Chimeric antigen receptors

Abstract

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

Description

TECHNICAL FIELD

[0001] The present disclosure relates to immune effector cells expressing one or more chimeric antigen receptors and their use in the treatment of gliomas. BACKGROUND

[0002] Immunotherapeutic approaches that induce tumor-specific immune responses are being considered for a variety of malignancies. For example, therapeutic T cells can be engineered to direct their cytotoxic effects toward a specific antigen of interest. In this way, T cells responsible for killing tumor cells can be engineered to be specific for a tumor antigen. The specificity of the T cells can be determined by endogenous or recombinant T cell receptors or chimeric antigen receptors (CARs).

[0003] CARs are synthetic receptors that include an extracellular domain, usually 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 be redirected to a desired antigen. Anti-CD 19 CAR T cells have led to a paradigm shift in cancer treatment based on response rates in relapsed / refractory diffuse large B-cell lymphoma (DLBCL) or pediatric refractory B-cell acute lymphoblastic leukemia (B-ALL) in adult patients. Two CAR T cell products, Kymriah (Novartis) and Yearcta (Kite Pharma), specific for the B-cell marker CD 19, became the first FDA-registered therapeutic products, including genetic engineering elements for the treatment of B-ALL and DLBCL.

[0004] Glioblastoma (GBM, grade IV astrocytoma) is the most common and most aggressive primary malignancy of brain origin. Despite treatment involving a combination of surgery, chemotherapy, and radiotherapy, the overall survival of GBM patients is 14.6 to 16 months post-treatment. Several phase I / II studies using polypeptide vaccines or novel adjuvants immune checkpoint blockers have been tested with limited success to date. Currently, CAR T cell approaches have been investigated for GBM targeting EGFRvIII, IL-13Ra2, and Her2 ((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 develop additional glioma antigen specific CARs and improved immune effector cells expressing the CARs for the treatment of glioma. SUMMARY

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

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

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

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

[0010] The present invention additionally provides a CAR specific for PTPRZ1. The present invention also provides a CAR specific for BCAN. The present invention also provides a CAR specific for a glioma-associated antigen selected from the group consisting of 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: 21-40, or (b) complementarity determining regions (CDR1, CDR2, and CDR3) from an amino acid sequence selected from any one of SEQ ID NOs: 83-93.

[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 is selected from the group consisting of PTPRZ1, BCAN, CSPG4, and / or TNC; and (b) an intracellular signaling domain.

[0012] The present invention also provides a nucleic acid encoding a CAR or a multivalent CAR of the present invention. The present invention also provides a vector comprising one or more of the nucleic acids.

[0013] The present application also provides antigen binding molecules specific for one or more glioma-associated antigens, including polypeptides having complementarity determining regions (CDR1, CDR2 and CDR3) from an amino acid sequence selected from any one of SEQ ID NOs: 83-93. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 Generation of GBM cell line overexpressing PTPRZ1. A) GBM cell line Ge518 was transduced with a 3rd passage lentivirus to introduce the 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) Ge518_PTPRZ1-KI staining by flow cytometry using six different anti-PTPRZ1 scFvs.

[0015] 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 by flow cytometry and expressed as percentage of specific lysis. E:T ratio = effector to target ratio.

[0016] Figure 3 Comparison of killing capacity of BBz and 28z variants of anti-PTPRZ1 RNA CAR T cells. A) Killing activity of anti-PTPRZ1 RNA CAR T cells containing scFvs 473 and 476 and 28z or BBz against Ge518_PTPRZ1-KI tumor cells measured by flow cytometry and expressed as percentage of specific lysis. E:T ratio = effector to target ratio. B) Comparison of killing activity of anti-PTPRZ1 scFvs 471 28z and BBz RNA CAR T cells against Ge518_PTPRZ1-KI tumor cells at two different E:T ratios.

[0017] Figure 4 Differences in killing activity of three different anti-PTPRZ1_28z RNA CAR T cells. Killing capacity of anti-PTPRZ1 28z RNA CAR T cells containing scFvs 470, 471 and 476 against Ge518_PTPRZ1-KI tumor cells was measured at three different E:T ratios. E:T ratio = effector to target ratio.

[0018] 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 control anti-IL13Ra2_BBz RNA CAR. NTD: non-transduced T cells. B) Killing activity of anti-CSPG4_BBz RNA CAR T cells on A375 melanoma cells measured by flow cytometry and expressed as percentage of specific lysis. E:T ratio = effector to target ratio. C) Killing activity of anti-CSPG4_BBz RNA CAR T cells on Ge518 GBM cells. D) IFN-g secretion in supernatant of anti-CSPG4_BBz RNA CAR T cells incubated with Ge518 GBM cells.

[0019] Figure 6 Generation of Ge518 variants KO for IL13Ra2, Her2 and CSPG4 antigens. A) IL13Ra2, Her2 and CSPG4 antigen expression was eliminated in Ge518 cells using the CRISPR-Cas9 system. Loss of antigen expression was verified by flow cytometry. B) Killing effect of wt Ge518 cell line and corresponding RNA BBz CAR T cells for Ge518 KO cell lines was evaluated by flow cytometry at E:T ratio 3:1.

[0020] Figure 7 Cytotoxic activity of a combination of three anti-GBM RNA CAR T cells on a heterogeneous mixture of tumor cells. A) Mechanism of better killing and less risk of antigen loss escape of a mixture of different anti-GBM CAR T cells compared to monospecific CAR T cells. B) Killing capacity of different mixtures of anti-CSPG4_BBz, anti-Her2_BBz and anti-IL13Ra2_BBz CAR T cells on a heterogeneous mixture of three different Ge518 KO cells (IL13Ra2-KO, Her2-KO and CSPG4-KO) and Ge518 wt cells. E:T ratio = effector to target ratio.

[0021] Figure 8: Generation and in vitro killing assays of triple RNA CAR-T cells. (A) CAR expression on 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_BCANv2TM-KI, and Ge518_PTPRZ1-KI cell lines by triple CAR or individual monovalent CAR-T cells. E:T ratio: 3:1 and 1:1. Mock EP cells were used as negative control. (C) In vitro killing of a mixture (2:2:1 ratio) of Ge518, Ge518_BCANv2TM-KI, and Ge518_PTPRZ1-KI cell lines by triple CAR or individual monovalent CAR-T cells. E:T ratio: 3:1 and 1:1. Mock EP cells were used as negative control. (D) In vitro inhibition of tumor growth by triple CAR or individual monovalent CAR-T cells on Ge518, Ge518_BCANv2TM-KI, and Ge518_PTPRZ1-KI cell lines measured by Incucyte. E:T ratio: 3:1. Mock EP cells were used as negative control. (E) In vitro inhibition of tumor growth by triple CAR or individual monovalent CAR-T cells on a mixture (2:2:1 ratio) of Ge518, Ge518_BCANv2TM-KI, and Ge518_PTPRZ1-KI cell lines measured by Incucyte. E:T ratio: 3:1. Mock EP cells were used as negative control.

[0022] Figure 9 : 471_28z CAR-T cells do not show bystander killing capacity on non-tumor human macrophages. (A) CD14+ monocytes were purified from human blood at day 0 and differentiated into macrophages by culture with M-CSF for 6 days. At day 6, Ge518_PTPRZ1-KI cells (FarRed stained) were added to macrophage cultures at E:T ratio 3:1, followed by anti-PTPRZ1 RNA 471_28z CAR-T cells or mock EP control cells. After 72h, cells were collected and cell death of tumor and macrophages was evaluated by FACS. (B) CAR expression of anti-PTPRZ1 RNA 471_28z CAR-T cells. (C) Gating strategy of differentiated tumor Ge518_PTPRZ1-KI cells (CD14-FarRed + ) and 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.

[0023] Figure 10 471_28z CAR-T cells demonstrated bystander killing ability via soluble media. (A) On day 0, Ge518_PTPRZ1-KO cells were seeded at the bottom of Transwell plates, while Ge518_PTPRZ1-KI cells were seeded at the top of the Transwell. On day 1, anti-PTPRZ1 RNA 471_28z CAR-T cells or EP-mimicking control cells were added to the top of the Transwell at an E:T ratio of 5:1. After 72 hours, cells were collected from the bottom of the wells and cell death was evaluated by FACS. (B) The indirect, soluble media-dependent killing of Ge518_PTPRZ1-KO cells (stained with yellow dye) by anti-PTPRZ1 RNA 471_28z CAR-T cells was evaluated. (C) The absence of Ge518_PTPRZ1-KI cells (stained with FarRed) or T cells (CD3+) at the bottom of the wells was confirmed. + ).

[0024] Figure 11 Correlation analysis between different antigens. Correlation between antigens (A) and (B). Lower diagonal: Scatter plot with regression lines. Middle diagonal: Density plot. Upper diagonal: Significant Pearson correlation coefficient: ***<0.001, **<0.01, *<0.05. Test statistics are based on Pearson product-moment correlation coefficients. (A) Expression data from TCGA, bulk RNA-seq (primary GBM). (B) Expression data from CGGA (recurrent GBM). (C) Heatmap of expression data from TCGA, bulk RNA-seq (primary GBM), showing Pearson correlation coefficients between antigens.

[0025] Figure 12 Anti-PTPRZ1 V HH Isolation, RNA CAR-T cell production, and in vitro killing assays. (A) Through different anti-PTPRZ1 V HH (B) To recognize the extracellular domain of PTPRZ1 by ELISA. HH -Fc (5 μg / mL) conjugated anti-PTPRZ1 V HH Identify Ge518_PTPRZ1-KI and Ge738 cell lines. (C) In cells expressing different anti-PTPRZ1 V... HH RB832 and RB833 RNA-CAR CAR expression on T cells, with short (-28z) or long (-IgG1H-28z) hinges followed by CD28 and CD3ζ domains. (D) Four different VHH Anti-PTPRZ1 RNA CAR-T cells kill Ge518_PTPRZ1-KI and Ge738 cell lines in vitro. E:T ratio: 3:1. Anti-PTPRZ1 RNA CAR T cells based on scFv RRB471 were used as positive control, while mock EP cells were used as negative control. (E) Killing of four different V HH Anti-PTPRZ1 RNA CAR-T cells inhibit tumor growth of Ge518_PTPRZ1-KI and Ge738 cell lines in vitro. E:T ratio: 3:1. Anti-PTPRZ1 RNA CAR T cells based on scFv RRB471 were used as positive control, while mock EP cells were used as negative control.

[0026] Figure 13 Anti-CSPG4 V HH Isolation, RNA CAR-T cell generation, and in vitro killing assay. (A) Recognition of the extracellular domain of CSPG4 by different anti-CSPG4 V HH were recognized by ELISA. (B) Anti-CSPG4 V HH -Fc conjugated to human IgGl Fc domain (10 pg / mL) were recognized by ELISA. (C) A375 and Ge738 cell lines were recognized by anti-CSPG4 V HH were recognized by ELISA. (B) Anti-CSPG4 V HH CAR expression on RNA-CAR T cells of different anti-CSPG4 V HH Anti-CSPG4 RB830 RNA CAR-T cells kill A375, Ge518 and Ge738 cell lines in vitro. E:T ratio: 3:1. Anti-CSPG4 RNA CAR T cells based on scFv HRB301 were used as positive control, while mock EP cells were used as negative control. (E) Killing of two different V HH Anti-CSPG4 RB830 RNA CAR-T cells inhibit tumor growth of A375, Ge518 and Ge738 cell lines in vitro. E:T ratio: 5:1. Anti-CSPG4 RNA CAR T cells based on scFv HRB301 were used as positive control, while mock EP cells were used as negative control.

[0027] Figure 14 Anti-Tenascin C V HH Isolation, RNA CAR-T cell generation, and in vitro killing assay. (A) Recognition of the extracellular domain of Tenascin C by different anti-Tenascin C VHH to the extracellular domain of Tenascin C. (B) CAR expression on RNA-CAR T cells expressing different anti-Tenascin C V HH -Fc at 10 pg / mL) conjugated anti-Tenascin C V HH recognize Ge518 and Ge738 cell lines. (C) CAR expression on RNA-CAR T cells expressing different anti-Tenascin C V HH RB835 and RB836, with either short (28z) or long (IgG1H 28z) hinge followed by CD28 and CD3 zeta domains. (D) In vitro killing of Ge518 and Ge738 cell lines by eight different V HH anti-Tenascin C RNA CAR-T cells. E:T ratio: 3:1. Anti-Tenascin C RNA CAR T cells based on scFv R6N were used as positive control, while mock EP cells were used as negative control. (E) In vitro inhibition of tumor growth of Ge518 and Ge738 cell lines by two different V HH anti-Tenascin C RNA CAR-T cells. E:T ratio: 5:1. Anti-Tenascin C RNA CAR T cells based on scFv R6N were used as positive control, while mock EP cells were used as negative control.

[0028] Figure 15 : anti-CSPG4 V HH Isolation, RNA CAR-T cell production, and in vitro killing assays. (A) BCAN recognition by different anti-BCAN V HH (B) CAR expression on RNA-CAR T cells expressing different anti-BCAN V HH RB826, RB827, RB828, and RB829, with either short (28z) or long (IgG1H 28z) hinge followed by CD28 and CD3 zeta domains. (C) In vitro killing of Ge518_BCANv2TM-KI cell line by eight different V HH anti-Tenascin C RNA CAR-T cells. E:T ratio: 3:1. Anti-Tenascin C RNA CAR T cells based on scFv R6N were used as positive control, while mock EP cells were used as negative control. (D) In vitro inhibition of tumor growth of Ge518 and Ge738 cell lines by two different V HHInhibition of tumor growth of Ge518_BCANv2TM-KI cell line by anti-BCAN RNA CAR-T cells in vitro. E:T ratio: 5:1. Anti-BCAN RNA CAR-T cells based on scFv HRB295 were used as positive control, while mock EP cells were used as negative control.

[0029] Figure 16 : Anti-PTPRZ1 RNA CAR-T cells based on anti-PTPRZ1 V HH Bispecific RNA CAR-T cells of RB832 in combination with a second CAR against CSPG4, BCAN or Tenascin C. The histograms in the left column show the increase in killing capacity of the bispecific RNA CAR-T cells compared to the killing of a monovalent anti-PTPRZ1 CAR against cell lines with low PTPRZ1 expression. The histograms in the right column show the increase in killing capacity of the bispecific RNA CAR-T cells compared to the killing of a second monovalent CAR against cell lines with high PTPRZ1 expression. (A) Bispecific CAR-T cells anti-PTPRZ1 (V HH RB832) plus anti-CSPG4 (V HH RB830).(B) Bispecific CAR-T cells anti-PTPRZ1 (V HH RB832) plus anti-BCAN (scFv HRB295).(C) Bispecific CAR-T cells anti-PTPRZ1 (V HH RB832) plus anti-Tenascin C (V HH RB835).

[0030] SEQUENCE LISTING

[0031] SEQ ID NO: 1 - Anti-PTPRZ1 scFv "RRB469" nucleotide sequence ATGGCCCAGGTGCAACTGGTGGAATCTGGGGGAGGCGTGGTTCAGCCTGGGAGGTCCCTGCGGCTCTCCTGTGCAGCCTCAGGATTTACCTTCAGTAGCTACGCCATGCACTGGGTCCGCCAGGCTCCAGGCAAAGGGTTGGAATGGGTTGCAGTTATATCATATGATGGAAGTAATAAATACTACGCAGACTCCGTGAAGGGCCGATTCACCATTTCACGTGACAATTCCAAGAACACGCTTTATCTGCAAATGAACAGCTTGAGAGCTGAAGATACGGCTGTGTATTACTGCGCGAGGGGTAGTGGATACAGCTATGGTCCGGGTTATGATGCATTTGATATTTGGGGCCAGGGAACCCTTGTCACAGTCTCAAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGGGGCGGATCTACAAATTTTATGCTGACTCAGCCTCATTCTGTATCGGAGTCTCCAGGGAAGACAGTAACCATCTCCTGCACACGCAGCAGTGGCAGCATCGCCAGCAACTATGTGCAGTGGTACCAGCAGAGACCAGGCAGTTCACCCACTACTGTGATTTATGAGGATAACCAAAGACCCTCTGGGGTCCCTGATCGGTTTTCTGGCTCCATCGACAGCAGTTCCAATTCGGCCTCCCTCACCATCTCTGGACTAAAAACTGAGGACGAGGCTGACTACTACTGTCAGTCCTGGGACCCCGTGTTCGGGGTGTTCGGCGGAGGGACAAAGCTGACCGTCTTAGGGGCGGCC

[0032] SEQ ID NO: 2 - Anti-PTPRZ1 scFv "RRB470" nucleotide sequence ATGGCACAAGTGCAGTTAGTTCAGTCTGGGGCTGAAGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTAGTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGCCTTGAATGGATGGGAGGGATCATTCCGATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAAGGCAGAGTCACTATTACCGCGGACGAATCCACAAGCACAGCATACATGGAGCTGAGCAGCCTGAGGTCTGAAGATACGGCCGTGTATTACTGTGCGAGAGAGGGGGGGGCCGTGGGGTACTACTACGGTATGGACGTCTGGGGCCAGGGAACACTTGTGACAGTCTCCAGCGGTGGAGGCGGTTCAGGCGGAGGCGGCTCAGGCGGTGGCGGATCTACTCAGAGTGCCTTGACTCAGCCAGCCTCCGTGTCTGGGTCACCCGGACAGTCGATAACCATCTCCTGCACTGGAACCAGCAGTGACGTTGGTGGTTATAATTATGTATCCTGGTACCAACAGCACCCAGGCAAAGCCCCCAAACTCATGATTTATGAGGTCAGTAATCGGCCCTCAGGGGTTTCTAATCGTTTCTCTGGCTCCAAATCTGGCAACACGGCCTCCCTGACTATATCAGGGCTCCAGGCTGAGGACGAGGCTGATTATTACTGTAGTTCATATGATAGGAGCAACCGCAGTATGGTGTTTGGCGGAGGGACCAAACTGACCGTACTAGGGGCAGCC

[0033] SEQ ID NO: 3 - Anti-PTPRZ1 scFv "RRB471" nucleotide sequence ATGGCCGAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCTGGATTTACCTTCAGTAGCTATAGCATGAACTGGGTCAGGCAGGCTCCAGGGAAGGGGCTTGAGTGGGTTTCATACATTAGTAGCAGTAGTAGCACAATATACTACGCAGACTCTGTGAAGGGCCGATTCACAATCTCCAGGGATAATGCCAAGAACTCACTGTATTTACAAATGAATAGCCTTAGAGCCGAAGACACGGCTGTGTATTACTGTGCGAGACCAGGCTACGGTGACTTTCCCGGTGCTTTTGATATCTGGGGCCAGGGAACCCTGGTCACAGTGTCGAGCGGTGGAGGCGGCTCAGGCGGAGGTGGCAGCGGCGGTGGCGGATCTACGCAGTCTGCATTGACTCAGCCTGCCTCCGTGTCTGGATCACCTGGACAAAGCATTACCATCTCCTGCACTGGAACCAGCAGTGACGTTGGTGGTTATAACTATGTCTCCTGGTACCAACAGCACCCGGGCAAAGCCCCCAAACTCATGATTTACGAAGTAAGTAATCGGCCCTCAGGGGTTTCTAATCGCTTCTCTGGCTCCAAATCCGGCAACACTGCCTCCCTGACAATCAGTGGGCTCCAGGCTGAGGACGAGGCTGATTATTACTGCAGCTCATATGATTGGGCCACCTACGGGTCGGTGTTCGGCGGAGGGACCAAGCTGACTGTCCTAGGGGCGGCA

