WO2019144091A1 - Methods of use for car t cells - Google Patents

Methods of use for car t cells Download PDF

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Publication number
WO2019144091A1
WO2019144091A1 PCT/US2019/014472 US2019014472W WO2019144091A1 WO 2019144091 A1 WO2019144091 A1 WO 2019144091A1 US 2019014472 W US2019014472 W US 2019014472W WO 2019144091 A1 WO2019144091 A1 WO 2019144091A1
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WIPO (PCT)
Prior art keywords
car
cells
dose
compound
pharmaceutically acceptable
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
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PCT/US2019/014472
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English (en)
French (fr)
Inventor
Philip Stewart Low
Haiyan CHU
Yingjuan June Lu
Christopher Paul Leamon
Leroy W. II WHEELER
Michael C. Jensen
James MATTHAEI
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Seattle Childrens Hospital
Purdue Research Foundation
Endocyte Inc
Original Assignee
Seattle Childrens Hospital
Purdue Research Foundation
Endocyte Inc
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Publication date
Application filed by Seattle Childrens Hospital, Purdue Research Foundation, Endocyte Inc filed Critical Seattle Childrens Hospital
Priority to CN201980021107.5A priority Critical patent/CN112055595B/zh
Priority to CN202411859267.6A priority patent/CN120154716A/zh
Priority to AU2019209428A priority patent/AU2019209428B2/en
Priority to EP19740881.8A priority patent/EP3740217A4/en
Priority to CA3089051A priority patent/CA3089051A1/en
Priority to JP2020561570A priority patent/JP7549303B2/ja
Publication of WO2019144091A1 publication Critical patent/WO2019144091A1/en
Anticipated expiration legal-status Critical
Priority to JP2024138411A priority patent/JP2024174886A/ja
Priority to AU2025237960A priority patent/AU2025237960A1/en
Ceased legal-status Critical Current

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Definitions

  • First generation CARs are composed of a recognition region, e.g., a single chain fragment variable (scFv) region derived from an antibody for recognition and binding to the antigen expressed by the tumor, and an activation signaling domain, e.g., the CD3z chain of T cells can serve as a T cell activation signal in CARs.
  • a recognition region e.g., a single chain fragment variable (scFv) region derived from an antibody for recognition and binding to the antigen expressed by the tumor
  • an activation signaling domain e.g., the CD3z chain of T cells can serve as a T cell activation signal in CARs.
  • a co-stimulation domain (e.g., CD137, CD28 or CD 134) has been included in second generation CARs to achieve prolonged activation of T cells in vivo. Addition of a co-stimulation domain enhances the in vivo proliferation and survival of T cells containing CARs, and initial clinical data have shown that such constructs are promising therapeutic agents in the treatment of diseases, such as cancer.
  • CAR T cell therapies Although improvements have been made in CAR T cell therapies, several problems remain. First,‘off-target’ toxicity may occur due to normal cells that express the antigen targeted by the CAR T cells (e.g., a tumor-associated antigen). Second, unregulated CAR T cell activation may be found where the rapid and uncontrolled elimination of diseased cells (e.g., cancer cells) by CAR T cells induces a constellation of metabolic disturbances, called tumor lysis syndrome, or cytokine release syndrome (CRS), which can be fatal to patients. Tumor lysis syndrome and CRS can result due to administered CAR T cells that cannot be easily regulated, and are activated uncontrollably. Accordingly, although CAR T cells show great promise as a tool in the treatment of diseases, such as cancer, additional CAR T cell therapies are needed that provide reduced off-target toxicity, and more precise control of CAR T cell activation.
  • diseased cells e.g., cancer cells
  • CRS cytokine release syndrome
  • the“small molecule ligand” can be, for example, a folate, DUPA, an NK-1R ligand, a CAIX ligand, a ligand of gamma glutamyl transpeptidase, an NKG2D ligand, or a CCK2R ligand, each of which is a small molecule ligand that binds specifically to cancer cells (i.e., the receptor for these ligands is overexpressed on cancers compared to normal tissues).
  • the“small molecule ligand” is linked to a“targeting moiety” that binds to the CAR expressed by CAR T cells.
  • the “targeting moiety” can be selected, for example, from fluorescein, fluorescein isothiocyanate (FITC), NHS-and/or fluorescein.
  • The“targeting moiety” binds to the recognition region of the genetically engineered CAR, expressing an E2 anti-fluorescein antibody fragment. Accordingly, the recognition region of the CAR (e.g., a single chain variable region (scFv) of an E2 anti fluorescein antibody fragment, an Fab, Fv, Fc, (Fab’)2 fragment, and the like) is directed to the “targeting moiety.”
  • the small molecule ligand linked to a targeting moiety by a linker acts as a bridge between the cancer and the CAR T cells, expressing an E2 anti-fluorescein antibody fragment, directing the CAR T cells to the cancer for amelioration of the cancer.
  • a method of treatment of a cancer comprises i) administering to a patient a compound, or a pharmaceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked to a targeting moiety by a linker, ii) administering to the patient a first dose of a CAR T cell composition comprising CAR T cells wherein the CAR T cells comprise a CAR directed to the targeting moiety and wherein the CAR comprises an E2 anti-fluorescein antibody fragment, and iii) administering to the patient a second dose of a CAR T cell composition comprising CAR T cells wherein the CAR T cells comprise the CAR directed to the targeting moiety and wherein the CAR comprises an E2 anti-fluorescein antibody fragment.
  • a method of treatment of a cancer comprises i) administering to a patient a compound, or a pharmaceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked to a targeting moiety by a linker, and ii) administering to the patient a CAR T cell composition wherein the CAR T cell composition comprises CAR T cells, wherein the CAR T cells comprise a CAR directed to the targeting moiety wherein the CAR comprises an E2 anti-fluorescein antibody fragment, and wherein the CAR T cell composition comprises a mixture of the CAR T cells and non-transformed T cells.
  • a method of treatment of a cancer comprises i) administering to a patient a compound, or a pharmaceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked to a targeting moiety by a linker, ii) administering to the patient a CAR T cell composition wherein the CAR T cell composition comprises CAR T cells wherein the CAR T cells comprise a CAR directed to the targeting moiety and wherein the CAR comprises an E2 anti-fluorescein antibody fragment; and iii) administering to the patient a folate, a conjugate comprising a folate wherein the conjugate comprising a folate does not comprise a targeting moiety, or an agent that inhibits activation of the CAR T cells.
  • a method of treatment of a cancer comprises i) administering to a patient a compound, or a pharmaceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked to a targeting moiety by a linker, wherein at least a first dose and a second dose of the compound, or the pharmaceutically acceptable salt thereof, are administered to the patient, wherein the first dose and the second dose are different, wherein the second dose of the compound, or the pharmaceutically acceptable salt thereof, is about 2-fold to about l5000-fold greater in amount than the first dose of the compound, or the pharmaceutically acceptable salt thereof, and ii) administering to the patient a CAR T cell composition comprising CAR T cells wherein the CAR T cells comprise a CAR directed to the targeting moiety and wherein the CAR comprises an E2 anti-fluorescein antibody fragment.
  • a method of treatment of a cancer comprises i) administering to a patient a first dose of a compound, or a pharmaceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked to a targeting moiety by a linker, ii) administering to the patient at least a second dose of the compound, or a pharmaceutically acceptable salt thereof, wherein the second dose of the compound, or the pharmaceutically acceptable salt thereof, is at least about 50 percent lower in amount than the first dose of the compound, or the pharmaceutically acceptable salt thereof; and iii) administering to the patient a dose of a CAR T cell composition comprising CAR T cells wherein the CAR T cells comprise a CAR directed to the targeting moiety and wherein the CAR comprises an E2 anti-fluorescein antibody fragment.
  • a method of treatment of a cancer comprises i) administering to a patient a compound, or a pharmaceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked to a targeting moiety by a linker, and wherein the compound, or the pharmaceutically acceptable salt thereof, is administered at a dose of about 10 nmoles/kg of body weight of the patient to about 2500 nmoles/kg of body weight of the patient, and ii) administering to the patient a CAR T cell composition comprising CAR T cells, wherein the CAR T cells comprise a CAR, wherein the CAR comprises an E2 anti-fluorescein antibody fragment, and wherein the CAR T cells are at a dose of about 1 million of the CAR T cells to about 15 million of the CAR T cells.
  • a method of treatment of a cancer comprises i) administering to a patient a compound, or a pharmaceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked to a targeting moiety by a linker and wherein the compound, or the pharmaceutically acceptable salt thereof, is administered once weekly to the patient, and ii) administering to the patient a CAR T cell composition comprising CAR T cells wherein the CAR T cells comprise a CAR, wherein the CAR comprises an E2 anti-fluorescein antibody fragment.
  • a method of treatment of a cancer comprises i) administering to a patient a first dose of a compound, or a
  • the compound comprises a small molecule ligand linked to a targeting moiety by a linker and wherein the compound, or the
  • CAR T cell composition comprising CAR T cells wherein the CAR T cells comprise a CAR directed to the targeting moiety, ii) then administering to the patient a dose of the CAR T cell composition, and iii) then administering to the patient a second dose of the compound, or the pharmaceutically acceptable salt thereof, wherein the CAR comprises an E2 anti-fluorescein antibody fragment.
  • a method of treatment of a cancer comprising i) administering to a patient a compound, or a pharmaceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked to a targeting moiety by a linker; ii) administering to the patient a first dose of a CAR T cell composition comprising CAR T cells wherein the CAR T cells comprise a CAR directed to the targeting moiety and wherein the CAR comprises an E2 anti-fluorescein antibody fragment; and
  • a CAR T cell composition comprising CAR T cells wherein the CAR T cells comprise the CAR directed to the targeting moiety and wherein the CAR comprises an E2 anti-fluorescein antibody fragment.
  • a method of treatment of a cancer comprising i) administering to a patient a compound, or a pharmaceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked to a targeting moiety by a linker;
  • CAR T cell composition comprises CAR T cells, wherein the CAR T cells comprise a CAR directed to the targeting moiety wherein the CAR comprises an E2 anti-fluorescein antibody fragment, and wherein the CAR T cell composition comprises a mixture of the CAR T cells and non-transformed T cells.
  • a method of treatment of a cancer comprising i) administering to a patient a compound, or a pharmaceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked to a targeting moiety by a linker;
  • CAR T cell composition comprises CAR T cells wherein the CAR T cells comprise a CAR directed to the targeting moiety and wherein the CAR comprises an E2 anti-fluorescein antibody fragment;
  • a conjugate comprising a folate wherein the conjugate comprising a folate does not comprise a targeting moiety, or an agent that inhibits activation of the CAR T cells.
  • step iii comprises administering a folate.
  • step iii comprises administering folic acid or leucovorin.
  • step iii comprises administering the conjugate comprising a folate.
  • X 1 and Y 1 are each-independently selected from the group consisting of halo, R 2 , OR 2 , SR 3 , and NR 4 R 5 ;
  • Q is selected from the group consisting of C and CH;
  • X 2 and X 3 are each independently selected from the group consisting of oxygen, sulfur, -C(Z , -C(Z)0-, -OC(Z)-, -N(R 4b )-, -C(Z)N(R 4b , -N(R 4b )C(Z)-,
  • R 1 is selected-from the group consisting of hydrogen, halo, C1-C12 alkyl, and Ci-
  • 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, halo, C1-C12 alkyl, C1-C12 alkoxy, C1-C12 alkanoyl, C1-C12 alkenyl, C 1 -C 12 alkynyl, (Ci-C 12 alkoxy)carbonyl, and (C 1 -C 12 alkylamino)carbonyl;
  • R 6 and R 7 are each independently selected from the group consisting of hydrogen, halo, C 1 -C 12 alkyl, and C 1 -C 12 alkoxy; or, R 6 and R 7 are taken together to form a carbonyl group;
  • R 6a and R 7a are each independently selected from the group consisting of hydrogen, halo, C 1 -C 12 alkyl, and C 1 -C 12 alkoxy; or R 6a and R 7a are taken together to form a carbonyl group;
  • p, r, s, and t are each independently either 0 or 1;
  • * represents an optional covalent bond to the rest of the conjugate.
  • the conjugate comprising a folate does not comprise a targeting moiety, or the agent that inhibits activation of the CAR T cells.
  • the conjugate comprising a folate does not comprise a targeting moiety, or the agent that inhibits activation of the CAR T cells, even though cytokine levels in the treated patient are reduced.
  • a method of treatment of a cancer comprising i) administering to a patient a compound, or a pharmaceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked to a targeting moiety by a linker, wherein at least a first dose and a second dose of the compound, or the pharmaceutically acceptable salt thereof, are administered to the patient, wherein the first dose and the second dose are different, wherein the second dose of the compound, or the pharmaceutically acceptable salt thereof, is about 2-fold to about l5000-fold greater in amount than the first dose of the compound, or the pharmaceutically acceptable salt thereof; and
  • the third dose of the compound, or the pharmaceutically acceptable salt thereof is about 800-fold to about lOOOO-fold greater in amount than the first dose of the compound, or the pharmaceutically acceptable salt thereof.
  • a compound or a pharmaceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked to a targeting moiety by a linker;
  • a CAR T cell composition comprising CAR T cells wherein the CAR T cells comprise a CAR directed to the targeting moiety and wherein the CAR comprises an E2 anti-fluorescein antibody fragment.
  • ligand is selected from the group consisting of a folate, DUPA, an NK-1R ligand, a CAIX ligand, a ligand of gamma glutamyl transpeptidase, an NKG2D ligand, and a CCK2R ligand.
  • L represents the linker
  • T represents the targeting moiety
  • L comprises a structure having the formula
  • n is an integer from 0 to 200.
  • any one of clauses 1 to 125 wherein the cancer is selected from the group consisting of lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head, cancer of the neck, cutaneous melanoma, intraocular melanoma uterine cancer, ovarian cancer, endometrial cancer, rectal cancer, stomach cancer, colon cancer, breast cancer, triple negative breast cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin’s Disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, non-small cell lung cancer, cancer of the adrenal gland, sarcoma of soft tissue, osteosarcoma, cancer of the urethra, prostate cancer, chronic leukemia, acute leukemia, acute myelocytic leukemia, lymphocytic lymphom
  • compound, or the pharmaceutically acceptable salt thereof is not an antibody, and does not comprise a fragment of an antibody.
  • FIG. 1 shows the E2 construct vs. the 4M5.3 construct diagrammatically and shows a map of the E2 construct.
  • FIG. 2 shows an EC 17 fixed dose and dose de-escalation schema.
  • FIG. 3A and 3B show E2-CAR-T anti-tumor activity (30 million cells) with EC17 dose de-escalation and body weight changes. As shown, antitumor activity is maintained after NaFL rescue.
  • FIG. 4A and 4B show 4M5.3-CAR-T anti-tumor activity (30 million cells) with EC17 dose de-escalation and body weight changes. As shown, 4M5.3-CAR-T was less active than E2-CAR-T. In addition, EC 17 dose dependent anti-tumor activity and body weight loss were observed.
  • FIG. 5A and 5B show E2-CAR-T anti-tumor activity (10 and 20 million cells) and body weight changes at a fixed EC 17 dosing regimen (500 nmol/kg, SIW). As shown, antitumor activity was maintained after NaFE rescue.
  • FIG. 6A and 6B show 4M5.3-CAR-T anti-tumor activity (10 and 20 million cells) and body weight changes at a fixed EC 17 dosing regimen (500 nmol/kg, SIW). As shown, 4M5.3-CAR-T was less active than E2-CAR-T.
  • FIG. 7A and 7B show the phenotypic characterization of E2 CAR T cells prior to infusion into NSG mice.
  • FIG. 8A and 8B show the comparison of CAR-T differentiation phenotypes in different preparations including E2-CAR-T cells and 4M5.3-CAR-T cells, and GFP + 4M5.3 CAR-T cells.
  • FIG. 9A and 9B show binding of DIG-labeled E2-IgG to FITC.
  • FIG. 10A and 10B show IHC staining of DIG-labeled E2 antibody on FITC- labeled KB cells.
  • Fig. 11 shows IHC staining of the bound DIG-E2 antibody in normal human organ tissues: Adrenal gland. Panel A: testing tissue section pre-incubated with DIG-E2 antibody; Panel B: control tissue section without pre-incubation with DIG-E2 antibody.
  • Fig. 12 shows IHC staining of the bound DIG-E2 antibody in normal human organ tissues: Bone marrow. Panel A: testing tissue section pre-incubated with DIG-E2 antibody; Panel B: control tissue section without pre-incubation with DIG-E2 antibody.
  • Fig. 13 shows IHC staining of the bound DIG-E2 antibody in normal human organ tissues: Breast.
  • Panel A testing tissue section pre-incubated with DIG-E2 antibody
  • Panel B control tissue section without pre-incubation with DIG-E2 antibody.
  • Fig. 14 shows IHC staining of the bound DIG-E2 antibody in normal human organ tissues: Cerebellum tissue. Panel A: testing tissue section pre-incubated with DIG-E2 antibody; Panel B: control tissue section without pre-incubation with DIG-E2 antibody.
  • Fig. 16 shows IHC staining of the bound DIG-E2 antibody in normal human organ tissues: Colon.
  • Panel A testing tissue section pre-incubated with DIG-E2 antibody
  • Panel B control tissue section without pre-incubation with DIG-E2 antibody.
  • Fig. 17 shows IHC staining of the bound DIG-E2 antibody in normal human organ tissues: Esophagus.
  • Panel A testing tissue section pre-incubated with DIG-E2 antibody
  • Panel B control tissue section without pre-incubation with DIG-E2 antibody.
  • Fig. 18 shows IHC staining of the bound DIG-E2 antibody in normal human organ tissues: Eye.
  • Panel A testing tissue section pre-incubated with DIG-E2 antibody
  • Panel B control tissue section without pre-incubation with DIG-E2 antibody.
  • Fig. 19 shows IHC staining of the bound DIG-E2 antibody in normal human organ tissues: Heart.
  • Panel A testing tissue section pre-incubated with DIG-E2 antibody
  • Panel B control tissue section without pre-incubation with DIG-E2 antibody.
  • Fig. 20 shows IHC staining of the bound DIG-E2 antibody in normal human organ tissues: Hypophysis. Panel A: testing tissue section pre-incubated with DIG-E2 antibody; Panel B: control tissue section without pre-incubation with DIG-E2 antibody.
  • Fig. 21 shows IHC staining of the bound DIG-E2 antibody in normal human organ tissues: Kidney.
  • Panel A testing tissue section pre-incubated with DIG-E2 antibody;
  • Panel B control tissue section without pre-incubation with DIG-E2 antibody.
