Chimeric antigen receptors comprising glypican 2 binding domain

Abstract

The present invention relates to a chimeric antigen receptor comprising a phosphatidylinositol proteoglycan 2 binding domain. The present disclosure relates to chimeric antigen receptors that bind to phosphatidylinositol proteoglycan 2, nucleic acids encoding the same, and cells that express the same, and methods of using such cells to treat cancers that express or overexpress a phosphatidylinositol proteoglycan 2 antigen.

Description

[0001] This application is a divisional application of the Chinese patent application with the application date of July 17, 2020, application number 202080052372.2, and invention name “Chimeric Antigen Receptor Comprising a Glypican 2 Binding Domain”.

[0002] Priority claim

[0003] This application claims the benefit of priority to U.S. Provisional Application Serial No. 62 / 876,483, filed on July 19, 2019, which is hereby incorporated by reference in its entirety.

[0004] Statement Regarding Federal Funding

[0005] This invention was made with government support under Grant No. NCI U54 CA232568-01 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0006] Pursuant to 37 CFR §1.821(c), the Sequence Listing is submitted herein as an ASCII-compatible text file, entitled "CHOPP0034WO.txt," which was created on July 17, 2020, and has a size of approximately 27 kilobytes. The contents of the above-mentioned file are hereby incorporated by reference in their entirety. Technical Field

[0007] The present disclosure relates generally to the fields of medicine, oncology, and immunotherapy, and more particularly to the development of chimeric antigen receptor immunoassays with binding specificity for glypican 2 (GPC2) and their use in treating GPC2-positive cancers. Background Art

[0008] Despite intensive multimodal chemoradiotherapy, children with high-risk neuroblastoma have a poor prognosis. Although monoclonal antibodies targeting the disialoganglioside GD2 have improved neuroblastoma outcomes, this treatment is associated with significant on-target and off-tumor toxicities. Therefore, a major challenge remains to identify new cell surface molecules that meet the stringent criteria of modern immunotherapy, including unique tumor expression compared to normal pediatric tissue, and preferably, that are essential for tumor maintenance.

[0009] Pharmaceutical and biotechnology companies are currently developing many biological drugs for the treatment of diseases or health conditions. For example, in cancer immunotherapy, the development of agents that activate the host immune system's T cells to prevent cancer cell proliferation or kill cancer cells has become a promising treatment method to supplement existing standards of care. T cells, especially adoptive transfer of T cells modified by chimeric antigen receptors (CARs), has become another promising method in cancer immunotherapy. Unlike naturally occurring T cell receptors, CARs can directly recognize their target antigens without being restricted by major histocompatibility complex (MHC) molecules and can potentially mediate high levels of cell killing activity. A common method is to genetically modify T cells in vitro to express CARs, which can recognize target antigens without the need for MHC presentation. These CAR-T cells have the potential to produce very high levels of anti-tumor activity, but they can also show improved off-target cell killing of CAR-T cells. Therefore, there is still an urgent need for alternative methods to minimize such side effects and supplement existing immunotherapy methods. Summary of the Invention

[0010] Thus, according to the present disclosure, a chimeric antigen receptor is provided, comprising (i) an extracellular domain comprising a single-chain antibody variable region fragment (scFv) region comprising a variable heavy chain (VH) and a variable light chain (VL) that selectively bind to glypican 2, (ii) a transmembrane domain; and (iii) an endodomain, wherein when the scFv engages with glypican 2, the endodomain comprises a signal transduction function.

[0011] The receptor may be characterized by the VH and VL sequences of SEQ ID NOs: 5 and 6, respectively; by the VH and VL sequences of SEQ ID NOs: 7 and 8, respectively, or by the VH and VL sequences of SEQ ID NOs: 9 and 10, respectively.

[0012] The scFv may be characterized by VH and VL sequences that are 80% homologous to SEQ ID NOs: 5 and 6, respectively, and have VH CDRs of SEQ ID NOs: 11 to 13 and VL CDRs of SEQ ID NOs: 14 to 16; or by VH and VL sequences that are 80% homologous to SEQ ID NOs: 7 and 8, respectively, and have VH CDRs of SEQ ID NOs: 17 to 19 and VL CDRs of SEQ ID NOs: 20 to 22; or by VH and VL sequences that are 80% homologous to SEQ ID NOs: 9 and 10, respectively, and have VH CDRs of SEQ ID NOs: 23 to 25 and VL CDRs of SEQ ID NOs: 26 to 28.

[0013] The receptor can be characterized by VH and VL sequences that are 90% homologous to SEQ ID NOs: 5 and 6, respectively, and have VH CDRs of SEQ ID NOs: 11 to 13 and VL CDRs of SEQ ID NOs: 14 to 16; or by VH and VL sequences that are 90% homologous to SEQ ID NOs: 7 and 8, respectively, and have VH CDRs of SEQ ID NOs: 17 to 19 and VL CDRs of SEQ ID NOs: 20 to 22; or by VH and VL sequences that are 90% homologous to SEQ ID NOs: 9 and 10, respectively, and have VH CDRs of SEQ ID NOs: 23 to 25 and VL CDRs of SEQ ID NOs: 26 to 28.

[0014] The receptor may comprise a sequence selected from SEQ ID NOs: 1, 2, and 3; or may comprise a sequence having 80% homology to SEQ ID NOs: 1, 2, or 3 and having VH CDRs of SEQ ID NOs: 11 to 13, 17 to 19, and 23 to 25, respectively, and VL CDRs of SEQ ID NOs: 14 to 16, 20 to 22, and 26 to 28, respectively; or may comprise a sequence having 90% homology to SEQ ID NOs: 1, 2, or 3 and having VH CDRs of SEQ ID NOs: 11 to 13, 17 to 19, and 23 to 25, respectively, and VL CDRs of SEQ ID NOs: 14 to 16, 20 to 22, and 26 to 28, respectively.

[0015] The transmembrane and endodomains can be derived from the same molecule. The endodomain can comprise a CD3-ζ domain or high-affinity FcεRI. The scFv can comprise a flexible linker between the VH and VL, for example, wherein the flexible linker is derived from CD8α, Ig, or SEQ ID NO: 4. The scFv can be arranged as VH-linker-VL or VL-linker-VH.

[0016] Also provided are nucleic acids, such as mRNA or DNA, encoding a chimeric antigen receptor as defined above, or cells, such as prokaryotic or eukaryotic cells, and in particular engineered T cells, expressing a chimeric antigen receptor as defined above.

[0017] In another embodiment, a method of treating a subject having a cancer that expresses or overexpresses Glypican 2 is provided, comprising administering to the subject a chimeric antigen receptor as defined above, a nucleic acid as defined above, or a cell as defined above, e.g., a T cell, e.g., an autologous T cell of the subject.

[0018] The method may further comprise administering a second anti-cancer therapy to the subject. The second cancer therapy may be radiation, chemotherapy, radiotherapy, hormone therapy, immunotherapy, toxin therapy, or surgery. The immunotherapy may be a checkpoint inhibitor therapy. The second cancer therapy may be administered simultaneously with the receptor, nucleic acid, or cell, or may be administered before or after the receptor, nucleic acid, or cell. The second cancer therapy may be administered more than once. The receptor, nucleic acid, or cell may be administered more than once.

[0019] Cancer can be drug-resistant, metastatic or recurrent. The subject can be a human or non-human mammal. The cancer can be a pediatric cancer or an adult cancer. The cancer can be a leukemia, for example, selected from the following leukemias: acute lymphoblastic leukemia (ALL), acute lymphocytic B cell leukemia, acute lymphocytic T cell leukemia, acute myeloid leukemia (AML), acute promyelocytic leukemia (APL), acute monocytic leukemia, acute erythroleukemic leukemia, acute megakaryocytic leukemia, acute myelomonocytic leukemia, acute non-lymphocytic leukemia, acute undifferentiated leukemia, chronic myelocytic leukemia (CML), chronic lymphocytic leukemia (CLL) and hairy cell leukemia.

[0020] The cancer can be a solid tumor cancer, such as lung cancer, liver cancer, pancreatic cancer, stomach 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. The cancer can also include sarcoma cells, rhabdoid cancer cells, neuroblastoma cells, retinoblastoma cells or medulloblastoma cells. The cancer may be uterine carcinosarcoma (UCS), brain lower grade glioma (LGG), thymoma (THYM), testicular germ cell tumor (TGCT), glioblastoma multiforme (GBM), skin cutaneous melanoma (SKCM), liver hepatocellular carcinoma (LIHC), uveal melanoma (UVM), kidney chromophobe (KICH), thyroid cancer (THCA), kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), stomach adenocarcinoma (SCC), or thyroid cancer (THCA). adenocarcinoma (STAD), cholangiocarcinoma (CHOL), adenoid cystic carcinoma (ACC), prostate adenocarcinoma (PRAD), pheochromocytoma and paraganglioma (PCPG), DLBC, lung adenocarcinoma (LUAD), head-neck squamous cell carcinoma (HNSC), pancreatic adenocarcinoma (PAAD), breast cancer (BRCA), mesothelioma (MESO), colon and rectaladenocarcinoma (COAD), rectum adenocarcinoma (READ), esophageal carcinoma (ESCA), ovarian cancer (OV), lung squamous cell carcinoma (LUSC), bladder urothelial carcinoma (BLCA), sarcoma (SARC), or uterine corpus endometrial carcinoma (UCEC).

[0021] Also provided are isolated nucleic acid molecules encoding chimeric antigen receptors (CARs), wherein the CAR comprises an antigen binding domain, a flexible hinge domain, a transmembrane domain, a costimulatory signaling region, and an intracellular signaling domain, and wherein the antigen binding domain selectively binds to cancer cell-associated glypican 2 (GPC2). 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.

[0022] The encoded antigen-binding domain 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. 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.

[0023] The encoded antigen-binding domain may comprise 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 the C-terminus of the light chain variable domain may be fused to the N-terminus of the heavy chain variable domain via a flexible linker. The linker may be a peptide linker, e.g., having a length of at least 15 amino acids, and / or the peptide linker may be a glycine-serine linker.

[0024] The isolated nucleic acid molecule can have (a) a flexible hinge domain from CD8α, CD28, or an immunoglobulin (Ig), (b) a transmembrane domain comprising the CD28 transmembrane domain, (c) a co-stimulatory signaling region comprising a domain from CD28, 4-1BB (CD137), OX40, or ICOS, and (d) an intracellular signaling domain comprising a CD3-ζ domain or high affinity FcεRI.

[0025] In another embodiment, a chimeric antigen receptor (CAR) polypeptide is provided, wherein (a) CAR comprises an antigen binding domain, a flexible hinge domain, a transmembrane domain, a costimulatory 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 can be a Fab, a single-chain variable fragment (scFv), or a single-domain antibody.

[0026] 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.

[0027] The encoded antigen-binding domain 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. 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.

[0028] The chimeric antigen receptor polypeptide may comprise (a) an antigen binding domain encoded by 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) a C-terminus of the light chain variable domain fused to the N-terminus of the heavy chain variable domain via a flexible linker.

[0029] 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 comprising a genetically modified T cell comprising a chimeric antigen receptor as defined herein. The genetically modified T cell may have the following characteristics:

[0030] (a) CAR that induces secretion of interferon-γ and interleukin-2, and

[0031] (b) When genetically modified T cells are exposed to cancer cell-associated GPC2, they exhibit cytotoxicity against GPC2-expressing cancers.

[0032] The cancer expressing GPC2 may be selected from the group consisting of sarcoma cells, rhabdoid carcinoma cells, neuroblastoma cells, retinoblastoma cells or medulloblastoma cells, uterine carcinosarcoma (UCS), brain low-grade glioma (LGG), thymoma (THYM), testicular germ cell tumor (TGCT), glioblastoma multiforme (GBM) and cutaneous melanoma (SKCM), hepatocellular carcinoma (LIHC), uveal melanoma (UVM), renal chromophobe cell carcinoma (KICH), thyroid carcinoma (THCA), renal clear cell renal carcinoma (KIRC), renal papillary cell renal carcinoma (KIRP), gastric glandular carcinoma (GC), thyroid carcinoma (THCA), renal clear cell renal carcinoma (KIRC), renal papillary cell renal carcinoma (KIRP), gastric glandular carcinoma (GC), thyroid glandular carcinoma (THCA ... cancer (STAD), bile duct cancer (CHOL), adenoid cystic carcinoma (ACC), prostate adenocarcinoma (PRAD), pheochromocytoma and paraganglioma (PCPG), DLBC, lung adenocarcinoma (LUAD), head and neck squamous cell carcinoma (HNSC), pancreatic cancer (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 uterine corpus endometrial cancer (UCEC).

[0033] Also provided are methods for preparing genetically modified T cells, comprising transducing immune effector cells with a chimeric antigen receptor as defined herein. Also provided are methods for providing anti-tumor immunity in a mammal, comprising administering to the mammal an effective amount of a population of genetically modified T cells as defined herein. Also provided are methods for treating a mammal suffering from a disease associated with GPC2 overexpression, comprising administering to the mammal an effective amount of a population of genetically modified T cells as defined herein.

[0034] It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein.

[0035] When used in conjunction with the term "comprising" in the claims and / or the specification, a noun without a quantifier may mean "a / kind," but it is also consistent with "one / kinds or more / kinds," "at least one / kinds," and "one / kinds or more than one / kinds." The word "about" means plus or minus 5% of the stated number.

[0036] Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples, while indicating some specific embodiments of the present disclosure, are given by way of illustration only, as many changes and modifications within the spirit and scope of the present disclosure will become apparent to those skilled in the art through this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0038] The following drawings constitute part of this specification and are included to further illustrate certain aspects of the present disclosure. The present disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0039] Figure 1 . Heavy and light chain amino acid and nucleic acid sequences of human antibody m201 The CDRs are shown in bold italics.

[0040] Figure 2 . Heavy and light chain amino acid and nucleic acid sequences of human antibody m202 The CDRs are shown in bold italics.

[0041] Figure 3 . Heavy and light chain amino acid and nucleic acid sequences of human antibody m203 The CDRs are shown in bold italics.

