2 signal sequence set forth in SEQ ID NO:

[0079] 43), a mouse IL-2 signal sequence (e.g., a mouse IL-2 signal sequence set forth in SEQ ID NO:

[0080] 44), a human kappa leader sequence (e.g., a human kappa leader sequence set forth in SEQ ID NO: 45), a mouse kappa leader sequence (e.g., a mouse kappa leader sequence set forth in SEQ ID NO: 46), a human CD8 leader sequence (e.g., a human CD8 leader sequence set forth in SEQ ID NO: 47), a truncated human CD8 signal peptide (e.g., a truncated human CD8 signal peptide set forth in SEQ ID NO: 48), a human albumin signal sequence (e.g., a human albumin signal sequence set forth in SEQ ID NO: 49), and a human prolactin signal sequence (e.g., a human prolactin signal sequence set forth in SEQ ID NO: 50). SEQ ID Nos: 43-50 are provided below.

[0081] MYRMQLLSCIALSLALVTNS [ SEQ ID NO : 43 ]

[0082] MYSMQLASCVTLTLVLLVNS [ SEQ ID NO : 44 ]

[0083] METPAQLLFLLLLWLPDTTG [ SEQ ID NO : 45 ]

[0084] METDTLLLWVLLLWVPGSTG [ SEQ ID NO : 46 ]

[0085] MALPVTALLLPLALLLHAARP [ SEQ ID NO : 47 ] ) MALPVTALLLPLALLLHA [ SEQ ID NO : 48 ] MKWVTFISLLFSSAYS [ SEQ ID NO : 49 ] MDSKGSSQKGSRLLLLLWSNLLLCQGWS [ SEQ ID NO : 50 ]

[0086] By “specifically binds” or “specifically binds to” or “specifically target” is meant a polypeptide or a fragment thereof that recognizes and / or binds to a biological molecule of interest e.g., a polypeptide, e.g., a mesothelin polypeptide), but which does not substantially recognize and / or bind to other molecules in a sample, for example, a biological sample, which naturally includes a presently disclosed polypeptide (e.g., a mesothelin polypeptide).

[0087] The terms “comprises”, “comprising”, and are intended to have the broad meaning ascribed to them in U.S. Patent Law and can mean “includes”, “including” and the like.

[0088] As used herein, “treatment” refers to clinical intervention in an attempt to alter the disease course of the individual or cell being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Therapeutic effects of treatment include, without limitation, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastases, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. By preventing progression of a disease or disorder, a treatment can prevent deterioration due to a disorder in an affected or diagnosed subject or a subject suspected of having the disorder, but also a treatment may prevent the onset of the disorder or a symptom of the disorder in a subject at risk for the disorder or suspected of having the disorder.

[0089] An “individual” or “subject” herein is a vertebrate, such as a human or non-human animal, for example, a mammal. Mammals include, but are not limited to, humans, primates, farm animals, sport animals, rodents and pets. Non-limiting examples of non-human animal subjects include rodents such as mice, rats, hamsters, and guinea pigs; rabbits; dogs; cats; sheep; pigs; goats; cattle; horses; and non-human primates such as apes and monkeys.

[0090] Other aspects of the presently disclosed subject matter are described in the following disclosure and are within the ambit of the presently disclosed subject matter.

[0091] 2. Mesothelin

[0092] Mesothelin is an immunogenic cell surface antigen that is highly expressed in solid cancers. Mesothelin is involved in cell proliferation, adhesion, invasion, cell signaling, and metastasis. Studies have demonstrated that serum soluble mesothelin-related peptide secreted by mesothelin-expressing tumors can be measured in both humans and mice, and has been shown to correlate with therapy response and prognosis. In normal tissues, mesothelin is expressed only in the pleura, pericardium, and peritoneum, at low levels. The anti-mesothelin recombinant immunotoxin SS1P has shown in vivo specificity and significant antitumor activity in patients. In a pancreatic cancer vaccine trial, patients with survival advantage had consistent CD8+ T cell responses to mesothelin associated with vaccine-induced delay ed-type hypersensitivity response. Specific T cell epitopes derived from mesothelin were shown to activate human T cells to efficiently lyse human tumors expressing mesothelin. Thus, there is strong supportive evidence that adoptive immunotherapy targeting mesothelin can target mesothelin-expressing tumors.

[0093] In certain embodiments, the antigen-recognizing receptor binds to human mesothelin. In certain embodiments, the human mesothelin comprises or consists of the amino acid sequence with a NCBI Reference No: AAV87530.1 (SEQ ID NO: 5) or a fragment thereof. SEQ ID NO: 5 is provided below: MALPTARPLLGSCGTPALGSLLFLLFSLGWVQPSRTLAGETGQEAAPLDGVLANPPNISSLS PRQLLGFPCAEVSGLSTERVRELAVALAQKNVKLSTEQLRCLAHRLSEPPEDLDALPLDLLL FLNPDAFSGPQACTHFFSRITKANVDLLPRGAPERQRLLPAALACWGVRGSLLSEADVRALG GLACDLPGRFVAESAEVLLPRLVSCPGPLDQDQQEAARAALQGGGPPYGPPSTWSVSTMDAL RGLLPVLGQPI IRS IPQGIVAAWRQRSSRDPSWRQPERTILRPRFRREVEKTACPSGKKARE IDESLI FYKKWELEACVDAALLATQMDRVNAIPFTYEQLDVLKHKLDELYPQGYPESVIQHL GYLFLKMSPEDIRKWNVTSLETLKALLEVNKGHEMSPQVATLIDRFVKGRGQLDKDTLDTLT AFYPGYLCSLSPEELSSVPPSS IWAVRPQDLDTCDPRQLDVLYPKARLAFQNMNGSEYFVKI QSFLGGAPTEDLKALSQQNVSMDLATFMKLRTDAVLPLTVAEVQKLLGPHVEGLKAEERHRP VRDWILRQRQDDLDTLGLGLQGGIPNGYLVLDLSVQEALSGTPCLLGPGPVLTVLALLLAST LA [ SEQ ID NO : 5 ] In certain embodiments, the human mesothelin comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence set forth in SEQ ID NO: 5 or a portion thereof.

[0094] 3. Antigen-Recognizing Receptors

[0095] The presently disclosed antigen-recognizing receptors specifically target or bind to mesothelin. In certain embodiments, the antigen-recognizing receptor is a chimeric antigen receptor (CAR). In certain embodiments, the antigen-recognizing receptor is a T-cell receptor (TCR). In certain embodiments, the antigen-recognizing receptor is a TCR like fusion molecule.

[0096] The presently disclosed subject matter also provides nucleic acid molecules that encode the presently disclosed antigen-recognizing receptors.

[0097] 3.1. T-Cell Receptor (TCR)

[0098] In certain embodiments, the antigen-recognizing receptor is a TCR. A TCR is a disulfide-linked heterodimeric protein consisting of two variable chains expressed as part of a complex with the invariant CD3 chain molecules. A TCR found on the surface of T-cells is responsible for recognizing antigens as peptides bound to major histocompatibility complex (MHC) molecules. In certain embodiments, a TCR comprises an alpha chain and a beta chain (encoded by TRA and TRB, respectively). In certain embodiments, a TCR comprises a gamma chain and a delta chain (encoded by TRG and TRD, respectively).

[0099] Each chain of a TCR is composed of two extracellular domains: Variable (V) region and a Constant (C) region. The Constant region is proximal to the cell membrane, followed by a transmembrane region and a short cytoplasmic tail. The Variable region binds to the peptide / MHC complex. The variable domain of both chains each consist of three complementarity determining regions (CDRs).

[0100] In certain embodiments, a TCR can form a receptor complex with three dimeric signaling modules CD35 / s, CD3y / s and CD247 / , or £ / r|. When a TCR complex engages with its antigen and MHC (peptide / MHC), the T-cell expressing the TCR complex is activated.

[0101] In certain embodiments, the TCR is an endogenous TCR. In certain embodiments, the antigen-recognizing receptor is naturally occurring TCR.

[0102] In certain embodiments, the antigen-recognizing receptor is an exogenous TCR. In certain embodiments, the antigen-recognizing receptor is a recombinant TCR. In certain embodiments, the antigen-recognizing receptor is a recombinant TCR. In certain embodiments, the recombinant TCR differs from any naturally occurring TCR by at least one amino acid residue. In certain embodiments, the recombinant TCR differs from any naturally occurring TCR by at least about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 20, about 25, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100 or more amino acid residues. In certain embodiments, the recombinant TCR is modified from a naturally occurring TCR by at least one amino acid residue. In certain embodiments, the recombinant TCR is modified from a naturally occurring TCR by at least about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 20, about 25, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100 or more amino acid residues.

[0103] 3.2. _ Chimeric Antisen Receptor (CAR)

[0104] In certain embodiments, the antigen-recognizing receptor is a CAR. CARs are engineered receptors, which graft or confer a specificity of interest onto an immune effector cell. CARs can be used to graft the specificity of a monoclonal antibody onto a T-cell; with transferring of their coding sequence facilitated by retroviral vectors.

[0105] There are three generations of CARs. “First generation” CARs are typically composed of an extracellular antigen-binding domain (e.g., an scFv), which is fused to a transmembrane domain, which is fused to cytoplasmic / intracellular signaling domain. “First generation” CARs can provide de novo antigen recognition and cause activation of both CD4+and CD8+T-cells through their CD3(^ chain signaling domain in a single fusion molecule, independent of HLA-mediated antigen presentation. “Second generation” CARs add intracellular signaling domains from various co-stimulatory molecules (e.g., CD28, 4- IBB, ICOS, 0X40) to the cytoplasmic tail of the CAR to provide additional signals to the T-cell. “Second generation” CARs comprise those that provide both co-stimulation (e.g., CD28 or 4-1BB) and activation (CD3Q. “Third generation” CARs comprise those that provide multiple co-stimulation (e.g., CD28 and 4-1BB) and activation (CD3Q. In certain embodiments, the antigen-recognizing receptor is a first-generation CAR. In certain embodiments, the antigen-recognizing receptor is a CAR that does not comprise an intracellular signaling domain of a co-stimulatory molecule. In certain embodiments, the antigen-recognizing receptor is a second-generation CAR. In certain embodiments, the CAR comprises an extracellular antigen-binding domain that specifically binds to mesothelin, a transmembrane domain, and an intracellular signaling domain.

[0106] 3,2.1. Extracellular Antigen-Binding Domain of A CAR

[0107] In certain embodiments, the extracellular antigen-binding domain is an scFv. The scFv can be a human scFv, a humanized scFv, or a murine scFv. In certain embodiments, the scFv is a humanized scFv.

[0108] In certain embodiments, the extracellular antigen-binding domain is a Fab. In certain embodiments, the Fab is crosslinked. In certain embodiments, the extracellular antigenbinding domain is a F(ab)2.

[0109] Any of the foregoing molecules may be comprised in a fusion protein with a heterologous sequence to form the extracellular antigen-binding domain.

[0110] In certain embodiments, the extracellular antigen-binding domain of the CAR (embodied, for example, in a scFv or an analog thereof) binds to human mesothelin with an EC50 value of from about 1 nM to about 25 nM as measured by enzyme-linked immunosorbent assay (ELISA). In certain embodiments, the extracellular antigen-binding domain of the CAR has an EC50 value of about 20 nM as measured by ELISA. In certain embodiments, the extracellular antigen-binding domain of the CAR is derived from Fab’s (e.g., from human or mouse Fab libraries).

[0111] Binding of the extracellular antigen-binding domain (embodiment, for example, in a scFv or an analog thereof) of the CAR can be confirmed by, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growth inhibition), or Western Blot assay. Each of these assays generally detect the presence of protein-antibody complexes of particular interest by employing a labeled reagent (e.g., an antibody, or a scFv) specific for the complex of interest. For example, the scFv can be radioactively labeled and used in a radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986, which is incorporated by reference herein). The radioactive isotope can be detected by such means as the use of a y counter or a scintillation counter or by autoradiography. In certain embodiments, the mesothelin targeted extracellular antigen-binding domain is labeled with a fluorescent marker. Non-limiting examples of fluorescent markers include green fluorescent protein (GFP), blue fluorescent protein (e.g., EBFP, EBFP2, Azurite, and mKalamal), cyan fluorescent protein (e.g., ECFP, Cerulean, and CyPet), and yellow fluorescent protein (e.g., YFP, Citrine, Venus, and YPet). In certain embodiments, the mesothelin-targeted human scFv is labeled with GFP.

[0112] In certain embodiments, the extracellular antigen-binding domain of the CAR binds to human mesothelin with a mesothelin level of about 1,000 or more mesothelin binding sites / cell. In certain embodiments, the extracellular antigen-binding domain of the CAR binds to human mesothelin with a mesothelin level of from about 1,000 to about 50,000 mesothelin binding sites / cell. In certain embodiments, the extracellular antigen-binding domain of the CAR does not bind to human mesothelin with a mesothelin expression level of less than 1,000 mesothelin binding sites / cell, e.g., the human mesothelin expressed normal tissues, e.g., normal pleura, pericardium, and peritoneum tissues. In certain embodiments, the extracellular antigenbinding domain of the CAR does not bind to human mesothelin with a mesothelin expression level of more than 50,000 mesothelin binding sites / cell. In certain embodiments, a human scFV comprised in the CAR binds to human mesothelin with a mesothelin expression level of from about 1,000 to about 50,000 mesothelin binding sites / cell. In certain embodiments, a human scFV comprised in the CAR does not bind to human mesothelin with a mesothelin expression level of more than 50,000 or less than 1,000 mesothelin binding sites / cell.

[0113] In certain embodiments, the extracellular antigen-binding domain of the CAR (e.g., an scFv) comprises a variable fragment heavy chain (VH) comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 6 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8 or a conservative modification thereof. SEQ ID NOs: 6-8 are provided in Table 1.

[0114] In certain embodiments, the extracellular antigen-binding domain of the CAR (e.g., an scFv) comprises a variable fragment light chain (VL) comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 9 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11 or a conservative modification thereof. SEQ ID NOs: 9-11 are provided in Table 1.

[0115] In certain embodiments, the extracellular antigen-binding domain of the CAR (e.g., an scFv) comprises a VH comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 6 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8 or a conservative modification thereof; and a VL comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 9 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10 or a conservative modification, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11 or a conservative modification thereof.

[0116] In certain embodiments, the extracellular antigen-binding domain of the CAR (e.g., an scFv) comprises a VH comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 6, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8; and a VL comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 9, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11.

[0117] In certain embodiments, the extracellular antigen-binding domain of the CAR (e.g., an scFv) comprises a VH comprising an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO: 12. For example, the extracellular antigenbinding domain of the CAR (e.g., an scFv) comprises a VH comprising 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: 12. In certain embodiments, the extracellular antigen-binding domain comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 12. SEQ ID NO: 12 is provided in Table 1 below.

[0118] In certain embodiments, the extracellular antigen-binding domain of the CAR (e.g., an scFv) comprises a VL comprising an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO: 13. For example, the extracellular antigenbinding domain of the CAR (e.g., an scFv) comprises a VL comprising 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: 13. In certain embodiments, the extracellular antigen-binding domain comprises a VL comprising the amino acid sequence set forth in SEQ ID NO: 13. SEQ ID NO: 13 is provided in Table 1 below. In certain embodiments, the extracellular antigen-binding domain of the CAR (e.g., an scFv) comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 12, and a VL comprising the amino acid sequence set forth in SEQ ID NO: 13.

[0119] In certain embodiments, the VH and VL are linked via a linker. In certain embodiments, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. In certain embodiments, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1.

[0120] In certain embodiments, the variable regions within the extracellular antigen-binding domain of the CAR have to be linked one after another such that at the N-terminus of the extracellular antigen-binding domain, a heavy chain variable region (VH) is positioned. In certain embodiments, the extracellular antigen-binding domain of the CAR is an scFv, and the variable regions are positioned from the N- to the C-terminus: VH-VL.

