Chimeric antigen receptor targeting GPC2 and containing CD28 hinge and transmembrane structure and application of chimeric antigen receptor

Abstract

Optimized chimeric antigen receptors (CARs) that target phosphatidylinositol proteoglycan-2 (GPC2) and have a CD28 hinge region and a CD28 transmembrane domain are described. The antigen binding domain of the disclosed CAR is derived from a GPC2 specific antibody CT3 or a humanized form thereof. The optimized CAR also includes an intracellular co-stimulation domain and an intracellular signaling domain. Immune cells or induced pluripotent stem cells expressing the optimized CAR can be used to treat GPC2 positive solid tumors, such as neuroblastoma.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 310,456, filed on February 15, 2022, which is incorporated herein by reference in its entirety.

[0003] U.S. Government Funding Statement

[0004] This invention was made with government support under Grant Nos. Z01BC010891, ZIABC010891, Z01 Z1ABC010788, Z1ABC011334, and ZIABC 012066 awarded by the National Cancer Institute of the National Institutes of Health. The U.S. Government has certain rights in this invention.

[0005] Field of the Disclosure

[0006] The present disclosure relates to an optimized chimeric antigen receptor (CAR) specific for the tumor antigen Glypican-2 (GPC2), which includes a hinge region and a transmembrane domain derived from CD28. The present disclosure further relates to the use of CAR targeting GPC2, such as for the treatment of solid tumors.

[0007] Incorporate into electronic sequence listing

[0008] The electronic version of the sequence listing is submitted as an XML file, with the file name 4239-107434-02.xml (63,252 bytes), created on January 26, 2023, and is incorporated herein by reference in its entirety.

[0009] background

[0010] CART cell therapy has become an important class of cancer treatments that are being actively developed and tested around the world. After initial success using CD19-targeted CAR T cells in blood cancers, multiple CAR strategies have been rigorously engineered and tested with the goal of treating solid tumors.

[0011] Glypican-2 (GPC2) is a member of the six-member glypican family of heparan sulfate proteoglycans that bind to the cell surface via a glycosylphosphatidylinositol (GPI) anchor (Li et al., Trends Cancer 4(11):741-754, 2018). GPC2 mRNA and protein are elevated in neuroblastoma and other pediatric cancers (Orentas et al., Front Oncol 2:194, 2012; Li et al., Proc Natl Acad Sci USA

[0012] 114(32):E6623-E6631, 2017; WO 2020 / 033430; and WO 2018 / 026533).

[0013] Neuroblastoma is the most common type of extracranial solid tumor in children. It originates from the neuroendocrine tissue of the sympathetic nervous system and accounts for about 8-10% of childhood cancers in the United States (Maris and Hogarty, Lancet 369:2106-2120, 2007). Neuroblastoma is a complex and heterogeneous disease, with nearly 50% of patients having a high-risk phenotype characterized by widespread dissemination of cancer and poor long-term survival, even with intensive multimodal therapy (Yu et al., New Engl J Med 363:1324-1334, 2010). Approximately 45% of patients receiving standard therapy relapse and eventually die of metastatic disease (Matthay et al., New Engl J Med 341:1165-1173, 1999). Therefore, the need for safe and effective treatment of neuroblastoma is urgent and unmet.

[0014] Overview

[0015] Disclosed herein is an optimized chimeric antigen receptor (CAR) specific for GPC2, comprising a hinge region and a transmembrane domain derived from human CD28. It is shown herein that, relative to a GPC2-specific CAR having a hinge region derived from CD8 and a transmembrane (TM) domain derived from CD8 or CD28, a GPC2-specific CAR having a CD28 hinge and a CD28 transmembrane domain is unexpectedly more effective in killing GPC2-positive cells in vitro and eradicating GPC2-positive tumors in animal models.

[0016] CAR is provided herein, which includes an extracellular antigen binding domain specific to GPC2; CD28 hinge region; CD28 transmembrane domain; Intracellular co-stimulatory domain; and intracellular signaling domain. In some aspects, the antigen binding domain includes a heavy chain variable (VH) domain and a light chain variable (VL) domain, and the VH domain and the VL domain include CDR sequences of GPC2-specific antibodies CT3 or its humanized form (such as hCT3-1, hCT3-2, hCT3-3 or hCT3-4). In some examples, the antigen binding domain includes a joint sequence between the VH domain and the VL domain, and the antigen binding domain can be in a VH-joint-VL direction or a VL-joint-VH direction.

[0017] Further provided are nucleic acid molecules encoding the disclosed CAR. In some aspects, the nucleic acid molecule includes a nucleic acid encoding a first granulocyte-macrophage colony stimulating factor receptor signal sequence (GMCSFRss) in the 5' to 3' direction; nucleic acid encoding an antigen binding domain; nucleic acid encoding a CD28 hinge region; nucleic acid encoding a CD28 transmembrane domain; nucleic acid encoding a co-stimulatory domain; nucleic acid encoding a signaling domain; nucleic acid encoding a self-cleaving 2A peptide; nucleic acid encoding a second GMCSFRss; and nucleic acid encoding a truncated human epidermal growth factor receptor (hEGFRt). In some examples, the nucleic acid molecule further includes a human elongation factor 1 alpha (EF1 alpha) promoter sequence at 5' of the nucleic acid encoding the first GMCSFRss. A vector (such as a lentiviral vector) comprising the disclosed nucleic acid molecule is further provided.

[0018] Also provided are isolated immune cells (such as T cells, NK cells, B cells or macrophages) and induced pluripotent stem cells (iPSCs) that express the CAR disclosed herein and / or contain an isolated nucleic acid molecule or vector encoding the CAR disclosed herein.

[0019] Further provided are compositions comprising a pharmaceutically acceptable carrier and a CAR, nucleic acid molecule, vector or cell disclosed herein.

[0020] Also provided is a method for treating GPC2 positive cancer in a subject or inhibiting tumor growth or metastasis of GPC2 positive cancer. In some aspects, these methods include administering to the subject a therapeutically effective amount of CAR, nucleic acid molecules, vectors, cells or compositions disclosed herein. In some examples, GPC2 positive cancer is a solid tumor, such as neuroblastoma, medulloblastoma or retinoblastoma.

[0021] The foregoing and other features of the present disclosure will become more apparent from the following detailed description of several aspects made with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1A-1B : CAR construct. ( Figure 1A ) Schematic diagram of CAR constructs CT3.8H.BBz, CT3.8H.28BBz and CT3.28H.BBz. ( Figure 1B ) Detailed schematic diagram of the CT3.28H.BBz CAR design.

[0024] Figure 2A-2B :IMR5 cells expressing GPC2 ( Figure 2A ) and GPC2 knockout (KO) IMR5 cells ( Figure 2B) in vitro cell killing assay. CT3.28H.BBz CAR T cells were more potent than CT3.8H.BBz CAR T cells in killing IMR5 cells. Both types of CART cells had minimal effects on GPC2-KO IMR5 cells, showing that they were GPC2 specific.

[0025] Figure 3A-3D :Comparison of CT3.8H.BBz CAR T cells and CT3.28H.BBz CAR T cells in the IMR5 metastasis model. Figure 3A ) Experimental design. Mice were inoculated intravenously with IMR5-luc and infused with 10 million CAR T cells 28 days later. Mice were imaged once a week after infusion. ( Figure 3B ) Bioluminescence images of mock-treated mice and CAR T cell-treated mice. ( Figure 3C ) Bioluminescence measured two, four, six, and eight weeks after CAR T cell infusion. ( Figure 3D ) Survival of mock-treated mice and CAR T cell-treated mice after infusion of CAR T cells. CT3.28H.BBz CART cells were significantly more potent than CT3.8H.BBz CART cells in regressing neuroblastoma tumors in mice. All mice in the CT3.28H.BBz-treated group survived at the end of the study.

[0026] Figure 4A-4G :Comparison of CT3.28H.BBz CAR T cells and CT3.8H.28BBz CART cells in the orthotopic IMR5 mouse model. Figure 4A-4C ) using A26M from three different human donors ( Figure 4A )、A59F( Figure 4B ) and A25F( Figure 4C ) T cells. Five million human T cells expressing either CT3.28H.BBz CAR or CT3.8H.28BBz CAR were administered to mice with moderate tumor burden. CT3.28H.BBz CAR T cells from all three donors outperformed CT3.8H.28BBz CART cells. ( Figure 4D-4E ) mock-treated mice and A26M ( Figure 4D ) and donor A59F( Figure 4E Bioluminescent images of mice treated with CT3.28H.BBz CAR T cells or CT3.8H.28BBz CAR T cells. CT3.28H.BBz CAR T cells were more potent than CT3.8H.28BBz CAR T cells in eradicating medium-sized IMR5 tumors. Figure 4F) Flow cytometry curve showing the gating strategy for flow cytometry samples. Live cells were gated on CD3+ human cells and the percentage of CAR-positive cells was determined. ( Figure 4G ) Graph showing the percentage of CD3+ cells expressing CT3.28H.BBz CAR or CT3.8H.28BBz CAR for donor A26M and donor A59F. For both donors, CT3.28H.BBz CAR T cells retained higher CAR expression levels than CT3.8H.28BBz CAR T cells.

[0027] Figure 5A-Figure 5B : Detection of CAR phosphorylation as a measure of CAR activation. ( Figure 5A )CT3.8H.BBz CAR T cells, CT3.8H.28BBz CAR T cells, and CT3.28H.BBz CART cells were unstimulated or stimulated with protein L or GPC2-Fc and CAR phosphorylation was detected by Western blotting. ( Figure 5B ) shows a graph of CAR phosphorylation fold change. When tested without stimulation, CT3.8H.28BBz CAR has a higher phosphorylation level than CT3.28H.BBz CAR. With GPC2 stimulation, both CARs upregulate their own phosphorylation levels. These results show that CT3.8H.28BBz has more tonic CAR signaling (phosphorylation), which is known to cause CAR exhaustion. CT3.28H.BBz CAR T cells have less tonic CAR signaling, but once antigen is presented (GPC2-Fc), they show appropriate CAR activation.

[0028] Figure 6A-6B : Comparison of CT3.28H.BBz CART cells and CT3.8H.28BBz CART cells in an orthotopic IMR5 animal model with low-dose or high-dose chemotherapy. Mice with large tumor burden were treated with no chemotherapy, low-dose chemotherapy, or high-dose chemotherapy (fludarabine / cyclophosphamide) for three days, followed by an infusion of 5 million CAR T cells. ( Fig. 6A )Tumor size measured by bioluminescence. Figure 6B ) Tumor weights 10 weeks after chemotherapy and CAR T cell infusion. CT3.28H.BBz was superior to CT3.8H.28BBz when low-dose conditioning chemotherapy was given in mice with high tumor burden.

[0029] Figure 7A-7B: Binding affinity of humanized CT3 (hCT3) antibodies. A set of graphs are shown, which illustrate that the binding affinity of four humanized CT3 antibodies (4.0 nM, 3.6 nM, 2.5 nM and 3.3 nM) is similar to that of the parental CT3 antibody (2.2 nM).

[0030] Figure 8 : Humanized CT3 antibodies bind to cell surface GPC2. Flow cytometry curves are shown, indicating that all four humanized CT3 antibodies maintain the ability to bind to cell surface GPC2.

[0031] Fig. 9 : Cell killing based on humanized CT3 CAR T cells. The figure shows that IMR5 cells expressing GPC2 are specifically lysed by CT3-8H-BBz, hCT3-1-8H-BBz, hCT3-2-8H-BBz, hCT3-3-8H-BBz and hCT3-4-8H-BBz CAR T cells. Compared to CT3-8H-BBz CAR T cells, all four humanized CT3-8H-BBz CAR T cells showed improved killing activity against IMR5 cells.

[0032] Fig.10 : Schematic representation of two humanized CT3-28H-BBz CAR constructs in which the GPC2 binding domain has a VH-linker-VL orientation (upper panel) or a VL-linker-VH orientation (lower panel).

[0033] Figures 11A-11D : In vitro comparison of three GPC2-CAR constructs. ( Fig.11A ) Schematic diagram of three CAR constructs with variable hinge, TM, and co-stimulatory domains used in preclinical studies. ( Fig. 11B ) Cytokine secretion characteristics of GPC2-CART cells when co-cultured with tumor cells for 24 hours. Human T cells transduced with three different GPC2-CARs were cultured with GPC2-KO or GPC2-WT IMR-5 tumor cells. Interferon gamma (IFNγ) levels, granzyme B (GZMB) levels, and soluble Fas ligand (sFASL) levels were significantly increased in the presence of GPC2, but the levels were comparable between the three tested CARs. ( Fig. 11C ) shows Western blot analysis of CAR signaling in resting T cells and CAR-crosslinked T cells. ( Fig.11D )From Fig. 11CDensitometric quantification of western blot signals. CT3.8H.28BBζ showed clear CAR tonic signaling at rest, whereas CT3.28H.BBζ and CT3.8H.BBζ lacked this signal. However, antigen-specific CAR activation induced higher phosphorylation levels in the case of CT3.28H.BBζ compared to CT3.8H.BBζ.

[0034] Figure 12A-12E :CT3.28H.BBζ is superior to CT3.8H.CD28BBζ in vivo. ( Fig. 12A ) Tumor weight on day 50 after tumor injection. CT3.28H.BBζ induced the most significant tumor regression among all treatment groups. ( Fig. 12B ) Model systems and experimental treatment protocols. Fig. 12C ) Longitudinal bioluminescence imaging (BLI) signals of mice treated with untransduced (UT) mock T cells or untreated controls and CAR T cells targeting GPC2. Weekly BLI revealed that animals treated with CT3.28H.BBζ-CART showed a substantial decrease in their BLI signals, and this substantial decrease was maintained during the duration of the study. In contrast, mice treated with CT3.8H.28BBζ or each CAR T cell targeting GPC2 at a lower cell dose showed a transient response, but eventually progressed. ( Fig.12D )From Fig. 12C Survival curve of mice. Fig.12E ) were analyzed by flow cytometry on day 80. Fig. 12C CAR isolated from tumors of treated mice + T cell persistence.

[0035] Figures 13A-13K :GPC2-CAR T cell production process generates proliferative and cytotoxic effector T cells. ( Fig.13A ) The proportion of immune cells captured on day 8 of CAR T production. The produced cells from donor 1 ( Fig. 13B ) unified manifold approximation (UMAP), ( Fig. 13C ) CD8 and CD4 protein expression levels detected by TotalSeq, ( Fig.13D ) Cluster annotation and ( Fig.13E ) each subpopulation fraction. Fig.13F )UMAP, ( Figure 13G )CD8 and CD4 protein expression levels, ( Fig.13H ) Cluster annotation and ( Fig.13I )The scores of each subgroup. Figure 13J-13K ) were used to analyze the differentially expressed genes (DEGs) of cells generated from the 3D-expressing cells.

[0036] Figure 14A-14J :GPC2-CAR T cells home to the tumor microenvironment (TME) and are enriched as a cytotoxic effector population in vivo. ( Fig.14A ) In vivo tracking of CAR T cells targeting GPC2 using BLI. T cells were transduced to express firefly luciferase (ffLUC)-GFP and homing and expansion were continuously monitored. ( Figure 14B-Figure 14C ) All three CARs were enriched and amplified in the TME compared to UT mock cells. *p<0.05; Student's t-test. ( Figure 14D-14E ) Mice carrying IMR-5 received T cell injections. Eight days later, tumors were isolated and single-cell RNA-seq was performed. Quantification of tumor and immune cells derived from the tumors is shown. ( Figure 14F-14G ) shows the UMAP images of tumor cells and five immune subsets. The bar graph quantifies the immune ratio. ( Fig.14H ) Volcano plot comparing differentially expressed genes between CT3.28H.BBζ vs CT3.8H.BBζ (upper panel) and CT3.28H.BBζ vs CT3.8H.28BBζ (lower panel). ( Fig.14I ) are grouped by function and from Fig.14H The differentially expressed genes obtained from the two comparisons made in ( Fig.14J ) Composite pathway analysis revealed that CT3.28H.BBζCART cells upregulated the granzyme A pathway, but downregulated pathways related to mitochondrial oxidative phosphorylation (OXPHOS) and eukaryotic initiation factor 2 (EIF2). Abbreviations: CTLA-4, cytotoxic T lymphocyte-associated protein 4; mTOR, mammalian target of rapamycin; TAM, tumor-associated macrophage; pDC, plasmacytoid dendritic cell; PD-1, programmed cell death protein-1; PD-L1, programmed death ligand 1; seq, sequencing.

[0037] Figures 15A-15F : Head-to-head comparison of GPC2-CAR (CT3.28H.BBζ) and GD2-CAR (K666.28H.BBζ). Fig.15A ) Transduction efficiency of CT3.28H.BBζCAR T cells and K666.28H.BBζCAR T cells. ( Fig. 15B ) In vitro cytotoxicity assays of two CARs tested against three NB strains at different E:T ratios. ***p<0.001; ****p<0.0001; two-way ANOVA. ( Fig. 15C ) In vitro tumor restimulation assay. CAR T cells were restimulated every 24 hours. Cytotoxic activity was measured at 24 hours, 96 hours and 7 days. *p<0.05; paired t-test. ( Fig.15D ) Tumor weight on day 50 after tumor implantation. *p<0.05; Student's t test. Fig.15E ) Image of a tumor harvested on day 50 after implantation. Scale bar = 1.0 cm. ( Fig.15F ) Flow cytometric analysis of bone marrow cells derived from one femur. The tumor cells were identified as hCD45 - mCD45 - GD2 + GFP + The total number of cells per femur is plotted, and each point represents one mouse.

[0038] Sequence Listing

[0039] As specified in 37 C.FR 1.822, the nucleic acid sequences and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases and single-letter codes for amino acids. Only one strand of each nucleic acid sequence is shown, but it should be understood that by any reference to a displayed strand, the complementary strand is also included. In the accompanying sequence listing:

[0040] SEQ ID NO: 1 is the nucleotide sequence encoding the CT3 VH domain.

[0041] SEQ ID NO: 2 is the amino acid sequence of the CT3 VH domain.

[0042] SEQ ID NO: 3 is the nucleotide sequence encoding the CT3 VL domain.

[0043] SEQ ID NO:4 is the amino acid sequence of the CT3 VL domain.

[0044] SEQ ID NO:5 is the nucleotide sequence encoding CT3 scFv.

[0045] SEQ ID NO:6 is the amino acid sequence of CT3 scFv.

[0046] SEQ ID NO: 7 is the nucleotide sequence encoding hCT3-1 (VH-VL) scFv.

[0047] SEQ ID NO: 8 is the amino acid sequence of hCT3-1 (VH-VL) scFv.

[0048] SEQ ID NO:9 is the nucleotide sequence encoding hCT3-1 (VL-VH) scFv.

[0049] SEQ ID NO: 10 is the amino acid sequence of hCT3-1 (VL-VH) scFv.

[0050] SEQ ID NO: 11 is the nucleotide sequence encoding hCT3-2 (VH-VL) scFv.

[0051] SEQ ID NO: 12 is the amino acid sequence of hCT3-2 (VH-VL) scFv.

[0052] SEQ ID NO: 13 is the nucleotide sequence encoding hCT3-2 (VL-VH) scFv.

[0053] SEQ ID NO: 14 is the amino acid sequence of hCT3-2 (VL-VH) scFv.

[0054] SEQ ID NO: 15 is the nucleotide sequence encoding hCT3-3 (VH-VL) scFv.

[0055] SEQ ID NO: 16 is the amino acid sequence of hCT3-3 (VH-VL) scFv.

[0056] SEQ ID NO: 17 is the nucleotide sequence encoding hCT3-3 (VL-VH) scFv.

[0057] SEQ ID NO: 18 is the amino acid sequence of hCT3-3 (VL-VH) scFv.

[0058] SEQ ID NO: 19 is the nucleotide sequence encoding hCT3-4 (VH-VL) scFv.

[0059] SEQ ID NO:20 is the amino acid sequence of hCT3-4 (VH-VL) scFv.

[0060] SEQ ID NO:21 is the nucleotide sequence encoding hCT3-4 (VL-VH) scFv.

[0061] SEQ ID NO:22 is the amino acid sequence of hCT3-4 (VL-VH) scFv.

[0062] SEQ ID NO: 23 is the nucleotide sequence encoding the hinge region of CD28.

[0063] SEQ ID NO: 24 is the amino acid sequence of the hinge region of CD28.

[0064] SEQ ID NO: 25 is the nucleotide sequence encoding the CD28 transmembrane domain.

[0065] SEQ ID NO:26 is the amino acid sequence of the CD28 transmembrane domain.

[0066] SEQ ID NO:27 is a nucleotide sequence encoding a 4-1BB signaling portion.

[0067] SEQ ID NO:28 is the amino acid sequence of the 4-1BB signaling portion.

[0068] SEQ ID NO:29 is the nucleotide sequence encoding the CD3 zeta signaling domain.

[0069] SEQ ID NO:30 is the amino acid sequence of the CD3 zeta signaling domain.

[0070] SEQ ID NO:31 is a nucleotide sequence encoding the GMCSFR signal sequence.

[0071] SEQ ID NO:32 is the amino acid sequence of the GMCSFR signal sequence.

[0072] SEQ ID NO: 33 is a nucleotide sequence encoding a T2A self-cleaving peptide.

