Methods and compositions for car t cell therapy

Abstract

To provide methods of treating a patient with a cancer by administering to the patient a composition comprising CAR T cells and a small molecule linked to a targeting moiety by a linker.SOLUTION: The present invention provides a method of treatment of a cancer, the method comprising i) administering to a patient a first dose of a compound, or a pharmaceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked to a targeting moiety by a linker; ii) administering to the patient a CAR T cell composition, wherein the CAR T cell comprises a CAR directed to the targeting moiety; ii) administering to the patient a second dose of the compound, or the pharmaceutically acceptable salt thereof, wherein the second dose is different than the first dose; and iv) treating the patient to ameliorate the cancer.SELECTED DRAWING: Figure 1A-1B

Description

Technical Field

[0001] Cross - reference to related applications This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 62 / 320,183, filed on April 8, 2016, and U.S. Provisional Application No. 62 / 323,971, filed on April 18, 2016. The entire disclosures of both applications are incorporated herein by reference. Technical field The present disclosure relates to methods of treating a patient having cancer by administering CAR T cells to the patient and by administering to the patient a composition comprising a small molecule linked to a targeting moiety by a linker. The present disclosure also relates to compositions for use in such methods.

Background Art

[0002] Immunotherapies based on adoptive transfer of lymphocytes (e.g., T cells) into patients are valuable treatments in the treatment of cancer and other diseases. Many important advances have been made in the development of adoptive transfer-based immunotherapies. Among the many different types of immunotherapeutic agents, one of the most promising immunotherapeutic agents being developed is the T cell-expressing chimeric antigen receptor (CAR T cell). A chimeric antigen receptor (CAR) is a genetically engineered receptor designed to target a specific antigen, e.g., a tumor antigen. This targeting can result in cytotoxicity to a tumor, such that, for example, CAR T cells expressing the CAR can target and kill the tumor via the specific tumor antigen.

[0003] The first-generation CARs consisted of a recognition region, such as a single-chain variable fragment (scFv) derived from an antibody for the recognition and binding to an antigen expressed by a tumor, and an activation signaling domain. For example, the CD3ζ chain of T cells can function as a T cell activation signal in CARs. CAR T cells showed positive results in vitro, but had limitations in eliminating diseases (e.g., cancer) in clinical trials. One problem was the inability to extend activation and expand the CAR T cell population in vivo.

[0004] To address this problem, second-generation CARs include a co-stimulatory domain (e.g., CD137, CD28, or CD134) to achieve long-term activation of T cells in vivo. The addition of the co-stimulatory domain enhanced the in vivo proliferation and survival of T cells containing the CAR, and initial clinical data indicated that such constructs are promising therapeutic agents in the treatment of diseases such as cancer.

Summary of the Invention

Problems to be Solved by the Invention

[0005] Although improvements have been made in CAR T cell therapy, some problems remain. First, "off-target" toxicity can occur due to normal cells expressing the antigen targeted by CAR T cells (e.g., tumor-associated antigens). Second, unregulated CAR T cell activation can lead to the rapid and uncontrolled elimination of diseased cells (e.g., cancer cells) by CAR T cells, which may result in a set of metabolic disorders called tumor lysis syndrome or cytokine release syndrome (CRS) when the tumor is being treated, and this can be life-threatening to patients. Tumor lysis syndrome and CRS cannot be easily regulated and can be caused by the administration of CAR T cells that are activated uncontrollably. Therefore, although CAR T cells are highly promising as tools in the treatment of diseases such as cancer, additional CAR T cell therapies are needed that provide a reduction in off-target toxicity and more precise control of CAR T cell activation.

Means for Solving the Problem

[0006] Summary of the invention The inventors have discovered a method that provides an important advance in CAR T cell therapy, reducing off-target toxicity and more precisely controlling CAR T cell activation. In various embodiments described herein, a small molecule ligand linked to a targeting moiety by a linker is used as a bridge between the cancer and the CAR T cells, directing the CAR T cells to the cancer for cancer amelioration. In one embodiment, the "small molecule ligand" can be, for example, folic acid, DUPA, an NK-1R ligand, a CAIX ligand, a ligand for gamma-glutamyl transpeptidase, or a CCK2R ligand, each of which is a small molecule ligand that specifically binds to cancer cells (i.e., the receptors for these ligands are overexpressed compared to normal tissue).

[0007] In one embodiment, the "small molecule ligand" is linked to a "targeting moiety" that binds to the CAR expressed by the CAR T cells. In various embodiments, the "targeting moiety" can be selected from, for example, 2,4-dinitrophenol (DNP), 2,4,6-trinitrophenol (TNP), biotin, digoxigenin, fluorescein, fluorescein isothiocyanate (FITC), NHS-fluorescein, pentafluorophenyl ester (PFP), tetrafluorophenyl ester (TFP), knottin, centyrin, and DARPin.

[0008] The "targeting moiety" binds to the recognition region of the genetically engineered CAR expressed by the CAR T cells. Thus, the recognition region of the CAR (e.g., a single-chain variable fragment (scFv) of an antibody) is directed towards the "targeting moiety". Thus, the small molecule ligand linked to the targeting moiety by a linker acts as a "bridge" between the cancer and the CAR T cells, directing the CAR T cells towards the cancer for cancer amelioration.

[0009] In one exemplary embodiment, the inventors have discovered that varying the dose of a small molecule ligand linked to a targeting moiety by a linker (i.e., a bridge) can result in the ability to control CRS in vivo. In another embodiment, the inventors have discovered that varying the dose of a small molecule ligand linked to a targeting moiety by a linker (i.e., a bridge) can result in the ability to control CRS in vivo upon CAR T cell activation. In yet another embodiment, a combination of these methods can be used for the precise control of CAR T cell activation and cytokine release in vivo. In another embodiment, the affinity of the small molecule ligand for its receptor can be altered to control CAR T cell activation or to achieve specificity to avoid toxicity to normal tissues.

[0010] In one embodiment, a method of treating cancer is provided. The method includes: i) administering to a patient a first dose of a compound or a pharmaceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked to a targeting moiety by a linker; ii) administering to the patient a CAR T cell composition, wherein the CAR T cells comprise a CAR directed to the targeting moiety; ii) administering to the patient a second dose of the compound or a pharmaceutically acceptable salt thereof, wherein the second dose is different from the first dose; and treating the patient to ameliorate the cancer.

[0011] In another embodiment, a method of treating cancer is provided. The method includes: i) administering to a patient a first complex or a pharmaceutically acceptable salt thereof; ii) administering to the patient a CAR T cell composition, wherein the CAR T cells comprise a CAR directed to the targeting moiety; iii) administering to the patient a second complex or a pharmaceutically acceptable salt thereof, wherein the first and second complexes each comprise a small molecule ligand linked to a targeting moiety by a linker and the first complex and the second complex are different; and iv) treating the patient to ameliorate the cancer.

[0012] In yet another embodiment, a method of treating cancer is provided. The method includes: i) administering to a patient a first dose of a first complex or a pharmaceutically acceptable salt thereof, where the first complex includes a small molecule ligand linked to a targeting moiety by a linker; ii) administering to the patient a CAR T cell composition, where the CAR T cells include a CAR directed to the targeting moiety; iii) administering to the patient a second dose of a second complex or a pharmaceutically acceptable salt thereof, where the first complex and the second complex each include a small molecule ligand linked to a targeting moiety, the first complex and the second complex are different, and the first dose and the second dose are different; and iv) treating the patient to ameliorate the cancer.

[0013] In yet another exemplary embodiment, CAR T cells are provided that include a nucleic acid comprising SEQ ID NO: 1. In another embodiment, CAR T cells are provided that include a polypeptide comprising SEQ ID NO: 2. In another embodiment, an isolated nucleic acid is provided that includes SEQ ID NO: 1 and encodes a chimeric antigen receptor. In yet another embodiment, a chimeric antigen receptor polypeptide is provided that includes SEQ ID NO: 2. In another embodiment, a vector is provided that includes SEQ ID NO: 1. In yet another exemplary embodiment, a vector is provided that includes SEQ ID NO: 1 and is a lentiviral vector.

[0014] Some embodiments are also described by the following listed items: 1. A method of treating cancer, comprising: i) administering to a patient a first dose of a compound or a pharmaceutically acceptable salt thereof, where the compound includes a small molecule ligand linked to a targeting moiety by a linker; ii) administering to the patient a CAR T cell composition, where the CAR T cells include a CAR directed to the targeting moiety; iii) administering to the patient a second dose of a compound or a pharmaceutically acceptable salt thereof, where the second dose is different from the first dose; and iv) treating the patient to ameliorate the cancer. 2. A method for treating cancer, comprising: i) administering to a patient a first complex or a pharmaceutically acceptable salt thereof; ii) administering to the patient a CAR T cell composition (wherein the CAR T cells comprise a CAR directed to a targeting moiety); iii) administering to the patient a second complex or a pharmaceutically acceptable salt thereof (wherein the first and second complexes each comprise a small molecule ligand linked to a targeting moiety by a linker, and the first complex and the second complex are different); and iv) treating the patient to ameliorate the cancer. 3. A method for treating cancer, comprising: i) administering to a patient a first dose of a first complex or a pharmaceutically acceptable salt thereof; ii) administering to the patient a CAR T cell composition (wherein the CAR T cells comprise a CAR directed to a targeting moiety); ii) administering to the patient a second dose of a second complex or a pharmaceutically acceptable salt thereof (wherein the first complex and the second complex each comprise a small molecule ligand linked to a targeting moiety by a linker, the first complex and the second complex are different, and the first dose and the second dose are different); and iv) treating the patient to ameliorate the cancer. 4. The method according to item 2 or 3, wherein the linker in the first complex or a pharmaceutically acceptable salt thereof is different from the linker in the second complex or a pharmaceutically acceptable salt thereof. 5. The method according to item 2 or 3, wherein the linker in the first complex or a pharmaceutically acceptable salt thereof is the same as the linker in the second complex or a pharmaceutically acceptable salt thereof. 6. The method according to any one of items 2 to 5, wherein the ligand in the first complex or a pharmaceutically acceptable salt thereof is different from the ligand in the second complex or a pharmaceutically acceptable salt thereof. 7. The method according to any one of items 2 to 5, wherein the ligand in the first complex or its pharmaceutically acceptable salt and the ligand in the second complex or its pharmaceutically acceptable salt are the same. 8. The method according to any one of items 2 to 7, wherein the targeting moiety in the first complex or its pharmaceutically acceptable salt and the targeting moiety in the second complex or its pharmaceutically acceptable salt are different. 9. The method according to any one of items 2 to 7, wherein the targeting moiety in the first complex or its pharmaceutically acceptable salt and the targeting moiety in the second complex or its pharmaceutically acceptable salt are the same. 10. The method according to any one of items 1 to 9, wherein the ligand is selected from folic acid, DUPA, NK-1R ligand, CAIX ligand, ligand for gamma-glutamyl transpeptidase, and CCK2R ligand. 11. The method according to item 10, wherein the ligand is folic acid. 12. The method according to item 10, wherein the ligand is NK-1R ligand. 13. The method according to item 10, wherein the ligand is DUPA. 14. The method according to item 10, wherein the ligand is CCK2R ligand. 15. The method according to item 10, wherein the ligand is ligand for gamma-glutamyl transpeptidase. 16. The method according to any one of items 1 to 15, wherein the targeting moiety is selected from 2,4-dinitrophenol (DNP), 2,4,6-trinitrophenol (TNP), biotin, digoxigenin, fluorescein, fluorescein isothiocyanate (FITC), NHS-fluorescein, pentafluorophenyl ester (PFP), tetrafluorophenyl ester (TFP), knottin, centyrin, and DARPin. 17. The method according to item 16, wherein the targeting moiety is FITC. 18. The method according to item 16, wherein the targeting moiety is DNP. 19. The method according to item 16, wherein the targeting moiety is TNP. 20. The method according to any one of items 1 to 19, wherein the linker comprises polyethylene glycol (PEG), polyproline, hydrophilic amino acids, sugars, non-natural peptidoglycans, polyvinylpyrrolidone, and / or pluronic (registered trademark) F-127. 21. The method according to item 20, wherein the linker comprises PEG. 22. The compound or a pharmaceutically acceptable salt thereof, the first complex or a pharmaceutically acceptable salt thereof, or the second complex or a pharmaceutically acceptable salt thereof has the formula:

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Brief description of the drawings