[0034] SEQ ID NO: 4 - Anti-PTPRZ1 scFv "RRB473" nucleotide sequence ATGGCCCAAGTGCAGCTGGTGGAGTCTGGGGGAGGCGTTGTCCAGCCTGGGAGGTCACTGAGACTCTCCTGTGCAGCCTCTGGGTTTACATTCAGTAGCTATGCTATGCACTGGGTACGCCAAGCTCCTGGCAAAGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGTAGTAACAAATACTACGCAGACAGTGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACTCTTTATCTGCAAATGAACAGCCTGCGGGCTGAAGACACAGCTGTGTACTACTGTGCGCGGGACCAGGATGACTCCAGTGATGCTTTTGATATCTGGGGCCAGGGAACCCTGGTCACAGTCTCGAGCGGTGGAGGCGGTAGCGGCGGAGGTGGCAGCGGCGGTGGCGGATCGACGCAGAGCGTTTTGACGCAACCGCCCTCAGTGTCTGCAGCCCCAGGACAGAAAGTCACCATATCCTGCTCTGGAAGCAGCTCCAACATTGGGAACAATTATGTATCCTGGTACCAGCAGTTGCCAGGGACAGCCCCCAAACTCCTCATTTACGACAATAATAAGCGTCCCTCAGGGATTCCTGACCGCTTTTCTGGCAGTAAGTCTGGCACTTCAGCCACTCTGGGCATCACCGGACTTCAGACTGGGGACGAAGCCGATTATTACTGCGGAACATATGATTACATCGCGACCAGGGCCGTGTTCGGTGGCGGGACCAAGTTAACTGTGCTAGGGGCAGCC

[0035] SEQ ID NO: 5 - Anti-PTPRZ1 scFv "RRB474" nucleotide sequence ATGGCCCAGGTGCAGCTTGTTCAGTCTGGGGCTGAGGTGAAGAAGCCAGGGTCCTCGGTGAAGGTTTCCTGCAAGGCTTCAGGAGGCACCTTCAGCAGCTATGCTATCAGTTGGGTGCGGCAAGCACCTGGCCAAGGGCTTGAGTGGATGGGAGGGATAATCCCTATCTTTGGTACAGCAAACTACGCACAAAAGTTCCAGGGCCGCGTCACGATTACCGCCGACGAATCCACCAGCACAGCCTACATGGAACTGTCCAGCCTGAGAAGTGAAGACACTGCCGTGTATTACTGTGCGCGTGGGACGTATTACGATTTTTGGAGTGGTTATTATGATGCTTTTGATATCTGGGGCCAGGGAACCCTGGTTACAGTCTCTAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGCTCTACTCAGTCTGTGTTGACACAGCCGCCCTCAGTGTCTGCAGCCCCAGGACAGAAGGTCACCATCTCCTGTTCTGGTAGCAGCTCCAACATTGGGAATAATTACGTATCCTGGTACCAGCAGTTGCCAGGAACAGCCCCCAAACTCTTAATATATGACAATAACAAAAGGCCCTCAGGGATTCCTGACCGATTCAGTGGCTCCAAATCTGGCACTTCAGCTACCCTGGGCATTACCGGACTCCAGACTGGGGACGAGGCCGATTATTACTGCGGAACATGGGATAGTTCGTACTGGCAACCCGTATTCGGCGGAGGGACTAAACTGACCGTCCTAGGGGCGGCC

[0036] SEQ ID NO: 6 - Anti-PTPRZ1 scFv "RRB476" nucleotide sequence ATGGCCCAGGTGCAACTGGTGGAATCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCGTTGCGGCTCTCCTGTGCAGCCTCTGGATTCACCTTTAGTAGCTATGCTATGCACTGGGTCCGCCAAGCTCCAGGCAAGGGGCTGGAATGGGTGGCAGTTATATCATACGATGGAAGTAATAAATACTACGCAGACTCCGTGAAGGGCCGCTTCACCATCTCCAGAGACAATTCCAAGAACACACTGTATCTGCAAATGAACAGCCTGAGGGCTGAGGACACTGCTGTGTATTACTGTGCGAGAGACCAGGATGACTCCAGTGATGCTTTTGATATCTGGGGGCAGGGAACCCTGGTAACAGTCAGTAGCGGTGGAGGCGGTTCAGGCGGCGGTGGCAGCGGCGGTGGCGGTAGCACGCAGTCTGTGTTGACGCAGCCGCCCTCAGTGTCTGCAGCCCCAGGACAAAAGGTCACTATCTCCTGCTCTGGAAGCAGCTCCAACATTGGGAACAACTATGTATCCTGGTACCAGCAGTTACCTGGTACAGCCCCCAAACTCCTCATTTATGACAATAATAAGCGACCCTCAGGGATTCCTGACCGTTTTAGTGGCAGCAAATCTGGCACTTCAGCCACCCTTGGCATCACCGGACTCCAGACTGGGGACGAGGCCGATTATTACTGCGGAACATGGGATTACAAAGTTTCGCGGCTTGTCTTCGGCGGAGGGACCAAGCTGACAGTTCTAGGGGCGGCC

[0037] SEQ ID NO: 7 - Anti-CSPG4 scFv "HRB298" nucleotide sequence ATGGCCCAGGTGCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTTAAAGTTTCCTGTAAGGCTAGTGGTTACACCTTTACCAGCTATGGTATCAGTTGGGTGAGGCAGGCCCCTGGACAAGGGCTTGAATGGATGGGATGGATCAGCGCTTACAATGGTAACACAAACTATGCACAGAAGCTCCAGGGCAGAGTCACCATGACCACAGACACATCCACTAGCACAGCATACATGGAGCTGAGGAGTTTGAGATCTGACGAGACGGCCGTTTATTACTGTGCGCGGCGAGATTACTATGATGGTAGTGGATTTGACTACTGGGGCCAAGGAACTCTGGTCACAGTCTCGAGCGGTGGAGGCGGTTCCGGCGGAGGGGGCAGCGGCGGTGGCGGATCAACTCAGTCTGTGTTGACGCAGCCGCCCTCAGTGAGTGCAGCCCCAGGCCAAAAAGTCACCATCTCCTGCTCTGGAAGCAGCTCCAACATTGGGAATAATTACGTATCCTGGTACCAGCAGCTCCCAGGCACAGCTCCCAAATTACTTATTTATGATAATAACAAGCGTCCCTCAGGGATTCCTGACCGGTTCTCTGGCTCCAAATCTGGCACGTCAGCCACCCTGGGCATAACTGGGCTCCAAACTGGGGACGAAGCCGATTATTACTGCGGAACTTATGATGGCGAAGGGCGCCACGAGGTGTTCGGCGGAGGGACCAAGCTGACCGTACTAGGGGCGGCA

[0038] SEQ ID NO: 8 - Anti-CSPG4 scFv "HRB299" nucleotide sequence ATGGCCCAGGTGCAACTGGTGGAATCGGGCGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTTTCCTGTGCAGCCTCTGGATTTACCTTCAGTAGCTATGCTATGCACTGGGTGCGCCAAGCTCCAGGCAAGGGGCTGGAATGGGTTGCAGTTATATCATATGATGGAAGTAATAAATACTACGCAGACTCCGTGAAGGGCCGTTTTACCATCTCCCGGGACAATTCCAAAAACACTCTGTATCTGCAAATGAACAGCCTTAGAGCTGAAGACACTGCCGTGTACTACTGCGCGCGCGATCCGTGGGGTGGTTGGTTAGGGAGCGATGCTTTTGACATTTGGGGCCAAGGAACCTTGGTCACAGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGCAGCACGCAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACACCCGGGCAGAGGGTCACCATTTCTTGTTCTGGAAGCAGCAGCAACATTGGAAGTAACACTGTAAACTGGTACCAGCAGTTGCCAGGAACGGCCCCCAAACTCCTCATCTATAGTAATAATCAGCGGCCTTCAGGGGTACCTGACCGATTCTCCGGCTCCAAGTCTGGCACCTCAGCCTCCCTCGCCATCAGTGGGCTCCAGTCTGAGGATGAGGCTGATTATTACTGCGCAGCATACGATGGGGACGGGGGGGAGGACGTGTTCGGCGGAGGTACAAAGCTGACAGTTCTAGGGGCCGCC

[0039] SEQ ID NO: 9 - Anti-CSPG4 scFv "HRB300" nucleotide sequence ATGGCCGAGGTGCAGCTGTTGGAATCTGGGGGAGGCTTGGTACAGCCGGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCTATGAGCTGGGTGCGCCAGGCTCCAGGGAAGGGGCTGGAATGGGTGTCAGCTATTAGTGGTAGTGGTGGCAGCACATACTACGCAGATTCCGTGAAAGGCCGGTTCACAATCTCCAGAGATAATAGTAAGAACACACTGTACCTTCAAATGAACAGCTTACGCGCCGAGGACACGGCGGTGTATTACTGTGCAAGACGATATAGCAGTGGCTGGTCATACTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACAGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGATCTACGCAGTCTGCCCTGACTCAACCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATATCCTGCACTGGAACATCCAGTGACGTTGGAGGTTATAACTATGTTTCCTGGTACCAACAGCACCCAGGCAAAGCCCCCAAACTCATGATTTATGAAGTAAGTAATCGGCCCTCAGGGGTTTCTAATAGGTTCTCAGGCTCCAAGAGTGGCAACACTGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGACGAGGCTGATTACTACTGCAGCTCATATGATACTTTTGAGAGGATTAGCGTGTTCGGCGGAGGGACCAAGCTTACCGTCCTAGGGGCGGCA

[0040] SEQ ID NO: 10 - Anti-CSPG4 scFv "HRB301" nucleotide sequence ATGGCACAAGTGCAACTGGTGCAGTCTGGAGCTGAAGTGAAGAAACCGGGGGCCTCAGTTAAGGTCTCCTGCAAAGCTTCTGGTTACACCTTTACTAGCTATGGTATCAGCTGGGTGAGACAAGCCCCTGGACAAGGGCTTGAGTGGATGGGCTGGATCAGCGCTTACAATGGTAACACAAACTATGCACAGAAGCTCCAGGGCCGTGTCACCATGACCACAGACACATCCACCTCCACAGCCTACATGGAGCTGAGGAGCCTGAGATCGGACGACACGGCTGTGTATTACTGTGCGAGACGGAGTTATGATAGTAGTGGACTTGACTACTGGGGCCAGGGAACATTGGTTACAGTTTCGAGCGGCGGCGGCGGTTCAGGCGGAGGGGGCAGCGGCGGTGGCGGTTCTACGCAGTCCGTGTTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATTTCTTGTTCTGGAAGCAGCTCCAACATTGGAAGTAATACTGTAAACTGGTACCAGCAGCTCCCAGGAACTGCCCCTAAATTACTCATATATAGTAATAATCAGCGGCCCTCAGGGGTACCTGACCGATTCTCCGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTGCAGAGCGAAGATGAGGCTGATTATTACTGCGCAGCATGGGATCGCAGGTGGCGCCTGGTGTTCGGCGGAGGGACCAAGCTGACTGTCCTAGGGGCCGCC

[0041] SEQ ID NO: 11 - Anti-CSPG4 scFv "HRB302" nucleotide sequence ATGGCCCAGGTGCAGCTTGTGCAGTCCGGTGCTGAGGTGAAGAAACCTGGGGCCTCAGTTAAGGTTTCCTGCAAGGCTTCTGGTTACACCTTTACCAGCTATGGTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGCTGGATAAGCGCTTACAATGGTAACACAAACTATGCACAAAAGTTGCAGGGCCGTGTCACCATGACCACAGACACATCCACAAGCACAGCCTACATGGAGCTGAGGTCCTTGAGATCTGACGACACGGCCGTGTATTACTGCGCAAGACAGGTGGGCGCTCCGACTCGCTTTGACTACTGGGGCCAGGGAACCCTGGTGACAGTCTCGAGCGGTGGAGGCGGGTCAGGCGGAGGCGGCAGCGGCGGTGGCGGATCGACGCAGTCTGTGCTGACTCAACCACCATCAGCGTCCGGGACCCCCGGGCAAAGGGTCACTATTAGTTGTAGTGGAAGCAGCTCCAACATTGGAAGTAATACTGTAAACTGGTACCAGCAGCTCCCAGGAACTGCCCCCAAACTCTTAATCTATAGTAATAATCAGCGGCCCTCAGGGGTTCCTGATCGGTTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCAATCAGTGGGCTCCAGTCTGAAGATGAAGCTGATTATTACTGTGCAGCATGGGATACGCACGCCTGGGCCCCCGTATTCGGCGGAGGGACTAAACTGACCGTCCTAGGGGCGGCT

[0042] SEQ ID NO: 12 - Anti-CSPG4 scFv "HRB303" nucleotide sequence ATGGCCCAGGTGCAACTGGTGCAGTCTGGGGCTGAGGTCAAGAAGCCAGGGTCCTCGGTGAAGGTCTCCTGTAAGGCTAGTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGAGACAAGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATATTTGGTACAGCAAACTACGCACAGAAATTTCAGGGCAGAGTTACGATAACTGCAGACGAATCCACTAGCACAGCATACATGGAGCTGAGTAGTTTAAGGTCTGAAGACACTGCAGTGTATTACTGTGCTCGTTCTAAATATAACTGGGCCTACAAAAATGATTACTGGGGCCAGGGAACCCTGGTTACAGTTTCAAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGCTCTACACAGAGCGTGTTGACGCAGCCGCCCTCAGTATCGGCGGCCCCAGGGCAGAAGGTCACCATCTCCTGCTCTGGAAGCAGTTCCAACATTGGGAATAACTATGTATCCTGGTACCAGCAGCTCCCAGGTACAGCCCCCAAATTGCTCATTTACGACAATAATAAGCGACCCTCAGGGATTCCTGATCGCTTCAGTGGCTCCAAATCTGGCACCTCAGCCACCCTGGGCATCACCGGACTTCAAACTGGGGACGAAGCTGATTATTACTGCGGAACATATGATCCCTGGGCTCGGACTGCCGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGGGCGGCC

[0043] SEQ ID NO: 13 - Anti-CSPG4 scFv "225.28S" nucleotide sequence CAGGTTAAACTCCAACAAAGTGGCGGAGGCTTGGTTCAGCCTGGAGGGAGTATGAAACTGTCTTGTGTCGTATCTGGTTTTACATTCTCAAATTATTGGATGAATTGGGTTAGGCAATCACCGGAGAAGGGATTGGAATGGATCGCTGAGATTCGGTTGAAATCAAACAACTTCGGTCGCTATTATGCGGAATCCGTGAAAGGTCGGTTCACGATTTCCCGCGACGATTCAAAGTCCAGTGCTTATCTGCAAATGATTAATCTTCGGGCAGAAGATACAGGAATATACTATTGTACCTCCTATGGTAACTATGTTGGTCACTATTTCGATCATTGGGGGCAGGGAACCACTGTCACCGTATCCAGCggtggcggagggagcggcggtggaggaagcggaggcggaggttccGACATTGAACTGACTCAATCTCCCAAATTTATGTCAACGAGCGTCGGGGACCGCGTGAGCGTTACGTGTAAGGCTTCACAAAACGTAGACACCAATGTGGCCTGGTATCAACAAAAGCCGGGACAATCTCCAGAGCCCCTGCTCTTTTCAGCAAGTTACAGGTACACCGGTGTTCCAGATAGATTCACAGGTAGTGGATCTGGTACTGATTTTACTCTCACCATAAGTAACGTGCAGTCCGAAGACCTCGCCGAGTACTTTTGTCAACAGTATAATAGTTACCCACTTACATTTGGGGGTGGAACAAAACTGGAAATCAAG

[0044] SEQ ID NO: 14 - Anti-BCAN scFv "HRB294" nucleotide sequence ATGGCCCAGGTACAACTGCAACAGTCAGGGCCAGGACTGGTTAAGCCCTCGCAGACCTTATCACTTACCTGTGCCATCTCCGGGGACAGTGTCTCTAGCAACAGTGCTGCTTGGAACTGGATAAGGCAGTCCCCAAGCCGCGGCCTTGAATGGCTGGGAAGAACATACTACAGGTCCAAGTGGTATAATGATTATGCAGTTAGTGTGAAAAGTCGAATAACTATCAACCCTGATACATCCAAGAACCAGTTCTCCCTGCAGCTGAACTCTGTGACTCCTGAGGACACAGCTGTGTATTACTGTGCGAGAGATCAGCGGAATTACGATTTTTGGAGTGGTTATTATCCGCCCGCAGAATTAGGGTACTACGGTATGGACGTCTGGGGCCAGGGAACCCTGGTCACAGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGCGGCAGCGGCGGTGGCGGCTCTACGCAGTCTGTGTTGACGCAGCCGCCTAGCGTGTCTGCTGCCCCAGGTCAGAAAGTGACCATCTCCTGCTCTGGAAGCAGCTCCAACATTGGGAATAATTATGTATCCTGGTACCAGCAACTCCCAGGAACAGCACCCAAACTCCTCATTTATGACAATAATAAGCGGCCCTCAGGGATTCCTGACCGTTTTTCTGGCAGTAAAAGCGGCACTTCAGCCACTCTGGGCATCACCGGGCTCCAAACTGGGGACGAGGCCGATTACTACTGCGGAACATGGGATTGGAGCGCATTGGTGGTGTTCGGCGGAGGGACCAAGCTGACCGTTCTAGGGGCGGCC

[0045] SEQ ID NO: 15 - Anti-BCAN scFv "HRB295" nucleotide sequence ATGGCCCAGGTGCAGCTGGTGGAGTCTGGGGGTGGCGTGGTCCAGCCTGGGAGGTCCTTGCGTCTCTCCTGCGCAGCCTCTGGATTCACTTTCAGTAGCTATGCTATGCACTGGGTCCGCCAAGCTCCGGGCAAGGGGCTGGAATGGGTGGCAGTTATAAGCTATGATGGAAGTAATAAATACTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTTTATCTGCAAATGAACAGCCTTCGCGCTGAAGACACAGCTGTGTATTACTGTGCCAGAGTATCAGACTGGAACGACGCCGCTTTTGATATTTGGGGCCAGGGAACTCTGGTTACAGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGATCGACCCAGTCTGTGCTGACTCAGCCACCCTCAGCGAGCGGGACACCAGGGCAGCGGGTCACCATTTCTTGTTCTGGAAGCAGCTCCAACATCGGTAGTAATACTGTAAACTGGTACCAGCAACTGCCAGGAACGGCCCCCAAACTCCTCATCTACAGTAATAATCAACGGCCTTCAGGGGTTCCTGATAGATTTTCTGGCTCCAAAAGCGGCACCTCAGCCTCCCTGGCCATTAGTGGGTTACAGTCTGAAGATGAGGCTGATTATTACTGCGCAGCATGGGACCCCGAGACCGCCAGGTGGGTGTTTGGCGGAGGGACAAAGTTGACCGTCCTAGGGGCGGCC

[0046] SEQ ID NO: 16 - Anti-BCAN scFv "HRB296" nucleotide sequence ATGGCCCAGGTACAGTTGCAGCAATCAGGTCCAGGACTGGTGAAGCCCAGCCAAACCTTATCATTAACTTGTGCAATCTCCGGGGACAGTGTTTCTAGCAACAGTGCTGCTTGGAACTGGATCAGGCAGTCCCCTTCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACCGGAGCAAGTGGTATAATGATTATGCAGTTAGCGTGAAAAGTCGGATAACCATCAACCCTGACACATCCAAGAACCAGTTCTCCCTGCAACTGAACTCTGTGACTCCCGAAGACACGGCTGTGTATTACTGTGCACGCAGAGGGGAACACTATGATAGTAGTGGTTATTACTACGGCCTTGATTACTGGGGCCAGGGAACCCTGGTCACAGTCAGCAGCGGTGGAGGCGGTTCAGGCGGCGGTGGCAGCGGCGGTGGCGGATCTACGCAGTCGGTGTTGACACAGCCGCCCTCAGTGTCTGCGGCCCCAGGGCAAAAAGTTACCATATCCTGCTCTGGAAGCAGCTCCAACATTGGGAATAATTATGTATCCTGGTACCAGCAGCTCCCAGGAACAGCCCCCAAACTCCTCATTTATGACAATAATAAGCGTCCTTCAGGGATTCCTGACCGATTCTCTGGCTCCAAGTCTGGCACTTCAGCCACTCTGGGCATCACCGGACTCCAGACTGGGGACGAGGCCGATTACTACTGCGGAACATATGATGTCGCGGCTGGGTACGTGTTTGGCGGAGGGACCAAACTGACCGTCCTAGGGGCAGCC

