Chimeric antigen receptors targeting glypican-2 and / or CD276 and their use for treating solid tumors

Abstract

Chimeric antigen receptors (CARs) and bicistronic chimeric antigen receptors (BiCisCARs) that target glypican-2 (GPC2) or both GPC2 and CD276 are disclosed. The GPC2-targeted CARs include a hinge and transmembrane (HTM) domain from CD28 and a co-stimulatory domain (CSD) from CD28. The CD276-targeted portion of the BiCisCARs includes a CD8 HTM domain and a 4-1BB CSD. The CARs and BiCisCARs can further include amino acid substitutions in one or more immunoreceptor tyrosine-based activation motifs (ITAMs) of a CD3ζ intracellular signaling domain. Cells expressing the CARs or BiCisCARs can be used for the treatment of solid tumors, such as those that express one or both of GPC2 and CD276.

Description

[0001] CHIMERIC ANTIGEN RECEPTORS TARGETING GLYPICAN-2 AND / OR CD276 AND THEIR USE FOR TREATING SOLID TUMORS

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Application No. 63 / 644,728, filed May 9, 2024, which is herein incorporated by reference in its entirety.

[0004] FIELD

[0005] This disclosure concerns chimeric antigen receptors (CARs) and bicistronic CARs targeting glypican-2 (GPC2) or targeting both GPC2 and CD276, and their use for treating solid tumors, such as GPC2-expressing and / or CD276-expressing tumors.

[0006] ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

[0007] This invention was made with government support under project numbers ZIA BC 010806 and Z01 BC 010891 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0008] INCORPORATION OF ELECTRONIC SEQUENCE LISTING

[0009] The electronic sequence listing, submitted herewith as an XML file named 4239-111444- O2.xml (75,185 bytes), created on April 17, 2025, is herein incorporated by reference in its entirety.

[0010] BACKGROUND

[0011] Chimeric antigen receptor (CAR) T cell therapies targeting cancer-specific antigens have impressive successes in treating refractory and relapsed leukemia and lymphoma (Majzner et al., Nat Med 25: 1341-1355, 2019; June et al., Science 359: 1361-1365, 2018). However, they have thus far displayed poor efficacy in treating solid tumors due to several challenges, including heterogenous expression of tumor-associated antigens, loss of target expression under selection pressure, limited T cell potency, inadequate trafficking, a hostile tumor microenvironment, the propensity of exhaustion and lack of persistence of the CAR T cells (Wagner et al., Mol Titer 28:2320-2339, 2020; Srivastava et al., J Immunol 200:459-468, 2018; Labanieh et al., Nat Biomed Eng 2:377-391, 2018). Overcoming these hurdles for the treatment of solid tumors remains a significant challenge.

[0012] Neuroblastoma is the most common extracranial solid tumor in children. Derived from neuroendocrine tissue of the sympathetic nervous system, it accounts for 8-10% of childhood cancers in the USA (Maris and Hogarty, Lancet 369:2106-2120, 2007). Neuroblastoma is a complex and heterogeneous disease, with nearly 50% of patients having a high-risk phenotype characterized by widespread dissemination of the cancer and poor long-term survival even if intensive multimodal treatments are used (Yu et al., New Engl J Med 363: 1324-1334, 2010). Approximately 45% of patients receiving standard therapy have a relapse and ultimately succumb to metastatic disease (Matthay et al., New Engl J Med 341 :1165-1173, 1999). As such, there is an unmet urgent need for a safe and effective treatment of neuroblastoma.

[0013] One of the most difficult challenges for the treatment of neuroblastoma and other deadly solid tumors is the lack of tumor- specific targets. Glypican-2 (GPC2) mRNA is highly expressed in neuroblastoma and other pediatric cancers (Orentas et al., Front Oncol 2: 194, 2012). GPC2 belongs to the six-member human glypican family of proteins that are attached to the cell surface by a glycosylphosphatidylinositol (GPI) anchor (Filmus et al., Genome Biol 9:224, 2008). Unlike other known glypicans, GPC2 is uniquely expressed in the nervous system (Stipp et al., J Cell Biol 124: 149-160, 1994), participates in cell adhesion and is thought to regulate the growth and guidance of axons.

[0014] SUMMARY

[0015] The present disclosure provides optimized chimeric antigen receptors (CARs) that target GPC2 and bicistronic CARs (BiCisCARs) that target both GPC2 and CD276. The disclosed GPC2- targeted CARs and BiCisCARs include a CD28 hinge and transmembrane (HTM) domain, a CD28 co-stimulatory domain (CSD), and a CD3^ signaling domain. In the disclosed BiCisCARs, the CD276-targeted CAR includes a CD8 HTM and a 4-1BB CSD, and in some instances, further includes a CD3C signaling domain. The CARs and BiCisCARs can optionally further include amino acid substitutions in one or more immunoreceptor tyrosine-based activation motifs (IT AMs) of a CD3L intracellular signaling domain. Cells expressing the CARs or BiCisCARs can be used for the treatment of cancers that express one or both of GPC2 and CD276.

[0016] Provided herein are GPC2-targeted CARs that include an extracellular antigen-binding domain that specifically binds GPC2. In some aspects, the CAR includes: an antigen-binding domain that includes a variable heavy (VH) domain and a variable light (VL) domain, wherein the VH domain includes the complementarity determining region 1 (CDR1), CDR2 and CDR3 sequences of SEQ ID NO: 1 and the VL domain includes the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 2; a HTM domain; an intracellular CSD; and a CD3 intracellular signaling domain. In some aspects, the HTM is a CD28 HTM domain and the intracellular CSD is a CD28 intracellular CSD. In some aspects, the CD3^ intracellular signaling domain is a wild-type CD3^ intracellular signaling domain. In other aspects, the CD3^ intracellular signaling domain includes at least one amino acid substitution in one or more ITAMs. Nucleic acid molecules and vectors encoding the GPC2-targeted CARs, and isolated cells expressing the GPC2-targeted CARs are also provided.

[0017] Also provided herein are isolated cells that express a first CAR and a second CAR (a BiCisCAR), wherein the first CAR is a GPC2-targeted CAR as disclosed herein and the second CAR is a CD276-targeted CAR that includes an extracellular antigen-binding domain that specifically binds CD276. In some aspects, the CD276-targeted CAR includes: an antigen-binding domain that includes a VH domain and a VL domain, wherein the VH domain includes the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 9 and the VL domain includes the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 10; a CD8 HTM domain; and a 4-1BB intracellular CSD. In some aspects, the CD276- targeted CAR further includes a CD3^ intracellular signaling domain, such as a wild-type CD3^ intracellular signaling domain or a CD3^ intracellular signaling domain having at least one amino acid substitution in one or more ITAMs. In some aspects, the cell is an immune cell, such as a T cell, a B cell, a natural killer (NK) cell, or a monocyte / macrophage. In other aspects, the cell is an induced pluripotent stem cell (iPSC).

[0018] Further provided herein are nucleic acid molecules that encode a first CAR and a second CAR (a BiCisCAR), wherein the first CAR is a GPC2-targeted CAR as disclosed herein and the second CAR is a CD276-targeted CAR that includes an extracellular antigen-binding domain that specifically binds CD276. In some aspects, the CD276-targeted CAR includes: an antigen-binding domain that includes a VH domain and a VL domain, wherein the VH domain includes the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 9 and the VL domain includes the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 10; a CD8 HTM domain; and a 4-1BB intracellular CSD. In some aspects, the CD276-targctcd CAR further includes a CD3^ intracellular signaling domain, such as a wild-type CD3L intracellular signaling domain or a CD3^ intracellular signaling domain having at least one amino acid substitution in one or more ITAMs. In some aspects of the nucleic acid molecules, the coding sequences for the first CAR and the second CAR are separated by a sequence encoding a 2A site, such as a T2A site. In some examples, the nucleic acid molecule further includes a first leader sequence (such as a CD8 or human IgG leader sequence) preceding the coding sequence for the first CAR and / or a second leader sequence (such as a GM-CSF leader sequence) preceding the coding sequence for the second CAR. Vectors, such as lentivirus vectors, that include a disclosed nucleic acid molecule are also provided.

[0019] Compositions that include a pharmaceutically acceptable carrier and a GPC2-targeted CAR, a GPC2 / CD276-targeted BiCisCAR, a nucleic acid molecule, a vector or an isolated cell disclosed herein are further provided.

[0020] Also provided are methods for treating a GPC2-expressing and / or CD276-expressing cancer in a subject, and methods of inhibiting tumor growth or metastasis of a GPC2-expressing and / or CD276-expressing cancer in a subject. In some aspects, the methods include administering to the subject a therapeutically effective amount of a CAR, BiCisCAR, isolated cell, nucleic acid molecule, vector, or composition disclosed herein. In some examples, the cancer expresses both GPC2 and CD276. The foregoing and other features of this disclosure will become more apparent from the following detailed description of several aspects which proceeds with reference to the accompanying figures.

[0021] BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIGS. 1A-1B: (FIG. 1A) Schematics of an optimized CAR construct targeting GPC2 (top) and a BiCisCAR construct targeting both GPC2 and CD276 (bottom). The GPC2-targeted CAR includes the VH and VL domains of GPC2-specific monoclonal antibody CT3, a CD28 hinge and transmembrane (HTM) domain, a CD28 co-stimulatory domain, and a CD3^ signaling domain. The BiCisCAR construct includes the GPC2-targeted CAR components as well as the VH and VL domains of CD276-specific antibody MGA271, a CD8 HTM, a 4- IBB co-stimulatory domain, and a CD3C signaling domain. (FIG. 1 B) Schematics of a GPC2-targeted CAR and a GPC2 / CD276 dualtargeted BiCisCAR containing amino acid substitutions in the IT AMs of the CD3^ domain for the GPC2-targctcd CAR cassette, and lacking a CD3^ domain for CD276-targctcd CAR cassette (top and bottom constructs). Also shown is a GPC2 / CD276 dual-targeted BiCisCAR lacking the IT AM modifications in the CD3^ domain of the GPC2-targeted CAR cassette, and lacking a CD3^ domain for CD276-targeted CAR cassette (middle).

[0023] FIG. 2: Representative flow cytometric plots showing the percentage of CAR+ T cells, the percentage of CD4+ or CD8+ T cells, and the percentage of CAR T cells in different memory states (right column) at day 9 post-manufacturing. Human peripheral blood mononuclear cells (PBMCs) from a healthy donor were activated with anti-CD3 / CD28 microbeads and then un-transduced (UTD) or transduced with lentivirus expressing GPC2.8HTM.BBz-CD276.8HTM.BBz BiCisCAR or GPC2.28HTM.28z-CD276.8HTM.BBz BiCisCAR construct separately. An Fc-GPC2 chimeric protein (first column) or a biotinylated human CD276 protein (second column) was separately used to detect the expression of cell surface chimeric antigen receptor (CAR). Anti-CD4 and anti-CD8 antibody were used to distinguish CD4+ or CD8+ T cells from CAR+ T cells. CD45RA and CD62L were used to distinguish different memory T cell subsets. CM, central memory T cells (CD45RA- CD62L+); EM, effector memory T cells (CD45RA-CD62L-); EMRA, terminally differentiated effector memory cells (CD45RA+CD62L-); SCM, stem cell memory T cells (CD45RA+CD62L+).

[0024] FIG. 3: GPC2.28HTM.28z-CD276.8HTM.BBz BiCisCAR showed better efficacy than GPC2.8HTM.BBz-CD276.8HTM.BBz BiCisCAR in vitro. The graphs show cytotoxicity of T cells expressing either GPC2.28HTM.28z-CD276.8HTM.BBz BiCisCAR or GPC2.8HTM.BBz- CD276.8HTM.BBz BiCisCAR after coculturing with neuroblastoma NBEB (top), IMR5 (middle) or SKNAS (bottom) cells at an E:T ratio of 1:5 in an xCELLigcncc real-time cell analysis (RTCA) assay. Statistical analysis was performed with two-way repeated measures (RM) ANOVA. **** p <0.0001. FIGS. 4A-4D: GPC2.28HTM.28z-CD276.8HTM.BBz BiCisCAR showed increased efficacy compared with GPC2.8HTM.BBz-CD276.8HTM.BBz BiCisCAR in an aggressive subcutaneous neuroblastoma (NB) PDX model. (FIG. 4A) Schematic of an NB PDX model infused with CAR T cells on day 14 after tumor inoculation. (FIG. 4B) Tumor volumes following CAR T cell infusion. Data indicates tumor volume for each mouse (n = 5). p = 0.0689, as determined by mixed effect analysis. (FIG. 4C) Bioluminescence kinetics of NB PDX showed tumor regression after CAR T cell treatment using total flux values (photons per second). *p = 0.0206 and ****p < 0.0001, ns, not significant, by 2- way repeated- measures (RM) ANOVA. (FIG. 4D) Kaplan-Meier survival analysis of mice treated with CAR T cells (n = 5 mice / group). **p = 0.0025 and ns by log-rank test.

[0025] FIG. 5: Schematic representations of GPC2 and CD276 CAR / BiCisCAR constructs. (CARs 1 - 3) GPC2 CARs with variations in hinge and transmembrane (HTM) regions, co-stimulatory domains (CSD), or CD3C signaling calibration. (CAR 4) CD276 CAR with CD8 HTM, 4-1BB CSD, and CD3C. (BiCisCARs 5 - 8) Dual-targeting GPC2 / CD276 CARs (BiCisCARs) with different CD3t calibrations, including CD3C deletion (BiCisCAR 7) or ITAM mutations in the CD3C domain (BiCisCAR 8). In 1XX CARs, the two tyrosine (Y) residues within the respective ITAM are mutated to phenylalanine (F) for the indicated ITAMs.

[0026] FIG. 6: Transduction efficiency of GPC2-targeted CAR or CD276-targeted CAR T cells. Representative histograms showing CAR surface expression across different CAR constructs, assessed by binding to GPC2 or CD276 protein on day 9 post-manufacturing.

[0027] FIG. 7 : Cytolytic activity of single GPC2 or CD276 single-targeting CAR T cells following CD3^ calibration. The killing efficiency of GPC2 or CD276 single-targeting CAR T cells was evaluated against neuroblastoma (NB) cell lines (IMR5, NBEB, or NB1691) at an effector-to-target (E: T) ratio of 1:5, using RTCA. The results demonstrate that CD3t calibration reduced the killing efficiency of GPC2.28HTM.28z CAR T cells.

[0028] FIG. 8: Cytolytic activity of GPC2 / CD276 dual-targeting BiCisCAR T cells following CD3C calibration. The cytotoxicity of GPC2 and CD276 dual-targeting CAR T cells was assessed against NB cell lines (IMR5, NBEB, NB1691) at an effector-to-target (E: T) ratio of 1:5. Killing activity was measured using RTCA. The results showed that CD3^ calibration led to a reduction in cytotoxic potency.

[0029] FIG. 9: Characterization of GPC2 / CD276 BiCisCAR T cells after CD3i calibration. BiCisCAR T cells demonstrated a higher CD4 / CD8 ratio and reduced PD-1 and LAG-3 expression following repeated stimulation. Black arrows indicate the stimulation time points of NB1691 cells at an effector-to-target (E: T) ratio of 1:5.

[0030] SEQUENCES

[0031] The nucleic acid and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and single letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. In the accompanying sequence listing:

[0032] SEQ ID NO: 1 is the amino acid sequence of the CT3 VH domain.

[0033] SEQ ID NO: 2 is the amino acid sequence of the CT3 VL domain.

[0034] SEQ ID NOs: 3-8 are the amino acid sequences of the CT3 CDRs (IMGT).

[0035] SEQ ID NO: 9 is the amino acid sequence of the MGA271 VH domain.

[0036] SEQ ID NO: 10 is the amino acid sequence of the MGA271 VL domain.

[0037] SEQ ID NOs: 11-16 are the amino acid sequences of the MGA271 CDRs (IMGT).

[0038] SEQ ID NO: 17 is a nucleotide sequence encoding GPC2.28HTM.28z CAR.

[0039] SEQ ID NO: 18 is the amino acid sequence of GPC2.28HTM.28z CAR.

[0040] SEQ ID NO: 19 is a nucleotide sequence encoding GPC2.28HTM.28z-CD276.8HTM.BBz BiCisCAR.

[0041] SEQ ID NO: 20 is the amino acid sequence of GPC2.28HTM.28z-CD276.8HTM.BBz BiCisCAR.

[0042] SEQ ID NO: 21 is a nucleotide sequence encoding GPC2.28HTM.28z(lXX) CAR.

[0043] SEQ ID NO: 22 is the amino acid sequence of GPC2.28HTM.28z(lXX) CAR.

[0044] SEQ ID NO: 23 is a nucleotide sequence encoding GPC2.28HTM.28z / CD276.8HTM.BB BiCisCAR.

[0045] SEQ ID NO: 24 is the amino acid sequence of GPC2.28HTM.28z / CD276.8HTM.BB BiCisCAR.

[0046] SEQ ID NO: 25 is a nucleotide sequence encoding GPC2.28HTM.28z(lXX) / CD276.8HTM.BB BiCisCAR.

[0047] SEQ ID NO: 26 is the amino acid sequence of GPC2.28HTM.28z(lXX) / CD276.8HTM.BB BiCisCAR.

[0048] SEQ ID NO: 27 is a nucleotide sequence encoding a human IgG leader sequence.

[0049] SEQ ID NO: 28 is a nucleotide sequence encoding a CD8 leader sequence.

[0050] SEQ ID NO: 29 is a nucleotide sequence encoding a CT3 scFv (VH-linker-VL).

[0051] SEQ ID NO: 30 is a nucleotide sequence encoding a MGA271 scFv (VL-linker-VH).

[0052] SEQ ID NO: 31 is a nucleotide sequence encoding a CD28 HTM.

[0053] SEQ ID NO: 32 is a codon-optimized nucleotide sequence encoding a CD8 HTM.

[0054] SEQ ID NO: 33 is a codon-optimized nucleotide sequence encoding a 4- IBB co-stimulatory domain.

[0055] SEQ ID NO: 34 is a nucleotide sequence encoding a CD28 co-stimulatory domain.

[0056] SEQ ID NO: 35 is a codon-optimized nucleotide sequence encoding a CD3q signaling domain.

[0057] SEQ ID NO: 36 is a nucleotide sequence encoding CD3^(1 XX). SEQ ID NO: 37 is a nucleotide sequence encoding a T2A site.

[0058] SEQ ID NO: 38 is a codon-optimized nucleotide sequence encoding a T2A site.

[0059] SEQ ID NO: 39 is a nucleotide sequence encoding a GM-CSF leader sequence.

[0060] SEQ ID NO: 40 is a nucleotide sequence encoding truncated EGFR (tEGFR).

[0061] SEQ ID NO: 41 is the amino acid sequence of a human IgG leader sequence.

