RU2792653C2 - Methods and compositions for car-t-cell therapy - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: present invention relates to the field of biotechnology, in particular to a new therapeutic method; it can be used in medicine. The invention discloses a method for the treatment of malignant neoplasm expressing NK-1 receptor, using a composition containing CAR-T-cells and NK-1 receptor-binding ligand (NK1RL) conjugate with fluorescein isothiocyanate (FITC) as active therapeutic agents.

EFFECT: obtainment of methods and compositions for CAR-T-cell therapy.

40 cl, 19 dwg, 1 tbl, 19 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

According to the present application under 35 U.S.C. 119(e) claims the priority of U.S. Provisional Application Serial No. 62/320183, filed April 8, 2016, and U.S. Provisional Application Serial No. 62/323971, filed April 18, 2016, both of which are incorporated herein by reference in their entirety.

FIELD OF TECHNOLOGY

The present disclosure relates to methods for treating a cancer patient by administering to the patient a composition comprising CAR-T cells and administering to the patient a small molecule linked to a targeting group via a linker. The description also relates to compositions for use in such methods.

BACKGROUND OF THE INVENTION

Immunotherapy based on the adoptive transfer of lymphocytes (eg, T cells) to a patient is a very useful therapy for the treatment of malignant tumors and other diseases. Many important improvements have been made in the development of immunotherapy methods based on adoptive lymphocyte transfer. Among the many different types of immunotherapeutic agents, one of the most promising immunotherapeutic agents developed are T-cells expressing chimeric antigen receptors (CAR-T cells). A chimeric antigen receptor (CAR) is a genetically engineered receptor designed to target a specific antigen, such as a tumor antigen. This targeting can result in cytotoxicity against a tumor, such that CAR-T cells expressing CAR can target tumors and kill tumors via tumor-specific antigens, for example. First-generation CARs consist of a recognition region, such as a single chain fragment of an antibody-derived variable region (scFv), for recognition and binding of tumor-expressed antigen, and an activation signaling domain, such as the CD3ζ chain of T cells, can signal T activation. -cells in CAR. Although CAR-T cells have shown positive results in vitro , they have had limited success in eradicating disease (eg, cancer) in clinical studies. One problem has been the inability to prolong the activation and expansion of the CAR-T cell population in vivo . To address this problem, a co-stimulatory domain (eg, CD137, CD28, or CD134) is included in second-generation CARs to achieve prolonged in vivo T-cell activation. The addition of a co-stimulatory domain increases the proliferation and in vivo survival of CAR-containing T cells, and initial clinical data have shown that such constructs are promising drugs for the treatment of diseases such as cancer. Although improvements have been made in CAR-T cell therapies, several problems remain. First, "off-target" toxicity may occur due to normal cells expressing an antigen targeted by CAR-T cells (eg, tumor-associated antigen). Second, unregulated CAR-T cell activation can be detected, where the rapid and uncontrolled killing of diseased cells (e.g., cancer cells) by CAR-T cells induces a set of metabolic disorders called tumor lysis syndrome, in the case of tumor treatment, or cytokine release syndrome (CRS), which can be lethal to patients. Tumor lysis syndrome and CRS can result from the introduction of CAR-T cells that cannot be easily regulated and are activated uncontrollably. Accordingly, while CAR-T cells have shown promise as a tool for treating diseases such as cancer, additional CAR-T cell therapies are needed that provide reduced off-target toxicity and more precise control of CAR-T cell activation. .

SUMMARY OF THE INVENTION

The present inventors have discovered ways to reduce off-target toxicity and more precisely control CAR-T cell activation, providing important benefits for CAR-T cell therapy. In various embodiments described herein, a small molecule ligand linked to a targeting group via a linker is used as a bridge between the cancer and CAR-T cells, targeting the CAR-T cells to the cancer to alleviate the cancer. In one embodiment, the "small molecular weight ligand" may be, for example, folate, DUPA, NK-1R ligand, CAIX ligand, gamma-glutamyl transpeptidase ligand, or CCK2R ligand, each of which is a small molecule ligand that specifically binds to cancer cells. (ie, the receptor for these ligands is overexpressed on malignant tumors compared to normal tissues).

In one embodiment, the "small molecular weight ligand" is linked to a "targeting group" that binds to a CAR expressed by CAR-T cells. In various embodiments, the "targeting group" may be selected from, for example, 2,4-dinitrophenol (DNP), 2,4,6-trinitrophenol (TNP), biotin, digoxigenin, fluorescein, fluorescein isothiocyanate (FITC), NHS- fluorescein, pentafluorophenyl ester (PFP), tetrafluorophenyl ester (TFP), nottin, centirin and darpin. The "targeting group" binds to the recognition region of the genetically engineered CAR expressed by CAR-T cells. Accordingly, a CAR recognition region (eg, a single chain variable region (scFv) fragment of an antibody) is directed to a "target group". Thus, the small molecule ligand linked to the targeting group via a linker acts as a "bridge" between the cancer and CAR-T cells, targeting the CAR-T cells to the cancer to alleviate the cancer.

In one exemplary embodiment, the inventors have discovered that changing the dose of a small molecule ligand linked to a targeting group via a linker (ie, a bridge) can result in the ability to control CRS in vivo . In another embodiment, the inventors discovered that changing the linker in the small molecule ligand associated with the targeting group (bridge) can provide control of CRS in vivo after activation of CAR-T cells. In another embodiment, combinations of these methods can be used to precisely control CAR-T cell activation and cytokine release in vivo . In another embodiment, the affinity of the small molecule ligand for its receptor on cancer can be altered to control CAR-T cell activation, or to achieve cancer specificity while avoiding toxicity to normal tissues.

In one embodiment, the invention relates to a method for treating cancer. The method includes i) administering to the patient a first dose of a compound or a pharmaceutically acceptable salt thereof, wherein the compound contains a low molecular weight ligand linked to the targeting group via a linker, ii) administering to the patient a CAR-T cell composition, wherein the CAR-T cell contains a CAR directed to a targeting group, ii) administering to the patient a second dose of the compound, or a pharmaceutically acceptable salt thereof, wherein the second dose is different from the first dose, and treating the patient to alleviate the cancer.

In another embodiment, the invention relates to a method for treating cancer. The method includes i) administering to the patient a first conjugate or a pharmaceutically acceptable salt thereof, ii) administering to the patient a CAR-T cell composition, wherein the CAR-T cell contains a CAR directed to a targeting group, iii) administering to the patient a second conjugate or a pharmaceutically acceptable salt thereof where each of the first and second conjugates contains a small molecule ligand linked to the targeting group via a linker, and where the first conjugate and the second conjugate are different, and iv) treating the patient to alleviate the condition of a malignant tumor.

In another embodiment, the invention relates to a method for treating cancer. The method comprises i) administering to the patient a first dose of the first conjugate or a pharmaceutically acceptable salt thereof, ii) administering to the patient a CAR-T cell composition, wherein the CAR-T cell contains a CAR directed to a targeting group, ii) administering to the patient a second dose of the second conjugate, or a pharmaceutically acceptable salt thereof, wherein the first conjugate and the second conjugate each contain a small molecule ligand associated with a targeting group, wherein the first conjugate and the second conjugate are different, and wherein the first dose and the second dose are different, and iv) treating the patient to alleviate the condition malignant tumor.

In another exemplary embodiment, the invention relates to a CAR-T cell comprising a nucleic acid comprising SEQ ID NO: 1. In another aspect, the invention relates to a CAR-T cell comprising a polypeptide comprising SEQ ID NO: 2. In another aspect, the invention relates to a CAR-T cell comprising a polypeptide comprising SEQ ID NO: 2. embodiment, the invention relates to an isolated nucleic acid containing SEQ ID NO: 1 and encoding a chimeric antigen receptor. In another embodiment, the invention relates to a chimeric antigen receptor polypeptide comprising SEQ ID NO: 2. In another aspect, the invention relates to a vector comprising SEQ ID NO: 1. In another exemplary embodiment, the invention relates to a vector comprising SEQ ID NO: : 1, where the vector is a lentiviral vector.

Several embodiments are also described by means of the following numbered paragraphs:

1. A method for treating a malignant tumor, comprising

i) administering to the patient a first dose of a compound or a pharmaceutically acceptable salt thereof, wherein the compound contains a low molecular weight ligand linked to the targeting group via a linker;

ii) administering to the patient a CAR-T cell composition, wherein the CAR-T cell contains a CAR directed to the targeting group;

ii) administering to the patient a second dose of the compound, or a pharmaceutically acceptable salt thereof, wherein the second dose is different from the first dose; And

iv) treating the patient to alleviate the condition of the malignancy.

2. A method for treating a malignant tumor, comprising

i) administering to the patient the first conjugate or a pharmaceutically acceptable salt thereof;

ii) administering to the patient a CAR-T cell composition, wherein the CAR-T cell contains a CAR directed to the targeting group;

iii) administering to the patient the second conjugate or a pharmaceutically acceptable salt thereof,

where each of the first and second conjugate contains a low molecular weight ligand associated with the targeting group through a linker, and where the first conjugate and the second conjugate are different; And

iv) treating the patient to alleviate the condition of the malignancy.

3. A method for treating a malignant tumor, comprising

i) administering to the patient a first dose of the first conjugate or a pharmaceutically acceptable salt thereof;

ii) administering to the patient a CAR-T cell composition wherein the CAR-T cell contains a CAR directed to a targeting group;

ii) administering to the patient a second dose of the second conjugate or a pharmaceutically acceptable salt thereof,

where each of the first conjugate and the second conjugate contains a small molecular weight ligand associated with the targeting group, where the first conjugate and the second conjugate are different, and where the first dose and second dose are different; And

iv) treating the patient to alleviate the condition of the malignancy.

4. The method of claim 2 or 3, wherein the linker in the first conjugate or a pharmaceutically acceptable salt thereof and the linker in the second conjugate or a pharmaceutically acceptable salt thereof are different.

5. The method of claim 2 or 3, wherein the linker in the first conjugate or a pharmaceutically acceptable salt thereof and the linker in the second conjugate or a pharmaceutically acceptable salt thereof are the same.

6. The method according to any one of paragraphs 2-5, wherein the ligand in the first conjugate, or a pharmaceutically acceptable salt thereof, and the ligand in the second conjugate, or a pharmaceutically acceptable salt thereof, are different.

7. The method according to any one of paragraphs 2-5, wherein the ligand in the first conjugate or a pharmaceutically acceptable salt thereof and the ligand in the second conjugate or a pharmaceutically acceptable salt thereof are the same.

8. The method of any one of paragraphs 2-7, wherein the targeting group in the first conjugate or pharmaceutically acceptable salt thereof and the targeting group in the second conjugate or pharmaceutically acceptable salt thereof are different.

9. The method of any one of paragraphs 2-7, wherein the targeting group in the first conjugate or pharmaceutically acceptable salt thereof and the targeting group in the second conjugate or pharmaceutically acceptable salt thereof are the same.

10. The method of any one of items 1-9, wherein the ligand is selected from folate, DUPA, NK-1R ligand, CAIX ligand, gamma-glutamyl transpeptidase ligand, and CCK2R ligand.

11. The method of claim 10 wherein the ligand is folate.

12. The method of claim 10 wherein the ligand is an NK-1R ligand.

13. The method of claim 10 wherein the ligand is DUPA.

14. The method of claim 10 wherein the ligand is a CCK2R ligand.

15. The method of claim 10, wherein the ligand is a gamma-glutamyl transpeptidase ligand.

16. The method of any one of 1-15 wherein the targeting group is selected from 2,4-dinitrophenol (DNP), 2,4,6-trinitrophenol (TNP), biotin, digoxigenin, fluorescein, fluorescein isothiocyanate (FITC), NHS- fluorescein, pentafluorophenyl ester (PFP), tetrafluorophenyl ester (TFP), nottin, centirin and darpin.

17. The method of claim 16 wherein the targeting group is FITC.

18. The method of claim 16 wherein the targeting group is DNP.

19. The method of claim 16 wherein the targeting group is TNP.

20. The method of any one of claims 1-19, wherein the linker comprises polyethylene glycol (PEG), polyproline, hydrophilic amino acid, sugar, non-natural peptidoglycan, polyvinylpyrrolidone, and/or pluronic F-127.

21. The method of claim 20 wherein the linker contains PEG.

22. The method according to any one of paragraphs 1-21, where the compound or its pharmaceutically acceptable salt, the first conjugate or its pharmaceutically acceptable salt, or the second conjugate or its pharmaceutically acceptable salt has the formula

,

,

where B is a low molecular weight ligand, L is a linker, and T is a targeting group, and where L contains a structure having the formula

,

,

where n is an integer from 0 to 200.

23. The method of claim 22, where n is an integer from 0 to 150.

24. The method of claim 22, where n is an integer from 0 to 110.

25. The method of claim 22, where n is an integer from 0 to 20.

26. The method according to paragraph 22, where n is an integer from 15 to 20.

27. The method according to paragraph 22, where n is an integer from 15 to 110.

28. The method of any one of paragraphs 1-27, wherein the linker comprises PEG and the targeting group is FITC or a pharmaceutically acceptable salt thereof.

29. The method according to any one of paragraphs 1-28, where the dose of the compound or its pharmaceutically acceptable salt, the first conjugate or its pharmaceutically acceptable salt, or the second conjugate or its pharmaceutically acceptable salt, is from about 10 nmol/kg to about 3000 nmol/kg body weight of the patient.

30. The method according to any one of paragraphs 1-29, where the dose of the compound or its pharmaceutically acceptable salt, the first conjugate or its pharmaceutically acceptable salt, or the second conjugate or its pharmaceutically acceptable salt, is from about 50 nmol/kg to about 2000 nmol/kg body weight of the patient.

31. The method according to any one of paragraphs 1-30, where the dose of the compound or its pharmaceutically acceptable salt, the first conjugate or its pharmaceutically acceptable salt, or the second conjugate or its pharmaceutically acceptable salt is from about 100 nmol/kg to about 1000 nmol/kg body weight of the patient.

32. The method according to any one of paragraphs 1-31, where the dose of the compound or its pharmaceutically acceptable salt, the first conjugate or its pharmaceutically acceptable salt, or the second conjugate or its pharmaceutically acceptable salt, is from about 100 nmol/kg to about 600 nmol/kg body weight of the patient.

33. The method according to any one of paragraphs 1-32, where the dose of the compound or its pharmaceutically acceptable salt, the first conjugate or its pharmaceutically acceptable salt, or the second conjugate or its pharmaceutically acceptable salt, is from about 200 nmol/kg to about 500 nmol/kg body weight of the patient.

34. The method according to any one of paragraphs 1-33, where the dose of the compound or its pharmaceutically acceptable salt, the first conjugate or its pharmaceutically acceptable salt, or the second conjugate or its pharmaceutically acceptable salt is from about 250 nmol/kg to about 500 nmol/kg body weight of the patient.

35. The method of any one of 1-34, wherein the cancer is selected from lung cancer, bone cancer, pancreatic cancer, skin cancer, head cancer, neck cancer, skin melanoma, intraocular melanoma, uterine cancer, ovarian cancer, endometrial cancer , rectal cancer, gastric cancer, colon cancer, breast cancer, triple negative breast cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, malignant endocrine system tumors, thyroid cancer, parathyroid cancer, non-small cell lung cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, prostate cancer, chronic leukemia, acute leukemia, lymphocytic lymphoma, pleural mesothelioma, bladder cancer , Burkitt's lymphoma, malignant tumor rosacea, kidney cancer, renal cell carcinoma, renal pelvis carcinoma, central nervous system (CNS) neoplasia, primary CNS lymphoma, spinal cord tumors, brainstem glioma, pituitary adenoma, and adenocarcinoma of the gastroesophageal junction.

36. The method of any one of 1-11 or 16-35, wherein the cancer is a folate receptor-expressing cancer.

37. The method of claim 35 wherein the cancer is endometrial cancer.

38. The method of claim 35 wherein the cancer is non-small cell lung cancer.

39. The method of claim 35 wherein the cancer is ovarian cancer.

40. The method of claim 35 wherein the cancer is triple negative breast cancer.

41. The method of any one of items 1-40, wherein the CAR has a recognition region and the recognition region is a single chain fragment of an antibody variable region (scFv).

42. The method of any one of 1-11, 16-17, or 20-41, wherein the CAR has a recognition region and the CAR recognition region is a single chain fragment of the variable region (scFv) of an anti-FITC antibody.

43. The method of any one of 1-42, wherein CAR has a costimulatory domain and the costimulatory domain is selected from CD28, CD137 (4-1BB), CD134 (OX40), and CD278 (ICOS).

