Methods of treatment employing targeting small molecules and anti-targeting CAR T cells

Abstract

Some embodiments of the methods and compositions provided herein include therapies for treating, ameliorating, or inhibiting cancer (e.g., osteosarcoma) in a human subject. Some embodiments include administering to a subject a fluorescein isothiocyanate (FITC)-folate conjugate, or a pharmaceutically acceptable salt thereof, in an incremental dose regimen following or concurrently with administering to the subject a chimeric antigen receptor (CAR) T cell therapy (e.g., T cells comprising anti-fluorescein CAR). In some such embodiments, the dose increment regimen includes a dose increment period to confirm a maximum tolerable dose (MTD) of the FITC-folate conjugate, or a pharmaceutically acceptable salt thereof.

Description

Cross - Reference to Related Applications

[0001] This application claims the priority of U.S. Provisional Application No. 63 / 380,124, filed on October 19, 2022, which is incorporated herein by reference in its entirety. Reference to Sequential List

[0002] This application is filed together with a sequential list in electronic format. The sequential list is provided as a file named SCRI458WOSEQLIST created on October 11, 2023, with a size of approximately 25,372 bytes. The information within the electronic format of the sequential list is incorporated herein by reference in its entirety. Technical Field

[0003] Some embodiments of the methods and compositions provided herein include therapies for treating, ameliorating, or inhibiting cancer (e.g., osteosarcoma) in a human subject. Some embodiments include administering to the subject a fluorescein isothiocyanate (FITC)-folate conjugate or a pharmaceutically acceptable salt thereof in a dosing escalation regimen after or simultaneously with administering a chimeric antigen receptor (CAR) T cell therapy (e.g., T cells comprising an anti - fluorescein CAR) to the subject. In some such embodiments, the dosing escalation regimen includes dosing escalation cycles to confirm the maximum tolerated dose (MTD) of the FITC - folate conjugate or a pharmaceutically acceptable salt thereof. Background Art

[0004] Immunotherapies based on the adoptive transfer of lymphocytes (e.g., T cells) into patients are valuable therapies for treating cancer and other diseases. Significant progress has been made in the development of immunotherapies based on the adoptive transfer of lymphocytes. Among the many different types of immunotherapeutic agents, one of the most promising immunotherapeutic agents being developed is T cells expressing chimeric antigen receptors (CAR T cells). A chimeric antigen receptor (CAR) is a genetically engineered receptor that is designed to target a specific antigen, such as a tumor antigen. This targeting can elicit cytotoxicity against the tumor, such that CAR T cells expressing the CAR can target and kill tumors through a specific tumor antigen.

[0005] The first - generation CAR consists of an antigen - recognition region (e.g., a single - chain variable fragment (scFv) region, which is derived from an antibody and is used to recognize and bind an antigen expressed by the tumor) and an activation signaling domain (e.g., the CD3ζ chain of a T cell can serve as a T - cell activation signal in the CAR). Although CAR T cells show positive results in vitro, their success in eliminating diseases (e.g., cancer) in clinical trials has been limited. One problem is the inability to prolong the activation and expansion of CAR T cell populations in vivo.

[0006] To address this issue, second-generation CARs incorporate co-stimulatory domains (e.g., CD137, CD28, or CD134) to achieve prolonged activation of T cells in vivo. The addition of co-stimulatory domains enhances the in vivo proliferation and survival of CAR-containing T cells, and preliminary clinical data suggest that such constructs are promising therapeutic agents in the treatment of diseases such as cancer.

[0007] Although CAR T cell therapy has seen improvements, several issues remain. First, "off-target" toxicity can occur because normal cells express antigens targeted by CAR T cells (e.g., tumor-associated antigens). Second, unregulated CAR T cell activation can occur, where the rapid and uncontrolled clearance of diseased cells (e.g., cancer cells) by CAR T cells induces a series of metabolic disorders called tumor lysis syndrome or cytokine release syndrome (CRS), which can be fatal to the patient. Tumor lysis syndrome and CRS can result from the inability of administered CAR T cells to be easily regulated and their uncontrolled activation. Thus, while CAR T cells show great promise as tools in the treatment of diseases such as cancer, additional CAR T cell treatment regimens are needed to provide reduced off-target toxicity and more precise control of CAR T cell activation. SUMMARY OF THE INVENTION

[0008] Some embodiments provided herein relate to therapies comprising compositions and methods for treating, ameliorating, or inhibiting osteosarcoma in a human subject, comprising administering to the subject a fluorescein isothiocyanate (FITC)-folate compound conjugate or a pharmaceutically acceptable salt thereof in a dosing regimen comprising: a dosing escalation period to confirm the maximum tolerated dose (MTD) of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof, wherein the dosing escalation period comprises administering: (a) a first dose of about 0.5% to about 5% of the full dose of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof; (b) a second dose of about 5% to about 50% of the full dose of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof; (c) a third dose of about 50% to about 500% of the full dose of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof, wherein the MTD is confirmed after the third dose; and (d) a fourth dose of the MTD of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof, wherein the full dose of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof is from about 1x10 -2 mg / kg to about 5x10 -2mg / kg; wherein the human subject has received or is receiving a chimeric antigen receptor (CAR) T cell composition comprising a population of T cells expressing an anti-fluorescein CAR or an anti-fluorescein derivative CAR, thereby treating, ameliorating, or inhibiting the osteosarcoma in the human subject. In some embodiments, the osteosarcoma is recurrent or refractory. In some embodiments, the time period between administering the first dose and administering the second dose is between about 1 day and about 7 days. In some embodiments, the time period between administering the first dose and administering the second dose is about 2 days. In some embodiments, the time period between administering the second dose and administering the third dose is between about 1 day and about 7 days. In some embodiments, the time period between administering the second dose and administering the third dose is about 3 days. In some embodiments, the total dose of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof is about 3.1x10 -2 mg / kg. In some embodiments, the method further comprises administering a fifth dose of the MTD of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof. In some embodiments, the method further comprises a restaging period after the fifth dose, wherein the restaging period comprises: evaluating the subject to determine whether one or more criteria for receiving at least one subsequent dosing cycle of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof are met, provided that the subject is not administered the FITC-folate compound conjugate during the restaging period. In some embodiments, the subject meets more than one criterion, and wherein the subject is administered at least one subsequent dosing cycle, the dosing cycle comprising administering the MTD of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof at a dosing interval of about every 5-10 days for a period of time. In some embodiments, the CAR comprises a full-length humanized anti-fluorescein antibody or an antigen-binding fragment thereof. In some embodiments, the CAR comprises a full-length humanized anti-fluorescein scFv. In some embodiments, the CAR comprises a full-length humanized E2 anti-fluorescein scFv. In some embodiments, the CAR T cells express a cell surface selectable marker comprising a truncated EGFR (EGFRt) polypeptide. In some embodiments, the dose of the CAR T cell composition is about 1x10 5 cells / kg to about 1x10 7 cells / kg. In some embodiments, the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof is administered intravenously.

[0009] Some embodiments include methods of treating, ameliorating, or inhibiting osteosarcoma in a human subject, comprising administering to the subject a fluorescein isothiocyanate (FITC)-folate compound conjugate or a pharmaceutically acceptable salt thereof according to the following dosing regimen: (i) a dosing escalation period to confirm the maximum tolerated dose (MTD) of the FITC-folate compound conjugate, wherein the dosing escalation period comprises administering: (a) a first dose of about 0.5% to about 5% of the full dose of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof, (b) a second dose of about 5% to about 50% of the full dose of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof, and (c) a third dose of about 50% to about 500% of the full dose of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof, wherein the full dose of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof is about 1x10 -2 mg / kg to about 5x10 -2 mg / kg; and (ii) a maintenance dosing period, the maintenance dosing period comprising administering the MTD of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof at a dosing interval of about every 5 - 10 days for a period of time; wherein the human subject has received or is receiving a CAR T cell composition comprising a population of T cells expressing an anti-fluorescein CAR to treat, ameliorate, or inhibit the osteosarcoma in the human subject. In some embodiments, the osteosarcoma is recurrent or refractory. In some embodiments, the time period between administering the first dose and administering the second dose is between about 1 day and about 7 days. In some embodiments, the time period between administering the first dose and administering the second dose is about 2 days. In some embodiments, the time period between administering the second dose and administering the third dose is between about 1 day and about 7 days. In some embodiments, the time period between administering the second dose and administering the third dose is about 3 days. In some embodiments, the full dose of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof is about 3.1x10 -2mg / kg. In some embodiments, the method further comprises administering a fifth dose of the MTD of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof. In some embodiments, the method further comprises a restaging phase after the fifth dose, wherein the restaging phase comprises: evaluating the subject to determine whether one or more criteria for receiving at least one subsequent dosing cycle of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof are met, provided that the subject is not administered the FITC-folate compound conjugate during the restaging phase. In some embodiments, the subject meets more than one criterion, and wherein the subject is administered at least one subsequent dosing cycle, the subsequent dosing cycle comprising administering the MTD of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof at a dosing interval of about once every 5 - 10 days for a period of time. In some embodiments, the CAR comprises a full-length humanized anti-fluorescein antibody or an antigen-binding fragment thereof. In some embodiments, the CAR comprises a full-length humanized anti-fluorescein scFv. In some embodiments, the CAR comprises a full-length humanized E2 anti-fluorescein scFv. In some embodiments, the CAR T cells express a cell surface selection marker comprising a truncated EGFR (EGFRt) polypeptide. In some embodiments, the dose of the CAR T cell composition is about 1x10 5 cells / kg to about 1x10 7 cells / kg. In some embodiments, the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof is administered intravenously.

[0010] Some embodiments include methods of treating, ameliorating, or inhibiting cancer in a human subject, the methods comprising: (i) administering to the subject a first dose of a fluorescein isothiocyanate (FITC)-folate compound conjugate or a pharmaceutically acceptable salt thereof; (ii) administering to the subject a second dose of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof, wherein the second dose is higher than the first dose; (iii) administering to the subject a third dose of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof, wherein the third dose is higher than the second dose, wherein the maximum tolerated dose (MTD) of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof is confirmed after the third dose; and (iv) administering to the subject a fourth dose that is the MTD of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof; wherein the human subject has received or is receiving a CAR T cell composition, the CAR T composition comprising a population of T cells expressing an anti-fluorescein CAR, thereby treating, ameliorating, or inhibiting the cancer in the human subject. In some embodiments, the cancer comprises osteosarcoma.

[0011] Some embodiments include methods of treating, ameliorating, or inhibiting osteosarcoma in a human subject, the methods comprising administering to the subject a combination of a fluorescein isothiocyanate (FITC)-folate compound conjugate or a pharmaceutically acceptable salt thereof and CAR T cell therapy, wherein the FITC-folate compound conjugate is administered to the subject in a dosing escalation regimen after or simultaneously with the administration of the CAR T cell therapy, thereby treating the osteosarcoma in the human subject. In some embodiments, the dosing escalation regimen comprises: a dosing escalation cycle to confirm the maximum tolerated dose (MTD) of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof, wherein the dosing escalation cycle comprises administering: (a) a first dose of from about 0.5% to about 5% of the full dose of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof, (b) a second dose of from about 5% to about 50% of the full dose of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof, (c) a third dose of from about 50% to about 500% of the full dose of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof, wherein the MTD is confirmed after the third dose, and (d) a fourth dose that is the MTD of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof, wherein the full dose of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof is from about 1x10 -2 mg / kg to about 5x10 -2mg / kg. In some embodiments, the escalating dosing regimen comprises: (i) an escalating dosing period to confirm the maximum tolerated dose (MTD) of the FITC-folate conjugate, wherein the escalating dosing period comprises administering: (a) a first dose of from about 0.5% to about 5% of the full dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof, (b) a second dose of from about 5% to about 50% of the full dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof, and (c) a third dose of from about 50% to about 500% of the full dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof, wherein the full dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof is from about 1x10 -2 mg / kg to about 5x10 -2 mg / kg; and (ii) a maintenance dosing period, the maintenance dosing period comprising administering the MTD of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof at a dosing interval of about once every 5 - 10 days for a period of time. In some embodiments, the CAR T cell therapy comprises administering to the subject a population of CAR T cells expressing an anti-fluorescein CAR.

[0012] Some embodiments include the use of a fluorescein isothiocyanate (FITC)-folate conjugate or a pharmaceutically acceptable salt thereof in combination with CAR T cell therapy for the treatment, amelioration, or inhibition of osteosarcoma in a human subject, wherein the FITC-folate conjugate is administered to the subject in an escalating dosing regimen after or simultaneously with the administration of the CAR T cell therapy. In some embodiments, the escalating dosing regimen comprises an escalating dosing period to confirm the maximum tolerated dose (MTD) of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof, wherein the escalating dosing period comprises administering: (a) a first dose of from about 0.5% to about 5% of the full dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof, (b) a second dose of from about 5% to about 50% of the full dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof, (c) a third dose of from about 50% to about 500% of the full dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof, wherein the MTD is confirmed after the third dose, and (d) a fourth dose of the MTD of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof, wherein the full dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof is from about 1x10 -2 mg / kg to about 5x10 -2mg / kg. In some embodiments, the escalating dosing regimen comprises: (i) an escalating dosing period to confirm the maximum tolerated dose (MTD) of the FITC-folate conjugate, wherein the dose escalation period comprises administering: (a) a first dose of about 0.5% to about 5% of the full dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof, (b) a second dose of about 5% to about 50% of the full dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof, and (c) a third dose of about 50% to about 500% of the full dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof, wherein the full dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof is about 1x10 -2 mg / kg to about 5x10 -2 mg / kg; and (ii) a maintenance dosing period, the maintenance dosing period comprising administering the MTD of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof at a dosing interval of about every 5 - 10 days for a period of time.

[0013] Some embodiments include the use of a fluorescein isothiocyanate (FITC)-folate conjugate or a pharmaceutically acceptable salt thereof in combination with CAR T cell therapy for the treatment, amelioration, or inhibition of cancer in a human subject, wherein the FITC-folate conjugate or a pharmaceutically acceptable salt thereof is administered by a method comprising: (i) administering a first dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof to the subject; (ii) administering a second dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof to the subject, wherein the second dose is higher than the first dose; (iii) administering a third dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof to the subject, wherein the third dose is higher than the second dose, wherein the maximum tolerated dose (MTD) of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof is confirmed after the third dose; and (iv) administering a fourth dose of the MTD of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof to the subject. In some embodiments, the cancer comprises osteosarcoma. In some embodiments, the FITC-folate conjugate is administered to the subject in an escalating dosing regimen after or simultaneously with the administration of CAR T cell therapy to the subject. In some embodiments, the CAR T cell therapy comprises administering to the subject a population of CAR T cells expressing an anti-fluorescein CAR.

[0014] Some embodiments include a kit that contains a container with a fluorescein isothiocyanate (FITC)-folate compound conjugate or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutical carrier, wherein the kit contains instructions for use in treating, ameliorating, or inhibiting osteosarcoma in a subject who has received or is receiving a CAR T cell composition comprising CAR T cells, wherein treating, ameliorating, or inhibiting comprises administering the FITC-folate compound conjugate according to a dosing regimen that includes: a dosing escalation period to confirm the maximum tolerated dose (MTD) of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof, wherein the dosing escalation period includes administering: (a) a first dose of about 0.5% to about 5% of the full dose of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof, (b) a second dose of about 5% to about 50% of the full dose of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof, (c) a third dose of about 50% to about 500% of the full dose of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof, wherein the MTD is confirmed after the third dose, and (d) a fourth dose of the MTD of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof, wherein the full dose of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof is from about 1x10 -2 mg / kg to about 5x10 -2 mg / kg, and wherein the CAR T cells express an anti-fluorescein CAR. In some embodiments, the container is a vial.

[0015] Some embodiments include a system for treating, ameliorating, or inhibiting osteosarcoma in a subject in combination with CAR T cell therapy with a fluorescein isothiocyanate (FITC)-folate conjugate or a pharmaceutically acceptable salt thereof, wherein the FITC-folate conjugate is administered to the subject in a dosing escalation regimen after or simultaneously with the administration of CAR T cell therapy to the subject, the system comprising: (a) a first dose of about 0.5% to about 5% of the full dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof; (b) a second dose of about 5% to about 50% of the full dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof; (c) a third dose of about 50% to about 500% of the full dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof, wherein the maximum tolerated dose (MTD) of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof is confirmed after the third dose; and (d) a fourth dose of the MTD of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof, wherein the full dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof is from about 1x10 -2 mg / kg to about 5x10 -2 mg / kg. In some embodiments, the system further comprises a population of CAR T cells that express an anti-fluorescein CAR. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1A schematic diagram showing an exemplary dosing schedule according to the claimed method is presented. In this exemplary schedule, the administration of E2 CAR T cells (CAR T cells expressing SEQ ID NO:04) occurs on Monday and Thursday of week 1, and then at the beginning of week 2, a small molecule ligand (e.g., EC17) linked to a targeting moiety via a linker is administered. In week 2, the small molecule ligand (e.g., EC17) linked to a targeting moiety via a linker is administered on Monday and Thursday, and then on Monday of week 3 in a dose escalation sequence (i.e., sequence 1). During Tuesday to Sunday of week 3, the small molecule ligand (e.g., EC17) linked to a targeting moiety via a linker is not administered. Weeks 2 to 3 are referred to as "cycle 1". In week 4, the small molecule ligand (e.g., EC17) linked to a targeting moiety via a linker is administered on Monday and Thursday, and then on Monday of week 5 in a dose escalation sequence (i.e., sequence 2). During Tuesday to Sunday of week 5, the small molecule ligand (e.g., EC17) linked to a targeting moiety via a linker is not administered. Weeks 4 to 5 are referred to as "cycle 2". In week 6, the small molecule ligand (e.g., EC17) linked to a targeting moiety via a linker is administered on Monday and Thursday, and then on Monday of week 7 in a dose escalation sequence (i.e., sequence 3). During Tuesday to Sunday of week 7, the small molecule ligand (e.g., EC17) linked to a targeting moiety via a linker is not administered. Weeks 6 to 7 are referred to as "cycle 3". Weeks 2 to 7 are referred to as "process 1". In sequence 1, the small molecule ligand (e.g., EC17) linked to a targeting moiety via a linker is administered on Monday and Thursday of week 2, and then on Monday of week 3, the small molecule ligand (e.g., EC17) linked to a targeting moiety via a linker is administered at the following doses: 1%, 10%, and 100% of the full dose (e.g., 31 μg / kg) of the small molecule ligand (e.g., EC17) linked to a targeting moiety, respectively. In sequence 2, the small molecule ligand (e.g., EC17) linked to a targeting moiety via a linker is administered on Monday and Thursday of week 4, and then on Monday of week 5, the small molecule ligand (e.g., EC17) linked to a targeting moiety via a linker is administered at the following doses: 1%, 30%, and 300% of the full dose (e.g., 31 μg / kg) of the small molecule ligand (e.g., EC17) linked to a targeting moiety, respectively. In sequence 3, the small molecule ligand (e.g., EC17) linked to a targeting moiety via a linker is administered on Monday and Thursday of week 6, and then on Monday of week 7, the small molecule ligand (e.g., EC17) linked to a targeting moiety via a linker is administered at the following doses: 1%, 50%, and 500% of the full dose (e.g., 31 μg / kg) of the small molecule ligand (e.g., EC17) linked to a targeting moiety, respectively.Given the clinical benefit and / or tolerable toxicity, the process 1 is then repeated.

[0017] Figure 2 A general schematic diagram of the construct for CAR T cell transduction is shown.

[0018] Figure 3 The comparison between the E2 construct and the 4M5.3 construct is shown diagrammatically, and the plasmid map of the E2 construct is shown.

[0019] Figure 4 (Top subfigure) is a graph showing the tumor volume of HOS-FRα (human osteosarcoma-folate receptor α) tumors when treated with E2 Car-T cells alone (·) and E2 Car-T cells + EC-17 (o). Figure 4 (Bottom subfigure) is a graph showing the change in body weight of mice bearing HOS-FRα tumors when treated with E2 Car-T cells alone (·) and E2 Car-T cells + EC-17 (o).

[0020] Figure 5 Is a graph showing the change in body weight of mice bearing THP-1-FRβ (folate receptor β) tumors expressing AMF (autocrine motility factor) when treated without Car-T (·); without EC-17 (■); with EC-17 SIW 500 nmol / kg (Δ); with EC-17 TIW (5, 50, 500 nmol / kg on / off) (▽); with (◆) EC-17 dose escalation (M / Th / M on / off).

[0021] Figure 6 (Left subfigure) is a graph showing the hepatic metastasis tumor burden. Figure 6 (Right subfigure) is a graph showing the non-hepatic metastasis tumor burden.

[0022] Figure 7 Is a graph showing the count of circulating THP1-FRb cells per 100 μL of blood on day 39 on a logarithmic scale.

[0023] Figure 8 Is a graph showing the percentage (y-axis) of total E2 CAR T cells isolated from solid liver tumors under different EC-17 dosing regimens. For each dosing regimen, the leftmost bar is PD1+LAG3+TIM3+; the second bar from the left is PD1+LAG3+TIM3-; the middle bar is PD1+LAG3-TIM3+; the second bar from the right is PD1+LAG3-TIM3-; the rightmost bar is PD1-LAG3-TIM3-.

[0024] Figure 9Shows the complete human CAR construct, which includes an anti-FITC scFv (clone E2), a full-length IgG4 spacer (Fc-derived hinge-CH2 (F235D, N297Q)-CH3), a CD28tm transmembrane domain, a 4-1BB / CD3ζ cytoplasmic activation domain, and a non-functional truncated cell surface polypeptide of epidermal growth factor receptor (EGFRt). Bottom: Example of CD4 / CD8 T cell phenotype analysis by flow cytometry for an EGFRt-sorted (left pie chart) CAR-T cell preparation and an unsorted "clinical facsimile" (right pie chart). Color legend is as shown.

[0025] Figure 10, Panel A: Kd value of 3H-EC17 uptake (calculated from the number of molecules bound per cell) after incubation of FR+ target cells at 37 °C for 2 hours. Panel B: Kd value of 3H-EC17 uptake (calculated from total cell-associated radioactivity, DPM) after incubation of E2-CAR-T cells (-24% EGFRt+, -95:5 CD8 / CD4 ratio) at room temperature for 2 hours. 3 H-EC17 uptake (calculated from total cell-associated radioactivity, DPM).

[0026] Figure 11 Shows the functional FR levels on tumor cells measured by a 3H-FA-based binding assay (100 nM, 1 hour at 37 °C).

[0027] Figure 12 shows the EC17 dose finding and CRS assessment in tumor-bearing and tumor-free mice. Panel A: Schematic of the dosing schedule of 3 different administrations of CAR-T cells (-10 million "clinical copies") plus EC17 in NSG mice with or without pre-established MDA-MB-231 xenografts. Tumor-free mice received EC17 SIW 500 nmol / kg (2 doses on days 2 and 10). Tumor-bearing mice received EC17 as follows: EC17 SIW 500 nmol / kg (5 doses on days 2, 10, 17, 24, and 31), EC17 M / Th / M_increasing-1 (repeated at 5 / 50 / 500 nmol / kg on Monday / Thursday / Monday, then with a 1-week interval, i.e., on days 2, 6, 10, 17, 20, 24, and 31), or EC17 M / Th / M_increasing-2 (repeated at 5 / 100 / 1000 nmol / kg on Monday / Thursday / Monday, then with a 1-week interval, i.e., on days 2, 6, 10, 17, 20, 24, and 31). Panel B: Systemic levels of human IFNγ on a log2 scale were detected in mouse plasma on days 11 and 12 (i.e., ~20 hours and 42 hours after the previous EC17 administration in all treatment cohorts) after injection of CAR-T cells in tumor-bearing and tumor-free mice. Panel C: CAR-T cells circulating in mouse blood were confirmed as human CD3ε+EGFRt+ events by flow cytometry and counted per 100 μL of whole blood. Panel D: Measurement of tumor volume and body weight changes in tumor-bearing mice that received either CAR-T cells alone or CAR-T cells plus EC17 administered in 3 different ways (dashed lines indicate each EC17 dose). n = 5 mice per group. All data are represented as mean ± s.e.m. *p < 0.05, tested by one-way ANOVA.

[0028] Figure 13 shows the EC17 dose escalation in terms of in vivo safety and anti-leukemia activity. Panel A: Schematic of the dosing schedule of EC17 plus unsorted EGFRt CAR-T cells (∼6 million, day 0) in NSG mice with 1-day-old intravenous THP-1-FRβ xenografts. Starting 3 days after CAR-T cell injection, EC17 was administered in 3 different ways: EC17 SIW 500 nmol / kg (6 doses, on days 3, 10, 17, 24, 31, and 38), EC17 TIW 5 / 50 / 500 nmol / kg (repeated 5 / 50 / 500 nmol / kg on Monday / Wednesday / Friday 3 times, then with a 9-day interval, i.e., on days 3 / 5 / 7, 17 / 19 / 21, and 31 / 33 / 35), or EC17 M / Th / M_dose escalation on Monday / Thursday / Monday (3 escalation cycles, 5 / 10 / 100 nmol / kg in cycle 1, 5 / 30 / 300 nmol / kg in cycle 2, and 5 / 50 / 500 nmol / kg in cycle 3, i.e., on days 3 / 6 / 10, 17 / 20 / 24, and 31 / 34 / 38). Panel B: Measurement of body weight change (n = 5). Panel C: Circulating CAR-T cells as human CD3s+EGFRt+ events per 100 μL of mouse whole blood on day 31 (on a logarithmic scale). Panel D: Left bar graph: Circulating tumor cells (GFP+) per 100 μL of whole blood in all cohorts measured at the end of the study (day 39); middle bar graph: Weight of the liver infiltrated with THP1-Erβ, representing the liver metastasis burden; right bar graph: Total tumor weight of all non-liver macrometastases. Panel E: Flow cytometry analysis of T cell exhaustion markers PD1, LAG3, TIM3 on tumor-infiltrating CAR-T cells isolated from liver metastases and the pre-infusion CAR-T cell product (triple-negative). The main feature of fully exhausted T cells is the co-expression of multiple inhibitory receptor markers (i.e., triple-positive).

[0029] Figure 14 shows anti-tumor activity and CRS rescue in an aggressive model of pediatric osteosarcoma. Panel A: Schematic of the dosing schedule of CAR-T cells (∼6 million, day 0) plus EC17 in NSG mice with 3-day-old subcutaneous HOS-FRα xenografts (n = 5). Starting 3 days after CAR-T cell injection (6 days after tumor engraftment), 3 cycles of EC17 M / Th / M “within-patient” dose escalation (on Monday / Thursday / Monday), 5 / 10 / 100 nmol / kg in cycle 1, 5 / 30 / 300 nmol / kg in cycle 2, and 5 / 50 / 500 nmol / kg in cycle 3, i.e., on days 3 / 6 / 10, days 17 / 20 / 24, and days 31 / 34 / 38. Panel B: Measurement of body weight change and tumor volume. Five mice receiving only CAR-T cells (without EC17) were euthanized on days 23 - 31 due to tumor progression, and five EC17-treated mice were euthanized on days 33 (2 mice) and 47 (3 mice), respectively. Panel C: Flow cytometry analysis of CAR-T cells (human CD3ε+EGFRt+) in 100 μL of whole blood (plotted on a logarithmic scale) (left) and tumor-infiltrating CAR-T cells at the time of euthanasia (right).

[0030] Figure 15 Describes the experimental design plan for clinical trials.

[0031] Figure 16 Describes the dose modification plan.

[0032] Figure 17 Describes the dosing methodology of dose regimen 0. In process 1 of this methodology, patients are given CAR T infusion at 1x10 6 cells / kg on day 0, and then the MTD-determined doses of compound UB-TT170 are administered at escalating phases of 0.5%, 5%, or 50% of the full dose on days 4, 7, 11, 18, and 25. After this dose escalation, re-staging will be performed. In processes 2 - 4, the MTD-determined doses of compound UB-TT170 are administered to patients at escalating phases (0.5%, 5%, or 50% of the full dose) on days 1, 8, 15, 22, 29, 36, and 43. The full dose of UB-TT170 is 3.1x10 -2 mg / kg.

[0033] Figure 18 Describes the dosing methodology of dose regimen 1. In process 1 of this methodology, patients are given CAR T infusion at 1x10 6Patients are given CAR T infusion at 1 x 10 cells / kg, and then the MTD-determined doses of compound UB-TT170 are administered to the patients at escalating stages of 1%, 10%, or 100% of the full dose on days 4, 7, 11, 18, and 25. After this dose escalation, re-staging will be carried out. In Processes 2 - 4, the MTD-determined doses of compound UB-TT170 are administered to the patients at escalating stages (1%, 10%, or 100% of the full dose) on days 1, 8, 15, 22, 29, 36, and 43. The full dose of UB-TT170 is 3.1x10 -2 mg / kg.

[0034] Figure 19 The dosing methodology for Dose Regimen 2 is described. In Process 1 of this methodology, patients are given CAR T infusion at 1 x 10 cells / kg on day 0, and then the MTD-determined doses of compound UB-TT170 are administered to the patients at escalating stages of 1%, 30%, or 300% of the full dose on days 4, 7, 11, 18, and 25. After this dose escalation, re-staging will be carried out. In Processes 2 - 4, the MTD-determined doses of compound UB-TT170 are administered to the patients at escalating stages (1%, 30%, or 300% of the full dose) on days 1, 8, 15, 22, 29, 36, and 43. The full dose of UB-TT170 is 3.1x10 6 mg / kg. -2 mg / kg.

[0035] Figure 20 The dosing methodology for Dose Regimen 3 is described. In Process 1 of this methodology, patients are given CAR T infusion at 1 x 10 cells / kg on day 0, and then the MTD-determined doses of compound UB-TT170 are administered to the patients at escalating stages of 1%, 50%, or 500% of the full dose on days 4, 7, 11, 18, and 25. After this dose escalation, re-staging will be carried out. In Processes 2 - 4, the MTD-determined doses of compound UB-TT170 are administered to the patients at escalating stages (1%, 50%, or 500% of the full dose) on days 1, 8, 15, 22, 29, 36, and 43. The full dose of UB-TT170 is 3.1x10 6 mg / kg. -2 mg / kg.

[0036] Figure 21Describes an in-host dose escalation treatment of EC17 (3 cycles), which includes administering approximately 5 million E2 CAR T cells on day -3; administering EC17 on days 0, 3, 7, 14, 17, 21, 28, 31, and 35.

[0037] Figure 22 Describes an in-host dose escalation treatment of EC17 (3 cycles).

[0038] Figure 23A Describes the research protocol for Group 2 (CAR-T only).

[0039] Figure 23B Describes the research protocol for Group 3 (EC17 SIW).

[0040] Figure 23C Describes the research protocol for Group 4 (EC17 exact TIW).

[0041] Figure 23D Describes the research protocol for Group 5 (EC17 low dose "TIW" (M / Th / M, 6-day drug holiday) with / effectiveness).

[0042] Figure 24A Describes the EC17 dosing regimen for Cohort 3.

[0043] Figure 24B Describes the EC17 dosing regimen for Cohort 4.

[0044] Figure 24C Describes the EC17 dosing regimen for Cohort 5. Detailed implementation

[0045] Over the past few decades, the survival rates of cancers in children and adolescents have increased significantly, with the largest decline in mortality observed in patients with hematologic malignancies. Despite the use of aggressive multimodal therapies, the treatment of solid tumors in children and young adults remains challenging. With the exception of primary CNS malignancies, the most common diagnoses in this population include neuroblastoma, bone and soft tissue sarcomas, and renal tumors. Recent attempts to improve outcomes through intensified cytotoxic therapies have had limited success. Patients with the highest-risk diseases, namely those with metastatic, recurrent, or refractory disease, are particularly resistant to these strategies, and their outcomes remain poor. Patients with recurrent osteosarcoma have a poor prognosis. Subjects who experienced recurrence in the upfront phase 3 international trial INT-0133 had a 10-year overall survival rate of 20%, which is consistent with results reported in other groups. Although several agents have activity in recurrent or refractory osteosarcoma, no chemotherapeutic agent or targeted therapy has been shown to improve overall survival in patients with recurrent osteosarcoma. Therefore, treatment options for this group of patients are extremely limited. For resectable osteosarcoma recurrences confined to the lung, surgical resection by aggressive thoracotomy is generally considered the standard treatment. Patients who experience recurrence in bony or other visceral sites (e.g., the brain) also have a poor prognosis, usually because surgical intervention is not feasible. Survivors from all risk groups often face chronic and debilitating long-term treatment complications, which include infertility, cardiotoxicity, and secondary malignancies. Therefore, new approaches targeting the pathobiology of these diseases must be explored.

[0046] The potential to target tumor burden with immune-based therapies is attractive because of the opportunity to engage immunologic effector mechanisms in which chemotherapy / radiation-resistant tumor cells are susceptible and because of the potentially limited toxicity of tumor-specific immunologic effector mechanisms. Ideally, cell surface epitope targets would be expressed only on tumor cells and would be uniformly expressed on all tumors within a specific cohort of more than one patient. To date, few ideal tumor-specific epitopes have been defined. To date, the approach that has provided the most experience in pediatric solid tumors is the use of tumor-directed monoclonal antibody therapies, which can target growth factors as well as other cell surface and immunomodulatory molecules. A related strategy is the adoptive transfer of T lymphocytes that are engineered to recognize tumor antigens with monoclonal antibody specificity through the expression of chimeric antigen receptors (CARs). T cells expressing tumor antigen-specific CARs specifically lyse target tumor cells and secrete proinflammatory cytokines. The recent success of CD19-specific CAR T cell therapy in high-risk B cell malignancies has stimulated interest in studying this approach in solid tumors. Clinical trials using CAR T cells directly targeting L1-CAM in neuroblastoma, GD-2, and HER2 in sarcoma have provided some promising preliminary reports.