[0062] SEQ ID NO: 42 is the amino acid sequence of a CD8 leader sequence.

[0063] SEQ ID NO: 43 i s the amino acid sequence of a CT3 scFv (VH-linker VL).

[0064] SEQ ID NO: 44 i s the amino acid sequence of a MGA271 scFv (VL-linker-VH).

[0065] SEQ ID NO: 45 i: s the amino acid sequence of a CD28 HTM.

[0066] SEQ ID NO: 46 i: s the amino acid sequence of a CD8 HTM.

[0067] SEQ ID NO: 47 is the amino acid sequence of 4- IBB.

[0068] SEQ ID NO: 48 is the amino acid sequence of a CD28 co-stimulatory domain.

[0069] SEQ ID NO: 49 is the amino acid sequence of a CD3 signaling domain

[0070] SEQ ID NO: 50 is the amino acid sequence of CD3^(1XX).

[0071] SEQ ID NO: 51 is the amino acid sequence of a T2A site.

[0072] SEQ ID NO: 52 is the amino acid sequence of a GM-CSF leader sequence.

[0073] SEQ ID NO: 53 is the amino acid sequence of tEGFR.

[0074] SEQ ID NO: 54 i s the amino acid sequence of the hCT3-l VH domain.

[0075] SEQ ID NO: 55 is the amino acid sequence of the hCT3-l VL domain.

[0076] SEQ ID NO: 56 is the amino acid sequence of the hCT3-2 VH domain.

[0077] SEQ ID NO: 57 is the amino acid sequence of the hCT3-2 VL domain.

[0078] SEQ ID NO: 58 is the amino acid sequence of the hCT3-3 VH domain. SEQ ID NO: 59 is the amino acid sequence of the hCT3-3 VL domain.

[0079] DETAILED DESCRIPTION

[0080] I. Introduction

[0081] Pediatric neuroblastoma (NB) is a lethal extracranial solid cancer of childhood. Despite the recent improvements in outcomes with anti-disialoganglioside (GD2) immunotherapy, overall survival remains low at approximately 50%. Hence, additional therapies are needed to treat this highly aggressive cancer. Glypican-2 (GPC2) or CD276 (B7-H3) are highly but heterogeneously expressed in NB, with low or undetectable expression in normal tissues, making them promising cell-surface targets for immunotherapy. However, CAR T cell therapies targeting single antigens have thus far performed poorly in clinical trials for solid tumors, in part due to heterogenous expression of tumor- associated antigens (TAAs), limited T cell persistence, and T cell exhaustion. Thus, there remains a need for developing and optimizing GPC2-targeted or GPC2 / CD276 dual-targeted CARs with improved cytotoxicity, reduced exhaustion, and increased persistence. Moreover, GPC2 and CD276 are not only highly expressed in NB, but also in medulloblastoma, glioma, and glioblastoma. Thus, the optimized GPC2 / CD276 CAR constructs disclosed herein can be broadly applied to GPC2- and CD276-expressing solid tumors.

[0082] II. Abbreviations

[0083] ALL acute lymphoblastic leukemia

[0084] ARMS alveolar rhabdomyosarcoma

[0085] BiCisCAR bicistronic chimeric antigen receptor

[0086] CAR chimeric antigen receptor

[0087] CDR complementarity determining region

[0088] CM central memory

[0089] CSD co-stimulatory domain

[0090] DSRCT desmoplastic small round cell tumor

[0091] E:T effector to target ratio

[0092] EM effector memory

[0093] ERMS embryonal rhabdomyosarcoma

[0094] FACS fluorescence activated cell sorting

[0095] GPC2 glypican-2

[0096] GPI glycosylphosphatidylinositol

[0097] HBL hepatoblastoma

[0098] HCC hepatocellular carcinoma

[0099] HTM hinge and transmembrane

[0100] IL-2 interleukin-2

[0101] ITAM immunoreceptor tyrosine -based activation motif

[0102] IU international unit

[0103] MOI multiplicity of infection

[0104] NB neuroblastoma

[0105] NSG NOD scid gamma

[0106] PBMC peripheral blood mononuclear cell

[0107] PDX patient-derived xenograft

[0108] RMS rhabdomyosarcoma

[0109] RTCA Real-Time Cell Analysis scFv single-chain variable fragment

[0110] SCM stem cell memory

[0111] TCR T cell receptor tEGFR truncated epidermal growth factor receptor

[0112] UTD untransduced VH variable heavy

[0113] VL variable light

[0114] III. Summary of Terms

[0115] Unless otherwise noted, technical terms are used according to conventional usage. Definitions of many common terms in molecular biology may 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 as well as plural, unless the context clearly indicates otherwise. For example, the term “an antigen” includes singular or plural antigens and can be considered equivalent to the phrase “at least one antigen.” As used herein, the term “comprises” means “includes.” It is further to be understood that any and all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for descriptive purposes, unless otherwise indicated. Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described herein. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. To facilitate review of the various aspects, the following explanations of terms are provided:

[0116] 4-1BB: A co-stimulatory molecule expressed by T cell receptor (TCR)-activated lymphocytes, and by other cells including natural killer cells. Ligation of 4- IBB induces a signaling cascade that results in cytokine production, expression of anti-apoptotic molecules and an enhanced immune response. An exemplary amino acid sequence of 4- IBB is set forth herein as SEQ ID NO: 47.

[0117] Acute lymphoblastic leukemia (ALL): An acute form of leukemia characterized by the overproduction of lymphoblasts. ALL is most common in childhood, peaking at ages 2-5.

[0118] Administration: To provide or give a subject an agent, such as a CAR- or BiCisCAR- expressing cell provided herein, by any effective route. Exemplary routes of administration include, but are not limited to, oral, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, intravenous, intraprostatic, and intratumoral), sublingual, rectal, transdermal, intranasal, vaginal, and inhalation routes.

[0119] Antibody: A polypeptide ligand comprising at least one variable region that recognizes and binds (such as specifically recognizes and specifically binds) an epitope of an antigen (such as GPC2 or CD276). Mammalian immunoglobulin molecules are composed of a heavy (H) chain and a light (L) chain, each of which has a variable region, termed the variable heavy (VH) domain and the variable light (VL) domain, respectively. Together, the VH domain and the VL domain are responsible for binding the antigen recognized by the antibody. There are five main heavy chain classes (or isotypes) of mammalian immunoglobulin, which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Antibody isotypes not found in mammals include IgX, IgY, IgW and IgNAR. IgY is the primary antibody produced by birds and reptiles and is functionally similar to mammalian IgG and IgE. IgW and IgNAR antibodies are produced by cartilaginous fish, while IgX antibodies are found in amphibians.

[0120] 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 serve to position and align the CDRs in three-dimensional space. The amino acid sequence boundaries of a given CDR can be readily determined using any of a number of well-known numbering schemes, including those described by Kabat et al. (Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991; the “Kabat” numbering scheme), Chothia et al. (see Chothia and Lesk, J Mol Biol 196:901-917, 1987; Chothia et al., Nature 342:877, 1989; and Al-Lazikani et al., JMB 273,927-948, 1997; the “Chothia” numbering scheme), Kunik et al. (see Kunik et al., PLoS Comput Biol 8:el002388, 2012; and Kunik et al., Nucleic Acids Res 40(Web Server issue):W521-524, 2012; “Paratome CDRs”) and the ImMunoGeneTics (IMGT) database (see, Lefranc, Nucleic Acids Res 29:207-9, 2001; the “IMGT” numbering scheme). The Kabat, Paratome and IMGT databases are maintained online. In addition, the AbRSA tool can be used to determine the CDR boundaries according to Kabat, IMGT or Chothia (online at aligncdr.labshare.cn / aligncdr / abrsa.php).

[0121] A “single-domain antibody” refers to an antibody having a single domain (a variable domain) that is capable of specifically binding an antigen, or an epitope of an antigen, in the absence of an additional antibody domain. Single-domain antibodies include, for example, Vn domain antibodies, VNAR antibodies, camelid VHH antibodies, and VL domain antibodies. VNAR antibodies are produced by cartilaginous fish, such as nurse sharks, wobbegong sharks, spiny dogfish and bamboo sharks. Camelid VHH antibodies are produced by several species including camel, llama, alpaca, dromedary, and guanaco, which produce heavy chain antibodies that are naturally devoid of light chains.

[0122] A “monoclonal antibody” is an antibody produced by a single clone of lymphocytes or by a cell into which the coding sequence of a single antibody has been transfected. Monoclonal antibodies can be produced by known methods. Monoclonal antibodies include humanized monoclonal antibodies.

[0123] A “chimeric antibody” has framework residues from one species, such as human, and CDRs (which generally confer antigen binding) from another species (such as mouse).

[0124] A “humanized” antibody is an immunoglobulin including a human framework region and one or more CDRs from a non-human (for example a mouse, rabbit, rat, shark or synthetic) immunoglobulin. The non-human immunoglobulin providing the CDRs is termed a “donor,” and the human immunoglobulin providing the framework is termed an “acceptor.” In one aspect, all CDRs are from the donor immunoglobulin in a humanized immunoglobulin. Constant regions need not be present, but if they are, they must be substantially identical to human immunoglobulin constant regions, such as at least about 85-90%, such as about 95% or more identical. Hence, all parts of a humanized immunoglobulin, except possibly the CDRs, are substantially identical to corresponding parts of natural human immunoglobulin sequences. A humanized antibody binds to the same antigen as the donor antibody that provides the CDRs. Humanized or other monoclonal antibodies can have additional conservative amino acid substitutions which have substantially no effect on antigen binding or other immunoglobulin functions.

[0125] Binding affinity: Affinity of an antibody (or CAR) for an antigen. In one aspect, affinity is calculated by a modification of the Scatchard method described by Frankel et al., Mol. Immunol., 16: 101-106, 1979. In another aspect, binding affinity is measured by an antigen / antibody dissociation rate. In another aspect, a binding affinity is measured by a competition radioimmunoassay. In another aspect, binding affinity is measured by ELISA. In other aspects, antibody affinity is measured by flow cytometry, surface plasmon reference, or biolayer interferometry (BLI). An antibody that “specifically binds” an antigen (such as GPC2 or CD276) is an antibody that binds the antigen with high affinity and does not significantly bind other unrelated antigens.

[0126] In some examples, a CAR (such as a GPC2-targeted CAR provided herein) specifically binds to a target (such as a GPC2) with a binding constant that is at least 103M'1greater, 104M-1greater or 105M1greater than a binding constant for other molecules in a sample or subject. In some examples, a CAR has an equilibrium constant (KD) of 5 (tM or less, such as 5,000 nM or less, 900 nM or less, 500 nM or less, 250 nM or less, 100 nM or less, 50 nM or less, 10 nM or less, 5 nM or less, or 1 nM or less. For example, a CAR binds to a target, such as GPC2, with a binding affinity of at least about 1 x 10'6M, at least about 0.5 x 10'6M, at least about 1 x 10'7M, at least about 0.5 x 10’7M, at least about 1 x 10’8M, at least about 0.5 x 10’8M, at least about 1 x 10’9M, at least about 0.5 x 10’9M, or at least about 0.1 x 10'9. In certain aspects, a specific binding agent that binds to its target has a dissociation constant (Kd) of <1000 nM, <750 nM, 500 nM, <250 nM, <100 nM, <50 nM, <25 nM, <10 nM, <5 nM, <2.5 nM, <1 nM, <0.5 nM, <0.25 nM, <0.01 nM, or <0.001 nM (e.g., 10'6M or less, e.g., from 10’6M to 1010M, e.g., from 1010M to 1012M). In some examples, binding affinity is measured using the Octet system (Creative Biolabs), which is based on BLI technology. In some examples, Kd is measured using surface plasmon resonance assays using a BIACORES-2000 or a BIACORES-3000 (BIAcore, Inc., Piscataway, N.J.).

[0127] Brain cancer or tumor: A type of cancer or tumor that develops from brain tissue. Brain cancers include, but are not limited to, medulloblastoma, glioma, glioblastoma, meningioma, pituitary adenoma, astrocytoma, choroid plexus carcinoma, ependymoma and pineoblastoma.

[0128] Breast cancer: A type of cancer that forms in tissues of the breast, usually the ducts and lobules. Types of breast cancer include, for example, ductal carcinoma in situ, invasive ductal carcinoma, triple negative breast cancer, inflammatory breast cancer, metastatic breast cancer, medullary carcinoma, tubular carcinoma and mucinous carcinoma. Triple negative breast cancer refers to a type of breast cancer in which the cancer cells do not express estrogen receptors, progesterone receptors or significant levels of HER2 / neu protein. Triple negative breast cancer is also called ER-negative PR-negative HER2 / neu-negative breast cancer.

[0129] CD276: An immune checkpoint molecule that is expressed in the stroma of most or all solid tumors and may also be expressed by solid tumor cells. This protein is a member of the B7 superfamily of co-stimulatory molecules. CD276 is also known as B7H3.

[0130] CD276-expressing cancer: A cancer that expresses or overexpresses CD276. Examples of CD276-expressing cancers include, but are not limited to, liver cancers (such as hepatocellular carcinoma), pancreatic cancers, kidney cancers, bladder cancers, cervical cancers, esophageal cancers, prostate cancers, breast cancers, ovarian cancers, colon cancers, lung cancers, brain cancers (such as glioma, glioblastoma, or medulloblastoma), pediatric cancers (such as osteosarcoma, neuroblastoma, rhabdomyosarcoma or Ewing’s sarcoma), melanoma and mesothelioma (see, for example, Seaman et al., Cancer Cell 31(4):501-505, 2017). In some instances, a CD276-expressing cancer refers to a cancer in which CD276 is expressed in the tumor stroma, and may also be expressed by the tumor cells.

[0131] Chemotherapeutic agent: Any chemical agent with therapeutic usefulness in the treatment of diseases characterized by abnormal cell growth. Such diseases include tumors, neoplasms, and cancer as well as diseases characterized by hyperplastic growth. In one aspect, a chemotherapeutic agent is an agent of use in treating a GPC2-expressing tumor, a CD276-expressing tumor, or both. In one aspect, a chemotherapeutic agent is a radioactive compound. A skilled person can readily identify a chemotherapeutic agent of use (see for example, 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 2nded., © 2000 Churchill Livingstone, Inc; Baltzer, L., Berkery, R. (eds.y. Oncology Pocket Guide to Chemotherapy, 2nd ed. St. Louis, Mosby- Year Book, 1995; Fischer, D.S., Knobf, M.F., Durivage, H.J. (eds): The Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 1993). Combination chemotherapy is the administration of more than one agent to treat cancer. One example is the administration of an antibody or CAR / BiCisCAR that binds GPC2, CD276, or both, used in combination with a radioactive or chemical compound. In one example, a chemotherapeutic agent is a biologic, such as a therapeutic antibody (e.g., therapeutic monoclonal antibody), such as anti-PDl or anti-PDLl (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).

[0132] Chimeric antigen receptor (CAR): A chimeric molecule that includes an antigen-binding portion (such as a scFv) and a signaling domain, such as a signaling domain from a T cell receptor (for example, CD3Q. Typically, CARs are comprised of an antigen-binding moiety, a hinge and transmembrane domain (HTM) and an endodomain. The endodomain typically includes a signaling chain having an immunoreceptor tyrosine -based activation motif (ITAM), such as CD3C or FceRIy. In some instances, the endodomain further includes the intracellular portion of at least one additional co-stimulatory domain, such as a CD28, 4-1BB (CD137), ICOS, 0X40 (CD134), CD27 and / or DAP10 co-stimulatory domain. In some examples, the CAR is multispecific (such as bispecific) or bicistronic. A multispecific CAR is a single CAR molecule comprised of at least two antigen-binding domains (such as scFvs) that each bind a different antigen or a different epitope on the same antigen (see, for example, US 2018 / 0230225). For example, abispecific CAR refers to a single CAR molecule having two antigen-binding domains that each bind a different antigen. A bicistronic CAR refers to two complete CAR molecules, each containing an antigen-binding moiety that binds a different antigen, an HTM and an endodomain. In some cases, a bicistronic CAR construct expresses two complete CAR molecules that are linked by a cleavage linker. T cells or NK cells expressing a bispecific or bicistronic CAR can bind cells that express both antigens to which the binding moieties are directed (see, for example, Qin et al., Blood 130:810, 2017; and WO / 20I8 / 2I 337).

[0133] Colon cancer: A type of cancer that develops in the colon or the rectum. The most common type of colon cancer (also known as “colorectal cancer”) is colorectal adenocarcinoma, which accounts for approximately 95% of all colon cancers. Adenocarcinomas develop in the cells lining the inside of the colon and / or rectum. Other types of colorectal cancers include gastrointestinal carcinoid tumors, metastatic colorectal cancer, primary colorectal lymphoma (a type of nonHodgkin’s lymphoma), gastrointestinal stromal tumors (classified as a sarcoma and arising from interstitial cells of Cajal), leiomyosarcoma (arising from smooth muscle cells) and colorectal melanoma.

[0134] Complementarity determining region (CDR): Amino acid sequences which together define the binding affinity and specificity of the natural Fv region of a native Ig binding site. The light and heavy chains of an Ig each have three CDRs, designated LCDR1, LCDR2, LCDR3 and HCDR1, HCDR2 and HCDR3, respectively.

[0135] Conservative variant: A protein containing conservative amino acid substitutions that do not substantially affect or decrease the activity or affinity of a protein, such as the affinity of an antibody or CAR to GPC2, CD276, or both. For example, a monoclonal antibody or CAR that specifically binds GPC2, CD276, or both can include at most about 1, at most about 2, at most about 5, and most about 10, or at most about 15 conservative substitutions and specifically bind the GPC2 polypeptide. The term “conservative variant” also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid, provided that the antibody or CAR specifically binds GPC2, CD276, or both. Non-conservative substitutions are those that reduce an activity or binding to GPC2, CD276, or both.

[0136] Conservative amino acid substitution tables providing functionally similar amino acids are known. The following six groups are examples of amino acids that are considered to be conservative substitutions for one another:

[0137] 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E);

[0138] 3) Asparagine (N), Glutamine (Q);

[0139] 4) Arginine (R), Lysine (K);

[0140] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

[0141] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0142] Degenerate variant: A polynucleotide encoding a polypeptide that includes a sequence that is degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences are included as long as the amino acid sequence of the polypeptide is unchanged.

[0143] Desmoplastic small round cell tumor (DSRCT): A type of tumor that grows in the abdomen and pelvic area. DSRCT is typically a soft tissue sarcoma, which is a type of cancer that forms in the connective tissue of the body, such as fat, muscles, tendons, blood vessels and nerves. This type of cancer is rare and typically occurs in young white males between the ages of 10 and 30.

[0144] Epitope: An antigenic determinant. These are particular chemical groups or peptide sequences on a molecule that are antigenic (that elicit a specific immune response). An antibody specifically binds a particular antigenic epitope on a polypeptide, such as GPC2.