  • Fig. 22 shows IHC staining of the bound DIG-E2 antibody in normal human organ tissues: Larynx.
  • Panel A testing tissue section pre-incubated with DIG-E2 antibody;
  • Panel B control tissue section without pre-incubation with DIG-E2 antibody.
  • Fig. 23 shows IHC staining of the bound DIG-E2 antibody in normal human organ tissues: Spleen.
  • Panel A testing tissue section pre-incubated with DIG-E2 antibody
  • Panel B control tissue section without pre-incubation with DIG-E2 antibody.
  • Fig. 24 shows IHC staining of the bound DIG-E2 antibody in normal human organ tissues: Liver. Panel A: testing tissue section pre-incubated with DIG-E2 antibody; Panel B : control tissue section without pre-incubation with DIG-E2 antibody.
  • Fig. 25 shows IHC staining of the bound DIG-E2 antibody in normal human organ tissues: Lung.
  • Panel A testing tissue section pre-incubated with DIG-E2 antibody;
  • Panel B control tissue section without pre-incubation with DIG-E2 antibody.
  • Fig. 26 shows IHC staining of the bound DIG-E2 antibody in normal human organ tissues: Lymph node.
  • Panel A testing tissue section pre-incubated with DIG-E2 antibody
  • Panel B control tissue section without pre-incubation with DIG-E2 antibody.
  • Fig. 27 shows IHC staining of the bound DIG-E2 antibody in normal human organ tissues: Nerve.
  • Panel A testing tissue section pre-incubated with DIG-E2 antibody
  • Panel B control tissue section without pre-incubation with DIG-E2 antibody.
  • Fig. 29 shows IHC staining of the bound DIG-E2 antibody in normal human organ tissues: Pancreas. Panel A: testing tissue section pre-incubated with DIG-E2 antibody; Panel B : control tissue section without pre-incubation with DIG-E2 antibody.
  • Fig. 31 shows IHC staining of the bound DIG-E2 antibody in normal human organ tissues: Skin.
  • Panel A testing tissue section pre-incubated with DIG-E2 antibody
  • Panel B control tissue section without pre-incubation with DIG-E2 antibody.
  • Fig. 32 shows IHC staining of the bound DIG-E2 antibody in normal human organ tissues: Small intestine.
  • Panel A testing tissue section pre-incubated with DIG-E2 antibody
  • Panel B control tissue section without pre-incubation with DIG-E2 antibody.
  • Fig. 33 shows IHC staining of the bound DIG-E2 antibody in normal human organ tissues: Stomach.
  • Panel A testing tissue section pre-incubated with DIG-E2 antibody
  • Panel B control tissue section without pre-incubation with DIG-E2 antibody.
  • Fig. 34 shows IHC staining of the bound DIG-E2 antibody in normal human organ tissues: Striated muscle.
  • Panel A testing tissue section pre-incubated with DIG-E2 antibody
  • Panel B control tissue section without pre-incubation with DIG-E2 antibody.
  • Fig. 35 shows IHC staining of the bound DIG-E2 antibody in normal human organ tissues: Testis. Panel A: testing tissue section pre-incubated with DIG-E2 antibody; Panel B: control tissue section without pre-incubation with DIG-E2 antibody.
  • Fig. 36 shows IHC staining of the bound DIG-E2 antibody in normal human organ tissues: Thymus gland.
  • Panel A testing tissue section pre-incubated with DIG-E2 antibody
  • Panel B control tissue section without pre-incubation with DIG-E2 antibody.
  • Fig. 37 shows IHC staining of the bound DIG-E2 antibody in normal human organ tissues: Tongue.
  • Panel A testing tissue section pre-incubated with DIG-E2 antibody
  • Panel B control tissue section without pre-incubation with DIG-E2 antibody.
  • Fig. 38 shows IHC staining of the bound DIG-E2 antibody in normal human organ tissues: Uterus.
  • Panel A testing tissue section pre-incubated with DIG-E2 antibody
  • Panel B control tissue section without pre-incubation with DIG-E2 antibody.
  • FIG. 39 is a chart showing T-cell activation in one day cocultures with various target cells.
  • the percentage of E2 CAR T cells which are activated after the one-day coculture (y-axis) is graphed against FR expression level of the target cells at the time of the assay (x- axis).
  • OV90 cells ⁇
  • SKOV3 cells ⁇
  • IGROV1 cells IGROV1 cells
  • HOS-Fr a cells ⁇
  • MDA- MB-231 cells MDA- MB-231 cells.
  • FIG. 40 is a graph showing target cell apoptosis in 2 day coculture with the 5 different cell types (OV90 cells; SKOV3 cells; IGROV1 cells; HOS-Fr a cells; MDA-MB-231 cells) under three different conditions. For each cell type the conditions are as follows: Left bar target cells alone +EC17; Middle bar CAR T plus target cell cocultures without EC 17 pre treatment; Right bar CAR T plus treated target cells with EC 17 pre-treatment.
  • FIG. 41 is a chart showing functional folate receptor expression levels in 5 different cell types (OV90 cells; SKOV3 cells; IGROV1 cells; HOS-Fr a cells; MDA-MB-231 cells).
  • FIG. 42 is a cartoon showing the experimental timeline for tumor bearing and naive mice.
  • FIG. 43 (left panel) is a chart showing levels of cytokine IFN-Y was EC- 17 dependent in both naive and tumor bearing mice, and that the increase in IFN-Y was much lower in naive mice compared to mice bearing MDA-MB-231 tumors (23 -fold lower).
  • FIG. 43 (right panel) is a chart showing FACS analysis CAR-T expansion in mice bearing MDA-MB- 231 tumors and no detectable CAR-T cell expansion in naive mice.
  • FIG. 44 is a chart showing cytokine (IL-2) production from Mock T- Cells and anti-FLCAR T-cells at quantitatively similar levels in co-culture with the positive control cell line K562-OKT3 (left pair of bars, where Mock is the left bar and anti-FLCAR T- cells is the right bar); no cytokine was produced by either Mock or anti-FLCAR T-cells upon co-culture with K562 (middle); and anti-FLCAR T-cells were the only cells able to elicit secretion of the cytokine IL-2, but only with pre treatment of EC- 17 (right, where only anti- FLCAR T-cells show a result).
  • FIG. 44 is a chart showing cytokine (IL-2) production from Mock T- Cells and anti-FLCAR T-cells at quantitatively similar levels in co-culture with the positive control cell line K562-OKT3 (left pair of bars, where Mock is the left bar and anti-FLCAR
  • FIG. 45 (upper left) is a chart showing percentage lysis when CD8+ Mock T cells and anti-FLCAR T-cells are co-cultured with negative control K562 cells at a ratio of 30:1, 10:1, 3:1, or 1:1.
  • FIG. 45 (upper right) is a chart showing percentage lysis when CD8+ Mock T cells and anti-FLCAR T-cells are co-cultured with K562+OKT3 target cells at a ratio of 30:1, 10:1, 3: 1, or 1:1.
  • FIG. 45 (upper left) is a chart showing percentage lysis when CD8+ Mock T cells and anti-FLCAR T-cells are co-cultured with K562+OKT3 target cells at a ratio of 30:1, 10:1, 3: 1, or 1:1.
  • FIG. 45 (lower left) is a chart showing percentage lysis when CD8+ Mock T cells and anti-FLCAR T-cells are co-cultured with unlabeled MDA-MB-231 cells at a ratio of 30: 1, 10:1, 3:1, or 1:1.
  • FIG. 45 (lower right) is a chart showing percentage lysis when CD8+ Mock T cells and anti-FLCAR T-cells are co-cultured with EC17 labeled MDA-MB-231 cells at a ratio of 30:1, 10: 1, 3: 1, or 1:1.
  • FIG. 46 (top) shows the chemical structure of FITC-folate.
  • FIG. 46 (bottom) is a chart showing the dose response curve for FITC-folate.
  • FIG. 47 (top) shows the chemical structure of FITC-DUPA.
  • FIG. 47 (bottom) is a chart showing the dose response curve for FITC-DUPA.
  • FIG. 48 (top) shows the chemical structure of FITC-CA9.
  • FIG. 48 (bottom) is a chart showing the dose response curve for FITC-CA9.
  • FIG. 49 shows the chemical structure of FITC-NK1R.
  • FIG. 49 (bottom) is a chart showing the dose response curve for FITC- NK1R.
  • FIG. 50 shows binding (by FACS analysis) of bridges to tumor cells used in an in vivo model and expressing the receptor corresponding to the small molecule ligand of the bridge.
  • FIG. 51 shows EC 17 induction of a potent FR-dependent tumor cell killing by
  • FIG. 52 shows the correlation of cytolytic activity of CAR T cells with functional FR levels on tumor cells.
  • FIG. 53 shows ECl7/FR-dependent CAR T cell activation and exhaustion.
  • the first two open bars represent CAR-T and Mock transduced T cells only without target cells.
  • Legend top to bottom for second to fourth panels bars left to right.
  • FIG. 54 shows ECl7/FR-dependent CAR T cell exhaustion profiles.
  • Top row Colored pie charts representing change in the differentiation status of CAR-T cells in culture without target cells on days 0-3.
  • FIG. 55 shows a fully human CAR construct comprised of anti-FITC scFv (clone E2), a full-length IgG4 spacer (Fc derived hinge-CH2(L235D, N297Q)-CH3), CD28tm transmembrane domain, 4- 1 BB/ € ⁇ 3z cytoplasmic activation domains, and a non-functional truncated cell surface polypeptide of epidermal growth factor receptor (EGFRt).
  • EGFRt epidermal growth factor receptor
  • FIG. 56 Panel A: Kd values of 3 H-ECl7 uptake by FR+ target cells after a 2 hour incubation at 37°C (calculated from the numbers of molecules bound per cell).
  • FIG. 58 shows functional FR levels on tumor cell measured by a 3 H-FA (folic acid)-based binding assay (100 nM, 1 hour at 37°C).
  • FIG. 59 shows (Panel A) full-range EC17 dose response in specific lysis (%) of 5 FR (folate receptor)+ tumor cell lines co-cultured with EGFRt-sorted CAR-T cells for 24 hours at 1:1 E/T (Effector/Target) ratio; (panel B) maximum lysis (%) and EC50 values were obtained from the dose-response curves fitted up to 100 nM.
  • FIG. 60 shows the correlation of CAR-T cell activity with FR levels and tumor cells’ natural sensitivity.
  • Panel A Kinetics of specific lysis (%) at varying E/T ratios in FR+ (MDA-MB-231, KB, THPl-FR , OV90, HOS-FRoc) and FR-negative (HOS-l43b) cell lines after 16 and 48 hours of co-culture in the presence of 10 nM EC17.
  • Panel B Specific lysis (%) of target cells plotted against a linear scale of E/T ratios. High FR+ KB cells demonstrated an early resistance at 16 hours while FR-negative HOS-l43b failed to respond.
  • Panel C Excluding KB cells, a semi-log correlation was established between specific lysis (%) at 16 hours of co culture and functional FR levels on tumor cells.
  • Panel D 3D diagrams depicting the relationships between the levels of CAR-T cell derived Thl cytokines (IFNy, IL-2 and TNFoc) after 44 hours of co-culture at 10 nM EC17 with varying E/T ratios.
  • Panel E CAR-T cell derived Thl cytokine levels plotted against FR levels of FR+ target cell in a LoglO scale.
  • FIG. 61 shows (panel A) bar graph to show EC 17 loading status of target cells confirmed by flow cytometry and expressed as MFI (EC 17 was undetectable on FR-negative cell lines) and (panel B) target cell apoptosis (%) detected by Annexin V staining after 2 days of co-culture as described in Figure 54.
  • FIG. 62 shows pharmacokinetics and tumor uptake of CAR-T cells in vivo.
  • Panel A Schematic diagram of the experimental layout to show animal collection schedule in relation to CAR-T cell injection and weekly EC 17 doses (500 nmol/kg) in MDA-MB-231 tumor- bearing mice. A total of 15 mice received ⁇ 4.8 million of EGFRt-sorted CAR-T cells on day 0 (3 mice had large tumors to begin with).
  • Panel B Left panel showing measurements of tumor volume and change in body weight; Middle plot showing CAR-T cell expansion in the blood 7 days after a single dose of EC17; Right bar graph showing differentiation profiles of circulating CD4/CD8 CAR-T cell subsets in mice with small and larger tumors.
  • Panel C Measurements of change in tumor volume and body weight.
  • Panel D Top plot showing CAR-T cell kinetics in the blood (solid line) versus that of tumor (dotted line). Bottom bar graph showing changes in CAR-T cell phenotypes in the blood.
  • Panel E Kinetic changes in the surface expression of activation markers (4-1BB, PD1) on tumor-infiltrating CAR-T cells.
  • FIG. 63 shows dietary folate effect on antitumor activity and CRS toxicity.
  • Panel B Bar graph to show circulating CAR-T cells (human CD3s+ EGFRt+) measured at the end of study on day 52 (FA-deficient) and day 59 (FA- replete), respectively.
  • “a” or“an” may mean one or more.
  • “about” in reference to a numeric value including, for example, whole numbers, fractions, and
  • percentages generally refers to a range of numerical values (e.g., +/- 5 % to 10% of the recited value) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result).
  • the terms“treat,”“treating,”“treated,” or“treatment” refer to both therapeutic treatment and prophylactic or preventative treatment.
  • the terms“ameliorate,”“ameliorating,”“amelioration,” or “ameliorated” in reference to cancer can mean reducing the symptoms of the cancer, reducing the size of a tumor, completely or partially removing the tumor (e.g., a complete or partial response), causing stable disease, preventing progression of the cancer (e.g., progression free survival), or any other effect on the cancer that would be considered by a physician to be a therapeutic, prophylactic, or preventative treatment of the cancer.
  • off-target toxicity means organ damage or a reduction in the patient’ s weight that is unacceptable to the physician treating the patient, or any other effect on the patient that is unacceptable to the physician treating the patient, for example, B cell aplasia, fever, a drop in blood pressure, or pulmonary edema.
  • transduction and“transfection” are used equivalently and the terms mean introducing a nucleic acid into a cell by any artificial method, including viral and non- viral methods.
  • a small molecule ligand linked to a targeting moiety by a linker is used as a bridge between a cancer and CAR T cells (i.e, T cells expressing a chimeric antigen receptor), wherein the CAR T cells comprise a genetically engineered CAR directed to the targeting moiety, wherein the CAR comprises an E2 anti fluorescein antibody fragment.
  • the bridge directs the CAR T cells to the cancer for amelioration of the cancer.
  • the“small molecule ligand” can be a folate, a CAIX ligand, DUPA, an NK-1R ligand, a ligand of gamma glutamyl transpeptidase, an NKG2D ligand, or a CCK2R ligand, each of which is a small molecule ligand that binds specifically to a cancer cell type (i.e., the receptor for each of these ligands is overexpressed on cancers compared to normal tissues).
  • The“targeting moiety” linked to the small molecule ligand binds to the recognition region of the genetically engineered CAR expressed by CAR T cells, wherein the CAR comprises an E2 anti-fluorescein antibody fragment. Accordingly, the recognition region of the CAR (e.g., a single chain fragment variable region (scFv) of an E2 anti-fluorescein antibody) is directed to the“targeting moiety.”
  • the small molecule ligand linked to a targeting moiety by a linker acts as a bridge between the cancer and the CAR T cells, wherein the CAR T cells comprise the genetically engineered CAR, directing the CAR T cells to the cancer for amelioration of the cancer.
  • the targeting moiety recognized by the CAR T cell may remain constant so that one type of CAR T cell, can be used, while the small molecule ligand that binds to the cancer can be altered to allow targeting of a wide variety of cancers.
  • the small molecule ligand linked to a targeting moiety by a linker is referred to as a“compound.”
  • the clause“E2 anti-fluorescein antibody fragment” means a CAR comprising a fragment (e.g., an scFv fragment) of the E2 anti-fluorescein antibody.
  • the E2 anti-fluorescein antibody is described, for example, in Vaughan, et ak,
  • the CAR can further comprise an IgG4 hinge domain, a CD3z activation domain, and/or a 4-1BB co-stimulation domain, or any other suitable domain such as the EGFRt domain.
  • the CAR can be encoded by a polynucleotide having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity to SEQ ID NO:l.
  • the CAR can be encoded by a polynucleotide that hybridizes under high stringency conditions to a polynucleotide having SEQ ID NO:l.
  • the CAR can be encoded by a polynucleotide having SEQ ID NO:l, or by a degenerate variant of SEQ ID NO:l.
  • the CAR protein sequence can have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:2.
  • the CAR protein sequence can have up to about 50 conservative amino acid substitutions.
  • the CAR binds fluorescein.
  • a method of treatment of a cancer comprises i) administering to a patient a compound, or a pharmaceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked to a targeting moiety by a linker, ii) administering to the patient a first dose of a CAR T cell composition, wherein the CAR T cell composition comprises CAR T cells, wherein the CAR T cells comprise a CAR, and wherein the CAR comprises an E2 anti-fluorescein antibody fragment, and iii)
  • a method of treatment of a cancer comprises i) administering to a patient a compound, or a pharmaceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked to a targeting moiety by a linker, and ii) administering to the patient a CAR T cell composition comprising CAR T cells, wherein the CAR T cells comprise a CAR, wherein the CAR comprises an E2 anti-fluorescein antibody fragment, and wherein the CAR T cell composition comprises a mixture of the CAR T cells and non-transformed T cells.
  • a method of treatment of a cancer comprises i) administering to a patient a compound, or a pharmaceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked to a targeting moiety by a linker, ii) administering to the patient a CAR T cell composition wherein the CAR T cell composition comprises CAR T cells wherein the CAR T cells comprise a CAR, wherein the CAR comprises an E2 anti-fluorescein antibody fragment, and iii) administering to the patient a folate, a conjugate comprising a folate wherein the conjugate comprising a folate does not comprise a targeting moiety, or a drug that inhibits activation of the CAR T cells.
  • a method of treatment of a cancer comprises i) administering to a patient a compound, or a pharmaceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked to a targeting moiety by a linker, and wherein the compound, or the pharmaceutically acceptable salt thereof, is administered at a dose of about 10 nmoles/kg of body weight of the patient to about 2500 nmoles/kg of body weight of the patient, and ii) administering to the patient a CAR T cell composition comprising CAR T cells, wherein the CAR T cells comprise a CAR, wherein the CAR comprises an E2 anti-fluorescein antibody fragment, and wherein the CAR T cells are at a dose of about 1 million of the CAR T cells to about 15 million of the CAR T cells.
