WO2025117851A1

WO2025117851A1 - Meso-fap with adam17 inhibitor or itk inhibitor

Info

Publication number: WO2025117851A1
Application number: PCT/US2024/057896
Authority: WO
Inventors: Marcela V. Maus; Trisha R. BERGER
Original assignee: General Hospital Corp
Current assignee: General Hospital Corp
Priority date: 2023-11-30
Filing date: 2024-11-27
Publication date: 2025-06-05
Anticipated expiration: 2026-05-30

Abstract

The present disclosure relates to methods of treating patients using anti-mesothelin- CAR-T cells comprising anti-fibroblast activation protein (FAP) TEAMs, and ADAM 17 or ITK inhibitors.

Description

MESO-FAP WITH ADAM17 INHIBITOR OR ITK INHIBITOR

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 63/604,730, filed November 30, 2023, entitled “Meso-FAP with ADAM17 Inhibitor or ITK Inhibitor”, the entire contents of which are incorporated herein by reference.

FEDERALLY SPONSORED RESEARCH

This invention was made with government support under 5R01CA238268-04 awarded by National Institutes of Health. The Government has certain rights in the invention.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing (M105370045WO00-SEQ-ARM.xml; Size: 52,310 bytes; and Date of Creation: November 20, 2024) is herein incorporated by reference in its entirety.

BACKGROUND

Cell-based gene therapies have shown tremendous promise in treating diseases like hematological cancers. For example, chimeric antigen receptor-T cells (CAR-T cells) are cellbased gene therapies for cancer that have had great success in the treatment of hematologic malignancies. Autologous CAR-T cells are made by collecting a patient’s T cells and genetically modifying them to express a chimeric antigen receptor (a CAR), which confers a novel specificity to the T cells: recognition of a tumor surface antigen through the CAR activates the T cell and initiates tumor killing and expansion of the CAR-T cells. While this therapy has transformed the landscape of treatment options for patients with hematologic cancers, patients with solid tumors have yet to truly benefit from CAR-T cell therapy.

SUMMARY

Prior studies have shown that chimeric antigen receptor T cells (CAR-T cells) are effective in treating hematological malignancies, but treatment of solid tumors with CAR-T cell therapy has not yet shown the same success. This disparity is likely due to a combination of several differences between solid tumors and blood cancers, from identification of target antigens to how CAR-T cells interact with and kill different kinds of tumor cells. While much is known about the expression of surface markers that could serve as CAR targets for blood cancers, solid tumors are not generally diagnosed or characterized by their expression of surface markers, and solid tumors tend to have more heterogeneity, which could lead to outgrowth of tumors with low or no target antigen expression. The parenchymal nature of solid tumors requires CAR-T cell extravasation and migration through a hostile tumor microenvironment (TME). In particular, the collagen-rich extracellular matrix (ECM) of the TME is a non-trivial obstacle for CAR-T cells as they navigate the environment. Cancer-associated fibroblasts (CAFs) in the TME deposit collagen to form the ECM, creating a physical barrier for the infiltration of both drugs and CAR-T cells, in addition to promoting the survival and migration of cancer cells. The development of CAR-T cells that can induce ablation of CAFs in addition to targeting cancer cells represents a promising development in treatment of solid cancers. Furthermore, the use of such CAR-T cells in combination with pharmacological agents that can facilitate their activity - e.g., inhibitors that result in increased expression of the CAR-T cell’s target antigen or promote the polarization of T cells to a tumor-killing phenotype - can further improve the efficacy of CAR-T cells against solid cancers. For example, one consideration in the use of CAR-T cells for solid tumors is the stability of the target antigen expression on cancer cells. Mesothelin is among the most highly-expressed cancer-associated antigens, but its expression on the surface of tumor cells can vary due to proteases in the TME that cleave mesothelin, such as ADAM 17. Cleavage of membrane-bound mesothelin by ADAM 17 decreases mesothelin expression by tumor cells and leads to an increase in soluble mesothelin in the interstitium. Cell-free mesothelin can bind receptors directed against mesothelin or fragments thereof and interfere with their intended purposes, e.g., a T cell containing a CAR directed against mesothelin, such as those provided by the present disclosure, may be unable to target mesothelin-expressing cancer cells due to blockage of the receptor by soluble mesothelin. Accordingly, the administration of an ADAM 17 inhibitor in combination with administration of CAR-T cells containing a mesothelin-binding CAR is contemplated herein.

Among other inhibitors contemplated by the present disclosure are inhibitors of interleukin-2 inducible T cell kinase (ITK). ITK is involved in, among other processes, the activation and regulation of T cell receptor (TCR) signaling, which influences the differentiation and polarization of effector T cells. Different T effector subtypes are desirable for different immunological insults, and Thl and Thl7 cells (characterized by IFNy and IL-17 cytokine production, respectively, and/or TBET and RORyt expression, respectively) are more efficacious against cancer than, e.g., Th2 cells (characterized by IL-4 cytokine production). Inhibition of ITK has been shown to polarize T cells to a Thl/Thl7 phenotype, in addition to increasing T cell numbers and decreasing checkpoint molecule expression.

Accordingly, some aspects of the present disclosure provide a method of treating a patient having a mesothelin-expressing cancer, the method comprising administering to the patient a chimeric antigen receptor (CAR)-T cell in combination with an a disintegrin and metalloprotease 17 (ADAM 17) inhibitor and/or interleukin-2-inducible T cell kinase (ITK) inhibitor, wherein the CAR-T cell comprises: a mesothelin-binding CAR and a T cell engaging molecule (TEAM) comprising a fibroblast activation protein (FAP) binding domain (meso-FAP CAR TEAM cell).

In some embodiments, the TEAM further comprises a CD3 binding domain. In some embodiments, the FAP binding domain and the CD3 binding domain of the TEAM are linked by a linker. In some embodiments, the linker comprises a sequence of SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, or SEQ ID NO: 40.

In some embodiments, the mesothelin-binding CAR comprises a VH domain of SEQ ID NO: 9 and a VL domain of SEQ ID NO: 10.

In some embodiments, the mesothelin-binding CAR comprises: (a) a VH domain comprising three complementarity determining regions (CDR-H1, CDR-H2, and CDR-H3), wherein CDR-H1 comprises SEQ ID NO: 3, CDR-H2 comprises SEQ ID NO: 4, and CDR-H3 comprises SEQ ID NO: 5; and (b) a VL domain comprising three CDRs (CDR-L1, CDR-L2, and CDR-L3), wherein CDR-L1 comprises SEQ ID NO: 6, CDR-L2 comprises SEQ ID NO: 7, and CDR-L3 comprises SEQ ID NO: 8.

In some embodiments, the mesothelin-binding CAR comprises: (a) a VH domain comprising three complementarity determining regions (CDR-H1, CDR-H2, and CDR-H3), wherein CDR-H1 comprises SEQ ID NO: 11, CDR-H2 comprises SEQ ID NO: 12, and CDR- H3 comprises SEQ ID NO: 13; and (b) a VL domain comprising three CDRs (CDR-L1, CDR- L2, and CDR-L3), wherein CDR-L1 comprises SEQ ID NO: 14, CDR-L2 comprises SEQ ID NO: 15, and CDR-L3 comprises SEQ ID NO: 16. In some embodiments, the mesothelin-binding CAR further comprises a hinge/transmembrane domain. In some embodiments, the hinge/transmembrane domain is selected from a group consisting of CD8, CD18, or CD28. In some embodiments, the hinge/transmembrane domain is a CD8 hinge/transmembrane domain. In some embodiments, the hinge/transmembrane domain comprises a sequence of SEQ ID NO: 35.

In some embodiments, the mesothelin-binding CAR comprises an intracellular signaling domain comprising a CD3^ intracellular signaling domain. In some embodiments, the CD3^ intracellular signaling domain comprises the sequence of SEQ ID NO: 33.

In some embodiments, the mesothelin-binding CAR further comprises a co- stimulatory domain. In some embodiments, the co-stimulatory domain is selected from the group consisting of CD2, CD7, CD18, CD27, CD28, and 4-1BB. In some embodiments, the co-stimulatory domain is a 4- IBB costimulatory domain.

In some embodiments, the FAP binding domain of the TEAM comprises a VH domain of SEQ ID NO: 17 and a VL domain of SEQ ID NO: 18.

In some embodiments, the FAP binding domain of the TEAM comprises: (a) a VH domain comprising three CDRs (CDR-H1, CDR-H2, and CDR-H3), wherein CDR-H1 comprises SEQ ID NO: 19, CDR-H2 comprises SEQ ID NO: 20, and CDR-H3 comprises SEQ ID NO: 21; and (b) a VL domain comprising three CDRs (CDR-L1, CDR-L2, and CDR-L3), wherein CDR-L1 comprises SEQ ID NO: 22, CDR-L2 comprises SEQ ID NO: 23, and CDR-L3 comprises SEQ ID NO: 24.

In some embodiments, the CD3 binding domain of the TEAM comprises a VH domain of SEQ ID NO: 25 and a VL domain of SEQ ID NO: 26.

In some embodiments, the CD3 binding domain of the TEAM comprises: (a) a VH domain comprising three CDRs (CDR-H1, CDR-H2, and CDR-H3), wherein CDR-H1 comprises SEQ ID NO: 27, CDR-H2 comprises SEQ ID NO: 28, and CDR-H3 comprises SEQ ID NO: 29; and (b) a VL domain comprising three CDRs (CDR-L1, CDR-L2, and CDR-L3), wherein CDR-L1 comprises SEQ ID NO: 30, CDR-L2 comprises SEQ ID NO: 31, and CDR-L3 comprises SEQ ID NO: 32.

In some embodiments, the mesothelin-binding CAR comprises SEQ ID NO: 41 or SEQ

ID NO: 42.

In some embodiments, the TEAM comprises SEQ ID NO: 43. In some embodiments, the CAR-T cell comprises an amino acid sequence of SEQ ID NO: 44, SEQ ID NO: 45, or SEQ ID NO: 51 encoding the mesothelin-binding CAR and TEAM.

In some embodiments, the mesothelin-expressing cancer is mesothelioma, ovarian cancer, pancreatic cancer, lung cancer, breast cancer, gastric cancer, or colorectal cancer. In some embodiments, the mesothelin-expressing cancer is pancreatic cancer. In some embodiments, the pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC).

In some embodiments, the ADAM17 inhibitor is aderbasib, TAPI-0, TAPI-1, TAPI-2, GW280264X, marimastat, INCB3619, compound 22a (INCB9471), MED 13622, apratastat, DPC-333, SCH 900567, or KP-457.

In some embodiments, the ADAM 17 inhibitor is aderbasib.

In some embodiments, the ITK inhibitor is ibrutinib, CPI-818, BMS 509774, or PRN694. In some embodiments, the ITK inhibitor is ibrutinib.

In some embodiments, a method described herein comprises administering the CAR-T cell by a first route of administration and administering the ADAM 17 inhibitor and/or ITK inhibitor by a second route of administration that is different from the first route of administration. In some embodiments, the administration of the CAR-T cell comprises intravenous administration and the administration of the ADAM 17 inhibitor and/or the ITK inhibitor comprises intraperitoneal administration. In some embodiments, the administration of the CAR-T cell comprises intravenous administration and the administration of the ADAM 17 inhibitor and/or the ITK inhibitor comprises oral administration.

In some embodiments, the ADAM 17 inhibitor is not a protein-based inhibitor.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIG. 1 is a schematic of the tumor microenvironment (TME), illustrating the cell types present, e.g., myeloid cells, myeloid-derived suppressor cells (MDSCs), cancer-associated fibroblasts (CAFs), T cells, cancer cells, and other cell types. Also shown is the vasculature and targets of inhibition by the ITK inhibitor ibrutinib and the ADAM 17 inhibitor aderbasib.

FIGs. 2A-2E illustrate the effectiveness of cells that secrete T cell engaging molecules (TEAMs) against fibroblast activated protein (FAP) and that comprise anti-mesothelin chimeric antigen receptors (meso-FAP CAR- TEAM cells) cancer-associated fibroblasts (CAFs) and pancreatic ductal adenocarcinoma (PDAC) cells. FIG. 2A shows the tumor volume in immunodeficient NSG® mice with subcutaneous AsPC-1 tumors (mesothelin-expressing tumors derived from a human PDAC cell line) and treated intravenously with either untransduced (UTD) control cells, meso-FAP CAR- TEAM cells, or meso-CD19 CAR-TEAM cells (not specific for mesothelin-expressing tumors). AsPC-1 tumor-bearing mice showed a significant reduction in tumor volume (FIG. 2A) and presence of AsPC-1 tumor cells (FIG. 2B) following treatment with meso-FAP CAR- TEAM cells compared to AsPC- 1 tumor-bearing mice treated with either meso-CD19 CAR-TEAM cells or UTD cells. FIGs. 2C-2E show the effect of meso- FAP CAR-TEAM cells against patient-derived mesothelin-expressing tumor organoids grown in the presence of CAFs. Organoid-CAF cultures treated with meso-FAP CAR- TEAM cells had a marked reduction in organoid recovery compared to cultures treated with UTD cells (FIGs. 2C, 2E, top), and a significant reduction in CAF cells compared to cultures treated with UTD cells or meso-CD19 CAR-TEAM cells (FIGs. 2D, 2E, bottom).

