Combination of a low molecular weight drug conjugate and a CAR-expressing cytotoxic lymphocyte and method of use

Abstract

A method of treating cancer in a subject, comprising the step of administering to the subject: (i) a small molecule drug conjugate (SMDC) that targets a cell surface receptor on an immunosuppressive cell or a cancerous cell; (ii) a cytotoxic lymphocyte that expresses a chimeric antigen receptor (CAR); and a synergistic combination of the SMDC and the CAR-expressing cytotoxic lymphocyte.

Description

Technical Field

[0001] This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 346,152, filed May 26, 2022, the content of which is hereby incorporated by reference in its entirety into this disclosure.

Background Art

[0002] The present disclosure includes a combination (e.g., a synergistic combination) of a small molecule drug conjugate (or a pharmaceutically acceptable salt thereof) and a cytotoxic lymphocyte expressing a chimeric antigen receptor, as well as methods of treating cancer using such combinations and the use of such combinations for the preparation of a medicament.

Summary of the Invention

[0003] A method of treating cancer in a subject, comprising (e.g., in a therapeutically effective amount): (a) a drug moiety (E) selected from the group consisting of a phosphoinositide 3-kinase (PI3K) inhibitor, an interferon gene stimulator (STING) agonist, a nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) agonist, a retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) agonist, a melanoma differentiation-associated gene 2 (AIM2)-like receptor (ALR) agonist, a receptor for advanced glycation end products (RAGE) agonist, a Pelle kinase / interleukin-1 (IL-1) receptor-associated kinase 1 (IRAK1) agonist, an IRAK2 agonist, an IRAK4 agonist, an IRAK3 antagonist, a Src homology 2 domain-containing tyrosine phosphatase 1 and 2 (SHP1 / 2) antagonist, a T cell protein tyrosine phosphatase (TC-PTP) antagonist, a diacylglycerol kinase (DGK) antagonist, an enhancer of zeste homolog 2 (EZH2) antagonist, a transforming growth factor beta (TGFβ) antagonist, an activator of nuclear factor kappa B (NFκβ) or an inhibitor of I kappa B (Iκβ) kinase; (b) The radicals of raltitrexed, the radical of 5-methyltetrahydrofolic acid, and the group represented by formula (X):

[0004]

Chemical formula

[0005] In some embodiments, the ligand comprises the radical of raltitrexed or the radical of 5 - methyltetrahydrofolic acid. In some embodiments, the ligand is a group containing formula (X). In some embodiments, J is CH2, and in some embodiments, J is a bond (e.g., J is (C(R J )2)0). In some embodiments, ligand (A) comprises formula (XA).

[0006]

Chemical formula

[0007] In some embodiments, the ligand (A) comprises formula (XB).

[0008]

Chemical formula

[0009] In some embodiments, the ligand (A) comprises formula (XC).

[0010]

Chemical formula

[0011] In some embodiments, the ligand (A) comprises formula (XD).

[0012]

Chemical formula

[0013] In some embodiments, the ligand comprises formula (XA), formula (XB), formula (XC) or formula (XD):

[0014]

Chemical formula

[0015] In some embodiments, the drug moiety is a PI3K inhibitor and the ligand is a radical of raltitrexed. In some embodiments, the drug moiety is a PI3K inhibitor and the ligand is a radical of 5-methyltetrahydrofolic acid. In some embodiments, the drug moiety is a PI3K inhibitor and the ligand is a group containing formula (X). In some embodiments, the drug moiety is an NFκβ activator and the ligand is a radical of raltitrexed. In some embodiments, the drug moiety is an NFκβ activator and the ligand is a radical of 5-methyltetrahydrofolic acid. In some embodiments, the drug moiety is an NFκβ activator and the ligand is a group containing formula (X). In some embodiments, the drug moiety is an Iκβ kinase inhibitor and the ligand is a radical of raltitrexed. In some embodiments, the drug moiety is an Iκβ kinase inhibitor and the ligand is a radical of 5-methyltetrahydrofolic acid. In some embodiments, the drug moiety is an Iκβ kinase inhibitor and the ligand is a group containing formula (X). In some embodiments, the drug moiety is a STING agonist and the ligand is a radical of raltitrexed. In some embodiments, the drug moiety is a STING agonist and the ligand is a radical of 5-methyltetrahydrofolic acid. In some embodiments, the drug moiety is a STING agonist and the ligand is a group containing formula (X). In some embodiments, the drug moiety is an NLR agonist and the ligand is a radical of raltitrexed. In some embodiments, the drug moiety is an NLR agonist and the ligand (A) is a radical of 5-methyltetrahydrofolic acid. In some embodiments, the drug moiety is an NLR agonist and the ligand is a group containing formula (X). In some embodiments, the drug moiety is an RLR agonist and the ligand is a radical of raltitrexed. In some embodiments, the drug moiety is an RLR agonist and the ligand is a radical of 5-methyltetrahydrofolic acid. In some embodiments, the drug moiety is an RLR agonist and the ligand is a group containing formula (X). In some embodiments, the drug moiety is an ALR agonist and the ligand is a radical of raltitrexed.In some embodiments, the drug moiety is an ALR agonist and the ligand is a radical of 5-methyltetrahydrofolic acid. In some embodiments, the drug moiety is an ALR agonist and the ligand is a group containing formula (X). In some embodiments, the drug moiety is a RAGE agonist and the ligand is a radical of larotrectinib. In some embodiments, the drug moiety is a RAGE agonist and the ligand is a radical of 5-methyltetrahydrofolic acid. In some embodiments, the drug moiety is a RAGE agonist and the ligand is a group containing formula (X). In some embodiments, the drug moiety is an IRAK1 agonist, IRAK2 agonist, IRAK4 agonist or IRAK3 antagonist and the ligand is a radical of larotrectinib. In some embodiments, the drug moiety is an IRAK1 agonist, IRAK2 agonist, IRAK4 agonist or IRAK3 antagonist and the ligand is a radical of 5-methyltetrahydrofolic acid. In some embodiments, the drug moiety is an IRAK1 agonist, IRAK2 agonist, IRAK4 agonist or IRAK3 antagonist and the ligand is a group containing formula (X). In some embodiments, the drug moiety is an SHP1 / 2 inhibitor and the ligand is a radical of larotrectinib. In some embodiments, the drug moiety is an SHP1 / 2 inhibitor and the ligand is a radical of 5-methyltetrahydrofolic acid. In some embodiments, the drug moiety is an SHP1 / 2 inhibitor and the ligand is a group containing formula (X). In some embodiments, the drug moiety is a TC-PTP inhibitor and the ligand is a radical of larotrectinib. In some embodiments, the drug moiety is a TC-PTP inhibitor and the ligand is a radical of 5-methyltetrahydrofolic acid. In some embodiments, the drug moiety is a TC-PTP inhibitor and the ligand is a group containing formula (X). In some embodiments, the drug moiety is a DGK inhibitor and the ligand is a radical of larotrectinib. In some embodiments, the drug moiety is a DGK inhibitor and the ligand is a radical of 5-methyltetrahydrofolic acid. In some embodiments, the drug moiety is a DGK inhibitor and the ligand (A) is a group containing formula (X).In some embodiments, the drug moiety is a TGFβ inhibitor and the ligand is a radical of raltitrexed. In some embodiments, the drug moiety is a TGFβ inhibitor and the ligand is a radical of 5-methyltetrahydrofolic acid. In some embodiments, the drug moiety is a TGFβ inhibitor and the ligand is a group containing formula (X).

[0016] In some embodiments, the linker comprises polyethylene glycol (PEG) in a cleavable form, non-cleavable PEG, polyproline, hydrophilic amino acids, sugars, non-natural peptidoglycans, polyvinylpyrrolidone, or a triblock copolymer comprising a central hydrophobic block of polypropylene glycol with hydrophilic blocks of polyethylene glycol adjacent to each side.

[0017] In some embodiments, the linker is a cleavable linker. In some embodiments, the linker is a non-cleavable linker. In some embodiments, the linker has the structure:

[0018]

Chemical formula

[0019]

Chemical formula

[0020]

Chemical formula

[0021] [Chemical formula]

[0022] In some embodiments, each CAR comprises a recognition region, a co-stimulatory domain, and an activation signaling domain. In some embodiments, the recognition region of the CAR is a single-chain variable fragment (scFv) of an antibody that binds to a cell surface antigen with high specificity. In some embodiments, the CAR selectively binds to a cell surface antigen on an immunosuppressive cell or a cancer cell. In some embodiments, the CAR specifically and selectively binds to a cell surface antigen on an immunosuppressive cell or a cancer cell.

[0023] In some embodiments, the cell surface antigen is a tumor-associated antigen (TAA). In some embodiments, the TAA is selected from the group consisting of CD19, CD20, CD30, CD3, CD4, CD5, BCMA, GD2, LEY, MS4A1, CD22, TNFRSF17, CD38, SDC1, TNFRSF8, IL3RA, CD7, NCAM1, CD34, CLEC12A, CD4, MME, CD5, SLAMF7, IL1RAP, FCGR3A, ITGB7, TNFRSF13B, TRBC1, CD33, ROR1, MUC1, KLRK1, KIT, CD274, CD70, PROM1, AFP, AXL, CD80, CD86, DLL3, TNFRSF10B, FAP, MAGEA1, MAGEA4, MUC16, PMEL, ROR2, KDR, EPHA2, L1CAM, CLDN18, PSCA, FOLR1, IL13RA2, MET, EPCAM, EGFR, EGFRVIII, FOLH1, GPC3, CEACAM5, ERBB2, CAIX, B4GALNT1, NY-ESO-1, PD-L1, and MSLN. In some embodiments, the TAA is CD19. In some embodiments, the CAR-expressing cytotoxic lymphocyte is a T cell, and the co-stimulatory domain of the CAR is CD27, CD40L, CD70, 2B4, DNAM1, DAP12, DAP10, NKG2, NKG2D, CD28, CD137 (4-1BB), CD134 (OX40) or CD278 (ICOS).

[0024] In some embodiments, the CAR-expressing cytotoxic lymphocyte is a T cell, and the co-stimulatory domain of the CAR is CD27, CD40L, CD70, 2B4, DNAM1, DAP12, DAP10, NKG2, NKG2D, CD28, CD137 (4-1BB), CD134 (OX40) or CD278 (ICOS). In some embodiments, the CAR-expressing cytotoxic lymphocyte is an NK cell, and the TAA is selected from the group consisting of CD3, CD4, and CD5. In some embodiments, the CAR-expressing cytotoxic lymphocyte is an NK cell, and the co-stimulatory domain of the CAR is selected from the group consisting of 2B4, CD137 (4-1BB), DNAM1, DAP12, DAP10, NKG2, and NKG2D.

[0025] In some embodiments, the activation signaling domain of the CAR is the T cell CD3ζ chain or the Fc receptor γ. In some embodiments, the cytotoxic lymphocyte is autologous to the individual. In some embodiments, the cytotoxic lymphocyte is allogeneic. In some embodiments, the cytotoxic lymphocyte is xenogeneic to the individual. In some embodiments, the cancer is a solid tumor cancer. In some embodiments, the cancer (e.g., solid tumor cancer) is a cancer of the brain, thyroid, lung, pancreas, kidney, stomach, gastrointestinal stroma, endometrium, breast, cervix, ovary, colon, prostate, head or neck. In some embodiments, the cancer is leukemia, lymphoma or another blood-related cancer. In some embodiments, the cancer is a folate receptor-expressing cancer, or a folate receptor-expressing immune cell-mediated cancer. In some embodiments, the cancer is a folate receptor α-expressing cancer, a folate receptor β-expressing cancer, a folate receptor δ-expressing cancer, or a folate receptor α, β or δ-expressing immune cell-mediated cancer. In some embodiments, the ligand is a radical of raltitrexed or a radical of 5-methyltetrahydrofolate, and the cancer is a folate receptor-expressing cancer, or a folate receptor-expressing immune cell-mediated cancer.

[0026] In some embodiments, the cancer is a cancer that expresses fibroblast activation protein (FAP), or a cancer that has cancer-associated fibroblasts that express FAP. In some embodiments, the ligand is a group of formula (X), and the cancer is a cancer that expresses FAP, or a cancer that has cancer-associated fibroblasts that express FAP.

[0027] Administration of a therapeutically effective amount of the SMDC or a pharmaceutically acceptable salt thereof inhibits the activity of cancer-associated fibroblasts in a subject and improves the effectiveness of the administered CAR-expressing cytotoxic lymphocytes against cancer as compared to the effectiveness of the CAR-expressing cytotoxic lymphocytes administered in the absence of administration of a therapeutically effective amount of the SMDC or a pharmaceutically acceptable salt thereof.

[0028] Also provided is the use of an SMDC or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of cancer. In certain embodiments, the treatment comprises administering a therapeutically effective amount of the SMDC in combination with CAR T cell therapy. The SMDC or a pharmaceutically acceptable salt thereof is (a) a drug moiety selected from the group consisting of a PI3K inhibitor, a STING agonist, an NLR agonist, an RLR agonist, an ALR agonist, a RAGE agonist, an IRAK1 agonist, an IRAK2 agonist, an IRAK4 agonist, an IRAK3 antagonist, an SHP1 / 2 antagonist, a TC-PTP antagonist, a DGK antagonist, an EZH2 antagonist, a TGFβ antagonist, an NFκβ activator or an Iκβ kinase antagonist, (b) a radical of raltitrexed, a radical of 5-methyltetrahydrofolic acid, or of formula (X):

[0029]

Chemical formula

[0030] The ligand of the SMDC is of formula (XA), formula (XB), formula (XC) or formula (XD):

[0031]

Chem.

[0032] The drug moiety may be selected from the group consisting of a PI3K inhibitor, an NFκβ activator, an Iκβ kinase inhibitor, a STING agonist, an NLR agonist, an RLR agonist, an ALR agonist, a RAGE agonist, an agonist of IRAK1, an agonist of IRAK2, an agonist of IRAK4, an antagonist or inhibitor of IRAK3, an SHP1 / 2 inhibitor, a TC-PTP inhibitor, a DGK inhibitor, and a TGFβ inhibitor. In certain embodiments, the ligand may be a radical of raltitrexed, a radical of 5-methyltetrahydrofolic acid, or a group containing formula (X). The linker may include PEG in a releasable form, PEG in a non-releasable form, polyproline, hydrophilic amino acids, sugars, non-natural peptidoglycans, polyvinylpyrrolidone, a triblock copolymer comprising a central hydrophobic block of polypropylene glycol with hydrophilic blocks of PEG adjacent to each side, or a combination of any two or more of the foregoing. The linker may be a cleavable linker. The linker may be a non-cleavable linker. The linker has the structure:

[0033]

Chem.

[0034]

Chem.

[0035]

Chem.

[0036] In certain embodiments, the SMDC further comprises a PEG group, a peptide group, a glycopeptide group, a saccharide group, a radical of an albumin-binding group, or a combination of radicals of two or more of the foregoing groups, and the PEG group, the peptide group, the glycopeptide group, the saccharide group, the radical of the albumin-binding group, or a combination thereof is attached to a linker. The CAR T cell therapy may include administration of CAR-expressing cytotoxic lymphocytes to a subject, and each CAR includes a recognition region, a co-stimulatory domain, and an activation signaling domain.

[0037] The recognition region of the CAR can be an scFv of an antibody that binds to a cell surface antigen with high specificity. The CAR can specifically and selectively bind to a cell surface antigen on immunosuppressive cells or cancer cells. The cell surface antigen can be a TAA. The TAA can be selected from the group consisting of CD19, CD20, CD30, CD3, CD4, CD5, BCMA, GD2, LEY, MS4A1, CD22, TNFRSF17, CD38, SDC1, TNFRSF8, IL3RA, CD7, NCAM1, CD34, CLEC12A, CD4, MME, CD5, SLAMF7, IL1RAP, FCGR3A, ITGB7, TNFRSF13B, TRBC1, CD33, ROR1, MUC1, KLRK1, KIT, CD274, CD70, PROM1, AFP, AXL, CD80, CD86, DLL3, TNFRSF10B, FAP, MAGEA1, MAGEA4, MUC16, PMEL, ROR2, KDR, EPHA2, L1CAM, CLDN18, PSCA, FOLR1, IL13RA2, MET, EPCAM, EGFR, EGFRVIII, FOLH1, GPC3, CEACAM5, ERBB2, CAIX, B4GALNT1, NY-ESO-1, PD-L1, and MSLN. The TAA can be CD19.

[0038] The CAR-expressing cytotoxic lymphocytes may be T cells, and the co-stimulatory domain of the CAR may be CD27, CD40L, CD70, 2B4, DNAM1, DAP12, DAP10, NKG2, NKG2D, CD28, CD137 (4-1BB), CD134 (OX40) or CD278 (ICOS). The CAR-expressing cytotoxic lymphocytes may be NK cells, and the TAA may be selected from the group consisting of CD3, CD4, and CD5.

[0039] The CAR-expressing cytotoxic lymphocytes may be NK cells, and the co-stimulatory domain of the CAR may be selected from the group consisting of 2B4, CD137 (4-1BB), DNAM1, DAP12, DAP10, NKG2, and NKG2D. The activating signaling domain of the CAR may be the T cell CD3ζ chain or the Fc receptor γ. The cytotoxic lymphocytes may be autologous to the individual. The cytotoxic lymphocytes may be allogeneic. The cytotoxic lymphocytes may be xenogeneic to the individual. The cancer may be a solid tumor cancer. The solid tumor cancer may be a cancer of the brain, thyroid, lung, pancreas, kidney, stomach, gastrointestinal stroma, endometrium, breast, cervix, ovary, colon, prostate, head or neck. The cancer may be a leukemia, lymphoma or another blood-related cancer. The ligand may be a radical of raltitrexed or a radical of 5-methyltetrahydrofolate, and the cancer may be a cancer expressing folate receptor or a cancer mediated by folate receptor-expressing immune cells. The cancer may be a cancer expressing folate receptor α, folate receptor β, folate receptor δ, or a cancer mediated by folate receptor α, β or δ-expressing immune cells. The ligand may be a group of formula (X), and the cancer may be a cancer expressing FAP or a cancer having cancer-associated fibroblasts expressing FAP.