  • a method of treatment of a cancer comprises i) administering continuously to a patient a compound, or a pharmaceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked to a targeting moiety by a linker, ii) administering to the patient a CAR T cell composition comprising CAR T cells wherein the CAR T cells comprise a CAR, wherein the CAR comprises an E2 anti-fluorescein antibody fragment, and iii) ending the continuous administration of the compound, or the pharmaceutically acceptable salt thereof, to inhibit or prevent cytokine release syndrome in the patient.
  • a method of treatment of a cancer comprises i) administering to a patient a compound, or a pharmaceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked to a targeting moiety by a linker, wherein at least a first dose and a second dose of the compound, or the pharmaceutically acceptable salt thereof, are administered to the patient, wherein the first dose and the second dose are different, wherein the second dose of the compound, or the pharmaceutically acceptable salt thereof, is about 2-fold to about l5000-fold greater in amount than the first dose of the compound, or the pharmaceutically acceptable salt thereof, and ii) administering to the patient a CAR T cell composition comprising CAR T cells wherein the CAR T cells comprise a CAR, wherein the CAR comprises an E2 anti-fluorescein antibody fragment.
  • a method of treatment of a cancer comprises i) administering to a patient a compound, or a pharmaceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked to a targeting moiety by a linker and wherein the compound, or the pharmaceutically acceptable salt thereof, is administered once weekly to the patient, and ii) administering to the patient a CAR T cell composition comprising CAR T cells wherein the CAR T cells comprise a CAR, wherein the CAR comprises an E2 anti-fluorescein antibody fragment.
  • a method of treatment of a cancer comprises i) administering to a patient a first dose of a compound, or a
  • the compound comprises a small molecule ligand linked to a targeting moiety by a linker, ii) administering to the patient at least a second dose of the compound, or a pharmaceutically acceptable salt thereof, wherein the second dose of the compound, or the pharmaceutically acceptable salt thereof, is at least about 50 percent lower in amount than the first dose of the compound, or the pharmaceutically acceptable salt thereof, and iii) administering to the patient a dose of a CAR T cell composition comprising CAR T cells wherein the CAR T cells comprise a CAR and wherein the CAR comprises an E2 anti-fluorescein antibody fragment.
  • a method of treatment of a cancer comprising i) administering to a patient a compound, or a pharmaceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked to a targeting moiety by a linker;
  • a first dose of a CAR T cell composition comprising CAR T cells wherein the CAR T cells comprise a CAR directed to the targeting moiety and wherein the CAR comprises an E2 anti-fluorescein antibody fragment;
  • a CAR T cell composition comprising CAR T cells wherein the CAR T cells comprise the CAR directed to the targeting moiety and wherein the CAR comprises an E2 anti-fluorescein antibody fragment.
  • a method of treatment of a cancer comprising i) administering to a patient a compound, or a pharmaceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked to a targeting moiety by a linker;
  • CAR T cell composition comprises CAR T cells, wherein the CAR T cells comprise a CAR directed to the targeting moiety wherein the CAR comprises an E2 anti-fluorescein antibody fragment, and wherein the CAR T cell composition comprises a mixture of the CAR T cells and non-transformed T cells.
  • a method of treatment of a cancer comprising i) administering to a patient a compound, or a pharmaceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked to a targeting moiety by a linker; ii) administering to the patient a CAR T cell composition wherein the CAR T cell composition comprises CAR T cells wherein the CAR T cells comprise a CAR directed to the targeting moiety and wherein the CAR comprises an E2 anti-fluorescein antibody fragment; and
  • a conjugate comprising a folate wherein the conjugate comprising a folate does not comprise a targeting moiety, or an agent that inhibits activation of the CAR T cells.
  • step iii comprises administering a folate.
  • step iii comprises administering folic acid or leucovorin.
  • step iii comprises administering the conjugate comprising a folate.
  • X 1 and Y 1 are each-independently selected from the group consisting of halo, R 2 , OR 2 , SR 3 , and NR 4 R 5 ;
  • Q is selected from the group consisting of C and CH;
  • X 2 and X 3 are each independently selected from the group consisting of oxygen, sulfur, -C(Z)-, -C(Z)0-, -OC(Z)-, -N(R 4b )-, -C(Z)N(R 4b )-, -N(R 4b )C(Z)-,
  • R 1 is selected-from the group consisting of hydrogen, halo, C 1 -C 12 alkyl, and Ci-
  • 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, halo, C1-C12 alkyl, C1-C12 alkoxy, C1-C12 alkanoyl, C1-C12 alkenyl, C 1 -C 12 alkynyl, (Ci-C 12 alkoxy)carbonyl, and (C 1 -C 12 alkylamino)carbonyl;
  • R 6 and R 7 are each independently selected from the group consisting of hydrogen, halo, C 1 -C 12 alkyl, and C 1 -C 12 alkoxy; or, R 6 and R 7 are taken together to form a carbonyl group;
  • R 6a and R 7a are each independently selected from the group consisting of hydrogen, halo, C 1 -C 12 alkyl, and C 1 -C 12 alkoxy; or R 6a and R 7a are taken together to form a carbonyl group;
  • * represents an optional covalent bond to the rest of the conjugate.
  • lymphocyte-specific protein tyrosine kinase inhibitor a PI3 kinase inhibitor, an inhibitor of an IL-2 inducible T cell kinase, a JAK inhibitor, a BTK inhibitor, EC2319, and an agent that blocks CAR T cell binding to the compound, or the pharmaceutically
  • a method of treatment of a cancer comprising i) administering to a patient a compound, or a pharmaceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked to a targeting moiety by a linker, wherein at least a first dose and a second dose of the compound, or the pharmaceutically acceptable salt thereof, are administered to the patient, wherein the first dose and the second dose are different, wherein the second dose of the compound, or the pharmaceutically acceptable salt thereof, is about 2-fold to about l5000-fold greater in amount than the first dose of the compound, or the pharmaceutically acceptable salt thereof; and
  • CAR T cell composition comprising CAR T cells wherein the CAR T cells comprise a CAR directed to the targeting moiety and wherein the CAR comprises an E2 anti-fluorescein antibody fragment.
  • the third dose of the compound, or the pharmaceutically acceptable salt thereof is about 800-fold to about lOOOO-fold greater in amount than the first dose of the compound, or the pharmaceutically acceptable salt thereof.
  • a method of treatment of a cancer comprising i) administering to a patient a first dose of a compound, or a
  • the compound comprises a small molecule ligand linked to a targeting moiety by a linker;
  • a CAR T cell composition comprising CAR T cells wherein the CAR T cells comprise a CAR directed to the targeting moiety and wherein the CAR comprises an E2 anti-fluorescein antibody fragment.
  • linker comprises polyethylene glycol (PEG), polyproline, a hydrophilic amino acid, a sugar, an unnatural peptidoglycan, a polyvinylpyrrolidone, pluronic F-127, or a combination thereof.
  • L represents the linker
  • T represents the targeting moiety
  • L comprises a structure having the formula
  • any one of clauses 1 to 125 wherein the cancer is selected from the group consisting of lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head, cancer of the neck, cutaneous melanoma, intraocular melanoma uterine cancer, ovarian cancer, endometrial cancer, rectal cancer, stomach cancer, colon cancer, breast cancer, triple negative breast cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin’s Disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, non-small cell lung cancer, cancer of the adrenal gland, sarcoma of soft tissue, osteosarcoma, cancer of the urethra, prostate cancer, chronic leukemia, acute leukemia, acute myelocytic leukemia, lymphocytic lymphom
  • a method of treatment of a cancer comprises i) administering to a patient a compound, or a pharmaceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked to a targeting moiety by a linker, ii) administering to the patient a first dose of a CAR T cell composition, wherein the CAR T cell composition comprises CAR T cells, wherein the CAR T cells comprise a CAR, and wherein the CAR comprises an E2 anti-fluorescein antibody fragment, and iii) administering to the patient a second dose of the CAR T cell composition wherein the CAR T cell composition comprises CAR T cells, wherein the CAR T cells comprise a CAR, and wherein the CAR comprises an E2 anti-fluorescein antibody fragment.
  • a method of treatment of a cancer comprises i) administering to a patient a compound, or a pharmaceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked to a targeting moiety by a linker, and ii) administering to the patient a CAR T cell composition comprising CAR T cells, wherein the CAR T cells comprise a CAR, wherein the CAR comprises an E2 anti-fluorescein antibody fragment, and wherein the CAR T cell composition comprises a mixture of the CAR T cells and non-transformed T cells.
  • a method of treatment of a cancer comprises i) administering to a patient a compound, or a pharmaceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked to a targeting moiety by a linker, ii) administering to the patient a CAR T cell composition wherein the CAR T cell composition comprises CAR T cells wherein the CAR T cells comprise a CAR, wherein the CAR comprises an E2 anti-fluorescein antibody fragment, and iii) administering to the patient a folate, a conjugate comprising a folate wherein the conjugate comprising a folate does not comprise a targeting moiety, or a drug that inhibits activation of the CAR T cells.
  • a method of treatment of a cancer comprises i) administering to a patient a compound, or a pharmaceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked to a targeting moiety by a linker, and wherein the compound, or the pharmaceutically acceptable salt thereof, is administered at a dose of about 10 nmoles/kg of body weight of the patient to about 2500 nmoles/kg of body weight of the patient, and ii) administering to the patient a CAR T cell composition comprising CAR T cells, wherein the CAR T cells comprise a CAR, wherein the CAR comprises an E2 anti-fluorescein antibody fragment, and wherein the CAR T cells are at a dose of about 1 million of the CAR T cells to about 15 million of the CAR T cells.
  • a method of treatment of a cancer comprises i) administering continuously to a patient a compound, or a pharmaceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked to a targeting moiety by a linker, ii) administering to the patient a CAR T cell composition comprising CAR T cells wherein the CAR T cells comprise a CAR, wherein the CAR comprises an E2 anti-fluorescein antibody fragment, and iii) ending the continuous administration of the compound, or the pharmaceutically acceptable salt thereof, to inhibit or prevent cytokine release syndrome in the patient.
  • a method of treatment of a cancer comprises i) administering to a patient a compound, or a pharmaceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked to a targeting moiety by a linker, wherein at least a first dose and a second dose of the compound, or the pharmaceutically acceptable salt thereof, are administered to the patient, wherein the first dose and the second dose are different, wherein the second dose of the compound, or the pharmaceutically acceptable salt thereof, is about 2-fold to about l5000-fold greater in amount than the first dose of the compound, or the pharmaceutically acceptable salt thereof, and ii) administering to the patient a CAR T cell composition comprising CAR T cells wherein the CAR T cells comprise a CAR, wherein the CAR comprises an E2 anti-fluorescein antibody fragment.
  • a method of treatment of a cancer comprises i) administering to a patient a compound, or a pharmaceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked to a targeting moiety by a linker and wherein the compound, or the pharmaceutically acceptable salt thereof, is administered once weekly to the patient, and ii) administering to the patient a CAR T cell composition comprising CAR T cells wherein the CAR T cells comprise a CAR, wherein the CAR comprises an E2 anti-fluorescein antibody fragment.
  • a method of treatment of a cancer comprises i) administering to a patient a first dose of a compound, or a
  • the compound comprises a small molecule ligand linked to a targeting moiety by a linker, ii) administering to the patient at least a second dose of the compound, or a pharmaceutically acceptable salt thereof, wherein the second dose of the compound, or the pharmaceutically acceptable salt thereof, is at least about 50 percent lower in amount than the first dose of the compound, or the pharmaceutically acceptable salt thereof, and iii) administering to the patient a dose of a CAR T cell composition comprising CAR T cells wherein the CAR T cells comprise a CAR and wherein the CAR comprises an E2 anti-fluorescein antibody fragment.
  • the cancer may be lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head, cancer of the neck, cutaneous melanoma, intraocular melanoma uterine cancer, ovarian cancer, endometrial cancer, rectal cancer, stomach cancer, colon cancer, breast cancer, triple negative breast cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin’s Disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, non-small cell lung cancer, cancer of the adrenal gland, sarcoma of soft tissue, osteosarcoma, cancer of the urethra, prostate cancer, chronic leukemia, acute leukemia, including acute myelocytic leukemia, a lymphocytic lymphoma, myeloid leukemia, myelomono
  • the cancer is a folate receptor expressing cancer. In another embodiment, the cancer is a folate receptor a-expressing cancer. In yet another embodiment, the cancer is a folate receptor b-expressing cancer. In some aspects of these embodiments, the cancer is an endometrial cancer, a non-small cell lung cancer, an ovarian cancer, or a triple-negative breast cancer. In another embodiment, the cancer being treated is a tumor. In another embodiment, the cancer is malignant. In another embodiment, the cancer is acute myelocytic leukemia. In yet another embodiment, the cancer is acute myelocytic leukemia and the cancer expresses the folate receptor-b.
  • the cancer is acute myelocytic leukemia and the CAR-T cells have a central memory/effector memory phenotype.
  • the CD8:CD4 ratio of the CAR T cells is about 1: 1, about a 1.2 to 1 ratio, about a 1 to 1.2 ratio, about a 1.3 to 1 ratio, about a 1 to 1.3 ratio, about a 1.4 to 1 ratio, about a 1 to 1.4 ratio, about a 1.5 to 1 ratio, or about a 1 to 1.5 ratio.
  • the CAR T cells associated with the tumor can have increased CD25 expression relative to the CAR T cells not associated with the tumor.
  • the“small molecule ligand” can be a folate, DUPA (a ligand bound by PSMA-positive human prostate cancer cells and other cancer cell types), an NK-1R ligand (receptors for NK-1R the ligand found, for example, on cancers of the colon and pancreas), a CAIX ligand (receptors for the CAIX ligand found, for example, on renal, ovarian, vulvar, and breast cancers), a ligand of gamma glutamyl transpeptidase (the transpeptidase overexpressed, for example, in ovarian cancer, colon cancer, liver cancer, astrocytic gliomas, melanomas, and leukemias), an NKG2D ligand (receptors for the NKG2D ligand found, for example, on cancers of the lung, colon, kidney, prostate, and on T and B cell lymphomas), or a CCK2R ligand (receptor
  • the small molecule ligand may have a mass of less than about 10,000 Daltons, less than about 9000 Daltons, less than about 8,000 Daltons, less than about 7000 Daltons, less than about 6000 Daltons, less than about 5000 Daltons, less than about 4500 Daltons, less than about 4000 Daltons, less than about 3500 Daltons, less than about 3000 Daltons, less than about 2500 Daltons, less than about 2000 Daltons, less than about 1500 Daltons, less than about 1000 Daltons, or less than about 500 Daltons.
  • the small molecule ligand may have a mass of about 1 to about 10,000 Daltons, about 1 to about 9000 Daltons, about 1 to about 8,000 Daltons, about 1 to about 7000 Daltons, about 1 to about 6000 Daltons, about 1 to about 5000 Daltons, about 1 to about 4500 Daltons, about 1 to about
  • a DUPA derivative can be the ligand of the small molecule ligand linked to a targeting moiety, and DUPA derivatives are described in WO 2015/057852, incorporated herein by reference.
  • the small molecule ligand in the context of the“small molecule ligand linked to a linker” is a folate.
  • the folate can be folic acid, a folic acid analog, or another folate receptor-binding molecule.
  • analogs of folate that can be used include folinic acid, pteropolyglutamic acid, and folate receptor-binding pteridines such as tetrahydropterins, dihydrofolates, tetrahydrofolates, and their deaza and dideaza analogs.
  • the terms“deaza” and“dideaza” analogs refers to the art recognized analogs having a carbon atom substituted for one or two nitrogen atoms in the naturally occurring folic acid structure.
  • the deaza analogs include the 1 -deaza, 3- deaza, 5-deaza, 8-deaza, and lO-deaza analogs.
  • the dideaza analogs include, for example, 1,5 dideaza, 5,l0-dideaza, 8,l0-dideaza, and 5,8-dideaza analogs.
  • the foregoing folic acid analogs are conventionally termed“folates,” reflecting their capacity to bind to folate receptors.
  • folate receptor-binding analogs include aminopterin, amethopterin (methotrexate), N10- methylfolate, 2-deamino-hydroxyfolate, deaza analogs such as l-deazamethopterin or 3- deazamethopterin, and 3',5'-dichloro-4-amino-4-deoxy-NlO-methylpteroylglutamic acid (dichloromethotrexate) .
  • the small molecule ligand in the context of the“small molecule ligand linked to a linker” can have the formula
  • X 1 and Y 1 are each-independently selected from the group consisting of halo, R 2 , OR 2 , SR 3 , and NR 4 R 5 ;
  • Q is selected from the group consisting of C and CH;
  • X 2 and X 3 are each independently selected from the group consisting of oxygen, sulfur, -C(Z)-, -C(Z)0-, -OC(Z)-, -N(R 4b )-, -C(Z)N(R 4b )-, -N(R 4b )C(Z)-,
  • R 1 is selected-from the group consisting of hydrogen, halo, Ci-Ci 2 alkyl, and Ci-
  • 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, halo, C1-C12 alkyl, C1-C12 alkoxy, C1-C12 alkanoyl, C1-C12 alkenyl, C 1 -C 12 alkynyl, (Ci-C 12 alkoxy)carbonyl, and (C 1 -C 12 alkylamino)carbonyl;
  • R 6 and R 7 are each independently selected from the group consisting of hydrogen, halo, C 1 -C 12 alkyl, and C 1 -C 12 alkoxy; or, R 6 and R 7 are taken together to form a carbonyl group;
  • * represents an optional covalent bond to the rest of the conjugate.
  • the“targeting moiety” that binds to the CAR comprising an E2 anti-fluorescein antibody fragment and expressed by CAR T cells can be selected, for example, from fluorescein, fluorescein isothiocyanate (FITC), and NHS -fluorescein.
  • the identity of the targeting moiety is limited only in that it should be recognized and bound by the CAR comprisimg an E2 anti-fluorescein antibody fragment, preferably with specificity, and that it have a relatively low molecular weight.
  • exemplary targeting moieties are haptens, including small molecular weight organic molecules.
  • the targeting moiety can have the following illustrative structure:
  • the linker can comprise polyethylene glycol (PEG), polyproline, a hydrophilic amino acid, a sugar, an unnatural peptidoglycan, a
  • the linker in the compound, or pharmaceutically acceptable salt thereof, described herein can comprise a direct linkage (e.g., a reaction between the isothiocyanate group of FITC and a free amine group of a small molecule ligand) or the linkage can be through an intermediary linker.
  • an intermediary linker can be any biocompatible linker known in the art, such as a divalent linker.
  • the divalent linker can comprise about 1 to about 30 carbon atoms.
