Methods of using car t cells

Abstract

To provide methods of treating a patient having a cancer.SOLUTION: The present disclosure relates to methods of treating a patient having a cancer by administering to the patient a composition comprising CAR T cells, where the CAR T cells comprise a CAR and the CAR comprises an E2 anti-fluorescein antibody fragment, and administering to the patient a small molecule linked to a targeting moiety by a linker.SELECTED DRAWING: Figure 3

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 62 / 620,414, filed January 22, 2018, U.S. Provisional Application No. 62 / 620,706, filed January 23, 2018, U.S. Provisional Application No. 62 / 656,233, filed April 11, 2018, U.S. Provisional Application No. 62 / 724,171, filed August 29, 2018, U.S. Provisional Application No. 62 / 735,627, filed September 24, 2018, and U.S. Provisional Application No. 62 / 736,727, filed September 26, 2018, all of which are incorporated herein by reference in their entireties.

[0002] Technical Field The present invention relates to methods of treating a patient with cancer by administering to the patient a composition comprising a CAR T cell, wherein the CAR T cell comprises a CAR, the CAR comprising an E2 anti-fluorescein antibody fragment, and administering to the patient a small molecule attached to a targeting moiety by a linker. The present invention also relates to compositions for use in such methods. [Background technology]

[0003] background Immunotherapy based on adoptive transfer of lymphocytes (e.g., T cells) to patients is a valuable treatment for cancer and other diseases. Many important advances in the development of immunotherapy have been based on adoptive transfer of lymphocytes. Among many types of immunotherapy, one of the most promising immunotherapies under development is chimeric antigen receptor-expressing T cells (CAR T cells). Chimeric antigen receptors (CARs) are genetically engineered receptors that are designed to target specific antigens, such as tumor antigens. This targeting can result in cytotoxicity against tumors, for example, so that CAR-expressing CAR T cells can target tumors via the specific tumor antigens and kill tumor cells.

[0004] First generation CARs consist of an antibody-derived recognition region, such as a single-chain fragment variable (scFv) region, and an activation signaling domain to recognize and bind to antigens expressed by tumors; for example, the CD3ζ chain of T cells can act as a T cell activation signal in CARs. CAR T cells have shown good results in vitro, but have only limited success in eliminating disease (e.g., cancer) in clinical trials. One problem is the inability to activate and expand CAR T cell populations for a long time in vivo.

[0005] To address this issue, second generation CARs have incorporated costimulatory domains (e.g., CD137, CD28, or CD134) to achieve long-term activation of T cells in vivo. The addition of costimulatory domains enhances the in vivo proliferation and survival of CAR-containing T cells, and early clinical data indicates that such constructs are promising therapeutic agents for the treatment of diseases such as cancer. Summary of the Invention [Problem to be solved by the invention]

[0006] Although CAR T cell therapy has improved, several problems remain. First, 'off-target' toxicity can occur against normal cells expressing the antigen targeted by the CAR T cells (e.g., tumor-associated antigens). Second, unregulated CAR T cell activation can be seen, where rapid and uncontrolled elimination of diseased cells (e.g., cancer cells) by CAR T cells induces a series of metabolic dysfunctions called tumor lysis syndrome or cytokine release syndrome (CRS), which can be fatal to the patient. Tumor lysis syndrome and CRS can result from the administration of CAR T cells that are not easily controlled and are activated in an unregulated manner. Thus, although CAR T cells have shown great promise as a tool in the treatment of diseases such as cancer, further CAR T therapies with reduced off-target toxicity and more tightly controlled CAR T cell activation are needed. [Means for solving the problem]

[0007] Summary of the Invention The inventors have discovered a method to reduce off-target toxicity and more tightly control CAR T cell activation, which provides an important advancement in CAR T cell therapy. In various embodiments described herein, a small molecule ligand linked to a targeting moiety by a linker is used as a bridge between a cancer and a CAR-expressing CAR T cell, where the CAR comprises an E2 anti-fluorescein antibody fragment. The bridge directs the CAR T cell expressing a CAR comprising an E2 anti-fluorescein antibody fragment to the cancer for cancer cure. In some embodiments, the "small molecule ligand" can be, for example, folate, DUPA, NK-1R ligand, CAIX ligand, gamma glutamyl transpeptidase ligand, NKG2D ligand, or CCK2R ligand, each of which is a small molecule ligand that specifically binds to cancer cells (i.e., the receptors of these ligands are more highly expressed in cancer than in normal tissues).

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

[0009] The "targeting moiety" binds to the recognition region of the genetically engineered CAR expressing the E2 anti-fluorescein 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 E2 anti-fluorescein antibody fragment) is directed to the "targeting moiety". Thus, the small molecule ligand linked to the targeting moiety by the linker serves as a bridge between the cancer and the CAR T cells expressing the E2 anti-fluorescein antibody fragment, directing the CAR T cells to the cancer for cancer cure.

[0010] In one embodiment, a method for treating cancer is provided.The method includes: i) administering a compound or a pharma- ceutically acceptable salt thereof to a patient, wherein the compound comprises a small molecule ligand linked to a targeting moiety by a linker; ii) administering a first dose of a CAR T cell composition comprising a CAR T cell to the patient, wherein the CAR T cell comprises a CAR directed to a targeting moiety, and the CAR comprises an E2 anti-fluorescein antibody fragment; and iii) administering a second dose of a CAR T cell composition comprising the CAR T cell to the patient, wherein the CAR T cell comprises a CAR directed to a targeting moiety, and the CAR comprises an E2 anti-fluorescein antibody fragment.

[0011] In other embodiments, a method of treating cancer is provided, comprising: i) administering to a patient a compound or a pharma- ceutically 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 a CAR T cell, the CAR T cell comprises a CAR directed to a targeting moiety, the CAR comprises an E2 anti-fluorescein antibody fragment, and the CAR T cell composition comprises a mixture of CAR T cells and non-transformed T cells.

[0012] In yet another embodiment, a method for treating cancer is provided, comprising: i) administering to a patient a compound or a pharma- ceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked to a targeting moiety by a linker; ii) administering to a patient a CAR T cell composition, wherein the CAR T cell composition comprises a CAR T cell, the CAR T cell comprises a CAR directed to a targeting moiety, the CAR comprises an E2 anti-fluorescein antibody fragment; and iii) administering to a patient folate, a conjugate comprising folate without a 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, comprising: i) administering to a patient a compound or a pharma- ceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked to a targeting moiety by a linker, and the patient is administered at least a first dose and a second dose of the compound or a pharma- ceutically acceptable salt thereof, wherein the first dose and the second dose are different, and the amount of the second dose of the compound or a pharma- ceutically 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 pharma- ceutically acceptable salt thereof; and ii) administering to the patient a CAR T cell composition comprising a CAR T cell, wherein the CAR T cell composition comprises a CAR T cell, the CAR T cell comprises a CAR directed to a targeting moiety, and the CAR comprises an E2 anti-fluorescein antibody fragment.

[0014] In another illustrative embodiment, a method for treating cancer is provided, which includes: i) administering a first dose of a compound or a pharmaceutically acceptable salt to a patient, wherein the compound comprises a small molecule ligand linked to a targeting moiety by a linker; ii) administering at least a second dose of a compound or a pharmaceutically acceptable salt to a patient, wherein the second dose of the compound or a pharmaceutically acceptable salt is at least about 50% lower than the amount of the first dose of the compound or a pharmaceutically acceptable salt; and iii) administering a dose of a CAR T cell composition comprising a CAR T cell to a patient, wherein the CAR T cell composition comprises a CAR T cell, the CAR T cell comprises a CAR directed to a targeting moiety, and the CAR comprises an E2 anti-fluorescein antibody fragment.

[0015] In another embodiment, a method of treating cancer is provided, comprising: i) administering to a patient a compound or a pharma- ceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked to a targeting moiety by a linker, and the compound or a pharma- ceutically acceptable salt thereof is administered at a dose of about 10 nM / kg to about 2500 nM / kg of patient body weight; and ii) administering to a patient a CAR T cell composition comprising a CAR T cell, wherein the CAR T cell comprises a CAR, the CAR comprises an E2 anti-fluorescein antibody fragment, and the CAR T cell is at a dose of about 1 million to about 15 million CAR T cells.

[0016] In yet another embodiment, a method of treating cancer is provided, comprising: i) continuously administering a compound to a patient, wherein the compound comprises a small molecule ligand linked to a targeting moiety by a linker; ii) administering a CAR T cell composition comprising a CAR T cell to the patient, wherein the CAR T cell comprises a CAR, and the CAR comprises an E2 anti-fluorescein antibody fragment; and iii) terminating the continuous administration of the compound or a pharma- ceutically acceptable salt thereof to block or prevent cytokine release syndrome in the patient.

[0017] In another embodiment, a method of treating cancer is provided, comprising: i) administering to a patient a compound or a pharma- ceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked to a targeting moiety by a linker, and the compound or a pharma- ceutically acceptable salt thereof is administered to the patient once a week; and ii) administering to the patient a CAR T cell composition comprising a CAR T cell, wherein the CAR T cell comprises a CAR, and the CAR comprises an E2 anti-fluorescein antibody fragment.

[0018] In yet another embodiment, a method for treating cancer is provided, which comprises: i) administering a first dose of a compound or a pharmaceutically acceptable salt to a patient, wherein the compound comprises a small molecule ligand linked to a targeting moiety by a linker, and wherein the compound or a pharmaceutically acceptable salt is administered to the patient before administering a CAR T cell composition comprising a CAR T cell comprising a CAR directed to a targeting moiety; ii) then administering a dose of a CAR T cell composition to the patient; and iii) then administering a second dose of the compound or a pharmaceutically acceptable salt to the patient, wherein the CAR comprises an E2 anti-fluorescein antibody fragment.

[0019] Further embodiments are also described by the following enumerated clauses. Any of the following embodiments are considered in combination with any of the applicable embodiments described in the Summary section, Detailed Description of Illustrative Embodiments section, Examples section, or claims of this application.

[0020] 1. A method of treating cancer, comprising: i) administering to a patient a compound or a pharma- ceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand attached to a targeting moiety by a linker; ii) administering to the patient a first dose of a CAR T cell composition comprising a CAR T cell, wherein the CAR T cell comprises a CAR directed to a targeting moiety, the CAR comprising an E2 anti-fluorescein antibody fragment; and iii) administering to the patient a second dose of a CAR T cell composition comprising the CAR T cell, wherein the CAR T cell comprises a CAR directed to a targeting moiety, and the CAR comprises an E2 anti-fluorescein antibody fragment. A method comprising:

[0021] 2. A method of treating cancer, comprising: i) administering to a patient a compound or a pharma- ceutical acceptable salt thereof, wherein the compound comprises a small molecule ligand attached 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 a CAR T cell, the CAR T cell comprises a CAR directed to a targeting moiety, the CAR comprises an E2 anti-fluorescein antibody fragment, and the CAR T cell composition comprises a mixture of CAR T cells and non-transformed T cells. A method comprising:

[0022] 3. A method of treating cancer, comprising: i) administering to a patient a compound or a pharma- ceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand attached to a targeting moiety by a linker; ii) administering to the patient a CAR T cell composition, wherein the CAR T cell composition comprises a CAR T cell, the CAR T cell comprising a CAR directed to a targeting moiety, the CAR comprising an E2 anti-fluorescein antibody fragment; and iii) administering to the patient folate, a conjugate containing folate without a targeting moiety, or an agent that inhibits activation of the CAR T cell. A method comprising:

[0023] 4. The method of paragraph 3, wherein step iii comprises administering folate.

[0024] 5. The method of paragraph 3 or 4, wherein step iii includes administration of folic acid or leucovorin.

[0025] 6. The method of paragraph 3, wherein step iii comprises administering a conjugate comprising a folate.

[0026] 7. The method of claim 6, wherein the folate-containing conjugate comprises folate bound to one or more amino acids.

[0027] 8. Folate-containing conjugates are of the formula [ka] 8. The method of claim 7, wherein

[0028] 9. Folate is the formula [ka] [In the formula, X 1 and Y 1 each independently represents halo, R 2 , OR 2 , S.R. 3 and N.R. 4 R 5 selected from the group consisting of; U, V and W are each -(R 6a )C=, -N=, -(R 6a )C(R 7a )- and -N(R 4a )-; Q is selected from the group consisting of C and CH; T is selected from the group consisting of S, O, N, and -C=C-; X 2 and X 3 are each independently oxygen, sulfur, -C(Z)-, -C(Z)O-, -OC(Z)-, -N(R 4b )-, -C(Z)N(R 4b )-, -N(R 4b )C(Z)-, -OC(Z)N(R 4b )-, -N(R 4b )C(Z)O-, -N(R 4b )C(Z)N(R 5b )-, -S(O)-, -S(O)2-, -N(R 4a )S(O)2-, -C(R 6b )(R 7b )-, -N(C≡CH)-, -N(CH2C≡CH)-, C1-C 12 Alkylene and C1-C 12 alkyleneoxy, where Z is oxygen or sulfur; R 1 are hydrogen, halo, C1-C 12 Alkyl and C1-C 12 alkoxy; R 2 , R 3 , R 4 , R 4a , R 4b , R5 , R 5b , R 6b and R 7b each independently represents hydrogen, halo, C1-C 12 Alkyl, C1-C 12 Alkoxy, C1-C 12 Alkanoyl, C1-C 12 Alkenyl, C1-C 12 Alkynyl, (C1-C 12 alkoxy)carbonyl and (C1-C 12 alkylamino)carbonyl; R 6 and R 7 each independently represents hydrogen, halo, C1-C 12 Alkyl and C1-C 12 alkoxy; or R 6 and R 7 together to form a carbonyl group; R 6a and R 7a each independently represents hydrogen, halo, C1-C 12 Alkyl and C1-C 12 alkoxy; or R 6a and R 7a together to form a carbonyl group; p, r, s and t are each independently 0 or 1; and * represents an optional covalent bond to the remainder of the conjugate. 9. The method according to any one of items 3 to 8,

[0029] 10. The method of item 3, wherein an agent that inhibits activation of CAR T cells is administered and is selected from the group consisting of lymphocyte-specific protein tyrosine kinase inhibitors, PI3 kinase inhibitors, IL-2 inducible T cell kinase inhibitors, JAK inhibitors, BTK inhibitors, EC2319, and agents that block CAR T cell binding of the compound or a pharma- ceutically acceptable salt thereof but do not bind to the cancer.

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

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

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

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

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

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

[0036] 17. The method of paragraph 10, wherein an agent that inhibits activation of CAR T cells is administered, the agent blocking CAR T cell binding of the compound or a pharma- ceutically acceptable salt thereof but not binding to the cancer.

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

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

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

[0040] 21. Any of the methods of paragraphs 17 to 20, wherein the agent that inhibits CAR T cell activation is administered at a dose of about .01 to about 300 μM / kg of patient body weight.

[0041] 22. Any of the methods of paragraphs 17 to 20, wherein the agent that inhibits CAR T cell activation is administered at a dose of about .06 to about 100 μM / kg of patient body weight.

[0042] 23. Any of the methods of paragraphs 17 to 20, wherein the agent that inhibits CAR T cell activation is administered at a dose of about .06 to about 90 μM / kg of patient body weight.

[0043] 24. Any of the methods of paragraphs 17 to 20, wherein the agent that inhibits CAR T cell activation is administered at a dose of about .06 to about 80 μM / kg of patient body weight.

[0044] 25. Any of the methods of paragraphs 17 to 20, wherein the agent that inhibits CAR T cell activation is administered at a dose of about .06 to about 70 μM / kg of patient body weight.

[0045] 26. Any of the methods of paragraphs 17 to 20, wherein the agent that inhibits CAR T cell activation is administered at a dose of about .06 to about 60 μM / kg of patient body weight.

[0046] 27. Any of the methods of paragraphs 17 to 20, wherein the agent that inhibits CAR T cell activation is administered at a dose of about .06 to about 50 μM / kg of patient body weight.

[0047] 28. Any of the methods of paragraphs 17 to 20, wherein the agent that inhibits CAR T cell activation is administered at a dose of about .06 to about 40 μM / kg of patient body weight.

[0048] 29. Any of the methods of paragraphs 17 to 20, wherein the agent that inhibits CAR T cell activation is administered at a dose of about .06 to about 30 μM / kg of patient body weight.

[0049] 30. Any of the methods of paragraphs 17 to 20, wherein the agent that inhibits CAR T cell activation is administered at a dose of about .06 to about 20 μM / kg of patient body weight.

[0050] 31. Any of the methods of paragraphs 17 to 20, wherein the agent that inhibits CAR T cell activation is administered at a dose of about .06 to about 10 μM / kg of patient body weight.

[0051] 32. Any of the methods of paragraphs 17 to 20, wherein the agent that inhibits CAR T cell activation is administered at a dose of about .06 to about 8 μM / kg of patient body weight.

[0052] 33. Any of the methods of paragraphs 17 to 20, wherein the agent that inhibits CAR T cell activation is administered at a dose of about .06 to about 6 μM / kg of patient body weight.

[0053] 34. Any of the methods of paragraphs 3 to 33, wherein the folate, a conjugate containing the folate without a targeting moiety, or an agent that inhibits activation of CAR T cells is administered to the patient more than once.

[0054] 35. The method of any of paragraphs 3 to 34, wherein folate, a conjugate comprising folate without a targeting moiety, or an agent that inhibits activation of CAR T cells is administered to the patient before and / or after the compound or a pharma- ceutically acceptable salt thereof.

[0055] 36. The method of any of paragraphs 3 to 35, wherein administration of folate, a conjugate containing folate without a targeting moiety, or an agent that inhibits activation of CAR T cells reduces cytokine levels in the patient.

[0056] 37. The method of paragraph 36, wherein the reduction in cytokine levels occurs by about 3 hours after administering to the patient the folate, the conjugate containing the folate without a targeting moiety, or the agent that inhibits activation of CAR T cells.

[0057] 38. The method of paragraph 36, wherein the reduction in cytokine levels occurs by about 6 hours after administering to the patient the folate, the conjugate containing the folate without a targeting moiety, or the agent that inhibits activation of CAR T cells.

[0058] 39. The method of any of paragraphs 36 to 38, wherein the reduction in cytokine levels is approximately to the cytokine levels of an untreated patient.

[0059] 40. The method of any of paragraphs 3 to 39, wherein the compound or a pharma- ceutically acceptable salt thereof is administered before and after administration of a folate, a conjugate comprising a folate that does not include a targeting moiety, or an agent that inhibits activation of CAR T cells.

[0060] 41. The method of any of paragraphs 3 to 40, wherein after administration of folate, a conjugate containing folate without a targeting moiety, or an agent that inhibits activation of CAR T cells, the number of CAR T cells in the patient's blood increases, even if the patient's cytokine levels decrease.

[0061] 42. The method of any of paragraphs 3 to 41, wherein after administration of folate, a conjugate comprising folate without a targeting moiety, or an agent that inhibits CAR T cell activation, CAR T cell activation is enhanced or maintained compared to a patient not treated with a rescue agent, even though cytokine levels in the treated patient are reduced.

[0062] 43. Any of the methods of paragraphs 3 to 42, wherein the cancer comprises a tumor and the size of the patient's tumor does not increase upon administration to the patient of folate, a conjugate comprising folate that does not include a targeting moiety, or an agent that inhibits activation of CAR T cells.

[0063] 44. The method of paragraph 43, wherein a complete tumor response is obtained.

[0064] 45. Any of the methods of paragraphs 3 to 44, wherein folate, a conjugate containing folate without a targeting moiety, or an agent that inhibits CAR T cell activation is administered to the patient when CRS grade reaches 1, 2, 3, or 4.

[0065] 46. ​​The method of paragraph 45, wherein the agent that inhibits CAR T cell activation is administered to the patient when CRS grade reaches 3 or 4.

[0066] 47. Any of the methods under items 3 to 46, in which pulmonary edema is reduced.

[0067] 48. The method of any of paragraphs 1 to 47, wherein the compound or a pharma- ceutically acceptable salt thereof is administered at a dose of about 10 nM / kg patient body weight to about 2500 nM / kg patient body weight, and the CAR T cells are administered at a dose of about 1 million CAR T cells to about 15 million CAR T cells.

[0068] 49. The method of paragraph 48, wherein the compound or a pharma- ceutically acceptable salt thereof is administered at a dose of about 10 nM / kg to about 100 nM / kg of patient body weight.

[0069] 50. The method of any of paragraphs 48 to 49, wherein the compound or a pharma- ceutically acceptable salt thereof is administered at a dose of about 10 nM / kg to about 50 nM / kg of patient body weight.

[0070] 51. The method of any of paragraphs 48 to 50, wherein the compound or a pharma- ceutically acceptable salt thereof is administered at a dose of about 10 nM / kg to about 20 nM / kg of patient body weight.

[0071] 52. The method of paragraph 48, wherein the compound or a pharma- ceutically acceptable salt thereof is administered at a dose of about 10 nM / kg to about 600 nM / kg of patient body weight.

[0072] 53. The method of paragraph 48, wherein the compound or a pharma- ceutically acceptable salt thereof is administered at a dose of about 200 nM / kg to about 600 nM / kg of patient body weight.

[0073] 54. The method of paragraph 48, wherein the compound or a pharma- ceutically acceptable salt thereof is administered at a dose of about 400 nM / kg to about 600 nM / kg of patient body weight.

[0074] 55. The method of any of paragraphs 48 to 54, wherein the CAR T cells are at a dose of about 1 million CAR T cells to about 12.5 million CAR T cells.

[0075] 56. The method of any of paragraphs 48 to 55, wherein the CAR T cells are at a dose of about 1 million CAR T cells to about 7 million CAR T cells.

[0076] 57. The method of any of paragraphs 48 to 56, wherein the CAR T cells are at a dose of about 1 million CAR T cells to about 5 million CAR T cells.

[0077] 58. The method of any of paragraphs 48 to 57, wherein the CAR T cells are at a dose of about 2 million CAR T cells to about 5 million CAR T cells.

[0078] 59. The method of any of paragraphs 1 to 58, further comprising the step of terminating the continuous administration of the compound or a pharma- ceutically acceptable salt thereof to inhibit or prevent cytokine release syndrome in the patient.

[0079] 60. The method of any of paragraphs 1 to 59, wherein the compound or a pharma- ceutically acceptable salt thereof is administered to the patient continuously for at least one hour.

[0080] 61. The method of any of paragraphs 1 to 59, wherein the compound or a pharma- ceutically acceptable salt thereof is administered to the patient for at least 4 consecutive hours.

[0081] 62. The method of any of paragraphs 1 to 59, wherein the compound or a pharma- ceutically acceptable salt thereof is administered to the patient continuously for at least 6 hours.

[0082] 63. The method of any of paragraphs 1 to 62, wherein the compound or a pharma- ceutically acceptable salt thereof is administered to the patient every other day.

[0083] 64. The method of any of paragraphs 1 to 62, wherein the compound or a pharma- ceutically acceptable salt thereof is administered to the patient three times per week.

[0084] 65. The method of any of paragraphs 1 to 62, wherein the compound or a pharma- ceutically acceptable salt thereof is administered to the patient twice weekly.

[0085] 66. The method of any of paragraphs 1 to 62, wherein the compound or a pharma- ceutically acceptable salt thereof is administered to the patient once a week.

[0086] 67. The method of any of paragraphs 1 to 62, wherein the compound or a pharma- ceutically acceptable salt thereof is administered to the patient until unacceptable patient weight loss, fever, hypotension, or pulmonary edema occurs.

[0087] 68. A method of treating cancer, comprising: i) administering to the patient a compound or a pharma- ceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand attached to a targeting moiety by a linker, and wherein the patient is administered at least a first dose and a second dose of the compound or a pharma- ceutically acceptable salt thereof, wherein the first dose and the second dose are different, and the amount of the second dose of the compound or a pharma- ceutically 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 pharma- ceutically acceptable salt thereof; and ii) administering to the patient a CAR T cell composition comprising a CAR T cell, wherein the CAR T cell composition comprises a CAR T cell, the CAR T cell comprising a CAR directed to a targeting moiety, the CAR comprising an E2 anti-fluorescein antibody fragment. A method comprising:

[0088] 69. The method of item 68, wherein at least a first dose, a second dose, and a third dose of the compound or a pharma- ceutically acceptable salt thereof are administered to the patient, wherein the first dose, the second dose, and the third dose are different, the second dose of the compound or a pharma- ceutically acceptable salt thereof being about 2 to about 750 times greater than the amount of the first dose of the compound or a pharma- ceutically acceptable salt thereof, and the third dose of the compound or a pharma- ceutically acceptable salt thereof being about 800 to about 10,000 times greater than the amount of the first dose of the compound or a pharma- ceutical acceptable salt thereof.

[0089] 70. The method of item 68, wherein at least a first dose, a second dose, a third dose and a fourth dose of the compound or a pharma- ceutically acceptable salt thereof are administered to the patient, wherein the first dose, the second dose, the third dose and the fourth dose are different, the second dose of the compound or a pharma- ceutically acceptable salt thereof being about 2 to about 750 times more than the amount of the first dose of the compound or a pharma- ceutically acceptable salt thereof, the third dose of the compound or a pharma- ceutically acceptable salt thereof being about 800 to about 7500 times more than the amount of the first dose of the compound or a pharma- ceutical acceptable salt thereof, and the fourth dose of the compound or a pharma- ceutical acceptable salt thereof being about 8000 to about 15000 times more than the amount of the first dose of the compound or a pharma- ceutical acceptable salt thereof.

[0090] 71. The method of paragraph 70, wherein the second dose of the compound or its pharma- ceutically acceptable salt is about 100 times greater than the amount of the first dose of the compound or its pharma- ceutically acceptable salt, the third dose of the compound or its pharma- ceutically acceptable salt is about 1000 times greater than the amount of the first dose of the compound or its pharma- ceutically acceptable salt, and the fourth dose of the compound or its pharma- ceutically acceptable salt is about 10000 times greater than the amount of the first dose of the compound or its pharma- ceutically acceptable salt.

[0091] 72. The method of paragraph 68, wherein at least a first dose and a second dose of the compound or a pharma- ceutically acceptable salt thereof are administered to the patient, wherein the first dose and the second dose of the compound or a pharma- ceutically acceptable salt thereof are different, and the second dose is about 2-fold to about 15,000-fold greater than the amount of the first dose of the compound or a pharma- ceutically acceptable salt thereof.

[0092] 73. A method of treating cancer, comprising: i) administering to the patient a first dose of a compound or a pharma- ceutically acceptable salt thereof, where the compound comprises a small molecule ligand attached to a targeting moiety by a linker; ii) administering to the patient at least a second dose of the compound or a pharma- ceutically acceptable salt thereof, wherein the second dose of the compound or a pharma- ceutically acceptable salt thereof is at least about 50% lower than the amount of the first dose of the compound or a pharma- ceutically acceptable salt thereof; and iii) administering to the patient a dose of a CAR T cell composition comprising a CAR T cell, wherein the CAR T cell composition comprises a CAR T cell, the CAR T cell comprising a CAR directed to a targeting moiety, and the CAR comprises an E2 anti-fluorescein antibody fragment. A method comprising:

[0093] 74. The method of paragraph 73, wherein the second dose of the compound or a pharma- ceutically acceptable salt thereof is at least about 60% lower than the amount of the first dose of the compound or a pharma- ceutically acceptable salt thereof.

[0094] 75. The method of paragraph 73, wherein the second dose of the compound or a pharma- ceutically acceptable salt thereof is at least about 70% lower than the amount of the first dose of the compound or a pharma- ceutically acceptable salt thereof.

[0095] 76. The method of paragraph 73, wherein the second dose of the compound or a pharma- ceutically acceptable salt thereof is at least about 80% lower than the amount of the first dose of the compound or a pharma- ceutically acceptable salt thereof.

[0096] 77. The method of paragraph 73, wherein the second dose of the compound or a pharma- ceutically acceptable salt thereof is at least about 90% lower than the amount of the first dose of the compound or a pharma- ceutically acceptable salt thereof.

[0097] 78. The method of paragraph 73, wherein the second dose of the compound or a pharma- ceutically acceptable salt thereof is at least about 95% lower than the amount of the first dose of the compound or a pharma- ceutically acceptable salt thereof.

[0098] 79. The method of paragraph 73, wherein the amount of the second dose of the compound or a pharma- ceutically acceptable salt thereof is at least about 96% lower than the amount of the first dose of the compound or a pharma- ceutically acceptable salt thereof.

[0099] 80. The method of paragraph 73, wherein the amount of the second dose of the compound or a pharma- ceutically acceptable salt thereof is at least about 97% lower than the amount of the first dose of the compound or a pharma- ceutically acceptable salt thereof.

[0100] 81. The method of paragraph 73, wherein the amount of the second dose of the compound or a pharma- ceutically acceptable salt thereof is at least about 98% lower than the amount of the first dose of the compound or a pharma- ceutically acceptable salt thereof.

[0101] 82. The method of paragraph 73, wherein the amount of the second dose of the compound or a pharma- ceutically acceptable salt thereof is at least about 99% lower than the amount of the first dose of the compound or a pharma- ceutically acceptable salt thereof.

[0102] 83. The method of paragraph 73, wherein the amount of the second dose of the compound or a pharma- ceutically acceptable salt thereof is at least about 99.5% lower than the amount of the first dose of the compound or a pharma- ceutically acceptable salt thereof.

[0103] 84. The method of any of paragraphs 73 to 83, wherein the first dose of the compound or a pharma- ceutically acceptable salt thereof is about 100 nM / kg to about 1000 nM / kg of the patient's body weight.

[0104] 85. The method of any of paragraphs 73 to 83, wherein the first dose of the compound or a pharma- ceutically acceptable salt thereof is about 100 nM / kg to about 900 nM / kg of the patient's body weight.

[0105] 86. The method of any of paragraphs 73 to 83, wherein the first dose of the compound or a pharma- ceutically acceptable salt thereof is about 100 nM / kg to about 800 nM / kg of the patient's body weight.

[0106] 87. The method of any of paragraphs 73 to 83, wherein the first dose of the compound or a pharma- ceutically acceptable salt thereof is about 100 nM / kg to about 700 nM / kg of the patient's body weight.

[0107] 88. The method of any of paragraphs 73 to 83, wherein the first dose of the compound or a pharma- ceutically acceptable salt thereof is about 100 nM / kg to about 600 nM / kg of the patient's body weight.

[0108] 89. The method of any of paragraphs 73 to 83, wherein the first dose of the compound or a pharma- ceutically acceptable salt thereof is about 200 nM / kg to about 600 nM / kg of the patient's body weight.

[0109] 90. The method of any of paragraphs 73 to 83, wherein the first dose of the compound or a pharma- ceutically acceptable salt thereof is about 400 nM / kg to about 600 nM / kg of the patient's body weight.

[0110] 91. The method of any of paragraphs 73 to 83, wherein the first dose of the compound or a pharma- ceutically acceptable salt thereof is about 500 nM / kg of the patient's body weight.

[0111] 92. The method of paragraph 84, wherein the second dose of the compound or a pharma- ceutically acceptable salt thereof is about 0.5 nM / kg to about 500 nM / kg of the patient's body weight.

[0112] 93. The method of paragraph 85, wherein the second dose of the compound or a pharma- ceutically acceptable salt thereof is about 0.5 nM / kg to about 450 nM / kg of the patient's body weight.

[0113] 94. The method of paragraph 86, wherein the second dose of the compound or a pharma- ceutically acceptable salt thereof is about 0.5 nM / kg to about 400 nM / kg of the patient's body weight.

[0114] 95. The method of paragraph 87, wherein the second dose of the compound or a pharma- ceutically acceptable salt thereof is about 0.5 nM / kg to about 350 nM / kg of the patient's body weight.

[0115] 96. The method of paragraph 88, wherein the second dose of the compound or a pharma- ceutically acceptable salt thereof is about 0.5 nM / kg to about 300 nM / kg of the patient's body weight.

[0116] 97. The method of paragraph 89, wherein the second dose of the compound or a pharma- ceutically acceptable salt thereof is from about 1 nM / kg to about 300 nM / kg of the patient's body weight.

[0117] 98. The method of paragraph 90, wherein the second dose of the compound or a pharma- ceutically acceptable salt thereof is about 2 nM / kg to about 300 nM / kg of the patient's body weight.

[0118] 99. The method of paragraph 91, wherein the second dose of the compound or a pharma- ceutically acceptable salt thereof is from about 2 nM / kg to about 250 nM / kg of the patient's body weight.

[0119] 100. The method of any of paragraphs 92 to 99, wherein the second dose of the compound or a pharma- ceutically acceptable salt thereof is about 5 nM / kg to about 40 nM / kg of the patient's body weight.

[0120] 101. The method of any of paragraphs 92 to 99, wherein the second dose of the compound or a pharma- ceutically acceptable salt thereof is about 40 nM / kg to about 150 nM / kg of the patient's body weight.

[0121] 102. The method of any of paragraphs 73 to 101, further comprising administering a third dose of the compound or a pharma- ceutically acceptable salt thereof, wherein the third dose of the compound or a pharma- ceutically acceptable salt thereof is the same as the second dose of the compound or a pharma- ceutically acceptable salt thereof.

