MESO-FAP with ADAM17 inhibitors or ITK inhibitors

Abstract

The present disclosure relates to methods of treating a patient using anti-mesothelin-CAR-T cells comprising an anti-fibroblast activation protein (FAP) TEAM and an ADAM17 or ITK inhibitor.

Description

[0001] Related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 604,730, filed November 30, 2023, entitled “MESO-FAP with ADAM17 Inhibitors or ITK Inhibitors,” pursuant to 35 USC §119(e), the entire contents of which are incorporated herein by reference.

[0003] Federal government-funded research

[0004] This invention was completed with government support under license number 5R01CA238268-04 granted by the National Institutes of Health. The government holds certain rights to this invention.

[0005] Reference to the electronic sequence list

[0006] The contents of the electronic serial number (M105370045WO00-SEQ-ARM.xml; size: 52,310 bytes; creation date: November 20, 2024) are incorporated herein by reference in their entirety. Background Technology

[0007] Cell-based gene therapies have shown great promise in treating diseases such as hematologic malignancies. For example, chimeric antigen receptor T-cell (CAR-T) cells, a cell-based gene therapy for cancer, have achieved great success in treating hematologic malignancies. Autologous CAR-T cells are prepared by collecting a patient's T cells and genetically modifying them to express chimeric antigen receptors (CARs), which endows the T cells with new specificity: CARs recognize tumor surface antigens, activate T cells, and initiate tumor killing and CAR-T cell proliferation. While this therapy has transformed treatment options for patients with hematologic malignancies, patients with solid tumors have not yet truly benefited from CAR-T cell therapy. Invention Overview

[0009] Previous studies have shown that chimeric antigen receptor T cells (CAR-T cells) are effective in treating hematologic malignancies, but CAR-T cell therapy for solid tumors has not yet shown the same success. This difference is likely due to a combination of factors between solid tumors and hematologic cancers, from the identification of target antigens to how CAR-T cells interact with and kill different types of tumor cells. While there is considerable understanding of the expression of surface markers that can serve as CAR targets for hematologic cancers, solid tumors are not typically diagnosed or characterized by the expression of their surface markers, and solid tumors tend to be more heterogeneous, which can lead to the growth of tumors with low or no expression of target antigens. The solid nature of solid tumors requires CAR-T cells to extravasate and migrate through the hostile tumor microenvironment (TME). In particular, the collagen-rich extracellular matrix (ECM) in the TME is a major barrier for CAR-T cells to navigate through this environment. Cancer-associated fibroblasts (CAFs) deposit collagen in the TME to form the ECM, creating a physical barrier for drug and CAR-T cell infiltration and also promoting cancer cell survival and migration. Developing CAR-T cells that can both target cancer cells and induce CAF ablation is a promising advancement in the treatment of solid cancers. Furthermore, combining these CAR-T cells with agents that enhance their activity—such as inhibitors that increase the expression of target antigens on CAR-T cells or promote T cell polarization into a tumor-killing phenotype—can further improve the efficacy of CAR-T cells against solid cancers. For example, one consideration when using CAR-T cells for solid tumors is the stability of target antigen expression on cancer cells. Mesothelin is one of 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 ADAM17). ADAM17 cleavage of membrane-bound mesothelin reduces mesothelin expression in tumor cells and leads to an increase in soluble mesothelin in the stroma. Cells lacking mesothelin can bind to receptors targeting mesothelin or fragments thereof, interfering with their intended purpose. For example, T cells containing CARs targeting mesothelin (such as those provided in this disclosure) may be unable to target cancer cells expressing mesothelin due to receptor blockade by soluble mesothelin. Therefore, this document anticipates the combined administration of ADAM17 inhibitors with CAR-T cells containing mesothelin-binding CARs.

[0010] Other inhibitors considered in this disclosure include interleukin-2-induced T-cell kinase (ITK) inhibitors. ITK is involved in multiple processes, including the activation and regulation of T-cell receptor (TCR) signaling, which affects the differentiation and polarization of effector T cells. Different T effector subtypes are desired to target different immune injuries, and Th1 and Th17 cells (characterized by the production of IFN-γ and IL-17 cytokines and / or the expression of TBET and RORγt, respectively) are more effective in fighting cancer than, for example, Th2 cells (characterized by the production of IL-4 cytokine). Inhibition of ITK has been shown to polarize T cells to the Th1 / Th17 phenotype, in addition to increasing T cell numbers and reducing checkpoint molecule expression.

[0011] Therefore, some aspects of this disclosure provide a method for treating a patient with mesothelin-expressing cancer, the method comprising administering to the patient a combination of chimeric antigen receptor (CAR)-T cells with an integrin and metalloproteinase 17 (ADAM17) inhibitor and / or an interleukin-2-inducible T-cell kinase (ITK) inhibitor, wherein the CAR-T cells comprise: a mesothelin-binding CAR and a T-cell adaptor molecule (TEAM) comprising a fibroblast activation protein (FAP) binding domain (meso-FAPCAR TEAM cells).

[0012] In some embodiments, TEAM further includes a CD3 binding domain. In some embodiments, the FAP binding domain and the CD3 binding domain of TEAM are connected by a linker. In some embodiments, the linker includes a sequence of SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, or SEQ ID NO:40.

[0013] In some embodiments, the mesothelin-binding CAR includes the VH domain of SEQ ID NO:9 and the VL domain of SEQ ID NO:10.

[0014] 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.

[0015] 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.

[0016] In some embodiments, the mesothelin-binding CAR further includes a hinge / transmembrane domain. In some embodiments, the hinge / transmembrane domain is selected from the 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 includes the sequence of SEQ ID NO:35.

[0017] In some embodiments, the mesothelin-binding CAR comprises an intracellular signaling domain containing a CD3ζ intracellular signaling domain. In some embodiments, the CD3ζ intracellular signaling domain comprises the sequence of SEQ ID NO:33.

[0018] In some embodiments, the mesothelin-binding CAR further includes 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 4-1BB co-stimulatory domain.

[0019] In some implementations, the FAP binding domain of TEAM includes the VH domain of SEQ ID NO:17 and the VL domain of SEQ ID NO:18.

[0020] In some implementations, the FAP-binding domain of TEAM includes: (a) a VH domain containing three CDRs (CDR-H1, CDR-H2, and CDR-H3), wherein CDR-H1 contains SEQ ID NO:19, CDR-H2 contains SEQ ID NO:20, and CDR-H3 contains SEQ ID NO:21; and (b) a VL domain containing three CDRs (CDR-L1, CDR-L2, and CDR-L3), wherein CDR-L1 contains SEQ ID NO:22, CDR-L2 contains SEQ ID NO:23, and CDR-L3 contains SEQ ID NO:24.

[0021] In some implementations, the CD3 binding domain of TEAM includes the VH domain of SEQ ID NO:25 and the VL domain of SEQ ID NO:26.

[0022] In some implementations, the CD3 binding domain of TEAM includes: (a) a VH domain containing three CDRs (CDR-H1, CDR-H2, and CDR-H3), wherein CDR-H1 contains SEQ ID NO:27, CDR-H2 contains SEQ ID NO:28, and CDR-H3 contains SEQ ID NO:29; and (b) a VL domain containing three CDRs (CDR-L1, CDR-L2, and CDR-L3), wherein CDR-L1 contains SEQ ID NO:30, CDR-L2 contains SEQ ID NO:31, and CDR-L3 contains SEQ ID NO:32.

[0023] In some implementations, the mesothelin-binding CAR comprises SEQ ID NO:41 or SEQ ID NO:42.

[0024] In some implementations, TEAM includes SEQ ID NO:43.

[0025] In some embodiments, CAR-T cells contain amino acid sequences encoding mesothelin-binding CAR and TEAM, namely SEQ ID NO:44, SEQ ID NO:45, or SEQ ID NO:51.

[0026] In some embodiments, mesothelin-expressing cancers are mesothelioma, ovarian cancer, pancreatic cancer, lung cancer, breast cancer, gastric cancer, or colorectal cancer. In some embodiments, mesothelin-expressing cancers are pancreatic cancer. In some embodiments, pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC).

[0027] In some implementations, the ADAM17 inhibitor is aderbasib, TAPI-0, TAPI-1, TAPI-2, GW280264X, marimastat, INCB3619, compound 22a (INCB9471), MEDI3622, apratastat, DPC-333, SCH 900567, or KP-457.

[0028] In some implementations, the ADAM17 inhibitor is aderbasib.

[0029] In some embodiments, the ITK inhibitor is ibrutinib, CPI-818, BMS 509774, or PRN694. In some embodiments, the ITK inhibitor is ibrutinib.

[0030] In some embodiments, the method described herein includes administering CAR-T cells via a first administration route and administering an ADAM17 inhibitor and / or an ITK inhibitor via a second administration route different from the first administration route. In some embodiments, administration of CAR-T cells includes intravenous administration, and administration of the ADAM17 inhibitor and / or ITK inhibitor includes intraperitoneal administration. In some embodiments, administration of CAR-T cells includes intravenous administration, and administration of the ADAM17 inhibitor and / or ITK inhibitor includes oral administration.

[0031] In some implementations, ADAM17 inhibitors are not protein-based inhibitors.

[0032] Brief description of the attached figures

[0033] The following figures form part of this specification and are included to further illustrate certain aspects of this disclosure, which can be better understood by referring to one or more of these figures in conjunction with the detailed description of the specific embodiments presented herein.

[0034] Figure 1 This is a schematic diagram of the tumor microenvironment (TME), showing the cell types present, such as myeloid cells, myeloid-derived suppressor cells (MDSCs), cancer-associated fibroblasts (CAFs), T cells, cancer cells, and other cell types. It also shows the vascular system and the inhibitory targets of the ITK inhibitor ibrutinib and the ADAM17 inhibitor aderbasib.

[0035] Figures 2A-2E This study demonstrated the effectiveness of meso-FAP CAR-TEAM cells (cells that secrete T-cell adaptor molecules (TEAMs) targeting fibroblast activation protein (FAP) and contain anti-mesothelin chimeric antigen receptors) against cancer-associated fibroblasts (CAF) and pancreatic ductal adenocarcinoma (PDAC) cells. Figure 2A This demonstrates immunodeficient NSG treated with subcutaneous inoculation of AsPC-1 tumors (mesothelin-expressing tumors derived from human PDAC cell lines) followed by intravenous treatment with untransduced (UTD) control cells, meso-FAP CAR-TEAM cells, or meso-CD19 CAR-TEAM cells (non-mesothelin-expressing tumor-specific). ®Tumor volume in mice. AsPC-1 tumor-bearing mice treated with meso-CD19 CAR-TEAM cells or UTD cells showed a significant reduction in tumor volume. Figure 2A ) and the reduction in the presence of AsPC-1 tumor cells ( Figure 2B ). Figure 2C-2E The efficacy of meso-FAP CAR-TEAM cells in organoids from patient-derived mesothelin-expressing tumors grown in the presence of CAF was demonstrated. The recovery rate of organoids in organoid-CAF cultures treated with meso-FAP CAR-TEAM cells was significantly reduced compared to cultures treated with UTD cells. Figure 2C , 2E (above), and compared with cultures treated with UTD cells or meso-CD19 CAR-TEAM cells, CAF cells were significantly reduced ( Figure 2D , 2E (below).

[0036] Figures 3A-3C The effects of meso-FAP CAR-TEAM cells were demonstrated depending on the route of administration. AsPC-1 tumor cells were intraperitoneally injected (IP) into NSG mice, followed by IP administration of meso-FAP CAR-TEAM cells. Figure 3A ) or intravenous (IV) Figure 3B Treatment of mice Figure 3B In both cases, mice treated with meso-FAP CAR-TEAM cells had significantly longer survival than mice treated with UTD cells. These differences decreased or disappeared when AsPC-1 tumor cells were subcutaneously implanted and meso-FAP CAR-TEAM cells were IV-injected. Figure 3C ).

[0037] Figures 4A-4E The study demonstrated the killing efficacy of meso-FAP CAR-TEAM cells against pancreatic cancer cells (AsPC-1 or BxPC-3) and ovarian cancer cells (SKOV3). Figure 4A The expression of ADAM17, ADAM10 and mesothelin in pancreatic cancer cell lines (ASPC1, CAPAN-2 and BxPC3) was shown. Figure 4B , 4D The images show pancreatic cancer cells (AsPC-1, BxPC-3). Figure 4B ) and ovarian cancer cells ( Figure 4D Mesothelin expression on pancreatic cancer cells (AsPC-1, BxPC-3) by meso-FAP CAR-TEAM cells. Figure 4C) and ovarian cancer cells ( Figure 4E The cell lysis capacity of ) was significantly higher than that of UTD cells.

[0038] Figure 5 This is a timeline of candidate subjects treated with meso-FAP CAR-TEAM cells in clinical trials. The bottom time points represent patient monitoring and study sample collection.

[0039] Figures 6A-6D The expression of mesothelin in pancreatic cancer cells treated with aderbasib (INCB7839). Figure 6A The fluorescence curves of mesothelin expression in ASPC-1 cells treated with aderbasib are shown (summarized as histograms in...). Figure 6B middle). Figure 6C The expression of mesothelin in CAPAN-2 cells treated with aderbasib is shown. Figure 6D Mesothelin expression on BxPC3 cells treated with aderbasib is shown.

