Methods and Compositions for CAR T Cell Therapy

Abstract

To provide methods of treating a patient with a cancer.SOLUTION: The method comprises: i) administering to a patient a first dose of a compound comprising a small molecule ligand linked to a targeting moiety by a linker, or a pharmaceutically acceptable salt thereof; ii) administering to the patient a CAR T cell composition comprising a CAR directed to the targeting moiety; and iii) administering to the patient a second dose of the compound, or the pharmaceutically acceptable salt thereof, the second dose being different from the first dose.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 April 8, 2016, and U.S. Provisional Application No. 62 / 323,971, filed April 18, 2016, the entire disclosures of both applications being incorporated herein by reference. Technical Field The present disclosure relates to methods of treating a patient with cancer by administering to the patient CAR T cells and 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 technology]

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

[0003] The first generation of CARs consists of a recognition domain, e.g., a single fragment variable region (scFv) derived from an antibody for recognition and binding to an antigen expressed by a tumor, and an activation signaling domain, e.g., the CD3ζ chain of a T cell can act as a T cell activation signal in a CAR. Although CAR T cells have shown positive results in vitro, they have been limited in eliminating disease (e.g., cancer) in clinical trials. One problem has been the inability to extend activation and expand the CAR T cell population in vivo.

[0004] To address this issue, second generation CARs have incorporated costimulatory domains (e.g., CD137, CD28, or CD134) to achieve long-term activation of T cells in vivo. The addition of costimulatory domains enhances the in vivo proliferation and survival of CAR-containing T cells, and early clinical data has demonstrated that such constructs are promising therapeutic agents in the treatment of diseases such as cancer. Summary of the Invention [Problem to be solved by the invention]

[0005] Although improvements have been made in CAR T cell therapy, several problems remain. First, "off-target" toxicity can occur due to normal cells expressing the antigen targeted by the CAR T cells (e.g., tumor-associated antigens). Second, unregulated CAR T cell activation can be found when rapid and uncontrolled elimination of diseased cells (e.g., cancer cells) by the CAR T cells induces tumor lysis syndrome, or a collection of metabolic disorders called cytokine release syndrome (CRS) if the tumor is being treated, which can be fatal to the patient. Tumor lysis syndrome and CRS can result from the administration of CAR T cells that cannot be easily regulated and are activated uncontrollably. Thus, although CAR T cells hold great promise as a tool in the treatment of diseases such as cancer, additional CAR T cell therapies are needed that provide reduced off-target toxicity and provide 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 cancer and CAR T cells to direct CAR T cells to cancer for cancer amelioration. In one embodiment, the "small molecule ligand" can be, for example, folic acid, DUPA, NK-1R ligand, CAIX ligand, gamma glutamyl transpeptidase ligand, or CCK2R ligand, each of which is a small molecule ligand that specifically binds to cancer cells (i.e., the receptors of these ligands are overexpressed compared to normal tissues).

[0007] In one embodiment, the "small molecule ligand" is linked to a "targeting moiety" that binds to the CAR expressed by the CAR T cell. In various embodiments, the "targeting moiety" may 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, centirin, and DARPin.

[0008] The "targeting moiety" binds to the recognition region of the engineered CAR expressed by the CAR T cell. Thus, the recognition region of the CAR (e.g., a single fragment variable region (scFv) of an antibody) is directed to 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 cell, directing the CAR T cell to 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 provide 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 provide 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 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 achieve specificity to control CAR T cell activation or to avoid toxicity to normal tissues.

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

[0011] In another embodiment, a method of treating cancer is provided, comprising: i) administering to a patient a first conjugate or a pharmaceutically 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 pharmaceutically acceptable salt thereof, wherein the first and second conjugates each comprise a small molecule ligand linked to the targeting moiety by a linker, and wherein the first and second conjugates are different; and iv) treating the patient to ameliorate the cancer.

[0012] In yet another embodiment, a method of treating cancer is provided, comprising: i) administering to a 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, wherein the first conjugate and the second conjugate each comprise a small molecule ligand linked to a targeting moiety, the first conjugate and the second conjugate 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, a CAR T cell is provided that comprises a nucleic acid comprising SEQ ID NO:1. In another aspect, a CAR T cell is provided that comprises a polypeptide comprising SEQ ID NO:2. In another embodiment, an isolated nucleic acid is provided that comprises SEQ ID NO:1 and encodes a chimeric antigen receptor. In yet another embodiment, a chimeric antigen receptor polypeptide is provided that comprises SEQ ID NO:2. In another aspect, a vector is provided that comprises SEQ ID NO:1. In another exemplary embodiment, a vector is provided that comprises SEQ ID NO:1, which is a lentiviral vector.

[0014] Some embodiments are also described by the following enumerated items: 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. 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. 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, the first conjugate and the second conjugate 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 conjugate or a pharma- ceutically acceptable salt thereof and the linker in the second conjugate or a pharma- ceutically acceptable salt thereof are different. 5. The method according to item 2 or 3, wherein the linker in the first conjugate or a pharma- ceutically acceptable salt thereof and the linker in the second conjugate or a pharma- ceutically 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 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 items 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 the same. 8. The method according to any one of items 2 to 7, wherein the targeting moiety in the first conjugate or a pharma- ceutically acceptable salt thereof and the targeting moiety in the second conjugate or a pharma- ceutically acceptable salt thereof are different. 9. The method according to any one of items 2 to 7, wherein the targeting moiety in the first conjugate or a pharma- ceutically acceptable salt thereof and the targeting moiety in the second conjugate or a pharma- ceutically 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, an NK-1R ligand, a CAIX ligand, a ligand of gamma glutamyl transpeptidase, and a 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 an NK-1R ligand. 13. The method of claim 10, wherein the ligand is DUPA. 14. The method according to item 10, wherein the ligand is a CCK2R ligand. 15. The method according to item 10, wherein the ligand is a 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, centirin, and DARPin. 17. The method of claim 16, wherein the targeting moiety is FITC. 18. The method of claim 16, wherein the targeting moiety is DNP. 19. The method of claim 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, a hydrophilic amino acid, a sugar, a non-natural peptidoglycan, polyvinylpyrrolidone, and / or Pluronic F-127. 21. The method according to item 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: [ka] wherein B represents a small molecule ligand, L represents a linker, and T represents a targeting moiety, where L has the formula: [ka] (In the formula, n is an integer of 0 to 200.) [including the structure represented by 22. The method according to any one of items 1 to 21, wherein the compound has a structure represented by the formula: 23. The method according to item 22, wherein n is an integer from 0 to 150. 24. The method according to item 22, wherein n is an integer from 0 to 110. 25. The method according to item 22, wherein n is an integer from 0 to 20. 26. The method according to item 22, wherein n is an integer from 15 to 20. 27. The method according to item 22, wherein n is an integer between 15 and 110. 28. The method according to any one of items 1 to 27, wherein the linker comprises PEG and the targeting moiety is FITC or a pharma- ceutically acceptable salt thereof. 29. The method according to any one of items 1 to 28, wherein the dose 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. The method according to any one of items 1 to 29, wherein the dose 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. The method according to any one of items 1 to 30, wherein the dose 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. The method according to any one of items 1 to 31, wherein the dose 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. The method according to any one of items 1 to 32, wherein the dose 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. The method according to any one of items 1 to 33, wherein the dose 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. Cancer: 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 according to any one of items 1 to 34, wherein the cancer is selected from the group consisting of cancer of the urethra, 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, and adenocarcinoma of the gastroesophageal junction. 36. The method according to any one of items 1 to 11 or 16 to 35, wherein the cancer is a folate receptor expressing cancer. 37. The method according to item 35, wherein the cancer is endometrial cancer. 38. The method according to item 35, wherein the cancer is non-small cell lung cancer. 39. The method according to item 35, wherein the cancer is ovarian cancer. 40. The method according to item 35, wherein the cancer is triple-negative breast cancer. 41. The method according to any one of items 1 to 40, wherein the CAR has a recognition region, and the recognition region is an antibody single fragment variable region (scFv). 42. The method according to any one of items 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 fragment variable region (scFv) of an anti-FITC antibody. 43. The method of any one of items 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 according to any one of items 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 items 1 to 11, 16 to 17, or 20 to 41, wherein the CAR has a recognition region, and the recognition region is a single variable fragment (scFv) of an anti-FITC antibody; the CAR has a costimulatory domain, and the costimulatory domain is CD137 (4-1BB); and the CAR has an activation signaling domain, and the activation signaling domain is a T cell CD3 zeta chain. 46. ​​The method according to any one of items 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. The method according to any one of items 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. The method according to any one of items 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 items 1 to 48, wherein the targeting moiety is not a peptide epitope. 50. The method according to any one of items 1 to 49, wherein the method results in CAR T cytotoxicity against cancer without cytokine release that results in "off-target" toxicity in the patient. 51. The method according to any one of items 1 to 50, which produces CAR T cytotoxicity against cancer without producing "off-target" tissue toxicity in the patient. 52. The method according to any one of items 1 to 51, wherein the cancer comprises a tumor and the tumor size in the patient is reduced and no "off-target" tissue toxicity occurs. 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. An isolated nucleic acid encoding a chimeric antigen receptor comprising SEQ ID NO:1. 56. A chimeric antigen receptor polypeptide comprising SEQ ID NO:2. 57. A vector comprising sequence number 1. 58. The vector according to item 57, wherein the vector is a lentiviral vector. 59. The method according to any one of items 1 to 56, the CAR T cell, the isolated nucleic acid encoding the chimeric antigen receptor (CAR), or the chimeric antigen receptor polypeptide, wherein the CAR comprises a human amino acid sequence. 60. The method according to any one of items 1 to 56, the CAR T cell, the isolated nucleic acid encoding the 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 folate, DUPA, CAIX ligands, NK-1R ligands, ligands for gamma glutamyl transpeptidase, and CCK2R ligands. 62. The kit according to item 61, wherein the ligand in at least one bridge is an NK-1R ligand. 63. The kit according to item 61, wherein the ligand in at least one bridge is a ligand for gamma glutamyl transpeptidase. 64. The kit according to item 61, wherein the ligand in at least one bridge is folic acid. 65. The bridge has the formula: [ka] wherein B represents a small molecule ligand, L represents a linker, and T represents a targeting moiety, where L has the formula: [ka] (In the formula, n is an integer of 0 to 200.) [including the structure represented by 65. The kit according to any one of Items 61 to 64, having a structure comprising: 66. The kit according to item 65, wherein n is an integer from 0 to 150. 67. The kit according to item 65, wherein n is an integer from 0 to 110. 68. The kit according to item 65, wherein n is an integer from 0 to 20. 69. The kit according to item 65, wherein n is an integer from 15 to 20. 70. The kit according to item 65, wherein n is an integer from 15 to 110. 71. The method according to any one of items 1 to 10, 16 to 52, or 59 to 60, or the kit according to any one of items 61 to 70, wherein the ligand is C, an AIX ligand. 72.Formula: [ka] , [ka] , [ka] ,or [ka] The complex shown in FIG. [Brief description of the drawings]

