Methods of use of CAR T cells

Abstract

The present invention relates to methods of using CAR T cells. The present disclosure relates to methods of treating a patient having cancer by administering to the patient a composition comprising CAR T cells, and administering to the patient a small molecule linked to a targeting moiety by a linker, where the CAR T cells comprise a CAR and the CAR comprises an E2 anti-fluorescein antibody fragment. The present disclosure also relates to compositions for use in such methods.

Description

[0001] Cross - Reference to Related Applications This application is a divisional application of the invention patent application titled "Methods of Use of CAR T Cells" with application number 201980021107.5, which entered China from the international application PCT / US2019 / 014472 with an international filing date of January 22, 2019. According to 35 U.S.C.§119(e), this application claims the priority of U.S. Provisional Application Serial No. 62 / 620,414 filed on January 22, 2018, U.S. Provisional Application Serial No. 62 / 620,706 filed on January 23, 2018, U.S. Provisional Application Serial No. 62 / 656,233 filed on April 11, 2018, U.S. Provisional Application Serial No. 62 / 724,171 filed on August 29, 2018, U.S. Provisional Application Serial 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 the above applications are hereby incorporated by reference in their entirety. Technical Field

[0002] The present disclosure relates to methods of treating a patient having cancer by administering to the patient a composition comprising CAR T cells and administering to the patient a small molecule attached to a targeting moiety via a linker, wherein the CAR T cells comprise a CAR and the CAR comprises an E2 anti - fluorescein antibody fragment. The present disclosure also relates to compositions for use in such methods. Background Art

[0003] Immunotherapies based on the adoptive transfer of lymphocytes (e.g., T cells) to a patient are valuable therapies for treating cancer and other diseases. Many important advances have been made in the development of adoptive transfer immunotherapies based on lymphocytes. Among many different types of immunotherapeutic agents, one of the most promising immunotherapeutic agents being developed is T cells expressing a chimeric antigen receptor (CAR T cells). A chimeric antigen receptor (CAR) is a genetically engineered receptor that is designed to target a specific antigen (e.g., a tumor antigen). This targeting can lead to cytotoxicity against the tumor, such that CAR T cells expressing the CAR can target and kill tumors via the specific tumor antigen.

[0004] First - generation CARs consist of an identification region (e.g., a single - chain fragment variable (scFv) region derived from an antibody) for recognizing and binding to an antigen expressed by a tumor, and an activation signal domain (e.g., the CD3ζ chain of a T cell can serve as a T - cell activation signal in the CAR). Although CAR T cells have shown positive results in vitro, their success in eliminating diseases (e.g., cancer) in clinical trials has been limited. One problem is the inability to prolong the activation and expansion of the CAR T cell population in vivo.

[0005] To address this issue, co-stimulatory domains (e.g., CD137, CD28, or CD134) have been included in second-generation CARs to achieve prolonged activation of T cells in vivo. The addition of co-stimulatory domains enhances the in vivo proliferation and survival of CAR-containing T cells, and initial clinical data have shown that such constructs are promising therapeutic agents in the treatment of diseases such as cancer.

[0006] Although improvements have been made in CAR T cell therapy, there are still several problems. First, "off-target" toxicity can occur due to normal cells that express the antigen targeted by CART cells (e.g., tumor-associated antigens). Second, unregulated CAR T cell activation can be found, where rapid and uncontrolled elimination of disease cells (e.g., cancer cells) by CAR T cells induces a series of metabolic disorders called tumor lysis syndrome or cytokine release syndrome (CRS), which can be fatal to patients. Tumor lysis syndrome and CRS can occur due to the administration of CAR T cells that cannot be easily regulated and are uncontrollably activated. Therefore, although CAR T cells show great promise as a tool in the treatment of diseases such as cancer, additional CAR T cell therapies are needed that provide reduced off-target toxicity and more precise control of CAR T cell activation. SUMMARY OF THE INVENTION

[0007] The inventors have discovered a method for reducing off-target toxicity and more precisely controlling CAR T cell activation, which provides an important advance in CAR T cell therapy. In various embodiments described herein, a small molecule ligand linked to a targeting moiety via a linker is used as a bridge between cancer and CAR T cells expressing a CAR, wherein the CAR comprises an E2 anti-fluorescein antibody fragment. The bridge directs CAR T cells expressing a CAR comprising an E2 anti-fluorescein antibody fragment to the cancer to ameliorate the cancer. In one embodiment, the "small molecule ligand" can be, for example, folic acid, DUPA, NK-1R ligand, CAIX ligand, γ-glutamyl transpeptidase, NKG2D ligand, or CCK2R ligand, each of which is a small molecule ligand that specifically binds to cancer cells (i.e., the receptors for these ligands are overexpressed on cancer compared to normal tissue).

[0008] In one embodiment, the "small molecule ligand" is linked to a "targeting moiety" that binds to the CAR expressed by the CAR T cell. In various embodiments, the "targeting moiety" can be selected from, for example, fluorescein, fluorescein isothiocyanate (FITC), NHS-, and / or fluorescein.

[0009] The "targeting moiety" binds to the recognition region of a genetically engineered CAR that expresses an anti-fluorescein E2 antibody fragment. Thus, the recognition region of the CAR (e.g., single-chain variable region (scFv), Fab, Fv, Fc, (Fab')2 fragment, etc. of the anti-fluorescein E2 antibody fragment) is directed to the "targeting moiety". Thus, a small molecule ligand linked to the targeting moiety via a linker serves as a bridge between the cancer and the CAR T cells expressing the anti-fluorescein E2 antibody fragment, directing the CAR T cells to the cancer to improve the cancer.

[0010] In one embodiment, a method of treating cancer is provided. The method comprises 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, ii) administering to the patient a first dose of a CAR T cell composition comprising CAR T cells, wherein the CAR T cells comprise a CAR directed to the targeting moiety and wherein the CAR comprises an anti-fluorescein E2 antibody fragment, and iii) administering to the patient a second dose of a CAR T cell composition comprising CAR T cells, wherein the CAR T cells comprise a CAR directed to the targeting moiety and wherein the CAR comprises an anti-fluorescein E2 antibody fragment.

[0011] In another embodiment, a method of treating cancer is provided. The method comprises 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 ii) administering to the patient a CAR T cell composition, wherein the CAR T cell composition comprises CAR T cells, wherein the CAR T cells comprise a CAR directed to the targeting moiety, wherein the CAR comprises an anti-fluorescein E2 antibody fragment, and wherein the CAR T cell composition comprises a mixture of the CAR T cells and non-transformed T cells.

[0012] In yet another embodiment, a method of treating cancer is provided. The method comprises 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, ii) administering to the patient a CAR T cell composition, wherein the CAR T cell composition comprises CAR T cells, wherein the CAR T cells comprise a CAR directed to the targeting moiety and wherein the CAR comprises an anti-fluorescein E2 antibody fragment, and iii) administering to the patient a folic acid compound, a conjugate comprising a folic acid compound (wherein the conjugate comprising a folic acid compound does not comprise the targeting moiety), or an agent that inhibits the activation of CAR T cells.

[0013] In yet another embodiment, a method of treating cancer is provided. The method comprises 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, 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 more than 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 CAR T cells, wherein the CAR T cells comprise a CAR directed to the targeting moiety and wherein the CAR comprises an E2 anti-fluorescein antibody fragment.

[0014] In another illustrative embodiment, a method of 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 compound or a pharmaceutically acceptable salt thereof in the second dose is at least about 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 CAR T cells, wherein the CAR T cells comprise a CAR directed to the targeting moiety and wherein the CAR comprises an E2 anti-fluorescein antibody fragment.

[0015] In another embodiment, a method of treating cancer is provided. The method comprises 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 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 to the patient a CAR T cell composition comprising CAR T cells, wherein the CAR T cells comprise a 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.

[0016] In yet another embodiment, a method of treating cancer is provided. The method comprises i) continuously 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, ii) administering to the patient a CAR T cell composition comprising CAR T cells, wherein the CAR T cells comprise 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.

[0017] In another embodiment, a method of treating cancer is provided. The method comprises 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 wherein the compound or a pharmaceutically acceptable salt thereof is administered to the patient once a week, and ii) administering to the patient a CAR T cell composition comprising CAR T cells, wherein the CAR T cells comprise a CAR, wherein the CAR comprises an E2 anti-fluorescein antibody fragment.

[0018] In yet another embodiment, a method of 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 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 to the patient a dose of the CAR T cell composition, 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.

[0019] Other embodiments are also described by the following enumerated clauses. Any one of the following embodiments is also contemplated in combination with any applicable embodiments described in the Summary of the Invention section, the Detailed Description of the Illustrative Embodiments section, the Examples section, or the Claims section of this application.

[0020] 1. A method of treating cancer, the method comprising: 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; ii) administering to the patient a first dose of a CAR T cell composition comprising CAR T cells, wherein the CAR T cells comprise a CAR directed to the 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 CAR T cells, wherein the CAR T cells comprise a CAR directed to the targeting moiety and wherein the CAR comprises an E2 anti-fluorescein antibody fragment.

[0021] 2. A method of treating cancer, the method comprising: 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 ii) Administering to the patient a CAR T cell composition, wherein the CAR T cell composition comprises CAR T cells, wherein the CAR T cells comprise a CAR directed to the 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 cells and non-transformed T cells.

[0022] 3. A method of treating cancer, the method comprising: 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; ii) Administering to the patient a CAR T cell composition, wherein the CAR T cell composition comprises CAR T cells, wherein the CAR T cells comprise a CAR directed to the targeting moiety and wherein the CAR comprises an E2 anti-fluorescein antibody fragment; and iii) Administering to the patient a folic acid compound, a conjugate comprising a folic acid compound (wherein the conjugate comprising a folic acid compound does not comprise a targeting moiety) or an agent that inhibits the activation of CAR T cells.

[0023] 4. The method of clause 3, wherein step iii comprises administering a folic acid compound.

[0024] 5. The method of any one of clauses 3 or 4, wherein step iii comprises administering folic acid or leucovorin.

[0025] 6. The method of clause 3, wherein step iii comprises administering the conjugate comprising a folic acid compound.

[0026] 7. The method of clause 6, wherein the conjugate comprising a folic acid compound comprises a folic acid compound linked to one or more amino acids.

[0027] 8. The method of clause 7, wherein the conjugate comprising a folic acid compound has the following formula

[0028] 9. The method of any one of clauses 3 to 8, wherein the folic acid compound has the following formula: wherein X 1 and Y 1 are each independently selected from halogen, R 2 , OR 2 , SR 3 and NR 4 R 5 ; U, V and W represent divalent moieties which are 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-; X 2 and X 3 are 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 alkoxy, wherein Z is oxygen or sulfur; R 1 is selected from hydrogen, halogen, C1-C 12 alkyl and C1-C 12 alkoxy; R 2 , R 3 , R 4 , R 4a , R 4b , R 5 , R 5b , R 6b and R 7b are 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 12Alkynyl, (C1-C 12 alkoxy)carbonyl, and (C1-C 12 alkylamino)carbonyl; R 6 and R 7 are each independently selected from hydrogen, halogen, C1-C 12 alkyl, and C1-C 12 alkoxy; or R 6 and R 7 together form a carbonyl group; R 6a and R 7a are each independently selected from hydrogen, halogen, C1-C 12 alkyl, and C1-C 12 alkoxy; or R 6a and R 7a together form a carbonyl group; p, r, s, and t are each independently 0 or 1; and * represents an optional covalent bond connecting to the remainder of the conjugate.

[0029] 10. The method according to clause 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 the binding of CAR T cells to the compound or a pharmaceutically acceptable salt thereof, but does not bind to the cancer.

[0030] 11. The method according to clause 10, wherein an agent that inhibits the activation of CAR T cells is administered and the agent is a lymphocyte-specific protein tyrosine kinase inhibitor.

[0031] 12. The method according to clause 11, wherein the lymphocyte-specific protein tyrosine kinase inhibitor is Dasatinib.

[0032] 13. The method according to clause 10, wherein an agent that inhibits the activation of CAR T cells is administered and the agent is a PI3 kinase inhibitor.

[0033] 14. The method according to clause 13, wherein the PI3 kinase inhibitor is GDC0980.

[0034] 15. The method according to clause 10, wherein an agent that inhibits the activation of CAR T cells is administered and the agent is an IL-2 inducible T cell kinase inhibitor.

[0035] 16. The method according to clause 15, wherein the IL-2 inducible T cell kinase inhibitor is BMS-509744.

[0036] 17. The method of clause 10, wherein an agent that inhibits the activation of CAR T cells is administered, and the agent is one that blocks the binding of CAR T cells to the compound or a pharmaceutically acceptable salt thereof, but does not bind to the cancer agent.

[0037] 18. The method of clause 17, wherein the agent is fluorescamine, FITC, or sodium fluorescein.

[0038] 19. The method of clause 18, wherein the agent is FITC.

[0039] 20. The method of clause 18, wherein the agent is sodium fluorescein.

[0040] 21. The method of any one of clauses 17 to 20, wherein the agent that inhibits the activation of CAR T cells is administered at a dose of about 0.01 to about 300 μmol / kg of patient body weight.

[0041] 22. The method of any one of clauses 17 to 20, wherein the agent that inhibits the activation of CAR T cells is administered at a dose of about 0.06 to about 100 μmol / kg of patient body weight.

[0042] 23. The method of any one of clauses 17 to 20, wherein the agent that inhibits the activation of CAR T cells is administered at a dose of about 0.06 to about 90 μmol / kg of patient body weight.

[0043] 24. The method of any one of clauses 17 to 20, wherein the agent that inhibits the activation of CAR T cells is administered at a dose of about 0.06 to about 80 μmol / kg of patient body weight.

[0044] 25. The method of any one of clauses 17 to 20, wherein the agent that inhibits the activation of CAR T cells is administered at a dose of about 0.06 to about 70 μmol / kg of patient body weight.

[0045] 26. The method of any one of clauses 17 to 20, wherein the agent that inhibits the activation of CAR T cells is administered at a dose of about 0.06 to about 60 μmol / kg of patient body weight.

[0046] 27. The method of any one of clauses 17 to 20, wherein the agent that inhibits the activation of CAR T cells is administered at a dose of about 0.06 to about 50 μmol / kg of patient body weight.

[0047] 28. The method of any one of clauses 17 to 20, wherein the agent that inhibits the activation of CAR T cells is administered at a dose of about 0.06 to about 40 μmol / kg of patient body weight.

[0048] 29. The method according to any one of clauses 17 to 20, wherein the agent for inhibiting the activation of CAR T cells is administered at a dose of about 0.06 to about 30 μmol / kg of patient body weight.

[0049] 30. The method according to any one of clauses 17 to 20, wherein the agent for inhibiting the activation of CAR T cells is administered at a dose of about 0.06 to about 20 μmol / kg of patient body weight.

[0050] 31. The method according to any one of clauses 17 to 20, wherein the agent for inhibiting the activation of CAR T cells is administered at a dose of about 0.06 to about 10 μmol / kg of patient body weight.

[0051] 32. The method according to any one of clauses 17 to 20, wherein the agent for inhibiting the activation of CAR T cells is administered at a dose of about 0.06 to about 8 μmol / kg of patient body weight.

[0052] 33. The method according to any one of clauses 17 to 20, wherein the agent for inhibiting the activation of CAR T cells is administered at a dose of about 0.06 to about 6 μmol / kg of patient body weight.

[0053] 34. The method according to any one of clauses 3 to 33, wherein more than one dose of the folic acid compound, the conjugate comprising a folic acid compound (wherein the conjugate comprising a folic acid compound does not include a targeting moiety), or the agent for inhibiting the activation of CAR T cells is administered to the patient.

[0054] 35. The method according to any one of clauses 3 to 34, wherein the folic acid compound, the conjugate comprising a folic acid compound (wherein the conjugate comprising a folic acid compound does not include a targeting moiety), or the agent for inhibiting the activation of CAR T cells is administered to the patient before and / or after the compound or its pharmaceutically acceptable salt.

[0055] 36. The method according to any one of clauses 3 to 35, wherein the administration of the folic acid compound, the conjugate comprising a folic acid compound (wherein the conjugate comprising a folic acid compound does not include a targeting moiety), or the agent for inhibiting the activation of CAR T cells results in a decrease in cytokine levels in the patient.

[0056] 37. The method according to clause 36, wherein the decrease in cytokine levels occurs about 3 hours after the administration of the folic acid compound, the conjugate comprising a folic acid compound (wherein the conjugate comprising a folic acid compound does not include a targeting moiety), or the agent for inhibiting the activation of CAR T cells to the patient.

[0057] 38. The method according to clause 36, wherein a decrease in cytokine level occurs about 6 hours after administering to the patient the folic acid compound, the conjugate comprising a folic acid compound (wherein the conjugate comprising a folic acid compound does not include a targeting moiety), or the agent that inhibits the activation of CAR T cells.

[0058] 39. The method according to any one of clauses 36 to 38, wherein the decrease in cytokine level is a decrease to nearly the cytokine level in an untreated patient.

[0059] 40. The method according to any one of clauses 3 to 39, wherein the compound or its pharmaceutically acceptable salt is administered before and subsequent to administering the folic acid compound, the conjugate comprising a folic acid compound (wherein the conjugate comprising a folic acid compound does not include a targeting moiety), or the agent that inhibits the activation of CAR T cells.

[0060] 41. The method according to any one of clauses 3 to 40, wherein after administering the folic acid compound, the conjugate comprising a folic acid compound (wherein the conjugate comprising a folic acid compound does not include a targeting moiety), or the agent that inhibits the activation of CAR T cells, the number of CAR T cells in the patient's blood increases, even though the cytokine level in the patient decreases.

[0061] 42. The method according to any one of clauses 3 to 41, wherein after administering the folic acid compound, the conjugate comprising a folic acid compound (wherein the conjugate comprising a folic acid compound does not include a targeting moiety), or the agent that inhibits the activation of CAR T cells, CAR T cell activation is enhanced or maintained relative to a patient not treated with a rescue agent, even though the cytokine level in the treated patient decreases.

[0062] 43. The method according to any one of clauses 3 to 42, wherein the cancer includes a tumor and when the folic acid compound, the conjugate comprising a folic acid compound (wherein the conjugate comprising a folic acid compound does not include a targeting moiety), or the agent that inhibits the activation of CAR T cells is administered to the patient, the size of the tumor in the patient does not increase.

[0063] 44. The method according to clause 43, wherein a complete response to the tumor is obtained.

[0064] 45. The method according to any one of clauses 3 to 44, wherein when the CRS grade reaches 1, 2, 3, or 4, the folic acid compound, the conjugate comprising a folic acid compound (wherein the conjugate comprising a folic acid compound does not include a targeting moiety), or the agent that inhibits the activation of CAR T cells is administered to the patient.

[0065] 46. The method according to clause 45, wherein when the CRS level reaches 3 or 4, an agent that inhibits the activation of CAR T cells is administered to the patient.

[0066] 47. The method according to clauses 3 to 46, wherein pulmonary edema is reduced.

[0067] 48. The method according to any one of clauses 1 to 47, wherein the compound or its pharmaceutically acceptable salt is administered at a dose of about 10 nmol / kg of patient body weight to about 2500 nmol / kg of 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.

[0068] 49. The method according to clause 48, wherein the compound or its pharmaceutically acceptable salt is administered at a dose of about 10 nmol / kg of patient body weight to about 100 nmol / kg of patient body weight.

[0069] 50. The method according to any one of clauses 48 to 49, wherein the compound or its pharmaceutically acceptable salt is administered at a dose of about 10 nmol / kg of patient body weight to about 50 nmol / kg of patient body weight.

[0070] 51. The method according to any one of clauses 48 to 50, wherein the compound or its pharmaceutically acceptable salt is administered at a dose of about 10 nmol / kg of patient body weight to about 20 nmol / kg of patient body weight.

[0071] 52. The method according to clause 48, wherein the compound or its pharmaceutically acceptable salt is administered at a dose of about 10 nmol / kg of patient body weight to about 600 nmol / kg of patient body weight.

[0072] 53. The method according to clause 48, wherein the compound or its pharmaceutically acceptable salt is administered at a dose of about 200 nmol / kg of patient body weight to about 600 nmol / kg of patient body weight.

[0073] 54. The method according to clause 48, wherein the compound or its pharmaceutically acceptable salt is administered at a dose of about 400 nmol / kg of patient body weight to about 600 nmol / kg of patient body weight.

[0074] 55. The method according to any one of clauses 48 to 54, wherein the dose of the CAR T cells is about 1 million CAR T cells to about 12.5 million CAR T cells.

[0075] 56. The method according to any one of clauses 48 to 55, wherein the dose of the CAR T cells is about 1 million CAR T cells to about 7 million CAR T cells.

[0076] 57. A method according to any one of clauses 48 to 56, wherein the dose of the CAR T cells is from about 1 million CAR T cells to about 5 million CAR T cells.

[0077] 58. A method according to any one of clauses 48 to 57, wherein the dose of the CAR T cells is from about 2 million CAR T cells to about 5 million CAR T cells.

[0078] 59. A method according to any one of clauses 1 to 58, further comprising the step of terminating the continuous administration of the compound or its pharmaceutically acceptable salt to inhibit or prevent cytokine release syndrome in the patient.

[0079] 60. A method according to any one of clauses 1 to 59, wherein the compound or its pharmaceutically acceptable salt is continuously administered to the patient for at least one hour.

[0080] 61. A method according to any one of clauses 1 to 59, wherein the compound or its pharmaceutically acceptable salt is continuously administered to the patient for at least four hours.

[0081] 62. A method according to any one of clauses 1 to 59, wherein the compound or its pharmaceutically acceptable salt is continuously administered to the patient for at least six hours.

[0082] 63. A method according to any one of clauses 1 to 62, wherein the compound or its pharmaceutically acceptable salt is administered to the patient every other day.

[0083] 64. A method according to any one of clauses 1 to 62, wherein the compound or its pharmaceutically acceptable salt is administered to the patient three times a week.

[0084] 65. A method according to any one of clauses 1 to 62, wherein the compound or its pharmaceutically acceptable salt is administered to the patient twice a week.

[0085] 66. A method according to any one of clauses 1 to 62, wherein the compound or its pharmaceutically acceptable salt is administered to the patient once a week.

[0086] 67. A method according to any one of clauses 1 to 62, wherein the compound or its pharmaceutically acceptable salt is administered to the patient until unacceptable weight loss, fever, blood pressure drop, or pulmonary edema occurs in the patient.

[0087] 68. A method of treating cancer, the method comprising: 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, 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, and wherein the amount of the second dose of the compound or a pharmaceutically acceptable salt thereof is about 2-fold to about 15,000-fold more than the amount of the first dose of the compound or a pharmaceutically acceptable salt thereof; and ii) Administering to the patient a CAR T cell composition comprising CAR T cells, wherein the CAR T cells comprise a CAR directed to the targeting moiety and wherein the CAR comprises an E2 anti-fluorescein antibody fragment.

[0088] 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, and wherein the amount of the second dose of the compound or a pharmaceutically acceptable salt thereof is about 2-fold to about 750-fold more than the amount of the first dose of the compound or a pharmaceutically acceptable salt thereof, and wherein the amount of the third dose of the compound or a pharmaceutically acceptable salt thereof is about 800-fold to about 10,000-fold more than the amount of the first dose of the compound or a pharmaceutically acceptable salt thereof.

[0089] 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, and wherein the amount of the second dose of the compound or a pharmaceutically acceptable salt thereof is about 2-fold to about 750-fold more than the amount of the first dose of the compound or a pharmaceutically acceptable salt thereof, wherein the amount of the third dose of the compound or a pharmaceutically acceptable salt thereof is about 800-fold to about 7500-fold more than the amount of the first dose of the compound or a pharmaceutically acceptable salt thereof, and wherein the amount of the fourth dose of the compound or a pharmaceutically acceptable salt thereof is about 8000-fold to about 15,000-fold more than the amount of the first dose of the compound or a pharmaceutically acceptable salt thereof.

[0090] 71. The method of clause 70, wherein the amount of the second dose of the compound or a pharmaceutically acceptable salt thereof is about 100-fold more than the amount of the first dose of the compound or a pharmaceutically acceptable salt thereof, wherein the amount of the third dose of the compound or a pharmaceutically acceptable salt thereof is about 1000-fold more than the amount of the first dose of the compound or a pharmaceutically acceptable salt thereof, and wherein the amount of the fourth dose of the compound or a pharmaceutically acceptable salt thereof is about 10,000-fold more than the amount of the first dose of the compound or a pharmaceutically acceptable salt thereof.

[0091] 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, and wherein the amount of the compound or a pharmaceutically acceptable salt thereof in the second dose is about 2 to about 15,000 times more than the amount of the compound or a pharmaceutically acceptable salt thereof in the first dose.

[0092] 73. A method of treating cancer, the method comprising: i) administering to a patient a first dose of a compound or a pharmaceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked to a targeting moiety 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 about 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 CAR T cells, wherein the CAR T cells comprise a CAR directed to the targeting moiety and wherein the CAR comprises an E2 anti-fluorescein antibody fragment.

[0093] 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% lower than the amount of the compound or a pharmaceutically acceptable salt thereof in the first dose.

[0094] 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% lower than the amount of the compound or a pharmaceutically acceptable salt thereof in the first dose.

[0095] 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% lower than the amount of the compound or a pharmaceutically acceptable salt thereof in the first dose.

[0096] 77. 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 90% lower than the amount of the compound or a pharmaceutically acceptable salt thereof in the first dose.

[0097] 78. 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 95% lower than the amount of the compound or a pharmaceutically acceptable salt thereof in the first dose.

[0098] 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% lower than the amount of the compound or a pharmaceutically acceptable salt thereof in the first dose.

[0099] 80. The method according to clause 73, wherein the amount of the second dose of the compound or its pharmaceutically acceptable salt is at least about 97% lower than the amount of the first dose of the compound or its pharmaceutically acceptable salt.

[0100] 81. The method according to clause 73, wherein the amount of the second dose of the compound or its pharmaceutically acceptable salt is at least about 98% lower than the amount of the first dose of the compound or its pharmaceutically acceptable salt.

[0101] 82. The method according to clause 73, wherein the amount of the second dose of the compound or its pharmaceutically acceptable salt is at least about 99% lower than the amount of the first dose of the compound or its pharmaceutically acceptable salt.

[0102] 83. The method according to clause 73, wherein the amount of the second dose of the compound or its pharmaceutically acceptable salt is at least about 99.5% lower than the amount of the first dose of the compound or its pharmaceutically acceptable salt.

[0103] 84. The method according to any one of clauses 73 to 83, wherein the first dose of the compound or its pharmaceutically acceptable salt is from about 100 nmol / kg of patient body weight to about 1000 nmol / kg of patient body weight.