[0047] SEQ ID NO: 17 - Anti-BCAN scFv "HRB297" nucleotide sequence ATGGCCCAGGTACAGCTGGTGCAGAGCGGGGCTGAGGTGAAAAAGCCCGGGTCCTCGGTGAAAGTGTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATAAGCTGGGTGCGACAAGCCCCGGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGAGTCACCATTACCGCGGACGAATCCACGAGCACAGCCTACATGGAACTGAGCAGCCTTAGGTCTGAGGACACTGCCGTGTATTACTGTGCGAGACCACGTACTGCAGGCTGGAGTTATGATGCTTTTGATGCCTGGGGCCAGGGAACATTGGTAACAGTCTCAAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGATCTACGCAATCTGCCTTAACTCAGCCTGCCAGTGTGTCTGGGAGTCCTGGACAGTCAATAACCATTTCCTGTACTGGAACCAGCAGTGACGTTGGCGGTTATAACTACGTTTCCTGGTACCAACAGCACCCAGGCAAAGCCCCCAAACTCATGATTTACGAAGTCAGTAATAGGCCCTCAGGGGTTTCTAATCGCTTCTCTGGCTCCAAGTCAGGCAACACAGCCTCCCTGACTATCTCTGGGCTCCAGGCTGAGGACGAGGCTGATTATTACTGCAGTTCATATGATTGGCGGTCCTCCGGGTCGGTGTTTGGCGGAGGGACCAAGCTGACCGTCCTAGGGGCAGCA

[0048] SEQ ID NO: 18 - Anti-TNC scFv "E10" nucleotide sequence

[0049] GAGGTACAGCTAGTGGAGTCAGGCGGGGGCCTGGTCCAGCCAGGCGGATCGTTAAGACTTAGTTGCGCAGCAAGCGGGTTTACGTTCTCAGGTAGCCGAATGGGGTGGGTGAGACAGGCCCCCGGGAAAGGACTCGAATGGGTTTCCGCGATCAACGAAGAAGGTGGACAAACTTACTACGCCGATAGCGTGAAGGGACGGTTTACAATTTCTCGTGACAACTCCAAGAATACCCTGTATCTGCAAATGAATAGTTTGAGGGCTGAGGACACCGCCGTCTATTATTGTGCTAAACATCCTCCGCACCGCCCCTTCGATTACTGGGGCCAGGGCACACTCGTGACTGTTTCTAGGGGTGGAGGCGGTTCCGGCGGAGGGGGCAGCGGCGGTGGCGGATCATCTTCCGAATTAACTCAAGACCCAGCCGTTTCTGTGGCCCTCGGCCAGACAGTCAGGATCACGTGCCAAGGGGATAGTCTGCGATCCTACTATGCAAGCTGGTACCAGCAGAAACCGGGCCAGGCTCCTGTATTGGTGATCTACGGAAAGAATAACAGACCCTCTGGTATACCCGACCGGTTCTCCGGTAGCAGTAGCGGCAACACCGCTTCACTTACTATTACCGGAGCACAGGCCGAGGATGAGGCGGACTATTATTGTAATTCATCGCACGGCCCACGTCGCCCTGTCGTGTTTGGGGGAGGCACCAAGCTGACAGTGCTGGGG

[0050] SEQ ID NO: 19 - Anti-TNC scFv "P12" nucleotide sequence

[0051] GAGGTGCAGCTGGTCGAGAGCGGTGGAGGCCTCGTGCAGCCCGGAGGGTCATTGCGTCTCTCCTGCGCCGCCAGCGGCTTTACCTTCGGTCAATATAGCATGAGTTGGGTCAGGCAGGCGCCTGGCAAGGGACTGGAATGGGTTTCCGCCATCACCGGTACAGGAGGGGAAACATACTACGCTGACTCAGTAGAGGGGAGATTCACTATTTCTCGAGATAACTCCAAGAACACGCTGTATCTACAAATGAATTCTTTACGCGCAGAAGACACTGCAGTTTATTACTGTGCTAAAGGGAGACGGATATTTGATTACTGGGGCCAGGGCACCCTTGTGACAGTGAGTAGGGGTGGAGGCGGTTCCGGCGGAGGGGGCAGCGGCGGTGGCGGATCATCCTCCGAACTGACACAGGACCCGGCTGTGAGCGTCGCCCTTGGTCAGACTGTGAGAATTACATGCCAGGGGGATTCACTCAGGCGGCAGCCTGCTTCTTGGTACCAACAAAAGCCTGGACAGGCCCCCGTGTTGGTGATATACTACAAAAAGCTGCGACCATCAGGAATCCCTGACCGCTTTTCTGGAAGCTCCAGTGGGAATACCGCAAGTCTCACCATCACTGGTGCCCAGGCAGAGGATGAGGCGGACTATTATTGTAACTCGTTCAGCCCCAAACCCAAACCAGTAGTCTTCGGCGGCGGGACCAAGCTGACGGTTTTAGGC

[0052] SEQ ID NO: 20 - Anti-TNC scFv "F16" Nucleotide Sequence

[0053] GAGGTCCAATTACTTGAATCAGGCGGAGGCCTGGTGCAGCCTGGAGGCAGCCTGAGACTGTCCTGCGCGGCAAGCGGTTTCACTTTCTCCAGATATGGCGCATCCTGGGTTAGGCAGGCACCCGGTAAAGGACTGGAGTGGGTATCTGCCATTTCTGGGAGTGGAGGGAGTACCTACTACGCTGATTCGGTGAAGGGGCGTTTTACAATCTCACGAGACAATAGCAAAAACACACTATATCTCCAGATGAATTCTCTCCGCGCCGAAGACACGGCTGTCTACTATTGTGCTAAGGCCCACAACGCCTTTGATTACTGGGGCCAGGGGACCTTGGTGACTGTGAGCCGGGGTGGAGGCGGTTCCGGCGGAGGGGGCAGCGGCGGTGGCGGATCATCGAGCGAACTGACACAGGACCCGGCGGTTTCCGTGGCACTGGGGCAGACAGTAAGAATAACTTGTCAAGGGGATAGCCTGCGCAGTTACTACGCCAGCTGGTACCAGCAGAAACCAGGCCAGGCCCCCGTTTTGGTGATTTATGGGAAGAATAACAGGCCTTCCGGCATCCCCGACCGGTTTTCTGGATCTAGTTCTGGAAACACCGCATCACTTACCATCACGGGAGCTCAAGCCGAGGATGAGGCTGACTACTATTGCAATTCATCCGTCTATACTATGCCTCCAGTGGTGTTCGGTGGCGGTACAAAGTTAACCGTCCTCGGC

[0054] SEQ ID NO: 21-40 are all scFv, orientation is V H - linker - V L , linker (G4S)3 is lowercase. Antigen binding region is underlined ABR1 ), bold and underlined ABR2 ), and underlined and italicized ABR3) highlighted. All ABRs were predicted using Paratome (Kunik V et al (2012). Structural Consensus among Antibodies Defines the Antigen Binding Site. PLoS Comput Biol 8(2): e1002388. doi: 10.1371 / journal.pcbi.1002388; and Kunik V et al (2012). Paratome: An online tool for systematic identification of antigen binding regions in antibodies based on sequence or structure. Nucleic Acids Res. 2012 Jul;40(Web Server issue):W521-4. doi: 10.1093 / nar / gks480. Epub 2012 Jun 6).

[0055] SEQ ID NO: 21 - Anti-PTPRZ1 scFv "RRB469" polypeptide sequence

[0056]

[0057] SEQ ID NO: 22 - Anti-PTPRZ1 scFv "RRB470" polypeptide sequence

[0058]

[0059] SEQ ID NO: 23 - Anti-PTPRZ1 scFv "RRB471 " polypeptide sequence

[0060]

[0061] SEQ ID NO: 24 - Anti-PTPRZ1 scFv "RRB473" polypeptide sequence

[0062]

[0063] SEQ ID NO: 25 - Anti-PTPRZ1 scFv "RRB474" polypeptide sequence

[0064]

[0065] SEQ ID NO: 26 - Anti-PTPRZ1 scFv "RRB476" polypeptide sequence

[0066]

[0067] SEQ ID NO: 27 - Anti-CSPG4 scFv "HRB298" polypeptide sequence

[0068]

[0069] SEQ ID NO: 28 - Anti-CSPG4 scFv "HRB299" polypeptide sequence

[0070]

[0071] SEQ ID NO: 29 - Anti-CSPG4 scFv "HRB300" polypeptide sequence

[0072]

[0073] SEQ ID NO: 30 - Anti-CSPG4 scFv "HRB301" polypeptide sequence

[0074]

[0075] SEQ ID NO: 31 - Anti-CSPG4 scFv "HRB302" polypeptide sequence

[0076]

[0077] SEQ ID NO: 32 - Anti-CSPG4 scFv "HRB303" polypeptide sequence

[0078]

[0079] SEQ ID NO: 33 - Anti-CSPG4 scFv "225.28S" polypeptide sequence

[0080]

[0081] SEQ ID NO: 34 - Anti-BCAN scFv "HRB294" polypeptide sequence

[0082]

[0083] SEQ ID NO: 35 - Anti-BCAN scFv "HRB295" polypeptide sequence

[0084]

[0085] SEQ ID NO: 36 - Anti-BCAN scFv "HRB296" polypeptide sequence

[0086]

[0087] SEQ ID NO: 37 - Anti-BCAN scFv "HRB297" polypeptide sequence

[0088]

[0089] SEQ ID NO: 38 - Anti-TNC scFv "E10" polypeptide sequence

[0090]

[0091] SEQ ID NO: 39 - Anti-TNC scFv "P12" polypeptide sequence

[0092]

[0093] SEQ ID NO: 40 - Anti-TNC scFv "F16" polypeptide sequence

[0094]

[0095] SEQ ID NO: 41 - Anti-PTPRZl scFv "RRB469" V H polypeptide sequence MAQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGSGYSYGPGYDAFDIWGQGTLVTVSS

[0096] SEQ ID NO: 42 - Anti-PTPRZl scFv "RRB469" V L polypeptide sequence TNFMLTQPHSVSESPGKTVTISCTRSSGSIASNYVQWYQQRPGSSPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSWDPVFGVFGGGTKLTVLGAA

[0097] SEQ ID NO: 43 - Anti-PTPRZl scFv "RRB470" V H polypeptide sequence MAQVQLVESGGGLVQPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSYISSSSSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPGYGDFPGAFDIWGQGTLVTVSS

[0098] SEQ ID NO: 44 - Anti-PTPRZl scFv "RRB470" V L Polypeptide sequence MAQVQLVESGGGLVQPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSYISSSSSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPGYGDFPGAFDIWGQGTLVTVSS

[0099] SEQ ID NO: 45 - Anti-PTPRZl scFv "RRB471" V H Polypeptide sequence MAQVQLVESGGGLVQPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSYISSSSSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPGYGDFPGAFDIWGQGTLVTVSS

[0100] SEQ ID NO: 46 - Anti-PTPRZl scFv "RRB471" V L Polypeptide sequence MAQVQLVESGGGLVQPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSYISSSSSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPGYGDFPGAFDIWGQGTLVTVSS

[0101] SEQ ID NO: 47 - Anti-PTPRZl scFv "RRB473" V H Polypeptide sequence MAQVQLVESGGGLVQPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSYISSSSSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPGYGDFPGAFDIWGQGTLVTVSS

[0102] SEQ ID NO:48 - Anti-PTPRZ1 scFv "RRB473" V L Polypeptide sequence TQSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTYDYIATRAVFGGGTKLTVLGAA

[0103] SEQ ID NO:49 - Anti-PTPRZ1 scFv "RRB474" V H Polypeptide sequence MAQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGTYYDFWSGYYDAFDIWGQGTLVTVSS

[0104] SEQ ID NO:50 - Anti-PTPRZ1 scFv "RRB474" V L Polypeptide sequence TQSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSYWQPVFGGGTKLTVLGAA

[0105] SEQ ID NO:51 - Anti-PTPRZ1 scFv "RRB476" V H Polypeptide sequence MAQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDQDDSSDAFDIWGQGTLVTVSS

[0106] SEQ ID NO:52 - Anti-PTPRZ1 scFv "RRB476" V L Polypeptide sequence TQSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDYKVSRLVFGGGTKLTVLGAA

[0107] SEQ ID NO: 53 - Anti-CSPG4 scFv "HRB298"V H Polypeptide sequence MAQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARRDYYDGSGFDYWGQGTLVTVSS

[0108] SEQ ID NO: 54 - Anti-CSPG4 scFv "HRB298"V L Polypeptide sequence TQSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTYDGEGRHEVFGGGTKLTVLGAA

[0109] SEQ ID NO: 55 - Anti-CSPG4 scFv "HRB299"V H Polypeptide sequence MAQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDPWGGWLGSDAFDIWGQGTLVTVSS

[0110] SEQ ID NO: 56 - Anti-CSPG4 scFv "HRB299"V L Polypeptide sequence TQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQLPGTAPKLLIYSNNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAYDGDGGEDVFGGGTKLTVLGAA

[0111] SEQ ID NO: 57 - Anti-CSPG4 scFv "HRB300"V H Polypeptide sequence MAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRYSSGWSYYFDYWGQGTLVTVSS

[0112] SEQ ID NO: 58 - Anti-CSPG4 scFv "HRB300"V L Polypeptide sequence TQSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYDTFERISVFGGGTKLTVLGAA

[0113] SEQ ID NO: 59 - Anti-CSPG4 scFv "HRB301"V H Polypeptide sequence MAQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARRSYDSSGLDYWGQGTLVTVSS

[0114] SEQ ID NO: 60 - Anti-CSPG4 scFv "HRB301"V L Polypeptide sequence TQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQLPGTAPKLLIYSNNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDRRWRLVFGGGTKLTVLGAA

[0115] SEQ ID NO: 61 - Anti-CSPG4 scFv "HRB302"V H Polypeptide sequence MAQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARQVGAPTRFDYWGQGTLVTVSS

[0116] SEQ ID NO: 62 - Anti-CSPG4 scFv "HRB302" V L Polypeptide sequence TQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQLPGTAPKLLIYSNNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDTHAWAPVFGGGTKLTVLGAA

[0117] SEQ ID NO: 63 - Anti-CSPG4 scFv "HRB303" V H Polypeptide sequence MAQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSKYNWAYKNDYWGQGTLVTVSS

[0118] SEQ ID NO: 64 - Anti-CSPG4 scFv "HRB303" V L Polypeptide sequence TQSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTYDPWARTAVFGGGTKLTVLGAA

[0119] SEQ ID NO: 65 - Anti-CSPG4 scFv "225.28S" V H Polypeptide sequence QVKLQQSGGGLVQPGGSMKLSCVVSGFTFSNYWMNWVRQSPEKGLEWIAEIRLKSNNFGRYYAESVKGRFTISRDDSKSSAYLQMINLRAEDTGIYYCTSYGNYVGHYFDHWGQGTTVTVSS

[0120] SEQ ID NO: 66 - Anti-CSPG4 scFv "225.28S" V L Polypeptide sequence DI EITQSPKFMSTSVGDRVSVTCKASQNVDTNVAWYQQKPGQSPEPLLFSASYRYTGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCQQYNSYPLTFGGGTKLEIK

[0121] SEQ ID NO: 67 - Anti-BCAN scFv "HRB294" V H Polypeptide sequence MAQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARDQRNYDFWSGYYPPAELGYYGMDVWGQGTLVTVSS

[0122] SEQ ID NO: 68 - Anti-BCAN scFv "HRB294" V L Polypeptide sequence TQSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDWSALVVFGGGTKLTVLGAA

[0123] SEQ ID NO: 69 - Anti-BCAN scFv "HRB295" V H Polypeptide sequence MAQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVSDWNDAAFDIWGQGTLVTVSS

[0124] SEQ ID NO: 70 - Anti-BCAN scFv "HRB295" V L Polypeptide sequence TQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQLPGTAPKLLIYSNNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDPETARWVFGGGTKLTVLGAA

[0125] SEQ ID NO: 71 - Anti-BCAN scFv "HRB296"V H Polypeptide sequence MAQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARRGEHYDSSGYYYGLDYWGQGTLVTVSS

[0126] SEQ ID NO: 72 - Anti-BCAN scFv "HRB296"V L Polypeptide sequence TQSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTYDVAAGYVFGGGTKLTVLGAA

[0127] SEQ ID NO: 73 - Anti-BCAN scFv "HRB297"V H Polypeptide sequence MAQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARPRTAGWSYDAFDAWGQGTLVTVSS

[0128] SEQ ID NO: 74 - Anti-BCAN scFv "HRB297"V L Polypeptide sequence TQSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYDWRSSGSVFGGGTKLTVLGAA

[0129] SEQ ID NO:75 - Anti-TNC scFv "E10"V H Polypeptide sequences EVQLVESGGGLVQPGGSLRLSCAASGFTFSGSRMGWVRQAPGKGLEWVSAINEEGGQ TY YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHPPHRPFDYWGQGTLV TVSR

[0130] SEQ ID NO:76 - Anti-TNC scFv "E10"V L Polypeptide sequences

[0131] SSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGI PDRFSGSSSGNTASLTITGAQAEDEADYYCNSSHGPRRPVVFGGGTKLTVLG

[0132] SEQ ID NO:77 - Anti-TNC scFv "P12"V H Polypeptide sequences EVQLVESGGGLVQPGGSLRLSCAASGFTFGQYSMSWVRQAPGKGLEWVSAITGTGGE TYYADSVEGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGRRIFDYWGQGTLVT VSR

[0133] SEQ ID NO:78 - Anti-TNC scFv "P12"V L Polypeptide sequences

[0134] SSELTQDPAVSVALGQTVRITCQGDSLRRQPASWYQQKPGQAPVLVIYYKKLRPSGI PDRFSGSSSGNTASLTITGAQAEDEADYYCNSFSPKPKPVVFGGGTKLTVLG

[0135] SEQ ID NO:79 - Anti-TNC scFv "F16"V H Polypeptide sequences EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYGASWVRQAPGKGLEWVSAISGSGGS TYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAHNAFDYWGQGTLVT VSR

[0136] SEQ ID NO: 80 - Anti-TNC scFv "F16" V L Polypeptide sequences

[0137] SSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSSVYTMPPVVFGGGTKLTVLG

[0138] The CDRs of SEQ ID NOs: 83-93 are annotated in the following manner: CDR1 underlined ), CDR2 bold and underlined ), and CDR3 italicized and underlined ). The CDRs were predicted using the antibody property prediction tool in Benchling, where the annotation was assigned using the North CDR definition based on the sequence-based Antibody CAnonical LOOP (SCALOP) structure annotation developed by the Oxford Protein Informatics Group (Dunbar et al, SAbPred: a structure-based antibody prediction server, Nucleic Acids Research, Volume 44, Issue Wl, 8 July 2016, Pages W474-W478, https: / / doi.org / 10.1093 / nar / gkw361).

