Ganglioside gm2 as a target for cancer immunotherapy

Abstract

The present disclosure provides compositions and methods for identifying and treating cancer. In particular, the disclosure provides methods of identifying and treating cancer having decreased sensitivity or resistance to ganglioside GD2 directed therapies.

Description

GANGLIOSIDE GM2 AS A TARGET FOR CANCER IMMUNOTHERAPYSTATEMENT OF RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 546,455, filed October 30, 2023, the entire contents of which are incorporated herein by reference for all purposes.SEQUENCE LISTING

[0002] The text of the computer readable sequence listing filed herewith, titled “STDU2- 42494-60 l_SQL.xml”, created October 29, 2024, having a file size of 101,785 bytes, is hereby incorporated by reference in its entirety.FIELD

[0003] The present disclosure provides compositions and methods related to identifying cancers and treating patients with the identified cancers. In particular, the disclosure identifies ganglioside GM2 as a viable target for immunotherapy in a patient having cancer. The disclosure also provides anti-ganglioside GM2 immunotherapies for treating a patient having cancer.BACKGROUND

[0004] The ganglioside GD2 is a validated target in several cancers including neuroblastoma. There are currently multiple clinically validated GD2 antibodies that have been used for treating cancer. However, in response to anti-GD2 immunotherapy, cancer patients have exhibited decreased sensitivity / reduced efficacy of GD2 directed therapies.SUMMARY

[0005] The present disclosure provides methods for identifying cancers and treating patients with the identified cancers. For example, in some embodiments, the disclosure provides a method of identifying a cancer that can be treated with (e.g., that will respond to treatment with) an anti-ganglioside GM2 directed treatment. In some embodiments, the method comprises providing a patient having cancer and determining whether the cancer can be treated with (e.g., will respond to treatment with) an anti-ganglioside GM2 directed treatment. In some embodiments, the patient having cancer displays resistance to anti- ganglioside GD2 therapy. In other embodiments, the patient having cancer displays reduced sensitivity to anti-ganglioside GD2 therapy (e.g., displays reduced sensitivity to anti-GD2 directed therapy due to low or heterogeneous GD2 expression). The disclosure is not limitedby the type of patient having cancer. Indeed, the disclosure is useful for a variety of types of patients having cancer including but not limited to patients with a neuroblastoma, a sarcoma, a retinoblastoma, a medulloblastoma, a Ewing sarcoma, a carcinoma, a glioma or a glioblastoma. In other embodiments, the patient is a patient with a sarcoma, a rhabdoid cancer, a neuroblastoma, retinoblastoma, medulloblastoma, Ewing sarcoma, lymphoma, melanoma, uterine carcinosarcoma (UCS), brain lower grade glioma (LGG), thymoma (THYM), testicular germ cell tumors (TGCT), glioblastoma multiforme (GBM) and skin cutaneous melanoma (SKCM), liver hepatocellular carcinoma (LIHC), uveal melanoma (UVM), kidney chromophobe (KICH), thyroid cancer (THCA), kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), stomach adenocarcinoma (STAD), cholangiocarcinoma (CHOL), adenoid cystic carcinoma (ACC), prostate adenocarcinoma (PR AD), pheochromocytoma and paraganglioma (PCPG), DLBC, lung adenocarcinoma (LUAD), head-neck squamous cell carcinoma (HNSC), pancreatic adenocarcinoma (PAAD), breast cancer (BRCA), mesothelioma (MESO), colon and rectal adenocarcinoma (COAD), rectum adenocarcinoma (READ), esophageal carcinoma (ESCA), ovarian cancer (OV), lung squamous cell carcinoma (LUSC), bladder urothelial carcinoma (BLCA), sarcoma (SARC), small cell lung cancer, or uterine corpus endometrial carcinoma (UCEC). In some embodiments, the method further comprises measuring the expression level of ganglioside synthase enzyme ST8SIA1 in the patient. In other implementations, the method comprises measuring the expression and / or level of ganglioside GD2 in the patient. In some embodiments, the method comprises measuring the expression and / or level of both ST8SIAland ganglioside GD2 in the patient. In some embodiments, the method comprises measuring the expression and / or level of ganglioside GM2 in the patient. In some embodiments, the expression and / or level of ganglioside synthase enzyme ST8SIA1 and / or ganglioside GD2 is measured in the patient’s cancer and / or tumor cells. In some embodiments, the expression and / or level of ganglioside GM2 is measured in the patient’s cancer and / or tumor cells. In some embodiments, the method comprises comparing the expression and / or level of ganglioside synthase enzyme ST8SIA1 and / or ganglioside GD2 in the patient (e.g., in the patient’s cancer and / or tumor cells) to the expression of ganglioside synthase enzyme ST8SIA1 and / or ganglioside GD2 in a control sample. In one embodiment, a patient displaying low expression of ganglioside synthase enzyme ST8SIA1 compared to the control sample identifies the cancer as one that will respond to treatment with antiganglioside GM2 immunotherapy. In other embodiments, a patient displaying low expression of ganglioside GD2 compared to the control sample identifies the cancer as one that willrespond to treatment with anti-ganglioside GM2 immunotherapy. In some embodiments, a patient displaying heterogeneous expression of ganglioside synthase enzyme ST8SIA1 compared to the control sample identifies the cancer as one that will respond to treatment with anti-ganglioside GM2 immunotherapy. In other embodiments, a patient displaying heterogeneous expression of ganglioside GD2 compared to the control sample identifies the cancer as one that will respond to treatment with anti-ganglioside GM2 immunotherapy. In some embodiments, the method identifies a patient as one to be treated with both GD2 and GM2 directed therapies (e.g., due to GD2 heterogeneous tumors or to overcome GD2-low antigen escape). In some embodiments, the method further comprises, once the cancer has been identified as one that will respond to treatment with anti-ganglioside GM2 immunotherapy, administering to the patient having cancer an anti-ganglioside GM2 immunotherapy. In some implementations, administering an anti-ganglioside GM2 immunotherapy comprises administering a therapeutically effective amount of an anti-GM2 specific antibody. The disclosure is not limited by the type of anti-GM2 specific antibody administered. Indeed a variety of anti-GM2 antibodies find use in the disclosure including but not limited to KM966 and DMF(10.62.3). In some implementations, administering an anti- ganglioside GM2 immunotherapy comprises administering a therapeutically effective amount of anti-GM2 specific chimeric antigen receptor (CAR) T cells. The disclosure is not limited by the type of anti-GM2 specific chimeric antigen receptor (CAR) T cells administered. Indeed a variety of anti-GM2 specific chimeric antigen receptor (CAR) T cells find use in the disclosure including but not limited to those disclosed herein. In some embodiments, the method further comprises measuring the expression level of ganglioside synthase enzyme ST8SIA1 and / or ganglioside GD2 and / or GM2 in the patient having cancer (e.g., in the patient’s cancer and / or tumor cells) at a time point after administering an anti-ganglioside GM2 immunotherapy to the patient. In some embodiments, the expression level of ganglioside synthase enzyme ST8SIA1 and / or ganglioside GD2 and / or GM2 comprises measuring gene expression levels. The disclosure is not limited by the method of measuring gene expression levels. Indeed a variety of methods for measuring gene expression may be utilized including one or more methods disclosed herein or known to those of skill in the art. In some embodiments, the expression level of ganglioside synthase enzyme ST8SIA1 and / or ganglioside GD2 and / or GM2 comprises measuring protein expression levels. The disclosure is not limited by the method of measuring protein expression levels. Indeed a variety of methods for measuring protein expression may be utilized including one or more methods disclosed herein or known to those of skill in the art.

[0006] The disclosure also provides a method for the treatment of cancer displaying decreased or lost sensitivity to ganglioside GD2 directed therapeutic treatment and / or decreased expression or activity of ganglioside synthase enzyme ST8SIA1 and / or decreased expression or activity of ganglioside GD2 comprising administering to a patient with the cancer displaying decreased or lost sensitivity to ganglioside GD2 directed therapeutic treatment and / or decreased expression or activity of ganglioside synthase enzyme ST8SIA1 and / or decreased expression or activity of ganglioside GD2 an immunotherapy targeting ganglioside GM2. In some embodiments, the method comprises administering an effective amount of any one or more of the anti-GM2 immunotherapeutics / immunotherapies described herein (e.g., anti-GM2 antibodies, anti-GM2 bispecific immune cell (e.g., T cell) engagers (e.g., anti-GM2 x anti-CD3 bispecific or anti-GM2 x anti-CD16 bispecific T cell engagers) and / or anti-GM2 CAR T cells). In some embodiments, the method comprises administering to the patient a therapeutically effective amount of a chemotherapeutic agent and / or radiation. The disclosure is not limited by type of cancer displaying decreased or lost sensitivity to ganglioside GD2 directed therapeutic treatment and / or decreased expression or activity of ganglioside synthase enzyme ST8S1A1 and / or decreased expression or activity of ganglioside GD2. Indeed, the disclosure is useful for any cancer displaying a decreased or lost sensitivity to ganglioside GD2 directed therapeutic treatment and / or a decreased expression or activity of ganglioside synthase enzyme ST8SIA1 and / or a decreased expression or activity of ganglioside GD2. Non-limiting types of cancer are described herein. In some embodiments, the method comprises administering to the patient a therapeutically effective amount of any one or more of the anti-GM2 antibodies described herein. In other embodiments, the method comprises administering to the patient a therapeutically effective amount of T-cells expressing a chimeric antigen receptor (CAR) specific for ganglioside GM2 under conditions sufficient to form an immune complex of an antigen binding domain on the chimeric antigen receptor and ganglioside GM2 in the patient.

[0007] The disclosure also provides a method of detecting minimal residual disease in a patient with cancer that has received treatment with a ganglioside GD2 directed immunotherapy comprising detecting the expression and / or level of ganglioside GM2 and / or ganglioside synthase enzyme ST8SIA1 in the patient (e.g., in a patient’s cancer and / or tumor cells using any one or more of the methods described herein).

[0008] These and other features, aspects, and advantages of the present disclosure will become better understood with regard to the following description, and accompanying drawings.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] FIGS. 1A and IB depict a graphical representation of the ganglioside synthesis pathways detailing the enzymes, substrates and products (See Sandhoff, R.; Sandhoff, K. Emerging Concepts of Ganglioside Metabolism. FEBS Lett. 2018, 592, 3835-3864). In both cases (A and B) the vignette shows a simplified version of the pathway highlighting the enzymes directly involved in GM2 and GD2 synthesis.

[0010] FIG. 2 shows CHLA255 cells were implanted in the kidney capsule of NSG mice. 4 days after tumor injection mice were treated with a combination of anti-GD2 (Dinutuximab) and anti-CD47 (B6-H12) antibody or control (NoRx). FIG. 2A shows tumor growth measured by BLI. FIG. 2B shows GD2 expression at endpoint measured by flow cytometry. FIG. 2C shows results after total RNA was extracted from xenograft tumors and gene expression analyzed by RT-qPCR.

[0011] FIG. 3 shows that when GD2 expression is reduced or lost, there is a compensatory increase in surface expression of GM2. (A) Schematic representation of the changes in ganglioside levels and ST8SIA1 enzyme expression before and after treatment. (B) Flow cytometry analysis of xenografts harvested at endpoint from mice that received anti- GD2 / anti-CD47 antibody treatment or GD2-CAR T cells. (C) GD2 and GM2 levels measured by flow cytometry in the isogenic cell lines CHLA255 and CHLA255 ST8SIA1 KO. (D) Tumor growth measured by BLI.

[0012] FIG. 4 shows surface expression levels of gangliosides GD2 and GM2 in three different neuroblastoma (NBL) cell lines measured by flow cytometry. FIG. 4A shows gangliosides GD2 and GM2 expression in CHLA255 (left), Kelly (center) and NB1 (right). FIG. 4B shows in vitro Antibody Dependent Cell Phagocytosis (ADCP) assay performed after the indicated cell lines were incubated with the corresponding antibodies.

[0013] FIG. 5 shows improved in vivo anti-tumor activity of co-targeting GD2 and GM2 by antibodies in a model of antigen escape. NSG mice baring CHLA255 xenografts were treated with anti-GD2 (Dinutuximab), anti-GM2 (DMF10.167.4) or both antibodies. (FIG. 5A) Tumor growth measured by BLI. (FIG. 5B) Kaplan-Meier curves showing survival of the different groups. (FIG. 5C) GD2 and GM2 levels at endpoint in xenografts measured by flow cytometry.

[0014] FIG. 6 shows results from NSG mice baring CHLA255 xenografts treated with anti- GD2 (Dinutuximab), anti-GM2 (KM966) or both antibodies. (FIG.6A) Tumor growth measured by BLI. (FIG. 6B) Kaplan-Meier curves showing survival of the different groups. (FIG. 6C) GD2 and GM2 levels at endpoint in xenografts measured by flow cytometry.

[0015] FIG. 7 shows results from NSG mice bearing Kelly cell xenografts treated with anti- GD2 (Dinutuximab) or anti-GM2 (KM966) antibodies. Kelly cells were injected into NSG mice to produce xenograft tumors. After confirming tumor engraftment, mice were treated with anti-GD2 (Dinutuximab) or anti-GM2 (KM966) antibodies. FIG. 7A shows tumor growth measured by BLI. FIG. 7B presents Kaplan-Meier curves showing survival of the different groups.

[0016] FIG. 8 shows GD2 and GM2 levels measured by flow cytometry in a panel of GD2 low neuroblastoma cell lines.

[0017] FIG. 9 shows in vivo activity of targeting GM2 in GM2-high, GD2-low neuroblastoma tumors. NSG mice were injected through the tail vein with SH-SY5Y cells and treated with anti-GD2 (Dinutuximab) or anti-GM2 (DMF10.167.4). FIG. 9A shows tumor growth measured by BLI. FIG. 9B provides Kaplan-Meier curves showing survival of the different groups. FIG. 9C shows GD2 and GM2 levels at endpoint in xenografts measured by flow cytometry.

[0018] FIG. 10 shows results from NSG mice inoculated through the tail vein with NB1 cells. After confirming tumor engraftment by BLI, mice were treated with anti-GD2 (Dinutuximab) or anti-GM2 (KM966) antibodies. FIG. 10A shows tumor growth measured by BLI. FIG.10B provides Kaplan-Meier curves showing survival of the different groups. FIG. 10C shows GD2 and GM2 levels at endpoint in xenografts measured by flow cytometry.

[0019] FIG. 11 shows that tandem GM2-GD2 CAR T cells bearing CARs with either the 4- IBB-zeta or the CD28-zeta fragment endodomains confer advantage over monospecific CARs against both GD2 and GM2-expressing tumor cells. (FIG. 11 A) Cytokine production by GD2-BBz, GM2-28z, or tandem GM2-GD2-targeting 4-lBB-zeta or CD28-zeta fragment CAR T cells when co-cultured with Nalm6-GD2 leukemia cells (GD2-high, GM2- negative / low), Nalm6-GM2 leukemia cells (GM2-high, GD2-negative / low), CHLA-255 neuroblastoma cells (GD2-high, GM2-low / medium), or SH-SY5Y (GM2-high, GD2- negative / low) neuroblastoma cells. (FIG. 1 IB) Schematic images for each CAR.

[0020] FIG. 12 provides a graph showing in vivo functionality of tandem GD2-GM2 CAR T cells bearing CARs with the 4-lBB-zeta fragment or CD28-zeta fragment endodomains and co-infusion of each monospecific GD2 and GM2-targeting CAR T cells against a mouse neuroblastoma model (Sy5y having GM2-high, GD2-negative / low expression) with two different doses: 3e6 (FIG. 12A) and le6 (FIG. 12B).

[0021] FIG. 13 provides graphs showing in vivo functionality of tandem GD2-GM2 CAR T cells bearing CARs against (A) CHLA255 or (B) Kelly, and ganglioside expression on tumorcells at endpoint (C). CHLA255 has high GD2 expression and intermediate / low GM2 expression while Kelly has intermediate GD2 and GM2 expression. GM2-GD2-28z tandem CAR showed the best efficacy / tumor inhibition compared to monospecific GM2-28z CAR T cells, GD2-28z CAR T cells or tandem GD2-GM2-28z CAR T cells in both CHLA255 (FIG. 13 A) or Kelly cells (FIG. 13B).

[0022] FIG. 14 is a set of graphs showing in vivo functionality of tandem GD2-GM2 CAR T cells bearing CARs with either the 4-lBB-zeta (FIG. 14A) or CD28-zeta (FIG. 14B) fragment endodomains in the Nalm-6-GD2 leukemia mice model.

[0023] FIG. 15 provides graphs showing the heterogeneity of expression of ST8SIA1 in neuroblastoma and Ewing sarcoma cell lines (right side panel) and high expression of B4GALNT1 in both neuroblastoma and Ewing sarcoma cell lines (left side panel).

[0024] FIG. 16 shows GM2 is highly expressed in a variety of Ewing sarcoma cell lines whereas GD2 is expressed heterogeneously. Graphs show GD2 (A) or GM2 (B) expressions on Ewing sarcoma cell lines.

[0025] FIG. 17 shows GM2-targeting KM966 CAR T cells induced the secretion of significantly higher levels of cytokines against GM2-high Ewing sarcoma cell lines than mock or GD2-targeting CAR T cells. Cytokine production by GD2 or GM2-targeting 4-1BB- zeta or CD28-zeta fragment CAR T cells when co-cultured with 5838 Ewing sarcoma cells (left graph), RD-ES Ewing sarcoma cells (middle graph), or SKNMC Ewing sarcoma cells (right graph).

[0026] FIG. 18 shows immunohistochemistry (IHC) for GD2 and GM2 performed on a neuroblastoma (NBL) sample obtained from a relapse patient previously treated with anti- GD2 immunotherapy.

[0027] FIG. 19 shows that GM2 is highly expressed on the surface of cancer cells, especially when GD2 is expressed at low levels. Graphs show GD2 and GM2 levels measured by flow cytometry on a panel of different neuroblastoma cell lines. FIG. 19A shows GM2 expression is high even when GD2 is low. FIG. 19B shows a simplified ganglioside synthesis pathway. FIG. 19C shows that neuroblastoma cell lines with high GD2 expression still show substantial GM2 expression levels.

[0028] FIG. 20 shows that GM2 is highly expressed in GD2-heterogeneous osteosarcoma patient-derived xenograft (PDX) cell lines (PSS cell lines). In FIGS. 20A and 20B, GD2 expression is shown in the left side panels, and GM2 expression in the right side panels.Despite heterogeneous GD2 surface expression, surface GM2 expression is high on virtually all Osteosarcoma lines.

[0029] These and other features, aspects, and advantages of the present disclosure will become better understood with regard to the following description, and accompanying drawings.DETAILED DESCRIPTIONDEFINITIONS

[0030] For purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth below shall control.

[0031] As used herein the terms “disease” and “pathologic condition” are used interchangeably, unless indicated otherwise herein, to describe a deviation from the condition regarded as normal or average for members of a species or group (e.g., humans), and which is detrimental to an affected individual under conditions that are not inimical to the majority of individuals of that species or group. Such a deviation can manifest as a state, signs, and / or symptoms (e.g., diarrhea, nausea, fever, pain, blisters, boils, rash, immune suppression, inflammation, etc.) that are associated with any impairment of the normal state of a subject or of any of its organs or tissues that interrupts or modifies the performance of normal functions. A disease or pathological condition may be caused by or result from contact with a microorganism (e.g., a pathogen or other infective agent (e.g., a virus or bacteria)), may be responsive to environmental factors (e.g., malnutrition, industrial hazards, and / or climate), may be responsive to an inherent or latent defect in the organism (e.g., genetic anomalies) or to combinations of these and other factors.

[0032] The terms “host,” “subject,” or “patient” are used interchangeably herein to refer to an individual to be treated by (e.g., administered) the compositions and methods of the present invention. Subjects include, but are not limited to, mammals (e.g., murines, simians, equines, bovines, porcines, canines, felines, and the like), and most preferably includes humans. In the context of the invention, the term “subject” generally refers to an individual who will be administered or who has been administered one or more compositions of the present invention (e.g., genetically modified immune cells described herein).

[0033] The term “solution” refers to an aqueous or non-aqueous mixture.

[0034] A “disorder” is any condition or disease that would benefit from treatment with a composition or method of the invention. This includes chronic and acute disorders includingthose pathological conditions which predispose the mammal to the disorder in question. Non-limiting examples of disorders to be treated herein include conditions such as cancer.

[0035] The terms “cell proliferative disorder,” and “proliferative disorder” refer to disorders that are associated with some degree of abnormal cell proliferation. For example, a “hyperproliferative disorder or disease” is a disease or disorder caused by excessive growth of cells. In one embodiment, the cell proliferative disorder is cancer.

[0036] As used herein, the terms “cancer” and “tumor” refer to a cell that exhibits a loss of growth control or tissue of uncontrolled growth or proliferation of cells. Cancer and tumor cells generally are characterized by a loss of contact inhibition, may be invasive, and may display the ability to metastasize. The present invention is not limited by the type of cancer or the type of treatment (e.g., prophylactically and / or therapeutically treated). Indeed, a variety of cancers may be treated with compositions and methods described herein including, but not limited to, brain cancer or other cancers of the central nervous system (e.g., diffuse midline glioma or diffuse intrinsic pontine glioma (DIPG, a highly aggressive glial tumor found at the base of the brain, see, e.g., Louis et al., Acta Neuropathol (2016) 131 :803-820), melanomas, lymphomas, epithelial cancer, breast cancer, ovarian cancer, endometrial cancer, colorectal cancer, lung cancer, renal cancer, melanoma, kidney cancer, prostate cancer, sarcomas, carcinomas, and / or a combination thereof.

[0037] “Metastasis” as used herein refers to the process by which a cancer spreads or transfers from the site of origin to other regions of the body with the development of a similar cancerous lesion at the new location. A “metastatic” or “metastasizing” cell is one that loses adhesive contacts with neighboring cells and migrates via the bloodstream or lymph from the primary site of disease to invade neighboring body structures.

[0038] The term “anticancer agent” as used herein, refer to any therapeutic agents (e.g., chemotherapeutic compounds and / or molecular therapeutic compounds), antisense therapies, radiation therapies, or surgical interventions, used in the treatment of hyperproliferative diseases such as cancer (e.g., in mammals, e.g., in humans).

[0039] An “effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic or prophylactic result.