[0015] [FIG. 1A - 1B] (CAR T cells): CAR T cell proliferation using the FITC-small molecule complex in different cell types with a target cell (cancer cell) ratio of 5:1 is shown. Figure 1A shows CAR T cell proliferation in KB (FR+) cells. Figure 1B shows CAR T cell proliferation in HEK293 (NK1R+) cells. [FIG. 2A - 2F]Shows the production of inflammatory cytokine IFN-γ by CAR T cells using FITC-small molecule complexes in different cell types. Figure 2A shows the production of inflammatory cytokine IFN-γ in KB (FR+) cells. Figure 2B shows the production of inflammatory cytokine IFN-γ in LNCaP (PSMA+) cells. Figure 2C shows the production of inflammatory cytokine IFN-γ in HEK293 (NK1R+) cells. Figure 2D shows the production of inflammatory cytokine IFN-γ in KB (FR+) cells using different concentrations of FITC-folic acid. Figure 2E shows the production of inflammatory cytokine IFN-γ in KB (FR+) cells using different complexes. Figure 2F shows the production of inflammatory cytokine IFN-γ in KB (FR+) cells using different complexes. [FIG. 3A - 3F] Shows the in vitro toxicity of tumor cells treated with FITC-small molecule complexes in different cell types. Figure 3A shows the in vitro toxicity in KB (FR+) cells. Figure 3B shows the in vitro toxicity in LNCaP (PSMA+) cells. Figure 3C shows the in vitro toxicity in HEK293 (NK1R+) cells. Figure 3D shows the in vitro toxicity in KB (FR+) cells as a function of different E:T (effector cell: target cell) ratios. Figure 3D shows the in vitro toxicity in KB (FR+) cells as a function of FITC-folic acid concentration. Figure 3F shows the in vitro toxicity in KB (FR+) cells using different complexes. [FIG. 4A - 4B] Shows that the activation of CAR T cells correlates with the expression level of tumor antigens on cancer cells. Figure 4A shows the tumor antigen FRα level. The highest peak is the peak of KB (FR+) cells. Figure 4B shows the activation of CAR T cells using FITC-small molecule complexes measured by IFN-γ production in MDA-MB-231 and KB cells. [FIG. 5A - 5C]Disclosed are HEK293 (NK1R+) tumor xenografts and CAR T cell therapies, including treating CAR T cells with the FITC-PEG11-NK1 complex or without the complex. Figure 5A shows the tumor volume measured over 24 days. Figure 5B shows the body weight measured over 22 days of therapy. Figure 5C shows the percentage of CAR T cells in CD3+ human T cells after injecting CAR T cells with FITC-PEG11-NK1. [FIG. 6A - 6B] Shown are organs harvested from exemplary mice of the models used in Figures 5A - 5C. Figure 6A shows the organs harvested from the non-treated group. Figure 6B shows the organs harvested 2 weeks after CAR T cell therapy. [FIG. 7A - 7C] Disclosed are MDA-MB-231 (FR+) xenografts under CAR T cell therapy, including treating cells with CAR T cells with either the FITC-PEG12-folic acid complex, the FITC-folic acid complex, or without the complex. Figure 7A shows the tumor volume measured over 23 days. Figure 7B shows the body weight measured over 21 days of therapy. Figure 7C shows the percentage of CAR T cells in CD3+ human T cells after injecting CAR T cells. [FIG. 8A - 8B] Shown are organs harvested from exemplary mice of the models shown in Figures 7A - 7C. Figure 8A shows the organs harvested from the non-treated group. Figure 8B shows the organs harvested 3 weeks after CAR T cell therapy including CAR T cells and 500 nmole / body weight kg of the FITC-PEG12-folic acid complex. [FIG. 9] Figure 9 shows the blood indices of the HEK293 (NK1R+) xenograft model of Figures 5 - 6 and the MDA-MB-231 (FR+) xenograft model of Figures 7 - 8. [FIG. 10] Shown are differences in cytotoxicity against KB (FR+) tumor cells treated with CAR T cells, depending on the FITC-small molecule complex used. [FIG. 11] Shown is the percentage change in body weight in a KB tumor xenograft model using different concentrations of the FITC-PEG-12-folic acid complex and CAR T cells. [FIG. 12A - 12C] Figures 12A-12C show organs collected from exemplary mice of the KB xenograft model shown in Figure 11. Figure 12A shows organs collected from the non-treated group. Figure 12B shows organs collected from the CAR T cell therapy group treated with 250 nmol / kg of FITC-PEG-12-folic acid. Figure 12C shows organs collected from the CAR T cell and CAR T cell therapy group treated with 500 nmol / kg of FITC-PEG-12-folic acid. [FIG. 13] Show the blood indices of mice from the KB xenograft model of Figures 11-12. [FIG. 14A - 14B] Show the constructs used for CAR transduction. Figure 14A shows the CAR4-1BBZ construct. Figure 14B shows the lentiviral vector. [FIG. 15A - 15B] Show the flow cytometry analysis of transduced T cells. Figure 15A shows non-transduced cells. Figure 15B shows transduced cells. [FIG. 16A - 16B] Show the use of a fluorescence microscope for transduced CAR T cells. Figure 16A shows GFP imaging showing transduction. Figure 16B shows FITC folic acid localized to positively transduced cells. [FIG. 17] Show the activation of CAR T cells measured by the relative expression of CD69 as a function of the complex used. [FIG. 18] Show the tumor heterogeneity of KB, LNCaP, and CAR T cells as a function of the complex used. [FIG. 19A - 19C]The same anti-FITC CAR T cells (10^7 cells) were introduced into mice having two different tumors arising from two different cell lines (i.e., MDA-MB-231 (FR+) and HEK (NK1R+)) in different flanks, and then either PBS only (Figure 19A), FITC-PEG11-NK1R (500 nmole / kg) (Figure 19B), or FITC-PEG11-NK1R (500 nmole / kg) + FITC-PEG12-folic acid (500 nmole / kg) (Figure 19C) was injected every other day to show the anti-tumor efficacy. Figure 19A: (black circles) FR+ (MDA-MB-231): CAR T cells + PBS, (black squares) NK1R+ (HEK): CAR T cells + PBS; Figure 19B: (black circles) FR+ (MDA-MB-231): CAR T cells + PBS, (black squares) NK1R+ (HEK): CAR T cells + FITC-PEG11-NK1R (500 nmole / kg); Figure 19C: (black circles) FR+ (MDA-MB-231): CAR T cells + FITC-PEG12-FA (500 nmole / kg), (black squares) NK1R+ (HEK): CAR T cells + PBS.

Mode for Carrying Out the Invention

[0016] Definitions As used herein, "a" or "an" can mean one or more. For example, with respect to numerical values such as integers, fractions, and percentages, "about" as used herein generally indicates a range of numerical values (e.g., ±5% to 10% of the recited value) that would be considered equivalent (e.g., having the same function or result) by one of ordinary skill in the art to the recited value. As used herein, the terms "treating", "being treated", "having been treated", or "treatment" mean both therapeutic treatment and prophylactic or preventative treatment.

[0017] As used herein, the terms "ameliorate," "ameliorating," "amelioration," or "ameliorated" with respect to cancer mean reducing cancer symptoms, reducing tumor size, completely or partially removing the tumor (e.g., complete remission or partial remission), stabilizing the disease, preventing cancer progression (e.g., progression-free survival), preventing the occurrence of the disease, preventing disease progression (e.g., relapse-free survival), or other effects on cancer that a physician deems to be a therapeutic or prophylactic treatment for cancer.

[0018] As used herein, the terms "administer," "administering," or "administered" mean, but are not limited to, all means for introducing into a patient a compound described herein or a pharmaceutically acceptable salt thereof, a first complex or a pharmaceutically acceptable salt thereof, or a second complex or a pharmaceutically acceptable salt thereof, or a CAR T cell composition, such as orally (po), intravenously (iv), intramuscularly (im), subcutaneously (sc), and transdermally.

[0019] As used herein, the term "off-target toxicity" means organ damage or weight loss in a patient that is unacceptable to the treating physician, or any other effect that is difficult for the treating physician to accept, such as B cell aplasia.

[0020] As used herein, the terms "transduction" and "transfection" are used interchangeably and mean introducing nucleic acid into a cell by any artificial method, such as viral and non-viral methods.

[0021] Detailed description of exemplary embodiments In various embodiments described herein, the small molecule ligand linked to the targeting moiety by a linker is used as a bridge between cancer and CAR T cells (i.e., cytotoxic T cells expressing a chimeric antigen receptor). The bridge directs the CAR T cells towards the cancer for the amelioration of the cancer. In one embodiment, the “small molecule ligand” can be folic acid, a CAIX ligand, DUPA, an NK-1R ligand, a ligand for gamma glutamyl transpeptidase, or a CCK2R ligand, each being a small molecule ligand that specifically binds to a cancer cell type (i.e., the respective receptor of each of these ligands is overexpressed on cancer cells compared to normal tissue).

[0022] The “targeting moiety” linked to the small molecule ligand binds to the recognition region of the genetically engineered CAR expressed by the CAR T cells. Thus, the recognition region of the CAR (e.g., a single chain variable fragment (scFv) of an antibody) is directed towards the “targeting moiety”. In this way, the small molecule ligand linked to the targeting moiety by a linker acts as a bridge between cancer and CAR T cells and directs the CAR T cells towards the cancer for the amelioration of the cancer. In various embodiments, the bridge between cancer and CAR T cells can be any of the complexes shown in Examples 5-12.

[0023] The bridge is a small organic molecule, and clearance from the bloodstream can be achieved rapidly (e.g., in about 20 minutes or less). In one embodiment, the CAR T cell response can target only cancer cells that express a receptor for the small molecule ligand portion of the "bridge", thereby reducing off-target toxicity to normal tissues. In another embodiment, activation of the CAR T cells can be controlled by the rapid clearance of the bridge from the bloodstream and by the ability to vary the dose and structure of the bridge that controls CAR T cell activation. Further, since one type of CAR T cell construct can be used to target various cancers, this system can be "universal". Exemplarily, the targeting moiety recognized by the CAR T cells can remain constant so that one type of CAR T cell construct can be used, while the small molecule ligand that binds to the cancer is altered to enable targeting of a wide variety of cancers.

[0024] In one embodiment, the inventors discovered that varying the dose of the small molecule ligand (i.e., the bridge) linked to the targeting moiety by a linker can result in the ability to control CRS in vivo in CAR T cell activation. In another embodiment, the inventors discovered that varying the linker in the small molecule ligand (bridge) linked to the targeting moiety can control CRS in vivo in CAR T cell activation. In yet another embodiment, a combination of these methods can be used to precisely control CAR T cell activation and cytokine release in vivo.

[0025] In the following list of items and in the various embodiments recited in the claims, a small molecule ligand linked to a targeting moiety by a linker is referred to as a "compound", a "first complex" or a "second complex". The term "compound" is used in embodiments where the dose of the small molecule ligand linked to the targeting moiety by the linker is varied to control cytokine release in vivo. The terms "first complex" and "second complex" are used in embodiments where two different complexes are administered to a patient. For example, the linker of the small molecule ligand linked to the targeting moiety can be varied to control cytokine release in vivo or the complex can be modified to include a different small molecule ligand or a different targeting moiety.

[0026] Some embodiments are described by the following enumerated items: 1. A method of treating cancer, comprising: i) administering to a patient a first dose of a compound or a pharmaceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked to a targeting moiety by a linker; ii) administering to the patient a CAR T cell composition, wherein the CAR T cells comprise a CAR directed to the targeting moiety; ii) administering to the patient a second dose of the compound or a pharmaceutically acceptable salt thereof, wherein the second dose is different from the first dose; and iv) treating the patient to ameliorate the cancer. 2. A method of treating cancer, comprising: i) administering to a patient a first complex or a pharmaceutically acceptable salt thereof; ii) administering to the patient a CAR T cell composition, wherein the CAR T cells comprise a CAR directed to the targeting moiety; iii) administering to the patient a second complex or a pharmaceutically acceptable salt thereof, wherein the first and second complexes each comprise a small molecule ligand linked to a targeting moiety by a linker and the first complex and the second complex are different; and iv) treating a patient to ameliorate cancer; A method comprising. 3. A method for treating cancer, i) administering to a patient a first dose of a first complex or a pharmaceutically acceptable salt thereof; ii) administering to the patient a CAR T cell composition (wherein the CAR T cells comprise a CAR directed to a targeting moiety); ii) administering to the patient a second dose of a second complex or a pharmaceutically acceptable salt thereof (wherein the first complex and the second complex each comprise a small molecule ligand linked to a targeting moiety by a linker, the first complex and the second complex are different, and the first dose and the second dose are different); and iv) treating a patient to ameliorate cancer; A method comprising. 4. The method according to item 2 or 3, wherein the linker in the first complex or a pharmaceutically acceptable salt thereof and the linker in the second complex or a pharmaceutically acceptable salt thereof are different. 5. The method according to item 2 or 3, wherein the linker in the first complex or a pharmaceutically acceptable salt thereof and the linker in the second complex or a pharmaceutically acceptable salt thereof are the same. 6. The method according to any one of items 2 to 5, wherein the ligand in the first complex or a pharmaceutically acceptable salt thereof and the ligand in the second complex or a pharmaceutically acceptable salt thereof are different. 7. The method according to any one of items 2 to 5, wherein the ligand in the first complex or a pharmaceutically acceptable salt thereof and the ligand in the second complex or a pharmaceutically acceptable salt thereof are the same. 8. The method according to any one of items 2 to 7, wherein the targeting moiety in the first complex or a pharmaceutically acceptable salt thereof and the targeting moiety in the second complex or a pharmaceutically acceptable salt thereof are different. 9. The method according to any one of items 2 to 7, wherein the targeting moiety in the first complex or a pharmaceutically acceptable salt thereof and the targeting moiety in the second complex or a pharmaceutically acceptable salt thereof are the same. 10. The method according to any one of items 1 to 9, wherein the ligand is selected from folic acid, DUPA, NK-1R ligand, CAIX ligand, ligand for gamma-glutamyl transpeptidase, and CCK2R ligand. 11. The method according to item 10, wherein the ligand is folic acid. 12. The method according to item 10, wherein the ligand is NK-1R ligand. 13. The method according to item 10, wherein the ligand is DUPA. 14. The method according to item 10, wherein the ligand is CCK2R ligand. 15. The method according to item 10, wherein the ligand is ligand for gamma-glutamyl transpeptidase. 16. The method according to any one of items 1 to 15, wherein the targeting moiety is selected from 2,4-dinitrophenol (DNP), 2,4,6-trinitrophenol (TNP), biotin, digoxigenin, fluorescein, fluorescein isothiocyanate (FITC), NHS-fluorescein, pentafluorophenyl ester (PFP), tetrafluorophenyl ester (TFP), notatin, centrin, and DARPin. 17. The method according to item 16, wherein the targeting moiety is FITC. 18. The method according to item 16, wherein the targeting moiety is DNP. 19. The method according to item 16, wherein the targeting moiety is TNP. 20. The method according to any one of items 1 to 19, wherein the linker comprises polyethylene glycol (PEG), polyproline, hydrophilic amino acids, sugars, non-natural peptidoglycans, polyvinylpyrrolidone, and / or pluronic F-127. 21. The method according to item 20, wherein the linker comprises PEG. 22. The compound or a pharmaceutically acceptable salt thereof, the first complex or a pharmaceutically acceptable salt thereof, or the second complex or a pharmaceutically acceptable salt thereof has the formula:

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[0027] As described herein, "patient" may be a human, or in the case of veterinary use, the patient may be an experimental animal, an agricultural animal, a domestic animal, or a wild animal. In one embodiment, the patient may be an experimental animal such as a rodent (e.g., mouse, rat, hamster, etc.), rabbit, monkey, chimpanzee; a pet animal such as a dog, cat, or rabbit; an agricultural animal such as a cow, horse, pig, sheep, goat; or a wild animal in captivity such as a bear, panda, lion, tiger, leopard, elephant, zebra, giraffe, gorilla, dolphin, or whale. In the methods described herein, the step of "treating a patient to improve cancer" may include or consist of the administration step in the method.

[0028] In one exemplary embodiment, The small molecule ligand (bridge) linked to the targeting moiety by a linker comprises a fluorescein isothiocyanate (FITC) linked to the small molecule ligand. Cancer overexpresses the receptor for the small molecule ligand. As a second component, for example, cytotoxic T cells are transformed to express a CAR that comprises an anti-FITC scFv. In this manner, the CAR targets the FITC that decorates the cancer with FITC molecules as a result of the small molecule ligand binding to the cancer. Thus, toxicity to normal non-target cells can be avoided. When the anti-FITC CAR-expressing T cells bind to FITC, the CAR T cells are activated and the cancer is ameliorated (e.g., by killing cancer cells).

