Chimeric antigen receptors containing phosphatidylinositol proteoglycan 2-binding domains

Abstract

The present disclosure relates to chimeric antigen receptors that bind to Glypican 2, nucleic acids encoding the same, and cells expressing the same, as well as methods of using such cells to treat cancers that express or overexpress the Glypican 2 antigen.

Description

[0001] Priority claim

[0002] This application claims the benefit of priority to U.S. Provisional Application Serial No. 62 / 876,483, filed on July 19, 2019, the entire contents of which are hereby incorporated by reference.

[0003] Statement Regarding Federal Funding

[0004] 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.

[0005] Pursuant to 37 C.FR §1.821(c), the Sequence Listing is submitted herein as an ASCII compatible text file, which is named "CHOPP0034WO.txt," which was created on July 17, 2020, and has a size of approximately 27 kilobytes. The contents of the above file are hereby incorporated by reference in their entirety. background 1. Technical Field

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

[0008] 2. Related technologies

[0009] Children with high-risk neuroblastoma have a poor prognosis despite intensive multimodal chemoradiotherapy. Although monoclonal antibodies targeting the disialoganglioside GD2 have improved outcomes in neuroblastoma, this treatment is associated with significant "on target-off tumor" toxicity. 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 tissues, and preferably these cell surface molecules are essential for tumor maintenance.

[0010] 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 T cells that activate the host immune system to prevent cancer cell proliferation or kill cancer cells has become a promising treatment method to supplement the existing standard 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

[0011] Therefore, according to the present disclosure, a chimeric antigen receptor is provided, which comprises (i) an extracellular domain comprising a single-chain antibody variable region fragment (scFv) region containing a variable heavy chain (VH) and a variable light chain (VL) that selectively bind to phosphatidylinositol proteoglycan 2, (ii) a transmembrane domain; and (iii) an endodomain, wherein when the scFv is engaged with phosphatidylinositol proteoglycan 2, the endodomain comprises a signal transduction function.

[0012] 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.

[0013] The scFv may be characterized by VH and VL sequences having 80% homology to SEQ ID NOs: 5 and 6, respectively, and having 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 having 80% homology to SEQ ID NOs: 7 and 8, respectively, and having 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 having 80% homology to SEQ ID NOs: 9 and 10, respectively, and having VH CDRs of SEQ ID NOs: 23 to 25 and VL CDRs of SEQ ID NOs: 26 to 28.

[0014] The receptor may be characterized by VH and VL sequences having 90% homology to SEQ ID NOs: 5 and 6, respectively, and having 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 having 90% homology to SEQ ID NOs: 7 and 8, respectively, and having 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 having 90% homology to SEQ ID NOs: 9 and 10, respectively, and having VH CDRs of SEQ ID NOs: 23 to 25 and VL CDRs of SEQ ID NOs: 26 to 28.

[0015] 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.

[0016] The transmembrane and endodomains may be derived from the same molecule. The endodomain may comprise a CD3-ζ domain or a high affinity FcεRI. The scFv may comprise a flexible linker between the VH and VL, for example wherein the flexible linker is from CD8α, Ig or SEQ ID NO: 4. The scFv may be arranged as VH-linker-VL or VL-linker-VH.

[0017] Also provided are nucleic acids, such as mRNA or DNA, encoding a chimeric antigen receptor as defined above, or cells, such as prokaryotic cells or eukaryotic cells, and in particular engineered T cells, expressing a chimeric antigen receptor as defined above.

[0018] 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, such as a T cell, such as an autologous T cell of the subject.

[0019] 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.

[0020] 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 myeloid leukemia (CML), chronic lymphocytic leukemia (CLL) and hairy cell leukemia.

[0021] 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), and 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 (STAD), cholangiocarcinoma (CHOL), adenoid cystic carcinoma (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 rectal adenocarcinoma (COAD), rectum adenocarcinoma (COAD), and ovarian adenocarcinoma (COAD).READ), esophageal carcinoma (ESCA), ovarian cancer (OV), lung squamous cell carcinoma (LUSC), bladder urothelial carcinoma (BLCA), sarcoma (SARC), or uterine corpus endometrial carcinoma (UCEC).

[0022] Also provided is an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen binding domain, a flexible hinge domain, a transmembrane domain, a co-stimulatory signaling region, and an intracellular signaling domain, and 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 an amino acid sequence of SEQ ID NO: 30 and a light chain variable domain comprising an amino acid sequence of SEQ ID NO: 32; or (b) a heavy chain variable domain comprising an amino acid sequence of SEQ ID NO: 34, and a light chain variable domain comprising an amino acid sequence of SEQ ID NO: 36, or (c) a heavy chain variable domain comprising an amino acid sequence of SEQ ID NO: 38 and a light chain variable domain comprising an amino acid sequence of SEQ ID NO: 40.

[0023] 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.

[0024] 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.

[0025] The isolated nucleic acid molecule may 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 a high affinity FcεRI.

[0026] 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 may be a Fab, a single-chain variable fragment (scFv), or a single-domain antibody.

[0027] 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.

[0028] 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.

[0029] 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) the C-terminus of the light chain variable domain fused to the N-terminus of the heavy chain variable domain via a flexible linker.

[0030] 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 a genetically modified T cell comprising a chimeric antigen receptor as defined herein. The genetically modified T cell may have the following characteristics:

[0031] (a) CAR that induces secretion of interferon-γ and interleukin-2, and

[0032] (b) When genetically modified T cells are exposed to cancer cell-associated GPC2, they exhibit cytotoxicity against GPC2-expressing cancers.

[0033] The cancer expressing GPC2 may be selected from the following: 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 of the skin (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), ovarian cystic fibrosis cell carcinoma (FCC), ovarian cystic fibrosis cell carcinoma (VFC ... 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 carcinoma (UCEC).

[0034] Also provided is a method for preparing genetically modified T cells, comprising transducing immune effector cells with a chimeric antigen receptor as defined herein. Also provided is a method 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 is a method for treating a mammal suffering from a disease associated with overexpression of GPC2, comprising administering to the mammal an effective amount of a population of genetically modified T cells as defined herein.

[0035] It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein.

[0036] When used in conjunction with the term "comprising" in the claims and / or the specification, a noun without a quantifier may mean "one", but it is also consistent with the meaning of "one or more", "at least one", and "one or more than one". The word "about" means plus or minus 5% of the stated number.

[0037] Through the following detailed description, other purposes, features and advantages of the present disclosure will become apparent. However, it should be understood that the detailed description and specific examples, although pointing out some specific embodiments of the present disclosure, are given by way of illustration only, because through the detailed description, multiple changes and modifications within the spirit and scope of the present disclosure will become apparent to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The 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.

[0039] 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 referring to one or more of these drawings in combination with the detailed description of specific embodiments given herein.

[0040] Figure 1 . Heavy and light chain amino acid and nucleic acid sequences of human antibody m201 The CDRs are shown in bold italics.

[0041] Figure 2 . Heavy and light chain amino acid and nucleic acid sequences of human antibody m202 The CDRs are shown in bold italics.

[0042] Figure 3 . Heavy and light chain amino acid and nucleic acid sequences of human antibody m203 The CDRs are shown in bold italics.

[0043] Figure 4 . GPC2 is expressed in a subset of pediatric brain tumorsThe RNA sequencing data came from the Childhood Brain Tumor Tissue Consortium (CBTTC), which contained 1,110 samples.

[0044] Figure 5A to C In vitro validation of binding and persistence of GPC2 RNACAR T cells (FIG. 5A) GPC2 RNA CAR binding specifically to GPC2 of four GPC2 RNA CAR T cell constructs, measured by flow cytometry. (FIG. 5B) Persistence of CAR for each construct over time, measured by flow cytometry. (FIG. 5C) Negative checkpoint regulators for each construct expressed PD1 and Lag3 four days after transfection.

[0045] Figure 6A to D D3V3 and D3V4 mRNAs produced the strongest cytotoxicity in vitro in PC2CART cells . (FIG. 6A) Cytotoxicity of four GPC2 CAR T cell constructs against SMS-SAN, a neuroblastoma cell line with endogenous high expression of GPC2. E:T ratio 10:1. (FIG. 6B) Interferon gamma degranulation by GPC2 CAR T cell constructs was measured by ELISA across multiple cell lines with different GPC2 expression at different E:T ratios. (FIG. 6C) Cytotoxicity and interferon gamma released by D3V3 and D3V4 CAR T cells against DAOY medulloblastoma cell line. E:T ratio 10:1. (FIG. 6D) Cytotoxicity and interferon gamma released by D3V3 and D3V4 CAR T cells against 7316-913 high-grade glioma cell line. E:T ratio 5:1.

[0046] Figure 7A to C D3V3 mRNAGPC2 CAR T cells in NB-1643 patient-derived xenografts (patient- Derived xenograft (PDX) models showed the greatest in vivo cytotoxicity . (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).

[0047] Figure 8 . Schematic diagram for CAR-T cell therapy and GPC2 RNA CAR construct design .

[0048] Figure 9A to C. Alignment of amino acid sequences of GPC2 single chain variable fragments and derived GPC2CAR constructs DA. (FIG. 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. (FIG. 9B) Schematic diagram of CAR T cell constructs for testing 2 different scFvs in the variable heavy chain-linker-variable light chain and variable light chain-linker-variable heavy chain orientations. (FIG. 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.

[0049] Fig.10 . Expression of GPC2 in neuroblastoma cell lines The cell surface expression of GPC2 on neuroblastoma cell lines and CHO negative control was stained with D3-IgG (labeled with Dylight650).