44. The method of any one of 1-43, wherein the CAR has an activation signaling domain and the activation signaling domain is a CD3ζ T cell chain or an Fcγ receptor.

45. The method of any one of 1-11, 16-17, or 20-41, wherein the CAR has a recognition region and the recognition region is a single chain variable region (scFv) fragment of an anti-FITC antibody, wherein the CAR has a costimulatory domain and the costimulatory the domain is CD137 (4-1BB), and wherein CAR has an activation signaling domain and the activation signaling domain is a CD3ζ T cell chain.

46. The method of any one of items 1-45, wherein multiple doses of a compound or a pharmaceutically acceptable salt thereof, a first conjugate or a pharmaceutically acceptable salt thereof, or a second conjugate or a pharmaceutically acceptable salt thereof, and a CAR-T cell composition are administered.

47. The method according to any one of paragraphs 1-46, wherein the patient is subjected to imaging prior to administration of the compound or a pharmaceutically acceptable salt thereof, the first conjugate or a pharmaceutically acceptable salt thereof, or the second conjugate or a pharmaceutically acceptable salt thereof, or prior to the administration of the CAR-T cell composition .

48. The method of any one of items 1-47, wherein the compound or a pharmaceutically acceptable salt thereof, the first conjugate or a pharmaceutically acceptable salt thereof, or the second conjugate or a pharmaceutically acceptable salt thereof, is not an antibody and does not contain an antibody fragment.

49. The method of any one of items 1-48, wherein the targeting group is not a peptide epitope.

50. The method of any one of items 1-49, wherein cytokine release resulting in "off-target" toxicity in the patient does not occur, and where toxicity of the CAR-T cell to the cancer occurs.

51. The method of any one of items 1-50, wherein "off-target" tissue toxicity does not occur in the patient, and where toxicity of the CAR-T cell to the cancer occurs.

52. The method of any one of items 1-51, wherein the cancer contains a tumor, where the size of the tumor is reduced in the patient, and where "off-target" toxicity does not occur.

53. CAR-T cell containing a nucleic acid containing SEQ ID NO: 1.

54. CAR-T cell containing a polypeptide containing SEQ ID NO: 2.

55. An isolated nucleic acid containing SEQ ID NO: 1 and encoding a chimeric antigen receptor.

56. Chimeric antigen receptor polypeptide comprising SEQ ID NO: 2.

57. Vector containing SEQ ID NO: 1.

58. The vector of item 57, wherein the vector is a lentiviral vector.

59. The method, a CAR-T cell, an isolated nucleic acid encoding a chimeric antigen receptor (CAR) or a chimeric antigen receptor polypeptide according to any one of 1-56, wherein the CAR comprises human amino acid sequences.

60. The method, a CAR-T cell, an isolated nucleic acid encoding a chimeric antigen receptor (CAR) or a chimeric antigen receptor polypeptide according to any one of 1-56, wherein the CAR is composed of human amino acid sequences.

61. A set containing at least two different types of bridges containing a small molecular weight ligand associated with a targeting group, where the ligands in at least two different types of bridges are different, and where the ligand is selected from folate, DUPA, CAIX ligand, NK- ligand 1R, gamma-glutamyl transpeptidase ligand and CCK2R ligand.

62. The kit of item 61, wherein the ligand in at least one of the bridges is an NK-1R ligand.

63. The kit of item 61, wherein the ligand in at least one of the bridges is a gamma-glutamyl transpeptidase ligand.

64. The kit of item 61 wherein the ligand in at least one of the bridges is folate.

65. The set according to any one of paragraphs 61-64, where the bridge has the formula

,

,

where B is a low molecular weight ligand, L is a linker, and T is a targeting group, and where L contains a structure having the formula

,

,

where n is an integer from 0 to 200.

66. The set of paragraph 65, where n is an integer from 0 to 150.

67. The set of paragraph 65, where n is an integer from 0 to 110.

68. The set of clause 65, where n is an integer from 0 to 20.

69. The set of paragraph 65, where n is an integer from 15 to 20.

70. The set of paragraph 65, where n is an integer from 15 to 110.

71. The method of any one of items 1-10, 16-52, or 59-60, or the kit of any of items 61-70, wherein the ligand is a CAIX ligand.

72. Formula conjugate

,

,

,

или

,

or

.

.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGURES 1A-B show the proliferation of CAR-T cells using FITC-small molecule conjugates in various cell types with a ratio of (CAR-T cells):target cells (cancer cells) of 5:1. Figure 1A shows the proliferation of CAR-T cells in KB (FR+) cells. Figure 1B shows the proliferation of CAR-T cells in HEK293 (NK1R+) cells.

FIGURES 2A-F show the production of the pro-inflammatory cytokine IFN-γ by CAR-T cells using FITC-small molecule conjugates in various cell types. Figure 2A shows the production of the pro-inflammatory cytokine IFN-γ in KB (FR+) cells. Figure 2B shows the production of the pro-inflammatory cytokine IFN-γ in LNCaP (PSMA+) cells. Figure 2C shows the production of the pro-inflammatory cytokine IFN-γ in HEK293 (NK1R+) cells. Figure 2D shows the production of the pro-inflammatory cytokine IFN-γ in KB (FR+) cells using various concentrations of FITC-folate. Figure 2E shows the production of the pro-inflammatory cytokine IFN-γ in KB (FR+) cells using various conjugates. Figure 2F shows the production of the pro-inflammatory cytokine IFN-γ in KB (FR+) cells using various conjugates.

FIGURES 3A-F show in vitro toxicity to tumor cells treated with FITC-small molecule conjugates in various cell types. Figure 3A shows in vitro toxicity to KB (FR+) cells. Figure 3B shows in vitro toxicity to LNCaP (PSMA+) cells. Figure 3C shows in vitro toxicity to HEK293 (NK1R+) cells. Figure 3D shows in vitro toxicity to KB (FR+) cells as a function of various E:T ratios (effector cells:target cells). Figure 3E shows in vitro toxicity to KB (FR+) cells as a function of FITC-folate concentration. Figure 3F shows in vitro toxicity to KB (FR+) cells using various conjugates.

FIGURES 4A-B show that CAR-T cell activation correlates with the level of tumor antigen expression on cancer cells. Figure 4A shows the level of tumor antigen FRα. The highest peak is present for KB (FR+) cells. Figure 4B shows CAR-T cell activation using FITC-small molecule conjugates as measured by IFN-γ production in MDA-MB-231 and KB cells.

FIGURES 5A-C show HEK293 (NK1R+) tumor xenografts and CAR-T cell therapy involving treatment with CAR-T cells with or without a FITC-PEG11-NK1 conjugate. Figure 5A shows tumor volume measured over 24 days. Figure 5B shows body weight measured over 22 days of therapy. Figure 5C shows the percentage of CAR-T cells in human CD3+ T cells after injection of CAR-T cells along with FITC-PEG11-NK1.

FIGURES 6A-B show organs harvested from exemplary mice from the models used in FIGS. 5A-C. Figure 6A shows organs harvested from the untreated group. Figure 6B shows organs harvested after two weeks of CAR-T cell therapy.

FIGURES 7A-C show MDA-MB-231 (FR+) xenografts under CAR-T cell therapy, which includes treatment of cells with CAR-T cells with FITC-PEG12-folate conjugate, with or without FITC-folate conjugate. Figure 7A shows tumor volume measured over 23 days. Figure 7B shows body weight measured over 21 days of therapy. Figure 7C shows the percentage of CAR-T cells in human CD3+ T cells after injection of CAR-T cells.

FIGURES 8A-B show organs harvested from exemplary mice from the models used in FIGS. 7A-C. Figure 8A shows organs harvested from the untreated group. Figure 8B shows organs harvested after three weeks of CAR-T cell therapy, including CAR-T cells and FITC-PEG12-folate conjugate at 500 nmol/kg body weight.

FIGURE 9 shows blood counts for the HEK293 (NK1R+) xenograft model of Figures 5-6 and for the MDA-MB-231 (FR+) xenograft model of Figures 7-8.

FIGURE 10 shows differences in cytotoxicity against CAR-T cell-treated KB (FR+) tumor cells depending on the FITC-small molecule conjugate used.

FIGURE 11 shows the percentage change in body weight in a KB tumor xenograft model using CAR-T cells with various concentrations of FITC-PEG-12-folate conjugate.

FIGURES 12A-C show organs harvested from exemplary mice from the KB xenograft models shown in Figure 11. Figure 12A shows organs harvested from the untreated group. Figure 12B shows organs harvested from the CAR-T cell therapy group treated with 250 nmol/kg FITC-PEG-12-folate. Figure 12C shows organs harvested from the CAR-T cell therapy group treated with CAR-T cells and 500 nmol/kg FITC-PEG-12-folate.

FIGURE 13 shows the blood counts of mice from the KB xenograft model of Figures 11-12.

FIGURES 14A-B show constructs used for CAR-T transduction. Figure 14A shows the construction of CAR4-1BBZ. Figure 14B shows a lentiviral vector.

FIGURES 15A-B show flow cytometry analysis of transduced T cells. Figure 15A shows non-transduced cells. Figure 15B shows transduced cells.

FIGURES 16A-B show fluorescence microscopy of transduced CAR-T cells. Figure 16A shows GFP imaging showing transduction. Figure 16B shows FITC-folate located on positively transduced cells.

FIGURE 17 shows CAR-T cell activation as measured by relative CD69 expression as a function of conjugate used.

FIGURE 18 shows tumor heterogeneity for KB, LNCaP and CAR-T cells as a function of conjugate used.

) NK1R+(HEK): CAR-T-клетки+PBS; Фигура 19B: (•) FR+ (MDA-MB-231): CAR-T-клетки+PBS, (

) NK1R+(HEK): CAR-T-клетки+FITC-PEG11-NK1R (500 нмоль/кг); Фигура 19C: (•) FR+ (MDA-MB-231): CAR-T-клетки+FITC-PEG12-FA (500 нмоль/кг), (

) NK1R+(HEK): CAR-T-клетки+PBS.FIGURES 19A-C show anti-tumor efficacy when identical anti-FITC CAR T cells (10 ⁷ cells) were injected into mice bearing two different tumors derived from two different cell lines (i.e. MDA-MB-231(FR+) and HEK (NK1R+)) on different sides, after which only PBS (Figure 19A), FITC-PEG11-NK1R (500 nmol/kg) (Figure 19B), or FITC-PEG11-NK1R (500 nmol/kg) plus FITC- PEG12-folate (500 nmol/kg) (Figure 19C) was injected every second day. Figure 19A: (•) FR+ (MDA-MB-231): CAR-T cells+PBS, (

) NK1R+(HEK): CAR-T cells+PBS; Figure 19B: (•) FR+ (MDA-MB-231): CAR-T cells+PBS, (

) NK1R+(HEK): CAR-T cells+FITC-PEG11-NK1R (500 nmol/kg); Figure 19C: (•) FR+ (MDA-MB-231): CAR-T cells + FITC-PEG12-FA (500 nmol/kg), (

) NK1R+(HEK): CAR-T cells+PBS.

DEFINITIONS

As used herein, the singular forms may refer to one or more. As used herein, "approximately" in relation to a numerical value, including, for example, whole numbers, fractions, and percentages, generally refers to a range of numerical values (for example, +/- 5% - 10% of the specified value) that a person skilled in the art in the field is considered equivalent to the specified value (eg, having the same function or result). As used herein, the terms "treat", "treatment", "treated", or "treatment" refer to both therapeutic treatment and prophylactic or preventative treatment.

As used herein, the terms "alleviate", "relieve", "alleviate", or "alleviate" in relation to a cancer may refer to a reduction in the symptoms of a cancer, a decrease in the size of a tumor, complete or partial removal of a tumor (e.g., complete or partial response), producing stable disease, preventing cancer progression (eg, progression-free survival), or any other effect on cancer that a therapist may consider to be a therapeutic or prophylactic treatment for cancer.

As used herein, the terms "administer", "administration" or "administered" refer to all methods of administering a compound or a pharmaceutically acceptable salt thereof, a first conjugate or a pharmaceutically acceptable salt thereof, a second conjugate or a pharmaceutically acceptable salt thereof, or a CAR-T composition. -cells described herein to a patient including, but not limited to, oral (po), intravenous (iv), intramuscular (im), subcutaneous (sc), and transdermal.

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

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

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

In various embodiments described herein, a small molecule ligand linked to a targeting group via a linker is used as a bridge between the cancer and CAR-T cells (ie, cytotoxic T cells expressing a chimeric antigen receptor). The bridge directs CAR-T cells to the cancer to alleviate the condition of the cancer. In one embodiment, the "small molecular weight ligand" may be folate, CAIX ligand, DUPA, NK-1R ligand, gamma-glutamyl transpeptidase ligand, or CCK2R ligand, each of which is a small molecule ligand that specifically binds to a cancer cell type (t i.e., the receptor for each of these ligands is overexpressed on malignant tumors compared to normal tissues). The "targeting group" associated with the small molecule ligand binds to the recognition region of the genetically engineered CAR expressed by CAR-T cells. Accordingly, a CAR recognition region (eg, a single chain variable region (scFv) fragment of an antibody) is directed to a "target group". Thus, the small molecule ligand linked to the targeting group via a linker acts as a bridge between the cancer and CAR-T cells, targeting the CAR-T cells to the cancer to alleviate the cancer. In various embodiments, the bridge between the cancer and CAR-T cells can be any of the conjugates shown in Examples 5-12.

The bridge is a small organic molecule such that clearance from the bloodstream can be rapidly achieved (eg, in about 20 minutes or less). In one aspect, the CAR-T cell response can target only those cancer cells that express the receptor for the small molecule ligand portion of the bridge, thus reducing off-target toxicity to normal tissues. In another aspect, CAR-T cell activation can be controlled by rapidly clearing the bridge from the circulation and the ability to vary the dose and structure of the bridge to regulate CAR-T cell activation. In addition, this system can be "universal" in that one type of CAR-T cell construct can be used to target a wide variety of cancers. By way of illustration, the targeting group recognized by the CAR-T cell can remain constant so that one type of CAR-T cell construct can be used while the small molecule ligand that binds to the cancer is altered to allow targeting to a wide variety of malignant tumors. In one embodiment, the inventors have discovered that changing the dose of a small molecule ligand linked to a targeting group via a linker (ie, a bridge) may result in the ability to control CRS in vivo upon activation of a CAR-T cell. In another embodiment, the inventors discovered that by changing the linker in the small molecular weight ligand associated with the targeting group (bridge), it is possible to control CRS in vivo after CAR-T cell activation. In another embodiment, combinations of these methods can be used to precisely control CAR-T cell activation and cytokine release in vivo . In the various embodiments described in the list of items below and in the claims, the small molecule ligand linked to the targeting group via a linker is referred to as a "compound", "first conjugate", or "second conjugate". The term "compound" is used in an embodiment where the dose of the small molecule ligand linked to the targeting group via a linker is varied to control the release of cytokines in vivo . The terms "first conjugate" and "second conjugate" are used in embodiments where two different conjugates are administered to a patient. For example, the linker in the small molecule ligand associated with the targeting group can be changed to control the release of cytokines in vivo , or the conjugates can be modified to contain different small molecular weight ligands or different targeting groups.

Several embodiments are described in the following numbered paragraphs:

1. A method for treating a malignant tumor, comprising

i) administering to the patient a first dose of a compound or a pharmaceutically acceptable salt thereof, wherein the compound contains a low molecular weight ligand linked to the targeting group via a linker;

ii) administering to the patient a CAR-T cell composition wherein the CAR-T cell contains a CAR directed to a targeting group;

ii) administering to the patient a second dose of the compound, or a pharmaceutically acceptable salt thereof, wherein the second dose is different from the first dose; And

iv) treating the patient to alleviate the condition of the malignancy.

2. A method for treating a malignant tumor, comprising

i) administering to the patient the first conjugate or a pharmaceutically acceptable salt thereof;

ii) administering to the patient a CAR-T cell composition wherein the CAR-T cell contains a CAR directed to a targeting group;

iii) administering to the patient the second conjugate or a pharmaceutically acceptable salt thereof,

where each of the first and second conjugate contains a low molecular weight ligand associated with the targeting group through a linker, and where the first conjugate and the second conjugate are different; And

iv) treating the patient to alleviate the condition of the malignancy.

3. A method for treating a malignant tumor, comprising

i) administering to the patient a first dose of the first conjugate or a pharmaceutically acceptable salt thereof;

ii) administering to the patient a CAR-T cell composition wherein the CAR-T cell contains a CAR directed to a targeting group;

ii) administering to the patient a second dose of the second conjugate or a pharmaceutically acceptable salt thereof,

where each of the first conjugate and the second conjugate contains a small molecular weight ligand associated with the targeting group, where the first conjugate and the second conjugate are different, and where the first dose and second dose are different; And

iv) treating the patient to alleviate the condition of the malignancy.