[0104] 85. The method according to any one of clauses 73 to 83, wherein the first dose of the compound or its pharmaceutically acceptable salt is from about 100 nmol / kg of patient body weight to about 900 nmol / kg of patient body weight.

[0105] 86. The method according to any one of clauses 73 to 83, wherein the first dose of the compound or its pharmaceutically acceptable salt is from about 100 nmol / kg of patient body weight to about 800 nmol / kg of patient body weight.

[0106] 87. The method according to any one of clauses 73 to 83, wherein the first dose of the compound or its pharmaceutically acceptable salt is from about 100 nmol / kg of patient body weight to about 700 nmol / kg of patient body weight.

[0107] 88. The method according to any one of clauses 73 to 83, wherein the first dose of the compound or its pharmaceutically acceptable salt is from about 100 nmol / kg of patient body weight to about 600 nmol / kg of patient body weight.

[0108] 89. The method according to any one of clauses 73 to 83, wherein the first dose of the compound or its pharmaceutically acceptable salt is from about 200 nmol / kg of patient body weight to about 600 nmol / kg of patient body weight.

[0109] 90. A method according to any one of clauses 73 to 83, wherein the first dose of the compound or a pharmaceutically acceptable salt thereof is from about 400 nmol / kg patient body weight to about 600 nmol / kg patient body weight.

[0110] 91. A method according to 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.

[0111] 92. A method according to 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.

[0112] 93. A method according to 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.

[0113] 94. A method according to 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.

[0114] 95. A method according to 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.

[0115] 96. A method according to 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.

[0116] 97. A method according to 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.

[0117] 98. A method according to 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.

[0118] 99. A method according to 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.

[0119] 100. A method according to any one of clauses 92 to 99, wherein the second dose of the compound or a pharmaceutically acceptable salt thereof is from about 5 nmol / kg patient body weight to about 40 nmol / kg patient body weight.

[0120] 101. A method according to any one of clauses 92 to 99, wherein the second dose of the compound or a pharmaceutically acceptable salt thereof is from about 40 nmol / kg patient body weight to about 150 nmol / kg patient body weight.

[0121] 102. A method according to 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.

[0122] 103. A method according to 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.

[0123] 104. A method according to 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 the growth of the cancer relative to the first dose of the compound or a pharmaceutically acceptable salt thereof.

[0124] 105. A method according to any one of clauses 73 to 104, wherein the CAR T cells are administered at a dose of from about 1 million CAR T cells to about 40 million CAR T cells.

[0125] 106. A method according to any one of clauses 73 to 105, 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 are administered once a week.

[0126] 107. A method according to 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 a week.

[0127] 108. A method according to 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.

[0128] 109. A method according to any one of clauses 1 to 108, wherein the E2 anti-fluorescein antibody fragment is a scFv fragment.

[0129] 110. A method according to any one of clauses 1 to 109, wherein the CAR protein sequence has at least about 90% identity with SEQ ID NO:2.

[0130] 111. A method according to any one of clauses 1 to 109, wherein the CAR protein sequence has at least about 95% identity with SEQ ID NO:2.

[0131] 112. A method according to any one of clauses 1 to 109, wherein the CAR protein sequence has at least about 98% identity with SEQ ID NO:2.

[0132] 113. A method according to any one of clauses 1 to 112, wherein the CAR binds fluorescein.

[0133] 114. A method according to any one of clauses 1 to 109, wherein the CAR protein sequence has at most about 50 conservative amino acid substitutions and wherein the CAR binds fluorescein.

[0134] 115. A method according to any one of clauses 1 to 109, wherein the CAR is encoded by a polynucleotide having at least about 90% identity with SEQ ID NO:1.

[0135] 116. A method according to 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.

[0136] 117. A method according to any one of clauses 1 to 109, wherein the CAR is encoded by a polynucleotide having at least about 98% identity with SEQ ID NO:1.

[0137] 118. A method according to any one of clauses 115 to 117, wherein the CAR binds fluorescein.

[0138] 119. A method according to any one of clauses 1 to 118, wherein the CAR is encoded by a polynucleotide that hybridizes with the polynucleotide having SEQ ID NO:1 under highly stringent conditions and wherein the CAR binds fluorescein.

[0139] 120. A method according to any one of clauses 1 to 119, wherein the CAR is encoded by the polynucleotide having SEQ ID NO:1 or by a degenerate variant of SEQ ID NO:1.

[0140] 121. A method according to any one of clauses 1 to 120, wherein the ligand is selected from the following: folate compounds, DUPA, NK-1R ligands, CAIX ligands, γ-glutamyl transpeptidase ligands, NKG2D ligands, and CCK2R ligands.

[0141] 122. A method according to any one of clauses 1 to 121, wherein the targeting moiety is fluorescein or a pharmaceutically acceptable salt thereof.

[0142] 123. The method according to any one of clauses 1 to 122, wherein the linker comprises polyethylene glycol (PEG), polyproline, hydrophilic amino acids, sugars, unnatural peptidoglycans, polyvinylpyrrolidone, pluronic F-127, or a combination thereof.

[0143] 124. The method according to any one of clauses 1 to 123, wherein the linker comprises PEG.

[0144] 125. The method according to any one of clauses 1 to 124, wherein the compound or its pharmaceutically acceptable salt has the following formula B-L-T, 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 following formula wherein n is an integer from 0 to 200.

[0145] 126. The method according to any one of clauses 1 to 125, wherein the cancer is selected from the following: lung cancer, bone cancer, pancreatic cancer, skin cancer, head cancer, neck cancer, cutaneous melanoma, uveal 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, 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 myeloid 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 pelvic cancer, neoplasms of the central nervous system (CNS), primary CNS lymphoma, spinal axis tumors, brainstem glioma, pituitary adenoma, and gastroesophageal junction adenocarcinoma.

[0146] 127. The method according to any one of clauses 1 to 126, wherein the cancer is a cancer that expresses the folate receptor.

[0147] 128. The method according to any one of clauses 1 to 127, wherein the cancer is endometrial cancer.

[0148] 129. The method according to any one of clauses 1 to 127, wherein the cancer is non-small cell lung cancer.

[0149] 130. The method according to any one of clauses 1 to 127, wherein the cancer is ovarian cancer.

[0150] 131. The method according to any one of clauses 1 to 127, wherein the cancer is triple-negative breast cancer.

[0151] 132. The method according to any one of clauses 1 to 127, wherein the cancer is acute myeloid leukemia.

[0152] 133. The method according to clause 132, wherein the cancer expresses the folate receptor-β.

[0153] 134. The method according to any one of clauses 1 to 133, wherein multiple doses of the CAR T cell composition are administered.

[0154] 135. The method according to any one of clauses 1 to 134, wherein at least two doses of the CAR T cell composition are administered.

[0155] 136. The method according to any one of clauses 1 to 135, wherein the patient is imaged before administering the compound or its pharmaceutically acceptable salt, or before administering the CAR T cell composition.

[0156] 137. The method according to any one of clauses 1 to 136, wherein the compound or its pharmaceutically acceptable salt is not an antibody and does not comprise an antibody fragment.

[0157] 138. The method according to any one of clauses 1 to 137, wherein the targeting moiety does not comprise a peptide epitope.

[0158] 139. The method according to any one of clauses 1 to 138, wherein cytokine release leading to off-target toxicity in the patient does not occur and wherein CAR T cell toxicity to the cancer occurs.

[0159] 140. The method according to 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.

[0160] 141. The method according to any one of clauses 1 to 138, wherein the cancer comprises a tumor, wherein the size of the patient's tumor decreases, and wherein off-target toxicity does not occur.

[0161] 142. The method according to any one of clauses 1 to 141, wherein CRS is reduced or prevented and the method results in a reduction in the tumor volume of the patient.

[0162] 143. The method according to any one of clauses 1 to 142, wherein weight loss due to CRS is reduced or prevented.

[0163] 144. The method according to any one of clauses 59 to 67, further comprising administering to the patient the compound or its pharmaceutically acceptable salt.

[0164] 145. The method of clause 144, wherein subsequent administration of the compound or a pharmaceutically acceptable salt thereof results in an increase in CAR T cell activation and cytokine levels in the patient.

[0165] 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 obtained.

[0166] 147. The method of any one of clauses 1 to 146, wherein the CAR T cells have a central memory / effector memory phenotype.

[0167] 148. The method of 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

[0168] Figure 1 The graphical method shows the E2 construct relative to the 4M5.3 construct and shows the map of the E2 construct.

[0169] Figure 2 Shows the EC17 fixed dose and dose de-escalation graph.

[0170] Figure 3A and 3B Shows the E2-CAR-T anti-tumor activity (30 million cells) and body weight change with decreasing EC17 dose. As shown, anti-tumor activity is maintained after NaFL rescue.

[0171] Figure 4A and 4B Shows the 4M5.3-CAR-T anti-tumor activity (30 million cells) and body weight change with decreasing EC17 dose. 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 are observed.

[0172] Figure 5A and 5B Shows the E2-CAR-T anti-tumor activity (10 and 20 million cells) and body weight change under a fixed EC17 dosing regimen (500 nmol / kg, SIW). As shown, anti-tumor activity is maintained after NaFL rescue.

[0173] Figure 6A and 6B Shows the 4M5.3-CAR-T anti-tumor activity (10 and 20 million cells) and body weight change under a fixed EC17 dosing regimen (500 nmol / kg, SIW). As shown, the activity of 4M5.3-CAR-T is lower than that of E2-CAR-T.

[0174] Figure 7 Figure A and Figure 7B show the phenotypic characterization of E2 CAR T cells before infusion into NSG mice.

[0175] Figure 8A and 8B show a comparison of the differentiated phenotypes of CAR-Ts in different formulations, which include E2-CAR-T cells, 4M5.3-CAR-T cells, and GFP+4M5.3 CAR-T cells.

[0176] Figure 9A and 9B show the binding of DIG-labeled E2-IgG to FITC.

[0177] Figure 10A and 10B show the IHC staining of DIG-labeled E2 antibody on FITC-labeled KB cells.

[0178] Figure 11 shows the IHC staining of the bound DIG-E2 antibody in normal human organ tissue: the adrenal gland. Panel A: Test tissue sections pre-incubated with the DIG-E2 antibody; Panel B: Control tissue sections not pre-incubated with the DIG-E2 antibody.

[0179] Figure 12 shows the IHC staining of the bound DIG-E2 antibody in normal human organ tissue: the bone marrow. Panel A: Test tissue sections pre-incubated with the DIG-E2 antibody; Panel B: Control tissue sections not pre-incubated with the DIG-E2 antibody.

[0180] Figure 13 shows the IHC staining of the bound DIG-E2 antibody in normal human organ tissue: the breast. Panel A: Test tissue sections pre-incubated with the DIG-E2 antibody; Panel B: Control tissue sections not pre-incubated with the DIG-E2 antibody.

[0181] Figure 14 shows the IHC staining of the bound DIG-E2 antibody in normal human organ tissue: the cerebellum. Panel A: Test tissue sections pre-incubated with the DIG-E2 antibody; Panel B: Control tissue sections not pre-incubated with the DIG-E2 antibody.

[0182] Figure 15 shows the IHC staining of the bound DIG-E2 antibody in normal human organ tissue: the cervix. Panel A: Test tissue sections pre-incubated with the DIG-E2 antibody; Panel B: Control tissue sections not pre-incubated with the DIG-E2 antibody.

[0183] 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.

[0184] 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.

[0185] 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.

[0186] 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.

[0187] 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.

[0188] 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.

[0189] 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.

[0190] 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.

[0191] 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.

[0192] Figure 25 shows IHC staining of DIG-E2 antibody bound in normal human organ tissue: the lung. Panel A: Test tissue sections pre-incubated with the DIG-E2 antibody; Panel B: Control tissue sections not pre-incubated with the DIG-E2 antibody.

[0193] Figure 26 shows IHC staining of DIG-E2 antibody bound in normal human organ tissue: the lymph node. Panel A: Test tissue sections pre-incubated with the DIG-E2 antibody; Panel B: Control tissue sections not pre-incubated with the DIG-E2 antibody.

[0194] Figure 27 shows IHC staining of DIG-E2 antibody bound in normal human organ tissue: the nerve. Panel A: Test tissue sections pre-incubated with the DIG-E2 antibody; Panel B: Control tissue sections not pre-incubated with the DIG-E2 antibody.

[0195] Figure 28 shows IHC staining of DIG-E2 antibody bound in normal human organ tissue: the ovary. Panel A: Test tissue sections pre-incubated with the DIG-E2 antibody; Panel B: Control tissue sections not pre-incubated with the DIG-E2 antibody.

[0196] Figure 29 shows IHC staining of DIG-E2 antibody bound in normal human organ tissue: the pancreas. Panel A: Test tissue sections pre-incubated with the DIG-E2 antibody; Panel B: Control tissue sections not pre-incubated with the DIG-E2 antibody.

[0197] Figure 30 shows IHC staining of DIG-E2 antibody bound in normal human organ tissue: the prostate. Panel A: Test tissue sections pre-incubated with the DIG-E2 antibody; Panel B: Control tissue sections not pre-incubated with the DIG-E2 antibody.

[0198] Figure 31 shows IHC staining of DIG-E2 antibody bound in normal human organ tissue: the skin. Panel A: Test tissue sections pre-incubated with the DIG-E2 antibody; Panel B: Control tissue sections not pre-incubated with the DIG-E2 antibody.

[0199] Figure 32 shows IHC staining of DIG-E2 antibody bound in normal human organ tissue: the small intestine. Panel A: Test tissue sections pre-incubated with the DIG-E2 antibody; Panel B: Control tissue sections not pre-incubated with the DIG-E2 antibody.

[0200] Figure 33 shows IHC staining of DIG-E2 antibody bound in normal human organ tissue: the stomach. Panel A: Test tissue sections pre-incubated with the DIG-E2 antibody; Panel B: Control tissue sections not pre-incubated with the DIG-E2 antibody.

[0201] Figure 34 shows normal human organ tissue: IHC staining of DIG-E2 antibody conjugated in 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.

[0202] Figure 35 shows normal human organ tissue: IHC staining of DIG-E2 antibody conjugated in testis. Panel A: Test tissue section pre-incubated with DIG-E2 antibody; Panel B: Control tissue section not pre-incubated with DIG-E2 antibody.

[0203] Figure 36 shows normal human organ tissue: IHC staining of DIG-E2 antibody conjugated in thymus. Panel A: Test tissue section pre-incubated with DIG-E2 antibody; Panel B: Control tissue section not pre-incubated with DIG-E2 antibody.

[0204] Figure 37 shows normal human organ tissue: IHC staining of DIG-E2 antibody conjugated in tongue. Panel A: Test tissue section pre-incubated with DIG-E2 antibody; Panel B: Control tissue section not pre-incubated with DIG-E2 antibody.

[0205] Figure 38 shows normal human organ tissue: IHC staining of DIG-E2 antibody conjugated in uterus. Panel A: Test tissue section pre-incubated with DIG-E2 antibody; Panel B: Control tissue section not pre-incubated with DIG-E2 antibody.

[0206] Figure 39 Graph showing T-cell activation in 1-day co-cultures with various target cells. The percentage of E2 CAR T cells activated after 1-day co-culture (y-axis) is plotted against the FR expression level of target cells at the time of assay (x-axis). (▼) OV90 cells; (◆) SKOV3 cells; (▲) IGROV1 cells; (■) HOS-Frα cells; (●) MDA-MB-231 cells.

[0207] Figure 40 Graph showing target cell apoptosis in 2-day co-cultures 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 co-culture with target cells without EC17 pre-treatment; right bar, CAR T co-culture with treated target cells with EC17 pre-treatment.

[0208] Figure 41A figure showing the expression levels of functional folate receptors in 5 different cell types (OV90 cells, SKOV3 cells, IGROV1 cells, HOS-Frα cells, MDA-MB-231 cells).

[0209] Figure 42 To show the experiment timeline for tumor-bearing and naive mice, a sketch.

[0210] Figure 43 (Left panel) A figure showing that the levels of the cytokine IFN-γ in both naive and tumor-bearing mice are EC-17 dependent, and that the increase in IFN-γ in naive mice is much lower (23-fold lower) compared to mice bearing MDA-MB-231 tumors. Figure 43 (Right panel) A figure showing FACS analysis of CAR-T expansion in mice bearing MDA-MB-231 tumors and no detectable CAR-T cell expansion in naive mice.

[0211] Figure 44 (Upper panel) A figure showing the following: In co-cultures with the positive control cell line K562-OKT3, cytokine (IL-2) production from mock T-cells and anti-FLCAR T-cells is at similar quantitative levels (left pair of bars, where mock is the left bar and anti-FLCAR T-cells are the right bar); no cytokine is produced by mock or anti-FLCAR T-cells after co-culture with K562 (middle); and anti-FLCAR T-cells are the only cells capable of inducing cytokine IL-2 secretion, but only when pre-treated with EC-17 (right, where only anti-FLCAR T-cells show results). Figure 44 (Middle panel) A figure showing the following: In co-cultures with the positive control cell line K562-OKT3, cytokine (IFN-γ) production from mock T-cells and anti-FLCAR T-cells is at similar quantitative levels (left pair of bars, where mock is the left bar and anti-FLCAR T-cells are the right bar); no cytokine is produced by mock or anti-FLCAR T-cells after co-culture with K562 (middle); and anti-FLCAR T-cells are the only cells capable of inducing cytokine IFN-γ secretion, but only when pre-treated with EC-17 (right, where only anti-FLCAR T-cells show results). Figure 44(The figure below) shows the following: in co-cultures with the positive control cell line K562-OKT3, cytokine (TNF-α) production from mock T-cells and anti-FLCAR T-cells was at similar quantitative levels (left pair of bars, where mock is the left bar and anti-FLCAR T-cells are the right bar); no cytokine was produced by mock or anti-FLCAR T-cells after co-culture with K562 (middle); and anti-FLCAR T-cells were the only cells capable of inducing cytokine TNF-α secretion, but only when pre-treated with EC-17 (right, where only results for anti-FLCAR T-cells are shown).

[0212] Figure 45 (Upper left) is a graph showing the percentage lysis when CD8+ mock T-cells and anti-FLCAR T-cells were co-cultured with the negative control K562 cells at ratios of 30:1, 10:1, 3:1, or 1:1. Figure 45 (Upper right) is a graph showing the percentage lysis when CD8+ mock T-cells and anti-FLCAR T-cells were co-cultured with K562+OKT3 target cells at ratios of 30:1, 10:1, 3:1, or 1:1. Figure 45 (Lower left) is a graph showing the percentage lysis when CD8+ mock T-cells and anti-FLCAR T-cells were co-cultured with unlabeled MDA-MB-231 cells at ratios of 30:1, 10:1, 3:1, or 1:1. Figure 45 (Lower right) is a graph showing the percentage lysis when CD8+ mock T-cells and anti-FLCAR T-cells were co-cultured with EC17-labeled MDA-MB-231 cells at ratios of 30:1, 10:1, 3:1, or 1:1. In all graphs, (x) CD8+ mock T-cells; (○) anti-FLCAR T-cells.

[0213] Figure 46 (Upper) shows the chemical structure of FITC-folate. Figure 46 (Lower) is a graph showing the dose-response curve of FITC-folate.

[0214] Figure 47 (Upper) shows the chemical structure of FITC-DUPA. Figure 47 (Lower) is a graph showing the dose-response curve of FITC-DUPA.

[0215] Figure 48 (Upper) shows the chemical structure of FITC-CA9. Figure 48 (Lower) is a graph showing the dose-response curve of FITC-CA9.

[0216] Figure 49 (Upper) shows the chemical structure of FITC-NK1R.Figure 49 (Bottom) is a graph showing the dose-response curve of FITC-NK1R.

[0217] Figure 50 Show the binding of the bridge to tumor cells in the in vivo model that express the receptor for the small molecule ligand corresponding to the bridge (via FACS analysis).

[0218] Figure 51 Show potent FR-dependent tumor cell killing by EC17-induced CAR T cells.

[0219] Figure 52 Show the correlation between the cytolytic activity of CAR T cells and the functional FR level on tumor cells.

[0220] Figure 53 Show EC17 / FR-dependent CAR T cell activation and exhaustion. Show 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 pre-loaded with or without EC17 (100 nM, 30-minute pulse at 37°C). The first two open bars represent CAR-T and mock-transduced T cells only without target cells. For the legends of the second to fourth figures, the bars from top to bottom = from left to right.

[0221] Figure 54 Show the EC17 / FR-dependent CAR T cell exhaustion profile. Top row: colored pie charts showing the changes in the differentiation state of CAR-T cells in cultures without target cells from day 0 to day 3. Bottom row: four sets of colored pie charts showing 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 pre-loaded with or without EC17 (0.1 or 10 nM, 30-minute pulse at 37°C).

[0222] Figure 55Display the all-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 truncated cell surface polypeptide of non-functional epidermal growth factor receptor (EGFRt). Bottom: Example of CD4 / CD8 T cell phenotype analysis performed by flow cytometry on EGFRt-sorted (left pie chart) CAR-T cell preparations and unsorted "clinical fax" (right pie chart). Color symbols are explained as shown.

[0223] Figure 56 , Panel A: Kd value of H-EC17 uptake by FR+ target cells (calculated from the number of bound molecules / cell) after incubation at 37 °C for 2 hours. Panel B: Kd value of H-EC17 uptake by E2-CAR-T cells (approximately 24% EGFRt+, approximately 95:5 CD8 / CD4 ratio) (calculated from total cell-associated radioactivity, DPM) after incubation at room temperature for 2 hours. 3 H-EC17 uptake by FR+ target cells (calculated from the number of bound molecules / cell) after incubation at 37 °C for 2 hours. Panel B: Kd value of H-EC17 uptake by E2-CAR-T cells (approximately 24% EGFRt+, approximately 95:5 CD8 / CD4 ratio) (calculated from total cell-associated radioactivity, DPM) after incubation at room temperature for 2 hours. 3 (Panels A and B) Show the CRS grading scale and experimental conditions applicable to fluorescein sodium rescue in HOS-FRα tumor-bearing mice. Show changes in T cell-derived cytokines with and without rescue. p values were calculated by Student's t-test.

[0224] Figure 57 (Panels A and B) Show the CRS grading scale and experimental conditions applicable to fluorescein sodium rescue in HOS-FRα tumor-bearing mice. Show changes in T cell-derived cytokines with and without rescue. p values were calculated by Student's t-test.

[0225] Figure 58 Show functional FR levels on tumor cells measured by binding assay based on 3 H-FA (folic acid) (100 nM, 1 hour at 37 °C).

[0226] Figure 59 Show (Panel A) the full range of EC17 dose-response in specific lysis (%) of 5 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) maximum lysis (%) and EC50 values obtained from dose-response curves fitted to 100 nM.

[0227] Figure 60Show the correlation of CAR-T cell activity with FR levels and the natural sensitivity of tumor cells. Figure A: Kinetics of specific lysis (%) at different E / T ratios in FR+(MDA-MB-231, KB, THP1-FRβ, OV90, HOS-FRα) and FR-negative (HOS-143b) cell lines after co-culture for 16 and 48 hours in the presence of 10 nM EC17. Figure B: Specific lysis (%) of target cells plotted against the E / T ratio in the linear range. High FR+ KB cells demonstrated early resistance at 16 hours, while FR-negative HOS-143b did not respond. Figure C: Excluding KB cells, a semi-logarithmic correlation was established between specific lysis (%) at 16-hour co-culture and the functional FR levels on tumor cells. Figure D: 3D plot depicting the relationship between Th1 cytokine (IFNγ, IL-2, and TNFα) levels derived from CAR-T cells and varying E / T ratios after co-culture for 44 hours with 10 nM EC17. Figure E: Th1 cytokine levels derived from CAR-T cells plotted against the FR levels of FR+ target cells on a Log10 scale.

[0228] Figure 61 Show (Figure A) a bar graph that shows the EC17 loading status of target cells verified by flow cytometry and expressed as MFI (EC17 is undetectable on FR-negative cell lines) and (Figure B) target cell apoptosis (%) detected by Annexin V staining after 2 days of co-culture as described Figure 54 above.

[0229] Figure 62 Show the pharmacokinetics and tumor uptake of CAR-T cells in vivo. Figure A: Schematic of the experimental layout to show the animal collection schedule related to CAR-T cell injection and weekly EC17 dose (500 nmol / kg) in MDA-MB-231 tumor-bearing mice. A total of 15 mice received approximately 4.8 million EGFRt-sorted CAR-T cells on day 0 (3 mice started with large tumors). Figure B: The left panel shows the measurement of tumor volume and body weight changes; the middle panel shows CAR-T cell expansion in the blood 7 days after a single dose of EC17; the right bar graph shows the differentiation profiles of circulating CD4 / CD8 CAR-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 panel shows CAR-T cell kinetics in the blood (solid line) relative to CAR-T cell kinetics in the tumor (dashed line). The bottom bar graph shows changes in the CAR-T cell phenotype in the blood. Figure E: Kinetic changes in the surface expression of activation markers (4-1BB, PD1) on tumor-infiltrating CAR-T cells.

[0230] Figure 63Show the effects of dietary folate on anti-tumor activity and CRS toxicity. Figure A: Measurement of changes in tumor volume and body weight of NSG mice with large MDA-MB-231 tumor xenografts treated with CAR-T cells (~10 million “clinical facsimiles”) plus EC17 SIW at 500 nmol / kg while maintaining a FA-sufficient or FA-deficient diet throughout the study (n = 5). Figure B: Bar graph showing circulating CAR-T cells (human CD3ε+EGFRt+) measured at the end of the study on day 52 (FA-deficient) and day 59 (FA-sufficient), respectively. Figure 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 an increase in the number of circulating CAR-T cells in FA-deficient diet mice treated with CAR-T cells plus EC17 compared to mice on the same treatment but with a FA-sufficient diet. Figure D: Loss of FR expression was detected in 2 / 3 of MDA-MB-231 tumors collected from FA-sufficient animals on day 59 and analyzed by flow cytometry (later confirmed by quantitative radioligand binding assay, data not shown).

[0231] Definitions As used herein, “a / an” can mean one or more. As used herein, “about” when referring to a numerical value (including, for example, integers, fractions, and percentages) generally refers to a range of numerical values that a person of ordinary skill in the art would consider equivalent to the recited value (e.g., + / −5% to 10% of the recited value) having the same function or result).

[0232] As used herein, the term “treat / treating / treated / treatment” refers to both therapeutic treatment and prophylactic / preventative treatment.