[0122] 103. The method of paragraph 102, further comprising administering a fourth dose of the compound or a pharma- ceutically acceptable salt thereof, wherein the fourth dose of the compound or a pharma- ceutically acceptable salt thereof is the same as the second dose of the compound or a pharma- ceutically acceptable salt thereof or the third dose of the compound or a pharma- ceutically acceptable salt thereof.

[0123] 104. The method of any of paragraphs 73 to 103, wherein a dose of the compound or a pharma- ceutically acceptable salt thereof administered after the first dose of the compound or a pharma- ceutically acceptable salt thereof maintains the inhibition of cancer growth associated with the first dose of the compound or a pharma- ceutically acceptable salt thereof.

[0124] 105. The method of any of paragraphs 73 to 104, wherein the CAR T cells are administered at a dose of about 1 million CAR T cells to about 40 million CAR T cells.

[0125] 106. The method of any of paragraphs 73 to 105, wherein the dose of the compound or a pharma- ceutically acceptable salt thereof administered after the first dose of the compound or a pharma- ceutically acceptable salt thereof is administered once a week.

[0126] 107. The method of any of paragraphs 73 to 105, wherein the dose of the compound or a pharma- ceutically acceptable salt thereof is administered twice weekly.

[0127] 108. The method of any of paragraphs 1 to 107, wherein the CAR further comprises an IgG4 hinge domain, a CD3ζ activation domain, and a 4-1BB costimulatory domain.

[0128] 109. The method of any of paragraphs 1 to 108, wherein the E2 anti-fluorescein antibody fragment is an scFv fragment.

[0129] 110. The method of any of paragraphs 1-109, wherein the CAR protein sequence has at least about 90% identity to SEQ ID NO:2.

[0130] 111. The method of any of paragraphs 1-109, wherein the CAR protein sequence has at least about 95% identity to SEQ ID NO:2.

[0131] 112. The method of any of paragraphs 1-109, wherein the CAR protein sequence has at least about 98% identity to SEQ ID NO:2.

[0132] 113. The method of any of paragraphs 1 to 112, wherein CAR is bound to fluorescein.

[0133] 114. The method of any of paragraphs 1 to 109, wherein the CAR protein sequence has up to about 50 conservative amino acid substitutions, and the CAR is conjugated to fluorescein.

[0134] 115. The method of any of paragraphs 1-109, wherein the CAR is encoded by a polynucleotide having at least about 90% identity to SEQ ID NO:1.

[0135] 116. The method of any of paragraphs 1-109, wherein the CAR is encoded by a polynucleotide having at least about 95% identity to SEQ ID NO:1.

[0136] 117. The method of any of paragraphs 1-109, wherein the CAR is encoded by a polynucleotide having at least about 98% identity to SEQ ID NO:1.

[0137] 118. The method of any of paragraphs 115 to 117, wherein the CAR is bound to fluorescein.

[0138] 119. The method of any of paragraphs 1 to 118, wherein the CAR is encoded by a polynucleotide that hybridizes under high stringency conditions to a polynucleotide having SEQ ID NO:1, and the CAR is bound to fluorescein.

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

[0140] 121. The method of any of paragraphs 1 to 120, wherein the ligand is selected from the group consisting of folate, DUPA, NK-1R ligands, CAIX ligands, gamma glutamyl transpeptidase ligands, NKG2D ligands, and CCK2R ligands.

[0141] 122. The method of any of paragraphs 1 to 121, wherein the targeting moiety is fluorescein or a pharma- ceutically acceptable salt thereof.

[0142] 123. The method of any of paragraphs 1 to 122, wherein the linker comprises polyethylene glycol (PEG), polyproline, a hydrophilic amino acid, a sugar, a non-natural peptidoglycan, polyvinylpyrrolidone, Pluronic F-127, or a combination thereof.

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

[0144] 125. A compound or a pharma- ceutically acceptable salt thereof having the formula [ka] wherein B is a small molecule ligand, L is a linker, and T is a targeting moiety, where L is of the formula [ka] and n is an integer of 0 to 200. Item 125. The method of any one of items 1 to 124,

[0145] 126. Cancer: Lung cancer, bone cancer, pancreatic cancer, skin cancer, head cancer, neck cancer, cutaneous melanoma, intraocular melanoma, uterine cancer, ovarian cancer, endometrial cancer, rectal cancer, stomach cancer, colon cancer, breast cancer, triple-negative breast cancer, fallopian tube cancer, endometrioma, 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 126. The method of any of paragraphs 1 to 125, wherein the cancer is selected from the group consisting of hematologic malignancies, lymphocytic lymphoma, myeloid leukemia, myelomonocytic leukemia, hairy cell leukemia, myelomonocytic leukemia, hairy cell leukemia, pleural mesothelioma, bladder cancer, Burkitt's lymphoma, ureteral cancer, kidney cancer, renal cell carcinoma, renal pelvis cancer, neoplasms of the central nervous system (CNS), primary CNS lymphoma, spinal axis tumor, brain stem glioma, pituitary adenoma, and esophagogastric junction adenocarcinoma.

[0146] 127. The method of any of paragraphs 1 to 126, wherein the cancer is a folate receptor-expressing cancer.

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

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

[0149] 130. Any of the methods of paragraphs 1 to 127, wherein the cancer is ovarian cancer.

[0150] 131. Any of the methods of paragraphs 1 to 127, wherein the cancer is triple-negative breast cancer.

[0151] 132. Any of the methods of paragraphs 1 to 127, wherein the cancer is acute myeloid leukemia.

[0152] 133. The method of paragraph 132, wherein the cancer expresses folate receptor-β.

[0153] 134. Any of the methods of paragraphs 1 to 133, wherein multiple doses of the CAR T cell composition are administered.

[0154] 135. Any of the methods of paragraphs 1 to 134, wherein at least two doses of the CAR T cell composition are administered.

[0155] 136. The method of any of paragraphs 1 to 135, wherein the patient is imaged prior to administration of the compound or a pharma- ceutically acceptable salt thereof or prior to administration of the CAR T cell composition.

[0156] 137. The method of any of paragraphs 1 to 136, wherein the compound or a pharma- ceutically acceptable salt thereof is not an antibody and does not include an antibody fragment.

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

[0158] 139. Any of the methods of paragraphs 1 to 138, wherein CAR T cell toxicity against the cancer occurs without causing cytokine release that results in off-target toxicity in the patient.

[0159] 140. Any of the methods of paragraphs 1 to 138, wherein off-target tissue toxicity does not occur in the patient and CAR T cell toxicity occurs against the cancer.

[0160] 141. The method of any of paragraphs 1 to 138, wherein the cancer comprises a tumor, the tumor size in the patient is reduced, and no off-target toxicity occurs.

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

[0162] 143. Any of the methods of paragraphs 1 to 142, wherein weight loss due to CRS is reduced or prevented.

[0163] 144. The method of any of paragraphs 59 to 67, further comprising re-administering the compound or a pharma- ceutically acceptable salt thereof to the patient.

[0164] 145. The method of paragraph 144, wherein subsequent administration of the compound or a pharma- ceutically acceptable salt thereof results in CAR T cell activation and increased cytokine levels in the patient.

[0165] 146. The method of any of paragraphs 1 to 145, wherein the cancer involves a tumor and a complete tumor response is obtained.

[0166] 147. Any of the methods of paragraphs 1 to 146, wherein the CAR T cells have a central memory / effector memory phenotype.

[0167] 148. Any of the methods of paragraphs 1 to 147, wherein the CD8:CD4 ratio in the CAR T cells is about 1:1. [Brief description of the drawings]

[0168] [Figure 1] FIG. 1 shows a schematic of the E2 construct versus the 4M5.3 construct and shows a map of the E2 construct.

[0169] [Diagram 2] FIG. 2 shows the EC17 fixed dose and dose tapering schema.

[0170] [Diagram 3] Figures 3A and 3B show E2-CAR-T antitumor activity (30 million cells) along with EC17 dose tapering and body weight change. Antitumor activity is shown to be maintained after NaFL rescue.

[0171] [Figure 4] Figures 4A and 4B show 4M5.3-CAR-T antitumor activity (30 million cells) along with EC17 dose tapering and body weight change. It is shown that 4M5.3-CAR-T was less active than E2-CAR-T. Furthermore, EC17 dose-dependent antitumor activity and body weight loss were observed.

[0172] [Diagram 5]Figures 5A and 5B show E2-CAR-T antitumor activity (10 and 20 million cells) and body weight change at a fixed EC17 dosing regimen (500 nmol / kg, SIW). It is shown that antitumor activity was maintained after NaFL rescue.

[0173] [Figure 6] Figures 6A and 6B show 4M5.3-CAR-T antitumor activity (10 and 20 million cells) and body weight change at a fixed EC17 dosing regimen (500 nmol / kg, SIW). It is shown that 4M5.3-CAR-T was less active than E2-CAR-T.

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

[0175] [Figure 8] Figures 8A and 8B show a comparison of CAR-T differentiation phenotypes in various preparations including E2-CAR-T cells, 4M5.3-CAR-T cells, and GFP+4M5.3 CAR-T cells.

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

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

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

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

[0180] [Figure 13] Figure 13 shows IHC staining of conjugated DIG-E2 antibody in normal human organ tissue: breast. Panel A: test tissue section pre-incubated with DIG-E2 antibody; Panel B: control tissue section not pre-incubated with DIG-E2 antibody.

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

[0182] [Figure 15] Figure 15 shows IHC staining of conjugated DIG-E2 antibody in normal human organ tissue: cervix. Panel A: test tissue section pre-incubated with DIG-E2 antibody; Panel B: control tissue section not pre-incubated with DIG-E2 antibody.

[0183] [Figure 16] 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] 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] Figure 18 shows IHC staining of conjugated 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] 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] 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] Figure 21 shows IHC staining of conjugated 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] Figure 22 shows IHC staining of conjugated 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] [Diagram 23]Figure 23 shows IHC staining of conjugated 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] 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] [Diagram 25] Figure 25 shows IHC staining of bound DIG-E2 antibody in normal human organ tissue: lung. Panel A: test tissue section pre-incubated with DIG-E2 antibody; Panel B: control tissue section not pre-incubated with DIG-E2 antibody.

[0193] [Figure 26] Figure 26 shows IHC staining of conjugated DIG-E2 antibody in normal human organ tissues: lymph nodes. Panel A: test tissue sections pre-incubated with DIG-E2 antibody; Panel B: control tissue sections not pre-incubated with DIG-E2 antibody.

[0194] [Figure 27] Figure 27 shows IHC staining of conjugated DIG-E2 antibody in normal human organ tissues: nerve. Panel A: test tissue sections pre-incubated with DIG-E2 antibody; Panel B: control tissue sections not pre-incubated with DIG-E2 antibody.

[0195] [Figure 28] Figure 28 shows IHC staining of conjugated DIG-E2 antibody in normal human organ tissue: ovary. Panel A: test tissue section pre-incubated with DIG-E2 antibody; Panel B: control tissue section not pre-incubated with DIG-E2 antibody.

[0196] [Figure 29] Figure 29 shows IHC staining of conjugated DIG-E2 antibody in normal human organ tissue: pancreas. Panel A: test tissue section pre-incubated with DIG-E2 antibody; Panel B: control tissue section not pre-incubated with DIG-E2 antibody.

[0197] [Diagram 30] Figure 30 shows IHC staining of bound DIG-E2 antibody in normal human organ tissue: prostate. Panel A: test tissue section pre-incubated with DIG-E2 antibody; Panel B: control tissue section not pre-incubated with DIG-E2 antibody.

[0198] [Diagram 31] Figure 31 shows IHC staining of conjugated DIG-E2 antibody in normal human organ tissue: skin. Panel A: test tissue section pre-incubated with DIG-E2 antibody; Panel B: control tissue section not pre-incubated with DIG-E2 antibody.

[0199] [Diagram 32] Figure 32 shows IHC staining of bound DIG-E2 antibody in normal human organ tissue: small intestine. Panel A: test tissue section pre-incubated with DIG-E2 antibody; Panel B: control tissue section not pre-incubated with DIG-E2 antibody.

[0200] [Diagram 33] Figure 33 shows IHC staining of bound DIG-E2 antibody in normal human organ tissue: stomach. Panel A: test tissue section pre-incubated with DIG-E2 antibody; Panel B: control tissue section not pre-incubated with DIG-E2 antibody.

[0201] [Diagram 34]Figure 34 shows IHC staining of conjugated DIG-E2 antibody in normal human organ tissue: striated muscle. Panel A: test tissue section pre-incubated with DIG-E2 antibody; Panel B: control tissue section not pre-incubated with DIG-E2 antibody.

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

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

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

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

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

[0207] [Diagram 40] Figure 40 is a graph showing target cell apoptosis in 2-day co-culture with five different cell types (OV90 cells; SKOV3 cells; IGROV1 cells; HOS-Frα cells; MDA-MB-231 cells) under various conditions. For each cell type, the conditions are as follows: left bar target cells alone + EC17; middle bar EC17 CAR T + target cell co-culture without pretreatment; right bar CAR T + target cells treated with EC17 pretreatment.

[0208] [Diagram 41] FIG. 41 is a chart showing functional folate receptor expression levels in five different cell types (OV90 cells; SKOV3 cells; IGROV1 cells; HOS-Frα cells; MDA-MB-231 cells).

[0209] [Diagram 42] FIG. 42 is a schematic showing the experimental timeline of tumor-bearing and naive mice.

[0210] [Diagram 43] Figure 43 (left panel) is a chart showing that cytokine IFN-γ levels were EC-17 dependent in both naive and tumor-bearing mice, with IFN-γ increases being much lower in naive mice compared to MDA-MB-231 tumor-bearing mice (23-fold lower). Figure 43 (right panel) is a chart showing FACS analysis of CAR-T expansion in MDA-MB-231 tumor-bearing mice, but no detectable CAR-T cell expansion in naive mice.

[0211] [Diagram 44] Figure 44 (top panel) is a chart showing quantitatively similar levels of cytokine (IL-2) production from mock and anti-FLCAR T cells in co-culture with the positive control cell line K562-OKT3 (pair of bars on the left, mock is the left bar and anti-FLCAR T cells is the right bar); no cytokine was produced by mock or anti-FLCAR T cells in co-culture with K562 (center); and anti-FLCAR T cells were the only cells that could be induced to secrete the cytokine IL-2, but only by pre-treatment with EC-17 (right, where only results for anti-FLCAR T cells are shown). Figure 44 (center panel) is a chart showing quantitatively similar levels of cytokine (IFN-γ) production from mock and anti-FLCAR T cells in co-culture with the positive control cell line K562-OKT3 (pair of bars on the left, mock is the left bar and anti-FLCAR T cells is the right bar); no cytokine was produced by mock or anti-FLCAR T cells in co-culture with K562 (center); and anti-FLCAR T cells were the only cells that could induce secretion of the cytokine IFN-γ, but only by pre-treatment with EC-17 (right, where only results for anti-FLCAR T cells are shown). Figure 44 (bottom panel) is a chart showing quantitatively similar levels of cytokine (TNF-α) production from mock and anti-FLCAR T cells in co-culture with the positive control cell line K562-OKT3 (pair of bars on the left, mock is the left bar and anti-FLCAR T cells is the right bar); no cytokine was produced by mock or anti-FLCAR T cells in co-culture with K562 (center); and anti-FLCAR T cells were the only cells that could be induced to secrete the cytokine TNF-α, but only by pre-treatment with EC-17 (right, here only results for anti-FLCAR T cells are shown).

[0212] [Diagram 45]Figure 45 (top left) is a chart showing the percentage lysis when CD8+ mock T cells and anti-FLCAR T cells are co-cultured with negative control K562 cells at ratios of 30:1, 10:1, 3:1 or 1:1. Figure 45 (top right) is a chart showing the percentage lysis when CD8+ mock T cells and anti-FLCAR T cells are co-cultured with K562+OKT3 target cells at ratios of 30:1, 10:1, 3:1 or 1:1. Figure 45 (bottom left) is a chart showing the percentage lysis when CD8+ mock T cells and anti-FLCAR T cells are co-cultured with unlabeled MDA-MB-231 cells at ratios of 30:1, 10:1, 3:1 or 1:1. Figure 45 (bottom right) is a chart 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. For all charts, (x) CD8+ mock T cells; (○) anti-FLCAR T cells.

[0213] [Diagram 46] Figure 46 (top) shows the chemical structure of FITC-folate, and Figure 46 (bottom) is a chart showing the dose-response curve for FITC-folate.

[0214] [Figure 47] Figure 47 (top) shows the chemical structure of FITC-DUPA. Figure 47 (bottom) is a chart showing the dose-response curve of FITC-DUPA.

[0215] [Figure 48] Figure 48 (top) shows the chemical structure of FITC-CA9. Figure 48 (bottom) is a chart showing the dose-response curve of FITC-CA9.

[0216] [Figure 49] Figure 49 (top) shows the chemical structure of FITC-NK1R. Figure 49 (bottom) is a chart showing the dose-response curve of FITC-NK1R.

[0217] [Figure 50]FIG. 50 shows binding of the bridge (by FACS analysis) to tumor cells expressing a receptor corresponding to the small molecule ligand of the bridge, used in an in vivo model.

[0218] [Figure 51] Figure 51 shows EC17 induction of potent FR-dependent tumor cell killing by CAR T cells.

[0219] [Figure 52] Figure 52 shows the correlation between cytolytic activity of CAR T cells and functional FR levels of tumor cells.

[0220] [Figure 53] Figure 53 shows EC17 / FR-dependent CAR T cell activation and exhaustion. Expression of early (CD69), intermediate (CD137) and late (PD1) T cell activation markers detected in CAR-T cells co-cultured (E / T=1:1) with FR+ tumor cell targets preloaded without or with EC17 (100 nM, 30 min pulse at 37°C). The first two open bars show only CAR-T and mock-transduced T cells without target cells. Annotation from top to bottom of second to fourth panels = bars from left to right.

[0221] [Figure 54] Figure 54 shows EC17 / FR-dependent CAR T cell exhaustion profiles. First row: Colored pie charts representing the differentiation state changes of CAR-T cells in culture without target cells from day 0 to day 3. Bottom row: Four sets of colored pie charts representing the differentiation state of CAR-T cells after 3 days of co-culture (E / T=1:2) with FR+ (MDA-MB-231, THP-1FRβ, HOS-FRα, KB) and FR-negative (THP1-FG12, HOS-143b) tumor cells preloaded without or with EC17 (0.1 or 10 nM, 30 min pulse at 37°C).

[0222] [Figure 55]Figure 55 shows a fully human CAR construct consisting of anti-FITC scFv (clone E2), full length IgG4 spacer (Fc-derived hinge-CH2(L235D, N297Q)-CH3), CD28tm transmembrane domain, 4-1BB / CD3ζ cytoplasmic activation domain and a non-functional truncated cell surface polypeptide of epidermal growth factor receptor (EGFRt). Bottom: Example of CD4 / CD8 T cell phenotyping performed by flow cytometry of EGFRt sorted (left pie chart) CAR-T cell preparations and non-sorted "clinical facsimile" (right pie chart). Color key as indicated.

[0223] [Figure 56] Figure 56, Panel A: Kd value of 3H-EC17 uptake by FR+ target cells after 2 hours incubation at 37°C (calculated from the number of molecules bound per cell). Panel B: Kd value of 3H-EC17 uptake by E2-CAR-T cells after 2 hours incubation at room temperature (-24% EGFRt+, -95:5 CD8 / CD4 ratio) (calculated from total cell-associated radioactivity, DPM).

[0224] [Figure 57] Figure 57 (Panels A and B) shows the CRS rating scale and experimental conditions applied to sodium fluorescein rescue of HOS-FRα tumor-bearing mice. Changes in cytokines derived from rescued and non-rescued T cells are shown. p values ​​were calculated by Student's t-test.

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

[0226] [Figure 59]FIG. 59 shows (Panel A) full range EC17 dose response in specific lysis (%) of five FR (Folate Receptor)+ tumor cell lines co-cultured with EGFRt sorted CAR-T cells at a 1:1 E / T (effector / target) ratio for 24 hours; (Panel B) Maximum lysis (%) and EC50 values ​​were obtained from dose-response curves fitted up to 100 nM.

[0227] [Figure 60] Figure 60 shows the correlation of CAR-T cell activity with FR levels and intrinsic sensitivity of tumor cells. Panel A: Kinetics of specific lysis (%) at various E / T ratios of FR+ (MDA-MB-231, KB, THP1-FRβ, OV90, HOS-FRα) and FR-negative (HOS-143b) cell lines after 16 and 48 hours of co-culture in the presence of 10 nM EC17. Panel B: Specific lysis (%) of target cells plotted against a linear scale of E / T ratio. High FR+ KB cells showed early resistance at 16 hours, while FR-negative HOS-143b did not respond. Panel C: Except for KB cells, semi-log correlations were established between specific lysis (%) at 16 hours of co-culture and functional FR levels of tumor cells. Panel D: 3D diagram showing the correlation between CAR-T cell-derived Th1 cytokine (IFN-γ, IL-2 and TNF-α) levels and different E / T ratios after 44 h co-culture with 10 nM EC17. Panel E: CAR-T cell-derived Th1 cytokine levels plotted against FR levels in FR+ target cells on a Log10 scale.

[0228] [Figure 61] FIG. 61 is a bar graph showing (Panel A) EC17 loading status of target cells as determined by flow cytometry, shown as MFI (EC17 was undetectable in FR-negative cell lines) and (Panel B) target cell apoptosis (%) as detected by Annexin V staining after 2 days of co-culture as described in FIG.

[0229] [Figure 62]Figure 62 shows the pharmacokinetics and tumor uptake of CAR-T cells in vivo. Panel A: Schematic of the experimental layout showing the animal harvest schedule associated with CAR-T cell injections and weekly EC17 dosing (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 had large tumors to initiate). Panel B: Left panel shows tumor volume measurements and body weight changes; center plot shows CAR-T cell expansion in blood 7 days after a single EC17 dose; right bar graph shows differentiation profile of circulating CD4 / CD8 CAR-T cell subsets in mice with small and large tumors. Panel C: Measurement of tumor volume and body weight changes. Panel D: Top plot shows CAR-T cell kinetics in blood (solid line) vs. tumor (dotted line). Bottom bar graph shows changes in CAR-T cell phenotype in blood. Panel E: Kinetic changes in surface expression of activation markers (4-1BB, PD1) on tumor-infiltrating CAR-T cells.

[0230] [Figure 63] Figure 63 shows the effect of dietary folate on antitumor activity and CRS toxicity. Panel A: Measurements of tumor volume and body weight change (n=5) of NSG mice xenografted with large MDA-MB-231 tumors and treated with CAR-T cells (approximately 10 million "clinical replicas") and 500 nmol / kg EC17 SIW, maintained on a FA-enriched or FA-deficient diet throughout the study. Panel B: Bar graphs showing circulating CAR-T cells (human CD3ε+EGFRt+) measured at the end of the study on day 52 (FA-deficient) and day 59 (FA-enriched), respectively. Panel C: Representative flow cytometry dot plots showing the absence of circulating CAR-T cells in FA-deficient mice treated with CAR-T cells only (no EC17) and increased numbers of circulating CAR-T cells in mice treated with CAR-T cells and EC17 on a FA-deficient diet compared to mice treated identically except for the FA-enriched diet. Panel D: Loss of FR expression was detected in two of three MDA-MB-231 tumors harvested on day 59 in FA-enhanced animals and analyzed by flow cytometry (later confirmed by quantitative radioligand binding assays, data not shown). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0231] definition As used herein, the singular term "a," "an," or "an" can mean 1 or more. As used herein, for example, with reference to numerical values, including integers, fractions, and percentages, "about" refers to a range of numerical values ​​(e.g., ±5% to 10% of the recited value) that one of skill in the art would generally consider equivalent to the recited value (e.g., having the same function or result).

[0232] As used herein, the terms "treatment," "treated," or "treating" include both therapeutic treatments and prophylactic or preventative treatments.

[0233] The terms "amelioration," "improved," or "ameliorating," as used herein with respect to cancer, can mean alleviation of symptoms of the cancer, reduction in tumor size, complete or partial removal of the tumor (e.g., complete or partial response), stabilizing the disease, preventing cancer progression (e.g., progression-free survival), or any other effect on the cancer that a physician considers to be a therapeutic, prophylactic, or preventative treatment of the cancer.

[0234] As used herein, the terms "administration," "administered," or "administering" refer to any means of introducing a compound or a pharma- ceutically acceptable salt thereof, or a CAR T cell composition (wherein the CAR T cell composition comprises a CAR T cell, and 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 a patient that is unacceptable to the physician treating the patient, such as B cell hypoplasia, fever, decreased blood pressure, or pulmonary edema.

[0236] As used herein, the terms "transduction" and "transfect" are used interchangeably and refer to the introduction of nucleic acid into a cell by any artificial method, including viral and non-viral methods.

[0237] DETAILED DESCRIPTION OF THE ILLUSTRATIVE EMBODIMENTS In various embodiments described herein, a small molecule ligand linked to a targeting moiety by a linker is used as a bridge between a cancer and a CAR T cell (i.e., a chimeric antigen receptor-expressing T cell), where the CAR T cell comprises a genetically modified CAR directed to a targeting moiety, where the CAR comprises an E2 anti-fluorescein antibody fragment. The bridge directs the CAR T cell to the cancer for amelioration of the cancer. In some embodiments, the "small molecule ligand" can be a folate, a CAIX ligand, a DUPA, an NK-1R ligand, a gamma glutamyl transpeptidase ligand, an NKG2D ligand, or a CCK2R ligand, each of which is a small molecule ligand that specifically binds to a cancer cell type (i.e., the receptor for each of these ligands is overexpressed in cancer compared to normal tissue).

[0238] The "targeting moiety" that binds to the small molecule ligand binds to the recognition region of the genetically modified CAR expressed by the CAR T cell, where the CAR comprises an E2 anti-fluorescein antibody fragment. Thus, the recognition region of the CAR (e.g., the single-chain fragment variable region (scFv) of the E2 anti-fluorescein antibody) is directed to the "targeting moiety". Thus, the small molecule ligand that is linked to the targeting moiety by the linker serves as a bridge between the cancer and the CAR T cell (where the CAR T cell comprises a genetically modified CAR) and directs the CAR T cell to the cancer for amelioration of the cancer.

[0239] The bridge is a small organic molecule, so clearance from the bloodstream can be achieved quickly (e.g., in about 20 minutes or less). In some embodiments, the CAR T cell response (wherein the CAR T cell comprises a genetically modified CAR that contains an E2 anti-fluorescein antibody fragment) can be targeted only to cancer cells that express a receptor for the small molecule ligand moiety of the 'bridge', thereby reducing off-target toxicity to normal tissues. Furthermore, this system can be 'universal', since one type of CAR T cell construct (wherein the CAR T cell comprises an E2 anti-fluorescein antibody fragment) can be used to target many cancers by using different 'bridges'. By way of illustration, the targeting moiety recognized by the CAR T cell (wherein the CAR T cell comprises a genetically modified CAR that contains an E2 anti-fluorescein antibody fragment) can remain constant to allow the use of one type of CAR T cell, while the small molecule ligand that binds to the cancer can be changed to allow the targeting of many cancers.

[0240] In various embodiments described herein, a small molecule ligand attached to a targeting moiety by a linker is referred to as a "compound."

[0241] In various embodiments, the term "E2 anti-fluorescein antibody fragment" refers to a CAR comprising a fragment (e.g., scFv fragment) of an E2 anti-fluorescein antibody. E2 anti-fluorescein antibodies are described, for example, in Vaughan, et al., Nature Biotechnol. Vol. 14(3), pp. 309-314, 1996, and are incorporated herein by reference. In various embodiments, the CAR may further comprise an IgG4 hinge domain, a CD3ζ activation domain, and / or any other suitable domain, such as a 4-1BB costimulatory domain or an EGFRt domain. In yet other embodiments, the CAR may be encoded by a polynucleotide having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identity to SEQ ID NO:1. In other illustrative embodiments, the CAR may be encoded by a polynucleotide that hybridizes under high stringency conditions to a polynucleotide having SEQ ID NO:1. In yet other embodiments, the CAR can be encoded by a polynucleotide having SEQ ID NO: 1 or 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 other embodiments, the CAR protein sequence can have up to about 50 conservative amino acid substitutions. In any of the embodiments described herein, the CAR is conjugated to fluorescein.

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

[0243] In another embodiment, a method of treating cancer is provided, comprising: i) administering to a patient a compound or a pharma- ceutically 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 comprising a CAR T cell, wherein the CAR T cell comprises a CAR, the CAR comprises an E2 anti-fluorescein antibody fragment, and the CAR T cell composition comprises a mixture of CAR T cells and non-transformed T cells.

[0244] In yet another embodiment, a method of treating cancer is provided, comprising: i) administering to a patient a compound or a pharma- ceutically 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 a CAR T cell, the CAR T cell comprises a CAR, and the CAR comprises an E2 anti-fluorescein antibody fragment; and iii) administering to the patient folate, a conjugate comprising folate without 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, comprising: i) administering to a patient a compound or a pharma- ceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked to a targeting moiety by a linker, and the compound or a pharma- ceutically acceptable salt thereof is administered at a dose of about 10 nM / kg to about 2500 nM / kg of patient body weight; and ii) administering to a patient a CAR T cell composition comprising a CAR T cell, wherein the CAR T cell comprises a CAR, the CAR comprises an E2 anti-fluorescein antibody fragment, and the CAR T cell is at a dose of about 1 million to about 15 million CAR T cells.

[0246] In yet another embodiment, a method of treating cancer is provided, comprising: i) continuously administering a compound to a patient, wherein the compound comprises a small molecule ligand linked to a targeting moiety by a linker; ii) administering a CAR T cell composition comprising a CAR T cell to the patient, wherein the CAR T cell comprises a CAR, and the CAR comprises an E2 anti-fluorescein antibody fragment; and iii) terminating the continuous administration of the compound or a pharma- ceutically acceptable salt thereof to block or prevent cytokine release syndrome in the patient.

[0247] In another illustrative embodiment, a method of treating cancer is provided, comprising: i) administering to a patient a compound or a pharma- ceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked to a targeting moiety by a linker, and wherein the patient is administered at least a first dose and a second dose of the compound or a pharma- ceutically acceptable salt thereof, wherein the first dose and the second dose are different, and the second dose of the compound or a pharma- ceutically 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 pharma- ceutically acceptable salt thereof; and ii) administering to the patient a CAR T cell composition comprising a CAR T cell, wherein the CAR T cell comprises a CAR, and the CAR comprises an E2 anti-fluorescein antibody fragment.

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

[0249] In yet another embodiment, a method for treating cancer is provided, comprising: i) administering to a patient a first dose of a compound or a pharmaceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked to a targeting moiety by a linker; ii) administering to a patient at least a second dose of a compound or a pharmaceutically acceptable salt thereof, wherein 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 a patient a dose of a CAR T cell composition comprising a CAR T cell, wherein the CAR T cell comprises a CAR, and the CAR comprises an E2 anti-fluorescein antibody fragment.

[0250] Several embodiments are described by the enumerated paragraphs below. Any of the following embodiments are contemplated in combination with any of the applicable embodiments described in the Summary section, Detailed Description of Illustrative Embodiments section, Examples section, or claims of this application.

[0251] 1. A method of treating cancer, comprising: i) administering to a patient a compound or a pharma- ceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand attached to a targeting moiety by a linker; ii) administering to the patient a first dose of a CAR T cell composition comprising a CAR T cell, wherein the CAR T cell comprises a CAR directed to a targeting moiety, the CAR comprising an E2 anti-fluorescein antibody fragment; and iii) administering to the patient a second dose of a CAR T cell composition comprising the CAR T cell, wherein the CAR T cell comprises a CAR directed to a targeting moiety, and the CAR comprises an E2 anti-fluorescein antibody fragment. A method comprising:

[0252] 2. A method of treating cancer, comprising: i) administering to a patient a compound or a pharma- ceutical acceptable salt thereof, wherein the compound comprises a small molecule ligand attached 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 a CAR T cell, the CAR T cell comprises a CAR directed to a targeting moiety, the CAR comprises an E2 anti-fluorescein antibody fragment, and the CAR T cell composition comprises a mixture of CAR T cells and non-transformed T cells. A method comprising:

[0253] 3. A method of treating cancer, comprising: i) administering to a patient a compound or a pharma- ceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand attached to a targeting moiety by a linker; ii) administering to the patient a CAR T cell composition, wherein the CAR T cell composition comprises a CAR T cell, the CAR T cell comprises a CAR directed to a targeting moiety, the CAR comprising an E2 anti-fluorescein antibody fragment; and iii) administering to the patient folate, a conjugate containing folate without a targeting moiety, or an agent that inhibits activation of the CAR T cell. A method comprising:

[0254] 4. The method of paragraph 3, wherein step iii comprises administering folate.

[0255] 5. The method of paragraph 3 or 4, wherein step iii includes administration of folic acid or leucovorin.

[0256] 6. The method of paragraph 3, wherein step iii comprises administering a conjugate comprising a folate.