4. The method of claim 2 or 3, wherein the linker in the first conjugate or a pharmaceutically acceptable salt thereof and the linker in the second conjugate or a pharmaceutically acceptable salt thereof are different.

5. The method of claim 2 or 3, wherein the linker in the first conjugate or a pharmaceutically acceptable salt thereof and the linker in the second conjugate or a pharmaceutically acceptable salt thereof are the same.

6. The method according to any one of paragraphs 2-5, wherein the ligand in the first conjugate, or a pharmaceutically acceptable salt thereof, and the ligand in the second conjugate, or a pharmaceutically acceptable salt thereof, are different.

7. The method according to any one of paragraphs 2-5, wherein the ligand in the first conjugate, or a pharmaceutically acceptable salt thereof, and the ligand in the second conjugate, or a pharmaceutically acceptable salt thereof, are the same.

8. The method of any one of paragraphs 2-7, wherein the targeting group in the first conjugate or pharmaceutically acceptable salt thereof and the targeting group in the second conjugate or pharmaceutically acceptable salt thereof are different.

9. The method of any one of paragraphs 2-7, wherein the targeting group in the first conjugate or pharmaceutically acceptable salt thereof and the targeting group in the second conjugate or pharmaceutically acceptable salt thereof are the same.

10. The method of any one of items 1-9, wherein the ligand is selected from folate, DUPA, CAIX ligand, NK-1R ligand, gamma-glutamyl transpeptidase ligand, and CCK2R ligand.

11. The method of claim 10 wherein the ligand is folate.

12. The method of claim 10 wherein the ligand is an NK-1R ligand.

13. The method of claim 10 wherein the ligand is DUPA.

14. The method of claim 10 wherein the ligand is a CCK2R ligand.

15. The method of claim 10, wherein the ligand is a gamma-glutamyl transpeptidase ligand.

16. The method of any one of 1-15 wherein the targeting group is selected from 2,4-dinitrophenol (DNP), 2,4,6-trinitrophenol (TNP), biotin, digoxigenin, fluorescein, fluorescein isothiocyanate (FITC), NHS- fluorescein, pentafluorophenyl ester (PFP), tetrafluorophenyl ester (TFP), nottin, centirin and darpin.

17. The method of claim 16 wherein the targeting group is FITC.

18. The method of claim 16 wherein the targeting group is DNP.

19. The method of claim 16 wherein the targeting group is TNP.

20. The method of any one of claims 1-19, wherein the linker comprises polyethylene glycol (PEG), polyproline, hydrophilic amino acid, sugar, non-natural peptidoglycan, polyvinylpyrrolidone, and/or pluronic F-127.

21. The method of claim 20 wherein the linker contains PEG.

22. The method according to any one of paragraphs 1-21, where the compound or its pharmaceutically acceptable salt, the first conjugate or its pharmaceutically acceptable salt, or the second conjugate or its pharmaceutically acceptable salt, has the formula

,

,

where B is a low molecular weight ligand, L is a linker, and T is a targeting group, and where L contains a structure having the formula

,

,

where n is an integer from 0 to 200.

23. The method of claim 22, where n is an integer from 0 to 150.

24. The method of claim 22, where n is an integer from 0 to 110.

25. The method of claim 22, where n is an integer from 0 to 20.

26. The method according to paragraph 22, where n is an integer from 15 to 20.

27. The method according to paragraph 22, where n is an integer from 15 to 110.

28. The method of any one of paragraphs 1-27, wherein the linker comprises PEG and the targeting group is FITC or a pharmaceutically acceptable salt thereof.

29. The method according to any one of paragraphs 1-28, where the dose of the compound or its pharmaceutically acceptable salt, the first conjugate or its pharmaceutically acceptable salt, or the second conjugate or its pharmaceutically acceptable salt is from about 10 nmol/kg to about 3000 nmol/kg of weight the patient's body.

30. The method according to any one of paragraphs 1-29, where the dose of the compound or its pharmaceutically acceptable salt, the first conjugate or its pharmaceutically acceptable salt, or the second conjugate or its pharmaceutically acceptable salt is from about 50 nmol/kg to about 2000 nmol/kg of body weight the patient's body.

31. The method according to any one of paragraphs 1-30, where the dose of the compound or its pharmaceutically acceptable salt, the first conjugate or its pharmaceutically acceptable salt, or the second conjugate or its pharmaceutically acceptable salt is from about 100 nmol/kg to about 1000 nmol/kg of weight the patient's body.

32. The method according to any one of paragraphs 1-31, where the dose of the compound or its pharmaceutically acceptable salt, the first conjugate or its pharmaceutically acceptable salt, or the second conjugate or its pharmaceutically acceptable salt, is from about 100 nmol/kg to about 600 nmol/kg body weight of the patient.

33. The method according to any one of paragraphs 1-32, where the dose of the compound or its pharmaceutically acceptable salt, the first conjugate or its pharmaceutically acceptable salt, or the second conjugate or its pharmaceutically acceptable salt is from about 200 nmol/kg to about 500 nmol/kg of body weight the patient's body.

34. The method according to any one of paragraphs 1-33, where the dose of the compound or its pharmaceutically acceptable salt, the first conjugate or its pharmaceutically acceptable salt, or the second conjugate or its pharmaceutically acceptable salt is from about 250 nmol/kg to about 500 nmol/kg of body weight the patient's body.

35. The method of any one of 1-34, wherein the cancer is selected from lung cancer, bone cancer, pancreatic cancer, skin cancer, head cancer, neck cancer, skin melanoma, intraocular melanoma, uterine cancer, ovarian cancer, endometrial cancer , rectal cancer, gastric cancer, colon cancer, breast cancer, triple negative breast cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, malignant endocrine system tumors, thyroid cancer, parathyroid cancer, non-small cell lung cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, prostate cancer, chronic leukemia, acute leukemia, lymphocytic lymphoma, pleural mesothelioma, bladder cancer , Burkitt's lymphoma, malignant tumor rosacea, kidney cancer, renal cell carcinoma, renal pelvis carcinoma, central nervous system (CNS) neoplasia, primary CNS lymphoma, spinal cord tumors, brainstem glioma, pituitary adenoma, and adenocarcinoma of the gastroesophageal junction.

36. The method of any one of 1-11 or 16-35, wherein the cancer is a folate receptor-expressing cancer.

37. The method of claim 35 wherein the cancer is endometrial cancer.

38. The method of claim 35 wherein the cancer is non-small cell lung cancer.

39. The method of claim 35 wherein the cancer is ovarian cancer.

40. The method of claim 35 wherein the cancer is triple negative breast cancer.

41. The method of any one of items 1-40, wherein the CAR has a recognition region and the recognition region is a single chain fragment of an antibody variable region (scFv).

42. The method of any one of 1-11, 16-17, or 20-41, wherein the CAR has a recognition region and the CAR recognition region is a single chain fragment of the variable region (scFv) of an anti-FITC antibody.

43. The method of any one of 1-42, wherein CAR has a costimulatory domain and the costimulatory domain is selected from CD28, CD137 (4-1BB), CD134 (OX40), and CD278 (ICOS).

44. The method of any one of 1-43, wherein the CAR has an activation signaling domain and the activation signaling domain is a CD3ζ T cell chain or an Fcγ receptor.

45. The method of any one of 1-11, 16-17, or 20-41, wherein the CAR has a recognition region, and the recognition region is a single chain variable region (scFv) fragment of an anti-FITC antibody, wherein the CAR has a co-stimulatory domain, and the co-stimulatory the domain is CD137 (4-1BB), and wherein CAR has an activation signaling domain and the activation signaling domain is a CD3ζ T cell chain.

46. The method of any one of items 1-45, wherein multiple doses of a compound or a pharmaceutically acceptable salt thereof, a first conjugate or a pharmaceutically acceptable salt thereof, or a second conjugate or a pharmaceutically acceptable salt thereof, and a CAR-T cell composition are administered.

47. The method according to any one of paragraphs 1-46, wherein the patient is subjected to imaging prior to administration of the compound or a pharmaceutically acceptable salt thereof, the first conjugate or a pharmaceutically acceptable salt thereof, or the second conjugate or a pharmaceutically acceptable salt thereof, or prior to the administration of the CAR-T cell composition .

48. The method of any one of paragraphs 1-47, wherein the compound, or a pharmaceutically acceptable salt thereof, the first conjugate, or a pharmaceutically acceptable salt thereof, or the second conjugate, or a pharmaceutically acceptable salt thereof, is not an antibody and does not contain an antibody fragment.

49. The method of any one of items 1-48, wherein the targeting group is not a peptide epitope.

50. The method of any one of items 1-49, wherein cytokine release resulting in "off-target" toxicity in the patient does not occur, and where toxicity of the CAR-T cell to cancer occurs.

51. The method of any one of items 1-50, wherein "off-target" tissue toxicity does not occur in the patient, and wherein toxicity of the CAR-T cell to the cancer occurs.

52. The method of any one of items 1-51, wherein the cancer contains a tumor, where the size of the tumor is reduced in the patient, and where "off-target" toxicity does not occur.

53. CAR-T cell containing a nucleic acid containing SEQ ID NO: 1.

54. CAR-T cell containing a polypeptide containing SEQ ID NO: 2.

55. An isolated nucleic acid containing SEQ ID NO: 1 and encoding a chimeric antigen receptor.

56. Chimeric antigen receptor polypeptide comprising SEQ ID NO: 2.

57. Vector containing SEQ ID NO: 1.

58. The vector of item 57, wherein the vector is a lentiviral vector.

59. The method, a CAR-T cell, an isolated nucleic acid encoding a chimeric antigen receptor (CAR) or a chimeric antigen receptor polypeptide according to any one of 1-56, wherein the CAR comprises human amino acid sequences.

60. The method, a CAR-T cell, an isolated nucleic acid encoding a chimeric antigen receptor (CAR) or a chimeric antigen receptor polypeptide according to any one of 1-56, wherein the CAR is composed of human amino acid sequences.

61. A set containing at least two different types of bridges containing a small molecular weight ligand associated with a targeting group, where the ligands in at least two different types of bridges are different, and where the ligand is selected from folate, DUPA, CAIX ligand, NK- ligand 1R, gamma-glutamyl transpeptidase ligand and CCK2R ligand.

62. The kit of item 61, wherein the ligand in at least one of the bridges is an NK-1R ligand.

63. The kit of item 61, wherein the ligand in at least one of the bridges is a gamma-glutamyl transpeptidase ligand.

64. The kit of item 61 wherein the ligand in at least one of the bridges is folate.

65. The set according to any one of paragraphs 61-64, where the bridge has the formula

,

,

where B is a low molecular weight ligand, L is a linker, and T is a targeting group, and where L contains a structure having the formula

,

,

where n is an integer from 0 to 200.

66. The set of paragraph 65, where n is an integer from 0 to 150.

67. The set of paragraph 65, where n is an integer from 0 to 110.

68. The set of clause 65, where n is an integer from 0 to 20.

69. The set of paragraph 65, where n is an integer from 15 to 20.

70. The set of paragraph 65, where n is an integer from 15 to 110.

71. The method of any one of items 1-10, 16-52, or 59-60, or the kit of any of items 61-70, wherein the ligand is a CAIX ligand.

72. Formula conjugate

,

,

,

или

,

or

.

.

As described herein, a "patient" may be a human or, in the case of veterinary applications, a patient may be a laboratory, farm, domestic, or wild animal. In one aspect, the patient may be a laboratory animal such as a rodent ( e.g. , mouse, rat, hamster, etc.), a rabbit, a monkey, a chimpanzee, a pet such as a dog, cat or rabbit, a farm animal such as a cow, horse, pig, sheep, goat, or a captive wild animal such as a bear, panda, lion, tiger, leopard, elephant, zebra, giraffe, gorilla, dolphin, or whale. In the methods described herein, the step of "treating a patient to alleviate the condition of a malignant tumor" may comprise or consist of administering steps in the method.

In one exemplary embodiment, the small molecule ligand linked to the targeting group via a linker (bridge) comprises fluorescein isothiocyanate (FITC) linked to the small molecule ligand. The malignant tumor overexpresses the receptor for the small molecular weight ligand. As a second component, for example, cytotoxic T cells are transformed to express a CAR containing an anti-FITC scFv. In this aspect, the CAR is targeted to FITC by coating the surface of the cancer with FITC molecules as a result of binding of the small molecule ligand to the cancer. Thus, toxicity to normal, non-target cells can be avoided. When anti-FITC CAR-expressing T cells bind FITC, the CAR-T cells are activated and the cancer is alleviated (eg, by killing the cancer cells). In one embodiment, the "small molecular weight ligand" can be folate, DUPA (a ligand associated with PSMA-positive human prostate cancer cells and other cancer cell types), NK-1R ligand (receptors for NK-1R ligand found, e.g. , on malignant tumors of the colon and pancreas), CAIX ligand (receptors for CAIX ligand are found, for example, on malignant tumors of the kidney, ovary, vulva, and breast), gamma-glutamyl transpeptidase ligand (transpeptidase is overexpressed, for example, in malignant tumors of the ovary, colon cancers, liver cancers, astrocytic gliomas, melanomas, and leukemias) or CCK2R ligand (receptors for CCK2R ligand are found on cancers of the thyroid, lung, pancreas, ovary, brain, stomach, gastrointestinal stroma, and colon). intestines, 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 malignant tumors compared to normal tissues). In one embodiment, the DUPA derivative may be a ligand of small molecular weight ligands associated with a targeting group, and DUPA derivatives are described in WO 2015/057852, the contents of which are incorporated herein by reference.

In one embodiment, the low molecular weight ligand is folate. Folate can be folic acid, a folic acid analog, or another folate receptor-binding molecule. In various embodiments, folate analogs that can be used include folinic acid, pteropolyglutamic acid, and folate receptor-binding pteridines such as tetrahydropterins, dihydrofolates, tetrahydrofolates, and their deaza and dideaza analogs. The terms "deaza" and "didea" analogs refer to analogs known in the art having a carbon replacement of one or two nitrogen atoms in the natural structure of folic acid. For example, deaza analogs include the 1-deaza, 3-deaza, 5-deaza, 8-deaza, and 10-deaza analogs. The dideaza analogs include, for example, the 1,5-dideaza, 5,10-dideaza, 8,10-dideaza and 5,8-dideaza analogues. The above folic acid analogues are commonly referred to as "folates", reflecting their ability to bind to folate receptors. Other folate receptor-binding analogs include aminopterin, amethopterin (methotrexate), N10-methylfolate, 2-deaminohydroxyfolate, deaza analogs such as 1-deazamethopterin or 3-deazamethopterin, and 3',5'-dichloro-4-amino-4- deoxy-N10-methylpteroylglutamic acid (dichloromethotrexate).

In one embodiment, the low molecular weight ligand may have a mass of less than about 10000 Daltons, less than about 9000 Daltons, less than about 8000 Daltons, less than about 7000 Daltons, less than about 6000 Daltons, less than about 5000 Less than about 4500 Daltons Less than about 4000 Daltons Less than about 3500 Daltons Less than about 3000 Daltons Less than about 2500 Daltons Less than about 2000 Daltons Less than about 1500 Daltons , less than about 1000 Daltons, or less than about 500 Daltons. In another embodiment, the low molecular weight ligand may have a mass of from about 1 to about 10,000 Daltons, from about 1 to about 9,000 Daltons, from about 1 to about 8,000 Daltons, from about 1 to about 7,000 Daltons, from about 1 to about 6,000 Daltons, about 1 to about 5000 Daltons, from about 1 to about 4500 Daltons, from about 1 to about 4000 Daltons, from about 1 to about 3500 Daltons, from about 1 to about 3000 Daltons, from about 1 to about 2500 Daltons, from about 1 to about 2000 Daltons, from about 1 to about 1500 Daltons, from about 1 to about 1000 Daltons, or from about 1 to about 500 Daltons.

In one aspect, the "targeting group" that binds to CAR expressed by CAR T cells can be selected from, for example, 2,4-dinitrophenol (DNP), 2,4,6-trinitrophenol (TNP), biotin, digoxigenin , fluorescein, fluorescein isothiocyanate (FITC), NHS-fluorescein, pentafluorophenyl ester (PFP), tetrafluorophenyl ester (TFP), nottin, centirin and darpin. The identity of the targeting group is limited only by the fact that it must be recognized and bound by CAR, preferably specifically, and that it has a relatively low molecular weight. In various aspects, illustrative targeting groups are haptens, including low molecular weight organic molecules.