[0040] Also provided is a synergistic combination for treating solid tumor cancer. In certain embodiments, the synergistic combination for treating solid tumor cancer comprises (i) a therapeutically effective amount of any of the SMDCs described herein or a pharmaceutically acceptable salt thereof, and (ii) a therapeutically effective amount of CAR-expressing cytotoxic lymphocytes. In certain embodiments, the SMDC of the synergistic combination is (a) A drug moiety selected from the group consisting of a PI3K inhibitor, a STING agonist, an NLR agonist, an RLR agonist, an ALR agonist, a RAGE agonist, an IRAK1 agonist, an IRAK2 agonist, an IRAK4 agonist, an IRAK3 antagonist, a SHP1 / 2 antagonist, a TC-PTP antagonist, a DGK antagonist, an EZH2 antagonist, a TGFβ antagonist, an NFκβ activator or an Iκβ kinase antagonist, (b) A radical of raltitrexed, a radical of 5-methyltetrahydrofolic acid, or a group of formula (X):

[0041]

Chemical formula

Mode for Carrying Out the Invention

[0042] The synergistic combinations and methods disclosed herein comprise a combination of (i) a small molecule drug conjugate (SMDC) comprising a drug moiety conjugated to a ligand or a pharmaceutically acceptable salt thereof, and (ii) chimeric antigen receptor (CAR)-expressing cytotoxic lymphocytes. Administration of the SMDC (or a pharmaceutically acceptable salt thereof) in combination with administration of CAR-expressing cytotoxic lymphocytes increases the efficacy of CAR cell therapy with little or no off-target toxicity.

[0043] The term "off-target toxicity" means damage to an organ or tissue, an undesirable decrease in the body weight of a subject for a physician or other individual treating the subject, or any other effect on a subject that is a potentially harmful indicator to the treating physician (e.g., B cell aplasia, fever, hypotension, or pulmonary edema).

[0044] The term "treat (treat, treating, treated or treatment)" (with respect to a disease or condition) is used to describe a procedure for obtaining a favorable or desired outcome, preferably including a clinical outcome. Such outcomes can include, but are not limited to: improvement of symptoms associated with a disease, cure of a disease, reduction of disease severity, improvement of the quality of life of a patient, extension of survival, and / or one or more of prophylactic or preventive treatments. With respect to cancer, in particular, the term "treat (treat, treating, treated or treatment)" can mean reduction of tumor size, complete or partial removal of a tumor (e.g., complete or partial response), stabilization of a disease, prevention of cancer progression (e.g., progression-free survival), or any other effect on cancer that is determined by a physician to be a therapeutic, prophylactic or preventive treatment of cancer. Neither term requires the creativity of a medical professional.

[0045] As used herein, "CAR therapy" and "CAR-T cells" refer to cytotoxic lymphocyte cells (e.g., T cells) modified by molecular biological methods to express CAR on the cell surface or a population thereof. CAR is a polypeptide having a predetermined binding specificity for a desired target and is operably bound to the inside of a cell activation domain (e.g., as a fusion, separate chains bound by one or more disulfide bonds, etc.). By circumventing the restrictions of MHC class I and MHC class II, both CD8+ and CD4+ subsets of CAR-modified lymphocyte cells can be mobilized to redirect the recognition of target cells.

[0046] When referring to a ligand / receptor, recognition region / target moiety, nucleic acid / complementary nucleic acid, antibody / antigen, or other binding pair, "specifically binds", "binds with high affinity", or "specifically" or "selectively" binds refers to a binding reaction that determines the presence of a protein in a heterogeneous population of proteins and other biological agents. Thus, under specified conditions, a specified ligand or recognition region binds to a specific receptor (e.g., a receptor present on a cancer cell) or target moiety, respectively, and does not bind in significant amounts to other proteins present in the sample (e.g., proteins associated with normal healthy cells). Also, specific binding or binding with high affinity means, for example, that a binding compound, ligand, antibody, or binding composition derived from the antigen-binding site of an antibody binds to its target with an affinity that is generally at least 25% greater, more generally at least 50% greater, most generally at least 100% (2-fold) greater, usually at least 10-fold greater, more usually at least 20-fold greater, and most usually at least 100-fold greater than its affinity for any other binding compound.

[0047] In a typical embodiment, a molecule that specifically binds to a target has, for example, at least about 10 6 liters / mol (K D = 10 -6M) Preferably, it has an affinity of at least about 10 liters / mol. Some binding compounds have the potential to specifically bind to two or more targets. For example, it is recognized by those skilled in the art that an antibody can specifically bind to its antigen, to lectin via the oligosaccharide of the antibody, and / or to the Fc receptor via the Fc region of the antibody.

[0048] Administering an SMDC or a pharmaceutically acceptable salt thereof in combination with the administration of CAR-expressing cytotoxic lymphocytes, thereby preventing the inactivation of such CAR-expressing lymphocytes in the tumor microenvironment (TME), which is recognized in conventional techniques, and / or inhibiting cancer-associated fibroblasts in the TME, thereby reducing or preventing the production of collagen therefrom. In some embodiments, the SMDC or a pharmaceutically acceptable salt thereof targets immunosuppressive cells (and / or cancerous cells) within the tumor to deliver the drug moiety to the target cells, thereby enhancing the infiltration and activity of CAR-expressing cytotoxic lymphocytes in the TME and, moreover, avoiding systemic toxicity. When an SMDC is administered together with CAR-expressing cytotoxic lymphocytes, better cytotoxicity against cancer cells within solid tumors occurs compared to the administration of CAR-expressing cytotoxic lymphocytes alone. Thus, when used to treat a subject having cancer (e.g., solid tumor cancer), administration by such a combination results in the cytotoxicity and / or effectiveness of the treatment being improved compared to the cytotoxicity and / or effectiveness of CAR-expressing cytotoxic lymphocytes administered without the administration of a therapeutically effective amount of the SMDC or a pharmaceutically acceptable salt thereof. In this regard, the combination of the present invention is synergistic.

[0049] In certain embodiments, provided herein is a method of treating cancer in a subject. The method comprises (E) a drug moiety selected from the group consisting of a phosphoinositide 3-kinase (PI3K) inhibitor, an interferon gene stimulator (STING) agonist, a nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) agonist, a retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) agonist, a melanoma antigen family A, member 2 (AIM2)-like receptor (ALR) agonist, a receptor for advanced glycation end products (RAGE) agonist, a pelle kinase / interleukin-1 (IL-1) receptor-associated kinase 1 (IRAK1) agonist, an IRAK2 agonist, an IRAK4 agonist, an IRAK3 antagonist, a Src homology 2 domain-containing tyrosine phosphatase 1 and 2 (SHP1 / 2) antagonist, a T cell protein tyrosine phosphatase (TC-PTP) antagonist, a diacylglycerol kinase (DGK) antagonist, an enhancer of zeste homolog 2 (EZH2) antagonist, a transforming growth factor beta (TGFβ) antagonist, an activator of nuclear factor kappa-B (NFκβ) or an inhibitor of Iκβ kinase (IκβK), (A) radicals of lartitrexed, radicals of 5-methyltetrahydrofolic acid, and of formula (X):

[0050]

Chemical formula

[0051] Small molecule drug conjugate In certain embodiments, the SMDC for use and methods according to the combination of the invention is (i) a drug moiety (E) selected from the group consisting of a PI3K inhibitor, a STING agonist, a NLR agonist, a RLR agonist, an ALR agonist, a RAGE agonist, an IRAK1 agonist, an IRAK2 agonist, an IRAK4 agonist, an IRAK3 antagonist, a SHP1 / 2 inhibitor, a TC-PTP inhibitor, a DGK inhibitor, an EZH2 inhibitor, a TGFβ inhibitor, an NFκβ activator, and an Iκβ kinase inhibitor, (ii) a radical of raltitrexed, a radical of 5-methyltetrahydrofolic acid, and a group of formula (X):

[0052]

Chemical formula

[0053] Drug moiety In certain embodiments, the SMDCs of the present disclosure include a drug moiety (E). Any drug useful for the treatment of cancer can be used with the SMDC. In some embodiments, the drug moiety (E) is a STING agonist, NLR agonist, RLR agonist, ALR agonist, RAGE agonist, IRAK1 agonist, IRAK2 agonist, IRAK4 agonist, IRAK3 antagonist, SHP1 / 2 antagonist, TC-PTP antagonist, DGK antagonist, EZH2 antagonist, TGFβ antagonist, PI3K inhibitor, NFκβ activator or Iκβ kinase antagonist. In some embodiments, the drug moiety (E) is a radical of a STING agonist, NLR agonist, RLR agonist, ALR agonist, RAGE agonist, IRAK1 agonist, IRAK2 agonist, IRAK4 agonist, IRAK3 antagonist, SHP1 / 2 antagonist, TC-PTP antagonist, DGK antagonist, EZH2 antagonist, TGFβ antagonist, PI3K inhibitor, NFκβ activator or Iκβ kinase antagonist, for example, a radical of a STING agonist, NLR agonist, RLR agonist, ALR agonist, RAGE agonist, IRAK1 agonist, IRAK2 agonist, IRAK4 agonist, IRAK3 antagonist, SHP1 / 2 antagonist, TC-PTP antagonist, DGK antagonist, EZH2 antagonist, TGFβ antagonist, PI3K inhibitor, NFκβ activator or Iκβ kinase antagonist as described herein.

[0054] The terms "activator" and "agonist" are used interchangeably herein to refer to a compound that interacts with a receptor and activates the receptor to produce a biological response. The terms "inhibitor" and "antagonist" are used interchangeably herein to refer to a compound that interacts with a receptor and blocks or reduces the ability of the receptor to produce a biological response.

[0055] In some embodiments, the drug moiety (E) is a PI3K inhibitor.

[0056] In some embodiments, the drug moiety (E) is an NFκβ activator.

[0057] In some embodiments, the drug moiety (E) is an Iκβ kinase antagonist.

[0058] In some embodiments, the drug moiety (E) is a STING agonist.

[0059] In some embodiments, the drug moiety (E) is an NLR agonist.

[0060] In some embodiments, the drug moiety (E) is an RLR agonist.

[0061] In some embodiments, the drug moiety (E) is an ALR agonist.

[0062] In some embodiments, the drug moiety (E) is a RAGE agonist.

[0063] In some embodiments, the drug moiety (E) is an agonist of IRAK1. In some embodiments, the drug moiety (E) is an agonist of IRAK2. In some embodiments, the drug moiety (E) is an agonist of IRAK4. In some embodiments, the drug moiety (E) is an antagonist of IRAK3.

[0064] In some embodiments, the drug moiety (E) is an SHP1 / 2 antagonist.

[0065] In some embodiments, the drug moiety (E) is a TC-PTP antagonist.

[0066] In some embodiments, the drug moiety (E) is a DGK antagonist.

[0067] In some embodiments, the drug moiety (E) is an EZH2 antagonist.

[0068] In some embodiments, the drug moiety (E) is a TGFβ antagonist.

[0069] Exemplary NFκβ activators are shown in Table 1.

[0070] [Table 1-1]

[0071] [Table 1-2]

[0072] In some embodiments, the drug moiety (E) is an NFκβ activator, and the ligand (A) is raltitrexed (radical). In some embodiments, the drug moiety (E) is an NFκβ activator, and the ligand (A) is 5-methyltetrahydrofolic acid (radical). In some embodiments, the drug moiety (E) is an NFκβ activator, and the ligand (A) is a group containing formula (X). In some embodiments, the drug moiety (E) is an NFκβ activator, and the ligand (A) is a group of formula (XA) or includes this. In some embodiments, the drug moiety (E) is an NFκβ activator, and the ligand (A) is a group containing formula (XB). In some embodiments, the drug moiety (E) is an NFκβ activator, and the ligand (A) is a group of formula (XC) or includes this. In some embodiments, the drug moiety (E) is an NFκβ activator, and the ligand (A) is a group of formula (XD) or includes this.

[0073] In some embodiments, the drug moiety (E) is a PI3K inhibitor. Non-limiting examples of PI3K inhibitors include, but are not limited to, the following.

[0074]

Chemical formula

[0075] In some embodiments, the drug moiety (E) is a PI3K inhibitor and the ligand (A) is (the radical of) raltitrexed. In some embodiments, the drug moiety (E) is a PI3K inhibitor and the ligand (A) is (the radical of) 5-methyltetrahydrofolic acid. In some embodiments, the drug moiety (E) is a PI3K inhibitor and the ligand (A) is a group containing formula (X). In some embodiments, the drug moiety (E) is a PI3K inhibitor and the ligand (A) is a group of formula (XA) or includes this. In some embodiments, the drug moiety (E) is a PI3K inhibitor and the ligand (A) is a group of formula (XB) or includes this. In some embodiments, the drug moiety (E) is a PI3K inhibitor and the ligand (A) is a group of formula (XC) or includes this. In some embodiments, the drug moiety (E) is a PI3K inhibitor and the ligand (A) is a group of formula (XD) or includes this.

[0076] In some embodiments, the drug moiety (E) is a STING agonist.

[0077] Non-limiting examples of STING agonists include the following.

[0078]

Chemical formula

[0079] In some embodiments, the drug moiety (E) is a STING agonist and the ligand (A) is a radical of raltitrexed. In some embodiments, the drug moiety (E) is a STING agonist and the ligand (A) is a radical of 5-methyltetrahydrofolic acid. In some embodiments, the drug moiety (E) is a STING agonist and the ligand (A) is a group comprising formula (X). In some embodiments, the drug moiety (E) is a STING agonist and the ligand (A) is a group of formula (XA) or comprises this. In some embodiments, the drug moiety (E) is a STING agonist and the ligand (A) is a group of formula (XB) or comprises this. In some embodiments, the drug moiety (E) is a STING agonist and the ligand (A) is a group of formula (XC) or comprises this. In some embodiments, the drug moiety (E) is a STING agonist and the ligand (A) is a group of formula (XD) or comprises this.

[0080] In some embodiments, the drug moiety (E) is an NLR agonist. Non-limiting examples of NLR agonists include the following.

[0081]

Chemical formula

[0082] In some embodiments, the drug moiety (E) is an NLR agonist, and the ligand (A) is the radical of raltitrexed. In some embodiments, the drug moiety (E) is an NLR agonist, and the ligand (A) is the radical of 5-methyltetrahydrofolic acid. In some embodiments, the drug moiety (E) is an NLR agonist, and the ligand (A) is a group containing formula (X). In some embodiments, the drug moiety (E) is an NLR agonist, and the ligand (A) is a group of formula (XA) or contains this. In some embodiments, the drug moiety (E) is an NLR agonist, and the ligand (A) is a group of formula (XB) or contains this. In some embodiments, the drug moiety (E) is an NLR agonist, and the ligand (A) is a group of formula (XC) or contains this. In some embodiments, the drug moiety (E) is an NLR agonist, and the ligand (A) is a group of formula (XD) or contains this.

[0083] In some embodiments, the drug moiety (E) is an Iκβ kinase inhibitor. In some embodiments, the drug moiety (E) is an Iκβ kinase inhibitor, and the ligand (A) is the radical of raltitrexed. In some embodiments, the drug moiety (E) is an Iκβ kinase inhibitor, and the ligand (A) is the radical of 5-methyltetrahydrofolic acid. In some embodiments, the drug moiety (E) is an Iκβ kinase inhibitor, and the ligand (A) is a group containing formula (X). In some embodiments, the drug moiety (E) is an Iκβ kinase inhibitor, and the ligand (A) is a group of formula (XA) or contains this. In some embodiments, the drug moiety (E) is an Iκβ kinase inhibitor, and the ligand (A) is a group of formula (XB) or contains this. In some embodiments, the drug moiety (E) is an Iκβ kinase inhibitor, and the ligand (A) is a group of formula (XC) or contains this. In some embodiments, the drug moiety (E) is an Iκβ kinase inhibitor, and the ligand (A) is a group of formula (XD) or contains this.

[0084] In some embodiments, the drug moiety (E) is an RLR agonist. In some embodiments, the drug moiety (E) is an RLR agonist and the ligand (A) is the radical of raltitrexed. In some embodiments, the drug moiety (E) is an RLR agonist and the ligand (A) is the radical of 5-methyltetrahydrofolic acid. In some embodiments, the drug moiety (E) is an RLR agonist and the ligand (A) is a group containing formula (X). In some embodiments, the drug moiety (E) is an RLR agonist and the ligand (A) is a group of formula (XA) or comprises the same. In some embodiments, the drug moiety (E) is an RLR agonist and the ligand (A) is a group of formula (XB) or comprises the same. In some embodiments, the drug moiety (E) is an RLR agonist and the ligand (A) is a group of formula (XC) or comprises the same. In some embodiments, the drug moiety (E) is an RLR agonist and the ligand (A) is a group of formula (XD) or comprises the same.

[0085] In some embodiments, the drug moiety (E) is an ALR agonist. In some embodiments, the drug moiety (E) is an ALR agonist and the ligand (A) is the radical of raltitrexed. In some embodiments, the drug moiety (E) is an ALR agonist and the ligand (A) is the radical of 5-methyltetrahydrofolic acid. In some embodiments, the drug moiety (E) is an ALR agonist and the ligand (A) is a group containing formula (X). In some embodiments, the drug moiety (E) is an ALR agonist and the ligand (A) is a group of formula (XA) or comprises the same. In some embodiments, the drug moiety (E) is an ALR agonist and the ligand (A) is a group of formula (XB) or comprises the same. In some embodiments, the drug moiety (E) is an ALR agonist and the ligand (A) is a group of formula (XC) or comprises the same. In some embodiments, the drug moiety (E) is an ALR agonist and the ligand (A) is a group of formula (XD) or comprises the same.

[0086] In some embodiments, the drug moiety (E) is a RAGE agonist. In some embodiments, the drug moiety (E) is a RAGE agonist and the ligand (A) is the radical of raltitrexed. In some embodiments, the drug moiety (E) is a RAGE agonist and the ligand (A) is the radical of 5-methyltetrahydrofolic acid. In some embodiments, the drug moiety (E) is a RAGE agonist and the ligand (A) is a group comprising formula (X). In some embodiments, the drug moiety (E) is a RAGE agonist and the ligand (A) is a group of formula (XA) or comprises the same. In some embodiments, the drug moiety (E) is a RAGE agonist and the ligand (A) is a group of formula (XB) or comprises the same. In some embodiments, the drug moiety (E) is a RAGE agonist and the ligand (A) is a group of formula (XC) or comprises the same. In some embodiments, the drug moiety (E) is a RAGE agonist and the ligand (A) is a group of formula (XD) or comprises the same.