[0145] Ewing’s sarcoma: A rare type of malignant tumor found in bone or soft tissue. Ewing’s sarcoma is a small, blue, round cell tumor.

[0146] Framework region: Amino acid sequences interposed between CDRs. Framework regions include variable light and variable heavy framework regions. The framework regions serve to hold the CDRs in an appropriate orientation for antigen binding.

[0147] Fusion protein: A protein comprising at least a portion of two different (heterologous) proteins.

[0148] Gastric adenocarcinoma: An adenocarcinoma of the stomach (also known as stomach cancer). Gastric adenocarcinoma begins in the mucus-producing cells in the innermost lining of the stomach.

[0149] Glioblastoma: A fast-growing tumor of the central nervous system that forms from glial tissue of the brain and spinal cord. Glioblastoma typically occurs in adults and affects the brain more often than the spinal cord. Glioblastoma is also known as glioblastoma multiforme (GBM) or grade IV astrocytoma.

[0150] Glioma: A type of tumor that originates in glial cells of the brain and / or spinal cord.

[0151] Glypican-2 (GPC2): A member of the six-member glypican family of heparan sulfate (HS) proteoglycans that are attached to the cell surface by a GPI anchor (Filmus et al., Genome Biol 9:224, 2008). GPC2 is uniquely expressed in the nervous system (Stipp et al., J Cell Biol 124:149-160, 1994), participates in cell adhesion and is thought to regulate the growth and guidance of axons. In addition, GPC2 mRNA is highly expressed in neuroblastoma and other pediatric cancers (Qrentas et al., Front Oncol 2: 194, 2012). GPC2 is also known as cerebroglycan proteoglycan and glypican proteoglycan 2. GPC2 genomic, mRNA and protein sequences are publicly available (see, for example, NCBI Gene ID 221914).

[0152] GPC2-positive cancer: A cancer that expresses or overexpresses GPC2. Examples of GPC2-positive cancers include, but are not limited to, glioma, glioblastoma, neuroblastoma, medulloblastoma, retinoblastoma, acute lymphoblastic leukemia, embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, Ewing’ s sarcoma, desmoplastic small round cell tumor or osteosarcoma.

[0153] Heterologous: Originating from a separate genetic source or species.

[0154] Immune response: A response of a cell of the immune system, such as a B cell, T cell, or monocyte, to a stimulus. In one aspect, the response is specific for a particular antigen (an “antigenspecific response”). In one aspect, an immune response is a T cell response, such as a CD4+response or a CD8+response. In another aspect, the response is a B cell response, and results in the production of specific antibodies.

[0155] Isolated: An “isolated” biological component, such as a nucleic acid, protein (including antibodies or CARs) or organelle, has been substantially separated or purified away from other biological components in the environment (such as a cell) in which the component naturally occurs, i.e., other chromosomal and extra-chromosomal DNA and RNA, proteins and organelles. Nucleic acids and proteins that have been “isolated” include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids and proteins.

[0156] Linker: In some cases, a linker is a peptide within an antibody binding fragment (such as an Fv fragment) which serves to indirectly bond the variable heavy chain to the variable light chain. “Linker” can also refer to a peptide serving to link a targeting moiety, such as an antibody, to an effector molecule, such as a drug or a detectable label. In some aspects herein, the linker connects a VH domain to a VL domain of an scFv (such as an scFv targeting GPC2 or CD276).

[0157] Liver cancer: Any type of cancer occurring in liver tissue. The most common type of liver cancer is hepatocellular carcinoma (HCC), which develops in hepatocytes. Other types of liver cancer include cholangiocarcinoma, which develops in the bile ducts; liver angiosarcoma, which is a rare form of liver cancer that begins in the blood vessels of the liver; and hepatoblastoma (HBL), which is a very rare type of liver cancer found most often in children.

[0158] Lung cancer: Cancer that forms in tissues of the lung, usually in the cells lining air passages. Most cancers that begin in the lung are carcinomas. The two primary types of lung carcinoma are small-cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC). Subclasses of NSCLC include adenocarcinoma, squamous-cell carcinoma and large-cell carcinoma.

[0159] Medulloblastoma: A fast-growing type of cancer that forms in the cerebellum. Medulloblastomas tend to spread through the cerebrospinal fluid to the spinal cord or to other parts of the brain. They may also spread to other parts of the body, but this is rare. Medulloblastomas are most common in children and young adults. They are a type of central nervous system embryonal tumor.

[0160] Neoplasia, malignancy, cancer or tumor: A neoplasm is an abnormal growth of tissue or cells that results from excessive cell division. Neoplastic growth can produce a tumor. The amount of a tumor in an individual is the “tumor burden” which can be measured as the number, volume, or weight of the tumor. A tumor that does not metastasize is referred to as “benign.” A tumor that invades the surrounding tissue and / or can metastasize is referred to as “malignant.”

[0161] Neuroblastoma: A solid tumor arising from embryonic neural crest cells. Neuroblastoma commonly arises in and around the adrenal glands, but can occur anywhere that sympathetic neural tissue is found, such as in the abdomen, chest, neck or nerve tissue near the spine. Neuroblastoma typically occurs in children younger than 5 years of age.

[0162] Operably linked: A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.

[0163] Osteosarcoma: A type of cancerous tumor found in the bone. Osteosarcoma is an aggressive cancer arising from primitive transformed cells of mesenchymal origin. This type of cancer is most prevalent in children and young adults.

[0164] Ovarian cancer: Cancer that forms in tissues of the ovary. Most ovarian cancers are either ovarian epithelial carcinomas (cancer that begins in the cells on the surface of the ovary) or malignant germ cell tumors (cancer that begins in egg cells). Another type of ovarian cancer is stromal cell cancer, which originates in cells that release hormones and connect the different structures of the ovaries.

[0165] Pancreatic cancer: A disease in which malignant cells are found in the tissues of the pancreas. Pancreatic tumors can be either exocrine tumors or neuroendocrine tumors, based on the cell origin of the cancer. The vast majority (-94%) of pancreatic cancers are exocrine tumors. Exocrine cancers include, for example, adenocarcinoma (the most common type of exocrine tumor), acinar cell carcinoma, intraductal papillary-mucinous neoplasm (IPMN), and mucinous cystadenocarcinoma. In some aspects, the pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC). Pancreatic neuroendocrine tumors, also referred to as islet cell tumors, are classified by the type of hormones they produce. Exemplary neuroendocrine tumors include gastrinoma, glucaganoma, insulinoma, somatostatinoma, VIPoma (vasoactive intestinal peptide) and nonfunctional islet cell tumor.

[0166] Pediatric cancer: A cancer that develops in children ages 0 to 14. The major types of pediatric cancers 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 (such as medulloblastoma), Wilm’s tumor, non-Hodgkin lymphoma, and retinoblastoma.

[0167] Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers of use are conventional. Remington: The Science and Practice of Pharmacy, The University of the Sciences in Philadelphia, Editor, Lippincott, Williams, & Wilkins, Philadelphia, PA, 21stEdition (2005), describes compositions and formulations suitable for pharmaceutical delivery of the CARs and other compositions disclosed herein. In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (such as powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

[0168] 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 a sign or symptom 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 of size of metastases. “Ameliorating” refers to the reduction in the number or severity of signs or symptoms of a disease, such as cancer.

[0169] Prostate Cancer: A malignant tumor, generally of glandular origin, of the prostate. Prostate cancers include adenocarcinomas and small cell carcinomas. Many prostate cancers express prostate specific antigen (PSA).

[0170] Recombinant: A recombinant nucleic acid or protein is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or by the artificial manipulation of isolated segments of nucleic acids, for example, by genetic engineering techniques.

[0171] Retinoblastoma: A type of cancer that forms in the tissues of the retina. Retinoblastoma usually occurs in children younger than 5 years. It may be hereditary or nonhereditary (sporadic).

[0172] Rhabdomyosarcoma (RMS): A soft tissue malignant tumor of skeletal muscle origin. The most common primary sites for rhabdomyosarcoma are the head and neck (e.g., parameningeal, orbit, pharyngeal, etc.), the genitourinary tract, and the extremities. Other less common primary sites include the trunk, chest wall, the abdomen (including the retroperitoneum and biliary tract), and the perineal / anal region. 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 noted in adolescents and in patients with primary sites involving the extremities, trunk, and perineum / perianal region. ARMS is associated with chromosomal translocations encoding a fusion gene involving FKHR on chromosome 13 and members of the PAX family. The embryonal subtype is the most frequently observed subtype in children, accounting for approximately 60-70% of rhabdomyosarcomas of childhood. Tumors with embryonal histology typically arise in the head and neck region or in the genitourinary tract, although they may occur at any primary site. ERMS is characterized by a younger age at diagnosis, loss of heterozygosity, and altered genomic imprinting.

[0173] 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 a polypeptide or nucleic acid molecule will possess a relatively high degree of sequence identity when aligned using standard methods.

[0174] Methods of alignment of sequences for comparison are well-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. U.S.A. 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. U.S.A. 85:2444, 1988. Altschul et al., Nature Genet. 6: 119, 1994, presents a detailed consideration of sequence alignment methods and homology calculations.

[0175] 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 in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. A description of how to determine sequence identity using this program is available on the NCBI website on the internet.

[0176] Homologs and variants of a VL or a VH of an antibody that specifically binds GPC2, CD276, or both, or a fragment thereof are typically characterized by possession of at least about 75%, for example 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 using the NCBI Blast 2.0, gapped blastp set to default parameters. For comparisons of amino acid sequences of greater than about 30 amino acids, the Blast 2 sequences function is employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per residue gap cost of 1). When aligning short peptides (fewer than around 30 amino acids), the alignment should be performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties). Proteins with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. When less than the entire sequence is being compared for sequence identity, homologs and variants will typically possess at least 80% sequence identity over short windows of 10-20 amino acids, and may possess sequence identities of at least 85% or at least 90% or 95% depending on their similarity to the reference sequence. Methods for determining sequence identity over such short windows are available at the NCBI website on the internet. A skilled person will appreciate that these sequence identity ranges are provided for guidance only; it is entirely possible that strongly significant homologs could be obtained that fall outside of the ranges provided.

[0177] Subject: Living multi-cellular vertebrate organisms, a category that includes both human and veterinary subjects, including human and non-human mammals. In some aspects, a subject is a human with a GPC2-expressing cancer. In some aspects, a subject is a human with a CD276-expressing cancer. In some aspects, a subject is a human with a cancer that expresses both GPC2 and CD276.

[0178] Therapeutically effective amount: A quantity of a specific substance sufficient to achieve a desired effect in a subject being treated. For instance, this can be the amount of a CAR or bicistronic CAR (or cell expressing same) necessary to inhibit or suppress growth of a tumor. In one aspect, a therapeutically effective amount is the amount necessary to eliminate, reduce the size, or prevent metastasis of a tumor (such as a GPC2-expressing cancer, a CD276-expressing cancer, or both), such as reduce a 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 as compared to a size / volume / number prior to treatment. In one aspect, a therapeutically effective amount is the amount necessary to increase the survival time of a subject with a tumor (such as a GPC2-expressing cancer, a CD276-expressing cancer, or both), such as increase survival time by at least 10%, at least 20%, at least 50%, at least 75%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, or at least 500%, for example as compared to a survival time compared to a subject with no treatment or a different treatment. In one aspect, a therapeutically effective amount is the amount necessary to increase the survival time of a subject with a tumor (such as a GPC2-expressing cancer, CD276-expressing cancer, or both), such as increase survival time 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 as compared a survival time compared to a subject with no treatment or a different treatment. In some aspects, combinations of these effects are achieved. When administered to a subject, a dosage will generally be used that will achieve target tissue concentrations (for example, in tumors) that has been shown to achieve a desired in vitro effect.

[0179] Vector: A nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell. A vector may include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector may also include one or more selectable marker genes and other genetic elements known in the art. In some aspects, the vector is a virus vector, such as a lentivirus vector, an adeno-associated virus (AAV) vector, or an adenovirus vector.

[0180] IV. Antibody, CAR and BiCisCAR Sequences for Targeting GPC2 and CD276

[0181] Disclosed herein are chimeric antigen receptors (CARs) and bicistronic CARs (BiCisCARs) that target GPC2, or target both GPC2 and CD276 (also known as B7-H3). The GPC2-targeted CARs have an antigen-binding domain based on GPC2-specific monoclonal antibody CT3 (see, e.g., WO 2020 / 033430). The CD276-targeted CARs have an antigen-binding domain based on CD276- specific antibody MGA271, also known as enoblituzumab (see, e.g., WO 2021 / 207171). The amino acid sequences of the VH and VL domains of the CT3 and MGA271 antibodies, along with their respective CDR sequences (as determined by IMGT), are provided below. Also provided below are nucleotide and amino acid sequences of exemplary GPC2 CAR constructs and GPC2 / CD276 BiCisCAR constructs. In some instances, the CAR and BiCisCAR constructs include humanized CT3 (hCT3) VH and / or VL domain sequences, such as those listed below.

[0182] A. Antibody sequences

[0183] CT3 VH domain (SEQ ID NO: 1)

[0184] EVOLOQSGPELVKPGASVKMSCKASRFTFTDYNIHWVKOSPGKTLEWIGYINPNNGDIFYK QKFNGKATLTINKSSNTAYMELRSLTSEDSAVYYCVRSSNIRYTFDRFFDVWGTGTTVTVSS

[0185] CT3 VL domain (SEQ ID NO: 2)

[0186] ENVLTOSPAIMSASLGEKVTMSCRASSSVNYIYWYOQKSDASPKLWIYYTSNLAPGVPARFS GSGSGNSYSLTISSMEGEDAATYYCQQFSSSPSTFGTGTKLELK

[0187] Table 1. CDR sequences of antibody CT3 (IMGT) hCT3-l VH domain (SEQ ID NO: 54)

[0188] QVQLVQSGAEVKKPGASVKVSCKASRFTFTDYNIHWVRQAPGQGLEWIGYINPNNGDIFYK

[0189] QKFNGRVTLTADKSTSTAYMELSSLTSEDTAVYYCVRSSNIRYTFDRFFDVWGQGTLVTVS hCT3-l VL domain (SEQ ID NO: 55)

[0190] DVVMTQSPLSLPVTPGEPASISCRASSSVNYIYWYLQKPGQSPQLWIYYTSNLAPGVPDRFSG

[0191] SGSGTDFTLKISRVEAEDVGVYYCQQFSSSPSTFGQGTKLEIK hCT3-2 VH domain (SEQ ID NO: 56)

[0192] QVQLVQSGAEVKKPGASVKVSCKASRFTFTDYNIHWVRQAPGQRLEWIGYINPNNGDIFYK

[0193] QKFNGRVTITRDTSASTAYMELSSLRSEDTAVYYCVRSSNIRYTFDRFFDVWGQGTLVTVS hCT3-2 VL domain (SEQ ID NO: 57)

[0194] DVVMTQSPAFLSVTPGEKVTITCRASSSVNYIYWYQQKPDQAPKLWIYYTSNLAPGVPSRFS

[0195] GSGSGTDFTFTISSLEAEDAATYYCQQFSSSPSTFGQGTKLEIK hCT3-3 VH domain (SEQ ID NO: 58)

[0196] QVQLVQSGAEVKKPGASVKVSCKASRFTFTDYNIHWVRQAPGQGLEWIGYINPNNGDIFYK

[0197] QKFNGKATMTVDTSTSTVYMELSSLRSEDTAVYYCVRSSNIRYTFDRFFDVWGQGTLVTVS hCT3-3 VL domain (SEQ ID NO: 59)

[0198] DIQMTQSPSSLSASVGDRVTITCRASSSVNYIYWYQQKSGKAPKLWIYYTSNLAPGVPSRFSG

[0199] SGSGTDFTLTISSLQPEDFATYYCQQFSSSPSTFGQGTKLEIK

[0200] MGA271 VH domain SEQ ID NO: 9)

[0201] EVQLVESGGGLVOPGGSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYISSDSSAIYYA

[0202] DTVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCGRGRENIYYGSRLDYWGOGTTVTVS

[0203] S

[0204] MGA271 VL domain (SEQ ID NO: 10)

[0205] DIOLTOSPSFLSASVGDRVTITCKASONVDTNVAWYQQKPGKAPKALIYSASYRYSGVPSRFS

[0206] GSGSGTDFTLTISSLQPEDFATYYCOOYNNYPFTFGOGTKLEIK

[0207] Table 2. CDR sequences of antibody MGA271 (IMGT)

[0208] B. CAR and BiCisCAR sequences

[0209] For each of the CAR and BiCisCAR nucleic acid sequences listed below, the name of the construct and the 5' to 3' order of the components of the CAR / BiCisCAR are provided. The tables following the amino acid sequences of each construct list the nucleotide and amino acid positions of each component of the construct.

