CD28 hinge and transmembrane-containing chimeric antigen receptors targeting GPC2 and uses thereof

Abstract

Described is an optimized chimeric antigen receptor (CAR) that targets glypican-2 (GPC2) and has a CD28 hinge region and a CD28 transmembrane domain. The antigen binding domain of the disclosed CAR is derived from GPC2-specific antibody CT3 or its humanized version. The optimized CAR also includes an intracellular co-stimulatory 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

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 310,456, filed February 15, 2022, which is incorporated by reference herein in its entirety.

[0002] Statement of government support This invention was made with Government support under Project Nos. Z01 BC010891, ZIA BC010891, Z01 Z1A BC010788, Z1A BC011334 and ZIA BC 012066 awarded by the National Institutes of Health, National Cancer Institute. The Government has certain rights in this invention.

[0003] Field of Disclosure The present disclosure relates to an optimized chimeric antigen receptor (CAR) specific for tumor antigen glypican-2 (GPC2), which comprises a hinge region and a transmembrane domain derived from CD28. The present disclosure further relates to the use of GPC2-targeting CARs, for example, to treat solid tumors.

[0004] Incorporation of Electronic Sequence Listings The electronic sequence listing submitted herewith as an XML file (63,252 bytes) named 4239-107434-02.xml, created on January 26, 2023, is hereby incorporated by reference in its entirety. [Background technology]

[0005] background CAR T-cell therapy has emerged as an important class of cancer therapeutics and is being actively developed and tested worldwide. After initial success in hematological cancers using CD19-targeting CAR T cells, various CAR strategies have been rigorously engineered and tested with the aim of treating solid tumors.

[0006] Glypican-2 (GPC2) is one member of the six-member glypican family of heparan sulfate proteoglycans that is attached to the cell surface by 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 114(32):E6623-E6631, 2017; WO 2020 / 033430; and WO 2018 / 026533).

[0007] Neuroblastoma is the most common type of extracranial solid tumor in children. It originates from the neuroendocrine tissues of the sympathetic nervous system and accounts for approximately 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 almost 50% of patients having a high-risk phenotype characterized by widespread dissemination of cancer and poor long-term survival, even when diverse intensive treatments are used (Yu et al., New Engl J Med 363:1324-1334, 2010). Approximately 45% of patients undergoing standard treatment have a recurrence and ultimately die due to metastatic disease (Matthay et al., New Engl J Med 341:1165-1173, 1999). Thus, there is an urgent and unmet need for safe and effective treatments for neuroblastoma. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] International Publication No. 2020 / 033430 [Patent Document 2] International Publication No. 2018 / 026533 [Non-patent literature]

[0009] [Non-Patent Document 1] Li et al., Trends Cancer 4(11):741-754, 2018 [Non-Patent Document 2] Orentas et al., Front Oncol 2:194, 2012 [Non-Patent Document 3] Li et al., Proc Natl Acad Sci USA 114(32):E6623-E6631, 2017 [Non-Patent Document 4] Maris and Hogarty, Lancet 369:2106-2120, 2007 [Non-Patent Document 5] Yu et al., New Engl J Med 363:1324-1334, 2010 [Non-Patent Document 6] Matthay et al., New Engl J Med 341:1165-1173, 1999 Summary of the Invention [Means for solving the problem]

[0010] Abstract Disclosed herein is an optimized GPC2-specific chimeric antigen receptor (CAR), which comprises hinge region and transmembrane domain derived from human CD28.Surprisingly, it is demonstrated herein that GPC2-specific CAR with CD28 hinge and CD28 transmembrane domain is more effective in killing GPC2-positive cells in vitro and eradicating GPC2-positive tumors in animal models, compared with GPC2-specific CAR with hinge region derived from CD8 and transmembrane (TM) domain derived from either CD8 or CD28.

[0011] Provided herein is a CAR that comprises an extracellular antigen binding domain specific for GPC2; a CD28 hinge region; a CD28 transmembrane domain; an intracellular costimulatory domain; and an intracellular signaling domain.In some embodiments, the antigen binding domain comprises a variable heavy (VH) domain and a variable light (VL) domain, and the VH and VL domain comprise the CDR sequence of GPC2-specific antibody CT3 or its humanized version (e.g., hCT3-1, hCT3-2, hCT3-3 or hCT3-4).In some examples, the antigen binding domain comprises a linker sequence between the VH domain and the VL domain, and the antigen binding domain can be in VH-linker-VL orientation or VL-linker-VH orientation.

[0012] Further provided is a nucleic acid molecule encoding the disclosed CAR. In some embodiments, the nucleic acid molecule comprises, 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 costimulatory domain; a nucleic acid encoding a signal transduction 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 comprises a human elongation factor 1 alpha (EF1 alpha) promoter sequence 5' to the nucleic acid encoding the first GMCSFRss. Further provided is a vector (e.g., a lentiviral vector) comprising the disclosed nucleic acid molecule.

[0013] Also provided are isolated immune cells (e.g., T cells, NK cells, B cells, or macrophages) and induced pluripotent stem cells (iPSCs) that express a CAR disclosed herein and / or contain an isolated nucleic acid molecule or vector encoding a CAR disclosed herein.

[0014] Further provided is a composition comprising a pharma- ceutically acceptable carrier and a CAR, nucleic acid molecule, vector, or cell disclosed herein.

[0015] Also provided is a method for treating GPC2 positive cancer or inhibiting tumor growth or metastasis of GPC2 positive cancer in a subject.In some embodiments, these methods comprise administering to a subject a therapeutically effective amount of CAR, nucleic acid molecule, vector, cell or composition disclosed herein.In some examples, the GPC2 positive cancer is a solid tumor, such as neuroblastoma, medulloblastoma or retinoblastoma.

[0016] The above and other features of the present disclosure will become more apparent from the following detailed description of several embodiments, which proceeds with reference to the accompanying drawings. [Brief description of the drawings]

[0017] [Figure 1] CAR constructs. (FIG. 1A) Schematic of CAR constructs CT3.8H.BBz, CT3.8H.28BBz and CT3.28H.BBz. (FIG. 1B) Detailed schematic of the CT3.28H.BBz CAR design. [Diagram 2] In vitro cell killing assay of GPC2-expressing IMR5 cells (Figure 2A) and GPC2 knockout (KO) IMR5 cells (Figure 2B). 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 effect on GPC2-KO IMR5 cells, demonstrating their GPC2 specificity. [Figure 3A-B]Comparison of CT3.8H.BBz and CT3.28H.BBz CAR T cells in the IMR5 metastasis model. (Figure 3A) Experimental design. Mice were inoculated iv with IMR5-luc 28 days prior to infusion of 10 million CAR T cells. Mice were imaged weekly after infusion. (Figure 3B) Bioluminescence images of mock and CAR T cell treated mice. (Figure 3C) Bioluminescence measured 2, 4, 6 and 8 weeks after CAR T cell infusion. (Figure 3D) Survival of mock and CAR T cell treated mice after CAR T cell infusion. CT3.28H.BBz CAR T cells were significantly more potent than CT3.8H.BBz CAR T cells in regressing neuroblastoma tumors in mice. All mice in the CT3.28H.BBz treatment group survived to the end of the study. [Figure 3C-D] Same as above. [Figure 4A-C]Comparison of CT3.28H.BBz and CT3.8H.28BBz CAR T cells in the orthotopic IMR5 mouse model. (Figure 4A-4C) T cells from three different human donors were used: A26M (Figure 4A), A59F (Figure 4B) and A25F (Figure 4C). Mice with moderate tumor burden were administered 5 million human T cells expressing CT3.28H.BBz CAR or CT3.8H.28BBz CAR. CT3.28H.BBz CAR T cells outperformed CT3.8H.28BBz CAR T cells for all three donors. (Figure 4D-4E) Bioluminescence images of mock-treated mice and mice treated with CT3.28H.BBz or CT3.8H.28BBz CAR T cells derived from donor A26M (Figure 4D) and donor A59F (Figure 4E). 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 plot showing gating strategy for flow samples. Live cells were gated for CD3+ human cells to determine the percentage of CAR-positive cells. (Figure 4G) Graph showing the percentage of CD3+ cells expressing CT3.28H.BBz CAR or CT3.8H.28BBz CAR for donors A26M and A59F. For both donors, CT3.28H.BBz CAR T cells retained higher levels of CAR expression than CT3.8H.28BBz CAR T cells. [Figure 4D] Same as above. [Figure 4E] Same as above. [Figure 4F] Same as above. [Figure 4G] Same as above. [Diagram 5]Detection of CAR phosphorylation as a measure of CAR activation. (Figure 5A) CT3.8H.BBz, CT3.8H.28BBz and CT3.28H.BBz CAR T cells were unstimulated or stimulated with protein L or GPC2-Fc, and CAR phosphorylation was detected by Western blot. (Figure 5B) Graph showing fold change in CAR phosphorylation. CT3.8H.28BBz CAR had a higher phosphorylation level than CT3.28H.BBz CAR when tested without stimulation. With GPC2 stimulation, both CARs upregulated their phosphorylation levels. These results demonstrate that CT3.8H.28BBz has more robust CAR signaling (phosphorylation), which is known to cause CAR exhaustion. CT3.28H.BBz CAR T cells have less robust CAR signaling, but show proper CAR activation upon antigen presentation (GPC2-Fc). [Figure 6A] Comparison of CT3.28H.BBz and CT3.8H.28BBz CAR T cells with low or high dose chemotherapy in an orthotopic IMR5 animal model. Mice with large tumor burdens were treated with no chemotherapy, low or high dose chemotherapy (Fludarabine / Cyclophosphamide) for 3 days prior to infusion of 5 million CAR T cells. (Figure 6A) Tumor size measured by bioluminescence. (Figure 6B) Tumor weight 10 weeks after chemotherapy and CAR T cell infusion. CT3.28H.BBz performed better than CT3.8H.28BBz when given low dose conditioning chemotherapy in high tumor burden mice. [Figure 6B] Same as above. [Figure 7A] Binding Affinity of Humanized CT3 (hCT3) Antibodies. Shown is a set of graphs demonstrating 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 parent CT3 antibody (2.2 nM). [Figure 7B] Same as above. [Figure 8]Humanized CT3 antibodies bind to cell surface GPC2. Shown are flow cytometry plots demonstrating that all four humanized CT3 antibodies maintain the ability to bind to cell surface GPC2. [Figure 9] Cell killing by humanized CT3-based CAR T cells. The graph shows the specific lysis of GPC2-expressing IMR5 cells by CT3-8H-BBz, hCT3-1-8H-BBz, hCT3-2-8H-BBz, hCT3-3-8H-BBz and hCT3-4-8H-BBz CAR T cells. All four humanized CT3-8H-BBz CAR T cells showed improved killing activity against IMR5 cells compared to CT3-8H-BBz CAR T cells. [Figure 10] Schematic diagram of two humanized CT3-28H-BBz CAR constructs, in which the GPC2 binding domain has a VH-linker-VL orientation (top) or a VL-linker-VH orientation (bottom). [Figure 11A-B] In vitro comparison of three GPC2-CAR constructs. (Figure 11A) Overview of the three CAR constructs used in preclinical studies, with variable hinge, TM and costimulatory domains. (Figure 11B) Cytokine secretion profile of GPC2-CAR T cells at 24 hours in co-culture with tumor cells. Human T cells transduced with three different GPC2-CARs were grown with GPC2-KO or GPC2-WT IMR-5 tumor cells. Levels of interferon-γ (IFNγ), granzyme B (GZMB) and soluble Fas ligand (sFASL) were significantly increased in the presence of GPC2 but were comparable across the three CARs tested. (Figure 11C) Western blot analysis to demonstrate CAR signaling in resting and CAR-crosslinked T cells. (Figure 11D) Densitometric quantification of the Western blot signals from Figure 11C. CT3.8H.28BBζ shows significant tonic signaling of CAR at rest that is absent for CT3.28H.BBζ and CT3.8H.BBζ. However, antigen-specific CAR activation induces higher phosphorylation levels in CT3.28H.BBζ compared to CT3.8H.BBζ. [Figure 11C]Same as above. [Figure 11D] Same as above. [Figure 12A-B] CT3.28H.BBζ outperforms CT3.8H.CD28BBζ in vivo. (Figure 12A) Tumor weight 50 days after tumor injection. CT3.28H.BBζ induced the most significant tumor regression across all treatment groups. (Figure 12B) Model system and experimental regimen. (Figure 12C) Long-term bioluminescence imaging (BLI) signals of mice treated with mock T cells without transduction (UT) or untreated control and mice treated with GPC2-targeting CAR T cells. Weekly BLI revealed that CT3.28H.BBζ-CAR T-treated animals demonstrated a strong decrease in their BLI signal that persisted for the duration of the study. In contrast, mice treated with CT3.8H.28BBζ or the lower cell dose of GPC2-targeting CAR T cells, respectively, showed a transient response but eventually progressed. (Figure 12D) Survival curves of mice from Figure 12C. (Figure 12E) Persistence of CAR+ T cells isolated from tumors of treated mice in Figure 12C at day 80 by flow cytometry analysis. [Figure 12C] Same as above. [Fig. 12D-E] Same as above. [Figure 13A-C] GPC2-CAR T cell manufacturing generates proliferative and cytotoxic effector T cells. (Figure 13A) Percentage of captured immune cells on day 8 of CAR T manufacturing. (Figure 13B) Uniform manifold approximation (UMAP), (Figure 13C) CD8 and CD4 protein expression levels detected by TotalSeq, (Figure 13D) cluster annotation, and (Figure 13E) fraction of subsets of cells manufactured from donor 1. (Figure 13F) UMAP, (Figure 13G) CD8 and CD4 protein expression levels, (Figure 13H) cluster annotation, and (Figure 13I) fraction of subsets of cells manufactured from donor 2. (Figures 13J-13K) Differentially expressed genes (DEG) analysis of generated cells. [Figure 13D] Same as above. [Figure 13EFGI] Same as above. [Figure 13H] Same as above. [Fig. 13J-K] Same as above. [Figure 14A-B] GPC2-CAR T cells home to the tumor microenvironment (TME) and are enriched in vivo as a cytotoxic effector population. (Figure 14A) In vivo GPC2-targeting CAR T cell tracking using BLI. T cells were transduced to express firefly luciferase (ffLUC)-GFP and continuously monitored for homing and expansion. (Figure 14B-14C) All three CARs enrich and expand in the TME compared to UT mock cells. *p<0.05; Student's t-test. (Figure 14D-14E) IMR-5-bearing mice received T cell injections. Eight days later, tumors were isolated and single-cell RNA-seq was performed. Quantification of tumors and tumor-derived immune cells is shown. (Figure 14F-14G) UMAP plots showing tumor cells and five immune subsets. Bar graphs quantify immune fractions. (Figure 14H) Volcano plot of differentially expressed genes comparing CT3.28H.BBζ vs. CT3.8H.BBζ (upper panel) and CT3.28H.BBζ vs. CT3.8H.28BBζ (lower panel). (Figure 14I) Differentially expressed genes grouped by function and extracted from both comparisons made in Figure 14H. (Figure 14J) Compound pathway analysis reveals that CT3.28H.BBζ CAR T cells upregulate the granzyme A pathway but downregulate 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. [Figure 14C-E] Same as above. [Figure 14F] Same as above. [Figure 14G] Same as above. [Figure 14H] Same as above. [Figure 14I-J] Same as above. [Figure 15A-B] Direct comparison of GPC2-CAR (CT3.28H.BBζ) vs. GD2-CAR (K666.28H.BBζ). (Figure 15A) Transduction efficiency of CT3.28H.BBζ and K666.28H.BBζ CAR T cells. (Figure 15B) In vitro cytotoxicity assay testing both CARs against three NB lines at varying E:T ratios. ***p<0.001; ****p<0.0001; two-way ANOVA. (Figure 15C) In vitro tumor re-challenge assay. CAR T cells were re-challenged every 24 hours. Cytotoxic activity was measured at 24 hours, 96 hours and 7 days. *p<0.05; paired t-test. (Figure 15D) Tumor weight 50 days after tumor implantation. *p<0.05; Student's t-test. (FIG. 15E) Image of tumor collected 50 days after implantation. Scale bar=1.0 cm. (FIG. 15F) Flow analysis of bone marrow cells derived from one femur. Tumor cells are identified as hCD45-mCD45-GD2+GFP+ cells. Total cell number per femur is plotted and each point represents one mouse. [Figure 15C] Same as above. [Fig. 15D-E] Same as above. [Figure 15F] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] array The nucleic acid and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases and one-letter codes for amino acids as defined in 37 CFR 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood to be included by any reference to the represented strand. In the accompanying sequence listing: SEQ ID NO:1 is the nucleotide sequence encoding the CT3 VH domain. SEQ ID NO:2 is the amino acid sequence of the CT3 VH domain. SEQ ID NO:3 is the nucleotide sequence encoding the CT3 VL domain. SEQ ID NO:4 is the amino acid sequence of the CT3 VL domain. SEQ ID NO:5 is the nucleotide sequence encoding the CT3 scFv. SEQ ID NO:6 is the amino acid sequence of CT3 scFv. SEQ ID NO:7 is the nucleotide sequence encoding the hCT3-1(VH-VL)scFv. SEQ ID NO: 8 is the amino acid sequence of hCT3-1(VH-VL)scFv. SEQ ID NO:9 is the nucleotide sequence encoding the hCT3-1(VL-VH)scFv. SEQ ID NO: 10 is the amino acid sequence of hCT3-1(VL-VH)scFv. SEQ ID NO:11 is the nucleotide sequence encoding the hCT3-2(VH-VL)scFv. SEQ ID NO: 12 is the amino acid sequence of hCT3-2(VH-VL)scFv. SEQ ID NO: 13 is the nucleotide sequence encoding the hCT3-2(VL-VH)scFv. SEQ ID NO: 14 is the amino acid sequence of hCT3-2(VL-VH)scFv. SEQ ID NO:15 is the nucleotide sequence encoding the hCT3-3(VH-VL)scFv. SEQ ID NO: 16 is the amino acid sequence of hCT3-3(VH-VL)scFv. SEQ ID NO:17 is the nucleotide sequence encoding the hCT3-3(VL-VH)scFv. SEQ ID NO: 18 is the amino acid sequence of hCT3-3(VL-VH)scFv. SEQ ID NO:19 is the nucleotide sequence encoding the hCT3-4(VH-VL)scFv. SEQ ID NO: 20 is the amino acid sequence of hCT3-4(VH-VL)scFv. SEQ ID NO:21 is the nucleotide sequence encoding the hCT3-4(VL-VH)scFv. SEQ ID NO: 22 is the amino acid sequence of hCT3-4(VL-VH)scFv. SEQ ID NO:23 is a nucleotide sequence encoding the CD28 hinge region. SEQ ID NO:24 is the amino acid sequence of the CD28 hinge region. SEQ ID NO:25 is a nucleotide sequence encoding the CD28 transmembrane domain. SEQ ID NO:26 is the amino acid sequence of the CD28 transmembrane domain. SEQ ID NO:27 is a nucleotide sequence encoding the 4-1BB signaling portion. SEQ ID NO:28 is the amino acid sequence of the 4-1BB signaling portion. SEQ ID NO:29 is a nucleotide sequence encoding the CD3 zeta signaling domain. SEQ ID NO:30 is the amino acid sequence of the CD3 zeta signaling domain. SEQ ID NO:31 is a nucleotide sequence encoding the GMCSFR signal sequence. SEQ ID NO:32 is the amino acid sequence of the GMCSFR signal sequence. SEQ ID NO:33 is a nucleotide sequence encoding the T2A self-cleaving peptide. SEQ ID NO:34 is the amino acid sequence of the T2A self-cleaving peptide. SEQ ID NO:35 is the nucleotide sequence encoding hEGFRt. SEQ ID NO: 36 is the amino acid sequence of hEGFRt. SEQ ID NO:37 is the nucleotide sequence encoding the CT3.28H.BBz CAR construct. 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 Residues 23-24 - restriction enzyme site Residues 25-268 - CT3 scFv Residues 269-270 - restriction enzyme site Residues 271-309 - CD28 hinge Residues 310-336 - CD28 transmembrane domain Residues 337-378 - 4-1BB costimulatory domain Residues 379-490 - CD3ζ signaling domain Residues 491-508 - T2A site Residues 509-530 - GMCSFR signal sequence Residues 531-865 - hEGFRt SEQ ID NO:39 is the amino acid sequence of the CT3.8H.BBz CAR construct, which includes the following features: Residues 1-22 - GMCSFR signal sequence Residues 23-24 - restriction enzyme site Residues 25-268 - CT3 scFv Residues 269-270 - restriction enzyme site Residues 271-315 - CD8 hinge Residues 316-336 - CD8 transmembrane domain Residues 337-378 - 4-1BB costimulatory domain Residues 379-490 - CD3ζ signaling domain Residues 491-508 - T2A site Residues 509-530 - GMCSFR signal sequence Residues 531-865 - hEGFRt SEQ ID NO:40 is the amino acid sequence of the CT3.8H.28BBz CAR construct, which includes the following features: Residues 1-22 - GMCSFR signal sequence Residues 23-24 - restriction enzyme site Residues 25-268 - CT3 scFv Residues 269-270 - restriction enzyme site Residues 271-315 - CD8 hinge Residues 316-342 - CD28 transmembrane domain Residues 343-383 - CD28 costimulatory domain Residues 384-425 - 4-1BB costimulatory domain Residues 426-537 - CD3ζ signaling domain Residues 538-555 - T2A site Residues 556-577 - GMCSFR signal sequence Residues 578-912 - hEGFRt

