Chimeric antigen receptors targeting monosialoganglioside gm2 and uses thereof

Abstract

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

Description

[0001] Statement of related applications This application claims priority to 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.

[0002] sequence list The accompanying computer-readable sequence list text, titled "STDU2-42495-601_SQL.xml", was created on October 29, 2024, and is 101,821 bytes in size. It is incorporated herein by reference in its entirety. Technical Field

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

[0004] In terms of lost life and healthcare costs, cancer is one of the most devastating diseases. It also presents unmet clinical needs. Cancer is typically treated with surgery, chemotherapy, radiation therapy, or a combination thereof. However, these treatments often have significant side effects, including immune system suppression, damage to normal cells, autoimmune disorders, abnormal cellular metabolism, and even metastasis and the development of secondary cancers. Summary of the Invention

[0005] This disclosure provides chimeric antigen receptors (CARs) that bind to monosialotetrahexosylganglioside GM2 (ganglioside GM2 or GM2), compositions comprising these CARs, and methods of utilizing these CARs (e.g., for therapeutic and / or prophylactic treatment). In some embodiments, 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 at least one heavy chain variable (VH) region and at least one light chain variable (VL) region pair. In some aspects, the VH and VL pairs are selected from the sequences listed in Table 1. In some embodiments, various CDR combinations of the VH and VL pairs are selected from the sequences listed in Table 1.

[0006] In some embodiments, the chimeric antigen receptor (CAR) that binds to ganglioside GM2 (GM2) comprises a GM2-binding single-chain Fv (scFv), a transmembrane domain, and one or more intracellular signal transduction 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) VH and VL are selected from: i. The VH region comprising heavy chain complementarity-determining region 1 (CDR-H1) having the amino acid sequence of SEQ ID NO: 1, heavy chain complementarity-determining region 2 (CDR-H2) having the amino acid sequence of SEQ ID NO: 2, and heavy chain complementarity-determining region 3 (CDR-H3) having the amino acid sequence of SEQ ID NO: 3, and The VL region includes light chain complementarity-determining region 1 (CDR-L1) with the amino acid sequence of SEQ ID NO: 18, light chain complementarity-determining region 2 (CDR-L2) with the amino acid sequence of SEQ ID NO: 19, and light chain complementarity-determining region 3 (CDR-L3) with the amino acid sequence of SEQ ID NO: 20. ii. The VH region comprising heavy chain complementarity-determining region 1 (CDR-H1) having the amino acid sequence of SEQ ID NO: 4, heavy chain complementarity-determining region 2 (CDR-H2) having the amino acid sequence of SEQ ID NO: 5, and heavy chain complementarity-determining region 3 (CDR-H3) having the amino acid sequence of SEQ ID NO: 6, and The VL region includes light chain complementarity-determining region 1 (CDR-L1) having the amino acid sequence of SEQ ID NO: 21, light chain complementarity-determining region 2 (CDR-L2) having the amino acid sequence of SEQ ID NO: 22, and light chain complementarity-determining region 3 (CDR-L3) having the amino acid sequence of SEQ ID NO: 23; and iii. A VH region comprising heavy chain complementarity-determining region 1 (CDR-H1) having the amino acid sequence of SEQ ID NO: 7, heavy chain complementarity-determining region 2 (CDR-H2) having the amino acid sequence of SEQ ID NO: 8, and heavy chain complementarity-determining region 3 (CDR-H3) having the amino acid sequence of SEQ ID NO: 9, and The VL region includes light chain complementarity-determining region 1 (CDR-L1) having the amino acid sequence of SEQ ID NO: 24, light chain complementarity-determining region 2 (CDR-L2) having the amino acid sequence of SEQ ID NO: 25, and light chain complementarity-determining region 3 (CDR-L3) having the amino acid sequence of SEQ ID NO: 26; or (B) The VH includes: Heavy chain complementarity-determining regions 1 (CDR-H1), 2 (CDR-H2), and 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 sequences selected from SEQ ID NO: 10-17, and The VL includes: Light chain complementarity-determining regions 1 (CDR-L1), 2 (CDR-L2), and 3 (CDR-L3), wherein the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are contained within the VL region of the amino acid sequences selected from SEQ ID NO: 27-35; or (C) The VH includes: Amino acid sequences having 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 with amino acid sequences selected from SEQ ID NO: 10-17; and The VL includes: Amino acid sequences having 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 with the amino acid sequences selected from SEQ ID NO: 27-35, or (D) The VH region includes: Amino acid sequences having 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 with amino acid sequences selected from SEQ ID NO: 10, 11, 12, 13, 14, 15, 16, and 17, and The VL region includes: Amino acid sequences having 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 with the amino acid sequences selected from SEQ ID NO: 27, 28, 29, 30, 31, 32, 33, 34, and 35. Optionally, the CAR includes one or more of a hinge domain, a spacer region, or a peptide linker.

[0007] In some implementations, the single-chain Fv (scFv) is selected from amino acid sequences having 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 with amino acid sequences selected from SEQ ID NO: 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 the CD8 transmembrane domain, CD28 transmembrane domain, 4-IBB transmembrane domain, CD3ζ chain transmembrane domain, PD-1 transmembrane domain, DAP10 transmembrane domain, CTLA-4 transmembrane domain, CD16a transmembrane domain, OX40 transmembrane domain, NKG2D transmembrane domain; CD4 transmembrane domain, LAG-3 transmembrane domain, OX40 transmembrane domain, NKp44 transmembrane domain, ICOS transmembrane domain, DAP12 transmembrane domain, BTLA transmembrane domain, KIR3DS1 transmembrane domain, 2B4 transmembrane domain, DNAM-1 transmembrane domain, FceRlg transmembrane domain, KIR2DS1 transmembrane domain, and NKp46 transmembrane domain.

[0009] In another aspect, the transmembrane domain is selected from amino acid sequences having 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 with amino acid sequences selected from SEQ ID NO: 36 and 37.

[0010] In some embodiments, one or more intracellular signal transduction domains are each selected from the following intracellular signal transduction domains: 4-1BB intracellular signal transduction domain, CD28 intracellular signal transduction domain, CD3ζ chain intracellular signal transduction domain, ZAP70(SRK) intracellular signal transduction domain, ZAP40 intracellular signal transduction domain, CD30 intracellular signal transduction domain, OX40 intracellular signal transduction domain, CD27 intracellular signal transduction domain, DAP12 intracellular signal transduction domain, KIR2DS1 intracellular signal transduction domain, NKG2D intracellular signal transduction domain, FceRlg intracellular signal transduction domain, MyD88 intracellular signal transduction domain, EAT-2 intracellular signal transduction domain, DA The intracellular signal transduction domains of P10, ICOS, DNAM-1, CD2, CD8, CD16a, CD97, CD154, GITR, NKp46, 2B4, CD11a-CD18, NKp44, KIR3DS1, and HVEM, and / or combinations of two or more intracellular signal transduction domains.

[0011] In another aspect, one or more intracellular signal transduction domains are each selected from amino acid sequences having 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 with amino acid sequences selected from SEQ ID NO:38, 39, 40, and 41.

[0012] In some embodiments, the VH and VL regions of the scFv are separated by a peptide linker. In other 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, this disclosure provides a chimeric antigen receptor (CAR) comprising a ganglioside GM2-binding domain. In some embodiments, the GM2 antigen-binding domain comprises an antibody, an antigen-binding fragment of an antibody, an F(ab) fragment, an F(ab') fragment, a single-chain variable fragment (scFv), or a single-domain antibody (sdAb). In some aspects, the CAR comprises one or more of a hinge domain, a spacer region, a frame region, and / or one or more peptide linkers. In some embodiments, the CAR comprises a spacer region between the scFv and a transmembrane domain. In other embodiments, the CAR comprises a single-domain antibody (sdAb) lacking an immunoglobulin light chain sequence. For example, in some embodiments, this document provides a single-domain antibody that binds to GM2, the single-domain antibody comprising one or more CDR regions from any one of SEQ ID NO: 10-17. In some embodiments, GM2sdAb includes heavy chain complementarity-determining region 1 (CDR-H1), heavy chain complementarity-determining region 2 (CDR-H2), and 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, or, in other embodiments, are contained within the VH region of an amino acid sequence having 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 with an amino acid sequence selected from SEQ ID NO: 10-17.

[0014] In some embodiments, this disclosure includes compositions comprising the CAR described herein, as well as pharmaceutically acceptable loads and / or pharmaceutically acceptable excipients.

[0015] This disclosure also provides engineered nucleic acids encoding the CARs 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 signal transduction domain, wherein the antigen-binding domain includes 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 includes heavy chain complementarity-determining region 1 (CDR-H1), heavy chain complementarity-determining region 2 (CDR-H2), and heavy chain complementarity-determining region 3 (CDR-H3), the VH region having an amino acid sequence having 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 with an amino acid sequence selected from SEQ ID NO: 10-17, and wherein the at least one light chain variable (VL) region includes light chain complementarity-determining region 1 (CDR-L1), light chain complementarity-determining region 2 (CDR-L2), and light chain complementarity-determining region 3 (CDR-L3), the VL ...2%, The amino acid sequence of 27-35 has 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% amino acid sequence identity. In some embodiments, at least one heavy chain variable region comprises or consists of the following: amino acid sequence shown in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, 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, at least one light chain variable region comprises or consists of the following amino acid sequences shown in SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, 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 scFv, wherein scFv comprises or consists of an amino acid sequence having 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 with an amino acid sequence selected from SEQ ID NO: 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 56, 57, and 58. In some embodiments, the intracellular signal transduction domain includes or is composed of the following: 4-1BB intracellular signal transduction domain, CD28 intracellular signal transduction domain, CD3ζ chain intracellular signal transduction domain, ZAP70(SRK) intracellular signal transduction domain, ZAP40 intracellular signal transduction domain, CD30 intracellular signal transduction domain, OX40 intracellular signal transduction domain, CD27 intracellular signal transduction domain, DAP12 intracellular signal transduction domain, KIR2DS1 intracellular signal transduction domain, NKG2D intracellular signal transduction domain, FceRlg intracellular signal transduction domain, MyD88 intracellular signal transduction domain, EAT-2 intracellular signal transduction domain, D... The 4-1BB intracellular signal transduction domain comprises or consists of the following intracellular signal transduction domains: AP10, ICOS, DNAM-1, CD2, CD8, CD16a, CD97, CD154, GITR, NKp46, 2B4, CD11a-CD18, NKp44, KIR3DS1, HVEM, and / or combinations of two or more intracellular signal transduction domains. In some embodiments, the 4-1BB intracellular signal transduction domain comprises or consists of the amino acid sequence shown in SEQ ID NO: 38. In some embodiments, the CD28 intracellular signal transduction domain comprises or consists of the amino acid sequence shown in SEQ ID NO: 39. In some embodiments, the CD3ζ chain intracellular signal transduction domain comprises or consists of the amino acid sequence shown in SEQ ID NO: 40. In some embodiments, the ZAP70(SRK) intracellular signal transduction domain comprises or consists of the amino acid sequence shown in SEQ ID NO: 41.In some embodiments, the transmembrane domain comprises or consists of the amino acid sequence shown in SEQ ID NO: 36. In other embodiments, the transmembrane domain comprises or consists of the amino acid sequence shown in SEQ ID NO: 37. In some embodiments, the nucleic acid molecule encoding a chimeric antigen receptor (CAR) comprises, from the N-terminus to the C-terminus, an antigen-binding domain, a transmembrane domain, and at least one intracellular T-cell signaling domain, and wherein the chimeric antigen receptor (CAR) further comprises a spacer domain between at least one heavy chain variable (VH) region and at least one light chain variable (VL) region. In some aspects, the engineered nucleic acid encoding the antigen-binding domain (scFv) of a chimeric antigen receptor (CAR) comprises or consists of the following: amino acid sequences shown in 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 embodiments, the nucleic acid molecule further includes codons optimized for expression in human T cells and / or operatively linked to expression control sequences. 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 a nucleic acid molecule includes a single-chain Fv (scFv) that binds to ganglioside GM2, and another chimeric antigen receptor encoded by a nucleic acid molecule includes a single-chain Fv (scFv) that binds to ganglioside GD2. In other embodiments, this disclosure provides engineered nucleic acids encoding a CAR comprising a single-domain antibody that binds to GM2, the single-domain antibody comprising one or more CDR regions from any one of SEQ ID NO: 10-17. In some embodiments, the nucleic acid encodes a GM2 sdAb comprising heavy chain complementarity-determining region 1 (CDR-H1), heavy chain complementarity-determining region 2 (CDR-H2), and heavy chain complementarity-determining region 3 (CDR-H3), wherein the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are contained within a VH region selected from the amino acid sequences of SEQ ID NO: 10-17.In some embodiments, the engineered nucleic acid encodes an amino acid sequence having 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 with an amino acid sequence selected from SEQ ID NO: 10-17. In some embodiments, this disclosure provides engineered nucleic acids encoding a CAR comprising a single-domain antibody that binds to GD2. This disclosure is not limited to sdAbs that bind to GD2. In fact, the sdAb of this disclosure may comprise any VH region of an antibody that binds to GD2. In some embodiments, the sdAb comprises the VH region of SEQ ID NO: 17.

[0016] This disclosure also provides expression vectors comprising the engineered nucleic acids described herein. In some embodiments, the vector is a recombinant DNA expression vector. In some embodiments, the vector is a viral vector. This disclosure is not limited to the type of vector, and any type of vector described herein or known in the art can be used. In some embodiments, the vector is a retroviral vector (e.g., 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, adeno-associated virus (AAV), or virus-like particle (VLP). In a preferred embodiment, the vector is a vector for preparing chimeric antigen receptor T cells.

[0017] This disclosure provides polypeptides comprising chimeric antigen receptors encoded by engineered nucleic acid molecules described herein.

[0018] This disclosure also provides isolated cells comprising the CAR described herein, cells modified to include the engineered nucleic acids disclosed herein, expression vectors comprising the engineered nucleic acids described herein, and methods for preparing them. For example, in some embodiments, this disclosure provides a method for preparing modified cells, the method comprising transducing isolated cells with the engineered nucleic acids or expression vectors described herein. In some embodiments, this disclosure provides cell populations comprising the CAR described herein. In some embodiments, the CAR is recombinantly expressed by cells or cell populations. In other embodiments, the CAR is expressed by a vector or selected locus of the cell genome. In further embodiments, compositions are provided comprising the engineered nucleic acids or expression vectors described herein, and pharmaceutically acceptable loads, pharmaceutically acceptable excipients, or combinations thereof. This disclosure is not limited to the cell types modified using the compositions and methods described herein. In some embodiments, the modified cells are autologous cells. In other embodiments, the cells are allogeneic cells. In some embodiments, the cells or cell populations are T cells, CD4 T cells, CD8 T cells, natural killer (NK) cells, natural killer T (NKT) cells, γ-δ T cells, cytotoxic T lymphocytes (CTLs), or regulatory T cells. In other embodiments, the cells or cell populations are dendritic cells, tumor-infiltrating lymphocytes (TILs), macrophages, monocytes, neutrophils, B cells, lymphoid cells, eosinophils, mast cells, basophils, erythrocytes, myeloid cells, platelet cells, stem cells, or mesenchymal matrix cells.

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

[0020] This disclosure provides a method for stimulating a subject to produce an immune response against tumor cells and / or cancer cells, the method comprising administering to a subject suffering from a tumor and / or cancer a therapeutically effective dose of the CAR described herein, the engineered nucleic acid described herein, the expression vector described herein, and / or the cells or cell populations described herein.

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

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

[0023] Embodiments of this disclosure include kits for treating and / or preventing tumors and / or cancers. For example, in some embodiments, this disclosure provides kits for preparing chimeric antigen receptor (CAR) T cells or treating tumors in a subject, wherein the tumor comprises cell surface expression of ganglioside GM2. In some embodiments, the kit includes a container comprising the CAR described herein, the engineered nucleic acid described herein, the expression vector described herein, and / or the cells or cell populations described herein, and instructions for using the kit. In some embodiments, the kit includes the CAR described herein. In some embodiments, the kit further includes written instructions regarding the use of the CAR to generate one or more antigen-specific cells for treating and / or preventing tumors in a subject. In some embodiments, the kit includes the cells or cell populations described herein. In some embodiments, the kit further includes written instructions regarding the use of cells to treat and / or prevent tumors in a subject. In some embodiments, the kit includes isolated nucleic acids described herein. In some aspects, the kit further includes written instructions regarding the use of nucleic acids to generate one or more antigen-specific cells for treating and / or preventing tumors in a subject. In some embodiments, the kit includes the vector described herein. In some embodiments, the kit further includes written instructions regarding the use of a carrier to generate one or more antigen-specific cells for the treatment and / or prevention of tumors in a subject. In some embodiments, the kit includes the compositions described herein. In some embodiments, the kit further includes written instructions regarding the use of the compositions to treat and / or prevent tumors in a subject.

[0024] These and other features, aspects and advantages of this disclosure will be better understood through the following description and accompanying drawings. Attached Figure Description

[0025] Figure 1 shows the expression of GD2 and GM2 in different neuroblastoma cell lines as measured by flow cytometry. Cells with low GD2 expression showed high levels of GM2 (Figure 1A). Cells with high GD2 expression showed medium to low levels of GM2 (Figure 1C). Figure 1B shows a simplified ganglioside synthesis pathway. GD2 and GM2 are highlighted because the same enzyme, B4GALNT1, catalyzes the synthesis of GD2 from GD3 or GM2 from GM3. This explains why cells with high GD2 expression express medium levels of GM2.

[0026] Figure 2 shows that GM2 is highly expressed in the GD2 heterogeneous Ewing sarcoma cell line. Surface GM2 expression (A) was higher than surface GD2 expression (B) in most of the analyzed Ewing sarcoma cell lines.

[0027] Figure 3 shows the expression of GD2 or GM2 in six different osteosarcoma patient-derived xenograft (PDX) cell lines (PSS cell lines). The left insets in Figures 3A and 3B show GD2 expression, and the right inset shows GM2 expression. Despite the heterogeneity of GD2 surface expression, almost all osteosarcoma cell lines showed high surface GM2 expression, suggesting that GM2 is a potential target for osteosarcoma therapy.