[0015] [Figure 1A-1B] Figure 1 shows CAR T cell proliferation using FITC-small molecule conjugates in different cell types at a 5:1 (CAR T cell): target cell (cancer cell) ratio. 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]Figure 2 shows inflammatory cytokine IFN-γ production by CAR T cells using FITC-small molecule conjugates in different cell types. Figure 2A shows inflammatory cytokine IFN-γ production in KB (FR+) cells. Figure 2B shows inflammatory cytokine IFN-γ production in LNCaP (PSMA+) cells. Figure 2C shows inflammatory cytokine IFN-γ production in HEK293 (NK1R+) cells. Figure 2D shows inflammatory cytokine IFN-γ production in KB (FR+) cells using different concentrations of FITC-folate. Figure 2E shows inflammatory cytokine IFN-γ production in KB (FR+) cells using different conjugates. Figure 2F shows inflammatory cytokine IFN-γ production in KB (FR+) cells using different conjugates. [Figure 3A-3F] Figure 3 shows the in vitro toxicity of tumor cells treated with FITC-small molecule conjugates 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-folate concentration. Figure 3F shows the in vitro toxicity in KB (FR+) cells with different conjugates. [Figure 4A-4B] Figure 4 shows that CAR T cell activation correlates with the expression level of tumor antigen 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 conjugates measured by IFN-γ production in MDA-MB-231 and KB cells. [Figure 5A-5C]Figure 5 shows HEK293 (NK1R+) tumor xenografts and CAR T cell therapy, including treatment of CAR T cells with or without FITC-PEG11-NK1 conjugates. Figure 5A shows tumor volume measured over 24 days. Figure 5B shows body weight measured over 22 days of therapy. Figure 5C shows the percentage of CAR T cells in CD3+ human T cells after injection of CAR T cells with FITC-PEG11-NK1. [Figure 6A-6B] 5A-5C show organs taken from an exemplary mouse of the model used in Fig. 6A shows organs taken from a non-treated group. Fig. 6B shows organs taken two weeks after CAR T cell therapy. [Figure 7A-7C] Figure 7 shows MDA-MB-231 (FR+) xenografts under CAR T cell therapy, including treating cells with CAR T cells with either FITC-PEG12-folic acid conjugate, FITC-folic acid conjugate, or no conjugate. Figure 7A shows tumor volume measured over 23 days. Figure 7B shows body weight measured over 21 days of therapy. Figure 7C shows the percentage of CAR T cells in CD3+ human T cells after CAR T cell injection. [Figure 8A-8B] 7A-7C show organs harvested from exemplary mice of the model shown in Figure 8A. Figure 8A shows organs harvested from a non-treated group. Figure 8B shows organs harvested 3 weeks after CAR T cell therapy with CAR T cells and 500 nmole / kg body weight of FITC-PEG12-folate conjugate. [Figure 9] FIG. 9 shows blood indices of the HEK293 (NK1R+) xenograft model of FIGS. 5 and 6 and the MDA-MB-231 (FR+) xenograft model of FIGS. 7 and 8. [Figure 10] Shows differences in cytotoxicity against CAR T cell-treated KB(FR+) tumor cells depending on the FITC-small molecule conjugate used. [Figure 11] Shows percent body weight change in the KB tumor xenograft model with different concentrations of FITC-PEG-12-folate conjugates and CAR T cells. [Figures 12A-12C] Figures 12A-12C show organs taken from exemplary mice of the KB xenograft model shown in Figure 11. Figure 12A shows organs taken from the untreated group. Figure 12B shows organs taken from the CAR T cell therapy group treated with 250 nmol / kg FITC-PEG-12-folate. Figure 12C shows organs taken from the CAR T cell therapy group treated with CAR T cells and 500 nmol / kg FITC-PEG-12-folate. [Figure 13] 11-12 show blood indices of mice from the KB xenograft model. [Figure 14A-14B] The constructs used for CAR T transduction are shown in Figure 14A, which shows the CAR4-1BBZ construct, and Figure 14B, which shows the lentiviral vector. [Figure 15A-15B] Figure 15 shows flow cytometry analysis of transduced T cells: Figure 15A shows non-transduced cells; Figure 15B shows transduced cells. [Figure 16A-16B] Fluorescence microscopy of transduced CAR T cells is shown. Figure 16A shows GFP imaging showing transduction. Figure 16B shows FITC-folate localized to positively transduced cells. [Figure 17] Shows CAR T cell activation measured by relative expression of CD69 as a function of the conjugate used. [Figure 18] KB, LNCaP, and CAR T cell tumor heterogeneity as a function of conjugate used. [Figures 19A-19C]Figure 19 shows anti-tumor efficacy when the same anti-FITC CAR T cells (107 cells) were introduced into mice bearing two different tumors arising from two different cell lines (i.e., MDA-MB-231 (FR+) and HEK (NK1R+)) in separate flanks and then injected every other day with either PBS only (Figure 19A), FITC-PEG11-NK1R (500 nmole / kg) (Figure 19B), or FITC-PEG11-NK1R (500 nmole / kg) + FITC-PEG12-Folate (500 nmole / kg) (Figure 19C). Figure 19A: (closed circle) FR+ (MDA-MB-231): CAR T cells + PBS, (closed square) NK1R+ (HEK): CAR T cells + PBS; Figure 19B: (closed circle) FR+ (MDA-MB-231): CAR T cells + PBS, (closed square) NK1R+ (HEK): CAR T cells + FITC-PEG11-NK1R (500 nmole / kg); Figure 19C: (closed circle) FR+ (MDA-MB-231): CAR T cells + FITC-PEG12-FA (500 nmole / kg), (closed square) NK1R+ (HEK): CAR T cells + PBS. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

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

[0017] As used herein, the terms "ameliorate," "improving," "amelioration," or "improved" in reference to cancer means alleviating the symptoms of cancer, reducing the size of a tumor, completely or partially removing a tumor (e.g., complete or partial remission), stabilizing the disease, preventing the progression of cancer (e.g., progression-free survival), preventing the onset of disease, preventing the progression of disease (e.g., progression-free survival), or other effect on cancer that a physician considers to be a therapeutic or prophylactic treatment of the disease.

[0018] As used herein, the terms "administer," "administering," or "administered" refer to all means of introducing a compound or a pharma- ceutically acceptable salt thereof, a first conjugate or a pharma- ceutically acceptable salt thereof, or a second conjugate or a pharma- ceutically acceptable salt thereof, or a CAR T cell composition described herein into a patient, including, but not limited to, oral (po), intravenous (iv), intramuscular (im), subcutaneous (sc), and transdermal.

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

[0020] As used herein, the terms "transduction" and "transfection" are used equivalently and refer to the introduction of nucleic acid into a cell by any artificial method, including viral and non-viral methods.

[0021] DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS In various embodiments described herein, a small molecule ligand linked to a targeting moiety by a linker is used as a bridge between cancer and CAR T cells (i.e., cytotoxic T cells expressing chimeric antigen receptors). The bridge directs CAR T cells to cancer for cancer amelioration. In one embodiment, the "small molecule ligand" can be folic acid, CAIX ligand, DUPA, NK-1R ligand, gamma glutamyl transpeptidase ligand, or CCK2R ligand, each of which is a small molecule ligand that specifically binds to a type of cancer cell (i.e., the receptor for 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 engineered CAR expressed by the CAR T cell. Thus, the recognition region of the CAR (e.g., a single fragment variable region (scFv) of an antibody) is directed to the "targeting moiety." In this way, the small molecule ligand linked to the targeting moiety by a linker acts as a bridge between the cancer and the CAR T cell, directing the CAR T cell to the cancer for cancer amelioration. In various embodiments, the bridge between the cancer and the CAR T cell can be any of the conjugates shown in Examples 5-12.

[0023] The bridge is a small organic molecule, and clearance from the bloodstream can be achieved quickly (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 to control activation of the CAR T cells. Furthermore, this system can be "universal," since one type of CAR T cell construct can be used to target a variety of cancers. Illustratively, the targeting moiety recognized by the CAR T cells can remain constant so that one type of CAR T cell construct can be used, but the small molecule ligand that binds to the cancer is altered to allow targeting of a wide variety of cancers.