[0050] Figure 11A to D Used to drive cytokine production, killing, and low-tonic signaling in the absence of antigen Capabilities of binder-based prioritization of CAR T cells based on the indication of antigen exposure . (FIG. 11A) IFNy secretion of all constructs in response to tumor cells with overexpression (Kelly-GPC2) and native GPC2 site density (NBSD), and (FIG. 11B) Baseline IFNy secretion of CAR T cells in the absence of antigen. (FIG. 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. (FIG. 11D) IL-2 secretion of GPC2 CAR T cells in response to overexpression (Kelly-GPC2) and native GPC2 site density (NBSD).

[0051] Figure 12A to D The engineered CAR constructs are ineffective against tumors expressing endogenous GPC2 antigen density (FIG. 12A) Site density of GPC2 on overexpressed engineered isogenic Kelly-GPC2 and endogenous GPC2 expressing neuroblastoma cell lines NBSD and SMS-SAN was measured using Quantibrite beads. (FIG. 12B) IFNy secretion of GPC2 CAR constructs in response to overexpressed and endogenous GPC2 site density. (FIG. 12C) Ability of GPC2 CAR T cells to kill isogenic Kelly-GPC2 and (FIG. 12D) native GPC2 cell lines when challenged with 5x excess tumor cells.

[0052] Figure 13A to E. CAR T cells containing a CH2CH3 spacer domain fail to improve GPC2CAR function. (FIG. 13A) Schematic diagram of GPC2 CAR constructs containing an IgG4-derived CH2CH3 spacer domain. (FIG. 13B) Expression of D3VLVH.GPC2 long and short CAR T cells assessed by staining with soluble recombinant GPC2. (FIG. 13C) In vitro expansion of short and long GPC2.19 CAR T cells shown as days post-activation. (FIG. 13D) Killing capacity 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.

[0053] Figure 14A to B GPC2 CAR T cell constructs incorporating 28 transmembrane and signaling domains effectively target natural killer cells GPC2 site density (FIG. 14A) Cytokine production (IFNy on the left, IL-2 on the right) of GPC2.D3VLVH CAR T cells compared to constructs incorporating the CD28 hinge / transmembrane domain and 4-1BBz or CD28 signaling domain. (FIG. 14B) Killing capacity of GPC2.D3VLVH CAR T cells compared to constructs incorporating the CD28 hinge / transmembrane domain and 4-1BBz or CD28 signaling domain.

[0054] Figure 15A to F D3(M201)-based GPC2 DNA CAR T cells have an inhibitory effect on preclinical models of neuroblastoma Potent cytotoxicity . (FIG. 15A) GPC2 CAR expression on T cells. D3(M201) long linker 28 / 28 / 4-1BB, a D3(M201)-based GPC2 CAR with a CD28-based hinge / CD28-based Tm domain / 4-1BB co-stimulatory domain and a long linker; 28 / 28 / 28, a D3(M201)-based GPC2 CAR with a CD28-based hinge / CD28-based Tm domain / CD28 co-stimulatory domain and a long linker. (FIG. 15B) Percent cytotoxicity of SY5Y-GPC2 cells of 8 different D3-based CAR constructs compared to UTD T cell controls. (FIG. 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) Xenograft tumor growth of neuroblastoma COG-N-421x patients after treatment with D3 / M201 based GPC2 CAR T cells. (FIG. 15F) The average weight of the mouse treatment group is shown in FIG. 15E. UTD, untransduced T cells.

[0055] Fig.16A To B. D3(M201)-VLVH-based CAR T cells in the SMS-SAN metastatic xenograft model Antitumor efficacy . ( Fig.16A) Research plan. Fig. 16B ) BLI data based on different D3(M201)-VLVH in SMS-SAN metastatic model (correlated with tumor volume). *, p<0.05'; **, p<0.005; ***, p<0.0005; ****, p<0.00005. DETAILED DESCRIPTION

[0056] The recent identification of 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 DNA constructs expressing CAR molecules targeting GPC2.

[0057] 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 toxicity for clinical testing.

[0058] These and other aspects of the disclosure are described in further detail below.

[0059] I. Glypican 2

[0060] Glypican-2 (GPC2) is a member of the six-member glypican family of heparan sulfate (HS) proteoglycans, which are attached to the cell surface via a glycosylphosphatidylinositol (GPI) anchor and play multiple roles in growth factor signaling and cancer cell growth. GPC2 is also known as cerebrospinal proteoglycan and glypican proteoglycan 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 numbers NM_152742 and NP_689955, respectively, which are incorporated herein by reference. Cell surface GPC2 proteins have been shown to be expressed in the developing nervous system, are involved in cell adhesion, and are thought to regulate the growth and guidance of axons.

[0061] GPC2 has recently been identified as a cell surface protein of several cancers, including pediatric cancers such as neuroblastoma, high-grade glioma (HGG), medulloblastoma, and several other pediatric cancers and adult malignancies, which represents an opportunity for the development of new targeted immunotherapy. For example, in pediatric cancers, GPC2 has been shown to be expressed at considerable levels on neuroblastoma, retinoblastoma, and medulloblastoma, while showing limited normal tissue expression. In addition, subsets of acute lymphoblastic leukemia, high-grade glioma, and rhabdomyosarcoma express GPC2. GPC2 is also highly expressed on small cell lung cancer, which is a common and almost universally lethal cancer. In addition, many adult malignancies can benefit from GPC2 targeted immunotherapy, such as using data derived from The Cancer Genome Atlas (TCGA) to evaluate GPC2 expression in adult cancers. 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 shows a high degree of differential expression between tumors and normal tissues.

[0062] II. Production of Monoclonal Antibodies

[0063] A. General Methods

[0064] 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. Pat. No. 4,196,265). Methods for producing monoclonal antibodies (MAbs) generally begin along the same routes as those used to prepare polyclonal antibodies. The first step in both methods is to immunize a suitable host or identify an object that has been 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, such as 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 non-specific stimulators of the immune response known as adjuvants. Exemplary and preferred adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response comprising killed Mycobacterium tuberculosis), incomplete Freund's adjuvant, and aluminum hydroxide adjuvant.

[0065] The amount of the immunogenic composition used to produce polyclonal antibodies varies according to the nature of the immunogen and the animal used for immunization. Immunogens can be administered using a variety of routes (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal). The production of polyclonal antibodies can be monitored by sampling blood from the immunized animals at different points after immunization. A second booster injection can also be given. The booster and titer determination process is repeated until a suitable titer is reached. When the desired immunogenicity level is obtained, blood can be taken from the immunized animals, the serum can be separated and stored, and / or the animals can be used to produce MAbs.

[0066] 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 B lymphocytes from the immunized animals producing antibodies are fused with cells of immortalized myeloma cells, which are usually immortalized myeloma cells of the same species as the immunized animals 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 defects, which subsequently prevents it from growing in certain selection media that only support the growth of the desired fusion cells (hybridomas).

[0067] 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, where 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 conjunction with human cell fusion. One particular murine myeloma cell 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 mouse myeloma cell line that can be used is the 8-azaguanine-resistant mouse 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.

[0068] Methods for producing hybrids of antibody-producing spleen cells or lymph node cells and myeloma cells generally include 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 the 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. It is also suitable to use an electrically induced fusion method (Goding, pp. 71 to 74, 1986).

[0069] Fusion procedures usually start with about 1×10 -6 Up to 1×10 -8 The low frequency of producing living heterozygotes. However, this does not cause problems, because by cultivating in the selection medium, the living fusion heterozygotes are distinguished from the unfused cells of the parents (particularly the unfused myeloma cells that will continue to divide indefinitely under normal circumstances). The selection medium is usually a culture medium containing a reagent that blocks the de novo synthesis of nucleotides in the tissue culture medium. Exemplary and preferred reagents are aminopterin, methotrexate and azaserine. Aminopterin and methotrexate block the de novo synthesis of both purine and pyrimidine, while azaserine only blocks purine synthesis. When using aminopterin or methotrexate, hypoxanthine and thymidine are supplemented in the culture medium as the source of nucleotides (HAT culture medium). When using azaserine, hypoxanthine is supplemented in the 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 system that is not fused with myeloma.

[0070] Preferred selection medium is HAT or HAT with ouabain. Only cells that can carry out nucleotide salvage pathway can survive in HAT medium. Myeloma cells are defective in the key enzyme of salvage pathway (e.g., hypoxanthine phosphoribosyl transferase (HPRT)), so they cannot survive. B cells can carry out this pathway, but they have a limited life span in culture and usually die within about two weeks. Therefore, only cells that can survive in the selection medium are those hybrids formed by myeloma and B cells. When the source of the B cell used for fusion is the B cell line transformed by EBV, ouabain is also used for the drug selection of the hybrid at this time, because the B cell transformed by EBV is susceptible to drug killing, and the myeloma partner used in the selection is resistant to ouabain.

[0071] Cultivation provides a population of hybridomas from which specific hybridomas are selected. Selection of hybridomas is usually performed by culturing cells by monoclonal dilution in microtiter plates, followed by testing of individual clone supernatants for desired reactivity (after about two to three weeks). The assay should be sensitive, simple and rapid, such as radioimmunoassay, enzyme immunoassay, cytotoxicity assay, plaque assay, spot immunobinding assay, etc.

[0072] 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 infinitely 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, the 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 that secrete specific monoclonal antibodies produced by the fused cell hybrids appear in the injected animals. Then, the body fluids of the animals, such as serum or ascites, can be released to provide high concentrations of MAbs. Single cell lines can also be cultured in vitro, where MAbs are naturally secreted into the culture medium, from which high concentrations of 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 to grow in serum-free medium to optimize the ability to recover highly pure human monoclonal immunoglobulins.