[0121] In certain embodiments, the extracellular antigen-binding domain of the CAR (e.g., an scFv) comprises or consists of the amino acid sequence set forth in SEQ ID NO: 16 and specifically binds to mesothelin (e.g., human mesothelin). In certain embodiments, the scFv set forth in SEQ ID NO: 16 is designated as “R6P1 -Bl” or “mM”. SEQ ID NO: 16 is provided in Table 1 below.

[0122] Table 1

[0123] In certain embodiments, the extracellular antigen-binding domain of the CAR (e.g., an scFv) comprises a variable fragment heavy chain (VH) comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 51 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 52 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 53 or a conservative modification thereof. SEQ ID NOs: 51-53 are provided in Table 2.

[0124] In certain embodiments, the extracellular antigen-binding domain of the CAR (e.g., an scFv) comprises a variable fragment light chain (VL) comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 54 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 55 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 56 or a conservative modification thereof. SEQ ID NOs: 54-56 are provided in Table 2.

[0125] In certain embodiments, the extracellular antigen-binding domain of the CAR (e.g., an scFv) comprises a VH comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 51 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 52 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 53 or a conservative modification thereof; and a VL comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 54 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 55 or a conservative modification, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 56 or a conservative modification thereof.

[0126] In certain embodiments, the extracellular antigen-binding domain of the CAR (e.g., an scFv) comprises a VH comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 51, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 52, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 53; and a VL comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 54, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 55, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 56.

[0127] In certain embodiments, the extracellular antigen-binding domain of the CAR (e.g., an scFv) comprises a VH comprising an amino acid sequence that is at least about 80% e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO: 57. For example, the extracellular antigenbinding domain of the CAR (e.g., an scFv) comprises a VH comprising 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: 57. In certain embodiments, the extracellular antigen-binding domain comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 57. SEQ ID NO: 57 is provided in Table 2 below.

[0128] In certain embodiments, the extracellular antigen-binding domain of the CAR (e.g., an scFv) comprises a VL comprising an amino acid sequence that is at least about 80% e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO: 58. For example, the extracellular antigenbinding domain of the CAR (e.g., an scFv) comprises a VL comprising 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: 58. In certain embodiments, the extracellular antigen-binding domain comprises a VL comprising the amino acid sequence set forth in SEQ ID NO: 58. SEQ ID NO: 58 is provided in Table 2 below.

[0129] In certain embodiments, the extracellular antigen-binding domain of the CAR (e.g., an scFv) comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 58, and a VL comprising the amino acid sequence set forth in SEQ ID NO: 58.

[0130] In certain embodiments, the VH and VL are linked via a linker. In certain embodiments, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. In certain embodiments, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1.

[0131] In certain embodiments, the variable regions within the extracellular antigen-binding domain of the CAR have to be linked one after another such that at the N-terminus of the extracellular antigen-binding domain, a heavy chain variable region (VH) is positioned. In certain embodiments, the extracellular antigen-binding domain of the CAR is an scFv, and the variable regions are positioned from the N- to the C-terminus: VH-VL.

[0132] In certain embodiments, the extracellular antigen-binding domain of the CAR (e.g., an scFv) comprises or consists of the amino acid sequence set forth in SEQ ID NO: 59 and specifically binds to mesothelin (e.g., human mesothelin). In certain, embodiments, the extracellular antigen-binding domain is a human scFv derived from a fully human anti-MSLN antibody m912. Various m912 scFvs are disclosed in International Patent Application Publication No. WO2015 / 188141, which is incorporated by reference hereby in its entirety. In certain embodiments, the scFv set forth in SEQ ID NO: 59 is designated as “M912 ” SEQ ID

[0133] NO: 59 is provided in Table 2 below.

[0134] Table 2

[0135] As used herein, the term “a conservative sequence modification” refers to an amino acid modification that does not significantly affect or alter the binding characteristics of the presently disclosed mesothelin-targeted CAR (e.g., the extracellular antigen-binding domain of the CAR) comprising the amino acid sequence. Conservative modifications can include amino acid substitutions, additions and deletions. Modifications can be introduced into the extracellular antigen-binding domain of the presently disclosed CAR by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Amino acids can be classified into groups according to their physicochemical properties such as charge and polarity. Conservative amino acid substitutions are ones in which the 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, histidine, negatively-charged amino acids include aspartic acid, glutamic acid, neutral charge amino acids include alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In addition, amino acids can be 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; non-polar amino acids include alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine. Thus, one or more amino acid residues within a CDR region can be replaced with other amino acid residues from the same group and the altered antibody can be tested for retained function (z.e., the functions set forth in (c) through (1) above) using the functional assays described herein. In certain embodiments, no more than one, no more than two, no more than three, no more than four, no more than five residues within a specified sequence or a CDR region are altered.

[0136] The VH and / or VL amino acid sequences having at least about 80%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% (e.g., about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) homology or identity to a specific sequence (e.g., SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 57, or SEQ ID NO: 58) may contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to the specified sequence(s), but retain the ability to bind to a target antigen (e.g., mesothelin). In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted and / or deleted in a specific sequence (e.g., SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 57, or SEQ ID NO: 58). In certain embodiments, substitutions, insertions, or deletions occur in regions outside the CDRs (e.g., in the FRs) of the extracellular antigen-binding domain. In certain embodiments, the extracellular antigen-binding domain comprises VH and / or VL sequence selected from SEQ ID NOs: 12, 13, 57, and 58, including post-translational modifications of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 57, or SEQ ID NO: 58.

[0137] In addition, the extracellular antigen-binding domain can comprise a leader or a signal peptide that directs the nascent protein into the endoplasmic reticulum. Signal peptide or leader can be essential if the CAR is to be glycosylated and anchored in the cell membrane. The signal sequence or leader can be a peptide sequence (about 5, about 10, about 15, about 20, about 25, or about 30 amino acids long) present at the N-terminus of newly synthesized proteins that directs their entry to the secretory pathway. In certain embodiments, the signal peptide is covalently joined to the 5’ terminus of the extracellular antigen-binding domain. In certain embodiments, the signal peptide comprises a CD8 polypeptide, e.g., the CAR comprises a truncated CD8 signal peptide.

[0138] 3,2.2. Transmembrane Domain of a CAR

[0139] In certain non-limiting embodiments, the transmembrane domain of the CAR comprises a hydrophobic alpha helix that spans at least a portion of the membrane. Different transmembrane domains result in different receptor stability. After antigen recognition, receptors cluster and a signal are transmitted to the cell. In accordance with the presently disclosed subject matter, the transmembrane domain of the CAR can comprise a native or modified transmembrane domain of CD8, CD28, CD3(^, CD4, 4-1BB, 0X40, ICOS, CD84, CD166, CD8a, CD8b, ICAM-1, CTLA-4, CD27, CD40, NKGD2, or a combination thereof.

[0140] In certain embodiments, the transmembrane domain of the CAR comprises a CD28 polypeptide (e.g., a transmembrane domain of CD28 or a portion thereof). In certain embodiments, the transmembrane domain of the CAR comprises a transmembrane domain of human CD28 or a portion thereof. In certain embodiments, the CD28 polypeptide comprises or consists of an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% homologous or identical to the sequence having a NCBI Reference No: NP_006130 (SEQ ID NO: 18), or a fragment thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. In certain embodiments, the CD28 polypeptide comprises or consists of an amino acid sequence that is a consecutive portion of SEQ ID NO: 18 which is at least 20, or at least 30, or at least 40, or at least 50, and up to 220 amino acids in length. Alternatively or additionally, in certain embodiments, the CD28 polypeptide comprises or consists of an amino acid sequence of amino acids 1 to 220, 1 to 50, 50 to 100, 100 to 150, 150 to 200, 153 to 179, or 200 to 220 of SEQ ID NO: 18. In certain embodiments, the transmembrane domain of the CAR comprises a CD28 polypeptide comprising or consisting of amino acids 153 to 179 of SEQ ID NO: 18. SEQ ID NO: 18 is provided below.

[0141] MLRLLLALNLFPS IQVTGNKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSA VEVCWYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDI YFCKIEVMYPPPYLD NEKSNGTI IHVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSLLVTVAFI I FWVRSKRSRL LHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS [ SEQ ID NO : 18 ]

[0142] An exemplary nucleotide sequence encoding the amino acids 153 to 179 of SEQ ID NO: 18 is set forth in SEQ ID NO: 19, which is provided below. TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGC CTTTATTATTTTCTGGGTG [ SEQ ID NO : 19 ]

[0143] In certain embodiments, the transmembrane domain of the CAR comprises a transmembrane domain of mouse CD28 or a portion thereof. In certain embodiments, the CD28 polypeptide comprises or consists of an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% homologous or identical to the sequence having a NCBI Reference No: NP 031668.3 (SEQ ID NO: 20), or a fragment thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. In certain embodiments, the CD28 polypeptide comprises or consists of an amino acid sequence that is a consecutive portion of SEQ ID NO: 20 which is at least 20, or at least 30, or at least 40, or at least 50, and up to 218 amino acids in length. Alternatively or additionally, in certain embodiments, the CD28 polypeptide comprises or consists of an amino acid sequence of amino acids 1 to 220, 1 to 50, 50 to 100, 100 to 150, 150 to 200, 151 to 177, or 200 to 218 of SEQ ID NO: 20. In certain embodiments, the transmembrane domain of the CAR comprises a CD28 polypeptide comprising or consisting of amino acids 151 to 177 of SEQ ID NO: 20. SEQ ID NO: 20 is provided below: MTLRLLFLALNFFSVQVTENKILVKQSPLLWDSNEVSLSCRYSYNLLAKEFRASLYKGVNS DVEVCVGNGNFTYQPQFRSNAEFNCDGDFDNETVTFRLWNLHVNHTDI YFCKIEFMYPPPYL DNERSNGTI IHIKEKHLCHTQSSPKLFWALVWAGVLFCYGLLVTVALCVIWTNSRRNRLLQ SDYMNMTPRRPGLTRKPYQPYAPARDFAAYRP [ SEQ ID NO : 20 ] In certain embodiments, the transmembrane domain of the CAR comprises a CD8 polypeptide (e.g., a transmembrane domain of CD8 or a portion thereof). In certain embodiments, the transmembrane domain of the CAR comprises a transmembrane domain of human CD8 or a portion thereof. In certain embodiments, the CD8 polypeptide comprises or consists of an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous or identical to the sequence having a NCBI Reference No: NP_001139345.1 (SEQ ID NO: 21) or a fragment thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. In certain embodiments, the CD8 polypeptide comprises or consists of an amino acid sequence that is a consecutive portion of SEQ ID NO: 21, which is at least 20, or at least 30, or at least 40, or at least 50, and up to 235 amino acids in length. Alternatively or additionally, in certain embodiments, the CD8 polypeptide comprises or consists of an amino acid sequence of amino acids 1 to 235, 1 to 50, 50 to 100, 100 to 150, 150 to 200, 137 to 209 or 200 to 235 of SEQ ID NO: 21. In certain embodiments, the transmembrane domain of the CAR comprises a CD8 polypeptide comprising or consisting of amino acids 137 to 209 of SEQ ID NO: 21. SEQ ID NO: 21 is provided below.

[0144] MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVELKCQVLLSNPTSGCSWLFQPRG AAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNS IMY FSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIW APLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPWKSGDKPSLSARYV [ SEQ ID NO : 21 ]

[0145] In certain embodiments, the transmembrane domain of the CAR comprises a transmembrane domain of mouse CD8 or a portion thereof. In certain embodiments, the CD8 polypeptide comprises or consists of an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous or identical to the sequence having a NCBI Reference No: AAA92533.1 (SEQ ID NO: 22) or a fragment thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. In certain embodiments, the CD8 polypeptide comprises or consists of an amino acid sequence that is a consecutive portion of SEQ ID NO: 22, which 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. Alternatively or additionally, in certain embodiments, the CD8 polypeptide comprises or consists of an amino acid sequence of amino acids 1 to 247, 1 to 50, 50 to 100, 100 to 150, 150 to 200, 151 to 219, or 200 to 247 of SEQ ID NO: 22. In certain embodiments, the transmembrane domain of the CAR comprises a CD8 polypeptide comprising or consisting of amino acids 151 to 219 of SEQ ID NO: 22. SEQ ID NO: 22 is provided below.

[0146] MASPLTRFLSLNLLLMGES I ILGSGEAKPQAPELRI FPKKMDAELGQKVDLVCEVLGSVSQG CSWLFQNSSSKLPQPTFWYMASSHNKITWDEKLNSSKLFSAVRDTNNKYVLTLNKFSKENE GYYFCSVISNSVMYFSSWPVLQKVNSTTTKPVLRTPSPVHPTGTSQPQRPEDCRPRGSVKG TGLDFACDI YIWAPLAGICVAPLLSLI ITLICYHRSRKRVCKCPRPLVRQEGKPRPSEKIV [ SEQ ID NO : 22 ]

[0147] In certain non-limiting 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 orient in different directions to facilitate antigen recognition while preserving the activating activity of the CAR.

[0148] In certain embodiments, the hinge / spacer region of the CAR comprises a native or modified hinge region of CD8, CD28, CD3^, CD40, 4-1BB, 0X40, CD84, CD166, CD8a, CD8b, ICOS, ICAM-1, CTLA-4, CD27, CD40, NKGD2, a synthetic polypeptide (not based on a protein associated with the immune response), or a combination thereof. The hinge / spacer region can be the hinge region from IgGl, or the CH2CH3 region of immunoglobulin and portions of CD3, a portion of a CD28 polypeptide (e.g., a portion of SEQ ID NO: 18 or SEQ ID NO: 20), a portion of a CD8 polypeptide (e.g., a portion of SEQ ID NO: 21 or SEQ ID NO: 22), a variation of any of the foregoing which is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% homologous or identical thereto, or a synthetic spacer sequence.

[0149] In certain embodiments, the hinge domain of the CAR comprises a native or modified hinge region of CD28. In certain embodiments, the hinge domain of the CAR comprises a native hinge region of CD28. In certain embodiments, the hinge domain of the CAR comprises the amino acid sequence of amino acids 114 to 152 of SEQ ID NO: 18.

[0150] 3,2.3. Intracellular Signaling Domain of a CAR

[0151] In certain embodiments, the CAR comprises an intracellular signaling domain. In certain non-limiting embodiments, the intracellular signaling domain of the CAR comprises a CD3^ polypeptide. CD3^ can activate or stimulate a cell (e.g., a cell of the lymphoid lineage, e.g., a T-cell). Wild type (“native”) CD3(^ comprises three functional immunoreceptor tyrosine-based activation motifs (ITAMs), three functional basic-rich stretch (BRS) regions (BRS1, BRS2 and BRS3). CD3(^ transmits an activation signal to the cell (e.g., a cell of the lymphoid lineage, e.g., a T-cell) after antigen is bound. The intracellular signaling domain of the CD3^-chain is the primary transmitter of signals from endogenous TCRs.

[0152] In certain embodiments, the intracellular signaling domain of the CAR comprises a native CD3(^. In certain embodiments, the CD3(^ polypeptide comprises or consists of an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous or identical to the sequence having a NCBI Reference No: NP_932170 (SEQ ID NO: 23), or a fragment thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. In certain embodiments, the CD3(^ polypeptide comprises or consists of an amino acid sequence that is a consecutive portion of SEQ ID NO: 23, which is at least 20, or at least 30, or at least 40, or at least 50, and up to 164 amino acids in length. Alternatively or additionally, in non-limiting various embodiments, the CD3(^ polypeptide comprises or consists of an amino acid sequence of amino acids 1 to 164, 1 to 50, 50 to 100, 52 to 164, 100 to 150, or 150 to 164 of SEQ ID NO: 23. In certain embodiments, the intracellular signaling domain of the CAR comprises a CD3(^ polypeptide comprising or consisting of amino acids 52 to 164 of SEQ ID NO: 23. SEQ ID NO: 23 is provided below: MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAP AYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYS EIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR [ SEQ ID NO : 23 ]

[0153] In certain embodiments, the intracellular signaling domain of the CAR comprises a CD3 polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 24. SEQ ID NO: 24 is provided below.