[0040] Figures 7A-7C Involves ASPC1 processed with aderbasib ( Figure 7A ), CAPAN-2 ( Figure 7B ) and BxPC3 ( Figure 7C Prevention of mesothelin shedding from cells.

[0041] Figures 8A-8C This involves the use of SS1 CAR-T cells or untransduced T cells (CAR-cells) with or without aderbasib treatment on pancreatic cancer cells. Figure 8A ASPC1 cells; Figure 8B CAPAN-2 cells; Figure 8C Cell lysis of BxPC3 cells. An asterisk indicates the following treatment groups: tumor only, no aderbasib and untransduced cells, aderbasib only, and aderbasib plus untransduced cells.

[0042] Figures 9A-9B This involves the combined treatment of mice with CAR-T cells and aderbasib. Figure 9A This is a schematic diagram of the experiment. Figure 9B Tumor growth was shown in mice that received subcutaneous (sc) injection of ASPC1 cells and were treated with SS1 CAR-T cells 14 days later (with or without aderbasib supplementation).

[0043] Figures 10A-10ETumor growth in mice involving subcutaneous (sc) injection of ASPC1 cells followed by SS1 CAR-T cell therapy 14 days later (with or without supplemental ibrutinib treatment) (experimental schematic). Figure 10A Summary chart, Figure 10B CAR-T therapy alone, Figure 10C CAR-T therapy combined with ibrutinib therapy Figure 10D The average value is displayed in the image. Figure 10E middle).

[0044] Figure 11 It is an exemplary construct encoding an anti-mesothelin CAR, a T-cell adaptor molecule (TEAM) targeting fibroblast activation protein (FAP) and CD3, and a truncated CD19 (tCD29). Detailed Implementation

[0045] In some aspects, this disclosure describes a method of treating a subject with mesothelin-expressing cancer, comprising administering to the patient a combination of chimeric antigen receptor (CAR)-T cells with an integrin and metalloproteinase 17 (ADAM17) inhibitor or an interleukin-2-inducible T-cell kinase (ITK) inhibitor, wherein the CAR-T cells comprise: a mesothelin-binding CAR; and a T-cell adaptor molecule (TEAM) comprising a fibroblast activation protein (FAP) binding domain.

[0046] General definition

[0047] The terms “reduction,” “decline,” or “reduction” are used herein to refer to a statistically significant reduction. In some implementations, “reduction,” “decline,” or “reduction” typically refers to a reduction of at least 10% compared to a reference level (e.g., in the absence of a specific treatment or drug), and may include, for example, a reduction of 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, the reduction may preferably be to an acceptable level within the normal range for individuals without a specific condition.

[0048] A "disease" is a state of health in an animal (such as a human) in which the animal is unable to maintain homeostasis, and its health continues to deteriorate if the disease is not treated. In contrast, an animal's "symptom" is a state of health in which the animal is able to maintain homeostasis, but its health is less favorable than it would be without the symptom. A symptom does not necessarily lead to a further decline in the animal's health if left untreated. In some implementations, the disease is cancer or a tumor.

[0049] As used herein, the terms “tumor antigen,” “tumor-associated antigen,” and “cancer antigen” are used interchangeably to refer to antigens differentially expressed by cancer cells and thus potentially used to target cancer cells. Cancer antigens are antigens that can potentially stimulate a tumor-specific immune response. Some of these antigens are encoded by normal cells, although not necessarily expressed. These antigens can be characterized as those that are normally silenced (i.e., not expressed) in normal cells, those expressed only at specific stages of differentiation, and those that are temporarily expressed, such as embryonic and fetal antigens. Other cancer antigens are encoded by mutated cellular genes such as oncogenes (e.g., activated Ras oncogenes), repressor genes (e.g., mutated p53), and fusion proteins resulting from internal deletions or chromosomal translocations. There are also other cancer antigens that can be encoded by viral genes, such as those carried by RNA and DNA tumor viruses. Many tumor antigens have been defined according to various solid tumors: MAGE 1, 2, and 3, defined by immunity; MART-1 / Melan-A, gp100, carcinoembryonic antigen (CEA), human epidermal growth factor receptor (HER2), mucin (i.e., MUC-1), prostate-specific antigen (PSA), and prostate acid phosphatase (PAP). In addition, viral proteins such as those encoded by hepatitis B virus (HBV), Epstein-Barr virus (EBV), and human papillomavirus (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.

[0050] As used herein, the term "chimera" refers to a product of the partial fusion of at least two or more distinct polynucleotide molecules. In some embodiments, the term "chimera" refers to a gene expression element generated by manipulation of known elements or other polynucleotide molecules.

[0051] In some embodiments, "activation" may refer to a T cell state that has been adequately stimulated to induce detectable cell proliferation. In some embodiments, activation may refer to induced cytokine production. In other embodiments, activation may refer to detectable effector function.

[0052] The “activated T cells” used in this article are at least proliferating T cells.

[0053] As used herein, the terms "specific binding" and "specifically binding" refer to a physical interaction between two molecules, compounds, cells, and / or particles, wherein the binding of a first entity to a second target entity has higher specificity and affinity than to a third non-target entity. In some embodiments, specific binding may mean that the affinity of the first entity for the second target entity is at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1000-fold, or more than that for the third non-target entity under the same conditions. A reagent specific to a particular target is a reagent that exhibits specific binding to that target under the assay conditions used. Non-limiting examples include antibodies or ligands that recognize and bind to homologous binding partner proteins (e.g., stimulatory molecules and / or co-stimulatory molecules present on T cells). As used herein, "stimulatory ligand" refers to a ligand that, when present on antigen-presenting cells (APCs) (e.g., macrophages, dendritic cells, B cells, artificial APCs, etc.), specifically binds to homologous binding partners (referred to herein as "stimulatory molecules" or "co-stimulatory molecules") on T cells, thereby mediating primary T cell responses, including but not limited to proliferation, activation, and initiation of immune responses. Stimulatory ligands are well-known in the art and include peptide-loaded MHC class I molecules, anti-CD3 antibodies, hyperagonist anti-CD28 antibodies, and hyperagonist anti-CD2 antibodies.

[0054] As used herein, the term "stimulatory molecule" refers to a molecule on T cells that specifically binds to homologous stimulatory ligands present on antigen-presenting cells. The term "costimulatory ligand" as used herein includes molecules on APCs that specifically bind to homologous costimulatory molecules on T cells, thereby providing signals that, in addition to the primary signals provided by, for example, the binding of the TCR / CD3 complex to MHC molecules carrying peptides, mediate T cell responses, including but not limited to proliferation, activation, and differentiation. Co-stimulatory ligands may include, but are 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 β receptor, 3 / TR6, IL T3, IL T4, HVEM, agonists or antibodies that bind to Toll-like receptors, and ligands that specifically bind to B7-H3. Costimulatory ligands may also include, but are not limited to, antibodies that specifically bind to costimulatory molecules present on T cells, such as, but not limited to, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83.

[0055] "Co-stimulatory molecules" refer to T cells that specifically bind to co-stimulatory ligands, thereby mediating the co-stimulatory response of T cells, such as, but not limited to, proliferating homologous binding partners. Co-stimulatory molecules include, but are not limited to, MHC class I molecules, BTLA, Toll-like receptors, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83.

[0056] In some embodiments, the term "engineered" and its grammatical equivalents as used herein may refer to one or more artificially designed alterations to nucleic acids (e.g., nucleic acids within an organism's genome). In another embodiment, "engineered" may refer to alterations, additions, and / or deletions of genes. "Engineered cell" may refer to a cell having added, deleted, and / or altered genes.

[0057] The term “cell” or “engineered cell” as used in this article, and its grammatical equivalents, may refer to cells of human or non-human animal origin.

[0058] As used herein, the term "operably linked" refers to the linking of a first polynucleotide molecule (such as a promoter) to a second transcribed polynucleotide molecule (such as a target gene), wherein the arrangement of the polynucleotide molecules such that the first polynucleotide molecule influences the function of the second polynucleotide molecule. The two polynucleotide molecules may or may not be part of a single, consecutive polynucleotide molecule, and may or may not be adjacent. For example, if a promoter regulates or mediates the transcription of a target gene in a cell, then the promoter is operably linked to the target gene.

[0059] In the various embodiments described herein, it is further contemplated that variants (naturally occurring or otherwise), alleles, homologs, conserved modified variants, and / or conserved substitution variants of any particular polypeptide described are included. Regarding amino acid sequences, those skilled in the art will recognize that variants in which a single substitution, deletion, or addition is made to a nucleic acid, peptide, polypeptide, or protein sequence, thereby altering a single amino acid or a small percentage of amino acids in the coding sequence, and where such alteration is made by substituting an amino acid with chemically similar properties and retaining the desired activity of the polypeptide, are referred to as “conserved modified variants.” Such conserved modified variants are complementary and do not exclude polymorphic variants, interspecies homologs, and alleles consistent with this disclosure.

[0060] Specific amino acids can be substituted with residues having similar physicochemical characteristics, such as replacing one aliphatic residue with another (e.g., with Ile, Val, Leu, or Ala substituted for each other), or replacing one polar residue with another (e.g., between Lys and Arg; between Glu and Asp; or between Gln and Asn). Other such conserved substitutions, such as substitutions of entire regions with similar hydrophobic characteristics, are well known. Peptides containing conserved amino acid substitutions can be tested in any of the assays described herein to confirm that the desired activity (e.g., ligand-mediated receptor activity and the specificity of the native or reference peptide) is retained. Amino acids can be grouped according to the similarity of their side chain properties (cited from Allehninger, Biochemistry, 2nd edition, pp. 73-75, Worth Publishers, New York (1975)): (1) Nonpolar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) Nonpolar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) Acidic: Asp (D), Glu (E); (4) Basic: Lys (K), Arg (R), His (H). Alternatively, naturally occurring residues can be grouped according to common side-chain properties: (1) hydrophobic: ortholeucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues affecting chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Non-conservative substitution would involve exchanging one member of these categories for another. Specific conservative substitutions include, for example: substituting Ala with Gly or Ser; substituting Arg with Lys; substituting Asn with Gln or His; substituting Asp with Glu; substituting Cys with Ser; substituting Gln with Asn; substituting Glu with Asp; substituting Gly with Ala or Pro; substituting His with Asn or Gln; substituting Ile with Leu or Val; substituting Leu with Ile or Val; substituting Lys with Arg, Gln, or Glu; substituting Met with Leu, Tyr, or Ile; substituting Phe with Met, Leu, or Tyr; substituting Ser with Thr; substituting Thr with Ser; substituting Trp with Tyr; substituting Tyr with Trp; and / or substituting Phe with Val, Ile, or Leu.

[0061] In some embodiments, the polypeptide (or nucleic acid encoding such polypeptide) described herein may be a functional fragment of one of the amino acid sequences described herein. As used herein, a “functional fragment” is a peptide fragment or region that retains at least 50% of the activity of a wild-type reference polypeptide according to assays known in the art or described below. A functional fragment may contain conserved substitutions of the sequences disclosed herein.

[0062] In some embodiments, the polypeptide described herein may be a variant of the polypeptide or molecule as described herein. In some embodiments, the variant is a conserved modification variant. Conserved substitution variants may be obtained, for example, by mutation of the natural nucleotide sequence. As used herein, a “variant” is a polypeptide that is substantially homologous to a natural or reference polypeptide but has an amino acid sequence different from the natural or reference polypeptide due to one or more deletions, insertions, or substitutions. The DNA sequence encoding the variant polypeptide encompasses sequences containing one or more nucleotide additions, deletions, or substitutions compared to the natural or reference DNA sequence, but the variant protein or fragment thereof encoded by these sequences retains the activity of the non-variant polypeptide. Various PCR-based site-specific mutagenesis methods are known in the art and can be applied by those skilled in the art.

[0063] The variant amino acid or DNA sequence may have 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 homology with the natural or reference sequence. The degree of homology (percentage of identity) between the natural and mutant sequences can be determined, for example, by comparing the two sequences using free computer programs commonly used for this purpose on the World Wide Web (e.g., using BLASTp or BLASTn with default settings).

[0064] Alterations to the natural amino acid sequence can be achieved using any of a variety of techniques known to those skilled in the art. For example, a mutation can be introduced at a specific locus by synthesizing an oligonucleotide containing the mutated sequence, flanked by a restriction site that allows ligation to a fragment of the natural sequence. After ligation, the resulting reconstructed sequence encodes an analogue with the desired amino acid insertion, substitution, or deletion. Alternatively, an oligonucleotide-directed site-specific mutagenesis procedure can be employed to provide an altered nucleotide sequence with specific codons changed according to the desired substitution, deletion, or insertion. Techniques for performing such alterations are well-established, including, for example, those disclosed in Walder et al. (Gene 42:133, 1986), Bauer et al. (Gene 37:73, 1985), Craik (BioTechniques, January 1985, 12-19), Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981), and U.S. Patent Nos. 4,518,584 and 4,737,462. Any cysteine ​​residues that do not participate in maintaining the correct conformation of the polypeptide can be substituted, usually with serine, to improve the oxidative stability of the molecule and prevent undesirable cross-linking. Conversely, cysteine ​​bonds can be added to the polypeptide to improve its stability or promote oligomerization.

