Targeting of multiple antigens with multiplex car t cells in solid and liquid malignancies

Abstract

To provide compositions and methods for treating cancers characterized by the presence of solid tumors, which simultaneously target multiple targets on cancer cells using a single CAR T construct.SOLUTION: The present invention provides a bifunctional compound comprising a first synthetic antigen covalently linked to a first targeting moiety that binds a first tumor associated antigen, or a pharmaceutically acceptable salt or a stereoisomer thereof.SELECTED DRAWING: Figure 1B

Description

[Technical field]

[0001] Related Applications This application claims the benefit of priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 62 / 691,486, filed June 28, 2018, and U.S. Provisional Application No. 62 / 697,526, filed July 13, 2018, the entireties of each of which are incorporated herein by reference.

[0002] Government Permission This invention was made with Government support under Grant Nos. DK105602-01 and T32AI007386 awarded by the National Institutes of Health. The United States Government has certain rights in this invention. [Background technology]

[0003] Clinical trials have demonstrated that cancer immunotherapy can induce durable responses in patients with advanced cancer. There have been numerous reports of successful treatment of B-cell-derived leukemia, lymphoma, and multiple myeloma (MM) with chimeric antigen receptor (CAR) T cells. These genetically modified T cells have specificity for cluster of differentiation 19 (CD19), CD22, and B-cell maturation antigen (BCMA) receptors, which are widely present on the surface of cancerous B cells, but also on normal non-cancerous B cells. Although response rates against these cancers are still high, there remains a need for more tunable CAR T cell lines (Kochenderfer et al. J. Clin Oncol. 33:540-9 (2015); Maude et al. Blood 125:4017-23 (2015); Friedman et al. Hum Gene Ther. 29(5):585-601 (2018); Raje et al. N Engl J Med. 380(18):1726-37 (2019); Fry et al. Nat Med 24:20-8 (2018)).

[0004] Brain cancer presents a particularly formidable opponent to meaningful and sustained treatment. In the case of glioblastoma (GBM), for example, all currently available FDA approved treatments are essentially palliative. The combination of radiation and temozolomide chemotherapy (Stupp et al. N Engl J Med, 352:987-96 (2005)), established as the current standard of care for newly diagnosed patients since 13 years ago, only provides a median survival of 15 months, while salvage therapy does not extend the survival of recurrent patients.

[0005] Remarkable insights have been gained into the mediators of key genetic alterations and dysregulated cell signaling pathways that drive GBM tumor growth. However, biologic therapies that block cancer-driving pathways have failed to consistently confer clinical benefit to GBM patients for multiple reasons, including overlapping mechanisms of pathway activation, intratumor heterogeneity, acquired resistance, and poor delivery through the blood-brain barrier into the central nervous system (CNS). Similarly, despite the fact that GBM is one of the most angiogenic cancers, effective inhibition of vascular endothelial growth factor did not improve patient survival, most likely due to compensatory upregulation of other pro-angiogenic factors as well as the ability of GBM tumors to adapt and progress in the hypoxic and nutrient-poor microenvironment that occurs after angiogenesis inhibition. Although disappointing, such findings are not unprecedented in GBM and thus underscore the complexity of this disease.

[0006] Various immunotherapeutic approaches, including immune checkpoint blockade, chimeric antigen receptor (CAR) T cells, vaccines and oncolytic viruses, have achieved interesting results across a wide range of cancer indications. Unfortunately, recent results of large randomized three-phase trials evaluating such approaches for GBM patients have been negative. These include a vaccine targeting a single tumor-specific mutation (epidermal growth factor receptor variant III (EGFRvIII)) for newly diagnosed patients (Weller et al. Lancet Oncol, 18:1373-85 (2017)) and administration of an anti-programmed death 1 (PD-1) antibody for recurrent patients (Reardon et al., World Federation of Neuro-Oncology Societies (WFNOS) Zurich, Switzerland: Oxford University Press (2017)).

[0007] Accumulating data indicates that the immunobiology of GBM is highly complex, with multiple intrinsic and adaptive factors contributing to antagonizing antitumor immune responses. Thus, successful immunotherapy treatment of GBM patients will require further discovery and deeper understanding of the key contributors to this complexity, followed by the design of optimized therapeutic strategies to overcome these challenging factors.

[0008] Another type of brain cancer, high-grade pediatric glioma, is one of the most aggressive tumors of childhood. Hundreds of clinical trials have been conducted in children with pediatric glioma, but there has been limited improvement in response rates or long-term outcomes. This is especially true for diffuse intrinsic pontine glioma (DIPG), a glioma found within the pons of the brainstem. DIPG is typically diagnosed in patients between the ages of 4 and 6, and the majority of these children die within one year of diagnosis.

[0009] Up to now, CAR T cell therapy has only limited success against solid tumors, including brain tumors.Moreover, regulating CAR T cell activity could enhance the treatment of hematological malignancies.Therefore, there is still a need for more effective CAR T cell-based anticancer therapy against hematological cancers such as MM and solid tumors, especially GBM and DIPG and other forms of brain cancer. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Kochenderfer et al.J.Clin Oncol.33:540-9(2015) [Non-Patent Document 2] Maude et al.Blood 125:4017-23(2015) [Non-Patent Document 3] Friedman et al.Hum Gene Ther.29(5):585-601(2018) [Non-Patent Document 4] Raje et al.N Engl J Med.380(18):1726-37(2019) [Non-Patent Document 5] Fry et al. Nat Med 24:20-8(2018) [Non-Patent Document 6] Stupp et al.N Engl J Med,352:987-96(2005) [Non-Patent Document 7] Weller et al.Lancet Oncol,18:1373-85(2017) [Non-Patent Document 8] Reardon et al., World Federation of Neuro-Oncology Societies (WFNOS) Zurich, Switzerland: Oxford University Press (2017) Summary of the Invention

[0011] A first aspect of the present invention is directed to a bifunctional compound comprising a small molecule, also referred to herein as a synthetic antigen, covalently linked to a targeting moiety (also referred to herein as a targeting ligand) that binds a tumor-associated antigen present on tumor cells.

[0012] In some embodiments, the targeting moiety specifically binds a brain tumor associated antigen. In some embodiments, the tumor associated antigen is selected from the group consisting of disialoganglioside GD2 (GD2), cyclic adenosine diphosphate (ADP) ribose hydrolase (CD38), signaling lymphocyte activation molecule (SLAM) family member 7 (SLAM7; CS1), interleukin 13 receptor alpha 2 (IL13Rα2), human epidermal growth factor receptor 2 (HER2), platelet-derived growth factor receptor alpha (PDGFRα), epidermal growth factor receptor variant III (EGFRvIII), chondroitin sulfate proteoglycan 4 (CSPG4), ephrin type A receptor 2 (EphA2), prominin-1 (CD133), B cell maturation antigen (BCMA), B lymphocyte antigen CD20 (CD20), B lymphocyte antigen CD19 (CD19) and B cell receptor CD22 (CD22).

[0013] In some embodiments, the synthetic antigen is a fluorescent dye such as fluorescein (FL). In other embodiments, the synthetic antigen is 4-[(6-methylpyrazin-2-yl)oxy]benzoate (MPOB), anthraquinone-2-carboxylate (AQ), or tetraxetane (DOTA).

[0014] In some embodiments, the synthetic antigen contains a protecting group that is removable under certain conditions, such as, for example, light (visible (VIS), near infrared (NIR) or ultraviolet (UV)) or the presence of reactive oxygen or nitrogen species (ROS, RNS). In such embodiments, the synthetic antigen is referred to herein as a pro-antigen. A pro-antigen is not accessible to a binding entity, such as an antibody or functional fragment thereof. Removal of the protecting group (e.g., by cleavage) renders the unmasked or uncaged synthetic antigen accessible to such binding.

[0015] Another aspect of the present invention is directed to a pharmaceutical composition comprising a therapeutically effective amount of a bifunctional compound and a pharma- ceutically acceptable carrier. In some embodiments, the pharmaceutical composition contains a plurality (two or more) subpopulations of bifunctional compounds, in which the synthetic antigen may be identical in each subpopulation, but each targeting moiety binds a different epitope of the same tumor-associated antigen. In some embodiments, the pharmaceutical composition contains a plurality (two or more) subpopulations of bifunctional compounds, in which the synthetic antigen may be identical in each subpopulation, but each targeting moiety binds a different tumor-associated antigen present on tumor cells. Thus, the pharmaceutical composition may contain a first subpopulation of bifunctional compounds having a first targeting moiety that specifically binds a first tumor-associated antigen, and a second subpopulation of bifunctional compounds each having a second targeting moiety that specifically binds a second tumor-associated antigen, the first and second targeting moieties binding different tumor-associated antigens. In other embodiments, the plurality of subpopulations of bifunctional compounds contains a third, fourth, fifth, etc. subpopulation of bifunctional compounds, each of which binds a different tumor-associated antigen present on tumor cells. Thus, in some embodiments, a given composition may target one or more epitopes of the same tumor-associated antigen. In other embodiments, a composition may target two or more tumor-associated antigens (and two or more epitopes of any one or more of the two or more tumor-associated antigens).

[0016] In some embodiments, the first and second targeting moieties specifically bind a brain tumor associated antigen. In some embodiments, the brain tumor associated antigen is selected from GD2, IL13Rα2, HER2, PDGFRα, EGFRvIII, CSPG4, EphA2, CD133, and the first and second targeting moieties bind the brain tumor associated antigen.

[0017] In some embodiments, the first and second targeting moieties specifically bind a hematological tumor associated antigen. In some embodiments, the hematological tumor associated antigen targeted by the subpopulation of bifunctional compounds is selected from the group consisting of CD38, CS1, BCMA, CD20, CD19, CD22, CD30, CD38, CD138, CD40, CD56, CD70 and CD74.

[0018] In some embodiments, the first or second targeting moiety specifically binds to HER2 on a HER2+ malignancy, such as breast cancer, lung cancer, colorectal cancer, brain cancer, ovarian cancer and pancreatic cancer. Another aspect of the invention is a method of treating cancer comprising: a) a subpopulation of a plurality of bifunctional molecules, said plurality comprising: a1) a first subpopulation of bifunctional compounds comprising a therapeutically effective amount of a synthetic antigen covalently linked to a first targeting moiety that specifically binds a first tumor associated antigen; and a2) a second subpopulation of bifunctional compounds comprising a therapeutically effective amount of a synthetic antigen covalently linked to a second targeting moiety that specifically binds a second tumor associated antigen, or a pharma- ceutically acceptable salt or stereoisomer thereof, wherein said first and second targeting moieties specifically bind different tumor associated antigens; b) a therapeutically effective number of CAR-T cells, the CAR-T cells comprising an extracellular ligand that specifically binds a synthetic antigen; and to a subject in need of treating cancer.

[0019] Therapeutically effective amounts of multiple, bifunctional compound subpopulations are administered simultaneously or sequentially. In some embodiments, subpopulations of bifunctional compounds are administered simultaneously, and targeting of multiple tumor antigens may result in increased efficacy due to heterogeneous expression of one or both of the tumor-associated antigens (within the same patient or among multiple patients). In other embodiments, multiple subpopulations of bifunctional compounds are given sequentially, for example, when a patient no longer responds to a first (set) of bifunctional compounds and a second series of bifunctional compounds is required to maintain therapeutic efficacy.

[0020] In some embodiments, the cancer is characterized by the presence of solid tumors.In some embodiments, the cancer is brain cancer, such as GBM or DIPG, and the targeting moiety of the bifunctional compound present in the first and second subpopulations specifically binds a brain tumor associated antigen.In some embodiments, the brain tumor associated antigen is selected from GD2, IL13Rα2, HER2, PDGFRα, EGFRvIII, CSPG4, EphA2 and CD133, and the first and second targeting moieties bind different brain tumor associated antigens.

[0021] In some embodiments, the cancer is a HER2+ malignancy such as brain cancer, lung cancer, colorectal cancer, brain cancer, ovarian cancer and pancreatic cancer.

[0022] In some embodiments, the cancer is a blood cancer. In some embodiments, the blood cancer is multiple myeloma, leukemia and lymphoma, and the targeting moiety of the bifunctional compound present in the first and second subpopulations specifically binds a blood tumor associated antigen. In some embodiments, the blood tumor associated antigen is selected from CD38, CS1, BCMA, CD20, CD19, CD22, CD30, CD138, CD40, CD56, CD70 and CD74, and the first and second targeting moieties bind different blood tumor associated antigens.

[0023] In some embodiments, these methods may involve more than one administration of a subpopulation of bifunctional compounds. In such cases, the respective targeting moieties in each of the multiple (e.g., first and second) subpopulations of bifunctional compounds may be identical in that they bind to the same epitope on the target tumor-associated antigen. In some other embodiments, any subpopulation of bifunctional compounds may be expanded or modified such that the subpopulation includes or further includes targeting moieties that bind different epitopes on the same target tumor-associated antigen. Thus, embodiments of the present invention may include a first administration of a subpopulation of bifunctional compounds and at least a second administration that differs from the first administration in that the targeting moieties of any one or more subpopulations bind different epitopes of the same tumor-associated antigen, and the first and second administrations may be simultaneous or sequential. This additional feature may mitigate antigen loss / escape or reduce toxicity. In some embodiments, the methods may involve administration of a third, fourth, fifth, etc. subpopulation of the bifunctional compound, where each subpopulation targets a different tumor-associated antigen.

[0024] A further aspect of the invention is directed to a system comprising multiple subpopulations of bifunctional compounds and CAR T cells.

[0025] A further aspect of the invention provides a therapeutically effective amount of a plurality of subpopulations of bifunctional compounds, or pharma- ceutically acceptable salts or stereoisomers thereof, wherein each bifunctional compound in a first subpopulation comprises a first synthetic antigen covalently linked to a first targeting moiety that specifically binds a first tumor associated antigen, and each bifunctional compound in a second subpopulation comprises said first synthetic antigen covalently linked to a second targeting moiety that specifically binds a second tumor associated antigen, wherein said first and second targeting moieties specifically bind different tumor associated antigens, and wherein said plurality of subpopulations of bifunctional compounds are disposed in said same or separate containers; b) a therapeutically effective amount of a plurality of subpopulations of the bifunctional compound; and instructions for co-administering to a cancer patient a therapeutically effective number of CAR-T cells, the CAR-T cells comprising an extracellular ligand that specifically binds the synthetic antigen. The present invention is directed to a kit comprising: As described above, in some embodiments, a pharmaceutical composition may contain multiple (two or more) subpopulations of bifunctional compounds, in which the synthetic antigens may be identical in each subpopulation, but each targeting moiety binds a different epitope of the same tumor-associated antigen. Thus, in some embodiments, a given composition may target one or more epitopes of the same tumor-associated antigen. In other embodiments, a composition may target two or more tumor-associated antigens (and two or more epitopes of any one or more of the two or more tumor-associated antigens).

