Sequencing method for car t cell therapy

Abstract

To provide CAR T cell therapeutic protocols that offer reduced off-target toxicity and more precise control of CAR T cell activation.SOLUTION: The present disclosure relates to methods of treating a patient with a cancer by administering to the patient a composition comprising CAR T cells and also administering to the patient a small molecule linked to a targeting moiety through a linker.SELECTED DRAWING: Figure 4

Description

[Technical field]

[0001] Cross-references to related applications This application claims priority to U.S. Provisional Application No. 62 / 634,573, filed February 23, 2018, U.S. Provisional Application No. 62 / 656,265, filed April 11, 2018, U.S. Provisional Application No. 62 / 724,345, filed August 29, 2018, and U.S. Provisional Application No. 62 / 736,730, filed September 26, 2018, the disclosures of all of which are expressly incorporated herein by reference in their entireties.

[0002] Technical Field The present invention relates to methods of treating a patient with cancer by administering to the patient a composition comprising CAR T cells and administering to the patient a small molecule attached to a targeting moiety by a linker. The present invention also relates to compositions for use in such methods. [Background technology]

[0003] background Immunotherapy based on adoptive transfer of lymphocytes (e.g., T cells) to patients is a valuable treatment in the treatment of cancer and other diseases. Significant progress has been made in the development of immunotherapy based on adoptive transfer of lymphocytes. Among various types of immunotherapeutic agents, one of the most useful immunotherapeutic agents under development is T cells expressing chimeric antigen receptors (CAR T cells). Chimeric antigen receptors (CARs) are genetically engineered receptors designed to target specific antigens, e.g., tumor antigens. This targeting can result in cytotoxicity against tumors, for example, CAR-expressing CAR T cells target and kill tumors via specific tumor antigens.

[0004] First generation CARs consist of a recognition region for recognizing and binding to antigens expressed by tumors, such as an antibody-derived single-chain fragment variable (scFv) region, and an activation signaling domain, for example, the CD3ζ chain of T cells can act as a T cell activation signal in CARs. CAR T cells have shown good results in vitro, but have only limited success in eradicating disease (e.g., cancer) in clinical trials. One problem is that CAR T cell populations cannot be activated and expanded long-term in vivo.

[0005] To address this issue, second generation CARs have been incorporated with 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 indicates that such constructs are promising therapeutic agents for the treatment of diseases such as cancer. Summary of the Invention [Problem to be solved by the invention]

[0006] Although improvements have been made in CAR T cell therapy, several problems remain. First, "off-target" toxicity may occur due to normal cells expressing the antigens (e.g., tumor-associated antigens) targeted by CAR T cells. Second, uncontrolled CAR T cell activation may be seen when triggered by a group of metabolic disorders called tumor lysis syndrome or cytokine release syndrome (CRS), in which rapid and uncontrolled elimination of diseased cells (e.g., cancer cells) by CAR T cells can be fatal to the patient. Tumor lysis syndrome and CRS may be due to the administered CAR T cells being activated uncontrollably, which cannot be easily controlled. Thus, although CAR T cells are very promising as a tool in the treatment of diseases such as cancer, additional CAR T cell therapy protocols are needed that provide reduced off-target toxicity and tighter control of CAR T cell activation. [Means for solving the problem]

[0007] Summary of the Invention In various embodiments described herein, a small molecule ligand attached to a targeting moiety by a linker is used as a bridge between the cancer and the CAR T cell to direct the CAR T cell to the cancer for cancer amelioration. In certain embodiments, the "small molecule ligand" can be, for example, folate, DUPA, an NK-1R ligand, a CAIX ligand, a ligand for gamma glutamyl transpeptidase, an NKG2D ligand, or a CCK2R ligand, each of which is a small molecule ligand that specifically binds to cancer cells (i.e., the receptors for these ligands are overexpressed in cancer compared to normal tissues).

[0008] In some embodiments, 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" 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.

[0009] The "targeting moiety" binds to the recognition region of the genetically modified CAR expressed by the CAR T cell. Thus, the recognition region of the CAR (e.g., single-chain fragment variable region (scFv), Fab, Fv, Fc, (Fab')2 fragment, etc. of an antibody) is directed to the "targeting moiety". Thus, the small molecule ligand linked to the targeting moiety by the linker serves as a bridge between the cancer and the CAR T cell, directing the CAR T cell to the cancer for amelioration of the cancer.

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

[0011] In another embodiment, a method for treating cancer is provided.The method comprises: i) administering at least one dose of CAR T cell composition to a patient, where the CAR T cell comprises a CAR directed to a targeting moiety; ii) administering a compound or its pharmaceutically acceptable salt to a patient, where the compound or its pharmaceutically acceptable salt comprises a small molecule ligand linked to a targeting moiety by a linker, where the compound or its pharmaceutically acceptable salt is administered in a first dose escalation course, and if severe CRS occurs in the first dose escalation course, the compound or its pharmaceutically acceptable salt is administered using a low dose escalation course, where the first dose of the compound or its pharmaceutically acceptable salt in the low dose escalation course is lower than the first dose of the compound or its pharmaceutically acceptable salt administered in the first dose escalation course.In some embodiments, in the low dose escalation course, the compound or its pharmaceutically acceptable salt can be administered in about 0.5%, about 5% and about 50% of the full dose of the compound or its pharmaceutically acceptable salt on three non-consecutive days.

[0012] Further embodiments are also described by the following numbered paragraphs. Any of the following embodiments in combination with any applicable embodiment described in the Summary section, Detailed Description of Illustrative Embodiments section, Examples section, or Claims of this application is also contemplated.

[0013] 1. A method of treating cancer, comprising: i) administering to the patient at least one dose of a CAR T cell composition, where the CAR T cell comprises a CAR directed to a targeting moiety; ii) administering to a patient a compound or a pharma- ceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand attached to a targeting moiety by a linker, wherein the compound or a pharma- ceutically acceptable salt thereof is administered by at least a first dose escalation course and a second dose escalation course.

[0014] 2. The method of claim 1, wherein the compound or a pharma- ceutically acceptable salt thereof is administered in at least a first dose-escalation course, a second dose-escalation course and a third dose-escalation course.

[0015] 3. The method of claim 1, wherein the compound or a pharma- ceutically acceptable salt thereof is administered at least a first dose escalation course, a second dose escalation course, a third dose escalation course and a fourth dose escalation course.

[0016] 4. The method of claim 1, wherein the compound or a pharma- ceutically acceptable salt thereof is administered at least a first dose escalation course, a second dose escalation course, a third dose escalation course, a fourth dose escalation course and a fifth dose escalation course.

[0017] 5. The method of claim 1, wherein the compound or a pharma- ceutically acceptable salt thereof is administered at least a first dose escalation course, a second dose escalation course, a third dose escalation course, a fourth dose escalation course, a fifth dose escalation course and a sixth dose escalation course.

[0018] 6. The method of any of paragraphs 1 to 5, wherein the patient is administered an initial dose of CAR T cells and a second dose of CAR T cells.

[0019] 7. The method of paragraph 6, wherein the initial dose of the CAR T cells is a test dose for monitoring the patient's tolerance to the CAR T cells.

[0020] 8. The method of paragraph 6, wherein the second dose of CAR T cells comprises a higher dose of CAR T cells than the first dose of CAR T cells.

[0021] 9. The initial dose of CAR T cells is approximately 0.5 × 10 per kg of patient body weight. 5 CAR T cells ~ approx. 1.5 x 10 per kg of patient body weight 6 The method according to any one of items 6 to 8, comprising a CAR T cell of the above-mentioned item 6.

[0022] 10. The second dose of CAR T cells is approximately 0.8 × 10 per kg of patient body weight. 6 CAR T cells ~ approximately 2 x 10 per kg of patient body weight 7 10. The method according to any one of items 6 to 9, comprising a CAR T cell of the above item.

[0023] 11. The method of any of items 1 to 10, wherein the first dose escalation course is followed by a period during which the compound or a pharma- ceutically acceptable salt thereof is not administered.

[0024] 12. The method of any of paragraphs 1 to 11, wherein the second dose-escalation course is followed by a period during which the compound or a pharma- ceutically acceptable salt thereof is not administered.

[0025] 13. The method according to any one of paragraphs 2 to 12, wherein the third dose escalation course is followed by a period during which the compound or a pharma- ceutically acceptable salt thereof is not administered.

[0026] 14. The method of any of paragraphs 3 to 13, wherein the fourth dose-escalation course is followed by a period during which the compound or a pharma- ceutically acceptable salt thereof is not administered.

[0027] 15. The method of any of paragraphs 4 to 14, wherein the fifth dose escalation course is followed by a period during which the compound or a pharma- ceutically acceptable salt thereof is not administered.

[0028] 16. The method according to any one of paragraphs 5 to 15, wherein the sixth dose escalation course is followed by a period during which the compound or a pharma- ceutically acceptable salt thereof is not administered.

[0029] 17. The method according to any one of items 11 to 16, wherein the period is about 7 days.

[0030] 18. The method of any of paragraphs 1 to 17, wherein the first dose escalation course comprises administering to the patient about 1%, about 10%, and about 100% of the total dose of the compound or a pharma- ceutically acceptable salt thereof on three non-consecutive days.

[0031] 19. The method of any of paragraphs 1 to 18, wherein the second dose escalation course comprises administering to the patient about 1%, about 30%, and about 300% of the total dose of the compound or a pharma- ceutically acceptable salt thereof on three non-consecutive days.

[0032] 20. The method of any of paragraphs 2 to 19, wherein the third dose escalation course comprises administering to the patient about 1%, about 50%, and about 500% of the total dose of the compound or a pharma- ceutically acceptable salt thereof on three non-consecutive days.

[0033] 21. The method of any of paragraphs 3 to 20, wherein the fourth dose escalation course comprises administering to the patient about 1%, about 10%, and about 100% of the total dose of the compound or a pharma- ceutically acceptable salt thereof on three non-consecutive days.

[0034] 22. The method of any of paragraphs 4 to 21, wherein the fifth dose escalation course comprises administering to the patient about 1%, about 30%, and about 300% of the total dose of the compound or a pharma- ceutically acceptable salt thereof on three non-consecutive days.

[0035] 23. The method of any of paragraphs 5 to 22, wherein the sixth dose escalation course comprises administering to the patient about 1%, about 50%, and about 500% of the total dose of the compound or a pharma- ceutically acceptable salt thereof on three non-consecutive days.

[0036] 24. The method according to any one of items 18 to 23, wherein the total dose of the compound or a pharma- ceutically acceptable salt thereof is about 10 μg / kg to about 50 μg / kg of the compound or a pharma- ceutically acceptable salt thereof.

[0037] 25. The method according to any one of items 18 to 24, wherein the total dose of the compound or a pharma- ceutically acceptable salt thereof is about 20 μg / kg to about 40 μg / kg of the compound or a pharma- ceutically acceptable salt thereof.

[0038] 26. The method according to any one of items 18 to 25, wherein the total dose of the compound or a pharma- ceutically acceptable salt thereof is about 25 μg / kg to about 35 μg / kg of the compound or a pharma- ceutically acceptable salt thereof.

[0039] 27. The method according to any one of items 18 to 26, wherein the total dose of the compound or a pharma- ceutically acceptable salt thereof is about 30 μg / kg of the compound or a pharma- ceutically acceptable salt thereof.

[0040] 28. The method of any of paragraphs 6 to 27, wherein a first dose of CAR T cells and a second dose of CAR T cells are administered to the patient during the first week.

[0041] 29. The method of any of paragraphs 6 to 28, wherein the first dose of CAR T cells and the second dose of CAR T cells are administered to the patient on Monday and Thursday of week 1.

[0042] 30. The method according to any one of paragraphs 1 to 29, wherein the first dose escalation course of the compound or a pharma- ceutically acceptable salt thereof is carried out during weeks 2 and 3.

[0043] 31. The method of any of paragraphs 1 to 30, wherein a second dose escalation course of the compound or a pharma- ceutically acceptable salt thereof is performed between weeks 4 and 5.

[0044] 32. The method according to any one of paragraphs 2 to 31, wherein a third dose escalation course of the compound or a pharma- ceutically acceptable salt thereof is administered between weeks 6 and 7.

[0045] 33. The method of paragraph 30, wherein the compound or a pharma- ceutically acceptable salt thereof is administered on three non-consecutive days, the three non-consecutive days being Monday and Thursday of the second week and Monday of the third week.

[0046] 34. The method of paragraph 31, wherein the compound or a pharma- ceutically acceptable salt thereof is administered on three non-consecutive days, the three non-consecutive days being Monday and Thursday of the fourth week and Monday of the fifth week.

[0047] 35. The method of paragraph 32, wherein the compound or a pharma- ceutically acceptable salt thereof is administered on three non-consecutive days, the three non-consecutive days being Monday and Thursday of the sixth week and Monday of the seventh week.

[0048] 36. The method of any of paragraphs 1 to 35, wherein the patient is lymphocyte-depleted prior to administering the CAR T cell composition to the patient.

[0049] 37. The method of any of paragraphs 1 to 36, further comprising administering platelets to the patient, administering packed red blood cells to the patient, administering cryoprecipitate to the patient, administering intravenous immunoglobulin to the patient, and / or administering antibacterial therapy to the patient.

[0050] 38. The method of any of paragraphs 1 to 37, wherein if CRS or neurotoxicity is not observed in the patient during the first dose escalation course, the method proceeds to a second dose escalation course.

[0051] 39. The method of any of paragraphs 2 to 38, wherein if CRS or neurotoxicity is not observed in the patient during the second dose escalation course, the method proceeds to a third dose escalation course.

[0052] 40. The method of any of paragraphs 3 to 39, wherein if CRS or neurotoxicity is not observed in the patient during the third dose escalation course, the method proceeds to a fourth dose escalation course.

[0053] 41. The method of any of paragraphs 4 to 40, wherein if CRS or neurotoxicity is not observed in the patient during the fourth dose escalation course, the method proceeds to a fifth dose escalation course.

[0054] 42. The method of any of paragraphs 5 to 41, wherein if CRS or neurotoxicity is not observed in the patient during the fifth dose escalation course, the method proceeds to a sixth dose escalation course.

[0055] 43. The method of any of paragraphs 1 to 42, wherein if fever without hypotension is observed in the patient at any point during the dose escalation process, and neurotoxicity is not observed in the patient, then all subsequent doses of the compound or a pharma- ceutically acceptable salt thereof are administered at the dose escalation level that caused fever without hypotension in the patient.

[0056] 44. The method of any of paragraphs 1 to 43, wherein if the patient experiences severe CRS or neurotoxicity during any dose escalation, all subsequent doses of the compound or a pharma- ceutically acceptable salt thereof are administered to the patient at a dose escalation level that is lower than the dose escalation level that caused the patient to experience severe CRS or neurotoxicity.

[0057] 45. A method of treating cancer, comprising: i) administering to the patient at least one dose of a CAR T cell composition, where the CAR T cell comprises a CAR directed to a targeting moiety; ii) administering to the patient a compound or a pharma- ceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand attached to a targeting moiety by a linker, wherein the compound or a pharma- ceutically acceptable salt thereof is administered during a first dose-escalation course, and if severe CRS occurs during the first dose-escalation course, administering the compound or a pharma- ceutically acceptable salt thereof using a low-dose escalation course, wherein the initial dose of the compound or a pharma- ceutically acceptable salt thereof in the low-dose escalation course is lower than the initial dose of the compound or a pharma- ceutically acceptable salt thereof administered during the first dose-escalation course.

[0058] 46. ​​The method of claim 45, wherein in a low-dose escalation course, the compound or a pharma- ceutically acceptable salt thereof is administered at about 0.5%, about 5%, and about 50% of the total dose of the compound or a pharma- ceutically acceptable salt thereof on three non-consecutive days.

[0059] 47. The method according to item 46, wherein the total dose of the compound or a pharma- ceutically acceptable salt thereof is about 10 μg / kg to about 50 μg / kg of the compound or a pharma- ceutically acceptable salt thereof.

[0060] 48. The method according to any one of items 46 to 47, wherein the total dose of the compound or a pharma- ceutically acceptable salt thereof is about 25 μg / kg to about 35 μg / kg of the compound or a pharma- ceutically acceptable salt thereof.

[0061] 49. The method according to any one of items 46 to 48, wherein the total dose of the compound or a pharma- ceutically acceptable salt thereof is about 30 μg / kg of the compound or a pharma- ceutically acceptable salt thereof.

[0062] 50. The method according to any one of items 1 to 49, wherein the ligand is selected from the group consisting of folate, DUPA, NK-1R ligands, CAIX ligands, ligands for gamma glutamyl transpeptidase, NKG2D ligands and CCK2R ligands.

[0063] 51. The method according to any one of items 1 to 50, wherein the ligand is folate.

[0064] 52. The method according to any one of items 1 to 50, wherein the ligand is an NK-1R ligand.

[0065] 53. The method according to any one of items 1 to 50, wherein the ligand is DUPA.

[0066] 54. The method according to any one of items 1 to 50, wherein the ligand is a CCK2R ligand.

[0067] 55. The method according to any one of items 1 to 50, wherein the ligand is a ligand for gamma glutamyl transpeptidase.

[0068] 56. The method of any of paragraphs 1 to 55, wherein the targeting moiety is selected from the group consisting of 2,4-dinitrophenol (DNP), 2,4,6-trinitrophenol (TNP), biotin, digoxigenin, fluorescein, fluorescein isothiocyanate (FITC), NHS-fluorescein, pentafluorophenyl ester, tetrafluorophenyl ester, knottin, centrin, and DARPin.

[0069] 57. The method of any of paragraphs 1 to 56, wherein the targeting moiety is FITC.

[0070] 58. The method of any of paragraphs 1 to 56, wherein the targeting moiety is DNP.

[0071] 59. The method of any of paragraphs 1 to 56, wherein the targeting moiety is TNP.

[0072] 60. The method of any one of items 1 to 59, wherein the linker comprises polyethylene glycol (PEG), polyproline, a hydrophilic amino acid, a sugar, a non-natural peptidoglycan, polyvinylpyrrolidone, Pluronic F-127, or a combination thereof.

[0073] 61. The method of any one of items 1 to 60, wherein the linker comprises PEG.

[0074] 62. A compound or a pharma- ceutically acceptable salt thereof having the formula [ka] wherein B is a small molecule ligand, L is a linker, and T is a targeting moiety, wherein L is of the formula [ka] (wherein n is an integer of 0 to 200). The structure includes the following structure: Item 62. The method according to any one of items 1 to 61, comprising:

[0075] 63. The method according to item 62, wherein n is an integer from 0 to 150.

[0076] 64. The method according to item 62, wherein n is an integer from 0 to 110.

[0077] 65. The method according to item 62, wherein n is an integer from 0 to 20.

[0078] 66. The method according to item 62, wherein n is an integer from 15 to 20.

[0079] 67. The method according to item 62, wherein n is an integer from 15 to 110.

[0080] 68. If your cancer is 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, adrenal cancer, soft tissue sarcoma, osteosarcoma including pediatric or non-pediatric osteosarcoma, urethral cancer, prostate cancer, chronic Item 68. The method according to any one of items 1 to 67, wherein the cancer is selected from the group consisting of leukemia, acute leukemia, acute myeloid leukemia, lymphocytic lymphoma, myeloid leukemia, myelomonocytic leukemia, hairy cell leukemia, pleural mesothelioma, bladder cancer, Burkitt's lymphoma, ureteral cancer, kidney cancer, renal cell carcinoma, renal pelvis cancer, central nervous system (CNS) neoplasms, primary CNS lymphoma, spinal axis tumor, brain stem glioma, pituitary adenoma, and esophagogastric junction adenocarcinoma.

[0081] 69. The method according to any one of items 1 to 51 or 56 to 68, wherein the cancer is a folate receptor-expressing cancer.

[0082] 70. The method according to any one of items 1 to 69, wherein the cancer is osteosarcoma.

[0083] 71. The method according to any one of items 1 to 70, wherein the CAR has a recognition region, and the recognition region is a single-chain fragment variable (scFv) region of an antibody.

[0084] 72. The method according to any one of items 1 to 57 or 60 to 71, wherein the CAR has a recognition region, and the recognition region of the CAR is a single-chain fragment variable (scFv) region of an anti-FITC antibody.

[0085] 73. The method of any of paragraphs 1 to 72, wherein the CAR has a costimulatory domain, and the costimulatory domain is selected from the group consisting of CD28, CD137 (4-1BB), CD134 (OX40) and CD278 (ICOS).

[0086] 74. The method of any of paragraphs 1 to 73, wherein the CAR has an activation signaling domain, and the activation signaling domain is T cell CD3 zeta chain or Fc receptor gamma.

[0087] 75. The method according to any one of items 1 to 57 or 60 to 74, wherein the CAR has a recognition region, the recognition region being a single-chain fragment variable (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 the T cell CD3 zeta chain.

[0088] 76. The method of any of paragraphs 1 to 75, wherein the patient is imaged prior to administration of the compound or a pharma- ceutically acceptable salt thereof or prior to administration of the CAR T cell composition.

[0089] 77. The method according to any one of items 1 to 76, wherein the compound or a pharma- ceutically acceptable salt thereof is not an antibody and does not include an antibody fragment.

[0090] 78. The method of any of paragraphs 1 to 77, wherein the targeting moiety does not comprise a peptide epitope.

[0091] 79. The method according to any of items 1 to 78, wherein CAR T cell toxicity against cancer is achieved without causing cytokine release that results in off-target toxicity in patients.

[0092] 80. The method of any of items 1 to 78, wherein off-target tissue toxicity does not occur in the patient and CAR T cell toxicity occurs against the cancer.

[0093] 81. The method of any of paragraphs 1 to 78, wherein the cancer comprises a tumor, the tumor size is reduced in the patient, and no off-target toxicity occurs.

[0094] 82. The method of any of paragraphs 1 to 81, wherein the CAR T cells comprise a nucleic acid comprising SEQ ID NO:1.

[0095] 83. The method of any of paragraphs 1 to 82, wherein the CAR T cells comprise a polypeptide comprising SEQ ID NO:2.

[0096] 84. The method of paragraph 82, wherein the nucleic acid encodes a chimeric antigen receptor.

[0097] 85. The method of any of paragraphs 1 to 81, wherein the CAR T cells comprise a nucleic acid comprising SEQ ID NO:4.

[0098] 86. The method of any of paragraphs 1 to 81 or 85, wherein the CAR T cells comprise a polypeptide comprising SEQ ID NO:5.

[0099] 87. The method of paragraph 85, wherein the nucleic acid encodes a chimeric antigen receptor.

[0100] 88. The method of any of paragraphs 1 to 87, wherein the CAR comprises a humanized amino acid sequence.

[0101] 89. The method of any of paragraphs 1 to 87, wherein the CAR consists of a humanized amino acid sequence.

[0102] 90. The method of any of paragraphs 1 to 89, further comprising administering to the patient a folate, a conjugate comprising a folate (wherein the conjugate comprising a folate does not comprise a targeting moiety), or an agent that inhibits activation of CAR T cells.

[0103] 91. The method of claim 90, wherein folate is administered.

[0104] 92. The method of paragraph 90, wherein folic acid or leucovorin is administered.

[0105] 93. The method of claim 90, wherein a conjugate containing folate is administered.

[0106] 94. The method of claim 93, wherein the folate-containing conjugate comprises folate bound to one or more amino acids.

[0107] 95. A conjugate containing folate has the formula [ka] 94. The method of claim 93, comprising the steps of:

[0108] 96. Folate is the formula [ka] [In the formula, X 1 and Y 1 each independently represents halo, R 2 , OR 2 , S.R. 3 and N.R. 4 R 5 selected from the group consisting of; U, V and W are each independently -(R 6a )C=, -N=, -(R 6a )C(R 7a )- and -N(R 4a )-; Q is selected from the group consisting of C and CH; T is selected from the group consisting of S, O, N, and -C=C-; X 2 and X 3 are each independently oxygen, sulfur, -C(Z)-, -C(Z)O-, -OC(Z)-, -N(R 4b )-, -C(Z)N(R 4b )-, -N(R 4b )C(Z)-, -OC(Z)N(R4b )-, -N(R 4b )C(Z)O-, -N(R 4b )C(Z)N(R 5b )-, -S(O)-, -S(O)2-, -N(R 4a )S(O)2-, -C(R 6b )(R 7b )-, -N(C≡CH)-, -N(CH2C≡CH)-, C1-C 12 Alkylene and C1-C 12 alkyleneoxy, where X is oxygen or sulfur; R 1 are hydrogen, halo, C1-C 12 Alkyl and C1-C 12 alkoxy; R 2 , R 3 , R 4 , R 4a , R 4b , R 5 , R 5b , R 6b and R 7b each independently represents hydrogen, halo, C1-C 12 Alkyl, C1-C 12 Alkoxy, C1-C 12 Alkanoyl, C1-C 12 Alkenyl, C1-C 12 Alkynyl, (C1-C 12 Alkoxy)carbonyl and (C1-C 12 alkylamino)carbonyl; R 6 and R 7 each independently represents hydrogen, halo, C1-C 12 Alkyl and C1-C 12 alkoxy; or R 6 and R 7 together to form a carbonyl group; R 6a and R 7a each independently represents hydrogen, halo, C1-C 12 Alkyl and C1-C 12 alkoxy; or R 6aand R 7a together to form a carbonyl group; p, r, s and t are each independently 0 or 1; and * is an optional covalent bond to the remainder of the conjugate if any additional chemical moieties are part of the folate. Item 92. The method of item 91, comprising the steps of:

[0109] 97. The method according to paragraph 90, wherein the agent that inhibits the activation of CAR T cells is selected from the group consisting of lymphocyte-specific protein tyrosine kinase inhibitors, PI3 kinase inhibitors, IL-2-inducible T cell kinase inhibitors, JAK inhibitors, BTK inhibitors, EC2319, and agents that block binding of CAR T cells to the compound or a pharma- ceutically acceptable salt thereof but do not bind to the cancer.

[0110] 98. The method of claim 90, wherein an agent that inhibits the activation of CAR T cells is administered, and the agent is a lymphocyte-specific protein tyrosine kinase inhibitor.

[0111] 99. The method according to paragraph 98, wherein the lymphocyte-specific protein tyrosine kinase inhibitor is dasatinib.

[0112] 100. The method of claim 90, wherein an agent that inhibits the activation of CAR T cells is administered, and the agent is a PI3 kinase inhibitor.

[0113] 101. The method according to paragraph 100, wherein the PI3 kinase inhibitor is GDC0980.

[0114] 102. The method of claim 90, wherein an agent that inhibits the activation of CAR T cells is administered, and the agent is an IL-2-inducible T cell kinase inhibitor.

[0115] 103. The method of claim 102, wherein the IL-2-inducible T cell kinase inhibitor is BMS-509744.

[0116] 104. The method of claim 90, wherein an agent that inhibits activation of CAR T cells is administered, the agent blocking binding of CAR T cells to the compound or a pharma- ceutically acceptable salt thereof but not to the cancer.

[0117] 105. The method of paragraph 104, wherein the agent is fluoresceinamine, FITC or sodium fluorescein.

[0118] 106. The method of claim 105, wherein the agent is sodium fluorescein.

[0119] 107. The method of any of paragraphs 90 to 106, wherein administration of folate, a conjugate comprising folate (wherein the conjugate comprising folate does not comprise a targeting moiety), or an agent that inhibits activation of CAR T cells results in a decrease in cytokine levels in the patient.

[0120] 108. The method of paragraph 107, wherein the reduction in cytokine levels is approximately to the cytokine levels of an untreated patient.

[0121] 109. The method of any of paragraphs 90 to 108, wherein the cancer comprises a tumor and the size of the tumor in the patient does not increase when the patient is administered folate, a conjugate comprising folate (wherein the conjugate comprising folate does not comprise a targeting moiety), or an agent that inhibits activation of CAR T cells.

[0122] 110. The method according to any of paragraphs 104 to 106, wherein the patient is administered a drug that inhibits CAR T cell activation when CRS grade reaches 1, 2, 3 or 4.

[0123] 111. The method of claim 110, wherein the patient is administered an agent that inhibits CAR T cell activation when CRS grade reaches 3 or 4.

[0124] 112. The method of any of paragraphs 104 to 106, wherein the agent that inhibits CAR T cell activation is administered at a dose of about .01 to about 300 μmole / kg of patient body weight.

[0125] 113. The method of any of paragraphs 1 to 112, wherein CRS is reduced or prevented in the patient and the method results in a reduction in tumor volume in the patient.