In one exemplary embodiment, the targeting group may have the following exemplary structure:

,

,

where X is oxygen, nitrogen or sulfur, and where X is attached to the linker L; Y is OR ^a , NR ^a ₂ or NR ^a ₃ ⁺ ; and Y' is O, NR ^a or NR ^a ₂ ⁺ ; where each R is independently selected at each occurrence from H, fluorine, sulfonic acid, sulfonate and their salts, and the like; and R ^a is hydrogen or alkyl.

In one illustrative aspect, the linker in a compound or pharmaceutically acceptable salt thereof, a first conjugate or a pharmaceutically acceptable salt thereof, a second conjugate or a pharmaceutically acceptable salt thereof described herein may be a direct linkage (e.g., by reacting an isothiocyanate group of FITC and the free amino group of the small molecular weight ligand), or the connection can be through an intermediate linker. In one embodiment, the intermediate linker, if present, may be any biocompatible linker known in the art, such as a divalent linker. In one exemplary embodiment, the divalent linker may contain from about 1 to about 30 carbon atoms. In another illustrative embodiment, the divalent linker may contain from about 2 to about 20 carbon atoms. In other embodiments, lower molecular weight divalent linkers are used (ie, linkers having an approximate molecular weight of about 30 to about 300). In another embodiment, useful 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.

In various embodiments, the small molecule ligand associated with the targeting group may have the formula

,

,

where B is a low molecular weight ligand, L is a linker, and T is a targeting group, and where L contains a structure having the formula

,

,

where n is an integer from 0 to 200. In another embodiment, n can be an integer from 0 to 150, 0 to 110, 0 to 100, 0 to 90, 0 to 80, 0 to 70, 0 to 60, 0 to 50, 0 to 40, 0 to 30, 0 to 20, 0 to 15, 0 to 14, 0 to 13, 0 to 12, 0 to 11, 0 to 10, 0 to 9, 0 to 8, 0 to 7, 0 to 6, 0 to 5, 0 to 4, 0 to 3, 0 to 2, 0 to 1, 15 to 16, 15 to 17, 15 to 18, 15 to 19, 15 to 20, 15 to 21, 15 to 22, 15 to 23, 15 to 24, 15 to 25, 15 to 26, 15 to 27, 15 to 28, 15 to 29, 15 to 30, 15 to 31, 15 to 32, 15 to 33, 15 to 34, 15 to 35, 15 to 36, 15 to 37, 15 to 38, 15 to 39, 15 to 40, 15 to 50, 15 to 60, 15 to 70, 15 to 80, 15 to 90, 15 to 100, 15 to 110, 15 to 120, 15 to 130, 15 to 140, 15 to 150, or n may be 1, 2, 3, 4, 5, 6, 7 , 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 50, 60, 70, 80, 90, 100, 108, 110, 120, 130, 140 or 150.

In another embodiment, the linker may be a divalent linker, which may include one or more spacers. Exemplary spacers are shown in the following table. The following non-limiting, illustrative spacers are described, where * denotes the point of attachment to the small molecular weight ligand or targeting group.

In other embodiments, the small molecule ligand associated with the targeting group (bridge) may have any of the following structures.

In other embodiments, the compound or a pharmaceutically acceptable salt thereof, the first conjugate or a pharmaceutically acceptable salt thereof, or the second conjugate or a pharmaceutically acceptable salt thereof, is not an antibody and does not contain an antibody fragment. In another embodiment, the targeting group is not a peptide epitope.

In one illustrative aspect, different types of conjugates (eg, first conjugate and second conjugate) can be administered to a patient. For example, the linker in the first conjugate, or a pharmaceutically acceptable salt thereof, and the linker in the second conjugate, or a pharmaceutically acceptable salt thereof, may be the same or different. In another aspect, the ligand in the first conjugate, or a pharmaceutically acceptable salt thereof, and the ligand in the second conjugate, or a pharmaceutically acceptable salt thereof, may be the same or different. In another exemplary embodiment, the targeting group in the first conjugate or pharmaceutically acceptable salt thereof and the targeting group in the second conjugate or pharmaceutically acceptable salt thereof may be the same or different. Any combinations of these embodiments are also contemplated along with any dose combinations described below.

In another embodiment, the present invention relates to a kit containing at least two different types of bridges, where the bridges contain a small molecular weight ligand associated with a targeting group, where the ligands in at least two different types of bridges are different, and where the ligand is selected from folate , DUPA, CAIX ligand, NK-1R ligand, gamma-glutamyl transpeptidase ligand and CCK2R ligand. In this embodiment, the ligand in at least one of the bridges can be a NK-1R ligand, a gamma-glutamyl transpeptidase ligand, folate, a CAIX ligand, a CCK2R ligand, or DUPA.

In another aspect, a bridge in a set may have the formula

,

,

where B is a low molecular weight ligand, L is a linker, and T is a targeting group, and where L contains a structure having the formula

,

,

where n is an integer from 0 to 200. In other embodiments, n can be an integer from 0 to 150, an integer from 0 to 110, an integer from 0 to 20, an integer from 15 to 20, an integer 15 to 110, or any other value or range of integers described herein for n.

A "pharmaceutically acceptable salt" of the low molecular weight ligand is contemplated linked to the targeting group via a linker. As used herein, the term "pharmaceutically acceptable salt" refers to salts whose counterions can be used as drugs. Such salts include 1) acid addition salts, which can be obtained by reacting the free base of the parent compound with inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, phosphoric acid, sulfuric acid and perchloric acid, etc., or with 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, and etc.; or 2) salts formed when an acid proton present in the parent compound is either replaced by a metal ion, such as an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinate with an organic base such as ethanolamine, diethanolamine, triethanolamine, trimetamine, N-methylglucamine, and the like. Pharmaceutically acceptable salts are well known to those skilled in the art, and any such pharmaceutically acceptable salt may be contemplated in connection with the embodiments described herein.

In various embodiments, useful acid addition salts are formed from acids forming non-toxic salts. Illustrative examples include acetate, aspartate, benzoate, besylate, bicarbonate/carbonate, bisulfate/sulfate, borate, camsylate, citrate, edisylate, esylate, format, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride/chloride, hydrobromide/ bromide, hydroiodide/iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methyl sulfate, naphthylate, 2-napsilate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate/hydrophosphate/dihydrogen phosphate, sucrose, stearate, succinate, tartrate, tosylate and trifluoroacetate.

In various embodiments, suitable base salts are formed from bases forming non-toxic salts. Illustrative examples include salts of arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine and zinc. It is also possible to form hemi-salts of acids and bases, for example the hemisulfate salt and hemicalcium salts.

In one illustrative aspect, a compound or a pharmaceutically acceptable salt thereof, a first conjugate or a pharmaceutically acceptable salt thereof, or a second conjugate or a pharmaceutically acceptable salt thereof described herein may contain one or more chiral centers, or may otherwise be capable of existing in form of multiple stereoisomers. Accordingly, various embodiments may include pure stereoisomers as well as mixtures of stereoisomers such as enantiomers, diastereomers, and enantiomerically or diastereomerically enriched mixtures. In one aspect, a compound or a pharmaceutically acceptable salt thereof, a first conjugate or a pharmaceutically acceptable salt thereof, a second conjugate or a pharmaceutically acceptable salt thereof described herein may be capable of existing in the form of geometric isomers. Accordingly, various embodiments may include pure geometric isomers or mixtures of geometric isomers.

In some aspects, a compound or a pharmaceutically acceptable salt thereof, a first conjugate or a pharmaceutically acceptable salt thereof, a second conjugate or a pharmaceutically acceptable salt thereof described herein may exist in unsolvated forms as well as in solvated forms, including hydrated forms. Generally, solvated forms are equivalent to unsolvated forms and are included within the scope of the present invention.

The methods described herein also use cytotoxic T lymphocytes engineered to express a chimeric antigen receptor (CAR) that recognizes and binds a targeting group (eg, FITC, DNP, or TNP) from the bridge. In one embodiment, the CARs described herein comprise three domains including 1) a recognition region (e.g., a single chain variable region (scFv) fragment of an antibody) that specifically recognizes and binds a targeting group, 2) a co-stimulatory domain that increases proliferation and T-lymphocyte survival; and 3) an activation signaling domain that generates an activation signal for cytotoxic T-lymphocytes. In various aspects, as non-limiting examples, scFv regions of antibodies that bind folate, DUPA, CAIX ligand, NK-1R ligand, gamma-glutamyl transpeptidase ligand, or CCK2R ligand can be used. In exemplary embodiments, scFv regions can be derived from (i) an antibody known in the art that binds a targeting group, (ii) an antibody first generated using a selected targeting group such as a hapten, and (iii) sequence variants, derived from scFv regions of such antibodies, e.g., scFv regions having at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least at least about 99%, or at least about 99.5% sequence identity with the amino acid sequence of the scFv region from which they originate.

In any of the embodiments described herein, the CAR binding portion may be, for example, a single chain variable region (scFv) fragment of an antibody, a Fab, Fv, Fc or (Fab')2 fragment, and the like.

In one aspect, the co-stimulatory domain serves to increase the proliferation and survival of cytotoxic T lymphocytes after CAR binding to a targeting group. Suitable costimulatory domains include: 1) CD28, 2) CD137 (4-1BB), a member of the tumor necrosis factor receptor (TNF) family, 3) CD134 (OX40), a member of the TNFR receptor superfamily, and 4) CD278 (ICOS), a superfamily of costimulatory molecules CD28 expressed on activated T cells, or combinations thereof. Suitable co-stimulatory domains also include, but are not limited to, CD27, CD30, CD150, DAP10, and NKG2D, or combinations thereof. One skilled in the art will appreciate that sequence variants of these co-stimulatory domains can be used without adversely affecting the invention, where the variants have the same or similar activity as the domain they are modeled on. In various embodiments, such variants have at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity with the amino acid sequence of the domain from which they originate. In an exemplary embodiment, the activation signaling domain serves to activate cytotoxic T lymphocytes upon binding of the CAR to the targeting group. Suitable activation signaling domains include the CD3ζ T cell chain and the Fcγ receptor. One skilled in the art will recognize that sequence variants of these marked activation signaling domains can be used, where the variants have the same or similar activity as the domain they are modeled on. In various embodiments, the variants are at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity with the amino acid sequence of the domain from which they originate. In one aspect, constructs encoding CARs are obtained using genetic engineering techniques. Such methods are described in detail in Sambrook et al., "Molecular Cloning: A Laboratory Manual", 3rd Edition, Cold Spring Harbor Laboratory Press, (2001), the contents of which are incorporated herein by reference. By way of example, a plasmid or viral expression vector (e.g., lentiviral vector, retroviral vector, sleeping beauty, and piggyback (transposon/transposase systems, including a non-viral mediated CAR gene delivery system)) can be generated that encodes a fusion protein containing a recognition region, one or more co-stimulatory domains and an activation signaling domain in frame and linked in the 5' to 3' direction. In other embodiments, other locations are acceptable and include a recognition region, an activation signaling domain, and one or more costimulatory domains. The location of the recognition region in the fusion protein is generally such as to expose the region to the outer surface of the cell. In one embodiment, CARs may include additional elements such as a signal peptide to ensure proper export of the fusion protein to the cell surface, a transmembrane domain to ensure that the fusion protein is maintained as a membrane-integrated protein, and a hinge domain to confer flexibility on the recognition region and allow strong binding to the target group. Diagrams of an exemplary CAR are shown in Figures 14A and B where the fusion protein sequence is inserted into a lentiviral expression vector and where "SP" is a signal peptide, CAR is an anti-FITC CAR, a CD8α hinge is present and a transmembrane (TM) region is present, the costimulatory domain is is 4-1BB and the activation signaling domain is CD3ζ. In one aspect, the nucleic acid sequence of the CAR insert is shown as SEQ ID NO:1 and the amino acid sequence of the insert is shown as SEQ ID NO:2.

In one embodiment, the CAR has a recognition region, and the recognition region is a single chain fragment of the variable region (scFv) of an anti-FITC antibody, the costimulatory domain, and the costimulatory domain is CD137 (4-1BB), and the activation signaling domain and the signaling The activation domain is the CD3ζ chain of the T cell. It is well known to one skilled in the art that anti-FITC scFv and anti-fluorescein scFv are equivalent terms.

In one embodiment, cytotoxic T lymphocytes can be genetically engineered to express CAR constructs by transfecting a population of cytotoxic T lymphocytes with an expression vector encoding the CAR construct. Suitable methods for obtaining a transduced population of T lymphocytes expressing the selected CAR construct are well known to those skilled in the art and are described in Sambrook et al., "Molecular Cloning: A Laboratory Manual", 3rd Edition, Cold Spring Harbor Laboratory Press, (2001) , the contents of which are incorporated herein by reference.

In various embodiments, the present invention provides CAR-T cells comprising the nucleic acid of SEQ ID NO: 1 or 3. In another embodiment, the present invention provides CAR-T cells comprising the polypeptide of SEQ ID NO: 2 In another illustrative aspect, the present invention provides an isolated nucleic acid comprising SEQ ID NO: 1 or 3 and encoding a chimeric antigen receptor. In another embodiment, the present invention provides a chimeric antigen receptor polypeptide comprising SEQ ID NO: 2. In another embodiment, the present invention relates to a vector comprising SEQ ID NO: 1 or 3. In another aspect, the present invention relates to a lentiviral vector containing SEQ ID NO: 1 or 3. In another embodiment, SEQ ID NO: 2 may contain or consist of human or humanized amino acid sequences. In each of these embodiments, a variant nucleic acid or amino acid sequences having at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98% %, at least about 99%, or at least about 99.5% sequence identity with SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In another embodiment, the nucleic acid sequence may be is a nucleic acid sequence variant having at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity with SEQ ID NO: 1 or 3, provided that the sequence variant encoded is a polypeptide of SEQ ID NO: 2. In another embodiment, the nucleic acid or amino acid sequence may be a nucleic acid or amino acid sequence variant having at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2, or SEQ ID NO: 3, together with a fragment of 200 nucleic acids or 200 amino acids from SEQ ID NO: 1 or SEQ ID NO: 2, or SEQ ID NO: 3. Determination of percent identity or similarity between sequences can be performed, for example, using the program GAP (software of Genetics Computer Group;; currently available through Accelrys at http://www.accelrys.com) and alignments can be performed using, for example, the ClustalW algorithm (VNTI software, InforMax Inc.). You can search the sequence database using the nucleic acid or amino acid sequence of interest. Database search algorithms are typically based on BLAST software (Altschul et al., 1990). In some embodiments, percent identity can be determined over the entire nucleic acid or amino acid sequence.

Also included within the scope of the invention are nucleic acids complementary to the nucleic acid represented by SEQ ID NO: 1 or 3 and nucleic acids which hybridize to the nucleic acid represented by SEQ ID NO: 1 or 3 or nucleic acids which hybridize to its complementary nucleic acid. acid under conditions of high severity. In accordance with the invention, "high stringency conditions" refers to hybridization at 65°C in 5X SSPE and 50% formamide, and washing at 65°C in 0.5X SSPE. High stringency, low stringency, and moderate stringency hybridization conditions are described in Sambrook et al., "Molecular Cloning: A Laboratory Manual", 3rd Edition, Cold Spring Harbor Laboratory Press, (2001), the contents of which are incorporated herein by reference. In some exemplary aspects, hybridization occurs throughout the entire nucleic acid. In one embodiment, the cytotoxic T lymphocytes used to generate CAR-T cells used in the methods described herein may be autologous cells, although heterologous cells may also be used, for example, when the patient being treated has been subjected to high dose of chemotherapy or radiation therapy to damage the patient's immune system. In one embodiment, allogeneic cells can be used.

In one aspect, cytotoxic T lymphocytes can be obtained from a patient by methods well known in the art. For example, cytotoxic T cells can be obtained by collecting peripheral blood from a patient, subjecting the blood to ficoll density gradient centrifugation, and then using a negative T cell isolation kit (such as the EasySep™ T Cell Isolation Kit) to isolate the cytotoxic T cell population. from peripheral blood. In one exemplary embodiment, the cytotoxic T lymphocyte population need not be pure and may contain other cells such as other T cells, monocytes, macrophages, natural killer cells, and B cells. In one aspect, the harvested population may contain at least about 90% of the selected cell type, at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% selected cell type. In one embodiment, after receiving cytotoxic T lymphocytes, the cells are cultured under conditions of cell activation. In this embodiment, culture conditions may be such that the cells can be administered to a patient without concerns about reactivity against components of the culture medium. For example, culture conditions may not include bovine serum products such as bovine serum albumin. In one exemplary aspect, activation can be accomplished by introducing known activators into the culture medium, such as anti-CD3 antibodies in the case of cytotoxic T cells. Other suitable activators include anti-CD28 antibodies. In one aspect, a population of lymphocytes can be cultured under activation-promoting conditions for about 1 to about 4 days. In one embodiment, an appropriate level of activation can be determined by cell size, proliferation rate, or activation markers determined by flow cytometry. In one exemplary embodiment, after culturing a population of cytotoxic T-lymphocytes under activation-promoting conditions, the cells can be transfected with an expression vector encoding a CAR. Suitable vectors and transfection methods are described above. In one aspect, after transfection, the cells may be immediately administered to a patient, or the cells may be cultured for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or more days, or between about 5 and about 12 days, between about 6 and about 13 days, between about 7 and about 14 days, or between about 8 and about 15 days, for example, to provide time to recover cells from transfection. Suitable culture conditions may be similar to those under which the cells were cultured for activation, either in the presence or absence of the agent used to promote activation.