[0233] As used herein, the term “ameliorate / ameliorating / amelioration / ameliorated” when referring to cancer can mean reducing the symptoms of cancer, reducing the size of a tumor, completely or partially removing a tumor (e.g., 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 would consider a therapeutic, prophylactic / preventative treatment of cancer.

[0234] As used herein, the term "administer" (including "administer", "administering", and "administered") means 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 CAR T cells 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.

[0235] As used herein, the term "off-target toxicity" means organ damage or weight loss in a patient that is unacceptable to the physician treating the patient, or any other effect on the patient that is unacceptable to the physician treating the patient, e.g., B cell hypoplasia, fever, blood pressure drop, or pulmonary edema.

[0236] As used herein, the terms "transduce" and "transfect" are used interchangeably and mean introducing nucleic acid into a cell via any artificial method, including viral and non-viral methods. Detailed Description

[0237] In various embodiments described herein, a small molecule ligand linked via a linker to a targeting moiety is used as a bridge between cancer and CAR T cells (i.e., T cells expressing a chimeric antigen receptor), wherein the CAR T cells comprise a genetically engineered CAR that is directed to the targeting moiety, and wherein the CAR comprises an E2 anti-fluorescein antibody fragment. The bridge directs the CAR T cells to the cancer to improve the cancer. In one embodiment, the "small molecule ligand" can be a folate compound, a CAIX ligand, DUPA, an NK-1R ligand, a ligand for gamma-glutamyl transpeptidase, an NKG2D ligand, or a CCK2R ligand, each of which is a small molecule ligand that specifically binds to a cancer cell type (i.e., the receptor for each of these ligands is overexpressed on the cancer compared to normal tissue).

[0238] The "targeting moiety" linked to the small molecule ligand binds to the recognition region of the genetically engineered CAR expressed by the CAR T cells, and the CAR comprises an E2 anti-fluorescein antibody fragment. Thus, the recognition region of the CAR (e.g., the single-chain variable fragment (scFv) of the E2 anti-fluorescein antibody) is directed to the "targeting moiety". Thus, the small molecule ligand linked via a linker to the targeting moiety acts as a bridge between cancer and CAR T cells, wherein the CAR T cells comprise a genetically engineered CAR, directing the CAR T cells to the cancer to improve the cancer.

[0239] The bridge is an organic small molecule and can thus achieve rapid clearance from the bloodstream (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 that comprises an E2 anti-fluorescein antibody fragment) can target only those cancer cells that express the receptor for the small molecule ligand portion of the "bridge", thereby reducing off-target toxicity to normal tissues. In addition, this system can be "universal" because one 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 moiety recognized by the CAR T cell (wherein the CAR T cell comprises a genetically engineered CAR that comprises an E2 anti-fluorescein antibody fragment) can be maintained constant such that one type of CAR T cell can be used while the small molecule ligand that binds to the cancer can be altered to allow targeting of a broad variety of cancers.

[0240] In various embodiments described herein, the small molecule ligand that is linked to the targeting moiety via a linker is referred to as a "compound".

[0241] In various embodiments, the phrase "E2 anti-fluorescein antibody fragment" means a CAR that comprises a fragment of an E2 anti-fluorescein antibody (e.g., an scFv fragment). The E2 anti-fluorescein antibody is described, for example, in Vaughan et al., Nature Biotechnol. Vol. 14(3), pp. 309-314, 1996, which is incorporated herein by reference. In various embodiments, the CAR can further comprise an IgG4 hinge domain, a CD3ζ activation domain, and / or a 4-1BB co-stimulatory domain, or any other suitable domain (such as an EGFRt domain). In still other embodiments, the CAR can 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 can be encoded by a polynucleotide that hybridizes to the polynucleotide having SEQ ID NO:1 under highly stringent conditions. In still another aspect, the CAR can 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 can 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 can have at most about 50 conservative amino acid substitutions. In any of the embodiments described herein, the CAR binds fluorescein.

[0242] In one embodiment, a method of treating cancer is provided. The method comprises i) administering to a patient a compound or a pharmaceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked via a linker to a targeting moiety, ii) administering to the patient a first dose of a CAR T cell composition, wherein the CAR T cell composition comprises CAR T cells, wherein the CAR T cells comprise a CAR, and wherein the CAR comprises an E2 anti-fluorescein antibody fragment, and iii) administering to the patient a second dose of the CAR T cell composition, wherein the CAR T cell composition comprises CAR T cells, wherein the CAR T cells comprise a CAR, and wherein the CAR comprises an E2 anti-fluorescein antibody fragment.

[0243] In another embodiment, a method of treating cancer is provided. The method comprises i) administering to a patient a compound or a pharmaceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked via a linker to a targeting moiety, and ii) administering to the patient a CAR T cell composition comprising CAR T cells, wherein the CAR T cells comprise 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 cells and non-transformed T cells.

[0244] In yet another embodiment, a method of treating cancer is provided. The method comprises i) administering to a patient a compound or a pharmaceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked via a linker to a targeting moiety, ii) administering to the patient a CAR T cell composition, wherein the CAR T cell composition comprises CAR T cells, wherein the CAR T cells comprise a CAR, wherein the CAR comprises an E2 anti-fluorescein antibody fragment, and iii) administering to the patient a folic acid compound, a conjugate comprising a folic acid compound (wherein the conjugate comprising a folic acid compound does not comprise a targeting moiety), or a drug that inhibits the activation of CAR T cells.

[0245] In another embodiment, a method of treating cancer is provided. The method comprises i) administering to a patient a compound or a pharmaceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked via a linker to a targeting moiety, and wherein the compound or a pharmaceutically acceptable salt thereof is administered at a dose of from about 10 nmol / kg patient body weight to about 2500 nmol / kg patient body weight, and ii) administering to the patient a CAR T cell composition comprising CAR T cells, wherein the CAR T cells comprise a CAR, wherein the CAR comprises an E2 anti-fluorescein antibody fragment, and wherein the dose of the CAR T cells is from about 1 million CAR T cells to about 15 million CAR T cells.

[0246] In yet another embodiment, a method of treating cancer is provided. The method comprises i) continuously administering to a patient a compound or a pharmaceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked via a linker to a targeting moiety, ii) administering to the patient a CAR T cell composition comprising CAR T cells, wherein the CAR T cells comprise 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.

[0247] In another illustrative aspect, a method of treating cancer is provided. The method comprises i) administering to a patient a compound or a pharmaceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked via a linker to a targeting moiety, wherein the patient is administered at least a first dose and a second dose of the compound or a pharmaceutically acceptable salt thereof, wherein the first dose and the second dose are different, and wherein the amount of the second dose of the compound or a pharmaceutically acceptable salt thereof is about 2-fold to about 15,000-fold more than the amount of the first dose of the compound or a pharmaceutically acceptable salt thereof, and ii) administering to the patient a CAR T cell composition comprising CAR T cells, wherein the CAR T cells comprise a CAR, and wherein the CAR comprises an E2 anti-fluorescein antibody fragment.

[0248] In another embodiment, a method of treating cancer is provided. The method comprises i) administering to a patient a compound or a pharmaceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked via a linker to a targeting moiety and wherein the compound or a pharmaceutically acceptable salt thereof is administered to the patient once a week, and ii) administering to the patient a CAR T cell composition comprising CAR T cells, wherein the CAR T cells comprise a CAR, wherein the CAR comprises an E2 anti-fluorescein antibody fragment.

[0249] In yet another embodiment, a method of 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 via a linker to a targeting moiety, 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 CAR T cells, wherein the CAR T cells comprise a CAR and wherein the CAR comprises an E2 anti-fluorescein antibody fragment.

[0250] Several embodiments are described by the clauses listed below. Any one of the following embodiments is also covered in combination with any applicable embodiments described in the Summary of the Invention section, the Detailed Embodiments section, the Examples section, or the claims of this patent application.

[0251] 1. A method of treating cancer, the method comprising: 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 by a linker; ii) administering to the patient a first dose of a CAR T cell composition comprising CAR T cells, wherein the CAR T cells comprise a CAR directed to the 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 CAR T cells, wherein the CAR T cells comprise a CAR directed to the targeting moiety and wherein the CAR comprises an E2 anti-fluorescein antibody fragment.

[0252] 2. A method of treating cancer, the method comprising: 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 by a linker; and ii) administering to the patient a CAR T cell composition, wherein the CAR T cell composition comprises CAR T cells, wherein the CAR T cells comprise a CAR directed to the 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 cells and non-transformed T cells.

[0253] 3. A method of treating cancer, the method comprising: 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 by a linker; ii) administering to the patient a CAR T cell composition, wherein the CAR T cell composition comprises CAR T cells, wherein the CAR T cells comprise a CAR directed to the targeting moiety and wherein the CAR comprises an E2 anti-fluorescein antibody fragment; and iii) administering to the patient a folic acid compound, a conjugate comprising a folic acid compound (wherein the conjugate comprising a folic acid compound does not comprise a targeting moiety), or an agent that inhibits the activation of CAR T cells.

[0254] 4. The method of clause 3, wherein step iii comprises administering a folic acid compound.

[0255] 5. A method according to any one of clauses 3 or 4, wherein step iii comprises administering folic acid or folinic acid.

[0256] 6. A method according to clause 3, wherein step iii comprises administering the conjugate comprising a folic acid compound.

[0257] 7. A method according to clause 6, wherein the conjugate comprising a folic acid compound comprises a folic acid compound linked to one or more amino acids.

[0258] 8. A method according to clause 7, wherein the conjugate comprising a folic acid compound has the following formula

[0259] 9. A method according to any one of clauses 3 to 8, wherein the folic acid compound has the following formula wherein X 1 and Y 1 are each independently selected from halogen, R 2 , OR 2 , SR 3 and NR 4 R 5 ; U, V and W represent divalent moieties, which are 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-; X 2 and X 3 are 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 alkoxy, where Z is oxygen or sulfur; R1 Selected from hydrogen, halogen, C1-C 12 alkyl, and C1-C 12 alkoxy; R 2 、R 3 、R 4 、R 4a 、R 4b 、R 5 、R 5b 、R 6b and R 7b each independently 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; R 6 and R 7 each independently selected from hydrogen, halogen, C1-C 12 alkyl, and C1-C 12 alkoxy; or R 6 and R 7 together form a carbonyl group; R 6a and R 7a each independently selected from hydrogen, halogen, C1-C 12 alkyl, and C1-C 12 alkoxy; or R 6a and R 7a together form a carbonyl group; p, r, s, and t are each independently 0 or 1; and * represents an optional covalent bond connecting to the remainder of the conjugate.

[0260] 10. The method according to clause 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 the binding of CAR T cells to the compound or its pharmaceutically acceptable salt but does not bind to the cancer.

[0261] 11. The method according to clause 10, wherein an agent that inhibits the activation of CAR T cells is administered and the agent is a lymphocyte-specific protein tyrosine kinase inhibitor.

[0262] 12. The method of clause 11, wherein the lymphocyte-specific protein tyrosine kinase inhibitor is dasatinib.

[0263] 13. The method of clause 10, wherein an agent that inhibits the activation of CAR T cells is administered and the agent is a PI3 kinase inhibitor.

[0264] 14. The method of clause 13, wherein the PI3 kinase inhibitor is GDC0980.

[0265] 15. The method of clause 10, wherein an agent that inhibits the activation of CAR T cells is administered and the agent is an IL-2 inducible T cell kinase inhibitor.

[0266] 16. The method of clause 15, wherein the IL-2 inducible T cell kinase inhibitor is BMS-509744.

[0267] 17. The method of clause 10, wherein an agent that inhibits the activation of CAR T cells is administered and it is an agent that blocks the binding of CAR T cells to the compound or a pharmaceutically acceptable salt thereof, but does not bind to the cancer.

[0268] 18. The method of clause 17, wherein the agent is fluorescamine, FITC or sodium fluorescein.

[0269] 19. The method of clause 18, wherein the agent is FITC.

[0270] 20. The method of clause 18, wherein the agent is sodium fluorescein.

[0271] 21. The method of any one of clauses 17 to 20, wherein the agent that inhibits the activation of CAR T cells is administered at a dose of about 0.01 to about 300 μmol / kg of patient body weight.

[0272] 22. The method of any one of clauses 17 to 20, wherein the agent that inhibits the activation of CAR T cells is administered at a dose of about 0.06 to about 100 μmol / kg of patient body weight.

[0273] 23. The method of any one of clauses 17 to 20, wherein the agent that inhibits the activation of CAR T cells is administered at a dose of about 0.06 to about 90 μmol / kg of patient body weight.

[0274] 24. The method of any one of clauses 17 to 20, wherein the agent that inhibits the activation of CAR T cells is administered at a dose of about 0.06 to about 80 μmol / kg of patient body weight.

[0275] 25. A method according to any one of clauses 17 to 20, wherein the agent that inhibits the activation of CAR T cells is administered at a dose of from about 0.06 to about 70 μmol / kg of patient body weight.

[0276] 26. A method according to any one of clauses 17 to 20, wherein the agent that inhibits the activation of CAR T cells is administered at a dose of from about 0.06 to about 60 μmol / kg of patient body weight.

[0277] 27. A method according to any one of clauses 17 to 20, wherein the agent that inhibits the activation of CAR T cells is administered at a dose of from about 0.06 to about 50 μmol / kg of patient body weight.

[0278] 28. A method according to any one of clauses 17 to 20, wherein the agent that inhibits the activation of CAR T cells is administered at a dose of from about 0.06 to about 40 μmol / kg of patient body weight.

[0279] 29. A method according to any one of clauses 17 to 20, wherein the agent that inhibits the activation of CAR T cells is administered at a dose of from about 0.06 to about 30 μmol / kg of patient body weight.

[0280] 30. A method according to any one of clauses 17 to 20, wherein the agent that inhibits the activation of CAR T cells is administered at a dose of from about 0.06 to about 20 μmol / kg of patient body weight.

[0281] 31. A method according to any one of clauses 17 to 20, wherein the agent that inhibits the activation of CAR T cells is administered at a dose of from about 0.06 to about 10 μmol / kg of patient body weight.

[0282] 32. A method according to any one of clauses 17 to 20, wherein the agent that inhibits the activation of CAR T cells is administered at a dose of from about 0.06 to about 8 μmol / kg of patient body weight.

[0283] 33. A method according to any one of clauses 17 to 20, wherein the agent that inhibits the activation of CAR T cells is administered at a dose of from about 0.06 to about 6 μmol / kg of patient body weight.

[0284] 34. A method according to any one of clauses 3 to 33, wherein more than one dose of the folic acid compound, the conjugate comprising a folic acid compound (wherein the conjugate comprising a folic acid compound does not include a targeting moiety), or the agent that inhibits the activation of CAR T cells is administered to the patient.

[0285] 35. A method according to any one of clauses 3 to 34, wherein before and / or after the compound or its pharmaceutically acceptable salt, the folic acid compound, the conjugate comprising a folic acid compound (wherein the conjugate comprising a folic acid compound does not include a targeting moiety), or the agent that inhibits the activation of CAR T cells is administered to the patient.

[0286] 36. A method according to any one of clauses 3 to 35, wherein administration of the folic acid compound, the conjugate comprising a folic acid compound (wherein the conjugate comprising a folic acid compound does not include a targeting moiety), or the agent that inhibits the activation of CAR T cells results in a decrease in cytokine levels in the patient.

[0287] 37. A method according to clause 36, wherein the decrease in cytokine levels occurs at about 3 hours after administration of the folic acid compound, the conjugate comprising a folic acid compound (wherein the conjugate comprising a folic acid compound does not include a targeting moiety), or the agent that inhibits the activation of CAR T cells to the patient.

[0288] 38. A method according to clause 36, wherein the decrease in cytokine levels occurs at about 6 hours after administration of the folic acid compound, the conjugate comprising a folic acid compound (wherein the conjugate comprising a folic acid compound does not include a targeting moiety), or the agent that inhibits the activation of CAR T cells to the patient.

[0289] 39. A method according to any one of clauses 36 to 38, wherein the decrease in cytokine levels is reduced to nearly the cytokine levels in an untreated patient.

[0290] 40. A method according to any one of clauses 3 to 39, wherein before and subsequently after administration of the folic acid compound, the conjugate comprising a folic acid compound (wherein the conjugate comprising a folic acid compound does not include a targeting moiety), or the agent that inhibits the activation of CAR T cells, the compound or its pharmaceutically acceptable salt is administered.

[0291] 41. A method according to any one of clauses 3 to 40, wherein after administration of the folic acid compound, the conjugate comprising a folic acid compound (wherein the conjugate comprising a folic acid compound does not include a targeting moiety), or the agent that inhibits the activation of CAR T cells, the number of CAR T cells in the patient's blood increases, even though the cytokine levels in the patient decrease.

[0292] 42. A method according to any one of clauses 3 to 41, wherein after administration of the folic acid compound, the conjugate comprising a folic acid compound (wherein the conjugate comprising a folic acid compound does not include a targeting moiety), or the agent that inhibits the activation of CAR T cells, CAR T cell activation is enhanced or maintained relative to a patient not treated with a rescue agent, even if cytokine levels in the treated patient are reduced.

[0293] 43. A method according to any one of clauses 3 to 42, wherein the cancer comprises a tumor and when the folic acid compound, the conjugate comprising a folic acid compound (wherein the conjugate comprising a folic acid compound does not include a targeting moiety), or the agent that inhibits the activation of CAR T cells is administered to the patient, the size of the patient's tumor does not increase.

[0294] 44. The method of clause 43, wherein a complete response to the tumor is obtained.

[0295] 45. A method according to any one of clauses 3 to 44, wherein when the CRS grade reaches 1, 2, 3, or 4, the folic acid compound, the conjugate comprising a folic acid compound (wherein the conjugate comprising a folic acid compound does not include a targeting moiety), or the agent that inhibits the activation of CAR T cells is administered to the patient.

[0296] 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.

[0297] 47. A method according to any one of clauses 3 to 46, wherein pulmonary edema is reduced.

[0298] 48. A method according to any one of clauses 1 to 47, wherein the compound or its pharmaceutically acceptable salt is administered at a dose of from about 10 nmol / kg patient body weight to about 2500 nmol / kg patient body weight; and the dose of the CAR T cells is from about 1 million CAR T cells to about 15 million CAR T cells.

[0299] 49. The method of clause 48, wherein the compound or its pharmaceutically acceptable salt is administered at a dose of from about 10 nmol / kg patient body weight to about 100 nmol / kg patient body weight.

[0300] 50. A method according to any one of clauses 48 to 49, wherein the compound or its pharmaceutically acceptable salt is administered at a dose of from about 10 nmol / kg patient body weight to about 50 nmol / kg patient body weight.

[0301] 51. A method according to any one of clauses 48 to 50, wherein the compound or a pharmaceutically acceptable salt thereof is administered at a dose of from about 10 nmol / kg patient body weight to about 20 nmol / kg patient body weight.

[0302] 52. A method according to clause 48, wherein the compound or a pharmaceutically acceptable salt thereof is administered at a dose of from about 10 nmol / kg patient body weight to about 600 nmol / kg patient body weight.

[0303] 53. A method according to clause 48, wherein the compound or a pharmaceutically acceptable salt thereof is administered at a dose of from about 200 nmol / kg patient body weight to about 600 nmol / kg patient body weight.

[0304] 54. A method according to clause 48, wherein the compound or a pharmaceutically acceptable salt thereof is administered at a dose of from about 400 nmol / kg patient body weight to about 600 nmol / kg patient body weight.

[0305] 55. A method according to any one of clauses 48 to 54, wherein the dose of the CAR T cells is from about 1 million CAR T cells to about 12.5 million CAR T cells.

[0306] 56. A method according to any one of clauses 48 to 55, wherein the dose of the CAR T cells is from about 1 million CAR T cells to about 7 million CAR T cells.

[0307] 57. A method according to any one of clauses 48 to 56, wherein the dose of the CAR T cells is from about 1 million CAR T cells to about 5 million CAR T cells.

[0308] 58. A method according to any one of clauses 48 to 57, wherein the dose of the CAR T cells is from about 2 million CAR T cells to about 5 million CAR T cells.

[0309] 59. A method according to 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.

[0310] 60. A method according to any one of clauses 1 to 59, wherein the compound or a pharmaceutically acceptable salt thereof is continuously administered to the patient for at least one hour.

[0311] 61. A method according to any one of clauses 1 to 59, wherein the compound or a pharmaceutically acceptable salt thereof is continuously administered to the patient for at least four hours.

[0312] 62. A method according to any one of clauses 1 to 59, wherein the compound or a pharmaceutically acceptable salt thereof is continuously administered to the patient for at least six hours.

[0313] 63. A method according to any one of clauses 1 to 62, wherein the compound or a pharmaceutically acceptable salt thereof is administered to the patient every other day.

[0314] 64. A method according to any one of clauses 1 to 62, wherein the compound or a pharmaceutically acceptable salt thereof is administered to the patient three times a week.

[0315] 65. A method according to any one of clauses 1 to 62, wherein the compound or a pharmaceutically acceptable salt thereof is administered to the patient twice a week.

[0316] 66. A method according to any one of clauses 1 to 62, wherein the compound or a pharmaceutically acceptable salt thereof is administered to the patient once a week.

[0317] 67. A method according to any one of clauses 1 to 62, wherein the compound or a pharmaceutically acceptable salt thereof is administered to the patient until an unacceptable loss of body weight, fever, a drop in blood pressure, or pulmonary edema occurs in the patient.

[0318] 68. A method of treating cancer, the method comprising: 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, 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, and wherein the amount of the second dose of the compound or a pharmaceutically acceptable salt thereof is about 2-fold to about 15,000-fold more than the amount of the first dose of the compound or a pharmaceutically acceptable salt thereof; and ii) administering to the patient a CAR T cell composition comprising CAR T cells, wherein the CAR T cells comprise a CAR directed to the targeting moiety and wherein the CAR comprises an E2 anti-fluorescein antibody fragment.

[0319] 69. A method according to 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, and wherein the amount of the second dose of the compound or a pharmaceutically acceptable salt thereof is about 2-fold to about 750-fold more than the amount of the first dose of the compound or a pharmaceutically acceptable salt thereof, and wherein the amount of the third dose of the compound or a pharmaceutically acceptable salt thereof is about 800-fold to about 10,000-fold more than the amount of the first dose of the compound or a pharmaceutically acceptable salt thereof.

[0320] 70. The method of clause 68, wherein the patient is administered at least a first dose, a second dose, a third dose, and a fourth dose of the compound or a pharmaceutically acceptable salt thereof, 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 to about 750 times more 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 to about 7500 times more 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 to about 15000 times more than the amount of the compound or a pharmaceutically acceptable salt thereof in the first dose.

[0321] 71. The method of clause 70, wherein the amount of the compound or a pharmaceutically acceptable salt thereof in the second dose is about 100 times more 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 1000 times more 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 10000 times more than the amount of the compound or a pharmaceutically acceptable salt thereof in the first dose.

[0322] 72. The method of clause 68, wherein the patient is administered at least a first dose and a second dose of the compound or a pharmaceutically acceptable salt thereof, 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 to about 15000 times more than the amount of the compound or a pharmaceutically acceptable salt thereof in the first dose.

[0323] 73. A method of treating cancer, the method comprising: i) administering to a patient a first dose of a compound or a pharmaceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked via a linker to a targeting moiety; 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% 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 CAR T cells, wherein the CAR T cells comprise a CAR directed to the targeting moiety and wherein the CAR comprises an E2 anti-fluorescein antibody fragment.

[0324] 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% lower than the amount of the compound or a pharmaceutically acceptable salt thereof in the first dose.

[0325] 75. A method according to clause 73, wherein the amount of the second dose of the compound or its pharmaceutically acceptable salt is at least about 70% lower than the amount of the first dose of the compound or its pharmaceutically acceptable salt.

[0326] 76. A method according to clause 73, wherein the amount of the second dose of the compound or its pharmaceutically acceptable salt is at least about 80% lower than the amount of the first dose of the compound or its pharmaceutically acceptable salt.

[0327] 77. A method according to clause 73, wherein the amount of the second dose of the compound or its pharmaceutically acceptable salt is at least about 90% lower than the amount of the first dose of the compound or its pharmaceutically acceptable salt.

[0328] 78. A method according to clause 73, wherein the amount of the second dose of the compound or its pharmaceutically acceptable salt is at least about 95% lower than the amount of the first dose of the compound or its pharmaceutically acceptable salt.

[0329] 79. A method according to clause 73, wherein the amount of the second dose of the compound or its pharmaceutically acceptable salt is at least about 96% lower than the amount of the first dose of the compound or its pharmaceutically acceptable salt.

[0330] 80. A method according to clause 73, wherein the amount of the second dose of the compound or its pharmaceutically acceptable salt is at least about 97% lower than the amount of the first dose of the compound or its pharmaceutically acceptable salt.

[0331] 81. A method according to clause 73, wherein the amount of the second dose of the compound or its pharmaceutically acceptable salt is at least about 98% lower than the amount of the first dose of the compound or its pharmaceutically acceptable salt.

[0332] 82. A method according to clause 73, wherein the amount of the second dose of the compound or its pharmaceutically acceptable salt is at least about 99% lower than the amount of the first dose of the compound or its pharmaceutically acceptable salt.

[0333] 83. A method according to clause 73, wherein the amount of the second dose of the compound or its pharmaceutically acceptable salt is at least about 99.5% lower than the amount of the first dose of the compound or its pharmaceutically acceptable salt.

[0334] 84. A method according to any one of clauses 73 to 83, wherein the first dose of the compound or its pharmaceutically acceptable salt is from about 100 nmol / kg of patient body weight to about 1000 nmol / kg of patient body weight.

[0335] 85. A method according to any one of clauses 73 to 83, wherein the first dose of the compound or its pharmaceutically acceptable salt is from about 100 nmol / kg of patient body weight to about 900 nmol / kg of patient body weight.

[0336] 86. A method according to any one of clauses 73 to 83, wherein the first dose of the compound or its pharmaceutically acceptable salt is from about 100 nmol / kg of patient body weight to about 800 nmol / kg of patient body weight.

[0337] 87. A method according to any one of clauses 73 to 83, wherein the first dose of the compound or its pharmaceutically acceptable salt is from about 100 nmol / kg of patient body weight to about 700 nmol / kg of patient body weight.

[0338] 88. A method according to any one of clauses 73 to 83, wherein the first dose of the compound or its pharmaceutically acceptable salt is from about 100 nmol / kg of patient body weight to about 600 nmol / kg of patient body weight.

[0339] 89. A method according to any one of clauses 73 to 83, wherein the first dose of the compound or its pharmaceutically acceptable salt is from about 200 nmol / kg of patient body weight to about 600 nmol / kg of patient body weight.