[0154] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNEL QKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR [ SEQ ID NO : 24 ]

[0155] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 24 is set forth in SEQ ID NO: 25, which is as provided below. AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTA TAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGG ACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTG CAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGG CAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCC TTCACATGCAGGCCCTGCCCCCTCGC [ SEQ ID NO : 25 ] In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3^ polypeptide. In certain embodiments, the modified CD3(^ polypeptide comprises one, two or three ITAMs. In certain embodiments, the modified CD3(^ polypeptide comprises a native ITAM1. In certain embodiments, the native IT AMI comprises or consists of the amino acid sequence set forth in SEQ ID NO: 29.

[0156] QNQLYNELNLGRREEYDVLDKR [ SEQ ID NO : 29 ]

[0157] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 29 is set forth in SEQ ID NO: 30, which is provided below. CAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAA GAGA [ SEQ ID NO : 30 ]

[0158] In certain embodiments, the modified CD3(^ polypeptide comprises an ITAM1 variant comprising one or more loss-of-function mutations. In certain embodiments, the ITAM1 variant comprises or consists of two loss-of-function mutations. In certain embodiments, each of the one or more (e.g., two) loss of function mutations comprises a mutation of a tyrosine residue in IT AMI . In certain embodiments, the IT AMI variant consists of two loss-of-function mutations. In certain embodiments, the IT AMI variant comprises or consists of the amino acid sequence set forth in SEQ ID NO: 31, which is provided below. QNQLFNELNLGRREEFDVLDKR [ SEQ ID NO : 31 ]

[0159] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 31 is set forth in SEQ ID NO: 32, which is provided below. CAGAACCAGCTCTTTAACGAGCTCAATCTAGGACGAAGAGAGGAGTTCGATGTTTTGGACAA GAGA [ SEQ ID NO : 32 ]

[0160] In certain embodiments, the modified CD3(^ polypeptide comprises a native ITAM2. In certain embodiments, the native ITAM2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 33, which is provided below.

[0161] QEGLYNELQKDKMAEAYSEIGMK [ SEQ ID NO : 33 ]

[0162] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 33 is set forth in SEQ ID NO: 34, which is provided below. CAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGG GATGAAA [ SEQ ID NO : 34 ]

[0163] In certain embodiments, the modified CD3(^ polypeptide comprises an ITAM2 variant. In certain embodiments, the ITAM2 variant comprises or consists of one or more loss-of- function mutations. In certain embodiments, the ITAM2 variant comprises or consists of two loss-of-function mutations. In certain embodiments, each of the one or more (e.g., two) the loss of function mutations comprises a mutation of a tyrosine residue in ITAM2. In certain embodiments, the ITAM1 variant consists of two loss-of-function mutations. In certain embodiments, the ITAM2 variant comprises or consists of the amino acid sequence set forth in SEQ ID NO: 35, which is provided below.

[0164] QEGLFNELQKDKMAEAFSEIGMK [ SEQ ID NO : 35 ]

[0165] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 35 is set forth in SEQ ID NO: 36, which is provided below.

[0166] CAGGAAGGCCTGTTCAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTTCAGTGAGATTGG GATGAAA [ SEQ ID NO : 36 ]

[0167] In certain embodiments, the modified CD3(^ polypeptide comprises a native ITAM3. In certain embodiments, the native ITAM3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 37, which is provided below.

[0168] HDGLYQGLSTATKDTYDALHMQ [ SEQ ID NO : 37 ]

[0169] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 37 is set forth in SEQ ID NO: 38, which is provided below.

[0170] CACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACAT

[0171] GCAG [ SEQ ID NO : 38 ]

[0172] In certain embodiments, the modified CD3(^ polypeptide comprises an ITAM3 variant. In certain embodiments, the ITAM3 variant comprises or consists of two loss-of-function mutations. In certain embodiments, each of the one or more (e.g., two) the loss of function mutations comprises a mutation of a tyrosine residue in ITAM3. In certain embodiments, the ITAM3 variant comprises or consists of two loss-of-function mutations. In certain embodiments, the ITAM3 variant comprises or consists of the amino acid sequence set forth in SEQ ID NO: 39, which is provided below.

[0173] HDGLFQGLSTATKDTFDALHMQ [ SEQ ID NO : 39 ]

[0174] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 39 is set forth in SEQ ID NO: 40, which is provided below.

[0175] CACGATGGCCTTTTCCAGGGGCTCAGTACAGCCACCAAGGACACCTTCGACGCCCTTCACAT

[0176] GCAG [ SEQ ID NO : 40 ]

[0177] Various modified CD3(^ polypeptides and CARs comprising modified CD3(^ polypeptides are disclosed in International Patent Application Publication No. WO2019 / 133969, which is incorporated by reference hereby in its entirety.

[0178] In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3(^ polypeptide comprising a native ITAM1, an ITAM2 variant comprising or consisting of one or more (e.g., two) loss-of-function mutations, and an ITAM3 variant comprising or consisting of one or more (e.g., two) loss-of-function mutations. In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3(^ polypeptide comprising a native IT AMI, an ITAM2 variant consisting of two loss-of-function mutations, and an ITAM3 variant consisting of two loss-of-function mutations. In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3(^ polypeptide comprising a native ITAM1 consisting of the amino acid sequence set forth in SEQ ID NO: 29, an ITAM2 variant consisting of the amino acid sequence set forth in SEQ ID NO: 35, and an ITAM3 variant consisting of the amino acid sequence set forth in SEQ ID NO: 39. In certain embodiments, the modified CD3(^ polypeptide is designated as “1XX”. In certain embodiments, the modified CD3(^ polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 41. SEQ ID NO: 41 is provided below.

[0179] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLFNEL QKDKMAEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTFDALHMQALPPR [ SEQ ID NO : 41 ]

[0180] In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3(^ polypeptide comprising or consisting 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% identical to SEQ ID NO: 162 or a fragment thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions.

[0181] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 41 is set forth in SEQ ID NO: 42, which is provided below.

[0182] AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTA TAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGG ACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTTCAATGAACTG CAGAAAGATAAGATGGCGGAGGCCTTCAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGG CAAGGGGCACGATGGCCTTTTCCAGGGGCTCAGTACAGCCACCAAGGACACCTTCGACGCCC TTCACATGCAGGCCCTGCCCCCTCGC [ SEQ ID NO : 42 ]

[0183] In certain non-limiting embodiments, the intracellular signaling domain of the CAR further comprises at least one co-stimulatory signaling region. In certain embodiments, the at least one co-stimulatory region comprises a co-stimulatory molecule or a portion thereof. In certain embodiments, the at least one co-stimulatory region comprises an intracellular domain of at least one co-stimulatory molecule or a portion thereof. As used herein, a “co-stimulatory molecule” refers to a cell surface molecule other than antigen receptor or its ligand that can provide an efficient response of lymphocytes to an antigen. In certain embodiments, a co-stimulatory molecule can provide optimal lymphocyte activation. Non-limiting examples of co-stimulatory molecules include CD28, 4-1BB, 0X40, ICOS, and DAP-10, and combinations thereof. The co-stimulatory molecule can bind to a co- stimulatory ligand, which is a protein expressed on cell surface that upon binding to its receptor produces a co-stimulatory response, z.e., an intracellular response that effects the stimulation provided when an antigen-recognizing receptor (e.g., a chimeric antigen receptor (CAR)) binds to its target antigen. As one example, a 4-1BB ligand (z.e., 4-1BBL) may bind to 4-1BB for providing an intracellular signal that in combination with a CAR signal induces an effector cell function of the CAR+T-cell.

[0184] In certain embodiments, the intracellular signaling domain of the CAR comprises a co- stimulatory signaling region that comprises a CD28 polypeptide, e.g., an intracellular domain of CD28 or a portion thereof. In certain embodiments, the intracellular signaling domain of the CAR comprises a co-stimulatory signaling region that comprises an intracellular domain of human CD28 or a portion thereof. In certain embodiments, the intracellular signaling domain of the CAR comprises a co-stimulatory signaling region comprising a CD28 polypeptide that comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 18, or a fragment thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. In certain embodiments, the CD28 polypeptide comprised in the co-stimulatory signaling region comprises or consists of an amino acid sequence that is a consecutive portion of SEQ ID NO: 18, which is at least 20, or at least 30, or at least 40, or at least 50, and up to 220 amino acids in length. Alternatively or additionally, in non-limiting various embodiments, the intracellular signaling domain of the CAR comprises a co-stimulatory signaling region comprising a CD28 polypeptide that comprises or consists of amino acids 1 to 220, 1 to 50, 50 to 100, 100 to 150, 114 to 220, 150 to 200, 180 to 220, or 200 to 220 of SEQ ID NO: 18. In certain embodiments, the intracellular signaling domain of the CAR comprises a co-stimulatory signaling region that comprises a CD28 polypeptide comprising or consisting of amino acids 180 to 220 of SEQ ID NO: 18.

[0185] An exemplary nucleotide sequence encoding amino acids 180 to 220 of SEQ ID NO: 18 is set forth in SEQ ID NO: 26, which is provided below. AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGG GCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC [ SEQ ID NO : 26 ]

[0186] In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region comprising a CD28 polypeptide that comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 20, or a fragment thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. In certain embodiments, the intracellular signaling domain of the CAR comprises a co-stimulatory signaling region comprising a CD28 polypeptide that comprises or consists of an amino acid sequence that is a consecutive portion of SEQ ID NO: 20 which is at least about 20, or at least about 30, or at least about 40, or at least about 50, and up to 218 amino acids in length. Alternatively or additionally, in nonlimiting various embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region comprising a CD28 polypeptide that comprises or consists of amino acids 1 to 218, 1 to 50, 50 to 100, 100 to 150, 150 to 218, 178 to 218, or 200 to 218 of SEQ ID NO: 20. In certain embodiments, the co-stimulatory signaling region of a presently disclosed CAR comprises a CD28 polypeptide that comprises or consists of amino acids 178 to 218 of SEQ ID NO: 20.

[0187] In certain embodiments, the intracellular signaling domain of the CAR comprises a co- stimulatory signaling region that comprises a 4-1BB polypeptide, e.g., an intracellular domain of 4- IBB or a portion thereof. In certain embodiments, the 4- IBB polypeptide comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to the sequence having a NCBI Ref. No.: NP_001552 (SEQ ID NO: 27) or a fragment thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. In certain embodiments, the 4-1BB polypeptide comprises or consists of an amino acid sequence that is a consecutive portion of SEQ ID NO: 25, which 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. Alternatively or additionally, in certain embodiments, the 4- IBB polypeptide comprises or consists of an amino acid sequence of amino acids 1 to 255, 1 to 50, 50 to 100, 100 to 150, 150 to 200, or 200 to 255 of SEQ ID NO: 27. In certain embodiments, the intracellular signaling domain of the CAR comprises a co-stimulatory signaling region that comprises a 4- IBB polypeptide comprising or consisting of amino acids 214 to 255 of SEQ ID NO: 27. SEQ ID NO: 27 is provided below.

[0188] MGNSCYNIVATLLLVLNFERTRSLQDPCSNCPAGTFCDNNRNQICSPCPPNS FSSAGGQRTC DICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGCKDCCFGT FNDQKRG I CRPWTNCS LDGKS VLVNGTKERDWCGPS PADLS PGAS S VT PPAPARE PGHS PQ I I S FFLALTSTALLFLLFFLTLRFSWKRGRKKLLYI FKQPFMRPVQTTQEEDGCSCRFPEE EEGGCEL [ SEQ ID NO : 27 ]

[0189] An exemplary nucleotide sequence encoding amino acids 214 to 255 of SEQ ID NO: 27 is set forth in SEQ ID NO: 28, which is provided below.

[0190] AAAC G G G G C AGAAAGAAAC TCCTGTATATATT C AAAC AAC C AT T T AT GAGAC GAG T AC AAAC TACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAAC TG [ SEQ ID NO : 28 ]

[0191] In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region that comprises intracellular domains of two or more costimulatory molecules or portions thereof, e.g., intracellular domains of CD28 and 4-1BB, or intracellular domains of CD28 and 0X40.

[0192] 3,2.4. Exemplified CARs

[0193] In certain embodiments, the CAR is designated as “mM28z”. In certain embodiments the CAR designated as “mM28z” comprises (a) an extracellular antigen-binding domain comprising (i) a VH that comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 6, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8, and (ii) a VL that comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 9, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11; (b) a transmembrane domain comprising a CD28 polypeptide (e.g., a transmembrane domain of human CD28 or a portion thereof), and (c) an intracellular signaling domain comprising (i) a CD3(^ polypeptide, and (ii) a costimulatory signaling region comprising a CD28 polypeptide (e.g., an intracellular domain of human CD28 or a portion thereof ). In certain embodiments, the VH and VL are linked via a linker consisting of the amino acid sequence set forth in SEQ ID NO: 1.

[0194] In certain embodiments, the CAR is designated as “mMz”. In certain embodiments the CAR designated as “mMz” comprises (a) an extracellular antigen-binding domain comprising (i) a VH that comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 6, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8, and (ii) a VL that comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 9, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11; (b) a transmembrane domain comprising a CD8 polypeptide (e.g., a transmembrane domain of human CD8 or a portion thereof), and (c) an intracellular signaling domain comprising (i) a CD3(^ polypeptide. In certain embodiments, the VH and VL are linked via a linker consisting of the amino acid sequence set forth in SEQ ID NO: 1.

[0195] In certain embodiments, the CAR is designated as “mMBBz” In certain embodiments the CAR designated as “mMBBz” comprises (a) an extracellular antigen-binding domain comprising (i) a VH that comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 6, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8, and (ii) a VL that comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 9, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11; (b) a transmembrane domain comprising a CD8 polypeptide (e.g., a transmembrane domain of human CD8 or a portion thereof), and (c) an intracellular signaling domain comprising (i) a CD3(^ polypeptide, and (ii) a co-stimulatory signaling region comprising a 4-1BB polypeptide (e.g., an intracellular domain of human 4- 1BB or a portion thereof). In certain embodiments, the VH and VL are linked via a linker consisting of the amino acid sequence set forth in SEQ ID NO: 1.

[0196] In certain embodiments, the CAR is designated as “M-mM28z”. In certain embodiments, the CAR designated as “M-mM28z” comprises (a) a first extracellular antigenbinding domain comprising (i) a VH that comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 51, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 52, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 53, and (ii) a VL that comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 54, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 55, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 56; (b) a second extracellular antigen-binding domain comprising (i) a VH that comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 6, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8, and (ii) a VL that comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 9, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11; (c) a transmembrane domain comprising a CD28 polypeptide (e.g., a transmembrane domain of human CD28 or a portion thereof), and (d) an intracellular signaling domain comprising (i) a CD3(^ polypeptide, and (ii) a co-stimulatory signaling region comprising a CD28 polypeptide (e.g., an intracellular domain of human CD28 or a portion thereof). In certain embodiments, the first extracellular antigen-binding domains is a single-chain variable fragment (scFv). In certain embodiments, the second extracellular antigen-binding domains is a single-chain variable fragment (scFv).

[0197] In certain embodiments, the CAR is designated as “mM-M28z”. In certain embodiments, the CAR designated as “mM-M28z”comprises (a) a first extracellular antigenbinding domain comprising (i) a VH that comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 6, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8, and (ii) a VL that comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 9, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11; (b) a second extracellular antigen-binding domain comprising (i) a VH that comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 51, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 52, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 53, and (ii) a VL that comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 54, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 55, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 56; (c) a transmembrane domain comprising a CD28 polypeptide (e.g., a transmembrane domain of human CD28 or a portion thereof), and (d) an intracellular signaling domain comprising (i) a CD3(^ polypeptide, and (ii) a co-stimulatory signaling region comprising a CD28 polypeptide (e.g., an intracellular domain of human CD28 or a portion thereof ). In certain embodiments, the first extracellular antigen-binding domain is a single-chain variable fragment (scFv). In certain embodiments, the second extracellular antigen-binding domain is a single-chain variable fragment (scFv).

