High-affinity monoclonal antibody targeting glypican-2 and its use

Abstract

Monoclonal antibodies that bind to glypican-2 (GPC2) with high affinity are described. Immunotoxins and chimeric antigen receptors (CARs) comprising the disclosed antibodies or antigen-binding fragments thereof are further described. In some cases, the antibodies or antigen-binding fragments are humanized. The disclosed GPC2-specific antibodies and conjugates can be used, for example, to diagnose or treat GPC2-positive cancers, including neuroblastoma, medulloblastoma, and retinoblastoma.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 716,169, filed August 8, 2018, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This disclosure relates to monoclonal antibodies that specifically bind to phosphatidylinositol proteoglycan-2 and their uses, such as for the treatment of pediatric cancers.

[0004] Thank you for the government's support

[0005] This invention was completed with government support, under project number Z01 BC010891 granted by the National Institutes of Health. The government holds certain rights to this invention. Background Technology

[0006] Neuroblastoma is the most common extracranial solid tumor in children. It originates from the neuroendocrine tissue of the sympathetic nervous system and accounts for 8-10% of childhood cancers in the United States (Maris and Hogarty, Lancet 369:2106-2120, 2007). Neuroblastoma is a complex and heterogeneous disease; even with intensive multimodal therapy, nearly 50% of patients have a high-risk phenotype characterized by widespread cancer spread and poor long-term survival (Yu et al., New Engl J Med 363:1324-1334, 2010). Approximately 45% of patients receiving standard therapy relapse and eventually die from metastatic disease (Matthay et al., New Engl J Med 341:1165-1173, 1999). Therefore, there is an urgent need for safe and effective treatments for neuroblastoma.

[0007] One of the most challenging aspects of treating neuroblastoma and other deadly solid tumors is the lack of tumor-specific targets. Phosphatidylinositol proteoglycan-2 (GPC2) mRNA has been shown to be highly expressed in neuroblastoma and other pediatric cancers (Orentas et al., Front Oncol 2:194, 2012). GPC2 belongs to the six-member human phosphatidylinositol proteoglycan family, which attaches to the cell surface via a glycosylphosphatidylinositol (GPI) anchor (Filmus et al., GenomeBiol 9:224, 2008). Unlike other known phosphatidylinositol proteoglycans, GPC2 is uniquely expressed in the nervous system (Stipp et al., JCellBiol 124:149-160, 1994), involved in cell adhesion, and is thought to regulate axonal growth and guidance. Summary of the Invention

[0008] This disclosure describes two high-affinity GPC2-specific monoclonal antibodies isolated using mouse hybridoma technology. The GPC2 antibodies (designated CT3 and CT5) specifically bind to GPC2 but not to other phosphatidylinositol proteoglycans. Immunotoxins composed of the disclosed antibodies and chimeric antigen receptor (CAR) T cells are able to effectively kill GPC2-positive tumor cells.

[0009] This document provides monoclonal antibodies (or antigen-binding fragments) that bind (e.g., specifically bind) to GPC2. In some embodiments, the monoclonal antibody or antigen-binding fragment includes a complementarity-determining region (CDR) sequence of CT3 or CT5. This document also provides conjugates comprising the disclosed monoclonal antibody or antigen-binding fragment. In some examples, immunoconjugates, CARs, multispecific antibodies, antibody-drug conjugates (ADCs), antibody-nanoparticles, conjugates, or fusion proteins comprising the monoclonal antibody or antigen-binding fragment disclosed herein are provided. This disclosure also provides compositions comprising a GPC-specific monoclonal antibody or antigen-binding fragment and a pharmaceutically acceptable carrier.

[0010] This article also provides nucleic acid molecules and vectors encoding the GPC2-specific monoclonal antibodies or antigen-binding fragments, immunoconjugates, CARs, multispecific antibodies, and fusion proteins disclosed herein.