Thus, as described above, in one illustrative aspect, the methods of treatment described herein may further include 1) obtaining a population of autologous or heterologous cytotoxic T lymphocytes, 2) culturing T lymphocytes under conditions that stimulate cell activation, and 3 ) transfection of lymphocytes with an expression vector encoding CAR to obtain CAR-T cells. In one exemplary embodiment, when the cells are transfected and activated, a composition containing CAR-T cells can be obtained and administered to a patient. In one embodiment, culture media free of any animal products such as bovine serum can be used. In another embodiment, cell culture conditions typically used by those skilled in the art can be used to avoid contamination by bacteria, fungi and mycoplasma. In an exemplary embodiment, prior to administration to a patient, the cells are pelleted, washed and resuspended in a pharmaceutically acceptable carrier or diluent. Exemplary compositions containing CAR-expressing cytotoxic T-lymphocytes include compositions containing cells in 290 mOsm sterile saline, in infused cryomedia (containing Plasma-Lyte A, dextrose, injectable sodium chloride, human serum albumin, and DMSO), in 0.9% NaCl with 2% human serum albumin or any other sterile infusible material 290 mOsm. Alternatively, depending on the identity of the culture medium, CAR-T cells can be introduced into the culture medium as a composition, or concentrated and resuspended in the culture medium prior to administration. The CAR-T cell composition can be administered to a patient by any suitable means, such as parenteral administration, for example intradermally, subcutaneously, intramuscularly, intraperitoneally, intravenously, or intrathecally. In one aspect, the total number of CAR-T cells and the concentration of cells in the composition administered to the patient can be varied depending on a number of factors, including the type of cytotoxic T-lymphocytes used, the specificity of CAR binding, the identity of the targeting group and small molecular weight ligand, the identity of the malignant tumor, the location of the cancer in the patient, the methods used to administer the compositions to the patient, and the general health, age and weight of the patient being treated. However, suitable compositions containing transduced CAR-T cells include compositions having a volume between about 5 ml and about 200 ml containing from about 1x10 ⁵ to about 1x10 ¹⁵ transduced CAR-T cells. Typical compositions have a volume between about 10 ml and about 125 ml, containing from about 1x10 ⁷ to about 1x10 ¹⁰ CAR-T cells. An exemplary composition contains approximately 1×10 ⁹ CAR-T cells in a volume of approximately 100 ml. In one aspect, a single dose or multiple doses of CAR-T cells can be administered to a patient. In various embodiments, the cancer being treated is a carcinoma, sarcoma, lymphoma, melanoma, mesothelioma, nasopharyngeal carcinoma, leukemia, adenocarcinoma, or myeloma. In other embodiments, the cancer may be lung cancer, bone cancer, pancreatic cancer, skin cancer, head cancer, neck cancer, skin melanoma, intraocular melanoma, uterine cancer, ovarian cancer, endometrial cancer, rectal cancer, cancer stomach, colon cancer, breast cancer, triple negative breast cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine system malignancy, malignant tumor thyroid, parathyroid cancer, non-small cell lung cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, prostate cancer, chronic leukemia, acute leukemia, lymphocytic lymphoma, pleural mesothelioma, bladder cancer, Burkitt's lymphoma, malignancy ureter, kidney cancer and, renal cell carcinoma, renal pelvis carcinoma, neoplasia of the central nervous system (CNS), primary CNS lymphoma, spinal cord tumor, brainstem glioma, pituitary adenoma, or adenocarcinoma of the gastroesophageal junction.

In some aspects of these embodiments, the cancer is a folate receptor-expressing cancer. In some aspects of these embodiments, the cancer is endometrial cancer, non-small cell lung cancer, ovarian cancer, or triple negative breast cancer. In another embodiment, the cancer being imaged is a tumor. In another embodiment, the cancer is a malignant tumor.

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

In one aspect, a compound or a pharmaceutically acceptable salt thereof, a first conjugate or a pharmaceutically acceptable salt thereof, a second conjugate or a pharmaceutically acceptable salt thereof, or a CAR-T cell composition as described herein may be administered directly into the bloodstream, into muscle, or into internal organ. Suitable routes for such parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, epidural, intracerebroventricular, intraurethral, intrasternal, intracranial, intratumoral, intramuscular and subcutaneous delivery. In one embodiment, the means for parenteral administration include needle (including microneedle) injectors, needleless injectors and infusion methods.

In one illustrative aspect, parenteral formulations are typically aqueous solutions which may contain carriers or excipients such as salts, carbohydrates, and buffering agents (preferably at a pH of 3 to 9), but may more suitably be formulated in in the form of a sterile non-aqueous solution, or in dried form for use in combination with a suitable vehicle such as sterile pyrogen-free water or sterile saline. In other embodiments, any of the liquid formulations described herein can be adapted for parenteral administration as described herein. Obtaining under sterile conditions, by lyophilization to obtain a sterile lyophilized powder for parenteral formulation, can be easily carried out using standard pharmaceutical methods, well known to specialists in this field. In one embodiment, the solubility of a compound or a pharmaceutically acceptable salt thereof, a first conjugate or a pharmaceutically acceptable salt thereof, a second conjugate or a pharmaceutically acceptable salt thereof, used to form a parenteral formulation can be increased by using suitable formulation methods such as the inclusion of solubility enhancing agents. .

In some embodiments, the rate of tumor lysis can be controlled by adjusting the concentration of the first conjugate, or a pharmaceutically acceptable salt thereof, the second conjugate, or a pharmaceutically acceptable salt thereof, or both the first and second conjugate, or pharmaceutically acceptable salts thereof. Accordingly, by varying the concentration of the first conjugate, or a pharmaceutically acceptable salt thereof, the second conjugate, or a pharmaceutically acceptable salt thereof, or both the first and second conjugate, or pharmaceutically acceptable salts thereof, the cytotoxicity of a CAR-T cell composition can be controlled. In some embodiments, the balance of cytotoxicity of a CAR-T cell composition and the risk of tumor lysis syndrome or cytokine release syndrome (CRS) as described herein can be maintained by adjusting the concentration of the first conjugate or a pharmaceutically acceptable salt thereof, the second conjugate or a pharmaceutically acceptable salt thereof. an acceptable salt, or both the first and second conjugates, or their pharmaceutically acceptable salts. It is understood that the concentration of the first conjugate, or a pharmaceutically acceptable salt thereof, the second conjugate, or a pharmaceutically acceptable salt thereof, or both the first and second conjugate, or pharmaceutically acceptable salts thereof, may be a function of the amount or dose of the first conjugate, or a pharmaceutically acceptable salt thereof, the second the conjugate, or a pharmaceutically acceptable salt thereof, or both the first and second conjugate, or pharmaceutically acceptable salts thereof, which is administered to the patient.

The amount of a compound or a pharmaceutically acceptable salt thereof, a first conjugate or a pharmaceutically acceptable salt thereof, a second conjugate or a pharmaceutically acceptable salt thereof to be administered to a patient can vary significantly depending on the cancer to be treated, the route of administration of the compound or its pharmaceutically acceptable salt, the first a conjugate or a pharmaceutically acceptable salt thereof, a second conjugate or a pharmaceutically acceptable salt thereof, and tissue distribution. The amount to be administered to a patient may be based on body surface area, weight, and physical assessment. In various embodiments, the amounts to be administered may range, for example, from about 0.05 mg to about 30 mg, from 0.05 mg to about 25.0 mg, from 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, from about 0.05 mg to about 7.0 mg, from about 0.05 mg to about 6.0 mg, from about 0.05 mg to about 5.0 mg, from about 0.05 mg to about 4.0 mg, from about 0.05 mg to about 3.0 mg, from about 0.05 mg to about 2.0 mg, from about 0.05 mg to about 1.0 mg, from about 0, 05 mg to about 0.5 mg, 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, from about 0.05 mg to about 0.1 mg, from about 0.01 mg to about 2 mg, from about 0.3 mg to about 10 mg, from about 0.1 mg to about 20 mg, or about 0.8 to about 3 mg. The person skilled in the art will appreciate that the dosage may be varied within the various ranges presented above based on the factors noted above, and this may be at the discretion of the physician.

In other embodiments, the dose of the compound, or a pharmaceutically acceptable salt thereof, the first conjugate, or a pharmaceutically acceptable salt thereof, the second conjugate, or a pharmaceutically acceptable salt thereof, may range from, for example, about 50 nmol/kg to about 3000 nmol/kg body weight. patient, from about 50 nmol/kg to about 2000 nmol/kg, from about 50 nmol/kg to about 1000 nmol/kg, from about 50 nmol/kg to about 900 nmol/kg, from about 50 nmol/kg to about 800 nmol/kg, from about 50 nmol/kg to about 700 nmol/kg, from about 50 nmol/kg to about 600 nmol/kg, from about 50 nmol/kg to about 500 nmol/kg, from about 50 nmol/kg to about 400 nmol/kg, from about 50 nmol/kg to about 300 nmol/kg, from about 50 nmol/kg to about 200 nmol/kg, from about 50 nmol/kg to about 10 0 nmol/kg, from about 100 nmol/kg to about 300 nmol/kg, from about 100 nmol/kg to about 500 nmol/kg, from about 100 nmol/kg to about 1000 nmol/kg, from about 100 nmol/kg up to about 2000 nmol/kg of the patient's body weight. In other embodiments, the dose may be about 100 nmol/kg, about 150 nmol/kg, about 200 nmol/kg, about 250 nmol/kg, about 300 nmol/kg, about 350 nmol/kg, about 400 nmol/kg, approximately 450 nmol/kg, approximately 500 nmol/kg, approximately 600 nmol/kg, approximately 700 nmol/kg, approximately 800 nmol/kg, approximately 900 nmol/kg, approximately 1000 nmol/kg, approximately 2000 nmol/kg or approximately 3000 nmol/kg patient body weight. In these embodiments, "kg" represents kilograms of the patient's body weight. In one aspect, a single dose or multiple doses of a compound or a pharmaceutically acceptable salt thereof, a first conjugate or a pharmaceutically acceptable salt thereof, a second conjugate or a pharmaceutically acceptable salt thereof, may be administered to a patient. In another embodiment, between about 20 µg/kg of patient body weight and about 3 mg/kg of patient body weight of the compound or a pharmaceutically acceptable salt thereof, a first conjugate or a pharmaceutically acceptable salt thereof, a second conjugate or a pharmaceutically acceptable salt thereof, is administered to the patient. In another aspect, the amounts may be between about 0.2 mg/kg of the patient's body weight and about 0.4 mg/kg of the patient's body weight, or may be about 50 μg/kg of the patient's body weight. In one aspect, a single dose or multiple doses of a compound or a pharmaceutically acceptable salt thereof, a first conjugate or a pharmaceutically acceptable salt thereof, a second conjugate or a pharmaceutically acceptable salt thereof, may be administered to a patient. In one embodiment, the small molecule ligand associated with the targeting group can be administered to the patient prior to the composition of the CAR-T cell. In another embodiment, the small molecule ligand associated with the targeting group can be administered to the patient simultaneously with the CAR-T cell composition, but in different formulations. In another embodiment, the low molecular weight ligand associated with the targeting group can be administered to the patient after the composition of the CAR-T cell.

In one illustrative aspect, the time periods between the introduction of CAR-T cells and a small molecule associated with a targeting group can be varied widely depending on factors including the type of CAR-T cells used, the specificity of CAR binding, the identity of the targeting group, and ligand, the identity of the cancer, the location of the cancer in the patient, the methods used to administer the CAR-T cells and the small molecule ligand associated with the targeting group to the patient, and the patient's general health, age, and weight. In one aspect, the small molecule ligand associated with the targeting group can be administered before or after CAR-T cells, for example, within about 3, 6, 9, 12, 15, 18, 21, or 24 hours, or within about 0, 5, 1, 1.5, 2, 2.5, 3, 4 5, 6, 7, 8, 9, 10 or more days.

In some embodiments, the rate of tumor lysis can be controlled by adjusting the frequency of administration of the first conjugate, or a pharmaceutically acceptable salt thereof, the second conjugate, or a pharmaceutically acceptable salt thereof, or both the first and second conjugate, or pharmaceutically acceptable salts thereof. Accordingly, by varying the frequency of administration of the first conjugate or a pharmaceutically acceptable salt thereof, the second conjugate or a pharmaceutically acceptable salt thereof, or both the first and second conjugate or pharmaceutically acceptable salts thereof, the cytotoxicity of a CAR-T cell composition can be controlled. In some embodiments, the balance of cytotoxicity of a CAR-T cell composition and the risk of tumor lysis syndrome or cytokine release syndrome (CRS) as described herein can be maintained by adjusting the frequency of administration of the first conjugate or a pharmaceutically acceptable salt thereof, the second conjugate or its a pharmaceutically acceptable salt, or both the first and second conjugate, or their pharmaceutically acceptable salts. It is understood that the frequency of administration of the first conjugate or a pharmaceutically acceptable salt thereof, the second conjugate or a pharmaceutically acceptable salt thereof, or both the first and second conjugate or pharmaceutically acceptable salts thereof, may be a function of any applicable dosing schedule known in the art. For example, the frequency of administration may be a function of a dosing schedule based on continuous dosing, dosing once a day (otherwise called qd), dosing twice a day (otherwise called bid), dosing three times a day (otherwise called tid), dosing two once a week (otherwise called BIW), dosing three times a week (otherwise called TIW), once a week, and the like. It is understood that a dosing schedule chosen for the first conjugate or a pharmaceutically acceptable salt thereof, the second conjugate or a pharmaceutically acceptable salt thereof, or both the first and second conjugate or a pharmaceutically acceptable salt thereof, can be used in connection with the concentration to regulate the cytotoxicity of the CAR composition. -T-cells. In some embodiments, it is possible to balance the cytotoxicity of the CAR-T cell composition and the risk of tumor lysis syndrome or cytokine release syndrome (CRS), as described herein, by adjusting the dosing schedule in relation to the concentration of the first conjugate or its pharmaceutically acceptable salt, the second conjugate or its pharmaceutically acceptable salt, or both the first and second conjugate, or their pharmaceutically acceptable salts.

In one embodiment of the methods described herein, the cancer is imaged prior to administering to the patient a compound or a pharmaceutically acceptable salt thereof, a first conjugate or a pharmaceutically acceptable salt thereof, a second conjugate or a pharmaceutically acceptable salt thereof, or prior to administering a CAR-T cell composition. patient. In one illustrative embodiment, the imaging is performed by PET imaging. In other exemplary embodiments, imaging is performed by MRI imaging or SPECT/CT imaging. One skilled in the art will appreciate that the imaging method may be any suitable imaging method known in the art.

In any of the embodiments described herein, cytokine release resulting in "off-target" toxicity in a patient may not occur even though CAR-T cell toxicity to cancer occurs. In any embodiment described herein, "off-target" tissue toxicity may not occur in a patient, even though CAR-T cell toxicity to cancer occurs. In any embodiment described herein, the cancer may contain a tumor and the size of the tumor may be reduced in the patient even though "off-target" toxicity does not occur.

EXAMPLES

EXAMPLE 1

Obtaining a lentiviral vector encoding the CAR gene

The overlapping PCR method was used to generate CAR constructs containing anti-fluorescein scFv. An anti-fluorescein scFV, 4M5.3 (Kd=270 fM, 762 bp) derived from an anti-fluorescein antibody (4-4-20) was synthesized. The sequence encoding the human CD8α signal peptide (SP, 63 bp), the hinge and transmembrane region (249 bp), the 4-1BB cytoplasmic domain (CD137, 141 bp) and the CD3ζ chain (336 bp). o.), as shown in Figure 14A, was fused to anti-fluorescein scFV by overlapping PCR. The resulting CAR construct (1551 bp) was inserted into the EcoRI/NotI digested lentiviral expression vector pCDH-EF1-MCS-(PGK-GFP) (Figure 14B, System Biosciences). The sequence of the CAR construct in the lentiviral vector was confirmed by DNA sequencing.