[0026] As shown diagrammatically in Figures 1A-1D, the present invention may be described as a multiplexable and doseable CAR T cell platform. Unlike current CAR T cell approaches to cancers characterized by the presence of solid or hematological tumors, including brain cancers such as DIPG and GBM, or hematological cancers such as multiple myeloma, leukemia, and lymphoma, the present invention may simultaneously target multiple tumor-associated antigens using only one CAR T construct. In so doing, the present invention may confer increased cytotoxicity, reduce the likelihood of antigen escape, and provide greater versatility in terms of clinically used targets, which in turn may increase the efficacy of CAR T cells across the diverse inter- and intra-patient heterogeneity observed in brain or hematological cancers, particularly GBM and multiple myeloma. Furthermore, the present invention obviates the need to engineer CAR T cells against each and every tumor-associated antigen that is the selected target. Furthermore, by combining multiple different tumor-targeting antibodies with different spatial distributions, the present invention may reduce or eliminate toxicity to healthy tissues by distributing low dose antigens throughout the body. Thus, this approach to treating solid or hematological tumors may increase efficacy, increase the duration of therapy, and reduce adverse side effects. [Brief description of the drawings]

[0027] [Figure 1]FIG. 1A-FIG. 1E are a series of diagrams illustrating the advantages of dose-tunable and multiplexed anti-small molecule CAR T cells. FIG. 1A shows a system of anti-small molecule CAR T cells that uncouples tumor cell recognition from tumor cell killing compared to traditional CAR T cell designs. FIG. 1B shows multiple antibody-small molecule conjugates from the system shown in FIG. 1A. FIG. 1C shows that this system allows dosing by allowing cells to work above inactivity and below toxic levels. FIG. 1D shows that the invention allows greater versatility to suppress and combat antigen escape mutants. Along with anti-small molecule CAR T cells, multiple antibodies can be given simultaneously or sequentially to prevent cancer recurrence. FIG. 1E shows that this system may be able to separately regulate the route of administration of antibody-small molecule conjugates and CAR T cells. [Diagram 2] 2A-H are a series of line and bar graphs showing the flexibility of CAR T cells using small molecules. FIG. 2A shows a representative example of two different CAR T cells (anti-MPOB and anti-FL CAR T cells) specifically killing their respective targets. FIG. 2B-D show that small molecule CAR T cells can be rerouted towards many different targets expressed in different types of tumor cells. FIG. 2E demonstrates CAR T cell dose-responsive cytotoxicity with increasing concentrations of CAR T cells. FIG. 2F and FIG. 2G show CAR T cell dose-responsive cytotoxicity with increasing concentrations of small molecule-conjugated antibody, demonstrating that different CAR T cell designs give similar increases in cytotoxicity as the amount of small molecule-conjugated antibody increases. FIG. 2H demonstrates that the same CAR T cells can be repurposed to target one or more tumor types. The bar graph shows a comparison in killing efficiency of anti-small molecule CAR T cells co-incubated with two tumor populations that do not express the same antigen: glioblastoma multiforme (GBM) and multiple myeloma (MM). When tumor cells are fully coated with small molecule-conjugated antibodies, both populations can be targeted and killed. [Diagram 3]Figures 3A-B are heat maps showing the expression levels of the indicated proteins. Figure 3A is a heat map showing the expression levels of GD2, PDGFRα, and IL13Rα2 in A172 (GBM cell line), BT145 and BT333 (adult GBM lines derived from an H3.3 WT patient), and BT869 (pediatric DIPG cell line derived from an H3-K27M patient). Figure 3B is a heat map showing the expression levels of CD38, CD20, and CS1 in multiple myeloma cell lines. [Figure 4] Figures 4A-B are chemical models and bar graphs, respectively, showing that small molecule antigens can be protected against site-specific CAR T cell activity. Figure 4A is a chemical model showing binding of an anti-fluorescein antibody (PBD ID: 1X9Q) to unshielded fluorescein. Anti-fluorescein antibody amino acid polar contacts are indicated by yellow dotted lines. Contacts with amino acid side chains, shown in purple, can be shielded with protecting moieties to prevent binding. Figure 4B is a graph showing in vitro cytotoxicity of human antibody-fluorescein CAR T cells using tumor-targeting antibodies conjugated to "caged / shielded" or "uncaged / unshielded" fluorescein derivatives, using photoreactive o-nitrobenzyl protecting groups. CAR T cells specifically killed target cells bound by commercially available antibodies conjugated to fluorescein isothiocyanate (FITC) or ultraviolet (UV) "uncaged / unshielded" fluorescein derivatives. However, CAR T cells were able to kill target cells that were bound by the “caged / shielded” fluorescein derivative. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. As used in this specification and the appended claims, unless expressly stated to the contrary, in order to facilitate understanding of the present invention, the following terms have the meanings set forth.

[0029] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a composition" includes mixtures of two or more such compositions, reference to "an inhibitor" includes mixtures of two or more such inhibitors, and so forth.

[0030] Unless otherwise specified, the term "about" means within 10% (eg, within 5%, 2%, or 1%) of the particular value that is modified by the term "about."

[0031] The transitional phrase "comprising," which is synonymous with "including," "containing," or "characterized by," is inclusive or open-ended and does not exclude further, unrecited elements or method steps. In contrast, the transitional phrase "consisting of" excludes any element, step, or ingredient not specified in the claim. The transitional phrase "consisting essentially of" limits the scope of the claim to the materials or steps specified and to those materials or steps that do not "materially affect the basic and novel characteristics" of the claimed invention.

[0032] With respect to the compounds of the present invention, to the extent the following terms are used herein to further describe the compounds of the present invention, the following definitions apply.

[0033] The term "specific binding", when referring to the interaction between a targeting moiety and a solid tumor-associated antigen, refers to an intermolecular interaction that is substantially specific in that the binding of the targeting moiety to other proteinaceous entities present on the cell surface may be functionally insignificant.

[0034] The term "specific binding" when referring to the interaction between a synthetic antigen (or unmasked pro-antigen) and an extracellular ligand on a CAR T cell refers to an intermolecular interaction that is substantially specific in that binding of these entities to any other endogenous proteins may be functionally insignificant.

[0035] Bifunctional compounds One key aspect of the present invention is the decoupling of tumor cell targeting from tumor cell killing.As shown in FIG. 1A, CAR T cells kill tumor cells without directly binding to tumor cells.This is achieved by using bifunctional compounds, one of which functional part (synthetic antigen or unmasked pro-antigen) is bound by CAR T cells, and the second functional part of which specifically binds solid tumor-associated antigens.

[0036] Synthetic antigens Synthetic antigens are small molecules or surrogate epitopes that serve as targets for CAR-T cells. The antigens are synthetic in the sense that they do not naturally occur on any normal cells in the body, and antigenic in the sense that CAR T cells can be engineered to bind to them.

[0037] Generally, a "small molecule" is understood in the art to be an organic molecule that is less than about 5 kilodaltons (kDa) in size. In some embodiments, a small molecule is less than about 4 kDa, about 3 kDa, about 2 kDa, or about 1 kDa. In some embodiments, a small molecule is less than about 800 Daltons (Da), about 600 Da, about 500 Da, about 400 Da, about 300 Da, about 200 Da, or about 100 Da.

[0038] Representative examples of synthetic antigens that may be suitable for use in the present invention include fluorescein, anthracene, rhodamine, rhodol, alexa fluor, acridine, xanthene, pyrazine, amphetamine, benzodiazepine, benzoylecgonine, buprenorphine, opioids, cannabinoids, phencyclidine, tricyclic antidepressants, dextromethorphan, fentanyl, meprobamate, methadone, methamphetamine, oxycodone, tetrahydrocannabinol (THC), tramadol, zolpidem, ketamine, lysergic acid diethylamide (LSD), 3,4-methylenedioxymethamphetamine (MDMA), methaqualone, propoxyphene or norketimine, and 4-[(6-methylpyrazine-2 Examples of synthetic antigens include L-alpha aspartyl-L-phenylalanine methyl ester or aspartame, 13-[(2-O-beta-D-glucopyranosyl-beta-D-glucopyranosyl)oxy]-ent-kaur-16-en-19-oic acid beta-D-glucopyranosyl ester or stevioside, and generic nucleotides such as nusinersen or spinraza. Further representative examples of synthetic antigens are known in the art.

[0039] Representative examples of fluoresceins (i.e., fluorescein and derivatives) that may be suitable for use in the present invention include 5-carboxyfluorescein, 6-carboxyfluorescein, 5-(iodoacetamido)fluorescein, 5-([4,6-dichlorotriazin-2-yl]amino)fluorescein hydrochloride, 5-(bromomethyl)fluorescein, fluorescein 5(6)-isothiocyanate, and fluorescein 5-carbamoylmethylthiopropanoate.

[0040] Thus, in some embodiments, when the synthetic antigen is fluorescein, the bifunctional compound has a structure represented by formula (I) or (II) (the linker (L), if present, and the targeting moiety are shown generically): [ka] wherein R1 and R2 are each independently O, OH, or H; R 1’ represents C=O, O, OH or H; and R3 represents [ka] [ka] [ka] represents [ka] is absent or a linker, and n is 1 to 12; or a pharma- ceutically acceptable salt or stereoisomer thereof.

[0041] Representative examples of anthracenes that may be suitable for use as synthetic antigens in the present invention include anthraquinone, anthraquinone-2-carboxylate, 2-aminoanthraquinone, 2-iodoanthraquinone, 2-chloroanthraquinone, 2-bromoanthraquinone, 2-ethynylanthraquinone, 2-cyanoanthraquinone, anthraquinone-2-sulfonate, anthraquinone-2-carbonyl chloride, and 2-hydroxyanthraquinone.

[0042] In some embodiments, the synthetic antigen is anthracene and the bifunctional compound has a structure represented by formula (III) (the linker (L), if present, and the targeting moiety are shown collectively): [ka] During the ceremony, R4, R 4’ each is independently O or OH; and R5 is [ka] [ka] [ka] and [ka] is absent or a linker, and n is 1 to 12; or a pharma- ceutically acceptable salt or stereoisomer thereof.

[0043] In some other embodiments where the small molecule is anthracene, the bifunctional compound has the structure represented by formula (III-1): [ka] During the ceremony, R4, R 4’ and each of R5 is as defined above; or a pharma- ceutically acceptable salt or stereoisomer thereof.

[0044] In some embodiments, when the synthetic antigen is MPOB, the bifunctional compound has the structure represented by formula (IV): [ka] During the ceremony, R6 is, [ka] [ka] [ka] and [ka] is absent or a linker, and n is 1 to 12; or a pharma- ceutically acceptable salt or stereoisomer thereof.

[0045] Pro-synthetic antigens (pro-antigens) In some embodiments, the synthetic antigen contains a protecting group. The protected synthetic antigen is referred to herein as a "pro-antigen". The protecting group is also referred to herein as a "shielding" or "cage". The pro-antigen is inactive in the sense that it does not bind or activate CAR-T cells until it is unshielded or uncaged upon exposure to appropriate stimuli, such as certain wavelengths of light and reactive chemical species that cause the removal of the protecting group. Thus, in these embodiments, the "shielding" or "cage" inhibits CAR T cell reactivity except at the site of solid tumors.

[0046] In some embodiments, the protecting group domain is a boronic ester that is sensitive to reactive species, i.e., reactive oxygen species (ROS) and / or reactive nitrogen species (RNS). When tissues are exposed to external radiation (e.g., X-rays, gamma rays, higher UV rays), high levels of ROS and / or RNS react with the proantigen, resulting in unshielded proantigen, thereby generating synthetic antigens accessible to CAR T cells.

[0047] In some embodiments, the boronic ester group is a pinacol boronic acid ester or a pinanediol boronic acid ester. Representative examples of boronic esters include: [ka] Examples include:

[0048] Thus, in some embodiments, when the proantigen is fluorescein, the bifunctional compound has a structure represented by formula (V) or (VI) (the linker (L), if present, and the targeting moiety are shown generically): [ka] or wherein R1 is independently O, OH or a protecting group; R 1’ is independently C=O, O, OH or a protecting group, R2 is independently O, OH or a protecting group; R3 is [ka] [ka] [ka] and [ka] is absent or a linker, and n is 1 to 12; provided that R, R 1’ and at least one of R2 is a protecting group; or a pharma- ceutically acceptable salt or stereoisomer thereof. In some embodiments, both R and R or R 1’ and R2 are both protecting groups.

[0049] In some embodiments, when the proantigen is a fluorescein containing a boronic ester protecting group, the bifunctional compound has the structure represented by formula (VII): [ka] During the ceremony, R1 is O, OH or a boronic ester protecting group; R2 is O, OH or a boronic ester protecting group; With the proviso that at least one of R1 and R2 is a boronic ester group, R3 is [ka] [ka] [ka] and [ka] is absent or a linker, and n is 1 to 12; The boronic ester protecting group is [ka] is; or a pharma- ceutically acceptable salt or stereoisomer thereof. In some embodiments, both R1 and R2 are boronic ester protecting groups.

[0050] In some embodiments, the bifunctional compound has the structure represented by formula (VIIa): [ka] or a pharma- ceutically acceptable salt or stereoisomer thereof.

[0051] In some embodiments, the bifunctional compound has the structure represented by formula (VIIa1): [ka] or a pharma- ceutically acceptable salt or stereoisomer thereof.

[0052] In some embodiments, the bifunctional compound has the structure represented by formula (VIIb): [ka] or a pharma- ceutically acceptable salt or stereoisomer thereof.

[0053] In some embodiments, the bifunctional compound has the formula [ka] or a pharma- ceutically acceptable salt or stereoisomer thereof.

[0054] In some embodiments, when the proantigen is an anthracene, the bifunctional compound has a structure represented by formula (VIII) (the linker (L), if present, and the targeting moiety are shown collectively): [ka] In the formula, R4 and R 4’ Each of is independently O or a protecting group, provided that R and R 4’ at least one of is a protecting group; and R5 is, [ka] [ka] [ka] and [ka] is absent or a linker, and n is 1 to 12; or a pharma- ceutically acceptable salt or stereoisomer thereof. In some embodiments, R and R 4’ are protecting groups.

[0055] In some embodiments, when the proantigen is an anthracene containing a boronic ester protecting group, the bifunctional compound has the structure represented by formula (VIII-1): [ka] In the formula, R4 and R 4’ each is independently O or a boronic ester protecting group; provided that R and R 4’ at least one of is a protecting group; and R5 is, [ka] [ka] [ka] and [ka] is absent or a linker, and n is 1 to 12; the boronic ester protecting group is [ka] [ka] is; or a pharma- ceutically acceptable salt or stereoisomer thereof.

