How CAR T cells are used

Abstract

The present disclosure relates to a method for treating a patient with cancer by administering a composition comprising a CAR T cell to the patient, and administering to the patient a small molecule connected to a targeting moiety by a linker, wherein the CAR T cell comprises a CAR and the CAR comprises an E2 anti-fluorescein antibody fragment. The present disclosure also relates to a composition for use in such a method.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] No. 62 / 735,627 filed on September 24, 2018, and U.S. Provisional Application Serial No. 62 / 736,727 filed on September 26, 2018, all of which are incorporated herein by reference in their entireties. Technical Field

[0003] The present disclosure relates to a method for treating a patient with cancer by administering a composition comprising a CAR T cell to the patient, and administering to the patient a small molecule connected to a targeting moiety by a linker, wherein the CAR T cell comprises a CAR and the CAR comprises an E2 anti-fluorescein antibody fragment. The present disclosure also relates to a composition for use in such a method. Background Art

[0004] Immunotherapy based on adoptive transfer of lymphocytes (e.g., T cells) to patients is a valuable therapy for the treatment of cancer and other diseases. In the development of immunotherapy based on adoptive transfer of lymphocytes, many important advances have been made. Among many different types of immunotherapeutics, one of the most promising immunotherapeutics being developed is T cells (CAR T cells) expressing chimeric antigen receptors. Chimeric antigen receptors (CAR) are genetically engineered receptors that are designed to target specific antigens (e.g., tumor antigens). This targeting can lead to cytotoxicity to tumors, such as allowing CAR T cells expressing CAR to target and kill tumors via specific tumor antigens.

[0005] The first generation of CAR consists of a recognition region for recognizing and binding to an antigen expressed by a tumor (e.g., a single-chain fragment variable (scFv) region derived from an antibody), and an activation signal domain (e.g., the CD3ζ chain of a T cell can serve as a T cell activation signal in a CAR). Although CAR T cells have shown positive results in vitro, their success in eliminating diseases (e.g., cancer) in clinical trials is limited. One problem is that the CAR T cell population cannot be activated and expanded for a long time in vivo.

[0006] To solve this problem, a costimulatory domain (e.g., CD137, CD28, or CD134) has been included in the second generation CAR to achieve prolonged activation of T cells in vivo. The addition of a costimulatory domain enhances the in vivo proliferation and survival of T cells containing CAR, and initial clinical data have shown that such constructs are promising therapeutic agents in the treatment of diseases such as cancer.

[0007] Although improvements have been made in CAR T cell therapy, there are still some problems. First, due to the expression of normal cells targeted by CART cells (e.g., tumor-associated antigens), "off-target" toxicity may occur. Secondly, unregulated CAR T cell activation can be found, wherein a series of metabolic disorders called tumor lysis syndrome or cytokine release syndrome (CRS) are induced by CAR T cells to eliminate disease cells (e.g., cancer cells) quickly and uncontrolled, which can be fatal to patients. Due to the administration of CAR T cells that cannot be easily adjusted and uncontrollably activated, tumor lysis syndrome and CRS can be produced. Therefore, although CAR T cells show great prospects as a tool in the treatment of diseases (such as cancer), it is necessary to provide reduced off-target toxicity and CAR T cell activation. Additional CAR T cell therapy with more precise control. Summary of the invention

[0008] The inventors have found methods for reducing off-target toxicity and more accurately controlling CAR T cell activation, which provide important progress in CAR T cell therapy. In various embodiments described herein, a small molecule ligand connected to a targeting moiety by a joint is used as a bridge between cancer and CAR T cells expressing CAR, wherein the CAR comprises an E2 anti-fluorescein antibody fragment. The bridge directs the CAR T cells expressing CARs comprising E2 anti-fluorescein antibody fragments to cancer to improve cancer. In one embodiment, the "small molecule ligand" can be, for example, folic acid, DUPA, NK-1R ligands, CAIX ligands, gamma glutamyl transpeptidase, NKG2D ligands, or CCK2R ligands, each of which is a small molecule ligand that specifically binds to cancer cells (i.e., the receptors of these ligands are overexpressed in cancer compared to normal tissues).

[0009] In one embodiment, the "small molecule ligand" is connected 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, fluorescein, fluorescein isothiocyanate (FITC), NHS- and / or fluorescein.

[0010] The "targeting moiety" is bound to the recognition region of the genetically engineered CAR expressing the E2 anti-fluorescein antibody fragment. Therefore, the recognition region of the CAR (e.g., single-chain variable region (scFv), Fab, Fv, Fc, (Fab')2 fragment, etc. of the E2 anti-fluorescein antibody fragment) is directed to the "targeting moiety". Therefore, the small molecule ligand connected to the targeting moiety by a linker acts as a bridge between the cancer and the CAR T cells expressing the E2 anti-fluorescein antibody fragment, directing the CAR T cells to the cancer to improve the cancer.

[0011] In one embodiment, a method for treating cancer is provided. The method comprises i) administering a compound or a pharmaceutically acceptable salt thereof to a patient, wherein the compound comprises a small molecule ligand connected to a targeting moiety via a linker, ii) administering to the patient a first dose of a CAR T cell composition comprising a CAR T cell, wherein the CAR T cell comprises a CAR directed to a targeting moiety and wherein the CAR comprises an E2 anti-fluorescein antibody fragment, and iii) administering to the patient a second dose of a CAR T cell composition comprising a CAR T cell, wherein the CAR T cell comprises a CAR directed to a targeting moiety and wherein the CAR comprises an E2 anti-fluorescein antibody fragment.

[0012] In another embodiment, a method for treating cancer is provided. The method comprises i) administering a compound or a pharmaceutically acceptable salt thereof to a patient, wherein the compound comprises a small molecule ligand connected to a targeting moiety via a linker, and ii) administering a CAR T cell composition to the patient, wherein the CAR T cell composition comprises a CAR T cell, wherein the CAR T cell comprises a CAR directed to a targeting moiety, wherein the CAR comprises an E2 anti-fluorescein antibody fragment, and wherein the CAR T cell composition comprises a mixture of the CAR T cell and a non-transformed T cell.

[0013] In yet another embodiment, a method for treating cancer is provided. The method comprises i) administering a compound or a pharmaceutically acceptable salt thereof to a patient, wherein the compound comprises a small molecule ligand connected to a targeting moiety by a linker, ii) administering a CAR T cell composition to the patient, wherein the CAR T cell composition comprises a CAR T cell, wherein the CART cell comprises a CAR directed to a targeting moiety and wherein the CAR comprises an E2 anti-fluorescein antibody fragment, and iii) administering a folate, a conjugate comprising a folate (wherein the conjugate comprising a folate does not include a targeting moiety) or an agent that inhibits activation of CAR T cells to the patient.

[0014] In yet another embodiment, a method for treating cancer is provided. The method comprises i) administering a compound or a pharmaceutically acceptable salt thereof to a patient, wherein the compound comprises a small molecule ligand connected to a targeting moiety by a linker, wherein at least a first dose and a second dose of the compound or a pharmaceutically acceptable salt thereof are administered to the patient, wherein the first dose is different from the second dose, wherein the amount of the compound or a pharmaceutically acceptable salt thereof in the second dose is about 2 times to about 15000 times greater than the amount of the compound or a pharmaceutically acceptable salt thereof in the first dose, and ii) administering a CAR T cell composition comprising a CAR T cell to the patient, wherein the CAR T cell comprises a CAR directed to a targeting moiety and wherein the CAR comprises an E2 anti-fluorescein antibody fragment.

[0015] In another illustrative embodiment, a method for treating cancer is provided. The method comprises i) administering to a patient a first dose of a compound or a pharmaceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked to a targeting moiety via a linker, ii) administering to the patient at least a second dose of the compound or a pharmaceutically acceptable salt thereof, wherein the amount of the second dose of the compound or a pharmaceutically acceptable salt thereof is at least about 50% lower than the amount of the first dose of the compound or a pharmaceutically acceptable salt thereof; and iii) administering to the patient a dose of a CAR T cell composition comprising a CAR T cell, wherein the CAR T cell comprises a CAR directed to the targeting moiety and wherein the CAR comprises an E2 anti-fluorescein antibody fragment.

[0016] In another embodiment, a method for treating cancer is provided. The method comprises i) administering a compound or a pharmaceutically acceptable salt thereof to a patient, wherein the compound comprises a small molecule ligand connected to a targeting moiety by a linker, and wherein the compound or a pharmaceutically acceptable salt thereof is administered at a dose of about 10 nmol / kg patient body weight to about 2500 nmol / kg patient body weight, and ii) administering a CAR T cell composition comprising CAR T cells to the patient, wherein the CAR T cells comprise CAR, wherein the CAR comprises an E2 anti-fluorescein antibody fragment, and wherein the dose of the CAR T cells is about 1 million CAR T cells to about 15 million CAR T cells.

[0017] In yet another embodiment, a method for treating cancer is provided. The method comprises i) continuously administering a compound or a pharmaceutically acceptable salt thereof to a patient, wherein the compound comprises a small molecule ligand connected to a targeting moiety by a linker, ii) administering a CAR T cell composition comprising a CAR T cell to the patient, wherein the CAR T cell comprises a CAR, wherein the CAR comprises an E2 anti-fluorescein antibody fragment, and iii) terminating the continuous administration of the compound or a pharmaceutically acceptable salt thereof to inhibit or prevent cytokine release syndrome in the patient.

[0018] In another embodiment, a method for treating cancer is provided. The method comprises i) administering a compound or a pharmaceutically acceptable salt thereof to a patient, wherein the compound comprises a small molecule ligand connected to a targeting moiety by a linker and wherein the compound or a pharmaceutically acceptable salt thereof is administered to the patient once a week, and ii) administering a CAR T cell composition comprising a CAR T cell to the patient, wherein the CAR T cell comprises a CAR, wherein the CAR comprises an E2 anti-fluorescein antibody fragment.

[0019] In yet another embodiment, a method for treating cancer is provided. The method comprises i) administering a first dose of a compound or a pharmaceutically acceptable salt thereof to a patient, wherein the compound comprises a small molecule ligand connected to a targeting moiety by a linker and wherein the compound or a pharmaceutically acceptable salt thereof is administered to the patient prior to administering a CAR T cell composition comprising CAR T cells, wherein the CAR T cells comprise a CAR directed to the targeting moiety, ii) then administering a dose of the CAR T cell composition to the patient, and iii) then administering a second dose of the compound or a pharmaceutically acceptable salt thereof to the patient, wherein the CAR comprises an E2 anti-fluorescein antibody fragment.

[0020] Other embodiments are also described by the following enumerated clauses.It is also contemplated that any of the following embodiments may be combined with any applicable embodiment described in the Summary section, Detailed Description of Illustrative Embodiments section, Examples section, or Claims of this application.

[0021] 1. A method for treating cancer, the method comprising:

[0022] i) administering to a patient a compound or a pharmaceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked to a targeting moiety via a linker;

[0023] ii) administering to the patient a first dose of a CAR T cell composition comprising a CAR T cell, wherein the CAR T cell comprises a CAR directed to the targeting moiety and wherein the CAR comprises an E2 anti-fluorescein antibody fragment; and

[0024] iii) administering to the patient a second dose of a CAR T cell composition comprising a CAR T cell, wherein the CART cell comprises a CAR directed to the targeting moiety and wherein the CAR comprises an E2 anti-fluorescein antibody fragment.

[0025] 2. A method for treating cancer, the method comprising:

[0026] i) administering to a patient a compound or a pharmaceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked to a targeting moiety via a linker, and

[0027] ii) administering a CAR T cell composition to the patient, wherein the CAR T cell composition comprises CAR T cells, wherein the CAR T cells comprise a CAR directed to the targeting portion, wherein the CAR comprises an E2 anti-fluorescein antibody fragment, and wherein the CAR T cell composition comprises a mixture of the CAR T cells and non-transformed T cells.

[0028] 3. A method for treating cancer, the method comprising:

[0029] i) administering to a patient a compound or a pharmaceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked to a targeting moiety via a linker;

[0030] ii) administering a CAR T cell composition to the patient, wherein the CAR T cell composition comprises a CAR T cell, wherein the CAR T cell comprises a CAR directed to the targeting moiety and wherein the CAR comprises an E2 anti-fluorescein antibody fragment; and

[0031] iii) administering to the patient a folate, a conjugate comprising a folate (wherein the conjugate comprising a folate does not include a targeting moiety), or an agent that inhibits activation of CAR T cells.

[0032] 4. The method of clause 3, wherein step iii comprises administering a folate.

[0033] 5. The method of any one of clauses 3 or 4, wherein step iii comprises administering folic acid or leucovorin.

[0034] 6. The method of clause 3, wherein step iii comprises administering the conjugate comprising a folate.

[0035] 7. The method of clause 6, wherein the conjugate comprising a folate comprises a folate linked to one or more amino acids.

[0036] 8. The method of clause 7, wherein the conjugate comprising a folate has the formula

[0037] .

[0038] 9. The method of any one of clauses 3 to 8, wherein the folate has the formula:

[0039]

[0040] Where X 1 and Y 1 are each independently selected from halogen, R 2 , OR 2 , SR 3 and NR 4 R 5 ;

[0041] U, V and W represent divalent moieties, each independently selected from -(R 6a )C=、-N=、-(R 6a )C(R 7a )- and -N(R 4a )-; Q is selected from C and CH; T is selected from S, O, N and -C=C-;

[0042] X 2 and X 3 Each independently selected from the following: 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, wherein Z is oxygen or sulfur;

[0043] R 1 Selected from hydrogen, halogen, C1-C 12 Alkyl and C1-C 12 Alkoxy;

[0044] R 2 , R 3 , R 4 , R 4a , R 4b, R 5 , R 5b , R 6b and R 7b Each independently selected from the following: hydrogen, halogen, C1-C 12 Alkyl, C1-C 12 Alkoxy, C1-C 12 Acyl, C1-C 12 Alkenyl, C1-C 12 Alkynyl, (C1-C 12 Alkoxy)carbonyl and (C1-C 12 alkylamino)carbonyl;

[0045] R 6 and R 7 are independently selected from hydrogen, halogen, C1-C 12 Alkyl and C1-C 12 Alkoxy; or R 6 With R 7 Together they form a carbonyl group;

[0046] R 6a and R 7a are independently selected from hydrogen, halogen, C1-C 12 Alkyl and C1-C 12 Alkoxy; or R 6a With R 7a Together they form a carbonyl group;

[0047] p, r, s and t are each independently 0 or 1; and

[0048] * indicates an optional covalent bond to the rest of the conjugate.

[0049] 10. A method as described in claim 3, wherein an agent that inhibits the activation of CAR T cells is administered and is selected from the following: lymphocyte-specific protein tyrosine kinase inhibitors, PI3 kinase inhibitors, IL-2-inducible T cell kinase inhibitors, JAK inhibitors, BTK inhibitors, EC2319, and an agent that blocks CAR T cells from binding to the compound or a pharmaceutically acceptable salt thereof, but not binding to the cancer.

[0050] 11. The method of clause 10, wherein an agent that inhibits activation of CAR T cells is administered and the agent is a lymphocyte-specific protein tyrosine kinase inhibitor.

[0051] 12. The method according to clause 11, wherein the lymphocyte-specific protein tyrosine kinase inhibitor is Dasatinib.

[0052] 13. The method of clause 10, wherein an agent that inhibits activation of CAR T cells is administered and the agent is a PI3 kinase inhibitor.

[0053] 14. The method of clause 13, wherein the PI3 kinase inhibitor is GDC0980.

[0054] 15. The method of clause 10, wherein an agent that inhibits activation of CAR T cells is administered and the agent is an IL-2 inducible T cell kinase inhibitor.

[0055] 16. The method of clause 15, wherein the IL-2 inducible T cell kinase inhibitor is BMS-509744.

[0056] 17. The method of clause 10, wherein an agent that inhibits activation of CAR T cells is administered and is an agent that blocks CAR T cells from binding to the compound or a pharmaceutically acceptable salt thereof, but not to the cancer.

[0057] 18. The method of clause 17, wherein the agent is fluoresceinamine, FITC or fluorescein sodium.

[0058] 19. The method of clause 18, wherein the agent is FITC.

[0059] 20. The method of clause 18, wherein the agent is fluorescein sodium.

[0060] 21. The method of any one of clauses 17 to 20, wherein the agent that inhibits activation of CAR T cells is administered at a dose of about .01 to about 300 μmol / kg patient body weight.

[0061] 22. The method of any one of clauses 17 to 20, wherein the agent that inhibits activation of CAR T cells is administered at a dose of about .06 to about 100 μmol / kg of patient body weight.

[0062] 23. The method of any one of clauses 17 to 20, wherein the agent that inhibits activation of CAR T cells is administered at a dose of about .06 to about 90 μmol / kg patient body weight.

[0063] 24. The method of any one of clauses 17 to 20, wherein the agent that inhibits activation of CAR T cells is administered at a dose of about .06 to about 80 μmol / kg patient body weight.

[0064] 25. The method of any one of clauses 17 to 20, wherein the agent that inhibits activation of CAR T cells is administered at a dose of about .06 to about 70 μmol / kg patient body weight.

[0065] 26. The method of any one of clauses 17 to 20, wherein the agent that inhibits activation of CAR T cells is administered at a dose of about .06 to about 60 μmol / kg patient body weight.

[0066] 27. The method of any one of clauses 17 to 20, wherein the agent that inhibits activation of CAR T cells is administered at a dose of about .06 to about 50 μmol / kg patient body weight.

[0067] 28. The method of any one of clauses 17 to 20, wherein the agent that inhibits activation of CAR T cells is administered at a dose of about .06 to about 40 μmol / kg patient body weight.

[0068] 29. The method of any one of clauses 17 to 20, wherein the agent that inhibits activation of CAR T cells is administered at a dose of about .06 to about 30 μmol / kg patient body weight.

[0069] 30. The method of any one of clauses 17 to 20, wherein the agent that inhibits activation of CAR T cells is administered at a dose of about .06 to about 20 μmol / kg patient body weight.

[0070] 31. The method of any one of clauses 17 to 20, wherein the agent that inhibits activation of CAR T cells is administered at a dose of about .06 to about 10 μmol / kg patient body weight.

[0071] 32. The method of any one of clauses 17 to 20, wherein the agent that inhibits activation of CAR T cells is administered at a dose of about .06 to about 8 μmol / kg patient body weight.

[0072] 33. The method of any one of clauses 17 to 20, wherein the agent that inhibits activation of CAR T cells is administered at a dose of about .06 to about 6 μmol / kg patient body weight.

[0073] 34. The method of any one of clauses 3 to 33, wherein more than one dose of the folate, the conjugate comprising the folate (wherein the conjugate comprising the folate does not include a targeting moiety) or the agent that inhibits activation of CAR T cells is administered to the patient.

[0074] 35. A method as described in any one of clauses 3 to 34, wherein the folate, the conjugate comprising the folate (wherein the conjugate comprising the folate does not include a targeting portion) or the agent that inhibits the activation of CAR T cells is administered to the patient before and / or after the compound or a pharmaceutically acceptable salt thereof.

[0075] 36. A method as described in any one of clauses 3 to 35, wherein administration of the folate, the conjugate comprising the folate (wherein the conjugate comprising the folate does not include a targeting portion), or the agent that inhibits activation of CAR T cells results in a reduction in cytokine levels in the patient.

[0076] 37. The method of clause 36, wherein a reduction in cytokine levels occurs about 3 hours after administering the folate, the conjugate comprising the folate (wherein the conjugate comprising the folate does not include a targeting portion), or the agent that inhibits activation of CAR T cells to the patient.

[0077] 38. The method of clause 36, wherein a reduction in cytokine levels occurs about 6 hours after administration of the folate, the conjugate comprising the folate (wherein the conjugate comprising the folate does not include a targeting moiety), or the agent that inhibits activation of CAR T cells to the patient.

[0078] 39. The method of any one of clauses 36 to 38, wherein the reduction in cytokine levels is to approximately the cytokine levels in untreated patients.

[0079] 40. A method as described in any one of clauses 3 to 39, wherein the compound or a pharmaceutically acceptable salt thereof is administered before and subsequently to the administration of the folate, the conjugate comprising the folate (wherein the conjugate comprising the folate does not include a targeting portion) or the agent that inhibits the activation of CAR T cells.

[0080] 41. A method as described in any one of clauses 3 to 40, wherein after administration of the folate, the conjugate comprising the folate (wherein the conjugate comprising the folate does not include a targeting portion) or the agent that inhibits the activation of CAR T cells, the number of CAR T cells in the patient's blood increases, even if the cytokine level in the patient is reduced.

[0081] 42. A method as described in any one of clauses 3 to 41, wherein after administration of the folate, the conjugate comprising the folate (wherein the conjugate comprising the folate does not include a targeting portion) or the agent that inhibits the activation of CAR T cells, CAR T cell activation is enhanced or maintained relative to patients not treated with a rescue agent, even if cytokine levels in the treated patient are reduced.

[0082] 43. A method as described in any one of clauses 3 to 42, wherein the cancer comprises a tumor and the tumor size in the patient does not increase when the folate, the conjugate comprising a folate (wherein the conjugate comprising a folate does not include a targeting portion) or the agent that inhibits the activation of CAR T cells is administered to the patient.

[0083] 44. The method of clause 43, wherein a complete response to the tumor is achieved.

[0084] 45. A method as described in any one of clauses 3 to 44, wherein when the CRS grade reaches 1, 2, 3 or 4, the folate, the conjugate comprising a folate (wherein the conjugate comprising a folate does not include a targeting portion) or the agent that inhibits the activation of CAR T cells is administered to the patient.

[0085] 46. ​​The method of clause 45, wherein when the CRS grade reaches 3 or 4, an agent that inhibits the activation of CAR T cells is administered to the patient.

[0086] 47. The method of clauses 3 to 46, wherein pulmonary edema is reduced.

[0087] 48. The method of any one of clauses 1 to 47, wherein the compound or a pharmaceutically acceptable salt thereof is administered at a dose of about 10 nmol / kg patient body weight to about 2500 nmol / kg patient body weight; and the dose of the CAR T cells is about 1 million CAR T cells to about 15 million CAR T cells.

[0088] 49. The method of clause 48, wherein the compound or a pharmaceutically acceptable salt thereof is administered at a dose of about 10 nmol / kg patient body weight to about 100 nmol / kg patient body weight.

[0089] 50. The method of any one of clauses 48 to 49, wherein the compound or a pharmaceutically acceptable salt thereof is administered at a dose of about 10 nmol / kg patient body weight to about 50 nmol / kg patient body weight.

[0090] 51. The method of any one of clauses 48 to 50, wherein the compound or a pharmaceutically acceptable salt thereof is administered at a dose of about 10 nmol / kg patient body weight to about 20 nmol / kg patient body weight.

[0091] 52. The method of clause 48, wherein the compound or a pharmaceutically acceptable salt thereof is administered at a dose of about 10 nmol / kg patient body weight to about 600 nmol / kg patient body weight.

[0092] 53. The method of clause 48, wherein the compound or a pharmaceutically acceptable salt thereof is administered at a dose of about 200 nmol / kg to about 600 nmol / kg of patient body weight.

[0093] 54. The method of clause 48, wherein the compound or a pharmaceutically acceptable salt thereof is administered at a dose of about 400 nmol / kg to about 600 nmol / kg of patient body weight.

[0094] 55. The method of any one of clauses 48 to 54, wherein the dose of CAR T cells is about 1 million CAR T cells to about 12.5 million CAR T cells.

[0095] 56. The method of any one of clauses 48 to 55, wherein the dose of CAR T cells is about 1 million CAR T cells to about 7 million CAR T cells.

[0096] 57. The method of any one of clauses 48 to 56, wherein the dose of CAR T cells is about 1 million CAR T cells to about 5 million CAR T cells.

[0097] 58. The method of any one of clauses 48 to 57, wherein the dose of CAR T cells is about 2 million CAR T cells to about 5 million CAR T cells.

[0098] 59. The method of any one of clauses 1 to 58, further comprising the step of terminating the continuous administration of the compound or a pharmaceutically acceptable salt thereof to inhibit or prevent cytokine release syndrome in the patient.

[0099] 60. The method of any one of clauses 1 to 59, wherein the compound or a pharmaceutically acceptable salt thereof is administered continuously to the patient for at least one hour.

[0100] 61. The method of any one of clauses 1 to 59, wherein the compound or a pharmaceutically acceptable salt thereof is administered continuously to the patient for at least four hours.

[0101] 62. The method of any one of clauses 1 to 59, wherein the compound or a pharmaceutically acceptable salt thereof is administered continuously to the patient for at least six hours.

[0102] 63. The method of any one of clauses 1 to 62, wherein the compound or a pharmaceutically acceptable salt thereof is administered to the patient every other day.

[0103] 64. The method of any one of clauses 1 to 62, wherein the compound or a pharmaceutically acceptable salt thereof is administered to the patient three times per week.

[0104] 65. The method of any one of clauses 1 to 62, wherein the compound or a pharmaceutically acceptable salt thereof is administered to the patient twice per week.

[0105] 66. The method of any one of clauses 1 to 62, wherein the compound or a pharmaceutically acceptable salt thereof is administered to the patient once a week.

[0106] 67. The method of any one of clauses 1 to 62, wherein the compound or a pharmaceutically acceptable salt thereof is administered to the patient until unacceptable loss of weight of the patient, fever, drop in blood pressure, or pulmonary edema occurs.

[0107] 68. A method for treating cancer, the method comprising:

[0108] i) administering a compound or a pharmaceutically acceptable salt thereof to a patient, wherein the compound comprises a small molecule ligand linked to a targeting moiety via a linker, wherein at least a first dose and a second dose of the compound or a pharmaceutically acceptable salt thereof are administered to the patient, wherein the first dose and the second dose are different, wherein the amount of the compound or a pharmaceutically acceptable salt thereof in the second dose is about 2 times to about 15,000 times greater than the amount of the compound or a pharmaceutically acceptable salt thereof in the first dose; and

[0109] ii) administering to the patient a CAR T cell composition comprising a CAR T cell, wherein the CAR T cell comprises a CAR directed to the targeting moiety and wherein the CAR comprises an E2 anti-fluorescein antibody fragment.

[0110] 69. The method of clause 68, wherein at least a first dose, a second dose, and a third dose of the compound or a pharmaceutically acceptable salt thereof are administered to the patient, wherein the first dose, the second dose, and the third dose are different, wherein the amount of the compound or a pharmaceutically acceptable salt thereof in the second dose is about 2 times to about 750 times more than the compound or a pharmaceutically acceptable salt thereof in the first dose, and wherein the amount of the compound or a pharmaceutically acceptable salt thereof in the third dose is about 800 times to about 10,000 times more than the compound or a pharmaceutically acceptable salt thereof in the first dose.

[0111] 70. The method of clause 68, wherein at least a first dose, a second dose, a third dose, and a fourth dose of the compound or a pharmaceutically acceptable salt thereof are administered to the patient, wherein the first dose, the second dose, the third dose, and the fourth dose are different, wherein the amount of the compound or a pharmaceutically acceptable salt thereof in the second dose is about 2 times to about 750 times greater than the compound or a pharmaceutically acceptable salt thereof in the first dose, wherein the amount of the compound or a pharmaceutically acceptable salt thereof in the third dose is about 800 times to about 7500 times greater than the compound or a pharmaceutically acceptable salt thereof in the first dose, and wherein the amount of the compound or a pharmaceutically acceptable salt thereof in the fourth dose is about 8000 times to about 15000 times greater than the compound or a pharmaceutically acceptable salt thereof in the first dose.

[0112] 71. The method of clause 70, wherein the amount of the compound or its pharmaceutically acceptable salt in the second dose is about 100 times greater than the amount of the compound or its pharmaceutically acceptable salt in the first dose, wherein the amount of the compound or its pharmaceutically acceptable salt in the third dose is about 1000 times greater than the amount of the compound or its pharmaceutically acceptable salt in the first dose, and wherein the amount of the compound or its pharmaceutically acceptable salt in the fourth dose is about 10000 times greater than the amount of the compound or its pharmaceutically acceptable salt in the first dose.

[0113] 72. The method of clause 68, wherein at least a first dose and a second dose of the compound or a pharmaceutically acceptable salt thereof are administered to the patient, wherein the first dose and the second dose are different, wherein the amount of the compound or a pharmaceutically acceptable salt thereof in the second dose is about 2 times to about 15,000 times greater than the amount of the compound or a pharmaceutically acceptable salt thereof in the first dose.

[0114] 73. A method for treating cancer, the method comprising:

[0115] i) administering to a patient a first dose of a compound or a pharmaceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked to a targeting moiety via a linker;

[0116] ii) administering to the patient at least a second dose of the compound or a pharmaceutically acceptable salt thereof, wherein the amount of the compound or a pharmaceutically acceptable salt thereof in the second dose is at least about 50% less than the amount of the compound or a pharmaceutically acceptable salt thereof in the first dose; and

[0117] iii) administering to the patient a dose of a CAR T cell composition comprising a CAR T cell, wherein the CART cell comprises a CAR directed to the targeting moiety and wherein the CAR comprises an E2 anti-fluorescein antibody fragment.

[0118] 74. The method of clause 73, wherein the amount of the compound or a pharmaceutically acceptable salt thereof in the second dose is at least about 60% less than the amount of the compound or a pharmaceutically acceptable salt thereof in the first dose.

[0119] 75. The method of clause 73, wherein the amount of the compound or a pharmaceutically acceptable salt thereof in the second dose is at least about 70% less than the amount of the compound or a pharmaceutically acceptable salt thereof in the first dose.

[0120] 76. The method of clause 73, wherein the amount of the compound or a pharmaceutically acceptable salt thereof in the second dose is at least about 80% less than the amount of the compound or a pharmaceutically acceptable salt thereof in the first dose.

[0121] 77. The method of clause 73, wherein the amount of the compound or pharmaceutically acceptable salt thereof in the second dose is at least about 90% less than the amount of the compound or pharmaceutically acceptable salt thereof in the first dose.

[0122] 78. The method of clause 73, wherein the amount of the compound or pharmaceutically acceptable salt thereof in the second dose is at least about 95% less than the amount of the compound or pharmaceutically acceptable salt thereof in the first dose.

[0123] 79. The method of clause 73, wherein the amount of the compound or a pharmaceutically acceptable salt thereof in the second dose is at least about 96% less than the amount of the compound or a pharmaceutically acceptable salt thereof in the first dose.

[0124] 80. The method of clause 73, wherein the amount of the compound or a pharmaceutically acceptable salt thereof in the second dose is at least about 97% less than the amount of the compound or a pharmaceutically acceptable salt thereof in the first dose.

[0125] 81. The method of clause 73, wherein the amount of the compound or a pharmaceutically acceptable salt thereof in the second dose is at least about 98% less than the amount of the compound or a pharmaceutically acceptable salt thereof in the first dose.

[0126] 82. The method of clause 73, wherein the amount of the compound or a pharmaceutically acceptable salt thereof in the second dose is at least about 99% less than the amount of the compound or a pharmaceutically acceptable salt thereof in the first dose.

[0127] 83. The method of clause 73, wherein the amount of the compound or pharmaceutically acceptable salt thereof in the second dose is at least about 99.5% less than the amount of the compound or pharmaceutically acceptable salt thereof in the first dose.

[0128] 84. The method of any one of clauses 73 to 83, wherein the first dose of the compound or a pharmaceutically acceptable salt thereof is from about 100 nmol / kg patient body weight to about 1000 nmol / kg patient body weight.

[0129] 85. The method of any one of clauses 73 to 83, wherein the first dose of the compound or a pharmaceutically acceptable salt thereof is from about 100 nmol / kg patient body weight to about 900 nmol / kg patient body weight.

[0130] 86. The method of any one of clauses 73 to 83, wherein the first dose of the compound or a pharmaceutically acceptable salt thereof is about 100 nmol / kg patient body weight to about 800 nmol / kg patient body weight.

[0131] 87. The method of any one of clauses 73 to 83, wherein the first dose of the compound or a pharmaceutically acceptable salt thereof is about 100 nmol / kg patient body weight to about 700 nmol / kg patient body weight.

[0132] 88. The method of any one of clauses 73 to 83, wherein the first dose of the compound or a pharmaceutically acceptable salt thereof is about 100 nmol / kg patient body weight to about 600 nmol / kg patient body weight.

[0133] 89. The method of any one of clauses 73 to 83, wherein the first dose of the compound or a pharmaceutically acceptable salt thereof is about 200 nmol / kg patient body weight to about 600 nmol / kg patient body weight.

[0134] 90. The method of any one of clauses 73 to 83, wherein the first dose of the compound or a pharmaceutically acceptable salt thereof is about 400 nmol / kg patient body weight to about 600 nmol / kg patient body weight.

[0135] 91. The method of any one of clauses 73 to 83, wherein the first dose of the compound or a pharmaceutically acceptable salt thereof is about 500 nmol / kg patient body weight.

[0136] 92. The method of clause 84, wherein the second dose of the compound or a pharmaceutically acceptable salt thereof is from about 0.5 nmol / kg patient body weight to about 500 nmol / kg patient body weight.

[0137] 93. The method of clause 85, wherein the second dose of the compound or a pharmaceutically acceptable salt thereof is from about 0.5 nmol / kg patient body weight to about 450 nmol / kg patient body weight.

[0138] 94. The method of clause 86, wherein the second dose of the compound or a pharmaceutically acceptable salt thereof is from about 0.5 nmol / kg patient body weight to about 400 nmol / kg patient body weight.

[0139] 95. The method of clause 87, wherein the second dose of the compound or a pharmaceutically acceptable salt thereof is from about 0.5 nmol / kg patient body weight to about 350 nmol / kg patient body weight.

[0140] 96. The method of clause 88, wherein the second dose of the compound or a pharmaceutically acceptable salt thereof is from about 0.5 nmol / kg patient body weight to about 300 nmol / kg patient body weight.

[0141] 97. The method of clause 89, wherein the second dose of the compound or a pharmaceutically acceptable salt thereof is from about 1 nmol / kg patient body weight to about 300 nmol / kg patient body weight.

[0142] 98. The method of clause 90, wherein the second dose of the compound or a pharmaceutically acceptable salt thereof is from about 2 nmol / kg patient body weight to about 300 nmol / kg patient body weight.

[0143] 99. The method of clause 91, wherein the second dose of the compound or a pharmaceutically acceptable salt thereof is from about 2 nmol / kg patient body weight to about 250 nmol / kg patient body weight.

[0144] 100. The method of any one of clauses 92 to 99, wherein the second dose of the compound or a pharmaceutically acceptable salt thereof is about 5 nmol / kg patient body weight to about 40 nmol / kg patient body weight.

[0145] 101. The method of any one of clauses 92 to 99, wherein the second dose of the compound or a pharmaceutically acceptable salt thereof is about 40 nmol / kg patient body weight to about 150 nmol / kg patient body weight.

[0146] 102. The method of any one of clauses 73 to 101, further comprising administering a third dose of the compound or a pharmaceutically acceptable salt thereof, wherein the third dose of the compound or a pharmaceutically acceptable salt thereof is the same as the second dose of the compound or a pharmaceutically acceptable salt thereof.

