Chimeric antigen receptor targeting glioma-associated antigens

Abstract

The disclosure relates to an immune effector cell or a population of immune effector cells expressing one or more chimeric antigen receptors (CARs) specific for one or more glioma-associated antigens. The disclosure also relates to an antigen-binding molecule, a CAR, an immune effector cell comprising the CAR, and use of the cell or population for treating cancer.

Description

[0001] CHIMERIC ANTIGEN RECEPTOR TARGETING GLIOMA-ASSOCIATED ANTIGENS

[0002] Field of the Invention

[0003] The disclosure relates to immune effector cells expressing one or more chimeric antigen receptors and their use in the treatment of glioma.

[0004] Background of Invention

[0005] Immunotherapeutic approaches that induce tumour-specific immune responses are being considered across multiple malignancies. For instance, therapeutic T cells may be engineered to direct their cytotoxic effects towards a particular antigen of interest. In this way, T cells responsible for killing tumour cells may be engineered to be specific for a tumour antigen. The specificity of a T cell may be directed by endogenous or recombinant T cell receptors or by chimeric antigen receptors (CARs).

[0006] CARs are synthetic receptors comprising an extracellular domain, often derived from an antibody single-chain variable fragment (scFv), and intracellular signalling and costimulatory domains derived from T cells. Genetic insertion of CARs into immune cells allows redirecting them to a desired antigen. Anti-CD19 CAR T cells led to a paradigm change in cancer therapy, based on their response rates in adult patients with recurrent / refractory diffuse large B cell lymphoma (DLBCL) or paediatric refractory B cell acute lymphoblastic leukaemia (B-ALL). Two CAR T cell products specific for the B-cell marker CD 19, Kymriah (Novartis) and Yescarta (Kite Pharma), became the first therapeutic products registered by the FDA comprising a genetic engineering element for the treatment of B-ALL and DLBCL.

[0007] Glioblastoma (GBM, grade IV astrocytoma) is the most frequent and aggressive primary malignant tumour originating in the brain. Despite treatments involving a combination of surgery, chemotherapy and radiotherapy, the overall survival of GBM patients is 14.6 to 16 months post-treatment. Several phase I / II studies using multipeptide vaccines or neoadjuvant immune checkpoint blockers have been tested, with limited success thus far. At present, CAR T cell approaches have been investigated for GBM targeting EGFRvIII, IL-13Ra2 and Her2 (Brown et al., New England Journal of Medicine NI526 (2016): 2561-2569; Ahmed et al., JAMA oncology 3.8 (2017): 1094- 1101; O’Rourke et al., Science translational medicine 9.399 (2017): eaaa0984). It is an object of the invention to develop further glioma antigen-specific CARs and improved immune effector cells expressing CARs, useful for treating glioma.

[0008] Summary of Invention

[0009] The invention provides an immune effector cell or a population of immune effector cells expressing one or more chimeric antigen receptors (CARs) specific for two or more glioma-associated antigens, wherein one or more of the glioma-associated antigens is a GD2 ganglioside.

[0010] The invention also provides an antigen-binding molecule specific for Tenascin C (TNC), which comprises a polypeptide having the complementary determining regions (CDR1, CDR2 and CDR3) from an amino acid sequence selected from any one of SEQ ID NOs: 31 to 36.

[0011] The invention further provides an antigen-binding molecule specific for Interleukin- 13 receptor subunit alpha-2 (IL13Ra2), which comprises a polypeptide having the complementary determining regions (CDR1, CDR2 and CDR3) from SEQ ID NO: 176.

[0012] The invention further provides an antigen-binding molecule specific for B7 Homolog 3 (B7-H3), which comprises a polypeptide having the complementary determining regions (CDR1, CDR2 and CDR3) from SEQ ID NO: 172.

[0013] The invention additionally provides a CAR comprising the antigen-binding molecule specific for TNC, IL13Ra2 or B7-H3. The invention also provides a CAR specific for a GD2 ganglioside, wherein the CAR comprises (i) an antigen binding domain comprising the CDRs of SEQ ID NO: 115, (ii) a hinge, (iii) a transmembrane domain, and (iv) an intracellular domain. The invention also provides an immune effector cell or population of immune effector cells comprising the CAR of the invention.

[0014] The invention further provides the immune effector cell or the population of immune effector cells of the invention for use in a method of treating cancer in a subject.

[0015] Brief Description of Figures

[0016] Figure 1: Specificity of human TNC-binding VHH. Specificity of the different anti-TNC VHH was measured by ELISA with coating of recombinant TNC (hTNCel7- TST) or CPSG4 (CSPG4-TST) as negative control. Results are given for VHH: A) RB895, B) RB896, C) RB897, D) RB898, E) RB899 and F) RB900. Figure 2: Comparison of the T cell-surface expression, killing activity and IFN-gamma secretion of anti-TNC VHH- and scFv-based CARs. A) CAR expression of 6 different VHH-based (895, 896, 897, 898, 899, 900) vs scFv-based control (R6N) anti- TNC_28z. Mock EP: non-transduced T cells. B) Killing activity, measured by flow cytometry as percentage of specific lysis, of the anti-TNC_28z RNA CAR T cells against Ge518 tumor cells. E:T ratio=effector to target ratio. C) JFN-y secretion in the supernatant of anti-TNC_28z RNA CAR T cells incubated with Ge518 GBM cells. E:T ratio=effector to target ratio. One-way ANOVA tests were used to compare the different experimental groups. For the post-tests, Dunnett’s multiple comparison test was used. The significance level was set at (*) p<0.05, (**) p<0.01, (***) p<0.001, and (****) p<0.0001.

[0017] Figure 3: CAR expression at the T cell surface of the triple valent (PTPRZ1- TNC-OAcGD2) CAR-T cells. CAR expression at the surface of individual monovalent or the triple CAR T cells was measured by flow cytometry. Expressions of A) anti-PTPRZl VHH-based CAR (832_28z), B) Anti-TNC (R6N_28z) and anti-OAcGD2 (h8B6_IgGlH_28z) scFv-based CAR were measured. Mock EP: Non-transduced T cells as control.

[0018] Figure 4: Cytotoxic activity and IFN-gamma secretion of the triple valent (PTPRZl-TNC-OAcGD2) CAR-T cells vs monovalent controls in PBT-22FH cells. A) Killing activity, measured by flow cytometry as percentage of specific lysis, of the anti- PTPRZl VHH-based CAR (832_28z), anti-TNC (R6N_28z), anti-OAcGD2 (h8B6_IgGlH_28z) and the trivalent (3v-CAR) RNA CAR T cells against PBT-22FH tumor cells. E:T ratio=effector to target ratio. B) IFN-y secretion in the supernatant of anti- PTPRZl VHH-based CAR (832_28z), anti-TNC (R6N_28z), anti-OAcGD2 (h8B6_IgGlH_28z) and the trivalent (3v-CAR) RNA CAR T cells incubated with PBT- 22FH GBM cells. E:T ratio=effector to target ratio. Mock EP: Non-electroporated (transduced) cells. One-way ANOVA tests were used to compare the different experimental groups. For the post-tests, Dunnett’s multiple comparison test was used. The significance level was set at (*) p<0.05, (**) p<0.01, (***) p<0.001, and (****) p<0.0001.

[0019] Figure 5: Cytotoxic activity and IFN-gamma secretion of the triple valent (PTPRZl-TNC-OAcGD2) CAR-T cells vs monovalent controls in Ge518 cells. A) Killing activity, measured by flow cytometry as percentage of specific lysis, of the anti- PTPRZ1 VHH-based CAR (832_28z), anti-TNC (R6N_28z), anti-0AcGD2 (h8B6_IgGlH_28z) and the trivalent (3v-CAR) RNA CAR T cells against Ge518 tumor cells. E:T ratio=effector to target ratio. B) IFN-y secretion in the supernatant of anti- PTPRZ1 VHH-based CAR (832_28z), anti-TNC (R6N_28z), anti-0AcGD2 (h8B6_IgGlH_28z) and the trivalent (3v-CAR) RNA CAR T cells incubated with Ge518 GBM cells. E:T ratio=effector to target ratio. Mock EP: Non-electroporated (transduced) cells. One-way ANOVA tests were used to compare the different experimental groups. For the post-tests, Dunnett’s multiple comparison test was used. The significance level was set at (*) p<0.05, (**) pO.Ol, (***) p<0.001, and (****) pO.OOOl.

[0020] Figure 6: Cytotoxic activity and IFN-gamma secretion of the triple valent (PTPRZl-TNC-OAcGD2) CAR-T cells vs monovalent controls in LAN-1 cells. A) Killing activity, measured by flow cytometry as percentage of specific lysis, of the anti- PTPRZ1 VHH-based CAR (832_28z), anti-TNC (R6N_28z), anti-OAcGD2 (h8B6_IgGlH_28z) and the trivalent (3v-CAR) RNA CAR T cells against LAN-1 tumor cells. E:T ratio=effector to target ratio. B) IFN-y secretion in the supernatant of anti- PTPRZ1 VHH-based CAR (832_28z), anti-TNC (R6N_28z), anti-OAcGD2 (h8B6_IgGlH_28z) and the trivalent (3v-CAR) RNA CAR T cells incubated with LAN-1 neuroblastoma cells. E:T ratio=effector to target ratio. Mock EP: Non-electroporated (transduced) cells. One-way ANOVA tests were used to compare the different experimental groups. For the post-tests, Dunnett’s multiple comparison test was used. The significance level was set at (*) p<0.05, (**) p<0.01, (***) p<0.001, and (****) pO.OOOl.

[0021] Figure 7: Cytotoxic activity of the triple valent (PTPRZl-TNC-OAcGD2) CAR-T cells vs monovalent controls. In vitro inhibition of A) PBT-22FH or B) LAN-1 tumor growth by the Triple CAR (3v-CAR) or individual monovalent CAR-T cells was measured by Incucyte. E:T ratio: effector to target ratio. Mock EP: Non-transduced T cells. One-way ANOVA tests were used to compare the different experimental groups. For the post-tests, Dunnett’s multiple comparison test was used. The significance level was set at (*) p<0.05, (**) pO.Ol, (***) p<0.001, and (****) pO.OOOl.

[0022] Figure 8: Cytotoxic activity of the triple valent (PTPRZl-TNC-OAcGD2) CAR-T cells vs monovalent controls. In vitro inhibition of A) Ge518_PTPRZl-KI or B) Ge518 tumor growth by the Triple CAR (3v-CAR) or individual monovalent CAR-T cells was measured by Incucyte. E:T ratio: effector to target ratio. Mock EP: Non-transduced T cells. One-way ANOVA tests were used to compare the different experimental groups. For the post-tests, Dunnett’s multiple comparison test was used. The significance level was set at (*) p<0.05, (**) p<0.01, (***) p<0.001, and (****) pO.OOOl.

[0023] Figure 9: CAR expression at the T cell surface of the triple valent (PTPRZ1- CSPG4-OAcGD2) CAR-T cells. Specific CAR expression at the surface of the triple CAR was measured by flow cytometry. Expressions of A) anti-PTPRZl vs anti-CSPG4, B) Anti-CSPG4 vs anti-0 AcGD2 and C) anti-PTPRZl vs anti-OAcGD2 were measured. NTD: Non-transduced T cells as control.

[0024] Figure 10: Cytotoxic activity of the triple valent (PTPRZl-CSPG4-OAcGD2) CAR-T cells on Ge518-PTPRZl-KI cells. A) Comparison of the killing activity, measured by flow cytometry as percentage of specific lysis, of the monovalent anti- PTPRZl_28z (471_28z), anti-CSPG4 (301_IgGlH_28z), anti-OAcGD2 (8B6_28z) and triple valent (TripleCAR) RNA CAR T cells against Ge518 tumor cells or B) Ge518 cells overexpressing PTPRZ1 (Ge518_PTPRZl-KI). E:T ratio=effector to target ratio. C) In vitro inhibition of Ge518_PTPRZl-KI tumor growth by the Triple CAR or individual monovalent CAR-T cells was measured by Incucyte. E:T ratio: 3: 1. NTD: Non-transduced T cells.

[0025] Figure 11: CAR expression at the T cell surface of the triple valent (B7- H3_IL13Ra2_OAcGD2) CAR-T cells. CAR expression at the surface of individual monovalent or triple CAR-T cells was measured by flow cytometry. Triple CAR-T cells were obtained by co-electroporation (co-EP) of the three individual CAR RNAs. Expressions of A) anti-B7-H3 and anti-IL13Ra2 VHH-based CARs (925_IgGlH_28z and 927_IgGlH_28z) and B) anti-OAcGD2 (h8B6_IgGlH_28z) scFv-based CAR were measured. Mock EP: Non-transduced T cells used as control. Numbers represent the fraction of T cells positive to the CAR expression.

[0026] Figure 12: Comparison of the killing activity of the triple valent (B7- H3_IL13Ra2_OAcGD2) CAR-T cells and monovalent anti-IL13R«2 CAR-T cells against different tumor cell lines. Killing activity, measured by flow cytometry as percentage of specific lysis, of the Triple CAR (B7-H3_IL13Ra2_OAcGD2) and anti- IL13Ra2 CAR-T cells against tumor cell lines Ge518 (glioblastoma), LN-229 (glioblastoma), LAN-1 (neuroblastoma), MDA-MB-231 (breast cancer), A375 (melanoma) and SKOV-3 (ovarian cancer). E:T ratio (effector to target ratio) = 2: 1. Mock EP: Nontransduced T cells used as control. One-way ANOVA tests were used to compare the different experimental groups. For the post-tests, Tukey’s multiple comparison test was used. The significance level was set at (*) p<0.05, (**) p<0.01, (***) p<0.001, and (****) p<0.0001.

[0027] Figure 13: Comparison of the tumor growth inhibition activity of the triple valent (B7-H3_IL13Ra2_OAcGD2) CAR-T cells and monovalent anti-IL13R«2 CAR- T cells against different tumor cell lines. Tumor growth inhibition, measured by Incucyte’s image analysis, of the Triple CAR (B7-H3_IL13Ra2_OAcGD2) and anti- IL13Ra2 CAR-T cells against tumor cell lines A) Ge518 (glioblastoma), B) LN-229 (glioblastoma), C) LAN-1 (neuroblastoma), D) MDA-MB-231 (breast cancer), E) A375 (melanoma) and F) SKOV-3 (ovarian cancer). E:T ratio (effector to target ratio) = 2: 1. Mock EP: Non-transduced T cells used as control. One-way ANOVA tests were used to compare the different experimental groups. For the post-tests, Dunnett’s multiple comparison test was used. The significance level was set at (*) p<0.05, (**) p<0.01, (***) p<0.001, and (****) pO.OOOl.

[0028] Figure 14: Comparison of the killing activity of the triple valent (B7- H3_IL13Ra2_OAcGD2) CAR-T cells and monovalent anti-B7-H3 CAR-T cells against different tumor cell lines. Killing activity, measured by flow cytometry as percentage of specific lysis, of the Triple CAR (B7-H3_IL13Ra2_OAcGD2) and anti-B7- H3 CAR-T cells against tumor cell lines Ge518 (glioblastoma), LN-229 (glioblastoma), LAN-1 (neuroblastoma), MDA-MB-231 (breast cancer), A375 (melanoma) and SKOV-3 (ovarian cancer). E:T ratio (effector to target ratio) = 2: 1. Mock EP: Non-transduced T cells used as control. One-way ANOVA tests were used to compare the different experimental groups. For the post-tests, Tukey’s multiple comparison test was used. The significance level was set at (*) p<0.05, (**) p<0.01, (***) p<0.001, and (****) p<0.0001.

[0029] Figure 15: Comparison of the tumor growth inhibition activity of the triple valent (B7-H3_IL13Ra2_OAcGD2) CAR-T cells and monovalent anti-B7-H3 CAR-T cells against different tumor cell lines. Tumor growth inhibition, measured by Incucyte’s image analysis, of the Triple CAR (B7-H3_IL13Ra2_OAcGD2) and anti-B7-H3 CAR-T cells against tumor cell lines A) Ge518 (glioblastoma), B) LN-229 (glioblastoma), C) LAN-1 (neuroblastoma), D) MDA-MB-231 (breast cancer), E) A375 (melanoma) and F) SKOV-3 (ovarian cancer). E:T ratio (effector to target ratio) = 2: 1. Mock EP: Nontransduced T cells used as control. One-way ANOVA tests were used to compare the different experimental groups. For the post-tests, Dunnett’s multiple comparison test was used. The significance level was set at (*) p<0.05, (**) p<0.01, (***) p<0.001, and (****) p<0.0001.

[0030] Figure 16: Comparison of the killing activity of the triple valent (B7- H3_IL13Ra2_OAcGD2) CAR-T cells and monovalent anti-OAcGD2 CAR-T cells against different tumor cell lines. Killing activity, measured by flow cytometry as percentage of specific lysis, of the Triple CAR (B7-H3_IL13Ra2_OAcGD2) and anti- 0AcGD2 CAR-T cells against tumor cell lines Ge518 (glioblastoma), LN-229 (glioblastoma), LAN-1 (neuroblastoma), MDA-MB-231 (breast cancer), A375 (melanoma) and SKOV-3 (ovarian cancer). E:T ratio (effector to target ratio) = 2: 1. Mock EP: Nontransduced T cells used as control. One-way ANOVA tests were used to compare the different experimental groups. For the post-tests, Tukey’s multiple comparison test was used. The significance level was set at (*) p<0.05, (**) p<0.01, (***) p<0.001, and (****) p<0.0001.

[0031] Figure 17: Comparison of the tumor growth inhibition activity of the triple valent (B7-H3_IL13Ra2_OAcGD2) CAR-T cells and monovalent anti-OAcGD2 CAR-T cells against different tumor cell lines. Tumor growth inhibition, measured by Incucyte’s image analysis, of the Triple CAR (B7-H3_IL13Ra2_OAcGD2) and anti- 0AcGD2 CAR-T cells against tumor cell lines A) Ge518 (glioblastoma), B) LN-229 (glioblastoma), C) LAN-1 (neuroblastoma), D) MDA-MB-231 (breast cancer), E) A375 (melanoma) and F) SKOV-3 (ovarian cancer). E:T ratio (effector to target ratio) = 2: 1. Mock EP: Non-transduced T cells used as control. One-way ANOVA tests were used to compare the different experimental groups. For the post-tests, Dunnett’s multiple comparison test was used. The significance level was set at (*) p<0.05, (**) p<0.01, (***) p<0.001, and (****) pO.OOOl.

[0032] Figure 18: Comparison of the killing activity of the triple valent (B7- H3_IL13Ra2_OAcGD2) CAR-T cells generated by co-electroporation or by pooling monovalent CAR-T cells, against different tumor cell lines. Killing activity, measured by flow cytometry as percentage of specific lysis, of the Triple CAR (B7- H3_IL13Ra2_OAcGD2) generated by co-electroporation (co-EP) or by pooling monovalent CAR-T cells (pool) against tumor cell lines Ge518 (glioblastoma), LN-229 (glioblastoma), LAN-1 (neuroblastoma), MDA-MB-231 (breast cancer), A375 (melanoma) and SKOV-3 (ovarian cancer). E:T ratio (effector to target ratio) = 2: 1. Mock EP: Nontransduced T cells used as control. One-way ANOVA tests were used to compare the different experimental groups. For the post-tests, Tukey’s multiple comparison test was used. The significance level was set at (*) p<0.05, (**) p<0.01, (***) p<0.001, and (****) p<0.0001.

[0033] Figure 19: Comparison of the tumor growth inhibition activity of the triple valent (B7-H3_IL13R«2_OAcGD2) CAR-T cells generated by co-electroporation or by pooling monovalent CAR-T cells, against different tumor cell lines. Tumor growth inhibition, measured by Incucyte’s image analysis, of the Triple CAR (B7- H3_IL13Ra2_OAcGD2) generated by co-electroporation (co-EP) or by pooling monovalent CAR-T cells (pool) against tumor cell lines A) Ge518 (glioblastoma), B) LN- 229 (glioblastoma), C) LAN-1 (neuroblastoma), D) MDA-MB-231 (breast cancer), E) A375 (melanoma) and F) SKOV-3 (ovarian cancer). E:T ratio (effector to target ratio) = 2: 1. Mock EP: Non-transduced T cells used as control. One-way ANOVA tests were used to compare the different experimental groups. For the post-tests, Dunnett’s multiple comparison test was used. The significance level was set at (*) p<0.05, (**) p<0.01, (***) p<0.001, and (****) pO.OOOl.

[0034] Figure 20: Comparison of the expression of the target antigens IL13R«2, B7- H3 and OAcGD2 on the surface of different tumor cell lines. Tumor cell lines Ge518 (glioblastoma), LN-229 (glioblastoma), LAN-1 (neuroblastoma), MDA-MB-231 (breast cancer), A375 (melanoma) and SKOV-3 (ovarian cancer) were stained with A) anti- IL13Ra2 (2.5 pg / mL of VHH 927 conjugate to rabbit Fc), B) anti-B7-H3 (2.5 pg / mL of VHH 925 conjugate to rabbit Fc) or C) anti-OAcGD2 (2.5 pg / mL of scFv h8B6 conjugate to rabbit Fc). As a secondary antibody, an anti-rabbit_AF647 was used in all cases.

