Chimeric Antigen Receptor

Abstract

The present disclosure relates to chimeric antigen receptors (CARs) specific for one or more glioma-associated antigens, and immune effector cells or populations of immune effector cells expressing one or more CARs specific for one or more glioma-associated antigens. The present disclosure also relates to methods for producing the immune cells or populations, and methods for treating cancer using the immune cells or populations.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present disclosure relates to immune effector cells expressing one or more chimeric antigen receptors and their use in the treatment of glioma. [Background technology]

[0002] Background of the Invention Immunotherapeutic approaches to induce tumor-specific immune responses have been investigated across multiple malignant tumors.For example, therapeutic T cells can be engineered to direct their cytotoxicity to specific antigens of interest.In this way, the T cells responsible for killing tumor cells can be engineered to be specific to tumor antigens.The specificity of T cells is directed by endogenous T cell receptors or recombinant T cell receptors, or chimeric antigen receptors (CARs).

[0003] CARs are synthetic receptors that often contain an extracellular domain derived from an antibody single-chain variable fragment (scFv) and intracellular signaling and costimulatory domains derived from T cells. Genetic insertion of CARs into immune cells allows them to target antigens of interest. Anti-CD19 CAR T cells have revolutionized cancer therapy, demonstrating high response rates in adult patients with relapsed / refractory diffuse large B-cell lymphoma (DLBCL) and pediatric patients with refractory B-cell acute lymphoblastic leukemia (B-ALL). Two CAR T cell products specific for the B-cell marker CD19, Kymriah (Novartis) and Yescarta (Kite Pharma), were approved by the FDA as the first therapeutic products containing genetically engineered components for the treatment of B-ALL and DLBCL.

[0004] Glioblastoma (GBM, grade IV astrocytoma) is the most common and aggressive primary malignant tumor of the brain. Despite combined treatment with surgery, chemotherapy, and radiation, the overall survival of GBM patients ranges from 14.6 to 16 months after treatment. Several phase I / II trials using multipeptide vaccines and neoadjuvant immune checkpoint inhibitors have been attempted, but to date, success has been limited. Currently, CAR T cell approaches targeting EGFRvIII, IL-13Rα2, and Her2 are being investigated for GBM (Brown et al., New England Journal of Medicine 375.26(2016):2561-2569; Ahmed et al., JAMA Oncology 3.8(2017):1094-1101; O'Rourke et al., Science Translational Medicine 9.399(2017):eaaa0984).

[0005] It is an object of the present invention to further develop glioma antigen-specific CARs and improved immune effector cells expressing CARs that are useful in the treatment of gliomas. Summary of the Invention

[0006] Summary of the Invention The present invention provides an immune effector cell or population of immune effector cells that express one or more chimeric antigen receptors (CARs) specific for two or more glioma-associated antigens, wherein one or more of the glioma-associated antigens are selected from PTPRZ1, BCAN, CSPG4, and TNC.

[0007] The present invention further provides an immune effector cell or population of immune effector cells that express a chimeric antigen receptor (CAR) specific for PTPRZ1. The present invention also provides an immune effector cell or population of immune effector cells that express a chimeric antigen receptor (CAR) specific for BCAN.

[0008] The present invention also provides a method of producing an immune effector cell or population of immune effector cells of the present invention, comprising transforming said cell or said population of cells with one or more nucleic acids encoding one or more CARs specific for one or more glioma-associated antigens.

[0009] The present invention further provides a method of treating cancer in a subject, comprising administering to the subject an effective amount of an immune effector cell or population of immune effector cells of the invention. The present invention also provides an immune effector cell or population of immune effector cells of the invention for use in a method of treating cancer in a subject.

[0010] The present invention further provides a CAR specific to PTPRZ1. The present invention also provides a CAR specific to BCAN. The present invention also provides a CAR specific to a glioma-associated antigen selected from PTPRZ1, BCAN, CSPG4, and TNC, comprising a polypeptide having (a) heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) and light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) from an amino acid sequence selected from any one of SEQ ID NOs: 21 to 40, or (b) complementary determining regions (CDR1, CDR2, and CDR3) from an amino acid sequence selected from any one of SEQ ID NOs: 83 to 93.

[0011] The present invention also provides a multivalent CAR comprising: (a) an extracellular domain specific for two or more glioma-associated antigens, wherein one or more of the glioma-associated antigens are selected from PTPRZ1, BCAN, CSPG4, and / or TNC; and (b) an intracellular signaling domain.

[0012] The present invention also provides nucleic acids encoding a CAR or multivalent CAR of the present invention. Vectors comprising one or more of said nucleic acids are also provided by the present invention.

[0013] The present invention also provides antigen-binding molecules specific for one or more glioma-associated antigens, comprising a polypeptide having complementarity-determining regions (CDR1, CDR2, and CDR3) selected from an amino acid sequence selected from any one of SEQ ID NOs: 83 to 93. [Brief explanation of the drawings]

[0014] [Figure 1] Generation of PTPRZ1-overexpressing GBM cell lines. A) The GBM cell line Ge518 was transfected with third-generation lentivirus to introduce extracellular domains 1 and 2 of human PTPRZ1. Antigen expression in Ge518 wt (light blue) and Ge518_PTPRZ1-KI (dark blue) was measured by flow cytometry. B) Staining of Ge518_PTPRZ1-KI by flow cytometry using six different anti-PTPRZ1 scFvs. [Figure 2] Killing activity of anti-PTPRZ1_BBz RNA CAR T cells against Ge518_PTPRZ1-KI cells. A) CAR expression of six different anti-PTPRZ1_BBz and control anti-IL13Rα2_BBz RNA CAR T cells. NTD: non-transduced T cells. B) Killing activity of anti-PTPRZ1_BBz RNA CAR T cells against Ge518_PTPRZ1-KI tumor cells measured as the percentage of specific lysis by flow cytometry. E:T ratio = effector to target ratio. [Figure 3] Comparison of the killing ability of BBz versus 28z variants of anti-PTPRZ1 RNA CAR T cells. A) Killing activity of anti-PTPRZ1 RNA CAR T cells containing scFv 473 and 476, and 28z or BBz against Ge518_PTPRZ1-KI tumor cells, measured as the percentage of specific lysis by flow cytometry. E:T ratio = effector to target ratio. B) Comparison of the killing activity of anti-PTPRZ1 scFv 471 28z and BBz RNA CAR T cells against Ge518_PTPRZ1-KI tumor cells at two different E:T ratios. [Figure 4]Differences in killing activity of three different anti-PTPRZ1_28z RNA CAR T cells. Measurement of the killing ability of anti-PTPRZ1 28z RNA CAR T cells containing 470, 471, and 476 scFv against Ge518_PTPRZ1-KI tumor cells at three different E:T ratios. E:T ratio = effector to target ratio. [Figure 5] Killing activity of anti-CSPG4_BBz RNA CAR T cells. A) Cell surface expression of six different anti-CSPG4_BBz CAR molecules and a control anti-IL13Rα2_BBz RNA CAR. NTD: non-transduced T cells. B) Killing activity of anti-CSPG4_BBz RNA CAR T cells against A375 melanoma cells, measured as the percentage of specific lysis by flow cytometry. E:T ratio = effector-to-target ratio. C) Killing activity of anti-CSPG4_BBz RNA CAR T cells against Ge518GBM cells. D) IFN-γ secretion in the supernatant of anti-CSPG4_BBz RNA CAR T cells incubated with Ge518GBM cells. [Figure 6] Generation of Ge518 variants with knockout of IL13Rα2, Her2, and CSPG4 antigens. A) Expression of IL13Rα2, Her2, and CSPG4 antigens was eliminated in Ge518 cells using the CRISPR-Cas9 system. Loss of antigen expression was confirmed by flow cytometry. B) The killing ability of wt Ge518 cell line and Ge518 KO cell line cognate RNA BBz CAR T cells was evaluated by flow cytometry at an E:T ratio of 3:1. [Figure 7] Cytotoxic activity of three anti-GBM RNA CAR T cell combinations against a heterogeneous mix of tumor cells. A) Schematic of the mechanism behind the superior killing and reduced risk of antigen loss escape by different anti-GBM CAR T cell mixes compared to monospecific CAR T cells. B) Killing capacity of different mixes of anti-CSPG4_BBz, anti-Her2_BBz, and anti-IL13Rα2_BBz CAR T cells against a heterogeneous mix of three different Ge518 KO cells (IL13Rα2-KO, Her2-KO, and CSPG4-KO) and Ge518 wt cells. E:T ratio = effector:target ratio. [Figure 8] Generation of triple RNA CAR-T cells and in vitro killing assay. (A) CAR expression in RNA-CAR T cells expressing anti-PTPRZ1 (471_28z), anti-CSPG4 (301_28z), and anti-BCAN (295_28z). (B) In vitro killing of Ge518, Ge518_BCANv2™-KI, and Ge518_PTPRZ1-KI cell lines by triple CAR or individual monovalent CAR-T cells at E:T ratios of 3:1 and 1:1. Mock EP cells served as a negative control. (C) In vitro killing of a mix of Ge518, Ge518_BCANv2™-KI, and Ge518_PTPRZ1-KI cell lines (2:2:1 ratio) by triple CAR or individual monovalent CAR-T cells at E:T ratios of 3:1 and 1:1. Mock EP cells served as a negative control. (D) In ​​vitro tumor growth inhibition of Ge518, Ge518_BCANv2TM-KI, and Ge518_PTPRZ1-KI cell lines by triple CAR or individual monovalent CAR-T cells was measured using an Incucyte™. The E:T ratio was 3:1. Mock EP cells were used as a negative control. (E) In vitro tumor growth inhibition of a mix of Ge518, Ge518_BCANv2TM-KI, and Ge518_PTPRZ1-KI cell lines (2:2:1 ratio) by triple CAR or individual monovalent CAR-T cells was measured using an Incucyte™. The E:T ratio was 3:1. Mock EP cells were used as a negative control. [Figure 9]471_28z CAR-T cells did not exhibit bystander killing against non-tumor-bearing human macrophages. (A) CD14+ monocytes were purified from human blood on day 0 and differentiated into macrophages by culturing with M-CSF for 6 days. On day 6, Ge518_PTPRZ1-KI cells (FarRed stained) were added to the macrophage culture, followed by the addition of anti-PTPRZ1 RNA 471_28z CAR-T cells or mock EP control cells at an E:T ratio of 3:1. After 72 hours, cells were harvested, and tumor and macrophage cell killing were assessed by FACS. (B) CAR expression in anti-PTPRZ1 RNA 471_28z CAR-T cells. (C) Gating strategy to distinguish tumor Ge518_PTPRZ1-KI cells (CD14- FarRed+) from human macrophages (CD14+ FarRed-) from wells treated with mock EP (left) or 471_28z CAR-T cells (right). (D) In ​​vitro evaluation of killing of Ge518_PTPRZ1-KI or human macrophages by anti-PTPRZ1 RNA 471_28z CAR-T cells. [Figure 10] 471_28z CAR-T cells exhibit bystander killing via soluble mediators. (A) On day 0, Ge518_PTPRZ1-KO cells were seeded in the bottom of a transwell plate, while Ge518_PTPRZ1-KI cells were seeded in the top of the transwell. On day 1, anti-PTPRZ1 RNA 471_28z CAR-T cells or mock EP control cells were added to the top of the transwell at an E:T ratio of 5:1. After 72 hours, cells in the bottom of the well were collected, and cell killing was assessed by FACS. (B) Assessment of indirect soluble mediator-dependent killing of Ge518_PTPRZ1-KO cells (stained with yellow dye) by anti-PTPRZ1 RNA 471_28z CAR-T cells. (C) The bottom of the well was confirmed to be free of Ge518_PTPRZ1-KI cells (stained with FarRed) or T cells (CD3+). [Figure 11]Correlation analysis between different antigens. (A) and (B) Correlation between antigens. Below the diagonal: scatter plot with regression line. Central diagonal: density plot. Above the diagonal: significant Pearson correlation coefficients: ***<0.001, **<0.01, *<0.05. Test statistics are based on Pearson's product-moment correlation coefficient. (A) Expression data from TCGA and bulk RNA-seq (primary GBM). (B) Expression data from CGGA (recurrent GBM). (C) Heatmap of expression data from TCGA and bulk RNA-seq (primary GBM) showing Pearson correlation coefficients between antigens. [Figure 12] Anti-PTPRZ1 VHH isolation, RNA CAR-T cell generation, and in vitro killing assay. (A) ELISA recognition of the extracellular domain of PTPRZ1 by different anti-PTPRZ1 VHHs. (B) Recognition of Ge518_PTPRZ1-KI and Ge738 cell lines by anti-PTPRZ1 VHHs conjugated to the human IgG1 Fc domain (5 μg / mL for each VHH-Fc). (C) CAR expression in RNA-CAR T cells expressing different anti-PTPRZ1 VHHs, RB832 and RB833, which harbor short (_28z) or long (_IgG1H_28z) hinges followed by CD28 and CD3ζ domains. (D) In ​​vitro killing of Ge518_PTPRZ1-KI and Ge738 cell lines by four different VHH anti-PTPRZ1 RNA CAR-T cells measured by flow cytometry at an E:T ratio of 3:1. Anti-PTPRZ1 RNA CAR T cells based on scFv RRB471 were used as a positive control, while Mock EP cells were used as a negative control. (E) In vitro tumor growth inhibition of Ge518_PTPRZ1-KI and Ge738 cell lines by four different VHH anti-PTPRZ1 RNA CAR-T cells was measured in an Incucyte system at an E:T ratio of 3:1. Anti-PTPRZ1 RNA CAR T cells based on scFv RRB471 were used as a positive control, while Mock EP cells were used as a negative control. [Figure 13]Isolation of anti-CSPG4 VHHs, generation of RNA CAR-T cells, and in vitro killing assays. (A) ELISA recognition of the extracellular domain of CSPG4 by different anti-CSPG4 VHHs. (B) Recognition of A375 and Ge738 cell lines by anti-CSPG4 VHHs conjugated to the human IgG1 Fc domain (10 μg / mL of each VHH-Fc). (C) CAR expression in RNA-CAR T cells expressing anti-CSPG4 VHH RB830, which contains a short (_28z) or long (_IgG1H_28z) hinge followed by CD28 and CD3ζ domains. (D) In ​​vitro killing of A375, Ge518, and Ge738 cell lines by two VHH anti-CSPG4 RB830 RNA CAR-T cells measured by flow cytometry at an E:T ratio of 3:1. Anti-CSPG4 RNA CAR T cells based on scFv HRB301 were used as a positive control, while Mock EP cells were used as a negative control. (E) In vitro tumor growth inhibition of A375, Ge518, and Ge738 cell lines by two VHH anti-CSPG4 RB830 RNA CAR-T cells was measured in an Incucyte system at an E:T ratio of 5:1. Anti-CSPG4 RNA CAR T cells based on scFv HRB301 were used as a positive control, while Mock EP cells were used as a negative control. [Figure 14]Anti-tenascin-C VHH isolation, RNA CAR-T cell generation, and in vitro killing assay. (A) ELISA recognition of the extracellular domain of tenascin-C by different anti-tenascin-C VHHs. (B) Recognition and intracellular staining of Ge518 and Ge738 cell lines by anti-tenascin-C VHHs conjugated with a human IgG1 Fc domain (each VHH-Fc at 10 μg / mL). (C) CAR expression in RNA-CAR T cells expressing different anti-tenascin-C VHHs, RB835 and RB836, carrying short (_28z) or long (_IgG1H_28z) hinges followed by CD28 and CD3ζ domains. (D) In ​​vitro killing of Ge518 and Ge738 cell lines by two different VHH anti-tenascin-C RNA CAR-T cells measured by flow cytometry. E:T ratio was 3:1. Anti-tenascin-C RNA CAR T cells based on scFv R6N were used as a positive control, while Mock EP cells were used as a negative control. (E) In vitro tumor growth inhibition of Ge518 and Ge738 cell lines by two different VHH anti-tenascin-C RNA CAR-T cells was measured in an Incucyte system at an E:T ratio of 5:1. Anti-tenascin-C RNA CAR T cells based on scFv R6N were used as a positive control, while Mock EP cells were used as a negative control. [Figure 15]Isolation of anti-BCAN VHHs, generation of RNA CAR-T cells, and in vitro killing assays. (A) ELISA recognition of BCAN by different anti-BCAN VHHs containing short (_28z) or long (_IgG1H_28z) hinges followed by CD28 and CD3ζ domains. (B) CAR expression in RNA-CAR T cells expressing different anti-BCAN VHHs, RB826, RB827, RB828, and RB829. (C) In vitro killing of the Ge518_BCANv2TM-KI cell line by eight different VHH anti-BCAN RNA CAR-T cells measured by flow cytometry. E:T ratio was 3:1. Anti-BCAN RNA CAR T cells based on scFv HRB295 were used as a positive control, while Mock EP cells were used as a negative control. (D) In ​​vitro tumor growth inhibition of the Ge518_BCANv2TM-KI cell line by eight different VHH anti-BCAN RNA CAR-T cells was measured in an Incucyte™ system at an E:T ratio of 5:1. Anti-BCAN RNA CAR T cells based on scFv HRB295 were used as a positive control, while Mock EP cells were used as a negative control. [Figure 16] Bispecific RNA CAR-T cells based on the combination of anti-PTPRZ1 VHH RB832 with a second CAR against CSPG4, BCAN, or tenascin-C. The bar graphs in the left column show the improved killing ability of the bispecific RNA CAR-T cells compared to the monovalent anti-PTPRZ1 CAR against cell lines with low PTPRZ1 expression. The bar graphs in the right column show the improved killing ability of the bispecific RNA CAR-T cells compared to the second monovalent CAR against cell lines with high PTPRZ1 expression. (A) Bispecific CAR-T cells: anti-PTPRZ1 (VHH RB832) and anti-CSPG4 (VHH RB830). (B) Bispecific CAR-T cells: anti-PTPRZ1 (VHH RB832) and anti-BCAN (scFv HRB295). (C) Bispecific CAR-T cells anti-PTPRZ1 (VHH RB832) and anti-tenascin C (VHH RB835). DETAILED DESCRIPTION OF THE INVENTION

[0015] Sequence Listing Description Array No. 1 - Nucleotide sequence of anti-PTPRZ1 scFv "RRB469" ATGGCCCAGGTGCAACTGGTGGAATCTGGGGGAGGCGTGGTTCAGCCTGGGAGGTCCCTGCGGCTCTCCTGTGCAGCCTCAGGATTTACCTTCAGTAGCTACGCCATGCACTGGGTCCGCCAGGCTCCAGGCAAAGGGTTGGAATGGGTTGCAGTTATATCATATGATGGAAGTAATAAATACTACGCAGACTCCGTGAAGGGCCGATTCACCATTTCACGTGACAATTCCAAGAACACGCTTTATCTGCAAATGAACAGCTTGAGAGCTGAAGATACGGCTGTGTATTACTGCGCGAGGGGTAGTGGATACAGCTATGGTCCGGGTTATGATGCATTTGATATTTGGGGCCAGGGAACCCTTGTCACAGTCTCAAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGGGGCGGATCTACAAATTTTATGCTGACTCAGCCTCATTCTGTATCGGAGTCTCCAGGGAAGACAGTAACCATCTCCTGCACACGCAGCAGTGGCAGCATCGCCAGCAACTATGTGCAGTGGTACCAGCAGAGACCAGGCAGTTCACCCACTACTGTGATTTATGAGGATAACCAAAGACCCTCTGGGGTCCCTGATCGGTTTTCTGGCTCCATCGACAGCAGTTCCAATTCGGCCTCCCTCACCATCTCTGGACTAAAAACTGAGGACGAGGCTGACTACTACTGTCAGTCCTGGGACCCCGTGTTCGGGGTGTTCGGCGGAGGGACAAAGCTGACCGTCTTAGGGGCGGCC