[0147] 103. The method of clause 102, further comprising administering a fourth dose of the compound or a pharmaceutically acceptable salt thereof, wherein the fourth dose of the compound or a pharmaceutically acceptable salt thereof is the same as the second dose or a pharmaceutically acceptable salt thereof and the third dose of the compound or a pharmaceutically acceptable salt thereof.

[0148] 104. The method of any one of clauses 73 to 103, wherein one or more doses of the compound or a pharmaceutically acceptable salt thereof administered after the first dose of the compound or a pharmaceutically acceptable salt thereof maintains inhibition of growth of the cancer relative to the first dose of the compound or a pharmaceutically acceptable salt thereof.

[0149] 105. The method of any one of clauses 73 to 104, wherein the CAR T cells are administered at a dose of about 1 million CAR T cells to about 40 million CAR T cells.

[0150] 106. The method of any one of clauses 73 to 105, wherein the one or more doses of the compound or a pharmaceutically acceptable salt thereof administered after the first dose of the compound or a pharmaceutically acceptable salt thereof is administered once a week.

[0151] 107. The method of any one of clauses 73 to 105, wherein the one or more doses of the compound or a pharmaceutically acceptable salt thereof are administered twice per week.

[0152] 108. The method of any one of clauses 1 to 107, wherein the CAR further comprises an IgG4 hinge domain, a CD3ζ activation domain, and a 4-1BB co-stimulatory domain.

[0153] 109. The method of any one of clauses 1 to 108, wherein the E2 anti-fluorescein antibody fragment is a scFv fragment.

[0154] 110. The method of any one of clauses 1 to 109, wherein the CAR protein sequence is at least about 90% identical to SEQ ID NO: 2.

[0155] 111. A method as described in any one of clauses 1 to 109, wherein the CAR protein sequence has at least about 95% identity with SEQ ID NO: 2.

[0156] 112. A method as described in any one of clauses 1 to 109, wherein the CAR protein sequence has at least about 98% identity with SEQ ID NO: 2.

[0157] 113. The method of any one of clauses 1 to 112, wherein the CAR is bound to fluorescein.

[0158] 114. The method of any one of clauses 1 to 109, wherein the CAR protein sequence has up to about 50 conservative amino acid substitutions and wherein the CAR binds to fluorescein.

[0159] 115. The method of any one of clauses 1 to 109, wherein the CAR is encoded by a polynucleotide having at least about 90% identity to SEQ ID NO: 1.

[0160] 116. The method of any one of clauses 1 to 109, wherein the CAR is encoded by a polynucleotide having at least about 95% identity to SEQ ID NO: 1.

[0161] 117. The method of any one of clauses 1 to 109, wherein the CAR is encoded by a polynucleotide having at least about 98% identity to SEQ ID NO: 1.

[0162] 118. The method of any one of clauses 115 to 117, wherein the CAR is conjugated to fluorescein.

[0163] 119. The method of any one of clauses 1 to 118, wherein the CAR is encoded by a polynucleotide that hybridizes to a polynucleotide having SEQ ID NO: 1 under high stringency conditions and wherein the CAR binds to fluorescein.

[0164] 120. The method of any one of clauses 1 to 119, wherein the CAR is encoded by a polynucleotide having SEQ ID NO: 1 or by a degenerate variant of SEQ ID NO: 1.

[0165] 121. The method of any one of clauses 1 to 120, wherein the ligand is selected from the group consisting of folates, DUPA, NK-1R ligands, CAIX ligands, gamma-glutamyl transpeptidase ligands, NKG2D ligands, and CCK2R ligands.

[0166] 122. The method of any one of clauses 1 to 121, wherein the targeting moiety is fluorescein or a pharmaceutically acceptable salt thereof.

[0167] 123. The method of any one of clauses 1 to 122, 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.

[0168] 124. The method of any one of clauses 1 to 123, wherein the linker comprises PEG.

[0169] 125. A method according to any one of clauses 1 to 124, wherein the compound or a pharmaceutically acceptable salt thereof has the formula

[0170] ,

[0171] Wherein B represents a small molecule ligand, L represents a linker, and T represents a targeting moiety, and wherein L comprises a structure having the formula

[0172]

[0173] wherein n is an integer from 0 to 200.

[0174] 126. The method of any one of clauses 1 to 125, wherein the cancer is selected from the group consisting of lung cancer, bone cancer, pancreatic cancer, skin cancer, head cancer, neck cancer, skin melanoma, intraocular melanoma, uterine cancer, ovarian cancer, endometrial cancer, rectal cancer, gastric cancer, colon cancer, breast cancer, triple-negative breast cancer, fallopian tube cancer, carcinoma of the endometrium, endometrium), 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, urethral cancer, prostate cancer, chronic leukemia, acute leukemia, acute myelocytic leukemia, lymphocytic lymphoma, myeloid leukemia, myelomonocytic leukemia, hairy cell leukemia, myelomonocytic leukemia, hairy cell leukemia, pleural mesothelioma, bladder cancer, Burkitt's lymphoma, ureteral cancer, kidney cancer, renal cell carcinoma, renal pelvis cancer, neoplasms of the central nervous system (CNS), primary CNS lymphoma, spinal axis tumors, brain stem gliomas, pituitary adenomas, and gastroesophageal junction adenocarcinoma.

[0175] 127. The method of any one of clauses 1 to 126, wherein the cancer is a cancer that expresses a folate receptor.

[0176] 128. The method of any one of clauses 1 to 127, wherein the cancer is endometrial cancer.

[0177] 129. The method of any one of clauses 1 to 127, wherein the cancer is non-small cell lung cancer.

[0178] 130. The method of any one of clauses 1 to 127, wherein the cancer is ovarian cancer.

[0179] 131. The method of any one of clauses 1 to 127, wherein the cancer is triple negative breast cancer.

[0180] 132. The method of any one of clauses 1 to 127, wherein the cancer is acute myeloid leukemia.

[0181] 133. The method of clause 132, wherein the cancer expresses the folate receptor-β.

[0182] 134. The method of any one of clauses 1 to 133, wherein multiple doses of the CAR T cell composition are administered.

[0183] 135. The method of any one of clauses 1 to 134, wherein at least two doses of the CAR T cell composition are administered.

[0184] 136. The method of any one of clauses 1 to 135, wherein the patient is imaged prior to administration of the compound or a pharmaceutically acceptable salt thereof, or prior to administration of the CAR T cell composition.

[0185] 137. The method of any one of clauses 1 to 136, wherein the compound or a pharmaceutically acceptable salt thereof is not an antibody, and does not comprise an antibody fragment.

[0186] 138. The method of any one of clauses 1 to 137, wherein the targeting moiety does not comprise a peptide epitope.

[0187] 139. The method of any one of clauses 1 to 138, wherein cytokine release that results in off-target toxicity in the patient does not occur and wherein CAR T cell toxicity to the cancer occurs.

[0188] 140. The method of any one of clauses 1 to 138, wherein off-target tissue toxicity does not occur in the patient and wherein CAR T cell toxicity to the cancer occurs.

[0189] 141. The method of any one of clauses 1 to 138, wherein the cancer comprises a tumor, wherein the patient's tumor size is reduced, and wherein off-target toxicity does not occur.

[0190] 142. The method of any one of clauses 1 to 141, wherein CRS is reduced or prevented and the method results in a reduction in tumor volume in the patient.

[0191] 143. The method of any one of clauses 1 to 142, wherein weight loss due to CRS is reduced or prevented.

[0192] 144. The method of any one of clauses 59 to 67, further comprising administering the compound or a pharmaceutically acceptable salt thereof to the patient.

[0193] 145. The method of clause 144, wherein subsequent administration of the compound or a pharmaceutically acceptable salt thereof results in increased CAR T cell activation and cytokine levels in the patient.

[0194] 146. The method of any one of clauses 1 to 145, wherein the cancer comprises a tumor and wherein a complete response to the tumor is achieved.

[0195] 147. A method as described in any one of clauses 1 to 146, wherein the CAR T cells have a central memory / effector memory phenotype.

[0196] 148. A method as described in any one of clauses 1 to 147, wherein the CD8:CD4 ratio of the CAR T cells is about 1:1. BRIEF DESCRIPTION OF THE DRAWINGS

[0197] Figure 1 The diagram shows the E2 construct relative to the 4M5.3 construct and a map of the E2 construct is shown.

[0198] Figure 2 EC17 fixed dose and dose reduction graphs are shown.

[0199] Figure 3A and 3B E2-CAR-T anti-tumor activity (30 million cells) and body weight changes with decreasing EC17 doses are shown. As shown, anti-tumor activity was maintained after NaFL rescue.

[0200] Figure 4A and 4B The anti-tumor activity of 4M5.3-CAR-T (30 million cells) and body weight changes with decreasing EC17 doses are shown. As shown, the activity of 4M5.3-CAR-T is lower than that of E2-CAR-T. In addition, EC17 dose-dependent anti-tumor activity and body weight loss were observed.

[0201] Figure 5A and 5B E2-CAR-T antitumor activity (10 and 20 million cells) and body weight changes at a fixed EC17 dosing regimen (500 nmol / kg, SIW) are shown. As shown, antitumor activity was maintained after NaFL rescue.

[0202] Fig. 6A and 6B The anti-tumor activity of 4M5.3-CAR-T (10 and 20 million cells) and body weight changes at a fixed EC17 dosing regimen (500 nmol / kg, SIW) are shown. As shown, the activity of 4M5.3-CAR-T is lower than that of E2-CAR-T.

[0203] Figure 7 A and 7B show phenotypic characterization of E2 CAR T cells before infusion into NSG mice.

[0204] Fig. 8A and 8BShown is a comparison of CAR-T differentiation phenotypes in different preparations, including E2-CAR-T cells and 4M5.3-CAR-T cells, and GFP + 4M5.3 CAR-T cells.

[0205] Fig.9A and 9B The binding of DIG-labeled E2-IgG to FITC is shown.

[0206] Fig. 10A and 10B IHC staining of DIG-labeled E2 antibody on FITC-labeled KB cells is shown.

[0207] Figure 11 shows IHC staining of bound DIG-E2 antibody in normal human organ tissue: adrenal gland. Panel A: test tissue section pre-incubated with DIG-E2 antibody; Panel B: control tissue section not pre-incubated with DIG-E2 antibody.

[0208] Figure 12 shows IHC staining of bound DIG-E2 antibody in normal human organ tissue: bone marrow. Panel A: test tissue section pre-incubated with DIG-E2 antibody; Panel B: control tissue section not pre-incubated with DIG-E2 antibody.

[0209] Figure 13 shows IHC staining of bound DIG-E2 antibody in normal human organ tissue: breast. Panel A: test tissue section pre-incubated with DIG-E2 antibody; Panel B: control tissue section not pre-incubated with DIG-E2 antibody.

[0210] Figure 14 shows IHC staining of bound DIG-E2 antibody in normal human organ tissue: cerebellum tissue. Panel A: test tissue section pre-incubated with DIG-E2 antibody; Panel B: control tissue section not pre-incubated with DIG-E2 antibody.

[0211] Figure 15 shows IHC staining of bound DIG-E2 antibody in normal human organ tissue: cervix. Panel A: test tissue section pre-incubated with DIG-E2 antibody; Panel B: control tissue section not pre-incubated with DIG-E2 antibody.

[0212] Figure 16 shows IHC staining of bound DIG-E2 antibody in normal human organ tissue: colon. Panel A: test tissue section pre-incubated with DIG-E2 antibody; Panel B: control tissue section not pre-incubated with DIG-E2 antibody.

[0213] Figure 17 shows IHC staining of bound DIG-E2 antibody in normal human organ tissue: esophagus. Panel A: test tissue section pre-incubated with DIG-E2 antibody; Panel B: control tissue section not pre-incubated with DIG-E2 antibody.

[0214] Figure 18 shows IHC staining of bound DIG-E2 antibody in normal human organ tissue: eye. Panel A: test tissue section pre-incubated with DIG-E2 antibody; Panel B: control tissue section not pre-incubated with DIG-E2 antibody.

[0215] Figure 19 shows IHC staining of bound DIG-E2 antibody in normal human organ tissue: heart. Panel A: test tissue section pre-incubated with DIG-E2 antibody; Panel B: control tissue section not pre-incubated with DIG-E2 antibody.

[0216] Figure 20 shows IHC staining of bound DIG-E2 antibody in normal human organ tissue: pituitary gland. Panel A: test tissue section pre-incubated with DIG-E2 antibody; Panel B: control tissue section not pre-incubated with DIG-E2 antibody.

[0217] Figure 21 shows IHC staining of bound DIG-E2 antibody in normal human organ tissue: kidney. Panel A: test tissue section pre-incubated with DIG-E2 antibody; Panel B: control tissue section not pre-incubated with DIG-E2 antibody.

[0218] Figure 22 shows IHC staining of bound DIG-E2 antibody in normal human organ tissue: larynx. Panel A: test tissue section pre-incubated with DIG-E2 antibody; Panel B: control tissue section not pre-incubated with DIG-E2 antibody.

[0219] Figure 23 shows IHC staining of bound DIG-E2 antibody in normal human organ tissue: spleen. Panel A: test tissue section pre-incubated with DIG-E2 antibody; Panel B: control tissue section not pre-incubated with DIG-E2 antibody.

[0220] Figure 24 shows IHC staining of bound DIG-E2 antibody in normal human organ tissue: liver. Panel A: test tissue section pre-incubated with DIG-E2 antibody; Panel B: control tissue section not pre-incubated with DIG-E2 antibody.

[0221] Figure 25 shows IHC staining of bound DIG-E2 antibody in normal human organ tissue: lung. Panel A: test tissue section pre-incubated with DIG-E2 antibody; Panel B: control tissue section not pre-incubated with DIG-E2 antibody.

[0222] Figure 26 shows IHC staining of bound DIG-E2 antibody in normal human organ tissue: lymph node. Panel A: test tissue section pre-incubated with DIG-E2 antibody; Panel B: control tissue section not pre-incubated with DIG-E2 antibody.

[0223] Figure 27 shows IHC staining of bound DIG-E2 antibody in normal human organ tissue: nerve. Panel A: test tissue section pre-incubated with DIG-E2 antibody; Panel B: control tissue section not pre-incubated with DIG-E2 antibody.

[0224] Figure 28 shows IHC staining of bound DIG-E2 antibody in normal human organ tissue: ovary. Panel A: test tissue section pre-incubated with DIG-E2 antibody; Panel B: control tissue section not pre-incubated with DIG-E2 antibody.

[0225] Figure 29 shows IHC staining of bound DIG-E2 antibody in normal human organ tissue: pancreas. Panel A: test tissue section pre-incubated with DIG-E2 antibody; Panel B: control tissue section not pre-incubated with DIG-E2 antibody.

[0226] Figure 30 shows IHC staining of bound DIG-E2 antibody in normal human organ tissue: prostate. Panel A: test tissue section pre-incubated with DIG-E2 antibody; Panel B: control tissue section not pre-incubated with DIG-E2 antibody.

[0227] Figure 31 shows IHC staining of bound DIG-E2 antibody in normal human organ tissue: skin. Panel A: test tissue section pre-incubated with DIG-E2 antibody; Panel B: control tissue section not pre-incubated with DIG-E2 antibody.

[0228] Figure 32 shows IHC staining of bound DIG-E2 antibody in normal human organ tissue: small intestine. Panel A: test tissue section pre-incubated with DIG-E2 antibody; Panel B: control tissue section not pre-incubated with DIG-E2 antibody.

[0229] Figure 33 shows IHC staining of bound DIG-E2 antibody in normal human organ tissue: stomach. Panel A: test tissue section pre-incubated with DIG-E2 antibody; Panel B: control tissue section not pre-incubated with DIG-E2 antibody.

[0230] Figure 34 shows IHC staining of bound DIG-E2 antibody in normal human organ tissue: striated muscle. Panel A: test tissue section pre-incubated with DIG-E2 antibody; Panel B: control tissue section not pre-incubated with DIG-E2 antibody.

[0231] Figure 35 shows IHC staining of bound DIG-E2 antibody in normal human organ tissue: testis. Panel A: test tissue section pre-incubated with DIG-E2 antibody; Panel B: control tissue section not pre-incubated with DIG-E2 antibody.

[0232] Figure 36 shows IHC staining of bound DIG-E2 antibody in normal human organ tissue: thymus. Panel A: test tissue section pre-incubated with DIG-E2 antibody; Panel B: control tissue section not pre-incubated with DIG-E2 antibody.

[0233] Figure 37 shows IHC staining of bound DIG-E2 antibody in normal human organ tissue: tongue. Panel A: test tissue section pre-incubated with DIG-E2 antibody; Panel B: control tissue section not pre-incubated with DIG-E2 antibody.

[0234] Figure 38 shows IHC staining of bound DIG-E2 antibody in normal human organ tissue: uterus. Panel A: test tissue section pre-incubated with DIG-E2 antibody; Panel B: control tissue section not pre-incubated with DIG-E2 antibody.

[0235] Fig.39 Figure 2 is a graph showing T-cell activation in one-day co-culture with various target cells. The percentage of E2 CAR T cells activated after one day of co-culture (y-axis) is plotted against the FR expression level of the target cells at the assay time (x-axis). (▼) OV90 cells; (♦) SKOV3 cells; (▲) IGROV1 cells; (■) HOS-Frα cells; (●) MDA-MB-231 cells.

[0236] Fig.40 Figure 2 shows target cell apoptosis in 2-day co-culture with 5 different cell types (OV90 cells, SKOV3 cells, IGROV1 cells, HOS-Frα cells, MDA-MB-231 cells) under three different conditions. For each cell type, the conditions are as follows: left bar target cells alone + EC17; middle bar CAR T plus target cell co-culture without EC17 pretreatment; right bar CAR T plus treated target cells with EC17 pretreatment.

[0237] Fig.41 The graph shows the expression level of functional folate receptor in five different cell types (OV90 cells, SKOV3 cells, IGROV1 cells, HOS-Frα cells, MDA-MB-231 cells).

[0238] Fig.42 A schematic diagram showing the experimental timeline for tumor-bearing and naïve mice.

[0239] Fig.43 (Left) is a graph showing that the level of the cytokine IFN-Ɣ in both naive and tumor-bearing mice is EC-17 dependent, and the increase in IFN-Ɣ in naive mice is much lower (23-fold lower) than that in mice bearing MDA-MB-231 tumors. Fig.43 (Right) is a graph showing FACS analysis of CAR-T expansion in MDA-MB-231 tumor-bearing mice and no detectable CAR-T cell expansion in naive mice.

[0240] Fig.44 (Top) is a graph showing that: in co-culture with the positive control cell line K562-OKT3, cytokine (IL-2) production from mock T-cells and anti-FLCAR T-cells was at quantitatively similar levels (left pair of bars, where mock is the left bar and anti-FLCAR T-cells are the right bar); no cytokine production by mock or anti-FLCAR T-cells after co-culture with K562 (middle); and anti-FLCAR T-cells were the only cells able to elicit cytokine IL-2 secretion, but only when pre-treated with EC-17 (right, where only anti-FLCAR T-cells show results). Fig.44 (Middle) is a graph showing that: in co-culture with the positive control cell line K562-OKT3, cytokines (IFN-Ɣ) from mock T-cells and anti-FLCAR T-cells were produced at quantitatively similar levels (left pair of bars, where mock is the left bar and anti-FLCAR T-cells are the right bar); no cytokine production by mock or anti-FLCAR T-cells after co-culture with K562 (middle); and anti-FLCAR T-cells are the only cells that can induce secretion of the cytokine IFN-Ɣ, but only when pre-treated with EC-17 (right, where only anti-FLCAR T-cells show results). Fig.44 (Bottom) is a graph showing that: in co-culture with the positive control cell line K562-OKT3, cytokine (TNF-α) production from mock T-cells and anti-FLCAR T-cells was at quantitatively similar levels (left pair of bars, where mock is the left bar and anti-FLCAR T-cells are the right bar); no cytokine production by mock or anti-FLCAR T-cells after co-culture with K562 (middle); and anti-FLCAR T-cells were the only cells that could elicit cytokine TNF-α secretion, but only when pre-treated with EC-17 (right, where only anti-FLCAR T-cells show results).

[0241] Fig.45 (Upper left) is a graph showing the percentage of lysis when CD8+ mimic T cells and anti-FLCAR T cells were co-cultured with negative control K562 cells at a ratio of 30:1, 10:1, 3:1 or 1:1. Fig.45(Upper right) is a graph showing the percentage of lysis when CD8+ mimic T cells and anti-FLCAR T-cells were co-cultured with K562+OKT3 target cells at a ratio of 30:1, 10:1, 3:1 or 1:1. Fig.45 (Lower left) is a graph showing the percentage of lysis when CD8+ mimic T cells and anti-FLCAR T cells were co-cultured with unlabeled MDA-MB-231 cells at a ratio of 30:1, 10:1, 3:1 or 1:1. Fig.45 (Lower right) is a graph showing the percentage of lysis when CD8+ mimetic T cells and anti-FLCAR T-cells were co-cultured with EC17-labeled MDA-MB-231 cells at a ratio of 30: 1, 10: 1, 3: 1, or 1: 1. In all figures, (x) CD8+ mimetic T cells; (○) anti-FLCAR T-cells.

[0242] Fig.46 (Top) shows the chemical structure of FITC-folic acid. Fig.46 (Bottom) is a graph showing the dose response curve of FITC-folate.

[0243] Fig.47 (Top) shows the chemical structure of FITC-DUPA. Fig.47 (Bottom) is a graph showing the dose response curve of FITC-DUPA.

[0244] Fig.48 (Top) shows the chemical structure of FITC-CA9. Fig.48 (Bottom) is a graph showing the dose response curve of FITC-CA9.

[0245] Fig.49 (Top) shows the chemical structure of FITC-NK1R. Fig.49 (Bottom) is a graph showing the dose response curve of FITC-NK1R.

[0246] Fig.50 Binding of the bridge to tumor cells used in the in vivo model and expressing the receptor corresponding to the small molecule ligand of the bridge is shown (via FACS analysis).

[0247] Fig.51 EC17-induced potent FR-dependent tumor cell killing by CAR T cells was demonstrated.

[0248] Fig.52 The correlation between the cytolytic activity of CAR T cells and the level of functional FR on tumor cells was shown.

[0249] Fig.53EC17 / FR dependent CAR T cell activation and exhaustion are shown. The expression of early (CD69), intermediate (CD137) and late (PD1) T cell activation markers detected on CAR T cells co-cultured (E / T = 1:1) with FR+ tumor cell targets preloaded with or without EC17 (100nM, 30 minutes pulse at 37°C). The first two open bars represent only CAR-T and mimetic transduced T cells without target cells. For the legends of the second to fourth figures, from top to bottom = bars from left to right.

[0250] Fig.54 The EC17 / FR-dependent CAR T cell exhaustion spectrum is shown. Top row: Colored pie charts representing the changes in the differentiation state of CAR-T cells in target cell-free cultures from day 0 to day 3. Bottom row: Four sets of colored pie charts representing the differentiation state of CAR-T cells after 3 days of co-culture (E / T = 1:2) with FR+ (MDA-MB-231, THP-1FRβ, HOS-FRα, KB) and FR-negative (THP1-FG12, HOS-143b) tumor cells preloaded without or with EC17 (0.1 or 10 nM, 30 min pulse at 37°C).

[0251] Fig.55 Shown is a fully human CAR construct consisting of: anti-FITC scFv (clone E2), full-length IgG4 spacer (hinge-CH2 (L235D, N297Q)-CH3 derived from Fc), CD28tm transmembrane domain, 4-1BB / CD3ζ cytoplasmic activation domain, and a shortened cell surface polypeptide (EGFRt) of the non-functional epidermal growth factor receptor. Bottom: Examples of CD4 / CD8 T cell phenotyping performed by flow cytometry on EGFRt-sorted (pie chart on the left) CAR-T cell preparations and unsorted "clinical fax" (pie chart on the right). Color symbol description as shown.

[0252] Fig.56 Figure A: After incubation at 37°C for 2 hours, the 3 Figure B: Kd values ​​for H-EC17 uptake (calculated from the number of bound molecules / cell). Figure B: After 2 hours of incubation at room temperature, E2-CAR-T cells (approximately 24% EGFRt+, approximately 95:5 CD8 / CD4 ratio) 3 Kd values ​​for H-EC17 uptake (calculated from total cell associated radioactivity, DPM).

[0253] Fig.57(Figures A and B) show the CRS grading scale and experimental conditions for sodium fluorescein rescue in HOS-FRα tumor-bearing mice. Shows changes in T cell-derived cytokines with and without rescue. Calculated by Student's t-test p value.

[0254] Fig.58 Display based on 3 Functional FR levels on tumor cells were measured by H-FA (folate) binding assay (100 nM, 1 hour at 37°C).

[0255] Fig.59 Shown (Panel A) is the full range of EC17 dose responses in specific lysis (%) of five FR (folate receptor)+ tumor cell lines co-cultured with EGFRt-sorted CAR-T cells at a 1:1 E / T (effector / target) ratio for 24 hours; (Panel B) is the maximum lysis (%) and EC50 values ​​obtained from the dose-response curves fitted to 100 nM.

[0256] Fig.60 The correlation of CAR-T cell activity with FR levels and the natural sensitivity of tumor cells is shown. Panel A: Kinetics of specific lysis (%) at different / T ratios in FR+ (MDA-MB-231, KB, THP1-FRβ, OV90, HOS-FRα) and FR-negative (HOS-143b) cell lines in the presence of 10 nM EC17 after 16 and 48 hours of co-culture. Panel B: Specific lysis (%) of target cells is plotted against the E / T ratio in the linear range. High FR+ KB cells demonstrate early resistance at 16 hours, while FR-negative HOS-143b fails to respond. Panel C: Excluding KB cells, a semi-logarithmic correlation is established between specific lysis (%) at 16 hours of co-culture and the level of functional FR on tumor cells. Panel D: 3D graph depicting the relationship between Th1 cytokine (IFNγ, IL-2, and TNFα) levels derived from CAR-T cells and changing E / T ratios after 44 hours of co-culture at 10 nM EC17. Panel E: Th1 cytokine levels derived from CAR-T cells are plotted against FR levels of FR+ target cells on a Log10 scale.

[0257] Fig.61 Shown are (Panel A) bar graphs showing the EC17 loading status of target cells confirmed by flow cytometry and expressed as MFI (EC17 was undetectable on FR-negative cell lines) and (Panel B) Fig.54 Target cell apoptosis (%) detected by Annexin V staining after 2 days of co-culture as described in.

[0258] Fig.62 Showing the pharmacokinetics and tumor uptake of CAR-T cells in vivo. Figure A: Schematic diagram of the experimental layout to show the animal collection schedule associated with CAR-T cell injections and weekly EC17 doses (500 nmol / kg) in mice bearing MDA-MB-231 tumors. A total of 15 mice received approximately 4.8 million EGFRt-sorted CAR-T cells on day 0 (3 mice had large tumors at the beginning). Figure B: The left figure shows the measurement of tumor volume and body weight changes; the middle figure shows the expansion of CAR-T cells in the blood 7 days after a single dose of EC17; the right bar graph shows the differentiation spectrum of circulating CD4 / CD8CAR-T cell subsets in mice with small and larger tumors. Figure C: Measurement of changes in tumor volume and body weight. Figure D: The upper figure shows the CAR-T cell dynamics in the blood (solid line) relative to the CAR-T cell dynamics in the tumor (dashed line). The bottom bar graph shows changes in the phenotype of CAR-T cells in the blood. Figure E: Kinetic changes in surface expression of activation markers (4-1BB, PD1) on tumor-infiltrating CAR-T cells.

[0259] Fig.63 The effects of dietary folate on antitumor activity and CRS toxicity are shown. Panel A: Measurement of changes in tumor volume and body weight in NSG mice with large MDA-MB-231 tumor xenografts and treated with CAR-T cells (~10 million "clinical fam") plus EC17 SIW at 500 nmol / kg while maintained on a FA-sufficient or FA-deficient diet throughout the study (n = 5). Panel B: Bar graph shows circulating CAR-T cells (human CD3ε+ EGFRt+) measured at the end of the study, on days 52 (FA-deficient) and 59 (FA-sufficient), respectively. Panel C: Representative flow cytometry dot plots showing the absence of circulating CAR-T cells in FA-deficient mice treated with CAR-T cells alone (no EC17) and the increase in the number of circulating CAR-T cells in mice on a FA-deficient diet treated with CAR-T cells plus EC17 compared to mice treated identically but on a FA-sufficient diet. Panel D: Loss of FR expression was detected in 2 / 3 of MDA-MB-231 tumors collected from FA-sufficient animals at day 59 and analyzed by flow cytometry (later confirmed by quantitative radioligand binding assay, data not shown).

[0260] definition

[0261] As used herein, "a" or "an" may mean one or more. As used herein, "about" in reference to a numerical value (including, for example, integers, fractions, and percentages) generally refers to a range of values ​​(e.g., + / - 5% to 10% of the value) that one of ordinary skill in the art would consider equivalent to the value (e.g., having the same function or result).

[0262] As used herein, the terms "treat," "treating," "treated," and "treatment" refer to both therapeutic and prophylactic or preventative treatment.

[0263] As used herein, the term "ameliorate / ameliorating / amelioration / ameliorated" in reference to cancer can mean reducing the symptoms of cancer, reducing the size of a tumor, completely or partially removing a tumor (e.g., a complete or partial response), causing stable disease, preventing the progression of cancer (e.g., progression-free survival), or any other effect on cancer that a physician considers to be a therapeutic, prophylactic, or preventative treatment of cancer.

[0264] As used herein, the term "administer", "administering", or "administered" refers to all ways of introducing a compound or a pharmaceutically acceptable salt thereof or a CAR T cell composition (wherein the CAR T cell composition comprises a CAR T cell and wherein the CAR comprises an E2 anti-fluorescein antibody fragment) into a patient, including but not limited to oral, intravenous, intramuscular, subcutaneous, and transdermal.

[0265] As used herein, the term "off-target toxicity" means organ damage or loss of weight in a patient that is unacceptable to the physician treating the patient, or any other effect on the patient that is unacceptable to the physician treating the patient, e.g., B-cell aplasia, fever, decreased blood pressure, or pulmonary edema.

[0266] As used herein, the terms "transduction" and "transfection" are used equivalently and mean the introduction of nucleic acid into a cell via any artificial method, including viral and non-viral methods. DETAILED DESCRIPTION

[0267] In various embodiments described herein, a small molecule ligand connected to a targeting moiety via a linker is used as a bridge between cancer and a CAR T cell (i.e., a T cell expressing a chimeric antigen receptor), wherein the CAR T cell comprises a genetically engineered CAR directed to a targeting moiety, wherein the CAR comprises an E2 anti-fluorescein antibody fragment. The bridge directs CAR T cells to cancer to improve cancer. In one embodiment, a "small molecule ligand" may be a folate, a CAIX ligand, a DUPA, a NK-1R ligand, a ligand of gamma glutamyl transpeptidase, a NKG2D ligand, or a CCK2R ligand, each of which is a small molecule ligand that specifically binds to a cancer cell type (i.e., a receptor for each of these ligands is overexpressed on cancer compared to normal tissue).

[0268] The "targeting moiety" connected to the small molecule ligand is bound to the recognition region of the genetically engineered CAR expressed by the CAR T cell, wherein the CAR comprises an E2 anti-fluorescein antibody fragment. Therefore, the recognition region of the CAR (e.g., a single-chain fragment variable region (scFv) of the E2 anti-fluorescein antibody) points to the "targeting moiety". Therefore, the small molecule ligand connected to the targeting moiety via a linker acts as a bridge between cancer and the CAR T cell, wherein the CAR T cell comprises a genetically engineered CAR, and the CAR T cell is directed to cancer to improve cancer.

[0269] The bridge is an organic small molecule, so it can quickly achieve self-bloodstream clearance (e.g., about 20 minutes or less). On the one hand, the CAR T cell response (wherein the CAR T cell comprises a genetically engineered CAR, which comprises an E2 anti-fluorescein antibody fragment) can only target those cancer cells that express the receptor of the small molecule ligand portion of the "bridge", thereby reducing the off-target toxicity to normal tissues. In addition, this system can be "universal" because a type of CAR T cell construct (wherein the CAR T cell comprises an E2 anti-fluorescein antibody fragment) can be used to target various cancers using different "bridges". Illustratively, the targeting portion recognized by the CART cell (wherein the CAR T cell comprises a genetically engineered CAR, which comprises an E2 anti-fluorescein antibody fragment) can be maintained constant, so that a type of CAR T cell can be used, while the small molecule ligand bound to the cancer can be changed to allow targeting of a wide variety of cancers.

[0270] In the various embodiments described herein, a small molecule ligand attached to a targeting moiety via a linker is referred to as a "compound."

[0271] In various embodiments, the clause "E2 anti-fluorescein antibody fragment" refers to a CAR comprising a fragment (e.g., a scFv fragment) of an E2 anti-fluorescein antibody. The E2 anti-fluorescein antibody is described, for example, in Vaughan et al., Nature Biotechnol. Volume 14 (3), pages 309 to 314, 1996, which are incorporated herein by reference. In various embodiments, the CAR may further include an IgG4 hinge domain, a CD3ζ activation domain, and / or a 4-1BB costimulatory domain, or any other suitable domain (such as an EGFRt domain). In still other embodiments, the CAR may be encoded by a polynucleotide having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity to SEQ ID NO: 1. In another illustrative embodiment, the CAR may be encoded by a polynucleotide hybridized with a polynucleotide having SEQ ID NO: 1 under highly stringent conditions. On the other hand, the CAR may be encoded by a polynucleotide having SEQ ID NO: 1 or by a degenerate variant of SEQ ID NO: 1. In other embodiments, the CAR protein sequence may have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 2. In yet another embodiment, the CAR protein sequence may have up to about 50 conservative amino acid substitutions. In any of the embodiments described herein, the CAR binds to fluorescein.

[0272] In one embodiment, a method for treating cancer is provided. The method comprises i) administering a compound or a pharmaceutically acceptable salt thereof to a patient, wherein the compound comprises a small molecule ligand connected to a targeting moiety via a linker, ii) administering a first dose of a CAR T cell composition to the patient, wherein the CAR T cell composition comprises a CAR T cell, wherein the CAR T cell comprises a CAR, and wherein the CAR comprises an E2 anti-fluorescein antibody fragment, and iii) administering a second dose of the CAR T cell composition to the patient, wherein the CAR T cell composition comprises a CAR T cell, wherein the CAR T cell comprises a CAR, and wherein the CAR comprises an E2 anti-fluorescein antibody fragment.

[0273] In another embodiment, a method for treating cancer is provided. The method comprises i) administering a compound or a pharmaceutically acceptable salt thereof to a patient, wherein the compound comprises a small molecule ligand linked to a targeting moiety via a linker, and ii) administering a CAR T cell composition comprising a CAR T cell to the patient, wherein the CAR T cell comprises a CAR, wherein the CAR comprises an E2 anti-fluorescein antibody fragment, and wherein the CAR T cell composition comprises a mixture of the CAR T cell and a non-transformed T cell.