[0040] The term “therapeutically effective amount,” as used herein, refers to that amount of the therapeutic agent sufficient to result in amelioration of one or more symptoms of a disorder, or prevent advancement of a disorder, or cause regression of the disorder. For example, with respect to the treatment of cancer, in one embodiment, a therapeutically effective amount will refer to the amount of a therapeutic agent that decreases the rate oftumor growth (e.g., reduces and / or clears tumor burden in the patient (e.g., reduces the number of H3K27M positive cancer cells in a patient)), decreases tumor mass, decreases the number of metastases, decreases tumor progression, or increases survival time by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%.

[0041] The terms “sensitize” and “sensitizing,” as used herein, refer to making, through the administration of a first agent, an animal or a cell within an animal more susceptible, or more responsive, to the biological effects (e.g., promotion or retardation of an aspect of cellular function including, but not limited to, cell division, cell growth, proliferation, invasion, angiogenesis, necrosis, or apoptosis) of a second agent. The sensitizing effect of a first agent on a target cell can be measured as the difference in the intended biological effect (e.g., promotion or retardation of an aspect of cellular function including, but not limited to, cell growth, proliferation, invasion, angiogenesis, or apoptosis) observed upon the administration of a second agent with and without administration of the first agent. The response of the sensitized cell can be increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least 300%, at least about 350%, at least about 400%, at least about 450%, or at least about 500% over the response in the absence of the first agent.

[0042] As used herein, the terms “purified” or “to purify” refer to the removal of contaminants or undesired compounds from a sample or composition. As used herein, the term “substantially purified” refers to the removal of from about 70 to 90%, up to 100%, of the contaminants or undesired compounds from a sample or composition.

[0043] As used herein, the terms “administration” and “administering” refer to the act of giving a composition of the present invention to a subject. Exemplary routes of administration to the human body include, but are not limited to, through the eyes (ophthalmic), mouth (oral), skin (transdermal), nose (nasal), lungs (inhalant), oral mucosa (buccal), ear, rectal, by injection (e.g., intravenously, subcutaneously, intraperitoneally, intratumorally, etc.), topically, and the like.

[0044] As used herein, the terms “co-administration” and “co-administering” refer to the administration of at least two agent(s) (e.g., genetically modified immune cells and one or more other agents - e.g., anti-cancer agents) or therapies to a subject. In some embodiments, the co-administration of two or more agents or therapies is concurrent. In otherembodiments, a first agent / therapy is administered prior to a second agent / therapy. In some embodiments, co-administration can be via the same or different route of administration. Those of skill in the art understand that the formulations and / or routes of administration of the various agents or therapies used may vary. The appropriate dosage for co-administration can be readily determined by one skilled in the art. In some embodiments, when agents or therapies are co-administered, the respective agents or therapies are administered at lower dosages than appropriate for their administration alone. Thus, co-administration is especially desirable in embodiments where the co-administration of the agents or therapies lowers the requisite dosage of a potentially harmful (e.g., toxic) agent(s), and / or when co-administration of two or more agents results in sensitization of a subject to beneficial effects of one of the agents via co-administration of the other agent.

[0045] The terms “pharmaceutically acceptable” or “pharmacologically acceptable,” as used herein, refer to compositions that do not substantially produce adverse reactions (e.g., toxic, allergic or other immunologic reactions) when administered to a subject.

[0046] As used herein, the term “pharmaceutically acceptable carrier” refers to any of the standard pharmaceutical carriers including, but not limited to, phosphate buffered saline solution, water, and various types of wetting agents (e.g., sodium lauryl sulfate), any and all solvents, dispersion media, coatings, sodium lauryl sulfate, isotonic and absorption delaying agents, disintegrants (e.g., potato starch or sodium starch glycolate), polyethylene glycol, and the like. The compositions also can include stabilizers and preservatives. Examples of carriers, stabilizers and adjuvants have been described and are known in the art (see, e.g., Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, Pa. (1975), incorporated herein by reference).

[0047] As used herein, the term “kit” refers to any delivery system for delivering materials. In the context of immunotherapeutic agents, such delivery systems include systems that allow for the storage, transport, or delivery of immunogenic agents and / or supporting materials (e.g., written instructions for using the materials, etc.) from one location to another. For example, kits include one or more enclosures (e.g., boxes) containing the relevant immunotherapeutic agents (e.g., genetically modified immune cells and / or supporting materials). As used herein, the term “fragmented kit” refers to delivery systems comprising two or more separate containers that each contain a subportion of the total kit components. The containers may be delivered to the intended recipient together or separately. For example, a first container may contain a composition comprising an immunotherapeutic composition for a particular use, while a second container contains asecond agent (e.g., a chemotherapeutic agent). Indeed, any delivery system comprising two or more separate containers that each contains a subportion of the total kit components are included in the term “fragmented kit.” In contrast, a “combined kit” refers to a delivery system containing all of the components of an immunogenic agent needed for a particular use in a single container (e.g., in a single box housing each of the desired components). The term “kit” includes both fragmented and combined kits.

[0048] As used herein, the term “gene transfer system” refers to any means of delivering a composition comprising a nucleic acid sequence to a cell or tissue. For example, gene transfer systems include, but are not limited to, vectors (e.g., retroviral, adenoviral, lentiviral, adeno-associated viral, and other nucleic acid-based delivery systems), microinjection of naked nucleic acid, polymer-based delivery systems (e.g., liposome-based and metallic particle-based systems), biolistic injection, and the like. As used herein, the term “viral gene transfer system” refers to gene transfer systems comprising viral elements (e.g., intact viruses, modified viruses and viral components such as nucleic acids or proteins) to facilitate delivery of the sample to a desired cell or tissue. Non-limiting examples of viral gene transfer systems useful in the compositions and methods of the invention are lentiviral- and retroviral-gene transfer systems.

[0049] As used herein, the term “site-specific recombination target sequences” refers to nucleic acid sequences that provide recognition sequences for recombination factors and the location where recombination takes place.

[0050] As used herein, the term “nucleic acid molecule” refers to any nucleic acid containing molecule, including but not limited to, DNA or RNA. The term encompasses sequences that include any of the known base analogs of DNA and RNA including, but not limited to, 4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxylmethyl)-uracil, 5 -fluorouracil, 5 -bromouracil, 5- carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1 -methyladenine, 1 -methylpseudouracil, 1-methylguanine, 1 -methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5- methoxy-aminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'- methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil- 5-oxyacetic acid methylester, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine, 2 thiocytosine, 5-methyl-2 thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, N-uracil-5- oxyacetic acid methylester, and 2,6-diaminopurine.

[0051] The term “gene” refers to a nucleic acid (e.g., DNA) sequence that comprises coding sequences necessary for the production of a polypeptide, precursor, or RNA (e.g., mRNA, rRNA, tRNA). The polypeptide can be encoded by a full length coding sequence or by any portion of the coding sequence so long as the desired activity or functional properties (e.g., enzymatic activity, ligand binding, signal transduction, immunogenicity, etc.) of the full-length gene product or fragment thereof are retained. The term also encompasses the coding region of a structural gene and the sequences located adjacent to the coding region on both the 5' and 3' ends for a distance of about 1 kb or more on either end such that the gene corresponds to the length of the full-length mRNA. Sequences located 5' of the coding region and present on the mRNA are referred to as 5' non- translated sequences. Sequences located 3’ or downstream of the coding region and present on the mRNA are referred to as 3’ non-translated sequences. The term “gene” encompasses both cDNA and genomic forms of a gene. A genomic form or clone of a gene contains the coding region interrupted with noncoding sequences termed “introns” or “intervening regions” or “intervening sequences.” Introns are segments of a gene that are transcribed into nuclear RNA (hnRNA); introns may contain regulatory elements such as enhancers. Introns are removed or “spliced out” from the nuclear or primary transcript; introns therefore are absent in the messenger RNA (mRNA) transcript. The mRNA specifies the sequence or order of amino acids in a nascent polypeptide during translation (e.g., protein synthesis).

[0052] As used herein, the term “heterologous gene” refers to a gene that is not in its natural environment. For example, a heterologous gene includes a gene from one species introduced into another species. A heterologous gene also includes a gene native to an organism that has been altered in some way (e.g., mutated, added in multiple copies, linked to non-native regulatory sequences, etc.). Heterologous genes are distinguished from endogenous genes in that the heterologous gene sequences are typically joined to DNA sequences that are not found naturally associated with the gene sequences in the chromosome or are associated with portions of the chromosome not found in nature (e.g., genes expressed in loci where the gene is not normally expressed).

[0053] As used herein, the terms “nucleic acid molecule encoding,” “DNA sequence encoding,” and “DNA encoding” refer to the order or sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of these deoxyribonucleotides determines the order of amino acids along the polypeptide (protein) chain. The DNA sequence thus codes for the amino acid sequence.

[0054] As used herein, the terms “an oligonucleotide having a nucleotide sequence encoding a gene” and “polynucleotide having a nucleotide sequence encoding a gene,” means a nucleic acid sequence comprising the coding region of a gene or in other words the nucleic acid sequence that encodes a gene product. The coding region may be present in a cDNA, genomic DNA or RNA form. When present in a DNA form, the oligonucleotide or polynucleotide may be single-stranded (i.e., the sense strand) or double-stranded. Suitable control elements such as enhancers / promoters, splice junctions, polyadenylation signals, etc. may he placed in close proximity to the coding region of the gene if needed to permit proper initiation of transcription and / or correct processing of the primary RNA transcript. Alternatively, the coding region utilized in the expression vectors of the present invention may contain endogenous enhancers / promoters, splice junctions, intervening sequences, polyadenylation signals, etc. or a combination of both endogenous and exogenous control elements.

[0055] “Amino acid sequence” and terms such as “polypeptide” or “protein” are not meant to limit the amino acid sequence to the complete, native amino acid sequence associated with the recited protein molecule.

[0056] “Percent "identity," in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to persons of skill) or by visual inspection. Depending on the application, the percent "identity" can exist over a region of the sequence being compared, e.g., over a functional domain, or, alternatively, exist over the full length of the two sequences to be compared. For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. Alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations ofthese algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et al., infra). An example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov).

[0057] “Sequence identity” refers to the degree two polymer sequences (e.g., peptide, polypeptide, nucleic acid, etc.) have the same sequential composition of monomer subunits. The term “sequence similarity” refers to the degree with which two polymer sequences (e.g., peptide, polypeptide, nucleic acid, etc.) have similar polymer sequences. For example, similar amino acids are those that share the same biophysical characteristics and can be grouped into the families, e.g., acidic (e.g., aspartate, glutamate), basic (e.g., lysine, arginine, histidine), non-polar (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) and uncharged polar (e.g., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). The “percent sequence identity” (or “percent sequence similarity”) is calculated by: (1) comparing two optimally aligned sequences over a window of comparison (e.g., the length of the longer sequence, the length of the shorter sequence, a specified window), (2) determining the number of positions containing identical (or similar) monomers (e.g., same amino acids occurs in both sequences, similar amino acid occurs in both sequences) to yield the number of matched positions, (3) dividing the number of matched positions by the total number of positions in the comparison window (e.g., the length of the longer sequence, the length of the shorter sequence, a specified window), and (4) multiplying the result by 100 to yield the percent sequence identity or percent sequence similarity. For example, if peptides A and B are both 20 amino acids in length and have identical amino acids at all but 1 position, then peptide A and peptide B have 95% sequence identity. If the amino acids at the non- identical position shared the same biophysical characteristics (e.g., both were acidic), then peptide A and peptide B would have 100% sequence similarity. As another example, if peptide C is 20 amino acids in length and peptide D is 15 amino acids in length, and 14 out of 15 amino acids in peptide D are identical to those of a portion of peptide C, then peptides C and D have 70% sequence identity, but peptide D has 93.3% sequence identity to an optimal comparison window of peptide C. For the purpose of calculating “percent sequence identity” (or “percent sequence similarity”) herein, any gaps in aligned sequences are treated as mismatches at that position.

[0058] The term “isolated” when used in relation to a nucleic acid, as in “an isolated oligonucleotide” or “isolated polynucleotide” refers to a nucleic acid sequence that is identified and separated from at least one component or contaminant with which it is ordinarily associated in its natural source. Isolated nucleic acid is such present in a form or setting that is different from that in which it is found in nature. In contrast, non-isolated nucleic acids as nucleic acids such as DNA and RNA found in the state they exist in nature. For example, a given DNA sequence (e.g., a gene) is found on the host cell chromosome in proximity to neighboring genes; RNA sequences, such as a specific mRNA sequence encoding a specific protein, are found in the cell as a mixture with numerous other mRNAs that encode a multitude of proteins. However, isolated nucleic acid encoding a given protein includes, by way of example, such nucleic acid in cells ordinarily expressing the given protein where the nucleic acid is in a chromosomal location different from that of natural cells, or is otherwise flanked by a different nucleic acid sequence than that found in nature. The isolated nucleic acid, oligonucleotide, or polynucleotide may be present in singlestranded or double- stranded form. When an isolated nucleic acid, oligonucleotide or polynucleotide is to be utilized to express a protein, the oligonucleotide or polynucleotide will contain at a minimum the sense or coding strand (i.e., the oligonucleotide or polynucleotide may be single-stranded), but may contain both the sense and anti-sense strands (i.e., the oligonucleotide or polynucleotide may be double- stranded).

[0059] As used herein, the term “purified” or “to purify” refers to the removal of components (e.g., contaminants) from a sample. For example, antibodies are purified by removal of contaminating non-immunoglobulin proteins; they are also purified by the removal of immunoglobulin that does not bind to the target molecule. The removal of non- immunoglobulin proteins and / or the removal of immunoglobulins that do not bind to the target molecule results in an increase in the percent of target-reactive immunoglobulins in the sample. In another example, recombinant polypeptides are expressed in bacterial host cells and the polypeptides are purified by the removal of host cell proteins; the percent of recombinant polypeptides is thereby increased in the sample.

[0060] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

[0061] The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and / or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0062] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0063] The term “about” as used herein means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within an acceptable standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to + 20 %, preferably up to ± 10 %, more preferably up to ± 5 %, and more preferably still up to ± 1 % of a given value. Where particular values are described in the application and claims, unless otherwise stated, the term “about” is implicit and in this context means within an acceptable error range for the particular value.

[0064] Compositions, methods, and practice of the present disclosure employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press; T.E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); Oligonucleotide Synthesis (M. J. Gait, ed. 1984); Methods in MolecularBiology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1989) Academic Press; Animal Cell Culture (R. I. Freshney, ed. 1987); A.L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds. 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.): Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel, et al. eds. 1987); PCR: The Polymerase Chain Reaction, (Mullis, et al., eds. 1994); Current Protocols in Immunology (J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practice approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds. Harwood Academic Publishers, 1995); DNA Cloning: A practical Approach, Volumes I and II (D.N. Glover ed. 1985); Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds.(1985»; Transcription and Translation (B.D. Hames & S.J. Higgins, eds.(1984»; Animal Cell Culture (R.I. Freshney, ed. ( 1986»; Immobilized Cells and Enzymes (IRL Press, ( 1986»; and B. Perbal, A practical Guide To Molecular Cloning (1984); F.M. Ausubel et al. (eds.).

[0065] Compositions and methods for targeting the ganglioside GM2 to overcome ganglioside GD2 loss and / or heterogeneity

[0066] The present disclosure provides methods for identifying cancers and treating patients with the identified cancers. For example, as detailed herein, experiments conducted during the development of the present disclosure resulted in the discovery that the monosialoganglioside GM2 (ganglioside GM2 or GM2) was found to be expressed (e.g., upregulated and / or highly expressed) in cancer cells that exhibited a loss of, or decreased levels / or expression of, ganglioside GD2 and / or heterogeneous expression of ganglioside GD2 and / or loss of, or decreased expression of, ganglioside synthase enzyme ST8SIA1 and / or heterogeneous expression of ganglioside synthase enzyme ST8SIA1 (See Example 1). Thus, in some embodiments, the disclosure provides that GD2loss / downregulation / heterogeneity in cancer (e.g., low or heterogeneous expression of GD2 (e.g., GD2 low antigen escape) in neuroblastoma, Ewing sarcoma, etc.) occurs through downregulation of ST8SIA1, and a concurrent increase in the expression of GM2. Moreover, the disclosure provides that treatment of GD2 expressing cancers with a therapeutic agent targeting GD2 (e.g., GD2-CAR T cells or anti-GD2 / anti-CD47 antibody treatment) resulted in a reduction in the levels of GD2 expression together with a compensatory increase in GM2 (See Example 1, FIG. 3B). Thus, the disclosure provides that when GD2 expression is reduced or lost in cancer (e.g., on the surface of cancer and / or tumor cells), there is a compensatory increase in surface expression of GM2. Thus, based on the discovery that GM2 expression was inversely correlated with GD2 expression, the disclosure provides that targeting GM2 exists as a therapeutic option in cases where tumors present low to heterogenous levels of GD2 (e.g., neuroblastomas, Ewing sarcoma, and other GD2- heterogeneous expressing cancer cells). The disclosure also provides that treatment of tumors with a combination of anti-GD2 and anti-GM2 antibodies showed better efficacy and tumor control compared to anti-ganglioside monotherapy (See Example 1, FIGS. 4A and 4B). The disclosure further provides, in some embodiments, that targeting GM2 with mono or bispecific CAR T cells overcomes resistance to GD2 CAR T cells in GD2 low / GM2 high neuroblastoma and that co-targeting GD2 and GM2 with CARs can prevent immune escape a priori that occurs in response to anti-GD2 CAR T cells.

[0067] Accordingly, based on the discoveries described herein, in some embodiments, the disclosure provides a method of identifying a cancer that can be treated with (e.g., that will respond to treatment with) an anti-ganglioside GM2 directed treatment. In some embodiments, the method comprises providing a patient having cancer and determining whether the cancer can be treated with (e.g., will respond to treatment with) an anti- ganglioside GM2 directed treatment. In some embodiments, the patient having cancer displays resistance to anti-ganglioside GD2 therapy (e.g., due to heterogeneous GD2 expression or low ST8SIA1, and / or due to GD2 low antigen escape). In other embodiments, the patient having cancer displays reduced sensitivity to anti-ganglioside GD2 therapy (e.g., due to heterogeneous GD2 expression or low ST8SIA1, and / or due to GD2 low antigen escape). The disclosure is not limited by the type of patient having cancer. Indeed, the disclosure is useful for a variety of types of patients having cancer including but not limited to patients with a neuroblastoma, a sarcoma, a retinoblastoma, a medulloblastoma, a Ewing sarcoma, glioma, or a glioblastoma. In other embodiments, the patient is a patient with a sarcoma, a rhabdoid cancer, a neuroblastoma, retinoblastoma, medulloblastoma, Ewingsarcoma, lymphoma, melanoma, uterine carcinosarcoma (UCS), brain lower grade glioma (LGG), thymoma (THYM), testicular germ cell tumors (TGCT), glioblastoma multiforme (GBM) and skin cutaneous melanoma (SKCM), liver hepatocellular carcinoma (LIHC), uveal melanoma (UVM), kidney chromophobe (KICH), thyroid cancer (THCA), kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), stomach adenocarcinoma (STAD), cholangiocarcinoma (CHOL), adenoid cystic carcinoma (ACC), prostate adenocarcinoma (PRAD), pheochromocytoma and paraganglioma (PCPG), DLBC, lung adenocarcinoma (LUAD), head-neck squamous cell carcinoma (HNSC), pancreatic adenocarcinoma (PAAD), breast cancer (BRCA), mesothelioma (MESO), colon and rectal adenocarcinoma (COAD). Rectum adenocarcinoma (READ), esophageal carcinoma (ESC A), ovarian cancer (OV), lung squamous cell carcinoma (LUSC), bladder urothelial carcinoma (BLCA), sarcoma (SARC), small cell lung cancer, or uterine corpus endometrial carcinoma (UCEC). In some embodiments, the method further comprises measuring the expression level of ganglioside synthase enzyme ST8SIA1 in the patient. In other implementations, the method comprises measuring the expression and / or level of ganglioside GD2 in the patient. In some embodiments, the method comprises measuring the expression and / or level of both ST8SIAland ganglioside GD2 in the patient. In some embodiments, the method comprises measuring the expression and / or level of ganglioside GM2 in the patient. In some embodiments, the expression and / or level of ganglioside synthase enzyme ST8SIA1 and / or ganglioside GD2 is measured in the patient’s cancer and / or tumor cells. In some embodiments, the expression and / or level of ganglioside GM2 is measured in the patient’s cancer and / or tumor cells. In some embodiments, the method comprises comparing the expression and / or level of ganglioside synthase enzyme ST8SIA1 and / or ganglioside GD2 in the patient (e.g., in the patient’s cancer and / or tumor cells) to the expression and / or level of ganglioside synthase enzyme ST8SIA1 and / or ganglioside GD2 in a control sample. In one embodiment, a patient displaying low expression of ganglioside synthase enzyme ST8SIA1 compared to the control sample identifies the cancer as one that will respond to treatment with anti-ganglioside GM2 immunotherapy. In other embodiments, a patient displaying low expression and / or level of ganglioside GD2 compared to the control sample identifies the cancer as one that will respond to treatment with anti-ganglioside GM2 immunotherapy. In some embodiments, a patient displaying heterogeneous expression of ganglioside synthase enzyme ST8SIA1 compared to the control sample identifies the cancer as one that will respond to treatment with anti-ganglioside GM2 immunotherapy. In other embodiments, a patient displaying heterogeneous expression and / or level of ganglioside GD2 compared to thecontrol sample identifies the cancer as one that will respond to treatment with antiganglioside GM2 immunotherapy. In some embodiments, the method further comprises, once the cancer has been identified as one that will respond to treatment with anti-ganglioside GM2 immunotherapy, administering to the patient having cancer an anti-ganglioside GM2 immunotherapy.

[0068] Accordingly, the present disclosure provides methods for identifying, diagnosing, and / or characterizing cancers (e.g., neuroblastomas, Ewing sarcoma, glioma, and other GD2 expressing cancer cells) by detecting the expression and / or level of ganglioside synthase enzyme ST8SIA1 and / or ganglioside GD2 and / or ganglioside GM2. As described herein, GM2 was identified to be a viable target in patients having cancer that have displayed decreased sensitivity and / or resistance to GD2 directed therapies.

[0069] Detection of ganglioside synthase enzyme ST8SIA1 and / or ganglioside GD2 and / or ganglioside GM2.

