Chimeric antigen receptors containing glypican 2 binding domains

Abstract

To provide an alternative approach to minimize side effects such as increased off-target cell killing by CAR-T cells, and to complement existing approaches for immunotherapy.SOLUTION: Provided is a chimeric antigen receptor comprising: (i) an ectodomain comprising a single-chain antibody variable region fragment (scFv) region including a variable heavy chain (VH) and a variable light chain (VL) that binds selectively to Glypican 2; (ii) a transmembrane domain; and (iii) an endodomain, wherein the endodomain comprises a signal transduction function when the scFv is bound to Glypican 2. Also provided are cells expressing the chimeric antigen receptor and methods of using such cells to treat cancers that express or overexpress the Glypican 2 antigen.SELECTED DRAWING: None

Description

Technical Field

[0001] Claim of Priority This application claims the benefit of priority of U.S. Provisional Application No. 62 / 876,483, filed Jul. 19, 2019. The entire content of this application is incorporated herein by reference.

[0002] Description of Government Funding This invention was made with government support under grant number NCI U54 CA232568-01 awarded by the National Institutes of Health. The United States government has certain rights in this invention.

[0003] In accordance with 37 C.F.R.§1.821(c), a Sequence Listing is filed herewith as an ASCII compliant text file named "CHOPP0034WO.txt", created on Jul. 17, 2020, and having a size of approximately 27 kilobytes. The entire contents of the above file are incorporated herein by reference.

Background Art

[0004] Background 1. Field The present disclosure generally relates to the fields of medicine, oncology, and immunotherapy. More particularly, the present disclosure relates to the development of chimeric antigen receptor immunoreagents having binding specificity for glypican 2 (GPC2), and the use of chimeric antigen receptor immunoreagents in the treatment of GPC2-positive cancers.

[0005] 2. Related Art Children with high-risk neuroblastoma have a poor prognosis even after receiving intensive multimodal chemoradiotherapy. Monoclonal antibodies targeting the disialoganglioside GD2 improve outcomes in neuroblastoma, but this therapy is associated with significant "on target-off tumor" toxicity. Thus, there remains a major challenge in identifying novel cell surface molecules that meet the stringent criteria of modern immunotherapeutic agents, including that tumor expression is unique compared to normal pediatric tissue and, preferably, that these cell surface molecules are required for tumor maintenance.

[0006] Numerous biologics for treating diseases or health disorders are currently under development by pharmaceutical and biotechnology companies. For example, in cancer immunotherapy, the development of agents that activate T cells of the host immune system to block the growth of cancer cells or kill cancer cells has emerged as a promising therapeutic approach to complement existing standard treatments. The adoptive transfer of T cells, particularly T cells engineered with chimeric antigen receptors (CARs), has emerged as another promising approach in cancer immunotherapy. Unlike natural T cell receptors, CARs can directly recognize the target antigen of the CAR without the constraints imposed by major histocompatibility complex (MHC) molecules and, in some cases, can mediate high levels of cell killing activity. One common approach is to genetically engineer T cells ex vivo to express a CAR that can recognize the target antigen without the need for MHC presentation. These CAR-T cells have the potential to generate very high levels of antitumor activity but may also exhibit increased off-target cell killing by CAR-T cells. Thus, alternative approaches are urgently needed to minimize such side effects and to complement existing approaches for immunotherapy. SUMMARY OF THE INVENTION

[0007] Summary Accordingly, according to the present disclosure, there is provided a chimeric antigen receptor comprising: (i) an ectodomain comprising a single-chain antibody variable region fragment (scFv) region comprising a variable heavy chain (VH) and a variable light chain (VL) that selectively binds to glypican 2; (ii) a transmembrane domain; and (iii) an endodomain, wherein the endodomain comprises a signal transduction function when the scFv binds to glypican 2.

[0008] The receptor may be characterized by the VH sequence of SEQ ID NO:5 and the VL sequence of SEQ ID NO:6; the VH sequence of SEQ ID NO:7 and the VL sequence of SEQ ID NO:8; or the VH sequence of SEQ ID NO:9 and the VL sequence of SEQ ID NO:10.

[0009] The scFv may be characterized by a VH sequence having 80% homology to SEQ ID NO:5 and having VH CDRs of SEQ ID NOs: 11-13 and a VL sequence having 80% homology to SEQ ID NO:6 and having VL CDRs of SEQ ID NOs: 14-16, or a VH sequence having 80% homology to SEQ ID NO:7 and having VH CDRs of SEQ ID NOs: 17-19 and a VL sequence having 80% homology to SEQ ID NO:8 and having VL CDRs of SEQ ID NOs: 20-22, or a VH sequence having 80% homology to SEQ ID NO:9 and having VH CDRs of SEQ ID NOs: 23-25 and a VL sequence having 80% homology to SEQ ID NO:10 and having VL CDRs of SEQ ID NOs: 26-28.

[0010] The receptor may be characterized by a VH sequence having 90% homology to SEQ ID NO:5 and having VH CDRs of SEQ ID NOs:11-13, and a VL sequence having 90% homology to SEQ ID NO:6 and having VL CDRs of SEQ ID NOs:14-16; or a VH sequence having 90% homology to SEQ ID NO:7 and having VH CDRs of SEQ ID NOs:17-19, and a VL sequence having 90% homology to SEQ ID NO:8 and having VL CDRs of SEQ ID NOs:20-22; or a VH sequence having 90% homology to SEQ ID NO:9 and having VH CDRs of SEQ ID NOs:23-25, and a VL sequence having 90% homology to SEQ ID NO:10 and having VL CDRs of SEQ ID NOs:26-28.

[0011] The receptor may include a sequence selected from SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, and may include a sequence having 80% homology to SEQ ID NO:1 and having VH CDRs of SEQ ID NOs:11-13 and VL CDRs of SEQ ID NOs:14-16, a sequence having 80% homology to SEQ ID NO:2 and having VH CDRs of SEQ ID NOs:17-19 and VL CDRs of SEQ ID NOs:20-22, and a sequence having 80% homology to SEQ ID NO:3 and having VH CDRs of SEQ ID NOs:23-25 and VL CDRs of SEQ ID NOs:26-28, and may include a sequence having 90% equivalence to SEQ ID NO:1 and having VH CDRs of SEQ ID NOs:11-13 and VL CDRs of SEQ ID NOs:14-16, a sequence having 90% homology to SEQ ID NO:2 and having VH CDRs of SEQ ID NOs:17-19 and VL CDRs of SEQ ID NOs:20-22, and a sequence having 90% equivalence to SEQ ID NO:3 and having VH CDRs of SEQ ID NOs:23-25 and VL CDRs of SEQ ID NOs:26-28.

[0012] The transmembrane domain and the endodomain may be derived from the same molecule. The endodomain may include a CD3-ζ domain or a high-affinity FcεRI. The scFv may include a flexible linker disposed between the VH and VL, for example, the flexible linker is derived from CD8α, Ig, or SEQ ID NO:4. The scFv may be arranged in the order of VH-linker-VL or in the order of VL-linker-VH.

[0013] Also provided are nucleic acids encoding the chimeric antigen receptor as defined above, such as mRNA or DNA, or cells expressing the chimeric antigen receptor as defined above, such as prokaryotic or eukaryotic cells, particularly engineered T cells.

[0014] In another aspect, provided is a method of treating a subject having a cancer that expresses or overexpresses glypican 2, the method comprising administering to the subject a chimeric antigen receptor as defined above, a nucleic acid as defined above, or a cell as defined above, such as a T cell, such as a T cell autologous to the subject.

[0015] The method may further comprise administering to the subject a second anti-cancer therapy. The second cancer therapy may be radiation, chemotherapy, radiotherapy, hormone therapy, immunotherapy, toxin therapy, or surgery. The immunotherapy may be checkpoint inhibitor therapy. The second cancer therapy may be administered at the same time as the receptor, nucleic acid, or cell, before or after the receptor, nucleic acid, or cell. The second cancer therapy may be administered multiple times. The receptor, nucleic acid, or cell may be administered multiple times.

[0016] The cancer may be drug-resistant, metastatic, or recurrent. The subject may be a human or non-human mammal. The cancer may be a pediatric cancer or an adult cancer. The cancer may be a leukemia, for example, acute lymphoblastic leukemia (ALL), acute lymphoblastic B-cell leukemia, acute lymphoblastic T-cell leukemia, acute myeloblastic leukemia (AML), acute promyelocytic leukemia (APL), acute monoblastic leukemia, acute erythroleukemia, acute megakaryoblastic leukemia, acute myelomonocytic leukemia, acute nonlymphocyctic leukemia, acute undifferentiated leukemia, chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), and hairy cell leukemia, selected from the group consisting of leukemia.

[0017] Cancer may be solid tumor cancer, such as lung cancer, liver cancer, pancreatic cancer, gastric cancer, colon cancer, kidney cancer, brain cancer, head and neck cancer, breast cancer, skin cancer, rectal cancer, uterine cancer, cervical cancer, ovarian cancer, testicular cancer, skin cancer, or esophageal cancer. Cancer may also include sarcoma cells, rhabdoid cancer cells, neuroblastoma cells, retinoblastoma cells, or medulloblastoma cells. Cancer may be uterine carcinosarcoma (UCS), brain lower grade glioma (LGG), thymoma (THYM), testicular germ cell tumor (TGCT), glioblastoma multiforme (GBM) and skin cutaneous melanoma (SKCM), liver hepatocellular carcinoma (LIHC), uveal melanoma (UVM), kidney chromophobe (KICH), thyroid cancer (THCA), kidney clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), stomach adenocarcinoma (STAD), cholangiocarcinoma (CHOL), adenoid cystic carcinoma (ACC), prostate adenocarcinoma (PRAD), chromaffin cell tumor and paraganglioma (PCPG), DLBC, lung adenocarcinoma (LUAD), head and neck squamous cell carcinoma (HNSC), pancreatic adenocarcinoma (PAAD), breast cancer (BRCA), mesothelioma (MESO), colon and rectal adenocarcinoma (COAD), rectal adenocarcinoma (READ), esophageal cancer (ESCA), ovarian cancer (OV), lung squamous cell carcinoma (LUSC), bladder urothelial carcinoma (BLCA), sarcoma (SARC), or corpus endometrial carcinoma (UCEC).

[0018] An isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen-binding domain, a flexible hinge domain, a transmembrane domain, a co-stimulatory signaling region, and an intracellular signaling domain, and the antigen-binding domain selectively binds to cancer cell-associated glypican 2 (GPC2), is also provided. The antigen-binding domain may comprise an antibody or an antigen-binding fragment thereof. The antigen-binding fragment may be a Fab, a single-chain variable fragment (scFv), or a single-domain antibody. The encoded antigen-binding domain may comprise (a) a heavy-chain variable domain comprising the amino acid sequence of SEQ ID NO:30 and a light-chain variable domain comprising the amino acid sequence of SEQ ID NO:32; or (b) a heavy-chain variable domain comprising the amino acid sequence of SEQ ID NO:34 and a light-chain variable domain comprising the amino acid sequence of SEQ ID NO:36; or (c) a heavy-chain variable domain comprising the amino acid sequence of SEQ ID NO:38 and a light-chain variable domain comprising the amino acid sequence of SEQ ID NO:40.

[0019] The encoded antigen-binding domain may comprise: (a) a heavy-chain variable domain comprising CDR1 having the amino acid sequence of SEQ ID NO:11, CDR2 having the amino acid sequence of SEQ ID NO:12, and CDR3 having the amino acid sequence of SEQ ID NO:13, and a light-chain variable domain comprising CDR1 having the amino acid sequence of SEQ ID NO:14, CDR2 having the amino acid sequence of SEQ ID NO:15, and CDR3 having the amino acid sequence of SEQ ID NO:16; or (b) a heavy-chain variable domain comprising CDR1 having the amino acid sequence of SEQ ID NO:17, CDR2 having the amino acid sequence of SEQ ID NO:18, and CDR3 having the amino acid sequence of SEQ ID NO:19, and a light-chain variable domain comprising CDR1 having the amino acid sequence of SEQ ID NO:20, CDR2 having the amino acid sequence of SEQ ID NO:21, and CDR3 having the amino acid sequence of SEQ ID NO:22; or (c) a heavy-chain variable domain comprising CDR1 having the amino acid sequence of SEQ ID NO:23, CDR2 having the amino acid sequence of SEQ ID NO:24, and CDR3 having the amino acid sequence of SEQ ID NO:25, and a light-chain variable domain comprising CDR1 having the amino acid sequence of SEQ ID NO:26, CDR2 having the amino acid sequence of SEQ ID NO:27, and CDR3 having the amino acid sequence of SEQ ID NO:28.

[0020] The encoded antigen-binding domain may comprise a heavy-chain variable domain having the amino acid sequence of SEQ ID NO:30 and a light-chain variable domain having the amino acid sequence of SEQ ID NO:32, and the C-terminus of the light-chain variable domain may be fused to the N-terminus of the heavy-chain variable domain by a flexible linker. The linker may be a peptide linker, for example, at least 15 amino acids in length, and / or the peptide linker may be a glycine-serine linker.

[0021] An isolated nucleic acid molecule may have: (a) a flexible hinge domain derived from CD8α, CD28, or immunoglobulin (Ig); (b) a transmembrane domain including the CD28 transmembrane domain; (c) a co-stimulatory signaling region including a domain derived from CD28, 4-1BB (CD137), OX40, or ICOS; and (d) an intracellular signaling domain including the CD3-ζ domain or high-affinity FcεRI.

[0022] In another aspect, provided is a chimeric antigen receptor (CAR) polypeptide, wherein (a) the CAR comprises an antigen-binding domain, a flexible hinge domain, a transmembrane domain, a co-stimulatory signaling region, and an intracellular signaling domain, and (b) the antigen-binding domain selectively binds to cancer cell-associated glypican 2 (GPC2). The antigen-binding fragment may be a Fab, a single-chain variable fragment (scFv), or a single-domain antibody.

[0023] The encoded antigen-binding domain may include: (a) a heavy-chain variable domain comprising the amino acid sequence of SEQ ID NO: 30 and a light-chain variable domain comprising the amino acid sequence of SEQ ID NO: 32; or (b) a heavy-chain variable domain comprising the amino acid sequence of SEQ ID NO: 34 and a light-chain variable domain comprising the amino acid sequence of SEQ ID NO: 36; or (c) a heavy-chain variable domain comprising the amino acid sequence of SEQ ID NO: 38 and a light-chain variable domain comprising the amino acid sequence of SEQ ID NO: 40.

[0024] The antigen-binding domain to be encoded may comprise: (a) a heavy-chain variable domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:11, a CDR2 comprising the amino acid sequence of SEQ ID NO:12, and a CDR3 comprising the amino acid sequence of SEQ ID NO:13, and a light-chain variable domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:14, a CDR2 comprising the amino acid sequence of SEQ ID NO:15, and a CDR3 comprising the amino acid sequence of SEQ ID NO:16; or (b) a heavy-chain variable domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:17, a CDR2 comprising the amino acid sequence of SEQ ID NO:18, and a CDR3 comprising the amino acid sequence of SEQ ID NO:19, and a light-chain variable domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:20, a CDR2 comprising the amino acid sequence of SEQ ID NO:21, and a CDR3 comprising the amino acid sequence of SEQ ID NO:22; or (c) a heavy-chain variable domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:23, a CDR2 comprising the amino acid sequence of SEQ ID NO:24, and a CDR3 comprising the amino acid sequence of SEQ ID NO:25, and a light-chain variable domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:26, a CDR2 comprising the amino acid sequence of SEQ ID NO:27, and a CDR3 comprising the amino acid sequence of SEQ ID NO:28.

[0025] The chimeric antigen receptor polypeptide may comprise: (a) an encoded antigen-binding domain comprising a heavy-chain variable domain comprising the amino acid sequence of SEQ ID NO:30 and a light-chain variable domain comprising the amino acid sequence of SEQ ID NO:32; and (b) the C-terminus of the light-chain variable domain fused to the N-terminus of the heavy-chain variable domain by a flexible linker.