[0274] In yet another embodiment, a method for treating cancer is provided. The method comprises i) administering a compound or a pharmaceutically acceptable salt thereof to a patient, wherein the compound comprises a small molecule ligand connected to a targeting moiety via a linker, ii) administering a CAR T cell composition to the patient, wherein the CAR T cell composition comprises a CAR T cell, wherein the CART cell comprises a CAR, wherein the CAR comprises an E2 anti-fluorescein antibody fragment, and iii) administering a folate, a conjugate comprising a folate (wherein the conjugate comprising a folate does not include a targeting moiety) or a drug that inhibits activation of CAR T cells to the patient.

[0275] In another embodiment, a method for treating cancer is provided. The method comprises i) administering a compound or a pharmaceutically acceptable salt thereof to a patient, wherein the compound comprises a small molecule ligand linked to a targeting moiety via a linker, and wherein the compound or a pharmaceutically acceptable salt thereof is administered at a dose of about 10 nmol / kg patient weight to about 2500 nmol / kg patient weight, and ii) administering a CAR T cell composition comprising CAR T cells to the patient, wherein the CAR T cells comprise CAR, wherein the CAR comprises an E2 anti-fluorescein antibody fragment, and wherein the dose of the CAR T cells is about 1 million CAR T cells to about 15 million CAR T cells.

[0276] In yet another embodiment, a method for treating cancer is provided. The method comprises i) continuously administering a compound or a pharmaceutically acceptable salt thereof to a patient, wherein the compound comprises a small molecule ligand connected to a targeting moiety via a linker, ii) administering a CAR T cell composition comprising a CAR T cell to the patient, wherein the CAR T cell comprises a CAR, wherein the CAR comprises an E2 anti-fluorescein antibody fragment, and iii) terminating the continuous administration of the compound or a pharmaceutically acceptable salt thereof to inhibit or prevent cytokine release syndrome in the patient.

[0277] In another illustrative aspect, a method for treating cancer is provided. The method comprises i) administering a compound or a pharmaceutically acceptable salt thereof to a patient, wherein the compound comprises a small molecule ligand linked to a targeting moiety via a linker, wherein at least a first dose and a second dose of the compound or a pharmaceutically acceptable salt thereof are administered to the patient, wherein the first dose is different from the second dose, wherein the amount of the compound or a pharmaceutically acceptable salt thereof in the second dose is about 2-fold to about 15,000-fold greater than the amount of the compound or a pharmaceutically acceptable salt thereof in the first dose, and ii) administering a CAR T cell composition comprising a CAR T cell to the patient, wherein the CAR T cell comprises a CAR, and wherein the CAR comprises an E2 anti-fluorescein antibody fragment.

[0278] In another embodiment, a method for treating cancer is provided. The method comprises i) administering a compound or a pharmaceutically acceptable salt thereof to a patient, wherein the compound comprises a small molecule ligand linked to a targeting moiety via a linker and wherein the compound or a pharmaceutically acceptable salt thereof is administered to the patient once a week, and ii) administering a CAR T cell composition comprising a CAR T cell to the patient, wherein the CAR T cell comprises a CAR, wherein the CAR comprises an E2 anti-fluorescein antibody fragment.

[0279] In yet another embodiment, a method for treating cancer is provided. The method comprises i) administering to a patient a first dose of a compound or a pharmaceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked to a targeting moiety via a linker, ii) administering to the patient at least a second dose of the compound or a pharmaceutically acceptable salt thereof, wherein the amount of the second dose of the compound or a pharmaceutically acceptable salt thereof is at least about 50% less than the amount of the first dose of the compound or a pharmaceutically acceptable salt thereof; and iii) administering to the patient a dose of a CAR T cell composition comprising a CAR T cell, wherein the CAR T cell comprises a CAR and wherein the CAR comprises an E2 anti-fluorescein antibody fragment.

[0280] Several implementations are described via the following enumerated clauses. It is also contemplated that any of the following implementations may be combined with any applicable implementation described in the Summary section, Detailed Description section, Examples section, or claims of this patent application.

[0281] 1. A method for treating cancer, comprising:

[0282] i) administering to a patient a compound or a pharmaceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked to a targeting moiety via a linker;

[0283] ii) administering to the patient a first dose of a CAR T cell composition comprising a CAR T cell, wherein the CAR T cell comprises a CAR directed to the targeting moiety and wherein the CAR comprises an E2 anti-fluorescein antibody fragment; and

[0284] iii) administering to the patient a second dose of a CAR T cell composition comprising a CAR T cell, wherein the CART cell comprises a CAR directed to the targeting moiety and wherein the CAR comprises an E2 anti-fluorescein antibody fragment.

[0285] 2. A method for treating cancer, the method comprising:

[0286] i) administering to a patient a compound or a pharmaceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked to a targeting moiety via a linker; and

[0287] ii) administering a CAR T cell composition to the patient, wherein the CAR T cell composition comprises CAR T cells, wherein the CAR T cells comprise a CAR directed to the targeting portion, wherein the CAR comprises an E2 anti-fluorescein antibody fragment, and wherein the CAR T cell composition comprises a mixture of the CAR T cells and non-transformed T cells.

[0288] 3. A method for treating cancer, the method comprising:

[0289] i) administering to a patient a compound or a pharmaceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked to a targeting moiety via a linker;

[0290] ii) administering a CAR T cell composition to the patient, wherein the CAR T cell composition comprises a CAR T cell, wherein the CAR T cell comprises a CAR directed to the targeting moiety and wherein the CAR comprises an E2 anti-fluorescein antibody fragment; and

[0291] iii) administering to the patient a folate, a conjugate comprising a folate (wherein the conjugate comprising a folate does not include a targeting moiety), or an agent that inhibits activation of CAR T cells.

[0292] 4. The method of clause 3, wherein step iii comprises administering a folate.

[0293] 5. The method of any one of clauses 3 or 4, wherein step iii comprises administering folic acid or leucovorin.

[0294] 6. The method of clause 3, wherein step iii comprises administering the conjugate comprising a folate.

[0295] 7. The method of clause 6, wherein the conjugate comprising a folate comprises a folate linked to one or more amino acids.

[0296] 8. The method of clause 7, wherein the conjugate comprising a folate has the formula

[0297] .

[0298] 9. A method according to any one of clauses 3 to 8, wherein the folate has the formula

[0299]

[0300] Where X 1 and Y 1 are each independently selected from halogen, R 2 , OR2 , SR 3 and NR 4 R 5 ;

[0301] U, V and W represent divalent moieties, each independently selected from -(R 6a )C=、-N=、-(R 6a )C(R 7a )- and -N(R 4a )-; Q is selected from C and CH; T is selected from S, O, N and -C=C-;

[0302] X 2 and X 3 Each independently selected from the following: 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, wherein Z is oxygen or sulfur;

[0303] R 1 Selected from hydrogen, halogen, C1-C 12 Alkyl and C1-C 12 Alkoxy;

[0304] R 2 , R 3 , R 4 , R 4a , R 4b , R 5 , R 5b , R 6b and R 7b Each independently selected from the following: hydrogen, halogen, C1-C 12 Alkyl, C1-C 12 Alkoxy, C1-C 12 Acyl, C1-C 12 Alkenyl, C1-C 12 Alkynyl, (C1-C 12Alkoxy)carbonyl and (C1-C 12 alkylamino)carbonyl;

[0305] R 6 and R 7 are independently selected from hydrogen, halogen, C1-C 12 Alkyl and C1-C 12 Alkoxy; or R 6 With R 7 Together they form a carbonyl group;

[0306] R 6a and R 7a are independently selected from hydrogen, halogen, C1-C 12 Alkyl and C1-C 12 Alkoxy; or R 6a With R 7a Together they form a carbonyl group;

[0307] p, r, s and t are each independently 0 or 1; and

[0308] * indicates an optional covalent bond to the rest of the conjugate.

[0309] 10. A method as described in claim 3, wherein an agent that inhibits the activation of CAR T cells is administered and is selected from the following: lymphocyte-specific protein tyrosine kinase inhibitors, PI3 kinase inhibitors, IL-2-inducible T cell kinase inhibitors, JAK inhibitors, BTK inhibitors, EC2319, and an agent that blocks CAR T cells from binding to the compound or a pharmaceutically acceptable salt thereof, but not binding to the cancer.

[0310] 11. The method of clause 10, wherein an agent that inhibits activation of CAR T cells is administered and the agent is a lymphocyte-specific protein tyrosine kinase inhibitor.

[0311] 12. The method according to clause 11, wherein the lymphocyte-specific protein tyrosine kinase inhibitor is dasatinib.

[0312] 13. The method of clause 10, wherein an agent that inhibits activation of CAR T cells is administered and the agent is a PI3 kinase inhibitor.

[0313] 14. The method of clause 13, wherein the PI3 kinase inhibitor is GDC0980.

[0314] 15. The method of clause 10, wherein an agent that inhibits activation of CAR T cells is administered and the agent is an IL-2 inducible T cell kinase inhibitor.

[0315] 16. The method of clause 15, wherein the IL-2 inducible T cell kinase inhibitor is BMS-509744.

[0316] 17. The method of clause 10, wherein an agent that inhibits activation of CAR T cells is administered and is an agent that blocks CAR T cells from binding to the compound or a pharmaceutically acceptable salt thereof, but not to the cancer.

[0317] 18. The method of clause 17, wherein the agent is fluoresceinamine, FITC or fluorescein sodium.

[0318] 19. The method of clause 18, wherein the agent is FITC.

[0319] 20. The method of clause 18, wherein the agent is fluorescein sodium.

[0320] 21. The method of any one of clauses 17 to 20, wherein the agent that inhibits activation of CAR T cells is administered at a dose of about .01 to about 300 μmol / kg patient body weight.

[0321] 22. The method of any one of clauses 17 to 20, wherein the agent that inhibits activation of CAR T cells is administered at a dose of about .06 to about 100 μmol / kg of patient body weight.

[0322] 23. The method of any one of clauses 17 to 20, wherein the agent that inhibits activation of CAR T cells is administered at a dose of about .06 to about 90 μmol / kg patient body weight.

[0323] 24. The method of any one of clauses 17 to 20, wherein the agent that inhibits activation of CAR T cells is administered at a dose of about .06 to about 80 μmol / kg patient body weight.

[0324] 25. The method of any one of clauses 17 to 20, wherein the agent that inhibits activation of CAR T cells is administered at a dose of about .06 to about 70 μmol / kg patient body weight.

[0325] 26. The method of any one of clauses 17 to 20, wherein the agent that inhibits activation of CAR T cells is administered at a dose of about .06 to about 60 μmol / kg patient body weight.

[0326] 27. The method of any one of clauses 17 to 20, wherein the agent that inhibits activation of CAR T cells is administered at a dose of about .06 to about 50 μmol / kg patient body weight.

[0327] 28. The method of any one of clauses 17 to 20, wherein the agent that inhibits activation of CAR T cells is administered at a dose of about .06 to about 40 μmol / kg patient body weight.

[0328] 29. The method of any one of clauses 17 to 20, wherein the agent that inhibits activation of CAR T cells is administered at a dose of about .06 to about 30 μmol / kg patient body weight.

[0329] 30. The method of any one of clauses 17 to 20, wherein the agent that inhibits activation of CAR T cells is administered at a dose of about .06 to about 20 μmol / kg patient body weight.

[0330] 31. The method of any one of clauses 17 to 20, wherein the agent that inhibits activation of CAR T cells is administered at a dose of about .06 to about 10 μmol / kg patient body weight.

[0331] 32. The method of any one of clauses 17 to 20, wherein the agent that inhibits activation of CAR T cells is administered at a dose of about .06 to about 8 μmol / kg patient body weight.

[0332] 33. The method of any one of clauses 17 to 20, wherein the agent that inhibits activation of CAR T cells is administered at a dose of about .06 to about 6 μmol / kg patient body weight.

[0333] 34. The method of any one of clauses 3 to 33, wherein more than one dose of the folate, the conjugate comprising the folate (wherein the conjugate comprising the folate does not include a targeting moiety) or the agent that inhibits activation of CAR T cells is administered to the patient.

[0334] 35. A method as described in any one of clauses 3 to 34, wherein the folate, the conjugate comprising the folate (wherein the conjugate comprising the folate does not include a targeting portion) or the agent that inhibits the activation of CAR T cells is administered to the patient before and / or after the compound or a pharmaceutically acceptable salt thereof.

[0335] 36. A method as described in any one of clauses 3 to 35, wherein administration of the folate, the conjugate comprising the folate (wherein the conjugate comprising the folate does not include a targeting portion), or the agent that inhibits activation of CAR T cells results in a reduction in cytokine levels in the patient.

[0336] 37. A method as described in claim 36, wherein the reduction in the level of the cytokine occurs about 3 hours after administering the folate, the conjugate comprising the folate (wherein the conjugate comprising the folate does not include a targeting portion), or the agent that inhibits the activation of CAR T cells to the patient.

[0337] 38. The method of clause 36, wherein the reduction in the cytokine level occurs about 6 hours after administering to the patient the folate, the conjugate comprising the folate (wherein the conjugate comprising the folate does not include a targeting portion), or the agent that inhibits activation of CAR T cells.

[0338] 39. The method of any one of clauses 36 to 38, wherein the reduction in cytokine levels is to approximately the cytokine levels in untreated patients.

[0339] 40. A method as described in any one of clauses 3 to 39, wherein the compound or a pharmaceutically acceptable salt thereof is administered before and subsequently to the administration of the folate, the conjugate comprising the folate (wherein the conjugate comprising the folate does not include a targeting portion) or the agent that inhibits the activation of CAR T cells.

[0340] 41. A method as described in any one of clauses 3 to 40, wherein after administration of the folate, the conjugate comprising the folate (wherein the conjugate comprising the folate does not include a targeting portion) or the agent that inhibits the activation of CAR T cells, the number of CAR T cells in the patient's blood increases, even if the cytokine level in the patient is reduced.

[0341] 42. A method as described in any one of clauses 3 to 41, wherein after administration of the folate, the conjugate comprising the folate (wherein the conjugate comprising the folate does not include a targeting portion) or the agent that inhibits the activation of CAR T cells, CAR T cell activation is enhanced or maintained relative to patients not treated with a rescue agent, even if cytokine levels in the treated patient are reduced.

[0342] 43. A method as described in any one of clauses 3 to 42, wherein the cancer comprises a tumor and the patient's tumor size does not increase when the folate, the conjugate comprising a folate (wherein the conjugate comprising a folate does not include a targeting portion) or the agent that inhibits the activation of CAR T cells is administered to the patient.

[0343] 44. The method of clause 43, wherein a complete response to the tumor is achieved.

[0344] 45. A method as described in any one of clauses 3 to 44, wherein when the CRS grade reaches 1, 2, 3 or 4, the folate, the conjugate comprising a folate (wherein the conjugate comprising a folate does not include a targeting portion) or the agent that inhibits the activation of CAR T cells is administered to the patient.

[0345] 46. ​​The method of clause 45, wherein when the CRS grade reaches 3 or 4, an agent that inhibits the activation of CAR T cells is administered to the patient.

[0346] 47. The method of clauses 3 to 46, wherein pulmonary edema is reduced.

[0347] 48. The method of any one of clauses 1 to 47, wherein the compound or a pharmaceutically acceptable salt thereof is administered at a dose of about 10 nmol / kg patient body weight to about 2500 nmol / kg patient body weight; and the dose of the CAR T cells is about 1 million CAR T cells to about 15 million CAR T cells.

[0348] 49. The method of clause 48, wherein the compound or a pharmaceutically acceptable salt thereof is administered at a dose of about 10 nmol / kg patient body weight to about 100 nmol / kg patient body weight.

[0349] 50. The method of any one of clauses 48 to 49, wherein the compound or a pharmaceutically acceptable salt thereof is administered at a dose of about 10 nmol / kg patient body weight to about 50 nmol / kg patient body weight.

[0350] 51. The method of any one of clauses 48 to 50, wherein the compound or a pharmaceutically acceptable salt thereof is administered at a dose of about 10 nmol / kg patient body weight to about 20 nmol / kg patient body weight.

[0351] 52. The method of clause 48, wherein the compound or a pharmaceutically acceptable salt thereof is administered at a dose of about 10 nmol / kg patient body weight to about 600 nmol / kg patient body weight.

[0352] 53. The method of clause 48, wherein the compound or a pharmaceutically acceptable salt thereof is administered at a dose of about 200 nmol / kg to about 600 nmol / kg of patient body weight.

[0353] 54. The method of clause 48, wherein the compound or a pharmaceutically acceptable salt thereof is administered at a dose of about 400 nmol / kg to about 600 nmol / kg of patient body weight.

[0354] 55. The method of any one of clauses 48 to 54, wherein the dose of CAR T cells is about 1 million CAR T cells to about 12.5 million CAR T cells.

[0355] 56. The method of any one of clauses 48 to 55, wherein the dose of CAR T cells is about 1 million CAR T cells to about 7 million CAR T cells.

[0356] 57. The method of any one of clauses 48 to 56, wherein the dose of CAR T cells is about 1 million CAR T cells to about 5 million CAR T cells.

[0357] 58. The method of any one of clauses 48 to 57, wherein the dose of CAR T cells is about 2 million CAR T cells to about 5 million CAR T cells.

[0358] 59. The method of any one of clauses 1 to 58, further comprising the step of terminating the continuous administration of the compound or a pharmaceutically acceptable salt thereof to inhibit or prevent cytokine release syndrome in the patient.

[0359] 60. The method of any one of clauses 1 to 59, wherein the compound or a pharmaceutically acceptable salt thereof is administered to the patient continuously for at least one hour.

[0360] 61. The method of any one of clauses 1 to 59, wherein the compound or a pharmaceutically acceptable salt thereof is administered to the patient continuously for at least four hours.

[0361] 62. The method of any one of clauses 1 to 59, wherein the compound or a pharmaceutically acceptable salt thereof is administered to the patient continuously for at least six hours.

[0362] 63. The method of any one of clauses 1 to 62, wherein the compound or a pharmaceutically acceptable salt thereof is administered to the patient every other day.

[0363] 64. The method of any one of clauses 1 to 62, wherein the compound or a pharmaceutically acceptable salt thereof is administered to the patient three times per week.

[0364] 65. The method of any one of clauses 1 to 62, wherein the compound or a pharmaceutically acceptable salt thereof is administered to the patient twice per week.

[0365] 66. The method of any one of clauses 1 to 62, wherein the compound or a pharmaceutically acceptable salt thereof is administered to the patient once a week.

[0366] 67. The method of any one of clauses 1 to 62, wherein the compound or a pharmaceutically acceptable salt thereof is administered to the patient until unacceptable loss of weight, fever, drop in blood pressure, or pulmonary edema occurs in the patient.

[0367] 68. A method for treating cancer, the method comprising:

[0368] i) administering a compound or a pharmaceutically acceptable salt thereof to a patient, wherein the compound comprises a small molecule ligand linked to a targeting moiety via a linker, wherein at least a first dose and a second dose of the compound or a pharmaceutically acceptable salt thereof are administered to the patient, wherein the first dose and the second dose are different, wherein the amount of the compound or a pharmaceutically acceptable salt thereof in the second dose is about 2-fold to about 15,000-fold greater than the amount of the compound or a pharmaceutically acceptable salt thereof in the first dose; and

[0369] ii) administering to the patient a CAR T cell composition comprising a CAR T cell, wherein the CAR T cell comprises a CAR directed to the targeting moiety and wherein the CAR comprises an E2 anti-fluorescein antibody fragment.

[0370] 69. The method of clause 68, wherein at least a first dose, a second dose, and a third dose of the compound or a pharmaceutically acceptable salt thereof are administered to the patient, wherein the first dose, the second dose, and the third dose are different, wherein the amount of the compound or a pharmaceutically acceptable salt thereof in the second dose is about 2 times to about 750 times greater than the amount of the compound or a pharmaceutically acceptable salt thereof in the first dose, and wherein the amount of the compound or a pharmaceutically acceptable salt thereof in the third dose is about 800 times to about 10,000 times greater than the amount of the compound or a pharmaceutically acceptable salt thereof in the first dose.

[0371] 70. The method of clause 68, wherein at least a first dose, a second dose, a third dose, and a fourth dose of the compound or a pharmaceutically acceptable salt thereof are administered to the patient, wherein the first dose, the second dose, the third dose, and the fourth dose are different, wherein the amount of the compound or a pharmaceutically acceptable salt thereof in the second dose is about 2 times to about 750 times greater than the amount of the compound or a pharmaceutically acceptable salt thereof in the first dose, wherein the amount of the compound or a pharmaceutically acceptable salt thereof in the third dose is about 800 times to about 7500 times greater than the amount of the compound or a pharmaceutically acceptable salt thereof in the first dose, and wherein the amount of the compound or a pharmaceutically acceptable salt thereof in the fourth dose is about 8000 times to about 15000 times greater than the amount of the compound or a pharmaceutically acceptable salt thereof in the first dose.

[0372] 71. The method of clause 70, wherein the amount of the compound or its pharmaceutically acceptable salt in the second dose is about 100 times greater than the amount of the compound or its pharmaceutically acceptable salt in the first dose, wherein the amount of the compound or its pharmaceutically acceptable salt in the third dose is about 1000 times greater than the amount of the compound or its pharmaceutically acceptable salt in the first dose, and wherein the amount of the compound or its pharmaceutically acceptable salt in the fourth dose is about 10000 times greater than the amount of the compound or its pharmaceutically acceptable salt in the first dose.

[0373] 72. The method of clause 68, wherein at least a first dose and a second dose of the compound or a pharmaceutically acceptable salt thereof are administered to the patient, wherein the first dose and the second dose are different, wherein the amount of the compound or a pharmaceutically acceptable salt thereof in the second dose is about 2 times to about 15,000 times greater than the amount of the compound or a pharmaceutically acceptable salt thereof in the first dose.

[0374] 73. A method for treating cancer, the method comprising:

[0375] i) administering to a patient a first dose of a compound or a pharmaceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked to a targeting moiety via a linker;

[0376] ii) administering to the patient at least a second dose of the compound or a pharmaceutically acceptable salt thereof, wherein the amount of the compound or a pharmaceutically acceptable salt thereof in the second dose is at least about 50% less than the amount of the compound or a pharmaceutically acceptable salt thereof in the first dose; and

[0377] iii) administering to the patient a dose of a CAR T cell composition comprising a CAR T cell, wherein the CART cell comprises a CAR directed to the targeting moiety and wherein the CAR comprises an E2 anti-fluorescein antibody fragment.

[0378] 74. The method of clause 73, wherein the amount of the compound or a pharmaceutically acceptable salt thereof in the second dose is at least about 60% less than the amount of the compound or a pharmaceutically acceptable salt thereof in the first dose.

[0379] 75. The method of clause 73, wherein the amount of the compound or a pharmaceutically acceptable salt thereof in the second dose is at least about 70% less than the amount of the compound or a pharmaceutically acceptable salt thereof in the first dose.

[0380] 76. The method of clause 73, wherein the amount of the compound or a pharmaceutically acceptable salt thereof in the second dose is at least about 80% less than the amount of the compound or a pharmaceutically acceptable salt thereof in the first dose.

[0381] 77. The method of clause 73, wherein the amount of the compound or pharmaceutically acceptable salt thereof in the second dose is at least about 90% less than the amount of the compound or pharmaceutically acceptable salt thereof in the first dose.

[0382] 78. The method of clause 73, wherein the amount of the compound or pharmaceutically acceptable salt thereof in the second dose is at least about 95% less than the amount of the compound or pharmaceutically acceptable salt thereof in the first dose.

[0383] 79. The method of clause 73, wherein the amount of the compound or a pharmaceutically acceptable salt thereof in the second dose is at least about 96% less than the amount of the compound or a pharmaceutically acceptable salt thereof in the first dose.

[0384] 80. The method of clause 73, wherein the amount of the compound or a pharmaceutically acceptable salt thereof in the second dose is at least about 97% less than the amount of the compound or a pharmaceutically acceptable salt thereof in the first dose.

[0385] 81. The method of clause 73, wherein the amount of the compound or a pharmaceutically acceptable salt thereof in the second dose is at least about 98% less than the amount of the compound or a pharmaceutically acceptable salt thereof in the first dose.

[0386] 82. The method of clause 73, wherein the amount of the compound or a pharmaceutically acceptable salt thereof in the second dose is at least about 99% less than the amount of the compound or a pharmaceutically acceptable salt thereof in the first dose.

[0387] 83. The method of clause 73, wherein the amount of the compound or pharmaceutically acceptable salt thereof in the second dose is at least about 99.5% less than the amount of the compound or pharmaceutically acceptable salt thereof in the first dose.

[0388] 84. The method of any one of clauses 73 to 83, wherein the first dose of the compound or a pharmaceutically acceptable salt thereof is from about 100 nmol / kg patient body weight to about 1000 nmol / kg patient body weight.

[0389] 85. The method of any one of clauses 73 to 83, wherein the first dose of the compound or a pharmaceutically acceptable salt thereof is from about 100 nmol / kg patient body weight to about 900 nmol / kg patient body weight.

[0390] 86. The method of any one of clauses 73 to 83, wherein the first dose of the compound or a pharmaceutically acceptable salt thereof is about 100 nmol / kg patient body weight to about 800 nmol / kg patient body weight.

[0391] 87. The method of any one of clauses 73 to 83, wherein the first dose of the compound or a pharmaceutically acceptable salt thereof is about 100 nmol / kg patient body weight to about 700 nmol / kg patient body weight.

[0392] 88. The method of any one of clauses 73 to 83, wherein the first dose of the compound or a pharmaceutically acceptable salt thereof is about 100 nmol / kg patient body weight to about 600 nmol / kg patient body weight.

[0393] 89. The method of any one of clauses 73 to 83, wherein the first dose of the compound or a pharmaceutically acceptable salt thereof is about 200 nmol / kg patient body weight to about 600 nmol / kg patient body weight.

[0394] 90. The method of any one of clauses 73 to 83, wherein the first dose of the compound or a pharmaceutically acceptable salt thereof is about 400 nmol / kg patient body weight to about 600 nmol / kg patient body weight.

[0395] 91. The method of any one of clauses 73 to 83, wherein the first dose of the compound or a pharmaceutically acceptable salt thereof is about 500 nmol / kg patient body weight.

[0396] 92. The method of clause 84, wherein the second dose of the compound or a pharmaceutically acceptable salt thereof is from about 0.5 nmol / kg patient body weight to about 500 nmol / kg patient body weight.

[0397] 93. The method of clause 85, wherein the second dose of the compound or a pharmaceutically acceptable salt thereof is from about 0.5 nmol / kg patient body weight to about 450 nmol / kg patient body weight.

[0398] 94. The method of clause 86, wherein the second dose of the compound or a pharmaceutically acceptable salt thereof is from about 0.5 nmol / kg patient body weight to about 400 nmol / kg patient body weight.

[0399] 95. The method of clause 87, wherein the second dose of the compound or a pharmaceutically acceptable salt thereof is from about 0.5 nmol / kg patient body weight to about 350 nmol / kg patient body weight.

[0400] 96. The method of clause 88, wherein the second dose of the compound or a pharmaceutically acceptable salt thereof is from about 0.5 nmol / kg patient body weight to about 300 nmol / kg patient body weight.

[0401] 97. The method of clause 89, wherein the second dose of the compound or a pharmaceutically acceptable salt thereof is from about 1 nmol / kg patient body weight to about 300 nmol / kg patient body weight.

[0402] 98. The method of clause 90, wherein the second dose of the compound or a pharmaceutically acceptable salt thereof is from about 2 nmol / kg patient body weight to about 300 nmol / kg patient body weight.

[0403] 99. The method of clause 91, wherein the second dose of the compound or a pharmaceutically acceptable salt thereof is from about 2 nmol / kg patient body weight to about 250 nmol / kg patient body weight.

[0404] 100. The method of any one of clauses 92 to 99, wherein the second dose of the compound or a pharmaceutically acceptable salt thereof is about 5 nmol / kg patient body weight to about 40 nmol / kg patient body weight.

[0405] 101. The method of any one of clauses 92 to 99, wherein the second dose of the compound or a pharmaceutically acceptable salt thereof is about 40 nmol / kg patient body weight to about 150 nmol / kg patient body weight.

[0406] 102. The method of any one of clauses 73 to 101, further comprising administering a third dose of the compound or a pharmaceutically acceptable salt thereof, wherein the third dose of the compound or a pharmaceutically acceptable salt thereof is the same as the second dose of the compound or a pharmaceutically acceptable salt thereof.

[0407] 103. The method of clause 102, further comprising administering a fourth dose of the compound or a pharmaceutically acceptable salt thereof, wherein the fourth dose of the compound or a pharmaceutically acceptable salt thereof is the same as the second dose of the compound or a pharmaceutically acceptable salt thereof and the third dose of the compound or a pharmaceutically acceptable salt thereof.

[0408] 104. The method of any one of clauses 73 to 103, wherein one or more doses of the compound or a pharmaceutically acceptable salt thereof administered after the first dose of the compound or a pharmaceutically acceptable salt thereof maintain inhibition of growth of the cancer relative to the first dose of the compound or a pharmaceutically acceptable salt thereof.

[0409] 105. The method of any one of clauses 73 to 104, wherein the CAR T cells are administered at a dose of about 1 million CAR T cells to about 40 million CAR T cells.

[0410] 106. The method of any one of clauses 73 to 105, wherein the one or more doses of the compound or a pharmaceutically acceptable salt thereof administered after the first dose of the compound or a pharmaceutically acceptable salt thereof is administered once a week.

[0411] 107. The method of any one of clauses 73 to 105, wherein the one or more doses of the compound or a pharmaceutically acceptable salt thereof are administered twice per week.

[0412] 108. The method of any one of clauses 1 to 107, wherein the CAR further comprises an IgG4 hinge domain, a CD3ζ activation domain, and a 4-1BB co-stimulatory domain.

[0413] 109. The method of any one of clauses 1 to 108, wherein the E2 anti-fluorescein antibody fragment is a scFv fragment.

[0414] 110. The method of any one of clauses 1 to 109, wherein the CAR protein sequence has at least about 90% identity with SEQ ID NO: 2.

[0415] 111. A method as described in any one of clauses 1 to 109, wherein the CAR protein sequence has at least about 95% identity with SEQ ID NO: 2.

[0416] 112. A method as described in any one of clauses 1 to 109, wherein the CAR protein sequence has at least about 98% identity with SEQ ID NO: 2.

[0417] 113. The method of any one of clauses 1 to 112, wherein the CAR is bound to fluorescein.

[0418] 114. The method of any one of clauses 1 to 109, wherein the CAR protein sequence has up to about 50 conservative amino acid substitutions and wherein the CAR binds to fluorescein.

[0419] 115. The method of any one of clauses 1 to 109, wherein the CAR is encoded by a polynucleotide having at least about 90% identity to SEQ ID NO: 1.

[0420] 116. A method as described in any one of clauses 1 to 109, wherein the CAR is encoded by a polynucleotide having at least about 95% identity with SEQ ID NO: 1.

[0421] 117. The method of any one of clauses 1 to 109, wherein the CAR is encoded by a polynucleotide having at least about 98% identity to SEQ ID NO: 1.

[0422] 118. The method of any one of clauses 115 to 117, wherein the CAR is conjugated to fluorescein.

[0423] 119. The method of any one of clauses 1 to 118, wherein the CAR is encoded by a polynucleotide that hybridizes to a polynucleotide having SEQ ID NO: 1 under high stringency conditions and wherein the CAR binds to fluorescein.

[0424] 120. The method of any one of clauses 1 to 119, wherein the CAR is encoded by a polynucleotide having SEQ ID NO: 1 or by a degenerate variant of SEQ ID NO: 1.

[0425] 121. The method of any one of clauses 1 to 120, wherein the ligand is selected from the group consisting of folates, DUPA, NK-1R ligands, CAIX ligands, gamma-glutamyl transpeptidase ligands, NKG2D ligands, and CCK2R ligands.

[0426] 122. The method of any one of clauses 1 to 121, wherein the targeting moiety is fluorescein or a pharmaceutically acceptable salt thereof.

[0427] 123. The method of any one of clauses 1 to 122, 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.

[0428] 124. The method of any one of clauses 1 to 123, wherein the linker comprises PEG.

[0429] 125. A method according to any one of clauses 1 to 124, wherein the compound or a pharmaceutically acceptable salt thereof has the formula

[0430] ,

[0431] Wherein B represents the small molecule ligand, L represents the linker, and T represents the targeting moiety, and wherein L comprises a structure having the formula

[0432]

[0433] wherein n is an integer from 0 to 200.

[0434] 126. The method of any one of clauses 1 to 125, wherein the cancer is selected from the group consisting of lung cancer, bone cancer, pancreatic cancer, skin cancer, head cancer, neck cancer, skin melanoma, intraocular melanoma, uterine cancer, ovarian cancer, endometrial cancer, rectal cancer, gastric cancer, colon cancer, breast cancer, triple-negative breast cancer, fallopian tube cancer, carcinoma of the endometrium, endometrium), 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, urethral cancer, prostate cancer, chronic leukemia, acute leukemia, acute myelocytic leukemia, lymphocytic lymphoma, myeloid leukemia, myelomonocytic leukemia, hairy cell leukemia, myelomonocytic leukemia, hairy cell leukemia, pleural mesothelioma, bladder cancer, Burkitt's lymphoma, ureteral cancer, kidney cancer, renal cell carcinoma, renal pelvis cancer, neoplasms of the central nervous system (CNS), primary CNS lymphoma, spinal axis tumors, brain stem gliomas, pituitary adenomas, and gastroesophageal junction adenocarcinoma.

[0435] 127. The method of any one of clauses 1 to 126, wherein the cancer is a cancer that expresses a folate receptor.

[0436] 128. The method of any one of clauses 1 to 127, wherein the cancer is endometrial cancer.

[0437] 129. The method of any one of clauses 1 to 127, wherein the cancer is non-small cell lung cancer.

[0438] 130. The method of any one of clauses 1 to 127, wherein the cancer is ovarian cancer.

[0439] 131. The method of any one of clauses 1 to 127, wherein the cancer is triple negative breast cancer.

[0440] 132. The method of any one of clauses 1 to 127, wherein the cancer is acute myeloid leukemia.

[0441] 133. The method of clause 132, wherein the cancer expresses the folate receptor-β.

[0442] 134. The method of any one of clauses 1 to 133, wherein multiple doses of the CAR T cell composition are administered.

[0443] 135. The method of any one of clauses 1 to 134, wherein at least two doses of the CAR T cell composition are administered.

[0444] 136. The method of any one of clauses 1 to 135, wherein the patient is imaged prior to administration of the compound or a pharmaceutically acceptable salt thereof, or prior to administration of the CAR T cell composition.

[0445] 137. The method of any one of clauses 1 to 136, wherein the compound or a pharmaceutically acceptable salt thereof is not an antibody, and does not comprise an antibody fragment.