FIGs. 3A-3C illustrate the differential effect of meso-FAP CAR-TEAM cells depending upon the route of administration. AsPC-1 tumor cells were injected in NSG mice intraperitoneally (IP), after which mice were treated with meso-FAP CAR-TEAM cells IP (FIG. 3A) or intravenously (IV) (FIG. 3B). In both instances, mice treated with meso-FAP CARTEAM cells survived significantly longer than mice treated with UTD cells. These differences were reduced or eliminated when AsPC- 1 tumor cells were implanted subcutaneously and meso- FAP CAR-TEAM cells were injected IV (FIG. 3C).

FIGs. 4A-4E show the efficacy of meso-FAP CAR-TEAM cell killing on pancreatic (AsPC-1 or BxPC-3) and ovarian (SKOV3) cancer cells. FIG. 4A shows expression of ADAM17, ADAM10, and mesothelin on pancreatic cancer cell lines (ASPC1, CAP AN-2, and BxPC3). FIG. 4B, 4D show mesothelin expression on pancreatic cancer cells (AsPC-1, BxPC- 3) (FIG. 4B) and ovarian cancer cells (FIG. 4D). The cytolytic capacity of meso-FAP CAR- TEAM cells against pancreatic cancer cells (AsPC-1, BxPC-3) (FIG. 4C) and ovarian cancer cells (FIG. 4E) is significantly higher than that of UTD cells.

FIG. 5 is a timeline of clinical treatment of candidate subjects with meso-FAP CARTEAM cells. The bottom timepoints indicate patient monitoring and research sample collection.

FIGs. 6A-6D relate to expression of mesothelin on pancreatic cancer cells treated with aderbasib (INCB7839). FIG. 6A shows fluorescent curves of mesothelin expression on ASPC-1 cells treated with aderbasib (summarized with histograms in FIG. 6B). FIG. 6C shows mesothelin expression on CAP AN-2 cells treated with aderbasib, and FIG. 6D shows mesothelin expression on BxPC3 cells treated with aderbasib.

FIGs. 7A-7C relate to the prevention of mesothelin shedding on ASPC1 (FIG. 7A), CAP AN-2 (FIG. 7B), and BxPC3 (FIG. 7C) cells treated with aderbasib.

FIGs. 8A-8C relate to cytolysis of pancreatic cancer cells (FIG. 8A, ASPC1 cells; FIG. 8B, CAP AN-2 cells; FIG. 8C, BxPC3 cells) treated with SSI CAR-T cells or untransduced T cells (CAR- cells) with or without aderbasib. The asterisk denotes the following treatment groups: tumor only, no aderbasib and untransduced cells, aderbasib only, and aderbasib with untransduced cells.

FIGs. 9A-9B relate to co-treatment of mice with CAR-T cells and aderbasib. FIG. 9A is an experimental schematic. FIG. 9B shows tumor growth in mice injected subcutaneously (s.c.) with ASPC1 cells and treated 14 days later with SSI CAR-T cells, with or without supplemental aderbasib treatment.

FIGs. 10A-10E relate to tumor growth in mice injected subcutaneously (s.c.) with ASPC1 cells and treated 14 days later with SSI CAR-T cells with or without supplemental ibrutinib treatment (experimental schematic, FIG. 10A; summary figure, FIG. 10B; CAR-T treatment only, FIG. IOC; CAR-T treatment in combination with ibrutinib treatment, FIG. 10D; averages shown in FIG. 10E).

FIG. 11 is an exemplary construct encoding an anti-mesothelin CAR, a T cell engaging molecule (TEAM) targeting fibroblast activated protein (FAP) and CD3, and truncated CD 19 (tCD29). DETAILED DESCRIPTION

In some aspects, this disclosure describes a method of treating a subject having a mesothelin-expressing cancer, the method comprising administering to the patient a chimeric antigen receptor (CAR)-T cell in combination with an a disintegrin and metalloprotease 17 (ADAM 17) inhibitor or interleukin-2-inducible T cell kinase (ITK) inhibitor, wherein the CAR- T cell comprises: a mesothelin-binding CAR; and a T cell engaging molecule (TEAM) comprising a fibroblast activation protein (FAP) binding domain.

General Definitions

The terms "decrease", "reduced", or "reduction" are all used herein to mean a decrease by a statistically significant amount. In some embodiments, "reduce", "reduction", or "decrease" typically means a decrease by at least 10% as compared to a reference level (e.g., the absence of a given treatment or agent) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% , or more. Where applicable, a decrease can be preferably down to a level accepted as within the range of normal for an individual without a given disorder.

A "disease" is a state of health of an animal, for example, a human, wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated, then the animal's health continues to deteriorate. In contrast, a "disorder" in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal's state of health. In some embodiments, the disease is a cancer or a tumor.

As used herein, the terms "tumor antigen", “tumor-associated antigen” and "cancer antigen" are used interchangeably to refer to antigens that are differentially expressed by cancer cells and can thereby be exploited in order to target cancer cells. Cancer antigens are antigens that can potentially stimulate tumor- specific immune responses. Some of these antigens are encoded, although not necessarily expressed, by normal cells. These antigens can be characterized as those which are normally silent (i.e., not expressed) in normal cells, those that are expressed only at certain stages of differentiation and those that are temporally expressed such as embryonic and fetal antigens. Other cancer antigens are encoded by mutant 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 on RNA and DNA tumor viruses. Many tumor antigens have been defined in terms of multiple solid tumors: MAGE 1, 2, & 3, defined by immunity; MART-l/Melan-A, gplOO, carcinoembryonic antigen (CEA), human epidermal growth factor receptor (HER2), mucins (i.e., MUC-1), pro state- 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-associated antigen is any one of CD19, CD79b, TACI, BCMA, MUC1, MUC16, B7H3, mesothelin, CD70, PSMA, PSCA, EGFRvIII, claudin6, or any pair of CD19/CD79b, or BCMA/TACI.

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

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

At a minimum, an "activated T cell" as used herein is a proliferative T cell.

As used herein, the terms "specific binding" and "specifically binds" refer to a physical interaction between two molecules, compounds, cells and/or particles wherein the first entity binds to the second, target, entity with greater specificity and affinity than it binds to a third entity which is a non-target. In some embodiments, specific binding can refer to an affinity of the first entity for the second target, entity, which 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 the affinity for the 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. A nonlimiting example includes an antibody, or a ligand, which recognizes and binds with a cognate binding partner (for example, a stimulatory and/or costimulatory molecule present on a T cell) protein.

A "stimulatory ligand," as used herein, refers to a ligand that when present on an antigen presenting cell (APC) (e.g., a macrophage, a dendritic cell, a B-cell, an artificial APC, and the like) can specifically bind with 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, and the like. Stimulatory ligands are well-known in the art and encompass, inter alia, an MHC Class I molecule loaded with a peptide, an antiCD3 antibody, a superagonist anti-CD28 antibody, and a superagonist anti-CD2 antibody.

A "stimulatory molecule," as the term is used herein, means a molecule on a T cell that specifically binds with a cognate stimulatory ligand present on an antigen presenting cell. "Costimulatory ligand," as the term is used herein, includes a molecule on an APC that specifically binds a cognate co- stimulatory molecule on a T cell, thereby providing a signal which, in addition to the primary signal provided by, for instance, binding of a TCR/CD3 complex with an MHC molecule loaded with peptide, mediates a T cell response, including, but not limited to, proliferation, activation, differentiation, and the like. A co- stimulatory ligand can include, but is not limited to, 4-1BBL, OX40L, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, inducible co-stimulatory 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, an agonist or antibody that binds Toll-like receptor and a ligand that specifically binds with B7-H3. A co-stimulatory ligand also can include, but is not limited to, an antibody that specifically binds with a co-stimulatory molecule present on a T cell, such as, but not limited to, CD27, CD28, 4- IBB, 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83.

A "co-stimulatory molecule" refers to the cognate binding partner on a T cell that specifically binds with a co-stimulatory ligand, thereby mediating a co-stimulatory response by the T cell, such as, but not limited to, proliferation. Co-stimulatory molecules include but are not limited to an MHC class I molecule, BTLA, a Toll-like receptor, CD27, CD28, 4- IBB, 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83.

In some embodiments, the term "engineered" and its grammatical equivalents as used herein can refer to one or more human-designed alterations of a nucleic acid, e.g., the nucleic acid within an organism's genome. In another embodiment, engineered can refer to alterations, additions, and/or deletion of genes. An "engineered cell" can refer to a cell with an added, deleted and/or altered gene.

The term "cell" or "engineered cell" and their grammatical equivalents as used herein can refer to a cell of human or non-human animal origin.

As used herein, the term "operably linked" refers to a first polynucleotide molecule, such as a promoter, connected with a second transcribable polynucleotide molecule, such as a gene of interest, where the polynucleotide molecules are so arranged 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 transcription of the gene of interest in a cell.

In the various embodiments described herein, it is further contemplated that variants (naturally occurring or otherwise), alleles, homologs, conservatively modified variants, and/or conservative substitution variants of any of the particular polypeptides described are encompassed. As to amino acid sequences, one of ordinary skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution 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.

A given amino acid can be replaced by a residue having similar physiochemical characteristics, e.g., substituting one aliphatic residue for another (such as He, Vai, Leu, or Ala for one another), or substitution of one polar residue for another (such as between Lys and Arg; Glu and Asp; or Gin and Asn). Other such conservative substitutions, e.g., substitutions of entire regions having similar hydrophobicity characteristics, are well known. Polypeptides comprising conservative amino acid substitutions can be tested in any one of the assays described herein to confirm that a desired activity, e.g., ligand-mediated receptor activity and specificity of a native or reference polypeptide is retained. Amino acids can be grouped according to similarities in the properties of their side chains (in A. L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)): (1) non-polar: Ala (A), Vai (V), Leu (L), He (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gin (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 sidechain properties: (1) hydrophobic: Norleucine, Met, Ala, Vai, Leu, He; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions will entail exchanging a member of one of these classes for another class.

Particular conservative substitutions include, for example: Ala into Gly or into Ser; Arg into Lys; Asn into Gin or into His; Asp into Glu; Cys into Ser; Gin into Asn; Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gin; He into Leu or into Vai; Leu into He or into Vai; Lys into Arg, into Gin or into Glu; Met into Leu, into Tyr or into He; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp; and/or Phe into Vai, into He or into Leu.

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 wildtype reference polypeptide's activity according to an assay known in the art or described below herein. A functional fragment can comprise conservative substitutions of the sequences disclosed herein.

In some embodiments, a polypeptide described herein can be a variant of a polypeptide or molecule as described herein. In some embodiments, the variant is a conservatively modified variant. Conservative substitution variants can be obtained by mutations of native nucleotide sequences, for example. A "variant," as referred to herein, is a polypeptide substantially homologous to a native or reference polypeptide, but which has an amino acid sequence different from that of the native or reference polypeptide because of one or a plurality of deletions, insertions, or substitutions. Variant polypeptide-encoding DNA sequences encompass sequences that comprise one or more additions, deletions, or substitutions of nucleotides when compared to a native or reference DNA sequence, but that encode a variant protein or fragment thereof that retains activity of the non-variant polypeptide. A wide variety of PCR-based sitespecific mutagenesis approaches are known in the art and can be applied by the ordinarily skilled artisan.

A variant amino acid or DNA sequence can 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 mutant sequence can be determined, for example, by comparing the two sequences using freely available computer programs commonly employed for this purpose on the world wide web (e.g., BLASTp or BLASTn with default settings).

Alterations of the native amino acid sequence can be accomplished by any of a number of techniques known to one of skill in the art. Mutations can be introduced, for example, at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites permitting ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion. Alternatively, oligonucleotide directed site-specific mutagenesis procedures can be employed to provide an altered nucleotide sequence having particular codons altered according to the substitution, deletion, or insertion required. Techniques for making such alterations are well established and include, for example, those disclosed by 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. Any cysteine residue not involved in maintaining the proper conformation of a polypeptide also can be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) can be added to a polypeptide to improve its stability or facilitate oligomerization.

The term "polynucleotide" is used herein interchangeably with "nucleic acid molecule" to indicate a polymer of nucleosides. Typically, a polynucleotide is composed of nucleosides that are naturally found in DNA or RNA (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine) joined by phosphodiester bonds. However, the term encompasses molecules comprising nucleosides or nucleoside analogs containing chemically or biologically modified bases, modified backbones, etc., whether or not found in naturally occurring nucleic acids, and such molecules may be preferred for certain applications. Where this disclosure refers to a polynucleotide it is understood that both DNA, RNA, and in each case both single- and double-stranded forms (and complements of each single- stranded molecule) are provided. "Polynucleotide sequence" as used herein can refer to the polynucleotide material itself and/or to the sequence information (i.e., the succession of letters used as abbreviations for bases) that biochemically characterizes a specific 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 exist within a given cell.