[0198] In certain embodiments, the CAR is designated as “MZ”. In certain embodiments, the CAR designated as “MZ” comprises (a) an extracellular antigen-binding domain comprising (i) a VH that comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 51, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 52, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 53, and (ii) a VL that comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 54, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 55, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 56; (b) a transmembrane domain comprising a CD8 polypeptide (e.g., a transmembrane domain of human CD8 or a portion thereof), and (d) an intracellular signaling domain comprising (i) a CD3(^ polypeptide.

[0199] In certain embodiments, the CAR is designated as “M28z”. In certain embodiments, the CAR designated as “M28z” comprises (a) an extracellular antigen-binding domain comprising (i) a VH that comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 51, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 52, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 53, and (ii) a VL that comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 54, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 55, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 56; (b) a transmembrane domain comprising a CD28 polypeptide (e.g., a transmembrane domain of human CD28 or a portion thereof), and (d) an intracellular signaling domain comprising (i) a CD3(^ polypeptide.

[0200] In certain embodiments, a presently disclosed CAR further comprises an inducible promoter (e.g., TRE), for expressing nucleotide sequences in human cells. Promoters for use in expressing CAR genes can be a constitutive promoter, such as ubiquitin C (UbiC) promoter.

[0201] 3.3. TCR like Fusion Molecules

[0202] In certain embodiments, the antigen-recognizing receptor is a TCR like fusion molecule. Non-limiting examples of TCR fusion molecules include HL A-Indep endent TCR-based Chimeric Antigen Receptor (also known as “HIT-CAR”, e.g., those disclosed in International Patent Application No. PCT / US19 / 017525, which is incorporated by reference in its entirety), and T cell receptor fusion constructs (TRuCs) (e.g., those disclosed in Baeuerle et al., “Synthetic TRuC receptors engaging the complete T cell receptor for potent anti -turn or response,” Nature Communications volume 10, Article number: 2087 (2019), which is incorporated by reference in its entirety).

[0203] In certain embodiments, the TCR like fusion molecule comprises an antigen binding chain that comprises an extracellular antigen-binding domain and a constant domain, wherein the TCR like fusion molecule binds to an antigen in an HLA-independent manner. In certain embodiments, the constant domain comprises a T cell receptor constant region selected from the group consisting of a native or modified TRAC peptide, a native or modified TRBC peptide, a native or modified TRDC peptide, a native or modified TRGC peptide and any variants or functional fragments thereof. In certain embodiments, the constant domain comprises a native or modified TRAC peptide. In certain embodiments, the constant domain comprises a native or modified TRBC peptide. In certain embodiments, the constant domain is capable of forming a homodimer or a heterodimer with another constant domain. In certain embodiments, the antigen binding chain is capable of associating with a CD3(^ polypeptide. In certain embodiments, the antigen binding chain, upon binding to an antigen, is capable of activating the CD3(^ polypeptide associated to the antigen binding chain. In certain embodiments, the activation of the CD3(^ polypeptide is capable of activating an immunoresponsive cell. In certain embodiments, the TCR like fusion molecule is capable of integrating with a CD3 complex and providing HLA-independent antigen recognition. In certain embodiments, the TCR like fusion molecule replaces an endogenous TCR in a CD3 / TCR complex. In certain embodiments, the extracellular antigen-binding domain of the TCR like fusion molecule is capable of dimerizing with another extracellular antigen-binding domain. In certain embodiments, the extracellular antigen-binding domain of the TCR like fusion molecule comprises a ligand for a cell-surface receptor, a receptor for a cell surface ligand, an antigen binding portion of an antibody or a fragment thereof or an antigen binding portion of a TCR. In certain embodiments, the extracellular antigen-binding domain of the TCR like fusion molecule comprises one or two immunoglobulin variable region(s). In certain embodiments, the extracellular antigen-binding domain of the TCR like fusion molecule comprises a heavy chain variable region (VH) of an antibody. In certain embodiments, the extracellular antigenbinding domain of the TCR like fusion molecule comprises a light chain variable region (VL) of an antibody. In certain embodiments, the extracellular antigen-binding domain of the TCR like fusion molecule is capable of dimerizing with another extracellular antigen-binding domain. In certain embodiments, the extracellular antigen-binding domain of the TCR like fusion molecule comprises a VH of an antibody, wherein the VH is capable of dimerizing with another extracellular antigen-binding domain comprising a VL of the antibody and form a fragment variable (Fv). In certain embodiments, the extracellular antigen-binding domain of the TCR like fusion molecule comprises a VL of an antibody, wherein the VL is capable of dimerizing with another extracellular antigen-binding domain comprising a VH of the antibody and form a fragment variable (Fv).

[0204] The TCR like fusion molecule can bind to a tumor antigen or a pathogen antigen. In certain embodiments, the TCR like fusion molecule binds to a tumor antigen. 4. Cells

[0205] The presently disclosed subject matter provides cells comprising a presently disclosed mesothelin-targeted antigen-recognizing receptor (e.g., one disclosed in Section 3). In certain embodiments, the cell is selected from the group consisting of cells of lymphoid lineage and cells of myeloid lineage. In certain embodiments, the cell is an immunoresponsive cell. In certain embodiments, the immunoresponsive cell is a cell of lymphoid lineage.

[0206] In certain embodiments, the cell is a cell of the lymphoid lineage. Cells of the lymphoid lineage can provide production of antibodies, regulation of cellular immune system, detection of foreign agents in the blood, detection of cells foreign to the host, and the like. Non-limiting examples of cells of the lymphoid lineage include T-cells, Natural Killer (NK) cells, B cells, dendritic cells, stem cells from which lymphoid cells may be differentiated. In certain embodiments, the stem cell is a pluripotent stem cell (e.g., embryonic stem cell).

[0207] In certain embodiments, the cell is a T-cell. T-cells can be lymphocytes that mature in the thymus and are chiefly responsible for cell-mediated immunity. T-cells are involved in the adaptive immune system. The T-cells of the presently disclosed subject matter can be any type of T-cells, including, but not limited to, helper T-cells, cytotoxic T-cells, memory T-cells (including central memory T-cells, stem-cell-like memory T-cells (or stem-like memory T- cells), and two types of effector memory T-cells: e.g., TEM cells and TEMRA cells, Regulatory T-cells (also known as suppressor T-cells), tumor-infiltrating lymphocyte (TIL), Natural killer T-cells, Mucosal associated invariant T-cells, and y5 T-cells. Cytotoxic T-cells (CTL or killer T-cells) are a subset of T lymphocytes capable of inducing the death of infected somatic or tumor cells. A patient’s own T-cells may be genetically modified to target specific antigens through the introduction of an antigen-recognizing receptor, e.g., a CAR or a TCR. In certain embodiments, the immunoresponsive cell is a T-cell. The T-cell can be a CD4+T-cell or a CD8+T-cell. In certain embodiments, the T-cell is a CD4+T-cell. In certain embodiments, the T-cell is a CD8+T-cell.

[0208] In certain embodiments, the cell is a NK cell. Natural killer (NK) cells can be lymphocytes that are part of cell-mediated immunity and act during the innate immune response. NK cells do not require prior activation in order to perform their cytotoxic effect on target cells.

[0209] Types of human lymphocytes of the presently disclosed subject matter include, without limitation, peripheral donor lymphocytes, e.g., those disclosed in Sadelain et al., Nat Rev Cancer (2003); 3:35-45 (disclosing peripheral donor lymphocytes genetically modified to express CARs), in Morgan, R. A., et al. 2006 Science 314: 126-129 (disclosing peripheral donor lymphocytes genetically modified to express a full-length tumor antigen-recognizing T-cell receptor complex comprising the a and P heterodimer), in 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 in Dupont et al., Cancer Res (2005);65:5417-5427; Papanicolaou et al., Blood (2003); 102:2498-2505 (disclosing selectively in vztro-expanded antigen-specific peripheral blood leukocytes employing artificial antigen-presenting cells (AAPCs) or pulsed dendritic cells).

[0210] The cells (e.g., T-cells) can be autologous, non-autologous (e.g., allogeneic), or derived in vitro from engineered progenitor or stem cells.

[0211] The cells of the presently disclosed subject matter can be cells of the myeloid lineage. Non-limiting examples of cells of the myeloid lineage include monocytes, macrophages, neutrophils, dendritic cells, basophils, neutrophils, eosinophils, megakaryocytes, mast cell, erythrocyte, thrombocytes, and stem cells from which myeloid cells may be differentiated. In certain embodiments, the stem cell is a pluripotent stem cell (e.g., an embryonic stem cell or an induced pluripotent stem cell).

[0212] In certain embodiments, the cells can be transduced with the presently disclosed mesothelin-targeted antigen-recognizing receptors such that the cells express the antigenrecognizing receptor (e.g., disclosed in Section 3.2.4).

[0213] In certain embodiments, the presently disclosed cells can be further transduced with a c-Kit mutant (e.g., cKIT D816V). c-Kit, known as CD117, is a cytokine receptor expressed on the surface of hematopoietic stem cells as well as other cell types. Signaling through c-Kit plays a role in cell survival, proliferation and differentiation (Ceredig et al., Nat Rev Immunol (2002); 2(11):888-97). c-Kit binds to stem cell factor (SCF). Upon binding, c-Kit and SCF form a dimer that activates its intrinsic tyrosine kinase activity and in turn phosphorylates and activates signal transduction molecules that propagate the signal in the cell. A presently disclosed c-Kit activating mutation (e.g., D816V mutation) results in constitutive activation absent SCF, e.g., without forming the cKit / SCF dimers (Hirota et al., Science (1998);279(5350):577-80; Kitamura et al., Mut Res (2001): 165-71).

[0214] Non-limiting examples of c-Kit mutants are disclosed in International Patent Application Publication No. WO 2021 / 113432, which is incorporated by reference hereby in its entirety.

[0215] In certain embodiments, the c-Kit mutant 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% identical to the amino acid sequence set forth in SEQ ID NO: 61 or a portion thereof. In certain embodiments, the c-Kit mutant comprises or consists of the amino acid sequence set forth in SEQ ID NO: 61 or a portion thereof. In certain embodiments, the c-Kit mutant comprises or consists of an amino acid sequence that is a consecutive portion of SEQ ID NO: 61, which is at least 50, or at least 100, or at least 150, or at least 200, or at least 250, or at least 300, or at least 350, or at least 400, or at least 450, or at least 500, or at least 550, or at least 600, or at least 650, or at least 700, or at least 750, or at least 800, or at least 850, or at least 900, or at least 950, and up to 976 amino acids in length. Alternatively or additionally, in nonlimiting various embodiments, the c-Kit mutant comprises or consists of an amino acid sequence of amino acids 1 to 976, 1 to 200, 400 to 976, 500 to 976, or 543 to 976 of SEQ ID NO: 61. In certain embodiments, the c-Kit mutant comprises or consists of amino acids 543 to 976 of SEQ ID NO: 61. SEQ ID NO: 61 is provided below.

[0216] MRGARGAWDFLCVLLLLLRVQTGSSQPSVSPGEPSPPS IHPGKSDLIVRVGDEIRLLCTDPG FVKWTFEILDETNENKQNEWITEKAEATNTGKYTCTNKHGLSNS IYVFVRDPAKLFLVDRSL YGKEDNDTLVRCPLTDPEVTNYSLKGCQGKPLPKDLRFIPDPKAGIMIKSVKRAYHRLCLHC SVDQEGKSVLSEKFILKVRPAFKAVPWSVSKASYLLREGEEFTVTCTIKDVSSSVYSTWKR ENSQTKLQEKYNSWHHGDFNYERQATLTISSARVNDSGVFMCYANNTFGSANVTTTLEWDK GFINI FPMINTTVFVNDGENVDLIVEYEAFPKPEHQQWI YMNRTFTDKWEDYPKSENESNIR YVSELHLTRLKGTEGGTYTFLVSNSDVNAAIAFNVYVNTKPEILTYDRLVNGMLQCVAAGFP EPTIDWYFCPGTEQRCSASVLPVDVQTLNSSGPPFGKLWQSS IDSSAFKHNGTVECKAYND VGKTSAYFNFAFKGNNKEQIHPHTLFTPLLIGFVIVAGMMCI IVMILTYKYLQKPMYEVQWK WEEINGNNYVYIDPTQLPYDHKWEFPRNRLSFGKTLGAGAFGKWEATAYGLIKSDAAMTV AVKMLKPSAHLTEREALMSELKVLSYLGNHMNIVNLLGACTIGGPTLVITEYCCYGDLLNFL RRKRDSFICSKQEDHAEAALYKNLLHSKESSCSDSTNEYMDMKPGVSYWPTKADKRRSVRI GSYIERDVTPAIMEDDELALDLEDLLSFSYQVAKGMAFLASKNCIHRDLAARNILLTHGRIT KICDFGLARVIKNDSNYWKGNARLPVKWMAPES I FNCVYTFESDVWSYGI FLWELFSLGSS PYPGMPVDSKFYKMIKEGFRMLSPEHAPAEMYDIMKTCWDADPLKRPTFKQIVQLIEKQISE STNHI YSNLANCSPNRQKPWDHSVRINSVGSTASSSQPLLVHDDV [ SEQ ID NO : 61 ]

[0217] In certain embodiments, the presently disclosed cells can be further transduced with a dominant negative form of programmed death 1 (referred to as “PD-1 DN”).

[0218] The PD-1 DN can enhance the therapeutic efficacy of an immunoresponsive cell comprising a CAR. In certain embodiments, the PD-1 DN comprises (a) at least a portion of an extracellular domain of programmed death 1 (PD-1) comprising a ligand binding region, and (b) a transmembrane domain.

[0219] Malignant cells adapt to generate an immunosuppressive microenvironment that protects the cells from immune recognition and elimination (Sharpe et al., Dis. Model Meeh. 8:337-350 (2015)). The immunosuppressive microenvironment puts limitations on immunotherapy methods. Details of DN forms of inhibitors of a cell-mediated immune response are disclosed in W02017 / 040945 and W02017 / 100428, the contents of each of which are incorporated herein in their entireties.

[0220] In certain embodiments, the PD-1 DN comprises the amino acid sequence set forth in SEQ ID NO: 62, which is provided below.

[0221] MQIPQAPWPWWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLWTEGDNATFTCSFSNTSES FVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSWRARRNDSGTYLCG AISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQAAAPTTTPAPRPPTPAPTIAS QPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCN [ SEQ ID NO : 62 ]

[0222] In certain embodiments, the PD-1 DN comprises the amino acid sequence set forth in SEQ ID NO: 63, which is provided below.

[0223] MQIPQAPWPWWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLWTEGDNATFTCSFSNTSES FVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSWRARRNDSGTYLCG AISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQAAAPTTTPAPRPPTPAPTIAS QPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHR [ SEQ ID NO : 63 ]

[0224] 4.1. Exempli fied Cells

[0225] In certain embodiments, the cell can be transduced with a mesothelin-targeted antigenrecognizing receptor designated as “mM28z” (disclosed in Section 3.2.4) such that the cells express the antigen-recognizing receptor.

[0226] In certain embodiments, the cell can be transduced with a mesothelin-targeted antigenrecognizing receptor designated as “mMz” (disclosed in Section 3.2.4) such that the cells express the antigen-recognizing receptor.

[0227] In certain embodiments, the cell can be transduced with a mesothelin-targeted antigenrecognizing receptor designated as “mMBBz” (disclosed in Section 3.2.4) such that the cells express the antigen-recognizing receptor. In certain embodiments, the cell can be transduced with a mesothelin-targeted antigenrecognizing receptor designated as “M-mM28z” (disclosed in Section 3.2.4) such that the cells express the antigen-recognizing receptor.

[0228] In certain embodiments, the cell can be transduced with a mesothelin-targeted antigenrecognizing receptor designated as “mM-M28z” (disclosed in Section 3.2.4) such that the cells express the antigen-recognizing receptor.

[0229] In certain embodiments, the cell can be transduced with a first mesothelin-targeted antigen-recognizing receptor designated as “mM28z” (disclosed in Section 3.2.4) and a second mesothelin-targeted antigen-recognizing receptor designated as “MZ” (disclosed in Section 3.2.4) such that the cells express the antigen-recognizing receptor.