[0047] Although adoptive transfer of tumor-specific T cells can result in tumor eradication in multiple animal models, adoptive T cell therapy for human malignancies presents more significant challenges and lower efficacy compared to viral diseases. Perhaps the most clinically reliable data on adoptive therapy come from the efforts of the Surgery Branch of the National Cancer Institute (NCI) to treat melanoma. Their studies have shown that administration of lymphodepleting chemotherapy followed by adoptive transfer of polyclonal tumor-infiltrating lymphocytes (TILs) against melanocyte lineage antigens / tumor testis antigens and high-dose rhuIL-2 resulted in visible tumor regression in nearly half of the treated subjects. Notably, the response rate was nearly 90% in subjects showing high-level persistent engraftment of transferred T cells, while no response was seen in approximately 50% of subjects without T cell engraftment. Interestingly, the persistent TILs represented only a small subset of the infused product repertoire, suggesting that the ability to engraft and persist may be linked to the intrinsic programming of rare populations of T cells derived from TILs. The generalization of the NCI group's adoptive transfer results for melanoma to other tumor types is an intense area of ongoing research.

[0048] Known obstacles to the clinical success of adoptive therapy: Tumor antigens presented by human leukocyte antigen (HLA) molecules and recognized by T cells have been identified and can originate from minor histocompatibility antigens in the allogeneic HCT setting, or from the processing of normal or overexpressed tissue-specific proteins, abnormally expressed oncofetal antigens, and mutant proteins. However, it is technically difficult to isolate and expand high-affinity major histocompatibility complex (MHC)-restricted tumor-reactive T cells from tumor-bearing subjects, and even when successful, these T cells often fail to eliminate tumors after adoptive transfer. Much of our evolving understanding of the counter-regulatory mechanisms that impede successful cancer therapy comes from mouse models. Since many tumor antigens are self-proteins expressed in some normal tissues, tolerance mechanisms affect the frequency, activity, and function of tumor-reactive T cells. Additionally, tumors evade immune recognition through multiple mechanisms, including local recruitment of regulatory T cells (Tregs) and other inhibitory cells, loss of antigen expression, downregulation of MHC and co-stimulatory molecules, and expression or secretion of inhibitory molecules or cytokines. Finally, cultured tumor-reactive T cells typically exhibit limited persistence in vivo after adoptive transfer, even when high doses of rhuIL-2 are administered to support their survival.

[0049] Some embodiments of the methods and compositions provided herein include therapies for treating, ameliorating, or inhibiting cancer (e.g., osteosarcoma) in a human subject. Some embodiments include administering to the subject a chimeric antigen receptor (CAR) T cell therapy, e.g., T cells comprising an anti-fluorescein CAR, and, after or simultaneously with the administration of the CAR T cells, administering to the subject a fluorescein isothiocyanate (FITC)-folate conjugate or a pharmaceutically acceptable salt thereof in a dosing escalation regimen. In some such embodiments, the dosing escalation regimen comprises dosing escalation cycles to confirm the maximum tolerated dose (MTD) of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof. Some embodiments of the methods and compositions provided herein include aspects disclosed in U.S. 2021 / 0346431, which is hereby expressly incorporated by reference in its entirety.

[0050] In various embodiments described herein, a small molecule ligand linked to a targeting moiety via a linker serves as a bridge between cancer and chimeric antigen receptor (CAR) T cells, which are T cells expressing a CAR. The bridge directs the CAR T cells to the cancer to ameliorate the cancer. In one embodiment, the "small molecule ligand" can be a folate compound, a CAIX ligand, DUPA, an NK-1R ligand, a ligand for γ-glutamyl transpeptidase, an NKG2D ligand, or a CCK2R ligand, each of which is a small molecule ligand that specifically binds to a cancer cell type (i.e., the receptor for each of these ligands is overexpressed on the cancer compared to normal tissue).

[0051] In some embodiments, the "small molecule ligand" is an NK-1 receptor ligand. In these embodiments, the NK-1 receptor binding moiety can be any one of a number of ligands, e.g., a small molecule ligand customized to have a high degree of selectivity or specificity for the NK-1 receptor. An exemplary NK-1 receptor binding ligand ("NKIRL") can be synthesized from the high-affinity NK-1 receptor antagonist (2S,3S)-3-{[3,5-bis(trifluoromethyl)benzyl]oxy}-2-phenylpiperidine ("L733060") and acetic acid (AcOH), and has the formula:

[0052] In an alternative embodiment of the fluorescence imaging conjugate, the NK-1 receptor binding moiety can be a small molecule ligand other than NKIRL. For example, the NK-1 receptor binding moiety can be a sulfur pentafluoride-containing small molecule ("NIRL-SF5") according to one of the following structures:

[0053] In some embodiments, the NK-1R ligand is conjugated to fluorescein or a fluorescein derivative. In some embodiments, the NK-1R ligand is conjugated to the fluorescein derivative FITC.

[0054] The "targeting moiety" linked to the small molecule ligand binds to the recognition region of the genetically engineered CAR expressed by the CAR T cell. Accordingly, the recognition region of the CAR (such as Fab, Fv, Fc, (Fab')2 fragment or single-chain variable fragment region (scFv) of an antibody, etc.) is directed to the "targeting moiety". Accordingly, the small molecule ligand linked to the targeting moiety via a linker serves as a bridge between the cancer and the CART cell, guiding the CAR T cell to the cancer to improve the cancer. In various embodiments, the bridge between the cancer and the CART cell can be any suitable conjugate shown in the examples.

[0055] The bridge is a small organic molecule and thus can be rapidly cleared from the bloodstream (e.g., about 20 minutes or less). In one aspect, the CAR T cell response can specifically target only those cancer cells that express the receptor for the small molecule ligand portion of the "bridge", thereby reducing off-target toxicity to normal tissues. Additionally, the system can be "universal" because one type of CAR T cell construct can use different "bridges" for targeting a wide variety of cancers. Illustratively, the targeting moiety recognized by the CAR T cell can be kept constant such that one type of CAR T cell construct can be used, while the small molecule ligand that binds to the cancer can be varied to allow targeting of a wide variety of cancers.

[0056] In one embodiment, a method of treating or inhibiting cancer is provided. The method comprises i) administering to a patient at least one dose of a CAR T cell composition comprising CAR T cells, wherein the CAR T cells comprise a CAR directed to a targeting moiety; ii) administering to the patient a compound or a pharmaceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked to the targeting moiety via a linker, and wherein the compound or its pharmaceutically acceptable salt is administered in at least a first dose escalation sequence. In one embodiment, the compound or its pharmaceutically acceptable salt is administered in a second dose escalation sequence.

[0057] In another embodiment, a method of treating or inhibiting cancer is provided. The method comprises i) administering to a patient at least one dose of a CAR T cell composition comprising CAR T cells, wherein the CAR T cells comprise a CAR directed to a targeting moiety; ii) administering to the patient a compound or a pharmaceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked to the targeting moiety by a linker, and wherein the compound or the pharmaceutically acceptable salt thereof is administered in a first dose escalation sequence, wherein if severe CRS occurs in the first dose escalation sequence, the compound or the pharmaceutically acceptable salt thereof is administered in a lower dose escalation sequence, wherein the first dose of the compound or the pharmaceutically acceptable salt thereof in the lower dose escalation sequence is lower than the first dose of the compound or the pharmaceutically acceptable salt thereof administered in the first dose escalation sequence. In another embodiment, in the lower dose escalation sequence, the compound or the pharmaceutically acceptable salt thereof may be administered at about 0.5%, about 5% and about 50% of the full dose of the compound or the pharmaceutically acceptable salt thereof over three separate days.

[0058] In the various embodiments described in the following list of clauses and in the claims and throughout the application, a small molecule ligand linked to a targeting moiety by a linker is referred to as a "compound". Some Definitions

[0059] As used herein, "a" or "an" can mean one or more. As used herein, with respect to a numerical value (including, for example, an integer, a fraction, and a percentage), "about" generally refers to a range of numerical values (e.g., + / -5% to 10% of the recited value) that a person of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result).

[0060] As used herein, the term "treat / treating / treated / treatment" refers to both therapeutic treatment and prophylactic or preventive treatment.

[0061] As used herein, with respect to cancer, the term "ameliorate / ameliorating / amelioration / ameliorated" can mean alleviating the symptoms of cancer, reducing the size of a tumor, completely or partially eliminating the tumor (e.g., complete or partial response), causing stable disease, preventing the progression of cancer (e.g., progression-free survival), or any other effect on cancer that would be considered by a physician to be a therapeutic, prophylactic, or preventive treatment for cancer.

[0062] As used herein, the term "administer / administering / administered" means all ways of introducing a compound or a pharmaceutically acceptable salt thereof or a CAR T cell composition as described herein into a patient, including (but not limited to) oral, intravenous, intramuscular, subcutaneous, and transdermal.

[0063] As used herein, the term "off-target toxicity" means organ damage unacceptable to the physician treating the patient or weight loss of the patient, or any other effect on the patient unacceptable to the physician treating the patient, such as B cell hypoplasia, fever, blood pressure drop, or pulmonary edema.

[0064] As used herein, the terms "transduce" and "transfect" are used interchangeably and these terms mean introducing nucleic acid into a cell by any artificial method (including viral and non-viral methods).

[0065] As used herein, the term "fluorescein" can refer to standard xanthene dye compounds, as well as any fluorescein derivatives. Thus, it will be understood that in some embodiments, an anti-fluorescein CAR can be an anti-fluorescein derivative CAR. In one embodiment, the CAR is an anti-FITC CAR. In one embodiment, the CAR is capable of binding to a FITC-folate conjugate (EC17).

[0066] As used herein, the term "stable dosing period" means administering a therapeutically effective compound or a pharmaceutically acceptable salt thereof at set dosing intervals for a period of time.

[0067] As used herein, the term "dose escalation sequence" means administering increasing doses of a compound or a pharmaceutically acceptable salt thereof over time. As used herein, reference to "a second dose escalation sequence", "a third dose escalation sequence", "a fourth dose escalation sequence", "a fifth dose escalation sequence", and "a sixth dose escalation sequence", etc. means that multiple dose escalation sequences occur, and for each individual dose escalation sequence after the first dose escalation sequence, the first dose of the compound or a pharmaceutically acceptable salt thereof in the subsequent dose escalation sequence is lower than the last dose of the compound or a pharmaceutically acceptable salt thereof in the previous dose escalation sequence (see, for example Figure 1 and the explanation in Figure 1 the description of the drawings).

[0068] Certain embodiments are described by the clauses listed below. Combinations of any of the following embodiments with any applicable embodiments described in the Summary of the Invention section, the Detailed Description section, the Examples section, or the claims of this patent application are also contemplated.

[0069] 1. A method of treating or inhibiting cancer, the method comprising: i) administering to a patient at least one dose of a CAR T cell composition comprising CAR T cells, wherein the CAR T cells comprise a CAR directed to a targeting moiety; and

[0070] ii) administering to the patient a compound or a pharmaceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked to the targeting moiety via a linker, and wherein the compound or a pharmaceutically acceptable salt thereof is administered in at least a first dose escalation sequence. In one embodiment, the compound or a pharmaceutically acceptable salt thereof is administered in at least a second dose escalation sequence.

[0071] 2. The method according to clause 1, wherein the compound or a pharmaceutically acceptable salt thereof is administered in at least a first dose escalation sequence, a second dose escalation sequence, and a third dose escalation sequence.

[0072] 3. The method according to clause 1, wherein the compound or a pharmaceutically acceptable salt thereof is administered in at least a first dose escalation sequence, a second dose escalation sequence, a third dose escalation sequence, and a fourth dose escalation sequence.

[0073] 4. The method according to clause 1, wherein the compound or a pharmaceutically acceptable salt thereof is administered in at least a first dose escalation sequence, a second dose escalation sequence, a third dose escalation sequence, a fourth dose escalation sequence, and a fifth dose escalation sequence.

[0074] 5. The method according to clause 1, wherein the compound or a pharmaceutically acceptable salt thereof is administered in at least a first dose escalation sequence, a second dose escalation sequence, a third dose escalation sequence, a fourth dose escalation sequence, a fifth dose escalation sequence, and a sixth dose escalation sequence.

[0075] 6. The method according to any one of clauses 1 - 5, wherein a first dose of the CAR T cells and a second dose of the CAR T cells are administered to the patient.

[0076] 7. The method according to clause 6, wherein the first dose of the CAR T cells is a test dose for monitoring the patient's tolerance to the CAR T cells.

[0077] 8. The method according to clause 6, wherein the second dose of the CAR T cells comprises a higher dose of the CAR T cells than the first dose of the CAR T cells.

[0078] 9. The method according to any one of clauses 6 - 8, wherein the first dose of the CAR T cells comprises about 0.5×10 5from about 1.0×10 of said CAR T cells / kg patient body weight to about 1.5×10 6 of said CAR T cells / kg patient body weight.

[0079] 10. The method according to any one of clauses 6 - 9, wherein the second dose of said CAR T cells comprises from about 0.8×10 6 of said CAR T cells / kg patient body weight to about 2×10 7 of said CAR T cells / kg patient body weight.

[0080] 11. The method according to any one of clauses 1 - 10, wherein after the first dose escalation sequence is a time period during which the compound or its pharmaceutically acceptable salt is not administered.

[0081] 12. The method according to any one of clauses 1 - 11, wherein after the second dose escalation sequence is a time period during which the compound or its pharmaceutically acceptable salt is not administered.

[0082] 13. The method according to any one of clauses 2 - 12, wherein after the third dose escalation sequence is a time period during which the compound or its pharmaceutically acceptable salt is not administered.

[0083] 14. The method according to any one of clauses 3 - 13, wherein after the fourth dose escalation sequence is a time period during which the compound or its pharmaceutically acceptable salt is not administered.

[0084] 15. The method according to any one of clauses 4 - 14, wherein after the fifth dose escalation sequence is a time period during which the compound or its pharmaceutically acceptable salt is not administered.

[0085] 16. The method according to any one of clauses 5 - 15, wherein after the sixth dose escalation sequence is a time period during which the compound or its pharmaceutically acceptable salt is not administered.

[0086] 17. The method according to any one of clauses 11 - 16, wherein the time period is about 7 days.

[0087] 18. The method according to any one of clauses 1 - 17, wherein the first dose escalation sequence comprises administering about 1%, about 10% and about 100% of the full dose of the compound or its pharmaceutically acceptable salt to the patient on separate days.

[0088] 19. The method according to any one of clauses 1 - 18, wherein the second dose escalation sequence comprises administering to the patient, on separate days, approximately 1%, approximately 30%, and approximately 300% of the full dose of the compound or a pharmaceutically acceptable salt thereof.

[0089] 20. The method according to any one of clauses 2 - 19, wherein the third dose escalation sequence comprises administering to the patient, on separate days, approximately 1%, approximately 50%, and approximately 500% of the full dose of the compound or a pharmaceutically acceptable salt thereof.

[0090] 21. The method according to any one of clauses 3 - 20, wherein the fourth dose escalation sequence comprises administering to the patient, on separate days, approximately 1%, approximately 10%, and approximately 100% of the full dose of the compound or a pharmaceutically acceptable salt thereof.

[0091] 22. The method according to any one of clauses 4 - 21, wherein the fifth dose escalation sequence comprises administering to the patient, on separate days, approximately 1%, approximately 30%, and approximately 300% of the full dose of the compound or a pharmaceutically acceptable salt thereof.

[0092] 23. The method according to any one of clauses 5 - 22, wherein the sixth dose escalation sequence comprises administering to the patient, on separate days, approximately 1%, approximately 50%, and approximately 500% of the full dose of the compound or a pharmaceutically acceptable salt thereof.

[0093] 24. The method according to any one of clauses 18 - 23, wherein the full dose of the compound or a pharmaceutically acceptable salt thereof is from about 10 μg / kg to about 50 μg / kg of the compound or a pharmaceutically acceptable salt thereof.

[0094] 25. The method according to any one of clauses 18 - 24, wherein the full dose of the compound or a pharmaceutically acceptable salt thereof is from about 20 μg / kg to about 40 μg / kg of the compound or a pharmaceutically acceptable salt thereof.

[0095] 26. The method according to any one of clauses 18 - 25, wherein the full dose of the compound or a pharmaceutically acceptable salt thereof is from about 25 μg / kg to about 35 μg / kg of the compound or a pharmaceutically acceptable salt thereof.

[0096] 27. The method according to any one of clauses 18 - 26, wherein the full dose of the compound or a pharmaceutically acceptable salt thereof is about 30 μg / kg of the compound or a pharmaceutically acceptable salt thereof.

[0097] 28. The method according to any one of clauses 6 - 27, wherein a first dose of the CAR T cells and a second dose of the CAR T cells are administered to the patient during week 1.

[0098] 29. The method according to any one of clauses 6 - 28, wherein a first dose of the CAR T cells and a second dose of the CAR T cells are administered to the patient on Monday and Thursday of week 1.

[0099] 30. The method according to any one of clauses 1 - 29, wherein the first dose escalation sequence for the compound or its pharmaceutically acceptable salt occurs during weeks 2 and 3.

[0100] 31. The method according to any one of clauses 1 - 30, wherein the second dose escalation sequence for the compound or its pharmaceutically acceptable salt occurs during weeks 4 and 5.

[0101] 32. The method according to any one of clauses 2 - 31, wherein the third dose escalation sequence for the compound or its pharmaceutically acceptable salt occurs during weeks 6 and 7.

[0102] 33. The method according to clause 30, wherein the compound or its pharmaceutically acceptable salt is administered on three separate days, and the three separate days are Monday and Thursday of week 2 and Monday of week 3.

[0103] 34. The method according to clause 31, wherein the compound or its pharmaceutically acceptable salt is administered on three separate days, and the three separate days are Monday and Thursday of week 4 and Monday of week 5.

[0104] 35. The method according to clause 32, wherein the compound or its pharmaceutically acceptable salt is administered on three separate days, and the three separate days are Monday and Thursday of week 6 and Monday of week 7.

[0105] 36. The method according to any one of clauses 1 - 35, wherein lymphocytes in the patient are depleted before administering the CAR T cell composition to the patient.

[0106] 37. The method according to any one of clauses 1 - 36, the method further comprising administering platelets to the patient, administering concentrated red blood cells to the patient, administering cryoprecipitate to the patient, administering intravenous immunoglobulin to the patient, and / or providing antibacterial therapy to the patient.

[0107] 38. The method according to any one of clauses 1 - 37, wherein if no CRS or neurotoxicity is observed in the patient during the first dose escalation sequence, the method proceeds to the second dose escalation sequence.

[0108] 39. The method according to any one of clauses 2 - 38, wherein if no CRS or neurotoxicity is observed in the patient during the second dose escalation sequence, the method proceeds to the third dose escalation sequence.

[0109] 40. The method according to any one of clauses 3 - 39, wherein if no CRS or neurotoxicity is observed in the patient during the third dose escalation sequence, the method proceeds to the fourth dose escalation sequence.

[0110] 41. The method according to any one of clauses 4 - 40, wherein if no CRS or neurotoxicity is observed in the patient during the fourth dose escalation sequence, the method proceeds to the fifth dose escalation sequence.

[0111] 42. The method according to any one of clauses 5 - 41, wherein if no CRS or neurotoxicity is observed in the patient during the fifth dose escalation sequence, the method proceeds to the sixth dose escalation sequence.

[0112] 43. The method according to any one of clauses 1 - 42, wherein if fever but no hypotension is observed in the patient during any of the dose escalation sequences and no neurotoxicity is observed, all subsequent doses of the compound or its pharmaceutically acceptable salt are administered to the patient at the dose escalation sequence level that results in the fever but no hypotension.

[0113] 44. The method according to any one of clauses 1 - 43, wherein if severe CRS or neurotoxicity occurs in the patient in any dose escalation sequence, all subsequent doses of the compound or its pharmaceutically acceptable salt are administered to the patient at a dose escalation sequence level lower than the dose escalation sequence level that resulted in the severe CRS or neurotoxicity in the patient.

[0114] 45. A method of treating or inhibiting cancer, the method comprising i) administering to a patient at least one dose of a CAR T cell composition comprising CAR T cells, wherein the CAR T cells comprise a CAR directed to a targeting moiety;

[0115] ii) administering to the patient a compound or a pharmaceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked to a targeting moiety via a linker, and wherein the compound or a pharmaceutically acceptable salt thereof is administered in a first dose escalation sequence, and wherein if severe CRS occurs in the first dose escalation sequence, the compound or a pharmaceutically acceptable salt thereof is administered using a lower dose escalation sequence, and wherein the first dose of the compound or a pharmaceutically acceptable salt thereof in the lower dose escalation sequence is lower than the first dose of the compound or a pharmaceutically acceptable salt thereof administered in the first dose escalation sequence.

[0116] 46. The method according to clause 45, wherein in the lower dose escalation sequence, the compound or a pharmaceutically acceptable salt thereof is administered at about 0.5%, about 5% and about 50% of the full dose of the compound or a pharmaceutically acceptable salt thereof over separate three days.

[0117] 47. The method according to clause 46, wherein the full dose of the compound or a pharmaceutically acceptable salt thereof is about the full dose of the compound or a pharmaceutically acceptable salt thereof, and is about 10 μg / kg to about 50 μg / kg of the compound or a pharmaceutically acceptable salt thereof.

[0118] 48. The method according to any one of clauses 46 - 47, wherein the full dose of the compound or a pharmaceutically acceptable salt thereof is about 25 μg / kg to about 35 μg / kg of the compound or a pharmaceutically acceptable salt thereof.

[0119] 49. The method according to any one of clauses 46 - 48, wherein the full dose of the compound or a pharmaceutically acceptable salt thereof is about 30 μg / kg of the compound or a pharmaceutically acceptable salt thereof.

[0120] 50. The method according to any one of clauses 1 - 49, wherein the ligand is selected from the group consisting of folate compounds, DUPA, NK-1R ligands, CAIX ligands, γ-glutamyl transpeptidase ligands, NKG2D ligands, and CCK2R ligands.

[0121] 51. The method according to any one of clauses 1 - 50, wherein the ligand is a folate compound.

[0122] 52. The method according to any one of clauses 1 - 50, wherein the ligand is an NK-1R ligand.

[0123] 53. The method according to any one of clauses 1 - 50, wherein the ligand is DUPA.

[0124] 54. The method according to any one of Clauses 1 - 50, wherein the ligand is a CCK2R ligand.

[0125] 55. The method according to any one of Clauses 1 - 50, wherein the ligand is a γ-glutamyl transpeptidase ligand.

[0126] 56. The method according to any one of Clauses 1 - 55, wherein the targeting moiety is selected from the group consisting of 2,4-dinitrophenol (DNP), 2,4,6-trinitrophenol (TNP), biotin, digoxin, fluorescein, fluorescein isothiocyanate (FITC), NHS-fluorescein, pentafluorophenyl ester, tetrafluorophenyl ester, knottin, centyrin, and DARPin.

[0127] 57. The method according to any one of Clauses 1 - 56, wherein the targeting moiety is FITC.

[0128] 58. The method according to any one of Clauses 1 - 56, wherein the targeting moiety is DNP.

[0129] 59. The method according to any one of Clauses 1 - 56, wherein the targeting moiety is TNP.

[0130] 60. The method according to any one of Clauses 1 - 59, wherein the linker comprises polyethylene glycol (PEG), polyproline, hydrophilic amino acids, sugars, unnatural peptidoglycans, polyvinylpyrrolidone, pluronic F-127, or a combination thereof.

[0131] 61. The method according to any one of Clauses 1 - 60, wherein the linker comprises PEG.

[0132] 62. The method according to any one of Clauses 1 - 61, wherein the compound or its pharmaceutically acceptable salt has the following formula B—L-T, wherein B represents the small molecule ligand, L represents the linker, and T represents the targeting moiety, and wherein L comprises a structure having the following formula wherein n is an integer from 0 to 200.

[0133] 63. The method according to Clause 62, wherein n is an integer from 0 to 150.

[0134] 64. The method according to Clause 62, wherein n is an integer from 0 to 110.

[0135] 65. The method according to clause 62, wherein n is an integer from 0 to 20.

[0136] 66. The method according to clause 62, wherein n is an integer from 15 to 20.

[0137] 67. The method according to clause 62, wherein n is an integer from 15 to 110.

[0138] 68. The method according to any one of clauses 1 - 67, wherein the cancer is selected from the group consisting of: lung cancer, bone cancer, pancreatic cancer, skin cancer, head cancer, neck cancer, cutaneous melanoma, uveal melanoma, uterine cancer, ovarian cancer, endometrial cancer, rectal cancer, gastric cancer, colon cancer, breast cancer, triple-negative breast cancer, fallopian tube cancer, endometrial carcinoma, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, non-small cell lung cancer, adrenal cancer, soft tissue sarcoma, osteosarcoma (including pediatric or non-pediatric osteosarcoma), urethral cancer, prostate cancer, chronic leukemia, acute leukemia, acute myeloid leukemia, lymphocytic lymphoma, myeloid leukemia, granulocytic-monocytic leukemia, hairy cell leukemia, pleural mesothelioma, bladder cancer, Burkitt's lymphoma, ureteral cancer, kidney cancer, renal cell carcinoma, renal pelvic cancer, central nervous system (CNS) neoplasm, primary CNS lymphoma, spinal axis tumor, brainstem glioma, pituitary adenoma, and gastroesophageal junction adenocarcinoma.

[0139] 69. The method according to any one of clauses 1 - 51 or clauses 56 - 68, wherein the cancer is a cancer that expresses folate receptor.

[0140] 70. The method according to any one of clauses 1 - 69, wherein the cancer is osteosarcoma.

[0141] 71. The method according to any one of clauses 1 - 70, wherein the CAR has an identification region, and the identification region is a single-chain variable fragment (scFv) region of an antibody.

[0142] 72. The method according to any one of clauses 1 - 57 or clauses 60 - 71, wherein the CAR has an identification region, and the identification region of the CAR is a single-chain variable fragment (scFv) region of an anti-FITC antibody.

[0143] 73. The method according to any one of clauses 1 - 72, wherein the CAR has a co-stimulatory domain, and the co-stimulatory domain is selected from the group consisting of CD28, CD137 (4-1BB), CD134 (OX40), and CD278 (ICOS).

[0144] 74. A method according to any one of clauses 1 - 73, wherein the CAR has an activation signaling domain, and the activation signaling domain is the T cell CD3ζ chain or the Fc receptor γ.

[0145] 75. A method according to any one of clauses 1 - 57 or clauses 60 - 74, wherein the CAR has an identification region, and the identification region is the single-chain variable fragment (scFv) region of an anti-FITC antibody, wherein the CAR has a co-stimulatory domain, and the co-stimulatory domain is CD137 (4-1BB), and wherein the CAR has an activation signaling domain, and the activation signaling domain is the T cell CD3ζ chain.

[0146] 76. A method according to any one of clauses 1 - 75, wherein the patient is imaged before administering the compound or its pharmaceutically acceptable salt, or before administering the CAR T cell composition.

[0147] 77. A method according to any one of clauses 1 - 76, wherein the compound or its pharmaceutically acceptable salt is not an antibody and does not contain fragments of an antibody.

[0148] 78. A method according to any one of clauses 1 - 77, wherein the targeting moiety does not contain a peptide epitope.

[0149] 79. A method according to any one of clauses 1 - 78, wherein cytokine release causing off-target toxicity does not occur in the patient, and wherein CAR T cell toxicity to the cancer appears.

[0150] 80. A method according to any one of clauses 1 - 78, wherein off-target tissue toxicity does not occur in the patient, and wherein CAR T cell toxicity to the cancer appears.

[0151] 81. A method according to any one of clauses 1 - 78, wherein the cancer includes a tumor, wherein the tumor size decreases in the patient, and wherein off-target toxicity does not occur.

[0152] 82. A method according to any one of clauses 1 - 81, wherein the CAR T cells comprise a nucleic acid containing SEQ ID NO:01.

[0153] 83. A method according to any one of clauses 1 - 82, wherein the CAR T cells comprise a polypeptide containing SEQ ID NO:02.

[0154] 84. The method according to clause 82, wherein the nucleic acid encodes a chimeric antigen receptor.

[0155] 85. The method according to any one of clauses 1 - 81, wherein the CAR T cell comprises a nucleic acid containing SEQ ID NO:04.

[0156] 86. The method according to any one of clauses 1 - 81 or clause 85, wherein the CAR T cell comprises a polypeptide containing SEQ ID NO:05.

[0157] 87. The method according to clause 85, wherein the nucleic acid encodes a chimeric antigen receptor.

[0158] 88. The method according to any one of clauses 1 - 87, wherein the CAR comprises a humanized amino acid sequence.

[0159] 89. The method according to any one of clauses 1 - 87, wherein the CAR consists of a humanized amino acid sequence.

[0160] 90. The method according to any one of clauses 1 - 89, the method further comprising administering to the patient a folic acid compound, a conjugate comprising a folic acid compound, or a reagent that inhibits the activation of the CAR T cell, wherein the conjugate comprising a folic acid compound does not comprise a targeting moiety.

[0161] 91. The method according to clause 90, wherein a folic acid compound is administered.

[0162] 92. The method according to clause 90, wherein folic acid or leucovorin is administered.

[0163] 93. The method according to clause 90, wherein the conjugate comprising a folic acid compound is administered.

[0164] 94. The method according to clause 93, wherein the conjugate comprising a folic acid compound comprises a folic acid compound linked to one or more amino acids.

[0165] 95. The method according to clause 93, wherein the conjugate comprising a folic acid compound has the following formula

[0166] 96. The method according to clause 91, wherein the folic acid compound has the following formula wherein X 1 and Y 1 are each independently selected from the group consisting of a halogen, R2 、 OR 2 、 SR 3 and NR 4 R 5 to form a group; U, V, and W each independently represent a divalent moiety selected from the group consisting of: -(R 6a )C═, -N═, -(R 6a )C(R 7a )-, and -N(R 4a )-; Q is selected from the group consisting of C and CH; T is selected from the group consisting of S, O, N, and -C═C-; X 2 and X 3 each independently selected from the group consisting of oxygen, sulfur, -C(Z)-, -C(Z)O-, -OC(Z)-, -N(R 4b )-, -C(Z)N(R 4b )-, -N(R 4b )C(Z)-, -OC(Z)N(R 4b )-, -N(R 4b )C(Z)O-, -N(R 4b )C(Z)N(R 5b )-, -S(O)-, -S(O)2-, -N(R 4a )S(O)2-, -C(R 6b )(R 7b )-, -N(C≡CH)-, -N(CH2C≡CH)-, C1-C 12 alkylene, and C1-C 12 alkoxy, where Z is oxygen or sulfur; R 1 is selected from the group consisting of hydrogen, halogen, C1-C 12 alkyl, and C1-C 12 alkoxy; R 2 , R 3 , R 4 , R 4a , R 4b , R 5 , R 5b , R 6b , and R 7b each independently selected from the group consisting of hydrogen, halogen, C1-C 12 alkyl, C1-C 12 alkoxy, C1-C 12 alkanoyl, C1-C 12 alkenyl, C1-C 12 alkynyl, (C1-C 12 alkoxy)carbonyl, and (C1-C12 the group consisting of (alkylamino)carbonyl; R 6 and R 7 each independently selected from the group consisting of hydrogen, halogen, C1-C 12 alkyl, and C1-C 12 alkoxy; or, R 6 and R 7 together form a carbonyl group; R 6a and R 7a each independently selected from the group consisting of hydrogen, halogen, C1-C 12 alkyl, and C1-C 12 alkoxy; or, R 6a and R 7a together form a carbonyl group; p, r, s, and t are each independently 0 or 1; and if any additional chemical moiety is part of the folic acid compound, * represents an optional covalent bond to the remainder of the conjugate.

[0167] 97. The method according to clause 90, wherein the reagent for inhibiting CAR T cell activation is selected from the group consisting of lymphocyte-specific

[0168] protein tyrosine kinase inhibitors, PI3 kinase inhibitors, inhibitors of IL-2-inducible T cell kinase, JAK inhibitors, BTK inhibitors, EC2319, and reagents that block the binding of CAR T cells to the compound or its pharmaceutically acceptable salts but not to the cancer.

[0169] 98. The method according to clause 90, wherein the reagent for inhibiting CAR T cell activation is administered and the reagent is a lymphocyte-specific protein tyrosine kinase inhibitor.

[0170] 99. The method according to clause 98, wherein the lymphocyte-specific protein tyrosine kinase inhibitor is dasatinib.

[0171] 100. The method according to clause 90, wherein the reagent for inhibiting CAR T cell activation is administered and the reagent is a PI3 kinase inhibitor.

[0172] 101. The method according to clause 100, wherein the PI3 kinase inhibitor is GDC0980.

[0173] 102. The method according to clause 90, wherein the reagent for inhibiting CAR T cell activation is administered and the reagent is an IL-2-inducible T cell kinase inhibitor.

[0174] 103. The method according to clause 102, wherein the IL-2 inducible T cell kinase inhibitor is BMS-509744.

[0175] 104. The method according to clause 90, wherein a reagent that inhibits CAR T cell activation is administered, and the reagent is a reagent that blocks the binding of CAR T cells to the compound or a pharmaceutically acceptable salt thereof but does not bind to the cancer.

[0176] 105. The method according to clause 104, wherein the reagent is fluorescamine, FITC, or sodium fluorescein.