[0035] Figure 21: Comparison of the killing activity of the double valent (PTPRZl_OAcGD2) CAR-T cells and monovalent CAR-T cells, against different tumor cell lines. Killing activity, measured by flow cytometry as percentage of specific lysis, of the PTPRZl-OAcGD2 CAR compared to monovalent anti-PTPRZland anti- 0AcGD2 CAR-T cells against tumor cell lines A) Ge518 (PTPRZ1', 0AcGD2+), B) Ge738_PTPRZl-KI (PTPRZ1+, 0AcGD2 ) and C) Ge518_PTPRZl-KI (PTPRZ1+, 0AcGD2+). E:T ratio (effector to target ratio) = 3: 1. Mock EP: Non-transduced T cells used as control. One-way ANOVA tests were used to compare the different experimental groups. For the post-tests, Dunnett’s multiple comparison test was used. The significance level was set at (*) p<0.05, (**) p<0.01, (***) p<0.001, and (****) p<0.0001.

[0036] Figure 22: Comparison of the tumor growth inhibition activity of the double valent (PTPRZl_OAcGD2) CAR-T cells and monovalent CAR-T cells, against different tumor cell lines. Tumor growth inhibition, measured by Incucyte’s image analysis, of the PTPRZl-OAcGD2 CAR compared to monovalent anti-PTPRZland anti- 0AcGD2 CAR-T cells against tumor cell lines A) Ge518 (PTPRZF, 0AcGD2+), B) Ge738_PTPRZl-KI (PTPRZ1+, 0AcGD2 ) and C) Ge518_PTPRZl-KI (PTPRZ1+, 0AcGD2+). E:T ratio (effector to target ratio) = 3: 1. Mock EP: Non-transduced T cells used as control. One-way ANOVA tests were used to compare the different experimental groups. For the post-tests, Dunnett’s multiple comparison test was used. The significance level was set at (*) p<0.05, (**) p<0.01, (***) p<0.001, and (****) p<0.0001.

[0037] Figure 23: Tumor-associated antigen (TAA) expression in healthy brain and GBM tumors. Frequency of cells positive for BCAN, TNC, PTPRZ1, or 0AcGD2 in normal brain tissue (left) and glioblastoma (GBM) tumor samples (right). Each data point represents an individual tissue microarray (TMA) core, with the median expression level indicated by a horizontal line.

[0038] Figure 24: Expression of tumour-associated antigens in cancer. Data in 0AcGD2 column generated in house (n between 22 and 122 for each cancer). Remaining data derived from published literature and patents. Values represent the percentage of cases / samples expressing the tumour associated antigen. ** = High expression, percentage of cases not reported. = No data available.

[0039] Figure 25: Expression of tumour-associated antigens in cancer. Data in 0AcGD2 column generated in house (n between 22 and 122 for each cancer). Remaining data derived The Human Protein Atlas (https: / / www.proteinatlas.org / ). Values represent the percentage of cases / samples expressing the tumour associated antigen. = No data available.

[0040] Description

[0041] The invention relates to an immune effector cell or a population of immune effector cells expressing one or more chimeric antigen receptors (CARs) specific for one or more glioma-associated antigens. The antigens receptor-type tyrosine-protein phosphatase zeta (PTPRZ1, alternatively designated tyrosine phosphatase receptor type Z polypeptide 1; UniProt No P23471), brevican core protein (BCAN; alternatively designated brain- enriched hyaluronan-binding protein (BEHAB), or Chondroitin sulfate proteoglycan 7; UniProt No Q96GW7), chondroitin sulfate proteoglycan 4 (CSPG4; alternatively designated chondroitin sulfate proteoglycan NG2, melanoma chondroitin sulfate proteoglycan, melanoma-associated chondroitin sulfate proteoglycan; UniProt No Q6UVK1) and tenascin C (TNC; alternatively designated cytotactin, GMEM, GP 150-225, glioma-associated-extracellular matrix antigen, hexabrachion, myotendinous antigen, neuronectin; UniProt No P24821) have recently been found to be expressed in glioma (Dutoit et al, Brain 135.4 (2012): 1042-1054). A multipeptide vaccine comprising peptides from these antigens has been created (Dutoit et al, Oncoimmunology 7.2 (2018): el391972.; Migliorini et al, Neuro-oncology 21.7 (2019): 923-933.) and is undergoing clinical trials (NCT03665545, NCT02924038). The inventors have also identified that GD2 gangliosides, such as (9-acetyl GD2 gangliosides, are also expressed in glioma in both paediatric and adult subjects.

[0042] The inventors have shown that immune effector cells expressing CARs specific for one or more glioma-associated antigens are able to generate a specific immune response to cells expressing the glioma-associated antigens as measured by cytotoxicity assays and T- cell activation.

[0043] Until now, CAR T cell approaches have been investigated clinically for recurrent GBM (targeting EGFRvIII, IL13Ra2, Her2) in the monovalent format, i.e., targeting one antigen at a time. Some patients displayed disease stabilisation but tumours invariably recurred and loss of the epitope targeted by the monovalent CAR-T cell used was seen in some instances. The present inventors have identified new glioma-associated antigens that have been targeted in a monovalent format and further demonstrated their efficacy in a multivalent targeting approach.

[0044] In one aspect, the invention provides an immune effector cell or a population of immune effector cells expressing one or more CARs specific for two or more glioma- associated antigens. One or more of the glioma-associated antigens is a GD2 ganglioside (CAS No.65988-71-8). The present inventors have found that GD2 gangliosides, such as (9-acetylated GD2 gangliosides (0AcGD2) are associated with cancer, particularly glioma, and that CARs targeting GD2 gangliosides are particularly efficacious in a multivalent targeting approach.

[0045] The immune effector cell of population of immune effector cells may express one or more CARs specific for three or more glioma-associated antigens. The immune effector cell of population of immune effector cells may express one or more CARs specific for four or more glioma-associated antigens. The immune effector cell of population of immune effector cells may express one or more CARs specific for five or more glioma- associated antigens.

[0046] The immune effector cell of population of immune effector cells may achieve the required multi-specificity for the two or more glioma-associated antigens through any means known to the skilled person.

[0047] In one aspect, the immune effector cell, or each cell in the population of immune effector cells expresses two or more CARs, wherein each CAR comprises specificity for a different glioma-associated antigen. Accordingly, the immune effector cell, or each cell in the population of immune effector cells, may express a CAR specific for a GD2 ganglioside and a CAR specific for a different glioma-associated antigen. The immune effector cell, or each cell in the population of immune effector cells, may express one or more further CARs. For example, the immune effector cell, or each cell in the population of immune effector cells, may express a CAR specific for a GD2 ganglioside, and two further CARs each specific for a different glioma-associated antigen. The immune effector cell, or each cell in the population of immune effector cells, may express three or more, four or more, or five or more CARs, each specific for a different glioma-associated antigen. The advantages of this approach are demonstrated in Figs. 18 and 19. The coelectroporation (co-EP) approach, in which T cells are transfected with three vectors each encoding a CAR specific for a different antigen to thereby express multiple different CARs within a single T cell, is shown to be more efficacious than an approach of pooling three populations of monovalent CAR T cells each specific for a different antigen. Without wishing to be bound by theory, it is believed that the use of multiple CARs specific for a different antigen within a single T cell may reduce the threshold level of antigen needed to achieve sufficient signalling for T cell activation, when compared to monovalent T cells.

[0048] One or more of the CARs may be a multivalent CAR specific for two or more glioma associated antigens. For example, the multivalent CAR may be specific for a GD2 ganglioside and another glioma-associated antigen. In some cases, the immune effector cell, or the population of immune effector cells, may express a CAR specific for a GD2 ganglioside and a multivalent CAR specific for two or more different glioma-associated antigens.

[0049] The population of immune effector cells may comprise two or more (i.e. at least two) different CAR-expressing immune effector cells, wherein each different CAR- expressing immune effector cell is specific for a different glioma-associated antigen. Accordingly, the population may comprise an immune effector cell expressing a CAR specific for a GD2 ganglioside and an immune effector cell expressing a CAR specific for a different glioma-associated antigen. The population may comprise one or more further different CAR-expressing immune effector cells. For example, the population may comprise an immune effector cell expressing a CAR specific for a GD2 ganglioside and two further different CAR-expressing immune effector cells, each expressing a CAR specific for a different glioma-associated antigen. The population may comprise three or more, four or more, or five or more different CAR-expressing immune effector cells, each expressing a CAR specific for a different glioma-associated antigen.

[0050] Suitable glioma-associated antigens to be targeted by the immune effector cell, or population of immune effector cells, are discussed in more detail herein.

[0051] In some cases, the invention makes use of the ‘bystander’ effect, as described in the Examples of the application. For example, the immune effector cells of the invention comprising two or more CARs specific for a glioma associated antigen may be capable of killing cancer cells comprising a mix of cancer cells that express the glioma-associated antigen and cancer cells that do not express the glioma-associated antigen. The cancer cells not expressing the glioma-associated antigen are believed to be killed via soluble factors, potentially allowing for greater therapeutic efficacy.

[0052] Furthermore, lentivirally-transduced CAR T cells are associated with toxicity, including cytokine release syndrome and neurotoxicity. In some aspects, the immune effector cells of the invention are transduced with RNA in order to overcome these issues.

[0053] Antigen-binding domain

[0054] The CARs discussed herein comprise an antigen-binding domain. The antigenbinding domain may be any domain that specifically binds to a glioma-associated antigen. For example, the anti gen -binding domain may be an scFv, a monoclonal antibody (comprising 2 heavy chains and 2 light chains), a polyclonal antibody, Fab, a Fab’, a F(ab’)2 fragment, a heavy chain variable domain (VH) or a nanobody (VHH). Each antigenbinding domain typically comprises three or more complementarity determining regions (CDRs) or antigen binding regions (ABRs)(these terms are used interchangeably herein). The CDRs or ABRs are typically responsible for antigen specificity, for example, by making direct interactions with the antigen. For example, the VHH sequences described herein each comprise three CDRs. The scFv sequences described herein each comprise six CDRs; three CDRs in the sequence corresponding to a VH domain and three CDRs in the sequence corresponding to a VL domain.

[0055] In some cases, the antigen-binding domain comprises one or more immunoglobulin variable domains. For example, the CAR may comprise one or more immunoglobulin variable domains. Each immunoglobulin variable domain typically comprises three complementarity determining regions (CDRs) or antigen binding regions (ABRs)(these terms are used interchangeably herein). The CDRs or ABRs are typically responsible for antigen specificity, for example, by making direct interactions with the antigen. The immunoglobulin variable domains also comprise framework regions, which provide the immunoglobulin-like structure of the domain and typically do not make direct interactions with an antigen. The immunoglobulin variable domains may be selected from an immunoglobulin variable domain from an scFv domain, an antibody domain (e.g. VH and / or VL) domains, typically human, a Fab, a Fab’, a F(ab’)2 fragment, a VHH domain and a VNAR domain. Accordingly, the antigen-binding domain may comprise an scFv domain, which typically comprises two immunoglobulin variable domains (e.g. a VH and a VL). In some cases, the antigen-binding domain may comprise a VHH domain, which typically comprises a single immunoglobulin variable domain.

[0056] Preferably, the antigen-binding domain is an scFv or a VHH domain (otherwise known as a VHH, a nanobody, a sdAb or a single domain antibody; said terms are used interchangeably herein).

[0057] A CAR described herein (e.g. the antigen-binding domain of a CAR) may comprise the CDRs (i.e. CDRs 1-3 for the VHH sequences, and for the scFv sequences, CDRs 1-3 for each of the VH and VL domains) from an amino acid sequence selected from 1 to 9, 31 to 42, 62 to 69, 86 to 94, 118, 172 and 176. A CAR described herein (e.g. the antigenbinding domain of a CAR) may comprise the CDRs (i.e. CDRs 1-3 for the VHH sequences, and for the scFv sequences, CDRs 1-3 for each of the VH and VL domains) from an amino acid sequence selected from 1 to 9, 31 to 42, 62 to 69, 86 to 94 and 118. These are scFv and VHH sequences. A CAR described herein may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 1 to 9, 31 to 42, 62 to 69, 86 to 94, 118, 172 and 176. A CAR described herein may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 1 to 9, 31 to 42, 62 to 69, 86 to 94 and 118. Typically, the CAR comprises the exact CDRs of the SEQ ID NOs from which the CAR is derived. A CAR described herein may comprise the CDRs from an amino acid sequence selected from 1 to 9, 31 to 42, 62 to 69, 86 to 94, 118, 172 and 176, and may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 1 to 9, 31 to 42, 62 to 69, 86 to 94, 118, 172 and 176, respectively. A CAR described herein may comprise the CDRs from an amino acid sequence selected from 1 to 9, 31 to 42, 62 to 69, 86 to 94 and 118, and may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 1 to 9, 31 to 42, 62 to 69, 86 to 94 and 118, respectively. In other words, a CAR described herein may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 1 to 9, 31 to 42, 62 to 69, 86 to 94, 118, 172 and 176 and may comprise the exact CDRs of the SEQ ID NO from which the CAR is derived. For example, a CAR may comprise at least 70% identity to SEQ ID NO: 1, and within this sequence, may comprise the CDRs of SEQ ID NO: 1.

[0058] The antigen-binding domain may be a VHH. VHH domains comprise a single heavy chain variable domain (VH) and lack the constant domain found in typical antibodies. Exemplary VHH domains of the invention are shown in SEQ ID NOs: 1 to 3, 31 to 38, 62 to 65, 86 to 87, 172 and 176. A CAR described herein (e.g. the VHH of a CAR) may comprise the CDRs (z.e. CDRs 1-3) from an amino acid sequence selected from 1 to 3, 31 to 38, 62 to 65, 86 to 87, 172 and 176. A CAR described herein (e.g. the VHH of a CAR) may comprise the CDRs (z.e. CDRs 1-3) from an amino acid sequence selected from 1 to 3, 31 to 38, 62 to 65 and 86 to 87. A CAR described herein may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 1 to 3, 31 to 38, 62 to 65, 86 to 87, 172 and 176. A CAR described herein may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 1 to 3, 31 to 38, 62 to 65 and 86 to 87. A CAR described herein may comprise the CDRs (i.e. CDRs 1-3) from an amino acid sequence selected from 1 to 3, 31 to 38, 62 to 65, 86 to 87, 172 and 176, and may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 1 to 3, 31 to 38, 62 to 65, 86 to 87, 172 and 176, respectively. A CAR described herein may comprise the CDRs (i.e. CDRs 1-3) from an amino acid sequence selected from 1 to 3, 31 to 38, 62 to 65 and 86 to 87, and may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 1 to 3, 31 to 38, 62 to 65 and 86 to 87, respectively. In other words, a CAR described herein may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 1 to 3, 31 to 38, 62 to 65, 86 to 87, 172 and 176and may comprise the exact CDRs of the SEQ ID NO from which the CAR is derived. For example, a CAR may comprise at least 70% identity to SEQ ID NO: 1, and within this sequence, may comprise the CDRs of SEQ ID NO: 1.

[0059] The antigen-binding domain may be an scFv. ScFv domains comprise a heavy chain variable domain (VH) and a light chain variable domain (VL) of an immunoglobulin and connected by a short linker peptide. Exemplary scFv domains of the invention are shown in SEQ ID NOs: 4 to 9, 39 to 42, 66 to 69, 88 to 94 and 118. The scFv may be derived from a human immunoglobulin. The scFv may be a derived from a murine immunoglobulin.

[0060] Whilst an scFv is typically arranged VH-Vrin an N-terminal to C-terminal orientation, antigen binding regions arranged VL-VH in an N-terminal to C-terminal orientation are also encompassed according to the invention.

[0061] Any linker peptide may be used to link the (VH) and (VL) domains of the scFv. In SEQ ID NOs: 4 to 9, 39 to 42, 66 to 69, 88 to 94 and 118, the linker peptide GGGGSGGGGSGGGGS (SEQ ID NO: 132) has been used. The linker peptide may comprise SSSGGGGSGGGGSGGGGSS (SEQ ID NO: 133).

[0062] A CAR described herein (e.g. the scFv of a CAR) may comprise the CDRs (z.e. CDRs 1-3 of each of the VH and VL domains of a CAR) from an amino acid sequence selected from 4 to 9, 39 to 42, 66 to 69, 88 to 94 and 118. A CAR described herein may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 4 to 9, 39 to 42, 66 to 69, 88 to 94 and 118. A CAR described herein may comprise the CDRs from an amino acid sequence selected from 4 to 9, 39 to 42, 66 to 69, 88 to 94 and 118, and may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 4 to 9, 39 to 42, 66 to 69, 88 to 94 and 118, respectively. In other words, a CAR described herein may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 4 to 9, 39 to 42, 66 to 69, 88 to 94 and 118 and may comprise the exact CDRs of the SEQ ID NO from which the CAR is derived. For example, a CAR may comprise at least 70% identity to SEQ ID NO: 4, and within this sequence, may comprise the CDRs of SEQ ID NO: 4.

[0063] In some cases, a CAR described herein comprises an scFv domain comprising a VH domain having an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to an amino acid selected from SEQ ID NOs: 10, 12, 14, 16, 18, 20, 43, 45, 47, 49, 70, 72, 74, 76, 95, 97, 99, 101, 103, 105, 107 and 120, and a VL domain having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to an amino acid selected from SEQ ID NOs: 11, 13, 15, 17, 19, 21, 44, 46, 48, 50, 71, 73, 75, 77, 96, 98, 100, 102, 104, 106 and 119, respectively. The scFv further comprises a linker peptide. The linker peptide may be, for example, any linker sequence discussed herein.

[0064] The term “antigen-binding regions” in an scFv is used to refer to the complementary determining regions (CDR) of the variable heavy chain and variable light chain domains that make up the scFv. For example, the scFvs of SEQ ID NOs: 4 to 9, 39 to 42, 66 to 69, 88 to 94 and 118 are labelled with six ABRs. For each scFv, the first ABR1, ABR2 and ABR3 correspond to the heavy chain CDR1, CDR2 and CDR3 respectively, and the second set of ABR1, ABR2 and ABR3 within the sequence correspond to the light chain CDR1, CDR2 and CDR3.

[0065] Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs within the specified sequenced disclosed herein. Specifically, the scFv sequences shown in SEQ ID NOs: 4 to 9, 39 to 42, 63 to 66, 85 to 91 and 115 comprise, from N-terminal to C-terminal, a heavy chain variable region of an antibody, a linker and a light chain variable region of an antibody. The separate heavy and light chain variable regions of SEQ ID NOs 4 to 9, 39 to 42, 66 to 69, 88 to 94 and 118 are provided in SEQ ID NOs: 10 to 21, 43 to 50, 78 to 85, 95 to 108 and 119 to 120. The ABRs of the scFv sequences disclosed herein were predicted using Paratome (Kunik V et al (2012). Structural Consensus among Antibodies Defines the Antigen Binding Site. PLoS Comput Biol 8(2): el002388. doi: 10.1371 / joumal.pcbi. l002388; and Kunik V et al (2012). Paratome: An online tool for systematic identification of antigen binding regions in antibodies based on sequence or structure. Nucleic Acids Res. 2012 Jul;40(Web Server issue):W521-4. doi: 10.1093 / nar / gks480. Epub 2012 Jun 6). The CDRs of the VHH sequences disclosed herein were predicted with the Benchling’s Antibody Property Prediction Tools, where annotations were assigned using the North CDR definition of the Sequence-based antibody CAnonical LOoP (SCALOP) structure annotation developed by the Oxford Protein Informatics Group (Dunbar et al, SAbPred: a structure-based antibody prediction server, Nucleic Acids Research, Volume 44, Issue Wl, 8 July 2016, Pages W474-W478, https: / / doi.org / 10.1093 / nar / gkw361). Other exemplary conventions that can be used to identify the boundaries of CDRs include the Kabat definition, the Chothia definition and the IMGT definition (see, for example, Kabat, Elvin Abraham. Sequences of proteins of immunological interest. No. 91. US Department of Health and Human Services, Public Health Service, National Institutes of Health, 1991; Lefranc, Marie-Paule, et al. "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains." Developmental & Comparative Immunology 27.1 (2003): 55-77). The CDRs may be the ABRs identified in the informal sequence listing provided above. For example, the heavy chain CDR1, CDR2 and CDR3 may be the first (i.e. preceding the linker) ABR1, ABR2 and ABR3 identified for an scFv, respectively, and the light chain CDR1, CDR2 and CDR3 may be the second (i.e. following the linker) ABR1, ABR2 and ABR3 identified for the scFv, respectively.