[0230] In certain embodiments, the cell comprises a mesothelin-targeted antigen-recognizing receptor and a cKit polypeptide. In certain embodiments, the mesothelin-targeted antigenrecognizing receptor is designated as “mM28z” (disclosed in Section 3.2.4). In certain embodiments, the cKit polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 61.

[0231] In certain embodiments, the cell comprises a mesothelin-targeted antigen-recognizing receptor and a cKit polypeptide. In certain embodiments, the mesothelin-targeted antigenrecognizing receptor is designated as “mMz” (disclosed in Section 3.2.4). In certain embodiments, the cKit polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 61.

[0232] In certain embodiments, the cell comprises a mesothelin-targeted antigen-recognizing receptor and a cKit polypeptide. In certain embodiments, the mesothelin-targeted antigenrecognizing receptor is designated as “mMBBz” (disclosed in Section 3.2.4). In certain embodiments, the cKit polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 61.

[0233] In certain embodiments, the cell comprises a mesothelin-targeted antigen-recognizing receptor and a cKit polypeptide. In certain embodiments, the mesothelin-targeted antigenrecognizing receptor is designated as “M-mM28z” (disclosed in Section 3.2.4). In certain embodiments, the cKit polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 61.

[0234] In certain embodiments, the cell comprises a mesothelin-targeted antigen-recognizing receptor and a cKit polypeptide. In certain embodiments, the mesothelin-targeted antigenrecognizing receptor is designated as “mM-M28z” (disclosed in Section 3.2.4). In certain embodiments, the cKit polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 61.

[0235] In certain embodiments, the cell comprises a mesothelin-targeted antigen-recognizing receptor, a cKit polypeptide, and a PD-1 DN polypeptide. In certain embodiments, the mesothelin-targeted antigen-recognizing receptor is designated as “mM28z” (disclosed in Section 3.2.4). In certain embodiments, the cKit polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 61. In certain embodiments, the PD-1 DN polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 62 or SEQ ID NO: 63.

[0236] In certain embodiments, the cell comprises a mesothelin-targeted antigen-recognizing receptor, a cKit polypeptide, and a PD-1 DN polypeptide. In certain embodiments, the mesothelin-targeted antigen-recognizing receptor is designated as “mMz” (disclosed in Section 3.2.4). In certain embodiments, the cKit polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 61. In certain embodiments, the PD-1 DN polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 62 or SEQ ID NO: 63.

[0237] In certain embodiments, the cell comprises a mesothelin-targeted antigen-recognizing receptor, a cKit polypeptide, and a PD-1 DN polypeptide. In certain embodiments, the mesothelin-targeted antigen-recognizing receptor is designated as “mMBBz” (disclosed in Section 3.2.4). In certain embodiments, the cKit polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 61. In certain embodiments, the PD-1 DN polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 62 or SEQ ID NO: 63.

[0238] In certain embodiments, the cell comprises a mesothelin-targeted antigen-recognizing receptor, a cKit polypeptide, and a PD-1 DN polypeptide. In certain embodiments, the mesothelin-targeted antigen-recognizing receptor is designated as “M-mM28z” (disclosed in Section 3.2.4). In certain embodiments, the cKit polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 61. In certain embodiments, the PD-1 DN polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 62 or SEQ ID NO: 63.

[0239] In certain embodiments, the cell comprises a mesothelin-targeted antigen-recognizing receptor, a cKit polypeptide, and a PD-1 DN polypeptide. In certain embodiments, the mesothelin-targeted antigen-recognizing receptor is designated as “mM-M28z” (disclosed in Section 3.2.4). In certain embodiments, the cKit polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 61. In certain embodiments, the PD-1 DN polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 62 or SEQ ID NO: 63.

[0240] In certain embodiments, the cell comprises a mesothelin-targeted antigen-recognizing receptor and a cKit polypeptide. In certain embodiments, the mesothelin-targeted antigen- recognizing receptor is designated as “MZ” (disclosed in Section 3.2.4). In certain embodiments, the cKit polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 61.

[0241] In certain embodiments, the cell comprises a first mesothelin-targeted antigenrecognizing receptor, a second mesothelin-targeted antigen-recognizing receptor, a cKit polypeptide. In certain embodiments, the first mesothelin-targeted antigen-recognizing receptor is designated as “mMZ” (disclosed in Section 3.2.4). In certain embodiments, the second mesothelin-targeted antigen-recognizing receptor is designated as “M28Z” (disclosed in Section 3.2.4). In certain embodiments, the cKit polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 61.

[0242] In certain embodiments, the cell comprises a first mesothelin-targeted antigenrecognizing receptor, a second mesothelin-targeted antigen-recognizing receptor, a cKit polypeptide, and a PD-1 DN polypeptide. In certain embodiments, the first mesothelin-targeted antigen-recognizing receptor is designated as “mMZ” (disclosed in Section 3.2.4). In certain embodiments, the second mesothelin-targeted antigen-recognizing receptor is designated as “M28Z” (disclosed in Section 3.2.4). In certain embodiments, the cKit polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 61. In certain embodiments, the PD-1 DN polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 62 or SEQ ID NO: 63.

[0243] 5. Compositions and Vectors

[0244] The presently disclosed subject matter provides compositions comprising a presently disclosed mesothelin-targeted antigen-recognizing receptor (e.g., one disclosed in Section 3). Also provided are cells comprising such compositions.

[0245] In certain embodiments, the presently disclosed mesothelin-targeted antigenrecognizing receptor is operably linked to a promoter.

[0246] Furthermore, the present discloses subject matter provides nucleic acid compositions comprising a polynucleotide encoding a presently disclosed mesothelin-targeted antigenrecognizing receptor (e.g., one disclosed in Section 3). Also provided are cells comprising such nucleic acid compositions.

[0247] In certain embodiments, the nucleic acid composition further comprises a promoter that is operably linked to the presently disclosed mesothelin-targeted antigen-recognizing receptor.

[0248] In certain embodiments, the promoter is endogenous or exogenous. In certain embodiments, the exogenous promoter is selected from an elongation factor (EF)-l promoter, a cytomegalovirus immediate-early promoter (CMV) promoter, a simian virus 40 early promoter (SV40) promoter, a phosphoglycerate kinase (PGK) promoter, and a metallothionein promoter. In certain embodiments, the promoter is an inducible promoter. In certain embodiment, the inducible promoter is selected from a NF AT transcriptional response element (TRE) promoter, a CD69 promoter, a CD25 promoter, and an IL-2 promoter.

[0249] In certain embodiments, the polynucleotide encoding a presently disclosed mesothelin- targeted antigen-recognizing receptor (e.g., one disclosed in Section 3) can include codons that are optimized for expression in a particular cell type or organism. These codon-optimized sequences are synthetic sequences that encode an identical polypeptide encoded by the non- codon-optimized parent polynucleotide. In certain embodiments, the coding region of the polynucleotide encoding a presently disclosed mesothelin-targeted antigen-recognizing receptor (e.g., one disclosed in Section 3) can include an altered sequence to optimize codon usage for a particular cell type (e.g., a eukaryotic). For example, the coding sequence for a human or humanized heavy (or light) chain variable region as described herein can be optimized for expression in human cells. The compositions and nucleic acid compositions can be administered to subjects or and / delivered into cells by art-known methods or as described herein. Genetic modification of a cell (e.g., a T-cell or a NK cell) can be accomplished by transducing a substantially homogeneous cell composition with a recombinant DNA construct. In certain embodiments, a retroviral vector (e.g., gammaretroviral vector or lentiviral vector) is employed for the introduction of the DNA construct into the cell. For example, a polynucleotide encoding an antigen-recognizing receptor can be cloned into a retroviral vector and expression can be driven from its endogenous promoter, from the retroviral long terminal repeat, or from a promoter specific for a target cell type of interest. Non-viral vectors may be used as well.

[0250] For initial genetic modification of a cell to include a presently disclosed mesothelin- targeted antigen-recognizing receptor (e.g., a CAR or a TCR), a retroviral vector can be employed for transduction, however any other suitable viral vector or non-viral delivery system can be used. The antigen-recognizing receptor can be constructed in a single, multi ci stronic expression cassette, in multiple expression cassettes of a single vector, or in multiple vectors. Examples of elements that create polycistronic expression cassette include, but is not limited to, various viral and non-viral Internal Ribosome Entry Sites (IRES, e.g., FGF-1 IRES, FGF-2 IRES, VEGF IRES, IGF-II IRES, NF-KB IRES, RUNX1 IRES, p53 IRES, hepatitis A IRES, hepatitis C IRES, pestivirus IRES, aphthovirus IRES, picomavirus IRES, poliovirus IRES and encephalomyocarditis virus IRES) and cleavable linkers (e.g., 2A peptides , e.g., P2A, T2A, E2A and F2A peptides). Combinations of retroviral vector and an appropriate packaging line are also suitable, where the capsid proteins 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 etal., Proc Natl Acad Sci USA (1988);85:6460-6464). Non-amphotropic particles are suitable too, e.g., particles pseudotyped with VSVG, RD114 or GALV envelope and any other known in the art.

[0251] Possible methods of transduction also include direct co-culture of the cells with producer cells (Bregni et al., Blood (1992);80: 1418-1422), or culturing with viral supernatant alone or concentrated vector stocks with or without appropriate growth factors and polycations(Xu etal., Exp Hemat (1994); 22: 223 -230; and Hughes et al. J Chn Invest (1992),' 89: 1817).

[0252] Other transducing viral vectors can be used to modify a cell. In certain embodiments, the chosen vector exhibits high efficiency of infection 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, adenoviral, lentiviral, and adena-associated viral vectors, vaccinia virus, a bovine papilloma virus, or a herpes virus, such as Epstein-Barr Virus (also see, for example, the vectors of 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; Cometta 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). Retroviral vectors are particularly well developed and have been 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).

[0253] Non-viral approaches can also be employed for genetic modification of a cell. For example, a nucleic acid molecule can be introduced into a cell by administering the nucleic acid 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 micro-injection 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 be potentially beneficial for delivery of DNA into a cell. Transplantation of normal genes into the affected tissues of a subject can also be accomplished by transferring a normal nucleic acid into a cultivatable cell type ex vivo (e.g., an autologous or heterologous primary cell or progeny thereof), after which the cell (or its descendants) are injected into a targeted tissue or are injected systemically. Recombinant receptors can also be derived or obtained using transposases or targeted nucleases (e.g. Zinc finger nucleases, meganucleases, or TALE nucleases, CRISPR). Transient expression may be obtained by RNA electroporation.

[0254] In certain embodiments, the vector is a low copy vector. In certain embodiments, a low copy vector is a vector that is present in an amount of at most 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 copy per host cell. Non-limiting examples of low copy vectors are yeast replicating plasmids or yeast centromeric plasmids.

[0255] Any targeted genome editing methods can also be used to deliver a presently disclosed antigen-recognizing receptor to a cell or a subject. In certain embodiments, a CRISPR system is used to deliver a presently disclosed antigen-recognizing receptor disclosed herein. In certain embodiments, zinc-finger nucleases are used to deliver the antigen-recognizing receptor. In certain embodiments, a TALEN system is used to deliver a presently disclosed antigenrecognizing receptor.

[0256] 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 Cas9 (a protein able to modify DNA utilizing crRNA as its guide), CRISPR RNA (crRNA, contains the RNA used by Cas9 to guide it to the correct section of host DNA along with a region that binds to tracrRNA (generally in a hairpin loop form) forming an active complex with Cas9), trans-activating crRNA (tracrRNA, binds to crRNA and forms an active complex with Cas9), and an optional section of DNA repair template (DNA that guides the cellular repair process allowing insertion of a specific DNA sequence). CRISPR / Cas9 often employs a plasmid to transfect the target cells. The crRNA needs to be designed for each application as this is the sequence that Cas9 uses to identify and directly bind to the target DNA in a cell. The repair template carrying CAR expression cassette need also be designed for each application, as it must overlap with the sequences on either side of the cut and code for the insertion sequence. Multiple crRNA's and the tracrRNA can be packaged together to form a single-guide RNA (sgRNA). This sgRNA can be joined together with the Cas9 gene and made into a plasmid in order to be transfected into cells.

[0257] A zinc-finger nuclease (ZFN) is an artificial restriction enzyme, which is generated by combining a zinc finger DNA-binding domain with a DNA-cleavage domain. A zinc finger domain can be engineered to target specific DNA sequences which allows a zinc-finger nuclease to target desired sequences within genomes. The DNA-binding domains of individual ZFNs typically contain a plurality of individual zinc finger repeats and can each recognize a plurality of base pairs. The most common method to generate new zinc-finger domain is to combine smaller zinc-finger "modules" of known specificity. The most common cleavage domain in ZFNs is the non-specific cleavage domain from the type Ils restriction endonuclease Fokl. Using the endogenous homologous recombination (HR) machinery and a homologous DNA template carrying CAR expression cassette, ZFNs can be used to insert the CAR expression cassette into genome. When the targeted sequence is cleaved by ZFNs, the HR machinery searches for homology between the damaged chromosome and the homologous DNA template, and then copies the sequence of the template between the two broken ends of the chromosome, whereby the homologous DNA template is integrated into the genome.

[0258] Transcription activator-like effector nucleases (TALEN) are restriction enzymes that can be engineered to cut specific sequences of DNA. TALEN system operates on almost the same principle as ZFNs. They are generated by combining a transcription activator-like effectors DNA-binding domain with a DNA cleavage domain. Transcription activator-like effectors (TALEs) are composed of 33-34 amino acid repeating motifs with two variable positions that have a strong recognition for specific nucleotides. By assembling arrays of these TALEs, the TALE DNA-binding domain can be engineered to bind desired DNA sequence, and thereby guide the nuclease to cut at specific locations in genome. cDNA expression for use in polynucleotide therapy methods can be directed from any suitable promoter (e.g., the human cytomegalovirus (CMV), simian virus 40 (SV40), or metallothionein promoters), and regulated by any appropriate mammalian regulatory element or intron (e.g. the elongation factor la enhancer / promoter / intron structure). For example, if desired, enhancers known to preferentially direct gene expression in specific cell types can be used to direct the expression of a nucleic acid. The enhancers used can include, without limitation, those that are characterized as tissue- or cell-specific enhancers. Alternatively, if a genomic clone is used as a therapeutic construct, regulation can be mediated by the cognate regulatory sequences or, if desired, by regulatory sequences derived from a heterologous source, including any of the promoters or regulatory elements described above. Methods for delivering the genome editing agents / systems can vary depending on the need. 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 is not limited to, electroporation, microinjection, gene gun, impalefection, hydrostatic pressure, continuous infusion, sonication, magnetofection, adeno-associated viruses, envelope protein pseudotyping of viral vectors, replication-competent vectors cis and trans-acting elements, herpes simplex virus, and chemical vehicles (e.g., oligonucleotides, lipoplexes, polymersomes, polyplexes, dendrimers, inorganic Nanoparticles, and cell-penetrating peptides).

[0259] In certain embodiments, the delivery methods include use of colloids. As used herein, the term “colloid” refers to systems in which there are two or more phases, with one phase (e.g., the dispersed phase) distributed in the other phase (e.g., the continuous phase). Moreover, at least one of the phases has small dimensions (in the range of about 109to about 106m). Nonlimiting examples of colloids encompassed by the presently disclosed subject matter include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems (e.g., micelles, liposomes, and lipid nanoparticles).

[0260] In certain embodiments, the delivery methods include use of liposomes. The term “liposome,” as used herein, refers to single- or multi-layered spherical lipid bilayer structures produced from lipids dissolved in organic solvents and then dispersed in aqueous media. Experimentally and therapeutically used for delivering an active pharmaceutical ingredient (e.g., nucleic acid compositions disclosed herein) to cells, liposomes fuse with cell membranes so the contents are transferred into the cytoplasm.

