Anti-mesothelin CART cells secreting TEAM and methods of use thereof

Abstract

The present disclosure relates to mesothelin chimeric antigen receptors (CARs), T cell engaging molecules (TEAMs), anti-mesothelin-CAR T cells, optionally comprising a TEAM, and methods of use thereof. Figure 12A

Description

[Technical field]

[0001] [Related Applications] This application claims the benefit under 35 U.S.C. §119(e) to U.S. Provisional Application No. 63 / 275911, filed November 4, 2021, entitled "ANTI-MESOTHELIN CAR T CELLS SECRETING TEAM AND METHODS OF USE THEREOF," and U.S. Provisional Application No. 63 / 278825, filed November 12, 2021, entitled "ANTI-MESOTHELIN CAR T CELLS SECRETING TEAM AND METHODS OF USE THEREOF," the entire contents of each of which are incorporated herein by reference.

[0002] [Federally Sponsored Research] This invention was made with government support under R01CA238268 awarded by the National Cancer Institute and T32CA009216 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0003] [Reference to Electronic Sequence Listing] The contents of the electronic sequence listing (M105370026WO00-SEQ-ARM.xml; size: 50238 bytes; creation date: November 3, 2022) are incorporated herein by reference in their entirety. [Background technology]

[0004] Many diseases, including cancer and autoimmune diseases, are associated with overexpression of mesothelin. Chimeric antigen receptor (CAR) T cells have previously been used to specifically bind to diseased cells and cause their death. Summary of the Invention

[0005] Described herein are mesothelin-targeted CARs and CAR T cells, which may be useful for treating a subject having a disease or disorder, such as, for example, cancer, a plasma cell dyscrasia or disorder, or an autoimmune disease or disorder. Also provided herein are nucleic acids and vectors encoding the CARs, as well as cells comprising the CARs, nucleic acids, or vectors described herein. Also provided herein are pharmaceutical compositions comprising the CARs, CAR T cells, and other compositions described herein. Also provided are methods of treating cancer or an autoimmune disease in a subject by administering the compositions described herein.

[0006] Accordingly, in one aspect, the present disclosure relates to a chimeric antigen receptor (CAR) T cell comprising a heterologous nucleic acid molecule, wherein the heterologous nucleic acid molecule comprises a first polynucleotide encoding a CAR comprising an extracellular antigen-binding domain that binds to a tumor antigen, comprising mesothelin, or a portion thereof; a transmembrane domain; and an intracellular signaling domain.

[0007] In some embodiments, the CAR T cells further comprise a second polynucleotide encoding a therapeutic agent. In some embodiments, the second polynucleotide is part of a heterologous nucleic acid molecule. In some embodiments, the CAR T cells further comprise a second heterologous nucleic acid molecule comprising the second polynucleotide.

[0008] In another aspect, the disclosure relates to a chimeric antigen receptor (CAR) comprising an extracellular antigen binding domain that binds to a tumor antigen comprising mesothelin or a portion thereof, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the CAR comprises a polynucleotide or polypeptide therapeutic.

[0009] In some embodiments, the therapeutic agent comprises an antibody reagent. In some embodiments, the antibody reagent comprises a single chain antibody or a single domain antibody. In some embodiments, the antibody reagent comprises a bispecific antibody reagent. In some embodiments, the bispecific antibody reagent comprises a T-cell engaging antibody molecule (TEAM). In some embodiments, the single domain antibody comprises a camelid antibody. In some embodiments, the therapeutic agent comprises a cytokine.

[0010] In some embodiments, the CAR and therapeutic agent are produced in the form of a single polypeptide that is cleaved to produce separate CAR and therapeutic agent molecules. In some embodiments, the single polypeptide comprises a cleavable moiety between the CAR and the therapeutic agent. In some embodiments, the cleavable moiety comprises a 2A peptide, a 2A ribosomal skipping sequence, or an IRES. In some embodiments, the 2A peptide comprises P2A or T2A.

[0011] In some embodiments, the CAR and therapeutic agent are each constitutively expressed. In some embodiments, expression of the CAR and therapeutic agent is driven by the elongation factor 1 alpha (EF1α) promoter. In some embodiments, the therapeutic agent is expressed under the control of an inducible promoter, optionally inducible by T cell receptor or CAR signaling. In some embodiments, the inducible promoter comprises an NFAT promoter. In some embodiments, the CAR is expressed under the control of a constitutive promoter and the therapeutic agent is expressed under the control of an inducible promoter, optionally inducible by T cell receptor or CAR signaling. In some embodiments, the CAR is expressed from a first vector and the therapeutic agent is expressed from a second vector.

[0012] In some embodiments, the CAR comprises one or more costimulatory domains, hi some embodiments, the costimulatory domain comprises a 4-1BB costimulatory domain, optionally comprising the sequence of SEQ ID NO:25 or a variant thereof.

[0013] In some embodiments, the antigen binding domain of the CAR comprises an antibody, a single chain antibody, a single domain antibody, or a ligand.

[0014] In some embodiments, the transmembrane domain of the CAR comprises a CD8 hinge / transmembrane domain, optionally comprising the sequence of SEQ ID NO: 24, or a variant thereof. In some embodiments, the intracellular signaling domain comprises a CD3 zeta intracellular signaling domain, optionally comprising the sequence of any one of SEQ ID NOs: 26-27, or a variant thereof.

[0015] In some embodiments, the therapeutic agent binds to a tumor antigen. In some embodiments, the tumor antigen to which the therapeutic agent binds is a solid tumor antigen. In some embodiments, the tumor antigen to which the therapeutic agent binds is an activated fibroblast marker. In some embodiments, the activated fibroblast marker comprises αSMA (ACTA2), fibroblast activation protein (FAP), platelet-derived growth factor receptors α and β (PDGFRA, PDGFRB), fibroblast specific protein 1 (FSP1 / S100A4), endoglin (ENG), transgelin (TAGLN), tenascin-C (TNC), periostin (POSTN), chondroitin sulfate proteoglycan 4 or neuron glial antigen 2 (CSPG4 / NG2), podoplanin (PDPN), or osteopontin (SPP1). In some embodiments, the activated fibroblast marker comprises FAP.

[0016] In some embodiments, the VH of the antibody reagent tumor antigen binding domain comprises the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:6. In some embodiments, the VL of the antibody reagent tumor antigen binding domain comprises the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:7. In some embodiments, the antibody reagent tumor antigen binding domain comprises the amino acid sequence of SEQ ID NO:8, or an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:8. In some embodiments, the VH of the antibody reagent tumor antigen binding domain is N-terminal to the VL of the antibody reagent tumor antigen binding domain. In some embodiments, the VL of the antibody reagent tumor antigen binding domain is N-terminal to the VH of the antibody reagent tumor antigen binding domain. In some embodiments, the antibody reagent tumor antigen binding domain comprises a single chain variable fragment (scFv) or a single domain antibody, optionally comprising the sequence of SEQ ID NO:8 or a variant thereof.

[0017] In some embodiments, the antibody reagent binds to a T cell surface antigen. In some embodiments, the T cell surface antigen comprises a T cell receptor (TCR), a TCR complex component, or a portion of any of these. In some embodiments, the TCR complex component is selected from CD3 (e.g., CD3ε subunit, CD3ζ subunit, or CD3γ subunit), a CD4 co-receptor, and a CD8 co-receptor. In some embodiments, the TCR complex component is CD3.

[0018] In some embodiments, the VH of the antibody reagent T cell surface antigen binding domain comprises the amino acid sequence of SEQ ID NO:9, or an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:9. In some embodiments, the VL of the antibody reagent T cell surface antigen binding domain comprises the amino acid sequence of SEQ ID NO:10, or an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:10. In some embodiments, the VH of the antibody reagent T cell surface antigen binding domain is N-terminal to the VL of the antibody reagent T cell surface antigen binding domain. In some embodiments, the VL of the antibody reagent T cell surface antigen binding domain is N-terminal to the VH of the antibody reagent T cell surface antigen binding domain. In some embodiments, the antibody reagent T cell surface antigen binding domain comprises an scFV or a single domain antibody, optionally comprising the sequence of any one of SEQ ID NOs:11-12, or a variant thereof.

[0019] In some embodiments, the antigen binding domain of the CAR comprises: (a) a heavy chain variable domain (VH) comprising three complementarity determining regions CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1 comprises the amino acid sequence of SEQ ID NO: 13, or an amino acid sequence with not more than one, two, or three amino acid substitutions of SEQ ID NO: 13; CDR-H2 comprises the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence with not more than one, two, or three amino acid substitutions of SEQ ID NO: 14; and CDR-H3 comprises the amino acid sequence of SEQ ID NO: 15, or an amino acid sequence with not more than one, two, or three amino acid substitutions of SEQ ID NO: 15. and (b) a light chain variable domain (VL) comprising three complementarity determining regions CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 comprises the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence with not more than one, two, or three amino acid substitutions of SEQ ID NO: 16; CDR-L2 comprises the amino acid sequence of SEQ ID NO: 17, or an amino acid sequence with not more than one, two, or three amino acid substitutions of SEQ ID NO: 17; and CDR-L3 comprises the amino acid sequence of SEQ ID NO: 18, or an amino acid sequence with not more than one, two, or three amino acid substitutions of SEQ ID NO: 18. In some embodiments, the VH of the antigen binding domain of the CAR comprises the amino acid sequence of SEQ ID NO: 19, or an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 19. In some embodiments, the VL of the antigen binding domain of the CAR comprises the amino acid sequence of SEQ ID NO:20, or an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:20. In some embodiments, the VH of the antigen binding domain of the CAR is N-terminal to the VL of the antigen binding domain of the CAR. In some embodiments, the VL of the antigen binding domain of the CAR is N-terminal to the VH of the antigen binding domain of the CAR. In some embodiments, the antigen binding domain of the CAR comprises an scFv or a single domain antibody, optionally comprising a sequence selected from the group consisting of SEQ ID NO:21 or 22, and variants thereof.

[0020] In some embodiments, the antigen binding domain of the CAR comprises: (a) a heavy chain variable domain (VH) comprising complementarity determining regions CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1 comprises the amino acid sequence of SEQ ID NO: 42, or an amino acid sequence with no more than one, two, or three amino acid substitutions therein; CDR-H2 comprises the amino acid sequence of SEQ ID NO: 43, or an amino acid sequence with no more than one, two, or three amino acid substitutions therein; and CDR-H3 comprises the amino acid sequence of SEQ ID NO: 44, or an amino acid sequence with no more than one, two, or three amino acid substitutions therein. and (b) a light chain variable domain (VL) comprising complementarity determining regions CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 comprises the amino acid sequence of SEQ ID NO:45, or an amino acid sequence with no more than 1, 2, or 3 amino acid substitutions thereto; CDR-L2 comprises the amino acid sequence of SEQ ID NO:46, or an amino acid sequence with no more than 1, 2, or 3 amino acid substitutions thereto; and CDR-L3 comprises the amino acid sequence of SEQ ID NO:47, or an amino acid sequence with no more than 1, 2, or 3 amino acid substitutions thereto. In some embodiments, the VH of the antigen binding domain of the CAR comprises the SS1 heavy chain sequence of SEQ ID NO:40, or an amino acid sequence with at least 90% sequence identity to the SS1 heavy chain sequence of SEQ ID NO:40. In some embodiments, the VL of the antigen binding domain of the CAR comprises the SS1 light chain sequence of SEQ ID NO: 41, or an amino acid sequence having at least 90% sequence identity to the SS1 light chain sequence of SEQ ID NO: 41. In some embodiments, the VH of the antigen binding domain of the CAR is N-terminal to the VL of the antigen binding domain of the CAR. In some embodiments, the VL of the antigen binding domain of the CAR is N-terminal to the VH of the antigen binding domain of the CAR. In some embodiments, the antigen binding domain of the CAR comprises an scFv or a single domain antibody, optionally comprising the SS1 VH amino acid sequence of SEQ ID NO: 40 and the SS1 VL amino acid sequence of SEQ ID NO: 41, and variants thereof.

[0021] In some embodiments, the heterologous nucleic acid molecule further comprises a suicide gene.

[0022] In another aspect, the disclosure relates to a nucleic acid molecule comprising a sequence encoding a CAR polypeptide described herein. In some embodiments, the nucleic acid molecule further comprises a sequence encoding a therapeutic agent described herein. In some embodiments, the nucleic acid molecule is a plasmid or vector.

[0023] In another aspect, the disclosure relates to a viral vector comprising the nucleic acid molecule described herein. In some embodiments, the viral vector is a lentiviral or adeno-associated viral vector.

[0024] In another aspect, the disclosure relates to a pharmaceutical composition comprising a CAR T cell, a CAR polypeptide, or a nucleic acid molecule or viral vector described herein. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of a CAR T cell, a CAR polypeptide, or a nucleic acid molecule or viral vector described herein. In some embodiments, the pharmaceutical composition comprises a pharma- ceutically acceptable excipient.

[0025] In another aspect, the disclosure relates to a method of treating a patient having cancer, comprising administering to the patient a pharmaceutical composition described herein. In some embodiments, targeting the tumor microenvironment reduces systemic toxicity. In some embodiments, the cancer is characterized by the presence of one or more solid tumors. In some embodiments, the cancer is characterized by expression of mesothelin. In some embodiments, the tumor microenvironment is characterized by the presence of activated fibroblasts. In some embodiments, the tumor microenvironment is characterized by the presence of fibroblast activation protein (FAP). In some embodiments, the cancer is pancreatic cancer, lung cancer, ovarian cancer, endometrial cancer, biliary tract cancer, gastric cancer, or mesothelioma.

[0026] In another aspect, the disclosure relates to a method of treating a patient with cancer, comprising administering to the patient a CAR T cell product genetically modified to secrete a tumor toxic antibody or cytokine, whereby systemic toxicity is reduced by targeting the cancer toxicity locally to the tumor microenvironment. In some embodiments, the CAR T cell is genetically modified to deliver an antibody or bispecific antibody against an activated fibroblast marker to the tumor microenvironment. In some embodiments, the CAR T cell comprises a polynucleotide encoding a CAR comprising an extracellular antigen binding domain that binds to a tumor antigen comprising mesothelin or a portion thereof. In some embodiments, the bispecific antibody is against an activated fibroblast marker and CD3.

[0027] In another aspect, the disclosure relates to a method of delivering a therapeutic agent to a tissue or organ of a patient to treat a disease or condition, comprising administering to said patient CAR T cells genetically modified to secrete a therapeutic antibody, toxin, or drug, where the therapeutic antibody, toxin, or drug is unable to enter or penetrate the tissue or organ by itself. In some embodiments, the tissue or organ is in the digestive system or endocrine system. In some embodiments, the tissue is in the pancreas or the organ is the pancreas. In some embodiments, the tissue or organ is in the reproductive system. In some embodiments, the tissue is in the ovary or the organ is the ovary. In some embodiments, the tissue or organ is in the respiratory system or pulmonary system. In some embodiments, the tissue is in the lung or the organ is the lung. In some embodiments, the therapeutic antibody is against an activated fibroblast marker.

[0028] In some embodiments of the methods described herein, the CAR T cell is a CAR T cell described herein, or the CAR T cell is comprised within a pharmaceutical composition described herein.

[0029] For convenience, the meanings of some terms and phrases used in the specification, examples, and appended claims are provided below. Unless otherwise specified or implied from the context, the following terms and phrases include the meanings set forth below. The meanings are provided to help describe certain embodiments and are not intended to limit the claimed technology, since the scope is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this technology belongs. In the event of an apparent discrepancy between the usage of a term in the art and its meaning provided herein, the meaning provided in the specification shall prevail.

[0030] As used herein, "subject" refers to a human or animal. Typically, an animal is a vertebrate, such as a primate, rodent, domestic animal, or game animal. Primates include, for example, chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, such as rhesus monkeys. Rodents include, for example, mice, rats, marmots, ferrets, rabbits, and hamsters. Domestic and game animals include, for example, cows, horses, pigs, deer, bison, buffalo, feline species, such as house cats, canine species, such as dogs, foxes, wolves, bird species, such as chickens, emus, ostriches, and fish, such as trout, catfish, and salmon. In some embodiments, the subject is a mammal, such as a primate, such as a human. The terms "individual," "patient," and "subject" are used interchangeably herein. The subject may be one who has previously been diagnosed with or has been determined to be suffering from or having a condition requiring treatment (e.g., mesothelioma, or other types of cancer, among others) or one or more complications associated with such a condition, and in some cases, has already been treated for the condition or one or more complications associated with the condition. Alternatively, the subject may also not have previously been diagnosed as having such a condition or associated complication. For example, the subject may be one who exhibits one or more risk factors for the condition or one or more complications associated with the condition, or one who does not exhibit risk factors.

[0031] A "subject in need" of treatment for a particular condition can be a subject who has the condition, has been diagnosed as having the condition, or is at risk for developing the condition.

[0032] As used herein, the terms "tumor antigen" and "cancer antigen" are used interchangeably to mean antigens that are differentially expressed by cancer cells and therefore available for targeting cancer cells (e.g., mesothelin). Cancer antigens are antigens that can potentially stimulate apparently tumor-specific immune responses. Some of these antigens are encoded by normal cells, although not necessarily expressed. These antigens can be characterized as those that are normally silent (i.e., not expressed) in normal cells, those that are expressed only at certain stages of differentiation, and those that are transiently expressed, such as embryonic and fetal antigens. Other cancer antigens are encoded by mutated cellular genes, such as oncogenes (e.g., activated Ras oncogene), suppressor genes (e.g., mutant p53), and fusion proteins resulting from internal deletions or chromosomal translocations. Still other cancer antigens can be encoded by viral genes, such as those carried by RNA and DNA tumor viruses. Many tumor antigens have been defined in terms of multiple solid tumors: immune-defined MAGE1, 2, and 3; MART-1 / Melan-A, gp100, carcinoembryonic antigen (CEA), human epidermal growth factor receptor (HER2), mucin (i.e., MUC-1), prostate-specific antigen (PSA), and prostatic acid phosphatase (PAP). In addition, viral proteins such as some encoded by Hepatitis B (HBV), Epstein-Barr (EBV), and human papilloma (HPV) have been shown to be important in the development of hepatocellular carcinoma, lymphoma, and cervical cancer, respectively. In some embodiments, the tumor antigen is mesothelin. Examples of tumor antigens are provided below and include, for example, mesothelin, EGFR, EGFRvIII, CD19, PSMA, B-cell maturation antigen (BCMA), interleukin-13 receptor subunit alpha-2 (IL13Ra2), and the like. In some embodiments, the tumor antigen is a tumor-associated antigen. A tumor-associated antigen may be present near a cancer cell or tumor, but not necessarily attached to the cancer cell or tumor. For example, a tumor-associated antigen may be a marker present in the extracellular matrix surrounding or adjacent to a tumor (e.g., at increased levels compared to normal tissue).

[0033] As used herein, the term "chimera" refers to a fusion product of portions of at least two or more different polynucleotide molecules. In some embodiments, the term "chimera" refers to a gene expression element produced through the manipulation of known elements or other polynucleotide molecules.

[0034] As used herein, the term "T cell engaging antibody molecule" or "TEAM" refers to a polypeptide comprising single chain variable fragments (scFvs) linked in tandem. Optionally, the scFvs are linked by a linker (e.g., a glycine-rich linker). One scFv of the TEAM binds to a T cell surface antigen (e.g., a T cell receptor (TCR) or a portion thereof (e.g., CD3ε subunit)) and the other binds to a target antigen (e.g., a tumor antigen). In some embodiments, the T cell surface antigen is a Treg antigen.