[0065] The term “polynucleotide” is used interchangeably with “nucleic acid molecule” herein and refers to a polymer of nucleosides. Typically, polynucleotides consist of nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine) linked by phosphodiester bonds and naturally occurring in DNA or RNA. However, the term encompasses molecules containing nucleosides or nucleoside analogs with chemically or biologically modified bases, modified backbones, etc., whether or not they are found in naturally occurring nucleic acids, and such molecules may be preferred in certain applications. When this disclosure refers to polynucleotides, it should be understood that both DNA and RNA are provided, and in each case, both single-stranded and double-stranded forms are provided (and the complementary strand of each single-stranded molecule). As used herein, “polynucleotide sequence” can refer to the polynucleotide material itself and / or the sequence information (i.e., the letter sequence used as a base abbreviation) that biochemically characterizes a particular nucleic acid. In some embodiments, the nucleic acid molecule is a heterologous nucleic acid molecule. As used herein, the term “heterologous nucleic acid molecule” refers to a nucleic acid molecule that is not naturally occurring in a particular cell.

[0066] The polynucleotide sequences presented in this article are shown in the 5' to 3' orientation unless otherwise stated.

[0067] As used herein, the term "peptide" refers to a polymer of amino acids. The terms "protein" and "peptide" are used interchangeably herein. A peptide can be a relatively short polypeptide, typically between about 2 and 60 amino acids in length. Peptides used herein generally contain amino acids, such as the 20 most common L-amino acids in proteins. However, other amino acids and / or amino acid analogs known in the art may be used. One or more amino acids in a polypeptide may be modified, for example by adding chemical entities such as carbohydrate groups, phosphate groups, fatty acid groups, linkers for coupling, functionalization, etc. A polypeptide having a non-peptide portion covalently or non-covalently associated with it should still be considered a "peptide". Exemplary modifications include glycosylation and palmitoylation. Peptides can be purified from natural sources, produced using recombinant DNA technology, or synthesized by chemical methods such as conventional solid-phase peptide synthesis. As used herein, the terms "peptide sequence" or "amino acid sequence" can refer to the polypeptide material itself and / or the sequence information (i.e., a letter sequence or a three-letter code used as an abbreviation for amino acid names). Peptide sequences presented herein are presented in an N-terminal to C-terminal orientation unless otherwise stated.

[0068] The term "gene" refers to a nucleic acid sequence (DNA) that, when operatively linked with appropriate regulatory sequences, is transcribed into RNA in vitro or in vivo. A gene may or may not include regions before and after the coding region, such as the 5' untranslated region (5'UTR) or "leader" sequence and the 3'UTR or "tail" sequence, as well as intercalation sequences (introns) between the individual coding regions (exons).

[0069] As used herein, "signal peptide" or "signal sequence" refers to a peptide located at the N-terminus of a newly synthesized protein, used to guide the nascent protein to the endoplasmic reticulum. In some embodiments, the signal peptide is a CD8 or IgK signal peptide.

[0070] In some embodiments, the peptides, polynucleotides, plasmids, and / or vectors described herein optionally further comprise a reporter molecule, such as one used to determine whether the vector is correctly expressed in cells. In some embodiments, the reporter molecule may be a fluorescent protein (e.g., GFP, YFP, RF), an antibody (e.g., CD34, tEGFR, tCD19, tCD20, tCD34, and tHer2), or a radioisotope. In some embodiments, the reporter molecule is a hygromycin phosphotransferase (hph) capable of imaging alone or in conjunction with a substrate or chemical substance (e.g., 9-[4-[18F]fluoro-3-(hydroxymethyl)butyl]guanine ([18F]FHBG)).

[0071] In some implementations, GFP and mCherry can be used as fluorescent tags for imaging CARs expressed on T cells (e.g., CAR-T cells). It is anticipated that virtually any fluorescent protein known in the art can be used as a fluorescent tag for this purpose. For clinical applications, the CAR does not need to contain a fluorescent tag or fluorescent protein. Therefore, any markers present in the construct in each specific construct provided herein can be removed.

[0072] Chimeric antigen receptor (CAR)

[0073] As used herein, the term "chimeric antigen receptor" or "CAR" refers to an engineered T-cell receptor that specifically transplants a ligand or antigen onto an immune cell. Immune cells are derived from the hematopoietic system 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 (e.g., naive T cells, central memory T cells, effector memory T cells, or combinations thereof). CARs are also called artificial T-cell receptors, chimeric T-cell receptors, or chimeric immune receptors. Any CAR described in this section can be a CAR in CAR TEAM cells.

[0074] CARs place an extracellular binding domain, which specifically binds to an antigen expressed on the surface of a cell to be targeted to elicit a T-cell response, onto a construct comprising a transmembrane domain and an intracellular domain of a T-cell receptor molecule. In some embodiments, the extracellular binding domain comprises an antigen domain of an antibody that specifically binds to an antigen expressed on the cell to be targeted to elicit a T-cell response. In some embodiments, the extracellular binding domain comprises a ligand that specifically binds to an antigen expressed on the cell to be targeted to elicit a T-cell response.

[0075] As used in this article, "CAR-T cells" or "CAR-T" refers to T cells that express CAR. When CAR is expressed in T cells, it can utilize the antigen-binding properties of monoclonal antibodies to redirect the specificity and reactivity of T cells in a non-MHC-restricted manner, targeting specific targets. Non-MHC-restricted antigen recognition allows CAR-expressing T cells to recognize antigens without antigen processing, thereby bypassing the main mechanisms of tumor escape.

[0076] As can be determined by those skilled in the art, the various functionally similar or equivalent components of these CARs may be interchanged or substituted with each other, or with other similar or functionally equivalent components known in the art or listed herein.

[0077] Any cell surface region can be targeted by CARs. Typically, the target will be a cell surface peptide that is differentially or preferentially expressed on cells to which a T-cell response is desired. In some implementations, the extracellular binding domain binds to the cancer-associated antigen mesothelin, as described in PCT / US2020 / 065733 and / or PCT / US2020 / 036108. Without being bound by theory, mesothelin (MSLN) (uniprot.org / uniprot / Q13421) is expressed in normal mesothelial cells of certain tissues (e.g., pleura, pericardium, peritoneum), in trace amounts in certain epithelial cells (e.g., ovary, vaginal tunica vaginalis, reticulum testis, and fallopian tubes), but in large quantities in various cancer cells. See, for example, 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 incorporated herein by reference. Mesothelin can be used as a marker for cells associated with various cancers (e.g., overexpressed in multiple cancers), and CAR and CAR-T cells bound to mesothelin or a portion thereof can be used to treat subjects with cancers such as those associated with mesothelin expression (mesothelin-expressing cancers). In some embodiments, CAR-T cells are bound to mesothelin or a portion thereof that has at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 50. In some embodiments, CAR-T cells are bound to mesothelin or a portion thereof that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 50. In some embodiments, CAR-T cells bind to mesothelin or a portion thereof that shares at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 50. In some embodiments, CAR-T cells bind to mesothelin or a portion thereof that shares at least 97% sequence identity with the amino acid sequence of SEQ ID NO: 50. In some embodiments, CAR-T cells bind to mesothelin or a portion thereof that shares at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 50. In some embodiments, CAR-T cells bind to mesothelin or a portion thereof that shares at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 50. In some embodiments, CAR-T cells bind to mesothelin or a portion thereof having the amino acid sequence of SEQ ID NO: 50.

[0078] extracellular binding domain

[0079] As used herein, the term "extracellular binding domain" refers to a polypeptide found outside the cell sufficient to facilitate binding to a target. In some embodiments, the CAR described herein comprises an extracellular binding domain. The extracellular binding domain specifically binds to its binding partner, i.e., the target. As a non-limiting example, the extracellular binding domain may include the antigen domain of an antibody or a ligand that recognizes and binds to a homologous binding partner protein. In this context, a ligand is a molecule that specifically binds to a portion of a protein and / or receptor. Homologous binding partners of ligands that can be used in the methods and compositions described herein are typically found on the cell surface. Ligand: Homologous partner binding can lead to alterations in the receptor carrying the ligand or activation of physiological responses, such as activation of signaling pathways. In some embodiments, the ligand may be genomically non-natural. In some embodiments, the ligand has a conserved function in at least two species.

[0080] Any cell surface region can be targeted by a CAR (e.g., the extracellular binding domain of a CAR). In some implementations, the target will be a cell surface peptide that is differentially or preferentially expressed on cells to which it is desired to target to elicit a T cell response. To target Tregs, antibodies can target proteins such as glycoprotein A repeat dominant protein (GARP), latent-related peptide (LAP), CD25, CTLA-4, ICOS, TNFR2, GITR, OX40, 4-1BB, and LAG-3.

[0081] In healthy individuals, mesothelin is expressed by structural cells lining many tissues, such as the lungs, heart, and peritoneum. However, in disease states such as cancer, mesothelin can be overexpressed, for example in mesothelioma, ovarian cancer, pancreatic cancer, and lung adenocarcinoma. The function of mesothelin in normal cells is not fully understood, but its prevalence in cancer makes it an attractive target for CAR-T cells, such as those disclosed herein.

[0082] In some embodiments, the CAR vector comprises a CAR containing an extracellular binding domain for binding mesothelin. In some embodiments, the mesothelin CAR comprises a polynucleotide encoding an extracellular binding domain for an antibody containing mesothelin (e.g., scFv). In some embodiments, the mesothelin scFv comprises the VH domain of SEQ ID NO:1 or SEQ ID NO:9 and the VL domain of SEQ ID NO:2 or SEQ ID NO:10, or variations thereof. In some embodiments, the mesothelin scFv comprises SEQ ID NO:41 or SEQ ID NO:42, or variations thereof. In some embodiments, the mesothelin scFv comprises the VH domain of SEQ ID NO:1 and the VL domain of SEQ ID NO:2, or variations thereof. In some embodiments, the mesothelin scFv comprises the VH domain of SEQ ID NO:9 and the VL domain of SEQ ID NO:10.

[0083] Hinges and transmembrane domains

[0084] In some embodiments, the CAR peptide further includes a transmembrane domain or hinge / transmembrane domain that links the extracellular binding domain to the intracellular signaling domain. In some embodiments, the binding domain of the CAR is followed by one or more "hinge domains" that function in positioning the extracellular binding domain away from the effector cell surface to enable appropriate cell / cell contact, antigen binding (via the extracellular binding domain), and activation. The CAR may include one or more hinge domains between the binding domain and the transmembrane domain (TM). The hinge domain may be derived from natural, synthetic, semi-synthetic, or recombinant sources. The hinge domain may include the amino acid sequence of a naturally occurring immunoglobulin hinge region or a modified amino acid sequence of an immunoglobulin hinge region. Exemplary hinge domains applicable in the CAR described herein include hinge regions derived from the extracellular regions of type I membrane proteins (such as CD8 (e.g., CD8α), CD4, CD28, 4-1BB, and CD7), which may be wild-type hinge regions from these molecules or may be modified hinge regions. In some embodiments, the CAR contains a polynucleotide encoding a CD8α hinge / transmembrane domain. In some embodiments, the CAR contains a polynucleotide encoding a 41BB intracellular domain.

[0085] 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 the CD8a hinge region.

[0086] As used herein, a “transmembrane domain” (TM domain) refers to the portion of a CAR that fuses the extracellular binding portion (in some embodiments via a hinge domain) to the intracellular portion (e.g., a co-stimulatory domain and an intracellular signaling domain) and anchors the CAR to the plasma membrane of immune effector cells. The transmembrane domain is typically a hydrophobic region of the CAR that crosses the cell's plasma membrane. The TM domain can be a transmembrane region or fragment of a transmembrane protein (e.g., type I transmembrane protein or other transmembrane proteins), an artificial hydrophobic sequence, or a combination thereof. While specific examples are provided and used herein, other transmembrane domains will be apparent to those skilled in the art and can be used in conjunction with alternative embodiments of the present technology. The selected transmembrane region or fragment thereof preferably does not interfere with the intended function of the CAR.

[0087] When used in connection with the transmembrane domains of proteins or peptides, "fraction of" refers to the portion of the transmembrane domain sufficient to anchor or attach the protein to the cell surface.

[0088] In some embodiments, the transmembrane domains or fragments thereof of the CAR described herein include transmembrane domains selected from the following: α, β or ζ chains of T cell receptors, CD2, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), 4-1BBL, GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFI), CD160, CD19, IL2Rβ, IL2Rγ and IL7Rα, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d , ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM Transmembrane domains of (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D and / or NKG2C.

[0089] As used herein, “hinge / transmembrane domain” refers to a domain that contains both a hinge domain and a transmembrane domain. For example, the hinge / transmembrane domain may be derived from the hinge / transmembrane domain of CD8, CD28, CD7, or 4-1BB. In some embodiments, the hinge / transmembrane domain of a CAR or a fragment thereof is derived from or contains the hinge / transmembrane domain of CD8 (e.g., SEQ ID NO: 35 or a variant thereof). CD8 is an antigen preferentially found on the 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 α chain (CD8α or CD8a) and a β chain (CD813 or CD8b). The CD8a sequence is known for many species, such as human CD8a (NCBI Gene ID: 925), peptides (e.g., NCBIRef Seq NP 001139345.1), and mRNA (e.g., NCBI Ref Seq NM_000002.12). CD8 can refer to human CD8, including its naturally occurring variants, molecules, and alleles. In some implementations of either aspect, such as in veterinary applications, CD8 can refer to CD8 in animals such as dogs, cats, cattle, horses, and pigs.