[0028] Figure 4 provides a set of graphs showing the surface expression of markers indicating T cell exhaustion and cytotoxicity between CAR-carrying T cells and human CDR-transplanted GM2 (huGM2) or mouse GM2 (KM966) single-chain variable fragments (scFv). Figure 4A shows CAR expression levels. Figure 4B shows the levels of surface exhaustion markers. Figures 4C and 4D show the killing of Nalm6-GM2 leukemia cells and Sy5y neuroblastoma cells, respectively.

[0029] Figure 5 shows that GM2-targeting DMF (10.62.3) CARs carrying CD8 hinge transmembrane domain CARs exhibit higher cytokine production compared to CAR T cells carrying CD8 hinge transmembrane domain CARs. Figure 5A shows CAR expression on CAR T cells carrying GM2-targeting 4-1BB-ζ CD8 hinge-transmembrane CARs, 4-1BB-ζ CD28 hinge-transmembrane CARs, CD28-ζ CD8 hinge-transmembrane CARs, or CD28-ζ CD28 hinge-transmembrane CARs. Figure 5B shows cytokines produced by GM2-targeting CAR T cells when co-cultured with Nalm6-GM2 leukemia cells, and Figure 5C shows the killing effect of CAR T cells on Nalm6-GM2 leukemia cells.

[0030] Figure 6 provides a set of graphs showing that KM966 CARs carrying CD8 hinge-transmembrane domain CARs targeting GM2 exhibit higher cytokine production compared to CAR T cells carrying CD28 hinge-transmembrane domain CARs. Figure 6A shows CAR expression on CAR T cells carrying GM2-targeting 4-1BB-ζ CD8 hinge-transmembrane CARs, 4-1BB-ζ CD28 hinge-transmembrane CARs, CD28-ζ CD8 hinge-transmembrane CARs, or CD28-ζ CD28 hinge-transmembrane CARs. Figure 6B shows the cytokines produced by GM2-targeting CAR T cells when co-cultured with Nalm6-GM2 leukemia cells. Figure 6C shows the killing effect of CAR T cells on Nalm6-GM2 leukemia cells.

[0031] Figure 7 provides a set of graphs showing that GM2-targeting CARs carrying scFv in the heavy-light chain direction (DMF(10.62.3-HL)) exhibited higher cytokine production and better tumor cell killing compared to CAR T cells carrying scFv in the light-heavy chain direction (DMF(10.62.3-LH)). Figure 7A presents a histogram showing the expression of CAR constructs on CAR T cells, with either GM2-targeting heavy-light chain direction DMF(10.62.3) CAR or light-heavy chain direction DMF(10.62.3) CAR compared to mock cells. Figure 7B shows the cytokines produced by GM2-targeting CAR T cells co-cultured with Nalm6-GM2 leukemia cells, and Figure 7C shows the killing effect of CAR T cells on Nalm6-GM2 leukemia cells.

[0032] Figure 8 provides a set of graphs showing that the GM2-targeting CAR (KM966-LH) carrying scFv in the light-to-heavy chain direction exhibits higher cytokine production and better tumor cell killing compared to the heavy-to-light chain CAR T cells (KM966-HL) carrying scFv. Figure 8A presents a histogram showing the expression of the CAR construct on CAR T cells, which, compared to blank control cells, contain either the GM2-targeting heavy-to-light chain KM966 CAR or the light-to-heavy chain KM966 CAR. Figure 8B shows the cytokines produced by GM2-targeting CAR T cells when co-cultured with Nalm6-GM2 leukemia cells, and Figure 8C shows the killing effect of CAR T cells on Nalm6-GM2 leukemia cells.

[0033] Figure 9 provides a set of graphs showing that KM966 CARs carrying the CD28-ζ intracellular domain, targeting GM2, exhibit higher cytokine production and better tumor cell killing compared to KM966 CARs carrying the 4-1BB-ζ intracellular domain. Figure 9A provides a histogram showing the expression of CAR constructs on CAR T cells carrying either the CD28-ζ CAR or the 4-1BB-ζ CAR targeting GM2. Figure 9B shows the cytokines produced by GM2-targeting CAR T cells when co-cultured with Nalm6-GM2 leukemia cells, and Figure 9C shows the killing effect of CAR T cells on Nalm6-GM2 leukemia cells (left) and Sy5y neuroblastoma cells (right).

[0034] Figure 10 provides a set of graphs showing that the GM2-targeting DMF (10.62.3) CAR carrying the CD28-ζ intracellular domain exhibits higher cytokine production and better tumor cell killing compared to the GM2-targeting DMF (10.62.3) CAR carrying the 4-1BB-ζ intracellular domain. Figure 10A presents a histogram showing the expression of CAR constructs on CAR T cells with either the GM2-targeting CD28-ζ CAR or the 4-1BB-ζ CAR. Figure 10B shows the cytokines produced by the GM2-targeting CAR T cells when co-cultured with Nalm6-GM2 leukemia cells, and Figure 10C shows the killing effect of CAR T cells on Nalm6-GM2 leukemia cells (left) and Sy5y neuroblastoma cells (right).

[0035] Figure 11 provides a set of graphs showing the surface expression and in vitro function of the markers, indicating that the expression of exhaustion markers (LAG3, PD1, TIM3) is reduced when the ZAP-70 signaling domain is used (the level of exhaustion markers expressed by CD28-ζ CAR is lower than that of 4-1BB-ζ), and that ZAP-70 has similar in vitro function / potency to CD28-ζ. Figure 11A shows the expression of the CAR construct. Figure 11B shows the surface expression of LAG-3, TIM-3, and PD-1 exhaustion markers on GM2-targeting DMF(10.62.3) / KM966 CAR T cells with 4-1BB-ζ, CD28-ζ, or ZAP70 KIDB intracellular domains. The killing effect of DMF(10.62.3) CAR (Figure 11C) or KM966 CAR (Figure 11D) with various intracellular domains on Nalm6-GM2 is shown.

[0036] Figure 12 provides a set of graphs showing the anti-tumor function of CD28-ζ KM966 CAR targeting GM2 against NBSD or Sy5y neuroblastoma cells in an in vivo mouse model. The expression levels of GD2 or GM2 on NBSD (Figure 12A) or Sy5y (Figure 12B) neuroblastoma cells were examined. Figure 12C shows the in vivo efficacy of KM966-28z CAR against NBSD (left inset) or Sy5y (right inset) neuroblastoma mouse models.

[0037] Figure 13 provides a set of graphs showing the enhanced antitumor function of the ZAP70 KIDB GM2-specific CAR compared to 4-1BB-ζ or CD28-ζ CARs. The in vivo efficacy / tumor measurements (Figure 13A) and survival (Figure 13B) are shown in mice inoculated with luciferase-expressing neuroblastoma xenografts (Sy5y) and treated with T cells expressing KM966-CD28-ζ CAR, KM966-4-1BB-ζ CAR, or KM966-ZAP70 KIDB CAR.

[0038] Figure 14 provides a set of graphs showing the enhanced antitumor function of the ZAP70 KIDB GM2-specific CAR compared to 4-1BB-ζ or CD28-ζ CARs. The in vivo efficacy / tumor measurements (Figure 14A) and survival (Figure 14B) are shown in mice inoculated with neuroblastoma xenografts expressing luciferase (Sy5y) and treated with T cells expressing DMF(10.62.3)-CD28-ζ CAR, DMF(10.62.3)-4-1BB-ζ CAR, or DMF(10.62.3)-ZAP70KIDB CAR.

[0039] Figure 15 provides a set of diagrams showing the in vitro function of tandem GD2-GM2 (DMF10.62.3) CAR T cells carrying a CAR with an intracellular domain of the 4-1BB-ζ fragment. Figure 15A shows the cytokines produced by CAR T cells when co-cultured with Nalm6-GD2 leukemia cells, Nalm6-GM2 leukemia cells, or CHLA-255 neuroblastoma cells, either GD2, GM2, or tandem GD2 / GM2-targeting 4-1BB-ζ fragment CARs. Figure 15B provides a schematic diagram of each CAR.

[0040] Figure 16 provides a set of diagrams illustrating the in vitro function of tandem GD2-GM2 CAR T cells, showing the ideal orientation where anti-GM2 is further away from anti-GD2 (anti-GM2 is further away from the membrane, and anti-GD2 is closer). T cells carrying CARs with intracellular domains of the 4-1BB-ζ or CD28-ζ fragments are generated. Figure 16A shows cytokine production when GD2, GM2, or tandem GD2 / GM2-targeting 4-1BB-ζ or CD28-ζ fragment CAR T cells are co-cultured with Nalm6-GD2 leukemia cells, Nalm6-GM2 leukemia cells, CHLA-255 neuroblastoma cells, or Sy5y neuroblastoma cells. Figure 16B shows a schematic diagram of each CAR.

[0041] Figure 17 illustrates the in vivo function of tandem GD2-GM2 CAR T cells carrying intracellular domains of either the 4-1BB-ζ (Figure 17A) or CD28-ζ (Figure 17B) fragments in a Sy5y neuroblastoma mouse model expressing 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.

[0042] Figure 18 provides an in vivo view of the function of tandem GD2-GM2CAR T cells carrying CARs targeting CHLA255 (Figure 18A) or Kelly (Figure 18B), and the expression of gangliosides on tumor cells at the endpoint (Figure 18C). CHLA255 showed high GD2 expression and moderate / low GM2 expression, while Kelly showed moderate expression of both GD2 and GM2. Figures 18A and 18B show that GM2-GD2-28z tandem CAR T cells exhibited the best efficacy compared to single-specific GM2-28z CAR T cells, GD2-28z CAR T cells, or tandem GD2-GM2-28z CAR T cells. Figure 18C shows a flow cytometry analysis of the xenograft harvested at the endpoint from CHLA255 xenograft mice that received GD2 or GM2 CAR T cells. Detailed Implementation

[0043] definition For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular form will also include the plural form, and vice versa. If any of the following definitions conflict with any document incorporated herein by reference, the following definitions shall prevail.

[0044] Unless otherwise stated herein, the terms “disease” and “pathological condition” are used interchangeably as used herein to describe a deviation from the normal or average condition of members of a species or population (e.g., humans) and that is harmful to the affected individual, provided that it is harmless to the majority of individuals of that species or population. This deviation may manifest as states, signs, and / or symptoms (e.g., diarrhea, nausea, fever, pain, blisters, boils, rashes, immunosuppression, inflammation, etc.) that are associated with impairment of the normal state of the subject or any of their organs or tissues, thereby disrupting or altering the performance of normal function. Diseases or pathological conditions may be caused by exposure to microorganisms (e.g., pathogens or other infectious agents (e.g., viruses or bacteria)), may be a response to environmental factors (e.g., malnutrition, industrial hazards, and / or climate), may be a response to inherent or potential defects in the organism (e.g., genetic abnormalities), or a combination of these factors with other factors.

[0045] The terms “host,” “subject,” or “patient” are used interchangeably herein and refer to an individual who receives treatment (e.g., administration) with the compositions and methods of the present invention. Subjects include, but are not limited to, mammals (e.g., rats, monkeys, horses, cattle, pigs, dogs, cats, etc.), and most preferably include humans. In the context of the present invention, the term “subject” generally refers to an individual who will be administered or has already been administered one or more compositions of the present invention (e.g., genetically modified immune cells as described herein).

[0046] The term "solution" refers to an aqueous or non-aqueous mixture.

[0047] "Disease" means any condition or disease that benefits from treatment with the compositions or methods of the present invention. This includes chronic and acute diseases, including pathological conditions that predispose mammals to related diseases. Non-limiting examples of diseases to be treated herein include conditions such as cancer.

[0048] The terms "cellular proliferative disorder" and "proliferative disorder" refer to disorders associated with a certain degree of abnormal cell proliferation. For example, "hyperproliferative disorder or disease" is a disease or disorder caused by excessive cell growth. In some implementations, a cellular proliferative disorder is cancer.

[0049] As used herein, the terms “cancer” and “tumor” refer to tissue exhibiting uncontrolled cell growth or uncontrolled cell growth or proliferation. Cancer cells and tumor cells are typically characterized by loss of contact inhibition, can be invasive, and can exhibit metastatic capacity. This invention is not limited to the type of cancer or the type of treatment (e.g., prophylactic and / or therapeutic treatment). In fact, the compositions and methods described herein can be used to treat a wide variety of cancers, including but not limited to brain cancer or other cancers of the central nervous system (e.g., diffuse midline glioma or diffuse endogenous pontine glioma (DIPG, a highly aggressive glioma found at the base of the brain, see, e.g., Louis et al., Acta Neuropathol (2016) 131:803–820), melanoma, lymphoma, epithelial cancer, breast cancer, ovarian cancer, endometrial cancer, colorectal cancer, lung cancer, kidney cancer, melanoma, prostate cancer, sarcoma, carcinoma, and / or combinations thereof.

[0050] As used in this article, “metastasis” refers to the process by which cancer spreads from its primary site or metastasizes to other areas of the body and develops into a cancer-like lesion in the new location. “Metastatic” cells or “metastatic” cells are those that have lost their adhesion to neighboring cells and have invaded adjacent body structures from the primary site of the disease via the bloodstream or lymphatic system.

[0051] As used herein, the term "anticancer agent" refers to any therapeutic agent (e.g., chemotherapy compounds and / or molecular therapeutic compounds), antisense therapy, radiation therapy, or surgical intervention used to treat hyperproliferative diseases such as cancer (e.g., in mammals, such as humans).

[0052] "Effective dose" refers to the amount that effectively achieves the desired therapeutic or preventative outcome within the necessary dosage and time period.

[0053] As used herein, the term "therapeuticly effective amount" refers to an amount of therapeutic agent sufficient to cause improvement in one or more symptoms of a disease, or to prevent the progression of a disease, or to induce the remission of a disease. For example, in relation to cancer treatment, in one embodiment, a therapeutically effective amount refers to an amount of therapeutic agent that reduces tumor growth rate (e.g., reduces and / or eliminates the patient's tumor burden (e.g., reduces the number of H3K27M-positive cancer cells in the patient)), reduces tumor mass, reduces the number of metastases, slows tumor progression, or prolongs survival 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% of the amount of therapeutic agent.

[0054] As used herein, the terms “sensitize” and “sensitizing” refer to the enhancement of the sensitivity or responsiveness of an animal or its cells to the biological effects of a second agent (e.g., aspects of promoting or inhibiting cellular function, including but not limited to cell division, cell growth, proliferation, invasion, angiogenesis, necrosis, or apoptosis) by administration of a first agent. The sensitizing effect of the first agent on target cells can be measured as the difference in the expected biological effects (e.g., aspects of promoting or inhibiting cellular function, including but not limited to cell growth, proliferation, invasion, angiogenesis, or apoptosis) observed when the second agent is administered in the presence or absence of the first agent. The response of sensitized cells 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% compared to the response without the first agent.

[0055] As used herein, the term "purified" or "purified" means the removal of contaminants or undesirable compounds from a sample or composition. As used herein, the term "substantially purified" means the removal of approximately 70% to 90% (up to 100%) of contaminants or undesirable compounds from a sample or composition.

[0056] As used herein, the terms “administration” and “administering” refer to the act of giving a subject the composition of the present invention. Exemplary routes of administration to the human body include, but are not limited to, via the eye (ophthalmic), mouth (oral), skin (transdermal), nose (nasal cavity), lungs (inhalation), oral mucosa (buccal), ear, rectum, via injection (e.g., intravenous, subcutaneous, intraperitoneal, intratumoral, etc.), local administration, etc.

[0057] As used herein, the terms "co-administration" and "co-administering" refer to administering at least two agents (e.g., genetically modified immune cells and one or more other agents, such as anticancer agents) or therapies to a subject. In some embodiments, co-administration of two or more agents or therapies is performed simultaneously. In other embodiments, the first agent / therapy is administered before the second agent / therapy. In some embodiments, co-administration may be via the same or different routes of administration. Those skilled in the art will understand that the formulations and / or routes of administration of the various agents or therapies used may vary. Those skilled in the art can readily determine the appropriate dosage for co-administration. In some embodiments, when agents or therapies are co-administered, each agent or therapy is administered at a lower dose than would be appropriate for its individual administration. Therefore, co-administration is particularly desirable in embodiments where co-administration of agents or therapies reduces the necessary dose of potentially harmful (e.g., toxic) agents, and / or when co-administration of two or more agents results in the subject becoming sensitive to the beneficial effects of one agent via co-administration of another agent.

[0058] As used herein, the terms “pharmaceutical acceptable” or “pharmacologically acceptable” mean a composition that, when administered to a subject, substantially does not produce adverse reactions (e.g., toxicity, allergic reactions, or other immune responses).

[0059] As used herein, the term “pharmaceutically acceptable load” means any standard drug carrier, including but not limited to phosphate-buffered saline solutions, water and various types of wetting agents (e.g., sodium dodecyl sulfate), any and all solvents, dispersion media, coatings, sodium dodecyl sulfate, isotonic agents and absorption delay agents, disintegrants (e.g., potato starch or sodium glycolate), polyethylene glycol, etc. These compositions may also include stabilizers and preservatives. Examples of carriers, stabilizers and adjuvants have been described and are known in the art (see, for example, Remington's PharmaceuticalSciences, 15th Ed., Mack Publ. Co., Easton, Pa. (1975), which is incorporated herein by reference).

[0060] 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 enable the storage, transport, or delivery of immunogenic agents and / or adjuvant materials (e.g., written instructions for using these materials, etc.) from one location to another. For example, a kit may include one or more shells (e.g., boxes) containing associated immunotherapeutic agents (e.g., genetically modified immune cells and / or adjuvant materials). As used herein, the term "fragmented kit" refers to a delivery system comprising two or more individual containers, each containing a sub-part of all kit components. Containers may be delivered together or separately to the intended recipient. For example, a first container may contain a composition comprising an immunotherapeutic composition for a specific purpose, while a second container contains a second agent (e.g., a chemotherapeutic agent). In practice, the term "fragmented kit" includes any delivery system comprising two or more individual containers, each containing a sub-part of all kit components. In contrast, a "combination kit" refers to a delivery system that includes all components of the immunogenic agent required for a specific purpose in a single container (e.g., placing each desired component in a single box). The term "kit" includes both segmented kits and combination kits.