A polynucleotide sequence presented herein is presented in a 5' to 3' direction unless otherwise indicated.

The term "polypeptide" as used herein refers to a polymer of amino acids. The terms "protein" and "polypeptide" are used interchangeably herein. A peptide may be a relatively short polypeptide, typically between about 2 and 60 amino acids in length. Polypeptides used herein typically contain amino acids such as the 20 L-amino acids that are 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 may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a phosphate group, a fatty acid group, a linker for conjugation, functionalization, etc. A polypeptide that has a nonpolypeptide moiety covalently or noncovalently associated therewith is still considered a "polypeptide." Exemplary modifications include glycosylation and palmitoylation. Polypeptides can be purified from natural sources, produced using recombinant DNA technology or synthesized through chemical means such as conventional solid phase peptide synthesis, etc. The term "polypeptide sequence" or "amino acid sequence" as used herein can refer to the polypeptide material itself and/or to the sequence information (i.e., the succession of letters or three letter codes used as abbreviations for amino acid names) that biochemically characterizes a polypeptide. A polypeptide sequence presented herein is presented in an N-terminal to C-terminal direction unless otherwise indicated. The term "gene" means the nucleic acid sequence which is transcribed (DNA) to RNA in vitro or in vivo when operably linked to appropriate regulatory sequences. The gene may or may not include regions preceding and following the coding region, e.g., 5' untranslated (5' UTR) or "leader" sequences and 3' UTR or "trailer" sequences, as well as intervening sequences (intrans) between individual coding segments (exons).

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

In some embodiments, a polypeptide, polynucleotide, plasmid and or/vector as described herein optionally further comprises a reporter molecule, e.g., to determine if the vector is properly expressed in a cell. In some embodiments, the reporter molecule may be a fluorescent protein (e.g., GFP, YFP, RF), antibody (e.g., CD34, tEGFR, tCD19, tCD20, tCD34, and tHer2), and a radioisotope. In some embodiments, the reporter molecule is hygromycin phosphotransferase (hph) that can be imaged alone or in combination with a substrate or chemical (for example 9-[4-[18F]fluoro-3-(hydroxymethyl)butyl]guanine ([18F]FHBG)).

In some embodiments, GFP and mCherry may be used as fluorescent tags for imaging a CAR expressed on a T cell (e.g., a CAR-T cell). 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, the CAR need not include a fluorescent tag or fluorescent protein. In each instance of particular constructs provided herein, therefore, any markers present in the constructs can be removed.

Chimeric Antigen Receptors (CARs)

The terms "chimeric antigen receptor" or "CAR" or "CARs", as used herein, refer to engineered T cell receptors, which graft a ligand or antigen specificity onto immune cells. Immune cells are of hematopoietic origin, and include lymphocytes, such as B cells and T cells; natural killer cells; and myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is a T-cell (for example, naive T cells, central memory T cells, effector memory T cells or combinations thereof). CARs are also known as artificial T cell receptors, chimeric T cell receptors or chimeric immunoreceptors. Any of the CARs described in this section may be the CAR in the CAR TEAM cells.

A CAR places an extra-cellular binding domain that specifically binds a target, e.g., a polypeptide, expressed on the surface of a cell to be targeted for a T cell response, onto a construct including a transmembrane domain and intracellular domain(s) of a T cell receptor molecule. In some embodiments, the extra-cellular binding domain includes the antigen domain(s) of an antibody that specifically binds an antigen expressed on a cell to be targeted for a T cell response. In some embodiments, the extra-cellular binding domain includes a ligand that specifically binds an antigen expressed on a cell to be targeted for a T cell response.

As used herein, a "CAR-T cell" or "CAR-T" refers to a T cell that expresses a CAR. When expressed in a T cell, CARs have the ability to redirect T cell specificity and reactivity toward a selected target in a non-MHC-restricted manner, exploiting the antigen binding properties of monoclonal antibodies. The non-MHC-restricted antigen recognition gives T cells expressing CARs the ability to recognize an antigen independent of antigen processing, thus bypassing a major mechanism of tumor escape.

As can be determined by those of skill in the art, various functionally similar or equivalent components of these CARs can be swapped or substituted with one another, as well as other similar or functionally equivalent components known in the art or listed herein.

Any cell-surface moiety can be targeted by a CAR. Often, the target will be a cellsurface polypeptide that may be differentially or preferentially expressed on a cell that one wishes to target for a T cell response. In some embodiments, the extra-cellular binding domain binds to the cancer-associated antigen mesothelin, e.g., as described in PCT/US2020/065733 and/or PCT/US2020/036108. Without wishing to be bound by theory, mesothelin (MSLN) (uniprot.org/uniprot/Q13421) is expressed on normal mesothelial cells in some tissues (e.g., pleura, pericardium, peritoneum) and in trace amounts in some epithelial cells (e.g., ovary, tunica vaginalis, rete testis, and 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 hereby incorporated by reference. Mesothelin may be used as a marker for cells associated with various cancers (e.g., over-expressed in various cancers), and CARs and CAR-T cells that bind to mesothelin or a portion thereof may be used to treat subjects having, e.g., cancers associated with mesothelin expression (mesothelin-expressing cancers). In some embodiments, a CAR-T cell binds to a mesothelin or a portion thereof of at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 50. In some embodiments, a CAR-T cell binds to a mesothelin or a portion thereof of at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 50. In some embodiments, a CAR-T cell binds to a mesothelin or a portion thereof of at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 50. In some embodiments, a CAR-T cell binds to a mesothelin or a portion thereof of at least 97% sequence identity to the amino acid sequence of SEQ ID NO: 50. In some embodiments, a CAR-T cell binds to a mesothelin or a portion thereof of at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 50. In some embodiments, a CAR-T cell binds to a mesothelin or a portion thereof of at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 50. In some embodiments, a CAR-T cell binds to a mesothelin or a portion thereof having the amino acid sequence of SEQ ID NO: 50.

Extra-Cellular Binding Domain

As used herein, the term "extra-cellular binding domain" refers to a polypeptide found on the outside of the cell that is sufficient to facilitate binding to a target. In some embodiments, the CARs described herein comprise an extra-cellular binding domain. The extra-cellular binding domain will specifically bind to its binding partner, i.e., the target. As non-limiting examples, the extra-cellular binding domain can include an antigen domain of an antibody, or a ligand, which recognizes and binds with a cognate binding partner protein. In this context, a ligand is a molecule that binds specifically to a portion of a protein and/or receptor. The cognate binding partner of a ligand useful in the methods and compositions described herein can generally be found on the surface of a cell. Ligand:cognate partner binding can result in the alteration of the ligand-bearing receptor, or activate a physiological response, for example, the activation of a signaling pathway. In some embodiments, the ligand can be non-native to the genome. In some embodiments, the ligand has a conserved function across at least two species.

Any cell-surface moiety can be targeted by a CAR (e.g., the extra-cellular binding domain of the CAR). In some embodiments, the target will be a cell-surface polypeptide that may be differentially or preferentially expressed on a cell that one wishes to target for a T cell response. To target Tregs, antibodies can be targeted against, e.g., Glycoprotein A Repetitions Predominant (GARP), latency-associated peptide (LAP), CD25, CTLA-4, ICOS, TNFR2, GITR, 0X40, 4-1BB, and LAG-3.

. In a healthy state, mesothelin is expressed by structural cells lining a number of tissues, e.g., the lungs, the heart, and the peritoneum. In a diseased state however, e.g., cancer, mesothelin can be overexpressed, e.g., in mesothelioma, ovarian cancer, pancreatic cancer, and lung adenocarcinoma, among others. The function of mesothelin in normal cells is not completely understood, but its prevalence in cancer makes it a compelling target for CAR-T cells, such as those provided in the present disclosure.

In some embodiments, the CAR vector comprises a CAR comprising an extra-cellular binding domain that binds mesothelin. In some embodiments, the mesothelin CAR comprises a polynucleotide encoding an extra-cellular binding domain comprising a mesothelin antibody (e.g., scFv). In some embodiments, the mesothelin scFv comprises a VH domain of SEQ ID NO: 1 or SEQ ID NO: 9 and a VL domain of SEQ ID NO: 2 or SEQ ID NO: 10, or a variant thereof. In some embodiments, the mesothelin scFv comprises SEQ ID NO: 41 or SEQ ID NO: 42, or a variant thereof. In some embodiments, the mesothelin scFv comprises a VH domain of SEQ ID NO: 1 and a VL domain of SEQ ID NO: 2, or a variant thereof. In some embodiments, the mesothelin scFv comprises a VH domain of SEQ ID NO: 9 and a VL domain of SEQ ID NO: 10.

Hinge and Transmembrane Domains

In some embodiments, the CAR polypeptide further comprises a transmembrane domain, or a hinge/transmembrane domain, which joins the extra-cellular binding domain to the intracellular signaling domain. The binding domain of the CAR is, in some embodiments, followed by one or more "hinge domains," which plays a role in positioning the extra-cellular binding domain away from the effector cell surface to enable proper cell/cell contact, antigen binding (by the extra-cellular binding domain) and activation. A CAR may include one or more hinge domains between the binding domain and the transmembrane domain (TM). The hinge domain may be derived either from a natural, synthetic, semi- synthetic, or recombinant source. The hinge domain may include the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region. Illustrative hinge domains suitable for use in the CARs described herein include the hinge region derived from the extracellular regions of type 1 membrane proteins such as CD8 (e.g., CD8alpha), CD4, CD28, 4- 1BB, and CD7, which may be wild- type hinge regions from these molecules or may be altered. In some embodiments, the CAR comprises polynucleotide encoding CD8alpha hinge/transmembrane domain. In some embodiments, the CAR comprises a polynucleotide encoding a 4- IBB intracellular domain.

In some embodiments, the hinge region is derived from the hinge region of an immunoglobulin like protein (e.g., IgA, IgD, IgE, IgG, or IgM), CD28, or CD8. In some embodiments, the hinge domain includes a CD8a hinge region.

As used herein, "transmembrane domain" (TM domain) refers to the portion of the CAR that fuses the extracellular binding portion, in some embodiments via a hinge domain, to the intracellular portion (e.g., the costimulatory domain and intracellular signaling domain) and anchors the CAR to the plasma membrane of the immune effector cell. The transmembrane domain is a generally hydrophobic region of the CAR, which crosses the plasma membrane of a cell. The TM domain can be the transmembrane region or fragment thereof of a transmembrane protein (for example a Type I transmembrane protein or other transmembrane protein), an artificial hydrophobic sequence, or a combination thereof. While specific examples are provided herein and used herein, other transmembrane domains will be apparent to those of skill in the art and can be used in connection with alternate embodiments of the technology. A selected transmembrane region or fragment thereof would preferably not interfere with the intended function of the CAR.

As used in relation to a transmembrane domain of a protein or polypeptide, "fragment thereof" refers to a portion of a transmembrane domain that is sufficient to anchor or attach a protein to a cell surface.

In some embodiments, the transmembrane domain or fragment thereof of the CAR described herein includes a transmembrane domain selected from the transmembrane domain of an alpha, beta or zeta chain of a T cell receptor, CD2, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, 0X40, CD2, CD27, LFA-1 (CDl la, CD18), ICOS (CD278), 4-1BB (CD137), 4- 1BBL, GITR, CD40, BAFFR, HVEM (EIGHTR), SEAMF7, NKp80 (KERFI), CD160, CD19, IE2R beta, IE2R gamma, IE7R a, ITGA1, VEA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VEA- 6, CD49f, ITGAD, CDl ld, ITGAE, CD103, ITGAL, CDl la, LFA-1, ITGAM, CDl lb, ITGAX, CD 11c, ITGB1, CD29, ITGB2, CD 18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, LylO8), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG/Cbp, NKp44, NKp30, NKp46, NKG2D, and/or NKG2C.

As used herein, a "hinge/transmembrane domain" refers to a domain including both a hinge domain and a transmembrane domain. For example, a hinge/transmembrane domain can be derived from the hinge/transmembrane domain of CD8, CD28, CD7, or 4- IBB. In some embodiments, the hinge/transmembrane domain of a CAR or fragment thereof is derived from or includes the hinge/transmembrane domain of CD8 (e.g., SEQ ID NO: 35, or variants thereof). CD8 is an antigen preferentially found on the cell surface of cytotoxic T lymphocytes. CD8 mediates cell-cell interactions within the immune system, and acts as a T cell co-receptor. CD8 consists of an alpha (CD8alpha or CD8a) and beta (CD813 or CD8b) chain. CD8a sequences are known for a number of species, e.g., human CD8a, (NCBI Gene ID: 925) polypeptide (e.g., NCBI Ref Seq NP 001139345.1) and mRNA (e.g., NCBI Ref Seq 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 the CD8 of, e.g., dog, cat, cow, horse, pig, and the like.

