WO2025096443A1

WO2025096443A1 - Chimeric antigen receptors targeting monosialoganglioside gm2 and methods of use thereof

Info

Publication number: WO2025096443A1
Application number: PCT/US2024/053413
Authority: WO
Inventors: Guillermo DALTON; Wonju Kim; Robbie MAJZNER
Original assignee: Leland Stanford Junior University
Current assignee: Leland Stanford Junior University
Priority date: 2023-10-30
Filing date: 2024-10-29
Publication date: 2025-05-08
Anticipated expiration: 2026-04-30

Abstract

The present disclosure provides compositions and methods related to chimeric antigen receptors (CARs). In particular, the present disclosure provides CAR-based immunotherapeutic compositions that target tumor cells expressing GM2 for the treatment and prevention of cancer.

Description

CHIMERIC ANTIGEN RECEPTORS TARGETING MONOSIALOGANGLIOSIDE GM2 AND METHODS OF USE THEREOF

STATEMENT OF RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63/546,454, 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- 42495-60 l_SQL.xml”, created October 29, 2024, having a file size of 101,821 bytes, is hereby incorporated by reference in its entirety.

FIELD

[0003] The present disclosure provides compositions and methods related to chimeric antigen receptors (CARs). In particular, the present disclosure provides CAR-based immunotherapeutic compositions that target tumor cells expressing ganglioside GM2 for the treatment and prevention of cancer.

BACKGROUND

[0004] Cancer is one of the most devastating diseases both in terms of human life opportunity loss and health care cost. It also presents unmet clinical needs. Cancer is typically treated with surgery, chemotherapy, radiation therapy, or a combination thereof. These treatments, however, often have significant side effects including immune system suppression, destruction of normal cells in the body, autoimmunity, aberrant cellular metabolism, and even metastasis and the onset of secondary cancer.

SUMMARY

[0005] The present 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. [0006] 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- H1 ) 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.

[0007] 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.

[0008] 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 1COS transmembrane domain, a DAP12 transmembrane domain, a BTLA transmembrane domain, a KIR3DS1 transmembrane domain, a 2B4 transmembrane domain, a DNAM-1 transmembrane domain, an FceRlg transmembrane domain, a KIR2DS 1 transmembrane domain, and an NKp46 transmembrane domain.

[0009] 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.

[0010] In some embodiments, the one or more intracellular signaling domains 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 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.

[0011] 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. [0012] 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.

[0013] In some embodiments, the disclosure provides a chimeric antigen receptor (CAR) that includes a ganglioside GM2 binding domain. In some embodiments, the GM2 antigenbinding 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 aspects, a CAR comprises one or more of a hinge domain, a spacer region, a framework region, and/or one or more peptide linkers. In some embodiments, a CAR comprises a spacer region between the scFV and the transmembrane domain. In other embodiments, a CAR comprises a single domain antibody (sdAb) devoid of immunoglobulin ligh chain sequences. For example, in some embodiments, provided herein is a single domain antibody that binds to GM2 comprising one or more CDR regions from any one of SEQ ID NOs: 10-17. In some implementations, a GM2 sdAb 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, or, in other embodiments, 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. [0014] In some embodiments, the disclosure includes compositions comprising a CAR described herein, and a pharmaceutically acceptable carrier and/or pharmaceutically acceptable excipient.

[0015] The disclosure also provides an engineered nucleic acid encoding a CAR disclosed herein. In some embodiments, the nucleic acid molecule encodes at least one chimeric antigen receptor, the at least one 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; and 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 at least one heavy chain variable region 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. In some embodiments, the at least one light chain variable region 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. In other embodiments, the antigen binding domain is a scFv, wherein 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. In some embodiments, the intracellular signaling domain comprises or consists of 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 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. In some embodiments, the 4- 1BB intracellular signaling domain comprises or consists of the amino acid sequence set forth as SEQ ID NO: 38. In some embodiments, the CD28 intracellular signaling domain comprises or consists of the amino acid sequence set forth as SEQ ID NO: 39. In some embodiments, the CD3zeta-chain intracellular signaling domain comprises or consists of the amino acid sequence set forth as SEQ ID NO: 40. In some embodiments, the ZAP70 (SRK) intracellular signaling domain comprises or consists of the amino acid sequence set forth as SEQ ID NO: 41. In some embodiments, the transmembrane domain comprises or consists of the amino acid sequence set forth as SEQ ID NO: 36. In other embodiments, the transmembrane domain comprises or consists of the amino acid sequence set forth as SEQ ID NO: 37. In some embodiments, the nucleic acid molecule encoding a chimeric antigen receptor (CAR) comprises, from N-terminus to C-terminus, the antigen binding domain, the transmembrane domain, and the at least one intracellular T-cell signaling domain and wherein the chimeric antigen receptor (CAR) further comprises a spacer domain between the at least one heavy chain variable (VH) region and the at least one light chain variable (VL) region. In some aspects, the engineered nucleic acid encoding the antigen binding domain (scFv) of the chimeric antigen receptor (CAR) comprises or consists of the amino acid sequence set forth as SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 53, SEQ ID NO: 56, SEQ ID NO: 57, or SEQ ID NO: 58. In some implementations, the nucleic acid molecule further comprises a codon optimized for expression in a human T cell, and/or operably linked to an expression control sequence. In some embodiments, the nucleic acid molecule encodes two or more chimeric antigen receptors. For example, in some embodiments, one of the chimeric antigen receptors encoded by the nucleic acid molecule comprises a single chain Fv (scFv) that binds to ganglioside GM2 and another one of the chimeric antigen receptors encoded by the nucleic acid molecule comprises a single chain Fv (scFv) that binds to ganglioside GD2. In other embodiments, the disclosure provides an engineered nucleic acid encoding a CAR comprising a single domain antibody that binds to GM2 comprising one or more CDR regions from any one of SEQ ID NOs: 10-17. In some implementations, a nucleic acid encodes a GM2 sdAb comprising 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. In some embodiments, an engineered nucleic acid encodes 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. In some embodiments, the disclosure provides an engineered nucleic acid encoding a CAR comprising a single domain antibody that binds to GD2. The disclosure is not limited by the sdAb that binds to GD2. Indeed, a sdAb of the the disclosure may include any VH region of an antibody that binds to GD2. In some ebodiments, a sdAb comprises a VH region of SEQ ID NO: 17.

[0016] The disclosure also provides an expression vector comprising an engineered nucleic acid described herein. In some embodiments, the vector is a recombinant DNA expression vector. In some embodiments, the vector is a viral vector. The disclosure is not limited by the type of vector and any type of vector describe herein or known in the art may be used. In some embodiments, the vector is a retroviral vector (e.g., a MSGV1 retroviral vector). In some embodiments, the viral vector is a lentiviral vector. In other embodiments, the vector is an oncolytic virus vector. In some embodiments, the vector is an adenovirus, an adeno-associated virus (AAV), or a virus-like particle (VLP). In a preferred embodiment, the vector is a vector used in making chimeric antigen receptor T-cells.

[0017] The disclosure provides a polypeptide comprising a chimeric antigen receptor encoded by an engineered nucleic acid molecule described herein. [0018] The disclosure also provides an isolated cell comprising a CAR described herein, a cell modified to include an engineered nucleic acid disclosed herein, an expression vector comprising an engineered nucleic acid described herein, as well as methods of making same. For example, in some embodiments, the disclosure provides a method of making a modified cell comprising transducing an isolated cell with an engineered nucleic acid as described herein or an expression vector as described herein. In some embodiments, the disclosure provides a population of cells comprising a CAR described herein. In some embodiments, the CAR is recombinantly expressed by the cell or population of cells. In other embodiments, the CAR is expressed from a vector or a selected locus from the genome of the cell. In further embodiments compositions comprising an engineered nucleic acid described herein or an expression vector described herein and a pharmaceutically acceptable carrier, pharmaceutically acceptable excipient, or a combination thereof are provided. The disclosure is not limited by the type of cell modified with the compositions and methods described herein. In some embodiments, the modified cell is an autologous cell. In other embodiments, the cell is an allogeneic cell. In some embodiments, the cell or population of cells is a T cell, a CD4 T cell, a CD8 T cell, a Natural Killer (NK) cell, a Natural Killer T (NKT) cell, a gamma-delta T cell, a cytotoxic T lymphocyte (CTL), or a regulatory T cell. In other embodiments the cell or population of cells is a dendritic cell, 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, or a mesenchymal stromal cell.

[0019] The disclosure also provides a pharmaceutical composition comprising an effective amount of a CAR described herein, an engineered nucleic acid described herein, an expression vector described herein, and/or a cell or population of cells described herein, together with a pharmaceutically acceptable carrier and/or pharmaceutically acceptable excipient.

[0020] The disclosure provides a method of stimulating an immune response to a tumor cell and/or cancer cell in a subject comprising administering to a subject having a tumor and/or cancer a therapeutically effective dose of a CAR described herein, an engineered nucleic acid described herein, an expression vector described herein, and/or a cell or population of cells described herein.

[0021] The disclosure also provides a method of treating a subject having a tumor or cancer, the method comprising administering a therapeutically effective dose of a CAR described herein, an engineered nucleic acid described herein, an expression vector described herein, and/or a cell or population of cells described herein.

[0022] The disclosure also provides a chimeric antigen receptor (CAR) encoded by a nucleic acid molecule described herein, for use in a method of treating a subject with a tumor or cancer, wherein the tumor or cancer comprises cell surface expression of ganglioside GM2 and/or ganglioside GD2. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of T-cells expressing a chimeric antigen receptor described herein, under conditions sufficient to form an immune complex of the antigen binding domain on the chimeric antigen receptor and ganglioside GM2 and/or ganglioside GD2 in the subject. In further embodiments, the T-cells are T cells from the subject that have been transformed with the engineered nucleic acid molecule encoding a chimeric antigen receptor or transduced with a vector comprising an engineered nucleic acid molecule described herein. For example, in some embodiments, the method comprises obtaining the T cells from the subject and transforming the T cells with an engineered nucleic acid molecule encoding the chimeric antigen receptor. In other embodiments, the method comprises obtaining the T cells from the subject and transducing the T cells with a vector comprising an engineered nucleic acid molecule. The disclosure is not limited by the type of cancer and/or tumor treated. Indeed, a variety of cancers and tumors may be treated including those described herein. In some embodiments, the cancer/tumor is a neuroblastoma, sarcoma, retinoblastoma, medulloblastoma, Ewing sarcoma, or glioblastoma. In some embodiments, the method further comprises selecting the subject for treatment by detecting cell-surface expression of ganglioside GM2 on the tumor.