[0073] SEQ ID NO: 34 is the amino acid sequence of the T2A self-cleaving peptide.

[0074] SEQ ID NO: 35 is a nucleotide sequence encoding hEGFRt.

[0075] SEQ ID NO:36 is the amino acid sequence of hEGFRt.

[0076] SEQ ID NO:37 is the nucleotide sequence encoding the CT3.28H.BBz CAR construct.

[0077] SEQ ID NO:38 is the amino acid sequence of the CT3.28H.BBz CAR construct, which includes the following features: Residues 1-22 - GMCSFR signal sequence

[0078] Residues 23-24 – restriction enzyme site

[0079] Residues 25-268 – CT3 scFv

[0080] Residues 269-270 – restriction enzyme site

[0081] Residues 271-309 – CD28 hinge

[0082] Residues 310-336 – CD28 transmembrane domain

[0083] Residues 337-378 – 4-1BB co-stimulatory domain

[0084] Residues 379-490 – CD3ζ signaling domain

[0085] Residues 491-508 – T2A site

[0086] Residues 509-530 – GMCSFR signal sequence

[0087] Residues 531-865–hEGFRt

[0088] SEQ ID NO:39 is the amino acid sequence of the CT3.8H.BBz CAR construct, which includes the following features:

[0089] Residues 1-22 – GMCSFR signal sequence

[0090] Residues 23-24 – restriction enzyme site

[0091] Residues 25-268 – CT3 scFv

[0092] Residues 269-270 – restriction enzyme site

[0093] Residues 271-315 – CD8 hinge region

[0094] Residues 316-336 – CD8 transmembrane domain

[0095] Residues 337-378 – 4-1BB co-stimulatory domain

[0096] Residues 379-490 – CD3ζ signaling domain

[0097] Residues 491-508 – T2A site

[0098] Residues 509-530 – GMCSFR signal sequence

[0099] Residues 531-865–hEGFRt

[0100] SEQ ID NO:40 is the amino acid sequence of the CT3.8H.28BBz CAR construct, which includes the following features:

[0101] Residues 1-22 – GMCSFR signal sequence

[0102] Residues 23-24 – restriction enzyme site

[0103] Residues 25-268 – CT3 scFv

[0104] Residues 269-270 – restriction enzyme site

[0105] Residues 271-315 – CD8 hinge

[0106] Residues 316-342 – CD28 transmembrane domain

[0107] Residues 343-383 – CD28 co-stimulatory domain

[0108] Residues 384-425 – 4-1BB co-stimulatory domain

[0109] Residues 426-537 – CD3ζ signaling domain

[0110] Residues 538-555 – T2A site

[0111] Residues 556-577 – GMCSFR signal sequence

[0112] Residues 578-912–hEGFRt

[0113] Detailed Description

[0114] I. Abbreviations

[0115] ALL Acute lymphoblastic leukemia

[0116] ARMS Alveolar rhabdomyosarcoma

[0117] BLI bioluminescence imaging

[0118] CAR Chimeric Antigen Receptor

[0119] CDR complementarity determining region

[0120] CNS Central Nervous System

[0121] DRCT Desmoplastic small round cell tumor

[0122] E:T effect-target ratio

[0123] EF1α Elongation factor 1α

[0124] EGF Epidermal Growth Factor

[0125] EGFR Epidermal Growth Factor Receptor

[0126] ERMS Embryonic rhabdomyosarcoma

[0127] ffLUC Firefly luciferase

[0128] GPC2 Glypican-2

[0129] GMCSFRss Granulocyte-macrophage colony-stimulating factor receptor signal sequence

[0130] hEGFRt Truncated human epidermal growth factor receptor

[0131] iPSC Induced Pluripotent Stem Cell

[0132] iv intravenous

[0133] KO knockout

[0134] NB Neuroblastoma

[0135] NK Natural Killer

[0136] PDX patient-derived xenografts

[0137] RMS Rhabdomyosarcoma

[0138] scFv single chain variable fragment

[0139] TM Transmembrane

[0140] TME Tumor Microenvironment

[0141] VH Heavy chain variable region

[0142] UT Untransduced

[0143] VL light chain variable region

[0144] WT Wild type

[0145] II. Summary of Terminology

[0146] Unless otherwise indicated, technical terms are used according to conventional usage. Definitions of many common terms in molecular biology can be found in the following literature: Krebs et al. (eds.), Lewin's genes XII, published by Jones & Bartlett Learning, 2017. As used herein, unless the context clearly indicates otherwise, the singular forms "a", "an" and "the" refer to the singular as well as the plural. For example, the term "antigen (an antigen)" includes a singular or plural antigen and can be regarded as equivalent to the phrase "at least one antigen". As used herein, the term "comprising" means "including". It should also be understood that, unless otherwise stated, any and all base sizes or amino acid sizes and all molecular weight or molecular mass values ​​given for nucleic acids or polypeptides are approximate and provided for descriptive purposes. Although many methods and materials similar or equivalent to those described herein can be used, specific suitable methods and materials are described herein. In the event of a conflict, this specification (including the explanation of the terms) will dominate. In addition, the materials, methods and examples are only illustrative and are not intended to be limiting. To facilitate review of various aspects, the following terminology is provided:

[0147] 4-1BB: A co-stimulatory molecule expressed by lymphocytes activated by T cell receptors (TCR) and other cells including natural killer cells. The connection of 4-1BB induces a signal transduction cascade, which leads to cytokine production, anti-apoptotic molecule expression, and immune response enhancement. The exemplary amino acid sequence of 4-1BB is described herein as SEQ ID NO: 28.

[0148] Acute lymphoblastic leukemia (ALL): An acute form of leukemia characterized by an overproduction of lymphoblasts. ALL most commonly presents in childhood, peaking at 2-5 years of age.

[0149] Administration: Provide or administer an agent to a subject by any effective route, such as a CAR or a cell expressing CAR as provided herein. Exemplary administration routes include, but are not limited to, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, intracerebral, intraventricular, intracranial, intramedullary, intravenous, intraarterial (including intrahepatic artery), intraosseous, intravitreal, and intratumoral), rectal, transdermal, intranasal, vaginal, and inhalation routes. In some examples, administration is local. In some examples, administration is systemic.

[0150] Antibody: A polypeptide ligand that contains at least one variable region that recognizes and binds (e.g., specifically recognizes and specifically binds) an antigen (e.g., GPC2) epitope. Mammalian immunoglobulin molecules are composed of heavy (H) chains and light (L) chains, each with a variable region, called the heavy chain variable (V H ) region and light chain variable (V L ) area. H Area and V L The heavy chain domains are together responsible for binding to the antigen recognized by the antibody. There are five major heavy chain classes (or isotypes) of mammalian immunoglobulins that determine the functional activity of the antibody molecule: IgM, IgD, IgG, IgA, and IgE. Some mammals, such as camels, alpacas, and llamas, have heavy chain antibodies that lack light chains. Antibody isotypes that do not exist in mammals include IgX, IgY, IgW, and IgNAR. IgY is the main antibody produced by birds and reptiles and has some functions similar to mammalian IgG and IgE. Cartilaginous fish such as sharks produce IgW antibodies and IgNAR antibodies, while IgX antibodies are found in amphibians. IgNAR antibodies are heavy chain antibodies.

[0151] The variable region of an antibody contains a "framework" region and a hypervariable region called a "complementarity determining region" or "CDR". The CDR is primarily responsible for binding to the epitope of an antigen. The antibody framework region serves to position and align the CDR in three-dimensional space. The amino acid sequence boundaries of a given CDR can be readily determined using any of a number of well-known numbering schemes, including those by Kabat et al. (Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, 1991; “Kabat numbering scheme), Chothia et al. (see Chothia and Lesk, J Mol Biol 196:901-917, 1987; Chothia et al., Nature 342:877, 1989; and Al-Lazikani et al., (JMB 273, 927-948, 1997; “Chothia” numbering scheme), Kunik et al. (see Kunik et al., PLoS Comput Biol 8:e1002388, 2012; and Kunik et al., Nucleic Acids Res 40:W521-524, 2012;

[0152] Kabat, Paratome and IMGT are maintained online.

[0153] "Single domain antibody" refers to an antibody with a single domain (the variable domain) that is capable of specifically binding to an antigen or antigenic epitope in the absence of additional antibody domains. Single domain antibodies include, for example, V H Domain antibodies, V NAR Antibodies, camelid V H H antibody and V L Domain antibodies. V NAR Antibodies are produced by cartilaginous fishes such as nurse sharks, wobbegong sharks, spiny dogfish, and bamboo sharks that produce heavy chain antibodies (IgNARs). Several species that produce heavy chain antibodies that naturally lack light chains produce camelid V H H antibody, the species include camel, llama, alpaca and guanaco.

[0154] A "monoclonal antibody" is an antibody produced by a single clone of lymphocytes or by a cell into which the coding sequence for a single antibody has been transfected. Monoclonal antibodies are produced by known methods. Monoclonal antibodies include humanized monoclonal antibodies.

[0155] "Chimeric antibodies" have framework residues from one species (eg, human) and the CDRs (which typically confer antigen binding) from another species.

[0156] "Humanized" antibodies are immunoglobulins that include human framework regions and one or more CDRs from non-human (e.g., mouse, rabbit, rat, shark, camel or synthetic) immunoglobulins. The non-human immunoglobulin that provides CDRs is called a "donor," and the human immunoglobulin that provides the framework is called an "acceptor." In one aspect, all CDRs are from the donor immunoglobulin of the humanized immunoglobulin. The constant region need not exist, but if it does, they are substantially identical to the human immunoglobulin constant region, i.e., at least about 85-90% identical, such as about 95% or more identical. Therefore, all parts of the humanized immunoglobulin (possibly except CDRs) are substantially identical to the corresponding parts of the natural human immunoglobulin sequence. Humanized antibodies are bound to the same antigen as the donor antibody that provides the CDRs. Humanized or other monoclonal antibodies may have additional conservative amino acid substitutions that do not substantially affect antigen binding or other immunoglobulin functions.

[0157] Binding affinity: The affinity of an antibody or other antigen-binding molecule to an antigen such as GPC2. In one aspect, affinity is calculated by the modified Scatchard method described by Frankel et al., Mol. Immunol., 16: 101-106, 1979. In another aspect, binding affinity is measured according to the antigen / antibody dissociation rate. In another aspect, high binding affinity is measured by competitive radioimmunoassay. In another aspect, binding affinity is measured by ELISA. In some aspects, binding affinity is measured using the Octet system (ForteBio) based on biolayer interferometry technology. In other aspects, surface plasmon resonance is used to measure, for example, using BIACORES-2000 or BIACORES-3000 (BIAcore, Inc., Piscataway, NJ), Kd is measured. In other aspects, antibody affinity is measured by flow cytometry. An antibody or CAR that "specifically binds" an antigen (such as GPC2) is an antibody or CAR that binds to an antigen with high affinity and does not significantly bind to other unrelated antigens.

[0158] Chemotherapeutic agent: Any chemical agent that has therapeutic use in treating a disease characterized by abnormal cell growth. Such diseases include tumors, neoplasms, and cancer. In one aspect, a chemotherapeutic agent is an agent used in treating a GPC2-positive tumor. In one aspect, a chemotherapeutic agent is a radioactive compound. Exemplary chemotherapeutic agents that can be used with the methods provided herein are disclosed in the following references: Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 in Harrison's Principles of Internal Medicine, 14th edition; Perry et al., Chemotherapy, Ch. 17 in Abeloff, Clinical Oncology 2 nd ed., 2000Churchill Livingstone, Inc; Baltzer, L., Berkery, R. (eds.): Oncology Pocket Guide to Chemotherapy, 2nd ed. St. Louis, Mosby-Year Book, 1995; Fischer, DS, Knobf, MF, Durivage, HJ (eds): The Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-YearBook, 1993. In one example, the chemotherapeutic agent is a biologic, such as a therapeutic antibody (e.g., a therapeutic monoclonal antibody), such as an anti-GPC2 antibody, and other anti-cancer antibodies, such as anti-PD1 or anti-PDL1 antibodies (e.g., pembrolizumab and nivolumab), anti-CTLA4 antibodies (e.g., ipilimumab), anti-EGFR antibodies (e.g., cetuximab), anti-VEGF antibodies (e.g., bevacizumab) or a combination thereof (e.g., anti-PD-1 and anti-CTLA-4). Combination chemotherapy is the administration of more than one agent to treat cancer. An example is the administration of immune cells expressing a CAR targeting GPC2, in combination with radioactive, biological or chemical compounds (or a combination thereof).

[0159] Chimeric Antigen Receptor (CAR): A chimeric molecule that comprises an antigen-binding portion, such as a single domain antibody (e.g., V NAR 、V H H or V H) or scFv, and a signaling domain, such as a signaling domain from a T cell receptor (e.g., CD3ζ). In many cases, CAR includes an antigen binding portion, a hinge region, a transmembrane domain, and an intracellular domain. The intracellular domain may include a signaling chain having an immunoreceptor tyrosine-based activation motif (ITAM), such as CD3ζ or FcεRIγ. In some cases, the intracellular domain further includes an intracellular portion of at least one additional costimulatory domain (such as CD28, 4-1BB (CD137), ICOS, OX40 (CD134), CD27, MYD88-CD40, KIR2DS2, and / or DAP10). In some examples, CAR has multispecificity (such as bispecificity) or bicistrans. Multispecific CAR is a single CAR molecule (see, e.g., US Pat. No. 5,837,676,854) comprising at least two antigen binding domains (such as scFv and / or single domain antibodies) that each binds to different antigens or different epitopes on the same antigen.

[0160] 2018 / 0230225). For example, a bispecific CAR refers to a single CAR molecule having two antigen binding domains that each bind to a different antigen. A bicistrans CAR refers to two complete CAR molecules, each containing an antigen binding portion that binds to a different antigen. In some cases, a bicistrans CAR construct expresses two complete CAR molecules connected by a cleavage linker. Immune cells (such as T cells, NK cells, B cells, or macrophages) or iPSCs expressing bispecific or bicistrans CARs can bind to cells expressing two antigens to which the binding portion is directed (see, e.g., Qin et al., Blood 130:810, 2017; and WO / 2018 / 213337). In some aspects, the CAR is a double-chain antibody-T cell receptor (AbTCR) as described in Xu et al. (Cell Discovery 4:62, 2018) or a synthetic T cell receptor and antigen receptor (STAR) as described in Liu et al. (Sci Transl Med 13(586):eabb5191, 2021).

[0161] Complementarity Determining Region (CDR): A highly variable region of amino acid sequence that determines the binding affinity and specificity of an antibody. The light and heavy chains of mammalian immunoglobulins each have three CDRs, named L-CDR1, L-CDR2, L-CDR3 and H-CDR1, H-CDR2, H-CDR3. Single-domain antibodies contain three CDRs (CDR1, CDR2, and CDR3).

[0162] Conservative variants: In the context of the present disclosure, "conservative" amino acid substitutions are those that do not substantially affect or reduce the affinity of a protein (such as an antibody) for GPC2. As an example, a monoclonal antibody that specifically binds to GPC2 may include up to about 1, up to about 2, up to about 5, up to about 10, up to about 15, up to about 20, or up to about 25 conservative substitutions and specifically bind to a GPC2 polypeptide. The term "conservative variant" also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid, provided that the variant retains activity. Non-conservative substitutions are those that reduce the activity (such as affinity) of a protein.

[0163] Conservative amino acid substitution tables that provide functionally similar amino acids are well known. The following six groups are examples of amino acids that are considered conservative substitutions for each other:

[0164] 1) Alanine (A), serine (S), threonine (T);

[0165] 2) Aspartic acid (D), glutamic acid (E);

[0166] 3) Asparagine (N), glutamine (Q);

[0167] 4) Arginine (R), Lysine (K);

[0168] 5) isoleucine (I), leucine (L), methionine (M), valine (V); and

[0169] 6) Phenylalanine (F), tyrosine (Y), tryptophan (W);

[0170] In some aspects herein, amino acid sequences are provided that comprise no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 amino acid substitution relative to any amino acid sequence disclosed herein.

[0171] Contact: In direct physical contact; includes both solid and liquid forms.

[0172] Degenerate variant: A polynucleotide encoding a polypeptide that contains a sequence that is degenerate due to the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, as long as the amino acid sequence of the polypeptide is unchanged, all degenerate nucleotide sequences are included.

[0173] Desmoplastic small round cell tumor (DRCT): A soft tissue sarcoma that occurs primarily in childhood, especially in boys. DRCT is an aggressive and rare type of cancer that appears primarily as a mass in the abdomen but can also be present in the lymph nodes, abdominal lining, diaphragm, spleen, liver, chest wall, skull, spinal cord, intestine, bladder, brain, lungs, testicles, ovaries, and pelvis.

[0174] Epitope: Antigenic determinant. It is a specific chemical group or peptide sequence on a molecule that is antigenic (provokes a specific immune response). Antibodies specifically bind to specific antigenic epitopes on polypeptides.

[0175] Framework region: amino acid sequence inserted between CDRs. The framework region includes the light chain variable framework region and the heavy chain variable framework region. The framework region plays a role in keeping the CDRs in the proper orientation for antigen binding.

[0176] Glioma: A type of tumor that arises in the brain and spinal cord. Glioma arises from glial cells that surround and support neurons in the brain, including astrocytes, oligodendrocytes, and ependymal cells. There are three types of gliomas based on the type of cell from which the tumor arises: astrocytomas, ependymomas, and oligodendrogliomas.

[0177] Glypican-2 (GPC2): A member of the six-member glypican family of heparan sulfate (HS) proteoglycans that binds to the cell surface via a GPI anchor (Li et al., Trends Cancer 4(11):741-754, 2018). GPC2 mRNA is highly expressed in neuroblastoma and other childhood cancers (Orentas et al., Front Oncol 2:194, 2012). GPC2 protein is highly expressed in about half of neuroblastoma cases and is associated with poor overall survival compared to patients with low GPC2 expression (Li et al., Proc Natl Acad Sci USA 114(32):E6623-E6631, 2017). GPC2 is also known as brain proteoglycan and glypican 2. The GPC2 genomic sequence, mRNA sequence, and protein sequence are publicly available (see, e.g., NCBI Gene ID 221914).

[0178] GPC2-positive cancer: A cancer that expresses or overexpresses GPC2. Examples of GPC2-positive cancers include, but are not limited to, neuroblastoma, medulloblastoma, retinoblastoma, acute lymphoblastic leukemia, embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, Ewing sarcoma, desmoplastic small round cell tumor, glioma, and osteosarcoma.

[0179] Heterologous: Derived from a separate genetic source or species.

[0180] Host cell: A cell in which a vector can be propagated and its DNA expressed. The cell can be prokaryotic or eukaryotic. In some examples, the prokaryotic cell is an Escherichia coli (E. coli) cell. In some examples, the eukaryotic cell is a human cell, such as a human embryonic kidney (HEK) cell. The term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parent cell because there may be mutations that occur during replication. However, such progeny are included when the term "host cell" is used.

[0181] Immune response: The response of an immune system cell, such as a B cell, T cell, or monocyte, to a stimulus. In one aspect, the response is specific for a particular antigen (an "antigen-specific response"). In one aspect, the immune response is a T cell response, such as a CD4 + Response or CD8 + In another aspect, the response is a B cell response and results in the production of specific antibodies.

[0182] Isolated: An "isolated" biological component, such as a nucleic acid, protein (including antibodies), or organelle, has been substantially separated or purified from other biological components (e.g., other chromosomal and extrachromosomal DNA and RNA, proteins, and organelles) in the environment (e.g., a cell) in which the component occurs. Nucleic acids and proteins that have been "isolated" include nucleic acids and proteins purified by standard purification methods. The term also encompasses nucleic acids and proteins prepared by recombinant expression in a host cell and chemically synthesized nucleic acids and proteins.

[0183] Label: A detectable compound or composition that is conjugated directly or indirectly to another molecule, such as an antibody or protein, to facilitate detection of that molecule. Specific, non-limiting examples of labels include fluorescent tags, enzymatic linkages, and radioisotopes. In one example, a "labeled antibody" refers to the incorporation of another molecule into an antibody. For example, a label is a detectable marker, such as a polypeptide incorporating a radiolabeled amino acid or conjugated to a biotinyl moiety that can be detected with the aid of labeled avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). A variety of methods for labeling polypeptides and glycoproteins are known and can be used. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionucleotides (such as 35 S. 11 C. 13 N. 15 O. 18 F. 19 F. 99m Tc, 131 I. 3 H.14 C. 15 N. 90 Y. 99 Tc, 111 In and 125 I), fluorescent markers (such as fluorescein isothiocyanate (FITC), rhodamine, lanthanide phosphors), enzyme markers (such as horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by secondary reporter molecules (such as leucine zipper pair sequences, binding sites of secondary antibodies, metal binding domains, epitope tags) or magnetic agents such as gadolinium chelates. In some aspects, the labels are connected by spacer arms of various lengths to reduce potential steric hindrance.