[0035] As used herein, "Treg antigen" or "Treg-associated antigen" are used interchangeably to mean antigens expressed by regulatory T (Treg) cells. These antigens may, in some cases, be targeted by the antibody reagents, antibody conjugates, and methods of the present disclosure. Examples of Treg antigens are provided below and include, for example, GARP, LAP, CD25, and CTLA-4.

[0036] In some embodiments, "activation" can refer to the state of a T cell that has been sufficiently stimulated to induce detectable cell proliferation. In some embodiments, activation can refer to induced cytokine production. In other embodiments, activation can refer to detectable effector function.

[0037] At a minimum, "activated T cells" as used herein are proliferating T cells.

[0038] As used herein, the term "therapeutically effective amount" refers to an amount necessary or sufficient to achieve a desired biological effect. For example, a therapeutically effective amount of a CAR, CAR T cell, or antibody reagent of the present disclosure may be an amount sufficient to ameliorate one or more symptoms of cancer. By selecting from among the various active compounds and weighing factors such as potency, relative bioavailability, patient weight, severity of adverse side effects, and preferred method of administration, in combination with the teachings provided herein, an effective prophylactic or therapeutic treatment regimen can be designed that does not cause substantial toxicity and is completely effective in treating a particular subject. The effective amount for any particular application may vary depending on factors such as the disease or condition being treated, the particular therapeutic compound being administered, the size of the subject, or the severity of the disease or condition. Those of ordinary skill in the art can empirically determine the effective amount of a particular therapeutic compound associated with the present disclosure without necessitating undue experimentation.

[0039] As used herein, the terms "specific binding" and "specifically bind" refer to a physical interaction between two molecules, compounds, cells and / or particles in which a first entity binds to a second target entity with greater specificity and affinity than it binds to a third, non-target entity. In some embodiments, specific binding can refer to an affinity of a first entity for a second target entity that is at least 10 times, at least 50 times, at least 100 times, at least 500 times, at least 1000 times or more greater than its affinity for a third, non-target entity under the same conditions. A reagent specific for a given target is one that exhibits specific binding for that target under the conditions of the assay being utilized. Non-limiting examples include antibodies or ligands that recognize and bind to their cognate binding partner (e.g., stimulatory and / or costimulatory molecule) proteins present on T cells.

[0040] As used herein, a "stimulatory ligand" refers to a ligand that, when present on an antigen-presenting cell (APC) (e.g., macrophage, dendritic cell, B cell, artificial APC, etc.), can specifically bind to a cognate binding partner (referred to herein as a "stimulatory molecule" or "costimulatory molecule") on a T cell, thereby mediating a primary response by the T cell, including, but not limited to, proliferation, activation, initiation of an immune response, etc. Stimulatory ligands are well known in the art and include, among others, peptide-loaded MHC class I molecules, anti-CD3 antibodies, superagonist anti-CD28 antibodies, and superagonist anti-CD2 antibodies.

[0041] "Stimulatory molecule," as the term is used herein, refers to a molecule on a T cell that specifically binds to a cognate stimulatory ligand present on an antigen presenting cell. "Co-stimulatory ligand," as the term is used herein, includes a molecule on an APC that specifically binds to a cognate costimulatory molecule on a T cell, thereby providing a signal that mediates T cell responses, including, but not limited to, proliferation, activation, differentiation, etc., in addition to the primary signal provided by engagement of the TCR / CD3 complex with a peptide-loaded MHC molecule. Costimulatory ligands can include, but are not limited to, 4-1BBL, OX40L, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3 / TR6, IL T3, IL T4, HVEM, agonists or antibodies that bind to Toll-like receptors, and ligands that specifically bind B7-H3. Costimulatory ligands can also include, but are not limited to, antibodies that specifically bind to costimulatory molecules present on T cells, such as, but not limited to, ligands that specifically bind to CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83.

[0042] "Costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA, Toll-like receptors, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83.

[0043] In some embodiments, the term "modified" and its grammatical equivalents as used herein can refer to one or more human-designed modifications of a nucleic acid, e.g., a nucleic acid within the genome of an organism. In other embodiments, modified can refer to a change, addition, and / or deletion of a gene. A "modified cell" can refer to a cell in which a gene has been added, deleted, and / or changed.

[0044] As used herein, the term "cells" or "modified cells" and their grammatical equivalents can refer to cells of human or non-human animal origin.

[0045] As used herein, the term "operably linked" refers to a first polynucleotide molecule, such as a promoter, linked to a second transcribable polynucleotide molecule, such as a gene of interest, where the polynucleotide molecules are arranged such that the first polynucleotide molecule affects the function of the second polynucleotide molecule. The two polynucleotide molecules may or may not be part of a single contiguous polynucleotide molecule, and may or may not be adjacent. For example, a promoter is operably linked to a gene of interest if the promoter regulates or mediates the transcription of the gene of interest in a cell.

[0046] It is further contemplated that the various embodiments described herein encompass any variant (naturally occurring or otherwise), allele, homolog, conservatively modified variant, and / or conservatively substituted variant of the specific polypeptide described. With respect to amino acid sequences, those skilled in the art will recognize that individual substitutions, deletions, or additions in a nucleic acid, peptide, polypeptide, or protein sequence that alter a single amino acid or a small percentage of amino acids in the encoded sequence are "conservatively modified variants" where the change results in the replacement of an amino acid with a chemically similar amino acid and retains the desired activity of the polypeptide. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles consistent with the disclosure.

[0047] A given amino acid can be replaced with a residue having similar physiochemical properties, for example, one aliphatic residue can be substituted for another aliphatic residue (e.g., Ile, Val, Leu, or Ala for one another), or one polar residue can be substituted for another polar residue (e.g., Lys for Arg; Glu for Asp; or Gln for Asn). Other such conservative substitutions, such as the substitution of entire regions with similar hydrophobic properties, are well known. Polypeptides containing conservative amino acid substitutions can be tested in any one of the assays described herein to confirm that the desired activity, e.g., ligand-mediated receptor activity and specificity, of the native or reference polypeptide is retained. Amino acids can be grouped according to the similarity of the properties of their side chains (A. L. Lehninger, in Biochemistry, 2nd ed., pp. 73-75, Worth Publishers, New York (1975)): (1) nonpolar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His (H). Alternatively, naturally occurring residues can be divided into groups based on common side chain properties: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that affect chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions involve exchanging a member of one of these classes for another class.Particular conservative substitutions include, for example, replacement of Ala with Gly or Ser; Arg with Lys; Asn with Gln or His; Asp with Glu; Cys with Ser; Gln with Asn; Glu with Asp; Gly with Ala or Pro; His with Asn or Gln; Ile with Leu or Val; Leu with Ile or Val; Lys with Arg, Gln, or Glu; Met with Leu, Tyr or Ile; Phe with Met, Leu or Tyr; Ser with Thr; Tyr with Ser; Trp with Tyr; Tyr with Trp; and / or Phe with Val, Ile, or Leu.

[0048] In some embodiments, a polypeptide described herein (or a nucleic acid encoding such a polypeptide) can be a functional fragment of one of the amino acid sequences described herein. As used herein, a "functional fragment" is a fragment or segment of a peptide that retains at least 50% of the activity of a wild-type reference polypeptide according to assays known in the art or described herein below. Functional fragments can include conservative substitutions of the sequences disclosed herein.

[0049] In some embodiments, the polypeptides described herein may be variants of the polypeptides or molecules described herein. In some embodiments, the variants are conservatively modified variants. Conservative substitution variants can be obtained, for example, by mutation of the native nucleotide sequence. A "variant" as referred to herein is a polypeptide that is substantially homologous to a native or reference polypeptide, but has an amino acid sequence that differs from that of the native or reference polypeptide due to one or more deletions, insertions, or substitutions. A DNA sequence encoding a variant polypeptide includes sequences encoding variant proteins or fragments thereof that contain one or more additions, deletions, or substitutions of nucleotides when compared to the native or reference DNA sequence, but that retain the activity of the non-variant polypeptide. A wide variety of PCR-based site-directed mutagenesis approaches are known in the art and can be applied by the skilled artisan.

[0050] A variant amino acid or DNA sequence may be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to a native or reference sequence. The degree of homology (percent identity) between a native and a variant sequence can be determined, for example, by comparing the two sequences using free publicly available computer programs commonly used for this purpose on the World Wide Web (e.g., BLASTp or BLASTn with default settings).

[0051] Modification of the native amino acid sequence can be accomplished by any of a number of techniques known to those of skill in the art. Mutations can be introduced at specific loci, for example, by synthesizing oligonucleotides containing the mutated sequence flanked by restriction sites that allow ligation to fragments of the native sequence. After ligation, the resulting reconstructed sequence encodes an analog with the desired amino acid insertion, substitution, or deletion. Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can be used to obtain modified nucleotide sequences with specific codons modified according to the required substitution, deletion, or insertion. Techniques for making such modifications are well established and include, for example, those disclosed in Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene 37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981); and U.S. Patent Nos. 4,518,584 and 4,737,462, which are incorporated herein by reference in their entireties. Any cysteine ​​residue not involved in maintaining the proper conformation of the polypeptide may also be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine ​​bond(s) can also be added to a polypeptide to improve its stability or promote oligomerization.

[0052] The term "polynucleotide" is used interchangeably herein with "nucleic acid molecule" to denote a polymer of nucleosides. Typically, polynucleotides are composed of nucleosides naturally found in DNA or RNA (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine) linked by phosphodiester bonds. However, the term encompasses molecules containing nucleosides or nucleoside analogs, including chemically or biologically modified bases, modified backbones, and the like, whether or not found in naturally occurring nucleic acids, and such molecules may be preferred for certain applications. When this application refers to polynucleotides, it is understood that both DNA, RNA, and in each case single- and double-stranded forms (and the complementary strand of each single-stranded molecule) are provided. As used herein, "polynucleotide sequence" can refer to the polynucleotide material itself and / or to sequence information (i.e., a series of letters used as abbreviations for bases) that biochemically characterize a particular nucleic acid. In some embodiments, the nucleic acid molecule is a heterologous nucleic acid molecule. As used herein, the term "heterologous nucleic acid molecule" refers to a nucleic acid molecule that does not naturally occur within a given cell.

[0053] Polynucleotide sequences presented herein are presented in a 5' to 3' orientation, unless otherwise indicated.

[0054] The term "polypeptide" as used herein means a polymer of amino acids. The terms "protein" and "polypeptide" are used interchangeably herein. A peptide is a relatively short polypeptide, typically about 2 to 60 amino acids in length. A polypeptide as used herein typically contains amino acids, such as the 20 L-amino acids most commonly found in proteins. However, other amino acids and / or amino acid analogs known in the art can be used. One or more of the amino acids in a polypeptide can be modified, for example, by adding chemical entities such as carbohydrate groups, phosphate groups, fatty acid groups, linkers for conjugation, functional groups, etc. A polypeptide to which a non-polypeptide moiety is covalently or non-covalently attached is still considered a "polypeptide". Exemplary modifications include glycosylation and palmitoylation. A polypeptide can be purified from a natural source, produced using recombinant DNA technology, or synthesized by chemical means such as conventional solid-phase peptide synthesis. The terms "polypeptide sequence" or "amino acid sequence" as used herein can refer to the polypeptide material itself and / or to sequence information that biochemically characterizes the polypeptide (i.e., a series of letters or three-letter codes used as abbreviations for the names of amino acids). Polypeptide sequences presented herein are presented in an N-terminal to C-terminal orientation, unless otherwise indicated.

[0055] In some embodiments, the nucleic acid encoding a polypeptide described herein (e.g., a CAR polypeptide) is contained by a vector. In some of the aspects described herein, a nucleic acid sequence encoding a given polypeptide described herein or any module thereof is operably linked to a vector. As used herein, the term "vector" refers to a nucleic acid construct designed for delivery to a host cell or for transfer between different host cells. As used herein, a vector can be viral or non-viral. The term "vector" encompasses any genetic element that can replicate when associated with the appropriate regulatory elements and transfer a genetic sequence to a cell. Vectors can include, but are not limited to, cloning vectors, expression vectors, plasmids, phages, transposons, cosmids, artificial chromosomes, viruses, virions, and the like.

[0056] As used herein, the term "expression vector" refers to a vector that directs the expression of an RNA or polypeptide from a sequence linked to a transcriptional regulatory sequence on the vector. The sequence to be expressed is often, but not necessarily, heterologous to the cell. An expression vector may contain additional elements, for example, an expression vector may have two replication systems, thereby allowing it to be maintained in two organisms, for example, in human cells for expression and in prokaryotic hosts for cloning and amplification. The term "expression" refers to the intracellular processes involved in the production of RNA and proteins, and suitably the secretion of proteins, including, but not limited to, transcription, transcript processing, translation, and protein folding, modification, and processing, where applicable. An "expression product" includes RNA transcribed from a gene and a polypeptide obtained by translation of mRNA transcribed from a gene.

[0057] The term "gene" refers to a nucleic acid sequence that is transcribed (DNA) into RNA in vitro or in vivo when operably linked to appropriate regulatory sequences. A gene may or may not include regions preceding and following the coding region, such as 5' untranslated (5'UTR) or "leader" sequences and 3'UTR or "trailer" sequences, as well as intervening sequences (introns) between individual coding segments (exons).

[0058] As used herein, the term "viral vector" refers to a nucleic acid vector construct that contains at least one element of viral origin and is capable of being packaged into a viral vector particle. The viral vector may contain a nucleic acid encoding a polypeptide as described herein in place of non-essential viral genes. The vector and / or particle may be utilized for the purpose of transferring the nucleic acid into cells either in vitro or in vivo. Many forms of viral vectors are known in the art.

[0059] By "recombinant vector" is meant a vector that contains a heterologous nucleic acid sequence or "transgene" that can be expressed in vivo. It is understood that the vectors described herein can, in some embodiments, be combined with other suitable compositions and therapeutics. In some embodiments, the vector is episomal. The use of a suitable episomal vector provides a means to maintain a nucleotide of interest in a high copy number of extrachromosomal DNA in a subject, thereby eliminating the potential effects of chromosomal integration.

[0060] As used herein, "signal peptide" or "signal sequence" refers to a peptide at the N-terminus of a newly synthesized protein that serves to direct the nascent protein to the endoplasmic reticulum. In some embodiments, the signal peptide is a CD8 or IgK signal peptide.

[0061] As used herein, the terms "treat", "treatment", "treating" or "amelioration" refer to therapeutic treatment whose purpose is to reverse, alleviate, ameliorate, inhibit, slow or halt the progression or severity of a condition associated with a disease or disorder, e.g., cancer or an autoimmune disease. The term "treating" includes reducing or alleviating at least one side effect or symptom of a disease, disease or disorder. Treatment is generally "effective" if one or more symptoms or clinical markers are reduced. Alternatively, treatment is "effective" if the progression of a disease is reduced or halted. That is, "treatment" includes not only the improvement of symptoms or markers, but also the halting or at least slowing of the progression or worsening of symptoms compared to symptoms that would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptoms, reduction in the extent of disease, stable disease (i.e., not getting worse), delay or slowing of disease progression, improvement or palliation of the disease state, remission (partial or complete), and / or reduced mortality, whether detectable or undetectable. The term "treatment" of a disease also includes providing relief from the symptoms or side effects of the disease (including palliative treatment).

[0062] As used herein, the term "about" or "approximately" when used in connection with a quantity refers to a range of values ​​around said quantity, ±1, 2, 3, 4, 5, 6, 7, 8, 9, or 10%, for example, ±1% or ±10%.

[0063] As used herein, the singular terms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly dictates otherwise. Other terms are defined in the description of various aspects and embodiments of the technology, as set forth below. [Brief description of the drawings]