[0257] 7. The method of claim 6, wherein the folate-containing conjugate comprises folate bound to one or more amino acids.

[0258] 8. Folate-containing conjugates are of the formula [ka] 8. The method of claim 7, wherein

[0259] 9. Folate is the formula [ka] [In the formula, X 1 and Y 1 each independently represents halo, R 2 , OR 2 , S.R. 3 and N.R. 4 R 5 selected from the group consisting of; U, V and W are each -(R 6a )C=, -N=, -(R 6a )C(R 7a )- and -N(R 4a )-; Q is selected from the group consisting of C and CH; T is selected from the group consisting of S, O, N, and -C=C-; X 2 and X 3 are each independently oxygen, sulfur, -C(Z)-, -C(Z)O-, -OC(Z)-, -N(R 4b )-, -C(Z)N(R 4b )-, -N(R 4b )C(Z)-, -OC(Z)N(R 4b )-, -N(R 4b )C(Z)O-, -N(R 4b )C(Z)N(R 5b )-, -S(O)-, -S(O)2-, -N(R 4a )S(O)2-, -C(R 6b )(R 7b)-, -N(C≡CH)-, -N(CH2C≡CH)-, C1-C 12 Alkylene and C1-C 12 alkyleneoxy, where Z is oxygen or sulfur; R 1 are hydrogen, halo, C1-C 12 Alkyl and C1-C 12 alkoxy; R 2 , R 3 , R 4 , R 4a , R 4b , R 5 , R 5b , R 6b and R 7b each independently represents hydrogen, halo, C1-C 12 Alkyl, C1-C 12 Alkoxy, C1-C 12 Alkanoyl, C1-C 12 Alkenyl, C1-C 12 Alkynyl, (C1-C 12 alkoxy)carbonyl and (C1-C 12 alkylamino)carbonyl; R 6 and R 7 each independently represents hydrogen, halo, C1-C 12 Alkyl and C1-C 12 alkoxy; or R 6 and R 7 together to form a carbonyl group; R 6a and R 7a each independently represents hydrogen, halo, C1-C 12 Alkyl and C1-C 12 alkoxy; or R 6a and R 7a together to form a carbonyl group; p, r, s and t are each independently 0 or 1; and * represents an optional covalent bond to the remainder of the conjugate. 9. The method according to any one of items 3 to 8,

[0260] 10. The method of item 3, wherein an agent that inhibits activation of CAR T cells is administered and is selected from the group consisting of lymphocyte-specific protein tyrosine kinase inhibitors, PI3 kinase inhibitors, IL-2 inducible T cell kinase inhibitors, JAK inhibitors, BTK inhibitors, EC2319, and agents that block CAR T cell binding of the compound or a pharma- ceutically acceptable salt thereof but do not bind to the cancer.

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

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

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

[0264] 14. The method according to item 13, wherein the PI3 kinase inhibitor is GDC0980.

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

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

[0267] 17. The method of paragraph 10, wherein an agent that inhibits activation of CAR T cells is administered, the agent blocking CAR T cell binding of the compound or a pharma- ceutically acceptable salt thereof but not binding to the cancer.

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

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

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

[0271] 21. Any of the methods of paragraphs 17 to 20, wherein the agent that inhibits CAR T cell activation is administered at a dose of about .01 to about 300 μM / kg of patient body weight.

[0272] 22. Any of the methods of paragraphs 17 to 20, wherein the agent that inhibits CAR T cell activation is administered at a dose of about .06 to about 100 μM / kg of patient body weight.

[0273] 23. Any of the methods of paragraphs 17 to 20, wherein the agent that inhibits CAR T cell activation is administered at a dose of about .06 to about 90 μM / kg of patient body weight.

[0274] 24. Any of the methods of paragraphs 17 to 20, wherein the agent that inhibits CAR T cell activation is administered at a dose of about .06 to about 80 μM / kg of patient body weight.

[0275] 25. Any of the methods of paragraphs 17 to 20, wherein the agent that inhibits CAR T cell activation is administered at a dose of about .06 to about 70 μM / kg of patient body weight.

[0276] 26. Any of the methods of paragraphs 17 to 20, wherein the agent that inhibits CAR T cell activation is administered at a dose of about .06 to about 60 μM / kg of patient body weight.

[0277] 27. Any of the methods of paragraphs 17 to 20, wherein the agent that inhibits CAR T cell activation is administered at a dose of about .06 to about 50 μM / kg of patient body weight.

[0278] 28. Any of the methods of paragraphs 17 to 20, wherein the agent that inhibits CAR T cell activation is administered at a dose of about .06 to about 40 μM / kg of patient body weight.

[0279] 29. Any of the methods of paragraphs 17 to 20, wherein the agent that inhibits CAR T cell activation is administered at a dose of about 0.06 to about 30 μM / kg of patient body weight.

[0280] 30. Any of the methods of paragraphs 17 to 20, wherein the agent that inhibits CAR T cell activation is administered at a dose of about .06 to about 20 μM / kg of patient body weight.

[0281] 31. Any of the methods of paragraphs 17 to 20, wherein the agent that inhibits CAR T cell activation is administered at a dose of about .06 to about 10 μM / kg of patient body weight.

[0282] 32. Any of the methods of paragraphs 17 to 20, wherein the agent that inhibits CAR T cell activation is administered at a dose of about .06 to about 8 μM / kg of patient body weight.

[0283] 33. Any of the methods of paragraphs 17 to 20, wherein the agent that inhibits CAR T cell activation is administered at a dose of about .06 to about 6 μM / kg of patient body weight.

[0284] 34. Any of the methods of paragraphs 3 to 33, wherein the folate, a conjugate containing the folate without a targeting moiety, or an agent that inhibits activation of CAR T cells is administered to the patient more than once.

[0285] 35. The method of any of paragraphs 3 to 34, wherein folate, a conjugate comprising folate without a targeting moiety, or an agent that inhibits activation of CAR T cells is administered to the patient before and / or after the compound or a pharma- ceutically acceptable salt thereof.

[0286] 36. Any of the methods of paragraphs 3 to 35, wherein administration of folate, a conjugate containing folate without a targeting moiety, or an agent that inhibits activation of CAR T cells reduces cytokine levels in the patient.

[0287] 37. The method of paragraph 36, wherein the reduction in cytokine levels occurs by about 3 hours after administering to the patient the folate, the conjugate containing the folate without a targeting moiety, or the agent that inhibits activation of CAR T cells.

[0288] 38. The method of paragraph 36, wherein the reduction in cytokine levels occurs by about 6 hours after administering to the patient the folate, the conjugate containing the folate without a targeting moiety, or the agent that inhibits activation of CAR T cells.

[0289] 39. The method of any of paragraphs 36 to 38, wherein the reduction in cytokine levels is approximately to the cytokine levels of an untreated patient.

[0290] 40. The method of any of paragraphs 3 to 39, wherein the compound or a pharma- ceutically acceptable salt thereof is administered before and after administration of a folate, a conjugate comprising a folate that does not include a targeting moiety, or an agent that inhibits activation of CAR T cells.

[0291] 41. The method of any of paragraphs 3 to 40, wherein after administration of folate, a conjugate containing folate without a targeting moiety, or an agent that inhibits activation of CAR T cells, the number of CAR T cells in the patient's blood increases, even if the patient's cytokine levels decrease.

[0292] 42. The method of any of paragraphs 3 to 41, wherein after administration of folate, a conjugate comprising folate without a targeting moiety, or an agent that inhibits CAR T cell activation, CAR T cell activation is enhanced or maintained compared to a patient not treated with a rescue agent, even though cytokine levels in the treated patient are reduced.

[0293] 43. Any of the methods of paragraphs 3 to 42, wherein the cancer comprises a tumor and the size of the patient's tumor does not increase upon administration to the patient of folate, a conjugate comprising folate that does not include a targeting moiety, or an agent that inhibits activation of CAR T cells.

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

[0295] 45. Any of the methods of paragraphs 3 to 44, wherein folate, a conjugate containing folate without a targeting moiety, or an agent that inhibits CAR T cell activation is administered to the patient when CRS grade reaches 1, 2, 3, or 4.

[0296] 46. ​​The method of paragraph 45, wherein the agent that inhibits CAR T cell activation is administered to the patient when CRS grade reaches 3 or 4.

[0297] 47. Any of the methods under items 3 to 46, in which pulmonary edema is reduced.

[0298] 48. The method of any of paragraphs 1 to 47, wherein the compound or a pharma- ceutically acceptable salt thereof is administered at a dose of about 10 nM / kg patient body weight to about 2500 nM / kg patient body weight, and the CAR T cells are administered at a dose of about 1 million CAR T cells to about 15 million CAR T cells.

[0299] 49. The method of paragraph 48, wherein the compound or a pharma- ceutically acceptable salt thereof is administered at a dose of about 10 nanomoles / kg to about 100 nanomoles / kg of patient body weight.

[0300] 50. The method of any of paragraphs 48 to 49, wherein the compound or a pharma- ceutically acceptable salt thereof is administered at a dose of about 10 nM / kg to about 50 nM / kg of patient body weight.

[0301] 51. The method of any of paragraphs 48 to 50, wherein the compound or a pharma- ceutically acceptable salt thereof is administered at a dose of about 10 nM / kg to about 20 nM / kg of patient body weight.

[0302] 52. The method of paragraph 48, wherein the compound or a pharma- ceutically acceptable salt thereof is administered at a dose of about 10 nM / kg to about 600 nM / kg of patient body weight.

[0303] 53. The method of paragraph 48, wherein the compound or a pharma- ceutically acceptable salt thereof is administered at a dose of about 200 nM / kg to about 600 nM / kg of patient body weight.

[0304] 54. The method of paragraph 48, wherein the compound or a pharma- ceutically acceptable salt thereof is administered at a dose of about 400 nM / kg to about 600 nM / kg of patient body weight.

[0305] 55. The method of any of paragraphs 48 to 54, wherein the CAR T cells are at a dose of about 1 million CAR T cells to about 12.5 million CAR T cells.

[0306] 56. The method of any of paragraphs 48 to 55, wherein the CAR T cells are at a dose of about 1 million CAR T cells to about 7 million CAR T cells.

[0307] 57. The method of any of paragraphs 48 to 56, wherein the CAR T cells are at a dose of about 1 million CAR T cells to about 5 million CAR T cells.

[0308] 58. The method of any of paragraphs 48 to 57, wherein the CAR T cells are at a dose of about 2 million CAR T cells to about 5 million CAR T cells.

[0309] 59. The method of any of paragraphs 1 to 58, further comprising the step of terminating the continuous administration of the compound or a pharma- ceutically acceptable salt thereof to inhibit or prevent cytokine release syndrome in the patient.

[0310] 60. The method of any of paragraphs 1 to 59, wherein the compound or a pharma- ceutically acceptable salt thereof is administered to the patient continuously for at least one hour.

[0311] 61. The method of any of paragraphs 1 to 59, wherein the compound or a pharma- ceutically acceptable salt thereof is administered to the patient for at least 4 consecutive hours.

[0312] 62. The method of any of paragraphs 1 to 59, wherein the compound or a pharma- ceutically acceptable salt thereof is administered to the patient continuously for at least 6 hours.

[0313] 63. The method of any of paragraphs 1 to 62, wherein the compound or a pharma- ceutically acceptable salt thereof is administered to the patient every other day.

[0314] 64. The method of any of paragraphs 1 to 62, wherein the compound or a pharma- ceutically acceptable salt thereof is administered to the patient three times per week.

[0315] 65. The method of any of paragraphs 1 to 62, wherein the compound or a pharma- ceutically acceptable salt thereof is administered to the patient twice weekly.

[0316] 66. The method of any of paragraphs 1 to 62, wherein the compound or a pharma- ceutically acceptable salt thereof is administered to the patient once a week.

[0317] 67. The method of any of paragraphs 1 to 62, wherein the compound or a pharma- ceutically acceptable salt thereof is administered to the patient until unacceptable patient weight loss, fever, hypotension, or pulmonary edema occurs.

[0318] 68. A method of treating cancer, comprising: i) administering to the patient a compound or a pharma- ceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand attached to a targeting moiety by a linker, and wherein the patient is administered at least a first dose and a second dose of the compound or a pharma- ceutically acceptable salt thereof, wherein the first dose and the second dose are different, and the amount of the second dose of the compound or a pharma- ceutically 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 pharma- ceutically acceptable salt thereof; and ii) administering to the patient a CAR T cell composition comprising a CAR T cell, wherein the CAR T cell composition comprises a CAR T cell, the CAR T cell comprising a CAR directed to a targeting moiety, the CAR comprising an E2 anti-fluorescein antibody fragment. A method comprising:

[0319] 69. The method of item 68, wherein at least a first dose, a second dose, and a third dose of the compound or a pharma- ceutically acceptable salt thereof are administered to the patient, wherein the first dose, the second dose, and the third dose are different, the second dose of the compound or a pharma- ceutically acceptable salt thereof being about 2 to about 750 times greater than the amount of the first dose of the compound or a pharma- ceutically acceptable salt thereof, and the third dose of the compound or a pharma- ceutically acceptable salt thereof being about 800 to about 10,000 times greater than the amount of the first dose of the compound or a pharma- ceutical acceptable salt thereof.

[0320] 70. The method of item 68, wherein at least a first dose, a second dose, a third dose and a fourth dose of the compound or a pharma- ceutically acceptable salt thereof are administered to the patient, wherein the first dose, the second dose, the third dose and the fourth dose are different, the second dose of the compound or a pharma- ceutically acceptable salt thereof being about 2 to about 750 times more than the amount of the first dose of the compound or a pharma- ceutically acceptable salt thereof, the third dose of the compound or a pharma- ceutically acceptable salt thereof being about 800 to about 7500 times more than the amount of the first dose of the compound or a pharma- ceutical acceptable salt thereof, and the fourth dose of the compound or a pharma- ceutical acceptable salt thereof being about 8000 to about 15000 times more than the amount of the first dose of the compound or a pharma- ceutical acceptable salt thereof.

[0321] 71. The method of paragraph 70, wherein the second dose of the compound or its pharma- ceutically acceptable salt is about 100 times greater than the amount of the first dose of the compound or its pharma- ceutically acceptable salt, the third dose of the compound or its pharma- ceutically acceptable salt is about 1000 times greater than the amount of the first dose of the compound or its pharma- ceutically acceptable salt, and the fourth dose of the compound or its pharma- ceutically acceptable salt is about 10000 times greater than the amount of the first dose of the compound or its pharma- ceutically acceptable salt.

[0322] 72. The method of paragraph 68, wherein at least a first dose and a second dose of the compound or a pharma- ceutically acceptable salt thereof are administered to the patient, wherein the first dose and the second dose are different, and the amount of the second dose of the compound or a pharma- ceutically 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 pharma- ceutically acceptable salt thereof.

[0323] 73. A method of treating cancer, comprising: i) administering to the patient a first dose of a compound or a pharma- ceutically acceptable salt thereof, where the compound comprises a small molecule ligand attached to a targeting moiety by a linker; ii) administering to the patient at least a second dose of the compound or a pharma- ceutically acceptable salt thereof, wherein the second dose of the compound or a pharma- ceutically acceptable salt thereof is at least about 50% lower than the amount of the first dose of the compound or a pharma- ceutically acceptable salt thereof; and iii) administering to the patient a dose of a CAR T cell composition comprising a CAR T cell, wherein the CAR T cell composition comprises a CAR T cell, the CAR T cell comprising a CAR directed to a targeting moiety, and the CAR comprises an E2 anti-fluorescein antibody fragment. A method comprising:

[0324] 74. The method of paragraph 73, wherein the second dose of the compound or a pharma- ceutically acceptable salt thereof is at least about 60% lower than the amount of the first dose of the compound or a pharma- ceutically acceptable salt thereof.

[0325] 75. The method of paragraph 73, wherein the second dose of the compound or a pharma- ceutically acceptable salt thereof is at least about 70% lower than the amount of the first dose of the compound or a pharma- ceutically acceptable salt thereof.

[0326] 76. The method of paragraph 73, wherein the second dose of the compound or a pharma- ceutically acceptable salt thereof is at least about 80% lower than the amount of the first dose of the compound or a pharma- ceutically acceptable salt thereof.

[0327] 77. The method of paragraph 73, wherein the second dose of the compound or a pharma- ceutically acceptable salt thereof is at least about 90% lower than the amount of the first dose of the compound or a pharma- ceutically acceptable salt thereof.

[0328] 78. The method of paragraph 73, wherein the second dose of the compound or a pharma- ceutically acceptable salt thereof is at least about 95% lower than the amount of the first dose of the compound or a pharma- ceutically acceptable salt thereof.

[0329] 79. The method of paragraph 73, wherein the amount of the second dose of the compound or a pharma- ceutically acceptable salt thereof is at least about 96% lower than the amount of the first dose of the compound or a pharma- ceutically acceptable salt thereof.

[0330] 80. The method of paragraph 73, wherein the amount of the second dose of the compound or a pharma- ceutically acceptable salt thereof is at least about 97% lower than the amount of the first dose of the compound or a pharma- ceutically acceptable salt thereof.

[0331] 81. The method of paragraph 73, wherein the amount of the second dose of the compound or a pharma- ceutically acceptable salt thereof is at least about 98% lower than the amount of the first dose of the compound or a pharma- ceutically acceptable salt thereof.

[0332] 82. The method of paragraph 73, wherein the amount of the second dose of the compound or a pharma- ceutically acceptable salt thereof is at least about 99% lower than the amount of the first dose of the compound or a pharma- ceutically acceptable salt thereof.

[0333] 83. The method of paragraph 73, wherein the amount of the second dose of the compound or a pharma- ceutically acceptable salt thereof is at least about 99.5% lower than the amount of the first dose of the compound or a pharma- ceutically acceptable salt thereof.

[0334] 84. The method of any of paragraphs 73 to 83, wherein the first dose of the compound or a pharma- ceutically acceptable salt thereof is about 100 nM / kg to about 1000 nM / kg of the patient's body weight.

[0335] 85. The method of any of paragraphs 73 to 83, wherein the first dose of the compound or a pharma- ceutically acceptable salt thereof is about 100 nM / kg to about 900 nM / kg of the patient's body weight.

[0336] 86. The method of any of paragraphs 73 to 83, wherein the first dose of the compound or a pharma- ceutically acceptable salt thereof is about 100 nM / kg to about 800 nM / kg of the patient's body weight.

[0337] 87. The method of any of paragraphs 73 to 83, wherein the first dose of the compound or a pharma- ceutically acceptable salt thereof is about 100 nM / kg to about 700 nM / kg of the patient's body weight.

[0338] 88. The method of any of paragraphs 73 to 83, wherein the first dose of the compound or a pharma- ceutically acceptable salt thereof is about 100 nM / kg to about 600 nM / kg of the patient's body weight.

[0339] 89. The method of any of paragraphs 73 to 83, wherein the first dose of the compound or a pharma- ceutically acceptable salt thereof is about 200 nM / kg to about 600 nM / kg of the patient's body weight.

[0340] 90. The method of any of paragraphs 73 to 83, wherein the first dose of the compound or a pharma- ceutically acceptable salt thereof is about 400 nM / kg to about 600 nM / kg of the patient's body weight.

[0341] 91. The method of any of paragraphs 73 to 83, wherein the first dose of the compound or a pharma- ceutically acceptable salt thereof is about 500 nM / kg of the patient's body weight.

[0342] 92. The method of paragraph 84, wherein the second dose of the compound or a pharma- ceutically acceptable salt thereof is about 0.5 nM / kg to about 500 nM / kg of the patient's body weight.

[0343] 93. The method of paragraph 85, wherein the second dose of the compound or a pharma- ceutically acceptable salt thereof is about 0.5 nM / kg to about 450 nM / kg of the patient's body weight.

[0344] 94. The method of paragraph 86, wherein the second dose of the compound or a pharma- ceutically acceptable salt thereof is about 0.5 nM / kg to about 400 nM / kg of the patient's body weight.

[0345] 95. The method of paragraph 87, wherein the second dose of the compound or a pharma- ceutically acceptable salt thereof is about 0.5 nM / kg to about 350 nM / kg of the patient's body weight.

[0346] 96. The method of paragraph 88, wherein the second dose of the compound or a pharma- ceutically acceptable salt thereof is about 0.5 nM / kg to about 300 nM / kg of the patient's body weight.

[0347] 97. The method of paragraph 89, wherein the second dose of the compound or a pharma- ceutically acceptable salt thereof is from about 1 nM / kg to about 300 nM / kg of the patient's body weight.

[0348] 98. The method of paragraph 90, wherein the second dose of the compound or a pharma- ceutically acceptable salt thereof is about 2 nM / kg to about 300 nM / kg of the patient's body weight.

[0349] 99. The method of paragraph 91, wherein the second dose of the compound or a pharma- ceutically acceptable salt thereof is from about 2 nM / kg to about 250 nM / kg of the patient's body weight.

[0350] 100. The method of any of paragraphs 92 to 99, wherein the second dose of the compound or a pharma- ceutically acceptable salt thereof is about 5 nM / kg to about 40 nM / kg of the patient's body weight.

[0351] 101. The method of any of paragraphs 92 to 99, wherein the second dose of the compound or a pharma- ceutically acceptable salt thereof is about 40 nM / kg to about 150 nM / kg of the patient's body weight.

[0352] 102. The method of any of paragraphs 73 to 101, further comprising administering a third dose of the compound or a pharma- ceutically acceptable salt thereof, wherein the third dose of the compound or a pharma- ceutically acceptable salt thereof is the same as the second dose of the compound or a pharma- ceutically acceptable salt thereof.

[0353] 103. The method of paragraph 102, further comprising administering a fourth dose of the compound or a pharma- ceutically acceptable salt thereof, wherein the fourth dose of the compound or a pharma- ceutically acceptable salt thereof is the same as the second dose of the compound or a pharma- ceutically acceptable salt thereof or the third dose of the compound or a pharma- ceutically acceptable salt thereof.

[0354] 104. The method of any of paragraphs 73 to 103, wherein a dose of the compound or a pharma- ceutically acceptable salt thereof administered after the first dose of the compound or a pharma- ceutically acceptable salt thereof maintains the inhibition of cancer growth associated with the first dose of the compound or a pharma- ceutically acceptable salt thereof.

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

[0356] 106. The method of any of paragraphs 73 to 105, wherein the dose of the compound or a pharma- ceutically acceptable salt thereof administered after the first dose of the compound or a pharma- ceutically acceptable salt thereof is administered once a week.

[0357] 107. The method of any of paragraphs 73 to 105, wherein the dose of the compound or a pharma- ceutically acceptable salt thereof is administered twice weekly.

[0358] 108. The method of any of paragraphs 1 to 107, wherein the CAR further comprises an IgG4 hinge domain, a CD3ζ activation domain, and a 4-1BB costimulatory domain.

[0359] 109. The method of any of paragraphs 1 to 108, wherein the E2 anti-fluorescein antibody fragment is an scFv fragment.

[0360] 110. The method of any of paragraphs 1-109, wherein the CAR protein sequence has at least about 90% identity to SEQ ID NO:2.

[0361] 111. The method of any of paragraphs 1-109, wherein the CAR protein sequence has at least about 95% identity to SEQ ID NO:2.

[0362] 112. The method of any of paragraphs 1-109, wherein the CAR protein sequence has at least about 98% identity to SEQ ID NO:2.

[0363] 113. The method of any of paragraphs 1 to 112, wherein CAR is bound to fluorescein.

[0364] 114. The method of any of paragraphs 1 to 109, wherein the CAR protein sequence has up to about 50 conservative amino acid substitutions, and the CAR is conjugated to fluorescein.

[0365] 115. The method of any of paragraphs 1-109, wherein the CAR is encoded by a polynucleotide having at least about 90% identity to SEQ ID NO:1.

[0366] 116. The method of any of paragraphs 1-109, wherein the CAR is encoded by a polynucleotide having at least about 95% identity to SEQ ID NO:1.

[0367] 117. The method of any of paragraphs 1-109, wherein the CAR is encoded by a polynucleotide having at least about 98% identity to SEQ ID NO:1.

[0368] 118. The method of any of paragraphs 115 to 117, wherein the CAR is bound to fluorescein.

[0369] 119. The method of any of paragraphs 1 to 118, wherein the CAR is encoded by a polynucleotide that hybridizes under high stringency conditions to a polynucleotide having SEQ ID NO:1, and the CAR is bound to fluorescein.

[0370] 120. The method of any of paragraphs 1 to 119, wherein the CAR is encoded by a polynucleotide having SEQ ID NO:1 or a degenerate variant of SEQ ID NO:1.

[0371] 121. The method of any of paragraphs 1 to 120, wherein the ligand is selected from the group consisting of folate, DUPA, NK-1R ligands, CAIX ligands, gamma glutamyl transpeptidase ligands, NKG2D ligands, and CCK2R ligands.

[0372] 122. The method of any of paragraphs 1 to 121, wherein the targeting moiety is fluorescein or a pharma- ceutically acceptable salt thereof.

[0373] 123. The method of any of paragraphs 1 to 122, wherein the linker comprises polyethylene glycol (PEG), polyproline, a hydrophilic amino acid, a sugar, a non-natural peptidoglycan, polyvinylpyrrolidone, Pluronic F-127, or a combination thereof.

[0374] 124. The method of any of paragraphs 1 to 123, wherein the linker comprises PEG.

[0375] 125. A compound or a pharma- ceutically acceptable salt thereof having the formula [ka] wherein B is a small molecule ligand, L is a linker, and T is a targeting moiety, where L is of the formula [ka] and n is an integer of 0 to 200. Item 125. The method of any one of items 1 to 124,

[0376] 126. Cancer: Lung cancer, bone cancer, pancreatic cancer, skin cancer, head cancer, neck cancer, cutaneous melanoma, intraocular melanoma, uterine cancer, ovarian cancer, endometrial cancer, rectal cancer, stomach cancer, colon cancer, breast cancer, triple-negative breast cancer, fallopian tube cancer, endometrioma, 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 126. The method of any of paragraphs 1 to 125, wherein the cancer is selected from the group consisting of hematologic malignancies, lymphocytic lymphoma, myeloid leukemia, myelomonocytic leukemia, hairy cell leukemia, myelomonocytic leukemia, hairy cell leukemia, pleural mesothelioma, bladder cancer, Burkitt's lymphoma, ureteral cancer, kidney cancer, renal cell carcinoma, renal pelvis cancer, neoplasms of the central nervous system (CNS), primary CNS lymphoma, spinal axis tumor, brain stem glioma, pituitary adenoma, and esophagogastric junction adenocarcinoma.

[0377] 127. The method of any of paragraphs 1 to 126, wherein the cancer is a folate receptor-expressing cancer.

[0378] 128. The method of any of paragraphs 1 to 127, wherein the cancer is endometrial cancer.

[0379] 129. The method of any of paragraphs 1 to 127, wherein the cancer is non-small cell lung cancer.

[0380] 130. Any of the methods of paragraphs 1 to 127, wherein the cancer is ovarian cancer.

[0381] 131. Any of the methods of paragraphs 1 to 127, wherein the cancer is triple-negative breast cancer.

[0382] 132. Any of the methods of paragraphs 1 to 127, wherein the cancer is acute myeloid leukemia.

[0383] 133. The method of paragraph 132, wherein the cancer expresses folate receptor-β.

[0384] 134. Any of the methods of paragraphs 1 to 133, wherein multiple doses of the CAR T cell composition are administered.

[0385] 135. Any of the methods of paragraphs 1 to 134, wherein at least two doses of the CAR T cell composition are administered.

[0386] 136. The method of any of paragraphs 1 to 135, wherein the patient is imaged prior to administration of the compound or a pharma- ceutically acceptable salt thereof or prior to administration of the CAR T cell composition.

[0387] 137. The method of any of paragraphs 1 to 136, wherein the compound or a pharma- ceutically acceptable salt thereof is not an antibody and does not include an antibody fragment.

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

[0389] 139. Any of the methods of paragraphs 1 to 138, wherein CAR T cell toxicity against the cancer occurs without causing cytokine release that results in off-target toxicity in the patient.

[0390] 140. Any of the methods of paragraphs 1 to 138, wherein off-target tissue toxicity does not occur in the patient and CAR T cell toxicity occurs against the cancer.

[0391] 141. The method of any of paragraphs 1 to 138, wherein the cancer comprises a tumor, the tumor size in the patient is reduced, and no off-target toxicity occurs.

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

[0393] 143. Any of the methods of paragraphs 1 to 142, wherein weight loss due to CRS is reduced or prevented.

[0394] 144. The method of any of paragraphs 59 to 67, further comprising re-administering the compound or a pharma- ceutically acceptable salt thereof to the patient.

[0395] 145. The method of paragraph 144, wherein subsequent administration of the compound or a pharma- ceutically acceptable salt thereof results in CAR T cell activation and increased cytokine levels in the patient.

[0396] 146. The method of any of paragraphs 1 to 145, wherein the cancer involves a tumor and a complete tumor response is obtained.

[0397] 147. Any of the methods of paragraphs 1 to 146, wherein the CAR T cells have a central memory / effector memory phenotype.

[0398] 148. Any of the methods of paragraphs 1 to 147, wherein the CD8:CD4 ratio in the CAR T cells is about 1:1.

[0399] Therefore, in one embodiment, a method for treating cancer is provided, which includes: i) administering to a patient a compound or a pharma- ceutically 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, wherein the CAR T cell composition comprises a CAR T cell, the CAR T cell comprises a CAR, and the CAR comprises an E2 anti-fluorescein antibody fragment; and iii) administering to the patient a second dose of a CAR T cell composition, wherein the CAR T cell composition comprises a CAR T cell, the CAR T cell comprises a CAR, and the CAR comprises an E2 anti-fluorescein antibody fragment.

[0400] In another embodiment, a method of treating cancer is provided, comprising: i) administering to a patient a compound or a pharma- ceutically 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 comprising a CAR T cell, wherein the CAR T cell comprises a CAR, the CAR comprises an E2 anti-fluorescein antibody fragment, and the CAR T cell composition comprises a mixture of CAR T cells and non-transformed T cells.

[0401] In yet another embodiment, a method of treating cancer is provided, comprising: i) administering to a patient a compound or a pharma- ceutically 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 a CAR T cell, the CAR T cell comprises a CAR, and the CAR comprises an E2 anti-fluorescein antibody fragment; and iii) administering to the patient folate, a conjugate comprising folate without 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, comprising: i) administering to a patient a compound or a pharma- ceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked to a targeting moiety by a linker, and the compound or a pharma- ceutically acceptable salt thereof is administered at a dose of about 10 nM / kg to about 2500 nM / kg of patient body weight; and ii) administering to a patient a CAR T cell composition comprising a CAR T cell, wherein the CAR T cell comprises a CAR, the CAR comprises an E2 anti-fluorescein antibody fragment, and the CAR T cell is at a dose of about 1 million to about 15 million CAR T cells.

[0403] In yet another embodiment, a method of treating cancer is provided, comprising: i) continuously administering a compound to a patient, wherein the compound comprises a small molecule ligand linked to a targeting moiety by a linker; ii) administering a CAR T cell composition comprising a CAR T cell to the patient, wherein the CAR T cell comprises a CAR, and the CAR comprises an E2 anti-fluorescein antibody fragment; and iii) terminating the continuous administration of the compound or a pharma- ceutically acceptable salt thereof to block or prevent cytokine release syndrome in the patient.

[0404] In another illustrative embodiment, a method of treating cancer is provided, comprising: i) administering to a patient a compound or a pharma- ceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked to a targeting moiety by a linker, and wherein the patient is administered at least a first dose and a second dose of the compound or a pharma- ceutically acceptable salt thereof, wherein the first dose and the second dose are different, and the second dose of the compound or a pharma- ceutically 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 pharma- ceutically acceptable salt thereof; and ii) administering to the patient a CAR T cell composition comprising a CAR T cell, wherein the CAR T cell comprises a CAR, and the CAR comprises an E2 anti-fluorescein antibody fragment.

[0405] In another embodiment, a method of treating cancer is provided, comprising: i) administering to a patient a compound or a pharma- ceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked to a targeting moiety by a linker, and the compound or a pharma- ceutically acceptable salt thereof is administered to the patient once a week; and ii) administering to the patient a CAR T cell composition comprising a CAR T cell, wherein the CAR T cell comprises a CAR, and the CAR comprises an E2 anti-fluorescein antibody fragment.

[0406] In yet another embodiment, a method for treating cancer is provided, which comprises: i) administering a first dose of a compound or a pharmaceutically acceptable salt to a patient, wherein the compound comprises a small molecule ligand linked to a targeting moiety by a linker; ii) administering at least a second dose of a compound or a pharmaceutically acceptable salt to a patient, wherein the second dose of the compound or a pharmaceutically acceptable salt is at least about 50% lower than the amount of the first dose of the compound or a pharmaceutically acceptable salt; and iii) administering a dose of a CAR T cell composition comprising a CAR T cell to a patient, wherein the CAR T cell comprises a CAR, and the CAR comprises an E2 anti-fluorescein antibody fragment.