[0019] Detailed Description I. Abbreviations ALL Acute Lymphoblastic Leukemia ARMS Alveolar rhabdomyosarcoma BLI Bioluminescence Imaging CAR Chimeric Antigen Receptor CDR Complementarity Determining Region CNS Central Nervous System DRCT Desmoplastic small round cell tumor Effector vs. target EF1α Elongation factor 1 alpha EGF epidermal growth factor EGFR epidermal growth factor receptor ERMS Embryonal rhabdomyosarcoma ffLUC Firefly luciferase GPC2 Glypican-2 GMCSFRss Granulocyte-macrophage colony-stimulating factor receptor signal sequence hEGFRt Human truncated epidermal growth factor receptor iPSC induced pluripotent stem cells iv KO Knockout NB Neuroblastoma NK Natural Killer PDX patient-derived xenografts RMS Rhabdomyosarcoma scFv single chain variable fragment TM transmembrane TME Tumor Microenvironment VH Variable Weight UT No transduction VL Variable Light WT wild type

[0020] II. Summary of Terms Unless otherwise noted, technical terms are used according to conventional usage. Definitions of many common terms in molecular biology can be found in Krebs et al. (eds.), Lewin's genes XII, published by Jones & Bartlett Learning, 2017. As used herein, the singular forms "a", "an" and "the" refer to both the singular and the plural, unless the context clearly indicates otherwise. For example, the term "an antigen" includes a single or multiple antigens and can be considered equivalent to the phrase "at least one antigen". As used herein, the term "comprises" means "includes". It should be further understood that any and all base 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, unless otherwise indicated. Although many methods and materials similar or equivalent to those described herein can be used, certain suitable methods and materials are described herein. In case of conflict, the present specification, including explanations of terms, will control. Furthermore, the materials, methods and examples are illustrative only and are not intended to be limiting. To facilitate review of various embodiments, the following explanations of terms are provided:

[0021] 4-1BB: a costimulatory molecule expressed by lymphocytes activated with T cell receptor (TCR) and by other cells, including natural killer cells. Ligation of 4-1BB induces a signal transduction cascade that results in cytokine production, expression of anti-apoptotic molecules, and enhanced immune response. An exemplary amino acid sequence of 4-1BB is shown herein as SEQ ID NO:28.

[0022] Acute lymphoblastic leukemia (ALL): An acute form of leukemia characterized by an overproduction of lymphoblasts. ALL is most common in children, peaking between the ages of 2 and 5.

[0023] Administration: Providing or giving an agent, such as a CAR or a CAR-expressing cell provided herein, to a subject by any effective route. Exemplary administration routes include, but are not limited to, injection (e.g., 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 cases, administration is local. In some cases, administration is systemic.

[0024] Antibody: A polypeptide ligand comprising at least one variable region that recognizes and binds (e.g., specifically recognizes and specifically binds) an epitope of an antigen, e.g., GPC2. Mammalian immunoglobulin molecules are composed of heavy (H) and light (L) chains, each of which has a respective variable heavy (V H ) area and variable light (V L ) region. Together, they form the V H Area and V L The region is 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 not found in mammals include IgX, IgY, IgW and IgNAR. IgY is the main antibody produced by birds and reptiles and has some functionality similar to mammalian IgG and IgE. IgW and IgNAR antibodies are produced by cartilaginous fish, such as sharks, while IgX antibodies are found in amphibians. IgNAR antibodies are heavy chain antibodies.

[0025] Antibody variable regions contain "framework" regions and hypervariable regions known as "complementarity determining regions" or "CDRs". The CDRs are primarily responsible for binding to an epitope of an antigen. The framework regions of an antibody function to position and arrange the CDRs in three dimensional space. The amino acid sequence boundaries of a given CDR may be determined by the methods described in Kabat et al. (Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, 1991; "Kabat" numbering scheme), Chothia et al. (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. (Kunik et al., PLoS Comput Biol 8:e1002388, 2012; and Kunik et al., Nucleic Acids Res 40:W521-524, 2012; "Paratome CDR) and the ImMunoGeneTics (IMGT) database (see Lefranc, Nucleic Acids Res 29:207-9, 2001; "IMGT" numbering scheme). The Kabat, Paratome and IMGT databases are maintained online.

[0026] A "single domain antibody" refers to an antibody having a single domain (a variable domain) that is capable of specifically binding to an antigen or an epitope of an antigen in the absence of further antibody domains. Single domain antibodies include, for example, H Domain antibodies, V NAR Antibody, Camelidae V H H antibodies and V L Domain antibodies are included.NAR Antibodies are produced by cartilaginous fishes that produce heavy chain antibodies (IgNAR), such as the nurse shark, wobbegong shark, spiny dogfish, and bamboo shark. H H antibodies are produced by several species, including camels, llamas, alpacas and guanacos, which produce heavy chain antibodies that are naturally devoid of light chains.

[0027] A "monoclonal antibody" is an antibody produced by a single clone of lymphocytes or by a cell transfected with a single antibody coding sequence. Monoclonal antibodies are produced by known methods. Monoclonal antibodies include humanized monoclonal antibodies.

[0028] A "chimeric antibody" has framework residues derived from one species, such as human, and CDRs (which generally confer antigen binding) from another species.

[0029] A "humanized" antibody is an immunoglobulin that includes a human framework region and one or more CDRs derived from a non-human (e.g., mouse, rabbit, rat, shark, camel or synthetic) immunoglobulin. The non-human immunoglobulin that provides the CDRs is called the "donor" and the human immunoglobulin that provides the framework is called the "acceptor". In one embodiment, all CDRs in a humanized immunoglobulin are derived from a donor immunoglobulin. Constant regions need not be present, but if they are present, they are substantially identical to human immunoglobulin constant regions, i.e., at least about 85-90%, e.g., about 95% or higher. Thus, all parts of a humanized immunoglobulin, except possibly the CDRs, are substantially identical to the corresponding parts of a natural human immunoglobulin sequence. A humanized antibody binds 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 have substantially no effect on antigen binding or other immunoglobulin functions.

[0030] Binding affinity: the affinity of an antibody or other antigen-binding molecule to an antigen, e.g., GPC2. In one embodiment, affinity is calculated by a modification of the Scatchard method described by Frankel et al., Mol. Immunol., 16:101-106, 1979. In another embodiment, binding affinity is measured by antigen / antibody dissociation rate. In another embodiment, high binding affinity is measured by competitive radioimmunoassay. In another embodiment, binding affinity is measured by ELISA. In some embodiments, binding affinity is measured using the Octet system (ForteBio) based on biolayer interference technology. In other embodiments, Kd is measured using a surface plasmon resonance assay, e.g., using BIACORES-2000 or BIACORES-3000 (BIAcore, Inc., Piscataway, NJ). In other embodiments, antibody affinity is measured by flow cytometry. An antibody or CAR that "specifically binds" to an antigen (e.g., GPC2) is an antibody or CAR that binds to the antigen with high affinity, but does not bind significantly to other, unrelated antigens.

[0031] Chemotherapeutic agent: Any chemical agent that has therapeutic utility in treating diseases characterized by abnormal cell growth. Such diseases include tumors, neoplasms, and cancers. In one embodiment, the chemotherapeutic agent is an agent used in treating GPC2-positive tumors. In one embodiment, the chemotherapeutic agent is a radioactive compound. Exemplary chemotherapeutic agents that can be used in the methods provided herein are those described in 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 nded., (C)2000 Churchill 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-Year Book, 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 (e.g., pembrolizumab and nivolumab), anti-CTLA4 (e.g., ipilimumab), anti-EGFR (e.g., cetuximab), anti-VEGF (e.g., bevacizumab), or combinations thereof (e.g., anti-PD-1 and anti-CTLA-4). Combination chemotherapy is the administration of more than one agent to treat cancer. One example is the administration of GPC2 targeting CAR-expressing immune cells used in combination with radioactive, biological or chemical compounds, or combinations thereof.