[0210] GPC2.28HTM.28z ( huIgG Leader-CT3 leader-tEGFR

[0211] ATGGAGTTTGGGCTGAGCTGGGTTTTCCTCGTAGCGCTGTTAAGAGGTGTCCAGTGTGAG GTCCAGCTGCAACAGTCTGGACCTGAACTGGTGAAGCCTGGGGCTTCAGTAAAGATGTC CTGCAAGGCTTCTAGATTCACATTCACTGACTACAACATACACTGGGTGAAGCAGAGCCC TGGAAAGACCCTTGAATGGATTGGATATATTAACCCTAACAATGGTGATATTTTCTACAA ACAGAAGTTCAATGGCAAGGCCACATTGACTATAAACAAGTCCTCCAACACAGCCTACA TGGAGCTCCGCAGCCTGACATCGGAGGATTCTGCAGTCTATTACTGTGTAAGATCCTCTA ATATTCGTTATACTTTCGACCGGTTCTTCGATGTCTGGGGCACAGGGACCACGGTCACCG TCTCCTCTGGAGGCGGAGGTTCCGGTGGCGGCGGCAGCGGTGGAGGAGGGAGCGAAAAT GTGCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCTCGGGGAGAAGGTCACCATGAGC TGCAGGGCCAGCTCAAGTGTAAATTACATTTACTGGTATCAGCAGAAGTCAGATGCCTCC CCCAAACTATGGATTTATTACACATCCAACCTGGCTCCTGGAGTCCCAGCTCGCTTCAGT GGCAGTGGGTCTGGGAACTCTTATTCTCTCACAATCAGCAGCATGGAGGGTGAAGATGCT GCCACTTATTACTGCCAGCAGTTTTCTTCTTCCCCATCCACGTTCGGTACTGGGACCAAGC TGGAGCTGAAAGCggccgcaattgaagttatgtatcctcctccttacctcgacaatgagaagagcaatggaaccattatccatgtgaaag ggaaacacctttgtccaagtcccctatttcccggaccttctaagcccttttgggtgctggtggtggttgggggagtcctggcttgctattccttgctagta acagtggcctttattattttctgggtgaggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgccgccccgggcccacc cgcaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctccAGAGTGAAGTTCAGCAGGAGCGCAG ACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGA AGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAA AGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGAT GGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCAC GATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATG CAGGCCCTGCCCCCTCGCTCTCGGGCTAAACGCTCTGGAAGCGGCgaaggacgaggtagccttcttacg tgcggagacgtggaggaaaacccaggacccatgctgctgcttgttacaagccttttgctctgcgaactcccccatccagcttttctcctgattccaag gaaggtttgcaatggaatcggtataggggagtttaaggattcacttagcataaacgctactaatattaaacacttcaaaaactgtacgagtataagtgg agatcttcacattttgccggttgcattccgaggcgattcattcacccacacgccaccgcttgacccacaagaattggatattcttaaaaccgttaaagaa ataacggggtttttgctcattcaagcgtggccagaaaatcgcactgacctccatgctttcgagaacctggagattataagaggacgaactaagcagc atggtcaattctcccttgctgtggtcagcctgaacatcaccagtcttggtttgcggtccctcaaggaaatttcagatggagatgtcatcataagcggca acaagaatttgtgctatgcaaataccataaactggaaaaaactgtttggcacttccggccagaaaaccaagattatttcaaatcggggcgagaacag ctgcaaagccaccggccaagtttgtcatgccttgtgctctccggagggctgttgggggccagaacccagggactgcgtcagttgcagaaacgtctc aagaggccgcgaatgcgttgacaagtgtaacctccttgagggcgagccacgagagtttgttgagaacagcgagtgtatacaatgtcaccctgaatg tttgccccaggctatgaatataacctgcacaggccgcgggcctgataactgcatccagtgtgctcattacatagatggacctcactgtgtgaaaacct gcccggccggagttatgggagaaaacaacactctggtgtggaaatacgctgatgcaggccacgtgtgccacctttgtcacccgaattgtacatatg ggtgtaccggtcctggacttgaaggttgccctaccaatggccctaaaatacccagtatcgcaactggcatggtaggcgctcttctcttgctcttggtgg ttgctctcggcataggtctttttatgtaa GPC2.28HTM.28z (SEQ ID NO: 18)

[0212] MEFGLSWVFLVALLRGVQCEVQLQQSGPELVKPGASVKMSCKASRFTFTDYNIHWVKQSPG

[0213] KTLEWIGYINPNNGDIFYKQKFNGKATLTINKSSNTAYMELRSLTSEDSAVYYCVRSSNIRYT

[0214] FDRFFDVWGTGTTVTVSSGGGGSGGGGSGGGGSENVLTQSPAIMSASLGEKVTMSCRASSSV

[0215] NYIYWYQQKSDASPKLWIYYTSNLAPGVPARFSGSGSGNSYSLTISSMEGEDAATYYCQQFS

[0216] SSPSTFGTGTKLELKAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVV

[0217] GGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSR

[0218] VKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNE

[0219] LQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRSRAKRSGSG

[0220] EGRGSLLTCGDVEENPGPMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKH

[0221] FKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENL

[0222] EURGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQK

[0223] TKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREF

[0224] VENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKY

[0225] ADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM

[0226] Table 3. GPC2.28HTM.28z CAR components

[0227] GPC2.28HTM.28 )

[0228] CD8 Leader-CT3 leader-MGA271 VL-VH-

[0229] CD8HTM-4-lBB atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgcccggccgGAGGTCCAGCTGCAACAGTC

[0230] TGGACCTGAACTGGTGAAGCCTGGGGCTTCAGTAAAGATGTCCTGCAAGGCTTCTAGATT

[0231] CACATTCACTGACTACAACATACACTGGGTGAAGCAGAGCCCTGGAAAGACCCTTGAAT

[0232] GGATTGGATATATTAACCCTAACAATGGTGATATTTTCTACAAACAGAAGTTCAATGGCA

[0233] AGGCCACATTGACTATAAACAAGTCCTCCAACACAGCCTACATGGAGCTCCGCAGCCTG

[0234] ACATCGGAGGATTCTGCAGTCTATTACTGTGTAAGATCCTCTAATATTCGTTATACTTTCG

[0235] ACCGGTTCTTCGATGTCTGGGGCACAGGGACCACGGTCACCGTCTCCTCTGGAGGCGGAG

[0236] GTTCCGGTGGCGGCGGCAGCGGTGGAGGAGGGAGCGAAAATGTGCTCACCCAGTCTCCA

[0237] GCAATCATGTCTGCATCTCTCGGGGAGAAGGTCACCATGAGCTGCAGGGCCAGCTCAAG

[0238] TGTAAATTACATTTACTGGTATCAGCAGAAGTCAGATGCCTCCCCCAAACTATGGATTTA

[0239] TTACACATCCAACCTGGCTCCTGGAGTCCCAGCTCGCTTCAGTGGCAGTGGGTCTGGGAA

[0240] CTCTTATTCTCTCACAATCAGCAGCATGGAGGGTGAAGATGCTGCCACTTATTACTGCCA

[0241] GCAGTTTTCTTCTTCCCCATCCACGTTCGGTACTGGGACCAAGCTGGAGCTGAAAGCGGC CGCAattgaagttatgtatcctcctccttacctcgacaatgagaagagcaatggaaccattatccatgtgaaagggaaacacctttgtccaagtccc ctatttcccggaccttctaagcccttttgggtgctggtggtggttgggggagtcctggcttgctattccttgctagtaacagtggcctttattattttctggg tgaggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgccgccccgggcccacccgcaagcattaccagccctatgc cccaccacgcgacttcgcagcctatcgctccAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACC AGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGAT GTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGA ACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGT GAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGG GTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTC GCTCTCGGGCTAAACGCTCTGGAAGCGGCGAGGGCAGAGGAAGTCTTCTAACATGCGGT GACGTGGAGGAGAATCCCGGCCCTATGCTGCTGCTTGTTACAAGCCTTTTGCTCTGCGAA CTCCCCCATCCAGCTTTTCTCCTGATTCCAAGGGATATCCAGCTGACCCAGTCCCCCTCCT TCCTGTCTGCCTCCGTGGGCGACAGAGTGACCATCACATGCAAGGCCTCCCAGAACGTGG ACACCAACGTGGCCTGGTATCAGCAGAAGCCTGGCAAGGCCCCTAAGGCGCTGATCTAC TCCGCCTCCTACCGCTACTCCGGCGTGCCTTCCCGGTTCTCCGGCTCCGGCTCTGGCACCG ACTTCACCCTGACCATCTCCAGCCTGCAGCCTGAGGACTTCGCCACCTACTACTGCCAGC AGTACAACAACTACCCTTTCACCTTCGGCCAGGGCACCAAGCTGGAAATCAAGggctccacaa gcggctctggcaagcctggatctggcgagggctctaccaagggcGAGGTGCAGCTGGTCGAGTCTGGCGGAGGAC TGGTGCAGCCTGGCGGCTCCCTGAGACTGTCTTGCGCCGCCTCCGGCTTCACCTTCTCCTC CTTCGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGACTGGAATGGGTGGCCTACA TCTCCTCCGACTCCTCCGCCATCTACTACGCCGACACCGTGAAGGGCAGGTTCACCATCT CCCGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGCGGGACGAGGAC ACCGCCGTGTACTACTGCGGCAGAGGCCGGGAGAATATCTACTACGGCTCCCGGCTGGA TTATTGGGGCCAGGGCACCACCGTGACCGTGTCTTCCGGATCCacaactactcccgcacctcggccccca acccctgctcctacaatcgcatcacagcctctgagcctgagacccgaagcttgtaggcccgcagctggaggcgccgtgcatactagaggactgga tttcgcttgcgatatctatatttgggcacctctggccggaacctgcggagtgctcctgctctccctggtcattaccctctactgcaagaggggaaggaa gaagctgctgtacatcttcaagcagcccttcatgaggcccgtccagaccacacaggaggaggacggctgctcctgccggttccccgaggaggag gagggcggctgcgagctgAGGGTGAAATTCTCCAGATCCGCCGATGCCCCTGCATATCAGCAGGG ACAGAATCAGCTGTACAATGAGCTGAACCTCGGCCGCAGGGAGGAGTATGACGTCCTGG ATAAAAGGCGCGGCAGAGATCCCGAGATGGGAGGCAAGCCTAGGCGGAAGAATCCCCA GGAGGGCCTGTATAACGAGCTGCAGAAGGACAAGATGGCAGAAGCCTATAGCGAGATC GGAATGAAGGGCGAAAGAAGGCGGGGCAAGGGCCATGACGGCCTCTATCAGGGCCTGA GCACCGCCACCAAAGATACCTATGATGCCCTCCACATGCAGGCTCTGCCTCCCAGATAA

[0242] GPC2.28HTM.28z-CD276.8HTM.BBz BiCisCAR (SEQ ID NO: 20)

[0243] MALPVTALLLPLALLLHAARPEVQLQQSGPELVKPGASVKMSCKASRFTFTDYNIHWVKQSP

[0244] GKTLEWIGYINPNNGDIFYKQKFNGKATLTINKSSNTAYMELRSLTSEDSAVYYCVRSSNIRY

[0245] TFDRFFDVWGTGTTVTVSSGGGGSGGGGSGGGGSENVLTQSPAIMSASLGEKVTMSCRASSS

[0246] VNYIYWYQQKSDASPKLWIYYTSNLAPGVPARFSGSGSGNSYSLTISSMEGEDAATYYCQQF

[0247] SSSPSTFGTGTKLELKAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVV

[0248] VGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

[0249] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYN

[0250] ELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRSRAKRSGS

[0251] GEGRGSLLTCGDVEENPGPMLLLVTSLLLCELPHPAFLLIPRDIQLTQSPSFLSASVGDRVTITC

[0252] KASQNVDTNVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATY

[0253] YCQQYNNYPFTFGQGTKLEIKGSTSGSGKPGSGEGSTKGEVQLVESGGGLVQPGGSLRLSCA

[0254] ASGFTFSSFGMHWVRQAPGKGLEWVAYISSDSSAIYYADTVKGRFTISRDNAKNSLYLQMNS

[0255] LRDEDTAVYYCGRGRENIYYGSRLDYWGQGTTVTVSSGSTTTPAPRPPTPAPTIASQPLSLRP

[0256] EACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMR

[0257] PVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTA

[0258] TKDTYDALHMQALPPR

[0259] Table 4. GPC2.28HTM.28z-CD276.8HTM.BBz BiCisCAR components

[0260] GPC2.28HTM.28z(lXX) CAR (SEQ ID NO: 21) hnlgG Leader-CT3 VH-VL-CD28HTM-CD28-CD3z(lXX)-T2A-GM-CSF leader-tEGFR

[0261] ATGGAGTTTGGGCTGAGCTGGGTTTTCCTCGTAGCGCTGTTAAGAGGTGTCCAGTGTGAG

[0262] GTCCAGCTGCAACAGTCTGGACCTGAACTGGTGAAGCCTGGGGCTTCAGTAAAGATGTC

[0263] CTGCAAGGCTTCTAGATTCACATTCACTGACTACAACATACACTGGGTGAAGCAGAGCCC

[0264] TGGAAAGACCCTTGAATGGATTGGATATATTAACCCTAACAATGGTGATATTTTCTACAA

[0265] ACAGAAGTTCAATGGCAAGGCCACATTGACTATAAACAAGTCCTCCAACACAGCCTACA

[0266] TGGAGCTCCGCAGCCTGACATCGGAGGATTCTGCAGTCTATTACTGTGTAAGATCCTCTA

[0267] ATATTCGTTATACTTTCGACCGGTTCTTCGATGTCTGGGGCACAGGGACCACGGTCACCG

[0268] TCTCCTCTGGAGGCGGAGGTTCCGGTGGCGGCGGCAGCGGTGGAGGAGGGAGCGAAAAT GTGCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCTCGGGGAGAAGGTCACCATGAGC TGCAGGGCCAGCTCAAGTGTAAATTACATTTACTGGTATCAGCAGAAGTCAGATGCCTCC CCCAAACTATGGATTTATTACACATCCAACCTGGCTCCTGGAGTCCCAGCTCGCTTCAGT

[0269] GGCAGTGGGTCTGGGAACTCTTATTCTCTCACAATCAGCAGCATGGAGGGTGAAGATGCT GCCACTTATTACTGCCAGCAGTTTTCTTCTTCCCCATCCACGTTCGGTACTGGGACCAAGC TGGAGCTGAAAGCggccgcaattgaagttatgtatcctcctccttacctcgacaatgagaagagcaatggaaccattatccatgtgaaag ggaaacacctttgtccaagtcccctatttcccggaccttctaagcccttttgggtgctggtggtggttgggggagtcctggcttgctattccttgctagta acagtggcctttattattttctgggtgaggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgccgccccgggcccacc cgcaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctccAGAGTGAAGTTCAGCAGGAGCGCAG

[0270] ACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGA

[0271] AGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAA

[0272] AGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTTCAATGAACTGCAGAAAGATAAGAT

[0273] GGCGGAGGCCTTCAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCAC GATGGCCTTTTCCAAGGTCTCAGTACAGCCACCAAGGACACCTTCGACGCCCTTCACATG

[0274] CAGGCCCTGCCCCCTCGCTCTCGGGCTAAACGCTCTGGAAGCGGCgaaggacgaggtagccttcttacg tgcggagacgtggaggaaaacccaggacccatgctgctgcttgttacaagccttttgctctgcgaactcccccatccagcttttctcctgattccaag gaaggtttgcaatggaatcggtataggggagtttaaggattcacttagcataaacgctactaatattaaacacttcaaaaactgtacgagtataagtgg agatcttcacattttgccggttgcattccgaggcgattcattcacccacacgccaccgcttgacccacaagaattggatattcttaaaaccgttaaagaa ataacggggtttttgctcattcaagcgtggccagaaaatcgcactgacctccatgctttcgagaacctggagattataagaggacgaactaagcagc atggtcaattctcccttgctgtggtcagcctgaacatcaccagtcttggtttgcggtccctcaaggaaatttcagatggagatgtcatcataagcggca acaagaatttgtgctatgcaaataccataaactggaaaaaactgtttggcacttccggccagaaaaccaagattatttcaaatcggggcgagaacag ctgcaaagccaccggccaagtttgtcatgccttgtgctctccggagggctgttgggggccagaacccagggactgcgtcagttgcagaaacgtctc aagaggccgcgaatgcgttgacaagtgtaacctccttgagggcgagccacgagagtttgttgagaacagcgagtgtatacaatgtcaccctgaatg tttgccccaggctatgaatataacctgcacaggccgcgggcctgataactgcatccagtgtgctcattacatagatggacctcactgtgtgaaaacct gcccggccggagttatgggagaaaacaacactctggtgtggaaatacgctgatgcaggccacgtgtgccacctttgtcacccgaattgtacatatg ggtgtaccggtcctggacttgaaggttgccctaccaatggccctaaaatacccagtatcgcaactggcatggtaggcgctcttctcttgctcttggtgg ttgctctcggcataggtctttttatgtaa

[0275] GPC2.28HTM.28z(lXX) CAR (SEQ ID NO: 22)

[0276] MEFGLSWVFLVALLRGVQCEVQLQQSGPELVKPGASVKMSCKASRFTFTDYNIHWVKQSPG KTLEWIGYINPNNGDIFYKQKFNGKATLTINKSSNTAYMELRSLTSEDSAVYYCVRSSNIRYT FDRFFDVWGTGTTVTVSSGGGGSGGGGSGGGGSENVLTQSPAIMSASLGEKVTMSCRASSSV NYIYWYQQKSDASPKLWIYYTSNLAPGVPARFSGSGSGNSYSLTISSMEGEDAATYYCQQFS SSPSTFGTGTKLELKAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVV GGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSR VKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLFNE LQKDKMAEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTFDALHMQALPPRSRAKRSGSGE GRGSLLTCGDVEENPGPMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHF KNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLE IIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKT

[0277] KIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFV ENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYA DAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM*

[0278] Table 5. GPC2.28HTM.28z(lXX) CAR components

[0279] GPC2.28HTM.28z / CD276.8HTM.BB BiCisCAR (SEQ ID NO: 23)

[0280] CD8 Leader-CT3 VH-VL-CD28HTM-CD28-CD3z-T2A-GM-CSF leader-MGA271 VL-VH-

[0281] CD8HTM-4-1BB atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgcccggccgGAGGTCCAGCTGCAACAGTC

[0282] TGGACCTGAACTGGTGAAGCCTGGGGCTTCAGTAAAGATGTCCTGCAAGGCTTCTAGATT CACATTCACTGACTACAACATACACTGGGTGAAGCAGAGCCCTGGAAAGACCCTTGAAT GGATTGGATATATTAACCCTAACAATGGTGATATTTTCTACAAACAGAAGTTCAATGGCA AGGCCACATTGACTATAAACAAGTCCTCCAACACAGCCTACATGGAGCTCCGCAGCCTG ACATCGGAGGATTCTGCAGTCTATTACTGTGTAAGATCCTCTAATATTCGTTATACTTTCG ACCGGTTCTTCGATGTCTGGGGCACAGGGACCACGGTCACCGTCTCCTCTGGAGGCGGAG GTTCCGGTGGCGGCGGCAGCGGTGGAGGAGGGAGCGAAAATGTGCTCACCCAGTCTCCA GCAATCATGTCTGCATCTCTCGGGGAGAAGGTCACCATGAGCTGCAGGGCCAGCTCAAG TGTAAATTACATTTACTGGTATCAGCAGAAGTCAGATGCCTCCCCCAAACTATGGATTTA TTACACATCCAACCTGGCTCCTGGAGTCCCAGCTCGCTTCAGTGGCAGTGGGTCTGGGAA CTCTTATTCTCTCACAATCAGCAGCATGGAGGGTGAAGATGCTGCCACTTATTACTGCCA GCAGTTTTCTTCTTCCCCATCCACGTTCGGTACTGGGACCAAGCTGGAGCTGAAAGCGGC CGCAattgaagttatgtatcctcctccttacctcgacaatgagaagagcaatggaaccattatccatgtgaaagggaaacacctttgtccaagtccc ctatttcccggaccttctaagcccttttgggtgctggtggtggttgggggagtcctggcttgctattccttgctagtaacagtggcctttattattttctggg tgaggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgccgccccgggcccacccgcaagcattaccagccctatgc cccaccacgcgacttcgcagcctatcgctccAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACC AGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGAT GTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGA ACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGT GAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGG GTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTC GCTCTCGGGCTAAACGCTCTGGAAGCGGCGAGGGCAGAGGAAGTCTTCTAACATGCGGT GACGTGGAGGAGAATCCCGGCCCTATGCTGCTGCTTGTTACAAGCCTTTTGCTCTGCGAA CTCCCCCATCCAGCTTTTCTCCTGATTCCAAGGGATATCCAGCTGACCCAGTCCCCCTCCT TCCTGTCTGCCTCCGTGGGCGACAGAGTGACCATCACATGCAAGGCCTCCCAGAACGTGG ACACCAACGTGGCCTGGTATCAGCAGAAGCCTGGCAAGGCCCCTAAGGCGCTGATCTAC TCCGCCTCCTACCGCTACTCCGGCGTGCCTTCCCGGTTCTCCGGCTCCGGCTCTGGCACCG ACTTCACCCTGACCATCTCCAGCCTGCAGCCTGAGGACTTCGCCACCTACTACTGCCAGC AGTACAACAACTACCCTTTCACCTTCGGCCAGGGCACCAAGCTGGAAATCAAGggctccacaa gcggctctggcaagcctggatctggcgagggctctaccaagggcGAGGTGCAGCTGGTCGAGTCTGGCGGAGGAC TGGTGCAGCCTGGCGGCTCCCTGAGACTGTCTTGCGCCGCCTCCGGCTTCACCTTCTCCTC CTTCGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGACTGGAATGGGTGGCCTACA TCTCCTCCGACTCCTCCGCCATCTACTACGCCGACACCGTGAAGGGCAGGTTCACCATCT CCCGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGCGGGACGAGGAC ACCGCCGTGTACTACTGCGGCAGAGGCCGGGAGAATATCTACTACGGCTCCCGGCTGGA