[0066] An scFv having the ABRs of SEQ ID NO: 4, or polypeptides having the CDRs of SEQ ID NOs 10 and 11, comprises a first ABR1 (HCDR1) having the sequence FTFSSYAMH (SEQ ID NO: 157), a first ABR2 (HCDR2) having the sequence WVAVISYDGSNKYY (SEQ ID NO: 158), a first ABR3 (HCDR3) having the sequence RGSGYSYGPGYDAFDI (SEQ ID NO: 159), a second ABR1 (LCDR1) having the sequence SGSIASNYVQ (SEQ ID NO: 160), a second ABR2 (LCDR2) having the sequence TTVIYEDNQRPS (SEQ ID NO: 161), and a second ABR3 (LCDR3) having the sequence QSWDPVFG (SEQ ID NO: 162). Similarly, an scFv having the ABRs of SEQ ID NO: 5, or polypeptides having the CDRs of SEQ ID NOs 12 and 13, comprises a first ABR1 (HCDR1) having the sequence GTFSSYAIS (SEQ ID NO: 163), a first ABR2 (HCDR2) having the sequence WMGGIIPIFGTANY (SEQ ID NO: 164), a first ABR3 (HCDR3) having the sequence REGGAVGYYYGMDV (SEQ ID NO: 165), a second ABR1 (LCDR1) having the sequence SSDVGGYNYVS (SEQ ID NO: 166), a second ABR2 (LCDR2) having the sequence LMIYEVSNRPS (SEQ ID NO: 167), and a second ABR3 (LCDR3) having the sequence SSYDRSNRSM (SEQ ID NO: 168). The same applies to any of SEQ ID NOs 6 to 21, 39 to 50, 66 to 77, 88 to 108 and 118 to 120 and similar considerations apply to CDRs 1-3 of each of SEQ ID NOs: 83 to 93.

[0067] Glioma-associated antigens

[0068] Glioma-associated antigens are antigens associated with malignant glioma (glioblastoma, GBM), e.g. expressed by cells of a malignant glioma. The glioma- associated antigens may be human glioma-associated antigens. An antigen is associated with malignant glioma when it is over-expressed in a malignant glioma sample when compared to normal brain tissues and non-CNS normal tissues. The glioma-associated antigen may be associated with gliomagenesis.

[0069] The immune effector cell or population of immune effector cells express one or more chimeric antigen receptors (CARs) specific for two or more glioma-associated antigens.

[0070] One or more of the glioma-associated antigens comprise a GD2 ganglioside. The GD2 ganglioside may be a O-acetyl-GD2 ganglioside (OAcGD2). The (9AcGD2 may comprise 9-(9AcGD2 and / or 7-OAcGD2. The two or more glioma-associated antigens may comprise a GD2 ganglioside and an (9AcGD2 ganglioside, e.g. the two or more glioma-associated antigens may comprise a non-O-acetylated-GD2 ganglioside and an (9AcGD2 ganglioside.

[0071] The two or more glioma-associated antigens may comprise one or more glioma- associated antigens selected from receptor-type tyrosine-protein phosphatase zeta (PTPRZ1), brevican core protein (BCAN), chondroitin sulfate proteoglycan 4 (CSPG4) and tenascin C (TNC). In other words, the two or more glioma-associated antigens may comprise a GD2 ganglioside (such as an (9AcGD2 ganglioside) and one or more glioma- associated antigens selected from PTPRZ1, BCAN, CSPG4 and TNC. Exemplary antigenbinding domains, CARs and immune effector cells specific for PTPRZ1, BCAN, CSPG4 and TNC are disclosed in PCT / EP2023 / 084961 (WO 2024 / 121414).

[0072] PTPRZ1, CSPG4 and BCAN may each be considered a cell surface (glioma) marker. TNC and BCAN may each be considered an extracellular matrix (ECM) marker, e.g. of tumour invasiveness. In some cases, it is advantageous for a CAR T cell of the invention to be specific to a cell surface marker and an ECM marker, such as the markers described herein.

[0073] In some cases, the immune effector cell or population of immune effector cells expresses one or more chimeric antigen receptors (CARs) specific for three or more glioma-associated antigens. The three or more glioma-associated antigens may comprise a GD2 ganglioside (such as an ( AcGD2 ganglioside) and two or more glioma-associated antigens selected from PTPRZ1, BCAN, CSPG4 and TNC.

[0074] In some cases, the immune effector cell or population of immune effector cells expresses one or more chimeric antigen receptors (CARs) specific for four or more glioma- associated antigens. The four or more glioma-associated antigens may comprise a GD2 ganglioside (such as an ( AcGD2 ganglioside) and three or more glioma-associated antigens selected from PTPRZ1, BCAN, CSPG4 and TNC.

[0075] In some cases, the immune effector cell or population of immune effector cells expresses one or more chimeric antigen receptors (CARs) specific for five or more glioma- associated antigens. The five or more glioma-associated antigens may comprise a GD2 ganglioside (such as an ( AcGD2 ganglioside) and all four of the glioma-associated antigens PTPRZ1, BCAN, CSPG4 and TNC.

[0076] In some cases, the immune effector cell or population of immune effector cells expresses one or more chimeric antigen receptors (CARs) specific for two or more glioma- associated antigens, wherein the two or more glioma-associated antigens comprise a GD2 ganglioside (such as an ( AcGD2 ganglioside) and TNC. In some cases, the immune effector cell or population of immune effector cells expresses one or more chimeric antigen receptors (CARs) specific for two or more glioma-associated antigens, wherein the two or more glioma-associated antigens comprise a GD2 ganglioside (such as an ( AcGD2 ganglioside) and PTPRZ1. In some cases, the immune effector cell or population of immune effector cells expresses one or more chimeric antigen receptors (CARs) specific for three or more glioma-associated antigens, wherein the three or more glioma-associated antigens comprise a GD2 ganglioside (such as an (9AcGD2 ganglioside), TNC and PTPRZ1.

[0077] The two or more glioma-associated antigens may comprise Interleukin- 13 receptor subunit alpha-2 (IL13Ra2) and B7 Homolog 3 (B7-H3). In other words, the two or more glioma-associated antigens may comprise a GD2 ganglioside (such as an (9AcGD2 ganglioside) and one or more glioma-associated antigens selected from IL13Ra2 and B7- H3. The immune effector cell or population of immune effector cells may express one or more chimeric antigen receptors (CARs) specific for three or more glioma-associated antigens, wherein the three or more glioma-associated antigens comprise a GD2 ganglioside (such as an (9AcGD2 ganglioside), IL13Ra2 and B7 Homolog 3 (B7-H3).

[0078] An exemplary CAR specific for an (9AcGD2 is set out in SEQ ID NO: 118. A CAR described herein may comprise the CDRs from SEQ ID NO: 118. A CAR described herein may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 118. A CAR described herein may comprise the CDRs from SEQ ID NO: 118, and may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 118.

[0079] Exemplary antigen-binding domains specific for PTPRZ1 are set out in SEQ ID NOs: 1 to 9. A CAR described herein may comprise the CDRs from an amino acid sequence selected from SEQ ID NOs: 1 to 9. A CAR described herein may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 1 to 9. A CAR described herein may comprise the CDRs from an amino acid sequence selected from 1 to 9, and may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 1 to 9, respectively.

[0080] Exemplary VHHS specific for PTPRZ1 are set out in SEQ ID NOs: 1 to 3. Preferably, a CAR described herein may be based on the VHH domain of RB832, RB833 or RB 834 (SEQ ID NO: 1 to 3, respectively). For example, a CAR described herein may comprise an antigen binding domain (such as an immunoglobulin variable domain and / or a VHH) comprising the CDRs from an amino acid sequence selected from SEQ ID NOs: 1 to 3. A CAR described herein may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 1 to 3. A CAR described herein may comprise the CDRs from an amino acid sequence selected from any one of SEQ ID NOs: 1 to 3, and may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 1 to 3, respectively. Preferably, a CAR described herein may comprise an antigen binding domain (such as an immunoglobulin variable domain and / or a VHH) comprising the CDRs of SEQ ID NO: 1. A CAR described herein may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 1. A CAR described herein may comprise the CDRs from SEQ ID NO: 1, and may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 1.

[0081] Exemplary scFvs specific for PTPRZ1 are set out in SEQ ID NOs: 4 to 9. The corresponding VH and VL domains are set out in SEQ ID NOs: 10 to 21. A CAR described herein may comprise the CDRs from an amino acid sequence selected from SEQ ID NOs: 4 to 9. A CAR described herein may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 4 to 9. A CAR described herein may comprise the CDRs from an amino acid sequence selected from 4 to 9, and may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 4 to 9, respectively. Preferably, a CAR as described herein may be based on the scFv domain of RRB470, RRB471 or RRB476 (SEQ ID NOs 5, 6 and 9, respectively). For example, the CAR may comprise the CDRs from an amino acid sequence selected from SEQ ID NOs: 5, 6 and 9. A CAR described herein may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 5, 6 and 9. A CAR described herein may comprise the CDRs from an amino acid sequence selected 5, 6 and 9, and may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 5, 6 and 9, respectively.

[0082] Exemplary antigen-binding domains specific for TNC are set out in SEQ ID NOs: 31 to 42. A CAR described herein may comprise the CDRs from an amino acid sequence selected from SEQ ID NOs: 31 to 42. A CAR described herein may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 31 to 42. A CAR described herein may comprise the CDRs from an amino acid sequence selected from 31 to 42, and may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 31 to 42, respectively.

[0083] Exemplary VHHS specific for TNC are set out in SEQ ID NOs: 31 to 38. A CAR described herein may comprise the CDRs from an amino acid sequence selected from SEQ ID NOs: 31 to 38. A CAR described herein may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 31 to 38. A CAR described herein may comprise the CDRs from an amino acid sequence selected from 31 to 38, and may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 31 to 38, respectively. In some aspects, a CAR described herein may be based on the VHH domain of RB895 to RB900 (SEQ ID NOs: 31 to 36, respectively). For example, a CAR described herein may comprise the CDRs from an amino acid sequence selected from SEQ ID NOs: 31 to 36. A CAR described herein may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 31 to 36. A CAR described herein may comprise the CDRs from an amino acid sequence selected from 31 to 36, and may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 31 to 36, respectively. In some aspects, a CAR described herein may be based on the VHH domain of RB835 or RB836 (SEQ ID NOs: 37 to 38, respectively). For example, the CAR may comprise the CDRs from an amino acid sequence selected from SEQ ID NOs: 37 and 38. A CAR described herein may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 37 and 38. A CAR described herein may comprise the CDRs from an amino acid sequence selected from any one of SEQ ID NOs: 37 and 38, and may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 37 and 38, respectively. Preferably, a CAR described herein may comprise an antigen binding domain (such as an immunoglobulin variable domain and / or a VHH) comprising the CDRs of SEQ ID NO: 34. A CAR described herein may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 34. A CAR described herein may comprise the CDRs from SEQ ID NO: 34, and may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 34. Exemplary scFvs specific for TNC are set out in SEQ ID NOs: 39 to 42. The corresponding VH and VL domains are set out in SEQ ID NOs 43 to 50. For example, the CAR may be selected from a CAR comprising (a) a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 39 to 42, (b) an amino acid sequence with at least 80%, 85%, 90%, 95% or 99% identity thereto, or (c) the HCDR1, HCDR2 and HCDR3 and the LCDR1, LCDR2 and LCDR3 from an amino acid sequence selected from any one of SEQ ID NOs: 39 to 42. A CAR described herein may comprise the CDRs from an amino acid sequence selected from SEQ ID NOs: 39 to 42. A CAR described herein may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 39 to 42. A CAR described herein may comprise the CDRs from an amino acid sequence selected from 39 to 42, and may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 39 to 42, respectively.

[0084] Exemplary antigen-binding domains specific for BCAN are set out in SEQ ID NOs: 62 to 69. A CAR described herein may comprise the CDRs from an amino acid sequence selected from SEQ ID NOs: 62 to 69. A CAR described herein may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 62 to 69. A CAR described herein may comprise the CDRs from an amino acid sequence selected from 62 to 69, and may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 62 to 69, respectively.

[0085] Exemplary VHHS specific for BCAN are set out in SEQ ID NOs: 62 to 65. Preferably, a CAR described herein may be based on the VHH domain of RB826, RB827, RB828 or RB829 (SEQ ID NOs: 62 to 65, respectively). For example, the CAR may comprise the CDRs from an amino acid sequence selected from SEQ ID NOs: 62 to 65. A CAR described herein may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 62 to 65. A CAR described herein may comprise the CDRs from an amino acid sequence selected from any one of SEQ ID NOs: 62 to 65, and may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 62 to 65, respectively. Preferably, a CAR described herein may comprise an antigen binding domain (such as an immunoglobulin variable domain and / or a VHH) comprising the CDRs of SEQ ID NO: 62. A CAR described herein may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 62. A CAR described herein may comprise the CDRs from SEQ ID NO: 62, and may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 62.

[0086] Exemplary scFvs specific for BCAN are set out in SEQ ID NOs: 66 to 69. The corresponding VH and VL domains are set out in SEQ ID NOs 70 to 77. For example, a CAR described herein may comprise the CDRs from an amino acid sequence selected from SEQ ID NOs: 66 to 69. A CAR described herein may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 66 to 69. A CAR described herein may comprise the CDRs from an amino acid sequence selected from 66 to 69, and may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 66 to 69, respectively. In some cases, a scFv specific for BCAN is selected from RBR295 (SEQ ID NO: 67). For example, a CAR described herein may comprise the CDRs from SEQ ID NO: 67. A CAR described herein may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 67. A CAR described herein may comprise the CDRs from SEQ ID NO: 67, and may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 67.

[0087] Exemplary antigen-binding domains specific for CSPG4 are set out in SEQ ID NOs: 86 to 94. A CAR described herein may comprise the CDRs from an amino acid sequence selected from SEQ ID NOs: 86 to 94. A CAR described herein may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 86 to 94. A CAR described herein may comprise the CDRs from an amino acid sequence selected from 86 to 94, and may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 86 to 94, respectively.

[0088] Exemplary VHHS specific for CSPG4 are set out in SEQ ID NOs: 86 to 87. Preferably, a CAR described herein may be based on the VHH domain of RB830 or RB831 (SEQ ID NOs: 83 to 84 respectively). For example, a CAR described herein may comprise the CDRs from an amino acid sequence selected from SEQ ID NOs: 86 to 87. A CAR described herein may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 86 to 87. A CAR described herein may comprise the CDRs from an amino acid sequence selected from 86 to 87, and may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 86 to 87, respectively. Preferably, a CAR described herein may comprise an antigen binding domain (such as an immunoglobulin variable domain and / or a VHH) comprising the CDRs of SEQ ID NO: 86. A CAR described herein may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 86. A CAR described herein may comprise the CDRs from SEQ ID NO: 86, and may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 86.

[0089] Exemplary scFvs specific for CSPG4 are set out in SEQ ID NOs: 88 to 94. The corresponding VH and VL domains are set out in SEQ ID NOs 95 to 108. For example, a CAR described herein may comprise the CDRs from an amino acid sequence selected from SEQ ID NOs: 88 to 94. A CAR described herein may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 88 to 94. A CAR described herein may comprise the CDRs from an amino acid sequence selected from 88 to 94, and may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 88 to 94, respectively. Preferably, a CAR as described herein may be based on the scFv domain of HRB299, HRB 301, HRB302 or HRB 303 (SEQ ID NOs 89 and 91 to 93, respectively). For example, a CAR described herein may comprise the CDRs from an amino acid sequence selected from SEQ ID NOs: 89 and 91 to 93. A CAR described herein may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 89 and 91 to 93. A CAR described herein may comprise the CDRs from an amino acid sequence selected from any one of SEQ ID NOs: 89 and 91 to 93, and may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 89 and 91 to 93, respectively. More preferably, a CAR as described herein may be based on the scFv domain of HRB301 or HRB302 (SEQ ID NOs 91 to 92, respectively). For example, a CAR described herein may comprise the CDRs from an amino acid sequence selected from SEQ ID NOs: 91 to 92. A CAR described herein may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 91 to 92. A CAR described herein may comprise the CDRs from an amino acid sequence selected from any one of SEQ ID NOs: 91 to 92, and may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 91 to 92, respectively.

[0090] An exemplary antigen-binding domain specific for IL13Ra2 is set out in SEQ ID NO: 176. SEQ ID NO: 176 is a VHH domain. For example, a CAR described herein may comprise the CDRs from SEQ ID NO: 176. A CAR described herein may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 176. A CAR described herein may comprise the CDRs from SEQ ID NO: 176, and may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 176.

[0091] An exemplary antigen-binding domain specific for B7-H3 is set out in SEQ ID NO: 172. SEQ ID NO: 172 is a VHH domain. For example, a CAR described herein may comprise the CDRs from SEQ ID NO: 172. A CAR described herein may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 172. A CAR described herein may comprise the CDRs from SEQ ID NO: 172, and may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 172. The inventors have identified combination of preferred CARs for use in the multivalent immune effector cell or multivalent population of immune effector cells of the invention. Specifically, a CAR specific for a GD2 ganglioside (such as an (9AcGD2 ganglioside) is preferably derived from the scFv sequence of 8B6 (SEQ ID NO: 118). A CAR specific for PTPRZ1 is preferably derived from the VHH sequence of RB832 (SEQ ID NO: 1). A CAR specific for TNC is preferably derived from the VHH sequence of RB898 (SEQ ID NO: 34). A CAR specific for BCAN is preferably derived from the VHH sequence of RB826 (SEQ ID NO: 62). A CAR specific for CSPG4 is preferably derived from the VHH sequence of RB830 (SEQ ID NO: 86). A CAR specific for IL13Ra2 is preferably derived from the VHH sequence of SEQ ID NO: 176. A CAR specific for B7-H3 is preferably derived from the VHH sequence of SEQ ID NO: 172. Accordingly, an immune effector cell or population of immune effector cells described herein may comprise one or more CARs specific for two or more glioma- associated antigens, wherein the glioma-associated antigens are selected from the features: (a) a GD2 ganglioside (such as an (9AcGD2 ganglioside) and the one or more CARs specific for the GD2 ganglioside comprise the CDRs from SEQ ID NO: 118; (b) PTPRRZ1, and the one or more CARs specific for PTPRZ1 comprise an amino acid sequence comprising the CDRs from SEQ ID NO: 1; (c) TNC, and the one or more CARs specific for TNC comprise an amino acid sequence comprising the CDRs from SEQ ID NO: 34; (d) BCAN, and the one or more CARs specific for BCAN comprise an amino acid sequence comprising the CDRs from SEQ ID NO: 62; and (e) CSPG4, and the one or more CARs specific for CSPG4 comprise an amino acid sequence comprising the CDRs from SEQ ID NO: 86. An immune effector cell or population of immune effector cells described herein may comprise one or more CARs specific for two or more glioma- associated antigens, wherein the glioma-associated antigens are selected from the features: (i) a GD2 ganglioside (such as an (9AcGD2 ganglioside) and the one or more CARs specific for the GD2 ganglioside comprise the CDRs from SEQ ID NO: 118; (ii) IL13Ra2, and the one or more CARs specific for PTPRZ1 comprise an amino acid sequence comprising the CDRs from SEQ ID NO: 176; and (iii) B7-H3, and the one or more CARs specific for TNC comprise an amino acid sequence comprising the CDRs from SEQ ID NO: 172. The one or more CARs specific for the GD2 ganglioside may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 118. The one or more CARs specific for the GD2 ganglioside may comprise the CDRs from SEQ ID NO: 118, and may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 118. The one or more CARs specific for PTPRZ1 may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 1. The one or more CARs specific for PTPRZ1 may comprise the CDRs from SEQ ID NO: 1, and may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 1. The one or more CARs specific for TNC may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 34. The one or more CARs specific for TNC may comprise the CDRs from SEQ ID NO: 34, and may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 34. The one or more CARs specific for BCAN may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 62. The one or more CARs specific for BCAN may comprise the CDRs from SEQ ID NO: 62, and may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 62. The one or more CARs specific for CSPG4 may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 86. The one or more CARs specific for CSPG4 may comprise the CDRs from SEQ ID NO: 86, and may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 86. The one or more CARs specific for IL13Ra2 may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 176. The one or more CARs specific for IL13Ra2 may comprise the CDRs from SEQ ID NO: 176, and may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 176. The one or more CARs specific for B7-H3 may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 172. The one or more CARs specific for B7-H3 may comprise the CDRs from SEQ ID NO: 172, and may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 172. Preferably, the immune effector cell or each cell of the population of immune effector cell comprises a separate CAR for each different glioma-associated antigen. The immune effector cell or population of immune effector cells may comprise feature (a) and one or more of features (b) to (e). The immune effector cell or population of immune effector cells may comprise (a), (b) and (c). The immune effector cell or population of immune effector cells may comprise three of features (a) to (e). The immune effector cell or population of immune effector cells may comprise four of features (a) to (e). The immune effector cell or population of immune effector cells may comprise all five of features (a) to (e). immune effector cell or population of immune effector cells may comprise features: (a) and (b); (a) and (c); (a) and (d); (a) and (e); (b) and (c); (b) and (d); (b) and (e); (c) and

[0092] (d); (c) and (e); (d) and (e); (a), (b) and (c); (a); (b) and (d); (a), (b) and (e); (a), (c) and (d); (a), (c) and (e); (a), (d) and (e); (b), (c) and (d); (b), (c) and (e); (b), (d) and (e); (c), (d) and

[0093] (e); (a), (b), (c) and (d); (a), (b), (c) and (e); (a), (b), (d) and (e); (a), (c), (d) and (e); (b), (c), (d) and (e); or (a), (b), (c), (d) and (e). The immune effector cell or population of immune effector cells may comprise feature (i) and one or more of features (ii) and (iii). The immune effector cell or population of immune effector cells may comprise features (i), (ii) and (iii). The immune effector cell or population of immune effector cells may express one or more CARs specific for one or more additional glioma-associated antigens, as described herein.