[0061] As used herein, the term "gene transfer system" refers to any method of delivering a composition comprising a nucleic acid sequence to cells or tissues. For example, gene transfer systems include, but are not limited to, vectors (e.g., retroviruses, adenoviruses, lentiviruses, adeno-associated viruses, and other nucleic acid-based delivery systems), microinjection of naked nucleic acids, polymer-based delivery systems (e.g., liposome-based and metal particle-based systems), gene gun injection, etc. As used herein, the term "viral gene transfer system" refers to a gene transfer system comprising viral elements (e.g., whole viruses, modified viruses, and viral components such as nucleic acids or proteins) to facilitate the delivery of samples to desired cells or tissues. Non-limiting examples of viral gene transfer systems available in the compositions and methods of the present invention are lentiviral gene transfer systems and retroviral gene transfer systems.

[0062] As used in this article, the term "site-specific recombination target sequence" refers to a nucleic acid sequence that provides a recognition sequence for recombination factors and the location where recombination occurs.

[0063] As used herein, the term "nucleic acid molecule" refers to any molecule containing nucleic acids, including but not limited to DNA or RNA. This term encompasses sequences of any known base analogues including DNA and RNA, including but not limited to, 4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinecytosine, pseudoisocytosine, 5-(carboxyhydroxymethyl)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, and 7-methylguanine. 5-Methylaminomethyluracil, 5-methoxy-aminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, methyl uracil-5-oxyacetic acid, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic acid methyl ester, and 2,6-diaminopurine.

[0064] The term "gene" refers to a nucleic acid (e.g., DNA) sequence that includes the coding sequence necessary to produce a polypeptide, precursor, or RNA (e.g., mRNA, rRNA, tRNA). A polypeptide may be encoded by a full-length coding sequence or any portion thereof, as long as the desired activity or functional property (e.g., enzymatic activity, ligand binding, signal transduction, immunogenicity, etc.) of the full-length gene product or a fragment thereof is retained. The term also encompasses the coding region of a structural gene and sequences adjacent to the 5' and 3' ends of the coding region, with a distance of approximately 1 kb or longer at either end, such that "gene" corresponds to the length of full-length mRNA. A sequence located at the 5' end of the coding region and present on the mRNA is called a 5' untranslated sequence. A sequence located at or downstream of the 3' end of the coding region and present on the mRNA is called a 3' untranslated sequence. The term "gene" encompasses both the cDNA and genomic forms of a gene. The genomic form or clone of a gene contains coding regions interrupted by non-coding sequences, referred to as "introns," "intercalation regions," or "intercalation sequences." Introns are gene segments transcribed into nuclear RNA (hnRNA); introns may contain regulatory elements such as enhancers. Introns are removed or "spliced ​​out" from nuclear transcripts or primary transcripts; therefore, introns are not present in messenger RNA (mRNA) transcripts. mRNA specifies the sequence or order of amino acids in nascent polypeptides during translation (e.g., protein synthesis).

[0065] As used herein, the term "heterogeneous gene" refers to a gene that is not found in the natural environment. For example, heterogeneous genes include genes introduced from one species into another. Heterogeneous genes also include genes inherent to an organism that have been altered in some way (e.g., through mutation, the addition of multiple copies, or the linking to a non-natural regulatory sequence). Heterogeneous genes differ from endogenous genes in that heterogeneous gene sequences are typically associated with DNA sequences that are not naturally present in chromosomes or with portions of chromosomes that are not naturally present (e.g., genes expressed at loci that are not normally expressed).

[0066] As used herein, the terms “nucleic acid molecule coding,” “DNA sequence coding,” and “DNA coding” refer to the sequence or order of deoxyribonucleotides along a chain of deoxyribonucleic acid (DNA). The sequence of these deoxyribonucleotides determines the sequence of amino acids along a polypeptide (protein) chain. Therefore, a DNA sequence codes for an amino acid sequence.

[0067] As used herein, the terms "oligonucleotide having a nucleotide sequence encoding a gene" and "polynucleotide having a nucleotide sequence encoding a gene" refer to nucleic acid sequences that include gene coding regions, or in other words, nucleic acid sequences encoding gene products. Coding regions can exist in the form of cDNA, genomic DNA, or RNA. When existing in DNA form, oligonucleotides or polynucleotides can be single-stranded (i.e., sense strands) or double-stranded. If it is necessary to allow proper initiation of transcription and / or proper processing of primary RNA transcripts, suitable control elements (such as enhancers / promoters, splicing junctions, polyadenylation signals, etc.) can be placed near the gene coding region. Alternatively, the coding region utilized in the expression vectors of the present invention can contain endogenous enhancers / promoters, splicing junctions, insertion sequences, polyadenylation signals, etc., or a combination of endogenous and exogenous control elements.

[0068] The term "amino acid sequence" and terms such as "peptide" or "protein" are not intended to limit the amino acid sequence to the complete, natural amino acid sequence associated with the protein molecule being referenced.

[0069] In the context of two or more nucleic acid or polypeptide sequences, the "identity" percentage refers to the percentage of identical nucleotide or amino acid residues between two or more sequences or subsequences when compared and aligned to achieve maximum correspondence. This percentage is measured by using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN, or other algorithms available to the technician) or by visual inspection. Depending on the application, the "identity" percentage may be present in a region of the compared sequences, such as a functional domain, or alternatively, across the entire length of the two sequences being compared. For sequence comparisons, typically one sequence serves as a reference sequence, against which the test sequence is compared. When using a sequence comparison algorithm, the test and reference sequences are input into the computer, with subsequence coordinates specified if necessary, and sequence algorithm program parameters specified. The sequence comparison algorithm then calculates the sequence identity percentage of the test sequence relative to the reference sequence based on the specified program parameters. Sequence alignment can be performed by comparisons such as, for example, 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 similarity search method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), computer 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 (usually see Ausubel et al. below). An example of an algorithm suitable for determining sequence identity and the percentage of sequence similarity is the BLAST algorithm, described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analysis is publicly available from the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov).

[0070] "Sequence identity" refers to the degree to which two polymer sequences (e.g., peptides, polypeptides, nucleic acids, etc.) have the same monomeric subunit sequence composition. The term "sequence similarity" refers to the degree to which two polymer sequences (e.g., peptides, polypeptides, nucleic acids, etc.) have similar polymer sequences. For example, similar amino acids are those amino acids that have the same biophysical characteristics and can be grouped into multiple families, such as acidic (e.g., aspartic acid, glutamic acid), basic (e.g., lysine, arginine, histidine), nonpolar (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (e.g., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). The “sequence identity percentage” (or “sequence similarity percentage”) is calculated by: (1) comparing two best-aligned sequences within a comparison window (e.g., the length of the longer sequence, the length of the shorter sequence, or a specified window); (2) determining the number of positions containing the same (or similar) monomers (e.g., the same amino acid in both sequences, or similar amino acids in both sequences) to obtain the number of matching positions; (3) dividing the number of matching 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, or a specified window); and (4) multiplying the result by 100 to obtain the sequence identity percentage or sequence similarity percentage. For example, if peptide A and peptide B are both 20 amino acids long and all but one of the positions contain the same amino acids, then peptide A and peptide B have 95% sequence identity. If the amino acids at non-identical positions have the same biophysical characteristics (e.g., both are acidic), then peptide A and peptide B will have 100% sequence similarity. For example, if peptide C is 20 amino acids long and peptide D is 15 amino acids long, and 14 of the 15 amino acids in peptide D are identical to some of the amino acids in peptide C, then peptide C and peptide D have 70% sequence identity. However, the optimal alignment window for peptide D and peptide C has 93.3% sequence identity. To calculate the “sequence identity percentage” (or “sequence similarity percentage”) in this paper, any gaps in the aligned sequences are considered mismatches at that position.

[0071] When used in relation to nucleic acids, such as "isolated oligonucleotides" or "isolated polynucleotides," the term refers to a nucleic acid sequence that has been identified and isolated from at least one component or contaminant typically associated with its natural source. Isolated nucleic acids exist in a manner different from their natural form or environment. In contrast, non-isolated nucleic acids are nucleic acids that exist in the state in which they exist in nature, such as DNA and RNA. For example, a given DNA sequence (e.g., a gene) is located on the host cell chromosome, close to adjacent genes; RNA sequences, such as specific mRNA sequences encoding a particular protein, exist in the cell as a mixture with many other mRNAs encoding multiple proteins. However, isolated nucleic acids encoding a specific protein include, for example, such nucleic acids in cells that typically express a given protein, where the nucleic acid is located on a chromosome at a location different from that in natural cells, or flanked by nucleic acid sequences different from those found in nature. Isolated nucleic acids (oligonucleotides or polynucleotides) can exist in single-stranded or double-stranded form. When using isolated nucleic acids, oligonucleotides, or polynucleotides to express proteins, the oligonucleotide or polynucleotide contains at least a sense strand or a coding strand (i.e., the oligonucleotide or polynucleotide may be single-stranded), but may also contain both a sense strand and an antisense strand (i.e., the oligonucleotide or polynucleotide may be double-stranded).

[0072] As used herein, the terms "purified" or "purified" refer to the removal of components (e.g., contaminants) from a sample. For example, antibodies can be purified by removing contaminating non-immunoglobulin proteins; they can also be purified by removing immunoglobulins that do not bind to target molecules. Removal of non-immunoglobulin proteins and / or removal of immunoglobulins that do not bind to target molecules results in an increase in the percentage of target-reactive immunoglobulins in the sample. In another example, a recombinant peptide is expressed in a bacterial host cell, and the peptide is purified by removing host cell proteins; thereby increasing the percentage of the recombinant peptide in the sample.

[0073] Unless otherwise expressly stated, the indefinite articles “a” and “an” in this specification and claims, as used herein, shall be understood to mean “at least one”.

[0074] As used herein in the specification and claims, the phrase “and / or” should be understood to mean “any one or two” of the elements so combined, that is, elements that exist together in some cases and separately in others. Multiple elements listed with “and / or” should be interpreted in the same way, that is, “one or more” elements so connected. In addition to the elements specifically identified by the “and / or” clause, other elements may optionally be present, whether or not they are related to those specifically identified elements. Thus, as a non-limiting example, when used in conjunction with open-ended language such as “comprising,” a reference to “A and / or B” may in one embodiment refer only to A (optionally including elements other than B); in another embodiment, only to B (optionally including elements other than A); in yet another embodiment, both A and B (optionally including other elements); and so on.

[0075] As used in the specification and claims herein, “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” should be interpreted as inclusive, that is, including at least one of many elements or a list of elements, but also including more than one, as well as optional other unlisted items. Only when the opposite terms are explicitly indicated, such as “only one” or “exactly one”, or when used in the claims, “consisting of” will refer to including exactly one of a plurality of elements or a series of elements. In general, when preceded by an exclusive term, such as “either of the two,” “one of them,” “only one,” or “exactly one,” the term “or” as used herein should only be interpreted as indicating an exclusive substitution (i.e., “one or the other but not both”). When “substantially constitutes” is used in the claims, it should have its general meaning as used in the field of patent law.

[0076] As used herein and as determined by one of ordinary skill in the art, the term "about" means within an acceptable range of error for a particular value, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, according to practice in the art, "about" may mean within an acceptable standard deviation. Alternatively, "about" may mean a given value up to ±20%, preferably up to ±10%, more preferably up to ±5%, and even more preferably up to ±1%. When a particular value is described in this application and claims, the term "about" is implied unless otherwise stated and in the context means an acceptable range of error for the particular value.

[0077] Unless otherwise stated, the compositions, methods, and practices of this disclosure employ conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, all of which are within the skill level of the art. These techniques are explained in detail in the literature, such as... Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press; TECreighton, Proteins: Structures and Molecular Properties (WH Freeman and Company, 1993); Oligonucleotide Synthesis (MJ Gait, ed. 1984); Methods in Molecular Biology Humana Press; Cell Biology: A Laboratory Notebook (JECellis, ed., 1989) Academic Press; Animal Cell Culture (RI Freshney, ed.1987); AL Lehninger, Biochemistry (Worth Publishers, Inc., currentaddition); Methods In Enzymology (S. Colowick and N. Kaplan eds., AcademicPress, Inc.); Remington’s Pharmaceutical Sciences, 18th Edition (Easton,Pennsylvania: Mack Publishing Company, 1990); Introduction to Cell and TissueCulture (J. P. Mather and P. E. Roberts, 1998) Plenum Press; Cell and TissueCulture: 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); ShortProtocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A.Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: apractice approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonalantibodies: a practical approach (P. Shepherd and C. Dean, eds., OxfordUniversity Press, 2000); Using antibodies: a laboratory manual (E. Harlow andD. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M.Zanetti and JD Capra, eds. Harwood Academic Publishers, 1995);. DNA Cloning: A practical Approach, Volumes I and II (DN Glover ed. 1985); Nucleic Acid Hybridization (BD Hames&S.J. Higgins eds.(1985»; Transcription and Translation (BD Hames&S.J. Higgins, eds. (1984); Animal Cell Culture (RIFreshney, ed. (1986) Immobilized Cells and Enzymes (lRL Press, (1986); and B. Perbal, A practical Guide To Molecular Cloning (1984); FM Ausubel et al. (eds.)

[0078] Chimeric antigen receptor (CAR) and genetically engineered cells expressing CAR Cells (e.g., T cells) can be genetically engineered to express chimeric antigen receptors (CARs) that target one or more antigens (e.g., GM2 and / or GD2).

[0079] Chimeric antigen receptor (CAR) Chimeric antigen receptors (CARs) are artificial immune cell receptors engineered to recognize and bind to antigens expressed by undesirable cells (e.g., diseased cells, such as cancer cells). T cells expressing CAR peptides are called CAR T cells. CARs can redirect T cell specificity and responsiveness to selected targets in a non-MHC-restricted manner. Non-MHC-restricted antigen recognition enables CAR-T cells to recognize antigens that are independent of antigen processing, thereby bypassing major mechanisms of tumor escape.

[0080] To date, multiple generations of CARs have been developed, each containing different components. First-generation CARs link the antibody-derived scFv to the intracellular signaling domain of the T cell receptor's CD3ζ (ζ or z) via hinges and transmembrane domains. Second-generation CARs incorporate additional co-stimulatory domains, such as CD28, 4-1BB (4-1BB), or ICOS, to provide co-stimulatory signaling. Third-generation CARs contain two or more co-stimulatory domains fused to the TCR CD3ζ chain (e.g., combinations of CD27, CD28, 4-1BB, ICOS, or OX40) (see, for example, Maude et al.). Blood. 2015; 125(26):4017-4023; Kakarlaand Gottschalk, Cancer J. 2014; 20(2):151-155). Any generation of CAR constructs is within the scope of this disclosure.

[0081] Typically, a CAR is a fusion peptide comprising an extracellular domain (e.g., a single-chain fragment of an antibody (scFv) or other antibody fragment) that recognizes a target antigen and an intracellular domain that includes a signaling domain of the T-cell receptor (TCR) complex (e.g., CD3ζ), and in most cases, a co-stimulatory domain. (See, for example, Enblad et al., Human Gene Therapy. 2015; 26(8):498-505). CAR constructs may further include a hinge and / or transmembrane domain between the extracellular and intracellular domains, and an N-terminal signal peptide for surface expression. Examples of signal peptides that may be used herein include signaling domains derived from 4-1BB, CD28, and / or CD3-ζ. Other signal peptides may be used.

[0082] Antigen-binding extracellular domain An antigen-binding extracellular domain is the region of a CAR peptide exposed to the extracellular fluid when expressing a CAR on the cell surface. In some embodiments, the signal peptide may be located at the N-terminus to promote cell surface expression. In some embodiments, the antigen-binding domain may be a single-chain variable fragment (scFv) that may include a variable (VH) region of the antibody heavy chain and a variable (VL) region of the antibody light chain (in either orientation). In some embodiments, the VH and VL fragments may be linked by a peptide linker. In some embodiments, the linker includes hydrophilic residues having glycine and serine fragments for flexibility and glutamate and lysine fragments for increasing solubility. This disclosure is not limited to any particular linker. Exemplary linkers are provided herein. The scFv fragment retains the antigen-binding specificity of its parent antibody, and the scFv fragment may be derived from the parent antibody. In some embodiments, the scFv may include humanized VH and / or VL domains. In other embodiments, the VH and / or VL domains of the scFv are fully human.

[0083] Embodiments of this disclosure include a chimeric antigen receptor (CAR) targeting cells (e.g., tumors and / or cancer cells) expressing monosialotetrahexosylganglioside GM2 (ganglioside GM2 or GM2), referred to herein as a GM2 CAR, and a nucleic acid molecule encoding the GM2 CAR. The GM2 CAR polypeptide and polynucleotide of this disclosure include an extracellular portion comprising a GM2 antigen-specific antigen-binding domain, a transmembrane domain, and one or more intracellular signaling domains. In some embodiments, the GM2 CAR includes a hinge domain, a spacer region, and / or one or more peptide linkers. When engineered for expression on the surface of immune cells (e.g., T lymphocytes, NK cells), the GM2 CAR of this disclosure targets GM2-expressing cells (e.g., tumor cells and / or cancer cells), resulting in targeted destruction of these cells.