  • the divalent linker can comprise about 2 to about 20 carbon atoms.
  • lower molecular weight divalent linkers i.e., those having an approximate molecular weight of about 30 to about 300 Daltons
  • linkers lengths that are suitable include, but are not limited to, linkers having 2, 3,
  • the small molecule ligand linked to a targeting moiety can be of the formula
  • L represents the linker
  • T represents the targeting moiety
  • L comprises a structure having the formula
  • the linker may be a divalent linker that may include one or more spacers.
  • 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 other divalent linker portions.
  • the compound, or the pharmaceutically acceptable salt thereof is not an antibody, and does not comprise a fragment of an antibody.
  • the targeting moiety does not comprise a peptide epitope.
  • the small molecule ligand linked to a targeting moiety by a linker comprises fluorescein isothiocyanate (FITC) linked to the small molecule ligand.
  • FITC fluorescein isothiocyanate
  • the cancer may overexpress a receptor for the small molecule ligand.
  • cytotoxic T cells, or another type of T cell can be transformed to express a CAR that comprises an E2 anti-fluorescein antibody fragment.
  • the CAR may target FITC
  • suitable acid addition salts are formed from acids which form non- toxic salts.
  • Illustrative examples include the acetate, aspartate, benzoate, besylate, bicarbonate/carbonate, bisulphate/sulphate, borate, camsylate, citrate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methylsulphate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate, saccharate, stearate, succinate, tartrate, to
  • suitable base salts are formed from bases which form non-toxic salts.
  • bases which form non-toxic salts.
  • Illustrative examples include the arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine and zinc salts.
  • Hemisalts of acids and bases may also be formed, for example, hemisulphate and hemicalcium salts.
  • the compound, or pharmaceutically acceptable salt thereof, described herein may exist in unsolvated forms as well as solvated forms, including hydrated forms.
  • the solvated forms are equivalent to unsolvated forms and are encompassed within the scope of the present invention.
  • Suitable co-stimulation domains include, but are not limited to, CD28, CD137 (4-1BB), a member of the tumor necrosis factor (TNF) receptor family, CD134 (0X40), a member of the TNFR- superfamily of receptors, CD27, CD30, CD150, DAP10, NKG2D, and CD278 (ICOS), a CD28-superfamily co- stimulatory molecule expressed on activated T cells, or combinations thereof.
  • the co- stimulation domains of the engineered CAR comprise CD 28 and CD137.
  • the activation signaling domain serves to activate T lymphocytes (e.g., cytotoxic T lymphocytes) upon binding of the CAR to a targeting moiety.
  • T lymphocytes e.g., cytotoxic T lymphocytes
  • suitable activation signaling domains include the T cell CD3z chain, CD3 delta receptor protein, mbl receptor protein, B29 receptor protein, and Fc receptor g. The skilled artisan 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 upon which they are modeled.
  • the variants have at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity with the amino acid sequence of the domain from which they are derived.
  • constructs encoding the CARs comprising the E2 anti-fluorescein antibody fragment are prepared using genetic engineering techniques. Such techniques are described in detail in Sambrook et a ,“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.
  • a plasmid or viral expression vector e.g., a lentiviral vector, a retrovirus vector, sleeping beauty, and piggyback (transposon/transposase systems that include a non- viral mediated CAR gene delivery system)
  • a fusion protein comprising a recognition region, one or more co- stimulation domains, and an activation signaling domain, in frame and linked in a 5’ to 3’ direction.
  • other arrangements are acceptable and include a recognition region, an activation signaling domain, and one or more co-stimulation domains.
  • the placement of the recognition region in the fusion protein will generally be such that display of the region on the exterior of the cell is achieved.
  • SEQ ID NO:1 E2 anti-fluorescein antibody fragment CAR nucleic acid sequence (insert)) atgcttctcctggtgacaagccttctgctctgtgagttaccacacccagcattcctcctgatcccaagcgtgctgacacagcctagctccgtg tctgccccctggccagaaagtgaccatcagctgtagcggcagcaccagcaacatcggcaacaactacgtgtcctggtatcagcagca cccggcaaggcccccaagctgatgatctacgacgtgtccaagcggcccagcggcgtgcccgatagatttccggcagcaagagcgg caacagcgccagccctggatatatcagcggcctgcaagag
  • the CAR expressing the E2 anti-fluorescein antibody fragment, comprises a recognition region and the recognition region is a single chain fragment variable (scFv) region of the E2 anti-fluorescein antibody, a co-stimulation domain and the co stimulation domain is CD137 (4-1BB), and an activation signaling domain and the activation signaling domain is a T cell O ⁇ 3z chain.
  • the CAR can further comprise any additional suitable domains. It is well-known to the skilled artisan that an anti- FITC scFv and an anti-fluorescein scFv are equivalent terms.
  • the“E2 anti-fluorescein antibody fragment” can be a CAR comprising a fragment (e.g., an scFv fragment) of the E2 anti-fluorescein antibody.
  • the E2 anti-fluorescein antibody is described, for example, in Vaughan, et al., Nature Biotechnol. Vol.14(3), pp.309-314, 1996, incorporated herein by reference.
  • the CAR expressing the E2 anti-fluorescein antibody fragment can have a binding affinity for fluorescein of about 0.7 nM to about 0.8 nM, about 0.72 nM to about 0.8 nM, about 0.73 nM to about 0.8 nM, about 0.72 nM to about 7.8 nM, about 0.73 to about 0.77 nM, or about 0.75 nM.
  • the CAR can further comprise an IgG4 hinge domain, a O ⁇ 3z activation domain, and/or a 4-1BB co-stimulation domain, and other suitable domains.
  • Suitable methods for preparing a transduced population of T lymphocytes expressing the E2 anti-fluorescein antibody fragment are well-known to the skilled artisan, and are described in Sambrook et ak,“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.
  • CAR T cells comprising a nucleic acid of SEQ ID NO:l can be used as described herein.
  • CAR T cells comprising a polypeptide of SEQ ID NO:2 can be used as described herein.
  • a nucleic acid e.g., an isolated nucleic acid
  • a chimeric antigen receptor polypeptide comprising SEQ ID NO:2 can be used to prepare the CAR T cells for use as described herein.
  • a vector comprising SEQ ID NO: 1 can be used to prepare the CAR T cells for use as described herein.
  • a lentiviral vector comprising SEQ ID NO: 1 can be used to prepare the CAR T cells for use as described herein.
  • SEQ ID NO:2 can comprise or consist of humanized, or human amino acid sequences.
  • variant nucleic acid sequences or amino acid sequences having at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity to SEQ ID NO:l are contemplated.
  • the nucleic acid sequence can be a variant nucleic acid sequence having at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity to SEQ ID NO:l as long as the variant sequence encodes a polypeptide of SEQ ID NO:2.
  • the nucleic acid sequence or the amino acid sequence can be a variant nucleic acid or amino acid sequence having at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity to SEQ ID NO:l along a stretch of 200 nucleic acids or, for SEQ ID NO:2, along a stretch of 200 amino acids.
  • determination of percent identity or similarity between sequences can be done, for example, by using the GAP program (Genetics Computer Group, software; now available via Accelrys on http://www.accelrys.com), and alignments can be done using, for example, the ClustalW algorithm (VNTI software, InforMax Inc.).
  • a sequence database can be searched using the nucleic acid or amino acid sequence of interest. Algorithms for database searching are typically based on the BLAST software (Altschul et al., 1990).
  • the percent identity can be determined along the full-length of the nucleic acid or amino acid sequence.
  • nucleic acids complementary to the nucleic acid represented by SEQ ID NO:l can be used to prepare the CAR T cells for use as described herein, and those that hybridize to the nucleic acid represented by SEQ ID NO:l, or those that hybridize to its complement under highly stringent conditions can be used.
  • “highly stringent conditions” means hybridization at 65 °C in 5X SSPE and 50% formamide, and washing at 65 °C in 0.5X SSPE.
  • hybridization occurs along the full-length of the nucleic acid.
  • the CAR for use in the methods described herein can be encoded by a polynucleotide having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity to SEQ ID NO:l.
  • the CAR can be encoded by a polynucleotide that hybridizes under high stringency conditions to a polynucleotide having SEQ ID NO:l.
  • the CAR can be encoded by a polynucleotide having SEQ ID NO:l, or by a degenerate variant of SEQ ID NO:l.
  • a degenerate variant refers to the genetic code having more than one codon to specify any particular amino acid.
  • the degenerate variant codons specifying each amino acid are well- known in the art.
  • a substitution can be made to optimize the level of production of the polypeptide in a particular prokaryotic or eukaryotic host cell (/. ⁇ ? ., a codon- usage variant).
  • the CAR protein sequence can have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:2. In yet another embodiment, the CAR protein sequence can have up to about 50 conservative amino acid substitutions.
  • the CAR protein sequence that expresses the E2 anti fluorescein antibody fragment can have up to about 5 conservative amino acid substitutions, up to about 10 conservative amino acid substitutions, up to about 15 conservative amino acid substitutions, up to about 20 conservative amino acid substitutions, up to about 25 conservative amino acid substitutions, up to about 30 conservative amino acid substitutions, up to about 35 conservative amino acid substitutions, up to about 40 conservative amino acid substitutions, up to about 45 conservative amino acid substitutions, up to about 50 conservative amino acid substitutions, up to about 55 conservative amino acid substitutions, up to about 60 conservative amino acid substitutions, up to about 65 conservative amino acid substitutions, up to about 70 conservative amino acid substitutions, or up to about 75 conservative amino acid substitutions.
  • the CAR binds fluorescein.
  • non-conservative substitutions are possible provided that these do not excessively affect the fluorescein-binding activity of the E2 anti-fluorescein antibody fragment polypeptide.
  • a“conservative substitution” of an amino acid or a“conservative substitution variant” of a polypeptide refers to an amino acid substitution which maintains: 1) the secondary structure of the polypeptide; 2) the charge or hydrophobicity of the amino acid; and 3) the bulkiness of the side chain or any one or more of these characteristics.
  • the conservative amino acid substitution can be with an amino acid analog.
  • the well-known terminologies“hydrophilic residues” relate to serine or threonine. “Hydrophobic residues” refer to leucine, isoleucine,
  • phenylalanine, valine or alanine, or the like refers to phenylalanine, valine or alanine, or the like.
  • “Positively charged residues” relate to lysine, arginine, ornithine, or histidine.
  • “Negatively charged residues” refer to aspartic acid or glutamic acid.
  • Residues having“bulky side chains” refer to phenylalanine, tryptophan or tyrosine, or the like.
  • An exemplary list of conservative amino acid substitutions is given in TABLE 1.
  • the T lymphocytes e.g., cytotoxic T lymphocytes used to prepare CAR T cells expressing the E2 anti-fluorescein antibody fragment, or non-transformed T cells
  • the T lymphocytes can be autologous cells, although heterologous cells can also be used, such as when the patient being treated has received high-dose chemotherapy or radiation treatment to destroy the patient’ s immune system.
  • allogenic cells can be used.
  • the T lymphocytes can be obtained from a patient by means well- known in the art.
  • T cells e.g., cytotoxic T cells or non-transformed T cells
  • a negative T cell isolation kit such as
  • the population of T lymphocytes (e.g., cytotoxic T cells or non- transformed T cells) need not be pure and may contain other cells such as other types of T cells (in the case of cytotoxic T cells, for example), monocytes, macrophages, natural killer cells, and B cells.
  • the population being collected can comprise 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.
  • the cells are cultured under conditions that promote the activation of the cells.
  • the culture conditions may be such that the cells can be administered to a patient without concern for reactivity against components of the culture medium.
  • the culture conditions may not include bovine serum products, such as bovine serum albumin.
  • the activation can be achieved by introducing known activators into the culture medium, such as anti-CD3 antibodies in the case of cytotoxic T cells. Other suitable activators include anti-CD28 antibodies.
  • the population of lymphocytes can be cultured under conditions promoting activation for about 1 to about 4 days. In one
  • the appropriate level of activation can be determined by cell size, proliferation rate, or activation markers determined by flow cytometry.
  • the cells can be transfected with an expression vector encoding a CAR comprising the E2 anti-fluorescein antibody fragment. Suitable vectors and transfection methods for use in various embodiments are described above. In one aspect, after transfection, the cells can be immediately
  • the cells can be cultured for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or more days, or between about 5 and about 12 days, between about 6 and about 13 days, between about 7 and about 14 days, or between about 8 and about 15 days, for example, to allow time for the cells to recover from the transfection.
  • suitable culture conditions can be similar to the conditions under which the cells were cultured for activation either with or without the agent that was used to promote activation.
  • the methods of treatment described herein can further comprise 1) obtaining a population of autologous or heterologous T lymphocytes (e.g., cytotoxic T lymphocytes used to prepare CAR T cells expressing the E2 anti-fluorescein antibody fragment), 2) culturing the T lymphocytes under conditions that promote the activation of the cells, and 3) transfecting the lymphocytes with an expression vector encoding a CAR comprising an E2 anti-fluorescein antibody fragment to form CAR T cells expressing the E2 anti-fluorescein antibody fragment.
  • cytotoxic T lymphocytes used to prepare CAR T cells expressing the E2 anti-fluorescein antibody fragment
  • a composition comprising the CAR T cells, wherein the CAR T cells comprise a CAR comprising an E2 anti-fluorescein antibody fragment can be prepared and administered to the patient, with or without non-transformed T cells.
  • culture media that lacks any animal products, such as bovine serum, can be used to culture the CAR T cells expressing the E2 anti-fluorescein antibody fragment and/or the non-transformed T cells.
  • tissue culture conditions typically used by the skilled artisan to avoid contamination with bacteria, fungi and mycoplasma can be used.
  • the cells prior to being administered to a patient, the cells (e.g., CAR T cells expressing the E2 anti fluorescein antibody fragment and/or non-transformed T cells) are pelleted, washed, and are resuspended in a pharmaceutically acceptable carrier or diluent.
  • compositions comprising CAR-expressing T lymphocytes expressing the E2 anti-fluorescein antibody fragment (e.g., cytotoxic T lymphocytes) or non-transformed T cells include compositions comprising the cells in sterile 290 mOsm saline, in infusible cryomedia (containing Plasma- Lyte A, dextrose, sodium chloride injection, human serum albumin and DMSO), in 0.9% NaCl with 2% human serum albumin, or in any other sterile 290 mOsm infusible materials.
  • infusible cryomedia containing Plasma- Lyte A, dextrose, sodium chloride injection, human serum albumin and DMSO
  • 0.9% NaCl with 2% human serum albumin or in any other sterile 290 mOsm infusible materials.
  • the CAR T cells expressing the E2 anti-fluorescein antibody fragment, or non-transformed T cells can be administered in the culture media as the composition, or concentrated and resuspended in the culture medium before administration ⁇
  • the CAR T cell composition wherein the CAR comprises the E2 anti-fluorescein antibody fragment, with or without non-transformed T cells can be administered to the patient via any suitable means, such as parenteral administration, e.g., intradermally, subcutaneously, intramuscularly, intraperitoneally, intravenously, or intrathecally.
  • the total number of CAR T cells expressing the E2 anti fluorescein antibody fragment and the concentration of the cells in the composition is the total number of CAR T cells expressing the E2 anti fluorescein antibody fragment and the concentration of the cells in the composition
  • suitable compositions comprising transduced CAR T cells expressing the E2 anti-fluorescein antibody fragment include those having a volume of about 0.1 ml to about 200 ml and about 0.1 ml to about 125 ml.
  • the transduced CAR T cells wherein the CAR T cells comprise a CAR comprising an E2 anti-fluorescein antibody fragment, administered to the patient can comprise from about 1 X 10 5 to about 1 X 10 15 or 1 X 10 6 to about 1 X 10 15 transduced CAR T cells expressing the E2 anti-fluorescein antibody fragment.
  • CAR T cells expressing the E2 anti fluorescein antibody fragment can be administered to the patient.
  • a single dose or multiple doses of the CAR T cells expressing the E2 anti-fluorescein antibody fragment can be administered to the patient.
  • the CAR T cell numbers can be in kg of patient body weight.
  • the CAR T cells expressing the E2 anti-fluorescein antibody fragment can be administered before the compound, or the pharmaceutically acceptable salt thereof.
  • the designations i), ii), and iii), etc. for steps of any method described herein do not indicate an order unless otherwise stated.
  • non-transformed T cells can also be administered with the CAR T cells expressing the E2 anti-fluorescein antibody fragment and can be administered in amounts described herein for the CAR T cells expressing the E2 anti fluorescein antibody fragment, and the non-transformed T cells.
  • a mixture of CAR T cells wherein the CAR T cells comprise a CAR comprising the E2 anti-fluorescein antibody fragment, and non-transformed T cells can be administered a single time or multiple times, or combinations of doses of pure CAR T cells expressing the E2 anti-fluorescein antibody fragment and mixtures of CAR T cells expressing the E2 anti-fluorescein antibody fragment, and non-transformed T cells, can be administered (e.g., a dose of CAR T cells expressing the E2 anti-fluorescein antibody fragment followed by one or more doses of a mixture of CAR T cells expressing the E2 anti-fluorescein antibody fragment and non- transformed T cells).
  • a“mixture” of CAR T cells expressing the E2 anti-fluorescein antibody fragment and non-transformed T cells as described herein means that CAR T cells expressing the E2 anti-fluorescein antibody fragment are mixed with non-transformed T cells that have not been exposed to a construct used for expression of a CAR comprising an E2 anti-fluorescein antibody fragment.
  • the dose of the CAR T cells expressing the E2 anti-fluorescein antibody fragment 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 the CAR T cells expressing the E2 anti-fluorescein antibody fragment.
  • the CAR T cell composition comprising CAR T cells expressing the E2 anti-fluorescein antibody fragment is administered by injection into the patient’s bloodstream, and the CAR T cells expressing the E2 anti-fluorescein antibody fragment in the patient’s bloodstream are at least 5 percent, at least 7 percent, at least 10 percent, at least 11 percent, at least 12 percent, at least 13 percent, at least 14 percent, or at least 15 percent of the patient’s total T cells in the patient’s bloodstream by about four weeks after injection of the CAR T cell composition, at least 20 percent, 25 percent, 30 percent, 35 percent, 40 percent, or 50 percent by about 3 weeks after injection of the CAR T cell composition, at least 60 percent, 65 percent, 70 percent, 75 percent, or 80 percent by about 2 weeks after injection of the CAR T cell composition, or at least 85 percent, 90 percent, or 95 by about 1 week after injection of the CAR T cell composition.