[0184] Linker: In some cases, a linker is a peptide inside an antibody binding fragment (such as an scFv fragment) that acts to indirectly bind the heavy chain variable region to the light chain variable region. In some aspects of the present invention, the disclosed scFv includes (G4S)3 linkers of different lengths that join the VH domain and the VL domain of the antigen binding domain. "Linker" can also refer to a peptide that acts to connect a targeting portion (such as an antibody) to an effector molecule (such as a cytotoxin or a detectable marker). The term "conjugation", "joining", "bonding" or "connection" refers to making two polypeptides a continuous polypeptide molecule, or refers to covalently joining a radionuclide or other molecule to a polypeptide such as scFv. In a specific context, the term includes joining a ligand (such as an antibody portion) to an effector molecule. The connection can be performed by chemical or recombinant means. "Chemical means" refers to the reaction between the antibody portion and the effector molecule, thereby forming a covalent bond between the two molecules to form one molecule.

[0185] Mammal: This term includes human and non-human mammals. Similarly, the term "subject" includes human subjects and veterinary subjects, such as mice, rats, cows, cats, dogs, pigs, and non-human primates.

[0186] Medulloblastoma: A fast-growing cancer that forms in the cerebellum. Medulloblastomas tend to spread through the cerebrospinal fluid to other parts of the spinal cord or brain. They can also spread to other parts of the body, but this is rare. Medulloblastomas are most common in children and young adults. They are a type of embryonal tumor of the central nervous system.

[0187] Neoplasia, malignancy, cancer, or tumor: A neoplasm is an abnormal growth of tissue or cells resulting from excessive cell division. Neoplastic growth can produce a tumor. The amount of tumor in an individual is the "tumor burden," which can be measured as the number, volume, or weight of tumors. Tumors that do not metastasize are called "benign." Tumors that invade surrounding tissues and / or can metastasize are called "malignant."

[0188] Neuroblastoma: A solid tumor that arises from cells of the embryonic neural crest. Neuroblastomas often arise in and around the adrenal glands, but can develop anywhere there is sympathetic nerve tissue, such as in the abdomen, chest, neck, or near the spine. Neuroblastomas usually occur in children under the age of 5.

[0189] Operably linked: A first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence. Typically, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.

[0190] Osteosarcoma: A type of cancerous tumor that is found in the bones. Osteosarcoma is an aggressive cancer that arises from primitive cells of mesenchymal origin that have become transformed. This type of cancer is most common in children and young adults.

[0191] Childhood cancer: Cancer that develops in children between the ages of 0 and 14. Major types of childhood cancer include, for example, neuroblastoma, acute lymphoblastic leukemia (ALL), embryonal rhabdomyosarcoma (ERMS), alveolar rhabdomyosarcoma (ARMS), Ewing sarcoma, desmoplastic small round cell tumor (DRCT), osteosarcoma, brain and other CNS tumors (such as medulloblastoma), Wilm's tumor, non-Hodgkin lymphoma, and retinoblastoma.

[0192] Pharmaceutically acceptable carriers: Useful pharmaceutically acceptable carriers are conventional. Remington: The Science and Practice of Pharmacy, 22 nd Ed., London, UK: Pharmaceutical Press, 2013 describes compositions and preparations suitable for pharmaceutical delivery of cells expressing CAR disclosed herein and other compositions. The nature of the carrier may depend on the specific mode of administration being adopted. For example, parenteral preparations generally include injectable fluids, which include pharmaceutically and physiologically acceptable fluids such as water, saline, balanced salt solutions, aqueous dextrose, glycerol, etc. as solvents. For solid compositions (such as powders, pellets, tablets or capsule forms), conventional non-toxic solid carriers may, for example, include pharmaceutical grade mannitol, lactose, starch or magnesium stearate. In addition to biological neutral carriers, the pharmaceutical composition to be administered may contain a small amount of non-toxic auxiliary substances, such as wetting or emulsifiers, preservatives and pH buffers, such as sodium acetate or sorbitan monolaurate.

[0193] Prevent, treat, or alleviate disease: "Preventing" a disease means inhibiting the disease from fully developing. "Treatment" refers to therapeutic intervention that relieves the signs or symptoms of a disease or pathological condition after it has already developed (e.g., a decrease in tumor burden or a decrease in the number or size of metastases). "Impact" refers to a decrease in the number or severity of signs or symptoms of a disease (e.g., cancer).

[0194] Purified: The term "purified" does not require absolute purity; on the contrary, it is intended as a relative term. Thus, for example, a purified peptide preparation is one in which the peptide or protein is more enriched than the peptide or protein in its natural environment inside a cell. Similarly, a purified cell is one in which the cell is more enriched than the cell in its natural environment inside a subject or in which the cell is substantially free of other cell types. In one aspect, the preparation is purified so that the protein or peptide accounts for at least 50% of the total peptide or protein content of the preparation. Substantially purified refers to purification from other proteins or cell components. Substantially purified proteins are at least 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.9% or 99.99% pure. Thus, in a specific non-limiting example, a substantially purified protein is at least 90% free of other proteins or cell components. Substantially purified cells (such as cells expressing CAR provided herein) can be at least 90%, 95%, 98%, 99%, 99.9% or 99.99% pure. Therefore, in a specific non-limiting example, substantially purified cells expressing CAR provided herein are at least 99% free of other cells (such as other immune cells or other cells not expressing CAR provided herein) or cell components.

[0195] Recombinant: A recombinant nucleic acid is a nucleic acid that has a sequence that does not exist in nature or has a sequence that is produced by artificially combining two otherwise separate sequence segments. This artificial combination is often achieved by chemical synthesis or by artificial manipulation of isolated nucleic acid segments (e.g., by genetic engineering techniques).

[0196] Retinoblastoma: A type of cancer that forms in the tissue of the retina. Retinoblastoma usually develops in children under the age of 5. It can be hereditary or non-hereditary (sporadic).

[0197] Rhabdomyosarcoma (RMS): A soft tissue malignancy of skeletal muscle origin. The most common primary sites of rhabdomyosarcoma are the head and neck (e.g., parameningeal, orbital, pharyngeal, etc.), genitourinary tract, and extremities. Other less common primary sites include the trunk, chest wall, abdomen (including the retroperitoneum and bile duct), and perineum / anus region. There are at least two types of RMS; the most common forms are alveolar RMS (ARMS) and embryonic tissue RMS (ERMS). Approximately 20% of children with rhabdomyosarcoma have the ARMS subtype. The increased frequency of this subtype is evident in adolescents and in patients whose primary sites involve the extremities, trunk, and perineum / perianal region. ARMS is associated with a chromosomal translocation encoding a fusion gene involving FKHR and PAX family members on chromosome 13. The embryonic subtype is the most frequently observed subtype in children, accounting for approximately 60-70% of childhood rhabdomyosarcoma. Tumors with embryonal histology generally arise in the head and neck region or in the genitourinary tract, although they may arise from any primary site. ERMS are characterized by a younger age at diagnosis, loss of heterozygosity, and alterations in genomic imprinting.

[0198] Sample (or biological sample): A biological specimen obtained from a subject containing genomic DNA, RNA (including mRNA), protein, or a combination thereof. Examples include, but are not limited to, peripheral blood, tissue, cells, urine, saliva, tissue biopsy samples, fine needle aspirates, surgical specimens, and autopsy materials. In one example, the sample includes a tumor biopsy sample, such as a tumor tissue biopsy sample.

[0199] Sequence identity: Similarity between amino acid sequences or nucleic acid sequences is expressed as similarity between sequences, also known as sequence identity. Sequence identity is often measured as a percentage of identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologues or variants of polypeptides or nucleic acid molecules will have a relatively high degree of sequence identity when aligned using standard methods.

[0200] Methods of alignment of sequences for comparison are known. A variety of programs and alignment algorithms are described in Smith and Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J. Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins and Sharp, Gene 73:237, 1988; Higgins and Sharp, CABIOS 5:151, 1989; Corpet et al., Nucleic Acids Research 16:10881, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988. Altschul et al., Nature Genet. 6:119, 1994 provides a detailed consideration of sequence alignment methods and homology calculations.

[0201] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the Internet, in conjunction with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. Instructions on how to use this program to determine sequence identity are available on the NCBI website on the Internet.

[0202] Homologs and variants of antibodies or CARs that specifically bind to GPC2 are generally characterized in that, when using NCBI Blast2.0, blastp with gaps (set to default parameters), they have at least about 75%, for example at least about 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity calculated over the entire length alignment with the amino acid sequence of the antibody or CAR. In order to compare amino acid sequences of more than about 30 amino acids, the Blast 2 sequence function is used, using the default BLOSUM62 matrix set to default parameters (the gap existence cost is 11, and the gap cost per residue is 1). When aligning short peptides (less than about 30 amino acids), the Blast 2 sequence function should be used for alignment, using the PAM30 matrix set to default parameters (open gap penalty is 9, and the extended gap penalty is 1). When evaluated in this way, proteins with even greater similarity to a reference sequence will show increasing percentages of identity, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. When the sequence identity of the entire sequence is less than that of the entire sequence, homologs and variants will generally have at least 80% sequence identity within a short window of 10-20 amino acids, and may have at least 85% or at least 90% or 95% sequence identity, depending on their similarity to the reference sequence. Methods for determining sequence identity within such short windows are available at the NCBI website on the Internet. These sequence identity ranges are provided for guidance only; it is entirely possible to obtain very significant homologs outside the ranges provided.

[0203] Subject: Living multicellular vertebrate organisms, a category that includes human subjects and veterinary subjects, including human and non-human mammals such as pigs, mice, rats, rabbits, sheep, horses, cows, dogs, cats, and non-human primates.

[0204] Synthetic: Produced by artificial means in a laboratory, e.g., a synthetic nucleic acid or protein (e.g., an antibody) can be chemically synthesized in a laboratory.

[0205] Therapeutically effective amount: The amount of a specific substance sufficient to achieve the desired effect in a subject being treated. For example, this amount can be the amount necessary to inhibit or suppress tumor growth. In one aspect, a therapeutically effective amount is the amount necessary to eliminate a tumor, reduce its size, or prevent its metastasis, such as by reducing tumor size and / or volume by at least 10%, at least 20%, at least 50%, at least 75%, at least 80%, at least 90%, at least 95% or even 100% and / or reducing the number of metastases and / or size / volume by at least 10%, at least 20%, at least 50%, at least 75%, at least 80%, at least 90%, at least 95% or even 100%. When administered to a subject, a dosage that will achieve a target tissue concentration (e.g., in a tumor) that has been shown to achieve the desired in vitro effect will generally be used.

[0206] Vector: A nucleic acid molecule introduced into a host cell to produce a transformed host cell. A vector may include a nucleic acid sequence that allows the vector to replicate in the host cell, such as an origin of replication. The vector may also include one or more selectable marker genes and other genetic elements. In some examples, the vector is a viral vector, such as a lentiviral vector, an adenoviral vector, or an adeno-associated virus (AAV) vector.

[0207] III. Overview of Several Aspects

[0208] CART cell therapy is a new class of cancer treatments that are being actively developed and tested around the world. After initial success using CD19-targeted CAR T cells in blood cancers, multiple CAR strategies have been engineered and tested with the goal of treating solid tumors. However, the success of CART cells in solid tumors is limited by several obstacles, including the lack of tumor-specific antigens, the inability of CART cells to efficiently expand at the tumor site, and heterogeneous antigen expression (Kochenderfer et al., Blood. 2012; 119(12):2709-2720; Porter et al., NEng J Med 2011; 365(8):725-733; Jiang et al., Front Immunol 2017; 7:690; Gao et al., Clin Cancer Res 2014; 20(24):6418-6428; Ishiguro et al., Cancer Res 2008; 68(23):9832-9838; Losic et al., Nat Commun 2020; 11(1):291; Li et al., Gastroenterology 158(8):2250-2265, 2020; Li et al., CellRep Med2(6):100297, 2021).

[0209] The present disclosure solves these problems and improves the efficacy of CAR-expressing cells for treating GPC2-positive tumors. The engineered CAR disclosed herein includes an antigen binding domain derived from GPC2-specific antibody CT3 (PCT Publication No. WO 2020 / 033430, incorporated herein by reference) or a humanized form thereof. It is disclosed herein that compared with CARs containing CD8 hinge regions and CD8 or CD28 transmembrane domains targeting GPC2, immune cells expressing CARs containing CD28 hinge regions and CD28 transmembrane domains targeting GPC2 significantly more powerfully kill GPC2-positive tumors. In addition, using an in situ neuroblastoma model, it was demonstrated that relative to CARs with CD8 hinges and transmembrane domains targeting GPC2, immune cells expressing CARs with CD28 hinges and transmembrane domains targeting GPC2 showed superior amplification in vitro and in vivo for GPC2-positive tumor cells, producing higher levels of tumor-infiltrating CAR+T cells, and resulting in increased survival. In addition, immune cells expressing CARs with CD28 hinge and transmembrane domains targeting GPC2 showed superior anti-tumor activity in a neuroblastoma model compared with existing CAR T cell therapies for neuroblastoma.

[0210] CARs are provided herein, which include an extracellular antigen binding domain that specifically binds to GPC2; a CD28 hinge region; a CD28 transmembrane domain; an intracellular co-stimulatory domain and an intracellular signaling domain. In some aspects, the GPC2-specific antigen binding domain is a scFv. The scFv can be VH-linker-VL or VL-linker-VH from the N-terminus to the C-terminus.

[0211] In some aspects, the antigen binding domain comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises a complementary determining region 1 (CDR1) sequence, a CDR2 sequence, and a CDR3 sequence of SEQ ID NO: 2 (CT3 VH domain sequence) and / or the VL domain comprises a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence of SEQ ID NO: 4 (CT3 VL domain sequence). In some examples, the CDR sequences are defined using the Kabat numbering scheme, the IMGT numbering scheme, or the Paratome numbering scheme, or a combination of the Kabat numbering scheme, the IMGT numbering scheme, and the Paratome numbering scheme. In other examples, the CDR sequences are determined using different numbering schemes (such as Chothia).

[0212] In some aspects, the VH domain CDR1 sequence, CDR2 sequence, and CDR3 sequence include residues 31-35, 50-66, and 99-112 of SEQ ID NO: 2, respectively, and / or the VL domain CDR1 sequence, CDR2 sequence, and CDR3 sequence include residues 24-33, 49-55, and 88-96 of SEQ ID NO: 4, respectively; the VH domain CDR1 sequence, CDR2 sequence, and CDR3 sequence include residues 26-33, 51-58, and 97-112 of SEQ ID NO: 2, respectively, and / or

[0213] The VL domain CDR1 sequence, CDR2 sequence and CDR3 sequence include residues 27-31, 49-51 and 88-96 of SEQ ID NO:4, respectively; the VH domain CDR1 sequence, CDR2 sequence and CDR3 sequence include residues 26-35, 47-61 and 97-112 of SEQ ID NO:2, respectively and / or the VL domain CDR1 sequence, CDR2 sequence and CDR3 sequence include residues 27-33, 45-55 and 88-95 of SEQ ID NO:4, respectively; or the VH domain CDR1 sequence, CDR2 sequence and CDR3 sequence include residues 26-35, 47-66 and 97-112 of SEQ ID NO:2, respectively and / or

[0214] The VL domain CDR1 sequence, CDR2 sequence, and CDR3 sequence include residues 24-33, 45-55, and 88-96, respectively, of SEQ ID NO: 4. In some examples, the amino acid sequence of the VH domain is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 2 (and includes the CDR1 sequence, CDR2 sequence, and CDR3 sequence of SEQ ID NO: 2) and / or the amino acid sequence of the VL domain is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 4 (and includes the CDR1 sequence, CDR2 sequence, and CDR3 sequence of SEQ ID NO: 4).

[0215] In some aspects, the VH domain sequence and the VL domain sequence are humanized. In some examples, the amino acid sequence of the humanized VH domain includes residues 1-123 of SEQ ID NO: 8, and / or the amino acid sequence of the humanized VL domain includes residues 139-244 of SEQ ID NO: 8; the amino acid sequence of the humanized VH domain includes

[0216] residues 1-122 of SEQ ID NO:12, and / or the amino acid sequence of the humanized VL domain includes residues 138-243 of SEQ ID NO:12; the amino acid sequence of the humanized VH domain includes residues 1-122 of SEQ ID NO:16, and / or the amino acid sequence of the humanized VL domain includes residues 138-244 of SEQ ID NO:16; or the amino acid sequence of the humanized VH domain includes residues 1-122 of SEQ ID NO:20, and / or the amino acid sequence of the humanized VL domain includes residues 138-243 of SEQ ID NO:20.

[0217] In some aspects, the amino acid sequence of the extracellular antigen binding domain is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, or SEQ ID NO: 22. In some examples, the amino acid sequence of the antigen binding domain comprises or consists of the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, or SEQ ID NO: 22.

[0218] In some aspects, the amino acid sequence of the hinge region of CD28 is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 24. In some examples, the amino acid sequence of the hinge region of CD28 comprises or consists of the amino acid sequence of SEQ ID NO: 24.

[0219] In some aspects, the amino acid sequence of the CD28 transmembrane domain is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 26. In some examples, the amino acid sequence of the CD28 transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO: 26.

[0220] In some aspects, the costimulatory domain includes a 4-1BB signaling portion. In some examples, the amino acid sequence of the 4-1BB signaling portion is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 28. In a specific example, the amino acid sequence of the 4-1BB signaling portion includes or consists of the amino acid sequence of SEQ ID NO: 28.

[0221] In some aspects, the signaling domain comprises a CD3 zeta signaling domain. In some examples, the amino acid sequence of the CD3 zeta signaling domain is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 30. In a specific example, the amino acid sequence of the CD3 zeta signaling domain comprises or consists of the amino acid sequence of SEQ ID NO: 30.

[0222] In certain aspects, the amino acid sequence of CAR is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 38. In a specific example, the amino acid sequence of CAR comprises or consists of the amino acid sequence of SEQ ID NO: 38.

[0223] In alternative aspects, the amino acid sequence of CAR is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 39 or SEQ ID NO: 40. In a specific example, the amino acid sequence of CAR comprises or consists of the amino acid sequence of SEQ ID NO: 39 or SEQ ID NO: 40.

[0224] Also provided herein are nucleic acid molecules encoding CAR disclosed herein. In some aspects, the sequence of the nucleic acid molecule is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to nucleotides 73-1470 of SEQ ID NO:37. In some examples, the sequence of the nucleic acid molecule includes nucleotides 73-1470 of SEQ ID NO:37 or consists of it. In a specific non-limiting example, the sequence of the nucleic acid molecule includes SEQ ID NO:37 or consists of it.

[0225] In some aspects, the nucleic acid molecule is operably linked to a promoter (eg, an inducible or constitutive promoter). In some examples, the promoter is the human elongation factor 1 alpha (EF1 alpha) promoter.

[0226] In some aspects, the nucleic acid molecule includes, in the 5' to 3' direction, a nucleic acid encoding a first granulocyte-macrophage colony stimulating factor receptor signal sequence (GMCSFRss); a nucleic acid encoding an antigen binding domain; a nucleic acid encoding a CD28 hinge region; a nucleic acid encoding a CD28 transmembrane domain; a nucleic acid encoding a co-stimulatory domain; a nucleic acid encoding a signaling domain; a nucleic acid encoding a self-cleaving 2A peptide; a nucleic acid encoding a second GMCSFRss; and a nucleic acid encoding a truncated human epidermal growth factor receptor (hEGFRt). In some examples, the nucleic acid molecule further includes a human elongation factor 1 alpha (EF1 alpha) promoter sequence 5' of the nucleic acid encoding the first GMCSFRss (see WO 2019 / 094482, which is incorporated herein by reference in its entirety).

[0227] Further provided are vectors comprising the nucleic acid molecules disclosed herein. In some examples, the vector is a viral vector, such as a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector.

[0228] Also provided is a separated cell, which includes a nucleic acid molecule (or vector) encoding a CAR disclosed herein and / or expresses a CAR disclosed herein. In some aspects, the cell is an immune cell, such as a T cell, a NK cell, a B cell or a macrophage. In other aspects, the cell is an induced pluripotent stem cell (iPSC).

[0229] A composition is further provided, comprising a pharmaceutically acceptable carrier (such as water or saline) and a CAR disclosed herein, a nucleic acid molecule, a vector or a cell. In some examples, the composition is frozen. In some examples, the composition is frozen and includes cells and DMSO or another cryopreservative. In some examples, the composition is lyophilized. In some examples, such compositions are in vials, such as glass vials or plastic vials. The disclosed composition can be part of a kit, such as a kit including one or more chemotherapeutic agents, a syringe, a cell culture medium, a pharmaceutically acceptable carrier or a combination thereof (wherein the additional agents in the kit can be in separate containers).

[0230] Also provided is a method for treating GPC2 positive cancer in a subject or inhibiting tumor growth or metastasis of GPC2 positive cancer. In some aspects, these methods include administering a therapeutically effective amount of CAR, nucleic acid molecules, vectors, cells or compositions disclosed herein to the subject. In some examples, GPC2 positive cancer is a solid tumor. In some examples, GPC2 positive cancer is a childhood cancer. In a specific non-limiting example, GPC2 positive cancer is neuroblastoma, medulloblastoma, retinoblastoma, acute lymphoblastic leukemia, embryonic rhabdomyosarcoma, alveolar rhabdomyosarcoma, Ewing sarcoma, desmoplastic small round cell tumor, glioma or osteosarcoma. In a specific example, GPC2 positive cancer is neuroblastoma. In some examples, these methods further include administering state-adjusting chemotherapy, such as fludarabine and cyclophosphamide, to the subject.