[0042] Figure 4 . GPC2 is expressed in a subset of pediatric brain tumorsRNA sequencing data were obtained from the Childhood Brain Tumor Tissue Consortium (CBTTC), which contains 1,110 samples.

[0043] Figure 5A-C . In vitro validation of GPC2 RNA CAR T cell binding and persistence (Figure 5A) GPC2 RNA CAR specific binding to GPC2 of four GPC2 RNA CAR T cell constructs, measured by flow cytometry. (Figure 5B) CAR persistence over time for each construct, measured by flow cytometry. (Figure 5C) Negative checkpoint regulator expression of PD1 and Lag3 for each construct four days after transfection.

[0044] Figure 6A-D . D3V3 and D3V4 mRNA GPC2 CAR T cells produced the strongest cytotoxicity in vitro (Figure 6A) Cytotoxicity of four GPC2 CAR T cell constructs against SMS-SAN, a neuroblastoma cell line with endogenous high GPC2 expression. E:T ratio 10:1. (Figure 6B) Interferon gamma degranulation by GPC2 CAR T cell constructs was measured by ELISA across multiple cell lines with varying GPC2 expression at different E:T ratios. (Figure 6C) Cytotoxicity and interferon gamma released by D3V3 and D3V4 CAR T cells against the DAOY medulloblastoma cell line. E:T ratio 10:1. (Figure 6D) Cytotoxicity and interferon gamma released by D3V3 and D3V4 CAR T cells against the 7316-913 high-grade glioma cell line. E:T ratio 5:1.

[0045] Figure 7A-C . D3V3 mRNAGPC2 CAR T cells in NB-1643 patient-derived xenografts (patient- showed the greatest in vivo cytotoxicity in PDX models (FIG. 7A) Tumor growth over time in mice treated with IV-delivered D3V3 CAR compared to CD19 CAR control. Each line represents one mouse. (FIG. 7B) Tumor growth over time in mice treated with IV-delivered D3V4 CAR compared to CD19 CAR control. Each line represents one mouse. (FIG. 7C) Tumor growth over time in mice treated with intratumorally delivered D3V3 and D3V4 CAR compared to CD19 CAR control (left) and Kaplan-Meier progression-free survival (right).

[0046] Figure 8 . Schematic diagram for CAR-T cell therapy and GPC2 RNA CAR construct design .

[0047] Figure 9A-C . Alignment of amino acid sequences of GPC2 single-chain variable fragments and expression of derived GPC2CAR constructs. (Figure 9A) Amino acid sequence alignment of GPC2-targeted single-chain variable fragments (scFv) in the variable heavy chain (VH)-linker-variable light chain (VL) orientation of GPC2.D4 (SEQ ID NO: 1) and GPC2.D3 (SEQ ID NO: 2). The complementarity-determining region (CDR) is shown in gray. (Figure 9B) Schematic diagram of CAR T cell constructs for testing two different scFvs in the variable heavy chain-linker-variable light chain and variable light chain-linker-variable heavy chain orientations. (Figure 9C) Expression of GPC2CAR T cell constructs on the surface of primary human T cells was assessed by the ability to bind fluorescently labeled soluble recombinant human GPC2.

[0048] Figure 10 . Expression of GPC2 in neuroblastoma cell lines The cell surface expression of GPC2 on neuroblastoma cell lines and CHO negative controls was stained with D3-IgG (labeled with Dylight 650).

[0049] Figure 11A-D . Used to drive cytokine production, killing, and hypotonic signaling in the absence of antigen Leading to the ability of binder-based prioritization of antigen-exposed CAR T cells (Figure 11A) IFNγ secretion of all constructs in response to tumor cells with overexpression (Kelly-GPC2) and native GPC2 site density (NBSD), and (Figure 11B) baseline IFNγ secretion of CAR T cells in the absence of antigen. (Figure 11C) Killing capacity of GPC2 CAR T cells against overexpression (Kelly-GPC2) and native GPC2 site density (NBSD) at a 1:1 effector to tumor cell ratio. (Figure 11D) IL-2 secretion of GPC2 CAR T cells in response to overexpression (Kelly-GPC2) and native GPC2 site density (NBSD).

[0050] Figure 12A-D . The engineered CAR constructs were ineffective against tumors expressing endogenous GPC2 antigen densities (FIG. 12A) GPC2 site density on overexpressed engineered isogenic Kelly-GPC2 and endogenous GPC2-expressing neuroblastoma cell lines NBSD and SMS-SAN was measured using Quantibrite beads. (FIG. 12B) IFNγ secretion by GPC2 CAR constructs in response to overexpressed and endogenous GPC2 site density. (FIG. 12C) GPC2 CAR T cells demonstrated the ability to kill isogenic Kelly-GPC2 and (FIG. 12D) native GPC2 cell lines when challenged with a 5x excess of tumor cells.

[0051] Figure 13A-E . CAR T cells containing a CH2CH3 spacer domain fail to improve GPC2CAR function(FIG. 13A) Schematic diagram of a GPC2 CAR construct containing an IgG4-derived CH2CH3 spacer domain. (FIG. 13B) Expression of D3VLVH.GPC2 long and short CAR T cells was assessed by staining with soluble recombinant GPC2. (FIG. 13C) In vitro expansion of short and long GPC2.19 CAR T cells is shown as days post-activation. (FIG. 13D) Cytotoxicity of short and long GPC2 CAR T cells against neuroblastoma cell lines. (FIG. 13E) Cytokine production of short and long GPC2 CAR T cells against neuroblastoma cell lines.

[0052] Figure 14A-B . GPC2 CAR T cell constructs incorporating 28 transmembrane and signaling domains effectively target natural killer cells. GPC2 site density (FIG. 14A) Cytokine production (IFNγ on the left, IL-2 on the right) by GPC2.D3VLVH CAR T cells compared to constructs incorporating the CD28 hinge / transmembrane domain and either 4-1BBz or the CD28 signaling domain. (FIG. 14B) Killing capacity of GPC2.D3VLVH CAR T cells compared to constructs incorporating the CD28 hinge / transmembrane domain and either 4-1BBz or the CD28 signaling domain.

[0053] Figure 15A-B 、 Figure 15C-D and Figure 15E-F . D3(M201)-based GPC2 DNA CAR T cells to neuroblasts Potent cytotoxicity in preclinical tumor models (Figure 15A) GPC2 CAR expression on T cells. D3 (M201) long linker 28 / 28 / 4-1BB, a GPC2 CAR based on D3 (M201) with a CD28-based hinge / CD28-based Tm domain / 4-1BB costimulatory domain and a long linker; 28 / 28 / 28, a GPC2 CAR based on D3 (M201) with a CD28-based hinge / CD28-based Tm domain / CD28 costimulatory domain and a long linker. (Figure 15B) Percent cytotoxicity of SY5Y-GPC2 cells for 8 different D3-based CAR constructs compared to UTD T cell controls. (FIGS. 15C to D) Percentage of INFg (FIG. 15C) and CD107A (FIG. 15D) positive GPC2 CAR T cells using 8 different D3(M201)-based CAR constructs after co-incubation with SY5Y-GPC2 cells. (FIG. 15E) Growth of neuroblastoma COG-N-421x patient-derived xenograft tumors after treatment with D3 / M201-based GPC2 CAR T cells. (FIG. 15F) The average weight of the mouse treatment groups is shown in FIG15E. UTD, untransduced T cells.

[0054] Figure 16A and Figure 16B . D3(M201)-VLVH-based CAR T cells in the SMS-SAN metastatic xenograft model Antitumor efficacy . ( Figure 16A ) research plan. Figure 16B ) BLI data based on different D3(M201)-VLVH in the SMS-SAN metastatic model (correlated with tumor volume). *, p < 0.05'; **, p < 0.005; ***, p < 0.0005; ****, p < 0.00005. DETAILED DESCRIPTION

[0055] The recent identification of glypican-2 (GPC2) as a cell surface oncoprotein in neuroblastoma, high-grade glioma (HGG), and medulloblastoma provides an opportunity for the development of targeted immunotherapy. The inventors hypothesized that chimeric antigen receptor (CAR) T cell therapy against GPC2 could be achieved by using in vitro transcribed RNA or by stable transduction of a DNA construct expressing a CAR molecule targeting GPC2.

[0056] The inventors created multiple CART cell constructs using D3 and D4 GPC2 binders with manipulated heavy and light chain orientations. The resulting data demonstrated the utility of efficiently designing and testing new CAR T cells using mRNA or DNA, providing a platform for demonstrating efficacy and screening for toxicity in clinical trials.

[0057] These and other aspects of the disclosure are described in further detail below.

[0058] I. Glypican 2

[0059] Glypican-2 (GPC2) is a member of the six-member glypican family of heparan sulfate (HS) proteoglycans that is attached to the cell surface via a glycosylphosphatidylinositol (GPI) anchor and plays a variety of roles in growth factor signaling and cancer cell growth. GPC2 is also known as cerebrospinal fluid and glypican 2. The GPC2 genome, mRNA, and protein sequences are publicly available. In addition, human glypican 2 mRNA and protein sequences can also be found in public databases, such as NCBI Gene ID 221914, Accession Nos. NM_152742 and NP_689955, respectively, which are incorporated herein by reference. 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 the growth and guidance of axons.

[0060] 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, representing an opportunity for the development of new targeted immunotherapies. For example, in pediatric cancers, GPC2 has been shown to be expressed at considerable levels in neuroblastoma, retinoblastoma, and medulloblastoma, while also showing limited normal tissue expression. In addition, a subset of acute lymphoblastic leukemia, high-grade glioma, and rhabdomyosarcoma expresses GPC2. GPC2 is also highly expressed in small cell lung cancer, a common and almost universally lethal cancer. In addition, many adult malignancies could benefit from GPC2-targeted immunotherapy, as evidenced by the evaluation of GPC2 expression in adult cancers using data from The Cancer Genome Atlas (TCGA). Due to this preferential expression, GPC2 represents a potential candidate for targeted immunotherapy. It is present on the cell surface of many pediatric and adult malignancies and exhibits a high degree of differential expression between tumors and normal tissues.

[0061] II. Production of Monoclonal Antibodies

[0062] A. General Methods

[0063] Antibodies to Glypican 2 can be produced by standard methods known in the art (see, e.g., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; U.S. Patent No. 4,196,265). Methods for producing monoclonal antibodies (MAbs) generally begin along the same lines as those used to prepare polyclonal antibodies. The first step in both methods is to immunize a suitable host or identify an object immunized due to a previous natural infection. As is known in the art, the immunogenicity of a given composition for immunization may vary. Therefore, it is usually necessary to strengthen the host's immune system, as can be 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, may also be used as carriers. Means for conjugating polypeptides to carrier proteins are well known in the art and include glutaraldehyde, m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide, and bis-biazotized benzidine. Also as is well known in the art, the immunogenicity of a particular immunogenic composition can be enhanced by the use of nonspecific stimulators of the immune response known as adjuvants. Exemplary and preferred adjuvants include complete Freund's adjuvant (a nonspecific stimulator of the immune response comprising killed Mycobacterium tuberculosis), incomplete Freund's adjuvant, and aluminum hydroxide adjuvant.

[0064] The amount of the immunogenic composition for producing polyclonal antibodies varies according to the properties of the immunogen and the animal used for immunization. Multiple approaches can be used to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal). The generation of polyclonal antibodies can be monitored by sampling blood from the immunized animal at different points after immunization. A second booster injection can also be given. Repeat the strengthening and titer determination process until a suitable titer is reached. When the desired immunogenicity level is obtained, blood can be taken from the immunized animal, serum can be separated and stored, and / or the animal can be used to produce MAbs.

[0065] After immunization, somatic cells with the potential to produce antibodies, particularly B lymphocytes (B cells) are selected for MAb production schemes. These cells can be obtained from biopsy spleen or lymph nodes or from circulating blood. Then, the cell fusion of the B lymphocytes and immortalized myeloma cells of the production of antibodies from the immunized animal is performed, which are typically immortalized myeloma cells of the same species as the immunized animal or human or human / mouse chimeric cells. The myeloma cell line suitable for the fusion program for producing hybridomas preferably does not produce antibodies, has high fusion efficiency and has enzyme deficiency, which subsequently makes it unable to grow in certain selection culture media that only support the growth of the desired fused cell (hybridoma).

[0066] As known to those skilled in the art, any of a variety of myeloma cells can be used (Goding, pp. 65-66, 1986; Campbell, pp. 75-83, 1984). For example, when the immunized animal is a mouse, P3-X63 / Ag8, X63-Ag8.653, NS1 / 1.Ag 4 1, Sp210-Ag14, FO, NSO / U, MPC-11, MPC11-X45-GTG 1.7, and S194 / 5XX0 Bul can be used; for rats, R210.RCY3, Y3-Ag 1.2.3, IR983F, and 4B210 can be used; and U-266, GM1500-GRG2, LICR-LON-HMy2, and UC729-6 can all be used in combination with human cell fusion. One particular murine myeloma cell line is the NS-1 myeloma cell line (also known as P3-NS-1-Ag4-1), which is readily available from the NIGMS Human Genetic Mutant Cell Repository by requesting cell line repository number GM3573. Another useful murine myeloma cell line is the 8-azaguanine-resistant murine myeloma SP2 / 0 non-producing cell line. Recently, additional fusion partner systems for 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 are produced using the SP2 / 0 / mIL-6 cell line, an IL-6-secreting derivative of the SP2 / 0 line.

[0067] Methods for producing hybrids of antibody-producing spleen cells or lymph node cells and myeloma cells generally involve mixing somatic cells with myeloma cells in a 2:1 ratio in the presence of an agent that promotes cell membrane fusion (chemical or electrical), but this ratio can vary from about 20:1 to about 1:1, respectively. Kohler and Milstein (1975; 1976) have described fusion methods using Sendai virus, and Gefter et al. (1977) have described fusion methods using polyethylene glycol (PEG), such as 37% (v / v) PEG. Electrically induced fusion methods are also suitable (Goding, pp. 71-74, 1986).