[0340] 90. A method according to any one of clauses 73 to 83, wherein the first dose of the compound or its pharmaceutically acceptable salt is from about 400 nmol / kg of patient body weight to about 600 nmol / kg of patient body weight.

[0341] 91. A method according to any one of clauses 73 to 83, wherein the first dose of the compound or its pharmaceutically acceptable salt is about 500 nmol / kg of patient body weight.

[0342] 92. A method according to clause 84, wherein the second dose of the compound or its pharmaceutically acceptable salt is from about 0.5 nmol / kg of patient body weight to about 500 nmol / kg of patient body weight.

[0343] 93. A method according to clause 85, wherein the second dose of the compound or its pharmaceutically acceptable salt is from about 0.5 nmol / kg of patient body weight to about 450 nmol / kg of patient body weight.

[0344] 94. A method according to clause 86, wherein the second dose of the compound or its pharmaceutically acceptable salt is from about 0.5 nmol / kg of patient body weight to about 400 nmol / kg of patient body weight.

[0345] 95. A method according to clause 87, wherein the second dose of the compound or its pharmaceutically acceptable salt is from about 0.5 nmol / kg of patient body weight to about 350 nmol / kg of patient body weight.

[0346] 96. The method of clause 88, wherein the second dose of the compound or its pharmaceutically acceptable salt is from about 0.5 nmol / kg patient body weight to about 300 nmol / kg patient body weight.

[0347] 97. The method of clause 89, wherein the second dose of the compound or its pharmaceutically acceptable salt is from about 1 nmol / kg patient body weight to about 300 nmol / kg patient body weight.

[0348] 98. The method of clause 90, wherein the second dose of the compound or its pharmaceutically acceptable salt is from about 2 nmol / kg patient body weight to about 300 nmol / kg patient body weight.

[0349] 99. The method of clause 91, wherein the second dose of the compound or its pharmaceutically acceptable salt is from about 2 nmol / kg patient body weight to about 250 nmol / kg patient body weight.

[0350] 100. The method of any one of clauses 92 to 99, wherein the second dose of the compound or its pharmaceutically acceptable salt is from about 5 nmol / kg patient body weight to about 40 nmol / kg patient body weight.

[0351] 101. The method of any one of clauses 92 to 99, wherein the second dose of the compound or its pharmaceutically acceptable salt is from about 40 nmol / kg patient body weight to about 150 nmol / kg patient body weight.

[0352] 102. The method of any one of clauses 73 to 101, further comprising administering a third dose of the compound or its pharmaceutically acceptable salt, wherein the third dose of the compound or its pharmaceutically acceptable salt is the same as the second dose of the compound or its pharmaceutically acceptable salt.

[0353] 103. The method of clause 102, further comprising administering a fourth dose of the compound or its pharmaceutically acceptable salt, wherein the fourth dose of the compound or its pharmaceutically acceptable salt is the same as the second dose of the compound or its pharmaceutically acceptable salt and the third dose of the compound or its pharmaceutically acceptable salt.

[0354] 104. The method of any one of clauses 73 to 103, wherein one or more doses of the compound or its pharmaceutically acceptable salt administered after the first dose of the compound or its pharmaceutically acceptable salt maintain inhibition of the growth of the cancer relative to the first dose of the compound or its pharmaceutically acceptable salt.

[0355] 105. The method of any one of clauses 73 to 104, wherein the CAR T cells are administered at a dose of from about 1 million CAR T cells to about 40 million CAR T cells.

[0356] 106. A method according to any one of clauses 73 to 105, wherein one or more doses of the compound or a pharmaceutically acceptable salt thereof are administered once a week after administration of the first dose of the compound or a pharmaceutically acceptable salt thereof.

[0357] 107. A method according to 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 a week.

[0358] 108. A method according to 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.

[0359] 109. A method according to any one of clauses 1 to 108, wherein the E2 anti-fluorescein antibody fragment is a scFv fragment.

[0360] 110. A method according to any one of clauses 1 to 109, wherein the CAR protein sequence has at least about 90% identity with SEQ ID NO:2.

[0361] 111. A method according to any one of clauses 1 to 109, wherein the CAR protein sequence has at least about 95% identity with SEQ ID NO:2.

[0362] 112. A method according to any one of clauses 1 to 109, wherein the CAR protein sequence has at least about 98% identity with SEQ ID NO:2.

[0363] 113. A method according to any one of clauses 1 to 112, wherein the CAR binds fluorescein.

[0364] 114. A method according to any one of clauses 1 to 109, wherein the CAR protein sequence has at most about 50 conservative amino acid substitutions and wherein the CAR binds fluorescein.

[0365] 115. A method according to any one of clauses 1 to 109, wherein the CAR is encoded by a polynucleotide having at least about 90% identity with SEQ ID NO:1.

[0366] 116. A method according to 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.

[0367] 117. A method according to any one of clauses 1 to 109, wherein the CAR is encoded by a polynucleotide having at least about 98% identity with SEQ ID NO:1.

[0368] 118. A method according to any one of clauses 115 to 117, wherein the CAR binds fluorescein.

[0369] 119. A method according to any one of clauses 1 to 118, wherein the CAR is encoded by a polynucleotide that hybridizes to the polynucleotide having SEQ ID NO:1 under highly stringent conditions and wherein the CAR binds fluorescein.

[0370] 120. A method according to 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.

[0371] 121. A method according to any one of clauses 1 to 120, wherein the ligand is selected from the following: folic acid compounds, DUPA, NK-1R ligands, CAIX ligands, γ-glutamyl transpeptidase ligands, NKG2D ligands, and CCK2R ligands.

[0372] 122. A method according to any one of clauses 1 to 121, wherein the targeting moiety is fluorescein or a pharmaceutically acceptable salt thereof.

[0373] 123. A method according to any one of clauses 1 to 122, wherein the linker comprises polyethylene glycol (PEG), polyproline, hydrophilic amino acids, sugars, unnatural peptidoglycans, polyvinylpyrrolidone, Pluronic F-127, or a combination thereof.

[0374] 124. A method according to any one of clauses 1 to 123, wherein the linker comprises PEG.

[0375] 125. A method according to any one of clauses 1 to 124, wherein the compound or a pharmaceutically acceptable salt thereof has the following formula B-L-T, 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 following formula wherein n is an integer from 0 to 200.

[0376] 126. A method according to any one of clauses 1 to 125, wherein the cancer is selected from the following: lung cancer, bone cancer, pancreatic cancer, skin cancer, head cancer, neck cancer, cutaneous melanoma, uveal 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, 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 myeloid 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 cancer, renal pelvic cancer, neoplasms of the central nervous system (CNS), primary CNS lymphoma, spinal axis tumors, brainstem glioma, pituitary adenoma, and gastroesophageal junction adenocarcinoma.

[0377] 127. A method according to any one of clauses 1 to 126, wherein the cancer is a cancer that expresses a folate receptor.

[0378] 128. A method according to any one of clauses 1 to 127, wherein the cancer is endometrial cancer.

[0379] 129. A method according to any one of clauses 1 to 127, wherein the cancer is non-small cell lung cancer.

[0380] 130. A method according to any one of clauses 1 to 127, wherein the cancer is ovarian cancer.

[0381] 131. A method according to any one of clauses 1 to 127, wherein the cancer is triple-negative breast cancer.

[0382] 132. A method according to any one of clauses 1 to 127, wherein the cancer is acute myeloid leukemia.

[0383] 133. The method according to clause 132, wherein the cancer expresses the folate receptor-β.

[0384] 134. A method according to any one of clauses 1 to 133, wherein multiple doses of the CAR T cell composition are administered.

[0385] 135. A method according to any one of clauses 1 to 134, wherein at least two doses of the CAR T cell composition are administered.

[0386] 136. The method according to any one of clauses 1 to 135, wherein the patient is imaged before administering the compound or its pharmaceutically acceptable salt, or before administering the CAR T cell composition.

[0387] 137. The method according to any one of clauses 1 to 136, wherein the compound or its pharmaceutically acceptable salt is not an antibody and does not comprise an antibody fragment.

[0388] 138. The method according to any one of clauses 1 to 137, wherein the targeting moiety does not comprise a peptide epitope.

[0389] 139. The method according to any one of clauses 1 to 138, wherein cytokine release leading to off-target toxicity in the patient does not occur and wherein CAR T cell toxicity to the cancer occurs.

[0390] 140. The method according to 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.

[0391] 141. The method according to any one of clauses 1 to 138, wherein the cancer comprises a tumor, wherein the size of the patient's tumor decreases, and wherein off-target toxicity does not occur.

[0392] 142. The method according to any one of clauses 1 to 141, wherein CRS is reduced or prevented and the method results in a reduction in the tumor volume of the patient.

[0393] 143. The method according to any one of clauses 1 to 142, wherein weight loss due to CRS is reduced or prevented.

[0394] 144. The method according to any one of clauses 59 to 67, further comprising administering to the patient the compound or its pharmaceutically acceptable salt.

[0395] 145. The method according to clause 144, wherein subsequent administration of the compound or its pharmaceutically acceptable salt results in CAR T cell activation and an increase in cytokine levels in the patient.

[0396] 146. The method according to any one of clauses 1 to 145, wherein the cancer comprises a tumor and wherein a complete response to the tumor is obtained.

[0397] 147. The method according to any one of clauses 1 to 146, wherein the CAR T cells have a central memory / effector memory phenotype.

[0398] 148. The method according to any one of clauses 1 to 147, wherein the CD8:CD4 ratio of the CAR T cells is about 1:1.

[0399] Thus, in one embodiment, a method of treating cancer is provided. The method comprises: 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; ii) administering to the patient a first dose of a CAR T cell composition, wherein the CAR T cell composition comprises CAR T cells, wherein the CAR T cells comprise a chimeric antigen receptor (CAR), and wherein the CAR comprises an E2 anti-fluorescein antibody fragment; and iii) administering to the patient a second dose of the CAR T cell composition, wherein the CAR T cell composition comprises CAR T cells, wherein the CAR T cells comprise a CAR, and wherein the CAR comprises an E2 anti-fluorescein antibody fragment.

[0400] In another embodiment, a method of treating cancer is provided. The method comprises: 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 ii) administering to the patient a CAR T cell composition comprising CAR T cells, wherein the CAR T cells comprise 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 cells and non-transformed T cells.

[0401] In yet another embodiment, a method of treating cancer is provided. The method comprises: 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; ii) administering to the patient a CAR T cell composition, wherein the CAR T cell composition comprises CAR T cells, wherein the CAR T cells comprise a CAR, wherein the CAR comprises an E2 anti-fluorescein antibody fragment; and iii) administering to the patient a folic acid compound, a conjugate comprising a folic acid compound (wherein the conjugate comprising a folic acid compound does not comprise a targeting moiety), or a drug that inhibits the activation of CAR T cells.

[0402] In another embodiment, a method of treating cancer is provided. The method comprises: 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 wherein the compound or a pharmaceutically acceptable salt thereof is administered at a dose of from about 10 nmol / kg of patient body weight to about 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 a CAR, wherein the CAR comprises an E2 anti-fluorescein antibody fragment, and wherein the dose of the CAR T cells is from about 1 million CAR T cells to about 15 million CAR T cells.

[0403] In yet another embodiment, a method of treating cancer is provided. The method comprises i) continuously 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, ii) administering to the patient a CAR T cell composition comprising CAR T cells, wherein the CAR T cells comprise 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.

[0404] In another illustrative aspect, a method of treating cancer is provided. The method comprises 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, wherein the patient is administered at least a first dose and a second dose of the compound or a pharmaceutically acceptable salt thereof, wherein the first dose and the second dose are different, and wherein the amount of the second dose of the compound or a pharmaceutically acceptable salt thereof is about 2-fold to about 15,000-fold greater than the amount of the first dose of the compound or a pharmaceutically acceptable salt thereof, and ii) administering to the patient a CAR T cell composition comprising CAR T cells, wherein the CAR T cells comprise a CAR, wherein the CAR comprises an E2 anti-fluorescein antibody fragment.

[0405] In another embodiment, a method of treating cancer is provided. The method comprises 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 wherein the compound or a pharmaceutically acceptable salt thereof is administered to the patient once a week, and ii) administering to the patient a CAR T cell composition comprising CAR T cells, wherein the CAR T cells comprise a CAR, wherein the CAR comprises an E2 anti-fluorescein antibody fragment.

[0406] In yet another embodiment, a method of 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 CAR T cells, wherein the CAR T cells comprise a CAR and wherein the CAR comprises an E2 anti-fluorescein antibody fragment.

[0407] Accordingly, the various embodiments provided in the paragraphs above and all applicable embodiments described in this "Detailed Description", the Summary of the Invention section, the Examples, and the claims apply to such embodiments.

[0408] As used herein, a "patient" can be a human or, in the case of veterinary applications, the patient can be a laboratory, agricultural, domestic, or wild animal. In various aspects, the patient can be a laboratory animal (such as a rodent (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).

[0409] 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, cutaneous melanoma, uveal 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, 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 myelogenous leukemia), lymphocytic lymphoma, myelogenous leukemia, myelomonocytic leukemia, hairy cell leukemia, pleural mesothelioma, bladder cancer, Burkitt's lymphoma, ureteral cancer, kidney cancer, renal cell carcinoma, renal pelvic cancer, neoplasms of the central nervous system (CNS), primary CNS lymphoma, spinal axis tumors, brainstem glioma, pituitary adenoma, and gastroesophageal junction adenocarcinoma.

[0410] In some aspects of such embodiments, the cancer is a cancer that expresses a folate receptor. In another embodiment, the cancer is a cancer that expresses folate receptor alpha. In yet another embodiment, the cancer is a cancer that expresses folate receptor beta. In some aspects of such embodiments, the 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-beta. In still another embodiment, the cancer is acute myeloid leukemia and the CAR-T cells have a central memory / effector 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, in the case where the cancer is acute myeloid leukemia or another cancer, the CAR T cells associated with the tumor may have increased CD25 expression relative to CAR T cells not associated with the tumor.

[0411] In one embodiment, the "small molecule ligand" can be a folate compound, DUPA (a ligand that binds via PSMA-positive human prostate cancer cells and other cancer cell types), an NK-1R ligand (a receptor for NK-1R (e.g., a ligand found on colon cancer and pancreatic cancer)), a CAIX ligand (a receptor for a CAIX ligand (e.g., found on renal cancer, ovarian cancer, vulvar cancer, and breast cancer)), a ligand for gamma-glutamyl transpeptidase (a transpeptidase overexpressed in ovarian cancer, colon cancer, liver cancer, astrocytoma, melanoma, and leukemia), an NKG2D ligand (a receptor for an NKG2D ligand (e.g., found on lung cancer, colon cancer, renal cancer, prostate cancer, and T and B cell lymphoma)), or a CCK2R ligand (a receptor for a CCK2R ligand found particularly 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 can be overexpressed in cancer compared to normal tissue).

[0412] In one embodiment, the small molecule ligand can have a mass of less than about 10,000 Daltons (Da), less than about 9000 Da, less than about 8,000 Da, less than about 7000 Da, less than about 6000 Da, less than about 5000 Da, less than about 4500 Da, less than about 4000 Da, less than about 3500 Da, less than about 3000 Da, less than about 2500 Da, less than about 2000 Da, less than about 1500 Da, less than about 1000 Da, or less than about 500 Da. In another embodiment, the small molecule ligand can have a mass of from about 1 to about 10,000 Da, from about 1 to about 9000 Da, from about 1 to about 8,000 Da, from about 1 to about 7000 Da, from about 1 to about 6000 Da, from about 1 to about 5000 Da, from about 1 to about 4500 Da, from about 1 to about 4000 Da, from about 1 to about 3500 Da, from about 1 to about 3000 Da, from about 1 to about 2500 Da, from about 1 to about 2000 Da, from about 1 to about 1500 Da, from about 1 to about 1000 Da, or from about 1 to about 500 Da.

[0413] In one embodiment, the DUPA derivative can be a ligand of a small molecule ligand linked to a targeting moiety, and the DUPA derivative is described in WO 2015 / 057852, which is incorporated herein by reference.

[0414] In one embodiment, in the case of "a small molecule ligand linked to a linker", the small molecule ligand is a folate compound. In various embodiments, the folate compound can be folic acid, a folic acid analogue, or another molecule that binds to the folate receptor. In various embodiments, folic acid analogues that can be used include formyltetrahydrofolic acid, pteroylpolyglutamic acid, and pteridines that bind to the folate receptor (such as tetrahydropterin, dihydrofolic acid, tetrahydrofolic acid, and their deaza and diaza analogues). The terms "deaza" and "diaza" analogues refer to analogues well known in the art having a carbon atom that replaces one or two nitrogen atoms in the structure of a naturally occurring folate. For example, deaza analogues include 1-deaza, 3-deaza, 5-deaza, 8-deaza, and 10-deaza analogues. Diaza analogues include, for example, 1,5-diaza, 5,10-diaza, 8,10-diaza, and 5,8-diaza analogues. The above folic acid analogues are commonly referred to as "folate compounds", reflecting their ability to bind to the folate receptor. Other analogues that bind to the folate receptor include aminopterin, amethopterin / methotrexate, N10-methylfolic acid, 2-deamino-hydroxyfolic acid, deaza analogues (such as 1-deazamethotrexate or 3-deazamethotrexate), and 3',5'-dichloro-4-amino-4-deoxy-N10-methylpteroylglutamic acid (dichloromethotrexate).

[0415] In another embodiment, in the context of "a small molecule ligand attached to a linker", the small molecule ligand may have the following formula wherein X 1 and Y 1 are each independently selected from halogen, R 2 、OR 2 、SR 3 and NR 4 R 5 ; U, V and W represent divalent moieties, which are 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-; X 2 and X 3 are 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 alkoxy, where Z is oxygen or sulfur; R 1 is selected from hydrogen, halogen, C1-C 12 alkyl and C1-C 12 alkoxy; R 2 、R 3 、R 4 、R 4a 、R 4b 、R 5 、R 5b 、R 6b and R 7b are each independently selected from the following: hydrogen, halogen, C1-C 12alkyl, 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; R 6 and R 7 are each independently selected from hydrogen, halogen, C1-C 12 alkyl and C1-C 12 alkoxy; or R 6 and R 7 together form a carbonyl group; R 6a and R 7a are each independently selected from hydrogen, halogen, C1-C 12 alkyl and C1-C 12 alkoxy; or R 6a and R 7a together form a carbonyl group; p, r, s and t are each independently 0 or 1; and * represents an optional covalent bond connecting to the rest of the conjugate.

[0416] On the one hand, the "targeting moiety" bound to the conjugate comprising the E2 anti-fluorescein antibody fragment and the CAR expressed by CAR T cells can be selected from, for example, fluorescein, fluorescein isothiocyanate (FITC), and NHS-fluorescein. The targeting moiety is characterized only by the requirement 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.

[0417] In an illustrative embodiment, the targeting moiety can have the following illustrative structure: 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, NR a or NR a 2 + ; wherein in each case, each R is independently selected from H, fluorine, sulfonic acid, sulfonate, and its salts, etc.; and R a is hydrogen or alkyl.

[0418] In an illustrative aspect, the linker may comprise polyethylene glycol (PEG), polyproline, hydrophilic amino acids, sugars, unnatural peptidoglycans, polyvinylpyrrolidone, Pluronic F-127, or combinations thereof.

[0419] In another illustrative aspect, the linker of the compounds or pharmaceutically acceptable salts thereof described herein may comprise a direct linkage (e.g., reaction between the isothiocyanate group of FITC and the free amino group of a small molecule ligand) or the linkage 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 contain from about 1 to about 30 carbon atoms. In another illustrative embodiment, the divalent linker may contain from about 2 to about 20 carbon atoms. In other embodiments, lower molecular weight divalent linkers are employed (i.e., those having an approximate molecular weight of from about 30 to about 300 daltons). 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.

[0420] In various embodiments, the small molecule ligand attached to the targeting moiety may have the formula B-L-T, 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 Where n is an integer from 0 to 200. In another embodiment, n can be an integer among the following: 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.

[0421] 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. Describe the following non-limiting, illustrative spacers, where * indicates the point of attachment of a small molecule ligand or targeting moiety or other bivalent linker moiety.

[0422] In other embodiments, the small molecule ligand (bridge) attached to the targeting moiety can have any of the following structures.

[0423]

[0424] In other embodiments, the compound or its pharmaceutically acceptable salt is not an antibody and does not contain antibody fragments. In another embodiment, the targeting moiety does not contain a peptide epitope.

[0425] In an illustrative embodiment, a small molecule ligand linked to a targeting moiety via a linker (bridge) includes fluorescein isothiocyanate (FITC) linked to the small molecule ligand. On the one hand, cancer may overexpress the receptor for the small molecule ligand. On the other hand, for example, cytotoxic T cells or another type of T cell may be transformed to express a CAR comprising an E2 anti-fluorescein antibody fragment. In this regard, the CAR may target FITC, as a result of the small molecule ligand binding to the cancer, decorating the cancer with FITC molecules. Thus, toxicity to normal, non-target cells can be avoided. In this embodiment, when a T cell expressing the E2 anti-fluorescein antibody fragment CAR binds to FITC, the CAR T cell is activated and the cancer is improved.

[0426] “Pharmaceutically acceptable salts” of small molecule ligands linked to a targeting moiety via a linker are encompassed. As used herein, the term “pharmaceutically acceptable salts” refers to those salts whose counterions are useful in medicine. In various embodiments, such salts include, but are not limited to: 1) acid addition salts, which can be obtained via 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, 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 encompass any such pharmaceutically acceptable salts relevant to the embodiments described herein.

[0427] 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, ethanedisulfonate, ethanesulfonate, formate, fumarate, glucoheptonate, gluconate, glucuronate, hexafluorophosphate, hippurate, hydrochloride / chloride, hydrobromide / bromide, hydroiodide / iodide, hydroxyethylsulfonate, 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.

[0428] 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 may also be formed, for example, hemisulfates and hemicalcium salts.

[0429] In an illustrative aspect, the compounds or their pharmaceutically acceptable salts described herein may contain one or more chiral centers or may otherwise be capable of existing as multiple stereoisomers. Accordingly, various embodiments may include pure stereoisomers as well as mixtures of stereoisomers, such as enantiomers, diastereomers, and mixtures enriched in enantiomers or diastereomers. In one aspect, the compounds or their pharmaceutically acceptable salts described herein may exist as geometric isomers. Accordingly, various embodiments may include pure geometric isomers or mixtures of geometric isomers.

[0430] In some aspects, the compounds or their pharmaceutically acceptable salts described herein may exist in unsolvated 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 invention.

[0431] 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 the targeting moiety of the bridge (e.g., fluorescein, fluorescein isothiocyanate (FITC), and NHS-fluorescein). In one embodiment, the CAR described herein comprises three domains that include 1) an identification region that specifically recognizes and binds to the targeting moiety (e.g., the single-chain variable fragment (scFv) region of an E2 anti-fluorescein antibody, a Fab fragment, etc.), 2) a co-stimulatory domain that enhances the proliferation and survival of T lymphocytes, and 3) an activation signal domain that generates a T lymphocyte activation signal.

[0432] In various aspects, as non-limiting examples, scFv regions of antibodies that bind fluorescein, fluorescein isothiocyanate (FITC), and NHS-fluorescein may be used. In illustrative non-limiting embodiments, the scFv region may be prepared from (i) an E2 antibody known in the art that binds the targeting moiety, and (iii) sequence variants derived from the scFv region of such antibodies, e.g., scFv regions 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 is derived.

[0433] On the one hand, the co-stimulatory domain helps to enhance the proliferation and survival of cytotoxic T lymphocytes after the CAR binds to the targeting moiety. Suitable co-stimulatory 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 co-stimulatory molecules expressed on activated T cells, or combinations thereof. In an illustrative embodiment, the co-stimulatory domain of the engineered CAR includes CD28 and CD137. Those skilled in the art will appreciate that sequence variants of such co-stimulatory domains can be used without adversely affecting the present invention, where the variants have the same or similar activity as the domain from which they were engineered. In various embodiments, such variants can 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 they were derived.

[0434] In an illustrative embodiment, the activation signal domain helps to activate T lymphocytes (e.g., cytotoxic T lymphocytes) after the CAR binds to the targeting moiety. In various embodiments, suitable activation signal domains include the T cell CD3ζ chain, CD3δ receptor protein, mbl receptor protein, B29 receptor protein, and Fc receptor γ. Those skilled in the art will appreciate that sequence variants of such activation signal domains can be used, where the variants have the same or similar activity as the domain from which they were engineered. In various embodiments, the variants have at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity to the amino acid sequence of the domain from which they were derived.

[0435] On the one hand, genetic engineering techniques are used to prepare constructs encoding chimeric antigen receptors (CARs) that comprise antibody fragments against fluorescein E2. 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.

[0436] 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 for non-viral mediated CAR gene delivery system)) encoding a fusion protein can be prepared, the fusion protein comprising a recognition region, one or more co-stimulatory domains, and an activation signal domain that are in-frame and linked in the 5' to 3' direction. In other embodiments, other arrangements are acceptable and comprise a recognition region, an activation signal domain, and one or more co-stimulatory 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, a CAR comprising an antibody fragment against fluorescein E2 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 the 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.

[0437] An illustration of an exemplary CAR construct in which the expressed CAR comprises an antibody fragment against fluorescein E2 is shown in Figure 1Among them, the fusion protein sequence is incorporated into an expression vector and the CAR comprises an E2 anti-fluorescein antibody fragment, an IgG4 hinge domain, a CD28 transmembrane domain, and the co-stimulatory domain is CD137 (4-1BB), and the activation signal domain is CD3ζ. The CAR may comprise additional suitable domains. An exemplary nucleic acid sequence of the CAR insert is provided as SEQ ID NO:1 and the exemplary encoded amino acid sequence is provided as SEQ ID NO:2. As used herein, "SEQ ID NO:1" means the sequence starting with the underlined "agc" codon and terminating with the underlined "ggc" codon. This portion of the longer sequence encodes the CAR inserted into the T cell membrane. Other portions of the longer sequence include portions that are not the CAR inserted into the membrane and the coding sequences of the signal peptide, EGFRt domain, etc. that function as a chimeric antigen receptor. As used herein, "SEQ ID NO:2" means the sequence starting with the underlined "S" and terminating with the underlined "G". This portion of the longer sequence is the amino acid sequence of the CAR inserted into the T cell membrane. Other portions of the longer sequence include portions that are not the CAR inserted into the membrane and the amino acid sequences of the signal peptide, EGFRt domain, etc. that function as a chimeric antigen receptor. In another embodiment, SEQ ID NO:2 may comprise 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 the methods described herein.