[0446] 138. The method of any one of clauses 1 to 137, wherein the targeting moiety does not comprise a peptide epitope.

[0447] 139. The method of any one of clauses 1 to 138, wherein cytokine release that results in off-target toxicity in the patient does not occur and wherein CAR T cell toxicity to the cancer occurs.

[0448] 140. The method of any one of clauses 1 to 138, wherein off-target tissue toxicity does not occur in the patient and wherein CAR T cell toxicity to the cancer occurs.

[0449] 141. The method of any one of clauses 1 to 138, wherein the cancer comprises a tumor, wherein the patient's tumor size is reduced, and wherein off-target toxicity does not occur.

[0450] 142. The method of any one of clauses 1 to 141, wherein CRS is reduced or prevented and the method results in a reduction in tumor volume in the patient.

[0451] 143. The method of any one of clauses 1 to 142, wherein weight loss due to CRS is reduced or prevented.

[0452] 144. The method of any one of clauses 59 to 67, further comprising administering the compound or a pharmaceutically acceptable salt thereof to the patient.

[0453] 145. The method of clause 144, wherein subsequent administration of the compound or a pharmaceutically acceptable salt thereof results in increased CAR T cell activation and cytokine levels in the patient.

[0454] 146. The method of any one of clauses 1 to 145, wherein the cancer comprises a tumor and wherein a complete response to the tumor is achieved.

[0455] 147. A method as described in any one of clauses 1 to 146, wherein the CAR T cells have a central memory / effector memory phenotype.

[0456] 148. A method as described in any one of clauses 1 to 147, wherein the CD8:CD4 ratio of the CAR T cells is about 1:1.

[0457] Therefore, in one embodiment, a method for treating cancer is provided. The method comprises i) administering a compound or a pharmaceutically acceptable salt thereof to a patient, wherein the compound comprises a small molecule ligand connected to a targeting moiety by a linker; ii) administering a first dose of a CAR T cell composition to the patient, wherein the CAR T cell composition comprises a CAR T cell, wherein the CAR T cell comprises a CAR, and wherein the CAR comprises an E2 anti-fluorescein antibody fragment; and iii) administering a second dose of the CAR T cell composition to the patient, wherein the CAR T cell composition comprises a CAR T cell, wherein the CAR T cell comprises a CAR, and wherein the CAR comprises an E2 anti-fluorescein antibody fragment.

[0458] In another embodiment, a method for treating cancer is provided. The method comprises i) administering a compound or a pharmaceutically acceptable salt thereof to a patient, wherein the compound comprises a small molecule ligand connected to a targeting moiety via a linker, and ii) administering a CAR T cell composition comprising a CAR T cell to the patient, wherein the CAR T cell comprises a CAR, wherein the CAR comprises an E2 anti-fluorescein antibody fragment, and wherein the CAR T cell composition comprises a mixture of the CAR T cell and a non-transformed T cell.

[0459] In yet another embodiment, a method for treating cancer is provided. The method includes i) administering a compound or a pharmaceutically acceptable salt thereof to a patient, wherein the compound includes a small molecule ligand connected to a targeting portion by a linker, ii) administering a CAR T cell composition to the patient, wherein the CAR T cell composition includes a CAR T cell, wherein the CART cell includes a CAR, wherein the CAR includes an E2 anti-fluorescein antibody fragment, and iii) administering a folate, a conjugate comprising a folate (wherein the conjugate comprising a folate does not include a targeting portion) or a drug that inhibits the activation of CAR T cells to the patient.

[0460] In another embodiment, a method for treating cancer is provided. The method comprises i) administering a compound or a pharmaceutically acceptable salt thereof to a patient, wherein the compound comprises a small molecule ligand connected to a targeting moiety by a linker, and wherein the compound or a pharmaceutically acceptable salt thereof is administered at a dose of about 10 nmol / kg patient weight to about 2500 nmol / kg patient weight, and ii) administering a CAR T cell composition comprising a CAR T cell to the patient, wherein the CAR T cell comprises a CAR, wherein the CAR comprises an E2 anti-fluorescein antibody fragment, and wherein the CAR T cell dose is about 1 million CAR T cells to about 15 million CAR T cells.

[0461] In yet another embodiment, a method for treating cancer is provided. The method comprises i) continuously administering a compound or a pharmaceutically acceptable salt thereof to a patient, wherein the compound comprises a small molecule ligand connected to a targeting moiety by a linker, ii) administering a CAR T cell composition comprising a CAR T cell to the patient, wherein the CAR T cell comprises a CAR, wherein the CAR comprises an E2 anti-fluorescein antibody fragment, and iii) terminating the continuous administration of the compound or a pharmaceutically acceptable salt thereof to inhibit or prevent cytokine release syndrome in the patient.

[0462] In another illustrative aspect, a method for treating cancer is provided. The method comprises i) administering a compound or a pharmaceutically acceptable salt thereof to a patient, wherein the compound comprises a small molecule ligand connected to a targeting moiety by a linker, wherein at least a first dose and a second dose of the compound or a pharmaceutically acceptable salt thereof are administered to the patient, wherein the first dose is different from the second dose, wherein the amount of the compound or a pharmaceutically acceptable salt thereof in the second dose is about 2 times to about 15,000 times greater than the amount of the compound or a pharmaceutically acceptable salt thereof in the first dose, and ii) administering a CAR T cell composition comprising a CAR T cell to the patient, wherein the CAR T cell comprises a CAR, wherein the CAR comprises an E2 anti-fluorescein antibody fragment.

[0463] In another embodiment, a method for treating cancer is provided. The method comprises i) administering a compound or a pharmaceutically acceptable salt thereof to a patient, wherein the compound comprises a small molecule ligand connected to a targeting moiety via a linker and wherein the compound or a pharmaceutically acceptable salt thereof is administered to the patient once a week, and ii) administering a CAR T cell composition comprising a CAR T cell to the patient, wherein the CAR T cell comprises a CAR, wherein the CAR comprises an E2 anti-fluorescein antibody fragment.

[0464] In yet another embodiment, a method for treating cancer is provided. The method comprises i) administering to a patient a first dose of a compound or a pharmaceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand connected to a targeting moiety via a linker, ii) administering to the patient at least a second dose of the compound or a pharmaceutically acceptable salt thereof, wherein the amount of the second dose of the compound or a pharmaceutically acceptable salt thereof is at least about 50% less than the amount of the first dose of the compound or a pharmaceutically acceptable salt thereof; and iii) administering to the patient a dose of a CAR T cell composition comprising a CAR T cell, wherein the CAR T cell comprises a CAR and wherein the CAR comprises an E2 anti-fluorescein antibody fragment.

[0465] Accordingly, various embodiments are provided in the preceding paragraph and in the clauses listed above, and all applicable embodiments described in this Detailed Description, Summary, Examples, and Claims apply to such embodiments.

[0466] As described herein, a "patient" may be a human or, in the case of veterinary applications, a patient may be a laboratory, agricultural, domestic, or wild animal. In various aspects, a patient may be a laboratory animal (such as a caries animal (e.g., mouse, rat, hamster, etc.), rabbit, monkey, chimpanzee), a domestic animal (such as a dog, cat, or rabbit), an agricultural animal (such as a cow, horse, pig, sheep, goat), or a captive wild animal (such as a bear, panda, lion, tiger, leopard, elephant, zebra, giraffe, gorilla, dolphin, or whale).

[0467] In various embodiments, the cancer to be treated can be selected from carcinoma, sarcoma, osteosarcoma, lymphoma, melanoma, mesothelioma, nasopharyngeal carcinoma, leukemia, adenocarcinoma or myeloma. In other embodiments, the cancer can be lung cancer, bone cancer, pancreatic cancer, skin cancer, head cancer, neck cancer, skin 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, endometrium), 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, 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, neoplasms of the central nervous system (CNS), primary CNS lymphoma, spinal axis tumors, brain stem glioma, pituitary adenoma, and gastroesophageal junction adenocarcinoma.

[0468] In some aspects of such embodiments, cancer is a cancer expressing folate receptors. In another embodiment, cancer is a cancer expressing folate receptor alpha. In yet another embodiment, cancer is a cancer expressing folate receptor beta. In some aspects of such embodiments, cancer is endometrial cancer, non-small cell lung cancer, ovarian cancer, or triple-negative breast cancer. In another embodiment, the cancer being treated is a tumor. In another embodiment, the cancer is malignant. In another embodiment, the cancer is acute myeloid leukemia. In yet another embodiment, the cancer is acute myeloid leukemia and the cancer expresses folate receptor-β. In still another embodiment, the cancer is acute myeloid leukemia and the CAR-T cells have a central memory / effect memory phenotype. In yet another embodiment, the CD8:CD4 ratio of the CAR T cells is about 1:1, about 1.2:1 ratio, about 1:1.2 ratio, about 1.3:1 ratio, about 1:1.3 ratio, about 1.4:1 ratio, about 1:1.4 ratio, about 1.5:1 ratio, or about 1:1.5 ratio. In another embodiment, where the cancer is acute myeloid leukemia or another cancer, CAR T cells associated with the tumor may have increased expression of CD25 relative to CAR T cells not associated with the tumor.

[0469] In one embodiment, the "small molecule ligand" may be a folate, DUPA (ligand bound by PSMA-positive human prostate cancer cells and other cancer cell types), NK-1R ligand (NK-1R The invention relates to a small molecule ligand that specifically binds to a cancer cell type (e.g., a receptor for a CAIX ligand found on kidney cancer, ovarian cancer, vulvar cancer, and breast cancer), a ligand for a gamma glutamyl transpeptidase (e.g., a transpeptidase overexpressed in ovarian cancer, colon cancer, liver cancer, astroglioma, melanoma, and leukemia), a NKG2D ligand (e.g., a receptor for a NKG2D ligand found on lung cancer, colon cancer, kidney cancer, prostate cancer, and T and B cell lymphomas), or a CCK2R ligand (particularly a receptor for a CCK2R ligand found on thyroid cancer, lung cancer, pancreatic cancer, ovarian cancer, brain cancer, gastric cancer, gastrointestinal stromal cancer, and colon cancer), each of which is a small molecule ligand that specifically binds to a cancer cell type (i.e., the receptor for each of such ligands may be overexpressed on cancer compared to normal tissue).

[0470] In one embodiment, the small molecule ligand may have a mass of less than about 10,000 Daltons, less than about 9000 Daltons, less than about 8,000 Daltons, less than about 7000 Daltons, less than about 6000 Daltons, less than about 5000 Daltons, less than about 4500 Daltons, less than about 4000 Daltons, less than about 3500 Daltons, less than about 3000 Daltons, less than about 2500 Daltons, less than about 2000 Daltons, less than about 1500 Daltons, less than about 1000 Daltons, or less than about 500 Daltons. In another embodiment, the small molecule ligand may have a mass of about 1 to about 10,000 Daltons, about 1 to about 9000 Daltons, about 1 to about 8,000 Daltons, about 1 to about 7000 Daltons, about 1 to about 6000 Daltons, about 1 to about 5000 Daltons, about 1 to about 4500 Daltons, about 1 to about 4000 Daltons, about 1 to about 3500 Daltons, about 1 to about 3000 Daltons, about 1 to about 2500 Daltons, about 1 to about 2000 Daltons, about 1 to about 1500 Daltons, about 1 to about 1000 Daltons, or about 1 to about 500 Daltons.

[0471] In one embodiment, the DUPA derivative can be a ligand of a small molecule ligand linked to a targeting moiety, and DUPA derivatives are described in WO 2015 / 057852, which is incorporated herein by reference.

[0472] In one embodiment, in the case of a "small molecule ligand attached to a linker", the small molecule ligand is a folate. In various embodiments, the folate may be folic acid, a folic acid analog, or another molecule that binds to a folate receptor. In various embodiments, usable folic acid analogs include formyltetrahydrofolate, pteroylpolyglutamate, and pteridines (such as tetrahydropterin, dihydrofolate, tetrahydrofolate, and deaza and dideaza analogs thereof) that bind to folate receptors. The terms "deaza" and "dideaza" analogs refer to art-recognized analogs having carbon atoms that replace one or two nitrogen atoms in the naturally occurring folate 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 above-mentioned folic acid analogs are often referred to as "folates", reflecting their ability to bind to the folate receptor. Other analogs that bind to the folate receptor include aminopterin, methotrexate, N10-methylfolate, 2-deamino-hydroxyfolate, deaza analogs (such as 1-deazamethotrexate or 3-deazamethotrexate), and 3',5'-dichloro-4-amino-4-deoxy-N10-methylpterylglutamate (dichloromethotrexate).

[0473] In another embodiment, in the context of a "small molecule ligand attached to a linker", the small molecule ligand may have the formula

[0474]

[0475] Where X 1 and Y 1 are each independently selected from halogen, R 2 , OR 2 , SR 3 and NR 4 R 5 ;

[0476] U, V and W represent divalent moieties, each independently selected from -(R 6a )C=、-N=、-(R 6a )C(R 7a )- and -N(R 4a )-; Q is selected from C and CH; T is selected from S, O, N and -C=C-;

[0477] X 2 and X 3 Each independently selected from the following: 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, wherein Z is oxygen or sulfur;

[0478] R 1 Selected from hydrogen, halogen, C1-C 12 Alkyl and C1-C 12 Alkoxy;

[0479] R 2 , R 3 , R 4 , R 4a , R 4b , R 5, R 5b , R 6b and R 7b Each independently selected from the following: hydrogen, halogen, C1-C 12 Alkyl, C1-C 12 Alkoxy, C1-C 12 Acyl, C1-C 12 Alkenyl, C1-C 12 Alkynyl, (C1-C 12 Alkoxy)carbonyl and (C1-C 12 alkylamino)carbonyl;

[0480] R 6 and R 7 are independently selected from hydrogen, halogen, C1-C 12 Alkyl and C1-C 12 Alkoxy; or R 6 With R 7 Together they form a carbonyl group;

[0481] R 6a and R 7a are independently selected from hydrogen, halogen, C1-C 12 Alkyl and C1-C 12 Alkoxy; or R 6a With R 7a Together they form a carbonyl group;

[0482] p, r, s and t are each independently 0 or 1; and

[0483] * indicates an optional covalent bond to the rest of the conjugate.

[0484] On the one hand, the "targeting moiety" bound to the CAR comprising the E2 anti-fluorescein antibody fragment and expressed by the CAR T cell can be selected from, for example, fluorescein, fluorescein isothiocyanate (FITC) and NHS-fluorescein. The characteristics of the targeting moiety are limited only to that it should be recognized and bound by the CAR comprising the E2 anti-fluorescein antibody fragment, preferably specifically recognized and bound, and that it has a relatively low molecular weight. In various aspects, exemplary targeting moieties are haptens, including small molecular weight organic molecules.

[0485] In one illustrative embodiment, the targeting moiety can have the following illustrative structure:

[0486]

[0487] wherein X is oxygen, nitrogen or sulfur, and wherein X is connected to the linker L; Y is OR a NR a 2 or NR a 3 + ; and Y' is O, NRa or NR a 2 + ; wherein at each occurrence, each R is independently selected from H, fluorine, sulfonic acid, sulfonate, and salts thereof, and the like; and R a is hydrogen or alkyl.

[0488] In an illustrative aspect, the linker can include polyethylene glycol (PEG), polyproline, a hydrophilic amino acid, a sugar, a non-natural peptidoglycan, polyvinylpyrrolidone, Pluronic F-127, or a combination thereof.

[0489] In another illustrative aspect, the linker of the compound described herein or its pharmaceutically acceptable salt may include a direct bond (e.g., a reaction between the isothiocyanate group of FITC and the free amino group of the small molecule ligand) or the bond may be through an intermediate linker. In one embodiment, if present, the intermediate linker may be any biocompatible linker known in the art, such as a divalent linker. In an illustrative embodiment, the divalent linker may include about 1 to about 30 carbon atoms. In another illustrative embodiment, the divalent linker may include about 2 to about 20 carbon atoms. In other embodiments, a lower molecular weight divalent linker (i.e., those with an approximate molecular weight of about 30 to about 300 Daltons) is used. In another embodiment, 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.

[0490] In various embodiments, the small molecule ligand attached to the targeting moiety may have the formula

[0491] ,

[0492] Wherein B represents the small molecule ligand, L represents the linker, and T represents the targeting moiety, and wherein L comprises a structure having the formula

[0493]

[0494] wherein n is an integer from 0 to 200. In another embodiment, n may be an integer from 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 to 1, 15 to 16, 15 to 17, 15 to 18, 15 to 19, 15 to 20, 15 to 21, 15 to 22, 15 to 23, 15 to 24, 15 to 25, 15 to 26, 15 to 27, 15 to 28, 15 to 29, 15 to 30, 15 to 31, 15 to 32, 15 to 33, 15 to 34, 15 to 35 , 15 to 36, 15 to 37, 15 to 38, 15 to 39, 15 to 40, 15 to 50, 15 to 60, 15 to 70, 15 to 80, 15 to 90, 15 to 100, 15 to 110, 15 to 120, 15 to 130, 15 to 140, 15 to 150, or n can be 1, 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, 40, 50, 60, 70, 80, 90, 100, 108, 110, 120, 130, 140, or 150.

[0495] In another embodiment, the linker can be a bivalent linker that can include one or more spacers. Illustrative spacers are shown in the table below. The following non-limiting, illustrative spacers are described, where * indicates the point of attachment of a small molecule ligand or targeting moiety or other bivalent linker moiety.

[0496]

[0497] In other embodiments, the small molecule ligand (bridge) attached to the targeting moiety can have any of the following structures.

[0498]

[0499]

[0500]

[0501] .

[0502] In other embodiments, the compound or a pharmaceutically acceptable salt thereof is not an antibody and does not comprise an antibody fragment. In another embodiment, the targeting moiety does not comprise a peptide epitope.

[0503] In an illustrative embodiment, the small molecule ligand connected to the targeting moiety via a joint (bridge) includes fluorescein isothiocyanate (FITC) connected to the small molecule ligand. On the one hand, cancer can overexpress receptors of small molecule ligands. On the other hand, for example, cytotoxic T cells or another type of T cells can be transformed to express a CAR comprising an E2 anti-fluorescein antibody fragment. In this regard, the CAR can target FITC, and as a result of the small molecule ligand binding to the cancer, the cancer is decorated with FITC molecules. Therefore, toxicity to normal, non-target cells can be avoided. In this embodiment, when the T cell expressing the E2 anti-fluorescein antibody fragment CAR binds to FITC, the CAR T cell is activated and the cancer is improved.

[0504] "Pharmaceutically acceptable salts" of small molecule ligands connected to the targeting moiety via a linker are contemplated. As used herein, the term "pharmaceutically acceptable salt" refers to those salts whose counterions can be used in medicine. In various embodiments, such salts include, but are not limited to: 1) acid addition salts, which can be obtained by reaction of the free base of the parent compound with an inorganic acid (such as hydrochloric acid, hydrobromic acid, nitric acid, phosphoric acid, sulfuric acid, and perchloric acid, etc.) or with an 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 the like), or 2) salts formed when an acidic proton present in the parent compound is replaced by a metal ion (e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion); or coordinated with an organic base (such as ethanolamine, diethanolamine, triethanolamine, trimethylamine, N-methylglucamine, etc.). Pharmaceutically acceptable salts are well known to those skilled in the art, and any such pharmaceutically acceptable salts are contemplated in connection with the embodiments described herein.

[0505] In various embodiments, suitable acid addition salts are formed from acids that form non-toxic salts. Illustrative examples include acetate, aspartate, benzoate, benzenesulfonate, bicarbonate / carbonate, bisulfate / sulfate, borate, camphorate, citrate, edisylate, ethanesulfonate, formate, fumarate, glucoheptonate, gluconate, glucuronate, hexafluorophosphate, hyaluronate, hydrochloride / chloride, hydrobromide / bromide, hydroiodide / iodide, isethionate, lactate, malate, maleate, malonate, methanesulfonate, methylsulfate, naphthoate, 2-naphthalenesulfonate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate / hydrogenphosphate / dihydrogenphosphate, saccharate, stearate, succinate, tartrate, toluenesulfonate, and trifluoroacetate.

[0506] In various embodiments, suitable base salts are formed from bases that form non-toxic salts. Illustrative examples include arginine, benzathine, calcium salts, choline, diethylamine, diethanolamine, glycine, lysine, magnesium salts, meglumine, ethanolamine, potassium salts, sodium salts, triethanolamine, and zinc salts. Hemisalts of acids and bases can also be formed, for example, hemisulphate and hemicalcium salts.

[0507] In one illustrative aspect, the compounds described herein or their pharmaceutically acceptable salts 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 mixtures of stereoisomers, such as enantiomers, diastereomers, and enantiomerically or diastereomerically enriched mixtures. In one aspect, the compounds described herein or their pharmaceutically acceptable salts may exist as geometric isomers. Thus, various embodiments may include pure geometric isomers or mixtures of geometric isomers.

[0508] In some aspects, the compounds described herein, or pharmaceutically acceptable salts thereof, may exist in unsolvated forms as well as solvated forms (including hydrated forms). In general, the solvated forms are equivalent to the unsolvated forms and are encompassed within the scope of the present invention.

[0509] The methods described herein also utilize T lymphocytes (e.g., cytotoxic T lymphocytes) engineered to express a chimeric antigen receptor (CAR) comprising an E2 anti-fluorescein antibody fragment that recognizes and binds to a targeting portion of the bridge (e.g., fluorescein, fluorescein isothiocyanate (FITC) and NHS-fluorescein). In one embodiment, the CAR described herein comprises three domains, the domains comprising 1) a recognition region that specifically recognizes and binds to a targeting portion (e.g., a single-chain fragment variable (scFv) region, a Fab fragment, etc. of an E2 anti-fluorescein antibody), 2) a co-stimulatory domain that enhances the proliferation and survival of T lymphocytes, and 3) an activation signaling domain that generates a T lymphocyte activation signal.

[0510] In various aspects, as non-limiting examples, scFv regions of antibodies that bind to fluorescein, fluorescein isothiocyanate (FITC), and NHS-fluorescein can be used. In illustrative non-limiting embodiments, the scFv region can be prepared from: (i) an E2 antibody known in the art that binds a targeting moiety, and (iii) a sequence variant derived from the scFv region of such an antibody, for example, a 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 scFv region amino acid sequence from which the scFv region was derived.

[0511] On the one hand, the costimulatory domain helps to bind CAR to the targeting portion, and enhances the proliferation and survival of cytotoxic T lymphocytes. Suitable costimulatory domains include but are not limited to CD28, CD137 (4-1BB), members of the tumor necrosis factor (TNF) receptor family, CD134 (OX40), members of the TNFR superfamily of receptors, CD27, CD30, CD150, DAP10, NKG2D and CD278 (ICOS), CD28 superfamily costimulatory molecules expressed on activated T cells, or combinations thereof. In an illustrative embodiment, the costimulatory domain of the engineered CAR includes CD 28 and CD137. Technicians will appreciate that sequence variants of such costimulatory domains can be used without adversely affecting the present invention, wherein the variant has the same or similar activity as the domain of the variant transformed. In various embodiments, such variants may 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 the variant is derived.

[0512] In an illustrative embodiment, the activation signal domain helps to bind CAR to the targeting portion, activating T lymphocytes (e.g., cytotoxic T lymphocytes). In various embodiments, suitable activation signal domains include T cell CD3 ζ chains, CD3 δ receptor proteins, mbl receptor proteins, B29 receptor proteins, and Fc receptor γ. Technicians will appreciate that sequence variants of such activation signal domains can be used, wherein the variant has the same or similar activity as the domain transformed from the variant. In various embodiments, the variant has 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 derived from the variant.

[0513] In one aspect, a construct encoding a CAR comprising an E2 anti-fluorescein antibody fragment is prepared using genetic engineering techniques. Such techniques are described in detail in Sambrook et al., "Molecular Cloning: A Laboratory Manual", 3rd edition, Cold Spring Harbor Laboratory Press, (2001), which is incorporated herein by reference, and Green and Sambrook, "Molecular Cloning: A Laboratory Manual", 4th edition, Cold Spring Harbor Laboratory Press, (2012), which is incorporated herein by reference.

[0514] As an example, a plasmid or viral expression vector (e.g., a lentiviral vector, a retroviral vector, a sleeping beauty, and a piggyback (including a transposon / transposase system of a non-viral mediated CAR gene delivery system) encoding a fusion protein can be prepared, the fusion protein comprising a recognition region, one or more costimulatory domains, and an activation signal domain connected in the same frame and in a 5' to 3' direction. In other embodiments, other settings are acceptable and include a recognition region, an activation signal domain, and one or more costimulatory domains. In one embodiment, the positioning of the recognition region in the fusion protein will generally be such that the display of the region outside the cell is achieved. In one embodiment, the CAR comprising the E2 anti-fluorescein antibody fragment may comprise additional components, such as a signal peptide (e.g., a CD8α signal peptide) that ensures proper export of the fusion protein to the cell surface, a transmembrane domain (e.g., a CD8α transmembrane domain, a CD28 transmembrane domain, or a CD3ζ transmembrane domain) that ensures maintenance of the fusion protein as an integral membrane protein, and a hinge domain (e.g., a CD8α hinge or an IgG4 hinge) that confers flexibility to the recognition region and allows strong binding to the targeting moiety, and any other suitable domains.

[0515] A schematic diagram of an exemplary CAR construct (in which the expressed CAR comprises an E2 anti-fluorescein antibody fragment) is shown in Figure 1In, wherein the fusion protein sequence is incorporated into an expression vector and wherein the CAR comprises an E2 anti-fluorescein antibody fragment, an IgG4 hinge domain, a CD28 transmembrane domain, and wherein the co-stimulatory domain is CD137 (4-1BB), and the activation signal domain is CD3ζ. The CAR may include additional suitable domains. An exemplary nucleic acid sequence of a CAR insert is provided as SEQ ID NO: 1 and an exemplary encoded amino acid sequence is provided as SEQ ID NO: 2. As used herein, "SEQ ID NO: 1" means a sequence starting with an underlined "agc" codon and terminating with an underlined "ggc" codon. This portion of the longer sequence encodes a CAR inserted into the T cell membrane. Other portions of the longer sequence include portions of the CAR that are not inserted into the membrane and coding sequences for signal peptides, EGFRt domains, etc. that function as chimeric antigen receptors. As used herein, "SEQ ID NO: 2" means a sequence starting with an underlined "S" and terminating with an underlined "G". This portion of the longer sequence is the amino acid sequence of the CAR that is inserted into the T cell membrane. Other portions of the longer sequence include amino acid sequences that are not part of the CAR that is inserted into the membrane and signal peptides, EGFRt domains, etc. that function as chimeric antigen receptors. In another embodiment, SEQ ID NO: 2 may include or consist of a humanized or human amino acid sequence. SEQ ID NOS: 1 and 2 are as described above. The start and stop codons of the longer nucleic acid sequence are underlined and the longer sequence is an exemplary sequence that can be used to transduce T cells for use in methods as described herein.

[0516] SEQ ID NO: 1 (E2 anti-fluorescein antibody fragment CAR nucleic acid sequence (insert))

[0517]

[0518] SEQ ID NO: 2 (E2 anti-fluorescein antibody fragment CAR amino acid sequence (insert))

[0519]

[0520] In one embodiment, the CAR expressing the E2 anti-fluorescein antibody fragment comprises a recognition region and the recognition region is a single-chain fragment variable (scFv) region of the E2 anti-fluorescein antibody, a costimulatory domain and the costimulatory domain is CD137 (4-1BB), and an activation signal domain and the activation signal domain is a T cell CD3ζ chain. In another embodiment, the CAR may also include any additional suitable domains. It is well known to technicians that anti-FITC scFv and anti-fluorescein scFv are equivalent terms.

[0521] In various embodiments, the "E2 anti-fluorescein antibody fragment" can be a CAR comprising a fragment (e.g., a scFv fragment) of the E2 anti-fluorescein antibody. The E2 anti-fluorescein antibody is described in, for example, Vaughan et al., Nature Biotechnol. Volume 14 (3), pages 309-314, 1996, which is incorporated herein by reference. In one embodiment, the CAR expressing the E2 anti-fluorescein antibody fragment can have a binding affinity for fluorescein of about 0.7 nM to about 0.8 nM, about 0.72 nM to about 0.8 nM, about 0.73 nM to about 0.8 nM, about 0.72 nM to about 7.8 nM, about 0.73 to about 0.77 nM, or about 0.75 nM.

[0522] In various embodiments, the CAR may also include an IgG4 hinge domain, a CD3ζ activation domain, and / or a 4-1BB co-stimulatory domain, and other suitable domains. In still other embodiments, the CAR may be encoded by a polynucleotide having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity to SEQ ID NO: 1. In another illustrative embodiment, the CAR may be encoded by a polynucleotide hybridized with a polynucleotide having SEQ ID NO: 1 under highly stringent conditions. On the other hand, the CAR may be encoded by a polynucleotide having SEQ ID NO: 1 or by a degenerate variant of SEQ ID NO: 1. In other embodiments, the CAR protein sequence may have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity to SEQ ID NO: 2. In yet another embodiment, the CAR protein sequence may have up to about 50 conservative amino acid substitutions. In any of the embodiments described herein, the CAR is conjugated to fluorescein.

[0523] In one embodiment, T lymphocytes (e.g., cytotoxic T lymphocytes) can be genetically engineered to express a CAR construct, wherein a T lymphocyte population is transfected with an expression vector encoding a CAR construct expressing an E2 anti-fluorescein antibody fragment, and the E2 anti-fluorescein antibody fragment is expressed by the CAR construct. Suitable methods for preparing a transduced population of T lymphocytes expressing an E2 anti-fluorescein antibody fragment are well known to technicians and are described in Sambrook et al., "Molecular Cloning: A Laboratory Manual", 3rd edition, Cold Spring Harbor Laboratory Press, (2001), which is incorporated herein by reference, and Green and Sambrook, "Molecular Cloning: A Laboratory Manual", 4th edition, Cold Spring Harbor Laboratory Press, (2012), which is incorporated herein by reference.

[0524] In one embodiment, a CAR T cell comprising a nucleic acid of SEQ ID NO: 1 may be used as described herein. In another embodiment, a CAR T cell comprising a polypeptide of SEQ ID NO: 2 may be used as described herein. In another illustrative aspect, a nucleic acid (e.g., an isolated nucleic acid) comprising SEQ ID NO: 1 and encoding a chimeric antigen receptor may be used to prepare CAR T cells for use as described herein. In yet another embodiment, a chimeric antigen receptor polypeptide comprising SEQ ID NO: 2 may be used to prepare CAR T cells for use as described herein. In another embodiment, a vector comprising SEQ ID NO: 1 may be used to prepare CAR T cells for use as described herein. On the other hand, a lentiviral vector comprising SEQ ID NO: 1 is provided, which can be used to prepare CAR T cells for use as described herein. In yet another embodiment, SEQ ID NO: 2 may comprise or consist of a humanized or human amino acid sequence.

[0525] In each of such embodiments, variant nucleic acid sequences or amino acid sequences 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 SEQ ID NO: 1 are contemplated. In another embodiment, the nucleic acid sequence may be a variant nucleic acid sequence 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 SEQ ID NO: 1, so long as the variant sequence encodes a polypeptide of SEQ ID NO: 2. In another embodiment, the nucleic acid sequence or amino acid sequence can be a variant nucleic acid or amino acid sequence 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 along a stretch of 200 nucleic acids for SEQ ID NO: 1, or along a stretch of 200 amino acids for SEQ ID NO: 2. In one embodiment, the determination of the percent identity or similarity between sequences can be performed, for example, via the use of the GAP program (Genetics Computer Group, software; now available via Accelrys at http: / / www.accelrys.com), and alignment can be performed using, for example, the ClustalW algorithm (VNTI software, InforMax Inc.). Sequence databases can be searched using the nucleic acid or amino acid sequence of interest. Algorithms for database searching are typically based on BLAST software (Altschul et al., 1990). In some embodiments, percent identity can be determined along the entire length of a nucleic acid or amino acid sequence.

[0526] It is also within the scope of the present invention that nucleic acids complementary to the nucleic acids represented by SEQ ID NO: 1 can be used to prepare CART cells for use as described herein, and those hybridized to the nucleic acids represented by SEQ ID NO: 1 can be used, or those hybridized to their complements under highly stringent conditions. According to the present invention, "highly stringent conditions" means hybridization at 65 ° C, in 5X SSPE and 50% formamide, and washing in 0.5X SSPE at 65 ° C. Highly stringent, lowly stringent and moderately stringent hybridization conditions are described in: Sambrook et al., "Molecular Cloning: A Laboratory Manual", 3rd edition, Cold Spring Harbor Laboratory Press, (2001), which is incorporated herein by reference, and Green and Sambrook, "Molecular Cloning: A Laboratory Manual", 4th edition, Cold Spring Harbor Laboratory Press, (2012), which is incorporated herein by reference. In some illustrative aspects, hybridization occurs along the full-length nucleic acid.

[0527] In still other embodiments, the CAR used in the methods described herein may be encoded by a polynucleotide having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity to SEQ ID NO: 1. In another illustrative embodiment, the CAR may be encoded by a polynucleotide hybridized with a polynucleotide having SEQ ID NO: 1 under highly stringent conditions. On the other hand, the CAR may be encoded by a polynucleotide having SEQ ID NO: 1 or by a degenerate variant of SEQ ID NO: 1. As used herein, a degenerate variant refers to a genetic code having more than one codon specifying any particular amino acid. The degenerate variant codons specifying each amino acid are well known in the art. On the other hand, substitutions may be made to optimize the level of polypeptide production in a specific prokaryotic or eukaryotic host cell (i.e., codon usage variant).

[0528] In other embodiments, the CAR protein sequence may have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 2. In yet another embodiment, the CAR protein sequence may have up to about 50 conservative amino acid substitutions. In one embodiment, the CAR protein sequence expressing the E2 anti-fluorescein antibody fragment may have up to about 5 conservative amino acid substitutions, up to about 10 conservative amino acid substitutions, up to about 15 conservative amino acid substitutions, up to about 20 conservative amino acid substitutions, up to about 25 conservative amino acid substitutions, up to about 30 conservative amino acid substitutions, up to about 35 conservative amino acid substitutions, up to about 40 conservative amino acid substitutions, up to about 45 conservative amino acid substitutions, up to about 50 conservative amino acid substitutions, up to about 55 conservative amino acid substitutions, up to about 60 conservative amino acid substitutions, up to about 65 conservative amino acid substitutions, up to about 70 conservative amino acid substitutions, or up to about 75 conservative amino acid substitutions. As is well known to those skilled in the art, any non-critical amino acid of a polypeptide that is changed via conservative amino acid substitution should not significantly change the activity of the polypeptide, because the side chain of the replaced amino acid should be able to form a similar bond and contact like the side chain of the replaced amino acid. In any embodiment described herein, the CAR binds to fluorescein.