[0090] For these species, those skilled in the art can easily identify human CD8 homologs and / or orthologs, for example, by using the NCBI ortholog search function or by searching for sequences similar to reference CD8 sequences in the available sequence data for a specific species.

[0091] In some embodiments, the CD8 hinge and transmembrane sequence correspond to the amino acid sequence of SEQ ID NO:35; or include sequences 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 with the sequence of SEQ ID NO:35.

[0092] Co-stimulatory domain

[0093] Each CAR described herein optionally includes one or more intracellular domains of co-stimulatory molecules or co-stimulatory domains. As used herein, the term "co-stimulatory domain" refers to the intracellular signaling domain of a co-stimulatory molecule. A co-stimulatory molecule is a cell surface molecule other than an antigen receptor or Fe receptor that provides a second signal required for the effective activation and function of T lymphocytes upon binding to an antigen. A co-stimulatory domain may be, for example, a co-stimulatory domain of 4-1BB, CD27, CD28, or OX40. Other illustrative examples of such co-stimulatory molecules include CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKD2C SLP76, TRIM, and ZAP70. In some embodiments, the intracellular domain is the intracellular domain of 4-1BB. 4-1BB (CD137; TNFRS9) is an activation-induced co-stimulatory molecule and an important regulator of the immune response.

[0094] 4-1BB is a membrane receptor protein, also known as CD137, and is a member of the tumor necrosis factor (TNF) receptor superfamily. 4-1BB is expressed on activated T lymphocytes. The 4-1BB sequence is known for many species, such as human 4-1BB, also known as TNFRSF9 (NCBI Gene 25 ID: 3604) and its mRNA (NCBI reference sequence: NM_001561.5). 4-1BB can refer to human 4-1BB, including its naturally occurring variants, molecules, and alleles. In some embodiments of any aspect, such as in veterinary applications, 4-1BB can refer to 4-1BB in animals such as dogs, cats, cattle, horses, pigs, etc. For these species, those skilled in the art can readily identify homologs and / or orthologs of human 4-1BB, e.g., by using the NCBI ortholog search function or by searching for sequences similar to the reference 4-1BB sequence in the available sequence data for a specific species.

[0095] Intracellular signal transduction domains

[0096] In some implementations, the CAR contains a polynucleotide encoding a CD3ζ intracellular signal transduction domain.

[0097] The properties of the intracellular signal transduction domains of CARs can vary as are known in the art and disclosed herein, but when the chimeric target / extracellular binding domain binds to the target / antigen on the surface of the target cell, the chimeric target / extracellular binding domain makes the receptor sensitive to signal transduction activation.

[0098] Regarding intracellular signal transduction domains, so-called "first-generation" CARs include those that provide only CD3-ζ signaling after binding to the antigen in their extracellular binding domain. So-called "second-generation" CARs include those that provide both a co-stimulatory domain (such as CD28 or CD137) and an activation domain (CD3ζ), and so-called "third-generation" CARs include those that provide multiple co-stimulatory domains (such as CD28 or CD137) and an activation domain (CD3ζ). In various implementations, CARs are selected to have high affinity or affinity for the target / antigen—for example, antibody-derived target or extracellular binding domains typically have higher affinity and / or affinity for the target antigen than naturally occurring T cell receptors. This characteristic, combined with the high specificity that can be selected for antibodies, enables CAR-T cells to target T cells with high specificity.

[0099] The CARs described herein include intracellular signal transduction domains. An “intracellular signal transduction domain” refers to a portion of the CAR polypeptide that participates in transducing information about the effective binding of the CAR to target antigens into immune effector cells to trigger effector cell functions (such as activation, cytokine production, proliferation, and cytotoxic activity), including the release of cytotoxic factors into CAR-bound target cells, or other cellular responses triggered by antigen binding to the extracellular CAR domain. In various instances, the intracellular signal transduction domain is derived from CD3-ζ (e.g., see below). Other non-limiting examples of intracellular signal transduction domains containing immune receptor tyrosine activation motifs (ITAMs) particularly suitable for this technology include those derived from TCR-ζ, FcR-γ, FcR-β, CD3γ, CD3θ, CD3σ, CD3η, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d.

[0100] CD3 is a T cell co-receptor that promotes T lymphocyte activation when it binds to an appropriate co-stimulus (e.g., the binding of a co-stimulatory molecule). The CD3 complex consists of four distinct chains; mammalian CD3 consists of a CD3γ chain, a CD3δ chain, and two CD3ε chains.

[0101] These chains bind to molecules called T-cell receptors (TCRs) and CD3ζ to generate activation signals in T lymphocytes. The complete TCR complex consists of TCR, CD3ζ, and the complete CD3 complex.

[0102] In some embodiments of any aspect, the CAR peptide described herein includes an intracellular signal transduction domain comprising an immune receptor tyrosine activation motif or ITAM derived from CD3ζ, including variants of CD3ζ such as ITAM-mutated CD3ζ, CD3η, or CD3θ. In some embodiments of any aspect, the ITAM comprises the three motifs (ITAM3) of the CD3ζ ITAM. In some embodiments of any aspect, the three motifs of the CD3ζ ITAM are not mutated and therefore comprise natural or wild-type sequences. In some embodiments, the CD3ζ sequence comprises the CD3ζ sequence shown in the sequences provided herein, such as the CD3ζ sequence of SEQ ID NO: 33, or a variant thereof.

[0103] For example, the CAR peptide described herein includes an intracellular signal transduction domain of CD3ζ. In some embodiments, the CD3ζ intracellular signal transduction domain corresponds to the amino acid sequence of SEQ ID NO:33; or includes a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity with the sequence of SEQ ID NO:33.

[0104] In some embodiments, the intracellular domain is the intracellular domain of 4-1BB. In some embodiments, the 4-1BB intracellular domain corresponds to an amino acid sequence selected from SEQ ID NO:46; or includes a sequence selected from SEQ ID NO:46; or includes a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identical to a sequence selected from SEQ ID NO:46. The individual CARs and other construct components described herein may be used together and repeatedly interchanged in the various constructs described herein, as can be determined by those skilled in the art. Each of these components may include any corresponding sequence listed herein or a variant thereof, or may consist of any corresponding sequence listed herein or a variant thereof.

[0105] More detailed descriptions of CAR 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., CancerRes 69:4559-4562, 2009; and Tamada et al., Clin. Cancer Res. 18:6436-6445, 2012; each of which is incorporated herein by reference in its entirety.

[0106] signal peptide

[0107] In some embodiments, the CAR polypeptide described herein includes a signal peptide. The signal peptide can be derived from any protein having an extracellular domain or being secreted. The CAR polypeptide described herein can include any signal peptide known in the art. In some embodiments, the CAR polypeptide includes a CD8 signal peptide, such as a CD8 signal peptide corresponding to the amino acid sequence of SEQ ID NO:47, or a CD8 signal peptide comprising 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 with the sequence of SEQ ID NO:47.

[0108] In a further embodiment, the CAR peptide described herein may optionally exclude one of the signal peptides described herein, such as the CD8 signal peptide of SEQ ID NO:47 or the IgK signal peptide of SEQ ID NO:47.

[0109] connector

[0110] In some embodiments, the CAR further includes a linker domain. As used herein, a "linker" refers to an oligopeptide or polypeptide region of about 2 to 100 amino acids in length that links together any domains / regions of the CAR described herein. In some embodiments, the linker may include or consist of flexible residues such as glycine and serine, allowing adjacent protein domains to move freely relative to each other. The linker sequence may be 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 includes any suitable linker known in the art. For example, linker sequences may include, but are not limited to, glycine / serine linkers, such as SEQ ID NO:36-38 and 40 described in Whitlow et al., Protein Eng. 6(8):989-95, 1993; the linker sequence SEQ ID NO:39 described in Andris-Widhopf et al., Cold Spring Harb. Protoc. 2011(9), 2011; and linker sequences with additional functions, such as epitope tags or coding sequences containing Cre-Lox recombination sites, as described in Sblattero et al., Nat. Biotechnol. 18(1):75-80, 2000. Longer linkers can be used when it is desirable to ensure that two adjacent domains do not spatially interfere with each other. Linkers may be cleavable or non-cleavable.

[0111] Furthermore, the linker can 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 their functional equivalents and combinations thereof. In various instances, linkers having the sequences described herein or variants thereof are used. It should be understood that indicating a particular linker at a specific location in a construct does not mean that the linker can only be used at that location. Rather, different linker sequences (e.g., P2A and T2A) can be interchanged with each other (e.g., in the context of constructs disclosed herein), as can be determined by those skilled in the art. In some embodiments, the linker region is T2A derived from the Thoseea asigna virus. Non-limiting examples of linkers that can be used in this technology include T2A, P2A, E2A, BmCPV2A, and BmlFV2A. Such linkers can be used in multi-protein systems, such as those described below. For example, they can be used to separate a multi-protein CAR component from a multi-protein therapeutic component (e.g., an antibody, such as scFv, a single-domain antibody (e.g., a camelid antibody), or a bispecific antibody (e.g., a TEAM)) (see below). In some embodiments, the P2A linker sequence comprises the amino acid sequence of SEQ ID NO:48. In some embodiments, the T2A linker sequence comprises the amino acid sequence of SEQ ID NO:49.

[0112] T-cell adaptor molecules (TEAM)

[0113] In some implementations, as described herein, the therapeutic agent delivered by CAR-T cells is a T-cell adaptor molecule (TEAM) (also referred to in the literature as a bispecific T-cell adaptor or BiTEs). TM The terms “T-cell adaptor molecule,” “TEAM antibody construct,” or “TEAM” refer to polypeptides, each comprising tandemly linked single-chain variable fragments (scFvs). Optionally, the scFvs are linked by linkers (such as glycine-rich linkers). One scFv of the TEAM binds to a T-cell receptor (TCR) (e.g., the CD3ε subunit), and the other binds to a target antigen (such as an antigen expressed by cancer-associated fibroblasts (CAF)). Such molecules can target T cells by binding to both the T-cell antigen (e.g., by binding to CD3) and the target antigen (such as the CAF antigen). Exemplary CAF antigens include fibroblast activation protein (FAP, see below). TEAMs can be used to enhance T-cell responses, such as in the tumor microenvironment. The two components of the TEAM may optionally be separated from each other by linkers (e.g., glycine-based linkers) as described herein, and may also be linked in either direction, such as the anti-CD3 component at the N-terminus of the anti-target antigen component, or vice versa. The anti-CD3 component or the anti-target antigen component of the TEAM may comprise any antibody reagent described herein.

[0114] CAR-T cell-secreted TEAMs can enhance the anticancer efficacy of CAR-T cell immunotherapy, for example, by stimulating the CAR-T cells themselves or by acting paracrinely through redirection of nonspecific bystander T cells to the tumor or CAF. CAR-T cell-mediated TEAM secretion can reduce the risk of adverse TEAM activity in systemic tissues by directing TEAM secretion to the tumor microenvironment. Exemplary TEAM constructs (e.g., anti-FAP TEAMs) are provided below; however, TEAMs other than those described herein may also be used in the CAR-T cells and methods of this disclosure.

[0115] An exemplary TEAM is an anti-FAP TEAM, comprising anti-FAP scFv and anti-CD3 scFv (also referred to herein as TEAM-FAP). The anti-FAP scFv may be arranged in a VH-VL or VL-VH orientation. Cancer-associated adenocarcinoma (CAF) cells are cancer-associated structural cells within the tumor microenvironment (TME). CAFs deposit collagen in the TME, forming the extracellular matrix (ECM), which creates a physical barrier preventing the infiltration of drugs and immune cells into the tumor site, while also providing a scaffold supporting cancer cell survival and migration. Furthermore, CAFs secrete growth factors, cytokines, and chemokines that support immunosuppression and tumor cell proliferation. CAF cells express the antigen FAP, and moderate to high FAP expression on CAFs is associated with poorer clinical outcomes in pancreatic cancer. To mitigate the role of CAFs in cancer, this disclosure provides a TEAM comprising a FAP-specific binding domain and a CD3-specific binding domain, which acts as a scaffold to aggregate effector T cells with CAFs to promote T cell-mediated cytotoxicity against CAF cells.

[0116] In some embodiments, the anti-CD3 scFv of any TEAM described herein may be arranged in a VH-VL or VL-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 higher sequence identity with the amino acid sequence of SEQ ID NO:25. In some embodiments, the anti-CD3 VL 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 higher sequence identity with the amino acid sequence of SEQ ID NO:26.

[0117] In some implementations, TEAM includes an antibody reagent that binds to fibroblast activation protein (FAP).

[0118] In some embodiments, the anti-FAP antibody reagent is sibrotuzumab. In some embodiments, the anti-FAP antibody reagent comprises the variable heavy chain (VH) of SEQ ID NO:17 or comprises a VH sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with the sequence of SEQ ID NO:17. In some embodiments, the anti-FAP antibody reagent comprises the variable light chain (VL) of SEQ ID NO:18 or comprises a VL sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with the sequence of SEQ ID NO:18. VH may be located at the N-terminus of VL, or VL may be located at the N-terminus of VH. In some embodiments, the anti-FAP antibody reagent comprises SEQ ID NO:43 or comprises a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with SEQ ID NO:43.