[0261] In certain embodiments, the delivery methods include use of lipid nanoparticles. As used herein, the term “lipid nanoparticle” refers to a particle having at least one dimension in the order of nanometers (e.g., from about 1 nm to about 1,000 nm) and including at least one lipid. In certain embodiments, the lipid nanoparticles can include an active pharmaceutical ingredient (e.g., nucleic acid compositions disclosed herein) for delivering to cells. The morphology of the lipid nanoparticles can be different from liposomes. While liposomes are characterized by a lipid bilayer surrounding a hydrophilic core, lipid nanoparticles have an electron-dense core where cationic lipids and / or ionizable lipids are organized into inverted micelles around an active pharmaceutical ingredient (e.g., nucleic acid compositions disclosed herein). Additional information on the morphology and properties of lipid nanoparticles and liposomes can be found in Wilczewska, et al., Pharmacological reports 64, no. 5 (2012): 1020-1037; Eygeris et al., Accounts of Chemical Research 55, no. 1 (2021): 2-12; Zhang et al., Chemical Reviews 121, no. 20 (2021): 12181-12277; and Fan et al., Journal of pharmaceutical and biomedical analysis 192 (2021): 113642.

[0262] In certain embodiments, the lipid nanoparticles have a mean diameter of from about 30 nm to about 150 nm, from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm.

[0263] In certain embodiments, the lipid nanoparticles can include a cationic lipid or an ionizable lipid. The term “cationic lipid” refers to lipids including a head group with permanent positive charges. Non-limiting examples of cationic lipids encompassed by the presently disclosed subject matter include l,2-di-O-octadecenyl-3-trimethylammonium-propane (DOTMA), l,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 2,3-dioleyloxy-N-[2- (sperminecarboxamido)ethyl]-N,N-dimethyl-l-propanaminium trifluoroacetate (DOSPA), and ethylphosphatidylcholine (ePC).

[0264] As used herein, the term “ionizable lipid” refers to lipids that are protonated at low pH and are neutral at physiological pH. The pH-sensitivity of ionizable lipids is particularly beneficial for delivery in vivo (e.g., delivery of nucleic acid compositions disclosed herein), because neutral lipids have less interactions with the anionic membranes of blood cells and, thus, improve the biocompatibility of the lipid nanoparticles. Once trapped in endosomes, ionizable lipids are protonated and promote membrane destabilization to allow the endosomal escape of the nanoparticles. Non-limiting example of ionizable lipids encompassed by the presently disclosed subject matter include tetrakis(8-methylnonyl) 3,3',3",3"'-(((methylazanediyl) bis(propane-3,l diyl))bis (azanetriyl))tetrapropionate; decyl (2-(dioctylammonio)ethyl) phosphate; ((4- hydroxybutyl)azanediyl)bis(hexane-6, 1 -diyl)bis(2-hexyldecanoate); bis(2-(dodecyldisulfanyl)ethyl) 3,3'-((3-methyl-9-oxo-10-oxa-13,14-dithia-3,6-diazahexacosyl)azanediyl)dipropionate; l,l'-((2-(4-(2- ((2-(bis(2-hydroxydodecyl)amino)ethyl) (2-hydroxydodecyl)amino)ethyl) piperazin- 1 - yl)ethyl)azanediyl) bis(dodecan-2-ol); cKK-E12, 3,6-bis(4-(bis(2- hydroxydodecyl)amino)butyl)piperazine-2, 5-dione; (6Z,9Z,28Z,3 lZ)-heptatriaconta-6,9,28,31 - tetraen-19-yl 4-(dimethylamino) butanoate; hexa(octan-3-yl) 9, 9', 9", 9"', 9'"', 9"'"- ((((benzene-1,3,5- tricarbonyl)yris(azanediyl)) tris (propane-3, 1 -diyl)) tris(azanetriyl))hexanonanoate; heptadecan-9-yl 8- ((2-hydroxyethyl)(6-oxo-6- (undecyloxy)hexyl)amino) octanoate; and (((3,6-dioxopiperazine-2,5- diy 1 )bi s(butane-4, 1 -diy l))bi s(azanetriyl))tetraki s(ethane-2, 1 -diyl)

[0265] (9Z,9'Z,9"Z,9"'Z, 12Z, 12'Z,12"Z, 12"'Z)-tetrakis (octadeca-9, 12-dienoate).

[0266] Additionally, in certain embodiments, the lipid nanoparticles can include other lipids. For example, but without any limitation, the lipid nanoparticles of the presently disclosed subject matter can include phospholipids, cholesterol, polyethylene glycol (PEG)- functionalized lipids (PEG-lipids). These lipids can improve certain properties of the lipid nanoparticles (e.g., stability, biodistribution, etc.). For example, cholesterol enhances the stability of the lipid nanoparticles by modulating the integrity and rigidity. Non-limiting examples of other lipids present in lipid nanoparticles include cholesterol, DC-cholesterol, P- sitosterol, BHEM-cholesterol, ALC-0159, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4- (N- maleimidom ethyl) -cyclohexane -1 -carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-0-monom ethyl PE, 16-O-dimethyl PE, 18-1 -trans PE, 1- stearioyl-2-oleoyl-phosphatidy ethanol amine (SOPE), and 1,2-dielaidoyl-sn- glycero-3- phophoethanolamine (transDOPE).

[0267] In certain embodiments, the lipid nanoparticles can include a targeting moiety that binds to a ligand. The use of the targeting moieties allows selective delivery of an active pharmaceutical ingredient (e.g., nucleic acid compositions disclosed herein) to target cells expressing the ligand (e.g., T cells). In certain embodiments, the targeting moiety can be an antibody or antigen-binding fragment thereof that binds to a cell surface receptor. For example, but without any limitation, the targeting domain is an antibody or antigen-binding fragment thereof that binds to a receptor expressed on the surface of a T cell (e.g., CD3, CD4, CD8, CD16, CD40L, CD95, FasL, CTLA-4, 0X40, GITR, LAG3, ICOS, and PD-1).

[0268] In certain embodiments, the delivery methods are in vivo delivery methods. In certain embodiments, the delivery methods are ex vivo delivery methods.

[0269] 6. Polypeptides

[0270] The presently disclosed subject matter provides methods for optimizing an amino acid sequence or a nucleotide sequence by producing an alteration in the sequence. Such alterations may include certain mutations, deletions, insertions, or post-translational modifications. The presently disclosed subject matter further includes analogs of any naturally-occurring polypeptides disclosed herein (including, but not limited to, CD8, CD28, 4- IBB, and CD3^,). Analogs can differ from a naturally-occurring polypeptide disclosed herein by amino acid sequence differences, by post-translational modifications, or by both. 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 homologous or identical to all or part of a naturally-occurring amino, acid sequence of the presently disclosed subject matter. The length of 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 to determining the degree of identity, a BLAST program may be used, with a probability score between e'3and e'100indicating a closely related sequence. Modifications include in vivo and in vitro chemical derivatization of polypeptides, e.g., acetylation, carboxylation, phosphorylation, or glycosylation; such modifications may occur during polypeptide synthesis or processing or following treatment with isolated modifying enzymes. Analogs can also differ from the naturally-occurring polypeptides by alterations in primary sequence. These include genetic variants, both natural and induced (for example, resulting from random mutagenesis by irradiation or exposure to ethanemethyl sulfate or by site-specific mutagenesis as described in Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual (2d ed.), CSH Press, 1989, or Ausubel et al., supra). Also included are cyclized peptides, molecules, and analogs which contain residues other than L-amino acids, e.g., D- amino acids or non-naturally occurring or synthetic amino acids, e.g., P or y amino acids.

[0271] In addition to full-length polypeptides, the presently disclosed subject matter also provides fragments of any of the polypeptides disclosed herein. As used herein, the term “a fragment” means at least 5, 10, 13, or 15 amino acids. In certain embodiments, a fragment comprises at least 20 contiguous amino acids, at least 30 contiguous amino acids, or at least 50 contiguous amino acids. In certain embodiments, a fragment comprises at least 60 to 80, 100, 200, 300 or more contiguous amino acids. Fragments can be generated by methods known to those skilled in the art or may result from normal protein processing (e.g., removal of amino acids from the nascent polypeptide that are not required for biological activity or removal of amino acids by alternative mRNA splicing or alternative protein processing events).

[0272] 7. Formulations and Administration

[0273] The presently disclosed subject matter also provides compositions comprising the presently disclosed cells. Compositions comprising the presently disclosed cells can be conveniently provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may be buffered to a selected pH. Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues. Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like) and suitable mixtures thereof.

[0274] Sterile injectable solutions can be prepared by incorporating the genetically modified cells in the required amount of the appropriate solvent with various amounts of the other ingredients, as desired. Such compositions may be in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like. The compositions can also be lyophilized. The compositions can contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. Standard texts, such as “REMINGTON’S PHARMACEUTICAL SCIENCE”, 17th edition, 1985, incorporated herein by reference, may be consulted to prepare suitable preparations, without undue experimentation.

[0275] Various additives which enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin. According to the presently disclosed subject matter, however, any vehicle, diluent, or additive used would have to be compatible with the genetically modified cells.

[0276] The compositions can be isotonic, i.e., they can have the same osmotic pressure as blood and lacrimal fluid. The desired isotonicity of the compositions may be accomplished 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 particularly for buffers containing sodium ions. Viscosity of the compositions, if desired, can be maintained at the selected level using a pharmaceutically acceptable thickening agent. For example, methylcellulose is readily and economically available and is easy to work with. Other suitable thickening agents include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, and the like. The concentration of the thickener can depend upon the agent selected. The important point is to use an amount that will achieve the selected viscosity. Obviously, the choice of suitable carriers and other additives will depend on the exact route of administration and the nature of the particular dosage form, e.g., liquid dosage form (e.g., whether the composition is to be formulated into a solution, a suspension, gel or another liquid form, such as a time release form or liquid-filled form).

[0277] Compositions comprising the presently disclosed cells can be provided systemically or directly to a subject for inducing and / or enhancing an immune response to an antigen and / or treating and / or preventing a neoplasia. In certain embodiments, the presently disclosed cells or compositions comprising thereof are directly injected into an organ of interest (e.g., an organ affected by a neoplasia). Alternatively, the presently disclosed cells or compositions comprising thereof are provided indirectly to the organ of interest, for example, by administration into the circulatory system (e.g., the tumor vasculature). Expansion and differentiation agents can be provided prior to, during or after administration of the cells or compositions to increase production of cells (e.g., T-cells or NK cells) in vitro or in vivo.

[0278] The presently disclosed cells can be administered in any physiologically acceptable vehicle, normally intravascularly, although they may also be introduced into bone or other convenient site where the cells may find an appropriate site for regeneration and differentiation (e.g., thymus).

[0279] The quantity of cells to be administered can vary for the subject being treated. In certain embodiments, between about 104and about IO10, between about 104and about 107, between about 105and about 107, between about 105and about 109, or between about 106and about 108of the presently disclosed cells are administered to a subject. More effective cells may be administered in even smaller numbers. Usually, at least about 1 x 105cells will be administered, eventually reaching about 1 x IO10or more. In certain embodiments, at least about U 105, at least about 5x 105, at least about 1 x 106, at least about 5x 106, at least about 1 x 107, at least about 5x l07, at least about U 108, or at least about 5x l08of the presently disclosed cells are administered to a subject. In certain embodiments, between about U 105and about 5x l05of the presently disclosed cells are administered to a subject. In certain embodiments, about 2x 105of the presently disclosed cells are administered to a subject. In certain embodiments, between about 1 x 106and about 5* 106of the presently disclosed cells are administered to a subject. In certain embodiments, about 1 * 106of the presently disclosed cells are administered to a subject. In certain embodiments, about 3*106of the presently disclosed cells are administered to a subject. In certain embodiments, between about l >< 107and about 5x l07of the presently disclosed cells are administered to a subject. In certain embodiments, about I x lO7of the presently disclosed cells are administered to a subject. The precise determination of what would be considered an effective dose can be based on factors individual to each subject, including their size, age, sex, weight, and condition of the particular subject. Dosages can be readily ascertained by those skilled in the art from this disclosure and the knowledge in the art.

[0280] The presently disclosed cells can comprise a purified population of cells. Those skilled in the art can readily determine the percentage of the presently disclosed cells in a population using various well-known methods, such as fluorescence activated cell sorting (FACS). Suitable ranges of purity in populations comprising the presently disclosed immunoresponsive cells are about 50% to about 55%, about 5% to about 60%, and about 65% to about 70%. In certain embodiments, the purity is about 70% to about 75%, about 75% to about 80%, or about 80% to about 85%. In certain embodiments, the purity is about 85% to about 90%, about 90% to about 95%, and about 95% to about 100%. Dosages can be readily adjusted by those skilled 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.

[0281] The skilled artisan can readily determine the amount of cells and optional additives, vehicles, and / or carrier in compositions and to be administered in methods. Typically, any additives (in addition to the active cell(s) and / or agent(s)) are present in an amount of 0.001 to 50% (weight) solution in phosphate buffered saline, and the active ingredient is present in the order of micrograms to milligrams, such as about 0.0001 to about 5 wt %, about 0.0001 to about 1 wt %, about 0.0001 to about 0.05 wt% or about 0.001 to about 20 wt %, about 0.01 to about 10 wt %, or about 0.05 to about 5 wt %. For any composition to be administered to an animal or human, the followings can be determined: toxicity such as by determining the lethal dose (LD) and LD50 in a suitable animal model e.g., rodent such as mouse; the dosage of the composition(s), concentration of components therein and timing of administering the composition(s), which elicit a suitable response. Such determinations do not require undue experimentation from the knowledge of the skilled artisan, this disclosure and the documents cited herein. And, the time for sequential administrations can be ascertained without undue experimentation. In certain embodiments, the composition is a pharmaceutical composition comprising the presently disclosed cells and a pharmaceutically acceptable carrier.

[0282] Administration of the compositions can be autologous or heterologous. For example, cells can be obtained from one subject, and administered to the same subject or a different, compatible subject. Peripheral blood derived cells or their progeny (e.g., in vivo, ex vivo or in vitro derived) can be administered. When administering a presently disclosed composition (e.g., a pharmaceutical composition comprising presently disclosed cells), it can be formulated in a unit dosage injectable form (solution, suspension, emulsion).

[0283] The presently disclosed cells and compositions can be administered by any method known in the art including, but not limited to, oral administration, intravenous administration, subcutaneous administration, intranodal administration, intratumoral administration, intrathecal administration, intrapleural administration, intraosseous administration, intraperitoneal administration, pleural administration, and direct administration to the subject.

[0284] 8. Methods of Treatment

[0285] The presently disclosed cells and compositions comprising thereof can be used for treating and / or preventing a tumor or a neoplasia. In certain embodiments, the tumor or neoplasia can be treated by the presently disclosed cells and compositions include a solid tumor (e.g., mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic carcinoma, endometrial carcinoma, stomach cancer, and / or cholangiocarcinoma). In certain embodiments, the cell is a T-cell. The T-cell can be a CD4+T-cell or a CD8+T-cell. In certain embodiments, the T-cell is a CD4+T-cell.

[0286] The presently disclosed subject matter provides methods for inducing and / or increasing an immune response in a subject in need thereof. The presently disclosed cells and compositions comprising thereof can be used in a therapy or medicament. The presently disclosed subject matter provides various methods of using the cells (e.g., T-cells) or compositions comprising thereof. For example, the presently disclosed cells and compositions comprising thereof can be used for reducing tumor burden in a subject. The presently disclosed cell can reduce the number of tumor cells, reduce tumor size, and / or eradicate the tumor in the subject. The presently disclosed cells and compositions comprising thereof can be used for treating and / or preventing a neoplasia in a subject. The presently disclosed cells and compositions comprising thereof can be used for prolonging the survival of a subject suffering from a neoplasia. Such methods comprise administering the presently disclosed cells or a composition (e.g., a pharmaceutical composition) comprising thereof to achieve the desired effect, e.g., palliation of an existing condition or prevention of recurrence. For treatment, the amount administered is an amount effective in producing the desired effect. An effective amount can be provided in one or a series of administrations. An effective amount can be provided in a bolus or by continuous perfusion.