[0032] Chimeric Antigen Receptor (CAR): An antigen-binding portion, such as a single domain antibody (e.g., a 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 examples, the CAR comprises an antigen-binding portion, a hinge region, a transmembrane domain, and an endodomain. The endodomain may comprise a signaling chain with an immunoreceptor tyrosine-based activation motif (ITAM), such as CD3ζ or FcεRIγ. In some examples, the endodomain further comprises at least one additional co-stimulatory domain, such as the intracellular portion of CD28, 4-1BB (CD137), ICOS, OX40 (CD134), CD27, MYD88-CD40, KIR2DS2, and / or DAP10. In some examples, the CAR is multispecific (e.g., bispecific) or bicistronic. A multispecific CAR is a single CAR molecule composed of at least two antigen binding domains (e.g., scFv and / or single domain antibodies) that each bind to a different antigen or to a different epitope on the same antigen (see, e.g., US Patent Application Publication No. 2018 / 0230225). For example, a bispecific CAR refers to a single CAR molecule with two antigen binding domains that each bind to a different antigen. A bicistronic CAR refers to two complete CAR molecules, each containing an antigen binding moiety that binds to a different antigen. In some examples, a bicistronic CAR construct expresses two complete CAR molecules linked by a cleavable linker. Immune cells (e.g., T cells, NK cells, B cells, or macrophages) or iPSCs expressing a bispecific or bicistronic CAR can bind to cells expressing both of the antigens to which the binding moieties are directed (see, e.g., Qin et al., Blood 130:810, 2017; and WO / 2018 / 213337).In some embodiments, the CAR is a two-chained antibody-T cell receptor (AbTCR) as described by Xu et al. (Cell Discovery 4:62, 2018), or a synthetic T cell receptor and antigen receptor (STAR) as described by Liu et al. (Sci Transl Med 13(586):eabb5191, 2021).

[0033] Complementarity determining region (CDR): A region of hypervariable amino acid sequence that defines the binding affinity and specificity of an antibody. The light and heavy chains of mammalian immunoglobulins each have three CDRs, called L-CDR1, L-CDR2, L-CDR3 and H-CDR1, H-CDR2, H-CDR3, respectively. Single domain antibodies contain three CDRs (CDR1, CDR2 and CDR3).

[0034] Conservative variant: In the context of this disclosure, a "conservative" amino acid substitution is a substitution that does not substantially affect or reduce the affinity of a protein, such as an antibody, to GPC2. As an example, a monoclonal antibody that specifically binds to GPC2 may contain at most about 1, at most about 2, at most about 5, at most about 10, at most about 15, at most about 20 or at most 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 a non-substituted parent amino acid, provided that the variant retains activity. A non-conservative substitution is a substitution that reduces the activity (e.g., affinity) of a protein.

[0035] Conservative amino acid substitution tables providing functionally similar amino acids are well known. The following six groups are examples of amino acids which are considered conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0036] In some aspects herein, provided are amino acid sequences that contain 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 compared to any amino acid sequence disclosed herein.

[0037] Contacting: To place into direct physical association; includes both solid and liquid forms.

[0038] Degenerate variant: A polynucleotide that encodes a polypeptide that contains a sequence that is degenerate as a result of the genetic code. There are 20 naturally occurring amino acids, most of which are specified by more than one codon. Thus, all degenerate nucleotide sequences are included as long as the amino acid sequence of the polypeptide is unchanged.

[0039] Desmoplastic small round cell tumor (DRCT): a soft tissue sarcoma that occurs predominantly in children, especially boys. DRCT is an aggressive, rare type of cancer that occurs primarily as a mass in the abdomen, but can also be found in the lymph nodes, abdominal lining, diaphragm, spleen, liver, chest wall, skull, spinal cord, intestine, bladder, brain, lungs, testes, ovaries, and pelvis.

[0040] Epitope: Antigenic determinant. These are particular chemical groups or peptide sequences on a molecule that are antigenic (elicit a specific immune response). Antibodies specifically bind to particular antigenic epitopes on a polypeptide.

[0041] Framework Region: Amino acid sequences interposed between the CDRs. Framework regions include variable light and variable heavy framework regions. Framework regions function to hold the CDRs in the proper orientation for antigen binding.

[0042] Glioma: A form of tumor that arises in the brain and spinal cord. Gliomas originate from glial cells that surround and support neurons in the brain, including astrocytes, oligodendrocytes, and ependymal cells. Based on the type of cell from which the tumor arises, there are three classes of gliomas: astrocytoma, ependymoma, and oligodendroglioma.

[0043] Glypican-2 (GPC2): A member of the six-member glypican family of heparan sulfate (HS) proteoglycans that is attached to the cell surface by 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 approximately half of neuroblastoma cases, and high GPC2 expression correlates 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 cerebroglycan proteoglycan and glypican proteoglycan 2. The genomic, mRNA and protein sequences of GPC2 are publicly available (see, e.g., NCBI Gene ID 221914).

[0044] GPC2 positive cancer: 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's sarcoma, desmoplastic small round cell tumor, glioma, and osteosarcoma.

[0045] Heterologous: originating from separate genetic sources or species.

[0046] Host cell: A cell into which a vector can be propagated and its DNA can be expressed. The cell can be prokaryotic or eukaryotic. In some examples, the prokaryotic cell is an E. coli cell. In some examples, the eukaryotic cell is a human cell, such as a human embryonic kidney (HEK) cell. This term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parent cell, since there may be mutations that occur during replication. However, when the term "host cell" is used, such progeny is included.

[0047] Immune response: A response of a cell of the immune system, e.g., a B cell, T cell, or monocyte, to a stimulus. In one embodiment, the response is specific for a particular antigen (an "antigen-specific response"). In one embodiment, the immune response is a T cell response, e.g., a CD4 + Response or CD8 + In another embodiment, the response is a B cell response, resulting in the production of specific antibodies.

[0048] Isolated: An "isolated" biological component, e.g., a nucleic acid, a protein (including an antibody), or an 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 it is present. "Isolated" nucleic acids and proteins 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 as well as chemically synthesized nucleic acids and proteins.

[0049] Label: A detectable compound or composition that is directly or indirectly conjugated 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 the incorporation of a detectable marker, such as a radiolabeled amino acid, or the attachment of a biotinyl moiety to a polypeptide that can be detected by a characteristic avidin (e.g., streptavidin that contains a fluorescent marker or an enzymatic activity that can be detected by optical or colorimetric methods). A variety of methods of 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 radionuclides (e.g., 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 labels (e.g., fluorescein isothiocyanate (FITC), rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags) or magnetic agents, e.g., gadolinium chelates. In some embodiments, the labels are attached by spacer arms of various lengths to reduce potential steric hindrance.

[0050] Linker: In some examples, a linker is a peptide in an antibody-binding fragment (e.g., an scFv fragment) that functions to indirectly link a variable heavy chain to a variable light chain. In some embodiments herein, the disclosed scFvs contain (G4S)3 linkers of different lengths that connect the VH and VL domains of the antigen-binding domain. "Linker" can also refer to a peptide that functions to link a targeting moiety, e.g., an antibody, to an effector molecule, e.g., a cytotoxin or a detectable label. The terms "conjugate," "connect," "couple," or "link" refer to making two polypeptides into one continuous polypeptide molecule, or covalently linking a radionuclide or other molecule to a polypeptide, e.g., an scFv. In certain contexts, these terms include reference to connecting a ligand, e.g., an antibody moiety, to an effector molecule. Linking can be by either chemical or recombinant means. "Chemical means" refers to a reaction between the antibody moiety and the effector molecule such that there is a covalent bond formed between the two molecules, forming one molecule.

[0051] Mammal: This term includes both human and non-human mammals. Similarly, the term "subject" includes both human and veterinary subjects, such as mice, rats, cows, cats, dogs, pigs and non-human primates.

[0052] Medulloblastoma: A fast-growing type of cancer that forms in the cerebellum. It tends to spread through the cerebrospinal fluid to the spinal cord or to other parts of the brain. It can also spread to other parts of the body, but this is rare. Medulloblastoma is most common in children and young adults. Medulloblastoma is a form of central nervous system embryonal tumor.

[0053] Neoplasm, malignancy, cancer or tumor: A neoplasm is an abnormal growth of tissue or cells resulting from excessive cell division. Neoplastic growth can give rise to 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 can invade surrounding tissues and / or metastasize are called "malignant."

[0054] Neuroblastoma: A solid tumor that arises from embryonic neural crest cells. Neuroblastomas generally arise in and around the adrenal glands, but can arise anywhere sympathetic nervous tissue is found, for example, in the abdominal, thoracic, cervical, or nervous tissue near the spine. Neuroblastomas typically arise in children younger than 5 years of age.

[0055] Operably linked: A first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence when the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, if necessary, connect two protein coding regions in the same reading frame.

[0056] Osteosarcoma: A form of cancerous tumor found in bone. Osteosarcoma is an aggressive cancer that arises from primitive transformed cells of mesenchymal origin. This type of cancer is most common in children and young adults.

[0057] Childhood cancer: Cancer occurring 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's sarcoma, desmoplastic small round cell tumor (DRCT), osteosarcoma, brain and other CNS tumors (e.g., medulloblastoma), Wilms' tumor, non-Hodgkin's lymphoma, and retinoblastoma.

[0058] Pharmaceutically acceptable carriers: The pharma- ceutically acceptable carriers used are conventional. Remington: The Science and Practice of Pharmacy, 22 nd ed., London, UK: Pharmaceutical Press, 2013, describes compositions and formulations suitable for pharmaceutical delivery of CAR-expressing cells and other compositions disclosed herein. The nature of the carrier may depend on the particular mode of administration being used. For example, parenteral formulations usually contain an injectable fluid that contains pharma- ceutical and pharmacologically acceptable fluids, such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, etc., as a vehicle. For solid compositions (e.g., powder, pill, tablet or capsule forms), conventional non-toxic solid carriers may include, for example, pharmaceutical grades of mannitol, lactose, starch or magnesium stearate. In addition to biologically neutral carriers, the pharmaceutical compositions to be administered may contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives and pH buffering agents, for example, sodium acetate or sorbitan monolaurate.

[0059] Preventing, Treating, or Ameliorating a Disease: "Preventing" a disease refers to inhibiting the full development of a disease. "Treating" refers to a therapeutic intervention that ameliorates the signs or symptoms of a disease or pathological condition after it has begun to develop, such as a reduction in tumor burden or a decrease in the number or size of metastases. "Ameliorating" refers to a reduction in the number or severity of signs or symptoms of a disease, such as cancer.

[0060] Purified: The term purified does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified peptide preparation is a preparation in which a peptide or protein is enriched more than the peptide or protein was in its natural environment in a cell. Similarly, a purified cell is a cell in which the peptide or protein is enriched more than the cell was in its natural environment in a subject, or the cell is substantially free of other cell types. In one embodiment, a preparation is purified such that the protein or peptide accounts for at least 50% of the total peptide or protein content of the preparation. Substantial purification refers to purification from other proteins or cellular components. A substantially purified protein is at least 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.9% or 99.99% pure. Thus, in one specific, non-limiting example, a substantially purified protein is at least 90% free of other proteins and cellular components. A substantially purified cell (e.g., a cell expressing a CAR provided herein) can be at least 90%, 95%, 98%, 99%, 99.9% or 99.99% pure. Thus, in one specific, non-limiting example, a substantially purified cell expressing a CAR provided herein is at least 99% free of other cells (e.g., other immune cells or other cells that do not express a CAR provided herein) or cellular components.

[0061] Recombinant: A recombinant nucleic acid is a nucleic acid having a sequence that is not found in nature or that is made by the artificial combination of two otherwise separated segments of sequence, which is often accomplished by chemical synthesis or by the artificial manipulation of isolated segments of nucleic acid, e.g., by genetic engineering techniques.

[0062] Retinoblastoma: A form of cancer that forms in the tissues of the retina. Retinoblastoma usually occurs in children younger than 5 years of age. Retinoblastoma can be hereditary or nonhereditary (sporadic).

[0063] 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 retroperitoneum and biliary tract), and perineal / anal regions. There are at least two types of RMS; the most common forms are alveolar RMS (ARMS) and embryonal histological RMS (ERMS). Approximately 20% of children with rhabdomyosarcoma have the ARMS subtype. An increased frequency of this subtype is seen in adolescents and in patients with primary sites involving the extremities, trunk, and perineal / perianal regions. ARMS is associated with a chromosomal translocation that encodes a fusion gene involving FKHR on chromosome 13 and a member of the PAX family. The embryonal subtype is the most frequently observed subtype in children, accounting for approximately 60-70% of pediatric rhabdomyosarcomas. Tumors with embryonal histology typically arise in the head and neck region or genitourinary tract, but can arise at any primary site. ERMS is characterized by younger age at diagnosis, loss of heterozygosity, and altered genomic imprinting.

[0064] Sample (or biological sample): A biological specimen containing genomic DNA, RNA (including mRNA), protein, or a combination thereof obtained from a subject. Examples include, but are not limited to, peripheral blood, tissue, cells, urine, saliva, tissue biopsy, fine needle aspirate, surgical specimen, and autopsy material. In one example, the sample includes a tumor biopsy, e.g., a tumor tissue biopsy.

[0065] Sequence identity: The similarity between amino acid or nucleic acid sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologs or variants of polypeptides or nucleic acid molecules have a relatively high degree of sequence identity when aligned using standard methods.

[0066] Methods for aligning sequences for comparison are known. Various 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, presents a detailed review of sequence alignment methods and homology calculations.

[0067] 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, for use with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. A description of how to use this program to determine sequence identity is available on the Internet at the NCBI website.

[0068] Homologs and variants of antibodies or CARs that specifically bind to GPC2 are typically characterized by the possession of at least about 75%, e.g., at least about 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity counted over the full length alignment with the amino acid sequence of the antibody or CAR using NCBI Blast 2.0, gapped blastp set to default parameters. For comparison of amino acid sequences larger than about 30 amino acids, the Blast 2 sequence function is used using the default BLOSUM62 matrix set to default parameters (gap existence cost of 11 and per residue gap cost of 1). When aligning short peptides (approximately less than 30 amino acids), the alignment should be performed using the Blast 2 sequence function using the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalty). Proteins with even higher similarity to the reference sequence will show increasing percentage identity, e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity, when assessed by this method. When less than the entire sequence is being compared for sequence identity, homologs and variants will typically have at least 80% sequence identity over a short window of 10-20 amino acids, and depending on their similarity to the reference sequence, may have at least 85% or at least 90% or 95% sequence identity. Methods for determining sequence identity over such short windows are available on the Internet at the NCBI website. These sequence identity ranges are provided for guidance only; it is entirely possible that strong and significant homologs may be obtained that fall outside the ranges provided.