[0023] Embodiments of the disclosure include kits for treating/and preventing a tumor and/or cancer. For example, in some embodiments, the disclosure provides a kit for making a chimeric antigen receptor (CAR) T-cell or treating a tumor in a subject wherein the tumor comprises cell surface expression of ganglioside GM2. In some embodiments, the kit comprises a container comprising a CAR described herein, an engineered nucleic acid described herein, an expression vector described herein, and/or a cell or population of cells described herein, and instructions for using the kit. In some embodiments, the kit comprises a CAR described herein. In some implementations, the kit further comprises written instructions for using the CAR for producing one or more antigen-specific cells for treating and/or preventing a tumor in a subject. In some embodiments, the kit comprises a cell or population of cells described herein. In some implementations, the kit further comprises written instructions for using the cell for treating and/or preventing a tumor in a subject. In some embodiments, the kit comprises an isolated nucleic acid described herein. In some aspects, the kit further comprises written instructions for using the nucleic acid for producing one or more antigen-specific cells for treating and/or preventing a tumor in a subject. In some embodiments, the kit comprises a vector described herein. In some implementations, the kit further comprises written instructions for using the vector for producing one or more antigenspecific cells for treating and/or preventing a tumor in a subject. In some embodiments, the kit comprises a composition described herein. In some implementations, the kit further comprises written instructions for using the composition for treating and/or preventing a tumor in a subject.

[0024] 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

[025] FIG. 1 shows GD2 and GM2 expression measure by flow cytometry on different neuroblastoma cell lines. Cells with low GD2 expression show high levels of GM2 (FIG.

1 A). Cells with high GD2 expression show intermediate to low GM2 levels (FIG. 1C). FIG. IB shows a simplified ganglioside synthesis pathway. GD2 and GM2 are highlighted, since the same enzyme, B4GALNT1, catalyzes the reaction that synthesis GD2 from GD3 or GM2 from GM3. This explains why cells with high GD2 expression express intermediate levels of GM2.

[026] FIG. 2 shows that GM2 is highly expressed in GD2-heterogeneous Ewing sarcoma cell lines. Most Ewing sarcoma cell lines analyzed had higher surface GM2 expression (A) than surface GD2 expression (B).

[027] FIG. 3 provides graphs showing GD2 or GM2 expressions in six different osteosarcoma patient-derived xenograft (PDX) cell lines (PSS cell lines). GD2 expression is shown in the left side panels and GM2 expression in the right side panels in both FIG 3A and 3B. Despite heterogeneous GD2 surface expression, surface GM2 expression is high on virtually all Osteosarcoma lines indicating GM2 as a potential target for osteosarcoma therapy.

[028] FIG. 4 provides a set of graphs showing surface expression of markers indicative of T cell exhaustion and cytotoxicity between CAR T cells bearing CARs with either the human CDR-grafted GM2 (huGM2) or the murine GM2 (KM966) single chain variable fragments (scFv). FIG. 4A shows CAR expression level. FIG. 4B shows surface exhaustion markers level. FIGS. 4C and 4D show the killing of Nalm6-GM2 leukemia cells and Sy5y neuroblastoma cells, respectively.

[029] FIG. 5 shows GM2-targeting DMF( 10.62.3) CARs bearing CD8 hinge- transmembrane domain CAR exhibit higher cytokine production compared to CD28 hinge- transmembrane domain CAR T cells. FIG. 5A shows CAR expression on CAR T cells with GM2-targeting 4-lBB-zeta CD8 hinge-transmembrane CAR, 4-lBB-zeta CD28 hinge- transmembrane CAR, CD28-zeta CD8 hinge-transmembrane CAR, or CD28-zeta CD28 hinge-transmembrane CAR. FIG. 5B shows cytokine production by GM2-targeting CAR T cells when co-cultured with Nalm6-GM2 leukemia cells and FIG. 5C shows killing of Nalm6-GM2 leukemia cells by the CAR T cells.

[030] FIG. 6 provides a set of graphs showing that GM2-targeting KM966 CARs bearing CD8 hinge-transmembrane domain CAR show higher cytokine production compared to CD28 hinge-transmembrane domain CAR T cells. FIG. 6A shows CAR expression on CAR T cells with GM2-targeting 4-lBB-zeta CD8 hinge-transmembrane CAR, 4-lBB-zeta CD28 hinge-transmembrane CAR, CD28-zeta CD8 hinge-transmembrane CAR, or CD28- zeta CD28 hinge-transmembrane CAR. FIG. 6B shows cytokine production by GM2- targeting CAR T cells when co-cultured with Nalm6-GM2 leukemia cells. FIG 6C shows killing of Nalm6-GM2 leukemia cells by the CAR T cells.

[031] FIG. 7 provides a set of graphs showing that GM2-targeting CARs bearing heavylight chain orientation of scFv (DMF(10.62.3-HL)) exhibit higher cytokine production and better tumor cell killing compared to CAR T cells bearing light-heavy chain orientation of scFv (DMF(10.62.3-LH)). FIG. 7A presents histograms showing expression of the CAR construct on CAR T cells with GM2-targeting heavy-light chain orientation DMF( 10.62.3) CAR, or light-heavy chain orientation DMF(10.62.3) CAR compared to mock cells. FIG. 7B shows cytokine production by GM2-targeting CAR T cells co-cultured with Nalm6-GM2 leukemia cells and FIG. 7C shows killing of Nalm6-GM2 leukemia cells by the CAR T cells. [032] FIG. 8 provides a set of graphs showing that GM2-targeting CARs bearing lightheavy chain orientation of scFv (KM966-LH) exhibit higher cytokine production and better tumor cell killing compared to CAR T cells bearing heavy-light chain orientation of scFv (KM966-HL). FIG. 8A presents histograms showing expression of the CAR construct on CAR T cells with GM2-targeting heavy-light chain orientation KM966 CAR, or light-heavy chain orientation KM966 CAR compared to mock cells. FIG. 8B shows cytokine production by GM2-targeting CAR T cells when co-cultured with Nalm6-GM2 leukemia cells and FIG. 8C shows killing of Nalm6-GM2 leukemia cells by the CAR T cells. [033] FIG. 9 provides a set of graphs showing that GM2-targeting KM966 CARs bearing CD28-zeta fragment endodomain exhibit higher cytokine production and better tumor cell killing compared to GM2-targeting KM966 CAR T cells bearing 4-lBB-zeta fragment endodomain. FIG. 9A provides histograms showing expression of the CAR construct on CAR T cells with GM2-targeting CD28-zeta CAR, or 4-lBB-zeta CAR. FIG. 9B shows cytokine production by GM2-targeting CAR T cells when co-cultured with Nalm6-GM2 leukemia cells by the CAR T cells, and FIG. 9C shows killing of Nalm6-GM2 leukemia cells (left) and Sy5y neuroblastoma cells (right) by the CAR T cells.

[034] FIG. 10 provides a set of graphs showing that GM2-targeting DMF(10.62.3) CARs bearing CD28-zeta fragment endodomain show higher cytokine production and better tumor cell killing compared to GM2-targeting DMF( 10.62.3) CAR T cells bearing 4-1BB- zeta fragment endodomain. FIG. 10A presents histograms showing expression of the CAR construct on CAR T cells with GM2-targeting CD28-zeta CAR, or 4-lBB-zeta CAR. FIG10B shows cytokine production by GM2-targeting CAR T cells when co-cultured with Nalm6- GM2 leukemia cells by the CAR T cells, and FIG. 10C shows killing of Nalm6-GM2 leukemia cells (left) and Sy5y neuroblastoma cells (right) by the CAR T cells.

[035] FIG. 11 provides a set of graphs showing surface expression of markers and in vitro functionalities demonstrating reduced exhaustion marker (LAG3, PD1, TIM3) expression when a ZAP-70 signaling domain is used (with CD28-zeta CARs also expressing lower exhaustion marker levels than 4-lBB-zeta) and similar in vitro functionality /potency for ZAP-70 vs CD28-zeta. FIG 11 A shows the expression of the CAR construct. FIG. 1 IB shows surface expression of LAG-3, TIM-3, and PD-1 exhaustion markers on GM2-targeting DMF(10.62.3)/KM966 CAR T cells with 4-lBB-zeta, CD28-zeta or ZAP70 KIDB endodomains. The killing of Nalm6-GM2 by DMF(10.62.3) CARs (FIG. 11C) or KM966 CARs (FIG. 11 D) with the various endodomains is shown.

[036] FIG. 12 provides a set of graphs showing that a GM2-targeting CD28-zeta KM966 CAR has anti-tumor function against NBSD or Sy5y neuroblastoma cells in a murine in vivo model. GD2 or GM2 expression levels were detected on NBSD (FIG. 12A) or Sy5y (FIG. 12B) neuroblastoma cells. FIG. 12 C shows in vivo efficacy of KM966-28z CAR against NBSD (left panel) or Sy5y (right panel) neuroblastoma mice models.

[037] FIG. 13 provides a set of graphs showing that ZAP70 KIDB GM2 specific CARs have enhanced anti-tumor function compared with 4-lBB-zeta or CD28-zeta CARs. In vivo efficacy I tumor measurements from (FIG. 13A), and survival of (FIG. 13B), mice inoculated with luciferase expressing neuroblastoma xenografts (Sy5y) and treated with T cells expressing the KM966-CD28-zeta CAR, KM966-4- IBB -zeta CAR, or the KM966-ZAP70 KIDB CAR are shown.

[038] FIG. 14 provides a set of graphs showing that ZAP70 KIDB GM2 specific CARs have enhanced anti-tumor function compared with 4-lBB-zeta or CD28-zeta CARs. In vivo efficacy / tumor measurements (FIG. 14A) and survival (FIG. 14B) from mice inoculated with luciferase expressing neuroblastoma xenografts (Sy5y) and treated with T cells expressing the DMF(10.62.3)-CD28-zeta CAR, DMF(10.62.3)-4-lBB-zeta CAR, or the DMF(10.62.3)-ZAP70 KIDB CAR are shown.

[039] FIG. 15 provides a set of graphs showing in vitro functionality of tandem GD2- GM2 (DMF10.62.3) CAR T cells bearing CARs with the 4-lBB-zeta fragment endodomain. FIG. 15A shows cytokine production by GD2, GM2, or tandem GD2/GM2-targeting 4-1BB- zeta fragment CAR T cells when co-cultured with Nalm6-GD2 leukemia cells, Nalm6-GM2 leukemia cells, or CHLA-255 neuroblastoma cells by the CAR T cells. FIG. 15B provides a schematic for each CAR.

[040] FIG. 16 provides a set of graphs showing the in vitro functionality of tandem GD2-GM2 CAR T cells demonstrating that an ideal orientation is anti-GM2 distal to anti- GD2 (anti-GM2 is further from the membrane and anti-GD2 is closer). T cells bearing CARs with either the 4-lBB-zeta or the CD28-zeta fragment endodomains were generated. FIG. 16A shows cytokine production by GD2, GM2, or tandem GD2/GM2-targeting 4-lBB-zeta or CD28-zeta fragment CAR T cells when co-cultured with Nalm6-GD2 leukemia cells, Nalm6-GM2 leukemia cells, CHLA-255 neuroblastoma cells, or Sy5y neuroblastoma cells. A schematic image for each CAR is shown in FIG. 16B.