[0231] IV. GPC2-specific antibody sequences

[0232] CAR disclosed herein includes antibodies (or antigen binding fragments thereof) that specifically bind to GPC2. In some aspects, the antibody is a humanized form of mouse monoclonal antibody CT3 or scFv style (e.g., hCT3-1, hCT3-2, hCT3-3 or hCT3-4). These antibodies are described in PCT Publication No. WO 2020 / 033430, which is incorporated herein by reference in its entirety. The nucleotide sequence and amino acid sequence of CT3 are provided below. Tables 1 and 2 respectively list the amino acid positions of CDR1, CDR2 and CDR3 of the VH domain and VL domain determined using Kabat, IMGT, Paratome and combinations thereof. Alternative numbering schemes such as Chothia numbering schemes can also be used to define CDR boundaries. Parent CT3 antibody and its four humanized forms of scFv nucleotide sequences and amino acid sequences are also listed below. In each scFv sequence, the VH domain and the VL domain are separated by (G4S)3 joints displayed in bold. For humanized antibodies, scFv sequences in both VH-linker-VL and VL-linker-VH orientations are provided.

[0233] CT3 VH nucleotide sequence (SEQ ID NO: 1)

[0234] GAGGTCCAGCTGCAACAGTCTGGACCTGAACTGGTGAAGCCTGGGGCTTCAGTAA

[0235] AGATGTCCTGCAAGGCTTCTAGATTCACATTCACTGACTACAACATACACTGGGTGA

[0236] AGCAGAGCCCTGGAAAGACCCTTGAATGGATTGGATATATTAACCCTAACAATGGTG

[0237] ATATTTTCTACAAACAGAAGTTCAATGGCAAGGCCACATTGACTATAAACAAGTCCT

[0238] CCAACACAGCCTACATGGAGCTCCGCAGCCTGACATCGGAGGATTCTGCAGTCTATT

[0239] ACTGTGTAAGATCCTCTAATATTCGTTATACTTTCGACAGGTTCTTCGATGTCTGGGG

[0240] CACAGGGACCACGGTCACCGTCTCCTCA

[0241] CT3 VH amino acid sequence (SEQ ID NO: 2)

[0242] EVQLQQSGPELVKPGASVKMSCKASRFTFTDYNIHWVKQSPGKTLEWIGYINPNNGDI

[0243] FYKQKFNGKATLTINKSSNTAYMELRRSLTSEDSAVYYCVRSSNIRYTFDRFFDVWGTGT

[0244] TVTVSS

[0245] Table 1. Location of CDRs in the CT3 VH domain (SEQ ID NO: 2)

[0246] plan CDR1 CDR2 CDR3 Kabat 31-35 50-66 99-112 IMGT 26-33 51-58 97-112 Paratome 26-35 47-61 97-112 combination 26-35 47-66 97-112

[0247] CT3 VL nucleotide sequence (SEQ ID NO: 3)

[0248] GAAAATGTGCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCTAGGGGAGAAGGTCACCATGAGCTGCAGGGCCAGCTCAAGTGTAAATTACATTTACTGGTACCAGCAGAAGTCAGATGCCTCCCCCAAACTATGGATTTATTACACATCCAACCTGGCTCCTGGAGTCCCAGCTCGCTTCAGTGGCAGTGGGTCTGGGAACTCTTATTCTCTCACAATCAGCAGCATGGAGGGTGAAGATGCTGCCACTTATTACTGCCAGCAGTTTTCTAGTTCCCCATCCACGTTCGGTACTGGGACCAAGCTGGAGCTGAAA

[0249] CT3 VL amino acid sequence (SEQ ID NO:4)

[0250] ENVLTQSPAIMSASLGEKVTMSCRASSSVNYIYWYQQKSDASPKLWIYYTSNLAPGVPARFSGSGSGNSYSLTISSMEGEDAATYYCQQFSSSPSTFGTGTKLELK

[0251] Table 2. Positions of CDRs in the CT3 VL domain (SEQ ID NO:4)

[0252] plan CDR1 CDR2 CDR3 Kabat 24-33 49-55 88-96 IMGT 27-31 49-51 88-96 Paratome 27-33 45-55 88-95 combination 24-33 45-55 88-96

[0253] CT3 (VH-VL) scFv nucleotide sequence (SEQ ID NO:5)

[0254] GAGGTCCAGCTGCAACAGTCTGGACCTGAACTGGTGAAGCCTGGGGCTTCAGTAA

[0255] AGATGTCCTGCAAGGCTTCTAGATTCACATTCACTGACTACAACATACACTGGGTGA

[0256] AGCAGAGCCCTGGAAAGACCCTTGAATGGATTGGATATATTAACCCTAACAATGGTG

[0257] ATATTTTCTACAAACAGAAGTTCAATGGCAAGGCCACATTGACTATAAACAAGTCCT

[0258] CCAACACAGCCTACATGGAGCTCCGCAGCCTGACATCGGAGGATTCTGCAGTCTATT

[0259] ACTGTGTAAGATCCTCTAATATTCGTTATACTTTCGACAGGTTCTTCGATGTCTGGGG

[0260] CACAGGGACCACGGTCACCGTCTCCTCAGGCGGAGGCGGATCAGGTGGTGGCG

[0261] GATCTGGAGGTGGCGGAAGCGAAAATGTGCTCACCCAGTCTCCAGCAATCATGTC

[0262] TGCATCTCTAGGGGAGAAGGTCACCATGAGCTGCAGGGCCAGCTCAAGTGTAAATT

[0263] ACATTTACTGGTACCAGCAGAAGTCAGATGCCTCCCCCAAACTATGGATTTATTACA

[0264] CATCCAACCTGGCTCCTGGAGTCCCAGCTCGCTTCAGTGGCAGTGGGTCTGGGAAC

[0265] TCTTATTCTCTCACAATCAGCAGCATGGAGGGTGAAGATGCTGCCACTTATTACTGCCAGCAGTTTTCTAGTTCCCCATCCACGTTCGGTACTGGGACCAAGCTGGAGCTGAAA

[0266] Amino acid sequence of CT3(VH-VL)scFv (SEQ ID NO:6)

[0267] EVQLQQSGPELVKPGASVKMSCKASRFTFTDYNIHWVKQSPGKTLEWIGYINPNNGDIFYKQKFNGKATLTINKSSNTAYMELRSLTSEDSAVYYCVRSSNIRYTFDRFFDVWGTGTTVTVSSGGGGSGGGGSGGGGSENVLTQSPAIMSASLGEKVTMSCRASSSVNYIYWYQQKSDASPKLWIYYTSNLAPGVPARFSGSGSGNSYSLTISSMEGEDAATYYCQQFSSSPSTFGTGTKLELK

[0268] Nucleotide sequence of hCT3-1 (VH-VL) (SEQ ID NO:7)

[0269] CAAGTACAGCTTGTACAATCCGGAGCGGAAGTAAAGAAGCCCGGGGCCTCCGTGAAAGTCAGCTGCAAAGCGTCTAGATTCACCTTCACTGACTATAACATCCACTGGGTGCGGCAAGCGCCTGGTCAGGGCCTCGAATGGATTGGCTATATAAACCCGAACAACGGGGACATTTTCTATAAGCAGAAATTCAATGGCAGAGTGACGCTCACCGCGGACAAAAGTACCAGTACTGCTTATATGGAACTGTCTAGTCTTACGAGTGAGGATACCGCTGTGTATTATTGCGTGAGGTCCTCCAATATACGCTATACGTTTGATAGATTCTTTGATGTATGGGGACAGGGAACCCTCGTCACGGTCAGCTCAGGCGGAGGCGGATCAGGTGGTGGCGGATCTGGAGGTGGCGGAAGCGACGTAGTAATGACTCAAAGCCCCCTCTCCTTGCCAGTGACGCCAGGTGAGCCGGCATCTATTTCTTGCAGGGCTAGTTCTTCAGTGAACTACATCTATTGGTATTTGCAGAAGCCAGGACAAAGTCCGCAGCTTTGGATATATTACACGAGCAACCTTGCGCCAGGTGTTCCAGATCGCTTCTCTGGCAGTGGATCAGGGACGGACTTCACACTGAAGATCTCTCGCGTTGAAGCTGAAGACGTCGGCGTGTACTATTGCCAACAATTCAGTAGTTCTCCGAGCACTTTTGGCCAGGGAACCAAGCTTGAGATTAAA

[0270] Amino acid sequence of hCT3-1 (VH-VL) (SEQ ID NO: 8)

[0271] QVQLVQSGAEVKKPGASVKVSCKASRFTFTDYNIHWVRQAPGQGLEWIGYINPNNGDIFYKQKFNGRVTLTADKSTSTAYMELSSLTSEDTAVYYCVRSSNIRYTFDRFFDVWGQGTLVTVSSGGGGSGGGGSGGGGSDVVMTQSPLSLPVTPGEPASISCRASSSVNYIYWYLQKPGQSPQLWIYYTSNLAPGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQFSSSPSTFGQGTKLEIK

[0272] hCT3-1 (VL-VH) nucleotide sequence (SEQ ID NO:9)

[0273] GACGTAGTAATGACTCAAAGCCCCCTCTCCTTGCCAGTGACGCCAGGTGAGCCGGCATCTATTTCTTGCAGGGCTAGTTCTTCAGTGAACTACATCTATTGGTATTTGCAGAAGCCAGGACAAAGTCCGCAGCTTTGGATATATTACACGAGCAACCTTGCGCCAGGTGTTCCAGATCGCTTCTCTGGCAGTGGATCAGGGACGGACTTCACACTGAAGATCTCTCGCGTTGAAGCTGAAGACGTCGGCGTGTACTATTGCCAACAATTCAGTAGTTCTCCGAGCACTTTTGGCCAGGGAACCAAGCTTGAGATTAAAGGCGGAGGCGGATCAGGTGGTGGCGGATCTGGAGGTGGCGGAAGCCAAGTACAGCTTGTACAATCCGGAGCGGAAGTAAAGAAGCCCGGGGCCTCCGTGAAAGTCAGCTGCAAAGCGTCTAGATTCACCTTCACTGACTATAACATCCACTGGGTGCGGCAAGCGCCTGGTCAGGGCCTCGAATGGATTGGCTATATAAACCCGAACAACGGGGACATTTTCTATAAGCAGAAATTCAATGGCAGAGTGACGCTCACCGCGGACAAAAGTACCAGTACTGCTTATATGGAACTGTCTAGTCTTACGAGTGAGGATACCGCTGTGTATTATTGCGTGAGGTCCTCCAATATACG

[0274] CTATACGTTTGATAGATTCTTTGATGTATGGGGACAGGGAACCCTCGTCACGGTCAG

[0275] CTCA

[0276] Amino acid sequence of hCT3-1(VL-VH) (SEQ ID NO:10)

[0277] DVVMTQSPLSLPVTPGEPASISCRASSSVNYIYWYLQKPGQSPQLWIYYTSNLAPGVPD

[0278] RFSGSGSGTDFTLKISRVEAEDVGVYYCQQFSSSPSTFGQGTKLEIKGGGGSGGGGSG

[0279] GGGSQVQLVQSGAEVKKPGASVKVSCKASRFTFTDYNIHWVRQAPGQGLEWIGYINP

[0280] NNGDIFYKQKFNGRVTLTADKSTSTAYMELSSLTSEDTAVYYCVRSSNIRYTFDRFFDV

[0281] WGQGTLVTVSS

[0282] Nucleotide sequence of hCT3-2(VH-VL) (SEQ ID NO:11)

[0283] CAGGTCCAGCTTGTCCAGTCTGGTGCTGAGGTTAAAAAACCTGGCGCAAGCGTGA

[0284] AGGTGAGCTGTAAGGCTAGCCGATTTACCTTTACCGACTACAACATACATTGGGTCA

[0285] GGCAAGCGCCTGGACAAAGGCTCGAGTGGATTGGTTACATAAACCCAAATAACGGG

[0286] GACATATTTTACAAACAGAAATTCAACGGCCGGGTAACTATAACACGAGACACAAG

[0287] TGCAAGTACCGCCTATATGGAGCTCTCTTCCCTTCGATCCGAAGACACGGCAGTCTA

[0288] CTACTGCGTTCGCAGCTCCAATATCCGCTATACCTTTGATAGGTTCTTTGATGTGTGG

[0289] GGTCAAGGGACGCTCGTAACCGTGAGCGGAGGAGGCGGTTCTGGTGGCGGGGG

[0290] CAGCGGTGGGGGAGGGTCTGATGTCGTTATGACTCAGAGTCCAGCGTTTCTCAGT

[0291] GTTACACCCGGTGAAAAGGTCACCATTACGTGCCGGGCGAGCTCTTCAGTTAACTA

[0292] CATTTATTGGTACCAGCAGAAGCCAGATCAGGCGCCCAAACTTTGGATTTACTATAC

[0293] CAGCAACCTTGCACCCGGTGTACCCAGTCGGTTTAGCGGCAGTGGGAGTGGTACTG

[0294] ACTTCACTTTTACTATTTCATCATTGGAGGCTGAAGATGCTGCGACATACTATTGTCA

[0295] GCAATTCAGCTCCTCTCCCAGCACCTTTGGCCAGGGCACCAAACTTGAAATTAAA

[0296] hCT3-2 (VH-VL) Amino Acid Sequence (SEQ ID NO:12)

[0297] QVQLVQSGAEVKKPGASVKVSCKASRFTFTDYNIHWVRQAPGQRLEWIGYINPNNGD

[0298] IFYKQKFNGRVTITRDTSASTAYMELSSLRSEDTAVYYCVRSSNIRYTFDRFFDVWGQG

[0299] TLVTVSGGGGSGGGGSGGGGSDVVMTQSPAFLSVTPGEKVTITCRASSSVNYIYWYQ

[0300] QKPDQAPKLWIYYTSNLAPGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCQQFSSSPST

[0301] FGQGTKLEIK

[0302] Nucleotide sequence of hCT3-2(VL-VH) (SEQ ID NO:13)

[0303] GATGTCGTTATGACTCAGAGTCCAGCGTTTCTCAGTGTTACACCCGGTGAAAAGGT

[0304] CACCATTACGTGCCGGGCGAGCTCTTCAGTTAACTACATTTATTGGTACCAGCAGAA

[0305] GCCAGATCAGGCGCCCAAACTTTGGATTTACTATACCAGCAACCTTGCACCCGGTGT

[0306] ACCCAGTCGGTTTAGCGGCAGTGGGAGTGGTACTGACTTCACTTTTACTATTTCATC

[0307] ATTGGAGGCTGAAGATGCTGCGACATACTATTGTCAGCAATTCAGCTCCTCTCCCAG

[0308] CACCTTTGGCCAGGGCACCAAACTTGAAATTAAAGGAGGAGGCGGTTCTGGTGG

[0309] CGGGGGCAGCGGTGGGGGAGGGTCTCAGGTCCAGCTTGTCCAGTCTGGTGCTG

[0310] AGGTTAAAAAACCTGGCGCAAGCGTGAAGGTGAGCTGTAAGGCTAGCCGATTTACC

[0311] TTTACCGACTACAACATACATTGGGTCAGGCAAGCGCCTGGACAAAGGCTCGAGTG

[0312] GATTGGTTACATAAACCCAAATAACGGGGACATATTTTACAAACAGAAATTCAACGG

[0313] CCGGGTAACTATAACACGAGACACAAGTGCAAGTACCGCCTATATGGAGCTCTCTTC

[0314] CCTTCGATCCGAAGACACGGCAGTCTACTACTGCGTTCGCAGCTCCAATATCCGCTA

[0315] TACCTTTGATAGGTTCTTTGATGTGTGGGGTCAAGGGACGCTCGTAACCGTGAGC

[0316] Nucleotide sequence of hCT3-2 (VL-VH) (SEQ ID NO:14)

[0317] DVVMTQSPAFLSVTPGEKVTITCRASSSVNYIYWYQQKPDQAPKLWIYYTSNLAPGVP

[0318] SRFSGSGSGTDFTFTISSLEAEDAATYYCQQFSSSPSTFGQGTKLEIKGGGGSGGGGSG

[0319] GGGSQVQLVQSGAEVKKPGASVKVSCKASRFTFTDYNIHWVRQAPGQRLEWIGYINP

[0320] NNGDIFYKQKFNGRVTITRDTSASTAYMELSSLRSEDTAVYYCVRSSNIRYTFDRFFDV

[0321] WGQGTLVTVS

[0322] Nucleotide sequence of hCT3-3 (VH-VL) (SEQ ID NO:15)

[0323] CAAGTGCAGTTGGTGCAATCAGGAGCGGAAGTCAAGAAACCGGGGGCATCTGTCA

[0324] AAGTGAGCTGCAAGGCGAGCCGGTTCACATTTACGGATTACAACATACATTGGGTG

[0325] CGCCAAGCCCCGGGACAGGGCCTCGAATGGATAGGCTACATCAATCCTAATAATGG

[0326] GGATATCTTCTATAAGCAGAAATTTAATGGAAAGGCAACCATGACAGTAGATACTTC

[0327] TACTAGCACAGTTTACATGGAGCTGTCCTCACTGCGGTCTGAGGATACGGCGGTGTA

[0328] CTATTGCGTTAGGAGCAGCAATATACGGTACACGTTCGATCGCTTCTTCGATGTTTGG

[0329] GGCCAGGGCACCCTCGTGACGGTATCTGGAGGAGGCGGGTCAGGTGGCGGTGG

[0330] CTCAGGCGGGGGGGGGAGTATGGACATCCAGATGACCCAGAGCCCTAGCAGTTT

[0331] GTCAGCTTCCGTGGGAGACAGAGTCACCATAACATGTCGAGCATCCAGTAGTGTCA

[0332] ACTATATATACTGGTATCAACAGAAGTCCGGCAAGGCGCCTAAACTGTGGATTTATTA

[0333] TACGTCCAACCTCGCACCCGGCGTTCCAAGCCGATTCTCCGGCAGTGGATCAGGAA

[0334] CCGACTTCACGCTGACGATCAGTAGTCTTCAGCCAGAGGATTTCGCTACATATTACT

[0335] GTCAACAGTTTTCCTCAAGCCCTAGTACCTTTGGGCAGGGAACCAAACTGGAGATA

[0336] AAG

[0337] Amino acid sequence of hCT3-3(VH-VL) (SEQ ID NO:16)

[0338] QVQLVQSGAEVKKPGASVKVSCKASRFTFTDYNIHWVRQAPGQGLEWIGYINPNNGD

[0339] IFYKQKFNGKATMTVDTSTSTVYMELSSLRSEDTAVYYCVRSSNIRYTFDRFFDVWGQ

[0340] GTLVTVSGGGGSGGGGSGGGGSMDIQMTQSPSSLSASVGDRVTITCRASSSVNYIYW

[0341] YQQKSGKAPKLWIYYTSNLAPGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFSSSP

[0342] STFGQGTKLEIK

[0343] Nucleotide sequence of hCT3-3 (VL-VH) (SEQ ID NO:17)

[0344] ATGGACATCCAGATGACCCAGAGCCCTAGCAGTTTGTCAGCTTCCGTGGGAGACAG

[0345] AGTCACCATAACATGTCGAGCATCCAGTAGTGTCAACTATATATACTGGTATCAACAG

[0346] AAGTCCGGCAAGGCGCCTAAACTGTGGATTTATTATACGTCCAACCTCGCACCCGGC

[0347] GTTCCAAGCCGATTCTCCGGCAGTGGATCAGGAACCGACTTCACGCTGACGATCAG

[0348] TAGTCTTCAGCCAGAGGATTTCGCTACATATTACTGTCAACAGTTTTCCTCAAGCCCT

[0349] AGTACCTTTGGGCAGGGAACCAAACTGGAGATAAAGGGAGGAGGCGGGTCAGGT

[0350] GGCGGTGGCTCAGGCGGGGGGGGGAGTCAAGTGCAGTTGGTGCAATCAGGAGC

[0351] GGAAGTCAAGAAACCGGGGGCATCTGTCAAAGTGAGCTGCAAGGCGAGCCGGTTC

[0352] ACATTTACGGATTACAACATACATTGGGTGCGCCAAGCCCCGGGACAGGGCCTCGA

[0353] ATGGATAGGCTACATCAATCCTAATAATGGGGATATCTTCTATAAGCAGAAATTTAAT

[0354] GGAAAGGCAACCATGACAGTAGATACTTCTACTAGCACAGTTTACATGGAGCTGTCC

[0355] TCACTGCGGTCTGAGGATACGGCGGTGTACTATTGCGTTAGGAGCAGCAATATACGG

[0356] TACACGTTCGATCGCTTCTTCGATGTTTGGGGCCAGGGCACCCTCGTGACGGTATCT

[0357] hCT3-3 (VL-VH) amino acid sequence (SEQ ID NO:18)

[0358] MDIQMTQSPSSLSASVGDRVTITCRASSSVNYIYWYQQKSGKAPKLWIYYTSNLAPGV

[0359] PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFSSSPSTFGQGTKLEIKGGGGSGGGGSG