[0026] Also provided are genetically modified T cells comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR), or comprising an isolated nucleic acid molecule as defined herein, or genetically modified T cells comprising a chimeric antigen receptor as defined herein. The genetically modified T cells are (a) A chimeric antigen receptor (CAR) that induces the secretion of interferon-γ and interleukin-2, and (b) is cytotoxic to GPC2-expressing cancer when genetically modified T cells are exposed to cancer cell-associated GPC2. It may be characterized by this.

[0027] GPC2-expressing cancers may be selected from the group consisting of sarcoma cells, rhabdoid cancer cells, neuroblastoma cells, retinoblastoma cells, or medulloblastoma cells, uterine carcinosarcoma (UCS), low-grade glioma of the brain (LGG), thymoma (THYM), testicular germ cell tumor (TGCT), glioblastoma multiforme (GBM) and cutaneous melanoma (SKCM), hepatocellular carcinoma (LIHC), uveal melanoma (UVM), kidney chromophobe cell (KICH), thyroid cancer (THCA), renal clear cell carcinoma (KIRC), renal papillary cell carcinoma (KIRP), gastric adenocarcinoma (STAD), cholangiocarcinoma (CHOL), adenoid cystic carcinoma (ACC), prostate adenocarcinoma (PRAD), chromaffin cell tumor and paraganglioma (PCPG), DLBC, lung adenocarcinoma (LUAD), head and neck squamous cell carcinoma (HNSC), pancreatic adenocarcinoma (PAAD), breast cancer (BRCA), mesothelioma (MESO), colorectal adenocarcinoma (COAD), rectal adenocarcinoma (READ), esophageal cancer (ESCA), ovarian cancer (OV), lung squamous cell carcinoma (LUSC), bladder urothelial carcinoma (BLCA), sarcoma (SARC), or endometrial carcinoma of the uterine body (UCEC).

[0028] Also provided is a method of producing genetically modified T cells comprising the step of transducing immune effector cells with a chimeric antigen receptor as defined herein. Also provided is a method of providing anti-tumor immunity in a mammal, comprising the step of administering to the mammal an effective amount of a population of genetically modified T cells as defined herein. Also provided is a method of treating a mammal having a disease associated with overexpression of GPC2, comprising the step of administering to the mammal an effective amount of a population of genetically modified T cells as defined herein.

[0029] Any method or composition described herein is intended to be practicable with any other method or composition described herein.

[0030] The use of the words "a" or "an" may mean "one" when used in conjunction with the term "comprising" in the claims and / or the specification, but is also consistent with the meanings of "one or more", "at least one", and "one or more than one". The word "about" means +5% or -5% of the stated number.

[0031] [The present invention 1001] An isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen-binding domain, a flexible hinge domain, a transmembrane domain, a co-stimulatory signaling region, and an intracellular signaling domain, the antigen-binding domain selectively binds to cancer cell-associated glypican 2 (GPC2), [[ID=…]] said isolated nucleic acid molecule. [The present invention 1002] The isolated nucleic acid molecule of the present invention 1001, wherein the antigen-binding domain comprises an antibody or an antigen-binding fragment thereof. [The present invention 1003] The isolated nucleic acid molecule of the present invention 1002, wherein the antigen-binding fragment is a Fab, a single-chain variable fragment (scFv), or a single-domain antibody. [The present invention 1004] The encoded antigen-binding domain is (a) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 30 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 32; (b) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 34 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 36; or (c) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 38 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 40 An isolated nucleic acid molecule of any one of the present inventions 1001 to 1003, comprising [Present invention 1005] The antigen-binding domain encoded is (a) A heavy chain variable domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:11, a CDR2 comprising the amino acid sequence of SEQ ID NO:12, and a CDR3 comprising the amino acid sequence of SEQ ID NO:13, and a CDR1 comprising the amino acid sequence of SEQ ID NO:14, a CDR2 comprising the amino acid sequence of SEQ ID NO:15, and a CDR3 comprising the amino acid sequence of SEQ ID NO:16, a light chain variable domain; (b) A heavy chain variable domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:17, a CDR2 comprising the amino acid sequence of SEQ ID NO:18, and a CDR3 comprising the amino acid sequence of SEQ ID NO:19, and a CDR1 comprising the amino acid sequence of SEQ ID NO:20, a CDR2 comprising the amino acid sequence of SEQ ID NO:21, and a CDR3 comprising the amino acid sequence of SEQ ID NO:22, a light chain variable domain; or (c) A heavy chain variable domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:23, a CDR2 comprising the amino acid sequence of SEQ ID NO:24, and a CDR3 comprising the amino acid sequence of SEQ ID NO:25, and a CDR1 comprising the amino acid sequence of SEQ ID NO:26, a CDR2 comprising the amino acid sequence of SEQ ID NO:27, and a CDR3 comprising the amino acid sequence of SEQ ID NO:28, a light chain variable domain An isolated nucleic acid molecule of any one of the present inventions 1001 to 1003, comprising [Present invention 1006] (a) The antigen-binding domain encoded comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:30 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO:32, and (b) The C-terminus of the light chain variable domain is fused to the N-terminus of the heavy chain variable domain by a flexible linker, The isolated nucleic acid molecule of the present invention 1002. [Isolated nucleic acid molecule of the present invention 1007] The isolated nucleic acid molecule of the present invention 1006, wherein the linker is a peptide linker. [Isolated nucleic acid molecule of the present invention 1008] The isolated nucleic acid molecule of the present invention 1007, wherein the peptide linker is at least 15 amino acids in length. [Isolated nucleic acid molecule of the present invention 1009] The isolated nucleic acid molecule of the present invention 1008, wherein the peptide linker is a glycine - serine linker. [Isolated nucleic acid molecule of the present invention 1010] (a) The flexible hinge domain is derived from CD8α, CD28, or immunoglobulin (Ig), (b) The transmembrane domain comprises the CD28 transmembrane domain, (c) The co - stimulatory signaling region comprises a domain derived from CD28, 4 - 1BB (CD137), OX40, or ICOS, and (d) The intracellular signaling domain comprises the CD3 - ζ domain or high - affinity FcεRI. The isolated nucleic acid molecule of the present invention 1001. [Isolated nucleic acid molecule of the present invention 1011] (a) The chimeric antigen receptor (CAR) comprises an antigen - binding domain, a flexible hinge domain, a transmembrane domain, a co - stimulatory signaling region, and an intracellular signaling domain, and (b) The antigen - binding domain selectively binds to cancer - cell - associated glypican 2 (GPC2). Chimeric antigen receptor (CAR) polypeptide. [Isolated nucleic acid molecule of the present invention 1012] The chimeric antigen receptor polypeptide of the present invention 1011, wherein the antigen - binding fragment is a Fab, a single - chain variable fragment (scFv), or a single - domain antibody. [Isolated nucleic acid molecule of the present invention 1013] The encoded antigen - binding domain is (a) A heavy - chain variable domain comprising the amino acid sequence of SEQ ID NO:30 and a light - chain variable domain comprising the amino acid sequence of SEQ ID NO:32; (b) A heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:34 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO:36; or (c) A heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:38 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO:40 The chimeric antigen receptor (CAR) polypeptide of the present invention 1011 or 1012, comprising. [The present invention 1014] (a) A heavy chain variable domain comprising CDR1 comprising the amino acid sequence of SEQ ID NO:11, CDR2 comprising the amino acid sequence of SEQ ID NO:12, and CDR3 comprising the amino acid sequence of SEQ ID NO:13, and a light chain variable domain comprising CDR1 comprising the amino acid sequence of SEQ ID NO:14, CDR2 comprising the amino acid sequence of SEQ ID NO:15, and CDR3 comprising the amino acid sequence of SEQ ID NO:16; (b) A heavy chain variable domain comprising CDR1 comprising the amino acid sequence of SEQ ID NO:17, CDR2 comprising the amino acid sequence of SEQ ID NO:18, and CDR3 comprising the amino acid sequence of SEQ ID NO:19, and a light chain variable domain comprising CDR1 comprising the amino acid sequence of SEQ ID NO:20, CDR2 comprising the amino acid sequence of SEQ ID NO:21, and CDR3 comprising the amino acid sequence of SEQ ID NO:22; or (c) A heavy chain variable domain comprising CDR1 comprising the amino acid sequence of SEQ ID NO:23, CDR2 comprising the amino acid sequence of SEQ ID NO:24, and CDR3 comprising the amino acid sequence of SEQ ID NO:25, and a light chain variable domain comprising CDR1 comprising the amino acid sequence of SEQ ID NO:26, CDR2 comprising the amino acid sequence of SEQ ID NO:27, and CDR3 comprising the amino acid sequence of SEQ ID NO:28 The chimeric antigen receptor (CAR) polypeptide of the present invention 1011 or 1012, comprising. [The present invention 1015] (a) The encoded antigen-binding domain comprises a heavy-chain variable domain comprising the amino acid sequence of SEQ ID NO:30 and a light-chain variable domain comprising the amino acid sequence of SEQ ID NO:32, and (b) the C-terminus of the light-chain variable domain is fused to the N-terminus of the heavy-chain variable domain by a flexible linker, The chimeric antigen receptor polypeptide of the present invention 1013. [The present invention 1016] A genetically modified T cell comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR), or a genetically modified T cell comprising any of the isolated nucleic acid molecules of the present invention 1001 to 1015. [The present invention 1017] A genetically modified T cell comprising any of the chimeric antigen receptors of the present invention 1011 to 1016. [The present invention 1018] A method for producing a genetically modified T cell comprising the step of transducing any of the chimeric antigen receptors of the present invention 1011 to 1016 into immune effector cells. [The present invention 1019] A method for providing anti-tumor immunity in a mammal, the method comprising administering to the mammal a population of the genetically modified T cells of the present invention 1016 in an effective amount. [The present invention 1020] A method for treating a mammal having a disease associated with overexpression of GPC2, the method comprising administering to the mammal a population of the genetically modified T cells of the present invention 1016 in an effective amount. [The present invention 1021] (a) The CAR induces the secretion of interferon γ and interleukin-2, and (b) the genetically modified T cell exhibits cytotoxicity against GPC-expressing cancer when exposed to cancer cell-associated GPC2, The genetically modified T cell of the present invention 1016. [The present invention 1022] The method of the present invention 1020, wherein the GPC2-expressing cancer is selected from the group consisting of sarcoma cells, rhabdoid cancer cells, neuroblastoma cells, retinoblastoma cells, or medulloblastoma cells, uterine carcinosarcoma (UCS), low-grade glioma of the brain (LGG), thymoma (THYM), testicular germ cell tumor (TGCT), glioblastoma multiforme (GBM) and cutaneous melanoma (SKCM), hepatocellular carcinoma (LIHC), uveal melanoma (UVM), clear cell renal cell carcinoma (KICH), thyroid cancer (THCA), clear cell renal cell carcinoma (KIRC), papillary renal cell carcinoma (KIRP), gastric adenocarcinoma (STAD), cholangiocarcinoma (CHOL), adenoid cystic carcinoma (ACC), prostate adenocarcinoma (PRAD), chromaffin cell tumor and paraganglioma (PCPG), DLBC, lung adenocarcinoma (LUAD), head and neck squamous cell carcinoma (HNSC), pancreatic adenocarcinoma (PAAD), breast cancer (BRCA), mesothelioma (MESO), colorectal adenocarcinoma (COAD), rectal adenocarcinoma (READ), esophageal cancer (ESCA), ovarian cancer (OV), lung squamous cell carcinoma (LUSC), bladder urothelial carcinoma (BLCA), sarcoma (SARC), or endometrial cancer of the uterine body (UCEC). Other objects, features, and advantages of the present disclosure will become apparent from the following detailed description. However, while the detailed description and specific examples show specific aspects of the present disclosure, it should be understood that these are merely illustrative, as various modifications and changes within the spirit and scope of the present disclosure will be apparent to those skilled in the art from this detailed description.

Brief Description of the Drawings

[0032] The patent or application file contains at least one drawing produced in color. Copies of this patent or patent application publication and the color drawings are provided by the Patent Office upon request or payment of the necessary fee.

[0033] The following drawings form part of this specification and are included to further demonstrate certain specific aspects of the present disclosure. The present disclosure can be more deeply understood by referring to one or more of these drawings in combination with the detailed description of the specific aspects shown herein.

[0034]

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Mode for Carrying Out the Invention

[0035] Description of Exemplary Embodiments The recent identification of glypican-2 (GPC2) as a cell surface oncoprotein in neuroblastoma, high-grade glioma (HGG), and medulloblastoma has created an opportunity to develop targeted immunotherapies. The inventors hypothesized that chimeric antigen receptor (CAR) T cell therapy against GPC2 could be achieved by using in vitro transcribed RNA or by stably transducing a DNA construct expressing a GPC2-targeted CAR molecule.

[0036] The inventors generated multiple CAR T cell constructs using D3 and D4 GPC2 binders engineered in the heavy and light chain directions. The resulting data indicate that the use of either mRNA or DNA is effective for efficiently designing and testing novel CAR T cells that serve as a platform for clinical trials to determine whether there is evidence of efficacy and to screen for toxicity.

[0037] These and other aspects of the present disclosure are described in further detail below.

[0038] I. Glypican 2 Glypican-2 (GPC2) is a member of the six-member glypican family of heparan sulfate (HS) proteoglycans that are attached to the cell surface by a glycosylphosphatidylinositol (GPI) anchor, and plays diverse roles in growth factor signaling and cancer cell proliferation. GPC2 is also known as cerebroglycan proteoglycan and glypican proteoglycan 2. The GPC2 genomic sequence, mRNA sequence, and protein sequence are publicly available. Furthermore, the mRNA sequence and protein sequence of human glypican 2, for example, NCBI Gene ID221914, accession number NM_l52742, and NP_689955, respectively, can also be found in public databases and are hereby incorporated by reference into this specification. The cell surface GPC2 protein has been shown to be expressed in the developing nervous system, is involved in cell adhesion, and is thought to regulate axon growth and guidance.

[0039] GPC2 has recently been identified as a cell surface protein in several cancers, including pediatric cancers such as neuroblastoma, high-grade glioma (HGG), medulloblastoma, and several other pediatric cancers and adult malignancies. This has created an opportunity to develop new targeted immunotherapies. For example, in pediatric cancers, GPC2 has been shown to be expressed at comparable levels in neuroblastoma, retinoblastoma, and medulloblastoma, whereas its expression in normal tissues was limited. Furthermore, some cases of acute lymphoblastic leukemia, high-grade glioma, and rhabdomyosarcoma express GPC2. GPC2 is also highly expressed on small cell lung cancer, a frequently seen and almost invariably lethal cancer. Additionally, when evaluating GPC2 expression in adult cancers using data provided by The Cancer Genome Atlas (TCGA), a large number of adult malignancies have the potential to benefit from GPC2-targeted immunotherapy. Due to this preferential expression, GPC2 is a potential targeted immunotherapy candidate. GPC2 is present on the cell surface of a large number of pediatric and adult malignancies and shows a high degree of differential expression between tumors and normal tissues.

[0040] II. Preparation of Monoclonal Antibodies A. General Method Antibodies against glypican 2 can be prepared by standard methods as well known in the art (see, for example, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; U.S. Patent No. 4,196,265). The method for preparing monoclonal antibodies (MAb) generally begins along the same lines as the method for preparing polyclonal antibodies. The first step in both of these methods is to immunize a suitable host or to identify a subject that is immune due to previous natural infection. As well known in the art, certain immunizing compositions may differ in terms of immunogenicity. Therefore, it is often necessary to boost the host immune system as achieved by coupling a peptide or polypeptide immunogen to a carrier. Exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such as ovalbumin, mouse serum albumin, or rabbit serum albumin can also be used as carriers. Means for conjugating a polypeptide to a carrier protein are well known in the art and include glutaraldehyde, m-maleimidobenzoyl-N-hydroxysuccinimide ester, carbodiimide, and bis-biazotized benzidine. Also as well known in the art, the immunogenicity of a particular immunogenic composition can be enhanced by using a non-specific stimulator of the immune response known as an adjuvant. Exemplary and preferred adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response containing inactivated Mycobacterium tuberculosis), incomplete Freund's adjuvant, and aluminum hydroxide adjuvant.