[0024] In one embodiment, the inventors have discovered that varying the dose of a small molecule ligand (i.e., bridge) linked to a targeting moiety by a linker can provide the ability to control CRS in vivo upon CAR T cell activation. In another embodiment, the inventors have discovered that varying the linker in the small molecule ligand (bridge) linked to a targeting moiety can control CRS in vivo upon 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 various embodiments described in the following item list and claims, the small molecule ligand linked to the targeting moiety by a linker is referred to as a "compound", "first complex" or "second complex". The term "compound" is used in embodiments in which the dose of the small molecule ligand linked to the targeting moiety by a linker is changed to control cytokine release in vivo. The terms "first complex" and "second complex" are used in embodiments in which two different complexes are administered to a patient. For example, the linker of the small molecule ligand linked to the targeting moiety can be changed 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 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. 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. 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, the first conjugate and the second conjugate 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 conjugate or a pharma- ceutically acceptable salt thereof and the linker in the second conjugate or a pharma- ceutically acceptable salt thereof are different. 5. The method according to item 2 or 3, wherein the linker in the first conjugate or a pharma- ceutically acceptable salt thereof and the linker in the second conjugate or a pharma- ceutically 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 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 items 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 the same. 8. The method according to any one of items 2 to 7, wherein the targeting moiety in the first conjugate or a pharma- ceutically acceptable salt thereof and the targeting moiety in the second conjugate or a pharma- ceutically acceptable salt thereof are different. 9. The method according to any one of items 2 to 7, wherein the targeting moiety in the first conjugate or a pharma- ceutically acceptable salt thereof and the targeting moiety in the second conjugate or a pharma- ceutically 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, an NK-1R ligand, a CAIX ligand, a ligand of gamma glutamyl transpeptidase, and a 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 an NK-1R ligand. 13. The method of claim 10, wherein the ligand is DUPA. 14. The method according to item 10, wherein the ligand is a CCK2R ligand. 15. The method according to item 10, wherein the ligand is a 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, centirin, and DARPin. 17. The method of claim 16, wherein the targeting moiety is FITC. 18. The method of claim 16, wherein the targeting moiety is DNP. 19. The method of claim 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, a hydrophilic amino acid, a sugar, a non-natural peptidoglycan, polyvinylpyrrolidone, and / or Pluronic F-127. 21. The method according to item 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: [ka] wherein B represents a small molecule ligand, L represents a linker, and T represents a targeting moiety, where L has the formula: [ka] (In the formula, n is an integer of 0 to 200.) [including the structure represented by 22. The method according to any one of items 1 to 21, wherein the compound has a structure represented by the formula: 23. The method according to item 22, wherein n is an integer from 0 to 150. 24. The method according to item 22, wherein n is an integer from 0 to 110. 25. The method according to item 22, wherein n is an integer from 0 to 20. 26. The method according to item 22, wherein n is an integer from 15 to 20. 27. The method according to item 22, wherein n is an integer between 15 and 110. 28. The method according to any one of items 1 to 27, wherein the linker comprises PEG and the targeting moiety is FITC or a pharma- ceutically acceptable salt thereof. 29. The method according to any one of items 1 to 28, wherein the dose 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. The method according to any one of items 1 to 29, wherein the dose 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. The method according to any one of items 1 to 30, wherein the dose 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. The method according to any one of items 1 to 31, wherein the dose 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. The method according to any one of items 1 to 32, wherein the dose 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. The method according to any one of items 1 to 33, wherein the dose 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. Cancer: 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 according to any one of items 1 to 34, wherein the cancer is selected from the group consisting of cancer of the urethra, 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, and adenocarcinoma of the gastroesophageal junction. 36. The method according to any one of items 1 to 11 or 16 to 35, wherein the cancer is a folate receptor expressing cancer. 37. The method according to item 35, wherein the cancer is endometrial cancer. 38. The method according to item 35, wherein the cancer is non-small cell lung cancer. 39. The method according to item 35, wherein the cancer is ovarian cancer. 40. The method according to item 35, wherein the cancer is triple-negative breast cancer. 41. The method according to any one of items 1 to 40, wherein the CAR has a recognition region, and the recognition region is an antibody single fragment variable region (scFv). 42. The method according to any one of items 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 fragment variable region (scFv) of an anti-FITC antibody. 43. The method of any one of items 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 according to any one of items 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 items 1 to 11, 16 to 17, or 20 to 41, wherein the CAR has a recognition region, and the recognition region is a single variable fragment (scFv) of an anti-FITC antibody; the CAR has a costimulatory domain, and the costimulatory domain is CD137 (4-1BB); and the CAR has an activation signaling domain, and the activation signaling domain is a T cell CD3 zeta chain. 46. ​​The method according to any one of items 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. The method according to any one of items 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. The method according to any one of items 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 items 1 to 48, wherein the targeting moiety is not a peptide epitope. 50. The method according to any one of items 1 to 49, wherein the method results in CAR T cytotoxicity against cancer without cytokine release that results in "off-target" toxicity in the patient. 51. The method according to any one of items 1 to 50, which produces CAR T cytotoxicity against cancer without producing "off-target" tissue toxicity in the patient. 52. The method according to any one of items 1 to 51, wherein the cancer comprises a tumor and the tumor size in the patient is reduced and no "off-target" tissue toxicity occurs. 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. An isolated nucleic acid encoding a chimeric antigen receptor comprising SEQ ID NO:1. 56. A chimeric antigen receptor polypeptide comprising SEQ ID NO:2. 57. A vector comprising sequence number 1. 58. The vector according to item 57, wherein the vector is a lentiviral vector. 59. The method according to any one of items 1 to 56, the CAR T cell, the isolated nucleic acid encoding the chimeric antigen receptor (CAR), or the chimeric antigen receptor polypeptide, wherein the CAR comprises a human amino acid sequence. 60. The method according to any one of items 1 to 56, the CAR T cell, the isolated nucleic acid encoding the 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 folate, DUPA, CAIX ligands, NK-1R ligands, ligands for gamma glutamyl transpeptidase, and CCK2R ligands. 62. The kit according to item 61, wherein the ligand in at least one bridge is an NK-1R ligand. 63. The kit according to item 61, wherein the ligand in at least one bridge is a ligand for gamma glutamyl transpeptidase. 64. The kit according to item 61, wherein the ligand in at least one bridge is folic acid. 65. The bridge has the formula: [ka] wherein B represents a small molecule ligand, L represents a linker, and T represents a targeting moiety, where L has the formula: [ka] (In the formula, n is an integer of 0 to 200.) [including the structure represented by 65. The kit according to any one of Items 61 to 64, having a structure comprising: 66. The kit according to item 65, wherein n is an integer from 0 to 150. 67. The kit according to item 65, wherein n is an integer from 0 to 110. 68. The kit according to item 65, wherein n is an integer from 0 to 20. 69. The kit according to item 65, wherein n is an integer from 15 to 20. 70. The kit according to item 65, wherein n is an integer from 15 to 110. 71. The method according to any one of items 1 to 10, 16 to 52, or 59 to 60, or the kit according to any one of items 61 to 70, wherein the ligand is C, an AIX ligand. 72.Formula: [ka] , [ka] , [ka] ,or [ka] The complex shown in FIG.

[0027] As described herein, a "patient" may be a human, or in the case of veterinary applications, the patient may be a laboratory animal, an agricultural animal, a livestock animal, or a wild animal. In one embodiment, the patient may be a laboratory 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 captive wild animal, such as a bear, panda, lion, tiger, leopard, elephant, zebra, giraffe, gorilla, dolphin, or whale. In the method 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 fluorescein isothiocyanate (FITC) linked to the small molecule ligand. The cancer overexpresses a receptor for the small molecule ligand. As a second component, for example, cytotoxic T cells are transformed to express a CAR comprising an anti-FITC scFv. In this embodiment, the CAR targets FITC, which decorates the cancer with FITC molecules as a result of the binding of the small molecule ligand 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 (for example, 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), NK-1R ligands (e.g., ligands for the receptor for NK-1R found on colon and pancreatic cancers), CAIX ligands (receptors for CAIX ligands are found on, e.g., renal, ovarian, vulvar, and breast cancers), gamma glutamyl transpeptidase ligands (transpeptidases are overexpressed in, e.g., ovarian, colon, liver, astrocytic glioma, melanoma, and leukemia), or CCK2R ligands (receptors for CCK2R ligands are found on thyroid, lung, pancreatic, ovarian, brain, stomach, gastrointestinal stromal, and colon cancers), each of which is a small molecule ligand that specifically binds to a cancer cell type (i.e., the receptors for each of these ligands are overexpressed on cancer cells compared to normal tissue). In one embodiment, the DUPA derivative can be a ligand that is a small molecule ligand linked to a targeting moiety, and DUPA derivatives are 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 folinic acid, pteropolyglutamic acid, and folic acid receptor binding pteridines such as tetrahydropterin, dihydrofolic acid, tetrahydrofolic acid, and their deaza and dideaza analogs. The terms "deaza" and "dideaza" analogs refer to art-recognized analogs that have one or two nitrogen atoms replaced by carbon 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 folate analogs are conventionally referred to as "folic acid", reflecting their ability to bind to the folate receptor. Other folate receptor binding analogs include aminopterin, amethopterin (methotrexate), N10-methylfolate, 2-deamino-hydroxybutyric acid, deaza analogs such as 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 9000 daltons, less than about 8,000 daltons, less than about 7000 daltons, less than about 6000 daltons, less than about 5000 daltons, less than about 4500 daltons, less than about 4000 daltons, less than about 3500 daltons, less than about 3000 daltons, less than about 2500 daltons, less than about 2000 daltons, less than about 1500 daltons, less than about 1000 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 9000 daltons, about 1 to about 8,000 daltons, about 1 to about 7000 daltons, about 1 to about 6000 daltons, about 1 to about 5000 daltons, about 1 to about 4500 daltons, about 1 to about 4000 daltons, about 1 to about 3500 daltons, about 1 to about 3000 daltons, about 1 to about 2500 daltons, about 1 to about 2000 daltons, about 1 to about 1500 daltons, about 1 to about 1000 daltons, or about 1 to about 500 daltons.

[0032] In one embodiment, 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, centrin, and DARPin. The identity of the targeting moiety is limited only in that it should be recognized and bound by the CAR, preferably with specificity, and have a relatively low molecular weight. In various embodiments, an exemplary targeting moiety is a hapten, such as a low molecular weight organic molecule.

[0033] The targeting moiety can have the following exemplary structures: [ka] wherein X is oxygen, nitrogen or sulfur, X is attached to a linker L; Y is ORa , N.R. a 2 or NR a 3 +; and Y' is O, NR a or NR a 2 + each R, at each occurrence, is independently selected from H, fluoro, sulfonic acid, sulfonate, salts thereof, and the like; and R a is hydrogen or alkyl.

[0034] In one exemplary embodiment, the linker in the compound or a pharma- ceutically acceptable salt thereof described herein, the first conjugate or a pharma- ceutically acceptable salt thereof, or the second conjugate or a pharma- ceutically acceptable salt thereof can be a direct bond (e.g., a reaction between an isothiocyanate group of FITC and a free amine group of a small molecule ligand), or the bond can be via an intermediary linker. In one embodiment, the intermediary linker, if present, can be a biocompatible linker known in the art, such as a bivalent linker. In one exemplary embodiment, the bivalent linker can include about 1 to about 30 carbon atoms. In another exemplary embodiment, the bivalent linker can include about 2 to about 20 carbon atoms. In other embodiments, low molecular weight bivalent linkers (i.e., those having an approximate molecular weight of about 30 to about 300) are used. In another embodiment, linkers of suitable length include, but are not limited to, 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.

[0035] In various embodiments, the small molecule ligand linked to the targeting moiety has the formula: [ka] wherein B represents a small molecule ligand, L represents a linker, and T represents a targeting moiety, where L has the formula: [ka] (In the formula, n is an integer of 0 to 200.) [including the structure represented by In another embodiment, n can be represented by 0 to 150, 0 to 110, 0 to 100, 0 to 90, 0 to 80, 0 to 70, 0 to 60, 0 to 50, 0 to 40, 0 to 30, 0 to 20, 0 to 15, 0 to 14, 0 to 13, 0 to 12, 0 to 11, 0 to 10, 0 to 9, 0 to 8, 0 to 7, 0 to 6, 0 to 5, 0 to 4, 0 to 3, 0 to 2 , 0~1, 15~16, 15~17, 15~18, 15~19, 15~20, 15~21, 15~22, 15~23, 15~24, 15~25, 15~26, 15~27, 15~28, 15~29, 15~30, 15~31, 15~32, 15~33, 15~34, 15~35, 15~36, 15~37, 15~39, 15~40, 15~41, 15~42, 15~43, 15~44, 15~45, 15~46, 15~47, 15~48, 15~49, 15~50, 15~51, 15~52, 15~53, 15~54, 15~55, 15~56, 15~57, 15~58, 15~59, 15~60, 15~61, 15~62, 15~63, 15~64, 15~65, 15~66, 15~67, 15~68, 15~69, 15~70, 15~71, 15~72, 15~73, 15~74, 15~75, 15~76, 15~77, 15~78, 15~79, 15~80, 15~81, 15~82, 15~83, 15~84, 15~85, 15~86, 15~87, 15~88, n may be an integer of 7, 15-38, 15-39, 15-40, 15-50, 15-60, 15-70, 15-80, 15-90, 15-100, 15-110, 15-120, 15-130, 15-140, or 15-150, or n may be an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, It can be 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, 40, 50, 60, 70, 80, 90, 100, 108, 110, 120, 130, 140, or 150.