[041] FIG. 17 provides graphs showing in vivo functionality of tandem GD2-GM2 CAR T cells bearing CARs with either the 4-lBB-zeta (FIG. 17A) or CD28-zeta (FIG. 17B) fragment endodomain in the Sy5y neuroblastoma mice model which expresses high GM2 and heterogeneous GD2 at baseline. Monospecific GM2-28z CAR and optimized tandem GM2- GD2-28z CAR showed improved efficacy compared to GD2 CAR T cells.

[042] FIG. 18 provides graphs showing in vivo functionality of tandem GD2-GM2 CAR T cells bearing CARs against CHLA255 (FIG. 18A) or Kelly (FIG. 18B), and ganglioside expression on tumor cells at endpoint (FIG. 18C). CHLA255 has high GD2 expression and intermediate/low GM2 expression while Kelly has intermediate GD2 and GM2 expression. FIGS. 18A and 18B show that GM2-GD2-28z tandem CAR T cells exhibited the best efficacy compared to monospecific GM2-28z CAR T cells, GD2-28z CAR T cells, or tandem GD2-GM2-28z CAR T cells. FIG 18C shows flow cytometry analysis of xenografts harvested at endpoint from CHLA255 xenograft mice that received GD2 or GM2 CAR T cells.

DETAILED DESCRIPTION

Definitions

[043] 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.

[044] 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.

[045] 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).

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

[047] 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 including those 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. [048] 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.

[049] 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.

[050] “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.

[051] 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).

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

[053] 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 of tumor 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%.

[054] 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. [055] 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.

[056] 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.

[057] 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 other embodiments, 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.

[058] 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.

[059] 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).

[060] 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 a second 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.

[061] 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.

[062] 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.

[063] 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.

[064] 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).

[065] 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).

[066] 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.

[067] 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 be 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.

[068] “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.

[069] “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 of these 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).

[070] “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.

[071] 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).

[072] 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.

[073] 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.” [074] 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” of the 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.

[075] 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.

[076] 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.

[077] 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 Molecular Biology, 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.).

Chimeric Antigen Receptors (CARs) and Cells Genetically Engineered to Express Same [078] Cells (e.g., T cells) may be genetically engineered to express a chimeric antigen receptor (CAR) targeting one or more antigens (e.g., GM2 and/or GD2).

[079] Chimeric Antigen Receptor ( CAR )

[080] A chimeric antigen receptor (CAR) refers to an artificial immune cell receptor that is engineered to recognize and bind to an antigen expressed by undesired cells, for example, disease cells such as cancer cells. A T cell that expresses a CAR polypeptide is referred to as a CAR T cell. CARs have the ability to redirect T-cell specificity and reactivity toward a selected target in a non-MHC -restricted manner. The non-MHC-restricted antigen recognition gives CAR-T cells the ability to recognize an antigen independent of antigen processing, thereby bypassing a major mechanism of tumor escape.

[081] To date, various generations of CARs have been generated, each of which contains different components. First generation CARs join an antibody-derived scFv to the CD3zeta ( or z) intracellular signaling domain of the T-cell receptor through hinge and transmembrane domains. Second generation CARs incorporate an additional co-stimulatory domain, e.g., CD28, 4-1BB (4- IBB), or ICOS, to supply a costimulatory signal. Third- generation CARs contain two or more costimulatory domains (e.g., a combination of CD27, CD28, 4-1BB, ICOS, or 0X40) fused with the TCR CD3 chain (See, e.g., Maude et al., Blood. 2015; 125(26):4017-4023; Kakarla and Gottschalk, Cancer J. 2014; 20(2): 151-155). Any generations of CAR constructs is within the scope of the present disclosure.

[082] Generally, a CAR is a fusion polypeptide comprising an extracellular domain that recognizes a target antigen (e.g. , a single chain fragment (scFv) of an antibody or other antibody fragment) and an intracellular domain comprising a signaling domain of the T-cell receptor (TCR) complex (e.g., CD3 and, in most cases, a co-stimulatory domain. (See, e.g., Enblad et al., Human Gene Therapy. 2015; 26(8):498-505). A CAR construct may further comprise a hinge and/or transmembrane domain between the extracellular domain and the intracellular domain, as well as a signal peptide at the N-terminus for surface expression. Examples of signal peptides include a signaling domain from 4-1BB, CD28 and/or CD3-zeta that may be used herein. Other signal peptides may be used.

[083] Antigen Binding Extracellular Domain

[084] The antigen-binding extracellular domain is the region of a CAR polypeptide that is exposed to the extracellular fluid when the CAR is expressed on cell surface. In some instances, a signal peptide may be located at the N-terminus to facilitate cell surface expression. In some embodiments, the antigen binding domain can be a single-chain variable fragment (scFv, which may include an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) (in either orientation). In some instances, the VH and VL fragment may be linked via a peptide linker. The linker, in some embodiments, includes hydrophilic residues with stretches of glycine and serine for flexibility as well as stretches of glutamate and lysine for added solubility. The disclosure is not limited to any particular linker. Exemplary linkers are provided herein. The scFv fragment retains the antigen-binding specificity of the parent antibody, from which the scFv fragment is derived. In some embodiments, the scFv may comprise humanized VH and/or VL domains. In other embodiments, the VH and/or VL domains of the scFv are fully human.

[085] Embodiments of the present disclosure include chimeric antigen receptors (CARs) that target cells (e.g., tumor and/or cancer cells) expressing monosialoganglioside GM2 (ganglioside GM2 or GM2), referred to herein as GM2 CARs, as well as nucleic acid molecules encoding GM2 CARs. The GM2 CAR polypeptides and polynucleotides of the present disclosure include an extracellular portion comprising an antigen binding domain specific for a GM2 antigen, a transmembrane domain, and one or more intracellular signaling domains. In some embodiments, the GM2 CARs include one or more of a hinge domain, a spacer region, and/or one or more peptide linkers. When engineered to be expressed on the surface of an immune cell (e.g., T lymphocyte, NK cell), the GM2 CARs of the present disclosure target GM2-expressing cells (e.g., tumor and/or cancer cells), which results in the targeted destruction of those cells.

[086] The disclosure provides, in some embodiments, a chimeric antigen receptor (CAR) that includes a ganglioside GM2 binding domain. In some embodiments, the antigenbinding 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, the antigen-binding domain comprises a single chain variable fragment (scFv). In some embodiments, the scFv comprises a heavy chain variable domain (VH) and a light chain variable domain (VL). In some embodiments, the VH and VL are separated by a peptide linker. Generally, an scFv has a variable domain of light chain (VL) connected from its C-terminus to the N-terminal end of a variable domain of heavy chain (VH) by a polypeptide chain. Alternately, the scFv comprises of polypeptide chain where the C-terminal end of the VH is connected to the N-terminal end of VL by a polypeptide chain. In some embodiments, the scFv comprises the structure VH-L-VL or VL- L-VH, wherein VH is the heavy chain variable domain, L is the peptide linker, and VL is the light chain variable domain. In a preferred embodiments, the disclosure provides a composition (e.g., T cells, stem cells) comprising molecules that bind GM2 (e.g., GM2 CARs, GM2 designed ankyrin repeat proteins (DARPINs), or any molecule that binds with specificity to GM2) and molecules that bind GD2 (e.g., GD2 CARs, GD2 designed ankyrin repeat proteins (DARPINs), or any molecule that binds with specificity to GD2).

[087] An sdAb is a molecule in which one variable domain of an antibody specifically binds to an antigen without the presence of the other variable domain. “Single domain antibody” or “sdAb” as used herein refer to antibody whose complementary determining regions are part of a single domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, antibodies naturally devoid of light chains, single domain antibodies derived from conventional antibodies (having two heavy and two light chains, a four chain immunoglobulin), engineered antibodies and single domain scaffolds other than those derived from antibodies. Single domain antibodies may be any of the art, or any future single domain antibodies. Single domain antibodies may be derived from any species including, but not limited to mouse, human, camel, llama, goat, rabbit, bovine. A F(ab) fragment contains the constant domain (CL) of the light chain and the first constant domain (CHI) of the heavy chain along with the variable domains VL and VH on the light and heavy chains respectively. F(ab') fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CHI domain including one or more cysteines from the antibody hinge region. F(ab’)2 fragments contain two Fab’ fragments joined, near the hinge region, by disulfide bonds.

[088] In some embodiments, the disclosure provides a chimeric antigen receptor (CAR) with an antigen recognition domain that binds with specificity to GM2. The disclosure provides examples of GM2-specific CARs. In some embodiments, the disclosure provides a chimeric antigen receptor (CAR) comprising a single chain variable fragment (scFv) that binds to GM2, wherein the scFv comprises a heavy chain variable (VH) region and a light chain variable (VL) region pair.

[089] 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 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.

[090] Table 1: CAR amino acid sequences.

*ARD - single-chain variable fragment (scFv) retaining GM2 and/or GD2 specificity

[091] In some embodiments, the chimeric antigen receptor (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 a heavy chain variable (VH) region and a light chain variable (VL), wherein the 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), 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 NO: 10-17.

[092] In some embodiments, 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 the amino acid sequence of SEQ ID NO: 10. In some embodiments, the 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), wherein the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are contained within the VH region of the amino acid sequence of SEQ ID NO: 10.

[093] In some embodiments, 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 the amino acid sequence of SEQ ID NO: 11. In some embodiments, the 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), wherein the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are contained within the VH region of the amino acid sequence of SEQ ID NO: 11.

[094] In some embodiments, 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 the amino acid sequence of SEQ ID NO: 12. In some embodiments, the 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), wherein the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are contained within the VH region of the amino acid sequence of SEQ ID NO: 12.

[095] In some embodiments, 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 the amino acid sequence of SEQ ID NO: 13. In some embodiments, the 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), wherein the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are contained within the VH region of the amino acid sequence of SEQ ID NO: 13.

[096] In some embodiments, 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 the amino acid sequence of SEQ ID NO: 14. In some embodiments, the 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), wherein the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are contained within the VH region of the amino acid sequence of SEQ ID NO: 14. [097] In some embodiments, 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 the amino acid sequence of SEQ ID NO: 15. In some embodiments, the 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), wherein the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are contained within the VH region of the amino acid sequence of SEQ ID NO: 15.

[098] In some embodiments, 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 the amino acid sequence of SEQ ID NO: 16. In some embodiments, the 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), wherein the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are contained within the VH region of the amino acid sequence of SEQ ID NO: 16.

[099] In some embodiments, 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 the amino acid sequence of SEQ ID NO: 17. In some embodiments, the 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), wherein the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are contained within the VH region of the amino acid sequence of SEQ ID NO: 17.

[0100] In some embodiments, 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, or 17.

[0101] In some embodiments, the chimeric antigen receptor (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 a heavy chain variable (VH) region and a light chain variable (VL), wherein the 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), 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 the group SEQ ID NO: 27-35.

[0102] In some embodiments, 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 the amino acid sequence of SEQ ID NO: 27. In some embodiments, the 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), and wherein the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are contained within the VL region of the amino acid sequence of SEQ ID NO: 27.

[0103] In some embodiments, 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 the amino acid sequence of SEQ ID NO: 28. In some embodiments, the 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), and wherein the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are contained within the VL region of the amino acid sequence of SEQ ID NO: 28.