[0064] The accompanying drawings are not intended to be drawn to scale. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Figure 1A-1C] Figures 1A-1E are graphs showing cytokine production in SS1 anti-mesothelin CAR T cells expressing the previously known anti-mesothelin SS1 CAR compared to exemplary CAR T cells of the present disclosure expressing the MGHmeso1 L / H anti-mesothelin CAR of the present disclosure (white is untransduced control). [Fig. 1D-1E] (the above) [Figure 1F] FIG. IF shows a graph of cell lysis assessed in an overnight luciferase-based killing assay using mesothelin-positive pancreatic cancer (Capan-2) (top) or mesothelin-negative lymphoma cell (JeKo-1) cell lines (bottom). [Figure 2A] Figures 2A-2B show an overview of the experimental mouse protocol used to evaluate the efficacy of the anti-mesothelin SS1 CAR or the anti-mesothelin MGHmeso1 L / H CAR. Figure 2A shows the schedule for administration of pancreatic cancer cells (AsPC-1 cells) to mice followed by administration of CAR T cells. [Figure 2B] Figure 2B shows the CAR T dose, days of treatment, and number of mice per treatment. [Figure 3A-3B]Figures 3A-3D compare anti-mesothelin CAR T cells, including the previously known anti-mesothelin SS1 CAR (C) or the disclosed anti-mesothelin MGHmeso1 L / H CAR (B), in mice injected with ASPC1 pancreatic cancer cells (grey indicates no CAR T cell injection). In each experiment, 1x106 (1e6) CAR T cells were administered. Each of Figures 3A-3D has a panel in the figure header showing flux and survival curves associated with administration of CAR-T doses. Figure 3A compares SS1 CAR T cells and MGHmeso1 L / H CAR T cells administered intravenously (IV) 7 days after cancer cell injection. Figure 3B compares SS1 CAR T cells and MGHmeso1 L / H CAR T cells administered IV 14 days after cancer cell injection. [Figure 3C-3D] Figure 3C compares SS1 and MGHmeso1 L / H CAR T cells administered by intraperitoneal (IP) injection 7 days after cancer cell injection, and Figure 3D compares SS1 and MGHmeso1 L / H CAR T cells administered by intraperitoneal (IP) injection 14 days after cancer cell injection. [Figure 4A-4B] Figures 4A-4D compare anti-mesothelin CAR T cells, including the previously known anti-mesothelin SS1 CAR (C) or the disclosed anti-mesothelin MGHmeso1 L / H CAR (B), in mice injected with ASPC1 pancreatic cancer cells (grey indicates no CAR T cell injection). In each experiment, 2x106 (2e6) CAR T cells were administered. Each of Figures 4A-4D has a panel in the figure header showing flux and survival curves associated with administration of CAR-T doses. Figure 4A compares SS1 CAR T cells and MGHmeso1 L / H CAR T cells administered intravenously (IV) 7 days after cancer cell injection. Figure 4B compares SS1 CAR T cells and MGHmeso1 L / H CAR T cells administered IV 14 days after cancer cell injection. [Fig. 4C-4D]Figure 4C compares SS1 and MGHmeso1 L / H CAR T cells administered by intraperitoneal (IP) injection 7 days after cancer cell injection, and Figure 4D compares SS1 and MGHmeso1 L / H CAR T cells administered by intraperitoneal (IP) injection 14 days after cancer cell injection. [Figure 5A-5B] Figures 5A-5D compare anti-mesothelin CAR T cells, including the previously known anti-mesothelin SS1 CAR (C) or the disclosed anti-mesothelin MGHmeso1 L / H CAR (B), in mice injected with BXPC3 pancreatic cancer cells (grey indicates no CAR T cell injection). In each experiment, 1x106 (1e6) CAR T cells were administered. Each of Figures 5A-5D has a panel in the figure header showing flux and survival curves associated with administration of CAR-T doses. Figure 5A compares SS1 CAR T cells and MGHmeso1 L / H CAR T cells administered intravenously (IV) 7 days after cancer cell injection. Figure 5B compares SS1 CAR T cells and MGHmeso1 L / H CAR T cells administered IV 14 days after cancer cell injection. [Fig. 5C-5D] Figure 5C compares SS1 and MGHmeso1 L / H CAR T cells administered by intraperitoneal (IP) injection 7 days after cancer cell injection, and Figure 5D compares SS1 and MGHmeso1 L / H CAR T cells administered by intraperitoneal (IP) injection 14 days after cancer cell injection. [Figure 6A-6B]Figures 6A-6D compare anti-mesothelin CAR T cells, including the previously known anti-mesothelin SS1 CAR (C) or the disclosed anti-mesothelin MGHmeso1 L / H CAR (B), in mice injected with BXPC3 pancreatic cancer cells (grey indicates no CAR T cell injection). In each experiment, 2x106 (2e6) CAR T cells were administered. Each of Figures 6A-6D has a panel in the figure header showing flux and survival curves associated with administration of CAR-T doses. Figure 6A compares SS1 CAR T cells and MGHmeso1 L / H CAR T cells administered intravenously (IV) 7 days after cancer cell injection. Figure 6B compares SS1 CAR T cells and MGHmeso1 L / H CAR T cells administered IV 14 days after cancer cell injection. [Figure 6C-6D] Figure 6C compares SS1 and MGHmeso1 L / H CAR T cells administered by intraperitoneal (IP) injection 7 days after cancer cell injection, and Figure 6D compares SS1 and MGHmeso1 L / H CAR T cells administered by intraperitoneal (IP) injection 14 days after cancer cell injection. [Figure 7A-7B] Figures 7A-7D compare anti-mesothelin CAR T cells, including the previously known anti-mesothelin SS1 CAR (C) or the disclosed anti-mesothelin MGHmeso1 L / H CAR (B), in mice injected with PANC1 pancreatic cancer cells (grey indicates no CAR T cell injection). In each experiment, 1x106 (1e6) CAR T cells were administered. Each of Figures 7A-7D has a panel in the figure header showing flux and survival curves associated with administration of CAR-T doses. Figure 7A compares SS1 CAR T cells and MGHmeso1 L / H CAR T cells administered intravenously (IV) 7 days after cancer cell injection. Figure 7B compares SS1 CAR T cells and MGHmeso1 L / H CAR T cells administered IV 14 days after cancer cell injection. [Figure 7C-7D]Figure 7C compares SS1 and MGHmeso1 L / H CAR T cells administered by intraperitoneal (IP) injection 7 days after cancer cell injection, and Figure 7D compares SS1 and MGHmeso1 L / H CAR T cells administered by intraperitoneal (IP) injection 14 days after cancer cell injection. [Figure 8A-8B] Figures 8A-8D compare anti-mesothelin CAR T cells, including the previously known anti-mesothelin SS1 CAR (C) or the disclosed anti-mesothelin MGHmeso1 L / H CAR (B), in mice injected with PANC1 pancreatic cancer cells (grey indicates no CAR T cell injection). In each experiment, 2x106 (2e6) CAR T cells were administered. Each of Figures 8A-8D has a panel in the figure header showing flux and survival curves associated with administration of CAR-T doses. Figure 8A compares SS1 CAR T cells and MGHmeso1 L / H CAR T cells administered intravenously (IV) 7 days after cancer cell injection. Figure 8B compares SS1 CAR T cells and MGHmeso1 L / H CAR T cells administered IV 14 days after cancer cell injection. [Fig. 8C-8D] Figure 8C compares SS1 and MGHmeso1 L / H CAR T cells administered by intraperitoneal (IP) injection 7 days after cancer cell injection, and Figure 8D compares SS1 and MGHmeso1 L / H CAR T cells administered by intraperitoneal (IP) injection 14 days after cancer cell injection. [Figure 9A-9B]Figures 9A-9D compare anti-mesothelin CAR T cells, including the previously known anti-mesothelin SS1 CAR (C) or the disclosed anti-mesothelin MGHmeso1 L / H CAR (B), in mice injected with CAPAN2 pancreatic cancer cells (grey indicates no CAR T cell injection). In each experiment, 2.5x105 (2.5e5) CAR T cells were administered. Each of Figures 9A-9D has a panel in the figure header showing flux and survival curves associated with administration of CAR-T doses. Figure 9A compares SS1 CAR T cells and MGHmeso1 L / H CAR T cells administered intravenously (IV) 7 days after cancer cell injection. Figure 9B compares SS1 CAR T cells and MGHmeso1 L / H CAR T cells administered IV 14 days after cancer cell injection. [Fig. 9C-9D] Figure 9C compares SS1 and MGHmeso1 L / H CAR T cells administered by intraperitoneal (IP) injection 7 days after cancer cell injection, and Figure 9D compares SS1 and MGHmeso1 L / H CAR T cells administered by intraperitoneal (IP) injection 14 days after cancer cell injection. [Figure 10A-10B] Figures 10A-10D compare anti-mesothelin CAR T cells, including the previously known anti-mesothelin SS1 CAR (C) or the disclosed anti-mesothelin MGHmeso1 L / H CAR (B), in mice injected with CAPAN2 pancreatic cancer cells (grey indicates no CAR T cell injection). 5x105 (5e5) CAR T cells were administered in each experiment. Each of Figures 10A-10D has a panel in the figure header showing flux and survival curves associated with administration of CAR-T doses. Figure 10A compares SS1 CAR T cells and MGHmeso1 L / H CAR T cells administered intravenously (IV) 7 days after cancer cell injection. Figure 10B compares SS1 CAR T cells and MGHmeso1 L / H CAR T cells administered IV 14 days after cancer cell injection. [Fig. 10C-10D]Figure 10C compares SS1 and MGHmeso1 L / H CAR T cells administered by intraperitoneal (IP) injection 7 days after cancer cell injection, and Figure 10D compares SS1 and MGHmeso1 L / H CAR T cells administered by intraperitoneal (IP) injection 14 days after cancer cell injection. [Figure 11A-11B] Figures 11A-11C compare the specific lysis of anti-mesothelin CART cells bearing a CAR containing the 41BBz intracellular signaling domain and a previously known SS1 anti-mesothelin antibody (C), the MGHmeso1 L / H antibody of the present disclosure (B-1), or the MGHmeso1 H / L antibody of the present disclosure (B-2) of different cancer cell lines (UTD control in white). Figure 11A compares SS1, MGHmeso1 L / H-BBz, and MGHmeso1 H / L-BBz specific lysis of BxPC-3 pancreatic cancer cells. Figure 11B compares SS1, MGHmeso1 L / H-BBz, and MGHmeso1 H / L-BBz CART cell specific lysis of Capan-2 cancer cells. [Figure 11C] FIG. 11C compares SS1, MGHmeso1 L / H-BBz, and MGHmeso1 H / L-BBz specific lysis of NCI-H226 lung cancer cells. [Figure 12A] Figures 12A-12B show schematics and transduction of anti-mesothelin CAR TEAM constructs and control constructs: Figure 12A shows a schematic of the SS1 anti-mesothelin CAR TEAM FAP construct and five additional control constructs. [Figure 12B] Figure 12B shows the transduction efficiency of the anti-mesothelin SS1 CAR TEAM construct and the control construct. [Figure 13] Figure 13 shows the results of a CART cell activation assay in plates with (dark grey) or without (light grey) mesothelin. CD69 expression on sorted CART cells 6 hours after mesothelin stimulation (day 14). [Figure 14A-14B]Figures 14A-14C show flow cytometry analysis (histograms) of supernatants of TEAM-secreting CART cells with secondary His-tag labeling of target cells. Figure 14A shows FAP-positive HFF cells, and Figure 14B shows CD19-positive K562 cells (representative of three individual donors, day 20). [Figure 14C] Figure 14C shows the mean fluorescence intensity (MFI) ratios of three individual healthy donors. Statistical significance was calculated by two-tailed paired t-test. [Figure 15A-15B] Figures 15A-15E show assays to quantify the cytotoxic effect of secreted TEAM. Figure 15A shows a scheme of the co-culture assay. Figures 15B, 15C, 15E show impedance-based cytotoxicity assays of CART secreting TEAM at a 1:1 ratio to human foreskin fibroblasts (HFF in Figures 15B-15C) and at a 1:1 ratio to cancer-associated fibroblasts (CAF-1 in Figure 15E). Figure 15D shows a scheme of the transwell assay. Key to Figure 15A: SS1 anti-mesothelin CAR FAP TEAM (1), supernatant of SS1 anti-mesothelin CAR FAP TEAM (2), SS1 anti-mesothelin CAR (3), and CD19 CAR (4). Key to Figure 15C: SS1 anti-mesothelin CAR CD19 TEAM (1), SS1 anti-mesothelin CAR (2), and CD19 CAR (3). Key to Figure 15E: SS1 anti-mesothelin CAR FAP TEAM (1), headless CAR FAP TEAM (2), anti-mesothelin CAR (3) CD19 CAR (4), CD19 CAR FAP TEAM (5), anti-mesothelin CAR CD19 TEAM (6), and UTD control (7). [Fig. 15C-15D] (the above) [Figure 15E] (the above) [Figure 16]FIG. 16 shows a timeline (top) and graphs (bottom) of an experiment evaluating the effect of anti-mesothelin CAR FAP TEAM on pancreatic tumor growth in mice. The top panel shows that pancreatic model cancer cells were injected into mice 3 days prior to treatment. The bottom panel shows tumor growth over time after treatment with anti-mesothelin CAR FAP TEAM (circles with horizontal line), anti-mesothelin CAR CD19 TEAM (circles with zigzag line), anti-CD19 CAR FAP TEAM (circles with vertical line), or UTD (open circles). Tumor sizes are shown as mean values, and error bars are standard deviations. Statistics were calculated by unpaired t-test, *P=0.0192. [Figure 17] Figure 17 is a graph showing that the avidity of FAP TEAM molecules increases the avidity between T cells and FAP-expressing fibroblasts. HFF-expressing FAP cells interacting with UTD (4, grey) and UTD CAR FAP TEAM (1) or anti-mesothelin CAR FAP TEAM (2) were analyzed using acoustic force microscopy. Anti-CD19 CAR FAP TEAM (3) was the control. The % of bound cells was measured as a function of acoustic force (pN) (left panel). The fold increase in bound cells compared to UTD is shown in the right panel (paired t-test, *P<0.05). MODE FOR CARRYING OUT THE DISCLOSURE

[0065] Provided herein are chimeric antigen receptors (CARs) and CAR T cells that target mesothelin. Without wishing to be bound by theory, MSLN (https: / / www.uniprot.org / uniprot / Q13421) is expressed in normal mesothelial cells in some tissues (e.g., pleura, pericardium, peritoneum), and in trace amounts in some epithelial cells (e.g., ovary, tunica vaginalis testis, rete testis, fallopian tube), but is abundantly expressed in various cancer cells. See, e.g., Lv, Jiang, and Li, Peng “Mesothelin as a biomarker for targeted therapy”. Biomark Res. 2019;7:18; and Hassan et al. “Mesothelin Immunotherapy for Cancer: Ready for Prime Time?” J Clin Oncol. 2016 Dec 1;34(34):4171-4179, each of which is incorporated herein by reference. Mesothelin can be used as a marker for cells associated with (e.g., overexpressed in) various cancers, and CARs and CAR T cells that bind mesothelin or portions thereof can be used to treat subjects having, for example, cancers associated with mesothelin expression.

[0066] [Chimeric Antigen Receptor (CAR)] The technology described here provides improved chimeric antigen receptors (CARs) for use in immunotherapy. Below, we discuss CARs and various improvements.

[0067] As used herein, the term "chimeric antigen receptor" or "CAR" or "CARs" refers to an engineered T cell receptor that transfers ligand or antigen specificity into a T cell (e.g., a naive T cell, a central memory T cell, an effector memory T cell, or a combination thereof). CARs are also known as artificial T cell receptors, chimeric T cell receptors, or chimeric immune receptors.

[0068] CARs are constructed by disposing a chimeric extracellular antigen binding domain that specifically binds to a target, e.g., a polypeptide, expressed on the surface of a cell targeted by a T cell response, in a construct that includes the transmembrane and intracellular domains of a T cell receptor molecule. In some embodiments, the chimeric extracellular antigen binding domain includes an antigen binding domain of an antibody reagent that specifically binds to an antigen expressed on a cell targeted by a T cell response. In some embodiments, the chimeric extracellular antigen binding domain includes a ligand that specifically binds to an antigen expressed on a cell targeted by a T cell response.

[0069] As used herein, "CAR T cell" or "CAR-T" refers to a T cell expressing a CAR. When expressed in a T cell, a CAR has the ability to redirect the specificity and reactivity of the T cell toward a selected target in an MHC-unrestricted manner, utilizing the antigen-binding properties of a monoclonal antibody. MHC-unrestricted antigen recognition endows the CAR-expressing T cell with the ability to recognize antigens independent of antigen processing, bypassing a major mechanism of tumor escape.

[0070] In some embodiments, the CAR polypeptide comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to a sequence selected from any one of SEQ ID NOs: 2-5. In some embodiments, the CAR polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 2-5. In some embodiments, the CAR does not comprise a CD8 signal peptide as described herein. As can be determined by one of skill in the art, various functionally similar or equivalent components of these CARs can be interchanged or substituted for each other and for other similar or functionally equivalent components known in the art or listed herein.

[0071] [Extracellular antigen-binding domain] As used herein, the term "extracellular antigen binding domain" refers to a polypeptide found outside of a cell sufficient to facilitate binding to a target. An extracellular target binding domain specifically binds to its binding partner, i.e., the target. As non-limiting examples, an extracellular antigen binding domain may include the antigen binding domain of an antibody or antibody reagent, or a ligand that recognizes and binds to a cognate binding partner protein. In this context, a ligand is a molecule that specifically binds to a portion of a protein and / or receptor. Cognate binding partners of a ligand useful in the methods and compositions described herein may generally be found on the surface of a cell. Ligand:cognate partner binding may result in a change in the receptor bearing the ligand or may activate a physiological response, such as activation of a signal transduction pathway. In some embodiments, the ligand may be non-native to the genome. In some cases, the ligand has a function that is conserved across at least two species.

[0072] Any cell surface moiety can be targeted by the CAR. Often the target is a cell surface polypeptide that can be differentially or preferentially expressed on cells to which one wishes to target a T cell response. To target Tregs, antibody reagents can be targeted, for example, to glycoprotein A repeat dominant (GARP), latency associated peptide (LAP), CD25, CTLA-4, ICOS, TNFR2, GITR, OX40, 4-1BB, and LAG-3. To target tumor or cancer cells, antibody domains can be targeted, for example, to mesothelin, as described herein. Targeting tumor-specific tumor antigens, such as mesothelin, can provide a means of targeting tumor cells while avoiding or at least limiting collateral damage to non-tumor cells or tissues.

[0073] In some embodiments, the target / antigen of the CAR is mesothelin.

[0074] [Hinge and transmembrane domains] Each CAR described herein comprises a transmembrane domain, e.g., a hinge / transmembrane domain, that connects the extracellular antigen binding domain to the intracellular signaling domain. The binding domain of the CAR is optionally followed by one or more "hinge domains," which serve to position the antigen binding domain away from the effector cell surface to allow for proper cell / cell contact, antigen binding, and activation. The CAR optionally comprises one or more hinge domains between the binding domain and the transmembrane domain (TM). The hinge domain may be derived from either natural, synthetic, semi-synthetic, or recombinant sources. The hinge domain may comprise the amino acid sequence of a naturally occurring immunoglobulin hinge region or a modified immunoglobulin hinge region. Exemplary hinge domains suitable for use in the CARs described herein include hinge regions derived from the extracellular regions of type 1 membrane proteins, such as CD8 (e.g., CD8α), CD4, CD28, 4-1BB, and CD7, which may be wild-type hinge regions from these molecules or may be modified.

[0075] In some embodiments, the hinge region is derived from an immunoglobulin-like protein (e.g., IgA, IgD, IgE, IgG, or IgM), CD28, or CD8. In some embodiments, the hinge domain comprises the CD8a hinge region.

[0076] As used herein, a "transmembrane domain" (TM domain) refers to a portion of a CAR that fuses the extracellular binding portion, optionally via a hinge domain, to the intracellular portion (e.g., the costimulatory domain and the intracellular signaling domain), anchoring the CAR to the cell membrane of an immune effector cell. The transmembrane domain is a generally hydrophobic region of the CAR that passes through the cell membrane of the cell. The TM domain can be a transmembrane region or fragment thereof of a transmembrane protein (e.g., a type I transmembrane protein or other transmembrane protein), an artificial hydrophobic sequence, or a combination thereof. Although specific examples are provided herein and used in the examples, other transmembrane domains will be apparent to those of skill in the art and can be used in connection with alternative embodiments of the technology. It is preferred that the selected transmembrane region or fragment thereof does not interfere with the intended function of the CAR.

[0077] When used in reference to a transmembrane domain of a protein or polypeptide, "fragment thereof" means a portion of the transmembrane domain sufficient to anchor or attach the protein to a cell surface.

[0078] In some embodiments, the transmembrane domain of a CAR described herein or a fragment thereof is selected from the group consisting of the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), 4-1BBL, GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80, (KLRF1), CD160, CD19, IL2R beta, IL2R gamma, IL7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT and / or NKG2C.

[0079] As used herein, "hinge / transmembrane domain" refers to a domain that includes both the hinge domain and the transmembrane domain. For example, the hinge / transmembrane domain can be derived from the hinge / transmembrane domain of CD8, CD28, CD7, or 4-1BB. In some embodiments, the hinge / transmembrane domain of the CAR or a fragment thereof is derived from or includes the hinge / transmembrane domain of CD8 (e.g., SEQ ID NO: 24, or any one of their variants). CD8 is an antigen found preferentially on the cell surface of cytotoxic T lymphocytes. CD8 mediates cell-cell interactions within the immune system and acts as a co-receptor for T cells. CD8 consists of an alpha (CD8α or CD8a) and a beta (CD813 or CD8b) chain. CD8a sequences are known for many species, e.g., human CD8a (NCBI Gene ID:925) polypeptide (e.g., NCBI Reference Sequence NP_001139345.1) and mRNA (e.g., NCBI Reference Sequence NM_000002.12). CD8 can refer to human CD8, including naturally occurring variants, molecules, and alleles thereof. In some embodiments of any of the aspects, e.g., in veterinary applications, CD8 can refer to CD8, e.g., canine, feline, bovine, equine, porcine, etc. In some embodiments, the hinge / transmembrane domain of the CAR or fragment thereof is derived from or comprises the hinge / transmembrane domain of CD28 (e.g., comprises the amino acid sequence of SEQ ID NO:38 or a variant thereof, or is encoded by the nucleic acid sequence of SEQ ID NO:39).

[0080] Homologs and / or orthologs of human CD8 are readily identified for a species by one of skill in the art using, for example, the NCBI ortholog search function, or by searching available sequence data for a given species for sequences similar to the reference CD8 sequence.

[0081] In some embodiments, the CD8 hinge and transmembrane sequence corresponds to the amino acid sequence of SEQ ID NO:24; or comprises the sequence of SEQ ID NO:24; or comprises a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to the sequence of SEQ ID NO:24.

[0082] [Costimulatory domain] Each CAR described herein optionally includes an intracellular domain or costimulatory domain of one or more costimulatory molecules. As used herein, the term "costimulatory domain" refers to the intracellular signaling domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or an Fc receptor that, upon binding to an antigen, provides a second signal required for efficient activation and function of T lymphocytes. The costimulatory domain can be, for example, a costimulatory domain of 4-1BB, CD27, CD28, or OX40. In one example, the 4-1BB intracellular domain (ICD) can be used (see, for example, below and SEQ ID NO: 25, or variants thereof). Additional illustrative examples of such costimulatory molecules include CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKD2C SLP76, TRIM, and ZAP70. In some embodiments, the intracellular domain is the intracellular domain of 4-1BB. 4-1BB (CD137; TNFRS9) is an activation-induced costimulatory molecule and a key regulator of the immune response.