[0177] 106. The method according to clause 105, wherein the reagent is sodium fluorescein.

[0178] 107. The method according to any one of clauses 90 - 106, wherein administering the folic acid compound, the conjugate comprising a folic acid compound (wherein the conjugate comprising a folic acid compound does not comprise a targeting moiety), or the reagent that inhibits CAR T cell activation results in a decrease in cytokine levels in the patient.

[0179] 108. The method according to clause 107, wherein the decrease in cytokine levels is a decrease to the cytokine levels in an untreated patient.

[0180] 109. The method according to any one of clauses 90 - 108, wherein the cancer comprises a tumor, and when the folic acid compound, the conjugate comprising a folic acid compound (wherein the conjugate comprising a folic acid compound does not comprise a targeting moiety), or the reagent that inhibits CAR T cell activation is administered to the patient, the tumor size in the patient does not increase.

[0181] 110. The method according to any one of clauses 104 - 106, wherein when the CRS level reaches 1, 2, 3, or 4, the reagent that inhibits CAR T cell activation is administered to the patient.

[0182] 111. The method according to clause 110, wherein when the CRS level reaches 3 or 4, the reagent that inhibits CAR T cell activation is administered to the patient.

[0183] 112. The method according to any one of clauses 104 - 106, wherein the reagent that inhibits CAR T cell activation is administered at a dose of about 0.01 μmoles / kg patient body weight to about 300 μmoles / kg patient body weight.

[0184] 113. The method according to any one of clauses 1 - 112, wherein CRS is reduced or prevented in the patient, and the method results in a reduction in tumor volume in the patient.

[0185] 114. The method according to any one of clauses 1 - 113, wherein weight loss caused by CRS is reduced or prevented.

[0186] 115. The method according to any one of clauses 90 - 114, the method further comprising readministering the compound or a pharmaceutically acceptable salt thereof to the patient.

[0187] 116. The method according to clause 115, wherein subsequent administration of the compound or a pharmaceutically acceptable salt thereof results in an increase in cytokine levels and CAR T cell activation in the patient.

[0188] 117. The method according to any one of clauses 1 - 116, wherein the CAR T cell composition is administered before the compound or a pharmaceutically acceptable salt thereof.

[0189] 118. The method according to any one of clauses 1 - 117, wherein the CAR T cells are autologous.

[0190] 119. The method according to any one of clauses 2 - 17, wherein the first dose escalation sequence comprises administering to the patient approximately 1%, approximately 2%, and approximately 20% of the full dose of the compound or a pharmaceutically acceptable salt thereof on separate days, wherein the second dose escalation sequence comprises administering to the patient approximately 1%, approximately 6%, and approximately 60% of the full dose of the compound or a pharmaceutically acceptable salt thereof on separate days, and wherein the third dose escalation sequence comprises administering to the patient approximately 1%, approximately 10%, and approximately 100% of the full dose of the compound or a pharmaceutically acceptable salt thereof on separate days.

[0191] 120. The method according to any one of clauses 2 - 17, wherein the first dose escalation sequence comprises administering to the patient approximately 1%, approximately 10%, and approximately 100% of the full dose of the compound or a pharmaceutically acceptable salt thereof on separate days, and wherein the second dose escalation sequence comprises administering to the patient approximately 1%, approximately 20%, and approximately 200% of the full dose of the compound or a pharmaceutically acceptable salt thereof on separate days.

[0192] 121. The method according to any one of clauses 2 - 17, wherein the first dose escalation sequence comprises administering to the patient, on separate days, approximately 1%, approximately 10%, and approximately 100% of the full dose of the compound or a pharmaceutically acceptable salt thereof, and wherein the second dose escalation sequence comprises administering to the patient, on separate days, approximately 1%, approximately 10%, and approximately 100% of the full dose of the compound or a pharmaceutically acceptable salt thereof.

[0193] 122. The method according to any one of clauses 2 - 17, wherein the first dose escalation sequence comprises administering to the patient, on separate days, approximately 1%, approximately 20%, and approximately 200% of the full dose of the compound or a pharmaceutically acceptable salt thereof, and wherein the second dose escalation sequence comprises administering to the patient, on separate days, approximately 1%, approximately 20%, and approximately 200% of the full dose of the compound or a pharmaceutically acceptable salt thereof.

[0194] 123. The method according to any one of clauses 119 - 122, wherein the full dose of the compound or a pharmaceutically acceptable salt thereof is approximately 500 nmoles / kg.

[0195] Accordingly, various embodiments are provided in the foregoing paragraphs and the list of clauses above, and all applicable embodiments described in this "Detailed Description", "Summary of the Invention" section, examples, and claims are applicable to these embodiments.

[0196] As described herein, a "patient" or "subject" can be a human, or in the case of veterinary applications, the patient or subject can be a laboratory animal, farm animal, livestock, or wild animal. In various aspects, the patient or subject can be a laboratory animal such as a rodent (e.g., mouse, rat, hamster, etc.), rabbit, monkey, chimpanzee, livestock (e.g., dog, cat, or rabbit), farm animal (e.g., cow, horse, pig, sheep, goat), or captive wild animal (e.g., bear, panda, lion, tiger, leopard, elephant, zebra, giraffe, gorilla, dolphin, or whale).

[0197] In various embodiments, the cancer to be treated / processed or inhibited may be selected from 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, cutaneous melanoma, uveal melanoma, uterine cancer, ovarian cancer, endometrial cancer, rectal cancer, gastric cancer, colon cancer, breast cancer, triple negative breast cancer, fallopian tube cancer, endometrial carcinoma, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid carcinoma, non-small cell lung cancer, adrenal cancer, soft tissue sarcoma, osteosarcoma (including pediatric or non-pediatric osteosarcoma), urethral cancer, prostate cancer, chronic leukemia, acute leukemia (including acute myeloid leukemia), lymphocytic lymphoma, myeloid leukemia, myelomonocytic leukemia, hairy cell leukemia, pleural mesothelioma, bladder cancer, Burkitt's lymphoma, ureteral cancer, kidney cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) neoplasm, primary CNS lymphoma, spinal axis tumor, brainstem glioma, pituitary adenoma, and gastroesophageal junction adenocarcinoma.

[0198] In some aspects of these embodiments, the cancer is a cancer that expresses a folate receptor. In another embodiment, the cancer is a cancer that expresses folate receptor a. In yet another embodiment, the cancer is a cancer that expresses folate receptor b. In some aspects of these embodiments, the cancer is endometrial cancer, non-small cell lung cancer, ovarian cancer, osteosarcoma (including pediatric or non-pediatric osteosarcoma), or triple negative breast cancer. In another embodiment, the cancer to be treated / processed or inhibited is a tumor. In another embodiment, the cancer is malignant. In another embodiment, the cancer is osteosarcoma, including pediatric or non-pediatric osteosarcoma.

[0199] In one embodiment, the "small molecule ligand" may be a folate compound, DUPA (a ligand that binds to PSMA-positive human prostate cancer cells and other cancer cell types), an NK-1R ligand (e.g., a receptor for the NK-1R ligand found in cancers of the colon and pancreas), a CAIX ligand (e.g., a receptor for the CAIX ligand found in kidney cancer, ovarian cancer, vulvar cancer, and breast cancer), a ligand for gamma-glutamyl transpeptidase (the transpeptidase being overexpressed, for example, in ovarian cancer, colon cancer, liver cancer, astrocytoma, melanoma, and leukemia), an NKG2D ligand (e.g., a receptor for the NKG2D ligand found in lung cancer, colon cancer, kidney cancer, prostate cancer, and in T cell and B cell lymphomas), or a CCK2R ligand (a receptor for the CCK2R ligand found in cancers of the thyroid, lung, pancreas, ovary, brain, stomach, gastrointestinal stroma, and colon, etc.), each of which is a small molecule ligand that specifically binds to a cancer cell type (i.e., the receptor for each of these ligands may be overexpressed in cancer compared to normal tissue).

[0200] In one embodiment, the small molecule ligand can have a mass of less than about 10,000 Daltons, less than about 9000 Daltons, less than about 8,000 Daltons, less than about 7000 Daltons, less than about 6000 Daltons, less than about 5000 Daltons, less than about 4500 Daltons, less than about 4000 Daltons, less than about 3500 Daltons, less than about 3000 Daltons, less than about 2500 Daltons, less than about 2000 Daltons, less than about 1500 Daltons, less than about 1000 Daltons or less than about 500 Daltons. In another embodiment, the small molecule ligand can have a mass of from about 1 to about 10,000 Daltons, from about 1 to about 9000 Daltons, from about 1 to about 8,000 Daltons, from about 1 to about 7000 Daltons, from about 1 to about 6000 Daltons, from 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.

[0201] In one embodiment, the DUPA derivative can be a ligand of a small molecule ligand attached to a targeting moiety, and the DUPA derivative is described in WO 2015 / 057852 and is incorporated herein by reference.

[0202] In one embodiment, the small molecule ligand in the context of "small molecule ligand linked to a linker" is a folic acid compound. In various embodiments, the folic acid compound can be folic acid, a folic acid analogue, or another folate receptor binding molecule. In various embodiments, folic acid analogues that can be used include folinic acid (e.g., leucovorin), pteroylpolyglutamic acids, and folate receptor binding pteridines such as tetrahydropteridines, dihydrofolates, tetrahydrofolates, or their deaza and dideaza analogues. The terms "deaza" and "dideaza" analogues refer to analogues known in the art in which one or two nitrogen atoms in the naturally occurring folic acid structure are replaced by carbon atoms. For example, deaza analogues include 1-deaza, 3-deaza, 5-deaza, 8-deaza, or 10-deaza analogues. Dideaza analogues include, for example, 1,5-dideaza, 5,10-dideaza, 8,10-dideaza, or 5,8-dideaza analogues. The above folic acid analogues are generally referred to as "folic acid compounds", reflecting their ability to bind to folate receptors. Other folate receptor binding analogues useful in the aspects described herein include aminopterin, methotrexate (amethopterin), N10-methylfolate, 2-deamino-hydroxyfolate, deaza analogues (e.g., 1-deazamethotrexate or 3-deazamethotrexate), and 3',5'-dichloro-4-amino-4-deoxy-N10-methylpteroylglutamic acid (dichloromethotrexate).

[0203] In another embodiment, the small molecule ligand in the context of "small molecule ligand linked to a linker" can have the following formula: Wherein, X 1 and Y 1 are each independently selected from the group consisting of halogen, R 2 , OR 2 , SR 3 and NR 4 R 5 ; U, V, and W represent divalent moieties each independently selected from the group consisting of: -(R 6a )C=, -N=, -(R 6a )C(R 7a )-, and -N(R 4a ); Q is selected from the group consisting of C and CH; T is selected from the group consisting of S, O, N, and -C=C-; X 2 and X 3 are each independently selected from the group consisting of oxygen, sulfur, -C(Z)-, -C(Z)O-, -OC(Z)-, -N(R 4b )-, -C(Z)N(R4b )-, -N(R 4b )C(Z)-, -OC(Z)N(R 4b )-, -N(R 4b )C(Z)O-, -N(R 4b )C(Z)N(R 5b )-, -S(O)-, -S(O)2-, -N(R 4a )S(O)2-, -C(R 6b )(R 7b )-, -N(C≡CH)-, -N(CH2C≡CH)-, C1-C 12 alkylene and C1-C 12 alkoxy groups, where Z is oxygen or sulfur; R 1 is selected from the group consisting of hydrogen, halogen, C1-C 12 alkyl and C1-C 12 alkoxy groups; R 2 , R 3 , R 4 , R 4a , R 4b , R 5 , R 5b , R 6b and R 7b are each independently selected from the group consisting of hydrogen, halogen, C1-C 12 alkyl, C1-C 12 alkoxy, C1-C 12 alkanoyl, C1-C 12 alkenyl, C1-C 12 alkynyl, (C1-C 12 alkoxy)carbonyl and (C1-C 12 alkylamino)carbonyl groups; R 6 and R 7 are each independently selected from the group consisting of hydrogen, halogen, C1-C 12 alkyl and C1-C 12 alkoxy groups; or, R 6 and R 7 together form a carbonyl group; R 6a and R 7a are each independently selected from the group consisting of hydrogen, halogen, C1-C 12 alkyl and C1-C 12 alkoxy groups; or, R 6a and R 7a together form a carbonyl group; p, r, s, and t are each independently 0 or 1; and If any additional chemical moiety is part of the folic acid compound, * represents an optional covalent bond to the remainder of the conjugate.

[0204] In one aspect, the "targeting moiety" that binds to the CAR expressed by CAR T cells can be selected from, for example, 2,4-dinitrophenol (DNP), 2,4,6-trinitrophenol (TNP), biotin, digoxin, fluorescein, fluorescein isothiocyanate (FITC), NHS-fluorescein, pentafluorophenyl ester, tetrafluorophenyl ester, avidin, centyrin, DARPin, affibody, affilin, anticalin, atrimer, avimer, bicyclic peptide, FN3 scaffold, cys-loop, fynomer, Kunitz domain or Obody. The properties of the targeting moiety are limited only in that it should be recognized by and bind to (preferably specifically bind to) the CAR, and it has a relatively low molecular weight. In various aspects, exemplary targeting moieties are haptens, which include small molecular weight organic molecules.

[0205] In an illustrative embodiment, the targeting moiety can have the following illustrative structure: wherein X is oxygen, nitrogen or sulfur, and wherein X is attached to linker L; Y is OR a , NR a 2 or NR a 3 + ; and Y' is O, NR a or NR a 2 + ; wherein in each case R is independently selected from H, fluorine, sulfonic acid, sulfonate and its salts, etc.; and R a is hydrogen or alkyl.

[0206] In an illustrative aspect, the linker can include polyethylene glycol (PEG), polyproline, hydrophilic amino acids, sugars, unnatural peptidoglycans, polyvinylpyrrolidone, Pluronic F-127 or a combination thereof.

[0207] In another illustrative aspect, the linker in the compounds or their pharmaceutically acceptable salts described herein can include a direct linkage (e.g., a reaction between the isothiocyanate group of FITC and the free amino group of a small molecule ligand) or the linkage can be via an intermediate linker. In one embodiment, if present, the intermediate linker can be any biocompatible linker known in the art, such as a divalent linker. In one illustrative embodiment, the divalent linker can contain from about 1 to about 30 carbon atoms. In another illustrative embodiment, the divalent linker can contain from about 2 to about 20 carbon atoms. In other embodiments, divalent linkers of lower molecular weight are employed (i.e., those having an approximate molecular weight of from about 30 to about 300 daltons). In another embodiment, suitable linker lengths include, but are not limited to, linkers having 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 or more atoms.

[0208] In various embodiments, the small molecule ligand attached to the targeting moiety can have the formula: B-L-T wherein B represents the small molecule ligand, L represents the linker, and T represents the targeting moiety, and wherein L comprises a structure having the formula: Wherein, n is an integer from 0 to 200. In another embodiment, n can be an integer from 0 to 150, from 0 to 110, from 0 to 100, from 0 to 90, from 0 to 80, from 0 to 70, from 0 to 60, from 0 to 50, from 0 to 40, from 0 to 30, from 0 to 20, from 0 to 15, from 0 to 14, from 0 to 13, from 0 to 12, from 0 to 11, from 0 to 10, from 0 to 9, from 0 to 8, from 0 to 7, from 0 to 6, from 0 to 5, from 0 to 4, from 0 to 3, from 0 to 2, from 0 to 1, from 15 to 16, from 15 to 17, from 15 to 18, from 15 to 19, from 15 to 20, from 15 to 21, from 15 to 22, from 15 to 23, from 15 to 24, from 15 to 25, from 15 to 26, from 15 to 27, from 15 to 28, from 15 to 29, from 15 to 30, from 15 to 31, from 15 to 32, from 15 to 33, from 15 to 34, from 15 to 35, from 15 to 36, from 15 to 37, from 15 to 38, from 15 to 39, from 15 to 40, from 15 to 50, from 15 to 60, from 15 to 70, from 15 to 80, from 15 to 90, from 15 to 100, from 15 to 110, from 15 to 120, from 15 to 130, from 15 to 140, from 15 to 150, or n can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 50, 60, 70, 80, 90, 100, 108, 110, 120, 130, 140 or 150.

[0209] In another embodiment, the linker can be a bivalent linker that may include more than one spacer. Illustrative spacers are shown in the following table. The following non-limiting, illustrative spacers are described, where * indicates the point of attachment to the small molecule ligand or to the targeting moiety or to another bivalent linker moiety.

[0210] In other embodiments, the small molecule ligand (the bridge) that is linked to the targeting moiety can have any of the following structures.

[0211] In other embodiments, the compound or its pharmaceutically acceptable salt is not an antibody and does not contain antibody fragments. In yet another embodiment, the targeting moiety does not contain a peptide epitope.

[0212] In an illustrative embodiment, a small molecule ligand (the bridge) linked to a targeting moiety via a linker includes fluorescein isothiocyanate (FITC) linked to the small molecule ligand. In one aspect, the cancer may overexpress the receptor for the small molecule ligand. In another aspect, for example, cytotoxic T cells or another type of T cell may be transformed to express a CAR comprising an anti-FITC scFv. In this aspect, as a result of the binding of the small molecule ligand to the cancer, the CAR may target the FITC, where the cancer is decorated with FITC molecules. Accordingly, toxicity to normal non-target cells may be avoided or reduced. In this embodiment, when a T cell expressing an anti-FITC CAR binds to the FITC, the CAR T cell is activated and the cancer is ameliorated or inhibited.

[0213] “Pharmaceutically acceptable salts” of the small molecule ligand linked to a targeting moiety via a linker are encompassed. As used herein, the term “pharmaceutically acceptable salts” refers to those salts whose counterions are useful in pharmaceuticals. In various embodiments, such salts include, but are not limited to, 1) acid addition salts, which may be obtained by reacting the free base of the parent compound with an inorganic acid such as hydrochloric acid, hydrobromic acid, nitric acid, phosphoric acid, sulfuric acid, perchloric acid, etc., or with an organic acid such as acetic acid, oxalic acid, (D) or (L) malic acid, maleic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, tartaric acid, citric acid, succinic acid, or malonic acid, etc.; or 2) salts formed when an acidic proton present in the parent compound is replaced by a metal ion (such as an alkali metal ion, an alkaline earth metal ion, or an aluminum ion); or salts formed by coordination with an organic base such as ethanolamine, diethanolamine, triethanolamine, trimethamine, N-methylglucosamine, etc. Pharmaceutically acceptable salts are well known to those skilled in the art and any such pharmaceutically acceptable salts relevant to the embodiments described herein are encompassed.

[0214] In various embodiments, suitable acid addition salts are formed from acids that form non-toxic salts. Illustrative examples include acetate, aspartate, benzoate, benzenesulfonate, bicarbonate / carbonate, bisulfate / sulfate, borate, camphorsulfonate, citrate, edisylate, esylate, formate, fumarate, glucoheptonate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride / chloride, hydrobromide / bromide, hydroiodide / iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methanesulfate, naphthoate, 2-naphthalenesulfonate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate / monohydrogenphosphate / dihydrogenphosphate, saccharate, stearate, succinate, tartrate, tosylate, or trifluoroacetate.

[0215] In various embodiments, suitable base salts are formed from bases that form non-toxic salts. Illustrative examples include salts of arginine, benzathine, calcium, choline, diethylamine, diethanolamine, glycine, lysine, magnesium, meglumine, ethanolamine, potassium, sodium, tromethamine, and zinc. Hemisalts of acids and bases may also be formed, such as hemisulfates and hemicalcium salts.

[0216] In an illustrative aspect, the compounds or their pharmaceutically acceptable salts described herein may contain more than one chiral center or may otherwise be capable of existing in multiple stereoisomers. Accordingly, various embodiments may include pure stereoisomers as well as mixtures of stereoisomers, such as enantiomers, diastereomers, and enantiomer- or diastereomer-enriched mixtures. In one aspect, the compounds or their pharmaceutically acceptable salts described herein are capable of existing as geometric isomers. Accordingly, various embodiments may include pure geometric isomers or mixtures of geometric isomers.

[0217] In some aspects, the compounds or their pharmaceutically acceptable salts described herein may exist in unsolvated as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to the unsolvated forms and are encompassed within the scope of the invention.

[0218] The methods described herein also utilize T lymphocytes, such as cytotoxic T lymphocytes, that are engineered to express a chimeric antigen receptor (CAR) that recognizes and binds to a targeting moiety of the bridge, such as FITC, DNP, or TNP. In one embodiment, the CAR described herein comprises three domains, including 1) an recognition domain (e.g., a single-chain variable fragment (scFv) region of an antibody, a Fab fragment, etc.) that recognizes and specifically binds to the targeting moiety, 2) a co-stimulatory domain that enhances the proliferation and survival of the T lymphocytes, and 3) an activation signaling domain that generates a T lymphocyte activation signal.

[0219] In various aspects, as non-limiting examples, scFv regions of antibodies conjugated to the following can be used: 2,4-dinitrophenol (DNP), 2,4,6-trinitrophenol (TNP), biotin, digoxin, fluorescein, fluorescein isothiocyanate (FITC), NHS-fluorescein, pentafluorophenyl ester, tetrafluorophenyl ester, tuberculin, centyrin, DARPin, affibody, affilin, anticalin, atrimer, avimer, bicyclic peptide, FN3 scaffold, cys-loop, fynomer, Kunitz domain, or Obody. In an illustrative non-limiting embodiment, the scFv region can be prepared from: (i) antibodies known in the art that bind a targeting moiety, (ii) antibodies newly prepared using a selected targeting moiety (such as a hapten), and (iii) sequence variants of the scFv region derived from such antibodies, such as scFv regions having at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity to the amino acid sequence of the scFv region from which they are derived.

[0220] In one aspect, the co-stimulatory domain serves to enhance the proliferation and survival of cytotoxic T lymphocytes after the CAR binds to the targeting moiety.

[0221] Suitable co-stimulatory domains include, but are not limited to, CD28, CD137 (4-1BB), members of the tumor necrosis factor (TNF) receptor family, CD134 (OX40), members of the TNFR receptor superfamily, CD27, CD30, CD150, DAP10, NKG2D, and CD278 (ICOS), CD28 superfamily co-stimulatory molecules expressed on activated T cells, or combinations thereof. Those skilled in the art will understand that sequence variants of these co-stimulatory domains can be used without adversely affecting the present invention, wherein the variants have the same or similar activity as the domain they mimic. In various embodiments, such variants can have at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity to the amino acid sequence of the domain from which they are derived.

[0222] In illustrative embodiments, an activation signaling domain is used to activate T lymphocytes (such as cytotoxic T lymphocytes) after the CAR binds to the targeting moiety. In various embodiments, suitable activation signaling domains include the T cell CD3ζ chain, CD3δ receptor protein, mbl receptor protein, B29 receptor protein, and Fc receptor γ. Those skilled in the art will understand that sequence variants of these activation signaling domains can be used, where the variants have the same or similar activity as the domain they mimic. In various embodiments, the variant has at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity to the amino acid sequence of the domain from which it is derived.

[0223] In one aspect, constructs encoding CARs are prepared using genetic engineering techniques. Such techniques are described in detail in Sambrook et al., "Molecular Cloning: A Laboratory Manual", 3rd Edition, Cold Spring Harbor Laboratory Press, (2001), which is incorporated herein by reference; and Green and Sambrook, "Molecular Cloning: A Laboratory Manual", 4th Edition, Cold Spring Harbor Laboratory Press, (2012), which is incorporated herein by reference.

[0224] As an example, a plasmid or viral expression vector (e.g., a lentiviral vector, a retroviral vector, sleeping beauty, and piggyback (a transposon / transposase system including non-viral mediated CAR gene delivery systems)) encoding a fusion protein can be prepared, the fusion protein comprising an recognition region, one or more co-stimulatory domains, and an activation signaling domain linked in-frame and in the 5' to 3' direction. In other embodiments, other arrangements are acceptable, including an antigen recognition domain, an activation signaling domain, and one or more co-stimulatory domains. In one embodiment, the placement of the recognition region in the fusion protein will generally be such that the region is displayed externally on the CAR T cell. In one embodiment, the CAR can include additional elements, such as a signal peptide (e.g., CD8α signal peptide) that ensures proper export of the fusion protein to the cell surface, a transmembrane domain (e.g., CD8α transmembrane domain, CD28 transmembrane domain, or CD3ζ transmembrane domain) that ensures the fusion protein remains an integral membrane protein, and a hinge domain (e.g., CD8α hinge) that confers flexibility to the recognition region and allows strong binding to the targeting moiety.

[0225] Schematic diagrams of exemplary CARs are shown in Figure 2 and Figure 3 . For Figure 2 , the fusion protein sequence can be incorporated into a lentiviral expression vector, where "SP" is a signal peptide, CAR is an anti-FITC CAR, there is a CD8α hinge and CD8α transmembrane domain, the co-stimulatory domain is 4-1BB, and the activation signaling domain is CD3ζ. Exemplary nucleic acid sequences of the CAR insert are provided as SEQ ID NO:01 and SEQ ID NO:03, and the encoded amino acid sequence is provided as SEQ ID NO:02. In another embodiment, SEQ ID NO:02 may comprise or consist of a humanized or human amino acid sequence.

[0226] For Figure 3 , a schematic diagram of an exemplary CAR construct is shown, where the expressed CAR comprises an E2 anti-fluorescein antibody fragment, where the fusion protein sequence can be incorporated into an expression vector, and where the CAR comprises an E2 anti-fluorescein antibody fragment, an IgG4 hinge domain, a CD28 transmembrane domain, and where the co-stimulatory domain is CD137 (4-1BB), and the activation signaling domain is CD3ζ. The CAR may comprise additional suitable domains. Exemplary nucleic acid sequences of such CAR inserts are provided as SEQ ID NO:04, and exemplary encoded amino acid sequences are provided as SEQ ID NO:05. SEQ ID NO:04 includes the sequence starting with the underlined "AGE" codon and ending with the underlined "GGC" codon (see Table 1). This portion of the longer sequence encodes the CAR inserted into the T cell membrane. Other portions of the longer sequence include the coding sequences for the signal peptide, EGFRt domain, etc., which are not part of the CAR inserted into the membrane and serving as a chimeric antigen receptor. SEQ ID NO:05 includes the sequence starting with the underlined "S" and ending with the underlined "G" (see Table 1). This portion of the longer sequence is the amino acid sequence of the CAR inserted into the T cell membrane. Other portions of the longer sequence include the amino acid sequences for the signal peptide, EGFRt domain, etc., which are not part of the CAR inserted into the membrane and serving as a chimeric antigen receptor. In another embodiment, SEQ ID NO:05 may comprise or consist of a humanized or human amino acid sequence. SEQ ID NO:04 and SEQ ID NO:05 are as described above and shown in Table 1. The start and stop codons in SEQ ID NO:04 (ATG, AGC, and GGC respectively) are underlined and the longer sequence (SEQ ID NO:08) is an exemplary sequence that can be used to transduce T cells for the methods described herein.

[0227] Another exemplary CAR construct is the 4M5.3 CAR, which is schematically shown in Figure 3 . SEQ ID NO:06 includes the sequence starting from the underlined "GAC" codon and ending at the underlined "GGC" codon (see Table 1). This portion of the longer sequence encodes the exemplary 4M5.3 CAR. The CAR is inserted into the T cell membrane. Other portions of the longer sequence include the coding sequences for the signal peptide, the EGFRt domain, etc., which are not inserted into the membrane and are not part of the CAR that serves as a chimeric antigen receptor. SEQ ID NO:07 includes the sequence starting from the underlined "D" and ending at the underlined "G" (see Table 1). This portion of the longer sequence is the amino acid sequence of the CAR inserted into the T cell membrane. Other portions of the longer sequence include the amino acid sequences for the signal peptide, the EGFRt domain, etc., which are not inserted into the membrane and are not part of the CAR that serves as a chimeric antigen receptor. In yet another embodiment, SEQ ID NO:07 may comprise or consist of a humanized or human amino acid sequence. SEQ ID NO:06 and SEQ ID NO:07 are as described above and are shown below. The start codon and stop codon in the longer nucleic acid sequence are underlined, and the longer sequence is an exemplary sequence that can be used to transduce T cells to prepare the 4M5.3 CAR.

[0228] In one embodiment, the CAR has: an identification region, and the identification region is the single-chain variable fragment (scFv) region of an anti-FITC antibody; a co-stimulatory domain, and the co-stimulatory domain is CD137 (4-1BB); and, an activation signal transduction domain, and the activation signal transduction domain is the T cell CD3ζ chain. Those skilled in the art are familiar with the fact that anti-FITC scFv and anti-fluorescein scFv are equivalent terms.

[0229] In one embodiment, T lymphocytes (such as cytotoxic T lymphocytes) can be genetically engineered to express the CAR construct by transfecting a population of T lymphocytes with an expression vector encoding the CAR construct. Methods suitable for preparing a transduced population of T lymphocytes expressing a selected CAR construct are well known to those skilled in the art and are described in the following: Sambrook et al., "Molecular Cloning: A Laboratory Manual", 3rd Edition, Cold Spring Harbor Laboratory Press, (2001), incorporated herein by reference; and Green and Sambrook, "Molecular Cloning: A Laboratory Manual", 4th Edition, Cold Spring Harbor Laboratory Press, (2012), incorporated herein by reference.

[0230] In one embodiment, provided are CAR T cells comprising a nucleic acid of SEQ ID NO:01, SEQ ID NO:03, SEQ ID NO:04 or SEQ ID NO:06. In another embodiment, provided are CAR T cells comprising a polypeptide of SEQ ID NO:02, SEQ ID NO:05 or SEQ ID NO:07. In another illustrative aspect, provided is a nucleic acid (such as an isolated nucleic acid) comprising SEQ ID NO:01, SEQ ID NO:03, SEQ ID NO:04 or SEQ ID NO:06 and encoding a chimeric antigen receptor. In yet another embodiment, provided is a chimeric antigen receptor polypeptide comprising SEQ ID NO:02, SEQ ID NO:05 or SEQ ID NO:07. In another embodiment, provided is a vector comprising SEQ ID NO:01, SEQ ID NO:03, SEQ ID NO:04 or SEQ ID NO:06. In another aspect, provided is a lentiviral vector comprising SEQ ID NO:01, SEQ ID NO:03, SEQ ID NO:04 or SEQ ID NO:06. In yet another embodiment, SEQ ID NO:02, SEQ ID NO:05 or SEQ ID NO:07 may comprise or consist of a humanized or human amino acid sequence.

[0231] In each of these embodiments, variant nucleic acid sequences or amino acid sequences having at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99% or at least about 99.5% sequence identity with SEQ ID NOs: 01 to 07 are also encompassed. In another embodiment, the nucleic acid sequence may be a variant nucleic acid sequence having at least about 80%, at least about 90%, at least about 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: 01, SEQ ID NO: 03, SEQ ID NO: 04 or SEQ ID NO: 06, provided that the variant sequence encodes SEQ ID NO: 02 (for SEQ ID NO: 01 and SEQ ID NO: 03), SEQ ID NO: 05 (for SEQ ID NO: 04) or SEQ ID NO: 07 (for SEQ ID NO: 06). In another embodiment, the nucleic acid sequence or amino acid sequence may be a variant nucleic acid or amino acid sequence 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: 01, SEQ ID NO: 03, SEQ ID NO: 04 or SEQ ID NO: 06 over a 200-nucleotide segment or with SEQ ID NO: 02, SEQ ID NO: 05 or SEQ ID NO: 07 over a 200-amino acid segment. In one embodiment, determination of the percentage of identity or similarity between sequences may be accomplished, for example, by using the GAP program (Genetics Computer Group, software; currently available through Accelrys); and alignment may be accomplished using, for example, the ClustalW algorithm (VNTI software, InforMax Int.). The nucleic acid or amino acid sequence of interest may be used to search sequence databases. Database search algorithms are typically based on the BLAST software (Altschul et al., 1990). In some embodiments, the percentage of identity may be determined over the full length of the nucleic acid or amino acid sequence.

[0232] Nucleic acids complementary to the nucleic acids represented by SEQ ID NO:01, SEQ ID NO:03, SEQ ID NO:04, or SEQ ID NO:06, and those nucleic acids that hybridize to the nucleic acids represented by SEQ ID NO:01, SEQ ID NO:03, SEQ ID NO:04, or SEQ ID NO:06, or those nucleic acids that hybridize to their complements under highly stringent conditions are also within the scope of the present invention. According to the present invention, "highly stringent conditions" means hybridization in 5X SSPE and 50% formamide at 65 °C, and washing in 0.5X SSPE at 65 °C. Conditions for highly stringent, low stringent, and medium stringent hybridization are described in the following: Sambrook et al., "Molecular Cloning: A Laboratory Manual", 3rd edition, Cold Spring Harbor Laboratory Press, (2001), which is incorporated herein by reference; and Green and Sambrook, "Molecular Cloning: A Laboratory Manual", 4th edition, Cold Spring Harbor Laboratory Press, (2012), which is incorporated herein by reference. In some illustrative aspects, hybridization occurs along the entire length of the nucleic acid.

[0233] In one embodiment, the T lymphocytes used in the methods described herein (e.g., cytotoxic T lymphocytes for the preparation of CAR T cells) can be autologous cells, but allogeneic cells can also be used, for example, when the patient being treated has received high-dose chemotherapy or radiotherapy that has damaged the patient's immune system. In one embodiment, allogeneic cells can be used.