[0068] Fusion procedures typically use approximately 1×10 -6 to 1×10 -8 The low frequency of producing living heterozygotes. However, this does not cause problems, because by cultivating in selective culture medium, living fusion heterozygotes are distinguished from the unfused cells of the parent (particularly the unfused myeloma cells that will continue to divide indefinitely under normal circumstances). Selective culture medium is typically a culture medium that includes a reagent that blocks the de novo synthesis of nucleotides in tissue culture medium. Exemplary and preferred reagents are aminopterin, methotrexate and azaserine. Aminopterin and methotrexate block the de novo synthesis of purines and pyrimidines, while azaserine only blocks purine synthesis. When using aminopterin or methotrexate, hypoxanthine and thymidine are supplemented in culture medium as the source (HAT culture medium) of nucleotides. When using azaserine, hypoxanthine is supplemented in culture medium. If the B cell source is the human B cell line transformed by Epstein Barr virus (Epstein Barr virus, EBV), ouabain is added to remove the EBV transformed line that is not fused with myeloma.

[0069] Preferred selection culture medium is HAT or HAT with ouabain. Only cells that can carry out nucleotide salvage pathway could survive in HAT culture medium. Myeloma cells are defective in the key enzyme of salvage pathway (for example, hypoxanthine phosphoribosyl transferase (HPRT)), so they cannot survive. B cells can carry out this pathway, but they have a limited lifespan in culture and usually die within about two weeks. Therefore, only cells that can survive in selection culture medium are those hybrids formed by myeloma and B cells. When the source of the B cell for fusion is the B cell line transformed by EBV, now ouabain is also used for the drug selection of hybrid, because the B cell transformed by EBV is susceptible to drug killing, and the myeloma partner used is selected to have resistance to ouabain.

[0070] Cultivation provides a population of hybridomas from which specific hybridomas are selected. Selection of hybridomas is typically performed by culturing cells by monoclonal dilution in microtiter plates and subsequently testing individual clone supernatants for the desired reactivity (after about two to three weeks). Assays should be sensitive, simple, and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunobinding assays, and the like.

[0071] The selected hybridomas are then serially diluted or sorted by flow cytometry for single cell sorting and cloned into separate antibody-producing cell lines, which can then be indefinitely propagated to provide mAbs. Cell lines can be used for MAb production in two basic ways. Hybridoma samples can be injected (usually into the peritoneal cavity) into animals (e.g., mice). Optionally, animals are primed with hydrocarbons, particularly oils (e.g., pristane (tetramethylpentadecane)) before injection. When using human hybridomas in this way, it is best to inject immunodeficient mice (e.g., SCID mice) to prevent tumor rejection. Tumors secreting specific monoclonal antibodies produced by the fused cell hybrids appear in the injected animals. Then, the animal's body fluids, such as serum or ascites, can be released to provide high-concentration MAbs. Individual cell lines can also be cultured in vitro, where MAbs are naturally secreted into the culture medium, from which high-concentration MAbs can be easily obtained. Alternatively, human hybridoma cell lines can be used in vitro to produce immunoglobulins in cell supernatants. The cell lines can be adapted for growth in serum-free medium to optimize the ability to recover highly pure human monoclonal immunoglobulins.

[0072] If desired, MAbs produced in either manner can be further purified using filtration, centrifugation, and various chromatographic methods (e.g., FPLC or affinity chromatography). Fragments of the monoclonal antibodies of the present disclosure can be obtained from the purified monoclonal antibodies by methods including digestion with enzymes (e.g., pepsin or papain), and / or by cleavage of disulfide bonds by chemical reduction. Alternatively, monoclonal antibody fragments encompassed by the present disclosure can be synthesized using an automated peptide synthesizer.

[0073] It is also contemplated that molecular cloning methods can be used to generate monoclonal clones. To this end, RNA can be isolated from the hybridoma line, the antibody gene obtained by RT-PCR, and cloned into an immunoglobulin expression vector. Alternatively, a combinatorial immunoglobulin phagemid library can be prepared from RNA isolated from the cell line, and phagemids expressing appropriate antibodies can be selected by panning using viral antigens. This approach has the advantage over conventional hybridoma technology that up to about 10 clones can be generated and screened in a single round. 4 The team also discovered that the H and L chains combined to create new specificities, which further increased the chances of finding suitable antibodies.

[0074] Other U.S. patents, each incorporated herein by reference, that teach the production of antibodies useful in the present disclosure include U.S. Patent 5,565,332, which describes the use of combinatorial methods to produce chimeric antibodies; U.S. Patent 4,816,567, which describes recombinant immunoglobulin production; and U.S. Patent 4,867,973, which describes antibody-therapeutic agent conjugates.

[0075] B. Single-chain / single-domain antibodies

[0076] Single-chain variable fragment (scFv) is a fusion of the immunoglobulin heavy chain and light chain variable regions connected together with a short (usually serine, glycine) linker. This chimeric molecule is also referred to as a single domain antibody. Although it has removed the constant region and introduced a linker peptide, it retains the specificity of the original immunoglobulin. This modification does not usually change the specificity. Historically, these molecules were produced to facilitate phage display, where it is very convenient to express the antigen-binding domain as a single peptide. Alternatively, scFv can be directly produced by subcloning heavy and light chains derived from hybridomas. Single domain or single-chain variable fragments lack the constant Fc region present in the complete antibody molecule, and therefore lack common binding sites (e.g., protein A / G) for purifying antibodies (single-chain antibodies comprising the Fc region). These fragments can usually be purified / immobilized using protein L because protein L interacts with the variable region of κ light chains.

[0077] Flexible linkers are typically composed of amino acid residues that promote helices and turns (e.g., alanine, serine, and glycine). However, other residues can also work well. Phage display can be used as a way to quickly select specialized linkers for single-chain antibodies (scFv) from a library of protein linkers. A random linker library is constructed in which the genes for the heavy and light chain variable domains are connected by segments encoding 18-amino acid polypeptides with variable compositions. The scFv library (approximately 5×10 6 The tethers were selected from 1054 different members and affinity selected with haptens. The population of selected variants showed significantly improved binding activity, but retained considerable sequence diversity. Subsequent screening of 1054 individual variants yielded catalytically active scFvs that were efficiently produced in soluble form. Sequence analysis revealed that the only common features of the selected tethers were: V H The two residues after the C-terminus are conserved prolines in the linker and there are numerous arginines and prolines at other positions.

[0078] The recombinant antibodies of the present disclosure may also relate to sequences or portions that allow receptor dimerization or multimerization. Such sequences include those derived from IgA, which allow the formation of multimers in combination with the J chain. Another multimerization domain is the Gal4 dimerization domain. In other embodiments, the chains may be modified with reagents that allow the combination of two antibodies (e.g., biotin / avidin).

[0079] In an independent embodiment, single-chain antibodies can be produced by connecting the receptor light chain and heavy chain using a non-peptide linker or chemical unit. Generally speaking, the light chain and heavy chain are produced in different cells, purified, and then connected together in a suitable manner (i.e., the N-terminus of the heavy chain is connected to the C-terminus of the light chain by a suitable chemical bridge).

[0080] Cross-linking agents are used to form molecular bridges that tether the functional groups of two different molecules, for example, stabilizing and coagulant agents. However, it is anticipated that dimers or multimers of the same analog or heteromeric complexes containing different analogs may be produced. To link two different compounds in a stepwise manner, hetero-bifunctional cross-linking agents can be used, which eliminate the unwanted formation of homopolymers.

[0081] An exemplary hetero-bifunctional cross-linker comprises two reactive groups: one that reacts with primary amine groups (e.g., N-hydroxysuccinimide) and the other that reacts with thiol groups (e.g., pyridyl disulfide, maleimide, halogen, etc.) Through the primary amine-reactive group, the cross-linker can react with lysine residues of one protein (e.g., a selected antibody or fragment), and through the thiol-reactive group, the cross-linker already tethered to the first protein reacts with cysteine residues (free sulfhydryl groups) of another protein (e.g., a selection agent).

[0082] Preferably, a cross-linking agent with reasonable stability in blood is used. Various types of disulfide-containing linkers are known to be successfully used to conjugate targeting agents to therapeutic / prophylactic agents. Linkers containing sterically hindered disulfide bonds may be shown to provide higher stability in vivo, thereby preventing the targeting peptide from being released before reaching the site of action. Therefore, these linkers are a group of connecting agents.

[0083] Another cross-linking reagent is SMPT, a bifunctional cross-linker containing a disulfide bond that is "sterically hindered" due to the adjacent phenyl ring and methyl groups. It is believed that the steric hindrance of the disulfide bond functions to protect the bond from attack by thiolate anions (e.g., glutathione) that may be present in tissues and blood, and thereby helps prevent the conjugate from uncoupling before the attached agent is delivered to the target site.

[0084] Like many other known cross-linking reagents, SMPT cross-linking reagents are also capable of cross-linking functional groups such as the SH of cysteine or primary amines (e.g., the epsilon amino group of lysine). Another possible cross-linking agent type includes hetero-bifunctional photoreactive phenylazido groups containing cleavable disulfide bonds, such as sulfosuccinimidyl-2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionate. The N-hydroxy-succinimide group reacts with primary amino groups, and the phenylazido group (after photolysis) reacts non-selectively with any amino acid residue.

[0085] In addition to hindered crosslinkers, non-hindered crosslinkers can also be used accordingly. Other useful crosslinkers, regardless of whether they contain or generate protected disulfides, include SATA, SPDP, and 2-iminothiolane. The use of such crosslinkers is well known in the art. Another embodiment involves the use of flexible linkers.

[0086] U.S. Patent No. 4,680,338 describes bifunctional linkers that can be used to conjugate ligands with amine-containing polymers and / or proteins, particularly for forming antibody conjugates with chelators, drugs, enzymes, detectable labels, and the like. U.S. Patent Nos. 5,141,648 and 5,563,250 disclose cleavable conjugates containing labile bonds that are cleavable under a variety of mild conditions. Such linkers are particularly useful because the desired agent can be directly bonded to the linker, and cleavage thereof results in the release of the active agent. Particular applications include the addition of free amino or free sulfhydryl groups to proteins, such as antibodies or drugs.

[0087] U.S. Patent No. 5,856,456 provides peptide linkers for linking polypeptide components to prepare fusion proteins (e.g., single-chain antibodies). The linkers are up to about 50 amino acids in length; contain at least one occurrence of a charged amino acid (preferably arginine or lysine) followed by a proline, and are characterized by increased stability and reduced aggregation. U.S. Patent No. 5,880,270 discloses aminooxy group-containing linkers that can be used in a variety of immunodiagnostic and separation techniques.

[0088] C. Chimeric Antigen Receptors and Their Encoding Nucleic Acid Sequences

[0089] Artificial T cell receptor (also referred to as chimeric T cell receptor, chimeric immune receptor, chimeric antigen receptor (CAR)) is a modified receptor that can be transplanted to immune effector cells with any specificity. Typically, these receptors are used to transplant the specificity of monoclonal antibodies to T cells and promote the transfer of their coding sequences by retroviral vectors. In this way, a large number of cancer-specific T cells can be produced for adoptive cell transfer. Phase I clinical studies of this method have shown efficacy.

[0090] The most common form of these molecules is the fusion of a single-chain variable fragment (scFv) derived from a monoclonal antibody, which is fused to the CD3-ζ transmembrane and endodomains. Such molecules result in the transmission of ζ signals in response to the identification of their targets by scFv. An example of such a construct is 14g2a-ζ, a fusion of the scFv derived from the hybridoma 14g2a (recognition of disialoganglioside GD2). When T cells express this molecule (usually achieved by oncoretroviral vector transduction), they recognize and kill target cells (e.g., neuroblastoma cells) expressing GD2. In order to target malignant B cells, researchers have redirected the specificity of T cells using a chimeric immune receptor specific for the B lineage molecule CD19.

[0091] The variable parts of the immunoglobulin heavy and light chains are fused by a flexible linker to form an scFv. The scFv is preceded by a signal peptide to direct the nascent protein to the endoplasmic reticulum and subsequently to surface expression (which is cleaved). The flexible spacer allows the scFv to be oriented in different directions to enable antigen binding. The transmembrane domain is a typical hydrophobic alpha helix of the original molecule that is usually derived from the signaling endodomain, which protrudes into the cell and transmits the desired signal.

[0092] Type I proteins are actually two protein domains connected by a transmembrane alpha helix. The transmembrane domains act to separate the inner portion (endodomain) from the outer portion (ectodomain) through the lipid bilayer of the cell membrane. Not surprisingly, the ectodomain from one protein is linked to the endodomain of another protein to produce a molecule that combines the recognition of the former with the signal of the latter.

[0093] Extracellular domain. A signal peptide that directs the nascent protein to the endoplasmic reticulum. This is essential if the receptor is to be glycosylated and anchored in the cell membrane. Any eukaryotic signal peptide sequence generally works well. Typically, a signal peptide naturally associated with the most amino-terminal component is used (e.g., in scFvs with a light chain-linker-heavy chain orientation, the native signal of the light chain is used).

[0094] Antigen recognition domains are typically scFv. However, there are many alternatives. Antigen recognition domains from natural T cell receptors (T-cellreceptor, TCR) α and β single chains have been described, such as having a simple extracellular domain (e.g., the CD4 extracellular domain of HIV infected cells) and a more unique recognition component such as a connected cytokine (which results in recognition of cells with cytokine receptors). In fact, almost anything that binds to a given target with high affinity can be used as an antigen recognition region.

[0095] The spacer connects the antigen-binding domain to the transmembrane domain. It should be flexible enough to allow the antigen-binding domain to be oriented in different directions to facilitate antigen recognition. The simplest form is the hinge region from IgG1. Alternatives include the CH2CH3 region of immunoglobulins and part of CD3. For most scFv-based constructs, the IgG1 hinge is sufficient. However, the optimal spacer must often be determined empirically.