[0529] In one illustrative aspect, non-conservative substitutions are possible as long as they do not unduly affect the fluorescein binding activity of the E2 anti-fluorescein antibody fragment polypeptide. As is well known in the art, "conservative substitutions" of amino acids or "conservative substitution variants" of polypeptides refer to amino acid substitutions that maintain the following: 1) the secondary structure of the polypeptide; 2) the charge or hydrophobicity of the amino acid; and 3) any one or more of the side chain volume or such features. In one embodiment, the conservative amino acid substitution can be with an amino acid analog. Illustratively, the well-known term "hydrophilic residue" refers to serine or threonine. "Hydrophobic residue" refers to leucine, isoleucine, phenylalanine, valine or alanine or the like. "Positively charged residue" refers to lysine, arginine, ornithine or histidine. "Negatively charged residue" refers to aspartic acid or glutamic acid. Residues with "bulky side chains" refer to phenylalanine, tryptophan or tyrosine or the like. An exemplary list of conservative amino acid substitutions is given in Table 1.

[0530] Table 1

[0531] Targeting amino acids Replace with Alanine D-Ala, Gly, Aib, β-Ala, L-Cys, D-Cys Arginine D-Arg, Lys, D-Lys, Orn D-Orn Aspartic acid D-Asn,Asp,D-Asp,Glu,D-Glu Gln,D-Gln Aspartic acid D-Asp, D-Asn, Asn, Glu, D-Glu, Gln, D-Gln Cysteine D-Cys, S-Me-Cys, Met, D-Met, Thr, D-Thr Glutamine D-Gln, Asn, D-Asn, Glu, D-Glu, Asp, D-Asp Glutamate D-Glu, D-Asp, Asp, Asn, D-Asn, Gln, D-Gln Glycine Ala, D-Ala, Pro, D-Pro, Aib, β-Ala Isoleucine D-Ile, Val, D-Val, Leu, D-Leu, Met, D-Met Leucine Val, D-Val, Met, D-Met, D-Ile, D-Leu, Ile Lysine D-Lys, Arg, D-Arg, Orn, D-Orn Methionine D-Met, S-Me-Cys, Ile, D-Ile, Leu, D-Leu, Val, D-Val Phenylalanine D-Phe, Tyr, D-Tyr, His, D-His, Trp, D-Trp Proline D-Pro Serine D-Ser, Thr, D-Thr, Allo-Thr, L-Cys, D-Cys Threonine D-Thr, Ser, D-Ser, other-Thr, Met, D-Met, Val, D-Val Tyrosine D-Tyr,Phe,D-Phe,His,D-His,Trp,D-Trp Valine D-Val, Leu, D-Leu, Ile, D-Ile, Met, D-Met

[0532] In one embodiment, the T lymphocytes used in the methods described herein (e.g., cytotoxic T lymphocytes or non-transformed T cells used to prepare CAR T cells expressing E2 anti-fluorescein antibody fragments) can be autologous cells, although allogeneic cells can also be used, such as when the patient being treated has received high-dose chemotherapy or radiation therapy to destroy the patient's immune system. In one embodiment, allogeneic cells can be used.

[0533] On the one hand, T lymphocytes are obtained from patients by methods well known in the art. For example, T cells (for example, cytotoxic T cells or non-converted T cells) can be obtained via the following: collect peripheral blood from patients, make the blood undergo Ficoll density gradient centrifugation and then use negative T cell separation kit (such as EasySep T cell separation kit) to separate T cell groups from peripheral blood. In an illustrative embodiment, the T lymphocyte colony (for example, cytotoxic T cells or non-converted T cells) does not need to be pure and can contain other cells, such as other types of T cells (for example, in the case of cytotoxic T cells), monocytes, macrophages, natural killer cells, and B cells. On the one hand, the colony being collected can include at least about 90% selected cell type, at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% selected cell type.

[0534] In one embodiment, after obtaining T lymphocytes (e.g., cytotoxic T cells for preparing CAR T cells expressing E2 anti-fluorescein antibody fragments), the cells are cultured under conditions that promote the activation of the cells. In this embodiment, the culture conditions may be such that the cells can be administered to the patient without concern for the reactivity of the components of the culture medium. For example, the culture conditions may not include bovine serum products, such as bovine serum albumin. In an illustrative aspect, activation can be achieved by introducing 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. On the one hand, lymphocyte colonies can be cultured for about 1 to about 4 days under conditions that promote activation. In one embodiment, the appropriate level of activation can be determined by cell size, proliferation rate, or activation markers determined by flow cytometry.

[0535] In an illustrative embodiment, after culturing a population of T lymphocytes (e.g., cytotoxic T lymphocytes for preparing CAR T cells expressing E2 anti-fluorescein antibody fragments) under conditions that promote activation, the cells can be transfected with an expression vector encoding a CAR comprising an E2 anti-fluorescein antibody fragment. Suitable vectors and transfection methods used in various embodiments are described above. On the one hand, after transfection, the cells can be immediately administered to the patient or the cells can be cultured for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or more days, or between about 5 and about 12 days, between about 6 and about 13 days, between about 7 and about 14 days, or between about 8 and about 15 days, for example, to allow the cells to recover from transfection. On the one hand, suitable culture conditions can be similar to the conditions in which the cells are cultured for activation, with or without an agent for promoting activation.

[0536] Therefore, as described above, in an illustrative aspect, the treatment method described herein may further include 1) obtaining an autologous or allogeneic T lymphocyte population (e.g., cytotoxic T lymphocytes for preparing CAR T cells expressing E2 anti-fluorescein antibody fragments), 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 comprising an E2 anti-fluorescein antibody fragment to form a CAR T cell expressing an E2 anti-fluorescein antibody fragment.

[0537] In an illustrative embodiment, when the cells have been transfected and activated, a composition comprising a CAR T cell can be prepared, wherein the CAR T cell includes a CAR comprising an E2 anti-fluorescein antibody fragment, and is administered to the patient with or without non-transformed T cells. In one embodiment, a culture medium lacking any animal product (such as bovine serum) can be used to culture CAR T cells and / or non-transformed T cells expressing E2 anti-fluorescein antibody fragments. In another embodiment, tissue culture conditions commonly used by technicians can be used to avoid contamination with bacteria, fungi, and mycoplasmas. In an exemplary embodiment, before administration to the patient, the cells (e.g., CAR T cells and / or non-transformed T cells expressing E2 anti-fluorescein antibody fragments) are precipitated, washed, and resuspended in a pharmaceutically acceptable carrier or diluent. Exemplary compositions comprising CAR-expressing T lymphocytes (e.g., cytotoxic T lymphocytes) or non-transformed T cells expressing E2 anti-fluorescein antibody fragments include compositions comprising cells in sterile 290 mOsm saline, in an infusible low-temperature medium (containing plasma-Lyte A, dextrose, sodium chloride injection, human serum albumin, and DMSO), 0.9% NaCl and 2% human serum albumin, or any other sterile 290 mOsm infusible substance. Alternatively, in another embodiment, depending on the characteristics of the culture medium, CAR T cells or non-transformed T cells expressing E2 anti-fluorescein antibody fragments may be administered as a composition in the culture medium, or concentrated and resuspended in the culture medium before administration. In various embodiments, a CAR T cell composition (wherein the CAR comprises an E2 anti-fluorescein antibody fragment) may be administered to a patient via any suitable means (such as parenteral administration, e.g., intradermal, subcutaneous, intramuscular, intraperitoneal, intravenous, or intrathecal), together with or without non-transformed T cells.

[0538] On the one hand, the total number and cell concentration of CAR T cells expressing E2 anti-fluorescein antibody fragments in the composition administered to the patient will vary depending on many factors including the type of T lymphocytes (e.g., cytotoxic T lymphocytes) being used, the binding specificity of the CAR comprising the E2 anti-fluorescein antibody fragment, the characteristics of the targeting moiety and the small molecule ligand, the characteristics of the cancer, the location of the cancer in the patient, the method used to administer the composition to the patient, and the health, age, and weight of the patient being treated. In various embodiments, suitable compositions comprising transduced CAR T cells expressing E2 anti-fluorescein antibody fragments include those having a volume of about 0.1 ml to about 200 ml and about 0.1 ml to about 125 ml.

[0539] In various embodiments, the transduced CAR T cells administered to the patient (wherein the CAR T cells comprise a CAR comprising an E2 anti-fluorescein antibody fragment) may comprise about 1 x 10 5 About 1 x 10 15 or 1 x 10 6 About 1 x 10 15 In various embodiments, about 1 x 10 5 About 1 x 10 10 , about 1 X 10 6 About 1 x 10 10 , about 1 X 10 6 About 1 x 10 9 , about 1 X 10 6 About 1X 10 8 , about 1 X 10 6 About 2 x 10 7 , about 1 X 10 6 About 3 x 10 7 , about 1 X 10 6 About 1.5 x 10 7 , about 1 X 10 6 About 1 x 10 7 , about 1 X 10 6 About 9 x 10 6 , about 1 X 10 6 About 8 x 10 6 , about 1 X 10 6 About 7 x 10 6 , about 1X 10 6 About 6 x 10 6 , about 1 X 10 6 About 5 x 10 6 , about 1 X 10 6 About 4 x 10 6 , about 1 X 10 6 About 3 x 10 6 , about 1 X 10 6 About 2 x 10 6 , about 2 X 10 6 About 6 x 10 6 , about 2 X 10 6 About 5 x 10 6 , about 3 x 10 6 About 6 x 10 6 , about 4 x 10 6About 6 x 10 6 , about 4 x 10 6 About 1 x 10 7 , about 1 X 10 6 About 1 x 10 7 , about 1 X 10 6 About 1.5 x 10 7 , about 1 X 10 6 About 2 x 10 7 , about 0.2 X 10 6 About 1 x 10 7 , about 0.2 X 10 6 About 1.5 x 10 7 , about 0.2 X 10 6 About 2 x 10 7 , or about 5 X 10 6 CAR T cells expressing E2 anti-fluorescein antibody fragments. On the one hand, in any of the embodiments described herein, a single dose or multiple doses of CAR T cells expressing E2 anti-fluorescein antibody fragments can be administered to the patient. In this paragraph or any of the embodiments described herein, the number of CAR T cells can be measured in kg of patient body weight. In any of the embodiments described herein, CAR T cells expressing E2 anti-fluorescein antibody fragments can be administered before the compound or a pharmaceutically acceptable salt thereof. It should be understood that unless otherwise indicated, the designation i), ii) and iii) etc. for the steps of any method described herein does not indicate an order.

[0540] In various embodiments described herein, non-converted T cells may also be administered with CAR T cells expressing E2 anti-fluorescein antibody fragments and may be administered in the amounts described herein for CAR T cells expressing E2 anti-fluorescein antibody fragments and non-converted T cells. On the one hand, a mixture of CAR T cells (wherein the CAR T cells include CAR, which includes E2 anti-fluorescein antibody fragments) and non-converted T cells may be administered once or multiple times, or a pure CAR T cell expressing E2 anti-fluorescein antibody fragment and a mixture of CAR T cells expressing E2 anti-fluorescein antibody fragments and non-converted T cells may be administered in a dosage combination (e.g., a dose of CAR T cells expressing E2 anti-fluorescein antibody fragments, followed by one or more doses of a mixture of CART cells expressing E2 anti-fluorescein antibody fragments and non-converted T cells). It should be clear to the technician from the present disclosure that a "mixture" of CAR T cells expressing E2 anti-fluorescein antibody fragments and non-converted T cells as described herein means mixing CAR T cells expressing E2 anti-fluorescein antibody fragments with non-converted T cells that have not yet been exposed to a construct for expressing a CAR comprising an E2 anti-fluorescein antibody fragment.

[0541] In other embodiments, the dose of CAR T cells expressing E2 anti-fluorescein antibody fragments in the CAR T cell composition administered to the patient is selected from the following: 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 expressing E2 anti-fluorescein antibody fragments.

[0542] In still other illustrative embodiments, the CAR T cell composition comprising CAR T cells expressing an E2 anti-fluorescein antibody fragment is administered via injection into the patient's bloodstream, and the CAR T cells expressing an E2 anti-fluorescein antibody fragment in the patient's bloodstream account for at least 5%, at least 7%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, or at least 15% of the patient's total T cells in the patient's bloodstream at about 4 weeks after injection of the CAR T cell composition, at least 20%, 25%, 30%, 35%, 40%, or 50% at about 3 weeks after injection of the CAR T cell composition, at least 60%, 70%, 75%, or 80% at about 2 weeks after injection of the CAR T cell composition, or at least 85%, 90%, or 95% at about 1 week after injection of the CAR T cell composition.

[0543] In the embodiments described herein, the CAR T cell composition may include CAR T cells expressing E2 anti-fluorescein antibody fragments without any other cell type, or non-transformed T cells may be administered to the patient in combination with CAR T cells expressing E2 anti-fluorescein antibody fragments. For embodiments of administering multiple doses of CAR T cell compositions, any dose may include a mixture of CAR T cells expressing E2 anti-fluorescein antibody fragments or CAR T cells expressing E2 anti-fluorescein antibody fragments and non-transformed T cells. In various embodiments, non-transformed T cells may be administered in the amounts described herein for CAR T cells expressing E2 anti-fluorescein antibody fragments.

[0544] In another embodiment, any dose of the CAR T cell composition may include a mixture of CAR T cells expressing E2 anti-fluorescein antibody fragments and non-transformed T cells at a ratio selected from the following: about 1:5 CAR T cells expressing E2 anti-fluorescein antibody fragments to non-transformed T cells, about 1:4 CAR T cells expressing E2 anti-fluorescein antibody fragments to non-transformed T cells, about 1:3 CAR T cells expressing E2 anti-fluorescein antibody fragments to non-transformed T cells, about 1:2 CAR T cells expressing E2 anti-fluorescein antibody fragments to non-transformed T cells, and about 1:1 CAR T cells expressing E2 anti-fluorescein antibody fragments to non-transformed T cells.

[0545] In still other embodiments, any dose of the CAR T cell composition may include a mixture of CART cells expressing E2 anti-fluorescein antibody fragments and non-transformed T cells at a ratio of about 1:1 to about 1:5 of CAR T cells expressing E2 anti-fluorescein antibody fragments to non-transformed T cells, or the CAR T cell composition may include a mixture of about 10 million CAR T cells expressing E2 anti-fluorescein antibody fragments and about 40 million non-transformed T cells, about 15 million CAR T cells expressing E2 anti-fluorescein antibody fragments and about 35 million non-transformed T cells, about 20 million CAR T cells expressing E2 anti-fluorescein antibody fragments and about 30 million non-transformed T cells, or about 25 million CAR T cells expressing E2 anti-fluorescein antibody fragments and about 25 million non-transformed T cells.

[0546] The compounds described herein or their pharmaceutically acceptable salts, or CAR T cell compositions (wherein the composition comprises CAR T cells, the CAR T cells comprising CARs comprising E2 anti-fluorescein antibody fragments) may be administered to patients using any suitable method known in the art. As described herein, the term "administering" includes all methods of introducing a compound or its pharmaceutically acceptable salt, or a CAR T cell composition comprising CAR T cells (the CAR T cells have CARs, the CARs comprising E2 anti-fluorescein antibody fragments) into a patient, including but not limited to oral, intravenous, intramuscular, subcutaneous, transdermal, etc. On the one hand, the compounds described herein or their pharmaceutically acceptable salts may be administered in unit dosage form and / or in formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles.

[0547] On the one hand, a compound as described herein or a pharmaceutically acceptable salt thereof, or a CAR T cell composition (wherein the CAR T cell composition comprises a CAR T cell expressing a CAR comprising an E2 anti-fluorescein antibody fragment) can be directly administered into the bloodstream, into the muscle, or into an internal organ. In various embodiments, suitable routes for this parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, supradural, intraventricular, intraurethral, ​​intrasternal, intracranial, intratumoral, intramuscular, and subcutaneous delivery. In one embodiment, parenteral administration includes needle (including microneedle) injection, needle-free injection, and infusion techniques.

[0548] In an illustrative aspect, parenteral preparations are generally aqueous solutions, which may contain carriers or excipients, such as salts, carbohydrates and buffers (preferably at a pH of 3 to 9), but may be more suitably formulated into sterile non-aqueous solutions or in dry form to be used in conjunction with suitable vehicles (such as sterile, pyrogen-free water or sterile saline). In other embodiments, any of the liquid preparations described herein may be suitable for parenteral administration as described herein. Standard pharmaceutical techniques well known to those skilled in the art can be used to easily achieve the preparation of sterile lyophilized powders for parenteral preparations via lyophilization under aseptic conditions. In one embodiment, the solubility of the compound or its pharmaceutically acceptable salt used in the preparation of parenteral preparations can be increased by using suitable formulation techniques (such as, incorporating solubility enhancing agents).

[0549] The amount of the compound or its pharmaceutically acceptable salt to be administered to the patient may vary significantly depending on the cancer being treated, the route of administration of the compound or its pharmaceutically acceptable salt, and tissue distribution. The amount to be administered to the patient may be based on body surface area, mass, and physician assessment. In various embodiments, the amount to be administered can range from, 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 4.0 mg, about 0.05 mg to about 3.0 mg, about 0.05 mg to about 2.0 mg, about 0.05 mg to about 1.0 mg, about 0.05 mg to about 0.5 mg, about 0.05 mg to about 0.4 mg, about 0.05 mg to about 0.3 mg, about 0.05 mg to about 0.1 mg, about 0.05 mg to about From about 0.1 mg to about 2 mg, from about 0.3 mg to about 10 mg, from about 0.1 mg to about 20 mg, or from about 0.8 to about 3 mg. One skilled in the art will readily appreciate that the dosage may vary within the various ranges provided above based on the factors noted above and may be at the physician's discretion.

[0550] In other embodiments, the dosage range of the compound or its pharmaceutically acceptable salt can be, for example, about 50 nmol / kg to about 3000 nmol / kg 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 500 nmol / kg, about 50 nmol / kg to about 400 nmol / kg, about 50 nmol / kg to about 300 nmol / kg, about 50 nmol / kg to about 200 nmol / kg, about 50 nmol / kg to about 100 nmol / kg, about 100 nmol / kg to about 300 nmol / kg, about 100 nmol / kg to about 5 ... nmol / kg, about 100 nmol / kg to about 1000 nmol / kg, about 100 nmol / kg to about 2000 nmol / kg patient body weight. In other embodiments, the dose can be 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 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, about nmol / kg or approximately 3000 nmol / kg patient body weight.

[0551] In various other embodiments, the dosage range of the compound or its pharmaceutically acceptable salt can be, for example, about 10 nmol / kg to about 10000 nmol / kg, about 10 nmol / kg to about 5000 nmol / kg, about 10 nmol / kg to about 3000 nmol / kg, about 10 nmol / kg to about 2500 nmol / kg, about 10 nmol / kg to about 2000 nmol / kg, about 10 nmol / kg to about 1000 nmol / kg, about 10 nmol / kg to about 900 nmol / kg, about 10 nmol / kg to about 800 nmol / kg, about 10 nmol / kg to about 700 nmol / kg, about 10 nmol / kg to about 600 nmol / kg, about 10 nmol / kg to about 500 nmol / kg, about 10 nmol / kg to about 400 nmol / kg, about 10 nmol / kg to about 3000 nmol / kg. nmol / kg, about 10 nmol / kg to about 200 nmol / kg, about 10 nmol / kg to about 150 nmol / kg, about 10 nmol / kg to about 100 nmol / kg, about 10 nmol / kg to about 90 nmol / kg, about 10 nmol / kg to about 80 nmol / kg, about 10 nmol / kg to about 70 nmol / kg, about 10 nmol / kg to about 60 nmol / kg, about 10 nmol / kg to about 50 nmol / kg, about 10 nmol / kg to about 40 nmol / kg, about 10 nmol / kg to about 30 nmol / kg, about 10 nmol / kg to about 20 nmol / kg, about 200 nmol / kg to about 900 nmol / kg, about 200 nmol / kg to about 800 nmol / kg, about 200 nmol / kg to about 700 nmol / kg, about 200 nmol / kg to about 600 nmol / kg, about 200 nmol / kg to about 500 nmol / kg, about 250 nmol / kg to about 600 nmol / kg, about 300 nmol / kg to about 600 nmol / kg, about 300 nmol / kg to about 500 nmol / kg, or about 400 nmol / kg to about 600 nmol / kg of patient body weight.In various other embodiments, the dosage range of the compound or its pharmaceutically acceptable salt can be, for example, from about 1 nmol / kg to about 10000 nmol / kg, from about 1 nmol / kg to about 5000 nmol / kg, from about 1 nmol / kg to about 3000 nmol / kg, from about 1 nmol / kg to about 2500 nmol / kg, from about 1 nmol / kg to about 2000 nmol / kg, from about 1 nmol / kg to about 1000 nmol / kg, from about 1 nmol / kg to about 900 nmol / kg, from about 1 nmol / kg to about 800 nmol / kg, from about 1 nmol / kg to about 700 nmol / kg, from about 1 nmol / kg to about 600 nmol / kg, from about 1 nmol / kg to about 500 nmol / kg, from about 1 nmol / kg to about 400 nmol / kg, from about 1 nmol / kg to about 300 nmol / kg, from about 1 nmol / kg to about 200 nmol / kg, from about 1 nmol / kg to about 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.In yet another embodiment, the dose can be 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 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 patient body weight. In other embodiments, the dosage can be from about 0.3 nmol / kg to about 1000 nmol / kg, from about 0.3 nmol / kg to about 900 nmol / kg, from about 0.3 nmol / kg to about 850 nmol / kg, from about 0.3 nmol / kg to about 800 nmol / kg, from about 0.3 nmol / kg to about 700 nmol / kg, from about 0.3 nmol / kg to about 600 nmol / kg, from about 0.3 nmol / kg to about 500 nmol / kg, from about 0.3 nmol / kg to about 400 nmol / kg, from about 0.3 nmol / kg to about 300 nmol / kg, from about 0.3 nmol / kg to about 200 nmol / kg, from about 0.3 nmol / kg to about 100 nmol / kg, from about 0.3 nmol / kg to about 50 nmol / kg, from about 0.3 nmol / kg to about 10 nmol / kg, or from about 0.3 nmol / kg to about 1 nmol / kg of patient body weight. In such embodiments, "kg" refers to the patient's body weight in kilograms. In one aspect, a single dose or multiple doses of a compound or a pharmaceutically acceptable salt thereof may be administered to a patient.

[0552] In another embodiment, between about 20 μg / kg and about 3 mg / kg of the patient's body weight, the compound or a pharmaceutically acceptable salt thereof may be administered to the patient. In another aspect, the amount may be from about 0.2 mg / kg to about 0.4 mg / kg of the patient's body weight, or may be about 50 μg / kg of the patient's body weight. In one aspect, a single dose or multiple doses of the compound or a pharmaceutically acceptable salt thereof may be administered to the patient.

[0553] In one embodiment, a small molecule ligand (compound) connected to a targeting portion may be administered to a patient before a CAR T cell composition comprising a CAR T cell (wherein the CAR T cell contains a CAR, and the CAR contains an E2 anti-fluorescein antibody fragment). In another embodiment, a small molecule ligand (bridge) connected to a targeting portion may be administered to a patient simultaneously with a CAR T cell composition comprising a CAR T cell (wherein the CAR T cell has a CAR, and the CAR contains an E2 anti-fluorescein antibody fragment), but in different preparations, or in the same preparation. In another embodiment, a small molecule ligand connected to a targeting portion may be administered to a patient after a CAR T cell composition comprising a CAR T cell (wherein the CAR T cell contains a CAR, and the CAR contains an E2 anti-fluorescein antibody fragment).

[0554] In one illustrative aspect, the time interval between administration of a CAR T cell containing a CAR comprising an E2 anti-fluorescein antibody fragment and a small molecule ligand (bridge) linked to a targeting moiety can vary widely depending on factors including the type of CAR T cells expressing an E2 anti-fluorescein antibody fragment being used, the binding affinity of the CAR containing the E2 anti-fluorescein antibody fragment, the characteristics of the targeting moiety and the small molecule ligand, the characteristics of the cancer, the location of the cancer in the patient, the method used to administer the CAR T cells expressing an E2 anti-fluorescein antibody fragment and the small molecule ligand linked to the targeting moiety to the patient, and the health, age and weight of the patient. In one aspect, the small molecule ligand linked to the targeting moiety can be administered before or after the CAR T cells, such as within about 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, or 51 hours, or within about 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10 or more days.

[0555] In one embodiment, any applicable dosing schedule known in the art can be used to administer the compound or a pharmaceutically acceptable salt thereof, or for a CAR T cell composition, wherein the CAR T cell composition comprises a CAR T cell containing a CAR, wherein the CAR comprises an E2 anti-fluorescein antibody fragment. For example, once-daily dosing (also referred to as qd), twice-daily dosing (also referred to as bid), three times-daily dosing (also referred to as tid), twice-weekly dosing (also referred to as BIW), three times-weekly dosing (also referred to as TIW), once-weekly dosing, etc. can be used. On the one hand, the dosing schedule selected for the compound or a pharmaceutically acceptable salt thereof, and the CAR T cell composition (wherein the CAR T cell composition comprises a CAR T cell containing a CAR, wherein the CAR comprises an E2 anti-fluorescein antibody fragment) can take into account the concentration of the compound or a pharmaceutically acceptable salt thereof, and the number of CAR T cells expressing the E2 anti-fluorescein antibody fragment administered to regulate the cytotoxicity of the CAR T cell composition (wherein the CAR T cell composition comprises a CAR T cell containing a CAR, wherein the CAR comprises an E2 anti-fluorescein antibody fragment) and to control CRS.

[0556] In one embodiment, to prevent or inhibit cytokine release syndrome (CRS) in patients, a method for treating cancer is provided. The method includes i) administering a compound or a pharmaceutically acceptable salt thereof to a patient, wherein the compound includes a small molecule ligand connected to a targeting portion via a linker, ii) administering a CAR T cell composition to the patient, wherein the CART cell composition comprises a CAR T cell, wherein the CAR T cell comprises a CAR, wherein the CAR comprises an E2 anti-fluorescein antibody fragment, and iii) administering a folate, a conjugate comprising a folate (wherein the conjugate comprising a folate does not include a targeting portion) or a drug that inhibits the activation of CAR T cells to the patient.

[0557] In this method embodiment, the step of administering a folate, a conjugate comprising a folate (wherein the conjugate comprising a folate does not include a targeting moiety) or a drug that inhibits the activation of CART cells expressing an E2 anti-fluorescein antibody fragment to the patient can be used to prevent or inhibit the patient's CRS. In this embodiment, any of the drugs that inhibit the activation of CAR T cells expressing an E2 anti-fluorescein antibody fragment, a folate, a conjugate comprising a folate (wherein the conjugate comprising a folate does not include a targeting moiety) or an E2 anti-fluorescein antibody fragment may be referred to herein as a "rescue agent". In one embodiment, a folate (such as folic acid) may be administered to prevent or inhibit the patient's CRS. In this embodiment, the folate inhibits the interaction of the bridge (ie, a small molecule ligand connected to the targeting moiety via a linker) with the receptor of the bridge on the tumor, inhibits tumor lysis and prevents or inhibits the patient's CRS.

[0558] In another embodiment, the rescue agent can reduce CRS as quickly as about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, or about 10 hours after administration of the rescue agent. For example, the grade of CRS can be reduced (e.g., grade 3 to grade 2, grade 4 to grade 3, etc.).

[0559] Columns 1 through 6 below (for levels 0, 1, 2, 3, 4, and 5) show an exemplary grading system:

[0560]

[0561] In one embodiment, the folate compound administered as an inhibitor of the binding of the bridge to the tumor can be, for example, folic acid, a folic acid analog, or another molecule that binds to the folic acid receptor. In various embodiments, the folic acid analogs that can be used include formyltetrahydrofolate, pterylpolyglutamate, and folic acid receptor binding pteridines, such as tetrahydropterin, dihydrofolate, tetrahydrofolate, and deaza and dideaza analogs thereof. The terms "deaza" and "dideaza" analogs refer to analogs recognized in the art that have carbon atoms that replace one or two nitrogen atoms in the naturally occurring folic acid structure. For example, deaza analogs include 1-deaza, 3-deaza, 5-deaza, 8-deaza, and 10-deaza analogs. Dideaza analogs include, for example, 1,5-dideaza, 5,10-dideaza, 8,10-dideaza, and 5,8-dideaza analogs. The above-mentioned folic acid analogs are generally referred to as "folate compounds", reflecting their ability to bind to folic acid receptors. Other folate receptor binding analogs include aminopterin, methotrexate, N10-methylfolate, 2-deamino-hydroxyfolate, deaza analogs (such as 1-deazamethotrexate or 3-deazamethotrexate), and 3',5'-dichloro-4-amino-4-deoxy-N10-methylpteroylglutamate (dichloromethotrexate).

[0562] In another embodiment, the folate compound administered as an inhibitor of the binding of the bridge to the tumor has the formula

[0563]

[0564] Where X 1 and Y 1 are each independently selected from halogen, R 2 , OR 2 , SR 3 and NR 4 R 5 ;

[0565] U, V and W represent divalent moieties, each independently selected from -(R 6a )C=、-N=、-(R 6a )C(R 7a )- and -N(R 4a )-; Q is selected from C and CH; T is selected from S, O, N and -C=C-;

[0566] X 2 and X 3 Each independently selected from the following: 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, wherein Z is oxygen or sulfur;

[0567] R 1 Selected from hydrogen, halogen, C1-C 12 Alkyl and C1-C 12 Alkoxy;

[0568] R 2 , R 3 , R 4 , R 4a , R 4b , R 5 , R5b , R 6b and R 7b Each independently selected from the following: hydrogen, halogen, C1-C 12 Alkyl, C1-C 12 Alkoxy, C1-C 12 Acyl, C1-C 12 Alkenyl, C1-C 12 Alkynyl, (C1-C 12 Alkoxy)carbonyl and (C1-C 12 alkylamino)carbonyl;

[0569] R 6 and R 7 are independently selected from hydrogen, halogen, C1-C 12 Alkyl and C1-C 12 Alkoxy; or R 6 With R 7 Together they form a carbonyl group;

[0570] R 6a and R 7a are independently selected from hydrogen, halogen, C1-C 12 Alkyl and C1-C 12 Alkoxy; or R 6a With R 7a Together they form a carbonyl group;

[0571] p, r, s and t are each independently 0 or 1; and

[0572] * indicates an optional covalent bond to the rest of the conjugate.

[0573] In yet another embodiment, the conjugate comprising a folate compound can be administered to prevent or inhibit cytokine release syndrome (CRS) in a patient.CRS is a well-known term in the art and this syndrome can cause adverse effects on the patient, including but not limited to weight loss, high fever, pulmonary edema, and a dangerous drop in blood pressure.

[0574] In this embodiment, the conjugate comprising a folate does not include a targeting moiety, and thus, the conjugate inhibits the interaction of the bridge with the tumor to prevent tumor lysis and reduce CRS in patients. In this embodiment, the folate moiety in the conjugate comprising a folate may include any of the folic acids described in the previous paragraphs linked to a chemical moiety (excluding a targeting moiety). On the one hand, the conjugate comprising a folate may include a folate linked to one or more amino acids (excluding a targeting moiety). Illustratively, the conjugate comprising a folate has the formula

[0575] .

[0576] This compound may also be referred to as "EC923". In such embodiments, the folate or conjugate comprising the folate may be administered to the patient in a molar excess relative to the bridge (i.e., the small molecule ligand connected to the targeting moiety via a linker) (such as a 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 the folate or conjugate comprising the folate relative to the small molecule ligand connected to the targeting moiety via a linker). The amount of folate or conjugate comprising the folate required to inhibit the interaction of the bridge with the tumor relative to the amount of the small molecule ligand connected to the targeting moiety via a linker can be determined by a skilled person.

[0577] In another embodiment, a rescue agent for inhibiting the activation of CAR T cells expressing E2 anti-fluorescein antibody fragments may be administered to the patient to inhibit CAR T cell activation and to inhibit or prevent the patient's CRS. On the one hand, the agent may be selected from the following: lymphocyte-specific protein tyrosine kinase inhibitors (e.g., dasatinib), PI3 kinase inhibitors (e.g., GDC0980), IL-2-induced T cell kinase inhibitors (e.g., BMS-509744), JAK inhibitors, BTK inhibitors, Tociluzumab, SIP agonists (e.g., Siponimod and Ozanimod) and blocking CAR T cells from binding to bridges, but not binding to cancer agents (e.g., fluorescein amine, FITC or sodium fluorescein). Technicians should understand that FITC (i.e., fluorescein) can be in the form of salts (e.g., sodium fluorescein) under physiological conditions, or in its non-salt form, or, for example, in a buffer at physiological pH. Thus, in one embodiment, when fluorescein is administered to a patient, it may be in equilibrium between its salt form (e.g., sodium fluorescein) and its non-salt form. In another embodiment, the rescue agent that inhibits the activation of CART cells may be a compound of the formula

[0578] .

[0579] In various embodiments, the rescue agent can be administered 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 any suitable 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. In other embodiments, the rescue agent can be administered at a dose of about .01 to about 300 μmol / kg patient body weight, about .06 to about 100 μmol / kg patient body weight, about .06 to about 90 μmol / kg patient body weight, about .06 to about 80 μmol / kg patient body weight, about .06 to about 70 μmol / kg patient body weight, about .06 to about 60 μmol / kg patient body weight, about .06 to about 50 μmol / kg patient body weight, about .06 to about 40 μmol / kg patient body weight, about .06 to about 30 μmol / kg patient body weight, about .06 to about 20 μmol / kg patient body weight, about .06 to about 10 μmol / kg patient body weight, about .06 to about 8 μmol / kg patient body weight, or about .06 to about 6 μmol / kg patient body weight.

[0580] In such embodiments, the rescue agent can be administered to the patient in a molar excess relative to the compound, or a pharmaceutically acceptable salt thereof (i.e., the small molecule ligand linked to the targeting moiety via 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 via a linker. The amount of rescue agent required to inhibit the interaction of the compound or a pharmaceutically acceptable salt thereof with tumors expressing E2 anti-fluorescein antibody fragments and / or CAR T cells relative to the amount of small molecule ligand attached to the targeting moiety via a linker can be determined by a skilled artisan.

[0581] In another embodiment, more than one dose of a folate, a conjugate comprising a folate (wherein the conjugate comprising a folate does not include a targeting moiety), or an agent that inhibits activation of CAR T cells expressing an E2 anti-fluorescein antibody fragment may be administered to the patient.

[0582] In the "rescue agent" embodiment described herein, a folate, a conjugate comprising a folate (wherein the conjugate comprising a folate does not include a targeting moiety), or an agent that inhibits activation of CAR T cells expressing an E2 anti-fluorescein antibody fragment may be administered to the patient before and / or after administration of the compound or a pharmaceutically acceptable salt thereof. On the other hand, the compound or a pharmaceutically acceptable salt thereof may be administered before and after administration of a folate, a conjugate comprising a folate (wherein the conjugate comprising a folate does not include a targeting moiety), or an agent that inhibits activation of CAR T cells expressing an E2 anti-fluorescein antibody fragment. In this embodiment, subsequent administration of the compound or a pharmaceutically acceptable salt thereof may result in activation of CAR T cells expressing an E2 anti-fluorescein antibody fragment and an increase in cytokine levels in the patient.