[0029] In one embodiment, the "small molecule ligand" can be folic acid, DUPA (a ligand bound by PSMA-positive human prostate cancer cells and other cancer cells), an NK-1R ligand (e.g., a ligand for the NK-1R receptor found on colon and pancreatic cancers), a CAIX ligand (the receptor for the CAIX ligand is found, e.g., on kidney cancer, ovarian cancer, vulvar cancer, and breast cancer), a ligand for gamma-glutamyl transpeptidase (the transpeptidase is overexpressed, e.g., in ovarian cancer, colon cancer, liver cancer, astrocytic glioma, melanoma and leukemia), or a CCK2R ligand (the receptor for the CCK2R ligand is found on cancers of the thyroid, lung, pancreas, ovary, brain, stomach, gastrointestinal stroma and colon), each being a small molecule ligand that binds specifically to a cancer cell type (i.e., the respective receptor for each of these ligands is overexpressed on cancer cells as compared to normal tissue). In one embodiment, the DUPA derivative can be a ligand that is a small molecule ligand linked to the targeting moiety, and the DUPA derivative is described in WO 2015 / 057852, which is incorporated herein by reference.

[0030] In one embodiment, the small molecule ligand is folic acid. Folic acid can be folic acid, a folic acid analog, or another folic acid receptor binding molecule. In various embodiments, folic acid analogs that can be used include folic acid receptor binding pteridines such as folinic acid, pteropolyg lutamic acid, and tetrahydropterin, dihydrofolic acid, tetrahydrofolic acid, and their deaza and dideaza analogs. The terms "deaza" and "dideaza" analogs refer to analogs recognized in the art having a carbon atom substituted for one or two nitrogen atoms in the naturally occurring folic acid structure. For example, deaza analogs include 1-deaza, 3-deaza, 5-deaza, 8-deaza, and 10-deaza analogs. Dideaza analogs include, for example, 1,5-dideaza, 5,10-dideaza, 8,10-dideaza, and 5,8-dideaza analogs. The aforementioned folic acid analogs have conventionally been called "folic acid" in reflection of their ability to bind to folic acid receptors. Other folic acid receptor binding analogs include deaza analogs such as aminopterin, amethopterin (methotrexate), N10-methylfolic acid, 2-deamino-hydroxybutyric acid, deazamethopterin or 3-deazamethopterin, and 3',5'-dichloro-4-amino-4-deoxy-N10-methylpteroylglutamic acid (dichloromethotrexate).

[0031] In one embodiment, the small molecule ligand may have a mass of less than about 10,000 daltons, less than about 9,000 daltons, less than about 8,000 daltons, less than about 7,000 daltons, less than about 6,000 daltons, less than about 5,000 daltons, less than about 4,500 daltons, less than about 4,000 daltons, less than about 3,500 daltons, less than about 3,000 daltons, less than about 2,500 daltons, less than about 2,000 daltons, less than about 1,500 daltons, less than about 1,000 daltons, or less than about 500 daltons. In another embodiment, the small molecule ligand may have a mass of about 1 to about 10,000 daltons, about 1 to about 9,000 daltons, about 1 to about 8,000 daltons, about 1 to about 7,000 daltons, about 1 to about 6,000 daltons, about 1 to about 5,000 daltons, about 1 to about 4,500 daltons, about 1 to about 4,000 daltons, about 1 to about 3,500 daltons, about 1 to about 3,000 daltons, about 1 to about 2,500 daltons, about 1 to about 2,000 daltons, about 1 to about 1,500 daltons, about 1 to about 1,000 daltons, or about 1 to about 500 daltons.

[0032] In one aspect, the "targeting moiety" that binds to the CAR expressed by the CAR T cell can be selected from, for example, 2,4-dinitrophenol (DNP), 2,4,6-trinitrophenol (TNP), biotin, digoxigenin, fluorescein, fluorescein isothiocyanate (FITC), NHS-fluorescein, pentafluorophenyl ester (PFP), tetrafluorophenyl ester (TFP), knottin, centyrin, and DARPin. The identity of the targeting moiety is preferably limited only in that it is recognized and bound by the CAR with specificity and has a relatively low molecular weight. In various aspects, exemplary targeting moieties are haptens such as low molecular weight organic molecules.

[0033] The targeting moiety can have the following exemplary structures: [Chemical formula] [wherein X is oxygen, nitrogen or sulfur, and X is bonded to the linker L; Y is ORa , NR a 2 or NR a 3 +; and Y’ is O, NR a or NR a 2 + ; each R is independently in each case selected from H, fluoro, sulfonic acid, sulfonate, and their salts, etc.; and R a is hydrogen or alkyl].

[0034] In one exemplary embodiment, the linker in the compounds described herein or their pharmaceutically acceptable salts, the first complex or its pharmaceutically acceptable salts, or the second complex or its pharmaceutically acceptable salts can be a direct bond (e.g., the reaction between the isothiocyanate group of FITC and the free amine group of the small molecule ligand), or the bond can be through an intermediary linker. In one embodiment, if present, the intermediary linker can be a biocompatible linker known in the art such as a divalent linker. In one exemplary embodiment, the divalent linker can contain from about 1 to about 30 carbon atoms. In another exemplary embodiment, the divalent linker can contain from about 2 to about 20 carbon atoms. In other embodiments, low molecular weight divalent linkers (i.e., those having an approximate molecular weight of about 30 to about 300) are used. In another embodiment, linkers having 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 atoms, among others, can be used as linkers of appropriate length, but are not limited thereto.

[0035] In various embodiments, the small molecule ligand linked to the targeting moiety has the formula:

Chemical Structure

[0036] In another embodiment, the linker may be a divalent linker that can include one or more spacers. Exemplary spacers are shown in the following table. The following non-limiting exemplary spacers are described, where * indicates the point of attachment to the small molecule ligand or the targeting moiety.

[0037] [Table 1]

[0038] In other embodiments, the small molecule ligand (bridge) linked to the targeting moiety can have any of the following structures.

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0039] In other embodiments, the compound or a pharmaceutically acceptable salt thereof, the first complex or a pharmaceutically acceptable salt thereof, or the second complex or a pharmaceutically acceptable salt thereof does not include an antibody or a fragment of an antibody. In yet another embodiment, the targeting moiety is not a peptide epitope.

[0040] In one exemplary embodiment, different types of conjugates (e.g., a first conjugate and a second conjugate) can be administered to a patient. For example, the linker in the first conjugate or a pharmaceutically acceptable salt thereof, and the linker in the second conjugate or a pharmaceutically acceptable salt thereof can be the same or different. In another embodiment, the ligand in the first conjugate or a pharmaceutically acceptable salt thereof, and the ligand in the second conjugate or a pharmaceutically acceptable salt thereof can be the same or different. In another exemplary embodiment, the targeting moiety in the first conjugate or a pharmaceutically acceptable salt thereof, and the targeting moiety in the second conjugate or a pharmaceutically acceptable salt thereof can be the same or different. Any combination of these embodiments is also contemplated together with any combination of the dosages described below.

[0041] In yet another embodiment, a kit is provided that includes at least two different types of bridges, where the bridge includes a small molecule ligand linked to a targeting moiety, where the ligands in at least two different types of bridges are different, and where the ligand is selected from folic acid, DUPA, a CAIX ligand, an NK-1R ligand, a ligand for gamma-glutamyl transpeptidase, and a CCK2R ligand. In this embodiment, the ligand in at least one bridge can be an NK-1R ligand, a ligand for gamma-glutamyl transpeptidase, folic acid, a CAIX ligand, a CCK2R ligand, or DUPA.

[0042] In another embodiment, the bridge in the kit has the formula:

Chemical formula

Chemical formula

[0043] “Pharmaceutically acceptable salts” of the small molecule ligand linked to the targeting moiety by a linker are contemplated. As used herein, the term “pharmaceutically acceptable salts” refers to salts whose counterions 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, or malonic acid; or 2) salts formed when 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. Pharmaceutically acceptable salts are well known to those skilled in the art, and such pharmaceutically acceptable salts can be contemplated in connection with the embodiments described herein.

[0044] In various embodiments, suitable acid addition salts are formed from acids that form non-toxic salts. Exemplary 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-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate / hydrogen phosphate / dihydrogen phosphate, saccharate, stearate, succinate, tartrate, tosylate, and trifluoroacetate.

[0045] In various embodiments, suitable base salts are formed from bases that form non-toxic salts. Exemplary examples include arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine, and zinc salts. Hemisalts of acids and bases, for example, hemisulfate and hemicalcium salts, may also be formed.

[0046] In one exemplary embodiment, the compounds described herein or pharmaceutically acceptable salts thereof, the first conjugate or pharmaceutically acceptable salts thereof, or the second conjugate or pharmaceutically acceptable salts thereof can contain one or more chiral centers or, alternatively, can exist as multiple stereoisomers. Accordingly, various embodiments can include pure stereoisomers as well as mixtures of stereoisomers such as enantiomers, diastereomers, and enantiomerically or diastereomerically enriched mixtures. In one embodiment, the compounds described herein or pharmaceutically acceptable salts thereof, the first conjugate or pharmaceutically acceptable salts thereof, or the second conjugate or pharmaceutically acceptable salts thereof can exist as geometric isomers. Accordingly, various embodiments can include pure geometric isomers or mixtures of geometric isomers.

[0047] In some embodiments, the compounds described herein or pharmaceutically acceptable salts thereof, the first conjugate or pharmaceutically acceptable salts thereof, or the second conjugate or pharmaceutically acceptable salts thereof can exist in solvated forms such as unsolvated and hydrated forms. Generally, solvated forms are equivalent to unsolvated forms and are encompassed within the scope of the present invention.

[0048] The methods described herein also utilize cytotoxic T lymphocytes engineered to express a chimeric antigen receptor (CAR) that recognizes and binds to a targeting moiety of the bridge (e.g., FITC, DNP, or TNP). In one embodiment, the CAR described herein comprises three domains such as: 1) a recognition domain (e.g., a single-chain variable fragment (scFv) region of an antibody) that recognizes and binds to a targeting moiety having specificity; 2) a co-stimulatory domain that promotes the proliferation and survival of T lymphocytes; and 3) an activation signaling domain that generates a cytotoxic T lymphocyte activation signal.

[0049] In various aspects, the scFv region of an antibody that binds to folic acid, DUPA, a CAIX ligand, an NK-1R ligand, a ligand of gamma-glutamyl transpeptidase, or a CCK2R ligand can be used. In an exemplary embodiment, the scFv region can be produced from (i) an antibody known in the art that binds to a targeting moiety, (ii) an antibody newly prepared using a selected target moiety such as a hapten, and (iii) sequence variants derived from the scFv region of such an antibody, for example, having at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity to the amino acid sequence of the scFv region from which they are derived.

[0050] In any of the embodiments described herein, the binding moiety of the CAR can be, for example, a single-chain variable region (scFv) such as an antibody, Fab, Fv, Fc, or (Fab’)2 fragment.

[0051] In one aspect, the co-stimulatory domain functions to enhance the proliferation and survival of cytotoxic T lymphocytes upon binding of the CAR to the targeting moiety. Suitable co-stimulatory domains include: 1) CD28, 2) CD137 (4-1BB), which is a member of the tumor necrosis factor (TNF) receptor family, 3) CD134 (OX40), which is a member of the TNFR superfamily of receptors, and 4) CD278 (ICOS), a CD28 superfamily co-stimulatory molecule expressed on activated T cells, or combinations thereof. Suitable co-stimulatory domains also include, but are not limited to, CD27, CD30, CD150, DAP10, and NKG2D, or combinations thereof. One of ordinary skill in the art will understand that sequence variants of these co-stimulatory domains can be used without adversely affecting the present invention, provided that they have the same or similar activity as the domain from which they are modeled. In various embodiments, such variants have at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity to the amino acid sequence of the domain from which they are derived.

[0052] In an exemplary embodiment, the activation signaling domain functions to activate cytotoxic T lymphocytes when the CAR binds to the targeting moiety. Suitable activation signaling domains include the T cell CD3ζ chain and the Fc receptor γ. One of ordinary skill in the art will understand that sequence variants of these noted activation signaling domains can be used if the variant has the same or similar activity as the domain from which it is modeled. In various embodiments, the variant has at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity to the amino acid sequence of the domain from which it is derived.

[0053] In one aspect, the construct encoding the CAR is produced using genetic engineering techniques. Such techniques are described in detail in Sambrook et al., "Molecular Cloning: A Laboratory Manual," 3rd ed., Cold Spring Harbor Laboratory Press (2001), which is incorporated herein by reference. As an example, a plasmid or viral expression vector (e.g., a lentiviral vector, a retroviral vector, Sleeping Beauty, and piggyback (a transposon / transposase system including a non-viral mediated CAR gene delivery system)) encoding a fusion protein containing a recognition region, one or more co-stimulatory domains, and an activation signaling domain in-frame and linked in the 5' to 3' direction can be produced. In other embodiments, other configurations are acceptable and include a recognition region, an activation signaling domain, and one or more co-stimulatory domains. The placement of the recognition region in the fusion protein is generally done such that the display of the region is achieved outside the cell. In one embodiment, the CAR includes a signal peptide that ensures proper transport of the fusion protein to the cell surface, a transmembrane domain that ensures the fusion protein is maintained as a complete membrane protein, and a hinge domain that provides flexibility to the recognition region and allows for strong binding to the targeting moiety and may include additional elements such as.

[0054] Diagrams of exemplary CARs are shown in FIGS. 14A and B, where the fusion protein sequence is incorporated into a lentiviral expression vector, "SP" is the signal peptide, the CAR is an anti-FITC CAR, the CD8α hinge and transmembrane (TM) regions are present, the co-stimulatory domain is 4-1BB, and the activation signaling domain is CD3ζ. In one aspect, the nucleic acid sequence of the CAR insert is provided as SEQ ID NO: 1, and the amino acid sequence of the insert is provided as SEQ ID NO: 2.

[0055] In one embodiment, the CAR has a recognition region, the recognition region is a single-chain fragment variable (scFv) region of an anti-FITC antibody, a co-stimulatory domain, the co-stimulatory domain is CD137 (4-1BB), an activation signaling domain and the activation signaling domain is the T cell CD3ζ chain. It is well known to those skilled in the art that anti-FITC scFv and anti-fluorescein scFv are equivalent terms.

[0056] In one embodiment, a population of cytotoxic T lymphocytes can be genetically engineered to express a CAR construct by transfection with an expression vector encoding the CAR construct. Suitable methods for preparing a transfected population of T lymphocytes that express the selected CAR construct are well known to those skilled in the art and are described in Sambrook et al., "Molecular Cloning: A Laboratory Manual," 3rd ed., Cold Spring Harbor Laboratory Press, (2001), which is incorporated herein by reference.