[0139] SEQ ID NO: 83 - Anti-BCAN "RB826" V HH Polypeptide sequences

[0140]

[0141] SEQ ID NO: 84 - Anti-BCAN "RB827" V HH Polypeptide sequences

[0142]

[0143] SEQ ID NO: 85 - Anti-BCAN "RB828" V HH Polypeptide sequences

[0144]

[0145] SEQ ID NO: 86 - Anti-BCAN "RB829" V HH Polypeptide sequences

[0146]

[0147] SEQ ID NO: 87 - Anti-CSPG4 "RB830" V HH Polypeptide sequence

[0148]

[0149] SEQ ID NO: 88 - Anti-CSPG4 "RB831" V HH Polypeptide sequence

[0150]

[0151] SEQ ID NO: 89 - Anti-PTPRZ1 "RB832" V HH Polypeptide sequence

[0152]

[0153] SEQ ID NO: 90 - Anti-PTPRZ1 "RB833" V HH Polypeptide sequence

[0154]

[0155] SEQ ID NO: 91 - Anti-PTPRZ1 "RB834" V HH Polypeptide sequence

[0156]

[0157] SEQ ID NO: 92 - Anti-TNC "RB835" V HH Polypeptide sequence

[0158]

[0159] SEQ ID NO: 93 - Anti-TNC "RB836" V HH Polypeptide sequence

[0160]

[0161] SEQ ID NO: 94 - Anti-BCAN "RB826" V HH Polynucleotide sequence GAGGTGCAATTGCAGGAGAGCGGCGGTGGATTTGTGCAGGCCGGAGGGTCCCTCCGACTTTCATGCGCTGCATCTGGGGAAAGCTTCGTTCCAGAAATGGGCTGGTTCCGCCAGGCACCTGGTAAAGAGCGGGAGTTTGTGGCCGCCATAAGCGTAGAACAGAGTCTGGATATGTATTACGCCGATTCCGTCAAGGGCAGGTTTACCATTAGTAGAGACAATGCGAAAAACACTGTCTATCTGCAGATGAATTCTTTAAAGCCCGAGGATACTGCTACATACTACTGTGCTATCGTTGCTATCGACGACTTCAACACATATTGGGGACAGGGGACCCAAGTGACGGTGTCATCC

[0162] SEQ ID NO: 95 - Anti-BCAN "RB827" V HH Polynucleotide sequence GAGGTGCAATTGCAGGAGAGCGGCGGTGGATTTGTGCAGGCCGGAGGGTCCCTCCGACTTTCATGCGCTGCATCTGGGGAAAGCTTCGTTCCAGAAATGGGCTGGTTCCGCCAGGCACCTGGTAAAGAGCGGGAGTTTGTGGCCGCCATAAGCGTAGAACAGAGTCTGGATATGTATTACGCCGATTCCGTCAAGGGCAGGTTTACCATTAGTAGAGACAATGCGAAAAACACTGTCTATCTGCAGATGAATTCTTTAAAGCCCGAGGATACTGCTACATACTACTGTGCTATCGTTGCTATCGACGACTTCAACACATATTGGGGACAGGGGACCCAAGTGACGGTGTCATCC

[0163] SEQ ID NO: 96 - Anti-BCAN "RB828" V HH Polynucleotide sequence GAGGTGCAATTACAGGAAAGCGGCGGTGGATTTGTCCAGGCTGGGGGGTCCCTCCGACTGTCATGTGCTGCAAGCGGTGTGCACGTCCCTCTGCAGAACATGGGCTGGTTCCGCCAAGCACCGGGGAAAGAGAGGGAGTTCGTTGCCGCCATTAGCCGGGATATGCCAGTCGATAACTATTACGCCGACTCCGTGAAGGGCAGGTTTACCATCAGTAGAGACAATGCGAAAAACACGGTGTACCTTCAGATGAATAGTTTGAAGCCCGAAGACACTGCTACATATTACTGCGCTGTTCGTGTGTACACTACCTCTCTGTGGTATTGGGGACAGGGAACCCAGGTAACAGTGTCATCT SEQ ID NO: 97 - Anti-BCAN "RB829" V HH Polynucleotide sequence GAGGTGCAATTACAGGAAAGCGGCGGTGGATTTGTCCAGGCTGGGGGGTCCCTCCGACTGTCATGTGCTGCAAGCGGTGTGCACGTCCCTCTGCAGAACATGGGCTGGTTCCGCCAAGCACCGGGGAAAGAGAGGGAGTTCGTTGCCGCCATTAGCCGGGATATGCCAGTCGATAACTATTACGCCGACTCCGTGAAGGGCAGGTTTACCATCAGTAGAGACAATGCGAAAAACACGGTGTACCTTCAGATGAATAGTTTGAAGCCCGAAGACACTGCTACATATTACTGCGCTGTTCGTGTGTACACTACCTCTCTGTGGTATTGGGGACAGGGAACCCAGGTAACAGTGTCATCT SEQ ID NO: 98 - Anti-CSPG4 "RB830" V HH Polynucleotide sequence GAGGTGCAGCTGCAGGAGAGCGGCGGGGGATTTGTCCAAGCTGGTGGGTCCTTGCGACTGTCATGCGCTGCATCGGGTAACGTTCAGAGACGGTTCAGAATGGGCTGGTTCCGCCAGGCACCTGGGAAAGAGAGGGAATTTGTTGCCGCCATTAGCACAAACCGGGATAGGCGCAACTATTACGCCGACTCCGTCAAGGGCAGGTTTACCATCAGTCGTGACAATGCGAAAAACACGGTGTACCTTCAGATGAATTCTCTCAAGCCCGAAGATACTGCTACATATTACTGTGCTGTGATGAATAAGAATTTCACTTACATGTATTGGGGACAGGGAACCCAAGTAACCGTGTCATCT SEQ ID NO: 99 - Anti-CSPG4 "RB831" V HH Polynucleotide sequence GAGGTACAGCTTCAAGAAAGCGGCGGGGGATTCGTCCAGGCGGGTGGGTCCTTGCGGCTGTCATGCGCTGCATCTGGGGAACCAGTGCACAGTACAAGTATGGGCTGGTTCCGCCAAGCACCGGGTAAAGAGAGGGAGTTTGTTGCCGCCATCAGCCTGAACGTTATGCACAGCAGATATTACGCCGATTCCGTCAAGGGCCGATTTACCATTTCGCGTGACAATGCAAAAAACACCGTGTATTTACAGATGAATTCCCTCAAGCCCGAAGACACTGCTACGTATTACTGTGCTTCTTACCCTCATTACATGACTCCCATGTATTGGGGACAGGGAACCCAGGTGACAGTGTCATCT SEQ ID NO: 100 - Anti-PTPRZ1 "RB832" V HH Polynucleotide sequence GAGGTGCAATTACAGGAAAGCGGCGGTGGATTTGTCCAGGCAGGTGGGTCCCTCCGACTCTCATGCGCCGCATCTGGGAGCGACGTCACACGTCTGAACATGGGCTGGTTCCGCCAGGCACCTGGGAAGGAGAGGGAGTTTGTTGCCGCCATTAGCAGAAGTGAACAGAACCGGCTGTACTACGCCGATTCCGTGAAGGGCAGGTTCACCATCAGTAGAGACAATGCGAAAAACACCGTTTATCTTCAGATGAATTCTTTGAAGCCCGAAGATACTGCTACCTATTACTGTGCAACAACAGCTCTGGCTGCTGTGACTAAAGCCACTCACTATTGGGGACAGGGAACCCAAGTAACGGTGTCATCC

[0164] SEQ ID NO: 101 - Anti-PTPRZl "RB833" V HH Polynucleotide sequence GAGGTACAGCTGCAAGAGAGCGGCGGGGGATTTGTCCAGGCTGGTGGGTCCCTCCGACTTTCATGTGCTGCATCTGGTAACGTTGTGTTCCTGACGTCGATGGGCTGGTTCCGCCAGGCACCCGGGAAGGAGAGGGAATTTGTTGCCGCCATAAGCCGGAGCTTCTTCGATGATCCATATTACGCCGATTCCGTCAAGGGCAGGTTTACCATTAGTAGAGACAATGCAAAAAACACAGTGTATTTACAGATGAATAGTTTGAAGCCCGAAGACACTGCTACATATTACTGCGCTCCGTACAAAAGAGACTACCGGCAGCACACTGTGCCTCGTCATATCTATTGGGGACAGGGAACCCAAGTGACCGTGTCATCT

[0165] SEQ ID NO: 102 - Anti-PTPRZl "RB834" V HH Polynucleotide sequence GAGGTGCAGCTTCAAGAGAGCGGCGGGGGATTTGTCCAAGCTGGTGGGTCCCTCCGATTATCATGCGCTGCATCTGGGAGCAGTAGTAGACTGTTCAACATGGGCTGGTTCCGCCAGGCACCTGGTAAAGAGCGGGAATTTGTGGCCGCCATTAGCCACATGGAAAACGATGTTGATTATTACGCCGATTCCGTGAAGGGCAGGTTCACCATCTCCAGAGACAATGCGAAGAACACAG TCTATTTGCAGATGAATTCTCTCAAGCCCGAAGACACTGCTACTTATTACTGTGCTCTGATGCTGAAAGGCTGGGACCATTCGACACGTTACTACTGGGGACAGGGAACCCAGGTAACGGTGTCATCT

[0166] SEQ ID NO: 103 - Anti-TNC "RB835" V HH Polynucleotide sequence GAGGTACAGCTTCAGGAGAGCGGCGGCGGATTTGTGCAAGCAGGTGGGTCCCTCCGATTATCATGCGCTGCATCCGGTAACCTGATGGTCCGTCGGGAAATGGGCTGGTTCCGCCAGGCACCTGGGAAAGAGAGGGAGTTCGTTGCCGCCATTAGCAGAAGCAGTCAGGAGGAAGTTTATTACGCCGACTCCGTCAAGGGCAGGTTTACCATCAGTAGAGACAATGCGAAAAACACAGTGTATCTGCAGATGAATTCTTTGAAGCCCGAAGATACTGCTACGTATTACTGTGCTATGGAAGGGTTCTACGTGTACAACCAGTATTGGGGACAGGGAACCCAAGTGACAGTGTCATCT SEQ ID NO: 104 - Anti-TNC "RB836" V HH Polynucleotide sequence GAGGTGCAACTGCAGGAGAGCGGCGGGGGATTTGTGCAGGCAGGGGGGTCCCTCCGACTTTCATGCGCTGCATCCGGTAGAAGAGTAACGGTCAGTGAAATGGGCTGGTTCCGCCAGGCACCTGGTAAAGAGAGGGAGTTTGTTGCCGCCATCAGCATGCGGGAACGGGAAAGCATGTATTACGCCGACTCCGTGAAGGGCAGGTTCACCATTAGTCGTGACAATGCTAAAAACACCGTCTATCTGCAGATGAACTCGTTGAAGCCCGAAGATACTGCTACATATTACTGTGCTGAGTACACTCACTGGTACTCTCATCCATATTGGGGACAGGGAACCCAAGTTACAGTGTCATCT

[0167] Hinge, transmembrane domain, and intracellular domain sequences are provided below.

[0168] SEQ ID NO: 117 - Human CD8a hinge polypeptide sequence TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

[0169] SEQ ID NO: 118 - Human IgG4 hinge polypeptide sequence ESKYGPPCPPCP

[0170] SEQ ID NO: 119 - Human IgGl hinge polypeptide sequence EPKSPDKTHTCP

[0171] SEQ ID NO: 120 - Human IgGl hinge polypeptide sequence EPKSCDKTHTCP

[0172] SEQ ID NO: 121 - Human IgGl long hinge polypeptide sequence

[0173] AEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPK

[0174] SEQ ID NO: 122 - human CD8a transmembrane domain polypeptide sequence IYIWAPLAGTCGVLLLSLVITLYC

[0175] SEQ ID NO: 123 - human CD28 transmembrane domain polypeptide sequence FWVLVVVGGVLACYSLLVTVAFIIFWV

[0176] SEQ ID NO: 124 - human 4-1BB intracellular region polypeptide sequence KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0177] SEQ ID NO: 125 - human CD28 intracellular region polypeptide sequence RSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

[0178] SEQ ID NO: 126 - human CD3z intracellular region polypeptide sequence 1

[0179] RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0180] SEQ ID NO: 127 - human CD3z intracellular region polypeptide sequence 2

[0181] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0182] SEQ ID NO: 128 - human CD3z intracellular region polypeptide sequence 3

[0183] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQARRA

[0184] SEQ ID NO: 129 - human CD8a hinge polynucleotide sequence

[0185] ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT

[0186] SEQ ID NO: 130 - human IgG4 hinge polynucleotide sequence GAGTCAAAGTATGGGCCTCCATGTCCTCCATGTCCG

[0187] SEQ ID NO: 131 - human IgGl hinge polynucleotide sequence GAGCCGAAGTCGCCAGACAAAACTCACACTTGTCCT

[0188] SEQ ID NO: 132 - human IgGl long hinge polynucleotide sequence

[0189] GCAGAGCCGAAGTCGCCAGACAAAACTCACACTTGTCCTCCTTGTCCAGCCCCCCCCGTGGCGGGTCCCAGCGTGTTCCTGTTTCCTCCGAAGCCAAAAGATACCCTGATGATCGCACGCACCCCCGAAGTAACGTGCGTGGTGGTCGATGTGTCACATGAGGACCCTGAGGTCAAATTCAATTGGTACGTTGACGGGGTAGAAGTTCACAACGCTAAAACCAAGCCAAGGGAGGAGCAGTACAACAGCACCTATCGAGTGGTGAGTGTACTGACCGTCCTACACCAAGATTGGTTGAATGGCAAGGAATACAAGTGTAAGGTGTCCAACAAGGCTTTACCTGCTCCTATCGAGAAGACAATTTCTAAGGCCAAAGGCCAGCCCAGAGAGCCACAGGTTTACACACTCCCACCATCACGTGACGAGCTTACGAAAAATCAGGTCAGTCTGACTTGCCTCGTTAAAGGATTTTACCCTAGTGACATAGCCGTGGAATGGGAAAGCAACGGCCAGCCCGAGAATAATTATAAAACAACACCGCCCGTGCTCGACTCTGATGGTTCTTTTTTCCTGTATTCCAAACTGACCGTCGATAAGAGCCGGTGGCAGCAGGGAAACGTGTTCTCCTGCTCCGTCATGCATGAAGCCTTGCATAACCACTATACTCAAAAGTCACTGTCTCTTAGCCCTGGGAAGAAAGATCCCAAG

[0190] SEQ ID NO:133 - Human CD8a Transmembrane Region Polynucleotide Sequence

[0191] ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC

[0192] SEQ ID NO:134 - Human CD28 Transmembrane Region Polynucleotide Sequence

[0193] TTCTGGGTGCTGGTGGTCGTGGGCGGCGTGCTGGCCTGTTACAGCCTGCTCGTGACCGTGGCCTTCATCATCTTTTGGGTC

[0194] SEQ ID NO: 135 - human 4-1BB intracellular region polynucleotide sequence

[0195] AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG

[0196] SEQ ID NO: 136 - human CD28 intracellular region polynucleotide sequence

[0197] CGAAGCAAGCGGAGCAGAGGCGGCCACAGCGACTACATGAACATGACCCCCAGACGGCCTGGCCCCACCCGGAAGCACTACCAGCCTTACGCCCCTCCCAGAGACTTCGCCGCCTACAGAAGC

[0198] SEQ ID NO: 137 - human CD3z intracellular region polynucleotide sequence 1

[0199] AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC

[0200] SEQ ID NO: 138 - human CD3z intracellular region polynucleotide sequence 2

[0201] AGAGTGAAGTTCAGCCGCAGCGCCGACGCCCCTGCCTACCAGCAGGGACAGAACCAGCTGTACAACGAGCTGAACCTGGGCAGACGGGAAGAGTACGACGTGCTGGACAAGCGGAGAGGCCGGGACCCTGAGATGGGCGGCAAGCCCCAGCGGCGGAAGAACCCCCAGGAAGGCCTGTATAACGAACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGCGGCGGAGAGGCAAGGGCCACGATGGCCTGTACCAGGGCCTGAGCACCGCCACCAAGGACACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCTAGG

[0202] SEQ ID NO: 139 - Human CD3z intracellular region polynucleotide sequence 3

[0203] AGAGTGAAGTTCAGCCGCAGCGCCGACGCCCCTGCCTACCAGCAGGGACAGAACCAGCTGTACAACGAGCTGAACCTGGGCAGACGGGAAGAGTACGACGTGCTGGACAAGCGGAGAGGCCGGGACCCTGAGATGGGCGGCAAGCCCCAGCGGCGGAAGAACCCCCAGGAAGGCCTGTATAACGAACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGCGGCGGAGAGGCAAGGGCCACGATGGCCTGTACCAGGGCCTGAGCACCGCCACCAAGGACACCTACGACGCCCTGCACATGCAGGCCCggcgcgcc DETAILED DESCRIPTION

[0204] 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. Antigen receptors type tyrosine-protein phosphatase zeta (PTPRZ1), short fibroprotein core protein (BCAN), chondroitin sulfate proteoglycan 4 (CSPG4), and tenascin (TNC) were recently found to be expressed in gliomas (Dutoit et al, Brain 135.4 (2012): 1042-1054). Polypeptide vaccines including peptides from these antigens have been generated (Dutoit et al, Oncoimmunology 7.2 (2018): el391972.; Migliorini et al, Neuro-oncology 21.7 (2019): 923-933.) and are currently undergoing clinical trials (NCT03665545, NCT02924038).

[0205] The present inventors have shown that immune effector cells expressing CARs specific for one or more glioma-associated antigens are able to generate a specific immune response against cells expressing the glioma-associated antigens, as measured by cytotoxicity assays and T cell activation.

[0206] To date, monovalent forms of the CAR T cell approach have been clinically investigated for recurrent GBM (targeting EGFRvIII, IL13Ra2, Her2), i.e. targeting one antigen at a time. Some patients show stable disease, but tumors always recur, and in some cases loss of the epitope targeted by the monovalent CAR-T cells used is seen. The present inventors have identified new glioma-associated antigens that have been targeted in monovalent forms and further demonstrated their efficacy in a multivalent targeting approach. Bielamouwicz et al. (Neuro-oncology 20.4 (2018): 506-518) describe a multivalent approach in which T cells are engineered to express a triple CAR molecule with different antigen specificities. Accordingly, in one aspect, the present invention provides immune effector cells expressing two or more CARs specific for different glioma-associated antigens. In one aspect, 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 a different glioma-associated antigen. Typically, one or more of the glioma-associated antigens is 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.

[0207] The present application also provides an immune effector cell or population of immune effector cells expressing a chimeric antigen receptor (CAR) specific for PTPRZ1. In addition, the present application provides an immune effector cell or population of immune effector cells expressing a chimeric antigen receptor (CAR) specific for BCAN. The present inventors have for the first time surprisingly shown that immune effector cells expressing a CAR specific for PTPRZ1 or BCAN have therapeutic utility, in particular for the treatment of glioblastoma. Any antigen binding domain specific for PTPRZ1 or for BCAN can be used, such as those known in the art. Preferably, the antigen binding domain is an antigen binding domain disclosed herein.

[0208] In some cases, the present application takes advantage of the "bystander" effect as described in the examples of the present application. For example, immune effector cells of the present application comprising one or more CARs specific for a glioma-associated antigen can be able to kill cancer cells, including a mixture of cancer cells expressing a glioma-associated antigen and cancer cells not expressing a glioma-associated antigen. Cancer cells not expressing a glioma-associated antigen are believed to be killed by soluble factors, potentially allowing for greater therapeutic efficacy.