[0041] The amount of the immunogenic composition used in the production of polyclonal antibodies varies depending on what the immunogen is and the animal used for immunization. Various routes (subcutaneous, intramuscular, intradermal, intravenous, and intraperitoneal) can be used to administer the immunogen. The production of polyclonal antibodies may be monitored by sampling the blood of the immunized animal at various time points after immunization. A second booster injection may also be given. The process of booster immunization and titer measurement is repeated until an appropriate titer is reached. Once the desired level of immunogenicity is obtained, the immunized animal can be bled, and the serum can be isolated and screened, and / or MAb can be produced using that animal.

[0042] After immunization, somatic cells capable of producing antibodies, specifically B lymphocytes (B cells), are selected for use in the MAb production protocol. These cells may be obtained from the spleen or lymph nodes obtained by biopsy, or from circulating blood. The antibody-producing B lymphocytes derived from the immunized animal are then fused with immortal myeloma cells, generally immortal myeloma cells of the same species as the immunized animal, or immortal myeloma cells of human cells or human / mouse chimeric cells. The myeloma cell line suitable for use in the hybridoma production fusion procedure preferably does not produce antibodies and has an enzyme deficiency that makes it impossible to grow in a specific selection medium, with a high fusion efficiency and supporting the growth of only the desired fused cells (hybridomas).

[0043] As is known to those skilled in the art (Goding, pp. 65-66, 1986; Campbell, pp. 75-83, 1984), any of a number of myeloma cells can be used. For example, if the immunized animal is a mouse, P3-X63 / Ag8, X63-Ag8.653, NS1 / 1.Ag4 1, Sp210-Ag14, FO, NSO / U, MPC-11, MPC11-X45-GTG1.7, and S194 / 5XX0Bul may be used. In the case of rats, R210.RCY3, Y3-Ag1.2.3, IR983F, and 4B210 may be used. With respect to human cell fusions, U-266, GM1500-GRG2, LICR-LON-HMy2, and UC729-6 are all useful. One particular mouse myeloma cell is the NS-1 myeloma cell line (also called P3-NS-1-Ag4-1), which can be readily obtained from the NIGMS Human Genetic Mutant Cell Repository by claiming the cell line repository number GM3573. Another mouse myeloma cell line that can be used is the 8-azaguanine-resistant mouse mouse myeloma SP2 / 0 non-secreting cell line. More recently, additional fusion partner strains for use with human B cells have been described, including KR12 (ATCC CRL-8658; K6H6 / B5 (ATCC CRL-1823 SHM-D33 (ATCC CRL-1668), and HMMA2.5 (Posner et al., 1987). The antibodies in the present disclosure were made using the SP2 / 0 / mIL-6 cell line, which is an IL-6-secreting derivative of the SP2 / 0 strain.

[0044] Methods for producing hybrids of spleen or lymph node cells that produce antibodies and myeloma cells generally involve mixing somatic cells with myeloma cells at a ratio of 2:1, although this ratio may be from about 20:1 to about 1:1, respectively, in the presence of agents (chemical or electrical) that promote cell membrane fusion. A fusion method using Sendai virus has been described by Kohler and Milstein (1975; 1976), and a fusion method using polyethylene glycol (PEG), such as 37% (v / v) PEG, has been described by Gefter et al (1977). The use of electrically induced fusion methods is also suitable (Goding, pp.71-74,1986).

[0045] The fusion procedure generally results in viable hybrids at a low frequency, about 1x10 -6 ~1x10 -8 However, this does not pose a problem since culturing in selective medium allows viable fusion hybrids to differentiate from the injected parental cells (particularly the injected myeloma cells, which usually continue to divide indefinitely). Selective medium is generally a medium containing an agent that blocks de novo nucleotide synthesis in tissue culture medium. Exemplary and preferred agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, while azaserine blocks only purine synthesis. When aminopterin or methotrexate is used, hypoxanthine and thymidine are added to the medium as sources of nucleotides (HAT medium). When azaserine is used, hypoxanthine is added to the medium. If the B cell source is an Epstein-Barr virus (EBV)-transformed human B cell line, ouabain is added to eliminate EBV-transformed strains that have not fused with the myeloma.

[0046] A preferred selection medium is HAT or HAT containing ouabain. In HAT medium, only cells capable of operating the nucleotide salvage pathway can survive. Myeloma cells are deficient in an important enzyme of the salvage pathway, such as hypoxanthine phosphoribosyl transferase (HPRT), and cannot survive. B cells can operate this pathway, but their lifespan is limited in culture and generally die within about two weeks. Therefore, the only cells that can survive in the selection medium are hybrids formed from myeloma cells and B cells. If the source of B cells used for fusion is a strain of B cells transformed with EBV as described herein, ouabain is also used for drug selection of the hybrids because B cells transformed with EBV are sensitive to drug killing. In contrast, the myeloma partner used is selected for ouabain resistance.

[0047] Culturing yields a hybridoma population from which specific hybridomas are selected. Typically, hybridoma selection is performed by culturing cells by single clone dilution in microtiter plates and then testing individual clone supernatants (after about 2 - 3 weeks) for the desired reactivity. This assay must be sensitive, simple, and rapid and includes, for example, radioimmunoassay, enzyme immunoassay, cytotoxicity assay, plaque assay, dot immunobinding assay, etc. 。

[0048] Next, the selected hybridomas are either serially diluted or single cell sorted by flow cytometry sorting and cloned into individual antibody-producing cell lines. The clones can then be grown indefinitely to supply the mAb. These cell lines can be used for MAb production in two basic ways. The hybridoma sample can be injected (often intraperitoneally) into an animal (e.g., a mouse). Optionally, prior to injection, the animal is primed with a hydrocarbon, particularly an oil such as pristane (tetramethylpentadecane). When human hybridomas are used in this way, it is optimal to inject into immunodeficient mice, e.g., SCID mice, to prevent tumor rejection responses. The injected animal develops a tumor that secretes the specific monoclonal antibody produced by the fused cell hybrid. Then, the body fluid of the animal, such as serum or ascites fluid, can be gently tapped to obtain a high concentration of MAb. Individual cell lines can also be cultured in vitro, in which case the MAb is secreted naturally into the culture medium and can be easily obtained at high concentration from the culture medium. Alternatively, human hybridoma cell lines can be used in vitro to produce immunoglobulins in the cell supernatant. This cell line can be adapted to grow in serum-free medium to optimize the ability to recover high-purity human monoclonal immunoglobulins.

[0049] MAbs produced by either means may, if desired, be further purified using filtration, centrifugation, and various chromatography methods, e.g., FPLC or affinity chromatography. Fragments of the monoclonal antibodies of the present disclosure can be obtained from the purified monoclonal antibody by methods involving digestion with enzymes such as pepsin or papain and / or by chemically reducing to cleave disulfide bonds. Alternatively, the monoclonal antibody fragments encompassed by the present disclosure can be synthesized using an automated peptide synthesizer.

[0050] It is also contemplated that a molecular cloning approach may be used to generate monoclonal antibodies. To this end, RNA can be isolated from a hybridoma strain, the antibody gene obtained by RT-PCR, and cloned into an immunoglobulin expression vector. Alternatively, a combinatorial immunoglobulin phagemid library is prepared from RNA isolated from a cell line, and phagemids expressing the appropriate antibody are selected by panning with a viral antigen. The advantages of this approach over traditional hybridoma techniques are that approximately 10 4 times more antibodies can be produced and screened at one time, new specificities are generated by the combination of H and L chains, thereby increasing the likelihood of finding the appropriate antibody even further.

[0051] Other U.S. patents that disclose the production of antibodies useful in the present disclosure include U.S. Patent No. 5,565,332, which describes the production of chimeric antibodies using a combinatorial approach; U.S. Patent No. 4,816,567, which describes recombinant immunoglobulin preparations; and U.S. Patent No. 4,867,973, which describes antibody-therapeutic conjugate, each of which is incorporated herein by reference.

[0052] B. Single-chain / single-domain antibodies A single-chain variable fragment (scFv) is a fusion of the variable regions of the heavy and light chains of an immunoglobulin, linked together with a short (usually serine, glycine) linker. This chimeric molecule, also known as a single-domain antibody, has had its constant region removed and retains the specificity of the original immunoglobulin even with the introduction of a linker peptide. This modification usually leaves the specificity unchanged. These molecules have been created throughout history to facilitate phage display. It is very convenient to express the antigen-binding domain as a single peptide in phage display. Alternatively, scFvs can be created directly from subcloned heavy and light chains derived from hybridomas. Single-domain or single-chain variable fragments lack the constant Fc region found in a complete antibody molecule and thus lack the common binding sites (e.g., protein A / G) used to purify antibodies (single-chain antibodies contain the Fc region). These fragments can often be purified / immobilized using protein L, as protein L interacts with the variable region of the κ light chain.

[0053] Flexible linkers generally consist of amino acid residues that promote helices and amino acid residues that promote turns, such as alanine, serine, and glycine. However, other residues can also function. Phage display can be used as a means to rapidly select linkers tailored to single-chain antibodies (scFvs) from a protein linker library. A random linker library was constructed in which the genes for the heavy-chain variable domain and the light-chain variable domain were linked by segments encoding 18-amino acid polypeptides with different compositions. The scFv repertoire (about 5x10 6Individual different members) were displayed on the filamentous phage and subjected to affinity selection using a hapten. The selected population of variants showed a significant increase in binding activity while retaining considerable sequence diversity. Subsequently, by screening 1054 variants one by one, a catalytically active scFv that was efficiently produced in soluble form was obtained. From sequence analysis, the only common feature of the selected tethers was that there was a conserved proline in the linker behind the 2 C-terminal residues and many arginines and prolines at other positions. H It was revealed that there is a conserved proline in the linker behind the 2 C-terminal residues and many arginines and prolines at other positions.

[0054] The recombinant antibodies of the present disclosure may also be accompanied by sequences or portions that allow dimerization or multimerization of the receptor. Such sequences include those derived from IgA that allow multimer formation together with the J chain. Another multimerization domain is the Gal4 dimerization domain. In other embodiments, these chains may be modified using agents such as biotin / avidin that allow combination of two antibodies.

[0055] In different embodiments, single-chain antibodies can be created by connecting the light and heavy chains of the receptor using non-peptide linkers or chemical units. Generally, the light and heavy chains are produced in different cells, purified, and then linked together in an appropriate manner (i.e., the heavy-chain N-terminus is attached to the light-chain C-terminus via an appropriate chemical crosslink).

[0056] Crosslinking reagents are used to form a molecular crosslink that binds the functional groups of two different molecules, such as stabilizers and coagulants. However, it is intended that dimers or multimers of the same analog or heteromeric complexes composed of different analogs can be created. To sequentially link two different compounds, hetero-bifunctional crosslinking agents that eliminate unnecessary homopolymer formation can be used.

[0057] Exemplary heterobifunctional crosslinkers contain two reactive groups, one that reacts with a primary amine group (e.g., N-hydroxysuccinimide), and the other that reacts with a thiol group (e.g., pyridyldisulfide, maleimide, halogen, etc.). The crosslinker can react with a lysine residue of a protein (e.g., a selected antibody or fragment) via the primary amine reactive group, and the crosslinker that is already bound to the first protein can react with a cysteine residue (free sulfhydryl group) of another protein (e.g., a selected agent) via the thiol reactive group.

[0058] It is preferred that a crosslinker having reasonable stability in blood be used. A very large number of types of disulfide bond-containing linkers are known that can be successfully used to bind a targeting agent and a therapeutic / prophylactic agent. Linkers containing sterically hindered disulfide bonds have sometimes been found to confer high stability in vivo, thus preventing the release of the targeting peptide before reaching the site of action. Therefore, these linkers are one of the groups for linking agents.

[0059] Another crosslinking reagent is SMPT, a bifunctional crosslinker containing a disulfide bond that is "sterically hindered" by adjacent benzene rings and a methyl group. The steric hindrance of the disulfide bond serves the function of protecting the bond from attack by thiolate anions that may be present in tissues and blood, such as glutathione, thereby preventing the conjugate from separating before the attached agent is delivered to the target site.

[0060] Like many other known cross-linking reagents, the SMPT cross-linking reagent confers the ability to cross-link functional groups, such as the SH of cysteine or a primary amine (e.g., the ε-amino group of lysine). Another possible type of cross-linking agent includes heterobifunctional photoreactive phenyl azides containing a cleavable disulfide bond, such as sulfosuccinimidyl-2-(p-azidosalicylamino)ethyl-1,3'-dithiopropionate. The N-hydroxysuccinimidyl group reacts with primary amino groups, and the phenyl azide reacts non-selectively (upon photolysis) with any amino acid residue.

[0061] In addition to the blocked cross-linking agents, unblocked linkers can also be used according to this specification. Other useful cross-linking agents that contain or are thought to generate protected disulfides include SATA, SPDP, and 2-iminothiolane. The use of such cross-linking agents is well understood in the art. Another aspect involves the use of cleavable linkers.

[0062] U.S. Patent No. 4,680,338 describes bifunctional linkers useful for generating conjugates of ligands with amine-containing polymers and / or proteins, and in particular for forming antibody conjugates with chelating agents, drugs, enzymes, detectable labels, etc. U.S. Patent Nos. 5,141,648 and 5,563,250 disclose cleavable conjugates containing labile bonds cleavable under various mild conditions. This linker is particularly useful because the agent of interest can be directly attached to the linker and the active agent is released upon cleavage. Certain applications involve adding free amino or free sulfhydryl groups to proteins, such as antibodies, or drugs.

[0063] U.S. Patent No. 5,856,456 provides a peptide linker for use in connecting polypeptide components for the purpose of making fusion proteins, such as single-chain antibodies. This linker is up to about 50 amino acids in length and is characterized by having at least one occurrence of proline following a charged amino acid (preferably arginine or lysine), and is highly stable and has low aggregation. U.S. Patent No. 5,880,270 discloses aminooxy-containing linkers useful in various immunoassay and separation methods.

[0064] C. Chimeric antigen receptors and nucleic acid sequences encoding the same Artificial T cell receptors (also known as chimeric T cell receptors, chimeric immune receptors, chimeric antigen receptors (CARs)) are engineered receptors that confer any specificity to immune effector cells. Typically, these receptors are used to transplant the specificity of monoclonal antibodies to T cells, and the introduction of the coding sequences of these receptors is facilitated by retroviral vectors. Thus, a large number of cancer-specific T cells can be generated for adoptive cell transfer. Phase I clinical studies of this approach have shown efficacy.

[0065] The most common form of these molecules is a fusion of a single-chain variable fragment (scFv) derived from a monoclonal antibody with the CD3ζ transmembrane and endodomains. Such molecules transmit ζ signals in response to recognition of their target by the scFv. An example of such a construct is 14g2aζ, which is a fusion of an scFv derived from the hybridoma 14g2a (which recognizes the disialoganglioside GD2). When T cells express this molecule (usually achieved by oncoretroviral vector transduction), they recognize and kill target cells expressing GD2 (e.g., neuroblastoma cells). To target malignant B cells, researchers redirected the specificity of T cells using a chimeric immune receptor specific for the B lineage molecule CD19.

[0066] To form the scFv, the variable portions of the immunoglobulin heavy and light chains are fused by a flexible linker. Prior to this scFv, there is a signal peptide (which is cleaved) that directs the nascent protein to the endoplasmic reticulum and then to be expressed on the surface. The flexible spacer allows the scFv to orient in various directions to bind to the antigen. The transmembrane domain is a typical hydrophobic α-helix usually obtained from the original molecule of the signaling end domain that protrudes into the cell and transmits the desired signal.

[0067] Type I proteins are actually two protein domains linked by a transmembrane α-helix. The cell membrane lipid bilayer through which the transmembrane domain passes serves to separate the outer part (ectodomain) from the inner part (endodomain). It is not very surprising that by attaching the ectodomain from one protein to the endodomain of another protein, a molecule is generated that combines the recognition of the former with the signal of the latter.