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

In some embodiments, the CD8 hinge and transmembrane sequence corresponds to the amino acid sequence of SEQ ID NO: 35; or includes a sequence with 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: 35.

Co-stimulatory Domains

Each CAR described herein optionally includes the intracellular domain of one or more co-stimulatory molecule or co-stimulatory domain. As used herein, the term "co-stimulatory domain" refers to an intracellular signaling domain of a co-stimulatory molecule. Co-stimulatory molecules are cell surface molecules other than antigen receptors or Fe receptors that provide a second signal required for efficient activation and function of T lymphocytes upon binding to antigen. The co-stimulatory domain can be, for example, the co-stimulatory domain of 4- IBB, CD27, CD28, or 0X40. Additional illustrative examples of such co-stimulatory molecules include CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (0X40), 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 is an important regulator of immune responses.

4-1BB is a membrane receptor protein, also known as CD137, which is a member of the tumor necrosis factor (TNF) receptor superfamily. 4- IBB is expressed on activated T lymphocytes. 4- IBB sequences are known for a number of species, e.g., human 4-1 BB, also known as TNFRSF9 (NCBI Gene 25 ID: 3604) and mRNA (NCBI Reference Sequence: NM_001561.5). 4-1BB can refer to human 4-1BB, including naturally occurring variants, molecules, and alleles thereof. In some embodiments of any of the aspects, e.g., in veterinary applications, 4-1BB can refer to the 4-1BB of, e.g., dog, cat, cow, horse, pig, and the like. Homologs and/or orthologs of human 4- IBB are readily identified for such species by one of skill in the art, e.g., using the NCBI ortholog search function or searching available sequence data for a given species for sequence similar to a reference 4-1 BB sequence.

Intracellular Signaling Domains

In some embodiments, the CAR comprises a polynucleotide encoding a CD3^ intracellular signaling domain.

The properties of the intracellular signaling domain(s) of the CAR can vary as known in the art and as disclosed herein, but the chimeric target/extra-cellular binding domains(s) render the receptor sensitive to signaling activation when the chimeric target/extra-cellular binding domain binds the target/antigen on the surface of a targeted cell.

With respect to intracellular signaling domains, so-called "first-generation" CARs include those that solely provide CD3^ signals upon antigen binding by the extra-cellular binding domain. So-called "second- generation" CARs include those that provide both costimulation (e.g., CD28 or CD137) and activation (CD3^) domains, and so-called "third- generation" CARs include those that provide multiple costimulatory (e.g., CD28 and CD 137) domains and activation domains (e.g., CD3^). In various embodiments, the CAR is selected to have high affinity or avidity for the target/antigen - for example, antibody-derived target or extra-cellular binding domains will generally have higher affinity and/or avidity for the target antigen than would a naturally occurring T cell receptor. This property, combined with the high specificity one can select for an antibody provides highly specific T cell targeting by CAR-T cells.

CARs as described herein include an intracellular signaling domain. An "intracellular signaling domain" refers to the part of a CAR polypeptide that participates in transducing the message of effective CAR binding to a target antigen into the interior of the immune effector cell to elicit effector cell function, e.g., activation, cytokine production, proliferation and cytotoxic activity, including the release of cytotoxic factors to the CAR-bound target cell, or other cellular responses elicited following antigen binding to the extracellular CAR domain. In various examples, the intracellular signaling domain is from CD3^ (see, e.g., below). Additional non-limiting examples of immunoreceptor tyrosine-based activation motif (ITAM)-containing intracellular signaling domains that are of particular use in the technology include those derived from TCR^, FcRy, FcRp, CD3y, CD30, CD3o, CD3r|, CD3e, CD3^, CD22, CD79a, CD79b, and CD66d.

CD3 is a T cell co-receptor that facilitates T lymphocyte activation when simultaneously engaged with the appropriate co-stimulation (e.g., binding of a co- stimulatory molecule). A CD3 complex consists of 4 distinct chains; mammalian CD3 consists of a CD3y chain, a CD38 chain, and two CD3e chains.

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

In some embodiments of any aspect, a CAR polypeptide described herein includes an intracellular signaling domain that includes an Immunoreceptor Tyrosine-based Activation Motif or ITAM from CD3^, including variants of CD3^ such as IT AM-mutated CD3^, CD3r|, or CD30. In some embodiments of any aspect, the ITAM includes three motifs of ITAM of CD3^ (ITAM3). In some embodiments of any aspect, the three motifs of IT AM of CD3^ are not mutated and, therefore, include native or wild-type sequences. In some embodiments, the CD3^ sequence includes the sequence of a CD3^ as set forth in the sequences provided herein, e.g., a CD3^ sequence of SEQ ID NO: 33, or variants thereof.

For example, a CAR polypeptide described herein includes the intracellular signaling domain of CD3^. In some embodiments, the CD3^ intracellular signaling domain corresponds to an amino acid sequence of SEQ ID NO: 33; or includes a sequence with 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 of SEQ ID NO: 33.

In some embodiments, the intracellular domain is the intracellular domain of a 4-1BB. In some embodiments, the 4- IBB intracellular domain corresponds to an amino acid sequence selected from SEQ ID NO: 46; or includes a sequence selected from SEQ ID NO: 46; or includes at least 75%, at least 80%, at least 85%, 35 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: 46. Individual CAR and other construct components as described herein can be used with one another and swapped in and out of various constructs described herein, as can be determined by those of skill in the art. Each of these components can include or consist of any of the corresponding sequences set forth herein, or variants thereof.

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.

Signal Peptide

In some embodiments, a CAR polypeptide as described herein includes a signal peptide. Signal peptides can be derived from any protein that has an extracellular domain or is secreted. A CAR polypeptide as described herein may include any signal peptides known in the art. In some embodiments, the CAR polypeptide includes a CD8 signal peptide, e.g., a CD8 signal peptide corresponding to the amino acid sequence of SEQ ID NO: 47 or including 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: 47.

In further embodiments, a CAR polypeptide described herein may optionally exclude one of the signal peptides described herein, e.g., a CD8 signal peptide of SEQ ID NO: 47 or an IgK signal peptide of SEQ ID NO: 47.

Linkers

In some embodiments, the CAR further includes a linker domain. As used herein, "linker" refers to an oligo- or polypeptide region from about 2 to 100 amino acids in length, which links together any of the domains/regions of the CAR as described herein. In some embodiment, linkers can include or be composed of flexible residues such as glycine and serine so that the adjacent protein domains are free to move relative to one another. Linker sequences may be from 2 to 100 amino acids, 5 to 50 amino acids, 10 to 15 amino acids, 15 to 20 amino acids, or 18 to 20 amino acids in length, and include any suitable linkers known in the art. For instance, linker sequences may include, but are not limited to, glycine/serine linkers, e.g., SEQ ID NOs: 36-38 and 40, as described by Whitlow et al., Protein Eng. 6(8) :989-95, 1993; the linker sequence of SEQ ID NO: 39, as described by Andris-Widhopf et al., Cold Spring Harb. Protoc. 2011 (9), 2011; as well as linker sequences with added functionalities, e.g., an epitope tag or an encoding sequence containing Cre-Lox recombination site as described by Sblattero et al., Nat. Biotechnol. 18(1 ):75-80, 2000. Longer linkers may be used when it is desirable to ensure that two adjacent domains do not sterically interfere with one another. Linkers may be cleavable or non-cleavable.

Furthermore, linkers may be cleavable or non-cleavable. Examples of cleavable linkers include 2A linkers (e.g., P2A (SEQ ID NO: 48) and T2A (SEQ ID NO: 49)), 2A-like linkers or functional equivalents thereof and combinations thereof. In various examples, linkers having sequences as set forth herein, or variants thereof, are used. It is to be understood that the indication of a particular linker in a construct in a particular location does not mean that only that linker can be used there. Rather, different linker sequences (e.g., P2A and T2A) can be swapped with one another (e.g., in the context of the constructs of this disclosure), as can be determined by those of skill in the art. In some embodiments, the linker region is T2A derived from Thosea asigna virus. Non-limiting examples of linkers that can be used in this technology include T2A, P2A, E2A, BmCPV2A, and BmlFV2A. 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 a CAR component of a polyprotein from a therapeutic agent (e.g., an antibody, such as a scFv, single domain antibody (e.g., a camelid antibody), or a bispecific antibody (e.g., a TEAM)) component of a polyprotein (see below). In some embodiments, a P2A linker sequence comprises the amino acid sequence of SEQ ID NO: 48. In some embodiments, a T2A linker sequence comprises the amino acid sequence of SEQ ID NO: 49.

T cell engaging molecules (TEAMs)

In some embodiments, the therapeutic agent delivered by a CAR-T cell as described herein is a T cell engaging molecule (TEAM) (also referred to in the literature as bispecific T cell engagers or BiTEs™). By "T cell engaging molecules," "TEAM antibody constructs," or "TEAMs" is meant polypeptides that each include tandemly linked single-chain variable fragments (scFvs). 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., to the CD3e subunit) and the other binds to a target antigen (e.g., an antigen expressed by a cancer-associated fibroblast (CAF)). Such molecules can target T cells by binding to a T cell antigen (e.g., by binding CD3) as well as a target antigen, e.g., a CAF antigen. Exemplary CAF antigens include fibroblast activation protein (FAP, also see below). The TEAMs can be used to augment the T cell response in, e.g., the tumor microenvironment. The two components of a TEAM can optionally be separated from one another by a inker as described herein (e.g., a glycine-based linker), and may also be connected in either orientation, e.g., with the anti-CD3 component N-terminal to the anti-target antigen component, or vice versa. The anti-CD3 component or the anti-target antigen component of the TEAM may include any of the antibody reagents described herein.

The CAR-T cell secreted TEAMs may, e.g., stimulate the CAR-T cell itself, or operate in a paracrine fashion by redirecting nonspecific bystander T cells against tumors or CAFs and therefore enhance the anti-cancer effects of CAR-T cell immunotherapy. CAR-T cell-mediated TEAM secretion may allow for the reduction of risk of undesired TEAM activity in systemic tissues by directing TEAM secretion to the tumor microenvironment. Exemplary TEAM constructs are provided below (e.g., anti-FAP TEAM); however, TEAMs other than those described herein may also be useful for the CAR-T cells and methods of the disclosure. An exemplary TEAM is an anti-FAP TEAM including an anti-FAP scFv and an anti- CD3 scFv (also referred to herein as TEAM-FAP). The anti-FAP scFv may be arranged in the VH-VE orientation, or in the VE-VH orientation. CAF cells are cancer-associated structural cells in the tumor microenvironment (TME). CAFs deposit collagen in the TME, creating an extracellular matrix (ECM) that creates a physical barrier preventing drug and immune cell infiltration at the tumor site, in addition to providing a scaffolding that supports the survival and migration of cancer cells. Additionally, CAFs secrete growth factors, cytokines, and chemokines that support the suppression of immune cells and the proliferation of tumor cells. CAF cells express the antigen FAP, and moderate to high FAP expression on CAFs is correlated with poorer clinical outcomes in pancreatic cancer. To undermine the role of CAFs in cancer, the present disclosure provides for TEAMs that contain a binding domain specific to FAP and a binding domain specific to CD3, which serve as a scaffold to bring effector T cells together with CAFs to promote T cell-mediated cytotoxicity against CAF cells.

In some embodiments, the anti-CD3 scFv of any of the TEAMs described herein may be arranged in the VH-VE orientation, or in the VE-VH orientation. In some embodiments, the anti-CD3 VH comprises the amino acid sequence of SEQ ID NO: 25 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 greater sequence identity to the amino acid sequence of SEQ ID NO: 25. In some embodiments, the anti-CD3 VE comprises the amino acid sequence of SEQ ID NO: 26 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 greater sequence identity to the amino acid sequence of SEQ ID NO: 26.

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

In some embodiments, the anti-FAP antibody reagent is Sibrotuzumab. In some embodiments, the anti-FAP antibody reagent comprises the variable heavy (VH) of SEQ ID NO: 17 or comprises VH sequences with 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 greater sequence identity to SEQ ID NOs: 17. In some embodiments, the anti-FAP antibody reagent comprises the variable light (VL) of SEQ ID NO: 18 or includes VL sequences with 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 greater sequence identity to the sequences of SEQ ID NOs: 18. The VH may be positioned N-terminal to the VL, or the VL may be positioned N-terminal to the VH. In some embodiments, the anti- FAP antibody reagent comprises SEQ ID NO: 43 or comprises a sequence with 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 greater sequence identity to SEQ ID NO: 43.

In some embodiments, the TEAM comprises an antibody reagent that binds to FAP and an antibody reagent that binds to CD3. In some embodiments, the FAP antibody reagent is encoded upstream of the CD3 scFv.