[0407] Thus, various embodiments are described in the preceding paragraphs and section listings above, and all applicable embodiments described in this Detailed Description of Illustrative Embodiments, Summary section, Examples, and Claims apply to these embodiments.

[0408] A "patient" as used herein may be a human, or in the case of veterinary applications, the patient may be a laboratory animal, an agricultural animal, a domestic animal, or a wild animal. In various embodiments, the patient may be a laboratory animal, such as a rodent (e.g., mouse, rat, hamster, etc.), rabbit, monkey, chimpanzee, or a domestic animal, such as a dog, cat, or rabbit, or 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 may be selected from carcinoma, sarcoma, osteosarcoma, lymphoma, melanoma, mesothelioma, nasopharyngeal carcinoma, leukemia, adenocarcinoma, or myeloma. In other embodiments, the cancer is lung cancer, bone cancer, pancreatic cancer, skin cancer, head cancer, neck cancer, cutaneous melanoma, intraocular melanoma, uterine cancer, ovarian cancer, endometrial cancer, rectal cancer, gastric cancer, colon cancer, breast cancer, triple-negative breast cancer, fallopian tube cancer, endometrioma, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, non-small cell lung cancer, adrenal cancer, soft tissue sarcoma, osteosarcoma, urethral cancer. , prostate cancer, chronic leukemia, acute leukemia including acute myeloid leukemia, lymphocytic lymphoma, myeloid leukemia, myelomonocytic leukemia, hairy cell leukemia, pleural mesothelioma, bladder cancer, Burkitt's lymphoma, ureteral cancer, kidney cancer, renal cell carcinoma, renal pelvis cancer, neoplasms of the central nervous system (CNS), primary CNS lymphoma, spinal axis tumors, brain stem glioma, pituitary adenoma and esophagogastric junction adenocarcinoma.

[0410] In some of these embodiments, the cancer is a folate receptor expressing cancer. In other embodiments, the cancer is a folate receptor alpha expressing cancer. In yet other embodiments, the cancer is a folate receptor beta expressing cancer. In some of these embodiments, the cancer is endometrial cancer, non-small cell lung cancer, ovarian cancer, or triple negative breast cancer. In other embodiments, the cancer being treated is a tumor. In other embodiments, the cancer is malignant. In other embodiments, the cancer is acute myeloid leukemia. In yet other embodiments, the cancer is acute myeloid leukemia and the cancer expresses folate receptor-beta. In yet other embodiments, the cancer is acute myeloid leukemia and the CAR-T cells have a central memory / effector memory phenotype. In yet other embodiments, the CD8:CD4 ratio in 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 other embodiments, where the cancer is acute myeloid leukemia or other cancers, CAR T cells that bind to the tumor may have increased CD25 expression relative to CAR T cells that do not bind to the tumor.

[0411] In certain embodiments, the "small molecule ligand" is a ligand that binds to folate, DUPA (a ligand bound by PSMA-positive human prostate cancer cells and other cancer cell types), NK-1R ligands (e.g., a receptor for NK-1R, a ligand found in colon and pancreatic cancers), CAIX ligands (e.g., a receptor for CAIX ligand found in renal, ovarian, vulvar and breast cancers), gamma glutamyl transpeptidase ligands (e.g., a ligand found in ovarian cancer, colon cancer, liver cancer, astrocytic glioma, melanoma and leukemia). The ligand may be a small molecule ligand that specifically binds to a cancer cell type (i.e., a receptor for an NKG2D ligand found in cancers of the lung, colon, kidney, prostate, and T and B cell lymphomas), an NKG2D ligand (e.g., a receptor for an NKG2D ligand found in cancers of the lung, colon, kidney, prostate, and T and B cell lymphomas), or a CCK2R ligand (a receptor for a CCK2R ligand found in cancers of the thyroid, lung, pancreas, ovary, brain, stomach, gastrointestinal stromal, and colon, among others), 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 may be overexpressed in cancer compared to normal tissue).

[0412] In certain embodiments, the small molecule ligand can have a mass of less than about 10,000 daltons, less than about 9000 daltons, less than about 8,000 daltons, less than about 7000 daltons, less than about 6000 daltons, less than about 5000 daltons, less than about 4500 daltons, less than about 4000 daltons, less than about 3500 daltons, less than about 3000 daltons, less than about 2500 daltons, less than about 2000 daltons, less than about 1500 daltons, less than about 1000 daltons or less than about 500 daltons. In other embodiments, the small molecule ligand can have a mass of about 1 to about 10,000 daltons, about 1 to about 9000 daltons, about 1 to about 8,000 daltons, about 1 to about 7000 daltons, about 1 to about 6000 daltons, about 1 to about 5000 daltons, about 1 to about 4500 daltons, about 1 to about 4000 daltons, about 1 to about 3500 daltons, about 1 to about 3000 daltons, about 1 to about 2500 daltons, about 1 to about 2000 daltons, about 1 to about 1500 daltons, about 1 to about 1000 daltons, or about 1 to about 500 daltons.

[0413] In certain embodiments, the DUPA derivative can be a ligand of a small molecule ligand linked to a targeting moiety, and the DUPA derivatives are described in WO2015 / 057852, which is incorporated herein by reference.

[0414] In some embodiments, the small molecule ligand in the "small molecule ligand attached to the linker" is folate. In various embodiments, folate can be folic acid, folic acid analogs or other folate receptor binding molecules. In various embodiments, folic acid analogs that can be used include folinic acid, pteropolyglutamic acid and folate receptor binding pteridines such as tetrahydropterin, dihydrofolic acid, tetrahydrofolic acid and their deaza and dideaza analogs. The terms "deaza" and "dideaza" analogs refer to art-recognized analogs that have carbon atoms replacing one or two nitrogen atoms of the naturally occurring folic acid structure. For example, deaza analogs include 1-deaza, 3-deaza, 5-deaza, 8-deaza and 10-deaza analogs. Dideaza analogs include, for example, 1,5-dideaza, 5,10-dideaza, 8,10-dideaza and 5,8-dideaza analogs. The folate analogs are commonly referred to as "folates," reflecting their ability to bind to the folate receptor. Other folate receptor binding analogs include aminopterin, amethopterin (methotrexate), N10-methylfolate, 2-deamino-hydroxyfolate, deaza analogs such as 1-deazamethopterin or 3-deazamethopterin, and 3',5'-dichloro-4-amino-4-deoxy-N10-methylpteroylglutamic acid (dichloromethotrexate).

[0415] In other embodiments, the small molecule ligand described as a "small molecule ligand attached to a linker" has the formula [ka] [In the formula, X 1 and Y 1 each independently represents halo, R 2 , OR 2 , S.R. 3 and N.R.4 R 5 selected from the group consisting of; U, V and W are each -(R 6a )C=, -N=, -(R 6a )C(R 7a )- and -N(R 4a )-; Q is selected from the group consisting of C and CH; T is selected from the group consisting of S, O, N, and -C=C-; X 2 and X 3 are each independently oxygen, sulfur, -C(Z)-, -C(Z)O-, -OC(Z)-, -N(R 4b )-, -C(Z)N(R 4b )-, -N(R 4b )C(Z)-, -OC(Z)N(R 4b )-, -N(R 4b )C(Z)O-, -N(R 4b )C(Z)N(R 5b )-, -S(O)-, -S(O)2-, -N(R 4a )S(O)2-, -C(R 6b )(R 7b )-, -N(C≡CH)-, -N(CH2C≡CH)-, C1-C 12 Alkylene and C1-C 12 alkyleneoxy, where Z is oxygen or sulfur; R 1 are hydrogen, halo, C1-C 12 Alkyl and C1-C 12 alkoxy; R 2 , R 3 , R 4 , R 4a , R 4b , R 5 , R 5b , R 6b and R 7b each independently represents hydrogen, halo, C1-C 12 Alkyl, C1-C 12 Alkoxy, C1-C 12 Alkanoyl, C1-C 12 Alkenyl, C1-C 12Alkynyl, (C1-C 12 alkoxy)carbonyl and (C1-C 12 alkylamino)carbonyl; R 6 and R 7 each independently represents hydrogen, halo, C1-C 12 Alkyl and C1-C 12 alkoxy; or R 6 and R 7 together to form a carbonyl group; R 6a and R 7a each independently represents hydrogen, halo, C1-C 12 Alkyl and C1-C 12 alkoxy; or R 6a and R 7a together to form a carbonyl group; p, r, s and t are each independently 0 or 1; and * represents an optional covalent bond to the remainder of the conjugate. may have:

[0416] In some embodiments, the "targeting moiety" that comprises the E2 anti-fluorescein antibody fragment and is bound to the CAR expressed by the CAR T cell can be selected from, for example, fluorescein, fluorescein isothiocyanate (FITC) and NHS-fluorescein. The characteristics of the targeting moiety are limited only by the fact that it must be specifically recognized and bound by the CAR that comprises the E2 anti-fluorescein antibody fragment and has a relatively low molecular weight. In various embodiments, an example of a targeting moiety is a hapten that comprises a small molecular weight organic molecule.

[0417] In one illustrative embodiment, the targeting moiety has the following exemplary structure: [ka] wherein X is oxygen, nitrogen or sulfur, X is attached to a linker L; Y is OR a , N.R.a 2 or NR a 3 + and Y' is O, NR a or NR a 2 + where each R is independently selected at each occurrence from H, fluoro, sulfonic acid, sulfonate, and salts thereof; and R a is hydrogen or alkyl. may have:

[0418] In certain illustrative embodiments, the linker may comprise polyethylene glycol (PEG), polyproline, a hydrophilic amino acid, a sugar, a non-natural peptidoglycan, polyvinylpyrrolidone, Pluronic F-127, or a combination thereof.

[0419] In other illustrative embodiments, the linker of the compounds described herein or pharma- ceutically acceptable salts thereof can be a direct bond (e.g., reaction of an isothiocyanate group of FITC with a free amine group of a small molecule ligand) or the bond can be via an intermediate linker. In certain embodiments, the intermediate linker, if present, can be any biocompatible linker known in the art, such as a bivalent linker. In certain illustrative embodiments, the bivalent linker can include about 1 to about 30 carbon atoms. In other illustrative embodiments, the bivalent linker can include about 2 to about 20 carbon atoms. In other embodiments, low molecular weight bivalent linkers (i.e., those having an approximate molecular weight of about 30 to about 300 Daltons) are used. In other embodiments, suitable linker lengths include, but are not limited to, linkers having 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 or more atoms.

[0420] In various embodiments, the small molecule ligand attached to the targeting moiety has the formula [ka] wherein B is a small molecule ligand, L is a linker, and T is a targeting moiety, where L is of the formula [ka] and n is an integer of 0 to 200. In other embodiments, n may be 0 to 150, 0 to 110, 0 to 100, 0 to 90, 0 to 80, 0 to 70, 0 to 60, 0 to 50, 0 to 40, 0 to 30, 0 to 20, 0 to 15, 0 to 14, 0 to 13, 0 to 12, 0 to 11, 0 to 10, 0 to 9, 0 to 8, 0 to 7, 0 to 6, 0 to 5, 0 to 4, 0 to 3, 0 to 2, 0~1, 15~16, 15~17, 15~18, 15~19, 15~20, 15~21, 15~22, 15~23, 15~24, 15~25, 15~26, 15~27, 15~28, 15~29, 15~30, 15~31, 15~32, 15~33, 15~34, 15~35, 15~36, 15~ 37, 15 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, or 15 to 150, or n may be an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, It can be 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 50, 60, 70, 80, 90, 100, 108, 110, 120, 130, 140 or 150.

[0421] In other embodiments, the linker may be a bivalent linker that may include one or more spacers. Illustrative spacers are shown in the following table. The following non-limiting illustrative spacers are listed, where * indicates the point of attachment to a small molecule ligand or targeting moiety or another bivalent linker moiety. [Table 1]

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

[0423] In other embodiments, the compound or a pharma- ceutically acceptable salt thereof is not an antibody and does not include an antibody fragment, hi yet other embodiments, the targeting moiety does not include a peptide epitope.

[0424] In one illustrative embodiment, the small molecule ligand (bridge) linked to the targeting moiety by the linker comprises fluorescein isothiocyanate (FITC) linked to the small molecule ligand. In some embodiments, the cancer can overexpress a receptor for the small molecule ligand. In other embodiments, for example, cytotoxic T cells or other types of T cells can be transformed to express a CAR comprising an E2 anti-fluorescein antibody fragment. In this embodiment, the CAR can target FITC, and as a result of the binding of the small molecule ligand to the cancer, the cancer is decorated with FITC molecules. Therefore, toxicity to normal, non-target cells can be avoided. In this embodiment, when the E2 anti-fluorescein antibody fragment CAR-expressing T cells bind to FITC, the CAR T cells are activated and the cancer is ameliorated.

[0425] The "pharmaceutical acceptable salt" of the small molecule ligand linked to the targeting moiety by the linker is considered. As used herein, the term "pharmaceutical acceptable salt" refers to a salt whose counter ion can be used in medicine. In various embodiments, such salts include, but are not limited to, 1) the acid addition salts that can be obtained by reacting the free base parent compound with inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, phosphoric acid, sulfuric acid and perchloric acid, or organic acids such as acetic acid, oxalic acid, (D) or (L) malic acid, maleic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, tartaric acid, citric acid, succinic acid or malonic acid; or 2) the salts that are formed when the acidic protons present in the parent compound are replaced with metal ions, for example, alkali metal ions, alkaline earth ions or aluminum ions; or the coordination with organic bases such as ethanolamine, diethanolamine, triethanolamine, trimethamine, N-methylglucamine. Pharmaceutically acceptable salts are well known to those of skill in the art, and all such pharma-ceutically acceptable salts are contemplated in connection with the embodiments described herein.

[0426] In various embodiments, suitable acid addition salts are formed from acids which form non-toxic salts. Illustrative examples include acetate, aspartate, benzoate, besylate, bicarbonate / carbonate, bisulfate / sulfate, borate, camsylate, citrate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hybenzate, hydrochloride / chloride, hydrobromide / bromide, hydroiodide / iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methylsulfate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate / hydrogenphosphate / dihydrogenphosphate, saccharate, stearate, succinate, tartrate, tosylate, and trifluoroacetate salts.

[0427] In various embodiments, suitable base salts are formed from bases which form non-toxic salts. Illustrative examples include arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine and zinc salts. Hemisalts of acids and bases can also be formed, such as hemisulfate and hemicalcium salts.

[0428] In some illustrative embodiments, the compounds described herein or pharmaceutical salts thereof may contain one or more chiral centers or may otherwise be capable of existing as multiple stereoisomers. Thus, various embodiments may include pure stereoisomers as well as stereoisomeric mixtures, such as enantiomers, diastereomers, and enantiomerically or diastereomerically enriched mixtures. In some embodiments, the compounds described herein or pharmaceutical acceptable salts thereof may exist as geometric isomers. Thus, various embodiments may include pure geometric isomers or geometric isomeric mixtures.

[0429] In certain embodiments, the compounds described herein or pharma- ceutically acceptable salts thereof can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to the unsolvated forms and are encompassed within the scope of the present invention.

[0430] 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, including 1) a recognition region (e.g., a single-chain fragment variable (scFv) region, Fab fragment, etc., of the E2 anti-fluorescein antibody) that recognizes and binds to the targeting moiety with specificity, 2) a costimulatory domain that enhances T lymphocyte proliferation and survival, and 3) an activation signaling domain that generates a T lymphocyte activation signal.

[0431] In various embodiments, as non-limiting examples, scFv regions of antibodies that bind fluorescein, fluorescein isothiocyanate (FITC), and NHS-fluorescein can be used. In illustrative, non-limiting embodiments, the scFv regions can be prepared from (i) E2 antibodies known in the art that bind targeting moieties, and (iii) sequence variants derived from the scFv regions of such antibodies, such as scFv regions 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 the amino acid sequence of the derived scFv region.

[0432] In some embodiments, the costimulatory domain acts to enhance the proliferation and survival of cytotoxic T lymphocytes by binding to the targeting portion of the CAR. Suitable costimulatory domains include, but are not limited to, CD28, CD137 (4-1BB), tumor necrosis factor (TNF) receptor family members, CD134 (OX40), TNFR superfamily members of receptors CD27, CD30, CD150, DAP10, NKG2D and CD278 (ICOS), CD28 superfamily costimulatory molecules expressed on activated T cells, or combinations thereof. In one illustrative embodiment, the costimulatory domain of the modified CAR comprises CD28 and CD137. Those skilled in the art will understand that sequence variants of these costimulatory domains can be used without adversely affecting the present invention, when the variants have the same or similar activity as the modeled domains. In various embodiments, such variants may have at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or at least about 99.5% sequence identity to the amino acid sequence of the domain from which it is derived.

[0433] In one illustrative embodiment, the activation signaling domain serves to activate T lymphocytes (e.g., cytotoxic T lymphocytes) by binding to the targeting portion of the CAR. In various embodiments, suitable activation signaling domains include T cell CD3 zeta chain, CD3 delta receptor protein, mbl receptor protein, B29 receptor protein, and Fc receptor gamma. Those skilled in the art will appreciate that sequence variants of these activation signaling domains can be used when the variants have the same or similar activity as the modeled domain. In various embodiments, the variants have at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity with the amino acid sequence of the domain from which it is derived.

[0434] In some embodiments, the construct encoding the CAR comprising the E2 anti-fluorescein antibody fragment is produced using genetic engineering techniques. Such techniques are described in detail in Sambrook et al., "Molecular Cloning: A Laboratory Manual", 3rd Edition, Cold Spring Harbor Laboratory Press, (2001) (incorporated herein by reference) and Green and Sambrook, "Molecular Cloning: A Laboratory Manual", 4th Edition, Cold Spring Harbor Labor (incorporated herein by reference).

[0435] As an example, a plasmid or viral expression vector (e.g., lentiviral vector, retroviral vector, sleeping beauty and piggyback (transposon / transposase system including non-viral mediated CAR gene delivery system)) can be prepared that contains a recognition region, one or more costimulatory domains and an activation signaling domain in frame and encodes a fusion protein linked in a 5' to 3' direction. In other embodiments, other arrangements are permissible, including a recognition region, an activation signaling domain and one or more costimulatory domains. In some embodiments, the placement of the recognition region on the fusion protein is such that it is generally presented to the extracellular region. In certain embodiments, a CAR comprising an E2 anti-fluorescein antibody fragment may include additional elements such as a signal peptide (e.g., CD8α signal peptide) to ensure proper export of the fusion protein to the cell surface, a transmembrane domain (e.g., CD8α transmembrane domain, CD28 transmembrane domain, or CD3ζ transmembrane domain) to ensure that the fusion protein is maintained as an integral membrane protein, and a hinge domain (e.g., CD8α hinge or IgG4 hinge) that provides flexibility to the recognition region and allows strong binding to the targeting moiety, and any other suitable domain.

[0436] A diagram of an example of a CAR construct in which the expressed CAR comprises an E2 anti-fluorescein antibody fragment is shown in FIG. 1, where 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, a costimulatory domain is CD137 (4-1BB), and an activation signaling domain is CD3ζ. The CAR may further comprise suitable domains. An example of a nucleic acid sequence of a CAR insert is provided as SEQ ID NO: 1, and an example of an encoded amino acid sequence is provided as SEQ ID NO: 2. As used herein, "SEQ ID NO: 1" refers to the sequence beginning with the underlined "agc" codon and ending with the underlined "ggc" codon. This portion of the long sequence encodes the CAR inserted into the T cell membrane. Other portions of the long sequence encode coding sequences such as a signal peptide, an EGFRt domain, etc., which are not part of the CAR inserted into the membrane and function as a chimeric antigen receptor. As used herein, "SEQ ID NO: 2" refers to the sequence beginning with the underlined "S" and ending with the underlined "G". This part of the long sequence is the amino acid sequence of the CAR inserted into the T cell membrane. The other part of the long sequence includes amino acid sequences such as signal peptide, EGFRt domain, etc., that are not part of the CAR inserted into the membrane and function as chimeric antigen receptor. In yet another embodiment, SEQ ID NO: 2 can comprise or consist of a humanized or human amino acid sequence. SEQ ID NOs: 1 and 2 are as above. The start and stop codons of the long nucleic acid sequence are underlined, and the long sequence is an example of a sequence that can be used to transduce T cells used in the methods described herein.

[0437] SEQ ID NO:1 (E2 anti-fluorescein antibody fragment CAR nucleic acid sequence (insert)) [ka]

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

[0439] In one embodiment, the CAR expressing E2 anti-fluorescein antibody fragment comprises a recognition region that is the single-chain fragment variable (scFv) region of E2 anti-fluorescein antibody, a costimulatory domain that is CD137 (4-1BB) and an activation signaling domain that is T cell CD3 zeta chain. In other embodiments, the CAR can further comprise any additional suitable domain. 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). E2 anti-fluorescein antibodies are described, for example, in Vaughan, et al., Nature Biotechnol. Vol. 14(3), pp. 309-314, 1996, which is incorporated herein by reference. In certain embodiments, a CAR expressing an 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 may further comprise an IgG4 hinge domain, a CD3 zeta activation domain and / or a 4-1BB costimulatory domain and other suitable domains. In yet other embodiments, the CAR may be encoded by a polynucleotide having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identity to SEQ ID NO: 1. In other illustrative embodiments, the CAR may be encoded by a polynucleotide that hybridizes under high stringency conditions to a polynucleotide having SEQ ID NO: 1. In yet other embodiments, the CAR may be encoded by a polynucleotide having SEQ ID NO: 1 or a degenerate variant of SEQ ID NO: 1. In other embodiments, the CAR protein sequence may have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identity to SEQ ID NO: 2. In yet other embodiments, the CAR protein sequence may have up to about 50 conservative amino acid substitutions. In any of the embodiments described herein, the CAR is conjugated to fluorescein.

[0442] In some embodiments, T lymphocytes (e.g., cytotoxic T lymphocytes) can be genetically modified to express CAR constructs expressing E2 anti-fluorescein antibody fragments by transfecting a population of T lymphocytes with an expression vector encoding a CAR construct expressing E2 anti-fluorescein antibody fragments.Suitable methods for preparing a transduced population of T lymphocytes expressing E2 anti-fluorescein antibody fragments are well known to those skilled in the art and are described in Sambrook et al., "Molecular Cloning: A Laboratory Manual", 3rd Edition, Cold Spring Harbor Laboratory Press, (2001) (incorporated herein by reference) and Green and Sambrook, "Molecular Cloning: A Laboratory Manual", 4th Edition, Cold Spring Harbor Labor (incorporated herein by reference).

[0443] In certain embodiments, CAR T cells comprising a nucleic acid of SEQ ID NO: 1 may be used as described herein. In other embodiments, CAR T cells comprising a polypeptide of SEQ ID NO: 2 may be used as described herein. In other illustrative embodiments, a nucleic acid (e.g., an isolated nucleic acid) comprising SEQ ID NO: 1 and encoding a chimeric antigen receptor may be used to prepare a CAR T cell for use as described herein. In yet other embodiments, a chimeric antigen receptor polypeptide comprising SEQ ID NO: 2 may be used to prepare a CAR T cell for use as described herein. In other embodiments, a vector comprising SEQ ID NO: 1 may be used to prepare a CAR T cell for use as described herein. In other embodiments, a lentiviral vector comprising SEQ ID NO: 1 may be used to prepare a CAR T cell for use as described herein. In yet other embodiments, SEQ ID NO: 2 may comprise or consist of a humanized or human amino acid sequence.

[0444] In each of these embodiments, variant nucleic acid sequences or amino acid sequences having at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity to SEQ ID NO:1 are contemplated. In other embodiments, 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, so long as the variant sequence encodes the polypeptide of SEQ ID NO:2. In other embodiments, the nucleic acid sequence or amino acid sequence may be a variant nucleic acid or amino acid sequence that has at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or at least about 99.5% sequence identity with SEQ ID NO:1 over a length of 200 nucleic acids or with SEQ ID NO:2 over a length of 200 amino acids. In some embodiments, the % identity or similarity determination between sequences can be performed, for example, using the GAP program (Genetics Computer Group, software; currently available through Accelrys at http: / / www.accelrys.com), and alignment can be performed, for example, using the ClustalW algorithm (VNTI software, InforMax Inc.). A sequence database can be searched using the nucleic acid or amino acid sequence of interest. The algorithm for database search is generally based on BLAST software (Altschul et al., 1990). In certain embodiments, the percent identity can be determined along the entire length of the nucleic acid or amino acid sequence.

[0445] Also within the scope of the present invention, a nucleic acid complementary to the nucleic acid set forth in SEQ ID NO: 1 may be used to prepare CAR T cells for use as described herein, and those that hybridize with the nucleic acid set forth in SEQ ID NO: 1 or its complement under highly stringent conditions may be used. According to the present invention, "highly stringent conditions" refers to hybridization at 5xSSPE and 50% formamide at 65°C and washing at 0.5xSSPE at 65°C. High stringency, low stringency and moderately stringent hybridization conditions are described in Sambrook et al., "Molecular Cloning: A Laboratory Manual", 3rd Edition, Cold Spring Harbor Laboratory Press, (2001) (incorporated herein by reference) and Green and Sambrook, "Molecular Cloning: A Laboratory Manual", 4th Edition, Cold Spring Harbor Labor (incorporated herein by reference). In one illustrative embodiment, hybridization occurs along the entire length of the nucleic acid.

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

[0447] In other embodiments, the CAR protein sequence may have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 2. In yet other embodiments, the CAR protein sequence may have up to about 50 conservative amino acid substitutions. In certain embodiments, the CAR protein sequence expressing an E2 anti-fluorescein antibody fragment may have up to about 5 conservative amino acid substitutions, up to about 10 conservative amino acid substitutions, up to about 15 conservative amino acid substitutions, up to about 20 conservative amino acid substitutions, up to about 25 conservative amino acid substitutions, up to about 30 conservative amino acid substitutions, up to about 35 conservative amino acid substitutions, up to about 40 conservative amino acid substitutions, up to about 45 conservative amino acid substitutions, up to about 50 conservative amino acid substitutions, up to about 55 conservative amino acid substitutions, up to about 60 conservative amino acid substitutions, up to about 65 conservative amino acid substitutions, up to about 70 conservative amino acid substitutions or up to about 75 conservative amino acid substitutions. As is well known to those skilled in the art, modification of any of the non-critical amino acids of a polypeptide by conservative amino acid substitution does not significantly alter the activity of the polypeptide, since the side chain of the replaced amino acid is capable of forming similar bonds and contacts with the side chain of the replaced amino acid. In any of the embodiments described herein, the CAR is conjugated to fluorescein.

[0448] In an illustrative embodiment, non-conservative substitutions are possible as long as they do not unduly affect the fluorescein binding activity of the E2 anti-fluorescein antibody fragment polypeptide. As is well known in the art, a "conservative substitution" of an amino acid or a "conservative substitution variant" of a polypeptide is 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 bulkiness of the side chain or any one or more of these characteristics. In an embodiment, a conservative amino acid substitution can be made with an amino acid analog. By way of illustration, the well-known term "hydrophilic residue" refers to serine or threonine. "Hydrophobic residue" refers to leucine, isoleucine, phenylalanine, valine, or alanine, etc. "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, etc. An exemplary list of conservative amino acid substitutions is shown in Table 1. [Table 2]

[0449] In some embodiments, the T lymphocytes used in the methods described herein (e.g., the cytotoxic T lymphocytes or non-transformed T cells used in the preparation of CAR T cells expressing E2 anti-fluorescein antibody fragments) can be autologous cells, but xenogeneic cells can also be used, such as when the patient being treated is undergoing high-dose chemotherapy or radiation therapy to destroy the patient's immune system.In some embodiments, allogeneic cells can be used.

[0450] In some embodiments, T lymphocytes can be obtained from a patient by means well known in the art. For example, T cells (e.g., cytotoxic T cells or non-transformed T cells) can be obtained by drawing peripheral blood from a patient, subjecting the blood to Ficoll density gradient centrifugation, and then isolating a T cell population from the peripheral blood using a negative T cell isolation kit (e.g., EasySep TMA population of T lymphocytes (e.g., cytotoxic T cells or non-transformed T cells) need not be pure, but may include other cells, such as other types of T cells (e.g., in the case of cytotoxic T cells), monocytes, macrophages, natural killer cells, and B cells. In some embodiments, the harvested population may include at least about 90% of the selected cell type, at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the selected cell type.

[0451] In certain embodiments, after obtaining T lymphocytes (e.g., cytotoxic T cells for use in preparing CAR T cells expressing E2 anti-fluorescein antibody fragments), 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 to components of the culture medium. For example, the culture conditions can be free of bovine serum products such as bovine serum albumin. In certain illustrative embodiments, activation can be achieved by introducing known activators into the culture medium, e.g., anti-CD3 antibodies in the case of cytotoxic T cells. Other suitable activators include anti-CD28 antibodies. In certain embodiments, the population of lymphocytes can be cultured under conditions that promote activation for about 1 to about 4 days. In certain embodiments, the appropriate level of activation can be determined by cell size, proliferation rate, or activation markers determined by flow cytometry.

[0452] In one illustrative embodiment, after a population of T lymphocytes (e.g., cytotoxic T lymphocytes used to prepare CAR T cells expressing an E2 anti-fluorescein antibody fragment) have been cultured 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 various embodiments are described above. In one embodiment, after transfection, the cells can be administered immediately to a patient, or the cells can be cultured for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or more days, or about 5 to about 12 days, about 6 to about 13 days, about 7 to about 14 days, or about 8 to about 15 days, e.g., to allow the cells time to recover from the transfection. In some embodiments, suitable culture conditions can be similar to the conditions under which the cells were cultured to activate, with or without the agent used to promote activation.

[0453] Thus, as noted above, in certain illustrative embodiments, the methods of treatment described herein may further include: 1) obtaining a population of autologous or heterologous T lymphocytes (e.g., the cytotoxic T lymphocytes used in the preparation of 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 the E2 anti-fluorescein antibody fragment to form a CAR T cell expressing the E2 anti-fluorescein antibody fragment.

[0454] In an illustrative embodiment, when the cells are transfected and activated, a composition comprising CAR T cells (wherein the CAR T cells comprise a CAR comprising an E2 anti-fluorescein antibody fragment) can be prepared with or without non-transformed T cells and administered to a patient. In an embodiment, a culture 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 the non-transformed T cells. In other embodiments, tissue culture conditions generally used by those skilled in the art to avoid contamination with bacteria, fungi and mycoplasma can be used. In an exemplary embodiment, the cells (e.g., the CAR T cells expressing the E2 anti-fluorescein antibody fragment and / or the non-transformed T cells) are pelleted, washed and resuspended in a pharma- ceutically acceptable carrier or diluent prior to administration to a patient. Examples of compositions comprising CAR-expressing T lymphocytes (e.g., cytotoxic T lymphocytes) or non-transformed T cells expressing E2 anti-fluorescein antibody fragments include compositions comprising cells in sterile 290 mOsm saline, insoluble freezing medium (Plasma-Lyte A, dextrose, sodium chloride injection, human serum albumin and DMSO), 0.9% NaCl with 2% human serum albumin or any other sterile 290 mOsm insoluble substance. Alternatively, in other embodiments, depending on the characteristics of the culture medium, CAR T cells expressing E2 anti-fluorescein antibody fragments or non-transformed T cells in culture medium can be administered as a composition or can be concentrated and resuspended in culture medium before administration. In various embodiments, CAR T cell compositions with or without non-transformed T cells (wherein the CAR comprises E2 anti-fluorescein antibody fragments) can be administered to patients by any suitable means, such as parenteral administration, e.g., intradermal, subcutaneous, intramuscular, intraperitoneal, intravenous or intrathecal.

[0455] In certain embodiments, the total number of CAR T cells expressing an E2 anti-fluorescein antibody fragment and the concentration of cells in the composition administered to the patient will vary depending on a number of factors, including the type of T lymphocyte (e.g., cytotoxic T lymphocyte) used, the binding specificity of the CAR comprising the E2 anti-fluorescein antibody fragment, the characteristics of the targeting moiety and small molecule ligand, the characteristics of the cancer, the location of the cancer in the patient, the means used to administer the composition to the patient, and the health, age, and weight of the patient being treated. In various embodiments, compositions comprising suitable transduced CAR T cells expressing an E2 anti-fluorescein antibody fragment include those having a volume of about 0.1 ml to about 200 ml and about 0.1 ml to about 125 ml.