[0234] In one aspect, T lymphocytes can be obtained from a patient by methods well known in the art. For example, T cells (e.g., cytotoxic T cells) can be obtained by: collecting peripheral blood from the patient, performing Ficoll density gradient centrifugation on the blood, and then using a negative T cell isolation kit (e.g., EasySep TM T cell isolation kit)) to isolate a population of T cells from the peripheral blood. In one illustrative embodiment, the population of T lymphocytes (e.g., cytotoxic T cells) need not be pure and can contain other cells, such as other types of T cells (e.g., in the case of cytotoxic T cells), monocytes, macrophages, natural killer cells, and B cells. In one aspect, the collected population can contain at least about 90% of the selected cell type, at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the selected cell type.

[0235] In one embodiment, after obtaining T lymphocytes (such as cytotoxic T cells for preparing CAR T cells), the cells are cultured under conditions that promote cell activation. In this embodiment, the culture conditions can be such that the cells can be administered to a patient without concern for reactivity against components of the culture medium. For example, the culture conditions may not include bovine serum products, such as bovine serum albumin. In an illustrative aspect, in the case of cytotoxic T cells, activation can be achieved by introducing a known activator, such as an anti-CD3 antibody, into the culture medium. Other suitable activators include anti-CD28 antibodies. In one aspect, a population of lymphocytes can be cultured for about 1 day to about 4 days under conditions that promote activation. In one embodiment, an appropriate level of activation can be determined by cell size, proliferation rate, or activation markers determined by flow cytometry.

[0236] In an illustrative embodiment, after culturing a population of T lymphocytes (such as cytotoxic T lymphocytes for preparing CAR T cells) under conditions that promote activation, the cells can be transfected with an expression vector encoding a CAR. Suitable vectors and transfection methods for various embodiments are described above. In one aspect, after transfection, the cells can be immediately administered to a patient, or the cells can be cultured for at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days or more, or between about 5 days and about 12 days, between about 6 days and about 13 days, between about 7 days and about 14 days, or between about 8 days and about 15 days, for example, to allow time for the cells to recover from transfection. In one aspect, suitable culture conditions can be similar to the conditions for culturing cells with or without reagents for promoting activation to activate the cells.

[0237] Thus, as described above, in an illustrative aspect, the treatment methods described herein can further comprise 1) obtaining a population of autologous or allogeneic T lymphocytes (such as cytotoxic T lymphocytes for preparing CAR T cells), 2) culturing the T lymphocytes under conditions that promote cell activation, and 3) transfecting the lymphocytes with an expression vector encoding a CAR to form CAR T cells.

[0238] In one embodiment, CART cells can be cultured using a medium lacking any animal products (such as bovine serum). In another embodiment, tissue culture conditions typically used by those skilled in the art to avoid contamination by bacteria, fungi, and mycoplasma can be used. In an exemplary embodiment, before being administered to a patient, the cells (such as CAR T cells) are precipitated, washed, and resuspended in a pharmaceutically acceptable carrier or diluent. Exemplary compositions comprising T lymphocytes expressing CAR (such as cytotoxic T lymphocytes) include compositions comprising the cells in sterile 290 mOsm saline, in an infusion-compatible cryomedium (containing Plasma-Lyte A, dextrose, sodium chloride injection, human serum albumin, and DMSO), in 0.9% NaCl with 2% human serum albumin, or in any other sterile 290 mOsm infusion-compatible substance. Alternatively, in another embodiment, depending on the medium itself, the CAR T cells can be administered as a composition in the medium or concentrated and resuspended in the medium before administration. In various embodiments, the CAR T cell composition can be administered to a patient by any suitable means, such as parenteral administration, such as intradermal, subcutaneous, intramuscular, intraperitoneal, intravenous, or intrathecal.

[0239] In one aspect, the total number of CAR T cells and the concentration of the cells in the composition administered to a patient will vary depending on a variety of factors, including the type of T lymphocytes being used (such as cytotoxic T lymphocytes), the binding specificity of the CAR, the characteristics of the targeting moiety and small molecule ligand, the characteristics of the cancer, the location of the cancer in the patient, the means by which the composition is administered to the patient, and the health, age, and weight of the patient being treated. In various embodiments, suitable compositions comprising transduced CAR T cells include compositions having a volume of from about 0.1 mL to about 200 mL and from about 0.1 mL to about 125 mL.

[0240] In various embodiments, the transduced CAR T cells administered to a patient can comprise from about 1x10 5 to about 1x10 15 or 1x10 6 to about 1x10 15 transduced CAR T cells. In various embodiments, from about 1x10 5 to about 1x10 10 , from about 1x10 6 to about 1x10 10 , from about 1x10 6 to about 1x10 9 , from about 1x10 6 to about 1x10 8 , from about 1x106 to about 2x10 7 、about 1x10 6 to about 3x10 7 、about 1x10 6 to about 1.5x10 7 、about 1x10 6 to about 1x10 7 、about 1x10 6 to about 9x10 6 、about 1x10 6 to about 8x10 6 、about 1x10 6 to about 7x10 6 、about 1x10 6 to about 6x10 6 、about 1x10 6 to about 5x10 6 、about 1x10 6 to about 4x10 6 、about 1x10 6 to about 3x10 6 、about 1x10 6 to about 2x10 6 、about 2x10 6 to about 6x10 6 、about 2x10 6 to about 5x10 6 、about 3x10 6 to about 6x10 6 、about 4x10 6 to about 6x10 6 、about 4x10 6 to about 1x10 7 、about 1x10 6 to about 1x10 7 、about 1x10 6 to about 1.5x10 7 、about 1x10 6 to about 2x10 7 、about 0.2x10 6 to about 1x10 7 、about 0.2x10 6 to about 1.5x10 7 、about 0.2x10 6 to about 2x10 7 、about 0.2x10 6 to about 3x10 7 、about 0.2x10 6 to about 4x10 7 、about 0.2x10 6 to about 5x10 7, about 0.2x10 5 to about 1.5x10 6 , about 0.5x10 5 to about 1.5x10 6 , about 0.2x10 5 to about 1.4x10 6 , about 0.2x10 5 to about 1.3x10 6 , about 0.5x10 5 to about 1.3x10 6 , about 0.8x10 6 to about 2x10 7 , about 0.8x10 6 to about 1.5x10 7 , about 0.9x10 6 to about 1.2x10 7 , or about 0.5x10 6 , 1x10 6 , 5x10 6 , 6x10 6 , 7x10 6 , 8x10 6 , 9x10 6 , 1x10 7 , 1.5x10 7 , 2x10 7 , 3x10 7 , 4x10 7 or 5x10 7 CAR T cells. These amounts can be per kg of patient body weight.

[0241] In other embodiments, the dose of CAR T cells administered to the patient in the CAR T cell composition is selected from the group consisting of: about 1 million, about 2 million, about 3 million, about 4 million, about 5 million, about 6 million, about 7 million, about 8 million, about 9 million, about 10 million, about 11 million, about 12 million, about 12.5 million, about 13 million, about 14 million and about 15 million of said CAR T cells.

[0242] In any of the embodiments described in this paragraph, the CAR T cell dose can be the number of CAR T cells per kg of patient body weight. In one aspect, in any of the embodiments described herein, a single dose or multiple doses of CAR T cells can be administered to a patient. In an illustrative embodiment, a first dose of CAR T cells and a second dose of CAR T cells can be administered to a patient. In one aspect, the first dose of CAR T cells can be a test dose used to monitor the patient's tolerance to CAR T cells, and the second dose of CAR T cells can contain a higher dose of CAR T cells compared to the first dose of CAR T cells. In one embodiment, the first dose of CAR T cells can contain from about 0.5x10 5 to about 1.5x10 6 CAR T cells. In one embodiment, the first dose of CAR T cells can contain about 1x10 6 CAR T cells or contain 1x10 6 CAR T cells. In another embodiment, the second dose of CAR T cells can contain from about 0.8x10 6 to about 2x10 7 CAR T cells. In these embodiments that include a first dose and a second dose of CAR T cells, any dose of the CAR T cells described herein can be administered.

[0243] In any of the embodiments described herein, the CAR T cells can be administered before or after the compound or its pharmaceutically acceptable salt. As will be understood, unless otherwise stated, the names i), ii), iii), etc. of the steps of any method described herein do not indicate an order.

[0244] Any suitable method known in the art can be used to administer the compound or its pharmaceutically acceptable salt or CAR T cell composition described herein to a patient. As used herein, the term "administering / administered" includes all means of introducing the compound or its pharmaceutically acceptable salt or CAR T cell composition to a patient, including, but not limited to, oral, intravenous, intramuscular, subcutaneous, transdermal, etc. In one aspect, the compound or its pharmaceutically acceptable salt described herein can be administered in unit dosage forms and / or in preparations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants, and vehicles.

[0245] In one aspect, a compound or a pharmaceutically acceptable salt thereof or a CAR T cell composition as described herein can be administered directly into the bloodstream, into a muscle, or into an internal organ. In various embodiments, suitable routes for such parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, epidural, intraventricular, intraurethral, intrasternal, intracranial, intratumoral, intramuscular, and subcutaneous delivery. In one embodiment, means for parenteral administration include needle (including microneedle) syringes, needleless syringes, and infusion techniques.

[0246] In an illustrative aspect, parenteral formulations are typically aqueous solutions that may contain carriers or excipients (such as salts, carbohydrates, and buffers, preferably at a pH of from 3 to 9), but they may be more desirably formulated as sterile non-aqueous solutions, or in a dry form for use in conjunction 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. The preparation of sterile lyophilized powders for parenteral formulations under sterile conditions and by lyophilization can be readily accomplished using standard pharmaceutical techniques well known to those skilled in the art. In one embodiment, the solubility of the compound or a pharmaceutically acceptable salt thereof used in preparing parenteral formulations can be increased by using appropriate formulation techniques (such as incorporating solubilizing agents).

[0247] In one embodiment, the amount of the compound or a pharmaceutically acceptable salt thereof to be administered to a patient can vary significantly depending on the cancer being treated, the route of administration of the compound or a pharmaceutically acceptable salt thereof, and tissue distribution. In one aspect, the amount to be administered to a patient can be based on body surface area, mass, and physician assessment.

[0248] In various embodiments, the compound or a pharmaceutically acceptable salt thereof can be administered in accordance with: 1) at least a first dose escalation sequence, 2) at least a first dose escalation sequence and a second dose escalation sequence, 3) at least a first dose escalation sequence, a second dose escalation sequence, and a third dose escalation sequence, 4) at least a first dose escalation sequence, a second dose escalation sequence, a third dose escalation sequence, and a fourth dose escalation sequence, 5) at least a first dose escalation sequence, a second dose escalation sequence, a third dose escalation sequence, a fourth dose escalation sequence, and a fifth dose escalation sequence, 6) at least a first dose escalation sequence, a second dose escalation sequence, a third dose escalation sequence, a fourth dose escalation sequence, a fifth dose escalation sequence, and a sixth dose escalation sequence, or 7) more than one additional dose escalation sequence.

[0249] In various embodiments, for a first, second, third, fourth, fifth, or sixth, etc. dose escalation sequence, the amount of the compound or a pharmaceutically acceptable salt thereof to be administered to a patient can be from 1 μg / kg to 50 μg / kg or about 1 μg / kg to 50 μg / kg, or about 31 μg / kg or 31 μg / kg. In various embodiments, for a first, second, third, fourth, fifth, or sixth, etc. dose escalation sequence, the amount of the compound or a pharmaceutically acceptable salt thereof to be administered to a patient can be from about 0.1 μg / kg to about 2000 μg / kg, 0.1 μg / kg to about 1500 μg / kg, 0.1 μg / kg to about 1000 μg / kg, 0.1 μg / kg to about 500 μg / kg, about 0.1 μg / kg to about 100 μg / kg, about 0.1 μg / kg to about 80 μg / kg, about 0.1 μg / kg to about 70 μg / kg, about 0.1 μg / kg to about 50 μg / kg, about 0.1 μg / kg to about 40 μg / kg, about 0.1 μg / kg to about 30 μg / kg, about 0.3 μg / kg to about 500 μg / kg, about 0.3 μg / kg to about 400 μg / kg, about 0.3 μg / kg to about 300 μg / kg, about 0.3 μg / kg to about 200 μg / kg, about 0.3 μg / kg to about 100 μg / kg, about 0.3 μg / kg to about 90 μg / kg, about 0.1 μg / kg to about 400 μg / kg, about 0.1 μg / kg to about 350 μg / kg, about 0.1 μg / kg to about 300 μg / kg, about 0.1 μg / kg to about 250 μg / kg, about 0.1 μg / kg to about 200 μg / kg, about 0.1 μg / kg to about 150 μg / kg, or about 0.3 μg / kg to about 150 μg / kg, or 5 μg / kg to about 2000 μg / kg, 5 μg / kg to about 1500 μg / kg, 5 μg / kg to about 1000 μg / kg, 5 μg / kg to about 500 μg / kg, about 5 μg / kg to about 100 μg / kg, about 5 μg / kg to about 80 μg / kg, about 5 μg / kg to about 70 μg / kg, about 5 μg / kg to about 50 μg / kg, about 5 μg / kg to about 40 μg / kg, or about 5 μg / kg to about 30 μg / kg.In another embodiment, for the first and fourth dose escalation sequences, the amount of the compound or a pharmaceutically acceptable salt thereof to be administered to the patient can be from about 0.1 μg / kg to about 100 μg / kg, from about 0.1 μg / kg to about 80 μg / kg, from about 0.1 μg / kg to about 70 μg / kg, from about 0.1 μg / kg to about 50 μg / kg, from about 0.1 μg / kg to about 40 μg / kg, from about 0.1 μg / kg to about 30 μg / kg, or from about 0.3 μg / kg to about 30 μg / kg, from about 5 μg / kg to about 100 μg / kg, from about 5 μg / kg to about 80 μg / kg, from about 5 μg / kg to about 70 μg / kg, from about 5 μg / kg to about 50 μg / kg, from about 5 μg / kg to about 40 μg / kg, from about 5 μg / kg to about 30 μg / kg, or from about 5 μg / kg to about 2000 μg / kg, or from about 5 μg / kg to about 1500 μg / kg, or from about 5 μg / kg to about 1000 μg / kg, or from about 5 μg / kg to about 500 μg / kg. In another embodiment, for the second and fifth dose escalation sequences, the amount of the compound or a pharmaceutically acceptable salt thereof to be administered to the patient can be from about 0.3 μg / kg to about 500 μg / kg, from about 0.3 μg / kg to about 400 μg / kg, from about 0.3 μg / kg to about 300 μg / kg, from about 0.3 μg / kg to about 200 μg / kg, from about 0.3 μg / kg to about 100 μg / kg, or from about 0.3 μg / kg to about 90 μg / kg, or from about 5 μg / kg to about 100 μg / kg, from about 5 μg / kg to about 80 μg / kg, from about 5 μg / kg to about 70 μg / kg, from about 5 μg / kg to about 50 μg / kg, from about 5 μg / kg to about 40 μg / kg, from about 5 μg / kg to about 30 μg / kg, or from about 5 μg / kg to about 2000 μg / kg, or from about 5 μg / kg to about 1500 μg / kg, or from about 5 μg / kg to about 1000 μg / kg, or from about 5 μg / kg to about 500 μg / kg.In yet another embodiment, for the third and sixth dose escalation sequences, the amount of the compound or its pharmaceutically acceptable salt to be administered to the patient can be from 0.1 μg / kg to about 400 μg / kg, from about 0.1 μg / kg to about 350 μg / kg, from about 0.1 μg / kg to about 300 μg / kg, from about 0.1 μg / kg to about 250 μg / kg, from about 0.1 μg / kg to about 200 μg / kg, from about 0.1 μg / kg to about 150 μg / kg, or from about 0.3 μg / kg to about 150 μg / kg, or from about 5 μg / kg to about 100 μg / kg, from about 5 μg / kg to about 80 μg / kg, from about 5 μg / kg to about 70 μg / kg, from about 5 μg / kg to about 50 μg / kg, from about 5 μg / kg to about 40 μg / kg, from about 5 μg / kg to about 30 μg / kg, or from about 5 μg / kg to about 2000 μg / kg, or from about 5 μg / kg to about 1500 μg / kg, or from about 5 μg / kg to about 1000 μg / kg, or from about 5 μg / kg to about 500 μg / kg. In these embodiments, "kg" is the number of kilograms of the patient's body weight.

[0250] In any of these embodiments, the range of the amount of the compound or its pharmaceutically acceptable salt can be a range of calculated percentages based on the "full dose" of the compound or its pharmaceutically acceptable salt, where the "full dose" of the compound or its pharmaceutically acceptable salt can be about 30 μg / kg, and where the percentage is from about 1% to about 100% of the "full dose" of the compound or its pharmaceutically acceptable salt (see Figure 1 Sequence 1), from about 1% to about 300% (see Figure 1 Sequence 2), and from about 1% to about 500% (see Figure 1 Sequence 3). In other embodiments, the percentage of the "full dose" of the compound or its pharmaceutically acceptable salt administered in a dose escalation sequence can be about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 200%, about 300%, about 400%, or about 500%, and the administered amounts can be about 0.3 μg / kg, about 3 μg / kg, about 9 μg / kg, about 15 μg / kg, about 30 μg / kg, about 90 μg / kg, or about 150 μg / kg, respectively, or can be about 17%, about 333%, about 1666%, or about 3333%, respectively, and the administered amounts can be about 5 μg / kg, about 100 μg / kg, about 500 μg / kg, or about 1000 μg / kg, respectively. In these embodiments, "kg" is the number of kilograms of the patient's body weight.

[0251] In another embodiment, the percentages of the "full dose" of the compound or its pharmaceutically acceptable salt administered in a dose-escalating order may be about 1%, about 2% and about 20% for the first dose-escalating order, about 1%, about 6% and about 60% for the second dose-escalating order, and about 1%, about 10% and about 100% for the third dose-escalating order, and the "full dose" of the compound or its pharmaceutically acceptable salt may be 500 nmoles / kg. In this embodiment, "kg" is the number of kilograms of the patient's body weight. In this embodiment, the compound or its pharmaceutically acceptable salt may be administered on different days, and may be repeated about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times, or have any appropriate number of days between each dose-escalation cycle. In this embodiment, the compound or its pharmaceutically acceptable salt may be administered on different days, with about 6 days between each dose-escalation cycle. In this embodiment, the compound or its pharmaceutically acceptable salt may be administered on the 4th, 7th, 11th, 18th and 25th days after the first dose. In this embodiment, the compound or its pharmaceutically acceptable salt may be administered on any day between the 26th and 32nd days after the first dose.

[0252] In another embodiment, for the first dose escalation sequence, the percentage of the "full dose" of the compound or its pharmaceutically acceptable salt administered in a dose escalation sequence can be about 1%, about 5%, about 10%, about 30%, about 50%, about 100%, about 200%, about 300%, about 400%, about 500%, about 600%, or any integer between 1% and 600%. In this embodiment, for the second dose escalation sequence, the percentage of the "full dose" of the compound or its pharmaceutically acceptable salt administered in a dose escalation sequence can be about 1%, about 5%, about 10%, about 20%, about 30%, about 50%, about 100%, about 200%, about 300%, about 400%, about 500%, about 600%, or any integer between 1% and 600%. In this embodiment, the "full dose" of the compound or its pharmaceutically acceptable salt can be 500 nmoles / kg. In this embodiment, "kg" is the number of kilograms of the patient's body weight. In this embodiment, the compound or its pharmaceutically acceptable salt can be administered on different days, and can be repeated about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times, or have any appropriate number of days between each dose escalation cycle. In this embodiment, the compound or its pharmaceutically acceptable salt can be administered on different days, with about 6 days between each dose escalation cycle. In this embodiment, the compound or its pharmaceutically acceptable salt can be administered on the 4th, 7th, 11th, 18th, and 25th days after the first dose. In this embodiment, the compound or its pharmaceutically acceptable salt can be administered on any day between the 26th and 32nd days after the first dose. In other embodiments, the dose escalation can be repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times or any suitable number of times.

[0253] In another embodiment, the percentages of the "full dose" of the compound or its pharmaceutically acceptable salt administered in the first or fourth dose escalation sequence may be about 1%, about 10%, and about 100% (in incremental amounts) of the "full dose" of the compound or its pharmaceutically acceptable salt (about 10 μg / kg to about 50 μg / kg, about 20 μg / kg to about 40 μg / kg, about 25 μg / kg to about 35 μg / kg, or about 30 μg / kg), and the amounts of the compound or its pharmaceutically acceptable salt administered may be about 0.3 μg / kg, about 3 μg / kg, and about 30 μg / kg, respectively. In yet another embodiment, the percentages of the "full dose" of the compound or its pharmaceutically acceptable salt administered in the second or fifth dose escalation sequence may be about 1%, about 30%, and about 300% (in incremental amounts) of the "full dose" of the compound or its pharmaceutically acceptable salt (about 10 μg / kg to about 50 μg / kg, about 20 μg / kg to about 40 μg / kg, about 25 μg / kg to about 35 μg / kg, or about 30 μg / kg), and the amounts of the compound or its pharmaceutically acceptable salt administered may be about 0.3 μg / kg, about 9 μg / kg, and about 90 μg / kg, respectively. In still another embodiment, the percentages of the "full dose" of the compound or its pharmaceutically acceptable salt administered in the third or sixth dose escalation sequence may be about 1%, about 50%, and about 500% (in incremental amounts) of the "full dose" of the compound or its pharmaceutically acceptable salt (about 10 μg / kg to about 50 μg / kg, about 20 μg / kg to about 40 μg / kg, about 25 μg / kg to about 35 μg / kg, or about 30 μg / kg), and the amounts of the compound or its pharmaceutically acceptable salt administered may be about 0.3 μg / kg, about 15 μg / kg, and about 150 μg / kg, respectively. In these embodiments, "kg" is the number of kilograms of the patient's body weight.

[0254] In various other embodiments, the amount to be administered to the patient can be in the range of, for example, from about 0.05 mg to about 30 mg, 0.05 mg to about 25.0 mg, about 0.05 mg to about 20.0 mg, about 0.05 mg to about 15.0 mg, about 0.05 mg to about 10.0 mg, about 0.05 mg to about 9.0 mg, about 0.05 mg to about 8.0 mg, about 0.05 mg to about 7.0 mg, about 0.05 mg to about 6.0 mg, about 0.05 mg to about 5.0 mg, about 0.05 mg to about 4.0 mg, about 0.05 mg to about 3.0 mg, about 0.05 mg to about 2.0 mg, about 0.05 mg to about 1.0 mg, about 0.05 mg to about 0.5 mg, about 0.05 mg to about 0.4 mg, about 0.05 mg to about 0.3 mg, about 0.05 mg to about 0.2 mg, about 0.05 mg to about 0.1 mg, about 0.01 mg to about 2 mg, about 0.3 mg to about 10 mg, about 0.1 mg to about 20 mg, or about 0.8 to about 3 mg.

[0255] In other embodiments, the dose of the compound or its pharmaceutically acceptable salt can be in the range of, for example, from about 50 nmoles / kg patient body weight to about 3000 nmoles / kg patient body weight, from about 50 nmoles / kg patient body weight to about 2000 nmoles / kg patient body weight, from about 50 nmoles / kg patient body weight to about 1000 nmoles / kg patient body weight, from about 50 nmoles / kg patient body weight to about 900 nmoles / kg patient body weight, from about 50 nmoles / kg patient body weight to about 800 nmoles / kg patient body weight, from about 50 nmoles / kg patient body weight to about 700 nmoles / kg patient body weight, from about 50 nmoles / kg patient body weight to about 600 nmoles / kg patient body weight, from about 50 nmoles / kg patient body weight to about 500 nmoles / kg patient body weight, from about 50 nmoles / kg patient body weight to about 400 nmoles / kg patient body weight, from about 50 nmoles / kg patient body weight to about 300 nmoles / kg patient body weight, from about 50 nmoles / kg patient body weight to about 200 nmoles / kg patient body weight, from about 50 nmoles / kg patient body weight to about 100 nmoles / kg patient body weight, from about 100 nmoles / kg patient body weight to about 300 nmoles / kg patient body weight, from about 100 nmoles / kg patient body weight to about 500 nmoles / kg patient body weight, from about 100 nmoles / kg patient body weight to about 1000 nmoles / kg patient body weight, from about 100 nmoles / kg patient body weight to about 2000 nmoles / kg patient body weight.In other embodiments, the dose may be about 1 nmoles / kg patient body weight, about 5 nmoles / kg patient body weight, about 10 nmoles / kg patient body weight, about 20 nmoles / kg patient body weight, about 25 nmoles / kg patient body weight, about 30 nmoles / kg patient body weight, about 40 nmoles / kg patient body weight, about 50 nmoles / kg patient body weight, about 60 nmoles / kg patient body weight, about 70 nmoles / kg patient body weight, about 80 nmoles / kg patient body weight, about 90 nmoles / kg patient body weight, about 100 nmoles / kg patient body weight, about 150 nmoles / kg patient body weight, about 200 nmoles / kg patient body weight, about 250 nmoles / kg patient body weight, about 300 nmoles / kg patient body weight, about 350 nmoles / kg patient body weight, about 400 nmoles / kg patient body weight, about 450 nmoles / kg patient body weight, about 500 nmoles / kg patient body weight, about 600 nmoles / kg patient body weight, about 700 nmoles / kg patient body weight, about 800 nmoles / kg patient body weight, about 900 nmoles / kg patient body weight, about 1000 nmoles / kg patient body weight, about 2000 nmoles / kg patient body weight, about 2500 nmoles / kg patient body weight, or about 3000 nmoles / kg patient body weight.In yet another alternative embodiment, the dose may be about 0.1 nmoles / kg patient body weight, about 0.2 nmoles / kg patient body weight, about 0.3 nmoles / kg patient body weight, about 0.4 nmoles / kg patient body weight, or about 0.5 nmoles / kg patient body weight, from about 0.1 nmoles / kg patient body weight to about 1000 nmoles / kg patient body weight, from about 0.1 nmoles / kg patient body weight to about 900 nmoles / kg patient body weight, from about 0.1 nmoles / kg patient body weight to about 850 nmoles / kg patient body weight, from about 0.1 nmoles / kg patient body weight to about 800 nmoles / kg patient body weight, from about 0.1 nmoles / kg patient body weight to about 700 nmoles / kg patient body weight, from about 0.1 nmoles / kg patient body weight to about 600 nmoles / kg patient body weight, from about 0.1 nmoles / kg patient body weight to about 500 nmoles / kg patient body weight, from about 0.1 nmoles / kg patient body weight to about 400 nmoles / kg patient body weight, from about 0.1 nmoles / kg patient body weight to about 300 nmoles / kg patient body weight, from about 0.1 nmoles / kg patient body weight to about 200 nmoles / kg patient body weight, from about 0.1 nmoles / kg patient body weight to about 100 nmoles / kg patient body weight, from about 0.1 nmoles / kg patient body weight to about 50 nmoles / kg patient body weight, from about 0.1 nmoles / kg patient body weight to about 10 nmoles / kg patient body weight, or from about 0.1 nmoles / kg patient body weight to about 1 nmoles / kg patient body weight. In other embodiments, the dose may be from about 0.3 nmoles / kg patient body weight to about 1000 nmoles / kg patient body weight, from about 0.3 nmoles / kg patient body weight to about 900.

[0256] nmoles / kg of patient body weight, from about 0.3 nmoles / kg of patient body weight to about 850 nmoles / kg of patient body weight, from about 0.3 nmoles / kg of patient body weight to about 800 nmoles / kg of patient body weight, from about 0.3 nmoles / kg of patient body weight to about 700 nmoles / kg of patient body weight, from about 0.3 nmoles / kg of patient body weight to about 600 nmoles / kg of patient body weight, from about 0.3 nmoles / kg of patient body weight to about 500 nmoles / kg of patient body weight, from about 0.3 nmoles / kg of patient body weight to about 400 nmoles / kg of patient body weight, from about 0.3 nmoles / kg of patient body weight to about 300 nmoles / kg of patient body weight, from about 0.3 nmoles / kg of patient body weight to about 200 nmoles / kg of patient body weight, from about 0.3 nmoles / kg of patient body weight to about 100 nmoles / kg of patient body weight, from about 0.3 nmoles / kg of patient body weight to about 50 nmoles / kg of patient body weight, from about 0.3 nmoles / kg of patient body weight to about 10 nmoles / kg of patient body weight, or from about 0.3 nmoles / kg of patient body weight to about 1 nmoles / kg of patient body weight. In these embodiments, "kg" is the number of kilograms of the patient's body weight.

[0257] In another embodiment, after the administration of the CAR-T cells (e.g., at any of the amounts described herein), a first dose escalation step and a second dose escalation step are performed using the compound (e.g., where each dose escalation step is 50 nmol / kg and then 500 nmol / kg) or a pharmaceutically acceptable salt thereof. In this embodiment, after the first dose escalation step and the second dose escalation step, for example, in the first and second weeks, the level of the compound or a pharmaceutically acceptable salt thereof may remain constant in the third week (e.g., remain constant at 500 nmol / kg) relative to the last dose administered in the second week. In any of the embodiments described herein, the level of the compound or a pharmaceutically acceptable salt thereof may remain constant in a subsequent week relative to the last dose administered in the previous week.

[0258] In various other embodiments, the dose of the compound or its pharmaceutically acceptable salt can be in the range of, for example, from about 10 nmoles / kg patient body weight to about 10,000 nmoles / kg patient body weight, from about 10 nmoles / kg patient body weight to about 5,000 nmoles / kg patient body weight, from about 10 nmoles / kg patient body weight to about 3,000 nmoles / kg patient body weight, from about 10 nmoles / kg patient body weight to about 2,500 nmoles / kg patient body weight, from about 10 nmoles / kg patient body weight to about 2,000 nmoles / kg patient body weight, from about 10 nmoles / kg patient body weight to about 1,000 nmoles / kg patient body weight, from about 10 nmoles / kg patient body weight to about 900 nmoles / kg patient body weight, from about 10 nmoles / kg patient body weight to about 800 nmoles / kg patient body weight, from about 10 nmoles / kg patient body weight to about 700 nmoles / kg patient body weight, from about 10 nmoles / kg patient body weight to about 600 nmoles / kg patient body weight, from about 10 nmoles / kg patient body weight to about 500 nmoles / kg patient body weight, from about 10 nmoles / kg patient body weight to about 400 nmoles / kg patient body weight, from aboutFrom about 200 nmoles / kg of patient body weight to about 600 nmoles / kg of patient body weight, from about 200 nmoles / kg of patient body weight to about 500 nmoles / kg of patient body weight, from about 250 nmoles / kg of patient body weight to about 600 nmoles / kg of patient body weight, from about 300 nmoles / kg of patient body weight to about 600 nmoles / kg of patient body weight, from about 300 nmoles / kg of patient body weight to about 500 nmoles / kg of patient body weight, or from about 400 nmoles / kg of patient body weight to about 600 nmoles / kg of patient body weight. In these embodiments, "kg" is the number of kilograms of the patient's body weight.

[0259] In all of the above dosage embodiments, the percentage of the "full dose" of the compound or its pharmaceutically acceptable salt administered at any step in the ascending order of dosage may be about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 200%, about 300%, about 400%, or about 500% of the "full dose" of the compound or its pharmaceutically acceptable salt. The "full dose" of the compound or its pharmaceutically acceptable salt may be any one of the doses of the compound or its pharmaceutically acceptable salt described in the preceding paragraphs as being administered in ascending order of dosage.

[0260] In various other embodiments, the dose of the compound or its pharmaceutically acceptable salt can be in the range of, for example, about 1 nmoles / kg to about 10,000 nmoles / kg, about 1 nmoles / kg to about 5,000 nmoles / kg, about 1 nmoles / kg to about 3,000 nmoles / kg, about 1 nmoles / kg to about 2,500 nmoles / kg, about 1 nmoles / kg to about 2,000 nmoles / kg, about 1 nmoles / kg to about 1,000 nmoles / kg, about 1 nmoles / kg to about 900 nmoles / kg, about 1 nmoles / kg to about 800 nmoles / kg, about 1 nmoles / kg to about 700 nmoles / kg, about 1 nmoles / kg to about 600 nmoles / kg, about 1 nmoles / kg to about 500 nmoles / kg, about 1 nmoles / kg to about 400 nmoles / kg, about 1 nmoles / kg to about 300 nmoles / kg, about 1 nmoles / kg to about 200 nmoles / kg, about 1 nmoles / kg to about 150 nmoles / kg, about 1 nmoles / kg to about 100 nmoles / kg, about 1 nmoles / kg to about 90 nmoles / kg, about 1 nmoles / kg to about 80 nmoles / kg, about 1 nmoles / kg to about 70 nmoles / kg, about 1 nmoles / kg to about 60 nmoles / kg, about 1 nmoles / kg to about 50 nmoles / kg, about 1 nmoles / kg to about 40 nmoles / kg, about 1 nmoles / kg to about 30 nmoles / kg, or about 1 nmoles / kg to about 20 nmoles / kg. In these embodiments, "kg" is the number of kilograms of the patient's body weight.

[0261] In another embodiment, a compound or its pharmaceutically acceptable salt can be administered to a patient at about 20 μg / kg of the patient's body weight to about 3 mg / kg of the patient's body weight. On the other hand, the amount can be about 0.2 mg / kg of the patient's body weight to about 0.4 mg / kg of the patient's body weight.

[0262] In any of the above dose embodiments, a single dose or multiple doses of the compound or its pharmaceutically acceptable salt can be administered to a patient.