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

[0037] [Table 1]

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

[0039] In other embodiments, 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.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 its pharmaceutically acceptable salt and the linker in the second conjugate or its pharmaceutically acceptable salt can be the same or different. In another embodiment, the ligand in the first conjugate or its pharmaceutically acceptable salt and the ligand in the second conjugate or its pharmaceutically acceptable salt can be the same or different. In another exemplary embodiment, the targeting moiety in the first conjugate or its pharmaceutically acceptable salt and the targeting moiety in the second conjugate or its pharmaceutically acceptable salt can be the same or different. Any combination of these embodiments is also contemplated, along with any combination of doses described below.

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

[0042] In another embodiment, the bridge in the kit has the formula: [ka] wherein B represents a small molecule ligand, L represents a linker, and T represents a targeting moiety, where L has the formula: [ka] (In the formula, n is an integer of 0 to 200.) [including the structure represented by In other embodiments, n can be an integer from 0 to 150, an integer from 0 to 110, an integer from 0 to 20, an integer from 15 to 20, an integer from 15 to 110, or any other integer value or range described herein for n.

[0043] The "pharmaceutical acceptable salt" of the small molecule ligand linked to the targeting moiety by the linker is intended. As used herein, the term "pharmaceutical acceptable salt" refers to the salt whose counterion can be used in medicine. Such salts include: 1) the 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) the salts that are formed when the acidic protons present in the parent compound are replaced by metal ions, such as alkali metal ions, alkaline earth ions, or aluminum ions, or coordinate with organic bases such as ethanolamine, diethanolamine, triethanolamine, trimethamine, N-methylglucamine, etc. Pharmaceutically acceptable salts are well known to those of skill in the art, and such pharma-ceutically acceptable salts may be contemplated in connection with the embodiments described herein.

[0044] In various embodiments, suitable acid addition salts are formed from acids which form non-toxic salts, illustrative examples of which include acetate, aspartate, benzoate, besylate, bicarbonate / carbonate, bisulfate / sulfate, borate, camsylate, citrate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hybenzate, hydrochloride / chloride, hydrobromide / bromide, hydroiodide / iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methylsulfate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate / hydrogenphosphate / dihydrogenphosphate, saccharate, stearate, succinate, tartrate, tosylate, and trifluoroacetate.

[0045] In various embodiments, suitable base salts are formed from bases from which non-toxic salts are formed. Illustrative examples include arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine and zinc salts. Hemisalts of acids and bases can also be formed, such as hemisulfate and hemicalcium salts.

[0046] In one exemplary embodiment, the compound described herein or a pharma- ceutically acceptable salt thereof, the first complex or a pharma- ceutically acceptable salt thereof, or the second complex or a pharma- ceutically acceptable salt thereof can contain one or more chiral centers or can otherwise exist as multiple stereoisomers. Thus, 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 compound described herein or a pharma- ceutically acceptable salt thereof, the first complex or a pharma- ceutically acceptable salt thereof, or the second complex or a pharma- ceutically acceptable salt thereof can exist as geometric isomers. Thus, various embodiments can include pure geometric isomers or mixtures of geometric isomers.

[0047] In some embodiments, the compound described herein or a pharma- ceutically acceptable salt thereof, the first complex or a pharma- ceutically acceptable salt thereof, or the second complex or a pharma- ceutically acceptable salt thereof may exist in a solvated form, such as an unsolvated form and a hydrated form. In general, the solvated form is equivalent to the unsolvated form and is included 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: 1) a recognition region (e.g., a single fragment variable (scFv) region of an antibody) that recognizes and binds to a targeting moiety with specificity; 2) a costimulatory domain that promotes T lymphocyte proliferation and survival; and 3) an activation signaling domain that generates a cytotoxic T lymphocyte activation signal.

[0049] In various embodiments, scFv regions of antibodies that bind folate, DUPA, CAIX ligands, NK-1R ligands, gamma glutamyl transpeptidase ligands, or CCK2R ligands can be used. In exemplary embodiments, scFv regions can be produced from (i) antibodies known in the art that bind targeting moieties, (ii) antibodies prepared de novo with a selected targeting moiety, such as a hapten, and (iii) sequence variants derived from the scFv regions of such antibodies, e.g., scFv regions 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 portion of the CAR can be, for example, an antibody, a Fab, Fv, Fc, or a single chain fragment variable region (scFv), such as a (Fab')2 fragment.

[0051] In one embodiment, the costimulatory domain serves to enhance the proliferation and survival of cytotoxic T lymphocytes upon binding of the CAR to the targeting moiety. Suitable costimulatory domains include: 1) CD28, 2) CD137 (4-1BB), a member of the tumor necrosis factor (TNF) receptor family, 3) CD134 (OX40), a member of the TNFR superfamily of receptors, and 4) CD278 (ICOS), a CD28 superfamily costimulatory molecule expressed on activated T cells, or combinations thereof. Suitable costimulatory domains also include, but are not limited to, CD27, CD30, CD150, DAP10, and NKG2D, or combinations thereof. Those skilled in the art will understand that sequence variants of these costimulatory domains can be used without adversely affecting the present invention and have the same or similar activity as the domains they are modeled on. 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 exemplary embodiments, the activation signaling domain acts to activate cytotoxic T lymphocytes upon binding of the CAR to the targeting moiety. Suitable activation signaling domains include T cell CD3 zeta chain and Fc receptor gamma. Those skilled in the art will understand that sequence variants of these noted activation signaling domains can be used if the variants have the same or similar activity as the domain they are modeled on. In various embodiments, the 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.

[0053] In one embodiment, 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 Edition, Cold Spring Harbor Laboratory Press (2001), incorporated herein by reference. By way of example, a plasmid or viral expression vector (e.g., lentiviral vectors, retroviral vectors, Sleeping Beauty, and PiggyBac (transposon / transposase systems, including non-viral mediated CAR gene delivery systems)) encoding a fusion protein containing a recognition region, one or more costimulatory domains, and an activation signaling domain linked in frame in a 5' to 3' direction. In other embodiments, other configurations are acceptable, including a recognition domain, an activation signaling domain, and one or more costimulatory domains. The placement of the recognition domain in the fusion protein is generally done to achieve display of the domain on the outside of the cell. In one embodiment, the CAR comprises a signal peptide to ensure proper transport of the fusion protein to the cell surface, a transmembrane domain to ensure that the fusion protein is maintained as an integral membrane protein, and a hinge domain that provides flexibility to the recognition domain and allows for strong binding to the targeting moiety. Additional elements such as may be included.

[0054] A diagram of an exemplary CAR is shown in Figures 14A and B, where the fusion protein sequence is incorporated into a lentiviral expression vector, where "SP" is the signal peptide, the CAR is an anti-FITC CAR, there is a CD8α hinge and transmembrane (TM) region, the costimulatory domain is 4-1BB, and the activation signaling domain is CD3ζ. In one embodiment, 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, which is a single-chain fragment variable (scFv) region of an anti-FITC antibody, a costimulatory domain, which is CD137 (4-1BB), an activation signaling domain, and an activation signaling domain, which is T cell CD3 zeta chain. Those skilled in the art are aware that anti-FITC scFv and anti-fluorescein scFv are equivalent terms.

[0056] In one embodiment, a population of cytotoxic T lymphocytes can be engineered to express a CAR construct by transfecting the population with an expression vector encoding the CAR construct. Suitable methods for preparing a transduced population of T lymphocytes expressing a 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 Edition, Cold Spring Harbor Laboratory Press, (2001), which is incorporated herein by reference.

[0057] In various embodiments, a CAR T cell is provided that comprises a nucleic acid of SEQ ID NO:1 or 3. In another embodiment, a CAR T cell is provided that comprises a polypeptide of SEQ ID NO:2. In another exemplary embodiment, an isolated nucleic acid is provided that comprises SEQ ID NO:1 or 3 and encodes a chimeric antigen receptor. In yet another embodiment, a chimeric antigen receptor polypeptide is provided that comprises SEQ ID NO:2. In another embodiment, a vector is provided that comprises SEQ ID NO:1 or 3. In another aspect, a lentiviral vector is provided that comprises SEQ ID NO:1 or 3. 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, variant nucleic acid or amino acid sequences are contemplated that 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 SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. In another embodiment, the nucleic acid sequence can be a variant nucleic acid sequence that 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 SEQ ID NO:1 or 3, and the variant sequence encodes the polypeptide of SEQ ID NO:2. In another embodiment, the variant nucleic acid or amino acid sequence may be a variant 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. Determination of percent identity or similarity between sequences can be performed, for example, using the GAP program (Genetics Computer Group, software; available from Accelrys on http: / / www.accelrys.com), and alignment can be performed, for example, using the ClustalW algorithm (VNTI software, InforMax Inc.). Sequence databases can be searched using the nucleic acid or amino acid sequence of interest. Algorithms for database searching are usually based on the BLAST software (Altschul et al., 1990). In some embodiments, percent identity can be determined along the entire length of the nucleic acid or amino acid sequence.

[0059] Also included within the scope of the present invention are nucleic acids complementary to the nucleic acids represented by SEQ ID NO: 1 or 3, and nucleic acids that hybridize to the nucleic acids represented by SEQ ID NO: 1 or 3, or hybridize to their complements under high stringency conditions. In the present invention, "high stringency conditions" refers to hybridization in 5xSSPE and 50% formamide at 65°C and washing in 0.5xSSPE at 65°C. High stringency, low stringency and moderate stringency 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, the cytotoxic T lymphocytes used to prepare the CAR T cells used in the methods described herein can be autologous cells, although xenogeneic cells can also be used, such as when the patient being treated has received high doses of chemotherapy or radiation therapy to destroy the patient's immune system. In one embodiment, allogeneic cells can be used.

[0061] In one embodiment, the cytotoxic T lymphocytes can be obtained from a patient by means well known in the art. For example, the cytotoxic T cells can be obtained by drawing 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 include other cells, such as other T cells, monocytes, macrophages, natural killer cells, and B cells. In one embodiment, the harvested population can include at least about 90% of the selected cell type, at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the selected cell type.

[0062] In one embodiment, after the cytotoxic T lymphocytes are obtained, the cells are cultured under conditions that promote activation of the cells. In this embodiment, the culture conditions may be such that the cells can be administered to a patient without consideration of reactivity to components of the culture medium. For example, the culture conditions may be free of 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 activating agent, such as an anti-CD3 antibody, into the medium. Other suitable activating agents include anti-CD28 antibodies. In one embodiment, the population of lymphocytes can be cultured under conditions that promote activation for about 1 to about 4 days. In one embodiment, the appropriate level of activation can be determined by cell size, proliferation rate, or activation markers determined by flow cytometry.

[0063] In one exemplary embodiment, a population of cytotoxic T lymphocytes can be cultured under conditions that promote activation, followed by transfection of the cells with an expression vector encoding a CAR. Suitable vectors and transfection methods are described above. In one embodiment, following transfection, the cells can be administered immediately to a patient, or the cells can be cultured 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 for 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, e.g., to allow time for the cells to recover from the transfection. Suitable culture conditions may be similar to those under which the cells were cultured for activation, either with or without the agent used to promote activation.