[0083] 4-1BB is a membrane receptor protein, also known as CD137, which is a member of the tumor necrosis factor (TNF) receptor superfamily. 4-1BB is expressed on activated T lymphocytes. 4-1BB sequences are known for many species, for example human 4-1BB, also known as TNFRSF9 (NCBI Gene 25 Number: 3604) and mRNA (NCBI Reference Sequence: NM_001561.5). 4-1BB may refer to human 4-1BB, including naturally occurring variants, molecules, and alleles thereof. In some embodiments of any of the aspects, for example, in veterinary applications, 4-1BB may refer to 4-1BB, for example, canine, feline, bovine, equine, porcine, etc. Homologs and / or orthologs of human 4-1BB are easily identified for such species by those skilled in the art, for example, using the NCBI ortholog search function or by searching available sequence data of a given species for sequences similar to the reference 4-1BB sequence.

[0084] In some embodiments, the intracellular domain is the intracellular domain of 4-1BB. In some embodiments, the 4-1BB intracellular domain corresponds to an amino acid sequence selected from SEQ ID NO:25; or comprises a sequence selected from SEQ ID NO:25; or comprises at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to a sequence selected from SEQ ID NO:25.

[0085] [Intracellular signaling domain] The properties of the intracellular signaling domain of the CAR can vary, as known in the art and as disclosed herein, but the chimeric target / antigen binding domain sensitizes the receptor to signaling activation upon binding of the chimeric target / antigen binding domain to a target / antigen on the surface of a target cell.

[0086] With respect to intracellular signaling domains, so-called "first generation" CARs include those that provide only a CD3 zeta signal upon antigen binding. So-called "second generation" CARs include those that provide both costimulatory (e.g., CD28 or CD137) and activation (CD3 zeta) domains, and so-called "third generation" CARs include those that provide multiple costimulatory (e.g., CD28 and CD137) and activation domains (e.g., CD3 zeta). In various embodiments, CARs are selected to have high affinity or avidity for a target / antigen - for example, target or antigen binding domains derived from antibodies generally have higher affinity and / or avidity for target antigens than naturally occurring T cell receptors. This property, combined with the high specificity that can be selected for antibodies, results in highly specific T cell targeting by CAR T cells.

[0087] The CAR described herein comprises an intracellular signaling domain. By "intracellular signaling domain" is meant the portion of a CAR polypeptide that is involved in transmitting the message of effective CAR binding to a target antigen inside an immune effector cell to induce effector cell functions, such as activation, cytokine production, proliferation and cytotoxic activity, such as release of cytotoxic factors to a target cell bound to the CAR, or other cellular responses elicited after antigen binding to the extracellular CAR domain. In various examples, the intracellular signaling domain is derived from CD3 zeta (see, e.g., below). Further non-limiting examples of intracellular signaling domains that contain immunoreceptor tyrosine-based activation motifs (ITAMs) that are particularly useful in the art include those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 theta, CD3 sigma, CD3 eta, CD3 epsilon, CD3 zeta; CD22, CD79a, CD79b, and CD66d.

[0088] CD3 is a T cell coreceptor that promotes T lymphocyte activation when engaged simultaneously with appropriate costimulation (e.g., binding of costimulatory molecules). The CD3 complex consists of four different chains; mammalian CD3 consists of the CD3γ chain, the CD3δ chain, and two CD3ε chains.

[0089] These chains associate with a molecule known as the T cell receptor (TCR) and CD3ζ to generate an activation signal in T lymphocytes. The complete TCR complex includes the TCR, CD3ζ, and the complete CD3 complex.

[0090] In some embodiments of any of the aspects, the CAR polypeptide described herein comprises an intracellular signaling domain that comprises an immunoreceptor tyrosine-based activation motif or ITAM from CD3ζ, including variants of CD3ζ, such as, for example, ITAM-mutated CD3ζ, CD3η, or CD3θ. In some embodiments of any of the aspects, the ITAM comprises an ITAM triad of CD3ζ (ITAM3). In some embodiments of any of the aspects, the ITAM triad of CD3ζ is not mutated and thus comprises a native or wild-type sequence. In some embodiments, the CD3ζ sequence comprises a CD3ζ sequence set forth in the sequences provided herein, e.g., SEQ ID NOs:26-27, or one of their variants.

[0091] For example, a CAR polypeptide described herein comprises an intracellular signaling domain of CD3 zeta. In some embodiments, the CD3 zeta intracellular signaling domain corresponds to an amino acid sequence selected from any one of SEQ ID NOs:26-27; or comprises a sequence selected from any one of SEQ ID NOs:26-27; or comprises a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to a sequence selected from any one of SEQ ID NOs:26-27.

[0092] The individual CAR and other construct components described herein can be used in conjunction with one another and substituted in the various constructs described herein, as can be determined by one of skill in the art. Each of these components can comprise or consist of any of the corresponding sequences described herein, or variants thereof.

[0093] A more detailed description of CARs and CAR T cells can be found in Maus et al., Blood 123:2624-2635, 2014; Reardon et al., Neuro-Oncology 16:1441-1458, 2014; Hoyos et al., Haematologica 97:1622, 2012; Byrd et al., J. Clin. Oncol. 32:3039-3047, 2014; Maher et al., Cancer Res 69:4559-4562, 2009; and Tamada et al., Clin. Cancer Res. 18:6436-6445, 2012; each of which is incorporated herein by reference in its entirety.

[0094] [Signal peptide] In some embodiments, the CAR polypeptides described herein include a signal peptide. The signal peptide can be from any protein that has an extracellular domain or is secreted. The CAR polypeptides described herein can include any signal peptide known in the art. In some embodiments, the CAR polypeptides include a CD8 signal peptide, for example, a CD8 signal peptide that corresponds to or includes the amino acid sequence of SEQ ID NO:29, or includes an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to the sequence of SEQ ID NO:29.

[0095] In further embodiments, the CAR polypeptides described herein can optionally exclude one of the signal peptides described herein, such as the CD8 signal peptide of SEQ ID NO:29 or the IgK signal peptide of SEQ ID NO:30.

[0096] [Linker domain] In some embodiments, the CAR further comprises a linker domain. As used herein, "linker domain" refers to an oligo- or polypeptide region of about 2 to 100 amino acids in length that links together any of the domains / regions of the CAR described herein. In some embodiments, the linker can comprise or consist of flexible residues such as glycine and serine, allowing adjacent protein domains to move freely relative to each other. Linker sequences useful for the CAR of the present disclosure can be 2 to 100 amino acids in length, 5 to 50 amino acids in length, 10 to 15 amino acids in length, 15 to 20 amino acids in length, or 18 to 20 amino acids in length, and include any suitable linker known in the art. For example, linker sequences useful in the CARs of the disclosure include, but are not limited to, glycine / serine linkers, such as GGGSGGGSGGGS (SEQ ID NO:31) and Gly4Ser(G4S) (SEQ ID NO:48) linkers, such as (G4S)3(GGGGSGGGGSGGGGS (SEQ ID NO:32)) and (G4S)4(GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:332)); the linker sequence GSTSGSGKPGSGEGSTKG (SEQ ID NO:34) described by Whitlow et al., Protein Eng. 6(8):989-95, 1993, the contents of which are incorporated herein by reference in their entirety; linker sequences of GGSSRSSSSGGGGSGGGG (SEQ ID NO:35) as described by Harb. Protoc. 2011(9), 2011; as well as linker sequences with added functionality, such as coding sequences containing Cre-Lox recombination sites or epitope tags, as described by Sblattero et al., Nat. Biotechnol. 18(1):75-80, 2000, the contents of which are incorporated herein by reference in their entirety. Longer linkers can be used when it is desirable to prevent two adjacent domains from sterically interfering with each other.

[0097] Further, the linker can be cleavable or non-cleavable. Examples of cleavable linkers include 2A linkers (e.g., P2A and T2A (SEQ ID NO:37)), 2A-like linkers or functional equivalents thereof and combinations thereof.

[0098] For example, the P2A linker sequence can correspond to the amino acid sequence of SEQ ID NO:38. In various examples, linkers having sequences described herein or variants thereof are used. It should be understood that the representation of a particular linker in a construct at a particular position does not mean that only that linker can be used therein. Rather, different linker sequences (e.g., P2A and T2A) can be interchanged (e.g., in the context of the constructs of the invention) as can be determined by one of skill in the art. In some embodiments, the linker region is T2A from Thosea asigna virus. Non-limiting examples of linkers that can be used in this technology include T2A, P2A, E2A, BmCPV2A, and BmIFV2A. Linkers such as these can be used in the context of polyproteins such as those described below. For example, they can be used to separate the CAR component of a polyprotein from the therapeutic (e.g., an antibody such as an scFv, single domain antibody (e.g., camelid antibody), or bispecific antibody (e.g., TEAM)) component of the polyprotein (see below).

[0099] [Reporter molecule] In some embodiments, the CARs described herein further comprise a reporter molecule, in some cases, for example, to allow for non-invasive imaging (e.g., positron emission tomography PET scans). In bispecific CARs that include a reporter molecule, the first and second extracellular binding domains can include different or the same reporter molecule. In bispecific CAR cells, the first and second CARs can express different or the same reporter molecule. In another embodiment, the CARs described herein further comprise a reporter molecule (e.g., hygromycin phosphotransferase (hph)) that can be imaged alone or in combination with a substrate or chemical (e.g., 9-[4-[18F]fluoro-3]-(hydroxymethyl)butyl]guanine ([18F]FHBG)). In another embodiment, the CARs described herein further comprise a nanoparticle that can be easily imaged using a non-invasive technique (e.g., gold nanoparticles (GNPs) functionalized with 64Cu2+). Labeling of CAR T cells for non-invasive imaging is reviewed, for example, in Bhatnagar et al., Integr. Biol. (Camb). 5(1):231-238, 2013, and Keu et al., Sci. Transl. Med. 18;9(373), 2017, which are incorporated by reference in their entireties.

[0100] In some embodiments, GFP and mCherry can be used as fluorescent tags to image CARs expressed on T cells (e.g., CAR T cells). It is expected that essentially any fluorescent protein known in the art can be used as a fluorescent tag for this purpose. For clinical applications, it is not necessary for the CAR to include a fluorescent tag or fluorescent protein. Thus, in each of the specific construct examples provided herein, any marker present in the construct can be removed. The present disclosure includes constructs with or without markers. Thus, when a specific construct is referred to herein, it can be considered to be included in the present disclosure with or without a marker or tag (e.g., including a histidine tag, such as the histidine tag of HHHHHH (SEQ ID NO: 39)).

[0101] [Antibody Reagents] In various embodiments, the CARs described herein comprise an antibody reagent or an antigen-binding domain thereof as the extracellular target binding domain.

[0102] As used herein, the term "antibody reagent" refers to a polypeptide that comprises at least one immunoglobulin variable domain or immunoglobulin variable domain sequence and specifically binds to a given antigen. In some embodiments, an antibody reagent may comprise an antibody or a polypeptide comprising an antigen-binding domain of an antibody. In some embodiments of any of the aspects, an antibody reagent may comprise a monoclonal antibody or a polypeptide comprising an antigen-binding domain of a monoclonal antibody. For example, an antibody may comprise a heavy (H) chain variable region (abbreviated herein as VH) and a light (L) chain variable region (abbreviated herein as VL). In some embodiments, an antibody comprises two heavy (H) chain variable regions and two light (L) chain variable regions. In some embodiments, an antibody reagent is a bispecific antibody reagent.

[0103] The term "antibody reagent" encompasses antigen-binding fragments of antibodies (e.g., single chain antibodies, Fab and sFab fragments, F(ab')2, Fd fragments, Fv fragments, scFv, CDR, and domain antibody (dAb) fragments (see, e.g., de Wildt et al., Eur. J. Immunol. 26(3):629-639, 1996, which is incorporated herein by reference in its entirety)) as well as complete antibodies. Antibodies can have the structural characteristics of IgA, IgG, IgE, IgD, or IgM (as well as combinations of subtypes thereof). Antibodies can be from any source, including mouse, rabbit, pig, rat, and primate (human and non-human primates) and primatized antibodies. Antibodies also include midibodies, humanized antibodies, chimeric antibodies, and the like. In some embodiments, a CAR comprises an antibody reagent. In some embodiments, a therapeutic agent comprises an antibody reagent.

[0104] Fully human antibody binding domains can be selected, for example, from phage display libraries using methods known to those of skill in the art. Additionally, antibody reagents include single domain antibodies, such as camelid antibodies.

[0105] The VH and VL regions can be further divided into regions of hypervariability called "complementarity determining regions" ("CDRs"), interspersed with more conserved regions called "framework regions" ("FRs"). The extent of the framework regions and CDRs has been precisely defined (see Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., USDepartment of Health and Human Services, NIH Publication No. 91-3242, and Chothia et al., J. Mol. Biol. 196:901-917, 1987; each of which is incorporated herein by reference in its entirety). Each VH and VL is typically composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0106] In some embodiments, the antibody or antibody reagent is not human (i.e., the antibody or antibody reagent is murine), but is humanized. By "humanized antibody or antibody reagent" is meant a non-human antibody or antibody reagent that has been modified at the protein sequence level to increase its similarity to the antibody or antibody reagent variants naturally produced in humans. One approach to humanizing antibodies employs grafting murine or other non-human CDRs onto a human antibody framework.

[0107] In some embodiments, the extracellular target binding domain of the CAR comprises or consists essentially of a single chain Fv (scFv) fragment generated by fusing the VH and VL domains of an antibody, typically a monoclonal antibody, via a flexible linker peptide. In various embodiments, the scFv is fused to a transmembrane domain and a T cell receptor intracellular signaling domain, e.g., an engineered intracellular signaling domain as described herein. In another embodiment, the extracellular target binding domain of the CAR comprises a camelid antibody.

[0108] In some embodiments, the CAR, antibody, or antigen binding domain binds to mesothelin. Mesothelin sequences are known for many species, for example, human mesothelin (NCBI gene ID: 10232) and polypeptides (e.g., preprotein NCBI reference sequence: NP_005814.2). Mesothelin can refer to human mesothelin, including its naturally occurring variants, molecules, and alleles. Homologs and / or orthologs of human mesothelin are easily identified for such species by one of skill in the art, for example, by using a mesothelin ortholog search function or by searching available sequence data for a given species for sequences similar to the reference mesothelin sequence. In some embodiments, a CAR, antibody, or antigen binding domain that specifically binds to mesothelin specifically binds to homologs and / or orthologs of human mesothelin in addition to, for example, human mesothelin.

[0109] The terms "anti-mesothelin antibody," "antibody that binds mesothelin," and "antibody that specifically binds mesothelin" refer to an antibody reagent that can bind to mesothelin with sufficient affinity such that the antibody is useful as a prophylactic, diagnostic, and / or therapeutic agent by targeting mesothelin. In one embodiment, the extent of binding of an anti-mesothelin antibody to unrelated, non-mesothelin proteins is less than about 10% of the binding of the antibody to mesothelin, e.g., as measured by radioimmunoassay (RIA). In certain embodiments, an antibody that binds mesothelin has an affinity of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, or ≦0.1 nM (e.g., 10-6 M or less, 10 -7 M or 10 -8 M or less, e.g. 10 -6 M to 10 -9 Dissociation constant (K D In certain embodiments, antibodies that bind mesothelin have a mAb of ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -9 , 10 -10 , 10 -11 , or 10 -12 M or less, e.g. 10 -9 M to 10 -12 Dissociation constant (K D ).

[0110] In some embodiments, the anti-mesothelin antibody reagent is MGHmesol. In some embodiments, the anti-mesothelin antibody reagent comprises a complementarity determining region (CDR) of SEQ ID NO:13-18, or comprises a CDR sequence having no more than 1, 2, or 3 amino acid substitutions relative to a given CDR of SEQ ID NO:13-18. In some embodiments, the anti-mesothelin antibody reagent comprises a variable heavy chain (VH) and / or variable light chain (VL) of SEQ ID NO:19-20, or comprises a VH and / or VL sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity to the sequences of SEQ ID NO:19-20. The VH can be located N-terminal to the VL, or the VL can be located N-terminal to the VH.

[0111] In some embodiments, the antibody reagent comprises daclizumab or an antigen-binding fragment thereof. The antibody reagent may also target any other antigen described herein or known in the art. In addition to optionally delivering an antibody reagent as described herein, the CART cells of the present disclosure may be used to deliver other therapeutic agents, including, but not limited to, cytokines and toxins. In some embodiments, the therapeutic reagent comprises an antibody reagent that binds to any one of epidermal growth factor receptor variant III (EGFRvIII), epidermal growth factor receptor (EGFR), CD19, prostate specific membrane antigen (PSMA), or IL-13 receptor alpha 2 (IL-13Rα2). In some embodiments, the therapeutic reagent comprises an antibody reagent that binds to two or more of epidermal growth factor receptor variant III (EGFRvIII), epidermal growth factor receptor (EGFR), CD19, prostate specific membrane antigen (PSMA), or IL-13 receptor alpha 2 (IL-13Rα2).

[0112] In some embodiments, the antibody reagent binds to a tumor associated antigen. Non-limiting examples of additional tumor antigens or other antigens of interest include activated fibroblast markers, CD19, CD37, BCMA (Tumor necrosis factor receptor superfamily member 17 (TNFRSF17); NCBI Gene ID: 608; NCBI Reference Sequence: NP001183.2 and mRNA (e.g., NCBI Reference Sequence: NM_001192.2)), CEA, immature laminin receptor, TAG-72, HPV E6 and E7, BING-4, calcium activated chloride channel 2, cyclin B1, 9D7, Ep-CAM, EphA3, 15her2 / neu, telomerase, EGFR, EGFRviii These include SAP-1, survivin, BAGE family, CAGE family, GAGE ​​family, MAGE family, SAGE family, XAGE family, NY-ESO-1 / LAGE-1, PRAME, SSX-2, Melan-NMART-1, gp100 / pmel17, tyrosinase, TRP-1 / -2, MC1R, BRCA1 / 2, CDK4, MART-2, p53, Ras, MUC1, TGF-BetaRII, IL-15, IL-13Ra2, and CSF1R. In some embodiments, the activated fibroblast marker comprises any one of αSMA (ACTA2), fibroblast activation protein (FAP), platelet-derived growth factor receptors α and β (PDGFRA, PDGFRB), fibroblast-specific protein 1 (FSP1 / S100A4), endoglin (ENG), transgelin (TAGLN), tenascin-C (TNC), periostin (POSTN), chondroitin sulfate proteoglycan 4 or neuron-glial antigen 2 (CSPG4 / NG2), podoplanin (PDPN), or osteopontin (SPP1).

[0113] [Multispecific antibody reagents] In some embodiments, the antibody reagent is multispecific and can specifically bind to two or more target antigens. In some embodiments, the antibody reagent is bispecific (i.e., a bispecific antibody reagent). In some embodiments, the bispecific antibody reagent specifically binds to a first antigen (e.g., mesothelin) and a second antigen. In some embodiments, the second antigen is a tumor antigen. In some embodiments, the second antigen is expressed by (e.g., on the surface of) a mesothelin-expressing cancer cell. In some embodiments, the second antigen is epidermal growth factor receptor (EGFR), BCMA, CD19, CD37, carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), EphA3, Her2 / neu, MUC1, TGF-βRII, colony-stimulating factor 1 receptor (CSF1R), prostate-specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), or any other tumor antigen described herein. In some embodiments, the multispecific antibody reagent is a TEAM, as described herein.