[0016] Array No. 2 - Nucleotide sequence of anti-PTPRZ1 scFv "RRB470" ATGGCACAAGTGCAGTTAGTTCAGTCTGGGGCTGAAGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTAGTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGCCTTGAATGGATGGGAGGGATCATTCCGATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAAGGCAGAGTCACTATTACCGCGGACGAATCCACAAGCACAGCATACATGGAGCTGAGCAGCCTGAGGTCTGAAGATACGGCCGTGTATTACTGTGCGAGAGAGGGGGGGGCCGTGGGGTACTACTACGGTATGGACGTCTGGGGCCAGGGAACACTTGTGACAGTCTCCAGCGGTGGAGGCGGTTCAGGCGGAGGCGGCTCAGGCGGTGGCGGATCTACTCAGAGTGCCTTGACTCAGCCAGCCTCCGTGTCTGGGTCACCCGGACAGTCGATAACCATCTCCTGCACTGGAACCAGCAGTGACGTTGGTGGTTATAATTATGTATCCTGGTACCAACAGCACCCAGGCAAAGCCCCCAAACTCATGATTTATGAGGTCAGTAATCGGCCCTCAGGGGTTTCTAATCGTTTCTCTGGCTCCAAATCTGGCAACACGGCCTCCCTGACTATATCAGGGCTCCAGGCTGAGGACGAGGCTGATTATTACTGTAGTTCATATGATAGGAGCAACCGCAGTATGGTGTTTGGCGGAGGGACCAAACTGACCGTACTAGGGGCAGCC

[0017] Sequence number 3 - Nucleotide sequence of anti - PTPRZ1 scFv "RRB471" ATGGCCGAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCTGGATTTACCTTCAGTAGCTATAGCATGAACTGGGTCAGGCAGGCTCCAGGGAAGGGGCTTGAGTGGGTTTCATACATTAGTAGCAGTAGTAGCACAATATACTACGCAGACTCTGTGAAGGGCCGATTCACAATCTCCAGGGATAATGCCAAGAACTCACTGTATTTACAAATGAATAGCCTTAGAGCCGAAGACACGGCTGTGTATTACTGTGCGAGACCAGGCTACGGTGACTTTCCCGGTGCTTTTGATATCTGGGGCCAGGGAACCCTGGTCACAGTGTCGAGCGGTGGAGGCGGCTCAGGCGGAGGTGGCAGCGGCGGTGGCGGATCTACGCAGTCTGCATTGACTCAGCCTGCCTCCGTGTCTGGATCACCTGGACAAAGCATTACCATCTCCTGCACTGGAACCAGCAGTGACGTTGGTGGTTATAACTATGTCTCCTGGTACCAACAGCACCCGGGCAAAGCCCCCAAACTCATGATTTACGAAGTAAGTAATCGGCCCTCAGGGGTTTCTAATCGCTTCTCTGGCTCCAAATCCGGCAACACTGCCTCCCTGACAATCAGTGGGCTCCAGGCTGAGGACGAGGCTGATTATTACTGCAGCTCATATGATTGGGCCACCTACGGGTCGGTGTTCGGCGGAGGGACCAAGCTGACTGTCCTAGGGGCGGCA

[0018] Sequence number 4 - Nucleotide sequence of anti-PTPRZ1 scFv "RRB473" ATGGCCCAAGTGCAGCTGGTGGAGTCTGGGGGAGGCGTTGTCCAGCCTGGGAGGTCACTGAGACTCTCCTGTGCAGCCTCTGGGTTTACATTCAGTAGCTATGCTATGCACTGGGTACGCCAAGCTCCTGGCAAAGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGTAGTAACAAATACTACGCAGACAGTGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACTCTTTATCTGCAAATGAACAGCCTGCGGGCTGAAGACACAGCTGTGTACTACTGTGCGCGGGACCAGGATGACTCCAGTGATGCTTTTGATATCTGGGGCCAGGGAACCCTGGTCACAGTCTCGAGCGGTGGAGGCGGTAGCGGCGGAGGTGGCAGCGGCGGTGGCGGATCGACGCAGAGCGTTTTGACGCAACCGCCCTCAGTGTCTGCAGCCCCAGGACAGAAAGTCACCATATCCTGCTCTGGAAGCAGCTCCAACATTGGGAACAATTATGTATCCTGGTACCAGCAGTTGCCAGGGACAGCCCCCAAACTCCTCATTTACGACAATAATAAGCGTCCCTCAGGGATTCCTGACCGCTTTTCTGGCAGTAAGTCTGGCACTTCAGCCACTCTGGGCATCACCGGACTTCAGACTGGGGACGAAGCCGATTATTACTGCGGAACATATGATTACATCGCGACCAGGGCCGTGTTCGGTGGCGGGACCAAGTTAACTGTGCTAGGGGCAGCC

[0019] SEQ ID NO: 5 - Nucleotide sequence of anti - PTPRZ1 scFv "RRB474" ATGGCCCAGGTGCAGCTTGTTCAGTCTGGGGCTGAGGTGAAGAAGCCAGGGTCCTCGGTGAAGGTTTCCTGCAAGGCTTCAGGAGGCACCTTCAGCAGCTATGCTATCAGTTGGGTGCGGCAAGCACCTGGCCAAGGGCTTGAGTGGATGGGAGGGATAATCCCTATCTTTGGTACAGCAAACTACGCACAAAAGTTCCAGGGCCGCGTCACGATTACCGCCGACGAATCCACCAGCACAGCCTACATGGAACTGTCCAGCCTGAGAAGTGAAGACACTGCCGTGTATTACTGTGCGCGTGGGACGTATTACGATTTTTGGAGTGGTTATTATGATGCTTTTGATATCTGGGGCCAGGGAACCCTGGTTACAGTCTCTAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGCTCTACTCAGTCTGTGTTGACACAGCCGCCCTCAGTGTCTGCAGCCCCAGGACAGAAGGTCACCATCTCCTGTTCTGGTAGCAGCTCCAACATTGGGAATAATTACGTATCCTGGTACCAGCAGTTGCCAGGAACAGCCCCCAAACTCTTAATATATGACAATAACAAAAGGCCCTCAGGGATTCCTGACCGATTCAGTGGCTCCAAATCTGGCACTTCAGCTACCCTGGGCATTACCGGACTCCAGACTGGGGACGAGGCCGATTATTACTGCGGAACATGGGATAGTTCGTACTGGCAACCCGTATTCGGCGGAGGGACTAAACTGACCGTCCTAGGGGCGGCC

[0020] Sequence number 6 - Nucleotide sequence of anti - PTPRZ1 scFv "RRB476" ATGGCCCAGGTGCAACTGGTGGAATCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCGTTGCGGCTCTCCTGTGCAGCCTCTGGATTCACCTTTAGTAGCTATGCTATGCACTGGGTCCGCCAAGCTCCAGGCAAGGGGCTGGAATGGGTGGCAGTTATATCATACGATGGAAGTAATAAATACTACGCAGACTCCGTGAAGGGCCGCTTCACCATCTCCAGAGACAATTCCAAGAACACACTGTATCTGCAAATGAACAGCCTGAGGGCTGAGGACACTGCTGTGTATTACTGTGCGAGAGACCAGGATGACTCCAGTGATGCTTTTGATATCTGGGGGCAGGGAACCCTGGTAACAGTCAGTAGCGGTGGAGGCGGTTCAGGCGGCGGTGGCGGTAGCACGCAGTCTGTGTTGACGCAGCCGCCCTCAGTGTCTGCAGCCCCAGGACAAAAGGTCACTATCTCCTGCTCTGGAAGCAGCTCCAACATTGGGAACAACTATGTATCCTGGTACCAGCAGTTACCTGGTACAGCCCCCAAACTCCTCATTTATGACAATAATAAGCGACCCTCAGGGATTCCTGACCGTTTTAGTGGCAGCAAATCTGGCACTTCAGCCACCCTTGGCATCACCGGACTCCAGACTGGGGACGAGGCCGATTATTACTGCGGAACATGGGATTACAAAGTTTCGCGGCTTGTCTTCGGCGGAGGGACCAAGCTGACAGTTCTAGGGGCGGCC

[0021] Sequence number 7 - Nucleotide sequence of anti - CSPG4 scFv "HRB298" ATGGCCCAGGTGCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTTAAAGTTTCCTGTAAGGCTAGTGGTTACACCTTTACCAGCTATGGTATCAGTTGGGTGAGGCAGGCCCCTGGACAAGGGCTTGAATGGATGGGATGGATCAGCGCTTACAATGGTAACACAAACTATGCACAGAAGCTCCAGGGCAGAGTCACCATGACCACAGACACATCCACTAGCACAGCATACATGGAGCTGAGGAGTTTGAGATCTGACGACACGGCCGTTTATTACTGTGCGCGGCGAGATTACTATGATGGTAGTGGATTTGACTACTGGGGCCAAGGAACTCTGGTCACAGTCTCGAGCGGTGGAGGCGGTTCCGGCGGAGGGGGCAGCGGCGGTGGCGGATCAACTCAGTCTGTGTTGACGCAGCCGCCCTCAGTGAGTGCAGCCCCAGGCCAAAAAGTCACCATCTCCTGCTCTGGAAGCAGCTCCAACATTGGGAATAATTACGTATCCTGGTACCAGCAGCTCCCAGGCACAGCTCCCAAATTACTTATTTATGATAATAACAAGCGTCCCTCAGGGATTCCTGACCGGTTCTCTGGCTCCAAATCTGGCACGTCAGCCACCCTGGGCATAACTGGGCTCCAAACTGGGGACGAAGCCGATTATTACTGCGGAACTTATGATGGCGAAGGGCGCCACGAGGTGTTCGGCGGAGGGACCAAGCTGACCGTACTAGGGGCGGCA

[0022] Sequence number 8 - Nucleotide sequence of anti - CSPG4 scFv "HRB299" ATGGCCCAGGTGCAACTGGTGGAATCGGGCGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTTTCCTGTGCAGCCTCTGGATTTACCTTCAGTAGCTATGCTATGCACTGGGTGCGCCAAGCTCCAGGCAAGGGGCTGGAATGGGTTGCAGTTATATCATATGATGGAAGTAATAAATACTACGCAGACTCCGTGAAGGGCCGTTTTACCATCTCCCGGGACAATTCCAAAAACACTCTGTATCTGCAAATGAACAGCCTTAGAGCTGAAGACACTGCCGTGTACTACTGCGCGCGCGATCCGTGGGGTGGTTGGTTAGGGAGCGATGCTTTTGACATTTGGGGCCAAGGAACCTTGGTCACAGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGCAGCACGCAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACACCCGGGCAGAGGGTCACCATTTCTTGTTCTGGAAGCAGCAGCAACATTGGAAGTAACACTGTAAACTGGTACCAGCAGTTGCCAGGAACGGCCCCCAAACTCCTCATCTATAGTAATAATCAGCGGCCTTCAGGGGTACCTGACCGATTCTCCGGCTCCAAGTCTGGCACCTCAGCCTCCCTCGCCATCAGTGGGCTCCAGTCTGAGGATGAGGCTGATTATTACTGCGCAGCATACGATGGGGACGGGGGGGAGGACGTGTTCGGCGGAGGTACAAAGCTGACAGTTCTAGGGGCCGCC

[0023] Sequence number 9 - Nucleotide sequence of anti-CSPG4 scFv "HRB" ATGGCCGAGGTGCAGCTGTTGGAATCTGGGGGAGGCTTGGTACAGCCGGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCTATGAGCTGGGTGCGCCAGGCTCCAGGGAAGGGGCTGGAATGGGTGTCAGCTATTAGTGGTAGTGGTGGCAGCACATACTACGCAGATTCCGTGAAAGGCCGGTTCACAATCTCCAGAGATAATAGTAAGAACACACTGTACCTTCAAATGAACAGCTTACGCGCCGAGGACACGGCGGTGTATTACTGTGCAAGACGATATAGCAGTGGCTGGTCATACTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACAGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGATCTACGCAGTCTGCCCTGACTCAACCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATATCCTGCACTGGAACATCCAGTGACGTTGGAGGTTATAACTATGTTTCCTGGTACCAACAGCACCCAGGCAAAGCCCCCAAACTCATGATTTATGAAGTAAGTAATCGGCCCTCAGGGGTTTCTAATAGGTTCTCAGGCTCCAAGAGTGGCAACACTGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGACGAGGCTGATTACTACTGCAGCTCATATGATACTTTTGAGAGGATTAGCGTGTTCGGCGGAGGGACCAAGCTTACCGTCCTAGGGGCGGCA

[0024] Sequence number 10 - Nucleotide sequence of anti-CSPG4 scFv "HRB301" ATGGCACAAGTGCAACTGGTGCAGTCTGGAGCTGAAGTGAAGAAACCGGGGGCCTCAGTTAAGGTCTCCTGCAAAGCTTCTGGTTACACCTTTACTAGCTATGGTATCAGCTGGGTGAGACAAGCCCCTGGACAAGGGCTTGAGTGGATGGGCTGGATCAGCGCTTACAATGGTAACACAAACTATGCACAGAAGCTCCAGGGCCGTGTCACCATGACCACAGACACATCCACCTCCACAGCCTACATGGAGCTGAGGAGCCTGAGATCGGACGACACGGCTGTGTATTACTGTGCGAGACGGAGTTATGATAGTAGTGGACTTGACTACTGGGGCCAGGGAACATTGGTTACAGTTTCGAGCGGCGGCGGCGGTTCAGGCGGAGGGGGCAGCGGCGGTGGCGGTTCTACGCAGTCCGTGTTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATTTCTTGTTCTGGAAGCAGCTCCAACATTGGAAGTAATACTGTAAACTGGTACCAGCAGCTCCCAGGAACTGCCCCTAAATTACTCATATATAGTAATAATCAGCGGCCCTCAGGGGTACCTGACCGATTCTCCGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTGCAGAGCGAAGATGAGGCTGATTATTACTGCGCAGCATGGGATCGCAGGTGGCGCCTGGTGTTCGGCGGAGGGACCAAGCTGACTGTCCTAGGGGCCGCC

[0025] Sequence number 11 - Nucleotide sequence of anti-CSPG4 scFv "HRB302" ATGGCCCAGGTGCAGCTTGTGCAGTCCGGTGCTGAGGTGAAGAAACCTGGGGCCTCAGTTAAGGTTTCCTGCAAGGCTTCTGGTTACACCTTTACCAGCTATGGTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGCTGGATAAGCGCTTACAATGGTAACACAAACTATGCACAAAAGTTGCAGGGCCGTGTCACCATGACCACAGACACATCCACAAGCACAGCCTACATGGAGCTGAGGTCCTTGAGATCTGACGACACGGCCGTGTATTACTGCGCAAGACAGGTGGGCGCTCCGACTCGCTTTGACTACTGGGGCCAGGGAACCCTGGTGACAGTCTCGAGCGGTGGAGGCGGGTCAGGCGGAGGCGGCAGCGGCGGTGGCGGATCGACGCAGTCTGTGCTGACTCAACCACCATCAGCGTCCGGGACCCCCGGGCAAAGGGTCACTATTAGTTGTAGTGGAAGCAGCTCCAACATTGGAAGTAATACTGTAAACTGGTACCAGCAGCTCCCAGGAACTGCCCCCAAACTCTTAATCTATAGTAATAATCAGCGGCCCTCAGGGGTTCCTGATCGGTTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCAATCAGTGGGCTCCAGTCTGAAGATGAAGCTGATTATTACTGTGCAGCATGGGATACGCACGCCTGGGCCCCCGTATTCGGCGGAGGGACTAAACTGACCGTCCTAGGGGCGGCT

[0026] Sequence number 12 - Nucleotide sequence of anti-CSPG4 scFv "HRB303" ATGGCCCAGGTGCAACTGGTGCAGTCTGGGGCTGAGGTCAAGAAGCCAGGGTCCTCGGTGAAGGTCTCCTGTAAGGCTAGTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGAGACAAGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATATTTGGTACAGCAAACTACGCACAGAAATTTCAGGGCAGAGTTACGATAACTGCAGACGAATCCACTAGCACAGCATACATGGAGCTGAGTAGTTTAAGGTCTGAAGACACTGCAGTGTATTACTGTGCTCGTTCTAAATATAACTGGGCCTACAAAAATGATTACTGGGGCCAGGGAACCCTGGTTACAGTTTCAAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGCTCTACACAGAGCGTGTTGACGCAGCCGCCCTCAGTATCGGCGGCCCCAGGGCAGAAGGTCACCATCTCCTGCTCTGGAAGCAGTTCCAACATTGGGAATAACTATGTATCCTGGTACCAGCAGCTCCCAGGTACAGCCCCCAAATTGCTCATTTACGACAATAATAAGCGACCCTCAGGGATTCCTGATCGCTTCAGTGGCTCCAAATCTGGCACCTCAGCCACCCTGGGCATCACCGGACTTCAAACTGGGGACGAAGCTGATTATTACTGCGGAACATATGATCCCTGGGCTCGGACTGCCGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGGGCGGCC

[0027] SEQ ID NO: 13 - Nucleotide sequence of anti - CSPG4 scFv "225.28S" CAGGTTAAACTCCAACAAAGTGGCGGAGGCTTGGTTCAGCCTGGAGGGAGTATGAAACTGTCTTGTGTCGTATCTGGTTTTACATTCTCAAATTATTGGATGAATTGGGTTAGGCAATCACCGGAGAAGGGATTGGAATGGATCGCTGAGATTCGGTTGAAATCAAACAACTTCGGTCGCTATTATGCGGAATCCGTGAAAGGTCGGTTCACGATTTCCCGCGACGATTCAAAGTCCAGTGCTTATCTGCAAATGATTAATCTTCGGGCAGAAGATACAGGAATATACTATTGTACCTCCTATGGTAACTATGTTGGTCACTATTTCGATCATTGGGGGCAGGGAACCACTGTCACCGTATCCAGCggtggcggagggagcggcggtggaggaagcggaggcggaggttccGACATTGAACTGACTCAATCTCCCAAATTTATGTCAACGAGCGTCGGGGACCGCGTGAGCGTTACGTGTAAGGCTTCACAAAACGTAGACACCAATGTGGCCTGGTATCAACAAAAGCCGGGACAATCTCCAGAGCCCCTGCTCTTTTCAGCAAGTTACAGGTACACCGGTGTTCCAGATAGATTCACAGGTAGTGGATCTGGTACTGATTTTACTCTCACCATAAGTAACGTGCAGTCCGAAGACCTCGCCGAGTACTTTTGTCAACAGTATAATAGTTACCCACTTACATTTGGGGGTGGAACAAAACTGGAAATCAAG

[0028] Sequence number 14 - Nucleotide sequence of anti - BCAN scFv "HRB294" ATGGCCCAGGTACAACTGCAACAGTCAGGGCCAGGACTGGTTAAGCCCTCGCAGACCTTATCACTTACCTGTGCCATCTCCGGGGACAGTGTCTCTAGCAACAGTGCTGCTTGGAACTGGATAAGGCAGTCCCCAAGCCGCGGCCTTGAATGGCTGGGAAGAACATACTACAGGTCCAAGTGGTATAATGATTATGCAGTTAGTGTGAAAAGTCGAATAACTATCAACCCTGATACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCTGAGGACACAGCTGTGTATTACTGTGCGAGAGATCAGCGGAATTACGATTTTTGGAGTGGTTATTATCCGCCCGCAGAATTAGGGTACTACGGTATGGACGTCTGGGGCCAGGGAACCCTGGTCACAGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGCGGCAGCGGCGGTGGCGGCTCTACGCAGTCTGTGTTGACGCAGCCGCCTAGCGTGTCTGCTGCCCCAGGTCAGAAAGTGACCATCTCCTGCTCTGGAAGCAGCTCCAACATTGGGAATAATTATGTATCCTGGTACCAGCAACTCCCAGGAACAGCACCCAAACTCCTCATTTATGACAATAATAAGCGGCCCTCAGGGATTCCTGACCGTTTTTCTGGCAGTAAAAGCGGCACTTCAGCCACTCTGGGCATCACCGGGCTCCAAACTGGGGACGAGGCCGATTACTACTGCGGAACATGGGATTGGAGCGCATTGGTGGTGTTCGGCGGAGGGACCAAGCTGACCGTTCTAGGGGCGGCC

[0029] SEQ ID NO:15 - Nucleotide sequence of anti - BCAN scFv "HRB295" ATGGCCCAGGTGCAGCTGGTGGAGTCTGGGGGTGGCGTGGTCCAGCCTGGGAGGTCCTTGCGTCTCTCCTGCGCAGCCTCTGGATTCACTTTCAGTAGCTATGCTATGCACTGGGTCCGCCAAGCTCCGGGCAAGGGGCTGGAATGGGTGGCAGTTATAAGCTATGATGGAAGTAATAAATACTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTTTATCTGCAAATGAACAGCCTTCGCGCTGAAGACACAGCTGTGTATTACTGTGCCAGAGTATCAGACTGGAACGACGCCGCTTTTGATATTTGGGGCCAGGGAACTCTGGTTACAGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGATCGACCCAGTCTGTGCTGACTCAGCCACCCTCAGCGAGCGGGACACCAGGGCAGCGGGTCACCATTTCTTGTTCTGGAAGCAGCTCCAACATCGGTAGTAATACTGTAAACTGGTACCAGCAACTGCCAGGAACGGCCCCCAAACTCCTCATCTACAGTAATAATCAACGGCCTTCAGGGGTTCCTGATAGATTTTCTGGCTCCAAAAGCGGCACCTCAGCCTCCCTGGCCATTAGTGGGTTACAGTCTGAAGATGAGGCTGATTATTACTGCGCAGCATGGGACCCCGAGACCGCCAGGTGGGTGTTTGGCGGAGGGACAAAGTTGACCGTCCTAGGGGCGGCC