An exemplary nucleic acid sequence encoding a CAR may comprise:

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGATGTCGTGATGACCCAGACCCCCCTCAGCCTCCCAGTGTCCCTCGGTGACCAGGCTTCTATTAGTTGCAGATCCAGCCAGTCCCTCGTGCACTCTAACGGTAATACCTACCTGAGATGGTATCTCCAGAAGCCCGGACAGAGCCCTAAGGTGCTGATCTACAAAGTCTCCAACCGGGTGTCTGGAGTCCCTGACCGCTTCTCAGGGAGCGGTTCCGGCACCGACTTCACCCTGAAGATCAACCGGGTGGAGGCCGAAGACCTCGGCGTCTATTTCTGCTCTCAGAGTACACATGTGCCCTGGACCTTCGGCGGAGGGACCAAGCTGGAGATCAAAAGCTCCGCAGACGATGCCAAGAAAGATGCCGCTAAGAAAGACGATGCTAAGAAAGACGATGCAAAGAAAGACGGTGGCGTGAAGCTGGATGAAACCGGAGGAGGTCTCGTCCAGCCAGGAGGAGCCATGAAGCTGAGTTGCGTGACCAGCGGATTCACCTTTGGGCACTACTGGATGAACTGGGTGCGACAGTCCCCAGAGAAGGGGCTCGAATGGGTCGCTCAGTTCAGGAACAAACCCTACAATTATGAGACATACTATTCAGACAGCGTGAAGGGCAGGTTTACTATCAGTA

GAGACGATTCCAAATCTAGCGTGTACCTGCAGATGAACAATCTCAGGGTCGAAGATACAGGCATCTACTATTGCACAGGGGCATCCTATGGTATGGAGTATCTCGGTCAGGGGACAAGCGTCACAGTCAGTTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAACCACAGGAACCGTTTCTCTGTTGTTAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA (SEQ ID NO: 1).

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

MALPVTALLLPLALLLHAARPDVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRVSGVPDRFSGSGSGTDFTLKINRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIKSSADDAKKDAAKKDDAKKDDAKKDGGVKLDETGGGLVQPGGAMKLSCVTSGFTFGHYWMNWVRQSPEKGLEWVAQFRNKPYNYETYYSDSVKGRFTISRDDSKSSVYLQMNNLRVEDTGIYYCTGASYGMEYLGQGTSVTVSFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 2)

An exemplary insert may include:

GCCACCATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGATGTCGTGATGACCCAGACCCCCCTCAGCCTCCCAGTGTCCCTCGGTGACCAGGCTTCTATTAGTTGCAGATCCAGCCAGTCCCTCGTGCACTCTAACGGTAATACCTACCTGAGATGGTATCTCCAGAAGCCCGGACAGAGCCCTAAGGTGCTGATCTACAAAGTCTCCAACCGGGTGTCTGGAGTCCCTGACCGCTTCTCAGGGAGCGGTTCCGGCACCGACTTCACCCTGAAGATCAACCGGGTGGAGGCCGAAGACCTCGGCGTCTATTTCTGCTCTCAGAGTACACATGTGCCCTGGACCTTCGGCGGAGGGACCAAGCTGGAGATCAAAAGCTCCGCAGACGATGCCAAGAAAGATGCCGCTAAGAAAGACGATGCTAAGAAAGACGATGCAAAGAAAGACGGTGGCGTGAAGCTGGATGAAACCGGAGGAGGTCTCGTCCAGCCAGGAGGAGCCATGAAGCTGAGTTGCGTGACCAGCGGATTCACCTTTGGGCACTACTGGATGAACTGGGTGCGACAGTCCCCAGAGAAGGGGCTCGAATGGGTCGCTCAGTTCAGGAACAAACCCTACAATTATGAGACATACTATTCAGACAGCGTGAAGGGCAGGTTTACTATCAGTA

GAGACGATTCCAAATCTAGCGTGTACCTGCAGATGAACAATCTCAGGGTCGAAGATACAGGCATCTACTATTGCACAGGGGCATCCTATGGTATGGAGTATCTCGGTCAGGGGACAAGCGTCACAGTCAGTTTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAACCACAGGAACCGTTTCTCTGTTGTTAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA (SEQ ID NO: 3)

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

ATMALPVTALLLPLALLLHAARPDVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRVSGVPDRFSGSGSGTDFTLKINRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIKSSADDAKKDAAKKDDAKKDDAKKDGGVKLDETGGGLVQPGGAMKLSCVTSGFTFGHYWMNWVRQSPEKGLEWVAQFRNKPYNYETYYSDSVKGRFTISRDDSKSSVYLQMNNLRVEDTGIYYCTGASYGMEYLGQGTSVTVSFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 4)

EXAMPLE 2

Obtaining a Lentivirus Containing the CAR Gene for Transduction of a Human T Cell

To generate a lentiviral virus containing a single chain variable region fragment (scFv) of the anti-fluorescein CAR, the 293TN packaging cell line was co-transfected with a lentiviral vector encoding the anti-fluorescein scFv CAR and a mixture of 2nd generation packaging plasmids (Cellecta). 24 and 48 hours after transfection, supernatants containing lentivirus with the CAR gene were harvested and viral particles were concentrated by a standard polyethylene glycol virus concentration method (Clontech) for future transduction using human T cells.

EXAMPLE 3

Isolation of human T cells from human PBMC

T cells were isolated from human peripheral blood mononuclear cells (PBMC) by ficoll density gradient centrifugation (GE Healthcare Lifesciences). After washing off the remaining ficoll solution, T cells were isolated using the EasySep™ human T cell isolation kit (STEM CELL technologies). Purified T cells were cultured in TexMACS™ medium (Miltenyi Biotech Inc) with 1% penicillin and streptomycin sulfate in the presence of human IL-2 (100 IU/ml, Miltenyi Biotech Inc). T cells were cultured at a density of 1×10 ⁶ cells/ml in multiwell plates. T cells were separated and refeeded every 2-3 days.

EXAMPLE 4

Human T cell transduction

Isolated T cells were activated using Dynabeads in combination with anti-CD3/CD28 antibodies (Life Technologies) for 12-24 hours in the presence of human IL-2 (100 IU/ml), then transduced with lentivirus encoding the anti-fluorescein CAR gene. Cells were harvested 72 hours later and CAR expression on transduced T cells was identified by measuring cells with fluorescent GFP using flow cytometry. As shown in Figure 15A, non-transduced T cells did not show GFP expression. As shown in Figure 15B, transduced T cells express GFP.

EXAMPLE 5

Synthesis of FITC-folate

Folate-γ-ethylenediamine was added to isomer I of fluorescein isothiocyanate (FITC) (Sigma-Aldrich) in anhydrous dimethyl sulfoxide (DMF) in the presence of tetramethylguanidine and diisopropylamine. The crude product was loaded onto an Xterra RP18 prep HPLC column (Waters) and eluted using gradient conditions starting with 99% 5 mM sodium phosphate (mobile phase A, pH 7.4) and 1% acetonitrile (mobile phase B) and working up to 90 % A and 10% B in 10 min at a flow rate of 20 ml/min. Under these conditions, the main peak of FITC-folate usually elutes after 27-50 minutes. The quality of the FITC-folate fraction was monitored by analytical reverse phase HPLC using a UV detector. Fractions greater than 98.0% pure (LCMS) were lyophilized to obtain the final product FITC-folate.

EXAMPLE 6

Synthesis of FITC-PEG12-folate

Polyethylene glycol (PEG) Nova Tag™ universal resin (0.2 g) was loaded into a peptide synthesis vessel and washed with isopropyl alcohol ( i -PrOH) (3×10 ml) and dimethylformamide (DMF, 3×10 ml). Deprotection of 9-fluorenylmethoxycarbonyl (Fmoc) was performed using 20% piperidine in DMF (3×10 ml). Kaiser tests were performed to evaluate the progression of the reaction. A solution of Fmoc-L-glutamic acid 5-tert-butyl ester (Fmoc-Glu-(Ot-Bu)-OH) (23.5 mg) in DMF, N,N-diisopropylethylamine ( i -Pr ₂ NEt) (4 equiv.) and benzotriazol-1-yl-hydroxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) (2 equiv.). Fmoc deprotection was performed using 20% piperidine in DMF (3×10 ml). Then a solution of N ¹⁰ -TFA-Pte-OH (22.5 mg), DMF, i -Pr ₂ NEt (4 equiv.) and PyBOP (2 equiv.) was introduced into the vessel. Argon was bubbled in for 2 hours 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 1M hydroxybenzotriazole (HOBT) in DCM/trifluoroethane (TFE) (1:1) (2×3 ml) was added. Argon was bubbled in for 1 hour, the solvent was removed and the resin was washed with DMF (3×3 ml) and i -PrOH (3×3 ml). After swelling the resin in DMF, a solution of Fmoc-NH-(PEG) ₁₂ -COOH (46.3 mg) in DMF, i -Pr ₂ NEt (4 equiv.), and PyBOP (2 equiv.) was added. Argon was bubbled in for 2 hours and the resin was washed with DMF (3×3 ml) and i -PrOH (3×3 ml). Fmoc deprotection was performed using 20% piperidine in DMF (3×10 ml). Kaiser tests were performed to evaluate the progression of the reaction. Then a solution of FITC (Life Technologies, 21.4 mg) in DMF and i -Pr ₂ NEt (4 equiv.) was introduced into the vessel, then argon was bubbled for 2 hours, and the resin was washed using DMF (3×3 ml) and i -PrOH (3×3 ml). Then 2% NH ₂ NH ₂ in DMF (2×2 ml) was added to the vessel. The final compound was cleaved from the resin using TFA:H ₂ O:triisopropylsilane (TIS) (95:2.5:2.5) (cleavage solution) and concentrated in vacuo. The concentrated product was precipitated into Et ₂ O and dried in vacuo. The crude product was purified using preparative RP-HPLC (mobile phase: A=10 mM ammonium acetate pH=7, B=ACN; method: 0% B - 30% B over 30 min at 13 ml/min). Purified fractions were pooled and lyophilized to give FITC-PEG12-folate.

EXAMPLE 7

Synthesis of FITC-PEG20-folate

Polymer-bound (200-400 mesh) resin with ethylenediamine (50 mg) was loaded into a peptide synthesis vessel and allowed to swell in DCM (3 ml) then DMF (3 ml). A solution of Fmoc-PEG ₂₀ -COOH (131 mg, 1.0 equiv.) in DMF, i -Pr ₂ NEt (6.0 equiv.) and PyBOP (4.0 equiv.) was then introduced into the vessel. Argon was bubbled in for 6 hours, the binding solution was drained, and the resin was washed with DMF (3×10 ml) and i -PrOH (3×10 ml). Kaiser tests were performed to evaluate the progression of the reaction. Fmoc deprotection was performed using 20% piperidine in DMF (3×10 ml) prior to addition of each amino acid. The above sequence was repeated until completion of the reaction using the coupling steps with Fmoc-Glu-OtBu (72 mg, 2.0 equiv.) and Tfa.pteroic acid (41 mg, 1.2 equiv.). The resin was washed with 2% hydrazine in DMF 3×10 ml (5 min) to cleave the trifluoroacetyl protecting group on pteric acid and washed with i -PrOH (3×10 ml) then with DMF (3×10 ml). The resin was dried under argon for 30 min. The folate peptide was cleaved from the resin using a cleavage solution. 10 ml of the digestion mixture was introduced and argon was bubbled in for 1.5 hours. The digestion mixture was drained into a clean flask. The resin was washed 3 times using more of the mixture for splitting. The combined mixture was concentrated under reduced pressure to a smaller volume (~5 ml) and precipitated in ethyl ether.

The precipitate was collected by centrifugation, washed with ethyl ether (3 times) and dried under high vacuum. Dried folate-PEG ₂₀ -EDA (1.0 equiv.) was treated with FITC (50 mg, 1.5 equiv.) in DMSO and DIPEA at room temperature. The progression of the reaction was monitored by LCMS. After 8 hours, the starting material was consumed to obtain the product. The crude reaction mixture was purified by preparative HPLC, (mobile phase A=10 mM ammonium acetate, pH=7; organic phase B=acetonitrile; Method: 0% B - 30% B in 35 minutes at 13 ml/min) to give FITC- PEG20-folate with 60% yield.

EXAMPLE 8

Synthesis of FITC-PEG108-folate

Polymer-bound (200-400 mesh) resin with ethylenediamine (50 mg) was loaded into a peptide synthesis vessel and allowed to swell in DCM (3 ml) then DMF (3 ml). Then a solution of Fmoc-PEG ₃₆ -COOH (161 mg, 1.0 equiv.) in DMF, i -Pr ₂ NEt (6.0 equiv.) and PyBOP (4.0 equiv.) was introduced into the vessel. Argon was bubbled in for 6 hours, the binding solution was drained, and the resin was washed with DMF (3×10 ml) and i -PrOH (3×10 ml). Kaiser tests were performed to evaluate the progression of the reaction. Fmoc deprotection was performed using 20% piperidine in DMF (3×10 ml) prior to addition of each amino acid. The above sequence was repeated until completion of the reaction using the steps of coupling with 2X Fmoc-PEG ₃₆ -COOH (161 mg, 1.0 equiv.), Fmoc-Glu-OtBu (72 mg, 2.0 equiv.) and Tfa.pteroic acid ( 41.0 mg, 1.2 equiv.). Finally, the resin was washed with 2% hydrazine in DMF 3×10 ml (5 min) to cleave the trifluoroacetyl protecting group on pteric acid and washed with i -PrOH (3×10 ml) then with DMF (3×10 ml) . The resin was dried under argon for 30 min. The folate peptide was cleaved from the resin using a cleavage solution. 10 ml of the digestion mixture was introduced and argon was bubbled in for 1.5 hours. The digestion mixture was drained into a clean flask. The resin was washed 3X with more cleavage solution. The combined mixture was concentrated under reduced pressure to a smaller volume (~5 ml) and precipitated in ethyl ether.

The precipitate was collected by centrifugation, washed with ethyl ether (3X) and dried under high vacuum. Dried Folate-PEG ₁₀₈ -EDA (1.0 equiv.) was treated with FITC (50 mg, 1.5 equiv.) in DMSO and DIPEA at room temperature. The progression of the reaction was monitored by LCMS. After 10 hours, the starting material was consumed to obtain the product. The crude reaction mixture was purified by preparative HPLC, (mobile phase A=10 mM ammonium acetate, pH=7; organic phase B=acetonitrile; Method: 0% B - 30% B in 35 minutes at 13 ml/min) to give FITC- PEG108-folate with 64% yield.

EXAMPLE 9

Synthesis of FITC-DUPA

DUPA-FITC was synthesized by the solid phase method as follows. Nova Tag™ Universal Resin (50 mg, 0.53 mmol) was allowed to swell in DCM (3 ml) then DMF, 3 ml). A solution of 20% piperidine in DMF (3 x 3 ml) was added to the resin and argon was bubbled in for 5 minutes. The resin was washed with DMF (3×3 ml) and isopropyl alcohol ( i -PrOH, 3×3 ml). After swelling the resin in DMF, a solution of DUPA-(OtBu)-OH (1.5 equiv.), HATU (2.5 equiv.) and i -Pr ₂ NEt (4.0 equiv.) in DMF was added. Argon was bubbled in for 2 hours and the resin was washed with DMF (3×3 ml) and i -PrOH (3×3 ml). After swelling the resin in DCM, a solution of 1 M HOBt in DCM/TFE (1:1) (2×3 ml) was added. Argon was bubbled in for 1 hour, the solvent was removed and the resin was washed with DMF (3×3 ml) and i -PrOH (3×3 ml). After swelling the resin in DMF, a solution of Fmoc-Phe-OH (2.5 equiv.), HATU (2.5 equiv.) and DIPEA (4.0 equiv.) in DMF was added. Argon was bubbled in for 2 hours and the resin was washed with DMF (3×3 ml) and i-PrOH (3×3 ml). The above sequence was repeated for 2 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 cleavage solution and concentrated in vacuo. The concentrated product was precipitated in diethyl ether and dried in vacuo. The crude product was purified using preparative RP-HPLC [λ=488 nm; solvent gradient: 1% B - 80% B over 25 min, wash 80% B over 30 min run; A=10 mM NH ₄ OAc, pH=7; B=acetonitrile (ACN)]. ACN was removed in vacuo and the purified fractions were lyophilized to give FITC-DUPA as an orange-brown solid. RP-HPLC: tR=8.0 min (A=10 mM NH ₄ OAc, pH=7.0; B=ACN, solvent gradient: 1% B - 50% B over 10 min, wash 80% B over 15 min run). ¹ H NMR (DMSO-d6/D ₂ O): δ 0.98-1.27 (ms, 9H); 1.45 (w, 3H); 1.68-1.85 (ms, 11H); 2.03 (m, 8H); 2.6-3.44 (ms, 12H); 3.82 (br, 2H); 4.35 (m, 1H); 6.53 (d, J=8.1 Hz, 2H), 6.61 (dd, J=5.3, 3.5 Hz, 2H); 6.64 (s, 2H); 7.05 (d, J=8.2 Hz, 2H), 7.19 (m, 5H); 7.76 (d, J=8.0 Hz, 1H); 8.38 (s, 1H). HRMS (ESI) (m/z): (M+H) ⁺ calculated for C ₅₁ H ₅₉ N ₇ O ₁₅ S, 1040.3712, found 1040.3702. UV/imaging: λ max=491 nm.