[0084] In some embodiments, this disclosure provides a chimeric antigen receptor (CAR) comprising a ganglioside GM2 binding domain. In some embodiments, the antigen-binding domain comprises an antibody, an antigen-binding fragment of an antibody, an F(ab) fragment, an 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. Typically, the scFv has a light chain variable domain (VL) linked from its C-terminus to the N-terminus of the heavy chain variable domain (VH) via a polypeptide chain. Alternatively, the scFv comprises a polypeptide chain wherein the C-terminus of the VH is linked to the N-terminus of the VL via the polypeptide chain. In some embodiments, scFv includes the structure VH-L-VL or VL-L-VH, wherein VH is a heavy chain variable domain, L is a peptide linker, and VL is a light chain variable domain. In a preferred embodiment, this disclosure provides compositions (e.g., T cells, stem cells) comprising a GM2-binding molecule (e.g., a GM2 CAR, a GM2-designed ankyrin repeat protein (DARPIN), or any molecule that specifically binds to GM2) and a GD2-binding molecule (e.g., a GD2 CAR, a GD2-designed ankyrin repeat protein (DARPIN), or any molecule that specifically binds to GD2).

[0085] An sdAb is a molecule in which one variable domain of an antibody binds specifically to an antigen, while other variable domains are absent. As used herein, a “single-domain antibody” or “sdAb” refers to an antibody whose complementarity-determining region is part of a single-domain polypeptide. Examples include, but are not limited to, heavy-chain antibodies, naturally occurring antibodies that do not contain light chains, single-domain antibodies derived from conventional antibodies (having two heavy chains and two light chains, a four-chain immunoglobulin), engineered antibodies, and single-domain scaffolds derived from sources other than antibodies. Single-domain antibodies can be any of the prior art or any future single-domain antibody. Single-domain antibodies can be derived from any species, including but not limited to mice, humans, camels, llamas, goats, rabbits, and cattle. The F(ab) fragment contains a constant domain (CL) of the light chain and a first constant domain (CH1) of the heavy chain, as well as variable domains VL and VH located on the light and heavy chains, respectively. The F(ab') fragment differs from the Fab fragment in that several residues are added to the carboxyl terminus of the CH1 domain of the heavy chain, including one or more cysteine ​​residues from the antibody hinge region. The F(ab')2 fragment contains two Fab' fragments connected by disulfide bonds near the hinge region.

[0086] In some embodiments, this disclosure provides a chimeric antigen receptor (CAR) having an antigen recognition domain that specifically binds to GM2. Examples of GM2-specific CARs are provided. In some embodiments, this disclosure provides a chimeric antigen receptor (CAR) comprising a single-chain variable fragment (scFv) that binds to GM2, wherein the scFv comprises a pair of heavy chain variable (VH) and light chain variable (VL) regions.

[0087] In some embodiments, the CAR of this disclosure includes the VH and / or VL amino acid sequences of Table 1. In some embodiments, the single-domain antibody (sdAb) disclosed herein includes a VH selected from the sequences disclosed in Table 1. In some embodiments, the VH and VL pairs of the GM2 CAR of this disclosure are selected from various sequences listed in Table 1. In some embodiments, various CDR combinations of VH and VL pairs are selected from the sequences listed in Table 1. Various embodiments of this disclosure may include one or more polypeptide sequences of chimeric antigen receptor (CAR) sequences mentioned in Table 1 below. Various embodiments of this disclosure may also include one or more polypeptide sequences associated with the GM2 antigen recognition domain, combined with polypeptide sequences associated with the GD2 antigen recognition domain, as described herein.

[0088] Table 1: CAR amino acid sequence.

[0089]

[0090] ARD—Single-stranded variable fragment (scFv) that retains GM2 and / or GD2 specificity In some embodiments, the chimeric antigen receptor (CAR) includes a single-chain Fv (scFv) that binds to GM2, a transmembrane domain, and one or more intracellular signal transduction domains, wherein the scFv includes a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region includes heavy chain complementarity-determining region 1 (CDR-H1), heavy chain complementarity-determining region 2 (CDR-H2), and 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.

[0091] In some embodiments, the VH region includes an amino acid sequence having 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 with the amino acid sequence of SEQ ID NO: 10. In some embodiments, the VH region includes heavy chain complementarity-determining region 1 (CDR-H1), heavy chain complementarity-determining region 2 (CDR-H2), and 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.

[0092] In some embodiments, the VH region includes an amino acid sequence having 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 with the amino acid sequence of SEQ ID NO: 11. In some embodiments, the VH region includes heavy chain complementarity-determining region 1 (CDR-H1), heavy chain complementarity-determining region 2 (CDR-H2), and 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.

[0093] In some embodiments, the VH region includes an amino acid sequence having 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 with the amino acid sequence of SEQ ID NO: 12. In some embodiments, the VH region includes heavy chain complementarity-determining region 1 (CDR-H1), heavy chain complementarity-determining region 2 (CDR-H2), and 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.

[0094] In some embodiments, the VH region includes an amino acid sequence having 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 with the amino acid sequence of SEQ ID NO: 13. In some embodiments, the VH region includes heavy chain complementarity-determining region 1 (CDR-H1), heavy chain complementarity-determining region 2 (CDR-H2), and 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.

[0095] In some embodiments, the VH region includes an amino acid sequence having 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 with the amino acid sequence of SEQ ID NO: 14. In some embodiments, the VH region includes heavy chain complementarity-determining region 1 (CDR-H1), heavy chain complementarity-determining region 2 (CDR-H2), and 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.

[0096] In some embodiments, the VH region includes an amino acid sequence having 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 with the amino acid sequence of SEQ ID NO: 15. In some embodiments, the VH region includes heavy chain complementarity-determining region 1 (CDR-H1), heavy chain complementarity-determining region 2 (CDR-H2), and 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.

[0097] In some embodiments, the VH region includes an amino acid sequence having 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 with the amino acid sequence of SEQ ID NO: 16. In some embodiments, the VH region includes heavy chain complementarity-determining region 1 (CDR-H1), heavy chain complementarity-determining region 2 (CDR-H2), and 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.

[0098] In some embodiments, the VH region includes an amino acid sequence having 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 with the amino acid sequence of SEQ ID NO: 17. In some embodiments, the VH region includes heavy chain complementarity-determining region 1 (CDR-H1), heavy chain complementarity-determining region 2 (CDR-H2), and 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.

[0099] In some embodiments, the VH region includes an amino acid sequence that is 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% identical to an amino acid sequence selected from SEQ ID NO: 10, 11, 12, 13, 14, 15, 16, or 17.

[0100] In some embodiments, the chimeric antigen receptor (CAR) includes a single-chain Fv (scFv) that binds to GM2, a transmembrane domain, and one or more intracellular signal transduction domains, wherein the scFv includes a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VL region 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), 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 of SEQ ID NO: 27-35.

[0101] In some embodiments, the VL region includes an amino acid sequence having 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 with the amino acid sequence of SEQ ID NO: 27. In some embodiments, the VL region includes light chain complementarity-determining region 1 (CDR-L1), light chain complementarity-determining region 2 (CDR-L2), and light chain complementarity-determining region 3 (CDR-L3), 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.

[0102] In some embodiments, the VL region includes an amino acid sequence having 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 with the amino acid sequence of SEQ ID NO: 28. In some embodiments, the VL region includes light chain complementarity-determining region 1 (CDR-L1), light chain complementarity-determining region 2 (CDR-L2), and light chain complementarity-determining region 3 (CDR-L3), 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.

[0103] In some embodiments, the VL region includes an amino acid sequence having 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 with the amino acid sequence of SEQ ID NO: 29. In some embodiments, the VL region includes light chain complementarity-determining region 1 (CDR-L1), light chain complementarity-determining region 2 (CDR-L2), and light chain complementarity-determining region 3 (CDR-L3), 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.

[0104] In some embodiments, the VL region includes an amino acid sequence having 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 with the amino acid sequence of SEQ ID NO: 30. In some embodiments, the VL region includes light chain complementarity-determining region 1 (CDR-L1), light chain complementarity-determining region 2 (CDR-L2), and light chain complementarity-determining region 3 (CDR-L3), 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.

[0105] In some embodiments, the VL region includes an amino acid sequence having 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 with the amino acid sequence of SEQ ID NO: 31. In some embodiments, the VL region includes light chain complementarity-determining region 1 (CDR-L1), light chain complementarity-determining region 2 (CDR-L2), and light chain complementarity-determining region 3 (CDR-L3), 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: 31.

[0106] In some embodiments, the VL region includes an amino acid sequence having 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 with the amino acid sequence of SEQ ID NO: 32. In some embodiments, the VL region includes light chain complementarity-determining region 1 (CDR-L1), light chain complementarity-determining region 2 (CDR-L2), and light chain complementarity-determining region 3 (CDR-L3), 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.

[0107] In some embodiments, the VL region includes an amino acid sequence having 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 with the amino acid sequence of SEQ ID NO: 33. In some embodiments, the VL region includes light chain complementarity-determining region 1 (CDR-L1), light chain complementarity-determining region 2 (CDR-L2), and light chain complementarity-determining region 3 (CDR-L3), 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.

[0108] In some embodiments, the VL region includes an amino acid sequence having 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 with the amino acid sequence of SEQ ID NO: 34. In some embodiments, the VL region includes light chain complementarity-determining region 1 (CDR-L1), light chain complementarity-determining region 2 (CDR-L2), and light chain complementarity-determining region 3 (CDR-L3), 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.

[0109] In some embodiments, the VL region includes an amino acid sequence having 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 with the amino acid sequence of SEQ ID NO: 35. In some embodiments, the VL region includes light chain complementarity-determining region 1 (CDR-L1), light chain complementarity-determining region 2 (CDR-L2), and light chain complementarity-determining region 3 (CDR-L3), 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.

[0110] In some embodiments, the VL region includes an amino acid sequence having 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 with an amino acid sequence selected from SEQ ID NO: 27, 28, 29, 30, 31, 32, 33, 34, and 35.

[0111] In some embodiments, VH includes heavy chain complementarity-determining region 1 (CDR-H1), heavy chain complementarity-determining region 2 (CDR-H2), and 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 amino acid sequences selected from SEQ ID NO: 10-17; and VL includes light chain complementarity-determining region 1 (CDR-L1), light chain complementarity-determining region 2 (CDR-L2), and light chain complementarity-determining region 3 (CDR-L3), wherein the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are contained within the VL region of amino acid sequences selected from SEQ ID NO: 27-35.

[0112] In some embodiments, VH comprises an amino acid sequence having 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 with an amino acid sequence selected from SEQ ID NO: 10-17; and VL comprises an amino acid sequence having 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 with an amino acid sequence selected from SEQ ID NO: 27-35.

[0113] In some embodiments, the CAR comprises an amino acid sequence having 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 with an amino acid sequence selected from SEQ ID NO: 59-88. In some embodiments, the CAR comprises an amino acid sequence selected from the amino acid sequences selected from SEQ ID NO: 59-88.

[0114] In some embodiments, the VH region includes an amino acid sequence having 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 with the amino acid sequence of SEQ ID NO: 10, and the VL region includes an amino acid sequence having 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 with the amino acid sequence of SEQ ID NO: 27. In some embodiments, VH includes heavy chain complementarity-determining region 1 (CDR-H1), heavy chain complementarity-determining region 2 (CDR-H2), and heavy chain complementarity-determining region 3 (CDR-H3), wherein the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are included within the VH region of the amino acid sequence of SEQ ID NO: 10; and VL includes light chain complementarity-determining region 1 (CDR-L1), light chain complementarity-determining region 2 (CDR-L2), and light chain complementarity-determining region 3 (CDR-L3), wherein the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are included within the VL region of the amino acid sequence of SEQ ID NO: 28. In some embodiments, the VH region includes an amino acid sequence having 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 with the amino acid sequence of SEQ ID NO: 11, and the VL region includes an amino acid sequence having 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 with the amino acid sequence of SEQ ID NO: 29. In some embodiments, VH includes heavy chain complementarity-determining region 1 (CDR-H1), heavy chain complementarity-determining region 2 (CDR-H2), and heavy chain complementarity-determining region 3 (CDR-H3), the VH having the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 contained in the VH region amino acid sequence of SEQ ID NO: 12; and VL includes light chain complementarity-determining region 1 (CDR-L1), light chain complementarity-determining region 2 (CDR-L2), and light chain complementarity-determining region 3 (CDR-L3), the VL having the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 contained in the VL region amino acid sequence of SEQ ID NO: 20.In some embodiments, VH includes heavy chain complementarity-determining region 1 (CDR-H1), heavy chain complementarity-determining region 2 (CDR-H2), and heavy chain complementarity-determining region 3 (CDR-H3), the VH having the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 contained in the VH region amino acid sequence of SEQ ID NO: 13; and VL includes light chain complementarity-determining region 1 (CDR-L1), light chain complementarity-determining region 2 (CDR-L2), and light chain complementarity-determining region 3 (CDR-L3), the VL having the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 contained in the VL region amino acid sequence of SEQ ID NO: 20. In some embodiments, VH includes heavy chain complementarity-determining region 1 (CDR-H1), heavy chain complementarity-determining region 2 (CDR-H2), and heavy chain complementarity-determining region 3 (CDR-H3), the VH having the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 contained in the VH region amino acid sequence of SEQ ID NO: 14; and VL includes light chain complementarity-determining region 1 (CDR-L1), light chain complementarity-determining region 2 (CDR-L2), and light chain complementarity-determining region 3 (CDR-L3), the VL having the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 contained in the VL region amino acid sequence of SEQ ID NO: 20. In some embodiments, VH includes heavy chain complementarity-determining region 1 (CDR-H1), heavy chain complementarity-determining region 2 (CDR-H2), and heavy chain complementarity-determining region 3 (CDR-H3), the VH having the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 contained in the VH region amino acid sequence of SEQ ID NO: 16; and VL includes light chain complementarity-determining region 1 (CDR-L1), light chain complementarity-determining region 2 (CDR-L2), and light chain complementarity-determining region 3 (CDR-L3), the VL having the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 contained in the VL region amino acid sequence of SEQ ID NO: 34. In some embodiments, VH includes heavy chain complementarity-determining region 1 (CDR-H1), heavy chain complementarity-determining region 2 (CDR-H2), and heavy chain complementarity-determining region 3 (CDR-H3), the VH having the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 contained in the VH region amino acid sequence of SEQ ID NO: 17; and VL includes light chain complementarity-determining region 1 (CDR-L1), light chain complementarity-determining region 2 (CDR-L2), and light chain complementarity-determining region 3 (CDR-L3), the VL having the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 contained in the VL region amino acid sequence of SEQ ID NO: 35.

[0115] In some embodiments, VH includes 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, VL includes 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.

[0116] In some embodiments, the VH region includes an amino acid sequence having 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 with the amino acid sequence of SEQ ID NO: 10, and the VL region includes an amino acid sequence having 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 with the amino acid sequence of SEQ ID NO: 27. In some embodiments, VH includes heavy chain complementarity-determining region 1 (CDR-H1), heavy chain complementarity-determining region 2 (CDR-H2), and heavy chain complementarity-determining region 3 (CDR-H3), wherein the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are included within the VH region of the amino acid sequence of SEQ ID NO: 10; and VL includes light chain complementarity-determining region 1 (CDR-L1), light chain complementarity-determining region 2 (CDR-L2), and light chain complementarity-determining region 3 (CDR-L3), wherein the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are included within the VL region of the amino acid sequence of SEQ ID NO: 27.

[0117] In some embodiments, the VH region includes an amino acid sequence having 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 with the amino acid sequence of SEQ ID NO: 11, and the VL region includes an amino acid sequence having 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 with the amino acid sequence of SEQ ID NO: 29. In some embodiments, VH includes heavy chain complementarity-determining region 1 (CDR-H1), heavy chain complementarity-determining region 2 (CDR-H2), and heavy chain complementarity-determining region 3 (CDR-H3), the VH having the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 contained in the VH region amino acid sequence of SEQ ID NO: 11; and VL includes light chain complementarity-determining region 1 (CDR-L1), light chain complementarity-determining region 2 (CDR-L2), and light chain complementarity-determining region 3 (CDR-L3), the VL having the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 contained in the VL region amino acid sequence of SEQ ID NO: 29.

[0118] In some embodiments, 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, 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.

[0119] In some embodiments, the VH region includes an amino acid sequence having 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 with the amino acid sequence of SEQ ID NO: 12, and the VL region includes an amino acid sequence having 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 with the amino acid sequence of SEQ ID NO: 20. In some embodiments, VH includes heavy chain complementarity-determining region 1 (CDR-H1), heavy chain complementarity-determining region 2 (CDR-H2), and heavy chain complementarity-determining region 3 (CDR-H3), the VH having the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 contained in the VH region amino acid sequence of SEQ ID NO: 12; and VL includes light chain complementarity-determining region 1 (CDR-L1), light chain complementarity-determining region 2 (CDR-L2), and light chain complementarity-determining region 3 (CDR-L3), the VL having the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 contained in the VL region amino acid sequence of SEQ ID NO: 20.

[0120] In some embodiments, 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, 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.

[0121] In some embodiments, the chimeric antigen receptor (CAR) includes a single-chain Fv (scFv) that binds to GM2, a transmembrane domain, and one or more intracellular signal transduction domains, wherein the scFv is selected from amino acid sequences having 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 with amino acid sequences selected from SEQ ID NO: 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 56, 57, and 58.

[0122] In some embodiments, VH and VL are separated by a peptide linker. Peptide linkers are well known in the art. In some embodiments, the peptide linker comprises an amino acid sequence selected from SEQ ID NO: 54-55.

[0123] In some embodiments, this disclosure provides engineered nucleic acids encoding the CARs disclosed herein. In some embodiments, the nucleic acid molecule encodes 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 heavy chain complementarity-determining region 1 (CDR-H1), heavy chain complementarity-determining region 2 (CDR-H2), and heavy chain complementarity-determining region 3 (CDR-H3), the VH region having an amino acid sequence having 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 with an amino acid sequence selected from SEQ ID NO: 10-17. In some embodiments, at least one light chain variable (VL) region includes light chain complementarity-determining region 1 (CDR-L1), light chain complementarity-determining region 2 (CDR-L2), and light chain complementarity-determining region 3 (CDR-L3), the VL region having an amino acid sequence having 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 with the amino acid sequences selected from SEQ ID NO: 27-35. In some embodiments, at least one heavy chain variable region includes or consists of the following: amino acid sequences shown in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, 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, at least one light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, 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 scFv.In some embodiments, scFv comprises or consists of an amino acid sequence having 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 with an amino acid sequence selected from SEQ ID NO: 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 a chimeric antigen receptor (CAR) comprises or consists of the following: amino acid sequences shown in 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 embodiments, the nucleic acid molecule further includes a codon optimized for expression in human T cells, and / or is operatively 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 a nucleic acid molecule includes a single-chain Fv (scFv) that binds to ganglioside GM2, and another chimeric antigen receptor encoded by a nucleic acid molecule includes a single-chain Fv (scFv) that binds to ganglioside GD2.