[0087] In some embodiments, the drug moiety (E) is an agonist of IRAK1, an agonist of IRAK2, an agonist of IRAK4 or an antagonist of IRAK3. In some embodiments, the drug moiety (E) is an agonist of IRAK1, an agonist of IRAK2, an agonist of IRAK4 or an antagonist of IRAK3, and the ligand (A) is (the radical of) raltitrexed. In some embodiments, the drug moiety (E) is an agonist of IRAK1, an agonist of IRAK2, an agonist of IRAK4 or an antagonist of IRAK3, and the ligand (A) is (the radical of) 5-methyltetrahydrofolic acid. In some embodiments, the drug moiety (E) is an agonist of IRAK1, an agonist of IRAK2, an agonist of IRAK4 or an antagonist of IRAK3, and the ligand (A) is a group comprising formula (X). In some embodiments, the drug moiety (E) is an agonist of IRAK1, an agonist of IRAK2, an agonist of IRAK4 or an antagonist of IRAK3, and the ligand (A) is a group of formula (XA) or comprises this. In some embodiments, the drug moiety (E) is an agonist of IRAK1, an agonist of IRAK2, an agonist of IRAK4 or an antagonist of IRAK3, and the ligand (A) is a group of formula (XB) or comprises this. In some embodiments, the drug moiety (E) is an agonist of IRAK1, an agonist of IRAK2, an agonist of IRAK4 or an antagonist of IRAK3, and the ligand (A) is a group of formula (XC) or comprises this. In some embodiments, the drug moiety (E) is an agonist of IRAK1, an agonist of IRAK2, an agonist of IRAK4 or an antagonist of IRAK3, and the ligand (A) is a group of formula (XD) or comprises this.

[0088] In some embodiments, the drug moiety (E) is an SHP1 / 2 inhibitor. In some embodiments, the drug moiety (E) is an SHP1 / 2 inhibitor and the ligand (A) is the radical of raltitrexed. In some embodiments, the drug moiety (E) is an SHP1 / 2 inhibitor and the ligand (A) is the radical of 5-methyltetrahydrofolic acid. In some embodiments, the drug moiety (E) is an SHP1 / 2 inhibitor and the ligand (A) is a group containing formula (X). In some embodiments, the drug moiety (E) is an SHP1 / 2 inhibitor and the ligand (A) is a group of formula (XA) or contains this. In some embodiments, the drug moiety (E) is an SHP1 / 2 inhibitor and the ligand (A) is a group of formula (XB) or contains this. In some embodiments, the drug moiety (E) is an SHP1 / 2 inhibitor and the ligand (A) is a group of formula (XC) or contains this. In some embodiments, the drug moiety (E) is an SHP1 / 2 inhibitor and the ligand (A) is a group of formula (XD) or contains this.

[0089] In some embodiments, the drug moiety (E) is a TC-PTP inhibitor. In some embodiments, the drug moiety (E) is a TC-PTP inhibitor and the ligand (A) is the radical of raltitrexed. In some embodiments, the drug moiety (E) is a TC-PTP inhibitor and the ligand (A) is the radical of 5-methyltetrahydrofolic acid. In some embodiments, the drug moiety (E) is a TC-PTP inhibitor and the ligand (A) is a group containing formula (X). In some embodiments, the drug moiety (E) is a TC-PTP inhibitor and the ligand (A) is a group of formula (XA) or contains this. In some embodiments, the drug moiety (E) is a TC-PTP inhibitor and the ligand (A) is a group of formula (XB) or contains this. In some embodiments, the drug moiety (E) is a TC-PTP inhibitor and the ligand (A) is a group of formula (XC) or contains this. In some embodiments, the drug moiety (E) is a TC-PTP inhibitor and the ligand (A) is a group of formula (XD) or contains this.

[0090] In some embodiments, the drug moiety (E) is a DGK inhibitor. In some embodiments, the drug moiety (E) is a DGK inhibitor and the ligand (A) is (the radical of) raltitrexed. In some embodiments, the drug moiety (E) is a DGK inhibitor and the ligand (A) is (the radical of) 5-methyltetrahydrofolic acid. In some embodiments, the drug moiety (E) is a DGK inhibitor and the ligand (A) is a group containing formula (X). In some embodiments, the drug moiety (E) is a DGK inhibitor and the ligand (A) is a group of formula (XA) or comprises the same. In some embodiments, the drug moiety (E) is a DGK inhibitor and the ligand (A) is a group of formula (XB) or comprises the same. In some embodiments, the drug moiety (E) is a DGK inhibitor and the ligand (A) is a group of formula (XC) or comprises the same. In some embodiments, the drug moiety (E) is a DGK inhibitor and the ligand (A) is a group of formula (XD) or comprises the same.

[0091] In some embodiments, the drug moiety (E) is an EZH2 inhibitor. Non-limiting examples of EZH2 inhibitors include

[0092]

Chemical formula

[0093] In some embodiments, the drug moiety (E) is a TGFβ inhibitor. In some embodiments, the drug moiety (E) is a TGFβ inhibitor and the ligand (A) is the radical of raltitrexed. In some embodiments, the drug moiety (E) is a TGFβ inhibitor and the ligand (A) is the radical of 5-methyltetrahydrofolic acid. In some embodiments, the drug moiety (E) is a TGFβ inhibitor and the ligand (A) is a group containing formula (X). In some embodiments, the drug moiety (E) is a TGFβ inhibitor and the ligand (A) is a group of formula (XA) or includes this. In some embodiments, the drug moiety (E) is a TGFβ inhibitor and the ligand (A) is a group of formula (XB) or includes this. In some embodiments, the drug moiety (E) is a TGFβ inhibitor and the ligand (A) is a group of formula (XC) or includes this. In some embodiments, the drug moiety (E) is a TGFβ inhibitor and the ligand (A) is a group of formula (XD) or includes this.

[0094] Ligand / target moiety The drug moiety of the SMDC is conjugated to a ligand (A) (e.g., the targeting moiety). As used herein, a "drug moiety" is a radical of a molecule that is a drug or has drug-like properties. The term "radical" means a chemical group having an open valence that forms a bond with another atom or molecule. A radical of a molecule can be formed by removing one or more atoms to put it in an open valence state. Unless otherwise stated, a radical is formed by removing a hydrogen atom from a molecule.

[0095] In certain embodiments, the ligand is conjugated to the drug moiety (E) via a linker (L). In some embodiments, the ligand (A) is a radical of raltitrexed, a radical of 5-methyltetrahydrofolic acid, or of formula (X):

[0096]

Chemical formula

[0097] In some embodiments, the ligand (A) comprises a radical of raltitrexed or a radical of 5-methyltetrahydrofolic acid. Raltitrexed has the following structure.

[0098]

Chemical formula

[0099] In some embodiments, the ligand (A) has the following structure.

[0100] [Chemical formula]

[0101] 5-Methyltetrahydrofolic acid has the following structure.

[0102] [Chemical formula]

[0103] In some embodiments, the ligand (A) has the following structure.

[0104] [Chemical formula]

[0105] In some embodiments, the ligand (A) is a group containing formula (X).

[0106] [Chemical formula]

[0107] In some embodiments, J in formula (X) is CH2. In some embodiments, J in formula (X) is C(O). In some embodiments, J in formula (X) is a bond (i.e., J is (C(R J 2)0)). In some embodiments, R in formula (X) 1 is CN. In some embodiments, R in formula (X) 10 is H. In some embodiments, R in formula (X) 3 and R 4 are each F. In some embodiments, R in formula (X) 3 is H and R in formula (X) 4 is F.

[0108] In some embodiments, the ligand (A) is of formula (XA) or comprises the same.

[0109]

Chemical formula

[0110] In some embodiments, the ligand (A) is of formula (XB) or comprises the same.

[0111]

Chemical formula

[0112] In some embodiments, the ligand (A) is of formula (XC) or comprises the same.

[0113]

Chemical formula

[0114] In some embodiments, the ligand (A) is of formula (XD) or comprises the same.

[0115]

Chemical formula

[0116] In some embodiments, the ligand (A) comprises the following.

[0117]

Chemical formula

[0118]

Chemical formula

[0119] Linker In some embodiments, the drug moiety and the ligand are conjugated via a linker (L). The terms "conjugate" and "compound" may be used interchangeably herein. More specifically, a "conjugate" is a type of compound formed by joining two or more small molecule compounds. The term "linker" includes a chain of atoms that is biochemically configured to form a chemical bond and connect the drug moiety and the ligand to form a conjugate. Exemplarily, the chain of atoms may include carbon, nitrogen, oxygen, sulfur, silicon (Si), and phosphorus (P), for example, C, N, O, S, and P or C, N, O, and S. The linker may include, for example, a wide range of bonds in the range of about 2 to about 100 atoms in a continuous backbone and may be cleavable or non-cleavable.

[0120] The linker may include a cleavable form of polyethylene glycol (PEG), a non-cleavable form of PEG, polyproline, a hydrophilic amino acid, a sugar, a non-natural peptidoglycan, polyvinylpyrrolidone, a triblock copolymer including a central hydrophobic block of polypropylene glycol with hydrophilic blocks of polyethylene glycol adjacent to each side, or a combination of any two or more of the foregoing. The linker may be (PEG)3.

[0121] The term "cleavability" in the context of a linker means a linker that contains at least one bond that can be cleaved (e.g., chemically or enzymatically hydrolyzed) under physiological conditions, such as reducing substance instability, pH instability, acid instability, base instability, oxidative instability, metabolic instability, biochemical instability or enzyme instability, or p-aminobenzyl-based, polyvalent, cleavable bonds. The physiological conditions under which bond cleavage occurs do not necessarily include biological or metabolic processes, but rather may include standard chemical reactions, such as hydrolysis reactions, for example, at physiological pH or as a result of compartmentalization into an endosome having a lower pH than the cytoplasmic pH of an organelle. The cleavable bond can connect two adjacent atoms in the cleavable linker and / or connect other linker moieties or target moiety / ligand and / or drug to either one or both ends of the cleavable linker. In some cases, the cleavable linker is cleaved into two or more fragments. In some cases, the cleavable linker is separated from the target moiety / ligand. In some embodiments, the target moiety / ligand and the drug are released from each other and the drug becomes active.

[0122] The term "non-cleavable" means, in the context of a linker, a linker that includes at least one bond that is not readily or rapidly cleaved under physiological conditions. A non-cleavable linker may include a stable backbone under physiological conditions (e.g., the backbone is resistant to hydrolysis (e.g., aqueous hydrolysis or enzymatic hydrolysis)). By conjugation with a non-cleavable linker, no component of the conjugate (e.g., a target ligand (e.g., a fully amorphous (FA) ligand) and a drug (e.g., a PI3K inhibitor)) dissociates. A non-cleavable linker has the possibility of containing neither a disulfide bond (e.g., S-S) nor an ester in the backbone. The conjugate may include a target moiety / ligand and a drug that are connected by a substantially stable backbone throughout the entire period of conjugate circulation (e.g., during endocytosis into a target cell endosome). A conjugate containing a non-cleavable linker may be partially advantageous when targeting NOD-like receptors and / or other pattern recognition receptors within a cell endosome with a drug. A non-cleavable linker may include amides, esters, ethers, amines, and / or thioethers (e.g., thio-maleimide). Specific examples are provided herein, but it is understood that any molecule(s) may be used for the non-cleavable linker as long as at least one bond is formed that is not readily or rapidly cleaved under physiological conditions.

[0123] The non-cleavable linker includes a linker that, at neutral pH, for example, less than 10 percent (10%), such as less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.1%, less than 0.01% or less than 0.001% thereof, hydrolyzes in an aqueous solution (e.g., a buffered (e.g., phosphate buffered) solution) within a certain period (e.g., 24 hours). When using a non-cleavable linker, less than about 10 percent (10%) of the administered conjugate, preferably less than 5 percent (5%), may release the free drug or not release it at all (e.g., in the systemic circulation before being taken up by the targeted cell / tissue). For example, less than 5 percent (5%) of the free drug can be released from the conjugate during the systemic circulation of the compound within 1 hour after administration. This can reduce the off-target toxicity of the free drug, so this can be advantageous.

[0124] Both cleavable and non-cleavable linkers can be modified, for example, by PEGylation, etc., according to methods generally known in the art or developed later to optimize the in vivo distribution, bioavailability, and PK / PD (e.g., of the compound), and / or to increase the uptake (e.g., of the conjugate or drug) into the targeted tissue. In some embodiments, the linker (L) is configured to avoid significant release of a pharmaceutically active amount of the drug in the circulation before being captured by a cell (e.g., a macrophage in the tissue to be treated, which is the target cell).

[0125] The conjugate containing a cleavable linker can be designed to diffuse through the membrane of the endosome and, for example, into the cytoplasm of the targeted cell. The cleavable linker can be designed so that the drug does not release until the conjugate reaches the cytoplasm.

[0126] In some embodiments, the ligand (A) is attached to the linker (L) of the compound via a nitrogen atom (e.g., of the linker (L)). The ligand (A) can be attached to the linker (L) via a triazolyl or amide (e.g., of the linker (L)).

[0127] In some embodiments, the linker (L) is a cleavable linker. In some embodiments, the linker (L) is a non-cleavable linker. In some embodiments, the linker (L) comprises an optionally substituted heteroalkyl. The optionally substituted heteroalkyl is substituted with at least one substituent selected from the group consisting of alkyl, hydroxyl, acyl, polyethylene glycol (PEG), carboxylic acid, and halo. In some embodiments, the linker (L) comprises a substituted heteroalkyl having at least one disulfide bond in its backbone. In some embodiments, the linker (L) comprises a peptide or peptidoglycan having at least one disulfide bond in its backbone. In some embodiments, the linker (L) is a cleavable linker that can be cleaved by an enzymatic reaction, reactive oxygen species (ROS), or reducing conditions. In some embodiments, the linker (L) is -NH-CH2-CR 60 R 70 -S-S-CH2-CH2-O-CO-, wherein R 60 and R 70 are each independently H, alkyl, or heteroalkyl.

[0128] In some embodiments, the linker (L) is of the formula:

[0129]

Chemical formula

[0130]

Chemical formula

[0131] In some embodiments, the linker (L) is or comprises an amino acid. In some embodiments, the linker (L) is or comprises a PEG group. In some embodiments, the linker (L) is or comprises a polysaccharide. In some embodiments, the linker (L) is a group represented by the structure:

[0132]

Chemical formula

[0133] In some embodiments, the linker (L) is

[0134]

Chemical formula

[0135]

Chemical formula

[0136]

Chemical formula

[0137] In some embodiments, the linker (L) has the following structure:

[0138]

Chemical formula

[0139] In some embodiments, the linker (L) has the following structure:

[0140]

Chemical formula

[0141] In some embodiments, the linker (L) has the following structure:

[0142]

Chemical formula

[0143] In some embodiments, the linker (L) includes one or more linker groups having the following structure.

[0144]

Chemical formula

[0145] In some embodiments, the linker (L) has the following structure:

[0146] [Chem.] comprising one or more linker groups having, wherein R 16 is H or C1-C6 alkyl, and R 14a , R 14b , R 15a , and R 15b can each independently be H or C1-C6 alkyl.

[0147] In some embodiments, the linker (L) comprises the following structure.

[0148] [Chem.]

[0149] In some embodiments, the linker (L) comprises a free form of polyethylene glycol (PEG), a non-free form of PEG, polyproline, a hydrophilic amino acid, a sugar, a non-natural peptidoglycan, polyvinylpyrrolidone, or a triblock copolymer comprising a central hydrophobic block of polypropylene glycol with hydrophilic blocks of polyethylene glycol adjacent to each side.

[0150] In some embodiments, the linker (L) is a cleavable linker.

[0151] In some embodiments, the linker (L) is a non-cleavable linker.

[0152] In some embodiments, the linker (L) has the following structure:

[0153] [Chem.] comprising one or more linker groups having, wherein n3 is from 0 to 15.

[0154] In some embodiments, the linker (L) has the following structure:

[0155]

Chemical formula

[0156]

Chemical formula

[0157] In some embodiments, the linker (L) is a substituted heteroalkyl comprising at least one substituent selected from the group consisting of alkyl, hydroxyl, oxo, PEG, carboxylic acid, and halo. “Halo” or “halogen” means a fluorine, chlorine, bromine, or iodine atom, alone or as part of another substituent, unless otherwise stated.

[0158] In some embodiments, the linker (L) includes a spacer (e.g., as described anywhere in this specification). In some embodiments, the spacer includes, for example, peptidoglycan or sugar. In some embodiments, the linker (L) is a substituted heteroalkyl having at least one disulfide bond in its backbone. In some embodiments, the linker (L) is a peptide having at least one disulfide bond in its backbone. In some embodiments, the linker (L) is -CONH-CH(COOH)-CH2-S-S-CH2-CR a R b -O-CO-, -CONH-CH(COOH)CR a R b -O-CO-, -C(O)NHCH(COOH)(CH2)2-CONH-CH(COOH)CR a R b -O-CO- or -C(O)NHCH(COOH)(CH2)2-CONH-CH(COOH)-CH2-S-S-CH2-CR a R b -O-CO- and where R a and R bis independently H, alkyl or heteroalkyl (e.g., PEG).

[0159] In some embodiments, the linker (L) has the structure:

[0160]

Chemical formula

[0161] In some embodiments, the linker (L) has the structure:

[0162]

Chemical formula

[0163] In some embodiments, the linker (L) has the structure:

[0164]

Chemical formula

[0165] In some embodiments, the linker (L) includes a cleavable form of PEG, a non-cleavable form of PEG, polyproline, a hydrophilic amino acid, a sugar, a non-natural peptidoglycan, polyvinylpyrrolidone, or a triblock copolymer comprising a central hydrophobic block of polypropylene glycol with hydrophilic blocks of polyethylene glycol adjacent to each side.

[0166] In some embodiments, the SMDC further comprises a PEG group, a peptide group, a glycopeptide group, a saccharide group, a radical of an albumin-binding group, or a combination of two or more radicals of the foregoing groups, and the PEG group, the peptide group, the glycopeptide group, the saccharide group, the radical of the albumin-binding group, or a combination thereof is attached to the linker (L).