[0360] GGGSQVQLVQSGAEVKKPGASVKVSCKASRFTFTDYNIHWVRQAPGQGLEWIGYINP

[0361] NNGDIFYKQKFNGKATMTVDTSTSTVYMELSSLRSEDTAVYYCVRSSNIRYTFDRFFD

[0362] VWGQGTLVTVS

[0363] hCT3-4 (VH-VL) nucleotide sequence (SEQ ID NO:19)

[0364] CAGGTGCAGCTGGTCCAGAGTGGGGCAGAAGTCAAGAAACCCGGAGCAAGTGTC

[0365] AAAGTGTCCTGTAAAGCCTCACGATTCACATTTACCGACTATAACATCCACTGGGTG

[0366] AGACAGGCACCAGGACAGAGGCTGGAGTGGATCGGCTATATCAACCCTAACAATGG

[0367] CGACATCTTCTACAAGCAGAAGTTTAATGGCCGGGTGACCATCACAAGAGATACCA

[0368] GCGCCTCCACAGCCTATATGGAGCTGAGCTCCCTGCGGAGCGAGGATACCGCCGTG

[0369] TACTATTGCGTGAGGTCTAGCAATATCCGCTACACATTCGACCGGTTCTTTGACGTGT

[0370] GGGGCCAGGGCACCCTGGTGACAGTGTCCGGCGGCGGCGGCTCTGGCGGAGGA

[0371] GGCAGCGGCGGAGGAGGCTCCGAGATCGTGCTGACCCAGTCTCCTGCCACACTG

[0372] TCTCTGAGCCCAGGAGAGAGGGCCACCCTGTCCTGTAGGGCCTCCTCTAGCGTGAA

[0373] CTACATCTATTGGTACCAGCAGAAGCCAGGCCAGGCCCCCAGACTGTGGATCTACTA

[0374] TACCTCCAATCTGGCACCAGGCATCCCTGCAAGGTTCTCCGGCTCTGGCAGCGGCA

[0375] CAGACTTTACCCTGACAATCTCCTCTCTGGAGCCCGAGGATTTTGCCGTGTACTATT

[0376] GTCAGCAGTTTAGCAGTTCACCAAGCACATTCGGGCAGGGCACCAAGCTGGAAATC

[0377] AAG

[0378] Amino acid sequence of hCT3-4(VH-VL) (SEQ ID NO:20)

[0379] QVQLVQSGAEVKKPGASVKVSCKASRFTFTDYNIHWVRQAPGQRLEWIGYINPNNGD

[0380] IFYKQKFNGRVTITRDTSASTAYMELSSLRSEDTAVYYCVRSSNIRYTFDRFFDVWGQG

[0381] TLVTVSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASSSVNYIYWYQQ

[0382] KPGQAPRLWIYYTSNLAPGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQFSSSPSTFG

[0383] QGTKLEIK

[0384] Nucleotide sequence of hCT3-4(VL-VH) (SEQ ID NO:21)

[0385] GAGATCGTGCTGACCCAGTCTCCTGCCACACTGTCTCTGAGCCCAGGAGAGAGGGC

[0386] CACCCTGTCCTGTAGGGCCTCCTCTAGCGTGAACTACATCTATTGGTACCAGCAGAA

[0387] GCCAGGCCAGGCCCCCAGACTGTGGATCTACTATACCTCCAATCTGGCACCAGGCAT

[0388] CCCTGCAAGGTTCTCCGGCTCTGGCAGCGGCACAGACTTTACCCTGACAATCTCCT

[0389] CTCTGGAGCCCGAGGATTTTGCCGTGTACTATTGTCAGCAGTTTAGCAGTTCACCAA

[0390] GCACATTCGGGCAGGGCACCAAGCTGGAAATCAAGGGCGGCGGCGGCTCTGGC

[0391] GGAGGAGGCAGCGGCGGAGGAGGCTCCCAGGTGCAGCTGGTCCAGAGTGGGG

[0392] CAGAAGTCAAGAAACCCGGAGCAAGTGTCAAAGTGTCCTGTAAAGCCTCACGATT

[0393] CACATTTACCGACTATAACATCCACTGGGTGAGACAGGCACCAGGACAGAGGCTGG

[0394] AGTGGATCGGCTATATCAACCCTAACAATGGCGACATCTTCTACAAGCAGAAGTTTA

[0395] ATGGCCGGGTGACCATCACAAGAGATACCAGCGCCTCCACAGCCTATATGGAGCTG

[0396] AGCTCCCTGCGGAGCGAGGATACCGCCGTGTACTATTGCGTGAGGTCTAGCAATATC

[0397] CGCTACACATTCGACCGGTTCTTTGACGTGTGGGGCCAGGGCACCCTGGTGACAGT

[0398] GTCC

[0399] Amino acid sequence of hCT3-4(VL-VH) (SEQ ID NO:22)

[0400] EIVLTQSPATLSLSPGERATLSCRASSSVNYIYWYQQKPGQAPRLWIYYTSNLAPGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQFSSSPSTFGQGTKLEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASRFTFTDYNIHWVRQAPGQRLEWIGYINPNNGDIFYKQKFNGRVTITRDTSASTAYMELSSLRSEDTAVYYCVRSSNIRYTFDRFFDVWGQGTLVTVS

[0401] V. CAR Sequences Targeting GPC2

[0402] CT3 scFv and CT3 humanized forms (hCT3-1, hCT3-2, hCT3-3 and hCT3-4) scFv are used to generate CAR constructs specifically targeting cells expressing GPC2. As disclosed herein, CAR constructs with CD28 hinge regions and CD28 transmembrane domains targeting GPC2 are superior to CAR constructs with CD8 hinge regions paired with CD8 transmembrane domains or CD28 transmembrane domains. The nucleotide sequences and amino acid sequences of CAR components and the complete amino acid sequences of three specific CAR constructs (CT3.28H.BBz, CT3.8H.BBz and CT3.8H.28BBz) are provided below.

[0403] Nucleotide sequence of CD28 hinge region (SEQ ID NO: 23)

[0404] ATCGAAGTCATGTATCCCCCCCCCTATCTGGACAACGAAAAGAGTAACGGAACTATCATTCACGTCAAAGGAAAACACCTGTGCCCTAGCCCACTGTTCCCCGGCCCTTCCAAGCCC

[0405] Amino acid sequence of the hinge region of CD28 (SEQ ID NO: 24)

[0406] IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPCD28 TM domain nucleotide sequence (SEQ ID NO: 25)

[0407] TTTTGGGTGCTGGTGGTGGTGGGCGGCGTGCTGGCTTGCTATTCCCTGCTGGTCACAGTCGCTTTTATTATTTTCTGGGTG

[0408] Amino acid sequence of CD28 TM domain (SEQ ID NO: 26)

[0409] FWVLVVVGGVLACYSLLVTVAFIIFWV

[0410] Nucleotide sequence of the 4-1BB signaling portion (SEQ ID NO: 27)

[0411] AAACGGGGCAGAAAGAAACTCCTGTATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG

[0412] Amino acid sequence of the 4-1BB signaling portion (SEQ ID NO: 28)

[0413] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0414] Nucleotide sequence of CD3ζ signaling domain (SEQ ID NO:29)

[0415] AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAA GGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC

[0416] Amino acid sequence of CD3ζ signaling domain (SEQ ID NO:30)

[0417] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0418] Nucleotide sequence of GMCSFR signal sequence (SEQ ID NO:31)

[0419] ATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCCTCCTGATCCCA

[0420] Amino acid sequence of GMCSFR signal sequence (SEQ ID NO: 32)

[0421] MLLLVTSLLLCELPHPAFLLIP

[0422] Nucleotide sequence of T2A self-cleaving peptide (SEQ ID NO: 33)

[0423] GAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCT

[0424] Amino acid sequence of T2A self-cleaving peptide (SEQ ID NO: 34)

[0425] EGRGSLLTCGDVEENPGP

[0426] hEGFRt nucleotide sequence (SEQ ID NO:35)

[0427]

[0428] hEGFRt amino acid sequence (SEQ ID NO: 36)

[0429] RKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM

[0430] CT3.28H.BBz nucleotide sequence (SEQ ID NO: 37)

[0431] (CT3 - CD28 hinge - CD28 TM - 4 - 1BB - CD3ζ underlined; nucleotides 73 - 1470)

[0432] CAGCCTGAACATAACATCCTTGGGATTACGCTCCCTCAAGGAGATAAGTGATGGAGA

[0433] TGTGATAATTTCAGGAAACAAAAATTTGTGCTATGCAAATACAATAAACTGGAAAAA

[0434] ACTGTTTGGGACCTCCGGTCAGAAAACCAAAATTATAAGCAACAGAGGTGAAAAC

[0435] AGCTGCAAGGCCACAGGCCAGGTCTGCCATGCCTTGTGCTCCCCCGAGGGCTGCTG

[0436] GGGCCCGGAGCCCAGGGACTGCGTCTCTTGCCGGAATGTCAGCCGAGGCAGGGAA

[0437] TGCGTGGACAAGTGCAACCTTCTGGAGGGTGAGCCAAGGGAGTTTGTGGAGAACT

[0438] CTGAGTGCATACAGTGCCACCCAGAGTGCCTGCCTCAGGCCATGAACATCACCTGC

[0439] ACAGGACGGGGACCAGACAACTGTATCCAGTGTGCCCACTACATTGACGGCCCCCA

[0440] CTGCGTCAAGACCTGCCCGGCAGGAGTCATGGGAGAAAACAACACCCTGGTCTGG

[0441] AAGTACGCAGACGCCGGCCATGTGTGCCACCTGTGCCATCCAAACTGCACCTACGG

[0442] ATGCACTGGGCCAGGTCTTGAAGGCTGTCCAACGAATGGGCCTAAGATCCCGTCCA

[0443] TCGCCACTGGGATGGTGGGGGCCCTCCTCTTGCTGCTGGTGGTGGCCCTGGGGATC

[0444] GGCCTCTTCATGTGA

[0445] CT3.28H.BBz amino acid sequence (SEQ ID NO:38)

[0446] MLLLVTSLLLCELPHPAFLLIPHMEVQLQQSGPELVKPGASVKMSCKASRFTFTDYNIH

[0447] WVKQSPGKTLEWIGYINPNNGDIFYKQKFNGKATLTINKSSNTAYMELRSLTSEDSAVY

[0448] YCVRSSNIRYTFDRFFDVWGTGTTVTVSSGGGGSGGGGSGGGGSENVLTQSPAIMSAS

[0449] LGEKVTMSCRASSSVNYIYWYQQKSDASPKLWIYYTSNLAPGVPARFSGSGSGNSYSL

[0450] TISSMEGEDAATYYCQQFSSSPSTFGTGTKLELKTSIEVMYPPPYLDNEKSNGTIIHVKG

[0451] KHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMR

[0452] PVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYD

[0453] VLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGL

[0454] YQGLSTATKDTYDALHMQALPPREGRGSLLTCGDVEENPGPMLLLVTSLLLCELPHPAF

[0455] LLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQE

[0456] LDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRS

[0457] LKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCS

[0458] PEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNI

[0459] TCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCT

[0460] YGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM

[0461] Amino acid sequence of CT3.8H.BBz (SEQ ID NO:39)

[0462] MLLLVTSLLLCELPHPAFLLIPHMEVQLQQSGPELVKPGASVKMSCKASRFTFTDYNIH

[0463] WVKQSPGKTLEWIGYINPNNGDIFYKQKFNGKATLTINKSSNTAYMELRSLTSEDSAVY

[0464] YCVRSSNIRYTFDRFFDVWGTGTTVTVSSGGGGSGGGGSGGGGSENVLTQSPAIMSAS

[0465] LGEKVTMSCRASSSVNYIYWYQQKSDASPKLWIYYTSNLAPGVPARFSGSGSGNSYSL

[0466] TISSMEGEDAATYYCQQFSSSPSTFGTGTKLELKTSTTTPAPRPPTPAPTIASQPLSLRPE

[0467] ACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITKRGRKKLLYIFKQPFMRP

[0468] VQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDV

[0469] LDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLY

[0470] QGLSTATKDTYDALHMQALPPREGRGSLLTCGDVEENPGPMLLLVTSLLLCELPHPAFL

[0471] LIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQEL

[0472] DILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSL

[0473] KEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSP

[0474] EGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNIT

[0475] CTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTY

[0476] GCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM

[0477] Amino acid sequence of CT3.8H.28BBz (SEQ ID NO:40)

[0478] MLLLVTSLLLCELPHPAFLLIPHMEVQLQQSGPELVKPGASVKMSCKASRFTFTDYNIHWVKQSPGKTLEWIGYINPNNGDIFYKQKFNGKATLTINKSSNTAYMELRSLTSEDSAVYYCVRSSNIRYTFDRFFDVWGTGTTTVTVSSGGGGSGGGGSGGGGSENVLTQSPAIMSASLGEKVTMSCRASSSVNYIYWYQQKSDASPKLWIYTSNLAPGVPARFSGSGS GNSYSLTISSMEGEDAATYYCQQFSSSPSTFGTGTKLELKTSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDWFVLVVVGGVLACYSLLVTVAFIIFWVRSKSRLLHSDYMNMTPRRPPGPTRKHYQPYAPPRDFAAYRSKRGRKKLLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREE YDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPREGRGSLLTCDGVEENPGPMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAV VSNLITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM

[0479] VI. Antigen receptor(CAR)

[0480] GPC2-specific CAR and cells engineered to express CAR (e.g., T cells, NK cells, B cells, macrophages, and iPSC) are disclosed herein. Typically, CAR includes a binding portion, an extracellular hinge / spacer element, a transmembrane domain, and an intracellular domain that performs a signal transduction function (Cartellieri et al., J Biomed Biotechnol 2010: 956304, 2010; Dai et al., J Natl Cancer Inst 108 (7): djv439, 2016). In many cases, the binding portion is an antigen-binding fragment of a monoclonal antibody, such as scFv or a single domain antibody. The spacer / hinge region generally includes sequences from IgG subclasses, such as IgG1, IgG4, IgD, and CD8 domains. In some aspects of this article, the hinge region is derived from human CD28. In a specific example, the amino acid sequence of the hinge region includes SEQ ID NO: 24 (or is composed thereof). Transmembrane (TM) domains can be derived from a variety of different T cell proteins, such as CD3ζ, CD4, CD8, CD28 or inducible T cell co-stimulator (T cell co-stimulator, ICOS). In some aspects of this article, the TM domain is derived from human CD28. In a specific example, the amino acid sequence of the TM domain includes SEQ ID NO: 26 (or is composed thereof). Several different intracellular domains have been used to produce CAR. For example, the intracellular domain can be composed of a signal transduction chain such as CD3ζ or FcεRIγ with ITAM. In some cases, the intracellular domain further includes at least one additional intracellular portion of a co-stimulatory domain, such as CD28, 4-1BB (CD137, TNFRSF9), OX-40 (CD134), ICOS, CD27, MYD88-CD40, killer cell immunoglobulin-like receptor 2DS2 (KIR2DS2) and / or DAP10.

[0481] CAR can also include a signal peptide sequence, for example, a signal peptide sequence at the N-terminus of the antigen binding domain. The signal peptide sequence can be any suitable signal peptide sequence, such as a signal sequence from a granulocyte-macrophage colony stimulating factor receptor (GMCSFR), an immunoglobulin light chain κ or IL-2. Although the signal peptide sequence can promote CAR to be expressed on the cell surface, it is not necessary for CAR to play a role in the expression of the signal peptide sequence in the CAR. When CAR is expressed on the cell surface, the signal peptide sequence can be cut off from CAR. Therefore, in some aspects, CAR lacks a signal peptide sequence.

[0482] In some aspects, the CAR disclosed herein is expressed from a construct (such as from a lentiviral vector) that also expresses a truncated form of human EGFR (hEGFRt; discussed in more detail in Part VII below). CAR and hEGFRt are separated by a self-cleaving peptide sequence (such as T2A), so that when expressed in a transduced cell, CAR is cut from hEGFRt (see WO2019 / 094482, which is incorporated herein by reference in its entirety).

[0483] In some aspects disclosed herein, the CAR construct encodes the following amino acid sequence in the N-terminal to C-terminal direction:

[0484] GMCSFRss: MLLLVTSLLCELPHPAFLLIP (SEQ ID NO:32)

[0485] NdeI:HM

[0486] Antigen binding: GPC2-specific scFv

[0487] SpeI:TS

[0488] CD28 hinge: IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP(SEQ ID NO:24)

[0489] CD28 TM: FWVLVVVGGVLACYSLLVTVAFIIFWV; SEQ ID NO: 26)

[0490] 4-1BB:KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL(SEQ ID NO:28)

[0491] CD3ζ:

[0492] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:30)

[0493] T2A:EGRGSLLTCGDVEENPGP(SEQ ID NO:34)

[0494] GMCSFRss: MLLLVTSLLCELPHPAFLLIP (SEQ ID NO:32)

[0495] hEGFRt:

[0496] RKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKAT GQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM(SEQ ID NO:36)

[0497] Immune cells (such as T cells, NK cells, B cells or macrophages) or iPSCs expressing CAR disclosed herein can be used to target specific cell types, such as tumor cells, such as GPC2-positive tumor cells. The use of immune cells (such as T cells) expressing CAR is more universal than standard immunotherapy based on CTL, because immune cells expressing CAR are not restricted by HLA and can therefore be used in any patient carrying a tumor expressing the target antigen.

[0498] Therefore, CAR comprising GPC2 specific antibody (or its binding fragment) is provided herein. Also provided are nucleic acid molecules and vectors encoding such CAR separations, and host cells expressing such CARs, such as T cells, NK cells, B cells, macrophages or iPSC. Cells expressing CARs comprising GPC2 specific monoclonal antibodies can be used to treat cancers expressing GPC2, such as neuroblastoma, medulloblastoma, retinoblastoma, acute lymphoblastic leukemia, embryonic rhabdomyosarcoma, alveolar rhabdomyosarcoma, Ewing sarcoma, desmoplastic small round cell tumors, gliomas or osteosarcomas.

[0499] VII. Truncated human EGFR (hEGFRt)

[0500] Human epidermal growth factor receptor consists of four extracellular domains, one transmembrane domain and three intracellular domains. The domains of EGFR exist in the following order from N-terminus to C-terminus: domain I-domain II-domain III-domain IV-transmembrane (TM) domain-near membrane domain-tyrosine kinase domain-C-terminal tail. Domain I and domain III are leucine-rich domains involved in ligand binding. Domain II and domain IV are cysteine-rich domains and do not contact EGFR ligands. Domain II mediates the formation of homodimers or heterodimers with similar domains from other EGFR family members, and domain IV can form a disulfide bond with domain II. The EGFR TM domain passes through the cell membrane once and can play a role in protein dimerization. The intracellular domain includes a juxtamembrane domain, a tyrosine kinase domain, and a C-terminal tail, which mediate EGFR signaling (Wee and Wang, Cancers 9(52), doi: 10.3390 / cancers9050052; Ferguson, Annu Rev Biophys 37:353-373, 2008; Wang et al., Blood 118(5):1255-1263, 2011).

[0501] The truncated form of human EGFR, referred to herein as "hEGFRt", includes only domain III, domain IV and TM domain. Therefore, hEGFRt lacks domain I, domain II and all three intracellular domains. hEGFRt is not able to bind EGF and lacks signal transduction activity. However, this molecule retains the ability to bind to specific EGFR-specific monoclonal antibodies (such as cetuximab (cetuximab) (PCT Publication No. WO 2011 / 056894) approved by the FDA).

[0502] Transducing immune cells (such as T cells, NK cells, B cells or macrophages) or iPSCs with constructs (such as lentiviral vectors) encoding hEGFRt and the GPC2-specific CAR disclosed herein allows for the use of labeled EGFR monoclonal antibody cetuximab (ERBITUX TM ) select transduced cells. For example, cetuximab can be labeled with biotin and commercially available (such as from Miltenyi Biotec) anti-biotin magnetic beads can be used to select transduced cells. The co-expression of hEGFRt also allows in vivo tracking of adoptively transferred CAR-expressing cells. In addition, cetuximab binds to cells expressing hEGFRt and induces the cytotoxicity of ADCC effector cells, thereby providing a mechanism for eliminating transduced immune cells or iPSCs in vivo (Wang et al., Blood 118 (5): 1255-1263, 2011), such as when treatment is completed.

[0503] In some aspects of the present invention, the amino acid sequence of hEGFRt is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 36. In some examples, the amino acid sequence of hEGFRt comprises or consists of SEQ ID NO: 36. In other aspects, the amino acid sequence of hEGFRt comprises no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 2 or no more than 1 amino acid substitution relative to SEQ ID NO: 36. In some examples, the amino acid substitution is a conservative substitution.

[0504] VIII. CAR-Expressing Cell Compositions and Administration Thereof

[0505] A composition is provided, comprising a cell expressing CAR in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. The cell expressing CAR can be iPSC, T cell, such as CD3 + T cells, such as CD4 + T cells and / or CD8 + T cells, NK cells, B cells, macrophages or any other suitable immune cells. Such compositions may include buffers such as neutral buffered saline, phosphate buffered saline, etc.; sugars such as glucose, mannose, sucrose, dextran or mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. In some examples, the composition containing cells includes a cryopreservative such as DMSO or glycerol. In some examples, the composition containing cells includes a culture medium such as DMEM or RPMI, and may further include serum such as FBS. In some examples, the composition containing cells is frozen or in liquid form. The cell can be an autologous cell of the recipient. However, the cell can also be heterologous (allogeneic).