[0070] In some embodiments, the present disclosure provides methods for detection of ST8SIA1, GD2 and / or GM2. In some embodiments, detection of ST8SIA1, GD2 and / or GM2 comprises detecting the expression and / or level of ST8S1A1, GD2 and / or GM2. In some embodiments, the expression and / or level is detected using an antibody or other moiety that displays specificity for GD2, GM2 and / or ST8SIA1. In some embodiments, the expression and / or level measured is gene or protein expression level (e.g., of ST8SIA1). In some embodiments, protein expression or gene expression (e.g., RNA expression) is measured directly. For example, in some embodiments, ST8SIA1, GD2 and / or GM2 levels and / or expression is detected in tissue samples (e.g., biopsy samples). In other embodiments, ST8SIA1, GD2 and / or GM2 is detected in bodily fluids e.g., serum, plasma, or urine). The present disclosure further provides kits for the detection of ST8SIA1, GD2 and / or GM2. In some embodiments, the expression level (e.g., presence or absence, or degree or amount of expression) of ST8SIA1, GD2 and / or GM2 is used to identify a course of treatment for a subject (e.g., whether or not to treat a patient with a GM2 directed therapy). In some embodiments, the level and / or expression level (e.g., presence or absence, or degree or amount of expression) of ST8SIA1, GD2 and / or GM2 is used to provide a diagnosis or prognosis to a subject.

[0071] ST8SIA1 may be detected at the nucleic acid (e.g., mRNA) level. In some embodiments, ST8SIAlgene expression is measured. There are many methods known to those of skill in the art for detecting gene expression levels. For example, gene expression may be measured by polymerase chain reaction (PCR) analysis, sequencing analysis,electrophoretic analysis, restriction fragment length polymorphism (RFLP) analysis, Northern blot analysis, quantitative PCR, reverse-transcriptase-PCR analysis (RT-PCR), allele- specific oligonucleotide hybridization analysis, comparative genomic hybridization, heteroduplex mobility assay (HMA), single strand conformational polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE), RNase mismatch analysis, mass spectrometry, tandem mass spectrometry, matrix assisted laser desorption / ionization-time of flight (MALDI-TOF) mass spectrometry, electrospray ionization (ESI) mass spectrometry, surface-enhanced laser desorption / ionization-time of flight (SELDT-TOF) mass spectrometry, quadrupole-time of flight (Q-TOF) mass spectrometry, atmospheric pressure photoionization mass spectrometry (APPI-MS), Fourier transform mass spectrometry (FTMS), matrix-assisted laser desorption / ionization-Fourier transform-ion cyclotron resonance (MALDI-FT-ICR) mass spectrometry, secondary ion mass spectrometry (SIMS), surface plasmon resonance, Southern blot analysis, in situ hybridization, fluorescence in situ hybridization (FISH), chromogenic in situ hybridization (CISH), immunohistochemistry (IHC), microarray, comparative genomic hybridization, karyotyping, multiplex ligation-dependent probe amplification (MLPA), Quantitative Multiplex PCR of Short Fluorescent Fragments (QMPSF), microscopy, methylation specific PCR (MSP) assay, Hpall tiny fragment Enrichment by Ligation-mediated PCR (HELP) assay, radioactive acetate labeling assays, colorimetric DNA acetylation assay, chromatin immunoprecipitation combined with microarray (ChlP-on-chip) assay, restriction landmark genomic scanning, Methylated DNA immunoprecipitation (MeDIP), molecular break light assay for DNA adenine methyltransferase activity, chromatographic separation, methylation-sensitive restriction enzyme analysis, bisulfite-driven conversion of non-methylated cytosine to uracil, methyl- binding PCR analysis, or a combination of any of the foregoing. Gene expression may also be measured by a sequencing technique such as direct sequencing, RNA sequencing, whole transcriptome shotgun sequencing, random shotgun sequencing, Sanger dideoxy termination sequencing, whole-genome sequencing, sequencing by hybridization, pyrosequencing, capillary electrophoresis, gel electrophoresis, duplex sequencing, cycle sequencing, singlebase extension sequencing, solid-phase sequencing, high-throughput sequencing, massively parallel signature sequencing, emulsion PCR, sequencing by reversible dye terminator, paired-end sequencing, near-term sequencing, exonuclease sequencing, sequencing by ligation, short-read sequencing, single-molecule sequencing, sequencing-by-synthesis, realtime sequencing, reverse-terminator sequencing, nanopore sequencing, 454 sequencing,Solexa Genome Analyzer sequencing, SOLID™ sequencing, MS-PET sequencing, mass spectrometry, and a combination of any of the foregoing.

[0072] In some embodiments, RNA is detected by measuring the expression of corresponding mRNA in a tissue sample (e.g., a neuroblastoma tissue sample, Ewing sarcoma tissue sample, or other GD2 expressing cancer tissue sample). mRNA expression may be measured by any suitable method, including but not limited to, those disclosed herein.

[0073] In some embodiments, RNA is detected by Northern blot analysis. Northern blot analysis involves the separation of RNA and hybridization of a complementary labeled probe. Methods for Northern blot analysis are well known in the art.

[0074] In other embodiments, RNA (or corresponding cDNA) may be detected by hybridization to a oligonucleotide probe. A variety of hybridization assays using a variety of technologies for hybridization and detection are available. For example, in some embodiments, TaqMan assay (Applied Biosystems, Foster City, CA; See e.g., U.S. Patent Nos. 5,962,233 and 5,538,848, each of which is herein incorporated by reference) is utilized. The assay is performed during a PCR reaction. The TaqMan assay exploits the 5'-3' exonuclease activity of the AMPLITAQ GOLD DNA polymerase. A probe consisting of an oligonucleotide with a 5'-reporter dye e.g., a fluorescent dye) and a 3'-quencher dye is included in the PCR reaction. During PCR, if the probe is bound to its target, the 5'-3' nucleolytic activity of the AMPLITAQ GOLD polymerase cleaves the probe between the reporter and the quencher dye. The separation of the reporter dye from the quencher dye results in an increase of fluorescence. The signal accumulates with each cycle of PCR and can be monitored with a fluorimeter.

[0075] In still other embodiments, reverse-transcriptase PCR (RT-PCR) is used to detect the expression of RNA. In RT-PCR, RNA is enzymatically converted to complementary DNA or "cDNA" using a reverse transcriptase enzyme. The cDNA is then used as a template for a PCR reaction. PCR products can be detected by any suitable method, including but not limited to, gel electrophoresis and staining with a DNA specific stain or hybridization to a labeled probe. In some embodiments, the quantitative reverse transcriptase PCR with standardized mixtures of competitive templates method described in U.S. Patents 5,639,606, 5,643,765, and 5,876,978 (each of which is herein incorporated by reference) is utilized.

[0076] Measuring ST8SIA1, GD2 and / or GM2 expression may also be performed utilizing a moiety with specificity for ST8SIA1, GD2 or GM2.. For example, antibodies with specificity for ST8SIA1, GD2 and / or GM2 may be used to detect the expression and / or level and / or distribution of ST8SIA1, GD2 and / or GM2. In some embodiments, ST8SIA1 ismeasured at the protein level. There are many methods that may be used to determine the level of protein expression. For example, protein expression may be measured by an immunohistochemistry assay, an enzyme-linked immunosorbent assay (ELISA), in situ hybridization, flow cytometry, chromatography, liquid chromatography, size exclusion chromatography, high performance liquid chromatography (HPLC), gas chromatography, mass spectrometry, tandem mass spectrometry, matrix assisted laser desorption / ionization- time of flight (MALDI-TOF) mass spectrometry, electrospray ionization (ESI) mass spectrometry, surface-enhanced laser desorption / ioniz.ation-time of flight (SELDI-TOF) mass spectrometry, quadrupole-time of flight (Q-TOF) mass spectrometry, atmospheric pressure photoionization mass spectrometry (APPLMS), Fourier transform mass spectrometry (FTMS), matrix-assisted laser desorption / ionization-Fourier transform-ion cyclotron resonance (MALDI-FT-ICR) mass spectrometry, secondary ion mass spectrometry (SIMS), radioimmunoassays, microscopy, microfluidic chip-based assays, surface plasmon resonance, sequencing, Western blotting assay, or a combination of any of the foregoing.

[0077] ST8SIA1, GD2 and / or GM2 may be detected by any suitable method. In some embodiments, ST8S1A1, GD2 and / or GM2 are detected by binding of an antibody specific for ST8SIA1, GD2 or GM2, respectively. The present disclosure is not limited to a particular antibody. Any antibody (monoclonal or polyclonal) that specifically detects ST8SIA1, GD2 and / or GM2 may by utilized. Methods for the generation of antibodies are known in the art and are described herein.

[0078] Antibody binding is detected by techniques known in the art. For example, in some embodiments where ST8SIA1, GD2 and / or GM2 is detected in bodily fluids, antibody binding is detected using a suitable technique, including but not limited to, radioimmunoassay, ELISA (enzyme-linked immunosorbant assay), "sandwich" immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays (e.g., using colloidal gold, enzyme or radioisotope labels, for example), Western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays, etc.), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays. In other embodiments, where ST8SIA1, GD2 and / or GM2 is detected in tissue samples, immunohistochemistry is utilized for the detection of antibody binding.

[0079] In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, thesecondary antibody is labeled. Many methods are known in the art for detecting binding in an immunoassay and are within the scope of the present disclosure.

[0080] In some embodiments, an automated detection assay is utilized. Methods for the automation of immunoassays include, but are not limited to, those described in U.S. Patents 5,885,530, 4,981,785, 6,159,750, and 5,358,691, each of which is herein incorporated by reference. In some embodiments, the analysis and presentation of results is also automated. For example, in some embodiments, software that generates a diagnosis and / or prognosis based on the presence or absence of ST8SIA1 , GD2 and / or GM2 is utilized.

[0081] In some embodiments, ST8SIA1, GD2 and / or GM2 expression is evaluated in a tumor or tumor sample. As used herein, a tumor or tumor sample may encompass part or all of the tumor area occupied by tumor cells. In some embodiments, a tumor or tumor sample may further encompass tumor area occupied by tumor associated intratumoral cells and / or tumor associated stroma (e.g., contiguous peri-tumoral desmoplastic stroma). Tumor-associated intratumoral cells and / or tumor associated stroma may include areas of immune infiltrates immediately adjacent to and / or contiguous with the main tumor mass.

[0082] As described herein, presence and / or expression levels / amount of a ST8SIA1, GD2 and / or GM2 can be determined qualitatively and / or quantitatively based on any suitable criterion known in the art, including but not limited to methods described herein. In certain embodiments, presence and / or expression levels / amount of ST8SIA1, GD2 and / or GM2 in a first sample is increased or elevated as compared to presence / absence and / or expression levels / amount in a second sample. In certain embodiments, presence / absence and / or expression levels / amount of ST8SIA1, GD2 and / or GM2 in a first sample is decreased or reduced as compared to presence and / or expression levels / amount in a second sample. In certain embodiments, the second sample is a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue.

[0083] Presence and / or expression level / amount of ST8SIA1, GD2 and / or GM2 in a sample can be analyzed by immunohistochemistry (“IHC”), Western blot analysis, immunoprecipitation, molecular binding assays, ELISA, ELIFA, fluorescence activated cell sorting (“FACS”), MassARRAY, proteomics, quantitative blood based assays (as for example serum ELISA), biochemical enzymatic activity assays, in situ hybridization, Southern analysis, Northern analysis, whole genome sequencing, polymerase chain reaction (“PCR”) including quantitative real time PCR (“qRT-PCR”) and other amplification type detection methods, such as, for example, branched DNA, SISBA, TMA and the like), RNA- Seq, FISH, microarray analysis, gene expression profiling, and / or serial analysis of geneexpression (“SAGE”), as well as any one of the wide variety of assays that can be performed by protein, gene, and / or tissue array analysis. Typical protocols for evaluating the status of genes and gene products (e.g., for detection of ST8SIA1) are found, for example in Ausubel et al., eds., 1995, Current Protocols In Molecular Biology, Units 2 (Northern Blotting), 4 (Southern Blotting), 15 (Immunoblotting) and 18 (PCR Analysis). Multiplexed immunoassays such as those available from Rules Based Medicine or Meso Scale Discovery (“MSD”) may also be used.

[0084] In alternative methods, the sample may be contacted with an antibody specific for ST8SIA1, GD2 and / or GM2 under conditions sufficient for an antibody- ST8SIA1, GD2 and / or GM2 complex to form, and then detecting said complex. The presence of ST8SIA1, GD2 and / or GM2 may be detected in a number of ways, such as by Western blotting and ELISA procedures for assaying a wide variety of tissues and samples, including plasma or serum. A wide range of immunoassay techniques using such an assay format are available, see, e.g., U.S. Pat. Nos. 4,016,043, 4,424,279 and 4,018,653. These include both single-site and two-site or “sandwich” assays of the non-competitive types, as well as in the traditional competitive binding assays. These assays also include direct binding of a labeled antibody to ST8SIA1, GD2 and / or GM2.

[0085] Presence and / or expression level / amount of ST8SIA1, GD2 and / or GM2 in a tissue or cell sample may also be examined by way of functional or activity-based assays. For instance, if for the enzyme ST8SIA1, one may conduct assays known in the art to determine or detect the presence of the given enzymatic activity in the tissue or cell sample.

[0086] In certain embodiments, the samples are normalized for both differences in the amount of ST8SIA1, GD2 and / or GM2 assayed and variability in the quality of the samples used, and variability between assay runs. Such normalization may be accomplished by detecting and incorporating the expression of certain normalizing biomarkers, including well known housekeeping genes. Alternatively, normalization can be based on the mean or median signal (global normalization approach). Measured normalized amount of a subject tumor mRNA or protein may be measured and compared to the amount found in a reference set. Normalized expression levels for each mRNA or protein per tested tumor per subject can be expressed as a percentage of the expression level measured in the reference set. The presence and / or expression level / amount measured in a particular subject sample to be analyzed will fall at some percentile within this range, which can be determined by methods well known in the art.

[0087] In one embodiment, the sample is a clinical sample. In another embodiment, the sample is used in a diagnostic assay. In some embodiments, the sample is obtained from a primary or metastatic tumor. Tissue biopsy is often used to obtain a representative piece of tumor tissue. Alternatively, tumor cells can be obtained indirectly in the form of tissues or fluids that are known or thought to contain the tumor cells of interest. ST8SIA1, GD2 and / or GM2 can be detected from cancer or tumor tissue or from other body samples such as urine, sputum, serum or plasma. The same techniques discussed above for detection of ST8SIA1, GD2 and / or GM2 in cancerous samples can be applied to other body samples. Cancer cells may be sloughed off from cancer lesions and appear in such body samples. By screening such body samples, a simple early diagnosis can be achieved for these cancers. In addition, the progress of therapy can be monitored more easily by testing such body samples for ST8SIA1, GD2 and / or GM2.

[0088] In certain embodiments, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a single sample or combined multiple samples from the same subject or individual that are obtained at one or more different time points than when the test sample is obtained. For example, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained at an earlier time point from the same subject or individual than when the test sample is obtained. Such reference sample, reference cell, reference tissue, control sample, control cell, or control tissue may be useful if the reference sample is obtained during initial diagnosis of cancer and the test sample is later obtained when the cancer becomes metastatic.

[0089] In certain embodiments, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a combined multiple samples from one or more healthy individuals who are not the subject or individual. In certain embodiments, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a combined multiple samples from one or more individuals with a disease or disorder (e.g., cancer) who are not the subject or individual. In certain embodiments, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is pooled RNA samples from normal tissues or pooled plasma or serum samples from one or more individuals who are not the subject or individual (e.g., for detection of ST8SIA1). In certain embodiments, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is pooled RNA samples from tumor tissues or pooled plasma or serum samples from one or more individuals with a disease or disorder (e.g., cancer) who are not the subject or individual.

[0090] In some embodiments, the sample is a tissue sample from the individual. In some embodiments, the tissue sample is a tumor tissue sample (e.g., biopsy tissue). In some embodiments, the tissue sample is CNS tissue. In some embodiments, the tissue sample is brain tissue (e.g., glial tissue).

[0091] A tumor sample may be obtained from a subject by any method known in the art, including without limitation a biopsy, endoscopy, or surgical procedure. In some embodiments, a tumor sample may be prepared by methods such as freezing, fixation (e.g., by using formalin or a similar fixative), and / or embedding in paraffin wax. In some embodiments, a tumor sample may be sectioned. In some embodiments, a fresh tumor sample (i.e., one that has not been prepared by the methods described above) may be used.

[0092] In some embodiments, responsiveness to treatment may refer to any one or more of: extending survival (including overall survival and progression free survival); resulting in an objective response (including a complete response or a partial response); or improving signs or symptoms of cancer. In some embodiments, responsiveness may refer to improvement of one or more factors according to the published set of RECIST guidelines for determining the status of a tumor in a cancer patient, i.e., responding, stabilizing, or progressing. For a more detailed discussion of these guidelines, see Eisenhauer et al., Eur I Cancer 2009;45: 228-47; Topalian et al., N Engl J Med 2012;366:2443-54; Wolchok et al., Clin Can Res 2009;15:7412-20; and Therasse, P„ et al. J. Natl. Cancer Inst. 92:205-16 (2000). A responsive subject may refer to a subject whose cancer(s) show improvement, e.g., according to one or more factors based on RECIST criteria. A non-responsive subject may refer to a subject whose cancer(s) do not show improvement, e.g., according to one or more factors based on RECIST criteria.

[0093] Conventional response criteria may not be adequate to characterize the anti-tumor activity of immunotherapeutic agents, which can produce delayed responses that may be preceded by initial apparent radiological progression, including the appearance of new lesions. Therefore, modified response criteria have been developed that account for the possible appearance of new lesions and allow radiological progression to be confirmed at a subsequent assessment. Accordingly, in some embodiments, responsiveness may refer to improvement of one of more factors according to immune-related response criteria2 (irRC). See, e.g., Wolchok et al., Clin Can Res 2009; 15:7412-20. In some embodiments, new lesions are added into the defined tumor burden and followed, e.g., for radiological progression at a subsequent assessment. In some embodiments, presence of non-target lesions are included in assessment of complete response and not included in assessment ofradiological progression. In some embodiments, radiological progression may be determined only on the basis of measurable disease and / or may be confirmed by a consecutive assessment >4 weeks from the date first documented.

[0094] In some embodiments, clinical monitoring and / or neurointensive management of edema is utilized together with immunotherapeutic compositions and methods of the disclosure in order to achieve successful clinical results. The disclosure is not limited by the type of monitoring or the type of management utilized. Indeed, any means of monitoring cranial inflammation and / or swelling may be used. In like manner, any means of managing cranial inflammation and / or swelling may be used. In one non-limiting example, monitoring for hydrocephalus and / or signs of increased intracranial pressure is performed during inpatient monitoring with frequent neurological and fundoscopic exams and / or neuroimaging. In some embodiments, neurosurgical interventions, such as intraventricular shunt placement for relief of hydrocephalus or even craniectomy for decompression, is utilized to support patients through tumoricidal neuroinflammation.

[0095] Antibodies

[0096] In some embodiments, the disclosure provides isolated antibodies. In preferred embodiments, the present disclosure provides monoclonal antibodies that specifically bind to ST8SIA1, GD2 and / or GM2 These antibodies find use in the diagnostic methods described herein. In some embodiments, GD2 antibodies known in the art may be used in combination with ST8SIA1 and / or GM2 antibodies. Non-limiting examples of anti-GD2 antibodies that find use in the disclosure include 14G2a, ch!4.18, hul4.18K322A, m3F8, hu3F8-IgGl, hu3F8-IgG4, HM3F8, UNITUXIN, DMAb-20 or any other antibody that binds with specificity to GD2.

[0097] An antibody against ST8SIA1, GD2 or GM2 of the present disclosure may be any monoclonal or polyclonal antibody, as long as it can recognize ST8SIA1, GD2 or GM2, respectively . Antibodies can be produced by using ST8SIA1 or GM2 as the antigen according to a conventional antibody or antiserum preparation process or other processes known in the art.

[0098] The present disclosure contemplates the use of both monoclonal and polyclonal antibodies. Any suitable method may be used to generate the antibodies used in the methods and compositions of the present disclosure, including but not limited to, those disclosed herein. For example, for preparation of a monoclonal antibody, antigen, as such, or together with a suitable carrier or diluent is administered to an animal {e.g., a mammal) under conditions that permit the production of antibodies. For enhancing the antibody productioncapability, complete or incomplete Freund's adjuvant may be administered. Normally, the antigen is administered once every 2 weeks to 6 weeks, in total, about 2 times to about 10 times. Animals suitable for use in such methods include, but are not limited to, primates, rabbits, dogs, guinea pigs, mice, rats, sheep, goats, etc.

[0099] For preparing monoclonal antibody -producing cells, an individual animal whose antibody titer has been confirmed (e.g., a mouse) is selected, and 2 days to 5 days after the final immunization, its spleen or lymph node is harvested and antibody -producing cells contained therein are fused with myeloma cells to prepare the desired monoclonal antibody producer hybridoma. Measurement of the antibody titer in antiserum can be carried out, for example, by reacting the labeled antigen, as described hereinafter and antiserum and then measuring the activity of the labeling agent bound to the antibody. The cell fusion can be carried out according to known methods, for example, the method described by Koehler and Milstein (Nature 256:495

[1975] ). As a fusion promoter, for example, polyethylene glycol (PEG) or Sendai virus (HVJ), preferably PEG is used.

[0100] Examples of myeloma cells include NS-1, P3U1, SP2 / 0, AP-1 and the like. The proportion of the number of antibody producer cells (spleen cells) and the number of myeloma cells to be used is preferably about 1 : 1 to about 20: 1. PEG (preferably PEG 1000-PEG 6000) is preferably added in concentration of about 10% to about 80%. Cell fusion can be carried out efficiently by incubating a mixture of both cells at about 20°C to about 40°C, preferably about 30°C to about 37°C for about 1 minute to 10 minutes.

[0101] Various methods may be used for screening for a hybridoma producing the antibody (e.g., against ST8SIA1 and / or GM2 of the present disclosure). For example, where a supernatant of the hybridoma is added to a solid phase (e.g., microplate) to which antibody is adsorbed directly or together with a carrier and then an anti-immunoglobulin antibody (if mouse cells are used in cell fusion, anti-mouse immunoglobulin antibody is used) or Protein A labeled with a radioactive substance or an enzyme is added to detect the monoclonal antibody against the antigen bound to the solid phase. Alternately, a supernatant of the hybridoma is added to a solid phase to which an anti-immunoglobulin antibody or Protein A is adsorbed and then the antigen labeled with a radioactive substance or an enzyme is added to detect the monoclonal antibody against the antigen bound to the solid phase.