[0119] 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.

[0120] CAR-TEAM building blocks

[0121] In some embodiments, the CAR and TEAM encode the same polypeptide. In some embodiments, the CAR and TEAM encoding the same polypeptide are separated by a linker domain (e.g., 2A peptide) as described above. In some embodiments, the linker domain is cleavable. In some embodiments, the CAR is an anti-mesothelin CAR, and the TEAM is an FAP and CD3 TEAM. In some embodiments, the CAR is an anti-mesothelin CAR, and the TEAM comprises anti-FAP scFv and anti-CD3 scFv. In some embodiments, the CAR and TEAM polypeptides comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity with SEQ ID NO:44, SEQ ID NO:45, or SEQ ID NO:51. In some embodiments, the CAR and TEAM polypeptides comprise the amino acid sequence of SEQ ID NO:44. In some embodiments, the CAR and TEAM polypeptides consist of the amino acid sequence of SEQ ID NO:45. In some embodiments, the CAR and TEAM peptides comprise the amino acid sequence of SEQ ID NO:51.

[0122] In some implementations, the TEAM enhances the binding of immune cells to cancer compared to the absence of a TEAM. In some implementations, the TEAM enhances the binding of immune cells to cancer compared to the absence of a TEAM and a CAR that binds to antigens on immune cells. In some implementations, the TEAM increases the binding of immune cells to cancer by at least 10% (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, or at least 250%). In some embodiments, TEAM increases the binding of immune cells to cancer by 10%-20%, 10%-30%, 10%-50%, 10%-100%, 10%-150%, 10%-200%, 10%-250%, 50%-100%, 50%-150%, 50%-200%, 50%-250%, 100%-150%, 100%-200%, 100%-250%, 20%-30%, 20%-40%, 20%-50%, 20%-60%, 20%-70%, 30%-40%, 30%-50%, 30%-60%, or 30%-70%. In some embodiments, TEAM increases the binding of immune cells to cancer by 20%-40%. In some embodiments, TEAM increases the binding of immune cells to cancer by 100%-250%. In some embodiments, the immune cells are T cells. In some implementations, the immune cells are CAR-T cells. In some implementations, the TEAM is a FAP TEAM.

[0123] Inhibitors

[0124] This article describes methods for combining CAR-T cells with integrin and metalloproteinase 17 (ADAM17) inhibitors or interleukin-2-induced T-cell kinase (ITK) inhibitors. Using these inhibitors with CAR-T cell therapy can enhance the efficacy of CAR-T cells against solid tumors. Inhibitors can be protein-based (e.g., antibodies or antigen-binding domains that inhibit the target), small molecules, RNA-based inhibitors, or any other form of inhibitor, depending on the target.

[0125] In some embodiments, to promote stable expression of the target antigen mesothelin in CAR-T cells provided herein, an ADAM17 inhibitor is administered in combination with meso-FAP CAR TEAM cells. In some embodiments, the ADAM17 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 ADAM17 inhibitor is aderbasib. Aderbasib is also known as INCB7839.

[0126] Other inhibitors provided in this disclosure, in some embodiments, are interleukin-2-induced T-cell kinase (ITK) inhibitors to promote T-cell polarization into a more effective cancer-killing phenotype. In some embodiments, the ITK inhibitor is administered in combination with CAR TEAM cell therapy. In some embodiments, the ITK inhibitor is ibrutinib, CPI-818, BMS509774, or PRN694. In some embodiments, the ITK inhibitor is ibrutinib.

[0127] In some embodiments, the ADAM17 inhibitor is administered simultaneously with the CAR TEAM cells. In some embodiments, the ADAM17 inhibitor is administered shortly after the CAR TEAM cells. In some embodiments, the ADAM17 inhibitor is administered via the same route of administration as the CAR TEAM cells. In some embodiments, the ADAM17 inhibitor is administered via intraperitoneal injection. In some embodiments, the ADAM17 inhibitor is administered orally.

[0128] In some embodiments, the ITK inhibitor is administered simultaneously with the CAR TEAM cells. In some embodiments, the ITK inhibitor is administered shortly after the CAR TEAM cells. In some embodiments, the ITK inhibitor is administered via 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.

[0129] Polynucleotides, plasmids and vectors

[0130] In some respects, this disclosure describes polynucleotides encoding any of the CAR polypeptides described herein. The term “polynucleotide” is used interchangeably herein with “nucleic acid molecule” and refers to a polymer of nucleosides. Typically, polynucleotides consist of nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine) naturally occurring in DNA or RNA linked by phosphodiester bonds. However, the term encompasses molecules containing nucleosides or nucleoside analogs with chemically or biologically modified bases, modified backbones, etc. (whether or not found in naturally occurring nucleic acids), and such molecules may be preferred in certain applications. When this application refers to polynucleotides, it should be understood that both DNA and RNA are provided, and in each case, both single-stranded and double-stranded forms (and complementary strands of each single-stranded molecule). As used herein, “polynucleotide sequence” can refer to the polynucleotide material itself and / or the sequence information (i.e., the letter sequence used as a base abbreviation) that biochemically characterizes a particular nucleic acid. In some embodiments, the nucleic acid molecule is a heterologous nucleic acid molecule. As used herein, the term "heterologous nucleic acid molecule" refers to a nucleic acid molecule that is not naturally present in a particular cell or a nucleic acid sequence that has been engineered into a cell. For example, a heterologous nucleic acid molecule can be a nucleic acid molecule that encodes a gene that has been engineered into a cell (e.g., via plasmids, vectors, or some other method). Unless otherwise stated, the polynucleotide sequences shown herein are displayed in the 5' to 3' orientation.

[0131] 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 and second nucleic acid sequences are operatively linked to different promoters. In some embodiments, the first and second nucleic acid sequences are operatively linked to the same promoter. In some embodiments, the polynucleotide further comprises a nucleic acid sequence encoding a self-cleaving peptide, said self-cleaving peptide being encoded between the first nucleic acid sequence encoding the CAR and the second nucleic acid sequence encoding the TEAM.

[0132] 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 with any of SEQ ID NO:44-45. In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding an amino acid sequence of any of SEQ ID NO:44-45.

[0133] In some implementations, the nucleic acid sequence is operatively linked to a promoter. As used herein, the term "operatively linked" refers to the linkage of a first polynucleotide molecule (such as a promoter) to a second transcribed polynucleotide molecule (such as a CAR, BCL-2 family protein, or CAR polypeptide), wherein the arrangement of the polynucleotide molecules allows the promoter to direct RNA polymerase to transcribe the second polynucleotide molecule. The two polynucleotide molecules may or may not be part of a single, consecutive polynucleotide molecule, and may or may not be adjacent. For example, if the promoter regulates or mediates the transcription of a target gene in a cell, then the promoter is operatively linked to the target gene.

[0134] In some embodiments, the promoter is a constitutively active promoter. In some embodiments, the U6 promoter is derived from a non-human species. In some embodiments, the promoter is selected from the group consisting of the CMV promoter, EF1a promoter, EF1a-short promoter, CAG promoter, PGK promoter, H1 promoter, or U6 promoter. In some embodiments, the U6 promoter is derived from the human U6 promoter. In some embodiments, the U6 promoter is derived from cattle, mice, rats, pigs, yeast, dogs, cats, fruit flies, or Caenorhabditis elegans. In some embodiments, the promoter is the H1 promoter. In some embodiments, the promoter is a tissue-specific promoter (e.g., HP1, CD14, CD43, CD45, C68, elastase, endothelial glycoprotein, fibronectin, Flt, GFAP, GPIIb, ICAM-2, mIFN-β, Mb, NphsI, OG-2, SP-B, SYN1, or WASP gene promoters). In some implementations, the promoter is an inducible promoter (e.g., a tet or lac enzyme promoter).

[0135] In some embodiments, the nucleic acid encoding the polypeptide described herein (e.g., a CAR polypeptide) is contained in a plasmid. The term "plasmid" can refer to a circular DNA fragment containing an origin of replication. In some embodiments, the plasmid contains a prokaryotic origin of replication. In some embodiments, the plasmid contains a bacterial origin of replication. In some embodiments, the plasmid contains a eukaryotic origin of replication. In some embodiments, the plasmid contains a mammalian origin of replication. In some embodiments, the plasmid contains both prokaryotic and eukaryotic origins of replication. In some embodiments, the plasmid contains an active origin of replication in the cell in which the plasmid is located. In some embodiments, the plasmid is a lentiviral plasmid (e.g., a second-generation lentiviral plasmid).

[0136] In some embodiments, the nucleic acid encoding the polypeptide described herein (e.g., a CAR polypeptide) is contained in a vector. As used herein, the term "vector" refers to a nucleic acid construct designed for delivery to a host cell or transfer between different host cells. As used herein, a vector may be viral or non-viral. The term "vector" encompasses any genetic element that, when bound to a suitable control element, is capable of replicating and transferring a gene sequence into a cell. Vectors may include, but are not limited to, cloning vectors, expression vectors, plasmids, bacteriophages, transposons, granules, artificial chromosomes, viruses, viral particles, etc.

[0137] As used herein, the term "expression vector" can refer to a vector that enables the direct expression of RNA or polypeptide from a sequence linked to a transcriptional regulatory sequence. The expressed sequence is usually (but not necessarily) heterologous to the cell. Expression vectors may contain other elements, such as having two replication systems, allowing them to be maintained in two organisms, for example, expressed in human cells and cloned and amplified in a prokaryotic host. The term "expression" refers to the cellular processes involved in the production of RNA and proteins, and, where applicable, the secretion of proteins, including, but not limited to, transcription, transcript processing, translation, and protein folding, modification, and processing. "Expression products" include RNA transcribed from a gene, as well as polypeptides obtained through translation of mRNA transcribed from a gene.

[0138] As used herein, the term "viral vector" can refer to a nucleic acid vector construct containing at least one viral source element and having the ability to be packaged into viral vector particles. A viral vector may contain a nucleic acid encoding a polypeptide described herein instead of a non-essential viral gene. The vector and / or particles can be used for the purpose of transferring nucleic acids into cells in vitro or in vivo. Various forms of viral vectors are known in the art. In some embodiments, the viral vector is an adeno-associated virus, adenovirus, lentivirus, or retroviral vector. In some embodiments, the lentiviral vector is a second-generation lentiviral vector.

[0139] A “recombinant vector” can be a vector containing a heterologous nucleic acid sequence or a “transgenic” capable of being expressed in vivo. It should be understood that the vectors described herein can be combined with other suitable compositions and therapies in some embodiments. In some embodiments, the vector is additive. Using a suitable additive vector provides a means of maintaining the target nucleotide in high copy numbers of extrachromosomal DNA in a subject, thereby eliminating the potential impact of chromosomal integration.

[0140] In some embodiments, the peptides, polynucleotides, plasmids, and / or vectors described herein optionally further comprise a reporter molecule, for example, to determine whether the vector is correctly expressed in cells. In some embodiments, the reporter molecule may be a fluorescent protein (e.g., GFP, YFP, RF, mCherry), an antibody (e.g., CD34, tEGFR, tCD19, tCD20, tCD34, and tHer2), or a radioisotope. In some embodiments, the reporter molecule is a hygromycin phosphotransferase (hph) capable of imaging alone or in combination with a substrate or chemical substance (e.g., 9-[4-[18F]fluoro-3-(hydroxymethyl)butyl]guanine ([18F]FHBG)).

[0141] In some embodiments, GFP and mCherry can be used as fluorescent tags for imaging CARs expressed on T cells (e.g., CAR-T cells). It is anticipated that virtually any fluorescent protein known in the art can be used for this purpose. For clinical applications, CARs do not need to contain fluorescent tags or fluorescent proteins. Therefore, any markers present in the construct provided herein are removable in each specific construct. This invention includes constructs with or without tags. Therefore, when a specific construct is referred to herein, it can be considered that it is included within the scope of this invention, whether it carries or does not carry any tags or labels (including, for example, histidine tags, such as the histidine tag HHHHHH (SEQ ID NO: 47)).

[0142] cell

[0143] One aspect of the technology described herein relates to mammalian cells (e.g., immune cells) comprising any meso-FAP CAR TEAM construct described herein. The mammalian cells may be derived from humans, primates, hamsters, rabbits, rodents, cattle, pigs, sheep, horses, goats, dogs, or cats, but any other mammalian cells may also be used. In a preferred embodiment in any aspect, the mammalian cell is a human cell.

[0144] In some embodiments of any aspect, the mammalian cells are immune cells. As used herein, "immune cell" refers to a cell that plays a role in an immune response. Immune cells originate from the hematopoietic system 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 cells are T cells.

[0145] In some embodiments, the immune cells are obtained from an individual who has already had or has been diagnosed with cancer, plasma cell disease, or an autoimmune disease. In some embodiments, the immune cells are allogeneic to the subject. In some embodiments, the immune cells are generated from stem cells (e.g., induced pluripotent stem cells or embryonic stem cells).