[0263] In one embodiment, the small molecule ligand linked to the targeting moiety can be administered to the patient prior to the CAR T cell composition. In another embodiment, the small molecule ligand linked to the targeting moiety can be administered to the patient simultaneously but in a different formulation or in the same formulation as the CAR T cell composition. In yet another embodiment, the small molecule ligand linked to the targeting moiety can be administered to the patient after the CAR T cell composition.

[0264] In an illustrative aspect, the time between administration of the CAR T cells and the small molecule linked to the targeting moiety can vary widely depending on factors including the type of CAR T cells used, the binding specificity of the CAR, the characteristics of the targeting moiety and the small molecule ligand, the characteristics of the cancer, the location of the cancer in the patient, the method used to administer the CAR T cells and the small molecule ligand linked to the targeting moiety to the patient, and the health, age, and weight of the patient. In one aspect, the small molecule ligand linked to the targeting moiety can be administered before or after the CAR T cells, for example, within about 3 hours, 6 hours, 9 hours, 12 hours, 15 hours, 18 hours, 21 hours, 24 hours, 27 hours, 30 hours, 33 hours, 36 hours, 39 hours, 42 hours, 45 hours, 48 hours, or 51 hours, or within about 0.5 days, 1 day, 1.5 days, 2 days, 2.5 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or more days.

[0265] In one embodiment, any suitable dosing schedule known in the art can be used for the administration of the compound or its pharmaceutically acceptable salt or the CAR T cell composition. In one aspect, the dosing schedule selected for the compound or its pharmaceutically acceptable salt and the CAR T cell composition can take into account the concentration of the compound or its pharmaceutically acceptable salt and the number of CAR T cells administered to modulate the cytotoxicity of the CAR T cell composition and control CRS.

[0266] In an exemplary embodiment, a first dose escalation sequence, a second dose escalation sequence, a third dose escalation sequence, a fourth dose escalation sequence, a fifth dose escalation sequence, a sixth dose escalation sequence, or any additional dose escalation sequence can be followed by a period of time during which the compound or its pharmaceutically acceptable salt is not administered. In various illustrative embodiments, the period of time during which the compound or its pharmaceutically acceptable salt is not administered can be 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days. In another embodiment, the period of time during which the compound or its pharmaceutically acceptable salt is not administered can be 6 days, 7 days, or 8 days. In yet another embodiment, the period of time during which the compound or its pharmaceutically acceptable salt is not administered can be 7 days.

[0267] In one aspect, a first dose of CAR T cells and a second dose of CAR T cells are administered to a patient during week 1, for example, on Monday and Thursday. In this embodiment, the first dose escalation of the compound or its pharmaceutically acceptable salt can then be carried out during weeks 2 and 3. For example, the compound or its pharmaceutically acceptable salt can be administered over three separate days, and the three separate days can be Monday and Thursday of week 2 and Monday of week 3 (see Figure 1 for sequence 1). In this embodiment, the second dose escalation of the compound or its pharmaceutically acceptable salt can then be carried out during weeks 4 and 5 (see Figure 1 for sequence 2). For example, the compound or its pharmaceutically acceptable salt can be administered over three separate days, and the three separate days can be Monday and Thursday of week 4 and Monday of week 5. In this embodiment, the third dose escalation of the compound or its pharmaceutically acceptable salt can then be carried out during weeks 6 and 7 (see Figure 1 for sequence 3). For example, the compound or its pharmaceutically acceptable salt can be administered over three separate days, and the three separate days can be Monday and Thursday of week 6 and Monday of week 7. In other embodiments, subsequent dose escalations can follow a similar sequence, or sequence 3 can be repeated approximately 7 days after the end of sequence 3 as "procedure 2", and no additional treatment with the compound or its pharmaceutically acceptable salt is carried out until "procedure 3" begins (the length of "procedure 2" is based on Figure 1 the time length shown for "procedure 1"). In one embodiment, a patient can receive one, two, three, or four treatment procedures.

[0268] In another illustrative embodiment, a method of treating or inhibiting cancer is provided. The method comprises i) administering to a patient at least one dose of a CAR T cell composition comprising CAR T cells, wherein the CAR T cells comprise a CAR directed to a target site, ii) administering to the patient a compound or a pharmaceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked to the target site by a linker, and wherein the compound or the pharmaceutically acceptable salt thereof is administered in a first dose escalation sequence, wherein if severe CRS occurs in the first dose escalation sequence, a lower dose escalation sequence is used to administer the compound or the pharmaceutically acceptable salt thereof, wherein the first dose of the compound or the pharmaceutically acceptable salt thereof in the lower dose escalation sequence is lower than the first dose of the compound or the pharmaceutically acceptable salt thereof administered in the first dose escalation sequence. In this embodiment, in the lower dose escalation sequence, the compound or the pharmaceutically acceptable salt thereof is administered at about 0.5%, about 5% and about 50% of the full dose of the compound or the pharmaceutically acceptable salt thereof over separate three days. In this embodiment, the full dose of the compound or the pharmaceutically acceptable salt thereof can be about 10 μg / kg to about 50 μg / kg, about 20 μg / kg to about 40 μg / kg, about 25 μg / kg to about 35 μg / kg or about 30 μg / kg. In these embodiments, "kg" is the number of kilograms of the patient's body weight.

[0269] In various related embodiments, prior to administering the CAR T cell composition to the patient, lymphocytes in the patient can be depleted using, for example, cyclophosphamide (cytoxan), fludarabine, and / or etopside, and the method can further comprise additional steps including, but not limited to, administering platelets to the patient, administering packed red blood cells to the patient, administering cryoprecipitate to the patient, administering intravenous immunoglobulin to the patient, treating with calcium supplements, acid citrate dextrose, and / or heparin, and / or providing antimicrobial therapy to the patient. In one embodiment, lymphocyte depletion occurs at least about 24 hours prior to CAR T cell administration.

[0270] In another aspect, the method may include a CRS monitoring step. In an illustrative aspect, if no CRS or neurotoxicity is observed in a patient during a first dose escalation sequence, the method may proceed to a second dose escalation sequence. If no CRS or neurotoxicity is observed in the patient during the second dose escalation sequence, the method may proceed to a third dose escalation sequence. If no CRS or neurotoxicity is observed during the third dose escalation sequence, the method may proceed to a fourth dose escalation sequence. If no CRS or neurotoxicity is observed in the patient during the fourth dose escalation sequence, the method may proceed to a fifth dose escalation sequence. If no CRS or neurotoxicity is observed in the patient during the fifth dose escalation sequence, the method may proceed to a sixth dose escalation sequence, and so on.

[0271] In another illustrative embodiment, if fever but no hypotension (i.e., non-severe CRS) is observed in a patient during any of the dose escalation sequences and no neurotoxicity is observed in the patient, all subsequent doses of the compound or its pharmaceutically acceptable salt may be administered to the patient at the dose escalation sequence level that results in fever but no hypotension. In another aspect, if CRS (i.e., severe CRS) or neurotoxicity occurs in a patient at any dose escalation sequence, all subsequent doses of the compound or its pharmaceutically acceptable salt may be administered to the patient at a dose escalation sequence level lower than the dose escalation sequence level that resulted in severe CRS or neurotoxicity in the patient.

[0272] In one aspect, severe CRS may include any toxicity requiring the use of cetuximab, any ≥ grade 3 autoimmune toxicity, any ≥ grade 3 toxicity attributable to the administration of CAR T cells or the compound or its pharmaceutically acceptable salt and occurring within 28 days after the start of treatment, other than ≤ grade 4 fever lasting less than 48 hours after the start of treatment, ≤ grade 3 rigors lasting less than 24 hours after the start of treatment, ≤ grade 3 cough lasting less than 24 hours after the start of treatment, ≤ grade 3 transaminases lasting less than 7 days after the start of treatment, ≤ grade 3 hypotension lasting less than 48 hours after the start of treatment, < grade 3 CRS lasting less than 48 hours after the start of treatment, ≤ grade 3 allergic reactions related to DMSO resolvable by diphenhydramine (Benadryl) and / or epinephrine, or ≤ grade 3 pain controlled with oral or IV anesthetics, or additionally toxicity (including ≤ grade 3 rigors lasting up to 5 days, ≤ grade 3 transaminases lasting up to 2 weeks, ≤ grade 3 CRS lasting up to 2 weeks, ≤ grade 4 lymphopenia, ≤ grade 4 leukopenia, or ≤ grade 3 pain lasting up to 2 weeks controlled with oral or IV anesthetics) occurring approximately 3 weeks after CART cell infusion.

[0273] In one embodiment, to prevent or inhibit CRS in a patient, the method may further comprise administering to the patient a folic acid compound, a conjugate comprising a folic acid compound (wherein the conjugate comprising a folic acid compound does not comprise a targeting moiety), or a drug that inhibits CAR T cell activation. In this embodiment, any folic acid compound, conjugate comprising a folic acid compound (wherein the conjugate comprising a folic acid compound does not comprise a targeting moiety), or drug that inhibits CAR T cell activation may be referred to herein as a "rescue agent". In one embodiment, a folic acid compound such as folic acid may be administered to prevent or inhibit CRS in a patient. In this embodiment, the folic acid compound inhibits the interaction of the bridge (i.e., a small molecule ligand linked to a targeting moiety via a linker) with the receptor for the bridge on the tumor, thereby inhibiting tumor lysis and preventing or inhibiting CRS in the patient.

[0274] In one embodiment, the folic acid compound administered as an inhibitor of bridge binding to the tumor may be, for example, folic acid, a folic acid analog, or another folate receptor binding molecule. In various embodiments, folate analogs that may be used include leucovorin, pteroylpolyglutamic acids, and folate receptor binding pteridines such as tetrahydropteridines, dihydrofolates, tetrahydrofolates, or their deaza and diaza analogs. The terms "deaza" and "diaza" analogs refer to analogs known in the art in which one or two nitrogen atoms in the naturally occurring folate structure are replaced by carbon atoms. For example, deaza analogs include 1-deaza, 3-deaza, 5-deaza, 8-deaza, and 10-deaza analogs. Diaza analogs include, for example, 1,5-dideaza, 5,10-dideaza, 8,10-dideaza, or 5,8-dideaza analogs. The above folic acid analogs are generally referred to as "folic acid compounds" to reflect their ability to bind to folate receptors. Other folate receptor binding analogs include aminopterin, methotrexate, N10-methylfolate, 2-deamino-hydroxyfolate, deaza analogs (such as 1-deazamethotrexate or 3-deazamethotrexate), and 3',5'-dichloro-4-amino-4-deoxy-N10-methylpteroylglutamic acid (dichloromethotrexate)

[0275] In another embodiment, the folic acid compound administered as an inhibitor of bridge binding to the tumor has the following formula: wherein X 1 and Y 1 are each independently selected from the group consisting of halogen, R 2 、OR 2 、SR 3 and NR 4 R 5 ; U, V, and W each independently represent a divalent moiety selected from the group consisting of: -(R 6a )C═, -N═, -(R 6a )C(R 7a )-, and -N(R 4a )-; Q is selected from the group consisting of C and CH; T is selected from the group consisting of S, O, N, and -C═C-; X 2 and X 3 each independently selected from the group consisting of oxygen, sulfur, -C(Z)-, -C(Z)O-, -OC(Z)-, -N(R 4b )-, -C(Z)N(R 4b )-, -N(R 4b )C(Z)-, -OC(Z)N(R 4b )-, -N(R 4b )C(Z)O-, -N(R 4b )C(Z)N(R 5b )-, -S(O)-, -S(O)2-, -N(R 4a )S(O)2-, -C(R 6b )(R 7b )-, -N(C≡CH)-, -N(CH2C≡CH)-, C1-C 12 alkylene, and C1-C 12 alkoxy groups, where Z is oxygen or sulfur; R 1 is selected from the group consisting of hydrogen, halogen, C1-C 12 alkyl, and C1-C 12 alkoxy groups; R 2 , R 3 , R 4 , R 4a , R 4b , R 5 , R 5b , R 6b and R 7b each independently selected from the group consisting of hydrogen, halogen, C1-C 12 alkyl, C1-C 12 alkoxy, C1-C 12 alkanoyl, C1-C 12 alkenyl, C1-C 12 alkynyl, (C1-C 12 alkoxy)carbonyl, and (C1-C 12 alkylamino)carbonyl groups; R 6 and R 7 each independently selected from the group consisting of hydrogen, halogen, C1-C12 alkyl and C1-C 12 alkoxy group; or, R 6 and R 7 together form a carbonyl group; R 6a and R 7a are each independently selected from the group consisting of hydrogen, halogen, C1-C 12 alkyl and C1-C 12 alkoxy group; or, R 6a and R 7a together form a carbonyl group; p, r, s and t are each independently 0 or 1; and if any additional chemical moiety is part of the folate compound, * represents an optional covalent bond to the remainder of the conjugate.

[0276] In yet another embodiment, a conjugate comprising a folate compound can be administered to prevent or inhibit cytokine release syndrome (CRS) in a patient. CRS can have deleterious effects on the patient, including but not limited to, weight loss, high fever, pulmonary edema, and a dangerous drop in blood pressure.

[0277] In this embodiment, the conjugate comprising a folate compound does not comprise a targeting moiety, and thus, the conjugate inhibits the interaction of the bridge with the tumor to prevent tumor lysis and reduce CRS in the patient. In this embodiment, the folate compound moiety in the conjugate comprising a folate compound can comprise any of the folate compounds described in the foregoing paragraphs attached to a chemical moiety that does not comprise a targeting moiety. In one aspect, the conjugate comprising a folate compound can comprise a folate compound attached to more than one amino acid that does not comprise a targeting moiety. Illustratively, the conjugate comprising a folate compound can have the following formula:

[0278] This compound can also be referred to as "EC923". In these embodiments, the folate compound or the conjugate comprising a folate compound can be administered to the patient in molar excess relative to the bridge (i.e., the small molecule ligand attached to the targeting moiety via a linker), e.g., the folate compound or the conjugate comprising a folate compound is in 10-fold excess, 100-fold excess, 200-fold excess, 300-fold excess, 400-fold excess, 500-fold excess, 600-fold excess, 700-fold excess, 800-fold excess, 900-fold excess, 1000-fold excess, or 10,000-fold excess relative to the small molecule ligand attached to the targeting moiety via a linker. The amount of the folate compound or the conjugate comprising a folate compound required to inhibit the interaction of the bridge with the tumor can be determined by one skilled in the art relative to the amount of the small molecule ligand attached to the targeting moiety via a linker.

[0279] In another embodiment, a reagent that inhibits CAR T cell activation can be administered to a patient to inhibit CAR T cell activation and inhibit or prevent CRS in the patient. In one aspect, the reagent can be selected from the group consisting of: lymphocyte-specific protein tyrosine kinase inhibitors (such as dasatinib), PI3 kinase inhibitors (such as GDC0980), tocilizumab, inhibitors of IL-2-induced T cell kinase (such as BMS-509744), JAK inhibitors, BTK inhibitors, SIP agonists (such as siponimod and ozanimod), and reagents that block CAR T cell binding to the bridge but do not bind to cancer (such as fluorescamine, FITC, or sodium fluorescein). Those skilled in the art understand that under physiological conditions or, for example, in a buffer at physiological pH, FITC (i.e., fluorescein) can be in the form of a salt (such as sodium fluorescein) or in its non-salt form. Thus, in one embodiment, when fluorescein is administered to a patient, it can be in equilibrium between its salt form (such as sodium fluorescein) and its non-salt form. In another embodiment, the rescue reagent that inhibits CAR T cell activation can be a compound of the following formula:

[0280] This compound can also be referred to as "EC2319".

[0281] In various embodiments, the rescue reagent may be administered at a concentration of from about 0.001 nM to about 100 mM, from about 0.01 nM to about 100 mM, from about 1 nM to about 100 mM, from about 10 nM to about 100 mM, from about 50 nM to about 100 mM, or from about 100 nM to about 100 mM in any suitable volume, including for example 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, 0.5 mL, 0.6 mL, 0.7 mL, 0.8 mL, 0.9 mL, 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 10 mL, 100 mL or 1000 mL. In other embodiments, the rescue reagent may be administered in a dose of from about 0.01 to about 300 umoles / kg patient body weight, from about 0.06 to about 100 umoles / kg patient body weight, from about 0.06 to about 90 umoles / kg patient body weight, from about 0.06 to about 80 umoles / kg patient body weight, from about 0.06 to about 70 umoles / kg patient body weight, from about 0.06 to about 60 umoles / kg patient body weight, from about 0.06 to about 50 umoles / kg patient body weight, from about 0.06 to about 40 umoles / kg patient body weight, from about 0.06 to about 30 umoles / kg patient body weight, from about 0.06 to about 20 umoles / kg patient body weight, from about 0.06 to about 10 umoles / kg patient body weight, from about 0.06 to about 8 umoles / kg patient body weight, or from about 0.06 to about 6 umoles / kg patient body weight.

[0282] In these embodiments, a molar excess of the rescue reagent relative to the compound or its pharmaceutically acceptable salt (i.e., the small molecule ligand linked to the targeting moiety via a linker) may be administered to the patient, for example, the rescue reagent is about 10-fold in excess, about 20-fold in excess, about 30-fold in excess, about 40-fold in excess, about 50-fold in excess, about 60-fold in excess, about 70-fold in excess, about 80-fold in excess, about 90-fold in excess, about 100-fold in excess, about 200-fold in excess, about 300-fold in excess, about 400-fold in excess, about 500-fold in excess, about 600-fold in excess, about 700-fold in excess, about 800-fold in excess, about 900-fold in excess, about 1000-fold in excess or about 10,000-fold in excess relative to the small molecule ligand linked to the targeting moiety via a linker. The amount of the rescue reagent required to inhibit the interaction of the compound or its pharmaceutically acceptable salt with the tumor and / or CAR T cells can be determined by one of ordinary skill in the art relative to the amount of the small molecule ligand linked to the targeting moiety via a linker.

[0283] In another embodiment, more than one dose of the folic acid compound, the conjugate comprising a folic acid compound (wherein the conjugate comprising a folic acid compound does not comprise a targeting moiety), or a reagent that inhibits CAR T cell activation may be administered to the patient.

[0284] In the "rescue reagent" embodiments described herein, the folic acid compound, the conjugate comprising a folic acid compound (wherein the conjugate comprising a folic acid compound does not comprise a targeting moiety), or a reagent that inhibits CAR T cell activation can be administered to a patient before and / or after the compound or a pharmaceutically acceptable salt thereof. In another aspect, the compound or a pharmaceutically acceptable salt thereof can be administered before and after administering the folic acid compound, the conjugate comprising a folic acid compound (wherein the conjugate comprising a folic acid compound does not comprise a targeting moiety), or a reagent that inhibits CAR T cell activation. In this embodiment, subsequent administration of the compound or a pharmaceutically acceptable salt thereof can result in increased CAR T cell activation and cytokine levels in the patient.

[0285] In another embodiment, administering the folic acid compound, the conjugate comprising a folic acid compound (wherein the conjugate comprising a folic acid compound does not comprise a targeting moiety), or a reagent that inhibits CAR T cell activation can result in a decrease in cytokine levels in the patient. In another embodiment, the decrease in cytokine levels is to the cytokine levels in an untreated patient. In other embodiments, a reagent that inhibits CAR T cell activation is administered to a patient when the CRS level reaches 1, 2, 3, or 4, or when the CRS level reaches 3 or 4.

[0286] In any of the embodiments in which the folate compound described herein is a ligand linked to a targeting moiety via a linker, the patient may be placed on a folate-depleted diet prior to treatment with the methods described herein, or the folate compound may be administered to the patient in the diet. In embodiments where the folate compound is administered to the patient, the dose may be in the range of, for example, from about 50 nmol / kg patient body weight to about 3000 nmol / kg patient body weight, from about 50 nmol / kg patient body weight to about 2000 nmol / kg patient body weight, from about 50 nmol / kg patient body weight to about 1000 nmol / kg patient body weight, from about 50 nmol / kg patient body weight to about 900 nmol / kg patient body weight, from about 50 nmol / kg patient body weight to about 800 nmol / kg patient body weight, from about 50 nmol / kg patient body weight to about 700 nmol / kg patient body weight, from about 50 nmol / kg patient body weight to about 600 nmol / kg patient body weight, from about 50 nmol / kg patient body weight to about 500 nmol / kg patient body weight, from about 50 nmol / kg patient body weight to about 400 nmol / kg patient body weight, from about 50 nmol / kg patient body weight to about 300 nmol / kg patient body weight, from about 50 nmol / kg patient body weight to about 200 nmol / kg patient body weight, from about 50 nmol / kg patient body weight to about 100 nmol / kg patient body weight, from about 100 nmol / kg patient body weight to about 300 nmol / kg patient body weight, from about 100 nmol / kg patient body weight to about 500 nmol / kg patient body weight, from about 100 nmol / kg patient body weight to about 1000 nmol / kg patient body weight, from about 100 nmol / kg patient body weight to about 2000 nmol / kg patient body weight. In other embodiments, the dose may be about 100 nmol / kg patient body weight, about 150 nmol / kg patient body weight, about 200 nmol / kg patient body weight, about 250 nmol / kg patient body weight, about 300 nmol / kg patient body weight, about 350 nmol / kg patient body weight, about 400 nmol / kg patient body weight, about 450 nmol / kg patient body weight, about 500 nmol / kg patient body weight, about 600 nmol / kg patient body weight, about 700 nmol / kg patient body weight, about 800 nmol / kg patient body weight, about 900 nmol / kg patient body weight, about 1000 nmol / kg patient body weight, about 2000 nmol / kg patient body weight or about 3000 nmol / kg patient body weight. In these embodiments, "kg" is the number of kilograms of the patient's body weight. In one aspect, the folate compound may be administered, for example, daily, weekly, biweekly, three times a week or using any suitable regimen for the administration of the folate compound.

[0287] In various embodiments described herein, the CAR T cells can maintain a high number of circulating CAR T cells for up to about 10 days, up to about 15 days, up to about 20 days, up to about 25 days, up to about 30 days, up to about 35 days, up to about 40 days, up to about 45 days, up to about 50 days, up to about 55 days, up to about 60 days, up to about 65 days, up to about 70 days, up to about 75 days, or up to about 80 days after CAR T cell administration.

[0288] In various embodiments described herein, the half maximal effective concentration (EC50) of the compound or its pharmaceutically acceptable salt can be from about 1 pM to about 2 nM, about 1 pM to about 5 nM, about 1 pM to about 10 nM, about 1 pM to about 20 nM, about 1 pM to about 30 nM, about 1 pM to about 40 nM, about 1 pM to about 50 nM, about 1 pM to about 60 nM, about 1 pM to about 70 nM, about 1 pM to about 80 nM, about 1 pM to about 90 nM, about 1 pM to about 100 nM, about 1 pM to about 200 nM, about 1 pM to about 300 nM, about 1 pM to about 400 nM, about 1 pM to about 500 nM, about 1 pM to about 600 nM, about 1 pM to about 700 nM, about 1 pM to about 800 nM, about 1 pM to about 900 nM, about 1 pM to about 1 nM, about 1 pM to about 900 pM, about 1 pM to about 800 pM, about 1 pM to about 700 pM, about 1 pM to about 600 pM, about 1 pM to about 500 pM, about 1 pM to about 400 pM, about 1 pM to about 300 pM, about 1 pM to about 200 pM, about 1 pM to about 100 pM, about 1 pM to about 90 pM, about 1 pM to about 80 pM, about 1 pM to about 70 pM, about 1 pM to about 60 pM, about 1 pM to about 50 pM, about 1 pM to about 40 pM, about 1 pM to about 30 pM, about 1 pM to about 20 pM, about 1 pM to about 10 pM, or about 1 pM to about 5 pM.

[0289] In various embodiments described herein, the Kd of the binding of the compound or its pharmaceutically acceptable salt to CAR T cells can be from about 1 nM to about 100 nM, about 1 nM to about 200 nM, about 1 nM to about 300 nM, about 1 nM to about 400 nM, about 1 nM to about 500 nM, about 1 nM to about 600 nM, about 1 nM to about 700 nM, about 1 nM to about 800 nM, about 1 nM to about 900 nM, from about 100 nM to about 500 nM, from about 100 nM to about 400 nM, from about 100 nM to about 300 nM, from about 100 nM to about 200 nM, from about 100 nM to about 150 nM, or about 130 nM.

[0290] In various embodiments described herein, CAR T cells that are EGFRt sorted or unsorted can be used. In another embodiment, “clinical replicate” batches of CAR T cells with low differentiation characteristics can be used. In another embodiment, “research batches” of CAR T cells can be used. The “clinical replicate” batches (~39% EGFRt+) can include CD4+ subsets that are approximately 66% TSCM and approximately 32% TSCM and CD8 subsets that are approximately 95% TSCM and approximately 3% TCM. The research batches (~23% EGFRt+) can include CD4 subsets that are approximately 32% TSCM, approximately 53% TCM, approximately 11% TEM, and approximately 3.7% TEFF and CD8 subsets that are approximately 44% TSCM, approximately 0.28% TCM, approximately 3.4% TEM, or approximately 52% TEFF.

[0291] In various illustrative embodiments described herein, the compound or a pharmaceutically acceptable salt thereof can be administered to a patient for the first time about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days before or after the CAR T cells or at any suitable time before or after the CAR T cells.

[0292] In one embodiment of the methods described herein, the cancer is imaged before administering the compound or a pharmaceutically acceptable salt thereof to the patient or before administering the CAR T cell composition to the patient. In one illustrative embodiment, the imaging is performed by PET imaging. In other illustrative embodiments, the imaging is performed by MRI imaging or SPECT / CT imaging. The imaging method can be any suitable imaging method known in the art. In one embodiment, the imaging method can involve the administration of a small molecule ligand described herein, but conjugated to an imaging reagent suitable for the type of imaging described herein, to determine whether the patient is positive for folate receptor expression. In another embodiment, immunohistochemical analysis can be used for this purpose.

[0293] In any of the embodiments described herein, even when CAR T cell toxicity against cancer occurs, cytokine release that causes off-target toxicity in the patient may not occur. In any of the embodiments described herein, even when CAR T cell toxicity against cancer occurs, off-target tissue toxicity may not occur in the patient. In any of the embodiments described herein, the cancer may include a tumor, and even when off-target toxicity does not occur, the tumor size in the patient may still decrease. In any of the embodiments described herein, CRS may be reduced or prevented, and the method may cause a decrease in tumor volume in the patient. In any of the embodiments described herein, weight loss and CAR T cell exhaustion caused by CRS may be reduced or prevented. In any of the embodiments described herein, the cancer may include a tumor, and a complete response to the tumor may be achieved. Certain Therapeutic Methods and Compositions

[0294] Some embodiments of the methods and compositions provided herein include methods of treating, ameliorating, or inhibiting osteosarcoma in a human subject. Some such methods include administering to the subject a fluorescein isothiocyanate (FITC)-folate conjugate or a pharmaceutically acceptable salt thereof in a dosing regimen comprising: a dosing escalation period to confirm the maximum tolerated dose (MTD) of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof, wherein the dosing escalation period includes administering: (a) a first dose of about 0.5% to about 5% of the full dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof, (b) a second dose of about 5% to about 50% of the full dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof, (c) a third dose of about 50% to about 500% of the full dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof, wherein the MTD is confirmed after the third dose, and (d) a fourth dose of the MTD of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof, wherein the full dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof is from about 1x10 -2 mg / kg to about 5x10 -2 mg / kg; wherein the human subject has received or is receiving a chimeric antigen receptor (CAR) T cell composition comprising a population of T cells expressing an anti-fluorescein CAR to treat osteosarcoma in the human subject.

[0295] Some embodiments of the methods and compositions provided herein include methods of treating, ameliorating, or inhibiting osteosarcoma in a human subject. Some such methods include administering to the subject a fluorescein isothiocyanate (FITC)-folate conjugate or a pharmaceutically acceptable salt thereof according to a dosing regimen comprising: (i) an escalating dosing period to confirm the maximum tolerated dose (MTD) of the FITC-folate conjugate, wherein the escalating dosing period includes administering: (a) a first dose of about 0.5% to about 5% of the full dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof, (b) a second dose of about 5% to about 50% of the full dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof, and (c) a third dose of about 50% to about 500% of the full dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof, wherein the full dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof is from about 1x10 -2 mg / kg to about 5x10 -2 mg / kg; and (ii) a maintenance dosing period that includes administering the MTD of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof at dosing intervals of about once every 5 - 10 days for a period of time; wherein the human subject has received or is receiving a CAR T cell composition comprising a population of T cells expressing an anti-fluorescein CAR, thereby treating, ameliorating, or inhibiting osteosarcoma in the human subject.

[0296] In some embodiments, the osteosarcoma is recurrent. In some embodiments, the osteosarcoma is refractory.

[0297] In some embodiments, the time period between administering the first dose and administering the second dose is between about 1 day and about 7 days. In some embodiments, the time period between administering the first dose and administering the second dose is about 2 days.

[0298] In some embodiments, the time period between administering the second dose and administering the third dose is between about 1 day and about 7 days. In some embodiments, the time period between administering the second dose and administering the third dose is about 3 days.

[0299] In some embodiments, the full dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof is about 3.1x10 -2 mg / kg.

[0300] Some embodiments also include administering a fifth dose of the MTD of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof. Some embodiments also include a restaging phase after the fifth dose, wherein the restaging phase includes: evaluating the subject to determine whether more than one criteria for receiving at least one subsequent dosing cycle of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof are met, provided that the FITC-folate compound conjugate is not administered to the subject during the restaging phase.

[0301] In some embodiments, the subject meets more than one criteria, and wherein at least one subsequent dosing cycle is administered to the subject, the at least one subsequent dosing cycle comprising administering the MTD of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof at a dosing interval of about once every 5 - 10 days for a period of time.

[0302] In some embodiments, the anti-fluorescein CAR comprises a full-length humanized anti-fluorescein antibody or an antigen-binding fragment thereof. In some embodiments, the anti-fluorescein CAR comprises a full-length humanized anti-fluorescein scFv. In some embodiments, the anti-fluorescein CAR comprises a full-length humanized E2 anti-fluorescein scFv. In some embodiments, the CAR T cells express a cell surface selectable marker, the cell surface selectable marker comprising a truncated EGFR (EGFRt) polypeptide.

[0303] In some embodiments, the dose of the CAR T cell composition is about 1x10 5 cells / kg to about 1x10 7 cells / kg.

[0304] In some embodiments, the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof is administered intravenously.

[0305] Some embodiments of the methods and compositions provided herein include methods of treating, ameliorating, or inhibiting cancer in a human subject, comprising: (i) administering to the subject a first dose of a fluorescein isothiocyanate (FITC)-folate conjugate or a pharmaceutically acceptable salt thereof; (ii) administering to the subject a second dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof, wherein the second dose is higher than the first dose; (iii) administering to the subject a third dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof, wherein the third dose is higher than the second dose, wherein the maximum tolerated dose (MTD) of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof is confirmed after the third dose; and (iv) administering to the subject a fourth dose of the MTD of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof; wherein the human subject has received or is receiving a CAR T cell composition comprising a population of T cells expressing an anti-fluorescein CAR, thereby treating, ameliorating, or inhibiting cancer in the human subject.

[0306] Some embodiments of the methods and compositions provided herein include methods of treating, ameliorating, or inhibiting osteosarcoma in a human subject, comprising administering to the subject a combination of a fluorescein isothiocyanate (FITC)-folate conjugate or a pharmaceutically acceptable salt thereof and CAR T cell therapy, wherein the FITC-folate conjugate is administered to the subject in an escalating dosing regimen after or simultaneously with administering the CAR T cell therapy, thereby treating osteosarcoma in the human subject.

[0307] In some embodiments, the escalating dosing regimen comprises: an escalating dosing period to confirm the maximum tolerated dose (MTD) of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof, wherein the escalating dosing period comprises administering: (a) a first dose of from about 0.5% to about 5% of the full dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof, (b) a second dose of from about 5% to about 50% of the full dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof, (c) a third dose of from about 50% to about 500% of the full dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof, wherein the MTD is confirmed after the third dose, and (d) a fourth dose of the MTD of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof, wherein the full dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof is from about 1 x 10 -2 mg / kg to about 5 x 10-2 mg / kg;

[0308] In some embodiments, the escalating dosing regimen comprises: (i) a dosing escalation period to confirm the maximum tolerated dose (MTD) of the FITC-folate compound conjugate, wherein the dosing escalation period comprises administering: (a) a first dose of about 0.5% to about 5% of the full dose of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof, (b) a second dose of about 5% to about 50% of the full dose of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof, and (c) a third dose of about 50% to about 500% of the full dose of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof, wherein the full dose of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof is about 1x10 -2 mg / kg to about 5x10 -2 mg / kg; and (ii) a steady dosing period, the steady dosing period comprising administering the MTD of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof at a dosing interval of about every 5 - 10 days for a period of time.

[0309] In some embodiments, the CAR T cell therapy comprises administering to a subject a population of CAR T cells expressing an anti-fluorescein CAR.

[0310] Some embodiments of the methods and compositions provided herein include the use of a fluorescein isothiocyanate (FITC)-folate compound conjugate or a pharmaceutically acceptable salt thereof in combination with CAR T cell therapy for treating, ameliorating, or inhibiting osteosarcoma in a human subject, wherein the FITC-folate compound conjugate is administered to the subject in an escalating dosing regimen after or simultaneously with administering the CAR T cell therapy to the subject.

[0311] In some embodiments, the escalating dosing regimen includes a dosing escalation period to confirm the maximum tolerated dose (MTD) of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof, wherein the dosing escalation period includes administering: (a) a first dose of about 0.5% to about 5% of the full dose of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof, (b) a second dose of about 5% to about 50% of the full dose of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof, (c) a third dose of about 50% to about 500% of the full dose of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof, wherein the MTD is confirmed after the third dose, and (d) a fourth dose of the MTD of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof, wherein the full dose of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof is about 1x10 -2 mg / kg to about 5x10 -2 mg / kg.