[0064] Thus, as noted above, in one exemplary embodiment, the therapeutic methods 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 a CAR T cell.

[0065] In an exemplary embodiment, once 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 with bacteria, fungi and mycoplasma can be used. In an exemplary embodiment, the cells are pelleted, washed and resuspended in a pharma- ceutically acceptable carrier or diluent prior to administration to a patient. Exemplary compositions comprising CAR-expressing cytotoxic T lymphocytes include compositions comprising cells in sterile 290 mOsm saline, in injectable cryo-medium (including Plasma-Lyte A, dextrose, sodium chloride injection, human serum albumin and DMSO), in 0.9% NaCl with 2% human serum albumin, or any other sterile 290 mOsm injectable material. Alternatively, depending on the identity of the culture medium, the CAR T cells may be administered in the culture medium as a composition or may be concentrated and resuspended in the medium prior to administration. The CAR T cell composition may 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 embodiment, the total number of CAR T cells and the concentration of cells in the composition administered to a patient will vary depending on many factors, such as the type of cytotoxic T lymphocyte used, the binding specificity of the CAR, the identity of the targeting moiety and small molecule ligand, the identity 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 comprising transduced CAR T cells will contain approximately 1×10 5 ~Approx. 1×10 15 A typical composition includes a composition having a volume of about 5 ml to about 200 ml, comprising about 1×10 transduced CAR T cells. 7 ~Approx. 1×10 10 An exemplary composition comprises about 1 x 10 CAR T cells in a volume of about 100 ml to about 125 ml. 9 In one embodiment, a single dose or multiple doses of the 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 may be lung cancer, bone cancer, pancreatic cancer, skin cancer, head cancer, neck cancer, cutaneous melanoma, intraocular melanoma, uterine cancer, ovarian cancer, endometrial cancer, rectal cancer, gastric 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, non-small cell lung cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, prostate cancer, chronic leukemia, acute leukemia, lymphocytic lymphoma, pleural mesothelioma, bladder cancer, Burkitt's lymphoma, ureteral cancer, kidney cancer, renal cell carcinoma, renal pelvis cancer, central nervous system (CNS) neoplasm, primary CNS lymphoma, spinal 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 folate receptors. 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 being imaged is a tumor. In another embodiment, the cancer is malignant.

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

[0070] In one embodiment, the compound or its pharmaceutically acceptable salt, the first conjugate or its pharmaceutically acceptable salt, the second conjugate or its pharmaceutically acceptable salt, or the CAR T cell composition described herein can be administered directly into the bloodstream, muscle, or internal organ.Suitable routes for such parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, epidural, intraventricular, intraurethral, ​​intrasternal, intracranial, intratumoral, intramuscular, and subcutaneous delivery.In one embodiment, means for parenteral administration include needle (including microneedle) injectors, needle-free injectors, and injection techniques.

[0071] In one exemplary embodiment, parenteral formulations are typically aqueous solutions that may contain carriers or excipients such as salts, carbohydrates, and buffers (preferably pH 3-9), although they may be more suitably formulated as sterile non-aqueous solutions or as dry forms used with an appropriate vehicle such as sterile, pyrogen-free water or sterile saline. In other embodiments, any of the liquid formulations described herein may 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 accomplished using standard pharmaceutical techniques well known to those skilled in the art. In one embodiment, the solubility of the compound or a pharma- ceutically acceptable salt thereof, the first conjugate or a pharma- ceutically acceptable salt thereof, the second conjugate or a pharma- ceutically acceptable salt thereof used in the preparation of the parenteral formulation may be increased by the use of appropriate formulation techniques, such as the incorporation of a solubility enhancer.

[0072] In some embodiments, the rate of tumor lysis can be controlled by adjusting the concentration of the first conjugate or a pharma- ceutically acceptable salt thereof, the second conjugate or a pharma- ceutically acceptable salt thereof, or both the first and second conjugates or a pharma- ceutically acceptable salt thereof. Thus, the cytotoxicity of the CAR T cell composition can be adjusted by varying the concentration of the first conjugate or a pharma- ceutically acceptable salt thereof, the second conjugate or a pharma- ceutically acceptable salt thereof, or both the first and second conjugates or a pharma- ceutically acceptable salt thereof. In some embodiments, the cytotoxicity of the CAR T cell composition can be balanced against the risk of tumor lysis syndrome or cytokine release syndrome (CRS) by adjusting the concentration of the first conjugate or a pharma- ceutically acceptable salt thereof, the second conjugate or a pharma- ceutically acceptable salt thereof, or both the first and second conjugates or a pharma- ceutically acceptable salt thereof, as described herein. It is understood that the concentration of the first conjugate or a pharma- ceutically acceptable salt thereof, the second conjugate or a pharma- ceutically acceptable salt thereof, or both the first and second conjugates or a pharma- ceutically acceptable salt thereof, can be a function of the amount or dose of the first conjugate or a pharma- ceutically acceptable salt thereof, the second conjugate or a pharma- ceutically acceptable salt thereof, or both the first and second conjugates or a pharma- ceutically acceptable salt thereof, administered to the patient.

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

[0074] In other embodiments, the dose of the compound or a pharma- ceutically acceptable salt thereof, the first complex or a pharma- ceutically acceptable salt thereof, or the second complex or a pharma- ceutically acceptable salt thereof is, for example, 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 800 nmol / kg, about 50 nmol / kg to about 900 nmol / kg, about 50 nmol / kg to about 1000 nmol / kg, about 50 nmol / kg to about 15 ... The range may be from about 1 / kg to about 600 nmol / kg, from about 50 nmol / kg to about 500 nmol / kg, from about 50 nmol / kg to about 400 nmol / kg, from about 50 nmol / kg to about 300 nmol / kg, from about 50 nmol / kg to about 200 nmol / kg, from about 50 nmol / kg to about 100 nmol / kg, from about 100 nmol / kg to about 300 nmol / kg, from about 100 nmol / kg to about 500 nmol / kg, from about 100 nmol / kg to about 1000 nmol / kg, or from about 100 nmol / kg to about 2000 nmol / kg. In other embodiments, the dosage may 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 of patient weight. In these embodiments, "kg" refers to kilograms of patient weight. In one aspect, a single or 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 may be administered to the patient.

[0075] In another embodiment, about 20 ug / kg of patient's body weight to about 3 mg / kg of patient's body weight of the compound or a pharma- ceutically acceptable salt thereof, the first conjugate or a pharma- ceutically acceptable salt thereof, the second conjugate or a pharma- ceutically acceptable salt thereof is administered to the patient. In another embodiment, the amount can be about 0.2 mg / kg of patient's body weight to about 0.4 mg / kg of patient's body weight, or about 50 ug / kg of patient's body weight. In one embodiment, a single or multiple doses of the compound or a pharma- ceutically acceptable salt thereof, the first conjugate or a pharma- ceutically acceptable salt thereof, the second conjugate or a pharma- ceutically acceptable salt thereof may be administered to the patient.

[0076] In one embodiment, the small molecule ligand linked to a targeting moiety may be administered to the patient prior to the CAR T cell composition. In another embodiment, the small molecule ligand linked to a 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 a targeting moiety may be administered to the patient after the CAR T cell composition.

[0077] In one exemplary embodiment, the timing between administration of the CAR T cells and the small molecule linked to a targeting moiety will vary widely depending on factors such as the type of CAR T cells used, the binding specificity of the CAR, the identity of the targeting moiety and the small molecule ligand, the identity 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 a 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 a targeting moiety can be administered before or after the CAR T cells, such as 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 administration rate of the first conjugate or a pharma- ceutically acceptable salt thereof, the second conjugate or a pharma- ceutically acceptable salt thereof, or both the first and second conjugates or a pharma- ceutically acceptable salt thereof. Thus, the cytotoxicity of the CAR T cell composition can be controlled by varying the administration rate of the first conjugate or a pharma- ceutically acceptable salt thereof, the second conjugate or a pharma- ceutically acceptable salt thereof, or both the first and second conjugates or a pharma- ceutically acceptable salt thereof. In some embodiments, the cytotoxicity of the CAR T cell composition can be balanced against the risk of tumor lysis syndrome or cytokine release syndrome (CRS) by adjusting the administration rate of the first conjugate or a pharma- ceutically acceptable salt thereof, the second conjugate or a pharma- ceutically acceptable salt thereof, or both the first and second conjugates or a pharma- ceutically acceptable salt thereof, as described herein. It is understood that the administration rate of the first conjugate or its pharma- ceutically acceptable salt, the second conjugate or its pharma- ceutically acceptable salt, or both the first and second conjugates or its pharma- ceutically acceptable salts, can be a function of any administration schedule known in the art. For example, the administration rate can be a function of an administration schedule based on continuous administration, once a day (also known as qd), twice a day (also known as bid), three times a day (also known as tid), twice a week (also known as BIW), three times a week (also known as TIW), once every week, and the like. It is understood that the administration schedule selected for the first conjugate or its pharma- ceutically acceptable salt, the second conjugate or its pharma- ceutically acceptable salt, or both the first and second conjugates or its pharma- ceutically acceptable salts, can be applied in conjunction with the concentration to control the cytotoxicity of the CAR T cell composition.In some embodiments, the cytotoxicity of the CAR T cell composition can be balanced against the risk of tumor lysis syndrome or cytokine release syndrome (CRS) by adjusting the administration schedule along with the concentration of the first conjugate or a pharma- ceutically acceptable salt thereof, the second conjugate or a pharma- ceutically acceptable salt thereof, or both the first and second conjugates or a pharma- ceutically acceptable salt thereof, as described herein.

[0079] In one embodiment of the method described herein, the cancer is imaged before administering the compound or a pharma- ceutically acceptable salt thereof, the first conjugate or a pharma- ceutically acceptable salt thereof, the second conjugate or a pharma- ceutically acceptable salt thereof, or before administering the CAR T cell composition to the patient.In one exemplary embodiment, the imaging occurs by PET imaging.In another exemplary embodiment, the imaging occurs by MRI imaging or SPECT / CT imaging.It is understood that the imaging method can be any suitable imaging method known in the art.