[0126] 114. A method according to any one of items 1 to 113, in which weight loss due to CRS is reduced or prevented.

[0127] 115. The method of any of paragraphs 90 to 114, further comprising re-administering the compound or a pharma- ceutically acceptable salt thereof to the patient.

[0128] 116. The method of paragraph 115, wherein subsequent administration of the compound or a pharma- ceutically acceptable salt thereof results in CAR T cell activation and increased cytokine levels in the patient.

[0129] 117. The method of any of paragraphs 1 to 116, wherein the CAR T cell composition is administered prior to the compound or a pharma- ceutically acceptable salt thereof.

[0130] 118. The method of any of paragraphs 1 to 117, wherein the CAR T cells are autologous.

[0131] 119. The method of any of paragraphs 2 to 17, wherein a first dose escalation course comprises administering to the patient about 1%, about 2%, and about 20% of the total dose of the compound or pharma- ceutically acceptable salt thereof on three non-consecutive days, a second dose escalation course comprises administering to the patient about 1%, about 6%, and about 60% of the total dose of the compound or pharma-ceutically acceptable salt thereof on three non-consecutive days, and a third dose escalation course comprises administering to the patient about 1%, about 10%, and about 100% of the total dose of the compound or pharma-ceutically acceptable salt thereof on three non-consecutive days.

[0132] 120. The method of any of paragraphs 2 to 17, wherein a first dose escalation course comprises administering to the patient about 1%, about 10%, and about 100% of the total dose of the compound or pharma- ceutically acceptable salt thereof on three non-consecutive days, and a second dose escalation course comprises administering to the patient about 1%, about 20%, and about 200% of the total dose of the compound or pharma- ceutically acceptable salt thereof on three non-consecutive days.

[0133] 121. The method of any of paragraphs 2 to 17, wherein a first dose escalation course comprises administering to the patient about 1%, about 10%, and about 100% of the total dose of the compound or pharma- ceutically acceptable salt thereof on three non-consecutive days, and a second dose escalation course comprises administering to the patient about 1%, about 10%, and about 100% of the total dose of the compound or pharma- ceutically acceptable salt thereof on three non-consecutive days.

[0134] 122. The method of any of paragraphs 2 to 17, wherein a first dose escalation course comprises administering to the patient about 1%, about 20%, and about 200% of the total dose of the compound or pharma- ceutically acceptable salt thereof on three non-consecutive days, and a second dose escalation course comprises administering to the patient about 1%, about 20%, and about 200% of the total dose of the compound or pharma- ceutically acceptable salt thereof on three non-consecutive days.

[0135] 123. The method according to any of items 119 to 122, wherein the total dose of the compound or a pharma- ceutically acceptable salt thereof is about 500 nmol / kg. [Brief description of the drawings]

[0136] [Figure 1]1 shows a schematic diagram of an exemplary administration scheme according to the present invention. In this exemplary scheme, E2 CAR T cells (CAR T cells expressing a protein encoded by the nucleic acid sequence of SEQ ID NO: 4) are administered on Monday and Thursday of the first week, followed by administration of a small molecule ligand (e.g., EC17) linked to a targeting moiety by a linker starting in the second week. In the second week, the small molecule ligand (e.g., EC17) linked to a targeting moiety by a linker is administered on Monday and Thursday, followed by Monday of the third week, using a dose escalation process (i.e., process 1). The small molecule ligand (e.g., EC17) linked to a targeting moiety by a linker is not administered from Tuesday to Sunday of the third week. Weeks 2-3 are referred to as "Cycle 1". In the fourth week, the small molecule ligand (e.g., EC17) linked to a targeting moiety by a linker is administered on Monday and Thursday, followed by Monday of the fifth week, using a dose escalation process (i.e., process 2). The small molecule ligand (e.g., EC17) linked to the targeting moiety by a linker is not administered from Tuesday to Sunday of the fifth week. Weeks 4-5 are referred to as "Cycle 2". In week 6, the small molecule ligand (e.g., EC17) linked to the targeting moiety by a linker is administered on Monday and Thursday, followed by Monday of the seventh week using a dose escalation course (i.e., Course 3). The small molecule ligand (e.g., EC17) linked to the targeting moiety by a linker is not administered from Tuesday to Sunday of the seventh week. Weeks 6-7 are referred to as "Cycle 3". Weeks 2-7 are referred to as "Course 1". During Course 1, the small molecule ligand (e.g., EC17) linked to the targeting moiety by a linker is administered on Monday and Thursday of the second week, followed by Monday of the third week, at doses of 1%, 10% and 100% of the total dose (e.g., 31 μg / kg) of the small molecule ligand (e.g., EC17) linked to the targeting moiety, respectively. During phase 2, a small molecule ligand (e.g., EC17) attached to a targeting moiety by a linker is administered on Monday and Thursday of week 4, then on Monday of week 5, at doses of 1%, 30%, and 300% of the total dose (e.g., 31 μg / kg) of the small molecule ligand (e.g., EC17) attached to the targeting moiety, respectively.During course 3, a small molecule ligand (e.g., EC17) linked to a targeting moiety by a linker is administered on Monday and Thursday of week 6, then on Monday of week 7, at doses of 1%, 50% and 500% of the total dose (e.g., 31 μg / kg) of the small molecule ligand (e.g., EC17) linked to a targeting moiety, respectively. Course 1 is then repeated, taking into account therapeutic benefit and acceptable toxicity.

[0137] [Diagram 2] Schematic diagram of constructs used for CAR T transduction.

[0138] [Diagram 3] Schematic representation of the E2 construct versus the 4M5.3 construct, with a map of the E2 construct shown.

[0139] [Figure 4] (Top panel) A chart showing the tumor volume of HOS-FRα tumors when treated with E2 CAR T cells alone (●) and E2 CAR T cells + EC-17 (◯). (Bottom panel) A chart showing the weight change of HOS-FRα tumor-bearing mice when treated with E2 CAR T cells alone (●) and E2 CAR T cells + EC-17 (◯).

[0140] [Diagram 5] A chart showing weight change in THP-1-FRβ AML-bearing mice treated with no Car-T (●); no EC-17 (■); EC-17 SIW 500 nmol / kg (▲); EC-17 TIW (5 nmol / kg, 50 nmol / kg, 500 nmol / kg on / off); (◆) EC-17 increasing doses (M / Th / M on / off).

[0141] [Figure 6] (Left panel) Chart showing liver metastatic tumor burden (right panel) Chart showing non-liver metastatic tumor burden.

[0142] [Figure 7]The number of circulating THP1-FRβ cells per 100 μL of blood on day 39 is shown on a logarithmic scale.

[0143] [Figure 8] 1 is a chart showing the percentage of total E2 CAR T cells isolated from solid liver tumors (y-axis) with different EC-17 dosing regimens. For each dosing regimen, the leftmost bar is PD1+LAG3+TIM3+, the second bar from the left is PD1+LAG3+TIM3-, the middle bar is PD1+LAG3-TIM3+; the second bar from the right is PD1+LAG3-TIM3-; the rightmost bar is PD1-LAG3-TIM3-.

[0144] [Figure 9] Shown is a fully human CAR construct containing anti-FITC scFv (clone E2), full length IgG4 spacer (Fc-derived hinge-CH2(L235D, N297Q)-CH3), CD28tm transmembrane domain, 4-1BB / CD3ζ cytoplasmic activation domain and a non-functional truncated cell surface polypeptide of epidermal growth factor receptor (EGFRt). Bottom: Example of CD4 / CD8 T cell phenotyping performed by flow cytometry of EGFRt-sorted (left pie chart) CAR T cell preparations and unsorted "clinical facsimile" (right pie chart). Color key as indicated.

[0145] [Figure 10] Panel A: Kd values ​​for 3H-EC17 uptake by FR+ target cells after 2 hours incubation at 37°C (calculated from the number of molecules bound per cell). Panel B: Kd values ​​for 3H-EC17 uptake by E2-CAR T cells (~24% EGFRt+, ~95:5 CD8 / CD4 ratio) after 2 hours incubation at room temperature (calculated from total cell-associated radioactivity, DPM).

[0146] [Figure 11] Functional FR levels on tumor cells measured by 3H-FA-based binding assay (100 nM, 1 h, 37° C.).

[0147] [Figure 12] EC17 dose setting and CRS assessment in tumor vs. tumor-free mice. Panel A: Schematic showing dosing schedule of CAR T cells (approximately 10 million "clinical facsimile") + EC17 administered in three different ways to NSG mice without or with pre-established MDA-MB-231 xenografts. Tumor-free mice received EC17 SIW 500 nmol / kg (2 doses on days 2 and 10). Tumor-bearing mice received EC17 as follows: EC17 SIW 500nmol / kg (5 doses on days 2, 10, 17, 24 and 31), EC17 M / Th / M_titration-1 (5 / 50 / 500nmol / kg on Monday / Thursday / Monday followed by a one week rest cycle i.e. days 2, 6, 10, 17, 20, 24 and 31) or EC17 M / Th / M_titration-2 (5 / 100 / 1000nmol / kg on Monday / Thursday / Monday followed by a one week rest cycle i.e. days 2, 6, 10, 17, 20, 24 and 31). Panel B: Systemic levels of human IFNγ in log2 memory in mouse plasma on days 11 and 12 after CAR T cell injection in tumor-bearing versus tumor-free mice (i.e., approximately 20 and 42 hours after the previous EC17 dose for all treatment cohorts). Panel C: Circulating CAR T cells in mouse blood identified as human CD3ε+EGFRt+ events in flow cytometry and enumerated per 100 μL of whole blood. Panel D: Measurement of body weight and tumor volume changes in tumor-bearing mice receiving CAR T cells alone or CAR T cells + EC17 administered in three different ways (dotted lines indicate each EC17 dose). n=5 mice / group. All data represent mean±sem. *p<0.05 by one-way ANOVA test.

[0148] [Figure 13]Showing EC17 dose escalation in vivo safety and anti-leukemic activity. Panel A: Schematic diagram showing dosing scheduling of EC17+ unsorted EGFRt CAR T cells (~6 million, day 0) in NSG mice bearing 1-day-old intravenous THP1-FRβ xenografts. Starting 3 days after CAR T-cell injection, EC17 was administered in three different ways: EC17 SIW 500nmol / kg (5 doses on days 3, 10, 17, 24, 31 and 38), EC17 TIW 5 / 50 / 500nmol / kg (5 / 50 / 500nmol / kg on Monday / Wednesday / Friday followed by 3 repetitions of 9 days off, i.e. days 3 / 5 / 7, 17 / 19 / 21 and 31 / 33 / 35) or EC17 M / Th / M_dose escalation on Monday / Thursday / Monday (3 escalating cycles of 5 / 10 / 100nmol / kg in cycle 1, 5 / 30 / 300nmol / kg in cycle 2 and 5 / 50 / 500nmol / kg in cycle 3, i.e. days 3 / 6 / 10, 17 / 20 / 24 and 31 / 34 / 38). Panel B: Measurement of body weight change (n=5). Panel C: Circulating CAR T cells as human CD3ε+EGFRt+ events found per 100 μL of whole blood from mice on day 31 on a logarithmic scale. Panel D: Left bar: Circulating tumor cells (GFP+) per 100 μL of whole blood in all cohorts measured on the last day of study (day 39); Center bar: THP1-FRβ infiltrated liver weight representing liver metastasis burden; Right bar: Total tumor weight of all non-hepatic macrometastases. Panel E: Flow cytometry analysis of T cell exhaustion markers, PD1, LAG3, TIM3, of pre-infused CAR T cell products (triple negative) and tumor-infiltrating CAR T cells isolated from liver metastases. A key characteristic of fully exhausted T cells is the co-expression of multiple inhibitory receptor markers (i.e. triple positive).

[0149] [Figure 14]Antitumor activity and CRS rescue in an aggressive model of pediatric osteosarcoma. Panel A: Schematic showing dosing schedule of CAR T cells (~6 million, day 0) + EC17 in NSG mice (n=5) bearing 3-day-old subcutaneous HOS-FRα xenografts. Starting 3 days after CAR T cell injection (6 days after tumor implantation), Monday / Thursday / Monday, 3 cycles of EC17 M / Th / M "intra-patient" dose escalation: 5 / 10 / 100 nmol / kg in cycle 1, 5 / 30 / 300 nmol / kg in cycle 2 and 5 / 50 / 500 nmol / kg in cycle 3, i.e. days 3 / 6 / 10, 17 / 20 / 24 and 31 / 34 / 38. Panel B: Measurement of tumor volume and body weight change. Due to tumor progression, five mice receiving CAR T cells only (no EC17) were sacrificed on days 23-31, and five EC17-treated mice were sacrificed on days 33 (2 mice) and 47 (3 mice), respectively. Panel C: Flow cytometry analysis of CAR T cells (human CD3ε+EGFRt+) per 100 μL of whole blood plotted on a logarithmic scale (left) and tumor-infiltrating CAR T cells at the time of sacrifice (right). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0150] definition As used herein, the term "a," "an," or "an" can mean one or more. As used herein, "about" in connection with numerical values, including, for example, integers, fractions, and percentages, refers to a range of numerical values ​​(e.g., ±5% to 10% of the recited value) that one of ordinary skill in the art would generally consider equivalent to the recited value (e.g., having the same function or result).

[0151] As used herein, the term "treatment" or "treating" refers to both therapeutic treatment and prophylactic or preventative treatment.

[0152] As used herein, the terms "amelioration," "ameliorate," or "improved" in relation to cancer can mean alleviation of symptoms of the cancer, reduction in tumor size, complete or partial elimination of the tumor (e.g., complete or partial response), induction of disease stabilization, prevention of cancer progression (e.g., progression-free survival), or any other effect on the cancer that a physician considers to be a therapeutic, prophylactic, or preventative treatment of the cancer.

[0153] As used herein, the term "administration" or "administering" refers to any means of introducing a compound described herein, or a pharma- ceutically acceptable salt thereof, or a CAR T cell composition into a patient, including, but not limited to, oral, intravenous, intramuscular, subcutaneous, and transdermal.

[0154] 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 hypoplasia, fever, decreased blood pressure, or pulmonary edema.

[0155] As used herein, the terms "transduction" and "transfection" are used interchangeably and refer to the introduction of nucleic acid into a cell by any artificial method, including viral and non-viral methods.

[0156] The term "dose escalation process" as used herein means that the dose of the compound or its pharma- ceutically acceptable salt is administered in increasing amounts over time. As used herein, the description of "first dose escalation process" in combination with "second dose escalation process", "third dose escalation process", "fourth dose escalation process", "fifth dose escalation process" and "sixth dose escalation process" etc. means that multiple dose escalation processes are performed, and for each separate dose escalation process, after the first dose escalation process, the initial dose of the compound or its pharma- ceutically acceptable salt in the subsequent dose escalation process is lower than the final dose of the compound or its pharma- ceutically acceptable salt in the previous dose escalation process (see, for example, FIG. 1 and the description of FIG. 1 in the brief description of the figures).

[0157] DETAILED DESCRIPTION OF THE ILLUSTRATIVE EMBODIMENTS In various embodiments described herein, a small molecule ligand attached to a targeting moiety by a linker is used as a bridge between the cancer and the CAR T cells (i.e., T cells expressing a chimeric antigen receptor). The bridge directs the CAR T cells to the cancer for amelioration of the cancer. In certain embodiments, the "small molecule ligand" can be a folate, a CAIX ligand, a DUPA, an NK-1R ligand, a ligand for gamma glutamyl transpeptidase, an NKG2D ligand, or a CCK2R ligand, each of which is a small molecule ligand that specifically binds to a cancer cell type (i.e., the receptor for each of these ligands is overexpressed in cancer compared to normal tissue).

[0158] The "targeting moiety" attached to the small molecule ligand binds to the recognition region of the genetically modified CAR expressed by the CAR T cell. Thus, the recognition region of the CAR (e.g., single-chain fragment variable region (scFv), Fab, Fv, Fc or (Fab')2 fragment of an antibody, etc.) is directed to the "targeting moiety". Thus, the small molecule ligand attached to the targeting moiety by a linker serves as a bridge between the cancer and the CAR T cell, directing the CAR T cell to the cancer for the amelioration of the cancer. In various embodiments, the bridge between the cancer and the CAR T cell can be any of the applicable conjugates shown in the examples.

[0159] The bridges are small organic molecules, so clearance from the bloodstream can be achieved quickly (e.g., within about 20 minutes). In some embodiments, the CAR T cell response can target only cancer cells that express receptors for the small molecule ligand moiety of the 'bridge', thereby reducing off-target toxicity to normal tissues. Furthermore, the system can be 'versatile', since one type of CAR T cell construct can be used to target a wide range of cancers using different 'bridges'. By way of illustration, the targeting moiety recognized by the CAR T cell 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 can be modified to allow targeting of a wide range of cancers.

[0160] In one embodiment, a method for treating cancer is provided.The method includes: i) administering at least one dose of a CAR T cell composition to a patient, wherein the CAR T cell comprises a CAR directed to a targeting moiety;ii) administering a compound or a pharmaceutically acceptable salt thereof to a patient, wherein the compound comprises a small molecule ligand linked to the targeting moiety by a linker, wherein the compound or a pharmaceutically acceptable salt thereof is administered by at least a first dose escalation process and a second dose escalation process.

[0161] In other embodiments, a method for treating cancer is provided.The method includes: i) administering at least one dose of CAR T cell composition to a patient, where the CAR T cell comprises a CAR that is directed to a targeting moiety; ii) administering a compound or its pharmaceutically acceptable salt to a patient, where the compound or its pharmaceutically acceptable salt is administered in a first dose escalation course, and if severe CRS occurs in the first dose escalation course, administer the compound or its pharmaceutically acceptable salt using a low dose escalation course, where the first dose of the compound or its pharmaceutically acceptable salt in the low dose escalation course is lower than the first dose of the compound or its pharmaceutically acceptable salt administered in the first dose escalation course.In other embodiments, in the low dose escalation course, the compound or its pharmaceutically acceptable salt can be administered in about 0.5%, about 5% and about 50% of the total dose of the compound or its pharmaceutically acceptable salt on three non-consecutive days.

[0162] In the sections below and in the claims, as well as in the various embodiments described herein, a small molecule ligand attached to a targeting moiety by a linker is referred to as a "compound."

[0163] Several embodiments are described by the following numbered paragraphs. Any of the following embodiments may be combined with any of the applicable embodiments described in the Summary section of this patent application, the Detailed Description of General Illustrative Embodiments section of this patent application, the Examples section, or the Claims.

[0164] 1. A method of treating cancer, comprising: i) administering to the patient at least one dose of a CAR T cell composition, where the CAR T cell comprises a CAR directed to a targeting moiety; ii) administering to a patient a compound or a pharma- ceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand attached to a targeting moiety by a linker, wherein the compound or a pharma- ceutically acceptable salt thereof is administered by at least a first dose escalation course and a second dose escalation course.

[0165] 2. The method of claim 1, wherein the compound or a pharma- ceutically acceptable salt thereof is administered in at least a first dose-escalation course, a second dose-escalation course and a third dose-escalation course.

[0166] 3. The method of claim 1, wherein the compound or a pharma- ceutically acceptable salt thereof is administered at least a first dose escalation course, a second dose escalation course, a third dose escalation course and a fourth dose escalation course.

[0167] 4. The method of claim 1, wherein the compound or a pharma- ceutically acceptable salt thereof is administered at least a first dose escalation course, a second dose escalation course, a third dose escalation course, a fourth dose escalation course and a fifth dose escalation course.

[0168] 5. The method of claim 1, wherein the compound or a pharma- ceutically acceptable salt thereof is administered at least a first dose escalation course, a second dose escalation course, a third dose escalation course, a fourth dose escalation course, a fifth dose escalation course and a sixth dose escalation course.

[0169] 6. The method of any of paragraphs 1 to 5, wherein the patient is administered an initial dose of CAR T cells and a second dose of CAR T cells.

[0170] 7. The method of paragraph 6, wherein the first dose of the CAR T cells is a test dose for monitoring the patient's tolerance to the CAR T cells.

[0171] 8. The method of paragraph 6, wherein the second dose of CAR T cells comprises a higher dose of CAR T cells than the first dose of CAR T cells.

[0172] 9. The initial dose of CAR T cells is approximately 0.5 × 10 per kg of patient body weight. 5 CAR T cells ~ approx. 1.5 x 10 per kg of patient body weight 6 The method according to any one of items 6 to 8, comprising a CAR T cell of the above-mentioned item 6.

[0173] 10. The second dose of CAR T cells is approximately 0.8 × 10 per kg of patient body weight. 6 CAR T cells ~ approximately 2 x 10 per kg of patient body weight 7 10. The method according to any one of items 6 to 9, comprising a CAR T cell of the above item.

[0174] 11. The method of any of items 1 to 10, wherein the first dose escalation course is followed by a period during which the compound or a pharma- ceutically acceptable salt thereof is not administered.

[0175] 12. The method of any of paragraphs 1 to 11, wherein the second dose-escalation course is followed by a period during which the compound or a pharma- ceutically acceptable salt thereof is not administered.

[0176] 13. The method according to any one of paragraphs 2 to 12, wherein the third dose escalation course is followed by a period during which the compound or a pharma- ceutically acceptable salt thereof is not administered.

[0177] 14. The method of any of paragraphs 3 to 13, wherein the fourth dose-escalation course is followed by a period during which the compound or a pharma- ceutically acceptable salt thereof is not administered.

[0178] 15. The method of any of paragraphs 4 to 14, wherein the fifth dose escalation course is followed by a period during which the compound or a pharma- ceutically acceptable salt thereof is not administered.

[0179] 16. The method according to any one of paragraphs 5 to 15, wherein the sixth dose escalation course is followed by a period during which the compound or a pharma- ceutically acceptable salt thereof is not administered.

[0180] 17. The method according to any one of items 11 to 16, wherein the period is about 7 days.

[0181] 18. The method of any of paragraphs 1 to 17, wherein the first dose escalation course comprises administering to the patient about 1%, about 10%, and about 100% of the total dose of the compound or a pharma- ceutically acceptable salt thereof on three non-consecutive days.

[0182] 19. The method of any of paragraphs 1 to 18, wherein the second dose escalation course comprises administering to the patient about 1%, about 30%, and about 300% of the total dose of the compound or a pharma- ceutically acceptable salt thereof on three non-consecutive days.

[0183] 20. The method of any of paragraphs 2 to 19, wherein the third dose escalation course comprises administering to the patient about 1%, about 50%, and about 500% of the total dose of the compound or a pharma- ceutically acceptable salt thereof on three non-consecutive days.

[0184] 21. The method of any of paragraphs 3 to 20, wherein the fourth dose escalation course comprises administering to the patient about 1%, about 10%, and about 100% of the total dose of the compound or a pharma- ceutically acceptable salt thereof on three non-consecutive days.

[0185] 22. The method of any of paragraphs 4 to 21, wherein the fifth dose escalation course comprises administering to the patient about 1%, about 30%, and about 300% of the total dose of the compound or a pharma- ceutically acceptable salt thereof on three non-consecutive days.

[0186] 23. The method of any of paragraphs 5 to 22, wherein the sixth dose escalation course comprises administering to the patient about 1%, about 50%, and about 500% of the total dose of the compound or a pharma- ceutically acceptable salt thereof on three non-consecutive days.

[0187] 24. The method according to any one of items 18 to 23, wherein the total dose of the compound or a pharma- ceutically acceptable salt thereof is about 10 μg / kg to about 50 μg / kg of the compound or a pharma- ceutically acceptable salt thereof.

[0188] 25. The method according to any one of items 18 to 24, wherein the total dose of the compound or a pharma- ceutically acceptable salt thereof is about 20 μg / kg to about 40 μg / kg of the compound or a pharma- ceutically acceptable salt thereof.

[0189] 26. The method according to any one of items 18 to 25, wherein the total dose of the compound or a pharma- ceutically acceptable salt thereof is about 25 μg / kg to about 35 μg / kg of the compound or a pharma- ceutically acceptable salt thereof.

[0190] 27. The method according to any one of items 18 to 26, wherein the total dose of the compound or a pharma- ceutically acceptable salt thereof is about 30 μg / kg of the compound or a pharma- ceutically acceptable salt thereof.

[0191] 28. The method of any of paragraphs 6 to 27, wherein a first dose of CAR T cells and a second dose of CAR T cells are administered to the patient during the first week.

[0192] 29. The method of any of paragraphs 6 to 28, wherein the first dose of CAR T cells and the second dose of CAR T cells are administered to the patient on Monday and Thursday of week 1.

[0193] 30. The method according to any one of paragraphs 1 to 29, wherein the first dose escalation course of the compound or a pharma- ceutically acceptable salt thereof is carried out during weeks 2 and 3.

[0194] 31. The method of any of paragraphs 1 to 30, wherein a second dose escalation course of the compound or a pharma- ceutically acceptable salt thereof is performed between weeks 4 and 5.

[0195] 32. The method according to any one of paragraphs 2 to 31, wherein a third dose escalation course of the compound or a pharma- ceutically acceptable salt thereof is administered between weeks 6 and 7.

[0196] 33. The method of paragraph 30, wherein the compound or a pharma- ceutically acceptable salt thereof is administered on three non-consecutive days, the three non-consecutive days being Monday and Thursday of the second week and Monday of the third week.

[0197] 34. The method of paragraph 31, wherein the compound or a pharma- ceutically acceptable salt thereof is administered on three non-consecutive days, the three non-consecutive days being Monday and Thursday of the fourth week and Monday of the fifth week.

[0198] 35. The method of paragraph 32, wherein the compound or a pharma- ceutically acceptable salt thereof is administered on three non-consecutive days, the three non-consecutive days being Monday and Thursday of the sixth week and Monday of the seventh week.

[0199] 36. The method of any of paragraphs 1 to 35, wherein the patient is lymphocyte-depleted prior to administering the CAR T cell composition to the patient.

[0200] 37. The method of any of paragraphs 1 to 36, further comprising administering platelets to the patient, administering packed red blood cells to the patient, administering cryoprecipitate to the patient, administering intravenous immunoglobulin to the patient, and / or administering antibacterial therapy to the patient.

[0201] 38. The method of any of paragraphs 1 to 37, wherein if CRS or neurotoxicity is not observed in the patient during the first dose escalation course, the method proceeds to a second dose escalation course.

[0202] 39. The method of any of paragraphs 2 to 38, wherein if CRS or neurotoxicity is not observed in the patient during the second dose escalation course, the method proceeds to a third dose escalation course.

[0203] 40. The method of any of paragraphs 3 to 39, wherein if CRS or neurotoxicity is not observed in the patient during the third dose escalation course, the method proceeds to a fourth dose escalation course.

[0204] 41. The method of any of paragraphs 4 to 40, wherein if CRS or neurotoxicity is not observed in the patient during the fourth dose escalation course, the method proceeds to a fifth dose escalation course.

[0205] 42. The method of any of paragraphs 5 to 41, wherein if CRS or neurotoxicity is not observed in the patient during the fifth dose escalation course, the method proceeds to a sixth dose escalation course.

[0206] 43. The method of any of paragraphs 1 to 42, wherein if fever without hypotension is observed in the patient at any point during the dose escalation process, and neurotoxicity is not observed in the patient, then all subsequent doses of the compound or a pharma- ceutically acceptable salt thereof are administered at the dose escalation level that caused fever without hypotension in the patient.

[0207] 44. The method of any of paragraphs 1 to 43, wherein if the patient experiences severe CRS or neurotoxicity during any dose escalation, all subsequent doses of the compound or a pharma- ceutically acceptable salt thereof are administered to the patient at a dose escalation level that is lower than the dose escalation level that caused the patient to experience severe CRS or neurotoxicity.

[0208] 45. A method of treating cancer, comprising: i) administering to the patient at least one dose of a CAR T cell composition, where the CAR T cell comprises a CAR directed to a targeting moiety; ii) administering to the patient a compound or a pharma- ceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand attached to a targeting moiety by a linker, wherein the compound or a pharma- ceutically acceptable salt thereof is administered during a first dose-escalation course, and if severe CRS occurs during the first dose-escalation course, administering the compound or a pharma- ceutically acceptable salt thereof using a low-dose escalation course, wherein the initial dose of the compound or a pharma- ceutically acceptable salt thereof in the low-dose escalation course is lower than the initial dose of the compound or a pharma- ceutically acceptable salt thereof administered during the first dose-escalation course.