[0068] Ectodomain The signal peptide directs the nascent protein to the endoplasmic reticulum. This is essential if the receptor is to be glycosylated and anchored to the cell membrane. Usually, any eukaryotic signal peptide sequence will work well. Generally, the signal peptide that is naturally attached to the most amino-terminal component is used (for example, in an scFv with the light-chain-linker-heavy-chain orientation, the natural signal of the light chain is used).

[0069] Usually, the antigen recognition domain is the scFv. However, there are many options. Antigen recognition domains derived from the natural T cell receptor (TCR) α and β single chains have been described. Similarly, simple ectodomains (such as the CD4 ectodomain that recognizes HIV-infected cells) and more unusual recognition components, such as linked cytokines (that recognize cells having cytokine receptors), have also been described. In fact, almost anything that binds to a specific target with high affinity can be used as the antigen recognition region.

[0070] The spacer region links the antigen-binding domain to the transmembrane domain. To facilitate antigen recognition, the spacer region must have sufficient flexibility for the antigen-binding domain to face in various directions. The simplest form is the hinge region derived from IgG1. Alternative options include the CH2CH3 region of immunoglobulins and part of CD3. In the case of most scFv-based constructs, the IgG1 hinge is sufficient. However, the best spacer often has to be determined empirically.

[0071] Transmembrane domain The transmembrane domain is a hydrophobic α-helix that traverses the membrane. Generally, transmembrane domains derived from the most membrane-proximal component of the endodomain are used. Interestingly, when using the CD3ζ transmembrane domain, artificial TCRs may be incorporated into natural TCRs, which are factors dependent on the presence of natural CD3ζ transmembrane charged aspartic acid residues. Different transmembrane domains result in different receptor stabilities. Using the CD28 transmembrane domain gives rise to a stably expressed receptor that is actively expressed.

[0072] Endodomain This is the "business-end" of the receptor. After the antigen is recognized, the receptor clusters and signals are transmitted to the cell. The most commonly used endodomain component is CD3ζ, which contains three ITAMs. This transmits activation signals to T cells after antigen binding. CD3ζ may not supply a sufficiently capable activation signal, and additional co-stimulatory signaling may be required. For example, chimeric CD28 and OX40 may be used together with CD3ζ to transmit proliferation / survival signals, or all three may be used together. [[ID=))

[0073] "First-generation" CARs typically had an intracellular domain derived from the CD3ξ chain, a primary signaling molecule derived from the endogenous TCR. In "second-generation" CARs, intracellular signaling domains derived from various costimulatory protein receptors (e.g., CD28, 41BB, ICOS) were added to the cytoplasmic tail of the CAR to supply additional signals to T cells. From preclinical studies, it has been found that second-generation CAR designs improve the antitumor activity of T cells. More recently, in "third-generation" CARs, multiple signaling domains, such as CD3z-CD28-41BB or CD3z-CD28-OX40, are combined to further enhance efficacy.

[0074] Adoptive transfer of T cells expressing chimeric antigen receptors is a promising anti-cancer therapy. This is because CAR-modified T cells can be engineered to target virtually any tumor-associated antigen. This approach has great potential to greatly improve individualized cancer therapy for patients. After the patient's T cells are collected, they are genetically engineered to express a CAR that is specifically directed against an antigen on the patient's tumor cells and then infused back into the patient. Although adoptive transfer of CAR-modified T cells is a unique and promising cancer therapy, there are significant safety issues. From clinical trials of this therapy, it has become clear that when healthy tissues express the same target antigen as tumor cells, these CARs have potential toxic effects, and as a result, the outcome resembles graft-versus-host disease (GVHD). A potential solution to this problem is to engineer and introduce a suicide gene into the modified T cells. Thus, when a prodrug designed to activate the suicide gene during GVHD is administered, apoptosis is induced in CAR T cells activated by the suicide gene. This method has been safely and effectively used in hematopoietic stem cell transplantation (HSCT). The adoption of suicide gene therapy for the clinical use of CAR-modified T cell adoptive transfer may reduce GVHD while improving overall antitumor efficacy.

[0075] In some aspects of the GPC2-targeted CARs disclosed herein, the VH sequence is operably linked downstream of the VL sequence. In some aspects, the VH sequence is operably linked upstream of the VL sequence. As used herein, the term "upstream" with respect to an amino acid sequence refers to a location distal from a reference point in the direction from the N-terminus to the C-terminus of the amino acid sequence. Similarly, the term "downstream" refers to a location distal from a reference point in the direction from the C-terminus to the N-terminus of the amino acid sequence.

[0076] Generally, a transmembrane domain suitable for the GPC2-targeted CARs disclosed herein can be any one of the transmembrane domains known in the art. Non-limiting examples of suitable transmembrane domains include transmembrane domains derived from the CD28 transmembrane domain, the CD8a transmembrane domain, the CTLA4 transmembrane domain, or the PD-I transmembrane domain. Thus, in some aspects, the GPC2-targeted CARs of the present disclosure include a transmembrane domain derived from the CD28 transmembrane domain, the CD8a transmembrane domain, the CTLA4 transmembrane domain, or the PD-I transmembrane domain. In some aspects, the GPC2-targeted CAR includes a transmembrane domain derived from the CD28 transmembrane domain.

[0077] In some embodiments, the intracellular signaling domain of the GPC2-targeted CAR disclosed herein includes a co-stimulatory domain. Generally, the co-stimulatory domain suitable for the GPC2-targeted CAR disclosed herein may be any one of the co-stimulatory domains known in the art. Examples of suitable co-stimulatory domains include, but are not limited to, co-stimulatory polypeptide sequences derived from 4-1BB (CD137), CD27, CD28, OX40 (CD134), and co-stimulatory inducible T cell co-stimulator (ICOS) polypeptide sequences. Thus, in some embodiments, the co-stimulatory domain of the GPC2-targeted CAR disclosed herein is selected from the group consisting of a co-stimulatory 4-1BB (CD137) polypeptide sequence, a co-stimulatory CD27 polypeptide sequence, a co-stimulatory CD28 polypeptide sequence, a co-stimulatory OX40 (CD134) polypeptide sequence, and a co-stimulatory inducible T cell co-stimulator (ICOS) polypeptide sequence. In some embodiments, the GPC2-targeted CAR includes a co-stimulatory domain derived from a co-stimulatory 4-1BB (CD137) polypeptide sequence. In some embodiments, the GPC2-targeted CAR includes a co-stimulatory domain derived from a co-stimulatory CD28 polypeptide sequence.

[0078] In some embodiments, the GPC2-targeted CAR further comprises an extracellular hinge domain (e.g., a hinge region) or a "linker". The term "hinge domain" generally refers to a flexible polypeptide connecting region or "linker" disposed between the targeting moiety and the transmembrane domain. These sequences are generally derived from IgG subclasses (e.g., IgG1 and IgG4), IgD, and the CD8 domain, among which IgG1 has been most widely used. In some embodiments, the hinge / linker domain provides structural flexibility to adjacent polypeptide regions. The hinge / linker domain may consist of a native polypeptide or a synthetic polypeptide. Those skilled in the art will understand that the hinge / linker domain may improve CAR function by promoting the optimal placement of the antigen-binding portion in relation to the antigen portion recognized by the antigen-binding portion. It is understood that in some embodiments, the hinge / linker domain may not be required for optimal CAR activity. In some embodiments, a beneficial hinge / linker domain comprising a short amino acid sequence promotes CAR activity by promoting antigen binding, e.g., by relaxing any steric constraints that can alter antibody binding kinetics. The sequence encoding the hinge / linker domain may be disposed between the antigen recognition portion and the transmembrane domain. In some embodiments, the hinge / linker domain is operably linked downstream of the antigen-binding portion and upstream of the transmembrane domain.

[0079] The hinge / linker sequence can be derived from any suitable molecule or any part or sequence obtained from any suitable molecule. For example, in some embodiments, the hinge / linker sequence may be derived from a human CD8a molecule or CD28 molecule, and any other receptor that functions similarly in conferring mobility to adjacent regions. The length of the hinge / linker domain can be from about 4 amino acids (aa) to about 50 aa, such as, for example, about 4 aa to about 10 aa, about 10 aa to about 15 aa, about aa to about 20 aa, about 20 aa to about 25 aa, about 25 aa to about 30 aa, about 30 aa to about 40 aa, or about 40 aa to about 50 aa. Suitable hinge / linker domains can be readily selected and can be of any length among a number of suitable lengths, including, for example, 4 aa to 10 aa, 5 aa to 9 aa, 6 aa to 8 aa, or 7 aa to 8 aa, and can be from 1 amino acid (e.g., Gly) to 20 aa, 2 aa to 15 aa, 3 aa to 12 aa, and can be 1 aa, 2 aa, 3 aa, 4 aa, 5 aa, 6 aa, or 7 aa.

[0080] The terms "long linker" and "short linker" are used throughout this application and are intended to refer to the following. "Long linker" amino acid sequence: GGGGSGGGGSGGGGS (SEQ ID NO:4) "Short linker" amino acid sequence: GGGGS (SEQ ID NO:41)

[0081] Non-limiting examples of suitable hinge / linker domains include the CD8 hinge domain, the CD28 hinge domain, the CTLA4 hinge domain, or the IgG4 hinge domain. In some embodiments, the hinge / linker domain may include a region derived from a human CD8a (alias CD8a) molecule or CD28 molecule, and any other receptor that functions similarly in conferring mobility to adjacent regions. In some embodiments, the GPC2-targeted CAR disclosed herein includes a hinge domain derived from the CD8a hinge domain. In some embodiments, the GPC2-targeted CAR disclosed herein includes a hinge domain derived from the CD28 hinge domain.

[0082] In some embodiments, the CARs disclosed herein further include an extracellular spacer domain that includes one or more intervening amino acid residues disposed between the anti-GPC2 scFV region and the extracellular hinge / linker domain. In some embodiments, the extracellular hinge / linker domain is operably linked downstream of the anti-GPC2 scFV region and upstream of the hinge / linker domain. Basically, there are no particular restrictions on the length and / or amino acid composition of the extracellular spacer. In some embodiments, any single-chain peptide containing from about 1 to about 300 amino acid residues (e.g., amino acid residues such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) can be used as the extracellular spacer. In some embodiments, the extracellular spacer contains from about 5 to 50, from about 10 to 60, from about 20 to 70, from about 30 to 80, from about 40 to 90, from about 50 to 100, from about 60 to 120, from about 70 to 150, from about 100 to 200, from about 150 to 250, from about 200 to 300, from about 30 to 60, from about 20 to 80, from about 30 to 90 amino acid residues. In some embodiments, the extracellular spacer contains from about 1 to 10, from about 50 to 100, from about 100 to 150, from about 150 to 200, from about 200 to 300, from about 20 to 80, from about 40 to 120, from about 200 to 250 amino acid residues. In some embodiments, the extracellular hinge / linker contains from about 40 to 70, from about 50 to 80, from about 60 to 80, from about 70 to 90, or from about 80 to 100 amino acid residues. In some embodiments, the extracellular hinge / linker contains from about 1 to 10, from about 5 to 15, from about 10 to 20, from about 15 to 25 amino acid residues. In some embodiments, the extracellular hinge / linker contains about 220, 225, 230, 235, or 240 amino acid residues. In some embodiments, the extracellular hinge / linker contains 229 amino acid residues. In some embodiments, the length and amino acid composition of the extracellular hinge / linker can be optimized to alter the orientation and proximity of the anti-GPC2 scFV region and the extracellular hinge / linker domain relative to each other for the purpose of achieving the desired activity of the GPC2-targeted CAR.In some embodiments, the orientation and proximity of the anti-GPC2 scFV region and the extracellular hinge / linker domain relative to each other can be altered and / or optimized as a "tuning" tool, or as an action to enhance or reduce the potency of the GPC2 CAR. In some embodiments, the orientation and / or proximity of the anti-GPC2 scFV region and the extracellular hinge / linker domain relative to each other can be altered and / or optimized to create a partially functional, or a version of the GPC2 CAR that is partially functional. In some embodiments, the extracellular hinge / linker domain comprises an amino acid sequence corresponding to the IgG4 hinge domain and the IgG4 CH2-CH3 domains.

[0083] In some embodiments, the intracellular signaling domain of the GPC2-targeted CAR disclosed herein comprises a CD3ζ intracellular signaling domain. In some embodiments of the present disclosure, the GPC2-targeted CAR comprises: a) an anti-GPC2 scFv region; b) a CD28 hinge domain; c) a CD28 transmembrane domain; and d) an intracellular signaling domain comprising a co-stimulatory domain derived from the 4-1BBz co-stimulatory domain or the CD28 co-stimulatory domain.

[0084] In one aspect, some embodiments of the present disclosure relate to a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding the GPC2-targeted CAR disclosed herein, or an antibody disclosed herein.

[0085] The terms "nucleic acid molecule" and "polynucleotide" are used interchangeably herein and include both RNA molecules and DNA molecules, including cDNA, genomic DNA, synthetic DNA, and nucleic acid analogs containing DNA or RNA molecules. The nucleic acid molecule may be double-stranded or single-stranded (e.g., sense strand or antisense strand). The nucleic acid molecule may contain nucleotides different from the conventional ones or modified nucleotides. The terms "polynucleotide sequence" and "nucleic acid sequence" as used herein synonymously refer to the sequence of a polynucleotide molecule.

[0086] The nucleic acid molecules of the present disclosure are generally nucleic acid molecules of any length, including nucleic acid molecules from about 5 Kb to about 50 Kb, such as from about 5 Kb to about 40 Kb, from about 5 Kb to about 30 Kb, from about 5 Kb to about 20 Kb, or from about 10 Kb to about 50 Kb, such as from about 15 Kb to 30 Kb, from about 20 Kb to about 50 Kb, from about 20 Kb to about 40 Kb, from about 5 Kb to about 25 Kb, or from about 30 Kb to about 50 Kb.

[0087] In some embodiments, the recombinant nucleic acid molecule is operably linked to a heterologous nucleic acid sequence, such as a structural gene encoding a protein of interest or a regulatory sequence (e.g., a promoter sequence). In some embodiments, the recombinant nucleic acid molecule is further defined as an expression cassette or a vector. In some embodiments, the vector is a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, or a retroviral vector.

[0088] Some embodiments disclosed herein relate to vectors or expression cassettes comprising the recombinant nucleic acid molecules disclosed herein. As used herein, the term "expression cassette" refers to a construct of genetic material containing a coding sequence and regulatory information sufficient to direct proper transcription and / or translation of the coding sequence in recipient cells in vivo and / or ex vivo. The expression cassette may be inserted into a vector for targeting to a desired host cell and / or within a subject. Thus, the term "expression cassette" may be used synonymously with the term "expression construct".

[0089] Chimeric antigen receptor (CAR) antibodies according to the present disclosure may, in a first instance, be defined by binding specificity, in this case binding specificity for glypican 2. The CAR may also be defined by the sequences described herein, or optionally may vary from the sequences provided above using methods discussed in further detail below. For example, the amino acid sequence may (a) optionally have the variable region separated from the constant domain of the light chain, (b) have amino acids that differ from the amino acid sequences shown above but do not thereby dramatically affect the chemical properties of the residues (so-called conservative substitutions), (c) have amino acids that differ from the amino acids shown above by a certain percentage, for example, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology. Alternatively, the nucleic acid encoding the antibody may (a) optionally be separated from the constant domain of the light chain, (b) differ from the nucleic acid shown above but not thereby change the encoded residues, (c) differ from the nucleic acid shown above by a certain percentage, for example, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology, or (d) differ from the nucleic acid shown above by the ability to hybridize under high stringency conditions, as exemplified by low salt and / or high temperature conditions, for example, from about 0.02M to about 0.15M NaCl, at a temperature of about 50°C to about 70°C.

[0090] When making conservative changes to an amino acid sequence, the hydropathy index of the amino acid may be considered. The importance of the hydropathy amino acid index in conferring interactive biological functions to proteins is generally understood in the art (Kyte and Doolittle, 1982). The relative hydropathy characteristics of amino acids contribute to the secondary structure of the resulting protein, and as a result, it is accepted that this defines the interaction of the protein with other molecules, such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc.