[0146] In some implementations, mammalian cells (such as T cells) can be engineered to include any of the meso-FAP CAR TEAM constructs described herein. T cells can be obtained from a subject using standard techniques known in the art. For example, T cells can be isolated from peripheral blood of a donor or patient. T cells can be isolated from mammals. Preferably, T cells are isolated from humans.

[0147] In some aspects, this disclosure describes CAR-T cells comprising any meso-FAP CAR TEAM construct disclosed herein. In some embodiments, the CAR-T cells are generated from T cells extracted from a subject (e.g., a subject to be administered CAR-T cells). In some embodiments, the CAR-T cells are allogeneic CAR-T cells. In some embodiments, the CAR-T cells comprise a meso-FAP CAR TEAM construct containing an anti-mesothelin CAR. In some embodiments, the CAR-T cells comprise a meso-FAP CAR TEAM construct containing an anti-mesothelin CAR and a fibroblast activation protein (FAP) antigen-binding domain. In some embodiments, the CAR-T cells comprise a meso-FAP CAR TEAM construct as described herein. In some embodiments, the CAR-T cells comprise a construct encoding any one of SEQ ID NO:44-45.

[0148] Treatment

[0149] In some aspects, this disclosure describes a method for treating a subject with mesothelin-expressing cancer (as described herein), the method comprising administering to the subject cells (e.g., CAR-T cells) expressing a CAR (as described herein) and a T-cell adaptor antibody molecule (TEAM, as described herein). In some embodiments, the method comprises administering CAR-T cells (e.g., CAR-T cells expressing a CAR peptide). In some embodiments, the method comprises administering T cells engineered to express both a CAR and a TEAM. In some embodiments, the method comprises administering CAR-T cells comprising a meso-FAP CAR TEAM construct containing an anti-mesothelin CAR. In some embodiments, the method comprises administering CAR-T cells comprising a meso-FAP CAR TEAM construct containing an anti-mesothelin CAR and a fibroblast activation protein (FAP) binding domain. In some embodiments, the method comprises administering CAR-T cells comprising a meso-FAP CAR TEAM construct containing an anti-mesothelin CAR and a TEAM containing a fibroblast activation protein (FAP) binding domain and a CD3 binding domain. In some embodiments, the method includes administering CAR-T cells comprising a construct encoding any one of SEQ ID NO:44-45. In some embodiments, the method includes administering CAR-T cells in combination with an ADAM17 inhibitor or an ITK inhibitor.

[0150] cancer

[0151] In some embodiments, the methods described herein include treating a subject with mesothelin-expressing cancer. As used herein, “cancer” can refer to the excessive proliferation of cells, characterized by a loss of normal cellular control, resulting in uncontrolled growth, lack of differentiation, localized tissue invasion, and metastasis. Exemplary cancers include, but are not limited to, liquid cancers (also known as liquid tumors) and solid tumors. As used herein, “liquid cancer” or “liquid tumor” can refer to leukemia, lymphoma, and myeloma cancers. Non-limiting examples of leukemia include acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), and chronic lymphocytic leukemia (CLL). In some embodiments, the cancer is ALL or CLL. Non-limiting examples of lymphomas include diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), marginal zone lymphoma, Burkitt lymphoma, hairy cell leukemia (HCL), and T-cell lymphomas (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.

[0152] Non-limiting examples of solid tumors include adrenocortical tumors, alveolar soft tissue sarcomas, carcinomas, chondrosarcomas, colorectal cancer, desmoidomas, desmoplastic small round cell tumors, endocrine tumors, endodermal sinus tumors, epithelioid hemangioendotheliomas, Ewing sarcomas, glioblastomas, prostate cancer, gliomas, lung cancer, pancreatic cancer, germ cell tumors (solid tumors), bone and soft tissue giant cell tumors, hepatoblastomas, hepatocellular carcinomas, melanomas, nephroblastomas, neuroblastomas, non-rhabdomyosarcoma soft tissue sarcomas (NRSTS), osteosarcomas, paravertebral sarcomas, renal cell carcinomas, retinoblastomas, rhabdomyosarcomas, synovial sarcomas, and nephroblastomas. In some embodiments, the cancer expresses mesothelin (mesothelin-expressing cancers). In some embodiments, the cancer expresses mesothelin or a portion thereof that is at least 85% sequence identical to the amino acid sequence of SEQ ID NO:50. In some embodiments, the cancer expresses mesothelin or a portion thereof having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:50. In some embodiments, the cancer expresses mesothelin or a portion thereof having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:50. In some embodiments, the cancer expresses mesothelin or a portion thereof having at least 97% sequence identity with the amino acid sequence of SEQ ID NO:50. In some embodiments, the cancer expresses mesothelin or a portion thereof having at least 98% sequence identity with the amino acid sequence of SEQ ID NO:50. In some embodiments, the cancer expresses mesothelin or a portion thereof having at least 99% sequence identity with the amino acid sequence of SEQ ID NO:50. In some embodiments, the cancer expresses mesothelin or has a portion thereof of 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 exist in bones, muscles, or organs, and can be sarcomas or carcinomas. As used in this article, the term "tumor" refers to the abnormal growth of cells or tissues (such as malignant or benign types).

[0153] Subjects

[0154] In some embodiments, the methods described herein include treating a subject with cancer. As used herein, "subject" refers to a person or animal. Typically, an animal is a vertebrate, such as a primate, rodent, domesticated animal, or hunting animal. Primates include, for example, chimpanzees, cynomolgus monkeys, spider monkeys, and macaques (e.g., rhesus monkeys). Rodents include, for example, mice, rats, marmots, ferrets, rabbits, and hamsters. Domesticated and hunting animals include, for example, cattle, horses, pigs, deer, bison, buffalo, felines (e.g., domestic cats), canines (e.g., dogs), foxes, wolves, birds (e.g., chickens, emus, ostriches), and fish (e.g., trout, catfish, and salmon). In some embodiments, the subject is a mammal, such as a primate, such as a human. The terms "individual," "patient," and "subject" are used interchangeably herein. Preferably, the subject is a mammal. Mammals can be humans, non-human primates, mice, rats, dogs, cats, horses, or cattle, but are not limited to these examples. Non-human mammals can be advantageously used as subjects representing animal models of diseases (e.g., cancer). Subjects can be male or female.

[0155] Subjects may be subjects who have been previously diagnosed or identified as suffering from or having a condition requiring treatment (e.g., diagnosed with mesothelin-expressing cancer) or one or more complications associated with that condition, and optionally have received treatment for that condition or one or more complications associated with that condition.

[0156] Alternatively, the subject may be a subject who has not previously been diagnosed with such a condition or related complications. For example, a subject may be a subject who exhibits one or more risk factors for the condition or one or more complications associated with the condition, or a subject who does not exhibit any risk factors.

[0157] Subjects who are “in need” for treatment of a specific condition can be those who have the condition, have been diagnosed with the condition, or are at risk of developing the condition.

[0158] Pharmaceutical Composition

[0159] In some embodiments, the methods described herein include administering to a subject a pharmaceutical composition comprising CAR-T cells and / or a pharmaceutical composition comprising an ADAM17 inhibitor and / or an ITK inhibitor. As used herein, the term "pharmaceutical composition" refers to a combination of an active agent (e.g., the CAR-T cells described herein, such as CAR-T cells expressing a CAR peptide) and a pharmaceutically acceptable carrier (e.g., a carrier commonly used in the pharmaceutical industry).

[0160] The phrase "pharmaceutically acceptable carrier" is used herein to refer to compounds, materials, compositions, and / or dosage forms that, within reasonable medical judgment, are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, and in proportion to a reasonable benefit / risk ratio. In some embodiments of any aspect, a pharmaceutically acceptable carrier may be a carrier other than water. In some embodiments of any aspect, a pharmaceutically acceptable carrier may be an artificial or engineered carrier, such as a carrier in which the active ingredient is not found in nature.

[0161] In one aspect of this technology, the technology described herein relates to pharmaceutical compositions comprising activated CAR-T cells containing the CAR peptide described herein and optionally a pharmaceutically acceptable carrier. The active ingredient of the pharmaceutical composition comprises at least activated CAR-T cells containing the CAR peptide described herein. In some embodiments, the active ingredient of the pharmaceutical composition consists substantially of activated CAR-T cells containing the CAR peptide described herein. In some embodiments, the active ingredient of the pharmaceutical composition consists of activated CAR-T cells containing the CAR peptide described herein. Pharmaceutically acceptable carriers for cell-based therapeutic formulations include physiological saline and aqueous buffer solutions, Ringer's solution, and serum components such as serum albumin, HDL, and LDL. Terms such as "excipient," "carrier," "pharmaceutically acceptable carrier," and "pharmaceuticalally acceptable excipient" are used interchangeably herein.

[0162] In some embodiments, the pharmaceutical composition comprising activated CAR-T cells containing the CAR peptides described herein may be a parenteral dosage form. Since administration of parenteral dosage forms typically bypasses a patient's natural defenses against contaminants, the components other than the CAR-T cells themselves are preferably sterile or capable of being sterilized prior to administration to the patient. Examples of parenteral dosage forms include, but are not limited to, ready-to-use injections, dry preparations prepared to be dissolved or suspended in a pharmaceutically acceptable injectable medium, ready-to-use injectable suspensions, and emulsions. Any of these may be incorporated into the activated CAR-T cell formulation prior to administration. Suitable media for providing parenteral dosage forms of the activated CAR-T cells disclosed herein are well known to those skilled in the art. Examples include, but are not limited to: physiological saline solutions; glucose solutions; aqueous media including, but not limited to, sodium chloride injection, Ringer's solution, glucose injection, glucose and sodium chloride injection, and lactated Ringer's solution; water-miscible media such as, but not limited to, ethanol, polyethylene glycol, and propylene glycol; and non-aqueous media such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

[0163] dose

[0164] In some embodiments, activated CAR-T cells comprising the CAR peptide described herein are administered as a monotherapy, i.e., without concurrent administration of another treatment for the condition to the subject. Pharmaceutical compositions comprising the T cells described herein are typically available in 10... 4 Up to 10 9 The dosage is administered at cells / kg body weight, and in some cases, the dosage is 10. 5 Up to 10 6 Cells per kg body weight, including all integer values ​​within these ranges. If necessary, the T-cell composition may also be administered multiple times at these doses. Cells can be administered using infusion techniques commonly used in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. Med. 30 319:1676, 1988).

[0165] In some cases, it may be necessary to administer activated CAR-T cells containing CAR peptides to the subject, followed by a re-drawing of blood (or apheresis) to activate the T cells as described herein, and then reinfusing these activated and expanded T cells back into the patient. This process can be performed multiple times every few weeks. In some cases, T cells can be activated from blood drawn from 10 cc to 400 cc. In some cases, T cells can be activated from blood drawn from 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc, or 100 cc.

[0166] application

[0167] In some embodiments, the methods described herein relate to treating a subject with mesothelin-expressing cancer (e.g., as described herein), and include administering CAR-T cells to the subject in combination with an ADAM17 inhibitor or an ITK inhibitor, wherein the CAR-T cells comprise a mesothelin-binding CAR and a T-cell adaptor antibody molecule comprising an FAP-binding domain. CAR-T cells comprising the CAR peptides described herein comprise mammalian cells containing any CAR peptide described herein or any CAR peptide known in the art, or nucleic acids encoding any CAR peptide described herein.

[0168] In some embodiments, the methods described herein include administering an effective amount of activated CAR-T cells comprising the CAR peptide described herein to treat a subject with mesothelin-expressing cancer. As used herein, “treating a subject with mesothelin-expressing cancer” means improving any condition or symptom associated with mesothelin-expressing cancer. This reduction is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more, as measured by any standard technique, compared to an equivalent untreated control. Various methods of administering the compositions described herein to a subject are known to those skilled 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 tumor site.

[0169] As used herein, the term "effective amount" refers to the amount of activated CAR-T cells containing the CAR peptide described herein, which is a sufficient amount to treat at least one or more symptoms of mesothelin-expressing cancer and involves a cell preparation or composition that provides the desired effect. Therefore, the term "therapeutic effective amount" refers to the amount of activated CAR-T cells containing the CAR peptide described herein that is sufficient to provide a specific anti-condition effect when administered to a typical subject. As used herein, in various contexts, an effective amount also includes an amount sufficient to delay the development of mesothelin-expressing cancer symptoms, alter the course of mesothelin-expressing cancer (e.g., but not limited to slowing the progression of mesothelin-expressing cancer), or reverse the symptoms of mesothelin-expressing cancer. Therefore, an exact "effective amount" cannot usually be precisely specified. However, for any given situation, an appropriate "effective amount" can be determined by a person skilled in the art using only routine experiments.

[0170] Effective doses, toxicity, and therapeutic efficacy can be assessed using standard pharmaceutical procedures in cell culture or laboratory animals. Dosage can vary depending on the dosage form and route of administration. The dose ratio between toxicity and therapeutic effect is the therapeutic index and can be expressed as the LD50 / ED50 ratio. Compositions and methods exhibiting a large therapeutic index are preferred. The therapeutically effective dose can be estimated initially from cell culture assays. Furthermore, doses can be formulated in animal models to achieve a range of circulating plasma concentrations including the IC50 (i.e., the concentration of activated CAR cells containing the CAR peptide described herein that achieves half-maximal inhibition of symptoms), which can be determined in cell culture or a suitable animal model. Plasma levels can be measured, for example, by high-performance liquid chromatography. The effect of any particular dose can be monitored by appropriate bioassays, such as bone marrow assays. The dose can be determined by the physician and adjusted as needed to suit the observed therapeutic effect.