[0096] Transmembrane domain. The transmembrane domain is a hydrophobic alpha-helix that spans the membrane. Typically, the transmembrane domain from the component closest to the membrane in the endodomain is used. Interestingly, the use of the CD3-ζ transmembrane domain can lead to the incorporation of artificial TCRs into natural TCRs, a factor that depends on the presence of charged aspartate residues across the membrane of natural CD3-ζ. Different transmembrane domains lead to different receptor stabilities. The CD28 transmembrane domain produces a brightly expressed, stable receptor.

[0097] Endodomain. This is the "business-end" of the receptor. After antigen recognition, the receptor aggregates and transmits the signal to the cell. The most commonly used endodomain component is CD3-ζ, which contains three ITAMs. This transmits the activation signal to the T cell after antigen binding. CD3-ζ may not provide a fully effective activation signal and requires additional costimulatory signaling. For example, chimeric CD28 and OX40 can be used together with CD3-ζ to transmit proliferation / survival signals, or all three can be used together.

[0098] "First generation" CAR generally has an intracellular domain from CD3 ξ-chain, which is the main transmitter of the signal from endogenous TCR. "Second generation" CAR adds the intracellular signaling domain from a variety of costimulatory protein receptors (such as CD28, 4-1BB, ICOS) to the cytoplasmic tail of CAR to provide additional signals for T cells. Preclinical studies have shown that the second generation CAR design improves the anti-tumor activity of T cells. Recently, "third generation" CAR combines a variety of signaling domains, such as CD3z-CD28-4-1BB or CD3z-CD28-OX40, to further enhance effectiveness.

[0099] The adoptive transfer of T cells expressing chimeric antigen receptors is a promising anticancer treatment because CAR-modified T cells can be transformed to target almost any tumor-associated antigen. This method has great potential to improve patient-specific cancer treatment in a profound way. After collecting the patient's T cells, the cells are genetically modified to express CARs specifically for antigens on the patient's tumor cells and then transferred back into the patient. Although the adoptive transfer of CAR-modified T cells is a unique and promising cancer treatment, there are major safety issues. Clinical trials of this treatment reveal that when healthy tissues express the same target antigens as tumor cells, the potential toxic effects of these CARs lead to outcomes similar to graft-versus-host disease (GVHD). A potential solution to this problem is to transform suicide genes into modified T cells. In this way, the administration of prodrugs designed to activate suicide genes during GVHD can trigger apoptosis in the CAR T cells activated by suicide genes. This method has been safely and effectively used in hematopoietic stem cell transplantation (HSCT). The clinical application of suicide gene therapy in adoptive cell transfer of CAR-modified T cells has the potential to alleviate GVHD while improving overall antitumor efficacy.

[0100] In some embodiments of the CAR targeting GPC2 disclosed herein, the VH sequence is operably linked to the VL sequence downstream. In some embodiments, the VH sequence is operably linked to the VL sequence upstream. As used herein, the term "upstream" referring to an amino acid sequence refers to a position away from a reference point in the N-terminal to C-terminal direction of the amino acid sequence. Similarly, the term "downstream" refers to a position away from a reference point in the C-terminal to N-terminal direction of the amino acid sequence.

[0101] Typically, the transmembrane domain suitable for the CAR targeting GPC2 disclosed herein can be any transmembrane domain known in the art. Some non-limiting examples of suitable transmembrane domains include transmembrane domains derived from CD28 transmembrane domains, CD8a transmembrane domains, CTLA4 transmembrane domains, or PD-1 transmembrane domains. Therefore, in some embodiments, the CAR targeting GPC2 of the present disclosure includes a transmembrane domain derived from CD28 transmembrane domains, CD8a transmembrane domains, CTLA4 transmembrane domains, or PD-1 transmembrane domains. In some embodiments, the CAR targeting GPC2 includes a transmembrane domain derived from a CD28 transmembrane domain.

[0102] In some embodiments, the intracellular signaling domain of the CAR targeting GPC2 disclosed herein includes a costimulatory domain. Generally, the costimulatory domain suitable for the CAR targeting GPC2 disclosed herein can be any costimulatory domain known in the art. Some examples of suitable costimulatory domains include but are not limited to the costimulatory polypeptide sequences derived from 4-IBB (CD137), CD27, CD28, OX40 (CD 134) and costimulatory inducible T cell costimulatory (ICOS) polypeptide sequences. Therefore, in some embodiments, the costimulatory domain of the CAR targeting GPC2 disclosed herein is selected from costimulatory 4-IBB (CD137) polypeptide sequences, costimulatory CD27 polypeptide sequences, costimulatory CD28 polypeptide sequences, costimulatory OX40 (CD134) polypeptide sequences and costimulatory inducible T cell costimulatory (ICOS) polypeptide sequences. In some embodiments, the CAR targeting GPC2 includes a costimulatory domain derived from costimulatory 4-1BB (CD137) polypeptide sequences. In some embodiments, the CAR targeting GPC2 comprises a costimulatory domain derived from a costimulatory CD28 polypeptide sequence.

[0103] In some embodiments, the CAR targeting GPC2 further comprises an extracellular hinge domain (e.g., hinge region) or "joint". The term "hinge domain" generally refers to a flexible polypeptide connecting region or "joint" located between the targeting portion and the transmembrane domain. These sequences are generally derived from IgG subclasses (e.g., IgG1 and IgG4), IgD, and CD8 domains, of which IgG1 has been most widely used. In some embodiments, the hinge / joint domain provides structural flexibility for the flanking polypeptide region. The hinge / joint domain may be composed of natural or synthetic polypeptides. It will be understood by those skilled in the art that the hinge / joint domain can improve the function of CAR by promoting the optimal positioning of the antigen-binding portion relative to the antigen portion recognized by it. It should be understood that in some embodiments, a hinge / joint domain may not be required for optimal CAR activity. In some embodiments, a beneficial hinge / joint domain comprising a short amino acid sequence promotes CAR activity by, for example, reducing any spatial constraints that may additionally change antibody binding kinetics, thereby promoting antigen binding. The sequence encoding the hinge / joint domain may be located 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.

[0104] Hinge / joint sequence can be any part or sequence derived from or obtained from any suitable molecule. For example, in some embodiments, hinge / joint sequence can be derived from human CD8a molecule or CD28 molecule and any other receptor providing similar function in providing flexibility to the flanking region. The length of hinge / joint domain can be about 4 amino acids (aa) to about 50aa, such as about 4aa to about 10aa, about 10aa to about 15aa, about aa to about 20aa, about 20aa to about 25aa, about 25aa to about 30aa, about 30aa to about 40aa, or about 40aa to about 50aa. Suitable hinge / linker domains can be readily selected and can have any of a number of suitable lengths, such as 1 amino acid (e.g., Gly) to 20 aa, 2 aa to 15 aa, 3 aa to 12 aa, including 4 aa to 10 aa, 5 aa to 9 aa, 6 aa to 8 aa, or 7 aa to 8 aa, and can be 1, 2, 3, 4, 5, 6, or 7 aa.

[0105] The terms "long linker" and "short linker" are used throughout this application and mean the following:

[0106] "Long linker" amino acid sequence: GGGGSGGGGSGGGGS (SEQ ID NO: 4)

[0107] "Short linker" amino acid sequence: GGGGS (SEQ ID NO: 41).

[0108] Some non-limiting examples of suitable hinge / joint domains include a CD8 hinge domain, a CD28 hinge domain, a CTLA4 hinge domain, or an IgG4 hinge domain. In some embodiments, the hinge / joint domain may include a region derived from a human CD8a (akaCD8a) molecule or a CD28 molecule and any other receptor that provides similar functions in providing flexibility to the flanking regions. In some embodiments, the CAR targeting GPC2 disclosed herein comprises a hinge domain derived from a CD8a hinge domain. In some embodiments, the CAR targeting GPC2 disclosed herein comprises a hinge domain derived from a CD28 hinge domain.

[0109] In some embodiments, CAR disclosed herein further comprises an extracellular spacer domain containing one or more intermediate amino acid residues between an anti-GPC2 scFV region and an extracellular hinge / joint domain. In some embodiments, the extracellular hinge / joint domain is operably connected to the anti-GPC2 scFV region downstream and to the hinge / joint domain upstream. In principle, there are no particular restrictions on the length and / or amino acid composition of the extracellular spacer. In some embodiments, any arbitrary single-chain peptide comprising about 1 to about 300 amino acid residues (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. amino acid residues) can be used as an extracellular spacer. In some embodiments, the extracellular space comprises about 5 to 50, about 10 to 60, about 20 to 70, about 30 to 80, about 40 to 90, about 50 to 100, about 60 to 120, about 70 to 150, about 100 to 200, about 150 to 250, about 200 to 300, about 30 to 60, about 20 to 80, about 30 to 90 amino acid residues. In some embodiments, the extracellular space comprises about 1 to 10, about 50 to 100, about 100 to 150, about 150 to 200, about 200 to 300, about 20 to 80, about 40 to 120, about 200 to 250 amino acid residues. In some embodiments, the extracellular hinge / linker comprises about 40 to 70, about 50 to 80, about 60 to 80, about 70 to 90, or about 80 to 100 amino acid residues. In some embodiments, the extracellular hinge / linker comprises about 1 to 10, about 5 to 15, about 10 to 20, or about 15 to 25 amino acid residues. In some embodiments, the extracellular hinge / linker comprises about 220, 225, 230, 235, or 240 amino acid residues. In some embodiments, the extracellular hinge / linker comprises 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 / or proximity of the anti-GPC2 scFV region and the extracellular hinge / linker domain to each other to achieve the desired activity of the GPC2-targeted CAR. In some embodiments, the orientation and / or proximity of the anti-GPC2 scFV region and the extracellular hinge / linker domain to each other can be altered and / or optimized as a "modulatory" tool or effect that enhances or reduces the efficacy of the GPC2 CAR. In some embodiments, the orientation and / or proximity of the anti-GPC2 scFV region and the extracellular hinge / linker domain to each other can be altered and / or optimized to generate a partially functional or partially functional form of the GPC2 CAR. In some embodiments, the extracellular hinge / linker domain comprises an amino acid sequence corresponding to an IgG4 hinge domain and an IgG4 CH2-CH3 domain.

[0110] In some embodiments, the intracellular signaling domain of the CAR targeting GPC2 disclosed herein includes a CD3 zeta intracellular signaling domain. In some embodiments of the present disclosure, the CAR targeting GPC2 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 costimulatory domain derived from a 4-1BBz costimulatory domain or a CD28 costimulatory domain.

[0111] In one aspect, some embodiments of the present disclosure relate to a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a CAR targeting GPC2 as disclosed herein, or an antibody as disclosed herein.

[0112] The terms "nucleic acid molecule" and "polynucleotide" are used interchangeably herein and refer to both RNA and DNA molecules, including nucleic acid molecules comprising cDNA, genomic DNA, synthetic DNA, and DNA or RNA molecules comprising nucleic acid analogs. Nucleic acid molecules can be double-stranded or single-stranded (e.g., sense strand or antisense strand). Nucleic acid molecules can contain unconventional or modified nucleotides. As used herein, the terms "polynucleotide sequence" and "nucleic acid sequence" refer interchangeably to the sequence of a polynucleotide molecule.

[0113] The nucleic acid molecules of the present disclosure can be nucleic acid molecules of any length, including nucleic acid molecules that are generally about 5 Kb to about 50 Kb, for example, about 5 Kb to about 40 Kb, about 5 Kb to about 30 Kb, about 5 Kb to about 20 Kb, or about 10 Kb to about 50 Kb, for example, about 15 Kb to 30 Kb, about 20 Kb to about 50 Kb, about 20 Kb to about 40 Kb, about 5 Kb to about 25 Kb, or about 30 Kb to about 50 Kb.

[0114] In some embodiments, the recombinant nucleic acid molecule is operably linked to a heterologous nucleic acid sequence, such as a structural gene encoding a target protein 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 vector. In some embodiments, the vector is a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, or a retroviral vector.

[0115] Some embodiments disclosed herein relate to vectors or expression cassettes comprising recombinant nucleic acid molecules as disclosed herein. As used herein, the term "expression cassette" refers to a genetic material construct comprising a coding sequence and sufficient regulatory information to direct the correct transcription and / or translation of the coding sequence in a recipient cell in vivo and / or ex vivo. The expression cassette can be inserted into a vector for targeting to a desired host cell and / or into a subject. Thus, the term expression cassette can be used interchangeably with the term "expression construct."

[0116] Chimeric antigen receptors (CARs) according to the present disclosure can first be defined by their binding specificity, in this case, for glypican 2. CARs can also be defined by sequences disclosed herein, or can be different from the sequences provided above, optionally using methods discussed in more detail below. For example, the amino acid sequence may be different from those listed above in that (a) the variable region can be separated from the constant domain of the light chain, (b) the amino acid may be different from those listed above while not significantly affecting the chemical properties of the residues (so-called conservative substitutions), and (c) the amino acid may vary from those listed above with a given percentage, such as 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology. Alternatively, the nucleic acid encoding the antibody may (a) be separated from the constant domain of the light chain, (b) vary from those listed above without altering the residues encoded thereby, (c) vary from those listed above by a given percentage, e.g., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology, or (d) vary from those listed above by the ability to hybridize under high stringency conditions, as exemplified by low salt and / or high temperature conditions, e.g., provided by about 0.02 M to about 0.15 M NaCl at a temperature of about 50°C to about 70°C.

[0117] When making conservative changes to an amino acid sequence, the hydropathic index of the amino acids can be considered. The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte and Doolittle, 1982). It is accepted that the relative hydropathicity of amino acids contributes to the secondary structure of the resulting protein, which in turn defines the interaction of the protein with other molecules (e.g., enzymes, substrates, receptors, DNA, antibodies, antigens, etc.).