[0438] SEQ ID NO:1 (E2 anti-fluorescein antibody fragment CAR nucleic acid sequence (insert)) SEQ ID NO:2 (E2 anti-fluorescein antibody fragment CAR amino acid sequence (insert))

[0439] In one embodiment, the CAR expressing the E2 anti-fluorescein antibody fragment comprises a recognition region and the recognition region is the single-chain fragment variable (scFv) region of the E2 anti-fluorescein antibody, a co-stimulatory domain and the co-stimulatory domain is CD137 (4-1BB), and an activation signal domain and the activation signal domain is the T cell CD3ζ chain. In another embodiment, the CAR may further comprise any additional suitable domains. It is well known to those skilled in the art that anti-FITC scFv and anti-fluorescein scFv are equivalent terms.

[0440] In various embodiments, the "E2 anti-fluorescein antibody fragment" can be a CAR comprising a fragment of an E2 anti-fluorescein antibody (e.g., an scFv fragment). The E2 anti-fluorescein antibody is described, for example, in Vaughan et al., Nature Biotechnol. Vol. 14(3), pp. 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.

[0441] In various embodiments, the CAR can further comprise 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 can 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 can be encoded by a polynucleotide that hybridizes to the polynucleotide having SEQ ID NO:1 under highly stringent conditions. In still another aspect, the CAR can be encoded by the polynucleotide having SEQ ID NO:1 or by a degenerate variant of SEQ ID NO:1. In other embodiments, the CAR protein sequence can 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 can have at most about 50 conservative amino acid substitutions. In any of the embodiments described herein, the CAR binds fluorescein.

[0442] In one embodiment, T lymphocytes (e.g., cytotoxic T lymphocytes) can be genetically engineered to express a CAR construct, wherein the T lymphocyte population is transfected with an expression vector encoding a CAR construct that expresses 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 those skilled in the art and are described in Sambrook et al., "Molecular Cloning: A Laboratory Manual", 3rd edition, Cold Spring Harbor Laboratory Press, (2001), which is incorporated herein by reference, and Green and Sambrook, "Molecular Cloning: A Laboratory Manual", 4th edition, Cold Spring Harbor Laboratory Press, (2012), which is incorporated herein by reference.

[0443] In one embodiment, CAR T cells comprising the nucleic acid of SEQ ID NO:1 can be used as described herein. In another embodiment, CAR T cells comprising the polypeptide of SEQ ID NO:2 can be used as described herein. In another illustrative aspect, nucleic acids (e.g., isolated nucleic acids) comprising SEQ ID NO:1 and encoding a chimeric antigen receptor can be used to prepare CAR T cells for use as described herein. In yet another embodiment, CAR T cells can be prepared using a chimeric antigen receptor polypeptide comprising SEQ ID NO:2 for use as described herein. In another embodiment, CAR T cells can be prepared using a vector comprising SEQ ID NO:1 for use as described herein. In another aspect, 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 can comprise or consist of a humanized or human amino acid sequence.

[0444] In each of such embodiments, variant nucleic acid sequences or amino acid sequences are encompassed that 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 SEQ ID NO:1. In another embodiment, the nucleic acid sequence can 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, provided that the variant sequence encodes the 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 to SEQ ID NO:1 over a segment of 200 nucleic acids, or to SEQ ID NO:2 over a segment of 200 amino acids. In one embodiment, the percent identity or similarity between sequences can be determined, for example, via use of the GAP program (Genetics Computer Group, software; now available through 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 searches are generally based on the BLAST software (Altschul et al., 1990). In some embodiments, the percent identity can be determined over the full length of the nucleic acid or amino acid sequence.

[0445] Also within the scope of the present invention, nucleic acids complementary to the nucleic acid represented by SEQ ID NO:1 can be used to prepare CAR T cells for the uses described herein, and those hybridizing to the nucleic acid represented by SEQ ID NO:1, or those hybridizing to its complement under highly stringent conditions, can be used. According to the present invention, "highly stringent conditions" means hybridization in 5X SSPE and 50% formamide at 65°C, and washing in 0.5X SSPE at 65°C. Conditions for highly stringent, low stringency and medium stringency hybridization are described in: Sambrook et al., "Molecular Cloning: A Laboratory Manual", 3rd Edition, Cold Spring Harbor Laboratory Press, (2001), 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.

[0446] In still other embodiments, the CAR used in the methods described herein can 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 can be encoded by a polynucleotide that hybridizes to the polynucleotide having SEQ ID NO:1 under highly stringent conditions. In still another aspect, the CAR can be encoded by the polynucleotide having SEQ ID NO:1 or by a degenerate variant of SEQ ID NO:1. As used herein, a degenerate variant refers to the genetic code having more than one codon specifying any particular amino acid. Degenerate variant codons specifying each amino acid are well known in the art. In another aspect, substitutions can be made to optimize the level of polypeptide production in a particular prokaryotic or eukaryotic host cell (i.e., codon usage variant).

[0447] In other embodiments, the CAR protein sequence can 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 can have up to about 50 conservative amino acid substitutions. In one embodiment, the CAR protein sequence expressing the E2 anti-fluorescein antibody fragment can 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, changing any non-critical amino acids of a polypeptide via conservative amino acid substitutions should not significantly alter the activity of the polypeptide, as the side chains of the substituted amino acids should be able to form similar bonds and interact as the side chains of the amino acids that have been substituted. In any of the embodiments described herein, the CAR binds fluorescein.

[0448] In an illustrative aspect, non-conservative substitutions are possible, provided that they do not overly affect the fluorescein-binding activity of the E2 anti-fluorescein antibody fragment polypeptide. As is well known in the art, "conservative substitution" of an amino acid or "conservative substitution variant" of a polypeptide refers to an amino acid substitution that maintains: 1) the secondary structure of the polypeptide; 2) the charge or hydrophobicity of the amino acid; and 3) the side chain volume or any one or more of such characteristics. In one embodiment, the conservative amino acid substitution can be made with amino acid analogs. 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.

[0449] Table 1

[0450] In one embodiment, the T lymphocytes used in the methods described herein (e.g., cytotoxic T lymphocytes or non-transformed T cells for preparing CAR T cells expressing an E2 anti-fluorescein antibody fragment) can be autologous cells, although allogeneic cells can also be used, such as when the patient being treated has received high-dose chemotherapy or radiotherapy to destroy the patient's immune system. In one embodiment, allogeneic cells can be used.

[0451] In one aspect, the T lymphocytes are obtained from a patient by methods well known in the art. For example, T cells (e.g., cytotoxic T cells or non-transformed T cells) can be obtained by: collecting peripheral blood from a patient, subjecting the blood to Ficoll density gradient centrifugation and then using a negative T cell isolation kit (such as the EasySep TM T Cell Isolation Kit) to isolate a population of T cells from the peripheral blood. In an illustrative embodiment, the population of T lymphocytes (e.g., cytotoxic T cells or non-transformed T cells) need not be pure and can contain other cells, such as other types of T cells (e.g., in the case of cytotoxic T cells), monocytes, macrophages, natural killer cells, and B cells. In one aspect, the population being collected can contain at least about 90% of the selected cell type, at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the selected cell type.

[0452] In one embodiment, after obtaining the T lymphocytes (e.g., cytotoxic T cells for preparing CAR T cells expressing an E2 anti-fluorescein antibody fragment), the cells are cultured under conditions that promote activation of the cells. In this embodiment, the culture conditions can be such that the cells can be administered to a patient without concern for reactivity against the components of the culture medium. For example, the culture conditions can exclude bovine serum products, such as bovine serum albumin. In an illustrative aspect, activation can be achieved by introducing a known activator into the culture medium, such as an anti-CD3 antibody in the case of cytotoxic T cells. Other suitable activators include anti-CD28 antibody. In one aspect, the lymphocyte population can be cultured under activation-promoting conditions for about 1 to about 4 days. In one embodiment, the appropriate level of activation can be determined by cell size, proliferation rate, or activation markers as measured by flow cytometry.

[0453] In an illustrative embodiment, after culturing a population of T lymphocytes (e.g., cytotoxic T lymphocytes for preparing CAR T cells expressing an E2 anti-fluorescein antibody fragment) 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 for use in the various embodiments are described above. In one aspect, after transfection, the cells can be immediately administered to a patient or the cells can be cultured for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 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 time for the cells to recover from transfection. In one aspect, suitable culture conditions can be similar to those in which the cells were cultured for activation, with or without an agent for promoting activation.

[0454] Thus, as described above, in an illustrative aspect, the treatment methods described herein can further comprise 1) obtaining a population of autologous or allogeneic T lymphocytes (e.g., cytotoxic T lymphocytes for preparing CAR T cells expressing an E2 anti-fluorescein antibody fragment), 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 CAR T cells expressing an E2 anti-fluorescein antibody fragment.

[0455] In an illustrative embodiment, when the cells have been transfected and activated, a composition comprising CAR T cells can be prepared, wherein the CAR T cells comprise a CAR comprising an E2 anti-fluorescein antibody fragment, and are administered to a patient, with or without non-transformed T cells. In one embodiment, a medium lacking any animal products, such as bovine serum, can be used to culture the CAR T cells expressing the E2 anti-fluorescein antibody fragment and / or non-transformed T cells. In another embodiment, tissue culture conditions commonly used by those skilled in the art can be used to avoid contamination by bacteria, fungi, and mycoplasma. In an exemplary embodiment, prior to administration to a patient, the cells (e.g., CAR T cells expressing the E2 anti-fluorescein antibody fragment and / or non-transformed T cells) are precipitated, washed, and resuspended in a pharmaceutically acceptable carrier or diluent. Exemplary compositions comprising T lymphocytes expressing a CAR (e.g., cytotoxic T lymphocytes) expressing an E2 anti-fluorescein antibody fragment or non-transformed T cells include compositions comprising the cells in sterile 290 mOsm saline, in an infusion-compatible cryomedium (containing Plasma-Lyte A, dextrose, sodium chloride injection, human serum albumin, and DMSO), in 0.9% NaCl with 2% human serum albumin, or in any other sterile 290 mOsm infusible substance. Alternatively, in another embodiment, depending on the characteristics of the medium, the CAR T cells expressing the E2 anti-fluorescein antibody fragment or non-transformed T cells can be administered as a composition in the medium, or concentrated and resuspended in the medium prior to administration. In various embodiments, the CAR T cell composition (wherein the CAR comprises an E2 anti-fluorescein antibody fragment) can be administered to a patient, with or without non-transformed T cells, by any suitable means, such as parenteral administration, e.g., intradermal, subcutaneous, intramuscular, intraperitoneal, intravenous, or intrathecal.

[0456] On the one hand, the total number and cell concentration of the CAR T cells expressing the E2 anti-fluorescein antibody fragment in the composition administered to a patient will vary depending on a number of 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 manner in which the composition is administered 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 the E2 anti-fluorescein antibody fragment include those having a volume of from about 0.1 ml to about 200 ml and from about 0.1 ml to about 125 ml.

[0457] In various embodiments, the transduced CAR T cells administered to a patient (wherein the CAR T cells comprise a CAR that comprises an E2 anti-fluorescein antibody fragment) can comprise from about 1X 10 5 to about 1X 10 15 or 1X 10 6 to about 1X10 15 transduced CAR T cells that express the E2 anti-fluorescein antibody fragment. In various embodiments, from about 1X 10 5 to about 1X 10 10 , about 1X 10 6 to about 1X 10 10 , about 1X 10 6 to about 1X 10 9 , about 1X 10 6 to about 1X 10 8 , about 1X 10 6 to about 2X 10 7 , about 1X 10 6 to about 3X 10 7 , about 1X 10 6 to about 1.5X 10 7 , about 1X 10 6 to about 1X 10 7 , about 1X 10 6 to about 9X 10 6 , about 1X 10 6 to about 8X 10 6 , about 1X 10 6 to about 7X10 6 , about 1X 10 6 to about 6X 10 6 , about 1X 10 6 to about 5X10 6 , about 1X 10 6 to about 4X 10 6 , about 1X 10 6 to about 3X 10 6 , about 1X 10 6 to about 2X 10 6 , about 2X 10 6 to about 6X 10 6 , about 2X 10 6 to about 5X 10 6 , about 3X 10 6 to about 6X 10 6 , about 4X 10 6 to about 6X 10 6 , about 4X 10 6from about 1 X 10 7 to about 1 X 10 6 to about 1 X 10 7 to about 1 X 10 6 to about 1.5 X 10 7 to about 1 X 10 6 to about 2 X 10 7 to about 0.2 X 10 6 to about 1 X 10 7 to about 0.2 X 10 6 to about 1.5 X 10 7 to about 0.2 X 10 6 to about 2 X 10 7 or about 5 X 10 6 CAR T cells expressing an E2 anti - fluorescein antibody fragment. In one aspect, in any of the embodiments described herein, a single dose or multiple doses of CAR T cells expressing an E2 anti - fluorescein antibody fragment can be administered to a patient. In any of the embodiments described in this paragraph or herein, the number of said CAR T cells can be per kg of patient body weight. In any of the embodiments described herein, CAR T cells expressing an E2 anti - fluorescein antibody fragment can be administered prior to a compound or a pharmaceutically acceptable salt thereof. It should be understood that unless otherwise specified, the designations i), ii), iii), etc. for the steps of any of the methods described herein do not indicate an order.

[0458] In various embodiments described herein, non - transformed T cells can also be administered in combination with CAR T cells expressing an E2 anti - fluorescein antibody fragment and can be administered in the amounts described herein for CAR T cells expressing an E2 anti - fluorescein antibody fragment and non - transformed T cells. In one aspect, a mixture of CAR T cells (wherein the CAR T cells comprise a CAR that comprises an E2 anti - fluorescein antibody fragment) and non - transformed T cells can be administered once or multiple times, or a dose combination of pure CAR T cells expressing an E2 anti - fluorescein antibody fragment and a mixture of CAR T cells expressing an E2 anti - fluorescein antibody fragment and non - transformed T cells can be administered (e.g., a dose of CAR T cells expressing an E2 anti - fluorescein antibody fragment followed by one or more doses of a mixture of CAR T cells expressing an E2 anti - fluorescein antibody fragment and non - transformed T cells). One of ordinary skill in the art will appreciate from this disclosure that a "mixture" of CAR T cells expressing an E2 anti - fluorescein antibody fragment and non - transformed T cells as described herein means mixing CAR T cells expressing an E2 anti - fluorescein antibody fragment with non - transformed T cells that have not been exposed to a construct for expressing a CAR comprising an E2 anti - fluorescein antibody fragment.

[0459] In other embodiments, the dose of CAR T cells expressing an E2 anti-fluorescein antibody fragment in the CAR T cell composition administered to a 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 an E2 anti-fluorescein antibody fragment.

[0460] 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 bloodstream of a patient, and the CAR T cells expressing an E2 anti-fluorescein antibody fragment in the patient's bloodstream are 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.

[0461] In the embodiments described herein, the CAR T cell composition may comprise CAR T cells expressing an E2 anti-fluorescein antibody fragment, without any other cell type, or non-transformed T cells may be administered to a patient in combination with CAR T cells expressing an E2 anti-fluorescein antibody fragment. For embodiments of administering multiple doses of the CAR T cell composition, any dose may comprise CAR T cells expressing an E2 anti-fluorescein antibody fragment or a mixture of CAR T cells expressing an E2 anti-fluorescein antibody fragment and non-transformed T cells. In each embodiment, the non-transformed T cells may be administered in the amounts described herein for CAR T cells expressing an E2 anti-fluorescein antibody fragment.

[0462] In another embodiment, any dose of the CAR T cell composition may comprise a mixture of CAR T cells expressing an E2 anti-fluorescein antibody fragment and non-transformed T cells selected from the following ratios: about 1:5 CAR T cells expressing an E2 anti-fluorescein antibody fragment to non-transformed T cells, about 1:4 CAR T cells expressing an E2 anti-fluorescein antibody fragment to non-transformed T cells, about 1:3 CAR T cells expressing an E2 anti-fluorescein antibody fragment to non-transformed T cells, about 1:2 CAR T cells expressing an E2 anti-fluorescein antibody fragment to non-transformed T cells, and about 1:1 CAR T cells expressing an E2 anti-fluorescein antibody fragment to non-transformed T cells.

[0463] In still other embodiments, any dose of the CAR T cell composition can include a mixture of CAR T cells expressing an E2 anti-fluorescein antibody fragment and non-transformed T cells at a ratio of about 1:1 to about 1:5, or the CAR T cell composition can comprise a mixture of about 10 million CAR T cells expressing an E2 anti-fluorescein antibody fragment and about 40 million non-transformed T cells, about 15 million CAR T cells expressing an E2 anti-fluorescein antibody fragment and about 35 million non-transformed T cells, about 20 million CAR T cells expressing an E2 anti-fluorescein antibody fragment and about 30 million non-transformed T cells, or about 25 million CAR T cells expressing an E2 anti-fluorescein antibody fragment and about 25 million non-transformed T cells.

[0464] The compounds or their pharmaceutically acceptable salts, or CAR T cell compositions (wherein the composition comprises CAR T cells, and the CAR T cells comprise a CAR comprising an E2 anti-fluorescein antibody fragment) described herein can be administered to a patient using any suitable method known in the art. As described herein, the term "administering / administered" includes all manners of introducing the compounds or their pharmaceutically acceptable salts, or CAR T cell compositions comprising CAR T cells (wherein the CAR T cells have a CAR comprising an E2 anti-fluorescein antibody fragment) into a patient, including but not limited to oral, intravenous, intramuscular, subcutaneous, transdermal, etc. In one aspect, the compounds or their pharmaceutically acceptable salts described herein can be administered in unit dosage forms and / or in preparations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants, and vehicles.

[0465] In one aspect, the compounds or their pharmaceutically acceptable salts, or CAR T cell compositions (wherein the CAR T cell composition comprises CAR T cells that express a CAR comprising an E2 anti-fluorescein antibody fragment) described herein can be administered directly into the bloodstream, into muscle, or into internal organs. In various embodiments, suitable routes for such parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, epidural, intracerebroventricular, intraurethral, intrasternal, intracranial, intratumoral, intramuscular, and subcutaneous delivery. In one embodiment, the manner of parenteral administration includes needle (including microneedle) injection, needle-free injection, and infusion techniques.

[0466] In an illustrative aspect, a parenteral preparation is generally an aqueous solution, 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 as a sterile non-aqueous solution or in a dry form for use in combination with a suitable vehicle such as sterile, pyrogen-free water or sterile saline. In other embodiments, any of the liquid preparations described herein may be suitable for parenteral administration as described herein. The preparation of a sterile lyophilized powder for parenteral preparations via lyophilization under sterile conditions can be readily achieved using standard pharmaceutical techniques well known to those skilled in the art. In one embodiment, the solubility of the compound or its pharmaceutically acceptable salt used in the preparation of a parenteral preparation can be increased via the use of suitable formulation techniques such as incorporating solubility enhancing agents.

[0467] The amount of the compound or its pharmaceutically acceptable salt to be administered to a patient can 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 a patient can be based on body surface area, mass, and physician evaluation. In various embodiments, the amount to be administered can range, for example, from about 0.05 mg to about 30 mg, 0.05 mg to about 25.0 mg, about 0.05 mg to about 20.0 mg, about 0.05 mg to about 15.0 mg, about 0.05 mg to about 10.0 mg, about 0.05 mg to about 9.0 mg, about 0.05 mg to about 8.0 mg, about 0.05 mg to about 7.0 mg, about 0.05 mg to about 6.0 mg, about 0.05 mg to about 5.0 mg, about 0.05 mg to about 4.0 mg, about 0.05 mg to about 3.0 mg, about 0.05 mg to about 2.0 mg, about 0.05 mg to about 1.0 mg, about 0.05 mg to about 0.5 mg, about 0.05 mg to about 0.4 mg, about 0.05 mg to about 0.3 mg, about 0.05 mg to about 0.2 mg, about 0.05 mg to about 0.1 mg, about 0.01 mg to about 2 mg, about 0.3 mg to about 10 mg, about 0.1 mg to about 20 mg, or about 0.8 to about 3 mg. Those skilled in the art will readily understand that the dosage can vary within the various ranges provided above based on the factors indicated above and can be at the discretion of the physician.

[0468] In other embodiments, the dosage range of the compound or its pharmaceutically acceptable salt 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 500 nmol / kg, from about 100 nmol / kg to about 1000 nmol / kg, from about 100 nmol / kg to about 2000 nmol / kg of patient body weight. In other embodiments, the dosage 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 or about 3000 nmol / kg of patient body weight.

[0469] In various other embodiments, the dosage range of the compound or its pharmaceutically acceptable salt can be (for example) from about 10 nmol / kg to about 10,000 nmol / kg, from about 10 nmol / kg to about 5,000 nmol / kg, from about 10 nmol / kg to about 3,000 nmol / kg, from about 10 nmol / kg to about 2,500 nmol / kg, from about 10 nmol / kg to about 2,000 nmol / kg, from about 10 nmol / kg to about 1,000 nmol / kg, from about 10 nmol / kg to about 900 nmol / kg, from about 10 nmol / kg to about 800 nmol / kg, from about 10 nmol / kg to about 700 nmol / kg, from about 10 nmol / kg to about 600 nmol / kg, from about 10 nmol / kg to about 500 nmol / kg, from about 10 nmol / kg to about 400 nmol / kg, from about 10 nmol / kg to about 300 nmol / kg, from about 10 nmol / kg to about 200 nmol / kg, from about 10 nmol / kg to about 150 nmol / kg, from about 10 nmol / kg to about 100 nmol / kg, from about 10 nmol / kg to about 90 nmol / kg, from about 10 nmol / kg to about 80 nmol / kg, from about 10 nmol / kg to about 70 nmol / kg, from about 10 nmol / kg to about 60 nmol / kg, from about 10 nmol / kg to about 50 nmol / kg, from about 10 nmol / kg to about 40 nmol / kg, from about 10 nmol / kg to about 30 nmol / kg, from about 10 nmol / kg to about 20 nmol / kg, from about 200 nmol / kg to about 900 nmol / kg, from about 200 nmol / kg to about 800 nmol / kg, from about 200 nmol / kg to about 700 nmol / kg, from about 200 nmol / kg to about 600 nmol / kg, from about 200 nmol / kg to about 500 nmol / kg, from about 250 nmol / kg to about 600 nmol / kg, from about 300 nmol / kg to about 600 nmol / kg, from about 300 nmol / kg to about 500 nmol / kg, or from about 400 nmol / kg to about 600 nmol / kg per 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 150 nmol / kg, from about 1 nmol / kg to about 100 nmol / kg, from about 1 nmol / kg to about 90 nmol / kg, from about 1 nmol / kg to about 80 nmol / kg, from about 1 nmol / kg to about 70 nmol / kg, from about 1 nmol / kg to about 60 nmol / kg, from about 1 nmol / kg to about 50 nmol / kg, from about 1 nmol / kg to about 40 nmol / kg, from about 1 nmol / kg to about 30 nmol / kg, or from about 1 nmol / kg to about 20 nmol / kg. In yet another embodiment, the dosage 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, from about 0.1 nmol / kg to about 1000 nmol / kg, from about 0.1 nmol / kg to about 900 nmol / kg, from about 0.1 nmol / kg to about 850 nmol / kg, from about 0.1 nmol / kg to about 800 nmol / kg, from about 0.1 nmol / kg to about 700 nmol / kg, from about 0.1 nmol / kg to about 600 nmol / kg, from about 0.1 nmol / kg to about 500 nmol / kg, from about 0.1 nmol / kg to about 400 nmol / kg, from about 0.1 nmol / kg to about 300 nmol / kg, from about 0.1 nmol / kg to about 200 nmol / kg, from about 0.1 nmol / kg to about 100 nmol / kg, from about 0.1 nmol / kg to about 50 nmol / kg, from about 0.1 nmol / kg to about 10 nmol / kg, or from about 0.1 nmol / kg to about 1 nmol / kg patient body weight.In other embodiments, the dose can be from about 0.3 nmol / kg to about 1000 nmol / kg, about 0.3 nmol / kg to about 900 nmol / kg, about 0.3 nmol / kg to about 850 nmol / kg, about 0.3 nmol / kg to about 800 nmol / kg, about 0.3 nmol / kg to about 700 nmol / kg, about 0.3 nmol / kg to about 600 nmol / kg, about 0.3 nmol / kg to about 500 nmol / kg, about 0.3 nmol / kg to about 400 nmol / kg, about 0.3 nmol / kg to about 300 nmol / kg, about 0.3 nmol / kg to about 200 nmol / kg, about 0.3 nmol / kg to about 100 nmol / kg, about 0.3 nmol / kg to about 50 nmol / kg, about 0.3 nmol / kg to about 10 nmol / kg, or about 0.3 nmol / kg to about 1 nmol / kg of patient body weight. In such embodiments, "kg" is the number of kilograms of the patient's body weight. In one aspect, a single dose or multiple doses of the compound or a pharmaceutically acceptable salt thereof can be administered to the patient.

[0470] In another embodiment, a compound or a pharmaceutically acceptable salt thereof can be administered to the patient at between about 20 μg / kg and about 3 mg / kg of patient body weight. In another aspect, the amount can be from about 0.2 mg / kg to about 0.4 mg / kg of patient body weight, or can be about 50 μg / kg of patient body weight. In one aspect, a single dose or multiple doses of the compound or a pharmaceutically acceptable salt thereof can be administered to the patient.

[0471] In one embodiment, a small molecule ligand (compound) linked to a targeting moiety can be administered to the patient prior to a CAR T cell composition comprising CAR T cells (wherein the CAR T cells contain a CAR that comprises an E2 anti-fluorescein antibody fragment). In another embodiment, the small molecule ligand (bridge) linked to the targeting moiety can be administered to the patient simultaneously with the CAR T cell composition comprising CAR T cells (wherein the CAR T cells have a CAR that comprises an E2 anti-fluorescein antibody fragment), but in different formulations or in the same formulation. In another embodiment, the small molecule ligand linked to the targeting moiety can be administered to the patient after the CAR T cell composition comprising CAR T cells (wherein the CAR T cells contain a CAR that comprises an E2 anti-fluorescein antibody fragment).

[0472] In an illustrative aspect, the time interval between administration of CAR T cells (which contain a CAR that includes an E2 anti-fluorescein antibody fragment) and a small molecule ligand (the bridge) linked to a targeting moiety can vary widely depending on factors including: the type of CAR T cell expressing the 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 manner in which the CAR T cells expressing the E2 anti-fluorescein antibody fragment and the small molecule ligand linked to the targeting moiety are administered 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.