[0114] A multispecific antibody reagent can include a first binding domain comprising a full-length antibody or antibody fragment thereof that binds to a first antigen (e.g., mesothelin) and a second binding domain comprising a different full-length antibody or antibody fragment thereof that binds to a second antigen (e.g., a tumor antigen). The first and second binding domains can be linked by a linker sequence, such as a linker sequence described herein.

[0115] [Therapeutic drugs delivered by CAR T cells] As described above, the CAR T cells of the present disclosure may be used to deliver therapeutic agents, e.g., antibody reagents or other therapeutic molecules, e.g., cytokines, to the tumor (i.e., tumor microenvironment) in some cases. In some embodiments, the cytokine is an interferon, interleukin, or growth factor. In some embodiments, the therapeutic agent is encoded by the same nucleic acid molecule as the CAR, thus facilitating the transduction of the cell (e.g., T cell) to express both the CAR and the therapeutic agent, e.g., antibody reagent or cytokine. In such examples, the therapeutic agent (e.g., antibody reagent or cytokine) may be expressed such that it is separated from the CAR (and optionally other proteins, e.g., markers) by, for example, a cleavable linker sequence (e.g., a 2A ribosomal skip sequence and an internal ribosome entry site (IRS), or a 2A linker (2A peptide), e.g., P2A or T2A; see above). In some embodiments, the therapeutic agent is encoded on a different nucleic acid molecule than the CAR.

[0116] In some embodiments, the therapeutic agent (e.g., antibody reagent or cytokine) can be expressed under the control of the same promoter as the CAR (e.g., by the EF1α promoter) and can be constitutively expressed. In some embodiments, the therapeutic agent (e.g., antibody reagent or cytokine) is expressed under the control of an inducible promoter, e.g., a promoter that is expressed upon T cell activation (e.g., the NFAT promoter). Such an inducible promoter can be used, for example, to ensure that the antibody is expressed only upon T cell activation and thus only when, for example, the CAR T cell is within a tumor microenvironment where limiting antibody production therein can be advantageous. As understood in the art, the CAR coding sequence can be 5' or 3' to the therapeutic agent (e.g., antibody reagent or cytokine) coding sequence in the various vector designs within this disclosure.

[0117] In some embodiments, the therapeutic agent comprises an IgK signal peptide, e.g., an IgK signal peptide comprising an amino acid sequence that corresponds to or comprises the amino acid sequence of SEQ ID NO:30, or has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to the sequence of SEQ ID NO:30.

[0118] In various examples, the therapeutic agent is an antibody reagent. The antibody reagent expressed in the CAR T cells (e.g., from the same nucleic acid molecule as the CAR) can be a single chain antibody (e.g., scFv) or a single domain antibody (e.g., camelid antibody), as described herein. In the case of a single chain antibody, the light (L) and heavy (H) chains can be in LH or HL order (N-terminus to C-terminus), and can optionally be separated from each other by a linker (e.g., a glycine-based linker). In a further example, the antibody reagent is a bispecific antibody, e.g., including a T cell engaging molecule (TEAM), as described below.

[0119] [T cell engaging antibody molecule (TEAM)] In some embodiments, the therapeutic agent delivered by the CART cells described herein is a T cell engaging molecule (TEAM) (also known in the literature as a bispecific T cell engager or BiTE). TM"T cell engaging molecule", "TEAM antibody construct", or "TEAM" refers to a polypeptide comprising single chain variable fragments (scFvs) linked in tandem. Optionally, the scFvs are linked by a linker (e.g., a glycine-rich linker). One scFv of the TEAM binds to the T cell receptor (TCR) (e.g., CD3 subunit) and the other binds to a target antigen (e.g., a tumor antigen). Such molecules can target T cells by binding to the T cell antigen (e.g., by binding to CD3) as well as by binding to a target antigen, e.g., a tumor antigen. Exemplary tumor antigens include mesothelin (see also below). TEAMs can be used, for example, to enhance T cell responses in the tumor microenvironment. The two components of the TEAM can optionally be separated from each other by a linker (e.g., a glycine-based linker) as described herein and can be linked in either orientation, e.g., the anti-CD3 component to the N-terminus of the anti-target antigen component, or vice versa. The anti-CD3 component or the anti-target antigen component of the TEAM can include any of the antibody reagents described herein.

[0120] TEAM secreted by CART cells may act paracrinely, for example, by stimulating the CART cells themselves or redirecting non-specific bystander T cells to the tumor, thereby enhancing the anti-tumor effects of CART cell immunotherapy. CART cell-mediated TEAM secretion may allow for reduced risk of unwanted TEAM activity in systemic tissues by directing TEAM secretion to the tumor microenvironment. Exemplary TEAM constructs are provided below, although TEAMs other than those described herein may also be useful in the CAR T cells and methods of the disclosure.

[0121] An exemplary TEAM is an anti-CD19 TEAM comprising an anti-CD19 scFv and an anti-CD3 scFv (also referred to herein as TEAM-CD19). The anti-CD19 scFv can be arranged in a VH-VL orientation, or a VL-VH orientation. In certain embodiments, the anti-CD19 scFv corresponds to, comprises, or has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity to the amino acid sequence of SEQ ID NO:37.

[0122] In some embodiments, the anti-CD3 scFv of any of the TEAMs described herein can be arranged in a VH-VL orientation or a VL-VH orientation. In some embodiments, the anti-CD3 VH comprises an amino acid sequence of SEQ ID NO:9, or an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity to the amino acid sequence of SEQ ID NO:9. In some embodiments, the anti-CD3 VL comprises an amino acid sequence of SEQ ID NO:10, or an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity to the amino acid sequence of SEQ ID NO:10.

[0123] In some embodiments, the TEAM comprises an antibody that binds to repeat dominant glycoprotein A (GARP), latency associated peptide (LAP), CD25, cytotoxic T lymphocyte associated antigen 4 (CTLA-4). In some embodiments, the TEAM comprises an amino acid sequence of any one of SEQ ID NOs: 11-12, or an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity to an amino acid sequence of any one of SEQ ID NOs: 11-12.

[0124] In some embodiments, the TEAM comprises an antibody reagent that binds to a fibroblast activation protein (FAP).

[0125] In some embodiments, the anti-FAP antibody reagent is sibrotuzumab. In some embodiments, the anti-FAP antibody reagent comprises a variable heavy chain (VH) of SEQ ID NO:6, or a VH sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity to SEQ ID NO:6. In some embodiments, the anti-FAP antibody reagent comprises a variable light chain (VL) of SEQ ID NO:7, or a VL sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity to the sequence of SEQ ID NO:7. The VH may be disposed N-terminal to the VL, or the VL may be disposed N-terminal to the VH. In some embodiments, the anti-FAP antibody reagent comprises SEQ ID NO:8 or a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to SEQ ID NO:8.

[0126] In some embodiments, the TEAM comprises an antibody reagent that binds to fibroblast activation protein (FAP) and an antibody reagent that binds to CD3. In some embodiments, the FAP antibody reagent is encoded upstream of the CD3 scFv.

[0127] In some embodiments, the CAR and TEAM are encoded on the same polypeptide. In some embodiments, the CAR and TEAM are encoded on the same polypeptide and separated by a linker domain as described above. In some embodiments, the linker domain is cleavable. In some embodiments, the CAR is an anti-mesothelin CAR and the TEAM is a fibroblast activation protein (FAP) and a CD3 TEAM. In some embodiments, the CAR is an anti-mesothelin CAR and the TEAM comprises an anti-FAP scFv and an anti-CD3 scFv. In some embodiments, the CAR and TEAM polypeptides comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to SEQ ID NO:1. In some embodiments, the CAR and TEAM polypeptides comprise the amino acid sequence SEQ ID NO:1. In some embodiments, the CAR and TEAM polypeptides consist of the amino acid sequence SEQ ID NO:1.

[0128] In some embodiments, the TEAM enhances binding of immune cells to the cancer compared to binding of immune cells to the cancer in the absence of the TEAM. In some embodiments, the TEAM enhances binding of immune cells to the cancer compared to binding of immune cells to the cancer in the absence of the TEAM and the absence of a CAR that binds to an antigen on the immune cell. In some embodiments, the TEAM increases binding of immune cells to the cancer by at least 10% (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, or at least 250%). In some embodiments, TEAM increases immune cell binding to cancer by 10%-20%, 10%-30%, 10%-50%, 10%-100%, 10%-150%, 10%-200%, 10%-250%, 50%-100%, 50%-150%, 50%-200%, 50%-250%, 100%-150%, 100%-200%, 100%-250%, 20%-30%, 20%-40%, 20%-50%, 20%-60%, 20%-70%, 30%-40%, 30%-50%, 30%-60%, or 30%-70%. In some embodiments, TEAM increases immune cell binding to cancer by 20%-40%. In some embodiments, the TEAM increases binding of the immune cell to the cancer by 100%-250%. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is a CAR T cell. In some embodiments, the TEAM is a FAP TEAM.

[0129] [CAR T cells] One aspect of the presently described technology pertains to mammalian cells comprising any of the CAR polypeptides described herein (optionally together with another therapeutic agent (e.g., an antibody reagent (e.g., scFv, camelid antibody, or TEAM) or cytokine); or a nucleic acid encoding any of the CAR polypeptides described herein (optionally together with another therapeutic agent (e.g., an antibody reagent (e.g., scFv, camelid antibody, or TEAM) or cytokine). In some embodiments, the mammalian cells comprise an antibody, an antibody reagent, an antigen-binding portion thereof, any of the CARs described herein, or a cytokine, or a nucleic acid encoding such an antibody, antibody reagent, an antigen-binding portion thereof, any of the CARs described herein, or a cytokine. The mammalian cells or tissues may be of human, primate, hamster, rabbit, rodent, bovine, porcine, ovine, equine, caprine, canine, or feline origin, although any other mammalian cells may be used. In a preferred embodiment of any aspect, the mammalian cells are human.

[0130] In some embodiments of either aspect, the mammalian cell is an immune cell. As used herein, "immune cell" refers to a cell that plays a role in an immune response. Immune cells are of hematopoietic origin and include lymphocytes, such as B cells and T cells; natural killer cells; myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes. In some embodiments, the immune cell is a T cell; a NK cell; a NKT cell; a lymphocyte, such as B cells and T cells; and a myeloid cell, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes. In some embodiments, the immune cell is a T cell.

[0131] In some embodiments, the immune cells are obtained from an individual who has or has been diagnosed with cancer, a plasma cell disorder, or an autoimmune disease.

[0132] In some embodiments, mammalian cells, such as T cells, can be engineered to contain any of the CAR polypeptides described herein and a therapeutic agent described herein; or a nucleic acid encoding any of the CAR polypeptides described herein and a therapeutic agent. T cells can be obtained from a subject using standard techniques known in the art. For example, T cells can be isolated from peripheral blood taken from a donor or patient. T cells can be isolated from a mammal. Preferably, T cells are isolated from a human.

[0133] In some embodiments, the CAR T cells comprise an anti-mesothelin CAR described herein. In some embodiments, the CAR T cells comprise a nucleic acid sequence encoding a CAR of any one of SEQ ID NOs:2-5 or a CAR-TEAM polypeptide of SEQ ID NO:1.

[0134] [CAR T and therapeutic drug nucleic acid sequence] Also provided are nucleic acid constructs and vectors encoding (i) a CAR polypeptide (e.g., any one of SEQ ID NOs:2-5), or (ii) a polyprotein comprising a CAR polypeptide and a therapeutic agent (e.g., SEQ ID NO:1) described herein for use in producing CART cells.

[0135] In some embodiments, the present disclosure provides constructs that include separate coding sequences for multiple proteins, each of which is expressed in a CART cell of the present disclosure. These separate coding sequences can be separated from one another by a cleavable linker sequence, as described herein. For example, a sequence encoding a viral 2A protein (e.g., T2A and P2A) can be placed between separate genes, which, when transcribed, can direct cleavage of the resulting polyprotein. As described above, the constructs and vectors of the present disclosure can include any of a variety of combinations of a number of sequences. For example, a construct or vector of the present disclosure can include a sequence encoding one CAR as described herein, optionally in combination with a therapeutic agent (e.g., an antibody reagent (e.g., a single chain antibody, a single domain antibody (e.g., a camelid antibody), or a bispecific antibody (e.g., a TEAM)) or a cytokine) as described herein.

[0136] Efficient expression of a protein in a CART cell described herein can be assessed using standard assays that detect the mRNA, DNA, or gene product of the nucleic acid encoding the protein. For example, RT-PCR, FAGS, Northern blotting, Western blotting, ELISA, or immunohistochemistry can be used. The proteins described herein can be constitutively expressed or inducibly expressed. In some embodiments, the protein is encoded by a recombinant nucleic acid sequence. For example, the disclosure provides a vector comprising a first polynucleotide sequence encoding a CAR, the CAR comprising an extracellular domain comprising an antigen binding sequence that binds, for example, a tumor antigen or a Treg-associated antigen, and optionally a second polynucleotide sequence encoding a therapeutic agent (e.g., an antibody reagent (e.g., a single chain antibody, a single domain antibody (e.g., a camelid antibody), or a bispecific antibody (e.g., a TEAM)) or a cytokine).

[0137] In some embodiments, the first polynucleotide sequence and the second polynucleotide sequence are each operably linked to a promoter. In some embodiments, the first polynucleotide sequence is operably linked to a first promoter and the second polynucleotide sequence is operably linked to a second promoter. The promoters can be constitutively expressed promoters (e.g., EF1α promoter) or inducibly expressed promoters (e.g., NFAT promoter).

[0138] In some embodiments, expression of the CAR and the therapeutic agent are driven by the same promoter, such as a constitutively expressed promoter (e.g., the EF1 alpha promoter). In other embodiments, expression of the CAR and the therapeutic agent are driven by different promoters. For example, expression of the CAR can be driven by a constitutively expressed promoter (e.g., the EF1 alpha promoter), while expression of the therapeutic agent can be driven by an inducibly expressed promoter (e.g., the NFAT promoter). The polynucleotide sequence encoding the CAR can be located upstream of the polynucleotide sequence encoding the therapeutic agent, or the polynucleotide sequence encoding the therapeutic agent can be located upstream of the polynucleotide sequence encoding the CAR.

[0139] In some embodiments, the polynucleotide may include expression of a suicide gene. This may be done to facilitate external drug-mediated control of the administered cells. For example, the suicide gene may be used to deplete the modified cells from the patient, for example, in the case of an adverse event. In some embodiments, the FK506 binding domain is fused to the caspase 9 pro-apoptotic molecule. T cells modified in this way are made sensitive to the immunosuppressant drug tacrolimus. Other examples of suicide genes are thymidine kinase (TK), CD20, thymidylate kinase, truncated prostate specific membrane antigen (PSMA), truncated low affinity nerve growth factor receptor (LNGFR), truncated co·19, and modified Fas, which may be triggered with specific molecules (e.g., ganciclovir for TK+ cells) or conditional ablation by administration of antibodies or antibody-drug conjugates.

[0140] A construct containing a sequence encoding a protein to be expressed in CAR T cells can be included in a vector. In various examples, the vector is a retroviral vector. Retroviruses, such as lentiviruses, provide a convenient platform for delivering nucleic acid sequences encoding genes or chimeric genes of interest. A selected nucleic acid sequence can be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells, for example, in vitro or ex vivo. Retroviral systems are well known in the art and described, for example, in U.S. Pat. No. 5,219,740; Kurth and Bannert (2010) "Retroviruses: Molecular Biology, Genomics and Pathogenesis" Calster Academic Press (ISBN: 978-1-90455-55-4); and Hu et al., Pharmacological Reviews 52:493-512, 2000, which are incorporated herein by reference in their entirety. A lentiviral system for efficient DNA delivery is available commercially from OriGene (Rockville, MD). In various embodiments, proteins are expressed in T cells by transfection or electroporation of an expression vector containing a nucleic acid encoding the protein, using vectors and methods known in the art. In some embodiments, the vector is a viral vector or a non-viral vector.

[0141] In some embodiments, the viral vector is a retroviral vector (eg, a lentiviral vector), an adenoviral vector, or an adeno-associated viral vector.

[0142] In some embodiments, a composition comprising a vector comprises a first polynucleotide sequence encoding a CAR, the CAR comprising an extracellular domain comprising a sequence that specifically binds to a tumor antigen or a Treg-associated antigen, and optionally a second polynucleotide sequence encoding a therapeutic agent. In certain embodiments, when the therapeutic agent is an antibody reagent (e.g., a single chain antibody, a single domain antibody (e.g., a camelid antibody), or a bispecific antibody (e.g., a TEAM)), the antibody reagent specifically binds to a tumor antigen or a Treg-associated antigen.

[0143] [CAR and therapeutic drug development] In some embodiments of any aspect, any of the CAR polypeptides described herein (optionally together with an antibody reagent or cytokine described herein) are expressed from a lentiviral vector. A lentiviral vector is used to express the CAR polypeptide (and optionally an antibody reagent or cytokine) in a cell using standard infection techniques.

[0144] Retroviruses, such as lentiviruses, provide a convenient platform for delivery of nucleic acid sequences encoding genes of interest or chimeric genes. A selected nucleic acid sequence can be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells, for example, in vitro or ex vivo. Retroviral systems are well known in the art and have been described, for example, in U.S. Pat. No. 5,219,740; Kurth and Bannert (2010) "Retroviruses: Molecular Biology, Genomics and Pathogenesis" Calster Academic Press (ISBN: 978-1-90455-55-4); and Hu et al., Pharmacological Reviews 52:493-512, 2000, which are incorporated herein by reference in their entirety. Lentiviral systems for efficient DNA delivery can be purchased from OriGene (Rockville, MD). In some embodiments, a CAR polypeptide of any of the CARs described herein (and optionally an antibody reagent or cytokine) is expressed in a mammalian cell via transfection or electroporation of an expression vector containing a nucleic acid encoding the CAR.

[0145] Transfection and electroporation techniques are known in the art.

[0146] Efficient expression of a CAR polypeptide (and optionally an antibody reagent or cytokine) of any of the polypeptides described herein can be assayed using standard assays that detect mRNA, DNA, or gene products of the nucleic acid encoding the CAR (and optionally an antibody reagent or cytokine), such as RT-PCR, FACS, Northern blotting, Western blotting, ELISA, or immunohistochemistry.

[0147] In some embodiments, the CAR polypeptides (and optionally antibody reagents or cytokines) described herein are constitutively expressed. In other embodiments, the CAR polypeptides are constitutively expressed and the optional antibody reagents or cytokines are inducibly expressed. In some embodiments, the CAR polypeptides (and optionally antibody reagents or cytokines) described herein are encoded by recombinant nucleic acid sequences.

[0148] [array] TIFF2024540359000002.tif236170TIFF2024540359000003.tif255170TIFF20245403590 00004.tif251170TIFF2024540359000005.tif229170TIFF2024540359000006.tif242170

[0149] [Administration and Treatment] [subject] As used herein, "subject" refers to a human or animal. Typically, an animal is a vertebrate, such as a primate, rodent, domestic animal, or game animal. Primates include, for example, chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, such as rhesus monkeys. Rodents include, for example, mice, rats, marmots, ferrets, rabbits, and hamsters. Domestic and game animals include, for example, cows, horses, pigs, deer, bison, buffalo, feline species, such as house cats, canine species, such as dogs, foxes, wolves, bird species, such as chickens, emus, ostriches, and fish, such as trout, catfish, and salmon. In some embodiments, the subject is a mammal, such as a primate, such as a human. The terms "individual," "patient," and "subject" are used interchangeably herein. Preferably, the subject is a mammal. The mammal may be, but is not limited to, a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow. Mammals other than humans may be advantageously used as subjects that represent animal models of disease, such as cancer. The subject may be male or female.