[0094] Other known glioma-associated antigens include HER2, EGFRvIII, IL13Ra2, PDGFRA, NKG2D, MET, HGF, B7-H3. The glioma-associated antigens may be selected from the group consisting of a GD2 ganglioside, PTPRZ1, BCAN, CSPG4, TNC, HER2, EGFRvIII, IL13Ra2, PDGFRA, NKG2D, MET, HGF, and B7-H3. For example, two or more glioma-associated antigens may comprise a GD2 ganglioside and one or more glioma-associated antigens selected from the group consisting of PTPRZ1, BCAN, CSPG4, TNC, HER2, EGFRvIII, IL13Ra2, PDGFRA, NKG2D, MET, HGF, and B7-H3. Two or more glioma-associated antigens may comprise a GD2 ganglioside and one or more other glioma-associated antigens may be selected from the group consisting of HER2, EGFRvIII, IL13Ra2, PDGFRA, NKG2D, MET, HGF, and B7-H3. Three or more glioma-associated antigens may comprise a GD2 ganglioside, one or more glioma- associated antigens selected from the group consisting of PTPRZ1, BCAN, CSPG4 and TNC, and one or more other glioma-associated antigens selected from the group consisting of HER2, EGFRvIII, IL13Ra2, PDGFRA, NKG2D, MET, HGF, and B7-H3. Preferably, other glioma-associated antigens are selected from HER2, EGFRvIII, IL13Ra2 and B7-H3. For example, the other glioma-associated antigens may be selected from the group consisting of HER2, EGFRvIII and IL13Ra2, the group consisting of HER2, IL13Ra2 and B7-H3, or the group consisting of Her2 and IL13Ra2. The other glioma-associated antigen may be Her2. The other glioma-associated antigen may be EGFRvIII. The other glioma- associated antigen may be IL13Ra2. The other glioma-associated antigen may be B7-H3.

[0095] The glioma-associated antigens may be selected from the group consisting of a GD2 ganglioside, PTPRZ1, TNC, IL13Ra2, B7H3, BCAN, CSPG4, HER2, PDGFRA, CD70, MET, MMP-2 (Metalloprotease), EphA2, CD44, CD 133, CD 147, Mucl, PD-L1, TREM2, and SPP1. For example, two or more glioma-associated antigens may comprise a GD2 ganglioside and one or more glioma-associated antigens selected from the group consisting of PTPRZ1, TNC, IL13Ra2, B7H3, BCAN, CSPG4, HER2, PDGFRA, CD70, MET, MMP-2 (Metalloprotease), EphA2, CD44, CD 133, CD 147, Mucl, PD-L1, TREM2, and SPP1. Two or more glioma-associated antigens may comprise a GD2 ganglioside and one or more other glioma-associated antigens may be selected from the group consisting of IL13Ra2, B7H3, HER2, PDGFRA, CD70, MET, MMP-2 (Metalloprotease), EphA2, CD44, CD 133, CD 147, Mucl, PD-L1, TREM2, and SPP1. Three or more glioma- associated antigens may comprise a GD2 ganglioside, one or more glioma-associated antigens selected from the group consisting of PTPRZ1, BCAN, CSPG4 and TNC, and one or more other glioma-associated antigens selected from the group consisting of IL13Ra2, B7H3, HER2, PDGFRA, CD70, MET, MMP-2 (Metalloprotease), EphA2, CD44, CD 133, CD 147, Mucl, PD-L1, TREM2, and SPP1. The glioma-associated antigens may be selected from the group consisting of a GD2 ganglioside, PTPRZ1, TNC, IL13Ra2, B7H3, BCAN, CSPG4, HER2, PDGFRA, CD70, MET, MMP-2 (Metalloprotease), EphA2, CD44, CD 133, CD 147, Mucl, PD-L1, TREM2, SPP1, NKG2D, MIC / A, HGF, CD56 and PSMA. For example, two or more glioma-associated antigens may comprise a GD2 ganglioside and one or more glioma- associated antigens selected from the group consisting of PTPRZ1, TNC, IL13Ra2, B7H3, BCAN, CSPG4, HER2, PDGFRA, CD70, MET, MMP-2 (Metalloprotease), EphA2, CD44, CD133, CD147, Mucl, PD-L1, TREM2, SPP1, NKG2D, MIC / A, HGF, CD56 and PSMA. Two or more glioma-associated antigens may comprise a GD2 ganglioside and one or more other glioma-associated antigens may be selected from the group consisting of IL13Ra2, B7H3, HER2, PDGFRA, CD70, MET, MMP-2 (Metalloprotease), EphA2, CD44, CD133, CD147, Mucl, PD-L1, TREM2, SPP1, NKG2D, MIC / A, HGF, CD56 and PSMA. Three or more glioma-associated antigens may comprise a GD2 ganglioside, one or more glioma-associated antigens selected from the group consisting of PTPRZ1, BCAN, CSPG4 and TNC, and one or more other glioma-associated antigens selected from the group consisting of IL13Ra2, B7H3, HER2, PDGFRA, CD70, MET, MMP-2 (Metalloprotease), EphA2, CD44, CD133, CD147, Mucl, PD-L1, TREM2, SPP1, NKG2D, MIC / A, HGF, CD56 and PSMA.

[0096] The immune effector cell, or each cell in the population of immune effector cells may express two or more CARs, wherein each CAR comprises specificity for a different glioma-associated antigen. The immune effector cell, or each cell in the population of immune effector cells, may express a CAR specific for a GD2 ganglioside and a CAR specific for a different glioma-associated antigen. The different glioma-associated antigen may be selected from a group consisting of HER2, EGFRvIII, IL13Ra2, PDGFRA, NKG2D, MET, HGF and B7-H3, and / or a group consisting of PTPRZ1, TNC, BCAN and CSPG4. The different glioma-associated antigen may be selected from a group consisting of IL13Ra2, B7H3, HER2, PDGFRA, CD70, MET, MMP-2 (Metalloprotease), EphA2, CD44, CD 133, CD 147, Mucl, PD-L1, TREM2, and SPP1 and / or a group consisting of PTPRZ1, TNC, BCAN and CSPG4. The different glioma-associated antigen may be selected from a group consisting of IL13Ra2, B7H3, HER2, PDGFRA, CD70, MET, MMP-2 (Metalloprotease), EphA2, CD44, CD 133, CD 147, Mucl, PD-L1, TREM2, SPP1, NKG2D, MIC / A, HGF, CD56 and PSMA, and / or a group consisting of PTPRZ1, TNC, BCAN and CSPG4. The immune effector cell, or each cell in the population of immune effector cells may express three or more CARs, wherein each CAR comprises specificity for a different glioma-associated antigen. The immune effector cell, or each cell in the population of immune effector cells, may express a CAR specific for a GD2 ganglioside, a CAR specific for at least one of PTPRZ1, BCAN, CSPG4, and TNC, such as two CARs specific for two of PTPRZ1, BCAN, CSPG4, and TNC, three CARs specific for three of PTPRZ1, BCAN, CSPG4, and TNC, or four CARs specific for all of PTPRZ1, BCAN, CSPG4, and TNC. The immune effector cell, or each cell in the population of immune effector cells, may express a CAR specific for a GD2 ganglioside and at least one CAR specific for at least one of IL13Ra2 and B7H3, such as two CARs one of which is specific for IL13Ra2 and one of which is specific for B7H3. The immune effector cell may comprise additional CARs specific for other known glioma-associated antigens as described herein. For example, an immune effector cell may comprise a CAR specific for an (9AcGD2, a CAR specific for PTPRZ1 and a CAR specific for TNC. An immune effector cell may comprise a CAR specific for an (9AcGD2, a CAR specific for PTPRZ1, and a CAR specific for Her2 and / or a CAR specific for IL13Ra2.

[0097] One or more of the CARs may be a multivalent CAR specific for two or more glioma associated antigens. For example, the multivalent CAR may be specific for a GD2 ganglioside and a different glioma-associated antigen. The different glioma-associated antigen may be selected from a group consisting of HER2, EGFRvIII, IL13Ra2, PDGFRA, NKG2D, MET, HGF and B7-H3, and / or a group consisting of PTPRZ1, TNC, BCAN and CSPG4. The different glioma-associated antigen may be selected from a group consisting of IL13Ra2, B7H3, HER2, PDGFRA, CD70, MET, MMP-2 (Metalloprotease), EphA2, CD44, CD 133, CD 147, Mucl, PD-L1, TREM2, and SPP1, and / or a group consisting of PTPRZ1, TNC, BCAN and CSPG4. The different glioma-associated antigen may be selected from a group consisting of IL13Ra2, B7H3, HER2, PDGFRA, CD70, MET, MMP-2 (Metalloprotease), EphA2, CD44, CD 133, CD 147, Mucl, PD-L1, TREM2, SPP1, NKG2D, MIC / A, HGF, CD56 and PSMA, and / or a group consisting of PTPRZ1, TNC, BCAN and CSPG4. The population of immune effector cells may comprise two or more (i.e. at least two) different CAR-expressing immune effector cells, wherein each different CAR- expressing immune effector cell is specific for a different glioma-associated antigen. Accordingly, the population may comprise an immune effector cell expressing a CAR specific for a GD2 ganglioside and an immune effector cell expressing a CAR specific for a different glioma-associated antigen. The different glioma-associated antigen may be selected from a group consisting of HER2, EGFRvIII, IL13Ra2, PDGFRA, NKG2D, MET, HGF and B7-H3, and / or a group consisting of PTPRZ1, TNC, BCAN and CSPG4. The different glioma-associated antigen may be selected from a group consisting of IL13Ra2, B7H3, HER2, PDGFRA, CD70, MET, MMP-2 (Metalloprotease), EphA2, CD44, CD 133, CD 147, Mucl, PD-L1, TREM2, and SPP1, and / or a group consisting of PTPRZ1, TNC, BCAN and CSPG4. The different glioma-associated antigen may be selected from a group consisting of IL13Ra2, B7H3, HER2, PDGFRA, CD70, MET, MMP-2 (Metalloprotease), EphA2, CD44, CD 133, CD 147, Mucl, PD-L1, TREM2, SPP1, NKG2D, MIC / A, HGF, CD56 and PSMA, and / or a group consisting of PTPRZ1, TNC, BCAN and CSPG4.

[0098] Chimeric antigen receptors

[0099] Chimeric antigen receptors (CARs) are expressed in immune effector cells. CARs comprise an extracellular antigen-binding domain. CARs generally comprise an extracellular antigen-binding domain and an intracellular cytoplasmic signalling domain. CARs may comprise an extracellular antigen-binding domain, a transmembrane domain and an intracellular cytoplasmic signalling domain. The CAR may also comprise an extracellular spacer and / or a hinge between the transmembrane domain and the antigenbinding domain and / or between the transmembrane domain and the cytoplasmic signalling domain. Preferably, the CAR comprises a hinge between the transmembrane domain and the antigen-binding domain.

[0100] The cytoplasmic signalling domain may comprise an activation domain. The activation domain serves to activate the immune effector cell following engagement of the extracellular domain (e.g. scFv). The cytoplasmic signalling domain may comprise one or more of (i) a CD3(^ (zeta) activation domain, (ii) a 4-1BB (CD137) activation domain, (iii) a CD3s (epsilon) activation domain, (iv) an 0X40 (CD134) activation domain, (v) a CD28 activation domain, and / or (vi) a CD27 activation domain.

[0101] The CD3zeta activation is comprised in the signalling domain of first generation CARs. Preferably, the cytoplasmic domain comprises a CD3(^ (zeta) activation domain.

[0102] Second generation CARs comprise a CD3zeta activation domain and a CD28 activation domain. The cytoplasmic domain comprises a CD3(^ (zeta) activation domain and a CD28 activation domain.

[0103] Third generation CARs comprise additional domains such as the 4- IBB activation domain or 0X40 (CD 134) activation domain. Preferably, the cytoplasmic domain comprises a 4-1BB activation domain and a CD3(^ (zeta) activation domain.

[0104] The cytoplasmic signalling domain may comprise a 4-lBBz domain, which comprises CD3zeta and the 4-lBBz activation domains. The cytoplasmic signalling domain may comprise a CD28z domain, which comprises CD3zeta and the CD28 activation domains. As illustrated in the Examples, a CAR comprising a CD28z domain in particularly useful in the invention, for example, when the immune effector cell is transduced with RNA encoding the CAR. The cytoplasmic signalling domain may comprise a 4-lBBz + CD28z domain, which comprises CD3zeta, CD28 and the 4-lBBz activation domains.

[0105] The cytoplasmic signalling domain may comprise the CD3(^ (zeta) activation domain alone or in combination with a CD28, CD27, OX-40 (CD134) and / or 4-1BB (CD 137) domain.

[0106] Other activation domains include IL-15Ra, CD2, CDS, ICAM-1, LTA-1 and ICOS and may be used in combination with the activation domains described above.

[0107] If the immune effector cell in which the CAR is expressed is a phagocyte, the intracellular signalling domain may comprise the intracellular domain of MegflO or FcRv.

[0108] In some cases, the 4-1BB activation domain comprises or consists of an amino acid sequence having at least 70% identity to SEQ ID NO: 141, such at least 80%, at least 90%, at least 95%, at least 99% or 100% identity to SEQ ID NO: 141. In some cases, the CD28 activation domain comprises or consists of an amino acid sequence having at least 70% identity to SEQ ID NO: 142, such at least 80%, at least 90%, at least 95%, at least 99% or 100% identity to SEQ ID NO: 142. In some cases, the CD3zeta activation domain comprises or consists of an amino acid sequence having at least 70% identity to any one of SEQ ID NOs: 143 to 145, such at least 80%, at least 90%, at least 95%, at least 99% or 100% identity to any one of SEQ ID NOs: 143 to 145. For example, the 4-lBBz domain may comprise an amino acid sequence having at least 70% identity to SEQ ID NO: 141 (such at least 80%, at least 90%, at least 95%, at least 99% or 100% identity to SEQ ID NO: 141) and an amino acid sequence having at least 70% identity to any one of SEQ ID NOs: 143 to 145 (such at least 80%, at least 90%, at least 95%, at least 99% or 100% identity to any one of SEQ ID NOs: 143 to 145). Similarly, a CD28z domain may comprise an amino acid sequence having at least 70% identity to SEQ ID NO: 142 (such at least 80%, at least 90%, at least 95%, at least 99% or 100% identity to SEQ ID NO: 142) and an amino acid sequence having at least 70% identity to any one of SEQ ID NOs: 143 to 145 (such at least 80%, at least 90%, at least 95%, at least 99% or 100% identity to any one of SEQ ID NOs: 143 to 145).

[0109] In some cases, the 4-1BB activation domain comprises or consists of an amino acid sequence having at least 70% identity to the amino acid sequence encoded by SEQ ID NO:

[0110] 152, such at least 80%, at least 90%, at least 95%, at least 99% or 100% identity to the amino acid sequence encoded by SEQ ID NO: 152. In some cases, the CD28 activation domain comprises or consists of an amino acid sequence having at least 70% identity to the amino acid sequence encoded by SEQ ID NO: 153, such at least 80%, at least 90%, at least 95%, at least 99% or 100% identity to the amino acid sequence encoded by SEQ ID NO:

[0111] 153. In some cases, the CD3zeta activation domain comprises or consists of an amino acid sequence having at least 70% identity to the amino acid sequence encoded by any one of SEQ ID NOs: 154-156, such at least 80%, at least 90%, at least 95%, at least 99% or 100% identity to the amino acid sequence encoded by any one of SEQ ID NOs: 154-156. For example, the 4-lBBz domain may comprise an amino acid sequence having at least 70% identity to the amino acid sequence encoded by SEQ ID NO: 152 (such at least 80%, at least 90%, at least 95%, at least 99% or 100% identity to the amino acid sequence encoded by SEQ ID NO: 152) and an amino acid sequence having at least 70% identity to the amino acid sequence encoded by any one of SEQ ID NOs: 154-156 (such at least 80%, at least 90%, at least 95%, at least 99% or 100% identity to the amino acid sequence encoded by any one of SEQ ID NOs: 154-156). Similarly, a CD28z domain may comprise an amino acid sequence having at least 70% identity to the amino acid sequence encoded by SEQ ID NO: 153 (such at least 80%, at least 90%, at least 95%, at least 99% or 100% identity to the amino acid sequence encoded by SEQ ID NO: 153) and an amino acid sequence having at least 70% identity to the amino acid sequence encoded by any one of SEQ ID NOs: 154-156 (such at least 80%, at least 90%, at least 95%, at least 99% or 100% identity to the amino acid sequence encoded by any one of SEQ ID NOs: 154-156).

[0112] The transmembrane domain spans the cell membrane, for example the cell membrane of a eukaryotic cell. The transmembrane domain serves to transmit activation signals to the cytoplasmic signal transduction domains following ligand binding of the extracellular antigen-binding domains (e.g. scFv). The transmembrane domain may be derived from a naturally occurring transmembrane protein, such as a type-I transmembrane protein. The transmembrane domain is typically the transmembrane domain of CD28 or CD8a. The transmembrane domain may be a transmembrane domain of the a, P, 5 or y subunits of the T-cell receptor, CD3s, CD3< CD4, CD6, CD8a, CD28, CD86, OX-40, 4- 1BB or CD40L (CD 154). The transmembrane domain may be the transmembrane domain of CD8, for example, when the immune effector cell is an NK cell.

[0113] In some cases, the CD8a transmembrane domain comprises or consists of an amino acid sequence having at least 70% identity to SEQ ID NO: 139, such at least 80%, at least 90%, at least 95%, at least 99% or 100% identity to SEQ ID NO: 139. In some cases, the CD28 transmembrane domain comprises or consists of an amino acid sequence having at least 70% identity to SEQ ID NO: 140, such at least 80%, at least 90%, at least 95%, at least 99% or 100% identity to SEQ ID NO: 140.

[0114] In some cases, the CD8a transmembrane domain comprises or consists of an amino acid sequence having at least 70% identity to the amino acid sequence encoded by SEQ ID NO: 150, such at least 80%, at least 90%, at least 95%, at least 99% or 100% identity to the amino acid sequence encoded by SEQ ID NO: 150. In some cases, the CD28 transmembrane domain comprises or consists of an amino acid sequence having at least 70% identity to the amino acid sequence encoded by SEQ ID NO: 151, such at least 80%, at least 90%, at least 95%, at least 99% or 100% identity to the amino acid sequence encoded by SEQ ID NO: 151. The CAR may comprise a hinge that connects the transmembrane domain to the extracellular domain. The hinge may confer steric effects that influence the strength of activation, cytotoxicity and signalling from the target cell and its surface receptors. In some cases, the hinge may be from a region from another immune molecule such as IgGl, IgG2, IgG3, IgG4, CD8 (e.g. a CD8a hinge) or CD28. The hinge may be any suitable length. The hinge may be at least one amino acid in length, such as at least five, at least 10 or at least 20 amino acids in length. The hinge may be one hundred or fewer amino acids in length, such as 80 or fewer, 60 or fewer, 40 or fewer, 30 or fewer, or 20 or fewer amino acids in length. The hinge may be 1 to 40 amino acids in length, such as 2 to 30, 3 to 25, 4 to 20, or 5 to 15 amino acids in length. The hinge is may comprises glycine and serine, threonine and / or alanine residues. However, the hinge may comprise any suitable residues.

[0115] In some cases, the hinge is an IgGl hinge, such as a human IgGl hinge.

[0116] In some cases, the hinge is an IgGl hinge (e.g. human IgGl hinge) further comprising one or more constant domains, such as CH2 or CH3.

[0117] In some cases, the hinge is an IgG4 hinge, such as a human IgG4 hinge.

[0118] In some cases, the hinge is a CD8a hinge, such as a human CD8a hinge.