[0030] Sequence number 16 - Nucleotide sequence of anti - BCAN scFv "HRB296" ATGGCCCAGGTACAGTTGCAGCAATCAGGTCCAGGACTGGTGAAGCCCAGCCAAACCTTATCATTAACTTGTGCAATCTCCGGGGACAGTGTTTCTAGCAACAGTGCTGCTTGGAACTGGATCAGGCAGTCCCCTTCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACCGGAGCAAGTGGTATAATGATTATGCAGTTAGCGTGAAAAGTCGGATAACCATCAACCCTGACACATCCAAGAACCAGTTCTCCCTGCAACTGAACTCTGTGACTCCCGAAGACACGGCTGTGTATTACTGTGCACGCAGAGGGGAACACTATGATAGTAGTGGTTATTACTACGGCCTTGATTACTGGGGCCAGGGAACCCTGGTCACAGTCAGCAGCGGTGGAGGCGGTTCAGGCGGCGGTGGCAGCGGCGGTGGCGGATCTACGCAGTCGGTGTTGACACAGCCGCCCTCAGTGTCTGCGGCCCCAGGGCAAAAAGTTACCATATCCTGCTCTGGAAGCAGCTCCAACATTGGGAATAATTATGTATCCTGGTACCAGCAGCTCCCAGGAACAGCCCCCAAACTCCTCATTTATGACAATAATAAGCGTCCTTCAGGGATTCCTGACCGATTCTCTGGCTCCAAGTCTGGCACTTCAGCCACTCTGGGCATCACCGGACTCCAGACTGGGGACGAGGCCGATTACTACTGCGGAACATATGATGTCGCGGCTGGGTACGTGTTTGGCGGAGGGACCAAACTGACCGTCCTAGGGGCAGCC

[0031] Sequence number 17 - Nucleotide sequence of anti - BCAN scFv "HRB297" ATGGCCCAGGTACAGCTGGTGCAGAGCGGGGCTGAGGTGAAAAAGCCCGGGTCCTCGGTGAAAGTGTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATAAGCTGGGTGCGACAAGCCCCGGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGAGTCACCATTACCGCGGACGAATCCACGAGCACAGCCTACATGGAACTGAGCAGCCTTAGGTCTGAGGACACTGCCGTGTATTACTGTGCGAGACCACGTACTGCAGGCTGGAGTTATGATGCTTTTGATGCCTGGGGCCAGGGAACATTGGTAACAGTCTCAAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGATCTACGCAATCTGCCTTAACTCAGCCTGCCAGTGTGTCTGGGAGTCCTGGACAGTCAATAACCATTTCCTGTACTGGAACCAGCAGTGACGTTGGCGGTTATAACTACGTTTCCTGGTACCAACAGCACCCAGGCAAAGCCCCCAAACTCATGATTTACGAAGTCAGTAATAGGCCCTCAGGGGTTTCTAATCGCTTCTCTGGCTCCAAGTCAGGCAACACAGCCTCCCTGACTATCTCTGGGCTCCAGGCTGAGGACGAGGCTGATTATTACTGCAGTTCATATGATTGGCGGTCCTCCGGGTCGGTGTTTGGCGGAGGGACCAAGCTGACCGTCCTAGGGGCAGCA

[0032] Sequence number 18 - Nucleotide sequence of anti-TNC scFv "E10" GAGGTACAGCTAGTGGAGTCAGGCGGGGGCCTGGTCCAGCCAGGCGGATCGTTAAGACTTAGTTGCGCAGCAAGCGGGTTTACGTTCTCAGGTAGCCGAATGGGGTGGGTGAGACAGGCCCCCGGGAAAGGACTCGAATGGGTTTCCGCGATCAACGAAGAAGGTGGACAAACTTACTACGCCGATAGCGTGAAGGGACGGTTTACAATTTCTCGTGACAACTCCAAGAATACCCTGTATCTGCAAATGAATAGTTTGAGGGCTGAGGACACCGCCGTCTATTATTGTGCTAAACATCCTCCGCACCGCCCCTTCGATTACTGGGGCCAGGGCACACTCGTGACTGTTTCTAGGGGTGGAGGCGGTTCCGGCGGAGGGGGCAGCGGCGGTGGCGGATCATCTTCCGAATTAACTCAAGACCCAGCCGTTTCTGTGGCCCTCGGCCAGACAGTCAGGATCACGTGCCAAGGGGATAGTCTGCGATCCTACTATGCAAGCTGGTACCAGCAGAAACCGGGCCAGGCTCCTGTATTGGTGATCTACGGAAAGAATAACAGACCCTCTGGTATACCCGACCGGTTCTCCGGTAGCAGTAGCGGCAACACCGCTTCACTTACTATTACCGGAGCACAGGCCGAGGATGAGGCGGACTATTATTGTAATTCATCGCACGGCCCACGTCGCCCTGTCGTGTTTGGGGGAGGCACCAAGCTGACAGTGCTGGGG

[0033] Sequence number 19 - Nucleotide sequence of anti-TNC scFv "P12" GAGGTGCAGCTGGTCGAGAGCGGTGGAGGCCTCGTGCAGCCCGGAGGGTCATTGCGTCTCTCCTGCGCCGCCAGCGGCTTTACCTTCGGTCAATATAGCATGAGTTGGGTCAGGCAGGCGCCTGGCAAGGGACTGGAATGGGTTTCCGCCATCACCGGTACAGGAGGGGAAACATACTACGCTGACTCAGTAGAGGGGAGATTCACTATTTCTCGAGATAACTCCAAGAACACGCTGTATCTACAAATGAATTCTTTACGCGCAGAAGACACTGCAGTTTATTACTGTGCTAAAGGGAGACGGATATTTGATTACTGGGGCCAGGGCACCCTTGTGACAGTGAGTAGGGGTGGAGGCGGTTCCGGCGGAGGGGGCAGCGGCGGTGGCGGATCATCCTCCGAACTGACACAGGACCCGGCTGTGAGCGTCGCCCTTGGTCAGACTGTGAGAATTACATGCCAGGGGGATTCACTCAGGCGGCAGCCTGCTTCTTGGTACCAACAAAAGCCTGGACAGGCCCCCGTGTTGGTGATATACTACAAAAAGCTGCGACCATCAGGAATCCCTGACCGCTTTTCTGGAAGCTCCAGTGGGAATACCGCAAGTCTCACCATCACTGGTGCCCAGGCAGAGGATGAGGCGGACTATTATTGTAACTCGTTCAGCCCCAAACCCAAACCAGTAGTCTTCGGCGGCGGGACCAAGCTGACGGTTTTAGGC

[0034] SEQ ID NO: 20 - Nucleotide sequence of anti - TNC scFv "F16" GAGGTCCAATTACTTGAATCAGGCGGAGGCCTGGTGCAGCCTGGAGGCAGCCTGAGACTGTCCTGCGCGGCAAGCGGTTTCACTTTCTCCAGATATGGCGCATCCTGGGTTAGGCAGGCACCCGGTAAAGGACTGGAGTGGGTATCTGCCATTTCTGGGAGTGGAGGGAGTACCTACTAC GCTGATTCGGTGAAGGGGCGTTTTACAATCTCACGAGACAATAGCAAAAACACACTATATCTCCAGATGAATTCTCTCCGCGCCGAAGACACGGCTGTCTACTATTGTGCTAAGGCCCACAACGCCTTTGATTACTGGGGCCAGGGGACCTTGGTGACTGTGAGCCGGGTGGAGGCGGT TCCGGCGGAGGGGGCAGCGGCGGTGGCGGATCATCGAGCGAACTGACACAGGACCCGGCGGTTTCCGTGGCACTGGGGCAGACAGTAAGAATAACTTGTCAAGGGGATAGCCTGCGCAGTTACTACGCCAGCTGGTACCAGCAGAAACCAGGCCAGGCCCCCGTTTTGGTGATTTATGGG AAGAATAACAGGCCTTCCGGCATCCCCGACCGGTTTTCTGGATCTAGTTCTGGAAACACCGCATCACTTACCATCACGGGAGCTCAAGCCGAGGATGGCTGACTACTATTGCAATTCATCCGTCTATACTATGCCTCCAGTGGTGTTCGGTGGCGGTACAAAGTTAACCGTCCTCGGC

[0035] SEQ ID NOs: 21 to 40 are all scFvs in a VH-linker-VL orientation, with the linker (G4S)3 in lower case.

[0036] The antigen-binding region is

[0037] [ka]

[0038] All ABRs were predicted using Paratome (Kunik V et al (2012)). Structural consensus among antibodies defines antigen-binding sites. PLoS Comput Biol 8(2):e1002388.doi:10.1371 / journal.pcbi.1002388; and Kunik V et al (2012). Paratome: an online tool for systematically identifying antigen-binding regions of antibodies based on sequence and structure. Nucleic Acids Res. 2012 Jul;40(Web Server issue):W521-4.doi:10.1093 / nar / gks480. Epub 2012 Jun 6).

[0039] [ka]

[0040] [ka]

[0041] [ka]

[0042] [ka]

[0043] [ka]

[0044] [ka]

[0045] [ka]

[0046]

change

[0047]

change

[0048]

change

[0049]

change

[0050]

change

[0051]

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[0052]

change

[0053]

change

[0054]

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[0055]

change

[0056]

change

[0057] [ka]

[0058] [ka]

[0059] SEQ ID NO: 41 - Anti-PTPRZ1 scFv "RRB469" V H Polypeptide Sequence MAQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGSGYSYGPGYDAFDIWGQGTLVTVSS

[0060] SEQ ID NO: 42 - Anti-PTPRZ1 scFv "RRB469" V L Polypeptide Sequence TNFMLTQPHSVSESPGKTVTISCTRSSGSIASNYVQWYQQRPGSSPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSWDPVFGVFGGGTKLTVLGAA

[0061] SEQ ID NO: 43 - Anti-PTPRZ1 scFv "RRB470" V H Polypeptide Sequence MAQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAREGGAVGYYYGMDVWGQGTLVTVSS

[0062] SEQ ID NO: 44 - Anti-PTPRZ1 scFv "RRB470" V L Polypeptide Sequence TQSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYDRSNRSMVFGGGTKLTVLGAA

[0063] SEQ ID NO: 45 - Anti-PTPRZ1 scFv "RRB471" V H Polypeptide Sequence MAEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSYISSSSSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPGYGDFPGAFDIWGQGTLVTVSS

[0064] SEQ ID NO: 46 - Anti-PTPRZ1 scFv "RRB471" V L Polypeptide Sequence TQSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYDWATYGSVFGGGTKLTVLGAA

[0065] SEQ ID NO: 47 - Anti-PTPRZ1 scFv "RRB473" V H Polypeptide Sequence MAQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDQDDSSDAFDIWGQGTLVTVSS

[0066] SEQ ID NO: 48 - Anti-PTPRZ1 scFv "RRB473" V L Polypeptide Sequence TQSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTYDYIATRAVFGGGTKLTVLGAA

[0067] SEQ ID NO: 49 - Anti-PTPRZ1 scFv "RRB474" V H Polypeptide Sequence MAQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGTYYDFWSGYYDAFDIWGQGTLVTVSS

[0068] SEQ ID NO: 50 - Anti-PTPRZ1 scFv "RRB474" V L Polypeptide Sequence TQSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSYWQPVFGGGTKLTVLGAA

[0069] SEQ ID NO: 51 - Anti-PTPRZ1 scFv "RRB476" V H Polypeptide Sequence MAQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDQDDSSDAFDIWGQGTLVTVSS

[0070] SEQ ID NO: 52 - Anti-PTPRZ1 scFv "RRB476" V L Polypeptide Sequence TQSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDYKVSRLVFGGGTKLTVLGAA

[0071] SEQ ID NO: 53 - Anti-CSPG4 scFv "HRB298" V H Polypeptide Sequence MAQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARRDYYDGSGFDYWGQGTLVTVSS

[0072] SEQ ID NO: 54 - Anti-CSPG4 scFv "HRB298" V L Polypeptide Sequence TQSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTYDGEGRHEVFGGGTKLTVLGAA

[0073] SEQ ID NO: 55 - Anti-CSPG4 scFv "HRB299" V H Polypeptide Sequence MAQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDPWGGWLGSDAFDIWGQGTLVTVSS

[0074] SEQ ID NO: 56 - Anti-CSPG4 scFv "HRB299" V L Polypeptide Sequence TQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQLPGTAPKLLIYSNNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAYDGDGGEDVFGGGTKLTVLGAA

[0075] SEQ ID NO: 57 - Anti-CSPG4 scFv "HRB300" V H Polypeptide Sequence MAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRYSSGWSYYFDYWGQGTLVTVSS

[0076] SEQ ID NO: 58 - Anti-CSPG4 scFv "HRB300" V L Polypeptide Sequence TQSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYDTFERISVFGGGTKLTVLGAA

[0077] SEQ ID NO: 59 - Anti-CSPG4 scFv "HRB301" V H Polypeptide Sequence MAQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARRSYDSSGLDYWGQGTLVTVSS

[0078] SEQ ID NO: 60 - Anti-CSPG4 scFv "HRB301" V L Polypeptide Sequence TQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQLPGTAPKLLIYSNNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDRRWRLVFGGGTKLTVLGAA

[0079] SEQ ID NO: 61 - Anti-CSPG4 scFv "HRB302" V H Polypeptide Sequence MAQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARQVGAPTRFDYWGQGTLVTVSS

[0080] SEQ ID NO: 62 - Anti-CSPG4 scFv "HRB302" V L Polypeptide Sequence TQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQLPGTAPKLLIYSNNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDTHAWAPVFGGGTKLTVLGAA

[0081] SEQ ID NO: 63 - Anti-CSPG4 scFv "HRB303" V H Polypeptide Sequence MAQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSKYNWAYKNDYWGQGTLVTVSS

[0082] SEQ ID NO: 64 - Anti-CSPG4 scFv "HRB303" V L Polypeptide Sequence TQSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTYDPWARTAVFGGGTKLTVLGAA

[0083] SEQ ID NO: 65 - Anti-CSPG4 scFv "225.28S" V H Polypeptide Sequence QVKLQQSGGGLVQPGGSMKLSCVVSGFTFSNYWMNWVRQSPEKGLEWIAEIRLKSNNFGRYYAESVKGRFTISRDDSKSSAYLQMINLRAEDTGIYYCTSYGNYVGHYFDHWGQGTTVTVSS

[0084] SEQ ID NO: 66 - Anti-CSPG4 scFv "225.28S" V L Polypeptide Sequence DIELTQSPKFMSTSVGDRVSVTCKASQNVDTNVAWYQQKPGQSPEPLLFSASYRYTGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCQQYNSYPLTFGGGTKLEIK

[0085] SEQ ID NO: 67 - Anti-BCAN scFv "HRB294" V H Polypeptide Sequence MAQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARDQRNYDFWSGYYPPAELGYYGMDVWGQGTLVTVSS

[0086] SEQ ID NO: 68 - Anti-BCAN scFv "HRB294" V L Polypeptide Sequence TQSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDWSALVVFGGGTKLTVLGAA

[0087] SEQ ID NO: 69 - Anti-BCAN scFv "HRB295" V H Polypeptide Sequence MAQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVSDWNDAAFDIWGQGTLVTVSS

[0088] SEQ ID NO: 70 - Anti-BCAN scFv "HRB295" V L Polypeptide Sequence TQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQLPGTAPKLLIYSNNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDPETARWVFGGGTKLTVLGAA

[0089] SEQ ID NO: 71 - Anti-BCAN scFv "HRB296" V H Polypeptide Sequence MAQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARRGEHYDSSGYYYGLDYWGQGTLVTVSS

[0090] SEQ ID NO: 72 - Anti-BCAN scFv "HRB296" V L Polypeptide Sequence TQSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTYDVAAGYVFGGGTKLTVLGAA

[0091] SEQ ID NO: 73 - Anti-BCAN scFv "HRB297" V H Polypeptide Sequence MAQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARPRTAGWSYDAFDAWGQGTLVTVSS

[0092] SEQ ID NO: 74 - Anti-BCAN scFv "HRB297" V L Polypeptide Sequence TQSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYDWRSSGSVFGGGTKLTVLGAA

[0093] SEQ ID NO: 75 - Anti-TNC scFv "E10" V H Polypeptide Sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFSGSRMGWVRQAPGKGLEWVSAINEEGGQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHPPHRPFDYWGQGTLVTVSR

[0094] SEQ ID NO: 76 - Anti-TNC scFv "E10" V L Polypeptide Sequence SSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSSHGPRRPVVFGGGTKLTVLG

[0095] SEQ ID NO: 77 - Anti-TNC scFv "P12" V H Polypeptide Sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFGQYSMSWVRQAPGKGLEWVSAITGTGGETYYADSVEGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGRRIFDYWGQGTLVTVSR

[0096] SEQ ID NO: 78 - Anti-TNC scFv "P12" V L Polypeptide Sequence SSELTQDPAVSVALGQTVRITCQGDSLRRQPASWYQQKPGQAPVLVIYYKKLRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSFSPKPKPVVFGGGTKLTVLG

[0097] SEQ ID NO: 79 - Anti-TNC scFv "F16" V H Polypeptide Sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYGASWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAHNAFDYWGQGTLVTVSR

[0098] SEQ ID NO: 80 - Anti-TNC scFv "F16" V L Polypeptide Sequence SSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSSVYTMPPVVFGGGTKLTVLG

[0099] The CDRs of SEQ ID NOs: 83-93 are annotated as follows:

[0100] [ka]

[0101] CDRs were predicted with 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 W1, 8 July 2016, Pages W474-W478, https: / / doi.org / 10.1093 / nar / gkw361).