[0167] In some embodiments, the albumin-binding group is selected from the group consisting of.

[0168]

Chemical formula

[0169] Compounds and Administration Two or more SMDCs can be administered, and in some cases, the SMDC can contain various drugs. One or more SMDCs can be administered in a composition with one or more conjugates and / or unconjugated drugs. The SMDC (e.g., the drug moiety) can be used according to the methods described herein and, in some cases, can be combined with other drugs that deplete or inhibit myeloid-derived suppressor cells (e.g., by binding to a therapeutic agent for cancer) and / or other anti-cancer drugs and therapies, depending on the desired application. Also, the SMDC can be used in a synergistic combination with CAR-expressing cell cytotoxic lymphocytes as described.

[0170] Any SMDC can be prepared by conventional methods of organic synthesis practiced by those skilled in the art. The general reaction sequences outlined below represent general methods useful for the preparation of compounds and are not intended to be limiting in scope and utility.

[0171] The description of the compound is limited by the principles of chemical bonding known to those skilled in the art. Thus, when a group can be substituted by one or more of a number of substituents, such substitution is selected such that, in accordance with the principles of chemical bonding, it is not inherently unstable and / or has the potential to become unstable under environmental conditions such as aqueous conditions, neutral conditions, and some known physiological conditions. For example, heterocycloalkyl or heteroaryl is bonded to the rest of the molecule via a cyclic heteroatom in accordance with the principles of chemical bonding known to those skilled in the art, thereby avoiding the formation of an inherently unstable compound.

[0172] In some cases, the pharmaceutical composition comprises at least one SMDC that includes a conjugate, for example, a drug moiety conjugated to a ligand (e.g., a targeting moiety) via a linker such as PEG or a derivative thereof.

[0173] The conjugate can be administered by a composition that includes a unit dosage form and / or one or more pharmaceutically acceptable carriers, adjuvants, diluents, excipients, and / or vehicles, and combinations thereof. The term "administer" and this idiomatic element generally refer to any means for introducing a compound into a host subject and include, but are not limited to, routes of administration such as oral, intravenous, intramuscular, subcutaneous, transdermal, inhalation, buccal, intraocular, sublingual, intravaginal, rectal, etc.

[0174] Administration of the compound as a salt can be appropriate. Examples of acceptable salts include, but are not limited to, salts of alkali metals (e.g., sodium, potassium or lithium) or alkaline earth metals (e.g., calcium). However, any salt that is generally non-toxic and effective when administered to the subject being treated is acceptable. Similarly, "pharmaceutically acceptable salts" refer to salts with counterions that can be used in pharmaceuticals. Such salts include (1) acid addition salts that can be obtained by reacting the free base of the parent compound with inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, phosphoric acid, sulfuric acid, and perchloric acid, or organic acids such as acetic acid, oxalic acid, (D) or (L) malic acid, maleic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, tartaric acid, citric acid, succinic acid, and malonic acid, or (2) salts formed when any of the acidic protons present in the parent compound are replaced by metal ions such as alkali metal ions, alkaline earth metal ions or aluminum ions, or coordinated with organic bases such as ethanolamine, diethanolamine, triethanolamine, trimethamine, N-methylglucamine, etc., but are not limited thereto. Pharmaceutically acceptable salts are well known to those skilled in the art, and any such pharmaceutically acceptable salts are contemplated.

[0175] Permissible salts can be obtained using standard procedures known in the art, which include (by way of non-limiting example) procedures of reacting a sufficiently acidic compound with a suitable base to obtain a physiologically acceptable anion. Suitable acid addition salts are formed from acids that form non-toxic salts. Exemplary, but non-limiting, examples include acetate, aspartate, benzoate, besylate, bicarbonate / carbonate, bisulfate / sulfate, borate, camsylate, citrate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride / chloride, hydrobromide / bromide, hydroiodide / iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methylsulfate, naphthylate, 2-naphthylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate / hydrogenphosphate / dihydrogenphosphate, saccharinate, stearate, succinate, tartrate, tosylate, and trifluoroacetate. Basic salts suitable for the compounds described herein are formed from bases that form non-toxic salts. Exemplary, but non-limiting, examples include arginine salt, benzathine salt, calcium salt, choline salt, diethylamine salt, diolamine salt, glycine salt, lysine salt, magnesium salt, meglumine salt, olamine salt, potassium salt, sodium salt, tromethamine salt, and zinc salt. Acidic and basic hemisalts, such as hemisulfate and hemicalcium salt, can also be formed.

[0176] The conjugate can be formulated as a pharmaceutical composition and administered to a mammalian host, such as a human patient, in a variety of forms adapted to the selected route of administration. For example, the pharmaceutical composition can be formulated for oral or parenteral routes, intravenous route, intraarterial route, intraperitoneal route, intrathecal route, epidural route, intracerebroventricular route, intraurethral route, intrasternal route, intracranial route, intratumoral route, intramuscular route, topical route, inhalation route, and / or subcutaneous route and administered by these routes. Indeed, the conjugate or a composition containing the same can be administered directly into the bloodstream, muscle, or viscera.

[0177] For example, the conjugate can be administered systemically (e.g., orally) in combination with a pharmaceutically acceptable vehicle, such as an inert diluent or an assimilable edible carrier. For oral therapeutic administration, the conjugate can be combined with one or more excipients and used in the form of tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc. that are orally ingestible. The proportions of the compositions and preparations may vary and can be about 1% to about 99% by mass of the active ingredient(s), as well as binders, excipients, disintegrating substances, lubricants, and / or sweetening substances (known in the art). The amount of the conjugate in such therapeutically useful compositions is an amount such that an effective dosage level is obtained.

[0178] In some embodiments, the SMDC is administered as a composition comprising one or more pharmaceutically acceptable carriers, adjuvants, diluents, excipients, vehicles, or any combination of the foregoing.

[0179] For example, the preparation of the parenteral conjugate / composition under aseptic conditions by lyophilization can be readily achieved using standard pharmaceutical techniques well known to those skilled in the art. The solubility of the conjugate used in the preparation of the parenteral composition can be increased by using appropriate formulation techniques, such as the incorporation of solubility enhancing substances.

[0180] As described above, the conjugate / composition can be administered by infusion or injection (e.g., using a needle (including a micron needle) syringe and / or a needleless syringe). The solution of the composition is aqueous and may optionally be mixed with a non-toxic surfactant and / or may contain a carrier or excipient, such as salts, carbohydrates, and buffering substances (preferably at a pH of 3 to 9). However, in some applications, this solution may be more preferably formulated as a sterile non-aqueous solution or in a dry form for use in combination with a suitable vehicle, such as sterile pyrogen-free water or phosphate-buffered saline (PBS). For example, a dispersant can be prepared with glycerol, liquid PEG, triacetin, and mixtures thereof, as well as oils. Under normal conditions of storage and use, such preparations may further contain a preservative to prevent the growth of microorganisms.

[0181] Pharmaceutical dosage forms suitable for injection or infusion may include sterile aqueous solutions or dispersions containing the active ingredient, which are adapted for immediate preparation of a sterile injectable or infusible solution or dispersion, optionally encapsulated in liposomes, or sterile powders. In any case, the final dosage form must be sterile, fluid, and stable under the conditions of manufacture and storage. The liquid carrier or vehicle may be a solvent or a liquid dispersion medium, such as water, ethanol, polyols (e.g., glycerol, polyethylene glycol, liquid PEG(s), etc.), vegetable oils, non-toxic glyceryl esters, and / or suitable mixtures thereof, without limitation. In at least one embodiment, appropriate fluidity can be maintained by the formation of liposomes, maintenance of the required particle size in the case of a dispersion, or the use of a surfactant. Microbial activity can be prevented by the addition of various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In certain cases, it is desirable to include one or more isotonic substances, such as sugars, buffers, or sodium chloride. Sustained absorption of the injectable composition can be brought about by the incorporation of substances formulated to delay absorption, such as aluminum monostearate and gelatin.

[0182] The sterile injectable solution can be prepared by incorporating the conjugate into a requisite amount of a suitable solvent, optionally together with one or more of the other ingredients described above, and then filter sterilizing. In the case of a sterile powder for the preparation of a sterile injectable solution, preferred preparation methods are vacuum drying techniques and lyophilization techniques, by which powders of any further desired ingredients present in the prior sterile filtered solution in addition to the active ingredient are obtained.

[0183] For topical administration, it may be desirable to administer the conjugate to the skin as a composition or formulation in combination with a dermatologically acceptable carrier, which can be solid or liquid. In some embodiments, the solid carrier includes micronized solids such as talc, clay, microcrystalline cellulose, silica, alumina, etc. Similarly, useful liquid carriers can optionally include water, alcohol or glycol or water-alcohol / glycol mixtures in which the conjugate is soluble or dispersible to an effective level using a non-toxic surfactant. Additionally or alternatively, adjuvants such as fragrances and antibacterial substances can be added to optimize the properties for a given use. The resulting liquid composition can be applied from an absorbent pad used to impregnate a bandage and / or other dressing material, sprayed onto the target site using a pump-type or aerosol nebulizer, or simply applied directly to the desired site of the subject.

[0184] Also, thickening agents such as synthetic polymers, fatty acids, salts and esters of fatty acids, fatty alcohols, modified celluloses or modified inorganic substances can be utilized together with a liquid carrier to form an applicable paste, gel, ointment, soap, etc., which can be applied directly to the skin of the subject.

[0185] Chimeric antigen receptor (CAR)-modified cells CAR is an artificially constructed hybrid receptor protein or polypeptide, and includes an antigen-binding domain of an antibody (i.e., an antigen recognition domain), such as a single-chain variable fragment (scFv) that binds to a signaling or activation domain (i.e., a co-stimulatory domain).

[0186] Without being bound by a particular theory or mechanism, inducing an antigen-specific response against a TAA may result in one or more of the following by the CAR: targeting and destruction of TAA-expressing cancer cells, reduction or elimination of cancer cells, promotion of infiltration of immune cells into the tumor site(s), and enhancement / expansion of the anti-cancer response. Thus, the present disclosure provides a method of destroying malignant cancer cells, the method comprising contacting one or more cytotoxic lymphocytes (e.g., cytotoxic T cells, NK cells, and LAK cells) modified to express a CAR with a population of cancer cells expressing a TAA disclosed herein, whereby the CAR is produced, binds to the TAA on the cancer cells, and the cancer cells are destroyed. As discussed above, cancer treatment typically includes, among other things, chemotherapy, radiation therapy, therapeutic monoclonal antibodies, checkpoint inhibition therapy, but a high recurrence rate is common among patients receiving such treatment across various cancers. The provided composition comprises a cytotoxic lymphocyte (e.g., cytotoxic T cells, NK cells, and LAK cells) modified to express a CAR comprising a monoclonal antibody specific for a tumor-associated antigen (TAA) disclosed herein or an antigen recognition domain comprising an antigen-binding portion of this antigen. The antigen recognition domain of the CAR can be an intact antibody or an antibody fragment (e.g., scFv). An intact antibody typically consists of four polypeptides: two identical copies of a heavy (H) chain polypeptide and two identical copies of a light (L) chain polypeptide. Each of the heavy chains contains one N-terminal variable (VH) region and three C-terminal constant (CH1, CH2, and CH3) regions, and each light chain contains one N-terminal variable (VL) region and one C-terminal constant (CL) region. The variable regions of each pair of light and heavy chains form the antigen-binding site of the antibody. The VH region and the VL region have the same general structure, and each region contains four framework regions with relatively conserved sequences. The framework regions are connected by three complementarity-determining regions (CDRs). The three CDRs, known as CDR1, CDR2, and CDR3, form the "hypervariable regions" of the antibody that mediate antigen recognition and binding.

[0187] The terms "antibody fragment", "antibody fragment", "functional fragment of an antibody", and "antigen-binding portion" are used interchangeably herein and mean one or more fragments or portions of an antibody that retain the ability to specifically bind to an antigen. The antigen recognition domain of a CAR can include any TAA-binding antibody fragment capable of targeting a modified lymphocyte to a target cell. The antibody fragment preferably includes, for example, one or more CDRs, variable regions (or portions thereof), constant regions (or portions thereof), or combinations thereof. Examples of antibody fragments include: (i) a Fab fragment, which is a monovalent fragment consisting of a VL domain, a VH domain, a CL domain, and a CH1 domain; (ii) an F(ab')2 fragment, which is a divalent fragment containing two Fab fragments linked by a disulfide bridge in the hinge region; (iii) an Fv fragment consisting of a VL domain and a VH domain of a single arm of an antibody; (iv) a single-chain Fv (scFv), which is a monovalent molecule consisting of two domains of an Fv fragment (i.e., VL and VH) joined by a synthetic linker that enables the two domains to be synthesized as a single-chain polypeptide (see, for example, Bird et al., Science, 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA, 85:5879-5883 (1988); and Osbourn et al., Nat. Biotechnol., 16:778 (1998)); and (v) a diabody, which is a dimer of polypeptide chains, each polypeptide chain being too short to pair between VH and VL on the same polypeptide chain, thus promoting pairing between complementary domains on different VH-VL polypeptide chains to generate a dimer molecule having two functional antigen-binding sites, including a VH connected to a VL by a peptide linker, but not limited thereto. In some embodiments, the antigen recognition domain of a CAR includes an scFv that binds to a TAA disclosed herein.

[0188] The antigen-binding portion or fragment of the monoclonal antibody may be of any size as long as the portion binds to the target TAA. In this regard, the antigen-binding portion or fragment of the monoclonal antibody specific for the TAA desirably comprises one or more CDRs comprising about 5 to 18 (e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or a range defined by any two of the foregoing values) amino acids.

[0189] In some embodiments, the CAR comprises an antigen recognition domain that comprises the variable region of a monoclonal antibody or antigen-binding fragment thereof that binds to the TAA. The monoclonal antibody or antigen-binding fragment thereof can be obtained from or derived from a mammal including, but not limited to, a mouse, rat, or human. In some embodiments, the antigen recognition domain comprises the variable region of a mouse or human monoclonal antibody or antigen-binding fragment thereof that binds to the TAA. In this regard, the antigen recognition domain comprises the light chain variable region, the heavy chain variable region, or both the light chain variable region and the heavy chain variable region of a mouse or human monoclonal antibody or antigen-binding fragment thereof that binds to the TAA.

[0190] In some embodiments, the CARs disclosed herein comprise a signal sequence. The signal sequence can be located at the amino terminus of the antigen recognition domain (e.g., the variable region of an antibody or antigen-binding fragment thereof). The signal sequence can comprise any suitable signal sequence. In one embodiment, the signal sequence is the human granulocyte macrophage colony-stimulating factor (GM-CSF) receptor signal sequence or the CD8α signal sequence. For example, a CAR comprising a mouse scFv may comprise the GM-CSF signal sequence, while a CAR comprising a human scFv may comprise the CD8α signal sequence.

[0191] In some embodiments, the CARs disclosed herein include an extracellular spacer sequence. The extracellular spacer sequence is a short sequence of amino acids that promotes the mobility of the antibody (see, e.g., Woof et al., Nat. Rev. Immunol., 4(2): 89-99 (2004)) and can be positioned between the antigen recognition domain (e.g., scFv) and the T cell activation domain. The extracellular spacer sequence can include all or part of the extracellular region of any transmembrane protein. In some embodiments, for example, the extracellular spacer sequence is derived from the human CD8α molecule or the human CD28 molecule.

[0192] In some embodiments, the CARs disclosed herein also include a transmembrane domain. The transmembrane domain can be any transmembrane domain derived from or obtained from any molecule known in the art. For example, the transmembrane domain can be obtained from or derived from the CD8α molecule, CD28 molecule, 2B4 molecule, CD16 molecule, NKp44 molecule, NKp46 molecule, NKG2D molecule or DAP12 molecule. CD8 is a transmembrane glycoprotein that functions as a co-receptor for the T cell receptor (TCR) and is mainly expressed on the surface of cytotoxic T cells. The most common form of CD8 exists as a dimer composed of the CD8α chain and the CD8β chain. CD28 is expressed on T cells and provides a co-stimulatory signal necessary for T cell activation. CD28 is a receptor for CD80 and CD86. In some embodiments, CD8α and CD28 are human CD8α and CD28.

[0193] The CARs disclosed herein include a T cell activation domain. The T cell activation domain includes at least one intracellular (e.g., cytoplasmic) T cell signaling domain (also referred to as a "co-stimulatory domain"). The most common intracellular T cell signaling domain utilized in CARs is CD3 zeta (CD3ζ), which associates with the TCR to generate signals and contains immunoreceptor tyrosine-based activation motifs (ITAMs).

[0194] In some embodiments, the T cell activation domain comprises a plurality (e.g., two or more) of intracellular T cell signaling domains. The intracellular T cell signaling domains can be obtained from or derived from CD28 molecule, CD3ζ molecule or a modified version thereof, the gamma chain of the human high-affinity IgE receptor (FcεRI), CD27 molecule, OX40 molecule, 4-1BB molecule, ICOS molecule, CD2 molecule, CD154 molecule, DAP10 molecule, NKG2D molecule, 2B4 molecule, DAP10 molecule, DAP12 molecule, DNAM1 molecule, NKG2 molecule, NKG2D molecule, CD27 molecule, CD40L molecule, CD70 molecule, or other intracellular signaling molecules known in the art. As discussed above, CD28 is an important T cell marker in T cell co-stimulation. 4-1BB, also known as CD137, conveys a strong co-stimulatory signal to T cells, promotes differentiation, and enhances the long-term survival of T lymphocytes. CD27 is a member of the TNF receptor superfamily and is required for the generation and long-term maintenance of T cell immunity. The human high-affinity IgE receptor (FcεRI) is a tetrameric receptor complex consisting of one alpha chain, one beta chain, and two disulfide-bonded gamma chains. FcεRI is constitutively expressed on mast cells and basophils and is eosinophil-inducing. In some embodiments, the intracellular T cell signaling domain is a human intracellular T cell signaling domain.