[0312] In some embodiments, the escalating dosing regimen includes: (i) a dosing escalation period to confirm the maximum tolerated dose (MTD) of the FITC-folate compound conjugate, wherein the dosing escalation period includes administering: (a) a first dose of about 0.5% to about 5% of the full dose of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof, (b) a second dose of about 5% to about 50% of the full dose of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof, and (c) a third dose of about 50% to about 500% of the full dose of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof, wherein the full dose of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof is about 1x10 -2 mg / kg to about 5x10 -2 mg / kg; and (ii) a maintenance dosing period, the maintenance dosing period including administering the MTD of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof at a dosing interval of about once every 5 - 10 days for a period of time.

[0313] In some embodiments, the escalating dosing regimen comprises: (i) an escalating dosing period to confirm the maximum tolerated dose (MTD) of the FITC-folate conjugate, wherein the escalating dosing period comprises administering: (a) a first dose of about 0.5% or about 1% of the full dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof, (b) a second dose of about 5%, about 10%, about 30% or about 50% of the full dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof, and (c) a third dose of about 50%, about 100%, about 300% or about 500% of the full dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof, wherein the full dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof is about 1x10 -2 mg / kg to about 5x10 -2 mg / kg; and (ii) a maintenance dosing period that comprises administering the MTD of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof at a dosing interval of about every 5 - 10 days for a period of time. In these embodiments, the dosing regimen can be (1) a first dose of 0.5% of the full dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof, a second dose of 5% of the full dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof, and a third dose of 50% of the full dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof; (2) a first dose of 1% of the full dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof, a second dose of 10% of the full dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof, and a third dose of 100% of the full dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof; (3) a first dose of 1% of the full dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof, a second dose of 30% of the full dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof, and a third dose of 300% of the full dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof; or (4) a first dose of 1% of the full dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof, a second dose of 50% of the full dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof, and a third dose of 500% of the full dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof.

[0314] Some embodiments of the methods and compositions provided herein include the use of a fluorescein isothiocyanate (FITC)-folate compound conjugate or a pharmaceutically acceptable salt thereof in combination with CAR T cell therapy for the treatment, amelioration, or inhibition of cancer in a human subject, wherein the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof is administered by a method comprising: (i) administering a first dose of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof to the subject; (ii) administering a second dose of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof to the subject, wherein the second dose is higher than the first dose; (iii) administering a third dose of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof to the subject, wherein the third dose is higher than the second dose, wherein the maximum tolerated dose (MTD) of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof is confirmed after the third dose; and (iv) administering a fourth dose of the MTD of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof to the subject. In some embodiments, the cancer includes osteosarcoma. In some embodiments, the FITC-folate compound conjugate is administered to the subject in an escalating dosing regimen after or concurrently with the administration of CAR T cell therapy to the subject. In some embodiments, the CAR T cell therapy includes administering to the subject a population of CAR T cells that express an anti-fluorescein CAR. Certain Kits and Systems

[0315] Some embodiments of the methods and compositions provided herein include systems and kits that contain a container with a fluorescein isothiocyanate (FITC)-folate compound conjugate or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutical carrier, wherein the kit contains instructions for use in treating, ameliorating, or inhibiting osteosarcoma in a subject who has received or is receiving a CAR T cell composition comprising CAR T cells, wherein treating, ameliorating, or inhibiting comprises administering the FITC-folate compound conjugate in a dosing regimen that includes a dosing escalation period to confirm the maximum tolerated dose (MTD) of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof, wherein the dosing escalation period includes administering: (a) a first dose of about 0.5% to about 5% of the full dose of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof, (b) a second dose of about 5% to about 50% of the full dose of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof, (c) a third dose of about 50% to about 500% of the full dose of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof, wherein the MTD is confirmed after the third dose, and (d) a fourth dose of the MTD of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof, wherein the full dose of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof is from about 1x10 -2 mg / kg to about 5x10 -2 mg / kg, and wherein the CAR comprises an anti-fluorescein antibody or an antigen-binding fragment thereof. In some embodiments, the container is a vial.

[0316] Some embodiments of the methods and compositions provided herein include a system for treating, ameliorating, or inhibiting osteosarcoma in a subject in combination with CAR T cell therapy using a fluorescein isothiocyanate (FITC)-folate conjugate or a pharmaceutically acceptable salt thereof, wherein the FITC-folate conjugate is administered to the subject in an escalating dosing regimen after or simultaneously with the administration of CAR T cell therapy to the subject. In some embodiments, the system comprises: (a) a first dose of from about 0.5% to about 5% of the full dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof; (b) a second dose of from about 5% to about 50% of the full dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof; (c) a third dose of from about 50% to about 500% of the full dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof, wherein the maximum tolerated dose (MTD) of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof is confirmed after the third dose; and (d) a fourth dose of the MTD of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof. In some embodiments, the full dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof is from about 1x10 -2 mg / kg to about 5x10 -2 mg / kg. Some embodiments further include a population of CAR T cells expressing an anti-fluorescein CAR.

[0317] Certain polynucleotide and polypeptide sequences useful in the embodiments provided herein are listed in Table 1. Table 1 Examples Example 1 - Synthesis of FITC-Folic Acid

[0318] In the presence of tetramethylguanidine and diisopropylamine, folic acid-γ-ethylenediamine was coupled with fluorescein isothiocyanate (FITC) isomer I (Sigma-Aldrich) in anhydrous dimethyl sulfoxide (DMF). The crude product was loaded onto an Xterra RP18 preparative HPLC column (Waters) and eluted with the following gradient conditions: starting with 99% 5 mM sodium phosphate (mobile phase A, pH 7.4) and 1% acetonitrile (mobile phase B), reaching 90% A and 10% B in 10 min at a flow rate of 20 mL / min. Under these conditions, the FITC-folic acid main peak typically eluted at 27 - 50 min. The quality of the FITC-folic acid fractions was monitored by analytical reverse-phase HPLC and a UV detector. Fractions with a purity greater than 98.0% (LCMS) were lyophilized to obtain the final FITC-folic acid product. As known in the art, compounds with this structure are also referred to as EC17. Example 2 - Synthesis of FITC-PEG12-Folic Acid

[0319] Load the general-purpose polyethylene glycol (PEG) Nova Tag TM resin (0.2 g) into a peptide synthesis vessel and wash with isopropanol (i-PrOH) (3 x 10 mL) and dimethylformamide (DMF, 3 x 10 mL). Perform 9-fluorenylmethoxycarbonyl (Fmoc) deprotection using 20% piperidine in DMF (3 x 10 mF). Conduct a Kaiser test to evaluate the reaction progress. Subsequently, introduce a solution of N,N-diisopropylethylamine (i-Pr2NEt) (4 equivalents), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) (2 equivalents), and Fmoc-F-glutamic acid 5-tert-butyl ester (Fmoc-Glu-(O-t-Bu)-OH) (23.5 mg) in DMF into the vessel. Perform Fmoc deprotection using 20% piperidine in DMF (3 x 10 mF). Subsequently, introduce a solution of N 10 -TFA-Pte-OH (22.5 mg), DMF, i-Pr2NEt (4 equivalents), and PyBOP (2 equivalents). Bubble with argon for 2 h and wash the resin with DMF (3 x 3 mF) and i-PrOH (3 x 3 mF). After the resin swells in dichloromethane (DCM), add a solution of 1 M hydroxybenzotriazole (HOBT) in DCM / trifluoroethane (TFE) (1:1) (2 x 3 mF). Bubble with argon for 1 h, remove the solvent, and wash the resin with DMF (3 x 3 mF) and i-PrOH (3 x 3 mF). After the resin swells in DMF, add i-Pr2NEt (4 equivalents), PyBOP (2 equivalents), and Fmoc-NH-(PEG) 12A solution of -COOH (46.3 mg). The resin was bubbled with argon for 2 h and washed with DMF (3 x 3 mL) and i-PrOH (3 x 3 mL). Fmoc deprotection was carried out using 20% piperidine in DMF (3 x 10 mL). Kaiser test was performed to evaluate the reaction progress. Subsequently, a solution of z-Pr2NEt (4 equiv) and FITC (Fife Technologies 21.4 mg) in DMF was introduced into the vessel, followed by argon bubbling for 2 h, and the resin was washed with DMF (3 x 3 mL) and i-PrOH (3 x 3 mL). Subsequently, 2% NH2NH2 in DMF (2 x 2 mL) was added to the vessel. The final compound was cleaved from the resin using TFA / iPPO:triisopropylsilane (TIS) (95:2.5:2.5) (cleavage solution) and concentrated under vacuum. The concentrated product was precipitated in Et2O and dried under vacuum. The crude product was purified using preparative RP-HPLC (mobile phase: A = 10 mM ammonium acetate pH = 7, B = ACN; method: 0% B to 30% B in 30 min at 13 mL / min). The pure fractions were pooled and lyophilized to give FITC-PEG12-folic acid. Example 3 - Synthesis of FITC-PEG20-Folic Acid

[0320] Ethylenediamine, polymer-bound (200 - 400 mesh)-resin (50 mg) was charged into a peptide synthesis vessel and swollen with DCM (3 mL) and subsequently DMF (3 mL). Then a solution of Fmoc-PEG20-COOH (131 mg, 1.0 equivalent), i-Pr2NEt (6.0 equivalents), and PyBOP (4.0 equivalents) in DMF was introduced into the vessel. Argon was bubbled through for 6 h, the coupling solution was drained, and the resin was washed with DMF (3 x 10 mL) and i-PrOH (3 x 10 mL). A Kaiser test was performed to evaluate the progress of the reaction. Prior to each amino acid coupling, Fmoc deprotection was carried out using 20% piperidine in DMF (3 x 10 mL). The above sequence was repeated to complete the reaction with Fmoc-Glu-OtBu (72 mg, 2.0 equivalents) and the coupling step with Tfa. pteroic acid (41 mg, 1.2 equivalents). The resin was washed with 2% hydrazine in DMF 3 x 10 mL (5 min) to cleave the trifluoro-acetyl protecting group on pteroic acid and then washed successively with i-PrOH (3 x 10 mL) and DMF (3 x 10 mL). The resin was dried under argon for 30 min. The folic acid-peptide was cleaved from the resin using a cleavage solution. 10 mL of the cleavage mixture was introduced and argon was bubbled through for 1.5 h. The cleavage mixture was drained into a clean flask. The resin was washed 3 times with more cleavage mixture. The combined mixture was concentrated to a smaller volume (~5 mL) under reduced pressure and precipitated in diethyl ether.

[0321] The precipitate was collected by centrifugation, washed with diethyl ether (3 times), and dried under high vacuum. At room temperature, the dried folic acid-PEG 20 -EDA (1.0 equivalent) was treated with FITC (50 mg, 1.5 equivalents) and DIPEA in DMSO. The progress of the reaction was monitored by LCMS. After 8 h, the starting material was depleted and the product was obtained. The crude reaction mixture was purified by preparative HPLC (mobile phase A = 10 mM ammonium acetate, pH = 7; organic phase B = acetonitrile; method: 0% B to 30% B in 35 minutes at 13 mL / min) to give FITC-PEG20-folic acid in 60% yield. Example 4 - Synthesis of FITC-PEG108-Folic Acid

[0322] Ethylenediamine, polymer-bound (200 - 400 mesh)-resin (50 mg) was charged into a peptide synthesis vessel and swollen successively with DCM (3 mL) and DMF (3 mL). Then a solution of Fmoc-PEG 36-COOH solution (161 mg, 1.0 equivalent), i-Pr2NEt (6.0 equivalents), and PyBOP (4.0 equivalents). The coupling solution was bubbled with argon for 6 h, the coupling solution was drained, and the resin was washed with DMF (3 x 10 mL) and i-PrOH (3 x 10 mL). A Kaiser test was performed to evaluate the reaction progress. Before each amino acid coupling, Fmoc deprotection was carried out using 20% piperidine in DMF (3 x 10 mL). The above sequence was repeated to complete 2XFmoc-PEG 36 Reaction of -COOH (161 mg, 1.0 equivalent), Fmoc-Glu-OtBu (72 mg, 2.0 equivalents), and the coupling step of Tfa. pteroic acid (41.0 mg, 1.2 equivalents). The resin was washed with 2% hydrazine in DMF 3 x 10 mL (5 min) to cleave the trifluoro-acetyl protecting group on pteroic acid and then washed successively with i-PrOH (3 x 10 mL) and DMF (3 x 10 mL). The resin was dried under argon for 30 min. The folic acid-peptide was cleaved from the resin using a cleavage solution. 10 mL of the cleavage mixture was introduced and bubbled with argon for 1.5 h. The cleavage mixture was drained into a clean flask. The resin was washed 3X with more cleavage mixture. The combined mixture was concentrated to a smaller volume (~5 mL) under reduced pressure and precipitated in ether.

[0323] The precipitate was collected by centrifugation, washed with ether (3X), and dried under high vacuum. At room temperature, the dried folic acid-PEG108-EDA (1.0 equivalent) was treated with FITC (50 mg, 1.5 equivalents) and DIPEA in DMSO. The progress of the reaction was monitored by LCMS. After 10 h, the starting material was depleted and the product was obtained. The crude reaction mixture was purified by preparative HPLC (mobile phase A = 10 mM ammonium acetate, pH = 7; organic phase B = acetonitrile; method: 0% B to 30% B in 35 min at 13 mL / min) to give FITC-PEG108-folic acid in 64% yield. Example 5 - Synthesis of FITC-DUPA

[0324] DUPA-FITC was synthesized by the solid-phase method as follows. The general-purpose Nova Tag TMThe resin (50 mg, 0.53 mM) was swollen successively with dichloromethane (DCM, 3 mL) and dimethylformamide (DMF, 3 mL). A solution of 20% piperidine in DMF (3 x 3 mL) was added to the resin and bubbled with argon for 5 min. The resin was washed with DMF (3 x 3 mL) and isopropanol (i-PrOH, 3 x 3 mL). After the resin was swollen in DMF, a solution of DUPA-(OtBu)-OH (1.5 equiv), HATU (2.5 equiv) and z-Pr2NEt (4.0 equiv) in DMF was added. Argon was bubbled for 2 h and the resin was washed with DMF (3 x 3 mL) and i-PrOH (3 x 3 mL). After the resin was swollen in DCM, a 1 M HOBt solution in DCM / TFE (1:1) (2 x 3 mL) was added. Argon was bubbled for 1 h, the solvent was removed and the resin was washed with DMF (3 x 3 mL) and i-PrOH (3 x 3 mL). After the resin was swollen 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 for 2 h and the resin was washed with DMF (3 x 3 mL) and i-PrOH (3 x 3 mL). The above sequence was repeated for two additional coupling steps to add 8-aminocaprylic acid and fluorescein isothiocyanate or rhodamine B isothiocyanate. The final compound was cleaved from the resin using the cleavage solution and concentrated under vacuum. The concentrated product was precipitated in diethyl ether and dried under vacuum. The crude product was purified using preparative RP-HPLC [λ = 488 nm; solvent gradient: 1% B to 80% B in 25 min, 80% B wash for 30 min run; A = 10 mM NH4OAC, pH = 7; B = acetonitrile (ACN)]. The ACN was removed under vacuum and the purified fraction was lyophilized to yield FITC-DUPA as a light brown-orange solid. RP-HPLC: tR = 8.0 min (A = 10 mM NH4OAc, pH = 7.0; B = CAN, solvent gradient: 1% B to 50% B in 10 min, 80% B wash for 15 min run). 1H NMR (DMS0-d6 / D2O): δ 0.98 - 1.27 (ms, 9H); 1.45 (b, 3H); 1.68 - 1.85 (ms, 11H); 2.03 (m, 8H); 2.6 - 3.44 (ms, 12H); 3.82 (b, 2H); 4.35 (m, 1H); 6.53 (d, J = 8.1 Hz, 2H), 6.61 (dd, J = 5.3, 3.5 Hz, 2H); 6.64 (s, 2H); 7.05 (d, J = 8.2 Hz, 2H), 7.19 (m, 5H); 7.76 (d, J = 8.0 Hz, 1H); 8.38 (s, 1H).HRMS(ESI)(m / z): (M+H)+, calculated for C51H59N7O15S, 1040.3712, found 1040.3702. UV / vis: λmax = 491 nm. Example 6 - Synthesis of FITC-PEG12-DUPA

[0325] 1,2-Diaminoethane trityl-resin (0.025 g) was loaded into a peptide synthesis vessel and washed successively with i-PrOH (3 x 10 mL) and DMF (3 x 10 mL). Then, z-Pr2NEt (2.5 equiv), PyBOP (2.5 equiv), and a solution of Fmoc-NH-(PEG) 12 -COOH (42.8 mg) in DMF were introduced into the vessel. The resulting solution was bubbled with Ar for 1 h, the coupling solution was drained, and the resin was washed with DMF (3 x 10 mL) and i-PrOH (3 x 10 mL). Kaiser tests were performed to assess the progress of the reaction. Fmoc deprotection was carried out using 20% piperidine in DMF (3 x 10 mL). This procedure was repeated to complete all coupling steps (2 x 1.5 equiv Fmoc-Phe-OH and 1.5 equiv 8-aminooctanoic acid and 1.2 equiv DUPA were used for each of their respective coupling steps). After DUPA coupling, the resin was washed with DMF (3 x 10 mL) and i-PrOH (3 x 10 mL) and dried under reduced pressure. The peptide was cleaved from the resin in the peptide synthesis vessel using a cleavage solution. 15 mL of the cleavage solution was added to the peptide synthesis vessel and the reaction was bubbled with Ar for 15 min. The resin was treated with two additional 10 mL portions of the cleavage solution, 5 min each. The cleavage mixture was concentrated to approximately 5 mL and precipitated with ether. The precipitate was collected by centrifugation, washed with ether (3X), and dried under high vacuum to recover the crude material. To a stirred solution of crude DUPA-(PEG)12-EDA (10 mg) and FITC (5.6 mg) in dimethyl sulfoxide (DMSO, 1 mL) at room temperature, z-Pr2NEt (5 equiv) was added and the mixture was stirred under argon for 6 h. The reaction was monitored by LCMS and purified by preparative HPLC (mobile phase: A = 10 mM ammonium acetate, pH = 7, B = ACN; method: 0% B to 50% B in 30 min at 13 mL / min). The purified fractions were pooled and lyophilized to give FITC-PEG12-DUPA. Example 7 - Synthesis of FITC-PEG11-NK1

[0326] Under argon at room temperature, to a stirred solution of NK-1 (0.02 g, 0.0433 mmol, 1.0 equiv), O-(2-aminoethyl)-O'-[2-(Boc-amino)ethyl] decaethylene glycol (BocNH-PEG 11 -NH2) (Sigma, 0.0336 g, 0.0521 mmol, 1.2 equiv), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) (0.027 g, 0.0521 mmol, 1.2 equiv) in dry CH2Cl2 was added diisopropylethylamine (DIPEA) (0.076 mL, 0.4338 mmol, 10 equiv). The reaction progress was monitored by LCMS and purified by preparative RP-HPLC (Waters, XBridge TM Prep C18, 5 μm; 19×100 mm column, mobile phase A = 20 mM ammonium acetate buffer, pH 7, B = acetonitrile, gradient 10 - 100% B in 30 min, 13 mL / min, λ = 220 nm, 254 nm). The pure fractions were collected, all organic solvents were evaporated, and the sample was lyophilized for 48 h to give NK1-PEG 11 -NHBoc. Yield: 40.13 mg (97%). To NK1-PEG 11 -NHBoc (0.0165 g, 0.015 mmol) in dry DCM was added trifluoroacetic acid (TFA, 20 equiv), and the reaction mixture was stirred at room temperature for 4 h. The excess TFA was removed, and the remaining solution was diluted with water and extracted with CH2Cl2 (3x5 mL). The combined organic layers were washed with brine, dried (Na2SO4) and concentrated. The resulting residue was dried under vacuum and used in the next step without further purification. Under argon at room temperature, a stirred solution of NK1-PEG 11 -NH2 (0.008 g, 0.0081 mmol, 1.0 equiv), fluorescein isothiocyanate (FITC) (Sigma, 0.0037 g, 0.0097 mmol, 1.2 equiv) in dry dimethyl sulfoxide (DMSO, 0.3 mL) was added to diisopropylethylamine (0.0028 mL, 0.0162 mmol, 2.0 equiv).

[0327] The reaction progress was monitored by LCMS and the product was purified by preparative RP-HPLC (Waters, XBridge TMPurified by PrepC18, 5 μm; 19×100 mm column, 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). The pure fractions were collected, all organic solvents were evaporated, and the sample was lyophilized for 48 h to give FITC-PEG11-NK1 in a yield of 8.54 mg (77%).

[0328] The NK-1 compound was synthesized by a two-step procedure starting from a base ligand and prepared using the procedures in the literature (e.g., U.S. Pat. No. 11,141,494, incorporated herein by reference). Example 8 - Synthesis of FITC-CA9

[0329] In a 50 mL round-bottom flask, the CA9 ligand (53.6 mg, synthesized in the laboratory) was dissolved in the required amount of N,N-dimethylformamide (DMF) (2 - 3 mL) using a Teflon magnetic stir bar. The ambient air was removed using a vacuum and replaced with nitrogen, for three cycles. Subsequently, the round-bottom flask was maintained under a constant nitrogen atmosphere. 28.9 mg of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) was added to the flask, then 21.6 mg of 1-hydroxybenzotriazole hydrate (HOBt) and 18.9 μL of Boc-PEG2-NH2 (purchased from Sigma Aldrich). Finally, 5.4 μL of triethylamine (TEA) was added and the reaction was stirred overnight. The reaction mixture was purified by HPLC and verified by UHPLC-MS (target m / z 831). Acetonitrile was removed using a high-vacuum rotary evaporator and placed on a lyophilizer for 48 h. Boc deprotection was carried out with 1:1 TFA:DCM for 30 min. TFA / DCM was removed using a high-vacuum rotary evaporator, followed by 30 min under high vacuum. Subsequently, the compound was dissolved in DMF and combined with 5 molar equivalents of N,N-diisopropylethylamine (DIPEA). 16 mg of fluorescein isothiocyanate (purchased from Life Technologies) was added to the solution and stirred for 1 h. The reaction mixture was purified by HPLC and the target compound was confirmed by UHPLC-MS (target m / z 1120). The sample was placed on a lyophilizer for 48 h and the compound was stored at -20 °C. Example 9 - Synthesis of FITC-PEG2-CA9

[0330] In a 50 mL round-bottom flask, the CA9 ligand (53.6 mg) was dissolved in DMF (2 - 3 mL) using a Teflon magnetic stir bar. The ambient air was removed using a vacuum and replaced with nitrogen, and this was carried out for three cycles. The round-bottom flask was maintained under a constant nitrogen atmosphere. 28.9 mg of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) was added to the flask, followed by 21.6 mg of 1-hydroxybenzotriazole hydrate (HOBt) and 18.9 μL of Boc-PEG2-NH2 (Sigma Aldrich). 5.4 μL of triethylamine (TEA) was added and the reaction was stirred overnight. The reaction mixture was purified using HPLC and verified using UHPLC-MS (target m / z of 831). Acetonitrile was removed using a high-vacuum rotary evaporator and the product was lyophilized. The compound was mixed with 1:1 TFA:DCM for 30 minutes. TFA / DCM was removed using a high-vacuum rotary evaporator, followed by 30 minutes under high vacuum. Subsequently, the compound was dissolved in DMF and combined with 5 molar equivalents of z-Pr2Net, 16 mg of fluorescein isothiocyanate (Life Technologies), and stirred for 1 h. The reaction mixture was purified using HPLC and the target compound was confirmed using UHPLC-MS (target m / z of 1120). The sample was lyophilized and stored at -20 °C. Example 10 - T Cell Preparation

[0331] Human peripheral blood mononuclear cells (PBMCs) were isolated from the whole blood of healthy donors by using Ficoll density gradient centrifugation (GE Healthcare Lifesciences). Subsequently, by using EasySep TMThe human T cell isolation kit (STEM CELL technologies) was used to isolate T cells from PBMC. The T cells were cultured in TexMACS medium (Miltenyi Biotech Inc) with 40 - 100 IU / mL human IL-2 (Miltenyi Biotech), 2% human AB serum, and 1% penicillin / streptomycin sulfate. Dynabeads human T-activator CD3 / CD28 (ThermoFisher Scientific) was added to the T cells at a 1:1 ratio to activate the T cells. 12 - 24 hours after activation, in the presence of 8 μg / mL polybrene (Santa Cruiz Biotech), the T cells were transduced with FITC-CAR lentiviral particles by centrifugation at 1,200 g for 90 minutes at 22 - 32°C. The T cell mixture containing CAR-modified T cells (CAR-T) and non-CAR-modified T cells (untransduced T) was cultured for 6 days in the presence of activation beads, and then the activation beads were removed. Fluorescence-activated cell sorting was used to sort out CAR-T cells (GFP positive) and untransduced T cells (GFP negative) based on their GFP expression. The sorted T cells were cultured for 7 - 15 days before injection into mice. When using the T cell mixture, CAR-T cells were mixed with untransduced T cells at the desired ratio before mouse injection. Example 11 - Generation of Lentiviral Vectors Encoding CAR Genes

[0332] The overlapping PCR method was used to generate a CAR construct containing the scFv against fluorescein. The scFV against fluorescein derived from the anti-fluorescein (4-4-20) antibody, 4M5.3 (Kd = 270 fM, 762 bp) was synthesized. The sequences encoding the human CD8α signal peptide (SP, 63 bp), hinge, and transmembrane region (249 bp), the cytoplasmic domain of 4-1BB (CD137, 141 bp), and the CD3ζ chain (336 bp) were fused with the anti-fluorescein scFV by overlapping PCR (as Figure 2 shown). The resulting CAR construct (1551 bp) was inserted into the EcoRI / NotI digested lentiviral expression vector pCDH-EF1-MCS-(PGK-GFP) ( Figure 2, SystemBiosciences). The sequence of the CAR construct in the lentiviral vector was confirmed by DNA sequencing. Exemplary CAR nucleic acid coding sequences can include SEQ ID NO:01. In SEQ ID NO:01, the first ATG is the start codon. Exemplary CAR amino acid sequences can include SEQ ID NO:02. Exemplary CAR amino acid sequence inserts can include SEQ ID NO:03. In SEQ ID NO:03, the first GCCACC sequence can include a restriction enzyme cleavage site, followed by an ATG start codon. The encoded amino acid sequence can include SEQ ID NO:02. More example sequences of the CAR or parts thereof are listed in Table 1. Example 12 - Generation of Lentivirus Containing CAR Gene for Human T Cell Transduction

[0333] To prepare lentivirus containing an anti-fluorescein (i.e., anti-FITC) single-chain variable fragment (scFv) CAR, the HEK-293TN packaging cell line was co-transfected with a lentiviral vector encoding the anti-fluorescein scFv CAR and a second-generation lentiviral packaging plasmid mixture (Cellecta) or ViraPower TM Lentiviral packaging mixture (ThermoFisher). After 24 and 48 hours of transfection, the supernatant containing lentivirus with the CAR gene was harvested and the viral particles were concentrated by standard polyethylene glycol virus concentration method to concentrate the viral particles for future human T cell transduction. Example 13 - Isolation of Human T Cells from Human PBMC

[0334] T cells were isolated from human peripheral blood mononuclear cells (PBMC) by Ficoll density gradient centrifugation (GE Healthcare Lifesciences). After washing away the remaining Ficoll solution, T cells were isolated by using the EasySep TM Human T Cell Isolation Kit (STEMCELL technologies). The purified T cells were cultured in TexMACS TM medium (MiltenyiBiotechInc) with 1% penicillin and streptomycin sulfate in the presence of human IL-2 (100 IU / mL, MiltenyiBiotechInc). The T cells were cultured in a multi-well plate at a density of 1x10 6 cells / mL. The T cells were passaged and the medium was changed every 2 - 3 days. Example 14 - Transduction of Human T Cells

[0335] In the presence of human IL-2 (100 IU / mL), isolated T cells were activated with Dynabeads conjugated to anti-CD3 / CD28 antibodies (Life Technologies) for 12 - 24 hours and then transduced with lentivirus encoding the anti-fluorescein CAR gene. Cells were harvested 72 hours later and CAR expression on the transduced T cells was confirmed by measuring GFP-fluorescent cells using flow cytometry. Example 15 - Overview of Human Osteosarcoma-Folate Receptor α (HOS-FR-α) and AML Studies

[0336] The dosing schedule is shown in Figure 21 and includes administration of approximately 5 million E2 CAR T cells on day - 3; EC17 on days 0, 3, 7, 14, 17, 21, 28, 31, and 35. Successive escalating doses of EC17 will be administered according to the following rules: If there is no CRS or neurotoxicity, the EC17 dose escalation will proceed as planned. If the CRS grade ≤ 2, all subsequent EC17 doses will be administered at the order level that causes a non-severe CRS in a fixed cycle (stone cycle). If the CRS grade > 2, further EC17 doses in the current cycle will be cancelled and all subsequent cycles will be started at the next highest order below that which caused the severe CRS (sCRS). A. Tumor Implantation

[0337] HOS-FRα (i.e., HOS-143b-LV-FRα) is a subclone of HOS-143B (ATCC CRL-8303) stably transfected with human FRα. HOS-143B was originally derived from a 13-year-old Caucasian female with osteosarcoma. Tumor cells were grown in folate-free RPMI 1640 with 5% FBS at 37°C in a 5% CO2 humid atmosphere. For in vivo studies, tumor cells were subcutaneously inoculated at 1x10 6 cells / animal. B. CAR-T Cell Administration

[0338] Anti-FITC E2 scFv-CAR T cells sorted by EGFRt were frozen in T cell freezing medium. Upon arrival, vials of frozen CAR-T cells were immediately stored at -80°C. CAR-T cells were rapidly thawed at 37°C, washed twice with PBS, and used for animal injection at 6 million live EGFRt+ E2 CAR-T cells (CD4 / CD8 ~ 1:1) / animal. Small aliquots were taken on the day of infusion for flow cytometry analysis of the E2-CAR T cell phenotype. C. Preparation of the Administered Drug Solution

[0339] At the start of dosing, the EC17 dosing solution was prepared as follows: Appropriate amounts of each compound were weighed, reconstituted in PBS (pH 7.4), sterile filtered through a 0.22 µm PVDF syringe filter, and frozen at -20 °C into aliquots for daily dosing. D. Compound Administration and Evaluation

[0340] All EC17 doses were administered at the end of the day (3 - 4 PM) to allow possible overnight manifestation of CRS (cytokine release syndrome). In the morning of the next day, the animals were scored according to the CRS grading system (shown in Table 2). Table 2

[0341] Tumor growth, body weight and overall assessment: Tumor size was monitored 2 - 3 times / week and body weight was measured 2 - 3 times / week. In the days immediately following any EC17 dose, body weight was measured daily and attention was focused on the overall animal morphology and behavior.

[0342] Euthanasia: Euthanasia was performed when the weight loss of the mice ≥ 20%, when the tumor reached ≥ 1500 mm 3 . Euthanasia was also performed if the mice lost a lot of weight in a short period of time (e.g., due to severe head tilt), or when they were close to a moribund state according to the CRS grading system.

[0343] EC17 in - vivo dose escalation treatment (3 cycles) is described in Figure 22 .

[0344] As Figure 4 seen: Cohort 1 (only E2 - CAR T cells) showed strong growth of HOS - FRα without EC17 treatment. Cohort 2 (EC17 5 / 100 / 1000 nmol / kg, M / Th / M) showed significant tumor regression in this study and no weight loss or CRS. The slow EC17 dose escalation (M / Th / M) regimen was safe. No symptoms of weight loss or cytokine release syndrome (CRS) were observed in mice bearing HOS - FRα tumors.

[0345] One objective of this E2-CAR T cell study was to test EC17 / E2 CAR-T cell activity and related toxicities (such as sCRS) using multiple dosing regimens starting 3 days after CAR-T infusion. Three different EC17 dosing regimens included (a) once weekly (SIW), 500 nmol / kg, (b) "TIW-like" at 5 nmol / kg, 50 nmol / kg, and 500 nmol / kg on Monday / Wednesday / Friday with a 9-day interval, and (c) "TIW-like" at 5 nmol / kg, 100 nmol / kg, and 1000 nmol / kg on Monday / Thursday / Monday with a 7-day interval. The experimental groups are listed in Table 3 Table 3

[0346] The study summary included: Group 1 (untreated): only for efficacy comparison. Group 2 (CAR-T only) is depicted in Figure 23A Group 3 (EC17 SIW) is depicted in Figure 23B Group 4 (EC17 exact TIW) is depicted in Figure 23C Group 5 - EC17 low-dose "TIW" (M / Th / M, off 6 days) has / efficacy is depicted in Figure 23D A. Tumor Implantation

[0347] THP-1-FRβ AML tumor cells were grown in folate-free RPMI 1640 with 5 - 10% FBS at 37°C in a humidified atmosphere of 5% CO2. THP-1-FRO tumor cells in serum-free folate-free RPMI1640 medium were injected i.v. at 5x10 6 cells / animal. B. CAR-T Cell Administration

[0348] Anti-FITC E2 scFv-CAR T cells were frozen in T cell freezing medium. Upon arrival, vials of frozen CAR-T cells were immediately stored at -80°C. CAR-T cells were rapidly thawed at 37°C, washed twice with CAR-T cell medium, and 6 million live EGFRt+E2 CAR-T cells (CD4 / CD8 at ~1:1) / animal were used for animal injection. Small aliquots were taken on the day of infusion for flow cytometry analysis of E2-CAR T cell phenotype. C. Preparation of the Administered Drug Solution

[0349] ​At the start of dosing, the EC17 and bis-EDA-FITC dosing solutions were prepared as follows: Appropriate amounts of each compound were weighed, reconstituted in PBS (pH 7.4), sterile filtered through a 0.22 µm PVDF syringe filter, and frozen at -20 °C for daily dosing aliquots. D. Compound Administration

[0350] All EC17 doses were administered by the end of the day (3 - 4 PM) to allow possible CRS (cytokine release syndrome) to manifest overnight. In the morning of the next day, the animals were scored according to the CRS grading system (see below).