[0118] It is also understood in the art that similar amino acid substitutions can be effectively made based on hydrophilicity. U.S. Patent 4,554,101, incorporated herein by reference, states that the maximum local average hydrophilicity of a protein (as controlled by the hydrophilicity of its neighboring amino acids) is correlated with the biological properties of the protein. As detailed in U.S. Patent 4,554,101, the following hydrophilicity values have been 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 nonionic amino acids: serine (+0.3), asparagine (+0.2), glutamine (+0.3), and arginine (-0.5). The amino acids in the 100 amino acids were cysteine (-1.0), methionine (-1.3), leucine (-1.8), proline (-0.5±1), alanine (-0.5), and glycine (0). The amino acids in the 100 amino acids were cysteine (-1.0), methionine (-1.3), leucine (-1.8), valine (-1.5), isoleucine (-1.8), proline (-0.5±1), alanine (-0.5), and glycine (0). The amino acids in the 100 amino acids were tryptophan (-3.4), phenylalanine (-2.5), and tyrosine (-2.3).

[0119] It is understood that an amino acid can be replaced with another amino acid of similar hydrophilicity and produce a biologically or immunologically modified protein. In such changes, substitutions of amino acids whose hydrophilicity values are within ±2 are preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[0120] As outlined above, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions that take into account the various characteristics mentioned above are well known to those skilled in the art and include arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine.

[0121] D. Expression

[0122] According to the nucleic acid of the present disclosure will encode CAR. As used in this application, the term "nucleic acid encoding Glypican 2 CAR" refers to a nucleic acid molecule that has been isolated and does not contain total cell nucleic acid. In certain embodiments, the present disclosure relates to a receptor encoded by any sequence described herein.

[0123] Table 2 - Codons

[0124]

[0125] DNA segments of the present disclosure include those encoding biologically functional equivalent proteins of the above sequences. Such sequences may arise due to codon redundancy and amino acid functional equivalence known to occur naturally in nucleic acid sequences and the proteins encoded thereby. Alternatively, functionally equivalent proteins may be produced by the application of recombinant DNA techniques, wherein variations in protein structure may be engineered based on considerations of the properties of the exchanged amino acids. As described below, artificially designed variations may be introduced by the application of site-directed mutagenesis techniques, or they may be introduced randomly and subsequently screened for desired function.

[0126] Throughout this application, the term "expression construct" is intended to include any type of genetic construct comprising a nucleic acid encoding a gene product, wherein some or all of the nucleic acid coding sequence is capable of being transcribed. The transcript can, but is not required to, be translated into a protein. In certain embodiments, expression includes both gene transcription and translation of mRNA into a gene product. In other embodiments, expression includes only transcription of the nucleic acid encoding the gene of interest.

[0127] The term "vector" is used to refer to a carrier nucleic acid molecule into which a nucleic acid sequence can be inserted to be introduced into a cell into which it can replicate. The nucleic acid sequence can be "exogenous," meaning that it is foreign to the cell into which the vector is introduced, or that the sequence is homologous to a sequence in the cell but is in a position in the host cell nucleic acid in which the sequence is not normally present. Vectors include plasmids, cosmids, viruses (bacteriophages, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs). Those skilled in the art will readily construct vectors using standard recombinant techniques, as described in Sambrook et al. (1989) and Ausubel et al. (1994), both of which are incorporated herein by reference.

[0128] The term "expression vector" refers to a vector that contains a nucleic acid sequence encoding at least a portion of a gene product that can be transcribed. 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, in the production of antisense molecules or ribozymes. An expression vector may contain a variety of "control sequences," which refer to nucleic acid sequences required for transcription and translation of an operably linked coding sequence in a particular host organism. In addition to control sequences that control transcription and translation, vectors and expression vectors may also contain nucleic acid sequences that serve other functions, and are described below.

[0129] 1. Regulatory elements

[0130] A "promoter" is a control sequence that is a region of a nucleic acid sequence that controls the initiation and rate of transcription. A promoter may comprise genetic elements to which regulatory proteins and molecules (e.g., RNA polymerase and other transcription factors) can bind. The phrases "operably positioned," "operably linked," "under control," and "under transcriptional control" mean that the promoter is in the correct functional position and / or orientation relative to a nucleic acid sequence to control the initiation and / or expression of transcription of the sequence. A promoter may or may not be used in conjunction with an "enhancer," which refers to a cis-acting regulatory sequence that participates in the transcriptional activation of a nucleic acid sequence.

[0131] A promoter may be one naturally associated with a gene or sequence, as it can be obtained by isolating 5' non-coding sequences upstream of the coding segment and / or exons. Such promoters may be referred to as "endogenous." Similarly, an enhancer may be one naturally associated with a nucleic acid sequence, located upstream or downstream of the sequence. Alternatively, certain advantages may be obtained by placing a coding nucleic acid segment under the control of a recombinant or heterologous promoter, which is a promoter not normally associated with a nucleic acid sequence in its natural environment.

[0132] 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, promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, and promoters or enhancers that are not "naturally occurring" (i.e., contain different elements of different transcriptional regulatory regions and / or mutations that alter expression). In addition to synthetically generating nucleic acid sequences for promoters and enhancers, recombinant cloning and / or nucleic acid amplification techniques (including PCR) may be used in conjunction with the compositions disclosed herein. TM ) production sequences (see U.S. Pat. No. 4,683,202, U.S. Pat. No. 5,928,906, each of which is incorporated herein by reference). In addition, it is contemplated that control sequences that direct transcription and / or expression of sequences within non-nuclear organelles (e.g., mitochondria, chloroplasts, etc.) may also be used.

[0133] Of course, it will be very important to utilize promoters and / or enhancers that effectively direct the expression of the DNA segment in the cell type, organelle, and organism selected for expression. The use of promoters, enhancers, and cell type combinations for protein expression is generally known to those skilled in the art of molecular biology, for example, see Sambrook et al. (1989), incorporated herein by reference. The promoters used can be constitutive, tissue-specific, inducible, and / or useful under appropriate conditions to direct high-level expression of the introduced DNA segment, for example, to facilitate large-scale production of recombinant proteins and / or peptides. The promoter can be heterologous or endogenous. The properties of tissue-specific promoters or elements and assays to characterize their activity are well known to those skilled in the art. Some examples of such regions include: human LIMK2 gene (Nomoto et al., 1999), somatostatin receptor 2 gene (Kraus et al., 1998), mouse epididymal retinoic acid binding gene (Lareyre et al., 1999), human CD4 (Zhao-Emonet et al., 1998), mouse α2(XI) collagen (Tsumaki, et al., 1998), D1A dopamine receptor gene (Lee, et al., 1997), insulin-like growth factor II (Wu et al., 1997), and human platelet endothelial cell adhesion molecule-1 (Almendro et al., 1996).

[0134] The effective translation of the coding sequence may also require a specific initiation signal. These signals include the ATG initiation codon or adjacent sequences. It may be necessary to provide an exogenous translation control signal including the ATG initiation codon. One of ordinary skill in the art will be able to easily determine this and provide the required signal. As is well known, the initiation codon must be "in frame" with the reading frame of the desired coding sequence to ensure translation of the entire insert. Exogenous translation control signals and initiation codons can be natural or synthetic. Expression efficiency can be enhanced by comprising suitable transcription enhancer elements.

[0135] 2.IRES

[0136] In certain embodiments of the present disclosure, an internal ribosome entry site (IRES) element is used to generate polygenic or polycistronic messages. The IRES element can bypass the ribosome scanning model of 5' methylated Cap-dependent translation and start translation at the internal site (Pelletier and Sonenberg, 1988). IRES elements from two members of the Picornaviridae family (poliomyelitis and encephalomyocarditis) have been described (Pelletier and Sonenberg, 1988), as well as IRES from mammalian messages (Macejak and Sarnow, 1991). The IRES element can be connected to a heterologous open reading frame. Multiple open reading frames can be transcribed together, each separated by an IRES, thereby generating polycistronic messages. By virtue of the IRES element, each open reading frame is accessible to ribosomes for efficient translation. Multiple genes can be efficiently expressed using a single promoter / enhancer to transcribe a single message (see US Patents 5,925,565 and 5,935,819, incorporated herein by reference).

[0137] 3. Multipurpose cloning site

[0138] The vector may contain a multiple cloning site (MCS), which is a nucleic acid region containing multiple restriction enzyme sites, any one of which can be used in conjunction with standard recombinant techniques to digest the vector. See Carbonelli et al., 1999; Levenson et al., 1998; and Cocea, 1997, incorporated herein by reference. "Restriction enzyme digestion" refers to the catalytic cutting of a nucleic acid molecule with an enzyme that functions only at a specific position 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. Typically, a restriction enzyme that cuts within the MCS is used to linearize or fragment the vector so that the exogenous sequence can be connected to the vector. "Ligation" refers to the process of forming a phosphodiester bond between two nucleic acid fragments, which may or may not be continuous with each other. Techniques involving restriction enzymes and ligation reactions are well known to those skilled in the art of recombinant technology.

[0139] 4. Splice Sites

[0140] Most transcribed eukaryotic RNA molecules will undergo RNA splicing to remove introns from the primary transcript. Vectors containing genomic eukaryotic sequences may require donor and / or acceptor splice sites to ensure correct processing of the transcript for protein expression (see Chandler et al., 1997, incorporated herein by reference).

[0141] 5. Termination Signal

[0142] The vectors or constructs of the present disclosure will typically contain at least one termination signal. A "termination signal" or "terminator" consists of a DNA sequence that participates in the specific termination of an RNA transcript by RNA polymerase. Thus, in certain embodiments, termination signals that terminate the production of RNA transcripts are contemplated. A terminator may be necessary to achieve desired messenger levels in vivo.

[0143] In eukaryotic systems, the terminator region can also comprise a specific DNA sequence that allows site-specific cutting of new transcripts to expose a polyadenylation site. It signals a specialized endogenous polymerase to add a section of approximately 200 A residues (polyA) to the 3' end of the transcript. RNA molecules modified with this poly A tail demonstrate more stable and more effectively translation. Therefore, in other embodiments relating to eukaryotic organisms, preferably, the terminator comprises a signal for RNA cutting, and more preferably, the terminator signal promotes the polyadenylation of the messenger. Terminator and / or polyadenylation site elements can be used to increase messenger levels and / or minimize read-through from the box into other sequences.

[0144] Terminators contemplated for use in the present disclosure include any known transcription terminator described herein or known to those of ordinary skill in the art, including but not limited to, for example, a termination sequence of a gene, such as the bovine growth hormone terminator, or a viral termination sequence, such as the SV40 terminator. In certain embodiments, the termination signal may lack a transcribable or translatable sequence, for example, due to sequence truncation.

[0145] 6. Polyadenylation signal

[0146] In expression, particularly in eukaryotic expression, a polyadenylation signal will typically be included to achieve proper polyadenylation of the transcript. The nature of the polyadenylation signal is not believed to be critical to the successful practice of the present disclosure, and / or any such sequence may be employed. Some 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 transcript stability or promote cytoplasmic trafficking.

[0147] 7. Initiation of Replication

[0148] In order to propagate the vector in the host cell, it may contain one or more replication origin sites (commonly referred to as "ori"), which are specific nucleic acid sequences at which replication is initiated. Alternatively, if the host cell is yeast, an autonomously replicating sequence (ARS) may be used.

[0149] 8. Marker selection and screening

[0150] In certain embodiments of the present disclosure, cells comprising a nucleic acid construct of the present disclosure can be identified in vitro or in vivo by including a marker in the expression vector. Such a marker will impart identifiable changes to the cell, thereby allowing easy identification of cells comprising the expression vector. In general, a selection marker is a marker that imparts a characteristic that allows selection. A positive selection marker is a marker in which the presence of the marker allows selection, while a negative selection marker is a marker in which the presence of the marker prevents selection. An example of a positive selection marker is a drug resistance marker.

[0151] Generally comprising drug selection markers helps 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 conferring markers that allow the phenotype of transformants to be distinguished based on conditional implementation, other types of markers are also contemplated, including screening markers, such as GFP, which is based on colorimetric analysis. Alternatively, screening enzymes such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) can be utilized. Those skilled in the art also know how to use immune markers, which may be combined with FACS analysis. It is believed that the marker used is unimportant, as long as it can be expressed simultaneously with the nucleic acid encoding the gene product. Other examples of selection markers and screening markers are well known to those skilled in the art.

[0152] 9. Viral vectors

[0153] The ability of certain viral vectors to effectively infect or enter cells, integrate into the host cell genome, and stably express viral genes has led to the development and application of many different viral vector systems (Robbins et al., 1998). Viral systems currently under development are used as vectors for ex vivo and in vivo gene transfer. For example, adenovirus, herpes simplex virus, retrovirus, and adeno-associated virus vectors are currently being evaluated for the treatment of diseases such as cancer, cystic fibrosis, Gaucher disease, kidney disease, and arthritis (Robbins and Ghivizzani, 1998; Imai et al., 1998; U.S. Patent No. 5,670,488). Other viral vectors, such as poxviruses; e.g., vaccinia virus (Gnant et al., 1999; Gnant et al., 1999), alphaviruses; e.g., Sindbis virus, Semliki Forest virus (Lundstrom, 1999), reoviruses (Coffey et al., 1998), and influenza A virus (Neumann et al., 1999) are contemplated for use in the present disclosure and can be selected based on the necessary properties of the target system.

[0154] 10. Non-viral transformation

[0155] Suitable methods of nucleic acid delivery for transforming an organelle, cell, tissue, or organism for use in the present disclosure are contemplated to include virtually any method by which a nucleic acid (e.g., DNA) can be introduced into an organelle, cell, tissue, or organism, as described herein or known to those of ordinary skill in the art. Such methods include, but are not limited to, direct delivery of DNA, for example, by injection (U.S. Patents 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 of which is incorporated herein by reference), including microinjection (Harland and Weintraub, 1985; U.S. Patent 5,789,215, incorporated herein by reference); by electroporation (U.S. Patent 5,384,253, incorporated herein by reference); by calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990); by using DEAE-dextran followed by polyethylene glycol (Gopal, 1985); by direct sonication (Fechheimer et al., 1990); et al., 1987); by liposome-mediated transfection (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987; Wong et al., 1980; Kaneda et al., 1989; Kato et al., 1991); by microparticle bombardment (PCT Application Nos. WO 94 / 09699 and 95 / 06128; U.S. Patents 5,610,042; 5,322,783; 5,563,055; 5,550,318; 5,538,877; and 5,538,880, each of which is incorporated herein by reference); by stirring with silicon carbide fibers (Kaeppler et al., 1991); et al., 1990; U.S. Patents 5,302,523 and 5,464,765, each of which is incorporated herein by reference); or by PEG-mediated transformation of protoplasts (Omirulleh et al., 1993; U.S. Patents 4,684,611 and 4,952,500, each of which is incorporated herein by reference); by desiccation / inhibition-mediated DNA uptake (Potrykus et al., 1985). By applying techniques such as these, organelles, cells, tissues or organisms can be stably or transiently transformed.