[0102] [ka]

[0103] [ka]

[0104] [ka]

[0105] [ka]

[0106] [ka]

[0107] [ka]

[0108] [ka]

[0109] [ka]

[0110] [ka]

[0111] [ka]

[0112] [ka]

[0113] SEQ ID NO: 94 - Anti-BCAN "RB826" VHH polynucleotide sequence GAGGTTCAACTGCAGGAGAGCGGCGGTGGATTTGTGCAAGCAGGGGGGTCCTTGCGATTATCATGTGCTGCATCGGGGCGGCTGAGACCATTCGAACGTATGGGCTGGTTCCGCCAGGCACCTGGTAAAGAGAGGGAGTTTGTGGCCGCCATAAGCGTGCACGATGAAGTGTTTCCGTACTACGCCGATTCCGTCAAGGGCAGGTTTACCATTAGTAGAGACAATGCGAAAAACACAGTCTATCTTCAGATGAACTCTCTCAAGCCCGAAGATACTGCTACATATTACTGCGCTTTCTTCATCATGGACGACGTTAAGTATTGGGGACAGGGAACCCAGGTAACGGTGTCATCT

[0114] SEQ ID NO: 95 - Anti - BCAN "RB827" VHH Polynucleotide Sequence GAGGTGCAATTGCAGGAGAGCGGCGGTGGATTTGTGCAGGCCGGAGGGTCCCTCCGACTTTCATGCGCTGCATCTGGGGAAAGCTTCGTTCCAGAAATGGGCTGGTTCCGCCAGGCACCTGGTAAAGAGCGGGAGTTTGTGGCCGCCATAAGCGTAGAACAGAGTCTGGATATGTATTACGCCGATTCCGTCAAGGGCAGGTTTACCATTAGTAGAGACAATGCGAAAAACACTGTCTATCTGCAGATGAATTCTTTAAAGCCCGAGGATACTGCTACATACTACTGTGCTATCGTTGCTATCGACGACTTCAACACATATTGGGGACAGGGGACCCAAGTGACGGTGTCATCC

[0115] SEQ ID NO: 96 - Anti - BCAN "RB828" VHH Polynucleotide Sequence GAGGTGCAATTGCAGGAGAGTGGCGGGGGATTTGTGCAAGCAGGAGGGTCCCTCCGACTTTCATGCGCTGCATCTGGTGTGAGCCTGAGAACCCGGAGCATGGGCTGGTTCCGCCAGGCACCAGGTAAAGAGAGGGAATTTGTTGCCGCCATCAGCCGTCACAACGATCATGAATTCTATTACGCCGATTCCGTCAAGGGCAGGTTCACCATTAGTAGAGACAATGCTAAGAACACAGTCTATTTACAGATGAATTCCCTCAAGCCCGAAGACACTGCTACGTACTACTGTGCTAAACTGCCTGTTAACCCGAATCTGCACTATTGGGGACAGGGGACACAGGTAACTGTGTCATCT

[0116] SEQ ID NO: 97 - Anti - BCAN "RB829" VHH Polynucleotide Sequence GAGGTGCAATTACAGGAAAGCGGCGGTGGATTTGTCCAGGCTGGGGGGTCCCTCCGACTGTCATGTGCTGCAAGCGGTGTGCACGTCCCTCTGCAGAACATGGGCTGGTTCCGCCAAGCACCGGGGAAAGAGAGGGAGTTCGTTGCCGCCATTAGCCGGGATATGCCAGTCGATAACTATTACGCCGACTCCGTGAAGGGCAGGTTTACCATCAGTAGAGACAATGCGAAAAACACGGTGTACCTTCAGATGAATAGTTTGAAGCCCGAAGACACTGCTACATATTACTGCGCTGTTCGTGTGTACACTACCTCTCTGTGGTATTGGGGACAGGGAACCCAGGTAACAGTGTCATCT

[0117] SEQ ID NO: 98 - Anti - CSPG4 "RB830" VHH Polynucleotide Sequence GAGGTGCAGCTGCAGGAGAGCGGCGGGGGATTTGTCCAAGCTGGTGGGTCCTTGCGACTGTCATGCGCTGCATCGGGTAACGTTCAGAGACGGTTCAGAATGGGCTGGTTCCGCCAGGCACCTGGGAAAGAGAGGGAATTTGTTGCCGCCATTAGCACAAACCGGGATAGGCGCAACTATTACGCCGACTCCGTCAAGGGCAGGTTTACCATCAGTCGTGACAATGCGAAAAACACGGTGTACCTTCAGATGAATTCTCTCAAGCCCGAAGATACTGCTACATATTACTGTGCTGTGATGAATAAGAATTTCACTTACATGTATTGGGGACAGGGAACCCAAGTAACCGTGTCATCT

[0118] Accession No. 99 - Anti - CSPG4 "RB831" VHH Polynucleotide Sequence GAGGTACAGCTTCAAGAAAGCGGCGGGGGATTCGTCCAGGCGGGTGGGTCCTTGCGGCTGTCATGCGCTGCATCTGGGGAACCAGTGCACAGTACAAGTATGGGCTGGTTCCGCCAAGCACCGGGTAAAGAGAGGGAGTTTGTTGCCGCCATCAGCCTGAACGTTATGCACAGCAGATATTACGCCGATTCCGTCAAGGGCCGATTTACCATTTCGCGTGACAATGCAAAAAACACCGTGTATTTACAGATGAATTCCCTCAAGCCCGAAGACACTGCTACGTATTACTGTGCTTCTTACCCTCATTACATGACTCCCATGTATTGGGGACAGGGAACCCAGGTGACAGTGTCATCT

[0119] Accession No. 100 - Anti - PTPRZ1 "RB832" VHH Polynucleotide Sequence GAGGTGCAATTACAGGAAAGCGGCGGTGGATTTGTCCAGGCAGGTGGGTCCCTCCGACTCTCATGCGCCGCATCTGGGAGCGACGTCACACGTCTGAACATGGGCTGGTTCCGCCAGGCACCTGGGAAGGAGAGGGAGTTTGTTGCCGCCATTAGCAGAAGTGAACAGAACCGGCTGTACTACGCCGATTCCGTGAAGGGCAGGTTCACCATCAGTAGAGACAATGCGAAAAACACCGTTTATCTTCAGATGAATTCTTTGAAGCCCGAAGATACTGCTACCTATTACTGTGCAACAACAGCTCTGGCTGCTGTGACTAAAGCCACTCACTATTGGGGACAGGGAACCCAAGTAACGGTGTCATCC

[0120] SEQ ID NO: 101 - Anti-PTPRZ1 "RB833" VHH Polynucleotide Sequence GAGGTACAGCTGCAAGAGAGCGGCGGGGGATTTGTCCAGGCTGGTGGGTCCCTCCGACTTTCATGTGCTGCATCTGGTAACGTTGTGTTCCTGACGTCGATGGGCTGGTTCCGCCAGGCACCCGGGAAGGAGAGGGAATTTGTTGCCGCCATAAGCCGGAGCTTCTTCGATGATCCATATTACGCCGATTCCGTCAAGGGCAGGTTTACCATTAGTAGAGACAATGCAAAAAACACAGTGTATTTACAGATGAATAGTTTGAAGCCCGAAGACACTGCTACATATTACTGCGCTCCGTACAAAAGAGACTACCGGCAGCACACTGTGCCTCGTCATATCTATTGGGGACAGGGAACCCAAGTGACCGTGTCATCT

[0121] SEQ ID NO: 102 - Anti-PTPRZ1 "RB834" VHH Polynucleotide Sequence GAGGTGCAGCTTCAAGAGAGCGGCGGGGGATTTGTCCAAGCTGGTGGGTCCCTCCGATTATCATGCGCTGCATCTGGGAGCAGTAGTAGACTGTTCAACATGGGCTGGTTCCGCCAGGCACCTGGTAAAGAGCGGGAATTTGTGGCCGCCATTAGCCACATGGAAAACGATGTTGATTATTACGCCGATTCCGTGAAGGGCAGGTTCACCATCTCCAGAGACAATGCGAAGAACACAGTCTATTTGCAGATGAATTCTCTCAAGCCCGAAGACACTGCTACTTATTACTGTGCTCTGATGCTGAAAGGCTGGGACCATTCGACACGTTACTACTGGGGACAGGGAACCCAGGTAACGGTGTCATCT

[0122] SEQ ID NO: 103 - Anti-TNC "RB835" VHH Polynucleotide Sequence GAGGTACAGCTTCAGGAGAGCGGCGGCGGATTTGTGCAAGCAGGTGGGTCCCTCCGATTATCATGCGCTGCATCCGGTAACCTGATGGTCCGTCGGGAAATGGGCTGGTTCCGCCAGGCACCTGGGAAAGAGAGGGAGTTCGTTGCCGCCATTAGCAGAAGCAGTCAGGAGGAAGTTTATTACGCCGACTCCGTCAAGGGCAGGTTTACCATCAGTAGAGACAATGCGAAAAACACAGTGTATCTGCAGATGAATTCTTTGAAGCCCGAAGATACTGCTACGTATTACTGTGCTATGGAAGGGTTCTACGTGTACAACCAGTATTGGGGACAGGGAACCCAAGTGACAGTGTCATCT

[0123] SEQ ID NO: 104 - Anti-TNC "RB836" VHH Polynucleotide Sequence GAGGTGCAACTGCAGGAGAGCGGCGGGGGATTTGTGCAGGCAGGGGGGTCCCTCCGACTTTCATGCGCTGCATCCGGTAGAAGAGTAACGGTCAGTGAAATGGGCTGGTTCCGCCAGGCACCTGGTAAAGAGAGGGAGTTTGTTGCCGCCATCAGCATGCGGGAACGGGAAAGCATGT ATTACGCCGACTCCGTGAAGGGCAGGTTCACCATTAGTCGTGACAATGCTAAAAACACCGTCTATCTGCAGATGAACTCGTTGAAGCCCGAAGATACTGCTACATATTACTGTGCTGAGTACACTCACTGGTACTCTCATCCATATTGGGGACAGGGAACCCAAGTTACAGTGTCATCT

[0124] The hinge sequence, transmembrane domain sequence, and intracellular domain sequence are shown below.

[0125] SEQ ID NO:117 - Human CD8a hinge polypeptide sequence TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

[0126] SEQ ID NO:118 - Human IgG4 hinge polypeptide sequence ESKYGPPCPPCP

[0127] SEQ ID NO:119 - Human IgG1 hinge polypeptide sequence EPKSPDKTHTCP

[0128] SEQ ID NO:120 - Human IgG1 hinge polypeptide sequence EPKSCDKTHTCP

[0129] SEQ ID NO: 121 - Human IgG1 long hinge polypeptide sequence AEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPK

[0130] SEQ ID NO: 122 - Human CD8a transmembrane domain polypeptide sequence IYIWAPLAGTCGVLLLSLVITLYC

[0131] SEQ ID NO: 123 - Human CD28 transmembrane domain polypeptide sequence FWVLVVVGGVLACYSLLVTVAFIIFWV

[0132] SEQ ID NO:124 - Human 4-1BB intracellular domain polypeptide sequence KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0133] SEQ ID NO: 125 - Human CD28 intracellular domain polypeptide sequence RSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

[0134] SEQ ID NO: 126 - Human CD3z intracellular domain polypeptide sequence No. 1 RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0135] SEQ ID NO: 127 - Human CD3z intracellular domain polypeptide sequence No. 2 RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0136] SEQ ID NO:128 - Human CD3z intracellular domain polypeptide sequence No. 3 RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQARRA

[0137] SEQ ID NO: 129 - Human CD8a hinge polynucleotide sequence ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT

[0138] SEQ ID NO: 130 - Human IgG4 hinge polynucleotide sequence GAGTCAAAGTATGGGCCTCCATGTCCTCCATGTCCG

[0139] SEQ ID NO:131 - Human IgG1 hinge polynucleotide sequence GAGCCGAAGTCGCCAGACAAAACTCACACTTGTCCT

[0140] SEQ ID NO: 132 - Human IgG1 long hinge polynucleotide sequence GCAGAGCCGAAGTCGCCAGACAAAACTCACACTTGTCCTCCTTGTCCAGCCCCCCCCGTGGCGGGTCCCAGCGTGTTCCTGTTTCCTCCGAAGCCAAAAGATACCCTGATGATCGCACGCACCCCCGAAGTAACGTGCGTGGTGGTCGATGTGTCACATGAGGACCCTGAGGTCAAATTCAATTGGTACGTTGACGGGGTAGAAGTTCACAACGCTAAAACCAAGCCAAGGGAGGAGCAGTACAACAGCACCTATCGAGTGGTGAGTGTACTGACCGTCCTACACCAAGATTGGTTGAATGGCAAGGAATACAAGTGTAAGGTGTCCAACAAGGCTTTACCTGCTCCTATCGAGAAGACAATTTCTAAGGCCAAAGGCCAGCCCAGAGAGCCACAGGTTTACACACTCCCACCATCACGTGACGAGCTTACGAAAAATCAGGTCAGTCTGACTTGCCTCGTTAAAGGATTTTACCCTAGTGACATAGCCGTGGAATGGGAAAGCAACGGCCAGCCCGAGAATAATTATAAAACAACACCGCCCGTGCTCGACTCTGATGGTTCTTTTTTCCTGTATTCCAAACTGACCGTCGATAAGAGCCGGTGGCAGCAGGGAAACGTGTTCTCCTGCTCCGTCATGCATGAAGCCTTGCATAACCACTATACTCAAAAGTCACTGTCTCTTAGCCCTGGGAAGAAAGATCCCAAG

[0141] SEQ ID NO: 133 - Human CD8a transmembrane domain polynucleotide sequence ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC

[0142] SEQ ID NO: 134 - Human CD28 transmembrane domain polynucleotide sequence TTCTGGGTGCTGGTGGTCGTGGGCGGCGTGCTGGCCTGTTACAGCCTGCTCGTGACCGTGGCCTTCATCATCTTTTGGGTC

[0143] SEQ ID NO:135 - Human 4-1BB intracellular domain polynucleotide sequence AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG

[0144] SEQ ID NO: 136 - Human CD28 intracellular domain polynucleotide sequence CGAAGCAAGCGGAGCAGAGGCGGCCACAGCGACTACATGAACATGACCCCCAGACGGCCTGGCCCCACCCGGAAGCACTACCAGCCTACGCCCCTCCCAGAGACTTCGCCGCCTACAGAAGC

[0145] SEQ ID NO: 137 - Human CD3z intracellular domain polynucleotide SEQ ID NO: 1 AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAA GGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC

[0146] SEQ ID NO: 138 - Human CD3z intracellular domain polynucleotide SEQ ID NO: 2 AGAGTGAAGTTCAGCCGCAGCGCCGACGCCCCTGCCTACCAGCAGGGACAGAACCAGCTGTACAACGAGCTGAACCTGGGCAGACGGGAAGAGTACGACGTGCTGGACAAGCGGAGAGGCCGGGACCCTGAGATGGGCGGCAAGCCCCAGCGGCGGAAGAACCCCCAGGAAGGCCTGTATAACGAACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGCGGCGGAGAGGCAAGGGCCACGATGGCCTGTACCAGGGCCTGAGCACCGCCACCAAGGACACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCTAGG

[0147] SEQ ID NO: 139 - Human CD3z intracellular domain polynucleotide SEQ ID NO: 3 AGAGTGAAGTTCAGCCGCAGCGCCGACGCCCCTGCCTACCAGCAGGGACAGAACCAGCTGTACAACGAGCTGAACCTGGGCAGACGGGAAGAGTACGACGTGCTGGACAAGCGGAGAGGCCGGGACCCTGAGATGGGCGGCAAGCCCCAGCGGCGGAAGAACCCCCAGGAAGGCCTGTATAACGAACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGCGGCGGAGAGGCAAGGGCCACGATGGCCTGTACCAGGGCCTGAGCACCGCCACCAAGGACACCTACGACGCCCTGCACATGCAGGCCCggcgcgcc

[0148] Detailed Description of the Invention The present invention relates to immune effector cells or populations of immune effector cells expressing one or more chimeric antigen receptors (CARs) specific for one or more glioma-associated antigens. The antigens receptor tyrosine-protein phosphatase zeta (PTPRZ1), brevican core protein (BCAN), chondroitin sulfate proteoglycan 4 (CSPG4), and tenascin (TNC) have recently been found to be expressed in gliomas (Dutoit et al., Brain 135.4 (2012):1042-1054). Multipeptide vaccines containing peptides from these antigens have been created (Dutoit et al., Oncoimmunology 7.2 (2018):e1391972; Migliorini et al., Neuro-oncology 21.7 (2019):923-933) and are currently in clinical trials (NCT03665545, NCT02924038).

[0149] The inventors have shown that immune effector cells expressing CARs specific for one or more glioma-associated antigens can generate specific immune responses against cells expressing the glioma-associated antigens, as measured by cytotoxicity assays and T cell activation.

[0150] To date, CAR T cell approaches for recurrent GBM (targeting EGFRvIII, IL13Rα2, and Her2) have been clinically studied in a monovalent format, i.e., targeting one antigen at a time. While some patients demonstrated disease stabilization, tumors invariably recurred, and in some cases, the epitopes targeted by the monovalent CAR T cells used were lost. The present inventors have identified new glioma-associated antigens that were targeted in a monovalent format and also demonstrated efficacy in multivalent targeting approaches. Bielamowicz et al. (Neuro-oncology 20.4(2018):506-518) described a multivalent approach in which T cells were engineered to express three CAR molecules with different antigen specificities. Thus, in one aspect, the present invention provides immune effector cells expressing two or more CARs specific for different glioma-associated antigens. In one embodiment, the present invention provides a population of immune effector cells, comprising at least two different CAR-expressing immune effector cells, wherein each different CAR-expressing immune effector cell is specific for different glioma-associated antigens.Typically, one or more of the glioma-associated antigens are selected from PTPRZ1, BCAN, CSPG4 and TNC.In some cases, two or more of the glioma-associated antigens are selected from PTPRZ1, BCAN, CSPG4 and TNC.

[0151] The present invention also provides immune effector cells or populations of immune effector cells expressing a chimeric antigen receptor (CAR) specific to PTPRZ1. Furthermore, the present invention provides immune effector cells or populations of immune effector cells expressing a chimeric antigen receptor (CAR) specific to BCAN. Surprisingly, the present inventors have demonstrated for the first time that immune effector cells expressing a CAR specific to PTPRZ1 or BCAN are therapeutically useful, particularly for the treatment of glioblastoma. Any antigen-binding domain specific to PTPRZ1 or BCAN known in the art may be used. Preferably, the antigen-binding domain is an antigen-binding domain disclosed herein.

[0152] In some cases, as described in the Examples of this application, the present invention utilizes the "bystander" effect. For example, immune effector cells of the present invention comprising one or more CARs specific for glioma-associated antigens may be able to kill a mixture of cancer cells that express glioma-associated antigens and cancer cells that do not express glioma-associated antigens. Cancer cells that do not express glioma-associated antigens are thought to be killed via soluble factors, which may lead to a more effective treatment.

[0153] Furthermore, lentivirally transduced CAR T cells are associated with toxicities such as cytokine release syndrome and neurotoxicity. In certain embodiments, to overcome these problems, the immune effector cells of the present invention are transduced with RNA.

[0154] Chimeric Antigen Receptor 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 signaling domain. CARs may comprise an extracellular antigen-binding domain, a transmembrane domain, and an intracellular cytoplasmic signaling domain. CARs may also comprise an extracellular spacer and / or a hinge between the transmembrane domain and the antigen-binding domain and / or between the transmembrane domain and the cytoplasmic signaling domain. Preferably, CARs comprise a hinge between the transmembrane domain and the antigen-binding domain.

[0155] The cytoplasmic signaling domain may comprise an activation domain, which, upon association with the extracellular domain (e.g., scFv), serves to activate immune effector cells. The cytoplasmic signaling domain may comprise one or more of: (i) a CD3ζ (zeta) activation domain, (ii) a 4-1BB (CD137) activation domain, (iii) a CD3ε (epsilon) activation domain, (iv) an OX40 (CD134) activation domain, (v) a CD28 activation domain, and / or (vi) a CD27 activation domain.

[0156] The CD3 zeta activation domain is included in the signaling domain of the first generation CAR. Preferably, the cytoplasmic domain includes a CD3 zeta activation domain.

[0157] The second generation CAR contains a CD3 zeta activation domain and a CD28 activation domain. The cytoplasmic domain contains a CD3 zeta activation domain and a CD28 activation domain.

[0158] Third generation CARs contain additional domains such as the 4-1BB activation domain or the OX40 (CD134) activation domain. Preferably, the cytoplasmic domain contains the 4-1BB activation domain and the CD3ζ (zeta) activation domain.

[0159] The cytoplasmic signaling domain may comprise a 4-1BBz domain, which includes CD3 zeta and a 4-1BBz activation domain. The cytoplasmic signaling domain may comprise a CD28z domain, which includes CD3 zeta and a CD28 activation domain. As shown in this example, a CAR comprising a CD28z domain is particularly useful in the present invention, for example, when immune effector cells are transduced with RNA encoding the CAR. The cytoplasmic signaling domain may comprise a 4-1BBz + CD28z domain, which includes CD3 zeta, CD28, and a 4-1BBz activation domain.

[0160] The cytoplasmic signaling domain may comprise a CD3ζ (zeta) activation domain alone or in combination with a CD28, CD27, OX-40 (CD134) and / or 4-1BB (CD137) domain.

[0161] Other activation domains include IL-15Rα, CD2, CDS, ICAM-1, LTA-1 and ICOS and can be used in combination with the above activation domains.

[0162] When the immune effector cell expressing the CAR is a phagocyte, the intracellular signaling domain can include the intracellular domain of Megf10 or FcRv.

[0163] 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: 124, e.g., at least 80%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO: 124. In some cases, the CD28 activation domain comprises or consists of an amino acid sequence having at least 70% identity to SEQ ID NO: 125, e.g., at least 80%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO: 125. In some cases, the CD3 zeta activation domain comprises or consists of an amino acid sequence having at least 70% identity to any one of SEQ ID NOs: 126-128, e.g., at least 80%, at least 90%, at least 95%, at least 99%, or 100% identity to any one of SEQ ID NOs: 126-128. For example, the 4-1BBz domain may include an amino acid sequence having at least 70% identity to SEQ ID NO: 124 (e.g., at least 80%, at least 90%, at least 95%, at least 99% or 100% identity to SEQ ID NO: 124) and an amino acid sequence having at least 70% identity to any one of SEQ ID NOs: 126-128 (e.g., at least 80%, at least 90%, at least 95%, at least 99% or 100% identity to any one of SEQ ID NOs: 126-128). Similarly, the CD28z domain may comprise an amino acid sequence having at least 70% identity to SEQ ID NO: 125 (e.g., at least 80%, at least 90%, at least 95%, at least 99% or 100% identity to SEQ ID NO: 125), and an amino acid sequence having at least 70% identity to any one of SEQ ID NOs: 126-128 (e.g., at least 80%, at least 90%, at least 95%, at least 99% or 100% identity to any one of SEQ ID NOs: 126-128).