[0069] Subject: Living multi-cellular vertebrate organisms, a category that includes both human and veterinary subjects, including humans and non-human mammals, e.g., pigs, mice, rats, rabbits, sheep, horses, cows, dogs, cats and non-human primates.

[0070] Synthetic: Produced by artificial means in a laboratory, for example, a synthetic nucleic acid or protein (e.g., an antibody) can be chemically synthesized in the laboratory.

[0071] Therapeutically effective amount: The amount of a particular substance that is sufficient to achieve a desired effect in a subject being treated. For example, this may be the amount required to inhibit or suppress tumor growth. In one embodiment, a therapeutically effective amount is the amount required to eliminate a tumor, reduce the size of a tumor, or prevent tumor metastasis, for example, to reduce 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 reduce the number and / or size / volume of metastases 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%, for example, compared to the size / volume / number before treatment. A dosage that achieves a target tissue concentration (e.g., in a tumor) that has been shown to achieve the desired in vitro effect when administered to a subject is generally used.

[0072] Vector: A nucleic acid molecule that is introduced into a host cell, thereby producing a transformed host cell. A vector can contain a nucleic acid sequence that allows it to replicate in a host cell, such as an origin of replication. A vector can also contain 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 viral (AAV) vector.

[0073] III. Overview of Certain Aspects CAR T-cell therapy is a new class of cancer therapeutics that is being actively developed and tested worldwide. After initial success in hematological cancers using CD19-targeting CAR T cells, various CAR strategies have been engineered and tested with the aim of treating solid tumors. However, the success of CAR T cells in solid tumors has been limited by several barriers, including a lack of tumor-specific antigens, the inability of CAR T cells to efficiently expand at the tumor site, and heterogeneous antigen expression (Kochenderfer et al., Blood. 2012;119(12):2709-2720;Porter et al., N Eng 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., Cell Rep Med 2(6):100297, 2021).

[0074] The present disclosure addresses these challenges and improves the efficacy of CAR-expressing cells for treating GPC2-positive tumors. The engineered CAR disclosed herein comprises an antigen-binding domain derived from the GPC2-specific antibody CT3 (PCT Publication No. WO 2020 / 033430, incorporated herein by reference) or a humanized version thereof. It is disclosed herein that immune cells expressing GPC2-targeting CARs containing a CD28 hinge region and a CD28 transmembrane domain are significantly more potent in killing GPC2-positive tumors than GPC2-targeting CARs containing a CD8 hinge region and a transmembrane domain of either CD8 or CD28. Furthermore, using an orthotopic neuroblastoma model, it was demonstrated that immune cells expressing a GPC2-targeting CAR with a CD28 hinge and transmembrane domain showed superior expansion against GPC2-positive tumor cells in vitro and in vivo, yielded higher levels of tumor-infiltrating CAR+ T cells, and resulted in increased survival, compared with GPC2-targeting CARs with a CD8 hinge and transmembrane domain. Furthermore, immune cells expressing a GPC2-targeting CAR with a CD28 hinge and transmembrane domain showed superior antitumor activity in a neuroblastoma model compared with existing CAR T cell therapies for neuroblastoma.

[0075] Provided herein is a CAR that comprises an extracellular antigen binding domain that specifically binds to GPC2; a CD28 hinge region; a CD28 transmembrane domain; an intracellular costimulatory domain; and an intracellular signaling domain.In some embodiments, the GPC2 specific antigen binding domain is a scFv.The scFv can have the orientation of VH-linker-VL or VL-linker-VH from N-terminus to C-terminus.

[0076] In some embodiments, the antigen-binding domain comprises a variable heavy (VH) domain and a variable light (VL) domain, wherein the VH domain comprises the complementarity determining region 1 (CDR1), CDR2 and CDR3 sequences of SEQ ID NO:2 (CT3 VH domain sequence), and / or the VL domain comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NO:4 (CT3 VL domain sequence). In some examples, the CDR sequences are defined using Kabat, IMGT or Paratome numbering schemes, or a combination of Kabat, IMGT and Paratome numbering schemes. In other examples, the CDR sequences are determined using a different numbering scheme, e.g., Chothia.

[0077] In some embodiments, the CDR1, CDR2 and CDR3 sequences of the VH domain comprise residues 31-35, 50-66 and 99-112, respectively, of SEQ ID NO:2, and / or the CDR1, CDR2 and CDR3 sequences of the VL domain comprise residues 24-33, 49-55 and 88-96, respectively, of SEQ ID NO:4; the CDR1, CDR2 and CDR3 sequences of the VH domain comprise residues 26-33, 51-58 and 97-112, respectively, of SEQ ID NO:2, and / or the CDR1, CDR2 and CDR3 sequences of the VL domain comprise residues 27-31, 49-51 and 88-96, respectively, of SEQ ID NO:4. the CDR1, CDR2 and CDR3 sequences of the VH domain comprise residues 26-35, 47-61 and 97-112, respectively, of SEQ ID NO:2, and / or the CDR1, CDR2 and CDR3 sequences of the VL domain comprise residues 27-33, 45-55 and 88-95, respectively, of SEQ ID NO:4; or the CDR1, CDR2 and CDR3 sequences of the VH domain comprise residues 26-35, 47-66 and 97-112, respectively, of SEQ ID NO:2, and / or the CDR1, CDR2 and CDR3 sequences of the VL domain comprise 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 (including the CDR1, CDR2 and CDR3 sequences 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 (including the CDR1, CDR2 and CDR3 sequences of SEQ ID NO:4).

[0078] In some embodiments, the sequences of the VH and VL domains are humanized. In some examples, the amino acid sequence of the humanized VH domain comprises residues 1-123 of SEQ ID NO:8, and / or the amino acid sequence of the humanized VL domain comprises residues 139-244 of SEQ ID NO:8; the amino acid sequence of the humanized VH domain comprises residues 1-122 of SEQ ID NO:12, and / or the amino acid sequence of the humanized VL domain comprises residues 138-243 of SEQ ID NO:12; the amino acid sequence of the humanized VH domain comprises residues 1-122 of SEQ ID NO:16, and / or the amino acid sequence of the humanized VL domain comprises residues 138-244 of SEQ ID NO:16; or the amino acid sequence of the humanized VH domain comprises residues 1-122 of SEQ ID NO:20, and / or the amino acid sequence of the humanized VL domain comprises residues 138-243 of SEQ ID NO:20.

[0079] In some embodiments, 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.

[0080] In some embodiments, the amino acid sequence of the CD28 hinge region 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 CD28 hinge region comprises or consists of the amino acid sequence of SEQ ID NO:24.

[0081] In some embodiments, 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.

[0082] In some embodiments, the costimulatory domain comprises a 4-1BB signaling moiety. In some examples, the amino acid sequence of the 4-1BB signaling moiety 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 specific examples, the amino acid sequence of the 4-1BB signaling moiety comprises or consists of the amino acid sequence of SEQ ID NO: 28.

[0083] In some embodiments, 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 specific examples, the amino acid sequence of the CD3 zeta signaling domain comprises or consists of SEQ ID NO: 30.

[0084] In certain embodiments, the amino acid sequence of the 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 specific examples, the amino acid sequence of the CAR comprises or consists of the amino acid sequence of SEQ ID NO:38.

[0085] In alternative embodiments, the amino acid sequence of the 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 specific examples, the amino acid sequence of the CAR comprises or consists of the amino acid sequence of SEQ ID NO: 39 or SEQ ID NO: 40.

[0086] Further provided herein is a nucleic acid molecule encoding a CAR disclosed herein. In some embodiments, 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 comprises or consists of nucleotides 73-1470 of SEQ ID NO: 37. In a specific non-limiting example, the sequence of the nucleic acid molecule comprises or consists of SEQ ID NO: 37.

[0087] In some embodiments, the nucleic acid molecule is operably linked to a promoter (e.g., an inducible or constitutive promoter). In some examples, the promoter is the human elongation factor 1 alpha (EF1α) promoter.

[0088] In some embodiments, the nucleic acid molecule comprises, 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 costimulatory domain; a nucleic acid encoding a signal transduction 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 comprises a human elongation factor 1 alpha (EF1 alpha) promoter sequence 5' to the nucleic acid encoding the first GMCSFRss (see WO 2019 / 094482, the entire contents of which are incorporated herein by reference).

[0089] Further provided is a vector comprising the nucleic acid molecule 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.

[0090] Also provided is an isolated cell that comprises the nucleic acid molecule (or vector) encoding the CAR disclosed herein and / or expresses the CAR disclosed herein.In some embodiments, the cell is an immune cell, such as a T cell, a NK cell, a B cell, or a macrophage.In other embodiments, the cell is an induced pluripotent stem cell (iPSC).

[0091] Further provided is a composition comprising a CAR, a nucleic acid molecule, a vector or a cell disclosed herein, and a pharmaceutically acceptable carrier (e.g., water or saline). In some cases, the composition is frozen. In some cases, the composition is frozen and comprises cells and DMSO or another cryoprotectant. In some cases, the composition is lyophilized. In some cases, such compositions are in a vial, e.g., a glass or plastic vial. The disclosed compositions can be part of a kit, e.g., a kit comprising one or more chemotherapeutic agents, a syringe, a cell culture medium, a pharmaceutically acceptable carrier or a combination thereof (additional agents in the kit can be in separate containers).

[0092] Also provided are methods of treating GPC2-positive cancer in a subject or inhibiting tumor growth or metastasis of GPC2-positive cancer. In some embodiments, these methods comprise administering to the subject a therapeutically effective amount of CAR, nucleic acid molecule, vector, cell or composition disclosed herein. In some examples, the GPC2-positive cancer is a solid tumor. In some examples, the GPC2-positive cancer is a childhood cancer. In certain non-limiting examples, the GPC2-positive cancer is neuroblastoma, medulloblastoma, retinoblastoma, acute lymphoblastic leukemia, embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, Ewing's sarcoma, desmoplastic small round cell tumor, glioma or osteosarcoma. In a specific example, the GPC2-positive cancer is neuroblastoma. In some examples, these methods further comprise administering to the subject a conditioning chemotherapy, for example, fludarabine and cyclophosphamide.

[0093] IV. GPC2-specific antibody sequences The CAR disclosed herein comprises an antibody (or an antigen-binding fragment thereof) that specifically binds to GPC2. In some embodiments, the antibody is a CT3 mouse monoclonal antibody, or a humanized version thereof (e.g., hCT3-1, hCT3-2, hCT3-3, or hCT3-4) in scFv format. These antibodies are described in PCT Publication No. WO 2020 / 033430, which is incorporated herein by reference in its entirety. The nucleotide and amino acid sequences of CT3 are provided below. Tables 1 and 2 list the amino acid positions of CDR1, CDR2, and CDR3 of the VH and VL domains, respectively, as determined using Kabat, IMGT, Paratome, and combinations thereof. The CDR boundaries can also be defined using alternative numbering schemes, such as the Chothia numbering scheme. The scFv nucleotide and amino acid sequences of the parent CT3 antibody, as well as four of its humanized versions, are also listed below. In each scFv sequence, the VH and VL domains are separated by a (G4S)3 linker, shown in bold. For humanized antibodies, scFv sequences in VH-linker-VL and VL-linker-VH orientations are provided. [ka] [Table 1] [ka] [Table 2] [ka] [ka] [ka] [ka]

[0094] V. GPC2-targeting CAR sequence CT3 scFv and the scFv of humanized versions of CT3 (hCT3-1, hCT3-2, hCT3-3 and hCT3-4) were used to generate CAR constructs that specifically target GPC2-expressing cells. As disclosed herein, GPC2-targeting CAR constructs with CD28 hinge region and CD28 transmembrane domain were superior to CAR constructs with CD8 hinge region paired with either CD8 transmembrane domain or CD28 transmembrane domain. The nucleotide and amino acid sequences of the CAR components, as well as the complete amino acid sequences of three specific CAR constructs (CT3.28H.BBz, CT3.8H.BBz and CT3.8H.28BBz) are provided below. [ka] [ka] [ka] [ka] [ka]

[0095] VI. Chimeric Antigen Receptors (CARs) Disclosed herein are GPC2-specific CARs and cells engineered to express the CARs (e.g., T cells, NK cells, B cells, macrophages and iPSCs). In general, CARs include a binding moiety, an extracellular hinge / spacer element, a transmembrane region, and an intracellular domain that performs signaling functions (Cartellieri et al., J Biomed Biotechnol 2010:956304, 2010; Dai et al., J Natl Cancer Inst 108(7):djv439, 2016). In many examples, the binding moiety is an antigen-binding fragment of a monoclonal antibody, e.g., an scFv or single-domain antibody. The spacer / hinge region typically includes sequences from IgG subclasses, e.g., IgG1, IgG4, IgD, and CD8 domains. In some embodiments herein, the hinge region is derived from human CD28. In a specific example, the amino acid sequence of the hinge region includes (or consists of) SEQ ID NO:24. The transmembrane (TM) domain can be derived from a variety of different T cell proteins, such as CD3ζ, CD4, CD8, CD28, or inducible T cell costimulator (ICOS). In some embodiments herein, the TM domain is derived from human CD28. In a specific example, the amino acid sequence of the TM domain comprises (or consists of) SEQ ID NO: 26. Several different endodomains have been used to generate CARs. For example, the endodomain can comprise a signaling chain with ITAM, such as CD3ζ or FcεRIγ. In some examples, the endodomain further comprises at least one additional costimulatory domain, such as the intracellular portion of CD28, 4-1BB (CD137, TNFRSF9), OX-40 (CD134), ICOS, CD27, MYD88-CD40, killer cell immunoglobulin-like receptor 2DS2 (KIR2DS2), and / or DAP10.

[0096] CAR may also include a signal peptide sequence, for example, N-terminal to the antigen binding domain. The signal peptide sequence may be any suitable signal peptide sequence, for example, a signal sequence derived from granulocyte-macrophage colony-stimulating factor receptor (GMCSFR), immunoglobulin light chain kappa or IL-2. The signal peptide sequence may facilitate the expression of the CAR on the surface of a cell, but the presence of the signal peptide sequence in the expressed CAR is not necessary for the CAR to function. Upon expression of the CAR on the surface of a cell, the signal peptide sequence may be cleaved from the CAR. Thus, in some embodiments, the CAR lacks a signal peptide sequence.