[0506] With respect to cells, a variety of aqueous carriers (e.g., buffered saline, etc.) can be used for cell introduction. These solutions are sterile and generally do not contain unwanted substances. These compositions can be sterilized with conventional sterilization techniques. The compositions can contain pharmaceutically acceptable auxiliary substances such as pH adjustment and buffering agents, toxicity regulators, etc., as required for close physiological conditions, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration in these preparations can vary widely, and will be selected mainly based on fluid volume, viscosity, body weight, etc., according to the selected specific mode of administration and the needs of the subject.

[0507] The exact amount of the composition to be administered can be determined by the physician taking into account individual differences in age, weight, tumor size / burden, degree of metastasis, and condition of the patient (subject). It can generally be specified that the pharmaceutical composition comprising the CAR-expressing immune cells (T cells, B cells, macrophages and / or NK cells) or iPSCs described herein can be administered in 10 4 Up to 10 9 The dose of cells / kg body weight is 10 5 Up to 10 6 An exemplary dose is 10 cells / kg body weight. 6 cells / kg to about 10 8 cells / kg, such as about 5 x 10 6 cells / kg to about 7.5 x 10 7 cells / kg, such as about 2.5 x 10 7 cells / kg, or approximately 5.0 x 10 7 cells / kg.

[0508] The composition can be administered once or more times, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 times at these dosages. The composition can be administered using known immunotherapy infusion techniques (see, for example, Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988). The composition can be administered once a day, once a week, twice a month (bimonthly) or once a month. In some non-limiting examples, the composition is formulated for intravenous administration and is administered multiple times. The amount and frequency of administration can be determined based on such factors as the type and severity of the subject's condition and the subject's disease, but the appropriate dosage can be determined by clinical trials.

[0509] In some aspects, the nucleic acid molecules encoding CAR are introduced into cells, such as T cells, NK cells, B cells, macrophages or iPSC, and the subject receives initial cell administration and subsequent one or more cell administrations, wherein subsequent one or more administrations are less than 15 days after previous administration, for example, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 days. In one aspect, more than one application of CAR expression cells is performed to the subject weekly, for example, 2, 3 or 4 applications of the disclosed expression CAR cells are performed weekly. In one aspect, the subject receives more than one application of CAR expression cells (for example, 2, 3 or 4 applications per week) (also referred to as a cycle), then a week does not apply the expression CAR cells, and then the subject is additionally applied one or more CAR expression cells (for example, more than one expression CAR cells are applied weekly). In another aspect, the subject (such as a human subject) receives more than one cycle of expression CAR cells, and the time between each cycle is less than 10, 9, 8, 7, 6, 5, 4 or 3 days. In one aspect, cells expressing CAR are administered every two days, 3 times a week. In another aspect, cells expressing CAR are administered for at least two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, eight weeks or more. The dosage of the above-mentioned therapy to be administered to the patient will vary with the exact nature of the condition being treated and the recipient of the treatment. The adjustment of the dosage administered to a person can be carried out according to a recognized method.

[0510] In some aspects, cells expressing CAR can replicate in vivo, producing long-term persistence that can lead to persistent control of tumors. In many aspects, iPSC, T cells, macrophages, B cells or NK cells or descendants of these cells applied to subjects continue to exist in subjects for at least four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, thirteen months, fourteen months, fifteen months, sixteen months, seventeen months, eighteen months, nineteen months, twenty months, twenty-one months, twenty-two months, twenty-three months or apply cells to subjects after several years. In other aspects, after applying cells expressing CAR to subjects, cells and their descendants exist less than six months, five months, four months, three months, two months or one month, for example, three weeks, two weeks, one week.

[0511] The use of the disclosed composition can be implemented in any convenient manner, including injection, intake, infusion, implantation or transplantation. The disclosed composition can be applied to the patient in the following manner: via artery, subcutaneous, intradermal, intratumoral, intralymph node, intramedullary, intracerebral, intraventricular, intracranial, intramuscular, intraarterial (including entering the hepatic artery (such as HAI) or femoral artery), intravenous (iv) injection, intraprostatic (such as for prostate cancer), intrabone, intravitreal or intraperitoneal. In some aspects, the composition is applied to the patient by intradermal or subcutaneous injection. In other aspects, the composition of the present disclosure is applied by iv injection. In other aspects, the composition of the present disclosure is applied by intraarterial injection. The composition can also be directly injected into a tumor or lymph node. In one example, it is applied as intrabone administration, and the cancer treated is a cancer of the bone (such as osteosarcoma). In one example, it is applied as intracerebral, intraventricular or intracranial administration and the cancer treated is a cancer of the brain (such as neuroblastoma or medulloblastoma). In one example, the administration is intravitreal and the cancer treated is a cancer of the eye (eg, retinoblastoma).

[0512] In some aspects, the subject may undergo leukapheresis, wherein leukocytes are collected, enriched or depleted in vitro to select and / or separate cells of interest, such as T cells, B cells, macrophages and / or NK cells. These cell isolates can be amplified by known methods and treatments, so that one or more CAR constructs can be introduced to produce autologous cells expressing CAR. In some aspects of this article, a lentiviral vector expressing CAR and a truncated form of human EGFR (hEGFRt) is used to generate cells expressing CAR. The co-expression of hEGFRt allows the use of antibodies (e.g., cetuximab, see PCT Publication No. WO 2011 / 056894) identifying hEGFRt to select and purify immune cells expressing CAR, which is described in Part V above.

[0513] In some aspects, the lysis of red blood cells and in some cases the depletion of monocytes is accomplished by, for example, PERCOLL TM Immune cells (such as T cells, NK cells, B cells and / or macrophages) are isolated from peripheral blood by gradient centrifugation or by counterflow centrifugal elutriation. Specific T cell subsets, such as CD3+, CD28+, CD4+, CD8+, CD45RA+ and CD45RO+ T cells, can be further isolated by positive or negative selection techniques. For example, T cells can be isolated by beads conjugated to anti-CD3 / anti-CD28 (e.g., 3×28) (e.g., In a non-limiting example, the time period is about 30 minutes. In other non-limiting examples, the time period ranges from 30 minutes to 36 hours or longer and all integer values ​​therebetween. In other non-limiting examples, the time period is at least 1, 2, 3, 4, 5 or 6 hours, 10 to 24 hours, 24 hours or longer. Longer incubation times can be used to separate T cells in any situation where T cells are less than other cell types (such as when separated from immunocompromised individuals). In addition, the efficiency of capturing CD8+T cells can be increased using longer incubation times. Therefore, by simply shortening or extending the time allowing T cells to bind to CD3 / CD28 beads and / or by increasing or decreasing the ratio of beads to T cells, T cell subsets can be preferentially selected at the beginning of culture or at other time points during the process. Additionally, by increasing or decreasing the ratio of anti-CD3 and / or anti-CD28 antibodies on the beads or other surface, T cell subsets can be preferentially selected at the beginning of the culture or at other desired time points. Multiple rounds of selection can also be performed.

[0514] Enrichment of cell colonies by negative selection can be achieved with a combination of antibodies, wherein the antibodies are directed to surface markers unique to the cells of negative selection. One method is to sort and / or select cells by means of negative magnetic immunoadhesion or flow cytometry, using a mixture of monoclonal antibodies for cell surface markers present on cells of negative selection. For example, in order to enrich CD4+T cells by negative selection, a mixture of monoclonal antibodies generally includes antibodies for CD14, CD20, CD11b, CD16, HLA-DR and CD8. A T cell colony expressing one or more cytokines can be selected. A method for screening cell expression is disclosed in PCT Publication No. WO 2013 / 126712.

[0515] In order to separate the desired cell colony by positive or negative selection, the cell and surface (e.g., particle, such as beads) concentration can be varied to ensure the maximum contact of cells and beads. In some aspects, a concentration of 1 billion cells / ml is used. In other aspects, a concentration greater than 100 million cells / ml is used. In other aspects, a cell concentration of 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 95 million or 100 million cells / ml is used. Not subject to theoretical constraints, using high concentrations can lead to increased cell yields, cell activation and cell expansion. Lower cell concentrations can also be used. Not subject to theoretical constraints, when the mixture of T cells and surfaces (e.g., particles, such as beads) is substantially diluted, the interaction between particles and cells is minimized. This selects for cells that express large amounts of the desired antigen that binds to the particle. For example, CD4+ T cells express higher levels of CD28 and are captured more efficiently than CD8+ T cells at dilute concentrations. In some aspects, the cell concentration used is 5×10 6 In other aspects, the concentration used can be about 1×10 5 / ml to 1×10 6 / ml and any integer value in between.

[0516] IX. Treatment Methods

[0517] Provided herein is a method for treating GPC2-positive cancer in a subject by the following manner: administering to the subject a therapeutically effective amount of immune cells (such as T cells, NK cells, B cells or macrophages) or iPSCs expressing CARs as disclosed herein that express CARs targeting GPC2. Also provided herein is a method for inhibiting tumor growth or metastasis in a subject, which is achieved by administering to the subject a therapeutically effective amount of cells expressing CARs targeting GPC2 disclosed herein. Thus, in some examples, for example, relative to the size, volume and / or weight of the tumor before treatment, these methods reduce the size, volume and / or weight of the tumor by at least 10%, at least 20%, at least 30%, at least 50%, at least 50%, at least 75%, at least 90%, at least 95%, at least 98%, at least 99% or 100%. In some examples, the methods reduce the size, volume, and / or weight of metastases by at least 10%, at least 20%, at least 30%, at least 50%, at least 50%, at least 75%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, for example relative to the size, volume, and / or weight of metastases before treatment. In some examples, the methods increase the survival time of subjects with GPC2-positive cancer by at least 3 months, at least 6 months, at least 9 months, at least 12 months, at least 18 months, at least 24 months, at least 36 months, at least 48 months, or at least 60 months, for example relative to the survival time in the absence of the treatment provided herein. In some examples, a combination of these effects is achieved.

[0518] Specifically, a method for treating GPC2 positive cancer in a subject is provided. In some aspects, the method includes administering to the subject a therapeutically effective amount of separated immune cells or iPSCs, the cells including nucleic acid molecules encoding CAR targeting GPC2 and hEGFRt, or administering a therapeutically effective amount of separated immune cells or iPSCs, the cells co-expressing CAR targeting GPC2 and hEGFRt. In some aspects, GPC2 positive cancer is a solid tumor. In a specific example, GPC2 positive cancer is neuroblastoma, medulloblastoma, retinoblastoma, acute lymphoblastic leukemia, embryonic rhabdomyosarcoma, alveolar rhabdomyosarcoma, Ewing sarcoma, desmoplastic small round cell tumor, glioma or osteosarcoma. In some aspects, GPC2 positive cancer is a childhood cancer.

[0519] In some aspects of the methods disclosed herein, the isolated immune cells are T lymphocytes. In some examples, the T lymphocytes are autologous T lymphocytes. In other aspects, the isolated host cells are NK cells, B cells or macrophages.

[0520] The therapeutically effective amount of CAR-expressing immune cells or iPSCs can depend on the severity of the disease, the type of disease, and the overall health of the patient. The therapeutically effective amount of CAR-expressing cells and compositions thereof is an amount that provides subjective relief of symptoms or objectively recognizable improvements (such as tumor volume or metastasis reduction) noted by clinical medical staff or other qualified observers.

[0521] The use of cells and compositions expressing CAR disclosed herein may also be accompanied by the administration of other anticancer agents or therapeutic treatments (such as surgical removal of tumors). Any suitable anticancer agent may be administered in combination with the compositions disclosed herein. Exemplary anticancer agents include, but are not limited to, chemotherapeutics, such as mitotic inhibitors, alkylating agents, antimetabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, antisurvival agents, biological response regulators, antihormones (such as anti-androgens) and anti-angiogenic agents. Other anticancer therapies include radiotherapy and antibodies (such as mAbs) that specifically target cancer cells or other cells (such as anti-PD-1, anti-CLTA4, anti-EGFR or anti-VEGF). In one example, cancer is treated by administering an immune cell (such as iPSC, T cell, NK cell, B cell, or macrophage) targeting a GPC2 CAR disclosed herein and one or more therapeutic mAbs, such as one or more PD-L1 antibodies (e.g., durvalumab, KN035, cosibelimab, BMS-936559, BMS935559, MEDI-4736, MPDL-3280A, or MEDI-4737) or a CLTA-4 antibody (e.g., ipilimumab or tremelimumab). In one example, cancer is treated by administering a cell (such as an iPSC, a T cell, a NK cell, a B cell, or a macrophage) expressing a CAR targeting GPC2 disclosed herein and one or more mAbs, such as 3F8, Abagovomab, Adecatumumab, Afutuzumab, Alacizumab, Alemtuzumab, Altumomab pentetate, Anatumomab mafenatox, Apolizumab, Arcitumomab, Bavituximab, Bectumomab, Belimumab, Besilesomab, Bevacizumab, Bivatuzumab mertansine), Blinatumomab, Brentuximab vedotin, Cantuzumab mertansine, Capromabpendetide, Catumaxomab, CC49, Cetuximab, Citatuzumab bogatox, Cixutumumab, Clivatuzumab tetraxetan), conatumumab, dacetuzumab, detumomab, ecomeximab, eculizumab, edrecolomab, epratuzumab, ertumaxomab, etaracizumab, farletuzumab, figitumumab, galiximab, gemtuzumab ozogamicin, girentuximab, glembatumumab vedotin, ibritumomab tiuxetan), Igovomab, Imciromab, Intetumumab, Inotuzumab ozogamicin, Ipilimumab, Iratumumab, Labetuzumab, Lexatumumab, Lintuzumab, Lorvotuzumab mertansine), Lucatumumab, Lumiliximab, Mapatumumab, Matuzumab, Mepolizumab, Metelimumab, Milatuzumab, Mitumomab, Morolimumab, Nacolomab tafenatox, Naptumomabestafenatox、Necitumumab、Nimotuzumab、Nofetumomabmerpentan、Ofatumumab、Olaratumab、Oportuzumabmonatox、Oregovomab、Panitumumab、Pemtumomab、Pertuzumab、Pintumomab、Pritumumab、Ramucirumab、Rilotumumab、Rituximab、Robatumumab、Satumomab pendetide, sibrotuzumab, sonepcizumab, tacatuzumab tetraxetan, tapitumomab paptox, tenatumomab, TGN1412, ticilimumab (tremelimumab), tigatuzumab, TNX-650, trastuzumab, tremelimumab, tucotuzumab celmoleukin, veltuzumab, volociximab, votumumab, zalutumumab, or a combination thereof.

[0522] In one example, cancer is treated by administering cells (such as iPSC, T cells, NK cells, B cells or macrophages) expressing CAR targeting GPC2 disclosed herein and one or more alkylating agents, such as nitrogen mustards (such as dichloromethane, cyclophosphamide, melphalan, uracil nitrogen mustard or chlorambucil), alkyl sulfonates (such as busulfan), nitrosoureas (such as carmustine, lomustine, semustine, streptozotocin or dacarbazine). In one example, cancer is treated by administering cells (such as iPSC, T cells, NK cells, B cells or macrophages) expressing CAR targeting GPC2 disclosed herein and cyclophosphamide.

[0523] In one example, cancer is treated by administering cells (such as iPSCs, T cells, NK cells, B cells, or macrophages) expressing a CAR targeting GPC2 disclosed herein and one or more antimetabolites, such as folate analogs (such as methotrexate), pyrimidine analogs (such as 5-FU or cytarabine), and purine analogs, such as mercaptopurine or thioguanine.

[0524] In one example, cancer is treated by administering cells (such as iPSCs, T cells, NK cells, B cells, or macrophages) expressing a CAR targeting GPC2 disclosed herein and one or more natural products, such as vinca alkaloids (such as vinblastine, vincristine, or vindesine), epipodophyllotoxins (such as etoposide or teniposide), antibiotics (such as dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, or mitomycin C), and enzymes (such as L-asparaginase).

[0525] In one example, cancer is treated by administering cells (such as iPSCs, T cells, NK cells, B cells, or macrophages) expressing a CAR targeting GPC2 disclosed herein and one or more platinum complexes (such as cis-dichlorodiammineplatinum II (also known as cisplatin)), substituted ureas (such as hydroxyurea), methylhydrazine derivatives (such as procarbazine), and adrenocortical inhibitors (such as mitotane and aminoglutethimide).

[0526] In one example, cancer is treated by administering cells (such as iPSCs, T cells, NK cells, B cells, or macrophages) expressing a CAR targeting GPC2 disclosed herein and one or more hormones or antagonists, such as adrenocortical steroids (such as prednisone), progestins (such as hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrol acetate), estrogens (such as diethylstilbestrol and ethinyl estradiol), antiestrogens (such as tamoxifen), and androgens (such as testosterone propionate and fluoxymesterone).

[0527] In one example, cancer is treated by administering a cell (such as iPSC, T cell, NK cell, B cell or macrophage) expressing a CAR targeting GPC2 disclosed herein and one or more chemotherapy drugs, such as doxorubicin hydrochloride, Alkeran, Ara-C, BiCNU, busulfan, CCNU, carboplatinum, cisplatin, Cytoxan, daunomycin, DTIC, 5-FU, fludarabine, Hydrea, idarubicin, ifosfamide, methotrexate, mitoxantrone, cytoxan ... Mycin, mitomycin, mitoxantrone, nitrogen mustard, taxol (or other taxanes, such as docetaxel), Velban, vincristine, VP-16, gemcitabine (Gemzar), Herceptin (Herceptin), irinotecan (Camptosar, CPT-11), cladribine (Leustatin), vinorelbine, rituximab (Rituxan) STI-571, taxotere, topotecan (Hycamtin), Xeloda (capecitabine), Zevelin and calcitriol. In one example, cancer is treated in the following manner: cells (such as iPSC, T cells, NK cells, B cells or macrophages) expressing CAR targeting GPC2 disclosed herein, cyclophosphamide and fludarabine are administered. In one example, cancer is treated by administering cells (such as iPSC, T cells, NK cells, B cells or macrophages) expressing CAR targeting GPC2 disclosed herein and one or more immunomodulators, such as AS-101 (Wyeth-Ayerst Labs.), bropiridamine (Upjohn), gamma interferon (Genentech), GM-CSF (granulocyte macrophage colony stimulating factor; Genetics Institute), IL-2 (Cetus or Hoffman-LaRoche), human immunoglobulin (Cutter Biological), IMREG (from Imreg of New Orleans, La.), SK&F 106528 and TNF (tumor necrosis factor). Another treatment that can be used in combination with those provided herein is surgical treatment, such as surgical removal of cancer or part thereof. Another example of treatment is radiotherapy, such as administering radioactive substances or energy (such as external beam therapy) to the tumor site to help eradicate the tumor or shrink it before surgical resection.

[0528] In a specific example, the method includes treating neuroblastoma in the following manner: administering to the subject a therapeutically effective amount of (1) separated immune cells or iPSCs, the cells including nucleic acid molecules encoding CAR targeting GPC2 and hEGFRt, or administering a therapeutically effective amount of separated immune cells or iPSCs, the cells co-expressing CAR targeting GPC2 and hEGFRt. In some examples, the method also includes administering to the subject a therapeutically effective amount of one or more other chemotherapeutic agents or biological agents. In some aspects, one or more other chemotherapeutic agents or biological agents are one or more of the following: 5-FU, cisplatin, gemcitabine, oxaliplatin, doxorubicin, capecitabine, floxuridine or mitoxantrone, such as gemcitabine plus oxaliplatin (GEMOS), floxuridine, cisplatin and oxaliplatin, or 5-FU, oxaliplatin and leucovovin (FOLFOX). In some aspects, the one or more other chemotherapeutic agents or biological agents are one or more of the following: sorafenib, lenvatinib, regorafenib, cabozantinib, and ramucirumab. In some aspects, the one or more other chemotherapeutic agents or biological agents are immunotherapy drugs, such as pembrolizumab and / or nivolumab. In some aspects, the one or more other chemotherapeutic agents or biological agents are cyclophosphamide, fludarabine, or both. Example

[0529] The following examples are provided to demonstrate specific features of certain aspects of the disclosure, but the scope of the claims should not be limited to those exemplified features.

[0530] Example 1: In vitro cell killing using CART cells targeting GPC2

[0531] Using GPC2-positive IMR5 cells and GPC2 knockout (KO) IMR5 cells as target cells, cell killing mediated by T cells expressing CT3.8H.BBz (also referred to herein as CT3.8H.BBζ) CAR and CT3.28H.BBz (also referred to herein as CT3.28H.BBζ) CAR targeting GPC2 was evaluated (see Figure 1A-1B ).like Figure 2A-2B As shown in Figure 2, CT3.28H.BBz CAR T cells were more potent than CT3.8H.BBz CAR T cells in killing IMR5 cells. Both types of CAR T cells had minimal effects on GPC2 KO-IMR5 cells, indicating that they were GPC2 specific.