[0209] 46. ​​The method of claim 45, wherein in a low-dose escalation course, the compound or a pharma- ceutically acceptable salt thereof is administered at about 0.5%, about 5%, and about 50% of the total dose of the compound or a pharma- ceutically acceptable salt thereof on three non-consecutive days.

[0210] 47. The method according to item 46, wherein the total dose of the compound or a pharma- ceutically acceptable salt thereof is about 10 μg / kg to about 50 μg / kg of the compound or a pharma- ceutically acceptable salt thereof.

[0211] 48. The method according to any one of items 46 to 47, wherein the total dose of the compound or a pharma- ceutically acceptable salt thereof is about 25 μg / kg to about 35 μg / kg of the compound or a pharma- ceutically acceptable salt thereof.

[0212] 49. The method according to any one of items 46 to 48, wherein the total dose of the compound or a pharma- ceutically acceptable salt thereof is about 30 μg / kg of the compound or a pharma- ceutically acceptable salt thereof.

[0213] 50. The method according to any one of items 1 to 49, wherein the ligand is selected from the group consisting of folate, DUPA, NK-1R ligands, CAIX ligands, ligands for gamma glutamyl transpeptidase, NKG2D ligands and CCK2R ligands.

[0214] 51. The method according to any one of items 1 to 50, wherein the ligand is folate.

[0215] 52. The method according to any one of items 1 to 50, wherein the ligand is an NK-1R ligand.

[0216] 53. The method according to any one of items 1 to 50, wherein the ligand is DUPA.

[0217] 54. The method according to any one of items 1 to 50, wherein the ligand is a CCK2R ligand.

[0218] 55. The method according to any one of items 1 to 50, wherein the ligand is a ligand for gamma glutamyl transpeptidase.

[0219] 56. The method of any of paragraphs 1 to 55, wherein the targeting moiety is selected from the group consisting of 2,4-dinitrophenol (DNP), 2,4,6-trinitrophenol (TNP), biotin, digoxigenin, fluorescein, fluorescein isothiocyanate (FITC), NHS-fluorescein, pentafluorophenyl ester, tetrafluorophenyl ester, knottin, centrin, and DARPin.

[0220] 57. The method of any of paragraphs 1 to 56, wherein the targeting moiety is FITC.

[0221] 58. The method of any of paragraphs 1 to 56, wherein the targeting moiety is DNP.

[0222] 59. The method of any of paragraphs 1 to 56, wherein the targeting moiety is TNP.

[0223] 60. The method of any one of items 1 to 59, wherein the linker comprises polyethylene glycol (PEG), polyproline, a hydrophilic amino acid, a sugar, a non-natural peptidoglycan, polyvinylpyrrolidone, Pluronic F-127, or a combination thereof.

[0224] 61. The method of any one of items 1 to 60, wherein the linker comprises PEG.

[0225] 62. A compound or a pharma- ceutically acceptable salt thereof having the formula [ka] wherein B is a small molecule ligand, L is a linker, and T is a targeting moiety, wherein L is of the formula [ka] (wherein n is an integer of 0 to 200). The structure includes the following structure: Item 62. The method according to any one of items 1 to 61, comprising:

[0226] 63. The method according to item 62, wherein n is an integer from 0 to 150.

[0227] 64. The method according to item 62, wherein n is an integer from 0 to 110.

[0228] 65. The method according to item 62, wherein n is an integer from 0 to 20.

[0229] 66. The method according to item 62, wherein n is an integer from 15 to 20.

[0230] 67. The method according to item 62, wherein n is an integer from 15 to 110.

[0231] 68. Cancer is 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, adrenal cancer, soft tissue sarcoma, osteosarcoma including pediatric or non-pediatric osteosarcoma, urethral cancer, prostate cancer, Item 68. The method according to any one of items 1 to 67, wherein the cancer is selected from the group consisting of chronic leukemia, acute leukemia, acute myeloid leukemia, lymphocytic lymphoma, myeloid leukemia, myelomonocytic leukemia, hairy cell leukemia, pleural mesothelioma, bladder cancer, Burkitt's lymphoma, ureteral cancer, kidney cancer, renal cell carcinoma, renal pelvis cancer, central nervous system (CNS) neoplasms, primary CNS lymphoma, spinal axis tumor, brain stem glioma, pituitary adenoma, and esophagogastric junction adenocarcinoma.

[0232] 69. The method according to any one of items 1 to 51 or 56 to 68, wherein the cancer is a folate receptor-expressing cancer.

[0233] 70. The method according to any one of items 1 to 69, wherein the cancer is osteosarcoma.

[0234] 71. The method according to any one of items 1 to 70, wherein the CAR has a recognition region, and the recognition region is a single-chain fragment variable (scFv) region of an antibody.

[0235] 72. The method according to any one of items 1 to 57 or 60 to 71, wherein the CAR has a recognition region, and the recognition region of the CAR is a single-chain fragment variable (scFv) region of an anti-FITC antibody.

[0236] 73. The method of any of paragraphs 1 to 72, wherein the CAR has a costimulatory domain, and the costimulatory domain is selected from the group consisting of CD28, CD137 (4-1BB), CD134 (OX40) and CD278 (ICOS).

[0237] 74. The method of any of paragraphs 1 to 73, wherein the CAR has an activation signaling domain, and the activation signaling domain is T cell CD3 zeta chain or Fc receptor gamma.

[0238] 75. The method according to any one of items 1 to 57 or 60 to 74, wherein the CAR has a recognition region, the recognition region being a single-chain fragment variable (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 the T cell CD3 zeta chain.

[0239] 76. The method of any of paragraphs 1 to 75, wherein the patient is imaged prior to administration of the compound or a pharma- ceutically acceptable salt thereof or prior to administration of the CAR T cell composition.

[0240] 77. The method according to any one of items 1 to 76, wherein the compound or a pharma- ceutically acceptable salt thereof is not an antibody and does not include an antibody fragment.

[0241] 78. The method of any of paragraphs 1 to 77, wherein the targeting moiety does not comprise a peptide epitope.

[0242] 79. The method according to any of items 1 to 78, wherein CAR T cell toxicity against cancer is achieved without causing cytokine release that results in off-target toxicity in patients.

[0243] 80. The method of any of items 1 to 78, wherein off-target tissue toxicity does not occur in the patient and CAR T cell toxicity occurs against the cancer.

[0244] 81. The method of any of paragraphs 1 to 78, wherein the cancer comprises a tumor, the tumor size is reduced in the patient, and no off-target toxicity occurs.

[0245] 82. The method of any of paragraphs 1 to 81, wherein the CAR T cells comprise a nucleic acid comprising SEQ ID NO:1.

[0246] 83. The method of any of paragraphs 1 to 82, wherein the CAR T cells comprise a polypeptide comprising SEQ ID NO:2.

[0247] 84. The method of paragraph 82, wherein the nucleic acid encodes a chimeric antigen receptor.

[0248] 85. The method of any of paragraphs 1 to 81, wherein the CAR T cells comprise a nucleic acid comprising SEQ ID NO:4.

[0249] 86. The method of any of paragraphs 1 to 81 or 85, wherein the CAR T cells comprise a polypeptide comprising SEQ ID NO:5.

[0250] 87. The method of paragraph 85, wherein the nucleic acid encodes a chimeric antigen receptor.

[0251] 88. The method of any of paragraphs 1 to 87, wherein the CAR comprises a humanized amino acid sequence.

[0252] 89. The method of any of paragraphs 1 to 87, wherein the CAR consists of a humanized amino acid sequence.

[0253] 90. The method of any of paragraphs 1 to 89, further comprising administering to the patient a folate, a conjugate comprising a folate (wherein the conjugate comprising a folate does not comprise a targeting moiety), or an agent that inhibits activation of CAR T cells.

[0254] 91. The method of claim 90, wherein folate is administered.

[0255] 92. The method of paragraph 90, wherein folic acid or leucovorin is administered.

[0256] 93. The method of claim 90, wherein a conjugate containing folate is administered.

[0257] 94. The method of claim 93, wherein the folate-containing conjugate comprises folate bound to one or more amino acids.

[0258] 95. A conjugate containing folate has the formula [ka] 94. The method of claim 93, comprising the steps of:

[0259] 96. Folate is the formula [ka] [In the formula, X 1 and Y 1 each independently represents halo, R 2 , OR 2 , S.R. 3 and N.R. 4 R 5 selected from the group consisting of; U, V and W are each independently -(R 6a )C=, -N=, -(R 6a )C(R 7a )- and -N(R 4a )-; Q is selected from the group consisting of C and CH; T is selected from the group consisting of S, O, N, and -C=C-; X 2 and X 3 are each independently oxygen, sulfur, -C(Z)-, -C(Z)O-, -OC(Z)-, -N(R 4b )-, -C(Z)N(R 4b )-, -N(R 4b )C(Z)-, -OC(Z)N(R4b )-, -N(R 4b )C(Z)O-, -N(R 4b )C(Z)N(R 5b )-, -S(O)-, -S(O)2-, -N(R 4a )S(O)2-, -C(R 6b )(R 7b )-, -N(C≡CH)-, -N(CH2C≡CH)-, C1-C 12 Alkylene and C1-C 12 alkyleneoxy, where X is oxygen or sulfur; R 1 are hydrogen, halo, C1-C 12 Alkyl and C1-C 12 alkoxy; R 2 , R 3 , R 4 , R 4a , R 4b , R 5 , R 5b , R 6b and R 7b each independently represents hydrogen, halo, C1-C 12 Alkyl, C1-C 12 Alkoxy, C1-C 12 Alkanoyl, C1-C 12 Alkenyl, C1-C 12 Alkynyl, (C1-C 12 alkoxy)carbonyl and (C1-C 12 alkylamino)carbonyl; R 6 and R 7 each independently represents hydrogen, halo, C1-C 12 Alkyl and C1-C 12 alkoxy; or R 6 and R 7 together to form a carbonyl group; R 6a and R 7a each independently represents hydrogen, halo, C1-C 12 Alkyl and C1-C 12 alkoxy; or R 6aand R 7a together to form a carbonyl group; p, r, s and t are each independently 0 or 1; and * is an optional covalent bond to the remainder of the conjugate if any additional chemical moieties are part of the folate. Item 92. The method of item 91, comprising the steps of:

[0260] 97. The method according to paragraph 90, wherein the agent that inhibits the activation of CAR T cells is selected from the group consisting of lymphocyte-specific protein tyrosine kinase inhibitors, PI3 kinase inhibitors, IL-2-inducible T cell kinase inhibitors, JAK inhibitors, BTK inhibitors, EC2319, and agents that block binding of CAR T cells to the compound or a pharma- ceutically acceptable salt thereof but do not bind to the cancer.

[0261] 98. The method of claim 90, wherein an agent that inhibits the activation of CAR T cells is administered, and the agent is a lymphocyte-specific protein tyrosine kinase inhibitor.

[0262] 99. The method according to paragraph 98, wherein the lymphocyte-specific protein tyrosine kinase inhibitor is dasatinib.

[0263] 100. The method of claim 90, wherein an agent that inhibits the activation of CAR T cells is administered, and the agent is a PI3 kinase inhibitor.

[0264] 101. The method according to paragraph 100, wherein the PI3 kinase inhibitor is GDC0980.

[0265] 102. The method of claim 90, wherein an agent that inhibits the activation of CAR T cells is administered, and the agent is an IL-2-inducible T cell kinase inhibitor.

[0266] 103. The method of claim 102, wherein the IL-2-inducible T cell kinase inhibitor is BMS-509744.

[0267] 104. The method of claim 90, wherein an agent that inhibits activation of CAR T cells is administered, the agent blocking binding of CAR T cells to the compound or a pharma- ceutically acceptable salt thereof but not to the cancer.

[0268] 105. The method of paragraph 104, wherein the agent is fluoresceinamine, FITC or sodium fluorescein.

[0269] 106. The method of claim 105, wherein the agent is sodium fluorescein.

[0270] 107. The method of any of paragraphs 90 to 106, wherein administration of folate, a conjugate comprising folate (wherein the conjugate comprising folate does not comprise a targeting moiety), or an agent that inhibits activation of CAR T cells results in a decrease in cytokine levels in the patient.

[0271] 108. The method of paragraph 107, wherein the reduction in cytokine levels is approximately to the cytokine levels of an untreated patient.

[0272] 109. The method of any of paragraphs 90 to 108, wherein the cancer comprises a tumor and the size of the tumor in the patient does not increase when the patient is administered folate, a conjugate comprising folate (wherein the conjugate comprising folate does not comprise a targeting moiety), or an agent that inhibits activation of CAR T cells.

[0273] 110. The method according to any of paragraphs 104 to 106, wherein the patient is administered a drug that inhibits CAR T cell activation when CRS grade reaches 1, 2, 3 or 4.

[0274] 111. The method of claim 110, wherein the patient is administered an agent that inhibits CAR T cell activation when CRS grade reaches 3 or 4.

[0275] 112. The method of any of paragraphs 104 to 106, wherein the agent that inhibits CAR T cell activation is administered at a dose of about .01 to about 300 μmole / kg of patient body weight.

[0276] 113. The method of any of paragraphs 1 to 112, wherein CRS is reduced or prevented in the patient and the method results in a reduction in tumor volume in the patient.

[0277] 114. A method according to any one of items 1 to 113, in which weight loss due to CRS is reduced or prevented.

[0278] 115. The method of any of paragraphs 90 to 114, further comprising re-administering the compound or a pharma- ceutically acceptable salt thereof to the patient.

[0279] 116. The method of paragraph 115, wherein subsequent administration of the compound or a pharma- ceutically acceptable salt thereof results in CAR T cell activation and increased cytokine levels in the patient.

[0280] 117. The method of any of paragraphs 1 to 116, wherein the CAR T cell composition is administered prior to the compound or a pharma- ceutically acceptable salt thereof.

[0281] 118. The method of any of paragraphs 1 to 117, wherein the CAR T cells are autologous.

[0282] 119. The method of any of paragraphs 2 to 17, wherein a first dose escalation course comprises administering to the patient about 1%, about 2%, and about 20% of the total dose of the compound or pharma- ceutically acceptable salt thereof on three non-consecutive days, a second dose escalation course comprises administering to the patient about 1%, about 6%, and about 60% of the total dose of the compound or pharma-ceutically acceptable salt thereof on three non-consecutive days, and a third dose escalation course comprises administering to the patient about 1%, about 10%, and about 100% of the total dose of the compound or pharma-ceutically acceptable salt thereof on three non-consecutive days.

[0283] 120. The method of any of paragraphs 2 to 17, wherein a first dose escalation course comprises administering to the patient about 1%, about 10%, and about 100% of the total dose of the compound or pharma- ceutically acceptable salt thereof on three non-consecutive days, and a second dose escalation course comprises administering to the patient about 1%, about 20%, and about 200% of the total dose of the compound or pharma- ceutically acceptable salt thereof on three non-consecutive days.

[0284] 121. The method of any of paragraphs 2 to 17, wherein a first dose escalation course comprises administering to the patient about 1%, about 10%, and about 100% of the total dose of the compound or pharma- ceutically acceptable salt thereof on three non-consecutive days, and a second dose escalation course comprises administering to the patient about 1%, about 10%, and about 100% of the total dose of the compound or pharma- ceutically acceptable salt thereof on three non-consecutive days.

[0285] 122. The method of any of paragraphs 2 to 17, wherein a first dose escalation course comprises administering to the patient about 1%, about 20%, and about 200% of the total dose of the compound or pharma- ceutically acceptable salt thereof on three non-consecutive days, and a second dose escalation course comprises administering to the patient about 1%, about 20%, and about 200% of the total dose of the compound or pharma- ceutically acceptable salt thereof on three non-consecutive days.

[0286] 123. The method according to any of items 119 to 122, wherein the total dose of the compound or a pharma- ceutically acceptable salt thereof is about 500 nmol / kg.

[0287] Accordingly, all applicable embodiments described in the preceding paragraphs and subsections recited above and in this Detailed Description of Illustrative Embodiments, Summary section, Examples and Claims apply to these embodiments.

[0288] As used herein, a "patient" may be a human, or in veterinary applications, the patient may be a laboratory animal, an agricultural animal, a domestic animal, or a wild animal.In various embodiments, the patient may be a laboratory animal such as a rodent (e.g., mouse, rat, hamster, etc.), rabbit, monkey, chimpanzee, or a domestic animal such as a dog, cat, or rabbit, or a farm 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.

[0289] In various embodiments, the cancer to be treated may be selected from carcinoma, sarcoma, lymphoma, melanoma, mesothelioma, nasopharyngeal carcinoma, leukemia, adenocarcinoma, or myeloma. In other embodiments, the cancer includes 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, 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, adrenal cancer, soft tissue sarcoma, pediatric or non-pediatric osteosarcoma. The cancer may be osteosarcoma, urethral cancer, prostate cancer, chronic leukemia, acute leukemia including acute myeloid leukemia, lymphocytic lymphoma, myeloid leukemia, myelomonocytic leukemia, hairy cell leukemia, pleural mesothelioma, bladder cancer, Burkitt's lymphoma, ureteral cancer, kidney cancer, renal cell carcinoma, renal pelvis cancer, central nervous system (CNS) neoplasms, primary CNS lymphoma, spinal axis tumors, brain stem glioma, pituitary adenoma, and esophagogastric junction adenocarcinoma.

[0290] In some of these embodiments, the cancer is a folate receptor expressing cancer. In other embodiments, the cancer is a folate receptor alpha expressing cancer. In yet other embodiments, the cancer is a folate receptor beta expressing cancer. In some of these embodiments, the cancer is endometrial cancer, non-small cell lung cancer, ovarian cancer, osteosarcoma, including pediatric or non-pediatric osteosarcoma, or triple negative breast cancer. In other embodiments, the cancer being treated is a tumor. In other embodiments, the cancer is malignant. In other embodiments, the cancer is osteosarcoma, including pediatric or non-pediatric osteosarcoma.

[0291] In certain embodiments, the "small molecule ligand" is selected from the group consisting of folate, DUPA (a ligand bound by PSMA-positive human prostate cancer cells and other cancer cell types), NK-1R ligands (receptors for NK-1R ligands are found, for example, in colon and pancreatic cancers), CAIX ligands (receptors for CAIX ligands are found, for example, in renal, ovarian, vulvar and breast cancers), ligands for gamma glutamyl transpeptidase (transpeptidases are found, for example, in ovarian cancer, colon cancer, liver cancer, astrocyte neuronal The ligands may be small molecule ligands that specifically bind to cancer cell types (i.e., receptors for each of these ligands may be overexpressed in cancer compared to normal tissues), NKG2D ligands (receptors for NKG2D ligands are found, for example, in cancers of the lung, colon, kidney, prostate, and T and B cell lymphomas), or CCK2R ligands (receptors for CCK2R ligands are found, among others, in cancers of the thyroid, lung, pancreas, ovary, brain, stomach, gastrointestinal stromal, and colon), each of which is a small molecule ligand that specifically binds to a cancer cell type (i.e., receptors for each of these ligands may be overexpressed in cancer compared to normal tissues).

[0292] In certain embodiments, the small molecule ligand can 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 other embodiments, the small molecule ligand can 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.

[0293] In certain embodiments, the DUPA derivative can be a ligand of a small molecule ligand linked to a targeting moiety, and the DUPA derivatives are described in WO2015 / 057852, which is incorporated herein by reference.

[0294] In some embodiments, the small molecule ligand in the description "small molecule ligand attached to a linker" is folate. In various embodiments, folate can be folic acid, folic acid analogs or other folate receptor binding molecules. In various embodiments, folate analogs that can be used can be folinic acid (e.g., leucovorin), pteropolyglutamic acid and folate receptor binding pteridines, such as tetrahydropterin, dihydrofolic acid, tetrahydrofolic acid and deaza and dideaza analogs thereof. The terms "deaza" and "dideaza" analogs refer to art-recognized analogs that have carbon atoms in place of one or two nitrogen atoms of 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 folate analogs are conventionally referred to as "folates," reflecting their ability to bind to the folate receptor. Other folate receptor binding analogs include aminopterin, amethopterin (methotrexate), N10-methylfolate, 2-deamino-hydroxyfolate, deaza analogs such as 1-deazamethopterin or 3-deazamethopterin, and 3',5'-dichloro-4-amino-4-deoxy-N10-methylpteroylglutamic acid (dichloromethotrexate).

[0295] In another embodiment, the small molecule ligand referred to as "a small molecule ligand attached to a linker" is represented by the formula [ka] [In the formula, X 1 and Y 1 each independently represents halo, R 2 , OR 2 , S.R. 3and N.R. 4 R 5 selected from the group consisting of; U, V and W are each independently -(R 6a )C=, -N=, -(R 6a )C(R 7a )- and -N(R 4a )-; Q is selected from the group consisting of C and CH; T is selected from the group consisting of S, O, N, and -C=C-; X 2 and X 3 are each independently oxygen, sulfur, -C(Z)-, -C(Z)O-, -OC(Z)-, -N(R 4b )-, -C(Z)N(R 4b )-, -N(R 4b )C(Z)-, -OC(Z)N(R 4b )-, -N(R 4b )C(Z)O-, -N(R 4b )C(Z)N(R 5b )-, -S(O)-, -S(O)2-, -N(R 4a )S(O)2-, -C(R 6b )(R 7b )-, -N(C≡CH)-, -N(CH2C≡CH)-, C1-C 12 Alkylene and C1-C 12 alkyleneoxy, where X is oxygen or sulfur; R 1 are hydrogen, halo, C1-C 12 Alkyl and C1-C 12 alkoxy; R 2 , R 3 , R 4 , R 4a , R 4b , R 5 , R 5b , R 6b and R 7b each independently represents hydrogen, halo, C1-C 12 Alkyl, C1-C 12 Alkoxy, C1-C 12 Alkanoyl, C1-C 12 Alkenyl, C1-C 12Alkynyl, (C1-C 12 Alkoxy)carbonyl and (C1-C 12 alkylamino)carbonyl; R 6 and R 7 each independently represents hydrogen, halo, C1-C 12 Alkyl and C1-C 12 alkoxy; or R 6 and R 7 together to form a carbonyl group; R 6a and R 7a each independently represents hydrogen, halo, C1-C 12 Alkyl and C1-C 12 alkoxy; or R 6a and R 7a together to form a carbonyl group; p, r, s and t are each independently 0 or 1; and * is an optional covalent bond to the remainder of the conjugate if any additional chemical moieties are part of the folate. may have:

[0296] In some embodiments, 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, tetrafluorophenyl ester, knottin, centirin, DARPin, affibody, affilin, anticalin, atrimer, avimer, bicyclic peptide, FN3 scaffold, cys knot, finomer, Kunitz domain, or Obody. The properties of the targeting moiety are limited only by the fact that it is recognized and bound by the CAR, preferably specifically, and is of relatively low molecular weight. In various embodiments, an exemplary targeting moiety is a hapten, including a small molecular weight organic molecule.

[0297] In one illustrative embodiment, the targeting moiety has the following illustrative structure: [ka] wherein X is oxygen, nitrogen or sulfur, X is attached to a linker L; Y is OR a , N.R. a 2 or NR a 3 + and Y' is O, NR a or NR a 2 + each R is independently selected at each occurrence from H, fluoro, sulfonic acid, sulfonate, and salts thereof, and the like; and R a is hydrogen or alkyl. may have:

[0298] In certain illustrative embodiments, the linker may comprise polyethylene glycol (PEG), polyproline, a hydrophilic amino acid, a sugar, a non-natural peptidoglycan, polyvinylpyrrolidone, Pluronic F-127, or a combination thereof.

[0299] In other illustrative embodiments, the linker in the compounds described herein or pharma- ceutically acceptable salts thereof may include a direct bond (e.g., a reaction between an isothiocyanate group of FITC of the small molecule ligand and a free amine group) or the bond may be via an intermediate linker. In certain embodiments, the intermediate linker, if present, may be any biocompatible linker known in the art, such as a bivalent linker. In certain illustrative embodiments, the bivalent linker may include about 1 to about 30 carbon atoms. In other illustrative embodiments, the bivalent linker may 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 Daltons) are used. In other embodiments, suitable linker lengths 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 or more atoms.

[0300] In various embodiments, the small molecule ligand that is attached to the targeting moiety has the formula [ka] wherein B is a small molecule ligand, L is a linker, and T is a targeting moiety, wherein L is of the formula [ka] (wherein n is an integer of 0 to 200). The structure includes the following structure: In other embodiments, n may be 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, 15-38, 15-39, 15-40, 15-50, 15-60, 15-70, 15-80, 15-90, 15-100, 15-110, 15-120, 15-130, 15-140, 15-150, or n may be an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 1 It can be 3, 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.

[0301] In other embodiments, the linker may be a bivalent linker that may include one or more spacers. Illustrative spacers are shown in the table below. The following non-limiting illustrative spacers are listed where * indicates the point of attachment to a small molecule ligand or targeting moiety or another bivalent linker moiety. [Table 1]

[0302] In other embodiments, the small molecule ligand (bridge) attached to the targeting moiety may have any of the following structures: [ka] [ka] [ka]

[0303] In other embodiments, the compound or a pharma- ceutically acceptable salt thereof is not an antibody and does not include a fragment of an antibody, hi yet other embodiments, the targeting moiety does not include a peptide epitope.

[0304] In one illustrative embodiment, the small molecule ligand (bridge) linked to the targeting moiety by a linker comprises fluorescein isothiocyanate (FITC) that binds to the small molecule ligand. In one embodiment, the cancer can overexpress a receptor for the small molecule ligand. In another embodiment, for example, cytotoxic T cells or other types of T cells can be transformed to express a CAR that comprises an anti-FITC scFv. In this embodiment, the CAR can be targeted FITC, where the cancer is decorated 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 or reduced. In this embodiment, when anti-FITC CAR-expressing T cells bind to FITC, the CAR T cells are activated and the cancer is ameliorated.

[0305] The "pharmaceutical acceptable salt" of the small molecule ligand linked to the targeting moiety by the linker is considered. As used herein, the term "pharmaceutical acceptable salt" refers to a salt whose counter ion can be used in medicine. In various embodiments, such salts include, but are not limited to, 1) the acid addition salts that can be obtained by reacting the parent compound of free base with inorganic acid, such as hydrochloric acid, hydrobromic acid, nitric acid, phosphoric acid, sulfuric acid and perchloric acid, or organic acid, 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 with metal ions, such as alkali metal ions, alkaline earth ions or aluminum ions; or by coordination 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 all such pharma-ceutically acceptable salts are contemplated in connection with the embodiments described herein.

[0306] In various embodiments, suitable acid addition salts are formed from acids which form non-toxic salts. Illustrative examples 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.

[0307] In various embodiments, suitable base salts are formed from bases that form non-toxic salts. Illustrative examples include arginine salts, benzathine salts, calcium salts, choline salts, diethylamine salts, diolamine salts, glycine salts, lysine salts, magnesium salts, meglumine salts, olamine salts, potassium salts, sodium salts, tromethamine salts and zinc salts. Hemisalts of acids and bases can also be formed, such as hemisulfate and hemicalcium salts.

[0308] In some illustrative embodiments, the compounds described herein or pharma- ceutically acceptable salts thereof may contain one or more chiral centers or may otherwise be capable of existing as multiple stereoisomers. Thus, various embodiments may include pure stereoisomers as well as stereoisomeric mixtures, such as enantiomers, diastereomers, and enantiomerically or diastereomerically enriched mixtures. In some embodiments, the compounds described herein or pharma- ceutically acceptable salts thereof may exist as geometric isomers. Thus, various embodiments may include pure geometric isomers or geometric isomeric mixtures.

[0309] In certain embodiments, the compounds described herein or pharma- ceutically acceptable salts thereof can exist in solvated forms, including unsolvated forms and hydrated forms. In general, the solvated forms are equivalent to the unsolvated forms and are encompassed within the scope of the present invention.

[0310] The methods described herein also utilize T lymphocytes (e.g., cytotoxic T lymphocytes) engineered to express a chimeric antigen receptor (CAR) that recognizes and binds to a crosslinked targeting moiety (e.g., FITC, DNP, or TNP). In one embodiment, the CAR described herein comprises three domains, including 1) a recognition region (e.g., a single-chain fragment variable (scFv) region of an antibody, a Fab fragment, etc.) that specifically recognizes and binds to a targeting moiety, 2) a costimulatory domain that enhances T lymphocyte proliferation and survival, and 3) an activation signaling domain that generates a T lymphocyte activation signal.