[0456] In various embodiments, the transduced CAR T cells (wherein the CAR T cells comprise a CAR comprising an E2 anti-fluorescein antibody fragment) administered to the patient are about 1×10 5 ~Approx. 1×10 15 or 1×10 6 ~Approx. 1×10 15 In various embodiments, the CAR T cells may comprise about 1×10 5 ~Approx. 1×10 10 , about 1×10 6 ~Approx. 1×10 10 , about 1×10 6 ~Approx. 1×10 9 , about 1×10 6 ~Approx. 1×10 8 , about 1×10 6 ~about 2×10 7 , about 1×10 6 ~Approx. 3×10 7 , about 1×10 6 ~Approx. 1.5×10 7 , about 1×10 6 ~Approx. 1×10 7 , about 1×10 6 ~Approx. 9×10 6 , about 1×10 6 ~Approx. 8×10 6 , about 1×10 6 ~Approx. 7×10 6 , about 1×106 ~about 6×10 6 , about 1×10 6 ~Approx. 5×10 6 , about 1×10 6 ~Approx. 4×10 6 , about 1×10 6 ~Approx. 3×10 6 , about 1×10 6 ~Approx. 2×10 6 , about 2×10 6 ~about 6×10 6 , about 2×10 6 ~Approx. 5×10 6 , about 3×10 6 ~about 6×10 6 , about 4×10 6 ~about 6×10 6 , about 4×10 6 ~Approx. 1×10 7 , about 1×10 6 ~Approx. 1×10 7 , about 1×10 6 ~Approx. 1.5×10 7 , about 1×10 6 ~Approx. 2×10 7 , about 0.2×10 6 ~Approx. 1×10 7 , about 0.2×10 6 ~Approx. 1.5×10 7 , about 0.2×10 6 ~Approx. 2×10 7 or about 5 x 10 6 The CAR T cells expressing the E2 anti-fluorescein antibody fragment may be administered to the patient. In some embodiments, in any of the embodiments described herein, the CAR T cells expressing the E2 anti-fluorescein antibody fragment may be administered to the patient in a single dose or multiple doses. In this paragraph or any of the embodiments described herein, the number of CAR T cells may be per kg of patient body weight. In any of the embodiments described herein, the CAR T cells expressing the E2 anti-fluorescein antibody fragment may be administered prior to the compound or a pharma- ceutically acceptable salt thereof. As will be understood, the designation i), ii), and iii), etc., of any of the steps of the methods described herein does not indicate an order unless otherwise indicated.

[0457] In various embodiments described herein, non-transformed T cells can also be administered 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 some embodiments, a mixture of CAR T cells (wherein the CAR T cells comprise a CAR that includes an E2 anti-fluorescein antibody fragment) and non-transformed T cells can be administered once or multiple times, and a combination of doses of pure E2 anti-fluorescein antibody fragment-expressing CAR T cells and a mixture of E2 anti-fluorescein antibody fragment-expressing CAR T cells and non-transformed T cells can be administered (e.g., one dose of E2 anti-fluorescein antibody fragment-expressing CAR T cells, followed by one or more doses of a mixture of E2 anti-fluorescein antibody fragment-expressing CAR T cells and non-transformed T cells). As will be apparent to one of skill in the art from the present disclosure, an "admixture" of CAR T cells expressing an E2 anti-fluorescein antibody fragment as described herein and non-transformed T cells means that the CAR T cells expressing an E2 anti-fluorescein antibody fragment are mixed with non-transformed T cells that have not been exposed to the construct used to express a CAR comprising the E2 anti-fluorescein antibody fragment.

[0458] In other embodiments, the dose of CAR T cells expressing an E2 anti-fluorescein antibody fragment administered to a patient in the CAR T cell composition is selected from the group consisting of about 1 million, about 2 million, about 3 million, about 4 million, about 5 million, about 6 million, about 7 million, about 8 million, about 9 million, about 10 million, about 11 million, about 12 million, about 12.5 million, about 13 million, about 14 million and about 15 million CAR T cells expressing an E2 anti-fluorescein antibody fragment.

[0459] In yet other illustrative embodiments, a CAR T cell composition comprising CAR T cells expressing an E2 anti-fluorescein antibody fragment is administered by injection into the patient's bloodstream, and the CAR T cells expressing the 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 about 4 weeks after injection of the CAR T cell composition, at least 20%, 25%, 30%, 35%, 40% or 50% about 3 weeks after injection of the CAR T cell composition, at least 60%, 65%, 70%, 75% or 80% about 2 weeks after injection of the CAR T cell composition, or at least 85%, 90% or 95% about 1 week after injection of the CAR T cell composition.

[0460] In the embodiments described herein, the CAR T cell composition may comprise CAR T cells expressing the E2 anti-fluorescein antibody fragment without any other cell type, or non-transformed T cells may be administered to the patient in combination with CAR T cells expressing the E2 anti-fluorescein antibody fragment. For embodiments in which the CAR T cell composition is administered multiple times, any one time may comprise CAR T cells expressing the E2 anti-fluorescein antibody fragment or a mixture of CAR T cells expressing the E2 anti-fluorescein antibody fragment and non-transformed T cells. In various embodiments, the non-transformed T cells may be administered in the amounts described herein for CAR T cells expressing the E2 anti-fluorescein antibody fragment.

[0461] In other embodiments, a dose of a CAR T cell composition may comprise a mixture of CAR T cells and non-transformed T cells, both expressing an E2 anti-fluorescein antibody fragment, in a ratio of 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.

[0462] In still other embodiments, a dose of a CAR T cell composition may comprise a mixture of CAR T cells and non-transformed T cells, either expressing an E2 anti-fluorescein antibody fragment, at about 1:1 to about 1:5 CAR T cells expressing an E2 anti-fluorescein antibody fragment to non-transformed T cells, or the CAR T cell composition may 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.

[0463] The compounds described herein or pharma- ceutically acceptable salts thereof or CAR T cell compositions (wherein the compositions include CAR T cells having a CAR comprising an E2 anti-fluorescein antibody fragment) can be administered to a patient using any suitable method known in the art. As described herein, the term "administer" or "administration" includes all means of introducing the compounds or pharma- ceutically acceptable salts thereof or CAR T cell compositions comprising CAR T cells having a CAR comprising an E2 anti-fluorescein antibody fragment into a patient, including, but not limited to, oral, intravenous, intramuscular, subcutaneous, transdermal, etc. In some embodiments, the compounds described herein or pharma- ceutically acceptable salts thereof can be administered in unit dosage forms and / or formulations containing conventional non-toxic pharma- ceutically acceptable carriers, adjuvants, and vehicles.

[0464] In some embodiments, the compound described herein or a pharma- ceutically acceptable salt thereof or a CAR T cell composition (wherein the CAR T cell composition comprises a CAR-expressing CAR T cell comprising an E2 anti-fluorescein antibody fragment) can be administered directly into the bloodstream, into a muscle, or into an internal organ. In various embodiments, suitable routes for such parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, epidural, intraventricular, intraurethral, ​​intrasternal, intracranial, intratumoral, intramuscular, and subcutaneous delivery. In some embodiments, means for parenteral administration include needle (including microneedle) injectors, needle-free injectors, and infusion techniques.

[0465] In one illustrative embodiment, the parenteral formulation is generally an aqueous solution that may contain carriers or additives such as salts, carbohydrates, and buffers (preferably at a pH of 3-9), but more preferably may be formulated as a sterile non-aqueous solution or a dry form used in combination with a suitable vehicle such as sterile, pyrogen-free water or sterile saline. In other embodiments, any of the liquid formulations described herein may be applied for parenteral administration as described herein. Formulation under sterile conditions by lyophilization to produce a sterile lyophilized powder for parenteral formulations may be readily accomplished using standard pharmaceutical techniques well known to those skilled in the art. In some embodiments, the solubility of the compound or a pharma-ceutically acceptable salt thereof for use in the formulation of the parenteral formulation may be increased by the use of appropriate formulation techniques, such as the incorporation of a solubility enhancer.

[0466] The amount of the compound or a pharma- ceutically acceptable salt thereof administered to a patient may vary significantly depending on the cancer to be treated, the route of administration and tissue distribution of the compound or a pharma- ceutically acceptable salt thereof. The amount to be administered to a patient may be based on body surface area, body weight and physician evaluation. In various embodiments, the amount administered may be, for example, about 0.05 mg to about 30 mg, 0.05 mg to about 25.0 mg, about 0.05 mg to about 20.0 mg, about 0.05 mg to about 15.0 mg, about 0.05 mg to about 10.0 mg, about 0.05 mg to about 9.0 mg, about 0.05 mg to about 8.0 mg, about 0.05 mg to about 7.0 mg, about 0.05 mg to about 6.0 mg, about 0.05 mg to about 5.0 mg, about 0.05 mg to about The dosage may range from 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 appreciate that dosages may vary within the various ranges above based on the factors listed above and may be at the discretion of the physician.

[0467] In other embodiments, the dose of the compound or a pharma- ceutically acceptable salt thereof is, for example, about 50 nM / kg to about 3000 nM / kg of patient body weight, about 50 nM / kg to about 2000 nM / kg, about 50 nM / kg to about 1000 nM / kg, about 50 nM / kg to about 900 nM / kg, about 50 nM / kg to about 800 nM / kg, about 50 nM / kg to about 700 nM / kg, about 50 nM / kg to about 600 nM / kg, about 50 nM / kg to about 700 nM / kg, about 50 nM / kg to about 800 nM / kg, about 50 nM / kg to about 900 nM / kg, about 50 nM / kg to about 1000 nM / kg, about 50 nM / kg to about 15 ... The range may be from about 100 nM / kg to about 500 nM / kg, about 50 nM / kg to about 400 nM / kg, about 50 nM / kg to about 300 nM / kg, about 50 nM / kg to about 200 nM / kg, about 50 nM / kg to about 100 nM / kg, about 100 nM / kg to about 300 nM / kg, about 100 nM / kg to about 500 nM / kg, about 100 nM / kg to about 1000 nM / kg, or about 100 nM / kg to about 2000 nM / kg of the patient's body weight. In other embodiments, the dose is about 1 nM / kg, about 5 nM / kg, about 10 nM / kg, about 20 nM / kg, about 25 nM / kg, about 30 nM / kg, about 40 nM / kg, about 50 nM / kg, about 60 nM / kg, about 70 nM / kg, about 80 nM / kg, about 90 nM / kg, about 100 nM / kg, about 150 nM / kg, about 200 nM / kg, about 25 The dose may be 0 nM / kg, about 300 nM / kg, about 350 nM / kg, about 400 nM / kg, about 450 nM / kg, about 500 nM / kg, about 600 nM / kg, about 700 nM / kg, about 800 nM / kg, about 900 nM / kg, about 1000 nM / kg, about 2000 nM / kg, about 2500 nM / kg or about 3000 nM / kg of the patient's body weight.

[0468] In various other embodiments, the dose of the compound or a pharma- ceutically acceptable salt thereof is, for example, about 10 nM / kg to about 10,000 nM / kg, about 10 nM / kg to about 5,000 nM / kg, about 10 nM / kg to about 3,000 nM / kg, about 10 nM / kg to about 2,500 nM / kg, about 10 nM / kg to about 2,000 nM / kg, about 10 nM / kg to about 1,000 nM / kg, about 10 nM / kg to about 900nM / kg, about 10nM / kg to about 800nM / kg, about 10nM / kg to about 700nM / kg, about 10nM / kg to about 600nM / kg, about 10nM / kg to about 500nM / kg, about 10nM / kg to about 400nM / kg, about 10nM / kg to about 300nM / kg, about 10nM / kg to about 200nM / kg, about 10nM / kg to about 150nM / kg, about 10nM / kg to about 10 0nM / kg, about 10nM / kg to about 90nM / kg, about 10nM / kg to about 80nM / kg, about 10nM / kg to about 70nM / kg, about 10nM / kg to about 60nM / kg, about 10nM / k g ~ about 50nM / kg, about 10nM / kg - about 40nM / kg, about 10nM / kg - about 30nM / kg, about 10nM / kg - about 20nM / kg, about 200nM / kg - about 900nM / kg, about The range may be from 200 nM / kg to about 800 nM / kg, from about 200 nM / kg to about 700 nM / kg, from about 200 nM / kg to about 600 nM / kg, from about 200 nM / kg to about 500 nM / kg, from about 250 nM / kg to about 600 nM / kg, from about 300 nM / kg to about 600 nM / kg, from about 300 nM / kg to about 500 nM / kg, or from about 400 nM / kg to about 600 nM / kg of the patient's body weight.In various other embodiments, the dose of the compound or a pharma- ceutically acceptable salt thereof is, for example, about 1 nM / kg to about 10,000 nM / kg, about 1 nM / kg to about 5,000 nM / kg, about 1 nM / kg to about 3,000 nM / kg, about 1 nM / kg to about 2,500 nM / kg, about 1 nM / kg to about 2,000 nM / kg, about 1 nM / kg to about 1,000 nM / kg, about 1 nM / kg to about 900 nM / kg, about 1 nM / kg to about 800 nM / kg, about 1 nM / kg to about 700 nM / kg, or about 1 nM / kg to about 1000 nM / kg. g, about 1nM / kg to about 600nM / kg, about 1nM / kg to about 500nM / kg, about 1nM / kg to about 400nM / kg, about 1nM / kg to about 300nM / kg, about 1nM / kg to about 200nM / kg, about 1nM / kg to about 150 nM / kg, about 1nM / kg to about 100nM / kg, about 1nM / kg to about 90nM / kg, about 1nM / kg to about 80nM / kg, about 1nM / kg to about 70nM / kg, about 1nM / kg to about 60nM / kg, about 1nM / kg to about 50nM In still other embodiments, the dose may range from about 0.1 nM / kg, about 0.2 nM / kg, about 0.3 nM / kg, about 0.4 nM / kg, or about 0.5 nM / kg, about 0.1 nM / kg to about 1000 nM / kg, about 0.1 nM / kg to about 900 nM / kg, about 0.1 nM / kg to about 850 nM / kg, about 0.1 nM / kg to about 800 nM / kg, and in still other embodiments, the dose may range from about 0.1 nM / kg, about 0.2 nM / kg, about 0.3 nM / kg, about 0.4 nM / kg, or about 0.5 nM / kg, about 0.1 nM / kg to about 1000 nM / kg, about 0.1 nM / kg to about 900 nM / kg, about 0.1 nM / kg to about 850 nM / kg, about 0.1 nM / kg to about 800 The amount of the compound may be about 0.1 nM / kg, about 0.1 nM / kg to about 700 nM / kg, about 0.1 nM / kg to about 600 nM / kg, about 0.1 nM / kg to about 500 nM / kg, about 0.1 nM / kg to about 400 nM / kg, about 0.1 nM / kg to about 300 nM / kg, about 0.1 nM / kg to about 200 nM / kg, about 0.1 nM / kg to about 100 nM / kg, about 0.1 nM / kg to about 50 nM / kg, about 0.1 nM / kg to about 10 nM / kg, or about 0.1 nM / kg to about 1 nM / kg.In other embodiments, the dose is from about 0.3 nM / kg to about 1000 nM / kg of patient body weight, from about 0.3 nM / kg to about 900 nM / kg, from about 0.3 nM / kg to about 850 nM / kg, from about 0.3 nM / kg to about 800 nM / kg, from about 0.3 nM / kg to about 700 nM / kg, from about 0.3 nM / kg to about 600 nM / kg, from about 0.3 nM / kg to about 50 The dose may be 0 nM / kg, about 0.3 nM / kg to about 400 nM / kg, about 0.3 nM / kg to about 300 nM / kg, about 0.3 nM / kg to about 200 nM / kg, about 0.3 nM / kg to about 100 nM / kg, about 0.3 nM / kg to about 50 nM / kg, about 0.3 nM / kg to about 10 nM / kg, or about 0.3 nM / kg to about 1 nM / kg of patient body weight. In these embodiments, "kg" refers to the patient's body weight in kilograms. In some embodiments, the compound or a pharma- ceutical acceptable salt thereof may be administered to the patient one or more times.

[0469] In other embodiments, the compound or its pharmaceutically acceptable salt can be administered to the patient at about 20 μg / kg patient weight to about 3 mg / kg patient weight.In other embodiments, the amount can be about 0.2 mg / kg patient weight and about 0.4 mg / kg patient weight or about 50 μg / kg patient weight.In some embodiments, the compound or its pharmaceutically acceptable salt can be administered to the patient once or multiple times.

[0470] In some embodiments, the small molecule ligand linked to the targeting moiety (compound) may be administered to the patient prior to the CAR T cell composition comprising the CAR T cells, where the CAR T cells have a CAR comprising an E2 anti-fluorescein antibody fragment. In other embodiments, the small molecule ligand linked to the targeting moiety (bridge) may be administered to the patient at the same time, but in a different formulation or in the same formulation, as the CAR T cell composition comprising the CAR T cells, where the CAR T cells have a CAR comprising an E2 anti-fluorescein antibody fragment. In yet other embodiments, the small molecule ligand linked to the targeting moiety may be administered to the patient after the CAR T cell composition comprising the CAR T cells, where the CAR T cells have a CAR comprising an E2 anti-fluorescein antibody fragment.

[0471] In one illustrative embodiment, the interval between administering the CAR T cells having a CAR comprising an E2 anti-fluorescein antibody fragment and the small molecule linked to a targeting moiety (bridge) can vary widely depending on factors including the type of CAR T cells expressing the E2 anti-fluorescein antibody fragment used, the binding specificity of the CAR having 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 means used to administer the CAR T cells expressing the E2 anti-fluorescein antibody fragment and the small molecule ligand linked to the targeting moiety to the patient, and the health, age, and weight of the patient. In certain embodiments, the small molecule ligand linked to a targeting moiety may be administered before or after the CAR T cells, such as within about 3 hours, 6 hours, 9 hours, 12 hours, 15 hours, 18 hours, 21 hours, 24 hours, 27 hours, 30 hours, 33 hours, 36 hours, 39 hours, 42 hours, 45 hours, 48 ​​or 51 hours or within about 0.5 days, 1 day, 1.5 days, 2 days, 2.5 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days or more days.

[0472] In some embodiments, any applicable dosing schedule known in the art may be used for administration of the compound or a pharma- ceutically acceptable salt thereof or a CAR T cell composition, wherein the CAR T cell composition comprises a CAR T cell having a CAR, the CAR comprising an E2 anti-fluorescein antibody fragment. For example, once daily dosing (qd), twice daily dosing (bid), three times daily dosing (tid), twice weekly dosing (BIW), three times weekly dosing (TIW), once weekly dosing, etc. may be used. In certain embodiments, the dosing schedule selected for administration of the compound or a pharma- ceutically acceptable salt thereof and the CAR T cell composition (wherein the CAR T cell composition comprises a CAR T cell having a CAR, the CAR comprises an E2 anti-fluorescein antibody fragment) may take into account the concentration of the compound or a pharma- ceutically acceptable salt thereof administered and the number of CAR T cells expressing an E2 anti-fluorescein antibody fragment, in order to control cytotoxicity of the CAR T cell composition (wherein the CAR T cell composition comprises a CAR T cell having a CAR, the CAR comprises an E2 anti-fluorescein antibody fragment) and to control CRS.

[0473] In one embodiment, a method for treating cancer is provided that prevents or inhibits cytokine release syndrome (CRS) in a patient. The method includes: i) administering a compound or a pharma- ceutically acceptable salt thereof to the patient, wherein the compound comprises a small molecule ligand linked to a targeting moiety by a linker; ii) administering a CAR T cell composition to the patient, wherein the CAR T cell composition comprises a CAR T cell, the CAR T cell comprises a CAR, and the CAR comprises an E2 anti-fluorescein antibody fragment; and iii) administering folate, a conjugate comprising folate without a targeting moiety, or a drug that inhibits the activation of CAR T cells to the patient.

[0474] In this method embodiment, administering to a patient folate, a conjugate comprising folate without a targeting moiety, or a drug that inhibits the activation of CAR T cells expressing E2 anti-fluorescein antibody fragments can be used to prevent or prevent CRS in the patient. In this embodiment, any of folate, a conjugate comprising folate without a targeting moiety, or a drug that inhibits the activation of CAR T cells expressing E2 anti-fluorescein antibody fragments can be referred to herein as a "rescue agent". In some embodiments, a folate such as folic acid can be administered to a patient to prevent or prevent CRS in the patient. In this embodiment, the folate prevents the interaction of the bridge (i.e., the small molecule ligand linked to the targeting moiety by a linker) with the receptor of the bridge on the tumor, preventing tumor lysis and preventing or preventing CRS in the patient.

[0475] In other embodiments, the rescue agent can reduce CRS for 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 administering 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.).

[0476] An example of a grading scheme is shown below with rows 1-6 from left to right being grades 0, 1, 2, 3, 4 and 5. [Table 3]

[0477] In some embodiments, the folate administered as an inhibitor of binding of bridge to tumor can be, for example, folic acid, folic acid analogs or other folic acid receptor binding molecules.In various embodiments, the folic acid analogs that can be used include folinic acid, pteropolyglutamic acid and folic acid receptor binding pteridines, such as tetrahydropterin, dihydrofolic acid, tetrahydrofolic acid and its deaza and dideaza analogs.The terms "deaza" and "dideaza" analogs refer to art-recognized analogs that have carbon atoms replacing one or two nitrogen atoms of naturally occurring folic acid structure.For example, deaza analogs include 1-deaza, 3-deaza, 5-deaza, 8-deaza and 10-deaza analogs.Dideaza analogs include, for example, 1,5-dideaza, 5,10-dideaza, 8,10-dideaza and 5,8-dideaza analogs. The folate analogs are commonly referred to as "folates," reflecting their ability to bind to the folate receptor. Other folate receptor binding analogs include aminopterin, amethopterin (methotrexate), N10-methylfolate, 2-deamino-hydroxyfolate, deaza analogs such as 1-deazamethopterin or 3-deazamethopterin, and 3',5'-dichloro-4-amino-4-deoxy-N10-methylpteroylglutamic acid (dichloromethotrexate).

[0478] In another embodiment, the folate administered as an inhibitor of binding of the bridge to the tumor has the formula [ka] [In the formula, X 1 and Y 1 each independently represents halo, R 2 , OR 2 , S.R. 3 and N.R. 4 R 5 selected from the group consisting of; U, V and W are each -(R 6a )C=, -N=, -(R 6a )C(R 7a )- and -N(R 4a)-; Q is selected from the group consisting of C and CH; T is selected from the group consisting of S, O, N, and -C=C-; X 2 and X 3 are each independently oxygen, sulfur, -C(Z)-, -C(Z)O-, -OC(Z)-, -N(R 4b )-, -C(Z)N(R 4b )-, -N(R 4b )C(Z)-, -OC(Z)N(R 4b )-, -N(R 4b )C(Z)O-, -N(R 4b )C(Z)N(R 5b )-, -S(O)-, -S(O)2-, -N(R 4a )S(O)2-, -C(R 6b )(R 7b )-, -N(C≡CH)-, -N(CH2C≡CH)-, C1-C 12 Alkylene and C1-C 12 alkyleneoxy, where Z is oxygen or sulfur; R 1 are hydrogen, halo, C1-C 12 Alkyl and C1-C 12 alkoxy; R 2 , R 3 , R 4 , R 4a , R 4b , R 5 , R 5b , R 6b and R 7b each independently represents hydrogen, halo, C1-C 12 Alkyl, C1-C 12 Alkoxy, C1-C 12 Alkanoyl, C1-C 12 Alkenyl, C1-C 12 Alkynyl, (C1-C 12 alkoxy)carbonyl and (C1-C 12 alkylamino)carbonyl; R 6 and R 7 each independently represents hydrogen, halo, C1-C12 Alkyl and C1-C 12 alkoxy; or R 6 and R 7 together to form a carbonyl group; R 6a and R 7a each independently represents hydrogen, halo, C1-C 12 Alkyl and C1-C 12 alkoxy; or R 6a and R 7a together to form a carbonyl group; p, r, s and t are each independently 0 or 1; and * represents an optional covalent bond to the remainder of the conjugate. has.

[0479] In yet another embodiment, the conjugate containing folate can be administered to prevent or inhibit cytokine release syndrome (CRS) in patients.CRS is a term well known in the art, and this syndrome can cause adverse effects in patients, including but not limited to weight loss, high fever, pulmonary edema, and dangerously low blood pressure.

[0480] In this embodiment, the conjugate containing folate does not contain a targeting moiety, and therefore the conjugate inhibits the interaction of the bridge with the tumor, preventing tumor lysis and reducing CRS in patients.In this embodiment, the folate portion of the conjugate containing folate can include any of the folates described in the previous paragraph in relation to the chemical moiety that does not contain a targeting moiety.In some embodiments, the conjugate containing folate can include a folate bonded to one or more amino acids that do not contain a targeting moiety.As an example, the conjugate containing folate can be represented by the formula [ka] may have:

[0481] This compound may also be referred to as "EC923". In these embodiments, folate or a conjugate comprising folate may be administered to a patient in molar excess relative to the bridge (i.e., the small molecule ligand attached to the targeting moiety by a linker), for example, 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 folate or a conjugate comprising folate relative to the small molecule ligand attached to the targeting moiety by a linker. The amount of folate or a conjugate comprising folate relative to the amount of small molecule ligand attached to the targeting moiety by a linker required to inhibit the interaction of the bridge with the tumor may be determined by one skilled in the art.

[0482] In other embodiments, a rescue agent that inhibits the activation of CAR T cells expressing E2 anti-fluorescein antibody fragments can be administered to the patient to inhibit CAR T cell activation and prevent or prevent CRS in the patient. In some embodiments, the agent can be selected from the group consisting of 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 agents that block CAR T cell binding to bridge but do not bind to cancer (e.g., fluoresceinamine, FITC, or sodium fluorescein). It will be understood by those skilled in the art that FITC (i.e., fluorescein) is in a salt form (e.g., sodium fluorescein) or in a non-salt form under physiological conditions or, for example, in a buffer at physiological pH. Thus, in some embodiments, when fluorescein is administered to a patient, there may be an equilibrium between the salt form (e.g., sodium fluorescein) and the non-salt form. In other embodiments, the rescue agent that inhibits activation of CAR T cells has the formula [ka] The compound may be:

[0483] In various embodiments, the rescue agent may be administered in an appropriate volume, including, for example, 0.1 ml, 0.2 ml, 0.3 ml, 0.4 ml, 0.5 ml, 0.6 ml, 0.7 ml, 0.8 ml, 0.9 ml, 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 10 ml, 100 ml, or 1000 ml, at a concentration of about .001 nM to about 100 mM, about .01 nM to about 100 mM, about 1 nM to about 100 mM, about 10 nM to about 100 mM, about 50 nM to about 100 mM, or about 100 nM to about 100 mM. In other embodiments, the rescue agent may be administered at a dose of about .01 to about 300 μM / kg patient body weight, about .06 to about 100 μM / kg patient body weight, about .06 to about 90 μM / kg patient body weight, about .06 to about 80 μM / kg patient body weight, about .06 to about 70 μM / kg patient body weight, about .06 to about 60 μM / kg patient body weight, about .06 to about 50 μM / kg patient body weight, about .06 to about 40 μM / kg patient body weight, about .06 to about 30 μM / kg patient body weight, about .06 to about 20 μM / kg patient body weight, about .06 to about 10 μM / kg patient body weight, about .06 to about 8 μM / kg patient body weight, or about .06 to about 6 μM / kg patient body weight.

[0484] In these embodiments, the rescue agent may be administered to the patient in molar excess relative to the compound or a pharma- ceutically acceptable salt thereof (i.e., the small molecule ligand attached to the targeting moiety by a linker), e.g., about a 10-fold excess, about a 20-fold excess, about a 30-fold excess, about a 40-fold excess, about a 50-fold excess, about a 60-fold excess, about a 70-fold excess, about a 80-fold excess, about a 90-fold excess, about a 100-fold excess, about a 200-fold excess, about a 300-fold excess, about a 400-fold excess, about a 500-fold excess, about a 600-fold excess, about a 700-fold excess, about a 800-fold excess, about a 900-fold excess, about a 1000-fold excess, or about a 10,000-fold excess of the rescue agent relative to the small molecule ligand attached to the targeting moiety by a linker. The amount of rescue agent relative to the amount of small molecule ligand (bridge) attached to a targeting moiety by a linker required to inhibit interaction of the compound or a pharma- ceutically acceptable salt thereof with a tumor and / or a CAR T cell expressing an E2 anti-fluorescein antibody fragment can be determined by one of skill in the art.

[0485] In other embodiments, the patient may be administered more than one dose of an agent that inhibits activation of CAR T cells expressing folate, a conjugate that includes folate without a targeting moiety, or an E2 anti-fluorescein antibody fragment.

[0486] In the embodiments of the 'rescue agent' described herein, the patient may be administered folate, a conjugate comprising folate without a targeting moiety, or an agent that inhibits activation of CAR T cells expressing an E2 anti-fluorescein antibody fragment before and / or after the compound or a pharma- ceutically acceptable salt thereof. In other embodiments, the compound or a pharma- ceutically acceptable salt thereof may be administered before and after administration of folate, a conjugate comprising folate without a targeting moiety, or an agent that inhibits activation of CAR T cells expressing an E2 anti-fluorescein antibody fragment. In this embodiment, subsequent administration of the compound or a pharma- ceutically acceptable salt thereof may result in activation of CAR T cells expressing an E2 anti-fluorescein antibody fragment in the patient, increasing cytokine levels.

[0487] In other embodiments, administration of an agent that inhibits the activation of CAR T cells expressing folate, a conjugate that comprises folate without a targeting moiety, or an E2 anti-fluorescein antibody fragment can result in a decrease in cytokine levels in patients.In yet other embodiments, the decrease in cytokine levels can occur by 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 an agent that inhibits the activation of CAR T cells expressing folate, a conjugate that comprises folate without a targeting moiety, or an E2 anti-fluorescein antibody fragment to a patient.In other embodiments, the decrease in cytokine levels is about the same as that of untreated patients.

[0488] In other illustrative embodiments, the number of CAR T cells expressing E2 anti-fluorescein antibody fragments may increase in the blood of a patient after administration of folate, a conjugate comprising folate without a targeting moiety, or an agent that inhibits activation of CAR T cells expressing E2 anti-fluorescein antibody fragments, even if cytokine levels in the patient are reduced. In other illustrative embodiments, the activation of CAR T cells expressing E2 anti-fluorescein antibody fragments may be enhanced or maintained after administration of folate, a conjugate comprising folate without a targeting moiety, or an agent that inhibits activation of rescue CAR T cells, compared to a patient not treated with a rescue agent, even if cytokine levels in the treated patient are reduced. In yet other embodiments, the cancer comprises a tumor, and the tumor size in the patient does not increase when the patient is administered folate, a conjugate comprising folate without a targeting moiety, or a rescue agent that inhibits activation of CAR T cells expressing E2 anti-fluorescein antibody fragments. In this embodiment, a complete tumor response may be obtained.

[0489] In other embodiments, a rescue agent that inhibits activation of CAR T cells expressing an E2 anti-fluorescein antibody fragment is administered to the patient when CRS grade reaches 1, 2, 3 or 4, or when CRS grade reaches 3 or 4. In other embodiments, the patient's pulmonary edema is reduced when the rescue agent is administered.

[0490] In certain embodiments described herein, a method of treating cancer is provided, the method comprising: i) continuously administering to a patient a compound, where the compound comprises a small molecule ligand attached to a targeting moiety by a linker; ii) administering to the patient a CAR T cell composition comprising a CAR T cell, where the CAR T cell comprises 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 block or prevent cytokine release syndrome in the patient.

[0491] In accordance with this embodiment, the term "continuously" may mean administering the compound or a pharma- ceutically acceptable salt thereof to a patient for, for example, at least 1 hour, at least 4 hours, at least 6 hours, at least 8 hours, at least 10 hours, at least 12 hours, or at least 24 hours, or may mean a daily or weekly administration regimen, 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 regimen that would be considered continuous administration by one of skill in the art. In other aspects, the term "continuously" may mean any combination of the embodiments described in this paragraph.

[0492] In an embodiment of this method, "terminating continuous administration" of the compound or its pharma- ceutically acceptable salt for preventing or preventing cytokine release syndrome in a patient can mean, for example, ceasing administration after a continuous period of administration of several hours or days, or ceasing treatment regimen, such as the daily or weekly regimen described above.In other embodiments, "terminating continuous administration" can mean, for example, administration until the patient experiences unacceptable weight loss or any other unacceptable side effect, such as high fever, low blood pressure, or pulmonary edema.In this embodiment, "terminating continuous administration" of the compound or its pharma- ceutically acceptable salt does not mean that treatment with the compound or its pharma- ceutically acceptable salt is a single treatment, followed by no treatment with the compound or its pharma- ceutically acceptable salt.In an embodiment of this method, "preventing or preventing" cytokine release syndrome (CRS) means eliminating CRS or reducing or improving symptoms of CRS.

[0493] In some embodiments, including the embodiment of "terminating the continuous administration" of the compound or its pharma- ceutically acceptable salt, the method may further include step iv) of re-administering the compound or its pharma- ceutically acceptable salt to the patient. In some embodiments, the compound or its pharma- ceutically acceptable salt may be administered, for example, once a week, and one dose may be omitted. In other embodiments, the compound or its pharma- ceutically acceptable salt may be administered every other day (i.e., every other day), and one or more doses (e.g., two, three, four, etc.) may be omitted. In other embodiments, the compound or its pharma- ceutically acceptable salt may be administered twice a week, and one or more doses (e.g., two, three, four, etc.) may be omitted. In other embodiments, the compound or its pharma- ceutically acceptable salt may be administered on Monday, Tuesday, and the following Monday, and then administration is discontinued for two weeks, and the cycle is repeated. In other embodiments, any of the above regimen embodiments may be used, and one or more doses (e.g., two, three, four, etc.) may be omitted. In these embodiments, the omitted administration may prevent or reduce CRS in the patient.