[0473] In one embodiment, a compound or its pharmaceutically acceptable salt, or for a CAR T cell composition, where the CAR T cell composition comprises CAR T cells that contain a CAR that includes an E2 anti-fluorescein antibody fragment, can be administered using any applicable dosing schedule known in the art. 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. In one aspect, the dosing schedule selected for the compound or its pharmaceutically acceptable salt, and the CAR T cell composition (where the CAR T cell composition comprises CAR T cells that contain a CAR that includes an E2 anti-fluorescein antibody fragment) can take into account the concentration of the compound or its pharmaceutically acceptable salt, and the number of CAR T cells expressing the E2 anti-fluorescein antibody fragment administered to modulate the cytotoxicity of the CAR T cell composition (where the CAR T cell composition comprises CAR T cells that contain a CAR that includes an E2 anti-fluorescein antibody fragment) and to control CRS.

[0474] In one embodiment, a method of treating cancer is provided for preventing or suppressing cytokine release syndrome (CRS) in a patient. The method comprises i) administering to the patient a compound or a pharmaceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked via a linker to a targeting moiety, ii) administering to the patient a CAR T cell composition, wherein the CAR T cell composition comprises CAR T cells, wherein the CAR T cells comprise a CAR, wherein the CAR comprises an E2 anti-fluorescein antibody fragment, and iii) administering to the patient a folic acid compound, a conjugate comprising a folic acid compound (wherein the conjugate comprising a folic acid compound does not comprise a targeting moiety), or a drug that inhibits the activation of CAR T cells.

[0475] In this method embodiment, the step of administering to the patient a folic acid compound, a conjugate comprising a folic acid compound (wherein the conjugate comprising a folic acid compound does not comprise a targeting moiety), or a drug that inhibits the activation of CAR T cells expressing an E2 anti-fluorescein antibody fragment can be used to prevent or suppress CRS in the patient. In this embodiment, any one of the folic acid compound, the conjugate comprising a folic acid compound (wherein the conjugate comprising a folic acid compound does not comprise a targeting moiety), or the drug that inhibits the activation of CAR T cells expressing an E2 anti-fluorescein antibody fragment can be referred to herein as a "rescue agent". In one embodiment, a folic acid compound (such as folic acid) can be administered to prevent or suppress CRS in the patient. In this embodiment, the folic acid compound inhibits the interaction of the bridge (i.e., the small molecule ligand linked via a linker to a targeting moiety) with the receptor of the bridge on the tumor, inhibits tumor lysis, and prevents or suppresses CRS in the patient.

[0476] In another embodiment, the rescue agent can reduce CRS as soon 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., from grade 3 to grade 2, from grade 4 to grade 3, etc.).

[0477] The following columns 1 to 6 (for grades 0, 1, 2, 3, 4, and 5) show an exemplary grading system:

[0478] 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 analogue, or another molecule that binds to the folate receptor. In various embodiments, folic acid analogues that can be used include formyltetrahydrofolic acid, pteroylpolyglutamic acid, and folate receptor-binding pteridines such as tetrahydropterin, dihydrofolic acid, tetrahydrofolic acid, and their deaza and dideaza analogues. The terms "deaza" and "dideaza" analogues refer to analogues well known in the art having a carbon atom that replaces one or two nitrogen atoms in the structure of naturally occurring folates. For example, deaza analogues include 1-deaza, 3-deaza, 5-deaza, 8-deaza, and 10-deaza analogues. Dideaza analogues include, for example, 1,5-dideaza, 5,10-dideaza, 8,10-dideaza, and 5,8-dideaza analogues. The above folic acid analogues are generally referred to as "folate compounds" to reflect their ability to bind to the folate receptor. Other folate receptor-binding analogues include aminopterin, methotrexate, N10-methylfolic acid, 2-deamino-hydroxyfolic acid, deaza analogues such as 1-deazamethotrexate or 3-deazamethotrexate, and 3',5'-dichloro-4-amino-4-deoxy-N10-methylpteroylglutamic acid (dichloromethotrexate).

[0479] In another embodiment, the folate compound administered as an inhibitor of the binding of the bridge to the tumor has the following formula wherein X 1 and Y 1 are each independently selected from halogen, R 2 , OR 2 , SR 3 , and NR 4 R 5 ; 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-; X 2 and X 3 are 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 alkoxy, where Z is oxygen or sulfur; R 1 is selected from hydrogen, halogen, C1-C 12 alkyl and C1-C 12 alkoxy; R 2 , R 3 , R 4 , R 4a , R 4b , R 5 , R 5b , R 6b and R 7b each independently is 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; R 6 and R 7 each independently is selected from hydrogen, halogen, C1-C 12 alkyl and C1-C 12 alkoxy; or R 6 and R 7 together form a carbonyl group; R 6a and R 7a each independently is selected from hydrogen, halogen, C1-C 12 alkyl and C1-C 12 alkoxy; or R 6a and R 7a together form a carbonyl group; p, r, s and t are each independently 0 or 1; and * represents an optional covalent bond connecting to the rest of the conjugate.

[0480] In yet another embodiment, a conjugate comprising a folic acid compound can be administered to prevent or inhibit cytokine release syndrome (CRS) in a patient. CRS is a term well known in the art and this syndrome can have deleterious effects on the patient, including but not limited to weight loss, high fever, pulmonary edema, and a dangerous drop in blood pressure.

[0481] In this embodiment, the conjugate comprising a folic acid compound 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 the patient. In this embodiment, the folic acid compound moiety in the conjugate comprising a folic acid compound can include any of the folic acids described in the previous paragraph attached to a chemical moiety (excluding the targeting moiety). In one aspect, the conjugate comprising a folic acid compound can comprise a folic acid compound attached to one or more amino acids (excluding the targeting moiety). Illustratively, the conjugate comprising a folic acid compound has the following formula

[0482] This compound can also be referred to as "EC923". In such embodiments, the folic acid compound or conjugate comprising a folic acid compound can be administered to the patient in a molar excess relative to the bridge (i.e., the small molecule ligand attached 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 folic acid compound or conjugate comprising a folic acid compound relative to the small molecule ligand attached to the targeting moiety via a linker. The amount of the folic acid compound or conjugate comprising a folic acid compound required to inhibit the interaction of the bridge with the tumor relative to the amount of the small molecule ligand attached to the targeting moiety via a linker can be determined by one of ordinary skill in the art.

[0483] In another embodiment, a rescue agent that inhibits the activation of CAR T cells expressing an E2 anti-fluorescein antibody fragment can be administered to a patient to inhibit CAR T cell activation and to inhibit or prevent CRS in the patient. In one aspect, the agent can be selected from the following: lymphocyte-specific protein tyrosine kinase inhibitors (e.g., dasatinib), PI3 kinase inhibitors (e.g., GDC0980), IL-2 inducible T cell kinase inhibitors (e.g., BMS-509744), JAK inhibitors, BTK inhibitors, tocilizumab, SIP agonists (e.g., siponimod and ozanimod), and an agent that blocks CAR T cell binding to the bridge but not to the cancer (e.g., fluorescamine, FITC, or sodium fluorescein). One of ordinary skill in the art will appreciate that FITC (i.e., fluorescein) can be in the form of a salt (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 can 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 can be a compound of the following formula

[0484] 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 the following doses: 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.

[0485] In such embodiments, the rescue agent can be in molar excess relative to the compound or its pharmaceutically acceptable salt (i.e., the small molecule ligand linked to the targeting moiety via a linker), such as the rescue agent being about 10-fold in excess, about 20-fold in excess, about 30-fold in excess, about 40-fold in excess, about 50-fold in excess, about 60-fold in excess, about 70-fold in excess, about 80-fold in excess, about 90-fold in excess, about 100-fold in excess, about 200-fold in excess, about 300-fold in excess, about 400-fold in excess, about 500-fold in excess, about 600-fold in excess, about 700-fold in excess, about 800-fold in excess, about 900-fold in excess, about 1000-fold in excess, or about 10,000-fold in excess relative to the small molecule ligand linked to the targeting moiety via a linker administered to the patient. The amount of the rescue agent required to inhibit the interaction of the compound or its pharmaceutically acceptable salt with the tumor and / or CAR T cells expressing the E2 anti-fluorescein antibody fragment relative to the amount of the small molecule ligand linked to the targeting moiety via a linker can be determined by one of ordinary skill in the art.

[0486] In another embodiment, more than one dose of a folate compound, a conjugate comprising a folate compound (wherein the conjugate comprising a folate compound does not include a targeting moiety), or an agent that inhibits the activation of CAR T cells expressing the E2 anti-fluorescein antibody fragment can be administered to the patient.

[0487] In the "rescue agent" embodiments described herein, a folate compound, a conjugate comprising a folate compound (wherein the conjugate comprising a folate compound does not include a targeting moiety), or an agent that inhibits the activation of CAR T cells expressing the E2 anti-fluorescein antibody fragment can be administered to the patient before and / or after the compound or its pharmaceutically acceptable salt. In another aspect, the compound or its pharmaceutically acceptable salt can be administered before and then after the administration of a folate compound, a conjugate comprising a folate compound (wherein the conjugate comprising a folate compound does not include a targeting moiety), or an agent that inhibits the activation of CAR T cells expressing the E2 anti-fluorescein antibody fragment. In this embodiment, the subsequent administration of the compound or its pharmaceutically acceptable salt can result in the activation of CAR T cells expressing the E2 anti-fluorescein antibody fragment and an increase in cytokine levels in the patient.

[0488] In another embodiment, administration of a folic acid compound, a conjugate comprising a folic acid compound (wherein the conjugate comprising a folic acid compound does not include a targeting moiety), or an agent that inhibits the activation of CAR T cells expressing an E2 anti-fluorescein antibody fragment can result in a decrease in cytokine levels in a patient. In yet another embodiment, the decrease in cytokine levels can 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 folic acid compound, a conjugate comprising a folic acid compound (wherein the conjugate comprising a folic acid compound does not include a targeting moiety), or an agent that inhibits the activation of CAR T cells expressing an E2 anti-fluorescein antibody fragment to a patient. In another embodiment, the decrease in cytokine levels is to a level approaching the cytokine levels in an untreated patient.

[0489] In another illustrative embodiment, after administration of a folic acid compound, a conjugate comprising a folic acid compound (wherein the conjugate comprising a folic acid compound does not include a targeting moiety), or an agent that inhibits the 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 increase, even though the cytokine levels in the patient decrease. In another illustrative aspect, after administration of a folic acid compound, a conjugate comprising a folic acid compound (wherein the conjugate comprising a folic acid compound does not include a targeting moiety), or a rescue agent that inhibits the activation of CAR T cells (wherein the CAR T cells comprise a CAR comprising an E2 anti-fluorescein antibody fragment), the 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 though the cytokine levels in the treated patient decrease. In still another embodiment, the cancer comprises a tumor and when a folic acid compound, a conjugate comprising a folic acid compound (wherein the conjugate comprising a folic acid compound does not include a targeting moiety), or a rescue agent that inhibits the activation of CAR T cells expressing an E2 anti-fluorescein antibody fragment is administered to a patient, the size of the patient's tumor does not increase. In this embodiment, a complete response to the tumor can be obtained.

[0490] In other embodiments, a rescue agent that inhibits the activation of CAR T cells expressing an E2 anti-fluorescein antibody fragment is administered to a 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 decreases.

[0491] In one embodiment described herein, a method of treating cancer is provided, and the method comprises i) continuously 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 by a linker, ii) administering to the patient a CAR T cell composition comprising CAR T cells, 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.

[0492] According to this embodiment, the term "continuous" can mean administering the compound or a pharmaceutically acceptable salt thereof to the patient for (e.g.) 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 can mean a daily or weekly administration schedule, such as once a day, twice a day, three times a day, daily, every other day, once a week, twice a week, three times a week, or any other suitable schedule considered continuous administration by those skilled in the art. In another aspect, the term "continuous" can mean any combination of the embodiments described in this paragraph.

[0493] In this method embodiment, the step of "terminating continuous administration" of the compound or a pharmaceutically acceptable salt thereof to inhibit or prevent cytokine release syndrome in the patient can mean, for example, terminating the administration after a continuous period of time (such as hours or days), or terminating a treatment schedule (such as the daily or weekly schedule described above). In another embodiment, the step of "terminating continuous administration" can mean, for example, administering until an unacceptable loss of the patient's body weight occurs, or until any other unacceptable side effect occurs, such as high fever, decreased blood pressure, or pulmonary edema. In this embodiment, the step of "terminating continuous administration" of the compound or a pharmaceutically acceptable salt thereof does not mean monotherapy with the compound or a pharmaceutically acceptable salt thereof without subsequent treatment with the 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 ameliorating the symptoms of CRS.

[0494] In one embodiment of an embodiment involving termination of continuous administration of a compound or a pharmaceutically acceptable salt thereof, the method may further comprise step iv) of readministering the compound or a pharmaceutically acceptable salt thereof to the patient. In one embodiment, the compound or a pharmaceutically acceptable salt thereof may be administered (e.g.,) once a week and one dose may be omitted. In another embodiment, the compound or a pharmaceutically acceptable salt thereof may be administered every other day (i.e., every second day) and one or more (e.g., two, three, four, etc.) doses may be omitted. In another embodiment, the compound or a pharmaceutically acceptable salt thereof may be administered twice a week and one or more (e.g., two, three, four, etc.) doses may be omitted. In another embodiment, the compound or a pharmaceutically acceptable salt thereof may be administered on Monday, Tuesday and the following Monday and then dosing may be stopped for two weeks and the cycle repeated. In another embodiment, any one of the above-described protocol embodiments may be used and one or more (e.g., two, three, four, etc.) doses may be omitted. In such embodiments, the omitted dose(s) may prevent or reduce CRS in the patient.

[0495] In another illustrative aspect, there is provided a method of treating cancer. The method comprises 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, wherein the patient is administered at least a first dose and a second dose of the compound or a pharmaceutically acceptable salt thereof, wherein the first dose and the second dose are different, wherein the amount of the second dose of the compound or a pharmaceutically acceptable salt thereof is about 2-fold to about 15,000-fold more than the amount of the first dose of the compound or a pharmaceutically acceptable salt thereof, and ii) administering to the patient a CAR T cell composition comprising CAR T cells, wherein the CAR T cells comprise a CAR comprising an E2 anti-fluorescein antibody fragment.

[0496] In this embodiment, the dose of the compound or its pharmaceutically acceptable salt can be incrementally increased to inhibit or prevent cytokine release syndrome in a patient. For example, at least a first dose and a second dose of the compound or its pharmaceutically acceptable salt can be administered to the patient, where the first dose is different from the second dose, and the amount of the compound or its pharmaceutically acceptable salt in the second dose is about 20-fold to about 15,000-fold, about 2-fold to about 15,000-fold more than the amount of the compound or its pharmaceutically acceptable salt in the first dose. In other embodiments, the amount of the second dose or subsequent doses can 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 15,000-fold, about 2-fold to about 10,000-fold, about 5-fold to about 9,000-fold, about 5-fold to about 8,000-fold, about 5-fold to about 7,000-fold, about 5-fold to about 6,000-fold, about 5-fold to about 5,000-fold, about 5-fold to about 4,000-fold, about 5-fold to about 3,000-fold, about 5-fold to about 4,000-fold, about 5-fold to about 3,000-fold, about 5-fold to about 2,000-fold, about 5-fold to about 1,000-fold, about 5-fold to about 750-fold, about 2-fold to about 750-fold, about 5-fold to about 500-fold, about 5-fold to about 100-fold, about 800-fold to about 15,000-fold, about 800-fold to about 10,000-fold, about 800-fold to about 9,000-fold, about 800-fold to about 8,000-fold, about 800-fold to about 7,000-fold, about 800-fold to about 6,000-fold, about 800-fold to about 5,000-fold, about 800-fold to about 4,000-fold, about 800-fold to about 3,000-fold, about 800-fold to about 2,000-fold, about 800-fold to about 1,000-fold, about 8,000-fold to about 15,000-fold, about 8,000-fold to about 10,000-fold, about 8,000-fold to about 9,000-fold, about 15,000-fold, about 10,000-fold, about 9,000-fold, about 8,000-fold, about 7,000-fold, about 6,000-fold, about 5,000-fold, about 4,000-fold, about 3,000-fold, about 2,000-fold, about 1,000-fold, about 500-fold, about 400-fold, about 300-fold, about 200-fold, about 100-fold, about 90-fold, about 80-fold, about 70-fold, about 60-fold, about 50-fold, about 40-fold, about 30-fold, about 20-fold, about 10-fold, about 5-fold, or about 2-fold more than the amount of the compound or its pharmaceutically acceptable salt in the first dose.

[0497] In another illustrative embodiment of the dose escalation method, a patient may be administered at least a first dose, a second dose, and a third dose of a compound or a pharmaceutically acceptable salt thereof, wherein the first dose, the second dose, and the third dose are different, wherein the amount of the second dose of the compound or a pharmaceutically acceptable salt thereof is about 2 to about 750 times greater than the amount of the first dose of the compound or a pharmaceutically acceptable salt thereof, and wherein the amount of the third dose of the compound or a pharmaceutically acceptable salt thereof is about 800 to about 10,000 times greater than the amount of the first dose of the compound or a pharmaceutically acceptable salt thereof.

[0498] In another aspect of the dose escalation method, a patient may be administered at least a first dose, a second dose, a third dose, and a fourth dose of a compound or a pharmaceutically acceptable salt thereof, wherein the first dose, the second dose, the third dose, and the fourth dose are different, wherein the amount of the second dose of the compound or a pharmaceutically acceptable salt thereof is about 2 to about 750 times greater than the amount of the first dose of the compound or a pharmaceutically acceptable salt thereof, wherein the amount of the third dose of the compound or a pharmaceutically acceptable salt thereof is about 800 to about 7,500 times greater than the amount of the first dose of the compound or a pharmaceutically acceptable salt thereof, and wherein the amount of the fourth dose of the compound or a pharmaceutically acceptable salt thereof is about 8,000 to about 15,000 times greater than the amount of the first dose of the compound or a pharmaceutically acceptable salt thereof.

[0499] In yet another embodiment, the amount of the second dose of the compound or a pharmaceutically acceptable salt thereof may be about 100 times greater than the amount of the first dose of the compound or a pharmaceutically acceptable salt thereof, the amount of the third dose of the compound or a pharmaceutically acceptable salt thereof may be about 1,000 times greater than the amount of the first dose of the compound or a pharmaceutically acceptable salt thereof, and the amount of the fourth dose of the compound or a pharmaceutically acceptable salt thereof may be about 10,000 times greater than the amount of the first dose of the compound or a pharmaceutically acceptable salt thereof. In an exemplary embodiment, the first dose of the compound or a pharmaceutically acceptable salt thereof is 0.05 nmol / kg, the second dose is 5 nmol / kg, the third dose is 50 nmol / kg, and the fourth dose is 500 nmol / kg. In the dose escalation embodiments described herein, the first, second, third, fourth, and any subsequent doses of the compound or a pharmaceutically acceptable salt thereof may be administered multiple times (e.g., the first dose of 0.05 nmol / kg may be administered several times before administering subsequent escalating doses).

[0500] In another embodiment described herein, a method of 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 CAR T cells, wherein the CAR T cells comprise a CAR comprising an E2 anti-fluorescein antibody fragment.

[0501] In various embodiments of this dose-decreasing embodiment, the amount of the second dose of the compound or a pharmaceutically acceptable salt thereof may be at least about 60% lower than the amount of the first dose of the compound or a pharmaceutically acceptable salt thereof, at least about 70% lower than the amount of the first dose of the compound or a pharmaceutically acceptable salt thereof, at least about 80% lower than the amount of the first dose of the compound or a pharmaceutically acceptable salt thereof, at least about 90% lower than the amount of the first dose of the compound or a pharmaceutically acceptable salt thereof, at least about 95% lower than the amount of the first dose of the compound or a pharmaceutically acceptable salt thereof, at least about 96% lower than the amount of the first dose of the compound or a pharmaceutically acceptable salt thereof, at least about 97% lower than the amount of the first dose of the compound or a pharmaceutically acceptable salt thereof, at least about 98% lower than the amount of the first dose of the compound or a pharmaceutically acceptable salt thereof, at least about 99% lower than the amount of the first dose of the compound or a pharmaceutically acceptable salt thereof, or at least about 99.5% lower than the amount of the first dose of the compound or a pharmaceutically acceptable salt thereof.

[0502] In various embodiments of the dose-decreasing embodiment described herein, the first dose of the compound or a pharmaceutically acceptable salt thereof may be from about 100 nmol / kg to about 1000 nmol / kg of patient body weight, from about 100 nmol / kg to about 900 nmol / kg of patient body weight, from about 100 nmol / kg to about 800 nmol / kg of patient body weight, from about 100 nmol / kg to about 700 nmol / kg of patient body weight, from about 100 nmol / kg to about 600 nmol / kg of patient body weight, from about 200 nmol / kg to about 600 nmol / kg of patient body weight, from about 400 nmol / kg to about 600 nmol / kg of patient body weight, or about 500 nmol / kg of patient body weight.

[0503] In various embodiments of the dose tapering regimens described herein, the second dose of the compound or its pharmaceutically acceptable salt 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.

[0504] In additional embodiments of the dose tapering regimens described herein, the method can further comprise administering a third dose of the compound or its pharmaceutically acceptable salt, wherein the third dose of the compound or its pharmaceutically acceptable salt is the same as the second dose of the compound or its pharmaceutically acceptable salt. In another embodiment, the method can further comprise administering a fourth dose of the compound or its pharmaceutically acceptable salt, wherein the fourth dose of the compound or its pharmaceutically acceptable salt is the same as the second dose of the compound or its pharmaceutically acceptable salt and the third dose of the compound or its pharmaceutically acceptable salt. In another embodiment, the dose of the compound or its pharmaceutically acceptable salt administered after the first dose of the compound or its pharmaceutically acceptable salt can maintain the inhibition of cancer growth relative to the first dose of the compound or its pharmaceutically acceptable salt.

[0505] In other embodiments of the dose tapering regimens described herein, CAR T cells expressing an E2 anti-fluorescein antibody fragment can be administered at a dose of from about 1 million to about 40 million CAR T cells expressing an E2 anti-fluorescein antibody fragment. In still other embodiments, the dose of the compound or its pharmaceutically acceptable salt administered after the first dose of the compound or its pharmaceutically acceptable salt can be administered once or twice a week.

[0506] In still other embodiments of the dose tapering embodiments described herein, the method may further comprise administering to the patient a folic acid compound, a conjugate comprising a folic acid compound (wherein the conjugate comprising a folic acid compound does not include a targeting moiety), or an agent that inhibits the activation of CAR T cells expressing an E2 anti-fluorescein antibody fragment. In another embodiment, an agent that inhibits the activation of CAR T cells expressing an E2 anti-fluorescein antibody fragment is administered to the patient and the agent is an agent that blocks the binding of CAR T cells expressing an E2 anti-fluorescein antibody fragment to a compound or a pharmaceutically acceptable salt thereof, but does not bind to cancer, and the agent is fluorescamine, sodium fluorescein, or fluorescein. In another embodiment, the agent is sodium fluorescein.

[0507] In another embodiment, a method of 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 and wherein the compound or a pharmaceutically acceptable salt thereof is administered to the patient at least about one hour prior to administering a CAR T cell composition comprising CAR T cells (wherein the CAR T cells comprise a CAR that comprises an E2 anti-fluorescein antibody fragment), ii) then administering to the patient a dose of a CAR T cell composition comprising CAR T cells, wherein the CAR T cells comprise a CAR that comprises an E2 anti-fluorescein antibody fragment, and iii) then administering to the patient a second dose of the compound or a pharmaceutically acceptable salt thereof. In various embodiments of this pretreatment embodiment, the first dose of the compound or a pharmaceutically acceptable salt thereof may be administered to the patient at least about two hours prior to administering the CAR T cell composition, at least about four hours prior to administering the CAR T cell composition, at least about eight hours prior to administering the CAR T cell composition, at least about twelve hours prior to administering the CAR T cell composition, at least about sixteen hours prior to administering the CAR T cell composition, at least about twenty hours prior to administering the CAR T cell composition, or at least about twenty-four hours prior to administering the CAR T cell composition, in each case wherein the CAR T cell composition comprises CAR T cells that comprise a CAR that comprises an E2 anti-fluorescein antibody fragment.

[0508] In various embodiments of this pre-treatment regimen, a second dose of the compound or a pharmaceutically acceptable salt thereof may 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 CAR T cells, the CAR T cells comprise a CAR, and the CAR comprises an E2 anti-fluorescein antibody fragment.

[0509] In any of the embodiments described herein, where the folate compound is a ligand linked to a targeting moiety via a linker, the patient may be placed on a folate-deficient diet prior to treatment using the methods described herein, or folate may be administered to the patient in the diet. In embodiments where folate is administered to the patient, the dosage range may be, for example, from about 50 nmol / kg to about 3000 nmol / kg of patient body weight, from about 50 nmol / kg to about 2000 nmol / kg, from about 50 nmol / kg to about 1000 nmol / kg, from about 50 nmol / kg to about 900 nmol / kg, from about 50 nmol / kg to about 800 nmol / kg, from about 50 nmol / kg to about 700 nmol / kg, from about 50 nmol / kg to about 600 nmol / kg, from about 50 nmol / kg to about 500 nmol / kg, from about 50 nmol / kg to about 400 nmol / kg, from about 50 nmol / kg to about 300 nmol / kg, from about 50 nmol / kg to about 200 nmol / kg, from about 50 nmol / kg to about 100 nmol / kg, from about 100 nmol / kg to about 300 nmol / kg, from about 100 nmol / kg to about 500 nmol / kg, from about 100 nmol / kg to about 1000 nmol / kg, from about 100 nmol / kg to about 2000 nmol / kg of patient body weight. In other embodiments, the dosage may be about 100 nmol / kg, about 150 nmol / kg, about 200 nmol / kg, about 250 nmol / kg, about 300 nmol / kg, about 350 nmol / kg, about 400 nmol / kg, about 450 nmol / kg, about 500 nmol / kg, about 600 nmol / kg, about 700 nmol / kg, about 800 nmol / kg, about 900 nmol / kg, about 1000 nmol / kg, about 2000 nmol / kg, or about 3000 nmol / kg of patient body weight. In such embodiments, "kg" is the number of kilograms of the patient's body weight. In one aspect, folate may be administered, for example, daily, weekly, bi-weekly, three times a week, or using any suitable regimen for administering folate.

[0510] In various embodiments described herein, after administration of the CAR T cells, the CAR T cells may 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.