[0195] In some embodiments, the CAR comprises any one of the transmembrane domains described above, and any one or more (e.g., 1, 2, 3, or 4) of the intracellular T cell signaling domains described above, in any combination. In some embodiments, the CAR comprises the CD28 transmembrane domain and the intracellular T cell signaling domains of CD28 and CD3ζ. In some embodiments, the CAR comprises the CD8α transmembrane domain, and the intracellular T cell signaling domains of CD28, CD3ζ, the gamma chain of FcεRI, and / or 4-1BB. In another embodiment, the CAR may comprise the CD8α transmembrane domain, and the intracellular T cell signaling domains of CD28, CD3ζ, and CD27. In some embodiments, the CAR comprises the CD28 transmembrane domain, and the intracellular T cell signaling domains of CD27, 4-1BB, and the gamma chain of FcεRI.

[0196] In some embodiments, the disclosed CARs and cytotoxic lymphocytes (e.g., cytotoxic T cells, NK cells, and LAK cells) that express them, which include any one of the above extracellular spacers, any one of the transmembrane domains, and any one of the intracellular T cell signaling domains in any combination, can be directed against any target molecule of interest (i.e., can incorporate any antigen recognition domain). In some embodiments, the antigen recognition domain specifically binds to a TAA. In some embodiments, the TAA is selected from the group consisting of CD19, CD20, CD30, CD3, CD4, CD5, BCMA, GD2, LEY, MS4A1, CD22, TNFRSF17, CD38, SDC1, TNFRSF8, IL3RA, CD7, NCAM1, CD34, CLEC12A, CD4, MME, CD5, SLAMF7, IL1RAP, FCGR3A, ITGB7, TNFRSF13B, TRBC1, CD33, ROR1, MUC1, KLRK1, KIT, CD274, CD70, PROM1, AFP, AXL, CD80, CD86, DLL3, TNFRSF10B, FAP, MAGEA1, MAGEA4, MUC16, PMEL, ROR2, KDR, EPHA2, L1CAM, CLDN18, PSCA, FOLR1, IL13RA2, MET, EPCAM, EGFR, EGFRVIII, FOLH1, GPC3, CEACAM5, ERBB2, CAIX, B4GALNT1, NY-ESO-1, PD-L1, and MSLN.

[0197] In some embodiments, the CAR comprises (i) an antigen recognition domain that binds to a TAA, (ii) an extracellular spacer, (iii) a transmembrane domain derived from the human CD8α molecule, and (iv) an intracellular T cell signaling domain derived from the human CD3 zeta (CD3ζ) molecule and the human CD28 molecule. In some embodiments, the CAR comprises (i) an antigen recognition domain that binds to a TAA, (ii) an extracellular spacer, (iii) a transmembrane domain derived from the human CD8α molecule, and (iv) an intracellular T cell signaling domain derived from the human CD28 molecule, the human CD27 molecule, and the human CD3ζ molecule. In some embodiments, the CAR comprises (i) an antigen recognition domain that binds to a TAA, (ii) an extracellular spacer, (iii) a transmembrane domain derived from the human CD8α molecule, and (iv) an intracellular T cell signaling domain derived from the human CD28 molecule, the human CD27 molecule, and the gamma chain of FcεRI. In some embodiments, the CAR comprises (i) an antigen recognition domain that binds to a TAA, (ii) an extracellular spacer, (iii) a transmembrane domain derived from the human CD8α molecule, and (iv) an intracellular T cell signaling domain derived from the human CD28 molecule and the gamma chain of FcεRI.

[0198] The CAR can comprise any number of amino acids, provided that the CAR retains its biological activity, such as the ability to specifically bind to an antigen, mediate cytotoxic activity, detect diseased cells in a mammal, or treat or prevent a disease in a mammal. In some embodiments, the CAR comprises more than 50 amino acids (e.g., more than 60 amino acids, more than 100 amino acids or more than 500 amino acids) and less than 1,000 amino acids (e.g., 900 amino acids or less, 800 amino acids or less, 700 amino acids or less or 600 amino acids or less). In some embodiments, the CAR comprises from about 50 to about 700 amino acids (e.g., about 70, about 80, about 90, about 150, about 200, about 300, about 400, about 550 or about 650 amino acids), from about 100 to about 500 amino acids (e.g., about 125, about 175, about 225, about 250, about 275, about 325, about 350, about 375, about 425, about 450 or about 475 amino acids) or a range defined by any two of the foregoing values.

[0199] Provides a functional portion of the CAR. The term "functional portion" when used in reference to a CAR refers to any portion or fragment of the CAR that retains the biological activity of the CAR of which it is a part (the parent CAR). Functional portions include, for example, CAR portions that recognize target cells to about the same extent, to the same extent or to a greater extent as the parent CAR, or retain the ability to detect, treat or prevent a disease. With respect to the nucleic acid sequence encoding the parent CAR, in some embodiments, the nucleic acid sequence encoding the functional portion of the CAR encodes a protein that comprises, for example, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95% or more of the parent CAR.

[0200] In some embodiments, the functional portion of the CAR includes additional amino acids at the amino or carboxy terminus or both termini of this portion, and these additional amino acids are not found in the amino acid sequence of the parental CAR. Desirably, the additional amino acids do not interfere with the biological function of the functional portion, such as recognition of target cells, mediation of cytotoxic activity, detection of cancer, treatment or prevention of cancer, etc. More desirably, the additional amino acids enhance the biological activity of the CAR as compared to the biological activity of the parental CAR.

[0201] Also provided are functional variants of the CAR. The term "functional variant" refers to a CAR, polypeptide or protein having substantial or significant sequence identity or similarity to the CAR, and this functional variant retains the biological activity of the CAR that is being variant. Functional variants include, for example, variants of the CAR (the parental CAR) that retain the ability to recognize target cells to approximately the same extent, the same extent or a higher degree as the parental CAR. With respect to the nucleic acid sequence encoding the parental CAR, in some embodiments, the nucleic acid sequence encoding the functional variant of the CAR is about 10%, about 25%, about 30%, about 50%, about 65%, about 80%, about 90%, about 95% or about 99% identical to the nucleic acid sequence encoding the parental CAR.

[0202] In some embodiments, the functional variant comprises an amino acid sequence of a CAR having a conservative substitution of at least one amino acid. The phrase "conservative substitution of an amino acid" or "conservative mutation" refers to the substitution of one amino acid with another amino acid having common characteristics. A functional way to define the common characteristics between individual amino acids is to analyze the normalized frequency of amino acid changes between corresponding proteins of homologous organisms (Schulz, G. E. and Schirmer, R. H., Principles of Protein Structure, Springer-Verlag, New York (1979)). According to such an analysis, groups of amino acids are defined when a certain group of amino acids are preferentially exchanged with each other, so that the effects on the overall protein structure can be most similar to each other (Schulz, G. E. and Schirmer, R. H., see above). Examples of conservative mutations include amino acid substitutions within the same subgroup of amino acids, for example, lysine for arginine or vice versa so that the positive charge can be maintained, glutamic acid for aspartic acid or vice versa so that the negative charge can be maintained, serine for threonine so that the free -OH can be maintained, and glutamine for asparagine so that the free -NH2 can be maintained.

[0203] Alternatively, or in addition, in some embodiments, the functional variant comprises an amino acid sequence of a parental CAR having a non-conservative substitution of at least one amino acid. "Non-conservative mutation" includes substitutions of amino acids between various groups, for example, lysine for tryptophan or phenylalanine for serine, etc. In this case, for non-conservative substitutions of amino acids, it is preferably not to interfere with or inhibit the biological activity of the functional variant. The biological activity of the functional variant can be enhanced by non-conservative substitutions of amino acids so that the biological activity of the functional variant is increased compared to the parental CAR.

[0204] In some embodiments, the CAR or this functional portion or variant also includes synthetic amino acids in place of one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, α-amino-n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3-hydroxyproline and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N'-,N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine.

[0205] The CAR (including this functional portion and functional variants) can be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized via, for example, disulfide bridging, or converted to an acid addition salt, and / or optionally, dimerized or polymerized, or conjugated.

[0206] CAR-T cells T cells are a major type of lymphocyte that mature in the thymus and have various roles in the immune system, including the identification of specific foreign antigens in the body and the MHC class I-restricted activation and inactivation of other immune cells. T cells can be genetically modified to express a chimeric antigen receptor (CAR). The CAR can redirect T cell specificity and reactivity to a selected target in a non-MHC-restricted manner, leveraging the antigen-binding properties of monoclonal antibodies. Non-MHC-restricted antigen recognition confers upon CAR-expressing T cells the ability to recognize antigens independent of antigen processing, thereby circumventing a major mechanism of immune evasion. Moreover, when expressed in T cells, the CAR preferably does not dimerize with the alpha and beta chains of the endogenous T cell receptor (TCR). Accordingly, provided are modified T cells engineered to express a CAR targeting a tumor-associated antigen (TAA).

[0207] T cells suitable for modification with a CAR construct can be any T cell, such as a cultured T cell, for example, a primary T cell, a T cell derived from a cultured T cell line, or a T cell obtained from a mammal. When obtained from a mammal, T cells can be obtained from a number of sources including, without limitation, blood, bone marrow, lymph nodes, the thymus, or other tissues or body fluids. Additionally, T cells can be concentrated or purified. T cells are preferably human T cells (e.g., isolated from a human). In some embodiments, the T cell is a T cell at any stage of development, CD4 + / CD8 + double positive T cells, CD4 + helper T cells, such as Th1 and Th2 cells, CD8 + T cells (e.g., cytotoxic T cells), tumor infiltrating cells, memory T cells, naive T cells, and the like, but are not limited thereto. In some embodiments, the T cell is a CD8 + T cell or a CD4 +It is a T cell. T cell lines are available, for example, from preservation institutions such as the American Type Culture Collection (ATCC, Manassas, VA) and the German Collection of Microorganisms and Cell Cultures (DSMZ), and examples include Jurkat cells (ATCC TIB-152), Sup-T1 cells (ATCC CRL-1942), RPMI8402 cells (DSMZ ACC-290), Karpas45 cells (DSMZ ACC-545), and derivatives thereof. Examples of TAA targets that can be CAR-T cells include, but are not limited to, CD19, CD20, CD30, BCMA, GD2, LEY, MS4A1, CD22, TNFRSF17, CD38, SDC1, TNFRSF8, IL3RA, CD7, NCAM1, CD34, CLEC12A, CD4, MME, CD5, SLAMF7, IL1RAP, FCGR3A, ITGB7, TNFRSF13B, TRBC1, CD33, ROR1, MUC1, KLRK1, KIT, CD274, CD70, PROM1, AFP, AXL, CD80, CD86, DLL3, TNFRSF10B, FAP, MAGEA1, MAGEA4, MUC16, PMEL, ROR2, KDR, EPHA2, L1CAM, CLDN18, PSCA, FOLR1, IL13RA2, MET, EPCAM, EGFR, EGFRVIII, FOLH1, GPC3, CEACAM5, ERBB2, CAIX, B4GALNT1, NY-ESO-1, PD-L1, and MSLN.

[0208] CAR-NK cells Natural killer (NK) cells represent an important part of innate immunity. Unlike T cells, NK cells can initiate antitumor cytotoxicity without prior sensitization and may have a lower potential for complications due to cytokine release syndrome and off-target / off-tumor effects. NK cells are a subpopulation of lymphocytes with spontaneous cytotoxicity against a variety of tumor cells, virus-infected cells, and some normal cells in the bone marrow and thymus. NK cells are important effectors of the early innate immune response against transformed and virus-infected cells. NK cells account for approximately 10% of lymphocytes in human peripheral blood. When lymphocytes are cultured in the presence of interleukin 2 (IL-2), a strong cytotoxic reactivity occurs. NK cells can be detected in humans by specific surface markers, such as CD16, CD56, and CD8. NK cells do not express the T cell antigen receptor, the pan-T marker CD3, or the surface immunoglobulin B cell receptor.

[0209] Stimulation of NK cells is achieved by cross-talk of signals derived from activating and inhibitory receptors on the cell surface. The activation state of NK cells is regulated by the balance of intracellular signals received from activating and inhibitory receptors encoded by numerous germline cells. When NK cells encounter abnormal cells (e.g., tumor or virus-infected cells) and activating signals predominate, NK cells can rapidly induce apoptosis of target cells by specific secretion of cytolytic granules containing perforin and granzyme or by association of death domain-containing receptors. Activated NK cells can also secrete type I cytokines, such as interferon γ, tumor necrosis factor α, and granulocyte macrophage colony-stimulating factor (GM-CSF), which activate both innate and adaptive immune cells as well as other cytokines. Production of such soluble factors by NK cells in the early innate immune response significantly affects the recruitment and function of other hematopoietic cells. Also, through physical contact and cytokine production, NK cells play a central role in the regulatory cross-talk network by dendritic cells and neutrophils, promoting or suppressing the immune response. Since the signaling activation mechanisms are shared by T cells and NK cells, CAR constructs containing the CD3-ζ activation domain can also activate NK cells.

[0210] Accordingly, provided are NK cells modified to express a CAR construct (CAR-NK cells). The CAR-NK cells can target any suitable TAA. In certain embodiments, the CAR-NK cells target a TAA selected from the group consisting of CD3, CD4, and CD5. In certain embodiments, the CAR-NK cells can include a co-stimulatory domain as described in the preceding section. In certain embodiments, the CAR-NK cells include a co-stimulatory domain selected from the group consisting of co-stimulatory domains derived from the CD28 molecule, 2B4 molecule, 4-1BB molecule, DNAM1 molecule, DAP10 molecule, DAP12 molecule, NKG2 molecule, and NKG2D molecule. In certain embodiments, the CAR-NK cells can include a transmembrane domain as described in the preceding section. In some embodiments, the CAR-NK cells include a transmembrane domain selected from the group consisting of transmembrane domains derived from the CD8 molecule, CD28 molecule, DAP12 molecule, 2B4 molecule, NKp44 molecule, NKp46 molecule, and NKG2D molecule.

[0211] The NK cells can be any NK cells, for example, NK cells derived from umbilical cord blood, peripheral blood, bone marrow, CD34 +It can be a cell or iPSC. In some embodiments, the NK cells are obtained by methods well known in the art from human peripheral blood mononuclear cells (PBMC), unstimulated leukapheresis preparations (PBSC), human embryonic stem cells (hESC), induced pluripotent stem cells (iPSC), bone marrow or umbilical cord blood. Specifically, NK cells can be isolated from umbilical cord blood (CB), peripheral blood (PB), bone marrow or stem cells. In some embodiments, the immune cells are isolated from stored CB. In some embodiments, the CB is stored from 2, 3, 4, 5, 6, 7, 8, 10 or more units. In some embodiments, the immune cells are autologous or allogeneic. In some embodiments, the isolated NK cells are of a haplotype compatible with the subject to whom the cell therapy is administered. NK cells can be detected by specific surface markers, such as CD16, CD56, and CD8, in humans without CD3 expression. In some embodiments, the starting population of NK cells is obtained by isolating mononuclear cells using Ficoll density gradient centrifugation. The cell culture may deplete any cells expressing CD3, CD14, and / or CD19 cells, and characterize to CD56 + / CD3 - and determine the proportion of cells or NK cells.

[0212] CAR-LAK cells Lymphokine-activated killer (LAK) cells can be any LAK cells, such as LAK cells derived from PBMC. LAK cells represent a complex population of CD3 - NK cells and CD3 + T cells. The characteristic of LAK cells is the ability to lyse various tumor cells in a non-MHC-restricted manner. In addition, LAK cells can kill class I-negative target cells, such as Daudi and K562, which serves as a general standard for the identification of non-MHC-restricted cytotoxic effector cells. Fractionate the LAK population by CD3 - fraction and CD3 +By separating into fractions, it has been shown that both NK cells and T cells are capable of lysing class I negative target cells and a wide variety of tumor cells. Moreover, LAK-T cells can also be inhibited by the expression of certain MHC class I molecules on the target cells. In some embodiments, the LAK cells are LAK-T cells. In some embodiments, the LAK-T cells are activated LAK-T cells of the CD4+ or CD8+ subtype.

[0213] Nucleic acid The present disclosure further provides an isolated or purified nucleic acid sequence encoding a CAR. The term "nucleic acid sequence" is intended to encompass polymers of DNA or RNA, i.e., polynucleotides, which may be single-stranded or double-stranded and may contain unnatural or modified nucleotides. The terms "nucleic acid" and "polynucleotide" refer to a polymeric form of nucleotides of any length, either ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). Such terms refer to the primary structure of the molecule and thus include double-stranded DNA and single-stranded DNA as well as double-stranded RNA and single-stranded RNA. The term includes any analogs of RNA or DNA generated from nucleotide analogs, as well as modified polynucleotides, for example, but not limited to, methylated and / or capped polynucleotides, as equivalents.

[0214] The CARs disclosed herein can be generated using methods known in the art. For example, nucleic acid sequences, polypeptides, and proteins can be recombinantly generated using standard recombinant DNA methods (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, N Y, 1994). Additionally, synthetically generated nucleic acid sequences encoding CARs can be isolated and / or purified from sources such as plants, bacteria, insects, or mammals, e.g., rats, humans, etc. Isolation and purification methods are well-known in the art. Alternatively, the nucleic acid sequences described herein can be commercially synthesized. In this regard, nucleic acid sequences can be synthesized, recombined, isolated, and / or purified.

[0215] Nucleic acid vector Also provided are vectors comprising nucleic acid sequences encoding CARs. In some embodiments, the vector is a plasmid, cosmid, viral vector (e.g., retrovirus or adenovirus), or phage. Suitable vectors and vector preparation methods are well-known in the art.

[0216] In addition to the nucleic acid sequence encoding the CAR, the vector, in some embodiments, includes regulatory sequences that effect the expression of the nucleic acid sequence in a host cell, such as, for example, a promoter, enhancer, polyadenylation signal, transcription terminator, internal ribosome entry site (IRES), etc. Examples of regulatory sequences suitable for incorporation into expression vectors are well-known in the art.

[0217] Numerous promoters, including constitutive promoters, inducible promoters, and repressive promoters from a variety of sources, are well known in the art. Representative sources of promoters include, for example, viruses, mammals, insects, plants, yeast, and bacteria, and suitable promoters from such sources are readily available from depositories such as the ATCC and other commercial or personal sources, or can be generated synthetically based on published sequences. Promoters can be unidirectional (i.e., initiate transcription in one direction) or bidirectional (i.e., initiate transcription in either the 3' or 5' direction). Non-limiting examples of promoters include, for example, the T7 bacterial expression system, the pBAD(araA) bacterial expression system, the cytomegalovirus (CMV) promoter, the SV40 promoter, and the RSV promoter. Examples of inducible promoters include, for example, the Tet system, the ecdysone induction system, the T-REX™ system (Invitrogen, Carlsbad, CA), the LACSWITCH™ system (Stratagene, San Diego, CA), and the Cre-ERT tamoxifen-inducible recombinase system.