[0311] In various embodiments, as non-limiting examples, scFv regions of antibodies that bind to 2,4-dinitrophenol (DNP), 2,4,6-trinitrophenol (TNP), biotin, digoxigenin, fluorescein, fluorescein isothiocyanate (FITC), NHS-fluorescein, pentafluorophenyl ester, tetrafluorophenyl ester, knottin, centirin, DARPin, affibody, affilin, anticalin, atrimer, avimer, bicyclic peptide, FN3 scaffold, cys knot, finomer, Kunitz domain, or Obody may be used. In illustrative, non-limiting embodiments, the scFv region can be produced from (i) antibodies known in the art to bind to a targeting moiety, (ii) antibodies prepared de novo using a selected targeting moiety, such as a hapten, and (iii) sequence variants derived from the scFv region of such antibodies, e.g., an scFv region having at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, 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 it is derived.

[0312] In some embodiments, the costimulatory domain acts to enhance the proliferation and survival of cytotoxic T lymphocytes by binding to the targeting portion of the CAR. Suitable costimulatory domains include, but are not limited to, CD28, CD137 (4-1BB), tumor necrosis factor (TNF) receptor family members expressed on activated T cells, CD134 (OX40), members of the TNFR superfamily of receptors, CD27, CD30, CD150, DAP10, NKG2D and CD278 (ICOS), CD28 superfamily costimulatory molecules, or combinations thereof. Those skilled in the art will understand that sequence variants of these costimulatory domains can be utilized without adversely affecting the present invention, where the variants 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 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or at least about 99.5% sequence identity to the amino acid sequence of the domain from which it is derived.

[0313] In an illustrative embodiment, the activation signaling domain serves to activate T lymphocytes (e.g., cytotoxic T lymphocytes) by binding to the targeting portion of the CAR. In various embodiments, suitable activation signaling domains include T cell CD3 zeta chain, CD3 delta receptor protein, mbl receptor protein, B29 receptor protein, and Fc receptor gamma. Those skilled in the art will recognize that sequence variants of these activation signaling domains can be used when the variants have the same or similar activity as the domains they model. In various embodiments, the variants have at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity with the amino acid sequence of the domain from which they are derived.

[0314] In some embodiments, the construct encoding the CAR is produced using genetic engineering techniques, such as those detailed in Sambrook et al., "Molecular Cloning: A Laboratory Manual", 3rd Edition, Cold Spring Harbor Laboratory Press, (2001), incorporated herein by reference, and Green and Sambrook, "Molecular Cloning: A Laboratory Manual", 4th Edition, Cold Spring Harbor Laboratory Press, (2012).

[0315] By way of example, a plasmid or viral expression vector (e.g., lentiviral vector, retroviral vector, sleeping beauty and piggyback (transposon / transposase systems including non-viral mediated CAR gene delivery systems)) can be produced that encodes a fusion protein comprising 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 arrangements are permissible, including the recognition region, the activation signaling domain and one or more costimulatory domains. In certain embodiments, the arrangement of the recognition region on the fusion protein is such that presentation of the region on the exterior of the CAR T cell is generally achieved. In certain embodiments, the CAR may include additional elements such as a signal peptide (e.g., CD8α 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 (e.g., CD8α transmembrane domain, CD28 transmembrane domain or CD3ζ transmembrane domain) and a hinge domain (e.g., CD8α hinge) that confers flexibility to the recognition region and allows for strong binding to the targeting moiety.

[0316] Schematic diagrams of exemplary CARs are shown in Figures 2 and 3. With reference to Figure 2, the fusion protein sequence can be incorporated into a lentiviral expression vector, where "SP" is the signal peptide, the CAR is an anti-FITC CAR, the CD8α hinge and CD8α transmembrane domains are present, the costimulatory domain is 4-1BB, and the activation signaling domain is CD3ζ. Exemplary nucleic acid sequences of the CAR insert are provided as SEQ ID NOs: 1 and 3, and the encoded amino acid sequence is provided as SEQ ID NO: 2. In yet other embodiments, SEQ ID NO: 2 can comprise or consist of a humanized or human amino acid sequence.

[0317] With reference to FIG. 3, a schematic diagram of an exemplary CAR construct is shown in which the expressed CAR comprises an E2 anti-fluorescein antibody fragment, where the fusion protein sequence can be incorporated into an expression vector, and the CAR comprises an E2 anti-fluorescein antibody fragment, an IgG4 hinge domain, a CD28 transmembrane domain, a costimulatory domain is CD137 (4-1BB), and an activation signaling domain is CD3ζ. The CAR can comprise additional suitable domains. An exemplary nucleic acid sequence of such a CAR insert is provided as SEQ ID NO: 4, and an exemplary encoded amino acid sequence is provided as SEQ ID NO: 5. As used herein, "SEQ ID NO: 4" refers to the sequence beginning with the underlined "agc" codon and ending with the underlined "ggc" codon. This portion of the long sequence encodes the CAR inserted into the T cell membrane. Other portions of the long sequence include coding sequences for signal peptides, EGFRt domains, etc., that are not part of the membrane-inserted CAR and function as a chimeric antigen receptor. As used herein, "SEQ ID NO: 5" refers to the sequence beginning with the underlined "S" and ending with the underlined "G". This part of the long sequence is the amino acid sequence of the CAR inserted into the T cell membrane. Other parts of the long sequence include amino acid sequences that are not part of the CAR inserted into the membrane, but function as chimeric antigen receptors, such as signal peptides, EGFRt domains, etc. In yet other embodiments, SEQ ID NO:5 can comprise or consist of humanized or human amino acid sequences. SEQ ID NOs:4 and 5 are as above and are shown below. The start and stop codons of the long nucleic acid sequence are underlined, and the long sequence is an exemplary sequence that can be used to transduce T cells used in the methods described herein.

[0318] SEQ ID NO:4 (E2 anti-fluorescein antibody fragment CAR nucleic acid sequence (insert)) [Table 2]

[0319] SEQ ID NO:5 (E2 anti-fluorescein antibody fragment CAR amino acid sequence (insert)) [Table 3]

[0320] Another exemplary CAR construct is the 4M5.3 CAR, which is shown diagrammatically in FIG. 3. As used herein, "SEQ ID NO: 6" refers to the following sequence beginning with the underlined "gac" codon and ending with the underlined "ggc" codon: This portion of the long sequence encodes an exemplary 4M5.3 CAR. The CAR is inserted into the T cell membrane. The other portion of the long sequence includes coding sequences for a signal peptide, EGFRt domain, etc., which are not part of the membrane-inserted CAR but function as a chimeric antigen receptor. As used herein, "SEQ ID NO: 7" refers to the following sequence beginning with the underlined "D" and ending with the underlined "G". This portion of the long sequence is the amino acid sequence of the CAR that is inserted into the T cell membrane. The other portion of the long sequence includes amino acid sequences for a signal peptide, EGFRt domain, etc., which are not part of the membrane-inserted CAR but function as a chimeric antigen receptor. In yet other embodiments, SEQ ID NO: 7 can comprise or consist of a humanized or human amino acid sequence. SEQ ID NOs: 6 and 7 are as described above and are shown below. The start and stop codons of the long nucleic acid sequences are underlined, and the long sequences are exemplary sequences that can be used to transduce T cells to produce the 4M5.3 CAR.

[0321] SEQ ID NO:7 [4M5.3-CAR amino acid sequence (insert)] [Table 4]

[0322] SEQ ID NO:6 [4M5.3-CAR nucleotide acid sequence (insert)] [Table 5]

[0323] In one embodiment, the CAR has a recognition region, which is a single-chain fragment variable (scFv) region of an anti-FITC antibody, the costimulatory domain and the costimulatory domain are CD137 (4-1BB), and the activation signaling domain is the T cell CD3 zeta chain. Those skilled in the art are aware that anti-FITC scFv and anti-fluorescein scFv are synonymous terms.

[0324] In some embodiments, T lymphocytes (e.g., cytotoxic T lymphocytes) can be genetically modified to express a CAR construct by transfecting a population of T lymphocytes with an expression vector encoding the CAR construct. Suitable methods for producing a population of transduced 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) (incorporated herein by reference) and Green and Sambrook, "Molecular Cloning: A Laboratory Manual", 4th Edition, Cold Spring Harbor Laboratory Press, (2012) (incorporated herein by reference).

[0325] In certain embodiments, CAR T cells are provided that comprise a nucleic acid of SEQ ID NO: 1, 3, 4 or 6. In other embodiments, CAR T cells are provided that comprise a polypeptide of SEQ ID NO: 2, 5 or 7. In other illustrative embodiments, a nucleic acid (e.g., an isolated nucleic acid) is provided that comprises SEQ ID NO: 1, 3, 4 or 6 and encodes a chimeric antigen receptor. In yet other embodiments, a chimeric antigen receptor polypeptide is provided that comprises SEQ ID NO: 2, 5 or 7. In other embodiments, a vector is provided that comprises SEQ ID NO: 1, 3, 4 or 6. In other embodiments, a lentiviral vector is provided that comprises SEQ ID NO: 1, 3, 4 or 6. In yet other embodiments, SEQ ID NO: 2, 5 or 7 can comprise or consist of a humanized or human amino acid sequence.

[0326] 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 NOs: 1-7. In other embodiments, 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 NOs: 1, 3, 4, or 6, so long as the variant sequence encodes a polypeptide of SEQ ID NO: 2 (for SEQ ID NOs: 1 and 3), SEQ ID NO: 5 (for SEQ ID NO: 4), or SEQ ID NO: 7 (for SEQ ID NO: 6). In other embodiments, the nucleic acid sequence or amino acid sequence may be a variant nucleic acid or amino 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 over 200 nucleic acids with respect to SEQ ID NO: 1, 3, 4 or 6 or over 200 amino acids with respect to SEQ ID NO: 2, 5 or 7. In some embodiments, the percent identity or similarity determination between sequences can be determined, for example, using the GAP program (Genetics Computer Group, software; currently available from Accelrys at http: / / www.accelrys.com), and alignment can be performed, for example, using the ClustalW algorithm (VNTI software, InforMax Inc.). A sequence database can be searched using the nucleic acid or amino acid sequence of interest. The algorithm for database search is generally based on the BLAST software (Altschul et al., 1990). In some embodiments, percent identity can be determined over the entire length of a nucleic acid or amino acid sequence.

[0327] Also within the scope of the present invention are nucleic acids complementary to the nucleic acids represented by SEQ ID NO: 1, 3, 4 or 6 and nucleic acids hybridizing to the nucleic acids represented by SEQ ID NO: 1, 3, 4 or 6 or their complements under high stringency conditions. According to the present invention, "high stringency conditions" refers to hybridization in 5xSSPE and 50% formamide at 65°C and washing with 0.5xSSPE at 65°C. Conditions for high stringency, low stringency and medium stringency hybridization are described in Sambrook et al., "Molecular Cloning: A Laboratory Manual", 3rd Edition, Cold Spring Harbor Laboratory Press, (2001) (incorporated herein by reference) and Green and Sambrook, "Molecular Cloning: A Laboratory Manual", 4th Edition, Cold Spring Harbor Laboratory Press, (2012) (incorporated herein by reference). In one illustrative embodiment, hybridization occurs over the entire length of the nucleic acid.

[0328] In some embodiments, the T lymphocytes used in the methods described herein (e.g., the cytotoxic T lymphocytes used in the production of CAR T cells) can be autologous cells, but xenogeneic cells can also be used, such as when the patient being treated is undergoing high-dose chemotherapy or radiation treatment to destroy the patient's immune system.In some embodiments, allogeneic cells can be used.

[0329] In some embodiments, T lymphocytes can be obtained from a patient by means well known in the art. For example, T cells (e.g., cytotoxic T cells) can be isolated by drawing peripheral blood from a patient, subjecting the blood to Ficoll density gradient centrifugation, and then isolating the T cells using a negative T cell isolation kit (e.g., EasySep TMThe T cell population can be obtained by isolating the T cell population from peripheral blood using a T cell isolation kit (T cell isolation kit). In certain illustrative embodiments, the population of T lymphocytes (e.g., cytotoxic T cells) need not be pure and may include other cells such as other types of T cells (e.g., in the case of cytotoxic T cells), monocytes, macrophages, natural killer cells, and B cells. In certain embodiments, the harvested population may 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.

[0330] In certain embodiments, after obtaining T lymphocytes (e.g., cytotoxic T cells for use in producing CAR T cells), the cells are cultured under conditions that promote activation of the cells. In this embodiment, the culture conditions can be such that the cells can be administered to a patient without concerns about reactivity with culture medium components. For example, the culture conditions do not include bovine serum products, such as bovine serum albumin. In certain illustrative embodiments, activation can be achieved by the introduction of known activators into the culture medium, such as anti-CD3 antibodies in the case of cytotoxic T cells. Other suitable activators include anti-CD28 antibodies. In certain embodiments, the population of lymphocytes can be cultured under conditions that promote activation for about 1 to about 4 days. In certain embodiments, the appropriate level of activation can be determined by cell size, proliferation rate, or activation markers determined by flow cytometry.

[0331] In one illustrative embodiment, after a population of T lymphocytes (e.g., cytotoxic T lymphocytes used to produce CAR T cells) have been cultured under conditions that promote activation, the cells can be transfected with an expression vector encoding a CAR. Suitable vectors and transfection methods for use in various embodiments are described above. In one embodiment, after transfection, the cells can be administered to a patient immediately 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 or more days, or about 5 to about 12 days, about 6 to about 13 days, about 7 to about 14 days, or about 8 to about 15 days, e.g., to allow the cells time to recover from the transfection. In one embodiment, suitable culture conditions can be similar to the conditions under which the cells were cultured for activation, with or without the agents used to promote activation.

[0332] Thus, as noted above, in certain illustrative embodiments, the methods of treatment described herein further include 1) obtaining a population of autologous or heterologous T lymphocytes (e.g., the cytotoxic T lymphocytes used to generate the CAR T cells), 2) culturing the T lymphocytes under conditions that promote activation of the cells, and 3) transfecting the lymphocytes with an expression vector encoding a CAR to form the CAR T cells.

[0333] In some embodiments, culture media free of any animal materials, such as bovine serum, may be used for culturing CAR T cells. In other embodiments, tissue culture conditions commonly used by those skilled in the art to avoid contamination with bacteria, fungi, and mycoplasma may be used. In exemplary embodiments, cells (e.g., CAR T cells) are pelleted, washed, and resuspended in a pharma- ceutically acceptable carrier or diluent before administration to a patient. Exemplary compositions that include CAR-expressing T lymphocytes (e.g., cytotoxic T lymphocytes) are compositions that include cells in sterile 290 mOsm saline, injectable cryopreservation medium (Plasma-Lyte A, dextrose, sodium chloride injection, containing human serum albumin and DMSO), 0.9% NaCl with 2% human serum albumin, or any other sterile 290 mOsm injectable material. Alternatively, in other embodiments, depending on the characteristics of the culture medium, CAR T cells can be administered as a composition in the culture medium or concentrated and resuspended in the culture medium before administration. In various embodiments, the CAR T cell composition may be administered to the patient by any suitable means, such as parenteral administration, e.g., intradermally, subcutaneously, intramuscularly, intraperitoneally, intravenously or intrathecally.

[0334] In certain embodiments, the total number of CAR T cells and the concentration of cells in the composition administered to a patient will vary depending on a number of factors, including the type of T lymphocyte (e.g., cytotoxic T lymphocyte) used, the binding specificity of the CAR, the characteristics of the targeting moiety and small molecule ligand, the characteristics of the cancer, the site 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. In various embodiments, compositions comprising suitable transduced CAR T cells have a volume of about 0.1 ml to about 200 ml and about 0.1 ml to about 125 ml.

[0335] In various embodiments, the transduced CAR T cells administered to the patient are about 1×10 5 ~Approx. 1×10 15 or 1×10 6 ~Approx. 1×10 15 In various embodiments, the transduced CAR T cells may consist of about 1×10 5~Approx. 1×10 10 , about 1×10 6 ~Approx. 1×10 10 , about 1×10 6 ~Approx. 1×10 9 , about 1×10 6 ~Approx. 1×10 8 , about 1×10 6 ~about 2×10 7 , about 1×10 6 ~Approx. 3×10 7 , about 1×10 6 ~Approx. 1.5×10 7 , about 1×10 6 ~Approx. 1×10 7 , about 1×10 6 ~Approx. 9×10 6 , about 1×10 6 ~Approx. 8×10 6 , about 1×10 6 ~Approx. 7×10 6 , about 1×10 6 ~about 6×10 6 , about 1×10 6 ~Approx. 5×10 6 , about 1×10 6 ~Approx. 4×10 6 , about 1×10 6 ~Approx. 3×10 6 , about 1×10 6 ~about 2×10 6 , about 2×10 6 ~about 6×10 6 , about 2×10 6 ~Approx. 5×10 6 , about 3×10 6 ~about 6×10 6 , about 4×10 6 ~about 6×10 6 , about 4×10 6 ~Approx. 1×10 7 , about 1×10 6 ~Approx. 1×10 7 , about 1×10 6 ~Approx. 1.5×10 7 , about 1×10 6 ~about 2×10 7 , about 0.2×10 6 ~Approx. 1×10 7 , about 0.2×10 6 ~Approx. 1.5×10 7 , about 0.2×10 6~about 2×10 7 , about 0.2×10 6 ~Approx. 3×10 7 , about 0.2×10 6 ~Approx. 4×10 7 , about 0.2×10 6 ~Approx. 5×10 7 , about 0.2×10 5 ~Approx. 1.5×10 6 , about 0.5×10 5 ~Approx. 1.5×10 6 , about 0.2×10 5 ~Approx. 1.4×10 6 , about 0.2×10 5 ~Approx. 1.3×10 6 , about 0.5×10 5 ~Approx. 1.3×10 6 , about 0.8×10 6 ~about 2×10 7 , about 0.8×10 6 ~Approx. 1.5×10 7 , about 0.9×10 6 ~Approx. 1.2×10 7 or about 0.5 x 10 6 , 1×10 6 , 5×10 6 , 6×10 6 , 7×10 6 , 8×10 6 , 9×10 6 , 1×10 7 , 1.5×10 7 , 2×10 7 , 3×10 7 , 4×10 7 or 5×10 7 The CAR T cells may be administered to the patient. These amounts are per kg of patient weight.

[0336] In other embodiments, the dose of CAR T cells administered to a patient in the CAR T cell composition is selected from the group consisting of about 1 million, about 2 million, about 3 million, about 4 million, about 5 million, about 6 million, about 7 million, about 8 million, about 9 million, about 10 million, about 11 million, about 12 million, about 12.5 million, about 13 million, about 14 million, and about 15 million CAR T cells. These amounts are per kg of patient body weight.

[0337] In any of the embodiments described in this paragraph, the CAR T cell dose can be the number of CAR T cells per kg of patient body weight. In an embodiment, in any of the embodiments described herein, a single dose or multiple doses of CAR T cells can be administered to the patient. In an illustrative embodiment, a first dose of CAR T cells and a second dose of CAR T cells can be administered to the patient. In an embodiment, the first dose of CAR T cells can be a test dose to monitor the patient's tolerance to the CAR T cells, and the second dose of CAR T cells can include a higher dose of CAR T cells than the first dose of CAR T cells. In an embodiment, the first dose of CAR T cells is about 0.5×10 5 CAR T cells ~ approximately 1.5 x 10 6 In other embodiments, the second dose of CAR T cells may comprise about 0.8 x 10 6 CAR T cells ~ approximately 2 x 10 7 In these embodiments involving a first and second dose of CAR T cells, any dose of CAR T cells described herein may be administered.

[0338] In any of the embodiments described herein, the CAR T cells may be administered before or after administration of the compound or a pharma- ceutically acceptable salt thereof. As will be understood, the designations i), ii), and iii), etc., of any method steps described herein do not imply any order unless specifically indicated.

[0339] The compounds described herein or their pharmaceutically acceptable salts or CAR T cell compositions may be administered to a patient using any suitable method known in the art. As used herein, the term "administer" or "administration" includes any means of introducing the compounds or their pharmaceutically acceptable salts or CAR T cell compositions into a patient, including, but not limited to, oral, intravenous, intramuscular, subcutaneous, transdermal, etc. In some embodiments, the compounds described herein or their pharmaceutically acceptable salts may be administered in unit dosage forms and / or formulations that include conventional non-toxic pharmaceutically acceptable carriers, adjuvants, and vehicles.

[0340] In some embodiments, the compound or its pharmaceutically acceptable salt or CAR T cell composition described herein can be directly administered into bloodstream, muscle or internal organs.In various embodiments, suitable routes for such parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, epidural, intraventricular, intraurethral, ​​intrasternal, intracranial, intratumoral, intramuscular and subcutaneous delivery.In some embodiments, parenteral administration means include needle (including microneedle) injectors, needle-free injectors and infusion techniques.

[0341] In one illustrative embodiment, the parenteral formulation is generally an aqueous solution that may contain carriers or additives such as salts, carbohydrates, and buffers (preferably pH 3-9), but may be more suitably formulated as a sterile non-aqueous solution or a dry form to be used in combination 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 sterile conditions by lyophilization to produce a sterile lyophilized powder for parenteral formulations may be readily accomplished using standard pharmaceutical techniques well known to those skilled in the art. In some embodiments, the solubility of the compound 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.

[0342] In some embodiments, the amount of the compound or its pharmaceutically acceptable salt administered to a patient may vary significantly depending on the cancer being treated, the route of administration and tissue distribution of the compound or its pharmaceutically acceptable salt. In some embodiments, the amount administered to a patient may be based on body surface area, body weight and physician evaluation.

[0343] In various embodiments, the compound or a pharma- ceutically acceptable salt thereof can be administered 1) at least a first dose escalation process and a second dose escalation process; 2) at least a first dose escalation process, a second dose escalation process and a third dose escalation process; 3) at least a first dose escalation process, a second dose escalation process, a third dose escalation process and a fourth dose escalation process; 4) at least a first dose escalation process, a second dose escalation process, a third dose escalation process, a fourth dose escalation process and a fifth dose escalation process; 5) at least a first dose escalation process, a second dose escalation process, a third dose escalation process, a fourth dose escalation process, a fifth dose escalation process and a sixth dose escalation process; or 6) one or more additional dose escalation processes can be included.

[0344] In various embodiments, for the first, second, third, fourth, fifth, or sixth, etc., dose escalation course, the amount of the compound or a pharma- ceutically acceptable salt thereof administered to the patient is from about 0.1 μg / kg to about 2000 μg / kg, 0.1 μg / kg to about 1500 μg / kg, 0.1 μg / kg to about 1000 μg / kg, 0.1 μg / kg to about 500 μg / kg, about 0.1 μg / kg to about 100 μg / kg, g, about 0.1 μg / kg to about 80 μg / kg, about 0.1 μg / kg to about 70 μg / kg, about 0.1 μg / kg to about 50 μg / kg, about 0.1 μg / kg to about 40 μg / kg, about 0.1 μg / kg to about 30 μg / kg, approximately 0.3μg / kg to approximately 500μg / kg, approximately 0.3μg / kg to approximately 400μg / kg, approximately 0.3μg / kg to approximately 300μg / kg, approximately 0.3μg / kg to approximately 200μg / kg, approximately 0.3 μg / kg to about 100 μg / kg, about 0.3 μg / kg to about 90 μg / kg, about 0.1 μg / kg to about 400 μg / kg, about 0.1 μg / kg to about 350 μg / kg, about 0.1 μg / kg to about 300 μg / kg, about 0.1 μg / kg to about 250 μg / kg, about 0.1 μg / kg to about 200 μg / kg, about 0.1 μg / kg to about 150 μg / kg, or about 0.3 μg / kg to about 150 μg / kg, or It may be 5 μg / kg to about 2000 μg / kg, 5 μg / kg to about 1500 μg / kg, 5 μg / kg to about 1000 μg / kg, 5 μg / kg to about 500 μg / kg, about 5 μg / kg to about 100 μg / kg, about 5 μg / kg to about 80 μg / kg, about 5 μg / kg to about 70 μg / kg, about 5 μg / kg to about 50 μg / kg, about 5 μg / kg to about 40 μg / kg, or about 5 μg / kg to about 30 μg / kg.In other embodiments, for the first and fourth dose escalation courses, the amount of the compound or a pharma- ceutically acceptable salt thereof administered to the patient is from about 0.1 μg / kg to about 100 μg / kg, from about 0.1 μg / kg to about 80 μg / kg, from about 0.1 μg / kg to about 70 μg / kg, from about 0.1 μg / kg to about 50 μg / kg, from about 0.1 μg / kg to about 40 μg / kg, from about 0.1 μg / kg to about 30 μg / kg, or from about 0.3 μg / kg to about 30 μg / kg. The amount of the compound may be about 5 μg / kg, about 5 μg / kg to about 100 μg / kg, about 5 μg / kg to about 80 μg / kg, about 5 μg / kg to about 70 μg / kg, about 5 μg / kg to about 50 μg / kg, about 5 μg / kg to about 40 μg / kg, about 5 μg / kg to about 30 μg / kg, or about 5 μg / kg to about 2000 μg / kg, or about 5 μg / kg to about 1500 μg / kg, or about 5 μg / kg to about 1000 μg / kg, or about 5 μg / kg to about 500 μg / kg. In other embodiments, for the second and fifth dose escalation courses, the amount of the compound or a pharma- ceutically acceptable salt thereof administered to the patient is about 0.3 μg / kg to about 500 μg / kg, about 0.3 μg / kg to about 400 μg / kg, about 0.3 μg / kg to about 300 μg / kg, about 0.3 μg / kg to about 200 μg / kg, about 0.3 μg / kg to about 100 μg / kg, or about 0.3 μg / kg to about 90 μg / kg, or about 5 μg / kg to about 60 μg / kg. The amount of the compound may be from about 5 μg / kg to about 100 μg / kg, from about 5 μg / kg to about 80 μg / kg, from about 5 μg / kg to about 70 μg / kg, from about 5 μg / kg to about 50 μg / kg, from about 5 μg / kg to about 40 μg / kg, from about 5 μg / kg to about 30 μg / kg, or from about 5 μg / kg to about 2000 μg / kg, or from about 5 μg / kg to about 1500 μg / kg, or from about 5 μg / kg to about 1000 μg / kg, or from about 5 μg / kg to about 500 μg / kg.In yet other embodiments, for the third and sixth dose escalation courses, the amount of the compound or a pharma- ceutically acceptable salt thereof administered to the patient is from 0.1 μg / kg to about 400 μg / kg, from about 0.1 μg / kg to about 350 μg / kg, from about 0.1 μg / kg to about 300 μg / kg, from about 0.1 μg / kg to about 250 μg / kg, from about 0.1 μg / kg to about 200 μg / kg, from about 0.1 μg / kg to about 150 μg / kg, or from about 0.3 μg / kg to about 150 μg / kg. μg / kg or about 5 μg / kg to about 100 μg / kg, about 5 μg / kg to about 80 μg / kg, about 5 μg / kg to about 70 μg / kg, about 5 μg / kg to about 50 μg / kg, about 5 μg / kg to about 40 μg / kg, about 5 μg / kg to about 30 μg / kg, or about 5 μg / kg to about 2000 μg / kg, or about 5 μg / kg to about 1500 μg / kg, or about 5 μg / kg to about 1000 μg / kg, or about 5 μg / kg to about 500 μg / kg. In these embodiments, "kg" refers to the patient's body weight in kilograms.

[0345] In any of these embodiments, the range of the amount of the compound or pharma- ceutically acceptable salt thereof can be a range based on a calculation of a percentage of the "total dose" of the compound or pharma- ceutically acceptable salt thereof, where the "total dose" of the compound or pharma- ceutically acceptable salt thereof can be about 30 μg / kg, and the percentage is about 1% to about 100% (see step 1 in FIG. 1), about 1% to about 300% (see step 2 in FIG. 1), and about 1% to about 500% (see step 3 in FIG. 1) of the "total dose" of the compound or pharma- ceutically acceptable salt thereof. In other embodiments, the percentage of the "total dose" of the compound or a pharma- ceutically acceptable salt thereof administered during the dose escalation process is about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 200%, about 300%, about 400%, or about 500%. and the dosage is about 0.3 μg / kg, about 3 μg / kg, about 9 μg / kg, about 15 μg / kg, about 30 μg / kg, about 90 μg / kg, or about 150 μg / kg, or about 17%, about 333%, about 1666%, or about 3333%, and the dosage can be about 5 μg / kg, about 100 μg / kg, about 500 μg / kg, or about 1000 μg / kg, respectively. In these embodiments, "kg" refers to the patient's body weight in kilograms.