[0097] In some embodiments, the CAR disclosed herein is expressed from a construct (e.g., from a lentiviral vector) that also expresses a truncated version of human EGFR (hEGFRt; discussed in more detail in Section VII below). The CAR and hEGFRt are separated by a self-cleaving peptide sequence (e.g., T2A) such that upon expression in transduced cells, the CAR is cleaved from the hEGFRt (see WO 2019 / 094482, which is incorporated herein by reference in its entirety).

[0098] In some embodiments disclosed herein, the CAR construct encodes, from N-terminus to C-terminus, the following amino acid sequence: [ka]

[0099] Immune cells (e.g., T cells, NK cells, B cells, or macrophages) or iPSCs expressing the CARs disclosed herein can be used to target specific cell types, such as tumor cells, e.g., GPC2-positive tumor cells. The use of immune cells (e.g., T cells) expressing CARs is more versatile than standard CTL-based immunotherapy, because immune cells expressing CARs are not HLA-restricted and therefore can be used in any patient with a tumor expressing the target antigen.

[0100] Thus, the present specification provides CAR that comprises GPC2-specific antibody (or its binding fragment).Also provided are isolated nucleic acid molecules and vectors that code CAR, and host cells that express CAR, such as T cells, NK cells, B cells, macrophages or iPSCs.Cells that express CAR that are composed of GPC2-specific monoclonal antibody can be used to treat cancers that express GPC2, such as neuroblastoma, medulloblastoma, retinoblastoma, acute lymphoblastic leukemia, embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, Ewing's sarcoma, desmoplastic small round cell tumor, glioma or osteosarcoma.

[0101] VII. Truncated human EGFR (hEGFRt) Human epidermal growth factor receptor is composed of four extracellular domains, one transmembrane domain and three intracellular domains. The EGFR domains are found in the following order from N-terminus to C-terminus: domain I-domain II-domain III-domain IV-transmembrane (TM) domain-juxtamembrane 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 disulfide bonds with domain II. The EGFR TM domain spans the cell membrane once and may play a role in protein dimerization. The intracellular domain contains the juxtamembrane domain, the tyrosine kinase domain and the 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).

[0102] The truncated version of human EGFR, referred to herein as "hEGFRt", contains only domain III, domain IV and TM domain.Thus, hEGFRt lacks domain I, domain II and all three intracellular domains.hEGFRt is unable to bind to EGF and lacks signal transduction activity.However, this molecule retains the ability to bind to certain EGFR-specific monoclonal antibodies, such as FDA-approved cetuximab (PCT Publication No. WO 2011 / 056894).

[0103] Transduction of immune cells (e.g., T cells, NK cells, B cells, or macrophages) or iPSCs with a construct (e.g., lentiviral vector) encoding both hEGFRt and the GPC2-specific CAR disclosed herein allows for the selection of transduced cells using labeled EGFR monoclonal antibody cetuximab (ERBITUX™). For example, cetuximab can be labeled with biotin, and transduced cells can be selected using commercially available anti-biotin magnetic beads (e.g., from Miltenyi Biotec). Co-expression of hEGFRt also allows for in vivo tracking of adoptively transferred CAR-expressing cells. Furthermore, binding of cetuximab to cells expressing hEGFRt induces cytotoxicity of ADCC effector cells, thereby providing a mechanism to eliminate transduced immune cells or iPSCs in vivo, for example, at the conclusion of treatment (Wang et al., Blood 118(5):1255-1263, 2011).

[0104] In some embodiments herein, 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 embodiments, the amino acid sequence of hEGFRt comprises 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, or 1 or less amino acid substitutions compared to SEQ ID NO: 36. In some examples, the amino acid substitutions are conservative substitutions.

[0105] VIII. CAR-Expressing Cell Compositions and Administration Thereof Compositions are provided that include CAR-expressing cells in combination with one or more pharma- ceutical or pharmacologically acceptable carriers, diluents, or excipients. CAR-expressing cells include iPSCs, T cells, e.g., CD3 + T cells, e.g., CD4 + and / or CD8 +The cell-containing composition may be a T cell, a NK cell, a B cell, a macrophage, or any other suitable immune cell. Such a composition may include a buffer, such as neutral buffered saline, phosphate buffered saline, etc.; a carbohydrate, such as glucose, mannose, sucrose, dextran, or mannitol; a protein; a polypeptide or an amino acid, such as glycine; an antioxidant; a chelating agent, such as EDTA or glutathione; an adjuvant (e.g., aluminum hydroxide); and a preservative. In some examples, the cell-containing composition includes a cryoprotectant, such as DMSO or glycerol. In some examples, the cell-containing composition includes a culture medium, such as DMEM or RPMI, and may further include serum, such as FBS. In some examples, the cell-containing composition is frozen or in liquid form. The cells may be autologous to the recipient. However, the cells may also be xenogeneic (allogeneic).

[0106] For cells, various aqueous carriers can be used to introduce cells, such as buffered saline. These solutions are sterile and generally free of undesirable matter. These compositions can be sterilized by conventional sterilization techniques. The compositions can contain pharma- ceutically acceptable auxiliary substances required to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, etc., such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentrations in these formulations can vary widely and are selected primarily based on fluid volumes, viscosities, body weight, etc., according to the particular mode of administration selected and the needs of the subject.

[0107] The exact amount of the composition to be administered can be determined by the physician, taking into account individual differences in the age, weight, tumor size / burden, extent of metastasis, and condition of the patient (subject). The pharmaceutical compositions comprising the CAR-expressing immune cells (T cells, B cells, macrophages and / or NK cells) or iPSCs described herein are administered in amounts of 10 to 200 mg / kg, including all integer values ​​within those ranges. 4 ~10 9 Cells / kg body weight, e.g., 10 5 ~10 6It can generally be stated that the cells may be administered in a dosage of 100 mg / kg body weight. Exemplary doses are 10 mg / kg body weight. 6 cells / kg~about 10 8 Cells / kg, e.g., about 5 x 10 6 cells / kg ~ approx. 7.5×10 7 Cells / kg, e.g., about 2.5 x 10 7 cells / kg, or approximately 5.0 x 10 7 cells / kg.

[0108] The composition can be administered at these dosages once or multiple times, for example, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times. 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 or once a month. In some non-limiting examples, the composition is formulated for intravenous administration and administered multiple times. The amount and frequency of administration can be determined by factors such as the condition of the subject and the type and severity of the subject's disease, but the appropriate dosage can be determined by clinical trials.

[0109] In some embodiments, a nucleic acid molecule encoding a CAR is introduced into a cell, e.g., a T cell, a NK cell, a B cell, a macrophage, or an iPSC, and the subject receives an initial administration of the cells and one or more subsequent administrations of the cells, the one or more subsequent administrations being administered less than 15 days after the previous administration, e.g., 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days after the previous administration. In one embodiment, the subject is administered more than one administration of a CAR-expressing cell per week, e.g., 2, 3, or 4 administrations of a CAR-expressing cell of the present disclosure per week. In one embodiment, the subject is administered more than one administration of a CAR-expressing cell per week (e.g., 2, 3, or 4 administrations per week) (also referred to as a cycle), followed by a week without a CAR-expressing cell administration, and then the subject is administered one or more additional administrations of a CAR-expressing cell (e.g., more than one administration of a CAR-expressing cell per week). In another embodiment, a subject (e.g., a human subject) receives more than one cycle of CAR-expressing cells, with the time between each cycle being less than 10, 9, 8, 7, 6, 5, 4, or 3 days. In one embodiment, the CAR-expressing cells are administered every other day, three times per week. In another embodiment, the CAR-expressing cells are administered for at least 2, 3, 4, 5, 6, 7, 8 weeks or longer. The dosage of the above treatment administered to a patient varies depending on the exact nature of the condition being treated and the recipient of the treatment. Scaling of dosages for human administration can be performed according to accepted practices.

[0110] In some embodiments, CAR-expressing cells can replicate in vivo, resulting in long-term persistence that can lead to sustained tumor control.In various embodiments, the iPSCs, T cells, macrophages, B cells or NK cells administered to a subject, or the progeny of these cells, persist in the subject for at least 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, or years after administration of the cells to the subject.In other embodiments, the cells and their progeny persist for 6 months, 5 months, 4 months, 3 months, 2 months, or less than 1 month, for example, 3 weeks, 2 weeks, 1 week, after administration of the CAR-expressing cells to the subject.

[0111] Administration of the disclosed compositions can be performed in any convenient manner, including by injection, ingestion, infusion, implantation or transplantation. The disclosed compositions can be administered to a patient intraarterially, subcutaneously, intradermally, intratumorally, intranodal, intramedullary, intracerebral, intraventricular, intracranial, intramuscularly, intraarterially (including into the hepatic artery (e.g., HAI) or femoral artery), by intravenous (iv) injection, intraprostatically (e.g., for prostate cancer), intraosseously, intravitreally or intraperitoneally. In some embodiments, the compositions are administered to a patient by intradermal or subcutaneous injection. In other embodiments, the compositions of the present disclosure are administered by iv injection. In other embodiments, the compositions of the present disclosure are administered by intraarterial injection. The compositions can also be directly injected into a tumor or lymph node. In one example, administration is intraosseous and the cancer being treated is a bone cancer (e.g., osteosarcoma). In one example, administration is intracerebral, intraventricular, or intracranial, and the cancer being treated is a brain cancer (e.g., neuroblastoma or medulloblastoma). In one example, administration is intravitreal, and the cancer being treated is an eye cancer (e.g., retinoblastoma).

[0112] In some embodiments, subjects can undergo leukapheresis, in which leukocytes are collected, enriched or depleted ex vivo to select and / or isolate cells of interest, e.g., T cells, B cells, macrophages and / or NK cells. These cell isolates can be expanded by known methods and treated so that one or more CAR constructs can be introduced, thereby creating autologous cells expressing the CAR. In some embodiments herein, CAR-expressing cells are generated using lentiviral vectors expressing CAR and a truncated form of human EGFR (hEGFRt). Co-expression of hEGFRt allows for the selection and purification of CAR-expressing immune cells using an antibody that recognizes hEGFRt (e.g., cetuximab, see PCT Publication No. WO 2011 / 056894), as described above in Section V.

[0113] In some embodiments, immune cells (e.g., T cells, NK cells, B cells and / or macrophages) are isolated from peripheral blood by lysing red blood cells and, in some cases, depleting monocytes, for example, by centrifugation through a PERCOLL™ gradient or by counterflow centrifugal elutriation. Specific subpopulations of T cells, for example, 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 incubation with anti-CD3 / anti-CD28 (e.g., 3×28) conjugated beads, for example, DYNABEADS® M-450 CD3 / CD28 T, for a period sufficient for positive selection of the desired T cells. See US Patent Publication No. 20140271635. In a non-limiting example, the 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 further 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. In any situation where there are fewer T cells compared to other cell types, such as isolation from an immunocompromised individual, longer incubation times can be used to isolate T cells. Furthermore, the use of longer incubation times can increase the efficiency of capture of CD8+ T cells. Thus, by simply shortening and lengthening the time, T cells can be allowed to bind to the CD3 / CD28 beads, and / or by increasing or decreasing the ratio of beads to T cells, subpopulations of T cells can be preferentially selected for or against at the beginning of the culture or at other times during the process. Furthermore, by increasing or decreasing the ratio of anti-CD3 and / or anti-CD28 antibodies on the beads or other surface, subpopulations of T cells can be selected for or against at the initiation of culture or at other desired time points. Multiple rounds of selection can also be used.

[0114] Enrichment of cell populations by negative selection can be achieved using a combination of antibodies against surface markers unique to the negatively selected cells. One method is cell sorting and / or selection via negative magnetic immunoadherence or flow cytometry using a cocktail of monoclonal antibodies against cell surface markers present on the negatively selected cells. For example, to enrich for CD4+ T cells by negative selection, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR and CD8. T cell populations expressing one or more cytokines can be selected. Methods for screening for cell expression are disclosed in PCT Publication No. WO 2013 / 126712.

[0115] For the isolation of a desired population of cells by positive or negative selection, the concentration of cells and surfaces (e.g., particles, e.g., beads) can be varied to ensure maximum contact between cells and beads. In some embodiments, a concentration of 1 billion cells / ml is used. In further embodiments, a concentration of more than 100 million cells / ml is used. In other embodiments, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 million cells / ml is used. Without being bound by theory, using a high concentration can result in increased cell yield, cell activation and cell expansion. Lower concentrations of cells can also be used. Without being bound by theory, by greatly diluting the mixture of T cells and surfaces (e.g., particles, e.g., beads), the interaction between the particles and the cells is minimized. This selects for cells that express high amounts of the desired antigen that is bound to the particles. For example, CD4+ T cells express higher levels of CD28 and are captured more efficiently than CD8+ T cells at lower concentrations. In some embodiments, the concentration of cells used is 5×10 6 In other embodiments, the concentration used is about 1×10 5 / ml~1×10 6 / ml, and any integer value therebetween.

[0116] IX. Treatment Methods Provided herein is a method for treating GPC2-positive cancer in a subject by administering to the subject a therapeutically effective amount of GPC2-targeting CAR-expressing immune cells (e.g., T cells, NK cells, B cells or macrophages) or CAR-expressing iPSCs disclosed herein.Also provided herein is a method for inhibiting tumor growth or metastasis in a subject by administering to the subject a therapeutically effective amount of GPC2-targeting CAR-expressing cells disclosed herein.Thus, in some examples, these methods reduce the size, volume and / or weight of 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%, for example, compared to the size, volume and / or weight of tumor before treatment. In some cases, these 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, compared to the size, volume and / or weight of metastases before treatment. In some cases, these 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, compared to the survival time in the absence of the treatment provided herein. In some cases, a combination of these effects is achieved.

[0117] Specifically provided is a method for treating GPC2-positive cancer in a subject.In some embodiments, this method comprises administering to a subject a therapeutically effective amount of isolated immune cells or iPSCs that comprise a nucleic acid molecule encoding GPC2 targeting CAR and hEGFRt, or administering to a subject a therapeutically effective amount of isolated immune cells or iPSCs that co-express GPC2 targeting CAR and hEGFRt.In some embodiments, the GPC2-positive cancer is a solid tumor.In a specific example, the GPC2-positive cancer is neuroblastoma, medulloblastoma, retinoblastoma, acute lymphoblastic leukemia, embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, Ewing's sarcoma, desmoplastic small round cell tumor, glioma or osteosarcoma.In some embodiments, the GPC2-positive cancer is a childhood cancer.