[0073] 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.

[0074] It is also contemplated that molecular cloning methods may be used to generate monoclonal clones. To this end, RNA may be isolated from the hybridoma line, the antibody genes obtained by RT-PCR, and cloned into an immunoglobulin expression vector. Alternatively, a combinatorial immunoglobulin phagemid library may be prepared from RNA isolated from the cell line, and phagemids expressing the appropriate antibodies may be selected by panning using viral antigens. This approach has the advantage over conventional hybridoma technology that up to about 10 clones may be generated and screened in a single round. 4 The antibodies are multiplied by 2.5 times, and new specificities are generated by combining H and L chains, which further improves the chances of discovering suitable antibodies.

[0075] 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.

[0076] B. Single-chain / single-domain antibodies

[0077] Single-chain variable fragment (scFv) is a fusion of immunoglobulin heavy chain and light chain variable region connected together with a short (usually serine, glycine) linker. This chimeric molecule is also referred to as a single domain antibody, which retains the specificity of the original immunoglobulin although the constant region has been removed and a linker peptide has been introduced. 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 subclone 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 the common binding sites (e.g., protein A / G) for purifying antibodies (single-chain antibodies comprising Fc regions). These fragments can usually be purified / fixed using protein L because protein L interacts with the variable region of κ light chains.

[0078] Flexible linkers are usually 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 protein linker library. A random linker library was constructed in which the genes for the heavy and light chain variable domains were connected by segments encoding 18-amino acid polypeptides with variable compositions. The scFv library (about 5×10 6 The selected population of variants showed significantly improved binding activity, but retained considerable sequence diversity. Subsequent screening of 1054 individual variants resulted in 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 abundant arginines and prolines at other positions.

[0079] The recombinant antibodies of the present disclosure may also involve sequences or portions that allow receptor dimerization or multimerization. Such sequences include those derived from IgA, which allow the formation of multimers in conjunction with the J chain. Another multimerization domain is the Gal4 dimerization domain. In other embodiments, the chains may be modified with reagents that allow two antibodies to combine (e.g., biotin / avidin).

[0080] In an independent embodiment, single-chain antibodies can be produced by connecting receptor light and heavy chains using non-peptide linkers or chemical units. Generally, light and heavy chains are produced in different cells, purified, and then connected together in a suitable manner (i.e., the N-terminal of the heavy chain is connected to the C-terminal of the light chain by a suitable chemical bridge).

[0081] Cross-linking agents are used to form molecular bridges that tether the functional groups of two different molecules, for example, stabilizing and coagulants. However, it is contemplated that dimers or multimers of the same analogs or heteromeric complexes containing different analogs may be produced. In order to link two different compounds in a stepwise manner, hetero-bifunctional cross-linking agents may be used, which eliminate unwanted homopolymer formation.

[0082] 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., the 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., the selection agent).

[0083] Preferably, a cross-linking agent with reasonable stability in blood is used. It is known that various types of disulfide-containing linkers can be successfully used to conjugate targeting agents and therapeutic / prophylactic agents. Linkers containing sterically hindered disulfide bonds can 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 linkers.

[0084] Another cross-linking reagent is SMPT, a bifunctional cross-linker containing a disulfide bond that is "sterically hindered" by the adjacent benzene 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 decoupling before the attached agent is delivered to the target site.

[0085] Like many other known cross-linking reagents, the SMPT cross-linking reagent is also able to cross-link functional groups such as the SH of cysteine ​​or primary amines (e.g., the epsilon amino group of lysine). Another possible type of cross-linking agent includes hetero-bifunctional photoreactive phenylazido groups containing cleavable disulfide bonds, such as sulfosuccinimidyl-2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionate. The N-hydroxy-succinimidyl group reacts with primary amino groups, and the phenylazido group (after photolysis) reacts non-selectively with any amino acid residue.

[0086] In addition to hindered crosslinkers, non-hindered crosslinkers may also be used accordingly. Other useful crosslinkers, without regard to the inclusion or generation of protected disulfides, include SATA, SPDP, and 2-iminothiolane. The use of such crosslinkers is well understood in the art. Another embodiment involves the use of a flexible linker.

[0087] U.S. Patent No. 4,680,338 describes bifunctional linkers that can be used to produce conjugates of 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 agent of interest can be directly bonded to the linker and its cleavage results in the release of the active agent. Particular uses include the addition of free amino groups or free sulfhydryl groups to proteins such as antibodies or drugs.

[0088] U.S. Patent No. 5,856,456 provides peptide linkers for connecting polypeptide components to prepare fusion proteins (e.g., single-chain antibodies). The linker is up to about 50 amino acids in length; contains at least one occurrence of a charged amino acid (preferably arginine or lysine) followed by a proline, and is characterized by greater stability and reduced aggregation. U.S. Patent No. 5,880,270 discloses aminooxy-containing linkers that can be used in a variety of immunodiagnostic and separation techniques.

[0089] C. Chimeric Antigen Receptor and Its Encoding Nucleic Acid Sequence

[0090] Artificial T cell receptors (also referred to as chimeric T cell receptors, chimeric immune receptors, chimeric antigen receptors (CAR)) are modified receptors 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.

[0091] The most common form of these molecules is the fusion of the single-chain variable fragment (scFv) derived from a monoclonal antibody, which is fused with the CD3-ζ transmembrane and endodomain. Such molecules cause the transmission of ζ signals in response to the identification of its target by scFv. An example of such a construct is 14g2a-ζ, which is a fusion of the scFv derived from hybridoma 14g2a (recognition of disialoganglioside GD2). When T cells express this molecule (usually achieved by oncoretroviral vector transduction), they identify and kill target cells (such as neuroblastoma cells) expressing GD2. In order to target malignant B cells, researchers use chimeric immune receptors specific for B lineage molecules CD19 to redirect the specificity of T cells.

[0092] 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.

[0093] Type I proteins are actually two protein domains connected by a transmembrane alpha helix between them. The cell membrane lipid bilayer through which the transmembrane domain passes serves to separate the inner part (endodomain) from the outer part (ectodomain). Not surprisingly, the ectodomain from one protein is connected to the endodomain of another protein to produce a molecule that combines the recognition of the former with the signal of the latter.

[0094] 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 attached to the most amino-terminal component is used (e.g., in a scFv with a light chain-linker-heavy chain orientation, the native signal of the light chain is used.

[0095] Antigen recognition domains are usually scFv. However, there are many alternatives. Antigen recognition domains from natural T cell receptors (T-cell receptor, TCR) α and β single chains have been described, such as having simple extracellular domains (e.g., CD4 extracellular domains that recognize HIV infected cells) and more unique recognition components such as connected cytokines (which lead to 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.

[0096] 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 best spacer must usually be determined empirically.

[0097] 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.

[0098] 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 3 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 co-stimulatory 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.

[0099] "First generation" CARs typically have intracellular domains from CD3 ξ-chains, which are the main transmitters of signals from endogenous TCRs. "Second generation" CARs add intracellular signaling domains from a variety of co-stimulatory 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" CARs combine a variety of signaling domains, such as CD3z-CD28-4-1BB or CD3z-CD28-OX40, to further enhance efficacy.

[0100] The adoptive transfer of T cells expressing chimeric antigen receptors is a promising anticancer therapy 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 to 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 triggers apoptosis in 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.

[0101] In some embodiments of the CAR targeting GPC2 disclosed herein, the VH sequence is operably connected to the VL sequence downstream. In some embodiments, the VH sequence is operably connected to the VL sequence upstream. The term "upstream" used herein to refer 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.

[0102] 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 a CD28 transmembrane domain, a CD8a transmembrane domain, a CTLA4 transmembrane domain, or a PD-1 transmembrane domain. In some embodiments, the CAR targeting GPC2 includes a transmembrane domain derived from a CD28 transmembrane domain.

[0103] 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 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 sequence, costimulatory CD27 polypeptide sequence, costimulatory CD28 polypeptide sequence, costimulatory OX40 (CD134) polypeptide sequence and costimulatory inducible T cell costimulatory (ICOS) polypeptide sequence. In some embodiments, the CAR targeting GPC2 includes a costimulatory domain derived from a costimulatory 4-1BB (CD137) polypeptide sequence. In some embodiments, the CAR targeting GPC2 comprises a costimulatory domain derived from a costimulatory CD28 polypeptide sequence.

[0104] 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 connection region or "joint" located between the targeting portion and the transmembrane domain. These sequences are generally derived from IgG subclasses (e.g., IgG 1 and IgG4), IgD, and CD8 domains, of which IgG 1 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 appreciated 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, the 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 the 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.

[0105] 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 flank 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 20aa, 2aa to 15aa, 3aa to 12aa, including 4aa to 10aa, 5aa to 9aa, 6aa to 8aa, or 7aa to 8aa, and can be 1, 2, 3, 4, 5, 6 or 7aa.

[0106] The terms "long linker" and "short linker" are used throughout the application and mean the following:

[0107] "Long linker" amino acid sequence: GGGGSGGGGSGGGGS (SEQ ID NO: 4)

[0108] "Short linker" amino acid sequence: GGGGS (SEQ ID NO: 41).

[0109] 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 terms of providing flexibility to the flanking regions. In some embodiments, the CAR targeting GPC2 disclosed herein includes a hinge domain derived from a CD8a hinge domain. In some embodiments, the CAR targeting GPC2 disclosed herein includes a hinge domain derived from a CD28 hinge domain.