[0583] In another embodiment, administration of a folate, a conjugate comprising a folate (wherein the conjugate comprising a folate does not include a targeting moiety), or an agent that inhibits activation of CAR T cells expressing an E2 anti-fluorescein antibody fragment may result in a reduction in cytokine levels in the patient. In yet another embodiment, a reduction in cytokine levels may occur about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, or about 8 hours after administration of a folate, a conjugate comprising a folate (wherein the conjugate comprising a folate does not include a targeting moiety), or an agent that inhibits activation of CAR T cells expressing an E2 anti-fluorescein antibody fragment to the patient. In another embodiment, the reduction in cytokine levels is a reduction to nearly the level of cytokines in untreated patients.

[0584] In another illustrative embodiment, after administration of a folate, a conjugate comprising a folate (wherein the conjugate comprising a folate does not include a targeting moiety), or an agent that inhibits activation of CAR T cells expressing an E2 anti-fluorescein antibody fragment, the number of CAR T cells expressing an E2 anti-fluorescein antibody fragment in the patient's blood can be increased, even if cytokine levels in the patient are reduced. In another illustrative aspect, after administration of a folate, a conjugate comprising a folate (wherein the conjugate comprising a folate does not include a targeting moiety), or a rescue agent that inhibits activation of CAR T cells (wherein the CAR T cells include a CAR comprising an E2 anti-fluorescein antibody fragment), activation of CAR T cells expressing an E2 anti-fluorescein antibody fragment can be enhanced or maintained relative to a patient not treated with the rescue agent, even if cytokine levels in the treated patient are reduced. In yet another embodiment, the cancer comprises a tumor and when a folate, a conjugate comprising a folate (wherein the conjugate comprising a folate does not include a targeting moiety), or a rescue agent that inhibits activation of CAR T cells expressing an E2 anti-fluorescein antibody fragment is administered to the patient, the patient's tumor size does not increase. In this embodiment, a complete response to the tumor can be obtained.

[0585] In other embodiments, a rescue agent that inhibits activation of CAR T cells expressing an E2 anti-fluorescein antibody fragment is administered to the patient when the CRS grade reaches 1, 2, 3, or 4 or when the CRS grade reaches 3 or 4. In another aspect, when the rescue agent is administered, the patient's pulmonary edema is reduced.

[0586] In one embodiment described herein, a method for treating cancer is provided, and the method comprises i) continuously administering a compound or a pharmaceutically acceptable salt thereof to a patient, wherein the compound comprises a small molecule ligand connected to a targeting moiety via a linker, ii) administering a CAR T cell composition comprising CAR T cells to the patient, wherein the CAR T cells comprise a CAR comprising an E2 anti-fluorescein antibody fragment, and iii) terminating the continuous administration of the compound or a pharmaceutically acceptable salt thereof to inhibit or prevent cytokine release syndrome in the patient.

[0587] According to this embodiment, the term "continuously" may mean administering the compound or a pharmaceutically acceptable salt thereof to a patient for, for example, at least one hour, at least four hours, at least six hours, at least eight hours, at least ten hours, at least twelve hours, or at least twenty-four hours, or may mean a daily or weekly administration regimen, such as once a day, twice a day, three times a day, every other day, once a week, twice a week, three times a week, or any other suitable regimen deemed continuous by one skilled in the art. On the other hand, the term "continuously" may mean any combination of the embodiments described in this paragraph.

[0588] In this method embodiment, the step of "terminating continuous administration" of a compound or a pharmaceutically acceptable salt thereof to inhibit or prevent the patient's cytokine release syndrome may mean, for example, terminating administration after administration for a continuous period of time (such as hours or days), or terminating a treatment regimen (such as the above-mentioned daily or weekly regimen). In another embodiment, the step of "terminating continuous administration" may mean, for example, administration until an unacceptable loss of patient weight occurs, or until any other unacceptable side effects occur, such as high fever, decreased blood pressure, or pulmonary edema. In this embodiment, the step of "terminating continuous administration" of a compound or a pharmaceutically acceptable salt thereof does not mean monotherapy with a compound or a pharmaceutically acceptable salt thereof, without subsequent treatment with a compound or a pharmaceutically acceptable salt thereof. In this method embodiment, "to inhibit or prevent" cytokine release syndrome (CRS) means eliminating CRS or reducing or improving the symptoms of CRS.

[0589] In one embodiment of the embodiment involving terminating the continuous administration of a compound or a pharmaceutically acceptable salt thereof, the method may further include a step iv of re-administering the compound or a pharmaceutically acceptable salt thereof to the patient). In one embodiment, the compound or its pharmaceutically acceptable salt may be administered once a week (for example) and one dose may be omitted. In another embodiment, the compound or its pharmaceutically acceptable salt may be administered every other day (ie, every other day) and one or more (eg, two, three, four, etc.) doses may be omitted. In another embodiment, the compound or its pharmaceutically acceptable salt may be administered twice a week and one or more (eg, two, three, four, etc.) doses may be omitted. In another embodiment, the compound or its pharmaceutically acceptable salt may be administered on Monday, Tuesday, and the following Monday and then administration may be stopped for two weeks and the cycle may be repeated. In another embodiment, any one of the above-mentioned scheme embodiments may be used and one or more (eg, two, three, four, etc.) doses may be omitted. In such embodiments, the omitted dose may prevent or reduce the patient's CRS.

[0590] In another illustrative aspect, a method for treating cancer is provided. The method comprises i) administering a compound or a pharmaceutically acceptable salt thereof to a patient, wherein the compound comprises a small molecule ligand connected to a targeting moiety by a linker, wherein at least a first dose and a second dose of the compound or a pharmaceutically acceptable salt thereof are administered to the patient, wherein the first dose is different from the second dose, wherein the amount of the compound or a pharmaceutically acceptable salt thereof in the second dose is about 2 times to about 15,000 times greater than the amount of the compound or a pharmaceutically acceptable salt thereof in the first dose, and ii) administering a CAR T cell composition comprising a CAR T cell to the patient, wherein the CAR T cell comprises a CAR comprising an E2 anti-fluorescein antibody fragment.

[0591] In this embodiment, the dose of the compound or a pharmaceutically acceptable salt thereof can be increased stepwise to inhibit or prevent cytokine release syndrome in the patient. For example, at least a first dose and a second dose of the compound or a pharmaceutically acceptable salt thereof can be administered to the patient, wherein the first dose is different from the second dose, wherein the amount of the compound or a pharmaceutically acceptable salt thereof in the second dose is about 20 times to about 15,000 times, about 2 times to about 15,000 times greater than the amount of the compound or a pharmaceutically acceptable salt thereof in the first dose. In other embodiments, the amount of the second or subsequent dose may be about 2-fold to about 5-fold, about 2-fold to about 10-fold, about 2-fold to about 20-fold, about 2-fold to about 30-fold, about 2-fold to about 40-fold, about 2-fold to about 50-fold, about 2-fold to about 60-fold, about 2-fold to about 70-fold, about 2-fold to about 80-fold, about 2-fold to about 90-fold, about 2-fold to about 100-fold, about 2-fold to about 15000-fold, about 2-fold to about 10000-fold, about 5-fold to about 15000-fold, about 5 ... times to about 9000 times, about 5 times to about 8000 times, about 5 times to about 7000 times, about 5 times to about 6000 times, about 5 times to about 5000 times, about 5 times to about 4000 times, about 5 times to about 3000 times, about 5 times to about 4000 times, about 5 times to about 3000 times, about 5 times to about 2000 times, about 5 times to about 1000 times, about 5 times to about 750 times, about 2 times to about 750 times, about 5 times to about 500 times, about 5 times to about 100 times, about 800 times to about 1500 times. 0 times, about 800 times to about 10000 times, about 800 times to about 9000 times, about 800 times to about 8000 times, about 800 times to about 7000 times, about 800 times to about 6000 times, about 800 times to about 5000 times, about 800 times to about 4000 times, about 800 times to about 3000 times, about 800 times to about 2000 times, about 800 times to about 1000 times, about 8000 times to about 15000 times, about 8000 times to about 10000 times, about 8000 times To about 9000 times, about 15000 times, about 10000 times, about 9000 times, about 8000 times, about 7000 times, about 6000 times, about 5000 times, about 4000 times, about 3000 times, about 2000 times, about 1000 times, about 500 times, about 400 times, about 300 times, about 200 times, about 100 times, about 90 times, about 80 times, about 70 times, about 60 times, about 50 times, about 40 times, about 30 times, about 20 times, about 10 times, about 5 times, or about 2 times.

[0592] In another illustrative embodiment of the dose escalation method, at least a first dose, a second dose, and a third dose of a compound, or a pharmaceutically acceptable salt thereof, may be administered to a patient, wherein the first dose, the second dose, and the third dose are different, wherein the amount of the compound, or a pharmaceutically acceptable salt thereof, in the second dose is about 2-fold to about 750-fold greater than the amount of the compound, or a pharmaceutically acceptable salt thereof, in the first dose, and wherein the amount of the compound, or a pharmaceutically acceptable salt thereof, in the third dose is about 800-fold to about 10,000-fold greater than the amount of the compound, or a pharmaceutically acceptable salt thereof, in the first dose.

[0593] In another aspect of the dose escalation method, at least a first dose, a second dose, a third dose, and a fourth dose of a compound, or a pharmaceutically acceptable salt thereof, may be administered to a patient, wherein the first dose, the second dose, the third dose, and the fourth dose are different, wherein the amount of the compound, or a pharmaceutically acceptable salt thereof, in the second dose is about 2-fold to about 750-fold greater than the amount of the compound, or a pharmaceutically acceptable salt thereof, in the third dose is about 800-fold to about 7500-fold greater than the amount of the compound, or a pharmaceutically acceptable salt thereof, in the first dose, and wherein the amount of the compound, or a pharmaceutically acceptable salt thereof, in the fourth dose is about 8000-fold to about 15000-fold greater than the amount of the compound, or a pharmaceutically acceptable salt thereof, in the first dose.

[0594] In still another embodiment, the amount of the second dose of the compound or its pharmaceutically acceptable salt may be about 100 times greater than the amount of the first dose of the compound or its pharmaceutically acceptable salt, the amount of the third dose of the compound or its pharmaceutically acceptable salt may be about 1000 times greater than the amount of the first dose of the compound or its pharmaceutically acceptable salt, and the amount of the fourth dose of the compound or its pharmaceutically acceptable salt may be about 10000 times greater than the amount of the first dose of the compound or its pharmaceutically acceptable salt. In an exemplary embodiment, the first dose of the compound or its pharmaceutically acceptable salt is .05 nmol / kg, the second dose is 5 nmol / kg, the third dose is 50 nmol / kg and the fourth dose is 500nmol / kg. In the dose escalation embodiments described herein, the first, second, third, fourth and any subsequent doses of the compound or its pharmaceutically acceptable salt may be administered multiple times (e.g., the first dose of .05nmol / kg may be administered several times before administering subsequent escalating doses).

[0595] In another embodiment described herein, a method for treating cancer is provided. The method comprises i) administering to a patient a first dose of a compound or a pharmaceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand connected to a targeting moiety via a linker, ii) administering to the patient at least a second dose of the compound or a pharmaceutically acceptable salt thereof, wherein the amount of the second dose of the compound or a pharmaceutically acceptable salt thereof is at least about 50% lower than the first dose of the compound or a pharmaceutically acceptable salt thereof, and iii) administering to the patient a dose of a CAR T cell composition comprising CAR T cells, wherein the CAR T cells comprise a CAR comprising an E2 anti-fluorescein antibody fragment.

[0596] In various embodiments of this dose-decreasing embodiment, the amount of the compound, or a pharmaceutically acceptable salt thereof, in the second dose may be at least about 60% less than the amount of the compound, or a pharmaceutically acceptable salt thereof, in the first dose, at least about 70% less than the amount of the compound, or a pharmaceutically acceptable salt thereof, in the first dose, at least about 80% less than the amount of the compound, or a pharmaceutically acceptable salt thereof, in the first dose, at least about 90% less than the amount of the compound, or a pharmaceutically acceptable salt thereof, in the first dose, at least about 95% less than the amount of the compound, or a pharmaceutically acceptable salt thereof, in the first dose, at least about 96% less than the amount of the compound, or a pharmaceutically acceptable salt thereof, in the first dose, at least about 97% less than the amount of the compound, or a pharmaceutically acceptable salt thereof, in the first dose, at least about 98% less than the amount of the compound, or a pharmaceutically acceptable salt thereof, in the first dose, at least about 99% less than the amount of the compound, or a pharmaceutically acceptable salt thereof, in the first dose, or at least about 99.5% less than the amount of the compound, or a pharmaceutically acceptable salt thereof, in the first dose.

[0597] In various embodiments of the dose-decreasing embodiments described herein, the first dose of the compound or a pharmaceutically acceptable salt thereof can be about 100 nmol / kg to about 1000 nmol / kg patient body weight, about 100 nmol / kg to about 900 nmol / kg patient body weight, about 100 nmol / kg to about 800 nmol / kg patient body weight, about 100 nmol / kg to about 700 nmol / kg patient body weight, about 100 nmol / kg to about 600 nmol / kg patient body weight, about 200 nmol / kg to about 600 nmol / kg patient body weight, about 400 nmol / kg to about 600 nmol / kg patient body weight, or about 500 nmol / kg patient body weight.

[0598] In various embodiments of the dose-decreasing embodiments described herein, the second dose of the compound or a pharmaceutically acceptable salt thereof can be from about 0.5 nmol / kg to about 500 nmol / kg patient body weight, from about 0.5 nmol / kg to about 450 nmol / kg patient body weight, from about 0.5 nmol / kg to about 400 nmol / kg patient body weight, from about 0.5 nmol / kg to about 350 nmol / kg patient body weight, from about 0.5 nmol / kg to about 300 nmol / kg patient body weight, from about 1 nmol / kg to about 300 nmol / kg patient body weight, from about 2 nmol / kg to about 300 nmol / kg patient body weight, from about 2 nmol / kg to about 250 nmol / kg patient body weight, from about 5 nmol / kg to about 40 nmol / kg patient body weight, or from about 40 nmol / kg to about 150 nmol / kg patient body weight.

[0599] In additional embodiments of the dose reduction embodiments described herein, the method may further include administering a third dose of the compound or a pharmaceutically acceptable salt thereof, wherein the third dose of the compound or a pharmaceutically acceptable salt thereof is the same as the second dose of the compound or a pharmaceutically acceptable salt thereof. In another embodiment, the method may further include administering a fourth dose of the compound or a pharmaceutically acceptable salt thereof, wherein the fourth dose of the compound or a pharmaceutically acceptable salt thereof is the same as the second dose of the compound or a pharmaceutically acceptable salt thereof and the third dose of the compound or a pharmaceutically acceptable salt thereof. In another embodiment, the dose of the compound or a pharmaceutically acceptable salt thereof administered after the first dose of the compound or a pharmaceutically acceptable salt thereof may maintain inhibition of cancer growth relative to the first dose of the compound or a pharmaceutically acceptable salt thereof.

[0600] In other embodiments of the dose reduction embodiments described herein, the CAR T cells expressing the E2 anti-fluorescein antibody fragment can be administered at a dose of about 1 million CAR T cells expressing the E2 anti-fluorescein antibody fragment to about 40 million CAR T cells expressing the E2 anti-fluorescein antibody fragment. In still other embodiments, the dose of the compound or a pharmaceutically acceptable salt thereof administered after the first dose of the compound or a pharmaceutically acceptable salt thereof can be administered once or twice a week.

[0601] In still other embodiments of the dose reduction embodiment described herein, the method may also include administering to the patient a folate, a conjugate comprising a folate (wherein the conjugate comprising a folate does not include a targeting moiety) or a step of inhibiting the activation of CAR T cells expressing E2 anti-fluorescein antibody fragments. In another embodiment, the patient is administered an agent that inhibits the activation of CAR T cells expressing E2 anti-fluorescein antibody fragments and the agent is an agent that blocks the binding of CAR T cells expressing E2 anti-fluorescein antibody fragments to a compound or a pharmaceutically acceptable salt thereof, but does not bind to cancer, and the agent is fluorescein amine, sodium fluorescein or fluorescein. In another embodiment, the agent is sodium fluorescein.

[0602] In another embodiment, a method for treating cancer is provided. The method comprises i) administering a first dose of a compound or a pharmaceutically acceptable salt thereof to a patient, wherein the compound comprises a small molecule ligand connected to a targeting moiety by a linker and wherein the compound or a pharmaceutically acceptable salt thereof is administered to the patient at least about one hour before administering a CAR T cell composition comprising a CAR T cell (wherein the CAR T cell comprises a CAR comprising an E2 anti-fluorescein antibody fragment), ii) then administering a dose of a CAR T cell composition comprising a CAR T cell to the patient, wherein the CAR T cell comprises a CAR comprising an E2 anti-fluorescein antibody fragment, and iii) then administering a second dose of the compound or a pharmaceutically acceptable salt thereof to the patient. In various embodiments of this pretreatment embodiment, the first dose of the compound or a pharmaceutically acceptable salt thereof can be administered to the patient at least about two hours prior to administration of the CAR T cell composition, at least about four hours prior to administration of the CAR T cell composition, at least about eight hours prior to administration of the CAR T cell composition, at least about twelve hours prior to administration of the CAR T cell composition, at least about sixteen hours prior to administration of the CAR T cell composition, at least about twenty hours prior to administration of the CAR T cell composition, or at least about twenty-four hours prior to administration of the CAR T cell composition, in each case, wherein the CAR T cell composition comprises a CAR T cell comprising a CAR comprising an E2 anti-fluorescein antibody fragment.

[0603] In various embodiments of this pretreatment embodiment, a second dose of the compound or a pharmaceutically acceptable salt thereof can be administered to the patient at least about twenty-four hours after administration of the CAR T cell composition, at least about twenty hours after administration of the CAR T cell composition, at least about eighteen hours after administration of the CAR T cell composition, at least about sixteen hours after administration of the CAR T cell composition, at least about fourteen hours after administration of the CAR T cell composition, at least about twelve hours after administration of the CAR T cell composition, at least about ten hours after administration of the CAR T cell composition, at least about eight hours after administration of the CAR T cell composition, at least about six hours after administration of the CAR T cell composition, at least about four hours after administration of the CAR T cell composition, or at least about two hours after administration of the CAR T cell composition, in each case, wherein the CAR T cell composition comprises a CAR T cell comprising a CAR comprising an E2 anti-fluorescein antibody fragment.

[0604] In any of the embodiments described herein, wherein the folate is a ligand attached to a targeting moiety via a linker, the patient can be placed on a folate deficient diet, or folate can be administered to the patient in the diet, prior to treatment using the methods described herein. In embodiments where folic acid is administered to a patient, the dosage range can 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 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 1500 nmol / kg, from about 1000 nmol / kg to about 2000 nmol / kg, from about 1000 nmol / kg to about 3000 nmol / kg, from about 1000 nmol / kg to about 4000 nmol / kg, from about 1000 nmol / kg to about 5000 nmol / kg, from about 10000 nmol / kg to about 50 ... nmol / kg to about 500nmol / kg, about 100 nmol / kg to about 1000 nmol / kg, about 100 nmol / kg to about 2000 nmol / kg patient body weight. In other embodiments, dosage can be about 100 nmol / kg, about 150 nmol / kg, about 200 nmol / kg, about 250nmol / 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 patient body weight. In such embodiments, "kg" is kilograms of patient body weight. In one aspect, folic acid can be administered, for example, daily, weekly, biweekly, three times a week, or using any suitable regimen for administering folic acid.

[0605] In various embodiments described herein, the CAR T cells can maintain an increased number 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.

[0606] In various embodiments described herein, the half maximal effective concentration (EC 50 ) can be from about 1 pM to about 2 nM, from about 1 pM to about 5 nM, from about 1 pM to about 10 nM, from about 1 pM to about 20 nM, from about 1 pM to about 30 nM, from about 1 pM to about 40 nM, from about 1 pM to about 50 nM, from about 1 pM to about 60 nM, from about 1 pM to about 70 nM, from about 1 pM to about 80 nM, from about 1 pM to about 90 nM, from about 1 pM to about 100 nM, from about 1 pM to about 200 nM, from about 1 pM to about 300 nM, from about 1 pM to about 400 nM, from about 1 pM to about 500 nM, from about 1 pM to about 600 nM, from about 1 pM to about 700 nM, from about 1 pM to about 800 nM, from about 1 pM to about 90 nM, from about 1 pM to about 100 nM, from about 1 pM to about 200 nM, from about 1 pM to about 300 nM, from about 1 pM to about 400 nM, from about 1 pM to about 500 nM, from about 1 pM to about 600 nM, from about 1 pM to about 700 nM, from about 1 pM to about 800 nM, from about 1 pM to about 900 nM, pM to about 700 pM, about 1 pM to about 600 pM, about 1 pM to about 500 pM, about 1 pM to about 400 pM, about 1 pM to about 300 pM, about 1 pM to about 200 pM, about 1 pM to about 100 pM, 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.

[0607] In various embodiments described herein, the compound, or a pharmaceutically acceptable salt thereof, binds to CAR T cells with a Kd of about 1 nM to about 100 nM, about 1 nM to about 200 nM, about 1 nM to about 300 nM, about 1 nM to about 400 nM, about 1 nM to about 500 nM, about 1 nM to about 600 nM, about 1 nM to about 700 nM, about 1 nM to about 800 nM, about 1 nM to about 900 nM, about 100 nM to about 500 nM, about 100 nM to about 400 nM, about 100 nM to about 300 nM, about 100 nM to about 200 nM, about 100 nM to about 150 nM, or about 130 nM.

[0608] In various illustrative embodiments described herein, the compound or a pharmaceutically acceptable salt thereof can be administered to the patient for the first time about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 days before or after the CAR T cells, or on any suitable day before or after the CAR T cells.

[0609] In various embodiments described herein, EGFRt sorted or unsorted CAR T cells may be used. In another embodiment, a "clinical facsimile" batch of CAR T cells with a low differentiation spectrum may be used. In another embodiment, a "research batch" of CAR T cells may be used. The "clinical facsimile" batch (about 39% EGFRt+) may contain about 66% T SCM and about 32% T CM CD4+ subsets and about 95% T SCM and about 3% T CM The study batch (about 23% EGFRt+) may contain about 32% T SCM , about 53% T CM , about 11% T EM and about 3.7% T EFF CD4 subsets and about 44% T SCM , about 0.28% T CM , about 3.4% T EM and about 52% T EFF CD8 subset.

[0610] In various additional embodiments of this pretreatment embodiment, cytokine release that causes off-target toxicity in the patient does not occur, but toxicity to cancer by CAR T cells expressing E2 anti-fluorescein antibody fragments in the patient occurs or off-target tissue toxicity does not occur, but toxicity to cancer by CAR T cells expressing E2 anti-fluorescein antibody fragments occurs, or the cancer comprises a tumor, and the patient's tumor size is reduced, but off-target toxicity does not occur, or before administration of a CAR T cell composition comprising a CAR T cell (comprising a CAR comprising an E2 anti-fluorescein antibody fragment), the patient's tumor size is reduced more than a patient not pretreated with the compound or a pharmaceutically acceptable salt thereof. The skilled person will appreciate that the "target" may be a cancer (e.g., a tumor).

[0611] In another embodiment, a method for treating cancer is provided. The method comprises i) administering a compound or a pharmaceutically acceptable salt thereof to a patient, wherein the compound comprises a small molecule ligand connected to a targeting moiety via a linker, and ii) administering a CAR T cell composition to the patient, wherein the CAR T cell composition comprises a CAR T cell and wherein the CAR T cell comprises a CAR, wherein the CAR comprises an E2 anti-fluorescein antibody fragment, and wherein the small molecule ligand is a PSMA ligand and the targeting moiety is FITC. In this embodiment, the small molecule ligand connected to the targeting moiety via a linker may have the formula

[0612] .

[0613] In yet another embodiment, a method for treating cancer is provided. The method comprises i) administering a compound or a pharmaceutically acceptable salt thereof to a patient, wherein the compound comprises a small molecule ligand connected to a targeting moiety via a linker, and ii) administering a CAR T cell composition to the patient, wherein the CAR T cell composition comprises a CAR T cell and wherein the CAR T cell comprises a CAR comprising an E2 anti-fluorescein antibody fragment, and wherein the small molecule ligand is a CAIX ligand and the targeting moiety is FITC. In this embodiment, the small molecule ligand connected to the targeting moiety via a linker may have the formula

[0614] .

[0615] In yet another embodiment, a method for treating cancer is provided. The method comprises i) administering a first compound or a pharmaceutically acceptable salt thereof to a patient, wherein the first compound or a pharmaceutically acceptable salt thereof comprises a PSMA ligand linked to FITC via a linker, ii) administering a second compound or a pharmaceutically acceptable salt thereof to the patient, wherein the second compound or a pharmaceutically acceptable salt thereof comprises a CAIX ligand linked to FITC via a linker, and iii) administering a CAR T cell composition to the patient, wherein the CAR T cell composition comprises a CAR T cell and wherein the CAR T cell comprises a CAR comprising an E2 anti-fluorescein antibody fragment. In this embodiment, the first compound may have the formula

[0616]

[0617] and the second compound may have the formula

[0618] .

[0619] In one embodiment of the methods described herein, the cancer is imaged before the compound or a pharmaceutically acceptable salt thereof is administered to the patient, or before the CAR T cell composition is administered to the patient, wherein the CAR T cell composition comprises a CAR T cell, the CAR T cell comprising a CAR, and the CAR comprises an E2 anti-fluorescein antibody fragment. In an illustrative embodiment, imaging occurs via PET imaging. In other illustrative embodiments, imaging occurs via MRI imaging or SPECT / CT imaging. The imaging method may be any suitable imaging method known in the art. In one embodiment, the imaging method may involve the use of a small molecule ligand described herein, but the ligand is attached to an imaging agent suitable for the imaging type described herein.

[0620] In any of the embodiments described herein, cytokine release that causes off-target toxicity in the patient may not occur, even if toxicity to the cancer of the CAR T cells expressing the E2 anti-fluorescein antibody fragment occurs. In any of the embodiments described herein, off-target tissue toxicity in the patient may not occur, even if toxicity to the cancer of the CAR T cells expressing the E2 anti-fluorescein antibody fragment occurs. In any of the embodiments described herein, the cancer may include a tumor, and the patient's tumor size may be reduced, but off-target toxicity does not occur. In any of the embodiments described herein, CRS can be reduced or prevented and the method can result in a reduction in the patient's tumor volume. In any of the embodiments described herein, weight loss due to CRS can be reduced or prevented. In any of the embodiments described herein, the cancer may include a tumor and a complete response to the tumor can be obtained.

[0621] In another embodiment of the methods described herein, any of the methods described herein can be used alone, or any of the methods described herein can be used in combination with any other method or methods described herein. Example

[0622] Example 1

[0623] EC17 dose reduction reduces therapy-related toxicity

[0624] The effect of EC17 dose reduction on the anti-tumor activity and toxicity (body weight change) of CAR-T therapy was studied. Two different anti-fluorescein scFvs were used for CAR constructs ( Figure 1). The first construct of CAR contains the following domains: anti-FL (FITC-E2) scFv-IgG4 hinge (CH2 (L235D, N297Q)-CH3)-CD28 TM-4-1BB-CD3z-T2A-EGFRt. The second construct of CAR contains the following domains: anti-FL (FITC-4M5.3) scFv-IgG4 hinge (CH2 (L235D, N297Q)-CH3)-CD28 TM-4-1BB-CD3z-T2A-EGFRt. The nucleic acid sequence and amino acid sequence of the CAR of each construct are shown in SEQ ID NOS: 1 to 4. Human T cells are isolated, activated and transduced with a lentiviral vector carrying an appropriate CAR gene. CD4+ T cells with CAR modification and CD8+ T cells with CAR modification are isolated and cultured separately in vitro for about 7 days. CD4+ CAR-T cells and CD8+ CAR-T cells were mixed at a 1:1 ratio prior to iv administration for animal studies.

[0625] MDA-MB-231 tumors (150 to 250 mm 3 ) mice were used for in vivo studies to evaluate E2-CAR-T cells. In the first part of the study, 10 or 20 million E2-CAR-T cells were administered, and after CAR-T administration, 500 nmol / kg EC17 was intravenously administered weekly on days 2, 9, 16, 23, etc. (eg, Figure 2 Mice administered 20 million E2-CAR-T cells showed severe CRS after the second EC17 dose and had to be rescued with 6 μmol / kg sodium fluorescein (NaFL). Both the 10 million and 20 million CAR-T groups showed severe CRS after the third and fourth EC17 doses and had to be rescued with NaFL (as shown in Figure 5A and 5B Both groups showed severe weight loss (approximately 20%) during E2-CAR-T therapy, indicating that 10 or 20 million E2 CAR-T / EC17 500 nmol / kg SIW therapy had severe toxicity. (Although 20 million E2-CAR-T cells alone also showed anti-tumor activity ( Figure 5A , open circles), but this may be caused by allogeneic HLA-mismatched TCRs that are often found in “young” T cells that have been cultured for a short time in vitro).

[0626] In the second part of the study, the E2-CAR-T cells used were cultured in vitro for about 2 weeks. 30 million E2-CAR-T cells were administered and the EC17 dosing was modified to a decreasing dose. Figure 2As shown in B, 500 nmol / kg EC17 was administered 2 days after CAR-T administration, and then lower doses of EC17 (5, 20, or 100 nmol / kg) were administered weekly (on days 9, 16, 23, etc. after CAR-T administration). Figure 3A As shown in the results, all three EC17 reduction groups achieved complete responses, indicating that bridge reduction did not affect the anti-tumor activity of CAR-T. More importantly, none of the three EC17 reduction groups showed any severe CRS. Figure 3B As shown in the results of the study, all the decrement groups showed very mild weight loss despite the administration of more CAR-T cells (30 million) compared to the first part of the study (10 or 20 million CAR-T cells). Such data indicate that bridge decrement (a high-level loading dose followed by a reduced-level maintenance dose) can reduce toxicity while maintaining good anti-tumor activity of CAR-T cells.

[0627] A second construct, 4M5.3-CAR (anti-FL (FITC-4M5.3) scFv-IgG4 hinge (CH2 (L235D, N297Q)-CH3)-CD28 TM-4-1BB-CD3z-T2A-EGFRt), was also evaluated. 3 ) mice were used for in vivo studies to evaluate 4M5.3-CAR-T cells. In the first part of the study, 10 or 20 million 4M5.3-CAR-T cells were administered, and after CAR-T administration, 500 nmol / kg EC17 was intravenously administered weekly on days 2, 9, 16, 23, etc. (eg, Figure 2 Mice administered 10 or 20 million 4M5.3-CAR-T cells did not show severe CRS or weight loss after EC17 doses ( Figure 6B ), but all of their tumors shrank and eventually disappeared ( Fig. 6A ). 20 million CAR-T cells (upper line) showed better anti-tumor activity than 10 million CAR-T cells (midline).

[0628] In the second part of the study, the 4M5.3-CAR-T cells used were cultured in vitro for about 2 weeks. 30 million 4M5.3-CAR-T cells were administered and the EC17 dosing was modified to a decreasing dose. Figure 2 As shown in B, 500 nmol / kg EC17 was administered 2 days after CAR-T administration, and then lower doses of EC17 (5, 20, or 100 nmol / kg) were administered weekly (day 9, day 16, day 23, etc. after CAR-T administration). Figure 4AAs shown in , the 100 nmol / kg EC17 decrement group showed the best anti-tumor activity and all tumors were cured (bottom line), however, neither the 5 nmol / kg EC17 group (second line from the top) nor the 20 nmol / kg EC17 group (third line from the top) showed a complete response to CAR-T therapy. The data indicate that CAR-T activity is controlled by the EC-17 dose used for decrement. In addition, as Figure 4B As shown in , the weight loss of mice in the three decrement groups also depended on the EC17 dose. The 100 nmol / kg EC17 dose decrement group showed more weight loss than the other two groups using 5 or 20 nmol / lg EC17. Such data indicate that bridge decrement (a high-level loading dose followed by a reduced-level maintenance dose) can control both the anti-tumor activity and toxicity of CAR-T therapy.

[0629] The only difference between E2-CAR and 4M5.3-CAR is the scFv sequence used. By comparing the two CARs under the same conditions, when testing EC17 dose reduction (5, 20 or 100 nmol / kg), it was found that 10 million E2-CAR-T cells had better anti-tumor activity than 10 million 4M5.3-CAR-T cells; 30 million E2-CAR-T cells also showed better anti-tumor activity than 30 million 4M5.3-CAR-T cells.

[0630] As used herein, "SEQ ID NO: 4" means a sequence that starts with an underlined "gac" codon and ends with an underlined "ggc" codon. This portion of the longer sequence encodes an exemplary 4M5.3 CAR. The CAR is inserted into the T cell membrane. Other portions of the longer sequence include coding sequences for signal peptides, EGFRt domains, etc. that are not part of the CAR inserted into the membrane and function as a chimeric antigen receptor. As used herein, "SEQ ID NO: 3" means a sequence that starts with an underlined "D" and ends with an underlined "G". This portion of the longer sequence is an amino acid sequence of a CAR inserted into the T cell membrane. Other portions of the longer sequence include amino acid sequences of signal peptides, EGFRt domains, etc. that are not part of the CAR inserted into the membrane and function as a chimeric antigen receptor. In yet another embodiment, SEQ ID NO: 3 may include or consist of a humanized or human amino acid sequence. SEQ ID NO: 3 and 4 are as described above. The start and stop codons of the longer nucleic acid sequence are underlined and the longer sequence is an exemplary sequence that can be used to transduce T cells to make 4M5.3 CAR.

[0631] SEQ ID NO: 3 [4M5.3-CAR amino acid sequence (insert)]

[0632]

[0633]

[0634] SEQ ID NO: 4 [4M5.3-CAR nucleic acid sequence (insertion)]

[0635]

[0636]

[0637] Example 2

[0638] Characterization of CAR T cell differentiation phenotype prior to infusion into NSG mice

[0639] Multicolor flow cytometry was used to determine the phenotype of the CAR T cell product to be infused into tumor-bearing mice. Figure 7 The differentiation status of CD4+ and CD8+ subsets was analyzed using specific combinations of surface markers shown in A. Unsorted E2 clinical facsimile CAR T cell products were primarily made of the self-renewing Tscm / Tcm phenotype ( Figure 7 B) In Figure 7 In B, the highest bar is "Tscm" and the lower bar is "Tcm". Fig. 8A In addition to the differences in CD8:CD4 ratios shown in , the current E2 clinical facsimile CAR T cells differ from previously sorted E2 / 4M5.3 CAR-T cells and early GFP+ 4M5.3 CAR T in vivo research preparations (e.g. Figure 8B ). Fig. 8A In the figure, among the 10 groups from left to right, the bars represent the following - 1 (high bar Tscm, lower bar Tcm), 2 - (high bar Tscm, lower bar Tcm), 3 - (high bar Teff, lower bar Tem), 4 - (high bar Tem, lower bar Teff, lowest bar Tcm), 5 - (high bar Teff, lower bar Tem), 6 - (high bar Tem, lower bar Teff, lowest bar Tcm), 7 - (high bar Teff, lower bar Tscm), 8 - (high bar Teff, lower bar Tem), 9 - (high bar Teff, lower bar Tscm), 10 - (high bar Teff, lower bar Tsm, next lower bar Tem, lowest bar Tcm). Therefore, when compared with past CAR T preparations with a more differentiated Tem / Teff phenotype, CAR T cells with a predominant Tscm / Tcm phenotype are expected to have a better anti-tumor phenotype. For the same reasons, considering the same EC17 dosing regimen, sCRS occurs later for current CAR-T agents than for previous CAR-T agents.