[0150] The subject may have previously been diagnosed with or been determined to be suffering from or having a condition requiring treatment (e.g., pancreatic cancer, lung cancer, ovarian cancer, endometrial cancer, biliary tract cancer, gastric cancer, or mesothelioma, among others), or one or more complications associated with such a condition, and in some cases may have already been treated for the condition or one or more complications associated with the condition.

[0151] Alternatively, the subject may also not have been previously diagnosed with such a condition or an associated complication, for example, the subject may be one who exhibits one or more risk factors for the condition or one or more complications associated with the condition, or one who exhibits no risk factors.

[0152] A "subject in need" of treatment for a particular condition can be a subject who has the condition, has been diagnosed as having the condition, or is at risk for developing the condition.

[0153] [Pharmaceutical Composition] As used herein, the term "pharmaceutical composition" means an active agent in combination with a pharma- ceutically acceptable carrier, such as a carrier commonly used in the pharmaceutical industry.

[0154] The phrase "pharmacologically acceptable" is used herein to refer to compounds, substances, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings or animals without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. In some embodiments of any of the aspects, the pharma- ceutically acceptable carrier can be a carrier other than water. In some embodiments of any of the aspects, the pharma- ceutically acceptable carrier can be a cream, emulsion, gel, liposome, nanoparticle, and / or ointment. In some embodiments of any of the aspects, the pharma- ceutically acceptable carrier can be an artificial or modified carrier, e.g., a carrier in which the active ingredient is not found naturally occurring.

[0155] In one aspect of the technology described herein, the technology described herein relates to a pharmaceutical composition comprising the activated CAR T cells described herein and, optionally, a pharma- ceutically acceptable carrier. The active ingredient of the minimal pharmaceutical composition comprises the activated CAR T cells described herein. In some embodiments, the active ingredient of the pharmaceutical composition consists essentially of the activated CAR T cells described herein. In some embodiments, the active ingredient of the pharmaceutical composition consists of the activated CAR T cells described herein. Pharmaceutically acceptable carriers for cell-based therapeutic formulations include saline and aqueous buffers, Ringer's solution, and serum components, such as serum albumin, HDL, and LDL. Terms such as "additives," "carriers," "pharma- ceutically acceptable carriers," "pharma- ceutically acceptable additives," and the like, are used interchangeably herein.

[0156] In some embodiments, pharmaceutical compositions comprising the activated CAR T cells described herein may be in parenteral dosage forms. Because administration of parenteral dosage forms typically circumvents the patient's natural defenses against contaminants, components other than the CAR T cells themselves are preferably sterile or can be sterilized prior to administration to a patient. Examples of parenteral dosage forms include, but are not limited to, solutions for injection, dry products dissolved or suspended in a pharma- ceutically acceptable vehicle for injection, suspensions for injection, and emulsions. Any of these can be added to the activated CAR T cell preparation prior to administration. Suitable vehicles that can be used to provide parenteral dosage forms of the disclosed activated CAR T cells are well known to those skilled in the art. Examples include, but are not limited to, saline; glucose solution; aqueous vehicles including, but not limited to, Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and propylene glycol; and non-aqueous vehicles such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

[0157] [Dosage] The term "unit dosage form" as used herein means a dosage amount suitable for single administration. By way of example, a unit dosage form can be an amount of a therapeutic agent disposed in a delivery device, such as a syringe or an intravenous infusion bag. In some embodiments, a unit dosage form is administered in a single administration. In other embodiments, more than one unit dosage form can be administered simultaneously.

[0158] In some embodiments, the activated CAR T cells described herein are administered as a monotherapy, i.e., the subject is not simultaneously administered another treatment for the condition. Pharmaceutical compositions comprising the T cells described herein are generally administered in a dose-dependent manner within 10 4 From 10 9 Cells / kg body weight, in some cases 10 5 From 10 6 The T cell composition can be administered in a dosage of cells / kg body weight, including all integer values ​​within these ranges. If necessary, the T cell composition can be administered multiple times at these dosages. The cells can be administered by using injection techniques commonly known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. Med. 319:1676, 1988).

[0159] In certain embodiments, it may be desirable to administer the activated CAR T cells to the subject, then redraw blood (or perform apheresis), activate T cells therefrom as described herein, and reinfuse the patient with these activated and expanded T cells. This method may be performed multiple times, every few weeks. In certain embodiments, the T cells may be activated from a blood draw of 35 10cc to 400cc. In certain embodiments, the T cells are activated from a blood draw of 20cc, 30cc, 40cc, 50cc, 60cc, 70cc, 80cc, 90cc, or 100cc.

[0160] [Administration] In some embodiments, the methods described herein relate to treating a subject having or diagnosed as having cancer, a plasma cell dyscrasia or disorder, or an autoimmune disease or disorder with any of the CAR polypeptides (and optional antibody reagents or cytokines) described herein, or a mammalian cell comprising a nucleic acid encoding any of the CAR polypeptides (and optional antibody reagents or cytokines) described herein. The CAR T cells described herein comprise a mammalian cell comprising any of the CAR polypeptides (and optional antibody reagents or cytokines) described herein, or a nucleic acid encoding any of the CAR polypeptides (and optional antibody reagents or cytokines) described herein. Subjects having a condition can be identified by a physician using the present methods of diagnosing a condition. Symptoms and / or complications of a condition that characterize and aid in the diagnosis of these conditions are well known in the art and include, but are not limited to, fatigue, persistent infection, and persistent bleeding. For example, tests that may aid in the diagnosis of a condition include, but are not limited to, blood tests and bone marrow tests, and are known in the art for a given condition. A family history of a condition, or exposure to a risk factor for a condition, can also help determine whether a subject is likely to have the condition, or in making a diagnosis of the condition.

[0161] The compositions described herein may be administered to a subject having or diagnosed with a condition. In some embodiments, the methods described herein include administering to a subject an effective amount of an activated CAR T cell described herein to alleviate symptoms of a condition. As used herein, "alleviating symptoms of a condition" refers to ameliorating any condition or symptoms associated with a condition. Such reduction, as measured by any standard technique, is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more compared to an equivalent untreated control. Various means for administering the compositions described herein to a subject are known to those of skill in the art. In some embodiments, the compositions described herein are administered systemically or locally. In a preferred embodiment, the compositions described herein are administered intravenously. In another embodiment, the compositions described herein are administered to the site of a tumor.

[0162] The term "effective amount" as used herein refers to the amount of activated CAR T cells necessary to alleviate at least one or more symptoms of a disease or disorder, and relates to a cell preparation or composition in an amount sufficient to produce a desired effect. Thus, the term "therapeutically effective amount" refers to an amount of activated CAR T cells sufficient to produce an effect against a particular condition when administered to a typical subject. An effective amount as used herein may also include an amount sufficient to slow the progression of a disease symptom, alter the course of a disease symptom (such as, but not limited to, slow the progression of a condition), or reverse a condition symptom, in various circumstances. Thus, it is generally not practical to specify an exact "effective amount". However, in any given case, an appropriate "effective amount" can be determined by one of ordinary skill in the art using only routine experimentation.

[0163] Effective doses, toxicity, and therapeutic effects can be evaluated by standard pharmaceutical procedures in cell cultures or experimental animals. Dosages can vary depending on the dosage form used and the route of administration utilized. The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD50 / ED50. Compositions and methods that exhibit large therapeutic indices are preferred. Therapeutically effective doses can be estimated initially from cell culture assays. A dose can also be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of activated CAR T cells that achieves half-maximal inhibition of symptoms) determined in cell culture or a suitable animal model. Plasma levels can be measured, for example, by high performance liquid chromatography. The effect of any particular dosage can be monitored by suitable bioassays, such as, inter alia, assays for bone marrow biopsy. Dosages can be determined by a physician and adjusted as necessary to accommodate observed therapeutic effects.

[0164] [Administration method] Methods of administration may include, for example, intravenous (iv) injection or infusion. The compositions described herein may be administered to a patient intraarterially, intratumorally, intranodally, intraperitoneally, or intrathecally. In some embodiments, the T cell compositions may be injected directly into a tumor, lymph node, or site of infection. In some embodiments, the compositions described herein are administered into a body cavity or fluid (e.g., ascites, pleural fluid, peritoneal fluid, or cerebrospinal fluid).

[0165] In certain exemplary aspects, the subject may undergo leukapheresis, where leukocytes are collected, enriched, or removed ex vivo to select and / or isolate cells of interest, e.g., T cells. These T cell isolates are expanded by contact with artificial APCs, e.g., aAPCs expressing anti-CD28 and anti-CD3 CDRs, and processed to allow for the introduction of one or more CAR constructs of the present technology, thereby generating CAR T cells. The subject in need thereof may then undergo standard treatment with high-dose chemotherapy, followed by a peripheral blood stem cell transplant. After or simultaneously with the transplant, the subject may receive an infusion of the expanded CAR T cells. In some embodiments, the expanded cells are administered before or after surgery. In some embodiments, lymphodepletion is performed on the subject prior to administration of one or more CAR T cells described herein. In such embodiments, lymphodepletion may include administering one or more of melphalan, cytoxan, cyclophosphamide, and fludarabine. Dosages of the above therapeutic agents administered to a patient will vary according to the exact nature of the condition being treated and the recipient of the treatment. Scaling of dosages for administration to humans can be performed in accordance with art-recognized practices.

[0166] In some embodiments, a single treatment regimen is required. In other cases, one or more subsequent doses or treatment regimen administrations may be performed. For example, after treatment every other week for three months, treatment may be repeated once a month for six months or for a year or more. In some embodiments, no additional treatment is administered after the first treatment.

[0167] The dosage of the compositions described herein can be determined by a physician and adjusted as necessary to suit the observed therapeutic effect. With regard to the duration and frequency of treatment, a skilled clinician will typically monitor the subject to determine when the treatment produces a therapeutic effect and determine whether to administer further cells, discontinue treatment, resume treatment, or make other changes to the treatment regimen. The dosage should not be so high as to cause adverse side effects, such as cytokine release syndrome. In general, dosage will vary according to the age, condition, and sex of the patient, and can be determined by one of skill in the art. Dosage can also be adjusted by the individual physician in the event of any complications.

[0168] [Combination therapy] The activated CAR T cells described herein can optionally be used in combination with each other and with other known drugs and therapies, as can be determined to be appropriate by one of skill in the art. In one example, two or more CAR T cells targeting different Treg markers (e.g., GARP, LAP, CD25, and cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) and the like) can be administered in combination. In another example, two or more CAR T cells targeting different cancer antigens are administered in combination. In a further example, one or more CAR T cells targeting a Treg marker (e.g., GARP, LAP, and the like) are administered in combination with one or more CAR T cells targeting one or more tumor antigens.

[0169] As used herein, administered "in combination" means that two (or more) different therapeutic agents are delivered to a subject during the course of the subject's disease affliction, e.g., two or more therapeutic agents are delivered after the subject is diagnosed with a disorder and before the disorder is cured or eliminated, or before treatment is discontinued for other reasons. In some embodiments, the delivery of one treatment is still occurring when the delivery of the second begins, and there is an overlap in administration. This is sometimes referred to herein as "simultaneous" or "concurrent delivery." In other embodiments, the delivery of one therapeutic agent ends before the delivery of the other begins. In some embodiments in either case, the treatment is more effective due to the combined administration. For example, the second therapeutic agent is more effective than would be observed if administered in the absence of the first therapeutic agent, e.g., the same effect is observed with less of the second therapeutic agent, or the second therapeutic agent significantly reduces symptoms, or a similar situation is observed as with the first therapeutic agent. In some embodiments, the delivery is such that the reduction in symptoms or other parameters associated with the disorder is greater than would be observed with one therapeutic agent delivered in the absence of the other therapeutic agent. The effects of the two therapeutic agents may be partially additive, fully additive, or greater than additive. Delivery may be such that the effect of the first therapeutic agent delivered is still detectable when the second therapeutic agent is delivered. The activated CAR T cells described herein and at least one additional therapeutic agent may be administered simultaneously, in the same or separate compositions, or sequentially. For sequential administration, the CAR-expressing cells described herein may be administered first and the additional agent may be administered a second time, or the order of administration may be reversed. CAR T therapy and / or other therapeutic agents, procedures, or modalities may be administered during periods of active disease or during periods of remission or less active disease. CAR T therapy may be administered prior to another treatment, concurrently with a treatment, after a treatment, or during remission of the disorder.

[0170] When administered in combination, the activated CAR T cells and additional agent (e.g., second or third agent), or all, may be administered in amounts or dosages that are greater than, less than, or the same as the amount or dosage of each agent used individually, e.g., as monotherapy. In certain embodiments, the amount or dosage of the activated CAR T cells, additional agent (e.g., second or third agent), or all administered is less than the amount or dosage of each agent used individually (e.g., at least 20%, at least 30%, at least 40%, or at least 50%). In other embodiments, the amount or dosage of the activated CAR T cells, additional agent (e.g., second or third agent), or all that results in a desired effect (e.g., treatment of cancer) is less than the amount or dosage of each agent individually required to achieve the same therapeutic effect (e.g., at least 20%, at least 30%, at least 40%, or at least 50% less). In further embodiments, the activated CAR T cells described herein may be used in a treatment regimen in combination with surgery, chemotherapy, radiation, mTOR pathway inhibitors, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies or other antibody therapies, cytotoxin, fludarabine, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, or peptide vaccines, such as those described in Izumoto et al., J. Neurosurg. 108:963-971, 2008.

[0171] In some embodiments, the activated CAR T cells described herein may be used in combination with checkpoint inhibitors. Exemplary checkpoint inhibitors include anti-PD-1 inhibitors (nivolumab, MK-3475, pembrolizumab, pidilizumab, AMP-224, AMP-514), anti-CTLA4 inhibitors (ipilimumab and tremelimumab), anti-POL1 inhibitors (atezolizumab, avelomab, MSB0010718C, MEDI4736, and MPDL3280A), and anti-TIM3 inhibitors.

[0172] In some embodiments, the activated CAR T cells described herein may be used in combination with a chemotherapeutic agent. Exemplary chemotherapeutic agents include anthracyclines (e.g., doxorubicin (e.g., liposomal doxorubicin)), vinca alkaloids (e.g., vinblastine, vincristine, vindesine, vinorelbine), alkylating agents (e.g., cyclophosphamide, decarbazine, melphalan, ifosfamide, temozolomide), immune cell antibodies (e.g., alemtuzamab, gemtuzumab, rituximab, tositumomab), antimetabolites, and the like. agents (including, for example, folate antagonists, pyrimidine analogues, purine analogues, and adenosine deaminase inhibitors (e.g., fludarabine)), mTOR inhibitors, TNFR glucocorticoid-induced TNFR-related protein (GITR) agonists, proteasome inhibitors (e.g., aclacinomycin A, gliotoxin, or bortezomib), immunomodulatory agents such as thalidomide or thalidomide derivatives (e.g., lenalidomide). Common chemotherapy agents that may be considered for use in combination therapy include anastrozole (Arimidex®), bicalutamide (Casodex®), bleomycin sulfate (Blenoxane®), busulfan (Myleran®), busulfan injection (Busulfex®), capecitabine (Xeloda®), N4-pentoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin (Paraplatin®), carmustine (BiCNU®), chlorambucil (Leukeran®), cisplatin ( Platinol®), cladribine (Leustatin®), cyclophosphamide (Cytoxan® or Neosar®), cytarabine, cytosine arabinoside (Cytosar-U®), cytarabine liposome injection (DepoCyt®), dacarbazine (DTIC-Dome®), dactinomycin (Actinomycin D, Cosmegan), daunorubicin hydrochloride (Cerbidine®), daunorubicin citrate liposome injection (DaunoXome®), dexamethasone, docetaxel (Taxotere®),Doxorubicin hydrochloride (Adriamycin®, Rubex®), etoposide (Bepesid®), fludarabine phosphate (Fludara®), 5-fluorouracil (Adolcil®, Efudex®), flutamide (Eulexin®), tezacitibine, gemcitabine (difluorodeoxycytidine), hydroxyurea (Hydrea®), idarubicin (Idamycin), ifosfamide (IFEX®), irinotecan (Camptosar®), L-asparaginase (ELSPAR®), leucovorin calcium, melphalan (Alkeran®), 6-mercaptopurine (Purinethol®), metoclopramide (Metoclopramide), methylprednisolone ... These include thotrexate (Folex®), mitoxantrone (Novantron®), Mylotarg, paclitaxel (Taxol®), Phoenix (Yttrium 90 / MX-DTPA), pentostatin, polipheprosan 20 with carmustine implant (Gliadel®), tamoxifen citrate (Nolvadex®), teniposide (Vumon®), 6-thioguanine, thiotepa, tirapazamine (Tirazone®), injectable topotecan hydrochloride (Hycamtin®), vinblastine (Velban®), vincristine (Oncovin®), and vinorelbine (Navelbine®). Exemplary alkylating agents include, but are not limited to, nitrogen mustards, ethylenimine derivatives, alkylsulfonates, nitrosoureas, and triazenes; uracil mustard (Aminouracil Mustard®, Chloretaminacil®, Demethyldopan®, Desmethyldopan®, Hemanthamine®, Nordpan®, Uracil Nitrogen Mustard®, Uracillost®, Uracilmostaza®, Uramustin®, Uramustine®),Chlormethine (Mustargen®), cyclophosphamide (Cytoxan®, Neosar®, Clafen®, Endoxan®, Procytox®, Revimmune, TM), ifosfamide (Mitoxana®), melphalan (Alkeran®), chlorambucil (Leukeran®), pipobroman (Amedel®, Vercyte®), triethylenemelamine (Hemel®, Hexalen®, Hexastat®), triethylenethiophosphoramine, temozolomide (Temodar®), thiotepa (Thioplex®), busulfan (Busilvex®, Myleran®), carmustine (BiCNU®), lomustine (CeeNU®), streptozocin (Zanosar®), and dacarbazine (DTIC-Dome®).Additional exemplary alkylating agents include, but are not limited to, oxaliplatin (Eloxatin®); temozolomide (Temodar® and Temodal®); dactinomycin (Actinomycin-D, also known as Cosmegen®); melphalan (L-PAM, L-sarcolysin, and phenylalanine mustard, also known as Alkeran®); altretamine (hexamethylmelamine (HMM), also known as Hexalen®); carmustine (BiCNU®); bendamustine (Treanda®); busulfan (Busulfex® and Myleran®); carboplatin (Paraplatin®); lomustine (CCNU, also known as CeeNU®); cisplatin (CDDP, also known as Platinol® and Platinol®-AO); chlorambucil. (Leukeran®); cyclophosphamide (Cytoxan® and Neosar®); dacarbazine (DTIC, also known as DIC and imidazole carboxamide, DTIC-Dome®); altretamine (hexamethylmelamine (HMM), also known as Hexalen®); ifosfamide (Ifex®); prednumstine; procarbazine (Matulane®); mechlorethamine (nitrogen mass) tard, mustine and mechlorethamine hydrochloride, also known as Mastergen®); streptozocin (Zanosar®); thiotepa (thiophosphoamide, TESPA and TSPA, also known as Thioplex®); cyclophosphamide (Endoxan®, Cytoxan®, Neosar®, Procytox®, Revimmune®); and bendamustine HC1 (Treanda®).Exemplary mTOR inhibitors include, for example, temsirolimus; ridaforolimus (formally also known as AP23573 and MK8669, and described in PCT Publication No. WO03 / 064383); deferolimus, (lR,2R,45)-4-[(2R)-2[(1R,95,125,15R,16E,18R,19R,21R,235,24E,26E,28Z,305,325,35R)-l,18-dihydro roxy-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-2,3,10,14,20-pentaoxo-ll,36-dioxa-4-azatricyclo[30.3.1.04'9]hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyl dimethylphosphinate; everolimus (Afinitor® or RADOOI); rapamycin (AY2 2989, Sirolimus®; simapimod (CAS 164301-51-3); emsirolimus, (5-{2,4-bis[(35,)-3-methylmorpholin-4-yl]pyrido[2,3-(i]pyrimidin-7-yl}-2-methoxyphenyl)methanol (AZD8055); 2-amino-8-[iraw5,-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4 -methyl-pyrido[2,3-JJ pyrimidin-7(8H)-one (PF04691502, CAS 1013101-36-4); and N2-[1,4-dioxo-4-[[4-(4-oxo-8-phenyl-4H-1-benzopyran-2-yl)morpholinium-4-yl]methoxy]butyl]-L-arginylglycyl-La-aspartyl L-serine-, inner salt (SF1126, CAS 936487-67-1), and XL765.Exemplary immunomodulatory agents include, for example, afutuzumab (available from Roche®), pegfilgrastim (Neulasta®); lenalidomide (CC-5013, Revlimid®); thalidomide (Thalomid®), actimid (CC4047); and IRX-2 (human cytokine mixture containing interleukin-1, interleukin-2, and interferon-y, CAS951209-71-5, available from IRX Therapeutics). Exemplary anthracyclines include, for example, doxorubicin (Adriamycin® and Rubex®); bleomycin (Lenoxan®); daunorubicin (daunorubicin hydrochloride, daunomycin, and rubidomycin hydrochloride, Cervizin®); daunorubicin liposome (daunorubicin citrate liposome, DaunoXome®); mitoxantrone (DHAD, Novantrone®); epirubicin (Elence. TM); idarubicin (Idamycin®, Idamycin PFS®); mitomycin C (Mutamycin®); geldanamycin; herbimycin; rabidomycin; and desacetylrabidomycin. Exemplary vinca alkaloids include, for example, vinorelbine tartrate (Navelbine®), vincristine (Oncovin®), and vindesine (Eldisine®); vinblastine (also known as vinblastine sulfate, vincaleukoblastine and VLB, Alkaban AQ® and Velban®); and vinorelbine (Navelbine®). Exemplary proteosome inhibitors include bortezomib (VELCADE®); carfilzomib (PX-171-007, (5)-4-methyl-N-((5)-l-(((5)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-((5,)-2-(2-morpholinoacetamido)-4-phenylbutanamido)-pentanamide); marizomib (NPT0052); ixazomib citrate (MLN-9708); delanzomib (CEP-18770); and O-methyl-N- and [(2-methyl-5-thiazolyl)carbonyl]-L-seryl-O-methyl-N-[(IIS')-2-[(2R)-2-methyl-2-oxiranyl]-2-oxo-1-(phenylmethyl)ethyl]-L-serinamide (ONX-0912).