[0119] In some cases, the CD8a hinge comprises or consists of an amino acid sequence having at least 70% identity to SEQ ID NO: 134, such at least 80%, at least 90%, at least 95%, at least 99% or 100% identity to SEQ ID NO: 134. In some cases, the IgG4 hinge comprises or consists of an amino acid sequence having at least 70% identity to SEQ ID NO: 135, such at least 80%, at least 90%, at least 95%, at least 99% or 100% identity to SEQ ID NO: 135. The IgG4 hinge set out in SEQ ID NO: 135 is otherwise referred to herein as the ‘short’ hinge. In some cases, the IgGl hinge comprises or consists of an amino acid sequence having at least 70% identity to any one of SEQ ID NOs: 136 to 138, such at least 80%, at least 90%, at least 95%, at least 99% or 100% identity to any one of SEQ ID NOs: 136 to 138. The IgGl hinge set out in SEQ ID NO: 138 is otherwise referred to herein as the Tong’ hinge or the ‘huIgGlmut’ hinge.

[0120] In some cases, the CD8a hinge comprises or consists of an amino acid sequence having at least 70% identity to the amino acid sequence encoded by SEQ ID NO: 146, such at least 80%, at least 90%, at least 95%, at least 99% or 100% identity to the amino acid sequence encoded by SEQ ID NO: 146. In some cases, the IgG4 hinge comprises or consists of an amino acid sequence having at least 70% identity to the amino acid sequence encoded by SEQ ID NO: 147, such at least 80%, at least 90%, at least 95%, at least 99% or 100% identity to the amino acid sequence encoded by SEQ ID NO: 147. In some cases, the IgGl hinge comprises or consists of an amino acid sequence having at least 70% identity to the amino acid sequence encoded by SEQ ID NO: 148 or 149, such at least 80%, at least 90%, at least 95%, at least 99% or 100% identity to the amino acid sequence encoded by SEQ ID NO: 148 or 149.

[0121] The CAR may comprise a transmembrane domain and a hinge derived from the same source. For example, the transmembrane domain and the hinge may be a CD8a transmembrane domain and a CD8a hinge. The transmembrane domain and the hinge may be a CD28 transmembrane domain and a CD28 hinge.

[0122] In some cases, the CAR may comprise a ‘short’ 28z construct, i.e. comprising an IgG4 hinge and a CD28z intracellular domain. The CD28z domain may comprise an amino acid sequence having at least 70% identity to SEQ ID NO: 142 (such at least 80%, at least 90%, at least 95%, at least 99% or 100% identity to SEQ ID NO: 142) and an amino acid sequence having at least 70% identity to any one of SEQ ID NOs: 143-145 (such at least 80%, at least 90%, at least 95%, at least 99% or 100% identity to any one of SEQ ID NOs: 143-145). The IgG4 hinge may comprises or consists of an amino acid sequence having at least 70% identity to SEQ ID NO: 135, such at least 80%, at least 90%, at least 95%, at least 99% or 100% identity to SEQ ID NO: 135.

[0123] In some cases, the CAR may comprise a Tong’ 28z construct, i.e. comprising the long IgGl hinge (comprising the CH2 and CH3 domains) and a CD28z intracellular domain. The CD28z domain may comprise an amino acid sequence having at least 70% identity to SEQ ID NO: 142 (such at least 80%, at least 90%, at least 95%, at least 99% or 100% identity to SEQ ID NO: 142) and an amino acid sequence having at least 70% identity to any one of SEQ ID NOs: 143-145 (such at least 80%, at least 90%, at least 95%, at least 99% or 100% identity to any one of SEQ ID NOs: 143-145). The IgGl hinge may comprise or consist of an amino acid sequence having at least 70% identity to SEQ ID NO: 138, such at least 80%, at least 90%, at least 95%, at least 99% or 100% identity to SEQ ID NO: 138. The ‘short’ and Tong’ 28z constructs typically comprise the CD28 transmembrane domain. For example, the short’ and Tong’ 28z constructs may further comprise an amino acid sequence having at least 70% identity to SEQ ID NO: 140, such at least 80%, at least 90%, at least 95%, at least 99% or 100% identity to SEQ ID NO: 140.

[0124] The CAR may comprise a signal peptide (otherwise referred to herein as a leader sequence). A nucleic acid molecule encoding the CAR typically encodes a signal peptide. A signal peptide targets the CAR to the endoplasmic reticulum during translation, and is typically co-translationally cleaved at a signal peptidase cleavage site. This enables cell surface expression of the CAR. Any suitable signal peptide may be used in the CAR. For example, the CAR may comprise a CD8 signal peptide as set out in SEQ ID NO: 130. After cleavage, the sequence corresponding to SEQ ID NO: 131 is removed from the mature CAR, leaving the sequence Gly-Ser at the N-terminal end of the CAR. Typically, the CAR referred to herein is mature and does not comprise the sequence cleaved by signal peptidase.

[0125] Accordingly, a CAR as described herein may comprise:

[0126] (a) optionally, a signal peptide sequence;

[0127] (b) an antigen-binding domain specific for a glioma-associated antigen, as described herein;

[0128] (c) a hinge;

[0129] (d) a transmembrane domain; and

[0130] (e) an intracellular domain.

[0131] The signal peptide sequence may be any amino acid signal peptide sequence as described herein. The signal peptide sequence may comprise SEQ ID NO: 130. The antigen-binding domain may be any antigen-binding domain as described herein. The antigen-binding domain may comprise an amino acid sequence having the CDRs from an amino acid sequence selected from SEQ ID NOs: 1 to 9, 31 to 42, 62 to 69, 86 to 94, 118, 172 and 176. The antigen-binding domain may comprise an amino acid sequence having the CDRs from an amino acid sequence selected from SEQ ID NOs: 1 to 9, 31 to 42, 62 to 69, 86 to 94 and 118. The antigen-binding domain may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 1 to 9, 31 to 42, 62 to 69, 86 to 94, 118, 172 and 176. The antigen-binding domain may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 1 to 9, 31 to 42, 62 to 69, 86 to 94 and 118. Typically, the CAR comprises the exact CDRs of the SEQ ID NOs from which the CAR is derived. The antigen-binding domain may comprise the CDRs from an amino acid sequence selected from SEQ ID NOs: 1 to 9, 31 to 42, 62 to 69, 86 to 94, 118, 172 and 176, and may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 1 to 9, 31 to 42, 62 to 69, 86 to 94, 118, 172 and 176, respectively. The antigen-binding domain may comprise the CDRs from an amino acid sequence selected from SEQ ID NOs: 1 to 9, 31 to 42, 62 to 69, 86 to 94 and 118, and may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 1 to 9, 31 to 42, 62 to 69, 86 to 94 and 118, respectively. The hinge may comprise any hinge as described herein. The hinge may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 134 to 138. The transmembrane domain comprise any transmembrane domain as described herein. The transmembrane domain may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 139 to 140. The intracellular domain may comprise any intracellular domain as described herein. The intracellular domain may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 141 to 145. The transmembrane domain may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 141 to 142 and an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 143 to 145.

[0132] The exemplary CARs used in the Examples are set out in SEQ ID NOs: 121 to 129, 170 and 174. The anti-0 AcGD2 CAR used in the Examples comprises the sequence as set out in SEQ ID NO: 121. The anti-0 AcGD2 CAR comprises in the direction from N- terminal to C-terminal, the CD8 leader sequence (SEQ ID NO 130), the 8B6 scFv sequence (SEQ ID NO: 118), an Ser-Gly linker, the huIgGlmut hinge (SEQ ID NO: 138), the CD28 transmembrane domain (SEQ ID NO: 140), a CD28 intracellular domain (SEQ ID NO: 142) and a CD3-zeta intracellular domain (SEQ ID NO: 145). Typically, the mature CAR does not comprise the cleaved sequence from the signal peptide, i.e. the N- terminus starts with Gly-Ser followed by the 8B6 scFv sequence. Accordingly, a CAR as described herein may comprise:

[0133] (a) optionally, a signal peptide sequence, optionally wherein the signal peptide sequence comprises the amino acid sequence set out in SEQ ID NO: 130 or is Gly-Ser;

[0134] (b) an antigen-binding domain comprising the CDRs from SEQ ID NO: 118, optionally wherein the antigen-binding domain comprises an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 118, optionally wherein the antigen-binding domain comprises the CDRs from SEQ ID NO: 118, and comprises an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 118;

[0135] (c) a hinge, optionally comprising an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 138;

[0136] (d) a transmembrane domain, optionally comprising an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 140; and

[0137] (e) an intracellular domain, optionally comprising an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 142 and / or an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 145.

[0138] The anti-PTPRZl CAR used in the Examples comprises the sequence as set out in SEQ ID NO: 122. The anti- PTPRZ1 CAR comprises in the direction from N-terminal to C -terminal, the CD8 leader sequence (SEQ ID NO 130), the RB832 VHH sequence (SEQ ID NO: 1), an Ser-Gly linker, the IgG4 hinge (SEQ ID NO: 135), the CD28 transmembrane domain (SEQ ID NO: 140), a CD28 intracellular domain (SEQ ID NO: 142) and a CD3-zeta intracellular domain (SEQ ID NO: 144). Typically, the mature CAR does not comprise the cleaved sequence from the signal peptide, i.e. the N-terminus starts with Gly-Ser followed by the RB832 VHH sequence. Accordingly, a CAR as described herein may comprise:

[0139] (a) optionally, a signal peptide sequence, optionally wherein the signal peptide sequence comprises the amino acid sequence set out in SEQ ID NO: 130 or is Gly-Ser;

[0140] (b) an antigen-binding domain comprising the CDRs from SEQ ID NO: 1, optionally wherein the antigen-binding domain comprises an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 1, optionally wherein the antigen-binding domain comprises the CDRs from SEQ ID NO: 1, and comprises an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 1;

[0141] (c) a hinge, optionally comprising an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 135;

[0142] (d) a transmembrane domain, optionally comprising an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 140; and

[0143] (e) an intracellular domain, optionally comprising an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 142 and / or an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 144.

[0144] An anti-TNC CAR used in the Examples comprises the sequence as set out in SEQ ID NO: 123. The anti-TNC CAR comprises in the direction from N-terminal to C- terminal, the CD8 leader sequence (SEQ ID NO 130), the R6N scFv sequence (SEQ ID NO: 42), an Ser-Gly linker, the IgG4 hinge (SEQ ID NO: 135), the CD28 transmembrane domain (SEQ ID NO: 140), a CD28 intracellular domain (SEQ ID NO: 142) and a CD3- zeta intracellular domain (SEQ ID NO: 144). Typically, the mature CAR does not comprise the cleaved sequence from the signal peptide, i.e. the N-terminus starts with Gly- Ser followed by the R6N scFv sequence. Accordingly, a CAR as described herein may comprise:

[0145] (a) optionally, a signal peptide sequence, optionally wherein the signal peptide sequence comprises the amino acid sequence set out in SEQ ID NO: 130 or is Gly-Ser;

[0146] (b) an antigen-binding domain comprising the CDRs from SEQ ID NO: 1, optionally wherein the antigen-binding domain comprises an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 1, optionally wherein the antigen-binding domain comprises the CDRs from SEQ ID NO: 1, and comprises an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 1;

[0147] (c) a hinge, optionally comprising an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 135;

[0148] (d) a transmembrane domain, optionally comprising an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 140; and

[0149] (e) an intracellular domain, optionally comprising an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 142 and / or an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 144.

[0150] Additional anti-TNC CARs used in the Examples comprise the sequences as set out in any one of SEQ ID NOs: 124 to 129. The anti-TNC CARs comprise in the direction from N-terminal to C-terminal, the CD8 leader sequence (SEQ ID NO: 130), one of the RB895 to RB900 VHH sequences (SEQ ID NOs: 31 to 36, respectively), an Ser-Gly linker, the IgG4 hinge (SEQ ID NO: 135), the CD28 transmembrane domain (SEQ ID NO: 140), a CD28 intracellular domain (SEQ ID NO: 142) and a CD3-zeta intracellular domain (SEQ ID NO: 144). Typically, the mature CAR does not comprise the cleaved sequence from the signal peptide, i.e. the N-terminus starts with Gly-Ser followed by the VHH sequence. Accordingly, a CAR as described herein may comprise:

[0151] (a) optionally, a signal peptide sequence, optionally wherein the signal peptide sequence comprises the amino acid sequence set out in SEQ ID NO: 130 or is Gly-Ser;

[0152] (b) an antigen-binding domain comprising the CDRs from an amino acid sequence selected from any one of SEQ ID NOs: 31 to 36, optionally wherein the antigen-binding domain comprises an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence selected from any one of SEQ ID NOs: 31 to 36, optionally wherein the antigen-binding domain comprises the CDRs from an amino acid sequence selected from any one of SEQ ID NOs: 31 to 36, and comprises an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to an amino acid sequence selected from any one of SEQ ID NOs: 31 to 36;

[0153] (c) a hinge, optionally comprising an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 135;

[0154] (d) a transmembrane domain, optionally comprising an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 140; and

[0155] (e) an intracellular domain, optionally comprising an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 142 and / or an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 144.

[0156] The anti-B7-H3 CAR used in the Examples comprises the sequence as set out in SEQ ID NO: 170. The anti-B7-H3 CAR comprises in the direction from N-terminal to C- terminal, the CD8 leader sequence (SEQ ID NO 130), the VHH sequence of SEQ ID NO: 172, an Ser-Gly linker, the human IgGl long hinge (SEQ ID NO: 138), the CD28 transmembrane domain (SEQ ID NO: 140), a CD28 intracellular domain (SEQ ID NO: 142) and a CD3-zeta intracellular domain (SEQ ID NO: 145). Typically, the mature CAR does not comprise the cleaved sequence from the signal peptide, i.e. the N-terminus starts with Gly-Ser followed by the VHH sequence. Accordingly, a CAR as described herein may comprise:

[0157] (a) optionally, a signal peptide sequence, optionally wherein the signal peptide sequence comprises the amino acid sequence set out in SEQ ID NO: 130 or is Gly-Ser;

[0158] (b) an antigen-binding domain comprising the CDRs from SEQ ID NO: 172, optionally wherein the antigen-binding domain comprises an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 172, optionally wherein the antigen-binding domain comprises the CDRs from SEQ ID NO: 172, and comprises an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 172;

[0159] (c) a hinge, optionally comprising an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 138;

[0160] (d) a transmembrane domain, optionally comprising an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 140; and

[0161] (e) an intracellular domain, optionally comprising an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 142 and / or an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 145.

[0162] The anti-IL13Ra2 CAR used in the Examples comprises the sequence as set out in SEQ ID NO: 174. The anti-IL13Ra2 CAR comprises in the direction from N-terminal to C-terminal, the CD8 leader sequence (SEQ ID NO 130), the VHH sequence of SEQ ID NO: 176, an Ser-Gly linker, the human IgGl long hinge (SEQ ID NO: 138), the CD28 transmembrane domain (SEQ ID NO: 140), a CD28 intracellular domain (SEQ ID NO: 142) and a CD3-zeta intracellular domain (SEQ ID NO: 145). Typically, the mature CAR does not comprise the cleaved sequence from the signal peptide, i.e. the N-terminus starts with Gly-Ser followed by the VHH sequence. Accordingly, a CAR as described herein may comprise:

[0163] (a) optionally, a signal peptide sequence, optionally wherein the signal peptide sequence comprises the amino acid sequence set out in SEQ ID NO: 130 or is Gly-Ser;

[0164] (b) an antigen-binding domain comprising the CDRs from SEQ ID NO: 176, optionally wherein the antigen-binding domain comprises an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 176, optionally wherein the antigen-binding domain comprises the CDRs from SEQ ID NO: 176, and comprises an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 176;

[0165] (c) a hinge, optionally comprising an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 138;

[0166] (d) a transmembrane domain, optionally comprising an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 140; and

[0167] (e) an intracellular domain, optionally comprising an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 142 and / or an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 145.

[0168] The CAR may comprise more than one extracellular antigen-binding domain, such as two extracellular antigen-binding domains or three extracellular antigen-binding domains. The two or more extracellular antigen binding domains may bind to different glioma associated antigens, i.e. the CAR may be bispecific or multispecific.

[0169] Immune effector cell

[0170] Reference to an immune effector cell as used herein is a cell capable of cell- mediated cytotoxicity against a target cell displaying a target antigen, e.g. a glioma- associated antigen. The immune effector cell may be a T cell, a y5 T cell, a natural killer (NK) cell, an NKT cell, an induced pluripotent stem cell (iPSC) derived NK cell (iPSC- NK), a y5 T cell, a phagocyte, or a macrophage. The immune effector cell is preferably a T cell.

[0171] Preferably, the T cell is a CD8+ T cell, or cytotoxic T cell. The T cell is preferably a CD4-CD8+ T cell.

[0172] The T cell may be a CD4+ T cell, or helper T cell (TH cell), such as a TH1, TH2, TH3, THU, TH9, or TFH cells. The T cell may be a regulatory T cell (Treg). The T cell may be a naive, effector, memory, effector memory, central memory, memory stem T cell. The T cell may be a peripheral lymphocyte.

[0173] The T cell may be expanded from peripheral blood mononuclear cells (PBMCs). The T cell may be autologous with respect to a subject into which it is to be administered. The T cell may be allogeneic with respect to a subject into which it is to be administered. The T cell may be partially HLA-mismatched with respect to a subject into which it is to be administered.

[0174] The NK cell may be a cell of the NK92 cell line. The NK cell may be isolated from peripheral blood mononuclear cells (PBMCs) of the subject to be treated or of a healthy donor. The NK cell may be isolated from cord blood. The NK cell may be differentiated from a CD34+haematopoietic progenitor cell (HPC).

[0175] The y5 T cell may be expanded from peripheral blood mononuclear cells (PBMCs). The y5 T cell may be autologous with respect to a subject into which it is to be administered. The T y6 cell may be allogeneic with respect to a subject into which it is to be administered.

[0176] The macrophage may be differentiated into the “Ml” phenotype. The Ml macrophage expresses pro-inflammatory cytokines and has strong anti-tumour activity. An undifferentiated macrophage expressing a CAR described herein may be induced to differentiate into the Ml phenotype by culturing in the presence of the glioma-associated antigen.

[0177] The immune effector cell may comprise a nucleic acid described herein. The immune effector cell may comprise a vector described herein. The immune effector cell preferably comprises an RNA nucleic acid or RNA vector described herein. A T cell comprising an RNA nucleic acid or RNA vector described herein is also referred to as RNA CAR T cell herein. The immune effector cell expresses a CAR specific for one or more glioma-associated antigens. The immune effector cell preferably transiently expresses the CAR.

[0178] Accordingly, the immune effector cell is preferably an RNA CAR T cell.

[0179] The immune effector cell may be engineered to transiently express a CAR specific for one or more glioma-associated antigens. This is to minimise on-target off-tumour toxicity as a result of expression of the antigen by normal healthy tissue. In some cases, the immune effector cell may be transfected with mRNA encoding a CAR specific for one or more glioma-associated antigens, for example by mRNA electroporation as demonstrated in Beatty et al, Gastroenterology 155.1 (2018): 29-32. (see supplementary materials 5) and Schutsky et al, Oncotarget 6.30 (2015): 28911.

[0180] In some cases, the invention relations to a population of immune effector cells that express one or more chimeric antigen receptors (CARs) specific for two or more glioma- associated antigens.

[0181] A population may comprise at least about IxlO6of the immune effector cells, such as at least about IxlO7, at least about IxlO8, at least about IxlO9or at least about IxlO10of the immune effector cells. A population may comprise at least about IxlO6to about IxlO12of the immune effector cells, such as about IxlO6to about IxlO11, about IxlO6to about IxlO10, about IxlO6to about IxlO9, about IxlO7to about IxlO11, about IxlO8to about IxlO10of the immune effector cells. The population may comprise about IxlO6of the immune effector cells, such as about 5xl06, about IxlO7, about 5xl07, about IxlO8, about 5xl08, about IxlO9, about 5xl09, about IxlO10, about 5xlO10, about IxlO11, about 5xlOn, or about IxlO12of the immune effector cells

[0182] In some cases, an immune effector cell of the invention expresses a multivalent CAR specific for two or more different glioma-associated antigens (z.e. a bispecific or multispecific CAR), as described herein. In some cases, the population may comprise at least two different CAR-expressing immune effector cells specific for at least two different glioma-associated antigens, as described herein. In some cases, an immune effector cell of the invention expresses at least two different CARs, wherein the at least two different CARs are specific for at least two different glioma-associated antigens.

[0183] A population of immune effector cells each expressing two or more CARs specific for different glioma-associated antigens is typically comprised in a larger population of cells with different antigen specificity as a result of the transfection process. As shown in Figures 9 and 11, transfection of a population of immune effector cells with three separate nucleic acid molecules each encoding a different CAR specific for a different antigen results in a heterogenous population of immune effector cells, some expressing a single CAR, some expressing different combinations of two CARs, and some expressing all three CARs. Accordingly, a population of immune effector cells may comprise a mix of immune effector cells with differential expression of the two or more CARs each specific for different glioma-associated antigens.