[0057] In various embodiments, CAR T cells comprising the nucleic acid of SEQ ID NO: 1 or 3 are provided. In another embodiment, CAR T cells comprising the polypeptide of SEQ ID NO: 2 are provided. In another exemplary embodiment, an isolated nucleic acid comprising SEQ ID NO: 1 or 3 and encoding a chimeric antigen receptor is provided. In yet another embodiment, a chimeric antigen receptor polypeptide comprising SEQ ID NO: 2 is provided. In another embodiment, a vector comprising SEQ ID NO: 1 or 3 is provided. In another embodiment, a lentiviral vector comprising SEQ ID NO: 1 or 3 is provided. In another embodiment, SEQ ID NO: 2 can comprise or consist of a human or humanized amino acid sequence.

[0058] In each of these embodiments, mutant nucleic acid sequences or amino acid sequences having at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 are contemplated. In another embodiment, the nucleic acid sequence can be a mutant nucleic acid sequence having at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity to SEQ ID NO: 1 or 3, and the mutant sequence encodes the polypeptide of SEQ ID NO: 2. In another embodiment, the mutant nucleic acid or amino acid sequence can be a mutant nucleic acid or amino acid sequence having at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 along a stretch of 200 nucleic acids or 200 amino acids of SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3. The determination of the percent identity or similarity between sequences can be made, for example, using the GAP program (Genetics Computer Group, software; available from Accelrys at http: / / www.accelrys.com), and the alignment can be made, for example, using the ClustalW algorithm (VNTI software, InforMax Inc.). A sequence database can be searched using the nucleic acid or amino acid sequence of interest. The algorithms for database searches are usually based on BLAST software (Altschul et al., 1990). In some embodiments, the percent identity can be determined along the entire length of the nucleic acid or amino acid sequence.

[0059] Also within the scope of the present invention are nucleic acids complementary to the nucleic acid represented by SEQ ID NO: 1 or 3, and nucleic acids that hybridize to the nucleic acid represented by SEQ ID NO: 1 or 3, or nucleic acids that hybridize to its complement under high stringency conditions. In the present invention, "high stringency conditions" mean hybridization at 65°C in 5×SSPE and 50% formamide and washing at 65°C in 0.5×SSPE. High stringency, low stringency, and moderately stringent hybridization conditions are described in Sambrook et al., "Molecular Cloning: A Laboratory Manual", 3rd edition, Cold Spring Harbor Laboratory Press, (2001), which is incorporated herein by reference. In some exemplary embodiments, hybridization occurs along the entire length of the nucleic acid.

[0060] In one embodiment, allogeneic cells can also be used, such as when the patient to be treated has received high-dose chemotherapy or radiation therapy to destroy the patient's immune system, but the cytotoxic T lymphocytes used to prepare CAR T cells, as used in the methods described herein, can be autologous cells. In one embodiment, allogeneic cells can be used.

[0061] In one aspect, cytotoxic lymphocyte T can be obtained from a patient by means well known in the art. For example, cytotoxic T cells can be obtained by collecting peripheral blood from a patient, subjecting the blood to Ficoll density gradient centrifugation, and then isolating a population of cytotoxic T cells from the peripheral blood using a negative T cell isolation kit (such as the EasySep™ T cell isolation kit). In one exemplary embodiment, the population of cytotoxic T lymphocytes need not be pure and can contain other cells such as other T cells, monocytes, macrophages, natural killer cells, and B cells. In one aspect, the collected population can contain at least about 90%, at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the selected cell type.

[0062] In one embodiment, after cytotoxic T lymphocytes are obtained, the cells are cultured under conditions that promote cell activation. In this embodiment, the culture conditions may be such that the cells can be administered to a patient without considering the reactivity to the components of the culture medium. For example, the culture conditions may not include bovine serum products such as bovine serum albumin. In one exemplary embodiment, in the case of cytotoxic T cells, activation can be achieved by introducing a known activator such as an anti-CD3 antibody into the medium. Other suitable activators include anti-CD28 antibodies. In one embodiment, the population of lymphocytes can be cultured for about 1 to about 4 days under conditions that promote activation. In one embodiment, an appropriate level of activation can be determined by cell size, growth rate, or activation markers determined by flow cytometry.

[0063] In one exemplary embodiment, after culturing a population of cytotoxic T lymphocytes under conditions that promote activation, the cells can be transfected with an expression vector encoding a CAR. Suitable vectors and transfection methods are described above. In one embodiment, after transfection, the cells can be administered to a patient immediately, or, for example, to allow time for the cells to recover from transfection, for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 days or more, or about 5 to about 12 days, about 6 to about 13 days, about 7 to about 14 days, or about 8 to about 15 days, the cells can be cultured. . Suitable culture conditions can be the same as the conditions under which the cells were cultured for activation, whether or not the cells use the agent used to promote activation.

[0064] Thus, as described above, in one exemplary embodiment, the treatment method described herein may further include: 1) obtaining a population of autologous or heterologous cytotoxic T lymphocytes; 2) culturing the T lymphocytes under conditions that promote cell activation; and 3) transfecting the lymphocytes with an expression vector encoding a CAR to form CAR T cells.

[0065] In one exemplary embodiment, when the cells are transfected and activated, a composition comprising CAR T cells can be prepared and administered to a patient. In one embodiment, a culture medium lacking animal products such as bovine serum can be used. In another embodiment, tissue culture conditions typically used by those skilled in the art to avoid contamination by bacteria, fungi, and mycoplasma can be used. In an exemplary embodiment, prior to administration to the patient, the cells are pelleted, washed, and resuspended in a pharmaceutically acceptable carrier or diluent. Exemplary compositions comprising cytotoxic T lymphocytes expressing a CAR include compositions comprising cells in sterile 290 mOsm saline, in an injectable cryopreservation medium (comprising Plasma-Lyte A, dextrose, sodium chloride injection, human serum albumin, and DMSO), in 0.9% NaCl containing 2% human serum albumin, or in any other sterile 290 mOsm injectable material. Alternatively, depending on the nature of the culture medium, the CAR T cells can be administered as a composition in the culture medium or concentrated and resuspended in the medium prior to administration. The CAR T cell composition can be administered to the patient via any suitable means such as parenteral administration (e.g., intradermal, subcutaneous, intramuscular, intraperitoneal, intravenous, or intrathecal).

[0066] In one aspect, the total number of CAR T cells and the concentration of the cells in the composition administered to the patient vary depending on many factors such as the type of cytotoxic T lymphocytes used, the binding specificity of the CAR, the nature of the targeting moiety and the small molecule ligand, the nature of the cancer, the location of the cancer in the patient, the means used to administer the composition to the patient, and the health, age and weight of the patient being treated. However, a suitable composition containing transduced CAR T cells comprises a composition having a volume of about 5 ml to about 200 ml and containing from about 1×10 5 to about 1×10 15 transduced CAR T cells. A typical composition comprises a volume of about 10 ml to about 125 ml and contains from about 1×10 7 to about 1×10 10 CAR T cells. An exemplary composition contains about 1×10 9 CAR T cells in a volume of about 100 ml. In one aspect, a single dose or multiple doses of CAR T cells can be administered to the patient.

[0067] In various embodiments, the cancer being treated is carcinoma, sarcoma, lymphoma, melanoma, mesothelioma, nasopharyngeal carcinoma, leukemia, adenocarcinoma, or myeloma. In other embodiments, the cancer can be lung cancer, bone cancer, pancreatic cancer, skin cancer, head cancer, neck cancer, cutaneous melanoma, uveal melanoma, uterine cancer, ovarian cancer, endometrial cancer, rectal cancer, stomach cancer, colon cancer, breast cancer, triple negative breast cancer, cervical cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, non-small cell lung cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, prostate cancer, chronic leukemia, acute leukemia, lymphocytic lymphoma, pleural mesothelioma, bladder cancer, Burkitt lymphoma, ureteral cancer, kidney cancer, renal cell carcinoma, renal pelvis cancer, central nervous system (CNS) neoplasm, primary CNS lymphoma, spinal cord axis tumor, brain stem glioma, pituitary adenoma or gastroesophageal junction adenocarcinoma.

[0068] In some aspects of these embodiments, the cancer is a cancer that expresses a folate receptor. In some aspects of these embodiments, the cancer is endometrial cancer, non-small cell lung cancer, ovarian cancer, or triple-negative breast cancer. In another embodiment, the cancer to be imaged is a tumor. In another embodiment, the cancer is malignant.

[0069] The compounds or pharmaceutically acceptable salts thereof, the first conjugate or pharmaceutically acceptable salts thereof, the second conjugate or pharmaceutically acceptable salts thereof, or the CAR T cell composition described herein can be administered to a patient using any suitable method known in the art. As described herein, the terms "administer" or "administered" include, but are not limited to, all means of introducing a compound or pharmaceutically acceptable salt thereof, the first conjugate or pharmaceutically acceptable salts thereof, the second conjugate or pharmaceutically acceptable salts thereof, or the CAR T cell composition to a patient, such as oral (po), intravenous (iv), intramuscular (im), subcutaneous (sc), and transdermal. The compounds or pharmaceutically acceptable salts thereof, the first conjugate or pharmaceutically acceptable salts thereof, or the second conjugate or pharmaceutically acceptable salts thereof described herein can be administered in unit dosage forms and / or formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants, and vehicles.

[0070] In one aspect, the compounds or pharmaceutically acceptable salts thereof, the first conjugate or pharmaceutically acceptable salts thereof, the second conjugate or pharmaceutically acceptable salts thereof, or the CAR T cell composition described herein can be administered directly into the bloodstream, muscle, or internal organs. Routes suitable for such parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, epidural, intracerebroventricular, intraurethral, intrasternal, intracranial, intratumoral, intramuscular, and subcutaneous delivery. In one embodiment, means for parenteral administration include needle (including microneedle) syringes, needleless syringes, and infusion techniques.

[0071] In one exemplary embodiment, the parenteral formulation is typically an aqueous solution that may contain carriers or excipients such as salts, carbohydrates, and buffers (preferably at pH 3 to 9), although they may be more suitably formulated as a sterile non-aqueous solution or in a dry form for use with a suitable vehicle such as sterile, pyrogen-free water or sterile saline. In other embodiments, any of the liquid formulations described herein can be adapted for parenteral administration as described herein. 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 ordinary skill in the art. In one embodiment, the solubility of the compound or its pharmaceutically acceptable salt, the first complex or its pharmaceutically acceptable salt, the second complex or its pharmaceutically acceptable salt used in the preparation of the parenteral formulation can be increased by the use of appropriate formulation techniques such as the incorporation of solubility enhancers.

[0072] In some embodiments, the rate of tumor lysis can be controlled by adjusting the concentration of the first complex or a pharmaceutically acceptable salt thereof, the second complex or a pharmaceutically acceptable salt thereof, or both the first and second complexes or a pharmaceutically acceptable salt thereof. Thus, the cytotoxicity of the CAR T cell composition can be adjusted by changing the concentration of the first complex or a pharmaceutically acceptable salt thereof, the second complex or a pharmaceutically acceptable salt thereof, or both the first and second complexes or a pharmaceutically acceptable salt thereof. In some embodiments, as described herein, the cytotoxicity of the CAR T cell composition can be balanced with the risk of tumor lysis syndrome or cytokine release syndrome (CRS) by adjusting the concentration of the first complex or a pharmaceutically acceptable salt thereof, the second complex or a pharmaceutically acceptable salt thereof, or both the first and second complexes or a pharmaceutically acceptable salt thereof. It is understood that the concentration of the first complex or a pharmaceutically acceptable salt thereof, the second complex or a pharmaceutically acceptable salt thereof, or both the first and second complexes or a pharmaceutically acceptable salt thereof can be a function of the amount or dosage of the first complex or a pharmaceutically acceptable salt thereof, the second complex or a pharmaceutically acceptable salt thereof, or both the first and second complexes or a pharmaceutically acceptable salt thereof administered to the patient.

[0073] The amount of the compound or a pharmaceutically acceptable salt thereof, the first complex or a pharmaceutically acceptable salt thereof, and the second complex or a pharmaceutically acceptable salt thereof to be administered to a patient can vary widely depending on the cancer being treated, the compound or a pharmaceutically acceptable salt thereof, the first complex or a pharmaceutically acceptable salt thereof, the second complex or a pharmaceutically acceptable salt thereof, the route of administration, and the tissue distribution. The amount to be administered to a patient can be based on body surface area, body weight, and the physician's assessment. In various embodiments, the amount administered can be, for example, in the range of about 0.05 mg to about 30 mg, 0.05 mg to about 25.0 mg, about 0.05 mg to about 20.0 mg, about 0.05 mg to about 15.0 mg, about 0.05 mg to about 10.0 mg, about 0.05 mg to about 9.0 mg, about 0.05 mg to about 8.0 mg, about 0.05 mg to about 7.0 mg, about 0.05 mg to about 6.0 mg, about 0.05 mg to about 5.0 mg, about 0.05 mg to about 4.0 mg, about 0.05 mg to about 3.0 mg, about 0.05 mg to about 2.0 mg, about 0.05 mg to about 1.0 mg, about 0.05 mg to about 0.5 mg, about 0.05 mg to about 0.4 mg, about 0.05 mg to about 0.3 mg, about 0.05 mg to about 0.2 mg, about 0.05 mg to about 0.1 mg, about 0.01 mg to about 2 mg, about 0.3 mg to about 10 mg, about 0.1 mg to about 20 mg, or about 0.8 to about 3 mg. Those skilled in the art will readily understand that the dosage can vary within the various ranges provided above based on the above factors and can be at the discretion of the physician.

[0074] In other embodiments, the dosage of the compound or a pharmaceutically acceptable salt thereof, the first complex or a pharmaceutically acceptable salt thereof, the second complex or a pharmaceutically acceptable salt thereof can be, for example, in the range of about 50 nmol / kg to about 3000 nmol / kg, about 50 nmol / kg to about 2000 nmol / kg, about 50 nmol / kg to about 1000 nmol / kg, about 50 nmol / kg to about 900 nmol / kg, about 50 nmol / kg to about 800 nmol / kg, about 50 nmol / kg to about 700 nmol / kg, about 50 nmol / kg to about 600 nmol / kg, about 50 nmol / kg to about 500 nmol / kg, about 50 nmol / kg to about 400 nmol / kg, about 50 nmol / kg to about 300 nmol / kg, about 50 nmol / kg to about 200 nmol / kg, about 50 nmol / kg to about 100 nmol / kg, about 100 nmol / kg to about 300 nmol / kg, about 100 nmol / kg to about 500 nmol / kg, about 100 nmol / kg to about 1000 nmol / kg, about 100 nmol / kg to about 2000 nmol / kg per patient body weight. In other embodiments, the dosage can be about 100 nmol / kg, about 150 nmol / kg, about 200 nmol / kg, about 250 nmol / kg, about 300 nmol / kg, about 350 nmol / kg, about 400 nmol / kg, about 450 nmol / kg, about 500 nmol / kg, about 600 nmol / kg, about 700 nmol / kg, about 800 nmol / kg, about 900 nmol / kg, about 1000 nmol / kg, about 2000 nmol / kg, or about 3000 nmol / kg per patient body weight. In these embodiments, "kg" is the kilogram of the patient's body weight. In one embodiment, a single or multiple dosages of the compound or a pharmaceutically acceptable salt thereof, the first complex or a pharmaceutically acceptable salt thereof, the second complex or a pharmaceutically acceptable salt thereof may be administered to the patient.