[0104] In some embodiments, 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 the amino acid sequence of SEQ ID NO: 29. In some embodiments, the 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), and wherein the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are contained within the VL region of the amino acid sequence of SEQ ID NO: 29.

[0105] In some embodiments, 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 the amino acid sequence of SEQ ID NO: 30. In some embodiments, the 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), and wherein the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are contained within the VL region of the amino acid sequence of SEQ ID NO: 30. [0106] In some embodiments, 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 the amino acid sequence of SEQ ID NO: 31. In some embodiments, the 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), and wherein the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are contained within the VL region of the amino acid sequence of SEQ ID NO: 1 .

[0107] In some embodiments, 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 the amino acid sequence of SEQ ID NO: 32. In some embodiments, the 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), and wherein the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are contained within the VL region of the amino acid sequence of SEQ ID NO: 32.

[0108] In some embodiments, 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 the amino acid sequence of SEQ ID NO: 33. In some embodiments, the 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), and wherein the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are contained within the VL region of the amino acid sequence of SEQ ID NO: 33.

[0109] In some embodiments, 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 the amino acid sequence of SEQ ID NO: 34. In some embodiments, the 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), and wherein the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are contained within the VL region of the amino acid sequence of SEQ ID NO: 34.

[0110] In some embodiments, 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 the amino acid sequence of SEQ ID NO: 35. In some embodiments, the 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), and wherein the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are contained within the VL region of the amino acid sequence of SEQ ID NO: 35.

[0111] In some embodiments, 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.

[0112] In some embodiments, 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 NO: 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 NO: 27-35.

[0113] In some embodiments, 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 NO: 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 NO: 27-35.

[0114] In some embodiments, the CAR 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 NO: 59-88. In some embodiments, the CAR comprises an amino acid sequence of an amino acid sequence selected from SEQ ID NO: 59-88.

[0115] In some embodiments, 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 the amino acid sequence of SEQ ID NO: 10 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 the amino acid sequence of SEQ ID NO: 27. In some embodiments, the VH comprises a heavy chain complementarity determining region 1 (CDR- Hl), 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 the amino acid sequence of SEQ ID NO: 10; 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 the amino acid sequence of SEQ ID NO: 28. In some embodiments, 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 the amino acid sequence of SEQ ID NO: 11 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 the amino acid sequence of SEQ ID NO: 29. In some embodiments, 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) having the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 contained within the VH region amino acid sequence of SEQ ID NO: 12; 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) having the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 contained within the VL region amino acid sequence of SEQ ID NO: 20. . In some embodiments, 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) having the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 contained within the VH region amino acid sequence of SEQ ID NO: 13; 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) having the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 contained within the VL region amino acid sequence of SEQ ID NO: 20. . In some embodiments, the VH comprises a heavy chain complementarity determining region 1 (CDR- Hl), a heavy chain complementarity determining region 2 (CDR-H2), and a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequences of CDR- Hl, CDR-H2, and CDR-H3 contained within the VH region amino acid sequence of SEQ ID NO: 14; and the VL comprises a light chain complementarity determining region 1 (CDR- Ll), a light chain complementarity determining region 2 (CDR-L2), and a light chain complementarity determining region 3 (CDR-L3) having the amino acid sequences of CDR- L1 , CDR-L2, and CDR-L3 contained within the VL region amino acid sequence of SEQ ID NO: 20. . In some embodiments, 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) having the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 contained within the VH region amino acid sequence of SEQ ID NO: 16; 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) having the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 contained within the VL region amino acid sequence of SEQ ID NO: 34. . In some embodiments, 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) having the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 contained within the VH region amino acid sequence of SEQ ID NO: 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) having the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 contained within the VL region amino acid sequence of SEQ ID NO: 35.

[0116] In some embodiments, the VH comprises a heavy chain complementarity determining region 1 (CDR-H1) 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, and a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of SEQ ID NO: 3. In some embodiments, the VL comprises 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. [0117] In some embodiments, 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 the amino acid sequence of SEQ ID NO: 10 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 the amino acid sequence of SEQ ID NO: 27. In some embodiments, the VH comprises a heavy chain complementarity determining region 1 (CDR- Hl), 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 the amino acid sequence of SEQ ID NO: 10; 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 the amino acid sequence of SEQ ID NO: 27.

[0118] In some embodiments, 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 the amino acid sequence of SEQ ID NO: 11 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 the amino acid sequence of SEQ ID NO: 29. In some embodiments, the VH includes a heavy chain complementarity determining region 1 (CDR- Hl), a heavy chain complementarity determining region 2 (CDR-H2), and a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequences of CDR- Hl, CDR-H2, and CDR-H3 contained within the VH region amino acid sequence of SEQ ID NO: 11; and the VL includes 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 the amino acid sequences of CDR- Ll, CDR-L2, and CDR-L3 contained within the VL region amino acid sequence of SEQ ID NO: 29.

[0119] In some embodiments, the VH includes a heavy chain complementarity determining region 1 (CDR-H1) 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, and a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of SEQ ID NO: 6. In some embodiments, the VL includes 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.

[0120] In some embodiments, 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 the amino acid sequence of SEQ ID NO: 12 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 the amino acid sequence of SEQ ID NO: 20. In some embodiments, the VH includes a heavy chain complementarity determining region 1 (CDR- Hl), a heavy chain complementarity determining region 2 (CDR-H2), and a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequences of CDR- Hl, CDR-H2, and CDR-H3 contained within the VH region amino acid sequence of SEQ ID NO: 12; and the VL includes 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 the amino acid sequences of CDR- Ll, CDR-L2, and CDR-L3 contained within the VL region amino acid sequence of SEQ ID NO: 20.

[0121] In some embodiments, the VH includes a heavy chain complementarity determining region 1 (CDR-H1) 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, and a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of SEQ ID NO: 9. In some embodiments, the VL includes 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.

[0122] In some embodiments, the chimeric antigen receptor (CAR) comprises a single chain Fv (scFv) that binds to GM2, a transmembrane domain, and one or more intracellular signaling domains, wherein the 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.

[0123] In some embodiments, the VH and the VL are separated by a peptide linker. Peptide linkers are well known in the art. In some embodiments, the peptide linker includes an amino acid sequence selected from SEQ ID NOs: 54-55.

[0124] In some implementations, the disclosure provides an engineered nucleic acid encoding a CAR disclosed herein. In some embodiments, the nucleic acid molecule encodes at least one chimeric antigen receptor, the at least one 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. In some embodiments, 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. In some embodiments, 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 at least one heavy chain variable region 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. In some embodiments, the at least one light chain variable region 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. In other embodiments, the antigen binding domain is a scFv. In some embodiments, 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. In some aspects, the engineered nucleic acid encoding the antigen binding domain (scFv) of the chimeric antigen receptor (CAR) comprises or consists of the amino acid sequence set forth as SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 53, SEQ ID NO: 56, SEQ ID NO: 57, or SEQ ID NO: 58. In some implementations, the nucleic acid molecule further comprises a codon optimized for expression in a human T cell, and/or operably linked to an expression control sequence. In some embodiments, the nucleic acid molecule encodes two or more chimeric antigen receptors. For example, in some embodiments, one of the chimeric antigen receptors encoded by the nucleic acid molecule comprises a single chain Fv (scFv) that binds to ganglioside GM2 and another one of the chimeric antigen receptors encoded by the nucleic acid molecule comprises a single chain Fv (scFv) that binds to ganglioside GD2.

[0125] In some embodiments, the disclosure provides an engineered nucleic acid encoding a CAR comprising a single domain antibody that binds to GM2 comprising one or more CDR regions from any one of SEQ ID NOs: 10-17. In some implementations, a nucleic acid encodes a GM2 sdAb comprising 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. In some embodiments, an engineered nucleic acid encodes 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. In some embodiments, the disclosure provides an engineered nucleic acid encoding a CAR comprising a single domain antibody that binds to GD2. The disclosure is not limited by the sdAb that binds to GD2. Indeed, a sdAb of the the disclosure may include any VH region of an antibody that binds to GD2. In some ebodiments, a sdAb comprises a VH region of SEQ ID NO: 17.

[0126] In some embodiments, the engineered nucleic acid molecule encodes an antigen binding domain comprising a first heavy chain variable (VH) region and a light chain variable (VL) region pair, and a second heavy chain variable (VH) region and a light chain variable (VL) region pair, wherein the first pair is different than the second pair (e.g., the first VH-VL and the second VH-VL pair are expressed by a vector containing the engineered nucleic acid molecule). For example, in some embodiments, an engineered nucleic acid molecule encodes VH-VL pair that binds with specificity to ganglioside GM2 and another VH-VL pair that binds with specificity to ganglioside GD2. In some embodiments, the VH and VL of the scFv are separated by a peptide linker (e.g., disclosed herein and/or known in the art). In some 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.

[0127] Thus, in some embodiments, a CAR disclosed herein comprise a bicistronic chimeric antigen receptor. In some embodiments, the bicistronic chimeric antigen receptor comprises a GM2 CAR and a GD2 CAR. In some embodiments, the bicistronic chimeric antigen receptor comprises any one of the GM2 CARs described herein and any one of the GD2 CARs described herein. In some embodiments, the bicistronic chimeric antigen receptor comprises a GM2 CAR. In some embodiments, the bicistronic chimeric antigen receptor comprises a CAR with an antigen binding domain targeting GM2. In some embodiments, the bicistronic chimeric antigen receptor comprises a CAR with an antigen binding domain targeting GD2.

[0128] In some embodiments, the bicistronic chimeric antigen receptor (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 a heavy chain variable (VH) region and a light chain variable (VL), wherein the 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), 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 NO: 10-17. In some embodiments, the bicistronic chimeric antigen receptor (CAR) 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, or 17. In some embodiments, the bicistronic chimeric antigen receptor (CAR) comprises a VH region of SEQ ID NO: 15. In other embodiments, the bicistronic chimeric antigen receptor (CAR) comprises a VH region of SEQ ID NO: 17.

[0129] In some embodiments, the bicistronic chimeric antigen receptor (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 a heavy chain variable (VH) region and a light chain variable (VL), wherein the 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), 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 the group SEQ ID NO: 27-35.

[0130] In some embodiments, the bicistronic chimeric antigen receptor (CAR) 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 or 35. In some embodiments, the bicistronic chimeric antigen receptor (CAR) comprises a VL region of SEQ ID NO: 33. In other embodiments, the bicistronic chimeric antigen receptor (CAR) comprises a VH region of SEQ ID NO: 35.

[0131] In some embodiments, the bicistronic chimeric antigen receptor (CAR) comprises a single chain Fv (scFv) comprising 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: 43 or 56.

[0132] In some embodiments, the bicistronic chimeric antigen receptor comprises a CAR with two or more antigen binding domains targeting GM2 and another antigen. In some embodiments, the bicistronic chimeric antigen receptor comprises a CAR with any combination of two or more antigen binding domains as described herein.