[0184] Multispecificity against two or more glioma-associated antigens is advantageous for a number of reasons. For example, due to interpatient and / or inter-tumour variability, the expression of glioma-associated antigens may vary within and between subjects. Multispecificity allows a single therapy to target a wider range of tumours. Furthermore, antigen escape is the phenomenon wherein an antigen targeted by, e.g., a CAR is no longer expressed by the tumour and the therapy loses its efficacy. By targeting a number of glioma-associated antigens, it is much more difficult for the tumour to ‘escape’ the therapy. Multispecificity also enhances immune effector cell effector functions such as cytotoxicity.

[0185] Also provided is a method of making an immune effector cell of the invention or a population of immune effector cells of the invention. The method comprises transforming the cell or the population of cells with one or more nucleic acids encoding one or more CARs specific for one or more glioma-associated antigens. The CARs may be any of the CARs discussed herein. The nucleic acids may be any of the nucleic acid or vectors of the invention.

[0186] Any method known in the art may be used to transform the cell or the population with the nucleic acid. The immune effector cell may be transfected or transduced with the nucleic acid. The CAR may be introduced to the immune effector cell using a vector.

[0187] The term “transduction” may be used to describe virus mediated nucleic acid transfer. A viral vector may be used to transduce the cell with the one or more constructs. Conventional viral based expression systems could include retroviral, alpha-retroviral, lentivirus, adenoviral, adeno-associated (AAV) and herpes simplex virus (HSV) vectors for gene transfer. Non-viral transduction vectors include transposon-based systems including PiggyBac and Sleeping Beauty systems. Methods for producing and purifying such vectors are known in the art. The vector is preferably a vector described herein. Immune effector cells may be transduced using any method known in the art. Transduction may be in vitro or ex vivo.

[0188] The term “transfection” may be used to describe non-virus-mediated nucleic acid transfer. The immune effector cells may be transfected using any method known in the art. Transfection may be in vitro or ex vivo. Any vector capable of transfecting immune effector cells may be used, such as conventional plasmid DNA or RNA transfection, preferably mRNA transfection. A human artificial chromosome and / or naked RNA may be used to transfect the cell with the nucleic acid sequence or nucleic acid construct. Human artificial chromosomes are described in e.g. Kazuki et al., Mol. Ther. 19(9): 1591- 1601 (2011), and Kouprina et al., Expert Opinion on Drug Delivery 11(4): 517-535 (2014). Alternative non-viral delivery systems include DNA plasmids, naked nucleic acid, and nucleic acid complexed with a delivery vehicle, such as a liposome. Methods of non-viral delivery of nucleic acids include lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipidmucleic acid conjugates, naked DNA, naked RNA, artificial virions, and agent-enhanced uptake of DNA.

[0189] Nanoparticle delivery systems may be used to transfect the immune effector cell with the nucleic acid sequence. Such delivery systems include, but are not limited to, lipid-based systems, liposomes, micelles, microvesicles and exosomes. With regard to nanoparticles that can deliver RNA, see, e.g., Alabi et al., Proc Natl Acad Sci U S A. 2013 Aug 6; 110(32): 12881-6; Zhang et al., Adv Mater. 2013 Sep 6;25(33):4641-5; Jiang et al., Nano Lett. 2013 Mar 13; 13(3): 1059-64; Karagiannis et al., ACS Nano. 2012 Oct 23;6(10):8484-7; Whitehead et al., ACS Nano. 2012 Aug 28;6(8):6922-9 and Lee et al., Nat Nanotechnol. 2012 Jun 3;7(6):389-93. Lipid Nanoparticles, Spherical Nucleic Acid (SNA™) constructs, nanoplexes and other nanoparticles (particularly gold nanoparticles) are also contemplated as a means for delivery of a nucleic acid or vector of the invention.

[0190] The immune effector cell may be transfected by electroporation. Preferably, the electroporation is mRNA electroporation. This has the advantage of allowing transient expression of the CAR.

[0191] The immune effector cell may be transfected by electroporation, such as RNA electroporation or mRNA electroporation. Where the immune effector cell expresses more than one CAR, the immune effector cell may be transfected by electroporation (e.g. RNA or mRNA electroporation) of a single polynucleotide (e.g. RNA or mRNA) or vector encoding the more than one CAR, or may be transfected by electroporation (e.g. RNA or mRNA electroporation) of two or more polynucleotides (e.g. RNA or mRNA) or vectors, each polynucleotide encoding at least one CAR. The electroporation of the two or more polynucleotides is typically performed simultaneously.

[0192] Uptake of nucleic acid constructs may be enhanced by several known transfection techniques, for example those including the use of transfection agents. Examples of these agents includes cationic agents, for example, calcium phosphate and DEAE-Dextran and lipofectants, for example, lipofectAmine, fugene and transfectam.

[0193] Other products

[0194] Also provided is an antigen binding molecule specific for TNC. The antigenbinding molecule comprises a polypeptide, e.g. an antigen binding domain as described herein, comprising the CDRs (CDR1, CDR2 and CDR3) from an amino acid sequence selected from any one of SEQ ID NOs: 31 to 36. Preferably the polypeptide is a VHH domain. The polypeptide may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 31 to 36. A CAR described herein may comprise the CDRs from an amino acid sequence selected from SEQ ID NOs: 31 to 36, and may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 31 to 36, respectively.

[0195] Also provided is an antigen binding molecule specific for B7-H3. The antigenbinding molecule comprises a polypeptide, e.g. an antigen binding domain as described herein, comprising the CDRs (CDR1, CDR2 and CDR3) from SEQ ID NO: 172. Preferably the polypeptide is a VHH domain. The polypeptide may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 172. A CAR described herein may comprise the CDRs from SEQ ID NO: 172, and may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 172.

[0196] Also provided is an antigen binding molecule specific for IL13Ra2. The antigenbinding molecule comprises a polypeptide, e.g. an antigen binding domain as described herein, comprising the CDRs (CDR1, CDR2 and CDR3) from SEQ ID NO: 176. Preferably the polypeptide is a VHH domain. The polypeptide may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 176. A CAR described herein may comprise the CDRs from SEQ ID NO: 176, and may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 176.

[0197] The antigen-binding molecule may be an antibody. The antigen-binding molecule may be an antibody-drug conjugate. The antigen-binding molecule may be used in an antibody-based therapy, for example, in a method of metabolite radiotherapy. The antigenbinding molecule may be used in a method of treatment of a cancer as described herein. Also provided is a CAR comprising the antigen-binding molecule specific for TNC. Accordingly, provided is a CAR specific for TNC comprising a polypeptide comprising the CDRs from an amino acid sequence selected from any one of SEQ ID NOs: 31 to 36, optionally which comprises a polypeptide comprising an amino acid sequence having at least 80% identity to the amino acid sequence of any one of SEQ ID NOs: 31 to 36. The CAR may comprise a hinge, a transmembrane domain and an intracellular domain as described herein, as well as any other features of a CAR as described herein.

[0198] Also provided is a CAR comprising the antigen-binding molecule specific for B7- H3. Accordingly, provided is a CAR specific for B7-H3 comprising a polypeptide comprising the CDRs from SEQ ID NO: 172 optionally which comprises a polypeptide comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 172. The CAR may comprise a hinge, a transmembrane domain and an intracellular domain as described herein, as well as any other features of a CAR as described herein.

[0199] Also provided is a CAR comprising the antigen-binding molecule specific for IL13Ra2. Accordingly, provided is a CAR specific for IL13Ra2 comprising a polypeptide comprising the CDRs from SEQ ID NO: 176 optionally which comprises a polypeptide comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 176. The CAR may comprise a hinge, a transmembrane domain and an intracellular domain as described herein, as well as any other features of a CAR as described herein. Also provided is an immune effector cell, or a population of immune effector cells comprising the CAR comprising the antigen-binding molecule specific for TNC. The immune effector cell may comprise any of the features of the immune effector cell as described herein. Accordingly, the immune effector cell, or population of immune effector cells, may express a CAR specific for TNC, wherein the CAR comprises a polypeptide comprising the CDRs from an amino acid sequence selected from any one of SEQ ID NOs: 31 to 36. The immune effector cell or population of immune effector cells may additionally express one or more CARs specific for one or more glioma-associated antigens, as described herein. For example, the one or more glioma-associated antigens may be selected from a group comprising PTPRZ1, BCAN and CSPG4. The one or more glioma-associated antigens may be selected from a group comprising a GD2 ganglioside (such as an 0AcGD2), PTPRZ1, BCAN and CSPG4. The one or more glioma-associated antigens (in addition to TNC) may comprise: PTPRZ1; BCAN; CSPG4; a GD2 ganglioside (such as an ( AcGD2); PTPRZ1 and BCAN; PTPRZ1 and CSPG4; BCAN and CPSG4; PTPRZ1, BCAN and CSPG4; a GD2 ganglioside (such as an ( AcGD2) and PTPRZ1; a GD2 ganglioside (such as an (9AcGD2) and BCAN; a GD2 ganglioside (such as an (9AcGD2) and CPSG4; a GD2 ganglioside (such as an 0AcGD2), PTPRZ1 and BCAN; a GD2 ganglioside (such as an 0AcGD2), PTPRZ1 and CSPG4; a GD2 ganglioside (such as an 0AcGD2), BCAN and CSPG4; or a GD2 ganglioside (such as an 0AcGD2), PTPRZ1, BCAN and CSPG4. Thus, the immune effector cell, or population of immune effector cells, may express one or more CARs specific for two or more glioma-associated antigens, wherein one or more of the glioma-associated antigens is TNC and a CAR comprises the CDRs from an amino acid sequence selected from any one of SEQ ID NOs: 31 to 36, and one or more of the glioma-associated antigens is optionally selected from a group comprising a GD2 ganglioside (such as an 0AcGD2), PTPRZ1, BCAN and CSPG4.

[0200] Also provided is an immune effector cell, or a population of immune effector cells comprising the CAR comprising the antigen-binding molecule specific for B7-H3. The immune effector cell may comprise any of the features of the immune effector cell as described herein. The immune effector cell or population of immune effector cells may additionally express one or more CARs specific for one or more glioma-associated antigens, as described herein. The one or more glioma-associated antigens may be selected from a group comprising a GD2 ganglioside (such as an 0AcGD2), IL13Ra2, PTPRZ1, BCAN, TNC and CSPG4. The one or more glioma-associated antigens may be selected from the group comprising a GD2 ganglioside (such as an (9AcGD2) and IL13Ra2.

[0201] Also provided is an immune effector cell, or a population of immune effector cells comprising the CAR comprising the antigen-binding molecule specific for IL13Ra2. The immune effector cell may comprise any of the features of the immune effector cell as described herein. The immune effector cell or population of immune effector cells may additionally express one or more CARs specific for one or more glioma-associated antigens, as described herein. The one or more glioma-associated antigens may be selected from a group comprising a GD2 ganglioside (such as an 0AcGD2), B7-H3, PTPRZ1, BCAN, TNC and CSPG4. The one or more glioma-associated antigens may be selected from the group comprising a GD2 ganglioside (such as an (9AcGD2) and B7-H3.

[0202] Also provided is a CAR comprising an antigen-binding domain specific for a GD2 ganglioside (such as an 0ACGD2). The CAR comprises an antigen-binding domain specific for the GD2 ganglioside, a hinge, a transmembrane domain and an intracellular domain. Such CARs are described herein. For example, the antigen-binding domain may comprise the CDRs from SEQ ID NO: 118. The antigen-binding domain may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 118. The antigen-binding domain may comprise the CDRs from SEQ ID NO: 118, and may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 118. The hinge, the transmembrane domain and the intracellular domain may be as described herein. The CAR may comprise any other features of a CAR as described herein. The hinge may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 138. The transmembrane domain may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 140. The intracellular domain may comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 142 and / or an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 145.

[0203] Also provided is an immune effector cell, or a population of immune effector cells comprising the CAR comprising an antigen-binding domain specific for a GD2 ganglioside (such as an (9ACGD2), described above. The immune effector cell, or population of immune effector cells, may or may not express a CAR specific for one or more further glioma-associated antigens (i.e. in addition to the GD2 ganglioside). Nucleic acids

[0204] Also provided is one or more isolated nucleic acid molecules (i.e. polynucleotides) encoding one or more CARs or one or more antigen binding molecules specific for TNC comprising at least one of SEQ ID NOs: 31 to 36 described herein. Also provided is one or more isolated nucleic acid molecules (i.e. polynucleotides) encoding one or more CARs or one or more antigen binding molecules specific for B7-H3 described herein, for example, a CAR or antigen-binding molecule based on SEQ ID NO: 172. Also provided is one or more isolated nucleic acid molecules (i.e. polynucleotides) encoding one or more CARs or one or more antigen binding molecules specific for IL13Ra2 described herein, for example, a CAR or antigen-binding molecule based on SEQ ID NO: 176. For example, provided herein is a single nucleic acid molecule encoding two or more of the CARs or antigen binding molecules described herein. Also provided are two or more nucleic acid molecules, wherein each nucleic acid molecule encodes one or more of the CARs or antigen binding molecules described herein. In some cases, the encoding nucleic acid sequence may be provided by two or more nucleic acid sequences, optionally present on two or more nucleic acid molecules, but collectively together they are able to encode a CAR or antigen binding molecules of the invention. The nucleic acid, or the two or more nucleic acids, may be provided as a composition. The composition may be used for transfecting a cell, as described herein, so that it expresses the one or more CARs or antigen binding molecules described herein.

[0205] Nucleic acids which encode a CAR or antigen binding molecules of the invention can be obtained by methods well known to those skilled in the art. For example, DNA sequences coding for part or all of the antibody heavy and light chains may be synthesised as desired from the corresponding amino acid sequences.

[0206] The nucleic acid may be a DNA sequence. The nucleic acid may be an RNA sequence, such as mRNA. A vector may comprise the nucleic acid.

[0207] The vector may be a viral vector. Conventional viral based expression systems could include retroviral, alpha-retroviral, lentivirus, adenoviral, adeno-associated (AAV) and herpes simplex virus (HSV) vectors for gene transfer. Non-viral transduction vectors include transposon-based systems including PiggyBac and Sleeping Beauty systems. Methods for producing and purifying such vectors are known in the art.

[0208] The vectors may be cloning vectors or expression vectors. A suitable vector may be any vector which is capable of carrying a sufficient amount of genetic information, and allowing expression of a polypeptide of the invention.

[0209] The vector is preferably an RNA vector. Suitable RNA vectors include the RNA vectors described in Schutsky, Keith, et al., Oncotarget 6.30 (2015): 28911 and Beatty, Gregory L., et al., Gastroenterology 155.1 (2018): 29-32.

[0210] General methods by which the vectors may be constructed, transfection methods and culture methods are well known to those skilled in the art. In this respect, reference is made to “Current Protocols in Molecular Biology”, 1999, F. M. Ausubel (ed), Wiley Interscience, New York and the Maniatis Manual produced by Cold Spring Harbor Publishing.

[0211] A nucleic acid may be provided in the form of an expression cassette, which includes control sequences operably linked to the inserted sequence, thus allowing for expression of the CAR or antigen binding molecules of the invention in vivo. Hence, also provided is one or more expression cassettes encoding the one or more nucleic acids that encode a CAR or antigen binding molecules described herein. These expression cassettes, in turn, are typically provided within vectors (e.g. plasmids or recombinant viral vectors). Hence, also provided is a vector encoding a CAR or antigen binding molecules described herein. Further provided are vectors which collectively encode a CAR described herein.

[0212] The vector may be a human artificial chromosome. Human artificial chromosomes are described in e.g. Kazuki et al., Mol. Ther. 19(9): 1591-1601 (2011), and Kouprina et al., Expert Opinion on Drug Delivery 11(4): 517-535 (2014).

[0213] The vector may be a non-viral delivery system, such as DNA plasmids, naked nucleic acid (e.g. naked RNA), and nucleic acid complexed with a delivery vehicle, such as a liposome or a nanoparticle.

[0214] The nucleic acids, expression cassettes or vectors described herein may be introduced into a host cell, e.g. by transfection. Hence, also provided is a host cell comprising the one or more nucleic acids, expression cassettes or vectors of the invention. The nucleic acids, expression cassettes or vectors described herein may be introduced transiently or permanently into the host cell, allowing expression of an antibody from the one or more nucleic acids, expression cassettes or vectors. Such host cells include transient, or preferably stable higher eukaryotic cell lines, such as mammalian cells or insect cells, lower eukaryotic cells, such as yeast, or prokaryotic cells, such as bacteria cells. Particular examples of cells include mammalian HEK293, such as HEK293F, HEK293T, HEK293S or HEK Expi293F, CHO, HeLa, NSO and COS cells, or any other cell line used herein. Preferred host cells are the immune effector cells described herein. Preferably, the nucleic acids, expression cassettes or vectors described herein are introduced transiently into the host cell.

[0215] Also provided is a kit suitable for transforming and / or transfecting an immune effector cell or a population of immune effector cells to generate an immune effector cell or population of immune effector cells of the invention. The kit comprises a nucleic acid or vector described herein. The kit may comprise further agents such as those discussed herein that improve transfection or transformation efficacy.

[0216] Also described are one or more isolated nucleic acid molecules encoding the specific antigen-binding molecules or CARs, as described herein. The one or more isolated nucleic acid molecules may encode an antigen-binding region of SEQ ID NOs: 1 to 9, 31 to 42, 62 to 69, 86 to 94, 118, 172 and 176. The one or more isolated nucleic acid molecules may encode an antigen-binding region of SEQ ID NOs: 1 to 9, 31 to 42, 62 to 69, 86 to 94 and 118. For example, the one or more isolated nucleic acid molecules may comprise a sequence as set out in SEQ ID NOs: 22 to 30, 51 to 61, 78 to 85, 109 to 117 and 169. The one or more isolated nucleic acid molecules may comprise a sequence as set out in SEQ ID NOs: 22 to 30, 51 to 61, 78 to 85, 109 to 117, 171, 173, 175 and 177. Whilst SEQ ID NOs: 22 to 30, 51 to 61, 78 to 85, 109 to 117, 171, 173, 175 and 177 are provided as a DNA sequence, the corresponding RNA sequence (replacing ‘T’ with ‘U’) is also encompassed. The one or more isolated nucleic acid molecules may encode the CAR of any one of SEQ ID NOs: 121 to 129, 170 and 174. The one or more isolated nucleic acid molecules may encode the CAR of any one of SEQ ID NOs: 121 to 129.

[0217] Also described are nucleic acids encoding the specific scFv antigen binding regions of SEQ ID NOs: 4 to 9, 39 to 42, 66 to 69, 88 to 94 and 118, which are provided in SEQ ID NOs: 25 to 30, 59 to 61, 82 to 85, 111 to 117 and 169, respectively. Whilst SEQ ID NOs: 25 to 30, 59 to 61, 82 to 85, 111 to 117 and 169 are provided as a DNA sequence, the corresponding RNA sequence (replacing ‘T’ with ‘U’) is also encompassed.

[0218] Also described are nucleic acids encoding the specific VHH antigen binding regions of SEQ ID NOs: 1 to 3, 31 to 38, 62 to 65, 86 to 87, 172 and 176, which are provided in SEQ ID NOs: 22 to 24, 51 to 58, 78 to 81, 109 to 110, 173 and 177, respectively. Also described are nucleic acids encoding the specific VHH antigen binding regions of SEQ ID NOs: 1 to 3, 31 to 38, 62 to 65 and 86 to 87, which are provided in SEQ ID NOs: 22 to 24, 51 to 58, 78 to 81 and 109 to 110, respectively. Whilst SEQ ID NOs: 22 to 24, 51 to 58, 78 to 81, 109 to 110, 173 and 177 are provided as a DNA sequence, the corresponding RNA sequence (replacing ‘T’ with ‘U’) is also encompassed.

[0219] Pharmaceutical composition

[0220] Also provided is a composition comprising an immune effector cell or population of immune effector cells of the invention. The immune effector cell or population of immune effector cells may be at least 50% of the total cells in the composition, such as at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.9% of the total cells in the composition. The total cells in the composition may consist or consist essentially of the immune effector cell or population of immune effector cells of the invention, i.e. no other cells are detectable in the composition.

[0221] The composition may comprise at least about IxlO6to about IxlO12of the immune effector cells of the invention, such as about IxlO6to about IxlO11, about IxlO6to about IxlO10, about IxlO6to about IxlO9, about IxlO7to about IxlO11, about IxlO8to about IxlO10of the immune effector cells. The composition may comprise about IxlO6of the immune effector cells of the invention, such as about 5xl06, about IxlO7, about 5xl07, about IxlO8, about 5xl08, about IxlO9, about 5xl09, about IxlO10, about 5xl010, about IxlO11, about 5xl0n, or about IxlO12of the immune effector cells. The composition may comprise a population of the immune effector cells of the invention in the amounts described above.

[0222] The composition may be a pharmaceutical composition. The pharmaceutical composition may comprise a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers comprise aqueous carriers or diluents. Examples of suitable aqueous carriers include water, buffered water and saline.