CAR-TEAM Constructs

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 are separated by a linker domain as described above (e.g., a 2A peptide). In some embodiments, the linker domain is cleavable. In some embodiments, the CAR is an anti-mesothelin CAR and the TEAM is a FAP and 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 polypeptide comprises an amino acid sequence with 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 greater sequence identity of SEQ ID NO: 44, SEQ ID NO: 45, or SEQ ID NO: 51. In some embodiments, the CAR and TEAM polypeptide comprises an amino acid sequence of SEQ ID NO: 44. In some embodiments, the CAR and TEAM polypeptide consists of an amino acid sequence of SEQ ID NO: 45. In some embodiments, the CAR and TEAM polypeptide comprise an amino acid sequence of SEQ ID NO: 51.

In some embodiments, the TEAM enhances binding of an immune cell to a cancer compared to binding of the immune cell to the cancer in the absence of the TEAM. In some embodiments, the TEAM enhances binding of an immune cell to a cancer compared to binding of the immune cell to the cancer without a TEAM and without a CAR-that binds to an antigen on the immune cell. In some embodiments, the TEAM increases binding of the immune cell 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, the TEAM increases immune cell binding to the 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, the TEAM increases immune cell binding to the cancer by 20%-40%. In some embodiments, the TEAM increases immune cell binding 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.

Inhibitors

Provided herein are methods for using CAR-T cells in combination with an a disintegrin and metalloprotease 17 (ADAM17) or interleukin-2-inducible T cell kinase (ITK) inhibitor. The use of such inhibitors alongside CAR-T cell therapy can enhance the efficacy of the CAR-T cells against solid tumors. The inhibitor can be a protein-based inhibitor (e.g., an antibody or an antigen-binding domain to inhibit the target), a small molecule, an RNA-based inhibitor, or any other form of inhibitor depending on the target.

In some embodiments, to promote stable expression of mesothelin, the target antigen of the CAR-T cells provided herein, an ADAM 17 inhibitor is administered in combination with meso-FAP CAR TEAM cells. In some embodiments, the ADAM 17 inhibitor is aderbasib, TAPI-0, TAPI-1, TAPI-2, GW280264X, marimastat, INCB3619, compound 22a (INCB9471), MEDI3622, apratastat, DPC-333, SCH 900567, or KP-457. In some embodiments, the ADAM 17 inhibitor is aderbasib. Aderbasib is also known as INCB7839.

Other inhibitors provided in the present disclosure, in some embodiments, are interleukin-2-inducible T cell kinase (ITK) inhibitors to facilitate polarization of T cells to a more effective cancer-killing phenotype. In some embodiments, an ITK inhibitor is administered in combination with CAR TEAM cell therapy. In some embodiments, the ITK inhibitor is ibrutinib, CPI-818, BMS 509774, or PRN694. In some embodiments, the ITK inhibitor is ibrutinib. In some embodiments, the ADAM17 inhibitor is administered at the same time as the CAR TEAM cells. In some embodiments, the ADAM17 inhibitor is administered at a time following administration of the CAR TEAM cells. In some embodiments, the ADAM 17 inhibitor is administered by the same route of administration as the CAR TEAM cells. In some embodiments, the ADAM 17 inhibitor is administered via intraperitoneal injection. In some embodiments, the ADAM 17 inhibitor is administered orally.

In some embodiments, the ITK inhibitor is administered at the same time as the CAR TEAM cells. In some embodiments, the ITK inhibitor is administered at a time following administration of the CAR TEAM cells. In some embodiments, the ITK inhibitor is administered by the same route of administration as the CAR TEAM cells. In some embodiments, the ITK inhibitor is administered via intraperitoneal injection. In some embodiments, the ITK inhibitor is administered orally.

Polynucleotides, Plasmids, and Vectors

In some aspects, this disclosure describes a polynucleotide encoding any one of the CAR polypeptides described herein. The term "polynucleotide" is used herein interchangeably with "nucleic acid molecule" to indicate a polymer of nucleosides. Typically, a polynucleotide is composed of nucleosides that are naturally found in DNA or RNA (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine) joined by phosphodiester bonds. However, the term encompasses molecules comprising nucleosides or nucleoside analogs containing chemically or biologically modified bases, modified backbones, etc., whether or not found in naturally occurring nucleic acids, and such molecules may be preferred for certain applications. Where this application refers to a polynucleotide it is understood that both DNA, RNA, and in each case both single- and doublestranded forms (and complements of each single-stranded molecule) are provided. "Polynucleotide sequence" as used herein can refer to the polynucleotide material itself and/or to the sequence information (i.e., the succession of letters used as abbreviations for bases) that biochemically characterizes a specific 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 exist within a given cell or a nucleic acid sequence that has been engineered into a cell. For example, a heterologous nucleic acid molecule may be a nucleic acid molecule encoding a gene that is engineered into a cell (e.g., via a plasmid, vector or some other method). A polynucleotide sequence presented herein is presented in a 5' to 3' direction unless otherwise indicated.

In some embodiments, this disclosure describes a polynucleotide comprising a first nucleic acid sequence encoding a CAR (as described herein) and a second nucleic acid sequence encoding a TEAM (as described herein). In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are operably linked to different promoters. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are operably linked to the same promoter. In some embodiments, the polynucleotide further comprises a nucleic acid sequence encoding a self-cleavable peptide encoded between the first nucleic acid sequence encoding the CAR and the second nucleic acid sequence encoding the TEAM.

In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%) sequence identity to any one of SEQ ID NOs: 44-45. In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding an amino acid sequence of any one of SEQ ID NOs: 44-45.

In some embodiments, the nucleic acid sequence is operably linked to a promoter. As used herein, the term "operably linked" refers to a first polynucleotide molecule, such as a promoter, connected with a second transcribable polynucleotide molecule, such as a CAR, a BCL-2 family protein, or a CAR polypeptide, where the polynucleotide molecules are so arranged that the promoter can direct a RNA polymerase to transcribe 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 transcription of the gene of interest in a cell.

In some embodiments, the promoter is a constitutively active promoter. In some embodiments, the U6 promoter is from a non-human species. In some embodiments, the promoter is selected from the group consisting of a CMV promoter, an EFla promoter, an EF la- short promoter, a CAG promoter, a PGK promoter, Hl promoter, or a U6 promoter. In some embodiments, the U6 promoter is from a human U6 promoter. In some embodiments, the U6 promoter is from cow, mice, rat, pig, yeast, dog, cat, drosophila, or C. elegans. In some embodiments, the promoter is a Hl promoter. In some embodiments, the promoter is a tissuespecific promoter (e.g., the HP1, CD14, CD43, CD45, C68, elastase, endoglin, fibronectin, Fit, GFAP, GPIIb, ICAM-2, mIFN-beta, Mb, NphsI, OG-2, SP-B, SYN1, or WASP gene promoter). In some embodiments, the promoter is an inducible promoter (e.g., a tet or lac promoter).

In some embodiments, a nucleic acid encoding a polypeptide as described herein (e.g., a CAR polypeptide) is comprised by a plasmid. The term “plasmid” may refer to a circular piece of DNA the comprises an origin of replication. In some embodiments, the plasmid comprises a prokaryotic origin of replication. In some embodiments, the plasmid comprises a bacterial origin of replication. In some embodiments, the plasmid comprises a eukaryotic origin of replication. In some embodiments, the plasmid comprises a mammalian origin of replication. In some embodiments, the plasmid comprises a prokaryotic and eukaryotic origin of replication. In some embodiments, the plasmid comprises an origin of replication that is active in a cell which the plasmid is located. In some embodiments, the plasmid is a lentiviral plasmid (e.g., a second generation lenti-viral plasmid).

In some embodiments, a nucleic acid encoding a polypeptide as described herein (e.g., a CAR polypeptide) is comprised by a vector. The term "vector," as used herein, 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 is capable of replication when associated with the proper control elements and that can transfer gene sequences to cells. A vector can include, but is not limited to, a cloning vector, an expression vector, a plasmid, phage, transposon, cosmid, artificial chromosome, virus, virion, etc.

As used herein, the term "expression vector" may refer to a vector that directs expression of an RNA or polypeptide from sequences linked to transcriptional regulatory sequences on the vector. The sequences expressed will often, but not necessarily, be heterologous to the cell. An expression vector may comprise additional elements, for example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example, in human cells for expression and in a prokaryotic host for cloning and amplification. The term "expression" refers to the cellular processes involved in producing RNA and proteins and as appropriate, secreting proteins, including where applicable, but not limited to, for example, transcription, transcript processing, translation and protein folding, modification and processing. "Expression products" include RNA transcribed from a gene, and polypeptides obtained by translation of mRNA transcribed from a gene.

As used herein, the term "viral vector" may refer to a nucleic acid vector construct that includes at least one element of viral origin and has the capacity to be packaged into a viral vector particle. The viral vector can 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 nucleic acids into cells either in vitro or in vivo. Numerous forms of viral vectors are known in the art. In some embodiments, the viral vector is an adeno-associated viral, adenoviral, lentiviral, or a retroviral vector. In some embodiments, the lentiviral vector is a second generation lentiviral vector.

By "recombinant vector" may be a vector that includes a heterologous nucleic acid sequence or "transgene" that is capable of expression in vivo. It should be understood that the vectors described herein can, in some embodiments, be combined with other suitable compositions and therapies. In some embodiments, the vector is episomal. The use of a suitable episomal vector provides a means of maintaining the nucleotide of interest in the subject in high copy number extra-chromosomal DNA thereby eliminating potential effects of chromosomal integration.

In some embodiments, a polypeptide, polynucleotide, plasmid and or/vector as described herein optionally further comprises a reporter molecule, e.g., to determine if the vector is properly expressed in a cell. In some embodiments, the reporter molecule may be a fluorescent protein (e.g., GFP, YFP, RF, mCherry), antibody (e.g., CD34, tEGFR, tCD19, tCD20, tCD34, and tHer2), or a radioisotope. In some embodiments, the reporter molecule is hygromycin phosphotransferase (hph) that can be imaged alone or in combination with a substrate or chemical (for example 9-[4-[18F]fluoro-3-(hydroxymethyl)butyl]guanine ([18F]FHBG)).

In some embodiments, GFP and mCherry may be used as fluorescent tags for imaging a CAR expressed on a T cell (e.g., a CAR-T cell). 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, the CAR need not include a fluorescent tag or fluorescent protein. In each instance of particular constructs provided herein, therefore, any markers present in the constructs can be removed. The invention includes the constructs with or without the markers. Accordingly, when a specific construct is referenced herein, it can be considered with or without any markers or tags (including, e.g., histidine tags, such as the histidine tag of HHHHHH (SEQ ID NO: 47)) as being included within the invention.

Cells

One aspect of the technology described herein relates to a mammalian cell (e.g., an immune cell) comprising any of the meso-FAP CAR TEAM constructs described herein. The mammalian cell can be of human, primate, hamster, rabbit, rodent, cow, pig, sheep, horse, goat, dog or cat origin, but any other mammalian cell may be used. In a preferred embodiment of any aspect, the mammalian cell is human.

In some embodiments of any aspect, the mammalian cell is an immune cell. As used herein, "immune cell" refers to a cell that plays a role in the 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

In some embodiments, the immune cell is obtained from an individual having or diagnosed as having cancer, a plasma cell disorder, or autoimmune disease. In some embodiments, the immune cell is allogenic to the subject. In some embodiments, the immune cell is produced from stem cell (e.g., an induced pluripotent stem cell or an embryonic stem cell).

In some embodiments, a mammalian cell, e.g., a T cell, can be engineered to include any of the meso-FAP CAR TEAM constructs, as described herein. T cells can be obtained from a subject using standard techniques known in the field. 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.

In some aspects, this disclosure describes a CAR-T cell comprising any of the meso-FAP CAR TEAM constructs disclosed herein. In some embodiments, the CAR-T cell is generated from T-cells extracted from a subject (e.g., the subject to whom the CAR-T cells will be administered). In some embodiments, the CAR-T cells are allogenic CAR-T cells. In some embodiments, the CAR-T cell comprises a meso-FAP CAR TEAM construct comprising an anti-mesothelin CAR. In some embodiments, the CAR-T cell comprises a meso-FAP CAR TEAM construct comprising an anti-mesothelin CAR and an fibroblast activation protein (FAP) antigen binding domain. In some embodiments, the CAR-T cell comprises a meso-FAP CAR TEAM construct as described herein. In some embodiments, the CAR-T cell comprises a construct encoding any one of SEQ ID NOs: 44-45.