[0080] In any of the embodiments described herein, the cytotoxicity of the CAR T cells on the cancer may not result in cytokine release resulting in "off-target" cytotoxicity in the patient. In any of the embodiments described herein, the cytotoxicity of the CAR T cells on the cancer may not result in "off-target" tissue cytotoxicity in the patient. In any of the embodiments described herein, the cancer may include a tumor, and the tumor size in the patient may be reduced, although "off-target" cytotoxicity may not occur. EXAMPLES

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

[0082] An exemplary CAR nucleic acid coding sequence may include:

[0083] ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGATGTCGTGATGACCCAGACCCCCCTCAGCCTCCCAGTGTCCCTCGGTGACCAGGCTTCTATTAGTTGCAGATCCAGCCAGTCCCTCGTGCACTCTAACGGTAATACCTACCTGAGATGGTATCTCCAGAAGCCCGGACAGAGCCCTAAGGTGCTGATCTACAAAGTCTCCAACCGGGTGTCTGGAGTCCCTGACCGCTTCTCAGGGAGCGGTTCCGGCACCGACTTCACCCTGAAGATCAACCGGGTGGAGGCCGAAGACCTCGGCGTCTATTTCTGCTCTCAGAGTACACATGTGCCCTGGACCTTCGGCGGAGGGACCAAGCTGGAGATCAAAAGCTCCGCAGACGATGCCAAGAAAGATGCCGCTAAGAAAGACGATGCTAAGAAAGACGATGCAAAGAAAGACGGTGGCGTGAAGCTGGATGAAACCGGAGGAGGTCTCGTCCAGCCAGGAGGAGCCATGAAGCTGAGTTGCGTGACCAGCGGATTCACCTTTGGGCACTACTGGATGAACTGGGTGCGACAGTCCCCAGAGAAGGGGCTCGAATGGGTCGCTCAGTTCAGGAACAAACCCTACAATTATGAGACATACTATTCAGACAGCGTGAAGGGCAGGTTTACTATCAGTA

[0084] GAGACGATTCCAAATCTAGCGTGTACCTGCAGATGAACAATCTCAGGGTCGAAGATACAGGCATCTACTATTGCACAGGGGCATCCTATGGTATGGAGTATCTCGGTCAGGGGACAAGCGTCACAGTCAGTTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAG CCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGCCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAACCACAGGAACCGTTTCTCTGTTGTTAAACGGGGCAGAAAGAAACTCCTGTATATATTCAA ACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGG ACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA (Sequence number: 1).

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

[0086] MALPVTALLLPLALLLHAARPDVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRVSGVPDRFSGSGSGTDFTLKINRVEAEDLGVYFCSQSTHVPWTFGGGTK LEIKSSADDAKKDAAKKDDAKKDDAKKDGGVKLDETGGGLVQPGGAMKLSCVTSGFTFGHYWMNWVRQSPEKGLEWVAQFRNKPYNYETYYSDSVKGRFTISRDDSKSSVYLQMNNLRVEDTGIYYCTG ASYGMEYLGQGTSVTVSFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 2)

[0087] Exemplary inserts may include:

[0088] GCCACCATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGATGTCGTGATGACCCAGACCCCCCTCAGCCTCCCAGTGTCCCTCGGTGACCAGGCTTCTATTAGTTGCAGATCCAGCCAGTCCCTCGTGCACTCTAACGGTAATACCTACCTGAGATGGTATCTCCAGAAGCCCGGACAGAGCCCTAAGGTGCTGATCTACAAAGTCTCCAACCGGGTGTCTGGAGTCCCTGACCGCTTCTCAGGGAGCGGTTCCGGCACCGACTTCACCCTGAAGATCAACCGGGTGGAGGCCGAAGACCTCGGCGTCTATTTCTGCTCTCAGAGTACACATGTGCCCTGGACCTTCGGCGGAGGGACCAAGCTGGAGATCAAAAGCTCCGCAGACGATGCCAAGAAAGATGCCGCTAAGAAAGACGATGCTAAGAAAGACGATGCAAAGAAAGACGGTGGCGTGAAGCTGGATGAAACCGGAGGAGGTCTCGTCCAGCCAGGAGGAGCCATGAAGCTGAGTTGCGTGACCAGCGGATTCACCTTTGGGCACTACTGGATGAACTGGGTGCGACAGTCCCCAGAGAAGGGGCTCGAATGGGTCGCTCAGTTCAGGAACAAACCCTACAATTATGAGACATACTATTCAGACAGCGTGAAGGGCAGGTTTACTATCAGTA

[0089] GAGACGATTCCAAATCTAGCGTGTACCTGCAGATGAACAATCTCAGGGTCGAAGATACAGGCATCTACTATTGCACAGGGGCATCCTATGGTATGGAGTATCTCGGTCAGGGGACAAGCGTCACAGTCAGTTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAG CCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGCCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAACCACAGGAACCGTTTCTCTGTTGTTAAACGGGGCAGAAAGAAACTCCTGTATATATTCAA ACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGG ACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA (Sequence number: 3)

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

[0091] ATMALPVTALLLPLALLLHAARPDVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRVSGVPDRFSGSGSGTDFTLKINRVEAEDLGVYFCSQSTHVPWTFGGG TKLEIKSSADDAKKDAAKKDDAKKDDAKKDGGVKLDETGGGLVQPGGAMKLSCVTSGFTFGHYWMNWVRQSPEKGLEWVAQFRNKPYNYETYYSDSVKGRFTISRDDSKSSVYLQMNNLRVEDTGIYYCT GASYGMEYLGQGTSVTVSFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 4) EXAMPLES

[0092] Generation of lentivocytes containing a CAR gene for human T cell transduction To prepare lentiviral virus containing anti-fluorescein single chain variable (scFv) CAR, 293TN packaging cell lines were co-transfected with the second generation of a mixture of lentiviral vectors and packaging plasmids encoding anti-fluorescein scFv CAR (Cellecta). 24 and 48 hours after transfection, supernatants containing lentivirus carrying the CAR gene were harvested and viral particles were concentrated by standard polyethylene glycol virus concentration methods (Clontech) for future transduction in human T cells. EXAMPLES

[0093] Isolation of human T cells from human PBMCs T cells were isolated from human peripheral blood mononuclear cells (PBMCs) by Ficoll density gradient centrifugation (GE Healthcare Lifesciences). After washing off the remaining Ficoll solution, T cells were isolated using the EasySep TM Human T Cell Isolation Kit (STEM CELL technologies). 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). T cells were cultured at 1 × 10 6 T cells were cultured at a density of 1000 cells / mL. T cells were split and refed every 2–3 days. EXAMPLES

[0094] Transduction of human T cells Isolated T cells were activated with Dynabeads coupled with anti-CD3 / CD28 antibodies (Life Technologies) for 12-24 h in the presence of human IL-2 (100 IU / mL) and then transduced with lentivirus encoding an anti-fluorescein CAR gene. Cells were harvested after 72 h and expression of CAR on transduced T cells was identified by measuring GFP fluorescent cells using flow cytometry. As shown in Figure 15A, non-transduced T cells do not show GFP expression. As shown in Figure 15B, transduced T cells express GFP. EXAMPLES

[0095] Synthesis of FITC-folate [ka]

[0096] Folate-γ-ethylenediamine was coupled to fluorescein isothiocyanate (FITC) isomer I (Sigma-Aldrich) in anhydrous dimethyl sulfoxide (DMF) in the presence of tetramethylguanidine and diisopropylamine. The crude product was loaded onto an Xterra RP18 preparative HPLC column (Waters) and eluted with 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, reaching 90% A and 10% B in 10 min. Under these conditions, the main peak of FITC-folate typically eluted at 27–50 min. The quality of the FITC-folate fractions was monitored by analytical reversed-phase HPLC with a UV detector. Fractions with purity >98.0% (LCMS) were lyophilized to obtain the final FITC-folate product. EXAMPLES

[0097] Synthesis of FITC-PEG12-Folate [ka]

[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 x 10 mL) and dimethylformamide (DMF, 3 x 10 mL). 9-Fluorenylmethoxycarbonyl (Fmoc) deprotection was performed using 20% ​​piperidine in DMF (3 x 10 mL). A Kaiser test was performed to assess the progress of the reaction. The vessel was then charged with 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-PrOH) (2 x 10 mL), and 100 mL of dimethylformamide (DMF, 3 x 10 mL). 2 NEt) (4 eq.) and benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) (2 eq.). Fmoc deprotection was carried out using 20% ​​piperidine in DMF (3×10 mL). The vessel was then charged with N 10-TFA-Pte-OH (22.5mg), DMF, i-Pr 2 A solution of NEt (4 equiv.) and PyBOP (2 equiv.) was charged. Argon was bubbled through for 2 h 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 h, 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, Fmoc-NH-(PEG) in DMF was added. 12 -COOH (46.3 mg) solution, i-Pr 2 NEt (4 equiv.) and PyBOP (2 equiv.) were added. Argon was bubbled through for 2 h and the resin was washed with DMF (3×3 mL) and i-PrOH (3×3 mL). Fmoc deprotection was carried out with 20% piperidine in DMF (3×10 mL). A Kaiser test was performed to assess the progress of the reaction. The vessel was then filled with DMF and i-PrOH. 2 A solution of FITC (Life Technologies 21.4 mg) in NEt (4 equiv.) was then bubbled with argon for 2 h and the resin was washed with DMF (3 × 3 mL) and i-PrOH (3 × 3 mL). The vessel was then filled with 2% NH 2 NH 2 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 dissolved in Et 2 The crude product was purified using preparative RP-HPLC (mobile phase: A = 10 mM ammonium acetate, pH = 7, = ACN; method: 0% B to 30% B in 30 min at 13 mL / min). Pure fractions were pooled, lyophilized, and purified to obtain FITC-PEG. 12 - I got folic acid. EXAMPLES

[0099] Synthesis of FITC-PEG20-folate [ka]

[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). The vessel was then filled with Fmoc-PEG in DMF. 20 -COOH solution (131 mg, 1.0 eq.), i-Pr 2 NEt (6.0 equiv.) and PyBOP (4.0 equiv.) were charged. Argon was bubbled for 6 h, 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. Before each amino acid coupling, Fmoc deprotection was performed with 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 equiv.) and Tfa.pteroic acid (41 mg, 1.2 equiv.). The resin was washed (5 min) with 2% hydrazine in DMF 3 × 10 mL to cleave the trifluoro-acetyl protecting group of pteroic acid, followed by washing with i-PrOH (3 × 10 mL) followed by DMF (3 × 10 mL). The resin was dried under argon for 30 min. The folate-peptide was cleaved from the resin using cleavage solution. 10 mL of cleavage mixture was charged and argon was bubbled through for 1.5 hours. The cleavage mixture was drained into a clean flask. The resin was washed three times with additional cleavage mixture. The combined mixture was concentrated under reduced pressure to a smaller volume (~5 mL) and precipitated in ethyl ether.