[0133] Accordingly, in some embodiments, engineered nucleic acids of the present disclosure can be multicistronic, i.e., more than one separate polypeptide (e.g., multiple exogenous polynucleotides or GM2 CARs) can be produced from a single mRNA transcript. Engineered nucleic acids can be multicistronic through the use of various linkers, e.g., a polynucleotide sequence encoding an exogenous polynucleotide or GM2 CAR can be linked to a nucleotide sequence encoding a second exogenous polynucleotide (another GM2 CAR or a GD2 CAR), such as in a first gene: linker: second gene 5’ to 3’ orientation.

[0134] A linker can be a combination of linkers. In general, a multicistronic system can use any number or combination of linkers, to express any number of genes or portions thereof (e.g., an engineered nucleic acid can encode a first, a second, and/or a third CAR, each separated by linkers such that separate polypeptides encoded by the first, second, and third CAR molecules are produced). [0135] “Linkers,” as used herein can refer to polypeptides that link a first polypeptide sequence and a second polypeptide sequence.

[0136] In some embodiments, the present disclosure provides an engineered nucleic acid comprising an expression cassette (e.g., vector) that includes a promoter operably linked to an exogenous polynucleotide sequence encoding a GM2 CAR, GM2-GD2 tandem CAR, and/or bicistronic GM2-GD2 CAR. As used herein, “promoter” generally refers to a control region of a nucleic acid sequence at which initiation and rate of transcription of the remainder of a nucleic acid sequence are controlled. A promoter may also contain sub-regions at which regulatory proteins and molecules may bind, such as RNA polymerase and other transcription factors. Promoters may be constitutive, inducible, repressible, tissue-specific or any combination thereof. A promoter drives expression or drives transcription of the nucleic acid sequence that it regulates. A promoter is considered to be “operably linked” when it is in a correct functional location and orientation in relation to a nucleic acid sequence it regulates to control transcriptional initiation and/or expression of that sequence.

[0137] Transmembrane Domain

[0138] A CAR disclosed herein may contain a transmembrane domain. For example, the transmembrane domain may be a hydrophobic alpha helix that spans the membrane. As used herein, a “transmembrane domain” refers to any protein structure that is thermodynamically stable in a cell membrane, preferably a eukaryotic cell membrane, and more preferably, a T cell membrane. The transmembrane domain can provide stability of the CAR containing same.

[0139] In some embodiments, the transmembrane domain of a CAR described herein can be a CD8 transmembrane domain. In other embodiments, the transmembrane domain can be a CD28 transmembrane domain. In yet other embodiments, the transmembrane domain is a chimera of a CD8 and CD28 transmembrane domain. In some embodiments, the transmembrane domain is selected from a CD8 transmembrane domain, a CD28 transmembrane domain a CD3zeta-chain transmembrane domain, a CD4 transmembrane domain, a 4- IBB transmembrane domain, an 0X40 transmembrane domain, an ICOS transmembrane domain, a CTLA-4 transmembrane domain, a PD-1 transmembrane domain, a LAG-3 transmembrane domain, a 2B4 transmembrane domain, a BTLA transmembrane domain, an 0X40 transmembrane domain, a DAP10 transmembrane domain, a DAP12 transmembrane domain, a CD 16a transmembrane domain, a DNAM-1 transmembrane domain, a KIR2DS1 transmembrane domain, a KIR3DS1 transmembrane domain, an NKp44 transmembrane domain, an NKp46 transmembrane domain, an FceRlg transmembrane domain, and an NKG2D transmembrane domain. Other transmembrane domains may be used.

[0140] Hinge or Spacer Domain

[0141] In some embodiments, a hinge or spacer domain may be located between an extracellular domain (comprising the antigen binding domain) and a transmembrane domain of a CAR, or between a cytoplasmic domain and a transmembrane domain of the CAR. A hinge domain can be any oligopeptide or polypeptide that functions to link the transmembrane domain to the extracellular domain and/or the cytoplasmic domain in the polypeptide chain. A spacer or hinge domain is any oligopeptide or polypeptide that functions to link the transmembrane domain to the extracellular domain and/or the intracellular signaling domain in the polypeptide chain. A hinge domain may function to provide flexibility to the CAR, or domains thereof, or to prevent steric hindrance of the CAR, or domains thereof.

[0142] In some embodiments, a hinge domain comprises up to 300 amino acids (e.g., 10 to 100 amino acids, or 5 to 20 amino acids). In some embodiments, one or more hinge domain(s) may be included in other regions of a CAR. In some embodiments, the hinge domain may be a CD8 hinge domain. Other hinge domains may be used.

[0143] Exemplary spacer or hinge domains include, without limitation an IgG domain (such as an IgGl hinge, an IgG2 hinge, an IgG3 hinge, or an IgG4 hinge), an IgD hinge domain, a CD8a hinge domain, and a CD28 hinge domain. In some embodiments, the spacer or hinge domain is an IgG domain, an IgD domain, a CD8a hinge domain, or a CD28 hinge domain.

[0144] Intracellular Signaling Domains

[0145] The CAR constructs disclosed herein may contain one or more intracellular signaling domains. In some embodiments, the CAR polypeptides disclosed herein comprise one or more co-stimulatory signaling domains. For example, the co-stimulatory domains of CD28 and/or 4- IBB may be used to transmit a full proliferative/survival signal, together with the primary signaling mediated by CD3zeta. (e.g. , CD3zeta, and optionally one or more co- stimulatory domains), which are the functional end of the receptor. Following antigen recognition, receptors cluster and a signal is transmitted to the cell.

[0146] CD3zeta is the cytoplasmic signaling domain of the T cell receptor complex. CD3zeta contains three (3) immunoreceptor tyrosine-based activation motif (ITAM)s, which transmit an activation signal to the T cell after the T cell is engaged with a cognate antigen. [0147] In some implementations, a CAR disclosed herein comprises a CD28 costimulatory molecule. In other implementations, a CAR disclosed herein comprises a 4- IBB co-stimulatory molecule. In some embodiments, a CAR includes a CD3zeta signaling domain and a CD28 co-stimulatory domain. In other embodiments, a CAR includes a CD3zeta signaling domain and 4-1BB co-stimulatory domain. In still other embodiments, a CAR includes a CD3zeta signaling domain, a CD28 co-stimulatory domain, and a 4- IBB co- stimulatory domain.

[0148] In some embodiments, the intracellular signaling domain comprises or consists of 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 DAP12 intracellular signaling domain, a KIR2DS 1 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 DAP 10 intracellular signaling domain, an 1COS 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 KIR3DS1 intracellular signaling domain, an HVEM intracellular signaling domain, and/or a combination of two or more intracellular signaling domains. In some embodiments, the 4-1BB intracellular signaling domain comprises or consists of the amino acid sequence set forth as SEQ ID NO: 38. In some embodiments, the CD28 intracellular signaling domain comprises or consists of the amino acid sequence set forth as SEQ ID NO: 39. In some embodiments, the CD3zeta-chain intracellular signaling domain comprises or consists of the amino acid sequence set forth as SEQ ID NO: 40. In some embodiments, the ZAP70 (SRK) intracellular signaling domain comprises or consists of the amino acid sequence set forth as SEQ ID NO: 41.

[0149] In some embodiments, the GM2 CARs, GM2-GD2 tandem CARs, and/or bicistronic GM2 CARs of the present disclosure comprise a GM2 binding domain of the present disclosure and one or more intracellular signaling domains, where the one or more intracellular signaling domains is selected from: a CD3zeta-chain intracellular signaling domain, a CD97 intracellular signaling domain, a CD1 la-CD18 intracellular signaling domain, a CD2 intracellular signaling domain, an ICOS intracellular signaling domain, a CD27 intracellular signaling domain, a CD 154 intracellular signaling domain, a CD8 intracellular signaling domain, an 0X40 intracellular signaling domain, a 4- IBB intracellular signaling domain, a CD28 intracellular signaling domain, a ZAP40 intracellular signaling domain, a CD30 intracellular signaling domain, a GITR intracellular signaling domain, an HVEM intracellular signaling domain, a DAP 10 intracellular signaling domain, a DAP 12 intracellular signaling domain, and a MyD88 intracellular signaling domain. In some embodiments, the CAR comprises a CD3zeta-chain intracellular signaling domain and one or more additional intracellular signaling domains (e.g., co-stimulatory domains) selected from a CD97 intracellular signaling domain, a CD1 la-CD18 intracellular signaling domain, a CD2 intracellular signaling domain, an ICOS intracellular signaling domain, a CD27 intracellular signaling domain, a CD 154 intracellular signaling domain, a CD8 intracellular signaling domain, an 0X40 intracellular signaling domain, a 4- IBB intracellular signaling domain, a CD28 intracellular signaling domain, a ZAP40 intracellular signaling domain, a CD30 intracellular signaling domain, a GITR intracellular signaling domain, an HVEM intracellular signaling domain, a DAP 10 intracellular signaling domain, a DAP 12 intracellular signaling domain, a MyD88 intracellular signaling domain, a 2B4 intracellular signaling domain, a CD16a intracellular signaling domain, a DNAM-1 intracellular signaling domain, a KIR2DS1 intracellular signaling domain, a KIR3DS 1 intracellular signaling domain, a NKp44 intracellular signaling domain, a NKp46 intracellular signaling domain, a FceRlg intracellular signaling domain, a NKG2D intracellular signaling domain, and an EAT-2 intracellular signaling domain.

[0150] Delivery of CAR constructs to cells (e.g., T cells)

[0151] In some embodiments, a nucleic acid encoding a CAR described herein can be introduced into any type of cell disclosed herein (e.g., T cells) by methods known to those of skill in the art. For example, a coding sequence of the CAR may be cloned into a vector, which may be introduced into the genetically engineered T cells for expression of the CAR. A variety of different methods known in the art can be used to introduce any of the nucleic acids or expression vectors disclosed herein into an immune effector cell. Non-limiting examples of methods for introducing nucleic acid into a cell include: lipofection, transfection (e.g., calcium phosphate transfection, transfection using highly branched organic compounds, transfection using cationic polymers, dendrimer-based transfection, optical transfection, particle-based transfection (e.g., nanoparticle transfection), or transfection using liposomes e.g., cationic liposomes)), microinjection, electroporation, cell squeezing, sonoporation, protoplast fusion, impalefection, hydrodynamic delivery, gene gun, magnetofection, viral transfection, and nucleofection.

[0152] In specific examples, a nucleic acid encoding a CAR construct can be delivered to a cell using an adeno-associated virus (AAV). A A Vs are small viruses which integrate site- specifically into the host genome and can therefore deliver a transgene, such as CAR.

Inverted terminal repeats (ITRs) are present flanking the AAV genome and/or the transgene of interest and serve as origins of replication. Also present in the AAV genome are rep and cap proteins which, when transcribed, form capsids which encapsulate the AAV genome for delivery into target cells. Surface receptors on these capsids which confer AAV serotype, which determines which target organs the capsids will primarily bind and thus what cells the AAV will most efficiently infect.