[0287] The presently disclosed subject matter provides various methods of using the cells (e.g., T-cells) or compositions comprising thereof. For example, the presently disclosed subject matter provides methods of reducing tumor burden in a subject. In certain embodiments, the method of reducing tumor burden comprises administering the presently disclosed cells or a composition comprising thereof to the subject. The presently disclosed cell can reduce the number of tumor cells, reduce tumor size, and / or eradicate the tumor in the subject.

[0288] The presently disclosed subject matter also provides methods of increasing or lengthening survival of a subject having a neoplasia. In certain embodiments, the method of increasing or lengthening survival of a subject having neoplasia comprises administering the presently disclosed immunoresponsive cells or a composition comprising thereof to the subject. The method can reduce or eradicate tumor burden in the subject. Additionally, the presently disclosed subject matter provides methods for increasing an immune response in a subject, comprising administering the presently disclosed cell or a composition comprising thereof to the subj ect. The presently disclosed subj ect matter further provides methods for treating and / or preventing a neoplasia in a subject, comprising administering the presently disclosed cells or a composition comprising thereof to the subject.

[0289] In certain embodiments, the tumor or neoplasia is associated with mesothelin. In certain embodiments, the tumor or neoplasia expresses mesothelin. In certain embodiments, the tumor is cancer.

[0290] In certain embodiments, the tumor or neoplasia is a solid tumor. Non-limiting examples of solid tumors include mesothelioma, renal cancer, bladder cancer, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic carcinoma, endometrial carcinoma, stomach cancer, and cholangiocarcinoma.

[0291] In certain embodiments, the neoplasm is a solid tumor. The neoplasm can a primary tumor or primary cancer. In addition, the neoplasm can be in metastatic status.

[0292] Cancers whose growth may be inhibited using the immunoresponsive cells of the presently disclosed subject matter comprise cancers typically responsive to immunotherapy. Non-limiting examples of cancers for treatment include mesothelioma, lung cancer (e.g., non- small cell lung cancer), pancreatic cancer, ovarian cancer, breast cancer (e.g., metastatic breast cancer, metastatic triple-negative breast cancer), colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic carcinoma, endometrial carcinoma, stomach cancer, cholangiocarcinoma, cervical cancer, and salivary gland cancer. Additionally, the presently disclosed subject matter comprises refractory or recurrent malignancies whose growth may be inhibited using the immunoresponsive cells of the presently disclosed subject matter.

[0293] Examples of other neoplasms or cancers that may be treated using the methods of the presently disclosed subject matter include bone cancer, intestinal cancer, liver cancer, skin cancer, cancer of the head or neck, melanoma (cutaneous or intraocular malignant melanoma), renal cancer (e.g. clear cell carcinoma), throat cancer, prostate cancer (e.g. hormone refractory prostate adenocarcinoma), blood cancers (e.g. leukemias, lymphomas, and myelomas), uterine cancer, rectal cancer, cancer of the anal region, bladder cancer, brain cancer, stomach cancer, testicular cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, leukemias (e.g., acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma (Hodgkin’s disease, non-Hodgkin’s disease), cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of childhood, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, include Waldenstrom’s macroglobulinemia, heavy chain disease, and solid tumors such as sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing’s tumor, leiomyosarcoma, rhabdomyosarcoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, hepatoma, nile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm’s tumor, cervical cancer, salivary gland cancer, uterine cancer, testicular cancer, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodenroglioma, schwannoma, meningioma, melanoma, neuroblastoma, and retinoblastoma).

[0294] The subjects can have an advanced form of disease, in which case the treatment objective can include mitigation or reversal of disease progression, and / or amelioration of side effects. The subjects can have a history of the condition, for which they have already been treated, in which case the therapeutic objective will typically include a decrease or delay in the risk of recurrence.

[0295] As a consequence of surface expression of a presently disclosed mesothelin-targeted antigen-recognizing receptor, adoptively transferred cells (e.g., immunoresponsive cells, e.g., T-cells or NK cells) are endowed with augmented and selective cytolytic activity at the tumor site. Furthermore, subsequent to their localization to tumor or viral infection and their proliferation, the cells turn the tumor or viral infection site into a highly conductive environment for a wide range of immune cells involved in the physiological anti-tumor or antiviral response (tumor infiltrating lymphocytes, NK-, NKT- cells, dendritic cells, and macrophages).

[0296] Further modification can be introduced to the presently disclosed cells (e.g., T-cells) to avert or minimize the risks of immunological complications (known as “malignant T-cell transformation”), e.g., graft versus-host disease (GvHD), or when healthy tissues express the same target antigens as the tumor cells, leading to outcomes similar to GvHD. A potential solution to this problem is engineering a suicide gene into the presently disclosed cells. Suitable suicide genes include, but are not limited to, Herpes simplex virus thymidine kinase (hsv-tk), inducible Caspase 9 Suicide gene (iCasp-9), and a truncated human epidermal growth factor receptor (EGFRt) polypeptide. In certain embodiments, the suicide gene is an EGFRt polypeptide. The EGFRt polypeptide can enable T-cell elimination by administering anti- EGFR monoclonal antibody (e.g. , cetuximab). EGFRt can be covalently j oined to the upstream of the antigen-recognizing receptor (e.g., CAR). The suicide gene can be included within the vector comprising nucleic acids encoding a presently disclosed antigen-recognizing receptor (e.g., CAR). In this way, administration of a prodrug designed to activate the suicide gene (e.g., a prodrug (e.g., API 903 that can activate iCasp-9) during malignant T-cell transformation (e.g., GVHD) triggers apoptosis in the suicide gene-activated cells expressing the antigenrecognizing receptor (e.g., CAR). The incorporation of a suicide gene into a presently disclosed antigen-recognizing receptor (e.g., CAR) gives an added level of safety with the ability to eliminate the majority of receptor-expressing cells within a very short time period. A presently disclosed cell (e.g., a T-cell) incorporated with a suicide gene can be pre-emptively eliminated at a given timepoint post the cell infusion, or eradicated at the earliest signs of toxicity.

[0297] 9. Exemplary Embodiments

[0298] Al. An antigen-recognizing receptor comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen-binding domain specifically binds to mesothelin and comprises a heavy chain variable region comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 6 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8 or a conservative modification thereof; and a light chain variable region comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 9 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11 or a conservative modification thereof.

[0299] A2. The antigen-recognizing receptor of Al, wherein the heavy chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 6, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8; and the light chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 9, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11.

[0300] A3. The antigen-recognizing receptor of Al or A2, wherein the extracellular antigenbinding domain is a single-chain variable fragment (scFv), a Fab, which is optionally crosslinked, or a F(ab)2.

[0301] A4. The antigen-recognizing receptor of A3, wherein the extracellular antigen-binding domain is a humanized scFv.

[0302] A5. The antigen-recognizing receptor of A3 or A4, wherein one or more of the scFv, Fab and F(ab)2 are comprised in a fusion protein with a heterologous sequence to form the extracellular antigen-binding domain. A6. The antigen-recognizing receptor of any one of A1-A5, wherein the heavy chain variable region comprises an amino acid sequence that is at least 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% or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 12.

[0303] A7. The antigen-recognizing receptor of any one of A1-A6, wherein the light chain variable region comprises an amino acid sequence that is at least 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% or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 13.

[0304] A8. The antigen-recognizing receptor of any one of A1-A7, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 12, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 13.

[0305] A9. The antigen-recognizing receptor of any one of A1-A8, wherein the extracellular antigen-binding domain comprises a linker between the heavy chain variable region and the light chain variable region of the extracellular antigen-binding domain.

[0306] A10. The antigen-recognizing receptor of A9, wherein the linker consists of the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.

[0307] Al 1. The antigen-recognizing receptor of A10, wherein the linker consists of the amino acid sequence set forth in SEQ ID NO: 1.

[0308] A12. The antigen-recognizing receptor of any one of Al-Al l, wherein a signal peptide is covalently joined to the 5' terminus of the extracellular antigen-binding domain.

[0309] Al 3. The antigen-recognizing receptor of any one of Al -Al 2, wherein the heavy chain variable region and the light chain variable region are positioned from the N- to the C-terminus: VH-VL.

[0310] Al 4. The antigen-recognizing receptor of any one of Al -Al 2, wherein the heavy chain variable region and the light chain variable region are positioned from the N- to the C-terminus: VL-VH.

[0311] Al 5. An antigen-recognizing receptor comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen-binding domain specifically binds to mesothelin and comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDR1, a CDR2, and a CDR3 of a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 12, and the light chain variable region comprises a CDR1, a CDR2, and a CDR3 of a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 13.

[0312] Al 6. An antigen-recognizing receptor comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen-binding domain specifically binds to mesothelin and comprises a single chain variable region (scFv) comprising the amino acid sequence set forth in SEQ ID NO: 16.

[0313] Al 7. The antigen-recognizing receptor of any one of Al -Al 6, wherein the transmembrane domain comprises a CD8 polypeptide, a CD28 polypeptide, a CD3^ polypeptide, a CD4 polypeptide, a 4-1BB polypeptide, an 0X40 polypeptide, an ICOS polypeptide, a CTLA-4 polypeptide, a PD-1 polypeptide, a LAG-3 polypeptide, a 2B4 polypeptide, a BTLA polypeptide, or a combination thereof.

[0314] A18. The antigen-recognizing receptor of any one of A1-A17, wherein the intracellular signaling domain comprises a CD3(^ polypeptide.

[0315] Al 9. The antigen-recognizing receptor of any one of Al -Al 8, wherein the intracellular signaling domain further comprises at least one co-stimulatory signaling region.

[0316] A20. The antigen-recognizing receptor of Al 9, wherein the at least one co-stimulatory signaling region comprises a CD28 polypeptide, a 4- IBB polypeptide, an 0X40 polypeptide, an ICOS polypeptide, a DAP-10 polypeptide, or a combination thereof.

[0317] A21. The antigen-recognizing receptor of any one of Al -20, wherein the antigenrecognizing receptor is a chimeric antigen receptor (CAR).

[0318] A22. The antigen-recognizing receptor of any one of A 1-21, wherein the antigenrecognizing receptor is recombinantly expressed or expressed from a vector.

[0319] A23. The antigen-recognizing receptor of A22, wherein the vector is a low copy number vector.

[0320] A24. A cell comprising the antigen-recognizing receptor of any one of A1-A23.

[0321] A25. The cell of A24, wherein the antigen-recognizing receptor is constitutively expressed on the surface of the cell.

[0322] A26. The cell of A24 or 25, wherein the cell is an immunoresponsive cell.

[0323] A27. The cell of any one of A24-A26, wherein the cell is a cell of the lymphoid lineage or a cell of the myeloid lineage. A28. The cell of any one of A24-A27, wherein the cell is selected from the group consisting of a T cell, a Natural Killer (NK) cell, and a stem cell from which a lymphoid cell may be differentiated.

[0324] A29. The cell of A28, wherein the cell is a T cell.

[0325] A30. The cell of A29, wherein the T cell is a cytotoxic T lymphocyte (CTL) or a regulatory T cell.

[0326] A31. The cell of A28, wherein the stem cell is a pluripotent stem cell.

[0327] A32. The cell of A31, wherein the pluripotent stem cell is an embryoid stem cell or an induced pluripotent stem cell.

[0328] A33. A nucleic acid molecule encoding the antigen-recognizing receptor of any one of A1-A23.

[0329] A34. A vector comprising the nucleic acid molecule of A33.

[0330] A35. The vector of A34, wherein the vector is a low copy number vector.

[0331] A36. The vector of A34 or A35, wherein the vector is a retroviral vector.

[0332] A37. The vector of A36, wherein the retroviral vector is a y-retroviral vector or a lentiviral vector.

[0333] A38. A lipid nanoparticle comprising the nucleic acid of A33.

[0334] A39. A composition comprising the cell of any one of A24-A32, the nucleic acid molecule of A33, the vector of any one of A34-A37, or the lipid nanoparticle of A38.

[0335] A40. The composition of A39, which is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

[0336] A41. A method of treating and / or preventing a tumor in a subject in need thereof, the method comprising administering to the subject an effective amount of the cell of any one of A24-A32 or the composition of A39 or A40.

[0337] A42. The method of A41, wherein the method reduces the number of tumor cells, reduces tumor size, and / or eradicates the tumor in the subject.

[0338] A43. A method of increasing or lengthening survival of a subject having a tumor, comprising administering to the subject an effective amount of the cell of any one of A24-A32 or the composition of A39 or A40.

[0339] A44. The method of any one of A41-A43, wherein the tumor is associated with mesothelin.

[0340] A45. The method of any one of A41-A44, wherein the tumor is a solid tumor.

[0341] A46. The method of A45, wherein the solid tumor is selected from mesothelioma, renal cancer, bladder cancer, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic carcinoma, endometrial carcinoma, stomach cancer, or cholangiocarcinoma.

[0342] A47. A method for producing a cell comprising an antigen-recognizing receptor of any one of A1-A23, comprising introducing into the cell a nucleic acid molecule that encodes the antigen-recognizing receptor.

[0343] A48. A kit for reducing tumor burden in a subject, treating and / or preventing a tumor in a subject, and / or increasing or lengthening survival of a subject having a tumor, comprising the cell of any one of A24-A32.

[0344] A49. The kit of A48, wherein the kit further comprises written instructions for using the cell for reducing tumor burden in a subject, treating and / or preventing a tumor or neoplasm in a subject, and / or increasing or lengthening survival of a subject having a tumor.

[0345] B 1. The cell of any one of A24-A32 or the composition of A39 or A40 for use in treating and / or preventing a tumor in a subject in need thereof.

[0346] B2. The cell or composition for use of Bl, wherein the method reduces the number of tumor cells, reduces tumor size, and / or eradicates the tumor in the subject.

[0347] B3. The cell of any one of A24-A32 or the composition of A39 or A40 for use increasing or lengthening survival of a subject having a tumor.

[0348] B4. The cell or composition for use of any one of B1-B3, wherein the tumor is associated with mesothelin.

[0349] B5. The cell or composition for use of any one of B1-B5, wherein the tumor is a solid tumor.

[0350] B6. The cell or composition for use of B5, wherein the solid tumor is selected from mesothelioma, renal cancer, bladder cancer, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic carcinoma, endometrial carcinoma, stomach cancer, or cholangiocarcinoma.

[0351] EXAMPLE

[0352] The presently disclosed subject matter will be better understood by reference to the following Example, which is provided as exemplary of the presently disclosed subject matter, and not by way of limitation.

[0353] Example 1 The present example illustrates scFv with cross-reactivity to human and mouse mesothelin developed using a phage based biopanning process. A phage display library with a diversity of 5.37xlO10different clones was subjected to six rounds of panning against human and mouse mesothelin. The identified binders were further analyzed by ELISA. Phage DNA was extracted for antibody sequencing and the binding specificity of the identified clone was validated in mesothelin expressing cell lines by flow cytometry analysis.

[0354] Biopanning

[0355] Six rounds of solid phase panning were carried out with peptide antigens as shown to Table 3. Briefly, the peptide antigens were coated onto tubes followed by standard washing and blocking steps. The tubes were incubated with phage library and washed to remove the unbound phages. The bound phages were eluted with glycine-HCl and the concentration of the eluted phages were determined by calculating plaque-forming units (pfu) in an E.coli TGI based assay. Eluted phages were amplified with helper phage and used in the subsequent rounds of biopanning or ELISA assays. As seen in Table 4, the biopanning data shows significant and progressive enrichment (bold) over the rounds of biopanning. Since significant enrichment was observed after six rounds, the assay was stopped in order to prevent a decrease in phage diversity.