Methods of Treatment

In some aspects, this disclosure describes a method of treating a subject having a mesothelin-expressing cancer (as described herein), the method comprising administering a cell (e.g., a CAR-T cell) expressing a CAR (as described herein) and a T cell engaging antibody molecule (TEAM, as described herein) to the subject. In some embodiments, the method comprises administering a CAR-T cell described herein (e.g., a CAR-T cell expressing a CAR polypeptide). In some embodiments, the method comprises administering a T-cell engineered to express a CAR and a TEAM. In some embodiments, the method comprises administering a CAR-T cell comprising a meso-FAP CAR TEAM construct comprising an anti-mesothelin CAR. In some embodiments, the method comprises administering a CAR-T cell comprising a meso-FAP CAR TEAM construct comprising an anti-mesothelin CAR and a fibroblast activation protein (FAP) binding domain. In some embodiments, the method comprises administering a CAR-T cell comprising a meso-FAP CAR TEAM construct comprising an anti- mesothelin CAR and a TEAM comprising a fibroblast activation protein (FAP)-binding domain and a CD3-binding domain. In some embodiments, the method comprises administering a CAR-T cell comprising a construct encoding any one of SEQ ID NOs: 44-45. In some embodiments, the method comprises administering a CAR-T cell in combination with an ADAM 17 inhibitor or an ITK inhibitor.

Cancer

In some embodiments, the methods described herein comprise treating a subject that has a mesothelin-expressing cancer. "Cancer" as used herein can refer to a hyperproliferation of cells whose unique trait, loss of normal cellular control, results in unregulated growth, lack of differentiation, local tissue invasion, and metastasis. Exemplary cancers include, but are not limited to liquid cancers (as referred to as liquid tumors, and solid tumors. A “liquid cancer” or “liquid tumor” as described herein may refer to a leukemia, lymphoma, and myeloma cancer. Nonlimiting examples of leukemia 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 lymphoma include diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), marginal zone lymphomas, 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. In some embodiments, the myeloma is multiple myeloma. In some embodiments, the multiple myeloma is smoldering and active multiple myeloma.

Non-limiting examples of solid tumors include adrenocortical tumor, alveolar soft part sarcoma, carcinoma, chondrosarcoma, colorectal carcinoma, desmoid tumors, desmoplastic small round cell tumor, endocrine tumors, endodermal sinus tumor, epithelioid hemangioendothelioma, Ewing sarcoma, glioblastoma, prostate cancer, glioma, lung cancer, pancreatic cancer, germ cell tumors (solid tumor), giant cell tumor of bone and soft tissue, hepatoblastoma, hepatocellular carcinoma, melanoma, nephroma, neuroblastoma, nonrhabdomyosarcoma soft tissue sarcoma (NRSTS), osteosarcoma, paraspinal sarcoma, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, synovial sarcoma, and Wilms tumor. In some embodiments, the cancer expresses mesothelin (a mesothelin-expressing cancer). In some embodiments, the cancer expresses a mesothelin or a portion thereof of at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 50. In some embodiments, the cancer expresses a mesothelin or a portion thereof of at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 50. In some embodiments, the cancer expresses a mesothelin or a portion thereof of at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 50. In some embodiments, the cancer expresses a mesothelin or a portion thereof of at least 97% sequence identity to the amino acid sequence of SEQ ID NO: 50. In some embodiments, the cancer expresses a mesothelin or a portion thereof of at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 50. In some embodiments, the cancer expresses a mesothelin or a portion thereof of at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 50. In some embodiments, the cancer expresses a mesothelin or a portion thereof having the amino acid sequence of SEQ ID NO: 50. In some embodiments, the mesothelin-expressing cancer is mesothelioma, ovarian cancer, pancreatic cancer, lung cancer, breast cancer, gastric cancer, or colorectal cancer. In some embodiments, the mesothelin-expressing cancer is pancreatic cancer. In some embodiments, the mesothelin-expressing cancer is pancreatic ductal adenocarcinoma (PDAC). Solid tumors can be found in bones, muscles, or organs, and can be sarcomas or carcinomas As used herein, the term "tumor" refers to an abnormal growth of cells or tissues, e.g., of malignant type or benign type.

Subject

In some embodiments, the methods described herein comprise treating a subject having cancer. As used herein, a “subject” means a human or animal. Usually, the 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, e.g., rhesus. Rodents include, for example, mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include, for example, cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. In some embodiments, the subject is a mammal, e.g., a primate, e.g., a human. The terms, “individual,” “patient,” and “subject” are used interchangeably herein. Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of disease, e.g., cancer. A subject can be male or female.

A subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment (e.g., diagnosed with a mesothelin-expressing cancer) or one or more complications related to such a condition, and optionally, have already undergone treatment for the condition or the one or more complications related to the condition.

Alternatively, a subject can also be one who has not been previously diagnosed as having such condition or related complications. For example, a subject can be one who exhibits one or more risk factors for the condition or one or more complications related to the condition or a subject who does not exhibit risk factors.

A “subject in need” of treatment for a particular condition can be a subject having that condition, diagnosed as having that condition, or at risk of developing that condition. Pharmaceutical Compositions

In some embodiments, the methods described herein comprise administering to the subject a pharmaceutical composition comprising the CAR-T cells and/or a pharmaceutical composition comprising the ADAM 17 inhibitor and/or the ITK inhibitor. As used herein, the term “pharmaceutical composition” refers to the active agent (e.g., a CAR-T described herein (e.g., a CAR-T cell expressing a CAR polypeptide) in combination with a pharmaceutically acceptable carrier e.g., a carrier commonly used in the pharmaceutical industry.

The phrase “pharmaceutically acceptable carrier” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be a carrier other than water. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be an artificial or engineered carrier, e.g., a carrier in which the active ingredient would not be found to occur in nature.

In one aspect of the technology, the technology described herein relates to a pharmaceutical composition including activated CAR-T cells comprising a CAR polypeptide described herein, and optionally a pharmaceutically acceptable carrier. The active ingredients of the pharmaceutical composition at a minimum include activated CAR-T cells comprising CAR polypeptide as described herein. In some embodiments, the active ingredients of the pharmaceutical composition consist essentially of activated CAR-T cells comprising a CAR polypeptide as described herein. In some embodiments, the active ingredients of the pharmaceutical composition consist of activated CAR-T cells comprising a CAR polypeptide as described herein. Pharmaceutically acceptable carriers for cell-based therapeutic formulation include saline and aqueous buffer solutions, Ringer’s solution, and serum component, such as serum albumin, HDL and LDL. The terms such as “excipient,” “carrier,” “pharmaceutically acceptable carrier”, “pharmaceutically acceptable excipient” or the like are used interchangeably herein.

In some embodiments, the pharmaceutical composition including activated CAR-T cells comprising CAR polypeptide as described herein can be a parenteral dose form. Since administration of parenteral dosage forms typically bypasses the patient’s natural defenses against contaminants, the components apart from the CAR-T cells themselves are preferably sterile or capable of being sterilized prior to administration to a patient. Examples of parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, and emulsions. Any of these can be added to the activated CAR-T cells preparation prior to administration. Suitable vehicles that can be used to provide parenteral dosage forms of activated CAR-T cells as disclosed within are well known to those skilled in the art. Examples include, without limitation: saline solution; glucose solution; aqueous vehicles including but not limited to, sodium chloride injection, Ringer’s injection, dextrose injection, dextrose and sodium chloride injection, and lactated Ringer’s injection; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and propylene glycol; and nonaqueous vehicles such as, but not limited to, com oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

Dosage

In some embodiments, the activated CAR-T cells comprising a CAR polypeptide described herein are administered as a monotherapy, i.e., another treatment for the condition is not concurrently administered to the subject. A pharmaceutical composition including the T cells described herein can generally be administered at a dosage of 104 to 109 cells/kg body weight, in some instances 105 to 106 cells/kg body weight, including all integer values within those ranges. If necessary, T cell compositions can also be administered multiple times at these dosages. The cells can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. Med. 30 319:1676, 1988).

In certain aspects, it may be desired to administer activated CAR-T cells comprising a CAR polypeptide to a subject and then subsequently redraw blood (or have an apheresis performed), activate T cells therefrom as described herein, and reinfuse the patient with these activated and expanded T cells. This process can be carried out multiple times every few weeks. In certain aspects, T cells can be activated from blood draws of from lOcc to 400cc. In certain aspects, T cells are activated from blood draws of 20cc, 30cc, 40cc, 50cc, 60cc, 70cc, 80cc, 90cc, or lOOcc. Administration

In some embodiments, the methods described herein relate to treating a subject having a mesothelin-expressing cancer (e.g., as described herein), the method comprising administering to the subject a CAR-T cell in combination with an ADAM17 inhibitor or an ITK inhibitor, wherein the CAR-T cell comprises a mesothelin-binding CAR and a T cell engaging antibody molecule comprising a FAP-binding domain. The CAR-T cells comprising a CAR polypeptide described herein include mammalian cells including any of the CAR polypeptide described herein and any of the CAR polypeptides described herein or known in the art, or a nucleic acid encoding any of the CAR polypeptides described herein.

In some embodiments, the methods described herein include administering an effective amount of activated CAR-T cells comprising a CAR polypeptide described herein to treat a subject having a mesothelin-expressing cancer. As used herein, “treating a subject having a mesothelin-expressing cancer” is ameliorating any condition or symptom associated with the mesothelin-expressing cancer. As compared with an equivalent untreated control, such reduction is by at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more as measured by any standard technique. A variety of means for administering the compositions described herein to subjects 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 at the site of a tumor.

The term "effective amount" as used herein refers to the amount of activated CAR-T cells comprising a CAR polypeptide. Described herein needed to treat at least one or more symptom of the mesothelin-expressing cancer and relates to a sufficient amount of the cell preparation or composition to provide the desired effect. The term “therapeutically effective amount” therefore refers to an amount of activated CAR-T cells comprising CAR polypeptide described herein that is sufficient to provide a particular anti-condition effect when administered to a typical subject. An effective amount as used herein, in various contexts, would also include an amount sufficient to delay the development of a symptom of mesothelin-expressing cancer, alter the course of mesothelin-expressing cancer (for example but not limited to, slowing the progression of the mesothelin-expressing cancer), or reverse a symptom of a mesothelin- expressing cancer. Thus, it is not generally practicable to specify an exact “effective amount.” However, for any given case, an appropriate “effective amount” can be determined by one of ordinary skill in the art using only routine experimentation.

Effective amounts, toxicity, and therapeutic efficacy can be evaluated by standard pharmaceutical procedures in cell cultures or experimental animals. The dosage can vary depending upon the dosage form employed and the route of administration utilized. The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50/ED50. Compositions and methods that exhibit large therapeutic indices are preferred. A therapeutically effective dose can be estimated initially from cell culture assays. Also, a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of activated CART cells comprising a CAR polypeptide described herein, which achieves a half-maximal inhibition of symptoms) as determined in cell culture, or in an appropriate animal model. Levels in plasma can be measured, for example, by high performance liquid chromatography. The effects of any particular dosage can be monitored by a suitable bioassay, e.g., assay for bone marrow testing, among others. The dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.

In some embodiments, the methods of treating a subject having a mesothelin-expressing cancer described herein comprise administering a meso-FAP CAR TEAM cell via intravenous administration. In some embodiments, the method comprises administering a meso-FAP CAR TEAM cell via intravenous administration in combination with an ADAM 17 inhibitor. In some embodiments, the method comprises administering a meso-FAP CAR TEAM cell via intravascular administration in combination with an ADAM 17 inhibitor administered intraperitoneally. In some embodiments, the method comprises administering a meso-FAP CAR TEAM cell via intravascular administration in combination with an ADAM 17 inhibitor administered orally. In some embodiments, the method comprises administering a meso-FAP CAR TEAM cell in combination with an ADAM 17 inhibitor via intravascular administration.

In some embodiments, the method comprises administering a meso-FAP CAR TEAM cell via intravascular administration in combination with an ITK inhibitor. In some embodiments, the method comprises administering a meso-FAP CAR TEAM cell via intravascular administration in combination with an ITK inhibitor administered intraperitoneally. In some embodiments, the method comprises administering a meso-FAP CAR TEAM cell via intravascular administration in combination with an ITK inhibitor administered orally. In some embodiments, the method comprises administering a meso-FAP CAR TEAM cell in combination with an ITK inhibitor via intravascular administration.

Modes of Administration

Modes of administration (e.g., of the CAR-T cell comprising a CAR polypeptide, the ADAM17 inhibitor, or the ITK inhibitor) can include, for example intravenous (iv) injection or infusion. The compositions described herein can be administered to a patient transarterially, intratumor ally, intranodally, intraperitoneally, intrathecally, intramedullary, or orally. In some embodiments, the compositions of CAR-T cells 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 body fluid (e.g., ascites, pleural fluid, peritoneal fluid, or cerebrospinal fluid).

In some embodiments, subjects may undergo leukapheresis, wherein leukocytes are collected, enriched, or depleted ex vivo to select and/or isolate the cells of interest, e.g., T cells. These T cell isolates can be expanded by contact with an artificial APC (aAPC), e.g., an aAPC expressing anti-CD28 and anti-CD3 CDRs, and treated such that one or more CAR constructs of the technology may be introduced, thereby creating a CAR-T cell.

Subjects in need thereof can subsequently undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation. Following or concurrent with the transplant, subjects can receive an infusion of the expanded CAR-T cells. In some embodiment, expanded cells are administered before or following surgery. In some embodiments, lymphodepletion is performed on a subject prior to administering one or more CAR-T cell as described herein. In such embodiments, the lymphodepletion can include administering one or more of melphalan, survivin, cyclophosphamide, and fludarabine. The dosage of the above treatments to be administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. The scaling of dosages for human administration can be performed according to art- accepted practices.