[0218] The term "enhancer" refers to, for example, a DNA sequence that increases the transcription of a nucleic acid sequence to which it is operably linked. An enhancer may be located many kilobases away from the coding region of a nucleic acid sequence and can mediate the binding of regulatory factors, the formation of DNA methylation patterns, or changes in DNA structure. A number of enhancers from a variety of sources are well known in the art and are available as cloned polynucleotides or within cloned polynucleotides (e.g., from depositories such as the ATCC and other commercial or personal sources). Many polynucleotides, including promoters (e.g., the commonly used CMV promoter), also contain enhancer sequences. An enhancer can be located upstream, within, or downstream of a coding sequence. The term "Ig enhancer" refers to enhancer elements derived from enhancer regions located within the immunoglobulin (Ig) locus (such enhancers include, for example, the heavy chain (mu) 5' enhancer, the light chain (kappa) 5' enhancer, the kappa intron enhancer and the mu intron enhancer, as well as the 3' enhancer).

[0219] In some embodiments, the vector contains a "selectable marker gene". The term "selectable marker gene" refers to a nucleic acid sequence that enables a cell expressing the nucleic acid sequence to be specifically selected for or against in the presence of a corresponding selection substance. Suitable selectable marker genes are known in the art.

[0220] In some embodiments, the vector is an "episomal expression vector" or "episome", which is capable of replicating within a host cell and persists as an extrachromosomal segment of DNA within the host cell in the presence of an appropriate selection pressure. Representative commercially available episomal expression vectors include, but are not limited to, episomal plasmids that utilize Epstein-Barr nuclear antigen 1 (EBNA1) and the Epstein-Barr virus (EBV) origin of replication (oriP). Vectors pREP4, pCEP4, pREP7, and pcDNA3.1 from Invitrogen (Carlsbad, CA) and pBK-CMV from Stratagene (La Jolla, CA) represent non-limiting examples of episomal vectors that use the T antigen and the SV40 origin of replication instead of EBNA1 and oriP.

[0221] Other suitable vectors include integration of an expression vector that can randomly integrate into the host cell's DNA or can contain recombination sites to allow for specific recombination between the expression vector and the host cell's chromosome. In such integration of an expression vector, endogenous expression control sequences of the host cell's chromosome can be utilized to effect expression of the desired protein. Examples of vectors that integrate site-specifically include, for example, components of the flp-in system from Invitrogen (Carlsbad, CA) (e.g., pcDNA™5 / FRT) or the cre-lox system, such as those found in pExchange-6CoreVectors from Stratagene (La Jolla, CA). Examples of vectors that integrate randomly into the host cell chromosome include, for example, pcDNA3.1 from Invitrogen (Carlsbad, CA) (when introduced in the absence of the T antigen) and pCI or pFN10A(ACT)FLEXI™ from Promega (Madison, WI).

[0222] Furthermore, the viral vectors can be used in combination with the methods and compositions disclosed herein. Representative viral expression vectors include, but are not limited to, adenovirus-based vectors (e.g., the Per.C6 system based on adenovirus available from Crucell, Inc. (Leiden, Netherland)), lentivirus-based vectors (e.g., pLP1 based on lentivirus from Life Technologies (Carlsbad, CA)), and retroviral vectors (e.g., pFB-ERV plus pCFB-EGSH from Stratagene (La Jolla, CA)). In some embodiments, the viral vector is a lentiviral vector.

[0223] A vector containing a nucleic acid encoding a CAR can be introduced into a host cell capable of expressing the CAR, including any suitable prokaryotic or eukaryotic cell. Preferred host cells are those that can be easily and reliably grown, have a reasonably rapid growth rate, have a well-characterized expression system, and can be easily and efficiently transformed or transfected.

[0224] host cell The term "host cell" refers to any type of cell that can contain an expression vector. The host cell can be a eukaryotic cell, such as a plant, animal, fungal, or algal cell, or a prokaryotic cell, such as a bacterial or protozoan cell. The host cell can be a cultured cell or a primary cell, i.e., can be isolated directly from an organism, such as a human. The host cell can be an adherent cell or a suspension cell, i.e., a cell grown by suspension. Suitable host cells are known in the art and include, by way of example, DH5α Escherichia coli (E. coli) cells, Chinese hamster ovary cells, simian VERO cells, COS cells, HEK293 cells, and the like. For the purpose of amplifying or replicating a recombinant expression vector, the host cell can be a prokaryotic cell, such as a DH5α cell. For the purpose of generating a recombinant CAR, the host cell can be a mammalian cell. In some embodiments, the host cell is a human cell. The host cell can be of any cell type, can be derived from any type of tissue, and can be at any developmental stage. In some embodiments, the host cell is a peripheral blood lymphocyte (PBL), a peripheral blood mononuclear cell (PBMC), a NK cell, a LAK cell, or a T cell. In some embodiments, the host cell is a T cell. In some embodiments, the host cell is a NK cell. In some embodiments, the host cell is a LAK cell. Methods for selecting suitable mammalian host cells, as well as methods for cell transformation, culture, amplification, screening, and purification are known in the art.

[0225] Provided are isolated cytotoxic lymphocytes (e.g., cytotoxic T cells, NK cells, and LAK cells) that express a nucleic acid sequence encoding a CAR.

[0226] Method for introducing a CAR-encoding nucleic acid into a host cell The nucleic acid sequence encoding the CAR can be introduced into cells by "transfection", "transformation" or "transduction". The terms "transfection", "transformation", and "transduction" refer to the introduction of one or more exogenous polynucleotides into a host cell by using physical or chemical methods. Many transfection techniques are known in the art, for example, calcium phosphate DNA coprecipitation (see, e.g., Murray E. J. (ed.), Methods in Molecular Biology, Vol. 7, Gene Transfer and Expression Protocols, Humana Press (1991)); DEAE-dextran; electroporation; cationic liposome-mediated transfection; tungsten particle-facilitated microparticle bombardment (Johnston, Nature, 346: 776-777 (1990)); and strontium phosphate DNA coprecipitation (Brash et al., Mol. Cell Biol., 7: 2031-2034 (1987)). Phage or viral vectors may be introduced into host cells after the growth of infectious particles in suitable packaging cells, and many of these are commercially available.

[0227] One or more isolated cytotoxic lymphocytes (e.g., T cells, NK cells or LAK cells) that express a nucleic acid sequence encoding a CAR can be contacted ex vivo, in vivo or in vitro with a population of cancer cells that express a TAA. In some embodiments, the method comprises an ex vivo component and an in vivo component. In this regard, for example, the isolated cytotoxic lymphocyte cells modified with a CAR may be cultured ex vivo under conditions that express the nucleic acid sequence encoding the CAR and then directly transplanted into a mammal (e.g., a human) suffering from cancer. Such a cell transplantation method is referred to in the art as "adoptive cell transfer (ACT)". These immune-derived cells are passively transplanted into a new recipient host to transfer the functionality of the donor immune-derived cells to the new host. Adoptive cell transfer methods for treating various cancers are known in the art, for example, as disclosed in Gattinoni et al., Nat. Rev. Immunol., 6(5): 383-393 (2006); June, C H, J. Clin. Invest., 117(6): 1466-76 (2007); Rapoport et al., Blood, 117(3): 788-797 (2011); and Barber et al., Gene Therapy, 18: 509-516 (2011).

[0228] Administration of CAR-Modified Cells When administering CAR-modified cells to a mammal, the cells can be allogeneic or autologous to the mammal. In an "autologous" administration method, cells (e.g., hematopoietic stem cells or lymphocytes) are removed from a mammal, stored (optionally, modified), and readministered to the same mammal. In an "allogeneic" administration method, a mammal is administered cells (e.g., hematopoietic stem cells or lymphocytes) from a donor that is genetically similar but not identical. Preferably, the cells are autologous to the mammal.

[0229] The CAR-modified cells are desirably administered to a human in the form of a composition, such as a pharmaceutical composition, which comprises a population of modified cells expressing the CAR and one or more (e.g., 1, 2, 3 or more) of the SMDC agents. Alternatively, the nucleic acid sequence encoding the CAR, or a vector comprising the CAR-encoding nucleic acid sequence, can be formulated into a composition, such as a pharmaceutical composition, and administered to a human. In addition to one or more (e.g., 1, 2, 3 or more) of the SMDC agents, the nucleic acid sequence encoding the CAR, and / or a population of cytotoxic lymphocytes modified to express the CAR, the pharmaceutical composition can further comprise other pharmaceutically active substances or drugs, such as chemotherapeutic agents, such as asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc. In certain embodiments, the pharmaceutical composition comprises isolated T cells expressing the CAR, more preferably a population of T cells expressing the CAR. In certain embodiments, the pharmaceutical composition comprises isolated NK cells expressing the CAR, more preferably a population of NK cells expressing the CAR. In certain embodiments, the pharmaceutical composition comprises isolated LAK cells expressing the CAR, more preferably a population of LAK cells expressing the CAR.

[0230] The choice of carrier is determined in part by the particular CAR, CAR-encoding nucleic acid sequence, vector or host cell expressing the CAR, as well as by the particular method used to administer the CAR-encoding nucleic acid sequence, vector or host cell expressing the CAR. Accordingly, a variety of suitable formulations of the pharmaceutical composition exist. For example, the pharmaceutical composition can contain a preservative. Suitable preservatives can include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. A mixture of two or more preservatives can optionally be used. The preservative or this mixture is typically present in an amount of about 0.0001% to about 2% by mass of the total composition.

[0231] Typical amounts of host cells to be administered to a mammal (e.g., a human) can range, for example, from 1 million to 100 billion cells, although amounts below or above this exemplary range are also within the scope of the present disclosure. For example, the daily dose of host cells can be from about 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values), preferably from about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the foregoing values), more preferably from about 100 million to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells, or a range defined by any two of the foregoing values).

[0232] Modified T cell receptor (TCR) T cells The T cell receptor (TCR) is CD4 + T cells and CD8 + naturally expressed by T cells. The TCR is designed to recognize short peptide antigens that complex with major histocompatibility complex (MHC) molecules and are presented on the surface of antigen-presenting cells (in humans, MHC molecules are also known as human leukocyte antigens (HLAs)). CD8 + T cells, also known as cytotoxic T cells, specifically recognize peptides that bind to MHC class I and generally mediate the discovery and destruction of diseased cells. CD8 + T cells can destroy cancerous cells as well as virus-infected cells, but the affinity of the TCR expressed by native repertoires of cancer-specific T cells is typically low due to thymic selection, meaning that cancerous cells frequently evade detection and destruction.

[0233] Natural TCRs are heterodimeric cell surface proteins of the immunoglobulin superfamily that associate with invariant proteins of the CD3 complex involved in mediating signal transduction. TCRs exist in αβ and γδ forms, which are structurally similar but have distinct anatomical locations and functions. MHC class I and class II ligands are also immunoglobulin superfamily proteins, specialized for antigen presentation and having highly polymorphic peptide-binding sites that enable the presentation of a diverse array of short peptide fragments on the surface of antigen-presenting cells (APCs). The extracellular portions of natural heterodimeric αβ TCRs and γδ TCRs consist of two polypeptides, each of which has a membrane-proximal extracellular constant domain and a membrane-distal variable region. Each of the extracellular constant domain and variable region contains intrachain disulfide bonds. The variable region contains highly polymorphic loops similar to the complementarity-determining regions (CDRs) of antibodies. The CDR3 of the αβ TCR interacts with the peptide presented by MHC, and the CDR1 and CDR2 of the αβ TCR interact with the peptide and MHC.

[0234] Immunotherapeutic approaches targeting the enhancement of cancer recognition by T cells offer a very promising strategy for the development of effective anticancer therapies. A recently proposed variant of adoptive T cell therapy is the use of gene therapy techniques to introduce TCRs specific for known cancer-specific MHC peptide complexes into T cells of cancer patients. Accordingly, provided are compositions and methods for immunotherapy using modified TCR-T cells in combination with one or more (e.g., 1, 2, 3 or more) SMDC agents. Compositions containing modified TCR-T cells can be used to target abnormal cells presenting cancer-specific MHC complexes.

[0235] The provided compositions and methods include an SMDC and a population of modified TCR-expressing cytotoxic lymphocytes (e.g., T cells, e.g., cytotoxic T cells, etc.) that specifically bind to and target TAAs disclosed herein.

[0236] TCRs can be naturally occurring, non-natural, and / or modified. A TCR can have two or more mutations in its α-chain variable domain and / or β-chain variable domain compared to a parental TCR. "Modified TCR" and "mutant TCR" are used interchangeably herein and generally refer to a TCR in which one or more mutations have been introduced into its α-chain variable domain and / or β-chain variable domain compared to a parental TCR. In some embodiments, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) mutations are incorporated into the α-chain variable domain and / or β-chain variable domain.

[0237] A TCR can be an αβ heterodimer. A TCR can be in a single-chain format. The single-chain format includes, without limitation, αβ TCR polypeptides of the Vα-L-Vβ, Vβ-L-Vα, Vα-Cα-L-Vβ, Vα-L-Vβ-Cβ, or Vα-Vα-L-Vβ-Cβ type, where Vα and Vβ are the TCRα variable region and TCRβ variable region, respectively, Cα and Cβ are the TCRα constant region and TCRβ constant region, respectively, and L is a linker sequence. One or both of the constant domains can be full-length or truncated as described above and / or can contain mutations. The α-chain extracellular constant domain can naturally have an asparagine (N) or lysine (K) residue at the fourth position due to polymorphism. In some embodiments, disulfide bonds are introduced between the residues of each constant domain in the single-chain TCR. As will be apparent to those skilled in the art, it may be possible to cleave, for example, 1, 2, 3, 4, 5 or more residues of the sequences located at the C-terminus and / or N-terminus of the TCR without substantially affecting its binding properties.

[0238] The α-β heterodimeric TCR typically includes the α-chain TRAC constant domain sequence and / or the β-chain TRBC1 or TRBC2 constant domain sequence. The α- and β-chain constant domain sequences can be modified by cleavage or substitution to remove the native disulfide bond between Cys4 of exon 2 of TRAC and Cys2 of exon 2 of TRBC1 or TRBC2. The α-chain constant domain sequence and / or the β-chain constant domain sequence(s) can be modified, for example, by substitution of cysteine residues for Thr48 of TRAC and Ser57 of TRBC1 or TRBC2, and the aforementioned cysteines form a disulfide bond between the α-constant domain and the β-constant domain of the TCR. In addition, TRBC1 or TRBC2 can include a mutation from cysteine to alanine at position 75 of the constant domain and a mutation from asparagine to aspartic acid at position 89 of the constant domain. Additionally, or alternatively, the constant domain can include further mutations, substitutions or deletions compared to the native TRAC sequence and / or TRBC1 / 2 sequence. The terms TRAC and TRBC1 / 2 include natural polymorphic variants, such as the substitution of N to K at position 4 of TRAC, etc. Also provided are variants, fragments, and derivatives of the TCR.

[0239] In some embodiments, the TCR targets TAAs that are expressed within cancer cells. In some embodiments, the TAA is a cell surface protein. In some embodiments, the TAA is a transmembrane protein. In some embodiments, the TAA is an intracellular protein. In some embodiments, the TAA is identified by isolating cancer cells from a subject (e.g., a human) and identifying TAAs that are selectively expressed or upregulated in the cancer cells. After identifying the target, a TCR display library can be constructed to screen for TCRs with high affinity and specificity for the TAA. Generally, any antigen that can be presented by MHC molecules, including intracellular and cell surface antigens, can be recognized by TCR-T cells. In some embodiments, the TAA is selected from the group consisting of CD19, CD20, CD30, CD3, CD4, CD5, BCMA, GD2, LEY, MS4A1, CD22, TNFRSF17, CD38, SDC1, TNFRSF8, IL3RA, CD7, NCAM1, CD34, CLEC12A, CD4, MME, CD5, SLAMF7, IL1RAP, FCGR3A, ITGB7, TNFRSF13B, TRBC1, CD33, ROR1, MUC1, KLRK1, KIT, CD274, CD70, PROM1, AFP, AXL, CD80, CD86, DLL3, TNFRSF10B, FAP, MAGEA1, MAGEA4, MUC16, PMEL, ROR2, KDR, EPHA2, L1CAM, CLDN18, PSCA, FOLR1, IL13RA2, MET, EPCAM, EGFR, EGFRVIII, FOLH1, GPC3, CEACAM5, ERBB2, CAIX, B4GALNT1, NY-ESO-1, PD-L1, and MSLN.

[0240] For some purposes, TCRs can be aggregated into complexes containing several TCRs to form multivalent TCR complexes. There are many human proteins containing multimerization domains that can be used in the generation of multivalent TCR complexes. For example, the tetramerization domain of p53 has been utilized to generate tetramers of scFv antibody fragments, which show improved serum persistence and significantly reduced off-rate toxicity compared to monomeric scFv fragments. Hemoglobin also has a tetramerization domain that can potentially be used for this type of application.

[0241] As is well known in the art, TCRs can undergo post-translational modifications. Glycosylation is one such modification and involves the covalent attachment of an oligosaccharide moiety to defined amino acids within the TCR chain. For example, asparagine residues or serine / threonine residues are well-known sites for oligosaccharide attachment. The glycosylation state of a particular protein depends on many factors, including the protein sequence, protein conformation, and the availability of specific enzymes. Moreover, the glycosylation state (e.g., the type of oligosaccharide, covalent linkages, and total number of linkages) can affect the function of the protein. Therefore, it is often desirable to control glycosylation when producing recombinant proteins. Control of glycosylation is used to improve antibody-based therapies. Such modifications are desirable because glycosylation improves pharmacokinetics, reduces immunogenicity, and better mimics natural human proteins.