[0110] In some embodiments, CAR disclosed herein also includes an extracellular spacer domain containing one or more intermediate amino acid residues between the anti-GPC2 scFV region and the 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 is no particular restriction on the length and / or amino acid composition of the extracellular interval. In some embodiments, any 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.) can be used as an extracellular interval. 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 / joint comprises about 1 to 10, about 5 to 15, about 10 to 20, about 15 to 25 amino acid residues. In some embodiments, the extracellular hinge / joint comprises about 220, 225, 230, 235 or 240 amino acid residues. In some embodiments, the extracellular hinge / joint comprises 229 amino acid residues. In some embodiments, the length and amino acid composition of the extracellular hinge / joint can be optimized to change the orientation and / or proximity of the anti-GPC2 scFV region and the extracellular hinge / joint domain to each other to achieve the desired activity of the CAR targeting GPC2. In some embodiments, the orientation and / or proximity of the anti-GPC2 scFV region and the extracellular hinge / joint domain to each other can be changed and / or optimized as a "regulatory" tool or effect, which 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 produce a partially functional or partially functional form of a 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.

[0111] In some embodiments, the intracellular signaling domain of the CAR targeting GPC2 disclosed herein includes a CD3 ζ 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.

[0112] 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.

[0113] 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.

[0114] The nucleic acid molecules of the present disclosure can be nucleic acid molecules of any length, including nucleic acid molecules of 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, nucleic acid molecules of 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.

[0115] 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.

[0116] 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 guide the correct transcription and / or translation of the coding sequence in a recipient cell in vivo and / or in vitro. The expression cassette can be inserted into a vector for targeting a desired host cell and / or into a subject. Therefore, the term expression cassette can be used interchangeably with the term "expression construct".

[0117] Chimeric antigen receptors (CARs) according to the present disclosure may first be defined by their binding specificity, in this case, for glypican 2. CARs may also be defined by sequences disclosed herein, or may 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 may be separated from the constant domain of the light chain, (b) the amino acids may be different from those listed above, while thus not significantly affecting the chemical properties of the residues (so-called conservative substitutions), (c) the amino acids may be changed with those listed above by 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 changing the residues encoded thereby, (c) may 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.

[0118] When making conservative changes to an amino acid sequence, the hydropathic index of the amino acids may 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.).

[0119] It is also understood in the art that similar amino acid substitutions can be effectively made based on hydrophilicity. U.S. Patent No. 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 related to the biological properties of the protein. As detailed in U.S. Patent No. 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 (+0.2); hydrophilic nonionic amino acids: serine (+0.3), asparagine (+0.2), glutamine (+0.2); hydrophilic nonionic amino acids: Amide (+0.2) and threonine (-0.4), sulfur-containing amino acids: cysteine ​​(-1.0) and methionine (-1.3); hydrophobic non-aromatic amino acids: valine (-1.5), leucine (-1.8), isoleucine (-1.8), proline (-0.5±1), alanine (-0.5) and glycine (0); hydrophobic aromatic amino acids: tryptophan (-3.4), phenylalanine (-2.5) and tyrosine (-2.3).

[0120] It is understood that an amino acid can be replaced by another amino acid of similar hydrophilicity and produce a biologically or immunologically modified protein. In such changes, substitution of amino acids whose hydrophilicity values ​​are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[0121] As summarized 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 described above are well known to those of skill in the art, and include arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine.

[0122] D. Expression

[0123] Nucleic acids according to 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.

[0124] Table 2 - Codons

[0125]

[0126] 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 proteins encoded therefrom. Alternatively, functionally equivalent proteins may be produced by the application of recombinant DNA techniques, wherein changes in protein structure may be engineered based on considerations of the properties of the exchanged amino acids. As described below, artificially designed changes may be introduced by the application of site-directed mutagenesis techniques, or may be introduced randomly and subsequently screened for desired functions.

[0127] 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 can be transcribed. The transcript can be translated into protein, but not necessarily. In certain embodiments, expression includes both gene transcription and translation of mRNA into a gene product. In other embodiments, expression only includes transcription of the nucleic acid encoding the gene of interest.

[0128] The term "vector" is used to refer to a carrier nucleic acid molecule into which a nucleic acid sequence can be inserted for introduction into a cell where it can be replicated. 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 which the sequence is not normally present in the host cell nucleic acid. Vectors include plasmids, cosmids, viruses (bacteriophages, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs). Those skilled in the art will be well-versed in constructing vectors by standard recombinant techniques, which are described in Sambrook et al. (1989) and Ausubel et al. (1994), both of which are incorporated herein by reference.

[0129] The term "expression vector" refers to a vector comprising 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, such as 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 transcribed and translatable operably linked coding sequences in a specific host organism. In addition to control sequences for transcription and translation, vectors and expression vectors may also contain nucleic acid sequences for other functions, and are described below.

[0130] 1. Regulatory elements

[0131] A "promoter" is a control sequence, which is a region of a nucleic acid sequence that controls the initiation and rate of transcription. A promoter may contain genetic elements to which regulatory proteins and molecules (e.g., RNA polymerases and other transcription factors) can bind. The phrases "operably positioned," "operably linked," "under control," and "under transcriptional control" mean that a promoter is in a 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.

[0132] A promoter may be one naturally associated with a gene or sequence, as it may be obtained by isolating 5' non-coding sequences upstream of the coding segment and / or exons. Such a promoter may be referred to as "endogenous". Similarly, an enhancer may be one naturally associated with a nucleic acid sequence, upstream or downstream of the sequence. Alternatively, certain advantages may be obtained by positioning a coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment.

[0133] 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 cells, 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.

[0134] 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 may 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 may 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), human platelet endothelial cell adhesion molecule-1 (Almendro et al., 1996).

[0135] Effective translation of the coding sequence may also require specific start signals. These signals include the ATG start codon or adjacent sequences. It may be necessary to provide an exogenous translation control signal including the ATG start 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 start 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 start codons can be natural or synthetic. Expression efficiency can be enhanced by including suitable transcription enhancer elements.

[0136] 2.IRES

[0137] In certain embodiments of the present disclosure, an internal ribosome entry site (IRES) element is used to generate a multi-gene or multi-cistronic message. 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 (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 a multi-cistronic message. With 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 Pat. Nos. 5,925,565 and 5,935,819, incorporated herein by reference).

[0138] 3. Multi-purpose cloning site

[0139] The vector may contain a multiple cloning site (MCS), which is a nucleic acid region containing multiple restriction enzyme sites, any 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 an exogenous sequence can be connected to the vector. "Connection" 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.

[0140] 4. Splice Sites

[0141] 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 transcripts for protein expression (see Chandler et al., 1997, incorporated herein by reference).

[0142] 5. Termination Signal

[0143] The vectors or constructs of the present disclosure will generally 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 an 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.

[0144] In eukaryotic systems, the terminator region may also include a specific DNA sequence that allows site-specific cutting of new transcripts to expose polyadenylation sites. It signals specialized endogenous polymerases 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 includes a signal for RNA cutting, and more preferably, the terminator signal promotes the polyadenylation of the messenger. Terminator and / or polyadenylation site elements may be used to increase messenger levels and / or minimize read-through from the box to other sequences.

[0145] Terminators contemplated for use in the present disclosure include any known transcription terminators described herein or known to those of ordinary skill in the art, including but not limited to, for example, termination sequences of genes, such as bovine growth hormone terminators or viral terminators, such as SV40 terminators. In certain embodiments, the termination signal may lack a transcribable or translatable sequence, such as due to sequence truncation.

[0146] 6. Polyadenylation signal

[0147] In expression, particularly in eukaryotic expression, a polyadenylation signal will generally 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 the stability of the transcript or may promote cytoplasmic transport.

[0148] 7. Initiation of replication

[0149] In order to propagate the vector in the host cell, it may contain one or more replication origin sites (generally 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.

[0150] 8. Selecting and screening markers

[0151] 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 an expression vector. Such a marker will confer identifiable changes to the cell, thereby allowing easy identification of cells comprising the expression vector. In general, a selective marker is a marker that confer a characteristic that allows selection. A positive selection marker is a marker in which the presence of the marker allows its selection, while a negative selection marker is a marker in which the presence of the marker prevents its selection. An example of a positive selection marker is a drug resistance marker.

[0152] Drug selection markers are usually included to help clone and identify transformants, for example, genes that confer 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 considered, 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 used. 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 not important 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.

[0153] 9. Viral vectors

[0154] 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 may be selected based on the necessary properties of the target system.

[0155] 10. Non-viral transformation

[0156] 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, such as by injection (U.S. Pat. Nos. 5,994,624, 5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466, and 5,580,859, each of which is incorporated herein by reference), including microinjection (Harland and Weintraub, 1985; U.S. Pat. No. 5,789,215, incorporated herein by reference); by electroporation (U.S. Pat. No. 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., 1991); 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. Pat. Nos. 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., 1992 ... al., 1990; U.S. Pat. Nos. 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. Pat. Nos. 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.

[0157] 11. Expression system

[0158] There are many expression systems that comprise at least part or all of the compositions discussed above. Systems based on prokaryotes and / or eukaryotes can be used for use 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.

[0159] Insect cell / baculovirus systems can produce high levels of protein expression of heterologous nucleic acid segments, such as described in U.S. Pat. Nos. 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.

[0160] Other examples of expression systems include Complete Control TM Inducible mammalian expression system, which involves 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 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 a vector, such as an expression construct, to produce a nucleic acid sequence or its homologous polypeptide, protein or peptide.

[0161] Primary mammalian cell cultures can be prepared in a variety of ways. In order for the cells to remain viable in vitro and in contact with the expression construct, it is necessary to ensure that the cells remain in contact with the correct ratio of oxygen and carbon dioxide and nutrients, but protected from microbial contamination. Cell culture techniques are well documented.