[0209] In addition, lentiviral transduced CAR T cells are associated with toxicity, including cytokine release syndrome and neurotoxicity. In some aspects, immune effector cells of the present application are transduced with RNA to overcome these issues.

[0210] chimeric antigen receptor

[0211] A chimeric antigen receptor (CAR) is expressed in an immune effector cell. A CAR comprises an extracellular antigen binding domain. A CAR typically comprises an extracellular antigen binding domain and an intracellular cytoplasmic signaling domain. A CAR can comprise an extracellular antigen binding domain, a transmembrane domain, and an intracellular cytoplasmic signaling domain. A CAR can 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, a CAR comprises a hinge between the transmembrane domain and the antigen binding domain.

[0212] A cytoplasmic signaling domain can comprise an activating domain. An activating domain serves to activate the immune effector cell upon engagement of the extracellular domain (e.g., scFv). A cytoplasmic signaling domain can comprise one or more of (i) a CD3 zeta activating domain, (ii) a 4-1BB (CD137) activating domain, (iii) a CD3 epsilon activating domain, (iv) an OX40 (CD134) activating domain, (v) a CD28 activating domain, and / or (vi) a CD27 activating domain.

[0213] CD3 zeta activation is included in the signaling domain of the first generation CAR. Preferably, the cytoplasmic domain includes a CD3 zeta activation domain.

[0214] The second generation CAR includes a CD3 zeta activation domain and a CD28 activation domain. The cytoplasmic domain includes a CD3 zeta activation domain and a CD28 activation domain.

[0215] The third generation CAR includes an additional domain, such as a 4-1BB activation domain or an OX40 (CD134) activation domain. Preferably, the cytoplasmic domain includes a 4-1BB activation domain and a CD3 zeta activation domain.

[0216] The cytoplasmic signaling domain can include a 4-1BBz domain, including a CD3 zeta and a 4-1BBz activation domain. The cytoplasmic signaling domain can include a CD28z domain, including a CD3 zeta and a CD28 activation domain. CARs including a CD28z domain are particularly useful in the application, as demonstrated in the Examples, for example, when the immune effector cells are transduced with RNA encoding the CAR. The cytoplasmic signaling domain can include a 4-1BBz+CD28z domain, including a CD3 zeta, a CD28, and a 4-1BBz activation domain.

[0217] The cytoplasmic signaling domain can include a CD3 zeta activation domain alone or in combination with a CD28, CD27, OX-40 (CD134), and / or 4-1BB (CD137) domain.

[0218] Other activation domains include IL-15R alpha, CD2, CDS, ICAM-1, LTA-1, and ICOS, and can be used in combination with the activation domains described above.

[0219] If the immune effector cell expressing the CAR is a phagocytic cell, the intracellular signaling domain can include the intracellular domain of MegflO or FcRv.

[0220] 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, a 4-1BBz domain can comprise 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, a CD28z domain can 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).

[0221] In some cases, the 4-1BB activation domain comprises or consists of an amino acid sequence that is at least 70% identical to the amino acid sequence encoded by SEQ ID NO: 135, for example at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical 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 that is at least 70% identical to the amino acid sequence encoded by SEQ ID NO: 136, for example at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical 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 that is at least 70% identical to the amino acid sequence encoded by any one of SEQ ID NOs: 137-139, for example at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequence encoded by any one of SEQ ID NOs: 137-139. For example, a 4-1BBz domain can comprise an amino acid sequence that is at least 70% identical 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% identical to the amino acid sequence encoded by SEQ ID NO: 135) and an amino acid sequence that is at least 70% identical to any one of SEQ ID NOs: 137-139 (e.g., at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to any one of SEQ ID NOs: 137-139). Similarly, a CD28z domain can comprise an amino acid sequence that is at least 70% identical 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% identical to the amino acid sequence encoded by SEQ ID NO: 136) and an amino acid sequence that is at least 70% identical to any one of SEQ ID NOs: 137-139 (e.g., at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to any one of SEQ ID NOs: 137-139).

[0222] A transmembrane domain spans a cell membrane, such as the cell membrane of a eukaryotic cell. A transmembrane domain serves to transmit an activation signal to a cytoplasmic signal transduction domain upon ligand binding by an extracellular antigen binding domain (e.g., scFv). A transmembrane domain can be derived from a naturally occurring transmembrane protein, such as a Type I transmembrane protein. A transmembrane domain is often a transmembrane domain of CD28 or CD8a. A transmembrane domain can be a transmembrane domain of a T cell receptor a, b, d or g subunit, CD3e, CD3z, CD4, CD6, CD8a, CD28, CD86, OX-40, 4-1BB, or CD40L (CD154). For example, when the immune effector cell is an NK cell, the transmembrane domain can be a transmembrane domain of CD8.

[0223] In some cases, the CD8a transmembrane domain comprises or consists of an amino acid sequence that is at least 70% identical to SEQ ID NO: 122, for example, at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 122. In some cases, the CD28 transmembrane domain comprises or consists of an amino acid sequence that is at least 70% identical to SEQ ID NO: 123, for example, at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 123.

[0224] In some cases, the CD8a transmembrane domain comprises or consists of an amino acid sequence that is at least 70% identical to the amino acid sequence encoded by SEQ ID NO: 133, for example, at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical 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 that is at least 70% identical to the amino acid sequence encoded by SEQ ID NO: 134, for example, at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequence encoded by SEQ ID NO: 134.

[0225] The CAR can include a hinge connecting the transmembrane domain to the extracellular domain. The hinge can confer a steric effect that influences the strength of activation, cytotoxicity, and signaling from the target cell and its surface receptors. In some cases, the hinge can be from a region of another immune molecule, such as IgGl, IgG2, IgG3, IgG4, CD8 (e.g., CD8a hinge), or CD28. The hinge can be any suitable length. The hinge can be at least one amino acid in length, such as at least 5, at least 10, or at least 20 amino acids. The hinge can be one hundred or fewer amino acids in length, such as 80 or fewer, 60 or fewer, 40 or fewer, 30 or fewer, or 20 or fewer amino acids in length. The hinge can be 1 to 40 amino acids in length, such as 2 to 30, 3 to 25, 4 to 20, or 5 to 15 amino acids in length. The hinge can include glycine and serine, threonine, and / or alanine residues. However, the hinge can include any suitable residues.

[0226] In some cases, the hinge is an IgGl hinge, such as a human IgGl hinge.

[0227] In some cases, the hinge is an IgGl hinge (such as a human IgGl hinge) that further includes one or more constant domains, such as CH2 or CH3.

[0228] In some cases, the hinge is an IgG4 hinge, such as a human IgG4 hinge.

[0229] In some cases, the hinge is a CD8a hinge, such as a human CD8a hinge.

[0230] In some cases, the CD8a hinge includes or consists of an amino acid sequence that is at least 70% identical to SEQ ID NO: 117, such as at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 117. In some cases, the IgG4 hinge includes or consists of an amino acid sequence that is at least 70% identical to SEQ ID NO: 118, such as at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 118. The IgG4 hinge as set forth in SEQ ID NO: 118 is also referred to herein as a “short” hinge. In some cases, the IgGl hinge includes or consists of an amino acid sequence that is at least 70% identical to any one of SEQ ID NOs: 119-121, such as at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to any one of SEQ ID NOs: 119-121. The IgGl hinge as set forth in SEQ ID NO: 121 is also referred to herein as a “long” hinge.

[0231] In some cases, the CD8a hinge comprises or consists of an amino acid sequence that is at least 70% identical to the amino acid sequence encoded by SEQ ID NO: 129, for example at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical 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 that is at least 70% identical to the amino acid sequence encoded by SEQ ID NO: 130, for example at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequence encoded by SEQ ID NO: 130. In some cases, the IgGl hinge comprises or consists of an amino acid sequence that is at least 70% identical to the amino acid sequence encoded by SEQ ID NO: 131 or 132, for example at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequence encoded by SEQ ID NO: 131 or 132.

[0232] The CAR can comprise a transmembrane domain and a hinge derived from the same source. For example, the transmembrane domain and the hinge can be a CD8a transmembrane domain and a CD8a hinge. The transmembrane domain and the hinge can be a CD28 transmembrane domain and a CD28 hinge.

[0233] In some cases, the CAR can comprise a“short” 28z construct, i.e., comprising an IgG4 hinge and a CD28z intracellular domain. The CD28z domain can comprise an amino acid sequence that is at least 70% identical to SEQ ID NO: 125 (e.g., at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 125) and an amino acid sequence that is at least 70% identical 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% identical to any one of SEQ ID NOs: 126-128). The IgG4 hinge can comprise or consist of an amino acid sequence that is at least 70% identical to SEQ ID NO: 118, for example at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 118.

[0234] In some cases, the CAR can include a“long” 28z construct, i.e., including a long IgGl hinge (including CH2 and CH3 domains) and a CD28z intracellular domain. The CD28z domain can include 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 IgGl hinge can include 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.

[0235] The“short” and“long” 28z constructs generally include a CD28 transmembrane domain. For example, the“short” and“long” 28z constructs can also include 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.

[0236] The CAR can include more than one extracellular antigen binding domain, e.g., two extracellular antigen binding domains or three extracellular antigen binding domains. The two or more extracellular antigen binding domains can bind to different glioma-associated antigens, i.e., the CAR can be bispecific or multispecific.

[0237] In some cases, the application relates to a CAR specific for PTPRZ1. In some cases, the application relates to a CAR specific for BCAN. The present inventors have for the first time surprisingly shown that immune effector cells expressing a CAR specific for PTPRZ1 or BCAN have therapeutic utility, in particular for the treatment of glioblastoma. Any antigen binding domain specific for PTPRZ1 or for BCAN can be used in a CAR, such as those known in the art. Preferably, the antigen binding domain is an antigen binding domain disclosed herein.

[0238] antigen binding domain

[0239] The CARs discussed herein include an antigen binding domain. The antigen binding domain can be any domain that specifically binds a glioma-associated antigen. For example, the antigen binding domain can be a scFv, a monoclonal antibody (including 2 heavy chains and 2 light chains), a polyclonal antibody, a Fab', a F(ab')2 fragment, a heavy chain variable domain (V H ) or a nanobody (V HH ).

[0240] In some cases, the antigen binding domain includes one or more immunoglobulin variable domains. For example, the CAR can include one or more immunoglobulin variable domains. Each immunoglobulin variable domain typically includes three complementarity determining regions (CDRs) or antigen binding regions (ABRs) (these terms are used interchangeably herein). The CDRs or ABRs are typically responsible for antigen specificity, e.g., by directly interacting with an antigen. The immunoglobulin variable domain also includes framework regions that provide an immunoglobulin-like structure to the domain and typically do not directly interact with an antigen. The immunoglobulin variable domain can be selected from immunoglobulin variable domains from scFv domains, antibody domains (e.g., V H and / or V L ) domains (typically human), Fab', F(ab')2 fragments, V HH domains and V NAR domains. Thus, the antigen binding domain can include a scFv domain that typically includes two immunoglobulin variable domains (e.g., V H and V L ). In some cases, the antigen binding domain can include a V HH domain that typically includes a single immunoglobulin variable domain.

[0241] Preferably, the antigen binding domain is a scFv or V HH domain. The scFv domain includes a heavy chain variable domain (V H ) and a light chain variable domain (V L ) of an immunoglobulin and is connected by a short linker peptide. Exemplary scFv domains of the application are shown in SEQ ID NOs: 21-40. The scFv can be derived from a human immunoglobulin. The scFv can be derived from a murine immunoglobulin.

[0242] While the scFv typically arranges the V H -V L in an N-terminal to C-terminal orientation, antigen binding regions that arrange the V L -V H in an N-terminal to C-terminal orientation are also included in accordance with the application.

[0243] Any linker can be used to connect (VH ) and (V L ) domains. In SEQ ID NOs:21-40, the linker GGGGSGGGGSGGGGS (SEQ ID NO:81) is used. The linker can include SSSGGGGSGGGGSGGGGSS (SEQ ID NO:82).

[0244] In some cases, the CAR described herein is selected from a CAR 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.

[0245] In some cases, the CAR described herein includes an scFv domain including a V H domain having an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid selected from the group consisting of SEQ ID NOs:42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, and 80, respectively, and a V L domain having an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid selected from the group consisting of SEQ ID NOs:42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, and 80, respectively. The scFv also includes a linker sequence. The linker sequence can be, for example, any of the linker sequences discussed above.

[0246] In some cases, the CAR described herein includes an scFv domain having the antigen binding region (ABR) of the V H and V L domains of an scFv selected from the group consisting of SEQ ID NOs:21-40. The framework regions of the V H and V L domains of an scFv selected from the group consisting of SEQ ID NOs:21-40 are regions other than the ABR and linker sequence of the scFv. In some cases, the CAR described herein includes an scFv domain having the V H and V LThe scFv domain of the ABR of the domain and has 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 with a scFv selected from the group consisting of SEQ ID NOs: 21-40. The term "antigen binding region," in the context of a scFv, refers to the complementarity determining regions (CDRs) of the variable heavy and variable light domains that make up the scFv. For example, SEQ ID NOs: 21-40 are labeled with six ABRs. For each scFv, the first ABR1, ABR2, and ABR3 correspond to the heavy chain CDR1, CDR2, and CDR3, respectively, while the second set of ABR1, ABR2, and ABR3 within the sequence correspond to the light chain CDR1, CDR2, and CDR3, respectively.

[0247] In some cases, the CAR described herein is selected from a CAR comprising a polypeptide, e.g., 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, the CAR is selected from a CAR comprising a polypeptide, e.g., one or more polypeptides comprising HCDR1-3 and LCDR1-3 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 CDRs within the particular sequences disclosed herein. Specifically, the scFv sequences such as those of SEQ ID NOs: 21-40 comprise, from N- to C-terminus, a heavy chain variable region of an antibody, a linker, and a light chain variable region of an antibody. The individual 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 (e.g., see 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 can be the ABRs identified in the informal sequence listing above. For example, the heavy chain CDR1, CDR2, and CDR3 can be the first (i.e., before the linker) ABR1, ABR2, and ABR3, respectively, identified for the scFV, while the light chain CDR1, CDR2, and CDR3 can be the second (i.e., after the linker) ABR1, ABR2, and ABR3, respectively, identified for the scFv.

[0248] The scFv of ABR having SEQ ID NO: 21, or polypeptide having CDRs of SEQ ID NOS: 41 and 42, includes 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, the scFv of ABR having SEQ ID NO: 22, or polypeptide having CDRs of SEQ ID NOS: 43 and 44, includes 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: 21-80, and similar considerations apply to CDRs 1-3 of each of SEQ ID NOS: 83-93.

[0249] The antigen binding domain can be a scFv or V HH (also referred to as V HH , Nanobody, sdAb, or single-domain antibody; the terms are used interchangeably herein).

[0250] In some cases, the antigen binding domain is preferably a V HH . The V HH domain includes a single heavy chain variable domain (V H ) and lacks the constant domains found in typical antibodies. Exemplary V HHThe domains are shown in SEQ ID NOs: 83-93.

[0251] In some cases, the CAR described herein comprises a polypeptide 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. The polypeptide can comprise two immunoglobulin variable domains, e.g., can comprise a scFv. In some cases, the CAR described herein comprises a polypeptide, typically a V HH complementarity determining regions (CDR1, CDR2, and CDR3) from an amino acid sequence selected from any one of SEQ ID NOs: 83-93.

[0252] 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 an 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.

[0253] glioma-associated antigen

[0254] A glioma-associated antigen is an antigen associated with malignant glioma (glioblastoma, GBM), e.g., an antigen expressed by a cell of a malignant glioma. A glioma-associated antigen can be a human glioma-associated antigen. An antigen is associated with malignant glioma when it is overexpressed in malignant glioma samples when compared to normal brain tissue and non-CNS normal tissue. A glioma-associated antigen can be associated with glioma development.

[0255] A glioma-associated antigen can be selected from receptor-type tyrosine-protein phosphatase zeta (PTPRZ1), short fibroprotein core protein (BCAN), chondroitin sulfate proteoglycan 4 (CSPG4), and tenascin (TNC).

[0256] PTPRZ1, CSPG4, and BCAN can each be considered a cell surface (glioma) marker. TNC and BCAN can each be considered an extracellular matrix (ECM) marker, e.g., a tumor-invasive ECM marker. In some cases, it is advantageous for a CAR T cell of the application to be specific for both a cell surface marker and an ECM marker, e.g., a marker described herein.

[0257] Exemplary scFvs specific for PTPRZ1 are shown in SEQ ID NOs: 21-26. The corresponding V H and VL The domains are set forth as SEQ ID NOs 41-52. Preferably, the CARs described herein can be based on the scFv domains of RRB470, RRB471, or RRB476 (SEQ ID NOs 22, 23, and 26, respectively). For example, the CAR can be selected from the group consisting of a polypeptide comprising (a) an amino acid sequence of any one of SEQ ID NOs: 22, 23, or 26, (b) an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical thereto, or (c) 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.

[0258] Exemplary scFv specific for PTPRZ1 are set forth as SEQ ID NOs 22-26. Preferably, the CARs described herein can be based on the scFv domains of RRB470, RRB471, or RRB476 (SEQ ID NOs 22, 23, and 26, respectively). For example, the CAR can be selected from the group consisting of a polypeptide comprising (a) an amino acid sequence of any one of SEQ ID NOs: 22, 23, or 26, (b) an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical thereto, or (c) 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. HH The domains are set forth as SEQ ID NOs 89-91. Preferably, the CARs described herein can be based on the V HH domains of RB832, RB833, or RB834 (SEQ ID NOs 89, 90, and 91, respectively). For example, the CAR can be selected from the group consisting of a polypeptide comprising CDR1, CDR2, and CDR3, of an amino acid sequence selected from any one of SEQ ID NOs: 89 to 91 (e.g., an immunoglobulin variable domain and / or V HH ) domain. The CAR can be selected from an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to an amino acid sequence selected from any one of SEQ ID NOs: 89 to 91, typically comprising CDRs 1-3 of SEQ ID NOs 89-91, respectively. The CAR can be selected from a CAR comprising an amino acid sequence of any one of SEQ ID NOs: 89-91.

[0259] Exemplary scFv specific for BCAN are set forth as SEQ ID NOs: 34-47. The corresponding V H and V L domains are set forth as 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 the group consisting of a polypeptide comprising (a) an amino acid sequence of any one of SEQ ID NOs: 34-47, (b) an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical thereto, or (c) HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3, from an amino acid sequence selected from any one of SEQ ID NOs: 34-47.

[0260] Exemplary V HHare shown in SEQ ID NOs: 83-86. Preferably, the CARs described herein can be based on the V HH domains of RB826, RB827, RB828, or RB829 (SEQ ID NOs: 83-86, respectively). For example, the CARs can be selected from CARs comprising a polypeptide (e.g., an immunoglobulin variable domain and / or V HH ) comprising CDR1, CDR2, and CDR3 from an amino acid sequence selected from any one of SEQ ID NOs: 83-86. The CARs can 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: 83-86, typically comprising CDRs 1-3 of SEQ ID NOs: 83-86, respectively. The CARs can be selected from CARs comprising an amino acid sequence of any one of SEQ ID NOs: 83-86.