[0124] In some embodiments, this disclosure provides an engineered nucleic acid encoding a CAR comprising a single-domain antibody that binds to GM2, the single-domain antibody comprising one or more CDR regions from any one of SEQ ID NO: 10-17. In some embodiments, the nucleic acid encodes a GM2 sdAb comprising heavy chain complementarity-determining region 1 (CDR-H1), heavy chain complementarity-determining region 2 (CDR-H2), and heavy chain complementarity-determining region 3 (CDR-H3), wherein the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are contained within a VH region of an amino acid sequence selected from SEQ ID NO: 10-17. In some embodiments, the engineered nucleic acid encodes an amino acid sequence having 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 with an amino acid sequence selected from SEQ ID NO: 10-17. In some embodiments, this disclosure provides engineered nucleic acids encoding CARs comprising single-domain antibodies that bind to GD2. This disclosure is not limited to sdAbs that bind to GD2. In fact, the sdAb of this disclosure may include any VH region of an antibody that binds to GD2. In some embodiments, the sdAb includes the VH region of SEQ ID NO: 17.

[0125] 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 from the second pair (e.g., the first VH-VL pair and the second VH-VL pair are expressed by a vector containing the engineered nucleic acid molecule). For example, in some embodiments, the engineered nucleic acid molecule encodes a VH-VL pair that specifically binds to ganglioside GM2, and another VH-VL pair that specifically binds 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.

[0126] Therefore, in some embodiments, the CARs disclosed herein include bicistronic chimeric antigen receptors. In some embodiments, the bicistronic chimeric antigen receptors include GM2 CARs and GD2 CARs. In some embodiments, the bicistronic chimeric antigen receptors include any of the GM2 CARs and any of the GD2 CARs described herein. In some embodiments, the bicistronic chimeric antigen receptors include GM2 CARs. In some embodiments, the bicistronic chimeric antigen receptors include CARs having an antigen-binding domain targeting GM2. In some embodiments, the bicistronic chimeric antigen receptors include CARs having an antigen-binding domain targeting GD2.

[0127] In some embodiments, the bicistronic chimeric antigen receptor (CAR) includes a single-chain Fv (scFv) that binds to GM2, a transmembrane domain, and one or more intracellular signal transduction domains, wherein the scFv includes a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region includes heavy chain complementarity-determining region 1 (CDR-H1), heavy chain complementarity-determining region 2 (CDR-H2), and 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 having 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 with an amino acid sequence selected from SEQ ID NO: 10, 11, 12, 13, 14, 15, 16, or 17. In some embodiments, the bicistronic chimeric antigen receptor (CAR) comprises the VH region of SEQ ID NO: 15. In other embodiments, the bicistronic chimeric antigen receptor (CAR) comprises the VH region of SEQ ID NO: 17.

[0128] In some embodiments, the bicistronic chimeric antigen receptor (CAR) includes a single-chain Fv (scFv) that binds to GM2, a transmembrane domain, and one or more intracellular signal transduction domains, wherein the scFv includes a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VL region includes light chain complementarity-determining region 1 (CDR-L1), light chain complementarity-determining region 2 (CDR-L2), and 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 of SEQ ID NO: 27-35.

[0129] In some embodiments, the bicistronic chimeric antigen receptor (CAR) comprises an amino acid sequence having 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 with an amino acid sequence selected from SEQ ID NO: 27, 28, 29, 30, 31, 32, 33, 34, or 35. In some embodiments, the bicistronic chimeric antigen receptor (CAR) comprises the VL region of SEQ ID NO: 33. In other embodiments, the bicistronic chimeric antigen receptor (CAR) comprises the VH region of SEQ ID NO: 35.

[0130] In some embodiments, the bicistronic chimeric antigen receptor (CAR) includes a single-chain Fv (scFv) comprising an amino acid sequence having 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 with an amino acid sequence selected from SEQ ID NO: 43 or 56.

[0131] In some embodiments, the bicistronic chimeric antigen receptor comprises a CAR having two or more antigen-binding domains targeting GM2 and another antigen. In some embodiments, the bicistronic chimeric antigen receptor comprises a CAR having any combination of the two or more antigen-binding domains described herein.

[0132] Therefore, in some embodiments, the engineered nucleic acid of this disclosure can be polycistronic, i.e., capable of producing more than one single polypeptide (e.g., multiple exogenous polynucleotides or GM2 CARs) from a single mRNA transcript. The engineered nucleic acid can be polycistronic by using various adapters; for example, 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 GD2 CAR), such as in a first gene: adapter: second gene 5' to 3' orientation.

[0133] Linkers can be combinations of multiple linkers. Typically, polycistronic systems can use any number or combination of linkers to express any number of genes or parts thereof (e.g., engineered nucleic acids can encode first, second, and / or third CARs, each separated by linkers, resulting in individual polypeptides encoded by the first, second, and third CAR molecules).

[0134] As used in this article, "connector" can refer to a polypeptide that links a first polypeptide sequence to a second polypeptide sequence.

[0135] In some embodiments, this disclosure provides engineered nucleic acids comprising expression cassettes (e.g., vectors) including a promoter operatively linked to an exogenous polynucleotide sequence encoding a GM2 CAR, a GM2-GD2 tandem CAR, and / or a bicistronic GM2-GD2 CAR. As used herein, a “promoter” generally refers to a control region of a nucleic acid sequence in which transcription initiation and rate are controlled for the remainder of the nucleic acid sequence. A promoter may also contain subregions in which regulatory proteins and molecules, such as RNA polymerases and other transcription factors, can bind. A promoter can be constitutive, inducible, repressive, tissue-specific, or any combination thereof. A promoter drives the expression or transcription of the nucleic acid sequence it regulates. A promoter is considered “operably linked” when it is in the correct functional position and orientation relative to the nucleic acid sequence it regulates to control transcription initiation and / or expression of that sequence.

[0136] Transmembrane domain The CAR disclosed herein may include a transmembrane domain. For example, the transmembrane domain may be a transmembrane hydrophobic α-helix. As used herein, "transmembrane domain" refers to any protein structure that has thermodynamic stability in the cell membrane (preferably the eukaryotic cell membrane, and more preferably the T cell membrane). The transmembrane domain can provide stability to the CAR containing that domain.

[0137] In some embodiments, the transmembrane domain of the CAR described herein may be a CD8 transmembrane domain. In other embodiments, the transmembrane domain may be a CD28 transmembrane domain. In still other embodiments, the transmembrane domain is a chimera of CD8 and CD28 transmembrane domains. In some embodiments, the transmembrane domain is selected from the CD8 transmembrane domain, CD28 transmembrane domain, CD3ζ chain transmembrane domain, CD4 transmembrane domain, 4-1BB transmembrane domain, OX40 transmembrane domain, ICOS transmembrane domain, CTLA-4 transmembrane domain, PD-1 transmembrane domain, LAG-3 transmembrane domain, 2B4 transmembrane domain, BTLA transmembrane domain, OX40 transmembrane domain, DAP10 transmembrane domain, DAP12 transmembrane domain, CD16a transmembrane domain, DNAM-1 transmembrane domain, KIR2DS1 transmembrane domain, KIR3DS1 transmembrane domain, NKp44 transmembrane domain, NKp46 transmembrane domain, FceRlg transmembrane domain, and NKG2D transmembrane domain. Other transmembrane domains may be used.

[0138] Hinge or spacer domain In some embodiments, the hinge or spacer domain may be located between the extracellular domains (including the antigen-binding domain) and the transmembrane domains of the CAR, or between the cytoplasmic domains and the transmembrane domains of the CAR. The hinge domain can be any oligopeptide or polypeptide, and its function is to connect the transmembrane domain to the extracellular and / or cytoplasmic domains in the polypeptide chain. The spacer or hinge domain is any oligopeptide or polypeptide, and its function is to connect the transmembrane domain to the extracellular and / or intracellular signal transduction domains in the polypeptide chain. The hinge domain can provide flexibility to the CAR or its domains, or prevent steric hindrance to the CAR or its domains.

[0139] In some embodiments, the 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 domains may be included in other regions of the CAR. In some embodiments, the hinge domain may be a CD8 hinge domain. Other hinge domains may be used.

[0140] Exemplary spacer or hinge domains include, but are not limited to, IgG domains (such as IgG1 hinges, IgG2 hinges, IgG3 hinges, or IgG4 hinges), IgD hinge domains, CD8α hinge domains, and CD28 hinge domains. In some embodiments, the spacer or hinge domain is an IgG domain, an IgD domain, a CD8α hinge domain, or a CD28 hinge domain.

[0141] Intracellular signal transduction domains The CAR constructs disclosed herein may include one or more intracellular signal transduction domains. In some embodiments, the CAR peptides disclosed herein include one or more co-stimulatory signal transduction domains. For example, the co-stimulatory domains of CD28 and / or 4-1BB can be used to deliver complete proliferation / survival signals, as well as the primary signal transduction mediated by CD3ζ (e.g., CD3ζ, and optionally one or more co-stimulatory domains), which are the functional terminals of the receptor. Upon antigen recognition, the receptor aggregates and transduces signals into the cell.

[0142] CD3ζ is the cytoplasmic signal transduction domain of the T cell receptor complex. CD3ζ contains three (3) immune receptor tyrosine-based activation motifs (ITAMs), which transmit activation signals to T cells after T cells bind to their homologous antigens.

[0143] In some embodiments, the CAR disclosed herein includes a CD28 co-stimulatory molecule. In other embodiments, the CAR disclosed herein includes a 4-1BB co-stimulatory molecule. In some embodiments, the CAR includes a CD3ζ signaling domain and a CD28 co-stimulatory domain. In other embodiments, the CAR includes a CD3ζ signaling domain and a 4-1BB co-stimulatory domain. In still other embodiments, the CAR includes a CD3ζ signaling domain, a CD28 co-stimulatory domain, and a 4-1BB co-stimulatory domain.

[0144] In some embodiments, the intracellular signal transduction domain includes or is composed of the following: 4-1BB intracellular signal transduction domain, CD28 intracellular signal transduction domain, CD3ζ chain intracellular signal transduction domain, ZAP70(SRK) intracellular signal transduction domain, ZAP40 intracellular signal transduction domain, CD30 intracellular signal transduction domain, OX40 intracellular signal transduction domain, CD27 intracellular signal transduction domain, DAP12 intracellular signal transduction domain, KIR2DS1 intracellular signal transduction domain, NKG2D intracellular signal transduction domain, FceRlg intracellular signal transduction domain, MyD88 intracellular signal transduction domain, EAT-2 intracellular signal transduction domain, D... The 4-1BB intracellular signal transduction domain comprises the following domains: AP10, ICOS, DNAM-1, CD2, CD8, CD16a, CD97, CD154, GITR, NKp46, 2B4, CD11a-CD18, NKp44, KIR3DS1, HVEM, and / or combinations of two or more intracellular signal transduction domains. In some embodiments, the 4-1BB intracellular signal transduction domain comprises or consists of the amino acid sequence shown in SEQ ID NO: 38. In some embodiments, the CD28 intracellular signal transduction domain comprises or consists of the amino acid sequence shown in SEQ ID NO: 39. In some embodiments, the CD3ζ chain intracellular signal transduction domain comprises or consists of the amino acid sequence shown in SEQ ID NO: 40. In some embodiments, the ZAP70(SRK) intracellular signal transduction domain comprises or consists of the amino acid sequence shown in SEQ ID NO: 41.

[0145] In some embodiments, the GM2 CAR, GM2-GD2 tandem CAR, and / or bicistronic GM2 of this disclosure are used. CAR includes the GM2 binding domain of this disclosure and one or more intracellular signal transduction domains, wherein the one or more intracellular signal transduction domains are selected from: CD3ζ chain intracellular signal transduction domain, CD97 intracellular signal transduction domain, CD11a-CD18 intracellular signal transduction domain, CD2 intracellular signal transduction domain, ICOS intracellular signal transduction domain, CD27 intracellular signal transduction domain, CD154 intracellular signal transduction domain, CD8 intracellular signal transduction domain, OX40 intracellular signal transduction domain, 4-1BB intracellular signal transduction domain, CD28 intracellular signal transduction domain, ZAP40 intracellular signal transduction domain, CD30 intracellular signal transduction domain, GITR intracellular signal transduction domain, HVEM intracellular signal transduction domain, DAP10 intracellular signal transduction domain, DAP12 intracellular signal transduction domain, and MyD88 intracellular signal transduction domain. In some embodiments, the CAR includes an intracellular signal transduction domain of the CD3ζ chain and one or more additional intracellular signal transduction domains (e.g., co-stimulatory domains) selected from the CD97, CD11a-CD18, CD2, ICOS, CD27, CD154, CD8, OX40, 4-1BB, CD28, ZAP40, and CD30 intracellular signal transduction domains. Intracellular signal transduction domains, including GITR, HVEM, DAP10, DAP12, MyD88, 2B4, CD16a, DNAM-1, KIR2DS1, KIR3DS1, NKp44, NKp46, FceRlg, NKG2D, and EAT-2.

[0146] Delivering CAR constructs to cells (e.g., T cells). In some embodiments, the nucleic acid encoding a CAR described herein can be introduced into any type of cell (e.g., T cells) disclosed herein using methods known to those skilled in the art. For example, the coding sequence of a CAR can be cloned into a vector, which can then be introduced into genetically engineered T cells to express the CAR. Various methods known in the art can be used to introduce any nucleic acid or expression vector disclosed herein into immune effector cells. Non-limiting examples of methods for introducing nucleic acids into cells include: liposome transfection, transfection (e.g., calcium phosphate transfection, transfection using highly branched organic compounds, transfection using cationic polymers, transfection based on dendritic polymers, optical transfection, particle-based transfection (e.g., nanoparticle transfection), or transfection using liposomes (e.g., cationic liposomes)), microinjection, electroporation, cell extrusion, sonopore, protoplast fusion, impalefection, hydrodynamic delivery, gene gun, magnetic transfection, viral transfection, and nuclear transfection.

[0147] In specific instances, adeno-associated virus (AAV) can be used to deliver nucleic acids encoding CAR constructs into cells. AAVs are small viruses that integrate site-specifically into the host genome, thus enabling the delivery of transgenes such as CARs. Inverted terminal repeats (ITRs) are present on the flanking sides of the AAV genome and / or the target transgene, serving as origins of replication. The AAV genome also contains rep and cap proteins, which, post-transcriptionally, form a capsid that encapsulates the AAV genome for delivery to target cells. Receptors on the surface of these capsids confer AAV serotypes, determining the primary target organs the capsid binds to, and consequently, which cells AAVs are most effective at infecting.

[0148] Adeno-associated viruses (AAVs) are among the most commonly used viruses in gene therapy for several reasons. First, AAVs do not elicit an immune response after administration to mammals, including humans. Second, AAVs are efficiently delivered to target cells, especially when considering the selection of a suitable AAV serotype. Finally, AAVs can infect both dividing and non-dividing cells because their genome can persist in the host cell without integration. This trait makes them ideal candidates for gene therapy.

[0149] Nucleic acids encoding CARs can be designed to insert into target genomic sites in host T cells (e.g., using CRISPR / CAS9 technology, TALE nucleases, and / or zinc finger nucleases). In some implementations, the target genomic site can be located in a safe harbor locus.

[0150] In some implementations, the nucleic acid encoding the CAR (e.g., carried via a donor template by a viral vector, such as a retroviral vector, lentiviral vector, or adeno-associated virus (AAV) vector) can be designed to insert into a target genomic site. In some cases, the nucleic acid may include a left and right homologous arm flanked by nucleotide sequences encoding the CAR. The left and right homologous arms are homologous to upstream and downstream sequences of the genomic site into which the CAR coding sequence is to be inserted. In some instances, the genomic site into which the CAR coding sequence is to be inserted is also the target site of the guide RNA, allowing the CAR-encoded nucleic acid to insert into the guide RNA target site. In some implementations, the left and right homologous arms may be homologous to sequences immediately flanking the guide RNA target site. In some cases, after gene editing, the guide RNA target site may be deleted and replaced by the CAR-encoded nucleic acid.

[0151] In some implementations, the nucleic acid encoding the CAR can be inserted into a genomic site via CRISPR / Cas9-mediated gene editing and homologous recombination. In other implementations, the donor template may not have a homologous region with the target site in the DNA, and integration can be achieved via NHEJ-dependent end joining after cleavage at the target site.

[0152] The donor template can be DNA or RNA, single-stranded and / or double-stranded, and can be introduced into the cell in a linear or circular form. If introduced in a linear form, the ends of the donor sequence can be protected (e.g., to prevent exonuclease degradation) by methods known to those skilled in the art. For example, adding one or more dideoxynucleotide residues to the 3' end of the linear molecule, and / or linking self-complementary oligonucleotides 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, adding one or more terminal amino groups and using modified internucleotide linkages, such as, for example, phosphate thioesters, phosphoramide esters, and O-methylribose or deoxyribose residues.

[0153] Donor templates can be introduced into cells as part of a vector molecule containing additional sequences, such as, for example, an origin of replication, a promoter, and a gene encoding antibiotic resistance. Furthermore, donor templates can be introduced into cells as naked nucleic acids, as nucleic acids complexed with drugs (such as liposomes or poloxamer), or delivered via viruses (e.g., adenoviruses, AAVs, herpesviruses, retroviruses, lentiviruses, and integrase-deficient lentiviruses (IDLVs)).

[0154] In some embodiments, the donor template can be inserted at a site near the 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 include an exogenous promoter and / or enhancer, such as a constitutive promoter, an inducible promoter, or a tissue-specific promoter, to control CAR expression.