In some embodiments, a single treatment regimen is required. In others, administration of one or more subsequent doses or treatment regimens can be performed. For example, after treatment biweekly for three months, treatment can be repeated once per month, for six months or a year or longer. In some embodiments, no additional treatments are administered following the initial treatment.

The dosage of CAR-T cells as described herein can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment. With respect to duration and frequency of treatment, it is typical for skilled clinicians to monitor subjects in order to determine when the treatment is providing therapeutic benefit, and to determine whether to administer further cells, discontinue treatment, resume treatment, or make other alterations to the treatment regimen. The dosage should not be so large as to cause adverse side effects, such as cytokine release syndrome. Generally, the dosage will vary with the age, condition, and sex of the patient and can be determined by one of skill in the art. The dosage can also be adjusted by the individual physician in the event of any complication.

Efficacy

The efficacy of activated CAR-T cells comprising a CAR polypeptide described herein in, e.g., the treatment of mesothelin-expressing cancer, or to induce a response as described herein (e.g., a reduction in cancer cells) can be determined by the skilled clinician. However, a treatment is considered “effective treatment,” as the term is used herein, if one or more of the signs or symptoms of a condition described herein is altered in a beneficial manner, other clinically accepted symptoms are improved, or even ameliorated, or a desired response is induced, e.g., by at least 10% following treatment according to the methods described herein. Efficacy can be assessed, for example, by measuring a marker, indicator, symptom, and/or the incidence of a mesothelin-expressing cancer treated according to the methods described herein or any other measurable parameter appropriate.

Treatment according to the methods described herein can reduce levels of a marker or symptom of a mesothelin-expressing cancer, e.g., 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.

Efficacy can also be measured by a failure of an individual to worsen as assessed by hospitalization, or need for medical interventions (i.e., progression of the disease is halted). Methods of measuring these indicators are known to those of skill in the art and/or are described herein. Treatment includes any treatment of a mesothelin-expressing cancer in an individual or an animal (some non-limiting examples include a human or an animal) and includes: (1) inhibiting the mesothelin-expressing cancer, e.g., preventing a worsening of symptoms (e.g., pain or inflammation); or (2) relieving the severity of the mesothelin-expressing cancer, e.g., causing regression of symptoms. An effective amount for the treatment of a mesothelin- expressing cancer means that amount which, when administered to a subject in need thereof, is sufficient to result in effective treatment as that term is defined herein, for mesothelin-expressing cancer. Efficacy of an agent can be determined by assessing physical indicators of mesothelin- expressing cancer or desired response. It is well within the ability of one skilled in the art to monitor efficacy of administration and/or treatment by measuring any one of such parameters, or any combination of parameters. Efficacy of a given approach can be assessed in animal models of a condition described herein. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant change in a marker is observed.

Sequences

The term "statistically significant" or "significantly" refers to statistical significance and generally means a two standard deviation (2SO) or greater difference.

Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term "about."

The terms "about" or “approximately” when used in connection with a value can mean that the value or a statement reciting the value encompasses a range within ±1%, ±2%, ±3%, ±4%, ±5%, ±6%, ±7%, ±8%, ±9%, ±10%, ±1-5%, ±2-7%, ±3-8%, ±4-9%, or ±5-10% of the value.

The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The abbreviation, "e.g." is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g." is synonymous with the term "for example."

Other terms are defined within the description of the various aspects and embodiments of the technology, as set forth herein.

In accordance with this disclosure, there may be employed conventional molecular biology, microbiology, biochemical, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. Embodiments of this disclosure are further described in the following examples, which do not limit the scope of the methods and compositions of matter described in the claims.

EXAMPLES

The tumor microenvironment (TME) is a heterogenous population of cells, including stromal cells, endothelial cells, immune cells, tumor cells, cancer-associated fibroblasts (CAFs), and others (FIG. 1). The density and diversity of cell types can function together to promote the proliferation and survival of tumor cells, while CAFs in the TME deposit collagen to form an extracellular matrix (ECM) that can make it difficult or impossible for immune cells and anticancer agents to penetrate and exert their cytolytic effects on the tumor. Here, cells comprising a chimeric antigen receptor (CAR) against mesothelin - an antigen expressed by many types of cancer - that secrete T cell engaging molecules (TEAMs) against fibroblast activated protein (FAP) (meso-FAP CAR- TEAM cells, construct shown in FIG. 11) - expressed on CAFs - were tested for their cytolytic capacity against pancreatic or ovarian cancer cells and CAFs. Additionally, the efficacy of meso-FAP CAR- TEAM cells in combination with inhibitors that prevent the cleavage of mesothelin from the tumor cell surface (e.g., ADAM 17 inhibitors) or promote the polarization of T cells to effector subtypes with enhanced anti-cancer capacity (e.g., ITK inhibitors) were also tested.

Example 1. Testing the efficacy of meso-FAP CAR- TEAM cells against pancreatic cancer cells

In vivo

Immunodeficient NSG® mice were injected subcutaneously with AsPC-1 tumor cells (mesothelin-expressing tumor cells derived from a human pancreatic ductal adenocarcinoma (PDAC) cell line) mixed 1:9 with CAF cells and later injected intravenously (IV) with untransduced (UTD) T cells, meso-FAP CAR- TEAM cells, or meso-CD19 CAR- TEAM cells, which secrete a TEAM not specific to the TME in these experiments. Reduction in tumor volume was significantly improved in mice that received meso-FAP CAR-TEAM cells as compared to mice that received UTD or meso-CD19 CAR-TEAM cells (FIG. 2A-B).

In vitro

As human CAF cells do not expand in mice, the efficacy of meso-FAP CAR- TEAM cells was next tested in vitro against patient-derived mesothelin-expressing tumor organoids grown in the presence of CAFs. CAFs were mixed with organoids to mimic the human TME. Mixed cultures were treated with either UTD cells, meso-FAP CAR- TEAM cells, or mesoCD 19 CAR-TEAM control cells. Organoid-CAF cultures treated with meso-FAP CAR- TEAM cells had a marked reduction in organoid recovery compared to cultures treated with UTD cells (FIGs. 2C, 2E, top), and a significant reduction in CAF cells compared to cultures treated with UTD cells or meso-CD19 CAR- TEAM cells (FIGs. 2D, 2E, bottom). Example 2. Testing the cytolytic capacity of meso-FAP CAR- TEAM cells against pancreatic and ovarian cancer cell lines.

Cultures of pancreatic cancer cell lines expressing mesothelin, ADAM17, and ADAM10 (FIGs. 4A-4B) AsPC-1 and CxPC-3 were treated with UTD T cells or meso-FAP CAR-TEAM cells. Meso-FAP CAR-TEAM cells had a significantly higher cytolytic effect against both cell lines compared to UTD T cells (FIG. 4D). The criticality of mesothelin expression for the cytolytic capacity of meso-FAP CAR- TEAM cells was demonstrated using SKOV3 ovarian cancer cells, which have low mesothelin baseline expression. SKOV3 cells were transduced to increase mesothelin expression (FIG. 4C), which increased the cytolytic capacity of meso-FAP CAR-TEAM cells as compared to untransduced SKOV3 cells or SKOV3 cells treated with UTD T cells (FIG. 4E). Thus, meso-FAP CAR-TEAM cells demonstrate robust killing of mesothelin-expressing cancer cells.

Example 3. Determining best mesothelin antigen binding domain for chimeric antigen receptor/T cell engaging molecule (CAR-TEAM) cells targeting intraperitoneal (IP) tumors.

Examples 1 and 2 demonstrated the efficacy of meso-FAP CAR- TEAM cells against mesothelin-expressing cancer models. However, there are multiple mesothelin-binding moieties that can be utilized in a meso-FAP CAR- TEAM cell. Here, the efficacy of two mesothelin- binding moieties is compared in vitro and in preclinical models.

In vitro

The efficacy of mesothelin antigen binding for mesothelin-fibroblast activation protein- targeted (meso-FAP) CAR-TEAM cells with either the SSI binding domain (SSI meso-FAP CAR TEAM cells) or the MGHlMesol binding domain (MGHlMesol meso-FAP CAR TEAM cells) is compared in vitro. TEAM binding to FAP on cancer-associated fibroblast 1 (CAF-1) cells and CD3 on untransduced (UTD) T cells is detected by flow cytometry. Next, MGHlMesol or SSI meso-FAP CAR TEAM cells are repeat stimulated with pancreatic ductal adenocarcinoma (PDAC) cells and CAF cells once per week for 4 weeks and their long-term proliferation and exhaustion phenotypes are measured. Additionally, PDAC cells or patient derived xenograft (PDX) cells are co-cultured with CAF cells and the cytotoxicity of MGHlMesol and SSI meso-FAP CAR TEAM cells is measured using a real-time impedancebased assay.

In vivo

PDAC cell lines (AsPC-1 or BXPC-3 cells) or PDX cells (1291 cells) are mixed 1:9 with CAF-1 cells in Matrigel and injected intraperitoneally (IP) into NOD.Cg-Prkdc scid 123rg tmlWjl/SzJ (NSG®) mice. Tumor growth is monitored via bioluminescent imaging. Two weeks after tumor implantation, mice are treated IP with UTD T cells or MGHlMesol or SSI meso- FAP CAR TEAM cells. Tumor growth is monitored weekly with in vivo bioluminescent imaging. Meso-FAP CAR TEAM cell expansion and phenotype in peritoneal cavity washes and peripheral blood is measured by flow cytometry.

Example 4. Optimizing the route of administration of meso-FAP CAR TEAM cells for in situ and metastatic tumors.

The efficacy of meso-FAP CAR TEAM cells depends upon the route by which they are administered to a subject (e.g., intravenously versus intraperitoneally). Tumor cells (either AsPCl or BXPC3 cells) were injected into NSG® mice intraperitoneally, and mice were thereafter treated with MGHlMesol or SSI meso-FAP CAR TEAM cells. MGHlMesol meso- FAP CAR TEAM cells had a superior tumor killing ability and survival when injected intraperitoneally compared to SSI meso-FAP CAR TEAM cells (FIG. 3A). This difference was less pronounced when meso-FAP CAR TEAM cells were injected intravenously (FIG. 3B) and even more modest when tumors were injected into the mice subcutaneously (FIG. 3C).

Next, the effect of either MGHlMesol or SSI meso-FAP CAR TEAM cells is tested on other cancer cells. PDAC cells are mixed 1:9 with CAF cells and injected into NSG® mice via injection into the pancreas (to form primary tumors) or via portal vein injection (to induce liver metastasis). Tumors are allowed to establish for two weeks. The most effective meso-FAP CAR TEAM cells (either MGHlMesol or SSI, as determined in Example 1) are injected intravenously, intraperitoneally, or both, and tumor growth is monitored once weekly by in vivo bioluminescent imaging. Additionally, meso-FAP CAR TEAM cell expansion and phenotype in peritoneal cavity washes and peripheral blood is measured by flow cytometry. Example 5. Increasing mesothelin density on tumor cells by preventing its cleavage using an a disintegrin and metalloprotease 17 (ADAM17) inhibitor.

Mesothelin expression on the surface of cancer cells can be decreased as a result of proteolytic cleavage, which leads to the release of mesothelin in its soluble form from the cell membrane. The cleavage and release of mesothelin from the surface of tumor cells can reduce the ability of meso-FAP CAR TEAM cells to target mesothelin-expressing cancer cells. Furthermore, soluble mesothelin can bind meso-FAP CAR TEAM cells and block the ability of meso-FAP CAR TEAM cells to recognize and bind mesothelin on the surface of tumor cells. Thus, the co-administration of inhibitors that prevent the proteolysis of mesothelin, e.g., by ADAM 17, may markedly improve the efficacy of meso-FAP CAR TEAM cells.

In vitro

To test this in vitro, pancreatic cancer cell lines ASPC1, CAP AN-2, and BxPC3 were treated with increasing doses of the ADAM17 inhibitor aderbasib. The levels of mesothelin expressed on the cell surface were measured by flow cytometry. Aderbasib was found to increase mesothelin expression on all three pancreatic cancer cell lines in a dose-dependent manner (FIGs. 6A-6D), as well as decrease mesothelin shedding by cancer cells (FIGs. 7A-7C), likely by preventing the cleavage of surface mesothelin by inhibiting ADAM- 17 activity.

Pancreatic cancer cell lines (ASPC1, CAP AN-2, and BxPC3) were next cultured and treated with SSI CAR-T cells only, untransduced cells (CAR- cells), 5 pm of aderbasib only, 5 pm of aderbasib and untransduced cells, or 5pm of aderbasib and SSI CAR-T cells. Treatment with SSI CAR-T cells resulted in a significant reduction in tumor area compared to controls or treatments that did not include SSI CAR-T cells, and a further reduction was observed when cancer cells were treated with SSI CAR-T cells in combination with 5 pM of aderbasib (FIGs. 8A-8C), indicating that the combination therapy provides a robust treatment against pancreatic cancer cells.