[0075] In another embodiment, a compound or a pharmaceutically acceptable salt thereof, a first complex or a pharmaceutically acceptable salt thereof, or a second complex or a pharmaceutically acceptable salt thereof, in an amount of from about 20 μg / kg of patient body weight to about 3 mg / kg of patient body weight, is administered to a patient. In another embodiment, the amount can be from about 0.2 mg / kg of patient body weight to about 0.4 mg / kg of patient body weight, or can be about 50 μg / kg of patient body weight. In one embodiment, a single or multiple doses of the compound or a pharmaceutically acceptable salt thereof, the first complex or a pharmaceutically acceptable salt thereof, or the second complex or a pharmaceutically acceptable salt thereof may be administered to the patient.

[0076] In one embodiment, the small molecule ligand linked to the targeting moiety can be administered to the patient prior to the CAR T cell composition. In another embodiment, the small molecule ligand linked to the targeting moiety is administered to the patient simultaneously with the CAR T cell composition but in a different formulation. In yet another embodiment, the small molecule ligand linked to the targeting moiety can be administered to the patient after the CAR T cell composition.

[0077] In one exemplary embodiment, the timing between the administration of the CAR T cells and the administration of the small molecule linked to the targeting moiety varies widely depending on factors such as the type of CAR T cells used, the binding specificity of the CAR, the nature of the targeting moiety and the small molecule ligand, the nature of the cancer, the location of the cancer in the patient, the means used to administer the CAR T cells and the small molecule ligand linked to the targeting moiety to the patient, and the health, age and weight of the patient being treated. In one embodiment, the small molecule ligand linked to the targeting moiety can be administered before or after the CAR T cells within about 3, 6, 9, 12, 15, 18, 21, or 24 hours, or within about 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10 days or more.

[0078] In some embodiments, the rate of tumor lysis can be controlled by adjusting the rate of administration of the first complex or a pharmaceutically acceptable salt thereof, the second complex or a pharmaceutically acceptable salt thereof, or both the first and second complexes or pharmaceutically acceptable salts thereof. Thus, the cytotoxicity of the CAR T cell composition can be controlled by varying the rate of administration of the first complex or a pharmaceutically acceptable salt thereof, the second complex or a pharmaceutically acceptable salt thereof, or both the first and second complexes or pharmaceutically acceptable salts thereof. In some embodiments, as described herein, the cytotoxicity of the CAR T cell composition can be balanced with the risk of tumor lysis syndrome or cytokine release syndrome (CRS) by adjusting the rate of administration of the first complex or a pharmaceutically acceptable salt thereof, the second complex or a pharmaceutically acceptable salt thereof, or both the first and second complexes or pharmaceutically acceptable salts thereof. It is understood that the rate of administration of the first complex or a pharmaceutically acceptable salt thereof, the second complex or a pharmaceutically acceptable salt thereof, or both the first and second complexes or pharmaceutically acceptable salts thereof can be a function of any dosing schedule well known in the art. For example, the rate of administration can be a function of a dosing schedule based on continuous administration, once daily (alias qd), twice daily (alias bid), three times daily (alias tid), twice a week (alias BIW), three times a week (alias TIW), once a week, and the like. It is understood that the dosing schedule selected for the first complex or a pharmaceutically acceptable salt thereof, the second complex or a pharmaceutically acceptable salt thereof, or both the first and second complexes or pharmaceutically acceptable salts thereof can be applied with the concentration to control the cytotoxicity of the CAR T cell composition.In some embodiments, as described herein, the cytotoxicity of the CAR T cell composition can be balanced with the risk of tumor lysis syndrome or cytokine release syndrome (CRS) by adjusting the dosing schedule with the concentration of the first complex or a pharmaceutically acceptable salt thereof, the second complex or a pharmaceutically acceptable salt thereof, or both the first and second complexes or pharmaceutically acceptable salts thereof.

[0079] In one embodiment of the methods described herein, the cancer is imaged before administration of the compound or a pharmaceutically acceptable salt thereof, the first complex or a pharmaceutically acceptable salt thereof, the second complex or a pharmaceutically acceptable salt thereof to the patient, or before administration of the CAR T cell composition to the patient. In one exemplary embodiment, the imaging is effected by PET imaging. In other exemplary embodiments, the imaging is effected by MRI imaging or SPECT / CT imaging. It is understood that the imaging method can be any suitable imaging method well known in the art.

[0080] In any of the embodiments described herein, cytokine release that causes "off-target" cytotoxicity in the patient may not occur, despite the cytotoxicity of the CAR T cells to the cancer. In any of the embodiments described herein, "off-target" tissue cytotoxicity may not occur in the patient, despite the cytotoxicity of the CAR T cells to the cancer. In any of the embodiments described herein, the cancer may include a tumor and "off-target" cytotoxicity may not occur, but the size of the patient's tumor may decrease.

Example

[0081] Preparation of a lentiviral vector encoding the CAR gene Using the overlap PCR method, a CAR construct containing scFv against fluorescein was prepared. The scFV against fluorescein, 4M5.3 (Kd = 270 fM, 762 bp) derived from the anti-fluorescein (4-4-20) antibody was synthesized. As shown in Figure 14A, the sequences encoding the human CD8α signal peptide (SP, 63 bp), hinge and transmembrane region (249 bp), 4-1BB (CD137, 141 bp) and the cytoplasmic domain of the CD3ζ chain (336 bp) were fused to the anti-fluorescein scFV by overlapping PCR. The resulting CAR construct (1551 bp) was inserted into the EcoRI / NotI digested lentiviral expression vector pCDH-EF1-MCS-(PGK-GFP) (Figure 14B, System Biosciences). The sequence of the CAR construct in the lentiviral vector was confirmed by DNA sequencing.

[0082] Exemplary CAR nucleic acid coding sequences may include the following:

[0083] ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGATGTCGTGATGACCCAGACCCCCCTCAGCCTCCCAGTGTCCCTCGGTGACCAGGCTTCTATTAGTTGCAGATCCAGCCAGTCCCTCGTGCACTCTAACGGTAATACCTACCTGAGATGGTATCTCCAGAAGCCCGGACAGAGCCCTAAGGTGCTGATCTACAAAGTCTCCAACCGGGTGTCTGGAGTCCCTGACCGCTTCTCAGGGAGCGGTTCCGGCACCGACTTCACCCTGAAGATCAACCGGGTGGAGGCCGAAGACCTCGGCGTCTATTTCTGCTCTCAGAGTACACATGTGCCCTGGACCTTCGGCGGAGGGACCAAGCTGGAGATCAAAAGCTCCGCAGACGATGCCAAGAAAGATGCCGCTAAGAAAGACGATGCTAAGAAAGACGATGCAAAGAAAGACGGTGGCGTGAAGCTGGATGAAACCGGAGGAGGTCTCGTCCAGCCAGGAGGAGCCATGAAGCTGAGTTGCGTGACCAGCGGATTCACCTTTGGGCACTACTGGATGAACTGGGTGCGACAGTCCCCAGAGAAGGGGCTCGAATGGGTCGCTCAGTTCAGGAACAAACCCTACAATTATGAGACATACTATTCAGACAGCGTGAAGGGCAGGTTTACTATCAGTA

[0084] GAGACGATTCCAAATCTAGCGTGTACCTGCAGATGAACAATCTCAGGGTCGAAGATACAGGCATCTACTATTGCACAGGGGCATCCTATGGTATGGAGTATCTCGGTCAGGGGACAAGCGTCACAGTCAGTTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAACCACAGGAACCGTTTCTCTGTTGTTAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA (SEQ ID NO: 1).

[0085] In the above exemplary nucleic acid sequence, the first ATG is the start codon. The exemplary CAR amino acid sequence may include the following:

[0086] MALPVTALLLPLALLLHAARPDVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRVSGVPDRFSGSGSGTDFTLKINRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIKSSADDAKKDAAKKDDAKKDDAKKDGGVKLDETGGGLVQPGGAMKLSCVTSGFTFGHYWMNWVRQSPEKGLEWVAQFRNKPYNYETYYSDSVKGRFTISRDDSKSSVYLQMNNLRVEDTGIYYCTGASYGMEYLGQGTSVTVSFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 2)

[0087] Exemplary inserts may include the following:

[0088] GCCACCATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGATGTCGTGATGACCCAGACCCCCCTCAGCCTCCCAGTGTCCCTCGGTGACCAGGCTTCTATTAGTTGCAGATCCAGCCAGTCCCTCGTGCACTCTAACGGTAATACCTACCTGAGATGGTATCTCCAGAAGCCCGGACAGAGCCCTAAGGTGCTGATCTACAAAGTCTCCAACCGGGTGTCTGGAGTCCCTGACCGCTTCTCAGGGAGCGGTTCCGGCACCGACTTCACCCTGAAGATCAACCGGGTGGAGGCCGAAGACCTCGGCGTCTATTTCTGCTCTCAGAGTACACATGTGCCCTGGACCTTCGGCGGAGGGACCAAGCTGGAGATCAAAAGCTCCGCAGACGATGCCAAGAAAGATGCCGCTAAGAAAGACGATGCTAAGAAAGACGATGCAAAGAAAGACGGTGGCGTGAAGCTGGATGAAACCGGAGGAGGTCTCGTCCAGCCAGGAGGAGCCATGAAGCTGAGTTGCGTGACCAGCGGATTCACCTTTGGGCACTACTGGATGAACTGGGTGCGACAGTCCCCAGAGAAGGGGCTCGAATGGGTCGCTCAGTTCAGGAACAAACCCTACAATTATGAGACATACTATTCAGACAGCGTGAAGGGCAGGTTTACTATCAGTA

[0089] GAGACGATTCCAAATCTAGCGTGTACCTGCAGATGAACAATCTCAGGGTCGAAGATACAGGCATCTACTATTGCACAGGGGCATCCTATGGTATGGAGTATCTCGGTCAGGGGACAAGCGTCACAGTCAGTTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAACCACAGGAACCGTTTCTCTGTTGTTAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA (SEQ ID NO: 3)

[0090] In the above exemplary insert, the first GCCACC sequence may be a restriction enzyme cleavage site followed by an ATG start codon. The exemplary insert amino acid sequence may include the following:

[0091] ATMALPVTALLLPLALLLHAARPDVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRVSGVPDRFSGSGSGTDFTLKINRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIKSSADDAKKDAAKKDDAKKDDAKKDGGVKLDETGGGLVQPGGAMKLSCVTSGFTFGHYWMNWVRQSPEKGLEWVAQFRNKPYNYETYYSDSVKGRFTISRDDSKSSVYLQMNNLRVEDTGIYYCTGASYGMEYLGQGTSVTVSFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 4)

Example

[0092] Production of lentivirus containing the CAR gene for human T - cell transduction To prepare a lentivirus containing an anti - fluorescein single - chain variable (scFv) CAR, the 293TN packaging cell line was co - transfected with a lentiviral vector encoding the anti - fluorescein scFv CAR and a second - generation (Cellecta) mixture of packaging plasmids. At 24 and 48 hours after transfection, supernatants containing lentivirus with the CAR gene were harvested, and virus particles were concentrated by a standard polyethylene glycol virus concentration method (Clontech) for future transduction of human T cells.

Example

[0093] Isolation of human T cells from human PBMC T cells were isolated from human peripheral blood mononuclear cells (PBMCs) by Ficoll density gradient centrifugation (GE Healthcare Lifesciences). After washing away the remaining Ficoll solution, T cells were isolated using the EasySep™ Human T Cell Isolation Kit (STEM CELL technologies). The purified T cells were cultured in TexMACS medium (Miltenyi Biotec Inc) containing 1% penicillin and streptomycin sulfate in the presence of human IL-2 (100 IU / mL, Miltenyi Biotech Inc). The T cells were cultured at a density of 1×10 6 cells / mL in a multi-well plate. The T cells were split and replenished every 2 - 3 days.

Example

[0094] Transduction of human T cells The isolated T cells were activated with Dynabeads coupled with anti-CD3 / CD28 antibody (Life Technologies) for 12 - 24 hours in the presence of human IL-2 (100 IU / mL), and then transduced with a lentivirus encoding the anti-fluorescein CAR gene. The cells were harvested 72 hours later, and the expression of CAR on the transduced T cells was identified by measuring GFP fluorescence cells using flow cytometry. As shown in Figure 15A, non-transduced T cells did not show GFP expression. As shown in Figure 15B, the transduced T cells expressed GFP.

Example

[0095] Synthesis of FITC - folic acid

Chemical

[0096] In the presence of tetramethylguanidine and diisopropylamine, folic acid-γ-ethylenediamine was conjugated to fluorescein isothiocyanate (FITC) isomer I (Sigma-Aldrich) in anhydrous dimethyl sulfoxide (DMF). The crude product was loaded onto an Xterra RP18 preparative HPLC column (Waters) and eluted under gradient conditions starting with 99% 5 mM sodium phosphate (mobile phase A, pH 7.4) and 1% acetonitrile (mobile phase B) at a flow rate of 20 mL / min to reach 90% A and 10% B in 10 minutes. Under these conditions, the main peak of FITC-folic acid typically eluted between 27 and 50 minutes. The quality of the FITC-folic acid fraction was monitored by analytical reverse-phase HPLC using a UV detector. Fractions with a purity greater than 98.0% (LCMS) were lyophilized to obtain the final FITC-folic acid product.