[0162] One embodiment of the foregoing relates to immortalizing cells using gene transfer for the production of proteins. The gene for the protein of interest may be transferred to a suitable host cell as described above, followed by culturing the cells under suitable conditions. In fact, the gene for any polypeptide may be used in this manner. The generation of recombinant expression vectors and the elements contained therein are discussed above. Alternatively, the protein to be produced may be an endogenous protein that is normally synthesized by the cell in question.

[0163] 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 the inserted sequence or modify and process the gene product in a desired manner can be selected. Such modification (e.g., glycosylation) and processing (e.g., cutting) of the protein product 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.

[0164] A number of 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. Likewise, antimetabolite resistance can be used as the basis for selection for: 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.

[0165] III. Pharmaceutical Preparations and Cancer Treatment

[0166] A. Cancer

[0167] Cancer is caused by the outgrowth of clonal populations of cells from tissues. The occurrence of cancer (called carcinogenesis) can be modeled and characterized in a variety of ways. The association between the occurrence 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, i.e., chronic inflammation can lead to dysplasia.

[0168] Cancer cells to which the methods of the present disclosure can be applied generally include any cell that expresses Glypican 2, and more specifically, a cell that overexpresses 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 to these: malignant neoplasms; 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; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular and cholangiocarcinoma; 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 adenocarcinoma; 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 w / 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 tumor; malignant lipid cell tumor; malignant ganglioneuroma; malignant extramammary paraganglioma; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented 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; Mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; malignant mesenchymal tumor; malignant Brenner tumor; malignant phyllodes tumor; synovial sarcoma; malignant mesothelioma; dysgerminoma; embryonal carcinoma; malignant teratoma; malignant ovarian goiter; choriocarcinoma; malignant mesonephroblastoma; 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 neurilemmoma; 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 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 may include osteosarcoma, angiosarcoma, rhabdomyosarcoma, leiomyosarcoma, Ewing's sarcoma, glioblastoma, medulloblastoma, neuroblastoma, or leukemia. ;

[0169] 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 re-induce 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.

[0170] B. Formulation and Administration

[0171] 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. Pharmacopeia or other generally recognized pharmacopoeia for use in animals and more particularly in humans. The term "carrier" refers to a diluent, excipient or vehicle with which a therapeutic agent is administered. Such a pharmaceutical carrier can be a sterile liquid, such as water and oil, 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.

[0172] The composition can be formulated as a 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.

[0173] The receptors, nucleic acids and cells of the present disclosure may include classical pharmaceutical preparations. Administration of these compositions according to the present disclosure may be by any common route, as long as the target tissue is accessible by the route. This includes oral, nasal, buccal, rectal, vaginal or surface. 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.

[0174] 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 appropriately 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.

[0175] C. Combination therapy

[0176] In the context of the present disclosure, it is also contemplated that the anti-Glypican 2 CAR T cells described herein may be similarly used in combination with immunological, chemo- or radiotherapeutic interventions or other treatments. In particular, the combination of anti-Glypican 2 CAR T cells with other treatments targeting different aspects of Glypican 2 function may also prove to be effective.

[0177] 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 generally contacted with anti-Glypican 2CAR T cells and at least one other agent according to the present disclosure. These compositions will be provided in a combined amount that effectively kills or inhibits cell proliferation. The process may include contacting cells with anti-Glypican 2CAR T cells and other agents or factors according to the present disclosure at the same time. This can be achieved by contacting cells with a single composition or pharmacological preparation containing both agents, or by contacting cells with two different compositions or preparations at the same time, wherein one composition contains anti-Glypican 2CAR T cells according to the present disclosure and the other contains other agents.

[0178] Alternatively, anti-Glypican 2CAR T cell therapy can be performed at intervals of several minutes to several weeks before or after other agents are treated. In some embodiments in which other agents and anti-Glypican 2CAR T cells are applied to cells separately, it should generally be ensured that the important time period between each delivery does not expire, so that the agent and the 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 each other, and the time is more preferably within about 6 to 12 hours 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 application 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).

[0179] It is also contemplated that more than one administration of anti-Glypican 2 CAR T cells or another agent is 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:

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

[0181] A / A / B / BA / B / A / BA / B / B / AB / B / A / AB / A / B / AB / A / A / BB / B / B / A

[0182] A / A / A / BB / A / A / AA / B / A / AA / A / B / AA / B / B / BB / A / B / BB / B / A / B

[0183] 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, etc. 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 may also include surgery. Various modes of these treatments are discussed below.

[0184] 1. Chemotherapy

[0185] 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 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.

[0186] 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 thiothiophosphoramide. and trimethylmelamine; acetogenins (especially bratacin and bratacinone); camptothecins (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its synthetic analogues adolesin, carzelesin and biszelesin); scutellarin (especially scutellarin 1 and scutellarin 8); dolastatin; duocarmycins (including the synthetic analogues KW-2189 and CB1-TM1); acanthosides; cypermethrin; scutellarin; spongestatin; nitrogen mustards, such as chlorambucil, naphthyl mustard, clofosamide, estramustine, ifosfamide, mechlorethamine, methoxychloramine hydrochloride, melphalan, nebiquinone, benzophenone mustard, prednimustine, trofosfamide, uracil nitrogen mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimustine; antibiotics, such as enediyne antibiotics (e.g., calicheamicins, especially calicheamicin γ1I and calicheamicin ω1I); dynemycins, including dynemycin Auncialamycin and its derivatives; bisphosphonates, such as clodronate; esperamicins; and neocarcinogens and related chromogenic protein enediyne antibiotic chromophores, aclarubicin, actinomycin, anthramycin, azaserine, bleomycin, cactinomycin, carrubicin, carmosin, Chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolinyl-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, mexilomycin, mitomycins (such as mitomycin C), mycophenolic acid, nogamycin, olive mycins, peplomycin, sphaminomycin, puromycin, triferric doxorubicin, rhodorubicin, streptozotocin, streptozotocin, tuberculocidin, ubenimex, zoloft, zorubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs, such as dimethylfolate, methotrexate, pteroxine, trimetrexate;Purine analogs, such as fludarabine, 6-mercaptopurine, thioimidazole, thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens, such as calosterone, drostanolone propionate, cyclothiodine, melastane, testolactone; antiadrenal agents, such as aminoglutethimide, mitotane, trilostane; folic acid supplements, such as folinic acid; aceglucuronolide; aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine; besbucil; bisantrene; edatrexate; defosfamide famine; colcemid; diazocine; ilonicet; elliptonium acetate; epothilones; etogluc; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansine alkaloids, such as maytansine and ansamitocin; mitoguanidine; mitoxantrone; mopidarol; nitrilotriamine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllic acid; 2-acetylhydrazine; procarbazine; PSK polysaccharide complex); razoxane; rhizoxin; sizoran; spirogermanium; tenuazonic acid acid); triazinon; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verrucosporin A, mycloracin A, and serpentin); urethane; vindesine; dacarbazine; mannomustine; dibromomannitol; dibromodulanol; pipobroman; garcitocin; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxanes, such as paclitaxel and doxetine. tadalafil; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum coordination complexes, such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan ; Retinoids, such as retinoic acid; Capecitabine; Cisplatin (CDDP); Carboplatin, procarbazine, nitrogen mustard, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosourea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, mitomycin, etoposide (VP-16), tamoxifen, raloxifene, estrogen receptor binding agents, paclitaxel, docetaxel, gemcitabine, navelbine, farnesyl-protein transferase inhibitors, transplatinum, 5-fluorouracil, vincristine, vinblastine and methotrexate, and pharmaceutically acceptable salts, acids or derivatives of any of the above. ;

[0187] 2. Radiation therapy

[0188] Radiotherapy, also known as radiation therapy, is the treatment of cancer and other diseases with ionizing radiation. Ionizing radiation deposits energy that damages or destroys cells in the area being treated by damaging their genetic material, making it impossible for these cells to continue to grow. Although radiation damages both cancer cells and normal cells, the latter are able to repair themselves and function properly.

[0189] 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 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 DNA, DNA precursors, DNA replication and repair, and to the assembly and maintenance of chromosomes. The dosage range of X-rays is a single dose of 2000 to 6000 roentgens for a period of time (3 to 4 weeks) 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.

[0190] Radiation therapy can include the use of radiolabeled antibodies to deliver radiation doses directly to the site of cancer (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.

[0191] Conformal radiation therapy uses the same radiation therapy machine, linear accelerator, as conventional radiation therapy, but metal blocks are 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 a substitute for metal blocks. A multi-leaf collimator consists of multiple metal sheets fixed to a 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.

[0192] 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 given. This method has the potential to achieve even more precise shaping of the treatment beam and to make the radiation therapy dose constant over the entire treatment area.

[0193] Although 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 with these specialized radiation therapy techniques.

[0194] 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 subjected to radiation. Radiosensitizers make tumor cells more likely to be destroyed, and radioprotectants protect normal tissue from the effects of radiation. Hyperthermia, which uses heat, is also being studied for its effectiveness in sensitizing tissue to radiation.

[0195] 3. Immunotherapy

[0196] 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 to some markers on the surface of tumor cells. A single antibody can be used as an effector of treatment or it can recruit other cells that actually affect cell killing. Antibodies can also be conjugated with drugs or toxins (chemotherapeutic agents, radionuclides, ricin A chains, 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 a tumor cell target. Various effector cells include cytotoxic T cells and NK cells. The combination of treatment modes (i.e., direct cytotoxic activity and inhibition or reduction of ErbB2) will provide therapeutic benefits in treating cancers overexpressed by ErbB2.