[0164] 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: 135, e.g., 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: 135. 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: 136, e.g., 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: 136. In some cases, the CD3 zeta 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: 137-139, e.g., 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: 137-139. For example, the 4-1BBz domain may include an amino acid sequence having at least 70% identity to the amino acid sequence encoded by SEQ ID NO: 135 (e.g., 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: 135), and an amino acid sequence having at least 70% identity to the amino acid sequence encoded by any one of SEQ ID NOs: 137-139 (e.g., 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: 137-139).Similarly, the CD28z domain may comprise an amino acid sequence having at least 70% identity to the amino acid sequence encoded by SEQ ID NO: 136 (e.g., 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: 136), and an amino acid sequence having at least 70% identity to the amino acid sequence encoded by any one of SEQ ID NOs: 137-139 (e.g., 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: 137-139).

[0165] The transmembrane domain spans the cell membrane, for example, the cell membrane of a eukaryotic cell. The transmembrane domain plays a role in transmitting an activation signal to the cytoplasmic signaling domain after ligand binding of the extracellular antigen-binding domain (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 CD8α. The transmembrane domain may also be the transmembrane domain of the α, β, δ, or γ subunit of the T cell receptor, CD3ε, CD3ζ, CD4, CD6, CD8α, CD28, CD86, OX-40, 4-1BB, or CD40L (CD154). The transmembrane domain may also be the transmembrane domain of CD8, for example, when the immune effector cell is an NK cell.

[0166] In some cases, the CD8α transmembrane domain comprises or consists of an amino acid sequence having at least 70% identity to SEQ ID NO: 122, e.g., at least 80%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO: 122. In some cases, the CD28 transmembrane domain comprises or consists of an amino acid sequence having at least 70% identity to SEQ ID NO: 123, e.g., at least 80%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO: 123.

[0167] In some cases, the CD8α 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: 133, e.g., 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: 133. 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: 134, e.g., 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: 134.

[0168] The CAR may include a hinge connecting the transmembrane domain and the extracellular domain. The hinge may provide steric effects that affect activation, cytotoxicity, and the strength of signaling from target cells and their surface receptors. In some cases, the hinge may be derived from a region of another immune molecule, such as IgG1, IgG2, IgG3, IgG4, CD8 (e.g., a CD8α hinge), or CD28. The hinge may be of any suitable length. The hinge may be at least 1 amino acid long, e.g., at least 5 amino acids long, at least 10 amino acids long, or at least 20 amino acids long. The hinge may be 100 amino acids or less long, e.g., 80 amino acids or less, 60 amino acids or less, 40 amino acids or less, 30 amino acids or less, or 20 amino acids or less. The hinge may be 1 to 40 amino acids long, e.g., 2 to 30 amino acids long, 3 to 25 amino acids long, 4 to 20 amino acids long, or 5 to 15 amino acids long. The hinge may include glycine and serine, threonine, and / or alanine residues. However, the hinge may comprise any suitable residues.

[0169] In some cases, the hinge is an IgG1 hinge, e.g., a human IgG1 hinge. In some cases, the hinge is an IgG1 hinge (e.g., a human IgG1 hinge) that further comprises one or more constant domains such as CH2 or CH3. In some cases, the hinge is an IgG4 hinge, e.g., a human IgG4 hinge. In some cases, the hinge is a CD8α hinge, e.g., a human CD8α hinge. In some cases, the CD8α hinge comprises or consists of an amino acid sequence having at least 70% identity to SEQ ID NO: 117, e.g., at least 80%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO: 117. In some cases, the IgG4 hinge comprises or consists of an amino acid sequence having at least 70% identity to SEQ ID NO: 118, e.g., at least 80%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO: 118. The IgG4 hinge set forth in SEQ ID NO: 118 is referred to herein as a "short" hinge. In some cases, the IgG1 hinge comprises or consists of an amino acid sequence having at least 70% identity to any one of SEQ ID NOs: 119-121, e.g., at least 80%, at least 90%, at least 95%, at least 99%, or 100% identity to any one of SEQ ID NOs: 119-121. The IgG1 hinge of SEQ ID NO: 121 is referred to herein as the "long" hinge.

[0170] In some cases, the CD8α hinge comprises or consists of an amino acid sequence having at least 70% identity to the amino acid sequence encoded by SEQ ID NO: 129, e.g., 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: 129. 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: 130, e.g., 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: 130. In some cases, the IgG1 hinge comprises or consists of an amino acid sequence having at least 70% identity to the amino acid sequence encoded by SEQ ID NO: 131 or 132, e.g., 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: 131 or 132.

[0171] 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 CD8α transmembrane domain and a CD8α hinge.The transmembrane domain and the hinge may be a CD28 transmembrane domain and a CD28 hinge.

[0172] In some cases, the CAR may comprise a "short" CD28z construct, i.e., a construct 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: 125 (e.g., at least 80%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO: 125) and an amino acid sequence having at least 70% identity to any one of SEQ ID NOs: 126-128 (e.g., at least 80%, at least 90%, at least 95%, at least 99%, or 100% identity to any one of SEQ ID NOs: 126-128). The IgG4 hinge may comprise or consist of an amino acid sequence having at least 70% identity to SEQ ID NO: 118, e.g., at least 80%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO: 118.

[0173] In some cases, the CAR may comprise a "long" CD28z construct, i.e., a construct comprising a long IgG1 hinge (including 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: 125 (e.g., at least 80%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO: 125) and an amino acid sequence having at least 70% identity to any one of SEQ ID NOs: 126-128 (e.g., at least 80%, at least 90%, at least 95%, at least 99%, or 100% identity to any one of SEQ ID NOs: 126-128). The IgG1 hinge may comprise or consist of an amino acid sequence having at least 70% identity to SEQ ID NO: 121, e.g., at least 80%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO: 121.

[0174] The "short" and "long" 28z constructs typically comprise a CD28 transmembrane domain. For example, the "short" and "long" 28z constructs may further comprise an amino acid sequence having at least 70% identity to SEQ ID NO: 123, such as at least 80%, at least 90%, at least 95%, at least 99%, or 100% identity to SEQ ID NO: 123.

[0175] A 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.

[0176] In some cases, the present invention relates to a CAR specific to PTPRZ1. In some cases, the present invention relates to a CAR specific to BCAN. The present inventors have surprisingly shown for the first time that immune effector cells expressing CAR specific to PTPRZ1 or BCAN are useful for treatment, particularly in the treatment of glioblastoma. Any antigen binding domain specific to PTPRZ1 or BCAN known in the art may be used for CAR. Preferably, the antigen binding domain is the antigen binding domain disclosed herein.

[0177] antigen-binding domain The CAR discussed herein comprises an antigen-binding domain. The antigen-binding domain can be any domain that specifically binds to a glioma-associated antigen. For example, the antigen-binding domain can be any domain that specifically binds to a glioma-associated antigen. For example, the antigen-binding domain can be any domain that specifically binds to a glioma-associated antigen, such as an scFv, a monoclonal antibody (comprising two heavy chains and two light chains), a polyclonal antibody, Fab, Fab', or F(ab')2 fragment, a heavy chain variable domain (V), or a glioma-associated antigen (ALA). H ), or nanobody (V HH ) may also be used.

[0178] In some cases, the antigen-binding domain comprises one or more immunoglobulin variable domains. For example, a 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). CDRs or ABRs typically are responsible for antigen specificity, for example, by directly interacting with an antigen. The immunoglobulin variable domain also comprises framework regions that provide the immunoglobulin-like structure of the domain and typically do not directly interact with an antigen. Immunoglobulin variable domains include scFv domains, antibody domains (e.g., V H and / or V L ) domains, typically human, Fab, Fab', F(ab')2 fragments, V HH Domains and V NAR The antigen-binding domain may be selected from an immunoglobulin variable domain from the V domain. Thus, the antigen-binding domain may comprise an scFv domain, which typically comprises two immunoglobulin variable domains (e.g., V H and V L In some cases, the antigen-binding domain comprises a V HH It may also comprise a domain, which typically comprises a single immunoglobulin variable domain.

[0179] Preferably, the antigen binding domain is an scFv or V HH The scFv domain is a heavy chain variable domain (V H ) and the light chain variable domain (V L ) and are linked by a short linker peptide. Exemplary scFv domains of the present invention are shown in SEQ ID NOS: 21 to 40. scFv may be derived from a human immunoglobulin. scFv may be derived from a mouse immunoglobulin.

[0180] scFv is typically V H -V L are arranged in the N-terminal-C-terminal direction, but V L -V HThe present invention also encompasses antigen-binding regions in which the following are arranged in the N-terminal-C-terminal direction:

[0181] The optional linker may be (V H ) and (V L ) domains. In SEQ ID NOs: 21 to 40, the linker GGGGSGGGGSGGGGS (SEQ ID NO: 81) is used. The linker may also include SSSGGGGSGGGGSGGGGSS (SEQ ID NO: 82).

[0182] In some embodiments, the CARs described herein are selected from CARs 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 the amino acid sequence of any one of SEQ ID NOs: 21-40.

[0183] In some embodiments, the CAR described herein comprises a V or VL 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: 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, and 79, respectively. H domain, and V 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: 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78 and 80. L The scFv further comprises a linker sequence, which may be, for example, any of the linker sequences described above.

[0184] In some embodiments, the CAR described herein comprises a V scFv selected from SEQ ID NOs: 21-40. H and V L It contains an scFv domain with an antigen-binding region (ABR) of the domain. H and V L The framework regions of a domain are regions of an scFv outside of the ABR and linker sequences. In some cases, the CARs described herein comprise a V domain of an scFv selected from SEQ ID NOs: 21-40. H and V L The scFv domains include ABRs of the domains, and scFv domains 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 scFv selected from SEQ ID NOS: 21-40. The term "antigen-binding region" in an scFv is used to refer to the complementarity-determining regions (CDRs) of the variable heavy and variable light chain domains that make up the scFv. For example, the scFvs of SEQ ID NOS: 21-40 are labeled with six ABRs. For each scFv, the first ABR1, ABR2, and ABR3 correspond to heavy chain CDR1, CDR2, and CDR3, respectively, and the second set of ABR1, ABR2, and ABR3 in the sequence correspond to light chain CDR1, CDR2, and CDR3.

[0185] In some cases, a CAR described herein is selected from a CAR comprising a polypeptide, such as one or more polypeptides comprising heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) and light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) from an amino acid sequence selected from any one of SEQ ID NOs: 21-40. In some cases, a CAR is selected from a CAR comprising a polypeptide, such as one or more polypeptides comprising HCDR1-3 and LCDR1-3 of an amino acid sequence selected from any one of SEQ ID NOs: 21-40, and 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 a polypeptide selected from any one of SEQ ID NOs: 21-40. 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 the specific sequenced CDRs disclosed herein. Specifically, the scFv sequences set forth in SEQ ID NOs: 21-40 comprise, from N-terminus to C-terminus, an antibody heavy chain variable region, a linker, and an antibody light chain variable region. The separate heavy and light chain variable regions of SEQ ID NOs: 21-40 are provided in SEQ ID NOs: 41-80. Exemplary conventions that can be used to identify CDR boundaries include the Kabat definition, the Chothia definition, and the IMGT definition (see, e.g., 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 above. For example, heavy chain CDR1, CDR2, and CDR3 may be the first ABR1, ABR2, and ABR3 identified for scFvs (i.e., preceding the linker), respectively, and light chain CDR1, CDR2, and CDR3 may be the second ABR1, ABR2, and ABR3 identified for scFvs (i.e., following the linker), respectively.

[0186] An scFv having an ABR of SEQ ID NO: 21, or a polypeptide having CDRs of SEQ ID NOs: 41 and 42, comprises a first ABR1 (HCDR1) having the sequence FTFSSYAMH (SEQ ID NO: 105), a first ABR2 (HCDR2) having the sequence WVAVISYDGSNKYY (SEQ ID NO: 106), a first ABR3 (HCDR3) having the sequence RGSGYSYGPGYDAFDI (SEQ ID NO: 107), a second ABR1 (LCDR1) having the sequence SGSIASNYVQ (SEQ ID NO: 108), a second ABR2 (LCDR2) having the sequence TTVIYEDNQRPS (SEQ ID NO: 109), and a second ABR3 (LCDR3) having the sequence QSWDPVFG (SEQ ID NO: 110). Similarly, an scFv having an ABR of SEQ ID NO: 22, or a polypeptide having CDRs of SEQ ID NOs: 43 and 44, comprises a first ABR1 (HCDR1) having the sequence GTFSSYAIS (SEQ ID NO: 111), a first ABR2 (HCDR2) having the sequence WMGGIIPIFGTANY ​​(SEQ ID NO: 112), a first ABR3 (HCDR3) having the sequence REGGAVGYYYGMDV (SEQ ID NO: 113), a second ABR1 (LCDR1) having the sequence SSDVGGYNYVS (SEQ ID NO: 114), a second ABR2 (LCDR2) having the sequence LMIYEVSNRPS (SEQ ID NO: 115), and a second ABR3 (LCDR3) having the sequence SSYDRSNRSM (SEQ ID NO: 116). The same applies to any of SEQ ID NOs: 21 to 80, and similar considerations apply to CDRs 1 to 3 of each of SEQ ID NOs: 83 to 93.

[0187] The antigen-binding domain is either scFv or V HH (also known as VHH, nanobody, sdAb or single domain antibody; the terms are used interchangeably herein).

[0188] In some cases, the antigen binding domain is preferably V HH V HH The domain consists of a single heavy chain variable domain (V H ) and lacks the constant domains found in typical antibodies. HHThe domains are shown in SEQ ID NOs: 83 to 93.

[0189] In some cases, the CARs described herein comprise a polypeptide comprising a heavy chain complementarity determining region (HCDR1, HCDR2, and HCDR3) and a light chain complementarity determining region (LCDR1, LCDR2, and LCDR3) from an amino acid sequence selected from any one of SEQ ID NOs: 21-40. The polypeptide may comprise two immunoglobulin variable domains, e.g., may comprise an scFv. In some cases, the CARs described herein comprise a polypeptide, typically a VFv, comprising a complementarity determining region (CDR1, CDR2, and CDR3) from an amino acid sequence selected from any one of SEQ ID NOs: 83-93. HH , including.

[0190] In some cases, the CAR 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 any one of SEQ ID NOs: 21-40 and 83-93. Typically, the CAR comprises the exact CDRs of the SEQ ID NO from which the CAR is derived.

[0191] glioma-associated antigens Glioma-associated antigens are antigens associated with malignant gliomas (glioblastomas, GBM), for example, expressed by malignant glioma cells.Glioma-associated antigens can be human glioma-associated antigens.Compared with normal brain tissues and non-CNS normal tissues, if the antigen is overexpressed in malignant glioma samples, the antigen is associated with malignant glioma.Glioma-associated antigens can be associated with glioma development.

[0192] The glioma-associated antigen may be selected from receptor tyrosine-protein phosphatase zeta (PTPRZ1), brevican core protein (BCAN), chondroitin sulfate proteoglycan 4 (CSPG4) and tenascin (TNC).

[0193] PTPRZ1, CSPG4, and BCAN can each be considered as a cell surface (glioma) marker. TNC and BCAN can each be considered as, for example, a tumor-infiltrating extracellular matrix (ECM) marker. In some cases, it is advantageous that the CAR T cells of the present invention are specific for cell surface markers and ECM markers, such as those described herein.

[0194] Exemplary scFvs specific for PTPRZ1 are set forth in SEQ ID NOs: 21-26. H and V L The domains are set forth in SEQ ID NOs: 41-52. Preferably, the CARs described herein may be based on the scFv domain of RRB470, RRB471, or RRB476 (SEQ ID NOs: 22, 23, and 26, respectively). For example, the CAR may be selected from (a) a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 22, 23, or 26; (b) an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% identity thereto; or (c) a CAR comprising HCDR1, HCDR2, and HCDR3 and LCDR1, LCDR2, and LCDR3 from an amino acid sequence selected from any one of SEQ ID NOs: 22, 23, or 26.

[0195] Exemplary V specific for PTPRZ1 HH are set forth in SEQ ID NOs: 89 to 91. Preferably, the CARs described herein are HH For example, a CAR can be a polypeptide (immunoglobulin variable domain and / or V domain) comprising CDR1, CDR2, and CDR3 from an amino acid sequence selected from any one of SEQ ID NOs: 89-91. HHのThe CAR may be selected from an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% identity to an amino acid sequence selected from any one of SEQ ID NOs: 89 to 91, and typically comprises CDRs 1 to 3 of SEQ ID NOs: 89 to 91, respectively. The CAR may be selected from a CAR comprising the amino acid sequence of any one of SEQ ID NOs: 89 to 91.

[0196] Exemplary scFvs specific for BCAN are set forth in SEQ ID NOS: 34-47. H and V L The domains are set forth in SEQ ID NOs: 67-74. In some cases, the scFv specific for BCAN is selected from RBR295 (SEQ ID NO: 35). For example, the CAR can be selected from (a) a polypeptide having an amino acid sequence of any one of SEQ ID NOs: 34-47, (b) an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% identity thereto, or (c) a CAR comprising HCDR1, HCDR2, and HCDR3 and LCDR1, LCDR2, and LCDR3 from an amino acid sequence selected from any one of SEQ ID NOs: 34-47.

[0197] Exemplary V specific to BCAN HH are set forth in SEQ ID NOs: 83 to 86. Preferably, the CARs described herein have the V of RB826, RB827, RB828 or RB829 (SEQ ID NOs: 83 to 86, respectively). HH For example, a CAR may be a polypeptide (immunoglobulin variable domain and / or V domain) comprising CDR1, CDR2, and CDR3 from an amino acid sequence selected from any one of SEQ ID NOs: 83-86. HHThe CAR may be selected from an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% identity to an amino acid sequence selected from any one of SEQ ID NOs: 83 to 86, and typically comprises CDRs 1 to 3 of SEQ ID NOs: 83 to 86, respectively. The CAR may be selected from a CAR comprising the amino acid sequence of any one of SEQ ID NOs: 83 to 86.

[0198] Exemplary scFvs specific for CSPG4 are set forth in SEQ ID NOs: 27-33. H and V L The domains are set forth in SEQ ID NOS: 53-66. Preferably, the CARs described herein can be based on the scFv domain of HRB299, HRB301, HRB302, or HRB303 (SEQ ID NOS: 28 and 30-32, respectively). For example, the CAR can be selected from (a) a polypeptide having the amino acid sequence of any one of SEQ ID NOS: 28, 30, 31, or 32; (b) an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% identity thereto; or (c) a CAR comprising HCDR1, HCDR2, and HCDR3 and LCDR1, LCDR2, and LCDR3 from an amino acid sequence selected from any one of SEQ ID NOS: 28, 30, 31, or 32. More preferably, the CARs described herein can be based on the scFv domain of HRB301 or HRB302 (SEQ ID NOS: 30-31, respectively). For example, the CAR may be selected from (a) a polypeptide having the amino acid sequence of SEQ ID NO: 30 or 31, (b) an amino acid sequence having at least 80%, 85%, 90%, 95% or 99% identity thereto, or (c) a CAR comprising HCDR1, HCDR2 and HCDR3 and LCDR1, LCDR2 and LCDR3 from an amino acid sequence selected from SEQ ID NO: 30 or 31.

[0199] Exemplary V specific for CSPG4 HHare set forth in SEQ ID NOs: 87-88. Preferably, the CARs described herein are HH For example, a CAR may be a polypeptide (an immunoglobulin variable domain and / or V domain) comprising CDR1, CDR2, and CDR3 from an amino acid sequence selected from any one of SEQ ID NOs: 87-88. HH The CAR may be selected from an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% identity to an amino acid sequence selected from any one of SEQ ID NOs: 87 to 88, and typically comprises CDRs 1 to 3 of SEQ ID NOs: 87 to 88, respectively. The CAR may be selected from a CAR comprising the amino acid sequence of any one of SEQ ID NOs: 87 to 88.

[0200] Exemplary scFvs specific for TNC are set forth in SEQ ID NOS: 38-40. H and V L The domains are set forth in SEQ ID NOs: 75 to 80. For example, the CAR can be selected from (a) a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 38 to 40, (b) an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% identity thereto, or (c) a CAR comprising an HCDR1, HCDR2, and HCDR3 and an LCDR1, LCDR2, and LCDR3 from an amino acid sequence selected from any one of SEQ ID NOs: 38 to 40.