[0351] The EC17 dose schedule included: Cohorts 1 - 2: No EC17 dose was administered. Cohort 3 is depicted in Figure 24A and Cohort 4 is depicted in Figure 24B and Cohort 5 is depicted in Figure 24C . Monitoring / Efficacy:

[0352] Daily body weight measurements were required for several days after the EC17 dose. Attention was focused on overall animal morphology and behavior. If a mouse lost a significant amount of weight in a short period of time, or when a mouse was near moribund according to the CRS grading system or neurotoxicity, then euthanasia was performed. The CRS grading system shown in Table 2 was used. CRS Grading System

[0353] The CRS grading system shown in Table 2 was used. Tumor growth, body weight, and overall assessment: Body weight was measured 2 - 3 times / week. In the days immediately following any EC17 dose, body weight was measured daily, and attention was focused on overall animal morphology and behavior. Flow Cytometry Analysis

[0354] Complete blood cell analysis: Plasma was removed from a pre-determined volume of all EDTA-treated blood, and RBCs were lysed with RBC lysis solution. The leukocyte pellet was then resuspended in a flow cytometry staining solution (1% bovine serum albumin, 50 mg / mL human IgG (Equitech Bio, cat# SLH56-0001), 0.9% sodium azide, in phosphate-buffered saline, pH = 7.4), and leukocyte surface marker staining was performed using the following antibodies: anti-human CD45 (clone HI30, eBioscience#47-0459-42, 1:20 (v / v) dilution), anti-human CD137 (clone 4B4-1, BD Bioscience#564092, 1:20 (v / v) dilution), anti-human CD8α [clone RPA-T8, BD Bioscience, catalog number#557746, 1:20 (v / v) dilution], anti-human CD4 [clone SK3, eBioscience catalog number#46-0047-42, 1:20 (v / v) dilution], anti-human EGFR [R&D systems, clone Hu1, catalog number#FAB9577B@1:10 (n / n)], anti-human PD1 [BD Biosciences, clone EH12.1, catalog number#562511@1:20 (v / v)], anti-human LAG3 [BD Biosciences, clone T47-530, catalog number#565616@1:20 (v / v)], anti-human TIM3 [BD Biosciences, clone 7D3, catalog number#565558@1:20 (v / v)], anti-human CD3ε [BD Biosciences, clone SK7, catalog number#557832@1:20 (v / v)]. After leukocyte staining, the cells were washed with PBS and resuspended in cold PBS containing 53,000 CountBright TM beads [Invitrogen catalog number#C36950] and transferred to a flow cytometry collection tube. Flow cytometry data was collected on a Gallios flow cytometer (Beckman Coulter, Brea, CA). The determination results of the concentration of CAR T cells in each blood sample were calculated according to the instructions of Invitrogen. CAR T cells were identified as human CD3ε+EGFRt+ events and were easily distinguished and counted using Kaluza TM flow cytometry software. Subsequently, the number of CAR T cells in the circulation of each infused mouse was represented on a graph as the total number of CAR T cells in 50 μL of analyzed whole blood. Statistical significance was determined by using an unpaired two-tailed Student's t-test, with significance set at p < 0.05. Tumor and Tissue Analysis

[0355] Harvest solid tumors (100 - 1000 mm 3 ), weigh them, and cut them into small pieces, then transfer them to a 50 mL tube containing 20 mL of tumor digestion mixture. The enzymatic tumor digestion mixture consists of 0.5 mg / mL type IV collagenase (Sigma - Aldrich, catalog number #C5138), 0.5 mg / mL hyaluronidase (Sigma - Aldrich, catalog number #H3506), and 0.1 mg / mL DNase I (Sigma - Aldrich, catalog number #DN25) in serum - free and folate - depleted RPMI 1640 medium supplemented with antibiotics. The tumor fragments are digested for one hour at 37 °C at 300 rpm on a horizontal shaker. Subsequently, the tumor digest is centrifuged at 400 x g for 5 minutes, and a red blood cell lysis step is performed on the tumor cell pellet, followed by washing with cold phosphate - buffered saline (PBS, pH 7.4) and finally filtering through a 40 pm nylon cell strainer. Results and Conclusions

[0356] A. Three different dosing schedules of EC17 administration showed different body weight loss patterns and different levels of CRS.

[0357] As Figure 5 shown, cohort 1 (untreated) and cohort 2 (CAR - T only) did not show much body weight loss and had no CRS. Cohort 3 (EC17 SIW) showed EC17 dose - dependent body weight loss after each EC17 dose and showed grade 1 - 2 CRS throughout the study. Cohort 5 (EC17 dose escalation) showed little body weight loss during the first two cycles, but started to show body weight loss similar to that of cohort 2 after the end of the second cycle of EC17 dose. Cohort 4 (EC17 TIW on / off) showed the mildest body weight loss among the three treatment groups. Neither cohort 4 nor cohort 5 showed any CRS greater than 1.

[0358] Three different dosing schedules of EC17 administration showed different anti - tumor activities.

[0359] As Figure 6 shown, liver and non - liver tumor burdens were evaluated on day 39 in all cohorts. Cohort 2 (CAR - T only) showed no difference compared to cohort 1 (untreated). Cohort 4 showed the best anti - tumor activity among the three different EC17 dosing schedules. Elimination of Circulating AML Cell Line THP1-FRB

[0360] In studies using CD19-specific anti-ALL therapies, expansion and persistence of CAR T cells in blood have been reported to correlate with elimination of circulating CD19+ ALL cells that elicit a complete response to the therapy. In this experiment, we performed a similar assay in our murine AML model to determine the anti-tumor activity of our aptamer-controlled E2 CAR T cells by looking for the disappearance of our GFP-labeled human AML cell line THP1-FRβ in response to different dosing regimens of the CAR T adaptor molecule EC17. Figure 7 Circulating THP1-FRβ cells are shown, with the y-axis showing the total number of GFP+ cells per 100 μL of whole blood on a log scale. Different columns represent different treatment cohorts. It is illustrated that 40 days after infusion of AML cells into mice that received neither CAR T cells nor EC17, a high AML cell burden was present in the circulation. In mice that received infusions of both AML cells and E2 CAR T cells but no EC17 treatment, no reduction in AML cells in the blood was seen. This indicates that anti-tumor activity generated by potential alloreactivity of human T cells with allogeneic THP1 cells is negligible. Interestingly, in all three cohorts of mice infused with E2 CAR T cells plus EC17 with different dosing regimens, the circulating THP1 cell burden was significantly reduced, thus demonstrating the effectiveness of CAR T therapy under three different aptamer dosing regimens. E2-CAR-T Exhaustion Phenotype

[0361] Under chronic antigen stimulation, such as in the case of chronic viral infection or cancer, T cells undergo a process of exhaustion, in which they are no longer able to proliferate, secrete inflammatory cytokines, or kill antigen-presenting target cells. CAR T cells also have the potential to become exhausted under chronic stimulation of the CAR resulting from persistent scFv-stimulated antigen. Since our E2 CAR T cells respond only to the presence of our bridging molecule EC17 that binds to the surface of FR+ tumor cells, we have the ability to prevent chronic antigen stimulation of E2 CAR T cells by stopping treatment with EC17 and thus removing the presence of surface-bound antigen. Intermittent periods characterized by the absence of surface antigen prevent chronic antigen exposure of E2 CAR T cells and prevent the possible resulting exhaustion.

[0362] To confirm that the EC17 dosing regimen does not cause exhaustion of E2 CAR T cells, flow cytometry analysis was performed on single cell preparations from THP1-FRβ liver metastases and surface markers specifically expressed on exhausted T cells. As T cells approach exhaustion, co-expression of the surface inhibitory receptors PD1, LAG3, and TIM3 increases. Figure 8A bar graph is shown, where the y-axis illustrates the percentage of total E2 CAR T cells isolated from solid liver tumors, which express various combinations of three surface markers represented by different bars. Notably, the fully exhausted T cells that express all three markers simultaneously are represented by the leftmost bars in each group. The left group shows E2 CAR T cells pre-infused into liver tumor CAR T cells isolated from three different EC17 treatment cohorts. The inhibitory receptor expression in the pre-infused CAR T cell product is nearly zero, as most of the T cells are negative for all three surface markers. Importantly, the E2 CAR T cells from all three EC17 treatment cohorts have exhausted triple-positive cells, and interestingly, cohort 4 (EC17 TIW) expresses the lowest number of double- and triple-positive events and is further composed of a significant number of triple-negative CAR T cells. These data suggest that all three EC17 dosing regimens employed in this mouse experiment can be used clinically, due to the presence of significant anti-tumor activity and the presence of little CAR T exhaustion induction. Example 16 - Cell Lines and Reagents

[0363] Unless otherwise indicated, all FR+ and FR- negative cancer cell lines were maintained in RPMI-1640 medium (Gibco BRL) supplemented with 10% heat-inactivated fetal bovine serum, which was either free of 2.4 mM folic acid (FA) (FFRPMI) or contained 2.4 mM folic acid (FA) (RPMI). KB (human cervical carcinoma expressing FRα with HeLa markers) and CHO-b (Chinese hamster ovary cells transfected with human FRβ) were used as sources of FRα and FRβ, respectively, for radioligand binding assays. MDA-MB-231 represents an FRα subclone of the human TNBC (triple-negative breast cancer) cell line. For AML studies, isogenic pairs expressing green fluorescent protein (GFP) of FRβ-positive (THP1-FRβ) and FR-negative (THP1-FG12) cell lines were provided. Both were established from THP-1 (ATCC, TIB-202), a commonly used cell model for studying pediatric AML, which was originally derived from a 1-year-old male infant with acute monocytic leukemia. For osteosarcoma studies, HOS-FRα was established by lentiviral transduction of the FR-negative HOS-143b (ATCC, CRL8303) with the FOLR1 gene encoding human FRα. HOS-143b was originally established from the primary tumor of a 13-year-old Caucasian female and was highly tumorigenic in NSG mice. Bioluminescent pairs expressing GFP of FR+HOS-FRαfLuc and FR-negative HOS-143bfLuc were transduced with lentiviral firefly luciferase. LEGENDplex TMThe human cytokine panel was purchased from BioLegend (San Diego, CA). The CytoTox non-radioactive cytotoxicity assay kit based on lactate dehydrogenase (LDH) was purchased from Promega (Madison, WI). Commercially available anti-human antibodies for multicolor flow cytometry were: CD69 (clone FN50), CD45RA (clone HI100), CD45RO (clone UCHL1), and CD4 (clone SK3) from Thermo Fisher Scientific (Waltham, MA); TIM3 (clone 7D3), CD3ε (clone SK7), CD8α (clone RPA-T8), CD137 / 4-1BB (clone 4B4-1), CD25 (clone M-A251), PD1 (clone EH12.1), and LAG3 (clone T47-530) from BD Bioscience (San Jose, CA); biotinylated anti-human EGFR (Cetuximab, clone Hu1) from R&D systems (Minneapolis, MN); and FRα (clone LK26) from BioLegend (San Diego, CA). Fluorophore-conjugated antibiotin was also purchased from BioLegend. APC-conjugated anti-FITC mouse IgG2a / kappa antibody (clone NAWESLEE), CountBright TM beads (Invitrogen), annexin V staining buffer, and Alexa Fluor-647-conjugated annexin V were purchased from Thermo Fisher Scientific. For enzymatic digestion of tumor tissues, type IV collagenase, hyaluronidase, and DNase I were all purchased from Sigma-Aldrich (St. Louis, MO).

[0364] EC17 or folic acid-FITC [FA-(γ)-ethylenediamine-FITC] was synthesized at Endocyte. 3H-EC17 was purchased from Moravek biochemicals (Brea, CA) with a specific activity of ∼0.952 Ci / mmol, or was prepared at Endocyte by conjugating FITC to 3H-FA-(γ)-ethylenediamine obtained from ViTrax (Placentia, CA) with a specific activity of ∼1.2 Ci / mmol. 3H-FA was also purchased from ViTrax with a specific activity of 59 Ci / mmol. For CRS rescue, the fluorescein sodium administration solution was diluted with AK- 25% (fluorescein injection, USP) purchased from Purdue Pharmacy (NDC 17478-250-25). Example 17 - Humanized CAR Constructs and CAR-Modified T Cells

[0365] Previous studies used a GFP+ second-generation anti-FITC scFv (clone 4M5.3) CAR with a hinge and transmembrane sequence containing CD8α and 4-1BB / CD3ζ signaling domains (i.e., FITC-4M5.3-scFfv-CD8α hinge-CD8αtm-4-1BB / CD3ζ). For translation into the first-in-human test, a second-generation fully human FITC-specific (clone E2) CAR construct (referred to herein as E2) ( Figure 9 , top figure) was developed. CAR-modified T cells are shown in Figure 9 , bottom pie chart.

[0366] The constructs described herein are FITC-specific CAR constructs comprising: (1) a fully human anti-FITC scFv (clone E2, Kd = 0.75 nM), (2) an IgG4 hinge-CH2 (F235D, N297Q)-CH3 spacer fused to a CD28-transmembrane domain, (3) a second-generation 4-1BB / CD3-intracellular domain, and (4) a cell surface human EGFRt tag ( Figure 9, top panel) (SEQ ID NO:04 and SEQ ID NO:05 are nucleic acid and amino acid sequences, respectively). To generate CAR-modified T cells, lentivirus was produced in 293T cells co-transfected with the epHIV7 lentiviral vector encoding the CAR. Donor CD4+ and CD8+ T cells were purified by immunomagnetic selection and transduced either individually or at a ratio of approximately 50:50. Generally, only one round of CD3 / CD28 bead activation was performed, followed by one or two rounds of rapid in vitro expansion. For preclinical evaluation, several batches of EGFRt-sorted CD4, CD8, and unsorted CD4 / CD8 CAR-T cells were used. All CAR-T cell preparations were analyzed before cryopreservation and after thawing to determine EGFRt expression and CD4 / CD8 ratio by flow cytometry. Using a combination of surface markers, the differentiation status of CD4+ and CD8+ CAR-T cell subsets on the day of infusion was analyzed and determined as TN, CD45RA+CD45RO-CD62F+CD95- naive T cells; TSCM, CD45RA+CD45RO-CD62F+CD95+ stem cell memory T cells; TCM, CD45RA-CD45RO+CD62F+CD95+ central memory T cells; TEM, CD45RA-CD45RO+CD62L-CD95+ effector memory cells; and TEFF, CD45RA+CD45RO-CD62L-CD95+ effector T cells. For the preclinical tests described below, the study included two batches of EGFRt-sorted pure CD4 and CD8 subsets (after mixing at a ratio of approximately 1:1) and several batches of unsorted ~1:1 EGFRt+CD4 / CD8 mixtures, including a "clinical replicate" preparation with low differentiation characteristics.

[0367] Among a series of different CAR constructs synthesized and evaluated, the fully human anti-FITC scFv (FITC-E2) CAR was selected for preclinical development ( Figure 9)。This second-generation fully human CAR consists of an anti-FITC scFv (clone E2), an IgG4-Fc spacer / hinge with double mutations (L235D and N297Q) in the CH2 region to reduce binding to FcyR, a CD28 transmembrane domain, and a 4-1BB / CD3ζ signaling domain attached to the cell surface EGFRt tag via a T2A ribosomal skipping sequence (i.e., FITC-E2-scFv-IgG4 hinge-CD28tm-4-1BB / CD3ζ-T2A-EGFRt). For preclinical studies, both EGFRt-sorted and unsorted E2 CAR-T cells were prepared at a ~1:1 CD4 / CD8 ratio and routinely analyzed for T cell subset phenotypes by flow cytometry at the time of CAR T cell infusion (day 0) in each in vivo experiment. The typical expression pattern of EGFRt-sorted CAR-T cells includes CD4 and CD8 subsets that are approximately 42% TSCM, 10% TCM, 12% TEM, and 34% TEFF each ( Figure 9 , pie chart on the upper left). Only EGFRt-sorted CAR-T cells were used for co-culture and pharmacokinetic studies. For tumor therapy, a "clinical replica" batch with the poorly differentiated characteristics of MDA-MB-231 ( Figure 9 , pie chart on the upper right) was used, and the study batch was used for THP1-FRβ and HOS-FRα studies. The "clinical replica" batch (~39% EGFRt+) included a CD4 subset that was -66% TSCM and -32% TCM and a CD8 subset that was ~95% TSCM and ~3% TCM. The study batch (~23% EGFRt+) was more differentiated and included a CD4 subset that was approximately 32% TSCM, approximately 53% TCM, approximately 11% TEM, and approximately 3.7% TEFF and a CD8 subset that was approximately 44% TSCM, approximately 0.28% TCM, approximately 3.4% TEM, and approximately 52% TEFF. Example 18 - Bispecific Affinity of EC17 CAM

[0368] The bispecific affinity of EC17 CAM (CAM is equivalent to "bridge" or "compound" in this application) was evaluated using 3H-EC17 in a cell-based radioligand binding assay. For binding to the FR+ target, KB and CHO-b cells were pre-seeded overnight in a 24-well tissue culture plate and incubated with 0.1, 0.5, 1, 5, 10, 20, and 40 nM of 3H-EC17 in FFRPMI at 37 °C for 2 h. Subsequently, the cells were rinsed with phosphate-buffered saline (PBS, pH 7.4) and lysed with 1% sodium dodecyl sulfate. The radioactivity levels and cellular protein content of the whole cell lysates were quantified by standard Pierce BCA protein assay. The number of 3H-EC17 molecules bound per cell was calculated to determine the dissociation constants (Kd) of FRα (KB) and FRβ (CHO-β), respectively (Figure 10). EC17 has been tested clinically for the purposes of immunotherapy and optical imaging. To directly quantify its bispecific binding affinity, 3 3H-EC17 was synthesized and a radioligand binding assay was performed on the KB and CHO-β cell lines, which represent FRα+ and FRβ+ target cells, respectively, and on unsorted EGFRt CAR-T cells, which represent effector cells. When bound to its target, EC17 demonstrated similar affinities for both FRα and FRβ, with low Kd values of 1.7 nM and 0.8 nM, respectively (Figure 10, panel A). When bound to unsorted E2-CAR-T cells (-24% EGFRt+, -95:5 CD8 / CD4 ratio), the estimated Kd value was -130 nM (Figure 10, panel B). Example 19 - Tumor Models

[0369] All animal care and use were conducted in accordance with NIH guidelines and followed a protocol approved by the Purdue University Animal Care and Use Committee. Female 4- to 5-week-old NOD / SCIDγ (NSG TM ) mice (stock number: 005557) were purchased from The Jackson Laboratory (Bar Harbor, ME). Unless specifically indicated, all animals were maintained on a FA-deficient diet (TestDiet, St. Louis, MO) upon arrival and throughout the study. To establish subcutaneous xenografts, MDA-MB-231 and HOS-FRα were implanted at 2.5x10 6 and 1x10 6 cells / animal, respectively, into the right flank area. For intravenous xenografts, 5x10 6THP1-FRβ cells were inoculated at a dose of [number of cells] cells per animal. Subcutaneous tumors were measured with calipers 2-3 times a week, and the volume was calculated using the ellipsoid formula (length x width2) / 2. Euthanasia was performed according to the study design or when any of the following occurred: (i) the animal had a weight loss ≥20% or was near moribund state, (ii) the size of the subcutaneous tumor reached ≥1500 mm 3 , or (iii) the animal showed signs of a swollen abdomen and severe distress (i.e., THP1-FRβ). All animals were given intravenous doses (CAR-T cells, EC17, sodium fluorescein). Example 20 - Tumor Therapies

[0370] In the treatment context, EC17CAM could be given before or after CAR-T cell injection. As described herein, the first dose of EC17 was administered 2-3.5 days after CART cells to allow a period for observing human T cells in tumor-bearing mice. Two batches of unsorted E2-CAR T cells (23% or 39% EGFRt+, 1:1 CD4 / CD8) were used for in vivo studies. On the infusion day (day 0) for each experiment, the cryopreserved CAR T cells were rapidly thawed at 37 °C, washed 2x with Dulbecco’s IX PBS (pH 7.4) and injected into the tail vein at the desired EGFRt+ E2-CAR-T cell dose. In addition, the differentiation status of CAR-T cells and the CD4 to CD8 ratio of small aliquots of CAR T cells were analyzed by flow cytometry. On the first day of the EC17 dose, the tumor-bearing animals were randomized into multiple groups based on the tumor size or the same number of days after intravenous transplantation (i.e., THP1-FRβ).

[0371] For the MDA-MB-231 study, mice received a high dose (∼10 million) of “clinical replica” E2-CAR T cells with low differentiation characteristics (∼39% EGFRt+)( Figure 9 ). Two days later, three different EC17 treatment regimens (Figure 12) were initiated at an average tumor size of -293 ± 39 mm 3 . EC17 was administered once a week (SIW) at 500 nmol / kg on Monday, or at escalating doses of 5, 50 or 100, and 500 or 1000 nmol / kg (i.e., 5 / 50 / 500 or 5 / 100 / 1000) on Monday, Thursday and Monday, with a 6-day rest between cycles. Control mice remained untreated (received CAR T cells but no EC17). For comparison, cohorts of tumor-free syngeneic partners also received the same number of CAR T cells with or without 500 nmol / kg of EC17 SIW.

[0372] For the AML study (Figure 13), THP1-FRβ tumor cells were intravenously infused into mice one day before receiving a low dose of approximately 6 million E2-CAR T cells (-23% EGFRt+). At ~3.5 days after CAR-T cell infusion, EC17 was administered in 3 different ways, including i) at 500 nmol / kg SIW, ii) three times at 5 / 50 / 500 nmol / kg on Monday / Wednesday / Friday, followed by a 9-day rest between cycles (TIW on / off), and iii) escalating doses of 5 / 10 / 100 nmol / kg in cycle 1, 5 / 30 / 300 nmol / kg in cycle 2, and 5 / 50 / 500 nmol / kg in cycle 3, all on Monday / Thursday / Monday, with a 6-day rest between cycles (M / Th / M on / off). On day 31, satellite samples of animals were harvested to quantify CAR-positive T cells identified as human CD3ε+EGFRt+ events in mouse blood and calculated as the absolute number per 100 μL of whole blood. After euthanasia or at the end of the study on day 38, the total tumor burden in mice bearing THP1-FRβ tumors was evaluated by measuring GFP+ tumor cells in the blood using flow cytometry and collecting the liver weight (with metastatic lesions) and the total weight of non-hepatic gross metastases found in the body. Tumor fragments of liver metastases were enzymatically digested to a single-cell suspension and the CAR-T cell exhaustion status of the live cell population was analyzed using anti-human PD1, LAG3, and TIM3 (clones EH12.1, T47-530, and 7D3, respectively).

[0373] For the osteosarcoma study (Figure 14), two cohorts of mice were subcutaneously implanted with HOS-FRα tumor cells 3 days before receiving the same CAR-T cell preparation of approximately 6 million used in the THP1-FRβ study (Figure 13). At approximately 3.5 days after CAR-T cell infusion, up to 3 cycles of EC17 were given to one cohort of mice: cycle 1 at 5 / 10 / 100 nmol / kg, cycle 2 at 5 / 30 / 300 nmol / kg, and cycle 3 at 5 / 50 / 500 nmol / kg, all following the Monday / Thursday / Monday protocol with a 6-day rest between cycles. At the end of the study, circulating CD3ε+EGFRt+ CAR-T cells per 100 μL of mouse blood were counted. At the same time, HOS-FRα tumors (+ / - EC17 treatment) were harvested and digested for flow cytometry analysis of tumor-infiltrating CAR-T cells. A. Dose Escalation Trial for Invasive Osteosarcoma Model

[0374] For our intended purpose, HOS-FRα has a low FR-expression but the most functional FR level of ~5.82 ± 1.45 pmol / mg protein InvasionA tumor model. Parallel to the THP1-FRβ study described above, the same E2-CAR-T cells were administered to two cohorts of mice with 3-day-old HOS-FRα tumors at the same dose (~6 million). One cohort was treated with the same accelerated EC17 dose escalation regimen: cycle 1 5 / 10 / 100 nmol / kg, cycle 2 5 / 30 / 300 nmol / kg, and cycle 3 5 / 50 / 500 nmol / kg, on a Monday / Thursday / Monday schedule, followed by a 6-day rest (Figure 14, subpanel A). Since the HOS-FRα tumors grow invasively without EC17 treatment, the accelerated EC17 dose escalation at this CAR-T cell dose was safe (no CRS or weight loss), and caused a significant delay in tumor growth during the first two cycles of treatment (Figure 14, subpanel B). When euthanasia was performed according to the protocol only due to tumor size (≥1500 mm 3 ), flow cytometry analysis of whole blood showed EC17-dependent CAR-T cell expansion up to day 47, but higher expansion at day 33 (Figure 14, subpanel C, left bar graph).

[0375] Ex vivo tumor analysis on day 33 showed a low but significant intratumoral CD3e+EGFRt+CAR-T cell population in EC17-treated animals, accounting for approximately 1% of the total viable digested tumor-derived cells (Figure 14, subpanel C, right bar graph). As the large HOS-FRα tumors stopped responding to cycle 3 treatment (Figure 14, subpanel B), intratumoral CAR T cells also decreased at day 47 (Figure 14, subpanel C). Notably, HOS-FRα tumors analyzed by 3H-FA radioligand assay after disease progression showed similar FRα levels with and without EC17 treatment. Thus, as shown by in vitro co-culture studies, the rapid growth of HOS-FRα tumors in NSG mice seems to exceed the tumor infiltration capacity of CAR T cells and may reduce cytolytic activity due to T cell exhaustion. EC17 Dose Exploration Study in Tumor-Bearing and Tumor-Free Mice

[0376] An initial EC17 dose exploration study was conducted in mice bearing MDA-MB-231 tumors, as shown in the schematic of the experimental design (Figure 12, Panel A). On day 0, approximately 10 million "clinical replica" batches of E2-CAR-T cells (-39% EGFRt+, 51:49 CD4 / CD8) were transplanted into NSG mice with or without MDA-MB-231 tumors (~211±65 mm 3 ). This batch of CAR-T cells consisted mostly of the TSCM and TCM phenotypes (see Figure 9)。Two days after CAR-T cell injection, one cohort of tumor-bearing mice remained untreated while three cohorts were treated with different EC17 regimens, including single injections weekly (SIW) at 500 nmol / kg, or given according to escalating EC17 dose levels of 5 / 50 / 500 (escalation-1) or 5 / 100 / 1000 nmol / kg (escalation-2) on a Monday / Thursday / Monday schedule, with a 1-week drug-free interval. For comparison, two tumor-free cohorts were either untreated or treated with 500 nmol / kg EC17 SIW.

[0377] Using satellite sample animals, human T cell-derived IFNγ levels in the blood of mice in all cohorts were measured on days 11 and 12 (20 and 42 hours after the previous EC17 dose). Compared to tumor-bearing mice receiving only CAR-T cells, all EC17-treated tumor cohorts had ~30x (day 11) and ~10x (day 12) higher IENγ production in the plasma of mice, and the levels of this cytokine decreased naturally later from 20 hours to 42 hours (Figure 12, subpanel B). According to IFNγ release, EC17 also triggered the expansion of CAR-T cells (identified as human CD3e+EGFRt+ events) in the blood of mice, and the cells persisted in tumor-bearing animals for up to 54 days (last measurement) (Figure 12, subpanel C). In tumor-free cohorts, CAR-T cell expansion was not detected by flow cytometry, but low levels of IFNγ were detected on days 11 and 12 in animals receiving the same number of CAR-T cells plus EC17 (Figure 12, subpanels B to C). Additionally, no CRS symptoms (grade 0 out of 0 to 5) or weight loss were observed in tumor-free mice receiving two weekly doses of EC17 at 500 nmol / kg.

[0378] Moderate to severe CRS symptoms (grades 2 to 3) and significant weight loss (Figure 12, Panel D) were observed in the tumor-bearing cohort receiving continuous EC17 administration independent of the protocol. An EC17 SIW of 500 nmol / kg resulted in the earliest onset of CRS as well as weight loss, while the aggressive EC17 escalation-2 protocol (up to 1000 nmol / kg) resulted in persistent weight loss with animal recovery occurring after EC17 dosing cessation (Figure 12, Panel D, top column). Notably, symptoms of graft-versus-host disease (GVHD) included skin redness and itching and hairlessness and became apparent at 1 month post CAR-T cell transplantation. Although animals receiving only CAR-T cells showed signs of non-specific CAR-T cell / tumor alloreactivity, the EC17-treated cohort alone produced 100% cures (Figure 12, Panel D, bottom column). Thus, EC17 administration in the presence of FR+ tumors was key to driving: i) CAR-T cell activation, ii) cytokine production, and iii) CAR-T cell expansion and persistence in vivo. However, under specific conditions, severe CRS (≥ grade 3) was triggered by the combination of high CAR-T cell doses with EC17 doses equal to or greater than 500 nmol / kg. EC17 CAM Administration Control for Anti-Leukemia Activity

[0379] Intravenously implanted GFP-expressing THP1-FRβ tumor cells produce systemic multiple diseases in NSG mice, with tumor cells in the circulation and liver / non-liver metastases. THP1-FRβ tumor cells can also localize to the mouse ovary, and the ovary appears inflamed at the initial stage of tumor progression. Therefore, the total tumor burden in each animal in the study cohort was evaluated by quantifying circulating GFP+ tumor cells in the blood, liver weight, and gross non-liver metastases visible to the naked eye. Although THP1-FRβ expresses low levels of FR in vitro, the THP1-FRβ tumor metastases were found to express a higher-than-expected functional FR level, at ~8.9 pmol / mg ± 2.8 pmol / mg membrane protein. Therefore, unsorted E2-CAR-T cells (-23% EGFRt+, 1:1 CD4:CD8) from the study batch were transplanted into THP1-FRβ tumor-bearing mice at approximately 6 million per animal, and then treated with 3 different EC17 dosing regimens (Figure 13). Starting 3 days after CAR-T cell injection, the EC17 dosing regimens were initiated, either continuously SIW at 500 nmol / kg, three times a week (TIW) on Monday / Wednesday / Friday at 5 / 50 / 500 nmol / kg followed by a 9-day rest, or by an accelerated dose escalation regimen, cycle 1 5 / 10 / 100 nmol / kg, cycle 2 5 / 30 / 300 nmol / kg, and cycle 3 5 / 50 / 500 nmol / kg, administered on Monday / Thursday / Monday (M / Th / M), followed by a 6-day rest (Figure 13, subpanel A). Some weight loss and grade 1-2 CRS were observed in cycles 2 and 3 in animals treated with EC17 SIW, while animals treated with EC17 TIW had the least weight loss and CRS was only grade 0-1 (Figure 13, subpanel B). Among animals receiving 3 cycles of EC17 M / Th / M dose escalation, grade 0-1 CRS and very mild weight loss were observed in cycle 2 after the final dose of 300 nmol / kg of EC17. On day 31, satellite sample animals were used to count CAR-T cells in the blood, and the data demonstrated EC17-dependent CAR-T cell expansion and persistent retention in all treated cohorts (Figure 13, subpanel C). Compared to control animals receiving only tumor cells or tumor cells plus CAR-T cells without EC17, EC17 administered in any of the 3 "intra-patient" escalation formats effectively reduced circulating THP1-FRβ tumor cells in the blood and showed similar activity against liver tumor metastases (Figure 13, subpanel D, left and middle bars). In mice receiving only CAR-T cells, only trace alloreactivity against THP1-FRβ liver metastases was found.Although EC17 SIW and TIW with a 10-fold dose escalation (on / off) successfully controlled non-hepatic macroscopic metastases, the EC17 M / Th / M dose escalation at a slow pace per cycle (Figure 13, sub-panel A) failed to control non-hepatic macroscopic metastases (Figure 13, sub-panel D, the rightmost sub-panel). At the end of the study (i.e., 39 days after CAR-T cell injection), CAR-T cells isolated from hepatic THP1-FRβ) tumor metastases seemed to have the least expression of the double-positive and triple-positive T cell inhibitory receptors PD1, LAG3, and TIM3 (Figure 13, sub-panel E). Overall, no severe CRS (i.e., grade ≥ 3) was observed in any EC17-treated cohort. However, the more aggressive EC17 TIW dose escalation (on / off) schedule tended to be the optimal regimen for reducing the overall THP1-FRβ) tumor burden in these mice. Example 21 - Functional FR Assessment

[0380] These functional FR evaluations were used in the examples described herein. In addition to the pediatric cancer cell lines transfected with FRα (HOS-FRα) and FRβ) (THP1-FRβ), cancer cell lines with different histologies and FR expression levels were included ( Figure 11 ). As estimated by radioligand binding assays (100 nM 3 H-FA, 1 h at 37 °C), the hierarchical order of the overall available FR on these cell lines was: 9x10 4 (OV90, an ovarian cancer cell line with low FR expression), 1.9x10 5 (THP1-FRβ), 2.4x10 5 (HOS-FRαfLuc), 7x10 5 (HOS-FRα), 2.1x10 6 (MDA-MB-231), and 4.8x10 6 (KB) FA molecules / cell. The HOS-143b (fLuc) and THP1-FG12 parental cell lines were also included as FR-negative controls. Thus, the general hierarchical order of functional FR expression on co-cultured FR+ cancer cell lines was: KB > MDA-MB-231 > HOS-FRα > HOS-FR αfLuc > THP1-FRβ (AML) > OV90. Example 22 - Statistics

[0381] Statistical analysis was performed using the computer program GraphPad Prism (GraphPad Software Inc., San Diego, CA). Data were analyzed using Student's t-test or one-way ANOVA. If applicable, appropriate multiple comparison post hoc tests were used to further analyze the data across treatment groups. * = p < 0.05 was considered statistically significant in all tests. Example 23 - Study of Fluorescein-Specific CAR T Cells and Parenteral Administration of Folic Acid-Fluorescein Combination for Osteosarcoma Tumor

[0382] This Phase 1 open-label non-randomized study will enroll study participants with recurrent or refractory osteosarcoma to examine the safety and feasibility of administering autologous peripheral blood-derived CD4 and CD8 T cells that have been genetically modified using a third-generation self-inactivating (SIN) lentiviral vector to express a fluorescein (FL)-specific human scFv second-generation (4-lBB:zeta) chimeric antigen receptor (CAR) and EGFRt, in combination with the conjugate UB-TT170 of folic acid and fluorescein (FL) (previously called EC17; BB-IND 010704, sponsor: Umoja Biopharma). Anti-FL (FITC-E2) CAR T cells are thought to be non-reactive to human cells, but can recognize and target folate receptor-positive target cells in the presence of a small molecule aptamer drug composed of folic acid conjugated to FL. Thus, control of the "on" phase reactivity of anti-FL (FITC-E2) CAR T cells depends on the dose of the small molecule drug and can be rapidly reversed by administration of free fluorescein. Thus, this system provides a new level of near real-time CAR T cell control based on physician-specified administration of UB-TT170.