[0242] For administration to a patient, the TCR, nucleic acid, and / or cell (usually associated with a detectable label or therapeutic substance) can be provided in a pharmaceutical composition with one or more SMDC substances and a pharmaceutically acceptable carrier or excipient. A therapeutic TCR or imaging TCR is typically supplied as part of a sterile pharmaceutical composition and typically contains a pharmaceutically acceptable carrier. This pharmaceutical composition can be in any suitable form (depending on the desired method of administration to the patient). This pharmaceutical composition can be provided in unit dosage form, is typically provided in a sealed container, and can be provided as part of a kit. Such a kit usually (but not necessarily) includes instructions for use. The kit can include a plurality of the aforementioned unit dosage forms.

[0243] Because TCRs have utility in adoptive T cell therapy, non-natural and / or purified and / or modified cells, particularly T cells, that present a TCR are provided. Also provided is an expanded population of T cells that present a TCR. There are many methods suitable for transfection of T cells with a nucleic acid encoding a TCR (such as DNA, cDNA, or RNA, etc.). T cells that express a TCR are suitable for use in cancer treatment based on adoptive therapy. As is known to those skilled in the art, there are many suitable methods by which adoptive therapy can be performed.

[0244] In some embodiments, each CAR is a fusion protein comprising a recognition region, a co-stimulatory domain, and an activation signaling domain, and the CAR specifically binds to a cell surface antigen on an immunosuppressive cell or a cancer cell.

[0245] In some embodiments, the recognition region is the scFv region of an anti-FITC (fluorescein isothiocyanate) antibody, the co-stimulatory domain is CD28, and the activation signaling domain is the T cell CD3ζ chain. In some embodiments, the recognition region is the scFv region of an anti-CD19 antibody, the co-stimulatory domain is CD137 (4-1BB), and the activation signaling domain is the T cell CD3ζ chain.

[0246] In some embodiments, the recognition region is the scFv region of an anti-CD19 antibody, the co-stimulatory domain is CD28, and the activation signaling domain is the T cell CD3ζ chain.

[0247] In some embodiments, the recognition region is the scFv region of an antibody that binds with high specificity to a cell surface antigen.

[0248] In some embodiments, each CAR comprises a recognition region, a co-stimulatory domain, and an activation signaling domain.

[0249] In some embodiments, the recognition region of the CAR is the scFv of an antibody that binds with high specificity to a cell surface antigen.

[0250] In some embodiments, the CAR selectively binds to a cell surface antigen on immunosuppressive cells or cancer cells.

[0251] In some embodiments, the CAR specifically and selectively binds to a cell surface antigen on immunosuppressive cells or cancer cells.

[0252] In some embodiments, the cell surface antigen is a tumor-associated antigen (TAA).

[0253] In some embodiments, the TAA is selected from the group consisting of CD19, CD20, CD30, CD3, CD4, CD5, BCMA, GD2, LEY, MS4A1, CD22, TNFRSF17, CD38, SDC1, TNFRSF8, IL3RA, CD7, NCAM1, CD34, CLEC12A, CD4, MME, CD5, SLAMF7, IL1RAP, FCGR3A, ITGB7, TNFRSF13B, TRBC1, CD33, ROR1, MUC1, KLRK1, KIT, CD274, CD70, PROM1, AFP, AXL, CD80, CD86, DLL3, TNFRSF10B, FAP, MAGEA1, MAGEA4, MUC16, PMEL, ROR2, KDR, EPHA2, L1CAM, CLDN18, PSCA, FOLR1, IL13RA2, MET, EPCAM, EGFR, EGFRVIII, FOLH1, GPC3, CEACAM5, ERBB2, CAIX, B4GALNT1, NY-ESO-1, PD-L1, and MSLN.

[0254] In some embodiments, the TAA is CD19.

[0255] In some embodiments, the co-stimulatory domain of the CAR is CD27, CD40L, CD70, 2B4, DNAM1, DAP12, DAP10, NKG2, NKG2D, CD28, CD137 (4-1BB), CD134 (OX40) or CD278 (ICOS).

[0256] In some embodiments, the activation signaling domain of the CAR is the T cell CD3ζ chain or the Fc receptor γ.

[0257] In some embodiments, the cytotoxic lymphocyte is autologous to the individual.

[0258] In some embodiments, the cytotoxic lymphocyte is allogeneic.

[0259] In some embodiments, the cytotoxic lymphocyte is xenogeneic to the individual.

[0260] Methods and Combinations of Use Provided are methods and combinations for treating cancer (e.g., solid tumors). The term "combination" generally refers to any formulation comprising two or more components, including one or more of a compound (e.g., an SMDC or a pharmaceutically acceptable salt thereof). It is understood that the compositions can be prepared from isolated compounds or salts of the compounds, solutions, hydrates, solvates, and other forms. It is understood that certain functional groups, such as hydroxy groups, amino groups, etc., can form complexes with water and / or various solvents in compounds of various physical forms. In certain situations, compounds (and compositions containing the compounds) can be prepared from amorphous, non-amorphous, partially crystalline, crystalline, and / or other various morphological forms of the compounds, and it is also understood that the compositions can be prepared from various hydrates and / or solvates of the compounds. Thus, pharmaceutical compositions listing the compounds include each of the various morphological forms and / or solvates or hydrate forms of the compounds, or any combination thereof, or these individual forms.

[0261] In certain embodiments, the combination can be a synergistic combination that, when administered to a subject (e.g., using a therapeutically effective amount), results in an increased cytotoxic effect against cancer in the subject as compared to administration of the individual compounds of the combination alone. In certain embodiments, administration of the combination (e.g., a therapeutically effective amount of one or more compounds (e.g., at least one SMDC or a pharmaceutically acceptable salt thereof) and CAR-expressing cytotoxic lymphocytes) inhibits cancer-associated fibroblast activity in the subject and improves the effectiveness of the CAR-expressing lymphocytes against cancer as compared to CAR-expressing cytotoxic lymphocytes administered alone.

[0262] Combinations for treating cancer include compositions comprising one or more compounds, including an SMDC (or a pharmaceutically acceptable salt thereof) and / or one or more compositions, and cytotoxic lymphocytes expressing a CAR, or a vector comprising a promoter operably linked to a nucleic acid sequence encoding a CAR.

[0263] Compounds and compositions can be administered by unit dosage forms, and / or compositions comprising one or more pharmaceutically acceptable carriers, adjuvants, diluents, excipients, and / or vehicles, and combinations thereof. The term "administer" and this element of the phrase generally refer to any means of introducing the compounds and compositions (e.g., CAR-expressing cytotoxic lymphocyte compositions and / or SMDC compounds or compositions) into the cells, tissues, organs or biological fluids of a subject.

[0264] In some embodiments, the SMDC is administered as a composition comprising one or more pharmaceutically acceptable carriers, adjuvants, diluents, excipients, vehicles or combinations of any of the foregoing.

[0265] Administration of the compounds and compositions as salts may be appropriate. Examples of acceptable salts include, but are not limited to, alkali metal (e.g., sodium, potassium or lithium) or alkaline earth metal (e.g., calcium) salts. However, any salt that is generally non-toxic and effective when administered to the subject being treated is acceptable.

[0266] Compounds and compositions can be formulated as pharmaceutical compositions and / or administered to a subject, e.g., a human patient, in a variety of forms adapted to the chosen route of administration. Indeed, the adapter compound or a pharmaceutically acceptable salt thereof, or the active modified compound or a pharmaceutically acceptable salt thereof, or the CAR-expressing cytotoxic lymphocyte composition, or the vector composition (e.g., comprising the lentiviral particles of the invention) can be administered to a subject using any suitable method known in the art. In one aspect, the SMDC compound or a pharmaceutically acceptable salt thereof can be administered by a formulation comprising a unit dosage form and / or a conventional non-toxic pharmaceutically acceptable carrier, adjuvant, and vehicle.

[0267] In addition, the SMDC compound(s) or pharmaceutically acceptable salt(s) thereof, or the CAR-expressing cytotoxic lymphocyte composition, or the vector composition can be administered orally or directly into the bloodstream, muscle, or viscera. In some embodiments, suitable routes for such parenteral administration include intravenous delivery, intraarterial delivery, intraperitoneal delivery, intrathecal delivery, epidural delivery, intracerebroventricular delivery, intraurethral delivery, intrasternal delivery, intracranial delivery, intratumoral delivery, intramuscular delivery, and subcutaneous delivery. In some embodiments, means for parenteral administration include needle (including microneedle) syringes, needleless syringes, and infusion techniques. It is evident that the compounds and compositions of the present invention can be formulated for the desired mode of administration.

[0268] In some embodiments, the SMDC and the CAR-expressing cytotoxic lymphocytes are administered to a subject by a method of administration selected from the group consisting of intravenous, intramuscular, intraperitoneal, and subcutaneous, and the method of administration of the SMDC is independent of the method of administration of the CAR-expressing cytotoxic lymphocytes.

[0269] In some embodiments, the SMDC and the CAR-expressing cytotoxic lymphocytes are administered to a subject by intravenous injection.

[0270] For example, parenteral formulations are typically aqueous solutions and may contain carriers or excipients such as salts, carbohydrates, and buffering substances (preferably pH 3-9), but may also be formulated, where appropriate, as sterile non-aqueous solutions or in dry form for use in combination with a suitable vehicle such as sterile pyrogen-free water or sterile saline. In some embodiments, any liquid formulation is adapted for parenteral administration. Preparation under aseptic conditions by lyophilization to produce a sterile lyophilized powder for parenteral formulations can be readily achieved using standard pharmaceutical techniques well known to those of skill in the art.

[0271] Pharmaceutical dosage forms of SMDC compound(s) suitable for injection or infusion may include sterile aqueous solutions or dispersions containing the active ingredient, optionally encapsulated in liposomes, which are suitable for immediate preparation of sterile injection or infusion solutions or dispersants, or sterile powders. In any case, the final dosage form must be sterile, fluid, and stable under the conditions of manufacture and storage. The liquid carrier or vehicle may be a solvent or liquid dispersion medium, for example, water, ethanol, polyols (such as glycerol, polyethylene glycol, liquid PEG(s), etc.), vegetable oils, non-toxic glyceryl esters, and / or suitable mixtures thereof, without limitation. In at least one embodiment, appropriate fluidity can be maintained by the formation of liposomes, maintenance of the required particle size in the case of dispersants, or the use of surfactants. Microbial activity can be prevented by the addition of various antibacterial and antifungal substances, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In certain cases, it is desirable to include one or more isotonic substances, such as sugars, buffers, or sodium chloride. Sustained absorption of the injectable composition can be achieved by incorporation of substances formulated to delay absorption, such as aluminum monostearate and gelatin.

[0272] Sterile injectable solutions can be prepared by incorporating the active ingredient in the required amount into a suitable solvent, optionally together with one or more of the other ingredients described above, and then filtering to sterilize. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying techniques and lyophilization techniques, by which powders of any further desired ingredients present in the previously sterile filtered solution in addition to the active ingredient are obtained.

[0273] Useful dosages of the compounds can be determined by comparing these in vitro and in vivo activities in animal models. Methods for extrapolating effective dosages for human subjects in mice and other animals are known in the art. In fact, the dosage of an SMDC compound can vary significantly depending on the condition of the subject, the type of cancer being treated, the degree of progression of the disease state, the route of administration and tissue distribution of the compound, and the potential for co-use of other therapeutic treatments (e.g., radiation therapy, or additional drugs in combination therapy). The amount of the composition and / or compound(s) (e.g., a therapeutically or prophylactically effective amount or dosage) required for use in therapy can vary not only with the particular application, but also with the salt selected (where applicable) and the characteristics of the subject (e.g., age, condition, gender, body surface area of the subject, and / or weight, drug tolerance, etc.), and will ultimately be determined by the judgment of the attending physician, clinician, or other. A “therapeutically effective amount,” “therapeutically effective dosage,” “therapeutically effective,” or “prophylactically effective amount” is defined (unless otherwise detailed) as the amount of the conjugate (e.g., an SMDC) or pharmaceutical composition that, when administered either once or over a course of treatment, affects the health, well-being, or mortality of the subject (e.g., without limitation, delay in the onset and / or decrease in the severity of one or more cancer-related symptoms).

[0274] Also provided is a method of treating cancer in a subject. The method includes administering to the patient, by any method, any one of a compound, a composition, and / or a combination, thereby treating the cancer in the patient. The compound, composition, and / or combination can be administered, for example, in a therapeutically effective amount. In certain embodiments, administration of an SMDC (or a pharmaceutically acceptable salt thereof) and a CAR-T cell therapy (e.g., CAR-expressing cytotoxic lymphocytes) and / or a combination of the present invention inhibits cancer-associated fibroblast activity in the patient, and improves the effectiveness of the administered CAR-T cell therapy and / or CAR-expressing cytotoxic lymphocytes against cancer as compared to the effectiveness of a CAR-T cell therapy and / or CAR-expressing cytotoxic lymphocytes administered in the absence of a therapeutically effective amount of an SMDC (or a pharmaceutically acceptable salt thereof).

[0275] In some embodiments, in addition, prior to administering an SMDC compound(s) or a pharmaceutically acceptable salt thereof, or a CAR-expressing cytotoxic lymphocyte composition to the subject, the cancer is imaged. Additionally, or alternatively, in some embodiments, during or after administration, the cancer is imaged to, for example, evaluate metastasis and the effectiveness of treatment. In some embodiments, the imaging can occur by positron emission tomography (PET) imaging, magnetic resonance imaging (MRI) or single photon emission computed tomography (SPECT) / computed tomography (CT) imaging. The imaging method can be any suitable imaging method known in the art.

[0276] In some embodiments, the method further includes imaging a solid tumor cancer prior to or during administration of an SMDC and a CAR-expressing cytotoxic lymphocyte.

[0277] In some embodiments, the cancer to be treated is a tumor. In some embodiments, the cancer is malignant. In some embodiments, the cancer is a solid tumor cancer.

[0278] In some embodiments, the cancer is a cancer of the brain, thyroid, lung, pancreas, kidney, stomach, gastrointestinal stroma, endometrium, breast, cervix, ovary, colon, prostate, or head and neck. In some embodiments, the cancer is leukemia, lymphoma, or other blood-related cancer.

[0279] In some embodiments, the cancer is a folate receptor-expressing cancer. In some embodiments, ligand (A) is a radical of raltitrexed or a radical of 5-methyltetrahydrofolate, and the cancer is a folate receptor-expressing cancer. In some embodiments, the cancer is a folate receptor alpha-expressing cancer, a folate receptor beta-expressing cancer, a folate receptor delta-expressing cancer, or a folate receptor alpha, beta, or delta-expressing immune cell-mediated cancer. In some embodiments, ligand (A) is a radical of raltitrexed or a radical of 5-methyltetrahydrofolate, and the cancer is a folate receptor alpha-expressing cancer, a folate receptor beta-expressing cancer, a folate receptor delta-expressing cancer, or a folate receptor alpha, beta, or delta-expressing immune cell-mediated cancer.

[0280] In some embodiments, the cancer is a cancer that expresses fibroblast activation protein (FAP), or a cancer that has cancer-associated fibroblasts that express FAP. In some embodiments, ligand (A) is a group that includes formula (X), and the cancer is a cancer that expresses FAP, or a cancer that has cancer-associated fibroblasts that express FAP.

[0281] In some embodiments, the method further comprises administering a first therapeutically effective amount of an SMDC and a second therapeutically effective amount of CAR-expressing cytotoxic lymphocytes.

[0282] In some embodiments, the step of administering the SMDC includes administering a dose of the SMDC to the subject at least three times a week.

[0283] In some embodiments, the step of administering the SMDC includes administering a dose of the SMDC to the subject five times a week.

[0284] In some embodiments, the first therapeutically effective amount of the SMDC and the second therapeutically effective amount of the CAR-expressing cytotoxic lymphocytes are administered simultaneously or sequentially over time in either order.

[0285] In some embodiments, the method increases the amount of myeloid cells exhibiting an immunostimulatory phenotype in the subject's tumor microenvironment (TME) as compared to the amount of myeloid cells exhibiting an immunosuppressive phenotype in the TME.

[0286] For the compounds, compositions, combinations, and methods, all embodiments of the SMDC (including without limitation the drug moiety or a pharmaceutically acceptable salt and / or ligand / target moiety thereof), the CAR-expressing cytotoxic lymphocyte composition, and the vector composition are applicable, including, but not limited to, embodiments of the linker.

[0287] Also provided is the use of an SMDC or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of cancer, the treatment comprising administering to a patient the SMDC(s) in combination with a CAR T cell therapy. The SMDC(s) may include any of the SMDCs described herein, and the CAR T cell therapy may include the administration of any of the CAR-expressing cytotoxic lymphocytes described herein.

[0288] General All patents, patent application publications, academic papers, textbooks, and other published documents referred to herein indicate the level of skill of those of ordinary skill in the art to which the present disclosure pertains.

[0289] Above, numerous specific details are set forth to provide a thorough understanding of the present disclosure. Specific examples can be practiced without using some or all of such specific details, and it is understood that the present disclosure is not limited to a particular biological system, a particular cancer, or a particular organ or tissue, which, of course, can vary, but remains applicable considering the data presented herein.

[0290] In addition, various techniques and mechanisms of the present disclosure may describe a connection or linkage between two components. Words such as coupling, linking, conjugating, connecting, and similar terms with inflectional morphemes are used interchangeably unless differences are recognized, occur, or are otherwise not apparent from the context. Such words and expressions do not necessarily indicate a direct connection and include connections through intervening components. It should be noted that, due to the possible presence of various other components between two components that should be noted, a connection between two components does not necessarily mean a direct unobstructed connection. Thus, a connection does not necessarily mean a direct unobstructed connection unless otherwise noted.

[0291] Furthermore, the present disclosure is presented in this way merely for illustrative purposes, and it is understood that the principles and embodiments described herein may be applied to the components of compounds and / or compositions having configurations other than those specifically described herein. In fact, it is explicitly contemplated that the components of the compositions and compounds of the present disclosure may be tailored to facilitate the desired applications.

[0292] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the chemical and biological arts. Any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the subject matter of this application, but the preferred methods and materials are described herein.

[0293] The term "about," when referring to a number or numerical value or range (e.g., including integers, fractions, and percentages), means that the number or numerical range being referred to is an approximation within experimental variability (or statistical experimental error), and thus the numerical value or range may vary by 1% to 15% (e.g., + / - 5% to 15% of the recited value) of the stated number or numerical range, provided that one of ordinary skill in the art would determine that the numerical value or range is equivalent (e.g., has the same function or result) to the recited value.