[0056] In some embodiments, R and R 4’ is a boronic ester protecting group. Thus, in some embodiments, the bifunctional compound has the structure represented by formula (VIII-1a): [ka] or a pharma- ceutically acceptable salt or stereoisomer thereof.

[0057] In some embodiments, the bifunctional compound has the structure represented by formula (VIII-1b): [ka] or a pharma- ceutically acceptable salt or stereoisomer thereof.

[0058] In some other embodiments, the protective group is entirely or partially photosensitive, i.e., photocleavable. As used herein, the terms "photosensitive" and "photocleavable" are interchangeable. In some embodiments, the protective domain is composed of one or more photocleavable groups. When the photocleavable group is exposed to light of an appropriate wavelength, the pro-antigen is unmasked, thereby generating a synthetic antigen. Those skilled in the art will be able to determine the appropriate photoprotecting group and the corresponding wavelength-selective deprotection according to techniques known in the art. See, for example, Hansen et al. Chem. Soc. Rev. 44: 3358-77 (2015).

[0059] Representative examples of photocleavable groups include ortho-nitrobenzyl-based groups, phenacyl ester-based groups, 8-quinolinylbenzenesulfonate groups, dicoumarin groups, 6-bromo-7-alkoxycoumarin-4-ylmethoxycarbonyl groups, bimane-based groups, and bis-arylhydrazone-based groups. General structures and cleavage conditions are as follows (dashed lines indicate the site of cleavage): [ka] [ka]

[0060] In certain embodiments, the photocleavable protecting group is [ka] During the ceremony, X is NH or O, R is C1-4 alkyl or H, and n is 0 to 3; In some embodiments, the ortho-nitrobenzyl based group is [ka] and is cut at 300-365 nm.

[0061] In some embodiments, the bifunctional compound having a photocleavable protecting group has the structure represented by formula (IX): [ka] During the ceremony, R6 is, [ka] [ka] [ka] and [ka] is absent or a linker, and n is an integer from 1 to 12; has.

[0062] In some embodiments, the bifunctional compound having a photocleavable protecting group has the structure represented by formula (IX-1): [ka] or a pharma- ceutically acceptable salt or stereoisomer thereof.

[0063] Methods known in the art that may be useful in the synthesis of the bifunctional compounds of the invention are described in Lin et al. Meth. Enzymol. 526:19-43 (2013) (and publications referenced therein); Chang et al. J. Am. Chem. Soc. 126(47):15392-3 (2004) (and publications referenced therein); Dickinson et al. J. Am. Chem. Soc. 132(16):5906-15 (2010) (and publications referenced therein); Debowska et al. Chemical Research in Toxicology 29(5):735-46 (2016); Rios et al. Free Radical Biology & Medicine 101:284-95 (2016); and Wang et al. ACS Applied Materials & Interfaces 7(43):24110-18 (2015).

[0064] targeting part The targeting moiety, which constitutes one of the functional modes of the bifunctional compounds of the invention, specifically binds a solid tumor-associated antigen.

[0065] The targeting moiety according to the invention has binding specificity for solid tumor-associated antigens that may be present on tumor cells. These tumor-associated antigens may be overexpressed in tumor cells relative to their expression in normal, non-diseased cells. The lower the expression on normal tissues, the more effectively the availability of synthetic antigens on normal tissues is reduced. Thus, the present invention not only increases the number of targetable antigens "on-target, on-tumor", but also allows for better regulation by reducing "on-target, off-tumor" toxicity.

[0066] In some embodiments, tumor antigens are cell surface molecules that are inappropriately synthesized in cancer cells, e.g., molecules that contain deletions, additions, or mutations compared to the molecules expressed on normal cells. In some embodiments, tumor-associated antigens are major histocompatibility complex (MHC)-presented peptides. Normally, peptides derived from endogenous proteins fill the pockets of MHC class I molecules and are recognized by T cell receptors (TCRs) on CD8+ T lymphocytes. MHC class I complexes are constitutively expressed by all nucleated cells. In cancer, virus-specific and / or tumor-specific peptide / MHC complexes represent a unique class of cell surface targets for immunotherapy. In some embodiments, tumor antigens are cell surface molecules that are overexpressed in cancer cells compared to normal cells, e.g., overexpressed 1-fold, 2-fold, 3-fold, or more compared to normal cells.

[0067] Representative examples of targeting moieties include antibody molecules and their functional (i.e., antigen-binding) fragments, receptor ligands, peptides, haptens, aptamers, affimers, T-cell receptor tetramers, and other targeting molecules known to those skilled in the art. For example, targeting moieties can include nucleic acids, polypeptides, glycoproteins, carbohydrates, or lipids.

[0068] In some embodiments, the targeting moiety is an antibody or an antibody fragment. Representative examples of antibodies include monoclonal antibodies, polyclonal antibodies, Fv, Fab, Fab' and F(ab')2 immunoglobulin fragments, synthetic stabilized Fv fragments, such as single chain Fv fragments (scFv), disulfide stabilized Fv fragments (dsFv), single variable region domain (dAbs) minibodies, combibodies and multivalent antibodies such as diabodies and multi-scFv, single domains from camelids or engineered human equivalents. The term "antibody" also includes any protein with a binding domain that is homologous or largely homologous to an immunoglobulin binding domain. Such proteins can be derived from natural sources or can be partially or completely synthetically produced.

[0069] In one embodiment, the targeting moiety is an Affimer. Affimer proteins are composed of a stable protein scaffold based on the cystatin protein fold. Affimer proteins display two peptide loops and an N-terminal sequence that can be randomized to bind different target proteins with high affinity and specificity similar to antibodies. Stabilization of the peptides on a protein scaffold constrains the possible conformations that the peptides can adopt, thus increasing binding affinity and specificity compared to libraries of free peptides.

[0070] In some embodiments, the targeting moiety is a nucleic acid molecule (e.g., aptamer) that binds to cell type-specific markers. Aptamers are short synthetic single-stranded oligonucleotides that specifically bind to various molecular targets, such as small molecules, peptides, proteins, nucleic acids, and even cells and tissues. These small nucleic acid molecules can form secondary and tertiary structures that can specifically bind proteins or other cellular targets, and are essentially the chemical equivalent of antibodies. Aptamers are highly specific, relatively small in size, and non-immunogenic. Aptamers are typically selected from a biopanning method known as SELEX (Systematic Evolution of Ligands by Exponential enrichment) (Ellington et al. Nature 346(6287):818-822(1990); Tuerk et al. Science 249(4968):505-510(1990); Ni et al. Curr Med Chem. (2011);18(27):4206-14). Methods for generating aptamers for any given target are well known in the art.

[0071] In some embodiments, a targeting moiety is a naturally occurring or synthetic ligand for a cell surface receptor.

[0072] In some embodiments, the targeting moiety is a carbohydrate. The carbohydrate may be natural or synthetic. The carbohydrate may be a derivatized natural carbohydrate. In some embodiments, the carbohydrate comprises a monosaccharide or disaccharide, including but not limited to glucose, fructose, galactose, ribose, lactose, sucrose, maltose, trehalose, cellbiose, mannose, xylose, arabinose, glucuronic acid, galactoronic acid, mannuronic acid, glucosamine, galatosamine, or neuraminic acid. In some embodiments, the carbohydrate is a polysaccharide, such as, but not limited to, pullulan, cellulose, microcrystalline cellulose, hydroxypropylmethylcellulose (HPMC), hydroxycellulose (HC), methylcellulose (MC), dextran, cyclodextran, glycogen, starch, hydroxyethyl starch, carrageenan, glycon, amylose, chitosan, N,O-carboxymethylchitosan, algin and alginic acid, starch, chitin, konjac, glucomannan, pustulan, heparin, hyaluronic acid, curdlan, and xanthan. In some embodiments, the carbohydrate is a sugar alcohol, such as, but not limited to, mannitol, sorbitol, xylitol, erythritol, maltitol, or lactitol.

[0073] In one embodiment, the targeting moiety is directed to the TAA expressed by solid tumors that produce elevated levels of ROS and / or RNS.Elevated production of reactive species (ROS / RNS) has been detected in almost all cancers, and reactive species (ROS / RNS) promote many aspects of tumor development and progression (Liou et al.Free Radic.Res.44:479-496(2010);Trachootham et al.Nat.Rev.Drug Discov.8:579-591(2009)).Altered cellular metabolism is considered a hallmark of cancer and is rapidly becoming a means for therapeutic intervention.Mitochondria have recently been viewed as a key cellular compartment that energizes the metabolic needs of cancer cells, as they are the main source of ATP and metabolites required to meet the bioenergetic and biosynthetic needs of cancer cells. Furthermore, mitochondria are central to cell death and a major source of reactive oxygen species (ROS; Chowdhury et al. Oxid. Med. Cell. Longev. 2018:1-10 (2018); Zhang et al., Oxid. Med. Cell. Longev. 2016:1616781 (2016)). Extensive analysis of tumor cell lines in vitro showed that they are typically characterized by i) extracellular superoxide anion generation and ii) expression of membrane-bound catalase that protects cells against intercellular ROS signaling and apoptosis.

[0074] Representative examples of targeting moieties and their corresponding receptors on tumor cells are listed in Table 1:

Table 1-1

Table 1-2

Table 1-3

Table 1-4

Table 1-5

Table 1-6

Table 1-7

[0075] In some embodiments, the targeting ligand binds hematological tumor-associated antigen.For example, the tumor-associated antigens present on multiple myeloma cells that can be targeted by the bifunctional compound include any combination of CD38, CS1, BCMA, CD20, CD19, CD22, CD30, CD138, CD40, CD56, CD70 and CD74, for example, any combination of two or more of CD38, CS1, BCMA, CD20, CD19, CD22, CD30, CD138, CD40, CD56, CD70 and CD74.

[0076] In some embodiments, the targeting ligand binds a brain tumor-associated antigen. For example, tumor-associated antigens present on GBM cells include ACVR1, EGFRvIII, IL13Rα2 and HER2. For example, FIG. 1B shows a schematic of a multiplex approach for treating brain cancer that simultaneously targets EGFRvIII, IL13Rα2 and HER2. Other proteins that are implicated in brain cancer and can be targeted by the bifunctional compounds of the present invention include EphA2, CSPG4, GD2, PDGFRα and GRP78. Antibodies and / or functional fragments thereof that bind brain tumor-associated antigens are known in the art. For example, see Table 1 above, which describes antibodies and / or fragments thereof that bind ACVR1, PDGFRα, GD2 and EphA2, among others. The targeting moiety that binds PDGFRα can include scFvs based on olaratumab (and its binding variants).

[0077] In some embodiments, the targeting ligand binds to HER2 on HER2+ malignancies such as breast, lung, colorectal, brain, ovarian and pancreatic cancers. Exemplary targeting ligands that bind HER and may be useful in the present invention include trastuzumab and pertuzumab, which bind the extracellular domains IV and II of HER, respectively, and their HER-binding fragments (e.g., scFv).

[0078] A binding fragment that binds EGFRvIII is described in O'Rourke et al. Sci. Transl. Med. 9(399):eaaa0984(2017). Other antibodies or fragments thereof that bind EGFRvIII are commercially available siltuximab and mAb DH8.3 (Novus Biologicals). Further representative examples of amino acid or gene sequences encoding scFvs targeting EGFRvIII that may be useful in the present invention are found in U.S. Patent Application Publication No. 2015 / 0259423.

[0079] Antibody fragments that bind IL13Rα2 are described in Brown et al. N. Engl J Med. 375(26):2561-2569 (2016). Other antibodies or fragments thereof that bind IL13Rα2 are commercially available from Abnova and Millipore.

[0080] Antibody fragments that bind HER2 are described in Ahmed, et al., JAMA Oncol. 3(8):1049-1101 (2017). Other antibodies or fragments thereof that bind HER2, such as trastuzumab and FRP5, are commercially available. Further representative examples of amino acid or gene sequences encoding scFvs that target HER2 that may be useful in the present invention can be found in U.S. Patent Application Publication No. 2011 / 0313137.

[0081] Another example of an antibody fragment that binds EphA2 is described in Chow et al., Mol Ther. 21(3):629-637 (2013). Still other antibodies or fragments thereof that bind EphA2 are commercially available from Thermo Fisher (mAb4H5 and mAb 1C11A12) and RND Systems. Further representative examples of amino acid or gene sequences encoding scFvs that target EphA2 that may be useful in the present invention are described in U.S. Patent Application Publication No. 2010 / 436783.

[0082] An antibody fragment that binds CSPG4 is described in Pellegatta et al., Sci Transl Med, 10:eaao2731 (2018). Another antibody that binds CSPG4 is described in Fenton et al., Oncol Res. 22(2):117-21 (2015). Other antibodies or fragments thereof that bind CSPG4 are commercially available bevacizumab and Creative Biolabs mAb 225.28. Still other antibodies or fragments thereof that bind CSPG4 are commercially available from Aviva Systems Biology. Further representative examples of amino acid or gene sequences encoding scFvs that target CSPG4 that may be useful in the present invention are described in U.S. Patent No. 9,801,928 and U.S. Patent Application Publication No. 2019 / 0008940.

[0083] Another example of an antibody fragment that binds GD2 is described in Mount et al., Nat Med. 24:572-579 (2018). Other antibodies or fragments thereof that bind GD2 include dinutuximab, mAb 3F8, mAb 14g2a and mAb 14.18. Further representative examples of amino acid or gene sequences encoding scFvs that target GD2 that may be useful in the present invention are described in U.S. Patent No. 4,675,287.

[0084] Another example of an antibody fragment that binds PDGFRα is described in Brennan et al., PLoS One, 4(11):e7752 (2009). Other antibodies or fragments thereof that bind PDGFRα are commercially available from Abcam, LifeSpan Bio, Santa Cruz (sc-338) and Thermo Fisher (mAb APA5). Further representative examples of amino acid or gene sequences encoding scFvs that target PDGFRα that may be useful in the present invention are described in U.S. Patent Application Publication No. 2012 / 0027767.

[0085] Antibody fragments that bind GRP78 are described in Kang et al., Sci Rep. 6:34922 (2016). Other antibodies or fragments thereof that bind GRP78 are commercially available from Thermo Fisher (PA1-014A) and Abcam (N-20). Further representative examples of amino acid or gene sequences encoding scFvs that target GRP78 that may be useful in the present invention are described in U.S. Patent No. 10,259,884.

[0086] Other proteins implicated in brain cancer and which may be targeted by the bifunctional compounds of the invention include neural cell adhesion molecule (NCAM), cluster of differentiation 276 (CD276) and the neuroectodermal stem cell marker (nestin).

[0087] Antibodies that bind NCAM are described in Modak et al., Cancer Res. 61:4048-4054 ​​(2001). Other antibodies or fragments thereof that bind NCAM are mAb UJ13A and mAb ERIC-1. Further representative examples of amino acid or gene sequences encoding scFvs that target NCAM that may be useful in the present invention are described in U.S. Patent No. 7,402,560.