[0101] The precipitate was collected by centrifugation, washed with ethyl ether (3 times), and dried under high vacuum. 20-EDA (1.0 equiv.) was treated with FITC (50 mg, 1.5 equiv.) in DMSO and DIPEA at room temperature. The progress of the reaction was monitored by LCMS. After 8 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, 35 min at 13 mL / min) to give the FITC-PEG. 20 -Folic acid was obtained in 60% yield. EXAMPLES

[0102] Synthesis of FITC-PEG108-Folate [ka]

[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). The vessel was then filled with Fmoc-PEG in DMF. 36 -COOH solution (161 mg, 1.0 eq.), i-Pr 2 NEt (6.0 equiv.) and PyBOP (4.0 equiv.) were charged. Argon was bubbled for 6 h, 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 assess the progress of the reaction. Before each amino acid coupling, Fmoc deprotection was performed with 20% piperidine in DMF (3×10 mL). The above procedure was repeated to obtain 2X Fmoc-PEG. 36The reaction was completed with the coupling step of -COOH (161 mg, 1.0 equiv.), Fmoc-Glu-OtBu (72 mg, 2.0 equiv.) and Tfa.pteroic acid (41.0 mg, 1.2 equiv.). The resin was washed with 2% hydrazine in DMF 3x10 mL (5 min) to cleave the trifluoro-acetyl protecting group on pteroic acid, followed by washing with i-PrOH (3x10 mL) followed by DMF (3x10 mL). Finally, the resin was washed with 2% hydrazine in DMF 3x10 mL (5 min) to cleave the trifluoroacetyl protecting group on pteroic acid, followed by washing with i-PrOH (3x10 mL) followed by DMF (3x10 mL). The resin was dried under argon for 30 min. The folate-peptide was cleaved from the resin using cleavage solution. 10 mL of cleavage mixture was charged and argon was bubbled through for 1.5 h. The cleavage mixture was drained into a clean flask. The resin was washed three times with additional cleavage mixture. The combined mixture was concentrated under reduced pressure to a smaller volume (~5 mL) and precipitated in ethyl ether.

[0104] The precipitate was collected by centrifugation, washed with ethyl ether (3 times), and dried under high vacuum. 108 -EDA (1.0 equiv.) was treated with FITC (50 mg, 1.5 equiv.) 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, 35 min at 13 mL / min) to give FITC-PEG-2-(2-methyl-2-propanediol). 108 -Folic acid was obtained in 64% yield. EXAMPLES

[0105] Synthesis of FITC-DUPA [ka]

[0106] DUPA-FITC was synthesized by the solid phase method as follows: Universal Nova Tag™ resin (50 mg, 0.53 mM) was swollen with DCM (3 mL) followed by 3 mL of DMF. A solution of 20% piperidine in DMF (3×3 mL) was added to the resin and argon was bubbled through for 5 min. The resin was washed with DMF (3×3 mL) and isopropyl alcohol (i-PrOH, 3×3 mL). Argon was bubbled through for 2 h and the resin was washed with DMF (3×3 mL) and i-PrOH (3×3 mL). After the resin was swollen in DCM, a solution of 1 M HOBt in DCM / TFE (1:1) (2×3 mL) was added. Argon was bubbled through for 1 h, 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-Phe-OH (2.5 equiv.), HATU (2.5 equiv.) and DIPEA (4.0 equiv.) in DMF was added. Argon was bubbled for 2 h and the resin was washed with DMF (3 × 3 mL) and i-PrOH (3 × 3 mL). The above procedure was repeated for two further 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 the 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 min, 80% B wash 30 min; A = 10 mM NH 4 The crude product was purified using 500 mL of 1000 mM NaCl, pH = 7; B = acetonitrile (ACN). The ACN was removed under vacuum and the purified fraction was lyophilized to give FITC-DUPA as a brownish orange solid. RP-HPLC: tR = 8.0 min (A = 10 mM NH 4 OAc, pH = 7.0; B = ACN, solvent gradient: 1% B to 50% B in 10 min, wash with 80% B, 15 min run). 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. EXAMPLES

[0107] Synthesis of FITC-PEG12-DUPA [ka]

[0108] 1,2-Diaminoethanetrityl resin (0.025 g) was placed in a peptide synthesis vessel and washed with i-PrOH (3 x 10 mL) followed by DMF (3 x 10 mL). Fmoc-NH-(PEG) in DMF was then added. 12 -COOH (42.8 mg), i-Pr 2A solution of NEt (2.5 equiv.) and PyBOP (2.5 equiv.) was introduced into the vessel. The resulting solution was bubbled with Ar for 1 h, 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. Fmoc deprotection was performed with 20% piperidine in DMF (3×10 mL). This procedure was repeated to complete all coupling steps (2×1.5 equiv. of Fmoc-Phe-OH and 1.5 equiv. of 8-aminooctanoic acid and 1.2 equiv. of DUPA were used in each of those coupling steps). After DUPA coupling, the resin was washed with DMF (3×10 mL) and i-PrOH (3×10 mL) and dried under reduced pressure. The peptide was cleaved from the resin in the peptide synthesis vessel using cleavage solution. 15 mL of cleavage solution was added to the peptide synthesis vessel, and the reaction was bubbled under Ar for 15 min. The resin was treated with two more 10 mL portions of cleavage solution for 5 min 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) in dimethyl sulfoxide (DMSO, 1 mL) 12 A stirred solution of -EDA (10 mg) and FITC (5.6 mg) was added at room temperature to i-Pr 2 NEt (5 equiv.) was added and stirred under argon for 6 h. 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 min at 13 mL / min). The purified fractions were pooled and lyophilized to obtain FITC-PEG. 12 The purified fractions were pooled and lyophilized to give FITC-PEG-DUPA. 12 -DUPA was obtained. EXAMPLES

[0109] Synthesis of FITC-PEG11-NK1 [ka]

[0110] Anhydrous CH 2 Cl 2 O-(2-aminoethyl)-O'-[2-(Boc-amino)ethyl]decaethylene glycol (BocNH-PEG 11 -NH 2 To a solution of NK-1 (0.02 g, 0.0433 mmol, 1.0 equiv) (Sigma, 0.0336 g, 0.0521 mmol, 1.2 equiv), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) (0.027 g, 0.0521 mmol, 1.2 equiv) under argon at room temperature was added N,N-diisopropylethylamine (DIPEA) (0.076 mL, 0.4338 mmol, 10 equiv). 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 min, 13 mL / min, λ=220 nm, 254 nm). Pure fractions were collected, all organic solvents were evaporated and the sample was lyophilized for 48 h to give NK1-PEG. 11 -NHBoc. Yield: 40.13 mg (97%). NK1-PEG in anhydrous DCM 11 To —NHBoc (0.0165 g, 0.015 mmol) was added trifluoroacetic acid (TFA, 20 equiv.) and the reaction mixture was stirred at room temperature for 4 h. Excess TFA was removed and the remaining solution was diluted with water and added to CH 2 Cl 2 (3×5 mL). The combined organic layers were washed with brine and dried (Na 2 SO 4 The resulting residue was dried under vacuum and used in the next step without further purification. 11 -NH 2A solution of FITC-PEG (0.008 g, 0.0081 mmol, 1.0 equiv.) and fluorescein isothiocyanate (FITC) (Sigma, 0.0037 g, 0.0097 mmol, 1.2 equiv.) was added to diisopropylethylamine (0.0028 mL, 0.0162 mmol, 2.0 equiv.) at room temperature under argon. 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 min, 13 mL / min, λ=280 nm). Pure fractions were collected, all organic solvents were evaporated and the sample was lyophilized for 48 h to obtain FITC-PEG. 11 -NK1 was obtained in a yield of 8.54 mg (77%).

[0111] *Note: NK-1 compounds were 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 COMPLEX, Application No: PCT / US2015 / 44229; incorporated herein by reference). EXAMPLES

[0112] Synthesis of FITC-PEG2-CA9 [ka]

[0113] CA9 ligand (53.6 mg) was dissolved in DMF (2-3 mL) in a 50 mL round bottom flask using a Teflon magnetic stir bar. The surrounding air was removed using vacuum and replaced with nitrogen gas for three cycles. The round bottom flask was kept under constant nitrogen gas. To this flask was added 28.9 mg of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), 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 was stirred overnight. The reaction mixture was purified using HPLC and confirmed by UHPLC-MS (target m / z of 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 min. The TFA / DCM was removed using high vacuum rotary evaporation followed by high vacuum for 30 min. The compound was then dissolved in DMF and 5 molar equivalents of i-Pr 2 NEt was mixed with 16 mg of fluorescein isothiocyanate (Life Technologies) and stirred for 1 h. 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. EXAMPLES

[0114] Assessment of anti-FITC scFv expression in transduced CAR-T cells To confirm whether functional anti-FITC scFv is expressed on the cell surface of transduced CAR T cells, CAR T cells were incubated with FITC-folate conjugates for 30 minutes on ice. After washing the cells, anti-folate antibody (Santa Cruz) was added and incubated on ice for 30 minutes. Finally, Alexa Fluor 647-labeled secondary antibody (Jackson ImmunoResearch) was used to visualize CAR T cell-expressing functional anti-FITC scFv. As shown in Figure 16A, the transduced CAR T cells show GFP expression. As shown in Figure 16B, these transduced CAR-T cells can bind to FITC-folate conjugates visualized by immunofluorescence labeling. EXAMPLES

[0115] In vitro CAR T cell cytotoxicity assay 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, 10 4 Cells were seeded into each well of a 96-well plate at a density of 100 μL and allowed to grow overnight. The next day, CAR T cells were prepared in various numbers to have different effector (CAR T cells): target cell (cancer cell) ratios (e.g., E:T = 20:1, 10:1, 5:1 and 1:1). Various concentrations of FITC-ligand were placed into each well and co-cultured for 6 to 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. Then, 50 μL of the supernatant of each sample was transferred to a new 96-well plate. To the transferred 50 μL of each sample, 50 μL of the prepared LDH reaction mixture was added and incubated for 30 minutes at room temperature. 50 μL of stop solution was added and the absorbance of each sample was measured at 490 nm and 680 nm. The percent cytotoxicity was calculated for each sample using the following formula: % Cytotoxicity = (experimental value - effector cells (spontaneous) - target cells (spontaneous)) / (target cells (maximum control) - target cells (spontaneous control)) x 100

[0116] As shown in Figures 3A-C, CAR T cells are activated only in combination with the conjugate that matches against the tumor antigen. Figure 3A shows the cytotoxicity of CAR T cells in the KB (FR+) model with 100 nM of each conjugate at E:T of 10:1. FITC-folate, FITC-PEG20-folate, and FITC-PEG108-folate activation is greater than activation with FITC-DUPA or in the absence of conjugate. Figure 3B shows the cytotoxicity of CAR T cells in the LNCaP (PSMA+) model with 100 nM of FITC-DUPA or FITC-PEG12-DUPA at E:T of 10:1. FITC-DUPA conjugates show greater activation than with FITC-folate or in the absence of conjugate. Figure 3C shows the cytotoxicity of CAR T cells in the HEK293 (NK1R+) model with 100 nM of FITC-PEG11-NK1 at E:T of 10:1. FITC-PEG11-NK1 shows greater activation than with FITC-folate or in the absence of conjugate.

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

[0118] As shown in Figure 10, CAR T 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 with an E:T of 10:1. EXAMPLES

[0119] Identification of CAR T cell proliferation and CAR T cell activation The proliferation of CAR T cells was primarily measured by flow cytometry. First, 10 4 Cancer cells (KB (FR+) or HEK (NK1R+)) were seeded at a density of cells / 100 μL and cultured overnight. The next day, CAR T cells were placed into 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, CAR T cells were stained with anti-human CD3 APC antibody (Biolegend) by standard immunostaining procedures (20 min on ice). Cells positive for both anti-human CD3 staining and GFP were counted for CAR T cell proliferation.