[0153] Adeno-associated viruses are among the most frequently used viruses for gene therapy for several reasons. First, AAVs do not provoke an immune response upon administration to mammals, including humans. Second, AAVs are effectively delivered to target cells, particularly when consideration is given to selecting the appropriate AAV serotype. Finally, AAVs have the ability to infect both dividing and non-dividing cells because the genome can persist in the host cell without integration. This trait makes them an ideal candidate for gene therapy.

[0154] A nucleic acid encoding a CAR can be designed to insert into a genomic site of interest in the host T cells (e.g., using CRISPR / CAS9 technology, TALE nucleases, and/or zinc-finger nucleases). In some embodiments, the target genomic site can be in a safe harbor locus.

[0155] In some embodiments, a nucleic acid encoding a CAR e.g., via a donor template, which can be carried by a viral vector such as a retroviral vector, lentivirus vector, or adeno- associated viral (AAV) vector) can be designed such that it can insert into a genomic site of interest. In some instances, the nucleic acid may comprise a left homologous arm and a right homologous arm flanking the nucleotide sequence encoding the CAR. The left and right homologous arms are homologous to the upstream and downstream sequences of the genomic site where the CAR-coding sequence is to be inserted. In some examples, the genomic site where the CAR-coding sequence is to be inserted is also the target site of a guide RNA such that the CAR-coding nucleic acid can be inserted at the guide RNA targeting site. In some embodiments, the left homologous arm and the right homologous arm may be homologous to the sequences immediately flank the guide RNA targeting site. In some instances, the guide RNA targeting site can be deleted and replaced by the CAR-encoding nucleic acid after gene editing.

[0156] In some embodiments, the nucleic acid encoding the CAR may be inserted at a genomic site via a CRISPR/Cas9-mediated gene editing and homologous recombination. In other embodiments, a donor template may have no regions of homology to the targeted location in the DNA and may be integrated by NHEJ-dependent end joining following cleavage at the target site.

[0157] A donor template can be DNA or RNA, single-stranded and/or double-stranded, and can be introduced into a cell in linear or circular form. If introduced in linear form, the ends of the donor sequence can be protected (e.g., from exonucleolytic degradation) by methods known to those of skill in the art. For example, one or more dideoxynucleotide residues are added to the 3' terminus of a linear molecule and/or self-complementary oligonucleotides are ligated to one or both ends. See, for example, Chang et al., (1987) Proc. Natl. Acad. Sci. USA 84:4959-4963; Nehls et al., (1996) Science 272:886-889. Additional methods for protecting exogenous polynucleotides from degradation include, but are not limited to, addition of terminal amino group(s) and the use of modified intemucleotide linkages such as, for example, phosphorothioates, phosphoramidates, and O-methyl ribose or deoxyribose residues.

[0158] A donor template can be introduced into a cell as part of a vector molecule having additional sequences such as, for example, replication origins, promoters and genes encoding antibiotic resistance. Moreover, a donor template can be introduced into a cell as naked nucleic acid, as nucleic acid complexed with an agent such as a liposome or poloxamer, or can be delivered by viruses (e.g. , adenovirus, AAV, herpesvirus, retrovirus, lentivirus and integrase defective lentivirus (IDLV)).

[0159] A donor template, in some embodiments, can be inserted at a site nearby an endogenous promoter (e.g., downstream or upstream) so that its expression can be driven by the endogenous promoter. In other embodiments, the donor template may comprise an exogenous promoter and/or enhancer, for example, a constitutive promoter, an inducible promoter, or tissue-specific promoter to control the expression of the CAR.

[0160] Furthermore, exogenous sequences may also include transcriptional or translational regulatory sequences, for example, promoters, enhancers, insulators, internal ribosome entry sites, sequences encoding 2A peptides and/or polyadenylation signals. If desired, additional gene editing (e.g., gene knock-in or knock-out) can be introduced into therapeutic T cells as disclosed herein to improve T cell function and therapeutic efficacy. Cells containing nucleic acid molecules encoding CARs

[0161] Also provided herein are cells modified with a nucleic acid sequence encoding a CAR disclosed herein, and methods of producing same. These modified cells containing one or more engineered nucleic acids do not occur in nature. In some embodiments, the cells are isolated cells that recombinantly express the one or more engineered nucleic acids. In some embodiments, the engineered one or more nucleic acids are expressed from one or more vectors or a selected locus from the genome of the cell. In some embodiments, the cells are engineered to include a nucleic acid comprising a promoter operable linked to a nucleotide sequence encoding a GM2-specific CAR (e.g., a GM2 CAR, a GM2-GD2 tandem CAR, and/or a GM2-GD2 bicistronic CAR) expressing any of the peptide sequences listed in Table 1.

[0162] 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.

[0163] 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.

[0164] The disclosure is not limited by the type of cell or population of cells modified to contain an engineered nucleic acid 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 or population of cells are stem cells (e.g., induced pluripotent stem cells).

[0165] The cell or population of cells may be human cell(s) (e.g., a primary T cell, tumor infiltrating lymphocyte, hematopoietic stem cell (HSC), or natural killer cell). In some embodiments, the cell is derived from a subject to be treated with the compositions and methods disclosed 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). As detailed herein, cells can be engineered to produce 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. One skilled in the art will appreciate the choice of delivery method can depend on the specific cell type to be engineered.

[0166] Therapeutic applications

[0167] 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).

[0168] In some embodiments, cells (e.g., T cells) modified to express GM2 CAR and/or GD2 CAR are administered to a patient. In some embodiments, a population of cells modified to express both GM2 CARs and GD2 CARs are administered. In other embodiments, a population of cells modified to express one or more GM2 CARs and a separate population of cells modified to express one more GD2 CARs are administered. For example, in some embodiments, treating a patient comprises administering a mixed pool of cells comprising GD2 CAR T cells and GM2 CAR T cells. As described herein, the cells or populations of cells may be allogeneic and/or autologous. In some embodiments, the cells are induced pluripotent stem cells. [0169] 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 T cells can be administered via a systemic route of administration, such as an intraperitoneal or intravenous route.

[0170] 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.

[0171] 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.

[0172] 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. [0173] In some embodiments, 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. 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.

[0174] 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.

[0175] 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.

[0176] 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.

[0177] Kits

[0178] The present disclosure also provides kits for use in producing the genetically engineered T cells, the therapeutic T cells, and for therapeutic uses.

[0179] 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.

[0180] 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.

[0181] 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 for administration 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.

[0182] 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.

[0183] 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 invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.

EXAMPLES

[0184] 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: Generation, testing, and characterization of GM2 CARs in T cells

[00185] Materials and methods

[00186] Culture conditions and cell lines

[00187] 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. [00188] Tumor cell line identity was regularly confirmed with STR fingerprinting, and cell lines tested negative for mycoplasma approximately every 6 months.

[00189] Generation of cell lines

[00190] 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) on using a FACSAria (BD Biosciences).

[00191] 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.

[00192] Production of retroviral and lentiviral supernatant

[00193] 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.

[00194] 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 (lend virus or retrovirus) supernatant was used fresh or frozen at -80 °C for later use.

[00195] Isolation of PBMCs and T cells

[00196] 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 IU ml-1 recombinant human IL-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-buff ered 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.

[00197] Construction of CAR constructs

[00198] 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. [00199] Flow Cytometry

[00200] 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).

[00201] Primary antibodies:

[00202] Dinutuximab (Anti-GD2, Ipg/ml), DMF10.167.4 (anti-GM2, Ipg/ml) [00203] Secondary antibodies:

[00204] Alexa Fluor® 647 AffiniPure Donkey Anti-Human IgG (Jackson ImmunoResearch) (1: 100 dilution).

[00205] 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 (BV5I0 or BV650, clone F38-2E2, BioLegend, 1 :50), LAG-3 (PE, clone 3DS223H, Invitrogen, 1 :50).

[00206] Cytotoxicity assays

[00207] CAR+ T cells (day 10 after activation) were co-cultured with 50,000 tumor cells at the specified E:T ratios in complete RPMI 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.

[00208] Cytokine assays

[00209] 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). [00210] In vivo experiments

[00211] All animal experiments were performed under IACUC approved protocols. Immunodeficient NSG (NOD.Cg-Prkdcscid I12rgtmlWjl/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.

[00212] Tumor cell lines (CHLA255, SH-SY5Y and NBSD) 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.

[00213] 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).

[00214] Anti-ganglioside CAR T cell treatment

[00215] Six-to-ten-week-old male or female NOD-scid IL2Rgnull (NSG, NOD.Cg- PrkdcscidI12rgtmlWjl/SzJl) mice were inoculated intravenously with IxlO⁶ CHLA255 cells 7 days, IxlO⁶ SY5Y cells 5 or 6 days, IxlO⁶ NBSD cells 5 days, IxlO⁶ Kelly cells 7 days or lxlO⁶ Nalm6-GM2 cells 3 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, SY5Y, Nalm6-GM2, or Kelly-bearing mice received 3xl0⁶ CAR+ T cells. NBSD or SY5Y-bearing mice received 5xl0⁶ CAR+ T cells in KM966-28z in vivo experiments. 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, SY5Y, or Kelly 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.

[00216] GM2 is expressed on the surface of cancer cells

[0217] 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. 1A). Neuroblastoma cell lines with high levels of GD2 surface expression displayed lower but substantial GM2 surface expression levels (See FIG. 1C). Since the same enzyme, B4GALNT1, catalyzes the reaction that synthesis GD2 from GD3 or GM2 from GM3, the present disclosure provides that cells with high GD2 expression express intermediate levels of GM2. The discovery of GM2 expression on neuroblastoma cell lines identifies GM2 as a potential target for therapy of this high-risk pediatric cancer. [00218] Additional experiments were conducted to determine the expression level of GD2 and GM2 on Ewing sarcoma cell lines. As shown in FIG. 2, it was discovered that most Ewing sarcoma cell lines displayed 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.

[00219] 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. 3), identifying GM2 as a target for osteosarcoma, whereas previously only GD2 was identified as a target.

[00220] CAR T cells and T cell exhaustion / cytotoxicity

[00221] Experiments were performed in order to evaluate and characterize T cell exhaustion and cytotoxicity between two different chimeric antigen receptor (CAR) T cells generated, one with the human CDR-grafted GM2 (huGM2) CAR and one with the murine GM2 (KM966) single chain variable fragments, both with specificity for GM2 ganglioside. The expression of each CAR was similar (See FIG. 4A), but KM966 CAR showed less exhausted phenotype as characterized by surface expression of LAG3, PD1, and TIM3 (FIG. 4B). When the CAR T cells were co-cultured with either GFP-expressing Nalm6-GM2 or Sy5y cell lines, KM966 CAR showed better cytotoxicity than huGM2 CAR T cells (See FIGS. 4C, 4D, respectively).

[00222] In order to optimize GM2-targeting DMF(10.62.3) CAR T cells, CD8 and CD28 hinge-transmembrane domains in either 4-lBBz or CD28z CAR T cells were generated and characterized. Each was found to have similar CAR surface expression (See FIG. 5A) but only CAR T cells with CD8 hinge-transmembrane domain secreted IL-2 when cocultured with Nalm6-GM2 cell lines (See FIG. 5B). Cytotoxicity was similar among each CARs against Nalm6-GM2 cell lines (See FIG. 5C). Thus, in some embodiments, the disclosure provides that GM2 specific CARs with a CD8 transmembrane domain confer an advantage over GM2 specific CARs with a CD28 transmembrane domain (e.g., induce higher cytokine (e.g., IL2) production).