[0283] TTATTGGGGCCAGGGCACCACCGTGACCGTGTCTTCCGGATCCacaactactcccgcacctcggccccca acccctgctcctacaatcgcatcacagcctctgagcctgagacccgaagcttgtaggcccgcagctggaggcgccgtgcatactagaggactgga tttcgcttgcgatatctatatttgggcacctctggccggaacctgcggagtgctcctgctctccctggtcattaccctctactgcaagaggggaaggaa gaagctgctgtacatcttcaagcagcccttcatgaggcccgtccagaccacacaggaggaggacggctgctcctgccggttccccgaggaggag gagggcggctgcgagctgTAA

[0284] GPC2.28HTM.28z / CD276.8HTM.BB BiCisCAR (SEQ ID NO: 24)

[0285] MALPVTALLLPLALLLHAARPEVQLQQSGPELVKPGASVKMSCKASRFTFTDYNIHWVKQSP GKTLEWIGYINPNNGDIFYKQKFNGKATLTINKSSNTAYMELRSLTSEDSAVYYCVRSSNIRY TFDRFFDVWGTGTTVTVSSGGGGSGGGGSGGGGSENVLTQSPAIMSASLGEKVTMSCRASSS VNYIYWYQQKSDASPKLWIYYTSNLAPGVPARFSGSGSGNSYSLTISSMEGEDAATYYCQQF SSSPSTFGTGTKLELKAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVV VGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYN ELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRSRAKRSGS GEGRGSLLTCGDVEENPGPMLLLVTSLLLCELPHPAFLLIPRDIQLTQSPSFLSASVGDRVTITC KASQNVDTNVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQYNNYPFTFGQGTKLEIKGSTSGSGKPGSGEGSTKGEVQLVESGGGLVQPGGSLRLSCA ASGFTFSSFGMHWVRQAPGKGLEWVAYISSDSSAIYYADTVKGRFTISRDNAKNSLYLQMNS LRDEDTAVYYCGRGRENIYYGSRLDYWGQGTTVTVSSGSTTTPAPRPPTPAPTIASQPLSLRP EACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMR

[0286] PVQTTQEEDGCSCRFPEEEEGGCEL

[0287] Table 6. GPC2.28HTM.28z / CD276.8HTM.BB BiCisCAR components

[0288] GPC2.28HTM.28z(lXX) / CD276.8HTM.BB BiCisCAR (SEQ ID NO: 25)

[0289] CD8 Leader-CT3 VH-VL-CD28HTM-CD28-CD3z(lXX)-T2A-GM-CSF leader-MGA271 VL-

[0290] VH-CD8HTM-4-1BB atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgcccggccgGAGGTCCAGCTGCAACAGTC TGGACCTGAACTGGTGAAGCCTGGGGCTTCAGTAAAGATGTCCTGCAAGGCTTCTAGATT CACATTCACTGACTACAACATACACTGGGTGAAGCAGAGCCCTGGAAAGACCCTTGAAT GGATTGGATATATTAACCCTAACAATGGTGATATTTTCTACAAACAGAAGTTCAATGGCA AGGCCACATTGACTATAAACAAGTCCTCCAACACAGCCTACATGGAGCTCCGCAGCCTG

[0291] ACATCGGAGGATTCTGCAGTCTATTACTGTGTAAGATCCTCTAATATTCGTTATACTTTCG ACCGGTTCTTCGATGTCTGGGGCACAGGGACCACGGTCACCGTCTCCTCTGGAGGCGGAG GTTCCGGTGGCGGCGGCAGCGGTGGAGGAGGGAGCGAAAATGTGCTCACCCAGTCTCCA GCAATCATGTCTGCATCTCTCGGGGAGAAGGTCACCATGAGCTGCAGGGCCAGCTCAAG TGTAAATTACATTTACTGGTATCAGCAGAAGTCAGATGCCTCCCCCAAACTATGGATTTA

[0292] TTACACATCCAACCTGGCTCCTGGAGTCCCAGCTCGCTTCAGTGGCAGTGGGTCTGGGAA CTCTTATTCTCTCACAATCAGCAGCATGGAGGGTGAAGATGCTGCCACTTATTACTGCCA GCAGTTTTCTTCTTCCCCATCCACGTTCGGTACTGGGACCAAGCTGGAGCTGAAAGCGGC CGCAattgaagttatgtatcctcctccttacctcgacaatgagaagagcaatggaaccattatccatgtgaaagggaaacacctttgtccaagtccc ctatttcccggaccttctaagcccttttgggtgctggtggtggttgggggagtcctggcttgctattccttgctagtaacagtggcctttattattttctggg tgaggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgccgccccgggcccacccgcaagcattaccagccctatgc cccaccacgcgacttcgcagcctatcgctccAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACC AGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGAT GTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGA

[0293] ACCCTCAGGAAGGCCTGTTCAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTTCAGT GAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTTCCAAG GTCTCAGTACAGCCACCAAGGACACCTTCGACGCCCTTCACATGCAGGCCCTGCCCCCTC

[0294] GCTCTCGGGCTAAACGCTCTGGAAGCGGCGAGGGCAGAGGAAGTCTTCTAACATGCGGT GACGTGGAGGAGAATCCCGGCCCTATGCTGCTGCTTGTTACAAGCCTTTTGCTCTGCGAA CTCCCCCATCCAGCTTTTCTCCTGATTCCAAGGGATATCCAGCTGACCCAGTCCCCCTCCT TCCTGTCTGCCTCCGTGGGCGACAGAGTGACCATCACATGCAAGGCCTCCCAGAACGTGG ACACCAACGTGGCCTGGTATCAGCAGAAGCCTGGCAAGGCCCCTAAGGCGCTGATCTAC

[0295] TCCGCCTCCTACCGCTACTCCGGCGTGCCTTCCCGGTTCTCCGGCTCCGGCTCTGGCACCG

[0296] ACTTCACCCTGACCATCTCCAGCCTGCAGCCTGAGGACTTCGCCACCTACTACTGCCAGC

[0297] AGTACAACAACTACCCTTTCACCTTCGGCCAGGGCACCAAGCTGGAAATCAAGggctccacaa gcggctctggcaagcctggatctggcgagggctctaccaagggcGAGGTGCAGCTGGTCGAGTCTGGCGGAGGAC TGGTGCAGCCTGGCGGCTCCCTGAGACTGTCTTGCGCCGCCTCCGGCTTCACCTTCTCCTC CTTCGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGACTGGAATGGGTGGCCTACA

[0298] TCTCCTCCGACTCCTCCGCCATCTACTACGCCGACACCGTGAAGGGCAGGTTCACCATCT

[0299] CCCGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGCGGGACGAGGAC ACCGCCGTGTACTACTGCGGCAGAGGCCGGGAGAATATCTACTACGGCTCCCGGCTGGA

[0300] TTATTGGGGCCAGGGCACCACCGTGACCGTGTCTTCCGGATCCacaactactcccgcacctcggccccca acccctgctcctacaatcgcatcacagcctctgagcctgagacccgaagcttgtaggcccgcagctggaggcgccgtgcatactagaggactgga tttcgcttgcgatatctatatttgggcacctctggccggaacctgcggagtgctcctgctctccctggtcattaccctctactgcaagaggggaaggaa gaagctgctgtacatcttcaagcagcccttcatgaggcccgtccagaccacacaggaggaggacggctgctcctgccggttccccgaggaggag gagggcggctgcgagctgTAA

[0301] GPC2.28HTM.28z(lXX) / CD276.8HTM.BB BiCisCAR (SEQ ID NO: 26)

[0302] MALPVTALLLPLALLLHAARPEVQLQQSGPELVKPGASVKMSCKASRFTFTDYNIHWVKQSP

[0303] GKTLEWIGYINPNNGDIFYKQKFNGKATLTINKSSNTAYMELRSLTSEDSAVYYCVRSSNIRY

[0304] TFDRFFDVWGTGTTVTVSSGGGGSGGGGSGGGGSENVLTQSPAIMSASLGEKVTMSCRASSS VNYIYWYQQKSDASPKLWIYYTSNLAPGVPARFSGSGSGNSYSLTISSMEGEDAATYYCQQF SSSPSTFGTGTKLELKAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVV VGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

[0305] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLFN

[0306] ELQKDKMAEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTFDALHMQALPPRSRAKRSGSG EGRGSLLTCGDVEENPGPMLLLVTSLLLCELPHPAFLLIPRDIQLTQSPSFLSASVGDRVTITCK ASQNVDTNVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYY CQQYNNYPFTFGQGTKLEIKGSTSGSGKPGSGEGSTKGEVQLVESGGGLVQPGGSLRLSCAA

[0307] SGFTFSSFGMHWVRQAPGKGLEWVAYISSDSSAIYYADTVKGRFTISRDNAKNSLYLQMNSL RDEDTAVYYCGRGRENIYYGSRLDYWGQGTTVTVSSGSTTTPAPRPPTPAPTIASQPLSLRPE ACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRP VQTTQEEDGCSCRFPEEEEGGCEL

[0308] Tabic 7. GPC2.28HTM.28z(lXX) / CD276.8HTM.BB BiCisCAR components

[0309] C. Nucleotide sequences of CAR and BiCisCAR components

[0310] Human IgG leader sequence (SEQ ID NO: 27) atggagtttgggctgagctgggttttcctcgtagcgctgttaagaggtgtccagtgt

[0311] CD8 leader sequence (SEQ ID NO: 28) atggcactgcccgtgaccgccctgcttctgccgcttgcacttctgctgcacgccgctaggccc

[0312] CT3 VH-linker-VL (SEQ ID NO: 29) gaggtccagctgcaacagtctggacctgaactggtgaagcctggggcttcagtaaagatgtcctgcaaggcttctagattcacattcactgactaca acatacactgggtgaagcagagccctggaaagacccttgaatggattggatatattaaccctaacaatggtgatattttctacaaacagaagttcaatg gcaaggccacattgactataaacaagtcctccaacacagcctacatggagctccgcagcctgacatcggaggattctgcagtctattactgtgtaag atcctctaatattcgttatactttcgaccggttcttcgatgtctggggcacagggaccacggtcaccgtctcctctggaggcggaggttccggtggcg gcggcagcggtggaggagggagcgaaaatgtgctcacccagtctccagcaatcatgtctgcatctctcggggagaaggtcaccatgagctgcag ggccagctcaagtgtaaattacatttactggtatcagcagaagtcagatgcctcccccaaactatggatttattacacatccaacctggctcctggagt cccagctcgcttcagtggcagtgggtctgggaactcttattctctcacaatcagcagcatggagggtgaagatgctgccacttattactgccagcagt tttcttcttccccatccacgttcggtactgggaccaagctggagctgaaa

[0313] MGA271 VL-VH (SEQ ID NO: 30) gatatccagctgacccagtccccctccttcctgtctgcctccgtgggcgacagagtgaccatcacatgcaaggcctcccagaacgtggacaccaac gtggcctggtatcagcagaagcctggcaaggcccctaaggcgctgatctactccgcctcctaccgctactccggcgtgccttcccggttctccggct ccggctctggcaccgacttcaccctgaccatctccagcctgcagcctgaggacttcgccacctactactgccagcagtacaacaactaccctttcac cttcggccagggcaccaagctggaaatcaagggctccacaagcggctctggcaagcctggatctggcgagggctctaccaagggcgaggtgca gctggtcgagtctggcggaggactggtgcagcctggcggctccctgagactgtcttgcgccgcctccggcttcaccttctcctccttcggcatgcac tgggtccgccaggctccaggcaagggactggaatgggtggcctacatctcctccgactcctccgccatctactacgccgacaccgtgaagggca ggttcaccatctcccgggacaacgccaagaactccctgtacctgcagatgaactccctgcgggacgaggacaccgccgtgtactactgcggcag aggccgggagaatatctactacggctcccggctggattattggggccagggcaccaccgtgaccgtgtcttcc

[0314] CD28 HTM (SEQ ID NO: 31) attgaagttatgtatcctcctccttacctcgacaatgagaagagcaatggaaccattatccatgtgaaagggaaacacctttgtccaagtcccctatttc ccggaccttctaagcccttttgggtgctggtggtggttgggggagtcctggcttgctattccttgctagtaacagtggcctttattattttctgggtg CD8 HTM (codon optimized) (SEQ ID NO: 32) acaactactcccgcacctcggcccccaacccctgctcctacaatcgcatcacagcctctgagcctgagacccgaagcttgtaggcccgcagctgg aggcgccgtgcatactagaggactggatttcgcttgcgatatctatatttgggcacctctggccggaacctgcggagtgctcctgctctccctggtca ttaccctctactgc

[0315] 4-1BB co-stimulatory domain (codon optimized) (SEQ ID NO: 33) aagaggggaaggaagaagctgctgtacatcttcaagcagcccttcatgaggcccgtccagaccacacaggaggaggacggctgctcctgccgg ttccccgaggaggaggagggcggctgcgagctg

[0316] CD28 co-stimulatory domain (SEQ ID NO: 34) aggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgccgccccgggcccacccgcaagcattaccagccctatgccc caccacgcgacttcgcagcctatcgctcc

[0317] CD3 (codon optimized) (SEQ ID NO: 35) agagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagagga gtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaact gcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtc tcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgc

[0318] CD3 (1XX) signaling domain (SEQ ID NO: 36) agagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagagga gtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgttcaatgaact gcagaaagataagatggcggaggccttcagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggccttttccaaggtct cagtacagccaccaaggacaccttcgacgcccttcacatgcaggccctgccccctcgc

[0319] T2A (SEQ ID NO: 37) gaaggacgaggtagccttcttacgtgcggagacgtggaggaaaacccaggaccc

[0320] T2A (codon optimized) (SEQ ID NO: 38) gagggcagaggaagtcttctaacatgcggtgacgtggaggagaatcccggccct

[0321] GM-CSF leader (SEQ ID NO: 39) atgctgctgcttgttacaagccttttgctctgcgaactcccccatccagcttttctcctgattccaagg tEGFR (SEQ ID NO: 40) aaggtttgcaatggaatcggtataggggagtttaaggattcacttagcataaacgctactaatattaaacacttcaaaaactgtacgagtataagtgga gatcttcacattttgccggttgcattccgaggcgattcattcacccacacgccaccgcttgacccacaagaattggatattcttaaaaccgttaaagaaa taacggggtttttgctcattcaagcgtggccagaaaatcgcactgacctccatgctttcgagaacctggagattataagaggacgaactaagcagcat ggtcaattctccctlgctgtggtcagcctgaacatcaccagtcttggtttgcggtccctcaaggaaatttcagatggagatgtcatcataagcggcaac aagaatttgtgctatgcaaataccataaactggaaaaaactgtttggcacttccggccagaaaaccaagattatttcaaatcggggcgagaacagctg caaagccaccggccaagtttgtcatgccttgtgctctccggagggctgttgggggccagaacccagggactgcgtcagttgcagaaacgtctcaa gaggccgcgaatgcgttgacaagtgtaacctccttgagggcgagccacgagagtttgttgagaacagcgagtgtatacaatgtcaccctgaatgttt gccccaggctatgaatataacctgcacaggccgcgggcctgataactgcatccagtgtgctcattacatagatggacctcactgtgtgaaaacctgc ccggccggagttatgggagaaaacaacactctggtgtggaaatacgctgatgcaggccacgtgtgccacctttgtcacccgaattgtacatatgggt gtaccggtcctggacttgaaggttgccctaccaatggccctaaaatacccagtatcgcaactggcatggtaggcgctcttctcttgctcttggtggttg ctctcggcataggtctttttatgtaa

[0322] D. Amino acid sequences of CAR and BiCisCAR components

[0323] Human IgG leader sequence (SEQ ID NO: 41)

[0324] MEFGLSWVFLVALLRGVQC

[0325] CD8 leader sequence (SEQ ID NO: 42)

[0326] MALPVTALLLPLALLLHAARP

[0327] CD3 VH-linker-VL (SEQ ID NO: 43)

[0328] EVQLQQSGPELVKPGASVKMSCKASRFTFTDYNIHWVKQSPGKTLEWIGYINPNNGDIFYKQ KFNGKATLTINKSSNTAYMELRSLTSEDSAVYYCVRSSNIRYTFDRFFDVWGTGTTVTVSSG GGGSGGGGSGGGGSENVLTQSPAIMSASLGEKVTMSCRASSSVNYIYWYQQKSDASPKLWI YYTSNLAPGVPARFSGSGSGNSYSLTISSMEGEDAATYYCQQFSSSPSTFGTGTKLELK

[0329] MGA271 VL-linker-VH (SEQ ID NO: 44)

[0330] DIQLTQSPSFLSASVGDRVTITCKASQNVDTNVAWYQQKPGKAPKALIYSASYRYSGVPSRFS GSGSGTDFTLTISSLQPEDFATYYCQQYNNYPFTFGQGTKLEIKGSTSGSGKPGSGEGSTKGE VQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYISSDSSAIYYADT VKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCGRGRENIYYGSRLDYWGQGTTVTVSS (VL-linker-VH)

[0331] CD28 HTM (SEQ ID NO: 45)

[0332] IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIF wv CD8 HTM (SEQ ID NO: 46)

[0333] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLV ITLYC

[0334] 4-1BB co-stimulatory domain (SEQ ID NO: 47)

[0335] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0336] CD28 co-stimulatory domain (SEQ ID NO: 48)

[0337] RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

[0338] CD3 signaling domain (SEQ ID NO: 49)

[0339] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYN ELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0340] CD3 (1XX) signaling domain (SEQ ID NO: 50)

[0341] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLFN ELQKDKMAEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTFDALHMQALPPR

[0342] T2A (SEQ ID NO: 51)

[0343] EGRGSLLTCGDVEENPGP

[0344] GM-CSF leader (SEQ ID NO: 52)

[0345] MLLLVTSLLLCELPHPAFLLIPR tEGFR (SEQ ID NO: 53)

[0346] KVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVK EITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISG NKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCR NVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPH CVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGM

[0347] VGALLLLLVVALGIGLFM

[0348] V. CARs and BiCisCARs Targeting GPC2 and / or CD276

[0349] Disclosed herein are GPC2-targeted chimeric antigen receptors (CARs) and bicistronic CARs (BiCisCARs) that target both GPC2 and CD276. The disclosed CARs and BiCisCARs include different combinations of hinge and transmembrane (HTM) domain and co-stimulatory domain (CSD) to identify CARs and BiCisCARs with the greatest potency against GPC2- and / or CD276-expressing tumors. The CARs and BiCisCARs can further include amino acid substitutions in one or more immunoreceptor tyrosine -based activation motifs (IT AMs) of a CD3^ intracellular signaling domain. Cells expressing the CARs or BiCisCARs can be used, for example, for the treatment of cancers that express one or both of GPC2 and CD276.