[0156] 11. Expression System

[0157] There are many expression systems that comprise at least part or all of the compositions discussed above. Prokaryotic and / or eukaryotic systems can be used in conjunction with the present disclosure to produce nucleic acid sequences or their homologous polypeptides, proteins, and peptides. Many such systems are commercially available and widely available.

[0158] Insect cell / baculovirus systems can produce high levels of protein expression of heterologous nucleic acid segments, as described, for example, in U.S. Patents 5,871,986 and 4,879,236, both of which are incorporated herein by reference, and which can be used, for example, from By name 2.0 and from BacPack TM Baculovirus Expression System purchase.

[0159] Other examples of expression systems include Complete Control TM Inducible mammalian expression system, which involves a synthetic ecdysone inducible receptor, or its pET expression system, which is an E. coli expression system. Another example of an inducible expression system can be obtained from Obtained, which carries T-Rex TM (tetracycline-regulated expression) system, which is an inducible mammalian expression system using the full-length CMV promoter. Also provided is a yeast expression system called the Pichia methanolica expression system, which is designed for high-level production of recombinant proteins in the methylotrophic yeast Pichia methanolica. Those skilled 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.

[0160] Primary mammalian cell cultures can be prepared in a variety of ways. In order to maintain viability of cells in vitro and in contact with expression constructs, it is necessary to ensure that the cells maintain contact with the correct ratio of oxygen and carbon dioxide and nutrients, but protect them from microbial contamination. Cell culture techniques are well documented.

[0161] One embodiment described above involves immortalizing cells using gene transfer for protein production. The gene for the protein of interest can be transferred into a suitable host cell as described above, followed by culturing the cells under appropriate conditions. Virtually any polypeptide gene can be used in this manner. The generation of recombinant expression vectors and the elements contained therein have been discussed above. Alternatively, the protein to be produced can be an endogenous protein normally synthesized by the cell in question.

[0162] Some examples of available mammalian host cell lines are Vero and HeLa cells and Chinese hamster ovary cell lines, W138, BHK, COS-7, 293, HepG2, NIH3T3, RIN and MDCK cells. In addition, host cell strains that regulate the expression of inserted sequences or modify and process gene products in a desired manner can be selected. Such modification (e.g., glycosylation) and processing (e.g., cutting) of protein products can be important for the function of the protein. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins. Suitable cell lines or host systems can be selected to ensure the correct modification and processing of the foreign protein expressed.

[0163] Many selection systems can be used, including but not limited to the HSV thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase, and adenine phosphoribosyltransferase genes in tk-, hgprt-, or aprt- cells, respectively. Similarly, antimetabolite resistance can be used as the basis for selection: dhfr, which confers resistance; gpt, which confers resistance to mycophenolic acid; neo, which confers resistance to the aminoglycoside G418; and hygro, which confers resistance to hygromycin.

[0164] III. Pharmaceutical Preparations and Cancer Treatment

[0165] A. Cancer

[0166] Cancer is caused by the outgrowth of clonal populations of cells from tissues. The development of cancer (called carcinogenesis) can be modeled and characterized in a variety of ways. The association between the development of cancer and inflammation has long been recognized. The inflammatory response involves the host's defense against microbial infection and also drives tissue repair and regeneration. Considerable evidence suggests a link between inflammation and the risk of cancer development, i.e., chronic inflammation can lead to developmental abnormalities.

[0167] Cancer cells to which the methods of the present disclosure can be applied generally include any cell that expresses Glypican 2, and more specifically, cells that overexpress Glypican 2. Cancer cells that can be treated according to the present disclosure include, but are not limited to, cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal tract, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, pancreas, testis, tongue, cervix, or uterus. Among other things, the cancer may specifically be of the following histological types, although it is not limited thereto: malignant neoplasm; carcinoma; undifferentiated carcinoma; giant cell and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatricoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; malignant gastrinoma; bile duct carcinoma; hepatocellular carcinoma; combined hepatocellular and bile duct carcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyps; adenocarcinoma, familial polyposis coli; solid carcinoma; malignant carcinoid tumor; bronchoalveolar adenocarcinoma; papillary adenocarcinoma; chromophobe cell carcinoma; oncocytic carcinoma; oncocytic adenocarcinoma; basophilic granulocyte carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinomas; unencapsulated sclerosing carcinoma; adrenocortical carcinoma; endometrioid carcinoma; carcinoma of the skin appendages; apocrine adenocarcinoma adenocarcinoma; sebaceous gland carcinoma; cerumen carcinoma; mucoepidermoid carcinoma; cystic carcinoma; papillary cystic carcinoma; papillary serous cystic carcinoma; mucinous cystic carcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease of the breast; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia; malignant thymoma; malignant ovarian stromal tumor; malignant thecoma cell tumor; malignant granulosa cell tumor; malignant androblastoma; sertoli cell carcinoma; malignant Leydig cell tumor malignant lipocytoma; malignant ganglioneuroma; malignant extramammary paraganglioma; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant nevus; epithelioid cell melanoma; malignant blue nevus; sarcoma; fibrosarcoma; malignant fibrous histiocytoma; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; malignant mixed tumor; Müllerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; malignant mesenchymal tumor; malignant Brenner tumor; malignant phyllodes tumor; synovial sarcoma; malignant mesothelioma; dysgerminoma; embryonal carcinoma; malignant teratoma; malignant goiter; choriocarcinoma; malignant mesonephrosarcoma; angiosarcoma; malignant hemangioendothelioma;Kaposi's sarcoma; malignant hemangiopericytoma; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; malignant chondroblastoma; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; malignant odontogenic tumor; ameloblastic odontosarcoma; malignant ameloblastoma; ameloblastic fibrosarcoma; malignant pinealoma; chordoma; malignant glioma; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroglioma; primary neuroectodermal; cerebellar sarcoma; ganglioblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; malignant meningioma; neurofibrosarcoma; malignant neurilemoma; malignant granular cell tumor; malignant lymphoma; Hodgkin's disease disease); paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia. In certain aspects, the tumor can include osteosarcoma, angiosarcoma, rhabdomyosarcoma, leiomyosarcoma, Ewing sarcoma, glioblastoma, medulloblastoma, neuroblastoma, or leukemia.

[0168] In addition, the methods of the present disclosure can be applied to a variety of species, such as humans, non-human primates (e.g., monkeys, baboons, or chimpanzees), horses, cattle, pigs, sheep, goats, dogs, cats, rabbits, guinea pigs, gerbils, hamsters, rats, and mice. Cancer can also be recurrent, metastatic, and / or multidrug-resistant, and the methods of the present disclosure can be particularly applied to such cancers to make them resectable, prolong or reinduce remission, inhibit angiogenesis, prevent or limit metastasis, and / or treat multidrug-resistant cancers. At the cellular level, this can translate into killing cancer cells, inhibiting cancer cell growth, or otherwise reversing or reducing the malignant phenotype of tumor cells.

[0169] B. Formulation and Administration

[0170] The present disclosure provides a pharmaceutical composition comprising an anti-glypican 2 receptor and a cell expressing the same. In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of a federal or state government or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for use in animals and more particularly for 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, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Other suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, saline, dextrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, water, ethanol, etc.

[0171] The compositions can be formulated in neutral or salt form. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, and the like; and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, and the like.

[0172] The receptors, nucleic acids and cells of the present disclosure may include classical pharmaceutical formulations. Administration of these compositions according to the present disclosure may be by any common route, as long as the target tissue is accessible via that route. This includes oral, nasal, buccal, rectal, vaginal or topical administration. Alternatively, administration may be by intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Such compositions are typically administered as the pharmaceutically acceptable compositions described above. Of particular interest are direct intratumoral administration, tumor infusion or local or regional administration to the tumor, for example, in the local or regional vasculature or lymphatic system, or in a resected tumor bed.

[0173] The active compound can also be administered parenterally or intraperitoneally. Solutions of the active compound as free alkali or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant (e.g., hydroxypropylcellulose). Dispersions can also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, and in oils. Under common storage and use conditions, these preparations contain preservatives to prevent the growth of microorganisms.

[0174] C. Combination therapy

[0175] In the context of the present disclosure, it is also contemplated that the anti-Glypican 2 CAR T cells described herein can be similarly used in combination with immunotherapy, chemotherapy, or radiotherapy interventions or other treatments. In particular, combining anti-Glypican 2 CAR T cells with other treatments targeting different aspects of Glypican 2 function may also prove effective.

[0176] In order to kill cells, inhibit cell growth, inhibit metastasis, inhibit angiogenesis, or otherwise reverse or reduce the malignant phenotype of tumor cells using the methods and compositions of the present disclosure, "target" cells are typically 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 or inhibit cell proliferation. The process may include contacting cells with anti-Glypican 2 CAR T cells according to the present disclosure and other agents or factors simultaneously. This can be achieved by contacting cells with a single composition or pharmacological preparation comprising both agents, or by contacting cells with two different compositions or preparations simultaneously, wherein one composition comprises anti-Glypican 2 CAR T cells according to the present disclosure and the other comprises other agents.

[0177] Alternatively, anti-Glypican 2CAR T cell therapy can be performed at intervals of several minutes to several weeks before or after other drug treatments. In some embodiments in which other agents and anti-Glypican 2CAR T cells are applied separately to cells, it should generally be ensured that the important time period between each delivery does not expire, so that the agent and expression construct will still be able to play a favorable combination effect on the cell. In such a case, it is considered to contact the cell with these two forms within about 12 to 24 hours of each other, and the time is more preferably within about 6 to 12 hours of each other, and most preferably the delay time is only about 12 hours. However, in some cases, it can be expected to significantly extend the treatment period, wherein the time between each administration is separated by several days (2, 3, 4, 5, 6 or 7 days) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8 weeks).

[0178] It is also contemplated that more than one administration of anti-Glypican 2 CAR T cells or another agent may be desirable. A variety of combinations may be employed, wherein anti-Glypican 2 CAR T cells according to the present disclosure are "A" and another treatment is "B," as shown below:

[0179] A / B / AB / A / BB / B / AA / A / BB / A / AA / B / BB / B / B / AB / B / A / B

[0180] A / A / B / BA / B / A / BA / B / B / AB / B / A / AB / A / B / AB / A / A / BB / B / B / A

[0181] A / A / A / BB / A / A / AA / B / A / AA / A / B / AA / B / B / BB / A / B / BB / B / A / B

[0182] Other combinations are contemplated. Again, to achieve cell killing, the two agents are delivered to the cells in a combined amount effective to kill the cells. Agents or factors suitable for cancer treatment include any chemical compound or treatment method that induces damage when applied to cells. Such agents and factors include radiation and waves that induce DNA damage, such as irradiation, microwaves, electron emission, and the like. A variety of chemical compounds can be used, which are also described as "chemotherapeutic agents" or "genotoxic agents." This can be achieved by irradiating the local tumor site; alternatively, tumor cells can be contacted with the agent by administering a therapeutically effective amount of a pharmaceutical composition to the subject. Combination therapy can also include surgery. Various modes of these treatments are discussed below.

[0183] 1. Chemotherapy

[0184] The term "chemotherapy" refers to the use of drugs to treat cancer. "Chemotherapeutic agent" is used to refer to a compound or composition administered in the treatment of cancer. These agents or drugs are classified by their mode of activity within the cell (e.g., whether and at what stage they affect the cell cycle). Alternatively, agents can be characterized based on their ability to directly crosslink DNA, intercalate into DNA, or induce chromosomal and mitotic aberrations by affecting nucleic acid synthesis. Most chemotherapeutic agents fall into the following categories: alkylating agents, antimetabolites, antitumor antibiotics, mitotic inhibitors, and nitrosoureas.

[0185] Some examples of chemotherapeutic agents include alkylating agents, such as thiotepa and cyclophosphamide; alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquinone, meturedopa, and uredopa; ethylenimines and methylmelamines, including hexamethylmelamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide, and trimethylmelamine; acetogenins (especially bratacin and bratacinone); camptothecins (including the synthetic analog topotecan); ); bryostatin; callystatin; CC-1065 (including its synthetic analogs adolesin, carzelesin, and biszelesin); nostoc (particularly nostoc 1 and nostoc 8); dolastatin; duocarmycin (including its synthetic analogs KW-2189 and CB1-TM1); acanthopanax; hymenocampine; stoloniferin; sponge inhibitors; nitrogen mustards, such as chlorambucil, naphthalene mustard, chlorambucil, Phosphamide, estramustine, ifosfamide, mechlorethamine, methoxychlor hydrochloride, melphalan, nembicevic, phenylephrine, prednimustine, trofosfamide, uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics, such as enediynes (e.g., calicheamicins, particularly calicheamicin gamma 1I and calicheamicin omega 1I); dynemycins, including dynemycin A uncialamycin and its derivatives; bisphosphonates, such as clodronate; esperamicins; and the neocarcin chromophore and related chromoprotein enediyne antibiotic chromophores, aclarubicin, dactinomycin, anthramycin, azaserine, bleomycin, cactinomycin, carrubicin, carminomycin, chromomycin, dactinomycin, daunorubicin, detoxibacin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino- doxorubicin, 2-pyrrolinyl-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, mexilomycin, mitomycins (e.g., mitomycin C), mycophenolic acid, nogamycin, olivomycins, peplomycin, sphingomycin, puromycin, triferon-adriamycin, rhodorubicin, streptozotocin, streptozotocin, tuberculin, ubenimex, zoloft, daunorubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs, such as leucovorin, methotrexate, pteropterin, trimetrexate;Purine analogs, such as fludarabine, 6-mercaptopurine, thioimidazole, and thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens, such as calosterone, drostanolone propionate, cyclothiocarbamate, melastane, and testolactone; antiadrenal agents, such as aminoglutethimide, mitotane, and trilostane; folic acid supplements, such as folinic acid; aceglucuronolactone; aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine; besbucil; bisantrene; edatrexate; defosfamide famine; colcemid; diazocine; elonicetate; elliptonium acetate; epothilones; etoglucagon; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansine alkaloids, such as maytansine and ansamitocin; mitoxantrone; mitoxantrone; mopidarol; niteracrine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllic acid; 2-acetylhydrazine; procarbazine; PSK polysaccharide complex; razoxane; rhizoxin; sizolan; spirogermanium; tenuazonic acid acid); triimidoquinone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verrucosporin A, myclosan, and serpentin); urethane; vindesine; dacarbazine; mannomustine; dibromomannitol; dibromodulcitol; pipobroman; garcitocin; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxanes, such as paclitaxel and doxetine. tretinoin; 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; daunorubicin; aminopterin; xeloda; ibandronate; irinotecan Retinoids such as retinoic acid; capecitabine; cisplatin (CDDP); carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosoureas, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, mitomycin, etoposide (VP-16), tamoxifen, raloxifene, estrogen receptor binding agents, taxol, paclitaxel, docetaxel, gemcitabine, navelbine, farnesyl-protein transferase inhibitors, transplatin, 5-fluorouracil, vincristine, vinblastine, and methotrexate, as well as pharmaceutically acceptable salts, acids, or derivatives thereof.