[0118] In some embodiments of the method disclosed herein, the isolated immune cell is a T lymphocyte.In some examples, the T lymphocyte is an autologous T lymphocyte.In other embodiments, the isolated host cell is a NK cell, a B cell or a macrophage.

[0119] The therapeutically effective amount of CAR-expressing immune cells or iPSCs may depend on the severity of the disease, the type of disease, and the general state of the patient's health. The therapeutically effective amount of CAR-expressing cells and compositions thereof is that amount that provides either a subjective relief of symptom(s) or an objectively identifiable improvement (e.g., a reduction in tumor volume or metastasis) noted by a clinician or other qualified observer.

[0120] Administration of the CAR-expressing cells and compositions disclosed herein may also be accompanied by administration of other anti-cancer agents or therapeutic treatments (e.g., surgical removal of tumors). Any suitable anti-cancer agent may be administered in combination with the compositions disclosed herein. Exemplary anti-cancer agents include, but are not limited to, chemotherapeutic agents, such as mitotic inhibitors, alkylating agents, antimetabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, anti-survival agents, biological response modifiers, anti-hormonal agents (e.g., anti-androgens), and anti-angiogenic agents. Other anti-cancer treatments include radiation therapy and antibodies (e.g., mAbs) that specifically target cancer cells or other cells (e.g., anti-PD-1, anti-CLTA4, anti-EGFR, or anti-VEGF). In one example, the cancer is treated by administering a GPC2-targeting CAR immune cell (e.g., iPSC, T cell, NK cell, B cell or macrophage) disclosed herein and one or more therapeutic mAbs, such as one or more of a PD-L1 antibody (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, the cancer is treated with a GPC2-targeting CAR-expressing cell (e.g., iPSC, T cell, NK cell, B cell, or macrophage) 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, or any combination thereof.mertansine, Blinatumomab, Brentuximab vedotin, Cantuzumab mertansine, Capromab pendetide, Catumaxomab, CC49, Cetuximab, Citatuzumab bogatox, Cixutumumab, Clivatuzumab tetraxetan Tetraxetan, Conatumumab, Dacetuzumab, Detumomab, Ecromeximab, Eculizumab, Edrecolomab, Epratuzumab, Ertumaxomab, Etaracizumab, Farletuzumab, Figitumumab, Galiximab, Gemtuzumab Ozogamicin, Direntuximab, Glembatumumab Vedotin vedotin, Ibritumomab Tiuxetan, Igovomab, Imciromab, Intetumumab, Inotuzumab Ozogamicin, Ipilimumab, Iratumumab, Labetuzumab, Lexatumumab, Lintuzumab, Lorvotuzumab Mertansine, Lucatumumab, Rumiliximab, Mapatuzumab, Matuzumab, Mepolizumab, Metelimumab, Milatuzumab, Mitumomab, Morolimumab, Nacolomab Tafenatox tafenatox, naptumomab estafenatox, necitumumab, nimotuzumab, nofetumomab merpentan, ofatumumab, olaratumab, oportuzumab monatox, oregovomab, panitumumab, pemtumomab, pertuzumab, pintumomab, pritumumab, ramucirumab, rilotumumab, rituximab, robatumumab, satumomab pendetidependetide, Sibrotuzumab, Sonepcizumab, Tacatuzumab tetraxetan, Taplitumomab paptox, Tenatumomab, TGN1412, Ticilimumab (Tremelimumab), Tigatuzumab, TNX-650, Trastuzumab, Tremelimumab, Tucotuzumab celmoleukin, Veltuzumab, Volociximab, Votumumab, Zalutumumab, or a combination thereof.

[0121] In one example, cancer is treated by administering a GPC2 targeting CAR expressing cell (e.g., iPSC, T cell, NK cell, B cell or macrophage) disclosed herein and one or more alkylating agents, such as nitrogen mustard (e.g., mechlorethamine, cyclophosphamide, melphalan, uracil mustard or chlorambucil), alkyl sulfonate (e.g., busulfan), nitrosourea (e.g., carmustine, lomustine, semustine, streptozocin or dacarbazine). In one example, cancer is treated by administering a GPC2 targeting CAR expressing cell (e.g., iPSC, T cell, NK cell, B cell or macrophage) disclosed herein and cyclophosphamide.

[0122] In one example, cancer is treated by administering a GPC3-targeting CAR-expressing cell (e.g., an iPSC, a T cell, a NK cell, a B cell, or a macrophage) disclosed herein and one or more antimetabolites, such as a folate analog (e.g., methotrexate), a pyrimidine analog (e.g., 5-FU or cytarabine), and a purine analog, such as mercaptopurine or thioguanine.

[0123] In one example, cancer is treated by administering a GPC2-targeting CAR-expressing cell (e.g., iPSC, T cell, NK cell, B cell, or macrophage) disclosed herein and one or more natural products including, for example, a vinca alkaloid (e.g., vinblastine, vincristine, or vindesine), an epipodophyllotoxin (e.g., etoposide or teniposide), an antibiotic (e.g., dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, or mitomycin C), and an enzyme (e.g., L-asparaginase).

[0124] In one example, cancer is treated by administering a GPC2-targeting CAR-expressing cell disclosed herein (e.g., iPSC, T cell, NK cell, B cell or macrophage) and one or more platinum coordination complexes (e.g., cis-diamine-dichloroplatinum II, also known as cisplatin), substituted ureas (e.g., hydroxyurea), methylhydrazine derivatives (e.g., procarbazine), and adrenal cortical suppressants (e.g., mitotane and aminoglutethimide).

[0125] In one example, cancer is treated by administering a GPC2-targeting CAR-expressing cell disclosed herein (e.g., iPSC, T cell, NK cell, B cell, or macrophage) and one or more hormones or antagonists, such as corticosteroids (e.g., prednisone), progestins (e.g., hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrol acetate), estrogens (e.g., diethylstilbestrol and ethinyl estradiol), antiestrogens (e.g., tamoxifen), and androgens (e.g., testerone proprionate and fluoxymesterone).

[0126] In one example, the cancer is treated with a GPC2-targeting CAR-expressing cell (e.g., iPSC, T cell, NK cell, B cell, or macrophage) disclosed herein and one or more chemotherapy drugs, such as Adriamycin, Alkeran, Ara-C, BiCNU, Busulfan, CCNU, Carboplatinum, Cisplatinum, Cytoxan, Daunorubicin, DTIC, 5-FU, Fludarabine, Hydrea, Idarubicin, Ifosfamide, Methotrexate, Mithramycin. , mitomycin, mitoxantrone, nitrogen mustard, taxol (or other taxanes, e.g., docetaxel), velban, vincristine, VP-16, gemcitabine (Gemzar), herceptin, irinotecan (Camptosar, CPT-11), leustatin, navelbine, rituxan STI-571, taxotere, topotecan (hycamtin), xeloda (capecitabine), zevelin, and calcitriol. In one example, cancer is treated by administering a GPC2 targeting CAR expressing cell (e.g., iPSC, T cell, NK cell, B cell, or macrophage) disclosed herein, cyclophosphamide, and fludarabine. In one example, the cancer is treated by administering the GPC2 targeting CAR expressing cells (e.g., iPSCs, T cells, NK cells, B cells or macrophages) disclosed herein and one or more immunomodulatory agents, such as AS-101 (Wyeth-Ayerst Labs.), bropirimine (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 a surgical treatment of the cancer or a portion thereof, such as surgical resection.Another example of a treatment is radiation therapy, eg, the administration of radioactive material or energy (eg, external beam radiation therapy) to a tumor site to eradicate the tumor or to help shrink the tumor prior to surgical removal.

[0127] In a specific example, the method includes (1) administering to the subject a therapeutically effective amount of isolated immune cells or iPSCs comprising a nucleic acid molecule encoding a GPC2 targeting CAR and hEGFRt, or administering a therapeutically effective amount of isolated immune cells or iPSCs co-expressing a GPC2 targeting CAR and hEGFRt, thereby treating neuroblastoma. In some examples, the method further includes administering to the subject a therapeutically effective amount of one or more other chemotherapeutic or biological agents. In some aspects, the one or more other chemotherapeutic or biological agents are one or more of 5-FU, cisplatin, gemcitabine, oxaliplatin, doxorubicin, capecitabine, floxuridine, or mitoxantrone, such as gemcitabine + oxaliplatin (GEMOS), floxuridine, cisplatin and oxaliplatin, or 5-FU, oxaliplatin and leucovorin (FOLFOX). In some embodiments, the one or more other chemotherapeutic or biological agents are one or more of sorafenib, lenvatinib, regorafenib, cabozantinib and ramucirumab.In some embodiments, the one or more other chemotherapeutic or biological agents are immunotherapy drugs, such as pembrolizumab and / or nivolumab.In some embodiments, the one or more other chemotherapeutic or biological agents are cyclophosphamide, fludarabine, or both. EXAMPLES

[0128] The following examples are provided to illustrate particular features of certain embodiments of the present disclosure, but the claims should not be limited to the exemplified features.

[0129] Example 1 In vitro cell killing by GPC2-targeting CAR T cells Cell killing mediated by T cells expressing GPC2-targeting CT3.8H.BBz (also referred to herein as CT3.8H.BBζ) and CT3.28H.BBz (also referred to herein as CT3.28H.BBζ) CARs (see Figures 1A-1B) was evaluated using GPC2-positive IMR5 cells and GPC2 knockout (KO) IMR5 cells as target cells. As shown in Figures 2A-2B, CT3.28H.BBz CAR T cells were more potent in killing IMR5 cells than CT3.8H.BBz CAR T cells. Both types of CAR T cells had minimal effects on GPC2 KO-IMR5 cells, demonstrating their GPC2 specificity.

[0130] Example 2 Comparison of CD8-hinge-containing GPC2-targeting CAR and CD28-hinge-containing GPC2-targeting CAR in an animal model of neuroblastoma metastasis CT3.8H.BBz and CT3.28H.BBz CAR T cells were compared in a mouse model of neuroblastoma (IMR5) metastasis. Mice were inoculated iv with IMR5-luc 28 days prior to infusion of 10 million CAR T cells and imaged weekly after infusion (Figure 3A). Bioluminescence images of mock and CAR T cell treated mice taken weekly up to 8 weeks after T cell infusion are shown in Figure 3B. Bioluminescence was measured 2, 4, 6 and 8 weeks after CAR T cell infusion and the results are shown in Figure 3C. Survival of mock and CAR T cell treated mice after CAR T cell infusion is shown in Figure 3D. The results demonstrated that CT3.28H.BBz CAR T cells were significantly more potent than CT3.8H.BBz CAR T cells in regressing neuroblastoma tumors in mice. All mice in the CT3.28H.BBz treatment group survived to the end of the study.

[0131] Example 3 Comparison of CT3.28H.BBz and CT3.8H.28BBz CAR T cells in the orthotopic IMR5 mouse model T cells from three different human donors (A26M, A59F and A25F) were used in this study. Mice with moderate tumor burden were administered 5 million human T cells expressing CT3.28H.BBz CAR or CT3.8H.28BBz CAR, and bioluminescence imaging was performed weekly for 4 (A26M and A59F) or 8 (A25F) weeks. As shown in Figures 4A-4C, CT3.28H.BBz CAR T cells outperformed CT3.8H.28BBz CAR T cells in reducing tumors for all three T cell donors. Bioluminescence images of mock-treated mice and mice treated with CT3.28H.BBz or CT3.8H.28BBz CAR T cells derived from donors A26M and A59F are shown in Figures 4D and 4E, respectively. CT3.28H.BBz CAR T cells were more potent than CT3.8H.28BBz CAR T cells in eradicating medium-sized IMR5 tumors.

[0132] Flow cytometry of dissociated spleen samples was performed to evaluate the maintenance of CAR expression in CAR T cell-treated animals. Live cells were gated on CD3+ human cells to determine the percentage of CAR-positive cells (Figure 4F). As shown in Figure 4G, CT3.28H.BBz CAR T cells maintained higher levels of CAR expression than CT3.8H.28BBz CAR T cells for both donors tested (A26M and A59F).

[0133] Example 4 CAR tonic signaling CAR phosphorylation was evaluated as a measure of CAR activation. CT3.8H.BBz, CT3.8H.28BBz and CT3.28H.BBz CAR T cells were unstimulated or stimulated with protein L or GPC2-Fc, and CAR phosphorylation was detected by Western blot (Figure 5A). The fold change in CAR phosphorylation is shown in Figure 5B. CT3.8H.28BBz CAR had a higher phosphorylation level than CT3.28H.BBz CAR when tested without stimulation. With GPC2 stimulation, both CARs upregulated their phosphorylation levels. These results demonstrate that CT3.8H.28BBz has more robust CAR signaling (phosphorylation), which is known to cause CAR exhaustion. CT3.28H.BBz CAR T cells have less robust CAR signaling, but show proper CAR activation upon antigen presentation (GPC2-Fc).

[0134] Example 5 Comparison of CT3.28H.BBz CAR T cells and CT3.8H.28BBz CAR T cells with low or high dose chemotherapy in an orthotopic IMR5 animal model Mice with large IMR5 tumor burdens were treated with no chemotherapy, low-dose chemotherapy, or high-dose chemotherapy (Fludarabine / Cyclophosphamide) for one week prior to infusion of 5 million CT3.28H.BBz or CT3.8H.28BBz CAR T cells. In this study, T cells were from a single donor. Tumor size measured by bioluminescence is shown in Figure 6A. Tumor weights 10 weeks after chemotherapy and CAR T cell infusion are shown in Figure 6B. These results demonstrate that CT3.28H.BBz performed better than CT3.8H.28BBz when given conditioning chemotherapy in high tumor burden mice.

[0135] Example 6 Characterization of humanized CT3 antibody and CAR Four humanized forms 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. As shown in Figures 7A-7B, the binding affinities of the four humanized CT3 antibodies (4.0 nM, 3.6 nM, 2.5 nM and 3.3 nM) were similar to that of the parent CT3 antibody (2.2 nM). Furthermore, all four humanized antibodies retained the ability to bind to GPC2 on the cell surface of G10 and IMR5 cells, but showed no binding to GPC2-KO IMR5 cells (Figure 8).

[0136] Cell killing by humanized CT3.8H.BBz CAR T cells was tested. The results demonstrated specific lysis of GPC2-expressing IMR5 cells by CT3-8H-BBz, hCT3-1-8H-BBz, hCT3-2-8H-BBz, hCT3-3-8H-BBz and hCT3-4-8H-BBz CAR T cells (Figure 9). All four humanized CT3-8H-BBz CAR T cells showed improved killing activity against IMR5 cells compared to CT3-8H-BBz CAR T cells.