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

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

[0122] IFN-γ production assay To test for IFN-γ production by CAR T cells, a standard ELISA assay was performed using a human IFN-γ detection ELISA kit from Biolegend. Briefly, cancer cells were plated in each well of a 96-well plate at 10 4 Cells were seeded at a density of 100 μL of cells / media and allowed to grow 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 harvested and centrifuged at 1000 × g and 4 °C for 10 minutes to remove cellular debris. The clear supernatant from each sample was then used to detect IFN-γ by ELISA or stored at -80 °C for future use. After preparation of each sample was completed, a standard ELISA was performed based on the manufacturer's instructions.

[0123] As shown in Figures 2A-2C, with an E:T ratio of 5:1 in KB cells (FR+) (Figure 2A), LNCaP (PSMA+) (Figure 2B), or HEK (NK1R+) (Figure 2C), CAR T cells produce significant amounts of inflammatory cytokines in the presence of 100 nM FITC-small molecule conjugates. As further shown, the amount of cytokines depends on the conjugate used. As shown in Figure 2D, the amount of inflammatory cytokines is related to the concentration of FITC-folate used in KB (FR+) cells. As shown in Figures 2E and 2F, different conjugates (100 nM) generate different IFN-γ responses in KB (FR+) cells.

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

[0125] Assessment of correlation between tumor antigen levels and CAR-T activation KB(FR+) cells and MDA-MB-231 cells were incubated with 100 nM FITC-folate for 30 min on ice. After washing, FITC-folate binding 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 in the presence of 100 nM FITC-folate, FITC-PEG20-folate and FITC-PEG108-folate at an E:T ratio of 10:1 for 24 hours. CAR T cell activation was detected by measuring IFN-γ production. The supernatants of cultured cells were harvested and IFN-γ production was measured using an ELISA kit. As shown in Figure 4B, with higher FRα levels, KB cells (FR+) cells activated CAR T cells much better than MDA-MB-231 cells. EXAMPLES

[0127] Antitumor effects of CAR T cells in vivo To identify the efficacy of CAR T cell antitumor 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. Approximately 50-100 mm 3 When the tumor volume reached 100 μg / kg, tumor-bearing mice were transfected with CAR T cells and the desired FITC-ligand was transfected every other day (iv). Control mice received PBS instead of FITC-ligand. Tumor volume and cytokine levels in blood (IL2, IL6, IL10, IFNγ, and TNFα) 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. Mouse organs were also evaluated.

[0128] A xenograft model using HEK293 (NK1R+) cells is shown in Figures 5A-5C and 6A-6B. As shown in Figure 5A, the tumor size was reduced in mice treated with FITC-PEG11-NK1 (500 nmol / kg), but continued to grow in control mice. As shown in Figure 5B, the weight of mice treated with FITC-PEG11-NK1 was unchanged relative to control mice. As shown in Figure 5C, the proportion of CAR T cells in human T cells increased after CAR T cell infusion. As shown in Figures 6A-6B, harvested organs from the treatment group (6B) appeared at normal size without signs of cytokine release syndrome after 2 weeks of treatment compared to mouse organs harvested from the control group (6A).

[0129] Xenograft models using MDA-MB-231(FR+) cells are shown in Figures 7A-7C and 8A-8B. As shown in Figure 7A, the size of tumors was reduced in mice treated with FITC-PEG12-Folate (500 nmol / kg) or FITC-Folate (500 nmoles / kg), while tumors continued to grow in control mice. As shown in Figure 7B, the weight of mice treated with FITC-PEG11-NK1 did not change during treatment. As shown in Figure 7C, the proportion of CAR T cells in human T cells increased after CAR T cell and conjugate treatment. As shown in Figures 8A-B, organs taken from mice treated with FITC-PEG12-Folate (8B) appeared normal in size, and no signs of cytokine release syndrome were observed after 3 weeks of treatment, compared to mouse organs taken 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 Figure 9, blood indicators of the FITC-PEG11-NK1 (500 nmoles / kg) HEK-NK1R model and the MDA-MB-231 model with FITC-PEG12-Folic Acid (500 nmoles / kg) also indicated that cytokine storm did not occur during the course of treatment.

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

[0132] Antitumor effects of CAR T cells in vivo To investigate whether the same anti-FITC CAR T cells could eradicate a heterogeneous mixture of cancer cells and a cocktail of FITC-ligands, 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 implanted into separate flanks of the same mouse. Anti-FITC CAR T cells (10 7 cells) in tumors with a volume of approximately 50 to 100 mm 3 The tumor cells were introduced by intravenous injection when the tumor volume reached 100 μg / kg. In addition, a mixture of FITC-ligands (i.e., FITC-PEG12-folate (500 nmole / kg) and FITC-PEG11-NK1R (500 nmole / kg)), single FITC-PEG11-NK-1R (500 nmole / kg) or PBS was administered by intravenous injection every other day. The antitumor effect of FITC-ligand-induced CAR T cells was analyzed by measuring the tumor volume every other day.

[0133] As shown in Figures 19A-19C, when both FITC-PEG11-NK1R and FITC-PEG12-Folate 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 FITC-PEG11-NK1R only, only the HEK (NK1R) tumor was eradicated. Without being bound by theory, it is believed that MDA-MB-231 continued to grow in the same mice that received only FITC-PEG11-NK1R because FITC-PEG12-Folate was not administered. As expected, both tumors did not show any response 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. A composition for treating cancer, comprising a first conjugate or a pharma- ceutically acceptable salt thereof, The first conjugate comprises an NK-1 receptor binding ligand (NK1RL) linked to fluorescein isothiocyanate (FITC) by a linker; the first complex is specific to a cancer cell expressing an NK-1 receptor; The first complex specifically binds to a chimeric antigen receptor (CAR) directed to FITC; NK1RL has the following formula: 【Chemistry 1】 Including, composition.

2. the composition comprises a second conjugate or a pharma- ceutically acceptable salt thereof; the second complex is different from the first complex; the second complex is specific to a cancer cell; The second complex comprises a ligand linked to FITC by a linker; The second complex specifically binds to the FITC-directed CAR. The composition of claim 1.

3. The composition of claim 2, wherein the ligand of the second complex is selected from folic acid, DUPA, a CCK2R ligand, a CAIX or CA9 ligand, and a ligand of gamma glutamyl transpeptidase.

4. 4. The composition of any of claims 1 to 3, wherein the ligand of the first conjugate and / or the second conjugate comprises polyethylene glycol (PEG), polyproline, a hydrophilic amino acid, a sugar, a non-natural peptidoglycan, polyvinylpyrrolidone, and / or Pluronic® F-127.

5. The first conjugate and / or the second conjugate, or a pharma- ceutically acceptable salt thereof, has the formula: 【Chemistry 2】 wherein B represents a ligand, L represents a linker, and T represents FITC, wherein L has the formula: 【Chemistry 3】 (wherein n is an integer from 0 to 200). [including the structure represented by The composition of any one of claims 1 to 4, having a structure represented by:

6. 6. The composition of claim 5, wherein n is an integer from 0 to 150, 0 to 110, 15 to 110, 0 to 20, or 15 to 20.

7. 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, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, non-small cell lung cancer, and adrenal gland cancer.

7. The composition of claim 1, wherein the cancer is selected from the group consisting of cancer of the bladder, soft tissue sarcoma, 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, and adenocarcinoma of the gastroesophageal junction.

8. The composition according to any one of claims 1 to 7, wherein the cancer comprises a cancer expressing an NK-1 receptor and a cancer expressing a folate receptor.

9. The composition according to any one of claims 1 to 8, wherein the CAR has a recognition region, and the recognition region comprises a single variable region fragment (scFv) of an anti-FITC antibody.

10. The composition of any one of claims 1 to 9, wherein the CAR has a costimulatory domain, and the costimulatory domain is selected from CD28, CD137 (4-1BB), CD134 (OX40), and CD278 (ICOS).

11. The composition according to any one of claims 1 to 10, wherein the CAR has an activation signaling domain, and the activation signaling domain is T cell CD3 ζ chain or Fc receptor γ.

12. The composition according to any one of claims 1 to 11, wherein the CAR has a recognition region, the recognition region being an scFv region 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.

13. The composition of any of claims 1 to 12, wherein the CAR comprises a polypeptide having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or at least about 100% sequence identity to SEQ ID NO:

2.

14. The composition according to any one of claims 1 to 13, wherein the first conjugate and / or the second conjugate, or a pharma- ceutically acceptable salt thereof, is not an antibody and does not include an antibody fragment.

15. The composition of any one of claims 1 to 14, wherein CAR T cell toxicity to cancer occurs when the composition is administered to a patient having CAR T cells directed against FITC.

16. The composition of claim 15, which does not cause cytokine release syndrome and / or "off-target" toxicity in the patient.

17. 17. The composition of claim 15 or 16, wherein the cancer comprises a tumor and the tumor size in the patient is reduced.

18. 18. The composition of any of claims 15 to 17, wherein the first conjugate and / or the second conjugate, or a pharma- ceutically acceptable salt thereof, is administered to the patient at a dose of about 50 nmol / kg to about 2,000 nmol / kg, about 100 nmol / kg to about 1,000 nmol / kg, about 100 nmol / kg to about 600 nmol / kg, about 200 nmol / kg to about 500 nmol / kg, or about 250 nmol / kg to about 500 nmol / kg of the patient's body weight.

19. The composition of any of claims 15 to 18, wherein multiple doses of the first conjugate and / or the second conjugate, or a pharma- ceutically acceptable salt thereof, are administered to the patient.

20. The composition of any of claims 15-19, wherein the CAR T cell comprises a polypeptide having at least about 90%, at least about 95%, at least about 98%, or at least about 100% sequence identity to SEQ ID NO:

2.

21. The composition of any of claims 15-20, wherein the CAR T cell comprises a nucleic acid having at least about 90%, at least about 95%, at least about 98%, or at least about 100% sequence identity to SEQ ID NO:1 or SEQ ID NO:

3.

22. The composition of any of claims 15-21, wherein the CAR T cell comprises a vector comprising a nucleic acid having at least about 90%, at least about 95%, at least about 98%, or at least about 100% sequence identity to SEQ ID NO:1 or SEQ ID NO:

3.

23. The composition of claim 22 , wherein the vector is a lentiviral vector.

24. The first conjugate has the formula: 【Chemistry 4】 The composition according to any one of claims 1 to 23,

25. The second complex has the formula: 【Chemistry 5】 【Chemistry 6】 【Chemistry 7】 【Chemistry 8】 【Chemistry 9】 【Chemistry 10】 or 【Chemistry 11】 The composition according to any one of claims 2 to 24, selected from

26. A pharmaceutical composition comprising the composition of any one of claims 1 to 25 and a pharma- ceutically acceptable carrier.

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