[0155] In addition, exogenous sequences may include transcriptional or translational regulatory sequences, such as promoters, enhancers, insulators, internal ribosome entry sites, sequences encoding 2A peptides and / or polyadenylation signals. If desired, as disclosed herein, additional gene editing (e.g., gene knock-in or knock-out) may be introduced into therapeutic T cells to improve T cell function and therapeutic efficacy.

[0156] Cells containing nucleic acid molecules encoding CAR This document also provides cells modified with nucleic acid sequences encoding the CARs disclosed herein, and methods for producing them. These modified cells, comprising one or more engineered nucleic acids, do not exist in nature. In some embodiments, these cells are isolated cells that recombinantly express one or more engineered nucleic acids. In some embodiments, the engineered one or more nucleic acids are expressed from selected loci in one or more vectors or cell genomes. In some embodiments, the cells are engineered to include nucleic acids comprising a promoter operatively 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) that expresses any peptide sequence listed in Table 1.

[0157] The modified cells disclosed herein may include engineered nucleic acids integrated into the cell's genome. Engineered cells may also include engineered nucleic acids capable of being expressed without integration into the cell's genome, for example, engineered using a transient expression system (such as a plasmid or mRNA).

[0158] In some embodiments, the polynucleotide encoding the GM2 CAR, GM2-GD2 tandem CAR, and / or GM2-GD2 bicistronic CAR is encoded by a single polynucleotide sequence in the engineered cell. For example, in some embodiments, the engineered cell comprises a single engineered nucleic acid that includes a polynucleotide sequence encoding the GM2 CAR, GM2-GD2 tandem CAR, and / or GM2-GD2 bicistronic CAR.

[0159] This disclosure is not limited to cell types or cell populations modified to include engineered nucleic acids encoding CARs. Cells or cell populations can be T cells, including but not limited to CD4 T cells, CD8 T cells, γ-δ T cells, cytotoxic T lymphocytes (CTLs), natural killer (NK) cells, natural killer T (NKT) cells, and regulatory T cells. In some embodiments, cells or cell populations are dendritic cells, tumor-infiltrating lymphocytes (TILs), macrophages, monocytes, neutrophils, B cells, lymphoid cells, eosinophils, mast cells, basophils, erythrocytes, myeloid cells, platelet cells, stem cells, or mesenchymal stromal cells. In some embodiments, cells or cell populations are stem cells (e.g., induced pluripotent stem cells).

[0160] The cells or cell populations may be one or more human cells (e.g., primary T cells, tumor-infiltrating lymphocytes, hematopoietic stem cells (HSCs), or natural killer cells). In some embodiments, the cells are derived from a subject to be treated with the compositions and methods disclosed herein (e.g., autologous cells). In other embodiments, the cells are derived from a donor (e.g., allogeneic cells). In some embodiments, the cells or cell populations are isolated from the subject using methods known in the art, including but not limited to cell sorting techniques based on cell surface marker expression, FACS sorting, positive separation techniques, negative separation techniques, magnetic separation, and combinations thereof. The cells may be cultured in vitro (e.g., primary cells may be isolated from the subject and cultured in vitro). As detailed herein, cells can be engineered to produce GM2 CARs, GM2-GD2 tandem CARs, and / or GM2-GD2 bicistronic CARs by introducing (delivering) one or more nucleic acid molecules of this disclosure, including 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 cytosol and / or nucleus of a cell. For example, the nucleic acid expression cassette encoding a GM2 CAR, a GM2-GD2 tandem CAR, and / or a GM2-GD2 bicistronic CAR can be any engineered nucleic acid described herein. Delivery methods include, but are not limited to, virus-mediated delivery, lipid-mediated transfection, nanoparticle delivery, electroporation, sonication, and cell membrane deformation achieved by physical means. Those skilled in the art will understand that the delivery method may be selected depending on the specific cell type to be engineered.

[0161] Therapeutic applications The cells or cell populations disclosed herein (e.g., T cells) can be administered to subjects for therapeutic purposes, such as treating cancer or tumors, such as solid tumors targeted by CAR constructs expressed by the cells (e.g., therapeutic CAR-T cells). As reported herein, GM2 has been found to be a viable target for treating cancer / tumors, and targeting cancer / tumor cells with GM2 CAR T cells (e.g., GM2CAR, GM2-GD2 tandem CAR, and / or GM2-GD2 bicistronic CAR) can improve T cell persistence (e.g., reduced T cell exhaustion observed with GD2 CAR), increase cytokine secretion, and / or enhance CAR potency, thereby leading to improved antitumor efficacy, as observed in animal models (see Example 1).

[0162] In some embodiments, cells modified to express GM2 CAR and / or GD2 CAR (e.g., T cells) are administered to a patient. In some embodiments, a cell population modified to express GM2 CAR and GD2 CAR is administered. In other embodiments, a cell population modified to express one or more GM2 CARs and a separate cell population modified to express one or more GD2 CARs are administered. For example, in some embodiments, treating a patient includes administering a mixed cell pool comprising GD2 CAR T cells and GM2 CAR T cells. As described herein, the cells or cell populations may be allogeneic cells and / or autologous cells. In some embodiments, these cells are induced pluripotent stem cells.

[0163] Administration steps may include placing (e.g., transplanting) therapeutic T cells into a subject via a method or route that results in the therapeutic T cells being at least partially localized at a desired site (such as a tumor site), thereby producing one or more desired effects. Therapeutic T cells may be administered via any suitable route, resulting in delivery to the desired site in the subject, where at least a portion of the implanted cells or cell components remain viable. The viability of the cells after administration to the subject can range from a few hours (e.g., twenty-four hours) to several days, to several years, or even the lifespan of the subject, i.e., long-term transplantation. For example, in some embodiments, an effective amount of therapeutic T cells may be administered via a systemic route (such as intraperitoneal or intravenous routes).

[0164] In some embodiments, therapeutic T cells are administered systemically. This means that the administered cell population is not administered directly to a target site, tissue, or organ, but rather the cell population is introduced into the subject's circulatory system, where it is affected by metabolism and other similar processes. Suitable routes of administration include injection, infusion, drip infusion, or ingestion. "Injection" includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, spinal, intramedullary, and intrasternal injection and infusion. In some embodiments, the route is intravenous.

[0165] The subject can be any subject desiring diagnosis, treatment, or therapy. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some cases, the human patient has cancer involving the expression of GM2 in cancer cells. CAR-T cells expressing anti-GM2 CARs (e.g., those disclosed herein) can be used to treat such patients.

[0166] As described herein, therapeutic T cells can be the subject's own ("self") cells, meaning the cells originate from the same subject. Alternatively, therapeutic T cells can be non-self cells of the subject ("non-self" cells, such as allogeneic, syngeneic, or xenogeneic cells). "Allogeneic" means that the therapeutic T cells are not derived from the treated subject, but from a different individual (donor) belonging to the same species as the subject. The donor is an individual who is not receiving treatment. The donor is a non-patient individual. In some embodiments, the donor is an individual who does not have or is not suspected of having the cancer being treated. In some embodiments, multiple donors are used, for example, two or more donors.

[0167] In some embodiments, GM2-specific and / or GD2-specific recognition portions (e.g., CARs) are delivered directly to T cells or other immune cells in a patient. This disclosure is not limited to any particular in vivo delivery method. In fact, a variety of methods can be used, including but not limited to any of the following: lipid nanoparticles containing DNA, RNA, or retrotransposons; infusion of wild-type lentiviruses or retroviruses or adenoviruses or adeno-associated viruses or niphaviruses; or pseudotyped lentiviruses or retroviruses or adenoviruses or adeno-associated viruses or niphaviruses that have a specific tropism for T cells or other immune cells; or infusion of virus-like particles derived from lentiviruses, retroviruses or adenoviruses or adeno-associated viruses or niphaviruses or other viruses.

[0168] An effective amount refers to the amount of engineered T cell population required to prevent or alleviate at least one sign or symptom of a medical condition (e.g., cancer), and is related to an adequate amount of the composition that provides the desired effect, for example, treating a subject's cancer signs or symptoms. An effective amount also includes an amount sufficient to prevent or delay the development of disease symptoms, alter the course of disease symptoms (e.g., but not limited to slowing the progression of disease symptoms), or reverse disease symptoms. It should be understood that, for any given situation, an appropriate effective amount can be determined by a person skilled in the art using routine experiments.

[0169] The therapeutic efficacy of the therapeutic T cells disclosed herein can be determined by an experienced clinician. Treatment is considered “effective” if any or all signs or symptoms of a functional target level change in a beneficial manner (e.g., an increase of at least 10%), or if other clinically accepted symptoms or markers of disease (e.g., cancer) are improved or alleviated. Efficacy can also be measured by the subject’s failure to worsen, such as by hospitalization or the need for medical intervention (e.g., cessation or at least slowing of disease progression). Methods for measuring these measures are known to those skilled in the art and / or described herein. Treatment includes any treatment of the subject’s disease and includes: (1) suppressing the disease, e.g., preventing or slowing the progression of symptoms; or (2) alleviating the disease, e.g., causing symptom resolution; and (3) preventing or reducing the likelihood of symptom development.

[0170] This disclosure also covers combination therapies. For example, the therapeutic T cells disclosed herein can be used in combination with other therapeutic agents to treat the same indication, or to enhance the efficacy of the therapeutic T cells and / or reduce their side effects. One or more of the compositions described herein can be administered alone or in combination with other treatments simultaneously or sequentially, depending on the condition to be treated. For example, the compositions of this disclosure (e.g., GM2 CAR T cells, GM2-GD2 tandem CAR T cells, and / or GM2-GD2 bicistronic CAR T cells) can be combined with one or more other types of cancer treatments (including, but not limited to, chemotherapy, radiotherapy, and / or surgery) for co-administration or sequential administration. The combined use of two or more treatments can be performed simultaneously, before and / or after administration of one of the two or more types of treatment.

[0171] Reagent test kit This disclosure also provides kits for generating genetically engineered T cells, therapeutic T cells, and for therapeutic purposes.

[0172] In some embodiments, the kits provided herein may include the genetically engineered T cell populations disclosed herein, and one or more components disclosed herein for generating therapeutic T cells. Such components may include nucleic acids encoding a target CAR construct. In some cases, the donor template may be carried by a viral vector (such as a retroviral vector, a lentiviral vector, or other vectors described herein or known in the art). In yet other embodiments, the kits disclosed herein may include the disclosed therapeutic T cell populations for an intended therapeutic purpose. Any kit disclosed herein may include instructions for preparing therapeutic T cells, or therapeutic applications of the therapeutic T cells. In some embodiments, the included instructions may include a description of how to introduce nucleic acids encoding a CAR construct into T cells to prepare therapeutic T cells.

[0173] In some embodiments, the kits disclosed herein may include a population of genetically engineered T cells (e.g., CAR-T cells) for eliminating unwanted cells targeted by the CAR construct (e.g., for treating cancers such as solid tumors). Such kits may include one or more containers in which genetically engineered T cells can be placed. The kit may further include instructions for administering the therapeutic T cells disclosed herein to achieve the desired activity, e.g., eliminating disease cells targeted by the CAR expressed on the therapeutic T cells. Alternatively or additionally, the kit may further include a description of selecting suitable subjects for treatment based on identifying whether a subject requires treatment. Instructions for use with the therapeutic T cells described herein typically include information about the intended treatment dose, dosing regimen, and route of administration. Containers may be unit dose, bulk packaging (e.g., multi-dose packaging), or subunit dose. Instructions provided in the kits disclosed herein are typically written instructions on a label or packaging insert. The label or packaging insert indicates that the therapeutic T cells are intended to treat, delay onset, and / or alleviate a subject's disease or condition.

[0174] The kits described herein are available in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, etc. Furthermore, packaging for use in conjunction with specific devices, such as infusion devices for therapeutic T-cell administration, is also envisioned. The kits may have sterile interfaces (e.g., the container may be an intravenous infusion bag or a vial with a stopper that can be punctured by a subcutaneous injection needle). The container may also have a sterile inlet.

[0175] The kit may optionally include additional components, such as buffer solutions and explanatory information. Typically, the kit includes a container and a label or packaging appendix on or associated with the container. In some embodiments, this disclosure provides an article of manufacture comprising the contents of the kit described above.

[0176] Those skilled in the art can make maximum use of the described compositions and methods based on this disclosure. Therefore, the following detailed description should be interpreted as illustrative only and does not limit the remainder of this disclosure in any way. All publications referenced herein for the purposes or subject matter are incorporated herein by reference. From the above description, those skilled in the art can readily determine the essential features of the invention, and various changes and modifications can be made to adapt it to various uses and conditions without departing from the spirit and scope of the invention. Therefore, other embodiments are also within the scope of the claims.

[0177] Example Examples of specific implementations of this disclosure are provided. These examples are for illustrative purposes only and are not intended to limit the scope of this disclosure in any way.

[0178] Example 1: Generation, testing, and characterization of GM2 CAR in T cells Materials and Methods Culture conditions and cell lines All tumor cell lines were cultured in complete RPMI-1640 medium. The basal RPMI-1640 was supplemented with 10% heat-inactivated FBS (Gibco), 10 mM HEPES, 100 U / ml penicillin, 100 μg / ml streptomycin, and 2 mM L-glutamine (Gibco). Cells were maintained in an incubator at 37°C under a 5% CO2 atmosphere.

[0179] The identity of the tumor cell line is regularly confirmed by STR fingerprinting, and the cell line is tested for mycoplasma approximately every 6 months to see if it is negative.

[0180] cell line generation Nalm6 lines expressing GD2 (Nalm6-GD2) or GM2 (Nalm6-GM2) were generated by transducing retroviruses or lentiviruses with cDNA encoded by codons of B4GALNT1 (Nalm6-GM2) or B4GALNT1 and ST8SIA1 (Nalm6-GD2). The expression of the desired gangliosides was confirmed by flow cytometry. To obtain uniform ganglioside levels, cells were sorted using fluorescence-activated cell sorting (FACS) with FACSAria (BD Biosciences).

[0181] CHAL255 ST8SIA1 KO cells were generated via CRISPR-Cas9 KO, guided by a specific sgRNA designed to maximize KO efficiency and minimize off-target effects. Cas9 and sgRNA were introduced into the cells via nuclear transfection using the P3 Primary Cell 4D-Nucleofector X Kit S (Lonza). Briefly, cells were resuspended in 18 μl of P3 buffer and mixed with 2 μl of the previously assembled ribonucleic acid particle complex (Cas9:sgRNA). Cells were then electroporated in 16-well cuvette strips using the 4D-Nucleofector XUnit (Lonza). Cell recovery was then performed, followed by flow cytometry to confirm GD2 downregulation. As previously described, the desired cells were sorted to generate a homogeneous GD2-negative cell population.

[0182] Production of supernatants from retroviruses and lentiviruses Lentiviral supernatant was generated by transient transfection of HEK293-FT cells. In short, 6.5 × 10⁶ cells were seeded into 100 mm poly-d-lysine-coated plates in complete DMEM medium (10% FBS (Gibco), 10 mM HEPES, 2 mM glutamine, 100 U / ml penicillin, and 100 μg / ml streptomycin (Gibco)). After 24 hours, cells were co-transfected with 9 μg of vector plasmid, 9 μg of pRSV-Rev, 9 μg of pMDLg / pRRe, and 3.5 μg of pMD2.G using Lipofectamine 2000 (Invitrogen) in Opti-MEM medium (Gibco). After 24 hours, the medium was replaced with complete DMEM medium, and viral supernatant was collected at 48 and 72 hours post-transfection.

[0183] Retroviral supernatant was generated in a similar manner. Briefly, 6.5 × 10⁶ HEK293-GP cells were seeded in 100 mm poly-d-lysine-coated plates in complete DMEM medium (10% FBS (Gibco), 10 mM HEPES, 2 mM glutamine, 100 U / ml penicillin, and 100 μg / ml streptomycin (Gibco)). After 24 hours, the cells were co-transfected with 9 μg of vector plasmid and 4.5 μg of RD114 in Opti-MEM medium (Gibco) using Lipofectamine 2000 (Invitrogen). After 24 hours, the medium was replaced with complete DMEM medium, and viral supernatant was collected at 48 and 72 hours post-transfection. In both cases (lentivirus or retrovirus), the supernatant was used fresh or frozen at -80°C for subsequent use.

[0184] Isolation of PBMCs and T cells Peripheral blood mononuclear cells (PBMCs) were isolated from blood, erythrocyte sedimentation rate (ESR) amber layer, or LRS chambers using Ficoll-Paque Plus (GE Healthcare, 17-1440) density gradient centrifugation and cryopreserved in CryoStor CS10 cryopreservation medium (Sigma-Aldrich) as aliquots of 1×10⁷–5×10⁷ cells. For CAR T cell transduction and culture, the cryopreserved PBMCs were thawed on day 0 and cultured in AIM-V medium (Gibco) with human T activator anti-CD3 / anti-CD28 Dynabeads (Gibco) at a 3:1 bead:cell ratio supplemented with 5% FBS, 10 mM HEPES, 2 mM GlutaMAX, 100 U ml⁻¹ penicillin, 100 μg ml⁻¹ streptomycin, and 100 IU ml⁻¹ recombinant human IL-2 (Peprotech). Retroviral transduction was performed on days 3 and 4 after activation on tissue culture plates coated with retrotronectin (Takara). Wells were coated overnight with 25 μg ml-1 retrotronectin in 1 ml of phosphate-buffered saline (PBS), and then blocked for 15 min with 2% BSA in PBS before transduction. 1 ml of thawed or 700 μl of fresh retroviral supernatant was added to each CAR construct, and the plates were centrifuged at 3,200 rpm for 2–3 h at 32°C. The viral supernatant was discarded, and 0.5 × 10⁶ T cells were added to 1 ml of complete AIM-V medium per well. On day 5 post-activation, anti-CD3 / anti-CD28 beads were magnetically removed, and the CAR T cells were cultured in AIM-V medium, with medium changed every two to three days at a density of 0.3 × 10⁶ cells / ml.