Example

[0097] Synthesis of FITC - PEG12 - folic acid

Chemical formula

[0098] Universal polyethylene glycol (PEG) Nova Tag™ resin (0.2 g) was placed in a peptide synthesis vessel and washed with isopropyl alcohol (i-PrOH) (3 × 10 mL) and dimethylformamide (DMF, 3 × 10 mL). 9-Fluorenylmethoxycarbonyl (Fmoc) deprotection was carried out using 20% piperidine in DMF (3 × 10 mL). A Kaiser test was performed to evaluate the progress of the reaction. Next, a solution of Fmoc-L-glutamic acid 5-tert-butyl ester (Fmoc-Glu-(Ot-Bu)-OH) (23.5 mg) in DMF, N,N-diisopropylethylamine (i-Pr 2 NEt) (4 equivalents) and benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) (2 equivalents) were added to the vessel. Fmoc deprotection was carried out using 20% piperidine in DMF (3 × 10 mL). Next, N 10-TFA-Pte-OH (22.5 mg), DMF, i-Pr 2 A solution of NEt (4 equivalents) and PyBOP (2 equivalents) was added. Argon was bubbled through for 2 hours, and the resin was washed with DMF (3 × 3 mL) and i-PrOH (3 × 3 mL). After swelling the resin in dichloromethane (DCM), a solution of 1 M hydroxybenzotriazole (HOBT) in DCM / trifluoroethane (TFE) (1:1) (2 × 3 mL) was added. Argon was bubbled through for 1 hour, the solvent was removed, and the resin was washed with DMF (3 × 3 mL) and i-PrOH (3 × 3 mL). After swelling the resin in DMF, a solution of Fmoc-NH-(PEG) 12 -COOH (46.3 mg), i-Pr 2 NEt (4 equivalents) and PyBOP (2 equivalents) were added. Argon was bubbled through for 2 hours, and the resin was washed with DMF (3 × 3 mL) and i-PrOH (3 × 3 mL). Fmoc deprotection was carried out using 20% piperidine in DMF (3 × 10 mL). A Kaiser test was performed to evaluate the progress of the reaction. Then, a solution of FITC (Life Technologies 21.4 mg) in DMF and i-Pr 2 NEt (4 equivalents) was placed in the vessel, then argon was bubbled through for 2 hours, and the resin was washed with DMF (3 × 3 mL) and i-PrOH (3 × 3 mL). Then, 2% NH 2 NH 2 was added to the vessel. The final compound was cleaved from the resin using TFA:H2O:triisopropylsilane (TIS) (95:2.5:2.5) (cleavage solution) and concentrated under vacuum. The concentrated product was precipitated in Et 2 O and dried under vacuum. The crude product was purified by preparative RP-HPLC (mobile phase: A = 10 mM ammonium acetate, pH = 7, = ACN; method: 0% B to 30% B in 30 minutes at 13 mL / min). The pure fractions were pooled and lyophilized to obtain FITC-PEG 12 -folic acid.

Example

[0099] Synthesis of FITC - PEG20 - folic acid [Chemistry]

[0100] Ethylenediamine, polymer bound (200 - 400 mesh) - resin (50 mg) was placed in a peptide synthesis vessel and swollen with DCM (3 mL), followed by DMF (3 mL). Then, an Fmoc - PEG 20 -COOH solution (131 mg, 1.0 equivalent), i-Pr 2 NEt (6.0 equivalents) and PyBOP (4.0 equivalents) were added. Argon was bubbled through for 6 hours, the coupling solution was drained, and the resin was washed with DMF (3 × 10 mL) and i-PrOH (3 × 10 mL). A Kaiser test was performed to evaluate the progress of the reaction. Prior to each amino acid coupling, Fmoc deprotection was carried out using 20% piperidine in DMF (3 × 10 mL). The above procedure was repeated to complete the reaction with the coupling steps of Fmoc - Glu - OtBu (72 mg, 2.0 equivalents) and Tfa. pteroic acid (41 mg, 1.2 equivalents). The resin was washed with 2% hydrazine in DMF 3 × 10 mL (for 5 minutes) to cleave the trifluoro - acetyl protecting group of pteroic acid and then washed with i-PrOH (3 × 10 mL), followed by DMF (3 × 10 mL). The resin was dried under argon for 30 minutes. The folic acid - peptide was cleaved from the resin using a cleavage solution. 10 mL of the cleavage mixture was added and argon was bubbled through for 1.5 hours. The cleavage mixture was drained into a clean flask. The resin was washed 3 times with additional cleavage mixture. The combined mixture was concentrated to a smaller volume (~5 mL) under reduced pressure and precipitated in ethyl ether.

[0101] The precipitate was collected by centrifugation, washed with ethyl ether (3 times), and dried under high vacuum. The dried folic acid - PEG 20-EDA (1.0 eq) was treated with FITC (50 mg, 1.5 eq) in DMSO and DIPEA at room temperature. The progress of the reaction was monitored by LCMS. After 8 hours, the starting material was consumed to give the product. The crude reaction mixture was purified by preparative HPLC (mobile phase A = 10 mM ammonium acetate, pH = 7; organic phase B = acetonitrile; method: 0% B to 30% B at 13 mL / min for 35 min) to give FITC-PEG 20 -folic acid in 60% yield.

Example

[0102] Synthesis of FITC - PEG108 - folic acid

Chem.

[0103] Ethylenediamine, polymer-bound (200 - 400 mesh)-resin (50 mg) was placed in a peptide synthesis vessel and swollen with DCM (3 mL), followed by DMF (3 mL). Then, a solution of Fmoc-PEG 36 -COOH (161 mg, 1.0 eq), i-Pr 2 NEt (6.0 eq) and PyBOP (4.0 eq) were added. Argon was bubbled through for 6 hours, the coupling solution was drained, and the resin was washed with DMF (3 × 10 mL) and i-PrOH (3 × 10 mL). A Kaiser test was performed to evaluate the progress of the reaction. Prior to each amino acid coupling, Fmoc deprotection was carried out using 20% piperidine in DMF (3 × 10 mL). The above procedure was repeated to give 2X Fmoc-PEG 36The reaction with the coupling step of -COOH (161 mg, 1.0 equivalent), Fmoc-Glu-OtBu (72 mg, 2.0 equivalents) and Tfa. pteroic acid (41.0 mg, 1.2 equivalents) was completed. The resin was washed (5 min) with 2% hydrazine in 3 × 10 mL of DMF to cleave the trifluoro-acetyl protecting group of pteroic acid, and then washed with i-PrOH (3 × 10 mL) and subsequently with DMF (3 × 10 mL). Finally, the resin was washed with 2% hydrazine in 3 × 10 mL of DMF (5 min) to cleave the trifluoroacetyl protecting group on pteroic acid, and then washed with i-PrOH (3 × 10 mL) and subsequently with DMF (3 × 10 mL). The resin was dried under argon for 30 min. The folic acid-peptide was cleaved from the resin using the cleavage solution. 10 mL of the cleavage mixture was added and argon was bubbled through for 1.5 h. The cleavage mixture was drained into a clean flask. The resin was washed 3 times with additional cleavage mixture. The combined mixture was concentrated to a smaller volume (~5 mL) under reduced pressure and precipitated in ethyl ether.

[0104] The precipitate was collected by centrifugation, washed with ethyl ether (3 times), and dried under high vacuum. The dried folic acid-PEG 108 -EDA (1.0 equivalent) was treated with FITC (50 mg, 1.5 equivalents) in DMSO and DIPEA at room temperature. The progress of the reaction was monitored by LCMS. After 10 h, the starting material was consumed to give the product. The crude reaction mixture was purified by preparative HPLC (mobile phase A = 10 mM ammonium acetate, pH = 7; organic phase B = acetonitrile; method: 0% B to 30% B at 13 mL / min for 35 min) to give FITC -PEG 108 -folic acid in 64% yield.

Example

[0105] Synthesis of FITC - DUPA

Chem.

[0106] DUPA-FITC was synthesized by a solid-phase method as follows. Universal Nova Tag (TM) resin (50 mg, 0.53 mM) was swollen with DCM (3 mL), followed by DMF 3 mL. A 20% piperidine solution in DMF (3 x 3 mL) was added to the resin, and argon was bubbled for 5 minutes. The resin was washed with DMF (3 x 3 mL) and isopropyl alcohol (i-PrOH, 3 x 3 mL). Argon was bubbled for 2 hours, and the resin was washed with DMF (3 x 3 mL) and i-PrOH (3 x 3 mL). After swelling the resin in DCM, a solution of 1 M HOBt in DCM / TFE (1:1) (2 x 3 mL) was added. Argon was bubbled for 1 hour, the solvent was removed, and the resin was washed with DMF (3 x 3 mL) and i-PrOH (3 x 3 mL). After swelling the resin in DMF, a solution of Fmoc-Phe-OH (2.5 equivalents), HATU (2.5 equivalents) and DIPEA (4.0 equivalents) in DMF was added. Argon was bubbled for 2 hours, and the resin was washed with DMF (3 x 3 mL) and i-PrOH (3 x 3 mL). The above procedure was repeated for two additional coupling steps for the addition of 8-aminooctanoic acid and fluorescein isothiocyanate or rhodamine B isothiocyanate. The final compound was cleaved from the resin using a cleavage solution and concentrated under vacuum. The concentrated product was precipitated in diethyl ether and dried under vacuum. Preparative RP-HPLC [λ = 488 nm; solvent gradient: 1% B to 80% B in 25 minutes, 80% B wash for 30 minutes; A = 10 mM NH 4 OAc, pH = 7; B = acetonitrile (ACN)] was used to purify the crude product. ACN was removed under vacuum, and the purified fractions were lyophilized to obtain FITC-DUPA as a brownish-orange solid. RP-HPLC: tR = 8.0 minutes (A = 10 mM NH 4 OAc, pH = 7.0; B = ACN, solvent gradient: 1% B to 50% B in 10 minutes, wash with 80% B, 15-minute run). 1H NMR (DMSO-d6 / D 2O): δ 0.98 - 1.27 (ms, 9H); 1.45 (b, 3H); 1.68 - 1.85 (ms, 11H); 2.03 (m, 8H); 2.6 - 3.44 (ms, 12H); 3.82 (b, 2H); 4.35 (m, 1H); 6.53 (d, J = 8.1 Hz, 2H), 6.61 (dd, J = 5.3, 3.5 Hz, 2H); 6.64 (s, 2H); 7.05 (d, J = 8.2 Hz, 2H), 7.19 (m, 5H); 7.76 (d, J = 8.0 Hz, 1H); 8.38 (s, 1H). HRMS (ESI) (m / z): (M + H) + Calculated for C51H59N7O15S, 1040.3712; found, 1040.3702. UV / vis: λ max = 491 nm.

Example

[0107] Synthesis of FITC - PEG12 - DUPA

Chem.

[0108] 1,2 - Diaminoethanetricityl resin (0.025 g) was placed in a peptide synthesis vessel and washed with i-PrOH (3 × 10 mL), followed by DMF (3 × 10 mL). Then, Fmoc-NH-(PEG) 12 -COOH (42.8 mg), i-Pr 2A solution of NEt (2.5 eq) and PyBOP (2.5 eq) was introduced into a container. The resulting solution was bubbled with Ar for 1 hour, the coupling solution was drained, and the resin was washed with DMF (3 x 10 mL) and i-PrOH (3 x 10 mL). A Kaiser test was performed to evaluate the progress of the reaction. Fmoc deprotection was carried out using 20% piperidine in DMF (3 x 10 mL). This procedure was repeated to complete all coupling steps (2x1.5 eq of Fmoc-Phe-OH and 1.5 eq of 8-aminooctanoic acid and 1.2 eq of DUPA were used in each of their respective coupling steps). After DUPA coupling, the resin was washed with DMF (3 x 10 mL) and i-PrOH (3 x 10 mL) and dried under reduced pressure. The peptide was cleaved from the resin in the peptide synthesis vessel using a cleavage solution. 15 mL of the cleavage solution was added to the peptide synthesis vessel and the reaction was bubbled with Ar for 15 minutes. The resin was further treated with 10 mL portions of the cleavage solution twice for 5 minutes each. The cleavage mixture was concentrated to approximately 5 mL and precipitated with ethyl ether. The precipitate was collected by centrifugation, washed with ethyl ether (3x), and dried under high vacuum to recover the crude material. Crude DUPA-(PEG) 12 -EDA (10 mg) and FITC (5.6 mg) in a stirred solution, at room temperature, i-Pr 2 NEt (5 eq) was added and the mixture was stirred under argon for 6 hours. The reaction was monitored by LCMS and purified by preparative HPLC (mobile phase: A = 10 mM ammonium acetate, pH = 7, B = ACN; method: 0% B to 50% B in 30 minutes at 13 mL / min). The purified fractions were pooled and lyophilized to obtain FITC-PEG 12 -DUPA. The purified fractions were pooled and lyophilized to obtain FITC-PEG 12 -DUPA was obtained.

Example

[0109] Synthesis of FITC - PEG11 - NK1

Chemical formula

[0110] Anhydrous CH 2 Cl 2 solution of O-(2-aminoethyl)-O’-[2-(Boc-amino)ethyl]decaethylene glycol (BocNH-PEG 11 -NH 2 ) of NK-1 (0.02 g, 0.0433 mmol, 1.0 equivalent) (Sigma, 0.0336 g, 0.0521 mmol, 1.2 equivalents), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) (0.027 g, 0.0521 mmol, 1.2 equivalents), under argon at room temperature, N,N-diisopropylethylamine (DIPEA) (0.076 mL, 0.4338 mmol, 10 equivalents) was added. The progress of the reaction was monitored by LCMS and purified by preparative RP-HPLC (Waters, XBridge™ PrepC18, 5 μm; 19×100 mm column, mobile phase A = 20 mM ammonium acetate buffer, pH 7, B = acetonitrile, 10 - 100% B in 30 minutes, 13 mL / min, λ = 220 nm, 254 nm). The pure fractions were collected, all organic solvents were evaporated, and the sample was lyophilized for 48 hours to obtain NK1-PEG 11 -NHBoc. Yield: 40.13 mg (97%). NK1-PEG in anhydrous DCM 11 -NHBoc (0.0165 g, 0.015 mmol) was added trifluoroacetic acid (TFA, 20 equivalents), and the reaction mixture was stirred at room temperature for 4 hours. Excess TFA was removed, the remaining solution was diluted with water, and extracted with CH 2 Cl 2 (3×5 mL). The combined organic layers were washed with brine, dried (Na 2 SO 4 ), and concentrated. The obtained residue was dried under vacuum and used in the next step without further purification. NK1-PEG in anhydrous dimethyl sulfoxide (DMSO, 0.3 mL) 11 -NH 2(0.008 g, 0.0081 mmol, 1.0 equivalent), fluorescein isothiocyanate (FITC) (Sigma, 0.0037 g, 0.0097 mmol, 1.2 equivalents) solution was added to diisopropylethylamine (0.0028 mL, 0.0162 mmol, 2.0 equivalents) under argon at room temperature. The progress of the reaction was monitored by LCMS, and the product was purified by preparative RP-HPLC (Waters, XBridge™ PrepC18, 5 μm; 19×100 mm column, mobile phase A = 20 mM ammonium acetate buffer, pH 7, B = acetonitrile, 10 - 100% B in 30 minutes, 13 mL / min, λ = 280 nm). The pure fractions were collected, all organic solvents were evaporated, and the sample was lyophilized for 48 hours to obtain FITC-PEG 11 -NK1 in a yield of 8.54 mg (77%).