[0532] Example 2: Comparison of CARs containing a CD8 hinge and a CD28 hinge targeting GPC2 in a neuroblastoma metastasis animal model

[0533] Comparison of CT3.8H.BBz CAR T cells and CT3.28H.BBz CAR T cells in a mouse model of neuroblastoma (IMR5) metastasis. Mice were vaccinated with IMR5-luc iv, infused with 10 million CAR T cells 28 days later, and imaged weekly after infusion ( Figure 3A ). Figure 3B Bioluminescence images of mock-treated mice and CAR T cell-treated mice taken weekly for up to eight weeks after T cell infusion are shown in Figure 2. Bioluminescence was measured at two, four, six, and eight weeks after CAR T cell infusion, and the results are shown in Figure 2. Figure 3C The survival of mock-treated mice and CAR T cell-treated mice after CAR T cell infusion is shown in Figure 3D The results showed that CT3.28H.BBz CART cells were significantly more potent than CT3.8H.BBz CART cells in regressing neuroblastoma tumors in mice. All mice in the CT3.28H.BBz-treated group were alive at the end of the study.

[0534] Example 3: Comparison of CT3.28H.BBz CAR T cells and CT3.8H.28BBz CAR T cells in the orthotopic IMR5 mouse model

[0535] T cells from three different human donors (A26M, A59F, and A25F) were used in this study. 5 million human T cells expressing CT3.28H.BBz CAR or CT3.8H.28BBz CAR were administered to mice carrying moderate tumor burden and bioluminescence imaging was performed once a week for four weeks (A26M and A59F) or eight weeks (A25F). Figure 4A-4C As shown in , for all three T cell donors, CT3.28H.BBz CAR T cells were superior to CT3.8H.28BBzCAR T cells in reducing tumors. Figure 4D and 4E Bioluminescent images of mock-treated mice and mice treated with CT3.28H.BBz CAR T cells or CT3.8H.28BBz CAR T cells derived from donors A26M and A59F are shown in Figure 1. CT3.28H.BBz CAR T cells were more potent than CT3.8H.28BBz CAR T cells in eradicating medium-sized IMR5 tumors.

[0536] Flow cytometry was performed on dissociated spleen samples to assess the maintenance of CAR expression in CAR T cell treated animals. Live cells were gated on CD3+ human cells and the percentage of CAR positive cells was determined ( Figure 4F ).like Figure 4GAs shown in , for both donors tested (A26M and A59F), CT3.28H.BBz CAR T cells retained higher CAR expression levels than CT3.8H.28BBz CAR T cells.

[0537] Example 4: CAR tonic signaling

[0538] CAR phosphorylation was evaluated as a measure of CAR activation. CT3.8H.BBz CAR T cells, CT3.8H.28BBz CART cells, and CT3.28H.BBz CAR T cells were unstimulated or stimulated with protein L or GPC2-Fc and CAR phosphorylation was detected by Western blotting ( Figure 5A ). The fold changes of CAR phosphorylation are shown in Figure 5B . When tested without stimulation, the CT3.8H.28BBz CAR had higher phosphorylation levels than the CT3.28H.BBz CAR. With GPC2 stimulation, both CARs upregulated their phosphorylation levels. These results show that CT3.8H.28BBz has more tonic CAR signaling (phosphorylation), which is known to lead to CAR exhaustion. CT3.28H.BBz CAR T cells have less tonic CAR signaling, but once antigen is presented (GPC2-Fc), they show appropriate CAR activation.

[0539] Example 5: Comparison of CT3.28H.BBz CART cells and CT3.8H.28BBz CAR T cells in an orthotopic IMR5 animal model using low-dose or high-dose chemotherapy

[0540] Mice bearing large IMR5 tumor burdens were treated with no chemotherapy, low-dose chemotherapy, or high-dose chemotherapy (fludarabine / cyclophosphamide) for one week before being infused with 5 million CT3.28H.BBz CAR T cells or CT3.8H.28BBz CAR T cells. In this study, T cells were from a single donor. Tumor size, as measured by bioluminescence, is shown in Fig. 6A Tumor weights at 10 weeks after chemotherapy and CAR T cell infusion are shown in Figure 6B These results show that CT3.28H.BBz is superior to CT3.8H.28BBz when given state-modifying chemotherapy in mice with high tumor burden.

[0541] Example 6: Characterization of humanized CT3 antibody and CAR

[0542] Four humanized versions of the murine antibody CT3 were generated: hCT3-1, hCT3-2, hCT3-3, and hCT3-4. The binding affinity of the humanized CT3 antibodies to GPC2 was tested. Figure 7A-7B As shown in , the binding affinity of the four humanized CT3 antibodies (4.0 nM, 3.6 nM, 2.5 nM and 3.3 nM) is similar to that of the parental CT3 antibody (2.2 nM). In addition, all four humanized antibodies retain the ability to bind to GPC2 on the surface of G10 and IMR5 cells, but do not show binding to GPC2-KO IMR5 cells ( Figure 8 ).

[0543] The cell killing effect of humanized CT3.8H.BBz CAR T cells was tested. The results showed that IMR5 cells expressing GPC2 were specifically lysed by CT3-8H-BBz, hCT3-1-8H-BBz, hCT3-2-8H-BBz, hCT3-3-8H-BBz and hCT3-4-8H-BBz CART cells ( Fig. 9 ). Compared with CT3-8H-BBz CAR T cells, all four humanized CT3-8H-BBz CART cells showed improved killing activity against IMR5 cells.

[0544] CAR constructs utilizing humanized CT3 scFv were generated with the CD28 hinge and CD28 transmembrane domain in either the VH-linker-VL orientation or the VL-linker-VH orientation ( Fig.10 ). It is expected that all eight CAR constructs will robustly reduce GPC2-positive tumors and show greater tumor reduction than the corresponding hCT3 CAR constructs with a CD8 hinge.

[0545] Example 7: Materials and Methods:

[0546] This example describes the materials and experimental procedures used in the studies described in Examples 8-11.

[0547] Cell lines

[0548] The resistant patient-derived xenograft (PDX) SJNBL012407_X1 (MYCN-amplified) was provided by the Children's Solid Tumor Network. IMR-5, CHP-212, SK-N-BE2C, Kelly (also MYCN-amplified), and SHIN, SK-N-FI, SK-N-AS, SH-SHEP, and SH-SY5Y (MYCN-wild-type (WT)) were obtained from the National Cancer Institute (NCI) Pediatric Oncology Division Cell Line Collection. NGP-GPC2 hi NBSD-GPC2 mod and SMS-SAN lo(all MYCN-amplified) provided by Stanford University. GPC2 expression was determined for all cell lines (Table 3). All cells were confirmed to be mycoplasma-free. Cell identity was determined by short-tandem repeat DNA profiling. Stable luciferase (ffLUC) green fluorescent protein (GFP) expressing cells were generated by lentiviral transduction and subsequent selection with 0.5 μg / mL puromycin (Thermo Fisher Scientific). PDX cells were passaged in mice. NB cell lines were cultured in RPMI (Roswell Park Memorial Institute) medium supplemented with 10% fetal bovine serum (FBS) and 100 U / mL penicillin / streptomycin (Gibco).

[0549] Table 3. GPC2 expression in neuroblastoma cell lines and xenograft lines

[0550] Cell / Xenograft Lines MFI Molecules / Cells <![CDATA[NGP-GPC2 hi ]]> 13,483 16,177 <![CDATA[NBSD-GPC2 mod ]]> 6,230 7,537 <![CDATA[SMS-SAN lo ]]> 3,008 3,698 SJNBL012407_X1 1,790 2,247 IMR-5 2,611 3,225 SHIN 1,950 2,437 SK-N-FI 1,197 1,540 CHP212 745 1,002 SK-N-BE2C 723 976 SK-N-AS 699 947 Kelly 29 471 SHEP 294 465

[0551] CAR constructs

[0552] As described above (Li et al., STAR Protoc 2: 100942, 2021), CT3 scFv is cloned into lentiviral vector pWPT (Addgene # 12255), and different hinge and TM domains (CD8 or CD28) and costimulatory domains (4-1BBζ and / or CD28) are added to produce CAR T cell construct variants. GD2 CAR sequences are retrieved from publicly available sources (Straath et al., Sci Transl Med 12: eabd6169, 2020; Pule et al., Nat Med 14: 1264-1270, 2008) and cloned into pWPT vectors containing CD8 or CD28 hinges and TM and 4-1BB costimulatory domains. Human truncated extracellular epithelial growth factor receptor domain (hEGFRt) is included as a tag and recognized by cetuximab.

[0553] Human T cells and CAR transduction

[0554] As previously described (Li et al., STAR Protoc 2:100942, 2021), cryopreserved human T cells from healthy volunteer donors (National Institutes of Health Blood Bank) were used to generate CAR T cells. Briefly, on day 0, Lenti-X293T cells were inoculated at 2×10 7Cells were plated at a density of 10 cells / poly-D-lysine coated 15 cm culture dish and subsequently transfected with CT3 CAR plasmid, envelope plasmid (pMD2.G) and packaging plasmid (psPAX2) at a ratio of 4:1:3 using Lipofectamine 2000 (Thermo Fisher Scientific). Lenti-X 293T culture supernatants containing lentivirus were harvested 48–72 hours after transfection and used to transduce human T cells by centrifugation. Cryopreserved human T cells were thawed and cultured in AIM-V medium (Gibco) supplemented with 10% FBS (Omega Scientific), 100 U / mL penicillin / streptomycin, 1X non-essential amino acids, 0.2 mM L-GlutaMAX, 0.1 mM sodium pyruvate (all from Gibco), CD3 / CD28-coated Dynabeads (1:1 beads / cell ratio, Thermo Fisher Scientific), and 40 IU / mL interleukin (IL)-2 (NCI Frederick BRB preclinical library). IL-2 concentration was increased to 100 IU / mL 48 hours after lentiviral transduction. On day 5 of T cell culture, Dynabeads were removed, and transduced CAR T cells were cultured and expanded until day 8–10 for subsequent downstream assays.

[0555] CAR western blot assay

[0556] The produced CAR T cells were grown in culture for 3–5 h while IL-2 was removed. To activate the CAR, 1.7 μg GPC2-Fc or 1 μg protein L (Acro Biosystems) was added to 2–3 × 10 6cells and then cross-linked at 37°C for different times. Subsequently, cells were lysed using radioimmunoprecipitation assay (RIPA) buffer supplemented with Halt protease and phosphatase inhibitor cocktail (ThermoFisher Scientific). Protein yield was quantified using Bradford assay (Bio-Rad Laboratories). Samples were denatured in a buffer containing sodium dodecyl sulfate (SDS) for 10 minutes. A total of 5–10 μg of protein was resolved by 4–20% SDS-polyacrylamide gel electrophoresis (PAGE) and electroblotted onto polyvinylidene difluoride membranes. The primary antibodies listed in Table 4 were incubated overnight at 4°C in Tris-buffered saline containing 5% bovine serum albumin (BSA), 0.1% Tween-20 (TBST) and 0.02% sodium azide. Secondary antibodies were incubated for 1 hour at room temperature in TBST containing 5% nonfat dry milk. Protein bands were visualized using goat anti-rabbit or anti-mouse IgG-HRP conjugated secondary antibodies (200 μg / mL; Santa Cruz Biotechnology) and SuperSignal West Femto Maximum Sensitivity Substrate (ThermoFisher Scientific). Bands were visualized using enhanced chemiluminescence (Bio-Rad Laboratories) and quantified using ImageJ.

[0557] Table 4. Antibodies

[0558] Antibody clone company Catalog Number Phospho-CD3ζ(Y142) EP265(2)Y Abcam ab68235 anti-CD3-ζ 6B10.2 SantaCruz sc-1239 Phospho-Zap-70(Y319) / Syk(Y352) 65E4 CST 2717 Anti-Zap70 1E7.2 SantaCruz sc-32760 Phospho-p44 / 42MAPK (Erk1 / 2) (T202 / Y204) D13.14.4E CST 4370 Anti-Erk1 / 2 137F5 CST 4695 Vinculin E1E9V CST 13901

[0559] CST:Cell Signaling Technology

[0560] In vitro cytotoxicity assay

[0561] CAR T cells and NB tumor cells expressing ffLUC-GFP were co-cultured as previously described at different effector / tumor (E:T) ratios (Nguyen et al., Cancer Immunol Immunother 67:615-626, 2018). Every 24 hours thereafter, the starting number of tumor cells was added to each well to re-stimulate the CAR T cells. The specific lysis of tumor cells was measured using the ONE-Glo assay. The results were normalized relative to the case of untransduced (UT) simulated T cells.

[0562] Mouse

[0563] For all studies, female NOD-SCID (NSG) mice, 4–6 weeks old, were obtained from the Animal Resources Program of the NCI Center for Cancer Research.

[0564] Bioluminescence imaging

[0565] IMR-5 stably expressing ffLUC was used for bioluminescent imaging (BLI). Tumor-bearing mice were injected with d-luciferin potassium salt (150 mg / kg, intraperitoneal (IP)) and imaged on IVIS LuminaXR system (PerkinElmer) 5 minutes after injection of d-luciferin (acquisition time 1 minute). Region of interest analysis was performed using Living Image software (PerkinElmer; V.4.3.1).

[0566] In vivo therapeutic models

[0567] NSG mice were orthotopically implanted with 2.5×10 5 )PDX cells or IMR-5 (Li et al., STAR Protoc 2:100942, 2021). Generally, 3 weeks after tumor implantation, the tumor meets the inclusion criteria (>10 7 Photons / second) were randomly assigned to receive either UT-mock control T cells or GPC-targeted CAR T cells. The number of T cells injected into the tail vein was based on the CAR + T cells. The total number of T cells in the mock group was adjusted to match the CAR group. For PDX studies, the experiment was terminated on day 50 after tumor injection. Since BLI signals are not associated with large tumor burdens, tumor weights were recorded at the end of the study to determine the efficacy of the treatment. For experiments using IMR-5, tumor-bearing animals were monitored with the aid of BLI. The survival of treated mice was monitored until day 80 (approximately 11 weeks after tumor implantation). The survival endpoint was death, weight loss >20% from baseline, or severe moribund status determined by an animal caregiver who was unaware of the study.

[0568] In vivo homing studies

[0569] Mice carrying IMR-5WT NB were injected with CAR T cells targeting GPC2 expressing ffLUC-GFP or UT mock T cells. After tail vein injection of T cells, animals were continuously monitored by BLI to evaluate T cell homing and expansion in vivo. At the end of the experiment, organs of mice injected with luciferin were removed at 5 minutes and imaged in 6-well petri dishes.

[0570] Flow cytometry

[0571] Use 1×10 6cells and stained the samples. Use fluorescence-minus-one control to set gates. Use single-color control to set compensation and voltage. Use the following antibodies to detect surface epitopes: CD45 (detected with clone HI-30), CD3 (OKT3), CD4 (OKT4), and CD8 (HIT8a). Use GPC2-F C Anti-human F C (M1310G05) or anti-EGFR antibody (AY13) was used to measure CAR transduction efficiency. GPC2 expression in tumor cells was detected using CT3 antibody and anti-mouse IgG1 (RMG1-1). The density of GPC2 expression was determined using the PE phycoerythrin fluorescence quantitative kit (BD) according to the manufacturer's instructions. Data were collected on a Fortessa LSR machine. Data analysis was performed using FlowJo V.10.

[0572] Cytokine bead assay

[0573] Cytokine bead assays (CBA) were performed following the manufacturer's instructions (BioLegend) to quantify secreted cytokines in the supernatants of T cell and tumor co-cultures.

[0574] Single-cell RNA-seq

[0575] CAR T cells were produced using two donors, and we injected the cells into mice carrying IMR-5 on day 8 of cell production. Cell injection products were stained with TotalSeq-C antibodies targeting CD8 (Catalog No. 344752) and CD4 (Catalog No. 300567) and subjected to 10X Genomics 5'V.3.1 chemical kit to produce libraries. Eight days after the mice were injected with T cells, the tumors were processed into single cell suspensions with viability>80%. Three tumor samples from each treatment group were pooled. About 10,000 cells / group were loaded to capture 6,000 cells. Complementary DNA libraries were sequenced on Illumina NextSeq 2000 and NovaSeq 6000, with a target depth of approximately 50,000 reads / cell.

[0576] Computational analysis

[0577] Single-cell RNA-seq FASTQ files were processed using the CellRanger software suite (V.6.1.2, 10X Genomics) with the help of the corresponding human GRCh38 genome control. Custom controls (GRCh38+GFP) were used to observe whether GFP sequences were detected in annotated tumor cells. Cell barcodes were determined based on the distribution of single-molecule tag (UMI) counts, and a filtered gene barcode matrix was generated by CellRanger for downstream analysis in Seurat (V.4.0.1, R software package) (Wolock et al., Cell Syst 8:281-291, 2019; Stuart et al., Cell 177:1888-1902, 2019). Cells with low UMI (<200 genes) and UMIs greater than 10% mapped to mitochondrial genes were removed. Data integration across different samples and treatment groups was performed using interactive principal component analysis (Hao et al., Cell 184:3573-3587, 2021) implemented in Seurat. The 'NormalizeData' function with the following parameters was used: normalization method (normalization.method) = 'LogNormalize', scale factor (scale.factor) = 10,000 to normalize the gene expression levels in each cell. 2000 highly variable genes were identified using the 'FindVariableFeatures' function with the 'vst' method. The 'ScaleData' function with default parameters was used to scale and center the gene expression matrix. In order to perform clustering, PCA dimensionality reduction was first implemented with the 'RunPCA' function. The first 20 principal components were selected to construct a common nearest neighbor graph with the 'FindNeighbors' function. Clusters were determined using the Louvain algorithm with the 'FindClusters' function. SingleR (V.1.8.1, R software package; Aran et al., NatImmunol 20: 163-172, 2019) was used to perform initial automatic annotation of cell types with known cell type labels from Blueprint / ENCODE reference and immune cell expression database to predict the identity of cell clusters. Subsequently, the annotations were manually checked to be reliable by checking the differentially expressed genes ranked first in each cluster, which were obtained by the 'FindAllMarkers' function with default parameters but min.pct=0.25 was set. Finally, unified manifold approximation and projection (UMAP) was applied to visualize single-cell transcriptional features in two-dimensional space.Tumor cells were confirmed by GFP sequence and copy number variation analysis using infercnv (V.1.10.1, R package; Tickle et al., inferCNV of the Trinity of CTAT project, Cambridge, MA, USA: Klarman Cell Observatory, Broad Institute of MIT and Harvard, 2019). CD45 was annotated using canonical gene markers. + Immune cells. Lymphoid cells were separated from tumor cells and mouse cells and re-clustered to obtain more refined cell clusters. Differential gene expression was calculated for all pairwise clusters and treatment groups. Samples across treatment groups were integrated using the standard anchor-based workflow in Seurat. For initial clustering and annotation, k-nearest neighbor (KNN) graph-based clustering was applied to weighted RNA similarities, aiming to calculate the Jaccard index (neighborhood overlap) between each pair of cells using high resolution and cluster merging. Results were visualized using UMAP plots using the Seurat software package. Tumor cells were confirmed based on green fluorescent protein sequence and copy number variation analysis. CD45 was annotated using canonical gene markers + Immune cells. QIAGEN IngenuityPathway Analysis was used for pathway enrichment analysis (QIAGEN) ( et al., Bioinformatics 30:523-530, 2014).

[0578] Statistical analysis

[0579] Two groups were compared using Student's t test (normally distributed data) or Mann-Whitney U test (skewed data), and more than two groups were compared using one-way ANOVA (normally distributed data) followed by Tukey's post hoc comparison test or one-way ANOVA on ranks followed by Dunn's test. For survival analysis, Kaplan-Meier curves were generated and survival rates were compared between groups using a two-sided log-rank test. All experiments were performed with biological replicates of at least two donors.

[0580] Example 8: In vitro comparison of CT3.28H.BBζ, CT3.8H.BBζ and CT3.8H.28BBζ

[0581] To identify the most effective GPC2-CAR construct for clinical translation, we first performed a head-to-head comparison of three different CAR backbone constructs in vitro using CT3 ( Fig.11A): (1) CT3 with CD8 hinge, CD8 TM and 4-1BB co-stimulatory domain (CT3.8H.BBζ, published CAR; Li et al., Cell Rep Med 2:100297, 2021), (2) CT3 with CD28 hinge, CD28 TM and 4-1BB co-stimulatory domain (CT3.28H.BBζ) and (3) CT3 with CD8 hinge, CD28 TM and CD28-4-1BB co-stimulatory domain (CT3.8H.28BBζ). When co-cultured with GPC2-WT or GPC2-knockout (KO) IMR-5 tumor cells, all three constructs showed increased levels of interferon-γ, granzyme B (GZMB) and soluble Fas ligand in the supernatant in the presence of the CAR antigen GPC2 ( Fig. 11B ). Cytokine levels were close to background levels in the presence of GPC2-KO cells, suggesting CAR specificity. However, of the three constructs, CT3.28H.BBζ exhibited the lowest tonic signaling, as indicated by low phosphorylation levels of CAR and downstream molecules such as ZAP70 or ERK at rest ( Figure 11C-11D ), but it is appropriately increased when CAR is cross-linked in vitro. Although CT3.8H.BBζ also shows lower tonic signaling at rest, CAR activation after antigen-specific cross-linking is not as robust as CT3.28H.BBζ. In addition, in the CAR framework of the present invention, CT3.28H.BBζ works better than another GPC2 scFv, GPC2.19 (Heitzeneder et al., Cancer Cell 32:295-309, 2022), especially at lower E:T ratios and for NB cell lines with lower antigen density, and has better cell expansion and CAR persistence when tumors are restimulated. These data show that all three CAR constructs have comparable in vitro function, but CT3.28H.BBζ lacks tonic signaling, which may positively affect anti-tumor activity in the context of persistent tumor exposure.