[0173] Those skilled in the art can readily identify chemotherapeutic agents to use (see, for example, Physicians' Cancer Chemotherapy Drug Manual 2014, Edward Chu, Vincent T. DeVita Jr., Jones & Bartlett Learning; Principles of Cancer Therapy, Chapter 85 in Harrison's Principles of Internal Medicine, 18th Edition; Therapeutic Targeting of Cancer Cells: Era of Molecularly Targeted Agents and Cancer Pharmacology, Chapters 28-29 in Abeloff's Clinical Oncology, 2013 Elsevier; and Fischer DS (ed.): The Cancer Chemotherapy Handbook, 4th Edition St. Louis, Mosby-Year Book, 2003).

[0174] In one embodiment, the activated CAR T cells described herein are administered to a subject in combination with a molecule that targets GITR and / or reduces the level and / or activity of a molecule that modulates GITR function, a molecule that reduces the Treg cell population, an mTOR inhibitor, a GITR agonist, a kinase inhibitor, a non-receptor tyrosine kinase inhibitor, a CDK4 inhibitor, and / or a BTK inhibitor.

[0175] [Effectiveness] For example, the efficacy of activated CAR T cells in treating a condition described herein or in inducing a response described herein (e.g., reduction in cancer cells) can be determined by a skilled clinician. However, a treatment is considered "effective treatment" as that term is used herein if, following treatment with the methods described herein, one or more of the signs or symptoms of a condition described herein are beneficially altered, other clinically acceptable symptoms are improved or even ameliorated, or a desired response is induced, for example, by at least 10%. Efficacy can be assessed, for example, by measuring markers, indicators, symptoms, and / or incidence or any other suitable measurable parameter of a condition treated by the methods described herein.

[0176] Treatment with the methods described herein can reduce the levels of a marker or symptom of a condition, for example, by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% or more.

[0177] Efficacy can also be measured by lack of deterioration of an individual as assessed by hospitalization or the need for medical intervention (i.e., halting of disease progression). Methods for measuring these indicators are known to those of skill in the art and / or described herein. Treatment includes any treatment of a disease in an individual or animal (some non-limiting examples include humans or animals) and includes (1) inhibiting the disease, e.g., preventing the worsening of symptoms (e.g., pain or inflammation); or (2) reducing the severity of symptoms, e.g., causing regression of symptoms. An effective amount for the treatment of a disease means an amount sufficient to provide effective treatment, as that term is defined herein, for that disease when administered to a subject in need thereof. The efficacy of an agent can be determined by assessing physical indicators of a condition or a desired response. It is well within the capabilities of one of skill in the art to monitor the efficacy of administration and / or treatment by measuring any one of such parameters, or any combination of parameters. The efficacy of a given approach can be evaluated in an animal model of a condition as described herein. When using an experimental animal model, the efficacy of the treatment is demonstrated when a statistically significant change in the marker is observed.

[0178] All patents and other publications, including literature references, issued patents, published patent applications, and co-pending patent applications, cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that may be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of this application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by reason of prior art or for any other reason. All statements as to the date or representation as to the contents of these documents are based on the information available to the applicants and do not constitute any admission as to the accuracy of the dates or contents of these documents.

[0179] The description of the embodiments of the present disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. Although specific embodiments and examples of the present disclosure are described herein for illustrative purposes, those skilled in the relevant art will recognize that various equivalent modifications are possible within the scope of the present disclosure. For example, while method steps or functions are presented in a given order, in alternative embodiments, the functions may be performed in a different order, or may be performed substantially simultaneously. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the present disclosure can be modified, as appropriate, to provide still further embodiments of the present disclosure using the compositions, functions, and concepts of the above references and applications. Furthermore, considerations of biological function equivalence allow for some modifications to protein structures without affecting biological or chemical action in type or amount. These and other modifications can be made to the present disclosure in light of the detailed description. All such modifications are intended to be within the scope of the appended claims.

[0180] Specific elements of any of the foregoing embodiments can be combined with or substituted for elements of other embodiments. Additionally, although advantages associated with certain embodiments of the present disclosure have been described in the context of those embodiments, other embodiments may also exhibit such advantages, and not all embodiments necessarily exhibit such advantages in order to fall within the scope of the present disclosure.

[0181] The technology described herein is further illustrated by the following examples, which should not be construed as further limiting in any way.

[0182] [cancer] In some embodiments, immune cells (e.g., T cells) comprising a CAR, such as a CART TEAM described herein, can also be used to treat cancers with heterologous antigen expression (e.g., mesothelin expression). For example, the CAR component of the CART TEAM construct can include an extracellular antigen binding domain that binds one antigen expressed by the cancer, and the TEAM component of the CART TEAM construct can bind a second antigen expressed by the cancer in addition to a T cell antigen (e.g., CD3). As used herein, "cancer" can refer to the hyperproliferation of cells whose unique phenotype, loss of normal cellular controls, results in unregulated growth, lack of differentiation, local tissue invasion, and metastasis. Exemplary cancers include, but are not limited to, glioblastoma, prostate cancer, glioma, leukemia, lymphoma, multiple myeloma, or solid tumors, such as lung cancer and pancreatic cancer. Non-limiting examples of leukemias include acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), and chronic lymphocytic leukemia (CLL). In some embodiments, the cancer is ALL or CLL. Non-limiting examples of lymphomas include diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), marginal zone lymphoma, Burkitt's lymphoma, hairy cell leukemia (HCL), and T-cell lymphoma (e.g., peripheral T-cell lymphoma (PTCL), including cutaneous T-cell lymphoma (CTCL) and anaplastic large cell lymphoma (ALCL)). In some embodiments, the cancer is DLBCL or follicular lymphoma. Non-limiting examples of solid tumors include adrenal cortical tumors, alveolar soft part sarcoma, carcinoma, chondrosarcoma, colorectal carcinoma, desmoid tumor, desmoplastic small round cell tumor, endocrine tumors, endodermal sinus tumor, epithelioid hemangioendothelioma, Ewing's sarcoma, germ cell tumors (solid tumors), giant cell tumor of bone and soft tissue, hepatoblastoma, hepatocellular carcinoma, melanoma, nephroma, neuroblastoma, non-rhabdomyosarcoma soft tissue sarcoma (NRSTS), osteosarcoma, paraspinal sarcoma, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, synovial sarcoma, and Wilms' tumor.

[0183] Solid tumors may be found in bone, muscle, or organs and may be sarcomas or carcinomas. It is believed that any aspect of the technology described herein may be used to treat all types of cancer, including cancers not listed in this application. As used herein, the term "tumor" refers to an abnormal growth of cells or tissue, for example of a malignant or benign type.

[0184] Cluster of differentiation (CD) molecules are cell surface markers present on white blood cells. As white blood cells differentiate and mature, their CD profile changes. When white blood cells become cancer cells (i.e., lymphoma), their CD profile is important for diagnosing the disease. The treatment and prognosis of a given type of cancer depends on determining the CD profile of the cancer cells. "CDX+", where "X" is a CD marker, indicates that the CD marker is present in the cancer cell, and "CDX-" indicates that the marker is not present. One skilled in the art can use standard techniques to assess the CD molecules present on cancer cells, for example, by using immunofluorescence to detect commercially available antibodies bound to the CD molecules.

[0185] [Autoimmune disease] As used herein, an "autoimmune disease or disorder" is characterized by the inability of the immune system to distinguish between foreign and normal cells. This results in the immune system targeting normal cells for programmed cell death. Non-limiting examples of autoimmune diseases or disorders include inflammatory arthritis, type 1 diabetes, multiple sclerosis, psoriasis, inflammatory bowel disease, SLE, and vasculitis, allergic inflammation, such as allergic asthma, atopic dermatitis, and contact hypersensitivity. Other examples of autoimmune related diseases or disorders, which should not be construed as limiting, include rheumatoid arthritis, multiple sclerosis (MS), systemic lupus erythematosus, Graves' disease (hyperthyroidism), Hashimoto's thyroiditis (hypothyroidism), celiac disease, Crohn's disease and ulcerative colitis, Guillain-Barre syndrome, primary biliary sclerosis / cirrhosis, sclerosing cholangitis, autoimmune hepatitis, Raynaud's phenomenon, scleroderma, Sjogren's syndrome, Goodpasture's syndrome, Wegener's granulomatosis, rheumatoid arthritis, and osteoporosis. These include polymyalgia, temporal arteritis / giant cell arteritis, chronic fatigue syndrome (CFS), psoriasis, autoimmune Addison's disease, ankylosing spondylitis, acute disseminated encephalomyelitis, antiphospholipid syndrome, aplastic anemia, idiopathic thrombocytopenic purpura, myasthenia gravis, opsoclonus-myoclonus syndrome, optic neuritis, Ord's thyroiditis, pemphigus, pernicious anemia, polyarthritis in dogs, Reiter's syndrome, Takayasu's arteritis, warm autoimmune hemolytic anemia, Wegener's granulomatosis, and fibromyalgia (FM). In some embodiments, the mammalian cells are obtained from patients with immune system disorders that result in abnormally low activity of the immune system, or immune deficiency disorders that interfere with the ability to fight foreign substances (e.g., viruses or bacterial cells). Plasma cells are white blood cells generated from B lymphocytes that function to produce and release antibodies needed to fight infections. As used herein, a "plasma cell disorder or disease" is characterized by an abnormal proliferation of plasma cells. The abnormal plasma cells can "crowd out" healthy plasma cells, reducing their ability to fight foreign substances such as viruses or bacterial cells. Non-limiting examples of plasma cell disorders include amyloidosis, Waldenstrom's macroglobulinemia, osteosclerosing myeloma (POEMS syndrome), monoclonal gammopathy of undetermined significance (MGUS), and plasma cell myeloma.

[0186] [CAR T cell therapy] One aspect of the technology described herein relates to a method of treating cancer, plasma cell disorder, or autoimmune disease in a subject in need thereof, comprising modifying T cells to include a CAR polypeptide (and optional antibody reagent or cytokine) described herein on the surface of the T cells, and administering the CAR T cells described herein to the subject. In the case of cancer, the method can be for treating a diagnosed cancer, preventing recurrence of cancer, or for use in an adjuvant or neoadjuvant setting. In some embodiments, the subject has a cancer that expresses mesothelin.

[0187] One aspect of the presently described technology relates to a method of treating cancer, a plasma cell disorder, or an autoimmune disease in a subject in need thereof, comprising administering any of the mammalian cells comprising any of the CAR polypeptides described herein (and optional antibody reagents or cytokines). In some embodiments of any of the aspects, the engineered CAR-T cells are stimulated and / or activated prior to administration to the subject.

[0188] In some embodiments, immune cells (e.g., T cells) comprising a CAR, such as a CART TEAM described herein, can be used to treat a cancer that expresses mesothelin, such as pancreatic cancer, lung cancer, ovarian cancer, endometrial cancer, biliary tract cancer, gastric cancer, or mesothelioma. In some embodiments, the cancer expresses mesothelin. In some embodiments, the CAR T cells or pharmaceutical compositions thereof are administered to a subject with cancer, as described above. In some embodiments, the CAR T cells administered to the subject with cancer comprise an anti-mesothelin CAR. In some embodiments, the CAR T cells administered to the subject with cancer comprise an anti-mesothelin CAR and a FAP TEAM. In some embodiments, the CAR T cells administered to the subject with cancer comprise a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 1-5.

[0189] In some embodiments, immune cells (e.g., T cells) comprising a CAR, such as a CART TEAM described herein, can be used to prevent or reduce immune suppression, e.g., by Tregs, in the tumor microenvironment. Furthermore, such CART TEAMs are useful for preventing or reducing T cell exhaustion in the tumor microenvironment. In some embodiments, the CAR T cells or pharmaceutical compositions thereof are administered to a subject having an autoimmune disease as described above. In some embodiments, the CAR T cells administered to a subject having cancer comprise the amino acid sequence of any one of SEQ ID NOs: 1-5. In some embodiments, the CAR T cells administered to a subject having an autoimmune disease comprise an anti-mesothelin CAR. In some embodiments, the CAR T cells administered to a subject having an autoimmune disease comprise an anti-mesothelin CAR and a FAP TEAM. In some embodiments, the CAR T cells administered to a subject having an autoimmune disease comprise the amino acid sequence of any one of SEQ ID NOs: 1-5. EXAMPLES

[0190] Example 1: Anti-mesothelin CAR-T cells for the treatment of cancer Mesothelin is a promising therapeutic target for many cancers, including pancreatic cancer. To target cancers expressing mesothelin, three different types of mesothelin chimeric antigen receptor (CAR) T cells were developed: type 1 expresses SS1-BBz CAR, type 2 expresses MGHmeso1 L / H-BBz CAR, and type 3 expresses MGHmeso1 H / L-BBz CAR. Each developed anti-mesothelin CAR contains a CD8 leader, VH and VL domains of scFv mesothelin antibody, CD8 hinge / transmembrane, 4-1BBz intracellular domain (ICD), CD3 ICD, and T2A. SS1-BBz contains the VH and VL of SS1 scFv antibody. MGHmeso1 L / H contains the VH and VL of MGHmeso1 scFv antibody, with the VL domain located N-terminal to the VH domain. MGHmeso1 H / L contains the VH and VL of the MGHmeso1 scFv antibody, and the VH domain is located at the N-terminus of the VL domain.

[0191] The cancer-killing efficacy of the developed anti-mesothelin CAR T cells was tested in three different cancer cell lines: BxPC-3 (pancreatic cancer), Capan-2 (pancreatic cancer), and NCI-H226 (lung cancer) (Figures 11A-11C). The results showed that all three anti-mesothelin CAR T cells induced specific lysis of each type of cancer cell. Because MGHmeso1 L / H-41BBz (containing the L / H-oriented anti-mesothelin CAR) consistently showed similar or higher specific lysis than MGHmeso1 H / L-41BBz (containing the H / L-oriented anti-mesothelin CAR), MGHmeso1L / H-41BBz CAR T cells were selected for further study.

[0192] The cytokine expression profiles of SS1-BBz and MGHmeso1 L / H-41BBz CAR-T cells were further analyzed (Figure 1A-1E, n=3 replicates). The results showed that expression of anti-mesothelin CARs (SS1-41BBz and MGHmeso1 L / H-41BBz) significantly increased the expression of INF-γ, IL-2, GM-CSF, TNF-α, and granzyme B in CAR T cells. In addition, the results showed that SS1-BBz and MGHmeso1 L / H-41BBz specifically lysed mesothelin-positive Capan-2 cells, but not mesothelin-negative JeKo-1 cells (mantle cell lymphoma) (Figure 1E-1F). The results in Figure 1 demonstrate that SS1-41BBz and MGHmeso1 L / H-41BBz cells exhibit similar cellular functions and cytotoxicity.

[0193] The efficacy of SS1-41BBz and MGHmeso1 L / H-41BBz CAR T cells was further tested in vivo in mice. Figures 2A-2B show details of a series of experiments in which CAR T was administered to mice, and the results of the series of experiments are shown in Figures 3A-10D. After injecting mice with AsPC-1 (pancreatic cancer) cells, CAR T cells were administered at different doses, for different periods (7 or 14 days), and in different ways (intravenous (IV) and intraperitoneal injection (IP)).

[0194] Figures 3A-3D compare administration of 1e6 SS1-BBz and MGHmeso1 L / H-BBz CAR-T cells to mice injected with AsPC-1. Figure 3A compares flux and viability after IV administration at day 7 post-treatment. Flux is defined as photons per second and is a measure of tumor burden. The cancer cell lines tested were transduced with luciferase, which emits light upon injection with D-luciferin substrate. Figure 3B compares flux and viability after IV administration at day 14 post-treatment. Figure 3C compares flux and viability after IP administration at day 7 post-treatment. Figure 3D compares flux and viability after IP administration at day 14 post-treatment. Taken together, the results in Figures 3A-3D show that MGHmeso1 CAR T cells are more effective than SS1 CAR T when administered by IP injection. These results demonstrate the potential utility and advantage of MGHmeso1 CAR T cells in treating mesothelin-expressing cancers, and that the method of administration of the CAR T cells may be a factor in the relative efficacy of the treatment.

[0195] Figures 4A-4D also compare the flux and survival of mice injected with AsPC-1 and administered a higher dose (2e6 cells) of SS1-BBz or MGHmeso1 L / H-BBz CAR-T cells. The results in Figures 4A-4D show that IP administration of MGHmeso1 CAR T cells is more effective than IV administration, and that when MGHmeso1 CAR T cells are administered on day 7, they are more effective than SS1 CAR T cells. These results further demonstrate the potential utility and advantage of MGHmeso1 CAR T cells in treating mesothelin-expressing cancers, and that both the timing and method of administration of the CAR T cells may be factors in the relative efficacy of the treatment.