[0350] A. GPC2-targeted CARs

[0351] Provided herein are GPC2-targeted CARs that include an extracellular antigen-binding domain that specifically binds GPC2, thereby targeting the CAR to GPC2-expressing cells. In some aspects, the extracellular antigen-binding domain of the CAR includes a variable heavy (VH) domain and a variable light (VL) domain, wherein the VH domain includes the complementarity determining region 1 (CDR1), CDR2 and CDR3 sequences of SEQ ID NO: 1 and the VL domain includes the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 2. In some aspects, the CAR further includes a hinge and transmembrane (HTM) domain; an intracellular co-stimulatory domain; and a CD3^ intracellular signaling domain.

[0352] In some aspects, the VH domain CDR1, CDR2 and CDR3 amino acid sequences respectively include or consist of SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5; and / or the VL domain CDR1, CDR2 and CDR3 amino acid sequences respectively include or consist of SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8. In some examples, the amino acid sequence of the VH domain is 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: 1 and includes the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 1; and / or the amino acid sequence of the VL domain is 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: 2 and includes the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 2. In specific non-limiting examples, the amino acid sequence of the VH domain includes or consists of SEQ ID NO: 1; and / or the amino acid sequence of the VL domain includes or consists of SEQ ID NO: 2.

[0353] In some aspects of the GPC2-targeted CAR, the extracellular antigen-binding domain of the CAR further includes a linker separating the VH domain and the VL domain. In some examples, the extracellular antigen-binding domain includes in the N-terminal to C-terminal orientation: VH domain-linker- VL domain. In other examples, the extracellular antigen-binding domain includes in the N-terminal to C-terminal orientation: VL domain-linker- VH domain.

[0354] In some aspects, the VH domain and / or the VL domain include human framework sequences. In some examples, the amino acid sequence of the VH domain is 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: 54, SEQ ID NO: 56, or SEQ ID NO: 58, or includes or consists of SEQ ID NO: 54, SEQ ID NO: 56, or SEQ ID NO: 58. In some examples, the amino acid sequence of the VL domain is 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: 55, SEQ ID NO: 57, or SEQ ID NO: 59, or includes or consists of SEQ ID NO: 55, SEQ ID NO: 57, or SEQ ID NO: 59.

[0355] In some aspects, the CAR includes a CD28 HTM domain and a CD28 co-stimulatory domain. In some examples, the amino acid sequence of the CD28 HTM is 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: 45. In particular examples, the amino acid sequence of the CD28 HTM includes or consists of SEQ ID NO: 45. In some examples, the amino acid sequence of the CD28 intracellular co-stimulatory domain is 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: 48. In particular examples, the amino acid sequence of the CD28 intracellular co- stimulatory domain includes or consists of SEQ ID NO: 48.

[0356] In some aspects, the CD3^ intracellular signaling domain is a wild-type CD3C. such as a wildtype human CD3^. In some examples, the amino acid sequence of the CD3^ intracellular signaling domain is 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: 49. In particular examples, the amino acid sequence of the CD3^ intracellular signaling domain includes or consists of SEQ ID NO: 49.

[0357] In other aspects, the CD3^ intracellular signaling domain includes one or more modifications, such as amino acid substitutions. For example, the modifications can result in an alteration in function or signaling activity of the CD3C intracellular signaling domain.

[0358] In some aspects, the CD3^ intracellular signaling domain includes a first, a second, and a third immunoreceptor tyrosine -based activation motif (ITAM), wherein at least one tyrosine is substituted with a phenylalanine in the second and / or third ITAM. In some examples, the second ITAM includes two tyrosine to phenylalanine substitutions or the third ITAM includes two tyrosine to phenylalanine substitutions. In some examples, the second ITAM and the third ITAM each have two tyrosine to phenylalanine substitutions. In particular examples, the amino acid sequence of the CD3^ intracellular signaling domain is 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: 50. In specific non-limiting examples, the amino acid sequence of the CD3q intracellular signaling domain includes or consists of SEQ ID NO: 50.

[0359] In some aspects, the amino acid sequence of the CAR is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to residues 20-485 of SEQ ID NO: 18 or residues 20-485 of SEQ ID NO: 22. In some examples, the amino acid sequence of the CAR includes or consists of residues 20-485 of SEQ ID NO: 18 or residues 20-485 of SEQ ID NO: 22.

[0360] Also provided are isolated cells expressing a GPC2-targeted CAR disclosed herein. In some aspects, the cell is an immune cell or an iPSC. In some examples, the immune cell is a T cell, a B cell, an NK cell, or a monocyte / macrophage. Further provided are nucleic acid molecules encoding a GPC2-targeted CAR disclosed herein. In some aspects, the nucleic acid molecule includes or consists of nucleotides 58-1455 of SEQ ID NO: 17 or nucleotides 58-1455 of SEQ ID NO: 21, or a degenerate variant thereof.

[0361] In some aspects, the nucleic acid molecule further includes a first leader sequence preceding the coding sequence of the extracellular antigen-binding domain, such as a CD8 leader sequence (e.g., a CD8 leader sequence including or consisting of SEQ ID NO: 28), or a human IgG leader sequence (e.g., a huIgG leader sequence including or consisting of SEQ ID NO: 27). In some aspects, the nucleic acid molecule further includes a nucleic acid sequence encoding a truncated epidermal growth factor receptor (tEGFR), such as a tEGFR that includes or consists of SEQ ID NO: 40. In some aspects, the nucleic acid molecule further includes a second leader sequence preceding the tEGFR coding sequence, such as a GM-CSF leader sequence (e.g., a GM-CSF leader sequence including or consisting of SEQ ID NO: 39).

[0362] In some aspects of the GPC2-targeted CARs, the nucleic acid molecule includes the following orientation in the 5’ to 3’ direction: the first leader sequence (such as a CD8 or huIgG leader sequence), the coding sequence for the extracellular antigen-binding domain, the coding sequence for the HTM (such as a CD28 HTM), the coding sequence for the co-stimulatory domain (such as a CD28 CSD), the coding sequence for the intracellular signaling domain (such as a wild-type CD3^ or an IT AM-modified CD3 , a 2A site, the second leader sequence, and the coding sequence for tEGFR. In some examples, the 2A site is a T2A site (such as a T2A site set forth as SEQ ID NO: 37).

[0363] In several aspects, the nucleic acid molecule encoding the GPC2-targeted CAR has a nucleotide sequence 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: 17 or SEQ ID NO: 21, or a degenerate variant thereof. In some examples, the nucleic acid molecule has a nucleotide sequence that includes or consists of SEQ ID NO: 17 or SEQ ID NO: 21, or a degenerate variant thereof.

[0364] Also provided are vectors that include a nucleic acid molecule encoding a GPC2-targeted CAR disclosed herein. In some aspects, the vector is a viral vector, such as a lentiviral vector. Isolated cells that include a nucleic acid molecule or vector disclosed herein are further provided. The cells can be, for example, immune cells (e.g., T cells, B cells, NK cells or monocytes / macrophages) or iPSCs.

[0365] B. GPC2-targeted and CD276-targeted BiCisCARs

[0366] Further provided herein are isolated cells that express a first CAR and a second CAR (a BiCisCAR), wherein the first CAR is a GPC2-targeted CAR disclosed herein and the second CAR is a CD276-targeted CAR that includes an extracellular antigen-binding domain that specifically binds CD276. In some aspects, the extracellular antigen-binding domain of the CAR includes a VH domain and a VL domain, wherein the VH domain includes the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 9 and the VL domain includes the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 10. In some aspects, the CAR further includes a CD8 HTM domain and a 4- IBB intracellular CSD. In some aspects, the CD276-targeted CAR further includes a CD3 intracellular signaling domain.

[0367] In some aspects of the second CAR, the VH domain CDR1, CDR2 and CDR3 amino acid sequences respectively include or consist of SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13; and / or the VL domain CDR1, CDR2 and CDR3 amino acid sequences respectively include or consist of SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16. In some examples, the amino acid sequence of the VH domain is 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: 9 and includes the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 9; and / or the amino acid sequence of the VL domain is 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: 10 and includes the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 10. In particular examples, the amino acid sequence of the VH domain includes or consists of SEQ ID NO: 9; and / or the amino acid sequence of the VL domain includes or consists of SEQ ID NO: 10.

[0368] In some aspects of the second CAR, the VH domain and / or the VL domain include human framework sequences.

[0369] In some aspects, the amino acid sequence of the CD8 HTM domain is 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: 46. In some examples, the amino acid sequence of the CD8 HTM domain includes or consists of SEQ ID NO: 46.

[0370] In some aspects, the amino acid sequence of the 4-1BB co-stimulatory domain is 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: 47. In some examples, the amino acid sequence of the 4-1BB co-stimulatory domain includes or consists of SEQ ID NO: 47.

[0371] In some aspects of the second CAR, the CD3C intracellular signaling domain is a wild-type CD3L such as a wild-type human CD3^. In some examples, the amino acid sequence of the CD3C intracellular signaling domain is 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: 49. In particular examples, the amino acid sequence of the CD3^ intracellular signaling domain includes or consists of SEQ ID NO: 49.

[0372] In other aspects of the second CAR, the CD3^ intracellular signaling domain includes one or more modifications, such as amino acid substitutions. For example, the modifications can result in an alteration in function or signaling activity of the CD3^ intracellular signaling domain.

[0373] In some aspects, the CD3^ intracellular signaling domain of the second CAR includes a first, a second, and a third IT AM, wherein at least one tyrosine is substituted with phenylalanine in the second and / or third ITAM. In some examples, the second ITAM includes two tyrosine to phenylalanine substitutions or the third ITAM includes two tyrosine to phenylalanine substitutions. In some examples, the second IT AM and the third IT AM each have two tyrosine to phenylalanine substitutions. In particular examples, the amino acid sequence of the CD3^ intracellular signaling domain is 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: 50. In specific non-limiting examples, the amino acid sequence of the CD3C intracellular signaling domain includes or consists of SEQ ID NO: 50.

[0374] In some aspects, the amino acid sequence of the second CAR is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to residues 538-1009 of SEQ ID NO: 20. In some examples, the amino acid sequence of the CAR includes or consists of residues 538-1009 of SEQ ID NO: 20.

[0375] In other aspects, the amino acid sequence of the second CAR is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to residues 538-897 of SEQ ID NO: 24. In some examples, the amino acid sequence of the CAR includes or consists of residues 538-897 of SEQ ID NO: 24.

[0376] Also provided herein are nucleic acid molecules that encode a first CAR and a second CAR (a BiCisCAR), wherein the first CAR is a GPC2-targeted CAR as disclosed herein and the second CAR is a CD276-targeted CAR that includes an extracellular antigen-binding domain that specifically binds CD276. In some aspects of the second CAR, the VH domain CDR1, CDR2 and CDR3 amino acid sequences respectively include or consist of SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13; and / or the VL domain CDR1, CDR2 and CDR3 amino acid sequences respectively include or consist of SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16. In some examples, the amino acid sequence of the VH domain is 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: 9 and includes the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 9; and / or the amino acid sequence of the VL domain is 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: 10 and includes the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 10. In particular examples, the amino acid sequence of the VH domain includes or consists of SEQ ID NO: 9; and / or the amino acid sequence of the VL domain includes or consists of SEQ ID NO: 10.

[0377] In some aspects of the second CAR, the VH domain and / or the VL domain include human framework sequences.

[0378] In some aspects of the second CAR, the amino acid sequence of the CD8 HTM domain is 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: 46. In some examples, the amino acid sequence of the CD8 HTM domain includes or consists of SEQ ID NO: 46.

[0379] In some aspects of the second CAR, the amino acid sequence of the 4- IBB co-stimulatory domain is 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: 47. In some examples, the amino acid sequence of the 4-1BB costimulatory domain includes or consists of SEQ ID NO: 47.

[0380] In some aspects of the second CAR, the CD3C intracellular signaling domain is a wild-type CD3L such as a wild-type human CD3^. In some examples, the amino acid sequence of the CD3C intracellular signaling domain is 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: 49. In particular examples, the amino acid sequence of the CD3^ intracellular signaling domain includes or consists of SEQ ID NO: 49.

[0381] In other aspects of the second CAR, the CD3^ intracellular signaling domain includes one or more modifications, such as amino acid substitutions. For example, the modifications can result in an alteration in function or signaling activity of the CD3^ intracellular signaling domain.

[0382] In some aspects, the CD3^ intracellular signaling domain of the second CAR includes a first, a second, and a third IT AM, wherein at least one tyrosine is substituted with phenylalanine in the second and / or third ITAM. In some examples, the second ITAM includes two tyrosine to phenylalanine substitutions or the third ITAM includes two tyrosine to phenylalanine substitutions. In some examples, the second ITAM and the third ITAM each have two tyrosine to phenylalanine substitutions. In particular examples, the amino acid sequence of the CD3^ intracellular signaling domain is 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: 50. In specific non-limiting examples, the amino acid sequence of the CD3C intracellular signaling domain includes or consists of SEQ ID NO: 50.

[0383] In some aspects, the amino acid sequence of the second CAR is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to residues 538-1009 of SEQ ID NO: 20. In some examples, the amino acid sequence of the CAR includes or consists of residues 538-1009 of SEQ ID NO: 20.

[0384] In other aspects, the amino acid sequence of the second CAR is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to residues 538-897 of SEQ ID NO: 24. In some examples, the amino acid sequence of the CAR includes or consists of residues 538-897 of SEQ ID NO: 24.

[0385] In some aspects, the nucleic acid molecule encoding the second CAR is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to nucleotides 1621-3027 of SEQ ID NO: 19 or nucleotides 1612-2691 of SEQ ID NO: 23. In some examples, the nucleic acid molecule encoding the second CAR includes or consists of nucleotides 1621-3027 of SEQ ID NO: 19 or nucleotides 1612-2691 of SEQ ID NO: 23.

[0386] In some aspects of the nucleic acid molecules, the coding sequences for the first CAR and the second CAR are separated by a 2A site sequence, such as a T2A site. In some examples, the nucleotide sequence of the T2A site includes or consists of SEQ ID NO: 37 or SEQ ID NO: 38. In some aspects, the nucleic acid molecule encoding the BiCisCAR further includes a first leader sequence preceding the coding sequence for the first CAR and / or a second leader sequence preceding the coding sequence for the second CAR. In some examples, the first leader sequence is a CD8 leader sequence, such as the CD8 leader sequence set forth as SEQ ID NO: 28, or a human IgG leader sequences, such as a huIgG leader sequence set forth as SEQ ID NO: 27. In some examples, the second leader sequence is a GM-CSF leader sequence, such as the GM-CSF leader sequence set forth as SEQ ID NO: 39.

[0387] In some aspects, the nucleotide sequence encoding the BiCisCAR is 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: 19, SEQ ID NO: 23, or SEQ ID NO: 25, or a degenerate variant thereof. In some examples, the nucleotide sequence encoding the BiCisCAR includes or consists of SEQ ID NO: 19, SEQ ID NO: 23, or SEQ ID NO: 25, or a degenerate variant thereof.

[0388] In some aspects, the nucleic acid molecules are operably linked to a promoter.

[0389] Also provided are vectors that include a BiCisCAR nucleic acid molecule disclosed herein. In some aspects, the vector is a viral vector, such as a lentivirus vector.

[0390] Isolated cells that include a BiCisCAR nucleic acid molecule or vector are further provided. In some aspects, the cell is an immune cell or an iPSC. In some examples, the immune cell is a T cell, a B cell, an NK cell, or a monocyte / macrophage.

[0391] C. Compositions and Methods of Use

[0392] Compositions that include a pharmaceutically acceptable carrier and a GPC2-targeted CAR, a GPC2 / CD276 BiCisCAR, a nucleic acid molecule, a vector or an isolated cell disclosed herein are further provided. CAR and BiCisCAR compositions are further described in section VI.

[0393] Also provided are methods for treating a GPC2-expressing and / or a CD276-expressing cancer in a subject. In some aspects, the method includes administering to the subject a therapeutically effective amount of a CAR, BiCisCAR, isolated cell, nucleic acid molecule, vector, or composition disclosed herein. Further provided are methods of inhibiting tumor growth or metastasis of a GPC2- expressing and / or a CD276-expressing cancer in a subject. In some aspects, the method includes administering to the subject a therapeutically effective amount of a CAR, BiCisCAR, isolated cell, nucleic acid molecule, vector, or composition disclosed herein. In some aspects of these methods, the GPC2-expressing cancer is a neuroblastoma, medulloblastoma, retinoblastoma, acute lymphoblastic leukemia, embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, Ewing’ s sarcoma, desmoplastic small round cell tumor or osteosarcoma. In some aspects, the CD276-expressing cancer is a pancreatic cancer, neuroblastoma, liver cancer, kidney cancer, bladder cancer, cervical cancer, esophageal cancer, prostate cancer, breast cancer, ovarian cancer, colon cancer, lung cancer, brain cancer, pediatric cancer, melanoma or mesothelioma. In some examples, the subject has a cancer that expresses both GPC2 and CD276. Methods of using the disclosed CAR and BiCisCAR compositions are further described in section VII.

[0394] VI. CAR and BiCisCAR Compositions

[0395] Compositions are provided that include a GPC2-targeted CAR or GPC2 / CD276-targeted BiCisCAR (such as a nucleic acid / vector encoding a GPC2-targeted CAR or GPC2 / CD276-targeted BiCisCAR, or cells expressing a GPC2-targeted CAR or GPC2 / CD276-targeted BiCisCAR), in a pharmaceutically acceptable carrier. The compositions can be prepared in unit dosage forms for administration to a subject. The amount and timing of administration are at the discretion of the treating clinician to achieve the desired outcome. The CAR / BiCisCAR composition can be formulated for systemic or local (such as intra-tumor) administration. In some aspects, the CAR / BiCisCAR composition is formulated for parenteral administration, such as intravenous administration.