[0346] In other embodiments, the percentage of "total dose" of the compound or its pharmaceutically acceptable salt administered in the dose escalation process is about 1%, about 2% and about 20% for the first dose escalation process, about 1%, about 6% and about 60% for the second dose escalation process, and about 1%, about 10% and about 100% for the third dose escalation process, and the "total dose" of the compound or its pharmaceutically acceptable salt can be 500nmol / kg.In this embodiment, "kg" is patient weight in kilograms.In this embodiment, the compound or its pharmaceutically acceptable salt can be administered on Monday, Thursday and Monday, with about 6 days between each dose escalation cycle.

[0347] In other embodiments, the percentage of "total dose" of the compound or its pharmaceutically acceptable salt administered in the dose escalation process can be about 1%, about 10% and about 100% for the first dose escalation process and about 1%, about 20% and about 200% for the second dose escalation process, and the "total dose" of the compound or its pharmaceutically acceptable salt can be 500nmol / kg.In this embodiment, "kg" is patient weight in kilograms.In this embodiment, the compound or its pharmaceutically acceptable salt can be administered on Monday, Thursday and Monday, with about 6 days between each dose escalation cycle.

[0348] In other embodiments, the percentage of the "total dose" of the compound or its pharma- ceutically acceptable salt administered in the dose escalation process may be about 1%, about 10% and about 100% for the first dose escalation process, and about 1%, about 10% and about 100% for the second dose escalation process, and the "total dose" of the compound or its pharma- ceutically acceptable salt may be 500 nmol / kg. In this embodiment, "kg" is the patient's body weight in kilograms. In this embodiment, the compound or its pharma- ceutically acceptable salt may be administered on Monday, Thursday and Monday with about 6 days between each dose escalation cycle. In other embodiments, the compound or its pharma- ceutically acceptable salt may be administered on Monday, Thursday and Monday with about 9 days between each dose escalation cycle. In other embodiments, the dose escalation may be repeated twice, twice, three times, four times, five times, six times, seven times, eight times, nine times or ten times, or any suitable number of times.

[0349] In yet another embodiment, the percentage of the "total dose" of the compound or its pharma- ceutically acceptable salt administered in the dose escalation process can be about 1%, about 20% and about 200% for the first dose escalation process, and about 1%, about 20% and about 200% for the second dose escalation process, and the "total dose" of the compound or its pharma- ceutically acceptable salt can be 500 nmol / kg. In this embodiment, "kg" is the patient's body weight in kilograms. In this embodiment, the compound or its pharma- ceutically acceptable salt can be administered on Monday, Thursday and Monday, with about 6 days between each dose escalation cycle. In other embodiments, the dose escalation can be repeated twice, twice, three times, four times, five times, six times, seven times, eight times, nine times or ten times, or any suitable number of times.

[0350] In other embodiments, the percentage of the "total dose" of the compound or a pharma- ceutically acceptable salt thereof administered in the first or fourth dose escalation course may be about 1%, about 10%, and about 100% (about 10 to about 50 μg / kg, about 20 to about 40 μg / kg, about 25 to about 35 μg / kg, or about 30 μg / kg) of the "total dose" of the compound or a pharma- ceutically acceptable salt thereof in the escalation dose, and the amount of the compound or a pharma- ceutically acceptable salt thereof administered may be about 0.3 μg / kg, about 3 μg / kg, and about 30 μg / kg, respectively. In yet other embodiments, the percentage of the "total dose" of the compound or a pharma- ceutically acceptable salt thereof administered in the second or fifth dose escalation course may be about 1%, about 30%, and about 300% (about 10 to about 50 μg / kg, about 20 to about 40 μg / kg, about 25 to about 35 μg / kg, or about 30 μg / kg) of the "total dose" of the compound or a pharma- ceutically acceptable salt thereof in the escalation dose, and the amount of the compound or a pharma- ceutically acceptable salt thereof administered may be about 0.3 μg / kg, about 9 μg / kg, and about 90 μg / kg, respectively. In yet other embodiments, the percentage of the "total dose" of the compound or its pharma- ceutically acceptable salt administered in the third or sixth dose escalation may be about 1%, about 50%, and about 500% (about 10 to about 50 μg / kg, about 20 to about 40 μg / kg, about 25 to about 35 μg / kg, or about 30 μg / kg) of the "total dose" of the compound or its pharma-ceutically acceptable salt in the escalation dose, and the amount of the compound or its pharma-ceutically acceptable salt administered may be about 0.3 μg / kg, about 15 μg / kg, and about 150 μg / kg, respectively. In these embodiments, "kg" refers to the patient's body weight in kilograms.

[0351] In various other embodiments, the amount to be administered to a patient is, for example, about 0.05 mg to about 30 mg, 0.05 mg to about 25.0 mg, about 0.05 mg to about 20.0 mg, about 0.05 mg to about 15.0 mg, about 0.05 mg to about 10.0 mg, about 0.05 mg to about 9.0 mg, about 0.05 mg to about 8.0 mg, about 0.05 mg to about 7.0 mg, about 0.05 mg to about 6.0 mg, about 0.05 mg to about 5.0 mg, about 0.05 mg to about 10.0 mg, about 0.05 mg to about 15.0 mg, about 0.05 mg to about 10.0 mg, about 0.05 mg to about 10.0 mg, about 0.05 mg to about 15 ... The amount of the active ingredient may range 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, 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.

[0352] In other embodiments, the dose of the compound or a pharma- ceutically acceptable salt thereof is, for example, about 50 nmol / kg to about 3000 nmol / kg of patient body weight, about 50 nmol / kg to about 2000 nmol / kg, about 50 nmol / kg to about 1000 nmol / kg, about 50 nmol / kg to about 900 nmol / kg, about 50 nmol / kg to about 800 nmol / kg, about 50 nmol / kg to about 700 nmol / kg, about 50 nmol / kg to about 600 nmol / kg, about 50 nmol / kg to about 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 1200 nmol / kg, about 50 nmol / kg to about 1400 nmol / kg, about 50 nmol / kg to about 1600 nmol / kg, about 50 nmol / kg to about 1800 nmol / kg, about 50 nmol / kg to about 2000 nmol / kg, about 50 nmol / kg to about 2200 nmol / kg, about 50 nmol / kg to about 3000 nmol / kg, about 50 nmol / kg to about 3200 nmol / kg, about 50 nmol / kg to about 3600 nmol / kg, about 50 nmol / kg to about 4000 nmol / kg, about 50 nmol / kg to about 4000 nmol / kg, about 50 nmol / kg to about 5000 nmol / kg, about 50 nmol / kg to about 6000 nmol / kg, about 50 nmol / The range may be from about 100 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 of the patient's body weight. In other embodiments, the dose is about 1 nmol / kg, about 5 nmol / kg, about 10 nmol / kg, about 20 nmol / kg, about 25 nmol / kg, about 30 nmol / kg, about 40 nmol / kg, about 50 nmol / kg, about 60 nmol / kg, about 70 nmol / kg, about 80 nmol / kg, about 90 nmol / kg, about 100 nmol / kg, about 150 nmol / kg, about 200 nmol / kg, about It can be 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, about 2500 nmol / kg or about 3000 nmol / kg of the patient's body weight.In still other embodiments, the dose is about 0.1 nmol / kg, about 0.2 nmol / kg, about 0.3 nmol / kg, about 0.4 nmol / kg or about 0.5 nmol / kg, about 0.1 nmol / kg to about 1000 nmol / kg, about 0.1 nmol / kg to about 900 nmol / kg, about 0.1 nmol / kg to about 850 nmol / kg, about 0.1 nmol / kg to about 800 nmol / kg, about 0.1 nmol / kg to about 700 nmol / kg, about 0.1 nmol / kg about 0.1 nmol / kg to about 600 nmol / kg, about 0.1 nmol / kg to about 500 nmol / kg, about 0.1 nmol / kg to about 400 nmol / kg, about 0.1 nmol / kg to about 300 nmol / kg, about 0.1 nmol / kg to about 200 nmol / kg, about 0.1 nmol / kg to about 100 nmol / kg, about 0.1 nmol / kg to about 50 nmol / kg, about 0.1 nmol / kg to about 10 nmol / kg, or about 0.1 nmol / kg to about 1 nmol / kg of the patient's body weight. In other embodiments, the dose is about 0.3 nmol / kg to about 1000 nmol / kg, about 0.3 nmol / kg to about 900 nmol / kg, about 0.3 nmol / kg to about 850 nmol / kg, about 0.3 nmol / kg to about 800 nmol / kg, about 0.3 nmol / kg to about 700 nmol / kg, about 0.3 nmol / kg to about 600 nmol / kg, about 0.3 nmol / kg to about 500 nmol / kg, The range may be about 0.3 nmol / kg to about 400 nmol / kg, about 0.3 nmol / kg to about 300 nmol / kg, about 0.3 nmol / kg to about 200 nmol / kg, about 0.3 nmol / kg to about 100 nmol / kg, about 0.3 nmol / kg to about 50 nmol / kg, about 0.3 nmol / kg to about 10 nmol / kg, or about 0.3 nmol / kg to about 1 nmol / kg of patient body weight. In these embodiments, all "kg" refers to patient body weight in kilograms.

[0353] In other embodiments, a first dose escalation step and a second dose escalation step of the compound (e.g., where each dose escalation step is 50 nmol / kg, then 500 nmol / kg) or a pharma- ceutically acceptable salt thereof are performed after administration of the CAR T cells (e.g., in any amount described herein). In this embodiment, for example, after the first and second dose escalation steps in weeks 1 and 2, the level of the compound or a pharma- ceutically acceptable salt thereof can be kept constant in week 3 (e.g., kept constant at 500 nmol / kg) relative to the final dose administered in week 2. In any embodiment described herein, the level of the compound or a pharma- ceutically acceptable salt thereof can be kept constant in the following week relative to the final dose administered in the previous week.

[0354] In various other embodiments, the dose of the compound or a pharma- ceutically acceptable salt thereof is, for example, about 10 nmol / kg to about 10,000 nmol / kg, about 10 nmol / kg to about 5,000 nmol / kg, about 10 nmol / kg to about 3,000 nmol / kg, about 10 nmol / kg to about 2,500 nmol / kg, about 10 nmol / kg to about 2,000 nmol / kg, about 10 nmol / kg to about 1,000 nmol / kg, about 10 nmol / kg to about 900 nmol / kg, or about 10 nmol / kg to about 1500 nmol / kg. / kg, about 10nmol / kg to about 800nmol / kg, about 10nmol / kg to about 700nmol / kg, about 10nmol / kg to about 600nmol / kg, about 10nmol / kg to about 500nmol / kg, about 10nmol / kg~about 400nmol / kg, about 10nmol / kg~about 300nmol / kg, about 10nmol / kg~about 200nmol / kg, about 10nmol / kg~about 150nmol / kg, about 10nmol / kg~about 100nmo l / kg, about 10nmol / kg to about 90nmol / kg, about 10nmol / kg to about 80nmol / kg, about 10nmol / kg to about 70nmol / kg, about 10nmol / kg to about 60nmol / kg, about 10nmol / k g~about 50nmol / kg, about 10nmol / kg~about 40nmol / kg, about 10nmol / kg~about 30nmol / kg, about 10nmol / kg~about 20nmol / kg, about 200nmol / kg~about 900nmol / kg, The range may be from about 200 nmol / kg to about 800 nmol / kg, from about 200 nmol / kg to about 700 nmol / kg, from about 200 nmol / kg to about 600 nmol / kg, from about 200 nmol / kg to about 500 nmol / kg, from about 250 nmol / kg to about 600 nmol / kg, from about 300 nmol / kg to about 600 nmol / kg, from about 300 nmol / kg to about 500 nmol / kg, or from about 400 nmol / kg to about 600 nmol / kg of patient weight. In these embodiments, "kg" refers to the patient's weight in kilograms.

[0355] In all of the above dosage embodiments, the percentage of the "total dose" of the compound or its pharma- ceutically acceptable salt administered at any stage of the dose escalation process can be about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 200%, about 300%, about 400%, or about 500% of the "total dose" of the compound or its pharma- ceutically acceptable salt. The "total dose" of the compound or its pharma- ceutically acceptable salt can be any of the doses of the compound or its pharma- ceutically acceptable salt described in the previous paragraph as the dose administered during the dose escalation process.

[0356] In various other embodiments, the dose of the compound or a pharma- ceutically acceptable salt thereof is, for example, about 1 nmol / kg to about 10,000 nmol / kg, about 1 nmol / kg to about 5,000 nmol / kg, about 1 nmol / kg to about 3,000 nmol / kg, about 1 nmol / kg to about 2,500 nmol / kg, about 1 nmol / kg to about 2,000 nmol / kg, about 1 nmol / kg to about 1,000 nmol / kg, about 1 nmol / kg to about 900 nmol / kg, about 1 nmol / kg to about 800 nmol / kg, about 1 nmol / kg to about 700 nmol / kg, about 1 nmol / kg to about 600 nmol / kg, about 1 nmol / kg to about 500 nmol / kg, about 1 nmol / kg to about 400 nmol / kg, about 1 nmol / kg to about 500 nmol / kg, about 1 nmol / kg to about 600 nmol / kg, about 1 nmol / kg to about 700 nmol / kg, about 1 nmol / kg to about 800 nmol / kg, about 1 nmol / kg to about 900 nmol / kg, about 1 nmol / kg to about 1000 nmol / kg, about 1 nmol / kg to about 1500 nmol / kg, about 1 nmol / kg to about 1600 nmol / kg, about 1 nmol / kg to about 1700 nmol / kg, about 1 nmol / kg to about 200 nmol / kg, about 1 nmol / kg to about 2500 nmol / kg, about 1 nmol / kg to about 3000 nmol / kg, about 1 nmol / kg to about 3500 nmol / kg, about 1 nmol / kg to about 400 mol / kg, about 1 nmol / kg to about 300 nmol / kg, about 1 nmol / kg to about 200 nmol / kg, about 1 nmol / kg to about 150 nmol / kg, about 1 nmol / kg to about 100 nmol / kg, about 1 nmol / kg to about 90 nmol / kg, about 1 nmol / kg to about 80 nmol / kg, about 1 nmol / kg to about 70 nmol / kg, about 1 nmol / kg to about 60 nmol / kg, about 1 nmol / kg to about 50 nmol / kg, about 1 nmol / kg to about 40 nmol / kg, about 1 nmol / kg to about 30 nmol / kg, or about 1 nmol / kg to about 20 nmol / kg, wherein in these embodiments, "kg" is the patient's body weight in kilograms.

[0357] In other embodiments, the patient may be administered about 20 μg / kg to about 3 mg / kg of the patient's body weight of the compound or a pharma- ceutically acceptable salt thereof, hi other embodiments, the amount may be about 0.2 mg / kg to about 0.4 mg / kg of the patient's body weight.

[0358] In any of the above dosage embodiments, a single dose or multiple doses of the compound or a pharma- ceutically acceptable salt thereof may be administered to the patient.

[0359] In some embodiments, the small molecule ligand bound to the targeting moiety can be administered to the patient before the CAR T cell composition.In other embodiments, the small molecule ligand bound to the targeting moiety can be administered to the patient simultaneously with the CAR T cell composition, in a different formulation or in the same formulation.In yet other embodiments, the small molecule ligand bound to the targeting moiety can be administered to the patient after the CAR T cell composition.

[0360] In certain illustrative embodiments, the timing between administration of the CAR T cells and the small molecule conjugated to the targeting moiety can vary widely depending on factors including the type of CAR T cells used, the binding specificity of the CAR, the characteristics of the targeting moiety and the small molecule ligand, the characteristics of the cancer, the site of the cancer in the patient, the means used to administer the CAR T cells and the small molecule ligand conjugated to the targeting moiety to the patient, and the health, age, and weight of the patient. In certain embodiments, the small molecule ligand conjugated to the targeting moiety can be administered within about 3 hours, 6 hours, 9 hours, 12 hours, 15 hours, 18 hours, 21 hours, 24 hours, 27 hours, 30 hours, 33 hours, 36 hours, 39 hours, 42 hours, 45 hours, 48 ​​hours, or 51 hours, or within about 0.5 days, 1 day, 1.5 days, 2 days, 2.5 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or more, before or after administration of the CAR T cells.

[0361] In some embodiments, any applicable administration schedule known in the art can be used for the administration of the compound or its pharmaceutically acceptable salt or CAR T cell composition.In some embodiments, the administration schedule selected for the compound or its pharmaceutically acceptable salt and CAR T cell composition can take into account the concentration of the compound or its pharmaceutically acceptable salt and the number of CAR T cells that are administered for the cytotoxicity control and CRS control of the CAR T cell composition.

[0362] In some exemplary embodiments, there is a period in which the compound or its pharmaceutically acceptable salt is not administered after the first dose escalation process, the second dose escalation process, the third dose escalation process, the fourth dose escalation process, the fifth dose escalation process, the sixth dose escalation process or further dose escalation process.In various illustrative embodiments, the period in which the compound or its pharmaceutically acceptable salt is not administered can be 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days.In other embodiments, the period in which the compound or its pharmaceutically acceptable salt is not administered can be 6 days, 7 days or 8 days.In still other embodiments, the period in which the compound or its pharmaceutically acceptable salt is not administered can be 7 days.

[0363] In some embodiments, the first dose of CAR T cells and the second dose of CAR T cells are administered to the patient during the first week, for example, on Monday and Thursday. In this embodiment, the first dose escalation process of the compound or its pharmaceutically acceptable salt can then be performed during the second and third week. For example, the compound or its pharmaceutically acceptable salt can be administered on three non-consecutive days, where the non-consecutive days can be Monday and Thursday of the second week and Monday of the third week (see process 1 in Figure 1). In this embodiment, the second dose escalation process of the compound or its pharmaceutically acceptable salt can then be performed during the fourth and fifth week (see process 2 in Figure 1). For example, the compound or its pharmaceutically acceptable salt can be administered on three non-consecutive days, where the non-consecutive days can be Monday and Thursday of the fourth week and Monday of the fifth week. In this embodiment, the third dose escalation process of the compound or its pharmaceutically acceptable salt can then be performed during the sixth and seventh week (see process 3 in Figure 1). For example, the compound or a pharma- ceutically acceptable salt thereof can be administered on three non-consecutive days, where the non-consecutive days can be Monday and Thursday of the sixth week and Monday of the seventh week. In other embodiments, subsequent dose escalation courses can follow a similar sequence, or course 3 can be repeated about seven days after course 3 is completed as "course 2," with no further treatment with the compound or a pharma- ceutically acceptable salt thereof until "course 3" begins (the length of "course 2" is based on the length of time shown in FIG. 1 for "course" 1). In some embodiments, patients can receive four courses of treatment.

[0364] In another illustrative embodiment, a method for treating cancer is provided.The method includes: i) administering at least one dose of CAR T cell composition to a patient, where the CAR T cell comprises a CAR directed to a targeting moiety; ii) administering a compound or its pharmaceutically acceptable salt to a patient, where the compound or its pharmaceutically acceptable salt comprises a small molecule ligand linked to a targeting moiety by a linker; wherein the compound or its pharmaceutically acceptable salt is administered in a first dose escalation course; if severe CRS occurs in the first dose escalation course, the compound or its pharmaceutically acceptable salt is administered using a low dose escalation course; wherein the first dose of the compound or its pharmaceutically acceptable salt in the low dose escalation course is lower than the first dose of the compound or its pharmaceutically acceptable salt administered in the first dose escalation course.In this embodiment, in the low dose escalation course, the compound or its pharmaceutically acceptable salt is administered in about 0.5%, about 5% and about 50% of the total dose of the compound or its pharmaceutically acceptable salt on three non-consecutive days. In this embodiment, the total dose of the compound or a pharma- ceutically acceptable salt thereof can be about 10 μg / kg to about 50 μg / kg, about 20 μg / kg to about 40 μg / kg, about 25 μg / kg to about 35 μg / kg, or about 30 μg / kg. In these embodiments, "kg" refers to the patient's body weight in kilograms.

[0365] In various related embodiments, lymphocytes can be depleted in the patient prior to administering the CAR T cell composition to the patient, for example, using cytoxan, fludarabine and / or etoposide, and the method can further include, but is not limited to, administering platelets to the patient, administering packed red blood cells to the patient, administering cryoprecipitate to the patient, administering intravenous immunoglobulin to the patient, treating with calcium supplements, acid-citrate-dextrose and / or heparin, and / or administering antibacterial therapy to the patient. In certain embodiments, lymphocyte depletion is performed at least about 24 hours prior to administration of the CAR T cells.

[0366] In other embodiments, the method may include a CRS monitoring step. In one illustrative embodiment, if CRS or neurotoxicity is not observed in the patient during the first dose escalation process, the method may proceed to a second dose escalation process. If CRS or neurotoxicity is not observed in the patient during the second dose escalation process, the method may proceed to a third dose escalation process. If CRS or neurotoxicity is not observed in the patient during the third dose escalation process, the method may proceed to a fourth dose escalation process. If CRS or neurotoxicity is not observed in the patient during the fourth dose escalation process, the method may proceed to a fifth dose escalation process. If CRS or neurotoxicity is not observed in the patient during the fifth dose escalation process, the method may proceed to a sixth dose escalation process, and so on.

[0367] In another illustrative embodiment, if fever without hypotension (i.e., non-severe CRS) is identified in the patient during any of the dose titration courses, and neurotoxicity is not identified in the patient, all subsequent doses of the compound or a pharma- ceutically acceptable salt thereof may be administered to the patient at the dose titration course level that caused fever without hypotension. In another embodiment, if CRS (i.e., severe CRS) or neurotoxicity occurs in the patient during any of the dose titration courses, all subsequent doses of the compound or a pharma-ceutically acceptable salt thereof may be administered to the patient at a dose titration course level that is lower than the dose titration course level that caused severe CRS or neurotoxicity in the patient.

[0368] In an embodiment, severe CRS can include any toxicity requiring the use of cetuximab, any > grade 3; autoimmune toxicity, any > grade 3 toxicity attributable to CAR T cell administration or compound or a pharma- ceutically acceptable salt thereof administration and occurring within 28 days after initiation of treatment, with the exception of, or in addition to, < grade 4 lasting less than 48 hours after initiation of treatment; fever, < grade 3 lasting less than 24 hours after initiation of treatment; chills, < grade 3 lasting less than 24 hours after initiation of treatment; cough, < grade 3 lasting less than 7 days after initiation of treatment; transaminases, < grade 3 lasting less than 48 hours after initiation of treatment; hypotension, < grade 3 lasting less than 48 hours after initiation of treatment; CRS, < grade 3 associated with DMSO that can be resolved with benadryl and / or epinephrine; anaphylaxis or < grade 3 controlled with oral or IV narcotic therapy; pain, with the exception of, or in addition to, < grade 3 lasting up to 5 days; chills, < grade 3 lasting up to 2 weeks. Toxicities occurring approximately 3 weeks after CAR T cell infusion may include transaminases, ≦Grade 3 CRS lasting up to 2 weeks, ≦Grade 4 lymphopenia, ≦Grade 4 leukopenia, or ≦Grade 3 pain lasting up to 2 weeks that is controlled with oral or IV narcotic treatment.

[0369] In some embodiments of preventing or preventing CRS in a patient, the method may further comprise administering to the patient folate, a conjugate comprising folate (wherein the conjugate comprising folate does not include a targeting moiety), or a drug that blocks the activation of CAR T cells. In this embodiment, any of folate, a conjugate comprising folate (wherein the conjugate comprising folate does not include a targeting moiety), or a drug that blocks the activation of CAR T cells may be referred to herein as a "rescue agent". In some embodiments, a folate, such as folic acid, may be administered to prevent or prevent CRS in a patient. In this embodiment, the folate inhibits the interaction of the bridge (i.e., the small molecule ligand linked to the targeting moiety by a linker) with the receptor of the bridge on the tumor, inhibiting tumor lysis and preventing or preventing CRS in the patient.

[0370] In some embodiments, the folate administered as an inhibitor of binding of crosslinks to tumors can be, for example, folic acid, folic acid analogs or other folic acid receptor binding molecules. In various embodiments, the folate analogs that can be used include folinic acid, pteropolyglutamic acid and folic acid receptor binding pteridines, such as tetrahydropterin, dihydrofolic acid, tetrahydrofolic acid and its deaza and dideaza analogs. The terms "deaza" and "dideaza" analogs refer to art-recognized analogs that have carbon atoms in place of one or two nitrogen atoms of the naturally occurring folic acid structure. For example, deaza analogs include 1-deaza, 3-deaza, 5-deaza, 8-deaza and 10-deaza analogs. For example, dideaza analogs include 1,5-dideaza, 5,10-dideaza, 8,10-dideaza and 5,8-dideaza analogs. The folic acid analogs are commonly referred to as "folates", reflecting their ability to bind to folic acid receptors. Other folate receptor binding analogs include aminopterin, amethopterin (methotrexate), N10-methylfolate, 2-deamino-hydroxyfolate, deaza analogs such as 1-deazamethopterin or 3-deazamethopterin and 3',5'-dichloro-4-amino-4-deoxy-N10-methylpteroylglutamic acid (dichloromethotrexate).

[0371] In another embodiment, the folate administered as an inhibitor of binding of the crosslink to the tumor has the formula [ka] [In the formula, X 1 and Y 1 each independently represents halo, R 2 , OR 2 , S.R. 3 and N.R. 4 R 5 selected from the group consisting of; U, V and W are each independently -(R 6a )C=, -N=, -(R 6a )C(R 7a )- and -N(R 4a)-; Q is selected from the group consisting of C and CH; T is selected from the group consisting of S, O, N, and -C=C-; X 2 and X 3 are each independently oxygen, sulfur, -C(Z)-, -C(Z)O-, -OC(Z)-, -N(R 4b )-, -C(Z)N(R 4b )-, -N(R 4b )C(Z)-, -OC(Z)N(R 4b )-, -N(R 4b )C(Z)O-, -N(R 4b )C(Z)N(R 5b )-, -S(O)-, -S(O)2-, -N(R 4a )S(O)2-, -C(R 6b )(R 7b )-, -N(C≡CH)-, -N(CH2C≡CH)-, C1-C 12 Alkylene and C1-C 12 alkyleneoxy, where X is oxygen or sulfur; R 1 are hydrogen, halo, C1-C 12 Alkyl and C1-C 12 alkoxy; R 2 , R 3 , R 4 , R 4a , R 4b , R 5 , R 5b , R 6b and R 7b each independently represents hydrogen, halo, C1-C 12 Alkyl, C1-C 12 Alkoxy, C1-C 12 Alkanoyl, C1-C 12 Alkenyl, C1-C 12 Alkynyl, (C1-C 12 Alkoxy)carbonyl and (C1-C 12 alkylamino)carbonyl; R 6 and R 7 each independently represents hydrogen, halo, C1-C12 Alkyl and C1-C 12 alkoxy; or R 6 and R 7 together to form a carbonyl group; R 6a and R 7a each independently represents hydrogen, halo, C1-C 12 Alkyl and C1-C 12 alkoxy; or R 6a and R 7a together to form a carbonyl group; p, r, s and t are each independently 0 or 1; and * is an optional covalent bond to the remainder of the conjugate if any additional chemical moieties are part of the folate. has.

[0372] In yet another embodiment, the conjugates containing folate can be administered to inhibit or prevent cytokine release syndrome (CRS) in patients, which can cause adverse effects in patients, including, but not limited to, weight loss, high fever, pulmonary edema, and dangerously low blood pressure.

[0373] In this embodiment, the conjugate containing folate does not contain a targeting moiety, and thus the conjugate inhibits the interaction of crosslinking with tumor, preventing tumor lysis and reducing CRS in patients.In this embodiment, the folate portion of the conjugate containing folate can comprise any of the folates described in the previous paragraphs bound to a chemical moiety that does not contain a targeting moiety.In some embodiments, the conjugate containing folate can comprise folate bound to one or more amino acids that do not contain a targeting moiety.As an example, the conjugate containing folate can be represented by the formula [ka] may have:

[0374] This compound is also referred to as "EC923". In these embodiments, folate or a conjugate comprising folate can be administered to a patient in molar excess relative to the crosslink (i.e., the small molecule ligand linked to the targeting moiety by a linker), such as 10-fold excess, 100-fold excess, 200-fold excess, 300-fold excess, 400-fold excess, 500-fold excess, 600-fold excess, 700-fold excess, 800-fold excess, 900-fold excess, 1000-fold excess, or 10,000-fold excess of folate or a conjugate comprising folate relative to the small molecule ligand linked to the targeting moiety by a linker, which is necessary to inhibit the interaction of the crosslink with the tumor, can be determined by those skilled in the art.