[0582] Example 9: CT3.28H.BBζ exhibits highly potent anti-NB activity in vivo

[0583] To determine which of the three GPC2-CAR constructs had the best anti-tumor activity against NB in ​​vivo, an orthotopic PDX model was used. 4–6-week-old NSG mice were orthotopically injected with SJNBL012407_X1. This PDX cell line has the molecular characteristics of high-risk NB (MYCN amplification), and most tumor-bearing mice treated with conventional chemotherapy and / or immunotherapy cannot be cured (Nguyen et al., Neoplasia 26:100776, 2022; Nguyen et al., Clin Cancer Res 28:3785-3796, 2022). Three weeks after tumor implantation, mice were randomly divided into groups to receive UT mock T cells or 2.5×10 6 CAR + T cells. Four weeks after infusion of CART cells (day 50 after tumor implantation), comparing all three CAR constructs, CT3.28H.BBζ induced the most significant tumor regression ( Fig. 12A ). Since CT3.8H.BBζ has been previously published (Li et al., Cell Rep Med 2:100297, 2021; Tian et al., J Clin Invest 132:e155621, 2022) and has comparable activity to CT3.8H.28BBζ, subsequent studies focused on CT3.28H.BBζ and CT3.8H.28BBζ. Survival studies were performed in vivo and tumor growth kinetics were evaluated after injection of GPC2-CAR T cells. 4–6 week old NSG mice bearing orthotopic IMR-5.ffLUC-GFP tumors were treated with high doses (5×10 6 ) or low dose (2.5×10 6 CAR T cell treatment ( Fig. 12B After high-dose CT3.28H.BBζ treatment, all animals showed a sharp drop in their BLI signals, which eventually reached background levels ( Fig. 12C ). Mice treated with high-dose CT3.8H.28BBζ responded transiently but could not maintain tumor control. 6 All mice treated with CAR T cells showed that their BLI signals initially decreased, but eventually increased in recurrent tumors as GPC2 was downregulated. The differences in tumor growth kinetics between mice receiving high-dose and lower-dose CAR T cells were reflected in the survival studies of these animals. Tumor-bearing mice treated with high-dose CT3.28H.BBζCAR T cells showed the longest survival ( Fig.12D ) and higher levels of tumor-infiltrating CAR based on flow cytometry analysis + T cells ( Fig.12E). There was no statistical difference in survival between the CAR groups at lower dose levels. However, when treated with CT3.28H.BBζCAR T cells, six of six mice never reached the study endpoint, and two of five mice treated with CT3.8H.28BBζCAR T cells died from tumors. Although in vitro experiments showed that the three different CARs targeting GPC2 functioned similarly, subsequent in vivo studies identified CT3.28H.BBζ as the most potent construct. The superior performance of CT3.28H.BBζ can be attributed to less tonic signaling, more CARs in the tumor microenvironment (TME), and the ability of the CAR to function in the tumor microenvironment (TME). + Higher levels of effector cells and / or antigen escape. To further evaluate the molecular differences between the three CAR constructs, tumor-infiltrating T cells were analyzed by single-cell RNA-seq.

[0584] Example 10: CT3.28H.BBζCAR T cells upregulate effector molecules in the TME

[0585] To understand the nature of T cells expressing different GPC2-CAR T constructs before infusion and after tumor encounter in vivo, single-cell RNA-seq was performed on produced GPC2-CAR Ts as well as on tumor-infiltrating T cells harvested at day 8, a time before tumor regression (generally seen at day 10).

[0586] CAR T cells from two donors were produced and the transcriptomes of these cells were analyzed using the droplet-based 10X genomics platform before injection into mice. After quality control and screening, a total of 14,169 and 13,515 single-cell transcriptomes were obtained for donor 1 and donor 2, respectively ( Fig.13A). Each subpopulation was uniformly reproduced for donor 1, but more CT3.8H.BBζ cells and fewer UT mock cells were captured for donor 2. To manually annotate cell subsets, a previously published annotation strategy was followed (Wilson et al., Cancer Discov 12:2098-2119, 2022; Patil et al., Sci Immunol 3:eaan8664, 2018). Graph-based unsupervised clustering was performed and CD8 and CD4 protein expression and traditional genes were used to define T cells (CD8A, CD4), NK cells (NKG7, GNLY) and B cells (MS4A1). T cell subsets were further defined as cytotoxic effector cells based on their robust expression of PRF1 and multiple genes encoding granzymes and as memory cells based on their expression of SELL, IL7R, CD27 and LEF1. Regulatory T cells (Treg) were identified based on IL2RA and FOXP3. Cell clusters were defined as proliferative when they expressed classical proliferation genes and cell cycle-related genes (e.g., TOP2A, MKI67, CCNB1 / 2, minichromosome maintenance (MCM) complex, or histone genes). The T cell injection product from donor 1 consisted of 12 cell clusters ( Fig. 13B ), these clusters were clustered according to their CD8 protein expression (22.3%) and CD4 protein expression (77.7%) ( Fig. 13C By using a composite gene signature, it was found that at the end of the CAR T production process, this donor contained mainly proliferating CD8 + and CD4 + T cells (72.8%) and CD8 + Cytotoxic effector cells (33.1%) ( Figure 13D-13E ). A portion of CD8 + Cells are cytotoxic effector cells ( Figure 13D-13E Donor 2 consists of 15 independent cell clusters ( Fig.13F ). As with donor 1, the total cell population contained more CD4 + T cells (62.1%; Figure 13G ) Fewer CD8 + T cells (37.9%), of which 25.6% were cytotoxic T cells and 1.7% were Tregs ( Figure 13H-13I ).

[0587] In order to determine the ideal time point for single-cell RNA-seq analysis of TME after injection of T cells into mice, the in vivo distribution and amplification of CART cells were evaluated. In order to track cells, GPC2-CAR T cells were transduced to express firefly luciferase-GFP (ffLUC-GFP). Three weeks after tumor implantation in the right adrenal fat pad, GPC2-CAR-ffLUC-GFP T cells were injected and continuous BLI was implemented. In all four experimental groups, T cells first accumulated in the lungs and femurs, and gradually expanded in the next 48 hours ( Fig.14A By day 7, the overall BLI signal had dimmed in the UT mock T group, while all three GPC2-CAR groups showed elevated signals localized to the tumor and spleen, consistent with localized expansion of T cells ( Figure 14B-Figure 14C ).

[0588] After injection of CAR T cells, tumors from IMR-5-bearing mice (day 8) were found to contain a large proportion of CD8 effector T cells and CD4 effector T cells, accounting for approximately 50% or more of cells from the TME ( Figure 14D-14G In contrast, UT-mock cells comprised <3% of cells in the TME. This is consistent with results from in vivo tracking experiments, in which UT-mock T cells failed to expand and engraft ( Fig.14A ). The residual cells in this group are almost exclusively M2 tumor-associated macrophages. Tumor cells are distinguished from immune cells based on their gene expression and copy number variation signatures.

[0589] Analysis of the in vivo trajectory of immune cells revealed that the few antigen-presenting cells present at the time of injection were quickly outnumbered by CD4 T cells and CD8 T cells. These cells developed from a highly proliferative state (characterized by expression of MKI67) to terminally differentiated and dysfunctional CD69-expressing, EOMES-expressing, and TOX-expressing effector cells or to a memory stage marked by high expression of IL7RA, LEF1, and CCL5. To better characterize the tumor-infiltrating CD8 + Transcriptome of effector cells, gene expression profile of CT3.28H.BBζ compared with the other two CARs targeting GPC2 ( Fig.14H ). In donor 1, 33 differentially expressed genes (DEGs) were found in common in both analyses (CT3.28H.BBζ vs CT3.8H.BBζ and CT3.28H.BBζ vs CT3.8H.28BBζ; Fig.14I In donor 2, there were 16 common DEGs ( Fig.14ICompared with the other two CAR T cell groups, CT3.28H.BBζCART cells showed upregulation of CXCR4, ARHGEF1, and LIME1, genes involved in chemokine-related T cell migration and T cell renewal (Bouafia et al., J Clin Invest 129:1047-1060, 2019; Chaix et al., J Immunol 193:1013-1016, 2014; Park et al., Mol Cells 43:921-934, 2020). Other DEGs include IL7R, JUND, ZFP36L and TXNIP, which are important in T cell homeostasis and memory formation (Schluns et al., Nat Immunol 1:426-432, 2000; Meixner et al., Embo J 23:1325-1335, 2004; Ruppert et al., PLoS One 7:e32262, 2012; Muri et al., Eur J Immunol 51:115-124, 2021; Petkau et al., Nat Commun 13(1):2274, 2022). CT3.28H.BBζCAR T cells also upregulated genes encoding effector molecules (e.g., GNLY, GZMB, ZNF683 and HMGN2) and genes encoding cell cycle components (e.g., STMN1, MCM5, MCM7 and PTTG1). Finally, pathway analysis confirmed these findings, showing activation of the granzyme A pathway (z score: 4.54; p value = 1.16E-35). CT3.28H.BBζCART cells also showed downregulation of the EIF2 pathway (z score: -3.628; p value = 6.19E-54) and the oxidative phosphorylation pathway (z score: -5.53; p value = 1.85E-56; Fig.14J ). Paired DEG analysis revealed that, compared with the other two CARs, CT3.28H.BBζ already expressed genes important in the regulation of T cell exhaustion pathways (e.g., NFKBIA, CISH), genes that promote T cell activation and proliferation (e.g., CD83, TXNIP, LDHA), and genes that can prevent apoptosis (e.g., MTRNR2L12; Fig.14J - Fig. 14K). These findings suggest that when these cells are produced, there are already transcriptomic differences that may influence the cytotoxic activity and survival divergence of the cells in vivo.

[0590] In summary, these findings show that all CAR T cells targeting GPC2 significantly expanded into the cytotoxic effector population in vivo. In addition, CT3.28H.BBζCAR T cells upregulated effector molecules and genes involved in T cell migration and memory homeostasis. These findings may be the reason for the superior antitumor cytotoxicity observed in mice treated with CT3.28H.BBζCAR T cells compared with other CAR treatment groups.

[0591] Example 11: CT3.28H.BBζ against GD2 + GPC2 低 Superior antitumor activity of NB over K666.28H.BBζ

[0592] Previous CAR T cell trials in NB were conducted with K666-based and 14.18-scFv-based GD2-CAR T cells (Straathof et al., Sci Transl Med 12: eabd6169, 2020; Heczey et al., Mol Ther 25: 2214-2224, 2017; Louis et al., Blood 118: 6050-6056, 2011; Pule et al., Nat Med 14: 1264-1270, 2008). Although the trial reported tolerance, very few treated patients achieved objective remission. Next, a study was conducted to evaluate how CT3.28H.BBζ performs compared to existing CAR T cell therapies targeting GD2 in terms of function. The goal is to compare the preclinical activity of K666-based and 14.G2a-based GD2-targeting CARs (Straath et al., Sci Transl Med 12: eabd6169, 2020) with that of CT3.28H.BBζ. In order to create comparable test conditions, scFvs were cloned into the same CAR constructs for CT3.28H.BBζ and CT3.8H.BBζ and compared head-to-head with continuous tumor restimulation in vitro. Subsequently, K666.28H.BBζ was selected and the anti-NB activity of CT3.28H.BBζ was further compared with K666.28H.BBζ in vitro and in vivo. These CAR T cells showed comparable conduction efficiency ( Fig.15A ). SJNBL012407_X1, IMR-5 (both MYCN-amplified), and SH-SY5Y (MYCN-WT) expressing ffLUC-GFP were incubated with CT3.28H.BBζCAR T cells or K666.28H.BBζCAR T cells at different E:T ratios. These NB cells have different GPC2 and GD2 expression levels. After 48 hours, tumor cell lysis was measured by applying a luciferase reporter assay ( Fig. 15B ). At an E:T ratio of 1:1, CT3.28H.BBζCAR T cells targeting GD2 中等 GPC2 低 PDX and GD2 低 GPC2 低 SH-SY5Y showed superior anti-NB cytotoxicity. GD2 of both groups 高 GPC2 高 Oncolysis in IMR-5 was comparable. Next, the test was extended to an in vitro tumor restimulation model using SJNBL012407_X1. After daily tumor restimulation, the cytotoxic capacity of the two CARs was measured at 24 hours, day 4, and day 7. Although CT3.28H.BBζCAR T cells initially showed better anti-NB cytotoxicity, their activity gradually decreased over time, making K666.28H.BBζCAR T cells superior on day 7 ( Fig. 15C Next, the antitumor activities of the two CARs were compared in vivo. On day 21 after tumor inoculation, mice carrying SJNBL012407_X1 were injected with 5×10 6 CAR + T cells. On day 50 after tumor injection, the primary tumor was weighed and the bone marrow was analyzed for residual NB cells. Compared with K666.28H.BBζCAR T, tumor weight was smaller after therapy with CT3.28H.BBζCART cells ( Figure 15D-15E In addition, 3 of 5 mice treated with K666.28H.BBζCAR T cells had higher levels of detectable tumor cells in their bone marrow than all mice treated with CT3.28H.BBζCAR T cells ( Fig.15F ). A possible reason for therapy resistance after K666.28H.BBζCAR T therapy may be downregulation of GD2 in the primary tumor. These findings show that CT3.28H.BBζCAR T cells outperform K666.28H.BBζCAR T cells in an early time window in vitro and in an orthotopic NB-PDX in vivo model, resulting in better tumor control of the primary tumor and a trend toward better control of metastatic disease burden in the bone marrow.

[0593] Obviously, the precise details of the methods or compositions may be changed or modified without departing from the spirit of the aspects described herein. We therefore claim all such changes and modifications that come within the scope and spirit of the following claims.

Claims

1. Chimeric antigen receptor (CAR), including: An extracellular antigen-binding domain that specifically binds to Glypican-2 (GPC2), comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises a complementarity determining region 1 (CDR1) sequence, a CDR2 sequence, and a CDR3 sequence of SEQ ID NO: 2, and the VL domain comprises a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence of SEQ ID NO: 4; CD28 hinge region; CD28 transmembrane domain; an intracellular co-stimulatory domain; and Intracellular signaling domain.

2. The CAR according to claim 1, wherein the CDR1 sequence, CDR2 sequence and CDR3 sequence are defined using the Kabat numbering scheme, the IMGT numbering scheme or the Paratome numbering scheme, or a combination of the Kabat numbering scheme, the IMGT numbering scheme and the Paratome numbering scheme.

3. The CAR according to claim 1 or claim 2, wherein: the VH domain CDR1, CDR2, and CDR3 sequences comprise residues 31-35, 50-66, and 99-112 of SEQ ID NO:2, respectively, and the VL domain CDR1, CDR2, and CDR3 sequences comprise residues 24-33, 49-55, and 88-96 of SEQ ID NO:4, respectively; the VH domain CDR1, CDR2, and CDR3 sequences comprise residues 26-33, 51-58, and 97-112 of SEQ ID NO:2, respectively, and the VL domain CDR1, CDR2, and CDR3 sequences comprise residues 27-31, 49-51, and 88-96 of SEQ ID NO:4, respectively; The VH domain CDR1, CDR2 and CDR3 sequences comprise residues 26-35, 47-61 and 97-112 of SEQ ID NO:2, respectively, and the VL domain CDR1, CDR2 and CDR3 sequences comprise residues 27-33, 45-55 and 88-95 of SEQ ID NO:4, respectively; or The VH domain CDR1, CDR2 and CDR3 sequences comprise residues 26-35, 47-66 and 97-112 of SEQ ID NO:2, respectively, and the VL domain CDR1, CDR2 and CDR3 sequences comprise residues 24-33, 45-55 and 88-96 of SEQ ID NO:4, respectively.

4. The CAR according to any one of claims 1 to 3, wherein: The amino acid sequence of the VH domain is at least 90% identical to SEQ ID NO: 2; and comprises the CDR1 sequence, CDR2 sequence, and CDR3 sequence of SEQ ID NO: 2; and The amino acid sequence of the VL domain is at least 90% identical to that of SEQ ID NO:4; and comprises the CDR1 sequence, CDR2 sequence, and CDR3 sequence of SEQ ID NO:

4.

5. The CAR according to any one of claims 1-3, wherein the VH domain sequence and the VL domain sequence are humanized.

6. The CAR according to claim 5, wherein: the amino acid sequence of the VH domain comprises residues 1-123 of SEQ ID NO:8, and the amino acid sequence of the VL domain comprises residues 139-244 of SEQ ID NO:8; the amino acid sequence of the VH domain comprises residues 1-122 of SEQ ID NO: 12, and the amino acid sequence of the VL domain comprises residues 138-243 of SEQ ID NO: 12; The amino acid sequence of the VH domain comprises residues 1-122 of SEQ ID NO: 16, and the amino acid sequence of the VL domain comprises residues 138-244 of SEQ ID NO: 16; or The amino acid sequence of the VH domain comprises residues 1-122 of SEQ ID NO:20, and the amino acid sequence of the VL domain comprises residues 138-243 of SEQ ID NO:

20.

7. The CAR according to any one of claims 1-6, wherein the extracellular antigen binding domain comprises or consists of the amino acid sequence of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20 or SEQ ID NO:

22.

8. The CAR according to any one of claims 1-7, wherein the CD28 hinge region comprises or consists of the amino acid sequence of SEQ ID NO:

24.

9. The CAR according to any one of claims 1-8, wherein the CD28 transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO:

26.

10. The CAR of any one of claims 1-9, wherein the co-stimulatory domain comprises a 4-1BB signaling portion.

11. The CAR of claim 10, wherein the amino acid sequence of the 4-1BB signaling portion comprises or consists of SEQ ID NO:

28.

12. The CAR of any one of claims 1-11, wherein the signaling domain comprises a CD3 zeta signaling domain.

13. The CAR of claim 12, wherein the amino acid sequence of the CD3ζ signaling domain comprises or consists of SEQ ID NO:

30.

14. The CAR of any one of claims 1-13, wherein the amino acid sequence of CAR comprises or consists of SEQ ID NO:

38.

15. An isolated cell expressing the CAR according to any one of claims 1-14.

16. The isolated cell of claim 15, wherein the cell is an immune cell or an induced pluripotent stem cell (iPSC).

17. The isolated cell of claim 16, wherein the immune cell is a T cell, a B cell, a natural killer (NK) cell or a macrophage.

18. An isolated nucleic acid molecule encoding the CAR according to any one of claims 1-14.

19. The isolated nucleic acid molecule of claim 18, comprising or consisting of nucleotides 73-1470 of SEQ ID NO:

37.

20. The isolated nucleic acid molecule of claim 18, comprising and consisting of SEQ ID NO:

37.

21. The isolated nucleic acid molecule of any one of claims 18-20, operably linked to a promoter.

22. The isolated nucleic acid molecule of claim 21, wherein the promoter is the human elongation factor 1 alpha (EF1 alpha) promoter.

23. A vector comprising the isolated nucleic acid molecule according to any one of claims 18 to 22.

24. The vector of claim 23, wherein the vector is a lentiviral vector.

25. An isolated cell comprising an isolated nucleic acid molecule according to any one of claims 18 to 22 or a vector according to claim 23 or claim 24.

26. The isolated cell of claim 20, wherein the cell is an immune cell or an induced pluripotent stem cell (iPSC).

27. The isolated cell of claim 26, wherein the immune cell is a T cell, a B cell, a natural killer (NK) cell or a macrophage.

28. A composition comprising a pharmaceutically acceptable carrier and a CAR according to any one of claims 1-14, an isolated cell according to any one of claims 15-17 and 25-27, an isolated nucleic acid molecule according to any one of claims 18-22, or a vector according to claim 23 or claim 24.

29. A method for treating a GPC2-positive cancer in a subject, comprising administering to the subject a therapeutically effective amount of a CAR according to any one of claims 1-14, an isolated cell according to any one of claims 15-17 and 25-27, an isolated nucleic acid molecule according to any one of claims 18-22, a vector according to claim 23 or claim 24, or a composition according to claim 28.

30. A method of inhibiting tumor growth or metastasis of a GPC2-positive cancer in a subject, comprising administering to the subject a therapeutically effective amount of a CAR according to any one of claims 1-14, an isolated cell according to any one of claims 15-17 and 25-27, an isolated nucleic acid molecule according to any one of claims 18-22, a vector according to claim 23 or claim 24, or a composition according to claim 28.

31. The method of claim 29 or claim 30, wherein the GPC2-positive cancer is a solid tumor.

32. The method of any one of claims 29-31, wherein the GPC2-positive cancer is a pediatric cancer.

33. The method of any one of claims 29-32, wherein the GPC2-positive cancer is neuroblastoma, medulloblastoma, retinoblastoma, acute lymphoblastic leukemia, embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, Ewing sarcoma, desmoplastic small round cell tumor, glioma, or osteosarcoma.

34. The method of any one of claims 29-33, further comprising administering chemotherapy to the subject.

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