[0223] The pharmaceutical composition may include one or more pharmaceutically acceptable salts. A "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects. Examples of such salts include acid addition salts and base addition salts.

[0224] The composition may comprise one or more additional therapeutic agent, such as a chemotherapeutic agent. The composition may comprise one or more preservative, such as an anti-fungal and / or anti-viral agent.

[0225] Therapeutic uses and methods

[0226] Also described herein is use of the immune effector cell or population of immune effector cells described herein, in a method of treatment of the human or animal body by therapy, or in a diagnostic method.

[0227] For instance, also provided is a method of treating cancer in a subject, the method comprising administering to the subject an effective amount of an immune effector cell or a population of immune effector cells of the invention.

[0228] Also provided is an immune effector cell or a population of immune effector cells of the invention for use in a method of treating cancer in a subject. Also provided is a use of an immune effector cell or a population of immune effector cells for the manufacture of a medicament for the treatment of cancer in a subject.

[0229] The therapeutic uses and methods may comprise administering a therapeutically effective amount of the immune effector cell or population of immune effector cells to the subject.

[0230] Also provided is a method of formulating a composition for treating cancer, wherein said method comprises mixing an immune effector cell or population of immune effector cells of the invention with an acceptable carrier to prepare said composition.

[0231] The cancer may be glioma, small cell lung cancer, neuroblastoma, glioblastoma, prostate cancer, esophageal cancer, ovarian cancer, endometrial cancer, adrenocortical cancer, renal cancer, skin cancer, neuroendocrine cancer, thyroid cancer, head & neck cancer, stomach cancer, non-small cell lung cancer, colorectal cancer, bladder cancer, liver cancer, melanoma, breast cancer, sarcoma, cervical cancer, pancreatic cancer or testicular cancer.

[0232] The cancer may be glioma, such as malignant glioma. The cancer may be glioblastoma. The cancer may be a recurrent cancer, such as recurrent glioblastoma. The subject may have been previously treated for the cancer, such as using CAR cell (e.g. T- cell) approaches target EGFRvIII, IL13Ra2 and / or Her2. The cancer may be glioblastoma multiforme (GBM). The cancer may be primary glioblastoma or second glioblastoma.

[0233] The subject may be a paediatric subject. The therapeutic uses described herein may therefore be for treating cancer, such as glioma, in a paediatric subject. GD2 gangliosides, and particularly (9AcGD2, are known to be expressed in gliomas of paediatric subjects. Surprisingly, the inventors have also identified that GD2 gangliosides, and particularly (9AcGD2, may also be expressed in a cancer, such as a glioma, in an adult subject. Accordingly, the subject may be an adult subject. The therapeutic uses described herein may therefore be for treating cancer, such as glioma, in an adult subject. The paediatric subject is typically up to the age of 18. The adult subject is typically over the age of 18, such as at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, or at least 65.

[0234] The cancer may be other solid tumours expressing the glioma-associated antigen(s). For example, the cancer may be a solid tumour expressing one or more of a GD2 ganglioside (such as (9AcGD2), TNC, PTPRZ1, BCAN and CSPG4. The cancer may be a solid tumour expressing one or more of a GD2 ganglioside (such as (9AcGD2), B7-H3 and IL13Ra2. The cancer may additionally express one or more of HER2, EGFRvIII, IL13Ra2, PDGFRA, NKG2D, MET, HGF, B7-H3. The cancer may additionally express one or more of TNC, PTPRZ1, BCAN, CSPG4, HER2, EGFRvIII, IL13Ra2, PDGFRA, MET, and B7-H3. The cancer may additionally express one or more of TNC, PTPRZ1, BCAN, CSPG4, IL13Ra2, B7H3, HER2, PDGFRA, CD70, MET, MMP-2 (Metalloprotease), EphA2, CD44, CD 133, CD 147, Mucl, PD-L1, TREM2, and SPP1. The cancer may additionally express one or more of TNC, PTPRZ1, BCAN, CSPG4, IL13Ra2, B7H3, HER2, PDGFRA, CD70, MET, MMP-2 (Metalloprotease), EphA2, CD44, CD 133, CD147, Mucl, PD-L1, TREM2, SPP1, NKG2D, MIC / A, HGF, CD56 and PSMA. The immune effector cells, populations of immune effector cells, CARs and antigen-binding fragments disclosed herein may be used for treating any solid cancers expressing the glioma-associated antigen(s).

[0235] The therapeutic methods and uses may comprise, prior to treatment with an immune effector cell or population of immune effector cells of the invention, determining whether the cancer expresses a glioma-associated antigen specifically targeted by immune effector cell or population of immune effector cells of the invention. For instance, the method may comprise determining whether the cancer expresses a GD2 ganglioside (such as 0AcGD2), PTPRZ1, BCAN, CSPG4 and / or TNC. The method may comprise determining whether the cancer expresses a GD2 ganglioside (such as 0AcGD2), B7-H3 and IL13Ra2. The method may comprise selecting an immune effector cell or population of immune effector cells based on the expression of glioma-associated antigens by the cancer, so that the immune effector cell or population of immune effector cell is specific for the cancer. The method may comprise transfecting or transforming an immune effector cell with a nucleic acid of the invention in response to information on the expression of glioma-associated antigens by the cancer.

[0236] At least 1% of the cancer cells from the tumour or from the individual may express a glioma-associated antigen. The glioma-associated antigen is typically selected from a GD2 ganglioside (such as 0AcGD2), PTPRZ1, BCAN, CSPG4 and / or TNC. The glioma- associated antigen may be selected from a GD2 ganglioside (such as 0AcGD2), B7-H3 and IL13Ra2. For example, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70% or at least 80% of the cancer cells from the tumour or from the individual may express the glioma-associated antigen. The percentage of cells that express the glioma-associated antigen may be determined by any means known to the skilled person, such as immunohistochemistry (H4C), flow-cytometry or enzyme-linked immunosorbent assay (ELISA).

[0237] The expression of the glioma-associated antigen may have an intensity of greater than or equal to (>) 1+, such as > 2+ or > 3+ The intensity score may be assessed by H4C staining of the tumour, with the scoring as follows: negative = no staining or staining in less than or equal to < 10% of the cells stained; 1+ = incomplete staining in > 10% of cells stained; 2+ = weak to moderate staining in > 10% of cells stained; strong and complete staining in > 10% of cells stained.

[0238] The therapeutic methods and uses described herein may comprise inhibiting the disease state (i.e. the cancer), for example by arresting its development and / / or causing regression of the disease state until a desired end point is reached. The therapeutic methods and uses of the invention may comprise achieving a partial response, a full response by the cancer. The therapeutic methods and uses of the invention may achieve remission of the cancer.

[0239] The therapeutic methods and uses described herein may delay the growth of the cancer, arrest the growth of the cancer and / or reverse the growth of the cancer. The therapeutic methods and uses of the invention may reduce the size of the cancer by at least 10%, such as at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or by 100%.

[0240] The therapeutic methods and uses described herein may comprise the induction of a bystander effect. The bystander effect may comprise the killing of cancer cells which do not express a glioma-associated antigen against which at least one of the CARs expressed by the immune effector cells administered to the subject in the method or use is directed.

[0241] Typically, the therapeutic methods and uses are for a human subject in need thereof. However, non-human animals such as non-human mammals are also contemplated. The non-human mammals may be rats, rabbits, sheep, pigs, cows, cats or dogs.

[0242] The dose of the immune effector cell or population of immune effector cells may vary depending on the age and size of a subject, as well as on the disease, conditions and route of administration. The immune effector cell or population of immune effector cells may be administered at a dose of about IxlO6to about IxlO12cells, such as about IxlO6to about IxlO11, about IxlO6to about IxlO10, about IxlO6to about IxlO9, about IxlO7to about IxlO11, about IxlO8to about IxlO10cells. The immune effector cell or population of immune effector cells may be administered at a dose of about IxlO6cells, such as about 5xl06cells, about IxlO7cells, about 5xl07cells, about IxlO8cells, about 5xl08cells, about IxlO9cells, about 5xl09cells, about IxlO10cells, about 5xl010cells, about IxlO11cells, about 5xl0ncells, or about IxlO12cells. The immune effector cell or population of immune effector cells may be administered at a dose of about IxlO5cells / kg to about IxlO11cells / kg, such as about IxlO5cells / kg to about IxlO10cells / kg, about IxlO5cells / kg to about IxlO9cells / kg, about IxlO5cells / kg to about IxlO8cells / kg, about IxlO6cells / kg to about IxlO11cells / kg, about IxlO6cells / kg to about IxlO10cells / kg, about IxlO6cells / kg to about IxlO9cells / kg, about IxlO7cells / kg to about IxlO11cells / kg, about IxlO7cells / kg to about IxlO10cells / kg, or about IxlO7cells / kg to about IxlO9cells / kg, The immune effector cell or population of immune effector cells may be administered at a dose of about IxlO5cells / kg, such as about 5xl05cells / kg, IxlO6cells / kg, 5xl06cells / kg, IxlO7cells / kg, 5xl07cells / kg, IxlO8cells / kg, 5xl08cells / kg, IxlO9cells / kg, 5xl09cells / kg, IxlO10cells / kg, 5xlO10cells / kg, or IxlO11cells / kg.

[0243] The immune effector cell or population of immune effector cells may be administered as a single dose. The immune effector cell or population of immune effector cells may be administered in a multiple dose regimen. For example, the initial dose may be followed by administration of a second or plurality of subsequent doses. The second and subsequent doses may be separated by an appropriate time. For example, the doses may be administered once about every week, once about every 2 weeks, once about every 3 weeks, once about every four weeks, or once about every month.

[0244] The immune effector cell or population of immune effector cells may be administered intravenously. The immune effector cell or population of immune effector cells may be administered intracranially. The immune effector cell or population of immune effector cells may be administered intraventricularly.

[0245] The immune effector cell or population of immune effector cells may be administered with one or more additional therapy, such as one or more additional therapeutic agents. The additional therapeutic agent may be an anti-tumour agent. The additional therapeutic agent may be oncolytic viruses. The additional therapeutic agent may be a CAR-enhancing drug. The additional therapeutic may be an additional immune effector cell.

[0246] Combined administration of the immune effector cell or population with the additional therapeutic agent may be achieved in a number of different ways. All the components may be administered together in a single composition. Each component may be administered separately as part of a combined therapy.

[0247] For example, the immune effector cell or the population of immune effector cells of the invention may be administered before, after or concurrently with the additional therapeutic agent.

[0248] The additional therapy may be chemotherapy, radiotherapy and / or surgery.

[0249] Prior to administration of the immune effector cell or population of immune effector cells of the invention, the subject may undergo lymphodepletion. Lymphodepletion may be achieved via administration to the subject with fluradabine, cyclophosphamide and / or bendamustine. Lymphodepletion may be carried out for at least about one day, such as about 2 days or about 3 days.

[0250] The biological activity and / or therapeutic efficacy of the administered immune effector cell or population of immune effector cells may be measured by known methods. For example, the method may comprise imaging, such as magnetic resonance imaging.

[0251] General definitions

[0252] It is to be understood that different applications of the disclosed CARs, cells, or pharmaceutical compositions of the invention may be tailored to the specific needs in the art. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting.

[0253] In addition, as used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the content clearly dictates otherwise. Thus, for example, reference to “a CAR” includes two or more “CARs”.

[0254] The terms “ comprising” or “ comprises" may be replaced by “ consisting of or “ consisting essentially of, unless the context dictates otherwise.

[0255] It is to be understood that the term “nucleic acid” and the term “polynucleotide” are used interchangeably herein.

[0256] For the purpose of this invention, in order to determine the percent identity of two sequences (such as two nucleic acids or two nucleic acids sequences), the sequences are aligned for optimal comparison purposes (e.g. gaps can be introduced in a first sequence for optimal alignment with a second sequence). The nucleotide or amino acid residues at each position are then compared. When a position in the first sequence is occupied by the same nucleotide or amino acid as the corresponding position in the second sequence, then the nucleotides or amino acids are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions in the reference sequence x 100).

[0257] Typically, the sequence comparison is carried out over the length of the reference sequence. For example, if the user wished to determine whether a given (“test”) sequence is 95% identical to SEQ ID NO: 1, SEQ ID NO: 1 would be the reference sequence. To assess whether a sequence is at least 95% identical to SEQ ID NO: 1 (an example of a reference sequence), the skilled person would carry out an alignment over the length of SEQ ID NO: 1, and identify how many positions in the test sequence were identical to those of SEQ ID NO: 1. If at least 95% of the positions are identical, the test sequence is at least 95% identical to SEQ ID NO: 1. If the sequence is shorter than SEQ ID NO: 1, the gaps or missing positions should be considered to be non-identical positions.

[0258] The skilled person is aware of different computer programs that are available to determine the homology or identity between two sequences. For instance, a comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In an embodiment, the percent identity between two amino acid or nucleic acid sequences is determined using the Needleman and Wunsch (1970) algorithm which has been incorporated into the GAP program in the Accelrys GCG software package (available at http: / / www.accelrys.com / products / gcg / ), using either a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0259] By “specific” or "specifically binds", it is meant that the antigen-binding region of a CAR binds to one or more antigenic determinants of the desired glioma-associated antigen and does not bind to other polypeptides. For example, a CAR specific for PTPRZ1 binds to an antigen of PTPRZ1 but does not bind to an antigen of a different polypeptide such as bovine serum albumin. A CAR may specifically bind if it binds to the desired glioma-associated antigen with a stronger affinity when compared to binding an antigen of a different polypeptide such as bovine serum albumin. Methods for measuring the affinity of binding are well known in the art.

[0260] As used herein, the term “about” may be interpreted to mean a value within + / - 10% of the recited value.

[0261] All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0262] The following examples illustrate the invention.

[0263] Examples

[0264] Materials and Methods

[0265] Flow cytometry-based killing assay

[0266] Target tumor cells were stained using the CellTrace™ Far Red Cell Proliferation Kit (Invitrogen, C34564) and 25’000 cells per well were seeded in a 96-well flat-bottom plate. The tumor cells were incubated for 2h at 37°C with 5% CO2 to allow them to adhere to the plate. Effector RNA CAR-T cells or control mock electroporated (Mock EP) T-cells were added to target cells at different effector-to-target (E:T) ratios. After 72h of coincubation, cells were collected and tumor cell death was measured by flow cytometry (BD FACSymphony™, BD Biosciences) using a LIVE / DEAD™ Fixable Violet Dead Cell Stain Kit (Invitrogen, L34964). Dead tumor cells were identified as LIVE / DEAD+ FarRed+ cells. The following formula was used to calculate the percentage of specific lysis:

[0267] (2)% Specific Lysis=((% Sample lysis - % Baseline lysis)) / ((l 00 - % Baseline lysis))* 100 where “% Sample lysis” corresponds to lysis in the presence of Mock EP or CAR-T cells and “% Baseline lysis” corresponds to lysis in the absence of T cells (tumor cells alone).

[0268] Incucyte-based tumor growth inhibition assay

[0269] Target tumor cells expressing a fluorophore (GFP or mCherry) were seeded in a 96-well plate at a concentration of 5’000 cells / well. Tumor cells were incubated for 2h at 37°C with 5% CO2 to allow adherence to the plate surface. Effector RNA CAR-T cells or control Mock EP T cells were added to target cells at different E:T ratios. Tumor cell growth was monitored for up to 120h by real-time imaging, measuring the total fluorophore area per well every 2h h using an Incucyte® S3 Live-Cell Analysis System (Sartorius, RRID:SCR_023147). The data from each well were normalized to the first measurement (Oh).

[0270] IFN-y detection by ELISA

[0271] IFN-y secreted in the supernatant after 72h of co-culture of tumor and effector T cells was measured using the Human IFN-gamma DuoSet ELISA kit (R&D Systems, DY285B) following the manufacturer's instructions. Absorbance of the ELISA plates at 450 and 540 nm was measured using a Spark® Multimode Microplate Reader (Tecan, RRID:SCR_021897).

[0272] Measure of CAR expression by FACS

[0273] CAR expression must be checked by flow cytometry taking a sample before freezing the cells. To measure the expression of scFv in cell membrane: a) Seed T cells in a 96-well round bottom plate, ~5xl04T cells must be good enough (usually this can be reached with 10-15 pL of T cells suspension after the overnight incubation with IL-2). b) Fill up to 200 pL / well with PBS IX. Centrifuge at 2000 rpm (-550 x g max.) for 3 min, at 4 °C. c) Add a Live-Dead Violet Dye, diluted 1 : 1000 in PBS, 60 pL / well, and resuspend by gently pipetting. Incubate for 15-20 min at 4 °C in the dark. d) Fill up to 200 pL / well with FACS buffer. Centrifuge at 2000 rpm (-550 x g max.) for 3 min, at 4 °C. e) Add the appropriate primary antibody diluted on FACS buffer, 60 pL / well, and resuspend by gently pipetting. Incubate for 15-20 min at 4 °C in the dark. i) Protein L: for most human scFv and some mouse scFv ii) Biotin Anti-Alpaca IgG, VHH specific: for nanobodies iii) Biotin Anti-Human IgG, F(ab')2 fragment specific: for most human scFv iv) Biotin Anti-Mouse IgG, F(ab')2 fragment specific: for most mouse scFv f) Fill up to 200 pL / well with FACS buffer. Centrifuge at 2000 rpm (-550 x g max.) for 3 min, at 4 °C. g) Add the Streptavidin-PE or APC on FACS buffer, 60 pL / well, and resuspend by gently pipetting. Incubate for 15-20 min at 4 °C in the dark. i) Note: Could be any other fluorophore combined with streptavidin. h) Fill up to 200 pL / well with FACS buffer. Centrifuge at 2000 rpm (-550 x g max.) for 3 min, at 4 °C. i) Resuspend cells on FACS fix (320 pL / well) and measure them in the FACS.

[0274] Measure of antigen expression by FACS

[0275] Antigen expression in tumor cells surface was checked by flow cytometry: a) Seed tumor cells in a 96-well round-bottom plate, ~5xl04T cells must be good enough. b) Fill up to 200 pL / well with PBS IX. Centrifuge at 2000 rpm (-550 x g max.) for 3 min, at 4 °C. c) Add a Live-Dead Violet Dye, diluted 1 : 1000 in PBS, 60 pL / well, and resuspend by gently pipetting. Incubate for 15-20 min at 4°C in the dark. d) Fill up to 200 pL / well with FACS buffer. Centrifuge at 2000 rpm (-550 x g max.) for 3 min, at 4 °C. e) Add the appropriate primary binder at 2.5 pg / mL in FACS buffer, 60 pL / well, and resuspend by gently pipetting. Incubate for 15-20 min at 4 °C in the dark. i) VHH 927 conjugated to rabbit Fc: for IL13Ra2 ii) VHH 925 conjugated to rabbit Fc: for B7-H3 iii) scFv h8B6 conjugated to rabbit Fc: for OAcGD2 f) Fill up to 200 pL / well with FACS buffer. Centrifuge at 2000 rpm (-550 x g max.) for 3 min, at 4 °C. g) Add the anti-rabbit_AF647 on FACS buffer, 60 pL / well, and resuspend by gently pipetting. Incubate for 15-20 min at 4 °C in the dark. h) Fill up to 200 pL / well with FACS buffer. Centrifuge at 2000 rpm (-550 x g max.) for 3 min, at 4 °C. i) Resuspend cells on FACS fix (320 pL / well) and measure them in the FACS. Multiplex Immunofluorescence Staining and Image Acquisition

[0276] Multiplex immunofluorescence staining was performed on 4 pm-thick formalin- fixed paraffin-embedded (FFPE) tissue microarray (TMA) sections, comprising 35 cases of WHO grade 4 glioblastoma and 5 normal cerebrum tissues, each represented in duplicate. TMA slides (GL806-L64, TissueArray.com) were processed on the automated Ventana Discovery Ultra platform (Ventana, Roche).

[0277] Slides underwent automated deparaffinization, followed by antigen retrieval at 98°C for 64 minutes and endogenous peroxidase blocking using Discovery Inhibitor for 8 minutes. Multiplex staining was performed in iterative cycles. Each cycle included a blocking step with Discovery Goat IgG and Discovery Inhibitor, incubation with a primary antibody, detection using a horseradish peroxidase (HRP)-conjugated secondary antibody (anti-rabbit or anti-mouse OmniMap HRP, Ventana), signal amplification using an OPAL reactive fluorophore (Akoya Biosciences), and heat-mediated antibody stripping before the next staining round. Fluorophores were covalently deposited, allowing successive rounds of staining without cross-reactivity.

[0278] The staining panel included the following targets and OPAL fluorophores: Tenascin-C (TNC, rabbit polyclonal, OPAL570), Brevican (BCAN, rabbit polyclonal, OPAL690), Protein tyrosine phosphatase receptor type Z1 (PTPRZ1, rabbit polyclonal, OPAL520), 0-acetyl-GD2 (0GD2, mouse monoclonal IgG3, clone 8B6, OPAL480). All primary antibodies were incubated for 1 hour at room temperature. Final nuclear counterstaining was performed using Spectral DAPI (1 : 10 dilution, Akoya Biosciences) for 12 minutes.