[0137] CAR constructs using humanized CT3 scFv in either VH-linker-VL or VL-linker-VH orientation were generated with a CD28 hinge and CD28 transmembrane domain (Figure 10). All eight CAR constructs are expected to potently reduce GPC2-positive tumors and show greater tumor reduction than the corresponding hCT3 CAR construct with a CD8 hinge.

[0138] Example 7 material and method This example describes the materials and experimental procedures used in the studies described in Examples 8-11.

[0139] cell line Drug-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 Branch Cell Line Repository. NGP-GPC2 hi , NBSD-GPC2 mod and SMS-SAN lo (all MYCN amplified) were provided by Stanford. GPC2 expression of all lines was determined (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 grown in RPMI (Roswell Park Memorial Institute) medium supplemented with 10% fetal bovine serum (FBS) and 100 U / mL penicillin / streptomycin (Gibco). [Table 3]

[0140] CAR constructs As previously described (Li et al., STAR Protoc 2:100942, 2021), the CT3 scFv was cloned into a lentiviral vector, pWPT (Addgene #12255), and different hinge and TM domains (either CD8 or CD28) and costimulatory domains (4-1BBζ and / or CD28) were added to generate variations of CAR T cell constructs. The GD2 CAR sequence was obtained from publicly available sources (Straathof et al., Sci Transl Med 12:eabd6169, 2020; Pule et al., Nat Med 14:1264-1270, 2008) and cloned into the pWPT vector containing the CD8 or CD28 hinge and TM and 4-1BB costimulatory domains. The human truncated extracellular epidermal growth factor receptor domain (hEGFRt) is included as a tag and is recognized by cetuximab.

[0141] Human T cells and CAR transduction Cryopreserved human T cells from healthy volunteer donors (National Institutes of Health Blood Bank) were used for CAR T cell production as previously described (Li et al., STAR Protoc 2:100942, 2021). Briefly, on day 0, Lenti-X 293T cells were cultured at 2 × 10 per poly-D-lysine-coated 15 cm dish. 7Cells were plated at a density of 100x and subsequently transfected with a 4:1:3 ratio of CT3 CAR plasmid, envelope plasmid (pMD2.G) and packaging plasmid (psPAX2) using Lipofectamine 2000 (Thermo Fisher Scientific). Lentivirus-containing supernatants of Lenti-X 293T cultures were collected 48-72 hours after transfection and used to spin-transduce human T cells. Cryopreserved human T cells were thawed and grown in AIM-V medium (Gibco) supplemented with 10% FBS (Omega Scientific), 100 U / mL penicillin / streptomycin, 1× non-essential amino acids, 0.2 mM L-GlutaMAX, 0.1 mM sodium pyruvate (all Gibco), CD3 / CD28 coated Dynabeads (1:1 bead-to-cell ratio, Thermo Fisher Scientific) and 40 IU / mL interleukin (IL)-2 (NCI Frederick BRB Preclinical Repository). IL-2 concentration was increased to 100 IU / mL 48 hours later, at the time of lentiviral transduction. On day 5 of T cell culture, Dynabeads were removed and transduced CAR T cells were expanded in culture until days 8–10 for subsequent downstream assays.

[0142] CAR Western blot assay The manufactured CAR T cells were grown in culture for 3–5 h while deprived of IL-2. To activate the CAR, either 1.7 μg of GPC2-Fc or 1 μg of Protein L (Acro Biosystems) was added to 2–3 × 10 cells in a 96-well round-bottom plate. 6The antibodies were added to the cells and subsequently cross-linked at 37°C for varying times. The cells were then lysed using radioimmunoprecipitation assay (RIPA) buffer supplemented with Halt protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific). Protein yield was quantified using Bradford Assay (Bio-Rad Laboratories). Samples in sodium dodecyl sulfate (SDS)-containing buffer were denatured for 10 min. A total of 5-10 μg of protein was resolved by 4-20% SDS-polyacrylamide gel electrophoresis (PAGE) and electroblotted onto polyvinylidene difluoride membranes. Primary antibodies listed in Table 4 were incubated overnight at 4°C in 5% bovine serum albumin (BSA) in Tris-buffered saline containing 0.1% Tween®-20 (TBST) and 0.02% sodium azide. Secondary antibodies were incubated in 5% nonfat dry milk in TBST for 1 h at room temperature. 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 (Thermo Fisher Scientific). Enhanced chemiluminescence (Bio-Rad Laboratories) was applied to visualize bands, which were quantified using ImageJ. [Table 4]

[0143] In vitro cytotoxicity assay CAR T cells and ffLUC-GFP-expressing NB tumor cells were co-cultured at varying effector-to-tumor (E:T) ratios as previously described (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-challenge the CAR T cells. Specific lysis of tumor cells was measured using the ONE-Glo assay. Results were normalized to conditions using untransduced (UT) mock T cells.

[0144] mouse For all studies, 4- to 6-week-old female NOD-SCID (NSG) mice were obtained from the NCI Center for Cancer Research Animal Resource Program.

[0145] Bioluminescence imaging IMR-5 with stable expression of ffLUC was used for bioluminescence imaging (BLI). Tumor-bearing mice were injected with d-luciferin potassium salt (150 mg / kg, intraperitoneally (IP)) and imaged 5 min after d-luciferin injection (1 min acquisition time) on an IVIS Lumina XR System (PerkinElmer). Region of interest analysis was performed using Living Image software (PerkinElmer; V.4.3.1).

[0146] In vivo therapeutic models Either PDX cells or IMR-5 were orthotopically implanted (2.5 × 10 5 ) (Li et al., STAR Protoc 2:100942, 2021). Typically, 3 weeks after tumor implantation surgery, patients who met the enrollment criteria (>10 7 Animals receiving 1000x the dose (1000x the dose of photons / s) were randomized to receive either UT mock control T cells or GPC-targeting CAR T cells. The number of tail veins injected with T cells was determined by the number of CAR +The total T cell count in the mock group was adjusted to match the total T cell count in the CAR group. For PDX studies, the experiment was terminated 50 days after tumor injection. Because BLI signal did not correlate with bulk tumor burden, tumor weight was recorded at the end of the study to determine treatment efficacy. For experiments with IMR-5, tumor-bearing animals were monitored via BLI. Survival of treated mice was monitored until day 80 (approximately 11 weeks after tumor implantation). Survival endpoints were death, >20% weight loss from baseline, or severe moribundity, as determined by an animal caretaker who was blinded to the study.

[0147] In vivo homing studies Mice bearing IMR-5 WT NB were injected with ffLUC-GFP expressing GPC2 targeting CAR T cells or UT mock T cells. After tail vein injection of T cells, animals were continuously monitored via BLI to assess T cell homing and expansion in vivo. At the end of the experiment, organs of luciferin-injected mice were removed at 5 min and imaged in 6-well Petri dishes.

[0148] Flow cytometry The sample was diluted to 1 x 10 6 Cells were stained using Fluorescence-minus-one controls. Gates were drawn using fluorescence-minus-one controls. Compensation and voltages were set using single-color controls. The following antibodies were used for detection of surface epitopes: CD45 (detected by clone HI-30), CD3 (OKT3), CD4 (OKT4) and CD8 (HIT8a). GPC2-F C and anti-human F CCAR transduction efficiency was measured using anti-EGFR antibody (M1310G05) or anti-EGFR antibody (AY13). CT3 antibody and anti-mouse IgG1 (RMG1-1) were used to detect GPC2 expression in tumor cells. The density of GPC2 expression was determined using a PE Phycoerythrin Fluorescence Quantitation Kit (BD) according to the manufacturer's instructions. Data were collected on a Fortessa LSR machine. Data analysis was performed using FlowJo V.10.

[0149] Cytokine bead assay Cytokine bead assays (CBA) were performed according to the manufacturer's instructions (BioLegend) to quantify secreted cytokines in the supernatants of T and tumor cocultures.

[0150] Single-cell RNA-seq Two donors were used to produce CAR T cells, which we injected into IMR-5-bearing mice on the 8th day of cell production. The injected cell products were stained with TotalSeq-C antibodies targeting CD8 (cat. no. 344752) and CD4 (cat. no. 300567) and subjected to 10X Genomics 5' V.3.1 chemistry kit for library generation. Eight days after T cell injection into mice, tumors were processed into single cell suspensions with >80% viability. Three tumor samples were pooled per treatment group. Approximately 10,000 cells were loaded per group and 6000 cells were captured. Complementary DNA libraries were sequenced on Illumina NextSeq 2000 and NovaSeq 6000 with a target depth of approximately 50,000 reads per cell.

[0151] computational analysis Single-cell RNA-seq FASTQ files were processed using the CellRanger software suite (V.6.1.2, 10X Genomics) with the corresponding human GRCh38 genome reference. A custom reference (GRCh38+GFP) was used to check whether GFP sequences were detected in annotated tumor cells. Cell barcodes were determined based on the distribution of unique molecular identifier (UMI) counts, and filtered gene-barcode matrices were generated by CellRanger for downstream analysis in Seurat (V.4.0.1, R package) (Wolock et al., Cell Syst 8:281-291, 2019; Stuart et al., Cell 177:1888-1902, 2019). Cells with low (<200 genes) and more than 10% of UMIs mapped to mitochondrial genes were removed. Data integration across different samples and treatment groups was performed using reciprocal principal component analysis (Hao et al., Cell 184:3573-3587, 2021) implemented in Seurat. The expression levels of genes in each cell were normalized by applying the “NormalizeData” function with the following parameters: normalization.method=“LogNormalize” and scale.factor=10,000. 2000 highly variable genes were identified using the “FindVariableFeatures” function with the “vst” method. The gene expression matrix was scaled and centered using the “ScaleData” function with default parameters. To perform clustering, PCA dimensionality reduction was first performed using the “RunPCA” function. The first 20 principal components were selected to construct a shared nearest neighbor graph using the “FindNeighbors” function. Clusters were determined using the Louvain algorithm with the “FindClusters” function.An initial automated cell type annotation was performed using SingleR (V.1.8.1, R package; Aran et al., Nat Immunol 20:163-172, 2019) with Blueprint / ENCODE references and known cell type markers from the Database of Immune Cell Expression to predict the identity of cell clusters. The annotation was then manually checked for reliability by testing the top-ranked differentially expressed genes of each cluster, obtained by the "FindAllMarkers" function with default parameters but with min.pct=0.25 set. Uniform manifold approximation and projection (UMAP) was finally applied to visualize single-cell transcriptional profiles in two-dimensional space. Tumor cells were identified by GFP sequence and copy number mutation 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) and CD45. +Immune cells were annotated using canonical gene markers. Lymphoid cells were separated from tumor and mouse cells and re-clustered to obtain more refined cell clusters. Variable gene expression was calculated for all pairs of clusters and treatment groups. Sample integration across treatment groups was performed using a standard anchor-based workflow in Seurat. For initial clustering and annotation, k-nearest neighbor (KNN) graph-based clustering was applied to weight RNA similarity to calculate Jaccard coefficients (neighborhood overlap) between all pairs of cells at high resolution and linkage of clusters. UMAP plots were used to visualize the results using the Seurat package. Tumor cells were confirmed by green fluorescent protein sequence and copy number mutation analysis. CD45 + Immune cells were annotated using canonical gene markers. QIAGEN Ingenuity Pathway Analysis was used for pathway enrichment analysis (QIAGEN) (Kramer et al., Bioinformatics 30:523-530, 2014).

[0152] statistical analysis Student's t test (normally distributed data) or Mann-Whitney U test (skewed data) were used to compare two groups, and for comparisons with more than two groups, one-way analysis of variance (ANOVA, normally distributed data) followed by Tukey's post-hoc comparison test or one-way ANOVA on ranks (skewed data) followed by Dunn's test were used. For survival analysis, Kaplan-Meier curves were generated and survival was compared between groups using the two-tailed log-rank test. All experiments were performed in biological replicates with at least two donors.

[0153] Example 8 In vitro comparison of CT3.28H.BBζ, CT3.8H.BBζ and CT3.8H.28BBζ To identify the most effective GPC2-CAR construct for clinical transfer, a direct comparison was first performed in vitro using three different CAR scaffold constructs, CT3 (Figure 11A): (1) CT3 with a CD8 hinge, CD8 TM and 4-1BB costimulatory domain (CT3.8H.BBζ, published CAR; Li et al., Cell Rep Med 2:100297, 2021), (2) CT3 with a CD28 hinge, CD28 TM and 4-1BB costimulatory domain (CT3.28H.BBζ), and (3) CT3 with a CD8 hinge, CD28 TM and CD28-4-1BB costimulatory domain (CT3.8H.28BBζ). In coculture 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 (Figure 11B). Cytokine levels approached background levels in conditions containing GPC2-KO cells, indicating the specificity of the CAR. However, of the three constructs, CT3.28H.BBζ demonstrated the lowest tonic signaling indicated by low phosphorylation levels of CAR and downstream molecules, such as ZAP70 or ERK, at rest (Figure 11C-11D), but demonstrated its appropriate increase in vitro upon CAR crosslinking. CT3.8H.BBζ also showed low tonic signaling at rest, but CAR activation was not as robust as CT3.28H.BBζ after antigen-specific crosslinking. Furthermore, CT3.28H.BBζ performed better than another GPC2 scFv in the CAR scaffold of the present invention, GPC2.19 (Heitzeneder et al., Cancer Cell 32:295-309, 2022), especially against the NB line at lower E:T ratios and with lower antigen density and better cell expansion growth and CAR persistence upon tumor rechallenge. These data indicate that all three CAR constructs have comparable functionality in vitro, but CT3.28H.BBζ lacks tonic signaling, which may positively impact antitumor activity with persistent tumor exposure.