[0102] Selection of the monoclonal antibody can be carried out according to any known method or its modification. Normally, a medium for animal cells to which HAT (hypoxanthine, aminopterin, thymidine) are added is employed. Any selection and growth medium can be employed as long as the hybridoma can grow. For example, RPMI 1640medium containing 1% to 20%, preferably 10% to 20% fetal bovine serum, GIT medium containing 1% to 10% fetal bovine serum, a serum free medium for cultivation of a hybridoma (SFM-101, Nissui Seiyaku) and the like can be used. Normally, the cultivation is carried out at 20°C to 40°C, preferably 37°C for about 5 days to 3 weeks, preferably 1 week to 2 weeks under about 5% CO2 gas. The antibody titer of the supernatant of a hybridoma culture can be measured according to the same manner as described above with respect to the antibody titer of the anti-antigen in the antiserum.

[0103] Separation and purification of a monoclonal antibody be carried out according to the same manner as those of conventional polyclonal antibodies such as separation and purification of immunoglobulins, for example, salting-out, alcoholic precipitation, isoelectric point precipitation, electrophoresis, adsorption and desorption with ion exchangers (e.g., DEAE), ultracentrifugation, gel filtration, or a specific purification method wherein only an antibody is collected with an active adsorbent such as an antigen-binding solid phase, Protein A or Protein G and dissociating the binding to obtain the antibody.

[0104] Polyclonal antibodies may be prepared by any known method or modifications of these methods including obtaining antibodies from patients. For example, a complex of an immunogen (an antigen) and a carrier protein is prepared, and an animal is immunized by the complex according to the same manner as that described with respect to the above monoclonal antibody preparation. A material containing the antibody against antigen is recovered from the immunized animal and the antibody is separated and purified.

[0105] As to the complex of the immunogen and the carrier protein to be used for immunization of an animal, any carrier protein and any mixing proportion of the carrier and a hapten / antigen can be employed as long as an antibody against the hapten, which is crosslinked on the carrier and used for immunization, is produced efficiently. For example, bovine serum albumin, bovine cycloglobulin, keyhole limpet hemocyanin, etc. may be coupled to an hapten in a weight ratio of about 0.1 part to about 20 parts, preferably, about 1 part to about 5 parts per 1 part of the hapten.

[0106] In addition, various condensing agents can be used for coupling of a hapten and a carrier. For example, glutaraldehyde, carbodiimide, maleimide activated ester, activated ester reagents containing thiol group or dithiopyridyl group, and the like find use with the present disclosure. The condensation product as such or together with a suitable carrier or diluent is administered to a site of an animal that permits the antibody production. For enhancing the antibody production capability, complete or incomplete Freund's adjuvant may beadministered. Normally, the antigen is administered once every 2 weeks to 6 weeks, in total, about 3 times to about 10 times.

[0107] The polyclonal antibody is recovered from blood, ascites and the like, of an animal immunized by the above method. The antibody titer in the antiserum can be measured according to the same manner as that described above with respect to the supernatant of the hybridoma culture. Separation and purification of the antibody can be carried out according to the same separation and purification method of immunoglobulin as that described with respect to the above monoclonal antibody.

[0108] The antigen used herein as the immunogen is not limited to any particular type of immunogen. For example, for ST8SIA1 protein (further including a gene having a nucleotide sequence partly altered) can be used as the immunogen. Further, fragments of the protein may be used. Fragments may be obtained by any methods including, but not limited to expressing a fragment of the gene, enzymatic processing of the protein, chemical synthesis, and the like.

[0109] In some embodiments, antibodies (<?.g., monoclonal antibodies) are humanized. Such humanized antibodies find particular use in the cancer immunotherapies described herein. Humanized antibodies are altered in order to make them less immunogenic to humans, e.g., by constructing chimeric antibodies in which a mouse antigen-binding variable domain is coupled to a human constant domain. Humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies. Methods for humanizing antibodies are well known in the art and include but are not limited to, those disclosed in U.S. patents 6,054,297, 4,816,567, 6,180,377, 5,871,907, 5,585,089, and 6,180,370, each of which is herein incorporated by reference.

[0110] Antibody Therapy

[0111] In some embodiments, the present disclosure provides antibodies that target GM2 and / or GD2 expressing tumors. In some embodiments, the antibody targets GM2. In some embodiments, the antibody target GD2. In some embodiments, a combination of antibodies targeting both GM2 and GD2 are used. In some embodiments, the antibodies used for cancer therapy are humanized antibodies.

[0112] In some embodiments, the therapeutic antibodies comprise an antibody generated against GM2, wherein the antibody is conjugated to a cytotoxic agent. In such embodiments, a tumor specific therapeutic agent is generated that does not target normal cells, thus reducing many of the detrimental side effects of traditional chemotherapy. For certain applications, itis envisioned that the therapeutic agents will be pharmacologic agents that will serve as useful agents for attachment to antibodies, particularly cytotoxic or otherwise anticellular agents having the ability to kill or suppress the growth or cell division of endothelial cells. The present disclosure contemplates the use of any pharmacologic agent that can be conjugated to an antibody, and delivered in active form. Exemplary anticellular agents include chemotherapeutic agents, radioisotopes, and cytotoxins. The therapeutic antibodies of the present disclosure may include a variety of cytotoxic moieties, including but not limited to, radioactive isotopes (e.g., iodine-131 , iodine-123, technicium-99m, indium-1 11 , rhenium-188, rhenium-186, gallium-67, copper-67, yttrium-90, iodine-125 or astatine-211), hormones such as a steroid, antimetabolites such as cytosines (e.g., arabinoside, fluorouracil, methotrexate or aminopterin; an anthracycline; mitomycin C), vinca alkaloids (e.g., demecolcine; etoposide; mithramycin), and antitumor alkylating agent such as chlorambucil or melphalan. Other embodiments may include agents such as a coagulant, a cytokine, growth factor, bacterial endotoxin or the lipid A moiety of bacterial endotoxin. For example, in some embodiments, therapeutic agents will include plant-, fungus- or bacteria-derived toxin, such as an A chain toxins, a ribosome inactivating protein, a-sarcin, aspergillin, restrictocin, a ribonuclease, diphtheria toxin or pseudomonas exotoxin. In some preferred embodiments, deglycosylated ricin A chain is utilized.

[0113] The above described agents may, if desired, be successfully conjugated to an antibody, in a manner that will allow their targeting, internalization, release or presentation to blood components at the site of the targeted tumor cells as required using known conjugation technology (See, e.g., Ghose et al., Methods Enzymol., 93:280

[1983] ).

[0114] For example, in some embodiments the present disclosure provides immunotoxins targeted to GM2. Immunotoxins are conjugates of a specific targeting agent typically a tumor-directed antibody or fragment, with a cytotoxic agent, such as a toxin moiety. The targeting agent directs the toxin to, and thereby selectively kills, cells carrying the targeted antigen. In some embodiments, therapeutic antibodies employ crosslinkers that provide high in vivo stability (Thorpe el al., Cancer Res., 48:6396

[1988] ).

[0115] In other embodiments, particularly those involving treatment of solid tumors, antibodies are designed to have a cytotoxic or otherwise anticellular effect against the tumor vasculature, by suppressing the growth or cell division of the vascular endothelial cells. This attack is intended to lead to a tumor-localized vascular collapse, depriving the tumor cells, particularly those tumor cells distal of the vasculature, of oxygen and nutrients, ultimately leading to cell death and tumor necrosis.

[0116] In preferred embodiments, antibody based therapeutics are formulated as pharmaceutical compositions as described herein. In preferred embodiments, administration of an antibody composition of the present disclosure results in a measurable decrease in cancer (e.g., decrease or elimination of tumor). In some embodiments, antibody based therapeutics are formulated with a T cell and / or NK cell engager (e.g., a bispecific T cell engager).

[0117] Chimeric antigen receptor (CAR) T cells and CAR T cell therapy

[0118] In some embodiments, the present disclosure provides chimeric antigen receptor (CAR) T cells that target GM2 and / or GD2 on tumor cells.

[0119] Thus, in one aspect, the disclosure provides novel methods for the treatment of cancers / tumors (e.g., GD2 resistant or desensitized cancers / tumors) using modified immune cells engineered to express a CAR targeting GM2 and / or targeting GM2 and GD2 (e.g., in a patient identified as one that will respond to GM2 therapy). In various aspects of the disclosure, methods of treating a cancer / tumors are provided, the methods comprising administering to a patient having such a cancer or tumor an effective amount of immune cells engineered to express a CAR targeting GM2 and / or targeting GM2 and GD2. In some embodiments, the cancer is a neuroblastoma, a sarcoma, a retinoblastoma, a medulloblastoma, a Ewing sarcoma, a glioma or a glioblastoma. In other embodiments, the cancer is a sarcoma, a rhabdoid cancer, a neuroblastoma, retinoblastoma, medulloblastoma, Ewing sarcoma, lymphoma, melanoma, uterine carcinosarcoma (UCS), brain lower grade glioma (LGG), thymoma (THYM), testicular germ cell tumors (TGCT), glioblastoma multiforme (GBM) and skin cutaneous melanoma (SKCM), liver hepatocellular carcinoma (LIHC), uveal melanoma (UVM), kidney chromophobe (KICH), thyroid cancer (THCA), kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), stomach adenocarcinoma (STAD), cholangiocarcinoma (CHOL), adenoid cystic carcinoma (ACC), prostate adenocarcinoma (PRAD), pheochromocytoma and paraganglioma (PCPG), DLBC, lung adenocarcinoma (LU AD), head-neck squamous cell carcinoma (HNSC), pancreatic adenocarcinoma (PAAD), breast cancer (BRCA), mesothelioma (MESO), colon and rectal adenocarcinoma (COAD), rectum adenocarcinoma (READ), esophageal carcinoma (ESCA), ovarian cancer (OV), lung squamous cell carcinoma (LUSC), bladder urothelial carcinoma (BLCA), sarcoma (SARC), small cell lung cancer, or uterine corpus endometrial carcinoma (UCEC).

[0120] In certain embodiments, the presence of significantly elevated levels of GM2 present on the surface of cancers / tumors (e.g., with decreased expression of GD2 and / or ST8SIA1)results in efficacious treatment (e.g., killing and / or inhibition of progression) of GM2 expressing cancers / tumors with GM2-targeted and / or GM2- and GD2-targeted CAR T cells. For example, the efficacy of the GM2-targeted and / or GM2- and GD2-targeted CAR T cells in cancers / tumors appeared to be driven largely by the homogeneously high expression of the GM2 in cancers / tumors with decreased expression of GD2 and / or ST8SIA1. Although an understanding of a mechanism is not needed to practice the present disclosure and while the present disclosure is not limited to any particular mechanism, high expression of the target GM2 antigen on cancers / tumors that have become resistant to GD2 directed therapy is related to the efficacy of treatment of the cancer / tumors with GM2-targeted and / or GM2- and GD2- targeted CAR T cells.

[0121] In certain embodiments, the disclosure provides methods of treating (e.g., inhibiting growth of and / or killing) tumors using immune cells (e.g., T cells (e.g., CD3+ T cells)) genetically engineered to express a receptor that recognizes GM2 and / or GM2 and GD2 (e.g., on the surface of neuroblastoma, Ewing sarcoma, or other cancer with enhanced GM2 expression) and transmit a signal that activates the immune cell to induce expansion of the immune cell and / or tumor killing.

[0122] Chimeric antigen receptors (CARs) are recombinant receptor constructs comprising an extracellular antigen-binding domain (e.g., a single-chain variable fragment (scFv) derived from an antibody) optionally joined to a hinge / spacer peptide, a transmembrane domain, and an intracellular signaling domain (e.g., an intracellular T cell signaling domain of a T cell receptor). Immune cells (e.g., T cells) genetically modified to express CARs display the specificity of an antibody (e.g., they are not MHC / HLA-restricted) with the functionality of effector cells (e.g., cytotoxic and / or memory functions of T cells).

[0123] The disclosure is not limited by the chimeric antigen receptor (CAR) specific for GM2 and / or GM2 and GD2 expressed in immune cells (e.g., the CAR constructs used in methods of the disclosure).

[0124] GM2 chimeric antigen receptors.

[0125] The disclosure provides, in some embodiments, a chimeric antigen receptor (CAR) that includes a ganglioside GM2 binding domain. In some embodiments, the antigen-binding domain comprises an antibody, an antigen-binding fragment of an antibody, a F(ab) fragment, a F(ab') fragment, a single chain variable fragment (scFv), or a single-domain antibody (sdAb). In some embodiments, a CAR of the disclosure comprise a VH and / or VL amino acid sequence of Table 1. In some embodiments, a single domain antibody (sdAb) disclosed herein comprises a VH selected from sequences disclosed in Table 1. In someembodiments, the VH and VL pairs of the GM2 CARs of the disclosure are selected from the various sequences listed in Table 1. In some embodiments, various combinations of CDRs of the VH and VL pairs are selected from the sequences listed in Table 1. The various embodiments of the disclosure may include one or more of the polypeptide sequences pertaining to chimeric antigen receptor (CAR) sequences referenced below in Table 1. Various embodiments of the disclosure may also include one or more of the polypeptide sequences pertaining to GM2 antigen recognition domains in combination with polypeptide sequences pertaining to GD2 antigen recognition domains, as described herein. Tn some embodiments, the disclosure provides chimeric antigen receptors (CARs) that bind to monosialoganglioside GM2 (ganglioside GM2 or GM2), compositions comprising the CARs and methods of utilizing same (e.g., for therapeutic and / or prophylactic treatment). In some implementations, a CAR comprises a single chain Fv (scFv) that binds to GM2, a transmembrane domain, and one or more intracellular signaling domains, wherein the scFv comprises at least one heavy chain variable (VH) region and at least one light chain variable (VL) region pair. In some aspects, a VH and VL pair is selected from the sequences listed in Table 1. In some embodiments, various combinations of CDRs of the VH and VL pairs are selected from the sequences listed in Table 1.

[0126] In some embodiments, a chimeric antigen receptor (CAR) that binds to ganglioside GM2 (GM2) comprises a single chain Fv (scFv) that binds to GM2, a transmembrane domain, and one or more intracellular signaling domains, wherein the scFv comprises at least one heavy chain variable (VH) region and at least one light chain variable (VL) region pair, and wherein(A) the VH and VL pair is selected from: i. a VH region comprising a heavy chain complementarity determining region 1 (CDR- Hl) having the amino acid sequence of SEQ ID NO: 1, a heavy chain complementarity determining region 2 (CDR-H2) having the amino acid sequence of SEQ ID NO: 2, a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of SEQ ID NO: 3, and a VL region comprising a light chain complementarity determining region 1 (CDR-L1) having the amino acid sequence of SEQ ID NO: 18, a light chain complementarity determining region 2 (CDR-L2) having the amino acid sequence of SEQ ID NO: 19, and a light chain complementarity determining region 3 (CDR-L3) having the amino acid sequence of SEQ ID NO: 20;ii. a VH region comprising a heavy chain complementarity determining region 1 (CDR- Hl) having the amino acid sequence of SEQ ID NO: 4, a heavy chain complementarity determining region 2 (CDR-H2) having the amino acid sequence of SEQ ID NO: 5, a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of SEQ ID NO: 6, and a VL region comprising a light chain complementarity determining region 1 (CDR-L1) having the amino acid sequence of SEQ ID NO: 21, a light chain complementarity determining region 2 (CDR-L2) having the amino acid sequence of SEQ ID NO: 22, and a light chain complementarity determining region 3 (CDR-L3) having the amino acid sequence of SEQ ID NO: 23; and iii. a VH region comprising a heavy chain complementarity determining region 1 (CDR- Hl) having the amino acid sequence of SEQ ID NO: 7, a heavy chain complementarity determining region 2 (CDR-H2) having the amino acid sequence of SEQ ID NO: 8, a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of SEQ ID NO: 9, and a VL region comprising a light chain complementarity determining region 1 (CDR-L1) having the amino acid sequence of SEQ ID NO: 24, a light chain complementarity determining region 2 (CDR-L2) having the amino acid sequence of SEQ ID NO: 25, and a light chain complementarity determining region 3 (CDR-L3) having the amino acid sequence of SEQ ID NO: 26; or(B) the VH comprises: a heavy chain complementarity determining region 1 (CDR-H1), a heavy chain complementarity determining region 2 (CDR-H2), and a heavy chain complementarity determining region 3 (CDR-H3), wherein the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are contained within the VH region of an amino acid sequence selected from SEQ ID NOs: 10-17, and the VL comprises: a light chain complementarity determining region 1 (CDR-L1), a light chain complementarity determining region 2 (CDR-L2), and a light chain complementarity determining region 3 (CDR-L3), and wherein the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are contained within the VL region of an amino acid sequence selected from SEQ ID NOs: 27- 35; or(C)the VH comprises: an amino acid sequence with at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence selected from SEQ ID NOs: 10-17; and the VL comprises: an amino acid sequence with at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence selected from SEQ ID NOs: 27-35; or(D) the VH region comprises: an amino acid sequence with at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence selected from SEQ ID NOs: 10, 11, 12, 13, 14, 15, 16, and 17, and the VL region comprises: an amino acid sequence with at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence selected from SEQ ID NOs: 27, 28, 29, 30, 31, 32, 33, 34 and 35, optionally wherein the CAR comprises one or more of a hinge domain, a spacer region, or one or more peptide linkers.

[0127] In some implementations, the single chain Fv (scFv) is selected from an amino acid sequence with at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence selected from SEQ ID NOs: 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 56, 57, and 58.

[0128] In some embodiments, the transmembrane domain is selected from a CD8 transmembrane domain, a CD28 transmembrane domain, a 4-IBB transmembrane domain, a CD3zeta-chain transmembrane domain, a PD-1 transmembrane domain, a DAP10 transmembrane domain, a CTLA-4 transmembrane domain, a CD 16a transmembrane domain, an 0X40 transmembrane domain, an NKG2D transmembrane domain; a CD4 transmembrane domain, a LAG-3 transmembrane domain, an 0X40 transmembrane domain, an NKp44 transmembrane domain, an ICOS transmembrane domain, a DAP12 transmembrane domain, a BTLA transmembrane domain, a KIR3DS 1 transmembrane domain, a 2B4 transmembrane domain, a DNAM-1 transmembrane domain, an FceRlgtransmembrane domain, a KIR2DS 1 transmembrane domain, and an NKp46 transmembrane domain.

[0129] In further aspects, the transmembrane domain is selected from an amino acid sequence with at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence selected from SEQ ID NOs: 36 and 37.

[0130] In some embodiments, the one or more intracellular signaling domains are each selected from a 4-1 BB intracellular signaling domain, a CD28 intracellular signaling domain, a CD3zeta-chain intracellular signaling domain, a ZAP70 (SRK) intracellular signaling domain, a ZAP40 intracellular signaling domain, a CD30 intracellular signaling domain, an 0X40 intracellular signaling domain, a CD27 intracellular signaling domain, a DAP 12 intracellular signaling domain, a KIR2DS1 intracellular signaling domain, a NKG2D intracellular signaling domain, a FceRlg intracellular signaling domain, a MyD88 intracellular signaling domain, an EAT-2 intracellular signaling domain, a DAP10 intracellular signaling domain, an ICOS intracellular signaling domain, a DNAM-1 intracellular signaling domain, a CD2 intracellular signaling domain, a CD8 intracellular signaling domain, a CD 16a intracellular signaling domain, a CD97 intracellular signaling domain, a CD 154 intracellular signaling domain, a GITR intracellular signaling domain, a NKp46 intracellular signaling domain, a 2B4 intracellular signaling domain, a CDl la-CD18 intracellular signaling domain, a NKp44 intracellular signaling domain, a KIR3DS1 intracellular signaling domain, an HVEM intracellular signaling domain, and / or a combination of two or more intracellular signaling domains.

[0131] In further aspects, the one or more intracellular signaling domains are each selected from an amino acid sequence with at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence selected from SEQ ID NOs: 38, 39, 40, and 41.

[0132] In some embodiments, the VH and VL of the scFv are separated by a peptide linker. In further embodiments, the scFV comprises the structure VH-L-VL or VL-L-VH, wherein VH is the heavy chain variable region, L is the peptide linker, and VL is the light chain variable region.

[0133] In some aspects, a CAR comprises one or more of a hinge domain, a spacer region, and / or one or more peptide linkers. In some embodiments, a CAR comprises a spacer region between the scFV and the transmembrane domain.

[0134] In some embodiments, the VH and VL pairs of the GM2 CARs of the disclosure are selected from the various sequences listed in Table 1. In some embodiments, various combinations of CDRs of the VH and VL pairs are selected from the sequences listed in Table 1. The various embodiments of the disclosure may include one or more of the polypeptide sequences pertaining to chimeric antigen receptor (CAR) sequences referenced below in Table 1. Various embodiments of the disclosure may also include one or more of the polypeptide sequences pertaining to GM2 antigen recognition domains in combination with polypeptide sequences pertaining to GD2 antigen recognition domains, as described herein.

[0135] Table 1: CAR amino acid sequences.*ARD - single-chain variable fragment (scFv) retaining GM2 and / or GD2 specificity

[0136] GD2 chimeric antigen receptors.

[0137] In some embodiments, the disclosure utilizes chimeric antigen receptors (CARs) that bind to GD2. Any antibody / immunoglobulin that binds with specificity to GD2 may be used to construct a CAR (e.g., using VH and VL regions to construct a fusion protein, scFv) for expression in immune cells (e.g., used in therapeutic methods of the disclosure). Examples of such antibodies / immunoglobulins include, but are not limited to, 14G2a, chl4.18, hul4.18K322A, m3F8, hu3F8-IgGl, hu3F8-IgG4, HM3F8, UNITUXIN, DMAb-20 or any other antibody that binds with specificity to GD2. In one embodiment, the CAR comprises a 14g2a scFv. A GD2 CAR may comprise a receptor incorporating variants within scFv of an anti-GD2 antibody (e.g., 14g2a scFv) generated to enhance affinity and / or diminish tonic signaling. The GD2 CAR may incorporate variable lengths of the hinge regions (e.g., between the scFv and the signaling domains) and / or varying transmembrane domains. The disclosure is not limited by the transmembrane domain used. Indeed, any transmembrane domain may be used including, but not limited to, all or part of the transmembrane domain of CD3-zeta chain (CD3Q, CD28, OX40 / CD134, 4-1BB / CD137 / TNFRSF9, FceRIy, ICOS / CD278, ILRB / CD122, IL-2RG / CD132, or CD40.