[0091] It is also understood in the art that based on hydrophilicity, similar amino acids can be effectively substituted. U.S. Patent No. 4,554,101, which is incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, governed by the hydrophilicity of adjacent amino acids, is correlated with the biological properties of the protein. As detailed in U.S. Patent No. 4,554,101, the following hydrophilicity values are assigned to amino acid residues: basic amino acids: arginine (+3.0), lysine (+3.0), and histidine (-0.5); acidic amino acids: aspartic acid (+3.0 ± 1), glutamic acid (+3.0 ± 1), asparagine (+0.2), and glutamine (+0.2); hydrophilic, non-ionic amino acids: serine (+0.3), asparagine (+0.2), glutamine (+0.2), and threonine (-0.4); sulfur-containing amino acids: cysteine (-1.0) and methionine (-1.3); hydrophobic non-aromatic amino acids: valine (-1.5), leucine (-1.8), isoleucine (-1.8), proline (-0.5 ± 1), alanine (-0.5), and glycine (0); hydrophobic aromatic amino acids: tryptophan (-3.4), phenylalanine (-2.5), and tyrosine (-2.3).

[0092] It is understood that an amino acid can be substituted with another amino acid having similar hydrophilicity to produce a biologically or immunologically modified protein. For such changes, amino acid substitutions with a hydrophilicity value within ±2 are preferred, amino acid substitutions with a hydrophilicity value within ±1 are particularly preferred, and amino acid substitutions within ±0.5 are even more particularly preferred.

[0093] As outlined above, amino acid substitutions generally are based on the relative similarity of the amino acid side chain substituents, for example, hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions taking into account the various foregoing characteristics are well known to those of skill in the art and include arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine.

[0094] D. Expression The nucleic acids according to the present disclosure encode a CAR. The term "nucleic acid encoding a glypican 2 CAR" as used herein refers to a nucleic acid molecule isolated in the absence of all cellular nucleic acids. In certain embodiments, the present disclosure relates to a receptor encoded by any of the sequences shown herein.

[0095] (Table 2) Codon TIFF2025111569000001.tif111138

[0096] The DNA segments of the present disclosure include those that encode biologically functional equivalent proteins of the foregoing sequences. Such sequences may occur as a result of the redundancy of codons and the functional equivalence of amino acids that are known to occur naturally within the nucleic acid sequence and the protein thus encoded. Alternatively, functionally equivalent proteins may be created by applying recombinant DNA techniques. In recombinant DNA techniques, changes in protein structure can be engineered based on the idea that amino acid properties are exchanged. Changes designed by humans may be introduced by applying site-directed mutagenesis methods, as described below, introduced randomly, and later screened for desirable functions.

[0097] Throughout this application, the term "expression construct" is intended to include any type of genetic construct that contains a nucleic acid encoding a gene product, and in which some or all of the nucleic acid coding sequences therein are capable of being transcribed. The transcript may or may not be translated into a protein. In certain embodiments, expression includes transcription of a gene and translation of the mRNA into a gene product. In other embodiments, expression includes only transcription of the nucleic acid encoding the gene of interest.

[0098] The term "vector" is used to refer to a carrier nucleic acid molecule into which a nucleic acid sequence can be inserted for introduction into a replicable cell. The nucleic acid sequence may be "exogenous". "Exogenous" means that the nucleic acid sequence is foreign to the cell into which the vector is introduced or is homologous to an intracellular sequence but is in a position within the host cell nucleic acid where it is not normally found. Vectors include plasmids, cosmids, viruses (bacteriophages, animal viruses, and plant viruses), and artificial chromosomes (e.g., YAC). One of ordinary skill in the art is considered to have sufficient equipment to construct vectors by standard recombinant techniques described in Sambrook et al., (1989) and Ausubel et al., (1994), both of which are incorporated herein by reference.

[0099] The term "expression vector" refers to a vector that contains a transcribable nucleic acid sequence encoding at least a portion of a gene product. In some cases, the RNA molecule is then translated into a protein, polypeptide, or peptide. In other cases, these sequences are not translated, for example, antisense molecules or ribozymes are produced. An expression vector may contain various "control sequences" that are necessary for the transcription of a coding sequence operably linked in a particular host organism and perhaps also for its translation. Vectors and expression vectors may also perform other functions and contain nucleic acid sequences as described below in addition to the control sequences that govern transcription and translation.

[0100] 1. Regulatory element A "promoter" is a control sequence that is a region of a nucleic acid sequence where the initiation and rate of transcription are controlled. A "promoter" may contain genetic elements to which regulatory proteins and regulatory molecules, such as RNA polymerase and other transcription factors, can bind. The phrases "functionally disposed", "functionally linked", "under control", and "under transcriptional control" mean that the promoter is in the correct functional position and / or orientation with respect to the nucleic acid sequence to control the initiation and / or expression of the nucleic acid sequence. A promoter may be used with an "enhancer" or may not be used with an "enhancer". An "enhancer" refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence.

[0101] The promoter may be a promoter that is naturally associated with a gene or sequence, or may likewise be obtained by isolating a 5' non-coding sequence located upstream of the coding segment and / or exon. Such a promoter is sometimes referred to as "endogenous". Similarly, an enhancer may be an enhancer that is naturally associated with a nucleic acid sequence and is located downstream or upstream of the nucleic acid sequence. Alternatively, certain benefits can be obtained by placing the coding nucleic acid segment under the control of a recombinant or heterologous promoter. A recombinant or heterologous promoter refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment.

[0102] A recombinant or heterologous enhancer also refers to an enhancer that is not normally associated with a nucleic acid sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, as well as promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, and promoters or enhancers that are not "natural", i.e., promoters or enhancers that contain different elements of different transcriptional regulatory regions and / or contain mutations that alter expression. In addition to synthesizing the nucleic acid sequences of promoters and enhancers, the sequences may also be made using recombinant cloning techniques and / or nucleic acid amplification techniques including PCR™ (see U.S. Pat. Nos. 4,683,202 and 5,928,906, each incorporated herein by reference). Furthermore, it is contemplated that regulatory sequences that induce transcription and / or expression of the sequences in non-nuclear organelles, such as mitochondria, chloroplasts, etc., can also be used.

[0103] Of course, it is important to use a promoter and / or enhancer that effectively expresses the DNA segment in the cell type, organelle, and organism selected for expression. Those skilled in the art of molecular biology are generally aware of using a combination of a promoter, an enhancer, and a cell type to express a protein. See, for example, Sambrook et al. (1989), which is incorporated herein by reference. The promoter used may be a constitutive promoter, a tissue-specific promoter, an inducible promoter, and / or a promoter useful under conditions appropriate for expressing the introduced DNA segment at a high level, such as, for example, advantageous in large-scale production of recombinant proteins and / or peptides. The promoter may be a heterologous promoter or an endogenous promoter. The identity of tissue-specific promoters or elements and assays for characterizing their activities are well known to those skilled in the art. Examples of such regions include the human LIMK2 gene (Nomoto et al. 1999), the somatostatin receptor 2 gene (Kraus et al., 1998), the mouse testicular retinoic acid-binding gene (Lareyre et al., 1999), human CD4 (Zhao-Emonet et al., 1998), mouse α2(XI) collagen (Tsumaki, et al., 1998), the D1A dopamine receptor gene (Lee, et al., 1997), insulin-like growth factor II (Wu et al., 1997), human platelet endothelial cell adhesion molecule-1 (Almendro et al., 1996).

[0104] Certain specific initiation signals may also be required for efficient translation of the coding sequence. These signals include the ATG initiation codon or adjacent sequences. It may be necessary to provide an exogenous translation control signal containing the ATG initiation codon. One skilled in the art will confirm this and be able to provide the necessary signals. It is well known that in order to ensure translation of the entire insert, the initiation codon must be "in-frame" with the desired coding sequence reading frame. The exogenous translation control signals and initiation codons may be natural or synthetic. The efficiency of expression may be enhanced by including appropriate transcriptional enhancer elements.

[0105] 2.IRES In certain embodiments of the present disclosure, the use of an internal ribosome entry site (IRES) element is employed to create a multi-gene message or a polycistronic message. The IRES element bypasses the ribosome scanning model of 5'-methylated Cap-dependent translation and can initiate translation at an internal site (Pelletier and Sonenberg, 1988). IRES elements derived from two members of the Picornaviridae family (poliovirus and encephalomyocarditis) have been described (Pelletier and Sonenberg, 1988), and IRESs derived from mammalian messages have also been described (Macejak and Sarnow, 1991). The IRES element can be linked to a heterologous open reading frame. Multiple open reading frames separated by IRESs can be transcribed together to create a polycistronic message. The IRES element allows each open reading frame to access the ribosome for efficient translation. Multiple genes can be efficiently expressed using one promoter / enhancer to transcribe one message (see U.S. Patent Nos. 5,925,565 and 5,935,819, which are hereby incorporated by reference in their entireties).

[0106] 3. Multiple Cloning Sites The vector may contain a multiple cloning site (MCS). A multiple cloning site is a nucleic acid region containing a plurality of restriction enzyme sites, each of which can be used with standard recombinant techniques to digest the vector. See Carbonelli et al., 1999, Levenson et al., 1998, and Cocea, 1997, which are incorporated herein by reference. "Restriction enzyme digestion" refers to the catalytic cleavage of a nucleic acid molecule by an enzyme that functions only at specific positions in the nucleic acid molecule. Many of these restriction enzymes are commercially available. The use of such enzymes is widely understood by those skilled in the art. Often, the vector is linearized or fragmented using a restriction enzyme that cleaves within the MCS so that an exogenous sequence can be ligated to the vector. "Ligation" refers to the process of forming a phosphodiester bond between two nucleic acid fragments, which may or may not be contiguous with each other. Techniques involving restriction enzymes and ligation reactions are well known to those skilled in recombinant techniques.

[0107] 4. Splicing Sites Most transcribed eukaryotic RNA molecules undergo RNA splicing to remove introns from the primary transcript. In order for the transcript to be correctly processed for protein expression, vectors containing genomic eukaryotic sequences may require donor splicing sites and / or acceptor splicing sites (see Chandler et al., 1997, which is incorporated herein by reference).

[0108] 5. Termination Signals The vectors or constructs of the present disclosure generally include at least one termination signal. A "termination signal" or "terminator" is composed of a DNA sequence involved in the specific termination of an RNA transcript by RNA polymerase. Thus, in certain embodiments, a termination signal that terminates the production of an RNA transcript is contemplated. A terminator may be necessary in vivo to achieve the desired message level.

[0109] In eukaryotic systems, the terminator region may also include specific DNA sequences that enable site-specific cleavage of the new transcript to expose the polyadenylation site. This signals a special endogenous polymerase to add a sequence of approximately 200 A residues (polyA) to the 3' end of the transcript. The RNA molecule modified with this polyA tail appears to be highly stable and is translated more efficiently. Thus, in other embodiments involving eukaryotes, it is preferred that the terminator include a signal for RNA cleavage, and more preferably the terminator signal promotes polyadenylation of the message. The terminator and / or polyadenylation site elements may serve to enhance the message level and / or minimize read-through from the cassette to other sequences.

[0110] Terminators intended for use in the present disclosure include any known transcriptional terminator described herein or known to those of skill in the art. This includes, for example, but is not limited to, gene termination sequences such as the bovine growth hormone terminator, or viral termination sequences such as the SV40 terminator. In certain embodiments, the termination signal may be one lacking a transcribable or translatable sequence, for example, one lacking a transcribable or translatable sequence due to the sequence being truncated.

[0111] 6. Polyadenylation signal In expression, particularly in eukaryotic expression, a polyadenylation signal is typically included to effect proper polyadenylation of the transcript. What the polyadenylation signal is has not been considered critical to the success of the practice of the present disclosure, and / or any such sequence can be used. Preferred embodiments include the SV40 polyadenylation signal and / or the bovine growth hormone polyadenylation signal, which are convenient and / or known to function well in a variety of target cells. Polyadenylation may increase the stability of the transcript and may facilitate transport to the cytoplasm.

[0112] 7. Replication origin To propagate the vector in a host cell, the vector may contain one or more replication origin sites (often referred to as "ori"). The replication origin is a specific nucleic acid sequence at which replication starts. Alternatively, if the host cell is yeast, an autonomously replicating sequence (ARS) can be used.

[0113] 8. Selection markers and screening markers In certain embodiments of the present disclosure, cells containing the nucleic acid constructs of the present disclosure can be identified in vitro or in vivo by including a marker in the expression vector. Such markers confer an identifiable change to the cells, thereby enabling easy identification of cells containing the expression vector. Generally, a selection marker confers a property that enables selection. A positive selection marker enables the selection of cells when the marker is present, whereas a negative selection marker prevents the selection of cells when the marker is present. An example of a positive selection marker is a drug resistance marker.

[0114] Typically, the inclusion of a drug selection marker aids in the cloning and identification of transformants. For example, genes conferring resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin, and histidinol are useful selection markers. In addition to markers that confer a phenotype allowing for the discrimination of transformants based on conditional implementation, other types of markers are contemplated, including screening markers such as GFP, which is based on colorimetric analysis. Alternatively, screenable enzymes such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) can be used. Those skilled in the art will likely also know methods of using immuno - markers, perhaps in conjunction with FACS analysis. The marker used is not considered important as long as it can be co - expressed with the nucleic acid encoding the gene product. Further examples of selection and screening markers are well - known to those skilled in the art.

[0115] 9. Viral Vector Because certain viral vectors can efficiently infect cells, enter, and integrate into the host cell genome, enabling stable expression of viral genes, many different viral vector systems have been developed and applied (Robbins et al., 1998). Viral systems for use as vectors for ex vivo gene transfer and in vivo gene transfer are currently being developed. For example, adenoviruses, herpes simplex viruses, retroviruses, and adeno-associated viral vectors are currently being evaluated for treating diseases such as cancer, cystic fibrosis, Gaucher's disease, kidney disease, and arthritis (Robbins and Ghivizzani, 1998; Imai et al., 1998; U.S. Patent No. 5,670,488). For use in the present disclosure, other viral vectors are contemplated, such as poxviruses; for example, vaccinia virus (Gnant et al., 1999; Gnant et al., 1999), alphaviruses; for example, Sindbis virus, Semliki Forest virus (Lundstrom, 1999), reoviruses (Coffey et al., 1998), and influenza A virus (Neumann et al., 1999), and can be selected according to the required properties of the system of interest.

[0116] 10. Non-viral transformation For use with the present disclosure, methods suitable for nucleic acid delivery to transform organelles, cells, tissues, or organisms include substantially any method by which a nucleic acid (e.g., DNA) can be introduced into an organelle, cell, tissue, or organism as described herein or as known to those of skill in the art. Such methods include, for example, injection, including microinjection (Harland and Weintraub, 1985, incorporated herein by reference; U.S. Patent No. 5,789,215), (U.S. Patent Nos. 5,994,624, 5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466, and 5,580,859, each incorporated herein by reference); electroporation (U.S. Patent No. 5,384,253, incorporated herein by reference); calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990); use of polyethylene glycol following DEAE-dextran (Gopal, 1985); direct sonic loading (Fechheimer et al., 1987); transfection via liposomes (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987; Wong et al., 1980; Kaneda et al., 1989; Kato et al., 1991); particle guns (PCT Application Nos. WO94 / 09699 and 95 / 06128; U.S. Patent Nos. 5,610,042; 5,322,783, 5,563,055, 5,550,318, 5,538,877, and 5,538,880, each incorporated herein by reference); agitation with silicon carbide fibers (Kaeppler et al., 1990; U.S. Patent Nos. 5,302,523 and 5,464,765.(each incorporated herein by reference); or protoplast transformation via PEG (Omirulleh et al., 1993; U.S. Pat. Nos. 4,684,611 and 4,952,500, each incorporated herein by reference); direct delivery of DNA by DNA uptake via drying / inhibition (Potrykus et al., 1985), among others. By applying techniques such as these, organelles, cells, tissues, or organisms may be stably transformed or transiently transformed.