[0111] * Note: The NK-1 compound was synthesized by a two-step procedure starting from a basic ligand prepared using the procedure in the literature (Ref: DESIGN AND DEVELOPMENT OF NEUROKININ-1 RECEPTOR-BINDING AGENT DELIVERY conjugate, application number: PCT / US2015 / 44229; incorporated herein by reference).

Example

[0112] Synthesis of FITC - PEG2 - CA9

Chem.

[0113] Using a Teflon (registered trademark) magnetic stir bar, CA9 ligand (53.6 mg) was dissolved in DMF (2 - 3 mL) in a 50 mL round bottom flask. The surrounding air was removed using vacuum and replaced with nitrogen gas, and this was done for 3 cycles. The round bottom flask was maintained under a constant nitrogen gas. To this flask, 28.9 mg of N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC) was added, followed by 21.6 mg of 1-hydroxybenzotriazole hydrate (HOBt) and 18.9 μL of Boc-PEG2-NH2 (Sigma Aldrich). 5.4 μL of triethylamine (TEA) was added and the reaction mixture was stirred overnight. The reaction mixture was purified using HPLC and confirmed by UHPLC-MS (target m / z = 831). Acetonitrile was removed using high vacuum rotary evaporation and the product was lyophilized. The compound was mixed with 1:1 TFA:DCM for 30 minutes. Using high vacuum rotary evaporation, TFA / DCM was removed subsequently over 30 minutes under high vacuum. Then, the compound was dissolved in DMF, and 5 molar equivalents of i-Pr 2 NEt, 16 mg of fluorescein isothiocyanate (Life Technologies) were mixed and stirred for 1 hour. The reaction mixture was purified by HPLC and the target compound was confirmed by UHPLC-MS (target m / z = 1120). The sample was lyophilized and stored at -20 °C.

Example

[0114] Evaluation of anti - FITC scFv expression in transduced CAR - T cells To confirm whether the functional anti-FITC scFv was expressed on the cell surface of the transduced CAR T cells, the CAR T cells were incubated with the FITC-folate conjugate on ice for 30 minutes. After washing the cells, anti-folate antibody (Santa Cruz) was added and incubated on ice for 30 minutes. Finally, the CAR T cell-expressed functional anti-FITC scFv was visualized using an Alexa Fluor 647-labeled secondary antibody (Jackson ImmunoResearch). As shown in Figure 16A, the transduced CAR T cells showed GFP expression. As shown in Figure 16B, these transduced CAR-T cells were able to bind to the FITC-folate conjugate visualized by immunofluorescent labeling.

Example

[0115] In vitro cytotoxicity assay of CAR T cells To test the cytotoxicity of CAR T cells with the desired FITC-ligand, a standard lactate dehydrogenase (LDH) release assay was performed using the Pierce™ LDH Cytotoxicity Assay Kit from ThermoFisher Scientific. To prepare samples for the LDH assay, cells were seeded into each well of a 96-well plate at a density of 10 4 cells / 100 μL and grown overnight. The next day, different numbers of CAR T cells were prepared to have different effector (CAR T cell):target cell (cancer cell) ratios (e.g., E:T = 20:1, 10:1, 5:1, and 1:1). Different concentrations of the FITC-ligand were placed in each well and co-cultured for 6 - 24 hours. After co-incubation, the plates containing CAR T cells and cancer cells were centrifuged at 350 × g for 10 minutes at room temperature to remove cell debris or remaining cells. Next, 50 μL of the supernatant from each sample was transferred to a new 96-well plate. To each transferred 50 μL sample, 50 μL of the prepared LDH reaction mixture was added and incubated at room temperature for 30 minutes. 50 μL of the stop solution was added, and the absorbance of each sample was measured at 490 nm and 680 nm. The percentage of cytotoxicity for each sample was calculated using the following formula: Cytotoxicity % = (Experimental value - Effector cells (spontaneous) - Target cells (spontaneous)) / (Target cells (maximum control) - Target cells (spontaneous control)) × 100

[0116] As shown in FIGS. 3A - C, CAR T cells are activated only in combination with a complex that is compatible with the tumor antigen. FIG. 3A shows the cytotoxicity of CAR T cells in the KB (FR+) model with an E:T of 10:1 using 100 nM of each complex. FITC - folic acid, FITC - PEG20 - folic acid, and FITC - PEG108 - folic acid activations are greater than those with FITC - DUPA or in the absence of the complex. FIG. 3B shows the cytotoxicity of CAR T cells in the LNCaP (PSMA+) model with a 10:1 E:T using 100 nM of FITC - DUPA or FITC - PEG12 - DUPA. The FITC - DUPA complex shows greater activation than using FITC - folic acid or in the absence of the complex. FIG. 3C shows the cytotoxicity of CAR T cells in the HEK293 (NK1R+) model with a 10:1 E:T using 100 nM of FITC - PEG11 - NK1. FITC - PEG11 - NK1 shows greater activation than using FITC - folic acid or in the absence of the complex.

[0117] As shown in FIG. 3D, the cytotoxicity of CAR T cells against tumor cells in the KB (FR+) model is a function of the E:T ratio used in the assay with 100 nM of FITC - folic acid and FITC - DUPA. As shown in FIG. 3E, the cytotoxicity of CAR T cells against tumor cells in the KB (FR+) model is a function of the concentration of FITC - folic acid used during co - incubation (E:T ratio 10:1).

[0118] As shown in FIG. 10, CAR T cell cytotoxicity in the KB (FR+) model can be controlled by adjusting the concentration of the complex bridge or by using linkers with different lengths of PEG having a 10:1 E:T.

Example

[0119] Identification of CAR T cell proliferation and CAR T cell activation The proliferation of CAR T cells was mainly measured by flow cytometry. First, cancer cells (KB(FR+) or HEK(NK1R+)) were seeded at a density of 10 4 cells / 100 μL in each well of a 96-well plate and cultured overnight. The next day, CAR T cells were added to each well in the presence or absence of the desired FITC-ligand, and the cells were co-cultured for 5 days (120 hours). After co-incubation, the CAR T cells were stained with an anti-human CD3 APC antibody (Biolegend) by standard immunostaining procedures (for 20 minutes on ice). Cells that were positive for both anti-human CD3 staining and GFP were counted for CAR T cell proliferation.

[0120] As shown in FIGS. 1A - B, CAR (T cell) cells proliferated as a result of targeting tumor cells with an FITC-small molecule complex (100 nM) using KB(FR+) cells and HEK293(NK1R+) cells (E:T = 5:1). FIG. 1A shows CAR T cell proliferation in the presence of KB(FR+) cells with different complexes. FIG. 1B shows CAR T cell proliferation in the presence of HEK (NK1R+) cells with different complexes. As further seen in FIG. 1, the presence of linkers with different lengths of PEG affects the level of CAR-T cell proliferation.

[0121] To measure the activation of CAR T cells, cancer cells were prepared using the same procedure as above. Cancer cells and CAR T cells were co-cultured at an E:T ratio of 10:1 for 24 hours in the presence or absence of 100 nM FITC-ligand and then harvested. After washing, the cells from each sample were stained with anti-human CD69 Alexa Fluor 647 (Biolegend). To quantify the activation of CAR T cells, the data were analyzed with a gate on GFP-positive T cells. As shown in FIG. 17, CD69 expression is related to the co-culture complex.

Example

[0122] IFN - γ production assay To test the production of IFN-γ by CAR T cells, a standard ELISA assay was performed using a human IFN-γ detection ELISA kit from Biolegend. Briefly, cancer cells were seeded at a density of 10 4 cells / 100 μL of medium in each well of a 96-well plate and grown overnight. CAR T cells were introduced into each cancer sample containing the desired FITC-ligand and co-cultured for 24 hours. After co-incubation, the supernatant of each sample was collected and centrifuged at 1000 × g and 4 °C for 10 minutes to remove cell debris. The clear supernatant from each sample was then either used for detecting IFN-γ by ELISA or stored at -80 °C for future use. After the preparation of each sample was completed, a standard ELISA was performed according to the manufacturer's instructions.

[0123] As shown in FIGS. 2A-2C, at an E:T ratio of 5:1 in KB cells (FR+)(FIG. 2A), LNCaP (PSMA+)(FIG. 2B), or HEK (NK1R+)(FIG. 2C), CAR T cells produce a significant amount of inflammatory cytokines in the presence of 100 nM FITC-small molecule complex. Further shown, the amount of cytokine depends on the complex used. As shown in FIG. 2D, the amount of inflammatory cytokine is related to the concentration of FITC-folic acid used in KB (FR+) cells. As shown in FIGS. 2E and 2F, different complexes (100 nM) produce different IFN-γ responses in KB (FR +) cells.

[0124] As shown in FIGS. 4A-4B, the activation of CAR T cells correlates with tumor antigen levels in KB (FR+) cells and MDA-MB-231 cells. Measuring IFN-γ production by incubating cells with the same complex dose shows that the activation of anti-FITC CAR T cells correlates with the expression level of tumor antigens on cancer cells.

Example

[0125] Evaluation of the correlation between tumor antigen levels and CAR - T activation KB(FR+) cells and MDA-MB-231 cells were incubated with 100 nM of FITC-folic acid on ice for 30 minutes. After washing, the FITC-folic acid bound to the FRα tumor antigen on the cells was measured by flow cytometry. As shown in Figure 4A, KB(FR+) cells have a higher level of FR expression and corresponding FITC binding than MDA-MB-231 cells.

[0126] These two cell lines were co-cultured with CAR T cells at an E:T ratio of 10:1 for 24 hours in the presence of 100 nM of FITC-folic acid, FITC-PEG20-folic acid, and FITC-PEG108-folic acid. CAR T cell activation was detected by measuring INF-γ production. The supernatant of the cultured cells was collected, and IFN-γ production was measured using an ELISA kit. As shown in Figure 4B, at a higher FRα level, KB cells (FR+) activated CAR T cells much better than MDA-MB-231 cells.

Example

[0127] Anti - tumor effect of CAR T cells in vivo To identify the efficacy of CAR T cell anti-tumor activity in vivo, immunodeficient NSG mice (Jackson Laboratory) were used. Cancer cell lines expressing each tumor-specific antigen were subcutaneously injected into the shoulders of NSG mice to establish solid tumor xenografts. When the tumor volume reached approximately 50 - 100 mm 3 CAR T cells were introduced into the mice with tumors, and the desired FITC-ligand was introduced every other day (i.v.). PBS was administered to the control mice instead of the FITC-ligand. Tumor volume and cytokine levels (IL2, IL6, IL10, IFNγ, and TNFα) in the blood were measured. General toxicity of the treatment was monitored by measuring weight loss. At the end of each treatment, mouse blood was collected to test for anemia, white blood cell count, and CAR T cell proliferation. The organs of the mice were also evaluated.

[0128] The xenograft models using HEK293 (NK1R+) cells are shown in FIGS. 5A-5C and FIGS. 6A-6B. As shown in FIG. 5A, the tumor size decreased in the mice treated with FITC-PEG11-NK1 (500 nmol / kg), while it continued to grow in the control mice. As shown in FIG. 5B, the body weight of the mice treated with FITC-PEG11-NK1 did not change compared to the control mice. As shown in FIG. 5C, the proportion of CAR T cells in human T cells increased after CAR T cell injection. As shown in FIGS. 6A-6B, the organs collected from the treatment group (6B) appeared normal-sized without showing signs of cytokine release syndrome after 2 weeks of treatment compared to the mouse organs collected from the control group (6A).

[0129] The xenograft models using MDA-MB-231 (FR+) cells are shown in FIGS. 7A-7C and FIGS. 8A-8B. As shown in FIG. 7A, the tumor size decreased in the mice treated with FITC-PEG12-folic acid (500 nmol / kg) or FITC-folic acid (500 nmoles / kg), while the tumor continued to grow in the control mice. As shown in FIG. 7B, the body weight of the mice treated with FITC-PEG11-NK1 did not change during the treatment. As shown in FIG. 7C, the proportion of CAR T cells in human T cells increased after CAR T cell and conjugate treatment. As shown in FIGS. 8A-B, the organs collected from the mice treated with FITC-PEG12-folic acid (8B) appeared normal-sized and no signs of cytokine release syndrome were observed after 3 weeks of treatment compared to the mouse organs collected from the control group (8A). As used in any of the embodiments of this patent application, "nmol / kg" and "nmoles / kg" are equivalent.

[0130] As shown in FIG. 9, the blood indices of the FITC-PEG11-NK1 (500 nmoles / kg) HEK-NK1R model and the MDA-MB-231 model using FITC-PEG12-folic acid (500 nmoles / kg) also indicated that no cytokine storm occurred during the treatment process.

[0131] As shown in FIGS. 11, 12A - 12C, and 13, KB(FR+) tumor xenografts were treated with two different concentrations of FITC - PEG12 - folic acid. As shown in FIG. 11, mice treated with the lower dose (250 nmoles / kg) showed a milder weight loss compared to mice treated with the higher dose (500 nmoles / kg). As shown in FIGS. 12A - 12C, organs harvested from untreated mice (FIG. 12A) and mice treated with the lower dose (FIG. 12B) showed a milder cytokine release syndrome compared to the higher dose (FIG. 12C). As shown in FIG. 13, KB xenograft mice receiving the lower dosing showed favorable blood indices indicative of milder cytokine release.

Example

[0132] Anti - tumor effect of CAR T cells in vivo To investigate whether the same anti - FITC CAR T cells could eradicate the mixture of heterologous cancer cells and FITC - ligand, immunodeficient NSG mice (Jackson laboratory) were utilized for in vivo studies. Two different cancer cell lines (e.g., MDA - MB - 231(FR+) and HEK(NK1R+)) were transplanted into separate flanks of the same mice. Then, anti - FITC CAR T cells (10 7 cells) were introduced by intravenous injection when the tumor volume reached approximately 50 - 100 mm 3 . Additionally, a mixture of FITC - ligands (i.e., FITC - PEG12 - folic acid (500 nmole / kg) and FITC - PEG11 - NK1R(500 nmole / kg)), single FITC - PEG11 - NK - 1R(500 nmole / kg) or PBS was administered every other day by intravenous injection. The anti - tumor effect of CAR T cells by FITC - ligand was analyzed by measuring the tumor volume every other day.