[0138] In some embodiments, a CAR expressed in immune cells is a CAR that specifically recognizes GD2 (e.g., that binds with specificity to an epitope of GD2 (e.g., GalNAc[31- 4(NeuAca2-8NeuAca2-3)Gal)). The disclosure is not limited by the type of GD2-specific CAR. Indeed, any CAR that binds with specificity to GD2 may be used to genetically modify (e.g., to be expressed in) immune cells. Exemplary CARs include, but are not limited to, CAR that includes the GD2 binding domain of a GD2-specific antibody such as, but not limited to, 14G2a, chl4.18, hul4.18K322A, m3F8, hu3F8-IgGl, hu3F8-IgG4, HM3F8, and DMAb-20. In certain embodiments, the antigen binding domain is a single-chain variable fragment (scFv) containing heavy and light chain variable regions that recognize or specifically bind an epitope of GD2. In some embodiments, the CAR further comprises a transmembrane domain (e.g., a T cell transmembrane domain (e.g., a CD28 transmembrane domain)) and a signaling domain comprising one or more immunoreceptor tyrosine-based activation motifs (ITAMs)(e.g., a T cell co-receptor signaling domain (e.g., a CD3-zeta chain (CD3 ). In some embodiments, the CAR comprises one or more co-stimulatory domains (e.g., domains that provide a second signal to stimulate T cell activation. The disclosure isnot limited by the type of co- stimulatory domain. Indeed, any co-stimulatory domain known in the art may be used including, but not limited to, CD28, OX40 / CD134, 4- 1BB / CD137 / TNFRSF9, FceRIy, ICOS / CD278, ILRB / CD122, IL-2RG / CD132, and CD40. In certain embodiments, the co-stimulatory domain is 4- IBB.

[0139] A modified cell of the disclosure can comprise an engineered nucleic acid integrated into the cell’s genome. An engineered cell can comprise an engineered nucleic acid capable of expression without integrating into the cell’s genome, for example, engineered with a transient expression system such as a plasmid or mRNA.

[0140] In some embodiments, polynucleotides encoding a GM2 CAR, a GM2-GD2 tandem CAR, and / or a GM2-GD2 bicistronic CAR are encoded by a single polynucleotide sequence in the engineered cells. For example, in some embodiments, the engineered cell comprises a single engineered nucleic acid comprising a polynucleotide sequence encoding a GM2 CAR, a GM2-GD2 tandem CAR, and / or a GM2-GD2 bicistronic CAR.

[0141] The disclosure is not limited by the type of cell or population of cells modified to express a CAR (e.g., to contain one or more engineered nucleic acids encoding a CAR). The cell or population of cells may be a T cell including, but not limited to, a CD4 T cell, a CD8 T cell, a gamma-delta T cell, a cytotoxic T lymphocyte (CTL), a Natural Killer (NK) cell, a Natural Killer T (NKT) cell, and a regulatory T cell. In some embodiments, the cell or population of cells is a dendritic cell, a tumor-infiltrating lymphocyte (TIL), a macrophage, a monocyte, a neutrophil, a B cell, a lymphoid cell, an eosinophil, a mast cell, a basophil, an erythrocyte, a myeloid cell, a platelet cell, a stem cell, and a mesenchymal stromal cell. In some embodiments, the cell is a stem cell. In some embodiments, the cell is modified to contain two or more separate CARs disclosed herein.

[0142] The cell or population of cells may be human cell(s) (e.g., a primary T cell, tumor infiltrating lymphocyte, hematopoietic stem cell (HSC) (e.g., induced pluripotent stem cell), or natural killer cell). In some embodiments, the cell is derived from a subject to be treated with the compositions and methods dis-closed herein (e.g., autologous cell). In other embodiments, the cell is derived from donor (e.g., an allogenic cell). In some embodiments, the cell or population of cells are isolated from a subject using methods known in the art including, but not limited to, cell sorting techniques based on cell-surface marker expression, FACS sorting, positive isolation techniques, negative isolation techniques, magnetic isolation, and combinations thereof. Cells may be cultured ex vivo (e.g., a primary cell may be isolated from a subject and cultured outside of the subject). Cells can be engineered toproduce a GM2 CAR, a GM2-GD2 tandem CAR, and / or a GM2-GD2 bicistronic CAR through introduction (delivery) of one or more nucleic acid molecules of the disclosure comprising a promoter and an exogenous polynucleotide sequence encoding a GM2 CAR, a GM2-GD2 tandem CAR, and / or a GM2-GD2 bicistronic CAR into the cell’s cytosol and / or nucleus. For example, nucleic acid expression cassettes encoding the GM2 CAR, a GM2- GD2 tandem CAR, and / or a GM2-GD2 bicistronic CAR can be any of the engineered nucleic acids described herein. Delivery methods include, but are not limited to, viral-mediated delivery, lipid-mediated transfection, nanoparticle delivery, electroporation, sonication, and cell membrane deformation by physical means. In some embodiments, the GM2-specific and / or GD2-speicific recognition moieties (e.g., CARs) are directly delivered to T cells or other immune cells in vivo in a patient. While the disclosure is not limited to any particular method of in vivo delivery. Indeed, a variety of methods may be used including but not limited to any of the following: lipid nanoparticles containing DNA, RNA, or retrotransposons, infusion of wildtype lentivirus or retrovirus or adenovirus or adeno- associated virus or niphavirus or pseudotyped lentivirus or retrovirus or adenovirus or adeno- associated virus or niphavirus with specific tropism for T cells or other immune cells, or infusion of viral like particles derived from lentivirus, retrovirus, or adenovirus or adeno- associated virus or niphavirus or other viruses. One skilled in the art will appreciate the choice of delivery method can depend on the specific cell type to be engineered.

[0143] Pharmaceutical compositions.

[0144] The present disclosure further provides pharmaceutical compositions (e.g., comprising the therapeutic compositions described herein). The pharmaceutical compositions of the present disclosure may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated.

[0145] Pharmaceutical compositions of the present disclosure include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids.

[0146] The pharmaceutical formulations of the present disclosure, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general the formulations are prepared by uniformly and intimately bringing into associationthe active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

[0147] The compositions of the present disclosure may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present disclosure may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances that increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and / or dextran. The suspension may also contain stabilizers.

[0148] In one embodiment of the present disclosure the pharmaceutical compositions may be formulated and used as foams. Pharmaceutical foams include formulations such as, but not limited to, emulsions, microemulsions, creams, jellies and liposomes. While basically similar in nature these formulations vary in the components and the consistency of the final product.

[0149] Agents that enhance uptake of oligonucleotides at the cellular level may also be added to the pharmaceutical and other compositions of the present disclosure. For example, cationic lipids, such as lipofectin (U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (WO 97 / 30731), also enhance the cellular uptake of oligonucleotides.

[0150] The compositions of the present disclosure may additionally contain other adjunct components conventionally found in pharmaceutical compositions. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present disclosure, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present disclosure. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and / or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.

[0151] Certain embodiments of the disclosure provide pharmaceutical compositions containing (a) one or more antibodies and / or CAR T cells specific for GM2 and / or GM2 and GD2 and (b) one or more other chemotherapeutic agents. Examples of suchchemotherapeutic agents include, but are not limited to, anticancer drugs such as daunorubicin, dactinomycin, doxorubicin, bleomycin, mitomycin, nitrogen mustard, chlorambucil, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine (CA), 5-fluorouracil (5-FU), floxuridine (5-FUdR), methotrexate (MTX), colchicine, vincristine, vinblastine, etoposide, teniposide, cisplatin and diethylstilbestrol (DES). Anti-inflammatory drugs, including but not limited to nonsteroidal anti-inflammatory drugs and corticosteroids, and antiviral drugs, including but not limited to ribivirin, vidarabine, acyclovir and ganciclovir, may also be combined in compositions of the disclosure. Other chemotherapeutic agents are also within the scope of this disclosure. Two or more combined compounds may be used together or sequentially.

[0152] Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. The administering physician can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary and can generally be estimated based on EC50S found to be effective in in vitro and in vivo animal models or based on the examples described herein. In general, dosage is from 0.01 pg to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly. The treating physician can estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the subject undergo maintenance therapy to prevent the recurrence of the disease state, wherein treatment is administered in maintenance doses, ranging from 0.01 pg to 100 g per kg of body weight, once or more daily, to once every 20 years.

[0153] In certain embodiments, the disclosure also provides methods for treating or delaying the progression of cancer in an individual comprising administering to the individual an effective amount of immune cells genetically modified to express GM2-, a tandem GM2- GD2-, and / or a bicistronic GM2-GD2 CARs. In some embodiments, the treatment results in a sustained response in the individual after cessation of the treatment. The methods described herein may find use in treating conditions where enhanced immunogenicity is desired such as increasing tumor immunogenicity for the treatment of cancer. Also provided herein are methods of enhancing immune function in an individual having cancer comprising administering to the individual an effective amount of immune cells genetically modified toexpress GM2-, a tandem GM2-GD2-, and / or a bicistronic GM2-GD2 CARs. Any immune cell (e.g., any T cell (e.g., a CD3+ T cell)) genetically modified to express a GD2-specific CAR known in the art or described herein may be used in these methods. In some embodiments, the individual is a human.

[0154] In some embodiments, the individual has cancer that is resistant (e.g., has been demonstrated to be resistant) to one or more other forms of anti-cancer treatment (e.g., anti- GD2 therapy, chemotherapy, immunotherapy, etc.). In some embodiments, resistance includes recurrence of cancer or refractory cancer. Recurrence may refer to the reappearance of cancer, in the original site or a new site, after treatment. In some embodiments, resistance includes progression of the cancer during treatment with chemotherapy. In some embodiments, resistance includes cancer that does not respond to traditional or conventional treatment with a chemotherapeutic agent. The cancer may be resistant at the beginning of treatment or it may become resistant during treatment. In some embodiments, the cancer is at early stage or at late stage.

[0155] In some embodiments, the disclosure provides a method of treating a mammal having a disease associated with increased expression of ganglioside GM2 and / or reduced expression of ganglioside GD2, the method comprising administering to the mammal an effective amount of a population of T cells genetically modified to express a nucleic acid molecule encoding a chimeric antigen receptor, the chimeric antigen receptor (CAR) comprising: an antigen binding domain, a transmembrane domain, and at least one intracellular signaling domain, wherein the antigen binding domain comprises at least one heavy chain variable (VH) region and at least one light chain variable (VL) region, wherein the at least one heavy chain variable (VH) region comprises a heavy chain complementarity determining region 1 (CDR-H1), a heavy chain complementarity determining region 2 (CDR-H2), and a heavy chain complementarity determining region 3 (CDR-H3) having an amino acid sequence with at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence selected from SEQ ID NOs: 10-17; wherein the at least one light chain variable (VL) region comprises a light chain complementarity determining region 1 (CDR-L1), a light chain complementarity determining region 2 (CDR-L2), and a light chain complementarity determining region 3 (CDR-L3) having an amino acid sequence with at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence selected from SEQ ID NOs: 27-35. In some embodiments, the chimeric antigen receptor (CAR) comprises or consists ofthe amino acid sequence set forth as SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 12 or SEQ ID NO: 13 or SEQ ID NO: 14 or SEQ ID NO: 15 or SEQ ID NO: 16 or SEQ ID NO: 17, and wherein the chimeric antigen receptor (CAR) specifically binds to ganglioside GM2 and / or ganglioside GD2. In some embodiments, the chimeric antigen receptor (CAR) comprises or consists of the amino acid sequence the amino acid sequence set forth as SEQ ID NO: 27 or SEQ ID NO: 28 or SEQ ID NO: 29 or SEQ ID NO: 30 or SEQ ID NO: 31 or SEQ ID NO: 32 or SEQ ID NO: 33 or SEQ ID NO: 34 or SEQ ID NO: 35, and wherein the chimeric antigen receptor (CAR) specifically binds to ganglioside GM2 and / or ganglioside GD2. In further embodiments, the antigen binding domain of the chimeric antigen receptor (CAR) is a scFv, particularly wherein a) the scFv comprises or consists of an amino acid sequence with at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence selected from SEQ ID NOs: 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 56, 57, and 58.

[0156] Therapeutic applications.

[0157] Antibodies and / or cells or populations of cells (e.g., T cells) disclosed herein can be administered to a subject for therapeutic purposes, for example, treatment of a cancer or tumor such as a solid tumor targeted by the CAR construct expressed by the cells (e.g., therapeutic CAR-T cells). As reported herein, GM2 was discovered to be a viable target for the treatment of cancers / tumors and targeting the cancer / tumor cells with GM2 CAR T cells (e.g., GM2 CARs, GM2-GD2 tandem CARs, and / or GM2-GD2 bicistronic CARs) improved T cell persistence (e.g., decreased T cell exhaustion observed with GD2 CARs), increased cytokine secretion, and / or enhanced CAR potency thereby leading to improved anti-tumor efficacy as observed in animal models (See Example 1).

[0158] The step of administering may include the placement e.g., transplantation) of the therapeutic T cells into a subject by a method or route that results in at least partial localization of the therapeutic T cells at a desired site, such as a tumor site, such that a desired effect(s) can be produced. Therapeutic T cells can be administered by any appropriate route that results in delivery to a desired location in the subject where at least a portion of the implanted cells or components of the cells remain viable. The period of viability of the cells after administration to a subject can be as short as a few hours, e.g., twenty-four hours, to a few days, to as long as several years, or even the lifetime of the subject, i.e., long-term engraftment. For example, in some embodiments, an effective amount of the therapeutic Tcells can be administered via a systemic route of administration, such as an intraperitoneal or intravenous route.

[0159] In some embodiments, the therapeutic T cells are administered systemically, which refers to the administration of a population of cells other than directly into a target site, tissue, or organ, such that it enters, instead, the subject’s circulatory system and, thus, is subject to metabolism and other like processes. Suitable modes of administration include injection, infusion, instillation, or ingestion. Injection includes, without limitation, intravenous, intramuscular, intra-arterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intracerebro spinal, and intrastemal injection and infusion. In some embodiments, the route is intravenous.

[0160] A subject may be any subject for whom diagnosis, treatment, or therapy is desired. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some instances, the human patient has a cancer involving cancer cells expressing GM2. CAR-T cells expressing an anti-GM2 CAR (e.g., disclosed herein) may be used to treat such a patient.

[0161] As described herein, the therapeutic T cells may be autologous (“self’) to the subject, i.e., the cells are from the same subject. Alternatively, the therapeutic T cells can be non- autologous (“non-self,” e.g., allogeneic, syngeneic or xenogeneic) to the subject.“Allogeneic” means that the therapeutic T cells are not derived from the subject who receives the treatment but from different individuals (donors) of the same species as the subject. A donor is an individual who is not the subject being treated. A donor is an individual who is not the patient. In some embodiments, a donor is an individual who does not have or is not suspected of having the cancer being treated. In some embodiments, multiple donors, e.g., two or more donors, are used.

[0162] An effective amount refers to the amount of a population of engineered T cells needed to prevent or alleviate at least one or more signs or symptoms of a medical condition (e.g., cancer), and relates to a sufficient amount of a composition to provide the desired effect, e.g., to treat a subject’s signs or symptoms of cancer. An effective amount also includes an amount sufficient to prevent or delay the development of a symptom of the disease, alter the course of a symptom of the disease (for example but not limited to, slow the progression of a symptom of the disease), or reverse a symptom of the disease. It is understood that for any given case, an appropriate effective amount can be determined by one of ordinary skill in the art using routine experimentation.

[0163] The efficacy of a treatment using the therapeutic T cells disclosed herein can be determined by a skilled clinician. A treatment is considered “effective”, if any one or all of the signs or symptoms of, as but one example, levels of functional target are altered in a beneficial manner (e.g., increased by at least 10%), or other clinically accepted symptoms or markers of disease e.g., cancer) are improved or ameliorated. Efficacy can also be measured by failure of a subject to worsen as assessed by hospitalization or need for medical interventions (e.g. , progression of the disease is halted or at least slowed). Methods of measuring these indicators are known to those of skill in the art and / or described herein. Treatment includes any treatment of a disease in subject and includes: (1) inhibiting the disease, e.g., arresting, or slowing the progression of symptoms; or (2) relieving the disease, e.g., causing regression of symptoms; and (3) preventing or reducing the likelihood of the development of symptoms.

[0164] Combination therapies are also encompassed by the present disclosure. For example, the therapeutic T cells disclosed herein may be co-used with other therapeutic agents, for treating the same indication, or for enhancing efficacy of the therapeutic T cells and / or reducing side effects of the therapeutic T cells. One or more of the compositions described herein may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated. For example, a composition of the present disclosure (e.g., GM2 CAR T cells, GM2-GD2 tandem CAR T cells, and / or GM2-GD2 bicistronic CAR T cells) may be co- administered or ordered to be combined with one or more other types of cancer treatment including, but not limited to, chemotherapy, radiation, and / or surgery. Combination of two or more treatments may occur at the same time, prior to, and / or after administration of one of the two or more types of treatment.

[0165] Thus, some embodiments of the present disclosure provide methods for administering an effective amount of CAR T cells and / or antibodies and at least one additional therapeutic agent (including, but not limited to, chemotherapeutic antineoplastics, apoptosis-modulating agents, antimicrobials, antivirals, antifungals, and anti-inflammatory agents) and / or therapeutic technique (e.g., surgical intervention, and / or radiotherapies). In a particular embodiment, the additional therapeutic agent(s) is an anticancer agent.

[0166] A number of suitable anticancer agents are contemplated for use in, or in combination with, the methods of the present disclosure. Indeed, the present disclosure contemplates, but is not limited to, administration of numerous anticancer agents such as: agents that induce apoptosis; polynucleotides (e.g., anti-sense, ribozymes, siRNA); polypeptides (e.g., enzymesand antibodies); biological mimetics; alkaloids; alkylating agents; antitumor antibiotics; antimetabolites; hormones; platinum compounds; monoclonal or polyclonal antibodies (e.g., antibodies conjugated with anticancer drugs, toxins, defensins), toxins; radionuclides; biological response modifiers (e.g., interferons (e.g., IFN-a) and interleukins (e.g., IL-2)); adoptive immunotherapy agents; hematopoietic growth factors; agents that induce tumor cell differentiation (e.g., all-trans-retinoic acid); gene therapy reagents (e.g., antisense therapy reagents and nucleotides); tumor vaccines; angiogenesis inhibitors; proteosome inhibitors: NF-KB modulators; anti-CDK compounds; HDAC inhibitors; and the like. Numerous other examples of chemotherapeutic compounds and anticancer therapies suitable for coadministration with the disclosed compounds are known to those skilled in the art.

[0167] In certain embodiments, anticancer agents comprise agents that induce or stimulate apoptosis. Agents that induce apoptosis include, but are not limited to, radiation (e.g., X- rays, gamma rays, UV); tumor necrosis factor (TNF)-related factors (e.g., TNF family receptor proteins, TNF family ligands, TRAIL, antibodies to TRAIL-R1 or TRAIL-R2); kinase inhibitors (e.g., epidermal growth factor receptor (EGFR) kinase inhibitor, vascular growth factor receptor (VGFR) kinase inhibitor, fibroblast growth factor receptor (FGFR) kinase inhibitor, platelet-derived growth factor receptor (PDGFR) kinase inhibitor, and Bcr- Abl kinase inhibitors (such as GLEEVEC®)); antisense molecules; antibodies (e.g., HERCEPTIN®, RITUXAN®, ZEVALIN ®, and AVASTIN®); anti-estrogens (e.g., raloxifene and tamoxifen); anti- androgens (e.g., flutamide, bicalutamide, finasteride, aminoglutethamide, ketoconazole, and corticosteroids); cyclooxygenase 2 (COX-2) inhibitors (e.g., celecoxib, meloxicam, NS-398, and non-steroidal anti-inflammatory drugs (NSAIDs)); anti-inflammatory drugs (e.g., butazolidin, DECADRON®, DELTASONE®, dexamethasone, dexamethasone intensol, DEXONE, HEXADROL®, hydroxychloroquine, METICORTEN®, ORADEXON®, ORASONE, oxyphenbutazone, PEDIAPRED®, phenylbutazone, PLAQUENIL®, prednisolone, prednisone, PRELONE®, and TANDEARIL); and cancer chemotherapeutic drugs (e.g., irinotecan (CAMPTOSAR®), CPT-11, fhidarabine (FLUDARA®), dacarbazine (DTIC), dexamethasone, mitoxantrone, MYLOTARG, VP- 16, cisplatin, carboplatin, oxaliplatin, 5-FU, doxorubicin, gemcitabine, bortezomib, gefitinib, bevacizumab, TAXOTERE® or TAXOL®); cellular signaling molecules; ceramides and cytokines; staurosporine, and the like.

[0168] In still other embodiments, the GM2 directed therapeutic compositions and methods of the present disclosure are used together with at least one anti-hyperproliferative orantineoplastic agent selected from alkylating agents, antimetabolites, and natural products (e.g., herbs and other plant and / or animal derived compounds).

[0169] Alkylating agents suitable for use in the present compositions and methods include, but are not limited to: 1) nitrogen mustards (e.g., mechlorethamine, cyclophosphamide, ifosfamide, melphalan (L-sarcolysin); and chlorambucil); 2) ethylenimines and methylmelamines (e.g., hexamethylmelamine and thiotepa); 3) alkyl sulfonates (e.g., busulfan); 4) nitrosoureas (e.g., carmustine (BCNU); lomustine (CCNU); semustine (methyl- CCNU); and streptozocin (streptozotocin)); and 5) triazenes (e.g., dacarbazine (DTIC; dimethyltriazenoimid-azolecarboxamide).

[0170] In some embodiments, antimetabolites suitable for use in the present compositions and methods include, but are not limited to: 1) folic acid analogs (e.g., methotrexate (amethopterin)); 2) pyrimidine analogs (e.g., fluorouracil (5-fluorouracil; 5-FU), floxuridine (fluorode-oxyuridine; FudR), and cytarabine (cytosine arabinoside)); and 3) purine analogs (e.g., mercaptopurine (6-mercaptopurine; 6-MP), thioguanine (6-thioguanine; TG), and pentostatin (2 ’ -deoxy coformy cin)) .