[0117] 11. Expression Systems There are a great many expression systems that include at least a portion or all of the compositions discussed above. Prokaryotic and / or eukaryotic-based systems can be used to generate nucleic acid sequences, or their cognate polypeptides, proteins, and peptides, in conjunction with the present disclosure. Many such systems are commercially and widely available.

[0118] The insect cell / baculovirus system can produce high-level protein expression of heterologous nucleic acid segments, as described, for example, in U.S. Pat. Nos. 5,871,986 and 4,879,236, each incorporated herein by reference, and can be purchased, for example, under the name MaxBac® 2.0 from Invitrogen® or under the name BacPack™ baculovirus expression system from Clontech®.

[0119] Other examples of expression systems include the Stratagene® Complete Control™ inducible mammalian expression system, which includes a synthetic exosome-inducible receptor, or its pET expression system, which is an E. coli expression system. Another example of an inducible expression system available from Invitrogen® is the T-Rex™ (tetracycline-regulated expression) system, an inducible mammalian expression system that uses a full-length CMV promoter. Invitrogen® also offers a yeast expression system called the Pichia methanolica expression system. This system is designed to produce high levels of recombinant protein in the methylotrophic yeast Pichia methanolica. One of ordinary skill in the art will know how to express vectors, such as expression constructs, to produce nucleic acid sequences or their cognate polypeptides, proteins, or peptides.

[0120] Primary mammalian cell cultures can be prepared in a variety of ways. In order for cells to survive in vitro and in contact with the expression construct, it is necessary to ensure that they maintain contact with the correct ratios of oxygen, carbon dioxide, and nutrients and are protected from microbial contamination. Cell culture methods are well documented.

[0121] One aspect described above involves immortalizing cells using gene transfer to produce a protein. The gene for the protein of interest can be introduced into an appropriate host cell as described above, and then the cells can be cultured under appropriate conditions. Thus, genes for substantially any polypeptide can be used. The construction of recombinant expression vectors and the elements contained therein were discussed above. Alternatively, the protein to be produced may be an endogenous protein that the cell in question normally synthesizes.

[0122] Examples of useful mammalian host cell lines include Vero cells, HeLa cells, and Chinese hamster ovary cell lines, W138, BHK, COS-7, 293, HepG2, NIH3T3, RIN, and MDCK cells. Further, a host cell line that modulates the expression of the inserted sequence, or a host cell line that modifies or processes the gene product in a desired manner may be selected. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of the protein product can be important for the function of the protein. Various host cells have characteristic and specific mechanisms for post-translational processing and post-translational modification of proteins. Selecting an appropriate cell line or host system can ensure the correct modification and processing of the expressed foreign protein.

[0123] Many selectable systems, including but not limited to the HSV thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase, and adenine phosphoribosyltransferase genes, can be used in tk− cells, hgprt− cells, or aprt− cells, respectively. Also, metabolic antagonist resistance can be used as a basis for selection against dhfr, which confers resistance to methotrexate, gpt, which confers resistance to mycophenolic acid, neo, which confers resistance to the aminoglycoside G418, and hygro, which confers resistance to hygromycin.

[0124] III. Pharmaceutical Formulations and Treatment of Cancer A. Cancer Cancer results from the proliferation of a clonal population of cells derived from a tissue. The development of cancer, called carcinogenesis, can be modeled and characterized in many ways. A link between cancer development and inflammation has long been recognized. The inflammatory response is involved in host defense against microbial infection and also serves as a driving force for tissue repair and regeneration. Considerable evidence has pointed to a relationship between inflammation and cancer development risk, i.e., that chronic inflammation can cause dysplasia.

[0125] Cancer cells to which the methods of the present disclosure can be applied generally include any cells that express glypican 2, and more particularly, any cells that overexpress glypican 2. Cancer cells that can be treated according to the present disclosure include, but are not limited to, cells derived from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal tract, gingiva, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, pancreas, testis, tongue, cervix, or uterus. Further, cancers specifically include cancers of the following histological types: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; carcinoma of giant and spindle cells; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; bronchioloalveolar adenocarcinoma; papillary adenocarcinoma; chromophobic carcinoma; eosinophilic carcinoma; eosinophilic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary adenocarcinoma and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenocortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous gland carcinoma; ceruminous gland adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease, breast; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w / squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; cystadenoma, malignant; granulosa cell tumor, malignant; androblastoma, malignant; sertoli cell tumor; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; chromaffin cell tumor; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant congenital nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; fetal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant;Muellerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymal tumor, malignant; Brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; undifferentiated embryonal cell tumor; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; angiosarcoma; vascular endothelioma, malignant; Kaposi sarcoma; perivascular cell tumor, malignant; lymphangiosarcoma; osteosarcoma; parosteal osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing sarcoma; odontogenic tumor, malignant; ameloblastic odontogenic sarcoma; ameloblastic epithelioma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; epithelioma; astrocytoma; protoplasmic astrocytoma; fibrous astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroglioblastoma; undifferentiated neuroectodermal; cerebellar sarcoma; ganglioblastoma; neuroblastoma; retinoblastoma; olfactory nerve tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin disease; side granuloma; malignant lymphoma, small lymphocyte; malignant lymphoma, diffuse large cell; malignant lymphoma, follicular; fungating polypoid tumor; other non-Hodgkin lymphoma as specified; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphocytic leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myelosarcoma; and hairy cell leukemia, but not limited thereto. In certain aspects, the tumor may include osteosarcoma, angiosarcoma, rhabdomyosarcoma, leiomyosarcoma, Ewing sarcoma, glioblastoma, medulloblastoma, neuroblastoma, or leukemia.;

[0126] Furthermore, the methods of the present disclosure can be applied to a wide range of species, such as humans, non-human primates (e.g., monkeys, baboons, or chimpanzees), horses, cows, pigs, sheep, goats, dogs, cats, rabbits, guinea pigs, gerbils, hamsters, rats, and mice. The cancer can also be recurrent, metastatic, and / or multi-drug resistant. The methods of the present disclosure can be applied to such cancers, particularly to make such cancers resectable, to extend or re-induce remission, to inhibit angiogenesis, to prevent or limit metastasis, and / or to treat multi-drug resistant cancers. At the cellular level, this can result in the death of cancer cells, inhibition of cancer cell proliferation, or otherwise the reversal or reduction of the malignant phenotype of tumor cells.

[0127] B. Formulations and Administration The present disclosure provides pharmaceutical compositions comprising an anti-glypican 2 receptor and cells expressing the same. In certain embodiments, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a State government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeias for use in animals and, more particularly, in humans. The term "carrier" refers to a diluent, excipient, or vehicle with which the therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, mineral oil, sesame oil and the like. Other suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, sodium chloride, dextrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, water, ethanol, and the like.

[0128] The composition may be formulated in neutral or salt form. Pharmaceutically acceptable salts include salts formed using anions such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and salts formed using cations such as those derived from sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0129] The receptors, nucleic acids, and cells of the present disclosure may include classical pharmaceutical preparations. Administration of these compositions according to the present disclosure is effected via any general route as long as the target tissue is accessible via that route. Such routes include oral, nasal, buccal, rectal, vaginal, or topical routes. Alternatively, administration may be by intradermal, subcutaneous, intramuscular, intraperitoneal, or intravenous injection. Such compositions will typically be administered as the pharmaceutically acceptable compositions described above. Particular interest lies in administration into, perfusion of, or topical or local administration to a tumor, e.g., to local or regional vasculature or lymphatics, or to an excised tumor bed.

[0130] The active compound may also be administered parenterally or intraperitoneally. Solutions of the active compound, which is a free base or a pharmaceutically acceptable salt, can be prepared by dissolving it in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared by dissolving in glycerol, liquid polyethylene glycols, and mixtures thereof, as well as in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0131] C. Combination Therapy In the context of the present disclosure, it is also contemplated that the anti-glypican 2 CAR T cells described herein can be used equally well in conjunction with chemotherapy intervention or radiation therapy intervention or other treatments. In particular, it may be found effective to combine the anti-glypican 2 CAR T cells with other therapies targeting different aspects of glypican 2 function.

[0132] [[ID = 4]]To kill cells, inhibit cell proliferation, inhibit metastasis, inhibit angiogenesis, otherwise reverse or reduce the malignant phenotype of tumor cells using the methods and compositions of the present disclosure, generally, the "target" cells will be contacted with anti-glypican 2 CAR T cells according to the present disclosure and at least one other agent. These compositions will be provided in a combined amount effective to kill cells or to inhibit cell proliferation. This process may involve contacting the cells simultaneously with anti-glypican 2 CAR T cells according to the present disclosure and other agents or factors. This may be achieved by contacting the cells with one composition or pharmaceutical formulation containing both agents, or by contacting the cells simultaneously with two separate compositions or formulations, one composition containing anti-glypican 2 CAR T cells according to the present disclosure and the other composition containing the other agent.

[0133] Alternatively, the anti-glypican 2 CAR T cell therapy may be administered before or after other pharmaceutical treatments at intervals ranging from several minutes to several weeks. In embodiments where the other pharmaceutical agent and the anti-glypican 2 CAR T cells are applied to the cells separately, generally, the effective period will not end between each delivery so that the pharmaceutical agent and the expression construct can still exert an advantageously combined effect on the cells. In such cases, it is contemplated that the cells are contacted with both modalities within about 12 to 24 hours, more preferably within about 6 to 12 hours, and most preferably within about 12 hours after the administration of either modality. Depending on the situation, it may be desirable to significantly extend the period for treatment. However, the period between each administration is from several days (2, 3, 4, 5, 6, or 7 days) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8 weeks).

[0134] It is also contemplated that it may be desirable to administer either the anti-glypican 2 CAR T cells or the other pharmaceutical agent multiple times. As illustrated below, various combinations can be used. In the formula, the anti-glypican 2 CAR T cells according to the present disclosure are "A" and the other therapy is "B". TIFF2025111569000002.tif17128

[0135] Other combinations are also contemplated. Again, both agents are delivered to the cells in a combined amount effective to kill the cells, for killing the cells. Agents or factors suitable for cancer therapy include any compound or method of treatment that induces damage when applied to cells. Such agents and factors include radiation and waves that induce DNA damage, such as radiation, microwaves, electroluminescence, etc. Various compounds, sometimes also referred to as "chemotherapeutic agents" or "genotoxic agents", may be used. This may be accomplished by irradiating the local tumor site with radiation. Alternatively, the tumor cells may be contacted with the agent by administering a therapeutically effective amount of the pharmaceutical composition to the subject. The combination therapy may also include surgery. Various ways of these therapies are discussed below.

[0136] 1. Chemotherapy The term "chemotherapy" refers to the use of drugs for treating cancer. The term "chemotherapeutic agent" is used to imply a compound or composition administered in the treatment of cancer. These agents or drugs are classified by their mechanism of action within the cell, e.g., whether they affect the cell cycle and at which stage they affect the cell cycle. Alternatively, agents may be characterized based on their ability to directly crosslink DNA, insert into DNA, or induce chromosomal and mitotic abnormalities by affecting nucleic acid synthesis. Most chemotherapeutic agents fall into the following categories: alkylating agents, antimetabolites, antitumor antibiotics, mitotic inhibitors, and nitrosoureas.

[0137] Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carbocone, meturedopa, and uredopa; altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamime, including ethyleneimine and methylamelamine; acetogenins (especially bullatacin and bullatacinone); camptothecin (including the synthetic analog topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogs); cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chloronaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin γ1I and calicheamicin ωI1; dynemicin including dynemicin A; uncialamycin and its derivatives); bisphosphonates such as clodronate; esperamicin;and neocarzinostatin chromophore and related chromoprotein engyin antibiotics chromophore, actinomycin, actinomycin, authrarnycin, azaserine, bleomycin, cactinomycin, carabicin, caminomycin, cardinophyllin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, for example, mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptozocin, streptozocin, tubercidin, ubenimex, dinostatin, or zorubicin; antimetabolites, for example, methotrexate and 5-fluorouracil (5-FU); folic acid analogs, for example, denopterin, methotrexate, pteropterin, trimetrexate; purine analogs, for example, fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs, for example, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, didoxyruridine, doxifluridine, enocitabine, floxuridine; androgens, for example, calusterone, drostanolone propionate, epithiostanol, mepitiostane, testolactone; antiadrenal agents, for example, aminoglutethimide, mitotane, trilostane; folic acid supplements, for example, folic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demeclocycline; diaziquone; elformithine; elliptinium acetate; epsilon; etoglucid; gallium nitrate;Hydroxyurea; Lentinan; Lonidamine; Maytansinoids, such as maytansine and ansamitocin; Mitoguazone; Mitoxantrone; Mopidanmol; Nitraerine; Pentostatin; Phenamet; Pirarubicin; Losoxantrone; Podophyllinic acid; 2-Ethylhydrazide; Procarbazine; PSK polysaccharide complex; Razoxane; Rhizoxin; Sizofiran; Spirogermanium; Tenuazonic acid; Triaziquone; 2,2',2''-Trichlorotriethylamine; Trichothecenes (especially, T-2 toxin, verracurin A, roridin A, and anguidine); Urethane; Vindesine; Dacarbazine; Mannomustine; Mitobronitol; Mitolactol; Pipobroman; Gacytosine; Arabinoside ("Ara-C"); Cyclophosphamide; Thiotepa; Taxoids, such as paclitaxel and docetaxel; Chlorambucil; Gemcitabine; 6-Thioguanine; Mercaptopurine; Methotrexate; Platinum coordination complexes, such as cisplatin, oxaliplatin, and carboplatin; Vinblastine; Platinum; Etoposide (VP-16); Ifosfamide; Mitoxantrone; Vincristine; Vinorelbine; Novantrone; Teniposide; Edatrexate; Daunomycin; Aminopterin; Zeloda; Ibandronate; Irinotecan (e.g., CPT-11); Topoisomerase inhibitor RFS 2000; Difluoromethylornithine (DMFO); Retinoids, such as retinoic acid; Capecitabine;Cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosourea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP16), tamoxifen, raloxifene, estrogen receptor binders, taxol, paclitaxel, docetaxel, gemcitabien, navelbine, farnesyl-protein transferase inhibitors, transplatinum, 5-fluorouracil, vincristine, vinblastine, and methotrexate, and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing are included.;

[0138] 2. Radiation Therapy Radiation therapy, also called radiotherapy, is the treatment of cancer and other diseases using ionizing radiation. Ionizing radiation deposits energy that damages or destroys cells in the area being treated by damaging the cells' genetic material, so that these cells can no longer continue to grow. Radiation damages both cancer cells and normal cells, but the latter can repair themselves and function properly on their own.

[0139] Radiation therapy used in accordance with the present disclosure may include, but is not limited to, the use of gamma rays, X-rays, and / or the directed delivery of radioisotopes to tumor cells. Other types of DNA damaging agents such as microwaves and UV radiation are also contemplated. It is almost certain that all of these agents induce extensive damage to DNA, DNA precursors, DNA replication and repair, and chromosome construction and maintenance. The radiation dose range of X-rays ranges from a daily dose of 50 to 200 roentgens for a long period (3 to 4 weeks) to a single dose of 2000 to 6000 roentgens. The radiation dose range of radioisotopes varies and depends on the half-life of the isotope, the intensity and type of radiation emitted, and the uptake by newly formed cells.

[0140] Radiation therapy may involve the use of radiolabeled antibodies to deliver a certain dose of radiation directly to the cancer site (radioimmunotherapy). Antibodies are highly specific proteins that the body makes in response to the presence of an antigen (a substance that the immune system recognizes as foreign). Some tumor cells contain specific antigens that induce the production of tumor-specific antibodies. Large amounts of these antibodies can be produced in the laboratory and attached to radioactive substances (a process known as radiolabeling). Once injected into the body, the antibodies actively seek out cancer cells, which are then destroyed by the cell-killing (cytotoxic) effects of the radiation. Using this approach, the risk of radiation damage to healthy cells can be minimized.