  • the CAR T cell composition can comprise CAR T cells expressing the E2 anti-fluorescein antibody fragment, without any other cell type, or non-transformed T cells can be administered to the patient in combination with CAR T cells expressing the E2 anti-fluorescein antibody fragment.
  • any dose can comprise CAR T cells expressing the E2 anti-fluorescein antibody fragment or a mixture of CAR T cells expressing the E2 anti-fluorescein antibody fragment and non-transformed T cells.
  • the non- transformed T cells can be administered in amounts described herein for the CAR T cells expressing the E2 anti-fluorescein antibody fragment.
  • any dose of the CAR T cell composition can comprise a mixture of the CAR T cells expressing the E2 anti-fluorescein antibody fragment, and non-transformed T cells in a ratio selected from about 1:5 of the CAR T cells expressing the E2 anti-fluorescein antibody fragment to the non-transformed T cells, about 1:4 of the CAR T cells expressing the E2 anti-fluorescein antibody fragment to the non-transformed T cells, about 1:3 of the CAR T cells expressing the E2 anti fluorescein antibody fragment to the non-transformed T cells, about 1:2 of the CAR T cells expressing the E2 anti-fluorescein antibody fragment to the non-transformed T cells, and about 1:1 of the CAR T cells expressing the E2 anti-fluorescein antibody fragment to the non-transformed T cells.
  • any dose of the CAR T cell composition can comprise a mixture of the CAR T cells expressing the E2 anti-fluorescein antibody fragment, and non-transformed T cells in a ratio of from about 1:1 to about 1:5 of the
  • the CAR T cells expressing the E2 anti-fluorescein antibody fragment to the non- transformed T cells can comprise a mixture of about 10 million of the CAR T cells expressing the E2 anti-fluorescein antibody fragment and about 40 million non-transformed T cells, about 15 million of the CAR T cells
  • fluorescein antibody fragment and about 30 million of the non-transformed T cells, or about 25 million of the CAR T cells expressing the E2 anti-fluorescein antibody
  • the compound, or pharmaceutically acceptable salt thereof, or CAR T cell composition, wherein the composition comprises CAR T cells comprising a CAR comprising an E2 anti-fluorescein antibody fragment, described herein can be administered to the patient using any suitable method known in the art.
  • the term“administering” or “administered” includes all means of introducing the compound, or pharmaceutically acceptable salt thereof, or CAR T cell composition comprising CAR T cells having a CAR comprising an E2 anti-fluorescein antibody fragment to the patient, including, but not limited to, oral, intravenous, intramuscular, subcutaneous, transdermal, and the like.
  • the compound, or pharmaceutically acceptable salt thereof, described herein may be administered in unit dosage forms and/or formulations containing conventional nontoxic pharmaceutically- acceptable carriers, adjuvants, and vehicles.
  • the compound, or pharmaceutically acceptable salt thereof, or CAR T cell composition wherein the CAR T cell composition comprises CAR T cells expressing a CAR comprising an E2 anti-fluorescein antibody fragment, as described herein may be administered directly into the blood stream, into muscle, or into an internal organ.
  • suitable routes for such parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, epidural, intracerebroventricular, intraurethral, intrasternal, intracranial, intratumoral, intramuscular and subcutaneous delivery.
  • means for parenteral administration include needle (including microneedle) injectors, needle-free injectors and infusion techniques.
  • parenteral formulations are typically aqueous solutions which may contain carriers or excipients such as salts, carbohydrates and buffering agents (preferably at a pH of from 3 to 9), but they may be more suitably formulated as a sterile non- aqueous solution or as a dried form to be used in conjunction with a suitable vehicle such as sterile, pyrogen-free water or sterile saline.
  • a suitable vehicle such as sterile, pyrogen-free water or sterile saline.
  • any of the liquid formulations described herein may be adapted for parenteral administration as described herein.
  • the preparation under sterile conditions, by lyophilization to produce a sterile lyophilized powder for a parenteral formulation may readily be accomplished using standard
  • solubility of the compound, or pharmaceutically acceptable salt thereof, used in the preparation of a parenteral formulation may be increased by the use of appropriate formulation techniques, such as the incorporation of solubility-enhancing agents.
  • the amount of the compound, or pharmaceutically acceptable salt thereof, to be administered to the patient can vary significantly depending on the cancer being treated, the route of administration of the compound, or pharmaceutically acceptable salt thereof, and the tissue distribution.
  • the amount to be administered to a patient can be based on body surface area, mass, and physician assessment.
  • amounts to be administered can range, for example, from about 0.05 mg to about 30 mg, 0.05 mg to about 25.0 mg, about 0.05 mg to about 20.0 mg, about 0.05 mg to about 15.0 mg, about 0.05 mg to about 10.0 mg, about 0.05 mg to about 9.0 mg, about 0.05 mg to about 8.0 mg, about 0.05 mg to about 7.0 mg, about
  • 0.05 mg to about 0.2 mg about 0.05 mg to about 0.1 mg, about .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.
  • dose may vary within the various ranges provided above based on the factors noted above, and may be at the physician’s discretion.
  • the dose of the compound, or pharmaceutically acceptable salt thereof can range, for example, from about 50 nmoles/kg to about 3000 nmoles/kg of patient body weight, about 50 nmoles/kg to about 2000 nmoles/kg, about 50 nmoles/kg to about 1000 nmoles/kg, about 50 nmoles/kg to about 900 nmoles/kg, about 50 nmoles/kg to about 800 nmoles/kg, about 50 nmoles/kg to about 700 nmoles/kg, about 50 nmoles/kg to about 600 nmoles/kg, about 50 nmoles/kg to about 500 nmoles/kg, about 50 nmoles/kg to about 400 nmoles/kg, about 50 nmoles/kg to about 300 nmoles/kg, about 50 nmoles/kg to about 200 nmoles/kg, about 50 nmoles/kg
  • the dose may be about 1 nmoles/kg, about 5 nmoles/kg, about 10 nmoles/kg, about 20 nmoles kg, about 25 nmoles/kg, about 30 nmoles/kg, about 40 nmoles/kg, about 50 nmoles/kg, about 60 nmoles/kg, about 70 nmoles/kg, about 80 nmoles/kg, about 90 nmoles/kg, about 100 nmoles/kg, about 150 nmoles/kg, about 200 nmoles/kg, about 250 nmoles/kg, about 300 nmoles/kg, about 350 nmoles/kg, about 400 nmoles/kg, about 450 nmoles/kg, about 500 nmoles/kg, about 600 nmoles/kg, about 700 nmoles/kg, about 800 nmoles/kg, about 900 nmol
  • the dose of the compound, or the pharmaceutically acceptable salt thereof may range from, for example, about 10 nmoles/kg to about 10000 nmoles/kg, from about 10 nmoles/kg to about 5000 nmoles/kg, from about 10 nmoles/kg to about 3000 nmoles/kg, about 10 nmoles/kg to about 2500 nmoles/kg, about 10 nmoles/kg to about 2000 nmoles/kg, about 10 nmoles/kg to about 1000 nmoles/kg, about 10 nmoles/kg to about 900 nmoles/kg, about 10 nmoles/kg to about 800 nmoles/kg, about 10 nmoles/kg to about 700 nmoles/kg, about 10 nmoles/kg to about 600 nmoles/kg, about 10 nmoles/kg to about 500 nmoles/kg, about
  • the dose of the compound, or the pharmaceutically acceptable salt thereof may range from, for example, about 1 nmoles/kg to about 10000 nmoles/kg, from about 1 nmoles/kg to about 5000 nmoles/kg, from about 1 nmoles/kg to about 3000 nmoles/kg, about 1 nmoles/kg to about 2500 nmoles/kg, about 1 nmoles/kg to about 2000 nmoles/kg, about 1 nmoles/kg to about 1000 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
  • the dose may be about 0.3 nmoles/kg to about 1000 nmoles/kg, about 0.3 nmoles/kg to about 900 nmoles/kg, about 0.3 nmoles/kg to about 850 nmoles/kg, about 0.3 nmoles/kg to about 800 nmoles/kg, about 0.3 nmoles/kg to about 700 nmoles/kg, about 0.3 nmoles/kg to about 600 nmoles/kg, about 0.3 nmoles/kg to about 500 nmoles/kg, about 0.3 nmoles/kg to about 400 nmoles/kg, about 0.3 nmoles/kg to about 300 nmoles/kg, about 0.3 nmoles/kg to about 200 nmoles/kg, about 0.3 nmoles/kg to about 100 nmoles/kg, about 0.3 nmoles/kg to about 50
  • between about 20 ug/kg of body weight of the patient and about 3 mg/kg of body weight of the patient of the compound, or the pharmaceutically acceptable salt thereof can be administered to the patient.
  • amounts can be from about 0.2 mg/kg of body weight of the patient and about 0.4 mg/kg of body weight of the patient, or can be about 50 ug/kg of body weight of the patient.
  • a single dose or multiple doses of the compound, or the pharmaceutically acceptable salt thereof, may be administered to the patient.
  • the small molecule ligand linked to the targeting moiety (the compound) can be administered to the patient before the CAR T cell composition comprising CAR T cells wherein the CAR T cells have a CAR comprising an E2 anti-fluorescein antibody fragment.
  • the small molecule ligand linked to the targeting moiety (bridge) can be administered to the patient at the same time as the CAR T cell composition comprising CAR T cells wherein the CAR T cells have a CAR comprising an E2 anti fluorescein antibody fragment, but in different formulations, or in the same formulation.
  • the small molecule ligand linked to the targeting moiety can be administered to the patient after the CAR T cell composition comprising CAR T cells wherein the CAR T cells have a CAR comprising an E2 anti-fluorescein antibody fragment.
  • the timing between the administration of the CAR T cells having a CAR comprising an E2 anti-fluorescein antibody fragment, and the small molecule linked to the targeting moiety (bridge) may vary widely depending on factors that include the type of CAR T cells expressing the E2 anti-fluorescein antibody fragment being used, the binding specificity of the CAR having the E2 anti-fluorescein antibody fragment, the identity of the targeting moiety and the small molecule ligand, the identity of the cancer, the location in the patient of the cancer, the means used to administer to the patient the CAR T cells expressing the E2 anti-fluorescein antibody fragment and the small molecule ligand linked to the targeting moiety, and the health, age, and weight of the patient.
  • the small molecule ligand linked to the targeting moiety can be administered before or after the CAR T cells, such as within about 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, or 51 hours, or within about 0.5, 1, 1.5, 2, 2.5, 3, 4 5, 6, 7, 8, 9, 10 or more days.
  • any applicable dosing schedule known in the art can be used for administration of the compound, or the pharmaceutically acceptable salt thereof, or for the CAR T cell composition wherein the CAR T cell composition comprises CAR T cells having a CAR, wherein the CAR comprises an E2 anti-fluorescein antibody fragment.
  • twice per day dosing (a.k.a qd)
  • twice per day dosing (a.k.a. bid)
  • three times per day dosing (a.k.a. tid)
  • twice per week dosing (a.k.a. BIW)
  • three times per week dosing (a.k.a. TIW)
  • once weekly dosing and the like, can be used.
  • the dosing schedule selected for the compound, or the pharmaceutically acceptable salt thereof, and the CAR T cell composition wherein the CAR T cell composition comprises CAR T cells having a CAR, wherein the CAR comprises an E2 anti-fluorescein antibody fragment can take into consideration the concentration of the compound, or the pharmaceutically acceptable salt thereof, and the number of CAR T cells expressing the E2 anti-fluorescein antibody fragment administered, to regulate the cytotoxicity of the CAR T cell composition wherein the CAR T cell composition comprises CAR T cells having a CAR, wherein the CAR comprises an E2 anti-fluorescein antibody fragment, and to control CRS.
  • a method of treatment of a cancer comprises i) administering to a patient a compound, or a pharmaceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked to a targeting moiety by a linker, ii) administering to the patient a CAR T cell composition wherein the CAR T cell composition comprises CAR T cells wherein the CAR T cells comprise a CAR, wherein the CAR comprises an E2 anti fluorescein antibody fragment, and iii) administering to the patient a folate, a conjugate comprising a folate wherein the conjugate comprising a folate does not comprise a targeting moiety, or a drug that inhibits activation of the CAR T cells.
  • the step of administering to the patient a folate, a conjugate comprising a folate wherein the conjugate comprising a folate does not comprise a targeting moiety, or a drug that inhibits activation of the CAR T cells expressing the E2 anti fluorescein antibody fragment can be used to prevent or inhibit CRS in the patient.
  • any of a folate, a conjugate comprising a folate wherein the conjugate comprising a folate does not comprise a targeting moiety, or a drug that inhibits activation of the CAR T cells expressing the E2 anti-fluorescein antibody fragment can be referred to herein as“a rescue agent”.
  • a folate such as folic acid
  • the folate inhibits interaction of the bridge (i.e., the small molecule ligand linked to the targeting moiety by a linker) with the receptors for the bridge on the tumor inhibiting tumor lysis and preventing or inhibiting CRS in the patient.
  • the bridge i.e., the small molecule ligand linked to the targeting moiety by a linker
  • the rescue agent can reduce CRS in as quickly as about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, or about 10 hours after administration of the rescue agent.
  • the grade of CRS can be reduced (e.g., grade 3 to grade 2, grade 4 to grade 3, etc.).
  • the folate administered as an inhibitor of binding of the bridge to the tumor can be, for example, folic acid, a folic acid analog, or another folate receptor-binding molecule.
  • analogs of folate that can be used include folinic acid, pteropolyglutamic acid, and folate receptor-binding pteridines such as
  • tetrahydropterins dihydrofolates, tetrahydrofolates, and their deaza and dideaza analogs.
  • the terms“deaza” and“dideaza” analogs refers to the art recognized analogs having a carbon atom substituted for one or two nitrogen atoms in the naturally occurring folic acid structure.
  • the deaza analogs include the l-deaza, 3-deaza, 5-deaza, 8-deaza, and lO-deaza analogs.
  • the dideaza analogs include, for example, 1,5 dideaza, 5,l0-dideaza, 8,l0-dideaza, and 5,8-dideaza analogs.
  • the foregoing folic acid analogs are conventionally termed“folates,” reflecting their capacity to bind to folate receptors.
  • folate receptor-binding analogs include aminopterin, amethopterin (methotrexate), NlO-methylfolate, 2-deamino- hydroxyfolate, deaza analogs such as l-deazamethopterin or 3-deazamethopterin, and 3', 5'- dichloro-4-amino-4-deoxy-NlO-methylpteroylglutamic acid (dichloromethotrexate).
  • the folate administered as an inhibitor of binding of the bridge to the tumor has the formula
  • X 2 and X 3 are each independently selected from the group consisting of oxygen, sulfur, -C(Z , -C(Z)0-, -OC(Z)-, -N(R 4b )-, -C(Z)N(R 4b )-, -N(R 4b )C(Z)-,
  • 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, halo, C 1 -C 12 alkyl, C 1 -C 12 alkoxy, C 1 -C 12 alkanoyl, C 1 -C 12 alkenyl, C1-C12 alkynyl, (Ci-C 12 alkoxy)carbonyl, and (C1-C12 alkylamino)carbonyl;
  • R 6 and R 7 are each independently selected from the group consisting of hydrogen, halo, C 1 -C 12 alkyl, and C 1 -C 12 alkoxy; or, R 6 and R 7 are taken together to form a carbonyl group;
  • R 6a and R 7a are each independently selected from the group consisting of hydrogen, halo, C 1 -C 12 alkyl, and C 1 -C 12 alkoxy; or R 6a and R 7a are taken together to form a carbonyl group;
  • p, r, s, and t are each independently either 0 or 1;
  • * represents an optional covalent bond to the rest of the conjugate.
  • a conjugate comprising a folate can be administered to prevent or inhibit cytokine release syndrome (CRS) in the patient.
  • CRS cytokine release syndrome
  • CRS is a term well- known in the art and this syndrome can cause detrimental effects to the patient, including, but not limited to weight loss, high fever, pulmonary edema, and a dangerous drop in blood pressure.
  • the conjugate comprising a folate does not comprise a targeting moiety, and, thus, the conjugate inhibits interaction of the bridge with the tumor to prevent tumor lysis and reduce CRS in the patient.
  • the folate moiety in the conjugate comprising a folate can comprise any of the folates described in the preceding paragraphs linked to a chemical moiety that does not comprise a targeting moiety.
  • the conjugate comprising a folate can comprise a folate linked to one or more amino acids that do not comprise a targeting moiety.
  • the conjugate comprising a folate can have the formula
  • a rescue agent that inhibits activation of the CAR T cells expressing the E2 anti-fluorescein antibody fragment can be administered to the patient to inhibit CAR T cell activation and to inhibit or prevent CRS in the patient.
  • the agent can be selected from the group consisting of a lymphocyte-specific protein tyrosine kinase inhibitor (e.g., Dasatinib), a PI3 kinase inhibitor (e.g., GDC0980), an inhibitor of an IL-2 inducible T cell kinase (e.g., BMS-509744), ), JAK inhibitors, BTK inhibitors, Tociluzumab, SIP agonists (e.g., Siponimod and Ozanimod) and an agent that blocks CAR T cell binding to the bridge, but does not bind to the cancer (e.g., fluoresceinamine, FITC, or sodium
  • FITC i.e., fluorescein
  • FITC can be in the form of a salt (e.g., sodium fluorescein), or in its unsalted form, under physiological conditions or, for example, in a buffer at physiological pH.
  • a rescue agent that inhibits activation of CAR T cells can be a compound of the formula
  • the rescue agent can be administered at a concentration of from about .001 nM to about 100 mM, about .01 nM to about 100 mM, about 1 nM to about 100 mM, about 10 nM to about 100 mM, about 50 nM to about 100 mM, or from about 100 nM to about 100 mM in any appropriate volume, including, for example, 0.1 ml, 0.2 ml, 0.3 ml, 0.4 ml, 0.5 ml, 0.6 ml, 0.7 ml, 0.8 ml, 0.9 ml, 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 10 ml, 100 ml, or 1000 ml.