[0375] In other embodiments, a drug that inhibits the activation of CAR T cells can be administered to a patient to inhibit CAR T cell activation and prevent or prevent CRS in the patient.In some embodiments, the drug is selected from the group consisting of lymphocyte-specific protein tyrosine kinase inhibitors (e.g., dasatinib), PI3 kinase inhibitors (e.g., GDC0980), tocilizumab, IL-2-inducible T cell kinase inhibitors (e.g., BMS-509744), JAK inhibitors, BTK inhibitors, SIP agonists (e.g., siponimod and ozanimod) and drugs that block CAR T cell binding to crosslink but do not bind to cancer (e.g., fluoresceinamine, FITC or sodium fluorescein).It will be understood by those skilled in the art that FITC (i.e., fluorescein) can be a salt (e.g., sodium fluorescein) or its non-salt form under physiological conditions or, for example, in a buffer at physiological pH. Thus, in some embodiments, when fluorescein is administered to a patient, it may be in equilibrium between a salt form (e.g., sodium fluorescein) and a non-salt form. In other embodiments, the rescue agent that inhibits activation of CAR T cells has the formula [ka] This compound may be compound of the formula: This compound is also called "EC2319".

[0376] In various embodiments, the rescue agent may be administered in any appropriate volume, including, for example, 0.1 ml, 0.2 ml, 0.3 ml, 0.4 ml, 0.5 ml, 0.6 ml, 0.7 ml, 0.8 ml, 0.9 ml, 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 10 ml, 100 ml, or 1000 ml, at a concentration of about .001 nM to about 100 mM, about .01 nM to about 100 mM, about 1 nM to about 100 mM, about 10 nM to about 100 mM, about 50 nM to about 100 mM, or about 100 nM to about 100 mM. In other embodiments, the rescue agent may be administered at a dose of about .01 to about 300 μmole / kg patient body weight, about .06 to about 100 μmole / kg patient body weight, about .06 to about 90 μmole / kg patient body weight, about .06 to about 80 μmole / kg patient body weight, about .06 to about 70 μmole / kg patient body weight, about .06 to about 60 μmole / kg patient body weight, about .06 to about 50 μmole / kg patient body weight, about .06 to about 40 μmole / kg patient body weight, about .06 to about 30 μmole / kg patient body weight, about .06 to about 20 μmole / kg patient body weight, about .06 to about 10 μmole / kg patient body weight, about .06 to about 8 μmole / kg patient body weight, or about .06 to about 6 μmole / kg patient body weight.

[0377] In these embodiments, the rescue agent may be administered to the patient in a molar excess relative to the compound or its pharma- ceutically acceptable salt (i.e., the small molecule ligand linked to the targeting moiety by a linker), such as about a 10-fold excess, about a 20-fold excess, about a 30-fold excess, about a 40-fold excess, about a 50-fold excess, about a 60-fold excess, about a 70-fold excess, about a 80-fold excess, about a 90-fold excess, about a 100-fold excess, about a 200-fold excess, about a 300-fold excess, about a 400-fold excess, about a 500-fold excess, about a 600-fold excess, about a 700-fold excess, about a 800-fold excess, about a 900-fold excess, about a 1000-fold excess, or about a 10,000-fold excess of the rescue agent relative to the small molecule ligand linked to the targeting moiety by a linker required to inhibit the interaction of the compound or its pharma-ceutically acceptable salt with the tumor and / or CAR T cell, may be determined by one of skill in the art.

[0378] In other embodiments, the folate, a conjugate comprising a folate (wherein the conjugate comprising a folate does not comprise a targeting moiety) or an agent that inhibits activation of CAR T cells may be administered to the patient more than once.

[0379] In the 'rescue agent' embodiments described herein, a folate, a conjugate comprising folate (wherein the conjugate comprising folate does not include a targeting moiety) or an agent that inhibits activation of CAR T cells may be administered to the patient before and / or after the compound or a pharma- ceutically acceptable salt thereof. In other embodiments, a compound or a pharma- ceutically acceptable salt thereof may be administered before and after administration of a folate, a conjugate comprising folate (wherein the conjugate comprising folate does not include a targeting moiety) or an agent that inhibits activation of CAR T cells. In this embodiment, subsequent administration of the compound or a pharma- ceutically acceptable salt thereof may cause CAR T cell activation and increased cytokine levels in the patient.

[0380] In other embodiments, administration of folate, a conjugate comprising folate (wherein the conjugate comprising folate does not comprise a targeting moiety) or an agent that inhibits activation of CAR T cells can cause a decrease in cytokine levels in patients.In other embodiments, the decrease in cytokine levels is approximately to the cytokine levels of untreated patients.In other embodiments, when CRS grade reaches 1, 2, 3 or 4 or when CRS grade reaches 3 or 4, the patient is administered an agent that inhibits activation of CAR T cells.

[0381] In any of the embodiments described herein in which folate is a ligand attached to a targeting moiety by a linker, the patient may be placed on a folate deficient diet prior to treatment in the manner described herein or the patient may be administered dietary folate. In embodiments in which the patient is administered folate, the dosage may be, for example, from about 50 nmol / kg to about 3000 nmol / kg of patient body weight, from about 50 nmol / kg to about 2000 nmol / kg, from about 50 nmol / kg to about 1000 nmol / kg, from about 50 nmol / kg to about 900 nmol / kg, from about 50 nmol / kg to about 800 nmol / kg, from about 50 nmol / kg to about 700 nmol / kg, from about 50 nmol / kg to about 600 nmol / kg, from about 50 nmol / kg to about 700 nmol / kg, from about 50 nmol / kg to about 800 nmol / kg, from about 50 nmol / kg to about 900 nmol / kg, from about 50 nmol / kg to about 1000 nmol / kg, from about 50 nmol / kg to about 15 ...500 nmol / kg, from about 50 nmol / kg to about 1500 nmol / kg, from about 50 nmol / kg to about The range may be from about 100 nmol / kg, about 50 nmol / kg to about 400 nmol / kg, about 50 nmol / kg to about 300 nmol / kg, about 50 nmol / kg to about 200 nmol / kg, about 50 nmol / kg to about 100 nmol / kg, about 100 nmol / kg to about 300 nmol / kg, about 100 nmol / kg to about 500 nmol / kg, about 100 nmol / kg to about 1000 nmol / kg, or about 100 nmol / kg to about 2000 nmol / kg of the patient's body weight. In other embodiments, the dosage can be about 100 nmol / kg, about 150 nmol / kg, about 200 nmol / kg, about 250 nmol / kg, about 300 nmol / kg, about 350 nmol / kg, about 400 nmol / kg, about 450 nmol / kg, about 500 nmol / kg, about 600 nmol / kg, about 700 nmol / kg, about 800 nmol / kg, about 900 nmol / kg, about 1000 nmol / kg, about 2000 nmol / kg or about 3000 nmol / kg of patient body weight. In these embodiments, "kg" refers to patient body weight in kilograms. In some embodiments, folate can be administered, for example, daily, weekly, twice weekly, three times weekly, or using any suitable regimen for administration of folate.

[0382] In various embodiments described herein, the CAR T cells may maintain high numbers of circulating CAR T cells for up to about 10 days, up to about 15 days, up to about 20 days, up to about 25 days, up to about 30 days, up to about 35 days, up to about 40 days, up to about 45 days, up to about 50 days, up to about 55 days, up to about 60 days, up to about 65 days, up to about 70 days, up to about 75 days, or up to about 80 days after administration of the CAR T cells.

[0383] In various embodiments described herein, the median effective concentration (EC 50 ) is about 1 pM to about 2 nM, about 1 pM to about 5 nM, about 1 pM to about 10 nM, about 1 pM to about 20 nM, about 1 pM to about 30 nM, about 1 pM to about 40 nM, about 1 pM to about 50 nM, about 1 pM to about 60 nM, about 1 pM to about 70 nM, about 1 pM to about 80 nM, about 1 pM to about 90 nM, about 1 pM to about 100 nM, about 1 pM to about 200 nM, about 1 pM to about 300 nM, about 1 pM to about 400 nM, about 1 pM to about 500 nM, about 1 pM to about 600 nM, about 1 pM to about 700 nM, about 1 pM to about 800 nM, about 1 pM to about 900 nM, about 1 pM to about 1 pM to about 1nM, about 1pM to about 900pM, about 1pM to about 800pM, about 1pM to about 700pM, about 1pM to about 600pM, about 1pM to about 500pM, about 1pM to about 400pM, about 1pM to about 300pM, about 1pM to about 200pM, about 1pM to about 100pM, It can be about 1 pM to about 90 pM, about 1 pM to about 80 pM, about 1 pM to about 70 pM, about 1 pM to about 60 pM, about 1 pM to about 50 pM, about 1 pM to about 40 pM, about 1 pM to about 30 pM, about 1 pM to about 20 pM, about 1 pM to about 10 pM, or about 1 pM to about 5 pM.

[0384] In various embodiments described herein, the Kd for binding of the compound, or a pharma- ceutically acceptable salt thereof, to a CAR T cell can be from about 1 nM to about 100 nM, from about 1 nM to about 200 nM, from about 1 nM to about 300 nM, from about 1 nM to about 400 nM, from about 1 nM to about 500 nM, from about 1 nM to about 600 nM, from about 1 nM to about 700 nM, from about 1 nM to about 800 nM, from about 1 nM to about 900 nM, from about 100 nM to about 500 nM, from about 100 nM to about 400 nM, from about 100 nM to about 300 nM, from about 100 nM to about 200 nM, from about 100 nM to about 150 nM, or about 130 nM.

[0385] In various embodiments described herein, EGFRt-selected or unselected CAR T cells may be used. In other embodiments, a "clinical replica" batch of CAR T cells with a less differentiated profile may be used. In other embodiments, a "research batch" of CAR T cells may be used. The "clinical replica" batch (about 39% EGFRt+) had a phenotype of about 66% T SCM and about 32%T CM CD4+ subset and approximately 95%T SCM and about 3%T CM The study batch (approximately 23% EGFRt+) contained approximately 32% T SCM , about 53%T CM , about 11%T EM and about 3.7%T EFF In the CD4 subset and approximately 44% SCM , about 0.28%T CM , about 3.4%T EM and about 52%T EFF and may include the CD8 subset.

[0386] In various illustrative embodiments described herein, the compound or a pharma- ceutically acceptable salt thereof may be administered to the patient about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days before or after the CAR T cells or any suitable day before or after the CAR T cells.

[0387] In some embodiments of the methods described herein, the cancer is imaged before administering the compound or a pharma- ceutically acceptable salt thereof to the patient or before administering the CAR T cell composition to the patient. In some illustrative embodiments, the imaging is performed by PET imaging. In other illustrative embodiments, the imaging is performed by MRI imaging or SPECT / CT imaging. The imaging method can be any suitable imaging method known in the art. In some embodiments, the imaging method can include the use of a small molecule ligand, as described herein, but conjugated to an imaging agent appropriate for the type of imaging described herein, to determine whether the patient is positive for folate receptor expression. In other embodiments, immunohistochemistry can be used for this purpose.

[0388] In any of the embodiments described herein, cytokine release resulting in off-target toxicity in the patient may not occur in the patient even if CAR T cell toxicity against the cancer occurs. In any of the embodiments described herein, off-target tissue toxicity may not occur in the patient even if CAR T cell toxicity against the cancer occurs. In any of the embodiments described herein, the cancer may include a tumor, and the tumor size may be reduced in the patient even if off-target toxicity does not occur. In any of the embodiments described herein, CRS may be reduced or prevented, and the method may result in tumor volume reduction in the patient. In any of the embodiments described herein, weight loss and CAR T cell exhaustion due to CRS may be reduced or prevented. In any of the embodiments described herein, the cancer may include a tumor, and a complete response against the tumor may be obtained. EXAMPLES

[0389] Example 1 Synthesis of FITC-Folate [ka] Folate-γ-ethylenediamine was coupled with fluorescein isothiocyanate (FITC) isomer I (Sigma-Aldrich) in the presence of tetramethylguanidine and diisopropylamine in anhydrous dimethylsulfoxide (DMF). 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) reaching 90% A and 10% B in 10 min at a flow rate of 20 mL / min. Under these conditions, the main FITC-folate peak generally eluted between 27 and 50 min. The quality of the FITC-folate fractions was monitored by analytical reversed-phase HPLC equipped with a UV detector. Fractions with greater than 98.0% purity (LCMS) were lyophilized to obtain the final FITC-folate product. As known in the art, compounds of this structure are also referred to as EC17.

[0390] Example 2 Synthesis of FITC-PEG12-Folate [ka] Versatile Polyethylene Glycol (PEG) Nova Tag TM The resin (0.2 g) was added to a peptide synthesizer and washed with isopropyl alcohol (i-PrOH) (3 x 10 mL) and dimethylformamide (DMF, 3 x 10 mL). 9-Fluorenylmethoxycarbonyl (Fmoc) deprotection was carried out using 20% ​​piperidine in DMF (3 x 10 mL). Kaiser test was performed to follow the reaction progress. The synthesizer was then charged with a DMF solution of Fmoc-L-glutamic acid 5-tert-butyl ester (Fmoc-Glu-(Ot-Bu)-OH) (23.5 mg), N,N-diisopropylethylamine (i-Pr2NEt) (4 eq.) and benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) (2 eq.). Fmoc deprotection was carried out using 20% ​​piperidine in DMF (3 x 10 mL). The synthesizer was then charged with N 10A solution of Fmoc-TFA-Pte-OH (22.5 mg), DMF, i-Pr2NEt (4 equiv.) and PyBOP (2 equiv.) was added. Argon was bubbled in for 2 h and the resin was washed with DMF (3 × 3 mL) and i-PrOH (3 × 3 mL). After swelling the resin with dichloromethane (DCM), a solution of 1 M hydroxybenzotriazole (HOBT) in DCM / trifluoroethane (TFE) (1:1) (2 × 3 mL) was added. Argon was bubbled in 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 with DMF, Fmoc-NH-(PEG) 12 A solution of -COOH (46.3 mg) in DMF, i-Pr2NEt (4 equiv.) and PyBOP (2 equiv.) were added. Argon was bubbled in for 2 h and the resin was washed with DMF (3 x 3 mL) and i-PrOH (3 x 3 mL). Fmoc deprotection was carried out using 20% ​​piperidine in DMF (3 x 10 mL). To follow the reaction progress, a Kaiser test was performed. The vessel was then charged with a solution of FITC (Life Technologies 21.4 mg) in DMF and i-Pr2NEt (4 equiv.), then argon was bubbled in for 2 h and the resin was washed with DMF (3 x 3 mL) and i-PrOH (3 x 3 mL). 2% NH2NH2 in DMF (2 x 2 mL) was then added to the vessel. The final compound was cleaved from the resin using TFA:H2O:triisopropylsilane (TIS) (95:2.5:2.5) (cleavage solution) and concentrated under reduced pressure. The concentrated product was precipitated with Et2O and dried under reduced pressure. The crude product was purified using preparative RP-HPLC (mobile phase: A = 10 mM ammonium acetate pH = 7, B = ACN; method: 0% B to 30% B at 13 mL / min for 30 min). The purified fractions were collected and lyophilized to give FITC-PEG12-Folate.

[0391] Example 3 Synthesis of FITC-PEG20-Folate [ka] Ethylenediamine, polymer-bound (200-400 mesh) resin (50 mg) was added to the peptide synthesizer 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 equiv), i-Pr2NEt (6.0 equiv) and PyBOP (4.0 equiv) were added. Argon was bubbled for 6 h, the coupling solution was discarded and the resin was washed with DMF (3 × 10 mL) and i-PrOH (3 × 10 mL). Kaiser test was performed to follow the reaction progress. Fmoc deprotection was performed using 20% ​​piperidine in DMF (3 × 10 mL) followed by each amino acid coupling. The above sequence was repeated to complete the reaction of Fmoc-Glu-OtBu (72 mg, 2.0 equiv) and Tfa.pteroic acid (41 mg, 1.2 equiv) coupling steps. The resin was washed with 2% hydrazine in DMF 3 × 10 mL (5 min) to cleave the trifluoro-acetyl protecting group of pteroic acid and washed with i-PrOH (3 × 10 mL) followed by DMF (3 × 10 mL). The resin was dried under argon for 30 minutes. The folate-peptide was cleaved from the resin using cleavage solution. 10 mL of cleavage mixture was added and argon was bubbled through for 1.5 hours. The cleavage mixture was drained into a new flask. The resin was washed three times with additional cleavage mixture. The combined mixture was concentrated under reduced pressure to a small volume (approximately 5 mL) and precipitated with ethyl ether. 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 reaction progress 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 at 13 mL / min for 35 min) to give FITC-PEG20-folate in 60% yield.

[0392] Example 4 Synthesis of FITC-PEG108-Folate [ka] Ethylenediamine, polymer-bound (200-400 mesh) resin (50 mg) was added to the peptide synthesizer and swollen with DCM (3 mL) followed by DMF (3 mL). The synthesizer was then loaded with Fmoc-PEG in DMF. 36 A solution of -COOH (161 mg, 1.0 equiv), i-Pr2NEt (6.0 equiv) and PyBOP (4.0 equiv) were added. Argon was bubbled for 6 h, the coupling solution was discarded and the resin was washed with DMF (3×10 mL) and i-PrOH (3×10 mL). A Kaiser test was performed to follow the reaction progress. Fmoc deprotection was performed using 20% ​​piperidine in DMF (3×10 mL) followed by each amino acid coupling. The above sequence was repeated for 2X Fmoc-PEG 36 -COOH (161 mg, 1.0 equiv), Fmoc-Glu-OtBu (72 mg, 2.0 equiv), and Tfa.pteroic acid (41.0 mg, 1.2 equiv) coupling steps were repeated to complete the reaction. Finally, the resin was washed with 2% hydrazine in DMF 3×10 mL (5 min) 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 the cleavage solution. 10 mL of cleavage mixture was added and argon was bubbled through for 1.5 h. The cleavage mixture was drained into a clean flask. The resin was washed three times with additional cleavage mixture. The combined mixture was concentrated under reduced pressure to a small volume (approximately 5 mL) and precipitated with ethyl ether. The precipitate was collected by centrifugation, washed with ethyl ether (3x) 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 reaction progress was monitored by LCMS. After 10 h the starting material was consumed to give the product. The crude reaction mixture was purified by preparative HPLC (mobile phase A=10 mM ammonium acetate, pH=7; organic phase B=acetonitrile; method: 0% B to 30% B at 13 mL / min for 35 min) to give FITC-PEG108-folate in 64% yield.

[0393] Example 5 Synthesis of FITC-DUPA [ka] DUPA-FITC was synthesized by solid phase synthesis as follows. TMThe resin (50 mg, 0.53 mM) was swollen with DCM (3 mL) followed by DMF (3 mL). 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). After swelling the resin with DMF, a solution of DUPA-(OtBu)-OH (1.5 equiv.), HATU (2.5 equiv.) and i-Pr2NEt (4.0 equiv.) in DMF was added. 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 with DCM, a solution of 1 M HOBt in DCM / TFE (1:1) (2 × 3 mL) was added. Argon was bubbled for 1 h, the solvent was removed, and the resin was washed with DMF (3 x 3 mL) and i-PrOH (3 x 3 mL). After swelling the resin with DMF, a DMF solution of Fmoc-Phe-OH (2.5 eq.), HATU (2.5 eq.) and DIPEA (4.0 eq.) was added. Argon was bubbled for 2 h, and the resin was washed with DMF (3 x 3 mL) and i-PrOH (3 x 3 mL). The above sequence was repeated for two more 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 reduced pressure. The concentrated product was precipitated with diethyl ether and dried under reduced pressure. The crude product was purified using preparative RP-HPLC [λ=488 nm; solvent gradient: 1% B to 80% B for 25 min, 80% B wash for 30 min; A=10 mM NH4OAc, pH=7; B=acetonitrile (ACN)]. ACN was removed under reduced pressure and the purified fractions were lyophilized to give FITC-DUPA as a brownish-orange solid. RP-HPLC: tR=8.0 min (A=10 mM NH4OAc, pH=7.0; B=ACN, solvent gradient: 1% B to 50% B for 10 min, 80% B wash for 15 min). 1H NMR (DMSO-d6 / D2O):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) + C 51 H 59 N7O 15 Calculated value of S: 1040.3712, measured value: 1040.3702. UV / vis: λ max=491nm

[0394] Example 6 Synthesis of FITC-PEG12-DUPA [ka] 1,2-Diaminoethanetrityl resin (0.025 g) was added to the peptide synthesizer and washed with i-PrOH (3 x 10 mL) followed by DMF (3 x 10 mL). The vessel was then charged with Fmoc-NH-(PEG). 12A solution of -COOH (42.8 mg), i-Pr2NEt (2.5 equiv.) and PyBOP (2.5 equiv.) was added. The resulting solution was bubbled with Ar for 1 h, the coupling solution was discarded and the resin was washed with DMF (3 x 10 mL) and i-PrOH (3 x 10 mL). A Kaiser test was performed to follow the reaction progress. Fmoc deprotection was performed using 20% ​​piperidine in DMF (3 x 10 mL). This process was repeated until all coupling steps were completed (2 x 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 coupling step). After DUPA coupling, the resin was washed with DMF (3 x 10 mL) and i-PrOH (3 x 10 mL) and dried under reduced pressure. The peptide was cleaved from the resin using cleavage solution in a peptide synthesizer. 15 mL of cleavage solution was added to the peptide synthesizer and the reaction was purged with Ar for 15 min. The resin was treated with two additional 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. The crude DUPA-(PEG) 12 To a solution of 10 mg of -EDA and 5.6 mg of FITC in dimethyl sulfoxide (DMSO, 1 mL) was added i-Pr2NEt (5 equiv.) at room temperature 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 at 13 mL / min for 30 min). The purified fractions were collected and lyophilized to give FITC-PEG12-DUPA.

[0395] Example 7 Synthesis of FITC-PEG11-NK1 [ka] A mixture of NK-1 (0.02 g, 0.0433 mmol, 1.0 equiv.), O-(2-aminoethyl)-O'-[2-(Boc-amino)ethyl]decaethylene glycol (BocNH-PEG 11To a solution of N,N-diisopropylethylamine (DIPEA) (0.076 mL, 0.4338 mmol, 10 equiv.) in dry CHCl (Sigma, 0.0336 g, 0.0521 mmol, 1.2 equiv.) and benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) (0.027 g, 0.0521 mmol, 1.2 equiv.) at room temperature under argon was added N,N-diisopropylethylamine (DIPEA) (0.076 mL, 0.4338 mmol, 10 equiv.). The reaction progress was monitored by LCMS and purified by preparative RP-HPLC (Waters, XBridge TM Purification was performed using a Prep C18, 5 μm; 19 × 100 mm column, mobile phase A = 20 mM ammonium acetate buffer, pH 7, B = acetonitrile, gradient 10 to 100% B for 30 min, 13 mL / min, λ = 220 nm, 254 nm). The purified fractions were taken, all organic solvents were evaporated, and the samples were lyophilized for 48 h to obtain NK1-PEG. 11 -NHBoc was obtained. Yield: 40.13 mg (97%). NK1-PEG 11 To a solution of -NHBoc (0.0165 g, 0.015 mmol) in dry DCM was added trifluoroacetic acid (TFA, 20 equiv.) and the reaction mixture was stirred for 4 h at rt. Excess TFA was removed and the remaining solution was diluted with water and extracted using CH2Cl2 (3 x 5 mL). The combined organic layers were washed with brine, dried (Na2SO4) and concentrated. The resulting residue was dried under reduced pressure and used in the next step without further purification. The stirred NK1-PEG 11 To a solution of -NH2 (0.008 g, 0.0081 mmol, 1.0 equiv.), fluorescein isothiocyanate (FITC) (Sigma, 0.0037 g, 0.0097 mmol, 1.2 equiv.) in dry dimethylsulfoxide (DMSO, 0.3 mL) was added diisopropylethylamine (0.0028 mL, 0.0162 mmol, 2.0 equiv.) at room temperature under argon. The reaction progress was monitored by LCMS and the product was purified by preparative RP-HPLC (Waters, XBridge TMPurification was performed on a Prep C18, 5 μm; 19 × 100 mm column, mobile phase A = 20 mM ammonium acetate buffer, pH 7, B = acetonitrile, gradient 10 to 100% B for 30 min, 13 mL / min, λ = 280 nm). Pure fractions were obtained, all organic solvents were evaporated, and the sample was lyophilized for 48 h to give FITC-PEG11-NK1 in a yield of 8.54 mg (77%). *Note: NK-1 compounds were synthesized by a two-step method starting from a basic ligand prepared using a literature method (DESIGN AND DEVELOPMENT OF NEUROKININ-1 RECEPTOR-BINDING AGENT DELIVERY CONJUGATES, Application No. PCT / US2015 / 44229; incorporated herein by reference).

[0396] Example 8 Synthesis of FITC-PEG2-CA9 [ka] 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. Air was removed using reduced pressure and nitrogen gas replacement, which was repeated three times. The round bottom flask was kept under nitrogen gas. To this flask, 28.9 mg of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) was added, followed by 21.6 mg 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 831). Acetonitrile was removed using high vacuum rotary evaporation and the product was lyophilized. The compound was mixed with 1:1 TFA:DCM for 30 minutes. 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 combined with 5 molar equivalents of i-Pr2NEt, 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.

[0397] Example 9 T cell preparation Human peripheral blood mononuclear cells (PBMCs) were isolated from whole blood of healthy donors using Ficoll density gradient centrifugation (GE Healthcare Lifesciences). T cells were then isolated using EasySep TMHuman T cell isolation kits (STEM CELL technologies) were used to isolate T cells from PBMCs. T cells were cultured in TexMACS medium (Miltenyi Biotech Inc) supplemented with 40–100 IU / mL human IL-2 (Miltenyi Biotech), 2% human AB serum, and 1% penicillin / streptomycin sulfate. T cells were activated by adding Dynabeads human T-activator CD3 / CD28 (ThermoFisher Scientific) to T cells at a 1:1 ratio. 12–24 h after activation, T cells were transduced with FITC-CAR lentiviral particles by spinfection at 1,200 g for 90 min at 22–32°C in the presence of 8 μg / mL polybrene (Santa Cruiz Biotech). T cell mixtures containing CAR-modified (CAR-Ts) and non-CAR-modified (non-transformed Ts) were cultured in the presence of activation beads for 6 days, after which the activation beads were removed. Fluorescence-activated cell sorting was used to sort CAR T cells (GFP positive) and non-transformed T cells (GFP negative) based on GFP expression. Sorted T cells were cultured for 7–15 days and then injected into mice. When T cell mixtures were used, CAR T cells and non-transformed T cells were mixed in the desired ratio before injection into mice.

[0398] Example 10 Production of lentiviral vector encoding the CAR gene A CAR construct containing a scFv against fluorescein was produced using overlap PCR method. 4M5.3 (Kd=270fM, 762bp), a scFv against fluorescein derived from anti-fluorescein (4-4-20) antibody, was synthesized. The sequences of human CD8α signal peptide (SP, 63bp), hinge and transmembrane region (249bp), cytoplasmic domain of 4-1BB (CD137, 141bp) and CD3ζ chain (336bp) shown in Figure 2 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) (Figure 2, System Biosciences). The sequence of the CAR construct in the lentiviral vector was confirmed by DNA sequencing.

[0399] An exemplary CAR nucleic acid coding sequence can include: [Table 6]

[0400] In the above exemplary nucleic acid sequence (SEQ ID NO:1), the first ATG is the start codon. An exemplary CAR amino acid sequence may include: [Table 7]

[0401] Exemplary inserts may include: [Table 8]

[0402] In the above exemplary insert (SEQ ID NO:3), the initial GCCACC sequence may include a restriction enzyme cleavage site followed by an ATG start codon. The encoded amino acid sequence may include: [Table 9]

[0403] Example 11 Production of lentivirus containing the CAR gene for human T cell transduction To produce lentiviral virus containing an anti-fluorescein (i.e., anti-FITC) single-chain fragment variable (scFv) CAR, the HEK-293TN packaging cell line was co-transfected with a lentiviral vector encoding an anti-fluorescein scFv CAR and a second-generation lentiviral packaging plasmid mix (Cellecta) or ViraPower Lentivrial Packaging Mix (ThermoFisher). 24 and 48 hours after transfection, the supernatant containing the lentivirus with the CAR gene was harvested and the viral particles were concentrated by standard polyethylene glycol virus concentration methods (Clontech) for subsequent transduction in human T cells.