[0261] Exemplary scFv specific for CSPG4 are shown in SEQ ID NOs: 27-33. The corresponding V H and V L domains are shown in SEQ ID NOs: 53-66. Preferably, the CARs described herein can be based on the scFv domains of HRB299, HRB 301, HRB302, or HRB 303 (SEQ ID NOs: 28 and 30-32, respectively). For example, the CARs can be selected from CARs comprising (a) a polypeptide having an amino acid sequence of any one of SEQ ID NO: 28, 30, 31, or 32, (b) an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% identity thereto, or (c) HCDR1, HCDR2, and HCDR3 and LCDR1, LCDR2, and LCDR3 from an amino acid sequence selected from any one of SEQ ID NO: 28, 30, 31, or 32. Preferably, the CARs described herein can be based on the scFv domains of HRB301 or HRB302 (SEQ ID NOs: 28 and 30-32, respectively). For example, the CARs can be selected from CARs comprising (a) a polypeptide having an 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) HCDR1, HCDR2, and HCDR3 and LCDR1, LCDR2, and LCDR3 from an amino acid sequence selected from SEQ ID NO: 30 or 31.

[0262] Exemplary V HH are shown in SEQ ID NOs: 87-88. Preferably, the CARs described herein can be based on the V HHThe CDRs can be selected from the CDRs of an amino acid sequence selected from any one of SEQ ID NOs: 87-88. For example, the CAR can be selected from a CAR comprising a polypeptide (e.g., an immunoglobulin variable domain and / or V HH ) having an amino acid sequence selected from any one of SEQ ID NOs: 87-88. The CAR can 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: 87-88, typically including the CDRs 1-3 of SEQ ID NOs: 87-88, respectively. The CAR can be selected from a CAR comprising an amino acid sequence of any one of SEQ ID NOs: 87-88.

[0263] Exemplary scFv specific for TNC are set forth as SEQ ID NOs: 38-40. The corresponding V H and V L domains are set forth as SEQ ID NOs 75-80. For example, the CAR can be selected from a CAR comprising (a) a polypeptide having an amino acid sequence of any one of SEQ ID NOs: 38-40, (b) an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% identity thereto, or (c) HCDR1, HCDR2, and HCDR3 and LCDR1, LCDR2, and LCDR3 from an amino acid sequence selected from any one of SEQ ID NOs: 38-40.

[0264] Exemplary V HH are set forth as SEQ ID NOs: 92-93. Preferably, the CAR described herein can be based on the V HH domains of RB835 or RB836 (SEQ ID NOs: 92-93, respectively). For example, the CAR can be selected from a CAR comprising a polypeptide (e.g., an immunoglobulin variable domain and / or V HH ) comprising CDR1, CDR2, and CDR3 from an amino acid sequence selected from any one of SEQ ID NOs: 92-93. The CAR can 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: 92-93, typically including the CDRs 1-3 of SEQ ID NOs: 92-93, respectively. The CAR can be selected from a CAR comprising an amino acid sequence of any one of SEQ ID NOs: 92-93.

[0265] Other known glioma-associated antigens include HER2, EGFRvIII, IL13Ra2, PDGFRA, NKG2D, MET, HGF, B7-H3. Preferably, the other glioma-associated antigen is selected from the group consisting of HER2, EGFRvIII, and IL13Ra2, more preferably Her2 and IL13Ra2.

[0266] A monospecific CAR can be specific for any of the above glioma-associated antigens. A bispecific or multispecific CAR can be specific for any combination of glioma-associated antigens. Preferably, a bispecific or multispecific CAR is specific for at least one of PTPRZ1, BCAN, CSPG4, and TNC, for example two, three, or all four of PTPRZ1, BCAN, CSPG4, and TNC. A bispecific or multispecific CAR can additionally be specific for other known glioma-associated antigens, such as those described herein. For example, a multispecific CAR can be specific for PTPRZ1 and CSPG4; CSPG4 and Her2; CSPG4 and IL13Ra2; CSPG4, Her2 and IL13Ra2; PTPRZ1 and Her2; PTPRZ1 and IL13Ra2; or PTPRZ1, Her2 and IL13Ra2.

[0267] In some cases, a bispecific or multispecific CAR is specific for PTPRZ1 and one or more other glioma-associated antigens. In some cases, a bispecific or multispecific CAR is specific for BCAN and one or more other glioma-associated antigens.

[0268] A population of immune effector cells can include two or more different immune effector cells, wherein each different immune effector cell is specific for a different glioma-associated antigen. Preferably, the population includes at least one immune effector cell that is specific for at least one of PTPRZ1, BCAN, CSPG4, and TNC, for example two immune effector cells that are specific for two of PTPRZ1, BCAN, CSPG4, and TNC, three immune effector cells that are specific for three of PTPRZ1, BCAN, CSPG4, and TNC, or four immune effector cells that are specific for all of PTPRZ1, BCAN, CSPG4, and TNC. The population can include additional immune effector cells that are specific for other known glioma-associated antigens. For example, a population of immune effector cells can include an immune effector cell that is specific for PTPRZ1 and an immune effector cell that is specific for CSPG4. A population of immune effector cells can include an immune effector cell that is specific for PTPRZ1, an immune effector cell that is specific for Her2, and an immune effector cell that is specific for IL13Ra2, as shown in the Examples.

[0269] In some cases, the population of immune effector cells can comprise two or more different immune effector cells, wherein one or more of the different immune effector cells is specific for a glioma-associated antigen selected from the group consisting of PTPRZl, BCAN, CSPG4, and TNC, e.g., at least two of PTPRZl, BCAN, CSPG4, and TNC, at least three of PTPRZl, BCAN, CSPG4, and TNC, or all four of PTPRZl, BCAN, CSPG4, and TNC. In some cases, one or more of the different immune effector cells is specific for PTPRZl. In some cases, one or more of the different immune effector cells is specific for BCAN. For example, the population of immune effector cells can comprise: an immune effector cell specific for PTPRZl and an immune effector cell specific for BCAN; an immune effector cell specific for PTPRZl and an immune effector cell specific for CSPG4; an immune effector cell specific for PTPRZl and an immune effector cell specific for TNC; an immune effector cell specific for BCAN and an immune effector cell specific for CSPG4; an immune effector cell specific for BCAN and an immune effector cell specific for TNC; an immune effector cell specific for CSPG4 and an immune effector cell specific for TNC; an immune effector cell specific for PTPRZl, an immune effector cell specific for BCAN, and an immune effector cell specific for CSPG4; an immune effector cell specific for PTPRZl, an immune effector cell specific for BCAN, and an immune effector cell specific for TNC; an immune effector cell specific for PTPRZl, an immune effector cell specific for CSPG4, and an immune effector cell specific for TNC; an immune effector cell specific for BCAN, an immune effector cell specific for CSPG4, and an immune effector cell specific for TNC; or an immune effector cell specific for PTPRZl, an immune effector cell specific for BCAN, an immune effector cell specific for CSPG4, and an immune effector cell specific for TNC.

[0270] In some cases, the immune effector cells of the application can comprise two or more different CARs, wherein each CAR is specific for a different glioma-associated antigen. Preferably, the immune effector cells comprise at least one CAR specific for at least one of PTPRZl, BCAN, CSPG4, and TNC, such as two CARs specific for two of PTPRZl, BCAN, CSPG4, and TNC, three CARs specific for three of PTPRZl, BCAN, CSPG4, and TNC, or four CARs specific for all of PTPRZl, BCAN, CSPG4, and TNC. The immune effector cells can comprise additional CARs specific for other known glioma-associated antigens described herein. For example, the immune effector cells can comprise a CAR specific for PTPRZl and a CAR specific for CSPG4. The immune effector cells can comprise a CAR specific for PTPRZl, a CAR specific for Her2, and a CAR specific for IL13Rα2.

[0271] In some cases, the immune effector cells of the application can comprise two or more different CARs, wherein each CAR is specific for a different glioma-associated antigen. In some cases, at least one glioma-associated antigen is PTPRZ1. In some cases, at least one glioma-associated antigen is BCAN. In some cases, at least one glioma-associated antigen is selected from PTPRZ1, BCAN, CSPG4, and TNC, e.g., 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, the immune effector cell can comprise: a CAR specific for PTPRZ1 and a CAR specific for BCAN; a CAR specific for PTPRZ1 and a CAR specific for CSPG4; a CAR specific for PTPRZ1 and a CAR specific for TNC; a CAR specific for BCAN and a CAR specific for CSPG4; a CAR specific for BCAN and a CAR specific for TNC; a CAR specific for CSPG4 and a CAR specific for TNC; a CAR specific for PTPRZ1, a CAR specific for BCAN, and a CAR specific for CSPG4; a CAR specific for PTPRZ1, a CAR specific for BCAN, and a CAR specific for TNC; a CAR specific for PTPRZ1, a CAR specific for CSPG4, and a CAR specific for TNC; a CAR specific for BCAN, a CAR specific for CSPG4, and a CAR specific for TNC; or a CAR specific for PTPRZ1, a CAR specific for BCAN, a CAR specific for CSPG4, and a CAR specific for TNC. The immune effector cell can comprise additional CARs specific for other known glioma-associated antigens described herein.

[0272] nucleic acid

[0273] Also provided are one or more isolated nucleic acids (i.e., polynucleotides) that encode a CAR of the application. In some cases, the encoding nucleic acid sequences can be provided by more than one nucleic acid sequence, optionally present on more than one nucleic acid molecule, but which collectively are capable of encoding a CAR of the application.

[0274] Nucleic acids that encode a CAR of the application can be obtained by methods well known to those of skill in the art. For example, DNA sequences encoding portions or all of the antibody heavy and light chains can be synthesized as desired from the corresponding amino acid sequences.

[0275] The nucleic acid can be a DNA sequence. The nucleic acid can be an RNA sequence, e.g., an mRNA. The vector can comprise the nucleic acid.

[0276] The vector can be a viral vector. Conventional viral-based expression systems can include retroviral, alpha- retroviral, lentiviral, adenoviral, adeno-associated viral (AAV), and herpes simplex viral (HSV) vectors for gene transfer. Non-viral transduction vectors include transposon-based systems, including PiggyBac and Sleeping Beauty systems. Methods for production and purification of such vectors are known in the art.

[0277] The vector can be a cloning vector or an expression vector. A suitable vector can be any vector capable of carrying sufficient amount of genetic information and allowing expression of the polypeptides of the application.

[0278] 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.

[0279] General methods of constructing vectors, transfection methods, and culturing methods are well known to those skilled in the art. In this regard, reference can be made to “Current Protocols in Molecular Biology”, 1999, F. M. Ausubel (ed), Wiley Interscience, New York and the Maniatis manual published by Cold Spring Harbor Laboratory Press.

[0280] The nucleic acid can be provided in the form of an expression cassette, comprising control sequences operably linked to the insert sequence, allowing expression of the CAR of the application in vivo. Thus, also provided is one or more expression cassettes encoding one or more nucleic acids of the CAR described herein. These expression cassettes are in turn typically provided within a vector, such as a plasmid or a recombinant viral vector. Thus, also provided is a vector encoding the CAR described herein. Further provided is a vector co-encoding the CAR described herein.

[0281] The vector can be a human artificial chromosome. 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).

[0282] The vector can be a non-viral delivery system, such as a DNA plasmid, naked nucleic acid (e.g. naked RNA), and nucleic acid complexed with a delivery vehicle, such as a liposome or a nanoparticle.

[0283] The nucleic acids, expression cassettes, or vectors described herein can be introduced into a host cell, e.g., by transfection. Accordingly, host cells comprising one or more nucleic acids, expression cassettes, or vectors of the application are also provided. The nucleic acids, expression cassettes, or vectors described herein can be introduced into a host cell transiently or permanently, allowing for expression of an antibody from the one or more nucleic acids, expression cassettes, or vectors. Such host cells include transient, or preferably stable, higher eukaryotic cell lines (such as mammalian cells or insect cells), lower eukaryotic cells (such as yeast), or prokaryotic cells (such as bacterial cells). Particular examples of cells include mammalian HEK293 (e.g., 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 introduced into a host cell transiently.

[0284] Also provided are kits suitable for transforming and / or transfecting an immune effector cell or population of immune effector cells to produce an immune effector cell or population of immune effector cells of the application. The kits include a nucleic acid or vector described herein. The kits can include other reagents, such as those discussed herein that improve the efficiency of transfection or transformation.

[0285] Also described are nucleic acids encoding particular scFv antigen binding regions of SEQ ID NOs: 21-40, which are provided in SEQ ID NOs: 1-20, respectively. SEQ ID NOs: 1-20 are provided as DNA sequences, but also include the corresponding RNA sequences (with “U” in place of “T”).

[0286] Also described are nucleic acids encoding particular scFv antigen binding regions of SEQ ID NOs: 83-93, which are provided in SEQ ID NOs: 94-104, respectively. SEQ ID NOs: 94-104 are provided as DNA sequences, but also include the corresponding RNA sequences (with “U” in place of “T”).

[0287] immune effector cell

[0288] An immune effector cell as used herein refers to a cell capable of cell-mediated cytotoxicity against a target cell displaying a target antigen, e.g., a glioma-associated antigen. The immune effector cell can be a T cell, a gd T cell, a natural killer (NK) cell, an NKT cell, an induced pluripotent stem cell (iPSC)-derived NK cell (iPSC-NK), a T cell, a phagocyte, or a macrophage. The immune effector cell is preferably a T cell.

[0289] Preferably, the T cell is a CD8 + T cell or cytotoxic T cell. The T cell is preferably a CD4-CD8+ T cells.

[0290] The T cells can be CD4 + The T cells, or helper T cells (TH cells), such as TH1, TH2, TH3, TH17, TH9, or TFH cells. The T cells can be regulatory T cells (Tregs). The T cells can be naive T cells, effector T cells, memory T cells, effector memory T cells, central memory T cells, memory stem T cells. The T cells can be peripheral lymphocytes.

[0291] The T cells can be expanded from peripheral blood mononuclear cells (PBMCs). The T cells can be autologous to the subject to which they are administered. The T cells can be allogeneic to the subject to which they are administered. The T cells can be partially HLA-mismatched to the subject to which they are administered.

[0292] The NK cells can be cells of the NK92 cell line. The NK cells can be isolated from peripheral blood mononuclear cells (PBMCs) of a subject to be treated or a healthy donor. The NK cells can be isolated from umbilical cord blood. The NK cells can be isolated from CD34 + Hematopoietic progenitor cells (HPCs) are differentiated.

[0293] The T cells can be expanded from peripheral blood mononuclear cells (PBMCs). The T cells can be autologous to the subject to which they are administered. The T cells can be allogeneic to the subject to which they are administered.

[0294] The macrophages can be differentiated into an "M1" phenotype. M1 macrophages express proinflammatory cytokines and have strong anti-tumor activity. Undifferentiated macrophages expressing a CAR described herein can be induced to differentiate into an M1 phenotype by culturing in the presence of a glioma-associated antigen.

[0295] The immune effector cells can comprise a nucleic acid described herein. The immune effector cells can comprise a vector described herein. The immune effector cells preferably comprise an RNA nucleic acid or RNA vector described herein. The immune effector cells express a CAR specific for one or more glioma-associated antigens. The immune effector cells preferably transiently express the CAR.

[0296] Thus, the immune effector cells are preferably RNA CAR T cells.

[0297] The immune effector cells can be engineered to transiently express a CAR specific for one or more glioma-associated antigens. This is to minimize on-target, off-tumor toxicity due to expression of the antigen by normal, healthy tissue. In some cases, the immune effector cells can be transfected with mRNA encoding a CAR against one or more glioma-associated antigens, for example by mRNA electroporation, as shown in Beatty et al., Gastroenterology 155.1 (2018): 29-32. (See Supplemental Material 5) and Schutsky et al., Oncotarget 6.30 (2015): 28911.

[0298] In some cases, the application relates to a population of immune effector cells expressing a CAR. The population can include at least two different immune effector cells expressing a CAR specific for at least two different glioma-associated antigens. The population can include at least three different immune effector cells expressing a CAR specific for at least three different glioma-associated antigens. In some cases, one or more of the glioma-associated antigens is selected from PTPRZ1, BCAN, CSPG4, and TNC. For example, two or more, such as three or four, of the glioma-associated antigens are selected from PTPRZ1, BCAN, CSPG4, and TNC.

[0299] The population can include at least about 1 x 10 6 immune effector cells, such as at least about 1 x 10 7 , at least about 1 x 10 8 , at least about 1 x 10 9 , or at least about 1 x 10 10 immune effector cells. The population can include at least about 1 x 10 6 to about 1 x 10 12 immune effector cells, such as about 1 x 10 6 to about 1 x 10 11 , about 1 x 10 6 to about 1 x 10 10 , about 1 x 10 6 to about 1 x 10 9 , about 1 x 10 7 to about 1 x 10 11 , about 1 x 10 7 to about 1 x 10 10 immune effector cells. The population can include about 1 x 10 6 immune effector cells, such as about 5 x 10 6 , about 1 x 10 7 , about 5 x 10 7 , about 1 x 10 8 , about 5 x 10 8about 1 x 10 9 about 5 x 10 9 about 1 x 10 10 about 5 x 10 10 about 1 x 10 11 about 5 x 10 11 about 1 x 10 12 immune effector cells.

[0300] In some cases, the immune effector cells of the application express a CAR, wherein the CAR is specific for at least two different glioma-associated antigens (i.e., a bi-specific or multi-specific CAR). For example, the CAR can include two different scFvs specific for two different glioma-associated antigens.

[0301] In some cases, the immune effector cells of the application express at least two different CARs, wherein 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 application express at least three different CARs, wherein the at least three different CARs are specific for at least three different glioma-associated antigens.

[0302] Multi-specificity against two or more glioma-associated antigens is advantageous for a variety of reasons. For example, due to inter-patient and / or inter-tumor variability, expression of glioma-associated antigens can vary within and between subjects. Multi-specificity allows a single therapy to target a wider range of tumors. Further, antigen escape is a phenomenon in which an antigen targeted by, for example, a CAR, is no longer expressed by a tumor and the therapy loses its efficacy. By targeting many glioma-associated antigens, it is more difficult for a tumor to "escape" therapy. Multi-specificity also enhances immune effector cell effector functions, such as cytotoxicity.

[0303] Also provided are methods of making an immune effector cell of the application or a population of immune effector cells of the application. The methods include transforming a cell or population of cells with one or more nucleic acids encoding one or more CARs specific for one or more glioma-associated antigens. The CARs can be any of the CARs discussed herein. The nucleic acids can be any of the nucleic acids or vectors of the application.

[0304] Any method known in the art can be used to transform a cell or population with a nucleic acid. The immune effector cell can be transfected or transduced with the nucleic acid. The CAR can be introduced into the immune effector cell using a vector.

[0305] The term“transduction” can be used to describe viral-mediated transfer of nucleic acid. Viral vectors can be used to transduce cells with one or more constructs. Conventional viral-based expression systems can include retroviral, alpha- retroviral, lentiviral, adenoviral, adeno-associated viral (AAV), and herpes simplex viral (HSV) vectors for gene transfer. Non-viral transduction vectors include transposon-based systems, including PiggyBac and Sleeping Beauty systems. Methods for production and purification of such vectors are known in the art. The vector is preferably a vector described herein. The immune effector cell can be transduced using any method known in the art. The transduction can be in vitro or ex vivo.

[0306] The term“transfection” can be used to describe non-viral-mediated transfer of nucleic acid. The immune effector cell can be transfected using any method known in the art. The transfection can be in vitro or ex vivo. Any vector capable of transfecting the immune effector cell can be used, such as conventional plasmid DNA or RNA transfection, preferably mRNA transfection. Human artificial chromosomes and / or naked RNA can be used to transfect nucleic acid sequences or nucleic acid constructs into cells. Human artificial chromosomes are described in, e.g., Kazuki et al., Mol. Ther. 19(9): 1591-1601 (2011), and Kouprina et al., Expert Opinion on Drug Delivery 11(4):517-535 (2014). Alternative non-viral delivery systems include DNA plasmids, naked nucleic acids, and nucleic acids complexed with delivery vehicles such as liposomes. Methods of non-viral delivery of nucleic acids include lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycations or lipid:nucleic acid conjugates, naked DNA, naked RNA, artificial virosomes, and agent-enhanced DNA uptake.