[0133] As shown in FIGS. 19A-19C, when both FITC-PEG11-NK1R and FITC-PEG12-folic acid were introduced, both tumors (i.e., MDA-MB-231 (MDA) and HEK (NK1R)) were eliminated by the same anti-FITC CAR T cells. In mice treated with only FITC-PEG11-NK1R, only the HEK (NK1R) tumor was eradicated. Without being bound by theory, it is thought that MDA-MB-231 continued to grow in the same mice administered only FITC-PEG11-NK1R because FITC-PEG12-folic acid was not administered. As expected, both tumors showed no reaction when PBS was introduced. These data suggest that the same anti-FITC CAR T cells can antigenically eradicate a heterogeneous tumor mixture via a cocktail of FITC-ligands.

Claims

1. 1. A method for treating cancer, comprising: i) administering to a patient a first dose of a compound or a pharma- ceutically acceptable salt thereof, where the compound comprises a small molecule ligand linked to a targeting moiety by a linker; ii) administering to the patient a CAR T cell composition, wherein the CAR T cell comprises a CAR directed to a targeting moiety; ii) administering to the patient a second dose of the compound or a pharma- ceutically acceptable salt thereof, wherein the second dose is different from the first dose; and iv) treating the patient to ameliorate the cancer.

2. 1. A method for treating cancer, comprising: i) administering to the patient a first conjugate or a pharma- ceutically acceptable salt thereof; ii) administering to the patient a CAR T cell composition, wherein the CAR T cell comprises a CAR directed to a targeting moiety; iii) administering to the patient a second conjugate or a pharma- ceutically acceptable salt thereof, wherein the first and second conjugates each comprise a small molecule ligand linked to a targeting moiety by a linker, and wherein the first and second conjugates are different; and iv) treating the patient to ameliorate the cancer.

3. 1. A method for treating cancer, comprising: i) administering to the patient a first dose of a first conjugate or a pharma- ceutically acceptable salt thereof; ii) administering to the patient a CAR T cell composition, wherein the CAR T cell comprises a CAR directed to a targeting moiety; ii) administering to the patient a second dose of a second conjugate or a pharma- ceutically acceptable salt thereof, where the first conjugate and the second conjugate each comprise a small molecule ligand linked to a targeting moiety by a linker, where the first conjugate and the second conjugate are different, and where the first dose and the second dose are different; and iv) treating the patient to ameliorate the cancer.

4. 4. The method of claim 2 or 3, wherein the linker in the first conjugate, or the pharma- ceutically acceptable salt thereof, and the linker in the second conjugate, or the pharma- ceutically acceptable salt thereof, are different.

5. 4. The method of claim 2 or 3, wherein the linker in the first conjugate, or the pharma- ceutically acceptable salt thereof, and the linker in the second conjugate, or the pharma- ceutically acceptable salt thereof, are the same.

6. The method according to any one of claims 2 to 5, wherein the ligand in the first complex, or a pharma- ceutically acceptable salt thereof, and the ligand in the second complex, or a pharma- ceutically acceptable salt thereof, are different.

7. The method according to any one of claims 2 to 5, wherein the ligand in the first conjugate, or the pharma- ceutically acceptable salt thereof, and the ligand in the second conjugate, or the pharma- ceutically acceptable salt thereof, are the same.

8. The method of any one of claims 2 to 7, wherein the targeting moiety in the first conjugate, or the pharma- ceutically acceptable salt thereof, and the targeting moiety in the second conjugate, or the pharma- ceutically acceptable salt thereof, are different.

9. The method of any one of claims 2 to 7, wherein the targeting moiety in the first conjugate, or the pharma- ceutically acceptable salt thereof, and the targeting moiety in the second conjugate, or the pharma- ceutically acceptable salt thereof, are the same.

10. The method according to any one of claims 1 to 9, wherein the ligand is selected from folic acid, DUPA, a NK-1R ligand, a CAIX ligand, a ligand of gamma glutamyl transpeptidase, and a CCK2R ligand.

11. The method of claim 10, wherein the ligand is folic acid.

12. The method of claim 10, wherein the ligand is an NK-1R ligand.

13. The method of claim 10, wherein the ligand is DUPA.

14. The method of claim 10, wherein the ligand is a CCK2R ligand.

15. The method of claim 10, wherein the ligand is a ligand for gamma glutamyl transpeptidase.

16. 16. The method of any one of claims 1 to 15, wherein the targeting moiety is selected from 2,4-dinitrophenol (DNP), 2,4,6-trinitrophenol (TNP), biotin, digoxigenin, fluorescein, fluorescein isothiocyanate (FITC), NHS-fluorescein, pentafluorophenyl ester (PFP), tetrafluorophenyl ester (TFP), knottin, centirin, and DARPin.

17. 17. The method of claim 16, wherein the targeting moiety is FITC.

18. 17. The method of claim 16, wherein the targeting moiety is DNP.

19. 17. The method of claim 16, wherein the targeting moiety is TNP.

20. 20. The method of any one of claims 1 to 19, wherein the linker comprises polyethylene glycol (PEG), polyproline, a hydrophilic amino acid, a sugar, a non-natural peptidoglycan, polyvinylpyrrolidone, and / or Pluronic F-127.

21. 21. The method of claim 20, wherein the linker comprises PEG.

22. The compound or a pharma- ceutically acceptable salt thereof, the first conjugate or a pharma- ceutically acceptable salt thereof, or the second conjugate or a pharma- ceutically acceptable salt thereof has the formula: 【Chemistry 1】 wherein B represents a small molecule ligand, L represents a linker, and T represents a targeting moiety, where L has the formula: 【Chemistry 2】 (wherein n is an integer from 0 to 200). [including the structure represented by 22. The method of any one of claims 1 to 21, having the structure shown below.

23. 23. The method of claim 22, wherein n is an integer from 0 to 150.

24. 23. The method of claim 22, wherein n is an integer from 0 to 110.

25. 23. The method of claim 22, wherein n is an integer from 0 to 20.

26. 23. The method of claim 22, wherein n is an integer from 15 to 20.

27. 23. The method of claim 22, wherein n is an integer from 15 to 110.

28. 28. The method of any one of claims 1 to 27, wherein the linker comprises PEG and the targeting moiety is FITC or a pharma- ceutically acceptable salt thereof.

29. 29. The method of any one of claims 1 to 28, wherein the dosage of the compound or a pharma- ceutically acceptable salt thereof, the first conjugate or a pharma- ceutically acceptable salt thereof, or the second conjugate or a pharma- ceutically acceptable salt thereof is from about 10 nmol / kg to about 3000 nmol / kg of the patient's body weight.

30. 30. The method of any one of claims 1 to 29, wherein the dosage of the compound or a pharma- ceutically acceptable salt thereof, the first conjugate or a pharma- ceutically acceptable salt thereof, or the second conjugate or a pharma- ceutically acceptable salt thereof is from about 50 nmol / kg to about 2000 nmol / kg of the patient's body weight.

31. 31. The method of any one of claims 1 to 30, wherein the dosage of the compound or a pharma- ceutically acceptable salt thereof, the first conjugate or a pharma- ceutically acceptable salt thereof, or the second conjugate or a pharma- ceutically acceptable salt thereof is from about 100 nmol / kg to about 1000 nmol / kg of the patient's body weight.

32. 32. The method of any one of claims 1 to 31, wherein the dosage of the compound or a pharma- ceutically acceptable salt thereof, the first conjugate or a pharma- ceutically acceptable salt thereof, or the second conjugate or a pharma- ceutically acceptable salt thereof is from about 100 nmol / kg to about 600 nmol / kg of the patient's body weight.

33. 33. The method of any one of claims 1 to 32, wherein the dosage of the compound or a pharma- ceutically acceptable salt thereof, the first conjugate or a pharma- ceutically acceptable salt thereof, or the second conjugate or a pharma- ceutically acceptable salt thereof is from about 200 nmol / kg to about 500 nmol / kg of the patient's body weight.

34. 34. The method of any one of claims 1 to 33, wherein the dosage of the compound or a pharma- ceutically acceptable salt thereof, the first conjugate or a pharma- ceutically acceptable salt thereof, or the second conjugate or a pharma- ceutically acceptable salt thereof is from about 250 nmol / kg to about 500 nmol / kg of the patient's body weight.

35. Cancers include lung cancer, bone cancer, pancreatic cancer, skin cancer, head cancer, neck cancer, cutaneous melanoma, intraocular melanoma, uterine cancer, ovarian cancer, endometrial cancer, rectal cancer, stomach cancer, colon cancer, breast cancer, triple-negative breast cancer, cervical cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, 35. The method of any one of claims 1 to 34, wherein the cancer is selected from non-small cell lung cancer, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, prostate cancer, chronic leukemia, acute leukemia, lymphocytic lymphoma, pleural mesothelioma, cancer of the bladder, Burkitt's lymphoma, cancer of the ureter, cancer of the kidney, renal cell carcinoma, cancer of the renal pelvis, central nervous system (CNS) neoplasms, primary CNS lymphoma, spinal axis tumors, brain stem glioma, pituitary adenoma, adenocarcinoma of the gastroesophageal junction.

36. The method of any one of claims 1 to 11 or 16 to 35, wherein the cancer is a folate receptor expressing cancer.

37. 36. The method of claim 35, wherein the cancer is endometrial cancer.

38. 36. The method of claim 35, wherein the cancer is non-small cell lung cancer.

39. 36. The method of claim 35, wherein the cancer is ovarian cancer.

40. 36. The method of claim 35, wherein the cancer is triple-negative breast cancer.

41. The method of any one of claims 1 to 40, wherein the CAR has a recognition region, and the recognition region is an antibody single variable region fragment (scFv).

42. The method of any one of claims 1 to 11, 16 to 17, or 20 to 41, wherein the CAR has a recognition region, and the recognition region of the CAR is a single variable region fragment (scFv) of an anti-FITC antibody.

43. The method of any one of claims 1 to 42, wherein the CAR has a costimulatory domain, and the costimulatory domain is selected from CD28, CD137 (4-1BB), CD134 (OX40), and CD278 (ICOS).

44. The method of any one of claims 1 to 43, wherein the CAR has an activation signaling domain, and the activation signaling domain is a T cell CD3 zeta chain or an Fc receptor gamma.

45. The method of any one of claims 1 to 11, 16 to 17, or 20 to 41, wherein the CAR has a recognition region, the recognition region being a single variable fragment (scFv) of an anti-FITC antibody; the CAR has a costimulatory domain, the costimulatory domain being CD137 (4-1BB); and the CAR has an activation signaling domain, the activation signaling domain being a T cell CD3 zeta chain.

46. 46. ​​The method of any one of claims 1 to 45, wherein multiple doses of the compound, or a pharma- ceutically acceptable salt thereof, the first conjugate, or a pharma- ceutically acceptable salt thereof, or the second conjugate, or a pharma- ceutically acceptable salt thereof, and the CAR T cell composition are administered.

47. 47. The method of any one of claims 1 to 46, wherein the patient is imaged prior to administration of the compound or a pharma- ceutically acceptable salt thereof, the first conjugate or a pharma- ceutically acceptable salt thereof, or the second conjugate or a pharma- ceutically acceptable salt thereof, or prior to administration of the CAR T cell composition.

48. 48. The method of any one of claims 1 to 47, wherein the compound or a pharma- ceutically acceptable salt thereof, the first conjugate or a pharma- ceutically acceptable salt thereof, or the second conjugate or a pharma- ceutically acceptable salt thereof is not an antibody and does not include a fragment of an antibody.

49. The method of any one of claims 1 to 48, wherein the targeting moiety is not a peptide epitope.

50. 50. The method of any one of claims 1-49, wherein the method results in CAR T cytotoxicity against cancer without cytokine release that results in "off-target" toxicity to the patient.

51. 51. The method of any one of claims 1 to 50, which produces CAR T cytotoxicity against cancer without causing "off-target" tissue toxicity in the patient.

52. 52. The method of any one of claims 1 to 51, wherein the cancer comprises a tumor and the tumor size in the patient is reduced and does not result in "off-target" tissue toxicity.

53. A CAR T cell comprising a nucleic acid comprising SEQ ID NO:

1.

54. A CAR T cell comprising a polypeptide comprising SEQ ID NO:

2.

55. 1. An isolated nucleic acid comprising SEQ ID NO:1 and encoding a chimeric antigen receptor.

56. A chimeric antigen receptor polypeptide comprising SEQ ID NO:

2.

57. A vector comprising sequence number 1.

58. The vector of claim 57, wherein the vector is a lentiviral vector.

59. 57. The method of any one of claims 1 to 56, the CAR T cell, the isolated nucleic acid encoding a chimeric antigen receptor (CAR), or the chimeric antigen receptor polypeptide, wherein the CAR comprises a human amino acid sequence.

60. 57. The method of any one of claims 1 to 56, the CAR T cell, the isolated nucleic acid encoding a chimeric antigen receptor (CAR), or the chimeric antigen receptor polypeptide, wherein the CAR consists of a human amino acid sequence.

61. A kit comprising at least two different types of bridges, the bridges comprising small molecule ligands linked to targeting moieties, the ligands in the at least two different types of bridges being different, and the ligands being selected from folic acid, DUPA, CAIX ligands, NK-1R ligands, ligands for gamma glutamyl transpeptidase, and CCK2R ligands.

62. 62. The kit of claim 61, wherein the ligand in at least one bridge is an NK-1R ligand.

63. 62. The kit of claim 61, wherein the ligand in at least one bridge is a ligand of gamma glutamyl transpeptidase.

64. 62. The kit of claim 61, wherein the ligand in at least one bridge is folic acid.

65. The bridge has the formula: 【Chemistry 3】 wherein B represents a small molecule ligand, L represents a linker, and T represents a targeting moiety, where L has the formula: 【Chemistry 4】 (wherein n is an integer from 0 to 200). [including the structure represented by The kit of any one of claims 61 to 64, having a structure comprising:

66. The kit of claim 65, wherein n is an integer from 0 to 150.

67. The kit of claim 65, wherein n is an integer from 0 to 110.

68. The kit of claim 65, wherein n is an integer from 0 to 20.

69. The kit of claim 65, wherein n is an integer from 15 to 20.

70. The kit of claim 65, wherein n is an integer from 15 to 110.

71. 71. The method of any one of claims 1 to 10, 16 to 52, or 59 to 60, or the kit of any one of claims 61 to 70, wherein the ligand is C, an AIX ligand.

72. formula: 【Chemistry 5】 、 【Chemistry 6】 、 【Chemistry 7】 ,or 【Chemistry 8】 The complex shown in FIG.

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