[0186] 2. Radiation therapy

[0187] Radiotherapy, also known as radiation therapy, is the use of ionizing radiation to treat cancer and other diseases. Ionizing radiation deposits energy that damages or destroys cells in the area being treated by damaging their genetic material, making it impossible for them to continue growing. Although radiation damages both cancer cells and normal cells, the latter are able to repair themselves and function properly.

[0188] The radiation therapy used according to the present disclosure may include, but is not limited to, the use of gamma-rays, X-rays and / or radioisotopes directed to the delivery of tumor cells. Other forms of DNA damaging factors, such as microwaves and UV irradiation, have also been considered. Most likely, all of these factors induce extensive damage to the DNA, the precursors of the DNA, the replication and repair of the DNA, and to the assembly and maintenance of chromosomes. The dosage range of X-rays is a single dose of 2000 to 6000 roentgens over a period of time (3 to 4 weeks) of continuous extension from a daily dose of 50 to 200 roentgens. The dosage range of radioisotopes varies greatly and depends on the half-life of the isotope, the intensity and type of the emitted radiation, and the uptake of neoplastic cells.

[0189] Radiation therapy may include using radiolabeled antibodies to deliver radiation doses directly to the cancer site (radioimmunotherapy). Antibodies are highly specific proteins produced by the body in response to the presence of antigens (substances recognized as foreign by the immune system). Some tumor cells contain specific antigens that trigger the production of tumor-specific antibodies. Large quantities of these antibodies can be prepared in the laboratory and linked to radioactive substances (a process called radiolabeling). Once injected into the body, the antibodies actively seek out cancer cells, which are destroyed by the cell-killing (cytotoxic) effects of the radiation. This approach minimizes the risk of radiation damage to healthy cells.

[0190] Conformal radiation therapy uses the same radiation therapy machine and linear accelerator as conventional radiation therapy, but a metal block is placed in the path of the x-ray beam to change its shape to match the shape of the cancer. This ensures that a higher radiation dose is given to the tumor. Healthy surrounding cells and nearby structures receive lower doses of radiation, thus reducing the possibility of side effects. A device called a multi-leaf collimator has been developed and can be used as an alternative to metal blocks. A multi-leaf collimator consists of multiple metal pieces fixed to the linear accelerator. The layers can be adjusted so that the radiation therapy beam can be shaped into the treatment area without the need for metal blocks. Precise positioning of the radiation therapy machine is very important for conformal radiation therapy, and a special scanner can be used to check the position of internal organs at the beginning of each treatment.

[0191] High-resolution intensity-modulated radiation therapy also uses a multi-leaf collimator. During this treatment, the layers of the multi-leaf collimator are moved while the treatment is being delivered. This approach has the potential to achieve even more precise shaping of the treatment beam and keep the radiation therapy dose constant across the entire treatment area.

[0192] While studies have shown that conformal radiation therapy and intensity-modulated radiation therapy can reduce the side effects of radiation therapy, shaping the treatment area so precisely can prevent microscopic cancer cells just outside the treatment area from being destroyed. This means that the risk of future cancer recurrence can be higher when these specialized radiation therapy techniques are used.

[0193] Scientists are also looking for ways to increase the effectiveness of radiation therapy. Two types of investigational drugs are being studied for their effects on cells undergoing radiation. Radiosensitizers make tumor cells more likely to be destroyed, and radioprotectants shield normal tissue from the effects of radiation. Hyperthermia, a method of using heat, is also being studied for its effectiveness in sensitizing tissue to radiation.

[0194] 3. Immunotherapy

[0195] In cancer treatment, immunotherapy generally relies on the use of immune effector cells and molecules to target and destroy cancer cells. TM ) is such an example. Immune effectors can be, for example, antibodies that are specific for some markers on the surface of tumor cells. A single antibody can be used as an effector for treatment or it can recruit other cells that actually affect cell killing. Antibodies can also be conjugated to drugs or toxins (chemotherapeutic agents, radionuclides, ricin A chain, cholera toxin, pertussis toxin, etc.) and used only as targeting agents. Alternatively, the effector can be a lymphocyte carrying a surface molecule that interacts directly or indirectly with the tumor cell target. Various effector cells include cytotoxic T cells and NK cells. The combination of treatment modalities (i.e., direct cytotoxic activity and inhibition or reduction of ErbB2) will provide therapeutic benefits in treating cancers in which ErbB2 is overexpressed.

[0196] In one aspect of immunotherapy, tumor cells must carry some markers suitable for targeting (i.e., not present on most 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 to combine anticancer effects with immunostimulatory effects. There are also immunostimulatory molecules, which include: 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. Combining immunostimulatory molecules (either as proteins or using gene delivery in combination with tumor suppressors) has been shown to enhance anti-tumor effects (Ju et al., 2000). In addition, antibodies against any of these compounds can be used to target the anti-cancer agents discussed herein.

[0197] Some examples of immunotherapies currently under investigation or use are immune adjuvants (e.g., Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds) (U.S. Patents 5,801,005 and 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al., 1998); cytokine therapy (e.g., interferon α, β, and γ; IL-1, GM-CSF, and TNF) (Bukowski et al., 1998; Davidson et al., 1998; Hellstrand et al., 1998); gene therapy (e.g., TNF, IL-1, IL-2, p53) (Qin et al., 1998); and immunotherapy (e.g., TNF, IL-1, IL-2, p53) (Qin et al., 1998). et al., 1998; Austin-Ward and Villaseca, 1998; U.S. Patents 5,830,880 and 5,846,945) and monoclonal antibodies (e.g., anti-ganglioside GM2, anti-HER-2, anti-p185) (Pietras et al., 1998; Hanibuchi et al., 1998; U.S. Patent 5,824,311). It is contemplated that one or more anti-cancer therapies can be used in conjunction with the gene silencing therapies described herein.

[0198] In active immunotherapy, antigenic peptides, polypeptides or proteins, or autologous or allogeneic tumor cell compositions or "vaccines" are usually administered with various bacterial adjuvants (Ravindranath and Morton, 1991; Morton et al., 1992; Mitchell et al., 1990; Mitchell et al., 1993).

[0199] In adoptive immunotherapy, circulating lymphocytes or tumor-infiltrating lymphocytes from patients are isolated in vitro, activated by lymphokines (eg, IL-2) or transduced with tumor necrosis genes, and re-administered (Rosenberg et al., 1988; 1989).

[0200] 4. Surgery

[0201] About 60% of people with cancer will undergo some type of surgery, which includes preventive, diagnostic or staging, curative, and palliative surgery. Curative surgery is a cancer treatment that can be used in conjunction with other treatments, such as the treatment of the present disclosure, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapies.

[0202] Curative surgery includes resection in which all or a portion of the cancerous tissue is physically removed, excised, and / or destroyed. Tumor resection refers to the physical removal of at least a portion of the tumor. In addition to tumor resection, surgical treatment also includes laser surgery, cryosurgery, electrosurgery, and microscopically controlled surgery (Mohs' surgery). It is also contemplated that the present disclosure may be used in conjunction with the removal of superficial cancers, precancers, or accompanying amounts of normal tissue.

[0203] After removing a part or all of cancer cells, tissue or tumor, a cavity can be formed in vivo. Treatment can be completed by perfusion, direct injection or other anticancer treatment of the region. Such treatment can be repeated, for example, every 1, 2, 3, 4, 5, 6 or 7 days, or every 1, 2, 3, 4 and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months. These treatments also can have multiple doses.

[0204] In some embodiments, adjuvant therapy with the compounds of the present disclosure following tumor removal is believed to be particularly effective in reducing tumor recurrence. Additionally, the compounds of the present disclosure may also be used in the neoadjuvant setting.

[0205] It should also be noted that any of the aforementioned treatments may prove useful in the treatment of cancer. Those skilled in the art will refer to Chapter 33 of "Remington's Pharmaceutical Sciences," 15th edition, particularly pages 624-652. Dosage will inevitably vary somewhat depending on the subject being treated. In any case, the person administering the drug will determine the appropriate dosage for the individual subject. Furthermore, for human administration, the formulation should meet the sterility, pyrogenicity, general safety, and purity standards required by the FDA's Office of Biologics Standards.

[0206] IV. Kit

[0207] In other embodiments, a kit for use in the above methods is provided. Thus, the kit will comprise a CAR, a nucleic acid encoding a CAR, or a cell that first expresses a CAR that binds to a Glypican 2 antigen in an appropriate container device.

[0208] The container means of the test kit generally includes at least one vial, test tube, flask, bottle, syringe or other container means in which the cells can be placed, or preferably the cells can be appropriately aliquoted. The test kit also includes a device for enclosing and restricting the CAR, nucleic acid or cells and any other reagents for commercial sale. Such a container may include an injection-molded or blow-molded plastic container in which the desired vial is held.

[0209] V. Examples

[0210] The following examples are included to demonstrate some preferred embodiments. It will be understood by those skilled in the art that the technology disclosed in the following examples represents the technology that the inventor has found to work well in the practice of the embodiments and can therefore be considered to constitute the preferred mode of its practice. However, based on the disclosure of the present invention, it will be understood by those skilled in the art that many changes can be made in the disclosed specific embodiments without departing from the spirit and scope of the present disclosure and still obtaining the same or similar results.

[0211] Example 1

[0212] A panel of three fully human antibodies (m201, m202, and m203) that specifically target cancer cell-associated GPC2 were isolated from a phage display antibody library and affinity matured. In vitro characterization showed that these antibodies have promising therapeutic activity in the development of CAR-T, antibody-drug conjugates (ADCs), and bispecific antibodies for cancer treatment. Figure 1 、 Figure 2 and Figure 3 Shown in.

[0213] GPC2 has recently been identified as a new oncogene and immunotherapy target in neuroblastoma and medulloblastoma. The inventors used GPC2-specific scFv paired with 4-1BB and CD3ζ costimulatory domains to create multiple different RNACAR constructs with different heavy and light chain orientations and linker lengths between chains. They evaluated CAR persistence, T cell exhaustion markers, and cytotoxicity against four primary and two isogenic neuroblastoma cell lines and three primary HGG cell lines. By flow cytometry, all four constructs showed >80% CAR expression and GPC2 specific binding. The CAR molecules of the light to heavy (VL-VH) configuration were shown to persist on the surface for more than seven days and had increased cytotoxicity compared to the heavy to light (VH-VL) configuration. The VH-VL configuration with a long linker provided the weakest cytotoxic effect, and evaluation of negative checkpoint regulators revealed the highest expression of PD1 and Lag3 (62% compared to 17 to 40% in other constructs, p<0.0001). Based on in vitro data, two VL-VH CAR constructs were selected for testing in a mouse flank model of neuroblastoma treated with IV GPC2 CAR T cells (once a week for a total of three doses). At day 14, animals treated with these two VL-VH CAR constructs had a reduced tumor burden (p<0.01) compared to the CD19CAR control, with several animals showing complete responses. Studies are currently underway to evaluate the efficacy of local delivery in a pediatric HGG orthotopic model.

[0214] Stable expression of multiple CAR T cell constructs was achieved by engineering a second-generation DNA-based CAR vector based on two reported GPC2 scFvs (D3 and D4) followed by retroviral transduction in primary human T cells. Initial constructs engineered with the CD8a hinge and transmembrane domains and the 4-1BBz signaling domain, with either an N-terminal variable heavy chain or an N-terminal variable light chain in two orientations (Figure 9B), showed stable cell surface expression and bound soluble recombinant GPC2 (Figure 9C). These constructs demonstrated efficient in vitro potency and cytokine production (IFNγ, IL2) against isogenic target cells engineered to express GPC2 at levels comparable to in vivo levels of GPC2 (Kelly-GPC2) at an effector to target ratio of 1:1 (Figure 9C). Figure 11A-D Furthermore, the inventors demonstrated that incorporation of CD28-H / TM and co-stimulatory domains into these CAR constructs exhibited additional CAR T cell potency advantages when targeting GPC2-expressing tumors ( Figure 14A-B). Taken together, these data show that using DNA-based CAR vectors and viral transduction, stable GPC2-targeting CAR T cells can be engineered to produce highly effective killing effects on GPC2-expressing cancer cells.

[0215] These data show that mRNA provides a rapid and iterative approach for testing new CAR T cells and that GPC2 is a promising CAR T cell target in a subset of neuroblastoma, medulloblastoma, and high-grade gliomas and other pediatric malignant brain tumors. In a mouse model, RNAGPC2CAR T cells expressing light to heavy D3 scFv chains with a long linker configuration provided the strongest cytotoxic effect with no evidence of toxicity.