[0404] Example 12 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 residual Ficoll solution, T cells were isolated using EasySep TM Human T cells were isolated using a human T cell isolation kit (STEM CELL technologies). Purified T cells were cultured in TexMACS™ medium (Miltenyi Biotech Inc.) supplemented with 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 in multi-well plates at a density of 1000 cells / mL. T cells were split and refed every 2-3 days.

[0405] Example 13 Transduction of human T cells Isolated T cells were activated with Dynabeads conjugated to 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 CAR expression in transduced T cells was confirmed by counting GFP fluorescent cells using flow cytometry.

[0406] Example 14 Synthesis of FITC-Folate Folate-γ-ethylenediamine was coupled with fluorescein isothiocyanate (FITC) isomer I (Sigma-Aldrich) in the presence of tetramethylguanidine and diisopropylamine in anhydrous dimethylsulfoxide (DMF). 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) and reaching 90% A and 10% B in 10 min at a flow rate of 20 mL / min. Under these conditions, the main FITC-folate peak generally eluted between 27 and 50 min. The quality of the FITC-folate fractions was monitored by analytical reversed-phase HPLC equipped with a UV detector. Fractions with greater than 98.0% purity (LCMS) were lyophilized to obtain the final FITC-folate product. [ka]

[0407] Example 15 Synthesis of FITC-DUPA DUPA-FITC was synthesized by solid-phase synthesis as follows: Universal NovaTag resin (50 mg, 0.53 mM) was swollen with dichloromethane (DCM) (3 mL) followed by dimethylformamide (DMF, 3 mL). A 20% solution of piperidine in DMF (3 × 3 mL) was added to the resin and argon was bubbled in for 5 min. The resin was washed with DMF (3 × 3 mL) and isopropyl alcohol (i-PrOH, 3 × 3 mL). After swelling the resin with DMF, a DMF solution of DUPA-(OtBu)-OH (1.5 equiv.), HATU (2.5 equiv.) and DIPEA (4.0 equiv.) was added. Argon was bubbled in for 2 h and the resin was washed with DMF (3 × 3 mL) and i-PrOH (3 × 3 mL). After swelling the resin with DCM, a solution of 1M HOBt in DCM / trifluoroethane (TFE) (1:1) (2×3 mL) was added. Argon was bubbled in 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 with 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 in for 2 h, and the resin was washed with DMF (3×3 mL) and i-PrOH (3×3 mL). The above sequence was repeated for two more couplings for the addition of 8-aminooctanoic acid and fluorescein isothiocyanate or rhodamine B isothiocyanate. The final compound was cleaved from the resin using a trifluoroacetic acid (TFA):H2O:triisopropylsilane:cocktail (95:2.5:2.5) and concentrated under reduced pressure. The concentrated product was precipitated with diethyl ether and dried under reduced pressure. The crude product was purified using preparative RP-HPLC [λ=488 nm; solvent gradient: 1% B to 80% B for 25 min, 80% B wash for 30 min; A=10 mM NHOAc, pH=7; B=acetonitrile (ACN)]. ACN was removed under reduced pressure and the purified fractions were lyophilized to give DUPA-FITC as a brownish-orange solid. RP-HPLC: tR=8.0 min (A=10 mM NHOAc, pH=7.0; B=ACN, solvent gradient: 1% B to 50% B for 10 min, 80% B wash for 15 min). 1H NMR (DMSO-d6 / D2O): 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)+ C 51 H 59 N7O 15 The calculated value of S is 1040.3712, the measured value is 1040.3702. UV / vis:λ max=491nm

change

[0408] Example 16 Synthesis of FITC-CA9 In a 50 mL round bottom flask, CA9 ligand (53.6 mg, synthesized in the lab) was dissolved in the desired amount of N,N-dimethylformamide (DMF) (2-3 mL) using a Teflon magnetic stir bar. The outside air was removed using reduced pressure and replacement with nitrogen gas, which was done three cycles. The round bottom flask was then maintained under constant nitrogen gas.To this flask, 28.9 mg of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) was added, followed by 21.6 mg 1-hydroxybenzotriazole hydrate (HOBt) and 18.9 μL of Boc-PEG2-NH2 (purchased from Sigma Aldrich). 5.4 μL of triethylamine (TEA) was finally added and the reaction was stirred overnight. The reaction mixture was purified using HPLC and confirmed by UHPLC-MS (target m / z 831). Acetonitrile was removed using high vacuum rotary evaporation and placed in a freeze dryer for 48 h. Boc deprotection was carried out with 1:1 TFA:DCM for 30 min. TFA / DCM was removed using high vacuum rotary evaporation followed by high vacuum for 30 min. The compound was then dissolved in DMF and combined with 5 molar equivalents of N,N-diisopropylethylamine (DIPEA). 16 mg of fluorescein isothiocyanate (purchased from Life Technologies) was added to the solution 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 placed in a lyophilizer for 48 h and the compound was stored at -20°C. [ka]

[0409] Example 17 Synthesis of FITC-NK1R To a stirring solution of NK-1 (0.02 g, 0.0433 mmol, 1.0 equiv.), O-(2-aminoethyl)-O'-[2-(Boc-amino)ethyl]decaethylene glycol (BocNH-PEG-NH) (Sigma, 0.0336 g, 0.0521 mmol, 1.2 equiv.), and benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PYBOP) (0.027 g, 0.0521 mmol, 1.2 equiv.) in dry CHCl at room temperature under argon was added N,N-diisopropylethylamine (DIPEA) (0.076 mL, 0.4338 mmol, 10 equiv.). The reaction progress was monitored by LCMS and purified by preparative RP-HPLC (Waters, XBridgeTM Prep C18, 5 μm; 19 × 100 mm column, mobile phase A = 20 mM ammonium acetate buffer, pH 7, B = acetonitrile, gradient 10 to 100% B for 30 min, 13 mL / min, λ = 220 nm, 254 nm). The purified fractions were taken, total organic solvents were evaporated and the sample was lyophilized for 48 h to give NK1-PEG11-NHBoc. Yield: 40.13 mg (97%). Trifluoroacetic acid (TFA, 20 equiv.) was added to a solution of NK1-PEG11-NHBoc (0.0165 g, 0.015 mmol) in dry CHCl and the reaction mixture was stirred for 4 h at rt. Excess TFA was removed, diluted with water and extracted using CHCl (3x5 mL). The combined organic layers were washed with brine, dried (Na2SO4) and concentrated. The resulting residue was dried under reduced pressure and used in the next step without further purification. To a stirred solution of NK1-PEG11-NH2 (0.008 g, 0.0081 mmol, 1.0 equiv), fluorescein isothiocyanate (FITC) (Sigma, 0.0037 g, 0.0097 mmol, 1.2 equiv) in dry dimethylsulfoxide (DMSO, 0.3 mL) at room temperature under argon was added diisopropylethylamine (0.0028 mL, 0.0162 mmol, 2.0 equiv).The reaction progress was monitored by LCMS and purified by preparative RP-HPLC (Waters, XBridgeTM Prep C18, 5 μm; 19 × 100 mm column, mobile phase A = 20 mM ammonium acetate buffer, pH 7, B = acetonitrile, gradient 10 to 100% B for 30 min, 13 mL / min, λ = 280 nm). The purified fractions were taken, all organic solvents were evaporated, and the sample was lyophilized for 48 h to give NK1-PEG11-FITC (5). Yield: 8.54 mg (77%). The NK-1 compound was synthesized by a two-step method starting from a basic ligand prepared using a literature method (DESIGN AND DEVELOPMENT OF NEUROKININ-1 RECEPTOR-BINDING AGENT DELIVERY CONJUGATES, Application No. PCT / US2015 / 44229, incorporated herein by reference in its entirety).

[0410] Example 18 HOS-FR-α and AML Study Overview [Table 10] Sequential ascending doses of EC17 will be administered as follows: · In the absence of CRS or neurotoxicity, proceed with EC17 dose escalation as planned. If CRS grade ≦2, administer all subsequent EC17 doses at the dose-escalation level that caused non-severe CRS in the same cycle. If CRS grade >2, discontinue further dosing of EC17 in the current cycle and administer all subsequent cycles at the highest dose course lower than that which caused sCRS.

[0411] A. Tumor Implantation HOS-FRα (i.e., HOS-143b-LV-FRα) is a subclone of HOS-143B (ATCC CRL-8303) stably transfected with human FRα. HOS-143B was originally derived from a 13-year-old Caucasian girl with osteosarcoma. Tumor cells were grown in folate-deficient RPMI 1640 supplemented with 5% FBS at 37°C in a humidified atmosphere of 5% CO2. For in vivo studies, tumor cells were grown at 1 × 10 per animal. 6 The cells were inoculated subcutaneously.

[0412] B. CAR T Cell Administration EGFRt-sorted anti-FITC E2 scFv-CAR T cells were frozen in T cell freezing medium. Upon arrival, vials of frozen CAR T cells were immediately stored at -80°C. CAR T cells were rapidly thawed at 37°C, washed twice with PBS, and used for animal injections at 6 million viable EGFRt+E2 CAR T cells (CD4 / CD8 approx. 1:1) per animal. An aliquot was taken on the day of injection for flow cytometric analysis of E2-CAR T cell phenotype.

[0413] C. Preparation of Drug Solution for Administration EC17 dosing solutions were prepared at the start of dosing by weighing out the appropriate amount of each compound, reconstituting with PBS, pH 7.4, filter sterilizing the drug solution through a 0.22 μm PVDF syringe filter, and freezing an aliquot at −20° C. on each dosing day.

[0414] D. Compound Administration All EC17 doses were administered at the end of the day (3-4 pm) to allow for potential CRS (cytokine release syndrome) to develop overnight. The following morning, animals were scored using the CRS grading system (see below).

[0415] E. Evaluation [Table 11]

[0416] Tumor growth, body weight and overall assessmentTumor size was monitored 2-3 times per week and body weights were measured 2-3 times per week. Body weights were measured daily immediately following administration of any EC17 dose, and general animal morphology and behavior were noted.

[0417] slaughter When mice lose ≥20% of their body weight, tumors grow to ≥1500 mm 3 Mice were also sacrificed when they lost significant weight in a short period of time (e.g., due to severe head tilt) or approached moribund status according to the CRS grading system.

[0418] EC17 in-host dose escalation treatment (3 cycles) [Table 12] Antitumor activity (N=5)

[0419] As shown in Figure 4: Cohort 1 (E2-CAR T cells only) showed aggressive proliferation of HOS-FRα without EC17 treatment. Cohort 2 (EC17 5 / 100 / 1000nmol / kg, M / Th / M) showed significant tumor regression throughout the study without weight loss or CRS. The slow EC17 dose escalation (M / Th / M) regimen was safe: no weight loss or symptoms of cytokine release syndrome (CRS) were observed in HOS-FRα tumor-bearing mice.

[0420] One goal of this E2-CAR T cell trial was to test EC17 / E2 CAR T cell activity and associated toxicities (e.g., sCRS) using multiple dosing regimens starting 3 days after CAR-T infusion. The three different EC17 dosing regimens included: (a) once weekly (SIW), 500 nmol / kg, (b) "TIW-like" at 5 nmol / kg, 50 nmol / kg, and 500 nmol / kg Monday / Wednesday / Friday, 9 days apart, and (c) "TIW-like" at 5 nmol / kg, 100 nmol / kg, and 1000 nmol / kg Monday / Thursday / Monday, 7 days apart. [Table 13] * Groups 4-5: No administration in the second week of each cycle.

[0421] Test diagram Group 1 (no treatment): for efficacy comparison only.

[0422] Group 2 (CAR-T only): [Table 14]

[0423] Group 3 (EC17 SIW): [Table 15]

[0424] Group 4 (EC17 True TIW): [Table 16]

[0425] Group 5 - EC17 low dose "TIW" (M / Th / M, 6 days off), with efficacy. [Table 17]

[0426] A. Tumor Implantation THP-1-FRβ AML tumor cells were grown in folate-deficient RPMI 1640 supplemented with 5–10% FBS at 37 °C in a 5% CO2 humidified atmosphere. THP-1-FRβ tumor cells in serum-free folate-deficient RPMI 1640 medium were grown at 5 × 10 per animal. 6 Cells were injected iv.

[0427] B. CAR T Cell Administration Anti-FITC E2 scFv-CAR T cells were frozen in T cell freezing medium. Upon arrival, vials of frozen CAR T cells were immediately stored at -80°C. CAR T cells were rapidly thawed at 37°C, washed twice with CAR T cell culture medium, and used for animal injections at 6 million viable EGFRt+E2 CAR T cells (CD4 / CD8 approximately 1:1) per animal. An aliquot was taken on the day of injection for flow cytometric analysis of E2-CAR T cell phenotype.

[0428] C. Preparation of Drug Solution for Administration EC17 and bis-EDA-FITC dosing solutions were prepared at the start of dosing by weighing out the appropriate amount of each compound, reconstituting in PBS, pH 7.4, filter sterilizing the drug solution with a 0.22 μm PVDF syringe filter, and freezing an aliquot at −20° C. on each dosing day.

[0429] D. Compound Administration All EC17 doses were administered at the end of the day (3-4 pm) to allow for potential CRS (cytokine release syndrome) to develop overnight. The following morning, animals were scored using the CRS grading system (see below).

[0430] EC17 administration schedule : Cohorts 1-2: No EC17 administration

[0431] Cohort 3: [Table 18]

[0432] Cohort 4: [Table 19]

[0433] Cohort 5: [Table 20]

[0434] Monitoring / Effectiveness : Daily body weight measurements were required after EC17 administration. Overall animal morphology and behavior were noted. Mice were sacrificed when they lost significant weight in a short period of time or approached moribund status according to the CRS grading system or neurotoxicity.

[0435] [Table 21]

[0436] Tumor growth, body weight and overall assessment Body weights were measured 2-3 times per week. Immediately following administration of any EC17 dose, body weights were measured daily and general animal morphology and behavior were noted.

[0437] Flow cytometry analysis Whole blood cell analysis:Plasma was removed from whole EDTA-treated blood in a predetermined volume, and RBCs were lysed with RBC lysis solution. The leukocyte pellet was then resuspended in flow cytometry staining solution (1% bovine serum albumin, 50 mg / mL human IgG (Equitech Bio, cat#SLH56-0001), 0.9% sodium azide in phosphate-buffered saline, pH=7.4), and leukocyte surface marker staining was performed using the following antibodies: anti-human CD45 (clone HI30, eBioscience #47-0459-42, diluted 1:20 (v / v)), anti-human CD137 (clone 4B4-1, BD Bioscience #564092, diluted 1:20 (v / v)), anti-human CD8α [clone RPA-T8, BD Bioscience, catalog #557746, diluted 1:20 (v / v)], anti-human CD4 [clone SK3, eBioscience catalog #1001621, diluted 1:20 (v / v)], anti-human CD5 [clone SK6, eBioscience catalog #1001636, diluted 1:20 (v / v)], anti-human CD6 [clone SK5, eBioscience catalog #1001645, diluted 1:20 (v / v)], anti-human CD7 [clone SK6, eBioscience catalog #100165, diluted 1:20 (v / v)], anti-human CD8 [clone SK7, eBioscience catalog #100166, diluted 1:20 (v / v)], anti-human CD4 [clone SK5, eBioscience catalog #100167, diluted 1:20 (v / v)], anti-human CD5 [clone SK6, eBioscience catalog #100168, diluted 1:2 #46-0047-42, diluted 1:20 (v / v)], anti-human EGFR [R&D systems, clone Hu1, catalog #FAB9577B @ 1:10 (v / v)], anti-human PD1 [BD Biosciences, clone EH12.1, catalog#562511 @ 1:20 (v / v)], anti-human LAG3 [BD Biosciences, clone T47-530, catalog #565616 @ 1:20 (v / v)], anti-human TIM3 [BD Biosciences, clone 7D3, catalog #565558 @ 1:20 (v / v)], and anti-human CD3ε [BD Biosciences, clone SK7, catalog #557832 @ 1:20 (v / v)]. After white blood cell staining, cells were washed with PBS and incubated with 53,000 CountBright TMThe beads [Invitrogen catalog #C36950] were resuspended in cold PBS and transferred to a flow cytometry collection tube. Flow cytometry data were collected on a Gallios flow cytometer (Beckman Coulter, Brea, CA). CAR T cell concentration determination for each blood sample was calculated according to Invitrogen's instructions. CAR T cells were identified as human CD3ε + EGFRt + events and were analyzed by Kaluza TM They were easily differentiated and counted using flow cytometry software. The number of circulating CAR T cells for each injected mouse was then graphed as the total number of CAR T cells per 50 μL of whole blood analyzed. Statistical significance was determined using an unpaired, two-tailed Student's t-test, with significance set at p<0.05.

[0438] Tumor and tissue analysis: Solid tumors (100-1000 mm 3 ) were harvested, weighed, cut into small pieces, and then transferred to a 50 mL tube containing 20 mL of tumor digestion cocktail. The enzymatic tumor digestion cocktail consisted of 0.5 mg / mL collagenase IV (Sigma-Aldrich, Catalog# C5138), 0.5 mg / mL hyaluronidase (Sigma-Aldrich, Catalog# H3506), and 0.1 mg / mL DNase I (Sigma-Aldrich, Catalog# DN25) in serum-free and folate-deficient RPMI1640 medium supplemented with antibiotics. The tumor fragments were digested for 1 h at 37°C at 300 rpm on a reciprocating shaker. The tumor digest was then centrifuged at 400×g for 5 min, and the tumor cell pellet was subjected to a red blood cell lysis step, then washed with cold phosphate-buffered saline (PBS, pH 7.4), and finally passed through a 40 μm nylon cell strainer.

[0439] Results and Conclusions The three different dosing schedules of EC17 administration showed different weight loss patterns and different levels of CRS. As shown in Figure 5, cohort 1 (no treatment) and cohort 2 (CAR-T only) did not show significant weight loss and were free of CRS. Cohort 3 (EC17 SIW) showed EC17 dose-dependent weight loss after each EC17 dose and grade 1-2 CRS throughout the study. Cohort 5 (EC17 dose escalation) showed little weight loss during the first two cycles but began to show weight loss similar to cohort 2 after the completion of the second cycle of EC17. Cohort 4 (EC17 TIW on / off) showed the mildest weight loss of the three treatment groups. Cohorts 4 or 5 did not show any CRS greater than grade 1.

[0440] Cohort 3 [Table 22]

[0441] Cohort 4 [Table 23]

[0442] Cohort 5 [Table 24]

[0443] Three different dosing schedules of EC17 administration showed different antitumor activities . As seen in Figure 6, liver and non-liver tumor burden was assessed across all cohorts at day 39. Cohort 2 (CAR-T only) did not show any differences compared to cohort 1 (no treatment). Cohort 4 showed the best anti-tumor activity across the three different EC17 dosing schedules.

[0444] Circulating AML cell lines, excluding THP1-FRβ In studies using CD19-specific anti-ALL therapy, it has been reported that the expansion and persistence of CAR T cells in the blood correlates with the clearance of circulating CD19+ ALL cells resulting in a complete response to therapy. In this study, a similar assay was performed in a mouse AML model to determine the antitumor activity of adaptor-controlled E2 CAR T cells by examining a GFP-labeled human AML cell line, THP1-FRβ, in response to different dosing regimens of the CAR T adaptor molecule, EC17. Figure 7 shows circulating THP1-FRβ cells, with the y-axis showing the total number of GFP+ cells per 100 μL of whole blood on a logarithmic scale. Different bars represent different treatment cohorts. A high AML cell burden was shown to be present in the circulation 40 days after AML cell infusion in mice that received neither CAR T cells nor EC17. No reduction in blood AML cells was seen in mice that received both AML cell and E2 CAR T cell infusions but no EC17 treatment. This indicated that the antitumor activity due to potential alloreactivity of allogeneic THP1 cells with human T cells was negligible. Interestingly, all three cohorts of mice injected with E2 CAR T cells+EC17 at various dosing regimens showed a significant reduction in circulating THP1 cell burden, thus demonstrating the efficacy of CAR T therapy under the three different adaptor dosing regimens.

[0445] E2-CAR-T exhaustion phenotype Under chronic antigen stimulation, such as in the case of chronic viral infection or cancer, T cells undergo a process of exhaustion in which they can no longer proliferate, secrete inflammatory cytokines, and kill antigen-presenting target cells. CAR T cells can also become exhausted under chronic CAR stimulation due to the chronic presence of scFv-stimulating antigen. Since E2 CAR T cells only respond in the presence of bridging molecule EC17 bound to the surface of FR+ tumor cells, E2 CAR T cells have the ability to prevent chronic antigen stimulation by ceasing treatment with EC17, thus removing the presence of surface-bound antigen. The remaining period characterized by the absence of surface antigen prevents chronic antigen exposure of E2 CAR T cells and the resulting exhaustion.

[0446] To confirm that the EC17 dosing regimen does not result in E2 CAR T cell exhaustion, flow cytometry analysis was performed on single cell preparations from THP1-FRβ liver metastases and surface markers specifically expressed on exhausted T cells. As T cells approach exhaustion, co-expression of surface inhibitory receptors, PD1, LAG3 and TIM3, increases. Figure 8 shows a bar graph showing the percentage of total E2 CAR T cells isolated from solid liver tumors expressing various combinations of the three surface markers, represented by different bars on the y-axis. Of note, fully exhausted T cells expressing all three markers simultaneously are shown on the left hand side of each group. The left group shows pre-infusion of E2 CAR T cells into liver tumor CAR T cells isolated from three different EC17-treated cohorts. Inhibitory receptor expression is nearly zero in the pre-infusion CAR T cell product, as the majority of T cells are negative for all three surface markers. Importantly, all 3EC17-treated cohorts of E2 CAR T cells had exhausted triple-positive cells, and interestingly, cohort 4 (EC17 TIW) had the lowest number of double- and triple-positive events, and furthermore consisted of a significant number of triple-negative CAR T cells. These data are clinically useful in that the entire 3EC17 dosing regimen used in this mouse study has substantial antitumor activity and induces minimal CAR T exhaustion.

[0447] Examples 19-25 - see Figures 9-14. Example 19 Cell lines and reagents Unless otherwise stated, all FR+ and FR-negative cancer cell lines were maintained in RPMI-1640 medium (Gibco BRL) supplemented with 10% heat-inactivated fetal bovine serum without (FFRPMI) or with (RPMI) 2.4 μM folic acid (FA). KB (FRα-expressing human cervical cancer with HeLa marker) and CHO-β (Chinese hamster ovary cells transfected with human FRβ) were used as sources of FRα and FRβ, respectively, for radioligand binding assays. MDA-MB-231 represents an FRα subclone of a human TNBC (triple-negative breast cancer) cell line. For AML testing, a green fluorescent protein (GFP)-expressing isogenic pair of FRβ-positive (THP1-FRβ) and FR-negative (THP1-FG12) cell lines were provided. Both were originally established from THP-1 (ATCC, TIB-202), a cell model commonly used for pediatric AML testing, derived from a 1-year-old boy with acute monocytic leukemia. For osteosarcoma testing, HOS-FRα was established by lentiviral transduction of FR-negative HOS-143b (ATCC, CRL8303) with the FOLR1 gene encoding human FRα. HOS-143b was originally established from a primary tumor in a 13-year-old Caucasian girl and is highly tumorigenic in NSG mice. FR+HOS-FRα fLuc and FR-negative HOS-143b fLuc The GFP-expressing bioluminescent pair was transduced with a lentivirus firefly luciferase.

[0448] LEGENDplex TM Human cytokine panels were purchased from BioLegend (San Diego, CA). (登録商標)A non-radioactive cytotoxicity assay kit was purchased from Promega (Madison, WI). Commercially available anti-human antibodies used for multicolor flow cytometry were CD45RA (clone HI100), CD45RO (clone UCHL1), CD4 (clone SK3), and CD69 (clone FN50) from BD Bioscience (San Jose, CA); CD3ε (clone SK7), CD8α (clone RPA-T8), CD137 / 4-1BB (clone 4B4-1), CD25 (clone M-A251), PD1 (clone EH12.1), LAG3 (clone T47-530), and TIM3 (clone 7D3) from BD Bioscience (San Jose, CA); biotinylated anti-human EGFR (cetuximab, clone Hu1) from R&D systems (Minneapolis, MN); and FRα (clone LK26) from BioLegend (San Diego, CA). Fluorophore-conjugated anti-biotin was purchased from BioLegend. APC-conjugated anti-FITC mouse IgG2a / kappa antibody (clone NAWESLEE), CountBright TM Beads (Invitrogen), Annexin V staining buffer and AlexaFluor-647-conjugated Annexin V were purchased from Thermo Fisher Scientific. For enzymatic digestion of tumor tissue, collagenase IV, hyaluronidase and DNase I were all purchased from Sigma-Aldrich (St. Louis, MO).

[0449] EC17 or folate-FITC [FA-(γ)-ethylenediamine-FITC] was synthesized in endocytes. 3 H-EC17 was purchased from Moravek biochemicals (Brea, CA) with a specific activity of approximately 0.952 Ci / mmol or produced by ViTrax (Placentia, CA) with a specific activity of approximately 1.2 Ci / mmol. 3 H-FA-(γ)-ethylenediamine conjugate was produced in endocytes. 3H-FA was also purchased from ViTrax with a specific activity of 59 Ci / mmol. For CRS rescue, sodium fluorescein dosing solution was purchased from Purdue Pharmacy (NDC 17478-250-25). (登録商標) Diluted at 25% (Fluorescein Injection, USP).

[0450] Example 20 Humanized CAR constructs and CAR-modified T cells Previous studies used a GFP+second-generation anti-FITC scFv (clone 4M5.3) CAR (i.e., FITC-4M5.3-scFv-CD8αhinge-CD8αtm-4-1BB / CD3ζ) that contains the hinge and transmembrane sequences of CD8α and the 4-1BB / CD3ζ signaling domain. To translate to first-in-human trials, a second-generation fully human FITC-specific (clone E2) CAR construct (herein referred to as E2) was developed (Figure 9, top diagram). CAR-modified T cells are shown in Figure 9, bottom pie chart. The construct described here is a FITC-specific CAR construct that contains (1) a fully human anti-FITC scFv (clone E2, Kd=0.75 nM), (2) an IgG4 hinge-CH2(L235D, N297Q)-CH3 spacer fused to a CD28-transmembrane domain, (3) a second generation 4-1BB / CD3ζ-endodomain, and (4) a cell surface human EGFRt tag (Figure 9, top diagram) (SEQ ID NOs: 4 and 5 are the nucleic acid and amino acid sequences, respectively). To transfect CAR-modified T cells, lentivirus was produced in 293T cells co-transfected with a CAR-encoding epHIV7 lentiviral vector. Donor CD4+ and CD8+ T cells were purified by immunomagnetic selection and transduced separately or in a 50:50 ratio. Typically, only one round of CD3 / CD28 bead activation was performed, followed by one or two rounds of rapid in vitro expansion. For preclinical evaluation, several batches of EGFRt-sorted CD4, CD8 and unsorted CD4 / CD8 CAR T cells were used. All CAR T cell preparations were analyzed by flow cytometry before cryopreservation and after thawing for EGFRt expression and CD4 / CD8 ratio determination. A combination of surface markers was used to analyze the differentiation status of CD4+ and CD8+ CAR T cell subsets on the day of infusion and to characterize T N , CD45RA+CD45RO- CD62L+CD95- naive T cells; T SCM , CD45RA+CD45RO- CD62L+CD95+stem cell memory T cell;T CM , CD45RA- CD45RO+CD62L+CD95+ central memory T cells;T EM , CD45RA- CD45RO+CD62L- CD95+ effector memory cells; and T EFF , defined as CD45RA+CD45RO- CD62L- CD95+ effector T cells. For the preclinical studies described below, studies included two batches of EGFRt sorted pure CD4 and CD8 subsets (after mixing at approximately 1:1 ratio) and several batches of unsorted approximately 1:1 EGFRt+CD4 / CD8 mixtures, including a "clinical replica" preparation with a poorly differentiated profile.