[0088] Antibodies that bind CD276 are described in Majzner et al., Clin Cancer Res. 25(8):2560-2574 (2019). Other antibodies or fragments thereof that bind CD276 are commercially available from Creative Biolabs (mAb 8H9). Further representative examples of amino acid or gene sequences encoding scFvs that target CD276 that may be useful in the present invention are described in U.S. Patent Application Publication No. 2018 / 0186890.

[0089] Antibodies or fragments thereof that bind nestin are commercially available from Abcam (ab6142) and Novus Biologicals (NB100-1604). Antibodies or fragments thereof that bind βIII-tubulin are available from Abcam (2G10) and RND Systems (mAB 1195).

[0090] Another example of an antibody fragment that binds CD38 is described in Mihara et al., J Hematol Oncol. 10:1-4 (2017). Other antibodies or fragments thereof that bind CD38 include commercially available antibodies from Miltenyi (REA572 and REA671), Biolegend (HIT2 and HB-7). Further representative examples of amino acid or gene sequences encoding scFvs that target CD38 that may be useful in the present invention are described in U.S. Patent No. 9,249,226.

[0091] Another example of an antibody fragment that binds CS-1 is described in Chu et al., Blood, 122:14 (2013). Other antibodies or fragments thereof that bind CS-1 are commercially available, including REA150 (Miltenyi) or 162.1 (Biolegend). Further representative examples of amino acid or gene sequences encoding scFvs that target CS-1 that may be useful in the present invention are described in WO 2004 / 100898 A2.

[0092] Another example of an antibody fragment that binds CD138 is described in Sun et al., Oncotarget 10(24):2369-2383 (2019). Other antibodies or fragments thereof that bind CD138 are commercially available, including 44F9 (Miltenyi) or DL-101 and MI15 (Biolegend). Further representative examples of amino acid or gene sequences encoding scFvs that target CD138 that may be useful in the present invention are described in WO 2009 / 080829 A1.

[0093] Another example of an antibody fragment that binds CD20 is described in Wang et al., Clin Immunol., 155(2):160-75 (2014). Other antibodies or fragments thereof that bind CD20 are commercially available, including LT20 and REA780 (Miltenyi) or 2H7 and SA271G2 (Biolegend). Further representative examples of amino acid or gene sequences encoding scFvs that target CD20 that may be useful in the present invention are described in WO 2004 / 056312 A2.

[0094] Another example of an antibody fragment that binds BCMA is described in Raje et al., N Engl J Med.380(18):1726-1737(2019). Other antibodies or fragments thereof that bind BCMA are commercially available and include REA315 (Miltenyi) and 19F2 (Biolegend). Further representative examples of amino acid or gene sequences encoding scFvs that target BCMA that may be useful in the present invention are described in WO 2010 / 104949 A2 and WO 2003 / 014294 A2.

[0095] Another example of an antibody fragment that binds CD19 is described in Lee et al., Lancet.385(9967):517-528(2015). Other antibodies or fragments thereof that bind CD19 are commercially available, including LT19, REA675 (Miltenyi) or 4G7, HIB19, SJ25C1 (Biolegend). Further representative examples of amino acid or gene sequences encoding scFvs that target CD19 that may be useful in the present invention are described in WO 2005 / 052004 A2.

[0096] Another example of an antibody fragment that binds CD22 is described in Haso et al., Blood 121(7):1165-1174 (2013). Other antibodies or fragments thereof that bind CD22 are commercially available, including REA340 (Miltenyi) or HIB22 and S-HCL-1 (Biolegend). Further representative examples of amino acid or gene sequences encoding scFvs that target CD22 that may be useful in the present invention are described in U.S. Patent No. 5,484,892.

[0097] Another example of an antibody fragment that binds CD30 is described in Ramos et al., Blood 132:680 (2018). Other antibodies or fragments thereof that bind CD30 are commercially available, including REA1085 and Ki-2 (Miltenyi) or BY88 (Biolegend). Further representative examples of amino acid or gene sequences encoding scFvs that target CD30 that may be useful in the present invention are described in U.S. Patent No. 7,090,843.

[0098] Another example of an antibody fragment that binds CD40 is described in Hussein et al., Haematologica 95:845-848 (2010). Other antibodies or fragments thereof that bind CD40 are commercially available, including REA733 and HB14 (Miltenyi) or 5C3 or HB14 (Biolegend). Further representative examples of amino acid or gene sequences encoding CD40-targeting scFvs that may be useful in the present invention are described in WO 2012 / 075111 A1 and WO 2016 / 069919 A1.

[0099] Another example of an antibody fragment that binds CD70 is described in Shaffer et al., Blood 117:4304-14 (2011). Other antibodies or fragments thereof that bind CD70 are commercially available, including REA292 (Miltenyi) or 113-16 (Biolegend). Further representative examples of amino acid or gene sequences encoding scFvs that target CD70 that may be useful in the present invention are described in WO 2004 / 073656 A2.

[0100] Another example of an antibody fragment that binds CD74 is described in Kaufman et al., Br J Haematol.163:478-486 (2013). Other antibodies or fragments thereof that bind CD74 are commercially available, including 5-329 and REA1103 (Miltenyi) or LN2 (Biolegend). Further representative examples of amino acid or gene sequences encoding scFvs that target CD74 that may be useful in the present invention are described in WO 2003 / 074567 A2.

[0101] The bifunctional compounds of the present invention can be synthesized according to methods known in the art. For example, see WO 2010 / 008519. For example, synthetic antigens can be derivatized to have chemical groups that react with primary amines (e.g., N-terminal amines or lysines of polypeptides) or with sulfhydryl groups (e.g., cysteines of polypeptides), and these polypeptides constitute the targeting moiety of the bifunctional compound. Bifunctional compounds can be prepared by conjugating targeting moieties to synthetic antigens (or pro-antigens) using techniques such as chemical conjugation and chemical cross-linking agents. In some embodiments, synthetic antigens can be conjugated to targeting moieties via linkers. Some of the factors that may require linkers include the need to recreate the microenvironment used to produce antibodies against unmasked pro-antigens (i.e., tags) used to engineer CAR-T cells, the need to expose small molecules to solvent and make them accessible to the extracellular tag-binding domains of CAR-T cells, and particularly hydrophobic small molecules may benefit from the use of hydrophilic / polar linkers, such as polyethylene glycol (PEG). The length of the linker can vary, but shorter linkers are preferred, since the tag that is proximal to the target cell can better elicit a CAR-T cell response to the target cell. Representative examples of hydrophobic / non-polar linkers include aliphatic linkers such as glycine, aminoheptanoic acid, aminohexanoic acid, aminopentanoic acid, and aminotetranic acid. Representative examples of polar linkers include, for example, polyethylene glycol moieties having 1-12 repeat units (e.g., 2, 4, 6, 8, 10, or 12 repeat units).

[0102] For example, 6-aminofluorescein can be reacted with maleimido-PEG-N-hydroxysuccinimide ester (NHS ester) to form compound (1), as shown in Scheme 1:

[0103] Scheme 1: [ka]

[0104] The maleimidofluorescein derivative, compound (1), then reacts with the sulfhydryl group of the targeting moiety to form a stable conjugate via a thioether bond as shown in Scheme 2.

[0105] Scheme 2: [ka]

[0106] Representative examples of the synthesis of bifunctional compounds with masked proantigens are shown in Schemes 3 and 4:

[0107] Scheme 3: [ka]

[0108] Scheme 4: [ka]

[0109] Each of compounds (2) and (3) can then be used in place of compound (1) to generate the bifunctional compounds of the invention, as shown in Scheme 2.

[0110] Another representative example of the synthesis of a bifunctional compound with a masked proantigen using an NHS ester derivative of fluorescein is shown in Scheme 5:

[0111] Scheme 5: [ka] where OR is substituted with a boronic ester and R' is a targeting moiety.

[0112] As described herein, the bifunctional compound may be in the form of a free acid or free base or a pharmaceutically acceptable salt. As used herein, the term "pharmaceutically acceptable" with respect to salt refers to a salt of a compound that does not suppress the biological activity or properties of the compound and is relatively non-toxic, i.e., the compound in salt form can be administered to a subject without causing undesirable biological effects (such as dizziness or upset stomach) or interacting in a harmful manner with any of the other components of the composition in which the salt is contained. The term "pharmaceutically acceptable salt" refers to the product obtained by reacting a compound described herein with a suitable acid or base. Examples of pharmaceutically acceptable salts of the compounds described herein include those derived from suitable inorganic bases, such as Li, Na, K, Ca, Mg, Fe, Cu, Al, Zn and Mn salts. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts of amino groups formed with inorganic acids such as hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, 4-methylbenzenesulfonate or p-toluenesulfonate. Certain compounds described herein can form pharmaceutically acceptable salts with various organic bases such as lysine, arginine, guanidine, diethanolamine or metformin. Suitable base salts include aluminum, calcium, lithium, magnesium, potassium, sodium or zinc salts.

[0113] As disclosed in various structures herein, the bifunctional compounds described herein may be in the form of stereoisomers, where stereoisomers refer to isomers of individual compounds that differ only in the arrangement of the atoms of the compounds in space, as known in the art.Thus, the term stereoisomer includes mirror images (enantiomers) of compounds, mixtures of mirror images of compounds (physical mixtures of enantiomers, racemates or racemic mixtures), geometric (cis / trans or E / Z, R / S) isomers of compounds, and isomers of compounds with more than one chiral center that are not mirror images of each other (diastereoisomers).The compounds described herein may be in the form of individual isomers, substantially free of other isomers, or in the form of mixtures of various isomers, for example racemic mixtures of stereoisomers.

[0114] Pharmaceutical Compositions The pharmaceutical composition comprises a therapeutically effective amount of a bifunctional compound and a pharma- ceutically acceptable carrier. In some embodiments, the pharmaceutical composition may further comprise another (second) bifunctional compound comprising a first synthetic antigen covalently linked to a second targeting moiety that binds a different epitope of the same (e.g., first) tumor-associated antigen, or a pharma- ceutically acceptable salt or stereoisomer thereof. Thus, the composition may comprise two or more bifunctional compounds, all of which bind the same tumor-associated antigen, but at least two of the bifunctional compounds bind different epitopes on the same tumor-associated antigen. In other embodiments, the pharmaceutical composition may further comprise another (e.g., second) bifunctional compound comprising a synthetic antigen covalently linked to a second targeting moiety that binds a different tumor-associated antigen (e.g., different from the first), or a pharma- ceutically acceptable salt or stereoisomer thereof. Thus, the composition may comprise two or more bifunctional compounds, all of which bind different tumor-associated antigens, and at least two of the bifunctional compounds bind different epitopes on different tumor-associated antigens.

[0115] A second important aspect of the present invention relates to the use of a therapeutically effective amount of a plurality of subpopulations of bifunctional compounds, each subpopulation having a specificity for a different tumor-associated antigen, but all subpopulations having the same specificity for CAR T cells (e.g., all bifunctional compounds contain the same synthetic antigen). Thus, in some embodiments, a pharmaceutical composition contains a therapeutically effective amount of a plurality of subpopulations of bifunctional compounds, in which the synthetic antigen (or pro-antigen) is the same in each subpopulation, but the targeting moieties differ in that they bind different tumor-associated antigens present on tumor cells. Thus, a pharmaceutical composition may contain a first subpopulation of bifunctional compounds having a synthetic antigen (or pro-antigen) covalently linked to a first targeting moiety that specifically binds a first tumor-associated antigen, and a second subpopulation of bifunctional compounds, each having a synthetic antigen (or pro-antigen) covalently linked to a second targeting moiety that specifically binds a second tumor-associated antigen, said first and second tumor-associated antigens being different.

[0116] In some embodiments, the first and second targeting moieties specifically bind brain tumor associated antigens. In some embodiments, the brain tumor associated antigens are selected from GD2, IL13Rα2, HER2, PDGFRα, EGFRvIII, CSPG4, EphA2, CD133, GRP78, NCAM, CD276 and nestin, and the first and second targeting moieties bind different epitopes on the same tumor associated antigen or different brain tumor associated antigens. In some embodiments, the multiple subpopulations of the bifunctional compound are designed to target two or more brain tumor associated antigens selected from GD2, IL13Rα2, HER2, PDGFRα, EGFRvIII, CSPG4, EphA2 and CD133. In some embodiments, the multiple subpopulations target two or more of IL13Rα2, EGFRvIII and HER2, and in some other embodiments, the multiple subpopulations target two or more of GD2, PDGFRα and CD133. In a further embodiment, multiple subpopulations of bifunctional compounds target EphA2 and CSPG4.

[0117] In some embodiments, the first and second targeting moieties specifically bind hematological tumor-associated antigens. In some embodiments, the hematological tumor-associated antigens are selected from CD38, CS1, BCMA, CD20, CD19, CD22, CD30, CD138, CD40, CD56, CD70 and CD74, and the first and second targeting moieties bind different epitopes on the same tumor-associated antigen or different hematological tumor-associated antigens. In some embodiments, the subpopulations of the bifunctional compound are designed to target two or more hematological tumor-associated antigens selected from CD38, CS1, BCMA, CD20, CD19, CD22, CD30, CD138, CD40, CD56, CD70 and CD74. In some embodiments, the subpopulations target two or more of CD38, CS1 and BCMA, and in some other embodiments, the subpopulations target two or more of CD19, CD20 and CD22. In a further embodiment, multiple subpopulations target CD30, CD40, CD56, CD70, CD74 and CD138.

[0118] In some embodiments, pharmaceutical composition can vary from administration to administration.Thus, the present invention can comprise a first composition as described above and a second composition, which is different from the first composition in that the targeting moiety of any one or more subpopulations binds to different epitopes of the same tumor-associated antigen.This additional feature can alleviate antigen loss / escape or reduce toxicity.

[0119] CAR T cells CAR T cells are engineered to bind synthetic or unmasked pro-antigens that do not naturally occur on any normal or cancer cells. Thus, by uncoupling tumor cell targeting from tumor cell killing, CAR T cells with a single specificity (for synthetic or unmasked pro-antigens) can simultaneously target multiple tumor-associated antigens.

[0120] In the method of the present invention, effector cells can be used. Effector cells can be autologous, syngeneic or allogeneic, and are selected according to the disease to be treated and the means available for treating the disease. Suitable populations of effector cells that can be used in these methods include any immune cell with cytolytic activity, such as T cells. Exemplary subpopulations of T cells include CD3 + CD8 + T cells, CD3 + CD4 + CD3, such as T cells and NKT cells + In some embodiments, the T cells are HLA-A2+ peripheral blood mononuclear cells (PBMCs), but the T cells can be of any HLA background from PBMCs and can be used in autologous, syngeneic or allogeneic systems. The T cells can also be isolated from any source, such as from tumor explants of the subject being treated or from intratumoral T cells of the subject being treated. For convenience, effector cells are referred to as T cells hereinafter, but it should be understood that all references to T cells herein refer to all effector cell types as defined herein, unless otherwise indicated.