[0494] In yet another illustrative embodiment, a method of treating cancer is provided, comprising: i) administering to a patient a compound or a pharma- ceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand attached to a targeting moiety by a linker, and wherein the patient is administered at least a first dose and a second dose of the compound or a pharma- ceutically acceptable salt thereof, wherein the first dose and the second dose are different, and the second dose of the compound or a pharma- ceutically 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 pharma- ceutically acceptable salt thereof; and ii) administering to the patient a CAR T cell composition comprising a CAR T cell, wherein the CAR T cell comprises a CAR comprising an E2 anti-fluorescein antibody fragment.

[0495] In this embodiment, the dose of compound or its pharmaceutically acceptable salt can be gradually increased to prevent or prevent cytokine release syndrome in patients.For example, at least a first dose and a second dose of compound or its pharmaceutically acceptable salt can be administered to patients, where the first dose and the second dose of compound or its pharmaceutically acceptable salt are different, and the second dose is about 20 times to about 15000 times more, 2 times to about 15000 times more than the amount of the first dose of compound or its pharmaceutically acceptable salt. In other embodiments, the second dose or any subsequent dose is 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 1,000-fold, about 2-fold to about 2,000-fold, about 2-fold to about 3,000-fold, about 2-fold to about 4,000-fold, about 2-fold to about 5,000-fold, about 2-fold to about 6,000-fold, about 2-fold to about 7,000-fold, about 2-fold to about 8,000-fold, about 2-fold to about 9,000-fold, about 2-fold to about 10,000-fold, about 2-fold to about 15 ...20,000-fold, about 2-fold to about 20,000-fold, about 2-fold to about 30,000-fold, about 2-fold to about 30,000-fold, about 2-fold to about 40,000-fold, about 2-fold to about 50,000-fold, about 2-fold to about 0 times, about 5 times to about 9000 times, about 5 times to about 8000 times, about 5 times to about 7000 times, about 5 times to about 6000 times, about 5 times to about 5000 times, about 5 times to about 4000 times, about 5 times to about 3000 times, about 5 times to about 4 000 times, approximately 5 times to approximately 3000 times, approximately 5 times to approximately 2000 times, approximately 5 times to approximately 1000 times, approximately 5 times to approximately 750 times, approximately 2 times to approximately 750 times, approximately 5 times to approximately 500 times, approximately 5 times to approximately 100 times, approximately 800 times to approximately 15 000 times, approximately 800 times to approximately 10,000 times, approximately 800 times to approximately 9,000 times, approximately 800 times to approximately 8,000 times, approximately 800 times to approximately 7,000 times, approximately 800 times to approximately 6,000 times, approximately 800 times to approximately 5,000 times, approximately 8 00x ~ 4000x, 800x ~ 3000x, 800x ~ 2000x, 800x ~ 1000x, 8000x ~ 15000x, 8000x ~ 10000x, 8000x ~ about 9000 times, about 15000 times, about 10000 times, about 9000 times, about 8000 times, about 7000 times, about 6000 times, about 5000 times, about 4000 times, about 3000 times, about 2000 times, about 1000 times, about 500 times, about 400 times, about 300 times, about 200 times, about 100 times, about 90 times, about 80 times, about 70 times, about 60 times, about 50 times, about 40 times, about 30 times, about 20 times, about 10 times, about 5 times, or about 2 times more.

[0496] In other illustrative embodiments of the dose escalation method, at least a first dose, a second dose, and a third dose of a compound or a pharma- ceutically acceptable salt thereof may be administered to a patient, wherein the first dose, the second dose, and the third dose are different, the second dose of the compound or a pharma- ceutically acceptable salt thereof being about 2-fold to about 750-fold greater than the amount of the first dose of the compound or a pharma- ceutically acceptable salt thereof, and the third dose of the compound or a pharma- ceutically acceptable salt thereof being about 800-fold to about 10,000-fold greater than the amount of the first dose of the compound or a pharma- ceutically acceptable salt thereof.

[0497] In other embodiments of the dose escalation method, at least a first dose, a second dose, a third dose, and a fourth dose of a compound or a pharma- ceutically acceptable salt thereof may be administered to a patient, wherein the first dose, the second dose, the third dose, and the fourth dose are different, and the second dose of the compound or a pharma- ceutically acceptable salt thereof is about 2 to about 750 times greater than the amount of the first dose of the compound or a pharma- ceutically acceptable salt thereof, the third dose of the compound or a pharma- ceutically acceptable salt thereof is about 800 to about 7500 times greater than the amount of the first dose of the compound or a pharma- ceutically acceptable salt thereof, and the fourth dose of the compound or a pharma- ceutically acceptable salt thereof is about 8000 to about 15000 times greater than the amount of the first dose of the compound or a pharma- ceutically acceptable salt thereof.

[0498] In yet other embodiments, the second dose of the compound or its pharma- ceutically acceptable salt may be about 100 times greater than the amount of the first dose of the compound or its pharma- ceutically acceptable salt, the third dose of the compound or its pharma- ceutically acceptable salt may be about 1000 times greater than the amount of the first dose of the compound or its pharma- ceutically acceptable salt, and the fourth dose of the compound or its pharma- ceutically acceptable salt may be about 10000 times greater than the amount of the first dose of the compound or its pharma- ceutically acceptable salt. In an exemplary embodiment, the first dose of the compound or its pharma- ceutically acceptable salt is .05 nM / kg, the second dose is 5 nM / kg, the third dose is 50 nM / kg, and the fourth dose is 500 nM / kg. In the dose escalation embodiments described herein, the first, second, third, fourth, and any subsequent doses of the compound or its pharma- ceutically acceptable salt may be administered multiple times (e.g., a first dose of .05 nM / kg is administered several times, followed by escalating doses).

[0499] In other embodiments described herein, a method for treating cancer is provided, which includes: i) administering to a patient a first dose of a compound or a pharmaceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked to a targeting moiety by a linker; ii) administering to a patient at least a second dose of a compound or a pharmaceutically acceptable salt thereof, wherein 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 a patient a dose of a CAR T cell composition comprising a CAR T cell, wherein the CAR T cell comprises a CAR comprising an E2 anti-fluorescein antibody fragment.

[0500] In various embodiments of this dose taper embodiment, the second dose of the compound or a pharma- ceutically acceptable salt thereof can be at least about 60% lower than the amount of the first dose of the compound or a pharma- ceutically acceptable salt thereof, at least about 70% lower than the amount of the first dose of the compound or a pharma- ceutically acceptable salt thereof, at least about 80% lower than the amount of the first dose of the compound or a pharma- ceutically acceptable salt thereof, at least about 90% lower than the amount of the first dose of the compound or a pharma- ceutically acceptable salt thereof, at least about 95% lower than the amount of the first dose of the compound or a pharma- ceutically acceptable salt thereof, at least about 96% lower than the amount of the first dose of the compound or a pharma- ceutically acceptable salt thereof, at least about 97% lower than the amount of the first dose of the compound or a pharma- ceutically acceptable salt thereof, at least about 98% lower than the amount of the first dose of the compound or a pharma- ceutically acceptable salt thereof, at least about 99% lower than the amount of the first dose of the compound or a pharma- ceutically acceptable salt thereof, or at least about 99.5% lower than the amount of the first dose of the compound or a pharma- ceutically acceptable salt thereof.

[0501] In various embodiments of the dose tapering embodiments described herein, the first dose of the compound or a pharma- ceutically acceptable salt thereof can be about 100 nM / kg to about 1000 nM / kg patient body weight, about 100 nM / kg to about 900 nM / kg patient body weight, about 100 nM / kg to about 800 nM / kg patient body weight, about 100 nM / kg to about 700 nM / kg patient body weight, about 100 nM / kg to about 600 nM / kg patient body weight, about 200 nM / kg to about 600 nM / kg patient body weight, about 400 nM / kg to about 600 nM / kg patient body weight, or about 500 nM / kg patient body weight.

[0502] In various embodiments of the dose tapering embodiments described herein, the second dose of the compound or a pharma- ceutically acceptable salt thereof can be from about 0.5 nM / kg to about 500 nM / kg patient body weight, from about 0.5 nM / kg to about 450 nM / kg patient body weight, from about 0.5 nM / kg to about 400 nM / kg patient body weight, from about 0.5 nM / kg to about 350 nM / kg patient body weight, from about 0.5 nM / kg to about 300 nM / kg patient body weight, from about 1 nM / kg to about 300 nM / kg patient body weight, from about 2 nM / kg to about 300 nM / kg patient body weight, from about 2 nM / kg to about 250 nM / kg patient body weight, from about 5 nM / kg to about 40 nM / kg patient body weight, or from about 40 nM / kg to about 150 nM / kg patient body weight.

[0503] In further embodiments of the dose tapering embodiments described herein, the method may further comprise administering a third dose of the compound or a pharma- ceutically acceptable salt thereof, wherein the third dose of the compound or a pharma- ceutically acceptable salt thereof is the same as the second dose of the compound or a pharma- ceutically acceptable salt thereof.In other embodiments, the method may further comprise administering a fourth dose of the compound or a pharma- ceutically acceptable salt thereof, wherein the fourth dose of the compound or a pharma- ceutically acceptable salt thereof is the same as the second dose or a pharma- ceutically acceptable salt thereof and the third dose of the compound or a pharma- ceutically acceptable salt thereof.In yet other embodiments, the dose of the compound or a pharma- ceutically acceptable salt thereof administered after the first dose of the compound or a pharma- ceutically acceptable salt thereof can maintain the inhibition of cancer growth associated with the first dose of the compound or a pharma- ceutically acceptable salt thereof.

[0504] In other embodiments of the dose-decreasing embodiments described herein, the CAR T cells expressing the E2 anti-fluorescein antibody fragment may be administered at a dose of about 1 million CAR T cells expressing the E2 anti-fluorescein antibody fragment to about 40 million CAR T cells expressing the E2 anti-fluorescein antibody fragment. In yet other embodiments, the dose of the compound or a pharma- ceutically acceptable salt thereof administered after the first dose of the compound or a pharma- ceutically acceptable salt thereof may be administered once or twice weekly.

[0505] In yet another embodiment of the dose-decreasing embodiment described herein, the method may further comprise administering to the patient folate, a conjugate comprising folate without a targeting moiety, or an agent that inhibits the activation of CAR T cells expressing E2 anti-fluorescein antibody fragments. In another embodiment, an agent that inhibits the activation of CAR T cells expressing E2 anti-fluorescein antibody fragments is administered to the patient, the agent being an agent that blocks the binding of the compound or a pharma- ceutically acceptable salt of the CAR T cells expressing E2 anti-fluorescein antibody fragments but does not bind to cancer, the agent being fluoresceinamine, sodium fluorescein, or fluorescein. In yet another embodiment, the agent is sodium fluorescein.

[0506] In another embodiment, a method for treating cancer is provided, comprising: i) administering a first dose of a compound to a patient, wherein the compound comprises a small molecule ligand linked to a targeting moiety by a linker, and the compound or a pharmaceutically acceptable salt is administered to the patient at least about 1 hour before administering a CAR T cell composition comprising a CAR T cell, wherein the CAR T cell comprises a CAR comprising an E2 anti-fluorescein antibody fragment; ii) then administering a dose of a CAR T cell composition comprising a CAR T cell to the patient, wherein the CAR T cell comprises a CAR comprising an E2 anti-fluorescein antibody fragment; and iii) then administering a second dose of the compound or a pharmaceutically acceptable salt to the patient. In various embodiments of this pretreatment embodiment, a first dose of the compound, or a pharma- ceutically acceptable salt thereof, can be administered to the patient at least about 2 hours prior to administration of the CAR T cell composition, at least about 4 hours prior to administration of the CAR T cell composition, at least about 8 hours prior to administration of the CAR T cell composition, at least about 12 hours prior to administration of the CAR T cell composition, at least about 16 hours prior to administration of the CAR T cell composition, at least about 20 hours prior to administration of the CAR T cell composition, or at least about 24 hours prior to administration of the CAR T cell composition, in each case where the CAR T cell composition comprises a CAR T cell comprising a CAR that comprises an E2 anti-fluorescein antibody fragment.

[0507] In various embodiments of this pretreatment embodiment, the second dose of the compound or a pharma- ceutically acceptable salt thereof can be administered to the patient at least about 24 hours after administration of the CAR T cell composition, at least about 20 hours after administration of the CAR T cell composition, at least about 18 hours after administration of the CAR T cell composition, at least about 16 hours after administration of the CAR T cell composition, at least about 14 hours after administration of the CAR T cell composition, at least about 12 hours after administration of the CAR T cell composition, at least about 10 hours after administration of the CAR T cell composition, at least about 8 hours after administration of the CAR T cell composition, at least about 6 hours after administration of the CAR T cell composition, at least about 4 hours after administration of the CAR T cell composition, or at least about 2 hours after administration of the CAR T cell composition, in each case where the CAR T cell composition comprises a CAR T cell comprising a CAR that comprises an E2 anti-fluorescein antibody fragment.

[0508] In any of the embodiments described herein in which folate is the ligand attached to the targeting moiety by a linker, the patient may be prescribed a folate deficient diet prior to treatment with the methods described herein or the patient may administer folate to the diet. In embodiments in which the patient is administered folate, the dosage may be, for example, from about 50 nM / kg to about 3000 nM / kg of patient body weight, from about 50 nM / kg to about 2000 nM / kg, from about 50 nM / kg to about 1000 nM / kg, from about 50 nM / kg to about 900 nM / kg, from about 50 nM / kg to about 800 nM / kg, from about 50 nM / kg to about 700 nM / kg, from about 50 nM / kg to about 600 nM / kg, from about 50 nM / kg to about 800 nM / kg, from about 50 nM / kg to about 900 nM / kg, from about 50 nM / kg to about 1000 nM / kg, from about 50 nM / kg to about 1500 nM / kg, from about 50 nM / kg to about 2000 nM / kg, from about 50 nM / kg to about 3000 nM / kg, from about 50 nM / kg to about 400 nM / kg, from about 50 nM / kg to about 600 nM / kg, from about 50 nM / kg to about 700 nM / kg, from about 50 nM / kg to about 800 nM / kg, from about 50 nM / kg to about 900 nM / kg, from about 50 nM / kg to about 1000 nM / kg, from about 50 nM / kg to about 1500 nM / kg, from about 50 nM / kg to about 2000 n The range may be about 500 nM / kg, about 50 nM / kg to about 400 nM / kg, about 50 nM / kg to about 300 nM / kg, about 50 nM / kg to about 200 nM / kg, about 50 nM / kg to about 100 nM / kg, about 100 nM / kg to about 300 nM / kg, about 100 nM / kg to about 500 nM / kg, about 100 nM / kg to about 1000 nM / kg, or about 100 nM / kg to about 2000 nM / kg of the patient's body weight. In other embodiments, the dose can be about 100 nM / kg, about 150 nM / kg, about 200 nM / kg, about 250 nM / kg, about 300 nM / kg, about 350 nM / kg, about 400 nM / kg, about 450 nM / kg, about 500 nM / kg, about 600 nM / kg, about 700 nM / kg, about 800 nM / kg, about 900 nM / kg, about 1000 nM / kg, about 2000 nM / kg, or about 3000 nM / kg of patient body weight. In these embodiments, "kg" refers to kilograms of patient body weight. In some embodiments, folate can be administered, for example, daily, weekly, twice weekly, three times weekly, or using any suitable regimen for administration of folate.

[0509] In various embodiments described herein, the CAR T cells may remain in increased numbers of circulating CAR T cells for up to about 10 days, up to about 15 days, up to about 20 days, up to about 25 days, up to about 30 days, up to about 35 days, up to about 40 days, up to about 45 days, up to about 50 days, up to about 55 days, up to about 60 days, up to about 65 days, up to about 70 days, up to about 75 days, or up to about 80 days after administration of the CAR T cells.

[0510] In various embodiments described herein, the 50% effective concentration (EC 50 ) is about 1 pM to about 2 nM, about 1 pM to about 5 nM, about 1 pM to about 10 nM, about 1 pM to about 20 nM, about 1 pM to about 30 nM, about 1 pM to about 40 nM, about 1 pM to about 50 nM, about 1 pM to about 60 nM, about 1 pM to about 70 nM, about 1 pM to about 80 nM, about 1 pM to about 90 nM, about 1 pM to about 100 nM, about 1 pM to about 200 nM, about 1 pM to about 300 nM, about 1 pM to about 400 nM, about 1 pM to about 500 nM, about 1 pM to about 600 nM, about 1 pM to about 700 nM, about 1 pM to about 800 nM, about 1 pM to about 900 nM, about 1 pM to about 1 pM to about 1nM, about 1pM to about 900pM, about 1pM to about 800pM, about 1pM to about 700pM, about 1pM to about 600pM, about 1pM to about 500pM, about 1pM to about 400pM, about 1pM to about 300pM, about 1pM to about 200pM, about 1pM to about 100pM, It can be about 1 pM to about 90 pM, about 1 pM to about 80 pM, about 1 pM to about 70 pM, about 1 pM to about 60 pM, about 1 pM to about 50 pM, about 1 pM to about 40 pM, about 1 pM to about 30 pM, about 1 pM to about 20 pM, about 1 pM to about 10 pM, or about 1 pM to about 5 pM.

[0511] In various embodiments described herein, the Kd for binding of a compound or a pharma- ceutically acceptable salt thereof to a CAR T cell can be 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.

[0512] In various illustrative embodiments described herein, the compound or a pharma- ceutically acceptable salt thereof may be administered to the patient first about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days before or after the CAR T cells, or on any suitable day before or after the CAR T cells.

[0513] In various embodiments described herein, EGFRt-sorted or non-sorted CAR T cells may be used. In other embodiments, a "clinical replica" batch of CAR T cells with a less differentiated profile may be used. In other embodiments, a "research batch" of CAR T cells may be used. The "clinical replica" batch (about 39% EGFRt+) mimicked about 66% T SCM and about 32%T CM CD4+ subset and approximately 95%T SCM and about 3%T CM The study batch (about 23% EGFRt+) may contain about 32% T SCM , about 53%T CM , about 11%T EM and about 3.7%T EFF of CD4 subsets and approximately 44%T SCM , about 0.28%T CM , about 3.4%T EM and about 52%T EFF The CD8 subset may include:

[0514] In various further embodiments of this pretreatment embodiment, the patient does not experience cytokine release resulting in off-target toxicity but does experience toxicity of the CAR T cells expressing the E2 anti-fluorescein antibody fragment to the cancer, or off-target tissue toxicity does not occur in the patient but does experience toxicity of the CAR T cells expressing the E2 anti-fluorescein antibody fragment to the cancer, or the cancer comprises a tumor and the tumor size is reduced in the patient but does not experience off-target toxicity, or the reduction in tumor size in the patient is greater than in a patient who has not been pre-treated with the compound or a pharma- ceutically acceptable salt thereof prior to administration of the CAR T cell composition comprising a CAR T cell comprising a CAR comprising an E2 anti-fluorescein antibody fragment. As will be appreciated by one of skill in the art, the "target" can be a cancer (e.g., a tumor).

[0515] In another embodiment, a method for treating cancer is provided. The method includes: i) administering a compound or a pharma- ceutically acceptable salt thereof to a patient, wherein the compound comprises a small molecule ligand linked to a targeting moiety by a linker; and ii) administering a CAR T cell composition to the patient, wherein the CAR T cell composition comprises a CAR T cell, wherein the CAR T cell comprises a CAR comprising an E2 anti-fluorescein antibody fragment, 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 by a linker is represented by the formula: [ka] may have:

[0516] In yet another embodiment, a method for treating cancer is provided.The method comprises: i) administering a compound or a pharma- ceutically acceptable salt thereof to a patient, wherein the compound comprises a small molecule ligand linked to a targeting moiety by a linker; and ii) administering a CAR T cell composition to the patient, wherein the CAR T cell composition comprises a CAR T cell, wherein the CAR T cell comprises a CAR comprising an E2 anti-fluorescein antibody fragment, 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 by a linker is represented by the formula: [ka] may have:

[0517] In yet another embodiment, a method for treating cancer is provided.The method comprises: i) administering a first compound or its pharmaceutically acceptable salt to a patient, wherein the first compound or its pharmaceutically acceptable salt comprises PSMA ligand linked to FITC by a linker; ii) administering a second compound or its pharmaceutically acceptable salt to a patient, wherein the second compound or its pharmaceutically acceptable salt comprises CAIX ligand linked to FITC by a linker; and iii) administering a CAR T cell composition to a patient, wherein the CAR T cell composition comprises a CAR T cell, and the CAR T cell comprises a CAR comprising E2 anti-fluorescein antibody fragment.In this embodiment, the first compound is represented by the formula: [ka] and the second compound can have the formula [ka] may have:

[0518] In some embodiments of the methods described herein, the cancer is imaged before administering the compound or a pharma- ceutically acceptable salt thereof to the patient or before administering the CAR T cell composition to the patient, wherein the CAR T cell composition comprises a CAR T cell comprising a CAR comprising an E2 anti-fluorescein antibody fragment. In some illustrative embodiments, the imaging is performed by PET imaging. In other illustrative embodiments, the imaging is performed by MRI imaging or SPECT / CT imaging. The imaging method can be any suitable imaging method known in the art. In some embodiments, the imaging method can include the use of a small molecule ligand conjugated to an imaging agent as described herein, but suitable for the type of imaging as described herein.

[0519] In any of the embodiments described herein, cytokine release resulting in off-target toxicity to the patient may not occur even if toxicity of the CAR T cells expressing the E2 anti-fluorescein antibody fragment to the cancer occurs. In any of the embodiments described herein, off-target tissue toxicity may not occur in the patient even if toxicity of the CAR T cells expressing the E2 anti-fluorescein antibody fragment to the cancer occurs. In any of the embodiments described herein, the cancer may include a tumor and the tumor size may be reduced in the patient even if off-target toxicity does not occur. In any of the embodiments described herein, CRS may be reduced or prevented and the method may result in a reduction in tumor volume in 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, the cancer may include a tumor and a complete tumor response may be obtained.

[0520] In other embodiments of the methods described herein, any of the methods described herein can be used alone or any of the methods described herein can be used in combination with any other method or methods described herein. EXAMPLES

[0521] Example 1 EC17 dose tapering may reduce treatment-related toxicity The effect of EC17 dose de-escalation was tested on the anti-tumor activity and toxicity (body weight change) of CAR-T treatment. Two different anti-fluorescein scFvs were used for CAR construction (Figure 1). The first construct of CAR contains the following domains: anti-FL (FITC-E2) scFv-IgG4 hinge (CH2 (L235D, N297Q)-CH3)-CD28TM-4-1BB-CD3z-T2A-EGFRt. The second construct of CAR contains the following domains: anti-FL (FITC-4M5.3) scFv-IgG4 hinge (CH2 (L235D, N297Q)-CH3)-CD28TM-4-1BB-CD3z-T2A-EGFRt. The nucleic acid and amino acid sequences of the CAR of each construct are shown as SEQ ID NOs: 1-4. Human T cells were isolated, activated, and transduced with lentiviral vectors carrying the appropriate CAR genes. CAR-modified CD4+ T cells and CAR-modified CD8+ T cells were isolated and cultured separately in vitro for about 7 days. CD4+ CAR-T cells and CD8+ CAR-T cells were mixed in a 1:1 ratio before iv administration for animal testing.

[0522] MDA-MB-231 tumor (150-250 mm 3)-bearing mice were used for in vivo studies evaluating E2-CAR-T cells. In the first part of the experiment, mice were administered 10 or 20 million E2-CAR-T cells and 500 nM / kg EC17 was administered iv weekly on days 2, 9, 16, 23, etc. after CAR-T administration (as shown in Figure 2A). Mice receiving 20 million E2-CAR-T cells showed severe CRS after the second EC17 and had to be rescued with 6 μmol / kg sodium fluorescein (NaFL). Both the 10 and 20 million CAR-T groups showed severe CRS after the third and fourth EC17 and had to be rescued with NaFL (as shown in Figures 5A and 5B). Both of these two groups showed severe weight loss (~20%) during E2-CAR-T treatment, indicating that 10 or 20 million E2 CAR-T / EC17 500 nM / kg SIW treatment had severe toxicity. (20 million E2-CAR-T cells alone also showed antitumor activity (Figure 5A, open circles), which could be due to the allogeneic HLA-mismatched TCR commonly seen in "young" T cells cultured in vitro for a short period of time.)

[0523] In the second part of the experiment, the E2-CAR-T cells used were cultured in vitro for about 2 weeks. Thirty million E2-CAR-T cells were administered, and EC17 administration was modified for tapering. As shown in Figure 2B, 500nM / kg EC17 was administered 2 days after CAR-T administration, and then low doses of EC17 (5nM / kg or 20nM / kg or 100nM / kg) were administered weekly (e.g., on days 9, 16, and 23 after CAR-T administration). As shown in Figure 3A, all three EC17 tapering groups reached a complete response, indicating that the bridge tapering dose did not affect the anti-tumor activity of CAR-T. More importantly, none of these three EC17 tapering groups showed any severe CRS. As shown in Figure 3B, although more CAR-T cells (30 million) were administered than in the first part of the study (10 or 20 million CAR-T cells), all tapering groups showed very mild weight loss. These data indicate that bridge tapering (one high-level loading dose followed by a reduced level of maintenance dose) can reduce toxicity while maintaining good antitumor activity of CAR-T cells.

[0524] A second construct, 4M5.3-CAR (anti-FL(FITC-4M5.3)scFv-IgG4 hinge(CH2(L235D,N297Q)-CH3)-CD28TM-4-1BB-CD3z-T2A-EGFRt), was also evaluated. Mice bearing MDA-MB-231 tumors (150-250 mm 3 ) was used in an in vivo study to evaluate 4M5.3-CAR-T cells. In the first part of the experiment, 10 or 20 million 4M5.3-CAR-T cells were administered and 500 nmol / kg EC17 was administered iv every week on days 2, 9, 16, 23, etc. after CAR-T administration (as shown in Figure 2A). Mice administered 10 or 20 million 4M5.3-CAR-T cells did not show severe CRS or weight loss after a single EC17 administration (Figure 6B), but all tumors were reduced and eventually disappeared (Figure 6A). 20 million CAR-T cells (top line) showed better antitumor activity than 10 million CAR-T cells (middle line).

[0525] In the second part of the experiment, the 4M5.3-CAR-T cells used were cultured in vitro for about 2 weeks. Thirty million 4M5.3-CAR-T cells were administered, and EC17 administration was modified for tapering. As shown in Figure 2B, 500nM / kg EC17 was administered 2 days after CAR-T administration, and then low doses of EC17 (5nM / kg or 20nM / kg or 100nM / kg) were administered weekly (days 9, 16, 23, etc. after CAR-T administration). As shown in Figure 4A, the 100nM / kg EC17 tapering group showed the best antitumor activity, with all tumors cured (bottom line), while neither the 5nM / kg EC17 group (second line from the top) nor the 20nM / kg EC17 group (third line from the top) showed a complete response to CAR-T treatment. The data indicate that CAR-T activity is controlled by the EC-17 dose used for taper. Furthermore, as shown in Figure 4B, the weight loss of mice in the three taper groups was also EC17 dose-dependent. The 100 nM / kg EC17 dose taper group showed greater weight loss than the other two groups of 5 nM / kg EC17 or 20 nM / kg EC17. These data indicate that bridge taper (one high-level loading dose, followed by a reduced level of maintenance dose) can control both the antitumor activity and toxicity of CAR-T therapy.

[0526] The only difference between E2-CAR and 4M5.3-CAR is the scFv sequence used. When comparing these two CARs under the same conditions, 10 million E2-CAR-T cells were shown to have better antitumor activity than 10 million 4M5.3-CAR-T cells, and when EC17 dose escalation (5nM / kg or 20nM / kg or 100nM / kg) was tested, 30 million E2-CAR-T cells also showed better antitumor activity than 30 million 4M5.3-CAR-T cells.

[0527] As used herein, "SEQ ID NO: 4" refers to the sequence beginning with the underlined "gac" codon and ending with the underlined "ggc" codon. This portion of the long sequence encodes an exemplary 4M5.3 CAR. The CAR is inserted into the T cell membrane. The other portion of the long sequence includes coding sequences for a signal peptide, EGFRt domain, etc., that are not part of the CAR that is inserted into the membrane, but function as a chimeric antigen receptor. As used herein, "SEQ ID NO: 3" refers to the sequence beginning with the underlined "D" and ending with the underlined "G". This portion of the long sequence is the amino acid sequence of the CAR that is inserted into the T cell membrane. The other portion of the long sequence includes amino acid sequences for a signal peptide, EGFRt domain, etc., that are not part of the CAR that is inserted into the membrane, but function as a chimeric antigen receptor. In yet other embodiments, SEQ ID NO: 3 can comprise or consist of a humanized or human amino acid sequence. SEQ ID NOs: 3 and 4 are as described above. The start and stop codons of the long nucleic acid sequence are underlined, and the long sequence is an exemplary sequence that can be used to transduce T cells to prepare a 4M5.3 CAR.

[0528] SEQ ID NO:3 [4M5.3-CAR amino acid sequence (insert)] [ka]

[0529] SEQ ID NO:4 [4M5.3-CAR nucleotide acid sequence (insert)] [ka]

[0530] Example 2 Characterization of CAR T cell differentiation phenotype prior to infusion into NSG mice Multicolor flow cytometry was used to confirm the phenotype of the CAR T cell product to be infused into tumor-bearing mice. The differentiation status of CD4+ and CD8+ subsets was analyzed using specific combinations of surface markers shown in Figure 7A. The unsorted E2 clinically replicated CAR T cell product consisted mainly of the self-renewing Tscm / Tcm phenotype (Figure 7B). In Figure 7B, the highest bar is "Tscm" and the lowest bar is "Tcm". In addition to the differences in CD8:CD4 ratios shown in Figure 8A, the current E2 clinically replicated CAR T cells differed from previously sorted E2 / 4M5.3 CAR-T cells and from early GFP+4M5.3 CAR T in vivo study preparations (summarized as a pie chart in Figure 8B). In Figure 8A, from left to right, the 10 groups, the bars represent the following - 1-(high bar Tscm, low bar Tcm), 2-(high bar Tscm, low bar Tcm), 3-(high bar Teff, low bar Tem), 4-(high bar Tem, low bar Teff, lowest bar Tcm), 5-(high bar Teff, low bar Tem), 6-(high bar Tem, low bar Teff, lowest bar Tcm), 7-(high bar Teff, low bar Tscm), 8-(high bar Teff, low bar Tem), 9-(high bar Teff, low bar Tscm), 10-(high bar Teff, low bar Tsm, then low bar Tem, lowest bar Tcm). Thus, CAR T cells with a predominantly Tscm / Tcm phenotype were expected to have a superior anti-tumor phenotype compared to previous CAR T preparations with a more differentiated Tem / Teff phenotype. For the same reason, sCRS occurs later with current CAR-T preparations than with previous CAR-T preparations, assuming the same EC17 dosing regimen.

[0531] CAR T cell recovery after cryopreservation Cryopreserved CAR T cells were rapidly thawed in a 37°C water bath and immediately transferred to T cell recovery medium (TexMACS 2% human AB serum supplemented with glutamine (Miltenyi Biotec #130-097-196)). TM The cells were placed in a 100-mL medium (medium) and supplemented with 50 U / mL recombinant human IL-2, and cultured in vitro for approximately 3 to 5 days for T cell recovery.

[0532] Flow cytometry analysis CAR T cells were harvested from the in vitro recovery medium and pelleted by centrifugation at 400g for 5 min. T cells were then resuspended in flow cytometry staining solution [1% bovine serum albumin, 50mg / mL human IgG (Equitech Bio, cat#SLH56-0001), 0.9% sodium azide in phosphate buffered saline, pH=7.4] supplemented with both anti-mouse FcγIII / II receptor (CD16 / CD32) block [clone 2.4G2; BD Bioscience, catalog#553142, diluted 1:100 (v / v)] and anti-human Fc block [BD Biosciences, catalog #564220, diluted 1:50 (v / v)]. Surface marker staining was performed with the following fluorochrome-conjugated monoclonal antibodies: anti-human CD45RA-APCeF780 [clone HI100, ThermoFisher #47-0458-42, 1:20 (v / v) dilution], anti-human CD45RO-eF450 [clone UCHL1, ThermoFisher #48-0457-42, 1:20 (v / v) dilution], anti-human CD8α-PECy7 [clone RPA-T8, BD Bioscience, catalog #557746, 1:20 (v / v) dilution], anti-human CD4-Percpe710 [clone SK3, eBioscience catalog #46-0047-42, 1:20 (v / v) dilution], biotinylated anti-human EGFR (cetuximab; R&D Systems Staining was performed by adding #FAB9577B-100, 1:10 (v / v), followed by PE-conjugated anti-biotin (BioLegend #53-9895-82, 1:400 (v / v)) to each sample for 20 min in the dark on ice. After surface marker staining, T cells were washed with PBS, resuspended in cold PBS containing 3 μM propidium iodide, and transferred to flow cytometry collection tubes. Flow cytometry data were collected on a Gallios flow cytometer (Beckman Coulter, Brea, CA), where a minimum of 20,000 events were collected, and data were analyzed using Kaluza v1.5 software.