[0216] Through lentivirus ( Figure 15A-B 、 Figure 15C-D and Figure 15E-F ) and retroviruses ( Figure 16A and Figure 16B )-transduced GPC2 DNACAR T cells based on D3 (M201) also had efficient cytotoxicity against preclinical models of neuroblastoma. GPC2 CAR was robustly expressed on T cells (Figure 15A) and was cytotoxic to isogenic SY5Y-GPC2 neuroblastoma cells (Figure 15B). Co-culture resulted in simultaneous T cell activation and increased expression of INFγ and CD107AT cells (Figure 15B). Figure 15C-D ). D3(M201) long linker 28 / 28 / 4-1BB (D3(M201)-based GPC2 CAR with CD28-based hinge / CD28-based Tm / 4-1BB co-stimulatory domain) and long linker 28 / 28 / 28 (D3(M201)-based GPC2 CAR with CD28-based hinge / CD28-based Tm / CD28 co-stimulatory domain) showed potent in vivo activity, inducing robust regression of COG-N-421x neuroblastoma patient-derived xenograft tumors and were very well tolerated ( Figure 15E-F In a metastatic SMS-SAN neuroblastoma model, D3(M201)-based GPC2 CAR T cells also induced tumor regression ( Figure 16A and Figure 16B )

[0217] *****************

[0218] According to the present disclosure, all compositions and methods disclosed and claimed herein can be prepared and implemented without excessive experimentation. Although the compositions and methods of the present disclosure have been described according to some preferred embodiments, it is obvious to those skilled in the art that, without departing from the concept, spirit and scope of the present disclosure, the steps or the sequence of steps of the compositions and methods and methods described herein can be changed. More specifically, it is obvious that certain reagents related to both chemistry and physiology can be substituted for the reagents described herein, and the same or similar results will be obtained simultaneously. All such similar substitutions and modifications obvious to those skilled in the art are considered to be within the spirit, scope and concept of the present disclosure as defined in the appended claims.

[0219] The following content corresponds to the original claims in the parent application and is incorporated herein as part of the specification:

[0220] 1. An isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR),

[0221] wherein the CAR comprises an antigen binding domain, a flexible hinge domain, a transmembrane domain, a costimulatory signaling region, and an intracellular signaling domain, and

[0222] The antigen-binding domain selectively binds to cancer cell-associated glypican 2 (GPC2).

[0223] 2. The isolated nucleic acid molecule of claim 1 , wherein the antigen binding domain comprises an antibody or an antigen binding fragment thereof.

[0224] 3. The isolated nucleic acid molecule of claim 2, wherein the antigen binding fragment is a Fab, a single-chain variable fragment (scFv), or a single-domain antibody.

[0225] 4. The isolated nucleic acid molecule of items 1 to 3, wherein the encoded antigen binding domain comprises:

[0226] (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

[0227] (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

[0228] (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.

[0229] 5. The isolated nucleic acid molecule of items 1 to 3, wherein the encoded antigen binding domain comprises:

[0230] (a) comprises the following heavy chain variable domain: 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 the following light chain variable domain: 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

[0231] (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

[0232] (c) comprises the following heavy chain variable domain: 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 comprises the following light chain variable domain: 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.

[0233] 6. The isolated nucleic acid molecule of claim 2, wherein:

[0234] (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

[0235] (b) The C-terminus of the light chain variable domain is fused to the N-terminus of the heavy chain variable domain via a flexible linker.

[0236] 7. The isolated nucleic acid molecule of claim 6, wherein the linker is a peptide linker.

[0237] 8. The isolated nucleic acid molecule of claim 7 , wherein the peptide linker is at least 15 amino acids in length.

[0238] 9. The isolated nucleic acid molecule of claim 8, wherein the peptide linker is a glycine-serine linker.

[0239] 10. The isolated nucleic acid molecule of claim 1 , wherein:

[0240] (a) the flexible hinge domain is derived from CD8α, CD28 or immunoglobulin (Ig),

[0241] (b) the transmembrane domain comprises a CD28 transmembrane domain,

[0242] (c) the co-stimulatory signaling region comprises a domain from CD28, 41BB (CD137), OX40 or ICOS, and

[0243] (d) The intracellular signaling domain comprises a CD3-ζ domain or a high affinity FcεRI.

[0244] 11. A chimeric antigen receptor (CAR) polypeptide, wherein:

[0245] (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

[0246] (b) The antigen-binding domain selectively binds to cancer cell-associated glypican 2 (GPC2).

[0247] 12. The chimeric antigen receptor polypeptide of claim 11, wherein the antigen binding fragment is a Fab, a single-chain variable fragment (scFv), or a single-domain antibody.

[0248] 13. The chimeric antigen receptor (CAR) polypeptide of claim 11 to 12, wherein the encoded antigen binding domain comprises:

[0249] (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

[0250] (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

[0251] (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.

[0252] 14. The chimeric antigen receptor (CAR) polypeptide of items 11 to 12, wherein the encoded antigen binding domain comprises:

[0253] (a) comprises the following heavy chain variable domain: 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 the following light chain variable domain: 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

[0254] (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

[0255] (c) comprises the following heavy chain variable domain: 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 comprises the following light chain variable domain: 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.

[0256] 15. The chimeric antigen receptor polypeptide of claim 13, wherein:

[0257] (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

[0258] (b) The C-terminus of the light chain variable domain is fused to the N-terminus of the heavy chain variable domain via a flexible linker.

[0259] 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 of items 1 to 15.

[0260] 17. A genetically modified T cell comprising the chimeric antigen receptor of any one of items 11 to 16.

[0261] 18. A method for preparing genetically modified T cells, comprising transducing immune effector cells with the chimeric antigen receptor of items 11 to 16.

[0262] 19. A method for providing anti-tumor immunity in a mammal, comprising administering to the mammal an effective amount of the population of genetically modified T cells of item 16.

[0263] 20. A method for treating a mammal suffering from a disease associated with overexpression of GPC2, the method comprising administering to the mammal an effective amount of the population of genetically modified T cells of item 16.

[0264] 21. The genetically modified T cell of claim 16, wherein:

[0265] (a) the CAR induces the secretion of interferon-γ and interleukin-2, and

[0266] (b) When the genetically modified T cells are exposed to cancer cell-associated GPC2, the genetically modified T cells exhibit cytotoxicity against cancer cells expressing GPC2.

[0267] 22. The method of claim 20, wherein the cancer expressing GPC2 is selected from the group consisting of sarcoma cells, rhabdoid carcinoma cells, neuroblastoma cells, retinoblastoma cells or medulloblastoma cells, uterine carcinosarcoma (UCS), brain low-grade glioma (LGG), thymoma (THYM), testicular germ cell tumor (TGCT), glioblastoma multiforme (GBM) and cutaneous melanoma (SKCM), hepatocellular carcinoma (LIHC), uveal melanoma (UVM), renal chromophobe cell carcinoma (KICH), thyroid carcinoma (THCA), renal clear cell renal carcinoma (KIRC), renal papillary cell renal carcinoma (KRC), The patients were classified as having ovarian cancer (OC), ovarian cancer (OV), ovarian squamous cell carcinoma (LUSC), bladder urothelial carcinoma (BLCA), sarcoma (SARC), or uterine corpus endometrial carcinoma (UCEC).

[0268] VII. References

[0269] The following references are specifically incorporated herein by reference, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein.

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Claims

1. A chimeric antigen receptor (CAR) that selectively binds to cancer cell-associated glypican 2 (GPC2), wherein the CAR comprises an antigen-binding domain, a flexible hinge domain, a transmembrane domain, a costimulatory signaling region, and an intracellular signaling domain. wherein the antigen binding domain comprises: (a) comprises the following heavy chain variable domain: a CDR1 consisting of the amino acid sequence of SEQ ID NO: 17, a CDR2 consisting of the amino acid sequence of SEQ ID NO: 18, and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 19, and comprises the following light chain variable domain: a CDR1 consisting of the amino acid sequence of SEQ ID NO: 20, a CDR2 consisting of the amino acid sequence of SEQ ID NO: 21, and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 22; or (b) a heavy chain variable domain comprising a CDR1 consisting of 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; wherein the C-terminus of the light chain variable domain is fused to the N-terminus of the heavy chain variable domain via a flexible peptide linker; and wherein the CAR exhibits enhanced persistence on the cell surface when compared to a CAR comprising a conventional VH_linker_VL configuration.

2. The CAR of claim 1, wherein the antigen binding domain is a single-chain variable fragment (scFv).

3. The CAR of claim 1 or 2, wherein the antigen binding domain comprises: (a) 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 (b) 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.

4. The CAR of claim 3, wherein the C-terminus of the light chain variable domain is fused to the N-terminus of the heavy chain variable domain via a flexible glycine-serine peptide linker.

5. The CAR of claim 4, wherein the flexible glycine-serine peptide linker is at least 15 amino acids in length.

6. The CAR of claim 1, wherein the flexible peptide linker is at least 15 amino acids in length.

7. The CAR of claim 1, wherein the flexible peptide linker is a glycine-serine linker.

8. The CAR of claim 1, wherein the flexible hinge domain is derived from CD8α, CD28, or immunoglobulin (Ig).

9. The CAR of claim 1, wherein the transmembrane domain comprises a CD28 transmembrane domain.

10. The CAR of claim 1, wherein the co-stimulatory signaling region comprises a domain from CD28, 4-1BB (CD137), OX40, or ICOS.

11. The CAR of claim 1, wherein the intracellular signaling domain comprises a CD3-ζ domain or a high affinity FcεRI domain.

12. The CAR of claim 1 or 3, wherein the CAR comprises a CD8α flexible hinge domain, a CD28 transmembrane domain, a CD28 or 4-1BB co-stimulatory signaling region, and a CD3-ζ intracellular signaling domain.

13. A vector comprising a nucleic acid encoding the CAR of any one of claims 1 to 12.

14. The vector of claim 13, wherein the antigen binding domain of the CAR comprises: (a) a heavy chain variable (VH) domain encoded by a nucleotide sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 33 and a light chain variable (VL) domain encoded by a nucleotide sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 35; or (b) a heavy chain variable (VH) domain encoded by a nucleotide sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 37 and a light chain variable (VL) domain encoded by a nucleotide sequence at least 70%, 80%, or 90% identical to SEQ ID NO:

39.

15. A vector comprising a nucleic acid encoding a CAR, wherein the CAR comprises an antigen binding domain, wherein the antigen binding domain comprises: (a) a heavy chain variable (VH) domain encoded by a nucleotide sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 29 and a light chain variable (VL) domain encoded by a nucleotide sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 31; or (b) a heavy chain variable (VH) domain encoded by a nucleotide sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 33 and a light chain variable (VL) domain encoded by a nucleotide sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 35; or (c) a heavy chain variable (VH) domain encoded by a nucleotide sequence at least 70%, 80%, or 90% identical to SEQ ID NO: 37 and a light chain variable (VL) domain encoded by a nucleotide sequence at least 70%, 80%, or 90% identical to SEQ ID NO:

39.

16. The vector of claim 13 or 15, wherein the vector is a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, or a retroviral vector.

17. A genetically modified cell comprising the CAR of any one of claims 1 to 12, or the vector of any one of claims 13 to 16.

18. The genetically modified cell of claim 17, wherein: (a) the CAR induces the secretion of interferon-γ and interleukin-2, and (b) when the genetically modified cells are exposed to the cancer cell-associated GPC2, the genetically modified cells exhibit cytotoxicity against cancer expressing GPC2.

19. The genetically modified cell of claim 17, wherein the genetically modified cell is a T cell or a NK cell.

20. A method for preparing genetically modified cells, comprising transducing immune effector cells with the vector of any one of claims 13 to 16.

21. A pharmaceutical composition comprising the genetically modified cell of any one of claims 17 to 19, or the vector of any one of claims 13 to 16 and a pharmaceutically acceptable carrier.

22. A pharmaceutical composition comprising an antibody derivative that selectively binds to cancer cell-associated glypican 2 (GPC2), wherein the antibody derivative comprises: (a) comprises the following heavy chain variable domain: a CDR1 consisting of the amino acid sequence of SEQ ID NO: 11, a CDR2 consisting of the amino acid sequence of SEQ ID NO: 12, and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 13, and comprises the following light chain variable domain: a CDR1 consisting of the amino acid sequence of SEQ ID NO: 14, a CDR2 consisting of the amino acid sequence of SEQ ID NO: 15, and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 16; or (b) a heavy chain variable domain comprising a CDR1 consisting of the amino acid sequence of SEQ ID NO: 17, a CDR2 consisting of the amino acid sequence of SEQ ID NO: 18, and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 19, and a light chain variable domain comprising a CDR1 consisting of the amino acid sequence of SEQ ID NO: 20, a CDR2 consisting of the amino acid sequence of SEQ ID NO: 21, and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 22; or (c) a heavy chain variable domain comprising a CDR1 consisting of 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; wherein the C-terminus of the light chain variable domain is fused to the N-terminus of the heavy chain variable domain via a flexible peptide linker; and wherein said antibody derivative exhibits enhanced persistence on the cell surface when compared to an antibody derivative comprising a conventional VH_linker_VL configuration.

23. The pharmaceutical composition of claim 22, wherein the antibody derivative is selected from the group consisting of: a single-chain variable fragment (scFv), a chimeric antigen receptor, an antibody drug conjugate (ADC), and a bispecific antibody.

24. Use of the pharmaceutical composition of any one of claims 21 to 23 in the preparation of a medicament for providing anti-tumor immunity in a mammal having a cancer cell-associated Glypican 2 (GPC2)-positive tumor.

25. The use of the pharmaceutical composition of claim 24, wherein the cancer expresses the cancer cell-associated GPC2.

26. The use of the pharmaceutical composition of claim 25, wherein the cancer is selected from the group consisting of neuroblastoma cells, retinoblastoma cells, or medulloblastoma cells, brain low-grade glioma (LGG), high-grade glioma (HGG), ATRT, DIPG, CPC, CPP, craniopharyngioma, DNET, ependymoma, MPNST, meningioma, pineoblastoma, PCET, and chordoma.

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