[0121] The genetically engineered T cells used herein have binding specificity for a specific unshielded pro-antigen (also referred to herein as tag) that is joined to a targeting moiety (such as an antibody or its functional fragment) that binds to a tumor-associated antigen. Additional features of CAR include an activation domain that induces efficient target lysis upon T cell binding and activation, and the ability to replace or replace the scFv portion of CAR with one that has specificity for any one of the unshielded pro-antigens or tags of the present invention. In light of the design and specificity of CAR T cells, CAR T cells can be referred to as universal CAR-T cells or binary activated T cells (BAT-CAR).

[0122] A BAT-CAR polypeptide typically comprises three domains: the first domain is an extracellular ligand or tag-binding domain, the second domain is a transmembrane (TM) domain, and the third domain is a T cell activation domain.

[0123] Extracellular Ligands The first domain is typically present at the amino-terminal end of the BAT-CAR polypeptide and is therefore outside the T cell, allowing the tag-binding domain free access to the tagged protein bound to the target cell. The tag-binding domain is typically an antibody or an antigen-binding fragment thereof. In some embodiments, the antibody is a human or humanized antibody or an antigen-binding fragment thereof.

[0124] The tag-binding domain is designed to specifically bind the synthetic antigen that is covalently linked to the targeting moiety that binds the target cell (e.g., cancer cell).For example, if the synthetic antigen is fluorescein or a fluorescein derivative that is derivatized with a protecting group, the tag-binding domain specifically binds the fluorescein or fluorescein derivative that is not caged or unshielded, but does not bind to the caged molecule.Examples of such binding moieties are known in the art and are, for example, 4M5.3 ScFv, disclosed in Midelfort et al. J. Mol. Biol. 343: 685-701 (2004), and 2D12.5, 2D12.5ds, or C8.2.5, disclosed in Orcutt et al. Nucl. Med. Biol. 38 (2): 223-233 (2011).

[0125] The type of antibody can be polyclonal, monoclonal, chimeric or humanized. The antibody can be obtained from any animal species, for example, human, monkey, mouse, rat, rabbit, guinea pig, horse, cow, sheep, goat, pig, dog or cat. There is also no restriction on the specific class of antibody that can be used, including IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD and IgE antibodies. Antibody fragments that can also be used include single chain variable fragments (scFv), single chain antibodies, F(ab')2 fragments, Fab fragments and fragments produced by a Fab expression library, provided that the antibody fragment retains the ability to bind the selected tag.

[0126] The BAT-CAR of the present invention can be produced using commercially available extracellular ligands, at least as long as the synthetic antigen is known. Alternatively, antibodies and fragments thereof that specifically bind synthetic antigens can be prepared using standard techniques, such as continuous cell lines in culture for monoclonal antibody production. Representative techniques include the hybridoma technique first described by Koehler and Milstein (Nature 256:495-497 (1975)), human B cell hybridoma technique (Kosbor et al., Immunol Today 4:72 (1983); Cote et al., Proc Natl. Acad. Sci 80:2026-2030 (1983)) and EBV hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss Inc, New York NY, pp 77-96 (1985)). Techniques developed for the production of "chimeric antibodies" can also be used, i.e., splicing mouse antibody genes into human antibody genes to obtain molecules with appropriate antigen specificity and biological activity (Morrison et al., Proc Natl. Acad. Sci 81:6851-6855 (1984); Neuberger et al., Nature 312:604-608 (1984); Takeda et al., Nature 314:452-454 (1985)). As is known in the art, a humanized antibody or antibody fragment typically has one or more amino acid residues from the variable domain of an antibody derived from a non-human source. A humanized antibody or antibody fragment may contain one or more CDRs from a non-human immunoglobulin molecule and a framework region derived entirely or largely from human germline. Techniques for humanizing antibodies or antibody fragments are well known and include CDR grafting, veneering or resurfacing and chain shuffling.See also Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988)).

[0127] In some embodiments, the tag-binding domain of BAT-CAR-T is a single chain variable fragment (scFv). scFvs include the variable regions of the heavy (VH) and light (VL) chains of an antibody, and typically include up to about 50, e.g., about 10, amino acid residues. The linker can connect the N-terminus of the VH to the C-terminus of the VL, or vice versa. scFvs can be prepared according to methods known in the art (see, e.g., Bird et al., Science 242:423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)). In some embodiments, the linker sequence includes the amino acids glycine and serine, and in some cases, (Gly4Ser) n (n is an integer equal to or greater than 1). The length and amino acid composition of the linker can be varied, for example, to achieve optimal folding and interaction between VH and VL to create a functional epitope. See, e.g., Hollinger et al., Proc Natl Acad. Sci. USA 90:6444-6448 (1993).

[0128] Other types of antibody fragments with specificity for unmasked proantigens that may be useful in the present invention include Fv, Fab and (Fab')2 fragments. See, e.g., U.S. Patent No. 4,946,788.

[0129] The second domain is a transmembrane (TM) domain that can anchor the BAT-CAR in the cell membrane of the T cell. The BAT-CAR can be designed to include a transmembrane domain attached to the extracellular domain of the CAR. The transmembrane domain can be derived from the same protein or a different protein from which the other domains of the CAR (e.g., signaling domain, costimulatory domain and hinge domain) are derived. The transmembrane domain can be derived from a natural or recombinant source. If the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. Representative examples of transmembrane domains that can be useful in the present invention include the transmembrane regions of the α, β or ζ chains of the T cell receptor, CD28, CD27, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154.

[0130] In some embodiments, the transmembrane domain is attached to the extracellular region of the CAR, e.g., the antigen-binding domain of the CAR, via a hinge, e.g., a hinge from a human protein. Sources of hinge domains include human Ig (immunoglobulin) hinges (e.g., IgG4 hinges, IgD hinges) and CD8 (e.g., CD8α hinges).

[0131] The third domain of BAT-CAR is a T cell activation domain, also known as an intracellular signaling domain, which aids in T cell activation upon binding of the CAR to a tagged protein bound to a target cell. The intracellular signaling domain is generally involved in the activation of at least one of the normal effector functions of the effector cell into which the CAR is introduced. Examples of intracellular signaling domains for use in the CAR of the present invention include the cytoplasmic sequence of the T cell receptor (TCR) and the co-receptors that act in concert to initiate signaling after antigen receptor engagement. The signal generated through the TCR alone is insufficient for full activation of the T cell, and therefore a secondary or costimulatory signal is also required. Thus, T cell activation is mediated by two distinct classes of cytoplasmic signaling sequences, namely, cytoplasmic signaling sequences that initiate antigen-dependent primary activation through the TCR (i.e., primary intracellular signaling domains) and cytoplasmic signaling sequences that act in an antigen-independent manner to provide secondary or costimulatory signals (i.e., secondary cytoplasmic or costimulatory domains). The primary signaling domain controls the primary activation of the TCR complex in either a stimulatory or inhibitory manner. The primary intracellular signaling domain that acts in a stimulatory manner may contain a signaling motif known as an immunoreceptor tyrosine-based activation motif (ITAM). Representative examples of ITAM-containing primary intracellular signaling domains that may be suitable for use in the present invention include CD3zeta, common FcRgamma (FCER1G), Fc-gamma RIIa, FcR-beta (Fc-epsilon R1b), CD3gamma, CD3delta and CD3epsilon. In some embodiments, the BAT-CAR includes an intracellular signaling domain that contains the primary signaling domain of CD3zeta.

[0132] The intracellular signaling domain of the BAT-CAR may also include at least one other intracellular signaling or costimulatory domain. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligand that is required for an efficient response of lymphocytes to antigens. Representative examples of costimulatory domains that may be useful in the BAT-CAR of the present invention include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, HVEM (LIGHTR), lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, and B7-H3. CD27 costimulation has been demonstrated, for example, to enhance the proliferation, effector function, and survival of human CART cells in vitro, and to enhance the persistence and antitumor activity of human T cells in vivo (Song, et al., Blood 119(3):696-706 (2012)).

[0133] The intracellular signaling domain can be designed to include one or more, e.g., 1, 2, 3, 4, 5 or more, costimulatory signaling domains, which can be linked together in a specified or random order, optionally via a linker molecule. A polypeptide linker that is about 1-10 amino acids in length can link consecutive intracellular signaling sequences. Examples of such linkers include doublets such as Gly-Ser and single amino acids, e.g., Ala and Gly. Combinations that can constitute a T cell activation domain can be based on the cytoplasmic regions of CD28, CD137 (4-1BB), OX40 and HVEM, which play a role in enhancing T cell survival and proliferation, as well as CD3 CD3ζ and FcRε, which induce T cell activation. For example, CD3ζ, which contains three ITAMs, is the most commonly used intracellular domain component of CARs and transmits activation signals to T cells after antigen is bound. However, to provide additional costimulatory signaling, the CD28 and OX40 domains can be used together with CD3ζ, allowing the BAT-CAR T cells to transmit proliferation / survival signals.

[0134] A representative example of a polynucleotide encoding anti-FL CAR-CD28-4-1BB-CD3ζ has the sequence set forth as SEQ ID NO:1.

[0135]

[0136] T cells can be engineered to express BAT-CAR according to known techniques. In general, a polynucleotide vector encoding BAT-CAR is constructed, and the vector is transfected into a population of T cells. The cells are then grown under conditions that promote the expression of the polynucleotide encoding BAT-CAR by T cells. Successful transfection (or transduction, which represents virus-mediated gene integration) and display of BAT-CAR by T cells can be performed via standard techniques.

[0137] In some embodiments, T cells can be engineered to produce BAT-CAR by first constructing a retroviral vector encoding a selected BAT-CAR. Retroviral transduction can be performed using known techniques (e.g., Johnson et al. Blood 114:535-546 (2009)). Surface expression of BAT-CAR on transduced T cells can be determined, for example, by flow cytometry.

[0138] The population of BAT-CAR T cells can be prepared for administration to a subject using known techniques. The formulation comprising a population of BAT-CAR-expressing T cells can comprise one or more pharma- ceutically acceptable excipients. The excipients included in the formulation can have different purposes, for example, depending on the nature of the tag-binding domain, the subpopulation of T cells used, and the mode of administration. Representative examples of excipients include saline, buffered saline, dextrose, water for injection, glycerol, ethanol, and combinations thereof, stabilizers, solubilizers and surfactants, buffers and preservatives, isotonicity agents, bulking agents, and lubricants. The formulation comprising a population of BAT-CAR T cells is typically prepared and cultured in the absence of any non-human components, such as animal serum (e.g., bovine serum albumin).

[0139] Systems and Kits A further aspect of the invention is directed to a system comprising multiple subpopulations of bifunctional compounds and CAR T cells, which may be autologous, allogeneic or syngeneic.

[0140] Any of the compositions described herein may be included in a kit, which comprises: a) a therapeutically effective amount of a plurality of subpopulations of bifunctional compounds or pharma- ceutically acceptable salts or stereoisomers thereof, each bifunctional compound in a first subpopulation comprising a first synthetic antigen covalently linked to a first targeting moiety that specifically binds a first tumor-associated antigen, and each bifunctional compound in a second subpopulation comprising said first synthetic antigen covalently linked to a second targeting moiety that specifically binds a second tumor-associated antigen, wherein said first and second targeting moieties specifically bind different tumor-associated antigens, and each subpopulation of bifunctional compounds administered to a patient contains said first synthetic antigen but specifically binds a different solid tumor-associated antigen, said plurality of subpopulations of bifunctional compounds being disposed in the same or separate containers; b) a therapeutically effective amount of a subpopulation of the plurality of bifunctional compounds; and instructions for co-administering to a cancer patient a therapeutically effective number of CAR-T cells, the CAR-T cells comprising an extracellular ligand that specifically binds the synthetic antigen. may include.

[0141] In some embodiments, the kit may further comprise one or more cells for use in cell therapy, and / or the kit or system may comprise reagents for generating one or more cells for use in cell therapy carrying the recombinant expression vector. In yet another embodiment, the kit further comprises a therapeutically effective number of allogeneic CAR-T cells. In any of the above embodiments, the kit optionally comprises a reagent (e.g., a ROS / RNS generating agent) that cleaves a protecting group contained in the pro-antigen. The kit components are provided in one or more suitable container means.

[0142] Some components of the kits may be packaged either in aqueous media or in lyophilized form. The container means of the kit will generally include at least one vial, test tube, flask, bottle, syringe or other container means into which a component may be placed, preferably in suitable aliquots. If there is more than one component in the kit, the kit will also generally contain a second, third or other additional container into which the additional component may be separately placed. However, various combinations of components may be included in a vial. The kits of the present invention will also typically include a means for containing the components in close confinement for commercial sale. Such containers may include injection or blow molded plastic containers into which the desired vials are retained.

[0143] When the components of the kit are provided in one and / or more liquid solutions, the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly useful. In some cases, the container means itself may be a syringe, pipette and / or other such similar instrument from which the formulation may be applied to an infected area of ​​the body, injected into an animal, and / or applied to and / or mixed with other components of the kit.

[0144] However, the components of the kit may be provided as a dry powder. When the reagents and / or components are provided as a dry powder, the powder may be reconstituted by the addition of a suitable solvent. It is also envisioned that the solvent may be provided in a separate solution means. The kit may also include a second container means for containing a sterile, pharma- ceutically acceptable buffer and / or diluent.

[0145] In certain embodiments, one or more devices suitable for extracting one or more samples from an individual are present in the kit. Devices may include syringes, scalpels, etc.

[0146] In some embodiments, the kit may further comprise, by way of a separate container, a second anti-cancer agent, which may be formulated therein with a pharma- ceutically acceptable carrier.

[0147] method The method of the invention is directed to a method of treating cancer, comprising: a) administering to a patient a subpopulation of bifunctional molecules, the subpopulation comprising: a1) a therapeutically effective amount of a first subpopulation of bifunctional compounds comprising a synthetic antigen covalently linked to a first targeting moiety that specifically binds a first tumor-associated antigen; and a2) a therapeutically effective amount of a second subpopulation of bifunctional compounds comprising a synthetic antigen covalently linked to a second targeting moiety that specifically binds a second tumor-associated antigen, or a pharma- ceutically acceptable salt or stereoisomer thereof, wherein the first and second targeting moieties specifically bind different tumor-associated antigens; b) a therapeutically effective number of CAR-T cells, the CAR-T cells comprising an extracellular ligand that specifically binds a synthetic antigen; and The term "co-administering," as used herein, when referring to multiple subpopulations of a bifunctional compound includes administering during the same treatment regimen. The multiple subpopulations can be administered simultaneously or sequentially (e.g., after the patient is no longer responsive to the combination of the first subpopulation of CAR T cells and the bifunctional compound).