  • the rescue agent can be administered at a dose of about .01 to about 300 umoles/kg of body weight of the patient, about .06 to about 100 umoles/kg of body weight of the patient, about .06 to about 90 umoles/kg of body weight of the patient, about .06 to about 80 umoles/kg of body weight of the patient, about .06 to about 70 umoles/kg of body weight of the patient, about .06 to about 60 umoles/kg of body weight of the patient, about .06 to about 50 umoles/kg of body weight of the patient, about .06 to about 40 umoles/kg of body weight of the patient, about .06 to about 30 umoles/kg of body weight of the patient, about .06 to about 20 umoles/kg of body weight of the patient, about .06 to about 10 umoles/kg of body weight of the patient, about .06 to about 8 umoles/kg of body weight of the patient, or about .06 to about 6 umoles
  • the rescue agent can be administered to the patient in molar excess relative to the compound, or its pharmaceutically acceptable salt (i.e., the small molecule ligand linked to a targeting moiety by a linker), such as about a lO-fold excess, about a 20-fold excess, about a 30-fold excess, about a 40-fold excess, about a 50-fold excess, about a 60-fold excess, about a 70-fold excess, about a 80-fold excess, about a 90-fold excess, about a lOO-fold excess, about a 200-fold excess, about a 300-fold excess, about a 400-fold excess, about a 500-fold excess, about a 600-fold excess, about a 700-fold excess, about a 800-fold excess, about a 900-fold excess, about a lOOO-fold excess, or about a 10, 000-fold excess of the rescue agent relative to the small molecule ligand linked to a targeting moiety by a linker.
  • the amount of the rescue agent relative to the amount of the small molecule ligand linked to a targeting moiety by a linker (bridge) needed to inhibit interaction of the compound, or its pharmaceutically acceptable salt, with the tumor and/or the CAR T cells expressing the E2 anti fluorescein antibody fragment can be determined by the skilled artisan.
  • the folate, the conjugate comprising a folate wherein the conjugate comprising a folate does not comprise a targeting moiety, or the agent that inhibits activation of the CAR T cells expressing the E2 anti fluorescein antibody fragment can be administered to the patient before and/or after the compound, or the pharmaceutically acceptable salt thereof.
  • the compound, or the pharmaceutically acceptable salt thereof can be administered before and subsequent to administration of the folate, the conjugate comprising a folate wherein the conjugate comprising a folate does not comprise a targeting moiety, or the agent that inhibits activation of the CAR T cells expressing the E2 anti-fluorescein antibody fragment.
  • the subsequent administration of the compound, or the pharmaceutically acceptable salt thereof can cause activation of the CAR T cells expressing the E2 anti-fluorescein antibody fragment and an increase in cytokine levels in the patient.
  • the reduction in cytokine levels can occur by about 1 hour, by about 2 hours, by about 3 hours, by about 4 hours, by about 5 hours, by about 6 hours, by about 7 hours, or by about 8 hours after administration to the patient of the folate, the conjugate comprising a folate wherein the conjugate comprising a folate does not comprise a targeting moiety, or the agent that inhibits activation of the CAR T cells expressing the E2 anti-fluorescein antibody fragment.
  • the reduction in cytokine levels is a reduction to about the cytokine levels in an untreated patient.
  • the number of the CAR T cells expressing the E2 anti-fluorescein antibody fragment can increase in the blood of the patient after administration of the folate, the conjugate comprising a folate wherein the conjugate comprising a folate does not comprise a targeting moiety, or the agent that inhibits activation of the CAR T cells expressing the E2 anti-fluorescein antibody fragment, even though cytokine levels in the patient are reduced.
  • activation of the CAR T cells expressing the E2 anti-fluorescein antibody fragment can be enhanced or maintained, relative to a patient not treated with a rescue agent, after administration of the folate, the conjugate comprising a folate wherein the conjugate comprising a folate does not comprise a targeting moiety, or the rescue agent that inhibits activation of the CAR T cells wherein the CAR T cells comprise a CAR comprising the E2 anti-fluorescein antibody fragment, even though cytokine levels in the treated patient are reduced.
  • the cancer comprises a tumor and tumor size in the patient is not increased when the folate, the conjugate comprising a folate wherein the conjugate comprising a folate does not comprise a targeting moiety, or the rescue agent that inhibits activation of the CAR T cells expressing the E2 anti-fluorescein antibody fragment is administered to the patient.
  • the rescue agent that inhibits activation of the CAR T cells expressing the E2 anti-fluorescein antibody fragment is administered to the patient.
  • a complete response for the tumor can be obtained.
  • the rescue agent that inhibits activation of the CAR T cells expressing the E2 anti-fluorescein antibody fragment is administered to the patient when the CRS grade reaches 1, 2, 3, or 4 or when the CRS grade reaches 3 or 4.
  • lung edema is reduced in the patient when the rescue agent is administered.
  • a method of treatment of a cancer comprises i) administering continuously to a patient a compound, or a pharmaceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked to a targeting moiety by a linker, ii) administering to the patient a CAR T cell composition comprising CAR T cells wherein the CAR T cells comprise a CAR comprising an E2 anti-fluorescein antibody fragment, and iii) ending the continuous administration of the compound, or the pharmaceutically acceptable salt thereof, to inhibit or prevent cytokine release syndrome in the patient.
  • the term“continuously” can mean administering the compound, or the pharmaceutically acceptable salt thereof, to the patient, for example, at least one hour, at least four hours, at least six hours, at least eight hours, at least ten hours, at least twelve hours, or at least twenty-four hours, or can mean a regimen of daily or weekly administration, such as once a day, two times a day, three times a day, every day, every other day, one time weekly, two times weekly, three times weekly, or any other suitable regimen that would be considered continuous administration by a person skilled in the art.
  • the term“continuously” can mean any combination of the embodiments described in this paragraph.
  • the step of“ending the continuous administration” of the compound, or the pharmaceutically acceptable salt thereof, to inhibit or prevent cytokine release syndrome in the patient can mean, for example, discontinuing administration after administration for a continuous period of time, such as hours or days, or discontinuing a treatment regimen, such as the daily or weekly regimens described above.
  • the step of“ending the continuous administration” can mean, for example, administration until an unacceptable loss of body weight for the patient occurs, or until any other unacceptable side effect occurs such as a high fever, a drop in blood pressure, or pulmonary edema.
  • the step of“ending the continuous administration” of the compound, or the pharmaceutically acceptable salt thereof does not mean a single treatment with the compound, or the pharmaceutically acceptable salt thereof, with no subsequent treatment with the compound, or the pharmaceutically acceptable salt thereof.
  • “to inhibit or prevent” cytokine release syndrome (CRS) means eliminating CRS or reducing or ameliorating the symptoms of CRS.
  • the pharmaceutically acceptable salt thereof can be administered, for example, once weekly and one dose can be omitted.
  • the compound, or the pharmaceutically acceptable salt thereof can be administered on alternate days (i.e., every other day) and one or more (e.g., two, three, fours, etc.) doses can be omitted.
  • the compound, or the pharmaceutically acceptable salt thereof can be administered twice weekly and one or more (e.g., two, three, fours, etc.) doses can be omitted.
  • the compound, or the pharmaceutically acceptable salt thereof can be administered Monday, Tuesday, and the following Monday, and then dosing can be stopped for two weeks and the cycle repeated.
  • any of the regimen embodiments described above can be used and one or more (e.g., two, three, four, etc.) doses can be omitted. In these
  • a method of treatment of a cancer comprises i) administering to a patient a first dose of a compound, or a pharmaceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked to a targeting moiety by a linker, ii) administering to the patient at least a second dose of the compound, or a pharmaceutically acceptable salt thereof, wherein the second dose of the compound, or the pharmaceutically acceptable salt thereof, is at least about 50 percent lower in amount than the first dose of the compound, or the pharmaceutically acceptable salt thereof, and iii) administering to the patient a dose of a CAR T cell composition comprising CAR T cells wherein the CAR T cells comprise a CAR comprising an E2 anti-fluorescein antibody fragment.
  • the first dose of the compound, or the pharmaceutically acceptable salt thereof can be about 100 nmoles/kg to about 1000 nmoles/kg of body weight of the patient, about 100 nmoles/kg to about 900 nmoles/kg of body weight of the patient, about 100 nmoles/kg to about 800 nmoles/kg of body weight of the patient, about 100 nmoles/kg to about 700 nmoles/kg of body weight of the patient, about 100 nmoles/kg to about 600 nmoles/kg of body weight of the patient, about 200 nmoles/kg to about 600 nmoles/kg of body weight of the patient, about 400 nmoles/kg to about 600 nmoles/kg of body weight of the patient, or about 500 nmoles/kg of body weight of the patient.
  • pharmaceutically acceptable salt thereof can be administered once or twice weekly.
  • the agent that inhibits activation of the CAR T cells expressing the E2 anti-fluorescein antibody fragment is administered to the patient and the agent is an agent that blocks binding of the CAR T cells expressing the E2 anti fluorescein antibody fragment to the compound, or the pharmaceutically acceptable salt thereof, but does not bind to the cancer, and the agent is fluoresceinamine, sodium fluorescein, or fluorescein. In yet another embodiment, the agent is sodium fluorescein.
  • a method of treatment of a cancer comprises i) administering to a patient a first dose of a compound, or a
  • the compound comprises a small molecule ligand linked to a targeting moiety by a linker and wherein the compound, or the
  • a CAR T cell composition comprising CAR T cells wherein the CAR T cells comprise a CAR comprising an E2 anti-fluorescein antibody fragment, ii) then administering to the patient a dose of the CAR T cell composition comprising CAR T cells wherein the CAR T cells comprise a CAR comprising an E2 anti-fluorescein antibody fragment, and iii) then administering to the patient a second dose of the compound, or the pharmaceutically acceptable salt thereof.
  • the second dose of the compound, or the pharmaceutically acceptable salt thereof can be administered to the patient by at least about twenty-four hours after the administration of the CAR T cell composition, by at least about twenty hours after the administration of the CAR T cell composition, by at least about eighteen hours after the administration of the CAR T cell composition, by at least about sixteen hours after the administration of the CAR T cell composition, by at least about fourteen hours after the administration of the CAR T cell composition, by at least about twelve hours after the administration of the CAR T cell composition, by at least about ten hours after the administration of the CAR T cell composition, by at least about eight hours after the administration of the CAR T cell composition, by at least about six hours after the administration of the CAR T cell composition, by at least about four hours after the administration of the CAR T cell composition, or by at least about two hours after the administration of the CAR T cell composition, in each case wherein the CAR T cell composition comprises CAR T cells comprising a CAR comprising an E2 anti-flu
  • the dose can range, for example, from about 50 nmol/kg to about 3000 nmol/kg of patient body weight, about 50 nmol/kg to about 2000 nmol/kg, about 50 nmol/kg to about 1000 nmol/kg, about 50 nmol/kg to about 900 nmol/kg, about 50 nmol/kg to about 800 nmol/kg, about 50 nmol/kg to about 700 nmol/kg, about 50 nmol/kg to about 600 nmol/kg, about 50 nmol/kg to about 500 nmol/kg, about 50 nmol/kg to about 400 nmol/kg, about 50 nmol/kg to about 300 nmol/kg, about 50 nmol/kg to about 200 nmol/kg, about 50 nmol/kg to about 100 nmol/kg, about 100 nmol/kg to about 300 nmol/kg, about 100 nmol/kg to about 500 nmol/kg, about 100 nmol/kg, about 100 nmol/kg
  • the dose may be about 100 nmol/kg, about 150 nmol/kg, about 200 nmol/kg, about 250 nmol/kg, about 300 nmol/kg, about 350 nmol/kg, about 400 nmol/kg, about 450 nmol/kg, about 500 nmol/kg, about 600 nmol/kg, about 700 nmol/kg, about 800 nmol/kg, about 900 nmol/kg, about 1000 nmol/kg, about 2000 nmol/kg, or about 3000 nmol/kg of patient body weight.
  • “kg” is kilograms of patient body weight.
  • the folate can be administered, for example, daily, weekly, biweekly, three times a week, or using any suitable regimen for administration of the folate.
  • the CAR T cells can persist in elevated numbers of circulating CAR T cells for as long as about 10 days, as long as about 15 days, as long as about 20 days, as long as about 25 days, as long as about 30 days, as long as about 35 days, as long as about 40 days, as long as about 45 days, as long as about 50 days, as long as about 55 days, as long as about 60 days, as long as about 65 days, as long as about 70 days, as long as about 75 days, or as long as about 80 days post CAR T cell administration ⁇
  • half-maximal effective concentrations (EC50) for the compound, or the pharmaceutically acceptable salt thereof can be 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
  • the compound, or the pharmaceutically acceptable salt thereof can be first administered to the patient about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 days before or after the CAR T cells, or on any appropriate day before or after the CAR T cells.
  • cytokine release resulting in off-target toxicity in the patient does not occur, but toxicity of the CAR T cells, expressing the E2 anti-fluorescein antibody fragment, to the cancer occurs or off-target tissue toxicity does not occur in the patient, but toxicity of the CAR T cells expressing the E2 anti-fluorescein antibody fragment to the cancer occurs, or the cancer comprises a tumor, and tumor size is reduced in the patient, but off-target toxicity does not occur, or reduction in tumor size in the patient is greater than in a patient not pre-treated with the compound, or the pharmaceutically acceptable salt thereof, prior to administration of the CAR T cell composition comprising CAR T cells comprising a CAR comprising an E2 anti-fluorescein antibody fragment.
  • the“target” can be the cancer (for example a tumor).
  • a method of treatment of a cancer comprises i) administering to a patient a compound, or a pharmaceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked to a targeting moiety by a linker, and ii) administering to the patient a CAR T cell composition wherein the CAR T cell composition comprises CAR T cells and wherein the CAR T cells comprise a CAR comprising an E2 anti-fluorescein antibody fragment, and wherein the small molecule ligand is a PSMA ligand and the targeting moiety is FITC.
  • the small molecule ligand linked to a targeting moiety by a linker can have the formula
  • a method of treatment of a cancer comprises i) administering to a patient a compound, or a pharmaceutically acceptable salt thereof, wherein the compound comprises a small molecule ligand linked to a targeting moiety by a linker, and ii) administering to the patient a CAR T cell composition wherein the CAR T cell composition comprises CAR T cells and wherein the CAR T cells comprise a CAR comprising an E2 anti-fluorescein antibody fragment, and wherein the small molecule ligand is a CAIX ligand and the targeting moiety is FITC.
  • the small molecule ligand linked to a targeting moiety by a linker can have the formula
  • the cancer is imaged prior to administration to the patient of the compound, or the pharmaceutically acceptable salt thereof, or prior to administration of the CAR T cell composition to the patient, wherein the CAR T cell composition comprises CAR T cells comprising a CAR comprising an E2 anti fluorescein antibody fragment.
  • imaging occurs by PET imaging. In other illustrative embodiments imaging occurs 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 use of the small molecule ligand described herein, but linked to an imaging agent suitable for the types of imaging described herein.
  • the effect of EC 17 dose de-escalation was investigated on the anti-tumor activity and the toxicity (body weight changes) of CAR-T therapy.
  • Two different anti fluorescein scFv were used for the CAR constructions (FIG. 1).
  • the first construct of CAR contains the following domains: antiFL(FITC-E2)scFv - IgG4 hinge (CH2 (E235D, N297Q)- CH3) -CD28 TM - 4-1BB - CD3z - T2A - EGFRt.
  • the second construct of CAR contains the following domains: antiFE(FITC-4M5.3)scFv - IgG4 hinge (CH2 (E235D, N297Q)-CH3) - CD28 TM - 4-1BB - CD3z - T2A - EGFRt.
  • the nucleic acid sequence and amino acid sequence of the CAR of each construct are shown as SEQ ID NOS:l to 4.
  • Human T cells were isolated, activated and transduced with a lentiviral vector carrying the appropriate CAR gene.
  • CD4+ T cells with the CAR-modification and CD8+ T cells with the CAR-modification were isolated and cultured separately in vitro for about 7 days.
  • CD4+ CAR-T cells and CD8+ CAR-T cells were mixed at a 1:1 ratio before i.v. administration for animal studies.
  • mice bearing MDA-MB-231 tumors were used for in vivo studies to evaluate E2-CAR-T cells.
  • 10 or 20 million E2-CAR-T cells were administered, and 500 nmol/kg EC17 was i.v. dosed weekly at days 2, 9, 16, 23, etc. post CAR-T administration (as shown in FIG. 2A).
  • Mice administered with 20 million E2- CAR-T cells showed severe CRS after the second EC 17 dose and had to be rescued with 6 umol/kg sodium fluorescein (NaFL).
  • E2-CAR-T cells used were cultured in vitro for about 2 weeks. 30 million E2-CAR-T cells were administered and EC 17 dosing was modified to de-escalation. As shown in FIG. 2B, 500 nmol/kg EC17 was dosed 2 days post CAR-T administration, then a lower dose of EC17 (5, or 20 or 100 nmol/kg) was administered weekly (at days 9, 16, 23, etc. post CAR-T administration). As shown in FIG. 3 A, all three EC 17 de- escalation groups reached a complete response, indicating that bridge de-escalation does not affect the anti-tumor activity of CAR-T.
  • the second construct 4M5.3-CAR (antiFL(FITC-4M5.3) scFv - IgG4 hinge (CH2 (L235D, N297Q)-CH3) -CD28 TM - 4-1BB - CD3z - T2A - EGFRt) was also evaluated.
  • Mice bearing MDA-MB-231 tumors (150-250 mm 3 ) were used for in vivo studies to evaluate 4M5.3-CAR-T cells.
  • 10 or 20 million 4M5.3-CAR-T cells were administered, and 500 nmol/kg EC17 was i.v. dosed weekly at days 2, 9, 16, 23, etc. post CAR- T administration (as shown in FIG. 2A).
  • mice administered with either 10 or 20 million 4M5.3-CAR-T cells did not show severe CRS or body weight loss after administering an EC17 dose (FIG. 6B), but their tumors were all reduced and eventually disappeared (FIG. 6A).
  • 20 million CAR-T cells (upper line) showed better anti-tumor activity than 10 million CAR-T cells (middle line).
  • 4M5.3-CAR-T cells used were cultured in vitro for about 2 weeks. 30 million 4M5.3-CAR-T cells were administered and EC17 dosing was modified to de-escalation.
  • 500 nmol/kg EC17 was dosed 2 days post CAR-T administration, then a lower dose of EC17 (5, or 20 or 100 nmol/kg) was administered weekly (at days 9, 16, 23, etc. post CAR-T administration).
  • EC17 500 nmol/kg EC17 was dosed 2 days post CAR-T administration, then a lower dose of EC17 (5, or 20 or 100 nmol/kg) was administered weekly (at days 9, 16, 23, etc. post CAR-T administration).
  • E2-CAR-T cells were found to have better anti-tumor activity than 10 million 4M5.3-CAR-T cells; 30 million E2-CAR-T cells also showed better anti-tumor activity than 30 million 4M5.3-CAR-T cells when EC 17 dose de-escalation (5, or 20, or 100 nmol/kg) was tested.
  • SEQ ID NOG can comprise or consist of humanized, or human amino acid sequences.