[0197] 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, including: cytokines, such as IL-2, IL-4, IL-12, GM-CSF, γ-IFN; chemokines, such as MIP-1, MCP-1, IL-8 and growth factors, such as FLT3 ligand. 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.

[0198] Some examples of immunotherapies currently under investigation or in use are immune adjuvants (e.g., Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds) (U.S. Pat. Nos. 5,801,005 and 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al., 1998); cytokine 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). 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 may be used in conjunction with the gene silencing therapies described herein.

[0199] 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).

[0200] In adoptive immunotherapy, circulating lymphocytes or tumor-infiltrating lymphocytes of the patient are isolated in vitro, activated by lymphokines (eg, IL-2) or transduced with tumor necrosis genes, and re-administered (Rosenberg et al., 1988; 1989).

[0201] 4. Surgery

[0202] 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 (e.g., treatment of the present disclosure, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapy).

[0203] 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, treatment by surgery 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 cancer, procancer, or an accompanying amount of normal tissue.

[0204] After removing a part or all of cancer cells, tissues or tumors, 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 can also have multiple doses.

[0205] 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. In addition, the compounds of the present disclosure may also be used in the neoadjuvant setting.

[0206] It should also be noted that any of the foregoing treatments may prove useful in the treatment of cancer. The skilled artisan will refer to "Remington's Pharmaceutical Sciences", 15th edition, Chapter 33, particularly pages 624-652. Depending on the condition of the subject being treated, some variation in dosage will necessarily occur. In any case, the person responsible for administration will determine the appropriate dosage for the individual subject. In addition, for human administration, the preparation should meet the sterility, pyrogenicity, general safety and purity standards required by the FDA Office of Biological Products Standards.

[0207] IV. Kit

[0208] In other embodiments, a kit for the above method is provided. Thus, the kit will contain 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.

[0209] The container device of the test kit generally includes at least one vial, test tube, flask, bottle, syringe or other container device, in which cells can be placed, or preferably the cells are appropriately aliquoted. The test kit also includes a device for closed and restricted containment of 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.

[0210] V. Examples

[0211] The following examples are included to demonstrate some preferred embodiments. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques that the inventors have found to work well in the practice of the embodiments and therefore can be considered to constitute preferred modes of their practice. However, based on the disclosure of the present invention, it will be appreciated 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.

[0212] Example 1

[0213] 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 therapy. The antibody sequences are available in Figures 1 to 3 Shown in.

[0214] 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 RNA CAR constructs with different heavy and light chain orientations and linker lengths between chains. They evaluated CAR persistence, T cell exhaustion markers, and cytotoxicity for 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. CAR molecules of light to heavy (VL-VH) configurations were shown to persist on the surface for more than seven days, and compared with heavy to light (VH-VL) configurations, cytotoxicity increased. The VH-VL configuration with a long linker provided the weakest cytotoxic effect, and the 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 the in vitro data, two VL-VH CAR constructs were selected for testing in the 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 reduced tumor burden (p<0.01) compared to CD19CAR controls, with several animals showing complete responses. Studies evaluating the efficacy of local delivery in pediatric HGG orthotopic models are currently underway.

[0215] Stable expression of multiple CAR T cell constructs was achieved by modifying 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. The initial constructs with CD8a hinge and transmembrane domains and 4-1BBz signaling domains, with N-terminal variable heavy chains or N-terminal variable light chains in two orientations (Figure 9B), showed stable cell surface expression and bound soluble recombinant GPC2 (Figure 9C). These constructs showed efficient in vitro efficacy and cytokine production (IFNy, IL2) against isogenic target cells, which were modified to express GPC2 at levels comparable to those in vivo for GPC2 (Kelly-GPC2), with an effector-target ratio of 1:1 (Figures 11A to D). In addition, the inventors demonstrated that incorporating CD28-H / TM and costimulatory domains into these CAR constructs exhibited additional CAR T cell potential advantages when targeting tumors expressing GPC2 (Figure 14A to B). In summary, these data show that using DNA-based CAR vectors and viral transduction, stable GPC2-targeted CAR T cells can be transformed, which produce efficient killing effects on cancer cells expressing GPC2.

[0216] These data show that mRNA provides a rapid and iterative approach to 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 murine model, RNAGPC2 CAR T cells with light to heavy D3 scFv chains in a long linker configuration provided the strongest cytotoxic effects with no evidence of toxicity.

[0217] Through lentivirus (Figure 15A to F) and retrovirus ( Fig.16ATo B) Transduced D3 (M201)-based GPC2 DNA CAR T cells also have efficient cytotoxicity in preclinical models of neuroblastoma. GPC2 CAR is robustly expressed on T cells (Figure 15A) and is cytotoxic to isogenic SY5Y-GPC2 neuroblastoma cells (Figure 15B), and co-culture leads to simultaneous T cell activation and increased INFγ and CD107A T cell expression (Figure 15C to 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 efficient in vivo activity, inducing robust regression of COG-N-421x neuroblastoma patient-derived xenograft tumors and being very well tolerated (Figure 15E to F). In the metastatic SMS-SAN neuroblastoma model, D3(M201)-based GPC2 CAR T cells also induced tumor regression ( Fig.16A to B)

[0218] *****************

[0219] 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 the steps or step sequences of the compositions and methods and methods described herein can be changed without departing from the concept, spirit and scope of the present disclosure. 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.

[0220] VII. References

[0221] 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.

[0222] U.S. Patent 3,817,837

[0223] U.S. Patent 3,850,752

[0224] U.S. Patent 3,939,350

[0225] U.S. Patent 3,996,345

[0226] U.S. Patent 4,196,265

[0227] U.S. Patent 4,275,149

[0228] U.S. Patent 4,277,437

[0229] U.S. Patent 4,366,241

[0230] U.S. Patent 4,472,509

[0231] U.S. Patent 4,554,101

[0232] U.S. Patent 4,680,338

[0233] U.S. Patent 4,683,202

[0234] U.S. Patent 4,684,611

[0235] U.S. Patent 4,816,567

[0236] U.S. Patent 4,867,973

[0237] U.S. Patent 4,879,236

[0238] U.S. Patent 4,938,948

[0239] U.S. Patent 4,952,500

[0240] U.S. Patent 5,021,236

[0241] U.S. Patent 5,141,648

[0242] U.S. Patent 5,196,066

[0243] U.S. Patent 5,302,523

[0244] U.S. Patent 5,322,783

[0245] U.S. Patent 5,384,253

[0246] U.S. Patent 5,464,765

[0247] U.S. Patent 5,538,877

[0248] U.S. Patent 5,538,880

[0249] U.S. Patent 5,550,318

[0250] U.S. Patent 5,563,055

[0251] U.S. Patent 5,563,250

[0252] U.S. Patent 5,565,332

[0253] U.S. Patent 5,580,859

[0254] U.S. Patent 5,589,466

[0255] U.S. Patent 5,610,042

[0256] U.S. Patent 5,656,610

[0257] U.S. Patent 5,670,488

[0258] U.S. Patent 5,702,932

[0259] U.S. Patent 5,736,524

[0260] U.S. Patent 5,780,448

[0261] U.S. Patent 5,789,215

[0262] U.S. Patent 5,824,544

[0263] U.S. Patent 5,856,456

[0264] U.S. Patent 5,858,744

[0265] U.S. Patent 5,871,982

[0266] U.S. Patent 5,871,986

[0267] U.S. Patent 5,880,270

[0268] U.S. Patent 5,925,565

[0269] U.S. Patent 5,928,906

[0270] U.S. Patent 5,935,819

[0271] U.S. Patent 5,945,100

[0272] U.S. Patent 5,981,274 U.S. Patent 5,994,624

[0273] "Antibodies: A Laboratory Manual," Cold Spring Harbor Press, Cold Spring Harbor, NY, 1988.

[0274] Abbondanzo et al,Am.J.Pediatr.Hematol.Oncol.,12(4),480-489,1990.

[0275] Allred et al.,Arch.Surg.,125(1),107-113,1990.

[0276] Almendro et al.,J Immunol.,157(12):5411-5421,1996.

[0277] Atherton et al.,Biol.of Reproduction,32,155-171,1985.

[0278] Ausubel et al.,CCurrent Protocols in Molecular Biology,GreenePublishing Associates and Wiley Interscience,N.Y.,1994.

[0279] Beidler et al.,J.Immunol.,141(11):4053-4060,1988.

[0280] Bosse et al.,Cancer Cell,32:295-309,e212,2017

[0281] Brown et al.,J.Immunol.Meth.,12;130(1),:111-121,1990.

[0282] Campbell,In:Monoclonal Antibody Technology,Laboratory Techniques inBiochemistry and Molecular Biology,Vol.13,Burden and Von Knippenberg,Eds.pp.75-83,Amsterdam,Elsevier,1984.

[0283] Capaldi et al.,Biochem.Biophys.Res.Comm.,74(2):425-433,1977.

[0284] Carbonelli et al., FEMS Microbiol. Lett., 177(1):75-82, 1999.

[0285] Chandler et al., Proc. Natl. Acad Sci. USA, 94(8): 3596-601, 1997.

[0286] Chen and Okayama, Mol. Cell Biol., 7(8):2745-2752, 1987.

[0287] Cocea, Biotechniques, 23(5):814-816, 1997.

[0288] Coffey et al., Science, 282(5392): 1332-1334, 1998.

[0289] De Jager et al., Semin. Nucl. Med 23(2), 165-179, 1993.

[0290] Dholakia et al., J. Biol. Chem., 264, 20638-20642, 1989.

[0291] Doolittle and Ben-Zeev, Methods Mol. Biol., 109, 215-237, 1999.