[0185] Construction of CAR building blocks All DNA constructs were visualized using SnapGene software (Dotmatics). CAR constructs were generated using a mixture of restriction enzymes and In-Fusion HD Cloning (Takara Bio) with codon-optimized gBlocks (purchased from Integrated DNA Technologies). Table 1 shows the amino acid sequences of the generated, tested, and characterized CARs.

[0186] Flow cytometry Cell lines were harvested using TrypLE Express (Gibco, Thermo Fisher Scientific), and single-cell suspensions were prepared. Cells were washed twice with PBS + 2% FBS, and then stained with the listed primary antibody, isotype, or secondary antibody only as a control. Antibodies were incubated at 4°C for 20 minutes. Data were collected using BD FACSDIVA v9.0 or NovoExpress on an LSR Fortessa X-20 (BD Biosciences) or NovoCyte Quanteon (Agilent), respectively. Data analysis was performed using FlowJo (v10.6.1).

[0187] First antibody: Dinutuximab (anti-GD2, 1 μg / ml), DMF10.167.4 (anti-GM2, 1 μg / ml) Second antibody: Alexa Fluor® 647 AffiniPure donkey anti-human IgG (Jackson ImmunoResearch) (1:100 dilution).

[0188] On the same day used for in vitro and in vivo assays, CAR expression on T cells was assessed. CARs targeting GD2 or GM2 were detected using anti-mouse IgG (H+L) antibody (1:100 dilution). The following antibodies were used for lymphocyte staining: PD-1 (PE-Cy7, clone EH12.2H7, BioLegend, 1:50), TIM-3 (BV510 or BV650, clone F38-2E2, BioLegend, 1:50), and LAG-3 (PE, clone 3DS223H, Invitrogen, 1:50).

[0189] Cytotoxicity assay CAR+ T cells (day 10 post-activation) were co-cultured with 50,000 tumor cells at a specified E:T ratio in 96-well flat-bottom plates in complete RPMI medium. The co-cultures were incubated at 37°C and imaged using the Incucyte S3 live-cell analysis system (Sartorius) for approximately 72 h. Using the basic analytical functions of the Incucyte S3 software, GFP+ tumor cell killing was quantified by measuring the total green target integral intensity over a period of time. The cytotoxicity index was calculated as the percentage of the total green target integral intensity at a specific time point relative to the total green target integral intensity at time 0.

[0190] Cytokine assay A total of 1 × 10⁵ CAR+ T cells (day 10 post-activation) were co-cultured with tumor cells at a 1:1 E:T ratio in complete RPMI medium and incubated at 37°C for approximately 24 hours. Following stimulation, the supernatant was collected, and IL-2 or IFNγ was measured by ELISA according to the manufacturer's protocol (BioLegend). Absorbance was measured using a Synergy H1 hybrid multimode microplate reader equipped with Gen5 software (BioTek).

[0191] In vivo experiments All animal experiments were conducted according to protocols approved by the IACUC. Immunodeficient NSG (NOD.Cg-PrkdcscidIl2rgtm1Wjl / SzJ) mice were ordered from Jackson Laboratories or bred in-house, and were 6 to 12 weeks old at the start of the experiments. Mice were housed under strictly controlled temperature and humidity conditions, with a 12-hour light / dark cycle, and had free access to food and water. Mice in all experimental groups were age- and sex-matched.

[0192] Tumor cell lines expressing green fluorescent protein (GFP) and luciferase (Luc) (CHLA255, SH-SY5Y, and NBSD) were amplified under standard cell culture conditions (described herein). For inoculation into mice, cells were harvested using TrypLE Express (Gibco, Thermo Fisher Scientific), washed with PBS, counted, and resuspended in PBS at a concentration of 5 × 10⁶ cells / mL. For all metastatic models, 200 μl (1 × 10⁶ cells) was injected via tail vein.

[0193] Four minutes after intraperitoneal injection of 3 mg d-fluorescein (PerkinElmer), tumor growth was monitored by bioluminescence imaging (BLI) on an IVIS Spectrum in vivo imaging system (PerkinElmer). BLI values ​​were quantified using Living Image v4.7.3 software (PerkinElmer).

[0194] Antiganglioside CAR T-cell therapy Male or female NOD-scid IL2Rgnull (NSG, NOD.Cg-PrkdcscidIl2rgtm1Wjl / SzJl) mice aged six to ten weeks were intravenously injected with 1×10 6 CHLA255 cells for 7 days, 1×10 6 SY5Y cells for 5 or 6 days, 1×10 6 NBSD cells for 5 days, 1×10 6One Kelly cell for 7 days, or 1×10 6 Nalm6-GM2 cells were injected intravenously for 3 days, followed by T-cell injection (in 200 μl PBS) and monitored by PBS. In all models, mice were randomly assigned to ensure homogeneous tumor burden between experimental and control groups before treatment. CAR T-cells were injected intravenously on day 10 post-activation: CHLA-255, SY5Y, Nalm6-GM2, or Kelly tumor-bearing mice received 3 × 10⁻⁶ cells. 6 CAR+ T cells. In in vivo experiments with KM966-28z, NBSD or SY5Y tumor-bearing mice received 5 × 10 CAR+ T cells. 6 CAR+ T cells. Disease progression in neuroblastoma model mice was monitored weekly using the IVIS imaging system (Perkin Elmer) and Living Image software (Perkin Elmer) via BLI. For CHLA255, SY5Y, or Kelly models, mice were humanely euthanized when they developed pathological or palpable solid tumor masses. Mice were randomized to groups prior to T cell infusion to ensure equal mean tumor burden. Technicians performing intravenous injection of T cells and tumor cells blinded the treatment and expected outcomes.

[0195] GM2 is expressed on the surface of cancer cells According to the embodiments disclosed herein, experiments were conducted to evaluate and determine the surface expression levels of GD2 and GM2 in cancer cells. GD2 and GM2 levels were measured in a series of different neuroblastoma cell lines by flow cytometry. It was found that GM2 surface expression was high when GD2 surface expression was low (see Figure 1A). Neuroblastoma cell lines with high GD2 surface expression levels exhibited lower but significant GM2 surface expression levels (see Figure 1C). Since the same enzyme B4GALNT1 catalyzes the synthesis of GD2 from GD3 or GM2 from GM3, this disclosure provides the expression levels of GM2 in cells with high GD2 expression. The discovery of GM2 expression in neuroblastoma cell lines suggests that GM2 is a potential therapeutic target for this high-risk childhood cancer.

[0196] Additional experiments were conducted to determine the expression levels of GD2 and GM2 in Ewing sarcoma cell lines. As shown in Figure 2, most Ewing sarcoma cell lines were found to exhibit higher surface GM2 expression than GD2 expression, identifying GM2 as a target for Ewing sarcoma treatment, whereas previously only GD2 had been identified as a target.

[0197] Next, osteosarcoma patient-derived xenograft (PDX) cell lines were stained to characterize the surface expression of GD2 and GM2. The expression levels of GD2 and GM2 were examined in six different osteosarcoma patient-derived xenograft cell lines, and most cell lines showed higher GM2 expression than GD2 expression (Figure 3), identifying GM2 as a target of osteosarcoma, whereas previously only GD2 had been identified as a target.

[0198] CAR T cells and T cell exhaustion / cytotoxicity To evaluate and characterize T cell exhaustion and cytotoxicity between two distinct chimeric antigen receptor (CAR) T cell lines, experiments were conducted using a human CDR-transplanted GM2 (huGM2) CAR and a CAR T cell line containing a single-chain variable fragment of mouse GM2 (KM966), both specific for GM2 gangliosides. Expression of each CAR was similar (see Figure 4A), but the KM966 CAR exhibited less exhaustion, as characterized by surface expression of LAG3, PD1, and TIM3 (Figure 4B). When CAR T cells were co-cultured with GFP-expressing Nalm6-GM2 or Sy5y cell lines, the KM966 CAR showed better cytotoxicity than the huGM2 CAR T cells (see Figures 4C and 4D, respectively).

[0199] To optimize GM2-targeting DMF (10.62.3) CAR T cells, CD8 and CD28 hinge transmembrane domains in 4-1BBz or CD28z CAR T cells were generated and characterized. Each was found to have similar CAR surface expression (see Figure 5A), but only CAR T cells with the CD8 hinge transmembrane domain secreted IL-2 when co-cultured with the Nalm6-GM2 cell line (see Figure 5B). The cytotoxicity of each CAR against the Nalm6-GM2 cell line was similar (see Figure 5C). Therefore, in some embodiments, this disclosure provides that GM2-specific CARs with the CD8 transmembrane domain have advantages over GM2-specific CARs with the CD28 transmembrane domain (e.g., inducing higher cytokine (e.g., IL2) production).

[0200] Further experiments were conducted to optimize GM2-targeting KM966 CAR T cells. The hinge-transmembrane domains of CD8 and CD28 were generated and characterized in 4-1BBz or CD28z CAR T cells. Each had similar CAR surface expression intensity (Fig. 6A), but only CAR T cells with the CD8 hinge-transmembrane domain secreted IL-2 when co-cultured with the Nalm6-GM2 cell line (Fig. 6B). Against the Nalm6-GM2 cell line, GM-2 cells with the CD8 hinge-transmembrane domain exhibited superior cytotoxicity against KM966 CAR T cells compared to those with the CD28 hinge-transmembrane domain (Fig. 6C).

[0201] Experiments were conducted to further optimize GM2-targeting DMF(10.62.3) CAR T cells. CAR constructs with heavy-light and light-heavy chain orientations were generated. CAR expression in each construct was characterized, and the results were similar (Fig. 7A). Notably, CAR T cells with the DMF(10.62.3) heavy-light chain (HL) orientation exhibited superior cytokine production and cytotoxicity compared to CAR T cells with the light-heavy chain (LH) orientation (see Figs. 7B and 7C, respectively). Therefore, in some embodiments, this disclosure provides that the heavy-light chain orientation DMF(10.62.3) CAR has advantages over the light-heavy chain orientation DMF(10.62.3) CAR (e.g., HL orientation CAR induces higher cytokine production and / or cytotoxicity compared to LH orientation CAR).

[0202] Experiments were also conducted to further optimize KM966 CAR T cells targeting GM2. CAR constructs with heavy-light and light-heavy chain orientations were generated. CAR expression in each construct was characterized (Figure 8A). Notably, KM966 CAR T cells with a light-heavy chain (LH) orientation exhibited superior cytokine production and cytotoxicity compared to CAR T cells with a heavy-light chain (HL) orientation (see Figures 8B and 8C, respectively). Therefore, in some embodiments, this disclosure provides that the light-heavy chain CAR of KM966 has advantages over the heavy-light chain CAR (e.g., the LH-oriented CAR induces higher cytokine production and / or cytotoxicity compared to the HL-oriented CAR).

[0203] To optimize GM2-targeting KM966 CAR T cells, 4-1BB-ζ and CD28-ζ intracellular domains were generated and compared in KM966 CAR T cells. CAR expression of each construct was characterized (Figure 9A). CAR T cells with the CD28-ζ intracellular domain exhibited better cytokine production and cytotoxicity in the GM2-high expression cell lines Nalm6-GM2 and Sy5y (see Figures 9B and 9C, respectively).

[0204] To optimize GM2-targeting DMF(10.62.3) CAR T cells, 4-1BB-ζ and CD28-ζ intracellular domains were generated and compared in DMF(10.62.3) CAR T cells. CAR expression of each construct was characterized (Fig. 10A). In the GM2-high expression cell lines Nalm6-GM2 and Sy5y, CAR T cells with the CD28-ζ intracellular domain exhibited better cytokine production and cytotoxicity compared to CAR T cells with the 4-1BB-ζ intracellular domain (see Figs. 10B and 10C, respectively).

[0205] To optimize intracellular domains in DMF (10.62.3) or KM966, the 4-1BB-ζ, CD28-ζ, and ZAP70KIDB intracellular domains were compared. CAR expression of each construct was characterized by comparison with blank control cells (Fig. 11A). In both DMF (10.62.3) and KM966, CAR T cells with the ZAP70 KIDB intracellular domain showed an advantage in exhaustion phenotype (characterized by surface expression of LAG3, PD1, and TIM3) (see Fig. 11B). Furthermore, the CD28-ζ intracellular domain-CAR showed lower exhaustion than the 4-1BB-ζ intracellular domain-CAR. When co-cultured with Nalm6-GM2 leukemia tumor cells, each CAR T intracellular domain exhibited similar killing activity without impairing CAR T cell efficacy (C, D). Therefore, in some embodiments, this disclosure provides that GM2 CAR T cells including the ZAP70 KIDB intracellular domain have advantages over CD28 and 4-1BBz CARs (e.g., in some embodiments, this disclosure provides that GM2-28z CAR T cells exhibit effective killing of tumor cells (e.g., Nalm6-GM2 leukemia tumor cells) in the event of exhaustion reduction or absence).

[0206] In vivo experiments A neuroblastoma mouse model was investigated using the compositions and methods disclosed herein. Specifically, a low GD2 / high GM2 neuroblastoma mouse model was used and treated with a GM2-targeting KM966 CAR. The GM2-targeting KM966 CAR exhibited anti-tumor function. Specifically, the GM2-targeting CD28-ζ KM966 CAR showed anti-tumor function in vivo against NBSD or Sy5y neuroblastoma cells. The expression levels of GD2 or GM2 on NBSD (Fig. 12A) or Sy5y (Fig. 12B) neuroblastoma cells were characterized. Fig. 12C shows the in vivo tumor control efficacy of GM2-28z CAR T cells against NBSD (left inset) or Sy5y (right inset) neuroblastoma mouse models.

[0207] To determine whether the in vivo function of KM966 CAR T cells depends on the intracellular domain used, KM966 CAR T cells carrying CD28-ζ, 4-1BB-ζ, or ZAP70 KIDB intracellular domains were injected into a GM2-overexpressing Sy5y neuroblastoma mouse model. Specifically, mice with luciferase-expressing neuroblastoma xenografts (Sy5y) were treated with T cells expressing KM966-CD28-ζ CAR, KM966-4-1BB-ζ CAR, or KM966-ZAP70 KIDB CAR. Compared to KM966 CARs with 4-1BB-ζ or CD28-ζ intracellular domains, KM966 CARs with ZAP70 KIDB exhibited superior in vivo efficacy / antitumor activity (Figure 13A) and survival rate (Figure 13B). Therefore, in some embodiments, this disclosure provides that CARs having the ZAP70 KIDB intracellular domain have enhanced in vivo efficacy / anti-tumor function and / or survival compared to CARs having the 4-1BB-ζ or CD28-ζ intracellular domains.

[0208] To determine whether the in vivo function of DMF(10.62.3) CAR T cells depends on the intracellular domain used, DMF(10.62.3) CAR T cells carrying the intracellular domains of CD28-ζ, 4-1BB-ζ, or ZAP70 KIDB were injected into a GM2-overexpressing Sy5y neuroblastoma mouse model. Specifically, mice inoculated with luciferase-expressing neuroblastoma xenografts (Sy5y) were treated with T cells expressing DMF(10.62.3)-CD28-ζ CAR, DMF(10.62.3)-4-1BB-ζ CAR, or DMF(10.62.3)-ZAP70 KIDB CAR. Compared to DMF (10.62.3) CARs with 4-1BB-ζ or CD28-ζ intracellular domains, the DMF (10.62.3) CAR with ZAP70 KIDB exhibited superior in vivo efficacy / antitumor activity (Figure 14A) and survival rate (Figure 14B). This further supports the findings of this disclosure that, in some embodiments, CARs with the ZAP70 KIDB intracellular domain have enhanced in vivo efficacy / antitumor function and / or survival rate compared to CARs with 4-1BB-ζ or CD28-ζ intracellular domains.

[0209] The efficacy of GD2-GM2 CAR T cells External efficacy Tandem GD2-GM2 CAR T cells were generated. Figure 15B shows a schematic diagram illustrating a non-limiting example of the tandem CAR of this disclosure. To identify the in vitro function of the tandem GD2-GM2 (DMF10.62.3) CAR T cells, CAR T cells were co-cultured with cell lines that were high in GD2 (Nalm6-GD2), high in GM2 (Nalm6-GM2), or high in both GD2 and GM2 (CHLA255). It was observed that the tandem GD2-GM2 (DMF10.62.3)-4-1BB-ζ fragment CAR T cells could secrete cytokines against all three cell lines, while the monospecific CARs (GD2-BBz or GM2(DMF10.62.3)-BBz) only secreted cytokines in their respective GD2 or GM2-expressing cell lines.

[0210] Additional tandem GD2-GM2 CAR T cells were generated. Figure 16B shows a schematic diagram illustrating these non-limiting examples of tandem CARs of the present disclosure. To identify and characterize the in vitro function of these tandem GD2-GM2 CAR T cells, tandem GD2-GM2 CAR T cells were co-cultured with cell lines that were high in GD2 (Nalm6-GD2), high in GM2 (Nalm6-GM2), high in both GD2 and GM2 (CHLA255), or medium in GD2 / high in GM2 (Sy5y). Figure 16A shows the cytokine production when GD2, GM2, or tandem GD2 / GM2-targeting 4-1BB-ζ or CD28-ζ fragment CAR T cells were co-cultured with Nalm6-GD2 leukemia cells, Nalm6-GM2 leukemia cells, CHLA-255 neuroblastoma cells, or Sy5y neuroblastoma cells. Tandem GD2 / GM2-targeting 4-1BB-ζ or CD28-ζ fragment CAR T cells secreted cytokines in all four cell lines, while monospecific CARs (GD2-BBz or GM2-28z) only secreted cytokines in their respective GD2 or GM2-expressing cell lines. Tandem CARs with anti-GM2 binding conjugates further from the membrane (anti-GD2 conjugates closer to the membrane) outperformed anti-GD2 conjugates with conjugates further from the membrane (anti-GM2 conjugates closer to the membrane), suggesting that this may be the optimal arrangement of scFvs for tandem GD2-GM2 CARs.