[0171] In still further embodiments, chemotherapeutic agents suitable for use in the compositions and methods of the present disclosure include, but are not limited to: 1) vinca alkaloids (e.g., vinblastine (VBL), vincristine); 2) epipodophyllotoxins (e.g., etoposide and teniposide); 3) antibiotics (e.g., dactinomycin (actinomycin D), daunorubicin (daunomycin; rubidomycin), doxorubicin, bleomycin, plicamycin (mithramycin), and mitomycin (mitomycin C)); 4) enzymes (e.g., L-asparaginase); 5) biological response modifiers (e.g., interferon-alfa); 6) platinum coordinating complexes (e.g., cisplatin (cis-DDP) and carboplatin); 7) anthracenediones (e.g., mitoxantrone); 8) substituted ureas (e.g., hydroxyurea); 9) methylhydrazine derivatives (e.g., procarbazine (N-methylhydrazine;MIH)); 10) adrenocortical suppressants (e.g., mitotane (o,p’-DDD) and aminoglutethimide); 11) adrenocorticosteroids (e.g., prednisone); 12) progestins (e.g., hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrol acetate); 13) estrogens (e.g., diethylstilbestrol and ethinyl estradiol); 14) antiestrogens (e.g., tamoxifen); 15) androgens (e.g., testosterone propionate and fluoxymesterone); 16) antiandrogens (e.g., fhitamide): and 17) gonadotropin-releasing hormone analogs (e.g., leuprolide).

[0172] Any oncolytic agent that is routinely used in a cancer therapy context may also be used in the compositions and methods of the present disclosure. For example, the U.S. Food and Drug Administration maintains a formulary of oncolytic agents approved for use in theUnited States. International counterpart agencies to the U.S. F.D.A. maintain similar formularies.

[0173] Anticancer agents further include compounds which have been identified to have anticancer activity. Examples include, but are not limited to, 3-AP, 12-0- tetradecanoylphorbol-13-acetate, 17AAG, 852A, ABI-007, ABR-217620, ABT-751, ADI- PEG 20, AE-941, AG-013736, AGRO 100, alanosine, AMG 706, antibody G250, antineoplastons, AP23573, apaziquone, APC8015, atiprimod, ATN-161, atrasenten, azacitidine, BB-10901 , BCX-1777, hevacizumab, BG00001 , bicalutamide, BMS 247550, bortezomib, bryostatin-1, buserelin, calcitriol, CCI-779, CDB-2914, cefixime, cetuximab, CG0070, cilengitide, clofarabine, combretastatin A4 phosphate, CP-675,206, CP-724,714, CpG 7909, curcumin, decitabine, DENSPM, doxercalciferol, E7070, E7389, ecteinascidin 743, efaproxiral, eflomithine, EKB-569, enzastaurin, erlotinib, exisulind, fenretinide, flavopiridol, fludarabine, flutamide, fotemustine, FR901228, G17DT, galiximab, gefitinib, genistein, glufosfamide, GTI-2040, histrelin, HKI-272, homoharringtonine, HSPPC-96, hu 14. 18 -interleukin-2 fusion protein, HuMax-CD4, iloprost, imiquimod, infliximab, interleukin- 12, IP1-504, irofulven, ixabepilone, lapatinib, lenalidomide, lestaurtinib, leuprolide, LMB-9 immunotoxin, lonafamib, luniliximab, mafosfamide, MB07133, MDX- 010, MLN2704, monoclonal antibody 3F8, monoclonal antibody J591, motexafin, MS-275, MVA-MUC1-IL2, nilutamide, nitrocamptothecin, nolatrexed dihydrochloride, nolvadex, NS- 9, O6-benzylguanine, oblimersen sodium, ONYX-015, oregovomab, OSI-774, panitumumab, paraplatin, PD-0325901, pemetrexed, PHY906, pioglitazone, pirfenidone, pixantrone, PS- 341, PSC 833, PXD101, pyrazoloacridine, R115777, RAD001, ranpirnase, rebeccamycin analogue, rhuAngiostatin protein, rhuMab 2C4, rosiglitazone, rubitecan, S-l, S-8184, satraplatin, SB-, 15992, SGN-0010, SGN-40, sorafenib, SR31747A, ST1571, SU011248, suberoylanilide hydroxamic acid, suramin, talabostat, talampanel, tariquidar, temsirolimus, TGFa-PE38 immunotoxin, thalidomide, thymalfasin, tipifarnib, tirapazamine, TLK286, trabectedin, trimetrexate glucuronate, TroVax, UCN-1, valproic acid, vinflunine, VNP40101M, volociximab, vorinostat, VX-680, ZD1839, ZD6474, zileuton, and zosuquidar trihydrochloride.

[0174] For a more detailed description of anticancer agents and other therapeutic agents, those skilled in the art are referred to any number of instructive manuals including, but not limited to, the Physician’s Desk Reference and to Goodman and Gilman's “Pharmaceutical Basis of Therapeutics” tenth edition, Eds. Hardman et al., 2002.

[0175] The present disclosure provides methods for administering GM2 directed therapeutic compositions and methods of the disclosure with (e.g., before, during, or after) radiation therapy. The disclosure is not limited by the types, amounts, or delivery and administration systems used to deliver the therapeutic dose of radiation to a subject. For example, a subject may receive photon radiotherapy, particle beam radiation therapy, other types of radiotherapies, and combinations thereof. In some embodiments, the radiation is delivered to a subject using a linear accelerator. In still other embodiments, the radiation is delivered using a gamma knife.

[0176] The source of radiation can be external or internal to a subject. External radiation therapy is most common and involves directing a beam of high-energy radiation to a tumor site through the skin using, for instance, a linear accelerator. While the beam of radiation is localized to the tumor site, it is nearly impossible to avoid exposure of normal, healthy tissue. However, external radiation is usually well tolerated. Internal radiation therapy involves implanting a radiation-emitting source, such as beads, wires, pellets, capsules, particles, and the like, inside the body at or near the tumor site including the use of delivery systems that specifically target cancer cells (e.g., using particles attached to cancer cell binding ligands). Such implants can be removed following treatment, or left in the body inactive. Types of internal radiation therapy include, but are not limited to, brachytherapy, interstitial irradiation, intracavity irradiation, radioimmunotherapy, and the like.

[0177] A subject may optionally receive radiosensitizers (e.g., metronidazole, misonidazole, intra-arterial Budr, intravenous iododeoxyuridine (ludR), nitroimidazole, 5-substituted-4- nitroimidazoles, 2H-isoindolediones, [[(2-bromoethyl)-amino]methyl]-nitro-lH-imidazole-l- ethanol, nitroaniline derivatives, DNA-affinic hypoxia selective cytotoxins, halogenated DNA ligand, 1,2,4 benzotriazine oxides, 2-nitroimidazole derivatives, fluorine-containing nitroazole derivatives, benzamide, nicotinamide, acridine-intercalator, 5-thiotretrazole derivative, 3-nitro-l,2,4-triazole, 4, 5-dinitroimidazole derivative, hydroxylated texaphrins, cisplatin, mitomycin, tiripazamine, nitrosourea, mercaptopurine, methotrexate, fluorouracil, bleomycin, vincristine, carboplatin, epirubicin, doxorubicin, cyclophosphamide, vindesine, etoposide, paclitaxel, heat (hyperthermia), and the like), radioprotectors (e.g., cysteamine, aminoalkyl dihydrogen phosphorothioates, amifostine (WR 2721), IL-1, IL-6, and the like). Radiosensitizers enhance the killing of tumor cells. Radioprotectors protect healthy tissue from the harmful effects of radiation.

[0178] Any type of radiation can be administered to a subject, so long as the dose of radiation is tolerated without unacceptable negative side-effects. Suitable types of radiotherapyinclude, for example, ionizing (electromagnetic) radiotherapy (e.g., X-rays or gamma rays) or particle beam radiation therapy (e.g., high linear energy radiation). Ionizing radiation is defined as radiation comprising particles or photons that have sufficient energy to produce ionization, i.e., gain or loss of electrons (as described in, for example, U.S. 5,770,581 incorporated herein by reference in its entirety). The effects of radiation can be at least partially controlled by the clinician. In one embodiment, the dose of radiation is fractionated for maximal target cell exposure and reduced toxicity.

[0179] In one embodiment, the total dose of radiation administered to a subject is about .01 Gray (Gy) to about 100 Gy. In another embodiment, about 10 Gy to about 65 Gy (e.g., about 15 Gy, 20 Gy, 25 Gy, 30 Gy, 35 Gy, 40 Gy, 45 Gy, 50 Gy, 55 Gy, or 60 Gy) are administered over the course of treatment. While in some embodiments a complete dose of radiation can be administered over the course of one day, the total dose is ideally fractionated and administered over several days. Desirably, radiotherapy is administered over the course of at least about 3 days, e.g., at least 5, 7, 10, 14, 17, 21, 25, 28, 32, 35, 38, 42, 46, 52, or 56 days (about 1-8 weeks). Accordingly, a daily dose of radiation will comprise approximately 1-5 Gy (e.g., about 1 Gy, 1.5 Gy, 1.8 Gy, 2 Gy, 2.5 Gy, 2.8 Gy, 3 Gy, 3.2 Gy, 3.5 Gy, 3.8 Gy, 4 Gy, 4.2 Gy, or 4.5 Gy), or 1-2 Gy (e.g., 1.5-2 Gy). The daily dose of radiation should be sufficient to induce destruction of the targeted cells. If stretched over a period, in one embodiment, radiation is not administered every day, thereby allowing the animal to rest and the effects of the therapy to be realized. For example, radiation desirably is administered on 5 consecutive days, and not administered on 2 days, for each week of treatment, thereby allowing 2 days of rest per week. However, radiation can be administered 1 day / week, 2 days / week, 3 days / week, 4 days / week, 5 days / week, 6 days / week, or all 7 days / week, depending on the animal’s responsiveness and any potential side effects. Radiation therapy can be initiated at any time in the therapeutic period. In one embodiment, radiation is initiated in week 1 or week 2, and is administered for the remaining duration of the therapeutic period. For example, radiation is administered in weeks 1-6 or in weeks 2-6 of a therapeutic period comprising 6 weeks for treating, for instance, a solid tumor. Alternatively, radiation is administered in weeks 1-5 or weeks 2-5 of a therapeutic period comprising 5 weeks. These exemplary radiotherapy administration schedules are not intended, however, to limit the present disclosure.

[0180] Antimicrobial therapeutic agents may also be used as therapeutic agents in the present disclosure. Any agent that can kill, inhibit, or otherwise attenuate the function of microbial organisms may be used, as well as any agent contemplated to have such activities.Antimicrobial agents include, but are not limited to, natural and synthetic antibiotics, antibodies, inhibitory proteins (e.g., defensins), antisense nucleic acids, membrane disruptive agents and the like, used alone or in combination. Indeed, any type of antibiotic may be used including, but not limited to, antibacterial agents, antiviral agents, antifungal agents, and the like.

[0181] In some embodiments of the present disclosure, immune cells (e.g., T cells (e.g., CD8 and / or CD4 T cells)) genetically modified to express CARs disclosed herein and one or more therapeutic agents or anticancer agents are administered to an animal under one or more of the following conditions: at different periodicities, at different durations, at different concentrations, by different administration routes, etc. In some embodiments, immune cells (e.g., T cells (e.g., CD8 and / or CD4 T cells)) genetically modified to express CARs disclosed herein are administered prior to the therapeutic or anticancer agent, e.g., 0.5, 1, 2, 3, 4, 5, 10, 12, 18 hours or more, 1, 2, 3, 4, 5, 6 or more days, or 1, 2, 3, 4, 5, 6 or more weeks prior to the administration of the therapeutic or anticancer agent. In some embodiments, immune cells (e.g., T cells (e.g., CD8 and / or CD4 T cells)) genetically modified to express CARs disclosed herein are administered after the therapeutic or anticancer agent, e.g., 0.5, 1, 2, 3, 4, 5, 10, 12, 18 or more hours, 1, 2, 3, 4, 5, 6 or more days, or 1, 2, 3, 4, 5, 6, or more weeks after the administration of the anticancer agent. In some embodiments, immune cells (e.g., T cells (e.g., CD8 and / or CD4 T cells)) genetically modified to express CARs disclosed herein and the therapeutic or anticancer agent are administered concurrently but on different schedules, e.g., modified immune cells are administered daily while the therapeutic or anticancer agent is administered once a week, once every two weeks, once every three weeks, once every four weeks, or more. In other embodiments, modified immune cells are administered once a week while the therapeutic or anticancer agent is administered daily, once a week, once every two weeks, once every three weeks, once every four weeks, or more.

[0182] Compositions within the scope of this disclosure include all compositions wherein the immune cells (e.g., T cells (e.g., CD8 and / or CD4 T cells)) genetically modified to express CARs disclosed herein are contained in an amount which is effective to achieve its intended purpose. While individual needs vary, determination of optimal ranges of effective amounts of each component is within the skill of the art. In one non-limiting example, immune cells (e.g., T cells (e.g., CD8 and / or CD4 T cells)) genetically modified to express CARs disclosed herein may be administered to mammals, e.g. humans, in order to provide the human between 1000 and 1010modified immune cells per day (e.g., for treating cancer). In another embodiment, between 1000 and 1010modified immune cells are administered to treat,ameliorate, or prevent cancer (e.g., prevent metastasis, recurrence, and / or progression of cancer). The unit dose may be administered in one or more administrations one or more times daily (e.g., for 1, 2, 3, 4, 5, 6, or more days or weeks).

[0183] Modified immune cells may be administered as part of a pharmaceutical preparation containing suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing and / or administration of the modified cells into preparations which can be used pharmaceutically. Modified immune cells and / or pharmaceutical preparations containing the same, or other treatments used in concurrently therewith, may be administered intravenously, intramuscularly, subcutaneously, intratumorally, intraperitoneally Jntrathecally, or intraventricularly.. An effective amount of modified immune cells and / or pharmaceutical preparations containing the same may be administered for prevention or treatment of disease. The appropriate dosage may be determined based on the type of disease to be treated, the type of modified immune cell, the severity and course of the disease, the clinical condition of the individual, the individual's clinical history and response to the treatment, and the discretion of the attending physician.

[0184] The efficacy of any of the methods described herein (e.g., treatment with immune cells engineered to express CARs disclosed herein alone in in combination with one or more chemotherapeutic agents described herein) may be tested in various models known in the art, such as clinical or pre-clinical models. Suitable pre-clinical models are exemplified herein. For any exemplary model, after developing tumors, mice are randomly recruited into treatment groups receiving treatment or control treatment. Tumor size (e.g., tumor volume) is measured during the course of treatment, and overall survival rate is also monitored.

[0185] In some embodiments, a sample is obtained prior to treatment with immune cells engineered to express CARs disclosed herein (e.g., alone or in combination with another therapy described herein). In some embodiments, the sample is a tissue sample (e.g., formalin-fixed and paraffin-embedded (FFPE), archival, fresh or frozen). In some embodiments, the sample is whole blood. In some embodiments, the whole blood comprises immune cells, circulating tumor cells and any combinations thereof.

[0186] In some embodiments, the present disclosure provides kits for the detection and characterization of cancer (e.g., neuroblastomas, Ewing sarcoma, or other GD - heterogeneous expressing cancer). In some embodiments, the kits contain antibodies specific for ST8SIA1, GD2 and / or GM2, in addition to detection reagents and buffers. In other embodiments, the kits contain reagents specific for the detection of ST8SIA1 mRNA or cDNA, GD2 mRNA or cDNA, and / or GM2 mRNA or cDNA (e.g., oligonucleotide probes orprimers). In preferred embodiments, the kits contain all of the components necessary to perform a detection assay, including all controls, directions for performing assays, and any necessary software for analysis and presentation of results.

[0187] The present disclosure also provides kits for use in producing the genetically engineered T cells, the therapeutic T cells, and for therapeutic uses.

[0188] In some embodiments, a kit provided herein may comprise a population of genetically engineered T cells as disclosed herein, and one or more components for producing the therapeutic T cells as also disclosed herein. Such components may comprise a nucleic acid coding for a CAR construct of interest. In some instances, the donor template may be carried by a viral vector such as a retroviral vector, a lentiviral vector, or other vector described herein or known in the art. In yet other embodiments, the kit disclosed herein may comprise a population of therapeutic T cells as disclosed for the intended therapeutic purposes. Any of the kits disclosed herein may comprise instructions for making the therapeutic T cells, or therapeutic applications of the therapeutic T cells. In some embodiments, the included instructions may comprise a description of how to introduce a nucleic acid encoding a CAR construct into the T cells for making therapeutic T cells.

[0189] In some embodiments, a kit as disclosed herein may comprise a population of genetically engineered T cells (e.g., CAR-T cells) for use to eliminate undesired cells targeted by the CAR construct (e.g., for treatment of cancer such as a solid tumor). Such a kit may comprise one or more containers in which the genetically engineered T cells can be placed. The kit may further comprise instructions for administration of the therapeutic T cells as disclosed herein to achieve the intended activity, e.g., eliminating disease cells targeted by the CAR expressed on the therapeutic T cells. Alternatively, or in addition, the kit may further comprise a description of selecting a subject suitable for treatment based on identifying whether the subject is in need of the treatment. The instructions relating to the use of the therapeutic T cells described herein generally include information as to dosage, dosing schedule, and route of administration for the intended treatment. The containers may be unit doses, bulk packages e.g., multi-dose packages) or sub-unit doses. Instructions supplied in the kits of the disclosure are typically written instructions on a label or package insert. The label or package insert indicates that the therapeutic T cells are used for treating, delaying the onset, and / or alleviating a disease or disorder in a subject.

[0190] The kits provided herein are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, and the like. Also contemplated are packages for use in combination with a specific device, such as an infusion device foradministration of the therapeutic T cells. A kit may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The container may also have a sterile access port.

[0191] Kits optionally may provide additional components such as buffers and interpretive information. Normally, the kit comprises a container and a label or package insert(s) on or associated with the container. In some embodiment, the disclosure provides articles of manufacture comprising contents of the kits described above.

[0192] One of ordinary skill in the art, based on the present disclosure, can utilize the compositions and methods described to their fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein. From the above description, one skilled in the art can easily ascertain the essential characteristics of the present disclosure, and without departing from the spirit and scope thereof, can make various changes and modifications of the disclosure to adapt it to various usages and conditions.EXAMPLES

[0193] Examples of specific embodiments for carrying out the present disclosure are provided. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present disclosure in any way.Example 1: GM2 is upregulated in GD2 low / antigen remodeled / heterogeneous cancers and is a viable target in GD2 resistant cancers

[0194] Materials and methods

[0195] Culture conditions and cell lines

[0196] All tumor cell lines were cultured in complete RPMI-1640 media. Base RPMI- 1640 was supplemented with 10% heat-inactivated FBS (Gibco), 10 mM HEPES, 100 U / ml of penicillin, 100 pg / ml of streptomycin and 2 mM L-glutamine (Gibco). Cells were kept at 37 °C in a 5% CO2 atmosphere incubator.

[0197] Tumor cell line identity was regularly confirmed with STR fingerprinting, and cell lines tested negative for mycoplasma approximately every 6 months.

[0198] Generation of cell lines

[0199] Generation of isogenic Nalm6 lines expressing GD2 (Nalm6-GD2) or GM2 (Nalm6-GM2) was done by retroviral or lentiviral transduction with vectors encoding codon optimized cDNA for B4GALNT1 (Nalm6-GM2) or B4GALNT1 and ST8SIA1 (Nalm6- GD2). Expression of the desired ganglioside was confirmed by flow cytometry. To obtain a homogeneous ganglioside level cells were sorted by Fluorescent-activated cell sorting (FACS) using a FACSAria (BD Biosciences).

[0200] CHAL255 ST8SIA1 KO cells were generated by CRISPR-Cas9 KO using specific sgRNA guides designed to maximize KO efficiency and minimize off-targets. Cas9 and sgRNA guides were introduced in cells by nucleofection using the P3 Primary Cell 4D- Nucleofector X Kit S (Lonza). Briefly, cells were resuspended in 18 pl of P3 buffer and mix with 2 pl of a, previously assembled, ribonucleic particle complex (Cas9:sgRNA). Then cells were electroporated in a 16- wells cuvette strip in a 4D-Nucleofector X Unit (Lonza). Cells were left to recover and later GD2 downregulation was confirmed by flow cytometry. When needed cells were sorted, as previously described, to generate a homogeneous GD2 negative population.

[0201] Production of retroviral and lentiviral supernatant

[0202] Lentiviral supernatant was generated by transient transfection of HEK293-FT cells. Briefly, 6.5x106 cells were seeded in 100 mm poly-d-lysine-coated plates in complete DMEM media (10% FBS (Gibco), 10 mM HEPES, 2 mM glutamine, 100 U / ml penicillin and 100 pg / ml streptomycin (Gibco)). 24 hours later cells were co-transfected with 9 pg vector plasmid, 9 pg pRSV-Rev, 9 pg pMDLg / pRRe and 3.5 pg pMD2.G with Lipofectamine 2000 (Invitrogen) in Opti-MEM medium (Gibco). After 24 hours media was replaced with complete DMEM, and viral supernatant was collected 48 hours and 72 hours post transfection.

[0203] Retroviral supernatant was generated in a similar fashion. Briefly, 6.5x106 HEK293-GP cells were seeded in a 100 mm poly-d-lysine-coated plates in complete DMEM media (10% FBS (Gibco), 10 mM HEPES, 2 mM glutamine, 100 U / ml penicillin and 100 pg / ml streptomycin (Gibco)). 24 hours later cells were co-transfected with 9 pg vector plasmid and 4.5 pg RD114 with Lipofectamine 2000 (Invitrogen) in Opti-MEM medium (Gibco). After 24 hours media was replaced with complete DMEM, and viral supernatant was collected 48 hours and 72 hours post transfection. In both cases (lenti virus or retrovirus) supernatant was used fresh or frozen at -80 °C for later use.