[0451] Among a series of different CAR constructs that were synthesized and evaluated, the fully human anti-FITC scFv (FITC-E2) CAR was selected for preclinical development (Figure 9). This second generation fully human CAR contained anti-FITC scFv (clone E2), double mutations (L235D and N297Q) in the IgG4-Fc spacer / hinge and CH2 region to reduce binding to FcγR, a CD28 transmembrane domain, and a 4-1BB / CD3ζ signaling domain linked to a cell surface EGFRt tag by a T2A ribosomal skipping sequence (i.e., FITC-E2-scFv-IgG4 hinge-CD28tm-4-1BB / CD3ζ-T2A-EGFRt). For preclinical studies, both EGFRt-selected and unselected E2 CAR T cells were prepared at approximately 1:1 CD4 / CD8 ratio, and T cell subtype phenotyping was routinely performed by flow cytometry at the time of CAR T cell infusion (day 0) for each in vivo experiment. The typical expression pattern of EGFRt-selected CAR T cells was approximately 42% T SCM , 10%T CM , 12%T EM and 34%T EFF EGFRt+, which included both CD4 and CD8 subsets (Figure 9, left pie chart). Only EGFRt-selected CAR T cells were used for co-culture and pharmacokinetic studies. For tumor therapy, a "clinical replica" batch with a poorly differentiated profile for MDA-MB-231 (Figure 9, right pie chart) was used, and the test batch was used for THP1-FRβ and HOS-FRα testing. The "clinical replica" batch (approximately 39% EGFRt+) contained approximately 66% T SCM and about 32%T CM CD4+ subset and approximately 95%T SCM and about 3%T CM The test batch (approximately 23% EGFRt+) was more fragmented, with approximately 32% T SCM , about 53%T CM , about 11%T EM and about 3.7%T EFF of CD4 subsets and approximately 44%T SCM , about 0.28%T CM , about 3.4%T EM and about 52%T EFFThe CD8 subset was included.

[0452] Example 21 Bispecific affinity of EC17 CAM The bispecific affinity of the EC17 CAM (CAM is equivalent to the "bridge" or "compound" in this application) was determined in a cell-based radioligand binding assay. 3 For binding to FR+ targets, KB and CHO-β cells were pre-seeded overnight in 24-well tissue culture plates and incubated at 0.1 nM, 0.5 nM, 1 nM, 5 nM, 10 nM, 20 nM and 40 nM. 3 H-EC17 was incubated in FFRPMI for 2 hours at 37°C. Cells were then rinsed with phosphate-buffered saline (PBS, pH 7.4) and lysed with 1% sodium dodecyl sulfate. Whole cell lysates were quantified for radioactivity levels and cellular protein content using a standard Pierce BCA protein assay. 3 The number of H-EC17 molecules was calculated to determine the dissociation constant (Kd) of each of FRα (KB) and FRβ (CHO-β) (FIG. 10). EC17 has already been tested in the clinic for immunotherapy and optical imaging purposes. 3 H-EC17 was synthesized and radioligand binding assays were performed with KB and CHO-β cell lines representing FRα+ and FRβ+ target cells, respectively, and unsorted EGFRt CAR T cells representing effector cells. Upon binding to the targets, EC17 showed similar affinity to both FRα and FRβ with low Kd values ​​of 1.7 nM and 0.8 nM (Figure 10, panel A). Upon binding to unsorted E2-CAR T cells (~24% EGFRt+, ~95:5 CD8 / CD4 ratio), the Kd value was estimated to be ~130 nM (Figure 10, panel B).

[0453] Example 22 Tumor models All animal care and use was performed in accordance with NIH guidelines and protocols approved by the Purdue University Animal Use and Care Committee. TM ) mice (stock number: 005557) were purchased from The Jackson Laboratory (Bar Harbor, ME). Unless otherwise stated, all animals were maintained on an FA-deficient diet (TestDiet, St. Louis, MO) from the time of arrival and throughout the study. To establish subcutaneous xenografts, MDA-MB-231 and HOS-FRα were each administered at 2.5 × 10 per animal. 6 and 1 × 10 6 For intravenous xenografting, THP1-FRβ cells were implanted at 5 × 10 per animal in the right flank region. 6 Subcutaneous tumors were measured with calipers 2-3 times a week and ellipsoidal (length × width) measurements were performed. 2 ) / 2. The mortality rate was calculated according to the study design or when (i) animals lost ≥ 20% of their body weight or approached moribund conditions, (ii) subcutaneous tumors grew ≥ 1500 mm 3 Sacrifice was performed when the animals reached a normal size or (iii) when they showed abdominal swelling and signs of severe distress (i.e., THP1-FRβ). All animal doses (CAR T cells, EC17, sodium fluorescein) were given intravenously.

[0454] Example 23 Tumor Treatment In a therapeutic setting, EC17 CAM may be given before or after CAR T cell injection. The first dose of EC17 used here was given 2–3.5 days after CAR T cells to allow for an observation period of human T cells in tumor-bearing mice. Two batches of unsorted E2-CAR T cells (23% or 39% EGFRt+, 1:1 CD4 / CD8) were used in the in vivo studies. On the day of injection (day 0) of each experiment, frozen CAR T cells were rapidly thawed at 37°C, washed twice with Dulbecco's 1X PBS (pH 7.4), and injected with the desired EGFRt+E2-CAR T cell dose into the tail vein. Additionally, a small amount of CAR T cells was analyzed by flow cytometry for the CD4 to CD8 ratio and differentiation status of CAR T cells. On the first day of EC17 administration, tumor-bearing animals were randomly divided into groups based on tumor size or being the same number of days since intravenous implantation (i.e., THP1-FRβ). For the MDA-MB-231 trial, mice received a high dose (~10 million) of a "clinical replicative line" of E2-CAR T cells (~39% EGFRt+) with a poorly differentiated profile (Figure 9). Two days later, mice received three different treatment regimens of EC17, with a mean tumor size of ~293±39 mm. 3 (Figure 12). EC17 dosing was given once weekly (SIW) at 500 nmol / kg on Mondays or at escalating doses of 5 nmol / kg, 50 nmol / kg or 100 nmol / kg and 500 or 1000 nmol / kg (i.e., 5 / 50 / 500 or 5 / 100 / 1000) on Mondays, Thursdays and Mondays with 6 days off between cycles. Control mice were left untreated (received CAR T cells but no EC17). For comparison, tumor-free littermate cohorts also received the same number of CAR T cells, with or without EC17 SIW, 500 nmol / kg.

[0455] For the AML study (Figure 13), mice were injected intravenously with THP1-FRβ tumor cells one day before receiving a low dose of approximately 6 million E2-CAR T cells (approximately 23% EGFRt+). Approximately 3.5 days after CAR T cell injection, EC17 was administered in three different ways, including i) SIW at 500 nmol / kg, ii) 5 / 50 / 500 nmol / kg three times Monday / Wednesday / Friday followed by 9 days off between cycles (TIW on / off) and iii) escalating doses of 5 / 10 / 100 nmol / kg in cycle 1, 5 / 30 / 300 nmol / kg in cycle 2 and 5 / 50 / 500 nmol / kg in cycle 3, all on Monday / Thursday / Monday with 6 days off between cycles (M / Th / M_on / off). On day 31, satellite animals were collected for quantification of CAR-positive T cells, identified as human CD3ε+EGFRt+ events in mouse blood, and absolute numbers per 100 μL of whole blood. At sacrifice or termination of the study on day 38, the total tumor burden of THP1-FRβ tumor-bearing mice was assessed by counting blood GFP+ tumor cells by flow cytometry, corrected for liver weight (including metastatic foci) and total weight of non-hepatic macrometastases found in the body. Liver metastatic tumor fragments were enzymatically digested into single cell suspensions, and viable cell populations were analyzed for CAR T cell exhaustion using anti-human PD1, LAG3 and TIM3 (clones EH12.1, T47-530 and 7D3, respectively). For the osteosarcoma study (Figure 14), two cohorts of mice were implanted subcutaneously with HOS-FRα tumor cells and 3 days later received approximately 6 million of the same CAR T cell preparation used for the THP1-FRβ study (Figure 13). Approximately 3.5 days after CAR T cell infusion, one cohort of mice received up to three cycles of EC17 at 5 / 10 / 100nmol / kg in cycle 1, 5 / 30 / 300nmol / kg in cycle 2, and 5 / 50 / 500nmol / kg in cycle 3, all following a Monday / Thursday / Monday regimen with 6 days off between cycles. At the end of the study, circulating CD3ε+EGFRt+CAR T cells were enumerated per 100 μL of mouse blood. HOS-FRα tumors (±EC17 treatment) were also harvested and digested for flow cytometric analysis of tumor-infiltrating CAR T cells.

[0456] Dose-escalation trial in an aggressive osteosarcoma model For the intended purpose, HOS-FRα is the most aggressive tumor model with low FR expression but functional FR level of about 5.82±1.45 pmol / mg protein. In parallel with the above THP1-FRβ study, two cohorts of mice with 3-day-old HOS-FRα tumors were given the same E2-CAR T cells at the same dose (about 6 million). One cohort was treated with the same accelerated EC17 dose escalation regimen of 5 / 10 / 100 nmol / kg in cycle 1, 5 / 30 / 300 nmol / kg in cycle 2, and 5 / 50 / 500 nmol / kg in cycle 3, Monday / Thursday / Monday schedule, followed by 6 days off (Figure 14, Panel A). Because HOS-FRα tumors grow aggressively without EC17 treatment, this CAR T cell dose-family EC17 dose escalation was safe (no CRS or weight loss) and resulted in a significant delay in tumor growth within the first two cycles of treatment (Figure 14, panel B). 3 Flow cytometry analysis performed on whole blood, with protocol-specified sacrifice by EC17 only, showed high EC17-dependent CAR T cell expansion by day 47, but not by day 33 (Figure 14, panel C, left bar graph). Ex vivo tumor analysis on day 33 showed a low, but significant, intratumoral CD3ε+EGFRt+CAR T cell population in EC17-treated animals, which consisted of approximately 1% total viable endothelial tumor-derived cells (Figure 14, panel C, right bar graph). Intratumoral CAR T cells also declined by day 47 (Figure 14, panel C), as large HOS-FRα tumors stopped responding to treatment at cycle 3 (Figure 14, panel B). Notably, disease progression led to a decline in intratumoral CAR T cells. 3 HOS-FRα tumors analyzed by H-FA radioligand assay showed similar FRα levels with and without EC17 treatment. Thus, HOS-FRα tumors in NSG mice appear to rapidly extend the tumor-infiltrating capacity of CAR T cells and reduce cytolytic activity due to T cell exhaustion, as suggested by in vitro coculture.

[0457] EC17 dose-finding study in tumor-bearing and tumor-free mice An initial EC17 dose-finding study was performed in MDA-MB-231 tumor-bearing mice as shown in the schematic diagram of the experimental layout (Figure 12, Panel A). MDA-MB-231 tumors (approximately 211 ± 65 mm 3 NSG mice lacking or bearing EGFRt were implanted with a "clinical replica" batch of approximately 10 million E2-CAR T cells (approximately 39% EGFRt+, 51:49 CD4 / CD8) on day 0. This batch of CAR T cells was predominantly T SCM and T CM The phenotypes consisted of (see Figure 9). Two days after CAR T cell injection, one cohort of tumor-bearing mice remained untreated, and three cohorts were treated with various regimens of EC17, including one injection per week (SIW) at 500 nmol / kg or escalating EC17 dose levels of 5 / 50 / 500 (escalation-1) or 5 / 100 / 1000 nmol / kg (escalation-2) on a Monday / Thursday / Monday schedule with a one-week rest interval. For comparison, two tumor-free cohorts included untreated or 500 nmol / kg treatment of EC17 SIW. Human T cell-derived IFNγ levels in mouse blood were measured in all cohorts using satellite animals on days 11 and 12 (approximately 20 and 42 hours after the previous EC17 dose). Compared to tumor-bearing mice receiving CAR T cells alone, the entire EC17-treated tumor cohort showed approximately 30× (day 11) and ~10× (day 12) higher IFNγ production in mouse plasma, with this cytokine level naturally decreasing 20–42 hours later. (Figure 12, Panel B). In line with IFNγ release, EC17 also induced CAR T cell expansion, identified as human CD3ε+EGFRt+ events in mouse blood, which persisted until day 54 (last measurement) in tumor-bearing animals (Figure 12, Panel C). In tumor-free cohorts, CAR-T cell expansion was not detected by flow cytometry, although low levels of IFNγ were detected on days 11 and 12 in animals receiving the same number of CAR T cells + EC17 (Figure 12, Panels B-C). Furthermore, no CRS symptoms (grade 0 out of a 0-5 scale) or weight loss were observed in tumor-free mice with twice-weekly administration of 500 nmol / kg EC17. Moderate to severe CRS symptoms (grade 2-3) and significant weight loss (Figure 12, panel D) were observed in the tumor-bearing cohort, which continued regimen-irrelevant EC17 administration. EC17 SIW, 500 nmol / kg caused early onset of CRS and weight loss, whereas the aggressive EC17 titration-2 regimen (up to 1000 nmol / kg) caused sustained weight loss, with animals recovering after EC17 dose cessation (Figure 12, panel D, top row). Notably, symptoms of graft-versus-host disease (GVHD), including red, itchy skin and hair loss, were evident approximately 1 month after CAR T cell engraftment. Animals receiving CAR T cells alone showed signs of nonspecific CAR T cell / tumor alloreactivity, whereas the EC17-only treated cohort resulted in 100% cure (Figure 12, panel D, bottom row). Therefore, EC17 administration in the presence of FR+ tumors was important to drive i) CAR T cell activation, ii) cytokine production, and iii) in vivo CAR T cell expansion and persistence. However, under specific conditions, severe CRS (grade ≥ 3) was driven by high CAR T cell doses in combination with EC17 doses of 500 nmol / kg or higher.

[0458] Administration of EC17 CAM for anti-leukemic activity Intravenously implanted GFP-expressing THP1-FRβ tumor cells developed disseminated disease in NSG mice with tumor cells in circulation and liver / body non-hepatic metastases. THP1-FRβ tumor cells also localized to mouse ovaries where inflammation appeared at early stages of tumor progression. Therefore, the total tumor burden of each animal in the test cohort was assessed by quantification of blood circulating GFP+ tumor cells, liver weight and total encompassing non-hepatic macrometastases visible to the naked eye. Although THP1-FRβ expressed low levels of FR in vitro, THP1-FRβ tumor metastases were found to express higher than expected functional FR at approximately 8.9±2.8 pmol / mg membrane protein levels. Therefore, THP1-FRβ tumor-bearing mice were implanted with a test batch of approximately 6 million / animal EGFRt-unsorted E2-CAR T cells (approximately 23% EGFRt+, 1:1 CD4:CD8) and then treated with three different EC17 dosing regimens (Figure 13). Beginning 3 days after CAR T cell injection, the EC17 dosing regimen was initiated either continuously at 500 nmol / kg in SIW, three times a week (TIW), at 5 / 50 / 500 nmol / kg on Monday / Wednesday / Friday followed by 9 days off or an accelerated dose escalation regimen of 5 / 10 / 100 nmol / kg in cycle 1, 5 / 30 / 300 nmol / kg in cycle 2, and 5 / 50 / 500 nmol / kg on Monday / Thursday / Monday (M / Th / M) followed by 6 days off (Figure 13, Panel A). In cycles 2 and 3, some weight loss and grade 1-2 CRS were observed in animals receiving the EC17 SIW treatment, whereas animals receiving EC17 TIW had minimal weight loss with only grade 0-1 CRS (Figure 13, Panel B). Among animals receiving 3 cycles of EC17 M / Th / M dose escalation, grade 0-1 CRS and very mild weight loss were observed following the final dose of 300 nmol / kg EC17 in cycle 2. Satellite animals were used to enumerate CAR T cells in the blood on day 31, and data demonstrated EC17-dependent CAR T cell expansion and persistence in all treatment cohorts (Figure 13, panel C).Compared to control animals that received tumor cells alone or tumor cells + CAR T cells without EC17, EC17 administered in any of the three "intrapatient" titration formats efficiently reduced circulating THP1-FRβ tumor cells and showed similar activity in liver tumor metastases (Figure 13, Panel D, left and center bars). Only mild alloreactivity was observed in THP1-FRβ liver metastases in mice that received CAR T cells alone. EC17 SIW and TIW with 10-fold dose escalation (on / off) were successful in completely controlling non-liver macrometastases, whereas EC17 M / Th / M dose escalation at a slower pace per cycle (Figure 13, Panel A) failed to control non-liver macrometastases (Figure 13, Panel D, far right panel). At the end of the study (i.e., 39 days after CAR T cell injection), CAR T cells isolated from hepatic THP1-FRβ tumor metastases were found to have minimal double and triple positive T cell inhibitory receptor, PD1, LAG3 and TIM3 expression (Figure 13, Panel E). No overall, severe CRS (i.e., grade > 3) was observed in any of the EC17-treated cohorts. Nevertheless, the more aggressive EC17 TIW dose escalation (on / off) schedule trended towards being the best regimen for reducing overall THP1-FRβ tumor burden in these mice.

[0459] Example 24 Functional FR evaluation These functional FR evaluations were applied in the examples described herein. Cancer cell lines of various histologies and FR expression levels (FIG. 11) were included, as well as pediatric cancer cell lines transfected with FRα (HOS-FRα) and FRβ (THP1-FRβ). Radioligand binding assays (100 nM 3 H-FA, 1 h, 37°C), the total available FR for these cell lines is estimated to be on the order of 9 × 10 4 (OV90, a low FR-expressing ovarian cancer cell line), 1.9 × 10 5 (THP1-FRβ), 2.4 × 10 5 (HOS-FRα fLuc ), 7×10 5 (HOS-FRα), 2.1 × 10 6 (MDA-MB-231) and 4.8 × 106 (KB) FA molecules / cell. As a FR negative control, HOS-143b (fLuc) and THP1-FG12 parental cell lines were also included. Thus, the order of functional FR expression in the co-cultured FR+ cancer cell lines is KB>MDA-MB-231>HOS-FRα>HOS-FRα. fLuc >THP1-FRβ(AML)>OV90.

[0460] Example 25 statistics Statistical analysis was performed with the computer program GraphPad Prism (GraphPad Software Inc., San Diego, CA). Data were analyzed using Student's t-test or one-way ANOVA. When applicable, data were further analyzed across multiple treatment groups using appropriate multiple post hoc tests. *=p<0.05 was considered statistically significant for all tests.

Claims

1. 1. A method of treating cancer, comprising: i) administering to the patient at least one dose of a CAR T cell composition, wherein the CAR T cell comprises a CAR directed to a targeting moiety; ii) administering to a patient a compound or a pharma- ceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand attached to a targeting moiety by a linker, wherein the compound or a pharma- ceutically acceptable salt thereof is administered with at least a first dose escalation course and a second dose escalation course.

2. 10. The method of claim 1, wherein the compound or a pharma- ceutically acceptable salt thereof is administered in at least a first dose-escalation course, a second dose-escalation course and a third dose-escalation course.

3. 10. The method of claim 1, wherein the compound or a pharma- ceutically acceptable salt thereof is administered in at least a first dose escalation course, a second dose escalation course, a third dose escalation course and a fourth dose escalation course.

4. 2. The method of claim 1, wherein the compound or a pharma- ceutically acceptable salt thereof is administered at least a first dose escalation course, a second dose escalation course, a third dose escalation course, a fourth dose escalation course and a fifth dose escalation course.

5. 2. The method of claim 1, wherein the compound or a pharma- ceutically acceptable salt thereof is administered at least a first dose escalation course, a second dose escalation course, a third dose escalation course, a fourth dose escalation course, a fifth dose escalation course and a sixth dose escalation course.

6. 6. The method of any of claims 1-5, wherein the patient is administered an initial dose of CAR T cells and a second dose of CAR T cells.

7. 7. The method of claim 6, wherein the initial dose of CAR T cells is a test dose for monitoring the patient's tolerance to the CAR T cells.

8. 7. The method of claim 6, wherein the second dose of CAR T cells comprises a higher dose of CAR T cells than the first dose of CAR T cells.

9. 9. The method of any of claims 1 to 8, wherein the first dose escalation course is followed by a period during which the compound or a pharma- ceutically acceptable salt thereof is not administered.

10. 10. The method of any of claims 1-9, wherein the second dose-escalation course is followed by a period during which the compound or a pharma- ceutically acceptable salt thereof is not administered.

11. 11. The method of any of claims 2-10, wherein after the third dose-escalation course there is a period during which the compound or a pharma- ceutically acceptable salt thereof is not administered.

12. 12. The method of any of claims 3 to 11, wherein after the fourth dose-escalation course there is a period during which the compound or a pharma- ceutically acceptable salt thereof is not administered.

13. 13. The method of any of claims 4 to 12, wherein after the fifth dose escalation course there is a period during which the compound or a pharma- ceutically acceptable salt thereof is not administered.

14. 14. The method of any of claims 5 to 13, wherein after the sixth dose-escalation course there is a period during which the compound or a pharma- ceutically acceptable salt thereof is not administered.

15. 15. The method of any of claims 9 to 14, wherein the period is about 7 days.

16. 16. The method of any of claims 1-15, wherein the first dose escalation course comprises administering to the patient about 1%, about 10%, and about 100% of the total dose of the compound or a pharma- ceutically acceptable salt thereof on three non-consecutive days.

17. 17. The method of any of claims 1-16, wherein the second dose escalation course comprises administering to the patient about 1%, about 30%, and about 300% of the total dose of the compound or a pharma- ceutically acceptable salt thereof on three non-consecutive days.

18. 18. The method of any of claims 2-17, wherein the third dose escalation course comprises administering to the patient about 1%, about 50%, and about 500% of the total dose of the compound or a pharma- ceutically acceptable salt thereof on three non-consecutive days.

19. 19. The method of any of claims 3-18, wherein the fourth dose escalation course comprises administering to the patient about 1%, about 10%, and about 100% of the total dose of the compound or a pharma- ceutically acceptable salt thereof on three non-consecutive days.

20. 20. The method of any of claims 4-19, wherein the fifth dose escalation course comprises administering to the patient on three non-consecutive days about 1%, about 30%, and about 300% of the total dose of the compound or a pharma- ceutically acceptable salt thereof.

21. 21. The method of any of claims 5-20, wherein the sixth dose escalation course comprises administering to the patient on three non-consecutive days about 1%, about 50%, and about 500% of the total dose of the compound or a pharma- ceutically acceptable salt thereof.

22. 22. The method of any of claims 16-21, wherein the total dosage of the compound or a pharma- ceutically acceptable salt thereof is from about 10 μg / kg to about 50 μg / kg of the compound or a pharma- ceutically acceptable salt thereof.

23. 23. The method of any of claims 16-22, wherein the total dosage of the compound or a pharma- ceutically acceptable salt thereof is from about 20 μg / kg to about 40 μg / kg of the compound or a pharma- ceutically acceptable salt thereof.

24. 24. The method of any of claims 16-23, wherein the total dosage of the compound or a pharma- ceutically acceptable salt thereof is from about 25 μg / kg to about 35 μg / kg of the compound or a pharma- ceutically acceptable salt thereof.

25. 25. The method of any of claims 16-24, wherein the total dosage of the compound or a pharma- ceutically acceptable salt thereof is about 30 μg / kg of the compound or a pharma- ceutically acceptable salt thereof.

26. 26. The method of any of claims 1 to 25, wherein if the patient does not observe CRS or neurotoxicity during the first dose escalation course, the method proceeds to a second dose escalation course.

27. 27. The method of any of claims 2 to 26, wherein if the patient does not observe CRS or neurotoxicity during the second dose escalation course, the method proceeds to a third dose escalation course.

28. 28. The method of any of claims 3 to 27, wherein if the patient does not observe CRS or neurotoxicity during the third dose-escalation course, the method proceeds to a fourth dose-escalation course.

29. 29. The method of any of claims 4 to 28, wherein if CRS or neurotoxicity is not observed in the patient during the fourth dose-escalation course, the method proceeds to a fifth dose-escalation course.

30. 30. The method of any of claims 5 to 29, wherein if no CRS or neurotoxicity is observed in the patient during the fifth dose escalation course, the method proceeds to a sixth dose escalation course.

31. 31. The method of any of claims 1-30, wherein if fever without hypotension is observed in the patient at any time during the dose escalation course, and neurotoxicity is not observed in the patient, then all subsequent doses of the compound, or a pharma- ceutically acceptable salt thereof, are administered at that dose escalation course level that caused fever without hypotension in the patient.

32. 31. The method of any of claims 1-30, wherein if the patient experiences severe CRS or neurotoxicity during any dose escalation course, all subsequent doses of the compound or a pharma- ceutically acceptable salt thereof are administered to the patient at a dose escalation course level that is lower than the dose escalation course level that caused the patient to experience severe CRS or neurotoxicity.

33. 1. A method of treating cancer, comprising: i) administering to the patient at least one dose of a CAR T cell composition, wherein the CAR T cell comprises a CAR directed to a targeting moiety; ii) administering to the patient a compound or a pharma- ceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand attached to a targeting moiety by a linker, wherein the compound or a pharma- ceutically acceptable salt thereof is administered during a first dose-escalation course, and if severe CRS occurs during the first dose-escalation course, administering the compound or a pharma- ceutically acceptable salt thereof using a low-dose escalation course, wherein the initial dose of the compound or a pharma- ceutically acceptable salt thereof in the low-dose escalation course is lower than the initial dose of the compound or a pharma- ceutically acceptable salt thereof administered during the first dose-escalation course.

34. 34. The method of claim 33, wherein in the low-dose escalation course, the compound or a pharma- ceutically acceptable salt thereof is administered at about 0.5%, about 5%, and about 50% of the total dose of the compound or a pharma- ceutically acceptable salt thereof on three non-consecutive days.

35. 35. The method of claim 34, wherein the total dose of the compound or a pharma- ceutically acceptable salt thereof is from about 10 μg / kg to about 50 μg / kg of the compound or a pharma- ceutically acceptable salt thereof.

36. 36. The method of any of claims 34-35, wherein the total dosage of the compound or a pharma- ceutically acceptable salt thereof is from about 25 μg / kg to about 35 μg / kg of the compound or a pharma- ceutically acceptable salt thereof.

37. 37. The method of any of claims 34-36, wherein the total dosage of the compound or a pharma- ceutically acceptable salt thereof is about 30 μg / kg of the compound or a pharma- ceutically acceptable salt thereof.

38. The method of any of claims 1 to 37, wherein the CAR T cell comprises a nucleic acid comprising SEQ ID NO:

4.

39. The method of any of claims 1-38, wherein the CAR T cell comprises a polypeptide comprising SEQ ID NO:

5.

40. 39. The method of claim 38, wherein the nucleic acid encodes a chimeric antigen receptor.

41. 16. The method of any of claims 2-15, wherein a first dose escalation course comprises administering to the patient about 1%, about 2%, and about 20% of the total dose of the compound or pharma- ceutically acceptable salt thereof on three non-consecutive days, a second dose escalation course comprises administering to the patient about 1%, about 6%, and about 60% of the total dose of the compound or pharma- ceutically acceptable salt thereof on three non-consecutive days, and a third dose escalation course comprises administering to the patient about 1%, about 10%, and about 100% of the total dose of the compound or pharma- ceutically acceptable salt thereof on three non-consecutive days.

42. 16. The method of any of claims 2-15, wherein a first dose escalation course comprises administering to the patient about 1%, about 10%, and about 100% of the total dose of the compound or a pharma- ceutically acceptable salt thereof on three non-consecutive days, and a second dose escalation course comprises administering to the patient about 1%, about 20%, and about 200% of the total dose of the compound or a pharma- ceutically acceptable salt thereof on three non-consecutive days.

43. 16. The method of any of claims 2-15, wherein a first dose escalation course comprises administering to the patient about 1%, about 10%, and about 100% of the total dose of the compound or a pharma- ceutically acceptable salt thereof on three non-consecutive days, and a second dose escalation course comprises administering to the patient about 1%, about 10%, and about 100% of the total dose of the compound or a pharma- ceutically acceptable salt thereof on three non-consecutive days.

44. 16. The method of any of claims 2-15, wherein a first dose escalation course comprises administering to the patient about 1%, about 20%, and about 200% of the total dose of the compound or a pharma- ceutically acceptable salt thereof on three non-consecutive days, and a second dose escalation course comprises administering to the patient about 1%, about 20%, and about 200% of the total dose of the compound or a pharma- ceutically acceptable salt thereof on three non-consecutive days.

45. 45. The method of any of claims 41 to 44, wherein the total dosage of the compound or its pharma- ceutically acceptable salt is about 500 nmol / kg.

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