[0148] As used herein, the terms "treat", "treating" and "treatment" have their usual and customary meanings, including one or more of preventing, alleviating, or reducing the severity and / or frequency of symptoms of cancer in a subject.In some embodiments, the subject undergoing treatment is a human.In other embodiments, the subject is a non-human animal, such as a non-human primate, bird, horse, cow, goat, sheep, dog, cat or rodent, or other mammal.

[0149] Cancers that may be suitable for treatment with the treatment modalities of the present invention are characterized by the presence of solid or hematological tumors. Broadly, solid or hematological tumors include both adult and pediatric adenomas, carcinomas, sarcomas, and hematological malignancies such as multiple myeloma, leukemia, and lymphoma. Cancers may be vascularized or not yet substantially vascularized or non-vascularized tumors.

[0150] The cancer to be treated includes primary tumors and secondary or metastatic tumors that have metastasized from, for example, lung, breast, brain or prostate, as well as recurrent or refractory tumors.Recurrent tumors include tumors that appear to be inhibited by treatment with such agents, but recur up to 5 years, or up to 10 years, or longer, after treatment has ceased.Refractory tumors are tumors that have been unresponsive or resistant to treatment with one or more conventional, approved, or experimental therapies for a particular tumor type.

[0151] The therapeutic method of the present invention may be a "first-line" treatment, i.e., an initial treatment in patients who have not yet received any anti-cancer treatment, alone or in combination with other treatments. The therapeutic method of the present invention may also be advantageously used as a "second-line" treatment, in the sense that it is administered to patients who have received at least one previous anti-cancer treatment regimen, such as chemotherapy, radioimmunotherapy, toxin therapy, prodrug-activated enzyme therapy, antibody therapy, surgical therapy, immunotherapy, radiation therapy, targeted therapy, or any combination thereof, alone or in combination with other treatments. In some cases, when a patient becomes intolerant to a particular treatment, especially when the first-line treatment is no longer effective due to antigen loss / escape, the previous treatment may have been unsuccessful or partially successful. The method of the present invention may also be used as an adjuvant treatment, for example, to inhibit the recurrence of cancer in patients who currently have undetectable disease or after surgical removal of a tumor.

[0152] Representative examples of cancers characterized by solid tumors that may be treated according to the present invention include breast (including HER2+ and metastatic), colorectal (e.g., colon), esophagus, bile duct, lung (including small cell and non-small cell lung tumors, adenocarcinoma of the lung and squamous cell carcinoma of the lung), liver, epidermoid tumors, squamous cell tumors, such as head and neck tumors, epithelial squamous cell carcinoma, thyroid, cervix, ovary, neuroendocrine tumors, pheochromacytoma, carcinoma of the peritoneum, hepatoblastoma, hepatocellular carcinoma, hepatocellular carcinoma, bladder cancer, hepatoma, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, bone cancer, soft tissue sarcomas (including embryonal and alveolar rhabdomyosarcoma, rectal, pancreatic, prostate, gastrointestinal (including gastric and stomach), alveolar soft part sarcoma and clear cell sarcoma), biliary Ductal cell carcinoma, gallbladder carcinoma, myeloma, vulvar cancer, penile carcinoma, retina, androgen-dependent tumors, androgen-independent tumors, Kaposi's sarcoma, synovial sarcoma, vasoactive intestinal peptide-secreting tumors, central nervous system (CNS) neoplasms, melanoma, Wilms' carcinoma, Ewing's carcinoma, osteosarcoma, PNT, rhabdoid, retinoblastoma, adrenal carcinoma, adrenal tumor, leiomyosarcoma, and embryonal rhabdomyosarcoma, alveolar striated myeloma. Rhabdomyosarcoma including pleomorphic rhabdomyosarcoma, botryoid rhabdomyosarcoma, multiple myeloma (MM), acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, and large granular lymphocytic (LGL) leukemia.

[0153] Representative examples of high ROS / RNS producing cancers that may be particularly suitable for treatment with bifunctional compounds containing a proantigen having one or more boronic ester protecting groups (e.g., boronic esters) that are reactive to ROS / RNS include pancreatic cancer, prostate cancer, Kaposi's sarcoma, liver cancer, breast cancer, cholangiocarcinoma, gastric cancer, lung adenocarcinoma, pancreatic ductal adenocarcinoma, breast carcinoma, lung carcinoma, thyroid carcinoma and sarcoma, melanoma, kidney, stomach, colon, liver, pancreatic and bladder carcinoma, neuroblastoma, prostate carcinoma, ovarian carcinoma, human papillomavirus (HPV) positive cervical carcinoma, osteogenic sarcoma, Ewing's sarcoma, rhabdomyosarcoma, fibrosarcoma, chondrosarcoma and neurosecretory tumors (Bauer, et al., Anticancer Res. 34:1467-1482 (2014)).

[0154] In some embodiments, the modalities of the present invention are used to treat brain cancers, representative examples of which include DIPG, capillary hemangioblastoma, meningioma and cerebral metastases, glioma, glioblastoma multiforme (GBM) and neuroblastoma, medulloblastoma and ependymoma.

[0155] In some embodiments, the modalities of the present invention are used to treat hematological cancers. Representative examples of blood cancers include multiple myeloma (MM), acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, and large granular lymphocytic (LGL) leukemia.

[0156] Representative examples of gliomas that may be treatable using the modalities of the present invention include recurrent high-grade gliomas, including glioblastoma, anaplastic astrocytoma, and anaplastic oligodendroglioma, as well as high-grade pediatric gliomas such as DIPG.

[0157] Representative examples of glioblastomas that may be treatable in the manner of the present invention include grade II (low-grade astrocytoma), grade III (anaplastic astrocytoma) and grade IV (glioblastoma) glioblastomas and glioblastoma multiforme (GBM).

[0158] In some embodiments, the treatment of brain cancer according to the present invention comprises simultaneous targeting of two or more brain tumor-associated antigens selected from GD2, IL13Rα2, HER2, PDGFRα, EGFRvIII, CSPG4, EphA2 and CD133. In some embodiments, the treatment of brain cancer may comprise simultaneous targeting of IL13Rα2, EGFRvIII and HER2. In some other embodiments, the treatment of brain cancer may comprise simultaneous targeting of GD2, PDGFRα and CD133. In some other embodiments, the treatment of brain cancer may comprise simultaneous targeting of EphA2 and CSPG4. In some embodiments, the multiple targeting is performed in a sequential manner.

[0159] Treatment of hematological cancers according to the present invention may, in some embodiments, include simultaneous targeting of two or more hematological tumor-associated antigens selected from CD38, CS1, BCMA, CD20, CD19, CD22, CD30, CD138, CD40, CD56, CD70, and CD74. In some embodiments, treatment of hematological cancers may include simultaneous targeting of CD38, CS1, and BCMA. In some other embodiments, treatment of hematological cancers may include simultaneous targeting of CD19, CD20, and CD22. In some other embodiments, treatment of hematological cancers may include simultaneous targeting of CD30, CD40, CD56, CD70, CD74, and CD138. In some embodiments, multiple targeting is performed in a sequential manner.

[0160] The formulation contains BAT-CAR T cells in a number that is effective for treating a particular cancer. Thus, a therapeutically effective population of BAT-CAR T cells is administered to the subject. The number of BAT-CAR T cells administered to the subject will vary between wide limits, depending on the location, type and severity of the cancer, the age and symptoms of the individual to be treated, etc. The physician will ultimately determine the appropriate dosage to be used. Generally, about 1×10 4 ~Approx. 1×10 10 In some embodiments, the formulation contains about 1×10 BAT-CAR T cells. 5 ~Approx. 1×10 9 BAT-CAR T cells, approximately 5 x 10 5 ~Approx. 5×10 8 BAT-CAR T cells or approximately 1 x 10 6 ~Approx. 1×10 7 Contains BAT-CAR T cells.

[0161] The BAT-CAR T cell formulation may be administered to a subject in need thereof according to accepted medical practice. An exemplary mode of administration is intravenous injection. Other modes include intratumoral, intradermal, subcutaneous (sc, sq, sub-Q, Hypo), intramuscular (im), intraperitoneal (ip), intraarterial, intramedullary, intracardiac, intraarticular (joint), intrasynovial (synovial fluid area), intracranial (including convection-enhanced transport), intraspinal and intrathecal (spinal fluid). To effect such a mode of administration, any known device useful for parenteral injection or infusion of a formulation can be used. Such formulations can include a buffer, such as neutral buffered saline, phosphate buffered saline, carbohydrates, such as glucose, mannose, sucrose, or dextran, mannitol, proteins, amino acids, such as polypeptides or glycine, antioxidants, chelating agents, such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), and preservatives.

[0162] The bifunctional compound and the BAT-CAR T cells are administered to the subject in combination, and in the present invention, combined administration includes administration during the same treatment regimen. The compound can be administered to the subject before, simultaneously (e.g., simultaneously) or after administration of the BAT-CAR T cells, so that the compound binds the target cells and the BAT-CAR cells bind the unmasked pro-antigen or tag. In some embodiments, the bifunctional compound is protected by a mask, and the BAT-CAR cells bind the compound only when the antigen becomes unmasked.

[0163] The formulation containing the bifunctional compound can be administered to a subject in an amount that is effective for treating a particular cancer.The compound can be formulated for administration to a subject using techniques known to those skilled in the art.The compound formulation can include a pharma-ceutically acceptable carrier that can be selected based on factors such as targeting moiety, nature of pro-antigen and mode of administration.Representative examples of commonly used carriers include saline, buffered saline, dextrose, water for injection, glycerol, ethanol and combinations thereof, stabilizers, solubilizers and surfactants, buffers and preservatives, isotonicity agents, bulking agents and lubricants.

[0164] In general, the therapeutically effective amount of the bifunctional compound administered to a subject will vary between wide limits, depending on the location, source, identity, extent and severity of the cancer, the age and symptoms of the individual to be treated, etc. The physician will ultimately determine the appropriate dosage to be used. Typically, the formulation may contain about 0.1 mg / kg to about 100 mg / kg body weight of the compound, in some embodiments about 1 mg / kg to about 10 mg / kg body weight of the compound, taking into account the route of administration, symptoms, etc. In general, the dosage of the compound of the present application administered to a subject to treat a disease or disorder such as cancer is in the range of 0.01 to 500 mg / kg of the subject's body weight, for example, in the range of 0.1 mg / kg to 100 mg / kg of the subject's body weight. For example, the dosage of the compound administered to a subject may range from 0.1 mg / kg to 50 mg / kg or 1 mg / kg to 50 mg / kg of the subject's body weight, and more preferably from 0.1 mg / kg to 25 mg / kg or 1 mg / kg to 25 mg / kg of the patient's body weight. In another example, the dosage of a compound of the invention administered to a subject to prevent, treat, and / or manage cancer in a patient is 500 mg / kg or less of the subject's body weight, preferably 250 mg / kg or less of the subject's body weight, 100 mg / kg or less of the subject's body weight, 95 mg / kg or less of the subject's body weight, 90 mg / kg or less of the subject's body weight, 85 mg / kg or less of the subject's body weight, 80 mg / kg or less of the subject's body weight, 75 mg / kg or less of the subject's body weight, 70 mg / kg or less of the subject's body weight, 65 mg / kg or less of the subject's body weight, 60 mg / kg or less of the subject's body weight, 55 mg / kg or less of the subject's body weight. or less, 50 mg / kg or less of the subject's body weight, 45 mg / kg or less of the subject's body weight, 40 mg / kg or less of the subject's body weight, 35 mg / kg or less of the subject's body weight, 30 mg / kg or less of the subject's body weight, 25 mg / kg or less of the subject's body weight, 20 mg / kg or less of the subject's body weight, 15 mg / kg or less of the subject's body weight, 10 mg / kg or less of the subject's body weight, 5 mg / kg or less of the subject's body weight, 2.5 mg / kg or less of the subject's body weight, 2 mg / kg or less of the subject's body weight, 1.5 mg / kg or less of the subject's body weight, or 1 mg / kg or less of the subject's body weight.

[0165] The bifunctional compound may be administered to a subject in need thereof according to accepted medical practice. An exemplary mode of administration is intravenous injection. Other modes include intratumoral, intradermal, subcutaneous (sc, sq, sub-Q, Hypo), intramuscular (im), intraperitoneal (ip), intraarterial, intramedullary, intracardiac, intraarticular (joint), intrasynovial (synovial area), intracranial, intraspinal and intrathecal (spinal fluid). Any known device useful for parenteral injection or infusion of a formulation may be used to carry out administration of the bifunctional compound.

[0166] In embodiments in which the bifunctional compound contains a proantigen that contains a protecting group that is removable by ROS / RNS, activation of the proantigen may be achieved in some embodiments of cancer treatment simply due to elevated levels of ROS / RNS in the tumor microenvironment, however, not all tumors naturally produce elevated amounts of ROS / RNS. Thus, in some embodiments of the invention, the methods may further include localized administration of one or more agents at or near the tumor site to increase ROS / RNS levels to activate or unmask proantigens. In some embodiments, the amount of ROS / RNS in the tumor microenvironment may be advantageously increased by radiation. Radiation may be administered in the form of extracorporeal light or via brachytherapy or administration of radionuclides. Representative examples of radionuclides that can increase the level of ROS / RNS when delivered to the tumor microenvironment include gallium-68, lutetium-177, carbon-11, indium-111, and yttrium-90. In other embodiments, the level of ROS / RNS may be increased by administration of lanthanide nanoparticles. The use of such nanoparticles may be advantageous in lowering the amount of radiation required to generate increased ROS / RNS levels. In some embodiments, lanthanide nanoparticles may be preferentially taken up by certain brain cells, such as microglia, resulting in increased ROS / RNS in these cells, thus making these cells preferential targets for BAT-CAR-T cells.

[0167] Alternatively or in combination with radiation, the method may include administration of an ROS / RNS generating agent. Such generating agents are known in the art. See, for example, U.S. Patent Application Publication Nos. 2014 / 0228290 and 2006 / 0235080.

[0168] In some embodiments, the ROS / RNS generating agent is an inhibitor of CD44. This protein, together with its splice variants, is often found to be overexpressed on tumors and tumor-initiating cells. Representative examples of tumors that express or overexpress CD44 or CD44 variants include cholangiocarcinoma, gastric cancer, glioblastoma, lung adenocarcinoma, stem and stem-like cancer cells, breast cancer, pancreatic ductal adenocarcinoma and neuroendocrine tumors. CD44 functions as a cysteine / glutamine exchanger, pumping glutamine out of cells and cysteine ​​into cells, resulting in the intracellular production of glutathione, which helps tumor cells deal with elevated ROS / RNS. In contrast to tumor cells, normal cells do not require extra glutathione due to the fact that the endogenous levels of antioxidants in normal cells are able to handle normal levels of ROS / RNS.