[0279] After staining, slides were imaged using the PhenoImager system (Akoya Biosciences), enabling high-resolution multispectral imaging across entire tissue sections.

[0280] Results

[0281] A phage display library was used to screen and select VHHs against the human TNC domain ADI. We obtained six different VHHs (RB895, RB896, RB897, RB898, RB899, and RB900) that showed by ELISA a specific recognition of the human TNC domain ADI while not recognizing a control irrelevant protein (Figure 1). We used the anti-TNC VHHs to generate 2nd generation RNA CAR constructs that contained the intracellular activation domains of human CD28 and CD3(^ molecules. All constructs generated showed a high level of expression of the CAR molecule measured by flow cytometry. A positive control anti- TNC CAR using the scFv R6N against TND domain D was also generated (Figure 2A). To evaluate the killing capacity of the different anti-TNC VHH-based CAR-T cells we incubated them for 72h with a GBM cell line, Ge518. Specific lysis was measured by flow cytometry meanwhile IFN-y secretion was measured by ELISA. The CAR-T cell with the VHH RB898 was the only one that showed a significant killing capacity, measured by both, flow cytometry-specific cell lysis and IFN-y secretion, compared to the rest of the anti-TNC VHH-based CAR-T cells (Figure 2B).

[0282] A trivalent CAR-T cells (3vCAR) co-expressing three different CAR molecules against 0AcGD2 (scFv h8B6), PTPRZ1 (VHH 832) and TNC (scFv R6N) were generated by simultaneous electroporation of the three independent mRNA. The 3vCAR showed a high expression of both VHH-based (Figure 3 A) and scFv-based (Figure 3B) CAR molecules measured by flow cytometry. To evaluate the cytotoxic capacity of the 3vCAR we incubated them, and the three control monovalent CAR-T cells, with different tumor cell lines. Killing activity was measured by flow cytometry and IFN-y secretion was measured by ELISA, additionally the capacity to inhibit tumor growth was measured using a real-time imaging system. 3vCAR showed a significant killing capacity against the brain tumor cell lines PBT- 22FH (Figure 4A), Ge518 (Figure 5 A) and LAN-1 (Figure 6A). In all cases, 3vCAR cytotoxic activity was not only significantly higher than control Mock EP cells but also than anti-PTPRZl (832_28z) CAR-T cells. Additionally, 3vCAR showed higher cytotoxic activity than anti-TNC (R6N_28z) CAR-T cells against PBT-22FH and LAN-1, and higher than anti-OAcGD2 (h8B6_IgGlH_28z) CAR-T cells against Ge518. The 3vCAR also was capable of producing a significant amount of IFN-y against PBT-22FH (Figure 4B), Ge518 (Figure 5B) and LAN-1 (Figure 6B). Similar to the killing capacity, the IFN-y secretion of the 3vCAR was significantly higher against the three cell lines in comparison to the Mock EP T cells and anti-PTPRZl CAR-T cells, and higher than anti-0 AcGD2 CAR-T cells against Ge518. higher in all conditions than anti-TNC CAR-T cells. In the case of anti-TNC CAR-T cells, the 3vCAR was able to produce a higher amount of IFN-y not only against PBT-22FH and LAN-1 but also against Ge518. The capacity of the 3vCAR to inhibit tumor growth, as a complementary measure of its anti-tumor activity, was measured using a realtime image assay. The 3vCAR showed the same tumor inhibition of anti-TNC and anti- 0AcGD2 CAR-T cells against the PBT-22FH cell line (Figure 7A), and significantly higher than anti-PTPRZl CAR-T cells or control Mock EP T cells. Any of the evaluated CAR-T cells showed tumor growth inhibition against the LAN-1 cell line (Figure 7B). We observed significant tumor growth inhibition by the 3vCAR, at the same level as the monovalent CAR- T cells, against the Ge518_PTPRZl-KI cell line (Figure 8 A). Also, we observed a significant tumor growth inhibition by the 3vCAR against the Ge518 cell line, similar to the anti-TNC and anti-0 AcGD2 CAR-T cells, but higher than anti-PTPRZl CAR-T cells (Figure 8B). The 3vCAR demonstrated to have equal or higher cytotoxic capacity than all monovalent CAR- T cells, with a wide range of targets that make it capable of killing a more heterogeneous tumor cell population.

[0283] A trivalent CAR-T cells (Triple CAR) co-expressing three different CAR molecules against 0AcGD2 (scFv h8B6), PTPRZ1 (scFv 471) and CSPG4 (scFv 301) were generated by simultaneous electroporation of the three independent mRNA. The co-expression of the different CAR molecules was measured in pairs by flow cytometry (Figure 9). We observed a high expression of anti-CSPG4 CAR (-94%) and medium levels for the anti-PTPRZl (46- 49%) and anti-0 AcGD2 (-30%). At least 26% of T cells express all three CARs (Figure 9C) and at least another 20% co-express anti-PTPRZl and anti-CSPG4 CAR molecules (Figure 9A). To evaluate the cytotoxic activity of the Triple CAR we incubated CAR-T cells, including control monovalent CAR-T cells, with the GBM cell lines Ge518 (Figure 10A) and Ge518_PTPRZl-KI (Figure 10B). In both cases, the Triple CAR showed a significant killing capacity compared to Mock EP T cells and the monovalent anti-CSPG4 (301_IgGlH_28z) and anti-OAcGD2 (8B6_28z) CAR-T cells. Additionally, we evaluated the capacity to inhibit tumor growth against the Ge518_PTPRZl-KI using a real-time imagining assay (Figure 10C). The Triple CAR showed a statistically higher capacity to inhibit tumor growth compared to Mock EP T cells and the monovalent anti-CSPG4 and anti-0 AcGD2, and a similar level compared to anti-PTPRZl (471_28z) CAR-T cells.

[0284] A trivalent CAR-T cell (Triple CAR / Triple CAR co-EP) co-expressing three different CAR molecules against OAcGD2 (scFv h8B6), B7-H3 (VHH 925) and IL13Ra2 (VHH 927) was generated by simultaneous electroporation of the three independent mRNAs. The Triple CAR showed a high expression of both VHH-based (Figure 11 A) and scFv-based (Figure 1 IB) CAR molecules, as measured by flow cytometry. To evaluate cytotoxic capacity, the Triple CAR, the three monovalent control CAR-T cells or a pool of the three monovalent CAR-T cells (Triple CAR pool) were incubated with different tumor cell lines: Ge518 (glioblastoma), LN-229 (glioblastoma), LAN-1 (neuroblastoma), MDA- MB-231 (breast cancer), A375 (melanoma) and SKOV-3 (ovarian cancer). Killing activity was measured by flow cytometry and the capacity to inhibit tumor growth was measured using a real-time imaging system (Incucyte). The Triple CAR showed a significant better killing capacity profile against tumor cell lines Ge518, LN-229, LAN-1, MDA-MB-231 and A375 in comparison to monovalent anti-IL13Ra2 CAR- T cells (Figure 12, cytometrybased killing, and Figure 13, Incucyte-based tumor growth inhibition), anti-B7-H3 CAR- T cells (Figure 14, cytometry-based killing, and Figure 15, Incucyte-based tumor growth inhibition), anti-OAcGD2 CAR- T cells (Figure 16, cytometry-based killing, and Figure

[0285] 17, Incucyte-based tumor growth inhibition) and the trivalent pool of CAR- T cells (Figure

[0286] 18, cytometry-based killing, and Figure 19, Incucyte-based tumor growth inhibition).

[0287] None of the CAR-T cells exhibited specific lysis against the SKOV-3 cell line. The expression of the three relevant antigens IL13Ra (Figure 20A), B7-H3 (Figure 20B) and 0AcGD2 (Figure 20C) was measured by flow cytometry using the corresponding VHHs or scFv conjugated to a rabbit Fc.

[0288] A bivalent CAR-T cell (PTPRZl-OAcGD2 co-EP) co-expressing two different CAR molecules against 0AcGD2 (scFv h8B) and PTPRZ1 (VHH832) was generated by simultaneous electroporation of the two independent mRNAs. To evaluate cytotoxic capacity, the PTPRZl-OAcGD2 co-EP and the two monovalent control CAR-T cells were incubated with different glioblastoma cell lines: Ge518 (PTPRZ1 0AcGD2+) (Figure 21A, cytometry-based killing, and Figure 22A, Incucyte-based tumor growth inhibition), Ge738_PTPRZl-KI (PTPRZl+OAcGD2 )(Figure 2 IB, cytometry-based killing, and Figure 22B, Incucyte-based tumor growth inhibition) and Ge518_PTPRZl-KI (PTPRZl+OAcGD2+)(Figure 21C, cytometry-based killing, and Figure 22C, Incucyte- based tumor growth inhibition). Killing activity was measured by flow cytometry, and the capacity to inhibit tumor growth was measured using a real-time imaging system (Incucyte). The PTPRZl-0AcGD2 co-EP CAR-T cell shows a better or equal killing capacity than the monovalent control CAR-T cells. The PTPRZl-OAcGD2 co-EP CAR-T cell was also capable of specific lysis against the three cell lines tested. In contrast, the monovalent control CAR-T cells were unable to kill the cell lines that didn’t express their respective antigen.

[0289] Tumor associated antigen expression in healthy brain and glioblastoma: Multiplex IF study

[0290] Tumor-associated antigen (TAA) expression in glioblastoma (GBM) demonstrates substantial heterogeneity, both across patients and within individual tumors. To identify the optimal combination of TAAs for maximizing tumor cell coverage across the largest number of patients, the expression patterns of 0AcGD2, PTPRZ1, TNC, and BCAN were analysed.

[0291] Multiplex immunofluorescence staining (IF) was performed on a formalin-fixed paraffin-embedded (FFPE) tissue microarray (TMA) consisting of 35 WHO grade 4 GBM cases and 5 normal cerebrum samples, each represented in duplicate. A threshold-based classification approach was used to categorize individual cells as positive or negative for each TAA. TAA coverage was evaluated based on the frequency of cells expressing one or more of the antigens.

[0292] Among the 35 GBM cases, 58% of tumors overexpressed BCAN, 83% TNC, 68% PTPRZ1, and 63% 0AcGD2. Notably, over 98% of tumors expressed at least one of the four TAAs. At the single-cell level, BCAN was overexpressed in an average of 28% of tumor cells, TNC in 41%, PTPRZ1 in 36%, and 0AcGD2 in 16%. Simultaneous targeting of all four TAAs would enable coverage of over 60% of tumor cells, supporting the rationale for a multivalent CAR-T approach in GBM (Figure 23).

[0293] TAA expression across different cancers was investigated. The expression of OAcGD2 in cases / samples for a range of different cancers was determined. These data were compared to expression of B7-H3, PTPRZ1, TNC, BCAN, CSPG4, IL13Ra2, HER2, EGFR, MET and PDGFRA as derived from patent / literature sources (Figure 24) or the Human Protein Atlas (Figure 25). Informal Sequence Listing

[0294] CDRs of SEQ ID NOs: 1-3, 31-38, 62-65, 86, 87, 171 and 175 are annotated as follows: CDR1 (underlined), CDR2 (bold and underlined) and CDR3 (italicised and underlined}. CDRs were predicted with the Benchling’s Antibody Property Prediction Tools, where annotations were assigned using the North CDR definition of the Sequence-based antibody CAnonical LOoP (SCALOP) structure annotation developed by the Oxford Protein Informatics Group (Dunbar etal, SAbPred: a structure-based antibody prediction server, Nucleic Acids Research, Volume 44, Issue Wl, 8 July 2016, Pages W474-W478, https: / / doi.org / 10.1093 / nar / gkw361)

[0295] SEQ ID NOs: 4-9, 39-41, 66-69, and 88-94 are scFv in orientation VH-linker-VL with the linker (G4S)s in lowercase. SEQ ID NO: 118 are scFv in orientation VL-linker-VH with the linker (G4S)s in lowercase. Antigen binding regions are highlighted in underlined (ABR1), bold and underlined (ABR2) and underlined and italicised (ABR3}. All ABRs are predicted using Paratome (Kunik V et al (2012). Structural Consensus among Antibodies Defines the Antigen Binding Site. PLoS Comput Biol 8(2): el002388. doi: 10.1371 / joumal.pcbi, 1002388; and Kunik V et al (2012). Paratome: An online tool for systematic identification of antigen binding regions in antibodies based on sequence or structure. Nucleic Acids Res. 2012 Jul;40(Web Server issue):W521-4. doi: 10.1093 / nar / gks480. Epub 2012 Jun 6).

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Claims

Claims1. An immune effector cell or a population of immune effector cells expressing one or more chimeric antigen receptors (CARs) specific for two or more glioma-associated antigens, wherein one or more of the glioma-associated antigens is a GD2 ganglioside.

2. An immune effector cell or a population of immune effector cells according to claim 1, wherein the GD2 ganglioside is an O-acetyl-GD2 ganglioside (< AcGD2).

3. An immune effector cell or a population of immune effector cells according to claim 1 or 2, which expresses one or more CARs specific for three or more glioma- associated antigens, optionally which expresses one or more CARs specific for four or more, or five or more glioma-associated antigens.

4. An immune effector cell or a population of immune effector cells according to any one of the preceding claims, wherein the two or more glioma-associated antigens comprise a GD2 ganglioside and one or more glioma-associated antigens selected from PTPRZ1, BCAN, CSPG4 and TNC, optionally wherein the two or more glioma associated antigens comprise a GD2 ganglioside and two or more, three or more, or all four glioma-associated antigens selected from PTPRZ1, BCAN, CSPG4 and TNC.

5. An immune effector cell or a population of immune effector cells according to any one of the preceding claims, wherein the two or more glioma-associated antigens comprise a GD2 ganglioside and IL13Ra2, optionally wherein the two or more glioma associated antigens comprise a GD2 ganglioside, IL13Ra2 and B7-H3.

6. An immune effector cell or a population of immune effector cells according to any one of the preceding claims, wherein the cell or each cell expresses two or more CARs specific for different glioma-associated antigens, optionally wherein the cell or each cell expresses three or more, four or more, or five or more CARs specific for different glioma- associated antigens.

7. An immune effector cell or a population of immune effector cells according to any one of the preceding claims:(a) which comprises at least two different CAR-expressing immune effector cells, wherein each different CAR-expressing immune effector cell is specific for a different glioma-associated antigen, optionally which comprises at least three, at least four or at least five different CAR-expressing immune effector cells, wherein each different CAR-expressing immune effector cell is specific for a different glioma-associated antigen; or(b) wherein one or more of the CARs is a multivalent CAR specific for two or more glioma-associated antigens, wherein one or more of the glioma-associated antigens is a GD2 ganglioside, optionally wherein one or more of the glioma-associated antigens is (i) selected from PTPRZ1, BCAN, CSPG4 and TNC, and / or (ii) selected from IL13Ra2 and B7-H3.

8. An immune effector cell or a population of immune effector cells according to any one of the preceding claims, wherein:(a) the one or more CARs specific for a GD2 ganglioside comprise an amino acid sequence comprising the complementary determining regions (CDRs) from the amino acid sequence of SEQ ID NO: 118;(b) the one or more CARs specific for PTPRZ1 comprise an amino acid sequence comprising the CDRs from an amino acid sequence selected from any one of SEQ ID NOs: 1 to 9;(c) the one or more CARs specific for TNC comprise an amino acid sequence comprising the CDRs from an amino acid sequence selected from any one of SEQ ID NOs: 31 to 41;(d) the one or more CARs specific for BCAN comprise an amino acid sequence comprising the CDRs from an amino acid sequence selected from any one of SEQ ID NOs: 62 to 69;(e) the one or more CARs specific for CSPG4 comprise an amino acid sequence comprising the CDRs from an amino acid sequence selected from any one of SEQ ID NOs: 86 to 94;(f) the one or more CARs specific for IL13Ra2 comprise an amino acid sequence comprising the CDRs from SEQ ID NO: 176; and / or(g) the one or more CARs specific for B7-H3 comprise an amino acid sequence comprising the CDRs from SEQ ID NO: 172.

9. An immune effector cell or a population of immune effector cells according to any one of the preceding claims, wherein:(a) the one or more CARs specific for a GD2 ganglioside comprise an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 118;(b) the one or more CARs specific for PTPRZ1 comprise an amino acid sequence having at least 80% identity to the amino acid sequence of any one of SEQ ID NOs: 1 to 9;(c) the one or more CARs specific for TNC comprise an amino acid sequence having at least 80% identity to the amino acid sequence of any one of SEQ ID NOs: 31 to 41;(d) the one or more CARs specific for BCAN comprise an amino acid sequence having at least 80% identity to the amino acid sequence of any one of SEQ ID NOs 62 to 69;(e) the one or more CARs specific for CSPG4 comprise an amino acid sequence having at least 80% identity to the amino acid sequence of any one of SEQ ID NOs: 86 to 94;(f) the one or more CARs specific for IL13Ra2 comprise an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 176; and / or(g) the one or more CARs specific for B7-H3 comprise an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 172.

10. An immune effector cell or a population of immune effector cells according to any one of the preceding claims, wherein:(a) the one or more CARs specific for a GD2 ganglioside comprise an amino acid sequence comprising the CDRs from SEQ ID NO: 118, optionally which comprise an amino acid sequence having at least 80% identity to the amino acid sequence SEQ ID NO: 118;(b) the one or more CARs specific for PTPRZ1 comprise an amino acid sequence comprising the CDRs from SEQ ID NO: 1, optionally which comprise an amino acid sequence having at least 80% identity to the amino acid sequence SEQ ID NO: 1;(c) the one or more CARs specific for TNC comprise an amino acid sequence comprising the CDRs from SEQ ID NO: 34, optionally which comprise an amino acid sequence having at least 80% identity to the amino acid sequence SEQ ID NO: 34;(d) the one or more CARs specific for BCAN comprise an amino acid sequence comprising the CDRs from SEQ ID NO: 62, optionally which comprise an amino acid sequence having at least 80% identity to the amino acid sequence SEQ ID NO: 62;(e) the one or more CARs specific for CSPG4 comprise an amino acid sequence comprising the CDRs from SEQ ID NO: 86, optionally which comprise an amino acid sequence having at least 80% identity to the amino acid sequence SEQ ID NO: 86;(f) the one or more CARs specific for IL13Ra2 comprise an amino acid sequence comprising the CDRs from SEQ ID NO: 176, optionally which comprise an amino acid sequence having at least 80% identity to the amino acid sequence SEQ ID NO: 176; and / or(g) the one or more CARs specific for B7-H3 comprise an amino acid sequence comprising the CDRs from SEQ ID NO: 172, optionally which comprise an amino acid sequence having at least 80% identity to the amino acid sequence SEQ ID NO: 172.

11. An immune effector cell or a population of immune effector cells according to any one of the preceding claims, which expresses one or more CARs specific for three or more glioma-associated antigens, wherein the three or more glioma-associated antigens comprise OAcGD2, PTPRZ1 and TNC.

12. An antigen-binding molecule specific for TNC, which comprises a polypeptide comprising the complementary determining regions (CDR1, CDR2 and CDR3) from an amino acid sequence selected from any one of SEQ ID NOs: 31 to 36, optionally which comprises a polypeptide comprising an amino acid sequence having at least 80% identity to the amino acid sequence of any one of SEQ ID NOs: 31 to 36.

13. An antigen-binding molecule specific for IL13Ra2, which comprises a polypeptide comprising the complementary determining regions (CDR1, CDR2 and CDR3) from SEQ ID NO: 176, optionally which comprises a polypeptide comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 176.

14. An antigen-binding molecule specific for B7-H3, which comprises a polypeptide comprising the complementary determining regions (CDR1, CDR2 and CDR3) from SEQ ID NO: 172, optionally which comprises a polypeptide comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 172.

15. A CAR compri sing :(a) an antigen-binding molecule according to any one of claims 12 to 14; and / or(b) an antigen-binding domain specific for a GD2 ganglioside, wherein the CAR comprising the antigen-binding domain specific for a GD2 ganglioside comprises:(i) an antigen binding domain comprising the CDRs of SEQ ID NO: 118; optionally which comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 118;(ii) a hinge, optionally wherein the hinge comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 138;(iii) a transmembrane domain, optionally wherein the transmembrane domain comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 140; and(iv) an intracellular domain, optionally wherein the intracellular domain comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 142 and / or an amino acid sequence having at least 80% identity to SEQ ID NO: 145.

16. An immune effector cell or a population of immune effector cells comprising a CAR according to claim 15.

17. An immune effector cell or a population of immune effector cells according to any one of claims 1 to 11 and 16, wherein the cell or cells are T cells, NK cells, iPSC-NK cells, y5 T cells, phagocytes or macrophages.

18. An immune effector cell or a population of immune effector cells according to any one of claims 1 to 11, 16 and 17 for use in a method of treating cancer in a subject, optionally wherein the cancer is glioma.

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