[0201] Exemplary V specific to TNC HH are set forth in SEQ ID NOs: 92-93. Preferably, the CARs described herein are HH For example, a CAR may be a polypeptide (immunoglobulin variable domain and / or V domain) comprising CDR1, CDR2, and CDR3 from an amino acid sequence selected from any one of SEQ ID NOs: 92-93. HHThe CAR may be selected from an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% identity to an amino acid sequence selected from any one of SEQ ID NOs: 92 to 93, and typically comprises CDRs 1 to 3 of SEQ ID NOs: 92 to 93, respectively. The CAR may be selected from a CAR comprising the amino acid sequence of any one of SEQ ID NOs: 92 to 93.

[0202] Other known glioma-associated antigens include HER2, EGFRvIII, IL13Rα2, PDGFRA, NKG2D, MET, HGF, and B7-H3. Preferably, the other glioma-associated antigens are selected from HER2, EGFRvIII, and IL13Rα2, more preferably HER2 and IL13Rα2.

[0203] Monospecific CARs may be specific to any one of the above-mentioned glioma-associated antigens. Bispecific or multispecific CARs may be specific to any combination of glioma-associated antigens. Preferably, bispecific or multispecific CARs are specific to at least one of PTPRZ1, BCAN, CSPG4, and TNC, for example, two, three, or all four of PTPRZ1, BCAN, CSPG4, and TNC. Bispecific or multispecific CARs may also be specific to other known glioma-associated antigens described herein. For example, multispecific CARs may be specific to PTPRZ1 and CSPG4; CSPG4 and Her2; CSPG4 and IL13Rα2; CSPG4, Her2, and IL13Rα2; PTPRZ1 and Her2; PTPRZ1 and IL13Rα2; or PTPRZ1, Her2, and IL13Rα2.

[0204] In some cases, the bispecific or multispecific CAR is specific for PTPRZ1 and one or more other glioma-associated antigens. In some cases, the bispecific or multispecific CAR is specific for BCAN and one or more other glioma-associated antigens.

[0205] The immune effector cell population can comprise two or more different immune effector cells, and each different immune effector cell is specific to a different glioma-associated antigen.Preferably, the population comprises at least one immune effector cell specific to at least one of PTPRZ1, BCAN, CSPG4, and TNC, for example, two immune effector cells specific to two of PTPRZ1, BCAN, CSPG4, and TNC, three immune effector cells specific to three of PTPRZ1, BCAN, CSPG4, and TNC, or four immune effector cells specific to all of PTPRZ1, BCAN, CSPG4, and TNC.The population can also comprise immune effector cells specific to other known glioma-associated antigens.For example, the immune effector cell population can comprise immune effector cells specific to PTPRZ1 and immune effector cells specific to CSPG4. The population of immune effector cells may comprise PTPRZ1-specific immune effector cells, Her2-specific immune effector cells, and IL13Rα2-specific immune effector cells, as shown in this example.

[0206] In some cases, the population of immune effector cells may include two or more different immune effector cells, and one or more of the different immune effector cells are specific for a glioma-associated antigen selected from at least one of PTPRZ1, BCAN, CSPG4, and TNC, such as at least two of PTPRZ1, BCAN, CSPG4, and TNC, at least three of PTPRZ1, BCAN, CSPG4, and TNC, or all four of PTPRZ1, BCAN, CSPG4, and TNC. In some cases, one or more of the different immune effector cells are specific for PTPRZ1. In some cases, one or more of the different immune effector cells are specific for BCAN. For example, the population of immune effector cells can include: PTPRZ1-specific immune effector cells and BCAN-specific immune effector cells; PTPRZ1-specific immune effector cells and CSPG4-specific immune effector cells; PTPRZ1-specific immune effector cells and TNC-specific immune effector cells; BCAN-specific immune effector cells and CSPG4-specific immune effector cells; BCAN-specific immune effector cells and TNC-specific immune effector cells; CSPG4-specific immune effector cells and TNC-specific immune effector cells; PTPRZ1-specific immune effector cells, BCAN-specific PTPRZ1-specific immune effector cells and CSPG4-specific immune effector cells; PTPRZ1-specific immune effector cells, BCAN-specific immune effector cells, and TNC-specific immune effector cells; PTPRZ1-specific immune effector cells, CSPG4-specific immune effector cells, and TNC-specific immune effector cells; BCAN-specific immune effector cells, CSPG4-specific immune effector cells, and TNC-specific immune effector cells; or PTPRZ1-specific immune effector cells, BCAN-specific immune effector cells, CSPG4-specific immune effector cells, and TNC-specific immune effector cells.

[0207] In some cases, the immune effector cells of the present invention can comprise two or more different CARs, each CAR being specific to a different glioma-associated antigen.Preferably, the immune effector cells comprise at least one CAR specific to at least one of PTPRZ1, BCAN, CSPG4, and TNC, for example, two CARs specific to two of PTPRZ1, BCAN, CSPG4, and TNC, three CARs specific to three of PTPRZ1, BCAN, CSPG4, and TNC, or four CARs specific to all of PTPRZ1, BCAN, CSPG4, and TNC.The immune effector cells can also comprise CARs specific to other known glioma-associated antigens described herein.For example, the immune effector cells can comprise a CAR specific to PTPRZ1 and a CAR specific to CSPG4.The immune effector cells can comprise a CAR specific to PTPRZ1, a CAR specific to Her2, and a CAR specific to IL13Rα2.

[0208] In some cases, the immune effector cell of the present invention can comprise two or more different CARs, and each CAR is specific to a different glioma-associated antigen.In some cases, at least one of the glioma-associated antigens is PTPRZ1.In some cases, at least one of the glioma-associated antigens is BCAN.In some cases, at least one of the glioma-associated antigens is selected from the group of PTPRZ1, BCAN, CSPG4 and TNC, for example, at least two of PTPRZ1, BCAN, CSPG4 and TNC, at least three of PTPRZ1, BCAN, CSPG4 and TNC, or all four of PTPRZ1, BCAN, CSPG4 and TNC. For example, immune effector cells can comprise: a PTPRZ1-specific CAR and a BCAN-specific CAR; a PTPRZ1-specific CAR and a CSPG4-specific CAR; a PTPRZ1-specific CAR and a TNC-specific CAR; a BCAN-specific CAR and a CSPG4-specific CAR; a BCAN-specific CAR and a TNC-specific CAR; a CSPG4-specific CAR and a TNC-specific CAR; a PTPRZ1-specific CAR, a BCAN-specific CAR and a CSPG4-specific CAR; a PTPRZ1-specific CAR, a BCAN-specific CAR, and a TNC-specific CAR; a PTPRZ1-specific CAR, a CSPG4-specific CAR, and a TNC-specific CAR; a BCAN-specific CAR, a CSPG4-specific CAR, and a TNC-specific CAR; or a PTPRZ1-specific CAR, a BCAN-specific CAR, a CSPG4-specific CAR, and a TNC-specific CAR. The immune effector cells may further comprise CARs specific for other known glioma-associated antigens described herein.

[0209] nucleic acid Also provided are one or more isolated nucleic acids (i.e., polynucleotides) encoding a CAR of the invention. In some cases, the encoding nucleic acid sequence can be provided by two or more nucleic acid sequences, optionally present on two or more nucleic acid molecules, but which together collectively encode a CAR of the invention.

[0210] Nucleic acids encoding the CARs of the invention can be obtained by methods well known to those skilled in the art. For example, DNA sequences encoding part or all of the heavy and light chains of an antibody can be synthesized from the corresponding amino acid sequences, if desired.

[0211] 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.

[0212] The vector may be a viral vector. Conventional viral expression systems include retroviral, alpha-retroviral, lentiviral, adenoviral, adeno-associated (AAV), and herpes simplex virus (HSV) vectors for gene transfer. Non-viral transfer vectors include transposon-based systems, including PiggyBac and Sleeping Beauty systems. Methods for producing and purifying such vectors are known in the art.

[0213] The vector may be a cloning vector or an expression vector. A suitable vector may be any vector that is capable of carrying a sufficient amount of genetic information to allow the expression of a polypeptide of the invention.

[0214] The vector is preferably an RNA vector. Suitable RNA vectors include those described in Schutsky, Keith, et al., Oncotarget 6.30 (2015): 28911 and Beatty, Gregory L., et al., Gastroenterology 155.1 (2018): 29-32.

[0215] The general methods by which vectors can be constructed, transfection methods and culture methods are well known to those skilled in the art, and in this regard, see "Current Protocols in Molecular Biology", 1999, FM Ausubel (ed), Wiley Interscience, New York and the Maniatis Manual, Cold Spring Harbor Publishing.

[0216] The nucleic acid can be provided in the form of an expression cassette comprising a control sequence operably linked to the inserted sequence, thus allowing the expression of the CAR of the present invention in vivo. Accordingly, one or more expression cassettes encoding one or more nucleic acids encoding the CARs described herein are also provided. These expression cassettes are typically provided in turn in a vector (e.g., a plasmid or a recombinant viral vector). Accordingly, vectors encoding the CARs described herein are also provided. Furthermore, vectors collectively encoding the CARs described herein are also provided.

[0217] The vector may be a human artificial chromosome, as described, for example, in Kazuki et al., Mol. Ther. 19(9):1591-1601 (2011) and Kouprina et al., Expert Opinion on Drug Delivery 11(4).517-535 (2014).

[0218] Vectors can be DNA plasmids, naked nucleic acids (eg, naked RNA), and non-viral delivery systems, such as nucleic acids complexed with delivery vehicles such as liposomes or nanoparticles.

[0219] The nucleic acids, expression cassettes, or vectors described herein can be introduced into host cells, for example, by transfection. Accordingly, host cells containing one or more nucleic acids, expression cassettes, or vectors of the present invention are also provided. The nucleic acids, expression cassettes, or vectors described herein can be transiently or permanently introduced into host cells, allowing for expression of antibodies from 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 bacterial cells. Specific examples of cells include mammalian HEK293, such as HEK293F, HEK293T, HEK293S, or HEK Expi293F, CHO, HeLa, NS0, and COS cells, or any other cell line used herein. Preferred host cells are immune effector cells described herein. Preferably, the nucleic acids, expression cassettes, or vectors described herein are transiently introduced into host cells.

[0220] Also provided are kits suitable for transforming and / or transfecting immune effector cells or populations of immune effector cells to produce the immune effector cells or populations of immune effector cells of the present invention. The kits include a nucleic acid or vector described herein. The kits may further include agents that improve the efficiency of transfection or transformation, as discussed herein.

[0221] Also described are nucleic acids encoding the specific scFv antigen-binding regions of SEQ ID NOs: 21 to 40, as provided in SEQ ID NOs: 1 to 20, respectively. SEQ ID NOs: 1 to 20 are provided as DNA sequences, but also encompass the corresponding RNA sequences (with "T" replaced by "U").

[0222] Also described are nucleic acids encoding the specific scFv antigen-binding regions of SEQ ID NOs: 83 to 93, which are provided as SEQ ID NOs: 94 to 104, respectively. SEQ ID NOs: 94 to 104 are provided as DNA sequences, but also encompass the corresponding RNA sequences (with "T" replaced by "U").

[0223] Immune effector cells As used herein, immune effector cells refer to cells that have cell-mediated cytotoxicity against target cells that present target antigens, such as glioma-associated antigens.Immune effector cells can be T cells, γδ T cells, natural killer (NK) cells, NKT cells, induced pluripotent stem cell (iPSC)-derived NK cells (iPSC-NK), γδ T cells, phagocytes, or macrophages.Immune effector cells are preferably T cells.

[0224] Preferably, the T cells are CD8+ T cells or cytotoxic T cells. The T cells are preferably CD4-CD8+ T cells.

[0225] The T cells may be CD4+ T cells or helper T cells (TH cells), such as TH1, TH2, TH3, TH17, TH9, or T FH cells. The T cells may be regulatory T cells (Tregs). The T cells may be naive T cells, effector T cells, memory T cells, effector memory T cells, central memory T cells, or memory stem T cells. The T cells may be peripheral lymphocytes.

[0226] The T cells may be expanded from peripheral blood mononuclear cells (PBMCs). The T cells may be autologous to the subject to be administered. The T cells may be allogeneic to the subject to be administered. The T cells may be partially HLA-mismatched to the subject to be administered.

[0227] The NK cells may be cells of the NK92 cell line. The NK cells may be isolated from peripheral blood mononuclear cells (PBMCs) of the subject to be treated or a healthy donor. The NK cells may be isolated from umbilical cord blood. The NK cells may be CD34+ They can be differentiated from hematopoietic progenitor cells (HPCs).

[0228] The γδ T cells may be expanded from peripheral blood mononuclear cells (PBMCs). The γδ T cells may be autologous with respect to the subject to whom they are administered. The Tγδ cells may be allogeneic with respect to the subject to whom they are administered.

[0229] The macrophages may be differentiated into an "M1" phenotype. M1 macrophages express pro-inflammatory cytokines and have strong anti-tumor activity. Undifferentiated macrophages expressing the CARs described herein can be induced to differentiate into an M1 phenotype by culturing them in the presence of a glioma-associated antigen.

[0230] The immune effector cells may comprise the nucleic acid described herein. The immune effector cells may comprise the vector described herein. Preferably, the immune effector cells comprise the RNA nucleic acid or RNA vector described herein. The immune effector cells express CARs specific to one or more glioma-associated antigens. The immune effector preferably transiently expresses the CAR.

[0231] Thus, the immune effector cells are preferably RNA CAR T cells.

[0232] Immune effector cells can be engineered to transiently express CARs specific for one or more glioma-associated antigens to minimize on-target off-tumor toxicity due to antigen expression in normal, healthy tissue. In some cases, immune effector cells can be transfected with mRNA encoding CARs specific for one or more glioma-associated antigens by mRNA electroporation, as demonstrated, for example, in Beatty et al., Gastroenterology 155.1 (2018):29-32 (see Supplementary Material 5) and Schutsky et al., Oncotarget 6.30 (2015):28911.

[0233] In some cases, the present invention relates to a population of immune effector cells expressing a CAR. The population may comprise at least two different CAR-expressing immune effector cells specific for at least two different glioma-associated antigens. The population may comprise at least three different CAR-expressing immune effector cells specific for at least three different glioma-associated antigens. In some cases, one or more of the glioma-associated antigens are selected from PTPRZ1, BCAN, CSPG4, and TNC. For example, two or more, for example, three or four, of the glioma-associated antigens are selected from PTPRZ1, BCAN, CSPG4, and TNC.

[0234] The population should be at least approximately 1x10 6 immune effector cells, e.g., at least about 1 x 10 7 , at least about 1x10 8 , at least about 1x10 9 , or at least about 1 x 10 10 The population may comprise at least about 1 x 10 immune effector cells. 6 ~approx. 1x10 12 immune effector cells, e.g., about 1 x 10 6 ~approx. 1x10 11 , approximately 1x10 6 ~approx. 1x10 10 , approximately 1x10 6 ~approx. 1x10 9 , approximately 1x10 7 ~approx. 1x10 11 , approximately 1x10 8 ~approx. 1x10 10 The population may comprise about 1 x 10 immune effector cells. 6 immune effector cells, e.g., about 5x10 6 , approximately 1x10 7 , about 5x10 7 , approximately 1x10 8 , about 5x10 8 , approximately 1x10 9 , about 5x10 9 , approximately 1x10 10 , about 5x10 10 , approximately 1x10 11 , about 5x1011 , or about 1x10 12 The immune effector cells may include:

[0235] In some cases, the immune effector cells of the present invention express a CAR, and the CAR is specific for at least two different glioma-associated antigens (i.e., a bispecific or multispecific CAR). For example, the CAR may comprise two different scFvs specific for two different glioma-associated antigens.

[0236] In some cases, the immune effector cells of the invention express at least two different CARs, and the at least two different CARs are specific for at least two different glioma-associated antigens. In some cases, the immune effector cells of the invention express at least three different CARs, and the at least three different CARs are specific for at least three different glioma-associated antigens.

[0237] Multispecificity against two or more glioma-associated antigens is advantageous for many reasons. For example, due to patient-to-patient and / or tumor-to-tumor variability, expression of glioma-associated antigens can vary within and between subjects. Multispecificity allows a single therapy to target a wider range of tumors. Furthermore, antigen escape is a phenomenon in which, for example, antigens targeted by a CAR are no longer expressed by the tumor, rendering the therapy ineffective. Targeting multiple glioma-associated antigens makes it more difficult for tumors to "escape" from the therapy. Multispecificity also enhances the effector functions of immune effector cells, such as cytotoxicity.

[0238] Also provided is a method for producing the immune effector cell of the present invention or a population of immune effector cells of the present invention.The method comprises transforming a cell or a population of cells with one or more nucleic acids encoding one or more CARs specific to one or more glioma-associated antigens.The CAR can be any of the CARs discussed herein.The nucleic acid can be any of the nucleic acids or vectors of the present invention.

[0239] Any method known in the art can be used to transform cells or populations with nucleic acid. Immune effector cells can be transfected or transduced with nucleic acid. CAR can be introduced into immune effector cells using a vector.

[0240] The term "transduction" may be used to describe viral-mediated nucleic acid transfer. Viral vectors may be used to introduce one or more constructs into cells. Conventional viral-based expression systems include retroviral, alpharetroviral, lentiviral, adenoviral, adeno-associated (AAV), and herpes simplex virus (HSV) vectors for gene transfer. Non-viral transfer vectors include transposon-based systems, including the PiggyBac and Sleeping Beauty systems. Methods for producing and purifying such vectors are known in the art. Preferably, the vector is 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.

[0241] The term "transfection" can be used to describe non-viral nucleic acid transfer. Immune effector cells can be transfected using any method known in the art. Transfection can be in vitro or ex vivo. Any vector capable of transfecting immune effector cells can be used, including traditional plasmid DNA or RNA transfection, preferably mRNA transfection. Human artificial chromosomes and / or naked RNA can also be used to transfect cells with nucleic acid sequences or nucleic acid constructs. Human artificial chromosomes are described, for example, in 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 acids, and nucleic acids complexed with delivery vehicles such as liposomes. Methods for non-viral delivery of nucleic acids include lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, naked RNA, artificial virions, and drug-enhanced uptake of DNA.

[0242] Nanoparticle delivery systems can be used to transfect immune effector cells with nucleic acid sequences. Such delivery systems include, but are not limited to, lipid-based systems, liposomes, micelles, microvesicles, and exosomes. For nanoparticles capable of delivering RNA, see, for example, Alabi et al., Proc Natl Acad Sci US 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. 2012 Oct 23;6(10):8484-7; Whitehead et al. 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 (SNAP) nanoparticles, and nanoparticles capable of delivering RNA (NAP) are also useful. TM ) constructs, nanoplexes and other nanoparticles (particularly gold nanoparticles) are also contemplated as delivery vehicles for the nucleic acids or vectors of the invention.

[0243] Immune effector cells can be transfected by electroporation. Preferably, electroporation is mRNA electroporation. This has the advantage that CAR can be transiently expressed.

[0244] Immune effector cells may be transfected by electroporation, such as RNA electroporation or mRNA electroporation. When immune effector cells express two or more CARs, they may be transfected by electroporation of a single polynucleotide (e.g., RNA or mRNA) or vector encoding two or more CARs (e.g., RNA or mRNA electroporation), or by electroporation of two or more polynucleotides (e.g., RNA or mRNA) or vectors (e.g., RNA or mRNA electroporation), each polynucleotide encoding at least one CAR. Electroporation of two or more polynucleotides is usually performed simultaneously.

[0245] Uptake of nucleic acid constructs can be enhanced by several known transfection techniques, including, for example, techniques involving the use of transfection agents, such as cationic agents, e.g., calcium phosphate and DEAE-dextran, and lipofectants, e.g., lipofectAmine, fugene, and transfectam.

[0246] Pharmaceutical Composition Also provided are compositions comprising immune effector cells or populations of immune effector cells of the invention. The immune effector cells or populations of immune effector cells can represent at least 50% of the total cells in the composition, e.g., 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. All cells in the composition can consist of, or essentially consist of, immune effector cells or populations of immune effector cells of the invention, i.e., no other cells are detectable in the composition.