[0511] In various embodiments described herein, the half maximal effective concentration (EC 50 ) of the compound or its pharmaceutically acceptable salt can be from about 1 pM to about 2 nM, about 1 pM to about 5 nM, about 1 pM to about 10 nM, about 1 pM to about 20 nM, about 1 pM to about 30 nM, about 1 pM to about 40 nM, about 1 pM to about 50 nM, about 1 pM to about 60 nM, about 1 pM to about 70 nM, about 1 pM to about 80 nM, about 1 pM to about 90 nM, about 1 pM to about 100 nM, about 1 pM to about 200 nM, about 1 pM to about 300 nM, about 1 pM to about 400 nM, about 1 pM to about 500 nM, about 1 pM to about 600 nM, about 1 pM to about 700 nM, about 1 pM to about 800 nM, about 1 pM to about 900 nM, about 1 pM to about 1 nM, about 1 pM to about 900 pM, about 1 pM to about 800 pM, about 1 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.

[0512] In various embodiments described herein, the Kd of the binding of the compound or its pharmaceutically acceptable salt to CAR T cells can be from 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.

[0513] In various illustrative embodiments described herein, the compound or its pharmaceutically acceptable salt can be administered to a 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 CAR T cells or on any suitable day before or after CAR T cells.

[0514] In various embodiments described herein, sorted or unsorted CAR T cells via EGFRt can be used. In another embodiment, “clinical fax” batches of CAR T cells with a low differentiation profile can be used. In another embodiment, “research batches” of CAR T cells can be used. The “clinical fax” batches (about 39% EGFRt+) can contain about 66% T SCM and about 32% T CM of the CD4+ subset and about 95% T SCM and about 3% T CM of the CD8 subset. The research batches (about 23% EGFRt+) can contain about 32% T SCM , about 53% T CM , about 11% T EM and about 3.7% T EFF of the CD4 subset and about 44% T SCM , about 0.28% T CM , about 3.4% T EM and about 52% T EFF of the CD8 subset.

[0515] In various additional embodiments of this pretreatment embodiment, cytokine release leading to off-target toxicity in the patient does not occur, but toxicity of CAR T cells expressing the E2 anti-fluorescein antibody fragment to cancer occurs or off-target tissue toxicity does not occur, but toxicity of CAR T cells expressing the E2 anti-fluorescein antibody fragment to cancer occurs, or the cancer includes a tumor, and the size of the patient's tumor decreases, but off-target toxicity does not occur, or before administering a CAR T cell composition comprising CAR T cells (comprising a CAR that comprises an E2 anti-fluorescein antibody fragment), the reduction in the size of the patient's tumor is greater than that of a patient not pretreated with the compound or a pharmaceutically acceptable salt thereof. One of ordinary skill in the art will appreciate that the “target” can be cancer (e.g., a tumor).

[0516] In another embodiment, a method of treating cancer is provided. The method comprises 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 ii) administering to the patient a CAR T cell composition, wherein the CAR T cell composition comprises CAR T cells and wherein the CAR T cells comprise a CAR that 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 linked to the targeting moiety via a linker can have the following formula

[0517] In yet another embodiment, a method of treating cancer is provided. The method comprises 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 ii) administering to the patient a CAR T cell composition, wherein the CAR T cell composition comprises CAR T cells and wherein the CAR T cells comprise 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 linked to the targeting moiety via a linker can have the following formula

[0518] In still another embodiment, a method of treating cancer is provided. The method comprises i) administering to a patient a first compound or a pharmaceutically acceptable salt thereof, wherein the first compound or the pharmaceutically acceptable salt thereof comprises a PSMA ligand linked to FITC via a linker, ii) administering to the patient a second compound or a pharmaceutically acceptable salt thereof, wherein the second compound or the pharmaceutically acceptable salt thereof comprises a CAIX ligand linked to FITC via a linker, and iii) administering to the patient a CAR T cell composition, wherein the CAR T cell composition comprises CAR T cells and wherein the CAR T cells comprise a CAR, the CAR comprising an E2 anti-fluorescein antibody fragment. In this embodiment, the first compound can have the following formula and the second compound can have the following formula

[0519] In one embodiment of the methods described herein, the cancer is imaged before administering to the patient the compound or a pharmaceutically acceptable salt thereof, or before administering to the patient the CAR T cell composition, wherein the CAR T cell composition comprises CAR T cells, the CAR T cells comprising a CAR, the CAR comprising an E2 anti-fluorescein antibody fragment. In an illustrative embodiment, the imaging occurs via PET imaging. In other illustrative embodiments, the imaging occurs via MRI imaging or SPECT / CT imaging. The imaging method can be any suitable imaging method known in the art. In one embodiment, the imaging method can involve using the small molecule ligand described herein, but the ligand is linked to an imaging agent suitable for the type of imaging described herein.

[0520] In any of the embodiments described herein, cytokine release leading to off-target toxicity in a patient may not occur, even if toxicity to cancer of CAR T cells expressing an E2 anti-fluorescein antibody fragment occurs. In any of the embodiments described herein, off-target tissue toxicity in a patient may not occur, even if toxicity to cancer of CAR T cells expressing an E2 anti-fluorescein antibody fragment occurs. In any of the embodiments described herein, cancer may include a tumor, and the size of the patient's tumor may be reduced, but off-target toxicity does not occur. In any of the embodiments described herein, CRS may be reduced or prevented and the method may result in a reduction in the tumor volume of the patient. In any of the embodiments described herein, weight loss due to CRS may be reduced or prevented. In any of the embodiments described herein, cancer may include a tumor and a complete response to the tumor may be obtained.

[0521] In another embodiment of the methods described herein, any one of the methods described herein may be used alone, or any one of the methods described herein may be used in combination with any other one or more methods as described herein. Examples

[0522] Example 1 EC17 dose de-escalation can reduce therapy-related toxicity Study the effect of EC17 dose de-escalation on the anti-tumor activity and toxicity (weight change) of CAR-T therapy. Two different anti-fluorescein scFvs were used for the CAR construct ( Figure 1 ). The first construct of the CAR contained 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 the CAR contained the following domains: anti-FL (FITC-4M5.3) scFv-IgG4 hinge (CH2 (L235D, N297Q)-CH3)-CD28TM-4-1BB-CD3z-T2A-EGFRt. The nucleic acid sequences and amino acid sequences of the CARs of each construct are shown in SEQ ID NOS: 1 to 4. Human T cells were isolated, activated and transduced with a lentiviral vector carrying the appropriate CAR gene. CAR-modified CD4+ T cells and CAR-modified CD8+ T cells were isolated and cultured separately in vitro for about 7 days. Before i.v. administration for animal studies, CD4+ CAR-T cells and CD8+ CAR-T cells were mixed at a 1:1 ratio.

[0523] MDA-MB-231 tumor-bearing (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 day 2, day 9, day 16, day 23, etc. (as shown in Figure 2 A). 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 indicated in Figure 5A and 5B ). Both groups showed severe weight loss (about 20%) during E2-CAR-T therapy, indicating that the 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), this may be caused by TCRs with allogeneic HLA mismatches found in "young" T cells that are usually cultured in vitro for a shorter time).

[0524] 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 administration was modified to a decreasing dose. As shown in Figure 2 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 day 9, day 16, day 23, etc. after CAR-T administration). As shown in Figure 3A , all three EC17 decreasing dose groups achieved complete responses, indicating that the bridge decreasing dose did not affect the anti-tumor activity of CAR-T. More importantly, none of the three EC17 decreasing dose groups showed any severe CRS. As shown in Figure 3B , all decreasing dose groups showed very slight weight loss, although more CAR-T cells (30 million) were administered compared to the first part of the study (10 or 20 million CAR-T cells). Such data indicate that the bridge decreasing dose (a high-level loading dose followed by a decreasing-level maintenance dose) can reduce toxicity while maintaining good anti-tumor activity of CAR-T cells.

[0525] The 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. Mice bearing MDA-MB-231 tumors (150 to 250 mm 3 ) 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. (as Figure 2 shown in A). Mice administered 10 or 20 million 4M5.3-CAR-T cells did not show severe CRS or weight loss ( Figure 6B ) after the EC17 dose was administered, but all of their tumors decreased and eventually disappeared ( Figure 6A ). 20 million CAR-T cells (upper line) showed better anti-tumor activity than 10 million CAR-T cells (middle line).

[0526] In the second part of the study, the 4M5.3-CAR-T cells used were cultured in vitro for approximately 2 weeks. 30 million 4M5.3-CAR-T cells were administered and the EC17 dosing was modified to be decreasing. As Figure 2 shown in B, 500 nmol / kg EC17 was administered 2 days after CAR-T administration, and then lower doses of EC17 (5, or 20 or 100 nmol / kg) were administered weekly (on days 9, 16, 23, etc. after CAR-T administration). As Figure 4A shown, the 100 nmol / kg EC17 decreasing group showed the best anti-tumor activity and all tumors were cured (bottom line), whereas 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 in a decreasing manner. In addition, as Figure 4B shown, the weight loss of the mice in the three decreasing groups also depended on the EC17 dose. The 100 nmol / kg EC17 dose decreasing group showed more weight loss than the other two groups using 5 or 20 nmol / lg EC17. Such data indicate that bridge decreasing (a high-level loading dose followed by a decreasing-level maintenance dose) can control both the anti-tumor activity and toxicity of CAR-T therapy.

[0527] The only difference between the E2-CAR and the 4M5.3-CAR is the scFv sequence used. By comparing the two CARs under the same conditions, when testing the 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.

[0528] As used herein, "SEQ ID NO:4" means the sequence starting with the underlined "gac" codon and ending with the underlined "ggc" codon. This part of the longer sequence encodes the exemplary 4M5.3 CAR. The CAR is inserted into the T cell membrane. The other parts of the longer sequence include the coding sequences of the signal peptide, the EGFRt domain, etc., which are not the parts of the CAR inserted into the membrane and act as chimeric antigen receptors. As used herein, "SEQ ID NO:3" means the sequence starting with the underlined "D" and ending with the underlined "G". This part of the longer sequence is the amino acid sequence of the CAR inserted into the T cell membrane. The other parts of the longer sequence include the amino acid sequences of the signal peptide, the EGFRt domain, etc., which are not the parts of the CAR inserted into the membrane and act as chimeric antigen receptors. In yet another embodiment, SEQ ID NO:3 may comprise 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 prepare 4M5.3 CAR.

[0529] SEQ ID NO:3 [4M5.3-CAR amino acid sequence (insertion)] SEQ ID NO:4 [4M5.3-CAR nucleic acid sequence (insertion)]

[0530] Example 2 Characterization of the CAR T cell differentiation phenotype before infusion into NSG mice The phenotype of the CAR T cell product to be infused into tumor-bearing mice was determined using multicolor flow cytometry. The differentiation status of the CD4+ and CD8+ subsets was analyzed using a specific combination of surface markers as shown in Figure 7 A. The unsorted E2 clinical fax CAR T cell product was mainly made up of the self-renewing Tscm / Tcm phenotype ( Figure 7 B). At Figure 7In B, the highest bar is "Tscm" and the lower bar is "Tcm". Except for Figure 8A the difference in the CD8:CD4 ratio shown in Figure 8B , the current E2 clinical fax CAR T cells are different from the previously sorted E2 / 4M5.3 CAR-T cells and the early GFP+4M5.3 CAR T in vivo study preparations (as Figure 8A outlined in the pie chart in Figure 8A . In Figure 8A , in 10 groups from left to right, the bars represent the following – 1 (highest bar Tscm, lower bar Tcm), 2 – (highest bar Tscm, lower bar Tcm), 3 – (highest bar Teff, lower bar Tem), 4 – (highest bar Tem, lower bar Teff, lowest bar Tcm), 5 – (highest bar Teff, lower bar Tem), 6 – (highest bar Tem, lower bar Teff, lowest bar Tcm), 7 – (highest bar Teff, lower bar Tscm), 8 – (highest bar Teff, lower bar Tem), 9 – (highest bar Teff, lower bar Tscm), 10 – (highest bar Teff, lower bar Tsm, next lower bar Tem, lowest bar Tcm). Thus, when compared to past CAR T preparations with a more differentiated Tem / Teff phenotype, CAR T cells with a predominantly Tscm / Tcm phenotype are expected to have a better anti-tumor phenotype. For the same reason, considering the same EC17 dosing regimen, sCRS for the current CAR-T preparation occurs later than for the previous CAR-T preparation.

[0531] Recovery of CAR T cells after cryopreservation Quickly thaw the cryopreserved CAR T cells in a 37°C water bath and then immediately place them in T cell recovery medium (TexMACS TM medium containing glutamine supplemented with 2% human AB serum (Miltenyi Biotec cat. no. 130-097-196)) and 50 U / mL recombinant human IL2 and culture in vitro for approximately 3 to 5 days for T cell recovery.

[0532] Flow cytometry analysis Harvest CAR T cells from ex vivo recovery medium and pellet by centrifugation at 400 g for 5 minutes. Then resuspend the T cells in flow cytometry staining solution [1% bovine serum albumin, 50 mg / mL human IgG (Equitech Bio, catalog number SLH56-0001), phosphate buffered saline with 0.9% sodium azide, pH = 7.4] supplemented with both anti-mouse FcγIII / II receptor (CD16 / CD32) block [clone 2.4G2; BD Bioscience, catalog number 553142 diluted 1:100 (v / v)] and anti-human Fc block [BD Biosciences, catalog number 564220 diluted 1:50 (v / v)]. Surface marker staining is performed using the following fluorescent dye-conjugated monoclonal antibodies added to each sample on ice in the dark (added over 20 minutes): anti-human CD45RA-APCeF780 [clone HI100, ThermoFisher number 47-0458-42 diluted 1:20 (v / v)], anti-human CD45RO-eF450 [clone UCHL1, Thermofisher number 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 Systems number FAB9577B-100 followed by PE-conjugated streptavidin (BioLegend number 53-9895-82 diluted 1:400 (v / v)). After surface marker staining, wash the T cells with PBS and resuspend in cold PBS containing 3 μM propidium iodide and transfer to a flow cytometry collection tube. Flow cytometry data is collected on a Gallios flow cytometer (Beckman Coulter, Brea, CA), where at least 20,000 events are collected and the data is analyzed using Kaluza v1.5 software.

[0533] Example 3 The E2 anti-FITC antibody does not bind to any normal human tissue The E2 anti-FITC IgG antibody having the same variable fragment sequences as those in the E2 anti-FITC CAR construct was used to test whether the E2 antibody has any binding to human normal tissues. Recombinant human E2 anti-FITC IgG1λ1 was expressed in HEK293 cells and purified using a protein A affinity column. By SEC-HPLC analysis, the purity of the antibody exceeded 98%. 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 the beads for 30 minutes. An anti-mouse secondary antibody conjugated with eF660 was added and incubated with the washed beads. The washed beads were analyzed by FACS for the eF660 signal. 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 shown in Figure 9. The binding affinity of the DIG-labeled E2 antibody to FITC was calculated using PRISM software. As Figure 9B shown in the insert, the Kd was approximately 0.66 nM, indicating that the DIG-labeled E2 anti-FITC antibody has a high binding affinity for FITC and can be used to evaluate the binding of E2 to human normal tissues.

[0534] An IHC assay was developed to test the binding of the E2 antibody to human tissues. Formalin-fixed paraffin-embedded (FFEP) tissue sections of agar blocks containing 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, then antigen retrieval was performed by incubating the tissue sections with antigen retrieval buffer (pH 6.0) at 91 °C for 24 minutes, then the 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 peroxidase-labeled anti-mouse secondary antibody. As Figure 10A shown, the FITC-labeled KB cells showed positive staining, indicating that the E2 antibody binds to FITC on these labeled KB cells. The unlabeled KB cells did not show any staining ( Figure 10B ).

[0535] The DIG-labeled E2 antibody was incubated with a human multi-organ normal tissue microarray (FDA999, USBiomax, Inc) and the binding of the E2 antibody to normal human tissues was evaluated using the same IHC procedure under the same conditions as above. This multi-organ normal tissue microarray has 99 cores with 28 normal human organs, including adrenal gland (Figure 11), bone marrow (Figure 12), breast (Figure 13), cerebellum tissue (Figure 14), cervix (Figure 15), colon (Figure 16), esophagus (Figure 17), eye (Figure 18), heart (Figure 19), pituitary gland (Figure 20), kidney (Figure 21), larynx (Figure 22), liver (Figure 24), lung (Figure 25), lymph node (Figure 26), nerve (Figure 27), ovary (Figure 28), pancreas (Figure 29), prostate (Figure 30), skin (Figure 31), small intestine (Figure 32), spleen (Figure 23), stomach (Figure 33), striated muscle (Figure 34), testis (Figure 35), thymus (Figure 36), tongue (Figure 37), and uterus (Figure 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 some 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 is non-specific.

[0536] In summary, the E2 anti-FITC antibody does not bind to any of the tested normal human organ tissues, indicating that the E2 anti-FITC CAR-T cells themselves should not bind to and attack any normal human tissues in the body.

[0537] Example 4 Synthesis of FITC-folic acid In anhydrous dimethyl sulfoxide (DMF), in the presence of tetramethylguanidine and diisopropylamine, folic acid-γ-ethylenediamine was coupled to fluorescein isothiocyanate (FITC) isomer I (Sigma-Aldrich). The crude product was loaded onto an Xterra RP18 preparative HPLC column (Waters) and eluted with gradient conditions (starting with 99% 5 mM sodium phosphate (mobile phase A, pH 7.4) and 1% acetonitrile (mobile phase B) and reaching 90% A and 10% B at a flow rate of 20 mL / min within 10 minutes). Under such conditions, the FITC-folic acid main peak typically elutes at 27 to 50 minutes. The quality of the FITC-folic acid eluate fractions was monitored by analytical reverse-phase HPLC with a UV detector. Fractions with a purity greater than 98.0% (LCMS) were lyophilized to obtain the final FITC-folic acid product. As is known in the art, compounds having this structure are also referred to as EC17.

[0538] Example 5 Synthesis of FITC-PEG12-folic acid Load the general polyethylene glycol (PEG) Nova Tag TM resin (0.2 g) into a peptide synthesis vessel and wash with isopropanol (i-PrOH) (3 x 10 mL) and dimethylformamide (DMF, 3 x 10 mL). Perform 9-fluorenylmethoxycarbonyl (Fmoc) deprotection using DMF containing 20% piperidine (3 x 10 mL). Perform a Kaiser test to evaluate the reaction progress. Then introduce into the vessel a DMF solution containing Fmoc-L-glutamic acid 5-tert-butyl ester (Fmoc-Glu-(O-t-Bu)-OH) (23.5 mg), N,N-diisopropylethylamine (i-Pr2NEt) (4 equivalents), and benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) (2 equivalents). Perform Fmoc deprotection using DMF containing 20% piperidine (3 x 10 mL). Then introduce into the vessel a solution of N 10 -TFA-Pte-OH (22.5 mg), DMF, i-Pr2NEt (4 equivalents), and PyBOP (2 equivalents). Bubble argon for 2 hours, and wash the resin with DMF (3 x 3 mL) and i-PrOH (3 x 3 mL). After the resin swells in dichloromethane (DCM), add a solution of DCM / trifluoroethane (TFE) (1:1) containing 1 M hydroxybenzotriazole (HOBT) (2 x 3 mL). Bubble argon for 1 hour, remove the solvent, and wash the resin with DMF (3 x 3 mL) and i-PrOH (3 x 3 mL). After the resin swells in DMF, add a solution containing Fmoc-NH-(PEG) 12A DMF solution of -COOH (46.3 mg), i-Pr2NEt (4 equivalents), and PyBOP (2 equivalents). Bubble argon for 2 hours, and wash the resin with DMF (3 x 3 mL) and i-PrOH (3 x 3 mL). Perform Fmoc deprotection using DMF containing 20% piperidine (3 x 10 mL). Perform the Kaiser test to evaluate the reaction progress. Then introduce into the vessel a solution of DMF and i-Pr2NEt (4 equivalents) containing FITC (Life Technologies 21.4 mg), then bubble argon for 2 hours, and wash the resin with DMF (3 x 3 mL) and i-PrOH (3 x 3 mL). Then add to the vessel DMF containing 2% NH2NH2 (2 x 2 mL). Cleave the final compound from the resin using TFA:H2O:triisopropylsilane (TIS) (95:2.5:2.5) (cleavage solution) and concentrate under vacuum. Precipitate the concentrated product in Et2O and dry under vacuum. Purify the crude product 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). Pool the pure fractions and lyophilize to afford FITC-PEG12-folic acid.

[0539] Example 6 Synthesis of FITC-PEG20-folic acid Load ethylenediamine, polymer-bound (200 - 400 mesh) resin (50 mg) into a peptide synthesis vessel and swell with DCM (3 mL) followed by DMF (3 mL). Then introduce into the vessel Fmoc-PEG in DMF 20-COOH solution (131 mg, 1.0 equiv), i-Pr2NEt (6.0 equiv) and PyBOP (4.0 equiv). Bubble argon for 6 h, empty the coupling solution, and wash the resin with DMF (3 x 10 mL) and i-PrOH (3 x 10 mL). Perform Kaiser test to evaluate the reaction progress. Before each amino acid coupling, perform Fmoc deprotection using DMF containing 20% piperidine (3 x 10 mL). Repeat the above sequence to complete the reaction with Fmoc-Glu-OtBu (72 mg, 2.0 equiv) and the coupling step of Tfa. folic acid (41 mg, 1.2 equiv). Wash the resin with 3 x 10 mL of DMF containing 2% hydrazine (5 min) to cleave the trifluoroacetyl protecting group on folic acid and wash with i-PrOH (3 x 10 mL) followed by DMF (3 x 10 mL). Dry the resin under argon for 30 min. Cleave the folic acid-peptide from the resin using the cleavage solution. Introduce 10 mL of the cleavage mixture and bubble argon for 1.5 h. Drain the cleavage mixture into a clean flask. Wash the resin with more cleavage mixture 3 times. Concentrate the combined mixture under reduced pressure to a smaller volume (ca. 5 mL) and precipitate in diethyl ether.

[0540] Collect the precipitate by centrifugation, wash with diethyl ether (3 times) and dry under high vacuum. At room temperature, treat the dried folic acid-PEG 20 -EDA (1.0 equiv) with DMSO and DIPEA containing FITC (50 mg, 1.5 equiv). Monitor the reaction progress via LCMS. After 8 h, the starting material was consumed to give the product. Purify the crude reaction mixture via preparative HPLC (mobile phase A = 10 mM ammonium acetate, pH = 7; organic phase B = acetonitrile; method: 0% B to 30% B in 35 min at 13 mL / min) and obtain FITC-PEG20-folic acid in 60% yield.

[0541] Example 7 Synthesis of FITC-PEG108-folic acid Load ethylenediamine, polymer-bound (200 - 400 mesh) resin (50 mg) into a peptide synthesis vessel and swell with DCM (3 mL) followed by DMF (3 mL). Then introduce into the vessel Fmoc-PEG in DMF 36-COOH solution (161 mg, 1.0 equivalent), i-Pr2NEt (6.0 equivalents), and PyBOP (4.0 equivalents). Argon was bubbled for 6 hours, the coupling solution was drained, and the resin was washed with DMF (3 x 10 mL) and i-PrOH (3 x 10 mL). Kaiser test was performed to evaluate the reaction progress. Before each amino acid coupling, Fmoc deprotection was carried out using DMF (3 x 10 mL) containing 20% piperidine. The above sequence was repeated to complete the reaction with 2X Fmoc-PEG 36 -COOH (161 mg, 1.0 equivalent), Fmoc-Glu-OtBu (72 mg, 2.0 equivalents), and the coupling step of Tfa. folic acid (41 mg, 1.2 equivalents). Finally, the resin was washed with 3 x 10 mL of DMF containing 2% hydrazine (5 minutes) to cleave the trifluoroacetyl protecting group on folic acid and then washed with i-PrOH (3 x 10 mL) followed by DMF (3 x 10 mL). The resin was dried under argon for 30 minutes. The folic acid-peptide was cleaved from the resin using the 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 to a smaller volume (about 5 mL) under reduced pressure and precipitated in ether.

[0542] The precipitate was collected by centrifugation, washed with ether (3X), and dried under high vacuum. At room temperature, the dried folic acid-PEG 108 -EDA (1.0 equivalent) was treated with DMSO and DIPEA containing FITC (50 mg, 1.5 equivalents). The reaction progress was monitored by LCMS. After 10 hours, the starting material was consumed to obtain 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) to obtain FITC-PEG108-folic acid in 64% yield.

[0543] Example 8 Synthesis of FITC-DUPA DUPA-FITC was synthesized via solid-phase method as follows. The general Nova Tag TMThe resin (50 mg, 0.53 mM) was swollen with DCM (3 mL) followed by DMF (3 mL). A solution of DMF (3 × 3 mL) containing 20% piperidine was added to the resin, and argon was bubbled through 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 equiv), HATU (2.5 equiv) and i-Pr2NEt (4.0 equiv) was added. Argon was bubbled through for 2 hours, and the resin was washed with DMF (3 × 3 mL) and i-PrOH (3 × 3 mL). After the resin was swollen in DCM, a solution of DCM / TFE (1:1) (2 × 3 mL) containing 1 M HOBt was added. Argon was bubbled through for 1 hour, the solvent was removed and the resin was washed with DMF (3 × 3 mL) and i-PrOH (3 × 3 mL). After the resin was swollen 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 through for 2 hours, 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-aminocaprylic acid and fluorescein isothiocyanate or rhodamine B isothiocyanate. The final compound was cleaved from the resin using a cleavage solution and concentrated under vacuum. The concentrated product was precipitated in diethyl ether and dried under vacuum. 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 purified fractions were lyophilized to give FITC-DUPA as a yellowish brown 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 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 C51 H 59 N7O 15 The calculated value of S is 1040.3712, and the measured value is 1040.3702. UV / vis: λmax = 491 nm.