[0533] Example 3 E2 anti-FITC antibody does not bind to any human normal tissues An E2 anti-FITC IgG antibody with the same variable fragment sequence as that of the E2 anti-FITC CAR construct was used to test whether the E2 antibody had any binding to human normal tissues. Recombinant human E2 anti-FITC IgG1 lambda 1 was expressed in HEK293 cells and purified using a protein A affinity column. The antibody purity was greater than 98% using SEC time-PLC analysis. The antibody was labeled with digoxigenin (DIG) using 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. Various concentrations of DIG-E2 IgG were incubated with FITC-immobilized beads for 30 minutes at room temperature. After washing, monoclonal anti-DIG Ab was added and incubated with the beads for 30 minutes. eF660-conjugated anti-mouse secondary antibody was added and incubated with the washed beads. The washed beads were analyzed by FACS for eF660 signal. PE-immobilized beads were used as negative control beads. The fluorescence intensity of eF660 was plotted against the added concentration of DIG-labeled E2-IgG and is shown in Figure 9. The binding affinity of DIG-labeled E2 antibody to FITC was calculated using PRISM software. As shown in the insert of Figure 9B, the Kd was about 0.66 nM, indicating that the DIG-labeled E2 anti-FITC antibody has high binding affinity to FITC and can be used for E2 binding to human normal tissues.

[0534] An IHC assay was developed to test the binding of E2 antibody to human tissues. Formalin-fixed, paraffin-embedded (FFEP) tissue sections of agar blocks with FITC-labeled KB cells were used as positive controls, and FFPE tissue sections of unlabeled KB cells were used as negative controls. The tissue sections were deparaffinized and rehydrated, then the antigens were retrieved by incubating the tissue sections with antigen retrieval buffer (pH 6.0) at 91°C for 24 minutes, then DIG-E2 antibody was incubated with the rehydrated tissue sections, and finally anti-DIG IHC staining was performed. Briefly, monoclonal anti-DIG antibody was added, followed by peroxidase-labeled anti-mouse secondary antibody, and incubated. As shown in Figure 10A, FITC-labeled KB cells showed positive staining, indicating that E2 antibody binds to the FITC on these labeled KB cells. Unlabeled KB cells did not show any staining (Figure 10B).

[0535] The DIG-labeled E2 antibody was incubated with a human multiple organ normal tissue microarray (FDA999, US Biomax, Inc.), and the binding of the E2 antibody to human normal tissue was evaluated using the same IHC method as above. This multi-organ normal tissue microarray has 99 cores with 28 types of 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 three normal human individuals were included in the microarray panel. As shown in Figures 11-38, no positive IHC staining was found in these 28 types of normal human organs tested, indicating that the E2 anti-FITC antibody had no binding to normal human organ tissues. Some plasma cells showed a certain level of staining intensity in both the test sections preincubated with DIG-E2 antibody and the negative control sections not preincubated with DIG-E2 antibody, indicating that this weak staining was nonspecific.

[0536] In conclusion, the E2 anti-FITC antibody does not bind to any of the human normal organ tissues tested, and the E2 anti-FITC CAR-T cells themselves do not bind to or attack any human normal tissues in vivo.

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

[0538] Example 5 Synthesis of FITC-PEG12-Folate [ka] General-purpose polyethylene glycol (PEG) Nova Tag TM The resin (0.2 g) was added to a peptide synthesis vessel and washed with isopropyl alcohol (i-PrOH) (3 x 10 mL) and dimethylformamide (DMF, 3 x 10 mL). 9-Fluorenylmethoxycarbonyl (Fmoc) deprotection was carried out using a 20% solution of piperidine in DMF (3 x 10 mL). A Kaiser test was performed to assess the progress of the reaction. The vessel was then charged with a DMF solution of Fmoc-L-glutamic acid 5-tert-butyl ester (Fmoc-Glu-(Ot-Bu)-OH) (23.5 mg), N,N-diisopropylethylamine (i-Pr2NEt) (4 eq.) and benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) (2 eq.). Fmoc deprotection was carried out using a 20% solution of piperidine in DMF (3 x 10 mL). The vessel was then washed with N 10A solution of Fmoc-TFA-Pte-OH (22.5 mg), DMF, i-Pr2NEt (4 equiv.) and PyBOP (2 equiv.) was added. Argon was bubbled for 2 h and the resin was washed with DMF (3×3 mL) and i-PrOH (3×3 mL). After swelling the resin in dichloromethane (DCM), a solution of 1 M hydroxybenzotriazole (HOBT) in DCM / trifluoroethane (TFE) (1:1) (2×3 mL) was added. Argon was bubbled for 1 h, the solvent was removed and the resin was washed with DMF (3×3 mL) and i-PrOH (3×3 mL). After swelling the resin in DMF, Fmoc-NH-(PEG) 12 A solution of -COOH (46.3 mg) in DMF, i-Pr2NEt (4 eq.) and PyBOP (2 eq.) were added. Argon was bubbled for 2 h and the resin was washed with DMF (3 x 3 mL) and i-PrOH (3 x 3 mL). Fmoc deprotection was carried out using a 20% solution of piperidine in DMF (3 x 10 mL). Kaiser test was performed to evaluate the progress of the reaction. A solution of FITC (Life Technologies, 21.4 mg) in DMF and i-Pr2NEt (4 eq.) were added to the vessel, then argon was bubbled for 2 h and the resin was washed with DMF (3 x 3 mL) and i-PrOH (3 x 3 mL). A solution of 2% NH2NH2 in DMF (2 x 2 mL) was then added to the vessel. The final compound was cleaved from the resin using TFA:H2O:triisopropylsilane (TIS) (95:2.5:2.5) (cleavage solution) and concentrated under reduced pressure. The concentrated product was precipitated with Et2O and dried under reduced pressure. The crude product was purified using preparative RP-ChPLC (mobile phase: A = 10 mM ammonium acetate pH = 7, B = ACN; method: 0% B to 30% B for 30 min, 13 mL / min). Pure fractions were collected and lyophilized to obtain FITC-PEG12-Folate.

[0539] Example 6 Synthesis of FITC-PEG20-Folate [ka] Ethylenediamine, polymer-bound (200-400 mesh) resin (50 mg) was added to a peptide synthesis vessel and swollen with DCM (3 mL) followed by DMF (3 mL). The vessel was then filled with Fmoc-PEG in DMF. 20 -COOH solution (131 mg, 1.0 equiv), i-Pr2NEt (6.0 equiv) and PyBOP (4.0 equiv). Argon was bubbled for 6 h, the coupling solution was drained and the resin was washed with DMF (3 × 10 mL) and i-PrOH (3 × 10 mL). Kaiser test was performed to evaluate the progress of the reaction. Fmoc deprotection was carried out using 20% ​​piperidine solution in DMF (3 × 10 mL) before each amino acid coupling. The above process was repeated to complete the reaction of Fmoc-Glu-OtBu (72 mg, 2.0 equiv) and Tfa.pteroic acid (41 mg, 1.2 equiv) coupling steps. The resin was washed with 2% hydrazine in DMF (3 × 10 mL) (5 min) to remove the trifluoro-acetyl protecting group of pteroic acid and then with i-PrOH (3 × 10 mL) followed by DMF (3 × 10 mL). The resin was dried under argon for 30 min. The folate-peptide was cleaved from the resin using cleavage solution. 10 mL of cleavage mixture was introduced and argon was bubbled through for 1.5 h. The cleavage mixture was poured into a new flask. The resin was washed three times with additional cleavage mixture. The combined mixture was concentrated under reduced pressure to a small volume (approximately 5 mL) and precipitated with ethyl ether. The precipitate was collected by centrifugation, washed with ethyl ether (3 times), and dried under high vacuum. 20 -EDA (1.0 equiv.) was treated with FITC (50 mg, 1.5 equiv.) in DMSO and DIPEA at room temperature. The progress of the reaction was monitored by LCMS. After 8 h, the starting material was consumed to the product. The crude reaction mixture was purified by preparative HPLC (mobile phase A=10 mM ammonium acetate, pH=7; mobile phase B=acetonitrile; method: 0% B to 30% B at 13 mL / min for 35 min) to give FITC-PEG20-folate in 60% yield.

[0540] Example 7 Synthesis of FITC-PEG108-Folate [ka] Ethylenediamine, polymer-bound (200-400 mesh) resin (50 mg) was placed in a peptide synthesis vessel and swollen with DCM (3 mL) followed by DMF (3 mL). The vessel was then filled with Fmoc-PEG in DMF. 36 -COOH solution (161 mg, 1.0 equiv), i-Pr2NEt (6.0 equiv) and PyBOP (4.0 equiv). Argon was bubbled for 6 h, the coupling solution was drained and the resin was washed with DMF (3×10 mL) and i-PrOH (3×10 mL). Kaiser test was performed to evaluate the progress of the reaction. Fmoc deprotection was performed using 20% ​​piperidine solution in DMF (3×10 mL) before each amino acid coupling. The above steps were repeated to obtain 2×Fmoc-PEG 36 The reaction of the coupling step was completed with -COOH (161 mg, 1.0 equiv.), Fmoc-Glu-OtBu (72 mg, 2.0 equiv.) and Tfa.pteroic acid (41.0 mg, 1.2 equiv.). Finally, the resin was washed with 2% hydrazine in DMF (3×10 mL) (5 min) to remove the trifluoro-acetyl protecting group of pteroic acid, followed by washing with i-PrOH (3×10 mL) and then DMF (3×10 mL). The resin was dried under argon for 30 min. The folate-peptide was cleaved from the resin using the cleavage solution. 10 mL of the cleavage mixture was introduced and bubbled with argon for 1.5 h. The cleavage mixture was poured into a new flask. The resin was washed three times with additional cleavage solution. The combined mixture was concentrated under reduced pressure to a small volume (approximately 5 mL) and precipitated with ethyl ether. The precipitate was collected by centrifugation, washed with ethyl ether (3x) and dried under high vacuum. 108-EDA (1.0 equiv.) was treated with FITC (50 mg, 1.5 equiv.) in DMSO and DIPEA at room temperature. The reaction progress was monitored by LCMS. After 10 h, the starting material was consumed to the product. The crude reaction mixture was purified by preparative HPLC (mobile phase A=10 mM ammonium acetate, pH=7; mobile phase B=acetonitrile; method: 0% B to 30% B at 13 mL / min for 35 min) to give FITC-PEG108-folate in 64% yield.

[0541] Example 8 Synthesis of FITC-DUPA [ka] DUPA-FITC was synthesized by the following solid phase method: Universal Nova Tag TMThe resin (50 mg, 0.53 mM) was swollen with DCM (3 mL) followed by DMF (3 mL). A solution of 20% piperidine in DMF (3 × 3 mL) was added to the resin and argon was bubbled in for 5 min. The resin was washed with DMF (3 × 3 mL) and isopropyl alcohol (i-PrOH, 3 × 3 mL). After swelling the resin in DMF, a DMF solution of DUPA-(OtBu)-OH (1.5 equiv.), HATU (2.5 equiv.) and i-Pr2NEt (4.0 equiv.) was added. After bubbling with argon for 2 h, the resin was washed with DMF (3 × 3 mL) and i-PrOH (3 × 3 mL). After swelling the resin with DCM, a solution of 1 M HOBt in DCM / TFE (1:1) (2 × 3 mL) was added. Argon was bubbled for 1 h, the solvent was removed, and the resin was washed with DMF (3 × 3 mL) and i-PrOH (3 × 3 mL). After swelling the resin in DMF, a DMF solution of Fmoc-Phe-OH (2.5 eq.), HATU (2.5 eq.) and DIPEA (4.0 eq.) was added. Argon was bubbled for 2 h, and the resin was washed with DMF (3 × 3 mL) and i-PrOH (3 × 3 mL). The above process was repeated for two more coupling steps to add 8-aminooctanoic acid and fluorescein isothiocyanate or rhodamine B isothiocyanate. The final compound was cleaved from the resin using the cleavage solution and concentrated under reduced pressure. The concentrated product was precipitated with diethyl ether and dried under reduced pressure. The crude product was purified using preparative RP Time PLC [λ = 488 nm; solvent gradient: 1% B to 80% B for 25 min run, 80% B wash for 30 min; A = 10 mM NH4OAc, pH = 7; B = acetonitrile (ACN)]. ACN was removed under reduced pressure and the purified fractions were lyophilized to give FITC-DUPA as a brownish-orange solid. RP Time PLC: tR = 8.0 min (A = 10 mM NH4OAc, pH = 7.0; B = ACN, solvent gradient: 1% B to 50% B for 10 min run, 80% B wash for 15 min). 1H NMR (DMSO-d6 / D2O): δ 0.98-1.27 (ms, 9H); 1.45 (b, 3H); 1.68-1.85 (ms, 11H); 2.03 (m, 8H); 2.6-3.44 (ms, 12H); 3.82 (b, 2H); 4.35 (m, 1H); 6.53 (d, J=8.1 Hz, 2H), 6.61 (dd, J=5.3, 3.5 Hz, 2H); 6.64 (s, 2H); 7.05 (d, J=8.2 Hz, 2H), 7.19 (m, 5H); 7.76 (d, J=8.0 Hz, 1H); 8.38 (s, 1H). HRMS (ESI)(m / z): (M+H) + C 51 H 59 N7O 15 S calculated 1040.3712, measured 1040.3702. UV / vis: λmax=491nm.

[0542] Example 9 Synthesis of FITC-PEG12-DUPA [ka] 1,2-Diaminoethanetrityl resin (0.025 g) was added to a peptide synthesis vessel and washed with i-PrOH (3×10 mL) followed by DMF (3×10 mL). The vessel was then charged with Fmoc-NH-(PEG). 12A solution of -COOH (42.8 mg) in DMF, i-Pr2NEt (2.5 equiv.) and PyBOP (2.5 equiv.) were added. The resulting solution was bubbled with Ar for 1 h, the coupling solution was drained and the resin was washed with DMF (3×10 mL) and i-PrOH (3×10 mL). A Kaiser test was performed to evaluate the progress of the reaction. Fmoc deprotection was carried out using a 20% piperidine solution in DMF (3×10 mL). This process was repeated until all coupling steps were completed (2×1.5 equiv. of Fmoc-Phe-OH and 1.5 equiv. of 8-aminooctanoic acid and 1.2 equiv. of DUPA were used in each of the coupling steps). After DUPA coupling, the resin was washed with DMF (3×10 mL) and i-PrOH (3×10 mL) and dried under reduced pressure. The peptide was cleaved from the resin in a peptide synthesis vessel using cleavage solution. 15 mL of cleavage solution was added to the peptide synthesis vessel and the reaction was bubbled under Ar for 15 min. The resin was treated with two additional 10 mL portions of cleavage solution for 5 min each. The cleavage mixture was concentrated to approximately 5 mL and precipitated with ethyl ether. The precipitate was collected by centrifugation, washed with ethyl ether (3x), and dried under high vacuum to recover the crude material. The crude DUPA-(PEG) 12 To a solution of -EDA (10 mg) and FITC (5.6 mg) in dimethyl sulfoxide (DMSO, 1 mL), i-Pr2NEt (5 equiv.) was added at room temperature and stirred under argon for 6 h. The reaction was monitored by LCMS and purified by preparative HPLC (mobile phase: A = 10 mM ammonium acetate pH = 7, B = ACN; method: 0% B to 50% B for 30 min, 13 mL / min). The purified fractions were collected and lyophilized to obtain FITC-PEG12-DUPA.

[0543] Example 10 Synthesis of FITC-PEG11-NK1 [ka] A mixture of NK-1 (0.02 g, 0.0433 mmol, 1.0 equiv.), O-(2-aminoethyl)-O'-[2-(Boc-amino)ethyl]decaethylene glycol (BocNH-PEG 11To a solution of benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) (0.027 g, 0.0521 mmol, 1.2 equiv.) and benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) (0.027 g, 0.0521 mmol, 1.2 equiv.) in dry CHCl was added N,N-diisopropylethylamine (DIPEA) (0.076 mL, 0.4338 mmol, 10 equiv.) at room temperature under argon. The reaction progress was monitored by LCMS and preparative RP-PLC (Waters, XBridge TM Purification was performed using a Prep C18, 5 μm; 19 × 100 mm column, mobile phase A = 20 mM ammonium acetate buffer, pH 7, B = acetonitrile, gradient 10 to 100% B for 30 min, 13 mL / min, λ = 220 nm, 254 nm). Pure fractions were collected, total organic solvent was evaporated, and the sample was lyophilized for 48 h to obtain NK1-PEG. 11 -NHBoc was obtained. Yield: 40.13 mg (97%). NK1-PEG 11 To a solution of -NHBoc (0.0165 g, 0.015 mmol) in dry DCM was added trifluoroacetic acid (TFA, 20 equiv.) and the reaction mixture was stirred for 4 h at rt. Excess TFA was removed and the remaining solution was diluted with water and extracted using CH2Cl2 (3 x 5 mL). The combined organic layers were washed with brine, dried (Na2SO4) and concentrated. The resulting residue was dried under reduced pressure and used in the next step without further purification. The stirred NK1-PEG 11 A solution of -NH2 (0.008 g, 0.0081 mmol, 1.0 equiv.), fluorescein isothiocyanate (FITC) (Sigma, 0.0037 g, 0.0097 mmol, 1.2 equiv.) in dry dimethylsulfoxide (DMSO, 0.3 mL) was added to diisopropylethylamine (0.0028 mL, 0.0162 mmol, 2.0 equiv.) at room temperature under argon. The reaction progress was monitored by LCMS and the products were separated by preparative RP-PLC (Waters, XBridge TMPurification was performed on a Prep C18, 5 μm; 19 × 100 mm column, mobile phase A = 20 mM ammonium acetate buffer, pH 7, B = acetonitrile, gradient 10 to 100% B for 30 min, 13 mL / min, λ = 280 nm). Pure fractions were collected, all organic solvents were evaporated, and the sample was lyophilized for 48 h to give FITC-PEG11-NK1 in 8.54 mg (77%) yield. *Note: NK-1 compounds were synthesized in a two-step process starting from base ligands prepared by methods described in the literature. (See DESIGN AND DEVELOPMENT OF NEUROKININ-1 RECEPTOR-BINDING AGENT DELIVERY CONJUGATES, Application No. PCT / US2015 / 44229; incorporated herein by reference.)

[0544] Example 11 FITC-PEG 2 Synthesis of -CA9 [ka] CA9 ligand (53.6 mg) was dissolved in DMF (2-3 mL) in a 50 mL round bottom flask using a Teflon magnetic stir bar. The outside air was removed using vacuum and replaced with nitrogen gas for three cycles. The round bottom flask was maintained under constant nitrogen gas. To the flask was added 28.9 mg of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), followed by 21.6 mg 1-hydroxybenzotriazole hydrate (HOBt) and 18.9 μL of Boc-PEG2-NH2 (Sigma Aldrich). 5.4 μL of triethylamine (TEA) was added and the reaction was stirred overnight. The reaction mixture was purified using HPLC and confirmed by UHPLC-MS (target m / z 831). Acetonitrile was removed by high vacuum rotary evaporation and the product was lyophilized. The compound was mixed with 1:1 TFA:DCM for 30 min. The TFA / DCM was removed by high vacuum rotary evaporation followed by high vacuum for 30 minutes. The compound was then dissolved in DMF and combined with 5 molar equivalents of i-Pr2NEt, 16 mg of fluorescein isothiocyanate (Life Technologies) and stirred for 1 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.

[0545] Example 12 FITC-DUPA [ka] DUPA-FITC was synthesized by the following solid phase method. Universal NovaTag resin (50 mg, 0.53 mM) was swollen with dichloromethane (DCM) (3 mL) followed by dimethylformamide (DMF, 3 mL). A 20% solution of piperidine in DMF (3 × 3 mL) was added to the resin and argon was bubbled in for 5 min. The resin was washed with DMF (3 × 3 mL) and isopropyl alcohol (i-PrOH, 3 × 3 mL). After swelling the resin in DMF, a DMF solution of DUPA-(OtBu)-OH (1.5 equiv.), HATU (2.5 equiv.) and DIPEA (4.0 equiv.) was added. Argon was bubbled in for 2 h and the resin was washed with DMF (3 × 3 mL) and i-PrOH (3 × 3 mL). After swelling the resin with DCM, a 1M solution of HOBt in DCM / trifluoroethane (TFE) (1:1) (2×3 mL) was added. Argon was bubbled for 1 h, the solvent was removed, and the resin was washed with DMF (3×3 mL) and i-PrOH (3×3 mL). After swelling the resin in DMF, a DMF solution of Fmoc-Phe-OH (2.5 equiv.), HATU (2.5 equiv.), and DIPEA (4.0 equiv.) was added. Argon was bubbled for 2 h, and the resin was washed with DMF (3×3 mL) and i-PrOH (3×3 mL). The above process was repeated for two more coupling steps to add 8-aminooctanoic acid and fluorescein isothiocyanate or rhodamine B isothiocyanate. The final compound was cleaved from the resin using a trifluoroacetic acid (TFA):H2O:triisopropylsilane:cocktail (95:2.5:2.5) and concentrated under reduced pressure. The concentrated product was precipitated with diethyl ether and dried under reduced pressure. The crude product was purified using preparative RP time PLC [λ=488 nm; solvent gradient: 1% B to 80% B for 25 min, 80% B wash for 30 min; A=10 mM NHOAc, pH=7; B=acetonitrile (ACN)]. The ACN was removed under reduced pressure and the pure fractions were lyophilized to give DUPA-FITC as a brownish-orange solid. RP time PLC: tR=8.0 min (A=10 mM NHOAc, pH=7.0; B=ACN, solvent gradient: 1% B to 50% B for 10 min, 80% B wash for 15 min). 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) + C 51 H 59 N7O 15 Calculated value for S is 1040.3712, measured value is 1040.3702. UV / vis: λmax = 491 nm.

[0546] Example 13 FITC-CA9

Chemical formula

[0547] Example 14 FITC-NK1R To a stirred solution of NK-1 (0.02 g, 0.0433 mmol, 1.0 equiv.), O-(2-aminoethyl)-O'-[2-(Boc-amino)ethyl]decaethylene glycol (BocNH-PEG-NH) (Sigma, 0.0336 g, 0.0521 mmol, 1.2 equiv.), and benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PYBOP) (0.027 g, 0.0521 mmol, 1.2 equiv.) in dry CHCl was added N,N-diisopropylethylamine (DIPEA) (0.076 mL, 0.4338 mmol, 10 equiv.) at room temperature under argon. Reaction progress was monitored by LCMS and preparative RP-time PLC (Waters, XBridge TM Purification was performed on a Prep C18, 5 μm; 19 × 100 mm column, mobile phase A = 20 mM ammonium acetate buffer, pH 7, B = acetonitrile, gradient 10 to 100% B for 30 min, 13 mL / min, λ = 220 nm, 254 nm). Pure fractions were collected, total organic solvent was evaporated, and the sample was freeze-dried for 48 h to give NK1-PEG11-NHBoc. Yield: 40.13 mg (97%). To a solution of NK1-PEG11-NHBoc (0.0165 g, 0.015 mmol) in dry CHCl was added trifluoroacetic acid (TFA, 20 equiv.) and the reaction mixture was stirred for 4 h at rt. Excess TFA was removed, diluted with water, and extracted using CHCl (3 × 5 mL). The combined organic layers were washed with brine, dried (NaSO), and concentrated. The resulting residue was dried under reduced pressure and used in the next step without further purification. To a stirring solution of NK1-PEG11-NH2 (0.008 g, 0.0081 mmol, 1.0 equiv.) and fluorescein isothiocyanate (FITC) (Sigma, 0.0037 g, 0.0097 mmol, 1.2 equiv.) in dry dimethylsulfoxide (DMSO, 0.3 mL) was added diisopropylethylamine (0.0028 mL, 0.0162 mmol, 2.0 equiv.) at room temperature under argon. Reaction progress was monitored by LCMS and preparative RP-PLC (Waters, XBridge). TMPurification was performed on a Prep C18, 5 μm; 19 × 100 mm column, mobile phase A = 20 mM ammonium acetate buffer, pH 7, B = acetonitrile, gradient 10 to 100% B for 30 min, 13 mL / min, λ = 280 nm). Pure fractions were collected, all organic solvents were evaporated, and the sample was lyophilized for 48 h to give NK1-PEG11-FITC (5). Yield: 8.54 mg (77%). The NK-1 compound was synthesized in a two-step process starting from the base ligand prepared by a method described in the literature (see DESIGN AND DEVELOPMENT OF NEUROKININ-1 RECEPTOR-BINDING AGENT DELIVERY CONJUGATES, Application No. PCT / US2015 / 44229, incorporated herein by reference in its entirety).

[0548] Example 15 In vitro co-culture E2 CAR T activation and target cell death assays E2 CAR T cells were thawed from cryopreservation at a cell density of 0.5–2 million T cells per milliliter of T cell growth medium (TexMACS medium + 2% human AB serum + 50 U / mL recombinant human IL-2) and allowed to recover for 4 days. Target cells expressing various levels of surface folate receptor were also thawed from cryopreservation and allowed to recover in growth medium (folate-deficient RPMI1640 + 10% FCS + pen / strep for 4 days). On day -1, 100,000 target cells were seeded in 2 ml of growth medium per well of a 12-well tissue culture plate to allow target cells to attach and recover. On day 0, wells containing EC17 bridge molecules were treated with 100 nM EC17 for 30 minutes in a tissue culture incubator. The entire medium was then washed out and replaced with fresh growth medium lacking folate and EC17. During the EC17 incubation, T cells were harvested and counted, and after completion of EC17 treatment, 100,000 E2 CAR T cells were added directly to each well of target cells in a total of 3 ml of growth medium at a 1:1 (E:T) ratio and cultured in standard tissue culture humidified conditions at 37°C and 5% CO2 for periods of 1 and 2 days, respectively. The 1-day co-culture condition was used to assay E2 CAR T cell activation by target cells, and the 2-day incubation was used to assay target cell death.

[0549] To determine E2 CAR T cell activity levels after 1 day of co-culture with target cells with various levels of surface folate receptors, floating cells were harvested together with the remaining adherent cells and saved together with the cells detached from the tissue culture plate using a 5 min 0.25% trypsin digestion. Saved floating and adherent cells were pelleted with a 5 min 400x gravity centrifugation step and resuspended in flow cytometry staining solution. Surface marker staining for E2 T cell identification by flow cytometry included anti-human EGFR and anti-human CD3, and detection of CAR T cell activation by flow cytometry used anti-human CD137 staining. E2 CAR T cells were identified as EGFR+CD3+, and activated CAR T cells also co-expressed CD137 (see Flow Cytometry Methods). To determine the efficiency of E2 CAR T cell killing of target cells expressing various levels of surface folate receptors after 2 days of co-culture, stored and adherent cells were pelleted in the same manner as in the 1-day co-culture assay described above and then stained for E2 CAR T markers EGFR and CD3, and then washed and stained for the apoptosis marker Annexin V by resuspending flow antibody stained samples in Alexa Fluor 647-conjugated recombinant Annexin V (Invitrogen cat# A23204, diluted 1:50 in 1× Annexin staining buffer provided) and 3 μM propidium iodide.

[0550] E2 CAR T cell activation is dependent on EC17 staining of target cells To determine the level of folate receptor required for activation of E2 CAR T cells, a one-day co-culture with target cells expressing different levels of surface folate receptor (FR) was performed (Figure 39). The percentage of E2 CAR T cells activated after one day of co-culture is graphed on the y-axis, and the FR expression level of the target cells at the time of the assay is graphed on the x-axis. Importantly, the FR expression level data was obtained on the same day as the assay performed using the same target cells treated with EC17 but cultured in the absence of CAR T cells to avoid any artifact of FR high expressing cells selected by CAR T cells. Four out of five target cell lines expressing a wide range of folate receptors activated CAR T cells to similar levels. Interestingly, the low FR expressing cell line OV90 (downward pointing triangle) expressed approximately 50% of the FRs of SKOV3 cells (diamonds) and activated approximately 50% of the CAR T cells of SKOV3 cells. These data suggest that a broad range of EC17s activate E2 CAR T cells to similar levels and that a certain threshold of EC17 binding to target cells is required for full activation of E2 CAR T cells. Furthermore, they suggest that there is a threshold of FR expression that confers suboptimal E2 CAR T responses.

[0551] E2 CAR T cell target cell killing To confirm whether E2 CAR T cell activation also translates into sufficient killing of target cells, a 2-day co-culture assay was performed, followed by monitoring target cell (EGFR- CD3-) events by flow cytometry and determining the percentage of target cells stained with the apoptotic surface marker, Annexin V staining. In Figure 40, the same five target cells (labeled on the x-axis) cultured for 2 days each under three different conditions are shown as three different bars. The first bar in each group represents the basal level of target cell apoptosis as target cells treated with EC17 but cultured in the absence of E2 CAR T cells. The second bar in each group represents CAR T + target cell co-culture without any prior EC17 treatment of the target cells, and the third bar in each group represents CAR T + EC17 pre-treated target cells. The y-axis represents the percentage of target cells that are apoptotic (Annexin V+). These data revealed that CAR T cell activation was successfully translated into target cell killing in the three highest folate receptor expressers (MDAMB231, HOS-FRα and IGROV1). However, for SKOV3 cells, which had the fourth lowest FR expression (Figure 39, x-axis), similar E2 CAR T activation did not translate into similar target cell death (Figure 40). It is possible that this separation of CAR T cell activation and target cell apoptosis in SKOV 3 cells may not be a function of low folate receptor expression. As mentioned earlier, each cancer cell may have evolved its own anti-apoptotic mechanisms, and FR expression levels in different cancer cell lines, but to a lesser extent in SKOV3 cells, may be sufficient for target cell apoptosis at high levels. Taken together, the data in Figures 39 and 40 show that EC17 binding to cancer cells is required for cell death. These data further suggest that below a certain threshold of target cell-bound EC17, there is a linear correlation between surface-bound EC17 and E2 CAR T activation and killing.

[0552] Example 16 3 H-Folate Binding Assay All tumor cell lines were seeded overnight in folate-free RPMI medium containing 10% heat-inactivated fetal bovine serum. The following day, cells were incubated for 15 min on ice with 100 nM 3 H-folate was incubated with or without 10 μM cold folic acid. After rinsing three times with cold 1× PBS, cells were lysed whole and total cell-associated radioactivity was counted in a scintillation counter. 3 FR-specific binding of H-folate was calculated by subtracting the counts from the folate-competitive sample. 3 The specific activity of H-folate was determined by calculating it as the number of molecules per cell. Figure 41 shows that these cell lines have different levels of FR expression, with the order of FR levels being IGROV1>MDA-MB-231, HOS-FRα>OV90, and SKOV3.

[0553] Example 17 EC17 / E2-CAR T cell therapy in NSG mice with and without subcutaneously implanted MDA-MB-231 tumors Tumor implantation MDA-MB-231 tumor cells were grown in folate-deficient RPMI 1640 containing 5–10% FBS at 37°C in a humidified 5% CO2 atmosphere. MDA-MB-231 tumor cells were cultured at 2.5 × 10 6 Cells were inoculated subcutaneously in 66 mice. Only 42 of 66 mice were inoculated, the remaining 22 mice remained tumor-free.

[0554] CAR-T cell administration EGFRt-sorted anti-FITC E2 scFv-CAR T cells were frozen in T cell freezing medium. Vials of frozen CAR-T cells were immediately stored at -80°C. CAR-T cells were rapidly thawed at 37°C, washed twice with PBS, and used for injection into animals at 10 million viable EGFRt+E2 CAR-T cells (CD4 / CD8, approximately 1:1) per animal. A small aliquot was taken on the day of infusion for flow cytometric analysis of E2-CAR T cell phenotype.

[0555] Human / Mouse Cytokine Analysis Mouse blood samples were processed for plasma and stored at -20°C until use. Human TH1 cytokine panel (Biolegend, Cat. No. 740009) and human hematopoietic stem cell cytokine panel (Biolegend, Cat. No. 740610) were used for detection of human cytokines in mouse blood. Mouse 13-plex inflammatory cytokine panel (Biolengend, No. 740150) was used for detection of mouse cytokines. All analyses were performed according to the manufacturer's instructions.