[0292] European applications 125,023

[0293] European applications 171,496

[0294] European applications 173,494

[0295] European applications 184, 187

[0296] European applications 273,085

[0297] Fechheimer et al., Proc Natl. Acad. Sci. USA, 84:8463-8467, 1987.

[0298] Fraley et al., Proc. Natl. Acad. Sci. USA, 76: 3348-3352, 1979.

[0299] Gerter et al.,Somatic Cell Genet.,3:231-236,1977

[0300] Gnant et al.,Cancer Res.,59(14):3396-403,1999.

[0301] Gnant et al.,J.NatlCancerInst.,91(20):1744-1750,1999.

[0302] Goding,In:Monoclonal AntibodiesPrinciples and Practice,2d ed.,Orlando,Fla.,Academic Press,60-61,65-66,71-74,1986.

[0303] Gopal,Mol.Cell Biol.,5:1188-1190,1985.

[0304] Graham and Van Der Eb,Virology,52:456-467,1973.

[0305] Greene et al.,Immunology Today,10:272,1989.

[0306] Gulbis and Galand,Hum.Pathol.24(12),1271-1285,1993.

[0307] Harland and Weintraub,J.CellBiol.,101(3):1094-1099,1985.

[0308] Imai et al.,Nephrologie,19(7):397-402,1998.

[0309] Jones et al.,Nature,321:522-525,1986.

[0310] Kaeppler et al.,Plant Cell Rep.,8:415-418,1990.

[0311] Kaneda et al.,Science,243:375-378,1989.

[0312] Kato et al,J.Biol.Chem.,266:3361-3364,1991.

[0313] Khatoon et al.,Ann.of Neurology,26,210-219,1989.

[0314] King et al.,J.Biol.Chem.,269,10210-10218,1989.

[0315] Kohler and Milstein,Eur.J.Immunol.,6,511-519,1976.

[0316] Kohler and Milstein,Nature,256,495-497,1975.

[0317] Kraus et al.FEBS Lett.,428(3):165-170,1998.

[0318] Kyte and Doolittle,J Mol.Biol.,157(1):105-132,1982.

[0319] Lareyre et al.,J.Biol.Chem.,274(12):8282-8290,1999.

[0320] Levenson et al,Hum.Gene Ther.,9(8):1233-1236,1998.

[0321] Lundstrom,J.Recept Signal Transduct.Res.,19(1-4):673-686,1999.

[0322] Macejak and Samow,Nature,353:90-94,1991.

[0323] Morrison,Science,229(4719):1202-1207,1985.

[0324] Nakamura et al.,In:Enzyme Immunoassays:Heterogeneous and HomogeneousSystems,Chapter 27,1987

[0325] Neumann et al., Proc. Natl. Acad. Sci. USA, 96(16): 9345 - 9350, 1999.

[0326] Nicolau and Sene, Biochim. Biophys. Acta, 721: 185 - 190, 1982.

[0327] Nicolau et al., Methods Enzymol., 149: 157 - 176, 1987.

[0328] Nomoto et al., Gene, 236(2): 259 - 271, 1999.

[0329] Omirulleh et al., Plant Mol. Biol., 21(3): 415 - 428, 1993.

[0330] O′Shannessy et al., J. Immun. Meth, 99, 153 - 161, 1987.

[0331] Owens and Haley, J. Biol. Chem., 259, 14843 - 14848, 1987.

[0332] PCT Application PCT / US 86 / 02269

[0333] PCT Application WO 86 / 01533

[0334] PCT Application WO 94 / 09699

[0335] PCT Application WO 95 / 06128

[0336] Pelletier and Sonenberg, Nature, 334(6180): 320 - 325, 1988.

[0337] Posner et al., Hybridoma 6, 611 - 625, 1987.

[0338] Potrykus et al., Mol. Gen. Genet., 199(2): 169 - 177, 1985.

[0339] Potter and Haley, Meth. Enzymol., 91, 613 - 633, 1983.

[0340] Remington’s Pharmaceutical Sciences,15 th Ed.,33:624-652,1990.

[0341] Rippe,et al.,Mol.Cell Biol.,10:689-695,1990.

[0342] Robbins and GhiviZZani,Pharmacol Ther,80(1):35-47,1998.

[0343] Robbins et al.,Trends Biotechnol.,16(1):35-40,1998.

[0344] Sambrook et al.,In:Molecular cloning:a laboratory manual,2 nd Ed.,ColdSpring Harbor Laboratory Press,Cold Spring Harbor,NY,1989.

[0345] Shaw et al.,J.Natl.Cancer Inst,80(19):1553-1559,1988.

[0346] Sun et al.,J.Steroid Biochem.,26(1):83-92,1987.

[0347] Tsumaki et al,J.BiolChem,273(36):22861-2286

[0348] Verhoeyen et al.,Science,239(4847):1534-1536,1988.

[0349] Wawrzynczak&Thorpe,Cancer Treat Res.,37:239-51,1988.

[0350] Wong et al.,Gene,10:87-94,1980.

[0351] Wood et al.,J.Clin.Lab.Immunol.,17(4):167-171,1985.

[0352] Wu et al., Biochem.Biophys.Res.Commun., 233(1):221-226, 1997.