[0171] In some embodiments, the method of treating a subject with mesothelin-expressing cancer described herein includes administering meso-FAP CAR TEAM cells via intravenous administration. In some embodiments, the method includes administering meso-FAP CAR TEAM cells via intravenous administration in combination with an ADAM17 inhibitor. In some embodiments, the method includes administering meso-FAP CAR TEAM cells via intravenous administration in combination with an intraperitoneally administered ADAM17 inhibitor. In some embodiments, the method includes administering meso-FAP CAR TEAM cells via intravenous administration in combination with an orally administered ADAM17 inhibitor. In some embodiments, the method includes administering meso-FAP CAR TEAM cells via intravenous administration in combination with an ADAM17 inhibitor.

[0172] In some embodiments, the method includes administering meso-FAPCAR TEAM cells intravenously in combination with an ITK inhibitor. In some embodiments, the method includes administering meso-FAP CAR TEAM cells intravenously in combination with an intraperitoneally administered ITK inhibitor. In some embodiments, the method includes administering meso-FAP CAR TEAM cells intravenously in combination with an orally administered ITK inhibitor. In some embodiments, the method includes administering meso-FAP CAR TEAM cells and an ITK inhibitor in combination via intravenous administration.

[0173] Application method

[0174] Administration methods (e.g., administration of CAR-T cells containing CAR peptides, ADAM17 inhibitors, or ITK inhibitors) may include, for example, intravenous (iv) injection or infusion. The compositions described herein can be administered to patients intra-arterial, intratumoral, intranodal, intraperitoneal, intrathecal, intramedullary, or orally. In some embodiments, the CAR-T cell composition may be injected directly into a tumor, lymph node, or site of infection. In some embodiments, the compositions described herein are administered into a body cavity or fluid (e.g., ascites, pleural fluid, peritoneal fluid, or cerebrospinal fluid).

[0175] In some implementations, the subject may undergo leukocyte ablation, in which leukocytes are collected, enriched, or depleted in vitro to select and / or isolate cells of interest, such as T cells. These T cell isolates may be amplified by contact with artificial APCs (aAPCs), such as aAPCs expressing anti-CD28 CDR and anti-CD3 CDR, and treated to allow the introduction of one or more CAR constructs into the present technology, thereby generating CAR-T cells.

[0176] Subjects in need may then receive standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. After or concurrently with transplantation, subjects may receive an infusion of expanded CAR-T cells. In some embodiments, the expanded cells are administered before or after the procedure. In some embodiments, lymphatic depletion is performed on the subject prior to administration of one or more of the CAR-T cells described herein. In such embodiments, lymphatic depletion may include administration of one or more of melphalan, survivin, cyclophosphamide, and fludarabine. The dosage of the above treatments administered to a patient will vary depending on the exact nature of the condition being treated and the recipient. Adjustments to the dosage for human administration may be made according to accepted practice in the art.

[0177] In some implementations, a single treatment regimen is required. In other implementations, one or more subsequent doses or treatment regimens may be administered. For example, after three months of treatment every two weeks, treatment may be repeated monthly for six months, a year, or longer. In some implementations, no further treatment is administered after the initial treatment.

[0178] The dosage of CAR-T cells described in this article can be determined by the physician and adjusted as needed to suit the observed therapeutic effect. Regarding the duration and frequency of treatment, skilled clinicians typically monitor subjects to determine when treatment provides a therapeutic benefit and to determine whether to administer more cells, discontinue treatment, resume treatment, or make other changes to the treatment regimen. The dosage should not be too high to cause adverse side effects, such as cytokine release syndrome. Generally, the dosage will vary depending on the patient's age, condition, and sex, and can be determined by someone skilled in the art. In the event of any complications, the dosage may also be adjusted by the individual physician.

[0179] effect

[0180] The efficacy of activated CAR-T cells containing the CAR peptides described herein in, for example, treating mesothelin-expressing cancers or inducing the responses described herein (e.g., reducing cancer cells) can be determined by a skilled clinician. However, treatment is considered “effective” (as used herein) if one or more signs or symptoms of the condition described herein change in a beneficial manner, other clinically accepted symptoms improve or even lessen, or a desired response is induced (e.g., at least 10% improvement after treatment according to the methods described herein). Efficacy can be assessed, for example, by measuring biomarkers, indicators, symptoms, and / or incidence of mesothelin-expressing cancers treated according to the methods described herein, or any other appropriate measurable parameter.

[0181] Treatment according to the methods described herein can reduce the levels of markers or symptoms of mesothelin-expressing cancers 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.

[0182] Efficacy can also be measured by assessing whether an individual's condition has not worsened (e.g., whether hospitalization or medical intervention is required) (i.e., whether disease progression has stopped). Methods for measuring these indicators are known to those skilled in the art and / or described herein. Treatment includes any treatment of mesothelin-expressing cancer in an individual or animal (some non-limiting examples include humans or animals) and includes: (1) inhibiting mesothelin-expressing cancer, e.g., preventing the worsening of symptoms (e.g., pain or inflammation); or (2) reducing the severity of mesothelin-expressing cancer, e.g., causing symptom resolution. An effective amount for treating mesothelin-expressing cancer means an amount sufficient, when administered to a subject in need, to result in an effective treatment as defined herein. The efficacy of an agent can be determined by assessing physical indicators or expected responses in mesothelin-expressing cancer. Those skilled in the art are fully capable of monitoring the efficacy of administration and / or treatment by measuring any one or any combination of such parameters. The efficacy of a particular method can be assessed in animal models of the condition described herein. When using experimental animal models, therapeutic efficacy is confirmed when a statistically significant change in a biomarker is observed.

[0183] sequence list

[0184]

[0185]

[0186]

[0187]

[0188]

[0189]

[0190] The terms “statistically significant” or “significantly” refer to statistical significance and typically indicate a difference of two standard deviations (2SO) or greater.

[0191] Unless otherwise indicated in the operational examples, all figures used herein representing amounts of components or reaction conditions should be understood to be modified by the term "about" in all cases.

[0192] When used with a numerical value, the term “about” or “approximately” may refer to the value or the statement that states the value and cover a range of ±1%, ±2%, ±3%, ±4%, ±5%, ±6%, ±7%, ±8%, ±9%, ±10%, ±1-5%, ±2-7%, ±3-8%, ±4-9%, or ±5-10%.

[0193] The singular forms “a,” “an,” and “the” include plural referents unless the context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. Although methods and materials similar to or equivalent to those described herein may be used in practice or testing of this disclosure, suitable methods and materials are described below. “As (abbreviation eg)” is derived from the Latin *exempli gratia* and is used herein to indicate a non-limiting example. Therefore, “as (abbreviation eg)” is synonymous with the term “for example.”

[0194] Other terms will be defined in the description of the various aspects and implementations of the technology described herein.

[0195] According to this disclosure, conventional molecular biology, microbiology, biochemistry, and recombinant DNA techniques in the art can be employed. Such techniques are well explained in the literature. Embodiments of this disclosure are further described in the following examples, which do not limit the scope of the methods and material compositions described in the claims.

[0196] Example

[0197] The tumor microenvironment (TME) is a heterogeneous population of cells, including stromal cells, endothelial cells, immune cells, tumor cells, cancer-associated fibroblasts (CAFs), and others. Figure 1 Cell type density and diversity can work together to promote tumor cell proliferation and survival, while CAF deposition in the TME forms extracellular matrix (ECM), which makes it difficult or impossible for immune cells and anticancer agents to penetrate the tumor and exert their cell-lysing effects. Here, the cell-lysing ability of cells containing a chimeric antigen receptor (CAR) targeting mesothelin (an antigen expressed in many types of cancer) against pancreatic or ovarian cancer cells, as well as CAF, was tested. These cells secreted a T-cell adaptor molecule (TEAM) targeting fibroblast activation protein (FAP) (meso-FAP CAR-TEAM cells, construct as shown in...). Figure 11 As shown in the figure, FAP is expressed on CAF. Furthermore, the efficacy of meso-FAP CAR-TEAM cells in combination with inhibitors that prevent mesothelin cleavage from the tumor cell surface (e.g., ADAM17 inhibitors) or inhibitors that promote T cell polarization to effector subtypes with enhanced anti-cancer capabilities (e.g., ITK inhibitors) was tested.

[0198] Example 1. Testing the efficacy of meso-FAP CAR-TEAM cells against pancreatic cancer cells.

[0199] in vivo

[0200] In these experiments, immunodeficient NSG ® Mice were subcutaneously injected with AsPC-1 tumor cells (mesothelin-expressing tumor cells derived from the human pancreatic ductal adenocarcinoma (PDAC) cell line) mixed with CAF cells at a 1:9 ratio, followed by intravenous (IV) injection of untransduced (UTD) T cells, meso-FAP CAR-TEAM cells, or meso-CD19 CAR-TEAM cells (the TEAM secreted by these cells is not TME-specific). Compared to mice receiving UTD or meso-CD19 CAR-TEAM cells, mice receiving meso-FAP CAR-TEAM cells showed significantly smaller tumor volume. Figure 2A -B).

[0201] in vitro

[0202] Since human CAF cells do not expand in mice, the efficacy of meso-FAP CAR-TEAM cells in the production of organoids from patient-derived mesothelin-expressing tumors grown in the presence of CAF was tested in vitro. CAF was mixed with organoids to mimic the human TME. The mixed cultures were treated with UTD cells, meso-FAP CAR-TEAM cells, or meso-CD19 CAR-TEAM control cells. Compared to UTD cell-treated cultures, the organoid recovery rate in meso-FAP CAR-TEAM cell-treated organoid-CAF cultures was significantly reduced. Figure 2C , 2E (above), and compared with cultures treated with UTD cells or meso-CD19 CAR-TEAM cells, CAF cells were significantly reduced ( Figure 2D , 2E (below).

[0203] Example 2. Testing the cell lysis ability of meso-FAP CAR-TEAM cells against pancreatic cancer and ovarian cancer cell lines.

[0204] The pancreatic cancer cell lines AsPC-1 and CxPC-3, which express mesothelin, ADAM17, and ADAM10, were compared. Figures 4A-4B Cultures of the cells were treated with either UTD T cells or meso-FAP CAR-TEAM cells. Compared to UTD T cells, meso-FAP CAR-TEAM cells exhibited a significantly higher cell lysis effect on both cell lines. Figure 4DUsing SKOV3 ovarian cancer cells with low baseline mesothelin expression, we demonstrated the critical role of mesothelin expression in the cytolytic ability of meso-FAP CAR-TEAM cells. Transducing SKOV3 cells to increase mesothelin expression (…) Figure 4C This increased the cell lysis capacity of meso-FAP CAR-TEAM cells compared to untransduced SKOV3 cells or SKOV3 cells treated with UTD T cells. Figure 4E Therefore, meso-FAPCAR-TEAM cells exhibit robust killing effects against mesothelin-expressing cancer cells.

[0205] Example 3. Determining the optimal mesothelin antigen-binding domain for chimeric antigen receptor / T cell adaptor molecule (CAR-TEAM) cell targeting of intraperitoneal (IP) tumors.

[0206] Examples 1 and 2 demonstrate the efficacy of meso-FAP CAR-TEAM cells in a mesothelin-expressing cancer model. However, multiple mesothelin-binding moieties are available for meso-FAP CAR-TEAM cells. Here, the efficacy of two mesothelin-binding moieties is compared in vitro and in preclinical models.

[0207] in vitro

[0208] The efficacy of mesothelin-fibroblast activation protein-targeting (meso-FAP) CAR-TEAM cells in binding to mesothelin antigen was compared in vitro with either the SS1-binding domain (SS1 meso-FAP CAR TEAM cells) or the MGH1Meso1-binding domain (MGH1Meso1 meso-FAP CAR TEAM cells). Flow cytometry was used to detect the binding of FAP on cancer-associated fibroblast 1 (CAF-1) cells and CD3 on untransduced (UTD) T cells. Next, MGH1Meso1 or SS1 meso-FAP CAR TEAM cells were repeatedly stimulated weekly with pancreatic ductal adenocarcinoma (PDAC) cells and CAF cells for 4 weeks, and their long-term proliferation and exhaustion phenotypes were measured. Furthermore, PDAC cells or patient-derived xenograft (PDX) cells were co-cultured with CAF cells, and cytotoxicity of MGH1Meso1 and SS1 meso-FAP CAR TEAM cells was measured using real-time impedance-based assays.

[0209] in vivo

[0210] PDAC cell lines (AsPC-1 or BXPC-3 cells) or PDX cells (1291 cells) were mixed with CAF-1 cells at a 1:9 ratio in Matrigel and intraperitoneally (IP) injected into NOD.Cg-Prkdc scid 123rg tm1Wjl / SzJ (NSG®) mice. Tumor growth was monitored by bioluminescence imaging. Two weeks after tumor implantation, mice were treated with UTD T cells or MGH1Meso1 or SS1 meso-FAP CAR TEAM cells via IP. Tumor growth was monitored weekly by in vivo bioluminescence imaging. The expansion and phenotype of meso-FAP CAR TEAM cells in peritoneal lavage fluid and peripheral blood were measured by flow cytometry.