[0011] Nucleic acid constructs encoding a GPC2-specific CAR and a truncated human epidermal growth factor receptor (huEGFRt) are also provided. The encoded CAR comprises a GPC2-specific monoclonal antibody fragment (e.g., scFv) fused to an extracellular hinge region, a transmembrane region, an intracellular costimulatory domain, and an intracellular signal transduction domain. huEGFRt contains two EGFR extracellular domains (domain III and domain IV) and an EGFR transmembrane domain, but lacks two distal extracellular domains and all intracellular domains. In some embodiments, the nucleic acid molecule includes, in the 5' to 3' orientation, a nucleic acid encoding a first signal sequence; a nucleic acid encoding a GPC2-specific antibody or an antigen-binding fragment thereof; a nucleic acid encoding the extracellular hinge region; a nucleic acid encoding the transmembrane domain; a nucleic acid encoding the intracellular costimulatory domain; a nucleic acid encoding the intracellular signal transduction domain; a nucleic acid encoding a self-cleaving 2A peptide; a nucleic acid encoding a second signal sequence; and a nucleic acid encoding huEGFRt. Vectors, such as viral vectors, comprising the nucleic acid molecules disclosed herein are also provided. It also disclosed isolated cells, such as T lymphocytes, that co-express the disclosed CAR and huEGFRt.

[0012] Methods for treating GPC2-positive cancers in subjects are also provided, as well as methods for inhibiting tumor growth or metastasis of GPC2-positive cancers in subjects. In some embodiments, the method includes administering to a subject a monoclonal antibody or antigen-binding fragment disclosed herein, or administering to a subject an immunoconjugate, CAR, ADC, multispecific antibody, antibody-nanoparticle conjugate, or fusion protein comprising a monoclonal antibody or antigen-binding fragment disclosed herein.

[0013] This document further provides a method for detecting GPC2 expression in a sample. In some embodiments, the method includes contacting the sample with a monoclonal antibody or antigen-binding fragment disclosed herein and detecting the binding of the antibody to the sample.

[0014] Methods for diagnosing GPC2-positive cancer in subjects are also provided. In some embodiments, the method includes contacting a sample obtained from the subject with a monoclonal antibody or antigen-binding fragment disclosed herein and detecting antibody binding to the sample.

[0015] The foregoing and other objects and features of this disclosure will become more apparent from the following detailed description with reference to the accompanying drawings. Attached Figure Description

[0016] Figure 1A ELISA analysis of the binding of CT3 and CT5 to GPC2 and other phosphatidylinositol proteoglycans. Both antibodies specifically bind to GPC2.

[0017] Figure 1B ELISA analysis of the binding of CT3 to human phosphatidylinositol proteoglycans (huGPC1, huGPC2, huGPC3, huGPC4, huGPC5, and huGPC6) and mouse GPC2 (msGPC2). CT3 antibody specifically binds to human GPC2.

[0018] Figure 1C Octet body kinetics analysis of the interaction between CT3 antibody and human GPC2. CT3 exhibits high affinity (K2) for human GPC2. D =0.5nM) combined with GPC2.

[0019] Figure 2A GPC2 expression on the cell surface of GPC2-positive neuroblastoma cell lines LAN1 and IMR5, as determined by flow cytometry. White peaks represent staining of the cell surface by allotype control antibody, and black peaks represent staining by CT5 antibody. CT5 at 10 μg / ml was used for staining.

[0020] Figure 2BCT3 antibody specifically binds to human GPC2 on the cell surface. A431 cells that do not express GPC2 were used as a control. G10 is an A431 cell line overexpressing GPC2. F8 is an IMR5 cell line overexpressing GPC2. The peak labeled "1" represents staining of the cell surface by the allotype control antibody, and the peak labeled "2" represents staining of the cell surface by the anti-GPC2 antibody CT3. CT3 specifically recognizes GPC2 expressed on the cell surface.

[0021] Figure 3 GPC2 expression in human neuroblastoma tumors. GPC2 expression in neuroblastoma (i to iv) and normal nerve (v and vi) tissues, as determined by immunohistochemistry. Tissues were labeled with 1 μg / ml CT3 antibody.

[0022] Figure 4 GPC2 expression in human medulloblastoma tumors. GPC2 expression in medulloblastoma tumors (i to iv) and normal brain tissue (v and vi) was determined by immunohistochemistry. Tissues were labeled with 1 μg / ml CT3 antibody.

[0023] Figure 5 GPC2 expression in human retinoblastoma tumors. GPC2 expression in retinoblastoma tumors (i to iv) and adjacent eye (v and vi) tissues was determined by immunohistochemistry. Tissues were labeled with 1 μg / ml CT3 antibody.

[0024] Figure 6 GPC2 expression in normal human tissues. GPC2 expression in normal human tissues (including brain, heart, lung, liver, stomach, small intestine, colon, pancreas, spleen, kidney, and skin) as determined by immunohistochemistry. Tissues were labeled with 1 μg / ml CT3 antibody. No significant GPC2 staining was detected in any normal tissue.