EXAMPLE 10

Synthesis of FITC-PEG12-DUPA

1,2-diaminoethanetrityl resin (0.025 g) was loaded into a peptide synthesis vessel and washed with i -PrOH (3×10 ml) followed by DMF (3×10 ml). Then a solution of Fmoc-NH-(PEG) ₁₂ -COOH (42.8 mg) in DMF, i -Pr ₂ NEt (2.5 equiv.), and PyBOP (2.5 equiv.) was introduced into the vessel. The resulting solution was sparged with Ar for 1 hour, the binding solution was drained, and the resin was washed with DMF (3×10 ml) and i -PrOH (3×10 ml). Kaiser tests were performed to evaluate the progression of the reaction. Fmoc deprotection was performed using 20% piperidine in DMF (3×10 ml). This procedure was repeated until all coupling steps were completed (2 x 1.5 equiv Fmoc-Phe-OH and 1.5 equiv 8-aminooctanoic acid and 1.2 equiv DUPA were used in each of their respective coupling steps). After binding DUPA, 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 a cleavage solution. 15 ml of the digestion solution was added to the peptide synthesis vessel and the reaction mixture was sparged under Ar for 15 minutes. The resin was treated with two additional amounts of 10 ml digestion solution for 5 minutes each. The digestion mixture was concentrated to approximately 5 ml and precipitated using ethyl ether. The precipitate was collected by centrifugation, washed with ethyl ether (3X) and dried under high vacuum, which resulted in the isolation of crude material. To a mixed solution of crude DUPA-(PEG) ₁₂ -EDA (10 mg) and FITC (5.6 mg) in dimethyl sulfoxide (DMSO, 1 ml) was added i -Pr ₂ NEt (5 equiv.) at room temperature and stirred for 6 hours under argon. 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 - 50% B over 30 min at 13 ml/min). Purified fractions were pooled and lyophilized to give FITC-PEG12-DUPA.

EXAMPLE 11

Synthesis of FITC-PEG11-NK1

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

*Note: The NK-1 compound was synthesized in a two-step process, starting from the base ligand, which was prepared using a method from the literature. (reference: DESIGN AND DEVELOPMENT OF NEUROKININ-1 RECEPTOR-BINDING AGENT DELIVERY CONJUGATES, application number: PCT/US2015/44229, the contents of which are incorporated herein by reference.

EXAMPLE 12

Synthesis of FITC-PEG2-CA9

The CA9 ligand (53.6 mg) was dissolved in DMF (2-3 ml) in a 50 ml round bottom flask using a Teflon stir bar. The circulating air was removed using vacuum and replaced with nitrogen gas, this was done in three cycles. The round bottom flask was constantly maintained under nitrogen gas. 28.9 mg of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) was added to the flask, followed by 21.6 mg of 1-hydroxybenzotriazole hydrate (HOBt) and 18.9 μl of Boc-PEG ₂ -NH ₂ (Sigma Aldrich). 5.4 μl of triethylamine (TEA) was added and the reaction mixture was stirred overnight. The reaction mixture was purified using HPLC and confirmed using UHPLC-MS (target m/z 831). The acetonitrile was removed using high vacuum rotary evaporation and the product was lyophilized. The compound was mixed with 1:1 TFA:DCM for 30 minutes. The TFA/DCM was removed using high vacuum rotary evaporation followed by 30 minutes under high vacuum. The compound was then dissolved in DMF and combined with 5 molar equivalents of i -Pr ₂ NEt, 16 mg of fluorescein isothiocyanate (Life Technologies) and stirred for 1 hour. The reaction mixture was purified by HPLC and the title compound was confirmed using UHPLC-MS (target m/z 1120). Samples were lyophilized and stored at -20°C.

EXAMPLE 13

Estimation of anti-FITC scFv expression on transduced CAR-T cells

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

EXAMPLE 14

CAR-T cell cytotoxicity assay in vitro

To test the cytotoxicity of CAR-T cells using the desired FITC ligands, a standard lactate dehydrogenase (LDH) release assay was performed using the Pierce™ LDH Cytotoxicity Assay Kit from ThermoFisher Scientific. To obtain samples for LDH analysis, malignant tumor cells were seeded at a density of 10 ⁴ cells/100 μl of medium into each well of a 96-well plate and grown overnight. CAR-T cells were prepared the next day in various amounts to obtain different ratios of effector cells (CAR-T cells):target cells (malignant tumor cells) (for example, E:T=20:1, 10:1, 5 :1 and 1:1). Various concentrations of FITC ligands were injected into each well and co-cultured for 6-24 hours. After co-incubation, the plate containing CAR-T cells and cancer cells was centrifuged at 350× g at room temperature for 10 min to remove cell debris or remaining cells. Then 50 μl of supernatants from each sample were transferred to a new 96-well plate. 50 µl of the resulting LDH reaction mixture was added to the transferred 50 µl of each sample and incubated at room temperature for 30 minutes. 50 μl of stop solution was added and the absorbance of each sample was measured at 490 nm and 680 nm. Percent cytotoxicity was calculated for each sample using the equation below:

% cytotoxicity=(experimental value - spontaneous for effector cells - spontaneous for target cells)/(maximum control for target cells - spontaneous control for target cells) × 100

As shown in Figures 3A-C, CAR-T cells are only activated when paired with matching tumor antigen conjugates. Figure 3A shows the cytotoxicity of CAR-T cells in the KB (FR+) model with E:T 10:1 using 100 nM of each conjugate. Activation with FITC-folate, FITC-PEG20-folate and FITC-PEG108-folate is greater than activation with FITC-DUPA or in the absence of the conjugate. Figure 3B shows the cytotoxicity of CAR-T cells in the LNCaP (PSMA+) model with E:T 10:1 using 100 nM FITC-DUPA or FITC-PEG12-DUPA. FITC-DUPA conjugates showed greater activation than with FITC-folate or no conjugate. Figure 3C shows the cytotoxicity of CAR-T cells in the HEK293 (NK1R+) model with E:T 10:1 using 100 nM FITC-PEG11-NK1. FITC-PEG11-NK1 showed greater activation than activation with FITC-folate or in the absence of the conjugate.

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

As shown in Figure 10, CAR-T cytotoxicity in the KB (FR+) model can be controlled by adjusting the bridge conjugate concentration, or by using linkers of various PEG lengths with E:T 10:1.

EXAMPLE 15

Identification of CAR-T Cell Proliferation and CAR-T Cell Activation

The proliferation of CAR-T cells was mainly measured by flow cytometry. First, cancer cells (KB (FR+) or HEK (NK1R+)) were seeded at a density of 10 ⁴ cells/100 μl of medium into each well of a 96-well plate and grown overnight. The following day, CAR-T cells were injected into each well, either in the presence or absence of the desired FITC ligands, and the cells were co-cultured for 5 days (120 hours). After co-incubation, CAR-T cells were stained with APC anti-human CD3 antibodies (Biolegend) by standard immunostaining methods (20 min on ice). Cells positive for both anti-human CD3 staining and GFP were counted for proliferation of CAR-T cells.

As shown in Figure 1A-B, CAR-T cells proliferated by targeting tumor cells (E:T=5:1) using FITC-small molecule conjugate (100 nM) using KB (FR+) cells and HEK293 cells (NK1R+). Figure 1A shows the proliferation of a CAR-T cell in the presence of KB (FR+) cells using various conjugates. Figure 1B shows the proliferation of a CAR-T cell in the presence of HEK (NK1R+) cells using various conjugates. As can also be seen in Figure 1, the presence of linkers with different PEG lengths affects the levels of proliferation of the CAR-T cell.

To measure the activation of CAR-T cells, cancer cells were obtained in the same way as described above. Cancer cells and CAR-T cells were co-cultured in the presence or absence of 100 nM FITC ligands at an E:T ratio of 10:1 for 24 hours and harvested. After washing, cells from each sample were stained with anti-human CD69 antibody with Alexa Fluor 647 (Biolegend). The data were analyzed by selection for GFP positive T cells to quantify CAR T cell activation. As shown in Figure 17, CD69 expression is associated with the co-cultured conjugate.

EXAMPLE 16

Analysis of IFN-γ production

To test IFN-γ production by CAR-T cells, a standard ELISA was performed using the human IFN-γ ELISA kit from Biolegend. Briefly, cancer cells were plated at a density of 10 ⁴ cells/100 μl of medium in each well of a 96-well plate and grown overnight. CAR-T cells were injected into each cancer sample with the desired FITC ligands and co-cultured for 24 hours. After co-incubation, the supernatants of each sample were collected and centrifuged at 1000× g and 4°C for 10 min to remove cell debris. The clarified supernatants from each sample were then either used for IFN-γ detection by ELISA or stored at -80° C. for future use. After completion of each sample, a standard ELISA was performed based on the manufacturer's instructions.

As shown in Figures 2A-C, when using a 5:1 E:T ratio in KB (FR+) (Figure 2A), LNCaP (PSMA+) (Figure 2B) or HEK (NK1R+) (Figure 2C) cells, CAR-T- cells produce a significant amount of pro-inflammatory cytokine in the presence of 100 nM FITC-small molecule conjugate. As can also be seen, the amount of cytokine depends on the conjugate used. As shown in Figure 2D, the amount of pro-inflammatory cytokine is related to the concentration of FITC-folate used with KB (FR+) cells. As shown in Figure 2E and 2F, different IFN-γ responses were obtained for different conjugates (100 nM) using KB (FR+) cells.

As shown in Figures 4A-B, CAR-T cell activation correlates with tumor antigen levels in KB (FR+) cells and MDA-MB-231 cells. Measurement of IFN-γ production following cell incubation with the same dose of conjugate shows that T cell activation with anti-FITC CAR correlates with the level of tumor antigen expression on cancer cells.

EXAMPLE 17

Assessing the Correlation between Tumor Antigen Level and CAR-T Activation

KB (FR+) cells and MDA-MB-231 cells were incubated with 100 nM FITC-folate for 30 min on ice. After washing, the binding of FITC-folate to tumor antigen FRα on cells was measured by flow cytometry. As shown in Figure 4A, KB (FR+) cells have a higher level of FR expression and corresponding FITC binding than MDA-MB-231 cells.

These two cell lines were co-cultured with CAR-T cells in the presence of 100 nM FITC-folate, FITC-PEG20-folate and FITC-PEG108-folate using an E:T ratio of 10:1 for 24 hours. The activation of CAR-T cells was detected by measuring their INF-γ production. The cultured cell supernatant was collected and IFN-γ production was measured using an ELISA kit. As shown in Figure 4B, using higher levels of FRα, KB (FR+) cells activated CAR-T cells much more than MDA-MB-231 cells.

EXAMPLE 18

Antitumor efficacy of CAR-T cells in vivo

Immunocompromised NSG mice (Jackson Laboratory) were used to identify the efficacy of the antitumor activity of the CAR-T cell in vivo . Each tumor-specific antigen-expressing cancer cell line was subcutaneously injected into the upper arm of NSG mice to develop solid tumor xenografts. When the tumor volume reached approximately 50-100 mm ³ CAR-T cells were injected into mice bearing tumors and the desired FITC ligands were also injected (iv) every other day. Control mice were injected with PBS instead of FITC ligands. Tumor volume and blood cytokine levels (IL2, IL6, IL10, IFNγ and TNFα) were measured. The overall toxicity of therapy was monitored by measuring weight loss. At the end of each treatment, mouse blood was collected for testing for anemia, white blood cell count, and CAR-T cell proliferation. The organs of mice were also evaluated.

A xenotransplantation model using HEK293 (NK1R+) cells is shown in Figures 5A-C and 6A-B. As shown in Figure 5A, tumors decreased in size in mice treated with FITC-PEG11-NK1 (500 nmol/kg), but they continued to grow in control mice. As shown in figure 5B, the body weight of mice treated with FITC-PEG11-NK1 did not change compared to the control mouse. As shown in Figure 5C, the percentage of CAR-T cells in human T cells increased after injection of CAR-T cells. As shown in Figures 6A-B, harvested organs from the treatment group (6B) appeared normal in size with no evidence of cytokine release syndrome after two weeks of therapy compared to harvested organs from control mice (6A).

A xenotransplantation model using MDA-MB-231 (FR+) cells is shown in Figures 7A-C and 8A-B. As shown in Figure 7A, tumor size decreased in mice treated with FITC-PEG12-folate (500 nmol/kg) or FITC-folate (500 nmol/kg), but tumors continued to grow in control mice. As shown in Figure 7B, the body weight of mice treated with FITC-PEG11-NK1 did not change during treatment. As shown in Figure 7C, the percentage of CAR-T cells in human T-cells increased after therapy with CAR-T cells and conjugate. As shown in Figures 8A-B, harvested organs from mice treated with FITC-PEG12-folate (8B) appeared normal in size and no signs of cytokine release syndrome were observed after 3 weeks of therapy compared to harvested organs from control mice. group (8A). As used in any of the embodiments in this patent application, "nmol/kg" and "nmol/kg" are equivalent.

As shown in Figure 9, blood counts for the HEK-NK1R model using FITC-PEG11-NK1 (500 nmol/kg) and the MDA-MB-231 model using FITC-PEG12-folate (500 nmol/kg) also show that no cytokine storm occurred during treatment.

As shown in Figures 11, 12A-C and 13, KB (FR+) tumor xenografts were treated with two different concentrations of FITC-PEG12-folate. As shown in Figure 11, mice treated with the lower dose (250 nmol/kg) had milder body weight loss compared to mice treated with the higher dose (500 nmol/kg). As shown in Figures 12A-C, harvested organs from untreated mice (Figure 12A) and lower dose mice (Figure 12B) showed a milder cytokine release syndrome compared to the higher dose (Figure 12C). As shown in Figure 13, KB xenograft mice given lower doses showed better blood counts indicating a milder release of cytokines.

EXAMPLE 19

Antitumor efficacy of CAR-T cells in vivo

To investigate whether identical T cells with anti-FITC CAR could kill a mixture of heterogeneous malignant tumor cells using a cocktail of FITC ligands, immunodeficient NSG mice (Jackson laboratory) were used for an in vivo study. Two different cell lines (eg, MDA-MB-231(FR+) and HEK(NK1R+)) were implanted in different flanks of the same mouse. Then T-cells with CAR against FITC (10 ⁷ cells) were administered by intravenous injection when tumor volumes reached approximately 50-100 mm ³ . In addition, a mixture of FITC ligands (i.e. FITC-PEG12-folate (500 nmol/kg) and FITC-PEG11-NK1R (500 nmol/kg)), one FITC-PEG11-NK-1R (500 nmol/kg ) or PBS was administered every other day by intravenous injection. The antitumor efficacy of CAR-T cells using FITC ligands was analyzed by measuring tumor volume every second day.

As shown in Figures 19A-C, both tumors (i.e., MDA-MB-231 (MDA) and HEK (NK1R)) were killed by the same anti-FITC CAR T cells when both FITC-PEG11-NK1R and FITC-PEG12-folate. Only the HEK (NK1R) tumor was killed in the mouse treated with FITC-PEG11-NK1R alone. Without being bound by theory, it is believed that MDA-MB-231 continued to grow in the same mouse when FITC-PEG11-NK1R alone was administered because FITC-PEG12-folate was not administered. As expected, both tumors showed no response when PBS was administered. These data suggest that identical T cells with anti-FITC CARs can eradicate an antigenically heterogamous mixture of tumors via a cocktail of FITC ligands.

--->

SEQUENCE LIST

<110> PURDUE RESEARCH FOUNDATION

ENDOCYTE, INC.