[0353] Zhao-Emonet et al., Biochim. Biophys. Acta, 1442(2-3): 109-119, 1998. Sequence Listing <110> THE CHILDREN'S HOSPITAL OF PHILADELPHIA THE UNITED STATES OF AMERICA, AS REPRESENTED BY THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SERVICES THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIVERSITY <120> Chimeric antigen receptor containing a glypican 2 binding domain <130> CHOP.P0034WO <140> Submit at the same time <141> 2020-07-17 <150> US 62 / 876,483 <151> 2019-07-19 <160> 41 <170> PatentIn Version 3.5 <210> 1 <211> 243 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 1 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Asn 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Val Ile Tyr Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp Ser Asn Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr 100 105 110 Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Leu Ser Leu Pro Val Thr 130 135 140 Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu 145 150 155 160 Tyr Ser Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly 165 170 175 Lys Ser Pro Gln Val Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly 180 185 190 Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu 195 200 205 Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met 210 215 220 Gln Ala Leu Gln Thr Pro Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu 225 230 235 240 Ile Lys Arg <210> 2 <211> 242 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 2 Glu Val Gln Leu Val Glu Thr Gly Gly Gly Val Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Ser Gly Tyr Asp Tyr Val Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Ala Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Thr 130 135 140 Leu Ser Ala Phe Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 145 150 155 160 Gln Ser Ile Ser Ser Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 165 170 175 Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val 180 185 190 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr 195 200 205 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 210 215 220 Leu Asn Ser Tyr Pro Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile 225 230 235 240 Lys Arg <210> 3 <211> 245 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 3 Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Ser 65 70 75 80 Leu Gln Met Asp Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ser Arg Asp Ser Gly Asn Tyr Leu Asp Ala Phe Asp Phe Trp 100 105 110 Gly Gln Gly Thr Met Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly 115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser 130 135 140 Pro Ser Thr Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys 145 150 155 160 Arg Ala Ser Gln Ser Ile Ser Ser Trp Leu Ala Trp Tyr Gln Gln Lys 165 170 175 Ala Gly Lys Ala Pro Arg Leu Leu Ile Tyr Asp Ala Ser Thr Leu Glu 180 185 190 Ser Gly Val Pro Ser Arg Phe Ser Gly Thr Gly Ser Gly Thr Tyr Phe 195 200 205 Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr 210 215 220 Cys Gln Gln Phe Asn Ser Phe Pro Leu Thr Phe Gly Gly Gly Thr Lys 225 230 235 240 Val Glu Ile Lys Arg 245 <210> 4 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 4 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 5 <211> 115 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 5 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Asn 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Val Ile Tyr Ser Gly Gly Ser Thr Tyr Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp Ser Asn Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr 100 105 110 Val Ser Ser 115 <210> 6 <211> 119 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 6 Glu Ile Val Leu Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Lys Ser 35 40 45 Pro Gln Val Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala 85 90 95 Leu Gln Thr Pro Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Gln Gly 100 105 110 Thr Arg Leu Glu Ile Lys Arg 115 <210> 7 <211> 119 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 7 Glu Val Gln Leu Val Glu Thr Gly Gly Gly Val Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Ser Gly Tyr Asp Tyr Val Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Ala Val Ser Ser 115 <210> 8 <211> 108 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 8 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Phe Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Leu Asn Ser Tyr Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg 100 105 <210> 9 <211> 122 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 9 Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Ser 65 70 75 80 Leu Gln Met Asp Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ser Arg Asp Ser Gly Asn Tyr Leu Asp Ala Phe Asp Phe Trp 100 105 110 Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 10 <211> 108 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 10 Asp Ile Gln Leu Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Ala Gly Lys Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Thr Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Thr Gly Ser Gly Thr Tyr Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Phe Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> 11 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 11 Gly Phe Thr Val Ser Ser Asn Tyr 1 5 <210> 12 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 12 Ile Tyr Ser Gly Gly Ser Thr 1 5 <210> 13 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 13 Ala Arg Asp Ser Asn Ala Phe Asp Ile 1 5 <210> 14 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 14 Gln Ser Leu Leu Tyr Ser Asn Gly Tyr Asn Tyr 1 5 10 <210> 15 <211> 3 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 15 Leu Gly Ser 1 <210> 16 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 16 Met Gln Ala Leu Gln Thr Pro Ile Thr 1 5 <210> 17 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 17 Gly Phe Thr Phe Ser Asp Tyr Tyr 1 5 <210> 18 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 18 Ile Ser Ser Ser Gly Ser Thr Ile 1 5 <210> 19 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 19 Ala Arg Glu Ser Gly Tyr Asp Tyr Val Phe Asp Tyr 1 5 10 <210> 20 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 20 Gln Ser Ile Ser Ser Trp 1 5 <210> twenty one <211> 3 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> twenty one Ala Ala Ser 1 <210> twenty two <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> twenty two Gln Gln Leu Asn Ser Tyr Pro Ile Thr 1 5 <210> twenty three <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> twenty three Gly Phe Thr Phe Ser Ser Tyr Ala 1 5 <210> twenty four <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> twenty four Ile Ser Gly Ser Gly Gly Ser Thr 1 5 <210> 25 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 25 Ala Lys Ser Arg Asp Ser Gly Asn Tyr Leu Asp Ala Asp Phe Asp Phe 1 5 10 15 <210> 26 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 26 Gln Ser Ile Ser Ser Trp 1 5 <210> 27 <211> 3 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 27 Asp Ala Ser 1 <210> 28 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 28 Gln Gln Phe Asn Ser Phe Pro Leu Thr 1 5 <210> 29 <211> 357 <212> DNA <213> Artificial sequence <220> <223> Synthetic primer <400> 29 gaggtgcagc tggtggagac tgggggaggc gtggtcaagc ctggagggtc cctgagactc 60 tcctgtgcag cctctggatt caccttcagt gactactaca tgagctggat ccgccaggct 120 ccagggaagg ggctggagtg ggtttcatac attagtagta gtggtagtac catatactac 180 gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agctgaggac acggctgtgt attactgtgc gagagagagt 300 ggctacgatt acgtgtttga ctactggggc cagggaaccc tggtcgccgt ctcctca 357 <210> 30 <211> 119 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 30 Glu Val Gln Leu Val Glu Thr Gly Gly Gly Val Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Ser Gly Tyr Asp Tyr Val Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Ala Val Ser Ser 115 <210> 31 <211> 324 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Primer <400> 31 gacatccaga tgacccagtc tccttccacc ctgtctgcat ttgtaggaga cagagtcacc 60 atcacttgcc gggccagtca gagtattagt agctggttgg cctggtatca gcaaaaacca 120 gggaaagccc ctaagctcct gatctatgct gcatccactt tgcaaagtgg ggtcccatca 180 aggttcagcg gcagtggatc tgggacagaa ttcactctca caatcagcag cctgcagcct 240 gaagattttg caacttatta ctgtcaacag cttaatagtt accctatcac cttcggccaa 300 gggacacgac tggagattaa acga 324 <210> 32 <211> 108 <212> PRT <213> artificial sequence <220> <223> Synthesis of peptides <400> 32 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Phe Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Leu Asn Ser Tyr Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg 100 105 <210> 33 <211> 345 <212> DNA <213> Artificial sequence <220> <223> Synthetic primer <400> 33 caggtgcagc tggtgcagtc tggaggaggc ttgatccagc ctggggggtc cctgagactc 60 tcctgtgcag cctctgggtt caccgtcagt agcaactaca tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcagtt atttatagcg gtggtagcac atactacgca 180 gactccgtga agggccgatt caccatctcc agagacaatt ccaagaacac gctgtatctt 240 caaatgaaca gcctgagagc cgaggacacg gccgtgtatt actgtgcgag agattcgaat 300 gcttttgata tctggggcca agggacaatg gtcaccgtct cttca 345 <210> 34 <211> 115 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 34 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Asn 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Val Ile Tyr Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp Ser Asn Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr 100 105 110 Val Ser Ser 115 <210> 35 <211> 339 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Primer <400> 35 gaaattgtgc tgactcagtc tccactctcc ctgcccgtca cccctggaga gccggcctcc 60 atctcctgca ggtctagtca gagcctcctg tatagtaatg gatacaacta tttggattgg 120 tacctgcaga agccagggaa gtctccacag gtcctgatct atttgggttc taatcgggcc 180 tccggggtcc ccgacaggtt cagtggcagt ggatcaggca cagatttcac actgaaaatc 240 agcagagtgg aggctgagga tgttggggtt tattactgca tgcaagctct acaaactccg 300 atcaccttcg gccaagggac acgactggag attaaacga 339 <210> 36 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Peptide <400> 36 Glu Ile Val Leu Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Lys Ser 35 40 45 Pro Gln Val Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala 85 90 95 Leu Gln Thr Pro Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105 110 Arg <210> 37 <211> 366 <212> DNA <213> Artificial sequence <220> <223> Synthetic primer <400> 37 gaggtgcagc tggtgcagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180 gcagactccg tgaagggccg cttcaccatc tccagagaca attccaagaa cacgctgtct 240 ctgcaaatgg acagcctgag acccgaggac acggccgtat attactgtgc gaaaagtcga 300 gatagtggga actaccttga tgcttttgat ttctggggcc aagggacaat ggtcaccgtc 360 tcttca 366 <210> 38 <211> 122 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 38 Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Ser 65 70 75 80 Leu Gln Met Asp Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ser Arg Asp Ser Gly Asn Tyr Leu Asp Ala Phe Asp Phe Trp 100 105 110 Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 39 <211> 324 <212> DNA <213> Artificial sequence <220> <223> Synthetic primer <400> 39 gacatccagt tgacccagtc tccttccacc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgcc gggccagtca gagtattagt agctggttgg cctggtatca gcagaaagca 120 gggaaagctc ctaggctcct gatctatgat gcctccactt tggaaagtgg agtcccatca 180 aggttcagcg gcactggatc tgggacatat ttcactctca ccatcagcag cctgcagcct 240 gaagattttg caacttatta ctgtcaacag tttaatagtt tcccgctcac tttcggcgga 300 gggaccaagg tggagatcaa acga 324 <210> 40 <211> 108 <212> PRT <213> artificial sequence <220> <223> Synthesis of peptides <400> 40 Asp Ile Gln Leu Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Ala Gly Lys Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Thr Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Thr Gly Ser Gly Thr Tyr Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Phe Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> 41 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 41 Gly Gly Gly Gly Ser 1 5

Claims

1. An isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen binding domain that selectively binds to cancer cell-associated glypican 2 (GPC2), a flexible hinge domain, a transmembrane domain, a co-stimulatory signaling region, and an intracellular signaling domain, and Wherein the antigen binding domain comprises an antibody or antigen binding fragment thereof comprising: A heavy chain variable domain comprising: 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 a light chain variable domain comprising: 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; 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 (VL_linker_VH configuration); 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 isolated nucleic acid molecule of claim 1, wherein: (a) the flexible hinge domain is derived from CD8α, (b) the co-stimulatory signal transduction region comprises a 4-1BB (CD137) co-stimulatory domain; (c) the intracellular signaling domain comprises a CD3-ζ intracellular signaling domain.

3. The isolated nucleic acid molecule of claim 1, wherein the antigen binding fragment is a scFv.

4. The isolated nucleic acid molecule of any one of claims 1 to 3, wherein the 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.

5. The isolated nucleic acid molecule of any one of claims 1 to 3, wherein the CAR exhibits increased cytotoxicity when compared to a CAR comprising a conventional VH_linker_VL configuration.

6. The isolated nucleic acid molecule of claim 4, wherein: (a) the antigen binding domain comprises a heavy chain variable domain consisting of the amino acid sequence of SEQ ID NO: 30 and a light chain variable domain consisting of the amino acid sequence of SEQ ID NO: 32; (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 glycine-serine peptide linker; (c) the CAR comprises a CD8α flexible hinge domain, (d) the CAR comprises a CD28 transmembrane domain, (e) the CAR comprises a CD28 or 4-1BB co-stimulatory signaling region, and (f) The CAR comprises a CD3-ζ intracellular signaling domain.

7. The isolated nucleic acid molecule of claim 6, wherein the flexible glycine-serine peptide linker is at least 15 amino acids in length.

8. The isolated nucleic acid molecule of claim 1, wherein the flexible peptide linker is at least 15 amino acids in length.

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

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

11. A chimeric antigen receptor (CAR) polypeptide that selectively binds to cancer cell-associated glypican 2 (GPC2), comprising an antigen binding domain, a flexible hinge domain, a transmembrane domain, a co-stimulatory signaling region, and an intracellular signaling domain; Wherein the antigen binding domain comprises an antibody or antigen binding fragment thereof comprising: comprising 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 A light chain variable domain comprising: 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; 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.

12. The chimeric antigen receptor polypeptide of claim 11, wherein the antigen binding fragment is a single chain variable fragment (scFv).

13. The chimeric antigen receptor (CAR) polypeptide of any one of claims 11 to 12, wherein the 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.

14. The chimeric antigen receptor (CAR) polypeptide of any one of claims 11 to 12, wherein the CAR exhibits increased cytotoxicity when compared to a CAR comprising a conventional VH_linker_VL configuration.

15. The chimeric antigen receptor polypeptide of claim 13, wherein: (a) the 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; (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 glycine-serine peptide linker; (c) the CAR comprises a CD8α flexible hinge domain, (d) the CAR comprises a CD28 transmembrane domain, (e) the CAR comprises a CD28 or 4-1BB co-stimulatory signaling region, and (f) The CAR comprises a CD3-ζ intracellular signaling domain.

16. A genetically modified T cell comprising an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR) as described in any one of claims 1 to 10.

17. The genetically modified T cell of claim 16, wherein: (a) the CAR induces secretion of interferon-γ and interleukin-2, and (b) When the genetically modified T cells are exposed to cancer cell-associated GPC2, the genetically modified T cells exhibit cytotoxicity against cancer expressing GPC2.

18. A genetically modified T cell comprising the chimeric antigen receptor of any one of claims 11 to 15.

19. A method for preparing genetically modified T cells, comprising transducing immune effector cells with an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR) as described in any one of claims 1 to 10.

Images