[0154] Example 9 CT3.28H.BBζ demonstrates highly potent anti-NB activity in vivo To determine which of the three GPC2-CAR constructs has the best antitumor activity in vivo against NB, an orthotopic PDX model was utilized. Four to six week old NSG mice were orthotopically injected with SJNBL012407_X1. This PDX line harbors the molecular hallmark of high-risk NB (MYCN amplification), and most tumor-bearing mice treated with conventional chemotherapy and / or immunotherapy are incurable (Nguyen et al., Neoplasia 26:100776, 2022; Nguyen et al., Clin Cancer Res 28:3785-3796, 2022). Three weeks after tumor implantation, mice were treated with either UT mock T cells or 2.5 × 10 6 CAR + Patients were randomized to receive either CT3.28H.BBζ or CT3.8H.28BBζ. Four weeks after CAR T cell infusion (50 days after tumor implantation), CT3.28H.BBζ induced the most significant tumor regression comparing all three CAR constructs (Figure 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 had comparable activity to CT3.8H.28BBζ, subsequent studies focused on CT3.28H.BBζ and CT3.8H.28BBζ. Survival studies were performed and in vivo tumor growth kinetics was evaluated after GPC2-CAR T cell injection. Four to six week old NSG mice bearing orthotopic IMR-5.ffLUC-GFP tumors were treated with CT3.28H.BBζ or CT3.8H.28BBζ at a high dose (5 × 10 6 ) or low dose (2.5 × 10 6) CAR T cells (Figure 12B). After high-dose treatment with CT3.28H.BBζ, all animals demonstrated a steep decrease in their BLI signal that eventually approached background levels (Figure 12C). Mice treated with high-dose CT3.8H.28BBζ responded transiently but were unable to maintain tumor control. 6 All mice in both GPC2-CAR T cell groups treated with CT3.28H.BBζ CAR T cells demonstrated an initial decrease in their BLI but eventually progressed, with downregulation of GPC2 in recurrent tumors. The differences in tumor growth kinetics between mice receiving high and lower doses of CAR T cells were mirrored in survival studies of these animals. Tumor-bearing mice treated with high-dose CT3.28H.BBζ CAR T cells exhibited the longest survival (Figure 12D) and higher levels of tumor-infiltrating CAR T cells by flow cytometry analysis. + T cells (Figure 12E). Survival between CAR groups was not statistically different at the lower dose levels. However, six of six mice never met the study endpoint when treated with CT3.28H.BBζ CAR T cells, whereas two of five mice treated with CT3.8H.28BBζ CAR T cells died due to tumors. In vitro experiments demonstrated similar function of the three different GPC2-targeting CARs, but subsequent in vivo studies identified CT3.28H.BBζ as the most potent construct. The superior performance of CT3.28H.BBζ was attributed to less tonic signaling, higher levels of CARs in the tumor microenvironment (TME), and increased responsiveness to CT3.28H.BBζ. + This may be due to effector cells and / or antigen escape. To further evaluate the molecular differences across the three CAR constructs, tumor-infiltrating T cells were analyzed by single-cell RNA-seq.

[0155] Example 10 CT3.28H.BBζ CAR T cells upregulate effector molecules in the TME To understand the properties of T cells expressing the different GPC2-CAR T constructs before infusion as well as after tumor encounter in vivo, single-cell RNA-seq was performed on manufactured GPC2-CAR T as well as on tumor-infiltrating T cells collected on day 8, a time point prior to tumor regression (which typically occurs on day 10).

[0156] CAR T cells from two donors were manufactured and their transcriptomes were analyzed prior to injection into mice using the droplet-based 10X Genomics platform. After quality control and filtering, a total of 14,169 single-cell transcriptomes were obtained for donor 1 and 13,515 single-cell transcriptomes were obtained for donor 2 (Figure 13A). Each subgroup was equally represented for donor 1, but more CT3.8H.BBζ cells and fewer UT mock cells were captured for donor 2. To manually annotate the cell subsets, we followed a previously published annotation strategy (Wilson et al., Cancer Discov 12:2098-2119, 2022; Patil et al., Sci Immunol 3:eaan8664, 2018). Graph-based unsupervised clustering was performed to define T (CD8A, CD4), NK (NKG7, GNLY) and B cells (MS4A1) using CD8 and CD4 protein expression as well as conventional genes. T cell subsets were further defined as cytotoxic effector cells by robust expression of PRF1 and various granzyme-encoding genes, and as memory cells by expression of SELL, IL7R, CD27 and LEF1. Regulatory T cells (Tregs) were identified by IL2RA and FOXP3. Cell clusters were defined as proliferative if they expressed classical proliferation and cell cycle-related genes (e.g., TOP2A, MKI67, CCNB1 / 2, minichromosome maintenance (MCM) complexes or histone genes). The T cell injection product of donor 1 consisted of 12 cell clusters (Figure 13B), which were clearly separated by their CD8 (22.3%) and CD4 (77.7%) protein expression (Figure 13C). By using a composite gene signature, it was determined that this donor had proliferating CD8 + and CD4 + T cells (72.8%) and CD8 + The CD8 cells were found to contain predominantly cytotoxic effector cells (33.1%) (Figures 13D-13E). +A portion of the cells were exhausted cytotoxic effector cells (Figures 13D-13E). Donor 2 consisted of 15 independent cell clusters (Figure 13F). Similar to donor 1, the total cell population consisted of CD4 + Fewer CD8 than T cells (62.1%; Fig. 13G) + (37.9%), of which 25.6% were cytotoxic T cells and 1.7% were Tregs (Figures 13H-13I).

[0157] To determine the ideal time point for single-cell RNA-seq analysis of the TME following T cell injection into mice, the in vivo distribution and expansion of CAR T cells was evaluated. To track cells, GPC2-CAR T cells were transduced to express firefly luciferase-GFP (ffLUC-GFP). Three weeks after tumor implantation into the right adrenal fat pad, GPC2-CAR-ffLUC-GFP T cells were injected and serial BLI was performed. T cells in all four test groups initially accumulated in the lungs and femurs and gradually expanded over the next 48 hours (Figure 14A). By day 7, the overall BLI signal faded in the UT mock T group, but all three GPC2-CAR groups demonstrated an increase in signal restricted to the tumor and spleen, consistent with local T cell expansion (Figures 14B-14C).

[0158] After CAR T cell injection, tumors from IMR-5-bearing mice (day 8) were found to contain a large proportion of CD8 and CD4 effector T cells, constituting approximately 50% or more of the cells from the TME (Figure 14D-14G). In contrast, UT Mock cells accounted for <3% of the cells in the TME. This is consistent with results from in vivo tracking experiments, where UT Mock T cells failed to expand and engraft (Figure 14A). The remaining cells in this group were almost exclusively M2 tumor-associated macrophages. Tumor cells were distinguished from immune cells by their gene expression and copy number mutation profiles.

[0159] In vivo immune cell trajectory analysis revealed that the small number of antigen-presenting cells present at the time of injection were quickly outnumbered by CD4 and CD8 T cells. These cells developed from a state of high proliferative potential (indicated by expression of MKI67) into terminally differentiated dysfunctional CD69-expressing, EOMES-expressing and TOX-expressing effector cells, or transitioned to a memory stage as evidenced by the abundance of IL7RA, LEF1 and CCL5. Tumor-infiltrating CD8 across the GPC2-CAR T cell population +To better characterize the transcriptome of effector cells, the gene expression profile of CT3.28H.BBζ was compared with that of two other GPC2-targeting CARs (Figure 14H). In donor 1, 33 differentially expressed genes (DEGs) were found that were shared by both analyses (CT3.28H.BBζ vs. CT3.8H.BBζ and CT3.28H.BBζ vs. CT3.8H.28BBζ; Figure 14I). In donor 2, 16 DEGs were shared (Figure 14I). Compared with the other two CAR T cell populations, CT3.28H.BBζ CAR T cells showed upregulation of CXCR4, ARHGEF1 and LIME1, which are involved in chemokine-associated T cell migration and T cell regeneration (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.BBζ CAR T cells also had upregulated genes encoding effector molecules (e.g., GNLY, GZMB, ZNF683 and HMGN2) and cell cycle components (e.g., STMN1, MCM5, MCM7 and PTTG1). Finally, pathway analysis supported these findings, demonstrating activation of the Granzyme A pathway (z-score: 4.54; p-value=1.16E-35).CT3.28H.BBζ CAR T cells also showed downregulation of EIF2 (z-score: -3.628; p-value = 6.19E-54) and oxidative phosphorylation pathways (z-score: -5.53; p-value = 1.85E-56; Figure 14J). Pairwise DEG analysis revealed that compared to the other two CARs, CT3.28H.BBζ already expressed genes important in regulating pathways of T cell exhaustion (e.g., NFKBIA, CISH), genes that promote T cell activation and proliferation (e.g., CD83, TXNIP, LDHA), and genes that may prevent apoptosis (e.g., MTRNR2L12; Figures 14J-14K). These findings indicate that at the time of manufacturing these cells, transcriptomic differences already exist that may affect the cells' differential cytotoxic activity and survival in vivo.

[0160] Together, these findings demonstrate that all GPC2-targeting CAR T cells substantially expand toward a cytotoxic effector population in vivo. Furthermore, CT3.28H.BBζ CAR T cells upregulate effector molecules and genes involved in T cell migration and memory homeostasis. These findings may account for the superior antitumor cytotoxicity observed in mice treated with CT3.28H.BBζ CAR T cells compared to other CAR treatment groups.

[0161] Example 11 CT3.28H.BBζ is GD2 + GPC2 低 Superior antitumor activity against NB, outperforming K666.28H.BBζ Previous CAR T cell trials in NB were performed with K666-based GD2-CAR T cells 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). These trials reported tolerability, but few treated patients achieved objective responses. Next, a study was performed to evaluate how comparable CT3.28H.BBζ was in function to existing CAR T cell therapies targeting GD2. The aim was to compare the preclinical activity of K666-based GD2-targeting CARs and 14.G2a-based GD2-targeting CARs (Straathof et al., Sci Transl Med 12:eabd6169, 2020) with that of CT3.28H.BBζ. To create comparable test conditions, scFvs were cloned into the same CAR constructs used for CT3.28H.BBζ and CT3.8H.BBζ, and a direct comparison in vitro with serial tumor rechallenge was performed. Subsequently, K666.28H.BBζ was selected, and the anti-NB activity of CT3.28H.BBζ was further compared to that of K666.28H.BBζ in vitro and in vivo. CAR T cells demonstrated comparable transduction efficiency (Figure 15A). ffLUC-GFP expressing SJNBL012407_X1, IMR-5 (both MYCN amplified), and SH-SY5Y (MYCN-WT) were incubated with CT3.28H.BBζ or K666.28H.BBζ CAR T cells at varying E:T ratios. These NB cells have different expression levels of GPC2 and GD2. After 48 hours, tumor cell lysis was determined by applying a luciferase reporter assay (Figure 15B). At an E:T ratio of 1:1, CT3.28H.BBζ CAR T cells suppressed GD2 expression, whereas GD2 expression was suppressed. 中等度GPC2 低 PDX and GD2 低 GPC2 低 CAR T cells showed superior anti-NB cytotoxicity against SH-SY5Y compared with K666.28H.BBζ CAR T cells. Tumor lysis was significantly higher in both groups than in GD2. 高 GPC2 高 The results were comparable in IMR-5. The study was then extended to an in vitro tumor rechallenge model with SJNBL012407_X1. The cytotoxic potential of the two CARs was measured 24 h, 4 days, and 7 days after daily tumor rechallenge. Although CT3.28H.BBζ CAR T cells initially showed better anti-NB cytotoxicity, their activity gradually decreased over time, with K666.28H.BBζ CAR T cells outperforming them on day 7 (Figure 15C). The antitumor activity of the two CARs was then compared in vivo. SJNBL012407_X1-bearing mice were treated with 5 × 10 6 CAR + T cells were injected into the primary tumors. 50 days after tumor injection, primary tumors were weighed and bone marrow was analyzed for residual NB cells. Tumor weight was smaller after treatment with CT3.28H.BBζ CAR T cells compared to K666.28H.BBζ CAR T (Figure 15D-15E). Furthermore, three out of five mice with K666.28H.BBζ CAR T cells had higher levels of detectable tumor cells in their bone marrow than all mice treated with CT3.28H.BBζ CAR T cells (Figure 15F). A possible cause of therapeutic resistance after K666.28H.BBζ CAR T treatment could be downregulation of GD2 in the primary tumors. These findings demonstrate that CT3.28H.BBζ CAR T cells outperformed K666.28H.BBζ CAR T within an early window in vitro as well as 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.

[0162] It will be apparent that the exact details of the methods or compositions described may be varied or modified without departing from the spirit of the described aspects of this disclosure, and we claim all such modifications and variations that come within the scope and spirit of the following claims.

Claims

1. an extracellular antigen-binding domain that specifically binds to glypican-2 (GPC2), comprising a variable heavy (VH) domain and a variable light (VL) domain, wherein the VH domain comprises the complementarity-determining region 1 (CDR1), CDR2, and CDR3 sequences of SEQ ID NO: 2, and the VL domain comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 4; CD28 hinge region; CD28 transmembrane domain; an intracellular costimulatory domain; and Intracellular signaling domains A chimeric antigen receptor (CAR) comprising:

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

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

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

5. The CAR of claim 1, wherein the sequences of the VH domain and the VL domain are humanized.

6. 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; The CAR according to claim 5.

7. The CAR of claim 1, 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 of claim 1, wherein the CD28 hinge region comprises or consists of the amino acid sequence of SEQ ID NO:

24.

9. The CAR of claim 1, wherein the CD28 transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO:

26.

10. The CAR of claim 1, wherein the costimulatory domain comprises a 4-1BB signaling moiety.

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 claim 1, wherein the signaling domain comprises a CD3ζ 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 claim 1, wherein the amino acid sequence of the CAR comprises or consists of SEQ ID NO:

38.

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

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

17. 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 of any one of claims 1 to 14.

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

37.

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

37.

21. 20. The isolated nucleic acid molecule of claim 18 operably linked to a promoter.

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

23. 20. A vector comprising the isolated nucleic acid molecule of claim 18.

24. 24. The vector of claim 23, which is a lentiviral vector.

25. An isolated cell comprising the vector described in claim 23.

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

27. 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. 15. A composition comprising a pharmaceutically acceptable carrier and the CAR of any one of claims 1 to 14, an isolated cell expressing the CAR, or an isolated nucleic acid molecule or vector encoding the CAR.

29. 15. A composition for treating GPC2-positive cancer in a subject, the composition comprising the CAR of any one of claims 1 to 14, an isolated cell expressing the CAR, or an isolated nucleic acid molecule or vector encoding the CAR.

30. 15. A composition for inhibiting tumor growth or metastasis of a GPC2-positive cancer in a subject, the composition comprising the CAR of any one of claims 1 to 14, an isolated cell expressing the CAR, or an isolated nucleic acid molecule or vector encoding the CAR.

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

32. 30. The composition of claim 29, wherein the GPC2-positive cancer is a childhood cancer.

33. 30. The composition of claim 29, wherein the GPC2-positive cancer is neuroblastoma, medulloblastoma, retinoblastoma, acute lymphoblastic leukemia, embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, Ewing's sarcoma, desmoplastic small round cell tumor, glioma, or osteosarcoma.

34. 30. The composition of claim 29, wherein chemotherapy is administered to the subject.

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