[0247] The composition comprises at least about 1 x 10 6 ~approx. 1x10 12 of immune effector cells of the invention, e.g., about 1 x 10 6 ~approx. 1x10 11 , approximately 1x10 6 ~approx. 1x10 10 , approximately 1x10 6 ~approx. 1x10 9 , approximately 1x10 7 ~approx. 1x10 11 , approximately 1x10 8 ~approx. 1x10 10 The composition may comprise about 1 x 10 immune effector cells. 6 of immune effector cells of the invention, e.g., about 5x10 6 , approximately 1x10 7 , about 5x10 7 , approximately 1x10 8 , about 5x10 8 , approximately 1x10 9 , about 5x10 9 , approximately 1x10 10 , about 5x10 10 , approximately 1x10 11 , about 5x10 11 , or about 1x10 12 The composition may comprise the above amounts of the population of immune effector cells of the invention.

[0248] The composition may be a pharmaceutical composition. The pharmaceutical composition may include a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers include aqueous carriers or diluents. Examples of suitable aqueous carriers include water, buffered water, and saline.

[0249] Pharmaceutical compositions may contain one or more pharmaceutically acceptable salts. "Pharmaceutically acceptable salts" refers to salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects. Examples of such salts include acid addition salts and base addition salts.

[0250] The composition may include one or more additional therapeutic agents, such as a chemotherapeutic agent. The composition may include one or more preservatives, such as an antifungal and / or antiviral agent.

[0251] Therapeutic Uses and Methods The use of the immune effector cells or populations of immune effector cells described herein in methods of treatment of the human or animal body by therapy or in diagnostic methods is also as described herein.

[0252] Also provided is a method of treating, for example, cancer in a subject, comprising administering to the subject an effective amount of an immune effector cell or population of immune effector cells of the invention.

[0253] Also provided is an immune effector cell or population of immune effector cells of the invention for use in a method of treating cancer, as well as the use of an immune effector cell or population of immune effector cells for the manufacture of a medicament for the treatment of cancer.

[0254] Therapeutic uses and methods may involve administering a therapeutically effective amount of an immune effector cell or population of immune effector cells.

[0255] Also provided is a method of formulating a composition for treating cancer, said method comprising mixing an immune effector cell or population of immune effector cells of the invention with an acceptable carrier to prepare said composition.

[0256] The cancer may be a glioma, such as a malignant glioma. The cancer may be a glioblastoma. The cancer may be a recurrent cancer, such as a recurrent glioblastoma. The subject may have previously been treated for the cancer, such as with a CAR cell (e.g., T cell) approach targeting EGFRvIII, IL13Rα2, and / or Her2. The cancer may be glioblastoma multiforme (GBM). The cancer is a primary glioblastoma or a secondary glioblastoma.

[0257] The cancer may be other solid cancers that express glioma-associated antigen(s). For example, the cancer may be a solid tumor that expresses one or more of PTPRZ1, BCAN, CSPG4, and / or TNC. The cancer may also express one or more of HER2, EGFRvIII, IL13Rα2, PDGFRA, NKG2D, MET, HGF, and B7-H3. The immune effector cells, populations of immune effector cells, CARs, and antigen-binding fragments disclosed herein can be used to treat any solid cancer that expresses glioma-associated antigen(s).

[0258] Therapeutic methods and uses may include, before treatment with the immune effector cells or a group of immune effector cells of the present invention, determining whether the cancer expresses a glioma-associated antigen that is specifically targeted by the immune effector cells or a group of immune effector cells of the present invention. For example, the method may include determining whether the cancer expresses PTPRZ1, BCAN, CSPG4, and / or TNC. The method may include selecting an immune effector cell or a group of immune effector cells based on the expression of a glioma-associated antigen by the cancer, so that the immune effector cell or group of immune effector cells is specific to the cancer. The method may include transfecting or transforming the immune effector cell with a nucleic acid of the present invention in response to information regarding the expression of a glioma-associated antigen by the cancer.

[0259] At least 1% of tumor- or individual-derived cancer cells may express glioma-associated antigen.Glioma-associated antigens are typically selected from PTPRZ1, BCAN, CSPG4 and / or TNC.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 tumor- or individual-derived cancer cells may express glioma-associated antigen.The proportion of cells expressing glioma-associated antigen can be determined by any means known to those skilled in the art, such as immunohistochemistry (IHC), flow cytometry, enzyme-linked immunosorbent assay (ELISA), etc.

[0260] Glioma-associated antigen expression may have an intensity of (≥) 1+ or greater, such as ≥ 2+ or ≥ 3+. Intensity scores can be assessed by IHC staining of tumors, with scoring as follows: negative = no staining or staining equal to or less than ≤ 10% of cells; 1+ = incomplete staining, ≥ 10% of cells; 2+ = weak to moderate staining, ≥ 10% of cells; strong and complete staining, ≥ 10% of cells.

[0261] The therapeutic methods and uses described herein may include inhibiting a disease state (i.e., cancer) by, for example, halting its development and / or causing regression of the disease state until a desired endpoint is reached. The therapeutic methods and uses of the present invention may include achieving a partial response, a complete response to cancer. The therapeutic methods and uses of the present invention may achieve remission of cancer.

[0262] The therapeutic methods and uses described herein may slow, stop, and / or reverse the growth of cancer. The therapeutic methods and uses of the present invention may reduce the size of cancer by at least 10%, for example, 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 100%.

[0263] The therapeutic methods and uses described herein may involve the induction of a bystander effect. The bystander effect may involve the killing of cancer cells that do not express the glioma-associated antigen directed by at least one of the CARs expressed by the immune effector cells administered to a subject in the method or use.

[0264] Typically, the therapeutic methods and uses are directed to human subjects in need thereof. However, non-human animals, such as non-human mammals, are also contemplated. The non-human mammal may be a rat, rabbit, sheep, pig, cow, cat, or dog.

[0265] The dose of the immune effector cell or population of immune effector cells may vary depending on the age and size of the subject, as well as the disease, condition, and route of administration. The immune effector cell or population of immune effector cells may be administered in an amount of about 1 x 10 6 ~approx. 1x10 12 cells, e.g., about 1x10 6 ~approx. 1x10 11 , approximately 1x10 6 ~approx. 1x10 10 , approximately 1x10 6 ~approx. 1x10 9 , approximately 1x10 7 ~approx. 1x10 11 , approximately 1x10 8 ~approx. 1x10 10 The immune effector cell or population of immune effector cells may be administered in a dose of about 1 x 10 6 cells, e.g., about 5x10 6 cells, approximately 1x10 7 cells, approximately 5x10 7 cells, approximately 1x10 8 cells, approximately 5x10 8 cells, approximately 1x10 9 cells, approximately 5x10 9 cells, approximately 1x10 10 cells, approximately 5x10 10 cells, approximately 1x10 11 cells, approximately 5x10 11 cells, or approximately 1x10 12 The dose may be in the form of a cell.

[0266] The immune effector cell or population of immune effector cells is about 1 x 10 5 cells / kg ~ approx. 1x10 11 cells / kg, e.g., about 1x10 5 cells / kg ~ approx. 1x10 10 cells / kg, approximately 1x10 5 cells / kg ~ approx. 1x10 9 cells / kg, approximately 1x10 5 cells / kg ~ approx. 1x10 8 cells / kg, approximately 1x10 6 cells / kg ~ approx. 1x10 11 cells / kg, approximately 1x10 6 cells / kg ~ approx. 1x10 10 cells / kg, approximately 1x10 6 cells / kg ~ approx. 1x10 9 cells / kg, approximately 1x10 7 cells / kg ~ approx. 1x10 11 cells / kg, approximately 1x10 7 cells / kg ~ approx. 1x10 10 cells / kg, or approximately 1x10 7 cells / kg ~ approx. 1x10 9 The immune effector cells or population of immune effector cells may be administered at a dose of about 1 x 10 cells / kg. 5 cells / kg, e.g., about 5x10 5 cells / kg, 1x10 6 cells / kg, 5x10 6 cells / kg, 1x10 7 cells / kg, 5x10 7 cells / kg, 1x10 8 cells / kg, 5x10 8 cells / kg, 1x10 9 cells / kg, 5x10 9 cells / kg, 1x10 10 cells / kg, 5x10 10 cells / kg, or 1x10 11 It may be administered at a dose of cells / kg.

[0267] The immune effector cells or population of immune effector cells can be administered as a single dose. The immune effector cells or population of immune effector cells can also be administered in a multiple dose regimen. For example, an initial dose can be administered followed by a second or multiple subsequent doses. The second and subsequent doses can be administered at appropriate intervals. For example, the doses can be administered about once a week, about once every two weeks, about once every three weeks, about once every four weeks, or about once a month.

[0268] 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 intracerebroventricularly.

[0269] The immune effector cells or population of immune effector cells may be administered with one or more additional therapies, such as one or more additional therapeutic agents. The additional therapeutic agent may be an anti-tumor agent. The additional therapeutic agent may be an oncolytic virus. The additional therapeutic agent may be a CAR-enhancing drug. The additional therapy may be additional immune effector cells.

[0270] The combined administration of the immune effector cells or populations and additional therapeutic agents can be achieved in a number of different ways: all components may be administered together in a single composition, or each component may be administered separately as part of a combination therapy.

[0271] For example, the immune effector cells or populations of immune effector cells of the invention may be administered before, after, or simultaneously with an additional therapeutic agent.

[0272] The additional therapy may be chemotherapy, radiation therapy and / or surgery.

[0273] Before administering the immune effector cells or population of immune effector cells of the present invention, the subject may be subjected to lymphodepletion. Lymphodepletion can be achieved by administering fludarabine, cyclophosphamide, and / or bendamustine to the subject. Lymphodepletion can be performed for at least about 1 day, for example, about 2 days or about 3 days.

[0274] The biological activity and / or therapeutic effect of the administered immune effector cell or population of immune effector cells can be measured by known methods, for example, the methods may include imaging, such as magnetic resonance imaging.

[0275] antigen binding molecule Also provided are antigen-binding molecules specific for one or more glioma-associated antigens. The antigen-binding molecules are selected from antigen-binding molecules comprising heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) and light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) from an amino acid sequence selected from any one of SEQ ID NOs: 21 to 32 and 34 to 37. In some cases, the antigen-binding molecules are selected from antigen-binding molecules comprising, for example, one or more polypeptides, comprising HCDR1 to 3 and LCDR1 to 3 of an amino acid sequence selected from any one of SEQ ID NOs: 21 to 32 and 34 to 37, and 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 SEQ ID NOs: 21 to 32 and 34 to 37.

[0276] 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 antibody-based therapy, such as metabolite radiotherapy. The antigen-binding molecule may be used in the cancer treatment methods described herein.

[0277] In some cases, the antigen-binding molecule comprises one or more of the antigen-binding domains disclosed herein. For example, the antigen-binding molecule may comprise a polypeptide disclosed herein. The antigen-binding molecule may comprise an scFv or V domain disclosed herein. HH For example, the antigen-binding molecule may comprise HCDR1-3 and LCDR1-3 of an scFv disclosed herein. HH It may also contain CDR1 to 3 of the above.

[0278] In some cases, the antigen-binding molecule comprises a V HH and comprises CDR1-3 of an amino acid sequence selected from any one of SEQ ID NOs: 83 to 93. The antigen-binding molecule may further comprise 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 SEQ ID NOs: 83-93.

[0279] In some cases, the antigen-binding molecule comprises a polypeptide having at least two immunoglobulin variable domains, such as an scFv. One variable domain is typically a V H and one variable domain is typically V L The variable domain may be an scFv or an antibody. H contains HCDR1-3, V L comprises LCDR1 to LCDR3 having an amino acid sequence selected from any one of SEQ ID NOs: 21 to 32 and 34 to 37. H The domain further comprises a V of an amino acid sequence selected from SEQ ID NOs: 21 to 32 and 34 to 37. H The V domain may have 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. L The domain is a V of an amino acid sequence selected from SEQ ID NOs: 21 to 32 and 34 to 37.L It may further comprise 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 domain.

[0280] definition It is understood that different uses of the disclosed CARs, cells, or pharmaceutical compositions of the present invention can be tailored to the particular needs of the art, and that the terminology used herein is for the purpose of describing particular embodiments of the present invention only, and is not intended to be limiting.

[0281] Furthermore, 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, a reference to "CAR" includes two or more "CARs."

[0282] It should be understood that the terms "nucleic acid" and "polynucleotide" are used interchangeably herein.

[0283] For the purpose of the present invention, to determine the percent identity of two sequences (such as two nucleic acids or two nucleic acid sequences), the sequences are aligned for optimal comparison purposes (for example, gaps can be introduced into the first sequence for optimal alignment with the second sequence).Then, the nucleotide or amino acid residue at each position is compared.If the same nucleotide or amino acid exists at a position in the first sequence as at the corresponding position in the second sequence, the nucleotide or amino acid is identical at that position.The percent identity between 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 × 100).

[0284] Usually, sequence comparison is performed over the length of the reference sequence. For example, if a user wants to determine whether a given ("test") sequence is 95% identical to SEQ ID NO: 1, SEQ ID NO: 1 is the reference sequence. To assess whether a sequence is at least 95% identical to SEQ ID NO: 1 (an example of a reference sequence), a person skilled in the art will align the length of SEQ ID NO: 1 and identify how many positions in the test sequence are identical to the positions 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, gaps or missing positions should be considered as non-identical positions.

[0285] Those skilled in the art are familiar with various computer programs that can be used to determine the homology or identity between two sequences.For example, the comparison of sequences and the determination of the percent identity between two sequences can be achieved using mathematical algorithms.In one embodiment, the percent identity between two amino acid or nucleic acid sequences is determined using the Needleman and Wunsch (1970) algorithm, which is incorporated into the GAP program in the Accelrys GCG software package (available at http: / / www.accelrys.com / products / gcg / ), using either Blosum62 matrix or PAM250 matrix, a gap weight of 16, 14, 12, 10, 8, 6 or 4, and a length weight of 1, 2, 3, 4, 5 or 6.

[0286] " Specific" or "specifically bind" means that the antigen-binding region of CAR binds to one or more antigenic determinants of desired glioma-associated antigens and does not bind to other polypeptides.For example, a CAR specific to PTPRZ1 binds to the antigen of PTPRZ1, but does not bind to the antigen of a different polypeptide, such as bovine serum albumin.A CAR can specifically bind if it binds to the desired glioma-associated antigen with stronger affinity than when it binds to the antigen of a different polypeptide, such as bovine serum albumin.Methods for measuring binding affinity are well known in the art.

[0287] As used herein, the term "about" may be interpreted to mean a value within ±10% of the stated value.

[0288] All publications, patents, and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0289] The following examples illustrate the invention. [Example]

[0290] Example 1 - Generation of anti-PTPRZ1 RNA CAR T cells Six different single-chain variable fragments (scFv) against PTPRZ1 were obtained by screening a human scFv phage display library. All scFvs were fused to either the hinge and transmembrane domains of human CD8α, followed by the intracellular domains of human 4-1BB (CD137) and human CD3-ζ (so-called BBz CARs), or to the hinge of human IgG4, followed by the transmembrane and intracellular domains of human CD28 and CD3-ζ (so-called 28z CARs). All CAR constructs were cloned into pDA plasmids designed for optimal production of mRNA molecules under the control of a T7 viral promoter.

[0291] Human T cells were purified from healthy donor blood using RosetteSep cocktail (StemCell Technologies). T cells were activated with anti-CD3 / anti-CD28 Dynabeads (Invitrogen) at a 1:1 ratio for 48 hours. The beads were then removed from the activated T cells, and mRNA for different CAR molecules was electroporated using a MaxCyte instrument. CAR T cells were allowed to recover overnight in the presence of 30 IU / mL IL-2 and then frozen.

[0292] To evaluate CAR T cell killing capacity, tumor target cells were stained with a cell tracker and seeded at 25,000 cells / well in a 96-well plate. CAR T cells were thawed the day before the experiment, allowed to recover overnight in the presence of 45 IU / mL IL-2, and then added to tumor cells at different effector-to-target (E:T) ratios. Prior to addition of CAR T cells, CAR molecule expression on the T cell surface was measured by flow cytometry.

[0293] As a tumor model to evaluate the efficacy of CAR T cells, we used the GBM cell line Ge518, which was obtained in our laboratory from a patient's tumor resection. Because PTPRZ1 expression was low in these cells, we overexpressed the extracellular portion of the human PTPRZ1 molecule (domains 1+2) under the EF1α promoter using a third-generation LV vector with puromycin selection. The new cell line, Ge518_PTPRZ1-KI, showed high cell surface expression of PTPRZ1, as measured by flow cytometry using our scFv conjugated to rabbit Fc followed by AlexaFluo488-conjugated anti-rabbit IgG (Figure 1A). Six scFvs generated against PTPRZ1 were able to specifically recognize PTPRZ1 on Ge518_PTPRZ1-KI cells, and scFvs 469, 471, 473, and 476 showed higher recognition levels (over 80%, clearly defined peaks) than scFvs 470 and 474 (Fig. 1B).

[0294] CAR T cell generation was efficient, with over 80% of T cells typically expressing CAR molecules and a post-thaw survival rate of 80–90% (Figure 2A). The only exception was 474_BBz CAR T cells, which showed low CAR expression. Six different CAR T cells and control non-transduced T cells (NTD) were incubated with Ge518_PTPRZ1-KI cells at a 5:1 E:T ratio for 72 hours. A positive control CAR T cell specific for IL13Rα2 was also used. Three anti-PTPRZ1 CAR T cells, 470_BBz, 471_BBz, and 476_BBz, were able to kill most tumor cells (75–85%, Figure 2B), as measured by flow cytometry. 469_BBz and 473_BBz CAR T cells exhibited low killing rates of Ge518_PTPRZ1-KI tumor cells. The expression level of 474_BBz CAR was too low to exhibit killing ability (Fig. 2B).

[0295] Second-generation CAR T cells can use different intracellular costimulatory domains, with 4-1BB and CD28 being more frequently used. 4-1BB appears to be more suitable for in vivo CAR T cell persistence, while CD28 has been reported to provide a more potent acute response. Because RNA CAR T cells have a relatively short CAR cell surface expression period (<7 days), the use of a costimulatory domain that generates a stronger immune response is desirable. Therefore, we evaluated the killing efficacy of CAR T cells with the same scFv but incorporating either the 4-1BB (BBz) or CD28 (28z) domain. Both CAR T cell versions, containing scFvs 473 and 476 at an E:T ratio of 5:1, were incubated with Ge518_PTPRZ1-KI cells. After 72 hours, the killing ability of CAR T cells was measured by flow cytometry. For both scFvs, the 28z variant exhibited higher cytotoxicity than the BBz variant (Figure 3A). We repeated the comparison using scFv 471 at two different E:T ratios, 3:1 and 1:1. Under these two conditions, the 471_28z variant exhibited higher killing activity than the 471_BBz variant, with the difference being more pronounced at 1:1, where 471_BBz exhibited reduced cytotoxicity (Figure 3B).

[0296] Next, to establish anti-PTPRZ1 CAR T cells with higher cytotoxicity, we performed killing assays using Ge518_PTPRZ1-KI target cells and CAR T cells, 470_28z, 471_28z, and 476_28z, at three different E:T ratios. The 471_28z CAR T cells exhibited high killing activity at all E:T ratios tested, reaching a maximum of ~80% at an E:T ratio of 3:1, while the other two showed 45–60% (Figure 4). Furthermore, the 471_28z CAR T cells maintained this activity even at a relatively low E:T ratio of 0.5:1, whereas the other two showed low killing (<20%) (Figure 4).

[0297] Example 2 - Anti-CSPG4 RNA CAR T cells Six different scFvs against CSPG4 were obtained from screening a human scFv phage display library. These scFvs were cloned in CAR BBz and 28z formats in pDA plasmids. The A375 melanoma cell line and GBM Ge518 cell line were used as tumor models to evaluate the cytotoxic activity of anti-CSPG4 CAR T cells. RNA CAR T cells were generated according to the protocol described in Example 1. All anti-CSPG4 CAR T cells highly expressed CAR molecules on their surface (>87%) (Figure 5A).

[0298] Anti-CSPG4_BBz CAR T cells were incubated with the A375 cell line, which highly expresses CSPG4, at an E:T ratio of 5:1 for 72 hours. Anti-IL13Rα2_BBz CAR T cells were used as a positive control, and untransduced T cells were used as a negative control; all were generated from the same donor. Four CSPG4-specific CAR T cells incorporating 299_BBz, 301_BBz, 302_BBz, and 303_BBz scFvs demonstrated high tumor cell killing rates (>75%, Figure 5B). Next, anti-CSPG4 CAR T cells were tested against Ge518 GBM cells at an E:T ratio of 5:1. Similarly, CAR T cells incorporating 299_BBz, 301_BBz, 302_BBz, and 303_BBz scFvs demonstrated the highest tumor cell killing rates against GBM cell lines, demonstrating 35–45% tumor cell death (Figure 5C). IFN-γ secretion was also measured in the supernatants of the killing experiments and was found to be at the highest levels in 301_BBz and 302_BBz CAR T cells (~2000 pg / mL) (Figure 5D).