[0141] In the case of conformal radiation therapy, a linear accelerator, which is the same radiation therapy device as used in normal radiation therapy procedures, is used, and metal blocks are placed in the path of the x-ray beam to change the shape of the x-ray beam to match the shape of the cancer. This ensures that a high radiation dose is definitely delivered to the tumor. A low dose of radiation is delivered to healthy surrounding cells and nearby structures, so the potential for side effects is reduced. A device called a multi-leaf collimator has been developed and may be used in place of the metal blocks. The multi-leaf collimator consists of a number of metal sheets fixed to the linear accelerator. Each layer can be adjusted so that the radiation therapy beam fits exactly into the treatment area without the need for metal blocks. The precise positioning of the radiation therapy device is very important in conformal radiation therapy procedures, and a special scanning device may be used to check the position of the internal organs at the start of each treatment.

[0142] High-resolution intensity modulated radiotherapy also uses a multi-leaf collimator. During this treatment, the layers of the multi-leaf collimator are moved while the treatment is being carried out. This method is likely to make the treatment beam fit more precisely and enable the radiation therapy dose to be uniform across the entire treatment area.

[0143] Research studies have shown that conformal radiation therapy and intensity-modulated radiation therapy may reduce the side effects of radiation therapy treatment, but by targeting the treatment area so precisely, tiny cancer cells just outside the treatment area may not be destroyed. This means that these specialized radiation therapy techniques may increase the risk of cancer recurrence in the future.

[0144] Scientists are also looking for ways to improve the effectiveness of radiation therapy. Two types of investigational drugs are being studied for their effect on cells receiving radiation: radiosensitizers, which make tumor cells more likely to be damaged, and radioprotectors, which protect normal tissue from the effects of radiation. Hyperthermia, the use of heat, is also being studied for its effectiveness in increasing tissue sensitivity to radiation.

[0145] 3. Immunotherapy In the context of cancer treatment, immunotherapy generally relies on the use of immune effector cells and molecules to target and destroy cancer cells. An example of this is trastuzumab (Herceptin™). The immune effector may be, for example, an antibody specific for some marker on the surface of tumor cells. The antibody may act alone as the therapeutic effector or may recruit other cells that actually affect cell killing. Antibodies may also be conjugated to drugs or toxins (e.g., chemotherapeutic agents, radionuclides, ricin A chain, cholera toxin, pertussis toxin, etc.) and act solely as targeting agents. Alternatively, the effector may be a lymphocyte carrying a surface molecule that interacts directly or indirectly with tumor cell targets. Various effector cells include cytotoxic T cells and NK cells. The use of a combination of therapeutic modalities, i.e., direct cytotoxic activity and ErbB2 inhibition or reduction, may provide therapeutic benefit in the treatment of ErbB2-overexpressing cancers.

[0146] In one aspect of immunotherapy, tumor cells should have some marker that is the target of targeting, i.e., that is not present in the majority of other cells. There are many tumor markers, and any of these may be suitable for targeting in the context of the present disclosure. Common tumor markers include carcinoembryonic antigen, prostate specific antigen, urinary tumor associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialyl Lewis antigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb B, and p155. Another aspect of immunotherapy is the combination of an anti-cancer effect and an immune-stimulating effect. There are also immune-stimulating molecules including cytokines such as IL-2, IL-4, IL-12, GM-CSF, γ-IFN, chemokines such as MIP-1, MCP-1, IL-8, and growth factors such as FLT3 ligand. The combined use of an immune-stimulating molecule as a protein or an immune-stimulating molecule using gene delivery and a tumor suppressor has been shown to enhance the anti-tumor effect (Ju et al., 2000). Further, antibodies against any of these compounds may be used to target the anti-cancer agents discussed herein.

[0147] Examples of immunotherapies that are currently being studied or used include immunoadjuvants such as Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds (U.S. Pat. Nos. 5,801,005 and 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al., 1998), cytokine therapies such as interferon α, β, and γ; IL-1, GM-CSF, and TNF (Bukowski et al., 1998; Davidson et al., 1998; Hellstrand et al., 1998), gene therapies such as TNF, IL-1, IL-2, p53 (Qin et al., 1998; Austin-Ward and Villaseca, 1998; U.S. Pat. Nos. 5,830,880 and 5,846,945), and monoclonal antibodies such as anti-ganglioside GM2, anti-HER-2, anti-p185 (Pietras et al., 1998; Hanibuchi et al., 1998; U.S. Pat. No. 5,824,311). It is contemplated that one or more anti-cancer therapies may be used in combination with the gene silencing therapy described herein.

[0148] In active immunotherapy, antigenic peptides, polypeptides, or proteins, or autologous or allogeneic tumor cell compositions, i.e., "vaccines", are generally administered together with a separate bacterial adjuvant (Ravindranath and Morton, 1991; Morton et al., 1992; Mitchell et al., 1990; Mitchell et al., 1993).

[0149] In adoptive immunotherapy, the patient's circulating lymphocytes or tumor-infiltrating lymphocytes are isolated in vitro, activated by lymphokines such as IL-2, or genes for tumor necrosis are introduced and readministered (Rosenberg et al., 1988; 1989).

[0150] 4. Surgery Approximately 60% of people with cancer undergo some type of surgery. Surgery includes prophylactic surgery, surgery for diagnosis or staging, curative surgery, and palliative surgery. Curative surgery is a cancer treatment that can be used in combination with other therapies such as the treatments of the present disclosure, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapies.

[0151] Curative surgery includes excisions in which all or part of the cancerous tissue is physically removed, excised, and / or destroyed. Tumor resection refers to the physical removal of at least part of the tumor. In addition to tumor resection, surgical procedures include laser surgery, cryosurgery, electrocautery, and microsurgically controlled surgery (Mohs surgery). Further, the present disclosure is intended to be used with the removal of superficial cancers, precancers, or incidental amounts of normal tissue.

[0152] A cavity may be formed in the body when removing some or all of the cancer cells, cancerous tissue, or tumor. The treatment may be achieved by perfusing, directly injecting, or topically applying additional anti-cancer therapies to this location. Such treatments may be repeated, for example, daily, every two days, every three days, every four days, every five days, every six days, or every seven days, or every week, every two weeks, every three weeks, every four weeks, and every five weeks, or every month, every two months, every three months, every four months, every five months, every six months, every seven months, every eight months, every nine months, every ten months, every eleven months, or every twelve months. These treatments may also be treatments with different dosages.

[0153] In certain embodiments, adjuvant treatment with the compounds of the present disclosure is believed to be particularly effective in reducing tumor recurrence after tumor removal. Further, the compounds of the present disclosure can also be used in the neoadjuvant setting.

[0154] It should also be pointed out that any of the foregoing therapies may be useful alone in cancer treatment. Those skilled in the art are directed to "Remington's Pharmaceutical Sciences," 15th Edition, Chapter 33, specifically pages 624 - 652. There will necessarily be some variation in dosage depending on the condition of the subject being treated. The person administering the treatment will determine the dosage appropriate for each individual subject, whatever the circumstances. Further, in the case of human administration, the preparation must meet the standards of sterility, pyrogenicity, general safety, and purity as required by the standards of the FDA's Office of Biologics.

[0155] IV. Kits In yet a further aspect, a kit for use with the above-described method is provided. Thus, the kit comprises, within suitable container means, a CAR, a nucleic acid encoding the CAR, and cells expressing a first CAR that binds to the glypican 2 antigen.

[0156] The container means of the kit generally comprises at least one vial, test tube, flask, bottle, syringe, or other container means, within which the cells may be disposed, preferably appropriately dispensed. The kit also comprises means for containing the CAR, nucleic acid, or cells, and other reagents, which are tightly enclosed for commercial sale. Such containers may comprise injection-molded plastic containers or blow-molded plastic containers that hold the desired vials.

Examples

[0157] V. Examples The following examples are included to demonstrate preferred embodiments. The techniques disclosed in the following examples show that the techniques discovered by the inventors function well enough in the implementation of the embodiments and should thus be understood by those skilled in the art to constitute preferred embodiments for carrying out the present invention. However, in view of the present disclosure, those skilled in the art will understand that many changes can be made to the specific embodiments disclosed without departing from the spirit and scope of the present disclosure and still obtain similar or comparable results.

[0158] Example 1 A panel of three fully human antibodies (m201, m202, and m203) that specifically target cancer cell-related GPC2 was isolated from a phage display antibody library and affinity matured. In vitro characterization demonstrated that these antibodies have promising therapeutic activities for use in the development of CAR-Ts, antibody-drug conjugates (ADCs), and bispecific antibodies for cancer therapy. The sequences of the antibodies are shown in FIGS. 1-3.

[0159] GPC2 has recently been identified as a novel cancer gene and as an immunotherapy target in neuroblastoma and medulloblastoma. The inventors generated multiple different RNA CAR constructs using GPC2-specific scFvs paired with 4-1BB and CD3ζ co-stimulatory domains, with different heavy and light chain orientations and different linker lengths between the chains. The inventors evaluated CAR persistence, T cell exhaustion markers, and cytotoxicity against four primary neuroblastoma cell lines, two isogenic neuroblastoma cell lines, and three primary HGG cell lines. All four constructs showed >80% CAR expression and GPC2-specific binding by flow cytometry. CAR molecules with a light chain-heavy chain (VL-VH) arrangement showed persistence on the surface over 7 days and increased cytotoxicity compared to a heavy chain-light chain (VH-VL) arrangement. The VH-VL arrangement with a long linker had the weakest cytotoxic effect. Evaluation of negative checkpoint regulators revealed the highest PD1 and Lag3 expression (62% vs 17-40% of other constructs, p<0.0001). Based on in vitro data, two VL-VH CAR constructs were selected for testing in a murine flank neuroblastoma model treated weekly with IV GPC2 CAR T cells, three times. On day 14, animals treated with both VL-VH CAR constructs had less tumor burden compared to CD19 CAR controls (p<0.01), and several animals showed complete remission. A study to evaluate efficacy using local delivery in a pediatric HGG orthotopic model is currently underway.

[0160] Based on two of the reported GPC2 scFvs (D3 and D4), DNA-based second-generation CAR vectors were engineered, and multiple CAR T cell constructs were stably expressed by following retroviral transduction in primary human T cells. The initial constructs engineered to have the CD8a hinge, transmembrane domain, and 41BBz signaling domain in either of two orientations of the N-terminal variable heavy chain or N-terminal variable light chain (Figure 9B) showed stable cell surface expression and bound to soluble recombinant GPC2 (Figure 9C). These constructs showed potent in vitro efficacy and cytokine production (IFNy, IL2) at an effector:target ratio of 1:1 against isogenic target cells (Kelly-GPC2) engineered to express GPC2 at levels comparable to in vivo levels of GPC2 (Figure 11A-D). Furthermore, the inventors showed the advantage that incorporation of the CD28-H / TM costimulatory domain into these CAR constructs added CAR T cell efficacy when targeting GPC2-expressing tumors (Figure 14A-B). In summary, these data show that the use of DNA-based CAR vectors and viral transduction allows for the engineering of stable CAR T cells targeting GPC2 that exert a potent killing effect against GPC2-expressing cancer cells.

[0161] From these data, it can be seen that mRNA provides a rapid and reproducible method for testing novel CAR T cells, and GPC2 is a promising CAR T cell target in neuroblastoma, medulloblastoma, and some high-grade gliomas and other pediatric malignant brain tumors. The RNA GPC2 CAR T cells with the light-chain - heavy-chain D3 scFv chain and long linker configuration were non-toxic in the mouse model and resulted in the strongest cytotoxic effect.

[0162] GPC2 DNA CAR T cells based on D3(M201) transduced via lentivirus (Figs. 15A - F) and retrovirus (Figs. 16A - B) also show potent cytotoxicity against neuroblastoma pre - clinical models. The GPC2 CAR is strongly expressed on T cells (Fig. 15A), shows cytotoxicity against isogenic SY5Y - GPC2 neuroblastoma cells (Fig. 15B), and when co - cultured, simultaneous T cell activation and increased T cell expression of INFγ and CD107A occur (Figs. 15C - D). D3(M201) long linker 28 / 28 / 41BB (GPC2 CAR based on D3(M201) and hinge based on CD28 / Tm based on CD28 / 41BB co - stimulatory domain) and long linker 28 / 28 / 28 (GPC2 CAR based on D3(M201) and hinge based on CD28 / Tm based on CD28 / CD28 co - stimulatory domain) show strong in vivo activity inducing tumor regression of strong COG - N - 421x neuroblastoma patient - derived xenografts and are very well - tolerated (Figs. 15E - F). GPC2 CAR T cells based on D3(M201) also induced tumor regression in the metastatic SMS - SAN neuroblastoma model (Figs. 16A - B).

[0163] All of the compositions and / or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of the present disclosure have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that changes can be made in the compositions and methods described herein and in the steps or the order of the steps of the methods described herein without departing from the concept, spirit, and scope of the present disclosure. More specifically, it will be apparent that certain chemically and physiologically related agents can be used in place of the agents described herein, and at the same time, the same or similar results can be obtained. All such similar substitutions and modifications apparent to those skilled in the art are considered to be within the scope of the spirit, scope, and concept of the present disclosure as defined by the appended claims.

[0164] VII. References The following references are hereby specifically incorporated by reference into this specification to the extent that they show details of exemplary procedures or other details that supplement those described herein. TIFF2025111569000003.tif19635TIFF2025111569000004.tif231150TIFF2025111569000005.tif231151TIFF2025111569000006.tif231150TIFF2025111569000007.tif59128