[0383] The response to a fixed dose of anti-FL (FITC-E2) CAR T cells followed by escalating doses of circulating UB-TT170 is expected to depend on the following factors: the tumor burden of the individual subject, T cell proliferation and cytokine production, and the degree to which the individual subject's tumor / tumor-associated myeloid cells and macrophages express receptor (FR) α and / or β. Thus, this study was designed to treat subjects with escalating doses of UB-TT170 within a subject to determine their individual UB-TT170 optimized dose, which is defined as the dose that elicits an anti-tumor response in a controllable and safe manner.

[0384] The primary objective of this study was to determine the recommended dose escalation sequence of UB-TT170 following administration of anti-FL (FITC-E2) CAR T cells for further clinical development. The safety objectives of the study included assessing the safety and tolerability of adoptive cellular immunotherapy using ex vivo expanded autologous T cells genetically modified to express anti-FL (FITC-E2) CAR when administered prior to UB-TT170 dosing. This study was a 3+3 design to explore the tolerability of three different dose escalation sequences of UB-TT170.

[0385] Secondary objectives included assessing the feasibility of manufacturing anti-FL (FITC-E2) CAR T cells from apheresis blood products obtained from study participants using a 7-day single-process (CD4 / 8 combination) T cell manufacturing platform, studying the in vivo engraftment of lentiviral vector-specific sequences and the persistence of transferred cells by flow cytometry and / or quantitative PCR, assessing the pharmacokinetics of UB-TT170, and quantifying the anti-tumor response by measuring changes in tumor burden using disease-specific evaluations. Exploratory objectives included evaluating the expression of folate receptor α or β in archived tumor or tissue samples or obtained from subjects during the conduct of the trial, evaluating the presence of adoptively transferred T cells in tumor tissue and / or normal tissue derived from subjects, and analyzing biomarkers of safety and / or anti-tumor activity in blood, bone marrow, CSF, normal tissue, and / or tumor tissue.

[0386] Following study enrollment, T cells will be isolated from apheresis blood products obtained from study participants. Following infusion of anti-FL (FITC-E2) CAR cells on cycle 1 day 0 (C1D0), subjects will receive escalating doses of UB-TT170 on C1D4, C1D7, and CID11 to determine the maximum tolerated dose of UB-TT170 for the subject. Subsequently, the subject-specific maximum tolerated dose will be administered weekly for a total of an additional 2 weeks, at which time re-staging will occur. Subjects with stable disease or disease responsive to treatment may continue in the study for up to 3 subsequent courses, each consisting of 7 weekly doses for a total of 4 complete courses. Figure 15 The experimental design protocol is depicted.

[0387] The Phase 1 study design will provide preclinical data on the safety and efficacy of administering anti-FL E2-CAR T cells, followed by repeated dosing with UB-TT170. The response to combination therapy of anti-FL (FITC-E2)-specific chimeric antigen receptor (CAR) in combination with UB-TT170 is expected to depend on the following factors: the tumor burden of individual subjects, T cell proliferation and cytokine production, and the degree to which the tumors of individual subjects express folate receptor α or β. Different from many “standard” oncology MTD / dose exploration studies, this study is designed to treat study participants with escalating doses of UB-TTT70 to determine their individual optimized dose of UB-TT170. Example 24 - Monotherapy UB-TT170 (Previously Known as EC17), Human Experience and Dose Rationality

[0388] The safety of administering UB-TT170 as a single agent in humans has been evaluated in multiple exploratory pilot studies and Phase 2 studies. Subjects received 0.1 - 0.3 mg / kg UB-TT170 prior to intraoperative tumor imaging. The most common adverse event reported in 10.7% of subjects (9 / 84) was mild hypersensitivity, with symptoms of urticaria, abdominal discomfort, itchy throat, and sneezing. Other adverse events after intravenous administration of UB-TT170 were vomiting, abdominal discomfort, and injection site reactions. No serious adverse events were reported. Overall, UB-TT170 administered alone was well tolerated for intraoperative tumor imaging. Principle of Administration

[0389] This study will examine the safety and feasibility of administering a combination of autologous peripheral blood-derived T cells, which have been genetically modified using a SIN lentiviral vector to express an anti-FL (FITC-E2)-specific chimeric antigen receptor (CAR), and UB-TT170 (a folate and fluorescein (FL) conjugate).

[0390] The response to combination therapy of anti-FL (FITC-E2)-specific chimeric antigen receptor in combination with UB-TT170 is expected to depend on the following factors: the tumor burden of individual subjects, T cell proliferation and cytokine production, and the degree to which individual subject tumors express FRα or FRβ. The successful implementation of the anti-FL (FITC-E2) CAR T cell and UB-TT170 dosing paradigm has the potential for the treatment of other FR-expressing cancers, including ovarian cancer, lung cancer, and other malignancies. EC17 Human Pharmacokinetics, Dose Adjustment and Previous Experience

[0391] Before surgery, the plasma pharmacokinetics of EC17 were determined in ovarian cancer subjects (n = 13) after a single 10-minute intravenous infusion of 0.1 mg / kg. Blood samples were collected before dosing and at 15, 30, 45, and 60 minutes after administration of EC17, and then hourly until the end of surgery. Immediately after collection, the blood samples were placed on ice and protected from light, and plasma was separated and stored at -20 °C until analysis. Bioanalysis was performed using liquid chromatography tandem mass spectrometry (LC-MS / MS), with a lower limit of quantification (LLOQ) and an upper limit of quantification (ULOQ) of 2.00 ng / mL and 500 ng / mL, respectively.

[0392] The biodistribution and excretion characteristics of EC17 in the subjects from this study were characterized by rapid distribution and specific uptake in folate receptor-positive cancerous lesions, followed by rapid clearance from the circulation. The maximum plasma concentration (C max ) was reached at the end of the 10-minute infusion and then decreased, with an elimination half-life of 86.8 minutes. EC17 fluorescence signals were observed at the tumor site up to 5 hours after administration of the compound, which was the latest visualization time point for surgical evaluation. The short terminal half-life and low background uptake in the circulation allowed real-time visualization of malignant lesions within 2 hours after EC17 administration. This short half-life further supported the planned treatment administration regimen proposed in this study.

[0393] EC17 binds to the tumor cell surface FR with sub-nanomolar affinity (internal report Endo 061401). It has good water solubility, is inherently small in size, and can easily penetrate solid tumors. Optical imaging studies have shown that EC17 can effectively deliver attached fluorescein molecules to almost all malignant cells in primary and metastatic tumor sites, while maintaining a sharp contrast between tumor cells and surrounding normal tissues. Using a folate imaging agent similar in size to EC17, a dose of ~2000 nmol / kg in mice appears to achieve FR saturation in vivo regardless of the FR level on the tumor. Based on studies in mice, the therapeutic effective dose range of EC17 is between 500 nmol / kg and 2000 nmol / kg to achieve opsonization of labeled tumor cells without competitive binding of excess EC17 to circulating antibodies (internal report 0003-PR-0010, internal report 0003-PR-0012)

[174] . Since the affinity of FR in mice and humans is similar, after conversion based on body surface area, it is estimated that a dose of 3.1×10 -2 mg / kg to 1.24×10 -1 mg / kg of EC17 will achieve saturation of tumor-associated FR in humans. Doses of 3.1×10 -2 mg / kg and 2.76×10 -1EC17 dose of mg / kg, and none reached the maximum tolerated dose (MTD). This study could use <1.55×10 -1 mg / kg of the EC17 dose, which is very low compared to the previous EC17 doses found safe in previous human studies.

[0394] Multiple exploratory pilot studies and phase 2 studies have evaluated the safety of administering EC17 as a single agent in humans. Six published studies evaluated whether EC17 was effective and safe in intraoperative imaging of tumors in 84 subjects with renal, ovarian, breast, or lung cancer. The most common adverse event reported by 10.7% of the subjects (9 / 84) was mild hypersensitivity reaction, with symptoms of urticaria, abdominal discomfort, itchy throat, and sneezing. Other adverse events after intravenous administration of EC17 were vomiting, abdominal discomfort, and mild injection site reactions. No serious adverse events were reported, and symptoms were well controlled using supportive care measures (such as administration of diphenhydramine). Overall, EC17 was well tolerated by intravenous injection.

[0395] In this study, the recruitment of child subjects will comply with 21 CFR 50, Subpart D. The initial subjects recruited in each dosing regimen will be >18 years old, and all other patients will be at least 15 years old to allow for assent / informed consent of all subjects and to maximize our ability to assess toxicity. Importantly, this complies with the scientific necessity principle of 21 CFR 50, Subpart D, as pediatric patients with recurrent / refractory osteosarcoma have a different tumor biology and anatomical location than other solid tumors that occur in adult patients. The peak age for osteosarcoma diagnosis is 13 to 16 years, and more than half of all cases diagnosed each year occur in patients under 20 years old. Therefore, the age range selected for this trial is absolutely necessary to answer important scientific questions regarding the health and welfare of pediatric osteosarcoma. In addition, all enrolled subjects will be the target pediatric population with refractory or recurrent osteosarcoma for which there is no known cure. In terms of fair selection, the recruitment of children is essential to answer the scientific objectives. We will implement fair and equitable subject selection in accordance with the Declaration of Helsinki (paragraph 24). As noted above, due to differences in disease biology, behavior, and anatomical tumor location, it is not possible to extrapolate from the adult population with other solid tumors. In this clinical trial, we have appropriately balanced risks and benefits and implemented additional protections for children in accordance with 21 CFR 50 Subpart D. We minimized risks to subjects by requiring only research procedures that directly contribute to the scientific objectives. Regarding the "significant" criteria of 21 CFR 50.52, although the intravenous administration of CAR T cells in children stores more than minimal risk, the eligibility criteria are clear in that these subjects have failed all standard of care regimens, thus providing a possible benefit. Finally, the expected potential benefits and possible risks will be discussed in depth with each subject, and adequate provisions will be made to obtain the subject's consent. Drug Information

[0396] The investigational product for SCRI-E2CAR_EGFRtvl consists of autologous CD4+ and CD8+ T cells that have been genetically modified to express anti-FL (FITC-E2) (which is an anti-fluorescein CAR). The investigational UB-TT170 drug product (BB-IND 010704) is provided in sterile, pyrogen-free, single-use vials for intravenous injection via a peripheral or central catheter over a 5-minute period.

[0397] The investigational product is defined as "a pharmaceutical form of an active ingredient or placebo that is tested or used as a reference in a clinical trial, which includes a product with a marketing authorization when used or assembled (formulated or packaged) in a manner different from the approved form, or for an unapproved indication, or for obtaining further information on the approved use" (from the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use [ICH] Harmonised Tripartite Guideline E6: Guideline for Good Clinical Practice).

[0398] The terms "investigational product" and "study drug" may be used interchangeably in the protocol. Dosing Regimen

[0399] Prior to administration of the CAR T cell product, subjects will be assigned a dosing schedule. Non-limiting examples of dosing schedules are Dosing Schedule 0, Dosing Schedule 1, Dosing Schedule 2, and Dosing Schedule 3, as Figure 17 、 Figure 18 、 Figure 19 and Figure 20 depicted and described below. Each dosing schedule will consist of a single, fixed, weight-based CAR T cell dose administration (Procedure 1, Day 0), followed by a dosing schedule-specific UB-TT170 dose escalation sequence (Procedure 1, Days 4, 7, and 11), followed by a fixed, weight-based weekly dose of UB-TT170 (Procedure 1, Days 18 and 25).

[0400] The full (100%) dose of UB-TT170 is 3.1x10 -2 mg / kg. Each dose described in the dosing schedule is expressed as a percentage of the full (100%) dose. See Table 4. Table 4

[0401] The initial subject cohort will be enrolled in Dosing Schedule 1, and subsequent cohorts will escalate to Dosing Schedule 3. If Dosing Schedule 1 is considered intolerable, subsequent subjects will be enrolled in Dosing Schedule 0. The UB-TT170 dose escalation sequence will be conducted in subjects without dose-limiting toxicity (DLT). If the dose escalation phase is tolerated without DLT, the subject will receive the maximum tolerated dose schedule UB-TT170 dose level as the subsequent UB-TT170 dose.

[0402] Dose regimen 0 will only be used if dose regimen 1 is considered intolerable. If the subject does reach dose regimen 1, after administration of the CAR T cell product, on day 0 of procedure 1 the subject will enter the UB-TT170 dose escalation phase. On day 4 of procedure 1, the subject will receive 0.5% of the full UB-TT170 dose (dose level 0 / A), then on day 7 of procedure 1 receive 5% of the full UB-TT170 dose (dose level 0 / B), and on day 11 of procedure 1, the subject will receive 50% of the full UB-TT170 dose (dose level 0 / C).

[0403] During the escalation phase, UB-TT170 dose escalation can only continue if the subject has no dose-limiting toxicity (DLT) or study-significant adverse event (ssAE). If the dose escalation is tolerated and there is no DLT or ssAE, the subject will receive 50% of the full UB-TT170 dose on days 18 and 25 of procedure 1 and for subsequent UB-TT170 administrations in subsequent procedures.

[0404] Dose regimen 1 will be the initial dose regimen to be employed. After administration of the CAR T cell product, on day 0 of procedure 1, the subject will enter the UB-TT170 dose escalation phase. On day 4 of procedure 1, the subject will receive 1% of the full UB-TT170 dose (dose level 1 / A), then on day 7 of procedure 1 receive 10% of the full UB-TT170 dose (dose level 1 / B), and on day 11 of procedure 1, the subject will receive 100% of the full UB-TT170 dose (dose level 1 / C). During the escalation phase, UB-TT170 dose escalation can only continue if the subject has no dose-limiting toxicity (DLT) or ssAE. If the dose escalation is tolerated and there is no DLT or ssAE, the subject will receive 100% of the full UB-TT170 dose on days 18 and 25 of procedure 1 and for subsequent UB-TT170 administrations in subsequent procedures.

[0405] Dose Regimen 2. After administration of the CAR T cell product, on Day 0 of Procedure 1, the subject will enter the UB-TT170 dose escalation phase. On Day 4 of Procedure 1, the subject will receive 1% of the full UB-TT170 dose (Dose Level 2 / A), then on Day 7 of Procedure 1, the subject will receive 30% of the full UB-TT170 dose (Dose Level 2 / B), and on Day 11 of Procedure 1, the subject will receive 300% of the full UB-TT170 dose (Dose Level 2 / C). During the escalation phase, UB-TT170 dose escalation can only continue if the subject has no dose-limiting toxicity (DLT) or ssAE. If the dose escalation is tolerated and there is no DLT or ssAE, the subject will receive 300% of the full UB-TT170 dose on Days 18 and 25 of Procedure 1 and for subsequent UB-TT170 administrations in subsequent procedures.

[0406] Dose Regimen 3. After administration of the CAR T cell product, on Day 0 of Procedure 1, the subject will enter the UB-TT170 dose escalation phase. On Day 4 of Procedure 1, the subject will receive 1% of the full UB-TT170 dose (Dose Level 3 / A), then on Day 7 of Procedure 1, the subject will receive 50% of the full UB-TT170 dose (Dose Level 3 / B), and on Day 11 of Procedure 1, the subject will receive 500% of the full UB-TT170 dose (Dose Level 3 / C). During the escalation phase, UB-TT170 dose escalation can only continue if the subject has no dose-limiting toxicity (DLT) or ssAE. If the dose escalation is tolerated and there is no DLT or ssAE, the subject will receive 500% of the full UB-TT170 dose on Days 18 and 25 of Procedure 1 and for subsequent UB-TT170 administrations in subsequent procedures.

[0407] For any given dose regimen, the subsequent dose escalation sequence can follow a similar order, as Figure 17 、 Figure 18 、 Figure 19 and Figure 20 depicted. For each additional procedure, the escalating dose is repeated approximately every 7 days (Days 1, 8, 15, 22, 29, 36, and 43). Patients can receive multiple additional dose procedures as needed; for example, patients can receive one, two, three, four, six, eight, or ten procedures of treatment.

[0408] If a subject experiences a toxic reaction that meets the definition of DLT at any time point, a dose adjustment is made. Figure 16 The dose adjustment scheme is depicted.

[0409] The maximum tolerated dose (MTD) regimen is defined as the highest dose regimen / cohort in which the cumulative DLT rate during Course 1 is closest to 20% among the dose regimens / cohorts tested during the trial, provided that it is also below 30% and at least 6 subjects have been evaluated in that dose cohort. Dose-limiting toxicity (DLT) is an event clearly, possibly, or probably attributable to CAR T-cell or UB-TT170 infusion and occurring within 28 days after CAR T-cell and within 7 days after the final UB-TT170 infusion. During Course 1 of each dose regimen, only DLTs occurring within 28 days after CAR T-cell and within 7 days after the final UB-TT170 infusion will be used for MTDR definition and for the purpose of dose regimen escalation or de-escalation.

[0410] If no DLT and no ssAE are observed during the dose escalation phase and the subject meets the criteria for subsequent UB-TT170 infusion, the subject will receive UB-TT170 dose level C for the remaining Course 1 UB-TT170 doses and all Course 2 - Course 4 UB-TT170 doses.

[0411] If no DLT occurs but the subject experiences ssAE, subsequent UB-TT170 infusions will be suspended until the ssAE has recovered to baseline or ≤ Grade 1, and then all subsequent UB-TT170 doses will be administered at the dose level that triggered the ssAE or at a lower level.

[0412] If DLT occurs after the subject receives the lowest UB-TT170 dose (Dose A) in each dose regimen, the subject should be withdrawn from the protocol treatment and no further doses of UB-TT170 should be given.

[0413] If the DLT is caused by UB-TT170 Dose B or Dose C, subsequent UB-TT170 infusions will be suspended until the DLT has recovered to baseline or ≤ Grade 1. Then, all subsequent UB-TT170 doses will be administered at a dose level lower than the dose that triggered the DLT. These policies are outlined as Figure 16 shown. Disease Response

[0414] All tumor measurements will be recorded in millimeters (or as a decimal of a centimeter). Previously irradiated lesions may be considered measurable and followed for response, provided that they show significant progression after completion of the most recent radiotherapy or that the disease is confirmed by biopsy after radiotherapy. Serial measurements should be made in the same manner (e.g., CT, MRI, PET) at each assessment. Response criteria include those listed in Table 5. Table 5

[0415] As used herein, the terms "comprising" and "including", "containing" or "characterized by" are synonymous and are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0416] The foregoing description discloses several methods and materials of the present invention. The present invention is susceptible to modifications in methods and materials as well as changes in manufacturing methods and equipment. Such modifications will become apparent to those skilled in the art by considering this disclosure or practicing the invention as disclosed herein. Accordingly, it is not intended to limit the invention to the specific embodiments disclosed herein, but rather to cover all modifications and alternatives that fall within the true scope and spirit of the invention.

[0417] All references cited herein (including but not limited to published and unpublished applications, patents, and literature references) are hereby incorporated by reference in their entirety and thus form a part of this specification. If the publications and patents or patent applications incorporated by reference conflict with the disclosure contained herein, this specification is intended to supersede and / or take precedence over any such conflicting material.

Claims

1. A method of treating, ameliorating or inhibiting osteosarcoma in a human subject, the method comprising administering a fluorescein isothiocyanate (FITC)-folate compound conjugate or a pharmaceutically acceptable salt thereof to the subject according to a dosing regimen comprising: The dosing escalation period is used to confirm the maximum tolerated dose (MTD) of the FITC-folic acid compound conjugate or a pharmaceutically acceptable salt thereof, wherein, The dosing escalation cycle comprises administering: (a) A first dose that is about 0.5% to about 5% of the full dose of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof, (b) A second dose that is about 5% to about 50% of the full dose of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof, (c) A third dose that is about 50% to about 500% of the full dose of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof, wherein the MTD is confirmed after the third dose, and (d) A fourth dose that is the MTD of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof, Wherein, the total dose of the FITC-folic acid compound conjugate or a pharmaceutically acceptable salt thereof is about 1x10 -2 mg / kg to about 5x10 -2 mg / kg; wherein the human subject has received or is receiving a chimeric antigen receptor (CAR) T cell composition, the CAR T composition comprising a population of T cells expressing an anti-fluorescein CAR or an anti-fluorescein derivative CAR, thereby treating, ameliorating or inhibiting the osteosarcoma in the human subject.

2. A method of treating, ameliorating or inhibiting osteosarcoma in a human subject, the method comprising administering a fluorescein isothiocyanate (FITC)-folate compound conjugate or a pharmaceutically acceptable salt thereof to the subject according to a dosing regimen comprising: (i) Administration escalation period to confirm the maximum tolerated dose (MTD) of the FITC-folate compound conjugate, wherein, The dosing escalation cycle comprises administering: (a) A first dose that is about 0.5% to about 5% of the full dose of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof, (b) A second dose that is about 5% to about 50% of the full dose of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof, and (c) A third dose that is about 50% to about 500% of the full dose of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof, Wherein, the total dose of the FITC-folic acid compound conjugate or a pharmaceutically acceptable salt thereof is about 1x10 -2 mg / kg to about 5x10 -2 mg / kg; and (ii) A maintenance dosing cycle, the maintenance dosing cycle comprising administering the MTD of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof at a dosing interval of about once every 5 - 10 days for a period of time; wherein the human subject has received or is receiving a CAR T cell composition, the CAR T composition comprising a population of T cells expressing an anti-fluorescein CAR, thereby treating, ameliorating or inhibiting the osteosarcoma in the human subject.

3. The method according to claim 1 or 2, wherein The osteosarcoma is recurrent or refractory.

4. The method according to any one of claims 1-3, wherein, The time period between administering the first dose and administering the second dose is between about 1 day and about 7 days.

5. The method according to any one of claims 1 - 4, wherein The time period between administering the first dose and administering the second dose is about 2 days.

6. The method according to any one of claims 1-5, wherein The time period between administering the second dose and administering the third dose is between about 1 day and about 7 days.

7. The method according to any one of claims 1-6, wherein, The time period between administration of the second dose and administration of the third dose is about 3 days.

8. The method according to any one of claims 1-7, wherein, The total dose of the FITC-folic acid compound conjugate or a pharmaceutically acceptable salt thereof is about 3.1x10 -2 mg / kg.

9. The method according to any one of claims 1-8, the method further comprising administering a fifth dose, the fifth dose being the MTD of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof.

10. The method according to claim 9, the method further comprising a re-staging phase after the end of the fifth dose, wherein, The restaging phase includes: Evaluating the subject to determine whether more than one criteria for receiving at least one subsequent dosing cycle of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof are met, provided that the FITC-folate conjugate is not administered to the subject during the restaging phase.

11. The method according to claim 10, wherein, The subject meets more than one criteria, and wherein at least one subsequent dosing cycle is administered to the subject, the at least one subsequent dosing cycle comprising administering the MTD of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof at a dosing interval of about every 5-10 days for a period of time.

12. The method according to any one of claims 1-11, wherein, The CAR comprises a full-length humanized anti-fluorescein antibody or an antigen-binding fragment thereof.

13. The method according to any one of claims 1 to 12, wherein, The CAR comprises a full-length humanized anti-fluorescein scFv.

14. The method according to claim 13, wherein, The CAR comprises a full-length humanized E2 anti-fluorescein scFv.

15. The method according to any one of claims 1-14, wherein, The CAR T cells express a cell surface selectable marker, the cell surface selectable marker comprising a truncated EGFR (EGFRt) polypeptide.

16. The method according to any one of claims 1-15, wherein, The dose of the CAR T cell composition is about 1x10 5 cells / kg to about 1x10 7 cells / kg.

17. The method according to any one of claims 1-16, wherein, The FITC-folate conjugate or a pharmaceutically acceptable salt thereof is administered intravenously.

18. A method of treating, ameliorating or inhibiting cancer in a human subject, the method comprising: (i) administering to the subject a first dose of a fluorescein isothiocyanate (FITC)-folate conjugate or a pharmaceutically acceptable salt thereof; (ii) administering to the subject a second dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof, wherein the second dose is higher than the first dose; (iii) administering to the subject a third dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof, wherein the third dose is higher than the second dose, wherein the maximum tolerated dose (MTD) of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof is confirmed after the third dose; and (iv) administering to the subject a fourth dose, the fourth dose being the MTD of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof; wherein the human subject has received or is receiving a CAR T cell composition, the CAR T cell composition comprising a population of T cells expressing an anti-fluorescein CAR, thereby treating, ameliorating or inhibiting the cancer in the human subject.

19. The method according to claim 18, wherein The cancer includes osteosarcoma.

20. A method of treating, ameliorating or inhibiting osteosarcoma in a human subject, the method comprising administering to the subject a fluorescein isothiocyanate (FITC)-folate compound conjugate or a pharmaceutically acceptable salt thereof in combination with CAR T cell therapy, wherein, After or simultaneously with administering the CAR T cell therapy to the subject, the FITC-folate conjugate is administered to the subject in an escalating dosing regimen to treat the osteosarcoma in the human subject.

21. The method according to claim 20, wherein, The escalating dosing regimen includes: a dosing escalation period to confirm the maximum tolerated dose (MTD) of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof, wherein the dosing escalation period includes administering: (a) A first dose that is about 0.5% to about 5% of the full dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof, (b) A second dose that is about 5% to about 50% of the full dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof, (c) A third dose that is about 50% to about 500% of the full dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof, wherein the MTD is confirmed after the third dose, and (d) A fourth dose that is the MTD of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof, Wherein, the total dose of the FITC-folic acid compound conjugate or a pharmaceutically acceptable salt thereof is about 1x10 -2 mg / kg to about 5x10 -2 mg / kg.

22. The method according to claim 20, wherein The escalating dosing regimen includes: (i) A dosing escalation period to confirm the maximum tolerated dose (MTD) of the FITC-folate conjugate, wherein the dosing escalation period includes administering: (a) A first dose that is about 0.5% to about 5% of the full dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof, (b) A second dose that is about 5% to about 50% of the full dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof, and (c) A third dose that is about 50% to about 500% of the full dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof, wherein, the total dose of the FITC-folic acid compound conjugate or a pharmaceutically acceptable salt thereof is about 1x10 -2 mg / kg to about 5x10 -2 mg / kg; and (ii) A steady dosing period that includes administering the MTD of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof at a dosing interval of about once every 5 - 10 days for a period of time.

23. The method according to any one of claims 20-22, wherein The CAR T cell therapy includes administering to the subject a population of CAR T cells expressing an anti-fluorescein CAR.

24. Use of a fluorescein isothiocyanate (FITC)-folate compound conjugate or a pharmaceutically acceptable salt thereof in combination with CAR T cell therapy for the treatment, amelioration or inhibition of osteosarcoma in a human subject, wherein, After or simultaneously with administering the CAR T cell therapy to the subject, the FTIC-folate conjugate is administered to the subject in an escalating dosing regimen.

25. The use according to claim 24, wherein, The escalating dosing regimen includes a dosing escalation period to confirm the maximum tolerated dose (MTD) of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof, wherein the dosing escalation period includes administering: (a) A first dose that is about 0.5% to about 5% of the full dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof, (b) A second dose that is about 5% to about 50% of the full dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof, (c) A third dose, wherein the third dose is about 50% to about 500% of the full dose of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof, wherein the MTD is confirmed after the third dose, and (d) A fourth dose, wherein the fourth dose is the MTD of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof, Wherein, the total dose of the FITC-folic acid compound conjugate or a pharmaceutically acceptable salt thereof is about 1x10 -2 mg / kg to about 5x10 -2 mg / kg.

26. The use according to claim 24, wherein The escalating dosing regimen comprises: (i) A dosing escalation period to confirm the maximum tolerated dose (MTD) of the FITC-folate compound conjugate, wherein the dosing escalation period comprises administering: (a) A first dose, wherein the first dose is about 0.5% to about 5% of the full dose of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof, (b) A second dose, wherein the second dose is about 5% to about 50% of the full dose of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof, and (c) A third dose, wherein the third dose is about 50% to about 500% of the full dose of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof, Wherein, the total dose of the FITC-folic acid compound conjugate or a pharmaceutically acceptable salt thereof is about 1x10 -2 mg / kg to about 5x10 -2 mg / kg; and (ii) A steady dosing period, wherein the steady dosing period comprises administering the MTD of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof at a dosing interval of about once every 5 - 10 days for a period of time.

27. Use of a fluorescein isothiocyanate (FITC)-folate compound conjugate or a pharmaceutically acceptable salt thereof in combination with CAR T cell therapy for the treatment, amelioration or inhibition of cancer in a human subject, wherein, The FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof is administered by a method comprising: (i) Administering a first dose of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof to the subject; (ii) Administering a second dose of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof to the subject, wherein the second dose is higher than the first dose; (iii) Administering a third dose of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof to the subject, wherein the third dose is higher than the second dose, wherein the maximum tolerated dose (MTD) of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof is confirmed after the third dose; and (iv) Administering a fourth dose to the subject, wherein the fourth dose is the MTD of the FITC-folate compound conjugate or a pharmaceutically acceptable salt thereof.

28. The use according to claim 27, wherein The cancer includes osteosarcoma.

29. Use according to claim 27 or 28, wherein, The FITC-folate compound conjugate is administered to the subject in an escalating dosing regimen after or simultaneously with administering the CAR T cell therapy to the subject.

30. Use according to any one of claims 24-29, wherein, The CAR T cell therapy comprises administering to the subject a population of CAR T cells expressing an anti-fluorescein CAR.

31. A kit, the kit comprising a container containing a fluorescein isothiocyanate (FITC)-folic acid compound conjugate or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutical carrier, wherein, The kit comprises instructions for use for treating, ameliorating or inhibiting osteosarcoma in a subject who has received or is receiving a CAR T cell composition comprising CAR T cells, wherein treating, ameliorating or inhibiting comprises administering the FITC-folate compound conjugate according to a dosing regimen comprising: A dosing escalation period to confirm the maximum tolerated dose (MTD) of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof, wherein the dosing escalation period includes administering: (a) A first dose that is about 0.5% to about 5% of the full dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof, (b) A second dose that is about 5% to about 50% of the full dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof, (c) A third dose that is about 50% to about 500% of the full dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof, wherein the MTD is confirmed after the third dose, and (d) A fourth dose that is the MTD of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof, Wherein, the total dose of the FITC-folic acid compound conjugate or a pharmaceutically acceptable salt thereof is about 1x10 -2 mg / kg to about 5x10 -2 mg / kg, and wherein the CAR T cells express an anti-fluorescein CAR.

32. The kit according to claim 31, wherein, The container is a vial.

33. A system for treating, ameliorating or inhibiting osteosarcoma in a subject in combination with CAR T cell therapy using a fluorescein isothiocyanate (FITC)-folic acid compound conjugate or a pharmaceutically acceptable salt thereof, wherein, After or simultaneously with administering the CAR T cell therapy to the subject, the FITC-folate conjugate is administered to the subject in an escalating dosing regimen, the system comprising: (a) A first dose that is about 0.5% to about 5% of the full dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof, (b) A second dose that is about 5% to about 50% of the full dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof, (c) A third dose that is about 50% to about 500% of the full dose of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof, wherein the maximum tolerated dose (MTD) of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof is confirmed after the third dose, and (d) A fourth dose that is the MTD of the FITC-folate conjugate or a pharmaceutically acceptable salt thereof, Wherein, the total dose of the FITC-folic acid compound conjugate or a pharmaceutically acceptable salt thereof is about 1x10 -2 mg / kg to about 5x10 -2 mg / kg.

34. The system according to claim 33, the system further comprising a population of CAR T cells expressing an anti-fluorescein CAR.

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