[0544] Example 9 Synthesis of FITC-PEG12-DUPA Load 1,2-diaminoethane triphenylmethyl resin (0.025 g) into a peptide synthesis vessel and wash with i-PrOH (3 x 10 mL), then DMF (3 x 10 mL). Then introduce into the vessel a DMF solution containing Fmoc-NH-(PEG) 12 -COOH (42.8 mg), i-Pr2NEt (2.5 equivalents), and PyBOP (2.5 equivalents). Bubble the resulting solution with Ar for 1 hour, drain the coupling solution, and wash the resin with DMF (3 x 10 mL) and i-PrOH (3 x 10 mL). Perform a Kaiser test to evaluate the reaction progress. Use DMF containing 20% piperidine (3 x 10 mL) for Fmoc deprotection. Repeat this procedure to complete all coupling steps (using 2 x 1.5 equivalents of Fmoc-Phe-OH, 1.5 equivalents of 8-aminooctanoic acid, and 1.2 equivalents of DUPA in their respective coupling steps). After DUPA coupling, wash the resin with DMF (3 x 10 mL) and i-PrOH (3 x 10 mL) and dry under reduced pressure. Use a cleavage solution to cleave the peptide from the resin in the peptide synthesis vessel. Add 15 mL of the cleavage solution to the peptide synthesis vessel, and bubble the reaction with Ar for 15 minutes. Treat the resin with two additional 10 mL portions of the cleavage solution for 5 minutes each. Concentrate the cleavage mixture to approximately 5 mL and precipitate with ether. Collect the precipitate by centrifugation, wash with ether (3X), and dry under high vacuum to give a crude recovery. At room temperature, add i-Pr2NEt (5 equivalents) to a stirred dimethyl sulfoxide (DMSO, 1 mL) solution containing crude DUPA-(PEG) 12 -EDA (10 mg) and FITC (5.6 mg) and stir under argon for 6 hours. Monitor the reaction by LCMS and purify by preparative HPLC (mobile phase: A = 10 mM ammonium acetate pH = 7, B = ACN; method: 0% B to 50% B in 30 minutes at 13 mL / min). Pool the purified fractions and lyophilize to obtain FITC-PEG12-DUPA.

[0545] Example 10 Synthesis of FITC-PEG11-NK1 At room temperature, under argon, to a stirred solution of NK-1 (0.02 g, 0.0433 mmol, 1.0 eq.) and O-(2-aminoethyl)-O'-[2-(Boc-amino)ethyl] decaethylene glycol (BocNH-PEG 11 -NH2) (Sigma, 0.0336 g, 0.0521 mmol, 1.2 eq.) and benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) (0.027 g, 0.0521 mmol, 1.2 eq.) in dry CH2Cl2 was added N,N-diisopropylethylamine (DIPEA) (0.076 mL, 0.4338 mmol, 10 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 minutes, 13 mL / min, λ = 220 nm, 254 nm). The pure fractions were collected, all organic solvents were evaporated and the sample was lyophilized for 48 hours to obtain NK1-PEG 11 -NHBoc. Yield: 40.13 mg (97%). To a solution of NK1-PEG 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. The excess TFA was removed, and the remaining solution was diluted with water and extracted with CH2Cl2 (3x 5 mL). The combined organic layers were washed with brine, dried (Na2SO4) and concentrated. The obtained residue was dried under vacuum and used for the next step without further purification. At room temperature, under argon, a stirred solution of NK1-PEG 11 -NH2 (0.008 g, 0.0081 mmol, 1.0 eq.) and fluorescein isothiocyanate (FITC) (Sigma, 0.0037 g, 0.0097 mmol, 1.2 eq.) in dry dimethyl sulfoxide (DMSO, 0.3 mL) 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 TMPurification was performed using a Prep C18, 5 μm; 19×100 mm column, with mobile phase A = 20 mM ammonium acetate buffer, pH 7, B = acetonitrile, gradient 10 to 100% B in 30 minutes, 13 mL / min, λ = 280 nm. The pure fractions were collected, all organic solvents were evaporated, and the sample was lyophilized for 48 hours to obtain 8.54 mg (77%) of FITC-PEG11-NK1 in yield.

[0546] *Note: The NK-1 compound was synthesized starting from a basic ligand via a two-step procedure, and the basic ligand was prepared using the procedures in 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).

[0547] Example 11 Synthesis of FITC-PEG2-CA9 In a 50 mL round-bottom flask, the CA9 ligand (53.6 mg) was dissolved in DMF (2 to 3 mL) using a Teflon magnetic stir bar. The ambient air was removed using a vacuum and replaced with nitrogen, which was done in three cycles. The round-bottom flask was maintained under a constant nitrogen atmosphere. 28.9 mg of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) was added to the flask, followed by 21.6 mg of 1-hydroxybenzotriazole hydrate (HOBt) and 18.9 μL of Boc-PEG2-NH2 (Sigma Aldrich). 5.4 μL of triethylamine (TEA) was added and the reaction was stirred overnight. The reaction mixture was purified by HPLC and confirmed by UHPLC-MS (target m / z 831). Acetonitrile was removed using a high-vacuum rotary evaporator and the product was lyophilized. The compound was mixed with 1:1 TFA:DCM for 30 minutes. TFA / DCM was removed using a high-vacuum rotary evaporator, followed by 30 minutes under high vacuum. Then the compound was dissolved in DMF and combined with 5 molar equivalents of i-Pr2Net, 16 mg of fluorescein isothiocyanate (Life Technologies), 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.

[0548] Example 12 FITC-DUPA DUPA-FITC was synthesized via solid-phase method as follows. The 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 DMF (3×3 mL) containing 20% piperidine 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 equivalents), HATU (2.5 equivalents), and DIPEA (4.0 equivalents) was added. Argon was bubbled for 2 hours, 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 DCM / trifluoroethane (TFE) (1:1) (2×3 mL) containing 1 M HOBt was added. Argon was bubbled for 1 hour, the solvent was removed, and the resin was washed with DMF (3×3 mL) and i-PrOH (3×3 mL). After the resin was swollen in DMF, a DMF solution containing Fmoc-Phe-OH (2.5 equivalents), HATU (2.5 equivalents), and DIPEA (4.0 equivalents) was added. Argon was bubbled for 2 hours, 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-aminocaprylic 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)]. ACN was removed under vacuum, and the pure fractions were lyophilized to obtain DUPA-FITC as a yellowish-brown 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). 11H 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: λmax = 491 nm.

[0549] Example 13 FITC-CA9 In a 50 mL round-bottom flask, the 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 stir bar. The ambient air was removed using a vacuum and replaced with nitrogen, which was completed in three cycles. Then the round-bottom flask was maintained under a constant nitrogen atmosphere. 28.9 mg of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) was added to the flask, followed by 21.6 mg of 1-hydroxybenzotriazole hydrate (HOBt) and 18.9 μL of Boc-PEG2-NH2 (purchased from Sigma Aldrich). Finally, 5.4 μL of triethylamine (TEA) was added and the reaction was allowed to stir overnight. The reaction mixture was purified by HPLC and confirmed by UHPLC-MS (target m / z 831). Acetonitrile was removed using a high-vacuum rotary evaporator and placed on a freeze dryer for 48 hours. Deprotection of Boc was carried out using 1:1 TFA:DCM for 30 minutes. TFA / DCM was removed using a high-vacuum rotary evaporator, followed by 30 minutes under high vacuum. Then the compound was 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 by 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.

[0550] Example 14 FITC-NK1R At room temperature, under argon, to a stirred solution of NK-1 (0.02 g, 0.0433 mmol, 1.0 eq.) in dry CH2Cl2, 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-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) (0.027 g, 0.0521 mmol, 1.2 eq.) was added N,N-diisopropylethylamine (DIPEA) (0.076 mL, 0.4338 mmol, 10 eq.). The reaction progress was monitored via LCMS and purified via 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 minutes, 13 mL / min, λ = 220 nm, 254 nm). The pure fractions were collected, all organic solvents were evaporated and the sample was lyophilized for 48 hours to obtain NK1-PEG 11 -NHBoc. Yield: 40.13 mg (97%). To dry CH2Cl2 containing NK1-PEG 11 -NHBoc (0.0165 g, 0.015 mmol) 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 (3x 5 mL). The combined organic layers were washed with brine, dried (Na2SO4) and concentrated. The residue obtained was dried under vacuum and used for the next step without further purification. At room temperature, under argon, the stirred solution of NK1-PEG 11 -NH2 (0.008 g, 0.0081 mmol, 1.0 eq.), fluorescein isothiocyanate (FITC) (Sigma, 0.0037 g, 0.0097 mmol, 1.2 eq.) in dry dimethyl sulfoxide (DMSO, 0.3 mL) was added to diisopropylethylamine (0.0028 mL, 0.0162 mmol, 2.0 eq.). The reaction progress was monitored via LCMS and purified via preparative RP-HPLC (Waters, XBridge TMPurified with a Prep C18, 5μm; 19×100mm column, mobile phase A = 20 mM ammonium acetate buffer, pH 7, B = acetonitrile, gradient 10 to 100% B in 30 minutes, 13 mL / min, λ = 280 nm). The pure fractions were collected, all organic solvents were evaporated and the sample was lyophilized for 48 hours to obtain NK1-PEG11-FITC(5). Yield: 8.54 mg (77%).

[0551] The NK-1 compound was synthesized from a basic ligand via a two-step procedure, and the basic ligand was prepared using literature procedures. (Reference: DESIGN AND DEVELOPMENT OF NEUROKININ-1 RECEPTOR-BINDING AGENT DELIVERY CONJUGATES, Application No.: PCT / US2015 / 44229; the full text of which is incorporated herein by reference).

[0552] Example 15 In vitro co-culture E2 CAR T activation and target cell killing assay At a cell density of 0.5 to 2 million T cells / mL of T cell growth medium (TexMACS medium + 2% human AB serum + 50 U / mL recombinant human IL2), the E2 CAR T cells were thawed and recovered from cryopreservation for 4 days. The target cells expressing different levels of surface folate receptor were also thawed and recovered from cryopreservation in growth medium (folate-deficient RPMI1640 + 10% FCS + penicillin / streptomycin (pen / strep)) for 4 days.

[0553] On day 1, 100,000 target cells were plated in 2 ml of growth medium / well in a 12-well tissue culture plate to allow the target cells to adhere and recover. On day 0, the wells receiving the EC17 bridging molecule were treated with 100 nM of EC17 in a tissue culture incubator for 30 minutes. After that, all the medium was washed off and replaced with fresh growth medium without folate and EC17. During the EC17 incubation, the T cells were harvested, counted and after the EC17 treatment was completed, 100,000 E2 CAR T cells were directly added to each well of the target cells contained in a total of 3 ml of growth medium and incubated for 1 and 2 days under standard tissue culture humidity conditions of 37 °C and 5% CO2. The one-day co-culture condition was used to assay the activation of E2 CAR T cells by the target cells and the two-day incubation was used to assay target cell death.

[0554] To determine the level of E2 CAR T cell activity after co - culturing with target cells having different levels of surface folate receptor for one day, floating cells and the remaining adherent cells were harvested and pooled, where the adherent cells were removed from the tissue culture plate using 5 - minute 0.25% trypsin digestion. The pooled floating and adherent cells were pelleted by a 400x g centrifugation step for 5 minutes and then resuspended in a 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 method).

[0555] To determine the efficacy of E2 CAR T cell killing of target cells with different levels of surface folate receptor expression after two - day co - culturing, the pooled and adherent cells were pelleted in the same manner as in the one - day co - culturing assay above, then stained for the E2 CAR T markers EGFR and CD3, then washed, and stained for the apoptosis marker Annexin V by resuspending the flow - antibody - stained sample 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.

[0556] Activation of E2 CAR T cells depends on EC17 staining of target cells To endeavor to understand the level of folate receptor necessary for activating E2 CAR T cells, co - culturing for one day with target cells expressing varying levels of surface folate receptor (FR) was performed ( Figure 39)。The percentage of E2 CAR T cells activated after a one-day co-culture was plotted on the y-axis, while the FR expression level of the target cells at the time of measurement was plotted on the x-axis. Importantly, on the same day that the measurement was performed using the same target cells, data on the FR expression level was obtained by treating the target cells with EC17, but culturing them in the absence of CAR T cells to avoid any artifacts of FR-high-expressing cells being eliminated via 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 (down triangles) expressing low FR expressed approximately 50% of the FR of SKOV3 cells (diamonds) and activated approximately 50% of the CAR T cells as 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 binding to the target cells that is required for complete 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.

[0557] Target cell killing by E2 CAR T cells 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 via flow cytometry and determined the percentage of target cells stained with an apoptosis surface marker (Annexin V staining). In Figure 40 , the same five target cells as shown (labeled 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 the CAR T plus target cell co-culture without any prior EC17 treatment of the target cells, while the third bar in each group represents CAR T plus target cells pre-treated with EC17. The y-axis shows the percentage of target cells that are apoptotic (Annexin V+). From these data, it was found that for the three highest folate receptor expressors (MDAMB231, HOS-FRα, and IGROV1), CAR T cell activation translated well into target cell killing. However, for SKOV3 cells ( Figure 39 , x-axis), which have the fourth lowest FR expression, similar E2 CAR T activation did not translate into similar target cell killing ( Figure 40 ). This disconnect between CAR T cell activation and target cell apoptosis in SKOV 3 cells may not be the result of low folate receptor expression. As previously mentioned, each cancer cell can evolve its own anti-apoptotic mechanism and the FR expression level may be sufficient for a higher level of target cell apoptosis in cells such as SKOV3 cells (but in different cancer cell lines).

[0558] In summary, data from Figure 39 and Figure 40 demonstrate that EC17 conjugated to cancer cells is required for cell killing. These data also show that below a certain threshold of EC17 bound to target cells, there is a linear relationship between surface-bound EC17 and E2 CAR T activation and killing.

[0559] Example 16 3 H-Folic Acid Binding Assay All tumor cell lines were seeded overnight in folate-free RPMI medium containing 10% heat-inactivated fetal bovine serum. On the next day, the cells were incubated on ice with 100 nM 3 H-folic acid with and without 10 μM cold folic acid for 15 minutes. After three washes with cold 1X PBS, whole cell lysis was performed and total cell-associated radioactivity was counted in a scintillation counter. Specific binding of 3 H-folic acid was determined by subtracting the counts of the folic acid competition samples and the specific activity of 3 H-folic acid was calculated as the number of molecules / cell.

[0560] Figure 41 Demonstrate various levels of FR expression in these cell lines. The order of FR levels is: IGROV1 > MDA-MB-231, HOS-FRα > OV90, SKOV3.

[0561] Example 17 EC17 / E2-CAR T-Cell Therapy Tumor Implantation in NSG Mice with and without Subcutaneous Implantation of MDA-MB-231 Tumors At 37 °C, in a 5% CO2 humidified atmosphere, MDA-MB-231 tumor cells were grown in folate-deficient RPMI 1640 containing 5 to 10% FBS. MDA-MB-231 tumor cells were subcutaneously inoculated at 2.5 x 10 6 cells / animal. Only 42 / 66 mice were inoculated and the remaining 22 mice were kept tumor-free.

[0562] CAR-T Cell Administration The EGFRt-sorted anti-FITC E2 scFv-CAR T cells were frozen in T-cell freezing medium. Vials of the frozen CAR-T cells were immediately stored at -80 °C. The CAR-T cells were rapidly thawed at 37 °C, washed twice with PBS and used for injection into animals at 10 million viable EGFRt+ E2 CAR-T cells (CD4 / CD8 ~1:1) / animal. Small aliquots were taken on the day of infusion for flow cytometry analysis of the E2-CAR T-cell phenotype.

[0563] Human / mouse cytokine analysis Mouse blood samples were processed into plasma and stored at -20 °C until use. Human cytokines in mouse blood were detected using the Human TH1 Cytokine Panel (Biolegend, catalog number 740009) and the Human Hematopoietic Stem Cell Cytokine Panel (Biolegend, catalog number 740610). Mouse cytokines were detected using the Mouse 13-plex Inflammatory Cytokine Panel (Biolengend, number 740150). All assays were performed according to the manufacturer's instructions.

[0564] Flow cytometry analysis Complete blood cell analysis: Plasma was taken from a predetermined volume of EDTA-treated whole blood, and RBCs were lysed with RBC lysis buffer. The leukocyte pellet was then resuspended in a 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 a 1:10 (v / v)], anti-human PD1 [BD Biosciences, clone EH12.1, catalog number 562511 at a 1:20 (v / v)], anti-human LAG3 [BD Biosciences, clone T47-530, catalog number 565616 at a 1:20 (v / v)], anti-human TIM3 [BD Biosciences, clone 7D3, catalog number 565558 at a 1:20 (v / v)], anti-human CD3ε [BD Biosciences, clone SK7, catalog number 557832 at a 1:20 (v / v)]. After leukocyte staining, the cells were washed with PBS and resuspended in cold PBS containing 53,000 CountBrightTM beads (Invitrogen catalog number C36950) and transferred to a flow cytometry collection tube. Flow cytometry data was collected on a Gallios flow cytometer (Beckman Coulter, Brea, CA). The determination of the concentration of CAR T cells in each blood sample was calculated according to the instructions of Invitrogen. CAR T cells were identified as human CD3ε+EGFRt+ events and were easily distinguishable and counted using KaluzaTM flow cytometry software. The number of CAR T cells in the circulation of each infused mouse was then plotted 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.

[0565] Tumor and tissue analysis: Harvest solid tumors (100 to 1000 mm3 ) were 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 consisted of serum-free and folate-deficient RPMI 1640 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. After that, the tumor digest was centrifuged at 400 x g for 5 minutes, and then the tumor cell pellet that had undergone the red blood cell lysis step was washed with cold phosphate-buffered saline (PBS, pH 7.4) and finally filtered through a 40 μm nylon cell strainer.

[0566] E2-CAR-T phenotype and in vivo proliferation It has been reported that the expansion and persistence of CAR T cells in the blood are related to the response to CD19-specific CAR T therapy 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 the EC17-directed attack 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 per 100 uL of whole blood and observed that the groups receiving EC17 stimulation (groups 2, 3, 4, and 5) had at least a greater than 10-fold increase in circulation when compared to the groups not receiving EC17 (groups 1 and 6). Importantly, this expansion of circulating CAR T cells in the blood conforms to the antigen-dependent manner of CAR T cell expansion. In addition, we also observed that after CAR T cell infusion, the elevated number of circulating CAR T cells persisted for up to 54 days and that CAR T persistence again depended on EC17 (groups 2, 3, 4, and 5) when compared to groups 1 and 6 that did not receive EC17.

[0567] Tumors versus tumor-free mice As Figure 42 shown, 10 million E2 CAR-T cells were injected intravenously into naïve NSG mice without tumors or NSG mice bearing MDA-MB-231 tumors. On the 2nd and 10th days after CAR-T injection, 500 nmol / kg of EC17 was administered intravenously, and the mice were euthanized 20 hours after the second EC17 dose (day 11) for organ evaluation and blood analysis. Plasma samples were immediately isolated from the blood and stored at -20 °C until cytokine analysis. On the 12th day after CAR-T injection, the isolated groups of animals were harvested for FACS analysis as described above.

[0568] As Figure 43 shown, the cytokine reduction in tumor-bearing mice is EC17-dependent. The levels of cytokines including IL2, IFN-γ, and IL-10 all increase in tumor-bearing mice containing EC17 relative to those without EC17 administration. In naïve mice without tumors, upregulation of cytokine production was also observed in those mice administered with EC17, but the increase in naïve mice was much lower compared to those mice with MDA-MB-231 tumors (e.g., 23-fold lower for IFN-γ).

[0569] Tumor-bearing mice have approximately 23-fold higher IFN-γ production compared to naïve mice. Although some cytokine production was shown in naïve mice (FD diet for approximately 2 months) after EC17 SIW500 x 2 doses, there was no CAR-T cell expansion detectable by FACS.

[0570] Example 18 Target-specific active cell lines demonstrated in vitro using a humanized anti-FITC scFv E2-CAR construct and CAR-modified T cells: 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 a gift from Dr. Stanley Riddell (FHCRC). The K562OKT3 cell line was generated by lentiviral transduction using a lentivirus containing the OKT3 transgene. OKT3 is a membrane tethered scFv targeting CD3ε. MDA-MB-231 was provided by Endocyte and cultured in folate-free DMEM. All cell lines were identified by STR profiling matching the DSMZ database of the University of Arizona Genetics Core.

[0571] Cytotoxicity and cytokine secretion: A four-hour chromium release assay was performed. Briefly, target cells were labeled overnight with 51 Cr (PerkinElmer), washed three times in PBS, and cultured in triplicate with T cells at various effector to target (E:T) ratios at 5x10 3 cells / well in a 96-well plate in RPMI. Supernatant S was harvested for γ-counting and calculation of specific lysis. For cytokine secretion, 5x10 5T cells and target cells were plated in triplicate at an E:T ratio of 2:1 in 96-well plates for 24 hours, and supernatants were analyzed via a bead-based cytokine array using the Bio-Plex Human Cytokine Group (Bio-Rad) according to the manufacturer's instructions. For EC17-labeled targets, cells were incubated with 100 nM EC17 in PBS for 1 hour at room temperature in the dark before plating. When using EC17, cells were incubated with folate-free RPMI during the assay.

[0572] Cytokine release assay: CD4+ T cells were co-cultured at a T cell to target ratio of 2:1 for 24 hours, and then supernatants were analyzed for the presence of effector cytokines IL-2, IFN-γ, and TNF-α. Assays were run in triplicate and data are shown as mean ± SE. As Figure 44 shown, we observed that mock and anti-FLCAR T cells responded to co-culture with the positive control cell line K562-OKT3, quantitatively producing similar levels of cytokine production. After co-culture with K562, neither mock nor anti-FLCAR T cells produced cytokines. Cytokine release was dependent on prior incubation of EC17 with the MDA-MB-231 cell line, and anti-FLCAR T cells were the only cells capable of eliciting secretion of the cytokines IL-2, IFN-γ, and TNF-α against EC17-labeled MDA-MB-231.

[0573] Chromium release assay: CD8+ T cells were co-cultured with target cells at ratios of 30:1, 10:1, 3:1, or 1:1. Percent lysis of triplicate wells is described (mean ± SE). As Figure 45 shown, anti-FLCAR T cells did not demonstrate cytotoxicity against the negative control (K562) or unlabeled MDA-MB-231 cell lines. However, both mock-transduced cells and anti-FLCAR T cells were able to induce similar levels of specific lysis against the positive control K562-OKT3 cell line. Additionally, EC17-labeled MDA-MB-231 cells were efficiently recognized and lysed by anti-FLCAR T cells, whereas mock T cells were unable to produce lysis.

[0574] Example 19 Structure of the CAR-T cell aptamer Figures 46 to 49 Shows the structure of the bridge used in the CAR-T cell experiment and its affinity in vitro for cell types against the bridge (e.g., folate-FITC for cancer cells expressing the folate receptor). Figure 50Show the binding of the bridge to tumor cells (by FACS analysis), where the tumor cells are used in an in vivo model and express a receptor for a small molecule ligand corresponding to the bridge.

[0575] For Examples 20 to 30 - see Figures 55 to 63 .

[0576] Example 20 Cell Lines and Reagents Unless otherwise specified, all FR+ and FR− negative cancer cell lines were maintained in RPMI-1640 medium (Gibco BRL) supplemented with 10% heat-inactivated fetal bovine serum and without (FFRPMI) or with (RPMI) 2.4 μM folic acid (FA). KB (human cervical carcinoma expressing FRα with HeLa markers) and CHO-β (Chinese hamster ovary cells transfected with human FRβ) were used as sources of FRα and FRβ, respectively, for radioligand binding assays. MDA-MB-231 represents a subclone of FRα of the human triple-negative breast cancer (TNBC) cell line. For AML studies, an isogenic pair of FRβ-positive (THP1-FRβ) and FR-negative (THP1-FG12) cell lines expressing green fluorescent protein (GFP) was 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 the primary tumor of a 13-year-old Caucasian female and was highly tumorigenic in NSG mice. Lentiviral firefly luciferase was used to transduce FR+ HOS-FRα expressing GFP fLuc and FR-negative HOS-143b fLuc for bioluminescence pairs.

[0577] LEGENDplex TM Human cytokine panel was purchased from BioLegend (San Diego, CA). CytoTox based on lactate dehydrogenase (LDH) The non-radioactive cytotoxicity assay kit was purchased from Promega (Madison, WI). Commercially available anti-human antibodies for multi-color 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 Biosciences (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 antibiotin was also purchased from BioLegend. APC-conjugated anti-FITC mouse IgG2a / κ antibody (clone NAWESLEE), CountBright TM Beads (Invitrogen), annexin V staining buffer, and Alexa Fluor-647-conjugated annexin V were purchased from Thermo Fisher Scientific. For enzymatic digestion of tumor tissues, collagenase IV, hyaluronidase, and DNase I were all purchased from Sigma-Aldrich (St. Louis, MO).

[0578] EC17 or folic acid-FITC [FA-(γ)-ethylenediamine-FITC] was synthesized at Endocyte. 3 H-EC17 was purchased from Moravek biochemicals (Brea, CA) with a specific activity of approximately 0.952 Ci / mmol or prepared at Endocyte by conjugation of FITC with 3 H-FA-(γ)-ethylenediamine (manufactured by ViTrax (Placentia, CA)) with a specific activity of approximately 1.2 Ci / mmol. 3 H-FA was also purchased from ViTrax with a specific activity of 59 Ci / mmol. For CRS rescue, the fluorescein sodium administration solution was diluted from 25% (fluorescein injection, USP), which was purchased from Purdue Pharmacy (NDC17478-250-25).

[0579] Example 21 Humanized CAR constructs and CAR-modified T cells Previous studies used GFP+ second-generation anti-FITC scFv (clone 4M5.3) CARs containing a CD8α hinge and transmembrane sequence and a 4-1BB / CD3ζ signaling domain (i.e., FITC-4M5.3-scFv-CD8α hinge-CD8αtm-4-1BB / CD3ζ). For the transition to the first-in-human test, a second-generation fully human FITC-specific (clone E2) CAR construct (referred to herein as E2) was developed ( Figure 55 , upper panel). CAR-modified T cells are shown in Figure 55 , the lower pie chart.

[0580] The constructs described herein are FITC-specific CAR constructs 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-intracellular domain, and (4) a cell surface human EGFRt tag ( Figure 55 , upper panel) (SEQ ID NOS: 1 and 2 are the nucleic acid and amino acid sequences, respectively). To generate CAR-modified T cells, lentiviruses were produced in 293T cells co-transfected with an epHIV7 lentiviral vector encoding the CAR. Donor CD4+ and CD8+ T cells were purified and separated via immunomagnetic selection or transduced at an approximate 50:50 ratio. 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 ratio via flow cytometry. Using a combination of surface markers, the differentiation status of CD4+ and CD8+ CAR-T cell subsets was analyzed on the day of infusion and defined as T N , CD45RA+CD45RO-CD62L+CD95- naive T cells; T SCM , CD45RA+CD45RO-CD62L+CD95+ stem cell memory T cells; ...

Claims

1. A method for treating cancer, the method comprising: i) Administering to a patient a compound or a pharmaceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked via a linker to a targeting moiety; ii) Administering to the patient a first dose of a CAR T cell composition comprising CAR T cells, wherein the CAR T cells comprise a CAR directed to the 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 CAR T cells, wherein the CAR T cells comprise a CAR directed to the targeting moiety and wherein the CAR comprises an E2 anti-fluorescein antibody fragment.

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