[0169] The frequency of administration of the formulation containing a population of BAT-CAR-T cells, the formulation of the compound and optionally the ROS / RNS generating agent will vary depending on factors that may include the disease being treated, the structure of the BAT-CAR-T cells and the compound and the mode of administration. Each formulation can be independently administered 4, 3, 2 or 1 times a day, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, once a week, every 8 days, every 9 days, every 10 days, every 2 weeks, every month and every 2 months. The duration of treatment also varies and is best determined by the attending physician, for example, based on the disease being treated. However, it is envisioned that the duration of treatment can last for days, weeks or months.

[0170] The methods of the present application may include separate or combined administration of the compound, BAT-CAR-T cells, and optionally the ROS / RNS generating agent to a subject in a single, one-time dose, or in multiple doses (e.g., 2, 3, 4, 5, 6, 7, 8, 10, 15, 20 or more doses). Thus, the frequency of combined administration may range from once to about once every 8 weeks up to about once every 8 weeks. In another example, the frequency of administration ranges from about once per week to about once per 6 weeks up to about once every 6 weeks. In some embodiments, the frequency of administration ranges from about once per 3 weeks to about once per 4 weeks up to about once every 4 weeks. In other embodiments, the BAT-CAR-T cells may be administered in a single, one-time dose, while the frequency of administration of the bifunctional compound and optionally the ROS-RNS generating agent may range from a single dose to once per day, once per week, or about once per 4-6 weeks up to about once every 4-6 weeks. Thus, in other embodiments, the BAT-CAR-T cells are administered more than once, i.e., in multiple doses (e.g., 2, 3, 4, 5, 6, 7, 8, 10, 15, 20 or more doses).

[0171] In some embodiments, these methods include the simultaneous administration of multiple pharmaceutical compositions, each comprising a therapeutically effective amount of a bifunctional compound and a pharma- ceutically acceptable carrier, in which the targeting moiety in each composition binds a different tumor-associated antigen. In some other embodiments, the simultaneous administration includes the use of a single pharmaceutical composition containing multiple (2 or more) subpopulations of bifunctional compounds, in which the composite antigen may be the same, but each targeting moiety binds a different tumor-associated antigen present on tumor cells. Thus, a pharmaceutical composition may contain a first subpopulation of bifunctional compounds having a first targeting moiety that specifically binds a first tumor-associated antigen, and a second subpopulation of bifunctional compounds, each having a second targeting moiety that specifically binds a second tumor-associated antigen, said first and second targeting moieties binding different tumor-associated antigens. In other embodiments, the multiple subpopulations of bifunctional compounds contain a third, fourth, fifth, etc. subpopulation of bifunctional compounds, each of which binds a different tumor-associated antigen present on tumor cells.

[0172] Some embodiments include any type of sequential administration of the composition, and in at least one such sequential administration, at least one of the subpopulations of the composition or bifunctional compound is modified to replace the targeting moiety that binds a different epitope on the same tumor-associated antigen compared to the epitope of the previous administration.Thus, an embodiment of the present invention may include a first administration of a "single" composition and a second administration that differs from the first administration in that the targeting moiety of any one or more subpopulations binds a different epitope on the same tumor-associated antigen.This additional feature may mitigate antigen loss / escape or reduce toxicity.

[0173] Combination therapy In certain embodiments, the method of the present invention for treating cancer can be part of a combination therapy in which the subject is also treated with another anti-cancer agent. An "anti-cancer" agent can adversely affect cancer in a subject, for example, by killing cancer cells, inducing apoptosis in cancer cells, reducing the rate of proliferation of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing blood supply to the tumor or cancer cells, promoting an immune response to the cancer cells or tumors, suppressing or inhibiting the progression of cancer, or increasing the life span of a subject with cancer. More generally, these other compositions will be given in a combined amount effective to kill or inhibit the proliferation of the cells. This process can include contacting the cancer cells with the expression construct and the agent or multiple factors simultaneously. This can be accomplished by contacting the cells with a single composition or pharmacological formulation that includes both agents, or by contacting the cells with two different compositions or formulations simultaneously, one composition that includes the expression construct and the other that includes the second agent.

[0174] Tumor cell resistance to chemotherapy and radiotherapy is a major problem in clinical oncology.One goal of current cancer research is to find a way to improve the effectiveness of chemotherapy and radiotherapy by combining chemotherapy and radiotherapy with other therapies.In the present invention, it is envisioned that cell therapy can be used in combination with chemotherapy, radiotherapy or immunotherapy intervention and apoptosis promoter or cell cycle regulator as well.

[0175] Alternatively, the therapy of the invention may precede or follow the other agent treatment by intervals ranging from minutes to weeks. In embodiments in which the other agent and the invention are applied separately to an individual, generally no significant period of time will pass between the time of each delivery, so that the agent and the therapy of the invention can still exert an advantageously combined effect on the cells. In such cases, it is contemplated that the cells may be contacted with both modalities within about 12-24 hours of each other, more preferably within about 6-12 hours of each other. In some circumstances, it may be desirable to significantly extend the period for treatment, with days (2, 3, 4, 5, 6, 7, or 8) to weeks (1, 2, 3, 4, 5, 6, 7, or 8) passing between each administration.

[0176] It is expected that treatment cycles will be repeated as necessary. It is also envisioned that various standard therapies and surgical interventions may be applied in combination with the cell therapy of the present invention.

[0177] chemotherapy Cancer therapy also includes a variety of combination therapies with both chemo- and radiation-based treatments. Combination chemotherapy includes, for example, Abraxane, altretamine, docetaxel, herceptin, methotrexate, novantrone, zoladex, cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin (pl Also included are icomycin, mitomycin, etoposide (VP16), tamoxifen, raloxifene, estrogen receptor binding agents, taxol, gemcitabien, navelbine, farnesyl protein transferase inhibitors, transplatinum, 5-fluorouracil, vincristine, vinblastine and methotrexate or analogs or derivative variants of any of the foregoing and combinations thereof.

[0178] In certain embodiments, chemotherapy to an individual is utilized in conjunction with the present invention, for example, before, during and / or after administration of the present invention.

[0179] Radiation therapy Other agents that cause DNA damage and have been widely used include gamma radiation, X-rays and / or what are commonly known as directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging agents such as microwave and UV radiation are also envisioned. All of these agents most likely result in a wide range of damage to DNA, to the precursors of DNA, to DNA replication and repair, and to chromosome assembly and maintenance. Dosage ranges for X-rays range from daily doses of 50-200 roentgens for extended periods (3-4 weeks) to single doses of 2000-6000 roentgens. Dosage ranges for radioisotopes vary widely and depend on the half-life of the isotope, the strength and type of radiation emitted, and uptake by the neoplastic cells.

[0180] immunotherapy Immunotherapy generally relies on the use of immune effector cells and molecules to target and destroy cancer cells. The immune effector can be, for example, an antibody specific for a certain marker on the surface of tumor cells. The antibody alone can act as the effector of therapy, or it can recruit other cells that actually cause cell death. The antibody can also be conjugated to a drug or toxin (chemotherapeutic agent, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and can simply act as a targeting agent. Alternatively, the effector can be a lymphocyte that carries a surface molecule that interacts directly or indirectly with the tumor cell target. Various effector cells include cytotoxic T cells and NK cells.

[0181] Immunotherapies other than the treatment of the present invention described herein can be used as part of a combination therapy in combination with the cell therapy of the present invention. Common tumor markers that can be targeted by monoclonal antibodies and the like include PD-1, PD-L1, CTLA4, carcinoembryonic antigen, prostate specific antigen, urinary tumor associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialyl Lewis antigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb B and p155.

[0182] gene In yet another embodiment, the secondary treatment is gene therapy in which a therapeutic polynucleotide is administered before, after, or simultaneously with the clinical embodiments of the invention. A variety of expression products are encompassed by the invention, including inducers of cell proliferation, inhibitors of cell proliferation, or regulators of programmed cell death.

[0183] surgery Approximately 60% of people with cancer will undergo some type of surgery, including preventative, diagnostic or staging, curative and palliative surgery. Curative surgery is a cancer treatment that may be used in combination with other therapies, such as the treatment of the present invention, chemotherapy, radiation therapy, hormonal therapy, gene therapy, immunotherapy and / or alternative therapies.

[0184] Therapeutic surgery includes resection, where all or part of the cancerous tissue is physically removed, excised, and / or destroyed. Tumor resection refers to the physical removal of at least part of the tumor. In addition to tumor resection, surgical treatment includes laser surgery, cryosurgery, electrosurgery, and microscopically controlled surgery (Mohs surgery). It is further envisioned that the present invention can be used in combination with the removal of incidental amounts of superficial cancer, pre-cancer, or normal tissue.

[0185] Upon removal of all of the cancer cells, tissues or tumors, cavities may be formed in the body. Treatment may be accomplished by perfusion, direct injection or local application of the area with additional anti-cancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6 or 7 days, or every 1, 2, 3, 4 and 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months. These treatments may also be in various dosages.

[0186] Other drugs It is envisioned that other agents may be combined with the present invention to improve the therapeutic efficacy of the treatment. These additional agents include immunomodulatory agents, agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adhesion, or agents that increase the sensitivity of hyperproliferative cells to apoptosis inducers. Immunomodulatory agents include tumor necrosis factor, interferon alpha, beta, and gamma; IL-2 and other cytokines; F42K and other cytokine analogs; or MIP-1, MIP-1 beta, MCP-1, RANTES, and other chemokines. It is further envisioned that upregulation of cell surface receptors or ligands of cell surface receptors, such as Fas / Fas ligand, DR4, or DR5 / TRAIL, will enhance the apoptosis-inducing ability of the present invention by establishing an autocrine or paracrine effect on hyperproliferative cells. Increased intercellular signaling by increasing the number of GAP junctions will increase the anti-hyperproliferative effect on adjacent hyperproliferative cell populations. In other embodiments, cytostatic or differentiation agents may be combined with the present invention to improve the anti-hyperproliferative efficacy of the treatment. It is assumed that the inhibitor of cell adhesion improves the efficacy of the present invention.Examples of cell adhesion inhibitors are focal adhesion kinase (FAK) inhibitors and lovastatin.It is further assumed that other agents that increase the sensitivity of hyperproliferative cells to apoptosis, such as antibody c225, can be used in combination with the present invention to improve the efficacy of treatment.

[0187] In some embodiments, when the present invention is used to treat brain cancer, such as DIPG or GBM, one or more additional active agents can be included as part of the overall treatment.For example, in the case of DIPG, treatment can also include radiation therapy.In the case of GBM, treatment can also include chemotherapy with temozolomide.Representative examples of additional drugs known to be used in the treatment of brain cancer and can be used in conjunction with the present invention include carmustine, BiCNU, procarbazine, Matulane, lapatinib ditoysylate, terameprocol, indoximod, melphalan, carboplatin, etoposide phosphate, mibefradil dihydrochloride, OKN-007, AQ4N and nelfinavir mesylate.

[0188] These and other aspects of the present invention will be further understood upon consideration of the following examples, which are intended to illustrate certain embodiments of the invention and are not intended to limit the scope of the invention as defined by the claims. EXAMPLES

[0189] Example 1: Titratable activation of CAR T cells with different small molecules

[0190] Peripheral blood mononuclear cells (PBMCs) from healthy donors were collected and transduced with retroviruses expressing third generation CARs against either a fluorescein molecule (4M5.3 scFv-CD8α hinge-CD28 transmembrane-CD28 intracellular domain-41BB intracellular domain-CD3z intracellular domain) or a 4-[(6-methylpyrazin-2-yl)oxy]benzoic acid molecule (αMPOB scFv-CD8α hinge-CD28 transmembrane-CD28 intracellular domain-41BB intracellular domain-CD3z intracellular domain) as described elsewhere (Newrzela et al., Methods Mol Biol. (2009)). Positively transduced cells that co-expressed the reporter gene were purified by FACS (BD FACSAria™ II, BD Biosciences, New Jersey (USA)) and co-incubated with CTV-labeled BT145 GBM tumor cells or EL4 lymphoma cells (CellTrace™ Violet, ThermoFisher Scientific) for 4 h at 37°C, 5% CO2, protected from light. Anti-GD2 or anti-mouse H-2K labeled with either fluorescein (FITC) or MPOB were incubated with 100 mM NaCl. b The antibodies coated the tumor cells. The assay was started when CAR T (effector cells, E) and tumor cells (target cells, T) were incubated in duplicates or triplicates at an E:T ratio of 0.5:1 to 20:1. After co-incubation, all cells were stained with eBioscience™ Fixable Viability Dye eFluor™ 780 (Invitrogen™, Massachusetts (USA)) and fixed with a 1:1 dilution of Fixation Buffer (BioLegend®, California (USA)) in sterile PBS / 5% FBS. The percentage of dead target cells was counted according to the following formula: Killing efficiency = [(% dead cells) 試料 -(% dead cells) 対照 ] / [100-(% dead cells) 対照 ] × 100. For controls, cells stained with the appropriate targeting antibody but without CAR T cells were used.

[0191] The results clearly showed that human T cells can be engineered with different CAR constructs to kill their tumor targets with equal efficiency and specificity. These results also demonstrated that CAR T cells can be engineered with specificity for different small molecules (Figures 2A-B).

[0192] Example 2: Identification and validation of targets for specific CAR T cells

[0193] CAR T cells were assayed for their killing properties as described in Example 1. In this case, CARs were co-incubated with cells derived from different tumor types: A673 (Ewing's sarcoma cells), HCC1954 (breast cancer cells), BT145 and BT286 (GBM cells), BT869 and DIPG13 (DIPG cells) and H929 (MM cells). The surface antigens targeted were HLA, CD99, HER2, GD2, CD133, IL13Rα2, CS-1 and CD38. The data demonstrated that human T cells engineered with third generation anti-fluorescein CAR T cells (see Example 1) efficiently killed fluorescein-coated tumor targets, regardless of the cancer type and the targeted target antigen (Figures 2C-E).

[0194] Example 3: Titratable activation of CAR T cells with small molecules

[0195] CAR T cells were prepared for the co-incubation killing assay as described in Example 1. DIPG, GBM or MM cells were co-incubated with anti-fluorescein third generation CAR T and increasing concentrations of fluorescein-labeled antibodies against GD2, CD133, IL13Rα2 and CD38. The results demonstrated the sensitivity and flexibility of the invention as a function of the amount of antigen coated. CAR T cells showed plateau killing efficiency over 2-4 orders of magnitude and titratable response over 4 orders of magnitude. This demonstrated that the invention can work with variable antibody concentrations and modulate its activity to optimize the therapeutic response and modulate efficacy versus toxicity (Figure 2F-Figure 2G).