[0396] The compositions for administration can include a solution of the CAR / BiCisCAR in a pharmaceutically acceptable carrier, such as an aqueous carrier. A variety of aqueous carriers can be used, for example, buffered saline and the like. These solutions are sterile and generally free of undesirable matter. These compositions can be sterilized by conventional sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of antibody in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the subject’s needs.

[0397] The compositions that include a CAR / BiCisCAR can be formulated in unit dosage form suitable for individual administration of precise dosages. In addition, the compositions can be administered in a single dose or in a multiple dose schedule. A multiple dose schedule is one in which a primary course of treatment may be with more than one separate dose, for instance 1-10 doses, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 doses, followed by other doses given at subsequent time intervals as needed to maintain or reinforce the action of the compositions. Treatment can involve daily or multidaily doses of compound(s) over a period of a few days to months, or even years. Thus, the dosage regime will also, at least in part, be determined based on the particular needs of the subject to be treated and will be dependent upon the judgment of the administering practitioner.

[0398] Typical dosages of the CAR / BiCisCAR compositions or additional agents can range from about 0.01 to about 30 mg / kg, such as from about 0.1 to about 10 mg / kg. In some examples, the dosage is at least about 0.1 mg / kg, at least about 0.2 mg / kg, at least about 0.3 mg / kg, at least about 0.4 mg / kg, at least about 0.5 mg / kg, at least about 1 mg / kg, at least about 4 mg / kg, at least about 3 mg / kg, at least about 5 mg / kg, at least about 6 mg / kg, at least about 7 mg / kg, at least about 8 mg / kg is at least about 9 mg / kg, at least about 10 mg / kg, at least about 11 mg / kg, at least about 12 mg / kg, at least about 13 mg / kg, at least about 14 mg / kg, at least about 15 mg / kg, at least about 16 mg / kg, at least about 17 mg / kg, at least about 18 mg / kg, at least about 19 mg / kg, at least about 20 mg / kg, at least about 21 mg / kg, at least about 22 mg / kg, at least about 23 mg / kg, at least about 24 mg / kg at least about 25 mg / kg, at least about 26 mg / kg, at least about 27 mg / kg, at least about 28 mg / kg, at least about 29 mg / kg, or at least about 30 mg / kg.

[0399] In particular aspects, the subject is administered a CAR / BiCisCAR or composition thereof, or additional agent(s), on a multiple daily dosing schedule, such as at least two consecutive days, 10 consecutive days, and so forth, for example for a period of weeks, months, or years. In one example, the subject is administered the CAR / BiCisCAR, composition or additional agent(s) for a period of at least 30 days, such as at least 2 months, at least 4 months, at least 6 months, at least 12 months, at least 24 months, or at least 36 months.

[0400] In some aspects, a CAR / BiCisCAR or composition is administered intravenously, subcutaneously or by another mode, daily or multiple times per week for a period of time, followed by a period of no treatment, then the cycle is repeated. In some aspects, the initial period of treatment (e.g., administration of the therapeutic agent daily or multiple times per week) is 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 1 1 weeks or 12 weeks. In a related aspect, the period of no treatment lasts for 3 days, 1 week, 2 weeks, 3 weeks or 4 weeks. In certain aspects, the dosing regimen of the therapeutic agent is daily for 3 days followed by 3 days off; or daily or multiple times per week for 1 week followed by 3 days or 1 week off; or daily or multiple times per week for 2 weeks followed by 1 or 2 weeks off; or daily or multiple times per week for 3 weeks followed by 1, 2 or 3 weeks off; or daily or multiple times per week for 4, 5, 6, 7, 8, 9, 10, 11 or 12 weeks followed by 1, 2, 3 or 4 weeks off.

[0401] The compositions disclosed herein can also be administered by other routes, including via inhalation, oral, topical or intraocular. In some examples, the composition is administered via fine- needle.

[0402] CAR / BiCisCAR compositions may be provided in lyophilized form and rehydrated with sterile water before administration, although they are also provided in sterile solutions of known concentration. The CAR / BiCisCAR solution is then added to an infusion bag containing 0.9% sodium chloride, USP, and in some cases administered at a dosage of from 0.5 to 15 mg / kg of body weight. Considerable experience is available in the art in the administration of antibody drugs, which have been marketed in the U.S. since the approval of RITUXAN™ in 1997. CAR / BiCisCAR compositions can be administered by slow infusion, rather than in an intravenous push or bolus. In one example, a higher loading dose is administered, with subsequent maintenance doses being administered at a lower level.

[0403] Controlled release parenteral formulations can be made as implants, oily injections, or as particulate systems. For a broad overview of protein delivery systems see, Banga, A.J., Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, Technomic Publishing Company, Inc., Lancaster, PA, (1995). Particulate systems include, for example, microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles. Microcapsules contain the therapeutic protein, such as a cytotoxin or a drug, as a central core. In microspheres the therapeutic is dispersed throughout the particle. Particles, microspheres, and microcapsules smaller than about 1 pm are generally referred to as nanoparticles, nanospheres, and nanocapsules, respectively. Capillaries have a diameter of approximately 5 Ltm so that only nanoparticles are administered intravenously. Microparticles are typically around 100 Ltm in diameter and are administered subcutaneously or intramuscularly. See, for example, Kreuter, J., Colloidal Drug Delivery Systems, J. Kreuter, ed., Marcel Dekker, Inc., New York, NY, pp. 219-342 (1994); and Tice & Tabibi, Treatise on Controlled Drug Delivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, NY, pp. 315-339, (1992).

[0404] Polymers can be used for ion-controlled release of the CAR / BiCisCAR compositions disclosed herein. Various degradable and nondegradable polymeric matrices for use in controlled drug delivery are known (e.g., see Langer, Accounts Chem. Res. 26:537-542, 1993). For example, the block copolymer, polaxamer 407, exists as a viscous yet mobile liquid at low temperatures but forms a semisolid gel at body temperature. Alternatively, hydroxyapatite has been used as a microcarrier for controlled release of proteins such as CARs / BiCisCARs (Ijntema et al., hit. J. Pharm. 112:215-224, 1994). In yet another aspect, liposomes are used for controlled release (Betageri et al., Liposome Drug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, PA (1993)). Numerous additional systems for controlled delivery of therapeutic proteins are known (see U.S. Patent Nos. 5,055,303; 5,188,837; 4,235,871; 4,501,728; 4,837,028; 4,957,735; 5,019,369; 5,055,303; 5,514,670; 5,413,797; 5,268,164; 5,004,697; 4,902,505; 5,506,206; 5,271,961; 5,254,342 and 5,534,496).

[0405] VII. Methods of Use

[0406] The CAR / BiCisCAR compositions disclosed herein can be administered to slow or inhibit the growth of tumor cells, inhibit the metastasis of tumor cells, and / or increase the survival of a subject having a tumor, such as a GPC2-expressing tumor and / or a CD276-expressing tumor, such as solid tumors. In these applications, a therapeutically effective amount of a composition is administered to a subject in an amount sufficient to inhibit growth, replication or metastasis of cancer cells, increase the survival of a subject having a tumor, and / or to inhibit a sign or a symptom of the cancer. Suitable subjects may include those diagnosed with a cancer that expresses GPC2, such as, but not limited neuroblastoma, medulloblastoma, retinoblastoma, acute lymphoblastic leukemia, embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, Ewing’s sarcoma, desmoplastic small round cell tumor or osteosarcoma. In some aspects, the subject has been diagnosed with a cancer that expresses CD276, such as but not limited to, a pancreatic cancer, a neuroblastoma, a liver cancer, a kidney cancer, a bladder cancer, a cervical cancer, an esophageal cancer, a prostate cancer, a breast cancer, an ovarian cancer, a colon cancer, a lung cancer, a brain cancer, a pediatric cancer, melanoma or mesothelioma.

[0407] Provided herein is a method of treating a GPC2-expressing cancer in a subject by administering to the subject a therapeutically effective amount of a GPC2-targeted CAR or BiCisCAR composition disclosed herein. Also provided herein is a method of inhibiting tumor growth or metastasis of a GPC2-expressing cancer in a subject by administering to the subject a therapeutically effective amount of a GPC2-targeted CAR or BiCisCAR composition disclosed herein. In some aspects, the GPC2-expressing cancer is neuroblastoma, medulloblastoma, retinoblastoma, acute lymphoblastic leukemia, embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, Ewing’s sarcoma, desmoplastic small round cell tumor or osteosarcoma.

[0408] Also provided herein is a method of treating a CD276-expressing cancer in a subject by administering to the subject a therapeutically effective amount of a CD276-targeted BiCisCAR composition disclosed herein. Further provided herein is a method of inhibiting tumor growth or metastasis of a CD276-expressing cancer in a subject by administering to the subject a therapeutically effective amount of a CD276-targeted BiCisCAR composition disclosed herein. In some aspects, the CD276-expressing cancer is a pancreatic cancer, neuroblastoma, liver cancer, kidney cancer, bladder cancer, cervical cancer, esophageal cancer, prostate cancer, breast cancer, ovarian cancer, colon cancer, lung cancer, brain cancer, pediatric cancer, melanoma or mesothelioma.

[0409] In some aspects of the methods, the cancer expresses both GPC2 and CD276.

[0410] A therapeutically effective amount of a GPC2-targeted CAR or a GPC2 / CD276-targeted BiCisCAR composition disclosed herein can depend upon the severity of the disease, the type of disease, and the general state of the patient’s health. A therapeutically effective amount of the CAR / BiCisCAR composition is that which provides either subjective relief of a symptom(s) or an objectively identifiable improvement as noted by the clinician or other qualified observer.

[0411] Administration of the CAR / BiCisCAR compositions disclosed herein can also be accompanied by administration of other anti-cancer agents or therapeutic treatments (such as surgical resection of a tumor). Any suitable anti-cancer agent can be administered in combination with the CAR / BiCisCAR compositions disclosed herein, such as administered prior to, concurrently with, or following administration of the CAR / BiCisCAR composition. Exemplary anti-cancer agents include, but are not limited to, chemotherapeutic agents, such as, for example, mitotic inhibitors, alkylating agents, anti-metabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, anti-survival agents, biological response modifiers, anti-hormones (e.g. anti-androgens) and anti-angiogenesis agents. Other anti-cancer treatments include radiation therapy and other antibodies that specifically target cancer cells.

[0412] Non-limiting examples of alkylating agents include nitrogen mustards (such as mechlorethamine, cyclophosphamide, melphalan, uracil mustard or chlorambucil), alkyl sulfonates (such as busulfan), nitrosoureas (such as carmustine, lomustine, semustine, streptozocin, or dacarbazine).

[0413] Non-limiting examples of antimetabolites include folic acid analogs (such as methotrexate), pyrimidine analogs (such as 5-FU or cytarabine), and purine analogs, such as mercaptopurine or thioguanine.

[0414] Non-limiting examples of natural products include vinca alkaloids (such as vinblastine, vincristine, or vindesine), epipodophyllotoxins (such as etoposide or teniposide), antibiotics (such as dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, or mitomycin C), and enzymes (such as L-asparaginase).

[0415] Non-limiting examples of miscellaneous agents include platinum coordination complexes (such as cis-diamine-dichloroplatinum II also known as cisplatin), substituted ureas (such as hydroxyurea), methyl hydrazine derivatives (such as procarbazine), and adrenocrotical suppressants (such as mitotane and aminoglutethimide).

[0416] Non-limiting examples of hormones and antagonists include adrenocorticosteroids (such as prednisone), progestins (such as hydroxyprogesterone caproate, medroxyprogesterone acetate, and magestrol acetate), estrogens (such as diethylstilbestrol and ethinyl estradiol), antiestrogens (such as tamoxifen), and androgens (such as testerone proprionate and fluoxymesterone). Examples of the most commonly used chemotherapy drugs include 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, such as docetaxel), Velban, Vincristine, VP-16, while some more newer drugs include Gemcitabine (Gemzar), Herceptin, Irinotecan (Camptosar, CPT-11), Leustatin, Navelbine, Rituxan STI-571, Taxotere, Topotecan (Hycamtin), Xeloda (Capecitabine), Zevelin and calcitriol.

[0417] Non-limiting examples of immunomodulators that can be used include 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 immune globulin (Cutter Biological), IMREG (from Imreg of New Orleans, La.), SK&F 106528, and TNF (tumor necrosis factor; Genentech).

[0418] Another common treatment for some types of cancer is surgical treatment, for example surgical resection of the cancer or a portion of it. Another example of a treatment is radiotherapy, for example administration of radioactive material or energy (such as external beam therapy) to the tumor site to help eradicate the tumor or shrink it prior to surgical resection.

[0419] EXAMPLES

[0420] The following examples are provided to illustrate particular features of certain aspects of the disclosure, but the scope of the claims should not be limited to those features exemplified. Example 1: Materials and Methods

[0421] This example provides the materials and experiment procedures for the studies described in Example 2.

[0422] Cell lines and cell culturing

[0423] Human NB cell lines, including NBEB, IMR5, and SKNAS were obtained from the NCI Pediatric Oncology Branch (POB).

[0424] Synthesis of GPC2 / CD276 BiCisCAR

[0425] The GPC2 / CD276 dual-targeted CAR was assembled using GPC2- and CD276-binding scFv regions derived from CT3 (WO 2020 / 033430) and MGA271-LH (WO 2021 / 207171). The optimal spacer domain and signaling domain including CD28 HTM, CD28 CSD and CD3C signaling domain were selected for the GPC2-targeted CAR, while CD8 HTM, 4- IBB CSD and CD3c signaling domain were selected for the CD276-targeted CAR. A cleavable T2A sequence was used to separate the two CAR constructs to enable co-expression upon transduction (FIG. 1 A). Any homologous sequences were codon-wobbled to avoid recombination. The optimized GPC2 / CD276 BiCisCAR was synthesized followed by cloning into the pELPS lentiviral transfer vector with an EF- la promoter.

[0426] CAR lentiviral production and T cell transduction

[0427] The BiCisCAR-encoding lentiviral supernatant was produced by transient transfection of the Lenti-X-293T lentiviral packaging cell line with the corresponding CAR plasmids. Thawed PBMCs were activated with CD3 and CD28 microbeads at a ratio of 1:1 (Dynabeads Human T-Expander CD3 / CD28, Thermo Fisher Scientific, Cat# 1114 ID) in AIM-V media (Invitrogen) containing 40 lU / mL recombinant IL-2 (rIL-2, Clinigen Inc.) and 5% heat-inactivated FBS for 48 hours. To generate CAR T cells, 12 million activated PBMCs were transduced with CAR-expressing lentiviral vector at a multiplicity of infection (MOI) of 20 and resuspended in a total of 15 mL fresh AIM-V media with 10 mg / mL protamine sulfate and 200 lU / mL rIL-2 in 6-well plates. T cells were then centrifuged at l,000xg for 2 hours at 32°C and incubated overnight at 37°C. A second transduction was performed on the following day by repeating the same transduction procedure as above described. The CD3:CD28 beads were removed on the third day following transduction and transduced T cells were cultured at 3xl05cells per milliliter in AIM-V medium containing 200 lU / mL IL-2, with fresh IL-2-containing media added every 2-3 days until harvest on day 8 or 9. Mock T cells, also called untransduced T cells (UTD), were treated the same as transduced T cells except during the transduction procedure. Antibodies and flow cytometry analysis

[0428] CAR expression on transduced T cells was measured by flow cytometry. Surface expression of GPC2 targeting (CT3) CAR in BiCisCAR transduced T cells was measured by staining with recombinant human GPC2-Fc Chimera Protein (Aero Biosystem, Cat# GP2-H5255), followed by R- Phycoerythrin-(Fab)2 specific for human IgG-Fc (Jackson ImmunoResearch Laboratories, Cat# 109- 116-170). Surface expression of CD276 targeting (MGA271) CAR in BiCisCAR transduced T cells was detected by staining with Biotinylated Human B7-H3 (41g) / B7-H3b Protein (Aero Biosystem, Cat# B73-H82F5), followed by incubation with R-Phycoerythrin-conjugated streptavidin.

[0429] For the T cell phenotyping panel analysis, T cells were detected using the following antihuman antibodies: CD45-FITC (BioLegend, Clone HI30), CD3-PE (BioLegend, Clone HIT3a), CD4- PE-Dazzle594 (BioLegend, Clone A161A1), CD8-APC (BioLegend, Clone RPA-T8), CD62L- Percp / Cy5.5 (BioLegend, Clone DREG-56), CD45RA- Brilliant Violet 605 (BioLegend, Clone HI100). All staining was performed in 0.1 mL FACS buffer (PBS+ 0.5% BSA+2 mM EDTA). Flow cytometry was performed using FACS Fortessa (BD Biosciences) and analyzed with FlowJo software (Tree Star).

[0430] Cytotoxicity assay of CAR T cells

[0431] For Cytolytic Assays by xCELLigence® real-time cell analysis (RTCA), 2xl04target human neuroblastoma cell lines (NBEB luc, IMR5_GL and SKNAS) were separately seeded into an E-Plate 96 (ACEA biosciences). After settling down for 4 hours, effector CAR T cells were added into the corresponding wells at an E:T ratio of 1: 1. Then the E-plate 96 was placed back to the xCELLigence RTCA SP, and impedance measurements were recorded every 15 minutes for about 70 additional hours at 37°C and 5% CO2. CAR T cell-mediated death of tumor cells was monitored in real-time and was indicated by a decrease in cell index. Data were analyzed with RTCA Software 2.0 (Acea Biosciences). Results were normalized before CAR T cell addition (about 4 hours after tumor cell addition).

[0432] Animal study

[0433] Five- to 8~week-old female NSG mice (NOD.Cg-PrkdcscidI12rgtm-lWjl / SzJ; NCI CCR Animal Resource Program, NCI Biological Testing Branch) were used for animal experiments. For the subcutaneous PDX NB model, luciferase expressing SJNB012407 PDX tumor cells (2 x 106cells) were resuspended in Matrigel (Corning) and subcutaneously injected into 1 flank of each mouse. After tumor establishment, the mice were randomized into 4 groups and separately infused via the tail vein with mock T cells or with 2.5 xlO6CAR T cells once on day 14. Total bioluminescent flux was measured using a Xenogen IVIS Lumina (PerkinElmer) every week. Mice were injected i.p. with 3 mg D-luciferin (PerkinElmer) and imaged 20 minutes later. Living Image software (PerkinElmer) was used to analyze the bioluminescence signal flux for each mouse as photons per second per square centimeter per steradian (photons / s / cm2 / sr), and tumor volume was measured by caliper and calculated using the formula (length x width2) / 2. Mice were euthanized when the tumor size reached 4000 mm3.