[00223] Further experiments were conducted in order to optimize GM2-targeting KM966 CAR T cells. CD8 and CD28 hinge-transmembrane domains in either 4-lBBz or CD28z CAR T cells were generated and characterized. Each had similar CAR surface expression intensity (FIG 6A) but only CAR T cells with CD8 hinge-transmembrane domain secreted IL-2 when cocultured with Nalm6-GM2 cell lines (FIG. 6B). GM-2 targeting KM966 CAR T cells with CD8 hinge-transmembrane domain displayed better cytotoxicity than CARs with CD28 hinge-transmembrane domain against Nalm6-GM2 cell lines (FIG. 6C).

[00224] Experiments were conducted to further optimize GM2-targeting DMF(10.62.3) CAR T cells. Heavy-light and light-heavy chain orientations of CAR constructs were generated. The CAR expression for each construct was characterized and observed to be similar (FIG. 7A). Notably, CAR T cells with heavy-light (HL) orientation of DMF(10.62.3) displayed better cytokine production and cytotoxicity than CAR T cells with light-heavy (LH) orientation (FIG. 7B and 7C, respectively). Thus, in some embodiments, the disclosure provides that heavy-light chain orientation DMF(10.62.3) CARs confer an advantage over light-heavy chain DMF( 10.62.3) CARs (e.g., HL orientation CARs induce higher cytokine production and/or cytotoxicity than LH orientation CARs).

[00225] Experiments were also conducted to further optimize GM2-targeting KM966 CAR T cells. Heavy-light and light-heavy chain orientations of CAR constructs were generated. The CAR expression for each construct was characterized (FIG. 8A). Notably, CAR T cells with light-heavy (LH) orientation of KM966 showed better cytokine production and cytotoxicity than CAR T cells with heavy-light (HL) orientation (FIG. 8B and 8C, respectively). Thus, in some embodiments, the disclosure provides that light-heavy chain orientation KM966 CARs confer an advantage over heavy-light chain KM966 CARs (e.g., LH orientation CARs induce higher cytokine production and/or cytotoxicity than HL orientation CARs).

[00226] In order to optimize GM2-targeting KM966 CAR T cells, 4-lBB-zeta and CD28- zeta endodomains were generated and compared in KM966 CAR T cells. The CAR expression for each construct was characterized (FIG. 9 A). CAR T cells with CD28-zeta endodomain displayed better cytokine production and cytotoxicity against GM2-high expressing cell lines Nalm6-GM2 and Sy5y (See FIG. 9B and 9C, respectively).

[00227] In order to optimize GM2-targeting DMF(10.62.3) CAR T cells, 4-lBB-zeta and CD28-zeta endodomains were generated and compared in DMF(10.62.3) CAR T cells. The CAR expression for each construct was characterized (FIG. 10A). CAR T cells with CD28- zeta endodomain displayed better cytokine production and cytotoxicity against GM2-high expressing cell lines Nalm6-GM2 and Sy5y than CAR T cells with 4-lBB-zeta endodomains (See FIG. 10B and 10C, respectively).

[00228] In order to optimize endodomains in DMF(10.62.3) or KM966, 4-lBB-zeta, CD28-zeta, and ZAP70 KIDB endodomains were compared. The CAR expression for each construct was characterized (FIG. HA) compared to mock cells. CAR T cells with ZAP70 KIDB endodomains displayed an advantage in exhaustion phenotype (characterized by the surface expression of LAG3, PD1 and TIM3) in both DMF(10.62.3) and KM966 (See FIG. 1 IB). Additionally, CD28-zeta endodomain-CARs were less exhausted than 4-lBB-zeta endodomain CARs. Each CAR T endodomain displayed similar killing without compromising CAR T cell efficacy when co-cultured with Nalm6-GM2 leukemia tumor cells (C, D). Thus, in some embodiments, the disclosure provides that GM2 CAR T cells comprising a ZAP70 KIDB endodomain confer an advantage over CD28 and 4-lBBz CARs (e.g., in some embodiments, the disclosure provides that GM2-28z CAR T cells display efficient killing of tumor cells (e.g., Nalm6-GM2 leukemia tumor cells) with reduced or absent exhaustion).

[00229] In vivo experiments

[00230] A mouse model of neuroblastoma was studied using compositions and methods of the disclosure. In particular, a GD2-low/GM2-high neuroblastoma mouse models were utilized and treated with GM2-targeting KM966 CARs. The GM2-targeting KM966 CARs displayed anti-tumor function. In particular, GM2-targeting CD28-zeta KM966 CAR displayed anti-tumor function against NBSD or Sy5y neuroblastoma cells in vivo. GD2 or GM2 expression levels on NBSD (FIG. 12 A) or Sy5y (FIG. 12B) neuroblastoma cells was characterized. FIG. 12 C shows in vivo tumor control efficacy of GM2-28z CAR T cells against NBSD (left panel) or Sy5y (right panel) neuroblastoma mice models.

[00231] In order to determine if in vivo functionality of KM966 CAR T cells depends on the endodomains used, KM966 CAR T cells bearing CD28-zeta, 4-lBB-zeta or ZAP70 KIDB endodomains were injected into GM2-high expressing Sy5y neuroblastoma mice model. In particular, mice inoculated with luciferase expressing neuroblastoma xenografts (Sy5y) were treated with T cells expressing the KM966-CD28-zeta CAR, KM966-4-lBB-zeta CAR, or the KM966-ZAP70 KIDB CAR. It was found that KM966 CARs with ZAP70 KIDB exhibited better in vivo efficacy I anti-tumor activity (FIG. 13A) and survival rate (FIG. 13B) than KM966 CARs with 4-lBB-zeta or CD28-zeta endodomains. Thus, in some embodiments, the disclosure provides that CARs with a ZAP70 KIDB endodomains have enhanced in vivo efficacy I anti-tumor function and/or survival compared to CARs with 4- IBB-zeta or CD28-zeta endodomains.

[00232] In order to determine if in vivo functionality of DMF(10.62.3) CAR T cells depends on endodomains used, DMF(10.62.3) CAR T cells bearing CD28-zeta, 4-lBB-zeta or ZAP70 KIDB endodomains were injected into GM2-high expressing Sy5y neuroblastoma mice model. In particular, mice inoculated with luciferase expressing neuroblastoma xenografts (Sy5y) were treated with T cells expressing the DMF(10.62.3)-CD28-zeta CAR, DMF(10.62.3)-4-lBB-zeta CAR, or the DMF(10.62.3)-ZAP70 KIDB CAR. It was found that DMF(10.62.3) CARs with ZAP70 KIDB exhibited better in vivo efficacy / anti-tumor activity (FIG. 14A) and survival rate (FIG. 14B) than DMF(10.62.3) CARs with 4-lBB-zeta or CD28-zeta endodomains. This further supports the finding of the disclosure that, in some embodiments, CARs with a ZAP70 KIDB endodomains have enhanced in vivo efficacy I anti-tumor function and/or survival compared to CARs with 4-lBB-zeta or CD28-zeta endodomains.

[00233] Efficacy of tandem GD2-GM2 CAR T cells

[00234] In vitro efficacy

[00235] Tandem GD2-GM2 CAR T cells were generated. A schematic showing nonlimiting examples of tandem CARs of the disclosure is shown in FIG. 15B. In order to identify in vitro functionality of tandem GD2-GM2(DMF10.62.3) CAR T cells, CAR T cells were co-cultured with GD2-high (Nalm6-GD2), GM2-high (Nalm6-GM2), or both GD2- and GM2-high (CHLA255) cell lines. Tandem GD2-GM2(DMF10.62.3)-4-lBB-zeta fragment CAR T cells were observed to secrete cytokines against all three cell lines while monospecific CARs, GD2-BBz or GM2(DMF10.62.3)-BBz, secreted cytokines only in each GD2 or GM2-expressing cell lines.

[00236] Additional tandem GD2-GM2 CAR T cells were generated. A schematic showing these non- limiting examples of tandem CARs of the disclosure is shown in FIG. 16B. In order to identify and characterize the in vitro functionality of these tandem GD2-GM2 CAR T cells, tandem GD2-GM2 CAR T cells were cocultured with GD2-high (Nalm6-GD2), GM2-high (Nalm6-GM2), GD2-high and GM2-high (CHLA255), or GD2- intermediate/GM2-high (Sy5y) cell lines. Cytokine production by GD2, GM2, or tandem GD2/GM2-targeting 4-lBB-zeta or CD28-zeta fragment CAR T cells when co-cultured with the Nalm6-GD2 leukemia cells, Nalm6-GM2 leukemia cells, CHLA-255 neuroblastoma cells, or Sy5y neuroblastoma cells in shown in FIG. 16A. The tandem GD2/GM2-targeting 4- IBB-zeta or CD28-zeta fragment CAR T cells secreted cytokines against all four cell lines while monospecific CARs, GD2-BBz or GM2-28z, secreted cytokines only in each GD2 or GM2-expressing cell lines. Tandem CARs in which the anti-GM2 binder was distal to the membrane (with anti-GD2 being closer to the membrane) outperformed those with anti-GD2 distal to the membrane (with anti-GM2 being closer to the membrane), indicating that this may be an optimal arrangement of scFv’s for tandem GD2-GM2 CARs. [00237] Thus, in some embodiments, the disclosure provides that tandem GD2-GM2 and/or GM2-GD2 CARs confer significant advantages (e.g., increased cytokine production and/or tumor cell killing) over monospecific CARs against both GD2- and GM2-expressing tumor cells.

[00238] In vivo efficacy

[00239] In order to determine and characterize in vivo functionality of tandem GD2-GM2 CAR T cells, tandem GD2-GM2-targeting 4-lBB-zeta or CD28-zeta fragment CAR T cells were injected into GD2-intermediate/GM2-high (Sy5y) cell line engrafted mice model. Tandem GM2-GD2-28z CAR showed similar efficacy compared to monospecific GM2-28z CAR T cells. In vivo functionality of tandem GD2-GM2 CAR T cells bearing CARs with either the 4-lBB-zeta (FIG. 17A) or CD28-zeta (FIG. 17B) fragment endodomain was observed in the Sy5y neuroblastoma mice model. Thus, in some embodiments, the disclosure provides that targeting GM2 with mono- or bi-specific/tandem CAR T cells overcomes resistance to GD2 CAR T cells in GD2 low/GM2 high neuroblastoma.

[00240] In order to determine and characterize the 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. Ganglioside levels at endpoint were affected by treatment. 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. 18C).

[0241] 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

1. A chimeric antigen receptor (CAR) that binds to the monosialoganglioside GM2

(GM2) wherein the 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 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 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

2. The CAR of claim 1 , 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.

3. The CAR of claims 1 or 2, wherein 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 DAP 10 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 FceRlg transmembrane domain, a KIR2DS 1 transmembrane domain, and an NKp46 transmembrane domain.