[0196] Example 4: Titratable activation of different generations of CAR T cells with small molecules

[0197] CAR T cells were prepared for co-incubation killing assay as described in Example 1. GBM cells coated with increasing concentrations of anti-human HLA antibodies conjugated to FITC were co-incubated with two different second generation CAR T cells (4M5.3 scFv-CD8α hinge-CD28 transmembrane-CD28 intracellular domain-CD3z intracellular domain and 4M5.3 scFv-CD8α hinge-CD28 transmembrane-41BB intracellular domain-CD3z intracellular domain). The results demonstrated that it is possible to confer a CAR T cell response, regardless of the CAR generation (Figure 2H).

[0198] CAR T cells were prepared for the co-incubation killing assay as described in Example 1. Anti-fluorescein third generation CAR T cells were co-incubated with GBM or MM tumor-derived cells that did not share the same tumor antigen at a ratio of 20:1. The data demonstrate the ease of retargeting the same CAR T cells to different tumor antigens by simply exchanging the fluorescein-labeled tumor targeting moiety: when only the MM targeting antibody is present (either anti-CD38 or antibody CS-1), only MM cells are killed (Figure 2I). Similarly, when only the GBM targeting moiety is present (either anti-GD2 or anti-IL13Rα2), only brain cells are targeted. Maximal killing of cells is obtained only when at least one antibody specific for each line is present. In agreement with this data, when the tumor-specific antibody is replaced with an antibody against a ubiquitous HLA molecule, all cells are targeted and killed. As a result, the present invention can be used to target highly heterogeneous tumors (Figure 2I).

[0199] Example 5: Identification and validation of targets for FL-specific CAR T cells

[0200] Tumor cell lines (GBM: A172; MM: ARP1, H929, U266, U266MS, JJN3, XG1, KMS12, H929-luc) and patient-derived cultures (GBM: BT145 and BT333; DIPG: BT869) were characterized for a panel of targets of interest and staining intensity was measured via flow cytometry. Approximately 100,000 cells of the target cell line were added in fixed aliquots into wells of a U-bottom 96-well plate and resuspended in a solution of 1 μg of tumor-targeting antibody in 100 μL of FACS buffer (sterile filtered PBS, 5% FBS). The cells were incubated with the antibody for 20 minutes at 4° C. in the dark. After incubation, the cells were washed with FACS buffer and fixed with a 1:1 dilution of Fixation Buffer (BioLegend®, California (USA)) in sterile PBS, 5% FBS. Each antibody / fluorophore combination was analyzed along with its corresponding isotype control. For each tumor marker, the formula log2((MFI) 試料 -(MFI) アイソタイプ Scores were assigned according to the formula (1): 1. Scores were rounded to the nearest tenth and a color code was assigned by GraphPad® software.

[0201] The results demonstrated the high variability of tumor antigens among cell lines and patient-derived cultures, especially brain tumors. Not only were "canonical" tumors non-ubiquitous, but their expression levels varied. The present invention may provide a solution to implement next-generation CAR T cell platforms that can efficiently adapt to antigens with intra- and inter-patient variability.

[0202] Example 6: Titratable activation of CAR T cells to unmasked small molecules alone

[0203] Human α-fluorescein CAR T cells were co-cultured with human CD99+ Ewing sarcoma cell line (A673) coated with anti-CD99 or negative control antibody at an effector-to-target ratio of 20:1 for approximately 4 hours as described in Example 1. A673 tumor cells were coated with commercially available αCD99-FITC, with its corresponding isotype, or with αCD99 antibody bound to caged fluorescein either alone (caged FL) or pretreated after pretreatment with 365 nm UV light (uncaged FL). A673 tumor cells were specifically killed when targeted via unshielded anti-CD99 fluorescein, but not if the cage on the anti-CD99 antibody was left intact. No statistically significant difference was observed between A673 cells targeted with caged anti-CD99 fluorescein and negative control antibody (IgG isotype control FITC). Statistical significance was calculated using Kruskal-Wallis one-way analysis of variance plus preselected pairwise comparisons (*p<0.05). The experiment was performed with four replicates.

[0204] The results, along with the specific in vitro cytotoxicity of human third generation anti-fluorescein CAR-T cells to the unshielded fluorescein derivative, are shown in Figure 4B. The results show that CAR-T cells specifically killed unshielded fluorescein but not targets bound by the protected fluorescein derivative.

[0205] All patent and non-patent publications are indicative of the level of skill of those skilled in the art. All such publications, including any specific portions thereof referred to, are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[0206] Although the invention herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present invention. It is therefore to be understood that numerous modifications can be made to the illustrative embodiments and that other arrangements may be devised without departing from the spirit and scope of the present invention as defined by the appended claims.

Claims

1. A bifunctional compound comprising a first synthetic antigen covalently linked to a first targeting moiety that binds a first tumor-associated antigen, or a pharma- ceutically acceptable salt or stereoisomer thereof.

2. The bifunctional compound of claim 1 , wherein the targeting moiety specifically binds a brain tumor associated antigen.

3. 3. The bifunctional compound of claim 2, wherein the targeting moiety specifically binds a brain tumor associated antigen selected from the group consisting of GD2, IL13Rα2, HER2, PDGFRα, EGFRvIII, CSPG4, EphA2 and CD133.

4. The bifunctional compound of claim 1 , wherein the targeting moiety specifically binds a hematological tumor associated antigen.

5. 5. The bifunctional compound of claim 4, wherein the targeting moiety specifically binds a hematological tumor associated antigen selected from the group consisting of CD38, CS1, BCMA, CD20, CD19, CD22, CD30, CD138, CD40, CD56, CD70 and CD74.

6. The bifunctional compound of claim 1 , wherein the targeting moiety specifically binds to HER2.

7. The bifunctional compound of claim 1 , wherein the synthetic antigen is a fluorescent molecule.

8. The bifunctional compound of claim 7 , wherein the fluorescent molecule is fluorescein or anthracene.

9. 2. The bifunctional compound of claim 1, wherein the synthetic antigen is 4-[(6-methylpyrazin-2-yl)oxy]benzoate (MPOB), anthraquinone-2-carboxylate (AQ) or tetraxetane (DOTA).

10. The bifunctional compound of claim 1 , wherein the synthetic antigen contains a removable protecting group.

11. The bifunctional compound of claim 10, wherein the removable protecting group is a boronic ester group.

12. The bifunctional compound of claim 10, wherein the removable protecting group is a photocleavable group.

13. The bifunctional compound of claim 1 , wherein the synthetic antigen is a peptide, glycoside or nucleotide antigen.

14. 11. A pharmaceutical composition comprising a therapeutically effective amount of the bifunctional compound of claim 1 or a pharma- ceutically acceptable salt or stereoisomer thereof and a pharma- ceutically acceptable carrier, wherein the bifunctional compound is a first bifunctional compound.

15. 15. The pharmaceutical composition of claim 14, further comprising a second bifunctional compound comprising the first synthetic antigen covalently linked to a second targeting moiety that binds a first tumor-associated antigen, or a pharma- ceutically acceptable salt or stereoisomer thereof.

16. 15. The pharmaceutical composition of claim 14, comprising a plurality of therapeutically effective amounts of a subpopulation of bifunctional compounds, the subpopulation comprising a first subpopulation of said first bifunctional compounds and further comprising at least a second subpopulation of a second bifunctional compound comprising said first synthetic antigen covalently linked to a second targeting moiety that specifically binds a second tumor-associated antigen, or a pharma- ceutically acceptable salt or stereoisomer thereof, wherein said first and second targeting moieties specifically bind different tumor-associated antigens, and each subpopulation of bifunctional compounds in said composition binds a different tumor-associated antigen than the bifunctional compounds contained in other subpopulations in said composition.

17. 17. The pharmaceutical composition of claim 16, wherein the first and second targeting moieties specifically bind a brain tumor associated antigen selected from the group consisting of GD2, IL13Rα2, HER2, PDGFRα, EGFRvIII, CSPG4, EphA2 and CD133.

18. 18. The pharmaceutical composition of claim 17, wherein each of the first and second targeting moieties specifically binds a brain tumor associated antigen selected from the group consisting of IL13Rα2, EGFRvIII and HER2, or each of the first and second targeting moieties specifically binds a brain tumor associated antigen selected from the group consisting of GD2, PDGFRα and CD133, or each of the first and second targeting moieties specifically binds a brain tumor associated antigen selected from the group consisting of EphA2 and CSPG4.

19. 15. The pharmaceutical composition of claim 14, wherein the first and second targeting moieties specifically bind a hematological tumor associated antigen selected from the group consisting of CD38, CS1, BCMA, CD20, CD19, CD22, CD30, CD138, CD40, CD56, CD70 and CD74.

20. 20. The pharmaceutical composition of claim 19, wherein each of the first and second targeting moieties specifically binds a hematological tumor associated antigen selected from the group consisting of CD38, CS1 and BCMA, or each of the first and second targeting moieties specifically binds a hematological tumor associated antigen selected from the group consisting of CD19, CD20 and CD22, or each of the first and second targeting moieties specifically binds a hematological tumor associated antigen selected from the group consisting of CD30, CD40, CD56, CD70, CD74 and CD138.

21. 1. A method of treating cancer, comprising: a) a subpopulation of a plurality of bifunctional molecules, said plurality comprising: a1) a first subpopulation of bifunctional compounds comprising a therapeutically effective amount of a synthetic antigen covalently linked to a first targeting moiety that specifically binds a first tumor associated antigen; and a2) a second subpopulation of bifunctional compounds comprising a therapeutically effective amount of a synthetic antigen covalently linked to a second targeting moiety that specifically binds a second tumor associated antigen, or a pharma- ceutically acceptable salt or stereoisomer thereof, wherein the first and second targeting moieties specifically bind different tumor associated antigens; b) a therapeutically effective number of CAR-T cells, the CAR-T cells comprising an extracellular ligand that specifically binds the synthetic antigen; and to a subject in need of treating cancer.

22. 22. The method of claim 21, wherein said therapeutically effective amounts of said plurality of subpopulations of bifunctional compounds are administered simultaneously.

23. 22. The method of claim 21, wherein the therapeutically effective amounts of the plurality of subpopulations of bifunctional compounds are administered sequentially.

24. 23. The method of claim 22, wherein the plurality of subpopulations of bifunctional compounds are administered in a single composition.

25. 23. The method of claim 22, wherein the plurality of subpopulations of bifunctional compounds are administered in different compositions.

26. 22. The method of claim 21, further comprising at least one subsequent administration of a therapeutically effective amount of said first and / or second subpopulations of said bifunctional compound.

27. 27. The method of claim 26, wherein in at least one subsequent administration of the first and / or second subpopulations of the bifunctional compound, the first targeting moiety binds a different epitope on the same tumor-associated antigen compared to the first targeting moiety in a previous administration, and the second targeting moiety binds a different epitope on the same tumor-associated antigen compared to the second targeting moiety in a previous administration.

28. 22. The method of claim 21, further comprising co-administering a third subpopulation of bifunctional compounds comprising a synthetic antigen covalently linked to a third targeting moiety that binds a different tumor associated antigen compared to the first and second targeting moieties.

29. 22. The method of claim 21 , wherein the synthetic antigen contains a removable protecting group and the extracellular ligand of the CAR T cell binds the synthetic antigen after removal of the removable protecting group.

30. 22. The method of claim 21, wherein the subject has brain cancer and the first and second targeting moieties specifically bind different brain cancer associated antigens.

31. 31. The method of claim 30, wherein each of the first and second targeting moieties specifically binds a brain tumor associated antigen selected from the group consisting of GD2, IL13Rα2, HER2, PDGFRα, EGFRvIII, CSPG4, EphA2 and CD133.

32. 31. The method of claim 30, wherein each of the first and second targeting moieties specifically binds a brain tumor associated antigen selected from the group consisting of IL13Rα2, EGFRvIII and HER2, or each of the first and second targeting moieties specifically binds a brain tumor associated antigen selected from the group consisting of GD2, PDGFRα and CD133, or each of the first and second targeting moieties specifically binds a brain tumor associated antigen selected from the group consisting of EphA2 and CSPG4.

33. 31. The method of claim 30, wherein the brain cancer is a glioma.

34. 34. The method of claim 33, wherein the glioma is diffuse intrinsic pontine glioma (DIPG).

35. 31. The method of claim 30, wherein the brain cancer is glioblastoma (GBM).

36. 22. The method of claim 21 , wherein the subject has a hematological cancer and the first and second targeting moieties specifically bind different hematological tumor associated antigens.

37. 37. The method of claim 36, wherein each of the first and second targeting moieties specifically binds a hematological tumor associated antigen selected from the group consisting of CD38, CS1, BCMA, CD20, CD19, CD22, CD30, CD138, CD40, CD56, CD70 and CD74.

38. 38. The method of claim 37, wherein each of the first and second targeting moieties specifically binds a hematological tumor associated antigen selected from the group consisting of CD38, CS1 and BCMA, or each of the first and second targeting moieties specifically binds a hematological tumor associated antigen selected from the group consisting of CD19, CD20 and CD22, or each of the first and second targeting moieties specifically binds a hematological tumor associated antigen selected from the group consisting of CD30, CD40, CD56, CD70, CD74 and CD138.

39. 37. The method of claim 36, wherein the hematological cancer is multiple myeloma (MM), acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, or large granular lymphocytic (LGL) leukemia.

40. 22. The method of claim 21, wherein the cancer is a HER2+ cancer.

41. 41. The method of claim 40, wherein the cancer is breast cancer, lung cancer, colorectal cancer, brain cancer, ovarian cancer or pancreatic cancer.

42. 22. The method of claim 21, wherein prior to a) and b), the subject has undergone a primary cancer treatment.

43. 43. The method of claim 42, wherein the primary cancer treatment became ineffective as a result of antigen escape.

44. 22. The method of claim 21, wherein the subject is a human.

45. a therapeutically effective amount of a plurality of subpopulations of bifunctional compounds or pharma- ceutically acceptable salts or stereoisomers thereof, wherein each bifunctional compound in a first subpopulation comprises a first synthetic antigen covalently linked to a first targeting moiety that specifically binds a first tumor associated antigen, and each bifunctional compound in a second subpopulation comprises said first synthetic antigen covalently linked to a second targeting moiety that specifically binds a second tumor associated antigen, wherein said first and second targeting moieties specifically bind different tumor associated antigens, and wherein each subpopulation of bifunctional compounds administered to a patient contains said first synthetic antigen but specifically binds a different tumor associated antigen, said plurality of subpopulations of bifunctional compounds being disposed in the same or separate containers, b) instructions for co-administering to a cancer patient a therapeutically effective amount of a subpopulation of bifunctional compounds, a therapeutically effective number of CAR-T cells, the CAR-T cells comprising an extracellular ligand that specifically binds the synthetic antigen; and A kit comprising:

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