[0204] Isolation of PBMCs and T cells

[0205] Peripheral blood mononuclear cells (PBMCs) were isolated from blood, buffy coats, or LRS chambers using Ficoll-Paque Plus (GE Healthcare, 17-1440) density gradient centrifugation according to the manufacturer’s instructions and cryopreserved with CryoStor CS10 freeze medium (Sigma- Aldrich) in 1 x 107-5 x 107 cell aliquots. For transduction and culture of CAR T cells, cryopreserved PBMCs were thawed on day 0 and cultured with Human T-Activator anti-CD3 / anti-CD28 Dynabeads (Gibco) at a 3:1 bead:cell ratio in AIM- V medium (Gibco) supplemented with 5% FBS, 10 mM HEPES, 2 mM GlutaMAX, 100 U ml-1 penicillin, 100 pg ml-1 streptomycin and 100 TU ml-1 recombinant human TL-2 (Peprotech). Retroviral transductions were performed on days 3 and 4 after activation on retronectin (Takara) -coated non-tissue culture treated plates. Wells were coated with 1 ml of 25 pg ml-1 retronectin in phosphate-buffered saline (PBS) overnight, then blocked with 2% BSA in PBS for 15 minutes before transduction. 1 ml of thawed or 700 ul of fresh retroviral supernatant per CAR construct was added and plates were then centrifuged at 3,200 rpm at 32 °C for 2-3 h. Viral supernatant was discarded, and 0.5x106 T cells were added to each well in 1 ml of complete AIM-V medium. On day 5 after activation, anti-CD3 / anti-CD28 beads were magnetically removed, and CAR T cells were maintained in culture with AIM-V medium changes every two to three days at a density of 0.3 x 106 cells per ml.

[0206] Construction of CAR constructs

[0207] All DNA constructs were visualized using SnapGene software (Dotmatics). CAR constructs were generated with a mix of restriction enzyme and In-Fusion HD Cloning (Takara Bio) using codon-optimized gB locks purchased from Integrated DNA Technologies. The amino acid sequences of CARs generated, tested and characterized are shown in Table 1.

[0208] Flow Cytometry

[0209] Cell lines were harvested with TrypLE Express (Gibco, Thermo Fisher Scientific), made into a single cell suspension and washed twice with PBS + 2% FBS before staining with primary antibodies listed or isotype or secondary only antibodies as a control.Antibodies were incubated for 20 minutes at 4 °C. Data were collected on a LSR Fortessa X- 20 (BD Biosciences) or a NovoCyte Quanteon (Agilent) using BD FACSDIVA v9.0 or NovoExpress respectively. Data analysis was performed using FlowJo (vlO.6.1).

[0210] Primary antibodies:

[0211] Dinutuximab (Anti-GD2, Ipg / ml), DMF10.167.4 (anti-GM2, Ipg / ml)

[0212] Secondary antibodies:

[0213] Alexa Fluor® 647 AffiniPure Donkey Anti-Human IgG (Jackson ImmunoResearch) (1: 100 dilution).

[0214] T cells were assessed for CAR expression on the same day they were used for in vitro and in vivo assays. GD2 or GM2-targeting CARs were detected using the anti-mouse IgG (H+L) antibody (1 :100 dilution). The following antibodies were used for lymphocyte staining: PD-1 (PE-Cy7, clone EH12.2H7, BioLegend, 1:50), TIM-3 (BV510 or BV650, clone F38-2E2, BioLegend, 1:50), LAG-3 (PE, clone 3DS223H, Invitrogen, 1 :50).

[0215] Real time PCR.

[0216] RNAeasy Mini Kit (Qiagen) was used for total RNA extraction from xenografted tumors, following manufacturer protocol. cDNA was synthesized from 2 pg of RNA using cDNA-IV kit (Thermo Fisher Scientific).

[0217] Gene expression of enzymes in the ganglioside synthesis pathway was measured using PowerUp SYBR Green Master Mix (Applied Biosystems, Thermo Fisher Scientific), using specific primers and analyzed using the AACT method. Gene expression was normalized to housekeeping genes and untreated tumor control.

[0218] Cytotoxicity assays

[0219] CAR+ T cells (day 10 after activation) were co-cultured with 50,000 tumor cells at the specified E:T ratios in complete RPM1 medium on 96-well flat-bottom plates. Cocultures were incubated at 37 °C and imaged with an Incucyte S3 Live-Cell Analysis System (Sartorius) for approximately 72 h. The basic analyzer feature on the Incucyte S3 software was used to quantify killing of GFP+ tumor cells by measuring the Total Green Object Integrated Intensity over time. Cytotoxicity index was calculated as the percentage of Total Green Object Integrated Intensity at a specific time point divided by the Total Green Object Integrated Intensity at time 0.

[0220] Cytokine assays

[0221] A total of 1x105 CAR+ T cells (day 10 after activation) were co-cultured with tumor cells in a 1 :1 E:T ratio in complete RPMI medium and incubated at 37 °C for approximately 24 hours. After stimulation, the supernatants were collected and IL-2 or IFNy were measured by ELISA following the manufacturer’s protocol (BioLegend). Absorbances were measured with a Synergy Hl Hybrid Multi-Mode Reader with Gen5 software (BioTek).

[0222] Antibody Dependent Cell Phagocytosis (ADCP) Assay

[0223] 50,000 GFP+ neuroblastoma (NBL) cells were incubated on ice for 30 minutes with the corresponding anti-ganglioside antibody (lug / ml), or vehicle. Afterwards, cells were washed with PBS and co incubated with 100,000 human derived macrophages at 37 C for 2 hours. Co-cultures were then washed with PBS and stained with anti-CDl lb (APC) and DAPI. Flow cytometry analysis of ADCP was conducted and the double positive cellpopulation (CD1 lb+ GFP+) were considered the macrophages that had phagocyted a GFP+ NBL cell. Finally, the phagocytosis index was calculated as the fold change from base line phagocytosis (vehicle).

[0224] In vivo experiments

[0225] All animal experiments were performed under protocols approved by the Stanford University Animal Care and Use committee. Immunodeficient NSG (NOD.Cg-Prkdcscid I12rgtml Wjl / SzJ), ordered from Jackson Laboratory or breed in-house, were between 6 and 12 weeks old at the start of the experiments. Mice were housed with strictly controlled temperature and humidity, kept on 12-h light / dark cycles and ad libitum food and water. Mice in all experimental groups were age and sex matched.

[0226] Tumor cell lines (CHLA255, SH-SY5Y and Kelly) expressing green fluorescent protein (GFP) and luciferase (Luc) were expanded under standard cell culture conditions (descried herein). For inoculation into mice, cells were harvested with TrypLE Express (Gibco, Thermo Fisher Scientific), washed with PBS, counted and resuspended in PBS at a concentration of 5x106 cells per milliliter. 200 pl (1x106 cells) were injected through the tail vein, for all metastatic models, or implanted in the kidney capsule.

[0227] Tumor growth was monitored by Bioluminescent Imaging (BLI) on an IVIS Spectrum In Vivo Imaging System (PerkinElmer) 4 min after 3 mg d-luciferin (PerkinElmer) was injected intraperitoneally. BLI values were quantified with the Living Image v4.7.3 software (PerkinElmer).

[0228] Anti-ganglioside antibody treatment

[0229] Mice were randomized based on BLI and antibody treatment was initiated 4-5 days after tumor engraftment (n = 5 per treatment group) and administered by intraperitoneal injection. For the kidney capsule model, treatment consisted of 300 pg anti-GD2 (dinutuximab) and 400 pg anti-CD47 (clone B6H12, Bio X Cell) administered every other day until completing 3 doses. For the metastatic models, mice were treated with 300 pg anti- GD2 (dinutuximab) and 300 pg of anti-GM2 (clone DMF10.167.4) alone or in combination, 3 doses a week for a period of 2 weeks.

[0230] Experimental endpoint was determined by reduced activity, swelling of the abdominal area and / or BLI measurements above lxl0A10 that triggered morbidity criteria for euthanasia.

[0231] Anti-ganglioside CAR T cell treatment

[0232] Six-to-ten-week-old male or female NOD-scid IL2Rgnull (NSG, NOD.Cg- PrkdcscidI12rgtmlWjl / SzJl) mice were inoculated intravenously with IxlO6CHLA255 cellsor Kelly cells 7 days before or SH-SY5Y cells 6 days before T cell injection in 200 pl PBS and monitored by BLI. In all models, mice were randomized to ensure even tumor burden between experimental and control groups before treatment began. CAR T cells were injected intravenously on day 10 after activation: CHLA-255, Kelly or SH-SY5Y-bearing mice received 3x106CAR+ T cells. Neuroblastoma model mice were monitored for disease progression once a week using BLI with an IVIS imaging system (Perkin Elmer) and Living Image software (Perkin Elmer). For CHLA255 models, mice were humanely euthanized when they showed morbidity or developed palpable solid tumor masses. Mice were randomized before T cell infusion to ensure equal mean tumor burden. The technician performing T cell and tumor cell intravenous injections was blinded to the treatments and expected outcomes.

[0233] Tumor processing, staining and analysis

[0234] Once endpoint criteria was met, mice were euthanized in a CO2 chamber, followed by cervical dislocation as a secondary method.

[0235] Mice livers were harvested, and tumors were immediately put on ice-cold PBS supplemented with 10% FBS. For flow cytometry, tumors were chopped in small pieces using scissors and processed using a combination of mechanical and chemical digestion. First, tumor pieces were processed on a gentleMACS (Miltenyi Biotec) using a preestablished program from the manufacturer (h_tumor_02), followed by 30 minutes incubation at 37 °C in agitation (80 rpm), with Img / ml Collagenase type IV (Gibco) and 0.2 mg / ml DNAse I (Sigma). Then cell suspension was once again run on a gentleMACS (Miltenyi Biotec) using the same preestablished program. The resulting cell suspension was filtered through a 100 pm cell strainer followed by a 70 pm cell strainer. Finally, the cell suspensions were treated twice with ACK lysing buffer (Quality Biological) for 10 minutes on ice, to deplete erythrocytes.

[0236] For ganglioside detection on tumor samples, primary and secondary antibodies (described herein) were used. Tumor cells were identified by excluding human CD45 and / or murine CD45.1+ (Invitrogen) cells and then gating on GFP+ and CD56+ (BioLegend) cell population.

[0237] Ganglioside synthesis pathway in cancer

[0238] The ganglioside synthesis pathway including the synthesis of gangliosides GD2 and GM2 is regulated by a network of sialyl and glycosyl transferases. A graphical representation of the ganglioside synthesis pathways detailing the enzymes, substrates and products is shown (See FIGS. 1A, and IB, 19B). Experiments were conducted duringdevelopment of embodiments of the disclosure in order to identify key components within the ganglioside synthesis pathway involved in tumor growth, development, inhibition and evasion of GD2 directed therapies.

[0239] GM2 is expressed on the surface of cancer cells

[0240] In accordance with embodiments disclosed herein, experiments were conducted to evaluate and to determine the surface expression level of GD2 and GM2 on cancer cells. GD2 and GM2 levels were measured by flow cytometry on a panel of different neuroblastoma cell lines. It was found that GM2 surface expression is high when GD2 surface expression is low (See FIG. 19A). Neuroblastoma cell lines with high levels of GD2 surface expression displayed lower but substantial GM2 surface expression levels (See FIG. 19C). The discovery of GM2 expression on neuroblastoma cell lines identifies GM2 as a potential target for therapy of this high-risk pediatric cancer.

[0241] Additional experiments were conducted to determine the expression level of GD2 and GM2 on Ewing sarcoma cell lines. As shown in FIG. 16, and as further described herein, it was discovered that most Ewing sarcoma cell lines had higher surface GM2 expression than GD2 expression, identifying GM2 as a target for Ewing sarcoma therapy, whereas previously only GD2 was identified as a target.

[0242] Next, osteosarcoma patient-derived xenograft (PDX) cell lines were stained for characterization of surface expression of GD2 and GM2. GD2 and GM2 expression levels were examined in six different osteosarcoma patient-derived xenograft cell lines and most of cell lines displayed higher GM2 expression than GD2 expression (FIG. 20), identifying GM2 as a target for osteosarcoma, whereas previously only GD2 was identified as a target.

[0243] CHLA255 cells were implanted in the kidney capsule of NSG mice. Four days after tumor injection mice were treated with a combination of anti-GD2 (Dinutuximab) and anti-CD47 (B6-H12) antibody or control (NoRx). Despite an initial anti-tumor effect, mice bearing CHLA255 tumors, treated with anti-GD2 therapy eventually relapse. Flow cytometry analysis of the xenograft at endpoint shows a reduction in the levels of GD2 which renders the therapy ineffective (FIG. 2A). It was discovered that the resistance mechanism related to the downregulation in the expression of ST8SIA1, the gene that codes for the enzyme that converts GM3 into GD3 (FIG. 2B). Since expression of other enzymes within the ganglioside synthesis pathway, including B4GALNT1 (GD2 synthase) were not affected (See FIG. 1), it was postulated that a compensatory increase in GM2 could be associated with these observations. Thus, in some embodiments, the disclosure provides that GD2loss / downregulation in neuroblastoma occurs through downregulation of ST8SIA1, and a concurrent increase in the surface levels of GM2.

[0244] Next, experiments were conducted to determine the consequence of anti-GD2 therapy (antibodies or CAR T cells) at the molecular level. Despite an initial anti-tumor effect, mice bearing CHLA255 treated with anti-GD2 therapy eventually relapse (See FIG. 2A). Flow cytometry analysis of the xenograft at endpoint shows a reduction in the levels of GD2, which renders the therapy ineffective (FIG. 2B). The resistance mechanism is explained by a downregulation in the expression of ST8SIA1 , the gene that codes for the enzyme that converts GD3 into GD2 (FIG. 2C). Since the levels of the other ganglioside enzymes including B4GALNT1 (GD2 synthase) were not affected (FIG. 2C), the pathway appeared to be redirected resulting in an increase in the level of GM2 and subsequent relapse (See FIG. 3D). Data generated and shown in FIG. 3B supported this hypothesis. Flow cytometry analysis of xenograft harvested from mice that received anti-GD2 therapy shows that, whether the therapeutic agent was GD2-CAR T cells or anti-GD2 / anti-CD47 antibody treatment, there was a reduction in the levels of GD2 and a compensatory increase in GM2 (FIG. 3B). Additionally, experiments conducted during development of embodiments of the disclosure revealed that ganglioside expression could be artificially altered by means of knocking out the ST8SIA1 gene (ST8SIA1 KO) which led to an increase in the levels of GM2 (See FIG. 3C). Thus, the disclosure provides that when GD2 expression is reduced or lost, there is a compensatory increase in surface levels of GM2.

[0245] Flow cytometry was utilized to measure the expression of gangliosides GD2 and GM2 in various neuroblastoma (NBL) cell lines. CHLA255, Kelly and NB1 present different levels of GD2 and GM2 on the cell surface (FIG. 4A). GFP+ cells were incubated with anti- GD2 (dinutuximab) or anti-GM2 (KM966) antibodies. Opsonized cells were then co-cultured with human macrophages for 2 hours. Antibody Dependent Cell Phagocytosis (ADCP) was determined by flow cytometry as the percentage of GFP+CD1 lb-1- cells in the CD1 lb-1- cell population. ADCP correlated with the level of ganglioside in the surface of the cells. CHLA255 (left) cells were actively phagocyte after incubating with anti-GD2 antibodies, while NB 1 cells were actively phagocyte after incubating with anti-GM2 antibodies (See FIG. 4B).

[0246] Next, NSG mice were inoculated through the tail vein with IxlO6CHLA255 cells expressing GFP and firefly luciferase. Tumor growth was measured by bioluminescence (FIG. 5A). Four days after tumor injection mice received anti-ganglioside treatment (see methods). A combination of anti-GD2 and anti-GM2 antibodies showed better efficacy andtumor control compared to anti-ganglioside monotherapy (See FIGS. 5A and 5B). Importantly, ganglioside levels at endpoint were affected by treatment (FIG. 5C). Xenografts treated with anti-GD2 antibodies showed a reduction in GD2 levels and a compensatory increase in GM2. Inversely, when anti-GM2 treatment was administered, GM2 was downregulated while GD2 levels were slightly increased. Finally, xenografts treated with both anti-ganglioside antibodies retain considerable levels of both GD2 and GM2.

[0247] NSG mice were inoculated through the tail vein with IxlO6CHLA255 cells expressing GFP and firefly luciferase as before (See FIG. 5). Tumor growth was measured by BLI (FIG. 6A). Three days after tumor injection mice received anti-ganglioside treatment (see methods). A combination of anti-GD2 and anti-GM2 (KM966) antibodies showed better efficacy and tumor control compared to anti-ganglioside monotherapy (See FIGS. 6A and 6B). Ganglioside levels at endpoint were affected by treatment (FIG. 6C). Similar to what was observed previously using a different anti-GM2 antibody (DMF10.167.4), xenografts treated with anti-GD2 antibodies showed a reduction in GD2 levels and a compensatory increase in GM2. Inversely, when anti-GM2 (KM966) treatment was administered, GM2 was downregulated. Again, xenografts treated with both anti-ganglioside antibodies retain considerable levels of both GD2 and GM2.

[0248] Next, IxlO6Kelly cells, expressing GFP and firefly luciferase, were injected through the teil vein of NSG mice. Four days after cell inoculation, tumor engraftment was confirmed by BLI, and mice were treated with anti-GD2 (Dinutuximab) or anti-GM2 (KM966) antibodies. While anti-GD2 antibodies were unable to control the disease, anti- GM2 antibodies showed increase efficacy and provided longer survival (See FIGS. 7A and 7B).

[0249] Because the results of the experiments conducted identified ganglioside levels at endpoint were affected by treatment, additional experiments were conducted in order to further identify and characterize the relationship between the expression levels of GD2 and GM2 within the ganglioside synthesis pathway. As detailed in FIGS. 3 and 4, an inverse correlation between GD2 and GM2 expression levels was discovered. Subsequent flow cytometry analysis of a panel of known GD2 low neuroblastoma cell lines (SH-SY5Y, SKNFI, SK-N-A, NB1, KPN-SI9S, Kelly, BE(2)-C) was performed and it was found that GM2 expression was intermediate to high in these cell lines See FIG. 8). Thus, in some embodiments, the disclosure provides that targeting GM2 exists as a therapeutic option in cases where tumors present low to heterogenous levels of GD2.

[0250] Next, NSG mice were inoculated with IxlO6SH-SY5Y cells (GD2 intermediate / low / heterogeneous, GM2 high) through the teil vein. Tumor growth was followed by BLI and 5 days after tumor injection, mice were treated with anti-GD2 or anti- GM2 antibodies (see methods). Anti-GM2 antibodies showed better efficacy and tumor control compared with anti-GD2 antibodies (FIGS. 9A and 9B). Ganglioside levels at endpoint were reduced or increased depending on the treatment (See FIG. 9C). Thus, based on the discovery that GM2 expression was inversely correlated with GD2 expression, the disclosure provides that targeting GM2 exists as a therapeutic option in cases where tumors present low to heterogenous levels of GD2 (e.g., neuroblastomas, and / or when ganglioside loss occurs due to treatment targeting the ganglioside).

[0251] NSG mice were inoculated with IxlO6NB1 cells (GD2 negative, GM2 high cell line) through the tail vein. BLI was used to follow tumor growth and 4 days after cell injection, mice were treated with anti-GD2 or anti-GM2 antibodies (see methods). While anti-GD2 antibodies presented no tumor control, anti-GM2 therapy showed better efficacy and prolonged mice survival (See FIGS. 10A and 10B). hi vitro, this cell line has no detectable GD2, while in vivo it gains a little expression. In this model, anti-gangliosides antibodies did not induce a significant loss of the target ganglioside (Figure 10C). Thus, in some embodiments, the disclosure provides that targeting GM2 can be accomplished and / or achieved with different agents.

[0252] Next, experiments were conducted in order to determine if T cells bearing chimeric antigen receptors (CARs) specific for GM2 and / or T cells bearing a tandem GM2- GD2 CAR could provide therapeutic potential / benefit. First, tandem GM2-GD2 CAR T cells bearing CARs with either the 4-lBB-zeta or the CD28-zeta fragment endodomains / intracellular domains were generated (See FIG. 1 IB) and tested for in vitro functionality. Tandem GM2-GD2 CAR with 4-lBB-zeta or CD28-zeta fragment CAR T cells secreted cytokines against Nalm6-GD2, Nalm6-GM2, CHLA255, and Sy5y tested even if the cell lines only expressed GD2 or GM2 (FIG. 11 A) whereas monospecific CARs, GD2-BBz or GM2-28z, secreted cytokines only in each GD2 or GM2 -expressing cell lines (FIG. 11 A).

[0253] In order to identify in vivo functionality of tandem GD2-GM2 CAR T cells bearing CARs with the CD28-zeta fragment endodomain, GM2-GD2-8HTM-28z, it was compared with each monospecific CAR T cells, GD2-8HTM-BBz and GM2-8HTM-28z, and co-infusion of each monospecific CAR T cells (See FIG. 12). Tandem GM2-GD2-8HTM- BBz CAR T cells showed similar or better in vivo efficacy compared to GM2-8HTM-28z or co-infusion of monospecific CAR T cells. Thus, the disclosure provides, in someembodiments, that targeting GM2 with mono or bispecific CAR T cells overcomes resistance to GD2 CAR T cells in GD2 low / GM2 high neuroblastoma. The disclosure also provides, in some embodiments, in vivo efficacy and functionality of tandem GD2-GM2-CAR-T cells with lower doses and / or that tandem GD2-GM2 CAR T cells display better in vivo efficacy than monospecific GM2 CAR T cells.

[0254] In order to identify in vivo functionality of tandem GD2-GM2 CAR T cells, GD2- , GM2-, GD2-GM2- or GM2-GD2-28z CAR T cells were injected into mice previously engrafted with a GD2-High / GM2-Intermideate / low (CHLA255) cell line or a GD2 / GM2- intermediate cell line (Kelly). GM2-GD2-28z tandem CAR displayed the best efficacy compared to monospecific GM2-28z, GD2-28z CAR T cells or tandem GD2-GM2-28z (See FIG. 13A and 13B). In the CHLA255 model, xenografts treated with GD2-CAR T cells showed a reduction in GD2 levels and a compensatory increase in GM2; Inversely, when GM2-CAR T cells treatment was administered, GM2 was downregulated while GD2 levels were slightly increased (See FIG. 13C). Thus, in some embodiments, the disclosure provides that tandem GM2-GD2 CAR T cells display the best efficacy / tumor inhibition compared to monospecific CAR T cells (e.g., demonstrating that, in some embodiments, tandem CAR T cells can be used to inhibit and / or prevent immune escape).