[0640] Recovery of CAR T cells after cryopreservation

[0641] The cryopreserved CAR T cells were quickly thawed in a 37°C water bath and immediately placed in T cell recovery medium (TexMACS™ medium containing glutamine (Miltenyi Biotec No. 130-097-196) supplemented with 2% human AB serum) and 50 U / mL recombinant human IL2 and cultured in vitro for approximately 3 to 5 days for T cell recovery.

[0642] Flow cytometric analysis

[0643] CAR T cells were harvested from in vitro recovery culture medium and precipitated by centrifugation at 400g for 5 minutes. The T cells were then resuspended in flow cytometry staining solution [1% bovine serum albumin, 50mg / mL human IgG (Equitech Bio, catalog number SLH56-0001), phosphate buffered saline containing 0.9% sodium azide, pH=7.4] supplemented with anti-mouse FcγIII / II receptor (CD16 / CD32) blocks [clone 2.4G2; BD Bioscience, catalog number 553142 diluted 1:100 (v / v)] and anti-human Fc blocks [BD Biosciences, catalog number 564220 diluted 1:50 (v / v)]. Surface marker staining was performed using the following fluorochrome-conjugated monoclonal antibodies added to each sample on ice in the dark (addition over 20 minutes): anti-human CD45RA-APCeF780 [clone HI100, ThermoFisher No. 47-0458-42 diluted 1:20 (v / v)], anti-human CD45RO-eF450 [clone UCHL1, Thermofisher No. 48-0457-42 diluted 1:20 (v / v)], anti-human CD8α-PECy7 [clone RPA-T8, BD Bioscience, catalog number 557746 diluted 1:20 (v / v)], anti-human CD4-Percpe710 [clone SK3, eBioscience catalog number 46-0047-42 diluted 1:20 (v / v)], biotinylated anti-human EGFR (Cetuximab; R&D Biosciences, Inc., MD Anderson Cancer Center, MD Anderson Cancer Center, 101006). Systems No. FAB9577B-100 at 1:10 (v / v) followed by PE-conjugated anti-biotin (BioLegend No. 53-9895-82 at 1:400 (v / v)). After surface marker staining, T cells were washed with PBS and resuspended in cold PBS containing 3 μM propidium iodide and transferred to flow cytometry collection tubes. Flow cytometry data were collected on a Gallios flow cytometer (Beckman Coulter, Brea, CA), with a minimum of 20,000 events collected and data analyzed using Kaluza v1.5 software.

[0644] Example 3

[0645] E2 anti-FITC antibody does not bind to any normal human tissue

[0646] E2 anti-FITC IgG antibodies with variable fragment sequences identical to those in the E2 anti-FITC CAR construct were used to test whether the E2 antibody had any binding to normal human tissue. Recombinant human E2 anti-FITC IgG1 λ1 was expressed in HEK293 cells and purified using a protein A affinity column. The purity of the antibody exceeded 98% using SEC-HPLC analysis. The antibody was labeled with digoxigenin (DIG) using the Mix-N-Stain DIG antibody labeling kit (Biotium). The binding affinity of the DIG-labeled E2 antibody to FITC immobilized beads was measured by FACS. At room temperature, various concentrations of DIG-E2 IgG were incubated with FITC immobilized beads for 30 minutes. After washing, monoclonal anti-DIG Ab was added and incubated with beads for 30 minutes. Anti-mouse secondary antibodies conjugated to eF660 were added and incubated with washed beads. For eF660 signals, washed beads were analyzed via FACS. PE immobilized beads were used as negative control beads. The fluorescence intensity of eF660 was plotted against the concentration of added DIG-labeled E2-IgG and is shown in Figure 9. The binding affinity of DIG-labeled E2 antibody to FITC was calculated using PRISM software. Fig. 9B As shown in the insert, the Kd is about 0.66 nM, indicating that the DIG-labeled E2 anti-FITC antibody has a high binding affinity to FITC and can be used to evaluate E2 binding to normal human tissues.

[0647] An IHC assay was developed to test the binding of the E2 antibody to human tissues. Formalin-fixed paraffin-embedded (FFEP) tissue sections containing agar blocks of FITC-labeled KB cells were used as positive controls, while FFPE tissue sections of unlabeled KB cells were used as negative controls. The tissue sections were deparaffinized and rehydrated, and then antigen retrieval was performed by incubating the tissue sections with antigen retrieval buffer (pH 6.0) at 91°C for 24 minutes, and then DIG-E2 antibody was incubated with the rehydrated tissue sections and finally anti-DIG IHC staining was performed. Briefly, monoclonal anti-DIG antibody was added and incubated, followed by oxidase-labeled anti-mouse secondary antibody. As Fig. 10A As shown in , FITC-labeled KB cells showed positive staining, indicating that the E2 antibody binds to FITC on these labeled KB cells. Unlabeled KB cells did not show any staining ( Fig. 10B ).

[0648] The DIG-labeled E2 antibody was incubated with a human multi-organ normal tissue microarray (FDA999, US Biomax, Inc) and the binding of the E2 antibody to human normal tissues was evaluated using the same IHC procedure using the same conditions as above. This multi-organ normal tissue microarray has 99 cores with 28 normal human organs, including adrenal gland (FIG. 11), bone marrow (FIG. 12), breast (FIG. 13), cerebellum (FIG. 14), cervix (FIG. 15), colon (FIG. 16), esophagus (FIG. 17), eye (FIG. 18), heart (FIG. 19), pituitary gland (FIG. 20), kidney (FIG. 21), larynx (FIG. 22), liver (FIG. 24), lung (FIG. 25), lymph node (FIG. 26), nerve (FIG. 27), ovary (FIG. 28), pancreas (FIG. 29), prostate (FIG. 30), skin (FIG. 31), small intestine (FIG. 32), spleen (FIG. 23), stomach (FIG. 33), striated muscle (FIG. 34), testis (FIG. 35), thymus (FIG. 36), tongue (FIG. 37), and uterus (FIG. 38). Organs from at least 3 normal human individuals were included in the microarray panel. As shown in Figures 11 to 38, no positive IHC staining was found in any of the 28 normal human organs tested, indicating that the E2 anti-FITC antibody does not have any binding to normal human organ tissues. Some plasma cells showed a certain level of staining intensity in both the test sections pre-incubated with the DIG-E2 antibody and the negative control sections not pre-incubated with the DIG-E2 antibody, indicating that this weak staining was non-specific.

[0649] In conclusion, the E2 anti-FITC antibody did not bind to any human normal organ tissue tested, indicating that the E2 anti-FITC CAR-T cells themselves should not bind to and attack any human normal tissue in vivo.

[0650] Example 4

[0651] Synthesis of FITC-Folic Acid

[0652]

[0653] In anhydrous dimethyl sulfoxide (DMF), in the presence of tetramethylguanidine and diisopropylamine, folate-γ-ethylenediamine was coupled to fluorescein isothiocyanate (FITC) isomer I (Sigma-Aldrich). The crude product was loaded on 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 minutes at a flow rate of 20 mL / min). Under such conditions, the main peak of FITC-folate is usually eluted at 27 to 50 minutes. The quality of the FITC-folate eluate was monitored by analytical reverse phase HPLC with a UV detector. The fractions with a purity greater than 98.0% (LCMS) were lyophilized to obtain the final FITC-folate product. As known in the art, compounds with this structure are also referred to as EC17.

[0654] Example 5

[0655] Synthesis of FITC-PEG12-folic acid

[0656]

[0657] The universal polyethylene glycol (PEG) Nova Tag TM The resin (0.2 g) was loaded into the peptide synthesis vessel and precipitated with isopropanol ( i -PrOH) (3 x 10 mL) and dimethylformamide (DMF, 3 x 10 mL) were used for washing. 9-Fluorenylmethoxycarbonyl (Fmoc) deprotection was performed using 20% ​​piperidine in DMF (3 x10 mL). Kaiser test was performed to evaluate the progress of the reaction. A DMF solution containing Fmoc-L-glutamic acid 5-tert-butyl ester (Fmoc-Glu-(Ot-Bu)-OH) (23.5 mg), N,N-diisopropylethylamine ( i -Pr2NEt) (4 equivalents), and benzotriazol-1-yl-oxytripyrrolidinephosphine hexafluorophosphate (PyBOP) (2 equivalents). Fmoc deprotection was performed using DMF containing 20% ​​piperidine (3 x 10 mL). Then, N 10 -TFA-Pte-OH (22.5 mg), DMF, i -Pr2NEt (4 eq.) and PyBOP (2 eq.) were prepared. Argon was bubbled for 2 h, and the resin was washed with DMF (3 x 3 mL) and i-PrOH (3 x 3 mL). After swelling the resin in dichloromethane (DCM), a solution of 1 M hydroxybenzotriazole (HOBT) in DCM / trifluoroethane (TFE) (1:1) (2 x 3 mL) was added. Argon was bubbled for 1 hour, the solvent was removed, and the resin was washed with DMF (3 x 3 mL) and i -PrOH (3 x 3 mL). After the resin was swollen in DMF, Fmoc-NH-(PEG) 12 -COOH (46.3 mg) in DMF, i -Pr2NEt (4 eq.) and PyBOP (2 eq.). Argon was bubbled for 2 h, and the resin was washed with DMF (3 x 3 mL) and i -PrOH (3 x 3 mL) was used for washing. Fmoc deprotection was performed using 20% ​​piperidine in DMF (3 x 10 mL). A Kaiser test was performed to assess the progress of the reaction. FITC (Life Technologies 21.4 mg) was then introduced into the vessel in DMF and i -Pr2NEt (4 equiv.) was added, then argon was bubbled for 2 h, and the resin was washed with DMF (3 x 3 mL) and i -PrOH (3 x 3 mL) was used for washing. Then DMF (2 x 2 mL) containing 2% NHNH was added to the container. The final compound was cleaved from the resin and concentrated under vacuum using TFA:H2O:triisopropylsilane (TIS) (95:2.5:2.5) (lysate). The concentrated product was precipitated in Et2O and dried under vacuum. 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 in 30 minutes at 13 mL / min). The pure fractions were pooled and lyophilized to provide FITC-PEG12-folate.

[0658] Example 6

[0659] Synthesis of FITC-PEG20-folic acid

[0660]

[0661] Ethylenediamine, polymer bound (200-400 mesh) resin (50 mg) was loaded into a peptide synthesis vessel and swollen with DCM (3 mL) followed by DMF (3 mL). Fmoc-PEG in DMF was then introduced into the vessel. 20 -COOH solution (131 mg, 1.0 equivalent), i-Pr2NEt (6.0 equiv.) and PyBOP (4.0 equiv.). Argon was bubbled for 6 h, the coupling solution was evacuated, and the resin was washed with DMF (3 x 10 mL) and i -PrOH (3 x 10 mL). Kaiser test was performed to assess the progress of the reaction. Before coupling each amino acid, Fmoc deprotection was performed using 20% ​​piperidine in DMF (3 x 10 mL). The above sequence was repeated to complete the reaction with Fmoc-Glu-OtBu (72 mg, 2.0 equiv) and Tfa. tetral acid (41 mg, 1.2 equiv) coupling step. The resin was washed with 2% hydrazine in DMF 3 x 10 mL (5 min) to cleave the trifluoroacetyl protecting group on the tetral acid and i -PrOH (3 x 10 mL) followed by DMF (3 x 10 mL) wash. The resin was dried under argon for 30 minutes. The folic acid-peptide was cleaved from the resin using a cleavage solution. 10 mL of cleavage mixture was introduced and argon was bubbled for 1.5 hours. The cleavage mixture was drained into a clean flask. The resin was washed 3 times with more cleavage mixture. The combined mixture was concentrated under reduced pressure to a smaller volume (approximately 5 mL) and precipitated in diethyl ether.

[0662] The precipitate was collected by centrifugation, washed with ether (3 times) and dried under high vacuum. 20 -EDA (1.0 eq.) was treated with DMSO and DIPEA containing FITC (50 mg, 1.5 eq.). The reaction progress was monitored by LCMS. After 8 hours, the starting material was consumed to give the product. The crude reaction mixture was purified by preparative HPLC (mobile phase A = 10 mM ammonium acetate, pH = 7; organic phase B = acetonitrile; method: 0% B to 30% B in 35 minutes at 13 mL / min) and FITC-PEG20-folate was obtained in 60% yield.

[0663] Example 7

[0664] Synthesis of FITC-PEG108-folic acid

[0665]

[0666] Ethylenediamine, polymer bound (200-400 mesh) resin (50 mg) was loaded into a peptide synthesis vessel and swollen with DCM (3 mL) followed by DMF (3 mL). Fmoc-PEG in DMF was then introduced into the vessel. 36 -COOH solution (161 mg, 1.0 equivalent), i-Pr2NEt (6.0 equiv.) and PyBOP (4.0 equiv.). Argon was bubbled for 6 h, the coupling solution was evacuated, and the resin was washed with DMF (3 x 10 mL) and i -PrOH (3 x 10 mL). Kaiser test was performed to assess the reaction progress. Before coupling each amino acid, Fmoc deprotection was performed using 20% ​​piperidine in DMF (3 x 10 mL). The above sequence was repeated to complete the reaction with 2X Fmoc-PEG 36 -COOH (161 mg, 1.0 equiv), Fmoc-Glu-OtBu (72 mg, 2.0 equiv) and Tfa.Pteranoic acid (41 mg, 1.2 equiv) coupling step. Finally, the resin was washed with 2% hydrazine in DMF 3 x 10 mL (5 min) to cleave the trifluoroacetyl protecting group on the pteranoic acid and to remove the protective group. i -PrOH (3 x 10 mL) followed by DMF (3 x10 mL) wash. The resin was dried under argon for 30 minutes. The folic acid-peptide was cleaved from the resin using a cleavage solution. 10 mL of the cleavage mixture was introduced and argon was bubbled for 1.5 hours. The cleavage mixture was drained into a clean flask. The resin was washed 3X with more cleavage solution. The combined mixture was concentrated under reduced pressure to a smaller volume (approximately 5 mL) and precipitated in diethyl ether.

[0667] The precipitate was collected by centrifugation, washed with ether (3X) and dried under high vacuum. 108 -EDA (1.0 eq.) was treated with DMSO and DIPEA containing FITC (50 mg, 1.5 eq.). 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 in 35 minutes at 13 mL / min) and FITC-PEG108-folate was obtained in 64% yield.

[0668] Example 8

[0669] Synthesis of FITC-DUPA

[0670]

[0671] DUPA-FITC was synthesized via solid phase method 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 for 5 min. The resin was swelled with DMF (3 × 3 mL) and isopropanol ( i -PrOH, 3 × 3 mL). After the resin was swollen in DMF, a mixture containing DUPA-(OtBu)-OH (1.5 eq.), HATU (2.5 eq.) and i -Pr2NEt (4.0 equiv) in DMF. Argon was bubbled for 2 h, and the resin was washed with DMF (3 × 3 mL) and i -PrOH (3 × 3 mL). After the resin was swollen in DCM, a solution of 1 M HOBt in DCM / TFE (1:1) (2 × 3 mL) was added. Argon was bubbled for 1 h, the solvent was removed and the resin was washed with DMF (3 × 3 mL) and i =The resin was washed with i-PrOH (3 × 3 mL). After swelling of the resin in DMF, a DMF solution containing Fmoc-Phe-OH (2.5 equiv), HATU (2.5 equiv) and DIPEA (4.0 equiv) was added. Argon was bubbled for 2 h, and the resin was washed with DMF (3 × 3 mL) and i-PrOH (3 × 3 mL). The above sequence was repeated for 2 additional coupling steps to add 8-aminooctanoic acid and fluorescein isothiocyanate or rhodamine B isothiocyanate. The final compound was cleaved from the resin using cleavage solution and concentrated under vacuum. The concentrated product was precipitated in diethyl ether and dried under vacuum. The crude product was purified using preparative RP-HPLC [λ = 488 nm; solvent gradient: 1% B to 80% B in 25 min, 80% B wash for 30 min run; A = 10 mM NH4OAc, pH = 7; B = acetonitrile (ACN)]. ACN was removed under vacuum, and the purified fractions were lyophilized to give FITC-DUPA as a tan solid. RP-HPLC: tR = 8.0 min (A = 10 mM NH4OAc, pH = 7.0; B = ACN, solvent gradient: 1% B to 50% B in 10 min, 80% B wash 15 min run). 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) + For C 51 H 59 N7O 15 S calculated value 1040.3712, measured value 1040.3702. UV / vis: λ max = 491nm.

[0672] Example 9

[0673] Synthesis of FITC-PEG12-DUPA

[0674]

[0675] 1,2-Diaminoethane trityl resin (0.025 g) was loaded into the peptide synthesis vessel and i -PrOH (3 x 10 mL), followed by DMF (3 x 10 mL) washing. Then, into the vessel was introduced a solution containing Fmoc-NH-(PEG) 12 -COOH (42.8 mg) in DMF, i -Pr2NEt (2.5 eq.) and PyBOP (2.5 eq.). The resulting solution was bubbled with Ar for 1 h, the coupling solution was drained, and the resin was treated with DMF (3 x 10 mL) and i -PrOH (3 x 10 mL). A Kaiser test was performed to assess the reaction progress. Fmoc deprotection was performed using 20% ​​piperidine in DMF (3 x 10 mL). This procedure was repeated to complete all coupling steps (2 x 1.5 equivalents of Fmoc-Phe-OH and 1.5 equivalents of 8-aminooctanoic acid and 1.2 equivalents of DUPA were used in their respective coupling steps). After DUPA coupling, the resin was washed with DMF (3 x 10 mL) and i-PrOH (3 x 10 mL) and dried under reduced pressure. The peptide was cleaved from the resin in the peptide synthesis vessel using the cleavage solution. 15 mL of the cleavage solution was added to the peptide synthesis vessel, and the reaction was bubbled under Ar for 15 minutes. The resin was treated with two additional 10 mL amounts of the cleavage solution for 5 minutes each. The cleavage mixture was concentrated to approximately 5 mL and precipitated with diethyl ether. The precipitate was collected by centrifugation, washed with diethyl ether (3X), and dried under high vacuum to produce a recovery of crude material. At room temperature, a stirred 4% tantalum ether containing crude DUPA-(PEG) 12 -EDA (10 mg) and FITC (5.6 mg) in dimethyl sulfoxide (DMSO, 1 mL) were added i -Pr2NEt (5 eq.) and stirred under argon for 6 h. The reaction was monitored by LCMS and purified by preparative HPLC (mobile phase: A = 10 mM ammonium acetate pH = 7, B = ACN; method: 0% B to 50% B in 30 min at 13 mL / min). The purified fractions were pooled and lyophilized to give FITC-PEG12-DUPA.

[0676] Example 10

[0677] Synthesis of FITC-PEG11-NK1

[0678]

[0679] At room temperature, under argon, to a stirred solution of NK-1 (0.02 g, 0.0433 mmol, 1.0 eq.), O -(2-aminoethyl)- O '-[2-(Boc-amino)ethyl]decaethylene glycol (BocNH-PEG 11 -NH2) (Sigma, 0.0336 g, 0.0521 mmol, 1.2 eq.), benzotriazol-1-yl-oxytripyrrolidinylphosphine hexafluorophosphate (PyBOP) (0.027 g, 0.0521 mmol, 1.2 eq.) were added to the solution. N , N - diisopropylethylamine (DIPEA) (0.076 mL, 0.4338 mmol, 10 eq.). The reaction progress was monitored by LCMS and the reaction was analyzed by preparative RP-HPLC (Waters, XBridge TMPrep C18, 5 μm; 19 × 100 mm column, mobile phase A = 20 mM ammonium acetate buffer, pH 7, B = acetonitrile, gradient 10 to 100% B in 30 min, 13 mL / min, λ = 220 nm, 254 nm). Collect the pure fractions, evaporate all organic solvents and lyophilize the sample for 48 hours to obtain NK1-PEG 11 -NHBoc. Yield: 40.13 mg (97%). 11 -NHBoc (0.0165 g, 0.015 mmol) in dry DCM was added trifluoroacetic acid (TFA, 20 eq.) and the reaction mixture was stirred at room temperature for 4 hours. Excess TFA was removed, and the remaining solution was diluted with water and extracted with CH2Cl2 (3 x 5 mL). The combined organic layers were washed with brine, dried (Na2SO4) and concentrated. The obtained residue was dried under vacuum and used in the next step without further purification. At room temperature, under argon, the stirred NK1-PEG in dry dimethyl sulfoxide (DMSO, 0.3 mL) was added to 4% MgCl2 (0.0165 g, 0.015 mmol). 11 A solution of -NH2 (0.008 g, 0.0081 mmol, 1.0 eq.) and fluorescein isothiocyanate (FITC) (Sigma, 0.0037 g, 0.0097 mmol, 1.2 eq.) was added to diisopropylethylamine (0.0028 mL, 0.0162 mmol, 2.0 eq.). The reaction progress was monitored by LCMS and the product was purified by preparative RP-HPLC (Waters, XBridge TM 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 in 30 min, 13 mL / min, λ = 280 nm). The pure fractions were collected, all organic solvents were evaporated and the sample was lyophilized for 48 hours to give 8.54 mg (77%) yield of FITC-PEG11-NK1.

[0680] *Note: The NK-1 compound was synthesized via a two-step procedure starting from a basic ligand, which was prepared using a procedure from the literature. (Reference: DESIGN AND DEVELOPMENT OF NEUROKININ-1 RECEPTOR-BINDING AGENT DELIVERY CONJUGATES, Application No.: PCT / US2015 / 44229; which is incorporated herein by reference).

[0681] Embodiment 11

[0682] Synthesis of FITC-PEG2-CA9

[0683]

[0684] In a 50 mL round-bottom flask, CA9 ligand (53.6 mg) was dissolved in DMF (2 to 3 mL) using a Teflon magnetic stirring bar. Ambient air was removed using vacuum and replaced with nitrogen, which was completed in three cycles. The round-bottom flask was kept under constant nitrogen. 28.9 mg N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) was added to the flask, followed by 21.6 mg 1-hydroxybenzotriazole hydrate (HOBt) and 18.9 μL Boc-PEG2-NH2 (SigmaAldrich). 5.4 μL triethylamine (TEA) was added and the reaction was stirred overnight. The reaction mixture was purified using HPLC and confirmed with 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. TFA / DCM was removed using high vacuum rotary evaporation, followed by 30 minutes under high vacuum. The compound was then dissolved in DMF and mixed with 5 molar equivalents of i -Pr2Net, 16 mg fluorescein isothiocyanate (Life Technologies) were combined and stirred for 1 hour. The reaction mixture was purified by HPLC and the target compound was confirmed by UHPLC-MS (target m / z 1120). The sample was lyophilized and stored at -20°C.

[0685] Example 12

[0686] FITC-DUPA

[0687] DUPA-FITC was synthesized via a solid phase method as follows. General Nova Tag resin (50 mg, 0.53 mM) was swollen with dichloromethane (DCM) (3 mL) followed by dimethylformamide (DMF, 3 mL). A solution of 20% piperidine in DMF (3 × 3 mL) was added to the resin and argon was bubbled for 5 minutes. The resin was washed with DMF (3 × 3 mL) and isopropanol ( i -PrOH, 3 × 3 mL). After the resin was swollen in DMF, a DMF solution containing DUPA-(OtBu)-OH (1.5 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 × 3 mL) and i-PrOH (3 × 3 mL). After the resin was swollen in DCM, a solution of 1 M HOBt in DCM / trifluoroethane (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 × 3 mL) and i The resin was washed with i-PrOH (3× 3 mL). After swelling of the resin in DMF, a DMF solution containing Fmoc-Phe-OH (2.5 equiv.), HATU (2.5 equiv.) and DIPEA (4.0 equiv.) was added. Argon was bubbled for 2 h, and the resin was washed with DMF (3× 3 mL) and i-PrOH (3× 3 mL). The above sequence of steps for 2 additional couplings was repeated to add 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 mixture (95:2.5:2.5) and concentrated under vacuum. The concentrated product was precipitated in diethyl ether and dried under vacuum. The crude product was purified using preparative RP-HPLC [λ = 488 nm; solvent gradient: 1% B to 80% B in 25 minutes, 80% B wash for 30 minutes run; A = 10 mM NH4OAc, pH = 7; B = acetonitrile (ACN)]. The ACN was removed under vacuum, and the pure fractions were lyophilized to give DUPA-FITC as a tan solid. RP-HPLC: tR = 8.0 minutes (A = 10 mM NH4OAc, pH = 7.0; B = ACN, solvent gradient: 1% B to 50% B in 10 minutes, 80% B wash for 15 minutes run). 1 H 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) + For C 51 H 59 N7O 15S calculated value 1040.3712, measured value 1040.3702. UV / vis: λ max = 491 nm.

[0688]

[0689] Embodiment 13

[0690] FITC-CA9

[0691] In a 50 mL round bottom flask, CA9 ligand (53.6 mg, synthesized in the laboratory) was dissolved in the required amount of N,N-dimethylformamide (DMF) (2 to 3 mL) using a Teflon magnetic stirring bar. Ambient air was removed using vacuum and replaced with nitrogen, which was completed in three cycles. The round bottom flask was then kept under constant nitrogen. 28.9 mg N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) was added to the flask, followed by 21.6 mg 1-hydroxybenzotriazole hydrate (HOBt) and 18.9 μL Boc-PEG2-NH2 (purchased from Sigma Aldrich). Finally, 5.4 μL triethylamine (TEA) was added and the reaction was allowed to stir overnight. The reaction mixture was purified using HPLC and confirmed with UHPLC-MS (target m / z 831). Acetonitrile was removed using high vacuum rotary evaporation and placed on a freeze dryer for 48 hours. Deprotection of Boc was performed using 1:1 TFA:DCM for 30 minutes. TFA / DCM was removed using high vacuum rotary evaporation, followed by 30 minutes under high vacuum. 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 hour. The reaction mixture was purified via HPLC and the target compound was confirmed by UHPLC-MS (target m / z 1120). The sample was placed on a freeze dryer for 48 hours and stored at -20°C.

[0692]

[0693] Embodiment 14

[0694] FITC-NK1R

[0695] At room temperature, under argon, a stirred solution of NK-1 (0.02 g, 0.0433 mmol, 1.0 eq.), O-(2-aminoethyl)-O'-[2-(Boc-amino)ethyl]decaglycol (BocNH-PEG 11-NH2) (Sigma, 0.0336 g, 0.0521 mmol, 1.2 eq.), benzotriazol-1-yl-oxytripyrrolidinylphosphine hexafluorophosphate (PyBOP) (0.027 g, 0.0521 mmol, 1.2 eq.) were added to the solution. N , N - diisopropylethylamine (DIPEA) (0.076 mL, 0.4338 mmol, 10 eq.). The reaction progress was monitored by LCMS and the reaction was analyzed by preparative RP-HPLC (Waters, XBridge TM 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 in 30 min, 13 mL / min, λ = 220 nm, 254 nm). Collect the pure fractions, evaporate all organic solvents and lyophilize the sample for 48 hours to obtain NK1-PEG 11 -NHBoc. Yield: 40.13 mg (97%). 11 -NHBoc (0.0165 g, 0.015 mmol) in dry CH2Cl2 was added trifluoroacetic acid (TFA, 20 eq.) and the reaction mixture was stirred at room temperature for 4 hours. Excess TFA was removed, diluted with water and extracted with CH2Cl2 (3 x 5 mL). The combined organic layers were washed with brine, dried (Na2SO4) and concentrated. The obtained residue was dried under vacuum and used in the next step without further purification. At room temperature, under argon, the stirred NK1-PEG in dry dimethyl sulfoxide (DMSO, 0.3 mL) was added to 4% MgCl2. 11 A solution of -NH2 (0.008 g, 0.0081 mmol, 1.0 eq.), fluorescein isothiocyanate (FITC) (Sigma, 0.0037 g, 0.0097 mmol, 1.2 eq.) was added to diisopropylethylamine (0.0028 mL, 0.0162 mmol, 2.0 eq.). The reaction progress was monitored by LCMS and the reaction was analyzed by preparative RP-HPLC (Waters, XBridge TM 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 in 30 min, 13 mL / min, λ = 280 nm) purification. The pure fractions were collected, all organic solvents were evaporated and the sample was lyophilized for 48 hours to give NK1-PEG11-FITC (5). Yield: 8.54 mg (77%).

[0696] The NK-1 compound was synthesized via a two-step procedure starting from a basic ligand prepared using a literature procedure (reference: DESIGN AND DEVELOPMENT OF NEUROKININ-1 RECEPTOR-BINDING AGENTDELIVERY CONJUGATES, application number: PCT / US2015 / 44229; the entire text of which is incorporated herein by reference).

[0697] Embodiment 15

[0698] In vitro co-culture E2 CAR T activation and target cell killing assay

[0699] E2 CAR T cells were thawed and recovered from cryopreservation for 4 days at a cell density of 0.5 million to 2 million T cells / mL T cell growth medium (TexMACS medium + 2% human AB serum + 50 U / mL recombinant human IL2). Target cells expressing different levels of surface folate receptors were also thawed and recovered from cryopreservation in growth medium (folate-deficient RPMI1640 + 10% FCS + penicillin / strep for 4 days).

[0700] On day 1, 100,000 target cells were plated in 2 ml growth medium / well of a 12-well tissue culture plate to allow target cells to adhere and recover. On day 0, the wells receiving the EC17 bridge molecule were treated with 100 nM EC17 for 30 minutes in a tissue culture incubator. Afterwards, all medium was washed off and replaced with fresh growth medium without folic acid and EC17. During the EC17 incubation period, T cells were harvested, counted, and after the EC17 treatment was completed, 100,000 E2 CAR T cells were added directly to each well of the target cells in a total of 3 ml growth medium at a ratio of 1:1 (E:T) and incubated for 1 and 2 days at 37°C, 5% CO2 under standard tissue culture humidity conditions. One day of co-culture conditions were used to determine E2 CAR T cell activation via target cells and two days of incubation were used to determine target cell death.

[0701] To determine the level of E2 CAR T cell activity after one day of co-culture with target cells having different levels of surface folate receptors, floating cells and remaining adherent cells were harvested and pooled, wherein the adherent cells were removed from the tissue culture plate using 5 minutes of 0.25% trypsin digestion. The pooled floating and adherent cells were precipitated with a 400 x gravity centrifugation step for 5 minutes and then resuspended in flow cytometry staining solution. Surface marker staining for E2 T cell recognition via flow cytometry included anti-human EGFR and anti-human CD3, while detection of CAR T cell activation via flow cytometry utilized anti-human CD137 staining. E2 CAR T cells were identified as EGFR+ CD3+, and activated CAR T cells also co-expressed CD137 (see flow cytometry methods).

[0702] To determine the efficacy of E2 CAR T cell killing of target cells with varying levels of surface folate receptor expression after two days of co-culture, pooled and adherent cells were pelleted in the same manner as the one-day co-culture assay above and then stained for E2 CAR T markers EGFR and CD3, then washed and stained for the apoptosis marker Annexin V by resuspending the flow antibody stained samples with Alexa Fluor 647-conjugated recombinant Annexin V (Invitrogen catalog number A23204 diluted 1:50 in the provided 1X Annexin staining buffer) and 3 µM propidium iodide.

[0703] E2 CAR T cell activation depends on EC17 staining of target cells

[0704] In an effort to understand the levels of folate receptor required for activation of E2 CAR T cells, one-day cocultures with target cells expressing varying levels of surface folate receptor (FR) were performed ( Fig.39). The percentage of E2 CAR T cells activated after one day of co-culture is plotted on the y-axis, while the FR expression level of the target cells at the time of the assay is plotted on the x-axis. Importantly, the FR expression level data was obtained on the same day of the assay using the same target cells, which were treated with EC17 but cultured in the absence of CAR T cells to avoid any artifacts of FR-high expressing cells being eliminated by the CAR T cells. Four of the five target cell lines expressing a wide range of folate receptors activated CAR T cells to similar levels. Interestingly, the cell line OV90 (lower triangle), which expresses low FRs, expresses about 50% of the FRs of SKOV3 cells (diamonds) and activates about 50% of the CAR T cells of SKOV3 cells. These data show that a wide range of EC17 will activate E2 CAR T cells to similar levels, indicating that there is a certain threshold of EC17 bound to target cells that is required to obtain full activation of E2 CAR T cells. In addition, it is also shown that there is a threshold of FR expression that will provide a suboptimal E2 CAR T response.

[0705] Target cell killing by E2 CAR T cells

[0706] To confirm that E2 CAR T cell activation also translates into successful killing of target cells, we performed a two-day co-culture assay and then observed target cell (EGFR-CD3-) events by flow cytometry and measured the % of target cells using apoptosis surface marker staining (Annexin V staining). Fig.40 In the , the same five target cells as shown (as marked on the x-axis) were each cultured for two days under three different conditions (shown as three different bars). The first bar in each group represents the basal level of target cell apoptosis, such as those of target cells treated with EC17 but cultured in the absence of E2 CAR T cells. The second bar in each group represents CAR T plus target cell co-cultures without any previous EC17 treatment of target cells, while the third bar in each group represents CAR T plus target cells pretreated with EC17. The y-axis shows the percentage of target cells that are apoptotic (Annexin V+). From these data, it was found that CAR T cell activation was well converted into target cell killing for the three highest folate receptor expressers (MDAMB231, HOS-FRα, and IGROV1). However, for SKOV3 cells ( Fig.39 , x-axis), similar E2 CAR T activation did not translate into similar target cell killing ( Fig.40). This disconnect between CAR T cell activation and target cell apoptosis in SKOV 3 cells may not be a result of low folate receptor expression. As mentioned previously, each cancer cell may evolve its own anti-apoptotic mechanisms and it is possible that the FR expression levels of SKOV3 cells (but in a different cancer cell line) may be sufficient for higher levels of target cell apoptosis.

[0707] In short, from Fig.39 and Fig.40 The data demonstrate that EC17 bound to cancer cells is required for cell killing. These data also indicate that below a certain threshold of target cell-bound EC17, there is a linear relationship between surface-bound EC17 and E2 CAR T activation and killing.