[0196] Figures 5A-5D compare the flux and survival of mice injected with a different pancreatic cancer cell line (BXPC3) and administered SS1-BBz or MGHmeso1 L / H-BBz CAR T cells at a dose of 1e6 CAR T cells. The results in Figures 5A-5D show that IP administration of 1e6 MGHmeso1 CAR T cells is as effective or more effective than IV administration when treating murine BXPC3 cancer in mice. The results in Figures 5A-5D also show that SS1 CAR T cells and MGHmeso1 CAR T cells have similar efficacy at day 14, but SS1 CAR T cells have slightly increased efficacy at day 7 when treating murine BXPC3 cancer.

[0197] Figures 6A-6D compare the flux and survival of mice injected with BXPC3 pancreatic cancer cells and administered SS1-BBz or MGHmeso1 L / H-BBz CAR T cells at a CAR T cell dose of 2e6. The results in Figures 6A-6D show that MGHmeso1 L / H CAR T cells showed improved efficacy compared to SS1 Car T cells when administered by IP injection to BXPC3 cancer-bearing mice at this higher dose compared to Figures 5A-5D. These results demonstrate the benefit of treating mesothelin-expressing cancers with MGHmeso1 CAR T cells and suggest a dose-dependent improvement over CAR T cells expressing a previously known CAR (SS1).

[0198] Figures 7A-7D compare the flux and survival of mice injected with a different pancreatic cancer cell line (PANC1) and administered SS1-BBz or MGHmeso1 L / H-BBz CAR T cells at a CAR T cell dose of 1e6. The results in Figures 7A-7D show that MGHmeso1 L / H CAR T cells have increased efficacy compared to SS1 Car T cells when administered by IP injection to mice with PANC1 cancer. These results demonstrate that MGHmeso1 CAR T cells have potential utility and advantages in the treatment of other mesothelin-expressing cancers, and that the method of administration of the CAR T cells may be a factor in the relative efficacy of the treatment.

[0199] Figures 8A-8D compare the flux and survival of mice injected with PANC1 pancreatic cancer cells and administered SS1-BBz or MGHmeso1 L / H-BBz CAR T cells at a CAR T cell dose of 2e6. The results in Figures 8A-8D show that MGHmeso1 L / H CAR T cells have improved efficacy when administered 14 days after cancer cell injection compared to SS1 CAR T cells when administered by IP injection in mice with PANC1 cancer. These results further demonstrate the potential utility and benefit of MGHmeso1 CAR T cells in treating mesothelin-expressing cancers, and that both the timing and method of administration of the CAR T cells may be factors in the relative efficacy of the treatment. A series of experiments were performed to assay the optimal dose, timing, and route of administration of CAR T cells (Figures 9A-10D). Figures 9A-9D compare the flux and survival of mice injected with a different pancreatic cancer cell line (CAPAN2) and administered SS1-BBz or MGHmeso1 L / H-BBz CAR T cells at a CAR T cell dose of 2.5e5. The results in Figures 9A-9D show that SS1 CAR T cells and MGHmeso1 CAR T cells have similar efficacy in treating CAPAN2 cancer in mice. Figures 10A-10D compare the flux and survival of mice injected with CAPAN2 cells and administered SS1-BBz or MGHmeso1 L / H-BBz CAR T cells at a CAR T cell dose of 5e5. The results in Figures 10A-10D show that SS1 CAR T cells and MGHmeso1 CAR T cells have similar efficacy in treating CAPAN2 cancer in mice.

[0200] Taken together, these results demonstrate the utility of MGHmeso1 CAR T cells in treating mesothelin-expressing cancers, particularly pancreatic cancer, suggest that IP administration of MGHmeso1 CAR-T is more effective than IV, and suggest that MGHmeso1 CAR T cells may be more potent than SS1 depending on the timing and method of administration. In addition, the results suggest that MGHmeso1 CAR T therapy may be more effective at lower doses than required for SS1 CAR T. The results also suggest that as few as 2.5e5 tumor cells can be implanted in mice, rather than millions.

[0201] Example 2: Anti-Mesothelin CAR-T Cells with TEAM FAP CAR T cells have revolutionized the treatment of hematological malignancies. However, targeting solid tumor types has proven more challenging due to their heterogeneous target antigen expression, antigen escape of single target antigens, and the tumor microenvironment. To address these challenges, we designed mesothelin-directed chimeric antigen receptor (CAR) T cells that secrete a T cell engaging molecule (TEAM) against fibroblast activation protein (FAP) that can redirect CAR T cells and recruit bystander T cells to FAP. This allows them to target the stroma and tumor microenvironment rather than the tumor cells themselves.

[0202] Mesothelin is associated with tumor invasion and is highly expressed in various cancer types, including mesothelioma, pancreatic, pleural, breast, and ovarian cancers, as well as other types of cancer. Fibroblast activation protein (FAP) is expressed by cancer-associated fibroblasts (CAFs) and is deposited and distributed in the extracellular matrix (ECM). CAFs are involved in different tumor-promoting functions. Using TEAMs that target FAP, it is possible to target CAFs and the ECM itself.

[0203] An anti-mesothelin CAR T construct was designed to include the SS1 anti-mesothelin CAR of Example 1, a TEAM including the FAP scFv antibody reagent sibrotuzumab, and a CD3 scFv antibody reagent (referred to herein as anti-mesothelin CAR FAP TEAM) (Figure 12A). The SS1 anti-mesothelin CAR FAP TEAM demonstrated efficient transduction of donor T cells (Figure 12B); an average transduction efficiency of 19.6% was achieved with the anti-mesothelin CAR FAP TEAM. To determine potential efficacy, a CD69 activation assay was used to quantitate SS1 anti-mesothelin CAR FAP TEAM T cell activation. Results showed that the SS1 anti-mesothelin CAR FAP Team CAR T cells were activated upon contact with mesothelin (Figure 13), indicating that the CAR was functioning properly (n=3 individual donors).

[0204] SS1 anti-mesothelin CAR FAP TEAM T cells were also shown to efficiently secrete FAP. Cells expressing FAP (human foreskin fibroblast (HFF) cells) or CD19 (K562 cells, negative control) were treated with supernatants from CAR FAP TEAM T cell cultures (Figures 14A-14C). Results show significant binding of FAP TEAM to HFF cells (Figures 14A, 14C), but not to K562 cells. FAP binding was detected using an anti-His antibody that binds to TEAM. These results suggest that 1) FAP TEAM is properly produced and secreted, and 2) FAP TEAM is specific to FAP-expressing cells.

[0205] SS1 anti-mesothelin CAR FAP TEAM T cells showed cytotoxicity against HFF cells in cell culture (Figure 15A-15B, n=3 individual T cell donors, each donor in duplicate). In contrast, SS1 anti-mesothelin CAR CD19 TEAM T cells showed no toxicity against HFF cells, suggesting that FAP TEAM contributes significantly to cytotoxicity. In a co-culture system using an impedance-based cytotoxicity assay, we were able to observe tumor lysis and redirection of CART cells by bystander T cells. Impedance assays using split cell cultures suggest that even in the absence of physical interaction with SS1 anti-mesothelin CAR FAP TEAM T cells, secretion of FAP TEAM is sufficient to generate activated bystander T cells and generate cytotoxicity against cancer-associated fibroblasts (CAFs) (Figure 15E, n=3 individual T cell donors, each donor in duplicate). However, physical interaction of CAR FAP TEAM T cells with cancer can increase the rate of cancer cell killing (Figures 15A, 15E).

[0206] The SS1 anti-mesothelin CAR FAP TEAM also reduced pancreatic cancer growth in vivo. Experiments were performed in a mouse model of subcutaneously implanted pancreatic cancer cell lines mixed with cancer-associated fibroblasts (CAFs), a mixture that mimics pancreatic cancer and the tumor microenvironment (TME). Injection of anti-mesothelin CAR FAP TEAM cells resulted in superior tumor control compared to control constructs and untransduced T cells (UTD) (Figure 16). These results demonstrate the efficacy of the anti-mesothelin CAR FAP TEAM in treating mesothelin-expressing cancers in combination with the TME, and the efficacy of the anti-mesothelin CAR FAP TEAM extends to in vivo use.

[0207] Next, the strength (e.g., avidity) of FAP TEAM-mediated physical interactions between FAP-expressing fibroblasts and CAR T cells was quantified using acoustic microscopy. HFFs (human foreskin fibroblasts) were attached to poly-L-lysine-coated microfluidic chips for at least 3 hours before testing on a z-Movi Cell Avidity Analyzer (Lumicks). CellTrace far-red-labeled (ThermoFisher Scientific) flow cytometry-sorted CAR T cells or untransduced T cells (UTD) co-incubated with isolated TEAM molecules were allowed to bind for 5 minutes before increasing the acoustic force. Experiments were performed with two different CAR T cell constructs, including UTD co-incubated with TEAM molecules produced from three normal donors. Results showed that FAP-specific TEAM molecules not only enhanced the avidity of anti-mesothelin CAR FAP TEAM cells compared to UTD, but also enhanced the avidity of UTD co-cultured with FAP-specific molecules (Figure 17). These results suggest that FAP TEAMs are capable of recruiting T cells independent of the presence of a CAR and may be broadly useful in targeting immune system activity to the TME.

[0208] Taken together, these results indicate that the addition of secreted TEAMS to CAR T cells opens two additional therapeutic avenues: first, to target not only the extracellular matrix but also the extracellular matrix-producing cancer-associated fibroblasts; and second, to recruit and redirect CART cells and bystander T cells. Based on these results, CART cells secreting TEAMs that target the extracellular matrix may increase the efficacy of CART cells in solid tumors in general.

Claims

1. (a) an extracellular antigen-binding domain that binds to a tumor antigen comprising mesothelin or a portion thereof; (b) a transmembrane domain, and (c) intracellular signaling domain A T cell comprising a chimeric antigen receptor (CAR) comprising:

2. The T cell described in claim 1, further comprising a therapeutic agent and / or a polynucleotide encoding the therapeutic agent.

3. The T cell of claim 2 , wherein the therapeutic agent comprises an antibody reagent or a cytokine.

4. The T cell of claim 3 , wherein the antibody reagent comprises a single chain antibody, a single domain antibody, or a bispecific antibody reagent.

5. A T cell as described in claim 4, wherein the single domain antibody comprises a camelid antibody.

6. The T cell of claim 4 , wherein the bispecific antibody reagent comprises a T cell engaging antibody molecule (TEAM).

7. (a) the CAR and therapeutic agent are constitutively expressed and / or the CAR and therapeutic agent are produced in the form of a single polypeptide comprising a cleavable moiety between the CAR and therapeutic agent; (b) expression of the CAR and therapeutic agent is driven by an inducible promoter, and / or the CAR and therapeutic agent are produced in the form of a single polypeptide that includes a cleavable moiety between the CAR and therapeutic agent; or (c) the CAR is expressed under the control of a constitutive promoter and the therapeutic agent is expressed under the control of an inducible promoter; The T cell of claim 2.

8. A T cell as described in claim 7, in which the CAR and the therapeutic agent are constitutively expressed and the expression of the CAR and the therapeutic agent is driven by the elongation factor 1 alpha (EF1α) promoter.

9. The T cell described in claim 7, wherein expression of the CAR and the therapeutic agent is driven by an inducible promoter and expression of the CAR and the therapeutic agent is induced by T cell receptor or CAR signaling.

10. The T cell of claim 9, wherein the inducible promoter comprises an NFAT promoter.

11. 8. The T cell of claim 7, wherein the CAR is expressed under the control of a constitutive promoter and the therapeutic agent is expressed under the control of an inducible promoter.

12. A T cell as described in claim 11, wherein expression of the therapeutic agent is induced by T cell receptor or CAR signaling.

13. A T cell described in any one of claims 7 to 12, wherein the CAR and therapeutic agent are produced in the form of a single polypeptide comprising a cleavable portion between the CAR and the therapeutic agent.

14. A T cell described in claim 13, wherein the single polypeptide comprises the amino acid sequence of SEQ ID NO:

1. (a) the transmembrane domain of the CAR comprises a CD8 hinge / transmembrane domain; and / or (b) the intracellular signaling domain comprises a CD37 intracellular signaling domain; and / or (c) the CAR further comprises one or more costimulatory domains. A T cell according to any one of claims 1 to 7.

16. The T cell of claim 15, wherein the costimulatory domain comprises a 4-1BB costimulatory domain. (a) the transmembrane domain of the CAR comprises the sequence of SEQ ID NO: 24 or a variant thereof; and / or (b) the intracellular signaling domain comprises the sequence of SEQ ID NO: 26 or 27 or a variant thereof; and / or (c) the costimulatory domain comprises the sequence of SEQ ID NO: 25 or a variant thereof; The T cell of claim 15.

18. The T cell of claim 2 , wherein the therapeutic agent binds to a tumor antigen and a T cell surface antigen. (a) the tumor antigen to which the therapeutic agent binds is a solid tumor antigen or an activated fibroblast marker; and (b) the T cell surface antigen comprises a T cell receptor (TCR), a TCR complex component, or any portion thereof; The T cell of claim 18.

20. (a) the activated fibroblast marker comprises αSMA (ACTA2), fibroblast activation protein (FAP), platelet-derived growth factor receptors α and β (PDGFRA, PDGFRB), fibroblast-specific protein 1 (FSP1 / S100A4), endoglin (ENG), transgelin (TAGLN), tenascin-C (TNC), periostin (POSTN), chondroitin sulfate proteoglycan 4 or neuron-glial antigen 2 (CSPG4 / NG2), podoplanin (PDPN), or osteopontin (SPP1); and (b) the TCR complex component is selected from CD3, CD4 co-receptors, and CD8 co-receptors; 20. The T cell of claim 19. (i) the tumor antigen-binding domain of the antibody reagent is (a) a VH comprising the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 6; and / or (b) a VL comprising the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 7; and (ii) the T cell surface antigen-binding domain of the antibody reagent is (a) a VH comprising the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 9; and / or (b) a VL comprising the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 10; Including, 20. The T cell of claim 19.

22. (i) the tumor antigen-binding domain of the antibody reagent comprises a single-chain variable fragment (scFv) or a single-domain antibody; and (ii) the T cell surface antigen of the antibody reagent comprises an scFv or a single domain antibody; The T cell of claim 20.

23. (i) the tumor antigen-binding domain of the antibody reagent comprises the amino acid sequence of SEQ ID NO: 21 or 22 or a variant thereof; and (ii) the T cell surface antigen-binding domain of the antibody reagent comprises the amino acid sequence of SEQ ID NO: 11 or 12 or a variant thereof; 23. The T cell of claim 22.

24. The antigen-binding domain of the CAR is (a) a heavy chain variable domain (VH) comprising three complementarity determining regions, CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1 comprises the amino acid sequence of SEQ ID NO: 13, or an amino acid sequence of SEQ ID NO: 13 with not more than one, two, or three amino acid substitutions; CDR-H2 comprises the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence of SEQ ID NO: 14 with not more than one, two, or three amino acid substitutions; and CDR-H3 comprises the amino acid sequence of SEQ ID NO: 15, or an amino acid sequence of SEQ ID NO: 15 with not more than one, two, or three amino acid substitutions; (b) a light chain variable domain (VL) comprising three complementarity determining regions CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 comprises the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence of SEQ ID NO: 16 with not more than one, two, or three amino acid substitutions; CDR-L2 comprises the amino acid sequence of SEQ ID NO: 17, or an amino acid sequence of SEQ ID NO: 17 with not more than one, two, or three amino acid substitutions; and CDR-L3 comprises the amino acid sequence of SEQ ID NO: 18, or an amino acid sequence of SEQ ID NO: 18 with not more than one, two, or three amino acid substitutions. The T cell of claim 1 , comprising:

25. the VH of the antigen-binding domain of the CAR comprises the amino acid sequence of SEQ ID NO: 19 or an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 19; and / or the VL of the antigen-binding domain of the CAR comprises the amino acid sequence of SEQ ID NO: 20 or an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:

20.

25. The T cell of claim 24.

26. whether the VH of the antigen-binding domain of the CAR is N-terminal to the VL of the antigen-binding domain of the CAR; 25. The T cell of claim 24, wherein the VL of the antigen-binding domain of the CAR is N-terminal to the VH of the antigen-binding domain of the CAR, or the antigen-binding domain of the CAR comprises an scFv or a single-domain antibody.

27. The T cell of claim 26, wherein the antigen-binding domain of the CAR comprises an scFv, and the scFv comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 21 or 22, and variants thereof, and amino acid sequences having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 21 or 22.

28. The antigen-binding domain of the CAR is (a) a heavy chain variable domain (VH) comprising complementarity determining regions CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1 comprises the amino acid sequence of SEQ ID NO: 42, or an amino acid sequence of SEQ ID NO: 42 with not more than one, two, or three amino acid substitutions; CDR-H2 comprises the amino acid sequence of SEQ ID NO: 43, or an amino acid sequence of SEQ ID NO: 43 with not more than one, two, or three amino acid substitutions; and CDR-H3 comprises the amino acid sequence of SEQ ID NO: 44, or an amino acid sequence of SEQ ID NO: 44 with not more than one, two, or three amino acid substitutions; (b) a light chain variable domain (VL) comprising complementarity determining regions CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 comprises the amino acid sequence of SEQ ID NO: 45, or an amino acid sequence of SEQ ID NO: 45 with not more than one, two, or three amino acid substitutions; CDR-L2 comprises the amino acid sequence of SEQ ID NO: 46, or an amino acid sequence of SEQ ID NO: 46 with not more than one, two, or three amino acid substitutions; and CDR-L3 comprises the amino acid sequence of SEQ ID NO: 47, or an amino acid sequence of SEQ ID NO: 47 with not more than one, two, or three amino acid substitutions. The T cell of claim 1 , comprising:

29. The T cell of claim 27, wherein the VH of the antigen-binding domain of the CAR comprises the SS1 heavy chain sequence of SEQ ID NO: 40 or an amino acid sequence having at least 90% sequence identity to the SS1 heavy chain sequence of SEQ ID NO: 40; and / or the VL of the antigen-binding domain of the CAR comprises the SS1 light chain sequence of SEQ ID NO: 41 or an amino acid sequence having at least 90% sequence identity to the SS1 light chain sequence of SEQ ID NO:

41.

30. The T cells are (a) a CAR polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 2 to 5; and (b) a therapeutic agent, (i) SEQ ID NOs: 6 and 7, and / or (ii) SEQ ID NOs: 9 and 10; a therapeutic agent comprising the amino acid sequence of Including, A T cell according to any one of claims 1 to 7.

31. The T cell described in claim 2, wherein the therapeutic agent is TEAM, and TEAM includes a FAP binding domain and a CD3 binding domain.

32. A nucleic acid molecule encoding the CAR of any one of claims 1 to 7.

33. 33. The nucleic acid molecule of claim 32, which is a plasmid or viral vector.

34. 1. A CAR T cell product for use in a method of treating a patient having cancer, the method comprising administering to the patient a CAR T cell product genetically modified to secrete a tumor toxic antibody or cytokine, wherein cancer toxicity is directed locally to the tumor microenvironment thereby reducing systemic toxicity, wherein the CAR T cell is genetically modified to deliver an antibody or bispecific antibody against an activated fibroblast marker to the tumor microenvironment, and further wherein the CAR T cell comprises a polynucleotide encoding a CAR comprising an extracellular antigen-binding domain that binds to a tumor antigen comprising mesothelin or a portion thereof, and / or the bispecific antibody is against an activated fibroblast marker and CD3.

35. A pharmaceutical for delivering a therapeutic agent to a patient's tissue or organ to treat a disease or condition, comprising CAR T cells, wherein the CAR T cells have been genetically modified to secrete a therapeutic antibody, toxin, or drug, which is incapable of entering or penetrating the tissue or organ by itself, and wherein the therapeutic antibody is directed against an activated fibroblast marker.

36. A polypeptide comprising the amino acid sequence of SEQ ID NO: 1.

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