[0211] Therefore, in some embodiments, this disclosure provides tandem GD2-GM2 and / or GM2-GD2 CARs, which have significant advantages over single-specific CARs when targeting tumor cells expressing both GD2 and GM2 (e.g., increased cytokine production and / or enhanced tumor cell killing).

[0212] Internal effects To determine and characterize the in vivo function of tandem GD2-GM2 CAR T cells, tandem GD2-GM2-targeting 4-1BB-ζ or CD28-ζ fragment CAR T cells were injected into mouse models transplanted with GD2-medium / GM2-high (Sy5y) cell lines. The tandem GM2-GD2-28z CAR cells exhibited similar efficacy to monospecific GM2-28z CAR T cells. In a Sy5y neuroblastoma mouse model, the in vivo function of tandem GD2-GM2 CAR T cells with intracellular domains of the 4-1BB-ζ (Fig. 17A) or CD28-ζ (Fig. 17B) fragments was observed. Therefore, in some embodiments, this disclosure provides for targeting GM2 with monospecific or bispecific / tandem CAR T cells, overcoming the resistance of GD2-low / GM2-high neuroblastoma to GD2 CAR T cells.

[0213] To determine and characterize the in vivo function of tandem GD2-GM2 CAR T cells, GD2-, GM2-, GD2-GM2-, or GM2-GD2-28z CAR T cells were injected into mice previously transplanted with GD2-high / GM2-medium / low (CHLA255) or GD2 / GM2-medium (Kelly) cell lines. The GM2-GD2-28z tandem CAR cell line showed the best efficacy compared to single-specific GM2-28z, GD2-28z CAR T cells, or tandem GD2-GM2-28z. Ganglioside levels at the endpoint were affected by treatment. In the CHLA255 model, xenografts treated with GD2-CAR T cells showed decreased GD2 levels and a compensatory increase in GM2. In contrast, when treated with GM2-CAR T cells, GM2 was downregulated while GD2 levels were slightly increased (see Figure 18C).

[0214] Although this disclosure has been shown and described with reference to preferred and various alternative embodiments, those skilled in the art will readily understand that various changes in form and details may be made without departing from the spirit and scope of this disclosure.

Claims

1. A chimeric antigen receptor (CAR) that binds to monosialotetrahexosylganglioside GM2 (GM2), wherein, The CAR includes a single-stranded Fv (scFv) that binds to GM2, a transmembrane domain, and one or more intracellular signal transduction domains. The scFv includes a pair of heavy chain variable (VH) regions and light chain variable (VL) regions, and in (A) VH and VL are selected from: i. The VH region comprising heavy chain complementarity-determining region 1 (CDR-H1) having the amino acid sequence of SEQ ID NO: 1, heavy chain complementarity-determining region 2 (CDR-H2) having the amino acid sequence of SEQ ID NO: 2, and heavy chain complementarity-determining region 3 (CDR-H3) having the amino acid sequence of SEQ ID NO: 3, and It includes the VL region of light chain complementarity-determining region 1 (CDR-L1) having the amino acid sequence of SEQ ID NO: 18, light chain complementarity-determining region 2 (CDR-L2) having the amino acid sequence of SEQ ID NO: 19, and light chain complementarity-determining region 3 (CDR-L3) having the amino acid sequence of SEQ ID NO: 20; ii. The VH region comprising heavy chain complementarity-determining region 1 (CDR-H1) having the amino acid sequence of SEQ ID NO: 4, heavy chain complementarity-determining region 2 (CDR-H2) having the amino acid sequence of SEQ ID NO: 5, and heavy chain complementarity-determining region 3 (CDR-H3) having the amino acid sequence of SEQ ID NO: 6, and This includes the VL region comprising the light chain complementarity-determining region 1 (CDR-L1) having the amino acid sequence of SEQ ID NO: 21, the light chain complementarity-determining region 2 (CDR-L2) having the amino acid sequence of SEQ ID NO: 22, and the light chain complementarity-determining region 3 (CDR-L3) having the amino acid sequence of SEQ ID NO: 23; and iii. A VH region comprising heavy chain complementarity-determining region 1 (CDR-H1) having the amino acid sequence of SEQ ID NO: 7, heavy chain complementarity-determining region 2 (CDR-H2) having the amino acid sequence of SEQ ID NO: 8, and heavy chain complementarity-determining region 3 (CDR-H3) having the amino acid sequence of SEQ ID NO: 9, and The VL region includes light chain complementarity-determining region 1 (CDR-L1) having the amino acid sequence of SEQ ID NO: 24, light chain complementarity-determining region 2 (CDR-L2) having the amino acid sequence of SEQ ID NO: 25, and light chain complementarity-determining region 3 (CDR-L3) having the amino acid sequence of SEQ ID NO: 26; or (B) The VH includes: Heavy chain complementarity-determining regions 1 (CDR-H1), 2 (CDR-H2), and 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 sequences selected from SEQ ID NO: 10-17, and The VL includes: Light chain complementarity-determining regions 1 (CDR-L1), 2 (CDR-L2), and 3 (CDR-L3), wherein the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are contained within the VL region of the amino acid sequences selected from SEQ ID NO: 27-35; or (C) The VH includes: Amino acid sequences having 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 with amino acid sequences selected from SEQ ID NO: 10-17; and The VL includes: Amino acid sequences having 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 with the amino acid sequences selected from SEQ ID NO: 27-35, or (D) The VH region includes: Amino acid sequences having 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 with amino acid sequences selected from SEQ ID NO: 10, 11, 12, 13, 14, 15, 16, and 17, and The VL region includes: Amino acid sequences having 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 with the amino acid sequences selected from SEQ ID NO: 27, 28, 29, 30, 31, 32, 33, 34, and 35. Optionally, the CAR includes one or more of a hinge domain, a spacer region, or a peptide linker.

2. The CAR according to claim 1, wherein, The single-chain Fv (scFv) is selected from amino acid sequences having 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 with amino acid sequences selected from SEQ ID NO: 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 56, 57, and 58.

3. The CAR according to claim 1 or 2, wherein, The transmembrane domains are selected from the following transmembrane domains: CD8 transmembrane domain, CD28 transmembrane domain, 4-IBB transmembrane domain, CD3ζ chain transmembrane domain, PD-1 transmembrane domain, DAP10 transmembrane domain, CTLA-4 transmembrane domain, CD16a transmembrane domain, OX40 transmembrane domain, NKG2D transmembrane domain; CD4 transmembrane domain, LAG-3 transmembrane domain, OX40 transmembrane domain, NKp44 transmembrane domain, ICOS transmembrane domain, DAP12 transmembrane domain, BTLA transmembrane domain, KIR3DS1 transmembrane domain, 2B4 transmembrane domain, DNAM-1 transmembrane domain, FceRlg transmembrane domain, KIR2DS1 transmembrane domain, and NKp46 transmembrane domain.

4. The CAR according to any one of claims 1-3, wherein, The transmembrane domain is selected from amino acid sequences having 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 with amino acid sequences selected from SEQ ID NO: 36 and 37.

5. The CAR according to any one of claims 1-4, wherein, The one or more intracellular signal transduction domains are each selected from the following intracellular signal transduction domains: 4-1BB intracellular signal transduction domain, CD28 intracellular signal transduction domain, CD3ζ chain intracellular signal transduction domain, ZAP70(SRK) intracellular signal transduction domain, ZAP40 intracellular signal transduction domain, CD30 intracellular signal transduction domain, OX40 intracellular signal transduction domain, CD27 intracellular signal transduction domain, DAP12 intracellular signal transduction domain, KIR2DS1 intracellular signal transduction domain, NKG2D intracellular signal transduction domain, FceRlg intracellular signal transduction domain, MyD88 intracellular signal transduction domain, EAT-2 intracellular signal transduction domain, and DAP10. Intracellular signal transduction domains, including the ICOS intracellular signal transduction domain, the DNAM-1 intracellular signal transduction domain, the CD2 intracellular signal transduction domain, the CD8 intracellular signal transduction domain, the CD16a intracellular signal transduction domain, the CD97 intracellular signal transduction domain, the CD154 intracellular signal transduction domain, the GITR intracellular signal transduction domain, the NKp46 intracellular signal transduction domain, the 2B4 intracellular signal transduction domain, the CD11a-CD18 intracellular signal transduction domain, the NKp44 intracellular signal transduction domain, the KIR3DS1 intracellular signal transduction domain, the HVEM intracellular signal transduction domain, and / or combinations of two or more intracellular signal transduction domains.

6. The CAR according to any one of claims 1-5, wherein, The one or more intracellular signal transduction domains are each selected from amino acid sequences having 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 with amino acid sequences selected from SEQ ID NO: 38, 39, 40, and 41.

7. The CAR according to any one of claims 1-6, wherein, The VH and VL of the scFv are separated by a peptide linker. Optionally, the scFv includes 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 according to 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. An engineered cell population 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 cells according to claim 10 or the cell population according to claim 11, wherein, The CAR is recombinantly expressed, optionally wherein the CAR is expressed from a vector or a selected locus in the genome of the cell.

13. The cell or cell population according to any one of claims 10-12, wherein, The cells or cell populations are selected from T cells, CD4 T cells, CD8 T cells, natural killer (NK) cells, natural killer T (NKT) cells, γ-δ T cells, cytotoxic T lymphocytes (CTLs), and regulatory T cells.

14. The cell or cell population according to any one of claims 10-12, wherein, The cells or cell groups are selected from dendritic cells, tumor-infiltrating lymphocytes (TILs), macrophages, monocytes, neutrophils, B cells, lymphoid cells, eosinophils, mast cells, basophils, erythrocytes, myeloid cells, platelet cells, stem cells, and mesenchymal matrix cells.

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

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

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

18. A kit for treating and / or preventing tumors, comprising the CAR of any one of claims 1-7, the engineered nucleic acid of claim 8, the expression vector of claim 9, any cell 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 (CAR), said at least one chimeric antigen receptor (CAR) comprising: The antigen-binding domain comprises an antigen-binding domain, a transmembrane domain, and at least one intracellular signal transduction domain, wherein the antigen-binding domain includes 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 includes heavy chain complementarity-determining region 1 (CDR-H1), heavy chain complementarity-determining region 2 (CDR-H2), and heavy chain complementarity-determining region 3 (CDR-H3), wherein the VH region has an amino acid sequence that is 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% identical to the amino acid sequence selected from SEQ ID NO: 10-17; Wherein, the at least one light chain variable (VL) region includes light chain complementarity-determining region 1 (CDR-L1), light chain complementarity-determining region 2 (CDR-L2), and light chain complementarity-determining region 3 (CDR-L3), wherein the VL region has an amino acid sequence that is 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% identical to the amino acid sequence selected from SEQ ID NO: 27-35.

20. The nucleic acid molecule according to claim 19, wherein, The at least one heavy chain variable region comprises or consists of the following: the amino acid sequence shown in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, 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 according to claim 19 or claim 20, wherein, The at least one light chain variable region comprises or consists of the following amino acid sequences shown in SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, 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 according to any one of claims 19-21, wherein, The antigen-binding domain is scFv, and in particular, wherein a) the scFv comprises or consists of an amino acid sequence having 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 with an amino acid sequence selected from SEQ ID NO: 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 56, 57, and 58.

23. The nucleic acid molecule according to any one of claims 19-22, wherein, The intracellular signal transduction domains include or consist of the following: 4-1BB intracellular signal transduction domain, CD28 intracellular signal transduction domain, CD3ζ chain intracellular signal transduction domain, ZAP70(SRK) intracellular signal transduction domain, ZAP40 intracellular signal transduction domain, CD30 intracellular signal transduction domain, OX40 intracellular signal transduction domain, CD27 intracellular signal transduction domain, DAP12 intracellular signal transduction domain, KIR2DS1 intracellular signal transduction domain, NKG2D intracellular signal transduction domain, FceRlg intracellular signal transduction domain, MyD88 intracellular signal transduction domain, EAT-2 intracellular signal transduction domain, and DAP10. Intracellular signal transduction domains, including the ICOS intracellular signal transduction domain, the DNAM-1 intracellular signal transduction domain, the CD2 intracellular signal transduction domain, the CD8 intracellular signal transduction domain, the CD16a intracellular signal transduction domain, the CD97 intracellular signal transduction domain, the CD154 intracellular signal transduction domain, the GITR intracellular signal transduction domain, the NKp46 intracellular signal transduction domain, the 2B4 intracellular signal transduction domain, the CD11a-CD18 intracellular signal transduction domain, the NKp44 intracellular signal transduction domain, the KIR3DS1 intracellular signal transduction domain, the HVEM intracellular signal transduction domain, and / or combinations of two or more intracellular signal transduction domains.

24. The nucleic acid molecule according to claim 23, wherein: The 4-1BB intracellular signal transduction domain includes or is composed of the amino acid sequence shown in SEQ ID NO:

38. The CD28 intracellular signal transduction domain includes or is composed of the amino acid sequence shown in SEQ ID NO:

39. The intracellular signal transduction domain of the CD3ζ chain includes or is composed of the amino acid sequence shown in SEQ ID NO: 40, or The intracellular signal transduction domain of the ZAP70(SRK) cell includes or is composed of the amino acid sequence shown in SEQ ID NO:

41.

25. The nucleic acid molecule according to any one of claims 19-24, wherein, The transmembrane domain includes or is composed of the amino acid sequence shown in SEQ ID NO: 36, or wherein, The transmembrane domain includes or consists of the amino acid sequence shown in SEQ ID NO:

37.

26. The nucleic acid molecule according to any one of claims 19-25, wherein, The chimeric antigen receptor (CAR) comprises an antigen-binding domain, a transmembrane domain, and at least one intracellular T-cell signaling domain from the N-terminus to the C-terminus, 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 according to any one of claims 19-26, wherein, The antigen-binding domain (scFv) of the chimeric antigen receptor (CAR) comprises or consists of the following amino acid sequences: 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 according to any one of claims 19-27, further comprising a codon optimized for expression in human T cells, and / or operatively 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 used to prepare chimeric antigen receptor T cells.

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

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

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

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

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

35. A method for preparing chimeric antigen receptor (CAR) T cells, the method comprising transducing T cells with the vector of claim 29 to prepare the chimeric antigen receptor (CAR) T cells.

36. Use of a method for treating a subject with a tumor by means of a chimeric antigen receptor (CAR) encoded by a nucleic acid molecule according to any one of claims 19-28, 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 with ganglioside GM2 and / or ganglioside GD2, particularly wherein the T cells are T cells derived from the subject, the T cells having been converted with a nucleic acid molecule encoding the chimeric antigen receptor according to any one of claims 19-28 or having been transduced with a vector comprising the nucleic acid molecule.

37. The chimeric antigen receptor (CAR) according to claim 36, wherein, The method further includes the following steps: T cells were obtained from the subject, and Transform the T cells with a nucleic acid molecule encoding the chimeric antigen receptor, or The T cells were transduced using a vector containing the nucleic acid molecules.

38. The chimeric antigen receptor (CAR) according to claim 36 or 37, wherein, The tumor is a neuroblastoma, sarcoma, or brain tumor.

39. The chimeric antigen receptor according to claim 36 or 37, wherein, The method further includes screening the subjects by detecting the cell surface expression of ganglioside GM2 on the tumor.

40. A kit for preparing chimeric antigen receptor (CAR) T cells or for treating tumors in a subject, wherein, The tumor includes cell surface expression of ganglioside GM2, the kit includes 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 the host cell of claim 31, and instructions for using the kit.

41. The nucleic acid molecule according to claim 19, wherein, The nucleic acid molecule is a component of a bicistronic carrier encoding an antigen-binding domain, the antigen-binding domain comprising a first heavy chain variable (VH) region and a light chain variable (VL) region pair specific to ganglioside GM2, and a second heavy chain variable (VH) region and a light chain variable (VL) region pair specific to ganglioside GD2.

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

43. The nucleic acid molecule according to claim 41, wherein, The nucleic acid molecule encodes a heavy chain variable (VH) region amino acid sequence that has 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 with an amino acid sequence selected from SEQ ID NO: 10, 11, 12, 13, 14, 15, 16, or 17.

44. The nucleic acid molecule according to claim 41, wherein, The nucleic acid molecule encodes a light chain variable (VL) region amino acid sequence that is 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% identical to an amino acid sequence selected from SEQ ID NO: 27, 28, 29, 30, 31, 32, 33, 34, or 35.

45. The nucleic acid molecule according to claim 41, wherein, The nucleic acid molecule encodes a single-stranded Fv (scFv) having an amino acid sequence that is 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% identical to an amino acid sequence selected from SEQ ID NO:43 or 56.

46. ​​A nucleic acid encoding a CAR, said CAR comprising a GM2-specific single-domain antibody (sdAb).

47. The nucleic acid according to claim 46, wherein, The CAR includes heavy chain complementarity-determining region 1 (CDR-H1), heavy chain complementarity-determining region 2 (CDR-H2), and 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 sequences selected from SEQ ID NO: 10-17.

48. The nucleic acid according to any one of claims 46-47, wherein, The CAR includes an amino acid sequence that is 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% identical to an amino acid sequence selected from SEQ ID NO:10-17.

49. A nucleic acid encoding the CAR according to 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. An engineered cell population expressing the nucleic acid of claim 49 or the expression vector of claim 50.

53. A composition comprising engineered cells, wherein, The engineered cells include a first engineered cell population and a second engineered cell population. The first engineered cell population expresses the nucleic acid of claim 49 or the expression vector of claim 50, and the second engineered cell population expresses 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 cell population according to any one of claims 10-12 or 51-53, wherein, The cells in question are stem cells.

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

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

57. The method according to claim 56, wherein, The cells in question are autologous.

58. The method according to claim 56, wherein, The cells are allogeneic.

59. The cell or cell population according to any one of claims 10-12 or 51-53, wherein, The cells or cell groups are selected from dendritic cells, tumor-infiltrating lymphocytes (TILs), macrophages, monocytes, neutrophils, B cells, lymphoid cells, eosinophils, mast cells, basophils, erythrocytes, myeloid cells, platelet cells, stem cells, and mesenchymal matrix cells.

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