[0708] Example 16

[0709] 3 H-Folic Acid Binding Assay

[0710] All tumor cell lines were seeded overnight in RPMI medium without folic acid containing 10% heat-inactivated fetal bovine serum. On the next day, cells were incubated on ice with 100 nM 1% Folic Acid with and without 10 μM cold folic acid. 3 H-Folic acid was incubated for 15 minutes. After rinsing 3 times with cold 1X PBS, whole cells were lysed and total cell-associated radioactivity was counted in a scintillation counter. The counts were determined by subtracting the folic acid competition samples. 3 FR-specific binding and use of H-folate 3 The specific activity of H-folate was calculated as the number of molecules / cell.

[0711] Fig.41 Various levels of FR expression in these cell lines were demonstrated. The order of FR levels was: IGROV1 > MDA-MB-231, HOS-FRα > OV90, SKOV3.

[0712] Embodiment 17

[0713] EC17 / E2-CAR T-cell therapy in NSG mice with and without subcutaneously implanted MDA-MB-231 tumors

[0714] Tumor implantation

[0715] MDA-MB-231 tumor cells were grown in RPMI 1640 deficient in folic acid containing 5 to 10% FBS at 37°C in a humidified atmosphere of 5% CO2. MDA-MB-231 tumor cells were plated at 2.5 x 10 6 Only 42 / 66 mice were vaccinated and the remaining 22 mice remained tumor-free.

[0716] CAR-T cell administration

[0717] The anti-FITC E2 scFv-CAR T cells sorted by EGFRt were frozen in T-cell freezing medium. The vials of frozen CAR-T cells were immediately stored at -80 ° C. The CAR-T cells were quickly thawed at 37 ° C, washed twice with PBS and used for animal injection with 10 million viable EGFRt+ E2 CAR-T cells (CD4 / CD8 about 1: 1) / animal. A small aliquot was obtained on the day of infusion for flow cytometric analysis of E2-CAR T-cell phenotype.

[0718] Human / Mouse Cytokine Analysis

[0719] Mouse blood samples were processed into plasma and stored at -20°C until use. Human cytokines in mouse blood were detected using a human TH1 cytokine panel (Biolegend, catalog number 740009) and a human hematopoietic stem cell cytokine panel (Biolegend, catalog number 740610). Mouse cytokines were detected using a mouse 13-plex inflammatory cytokine panel (Biolengend, number 740150). All analyses were performed according to the manufacturer's instructions.

[0720] Flow cytometric analysis

[0721] Whole blood cell analysis: Plasma was taken from a predetermined volume of EDTA-treated whole blood, and RBCs were lysed with RBC lysing solution. The leukocyte pellet was then resuspended in flow cytometry staining solution (1% bovine serum albumin, 50 mg / mL human IgG (Equitech Bio, catalog number SLH56-0001), phosphate buffered saline containing 0.9% sodium azide, pH = 7.4) and stained for leukocyte surface markers using the following antibodies: anti-human CD45 [clone HI30, eBioscience #47-0459-42 at a 1:20 (v / v) dilution], anti-human CD137 [clone 4B4-1, BD Bioscience #564092 at a 1:20 (v / v) dilution], anti-human CD8α [clone RPA-T8, BD Bioscience, catalog number 557746 at a 1:20 (v / v) dilution], anti-human CD4 [clone SK3, eBioscience catalog number 46-0047-42 at a 1:20 (v / v) dilution], anti-human EGFR [R&D systems, clone Hu1, catalog number FAB9577B at 1:10 (v / v)], anti-human PD1 [BD Biosciences, clone EH12.1, catalog number 562511 at 1:20 (v / v)], anti-human LAG3 [BD Biosciences, clone T47-530, catalog number 565616 at 1:20 (v / v)], anti-human TIM3 [BD Biosciences, clone 7D3, catalog number 565558 at 1:20 (v / v)], anti-human CD3ε [BD Biosciences, clone SK7, catalog number 557832 at 1:20 (v / v)]. After leukocyte staining, cells were washed with PBS and resuspended in cold PBS containing 53,000 CountBrightTM beads (Invitrogen catalog number C36950) and transferred to flow cytometry collection tubes. Flow cytometry data were collected on a Gallios flow cytometer (Beckman Coulter, Brea, CA). The concentration of CAR T cells in each blood sample was calculated according to Invitrogen's instructions. CAR T cells were identified as human CD3ε + EGFRt + events and were easily distinguished and counted using KaluzaTM flow cytometry software. The number of CAR T cells in the circulation of each infused mouse was then represented on the graph as the total number of CAR T cells / 100 μL of whole blood analyzed. Statistical significance was determined by using an unpaired, two-tailed Student's t-test, with significance set at p<0.05.

[0722] Tumor and tissue analysis: Harvest solid tumors (100 to 1000 mm 3 ), weighed and minced into small pieces, and then transferred to a 50 mL tube containing 20 mL of tumor digestion mixture. The enzymatic tumor digestion mixture consists of serum-free and folic acid-deficient RPMI1640 medium supplemented with antibiotics containing 0.5 mg / mL collagenase IV (Sigma-Aldrich, catalog number C5138), 0.5 mg / mL hyaluronidase (Sigma-Aldrich, catalog number H3506) and 0.1 mg / mL DNase I (Sigma-Aldrich, catalog number DN25). The tumor fragments were digested at 37°C on a horizontal shaker at 300 rpm for 1 hour. Afterwards, the tumor digest was centrifuged at 400 xg for 5 minutes, and the tumor cell pellet that underwent the red blood cell lysis step was then washed with cold phosphate-buffered saline (PBS, pH 7.4) and finally filtered through a 40 µm nylon cell filter.

[0723] E2-CAR-T phenotype and in vivo proliferation

[0724] It has been reported that the expansion and persistence of CAR T cells in the blood are associated with responses to CD19-specific CAR T therapies in clinical studies. It is reasonable to assume that in our in vivo model, a larger pool of circulating blood with E2 CAR T will allow a greater response to EC17-directed attacks on FR+ tumors. As a measure of the response of E2 CAR T cell therapy to EC17, we quantified the number of circulating E2 CAR T cells / 100 uL whole blood and observed that the groups stimulated by EC17 (groups 2, 3, 4, and 5) had at least a 10-fold increase in the circulation compared to the groups that did not receive EC17 (groups 1 and 6). Importantly, this expansion of circulating CAR T cells in the blood is consistent with the expansion of CAR T cells in an antigen-dependent manner. In addition, we also observed that after CAR T cell infusion, the elevated number of circulating CAR T cells lasted up to 54 days and when compared to groups 1 and 6 that did not receive EC17, CAR T persistence again depended on EC17 (groups 2, 3, 4, and 5).

[0725] Tumors vs. tumor-free mice

[0726] like Fig.42As shown in , 10 million E2 CAR-T cells were injected intravenously into tumor-free naive NSG mice or NSG mice bearing MDA-MB-231 tumors. On the 2nd and 10th day after CAR-T injection, 500 nmol / kg EC17 was administered intravenously, and mice were euthanized 20 hours after the second EC17 dose (day 11) for organ evaluation and blood analysis. Plasma samples were immediately separated from the blood and stored at -20°C until cytokine analysis. On the 12th day after CAR-T injection, separate groups of animals were harvested for FACS analysis as described above.

[0727] like Fig.43 As shown in , the reduction of cytokines in tumor-bearing mice is EC17-dependent. The levels of cytokines including IL2, IFN-γ, IL-10 were all increased in tumor-bearing mice with EC17 relative to those without EC17 administration. In tumor-free naive mice, upregulation of cytokine production was also observed in those mice dosed with EC17, but the increase in tumor-free mice was much lower than those with MDA-MB-231 tumors (e.g., 23-fold lower for IFN-γ).

[0728] Tumor-bearing mice had approximately 23-fold higher IFN-γ production than tumor-free mice. Although tumor-free mice (FD diet for approximately 2 months) showed some cytokine production after EC17 SIW500 x 2 doses, there was no detectable CAR-T cell expansion via FACS.

[0729] Embodiment 18

[0730] Target-specific activity demonstrated in vitro using humanized anti-FITC scFv E2-CAR constructs and CAR-modified T cells

[0731] Cell lines: Unless otherwise specified, all cell lines were maintained in RPMI1640 (Irvine Scientific) supplemented with 2 mM L-glutamine (Cellgro), 25 mM HEPES (Irvine Scientific) and 10% heat-inactivated FBS (Hyclone). The K562 target cell line was kindly provided by Dr. Stanley Riddell (FHCRC). The K562 OKT3 cell line was generated by transduction with a lentivirus containing an OKT3 transgene. OKT3 is a membrane tethered to a scFv targeting CD3ε. MDA-MB-231 was provided by Endocyte and cultured in folic acid-free DMEM. All cell lines were identified by STR profiles matching the DSMZ database of the University of Arizona Genetics Core.

[0732] Cytotoxicity and cytokine secretion: A four-hour chromium release assay was performed. Briefly, target cells were treated with 51 Cr (PerkinElmer) was labeled overnight, washed three times in PBS, and plated in 96-well plates at 5 × 10 3 10 cells / well were incubated with T cells at various effector to target (E:T) ratios in triplicate. S supernatants were harvested for γ-counting and calculation of specific lysis. For cytokine secretion, 5x10 5 T-cells and target cells were plated in triplicate in 96-well plates at an E:T ratio of 2:1 for 24 hours, and supernatants were analyzed via cytometric bead arrays using the Bio-Plex Human Cytokine Panel (Bio-Rad) according to the manufacturer's instructions. For targets labeled with EC17, cells were incubated with PBS containing 100 nM EC17 for 1 hour at room temperature in the dark before plating. When EC17 was used, cells were incubated with folic acid-free RPMI during the assay.

[0733] Cytokine release assay:

[0734] CD4+ T cells were co-cultured at a 2:1 T-cell to target ratio for 24 hours, and the supernatants were then analyzed for the presence of effector cytokines IL-2, IFN-γ, and TNF-α. The assays were run in triplicate and the data are shown as mean ± SE. Fig.44As shown in, we observed that the mocks (Mock) and anti-FLCAR T-cells reacted to co-culture with the positive control cell line K562-OKT3, and quantitatively produced similar levels of cytokine production. After co-culture with K562, the mocks or anti-FLCAR T-cells did not produce cytokines. Cytokine release depends on the previous cultivation of EC17 and MDA-MB-231 cell lines, and anti-FLCAR T-cells are the only cells that can cause the secretion of cytokines IL-2, IFN-γ and TNF-α for EC17-labeled MDA-MB-231.

[0735] Chromium release assay:

[0736] CD8+ T cells were co-cultured with target cells at a ratio of 30:1, 10:1, 3:1, or 1:1. Percent lysis of triplicate wells is depicted (mean ± SE). Fig.45 As shown in, anti-FLCAR T- cells did not show cytotoxicity against negative control (K562) or unlabeled MDA-MB-231 cell lines. However, both the cells transduced by the mimics and anti-FLCAR T- cells were able to induce similar levels of specific lysis against the positive control K562-OKT3 cell line. In addition, EC17-labeled MDA-MB-231 cells were effectively recognized and lysed by anti-FLCAR T- cells, whereas mimics T- cells were unable to produce lysis.

[0737] Embodiment 19

[0738] The structure of CAR-T cell aptamers

[0739] Figures 46 to 49 Shown are the structures of the bridges used in the CAR-T cell experiments and their in vitro determined affinity to the cell types to which the bridges were directed (e.g., folate-FITC to cancer cells expressing the folate receptor). Fig.50 The bridges were shown to bind (by FACS analysis) to tumor cells used in the in vivo model and expressing receptors corresponding to the small molecule ligands for the bridges.

[0740] For Examples 20 to 30 - see Figures 55 to 63 .

[0741] Embodiment 20

[0742] Cell lines and reagents

[0743] Unless otherwise specified, all FR+ and FR-negative cancer cell lines were maintained in RPMI-1640 medium (GibcoBRL) supplemented with 10% heat-inactivated fetal bovine serum without (FFRPMI) or with (RPMI) 2.4 μM folic acid (FA). KB (human cervical carcinoma expressing FRα with HeLa marker) and CHO-β (Chinese hamster ovary cells transfected with human FRβ) were used as sources of FRα and FRβ for radioligand binding assays, respectively. MDA-MB-231 represents a subclone of FRα of a human triple-negative breast cancer (TNBC) cell line. For AML research, isogenic pairs of FRβ-positive (THP1-FRβ) and FR-negative (THP1-FG12) cell lines expressing green fluorescent protein (GFP) are provided. Both were established from THP-1 (ATCC, TIB-202), a commonly used cell model for studying pediatric AML, which was originally derived from a 1-year-old male infant with monocytic leukemia. For osteosarcoma studies, 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 of a 13-year-old Caucasian female and is highly tumorigenic in NSG mice. FR+ HOS-FRα expressing GFP was transduced with lentiviral firefly luciferase fLuc and FR-negative HOS-143b fLuc of bioluminescent pairs.

[0744] LEGENDplex TM Human cytokine panel was purchased from BioLegend (San Diego, CA). Lactate dehydrogenase (LDH)-based CytoTox 96 ®Non-radioactive cytotoxicity assay kits were 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 Thermo Fisher Scientific (Waltham, MA); 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 also purchased from BioLegend. APC-conjugated anti-FITC mouse IgG2a / κ antibody (clone NAWESLEE), CountBright TM Beads (Invitrogen), Annexin V staining buffer, and AlexaFluor-647-conjugated Annexin V were purchased from ThermoFisher Scientific. For enzymatic digestion of tumor tissues, collagenase IV, hyaluronidase, and DNase I were purchased from Sigma-Aldrich (St. Louis, MO).

[0745] EC17 or folate-FITC [FA-(γ)-ethylenediamine-FITC] was synthesized in Endocyte. 3 H-EC17 was purchased from Moravek biochemicals (Brea, CA) with a specific activity of approximately 0.952 Ci / mmol or was purified by FITC in Endocyte. 3 It was prepared by conjugation with H-FA-(γ)-ethylenediamine (manufactured by ViTrax (Placentia, CA)) and had a specific activity of about 1.2 Ci / mmol. 3 H-FA was also purchased from ViTrax and had a specific activity of 59 Ci / mmol. For CRS rescue, sodium fluorescein dosing solution was purchased from AK-FLUOR ® 25% (Fluorescein Injection, USP) dilution, the AK-FLUOR ®Purchased from Purdue Pharmacy (NDC 17478-250-25).

[0746] Embodiment 21

[0747] Humanized CAR constructs and CAR-modified T cells

[0748] Previous studies used GFP+ second generation anti-FITC scFv (clone 4M5.3) CAR containing the hinge and transmembrane sequence of CD8α and the 4-1BB / CD3ζ signaling domain (i.e., FITC-4M5.3-scFv-CD8α hinge-CD8αtm-4-1BB / CD3ζ). In order to transition to the first human test, a second generation fully human FITC-specific (clone E2) CAR construct (referred to herein as E2) was developed ( Fig.55 , upper figure). CAR-modified T cells are shown in Fig.55 , in the pie chart below.

[0749] The construct described herein is a FITC-specific CAR construct comprising: (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 ( Fig.55 , above) (SEQ ID NOS: 1 and 2 are nucleic acid and amino acid sequences, respectively). To generate CAR-modified T cells, lentivirus was produced in 293T cells co-transfected with epHIV7 lentiviral vector encoding CAR. Donor CD4+ and CD8+ T cells were purified and separated via immunomagnetic selection or transduced at a ratio of approximately 50:50. In summary, 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 before cryopreservation and after thawing to determine EGFRt expression and CD4 / CD8 ratios by flow cytometry. Using a combination of surface markers, the differentiation state of CD4+ and CD8+ CAR-T cell subsets on the day of infusion was analyzed and defined as T N , CD45RA+ CD45RO- CD62L+ CD95- initial T cells; T SCM , CD45RA+ CD45RO- CD62L+ CD95+ stem cell memory T cells; T CM, CD45RA- CD45RO+ CD62L+CD95+ central memory T cells; T EM , CD45RA- CD45RO+ CD62L- CD95+ effector memory cells; and T EFF , CD45RA+CD45RO- CD62L- CD95+ effector T cells. For the preclinical testing described below, the study included two batches of EGFRt-sorted pure CD4 and CD8 subsets (after mixing at an approximately 1:1 ratio) and several batches of unsorted approximately 1:1 EGFRt+ CD4 / CD8 mixtures, which contained a "clinical facsimile" preparation with poor differentiation properties.

[0750] Among a series of different CAR constructs synthesized and evaluated, a fully human anti-FITC scFv (FITC-E2) CAR was selected for preclinical development ( Fig.55 ). This second-generation all-human CAR consists of an anti-FITC scFv (clone E2), a double mutation (L235D and N297Q) in the CH2 region to reduce the binding to FcγR IgG4-Fc spacer / hinge, a CD28 transmembrane domain, and a 4-1BB / CD3ζ signaling domain attached to the cell surface EGFRt tag via a T2A ribosomal skipping sequence (ie, FITC-E2-scFv-IgG4 hinge-CD28tm-4-1BB / CD3ζ-T2A-EGFRt). For preclinical studies, both EGFRt-sorted and unsorted E2 CAR-T cells were prepared at a ratio of about 1:1CD4 / CD8, and for each in vivo experiment, T cell subtype phenotype analysis was routinely performed by flow cytometry at the time of CART cell infusion (day 0). The typical expression pattern of EGFRt-sorted CAR-T cells includes about 42% T SCM , 10% T CM , 12% T EM and 34% T EFF Both CD4 and CD8 subsets ( Fig.55 , pie chart on the left). Only EGFRt-sorted CAR-T cells were used for co-culture ( Fig.53 , 54 , 56, 58, 59 and 61) and pharmacokinetic studies ( Fig.62 ). For oncology therapy, use a "clinical facsimile" batch with poorly differentiated properties ( Fig.55 , right pie chart) and this “clinical batch” was also used in the MDA-MB-231 study ( Fig.63 ), and the research batches were used for THP1-FRβ and HOS-FRα studies ( Fig.57The “clinical facsimile” batch (approximately 39% EGFRt+) included approximately 66% T SCM and about 32% T CM CD4+ subsets and about 95% T SCM and 3% T CM This study batch (approximately 23% EGFRt+) was more differentiated and included 32% T SCM 53% T CM , 11% T EM and 3.7% T EFF CD4 subsets and 44% T SCM , 0.28% T CM 3.4% T EM and 52% T EFF CD8 subset.

[0751] Embodiment 22

[0752] Dual-specific affinity of EC17 CAM

[0753] In cell-based radioligand binding assays, use 3 H-EC17 was used to evaluate the bispecific affinity of EC17 CAM. For binding to FR+ targets, KB and CHO-β cells were pre-seeded in 24-well tissue culture plates overnight and cultured with 0.1, 0.5, 1, 5, 10, 20, and 40 nM 3 H-EC17 were incubated in FFRPMI at 37°C for 2 hours. Afterwards, the cells were washed with phosphate-buffered saline (PBS, pH 7.4) and lysed with 1% sodium dodecyl sulfate. The radioactivity of whole cell lysates and the level of cellular protein content were quantified by standard Pierce BCA protein assay. 3 The number of H-EC17 molecules was used to determine the dissociation constants (Kd) of FRα (KB) and FRβ (CHO-β) ( Fig.56 ).

[0754] The main components of CAM-controlled CAR T cell therapy: The basic components of CAR-T cell therapy involve FITC as a pseudotumor antigen, high-affinity EC17 CAM (CAM is equivalent to the bridge or "compound" in this application) and rationally designed anti-FITC CAR and CAR-modified T cells. EC17 has been tested in the clinic for immunotherapy and opt...

Claims

1. Use of a compound or a pharmaceutically acceptable salt thereof and a CAR T cell composition comprising CAR T cells in the preparation of a medicament for treating cancer by the following method, wherein the method include: i) administering the compound or a pharmaceutically acceptable salt thereof to a patient, wherein the compound comprises a small molecule ligand linked to a targeting moiety via a linker; ii) administering a first dose of a CAR T cell composition to the patient; and iii) administering a second dose of the CAR T cell composition to the patient; wherein the CAR T cell comprises a CAR directed to the targeting moiety, wherein the CAR comprises an E2 anti-fluorescein antibody fragment, and wherein the CAR T cell comprises a polypeptide having a sequence as shown in (SEQ ID NO: 2), wherein the small molecule ligand is folic acid, and wherein the cancer is triple-negative breast cancer, human cervical cancer, ovarian cancer, acute myeloid leukemia or osteosarcoma.

2. The use according to claim 1, wherein the compound or a pharmaceutically acceptable salt thereof is not an antibody and does not comprise an antibody fragment.

3. The method of claim 1 or claim 2, wherein the targeting moiety does not comprise a peptide epitope.

4. The use of claim 1 or claim 2, wherein the targeting moiety comprises a folate and wherein the cancer is a cancer that expresses a folate receptor.

5. The use of claim 1 or claim 2, wherein cytokine release syndrome (CRS) is reduced or prevented and the method results in a reduction in tumor volume in the patient.

6. The use of claim 1 or claim 2, wherein the CAR T cells have a central memory / effector memory phenotype.

7. The use according to claim 1 or claim 2, wherein the CD8:CD4 ratio of the CAR T cells is 1:

1.

8. The use of claim 1 or claim 2, wherein weight loss due to CRS is reduced or prevented.

9. The use of claim 1 or claim 2, wherein the cancer comprises a tumor and wherein a complete response to the tumor is obtained.

10. Use of a compound or a pharmaceutically acceptable salt thereof and a CAR T cell composition in the preparation of a medicament for treating cancer by the following method: include: i) administering the compound or a pharmaceutically acceptable salt thereof to a patient, wherein the compound comprises a small molecule ligand linked to a targeting moiety via a linker; and ii) administering the CAR T cell composition to the patient, wherein the CAR T cell composition comprises a CAR T cell, wherein the CAR T cell comprises a CAR directed to the targeting moiety, wherein the CAR comprises an E2 anti-fluorescein antibody fragment, wherein the CAR T cell comprises a polypeptide with a sequence as shown in (SEQ ID NO: 2), and wherein the CAR T cell composition comprises a mixture of the CAR T cell and non-transformed T cells, wherein the small molecule ligand is folic acid, and wherein the cancer is triple-negative breast cancer, human cervical cancer, ovarian cancer, acute myeloid leukemia or osteosarcoma.

11. The use of claim 10, wherein the CAR T cell composition comprises a mixture of the CAR T cells and non-transformed T cells selected from the following ratios: 1:5 of the CAR T cells to non-transformed T cells, 1:4 of the CAR T cells to non-transformed T cells, 1:3 of the CAR T cells to non-transformed T cells, 1:2 of the CAR T cells to non-transformed T cells, and 1:1 of the CAR T cells to non-transformed T cells.

12. The use of claim 10, wherein the CAR T cell composition comprises a mixture of the CAR T cells and non-transformed T cells, wherein the ratio of the CAR T cells to the non-transformed T cells is 1:1 to 1:

5.

13. The use of claim 10, wherein the CAR T cells are administered in a mixture of 10 million of the CAR T cells and 40 million non-transformed T cells.

14. The use of claim 10, wherein the CAR T cells are administered in a mixture of 15 million of the CAR T cells and 35 million non-transformed T cells.

15. The use of claim 10, wherein the CAR T cells are administered in a mixture of 20 million of the CAR T cells and 30 million non-transformed T cells.

16. Use of a compound or a pharmaceutically acceptable salt thereof, a CAR T cell composition, a folate compound, a conjugate containing a folate compound, or an agent that inhibits activation of CAR T cells in the preparation of a medicament for treating cancer by the following method, wherein the method include: i) administering the compound or a pharmaceutically acceptable salt thereof to a patient, wherein the compound comprises a small molecule ligand linked to a targeting moiety via a linker; ii) administering the CAR T cell composition to the patient, wherein the CAR T cell composition comprises a CAR T cell, wherein the CAR T cell comprises a CAR directed to the targeting moiety and wherein the CAR comprises an E2 anti-fluorescein antibody fragment, wherein the CAR T cell comprises a polypeptide having a sequence as shown in (SEQ ID NO: 2); and iii) administering to the patient a folate; a conjugate comprising a folate, wherein the conjugate comprising a folate does not include a targeting moiety; or an agent that inhibits activation of CAR T cells, wherein the small molecule ligand is folic acid, and wherein the cancer is triple-negative breast cancer, human cervical cancer, ovarian cancer, acute myeloid leukemia or osteosarcoma.

17. The use according to claim 16, wherein a folate is administered and the folate is folic acid or leucovorin.

18. The use according to claim 16, wherein a conjugate comprising a folate is administered, wherein the conjugate comprising a folate does not comprise a targeting moiety.

19. The use according to claim 16, wherein an agent that inhibits the activation of CAR T cells is administered and the agent is an agent that blocks CAR T cells from binding to the compound or a pharmaceutically acceptable salt thereof, but not to the cancer.

20. The method of claim 16, wherein an agent that inhibits the activation of CAR T cells is administered.

21. The use according to claim 19, wherein the agent is fluorescein amine, FITC or fluorescein sodium.

22. The use of claim 16, wherein the patient is administered an agent that inhibits the activation of CAR T cells and when the CRS grade reaches 3 or 4, the agent that inhibits the activation of CAR T cells is administered.

23. Use of a compound or a pharmaceutically acceptable salt thereof and a CAR T cell composition comprising CAR T cells in the preparation of a medicament for treating cancer by the following method, wherein the method include: i) administering the compound or a pharmaceutically acceptable salt thereof to a patient, wherein the compound comprises a small molecule ligand linked to a targeting moiety via a linker, and wherein the compound or a pharmaceutically acceptable salt thereof is administered at a dose of 10 nmol / kg to 2500 nmol / kg of patient body weight, and ii) administering to the patient a CAR T cell composition comprising CAR T cells, wherein the CAR T cells comprise CAR, wherein the CAR comprises an E2 anti-fluorescein antibody fragment, wherein the CAR T cells comprise a polypeptide having a sequence as shown in (SEQ ID NO: 2), and wherein the dose of the CAR T cells is 1 million CAR T cells to 15 million CAR T cells, wherein the small molecule ligand is folic acid, and wherein the cancer is triple-negative breast cancer, human cervical cancer, ovarian cancer, acute myeloid leukemia or osteosarcoma.

24. The use according to claim 23, wherein the compound or a pharmaceutically acceptable salt thereof is administered at a dose of 10 nmol / kg to 100 nmol / kg of patient body weight.

25. The use according to claim 23, wherein the compound or a pharmaceutically acceptable salt thereof is administered at a dose of 10 nmol / kg to 50 nmol / kg of patient body weight.

26. The use according to claim 23, wherein the compound or a pharmaceutically acceptable salt thereof is administered at a dose of 10 nmol / kg to 20 nmol / kg of patient body weight.

27. The use according to claim 23, wherein the compound or a pharmaceutically acceptable salt thereof is administered at a dose of 10 nmol / kg to 600 nmol / kg of patient body weight.

28. The use according to claim 23, wherein the compound or a pharmaceutically acceptable salt thereof is administered at a dose of 200 nmol / kg to 600 nmol / kg of patient body weight.

29. Use of a compound or a pharmaceutically acceptable salt thereof and a CAR T cell composition comprising CAR T cells in the preparation of a medicament for treating cancer by the following method, wherein the method include: i) continuously administering a compound or a pharmaceutically acceptable salt thereof to a patient, wherein the compound comprises a small molecule ligand linked to a targeting moiety via a linker, ii) administering a CAR T cell composition comprising a CAR T cell to the patient, wherein the CAR T cell comprises a CAR, wherein the CAR comprises an E2 anti-fluorescein antibody fragment, wherein the CAR T cell comprises a polypeptide having a sequence as shown in (SEQ ID NO: 2), and iii) terminating the continuous administration of the compound or a pharmaceutically acceptable salt thereof to inhibit or prevent cytokine release syndrome in the patient, wherein the small molecule ligand is folic acid, and wherein the cancer is triple-negative breast cancer, human cervical cancer, ovarian cancer, acute myeloid leukemia or osteosarcoma.

30. The use of claim 29, wherein the compound or a pharmaceutically acceptable salt thereof is administered to the patient continuously for at least one hour.

31. The use of claim 29, wherein the compound or a pharmaceutically acceptable salt thereof is administered continuously to the patient for at least four hours.

32. The use of claim 29, wherein the compound or a pharmaceutically acceptable salt thereof is administered continuously to the patient for at least six hours.

33. The use of claim 29, wherein the compound or a pharmaceutically acceptable salt thereof is administered to the patient three times per week.

34. Use of a compound or a pharmaceutically acceptable salt thereof and a CAR T cell composition comprising CAR T cells in the preparation of a medicament for treating cancer by the following method, wherein the method include: i) administering the compound or a pharmaceutically acceptable salt thereof to a patient, wherein the compound comprises a small molecule ligand linked to a targeting moiety via a linker, wherein at least a first dose and a second dose of the compound or a pharmaceutically acceptable salt thereof are administered to the patient, wherein the first dose and the second dose are different, wherein the amount of the compound or a pharmaceutically acceptable salt thereof in the second dose is 2 to 15,000 times the amount of the compound or a pharmaceutically acceptable salt thereof in the first dose; and ii) administering to the patient a CAR T cell composition comprising a CAR T cell, wherein the CAR T cell comprises a CAR directed to the targeting moiety and wherein the CAR comprises an E2 anti-fluorescein antibody fragment, wherein the CAR T cell comprises a polypeptide having a sequence as shown in (SEQ ID NO: 2), wherein the small molecule ligand is folic acid, and wherein the cancer is triple-negative breast cancer, human cervical cancer, ovarian cancer, acute myeloid leukemia or osteosarcoma.

35. The method of claim 34, wherein at least a first dose, a second dose, and a third dose of the compound or a pharmaceutically acceptable salt thereof are administered to the patient, wherein the first dose, the second dose, and the third dose are different, wherein the amount of the compound or a pharmaceutically acceptable salt thereof in the second dose is 2 to 750 times that of the compound or a pharmaceutically acceptable salt thereof in the first dose, and wherein the amount of the compound or a pharmaceutically acceptable salt thereof in the third dose is 800 to 10,000 times that of the compound or a pharmaceutically acceptable salt thereof in the first dose.

36. The use of claim 34, wherein at least a first dose, a second dose, a third dose, and a fourth dose of the compound or a pharmaceutically acceptable salt thereof are administered to the patient, wherein the first dose, the second dose, the third dose, and the fourth dose are different, wherein the amount of the compound or a pharmaceutically acceptable salt thereof in the second dose is 2 to 750 times that of the compound or a pharmaceutically acceptable salt thereof in the first dose, wherein the amount of the compound or a pharmaceutically acceptable salt thereof in the third dose is 800 to 7500 times that of the compound or a pharmaceutically acceptable salt thereof in the first dose, and wherein the amount of the compound or a pharmaceutically acceptable salt thereof in the fourth dose is 8000 to 15000 times that of the compound or a pharmaceutically acceptable salt thereof in the first dose.

37. The use of claim 34, wherein the amount of the compound or its pharmaceutically acceptable salt in the second dose is 100 times the amount of the compound or its pharmaceutically acceptable salt in the first dose, wherein the amount of the compound or its pharmaceutically acceptable salt in the third dose is 1000 times the amount of the compound or its pharmaceutically acceptable salt in the first dose, and wherein the amount of the compound or its pharmaceutically acceptable salt in the fourth dose is 10000 times the amount of the compound or its pharmaceutically acceptable salt in the first dose.

38. Use of a compound or a pharmaceutically acceptable salt thereof and a CAR T cell composition comprising CAR T cells in the preparation of a medicament for treating cancer by the following method, wherein the method include: i) administering to a patient a first dose of a compound or a pharmaceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked to a targeting moiety via a linker; ii) administering to the patient at least a second dose of the compound or a pharmaceutically acceptable salt thereof, wherein the amount of the compound or a pharmaceutically acceptable salt thereof in the second dose is at least 50% lower than the amount of the compound or a pharmaceutically acceptable salt thereof in the first dose; and iii) administering to the patient a dose of a CAR T cell composition comprising a CAR T cell, wherein the CAR T cell comprises a CAR directed to the targeting moiety and wherein the CAR comprises an E2 anti-fluorescein antibody fragment, wherein the CART cell comprises a polypeptide having a sequence as shown in (SEQ ID NO: 2), wherein the small molecule ligand is folic acid, and wherein the cancer is triple-negative breast cancer, human cervical cancer, ovarian cancer, acute myeloid leukemia or osteosarcoma.

39. The use of claim 38, wherein the amount of the compound or a pharmaceutically acceptable salt thereof in the second dose is at least 60% lower than the amount of the compound or a pharmaceutically acceptable salt thereof in the first dose.

40. The use of claim 38, wherein the amount of the compound or a pharmaceutically acceptable salt thereof in the second dose is at least 70% less than the amount of the compound or a pharmaceutically acceptable salt thereof in the first dose.

41. The use of claim 38, wherein the amount of the compound or a pharmaceutically acceptable salt thereof in the second dose is at least 80% less than the amount of the compound or a pharmaceutically acceptable salt thereof in the first dose.

42. The use of claim 38, wherein the amount of the compound or a pharmaceutically acceptable salt thereof in the second dose is at least 90% less than the amount of the compound or a pharmaceutically acceptable salt thereof in the first dose.

43. The use of claim 38, wherein the amount of the compound or a pharmaceutically acceptable salt thereof in the second dose is at least 95% less than the amount of the compound or a pharmaceutically acceptable salt thereof in the first dose.

44. Use of a compound or a pharmaceutically acceptable salt thereof and a CAR T cell composition comprising CAR T cells in the preparation of a medicament for treating cancer by the following method, wherein the method include: i) administering a first dose of a compound or a pharmaceutically acceptable salt thereof to a patient, wherein the compound comprises a small molecule ligand linked to a targeting moiety via a linker and wherein the compound or a pharmaceutically acceptable salt thereof is administered to the patient prior to administering the CAR T cell composition, ii) then administering a dose of the CAR T cell composition to the patient, and iii) then administering to the patient a second dose of the compound or a pharmaceutically acceptable salt thereof, wherein the CAR comprises an E2 anti-fluorescein antibody fragment, The CAR T cell comprises a polypeptide having a sequence as shown in (SEQ ID NO: 2), wherein the small molecule ligand is folic acid, and wherein the cancer is triple-negative breast cancer, human cervical cancer, ovarian cancer, acute myeloid leukemia or osteosarcoma.

45. Use of a compound or a pharmaceutically acceptable salt thereof and a CAR T cell composition comprising CAR T cells in the preparation of a medicament for treating cancer by the following method, wherein the method include: i) administering the compound or a pharmaceutically acceptable salt thereof to a patient, wherein the compound comprises a small molecule ligand linked to a targeting moiety via a linker and wherein the compound or a pharmaceutically acceptable salt thereof is administered to the patient once a week, and ii) administering a CAR T cell composition comprising a CAR T cell to the patient, wherein the CAR T cell comprises a CAR, wherein the CAR comprises an E2 anti-fluorescein antibody fragment, wherein the CAR T cell comprises a polypeptide having a sequence as shown in (SEQ ID NO: 2), wherein the small molecule ligand is folic acid, and wherein the cancer is triple-negative breast cancer, human cervical cancer, ovarian cancer, acute myeloid leukemia or osteosarcoma.

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