[0556] Flow cytometry analysis Whole blood cell analysis: Plasma was removed from a planned volume of whole EDTA-treated blood, and RBCs were lysed with RBC lysis solution. The leukocyte pellet was then resuspended in flow cytometry staining solution (1% bovine serum albumin, 50 mg / mL human IgG (Equitech Bio, cat#SLH56-0001), 0.9% sodium azide in phosphate-buffered saline, pH=7.4), and leukocyte surface marker staining was performed using the following antibodies: anti-human CD45 [clone HI30, eBioscience #47-0459-42, diluted 1:20 (v / v)], anti-human CD137 [clone 4B4-1, BD Bioscience #564092, diluted 1:20 (v / v)], anti-human CD8α [clone RPA-T8, BD Bioscience, catalog #557746, diluted 1:20 (v / v)], anti-human CD4 [clone SK3, eBioscience catalog #1001621, diluted 1:20 (v / v)], anti-human CD5 [clone SK6, eBioscience catalog #1001636, diluted 1:20 (v / v)], anti-human CD6 [clone SK5, eBioscience catalog #1001645, diluted 1:20 (v / v)], anti-human CD7 [clone SK6, eBioscience catalog #100165, diluted 1:20 (v / v)], anti-human CD8α [clone RPA-T8, BD Bioscience, catalog #100166, diluted 1:20 (v / v)], anti-human CD4 [clone SK3, eBioscience catalog #100166, diluted 1:20 (v / v)], anti-human CD5 [clone SK6, eBioscience catalog #10 #46-0047-42, diluted 1:20 (v / v)], anti-human EGFR [R&D systems, clone Hu1, catalog #FAB9577B, 1:10 (v / v)], anti-human PD-1 [BD Biosciences, clone EH12.1, catalog #562511, 1:20 (v / v)], anti-human LAG-3 [BD Biosciences, clone T47-530, catalog #565616, 1:20 (v / v)], anti-human TIM-3 [BD Biosciences, clone 7D3, catalog #565558, 1:20 (v / v)], and anti-human CD3ε [BD Biosciences, clone SK7, catalog #557832, 1:20 (v / v)]. After leukocyte staining, cells were washed with PBS and incubated with 53,000 CountBright TM The beads (Invitrogen catalog #C36950) were resuspended in cold PBS and transferred to flow cytometry collection tubes. Flow cytometry data were collected on a Gallios flow cytometer (Beckman Coulter, Brea, CA). The concentration of CAR T cells in each blood sample was calculated according to the Invitrogen instructions. CAR T cells were identified as human CD3ε+EGFRt+ events, which were easily differentiated and analyzed using the KaluzaTM Flow cytometry software was used to count. The number of circulating CAR T cells for each injected mouse was then graphed as the total number of CAR T cells per 100 μL of whole blood analyzed. Statistical significance was determined using an unpaired two-tailed Student's t-test, with significance set at p<0.05.

[0557] Tumor and tissue analysis: Solid tumors (100–1000 mm 3 ) were harvested, weighed, chopped into small pieces, and then transferred to a 50 mL tube containing 20 mL of tumor digestion cocktail. The enzymatic tumor digestion cocktail consisted of 0.5 mg / mL collagenase IV (Sigma-Aldrich, Catalog# C5138), 0.5 mg / mL hyaluronidase (Sigma-Aldrich, Catalog# H3506), and 0.1 mg / mL DNase I (Sigma-Aldrich, Catalog# DN25) in antibiotic-supplemented serum-free and folate-deficient RPMI1640 medium. The tumor fragments were digested for 1 hour at 37°C and 300 rpm on a horizontal shaker. The tumor digest was then centrifuged at 400×g for 5 minutes, and the tumor cell pellet was subjected to a red blood cell lysis step, then washed with cold phosphate-buffered saline (PBS, pH 7.4), and finally filtered through a 40 μm nylon cell strainer.

[0558] E2-CAR-T phenotype and proliferation in vivo The expansion and persistence of CAR T cells in blood has been reported to correlate with response to CD19-specific CAR T therapy in clinical trials. It is not unreasonable to speculate that circulating blood-derived E2 CAR T from a large pool would enable a greater response to EC17-directed attack of FR+ tumors in this in vivo model. As an indicator of the response of E2 CAR T cell therapy to EC17, we quantified the number of circulating E2 CAR T cells per 100 μL of whole blood and observed that cohorts that received EC17 stimulation (cohorts 2, 3, 4 and 5) had at least 10-fold more circulating E2 CAR T cells compared to cohorts that did not receive EC17 (cohorts 1 and 6). Importantly, this expansion of circulating CAR T cells in blood is consistent with CAR T cell expansion in an antigen-dependent manner. Furthermore, we observed that persistence of expanded numbers of circulating CAR T cells was up to 54 days after CAR T cell infusion, and that CAR T persistence was also EC17-dependent (cohorts 2, 3, 4 and 5) when compared to cohorts 1 and 6, which did not receive EC17.

[0559] Tumor vs. tumor-free mice As shown in Figure 42, 10 million E2 CAR-T cells were injected iv into tumor-free naive NSG mice or NSGMDA-MB-231 tumor-bearing mice. 500 nmol / kg EC17 was administered iv on days 2 and 10 after CAR-T injection, and mice were sacrificed 20 hours after the second EC17 administration (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 day 12 after CAR-T injection, another set of animals was obtained for FACS analysis as described above. As shown in Figure 43, cytokine production in tumor-bearing mice was EC17-dependent. Levels of cytokines including IL-2, IFN-γ, and IL-10 were all increased in tumor-bearing mice with EC17 compared to mice not treated with EC17. In naive mice without tumors, upregulation of cytokine production was also seen in mice treated with EC17, but the increase in tumor-free mice was much lower (e.g., 23-fold lower for IFN-γ) compared to mice bearing MDA-MB-231 tumors. Tumor-bearing mice had approximately 23-fold higher IFN-γ production than tumor-free mice. Tumor-free mice (FD-fed for approximately 2 months) showed some cytokine production after 2× EC17 SIW500 but no FACS-detectable CAR-T cell expansion.

[0560] Example 18 Target-specific activity using humanized anti-FITC scFv E2-CAR constructs and CAR-modified T cells was demonstrated in vitro Cell Lines: All cell lines were maintained in RPMI 1640 (Irvine Scientific) supplemented with 2 mM L-glutamine (Cellgro), 25 mM HEPES (Irvine Scientific) and 10% heat-inactivated FBS (Hyclone) unless otherwise stated. The K562 target cell line was kindly provided by Dr. Stanley Riddell (FHCRC). The K562 OKT3 cell line was generated by transduction with lentivirus containing the OKT3 transgene. OKT3 is a membrane-tethered scFv targeting CD3 epsilon. MDA-MB-231 was provided by Endocyte and cultured in folate-free DMEM. All cell lines were authenticated by STR Profiling matched to the DSMZ database at the University of Arizona Genetics Core.

[0561] Cytotoxicity and cytokine secretion: A 4-hour chromium release assay was performed. Briefly, target cells were 51 Cr (PerkinElmer) overnight, washed three times with PBS, and plated in triplicate in RPMI in 96-well plates at 5 × 10 3 T cells were incubated at various effector to target (E:T) ratios at 10 cells / well. Supernatants were harvested for γ-counting and specific lysis was calculated. For cytokine secretion, 5×10 5T cells were seeded in triplicate in 96-well plates at a 2:1 E:T ratio with target cells for 24 h, and supernatants were analyzed by cytometric bead array using a Bio-Plex human cytokine panel (Bio-Rad) according to the manufacturer's instructions. For EC17-labeled targets, cells were incubated with 100 nM EC17 in PBS for 1 h at room temperature in the dark before seeding. When EC17 was used, cells were incubated with folate-free RPMI throughout the assay.

[0562] Cytokine release assay : CD4+ T cells were co-cultured at a 2:1 ratio of T cells to targets for 24 hours, and then the supernatants were assayed for the presence of the effector cytokines IL-2, IFN-γ, and TNF-α. Assays were performed in triplicate, and data are presented as mean ± SE. As shown in Figure 44, we observed that mock and anti-FLCAR T cells produced quantitatively similar levels of cytokines in response to co-culture with the positive control cell line K562-OKT3. Cytokines were not secreted by mock or anti-FLCAR T cells in co-culture with K562. Cytokine release was dependent on prior incubation with EC17, and the MDA-MB-231 cell line and anti-FLCAR T cells were the only cells capable of inducing secretion of the cytokines IL-2, IFN-γ, and TNF-α against EC17-labeled MDA-MB-231.

[0563] Chromium release assay : CD8+ T cells were co-cultured with target cells at 30:1, 10:1, 3:1 or 1:1 ratios. Percentage lysis (mean ± SE) of triplicate wells is shown. As shown in Figure 45, anti-FLCAR T cells did not show cytotoxicity against the negative control (K562) or unlabeled MDA-MB-231 cell lines. However, mock-transduced and anti-FLCAR T cells were able to induce similar levels of specific lysis against the positive control K562-OKT3 cell line. Furthermore, EC17-labeled MDA-MB-231 cells were efficiently recognized and lysed by anti-FLCAR T cells, whereas mock T cells were unable to lyse them.

[0564] Example 19 Adaptor structure of CAR-T cells Figures 46-49 show the bridge structures used in the CAR-T cell experiments and their affinity determined in vitro for the cell type to which the bridge is directed (e.g., Folate-FITC for folate receptor expressing cancer cells). Figure 50 shows the binding of the bridge to tumor cells used in the in vivo model expressing a receptor corresponding to the small molecule ligand of the bridge (by FACS analysis).

[0565] Examples 20 to 30 - see Figures 55 to 63 Example 20 Cell lines and reagents Unless otherwise stated, all FR+ and FR-negative cancer cell lines were maintained in RPMI-1640 medium (Gibco BRL) supplemented with 10% heat-inactivated fetal bovine serum without (FFRPMI) or with (RPMI) 2.4 μM folic acid (FA). KB (FRα-expressing human cervical cancer with HeLa marker) and CHO-β (Chinese hamster ovary cells transfected with human FRβ) were used as FRα and FRβ sources, respectively, for radioligand binding assays. MDA-MB-231 represents an FRα subclone of a human triple-negative breast cancer (TNBC) cell line. A green fluorescent protein (GFP)-expressing isogenic pair of FRβ-positive (THP1-FRβ) and FR-negative (THP1-FG12) cell lines was provided for AML testing. Both were established from THP-1 (ATCC, TIB-202), a cell model commonly used to study pediatric AML, originally derived from a 1-year-old boy with acute monocytic leukemia. For osteosarcoma studies, HOS-FRα was established by lentiviral transduction of FR-negative HOS-143b (ATCC, CRL8303) with the FOLR1 gene encoding human FRα. HOS-143b was originally established from a primary tumor in a 13-year-old Caucasian girl and is highly tumorigenic in NSG mice. FR+HOS-FRα fLuc and FR-negative HOS-143b fLuc The GFP-expressing bioluminescent pair was transduced with a lentivirus containing firefly luciferase.

[0566] LEGENDplex TM Human cytokine panels were purchased from BioLegend (San Diego, CA). (登録商標) A non-radioactive cytotoxicity assay kit was purchased from Promega (Madison, WI). Commercially available anti-human antibodies used for multicolor flow cytometry were CD45RA (clone HI100), CD45RO (clone UCHL1), CD4 (clone SK3), and CD69 (clone FN50) from Thermo Fisher Scientific (Waltham, MA); CD3ε (clone SK7), CD8α (clone RPA-T8), CD137 / 4-1BB (clone 4B4-1), CD25 (clone M-A251), PD-1 (clone EH12.1), LAG-3 (clone T47-530), and TIM-3 (clone 7D3) from BD Bioscience (San Jose, CA); biotinylated anti-human EGFR (cetuximab, clone Hu1) from R&D Systems (Minneapolis, MN); and FRα (clone LK26) from BioLegend (San Diego, CA). Fluorophore-conjugated anti-biotin was also purchased from BioLegend. APC-conjugated anti-FITC mouse IgG2a / kappa antibody (clone NAWESLEE), CountBright TM Beads (Invitrogen), Annexin V staining buffer and AlexaFluor-647-conjugated Annexin V were purchased from Thermo Fisher Scientific. For tumor tissue enzymatic digestion, collagenase IV, hyaluronidase and DNase I were purchased in total from Sigma-Aldrich (St. Louis, MO). EC17 or folate-FITC [FA-(γ)-ethylenediamine-FITC] was synthesized in endocytes. 3H-EC17 was purchased from Moravek biochemicals (Brea, CA) with a specific activity of approximately 0.952 Ci / mmol or prepared by Endocyte from ViTrax (Placentia, CA) with FITC. 3 H-FA-(γ)-ethylenediamine was prepared by conjugation 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, sodium fluorescein dosing solution was purchased from Purdue Pharmacy (NDC 17478-250-25) with AK-FLUORO. (登録商標) Diluted from 25% (Fluorescein Injection, USP).

[0567] Example 21 Humanized CAR constructs and CAR-modified T cells Previous studies used a GFP+second generation anti-FITC scFv (clone 4M5.3) CAR, containing the hinge and transmembrane sequences of CD8α and the 4-1BB / CD3ζ signaling domain (i.e., FITC-4M5.3-scFv-CD8αhinge-CD8αtm-4-1BB / CD3ζ). To translate to initial studies in humans, a second generation fully human FITC-specific (clone E2) CAR construct (referred to here as E2) was developed (Figure 55, top). CAR-modified T cells are shown in Figure 55, bottom pie chart. The construct described here is a FITC-specific CAR construct consisting of (1) a fully human anti-FITC scFv (clone E2, Kd=0.75 nM), (2) an IgG4 hinge-CH2(L235D, N297Q)-CH3 spacer fused to a CD28 transmembrane domain, (3) a second generation 4-1BB / CD3ζ endodomain, and (4) a cell surface human EGFRt tag (Figure 55, top) (SEQ ID NOs: 1 and 2 are the nucleic acid and amino acid sequences, respectively). To generate CAR-modified T cells, lentivirus was produced in 293T cells co-transfected with a CAR-encoding epHIV7 lentiviral vector. Donor CD4+ and CD8+ T cells were purified by immunomagnetic selection and transduced separately or in a 50:50 ratio. In general, 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 pre-cryopreservation and post-thaw to determine EGFRt expression and CD4 / CD8 ratios by flow cytometry. A combination of surface markers was used to analyze the differentiation status of CD4+ and CD8+ CAR-T cell subsets on the day of infusion and to characterize T N , CD45RA+CD45RO-CD62L+CD95- naive T cells; T SCM , CD45RA+CD45RO-CD62L+CD95+ stem cell memory T cells;T CM , CD45RA-CD45RO+CD62L+CD95+ central memory T cells;T EM , CD45RA-CD45RO+CD62L-CD95+ effector memory cells; and T EFF EGFRt was defined as CD45RA+CD45RO-CD62L-CD95+ effector T cells. For the preclinical studies described below, studies included two batches of EGFRt sorted pure CD4 and CD8 subsets (after mixing in an approximately 1:1 ratio) and several batches of unsorted approximately 1:1 EGFRt+CD4 / CD8 mixtures, including a "clinical replica" preparation with a poorly differentiated profile. Among a series of different CAR constructs that were synthesized and evaluated, a fully human anti-FITC scFv (FITC-E2) CAR was selected for preclinical development (Figure 55). This second generation fully human CAR consisted of an anti-FITC scFv (clone E2), an IgG4-Fc spacer / hinge with a double mutation in the CH2 region (L235D and N297Q) to reduce binding to FcγR, a CD28 transmembrane domain, and a 4-1BB / CD3ζ signaling domain appended to a cell surface EGFRt tag with a T2A ribosomal skip sequence (i.e., FITC-E2-scFv-IgG4 hinge-CD28tm-4-1BB / CD3ζ-T2A-EGFRt). For preclinical trials, both EGFRt-sorted and non-sorted E2 CAR-T cells were prepared at approximately 1:1 CD4 / CD8 ratio, and T cell subtype phenotyping was routinely performed by flow cytometry at the time of CAR T cell infusion (day 0) for each in vivo experiment. The typical expression pattern of EGFRt-sorted CAR-T cells was approximately 42% T SCM , 10%T CM , 12%T EM and 34%T EFF The EGFRt-sorted CAR-T cells consisted of both CD4 and CD8 subsets (Figure 55, left pie chart). Only EGFRt-sorted CAR-T cells were used for co-culture (Figures 53, 54, 56, 58, 59 and 61) and pharmacokinetic studies (Figure 62). For tumor therapy, a "clinical replica" batch (Figure 55, right pie chart) with a poorly differentiated profile was used, and this "clinical batch" was also used for MDA-MB-231 studies (Figure 63), and the research batch was used for THP1-FRβ and HOS-FRα studies (Figure 57). The "clinical replica" batch (approximately 39% EGFRt+) consisted of approximately 66% T SCM and about 32%T CM CD4+ subsets and approximately 95%T SCM and 3%T CM The study batch (approximately 23% EGFRt+) was more differentiated, with 32% T SCM , 53%T CM , 11%T EM and 3.7%T EFF CD4 subsets and 44%T SCM, 0.28%T CM , 3.4%T EM and 52%T EFF The CD8 subset consisted of

[0568] Example 22 Bispecific affinity of EC17 CAM Bispecific affinity of EC17 CAM was determined in a cell-based radioligand binding assay. 3 For binding to FR+ targets, KB and CHO-β cells were pre-seeded overnight in 24-well tissue culture plates and diluted with 0.1 nM, 0.5 nM, 1 nM, 5 nM, 10 nM, 20 nM and 40 nM in FFRPMI. 3 H-EC17 for 2 hours at 37°C. Cells were then rinsed with phosphate-buffered saline (PBS, pH 7.4) and lysed with 1% sodium dodecyl sulfate. Total cell lysates were quantified for radioactivity levels and cellular protein content by standard Pierce BCA protein assay. 3 The number of H-EC17 molecules was calculated to determine the dissociation constants (Kd) of FRα (KB) and FRβ (CHO-β), respectively (FIG. 56).

[0569] Main component of CAM-regulated CAR T cell therapy The basic components of CAR-T cell therapy include FITC as a pseudo-tumor antigen, the high affinity EC17 CAM (CAM is equivalent to bridge or "compound" herein) and rationally designed anti-FITC CAR and CAR-modified T cells. EC17 has already been tested in the clinic for immunotherapy and optical imaging purposes. To directly quantify its bispecific binding affinity, 3H-EC17 was synthesized and radioligand binding assays were performed on KB and CHO-β cell lines representing FRα+ and FRβ+ target cells, respectively, and on unsorted EGFRt CAR-T cells representing effector cells. Upon binding to targets, EC17 showed similar affinity to both FRα and FRβ with low Kd values ​​of 1.7 nM and 0.8 nM, respectively (Figure 56, Panel A). Upon binding to unsorted E2-CAR-T cells (approximately 24% EGFRt+, approximately 95:5 CD8 / CD4 ratio), the Kd value was estimated to be approximately 130 nM (Figure 56, Panel B).

[0570] Example 23 Tumor models All animal care and use was in accordance with NIH guidelines and protocols approved by the Purdue University Animal Use and Care Committee. Female 4-5 week old NOD / SCID gamma (NSG TM ) mice (stock number: 005557) were purchased from The Jackson Laboratory (Bar Harbor, ME). All animals were maintained on an FA-deficient diet (TestDiet, St. Louis, MO) from the time of arrival and throughout the study unless otherwise specified. To establish subcutaneous xenografts, MDA-MB-231 and HOS-FRα were each administered at 2.5 × 10 per animal. 6 cells and 1 x 10 6 For intravenous xenografting, THP1-FRβ cells were implanted at 5 × 10 per animal in the right flank region. 6 Subcutaneous tumors were measured with calipers 2-3 times a week and ellipsoidal (length × width) 2 ) / 2. The survival time was determined according to the study design or when (i) animals lost ≥ 20% of their body weight or approached moribund status, or (ii) subcutaneous tumor size was ≥ 1500 mm 3 or (iii) when animals showed abdominal distension and signs of severe distress (i.e., THP1-FRβ). All animal doses (CAR-T cells, EC17, sodium fluorescein) were given intravenously.

[0571] Example 24 Tumor Treatment In a therapeutic setting, EC17 CAM could theoretically be given before or after CAR-T cell injection. As described here, the first dose of EC17 was given 2-3.5 days after CAR-T cells to ensure an observation period of human T cells in tumor-bearing mice. Two batches of unsorted E2 CAR-T cells (23% or 39% EGFRt+, 1:1 CD4 / CD8) were used in the in vivo studies. On the day of injection (day 0) for each experiment, frozen CAR-T cells were rapidly thawed at 37°C, washed twice with Dulbecco's 1x PBS (pH 7.4), and injected into the tail vein at the desired EGFRt+E2-CAR-T cell dose. Additionally, a small amount of CAR-T cells was tested by flow cytometry for CD4 to CD8 ratio and CAR-T cell differentiation status. On the first day of EC17 administration, tumor-bearing animals were assigned to groups according to tumor size or the same number of days after intravenous implantation (i.e., THP1-FRβ).

[0572] Example 25 Toxicity and CRS rescue Depending on the CAR-T cell dose and the method of administration of EC17, tumor-bearing mice receiving FITC-specific CAR-T cells may experience severe CRS and various degrees of weight loss. Therefore, a CRS scoring system (0-5 scale) was developed to experimentally evaluate CRS toxicity from the macroscopic morphology and social behavior of animals to allow good timing of CRS rescue. Grades 0 and 5 were normal (no CRS) or death (due to severe CRS), respectively, and grades 1, 2, and 3-4 were considered as mild, mild to moderate, and severe CRS (Figure 57A, table description). Furthermore, all EC17 doses were intentionally given towards the end of the day so that possible CRS symptoms would develop at night. Immediately following each EC17 dose, animals were scored and rescue agents such as sodium fluorescein, folic acid, and leucovorin were used for CRS toxicity reduction. In this particular case, a batch of E2 CAR-T cells with a high EGFRt+CD4 / CD8 ratio (approximately 93:7) was given to HOS-FRα tumor-bearing mice as a mixture of approximately 2.5 million CD8 and approximately 33 million CD4 on day 0 (Figure 57A, schematic). When EC17 was given at 500 nmol / kg, 100 nmol / kg and 500 nmol / kg, respectively, on days 3, 10 and 12, low CRS (grades 1-2) was shown on days 4 and 11, while high CRS (grade 3) was shown on day 13. To mitigate severe CRS toxicity, sodium fluorescein was administered intravenously at approximately 96 mg / kg on the morning of day 13, and mouse plasma samples were collected 6 hours later for analysis of CRS-associated cytokines. To investigate the ability of sodium fluorescein to rescue CRS, a CRS scoring system was developed that was able to capture the onset of severe CRS (grade 3 / 4) after EC17 administration (Figure 57A). As shown by the challenge of rapid CRS onset in HOS-FRα tumor-bearing mice with a very high dose of 35 million total CAR-T cells (93:7 CD4 / CD8), intravenous sodium fluorescein at a human equivalent dose (approximately 95 mg / kg in mice) acted extremely fast in reducing CAR-T cell-derived human cytokines (Figure 57B).

[0573] Example 26 statistics Statistical analysis was performed using the computer program GraphPad Prism (GraphPad Software Inc., San Diego, CA). Data were analyzed using Student's t-test or one-way ANOVA. Where applicable, ...

Claims

1. 1. A method of treating cancer, comprising: i) administering to a patient a compound or a pharma- ceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand attached to a targeting moiety by a linker; ii) administering to the patient a first dose of a CAR T cell composition comprising a CAR T cell, wherein the CAR T cell comprises a CAR directed to a targeting moiety, the CAR comprising an E2 anti-fluorescein antibody fragment; and iii) administering to the patient a second dose of a CAR T cell composition comprising the CAR T cell, wherein the CAR T cell comprises a CAR directed to a targeting moiety, the CAR comprising an E2 anti-fluorescein antibody fragment. A method comprising:

2. The method of claim 1 , wherein the compound or a pharma- ceutically acceptable salt thereof is not an antibody and does not include an antibody fragment.

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

4. 2. The method of claim 1, wherein the cancer is a folate receptor-expressing cancer.

5. 10. The method of claim 1, wherein CRS is reduced or prevented and the method results in a reduction in tumor volume in the patient.

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

7. 2. The method of claim 1, wherein the CD8:CD4 ratio in the CAR T cells is about 1:

1.

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

9. 10. The method of claim 1, wherein the cancer comprises a tumor and a complete tumor response is obtained.

10. 1. A method of treating cancer, comprising: i) administering to a patient a compound or a pharma- ceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand attached to a targeting moiety by a linker; and ii) administering to the patient a CAR T cell composition, wherein the CAR T cell composition comprises a CAR T cell, the CAR T cell comprises a CAR directed to a targeting moiety, the CAR comprises an E2 anti-fluorescein antibody fragment, and the CAR T cell composition comprises a mixture of CAR T cells and non-transformed T cells. A method comprising:

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

12. 11. The method of claim 10, wherein the CAR T cell composition comprises a mixture of CAR T cells and non-transformed T cells at a ratio of about 1:1 to about 1:5 CAR T cells to non-transformed T cells.

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

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

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

16. 1. A method of treating cancer, comprising: i) administering to a patient a compound or a pharma- ceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand attached to a targeting moiety by a linker; ii) administering to the patient a CAR T cell composition, wherein the CAR T cell composition comprises a CAR T cell, the CAR T cell comprising a CAR directed to a targeting moiety, the CAR comprising an E2 anti-fluorescein antibody fragment; and iii) administering to the patient folate, a conjugate comprising folate without a targeting moiety, or an agent that inhibits activation of CAR T cells. A method comprising:

17. 17. The method of claim 16, wherein a folate is administered, the folate being folic acid or leucovorin.

18. The method of claim 16 , wherein a folate-containing conjugate is administered that does not include a targeting moiety.

19. 17. The method of claim 16, wherein an agent that inhibits activation of a CAR T cell is administered, wherein the agent blocks CAR T cell binding of the compound or a pharma- ceutically acceptable salt thereof but does not bind to the cancer.

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

21. 20. The method of claim 19, wherein the agent is fluoresceinamine, FITC, or sodium fluorescein.

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

23. 1. A method of treating cancer, comprising: i) administering to a patient a compound or a pharma- ceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand attached to a targeting moiety by a linker, and wherein the compound or a pharma- ceutically acceptable salt thereof is administered at a dose of about 10 nM / kg to about 2500 nM / kg of the patient's body weight; and ii) administering to the patient a CAR T cell composition comprising a CAR T cell, wherein the CAR T cell comprises a CAR, the CAR comprising an E2 anti-fluorescein antibody fragment, and wherein the CAR T cell is at a dose of about 1 million CAR T cells to about 15 million CAR T cells. A method comprising:

24. 24. The method of claim 23, wherein the compound or a pharma- ceutically acceptable salt thereof is administered at a dose of about 10 nM / kg to about 100 nM / kg of patient body weight.

25. 24. The method of claim 23, wherein the compound or a pharma- ceutically acceptable salt thereof is administered at a dose of about 10 nM / kg to about 50 nM / kg of patient body weight.

26. 24. The method of claim 23, wherein the compound or a pharma- ceutically acceptable salt thereof is administered at a dose of about 10 nM / kg to about 20 nM / kg of patient body weight.

27. 24. The method of claim 23, wherein the compound or a pharma- ceutically acceptable salt thereof is administered at a dose of about 10 nM / kg to about 600 nM / kg of patient body weight.

28. 24. The method of claim 23, wherein the compound or a pharma- ceutically acceptable salt thereof is administered at a dose of about 200 nM / kg to about 600 nM / kg of patient body weight.

29. 1. A method of treating cancer, comprising: i) continuously administering to the patient a compound, wherein the compound comprises a small molecule ligand attached to a targeting moiety by a linker; ii) administering to the patient a CAR T cell composition comprising a CAR T cell, wherein the CAR T cell comprises a CAR, the CAR comprising an E2 anti-fluorescein antibody fragment; and iii) terminating the continuous administration of the compound, or a pharma- ceutically acceptable salt thereof, to inhibit or prevent cytokine release syndrome in the patient. A method comprising:

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

31. 30. The method of claim 29, wherein the compound or a pharma- ceutically acceptable salt thereof is administered to the patient for at least 4 consecutive hours.

32. 30. The method of claim 29, wherein the compound or a pharma- ceutically acceptable salt thereof is administered to the patient for at least 6 consecutive hours.

33. 30. The method of claim 29, wherein the compound or a pharma- ceutically acceptable salt thereof is administered to the patient three times per week.

34. 1. A method of treating cancer, comprising: i) administering to a patient a compound or a pharma- ceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand attached to a targeting moiety by a linker, and wherein the patient is administered at least a first dose and a second dose of the compound or a pharma- ceutically acceptable salt thereof, wherein the first dose and the second dose are different, and wherein the second dose of the compound or a pharma- ceutically acceptable salt thereof is from about 2-fold to about 15,000-fold greater than the amount of the first dose of the compound or a pharma- ceutically acceptable salt thereof; and ii) administering to the patient a CAR T cell composition comprising a CAR T cell, wherein the CAR T cell composition comprises a CAR T cell, the CAR T cell comprising a CAR directed to a targeting moiety, the CAR comprising an E2 anti-fluorescein antibody fragment. A method comprising:

35. 35. The method of claim 34, wherein at least a first dose, a second dose, and a third dose of a compound or a pharma- ceutically acceptable salt thereof are administered to a patient, wherein the first dose, the second dose, and the third dose are different, the second dose of the compound or a pharma- ceutically acceptable salt thereof being about 2-fold to about 750-fold greater than the amount of the first dose of the compound or a pharma- ceutically acceptable salt thereof, and the third dose of the compound or a pharma- ceutical acceptable salt thereof being about 800-fold to about 10,000-fold greater than the amount of the first dose of the compound or a pharma- ceutical acceptable salt thereof.

36. 35. The method of claim 34, wherein at least a first dose, a second dose, a third dose, and a fourth dose of a compound or a pharma- ceutically acceptable salt thereof are administered to the patient, wherein the first dose, the second dose, the third dose, and the fourth dose are different, the second dose of the compound or a pharma- ceutically acceptable salt thereof being about 2-fold to about 750-fold greater than the amount of the first dose of the compound or a pharma- ceutical acceptable salt thereof, the third dose of the compound or a pharma- ceutical acceptable salt thereof being about 800-fold to about 7500-fold greater than the amount of the first dose of the compound or a pharma- ceutical acceptable salt thereof, and the fourth dose of the compound or a pharma- ceutical acceptable salt thereof being about 8000-fold to about 15000-fold greater than the amount of the first dose of the compound or a pharma- ceutical acceptable salt thereof.

37. 35. The method of claim 34, wherein the second dose of the compound or its pharma- ceutically acceptable salt is about 100 times greater than the amount of the first dose of the compound or its pharma- ceutically acceptable salt, the third dose of the compound or its pharma- ceutically acceptable salt is about 1000 times greater than the amount of the first dose of the compound or its pharma- ceutically acceptable salt, and the fourth dose of the compound or its pharma- ceutically acceptable salt is about 10000 times greater than the amount of the first dose of the compound or its pharma- ceutically acceptable salt.

38. 1. A method of treating cancer, comprising: i) administering to a patient a first dose of a compound or a pharma- ceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand attached to a targeting moiety by a linker; ii) administering to the patient at least a second dose of the compound or a pharma- ceutically acceptable salt thereof, wherein the second dose of the compound or a pharma- ceutically acceptable salt thereof is at least about 50% lower than the amount of the first dose of the compound or a pharma- ceutically acceptable salt thereof; and iii) administering to the patient a dose of a CAR T cell composition comprising a CAR T cell, wherein the CAR T cell composition comprises a CAR T cell, the CAR T cell comprising a CAR directed to a targeting moiety, the CAR comprising an E2 anti-fluorescein antibody fragment. A method comprising:

39. 39. The method of claim 38, wherein the second dose of the compound or a pharma- ceutically acceptable salt thereof is at least about 60% lower than the amount of the first dose of the compound or a pharma- ceutically acceptable salt thereof.

40. 39. The method of claim 38, wherein the second dose of the compound or a pharma- ceutically acceptable salt thereof is at least about 70% lower than the amount of the first dose of the compound or a pharma- ceutically acceptable salt thereof.

41. 39. The method of claim 38, wherein the second dose of the compound or a pharma- ceutically acceptable salt thereof is at least about 80% lower than the amount of the first dose of the compound or a pharma- ceutically acceptable salt thereof.

42. 39. The method of claim 38, wherein the second dose of the compound or a pharma- ceutically acceptable salt thereof is at least about 90% lower than the amount of the first dose of the compound or a pharma- ceutically acceptable salt thereof.

43. 39. The method of claim 38, wherein the second dose of the compound or a pharma- ceutically acceptable salt thereof is at least about 95% lower than the amount of the first dose of the compound or a pharma- ceutically acceptable salt thereof.

44. 1. A method of treating cancer, comprising: i) administering to the patient a first dose of a compound or a pharma- ceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand attached to a targeting moiety by a linker, and wherein the compound or a pharma- ceutically acceptable salt thereof is administered to the patient prior to administration of a CAR T cell composition comprising a CAR T cell comprising 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 pharma- ceutically acceptable salt thereof, wherein the CAR comprises an E2 anti-fluorescein antibody fragment. A method comprising:

45. 1. A method of treating cancer, comprising: i) administering to the patient a compound or a pharma- ceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand attached to a targeting moiety by a linker, and wherein the compound or a pharma- ceutically acceptable salt thereof is administered to the patient once a week; and ii) administering to the patient a CAR T cell composition comprising a CAR T cell, wherein the CAR T cell comprises a CAR, the CAR comprising an E2 anti-fluorescein antibody fragment. A method comprising:

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