[0294] When ranges are used herein for physical properties, such as molecular weight, or chemical properties, such as chemical formula, all combinations and sub - combinations of the ranges and specific embodiments are intended to be included.

[0295] The present disclosure may be practiced suitably in the absence of any element(s) or limitation(s) not specifically disclosed herein. Accordingly, for each case herein of any of the terms "comprising", "consisting essentially of", and "consisting of" (and related terms, such as "comprise" or "comprises" or "having" or "including"), it can be replaced with other terms referred to. Similarly, the singular forms of "a", "an", and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to "a method" includes one or more methods and / or steps of that kind which are described and / or apparent to one of ordinary skill in the art upon understanding the present disclosure. The term "substantially" may allow for some variability in a value or range, for example, within 90%, 95% or 99% of a defined value or defined range limitation.

[0296] The term "receptor" refers to a chemical structure in a biological system that transmits and receives signals.

[0297] Unless otherwise explicitly stated, the indicated structure includes all stereochemical forms of the structure, i.e., the right - hand (R) and left - hand (S) configurations of each chiral center. Accordingly, single stereochemical isomers as well as mixtures of enantiomers and diastereomers are within the scope of the present disclosure.

[0298] One of ordinary skill in the art will further appreciate that the above SMDCs can be “deuterated”. This means that one or more hydrogen atoms can be replaced with deuterium. Since deuterium and hydrogen have nearly identical physical properties, deuterium substitution is the smallest structural change that can be made. Substitution of hydrogen with deuterium can improve stability in the presence of other drugs, thereby reducing unwanted drug-drug interactions and significantly reducing the metabolic rate (due to the kinetic effects of the isotope). Reduction of the metabolic rate allows for an increase in half-life, a decrease in the formation of toxic metabolites, and a reduction in dosage and / or dosing frequency.

[0299] The term “alkylene”, alone or as part of another substituent, unless otherwise stated, means a divalent radical derived from alkyl, for example, -CH2CH2CH2CH2- (but not limited thereto). Typically, an alkyl (or alkylene) group has from 1 to 24 carbon atoms. “Lower alkyl” or “lower alkylene” are short-chain alkyl or alkylene groups generally having 8 or fewer carbon atoms.

[0300] The term "heteroalkyl", alone or in combination with another term, unless otherwise stated, means a stable straight-chain, branched-chain, or combination thereof (if plural) consisting of at least one carbon atom and at least one heteroatom selected from the group consisting of oxygen, nitrogen, phosphorus, silicon, and sulfur, where the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. This heteroatom (if plural) of oxygen, nitrogen, phosphorus, sulfur, and silicon may be located at any internal position of the heteroalkyl group or at the position where the alkyl group is attached to the remainder of the molecule. Examples include, but are not limited to, -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2-S(O)-CH3, -CH2-CH2-S(O)2-CH3, -CH2=CH-O-CH3, -Si(CH3)3, -CH2-CH=N-OCH3, -CH=CH-N(CH3)-CH3, -O-CH3, -O-CH2-CH3, and -CN. Up to two heteroatoms may be consecutive, for example, -CH2-NH-OCH3, etc.

[0301] Similarly, the term "heteroalkylene" means a divalent radical derived from heteroalkyl, alone or as part of another substituent (unless otherwise stated), and examples include, but are not limited to, -CH2-CH2-S-CH2-CH2 and -CH2-S-CH2-CH2-NH-CH2. In a heteroalkylene group, the heteroatom can also occupy one or both chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, etc.). Even further, in an alkylene linking group and a heteroalkylene linking group, the orientation of the linking group is not meant by the direction in which the formula of the linking group is written. For example, the formula -C(O)2R'- represents both -C(O)2R'- and -R'C(O)2-. As noted above, a heteroalkyl group includes a group that is bonded to the remainder of the molecule through a heteroatom, and examples include, but are not limited to, -C(O)R', -C(O)NR', -NR'R", -OR', -SR', and / or -SO2R'. When listing "heteroalkyl" followed by a specific heteroalkyl group, e.g., -NR'R", etc., it is understood that the terms heteroalkyl and -NR'R" are neither duplicative nor mutually exclusive. Rather, the specific heteroalkyl group is listed for clarity. Thus, the term "heteroalkyl" should not be construed in this specification as excluding specific heteroalkyl groups, e.g., -NR'R", etc.

[0302] The terms "polypeptide", "peptide", and "protein" are used interchangeably herein to refer to a polymer of amino acid residues, a polypeptide or fragment of a polypeptide, a peptide, or a fusion polypeptide. These terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers and non-natural amino acid polymers.

[0303] Also provided is a pharmaceutical composition. As used herein, the term "composition" generally refers to any formulation that comprises two or more components, such as one or more conjugates, such as an SMDC. It is understood that the composition can be prepared from isolated conjugates or salts, solutions, hydrates, solvates, and other forms. It is understood that certain functional groups, such as hydroxy groups, amino groups, etc., can form complexes with water and / or various solvents in conjugates in various physical forms. The composition can be prepared from amorphous, non-amorphous, partially crystalline, crystalline, and / or other various morphological forms of conjugates, and it is also understood that the composition can be prepared from various hydrates and / or solvates of the conjugate. Thus, the pharmaceutical composition listing the conjugate includes each of the various morphological forms and / or solvates or hydrate forms of the conjugate, or any combination thereof, or these individual forms.

[0304] As used herein, "subject" is a mammal, preferably a human, but can also be a non-human animal (including, without limitation, experimental, agricultural, breeding, or wild animals). Thus, the methods, compounds, and compositions of the present invention are applicable to diseases and applications in both humans and animals. In various embodiments, the subject can be an experimental animal, such as a rodent (e.g., mouse, rat, hamster, etc.), rabbit, monkey, chimpanzee, a breeding animal, such as a dog, cat, or rabbit, an agricultural animal, such as a cow, horse, pig, sheep, or goat, or a captured wild animal, such as a bear, panda, lion, tiger, leopard, elephant, zebra, giraffe, gorilla, dolphin, or whale. In some embodiments, the subject is a "patient", i.e., a living human or animal receiving medical treatment for a disease or condition, including a human or animal having an undefined disease and being evaluated for signs of the condition. In some embodiments, the subjects that can be treated using the methods of the present invention include subjects identified or selected as having or at risk of having cancer. Such identification and / or selection can be performed by clinical or diagnostic evaluation.

[0305] For radicals, substituents, and the specific values recited herein with respect to ranges, unless otherwise specified, are for illustrative purposes only, and such examples do not exclude other defined values or other values within the defined ranges for radicals and substituents. For example, (C1-C6)alkyl may be methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec-butyl, pentyl, 3-pentyl or hexyl, (C1-C3)alkyl may be iodomethyl, bromomethyl, chloromethyl, fluoromethyl, trifluoromethyl, 2-chloroethyl, 2-fluoroethyl, 2,2,2-trifluoroethyl or pentafluoroethyl, (C1-C3)alkoxy may be methoxy, ethoxy or propoxy, and (C2-C6)alkanoyloxy may be acetoxy, propanoyloxy, butanoyloxy, isobutanoyloxy, pentanoyloxy or hexanoyloxy.

[0306] When substituents are specified by the conventional chemical formula written from left to right, these equally encompass the chemically identified substituents resulting from writing the structure from right to left. For example, -CH2O- is equivalent to -OCH2-.

[0307] Alkyl, alkoxy, etc. represent straight-chain (i.e., unbranched), branched-chain or combinations thereof, and these may be fully saturated, mono-unsaturated or poly-unsaturated, and may include divalent and polyvalent radicals, and have the specified number of carbon (C) atoms (i.e., C1-C 10(wherein, C1-C10 means 1 to 10 carbon atoms). Examples of saturated hydrocarbon radicals include groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, and (cyclohexyl)methyl, and homologs and isomers such as, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, etc., but are not limited thereto. An unsaturated alkyl group is a group having one or more double bonds or triple bonds. Examples of unsaturated alkyl groups include vinyl, 2-propenyl, crotyl-2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1-propynyl and 3-propynyl, 3-butynyl, and higher homologs and isomers, but are not limited thereto. An alkoxy is an alkyl bonded to the remainder of the molecule via an oxygen linker (-O-). In some embodiments, alkoxy refers to a radical bonded through an oxygen atom of the formula -O-alkyl.

[0308] Generally, the terms "acyl" or "acyl substituent" refer to a substituent obtained by removal of one or more hydroxyl groups from an oxo acid, including an inorganic acid, and include a double-bonded oxygen atom and an alkyl group. Further, reference to an individual radical, for example, "propyl" includes only the straight-chain radical, and branched-chain isomers, for example, "isopropyl", are referred to in detail.

[0309] It is recognized that various modifications are possible within the scope of the present disclosure. Accordingly, although the present disclosure has been disclosed in detail in the context of preferred embodiments and any features, those skilled in the art can use modified and variant forms of the concepts disclosed herein. Such modified and variant forms are considered to be within the scope of the present disclosure claimed herein.

[0310] Accordingly, the specification and the appended claims are intended to cover all modifications and variations that are apparent to those skilled in the art based on this disclosure. For example, when a method of treatment or therapy involves administering two or more treatments, compounds, or compositions to a subject, it is understood that the order, timing, number, concentration, and volume of administration are limited only by medical requirements and treatment limitations (i.e., the two treatments can be administered to the subject, for example, simultaneously, sequentially, over time, alternately, or according to any other regimen).

[0311] In addition, in the description of representative embodiments, a method and / or process may be presented by this disclosure as steps in a particular order. Unless the method or process depends on the steps in the particular order described herein, the method or process should not be limited to the steps in the particular order described. As will be understood by those skilled in the art, steps in other orders may also be possible. Accordingly, the steps in the particular order disclosed herein should not be construed as a limitation on the claims. In addition, claims directed to a method and / or process should not be limited to the performance of these steps in the order described, and those skilled in the art can readily understand that this order can vary and still remain within the spirit and scope of this disclosure.

[0312] Furthermore, the use of headings and subheadings is for ease of reference in view of the length of the document. The description under a particular heading or subheading (e.g., a subheading in the "DETAILED DESCRIPTION OF THE INVENTION") is not intended to be limited to the subject matter described only under that particular heading or subheading.

Deposit Number

[0313] ATCC TIB-152 ATCC CRL-1942 DSMZ ACC-290 DSMZ ACC-545

Claims

1. A pharmaceutical composition for the treatment of cancer comprising a small molecule drug conjugate (SMDC) or a pharmaceutically acceptable salt thereof, wherein the treatment comprises the step of administering a therapeutically effective amount of the SMDC in combination with chimeric antigen receptor (CAR) T-cell therapy, and the SMDC or pharmaceutically acceptable salt thereof (a) Phosphoinositide 3-kinase (PI3K) inhibitors, interferon gene-stimulating factor (STING) agonists, nucleotide-binding oligomerized domain (NOD)-like receptor (NLR) agonists, retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) agonists, melanoma deficiency factor 2 (AIM2)-like receptor (ALR) agonists, advanced glycation end product receptor (RAGE) agonists, perekinase / interleukin-1 (IL-1) receptor-related kinase 1 (IRAK1) agonists, IRAK2 agonists, IRAK4 agonists, A drug moiety selected from the group consisting of IRAK3 antagonists, Src homology 2-domain tyrosine phosphatase 1 and 2 (SHP1 / 2) antagonists, T cell protein tyrosine phosphatase (TC-PTP) antagonists, diacylglycerol kinase (DGK) antagonists, zeste homolog 2 enhancer (EZH2) antagonists, transforming growth factor beta (TGFβ) antagonists, activated B cell nuclear factor kappa light chain enhancer (NFκβ) activators, or 1-kappa β (Iκβ) kinase antagonists. (b) The radical of larcitrexed, the radical of 5-methyltetrahydrofolate, or formula (X): 【Transformation 6】 A group containing, in the formula, J is (C(R Ｊ ) ２ ) ０－３ And in the formula, each R Ｊ However, it is either H or two or more R Ｊ However, they combine to form an oxo, R １ However, -CN, -CHO, and -B(OH) ２ Selected from the group consisting of, R ３ and R ４ are each independently selected from the group consisting of -H and F, R １０ However, H, -CF ３ A ligand selected from the group consisting of F, Cl, Br, and I, and (c) A linker connecting the ligand and the drug portion. A pharmaceutical composition containing the following:

2. The ligand is formula (XA), formula (XB), formula (XC), or formula (XD): 【Transformation 7】 including, or, The drug portion is selected from the group consisting of PI3K inhibitors, NFκβ activators, Iκβ kinase inhibitors, STING agonists, NLR agonists, RLR agonists, ALR agonists, RAGE agonists, IRAK1 agonists, IRAK2 agonists, IRAK4 agonists, IRAK3 antagonists or inhibitors, SHP1 / 2 inhibitors, TC-PTP inhibitors, DGK inhibitors, and TGFβ inhibitors. The pharmaceutical composition according to claim 1, wherein the ligand is a radical of larcitrexed, a radical of 5-methyltetrahydrofolate, or a group comprising formula (X).

3. The pharmaceutical composition according to claim 1, wherein the linker comprises polyethylene glycol (PEG) in a freeable form, PEG in a non-freeable form, polyproline, hydrophilic amino acids, sugars, unnatural peptidoglycans, polyvinylpyrrolidone, a triblock copolymer comprising a central hydrophobic block of polypropylene glycol on each side adjacent to a hydrophilic block of PEG, or any two or more of the above.

4. The pharmaceutical composition according to claim 1, wherein the linker is a releaseable linker or a non-releaseable linker.

5. The aforementioned linker has the following structure: 【Transformation 8】 Includes a group represented by, In the formula, w is between 0 and 5, and t is between 1 and 30, or The linker has the following structure: 【Chemistry 9-1】 【Chemistry 9-2】 It comprises one or more linker groups each independently having the following: The pharmaceutical composition according to claim 1, wherein n is between 0 and 15 in the formula.

6. The pharmaceutical composition according to claim 1, wherein the SMDC further comprises a radical of a PEG group, a peptide group, a glycopeptide group, a saccharide group, an albumin-binding group, or a combination of two or more radicals of the aforementioned groups, and the radical or combination of the PEG group, the peptide group, the glycopeptide group, the saccharide group, the albumin-binding group is bound to the linker.

7. The albumin-binding group is 【Chemistry 10】 A pharmaceutical composition according to claim 6, selected from the group consisting of the following.

8. The pharmaceutical composition according to claim 1, wherein the CAR T cell therapy comprises the administration of CAR-expressing cytotoxic lymphocytes to the subject, and each CAR comprises a recognition domain, a co-stimulatory domain, and an activation signaling domain.

9. The pharmaceutical composition according to claim 8, wherein the recognition region of the CAR is a single-chain variable fragment (scFv) of an antibody that binds with high specificity to cell surface antigens, or specifically to cell surface antigens on immunosuppressive cells or cancer cells, or to cell surface antigens that are tumor-associated antigens (TAAs).

10. The TAA is CD19, CD20, CD30, CD3, CD4, CD5, BCMA, GD2, LEY, MS4A1, CD22, TNFRSF17, CD38, SDC1, TNFRSF8, IL3RA, CD7, NCAM1, CD34, CL EC12A, CD4, MME, CD5, SLAMF7, IL1RAP, FCGR3A, ITGB7, TNFRSF13B, TRBC1, CD33, ROR1, MUC1, KLRK1, KIT, CD274, CD70, PROM1, AFP, A A pharmaceutical composition according to claim 9, selected from the group consisting of XL, CD80, CD86, DLL3, TNFRSF10B, FAP, MAGEA1, MAGEA4, MUC16, PMEL, ROR2, KDR, EPHA2, L1CAM, CLDN18, PSCA, FOLR1, IL13RA2, MET, EPCAM, EGFR, EGFRVIII, FOLH1, GPC3, CEACAM5, ERBB2, CAIX, B4GALNT1, NY-ESO-1, PD-L1, and MSLN.

11. The CAR-expressing cytotoxic lymphocyte is a T cell, and the costimulatory domain of the CAR is CD27, CD40L, CD70, 2B4, DNAM1, DAP12, DAP10, NKG2, NKG2D, CD28, CD137 (4-1BB), CD134 (OX40), or CD278 (ICOS), or The CAR-expressing cytotoxic lymphocytes are NK cells, and the TAA is selected from the group consisting of CD3, CD4, and CD5, or The pharmaceutical composition according to claim 1, wherein the CAR-expressing cytotoxic lymphocyte is an NK cell, and the costimulatory domain of the CAR is selected from the group consisting of 2B4, CD137 (4-1BB), DNAM1, DAP12, DAP10, NKG2, and NKG2D.

12. The pharmaceutical composition according to claim 8, wherein the activation signaling domain of the CAR is a T cell CD3ζ chain or an Fc receptor γ.

13. The pharmaceutical composition according to claim 1, wherein the cytotoxic lymphocytes are autologous or heterologous to the individual.

14. The pharmaceutical composition according to claim 1, wherein the cancer is a solid tumor cancer, leukemia, lymphoma, or another hematological cancer.

15. The ligand is a radical of larcitrexed or a radical of 5-methyltetrahydrofolate, and the cancer is a folate receptor-expressing cancer or a folate receptor-expressing immune cell-mediated cancer, or The pharmaceutical composition according to claim 1, wherein the ligand is a base of formula (X), and the cancer is a cancer expressing fibroblast-activating protein (FAP), or a cancer having cancer-associated fibroblasts expressing FAP.

16. (i) A therapeutically effective amount of the pharmaceutical composition according to any one of claims 1 to 15, (ii) A therapeutically effective amount of CAR-expressing cytotoxic lymphocytes and A synergistic combination for treating solid tumor cancers in the target area, including [specific ingredients / methods].

17. The synergistic combination according to claim 16, wherein administration of a therapeutically effective amount of the pharmaceutical composition inhibits cancer-associated fibroblast activity in the subject, and improves the effectiveness of the administered CAR-expressing cytotoxic lymphocytes against cancer compared to the effectiveness of the CAR-expressing cytotoxic lymphocytes administered without administration of a therapeutically effective amount of the pharmaceutical composition.

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