[0299] Example 3 - Activity of a mix of three CAR T cells against GBM cells We generated RNA CAR T cells containing scFv 302 against three different GBM targets: IL13Rα2, Her2, and CSPG4. To evaluate the efficacy of combination therapy against heterogeneous GBM, we created three variants of the Ge518 tumor cell line, each lacking one of the antigens of interest. Knockout (KO) variants were generated using the CRISPR-Cas9 system. After a cloning process, antigen expression was tested by flow cytometry, and the gene was sequenced to confirm the mutation (Figure 6A). The ability of each CAR T cell to kill the wild-type (wt) variant of Ge518 compared to the respective antigen KO variant was assessed by flow cytometry. In all cases, the CAR T cells were able to specifically kill the wt variant but not the KO variant (Figure 6B).

[0300] Next, a mix of three different Ge518 KO cells (IL13Rα2-KO, Her2-KO, and CSPG4-KO) and Ge518 wt cells was incubated with a mix of anti-Her2_BBz, anti-IL13Rα2-BBz, and anti-CSPG4_BBz CAR T cells for 72 hours. CAR T cells were seeded to obtain a final E:T ratio of 3:1 for both individual CAR T cells and the total of all CAR T cells in the mix. Equal mixes were used (Mix A, E:T ratio of each CAR T cell was 1:1) or two unbalanced mixes: Mix B, E:T ratios of 1.5:1 anti-Her2, 1:1 anti-IL13Rα2, and 0.5:1 anti-CSPG4, and Mix C, E:T ratios of 0.5:1 anti-Her2, 1:1 anti-IL13Rα2, and 1.5:1 anti-CSPG4. The killing potency of individual or mixed CAR T cells was measured by flow cytometry after 72 hours. All mixes demonstrated high cytotoxic activity, with >70% tumor cell death, similar to that obtained with individual anti-Her2_BBz CAR T cells at a 3:1 E:T ratio and higher than that demonstrated with individual anti-IL13Rα2_BBz or anti-CSPG4_BBz at a 3:1 E:T ratio (Figure 7B). In addition to addressing tumor heterogeneity, mixing CAR T cells also reduces the likelihood of tumor escape compared to individual CAR T cells.

[0301] Example 4 - Generation of triple CAR T cells We generated RNA CAR T cells against three different glioma-associated antigens: PTPRZ1 (471_28z), CSPG4 (301_28z), and BCAN (295_28z), using scFvs specific for each antigen. Expression of each CAR was confirmed individually (Figure 8A). In addition to the scFv sequence, the _28z construct contained a human IgG4 hinge sequence (SEQ ID NO: 118), CD28 transmembrane and intracellular domains (SEQ ID NOs: 123 and 125, respectively), and a CD3 zeta intracellular domain (SEQ ID NOs: 126-128).

[0302] Simultaneous RNA electroporation of human T cells generated CAR T cells transfected with RNA encoding and expressing all three CARs: anti-PTPRZ1 (471_28z), anti-CSPG4 (301_28z), and anti-BCAN (295_28z), producing "triple" RNA CAR T cells. To evaluate the efficacy of combination therapy against heterogeneous GBM, two variants of the Ge518 tumor cell line were generated: Ge518_BCANv2-TM KI, a knock-in for BCAN, and Ge518_PTPRZ1 KI, a knock-in for PTPRZ1. In all three target cell lines, triple CAR T cells increased cell killing compared with either monovalent CAR T cell (Figure 8B). When all three target cell lines were mixed, more representative of heterogeneous GBM, enhanced cell killing of the Ge518_wt and Ge518_BCANv2 cell lines was observed, particularly by monovalent anti-PTPRZ1 CAR T cells and triple CARs (Figure 8C), demonstrating a "bystander" effect. These results were also reflected in an in vitro model of tumor growth inhibition (Figures 8D and 8E).

[0303] Example 5 - Safety and analysis of bystander effects As shown in Example 4, monovalent anti-PTPRZ1 CAR T cells were found to efficiently kill GBM cell lines that do not significantly express PTPRZ1 when mixed with GBM cell lines that do express PTPRZ1. Next, we investigated whether this bystander effect was specific to GBM cells or whether it also affected healthy cells by mixing Ge518_PTPRZ1-KI cells with "healthy" macrophages. Upon addition of monovalent anti-PTPRZ1 CAR T cells, cell killing of Ge518_PTPRZ1-KI cells was observed, but no significant increase in macrophage killing was observed (Figure 9).

[0304] To determine whether the "bystander" effect was mediated by soluble factors, an experiment was set up according to Figure 10A. Cell killing of a PTPRZ1-knockout GBM cell line (Ge518_PTPRZ1-KO) was observed when Ge518_PTPRZ1-KI cells were contacted with monovalent anti-PTPRZ1 CAR T cells, even though there was no direct contact between the monovalent anti-PTPRZ1 CAR T cells and the Ge518_PTPRZ1-KO cell line.

[0305] Example 6 - Co-dependence of target expression in human glioblastoma Using bulk RNA-seq data from TCGA (primary GBM) and CGGA (recurrent GBM) data, we studied the correlation between the expression of pairs of antigens (Figures 11A and 11B).

[0306] While CAR T cell therapy can be tailored to patients based on antigen expression, this data could also be used to find optimal combinations that take into account patient-to-patient variability. That is, even without knowledge of glioma antigen expression, selecting combinations of antigens with low or negative correlations could increase the likelihood that multivalent CAR T cell therapy will target gliomas. These antigens tend to be expressed differently in different patients. Therefore, this combination should target a large number of patients. Possible combinations include BCAN, TNC, and CSPG4.

[0307] Conversely, if expression of one or more glioma-associated antigens is known in a patient, the use of multivalent CAR T cells that also target glioma-associated antigens known to positively correlate with the known antigens may also be useful, for example, to reduce the likelihood that the glioma will evade CAR T-cell therapy by reducing expression of a single antigen.

[0308] Example 7 - Generation of monovalent and multivalent CAR T cells using nanobodies Nanobodies (VHHs) against BCAN (RB826-829), CSPG4 (RB830-831), PTPRZ1 (832-834), and TNC (835-836) were generated. The specificity of the nanobodies was tested by ELISA against their target antigens and control antigens (Figures 12A, 13A, 14A, and 15A). The nanobodies were conjugated to human IgG1 Fc and their recognition against tumor cell lines was examined (Figures 12B, 13B, and 14B).

[0309] CARs were generated using nanobodies with short or long hinges, and the expression of each CAR by transfected T cells was examined to determine the expression level of each construct (Figures 12C, 13C, 14C, and 15B). The short "_28z" construct contained, in addition to the nanobody sequence, a human IgG4 hinge sequence (SEQ ID NO: 118), CD28 transmembrane and intracellular domains (SEQ ID NOs: 123 and 125, respectively), and CD3 zeta intracellular domains (SEQ ID NOs: 126-128). The long "_IgG1H_28z" construct contained, in addition to the nanobody sequence, a human IgG1 long hinge sequence (SEQ ID NO: 121), CD28 transmembrane and intracellular domains (SEQ ID NOs: 123 and 125, respectively), and CD3 zeta intracellular domains (SEQ ID NOs: 126-128). Cell killing of nanobody-based CAR T cells was determined by flow cytometry against multiple tumor cell lines (Figures 12D, 13D, 14D, and 15C). Corresponding data were also generated for in vitro tumor growth inhibition (Figures 12E, 13E, 14E and 15D).

[0310] Bispecific CAR T cells were generated expressing a CAR containing anti-PTPRZ1 nanobody 832 in combination with a CAR containing anti-CSPG4 nanobody 830, anti-BCAN scFc 295, or anti-TNC nanobody 835. The graphs in the left columns of Figures 16A-16C compare cell killing by monovalent anti-PTPRZ1 CAR T cells versus bispecific CAR T cells against cell lines with low PTPRZ1 expression but high CSPG4 (Figure 16A), BCAN (Figure 16B), or TNC (Figure 16C). The graphs in the right columns of Figures 16A-16C compare cell killing of highly PTPRZ1-expressing cell lines by monovalent anti-CSPG4 CAR T cells (Figure 16A), monovalent anti-BCAN CAR T cells (Figure 16B), or monovalent anti-TNC CAR T cells (Figure 16C) compared with bispecific CAR T cells also expressing an anti-PTPRZ1 CAR.

[0311] Further Numbered Embodiments 1. An immune effector cell or population of immune effector cells expressing one or more chimeric antigen receptors (CARs) specific for one or more glioma-associated antigens. 2. The immune effector cell or population of immune effector cells of embodiment 1, wherein the one or more glioma-associated antigens are PTPRZ1, BCAN, CSPG4 and / or TNC. 3. The population of CAR-expressing immune effector cells of embodiment 1 or 2, comprising 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, and optionally, wherein the population of CAR-expressing immune effector cells comprises at least three different CAR-expressing immune effector cells. 4. A method for producing an immune effector cell or population of immune effector cells according to any one of the preceding embodiments, comprising transforming said cell or population of said cells with one or more nucleic acids encoding one or more CARs specific for one or more glioma-associated antigens. 5. A method for treating cancer in a subject, the method comprising administering to the subject an effective amount of an immune effector cell or population of immune effector cells described in any one of embodiments 1 to 3. 6. An immune effector cell or population of immune effector cells according to any one of embodiments 1 to 3 for use in a method for treating cancer. 7. The method for treating a subject according to embodiment 5, or the immune effector cell or population of immune effector cells for use according to embodiment 6, wherein the cancer is glioma. 8. The immune effector cell or population of immune effector cells of any one of embodiments 1 to 3, the method of any one of embodiments 4, 5, or 7, or the immune effector cell or population of immune effector cells for use of embodiment 6 or 7, wherein one or more of said CARs are selected from CARs comprising a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 21-40, or an amino acid sequence with at least 80%, 85%, 90%, 95%, or 99% identity thereto. 9. The immune effector cell or population of immune effector cells of any one of embodiments 1 to 3 or 8, the method of any one of embodiments 4, 5, 7 or 8, or the immune effector cell or population of immune effector cells for use of any one of embodiments 6 to 8, wherein one or more of the CARs are selected from CARs comprising a polypeptide comprising a heavy chain complementarity determining region (HCDR1, HCDR2 and HCDR3) and a light chain complementarity determining region (LCDR1, LCDR2 and LCDR3) from an amino acid sequence selected from any one of SEQ ID NOs: 21 to 40. 10. The immune effector cell or population of immune effector cells of any one of embodiments 1 to 3, 8 or 9, the method of any one of embodiments 4 to 5 or 7 to 9, or the immune effector cell or population of immune effector cells for use of any one of embodiments 6 to 9, wherein the cell or cells are T cells, NK cells, iPSC-NK cells, γδ T cells, phagocytes or macrophages. 11. A CAR comprising a polypeptide having (a) an amino acid sequence of any one of SEQ ID NOs: 21-40, (b) an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% identity thereto, and an intracellular signaling domain, or (c) heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) and light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) from an amino acid sequence selected from any one of SEQ ID NOs: 21-40. 12. A multivalent CAR comprising: (a) an extracellular domain specific for two or more glioma-associated antigens, wherein optionally, the glioma-associated antigens are selected from PTPRZ1, BCAN, CSPG4, and / or TNC; and (b) an intracellular signaling domain. 13. The multivalent CAR of embodiment 12, wherein the extracellular domain is an ScFv, VH, or VHH. 14. The multivalent CAR of embodiment 13, wherein the extracellular domain comprises a polypeptide selected from (a) an amino acid sequence of SEQ ID NO: 21-40, (b) an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% identity thereto, or (c) an amino acid sequence comprising a heavy chain complementarity determining region (HCDR1, HCDR2, and HCDR3) and a light chain complementarity determining region (LCDR1, LCDR2, and LCDR3) from an amino acid sequence selected from SEQ ID NO: 21-40. 15. The CAR of embodiment 11 or the multivalent CAR of any one of embodiments 12 to 14, wherein the intracellular domain comprises a CD3 zeta signaling domain alone or in combination with a CD28, CD27, CD134 (OX40) and / or CD137 (4-1BB) intracellular domain. 16. A CAR according to embodiment 11 or 15, or a multivalent CAR according to any of embodiments 12 to 15, further comprising a transmembrane domain. 17. A CAR or multivalent CAR described in embodiment 16, wherein the transmembrane domain is a CD28 transmembrane domain. 18. A nucleic acid encoding a CAR described in any one of embodiments 11 and 15 to 17, or a multivalent CAR described in any one of embodiments 12 to 17. 19. The nucleic acid of embodiment 18, which is DNA or RNA. 20. A nucleic acid according to embodiment 18 or 19, comprising a sequence encoding any one of the sequences of SEQ ID NOs: 1 to 20 or any one of the amino acid sequences of SEQ ID NOs: 21 to 40. 21. A vector comprising one or more nucleic acids according to any one of embodiments 18 to 20. 22. The vector described in embodiment 21, which is a lentiviral vector, an RNA vector, a liposome, or a lipid nanoparticle for in vivo delivery of RNA. 23. An antigen-binding molecule specific for one or more glioma-associated antigens selected from: (a) a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 21 to 32 and 34 to 37; (b) an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% identity thereto; or (c) an antigen-binding molecule comprising the HCDR1, HCDR2, and HCDR3 and the LCDR1, LCDR2, and LCDR3 from an amino acid sequence selected from any one of SEQ ID NOs: 21 to 32 and 34 to 37.

Claims

1. 1. An immune effector cell or population of immune effector cells that expresses one or more chimeric antigen receptors (CARs) specific for two or more glioma-associated antigens, wherein one or more of the glioma-associated antigens are selected from PTPRZ1, BCAN, CSPG4, and TNC.

2. 2. The immune effector cell or population of immune effector cells of claim 1, wherein two or more of the glioma-associated antigens are selected from PTPRZ1, BCAN, CSPG4 and TNC.

3. 3. The immune effector cell or population of immune effector cells of claim 1 or 2, which expresses one or more CARs specific for three or more glioma-associated antigens.

4. 10. The immune effector cell or population of immune effector cells of any one of the preceding claims, wherein the or each cell expresses two or more CARs specific for different glioma-associated antigens.

5. 10. The immune effector cell or population of immune effector cells of any one of the preceding claims, wherein the or each cell expresses three or more CARs specific for different glioma-associated antigens.

6. 10. The population of any one of the preceding claims, comprising 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.

7. 7. The population of claim 6, comprising at least three different CAR-expressing immune effector cells, wherein each different CAR-expressing immune effector cell is specific for a different glioma-associated antigen.

8. 10. The immune effector cell or population of immune effector cells of any one of the preceding claims, wherein one or more of the glioma-associated antigens are cell surface markers and one or more of the glioma-associated antigens are extracellular matrix (ECM) markers.

9. 9. The immune effector cell or population of immune effector cells of claim 8, wherein the cell surface markers are selected from PTPRZ1, CSPG4 and BCAN, and / or the ECM markers are selected from TNC and BCAN.

10. An immune effector cell or population of immune effector cells that expresses a chimeric antigen receptor (CAR) specific for PTPRZ1.

11. An immune effector cell or population of immune effector cells that expresses a chimeric antigen receptor (CAR) specific for BCAN.

12. 10. A method for producing an immune effector cell or population of immune effector cells according to any one of the preceding claims, comprising transforming said cell or said population of cells with one or more nucleic acids encoding one or more CARs specific for one or more glioma associated antigens.

13. A method of treating cancer in a subject, comprising administering to the subject an effective amount of the immune effector cell or population of immune effector cells of any one of claims 1 to 11.

14. 12. An immune effector cell or population of immune effector cells according to any one of claims 1 to 11 for use in a method for the treatment of cancer.

15. 15. The method for treating a subject according to claim 13, or the immune effector cell or population of immune effector cells for use according to claim 14, wherein the cancer is glioma.

16. 16. The immune effector cell or population of immune effector cells according to any one of claims 1 to 11, the method of any one of claims 12 to 13 and 15, or the immune effector cell or population of immune effector cells for use according to claim 14 or 15, wherein the cell or cells are T cells, NK cells, iPSC-NK cells, γδ T cells, phagocytes or macrophages.

17. CAR specific for PTPRZ1.

18. BCAN-specific CAR.

19. A CAR specific for a glioma-associated antigen selected from PTPRZ1, BCAN, CSPG4, and TNC: (a) a heavy chain complementarity determining region (HCDR1, HCDR2, and HCDR3) and a light chain complementarity determining region (LCDR1, LCDR2, and LCDR3) from an amino acid sequence selected from any one of SEQ ID NOs: 21-40; or (b) a complementarity-determining region (CDR1, CDR2, and CDR3) from an amino acid sequence selected from any one of SEQ ID NOs: 83 to 93; A CAR comprising a polypeptide comprising:

20. the polypeptide comprising: (a) the amino acid sequence of any one of SEQ ID NOs: 21-40 and 83-93, and / or (b) an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% identity to any one of the amino acid sequences of SEQ ID NOs: 21 to 40 and 83 to 93; The CAR of claim 19, comprising:

21. The CAR according to any one of claims 17 to 20, further comprising an intracellular signaling domain.

22. the polypeptide comprising: (a) one or more immunoglobulin variable domains; and / or (b) scFv, VH or VHH domain The CAR according to any one of claims 17 to 21, comprising:

23. A multivalent CAR comprising: (a) an extracellular domain specific for two or more glioma-associated antigens, wherein one or more of the glioma-associated antigens are selected from PTPRZ1, BCAN, CSPG4, and TNC; and (b) an intracellular signaling domain.

24. The multivalent CAR of claim 23, wherein the extracellular domain is an ScFv, VH, or VHH.

25. the extracellular domain comprising: (a) a heavy chain complementarity determining region (HCDR1, HCDR2, and HCDR3) and a light chain complementarity determining region (LCDR1, LCDR2, and LCDR3) from an amino acid sequence selected from any one of SEQ ID NOs: 21-40; or (b) a complementarity-determining region (CDR1, CDR2, and CDR3) from an amino acid sequence selected from any one of SEQ ID NOs: 83 to 93; 25. The multivalent CAR of claim 23 or 24, comprising a polypeptide comprising:

26. the extracellular domain comprising: (a) the amino acid sequence of any one of SEQ ID NOs: 21-40 and 83-93, and / or (b) an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% identity to any one of the amino acid sequences of SEQ ID NOs: 21 to 40 and 83 to 93; The multivalent CAR according to any one of claims 23 to 26, comprising:

27. The CAR according to any one of claims 17 to 22, or the multivalent CAR according to any one of claims 23 to 26, wherein the intracellular domain comprises a CD3 zeta signaling domain alone, or a combination of CD28, CD27, CD134 (OX40) and / or CD137 (4-1BB) intracellular domains.

28. The CAR according to any one of claims 17 to 22 and 27, or the multivalent CAR according to any one of claims 23 to 27, further comprising a transmembrane domain.

29. 29. The CAR or multivalent CAR of claim 28, wherein the transmembrane domain is a CD28 transmembrane domain.

30. The immune effector cell or population of immune effector cells of any one of claims 1 to 11 and 16, the method of any one of claims 12, 13, 15 and 16, or the immune effector cell or population of immune effector cells for use according to any one of claims 14 to 16, wherein said one or more CARs comprise a CAR or a multivalent CAR of any one of claims 19 to 29.

31. A nucleic acid encoding the CAR according to any one of claims 17 to 22 and 27 to 29, or the multivalent CAR according to any one of claims 23 to 29.

32. 32. The nucleic acid of claim 31, which is DNA or RNA.

33. 33. The nucleic acid of claim 31 or 32, comprising a sequence encoding the amino acid sequence of any one of SEQ ID NOs: 1 to 20 and 94 to 104 or any one of SEQ ID NOs: 21 to 40 and 83 to 93.

34. A vector comprising one or more nucleic acids according to any one of claims 31 to 33.

35. 35. The vector of claim 34, which is a lentiviral vector, an RNA vector, a liposome, or a lipid nanoparticle for in vivo delivery of RNA.

36. An antigen-binding molecule specific for one or more glioma-associated antigens, comprising a polypeptide comprising the complementarity-determining regions (CDR1, CDR2, and CDR3) from an amino acid sequence selected from any one of SEQ ID NOs: 83 to 93.

37. The antigen-binding molecule of claim 36, wherein the polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% identity to any one of the amino acid sequences of SEQ ID NOs: 21 to 40 and 83 to 93.

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