[0165] Array information SEQUENCE LISTING <110> THE CHILDREN'S HOSPITAL OF PHILADELPHIA THE UNITED STATES OF AMERICA, AS REPRESENTED BY THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SERVICES THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIVERSITY <120> CHIMERIC ANTIGEN RECEPTORS CONTAINING GLYPICAN 2 BINDING DOMAINS <150> US 62 / 876,483 <151> 2019-07-19 <160> 41 <170> PatentIn version 3.5 <210> 1 <211> 243 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 1 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Asn 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Val Ile Tyr Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp Ser Asn Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr 100 105 110 Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Leu Ser Leu Pro Val Thr 130 135 140 Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu 145 150 155 160 Tyr Ser Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly 165 170 175 Lys Ser Pro Gln Val Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly 180 185 190 Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu 195 200 205 Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met 210 215 220 Gln Ala Leu Gln Thr Pro Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu 225 230 235 240 Ile Lys Arg <210> 2 <211> 242 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 2 Glu Val Gln Leu Val Glu Thr Gly Gly Gly Val Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Ser Gly Tyr Asp Tyr Val Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Ala Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Thr 130 135 140 Leu Ser Ala Phe Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 145 150 155 160 Gln Ser Ile Ser Ser Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 165 170 175 Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val 180 185 190 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr 195 200 205 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 210 215 220 Leu Asn Ser Tyr Pro Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile 225 230 235 240 Lys Arg <210> 3 <211> 245 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 3 Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 [[ID=3,4]]Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Ser 65 70 75 80 Leu Gln Met Asp Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ser Arg Asp Ser Gly Asn Tyr Leu Asp Ala Phe Asp Phe Trp 100 105 110 Gly Gln Gly Thr Met Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly 115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser 130 135 140 Pro Ser Thr Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys 145 150 155 160 Arg Ala Ser Gln Ser Ile Ser Ser Trp Leu Ala Trp Tyr Gln Gln Lys 165 170 175 Ala Gly Lys Ala Pro Arg Leu Leu Ile Tyr Asp Ala Ser Thr Leu Glu 180 185 190 Ser Gly Val Pro Ser Arg Phe Ser Gly Thr Gly Ser Gly Thr Tyr Phe 195 200 205 Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr 210 215 220 Cys Gln Gln Phe Asn Ser Phe Pro Leu Thr Phe Gly Gly Gly Thr Lys 225 230 235 240 Val Glu Ile Lys Arg<9999999>245 <210> 4 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 4 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 5 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 5 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Asn<l 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Val Ile Tyr Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp Ser Asn Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr 100 105 110 Val Ser Ser 115 <210> 6 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 6 Glu Ile Val Leu Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Lys Ser 35 40 45 Pro Gln Val Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala 85 90 95 Leu Gln Thr Pro Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Gln Gly 100 105 110 Thr Arg Leu Glu Ile Lys Arg 115 <210> 7 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 7 Glu Val Gln Leu Val Glu Thr Gly Gly Gly Val Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Ser Gly Tyr Asp Tyr Val Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Ala Val Ser Ser 115 <210> 8 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 8 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Phe Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Leu Asn Ser Tyr Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg 100 1'05 <210> 9 <211> 122 <212> PRT <213> Artificial Sequence It should be noted that there seems to be a typo in "1'05" in the original text which is translated as "1'05" here. It might be "105".<220> <223> Synthetic peptide <400> 9 Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Ser 65 70 75 80 Leu Gln Met Asp Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ser Arg Asp Ser Gly Asn Tyr Leu Asp Ala Phe Asp Phe Trp 100 105 110 Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 10 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 10 Asp Ile Gln Leu Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Ala Gly Lys Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Thr Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Thr Gly Ser Gly Thr Tyr Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Phe Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> 11 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 11 Gly Phe Thr Val Ser Ser Asn Tyr 1 5 <210> 12 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 12 Ile Tyr Ser Gly Gly Ser Thr 1 5 <210> 13 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 13 Ala Arg Asp Ser Asn Ala Phe Asp Ile 1 5 <210> 14 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 14 Gln Ser Leu Leu Tyr Ser Asn Gly Tyr Asn Tyr 1 5 10 <210> 15 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 15 Leu Gly Ser 1 <210> 16 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 16 Met Gln Ala Leu Gln Thr Pro Ile Thr 1 5 <210> 17 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 17 Gly Phe Thr Phe Ser Asp Tyr Tyr 1 5 <210> 18 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 18 Ile Ser Ser Ser Gly Ser Thr Ile 1 5 <210> 19 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 19 Ala Arg Glu Ser Gly Tyr Asp Tyr Val Phe Asp Tyr 1 5 10 <210> 20 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 20 Gln Ser Ile Ser Ser Trp 1 5 <210> 21 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 21 Ala Ala Ser 1 <210> 22 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 22 Gln Gln Leu Asn Ser Tyr Pro Ile Thr 1 5 <210> 23 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 23 Gly Phe Thr Phe Ser Ser Tyr Ala 1 5 <210> 24 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 24 Ile Ser Gly Ser Gly Gly Ser Thr 1 5 <210> 25 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 25 Ala Lys Ser Arg Asp Ser Gly Asn Tyr Leu Asp Ala Asp Phe Asp Phe 1 5 10 15 <210> 26 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 26 Gln Ser Ile Ser Ser Trp 1 5 <210> 27 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 27 Asp Ala Ser 1 <210> 28 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 28 Gln Gln Phe Asn Ser Phe Pro Leu Thr 1 5 <210> 29 <211> 357 <212> DNA <213> Artificial Sequence <220> <223> Synthetic primer <400> 29 gaggtgcagc tggtggagac tgggggaggc gtggtcaagc ctggagggtc cctgagactc 60 tcctgtgcag cctctggatt caccttcagt gactactaca tgagctggat ccgccaggct 120 ccagggaagg ggctggagtg ggtttcatac attagtagta gtggtagtac catatactac 180 gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agctgaggac acggctgtgt attactgtgc gagagagagt 300 ggctacgatt acgtgtttga ctactggggc cagggaaccc tggtcgccgt ctcctca 357 <210> 30 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 30 Glu Val Gln Leu Val Glu Thr Gly Gly Gly Val Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Ser Gly Tyr Asp Tyr Val Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Ala Val Ser Ser 115 <210> 31 <211> 324 <212> DNA <213> Artificial Sequence <220> <223> Synthetic primer <400> 31 gacatccaga tgacccagtc tccttccacc ctgtctgcat ttgtaggaga cagagtcacc 60 atcacttgcc gggccagtca gagtattagt agctggttgg cctggtatca gcaaaaacca 120 gggaaagccc ctaagctcct gatctatgct gcatccactt tgcaaagtgg ggtcccatca 180 aggttcagcg gcagtggatc tgggacagaa ttcactctca caatcagcag cctgcagcct 240 gaagattttg caacttatta ctgtcaacag cttaatagtt accctatcac cttcggccaa 300 gggacacgac tggagattaa acga 324 <210> 32 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 32 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Phe Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Leu Asn Ser Tyr Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg 100 105 <210> 33 <211> 345 <212> DNA <213> Artificial Sequence <220> <223> Synthetic primer <400> 33 caggtgcagc tggtgcagtc tggaggaggc ttgatccagc ctggggggtc cctgagactc 60 tcctgtgcag cctctgggtt caccgtcagt agcaactaca tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcagtt atttatagcg gtggtagcac atactacgca 180 gactccgtga agggccgatt caccatctcc agagacaatt ccaagaacac gctgtatctt 240 caaatgaaca gcctgagagc cgaggacacg gccgtgtatt actgtgcgag agattcgaat 300 gcttttgata tctggggcca agggacaatg gtcaccgtct cttca 345 <210> 34 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 34 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Asn 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Val Ile Tyr Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp Ser Asn Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr 100 105 110 Val Ser Ser 115 <210> 35 <211> 339 <212> DNA <213> Artificial Sequence <220> <223> Synthetic primer <400> 35 gaaattgtgc tgactcagtc tccactctcc ctgcccgtca cccctggaga gccggcctcc 60 atctcctgca ggtctagtca gagcctcctg tatagtaatg gatacaacta tttggattgg 120 tacctgcaga agccagggaa gtctccacag gtcctgatct atttgggttc taatcgggcc 180 tccggggtcc ccgacaggtt cagtggcagt ggatcaggca cagatttcac actgaaaatc 240 agcagagtgg aggctgagga tgttggggtt tattactgca tgcaagctct acaaactccg 300 atcaccttcg gccaagggac acgactggag attaaacga 339 <210> 36 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 36 Glu Ile Val Leu Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Lys Ser 35 40 45 Pro Gln Val Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala 85 90 95 Leu Gln Thr Pro Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105 110 Arg <210> 37 <211> 366 <212> DNA <213> Artificial Sequence <220> <223> Synthetic primer <400> 37 gaggtgcagc tggtgcagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180 gcagactccg tgaagggccg cttcaccatc tccagagaca attccaagaa cacgctgtct 240 ctgcaaatgg acagcctgag acccgaggac acggccgtat attactgtgc gaaaagtcga 300 gatagtggga actaccttga tgcttttgat ttctggggcc aagggacaat ggtcaccgtc 360 tcttca 366 <210> 38 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 38 Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Ser 65 70 75 80 Leu Gln Met Asp Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ser Arg Asp Ser Gly Asn Tyr Leu Asp Ala Phe Asp Phe Trp 100 105 110 Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 39 <211> 324 <212> DNA <213> Artificial Sequence <220> <223> Synthetic primer <400> 39 gacatccagt tgacccagtc tccttccacc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgcc gggccagtca gagtattagt agctggttgg cctggtatca gcagaaagca 120 gggaaagctc ctaggctcct gatctatgat gcctccactt tggaaagtgg agtcccatca 180 aggttcagcg gcactggatc tgggacatat ttcactctca ccatcagcag cctgcagcct 240 gaagattttg caacttatta ctgtcaacag tttaatagtt tcccgctcac tttcggcgga 300 gggaccaagg tggagatcaa acga 324 <210> 40 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 40 Asp Ile Gln Leu Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Ala Gly Lys Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Thr Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Thr Gly Ser Gly Thr Tyr Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Phe Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> 41 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 41 Gly Gly Gly Gly Ser 1 5

Claims

1. An isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen-binding domain, a flexible hinge domain, a transmembrane domain, a co-stimulatory signaling region, and an intracellular signaling domain, the antigen-binding domain selectively binds to cancer cell-associated glypican 2 (GPC2), said isolated nucleic acid molecule.

2. The isolated nucleic acid molecule according to claim 1, wherein the antigen-binding domain comprises an antibody or an antigen-binding fragment thereof.

3. The isolated nucleic acid molecule according to claim 2, wherein the antigen-binding fragment is a Fab, a single-chain variable fragment (scFv), or a single-domain antibody.

4. The encoded antigen-binding domain comprises (a) a heavy-chain variable domain comprising the amino acid sequence of SEQ ID NO:30 and a light-chain variable domain comprising the amino acid sequence of SEQ ID NO:32; (b) a heavy-chain variable domain comprising the amino acid sequence of SEQ ID NO:34 and a light-chain variable domain comprising the amino acid sequence of SEQ ID NO:36; or (c) a heavy-chain variable domain comprising the amino acid sequence of SEQ ID NO:38 and a light-chain variable domain comprising the amino acid sequence of SEQ ID NO:40 The isolated nucleic acid molecule according to any one of claims 1 to 3.

5. The encoded antigen-binding domain comprises (a) a heavy-chain variable domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:11, a CDR2 comprising the amino acid sequence of SEQ ID NO:12, and a CDR3 comprising the amino acid sequence of SEQ ID NO:13, and a light-chain variable domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:14, a CDR2 comprising the amino acid sequence of SEQ ID NO:15, and a CDR3 comprising the amino acid sequence of SEQ ID NO:16; (b) a heavy-chain variable domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:17, a CDR2 comprising the amino acid sequence of SEQ ID NO:18, and a CDR3 comprising the amino acid sequence of SEQ ID NO:19, and a light-chain variable domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:20, a CDR2 comprising the amino acid sequence of SEQ ID NO:21, and a CDR3 comprising the amino acid sequence of SEQ ID NO:22; or (c) A heavy chain variable domain comprising a CDR1 containing the amino acid sequence of SEQ ID NO:23, a CDR2 containing the amino acid sequence of SEQ ID NO:24, and a CDR3 containing the amino acid sequence of SEQ ID NO:25, and a light chain variable domain comprising a CDR1 containing the amino acid sequence of SEQ ID NO:26, a CDR2 containing the amino acid sequence of SEQ ID NO:27, and a CDR3 containing the amino acid sequence of SEQ ID NO:28 An isolated nucleic acid molecule according to any one of claims 1 to 3, comprising the same.

6. (a) The encoded antigen-binding domain comprises a heavy chain variable domain containing the amino acid sequence of SEQ ID NO:30 and a light chain variable domain containing the amino acid sequence of SEQ ID NO:32, and (b) The C-terminus of the light chain variable domain is fused to the N-terminus of the heavy chain variable domain by a flexible linker. The isolated nucleic acid molecule according to claim 2.

7. The isolated nucleic acid molecule according to claim 6, wherein the linker is a peptide linker.

8. The isolated nucleic acid molecule according to claim 7, wherein the peptide linker is at least 15 amino acids in length.

9. The isolated nucleic acid molecule according to claim 8, wherein the peptide linker is a glycine-serine linker.

10. (a) The flexible hinge domain is derived from CD8α, CD28, or immunoglobulin (Ig), (b) The transmembrane domain comprises the CD28 transmembrane domain, (c) The co-stimulatory signaling region comprises a domain derived from CD28, 4-1BB (CD137), OX40, or ICOS, and (d) The intracellular signaling domain comprises the CD3-ζ domain or high-affinity FcεRI. The isolated nucleic acid molecule according to claim 1.

11. (a) The chimeric antigen receptor (CAR) comprises an antigen-binding domain, a flexible hinge domain, a transmembrane domain, a co-stimulatory signaling region, and an intracellular signaling domain, and (b) The antigen-binding domain selectively binds to cancer cell-associated glypican 2 (GPC2). A chimeric antigen receptor (CAR) polypeptide.

12. The chimeric antigen receptor polypeptide according to claim 11, wherein the antigen-binding fragment is a Fab, a single-chain variable fragment (scFv), or a single-domain antibody.

13. The encoded antigen-binding domain is (a) A heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:30 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO:32; (b) A heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:34 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO:36; or (c) A heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:38 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO:40 The chimeric antigen receptor (CAR) polypeptide according to claim 11 or 12, comprising.

14. (a) A heavy chain variable domain comprising CDR1 comprising the amino acid sequence of SEQ ID NO:11, CDR2 comprising the amino acid sequence of SEQ ID NO:12, and CDR3 comprising the amino acid sequence of SEQ ID NO:13, and a light chain variable domain comprising CDR1 comprising the amino acid sequence of SEQ ID NO:14, CDR2 comprising the amino acid sequence of SEQ ID NO:15, and CDR3 comprising the amino acid sequence of SEQ ID NO:16; (b) A heavy chain variable domain comprising CDR1 comprising the amino acid sequence of SEQ ID NO:17, CDR2 comprising the amino acid sequence of SEQ ID NO:18, and CDR3 comprising the amino acid sequence of SEQ ID NO:19, and a light chain variable domain comprising CDR1 comprising the amino acid sequence of SEQ ID NO:20, CDR2 comprising the amino acid sequence of SEQ ID NO:21, and CDR3 comprising the amino acid sequence of SEQ ID NO:22; or (c) A heavy chain variable domain comprising CDR1 comprising the amino acid sequence of SEQ ID NO:23, CDR2 comprising the amino acid sequence of SEQ ID NO:24, and CDR3 comprising the amino acid sequence of SEQ ID NO:25, and a light chain variable domain comprising CDR1 comprising the amino acid sequence of SEQ ID NO:26, CDR2 comprising the amino acid sequence of SEQ ID NO:27, and CDR3 comprising the amino acid sequence of SEQ ID NO:28 The chimeric antigen receptor (CAR) polypeptide according to claim 11 or 12, comprising.

15. (a) The encoded antigen-binding domain comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:30 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO:32, and (b) The C-terminus of the light chain variable domain is fused to the N-terminus of the heavy chain variable domain by a flexible linker. The chimeric antigen receptor polypeptide according to claim 13.

16. A genetically modified T cell comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR), or a genetically modified T cell comprising the isolated nucleic acid molecule according to any one of claims 1 to 15.

17. A genetically modified T cell comprising the chimeric antigen receptor according to any one of claims 11 to 16.

18. A method for producing a genetically modified T cell, comprising the step of transducing an immune effector cell with the chimeric antigen receptor according to any one of claims 11 to 16.

19. A method for providing anti-tumor immunity in a mammal, comprising the step of administering to the mammal a population of the genetically modified T cells according to claim 16 in an effective amount.

20. A method for treating a mammal having a disease associated with overexpression of GPC2, comprising the step of administering to the mammal a population of the genetically modified T cells according to claim 16 in an effective amount.

21. (a) The CAR induces the secretion of interferon γ and interleukin-2, and (b) the genetically modified T cell exhibits cytotoxicity against GPC2-expressing cancer cells when exposed to cancer cell-associated GPC2. The genetically modified T cell according to claim 16.

22. The method according to claim 20, wherein the GPC2-expressing cancer is selected from the group consisting of sarcoma cells, rhabdoid cancer cells, neuroblastoma cells, retinoblastoma cells, or medulloblastoma cells, uterine carcinosarcoma (UCS), low-grade glioma of the brain (LGG), thymoma (THYM), testicular germ cell tumor (TGCT), glioblastoma multiforme (GBM) and cutaneous melanoma (SKCM), hepatocellular carcinoma (LIHC), uveal melanoma (UVM), kidney chromophobe (KICH), thyroid cancer (THCA), renal clear cell carcinoma (KIRC), renal papillary cell carcinoma (KIRP), gastric adenocarcinoma (STAD), cholangiocarcinoma (CHOL), adenoid cystic carcinoma (ACC), prostate adenocarcinoma (PRAD), chromaffin cell tumor and paraganglioma (PCPG), DLBC, lung adenocarcinoma (LUAD), head and neck squamous cell carcinoma (HNSC), pancreatic adenocarcinoma (PAAD), breast cancer (BRCA), mesothelioma (MESO), colorectal adenocarcinoma (COAD), rectal adenocarcinoma (READ), esophageal cancer (ESCA), ovarian cancer (OV), lung squamous cell carcinoma (LUSC), bladder urothelial carcinoma (BLCA), sarcoma (SARC), or endometrial carcinoma of the uterine corpus (UCEC).

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