<120> METHODS AND COMPOSITIONS FOR CAR-T CELL THERAPY

<130> 3220-264637

<140> PCT/US2017/026618

<141> 2017-04-07

<150> 62/323.971

<151> 2016-04-18

<150> 62/320.183

<151> 2016-04-08

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 1554

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polynucleotide

<400> 1

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggatgtcg tgatgaccca gacccccctc agcctcccag tgtccctcgg tgaccaggct 120

tctattagtt gcagatccag ccagtccctc gtgcactcta acggtaatac ctacctgaga 180

tggtatctcc agaagcccgg acagagccct aaggtgctga tctacaaagt ctccaaccgg 240

gtgtctggag tccctgaccg cttctcaggg agcggttccg gcaccgactt caccctgaag 300

atcaaccggg tggaggccga agacctcggc gtctatttct gctctcagag tacacatgtg 360

ccctggacct tcggcgggagg gaccaagctg gagatcaaaa gctccgcaga cgatgccaag 420

aaagatgccg ctaagaaaga cgatgctaag aaagacgatg caaagaaaga cggtggcgtg 480

aagctggatg aaaccgggagg aggtctcgtc cagccaggag gagccatgaa gctgagttgc 540

gtgaccagcg gattcacctt tgggcactac tggatgaact gggtgcgaca gtccccagag 600

aaggggctcg aatgggtcgc tcagttcagg aacaaaccct acaattatga gacatactat 660

tcagacagcg tgaagggcag gtttactatc agtagagacg attccaaatc tagcgtgtac 720

ctgcagatga acaatctcag ggtcgaagat acaggcatct actattgcac aggggcatcc 780

tatggtatgg agtatctcgg tcaggggaca agcgtcacag tcagtttcgt gccggtcttc 840

ctgccagcga agcccaccac gacgccagcg ccgcgaccac caacaccggc gcccaccatc 900

gcgtcgcagc ccctgtccct gcgccgag gcgtgccggc cagcggcggg gggcgcagtg 960

cacacgaggg ggctggactt cgcctgtgat atctacatct gggcgccctt ggccgggact 1020

tgtggggtcc ttctcctgtc actggttatc accctttact gcaaccacag gaaccgtttc 1080

tctgttgtta aacggggcag aaagaaactc ctgtatatat tcaaacaacc atttatgaga 1140

ccagtacaaa ctactcaaga ggaagatggc tgtagctgcc gatttccaga agaagaagaa 1200

ggaggatgtg aactgagagt gaagttcagc aggagcgcag acgcccccgc gtaccagcag 1260

ggccagaacc agctctataa cgagctcaat ctaggacgaa gagaggagta cgatgttttg 1320

gacaagagac gtggccggga ccctgagatg gggggaaagc cgagaaggaa gaaccctcag 1380

gaaggcctgt acaatgaact gcagaaagat aagatggcgg aggcctacag tgagattggg 1440

atgaaaggcg agcgccggag gggcaagggg cacgatggcc tttaccaggg tctcagtaca 1500

gccaccaagg acacctacga cgcccttcac atgcaggccc tgccccctcg ctaa 1554

<210> 2

<211> 517

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 2

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu

20 25 30

Pro Val Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln

35 40 45

Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu Arg Trp Tyr Leu Gln

50 55 60

Lys Pro Gly Gln Ser Pro Lys Val Leu Ile Tyr Lys Val Ser Asn Arg

65 70 75 80

Val Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp

85 90 95

Phe Thr Leu Lys Ile Asn Arg Val Glu Ala Glu Asp Leu Gly Val Tyr

100 105 110

Phe Cys Ser Gln Ser Thr His Val Pro Trp Thr Phe Gly Gly Gly Thr

115 120 125

Lys Leu Glu Ile Lys Ser Ser Ala Asp Asp Ala Lys Lys Asp Ala Ala

130 135 140

Lys Lys Asp Asp Ala Lys Lys Asp Asp Ala Lys Lys Asp Gly Gly Val

145 150 155 160

Lys Leu Asp Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Gly Ala Met

165 170 175

Lys Leu Ser Cys Val Thr Ser Gly Phe Thr Phe Gly His Tyr Trp Met

180 185 190

Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val Ala Gln

195 200 205

Phe Arg Asn Lys Pro Tyr Asn Tyr Glu Thr Tyr Tyr Ser Asp Ser Val

210 215 220

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser Val Tyr

225 230 235 240

Leu Gln Met Asn Asn Leu Arg Val Glu Asp Thr Gly Ile Tyr Tyr Cys

245 250 255

Thr Gly Ala Ser Tyr Gly Met Glu Tyr Leu Gly Glyn Gly Thr Ser Val

260 265 270

Thr Val Ser Phe Val Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr

275 280 285

Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro

290 295 300

Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val

305 310 315 320

His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro

325 330 335

Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu

340 345 350

Tyr Cys Asn His Arg Asn Arg Phe Ser Val Val Lys Arg Gly Arg Lys

355 360 365

Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr

370 375 380

Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu

385 390 395 400

Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro

405 410 415

Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly

420 425 430

Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro

435 440 445

Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr

450 455 460

Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly

465 470 475 480

Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln

485 490 495

Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln

500 505 510

Ala Leu Pro Pro Arg

515

<210> 3

<211> 1560

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polynucleotide

<400> 3

gccaccatgg ccttaccagt gaccgccttg ctcctgccgc tggccttgct gctccacgcc 60

gccaggccgg atgtcgtgat gacccagacc cccctcagcc tcccagtgtc cctcggtgac 120

caggcttcta ttagttgcag atccagccag tccctcgtgc actctaacgg taatacctac 180

ctgagatggt atctccagaa gcccggacag agccctaagg tgctgatcta caaagtctcc 240

aaccgggtgt ctggagtccc tgaccgcttc tcagggagcg gttccggcac cgacttcacc 300

ctgaagatca accgggtgga ggccgaagac ctcggcgtct atttctgctc tcagagtaca 360

catgtgccct ggaccttcgg cggagggacc aagctggaga tcaaaagctc cgcagacgat 420

gccaagaaag atgccgctaa gaaagacgat gctaagaaag acgatgcaaa gaaagacggt 480

ggcgtgaagc tggatgaaac cggaggaggt ctcgtccagc caggaggagc catgaagctg 540

agttgcgtga ccagcggatt cacctttggg cactactgga tgaactgggt gcgacagtcc 600

ccagagaagg ggctcgaatg ggtcgctcag ttcaggaaca aaccctacaa ttatgagaca 660

tactattcag acagcgtgaa gggcaggttt actatcagta gagacgattc caaatctagc 720

gtgtacctgc agatgaacaa tctcaggggtc gaagatacag gcatctacta ttgcacaggg 780

gcatcctatg gtatggagta tctcggtcag gggacaagcg tcacagtcag tttcgtgccg 840

gtcttcctgc cagcgaagcc caccacgacg ccagcgccgc gaccaccaac accggcgccc 900

accatcgcgt cgcagcccct gtccctgcgc ccagaggcgt gccggccagc ggcggggggc 960

gcagtgcaca cgagggggct ggacttcgcc tgtgatatct acatctgggc gcccttggcc 1020

gggacttgtg gggtccttct cctgtcactg gttatcaccc tttactgcaa ccacaggaac 1080

1140

atgagaccag tacaaactac tcaagaggaa gatggctgta gctgccgatt tccagaagaa 1200

gaagaaggag gatgtgaact gagagtgaag ttcagcagga gcgcagacgc ccccgcgtac 1260

cagcagggcc agaaccagct ctataacgag ctcaatctag gacgaagaga ggagtacgat 1320

1380

cctcaggaag gcctgtacaa tgaactgcag aaagataaga tggcggaggc ctacagtgag 1440

1500

agtacagcca ccaaggacac ctacgacgcc cttcacatgc aggccctgcc ccctcgctaa 1560

<210> 4

<211> 519

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 4

Ala Thr Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu

1 5 10 15

Leu Leu His Ala Ala Arg Pro Asp Val Val Met Thr Gln Thr Pro Leu

20 25 30

Ser Leu Pro Val Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser

35 40 45

Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu Arg Trp Tyr

50 55 60

Leu Gln Lys Pro Gly Gln Ser Pro Lys Val Leu Ile Tyr Lys Val Ser

65 70 75 80

Asn Arg Val Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly

85 90 95

Thr Asp Phe Thr Leu Lys Ile Asn Arg Val Glu Ala Glu Asp Leu Gly

100 105 110

Val Tyr Phe Cys Ser Gln Ser Thr His Val Pro Trp Thr Phe Gly Gly

115 120 125

Gly Thr Lys Leu Glu Ile Lys Ser Ser Ala Asp Asp Ala Lys Lys Asp

130 135 140

Ala Ala Lys Lys Asp Asp Ala Lys Lys Asp Asp Ala Lys Lys Asp Gly

145 150 155 160

Gly Val Lys Leu Asp Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Gly

165 170 175

Ala Met Lys Leu Ser Cys Val Thr Ser Gly Phe Thr Phe Gly His Tyr

180 185 190

Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val

195 200 205

Ala Gln Phe Arg Asn Lys Pro Tyr Asn Tyr Glu Thr Tyr Tyr Ser Asp

210 215 220

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser

225 230 235 240

Val Tyr Leu Gln Met Asn Asn Leu Arg Val Glu Asp Thr Gly Ile Tyr

245 250 255

Tyr Cys Thr Gly Ala Ser Tyr Gly Met Glu Tyr Leu Gly Gln Gly Thr

260 265 270

Ser Val Thr Val Ser Phe Val Pro Val Phe Leu Pro Ala Lys Pro Thr

275 280 285

Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser

290 295 300

Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly

305 310 315 320

Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp

325 330 335

Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile

340 345 350

Thr Leu Tyr Cys Asn His Arg Asn Arg Phe Ser Val Val Lys Arg Gly

355 360 365

Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val

370 375 380

Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu

385 390 395 400

Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp

405 410 415

Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn

420 425 430

Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg

435 440 445

Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly

450 455 460

Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu

465 470 475 480

Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu

485 490 495

Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His

500 505 510

Met Gln Ala Leu Pro Pro Arg

515

<---

Claims (52)

1. A method for treating a cancer expressing an NK-1 receptor, comprising i) administering to the patient the conjugate or a pharmaceutically acceptable salt thereof, where the conjugate contains NK-1 receptor-binding ligand (NK1RL) and fluorescein isothiocyanate (FITC); And wherein the conjugate is capable of binding to a cancer expressing the NK-1 receptor; And ii) administering to the patient a CAR-T cell composition, wherein the CAR-T cell contains a CAR directed to FITC; And where a patient is being treated for a malignant tumor. 2. The method of claim 1, wherein the NK1RL is linked to FITC via a linker, wherein the linker contains polyethylene glycol (PEG), polyproline, a hydrophilic amino acid, sugar, non-natural peptidoglycan, polyvinylpyrrolidone, and/or pluronic F-127. 3. The method of claim 2 wherein the linker contains PEG. 4. The method according to p. 2 or 3, where the conjugate or its pharmaceutically acceptable salt has the formula

, where B is a low molecular weight ligand, L is a linker, and T is a targeting group, and where L contains a structure having the formula

, where n is an integer from 0 to 200. 5. The method according to claim 4, where n is an integer from 0 to 150. 6. The method according to claim 4, where n is an integer from 0 to 110. 7. The method according to claim 4, where n is an integer from 0 to 20. 8. The method according to claim 4, where n is an integer from 15 to 20. 9. The method according to claim 4, where n is an integer from 15 to 110. 10. The method according to any one of paragraphs. 1-9, wherein the conjugate, or a pharmaceutically acceptable salt thereof, is administered at a dose of about 10 nmol/kg to about 3000 nmol/kg of the patient's body weight. 11. The method according to any one of paragraphs. 1-9, wherein the conjugate, or a pharmaceutically acceptable salt thereof, is administered at a dose of about 50 nmol/kg to about 2000 nmol/kg of the patient's body weight. 12. The method according to any one of paragraphs. 1-9, wherein the conjugate, or a pharmaceutically acceptable salt thereof, is administered at a dose of about 100 nmol/kg to about 1000 nmol/kg of the patient's body weight. 13. The method according to any one of paragraphs. 1-9, wherein the conjugate, or a pharmaceutically acceptable salt thereof, is administered at a dose of about 100 nmol/kg to about 600 nmol/kg of the patient's body weight. 14. The method according to any one of paragraphs. 1-9, wherein the conjugate, or a pharmaceutically acceptable salt thereof, is administered at a dose of about 200 nmol/kg to about 500 nmol/kg of the patient's body weight. 15. The method according to any one of paragraphs. 1-9, wherein the conjugate, or a pharmaceutically acceptable salt thereof, is administered at a dose of about 250 nmol/kg to about 500 nmol/kg of the patient's body weight. 16. The method according to any one of paragraphs. 1-15, where the malignant tumor is selected from lung cancer, bone cancer, pancreatic cancer, skin cancer, head cancer, neck cancer, skin melanoma, intraocular melanoma, uterine cancer, ovarian cancer, endometrial cancer, rectal cancer, gastric cancer , colon cancer, breast cancer, triple negative breast cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, 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, urethral cancer, prostate cancer, chronic leukemia, acute leukemia, lymphocytic lymphoma, pleural mesothelioma, bladder cancer, Burkitt's lymphoma, ureter cancer , kidney cancer, kidney cancer ancestral cancer, renal pelvis carcinoma, neoplasia of the central nervous system (CNS), primary CNS lymphoma, spinal cord tumors, brainstem glioma, pituitary adenoma and adenocarcinoma of the gastroesophageal junction. 17. The method of claim 16 wherein the cancer is endometrial cancer. 18. The method of claim 16 wherein the cancer is non-small cell lung cancer. 19. The method of claim 16 wherein the cancer is ovarian cancer. 20. The method of claim 16 wherein the cancer is triple negative breast cancer. 21. The method according to any one of paragraphs. 1-20, where CAR has a recognition region and the recognition region is a single chain fragment of the variable region (scFv) of an antibody. 22. The method according to any one of paragraphs. 1-21, where CAR has a recognition region and the CAR recognition region is a single chain fragment of the variable region (scFv) of an anti-FITC antibody. 23. The method according to any one of paragraphs. 1-22, where CAR has a costimulatory domain and the costimulatory domain is selected from CD28, CD137 (4-1BB), CD134 (OX40), and CD278 (ICOS). 24. The method according to any one of paragraphs. 1-23, wherein CAR has an activation signaling domain and the activation signaling domain is a CD3ζ T cell chain or an Fcγ receptor. 25. The method according to any one of paragraphs. 1-24, where CAR has a recognition region and the recognition region is a single chain fragment of the variable region (scFv) of an anti-FITC antibody, where CAR has a costimulatory domain and the costimulatory domain is CD137 (4-1BB), and where CAR has a signaling activation domain, and the activation signaling domain is the CD3ζ T cell chain. 26. The method according to any one of paragraphs. 1-25 wherein multiple doses of the conjugate or a pharmaceutically acceptable salt thereof and the CAR-T cell composition are administered. 27. The method according to any one of paragraphs. 1-26 wherein the patient is imaged prior to administration of the conjugate or a pharmaceutically acceptable salt thereof, or prior to administration of the CAR-T cell composition. 28. The method according to any one of paragraphs. 1-27, wherein the conjugate, or a pharmaceutically acceptable salt thereof, is not an antibody and does not contain an antibody fragment. 29. The method according to any one of paragraphs. 1-28, where cytokine release leading to "off-target" toxicity in the patient does not occur, and where CAR-T cell toxicity to cancer occurs. 30. The method according to any one of paragraphs. 1-29, where "off-target" tissue toxicity does not occur in the patient, and where CAR-T cell toxicity to cancer occurs. 31. The method according to any one of paragraphs. 1-30, where the cancer contains a tumor, where the size of the tumor is reduced in the patient, and where "off-target" toxicity does not occur. 32. The method according to any one of paragraphs. 1-31, where the CAR-T cell contains a nucleic acid containing SEQ ID NO:1. 33. The method according to any one of paragraphs. 1-32, where the CAR-T cell contains a polypeptide containing SEQ ID NO:2. 34. The method according to any one of paragraphs. 1-33, where the CAR-T cell contains an isolated nucleic acid containing SEQ ID NO:1 and encoding a chimeric antigen receptor. 35. The method according to any one of paragraphs. 1-34, where the CAR-T cell contains a chimeric antigen receptor polypeptide containing SEQ ID NO:2. 36. The method according to any one of paragraphs. 1-35, where the CAR-T cell is transformed with a vector containing SEQ ID NO:1. 37. The method of claim 36 wherein the vector is a lentiviral vector. 38. The method according to any one of paragraphs. 1-37, where CAR contains human amino acid sequences. 39. The method according to any one of paragraphs. 1-38, where CAR consists of human amino acid sequences. 40. The method according to any one of paragraphs. 1-39, where the conjugate has the formulas

,

,

or

.

Images