  • SEQ ID NOSG and 4 are as described above. The start and stop codons in the longer nucleic acid sequence are underlined and the longer sequence is an exemplary sequence that can be used for transduction of T cells to prepare the 4M5.3 CAR.
  • CAR T cells of predominately Tscm/Tcm phenotype were expected to possess a superior anti-tumor phenotype when compared to past CAR T preparations which possess a more differentiated Tem/Teff phenotype.
  • sCRS arises later for the current CAR-T preparation than the previous CAR-T preparations given the same EC 17 dosing regimens.
  • T cell recovery media TexMACSTM Media containing glutamine (Miltenyi Biotec #130-097-196) supplemented with 2% human AB serum
  • 50U/mL recombinant human IL2 cultured in vitro for approximately 3 to 5 days for T cell recovery.
  • T cells were washed with PBS and resuspended in cold PBS containing 3mM propidium iodide and transferred to flow cytometry collection tubes. Flow cytometry data was collected on the Gallios flow cytometer (Beckman Coulter, Brea,
  • E2 anti-FITC IgG antibody with the same variable fragment sequences as those in E2 anti-FITC CAR construct was used to test if E2 antibody has any binding with human normal tissues.
  • a recombinant human E2 anti-FITC IgGl Lambda 1 was expressed in HEK293 cells and purified by using a protein A affinity column. The purity of the antibody was over 98% by using SEC-HPLC analysis.
  • the antibody was labeled with digoxigenin (DIG) using Mix-N-Stain DIG antibody labeling kit (Biotium). The binding affinity of DIG- labeled E2 antibody for FITC-immobilized beads was measured by FACS.
  • An IHC assay was developed to test the binding of E2 antibody to human tissues.
  • Formalin-fixed, paraffin-embedded (FFEP) tissue section of the agar block with FITC- labeled KB cells was used as the positive control, while FFPE tissue section of unlabeled KB cells was used as the negative control.
  • the tissue sections were deparaffinizaed and rehydrated, then antigen retrieval was done by incubating tissue sections with an antigen retrieval buffer (pH6.0) at 91 °C for 24 minutes, then DIG-E2 antibody was incubated with rehydrated tissue sections, and finally an anti-DIG IHC staining was performed.
  • an antigen retrieval buffer pH6.0
  • a monoclonal anti-DIG antibody was added and incubated, followed by a peoxidase-labeled anti-mouse secondary antibody.
  • FITC-labeled KB cells showed positive staining, indicating that the E2 antibody binds with FITC on these labeled KB cells.
  • the unlabeled KB cells did not show any staining (FIG. 10B).
  • DIG-labeled E2 antibody was incubated with a human multiple organ normal tissue microarray (FDA999, US Biomax, Inc) and the binding of E2 antibody to human normal tissues was evaluated using the same IHC procedure with the same conditions described above.
  • This multiple organ normal tissue microarray has 99 cores with 28 types of normal human organs, including adrenal gland (FIG. 11), bone marrow (FIG. 12), breast (FIG. 13), cerebellum tissue (FIG. 14), cervix (FIG. 15), colon (FIG. 16), esophagus (FIG. 17), eye (FIG. 18), heart (FIG. 19), hypophysis (FIG. 20), kidney (FIG. 21), larynx (FIG.
  • FIGS. 22 liver (FIG. 24), lung (FIG. 25), lymph node (FIG. 26), nerve (FIG. 27), ovary (FIG. 28), pancreas (FIG. 29), prostate (FIG. 30), skin (FIG. 31), small intestine (FIG. 32), spleen (FIG. 23), stomach FIG. 33), striated muscle (FIG. 34), testis (FIG. 35), thymus gland (FIG. 36), tongue (FIG. 37), and uterus (FIG. 38). Organs taken from at least 3 normal human individuals were included in the microarray panel. As shown in FIGS.
  • E2 anti-FITC antibody does not bind with any tested human normal organ tissues, indicating E2 anti-FITC CAR-T cell itself should not bind to and attack any human normal tissues in vivo.
  • FITC fluorescein isothiocyanate
  • DMF dimethylsulfoxide
  • the crude product was loaded onto an Xterra RP18 preparative HPLC column (Waters) and eluted with gradient conditions starting with 99% 5 mM sodium phosphate (mobile phase A, pH 7.4) and 1% acetonitrile (mobile phase B) and reaching 90% A and 10% B in 10 min at a flow rate of 20 mL/min. Under these conditions, the FITC-folate main peak typically eluted at 27-50 min.
  • FITC-folate fraction The quality of the FITC-folate fraction was monitored by analytical reverse-phase HPLC with a UV detector. Fractions with greater than 98.0% purity (LCMS) were lyophilized to obtain the final FITC-folate product. As known in the art, the compound with this structure is also referred to as EC17.
  • Ethylenediamine, polymer-bound (200-400 mesh)-resin (50 mg) was loaded into a peptide synthesis vessel and swollen with DCM (3 mL) followed by DMF (3 mL).
  • DCM 3 mL
  • DMF 3 mL
  • Fmoc-PEG2o-COOH solution 131 mg, 1.0 equiv
  • i- PnNEt 6.0 equiv
  • PyBOP 4.0 equiv
  • Argon was bubbled for 6 h, the coupling solution was drained, and the resin was washed with DMF (3 x 10 mL) and z ' -PrOH (3 x 10 mL). Kaiser tests were performed to assess reaction progress.
  • Fmoc deprotection was carried out using 20% piperidine in DMF (3 x 10 mL), before each amino acid coupling. The above sequence was repeated to complete the reaction with Fmoc-Glu-OtBu (72 mg, 2.0 equiv) and Tfa.Pteroic-acid (41 mg, 1.2 equiv) coupling steps.
  • the resin was washed with 2% hydrazine in DMF 3 x 10 mL (5 min) to cleave the trifluoro-acetyl protecting group on pteroic acid and washed with i- PrOH (3 x 10 mL) followed by DMF (3 x lOmL). The resin was dried under argon for 30 min.
  • the folate -peptide was cleaved from the resin using the Cleavage Solution. 10 mL of the cleavage mixture was introduced and argon was bubbled 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 under reduced pressure to a smaller volume ( ⁇ 5 mL) and precipitated in ethyl ether.
  • Fmoc deprotection was carried out using 20% piperidine in DMF (3 x 10 mL), before each amino acid coupling. The above sequence was repeated to complete reaction with 2X Fmoc-PEG36-COOH (161 mg, 1.0 equiv), Fmoc-Glu- OtBu (72 mg, 2.0 equiv ) and Tfa.Pteroic-acid ( 41.0 mg, 1.2 equiv) coupling steps.
  • DUPA-FITC was synthesized by solid phase methodology as follows. Universal Nova TagTM resin (50 mg, 0.53 mM) was swollen with DCM (3 mL) followed by DMF 3 mL). A solution of 20% piperidine in DMF (3 x 3 mL) was added to the resin, and argon was bubbled for 5 min. The resin was washed with DMF (3 x 3 mL) and isopropyl alcohol (z ' -PrOH, 3 x 3 mL).
  • FITC-DUPA FITC-DUPA as a brownish-orange solid.
  • the pure fractions were collected, all organic solvents were evaporated and the sample was lyophilized for 48 h to provide the NK1 -PEGi l -NHBoc. Yield: 40.13 mg (97%).
  • NK-l compound was synthesized by a two-step procedure starting from the base ligand, which was prepared by using a procedure in the literature. (Ref: DESIGN AND DEVELOPMENT OF NEUROKININ- 1 RECEPTOR-BINDING AGENT DELIVERY CONJUGATES, Application Number: PCT/US2015/44229; incorporated herein by reference.
  • CA9 ligand (53.6mg) was dissolved in DMF (2-3mL) in a 50mL round bottom flask using a Teflon magnetic stir bar. Ambient air was removed using a vacuum and replaced with nitrogen gas, this was done in three cycles. The round bottom flask was kept under constant nitrogen gas. To the flask, 28.9mg of N-(3-Dimethylaminopropyl)-N'- ethylcarbodiimide hydrochloride (EDC) was added followed by 2l.6mg 1- Hydroxybenzotriazole hydrate (HOBt) and l8.9pL of B0C-PEG2-NH2 (Sigma Aldrich).
  • EDC N-(3-Dimethylaminopropyl)-N'- ethylcarbodiimide hydrochloride
  • DUPA-FITC was synthesized by solid phase methodology as follows. Universal NovaTag resin (50 mg, 0.53 mM) was swollen with dichloromethane (DCM) (3 mL) followed by dimethylformamide (DMF, 3 mL). A solution of 20% piperidine in DMF (3 x 3 mL) was added to the resin, and argon was bubbled for 5 min. The resin was washed with DMF (3 x 3 mL) and isopropyl alcohol (i-PrOH, 3 x 3 mL).
  • DCM dichloromethane
  • DMF dimethylformamide
  • CA9 ligand (53.6mg, synthesized in lab) was dissolved in a desired amount of N,N-Dimethylformamide (DMF) (2-3mL) using a Teflon magnetic stir bar. Ambient air was removed using vacuum and replaced with nitrogen gas, this was done in three cycles.
  • DMF N,N-Dimethylformamide
  • Acetonitrile was removed using high vacuum rotary evaporation and place on lyophilizer for 48 hours.
  • Deprotection of Boc was done with with 1:1 TFA:DCM for 30 minutes.
  • TFA/DCM was removed using high vacuum rotary evaporation followed by 30 minutes on high vacuum.
  • the compound was then dissolved in DMF and combined with 5 molar equivalents of N,N-Diisopropylethylamine (DIPEA).
  • DIPEA N,N-Diisopropylethylamine
  • l6mg of fluorescein isothiocyanate purchased from Life Technologies
  • Reaction mixture was purified by HPLC and target compound was confirmed with UHPLC-MS (target m/z of 1120).
  • the samples was placed on lyophilizer for 48 hours and store compound at -20°C.
  • DIPEA Diisopropylethylamine
  • NK1-PEG11- NH2 (0.008 g, 0.0081 mmol, 1.0 eq.), Fluorescein isothiocyanate (FITC) (Sigma, 0.0037 g, 0.0097 mmol, 1.2 eq.) in dry dimethylsulfoxide (DMSO, 0.3 mL) was added diisopropylethyl amine (0.0028 mL, 0.0162 mmol, 2.0 eq.) at room temperature under argon.
  • DMSO dry dimethylsulfoxide
  • the pure fractions were collected, evaporated all organic solvents and lyophilized the sample for 48 h to furnish the NK1 -PEG 11 -FITC (5). Yield: 8.54 mg (77%).
  • the NK-l compound was synthesized by a two-step procedure starting from base ligand, which was prepared by using a literature procedure. (Ref: DESIGN AND
  • E2 CAR T cells were thawed and recovered from cryopreservation at a cell density between 0.5million to 2million T cells per milliliter of T cell growth media (TexMACS media + 2% human AB serum + 50 U/mL recombinant human IL2) for 4 days.
  • Target cells which express different levels of surface folate receptor were also thawed and recovered from cryopreservation in growth media (folate deficient RPMI1640 + 10% FCS +pen/strep for 4 days).
  • E2 CAR T cell activity level after one day coculture with target cells possessing different levels of surface folate receptor, floating cells were harvested and pooled with the remaining adherent cells which were removed using a 5-minute 0.25% trypsin digest from the tissue culture plate. Pooled floating and adherent cells were pelleted with a 400 x gravity centrifugation step for 5 minutes then resuspended in flow cytometry staining solution.
  • Surface marker staining for E2 T cell identification via flow cytometry included anti-human EGFR and anti-human CD3 while detection of CAR T cell activation via flow cytometry utilized an anti-human CD 137 stain.
  • E2 CAR T cells were identified as EGFR+ CD3+ while activated CAR T cells also co-expressed CD137 (see flow cytometry methods).
  • E2 CAR T cell killing of target cells with different levels of surface folate receptor expression after a two day coculture pooled and adherent cells were pelleted in the same manner as the one day coculture assay above then stained for E2 CAR T markers EGFR and CD3 then washed and stained for the apoptosis marker, Annexin V, by resuspending the flow antibody stained samples with Alexa Fluor 647 conjugated recombinant Annexin V (Invitrogen cat# A23204 at 1:50 dilution in IX Annexin staining buffer provided) with 3mM propidium iodide.
  • E2 CAR T cell activation is dependent on EC 17 staining of target cells
  • E2 CAR T cell activation also translates to successful killing of the target cells
  • target cell EGFR- CD3-
  • FIG. 40 the same five target cells as shown (as labeled on x-axis), each cultured for two days under three different conditions, shown as three different bars.
  • the first bar in each group represents the basal level of target cell apoptosis as these were target cells treated with EC 17 but cultured in the absence of E2 CAR T cells.
  • the second bar in each group represents CAR T plus target cell cocultures without any previous EC 17 treatment of target cells while the third bar in each group represents the CAR T plus EC 17 pre-treated target cells.
  • the y-axis shows the percentage of target cells which are apoptotic (Annexin V+). From these data it was discovered that CAR T cell activation translates well into target cell killing for the three highest folate receptor expressers
  • All tumor cell lines were seeded overnight in folate-free RPMI medium containing 10% heat-inactivated fetal calf serum. On the following day, the cells were incubated for 15 min on ice with 100 nM 3 H-folic acid with and without 10 mM cold folic acid. After rising 3 times with cold IX PBS, whole cells were lysed and total cell- associated radioactivity was counted in a scintillation counter. FR-specific binding of 3 H-folic acid were determined by subtracting the counts of folic acid competed samples and calculated as number of molecules per cell using the specific activity of 3 H-folic acid.
  • Figure 41 demonstrates various levels of FR expression in these cell lines.
  • the order of FR levels are: IGROV1 > MDA-MB-231, HOS-FRa > OV90, SKOV3.
  • MDA-MB-231 tumor cells were grown in folate-deficient RPMI 1640 with 5-10% FBS at 37 °C in a 5% CO2 humidified atmosphere. MDA-MB-231 tumor cells were inoculated subcutaneously at 2.5 x 10 6 cells per animal. Only 42 out of the 66 mice were inoculated and the remaining 22 mice were kept tumor-free.
  • EGFRt- sorted anti-FITC E2 scFv-CAR T cells were frozen in a T-cell freezing medium. Vials of frozen CAR-T cells were immediately stored at -80°C. The CAR-T cells were quickly thawed at 37°C, washed twice with PBS, and used for animal injection at 10 million viable EGFRt-i- E2 CAR-T cells (CD4/CD8 at—1: 1) per animal. A small aliquot was taken on the day infusion for flow cytometric analysis of E2-CAR T-cell phenotypes.
  • Mouse blood samples were processed for plasma and stored at -20°C until use.
  • a human TH1 cytokine panel (Biolegend, Cat. No. 740009) and a human hematopoietic stem cell cytokine panel (Biolegend, Cat. No. 740610) was used to detect human cytokines in the mouse blood.
  • a mouse l3-plex inflammatory cytokine panel (Biolengend, No. 740150) was used to detect mouse cytokines. All analyses were performed per the manufacturer’s instructions.
  • the leukocyte pellets were then resuspended in flow cytometry staining solution (1% bovine serum albumin, 50mg/mL human IgG (Equitech Bio, cat#SLH56-000l), 0.9% sodium azide in a 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 at 1:20 (v/v) dilution], anti-human CD137 [clone 4B4-1, BD Bioscience #564092 at 1:20 (v/v) dilution], anti-human CD8a [clone RPA-T8, BD Bioscience, catalog #557746 at 1:20 (v/v) dilution], anti-human CD4 [clone SK3,
  • Flow cytometry data was collected on the Gallios flow cytometer (Beckman Coulter, Brea, CA). Determination of the concentration of CAR T cells in each blood sample was calculated according to Invitrogen’ s instructions. CAR T cells were identified as human CD3s + EGFRt-i- events and easily distinguished and counted using the KaluzaTM flow cytometry software. The number of CAR T cells in the circulation of each infused mouse was then represented on the graphs as the total number of CAR T cells per 100 pL of whole blood analyzed. Statistical significance was determined by utilizing an unpaired, two-tailed, students t-test with significance set at p ⁇ 0.05.
  • Tumor and tissue analysis Solid tumors (100-1000 mm3) were harvested, weighed, and minced into small pieces then transferred into 50 mL tubes containing 20 mL of a tumor digestion cocktail.
  • the enzymatic tumor digestion cocktail consisted of 0.5 mg/mL Collagenase IV (Sigma- Aldrich, Catalog# C5138), 0.5 mg/mL Hyaluronidase (Sigma- Aldrich, Catalog# H3506) and 0.1 mg/mL DNase I (Sigma- Aldrich, Catalog# DN25) in serum- free and folate- deficient RPMI1640 medium supplemented with antibiotics.
  • the tumor fragments were digested for one hour at 37°C at 300 rpm on a horizontal shaker.
  • the tumor digest was centrifuged at 400 x g for 5 minutes and tumor cell pellet underwent a red blood cell lysis step, was then washed with cold phosphate-buffered saline (PBS, pH 7.4) and finally filtered through a 40 pm nylon cell strainer.
  • PBS cold phosphate-buffered saline
  • E2 CAR-T cells were i.v. injected into naive NSG mice without tumor or NSG mice bearing MDA-MB-231 tumors.
  • 500 nmol/kg EC17 was i.v. dosed on days 2 and 10 post CAR-T injection, and mice were euthanized 20 hours after second EC17 dose (day 11) for organ evaluation and blood analysis.
  • the plasma samples were isolated from blood immediately and stored at -20°C until cytokine analysis.
  • a separate set of animals were harvested for FACS analysis as described above.
  • cytokine production in tumor-bearing mice was EC17- dependent.
  • the levels of cytokine including IL2, IFN-Y, IL-10 were all increased in tumor bearing mice with EC 17 relative to those without an EC 17 dose.
  • the up-regulation of cytokine production was also observed in those mice dosed with EC17, but the increase in tumor-free mice was much lower comparing to those mice with MDA-MB-231 tumors (e.g. 23-fold lower for IFN-g).
  • Tumor-bearing mice had ⁇ 23-fold higher IFN-g production than tumor-free mice.
  • mice While tumor-free mice (on FD-diet for ⁇ 2 months) showed some cytokine production post EC17 SIW500 x 2 doses, there was no detectable CAR-T cell expansion by FACS.
  • the MDA-MB-231 were provided by Endocyte and cultured in folate free DMEM. All cell lines were authenticated by STR Profiling matched to the DSMZ Database by the University of Arizona Genetics Core.