[0434] Example 2: Optimization of a GPC2 / CD276 BiCisCAR

[0435] This example describes the development of an optimized bicistronic CAR (BiCisCAR) against both GPC2 and CD276, using a different CSD for each CAR. Specifically, the GPC2-targeted CAR includes a CD28 HTM and a CD28 CSD, while the CD276-targeted CAR includes a CD8 HTM and a 4-1BB CSD. The optimized BiCisCAR is called GPC2.28HTM.28z-CD276.8HTM.BBz BiCisCAR (FIG. 1 A). The optimized BiCisCAR showed higher CAR+ percentages, and more stem memory T cell and central memory T cell differentiation phenotypes (FIG. 2) compared to a previously developed BiCisCAR in which both the GPC2-targeted and CD27-targeted CARs contained a CD8 HTM and a 4-1BB co-stimulatory domain (GPC2.8HTM.BBz-CD276.8HTM.BBz; Tian et al., J Clin Invest 132(16):el55621, 2022).

[0436] Optimized BiCisCAR showed better efficacy in vitro

[0437] Tn vitro NB cell killing activity of the optimized GPC2.28HTM.28z-CD276.8HTM.BBz BiCisCAR was compared to killing activity of the original GPC2.8HTM.BBz-CD276.8HTM.BBz BiCisCAR. This study included three different NB cell lines: NBEB, IMR5, and SKNAS. The results showed that the optimized GPC2.28HTM.28z-CD276.8HTM.BBz BiCisCAR induced greater cytotoxicity against all three NB cell lines compared to the original BiCisCAR (FIG. 3).

[0438] Optimized BiCisCAR showed increased efficacy in an aggressive s.c. NB PDX model

[0439] To further compare the therapeutic efficacy of BiCisCAR T cells in vivo, a GPC2- and CD276-coexpressing NB patient-derived xenograft (PDX) was used in a s.c. mouse model (FIG. 4A). GPC2.8HTM.BBz-CD276.8HTM.BBz BiCisCAR T cells moderately suppressed the tumor progression in 60% of the mice, while the optimized GPC2.28HTM.28z-CD276.8HTM.BBz BiCisCAR T cells significantly suppressed or even eliminated the tumor xenografts in all 5 mice (FIGS. 4B-4C). Mice treated with the optimized BiCisCAR also had an increased probability of survival compared to mice treated with untransduced T cells or the original BiCisCAR (FIG. 4D). These data demonstrate that the optimized BiCisCAR provides improved anti-tumor activity and extends survival (FIGS. 4B-4D). Example 3: Modification of the CD3 region of optimized CAR and BiCisCAR constructs

[0440] This example describes calibration of CAR activation by introducing modifications in the IT AMs of the CD3^ region of GPC2-targeted CAR and BiCisCAR constructs, as well as deletion of the CD3^ region in CD276-targeted BiCisCAR constructs.

[0441] Cytotoxicity assay of CAR T cells

[0442] Neuroblastoma cell lines (IMR5, NBEB, NB1691) were separately seeded at a density of 1 x 104cells per well into an E-Plate 16 (ACEA biosciences). After settling down for 4 hours, effector CAR T cells were added into the corresponding wells at an E:T ratio of 1:5. Then the E-plate 96 was placed back to the xCELLigence RTCA SP, and impedance measurements were recorded every 15 minutes for about 20 additional hours at 37°C and 5% COz- CAR T cell-mediated death of tumor cells was monitored in real time and was indicated by a decrease in cell index. Data were analyzed with RTCA Software 2.0 (Acea Biosciences). Results were normalized before CAR T cell addition (about 4 hours after tumor cell addition).

[0443] Repeated stimulation assay of CAR T cells

[0444] Tumor cells (NB1691) were seeded in 6-well plates at a concentration of 1 x 106cells per well, 1 day before the addition of T cells. T cells, normalized for transduction efficiency, were added at a CAR+ T cell to tumor cell ratio of 1:5 without the addition of exogenous cytokines. For each round of co-culture, four, three, and two duplicates were performed for the first, second, and third rounds, respectively. At the end of each co-culture round, one duplicate was collected for quantifying residual tumor cells (GFP+) and T cells (CD3+) by flow cytometry using CountBright absolute counting beads (Thermo Scientific). The remaining T cells in the other duplicates were collected and used for the next round of co-culture. For each round of co-culture, T cells were collected on days 2, 5, and 9, and transferred into a new well, where 1 x 106NB cells were seeded 1 day before T cell addition. Supernatants were collected 24 hours after the addition of T cells for cytokine measurements at each co-culture cycle. UTD cells were used as a negative control. Dead cells were excluded from analysis by staining with Ghost Dye Violet 510 (Tonbo Bioscience).

[0445] Results

[0446] Eight CAR and BiCisCAR constructs were produced (numbered 1 to 8), which are depicted in FIG. 5. After manufacturing GPC2 and CD276 CAR and BiCisCAR T cells, CAR and BiCisCAR transduction efficiency was assessed by staining with recombinant human GPC2 or CD276 protein (FIG. 6). GPC2 single-targeting CAR T cells, including GPC2.8HTM.BBz CAR, GPC2.28HTM.28z CAR (SEQ ID NO: 18), and GPC2.28HTM.28z(lXX) CAR (SEQ ID NO: 22), specifically bound to GPC2 protein, while CD276 single-targeting CAR T cells (CD276.8HTM.BBz CAR) exclusively bound to CD276 protein. In contrast, all GPC2 / CD276 dual-targeting CAR T cells exhibited binding to both GPC2 and CD276 proteins, indicating comparable expression levels of the GPC2 and CD276 CARs in these dual-targeting cells.

[0447] Next, in vitro killing activity against three neuroblastoma (NB) cell lines (IMR5, NBEB, NB1691) was tested using RTCA. Among the GPC2 and CD276 single-targeting CAR T cells, CD28- based GPC2 CAR T cells (GPC2.28HTM.28z CAR; SEQ ID NO: 18) exhibited greater cytotoxicity than 4-lBB-based GPC2 CAR T cells (GPC2.8HTM.BBz CAR). However, mutating two ITAMs in the CD3C domain of CD28-based GPC2 CAR T cells (GPC2.28HTM.28z(lXX) CAR; SEQ ID NO: 22) significantly reduced their killing activity (FIG. 7).

[0448] The killing activity of dual-targeting CAR T cells incorporating different co-stimulatory domains (CSDs) were tested. As shown in FIG. 8, GPC2 / CD276 dual-targeting CAR T cells with CD28 / 4-1BB CSDs (GPC2.28HTM.28z-CD276.8HTM.BBz; SEQ ID NO: 20) demonstrated greater killing activity than those with 4-1BB / 4-1BB CSDs (GPC2.8HTM.BBz-CD276.8HTM.BBz). However, CD3C deletion in the CD276 CAR cassette (GPC2.28HTM.28z-CD276.8HTM.BB; SEQ ID NO: 24) reduced the potency of GPC2.28HTM.28z-CD276.8HTM.BBz BiCisCAR (SEQ ID NO: 20). Additionally, mutating ITAMs in the CD28-based GPC2 CAR cassette in GPC2.28HTM.28z- CD276.8HTM.BB (SEQ ID NO: 24) further decreased its cytotoxicity (FIG. 8). This reduction in potency aligns with the known redundancy between CD28 and CD3C signaling, where attenuating CD3e reduces the overall activation signal.

[0449] To further characterize the phenotype of these CAR T cells, repeated stimulation experiments were performed to evaluate their expansion, proliferation, and exhaustion marker expression. As shown in FIG. 9, BiCisCAR T cells after CD3^ calibration (GPC2.28HTM.28z-CD276.8HTM.BB, SEQ ID NO: 24 and GPC2.28HTM.28z(lXX)-CD276.8HTM.BB, SEQ ID NO: 26) exhibited the highest CD4 / CD8 ratio, indicating improved long-term persistence. These cells also showed lower expression of PD-1 and LAG-3, indicating reduced exhaustion. These data indicate that CD3C calibration enhances the therapeutic potential of these CAR T cells in vivo by promoting prolonged persistence and reducing exhaustion.

[0450] It will be apparent that the precise details of the methods or compositions described may be varied or modified without departing from the spirit of the described aspects of the disclosure. We claim all such modifications and variations that fall within the scope and spirit of the claims below.

Claims

CLAIMS1. A chimeric antigen receptor (CAR), comprising: an extracellular antigen-binding domain that specifically binds 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: 1 and the VL domain comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 2; a CD28 hinge and transmembrane (HTM) domain: a CD28 intracellular co- stimulatory domain: and a CD3 intracellular signaling domain.

2. The CAR of claim 1, wherein: the VH domain CDR1, CDR2 and CDR3 amino acid sequences respectively comprise SEQID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5; and / or the VL domain CDR1 , CDR2 and CDR3 amino acid sequences respectively comprise SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8.

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

4. The CAR of any one of claims 1-3, wherein: the amino acid sequence of the VH domain comprises or consists of SEQ ID NO: 1; and / or the amino acid sequence of the VL domain comprises or consists of SEQ ID NO: 2.

5. The CAR of claim 1, wherein the VH domain and the VL domain comprise human framework sequences.

6. The CAR of claim 5, wherein: the amino acid sequence of the VH domain comprises or consists of SEQ ID NO: 54, SEQ ID NO: 56, or SEQ ID NO: 58; and / or the amino acid sequence of the VL domain comprises or consists of SEQ ID NO: 55, SEQ ID NO: 57, or SEQ ID NO: 59.

7. The CAR of any one of claims 1-6, wherein:the amino acid sequence of the CD28 HTM domain is at least 90% identical to SEQ ID NO:45; the amino acid sequence of the CD28 co-stimulatory domain is at least 90% identical to SEQ ID NO: 48; and / or the amino acid sequence of the CD3^ intracellular signaling domain is at least 90% identical to SEQ ID NO: 49.

8. The CAR of any one of claims 1-7, wherein: the amino acid sequence of the CD28 HTM domain comprises or consists of SEQ ID NO: 45; the amino acid sequence of the CD28 co-stimulatory domain comprises or consists of SEQ ID NO: 48; and / or the amino acid sequence of the CD3^ intracellular signaling domain comprises or consists of SEQ ID NO: 49.

9. The CAR of any one of claims 1-8, wherein the amino acid sequence of the CAR is at least 90% identical to residues 20-485 of SEQ ID NO: 18, or comprises or consists of residues 20-485 of SEQ ID NO: 18.

10. The CAR of any one of claims 1-6, wherein the CD3^ intracellular signaling domain comprises a first, a second, and a third immunoreceptor tyrosine-based activation motif (IT AM), wherein at least one tyrosine is substituted with phenylalanine in the second and / or third IT AM.

11. The CAR of claim 10, wherein: the second IT AM comprises two tyrosine to phenylalanine substitutions; the third ITAM comprises two tyrosine to phenylalanine substitutions; or the second ITAM and the third ITAM each have two tyrosine to phenylalanine substitutions.

12. The CAR of any one of claims 1-6 and 10-11, wherein the amino acid sequence of the CAR is at least 90% identical to residues 20-485 of SEQ ID NO: 22, or comprises or consists of residues 20-485 of SEQ ID NO: 22.

13. A nucleic acid molecule encoding the CAR of any one of claims 1-12.

14. The nucleic acid molecule of claim 13, comprising or consisting of: nucleotides 58-1455 of SEQ ID NO: 17, or a degenerate variant thereof; or nucleotides 58-1455 of SEQ ID NO: 21, or a degenerate variant thereof.

15. The nucleic acid molecule of claim 13 or claim 14, comprising or consisting of:SEQ ID NO: 17, or a degenerate variant thereof; or.SEQ ID NO: 21, or a degenerate variant thereof16. An isolated cell expressing a first chimeric antigen receptor (CAR) targeting glypican-2 (GPC2) and a second CAR targeting CD276, wherein the first CAR comprises the CAR of any one of claims 1-21, and the second CAR comprises: an extracellular antigen-binding domain that specifically binds CD276, 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: 9 and the VL domain comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 10; a CDS hinge and transmembrane (HTM) domain; and a 4- IBB intracellular co-stimulatory domain.

17. The isolated cell of claim 16, wherein the second CAR further comprises a CD3^ intracellular signaling domain.

18. The isolated cell of claim 16 or claim 17, wherein for the second CAR: the VH domain CDR1, CDR2 and CDR3 amino acid sequences respectively comprise SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13; and / or the VL domain CDR1, CDR2 and CDR3 amino acid sequences respectively comprise SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16.

19. The isolated cell of any one of claims 16-18, wherein for the second CAR: the amino acid sequence of the VH domain is at least 90% identical to SEQ ID NO: 9 and comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 9; and / or the amino acid sequence of the VL domain is at least 90% identical to SEQ ID NO: 10 and comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 10.

20. The isolated cell of any one of claims 16-19, wherein for the second CAR: the amino acid sequence of the VH domain comprises or consists of SEQ ID NO: 9; and / or the amino acid sequence of the VL domain comprises or consists of SEQ ID NO: 10.

21. The isolated cell of any one of claims 16-20, wherein for the second CAR, the VH domain and the VL domain comprise human framework sequences.

22. The isolated cell of any one of claims 16-21, wherein the amino acid sequence of the CD8 HTM is at least 90% identical to SEQ ID NO: 46, or comprises or consists of SEQ ID NO: 46; the amino acid sequence of the 4- IBB intracellular co-stimulatory domain is at least 90% identical to SEQ ID NO: 47, or comprises or consists of SEQ ID NO: 47; and / or the amino acid sequence of the CD3^ intracellular signaling domain is at least 90% identical to SEQ ID NO: 49, or comprises or consists of SEQ ID NO: 49.

23. The isolated cell of any one of claims 16-22, wherein: the amino acid sequence of the second CAR is at least 90% identical to residues 538-1009 of SEQ ID NO: 20, or comprises or consists of residues 538-1009 of SEQ ID NO: 20; or the amino acid sequence of the second CAR is at least 90% identical to residues 538-897 of SEQ ID NO: 24, or comprises or consists of residues 538-897 SEQ ID NO: 24.

24. A nucleic acid molecule encoding a first chimeric antigen receptor (CAR) targeting glypican-2 (GPC2) and a second CAR targeting CD276, wherein the first CAR comprises the CAR of any one of claims 1-21, and the second CAR comprises: an extracellular antigen-binding domain that specifically binds CD276, 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: 9 and the VL domain comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 10; a CD8 hinge and transmembrane (HTM) domain; and a 4- IBB intracellular co-stimulatory domain.

25. The nucleic acid molecule of claim 24, wherein the second CAR further comprises a CD3C intracellular signaling domain.

26. The nucleic acid molecule of claim 24 or claim 25, wherein for the second CAR: the VH domain CDR1, CDR2 and CDR3 amino acid sequences respectively comprise SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13; and / or the VL domain CDR1, CDR2 and CDR3 amino acid sequences respectively comprise SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16.

27. The nucleic acid molecule of any one of claims 24-26, wherein for the second CAR: the amino acid sequence of the VH domain is at least 90% identical to SEQ ID NO: 9 and comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 9; and / orthe amino acid sequence of the VL domain is at least 90% identical to SEQ ID NO: 10 and comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 10.

28. The nucleic acid molecule of any one of claims 24-27, wherein for the second CAR: the amino acid sequence of the VH domain comprises or consists of SEQ ID NO: 9; and / or the amino acid sequence of the VL domain comprises or consists of SEQ ID NO: 10.

29. The nucleic acid molecule of any one of claims 24-26, wherein for the second CAR, the VH domain and the VL domain comprise human framework sequences.

30. The nucleic acid molecule of any one of claims 24-29, wherein: the amino acid sequence of the CD8 HTM is at least 90% identical to SEQ ID NO: 46, or comprises or consists of SEQ ID NO: 46; the amino acid sequence of the 4- IBB intracellular co-stimulatory domain is at least 90% identical to SEQ ID NO: 47, or comprises or consists of SEQ ID NO: 47; and / or the amino acid sequence of the CD3^ intracellular signaling domain is at least 90% identical to SEQ ID NO: 49, or comprises or consists of SEQ ID NO: 49.

31. The nucleic acid molecule of any one of claims 24-30, wherein: the amino acid sequence of the second CAR is at least 90% identical to residues 538-1009 of SEQ ID NO: 20, or comprises or consists of residues 538-1009 of SEQ ID NO: 20; or the amino acid sequence of the second CAR is at least 90% identical to residues 538-897 of SEQ ID NO: 24, or comprises or consists of residues 538-897 SEQ ID NO: 24.

32. The nucleic acid molecule of any one of claims 24-31, comprising or consisting of SEQ ID NO: 19, SEQ ID NO: 23, or SEQ ID NO: 25.

33. The nucleic acid molecule of any one of claims 13-15 and 24-32, operably linked to a promoter.

34. A vector comprising the nucleic acid molecule of any one of claims 13-15 and 24-33.

35. The vector of claim 34, which is a lentivirus vector.

36. An isolated cell comprising the nucleic acid molecule of any one of claims 13-15 and 24-33 or the vector of claim 34 or claim 35.

37. The isolated cell of claim 36, wherein the cell is an immune cell or an induced pluripotent stem cell (iPSC).

38. The isolated cell of claim 37, wherein the immune cell is a T cell, a B cell, a natural killer (NK) cell or a monocyte / macrophage.

39. A composition comprising a pharmaceutically acceptable carrier and the CAR of any one of claims 1-12, the isolated cell of any one of claims 16-23 and 36-38, the nucleic acid molecule of any one of claims 13-15 and 24-33, or the vector of claim 34 or claim 35.

40. A method of treating a GPC2-expressing cancer in a subject, comprising administering to the subject a therapeutically effective amount of the CAR of any one of claims 1-12, the isolated cell of any one of claims 16-23 and 36-38, the nucleic acid molecule of any one of claims 13-15 and 24-33, the vector of claim 34 or claim 35, or the composition of claim 39, thereby treating the GPC2-expressing cancer.41 . A method of inhibiting tumor growth or metastasis of a GPC2-expressing cancer in a subject, comprising administering to the subject a therapeutically effective amount of the CAR of any one of claims 1-12, the isolated cell of any one of claims 16-23 and 36-38, the nucleic acid molecule of any one of claims 13-15 and 24-33, the vector of claim 34 or claim 35, or the composition of claim 39, thereby inhibiting tumor growth or metastasis of the GPC2-expressing cancer.

42. The method of claim 40 or claim 41, wherein the GPC2-expressing cancer is a neuroblastoma, medulloblastoma, retinoblastoma, acute lymphoblastic leukemia, embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, Ewing’s sarcoma, desmoplastic small round cell tumor or osteosarcoma.

43. The method of any one of claims 40-42, wherein the GPC2-expressing cancer also expresses CD276.

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