[0307] 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 that can deliver RNA, see, e.g., Alabi et al., Proc Natl Acad Sci U S A. 2013 Aug 6; 110(32): 12881-6; Zhang et al., Adv Mater. 2013 Sep 6; 25(33): 4641-5; Jiang et al., Nano Lett. 2013 Mar 13; 13(3): 1059-64; Karagiannis et al., ACS Nano. 2012 Oct 23; 6(10): 8484-7; Whitehead et al., ACS Nano. 2012 Aug 28; 6(8): 6922-9; and Lee et al., Nat Nanotechnol. 2012 Jun 3; 7(6): 389-93. Lipid nanoparticles, spherical nucleic acid (SNA TM ) construct, nanocomplexes, and other nanoparticles, particularly gold nanoparticles, are also contemplated as means of delivering nucleic acids or vectors of the application.

[0308] Immune effector cells can be transfected by electroporation. Preferably, the electroporation is mRNA electroporation. This has the advantage of allowing transient expression of the CAR.

[0309] Immune effector cells can be transfected by electroporation, e.g., RNA electroporation or mRNA electroporation. When immune effector cells express more than one CAR, the immune effector cells can be transfected by electroporation, e.g., RNA or mRNA electroporation, of a single polynucleotide, e.g., RNA or mRNA, or vector, encoding at least one CAR, or can be transfected by electroporation, e.g., RNA or mRNA electroporation, of two or more polynucleotides, e.g., RNAs or mRNAs, or vectors, encoding at least one CAR. Electroporation of two or more polynucleotides is typically performed simultaneously.

[0310] Uptake of nucleic acid constructs can be enhanced by several known transfection techniques, e.g., those involving the use of transfection agents. Examples of these agents include cationic agents, such as calcium phosphate and DEAE-dextran, and lipid transfection reagents, such as lipofectamine, Fugene, and transfectam.

[0311] pharmaceutical composition

[0312] Compositions comprising the immune effector cells or immune effector cell populations of the present invention are also provided. The immune effector cells or immune effector cell populations may constitute at least 50% of the total cells in the composition, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.9% of the total cells in the composition. The total cells in the composition may consist of or substantially consist of the immune effector cells or immune effector cell populations of the present invention, i.e., no other cells are detected in the composition.

[0313] The composition may include at least about 1×10 6 To approximately 1×10 12 One immune effector cell of the present invention, for example, about 1 × 10 6 To approximately 1×10 11 Approximately 1×10 6 To approximately 1×10 10 Approximately 1×10 6 To approximately 1×10 9 Approximately 1×10 7 To approximately 1×10 11 Approximately 1×10 8 To approximately 1×10 10 One immune effector cell. The composition may include about 1 × 102 6 One immune effector cell of the present invention, for example about 5 × 10 6 Approximately 1×10 7 Approximately 5×10 7 Approximately 1×10 8 Approximately 5×10 8 Approximately 1×10 9 Approximately 5×10 9 Approximately 1×10 10 Approximately 5×10 10 Approximately 1×10 11 Approximately 5×10 11 or approximately 1×10 12 A population of immune effector cells. The composition may include the above-described amount of the immune effector cell population of the present invention.

[0314] The composition may be a pharmaceutical composition. A 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 solution.

[0315] Pharmaceutical compositions may contain one or more pharmaceutically acceptable salts. A "pharmaceutically acceptable salt" is a salt that retains the desired biological activity of the parent compound without conferring any undesirable toxicological effects. Examples of such salts include acid addition salts and base addition salts.

[0316] The composition can include one or more additional therapeutic agents, such as a chemotherapeutic agent. The composition can include one or more preservatives, such as an antifungal agent and / or an antiviral agent.

[0317] therapeutic uses and methods

[0318] Also described herein are uses 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.

[0319] For example, also provided are methods of treating a cancer in a subject, the method comprising administering to the subject an effective amount of the immune effector cells or population of immune effector cells of the application.

[0320] Also provided are the immune effector cells or population of immune effector cells of the application for use in a method of treating a cancer. Also provided is the use of the immune effector cells or population of immune effector cells for the manufacture of a medicament for treating a cancer.

[0321] The therapeutic uses and methods can comprise administering a therapeutically effective amount of the immune effector cells or population of immune effector cells.

[0322] Also provided are methods of formulating a composition for treating a cancer, wherein the method comprises mixing the immune effector cells or population of immune effector cells of the application with an acceptable carrier to produce the composition.

[0323] The cancer can be a glioma, such as a malignant glioma. The cancer can be a glioblastoma. The cancer can be a recurrent cancer, such as a recurrent glioblastoma. The subject can have previously received treatment for the cancer, such as using a CAR cell (e.g., T cell) approach targeting EGFRvIII, IL13Rα2, and / or Her2. The cancer can be a glioblastoma multiforme (GBM). The cancer can be a primary glioblastoma or a secondary glioblastoma.

[0324] The cancer can be another solid tumor that expresses a glioma-associated antigen. For example, the cancer can be a solid tumor that expresses one or more of PTPRZ1, BCAN, CSPG4, and / or TNC. The cancer can additionally express one or more of HER2, EGFRvIII, IL13Rα2, PDGFRA, NKG2D, MET, HGF, 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 a glioma-associated antigen.

[0325] The methods of treatment and uses can comprise, prior to treatment with the immune effector cells or population of immune effector cells of the application, determining whether the cancer expresses a glioma-associated antigen that is specifically targeted by the immune effector cells or population of immune effector cells of the application. For example, the methods can comprise determining whether the cancer expresses PTPRZ1, BCAN, CSPG4, and / or TNC. The methods can comprise selecting the immune effector cells or population of immune effector cells based on expression of the glioma-associated antigen by the cancer, such that the immune effector cells or population of immune effector cells are specific for the cancer. The methods can comprise transfecting or transforming the immune effector cells with the nucleic acid of the application in response to information about expression of the glioma-associated antigen by the cancer.

[0326] At least 1% of the cancer cells from the tumor or from the individual can express the glioma-associated antigen. The glioma-associated antigen is 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 the cancer cells from the tumor or from the individual can express the glioma-associated antigen. The percentage of cells expressing the glioma-associated antigen can be determined by any method known to one of skill in the art, such as immunohistochemistry (IHC), flow cytometry, or enzyme-linked immunosorbent assay (ELISA).

[0327] Expression of the glioma-associated antigen can have an intensity of greater than or equal to (>) 1+, such as > 2+ or > 3+. Intensity scores can be assessed by IHC staining of the tumor, with scores as follows: negative = no staining or staining of less than or equal to (<) 10% of cells; 1+ = > 10% of cells incompletely stained; 2+ = weak to moderate staining in > 10% of cells; > 10% of cells strongly and completely stained.

[0328] The methods of treatment and uses described herein can comprise inhibiting a disease state (i.e., a cancer), such as by halting its progression and / or causing regression of the disease state until a desired endpoint is achieved. The methods of treatment and uses of the application can comprise achieving a partial response, a complete response by the cancer. The methods of treatment and uses of the application can achieve remission of the cancer.

[0329] The methods of treatment and uses described herein can delay growth of the cancer, halt growth of the cancer, and / or reverse growth of the cancer. The methods of treatment and uses of the application can reduce the size of the cancer by at least 10%, such as at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100%.

[0330] The treatments and uses described herein may include inducing a bystander effect. A bystander effect may include killing cancer cells that do not express glioma-associated antigens, wherein at least one of a CAR expressed by the immune effector cells of the subject in the methods or uses targets said antigen.

[0331] Typically, treatments and uses are intended for human subjects in need. However, non-human animals, such as non-human mammals, may also be considered. Non-human mammals may include rats, rabbits, sheep, pigs, cattle, cats, or dogs.

[0332] The dosage of immune effector cells or immune effector cell populations may vary depending on the subject's age and size, as well as the disease, condition, and route of administration. The dosage of immune effector cells or immune effector cell populations may be approximately 1 × 10⁻⁶. 6 To approximately 1×10 12 Cells, for example, about 1 × 10 6 To approximately 1×10 11 Approximately 1×10 6 To approximately 1×10 10 Approximately 1×10 6 To approximately 1×10 9 Approximately 1×10 7 To approximately 1×10 11 Approximately 1×10 8 To approximately 1×10 10 Administered at a dose per cell. Immune effector cells or immune effector cell populations can be approximately 1 × 10⁻⁶. 6 Cells, for example, about 5 × 10 6 One cell, approximately 1 × 10 7 1 cell, approximately 5 × 10 7 One cell, approximately 1 × 10 8 1 cell, approximately 5 × 10 8 One cell, approximately 1 × 10 9 1 cell, approximately 5 × 10 9 One cell, approximately 1 × 10 10 1 cell, approximately 5 × 10 10 One cell, approximately 1 × 10 11 1 cell, approximately 5 × 10 11 One cell, or approximately 1 × 10⁻⁶ 12 Administer a dose per cell.

[0333] Immune effector cells or immune effector cell populations can reach approximately 1 × 10⁻⁶. 5 cells / kg to approximately 1×10 11 cells / kg, for example, about 1×10 5 cells / kg to approximately 1×10 10 cells / kg, approximately 1×10 5 cells / kg to approximately 1×109 cells / kg, approximately 1×10 5 cells / kg to approximately 1×10 8 cells / kg, approximately 1×10 6 cells / kg to approximately 1×10 11 cells / kg, approximately 1×10 6 cells / kg to approximately 1×10 10 cells / kg, approximately 1×10 6 cells / kg to approximately 1×10 9 cells / kg, approximately 1×10 7 cells / kg to approximately 1×10 11 cells / kg, approximately 1×10 7 cells / kg to approximately 1×10 10 cells / kg, or approximately 1×10 7 cells / kg to approximately 1×10 9 At a dose of cells / kg, approximately 1 × 10⁻⁶ immune effector cells or immune effector cell populations can be achieved. 5 cells / kg, for example, about 5 × 10 5 cells / kg, 1×10 6 cells / kg, 5×10 6 cells / kg, 1×10 7 cells / kg, 5×10 7 cells / kg, 1×10 8 cells / kg, 5×10 8 cells / kg, 1×10 9 cells / kg, 5×10 9 cells / kg, 1×10 10 cells / kg, 5×10 10 cells / kg, or 1×10 11 A dose of cells / kg was administered.

[0334] Immune effector cells or immune effector cell populations can be administered in a single dose. They can also be administered in multiple-dose regimens. For example, a second or more subsequent doses can be administered after the initial dose. The second and subsequent doses can be administered at appropriate intervals. For example, the doses can be administered approximately once a week, approximately once every two weeks, approximately once every three weeks, approximately once every four weeks, or approximately once a month.

[0335] Immune effector cells or immune effector cell populations can be administered intravenously. Immune effector cells or immune effector cell populations can be administered intracranially. Immune effector cells or immune effector cell populations can be administered intraventricularly.

[0336] The immune effector cell or population of immune effector cells can be administered with one or more additional therapies, such as one or more additional therapeutic agents. The additional therapeutic agent can be an anti-tumor agent. The additional therapeutic agent can be an oncolytic virus. The additional therapeutic agent can be a CAR-enhancing drug. The additional therapeutic agent can be an additional immune effector cell.

[0337] The combined administration of the immune effector cell or population and the additional therapeutic agent can be achieved in many different ways. All components can be administered together in a single composition. Each component can be administered separately as part of a combination therapy.

[0338] For example, the immune effector cell or population of immune effector cells of the application can be administered prior to, after, or concurrently with the additional therapeutic agent.

[0339] The additional therapy can be chemotherapy, radiation therapy, and / or surgery.

[0340] The subject can undergo lymphodepletion prior to administration of the immune effector cell or population of immune effector cells of the application. Lymphodepletion can be achieved by administering fludarabine, cyclophosphamide, and / or bendamustine to the subject. Lymphodepletion can be performed for at least about one day, such as about 2 days or about 3 days.

[0341] The biological activity and / or therapeutic efficacy of the administered immune effector cell or population of immune effector cells can be measured by known methods. For example, the methods can include imaging, such as magnetic resonance imaging.

[0342] antigen binding molecule

[0343] Also provided are antigen binding molecules specific for one or more glioma-associated antigens. The antigen binding molecule is selected from the group consisting of antigen binding molecules comprising a heavy chain complementarity determining region (HCDR1, HCDR2, and HCDR3) and a light chain complementarity determining region (LCDR1, LCDR2, and LCDR3) selected from the amino acid sequence of any one of SEQ ID NOs: 21-32 and 34-37. In some cases, the antigen binding molecule is selected from the group consisting of antigen binding molecules comprising a polypeptide, e.g., one or more polypeptides comprising HCDR1-3 and LCDR1-3 selected from the amino acid sequence of any one of SEQ ID NOs: 21-32 and 34-37, 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 the amino acid sequence selected from SEQ ID NOs: 21-32 and 34-37.

[0344] The antigen binding molecule can be an antibody. The antigen binding molecule can be an antibody-drug conjugate. The antigen binding molecule can be used in an antibody-based therapy, e.g., in a method for metabolite radiotherapy. The antigen binding molecule can be used in a method of treatment of cancer as described herein.

[0345] In some cases, the antigen binding molecule comprises one or more antigen binding domains disclosed herein. For example, the antigen binding molecule can comprise a polypeptide disclosed herein. The antigen binding molecule can comprise an scFv or V HH domain disclosed herein. For example, the antigen binding molecule can comprise HCDR1-3 and LCDR1-3 of an scFv disclosed herein. The antigen binding molecule can comprise CDR1-3 of a V HH domain disclosed herein.

[0346] In some cases, the antigen binding molecule comprises an immunoglobulin variable domain, e.g., a V HH domain, and comprises CDR1-3 of an amino acid sequence selected from the group consisting of SEQ ID NOs: 83-93. The antigen binding molecule can further comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the V

[0347] In some cases, the antigen binding molecule comprises a polypeptide having at least two immunoglobulin variable domains, e.g., an scFv. One variable domain is typically a V H domain, and one variable domain is typically a V L domain. The variable region can be an scFv or an antibody. The V H domain can comprise HCDR 1-3, and the V L domain can comprise LCDR1-3 of an amino acid sequence selected from the group consisting of any one of SEQ ID NOs: 21-32 and 34-37. The V H domain can further comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the V H domain of an amino acid sequence selected from the group consisting of SEQ ID NOs: 21-32 and 34-37. The V L domain can further comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the V L domain of an amino acid sequence selected from the group consisting of SEQ ID NOs: 21-32 and 34-37.

[0348] definitions

[0349] It should be understood that the various applications of the CARs, cells, or pharmaceutical compositions disclosed herein can be adjusted according to the particular needs of the art. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments of the application only and is not intended to be limiting.

[0350] Furthermore, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a CAR" includes two or more such CARs.

[0351] It should be understood that the term "nucleic acid" and the term "polynucleotide" are used interchangeably herein.

[0352] For purposes of the present application, to determine the percent identity of two sequences (e.g., two nucleic acids or two nucleic acid sequences), the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first sequence for optimal alignment with a second sequence). Then the nucleotides or amino acid residues at each position are compared. When a position in the first sequence is occupied by the same nucleotide or amino acid as the corresponding position in the second sequence, then the nucleotides or amino acids are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = # of identical positions / # of positions in the reference sequence x 100).

[0353] In general, sequence comparisons are performed over the length of the reference sequence. For example, if a user wishes to determine whether a given ("test") sequence is 95% identical to SEQ ID NO: 1, then SEQ ID NO: 1 would be the reference sequence. To assess whether a certain sequence is at least 95% identical to SEQ ID NO: 1 (one example of a reference sequence), one would align the length of SEQ ID NO: 1 and identify how many positions in the test sequence are identical to the positions in SEQ ID NO: 1. If at least 95% of the positions are identical, then the test sequence is at least 95% identical to SEQ ID NO: 1. If the sequence is shorter than SEQ ID NO: 1, then the positions that are gapped or missing should be considered non-identical positions.

[0354] The skilled person knows different 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 percentage of identity between two sequences can be done using mathematical algorithms. In one embodiment, the percentage of identity between two amino acid or nucleic acid sequences is determined using the algorithm of Needleman and Wunsch (1970), which has been incorporated into the GAP program in the Accelrys GCG software package (available at http: / / www.accelrys.com / products / gcg / ), using either a Blosum 62 matrix or a PAM250 matrix, gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0355] "Specific" or "specifically binds" means that the antigen binding region of a CAR binds to one or more antigenic determinants of a desired glioma-associated antigen and does not bind to other polypeptides. For example, a CAR specific for PTPRZ1 binds to an antigen of PTPRZ1 but not to an 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 a stronger affinity than to an antigen of a different polypeptide, such as bovine serum albumin. Methods of measuring binding affinity are well known in the art.

[0356] As used herein, the term "about" can be construed as meaning a value that is within ±10% of the recited value.

[0357] All publications, patents, and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0358] The following examples illustrate the application.

[0359] Example

[0360] Example 1 - Generation of Anti-PTPRZ1 RNA CAR T cells

[0361] A human scFv phage display library was screened and six different single chain variable fragments (scFv) against PTPRZ1 were obtained therefrom. All scFv were fused to the hinge-transmembrane domain of the human CD8a molecule, followed by the intracellular domains of human 4-1BB (CD137) and human CD3-zeta (designated BBz CAR), or to the hinge of a human IgG4 molecule, followed by the transmembrane and intracellular domains of human CD28 and CD3-zeta (designated 28z CAR). All CAR constructs were cloned into the pDA plasmid designed to optimally produce mRNA molecules under a T7 viral promoter.

[0362] Human T cells were purified from healthy donor blood with RosetteSep cocktail (Stem Cell Technologies). T cells were activated with anti-CD3 / anti-CD28 Dynabeads (Invitrogen) at a cell to bead ratio of 1 : 1 for a 48 hour period. Beads were then removed from activated T cells and mRNA for different CAR molecules was electroporated using a MaxCyte device. CAR T cells were recovered overnight in the presence of 30 IU / mL of IL-2 before being frozen.

[0363] To evaluate CAR T cell killing capacity, tumor target cells were stained with Cell Tracker and plated in 96 well plates at a density of 25000 cells / well. CAR T cells were thawed one day before the experiment, recovered overnight in the presence of 45 IU / mL of IL-2 and added to tumor cells at different effector to target (E:T) ratios. CAR molecule expression on T cell surface was measured by flow cytometry before addition of CAR T cells.

[0364] The GBM cell line Ge518, obtained in our laboratory from a patient tumor resection, was used as a tumor model to evaluate CAR T cell efficacy. Since PTPRZ1 expression is low in these cells, the antigen was overexpressed by introducing (knocking in) the extracellular part of the human PTPRZ1 molecule (domains 1 plus 2) under the EFla promoter, using a third generation LV vector with puromycin selection. The new cell line Ge518_PTPRZ1-KI showed high expression of PTPRZ1 on the cell surface as measured by flow cytometry using a scFv coupled to rabbit Fc followed by an anti-rabbit IgG coupled to AlexaFluo 488 (A). Six scFvs generated against PTPRZ1 were able to specifically recognize PTPRZ1 on Ge518_PTPRZ1-KI cells, scFvs 469, 471, 473 and 476 showing higher recognition levels than scFvs 470 and 474 (B). Figure 1 Figure 1 B) more (80% or more with a clearly defined peak).