[0255] In order to identify and characterize in vivo functionality of tandem GD2-GM2 CAR T cells, GD2-GM2-targeting 4-lBB-zeta or CD28-zeta fragment CAR T cells were injected into GD2- high (Nalm-6-GD2) cell line engrafted mice model. It was discovered that GM2-GD2-28z tandem CAR showed better efficacy compared to monospecific GD2 CAR T cells (FIG. 14A-B).

[0256] Next, RNA expression of ST8SIA1 and B4GALNT1 enzymes was analyzed. As depicted in FIG. 1, each enzyme is necessary in the production of GD2. The expression of ST8SIA1 was found to be heterogenous in Ewing sarcoma compared to neuroblastoma, while the expression of B4GALNT1 was found to be high in both neuroblastoma and sarcoma cell lines (FIG. 15).

[0257] In order to further identify and characterize the expression of GD2 and GM2 in cancer, a variety of Ewing sarcoma cell lines (NCI-LG, NCI-LD, NCI-JD, NCI-AK, TC71, TC32, SK-N-MC, RD-ES, EW8, A673, 6647, 5938) were analyzed for surface expression of GD2 and GM2 using flow cytometry. As shown in FIG. 16, most of the Ewing sarcoma cell lines exhibited significantly higher surface GM2 expression than GD2 surface expression. Thus, in some embodiments, the disclosure provides that GM2 is a viable therapeutic targetfor Ewing sarcoma (e.g., that Ewing sarcoma be treated by targeting GM2 and / or other cell surface markers (e.g., GD2)) and cancers that display heterogeneous levels of GD2.

[0258] Next, GM2-targeting CAR T cells harboring the CD28-zeta fragment intracellular domain (KM966-8HTM-28z) were tested for in vitro functionality against GM2-high Ewing sarcoma cell lines. GD2-targeting CAR T cells (GD2-8HTM-BBz) were also tested in the same model. GM2-targeting KM966 CAR T cells induced the secretion of significantly higher levels of cytokines against GM2-high Ewing sarcoma cell lines than mock or GD2- targeting CAR T cells (FIG. 17).

[0259] Immunohistochemistry (IHC) was performed on a neuroblastoma (NBL) sample obtained from a relapse patient previously treated with anti-GD2 immonotherapy.Ganglioside heterogeneity was observed using IHC in a frozen NBL sample using Dinutuximab and DMF10.167.4 antibodies to detect GD2 and GM2, respectively. Areas of intense GD2 positive staining (DAB, brown) and areas of intense GM2 positive staining (RED, red) and areas where both gangliosides stained were observed (FIG. 18).

[0260] While the present disclosure has been shown and described with reference to preferred and various alternate embodiments, it will be readily understood by persons skilled in the art that various changes in form and details can be made without departing from the spirit and scope of the present disclosure

Claims

CLAIMS1. A method for identifying and treating an anti-ganglioside GM2 treatment sensitive cancer within a patient comprising:- providing a patient having cancer;- measuring the expression or level of ganglioside synthase enzyme ST8SIA1 and / or ganglioside GD2 in the patient’s cancer and / or tumor cells;- comparing the expression or level of ganglioside synthase enzyme ST8SIA1 and / or ganglioside GD2 in the patient’ s cancer and / or tumor cells to the expression or level of ganglioside synthase enzyme ST8SIA1 and / or ganglioside GD2 in a control sample;- wherein cancer and / or tumor cells displaying low to heterogeneous expression or level of ganglioside synthase enzyme ST8S1A1 and / or ganglioside GD2 compared to the control sample identifies the cancer as one that will respond to treatment with anti-ganglioside GM2 immunotherapy; and administering to a patient having cancer identified as one that will respond to treatment with anti-ganglioside GM2 immunotherapy an anti-ganglioside GM2 immunotherapy.

2. The method of claim 1, further comprising measuring the expression level of ganglioside GM2 in the patient’s cancer and / or tumor cells.

3. The method of claim 1, wherein the patient having cancer has a neuroblastoma, sarcoma, retinoblastoma, medulloblastoma, Ewing sarcoma, lung carcinoma, or glioblastoma.

4. The method of claim 1 , wherein the patient with cancer has a sarcoma, a rhabdoid cancer, a neuroblastoma, retinoblastoma, medulloblastoma, Ewing sarcoma, lymphoma, melanoma, uterine carcinosarcoma (UCS), brain lower grade glioma (LGG), thymoma (THYM), testicular germ cell tumors (TGCT), glioblastoma multiforme (GBM) and skin cutaneous melanoma (SKCM), liver hepatocellular carcinoma (LIHC), uveal melanoma (UVM), kidney chromophobe (KICH), thyroid cancer (THCA), kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), stomach adenocarcinoma (STAD), cholangiocarcinoma (CHOL), adenoid cystic carcinoma (ACC), prostate adenocarcinoma (PR AD), pheochromocytoma and paraganglioma (PCPG), DLBC, lung adenocarcinoma (LUAD), head-neck squamous cell carcinoma (HNSC), pancreatic adenocarcinoma (PAAD), breast cancer (BRCA), mesothelioma (MESO), colon and rectaladenocarcinoma (COAD), rectum adenocarcinoma (READ), esophageal carcinoma (ESCA), ovarian cancer (OV), lung squamous cell carcinoma (LUSC), bladder urothelial carcinoma (BLCA), sarcoma (SARC), small cell lung cancer, or uterine corpus endometrial carcinoma (UCEC).

5. The method of claim 1, wherein administering an anti-ganglioside GM2 immunotherapy comprises administering a therapeutically effective amount of an anti-GM2 specific antibody.

6. The method of claim 1 , wherein administering an anti-ganglioside GM2 immunotherapy comprises administering a therapeutically effective amount of anti-GM2 specific chimeric antigen receptor (CAR) T cells.

7. The method of any one of claims 1, 5 or 6, further comprising measuring the expression and / or level of ganglioside synthase enzyme ST8SIA1 and / or ganglioside GD2 in the patient’s cancer and / or tumor cells at a time point after administering an anti-ganglioside GM2 immunotherapy to the patient having cancer.

8. The method of claim 1, wherein measuring the expression and / or level of ganglioside synthase enzyme ST8SIA1 and / or ganglioside GD2 comprises measuring gene and / or protein expression levels and surface ganglioside levels.

9. The method of claim 8, wherein gene expression is measured by polymerase chain reaction (PCR) analysis, sequencing analysis, electrophoretic analysis, restriction fragment length polymorphism (RFLP) analysis, Northern blot analysis, quantitative PCR, reverse- transcriptase-PCR analysis (RT-PCR), allele-specific oligonucleotide hybridization analysis, comparative genomic hybridization, heteroduplex mobility assay (HMA), single strand conformational polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE), RNAase mismatch analysis, mass spectrometry, tandem mass spectrometry, matrix assisted laser desorption / ionization-time of flight (MALDI-TOF) mass spectrometry, electrospray ionization (ESI) mass spectrometry, surface-enhanced laser desorption / ionization-time of flight (SELDI-TOF) mass spectrometry, quadrupole-time of flight (Q-TOF) mass spectrometry, atmospheric pressure photoionization mass spectrometry (APPI-MS), Fourier transform mass spectrometry (FTMS), matrix-assisted laser desorption / ionization-Fouriertransform-ion cyclotron resonance (MALDI-FT-ICR) mass spectrometry, secondary ion mass spectrometry (SIMS), surface plasmon resonance, Southern blot analysis, in situ hybridization, fluorescence in situ hybridization (FISH), chromogenic in situ hybridization (CISH), immunohistochemistry (IHC), microarray, comparative genomic hybridization, karyotyping, multiplex ligation-dependent probe amplification (MLP A), Quantitative Multiplex PCR of Short Fluorescent Fragments (QMPSF), microscopy, methylation specific PCR (MSP) assay, Hpall tiny fragment Enrichment by Ligation-mediated PCR (HELP) assay, radioactive acetate labeling assays, colorimetric DNA acetylation assay, chromatin immunoprecipitation combined with microarray (ChlP-on-chip) assay, restriction landmark genomic scanning, Methylated DNA immunoprecipitation (MeDIP), molecular break light assay for DNA adenine methyltransferase activity, chromatographic separation, methylationsensitive restriction enzyme analysis, bisulfite-driven conversion of non-methylated cytosine to uracil, methyl-binding PCR analysis, or a combination of any of the foregoing.

10. The method of claim 8, wherein gene expression is measured by a sequencing technique selected from direct sequencing, RNA sequencing, whole transcriptome shotgun sequencing, random shotgun sequencing, Sanger dideoxy termination sequencing, wholegenome sequencing, sequencing by hybridization, pyrosequencing, capillary electrophoresis, gel electrophoresis, duplex sequencing, cycle sequencing, single-base extension sequencing, solid-phase sequencing, high-throughput sequencing, massively parallel signature sequencing, emulsion PCR, sequencing by reversible dye terminator, paired-end sequencing, near-term sequencing, exonuclease sequencing, sequencing by ligation, short-read sequencing, single-molecule sequencing, sequencing-by-synthesis, real-time sequencing, reverse- terminator sequencing, nanopore sequencing, 454 sequencing, Solexa Genome Analyzer sequencing, SOLID™ sequencing, MS-PET sequencing, mass spectrometry, and a combination of any of the foregoing.

11. The method of claim 8, wherein protein expression is measured by an immunohistochemistry assay, an enzyme-linked immunosorbent assay (ELISA), in situ hybridization, flow cytometry, chromatography, liquid chromatography, size exclusion chromatography, high performance liquid chromatography (HPLC), gas chromatography, mass spectrometry, tandem mass spectrometry, matrix assisted laser desorption / ionization- time of flight (MALDLTOF) mass spectrometry, electrospray ionization (ESI) mass spectrometry, surface-enhanced laser desorption / ionization-time of flight (SELDLTOF) massspectrometry, quadrupole-time of flight (Q-TOF) mass spectrometry, atmospheric pressure photoionization mass spectrometry (APPI-MS), Fourier transform mass spectrometry (FTMS), matrix-assisted laser desorption / ionization-Fourier transform-ion cyclotron resonance (MALDI-FT-ICR) mass spectrometry, secondary ion mass spectrometry (SIMS), radioimmunoassays, microscopy, microfluidic chip-based assays, surface plasmon resonance, sequencing, Western blotting assay, or a combination of any of the foregoing.

12. The method of any one of the preceding claims, wherein the patient having cancer displays resistance to anti-ganglioside GD2 therapy.

13. A method for the treatment of cancer comprising administering to a patient with the cancer an immunotherapy targeting ganglioside GM2 or an immunotherapy targeting both ganglioside GM2 and ganglioside GD2.

14. A method for treating a cancer displaying decreased expression or activity of ganglioside synthase enzyme ST8S1A1, the method comprising administering to a patient in need thereof an effective amount of an immunotherapy targeting ganglioside GM2.

15. The method of any one of claims 13 and 14, wherein the immunotherapy targeting ganglioside GM2 comprises an anti-GM2 specific antibody.

16. The method of any one of claims 13-15, wherein the immunotherapy targeting ganglioside GM2 comprises anti-GM2 specific chimeric antigen receptor (CAR) T cells.

17. The method of any one of claims 13-16, further comprising administering to the patient a therapeutically effective amount of a chemotherapeutic agent and / or radiation.

18. The method of any one of claims 13-17, wherein the cancer is a neuroblastoma, sarcoma, lymphoma, melanoma, glioblastoma, small cell lung cancer, rhabdoid cancer, retinoblastoma, medulloblastoma, Ewing sarcoma, uterine carcinosarcoma (UCS), brain lower grade glioma (LGG), thymoma (THYM), testicular germ cell tumors (TGCT), glioblastoma multiforme (GBM) and skin cutaneous melanoma (SKCM), liver hepatocellular carcinoma (LIHC), uveal melanoma (UVM), kidney chromophobe (KICH), thyroid cancer (THCA), kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma(KIRP), stomach adenocarcinoma (STAD), cholangiocarcinoma (CHOL), adenoid cystic carcinoma (ACC), prostate adenocarcinoma (PRAD), pheochromocytoma and paraganglioma (PCPG), DLBC, lung adenocarcinoma (LU AD), head-neck squamous cell carcinoma (HNSC), pancreatic adenocarcinoma (PAAD), breast cancer (BRCA), mesothelioma (MESO), colon and rectal adenocarcinoma (COAD), rectum adenocarcinoma (READ), esophageal carcinoma (ESCA), ovarian cancer (OV), lung squamous cell carcinoma (LUSC), bladder urothelial carcinoma (BLCA), sarcoma (SARC), or uterine corpus endometrial carcinoma (UCEC).

19. The method of any one of claims 13-18, comprising administering to the patient a therapeutically effective amount of DMF10.167.4, KM966, or chimeric or humanized versions of DMF10.167.4 or chimeric or humanized versions of KM966.

20. The method of any one of claims 13-19, comprising administering to the patient a therapeutically effective amount of T-cells expressing a chimeric antigen receptor (CAR) specific for ganglioside GM2 under conditions sufficient to form an immune complex of an antigen binding domain on the chimeric antigen receptor and ganglioside GM2 in the patient.

21. The method of claim 20, wherein the chimeric antigen receptor (CAR) specific for ganglioside GM2 comprises a single chain Fv (scFv) or other binding domain that binds to GM2, a transmembrane domain, and one or more intracellular signaling domains, wherein the scFv comprises a heavy chain variable (VH) region and a light chain variable (VL) region pair, and wherein(A) the VH and VL pair is selected from: i. a VH region comprising a heavy chain complementarity determining region 1 (CDR- Hl) having the amino acid sequence of SEQ ID NO: 1, a heavy chain complementarity determining region 2 (CDR-H2) having the amino acid sequence of SEQ ID NO: 2, a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of SEQ ID NO: 3, and a VL region comprising a light chain complementarity determining region 1 (CDR-L1) having the amino acid sequence of SEQ ID NO: 18, a light chain complementarity determining region 2 (CDR-L2) having the amino acid sequence of SEQ ID NO: 19, and alight chain complementarity determining region 3 (CDR-L3) having the amino acid sequence of SEQ ID NO: 20; ii. a VH region comprising a heavy chain complementarity determining region 1 (CDR- Hl) having the amino acid sequence of SEQ ID NO: 4, a heavy chain complementarity determining region 2 (CDR-H2) having the amino acid sequence of SEQ ID NO: 5, a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of SEQ ID NO: 6, and a VL region comprising a light chain complementarity determining region 1 (CDR-L1) having the amino acid sequence of SEQ ID NO: 21, a light chain complementarity determining region 2 (CDR-L2) having the amino acid sequence of SEQ ID NO: 22, and a light chain complementarity determining region 3 (CDR-L3) having the amino acid sequence of SEQ ID NO: 23; and iii. a VH region comprising a heavy chain complementarity determining region 1 (CDR- Hl) having the amino acid sequence of SEQ ID NO: 7, a heavy chain complementarity determining region 2 (CDR-H2) having the amino acid sequence of SEQ ID NO: 8, a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of SEQ ID NO: 9, and a VL region comprising a light chain complementarity determining region 1 (CDR-L1) having the amino acid sequence of SEQ ID NO: 24, a light chain complementarity determining region 2 (CDR-L2) having the amino acid sequence of SEQ ID NO: 25, and a light chain complementarity determining region 3 (CDR-L3) having the amino acid sequence of SEQ ID NO: 26; or(B) the VH comprises: a heavy chain complementarity determining region 1 (CDR-H1), a heavy chain complementarity determining region 2 (CDR-H2), and a heavy chain complementarity determining region 3 (CDR-H3), wherein the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are contained within the VH region of an amino acid sequence selected from SEQ ID NOs: 10-17, and the VL comprises: a light chain complementarity determining region 1 (CDR-L1), a light chain complementarity determining region 2 (CDR-L2), and a light chain complementarity determining region 3 (CDR-L3), and wherein the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 arecontained within the VL region of an amino acid sequence selected from SEQ ID NOs: 27- 35; or(C) the VH comprises: an amino acid sequence with at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence selected from SEQ ID NOs: 10-17, and the VL comprises: an amino acid sequence with at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence selected from SEQ ID NOs: 27-35; or(D) the VH region comprises: an amino acid sequence with at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence selected from SEQ ID NOs: 10, 11, 12, 13, 14, 15, 16, and 17, and the VL region comprises: an amino acid sequence with at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence selected from SEQ ID NOs: 27, 28, 29, 30, 31, 32, 33, 34 and 35, optionally wherein the CAR comprises one or more of a hinge domain, a spacer region, or one or more peptide linkers.

22. The method of claim 21, wherein the single chain Fv (scFv) is selected from an amino acid sequence with at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence selected from SEQ ID NOs: 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 56, 57, and 58.

23. The method of claims 21 or 22, wherein the CAR transmembrane domain is selected from a CD8 transmembrane domain, a CD28 transmembrane domain, a 4-IBB transmembrane domain, a CD3zeta-chain transmembrane domain, a PD-1 transmembrane domain, a DAP 10 transmembrane domain, a CTLA-4 transmembrane domain, a CD 16a transmembrane domain, an 0X40 transmembrane domain, an NKG2D transmembranedomain; a CD4 transmembrane domain, a LAG-3 transmembrane domain, an 0X40 transmembrane domain, an NKp44 transmembrane domain, an ICOS transmembrane domain, a DAP12 transmembrane domain, a BTLA transmembrane domain, a KIR3DS 1 transmembrane domain, a 2B4 transmembrane domain, a DNAM-1 transmembrane domain, an FceRlg transmembrane domain, a KIR2DS1 transmembrane domain, and an NKp46 transmembrane domain.

24. The method of any one of claim 21 -23, wherein the CAR transmembrane domain is selected from an amino acid sequence with at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence selected from SEQ ID NOs: 36 and 37.

25. The method of any one of claim 21-24, wherein the one or more intracellular signaling domains of the CAR are each selected from a 4- IBB intracellular signaling domain, a CD28 intracellular signaling domain, a CD3zeta-chain intracellular signaling domain, a ZAP70 (SRK) intracellular signaling domain, a ZAP40 intracellular signaling domain, a CD30 intracellular signaling domain, an 0X40 intracellular signaling domain, a CD27 intracellular signaling domain, a DAP 12 intracellular signaling domain, a KIR2DS1 intracellular signaling domain, a NKG2D intracellular signaling domain, a FceRlg intracellular signaling domain, a MyD88 intracellular signaling domain, an EAT-2 intracellular signaling domain, a DAP10 intracellular signaling domain, an ICOS intracellular signaling domain, a DNAM-1 intracellular signaling domain, a CD2 intracellular signaling domain, a CD8 intracellular signaling domain, a CD 16a intracellular signaling domain, a CD97 intracellular signaling domain, a CD 154 intracellular signaling domain, a GITR intracellular signaling domain, a NKp46 intracellular signaling domain, a 2B4 intracellular signaling domain, a CD1 la-CD18 intracellular signaling domain, a NKp44 intracellular signaling domain, a KIR3DS 1 intracellular signaling domain, an HVEM intracellular signaling domain, and / or a combination of two or more intracellular signaling domains.

26. A method of treating a mammal having a disease associated with increased expression of ganglioside GM2 and / or reduced expression of ganglioside GD2, the method comprising administering to the mammal an effective amount of a population of T cells genetically modified to express a nucleic acid molecule encoding a chimeric antigen receptor, the chimeric antigen receptor (CAR) comprising:an antigen binding domain, a transmembrane domain, and at least one intracellular signaling domain, wherein the antigen binding domain comprises at least one heavy chain variable (VH) region and at least one light chain variable (VL) region, wherein the at least one heavy chain variable (VH) region comprises a heavy chain complementarity determining region 1 (CDR-H1), a heavy chain complementarity determining region 2 (CDR-H2), and a heavy chain complementarity determining region 3 (CDR-H3) having an amino acid sequence with at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence selected from SEQ ID NOs: 10-17, wherein the at least one light chain variable (VL) region comprises a light chain complementarity determining region 1 (CDR-L1), a light chain complementarity determining region 2 (CDR-L2), and a light chain complementarity determining region 3 (CDR-L3) having an amino acid sequence with at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to an amino acid sequence selected from SEQ ID NOs: 27-35.

27. The method of claim 26, wherein the chimeric antigen receptor (CAR) comprises or consists of the amino acid sequence set forth as SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 12 or SEQ ID NO: 13 or SEQ ID NO: 14 or SEQ ID NO: 15 or SEQ ID NO: 16 or SEQ ID NO: 17, and wherein the chimeric antigen receptor (CAR) specifically binds to ganglioside GM2 and / or ganglioside GD2.

28. The method of claim 26 or claim 27, wherein the at least one light chain variable region of the chimeric antigen receptor (CAR) comprises or consists of the amino acid sequence set forth as SEQ ID NO: 27 or SEQ ID NO: 28 or SEQ ID NO: 29 or SEQ ID NO: 30 or SEQ ID NO: 31 or SEQ ID NO: 32 or SEQ ID NO: 33 or SEQ ID NO: 34 or SEQ ID NO: 35, and wherein the chimeric antigen receptor (CAR) specifically binds to ganglioside GM2 and / or ganglioside GD2.

29. The method of any one of claims 26-28, wherein the antigen binding domain of the chimeric antigen receptor (CAR) is a scFv, particularly wherein a) the scFv comprises or consists of an amino acid sequence with at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identity to an amino acid sequence selected from SEQ ID NOs: 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 56, 57, and 58.

30. A method of detecting minimal residual disease in a patient with cancer that has received treatment with a ganglioside GD2 directed immunotherapy comprising detecting the expression level of ganglioside GM2 in the patient’s cancer and / or tumor cells. 1 . The method of any one of claims 13-19, wherein the cancer displays decreased or lost sensitivity to ganglioside GD2 directed therapeutic treatment.

32. The method of claim 31 , comprising administering to the patient with the cancer an immunotherapy targeting both ganglioside GM2 and ganglioside GD2.

33. The method of claim 31 , wherein the immunotherapy targeting both ganglioside GM2 and ganglioside GD2 comprises a CAR specific for GM2 and a CAR specific for GD2.

34. The method of claim 33, wherein one or both of the CAR specific for GM2 and the CAR specific for GD2 comprise an antibody, an antigen-binding fragment of an antibody, a F(ab) fragment, a F(ab’) fragment, a single chain variable fragment (scFv), or a single-domain antibody (sdAb).

35. The method of claim 33 or 34, wherein the CAR comprises a VH and / or VL amino acid sequence of Table 1.

36. The method of any one of claims 33-35, wherein the CAR comprises a VH and VL pair from the VH and VL amino acid sequences of Table 1.

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