[0356] Table 3. Selection conditions for each round

[0357] Table 4. Biopanning results

[0358] Polyclonal phase ELISA (phase pools screenins)

[0359] Pooled phage screening was carried out with the eluted phages from each round of biopanning. Briefly, an ELISA plate was coated with antigen (5 pg / mL) or control followed by standard washing and blocking steps. The prepared ELISA plate was incubated with amplified phages followed by standard washing and incubation with a phage specific HRP antibody. The plate was then incubated with TMB solution followed by HC1 and the corresponding ELISA signal was read at 450 nm. As seen in Tables 5A and 5B, enrichment for antigen specific binders started slightly from Round 3, as indicated by the higher ELISA signal compared to the control (NC) at low dilutions of phage. Enrichment strengthened in Round 4 with very high specific ELISA signals from Round 5, with all concentrations nearing saturation at Round 6. Rounds 4, 5 and 6 were selected for monoclonal ELISA in which 384 single phage-binders were tested in order to maximize the chances of identifying all relevant clones. Table 5 A. Results of polyclonal phage ELISA Table 5B. Results of polyclonal phage ELISA

[0360] Agl : 4 pg / ml Human Mesothelin; Ag2: 4 pg / ml Mouse Mesothelin; NC: PBS. Monoclonal phage ELISA (single phage binders screening)

[0361] Single phage binders were generated by randomly picking and amplifying 288 single TGI clones from the pfu assay of Rounds 4, 5, and 6. ELISA plates were coated with antigen (5 pg / mL) or control followed by the washing, blocking steps, and incubation steps described above. The highest proportion of positive clones were observed from Round 6 (Tables 15-17), followed by Round 5 (Tables 12-14), and Round 4 (Table 5-10). Since a high proportion of positive clones were observed on the ELISA plates from Rounds 5 and 6, all clones highlighted in bold (Tables 12-17) were sent for sequencing. One unique sequence was obtained from the positive binders, indicating that the cross-panning was successful at eliminating the non-cross reactive binders. Clone R6P1-B1 was selected for further analysis.

[0362] Table 6. Results of R4P1 output phage ELISA for AG1 (4 pg / mL Human Mesothelin)

[0363] Table 7. Results of R4P1 output phage ELISA for AG2 (4 pg / mL Mouse Mesothelin)

[0364] Table 8. Results of R4P1 output phage ELISA for NC (PBS)

[0365] Table 9. Results of R4P2 output phage ELISA for AG1 (4 pg / mL Human Mesothelin) Table 10. Results of R4P2 output phage ELISA for AG2 (4 pg / mL Mouse Mesothelin)

[0366] Table 11. Results of R4P2 output phage ELISA for NC (PBS) Table 12. Results of R5P1 output phage ELISA for AG1 (4 pg / mL Human Mesothelin)

[0367] Table 13. Results of R5P1 output phage ELISA for AG2 (4 pg / mL Mouse Mesothelin)

[0368] Table 14. Results of R5P1 output phage ELISA for NC (PBS)

[0369] Table 15. Results of R6P1 output phage ELISA for AG1 (4 pg / mL Human Mesothelin)

[0370] Table 16. Results of R6P1 output phage ELISA for AG2 (4 pg / mL Mouse Mesothelin)

[0371] Table 17. Results of R6P1 output phage ELISA for NC (PBS)

[0372] Confirmation ELISA after sequencing A second verification ELISA was carried out on clone R6P1-B1. As seen in Table 18, the single identified unique clone shows human-mouse cross-reactivity against both recombinant protein antigens.

[0373] Table 18. Result of ELISA verification of 21055-R6P1-B1

[0374] AG1 : 4 pg / mL Human Mesothelin; AG2: 4 pg / mL Mouse Mesothelin; NC: PBS.

[0375] Validation by FACS

[0376] The binding specificity of clone R6P1-B1 was validated by FACS analysis in cultured human mesothelin expressing MGM cells, mouse mesothelin expressing AB 12 cells and non- mesothelin expressing MSTO-211H cells. As seen in Figure 1 and Table 19, the 21055-R6P1- B1 clone showed a strong positive signal in flow cytometry against the human mesothelin expressing MGM cells, confirming the activity of the clone against the native form of the protein. A weak positive signal was detected on the mouse mesothelin expressing AB 12 cells, which likely indicates that there is low expression of mouse mesothelin in AB 12 cells rather than a lack of activity of the phage clone, since an equivalent ELISA signal was observed against the recombinant human and mouse mesothelin proteins in Table 18.

[0377] Table 19. Result of FACS verification

[0378] % True positive Cells = (% Positive Cells Stained - % Positive Cells Unstained) - (% Positive Stained MSTO Cells - % Positive Unstained MSTO Cells)

[0379] Structure prediction analysis

[0380] The sequence identified from the biopanning process was compared to the previously characterized mesothelin (m912) scFv. The presently disclosed scFv was compared to m912 by sequence alignment analysis (Table 20). Further structural analysis highlights differences in the electrostatic forces, lipophilicity, and the predicted binding pocket between m912 and the presently disclosed scFv (Figures 2A-2E).

[0381] Table 20. Sequence alignment: m912 scFv vs. presently disclosed scFv

[0382] Example 2

[0383] The present example illustrates functional activity of the presently disclosed CAR expressed in T cells. Briefly, multiple constructs were designed and expressed in T cell in order to express the mM28z CAR disclosed herein (Figures 3 A-3F). As seen in Figure 4A, T cells have CAR expression levels comparable to the M28z CAR used herein as control. For clarity, the control cells include a CAR comprising the m912 scFv.

[0384] Next, it was determined if T cells expressing the mM28z CAR were effective in killing tumor cells expressing mesothelin. As seen in Figures 4A and 4B, cells expressing mM28z CAR were effective in killing both human and mouse tumor cells expressing mesothelin. Importantly, they had a higher killing activity as compared to the controls (i.e., T cells expressing M28z CAR including m912 scFv). Overall, the presently disclosed cells are effective in tumor killing.

[0385] Example 3: Enhanced CAR T Cell Transduction via Codon-Optimized Constructs

[0386] One of the main challenges in chimeric antigen receptor (CAR) T cell therapy is the variability in CAR transduction efficiency among donor-derived T cells. Clinical data indicate that transduction rates typically range from approximately 15% to 50%, with some instances reaching up to 75%. This variability can compromise therapeutic consistency and efficacy. The present example addresses this limitation through the use of the presently disclosed codon- optimized CAR constructs, which enabled consistently high levels of CAR expression on the T cell surface across multiple donors.

[0387] To evaluate the performance of the presently disclosed CAR constructs, T cells isolated from nine independent donors were transduced with the codon-optimized CAR construct targeting mesothelin mM28z. As shown in Figure 5, high levels of CAR expression were observed across all donor samples, with transduction efficiencies ranging from 30% to 73%. A positive correlation was observed between vector copy number (VCN) and transduction efficiency. Notably, robust CAR expression was achieved even at low VCNs (approximately 1), indicating that the codon optimization significantly enhances transgene expression. Without being bound by any theory, it is believed that this effect is attributable to the specific codon usage and the unique antigen-binding domain of the disclosed CAR constructs.

[0388] To assess functional activity, cytotoxicity assays were performed using human mesothelioma MSTO-GM cells. T cells expressing either the original M28z or the mM28z CAR were co-cultured with MSTO-GM cells at different effector-to-target ratios for 48 hours. As shown in Figure 6, both CAR T cell types effectively lysed MSTO-GM cells. In contrast, when tested against mouse mesothelioma AB 12 cells, only mM28z CAR T cells exhibited cytotoxic activity (Figure 7), indicating that mM28z can recognize both human and murine mesothelin, whereas M28z is specific to the human antigen.

[0389] T cell activation was further evaluated by culturing mM28z CAR T cells with soluble mesothelin (MSLN) derived from the supernatant of human MGM mesothelioma cells. Upon antigen engagement, mM28z CAR T cells exhibited increased expression of activation markers CD69, CD107a, PD-1, and CD25 (Figures 8A-8D). These results confirm that the disclosed CAR T cells are functionally responsive to antigen binding. Importantly, no cytotoxicity was observed when mM28z CAR T cells were pre-incubated with MSLN-containing supernatant prior to chromium release assays (Figures 9A and 9B), indicating that binding to soluble antigen does not trigger off-target cytotoxicity.

[0390] To further characterize antigen binding, mM28z CAR T cells were incubated with increasing concentrations of recombinant mesothelin (rMSLN). Flow cytometry analysis revealed a dose-dependent increase in antigen binding (Figure 10), confirming the specificity and sensitivity of the CAR construct.

[0391] Finally, the impact of vector concentration on transduction efficiency was assessed. T cells were transduced with serial dilutions of the mM28z vector (1 : 10 to 1 :30) using spinoculation. As shown in Figure 11, high transduction levels (up to 46%) were maintained even at a 1 :30 dilution, further demonstrating the efficiency of the codon-optimized construct. These findings indicate that both the antigen-binding domain and codon optimization contribute to the observed high transduction rates.

[0392] The data presented herein demonstrate that the disclosed CAR constructs enable high- efficiency T cell transduction across multiple donors, even at low vector copy numbers. This represents a significant advancement in CAR T cell manufacturing and therapeutic reliability, offering improved scalability and consistency for clinical applications.

[0393] Embodiments of the presently disclosed subject matter From the foregoing description, it will be apparent that variations and modifications may be made to the presently disclosed subject matter to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.

[0394] The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or sub-combination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

[0395] All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.

Claims

WHAT IS CLAIMED IS:

1. An antigen-recognizing receptor comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen-binding domain specifically binds to mesothelin and comprises a heavy chain variable region comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 6 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8 or a conservative modification thereof; and a light chain variable region comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 9 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11 or a conservative modification thereof.

2. The antigen-recognizing receptor of claim 1, wherein the heavy chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 6, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8; and the light chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 9, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11.

3. The antigen-recognizing receptor of claim 1, wherein the extracellular antigenbinding domain is a single-chain variable fragment (scFv), a Fab, which is optionally crosslinked, or a F(ab)2.

4. The antigen-recognizing receptor of claim 3, wherein the extracellular antigenbinding domain is a humanized scFv.

5. The antigen-recognizing receptor of claim 3, wherein one or more of the scFv, Fab and F(ab)2 are comprised in a fusion protein with a heterologous sequence to form the extracellular antigen-binding domain.

6. The antigen-recognizing receptor of claim 1, wherein the heavy chain variable region comprises an amino acid sequence that is at least 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% or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 12.

7. The antigen-recognizing receptor of claim 1, wherein the light chain variable region comprises an amino acid sequence that is at least 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% or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 13.

8. The antigen-recognizing receptor of claim 1, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 12, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 13.

9. The antigen-recognizing receptor of claim 1, wherein the extracellular antigenbinding domain comprises a linker between the heavy chain variable region and the light chain variable region of the extracellular antigen-binding domain.

10. The antigen-recognizing receptor of claim 9, wherein the linker consists of the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.

11. The antigen-recognizing receptor of claim 10, wherein the linker consists of the amino acid sequence set forth in SEQ ID NO: 1.

12. The antigen-recognizing receptor of claim 1, wherein a signal peptide is covalently joined to the 5’ terminus of the extracellular antigen-binding domain.

13. The antigen-recognizing receptor of claim 1, wherein the heavy chain variable region and the light chain variable region are positioned from the N- to the C-terminus: VH-VL.

14. The antigen-recognizing receptor of claim 1, wherein the heavy chain variable region and the light chain variable region are positioned from the N- to the C-terminus: VL-VH.

15. An antigen-recognizing receptor comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen-binding domain specifically binds to mesothelin and comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDR1, a CDR2, and a CDR3 of a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 12, and the light chain variable region comprises a CDR1, a CDR2, and a CDR3 of a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 13.

16. An antigen-recognizing receptor comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein theextracellular antigen-binding domain specifically binds to mesothelin and comprises a single chain variable region (scFv) comprising the amino acid sequence set forth in SEQ ID NO: 16.

17. The antigen-recognizing receptor of any one of claims 1-16, wherein the transmembrane domain comprises a CD8 polypeptide, a CD28 polypeptide, a CD3^ polypeptide, a CD4 polypeptide, a 4-1BB polypeptide, an 0X40 polypeptide, an ICOS polypeptide, a CTLA-4 polypeptide, a PD-1 polypeptide, a LAG-3 polypeptide, a 2B4 polypeptide, a BTLA polypeptide, or a combination thereof.

18. The antigen-recognizing receptor of any one of claims 1-16, wherein the intracellular signaling domain comprises a CD3(^ polypeptide.

19. The antigen-recognizing receptor of claim 18, wherein the intracellular signaling domain further comprises at least one co-stimulatory signaling region.

20. The antigen-recognizing receptor of claim 19, wherein the at least one costimulatory signaling region comprises a CD28 polypeptide, a 4-1BB polypeptide, an 0X40 polypeptide, an ICOS polypeptide, a DAP- 10 polypeptide, or a combination thereof.

21. The antigen-recognizing receptor of claim 1, wherein the antigen-recognizing receptor is a chimeric antigen receptor (CAR).

22. The antigen-recognizing receptor of claim 1, wherein the antigen-recognizing receptor is recombinantly expressed or expressed from a vector.

23. The antigen-recognizing receptor of claim 22, wherein the vector is a low copy number vector.

24. A cell comprising the antigen-recognizing receptor of any one of claims 1-23.

25. The cell of claim 24, wherein the antigen-recognizing receptor is constitutively expressed on the surface of the cell.

26. The cell of claim 24 or 25, wherein the cell is an immunoresponsive cell.

27. The cell of any one of claims 24-26, wherein the cell is a cell of the lymphoid lineage or a cell of the myeloid lineage.

28. The cell of any one of claims 24-27, wherein the cell is selected from the group consisting of a T cell, a Natural Killer (NK) cell, and a stem cell from which a lymphoid cell may be differentiated.

29. The cell of claim 28, wherein the cell is a T cell.

30. The cell of claim 29, wherein the T cell is a cytotoxic T lymphocyte (CTL) or a regulatory T cell.

31. The cell of claim 28, wherein the stem cell is a pluripotent stem cell.

32. The cell of claim 31, wherein the pluripotent stem cell is an embryoid stem cell or an induced pluripotent stem cell.

33. A nucleic acid molecule encoding the antigen-recognizing receptor of any one of claims 1-23.

34. A vector comprising the nucleic acid molecule of claim 33.

35. The vector of claim 34, wherein the vector is a low copy number vector.

36. The vector of claim 35, wherein the vector is a retroviral vector.

37. The vector of claim 36, wherein the retroviral vector is a y-retroviral vector or a lentiviral vector.

38. A lipid nanoparticle comprising the nucleic acid of claim 33.

39. A composition comprising the cell of any one of claims 24-32, the nucleic acid molecule of claim 33, the vector of any one of claims 34-37, or the lipid nanoparticle of claim 38.

40. The composition of claim 39, which is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

41. A method of treating and / or preventing a tumor in a subject in need thereof, the method comprising administering to the subject an effective amount of the cell of any one of claims 24-32 or the composition of claim 39 or 40.

42. The method of claim 41 , wherein the method reduces the number of tumor cells, reduces tumor size, and / or eradicates the tumor in the subject.

43. A method of increasing or lengthening survival of a subject having a tumor, comprising administering to the subject an effective amount of the cell of any one of claims 24-32 or the composition of claim 39 or 40.

44. The method of any one of claims 41-43, wherein the tumor is associated with mesothelin.

45. The method of any one of claims 41-44, wherein the tumor is a solid tumor.

46. The method of claim 45, wherein the solid tumor is selected from mesothelioma, renal cancer, bladder cancer, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic carcinoma, endometrial carcinoma, stomach cancer, or cholangiocarcinoma.

47. A method for producing a cell comprising an antigen-recognizing receptor of any one of claims 1-23, comprising introducing into the cell a nucleic acid molecule that encodes the antigen-recognizing receptor.

48. A kit for reducing tumor burden in a subject, treating and / or preventing a tumor in a subject, and / or increasing or lengthening survival of a subject having a tumor, comprising the cell of any one of claims 24-32.

49. The kit of claim 48, wherein the kit further comprises written instructions for using the cell for reducing tumor burden in a subject, treating and / or preventing a tumor or neoplasm in a subject, and / or increasing or lengthening survival of a subject having a tumor.

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