[0365] In general, CAR T cell production was efficient using more than 80% of T cells expressing the CAR molecule and viability after thawing was between 80 and 90% ( Figure 2 ​A). 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 an E:T ratio of 5:1 for 72 hours. A positive control CAR T cell specific for IL13Rα2 was also used. Three of the anti-PTPRZ1 CAR T cells, 470_BBz, 471_BBz and 476_BBz, were able to kill the majority of tumor cells (75-85%, p<0.001) as measured by flow cytometry, Figure 2 B). 469_BBz and 473_BBz CAR T cells showed a lower percentage of Ge518_PTPRZ1-KI tumor cells killed. 474_BBz CAR expressed at too low a level to show any killing capacity Figure 2 B).

[0366] Generation 2 CAR T cells can use different intracellular costimulatory domains, 4-1BB and CD28 are more frequently used. While 4-1BB seems more suitable for persistence of CAR T cells in vivo, CD28 is described to provide a more potent acute response. Since RNA CAR T cells have a relatively short CAR cell surface expression (less than 7 days), it can be desirable to use a costimulatory domain that generates a strong immune response. Therefore, the killing potency of CAR T cells carrying the same scFv but incorporating either the 4-1BB (BBz) or CD28 (28z) domain was evaluated. Both CAR T cell versions contained scFv 473 and 476, incubated with Ge518_PTPRZ1-KI cells at an E:T ratio of 5:1. After 72 hours, the killing capacity of the CAR T cells was measured by flow cytometry. For both scFv, the 28z variant showed higher cytotoxicity than the BBz variant Figure 3 A). The comparison was repeated using scFv 471 and two different E:T ratios were evaluated, 3:1 and 1:1. In both conditions, the 471_28z variant showed higher killing capacity than the 471_BBz variant, this difference was more pronounced at 1:1, where 471_BBz showed reduced cytotoxicity Figure 3 B).

[0367] To determine which anti-PTPRZ1 CAR T cells have higher cytotoxic capacity, a killing assay was performed using Ge518_PTPRZ1-KI target cells and CAR T cells 470_28z, 471_28z and 476_28z at three different E:T ratios. The comparison showed that 471_28z CAR T cells have higher killing capacity at any of the tested E:T ratios, up to ~80% at E:T ratio 3:1, while the other two were 45-60%Figure 4 ). Furthermore, 471_28z CAR T cells were able to maintain this activity at a relatively low E:T ratio of 0.5:1, while the other cells showed lower killing (<20%) ( Figure 4 ).

[0368] Example 2 - Anti-CSPG4 RNA CAR T cells

[0369] Six different scFvs against CSPG4 were obtained from the screening of a human scFv phage display library. These scFvs were cloned into the CAR BBz and 28z formats in the pDA plasmid. The A375 melanoma cell line and the GBM Ge518 cell line were used as tumor models to evaluate the cytotoxic activity of the anti-CSPG4 CAR T cells. RNA CAR T cells were generated following the protocol described in Example 1. All anti-CSPG4 CAR T cells had a high CAR molecule expression at the surface (>87%) ( Figure 5 A).

[0370] 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-IL13Ra2_BBz CAR T cells were used as a positive control and non-transduced T cells were used as a negative control, all generated from the same donor. The four CSPG4-specific CAR T cells incorporating the 299_BBz, 301_BBz, 302_BBz, and 303_BBz scFvs showed a high percentage of tumor cell killing (>75%, Figure 5 B). Next, anti-CSPG4 CAR T cells were tested against the Ge518 GBM cells at an E:T ratio of 5:1. Similarly, CAR T cells incorporating the 299_BBz, 301_BBz, 302_BBz, and 303_BBz scFvs showed the highest killing of the GBM cell line, with 35 to 45% tumor cell death ( Figure 5 C). IFN-g secretion was also measured in the supernatant of the killing experiments and the 301_BBz and 302_BBz CAR T cells produced the highest levels (~2000 pg / mL) Figure 5 D).

[0371] Example 3 - Activity of a mixture of three CAR T cells against GBM cells

[0372] RNA CAR T cells containing scFv302 were generated targeting three different GBM targets: IL13Rα2, Her2, and CSPG4. To evaluate the effect of combination therapy against heterogeneous GBM, three variants of the Ge518 tumor cell line, each lacking a target antigen, were generated. Knockout (KO) variants were generated using a CRISPR-Cas9 system, and antigen expression was tested by flow cytometry after cloning, and gene sequencing was performed to confirm the mutation. Figure 6 A). The differential ability of each CAR T cell to kill wild-type (wt) variants of Ge518 compared to their respective antigen KO variants was evaluated by flow cytometry. In each case, CAR T cells were able to specifically kill WT variants without killing KO variants. Figure 6 B).

[0373] Then, a mixture of three different Ge518 KO cells (IL13Rα2-KO, Her2-KO, and CSPG4-KO) and Ge518 wt cells was incubated with a mixture of anti-Her2_BBz, anti-IL13Rα2-BBz, and anti-CSPG4_BBz CAR T cells for 72 hours. The CAR T cells were plated to obtain a final E:T ratio of 3:1, both for individual CAR T cells and for the total number of CAR T cells in the mixture. For mixtures, either equal ratios (mixture A, E:T ratio of 1:1 for each CAR T cell) or two unbalanced mixtures were used: mixture B, with an E:T ratio of 1.5:1 for anti-Her2, 1:1 for anti-IL13Rα2, and 0.5:1 for anti-CSPG4; and mixture C, with an E:T ratio of 0.5:1 for anti-Her2, 1:1 for anti-IL13Rα2, and 1.5:1 for anti-CSPG4. The killing capacity of individual or CAR T cell mixtures was measured by flow cytometry after 72 hours. All mixtures exhibited high cytotoxic activity, with tumor cell mortality >70%, similar to and higher than the cytotoxic activity observed in individual anti-IL13Rα2_BBz or anti-CSPG4_BBz cells at an E:T ratio of 3:1. Figure 7 B). Compared to individual CAR T cells, hybrid CAR T cells not only address tumor heterogeneity but also reduce the likelihood of tumor escape.

[0374] Example 4 – Generation of Triple CAR T Cells

[0375] RNA CAR T cells were generated against three different glioma-associated antigens, PTPRZ1 (471_28z), CSPG4 (301_28z), and BCAN (295_28z), using scFv specific for each antigen. It was confirmed that each CAR was expressed individually Figure 8 A). In addition to the scFv sequence, the _28z construct includes a human IgG4 hinge sequence (SEQ ID NO: 118), a CD28 transmembrane and intracellular domain (SEQ ID NO: 123 and 125, respectively), and a CD3 zeta intracellular domain (SEQ ID NO: 126-128).

[0376] “Triple” RNA CAR T cells were generated by RNA electroporating human T cells with RNA encoding, and expressing, all three of the anti-PTPRZ1 (471_28z), anti-CSPG4 (301_28z), and anti-BCAN (295_28z) CARs simultaneously. To evaluate the effect of combination therapy on a heterogeneous GBM, two variants of the Ge518 tumor cell line were generated: Ge518_BCANv2-TM KI (BCAN knock-in), and Ge518_PTPRZ1 KI (PTPRZ1 knock-in). Triple CAR T cells resulted in increased cell killing compared to any monovalent CAR T cell in all three target cell lines Figure 8 B). Enhanced cell killing of the Ge518_wt and Ge518_BCANv2 cell lines was observed, particularly by the monovalent anti-PTPRZ1 CAR T cells and the triple CAR, when all three target cell lines were mixed, as is more representative of a heterogeneous GBM. Figure 8 C). This represents a “bystander” effect. These results were reflected in an in vitro model of tumor growth inhibition Figure 8 D and 8E).

[0377] Example 5 - Analysis of safety and bystander effect

[0378] As demonstrated in Example 4, it was found that monovalent anti-PTPRZ1 CAR T cells were effective at killing GBM cell lines that did not significantly express PTPRZ1 in the presence of mixed GBM cell lines expressing PTPRZ1. It was then investigated whether the bystander effect was specific to GBM cells or also affected healthy cells by mixing Ge518_PTPRZ1-KI cells with “healthy” macrophages. Cell killing of the Ge518_PTPRZ1-KI cells was observed, while no significant increase in macrophage killing was observed Figure 9 ) upon the addition of monovalent anti-PTPRZ1 CAR T cells.

[0379] To determine whether the "bystander" effect is mediated by soluble factors, according to Figure 10 Experiment A. When Ge518_PTPRZ1-KI cells were exposed to monovalent anti-PTPRZ1 CAR T cells, cell killing was observed in the PTPRZ1 knockout (Ge518_PTPRZ1-KO) GBM cell line, even though there was no direct contact between the monovalent anti-PTPRZ1 CAR T cells and the Ge518_PTPRZ1-KO cell line.

[0380] Example 6 – Co-dependence of target expression in human glioblastoma

[0381] The correlation between antigen pair expression was studied using extensive RNA-seq data from TCGA (primary GBM) and CGGA (recurrent GBM) data. Figure 11 A and 11B).

[0382] While CAR T-cell therapy can be tailored to patients based on antigen expression, this data can also be used to discover the optimal combination that takes into account inter-patient variability. That is, without knowing the specific antigen expression of the glioma, we can improve our chances of targeting gliomas with multivalent CAR T-cell therapy by selecting combinations of antigens with poor or negative correlations. These antigens exhibit different expression trends in different patients. Therefore, combinations should be tailored to a large number of patients. Possible combinations could include BCAN, TNC, and CSPG4.

[0383] Conversely, when the expression of one or more glioma-associated antigens is known in a patient, the use of multivalent CAR T cells that also target known glioma-associated antigens that are positively correlated with known antigens can also be used, for example, to reduce the chances of gliomas escaping CAR T cell therapy by reducing the expression of a single antigen.

[0384] Example 7 – Using nanobodies to generate monovalent and multivalent CAR T cells

[0385] Nanobodies (V) are generated targeting BCAN (RB826-829), CSPG4 (RB830-831), PTPRZ1 (832-834), and TNC (835-836). HH The specificity of nanobodies against their target and control antigens was tested using ELISA. Figure 12 A, 13A, 14A, and 15A). Nanobodies conjugated with human IgG1Fc were used to examine tumor cell line recognition (…). Figure 12 B, 13B and 14B).

[0386] CARs were generated using nanobodies with short or long hinges, and the expression of each CAR in transformed T cells was examined to determine the expression level of each construct.Figure 12 C, 13C, 14C, and 15B). The short “_28z” construct includes, in addition to the Nanobody sequence, a human IgG4 hinge sequence (SEQ ID NO: 118), a CD28 transmembrane and intracellular domain (SEQ ID NOs: 123 and 125, respectively), and a CD3 zeta intracellular domain (SEQ ID NOs: 126-128). The long “_IgGlH_28z” construct includes, in addition to the Nanobody sequence, a human IgGl long hinge sequence (SEQ ID NO: 121), a CD28 transmembrane and intracellular domain (SEQ ID NOs: 123 and 125, respectively), and a CD3 zeta intracellular domain (SEQ ID NOs: 126-128). Cell killing by the Nanobody-based CAR-T cells was determined by flow cytometry against a number of tumor cell lines Figure 12 D, 13D, 14D, and 15C). Corresponding data for in vitro inhibition of tumor growth were generated Figure 12 E, 13E, 14E, and 15D).

[0387] Bispecific CAR T cells were generated expressing a combination of a CAR including anti-PTPRZl Nanobody 832 with a CAR including anti-CSPG4 Nanobody 830, anti-BCAN scFc 295, or anti-TNC Nanobody 835. Figure 16 A - 16C left column graphs compare cell killing by monovalent anti-PTPRZl CAR T cells relative to bispecific CAR T cells against cell lines that are low in PTPRZl but high in CSPG4 Figure 16 A), BCAN Figure 16 B), or TNC Figure 16 C). Figure 16 A - 16C right column graphs compare cell killing by monovalent anti-CSPG4 CAR T cells Figure 16 A), monovalent anti-BCAN CAR T cells Figure 16 B), or monovalent anti-TNC CAR T cells Figure 16 C) against cell lines that are high in PTPRZl relative to bispecific CAR T cells that also express an anti-PTPRZl CAR.

[0388] Further embodiments with numbering

[0389] 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.

[0390] 2. The immune effector cell or population of immune effector cells of embodiment 1, wherein the one or more glioma-associated antigen is PTPRZl, BCAN, CSPG4, and / or TNC.

[0391] 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, optionally wherein the population of CAR-expressing immune effector cells comprises at least three different CAR-expressing immune effector cells.

[0392] 4. A method of making the immune effector cell or population of immune effector cells of any of the preceding embodiments, comprising transforming the cell or the population of cells with one or more nucleic acids encoding one or more CARs specific for one or more glioma-associated antigens.

[0393] 5. A method of treating a 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 of embodiments 1-3.

[0394] 6. The immune effector cell or population of immune effector cells of any of embodiments 1-3, for use in a method of treating a cancer.

[0395] 7. The method of treating a subject of embodiment 5, or the immune effector cell or population of immune effector cells of embodiment 6, wherein the cancer is a glioma.

[0396] 8. The immune effector cell or population of immune effector cells of any of embodiments 1-3, the method of any of embodiments 4, 5, or 7, or the immune effector cell or population of immune effector cells of embodiment 6 or 7, wherein the one or more CARs is selected from a CAR comprising a polypeptide having an amino acid sequence of any of SEQ ID NOs: 21-40 or an amino acid sequence at least 80%, 85%, 90%, 95%, or 99% identical thereto.

[0397] 9. The immune effector cell or population of immune effector cells of any of embodiments 1-3 or 8, the method of any of embodiments 4, 5, 7, or 8, or the immune effector cell or population of immune effector cells of any of embodiments 6-8, wherein the one or more CARs is selected from a CAR comprising a polypeptide 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 of SEQ ID NOs: 21-40.

[0398] 10. The immune effector cell or population of immune effector cells of any of embodiments 1-3, 8, or 9, the method of any of embodiments 4-5, or 7-9, or the immune effector cell or population of immune effector cells of any of embodiments 6-9, wherein the cell is a T cell, an NK cell, an iPSC-NK cell, a T cell, a phagocyte, or a macrophage.

[0399] 11. A CAR comprising a polypeptide having (a) an amino acid sequence of any of SEQ ID NOs: 21-40, (b) an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical 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 of SEQ ID NOs: 21-40.

[0400] 12. A multivalent CAR comprising (a) an extracellular domain specific for two or more glioma-associated antigens, optionally wherein the glioma-associated antigens are selected from PTPRZ1, BCAN, CSPG4, and / or TNC; and (b) an intracellular signaling domain.

[0401] 13. The multivalent CAR of embodiment 12, wherein the extracellular domain is a ScFv, a V H or a V HH .

[0402] 14. The multivalent CAR of embodiment 13, wherein the extracellular domain comprises a polypeptide selected from (a) an amino acid sequence of any of SEQ ID NOs: 21-40, (b) an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical thereto, 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 of SEQ ID NOs: 21-40.

[0403] 15. The CAR of embodiment 11 or the multivalent CAR of any of embodiments 12-14, 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.

[0404] 16. The CAR of embodiment 11 or 15, or the multivalent CAR of any of embodiments 12-15, further comprising a transmembrane domain.

[0405] 17. The CAR or multivalent CAR of embodiment 16, wherein the transmembrane domain is a CD28 transmembrane domain.

[0406] 18. A nucleic acid encoding the CAR of any one of embodiments 11 and 15 to 17, or the multivalent CAR of any one of embodiments 12 to 17.

[0407] 19. The nucleic acid of embodiment 18, which is DNA or RNA.

[0408] 20. The nucleic acid of embodiment 18 or 19, comprising the sequence of any one of SEQ ID NOs: 1-20 or encoding the amino acid sequence of any one of SEQ ID NOs: 21-40.

[0409] 21. A vector comprising one or more nucleic acids of any one of embodiments 18 to 20.

[0410] 22. The vector of embodiment 21, which is a lentiviral vector for in vivo delivery of RNA, an RNA vector, a liposome, or a lipid nanoparticle.

[0411] 23. An antigen binding molecule specific for one or more glioma-associated antigens selected from the group consisting of (a) a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 21-32 and 34-37, (b) an amino acid sequence at least 80%, 85%, 90%, 95%, or 99% identical thereto, or (c) HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3, from an amino acid sequence selected from any one of SEQ ID NOs: 21-32 and 34-37.

Claims

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

2. An immune effector cell or population of immune effector cells as claimed in claim 1, wherein two or more of the glioma-associated antigens is selected from PTPRZl, BCAN, CSPG4 and TNC.

3. An immune effector cell or population of immune effector cells as claimed in claim 1 or 2 expressing one or more CARs specific for three or more glioma-associated antigens.

4. An immune effector cell or population of immune effector cells as claimed in any preceding claim, wherein the or each cell expresses two or more CARs specific for different glioma-associated antigens.

5. An immune effector cell or population of immune effector cells as claimed in any preceding claim, wherein the or each cell expresses three or more CARs specific for different glioma-associated antigens.

6. A population as claimed in any preceding claim 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. A population as claimed in 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. An immune effector cell or population of immune effector cells as claimed in any preceding claim, wherein one or more of the glioma-associated antigens is a cell surface marker and one or more of the glioma-associated antigens is an extracellular matrix (ECM) marker.

9. An immune effector cell or population of immune effector cells as claimed in claim 8, wherein the cell surface marker is selected from PTPRZl, CSPG4 and BCAN and / or wherein the ECM marker is selected from TNC and BCAN.

10. An immune effector cell or population of immune effector cells expressing a chimeric antigen receptor (CAR) specific for PTPRZl.

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

12. A method of making an immune effector cell or population of immune effector cells as claimed in any preceding claim, comprising transforming the cell or 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 an immune effector cell or population of immune effector cells as claimed in any of claims 1-11.

14. An immune effector cell or population of immune effector cells as claimed in any of claims 1-11 for use in a method of treating cancer.

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

16. The immune effector cell or population of immune effector cells of any one of claims 1-11, the method of any one of claims 12-13 and 15, or the immune effector cell or population of immune effector cells of claim 14 or 15, wherein the cell is a T cell, an NK cell, an iPSC-NK cell, a T cell, a phagocyte, or a macrophage.

17. A CAR specific for PTPRZl.

18. A CAR specific for BCAN.

19. A CAR specific for a glioma-associated antigen selected from the group consisting of PTPRZl, BCAN, CSPG4, and TNC, comprising a polypeptide comprising: (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: 21-40; or (b) complementarity determining regions (CDR1, CDR2, and CDR3) from an amino acid sequence selected from any one of SEQ ID NOs: 83-93.

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

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

22. The CAR of any one of claims 17-21, wherein the polypeptide comprises: (a) one or more immunoglobulin variable domains; and / or 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 is selected from the group consisting of PTPRZl, BCAN, CSPG4, and TNC; and (b) an intracellular signaling domain.

25. The multivalent CAR of claim 23 or 24, wherein the extracellular domain comprises a polypeptide comprising: (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: 21-40; or (b) complementarity determining regions (CDR1, CDR2, and CDR3) from an amino acid sequence selected from any one of SEQ ID NOs: 83-93.

26. The multivalent CAR of any one of claims 23-26, wherein the extracellular domain comprises: (a) an amino acid sequence of any one of SEQ ID NOs: 21-40 and 83 to 93; and / or (b) an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to an amino acid sequence of any one of SEQ ID NOs: 21-40 and 83-93. ​ ​ (b) scFv, VH or V HH domain. ​ 24. The multivalent CAR of claim 23, wherein the extracellular domain is a ScFv, VH, or V HH . ​ ​ ​ ​ ​ (b) an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 21-40 and 83-93.

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

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

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 1-11 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 of any one of claims 14 to 16, wherein the one or more CARs comprise a CAR or multivalent CAR of any one of claims 19 to 29.

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

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

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

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

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 complementarity determining regions (CDR1, CDR2, and CDR3) from an amino acid sequence selected from any one of SEQ ID NOs: 83-93.

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

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