[0211] Example 4. Optimization of meso-FAP CAR TEAM cell administration routes in orthotopic and metastatic tumors.

[0212] The efficacy of meso-FAP CAR TEAM cells depends on the route of administration to the subject (e.g., intravenous or intraperitoneal). Tumor cells (AsPC1 or BXPC3 cells) are injected intraperitoneally into NSG. ® Mice were then treated with either MGH1Meso1 or SS1 meso-FAP CAR TEAM cells. Compared to SS1 meso-FAP CAR TEAM cells, MGH1Meso1 meso-FAP CAR TEAM cells exhibited superior tumor-killing ability and survival rate upon intraperitoneal injection. Figure 3A This difference was less pronounced when meso-FAP CARTEAM cells were administered intravenously. Figure 3B Furthermore, this difference was more subtle when the tumor was subcutaneously injected into mice. Figure 3C ).

[0213] Next, the effects of MGH1Meso1 or SS1 meso-FAP CAR TEAM cells were tested on other cancer cells. PDAC cells were mixed with CAF cells at a 1:9 ratio and injected into NSG via intrapancreatic injection (to form the primary tumor) or portal vein injection (to induce liver metastasis). ® In mice, tumors were allowed to establish for two weeks. The most effective meso-FAP CAR TEAM cells (such as MGH1Meso1 or SS1 as identified in Example 1) were injected intravenously, intraperitoneally, or both simultaneously, and tumor growth was monitored weekly by in vivo bioluminescence imaging. Furthermore, the expansion and phenotype of meso-FAP CAR TEAM cells in peritoneal lavage fluid and peripheral blood were measured by flow cytometry.

[0214] Example 5. Increasing mesothelin density on tumor cells by using integrin and metalloproteinase 17 (ADAM17) inhibitors to prevent mesothelin cleavage.

[0215] Mesothelin expression on the surface of cancer cells can be reduced due to proteolytic cleavage, leading to the release of mesothelin from the cell membrane in its soluble form. This cleavage and release of mesothelin from the tumor cell surface reduces the ability of meso-FAP CAR TEAM cells to target mesothelin-expressing cancer cells. Furthermore, soluble mesothelin can bind to meso-FAP CAR TEAM cells and block their ability to recognize and bind mesothelin to tumor cell surfaces. Therefore, co-administration of an inhibitor that prevents mesothelin proteolysis (such as ADAM17) can significantly enhance the efficacy of meso-FAP CAR TEAM cells.

[0216] in vitro

[0217] To test this in vitro, pancreatic cancer cell lines ASPC1, CAPAN-2, and BxPC3 were treated with escalating doses of the ADAM17 inhibitor aderbasib. Mesothelin levels expressed on the cell surface were measured by flow cytometry. Aderbasib was found to increase mesothelin expression in all three pancreatic cancer cell lines in a dose-dependent manner. Figures 6A-6D ), and reduce the shedding of mesothelin from cancer cells ( Figures 7A-7C It may prevent the cleavage of surface mesothelin by inhibiting ADAM-17 activity.

[0218] Next, pancreatic cancer cell lines (ASPC1, CAPAN-2, and BxPC3) were cultured and treated with SS1 CAR-T cells alone, untransduced cells (CAR-T cells), 5 μm aderbasib alone, 5 μm aderbasib and untransduced cells, or 5 μm aderbasib and SS1 CAR-T cells, respectively. Compared with control or treatments without SS1 CAR-T cells, SS1 CAR-T cell treatment resulted in a significant reduction in tumor area, and a further reduction was observed when cancer cells were treated with a combination of SS1 CAR-T cells and 5 μM aderbasib. Figures 8A-8C This indicates that the combination therapy provides robust treatment for pancreatic cancer cells.

[0219] In addition, PDX cells were treated with escalating doses of aderbasib, and surface mesothelin expression was assessed. Soluble mesothelin levels in the supernatant were measured by ELISA. Next, meso-FAP CAR TEAM cells were treated with lower aderbasib concentrations that induced the highest levels of mesothelin expression in PDAC and PDX cell lines. Proliferation and cell death of meso-FAP CAR TEAM cells in the presence of aderbasib were measured. PDAC cells or patient-derived xenograft (PDX) cells were then co-cultured with CAF cells, and meso-FAP CAR TEAM cells were added to the co-culture with or without aderbasib. Cytotoxicity of meso-FAP CAR TEAM cells under each condition was measured and compared.

[0220] in vivo

[0221] The efficacy of the combination of SS1 CAR-T cells and aderbasib was tested in vivo. Mice were subcutaneously injected (sc) with ASPC1 pancreatic cancer cells, followed by oral administration (og) of aderbasib at a dose of 60 mg / kg 11 days later, continuing aderbasib treatment until 14 days after SS1 CAR-T cell therapy. SS1 CAR-T cells were administered intravenously (iv) three days after aderbasib administration. Mice treated with SS1 CAR-T cells showed significantly improved tumor control nearly 50 days after CAR-T cell metastasis compared to mice receiving untransduced (CAR-) cells. Pretreatment with aderbasib in mice receiving SS1 CAR-T cells also significantly improved this tumor control compared to mice receiving CAR-T cells but not aderbasib (Figure 9). Therefore, the combination of SS1 CAR-T cells and aderbasib significantly improved tumor control both in vivo and in vitro.

[0222] PDAC cells and CAF cells were mixed at a 1:9 ratio in Matrigel and injected intraperitoneally (IP) into NSG® mice. Two weeks later, mice were treated with meso-FAP CAR TEAM cells via IP with or without aderbasib. Tumor growth was monitored by in vivo bioluminescence imaging, and survival was analyzed using Kaplan-Meier curves. The expansion and phenotype of meso-FAP CAR TEAM cells in peripheral blood were also measured.

[0223] Example 6. Enhancement of meso-FAP CAR TEAM cell killing by polarizing to Th1 / Th17 phenotype using an interleukin-2 induced T-cell kinase (ITK) inhibitor.

[0224] CD4 T cell phenotypes, such as helper T cell 1 (Th1) and Th17 cells, promote cytotoxicity and are therefore ideal in an anti-tumor context. The FDA-approved ITK inhibitor ibrutinib has previously been shown to increase T cell numbers, decrease checkpoint molecule expression, and increase Th1 / Th17 polarization. Therefore, co-administration of ibrutinib with meso-FAP CAR TEAM cells may enhance their tumor-killing efficacy and survival.

[0225] Mice were subcutaneously injected (sc) with ASPC1 cells, and fourteen days later received: (i) intravenous (iv) injection of SS1 CAR-T cells, (ii) untransduced cells, (iii) oral (og) administration of 25 mg / kg ibrutinib and untransduced cells, or (iv) the same ibrutinib and SS1 CAR-T cells as in (iii). Mice receiving ibrutinib continued treatment until day 35 after initial treatment. Mice receiving SS1 CAR-T cells showed improved tumor control compared to mice receiving untransduced cells, and mice additionally receiving ibrutinib showed significantly improved tumor control (i.e., tumor size reduction), indicating that the combination of anti-mesothelin CAR-T cell therapy and ibrutinib therapy improved antitumor efficacy. Figures 10A-10C Ibrutinib treatment was also observed to support CAR-T cell expansion and / or survival, as mice receiving ibrutinib in combination with SS1 CAR-T cells had significantly higher CAR-T cell counts up to 20 days post-injection. Figure 10D In summary, the data show that ibrutinib enhances the antitumor efficacy of anti-mesothelin CAR-T cells and supports the survival and / or expansion of said CAR-T cells.

[0226] To test this, PDAC cells were mixed with CAF cells at a 1:9 ratio in Matrigel and injected intraperitoneally (IP) into NSG® mice. Two weeks later, mice were treated with meso-FAP CAR TEAM cells via IP with or without ibrutinib. Tumor growth was monitored by in vivo bioluminescence imaging, and survival was analyzed using Kaplan-Meier curves. Peripheral blood meso-FAP CAR TEAM cell expansion and phenotype were also measured. Mouse serum was subjected to Luminex assays to measure cytokines associated with Th1, Th2, and Th17 phenotypes (e.g., IFNγ, IL-4, and IL-17).

[0227] Example 7. Treatment Plan and Assessment Type

[0228] This example describes the timeline of treatment and subsequent evaluations received by the subjects. All subjects received 3 days of lymphodepletion chemotherapy starting on day -5 (initially via IV infusion of 300 mg / m² over 30 minutes). 2 Cyclophosphamide, followed immediately by an IV infusion of 30 mg / m² over 30 minutes. 2 Fludarabine was administered, followed by meso-FAP CAR-TEAM cells on day 0. Lymphatic depletion can be performed on an outpatient or inpatient basis, depending on the investigator's judgment. Following CAR T-cell infusion, patients will be monitored for adverse events (AEs), clinical status, and laboratory indicators as described below. Study samples will be collected for related research, continuing for up to 24 months. Figure 5 All participants who complete the study, as well as those who withdraw from the study after receiving the Meso-FAP CAR-TEAM for reasons other than death, will be required to participate in FDA-mandated long-term follow-up studies of up to 15 years after meso-FAP CAR infusion, focusing on long-term efficacy and safety.

Claims

1. A method of treating a subject with mesothelin-expressing cancer, the method comprising administering to the subject in need an effective amount of chimeric antigen receptor (CAR)-T cell inhibitors combined with integrin and metalloproteinase 17 (ADAM17) inhibitors or interleukin-2-induced T-cell kinase (ITK) inhibitors. The CAR-T cells described herein comprise: Mesothelin-binding CAR; and T cell adaptor molecules (TEAMs) containing fibroblast activation protein (FAP) binding domains.

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

3. The method according to claim 1 or 2, wherein the FAP binding domain and the CD3 binding domain of TEAM are connected by connectors.

4. The method according to any one of claims 1-3, wherein the connector comprises the 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 according to claim 1 or 2, wherein the mesothelin-binding CAR comprises the VH domain of SEQ ID NO:1 and the VL domain of SEQ ID NO:

2.

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

10.

7. The method according to any one of claims 1-5, wherein the mesothelin-bound CAR comprises: (a) A VH domain comprising three complementary determinant regions (CDR-H1, CDR-H2, and CDR-H3), wherein CDR-H1 contains SEQ ID NO:3, CDR-H2 contains SEQ ID NO:4, and CDR-H3 contains 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 according to any one of claims 1-2 or 6, wherein the mesothelin-bound CAR comprises: (a) A VH domain comprising three complementary determinant regions (CDR-H1, CDR-H2, and CDR-H3), wherein CDR-H1 contains SEQ ID NO:11, CDR-H2 contains SEQ ID NO:12, and CDR-H3 contains 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 according to 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 structural domain is selected from the group consisting of CD8, CD18 and CD28.

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

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

35.

13. The method according to any one of claims 1-12, wherein the mesothelin-binding CAR comprises an intracellular signal transduction domain containing a CD3ζ intracellular signal transduction domain.

14. The method of claim 13, wherein the CD3ζ intracellular signal transduction domain comprises the sequence of SEQ ID NO:

33.

15. The method according to 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, CD18, CD27, CD28 and 4-1BB.

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

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

18.

19. The method according to any one of claims 1-18, wherein the FAP-binding domain of TEAM comprises: (a) A VH domain comprising three CDRs (CDR-H1, CDR-H2, and CDR-H3), wherein CDR-H1 contains SEQ ID NO:19, CDR-H2 contains SEQ ID NO:20, and CDR-H3 contains 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 according to any one of claims 2-19, wherein the CD3 binding domain of TEAM comprises the VH domain of SEQ ID NO:25 and the VL domain of SEQ ID NO:

26.

21. The method according to any one of claims 2-20, wherein the CD3 binding domain of TEAM comprises: (a) A VH domain comprising three CDRs (CDR-H1, CDR-H2, and CDR-H3), wherein CDR-H1 contains SEQ ID NO:27, CDR-H2 contains SEQ ID NO:28, and CDR-H3 contains 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 according to any one of claims 1-21, wherein the mesothelin-binding CAR comprises SEQ ID NO:41 or SEQ ID NO:

42.

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

43.

24. The method according to any one of claims 1-23, wherein the CAR-T cells comprise the amino acid sequence encoding the mesothelin-binding CAR and TEAM of SEQ ID NO:44, SEQ ID NO:45 or SEQ ID NO:

51.

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

26. The method according to 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 according to 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 according to any one of claims 1-28, wherein the ADAM17 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 ADAM17 inhibitor is aderbasib.

31. The method according to 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 according to any one of claims 1-32, comprising administering CAR-T cells via a first administration route and administering an ADAM17 inhibitor and / or an ITK inhibitor via a second administration route different from the first administration route.

34. The method according to any one of claims 1-33, wherein the administration of CAR-T cells comprises intravenous administration, and the administration of ADAM17 inhibitors and / or ITK inhibitors comprises intraperitoneal administration.

35. The method according to any one of claims 1-33, wherein the administration of CAR-T cells comprises intravenous administration, and the administration of ADAM17 inhibitors and / or ITK inhibitors comprises oral administration.

36. The method according to any one of claims 1-35, wherein the ADAM17 inhibitor is not a protein-based inhibitor.

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