4. The CAR of any one of claims 1-3, wherein 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.

5. The CAR of any one of claims 1-4, wherein the one or more intracellular signaling domains 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 CD11 a-CDl 8 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.

6. The CAR of any one of claims 1-5, wherein 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.

7. The CAR of any one of claims 1-6, wherein the VH and VL of the scFv are separated by a peptide linker, optionally wherein 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.

8. An engineered nucleic acid encoding the CAR of any one of claims 1-7.

9. An expression vector comprising the engineered nucleic acid of claim 8.

10. An isolated cell comprising the CAR of any one of claims 1-7, the engineered nucleic acid of claim 8, or the expression vector of claim 9.

11. A population of engineered cells expressing the CAR of any one of claims 1-7, the engineered nucleic acid of claim 8, or the expression vector of claim 9.

12. The isolated cell of claim 10 or population of cells of claim 11, wherein the CAR is recombinantly expressed, optionally wherein the CAR is expressed from a vector or a selected locus from the genome of the cell.

13. The cell or population of cells of any one of claims 10-12, wherein the cell or population of cells is selected from a T cell, a CD4 T cell, a CD8 T cell, a Natural Killer (NK) cell, a Natural Killer T (NKT) cell, a gamma-delta T cell, a cytotoxic T lymphocyte (CTL), and a regulatory T cell.

14. The cell or population of cells of any one of claims 10-12, wherein the cell or population of cells is selected from a dendritic cell, 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.

15. A pharmaceutical composition comprising an effective amount of the CAR of any one of claims 1-7, the engineered nucleic acid of claim 8, the expression vector of claim 9, or the cell or population of cells of any one of claims 10-12, and a pharmaceutically acceptable carrier, pharmaceutically acceptable excipient, or a combination thereof.

16. A method of stimulating an immune response to a tumor cell in a subject comprising administering to a subject having a tumor a therapeutically effective dose of the CAR of any one of claims 1-7, the engineered nucleic acid of claim 8, the expression vector of claim 9, any of the cells of any one of claims 10-12, or the composition of claim 15.

17. A method of treating a subject having a tumor, the method comprising administering to the subject a therapeutically effective dose of the CAR of any one of claims 1-7, the engineered nucleic acid of claim 8, the expression vector of claim 9, any of the cells of any one of claims 10-12, or the composition of claim 15.

18. A kit for treating and/or preventing a tumor, comprising the CAR of any one of claims 1-7, the engineered nucleic acid of claim 8, the expression vector of claim 9, any of the cells of any one of claims 10-12, or the composition of claim 15.

19. A nucleic acid molecule encoding at least one chimeric antigen receptor, the at least one 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.

20. The nucleic acid molecule of claim 19, wherein the at least one heavy chain variable region comprises or consists of the amino acid sequence set forth as SEQ ID NO: 10 or SEQ ID NO: 1 1 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.

21. The nucleic acid molecule of claim 19 or claim 20, wherein the at least one light chain variable region 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.

22. The nucleic acid molecule of any one of claim 19-21, wherein the antigen binding domain 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.

23. The nucleic acid molecule of any one of claim 19-22, wherein the intracellular signaling domain comprises or consists of 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 DAP 10 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 KIR3DS1 intracellular signaling domain, an HVEM intracellular signaling domain, and/or a combination of two or more intracellular signaling domains.

24. The nucleic acid molecule of claim 23 wherein: the 4- IBB intracellular signaling domain comprises or consists of the amino acid sequence set forth as SEQ ID NO: 38, the CD28 intracellular signaling domain comprises or consists of the amino acid sequence set forth as SEQ ID NO: 39, the CD3zeta-chain intracellular signaling domain comprises or consists of the amino acid sequence set forth as SEQ ID NO: 40, or the ZAP70 (SRK) intracellular signaling domain comprises or consists of the amino acid sequence set forth as SEQ ID NO: 41.

25. The nucleic acid molecule of any one of claims 19-24, wherein the transmembrane domain comprises or consists of the amino acid sequence set forth as SEQ ID NO: 36, or wherein the transmembrane domain comprises or consists of the amino acid sequence set forth as SEQ ID NO: 37.

26. The nucleic acid molecule of any one of claims 19-25, wherein the chimeric antigen receptor (CAR) comprises, from N-terminus to C-terminus, the antigen binding domain, the transmembrane domain, and the at least one intracellular T-cell signaling domain and wherein the chimeric antigen receptor (CAR) further comprises a spacer domain between the at least one heavy chain variable (VH) region and the at least one light chain variable (VL) region.

27. The nucleic acid molecule of any one of claims 19-26, wherein the antigen binding domain (scFv) of the chimeric antigen receptor (CAR) comprises or consists of the amino acid sequence set forth as SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 53, SEQ ID NO: 56, SEQ ID NO: 57, or SEQ ID NO: 58.

28. The nucleic acid molecule of any one of claims 19-27, further comprising a codon optimized for expression in a human T cell, and/or operably linked to an expression control sequence.

29. A vector comprising the nucleic acid molecule of any one of claims 19-28, particularly wherein the vector is a recombinant DNA expression vector, or wherein the vector is a viral vector, particularly wherein the viral vector is a lentiviral vector, particularly wherein the vector is for use in making a chimeric antigen receptor T-cell.

30. A polypeptide comprising the chimeric antigen receptor encoded by the nucleic acid molecule of any one of claims 19-29.

31. A host cell comprising the nucleic acid molecule or vector of any one of claims 19-29, particularly wherein the host cell is a T cell.

32. A composition comprising a pharmaceutically effective amount of the nucleic acid molecule of any one of claims 19-28, the vector of claim 29, polypeptide of claim 30, or host cell of claim 31 , and a pharmaceutically acceptable carrier and/or excipient.

33. A method of stimulating a cell-mediated immune response to a tumor cell in a subject, the method comprising administering to a subject having a tumor a therapeutically effective dose of the CAR of any one of claims 1-4, the nucleic acid molecule of any one of claims 19- 28, the vector of claim 29, polypeptide of claim 30, or host cell of claim 31, or the composition of claim 32.

34. A method of treating a subject having a tumor, the method comprising administering a therapeutically effective dose of the CAR of any one of claims 1 -4, the nucleic acid molecule of any one of claims 19-28, the vector of claim 29, polypeptide of claim 30, or host cell of claim 31, or the composition of claim 32.

35. A method of making a chimeric antigen receptor (CAR) T-cell comprising transducing a T cell with the vector of claim 29, thereby making the chimeric antigen receptor (CAR) T cell.

36. A chimeric antigen receptor (CAR) encoded by the nucleic acid molecule of any of claims 19-28, for use in a method of treating a subject with a tumor, wherein the tumor comprises cell surface expression of ganglioside GM2 and/or ganglioside GD2, the method comprising: administering to the subject a therapeutically effective amount of T-cells expressing the chimeric antigen receptor, under conditions sufficient to form an immune complex of the antigen binding domain on the chimeric antigen receptor and ganglioside GM2 and/or ganglioside GD2 in the subject, particularly wherein the T-cells are T cells from the subject that have been transformed with the nucleic acid molecule of any one of claims 19-28 encoding the chimeric antigen receptor or transduced with a vector comprising the nucleic acid molecule.

37. The chimeric antigen receptor (CAR) for use of claim 36, wherein the method further comprises the steps of: obtaining the T cells from the subject, and transforming the T cells with the nucleic acid molecule encoding the chimeric antigen receptor or transducing the T cells with a vector comprising the nucleic acid molecule.

38. The chimeric antigen receptor (CAR) for use of claim 36 or 37, wherein the tumor is a neuroblastoma, sarcoma or a brain tumor.

39. The chimeric antigen receptor for use of claim 36 or 37, wherein the method further comprises selecting the subject by detecting cell-surface expression of ganglioside GM2 on the tumor.

40. A kit for making a chimeric antigen receptor (CAR) T-cell or treating a tumor in a subject wherein the tumor comprises cell surface expression of ganglioside GM2, comprising a container comprising the CAR of any one of claims 1-4, the nucleic acid molecule of any one of claims 19-28, the vector of claim 29, the polypeptide of claim 30, and/or host cell of claim 31 , and instructions for using the kit.

41. The nucleic acid molecule of claim 19, wherein the nucleic acid molecule is a component of a bicistronic vector that encodes an antigen binding domain comprising a first heavy chain variable (VH) region and a light chain variable (VL) region pair with specificity for ganglioside GM2, and a second heavy chain variable (VH) region and a light chain variable (VL) region pair with specificity for ganglioside GD2.

42. The nucleic acid molecule of claim 41 , wherein one of the chimeric antigen receptors comprises a single chain Fv (scFv) that binds to ganglioside GM2 and one of the chimeric antigen receptors comprises a single chain Fv (scFv) that binds to ganglioside GD2.

43. The nucleic acid molecule of claim 41, wherein the nucleic acid molecule encodes a heavy chain variable (VH) region 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, or 17.

44. The nucleic acid molecule of claim 41 , wherein the nucleic acid molecule encodes a light chain variable (VL) region 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 or 35.

45. The nucleic acid molecule of claim 41, wherein the nucleic acid molecule encodes a single chain Fv (scFv) comprising 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: 43 or 56.

46. A nucleic acid encoding a CAR comprising a single domain antibody (sdAb) specific for GM2.

47. The nucleic acid of claim 46, wherein the CAR 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.

48. The nucleic acid of any one of claims 46-47, wherein the CAR 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.

49. A nucleic acid encoding the CAR of any one of claims 46-48.

50. An expression vector comprising the nucleic acid of claim 49.

51. An isolated cell comprising the nucleic acid of claim 49 or the expression vector of claim 50.

52. A population of engineered cells expressing the nucleic acid of claim 49 or the expression vector of claim 50.

53. A composition comprising engineered cells, wherein the engineered cells comprise a first population of engineered cells expressing the nucleic acid of claim 49 or the expression vector of claim 50, and a second population of engineered cells expressing the CAR of any one of claims 1-7, the engineered nucleic acid of claim 8, or the expression vector of claim 9.

54. The cell or population of cells of any one of claims 10-12 or 51-53, wherein the cells are stem cells.

55. A method of stimulating an immune response to a tumor cell in a subject comprising administering to a subject having a tumor a therapeutically effective dose of the CAR of any one of claims 1-7, the engineered nucleic acid of claim 8 or claim 49, the expression vector of claim 9 or claim 50, any of the cells of any one of claims 10-12 and 51-53, or the composition of claim 15.

56. A method of treating a subject having a tumor, the method comprising administering a a therapeutically effective dose of the CAR of any one of claims 1-7, the engineered nucleic acid of claim 8 or claim 49, the expression vector of claim 9 or claim 50, any of the cells of any one of claims 10-12 and 51-53, or the composition of claim 15.

57. The method of claim 56, wherein the cells are autologous.

58. The method of claim 56, wherein the cells are allogeneic.

59. The cell or population of cells of any one of claims 10-12 or 51-53, wherein the cell or population of cells is selected from a dendritic cell, 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.