  • Cytotoxicity and cytokine secretion Four-hour chromium release assays were performed. Briefly, target cells were labeled with 51 Cr (PerkinElmer) overnight, washed three times in PBS, and incubated in RPMI in triplicate at 5x103 cells/well with T-cells at various effector to target (E:T) ratios in a 96-well plate. S upernatants were harvested for g-counting and specific lysis was calculated.
  • cytokine secretion 5xl0 5 T-cells were plated in triplicate with target cells at an E:T ratio of 2:1 in a 96- well plate for 24-hours and supernatants were analyzed by cytometric bead array using a Bio-Plex Human Cytokine Panel (Bio-Rad) according to the manufacturer’s instructions.
  • Bio-Rad Bio-Rad
  • EC17 labeled targets the cells were incubated in the dark at room temperature for lhr with 100 nM EC17 in PBS prior to plating. When EC17 was used, cells were incubated with folate free RPMI during the assay.
  • CD4+ T cells were co-cultured at a 2:1 ratio T-cell to target for 24 hrs and then the supernatant was analyzed for the presence of effector cytokines IF-2, IFN-g, and TNF-a.
  • Chromium release assay CD8+ T cells were co-cultured with target cells at a 30:1, 10:1, 3:1, or 1:1 ratio.
  • Percentage lysis (mean ⁇ SE) of triplicate wells is depicted.
  • the anti- FLCAR T-cells did not exhibit cytotoxicity against the negative control (K562) or unlabeled MDA-MB-231 cell lines.
  • both the mock transduced and anti-FFCAR T-cells were able to induce similar levels of specific lysis against the positive control K562-OKT3 cell line.
  • EC17 labeled MDA-MB-231 cells were efficiently recognized and lysed by anti- FFCAR T-cells whereas the mock T-cells were unable to confer lysis.
  • Figures 46-49 shown the structures of bridges used in CAR-T cell experiments and their affinities, determined in vitro, for the types of cells the bridges are directed against (e.g., folate - FITC for folate receptor expressing cancer cells).
  • Figure 50 shows binding (by FACS analysis) of bridges to tumor cells used in an in vivo model and expressing the receptor corresponding to the small molecule ligand of the bridge.
  • FR+ and FR-negative cancer cell lines were maintained in RPMI-1640 medium (Gibco BRL) supplemented with 10% heat-inactivated fetal calf serum without (FFRPMI) or with (RPMI) 2.4 mM folic acid (FA).
  • KB FRoc-expressing human cervical carcinoma with HeLa markers
  • CHO-b Choinese hamster ovary cells transfected with human FR
  • MDA-MB-231 represents a FRoc subclone of human triple negative breast cancer (TNBC) cell line.
  • FR-positive (THPl-FR ) and FR-negative (THP1-FG12) cell lines were provided. Both were established from THP-l (ATCC, TIB-202), a commonly used cell model for researching pediatric AML which was originally derived from a 1 -year-old male infant with acute monocytic leukemia.
  • THP-l ATCC, TIB-202
  • HOS-FRa was established by lentiviral transduction of FR-negative HOS-l43b (ATCC, CRL8303) with FOLR1 gene encoding the human FRoc.
  • HOS-l43b is originally established from a primary tumor of a l3-year-old Caucasian female and highly tumorigenic in NSG mice.
  • the GFP-expressing bioluminescent pairs of FR+ HOS-FRa 11110 and FR-negative HOS-l43b fLuc were transduced with lentiviral firefly luciferase.
  • LEGENDplexTM human cytokine panels were purchased from BioLegend (San Diego, CA).
  • the lactate dehydrogenase (LDH) based CytoTox 96 ® non-radioactive cytotoxicity assay kit was purchased from Promega (Madison, WI).
  • CD45RA clone HI100
  • CD45RO clone UCHL1
  • CD4 clone SK3
  • CD69 clone FN50
  • CD3s clone SK7
  • CD8oc clone RPA-T8
  • CD137/4-1BB clone 4B4-1
  • CD25 clone M-A251
  • PD1 clone EH12.1
  • LAG3 clone T47-530
  • TIM3 clone 7D3
  • biotinylated anti-human EGFR Cetuximab, clone Hul
  • R&D systems Minneapolis, MN
  • FRa clone LK26
  • a fluorophore-conjugated anti-biotin was also purchased from BioLegend.
  • APC-conjugated anti- FITC mouse IgG2a/kappa antibody (clone NAWESLEE), CountBrightTM beads (Invitrogen), Annexin V staining buffer, and AlexaFluor-647-conjugated Annexin V were purchased from Thermo Fisher Scientific.
  • collagenase IV, hyaluronidase and DNase I were all purchased from Sigma- Aldrich (St. Louis, MO).
  • EC17 or folate-FITC [FA-(y)-ethylenediamine-FITC] was synthesized at Endocyte.
  • 3 H- EC17 was either purchased from Moravek biochemicals (Brea, CA) at a specific activity of -0.952 Ci/mmol or prepared at Endocyte by conjugating FITC with 3 H-FA-(y)-ethylenediamine made by ViTrax (Placentia, CA) at a specific activity of -1.2 Ci/mmol.
  • 3 H-FA was also purchased from ViTrax at a specific activity of 59 Ci/mmol.
  • sodium fluorescein dosing solution was diluted from AK-FLUOR ® 25% (fluorescein injection, USP) which was purchased from Purdue Pharmacy (NDC 17478-250-25).
  • lentivirus was produced in 293T cells co-transfected with CAR-encoding epHIV7 lentiviral vector.
  • Donor CD4+ and CD8+ T cells were purified by immunomagnetic selection and transduced separately or at about a 50:50 ratio. In general, only one round of CD3/CD28 bead activation followed by one or two rounds of rapid in vitro expansion were carried out.
  • T N CD45RA+ CD45RO- CD62L+ CD95- naive T cells
  • TSCM CD45RA+ CD45RO- CD62L+ CD95+ stem cell memory T cells
  • TCM CD45RA- CD45RO+ CD62L+ CD95+ central memory T cells
  • TEM CD45RA- CD45RO+ CD62L- CD95+ effector memory cells
  • TEFF CD45RA+ CD45RO- CD62L- CD95+ effector T cells.
  • FITC-E2 fully human anti-FITC scFv
  • This second-generation fully human CAR consisted of anti-FITC scFv (clone E2), an IgG4-Fc spacer/hinge with double mutations in the CH2 region (L235D and N297Q) to reduce binding to FcyR, a CD28 transmembrane domain, and 4- 1 BB/ € ⁇ 3z signaling domains appended to a cell-surface EGFRt tag by a T2A ribosomal skip sequence (i.e., FITC-E2-scFv-IgG4hinge- CD28tm-4-lBB/CD3z-T2A-EGFRt).
  • both EGFRt-sorted and unsorted E2 CAR-T cells were prepared at ⁇ l:l CD4/CD8 ratios, and T cell subtype phenotyping was routinely performed by flow cytometry at the time of CAR T cell infusion (day 0) for each in vivo experiment.
  • a typical expression pattern of EGFRt-sorted CAR-T cells was comprised of both CD4 and CD8 subsets at approximately 42% TSCM, 10% TCM, 12% TEM and 34% TEFF (Figure 55, pie charts on the left). Only EGFRt-sorted CAR-T cells were used for co-culture ( Figures 53, 54, 56, 58, 59, and 61) and pharmacokinetic studies ( Figure 62).
  • a“clinical facsimile” batch with a low differentiation profile (Figure 55, pie charts on the right) was used and this“clinical batch” was also used for MDA-MB-231 studies ( Figure 63), and a research batch was used for THPl-FR and HOS-FRoc studies ( Figure 57).
  • the “clinical facsimile” batch (-39% EGFRt+) was comprised of CD4+ subsets at -66% TSCM and -32% TCM and CD8 subsets at -95% TSCM and 3% TCM.
  • the research batch (-23% EGFRt+) was more differentiated and comprised of CD4 subsets at 32% TSCM, 53% TCM, 11% TEM and 3.7% TEFF and CD8 subsets at 44% TSCM, 0.28% TCM, 3.4% TEM and 52% TEFF ⁇
  • the bispecific affinities of EC 17 CAM were assessed using 3 H-ECl7 in cell-based radioligand binding assays.
  • KB and CHO-b cells were pre-seeded overnight in 24-well tissue culture plates and incubated with 0.1, 0.5, 1, 5, 10, 20, and 40 nM of 3 H-ECl7 in FFRPMI for 2 h at 37 °C. Afterwards, the cells were rinsed with phosphate- buffered saline (PBS, pH 7.4) and lysed with 1% sodium dodecylsulfate. The whole cell lysates were quantitated for the level of radioactivity and cellular protein content by PBS, pH 7.4.
  • the basic components of the CAR-T cell therapy involve FITC as the pseudo tumor antigen, the high affinity EC 17 CAM (a CAM is equivalent to a bridge or the“compound” in this application), and a rationally designed anti-FITC CAR and CAR-modified T cells.
  • the EC17 has already been tested in the clinic for immunotherapy and optical imaging purposes.
  • 3 H-ECl7 was synthesized and radioligand binding assays were carried out on KB and CHO-b cell lines representing FRa+ and FRP+ target cells, respectively, and on unsorted EGFRt CAR-T cells representing the effector cells.
  • mice Female 4 to 5-week-old NOD/SCID gamma (NSGTM) mice (stock number:
  • Euthanasia was performed per study design or when (i) the animals had lost >20% of body weight or approached moribund conditions, (ii) subcutaneous tumors reached >1500 mm 3 in size, or (iii) animals displayed signs of swollen belly and severe distress (i.e., THPl-FR ). All animal doses (CAR-T cells, EC17, sodium fluorescein) were given intravenously.
  • EC 17 CAM can theoretically be given before or after CAR-T cell injection.
  • the first dose of EC17 was administered 2-3.5 days after CAR-T cells to allow for an observation period of human T cells in tumor-bearing mice.
  • Two batches of unsorted E2 CAR-T cells (23% or 39% EGFRt+, 1:1 CD4/CD8) were used for in vivo studies.
  • frozen CAR-T cells were quickly thawed at 37°C, washed 2x with Dulbecco’s IX PBS (pH 7.4) and injected into the tail vein at desired EGFRt-i- E2-CAR-T cell doses.
  • CAR-T cells were analyzed by flow cytometry for CD4 to CD8 ratio and differentiation status of CAR-T cells.
  • THPl-FR tumor-bearing animals were randomly assigned into groups according to their tumor sizes or the same number of days post intravenous implantation (i.e., THPl-FR ).
  • CRS grading system (0-5 scale) was developed to empirically assess CRS toxicity from animals’ gross morphology and social behavior to allow for better timing of CRS rescue. While grades 0 and 5 indicated normal (no CRS) or death (due to severe CRS), respectively, grades 1, 2, and 3-4 were considered mild, light-to-moderate, and severe CRS ( Figure 57 A, table descriptions). In addition, all EC17 doses were intentionally given towards the end of any given day to allow potential CRS symptoms to develop overnight.
  • FR+ cell lines (KB, MDA-MB-231, HOS-FRoc, THPl-FR , OV90) were co-cultured with EGFRt-sorted E2-CAR- T cells at an effector-to-target (E/T) ratio of 1:1 in the presence of EC 17 ranging from 0.1 pM to 100 mM in lO-fold increments. After 24 hours of co-culture, supernatants were harvested to determine tumor cell killing.
  • FR+ KB, MDA-MB-231, HOS-FRa II uc , THPl-FR
  • FR-negative HOS- 143b ILuc , THP1-FG12 tumor cell lines
  • target cells were incubated on day 0 without or with 0.1 and 10 nM of EC17 for 30 min at 37°C and the status of EC 17“pre-loading” was assessed by counterstaining cell-surface EC 17 with an anti-FITC antibody ( Figure 61, panel A).
  • target cell apoptosis (%) was measured as Annexin V+ after 2 days of co-culture and normalized against background levels of apoptosis of target cells cultured under the same conditions in the absence of T cells ( Figure 61, panel B).
  • the ranking order of total available FRs on these cell lines was: 9 x 10 4 (OV90, a low-FR expressing ovarian cancer cell line), 1.9 x 10 5 (THPl-FR ), 2.4 x 10 5 (HOS-FRa ® ” 0 ), 7 x 10 5 (HOS-FRa), 2.1 x 10 6 (MDA-MB-231) and 4.8 x 10 6 (KB) FA molecules/cell. Also included as FR-negative controls were HOS-l43b (fLuc) and THP1-FG12 parent cell lines. Thus, the general ranking of functional FR expression on co-cultured FR+ cancer cell lines was: KB > MDA-MB-231 > HOS-FRa > HOS-FRa ® 110 > THPl-FR (AML) > OV90.
  • Monovalent EC 17 in its free form i.e. one FITC per FA ligand
  • monovalent EC 17 in its free form does not automatically activate anti-FITC CAR-T cells.
  • 5 FR+ cell lines KB, MDA-MB-231, HOS-FRoc, THPl-FR , OV90
  • 5 FR+ cell lines KB, MDA-MB-231, HOS-FRoc, THPl-FR , OV90
  • MDA-MB-231, THP1- FR and HOS-FRoc cells were preloaded with 100 nM EC17 over 30 min. The cells were washed and then incubated at 1:1 E/T ratio with either EGFRt-sorted E2-CAR-T cells (1:1 CD4/CD8), or mock transduced control T cells. After 24 hours of co-culture, we measured the surface expression of T cell activation markers CD69, CD 137 (4-1BB) and PD1 ( Figure 53). ECl7-preloaded MDA-MB-231 and HOS-FRoc were strong activators of our CAR-T cells.
  • THPl-FR cells which express l l-fold lower FR compared to the MDA-MB-231 cells, did trigger CAR-T cell activation, but at lower levels. Importantly, the mock controls T cells were not activated under these conditions.
  • Some FR+ tumor types e.g. KB
  • display a natural resistance to killing by ECl7-directed CAR T cells Figures 56, 58, 59, and 60
  • T cell activation versus exhaustion as a mechanism of EC17 CAM action were chosen for this purpose.
  • HOS-FRa ll uc expressed ⁇ 5.6x lower level of functional FR than its parent HOS-FRoc, and HOS- 143b ll uc was FR-negative similar to HOS-l43b ( Figure 59, panel A).
  • FR+ and FR-negative tumor cells without or with EC17 pre-loading (0.1 or 10 nM, 30-min pulse at 37 °C) were incubated with EGFRt-sorted CAR-T cells (1:1 CD4/CD8) at an E/T ratio of 1:2 ( Figure 54 and Figure 61, panel B).
  • EC17 dose-dependent pre-loading was confirmed on day 0 by counter-staining of membrane bound EC17 with an APC-conjugated anti-FITC antibody (clone NAWESLEE) ( Figure 61, panel A).
  • APC-conjugated anti-FITC antibody clone NAWESLEE
  • co-cultured CAR-T cells were measured for upregulation and co-expression of T cell activation/exhaustion markers PD1, LAG3 and TIM3.
  • E2-CAR-T cells underwent a low-grade differentiation in culture with low frequencies of double or triple-positive cells.
  • the presence of tumor cells alone (without EC 17 pre-loading) appeared to“relax” the CAR-T cells to various degrees.
  • E2-CAR-T cells underwent significant differentiation with increased co-expression of exhaustion markers. Based on increased frequencies of triple- and double-positive cells, a more exhausted phenotype appeared on co-cultured CAR-T cells in the order of KB > HOS-FRa ll uc > THPl-FR > MDA-MD-231. While EC17 dose-dependent apoptosis (Annexin V+) was seen in all FR+ tumor cell lines on day 2, high FR-expressing KB cells again showed disproportionally low apoptosis ( Figure 61, panel B).
  • plasma was removed from a predetermined volume of whole blood collected into tubes containing ethylenediaminetetraacetic acid anticoagulant. After lysing red blood cells, leukocyte pellets were re-suspended in a flow cytometry staining solution comprising 1% bovine serum albumin, 50 mg/mL human IgG (Equitech Bio) and 0.9% sodium azide. The samples were stained for human leukocyte surface markers (CD3s, CD4, CD8a, CD45RA, CD62L) and biotinylated anti-human EGFR
  • tumor-infiltrating CAR-T cells pre-weighed fresh tumor fragments were finely minced and enzymatically digested with a digestion cocktail consisting of 0.5 mg/mL collagenase IV, 0.5 mg/mL hyaluronidase and 0.1 mg/mL DNase I in serum- free FFRPMI with vigorous shaking for one hour at 37°C. Afterwards, tumor cell pellets underwent a red blood cell lysis step, washed with cold PBS and filtered through a 40 pm nylon cell strainer.
  • a digestion cocktail consisting of 0.5 mg/mL collagenase IV, 0.5 mg/mL hyaluronidase and 0.1 mg/mL DNase I in serum- free FFRPMI with vigorous shaking for one hour at 37°C.
  • the resulting single cell suspensions were stained for EGFRt and human leukocyte markers, CD137/4-1BB and PD1.
  • a minimum of 20,000 propidium iodide negative live cell events were collected on the Gallios flow cytometer and analyzed with the Kaluza software, version 2.1 (Beckman Coulter, Brea, CA).
  • CAR-positive T cells were identified in mouse blood as human CD3e+ EGFRt-l ⁇ - events and absolute numbers per volume of blood were calculated using equal numbers of CountBrightTM beads (Invitrogen, Carlsbad, CA) added to each sample.
  • the number of tumor-infiltrating CD3s+ EGFRt-i- CAR-T cells was expressed as % total viable tumor cells analyzed.
  • mice were placed on either a FA-replete diet (4 mg/kg, Envigo, Indianapolis, IN) or a FA-deficient diet (TestDiet, St. Louis, MO) upon arrival. On day 0, both sets of the mice received -10 million of the same“clinical facsimile” CAR-T cells followed by no EC17 or EC17 SIW at 500 nmol/kg starting on day 3.
  • mice were maintained on defined diets for -73 days and used when their tumors reached -549 ⁇ 184 mm 3 (280-918 mm 3 ) in FA-replete mice, and -559 ⁇ 165 mm 3 (356-961 mm 3 ) in FA-deficient mice ( Figure 63, panels A-D).
  • mice received -10 million of the same“clinical facsimile” of E2-CAR-T cells on day 0, and ECl7-treated cohorts received eight weekly doses of 500 nmol/kg starting on day 2 (FA-replete) or day 3 (FA-deficient).
  • the first full dose of EC17 was generally safe and did not cause any CRS or body weight loss in mice on either diet.
  • FA-replete animals did not show symptoms of CRS throughout EC 17 treatment, whereas FA-deficient animals experienced grades 2-3 CRS and body weight loss (up to -11.4%) with each of the subsequent EC 17 doses (Figure 63, panel A, bottom panels).

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