PDX cells are additionally treated with increasing doses of aderbasib and surface mesothelin expression is assessed. The levels of soluble mesothelin in the supernatant are measured by ELISA. Next, the lower concentration of aderbasib that induces the highest level of mesothelin expression on PDAC cell lines and PDX cells is used to treat meso-FAP CAR TEAM cells. The proliferation and cell death of meso-FAP CAR TEAM cells in the present of aderbasib is measured. PDAC cells or patient derived xenograft (PDX) cells are next co-cultured with CAF cells and meso-FAP CAR TEAM cells are added to co-cultures with or without aderbasib. The cytotoxicity of meso-FAP CAR TEAM cells is measured and compared among conditions.

In vivo

The efficacy of SSI CAR-T cells in combination with aderbasib was tested in vivo. Mice were injected subcutaneously (s.c.) with ASPC1 pancreatic cancer cells and eleven days later were administered aderbasib via oral gavage (o.g.) at a dose of 60 mg/kg and continued receiving aderbasib until 14 days post-treatment with SSI CAR-T cells, which were administered via intravenous (i.v.) injection three days following administration of aderbasib. Mice treated with SSI CAR-T cells exhibited markedly improved tumor control as far as nearly 50 days following CAR-T cell transfer compared to mice that received untransduced (CAR-) cells. Pre-treatment with aderbasib additionally significantly improved this tumor control in mice that received SSI CAR-T cells compared to mice that received CAR-T cells but did not receive aderbasib treatment (FIG. 9). Thus, combination treatment of SSI CAR-T cells and aderbasib significantly improves tumor control in vivo as well as in vitro.

PDAC cells are mixed 1:9 with CAF cells in Matrigel and injected IP into NSG® mice. Two weeks later, mice are treated IP with meso-FAP CAR TEAM cells with or without aderbasib. Tumor growth is monitored by in vivo bioluminescent imaging and survival is analyzed with a Kaplan-Meir curve. Meso-FAP CAR TEAM cell expansion and phenotype in the peripheral blood is also measured.

Example 6. Enhancing meso-FAP CAR TEAM cell killing by polarizing to a Thl/Thl7 phenotype using an interleukin-2-induced T cell kinase (ITK) inhibitor.

CD4 T cell phenotypes such as T helper 1 (Thl) and Th 17 cells promote cytotoxicity and are thus desirable in an anti-tumor context. The FDA-approved ITK inhibitor ibrutinib has been previously shown to increase T cell numbers, decrease checkpoint molecule expression, and increase Thl/Thl7 polarization. Thus, co-administration of ibrutinib with meso-FAP CAR TEAM cells may promote their tumor-killing efficacy and survival.

Mice were subcutaneously (s.c.) injected with ASPC1 cells and fourteen days later received (i) SSI CAR-T cells via intravenous (i.v.) injection, (ii) untransduced cells, (iii) ibrutinib via oral gavage (o.g.) at a dose of 25 mg/kg and untransduced cells, or (iv) ibrutinib as in (iii) and SSI CAR-T cells. Mice receiving ibrutinib received it up to day 35 post-initial treatment. Mice receiving SSI CAR-T cells exhibited improved tumor control compared to those that received untransduced cells, and tumor control was significantly improved (i.e., tumor area reduced) in mice that additionally received ibrutinib, demonstrating that the combination of anti-mesothelin CAR-T cell treatment with ibrutinib treatment improves anti-tumor efficacy (FIGs. 10A-10C). It was additionally observed that treatment with ibrutinib supported CAR-T cell expansion and/or survival, as mice that received ibrutinib in combination with SSI CAR-T cells had significantly higher CAR-T cell counts as far as 20 days post-injection (FIG. 10D). Taken together, the data show that ibrutinib enhances the anti-tumor efficacy of anti-mesothelin CAR-T cells and supports the survival and/or expansion of said CAR-T cells.

To test this, PDAC cells are mixed 1:9 with CAF cells in Matrigel and injected IP into NSG® mice. Two weeks later, mice are treated IP with meso-FAP CAR TEAM cells with or without ibrutinib. Tumor growth is monitored by in vivo bioluminescent imaging and survival is analyzed with a Kaplan-Meir curve. Meso-FAP CAR TEAM cell expansion and phenotype in the peripheral blood is also measured. Luminex is performed on mouse serum to measure cytokines associated with Thl, Th2, and Thl7 phenotypes (e.g., IFNy, IL-4, and IL-17, among others).

Example 7. Schedule of therapy and types of evaluations.

This Example describes the timeline of treatment and subsequent evaluations that subjects will undergo. All subjects will undergo 3 days of lymphodepletion chemotherapy (300 mg/m2 cyclophosphamide infused IV over 30 min immediately followed by 30 mg/m2 fludarabine infused IV over 30 min) starting Day -5, before the infusion of meso-FAP CARTEAM cells on Day 0. Lymphodepletion can be given outpatient or inpatient, per the investigator’s judgment. Following CAR T cell infusion, patients will be monitored for adverse events (AEs) as described below, clinical status, and laboratory parameters. Research samples will be collected for correlative studies for up to 24 months (FIG. 5). All subjects who complete the study, and those who withdraw from the study after receiving Meso-FAP CAR-TEAM for reasons other than death, will be asked to participate in an FDA-mandated long-term follow-up study for up to 15 years after their Meso-FAP CAR infusion, with a focus on long-term efficacy and safety.

Claims

CLAIMS What is claimed is:

1. A method of treating a subject having a mesothelin-expressing cancer, the method comprising administering to the subject in need thereof an effective amount of a chimeric antigen receptor (CAR)-T cell in combination with an a disintegrin and metalloprotease 17 (ADAM 17) inhibitor or interleukin-2-inducible T cell kinase (ITK) inhibitor, wherein the CAR-T cell comprises: a mesothelin-binding CAR; and a T cell engaging molecule (TEAM) comprising a fibroblast activation protein (FAP) binding domain.

2. The method of claim 1, wherein the TEAM further comprises a CD3 binding domain.

3. The method of claim 1 or 2, wherein the FAP binding domain and the CD3 binding domain of the TEAM are linked by a linker.

4. The method of any one of claims 1-3, wherein the linker comprises a sequence of SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, or SEQ ID NO: 40.

5. The method of claim 1 or 2, wherein the mesothelin-binding CAR comprises a VH domain of SEQ ID NO: 1 and a VL domain of SEQ ID NO: 2.

6. The method of claim 1 or 2, wherein the mesothelin-binding CAR comprises a VH domain of SEQ ID NO: 9 and a VL domain of SEQ ID NO: 10.

7. The method of any one of claims 1-5, wherein the mesothelin-binding CAR comprises: (a) a VH domain comprising three complementarity determining regions (CDR-H1,

CDR-H2, and CDR-H3), wherein CDR-H1 comprises SEQ ID NO: 3, CDR-H2 comprises SEQ ID NO: 4, and CDR-H3 comprises SEQ ID NO: 5; and (b) a VL domain comprising three CDRs (CDR-L1, CDR-L2, and CDR-L3), wherein CDR-L1 comprises SEQ ID NO: 6, CDR-L2 comprises SEQ ID NO: 7, and CDR-L3 comprises SEQ ID NO: 8.

8. The method of any one of claims 1-2 or 6, wherein the mesothelin-binding CAR comprises:

(a) a VH domain comprising three complementarity determining regions (CDR-H1, CDR-H2, and CDR-H3), wherein CDR-H1 comprises SEQ ID NO: 11, CDR-H2 comprises SEQ ID NO: 12, and CDR-H3 comprises SEQ ID NO: 13; and

(b) a VL domain comprising three CDRs (CDR-L1, CDR-L2, and CDR-L3), wherein CDR-L1 comprises SEQ ID NO: 14, CDR-L2 comprises SEQ ID NO: 15, and CDR-L3 comprises SEQ ID NO: 16.

9. The method of any one of claims 1-8, wherein the mesothelin-binding CAR further comprises a hinge/transmembrane domain.

10. The method of claim 9, wherein the hinge/transmembrane domain is selected from a group consisting of CD8, CD18, and CD28.

11. The method of claim 9 or 10, wherein the hinge/transmembrane domain is a CD8 hinge/transmembrane domain.

12. The method of any one of claims 9-11, wherein the hinge/transmembrane domain comprises a sequence of SEQ ID NO: 35.

13. The method of any one of claims 1-12, wherein the mesothelin-binding CAR comprises an intracellular signaling domain comprising a CD3^ intracellular signaling domain.

14. The method of claim 13, wherein the CD3^ intracellular signaling domain comprises the sequence of SEQ ID NO: 33.

15. The method of any one of claims 1-14, wherein the mesothelin-binding CAR further comprises a co-stimulatory domain.

16. The method of claim 15, wherein the co-stimulatory domain is selected from the group consisting of CD2, CD7, CD 18, CD27, CD28, and 4- IBB.

17. The method of claims 16, wherein the co-stimulatory domain is a 4-1BB costimulatory domain.

18. The method of any one of claims 1-17, wherein the FAP binding domain of the TEAM comprises a VH domain of SEQ ID NO: 17 and a VL domain of SEQ ID NO: 18.

19. The method of any one of claims 1-18, wherein the FAP binding domain of the TEAM comprises:

(a) a VH domain comprising three CDRs (CDR-H1, CDR-H2, and CDR-H3), wherein CDR-H1 comprises SEQ ID NO: 19, CDR-H2 comprises SEQ ID NO: 20, and CDR-H3 comprises SEQ ID NO: 21; and

(b) a VL domain comprising three CDRs (CDR-L1, CDR-L2, and CDR-L3), wherein CDR-L1 comprises SEQ ID NO: 22, CDR-L2 comprises SEQ ID NO: 23, and CDR-L3 comprises SEQ ID NO: 24.

20. The method of any one of claims 2-19, wherein the CD3 binding domain of the TEAM comprises a VH domain of SEQ ID NO: 25 and a VL domain of SEQ ID NO: 26.

21. The method of any one of claims 2-20, wherein the CD3 binding domain of the TEAM comprises:

(a) a VH domain comprising three CDRs (CDR-H1, CDR-H2, and CDR-H3), wherein CDR-H1 comprises SEQ ID NO: 27, CDR-H2 comprises SEQ ID NO: 28, and CDR-H3 comprises SEQ ID NO: 29; and (b) a VL domain comprising three CDRs (CDR-L1, CDR-L2, and CDR-L3), wherein CDR-L1 comprises SEQ ID NO: 30, CDR-L2 comprises SEQ ID NO: 31, and CDR-L3 comprises SEQ ID NO: 32.

22. The method of any one of claims 1-21, wherein the mesothelin-binding CAR comprises SEQ ID NO: 41 or SEQ ID NO: 42.

23. The method of any one of claims 1-22, wherein the TEAM comprises SEQ ID NO: 43.

24. The method of any one of claims 1-23, wherein the CAR-T cell comprises an amino acid sequence of SEQ ID NO: 44, SEQ ID NO: 45, or SEQ ID NO: 51 encoding the mesothelin- binding CAR and TEAM.

25. The method of any one of claims 1-24, wherein the TEAM is secreted by the CAR-T cell.

26. The method of any one of claims 1-25, wherein the mesothelin-expressing cancer is mesothelioma, ovarian cancer, pancreatic cancer, lung cancer, breast cancer, gastric cancer, or colorectal cancer.

27. The method of any one of claims 1-26, wherein the mesothelin-expressing cancer is pancreatic cancer.

28. The method of claim 27, wherein the pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC).

29. The method of any one of claims 1-28, wherein the ADAM 17 inhibitor is aderbasib, TAPI-0, TAPI-1, TAPI-2, GW280264X, marimastat, INCB3619, compound 22a (INCB9471), MEDI3622, apratastat, DPC-333, SCH 900567, or KP-457.

30. The method of claim 28, wherein the ADAM 17 inhibitor is aderbasib.

31. The method of any one of claims 1-28, wherein the ITK inhibitor is ibrutinib, CPI-818, BMS 509774, or PRN694.

32. The method of claim 28, wherein the ITK inhibitor is ibrutinib.

33. The method of any one of claims 1-32, comprising administering the CAR-T cell by a first route of administration and administering the ADAM 17 inhibitor and/or ITK inhibitor by a second route of administration that is different from the first route of administration.

34. The method of any one of claims 1-33, wherein the administration of the CAR-T cell comprises intravenous administration and the administration of the ADAM 17 inhibitor and/or the ITK inhibitor comprises intraperitoneal administration.

35. The method of any one of claims 1-33, wherein the administration of the CAR-T cell comprises intravenous administration and the administration of the ADAM 17 inhibitor and/or the ITK inhibitor comprises oral administration.

36. The method of any one of claims 1-35, wherein the ADAM 17 inhibitor is not a proteinbased inhibitor.