[0025] Figures 7A-7E CT3 CAR T cells targeting GPC2 kill neuroblastoma cells. Figure 7A A schematic diagram of a lentiviral construct expressing a GPC2-targeting CT3 CAR along with a truncated human EGFR (huEGFRt) using a T2A ribosomal skipping sequence. Figure 7B Flow cytometry was used to analyze CT3 CAR expression on human T cells transduced with lentiviral particles by detecting huEGFRt expression. Figures 7C-7E ) Cellular lysis activity of CAR T cells targeting GPC2 in cell assays. SKNBE2 expressing luciferase ( Figure 7C ), NBEB Figure 7D ) and LAN1 ( Figure 7E Neuroblastoma cells were co-cultured with either a mock control or CT3 CAR-transduced T cells at a specified effector (E):target (T) ratio for 20 hours, and specific lysis was measured using a chemiluminescence-based cell lysis assay. CT3 CAR-transduced cells induced effective lysis of GPC2-expressing neuroblastoma cells.

[0026] Figures 8A-8D The cytolytic activity of GPC2-targeting CAR T cells was measured after co-culturing GPC2-positive cells (G10, IMR5, F8) and GPC2-negative cells (A431) at a specified E:T ratio for 20 hours. Specific lysis was measured using a chemiluminescence-based cytolysis assay. hCT3#1, #2, and #3 represent three CAR versions with different humanized CT3 antibodies (as shown in SEQ ID NO: 10, 11, and 21, respectively). CT3 and humanized CT3 (hCT3) CAR T cells effectively lysed GPC2-positive tumor cells but not GPC2-negative A431 cells.

[0027] Figures 9A-9B :( Figure 9A A schematic diagram of experiments in which IMR5 tumor-bearing NSG mice were treated with 2.5 million, 5 million, and 10 million (CT3) CAR T cells via intravenous tail vein injection of blank control T cells (blank control). Tumor burden was monitored by bioluminescence imaging on days 0 (infusion), 3, 11, 10, and 28 after CAR T cell infusion. Figure 9B CT3CAR induces regression of metastatic IMR5 neuroblastoma tumors. Partial remission can be achieved with doses as low as 2.5 million CT3CAR T cells, while complete remission can be achieved with doses of 5 million and 10 million CT3CAR T cells.

[0028] Figure 10 CAR vector-positive cells were detected in mouse spleens 2-4 weeks after treatment. Spleens were harvested on day 34 (2.5M#737), day 14 (5M#733), and day 28 (10M#742) post-treatment. One month after treatment, CT3CAR T cells accounted for 21.2% to 41.2% of total mouse spleen cells.

[0029] Figure 11A-11G Characterization of CT3-PE38 and CT3-T20 immunotoxins. Figure 11AHuman GPC2-His (250 ng / ml) was loaded onto a Ni-NTA biosensor using the Octet Red96e system. Immunotoxins (CT3-PE38 and CT3-T20) were added at 100 nM to determine binding affinity. Association occurred at 600 seconds, and dissociation at 1800 seconds. The affinity of CT3-PE38 and CT3-T20 for human GPC2 (K0.05) was determined. D The values ​​were determined to be 0.22 nM and 0.16 nM, respectively. Figure 11B-11C CT3-PE38 immunotoxin was purified using anion exchange and size exclusion chromatography. Tris-glycine 4-20% gels were used to assess the purity of fractions during protein elution. Figure 11B This represents the fraction selected from the eluent of Qseph and MonoQ columns. Figure 11C This represents the fraction eluted from the TSK column. Figure 11D-11E CT3-T20 immunotoxin was purified using anion exchange and size exclusion chromatography. Tris-glycine 4-20% gels were used to assess the purity of fractions during protein elution. Figure 11D This represents the fraction selected from the eluent of Qseph and MonoQ columns. Figure 11E This represents the fraction eluted from the TSK column. Figure 11F-11G CT3-PE38 Figure 11F ) and CT3-T20 ( Figure 11G Immunotoxins selectively killed GPC2-expressing cell lines. Cells were incubated for three days with different concentrations of immunotoxin, and cell numbers were determined using the WST-8 cell proliferation assay. Cell proliferation in untreated wells was set to 100% when calculating cell proliferation inhibition. Treatment with CT3-PE38 and CT3-T20 resulted in inhibition of GPC2-positive cells, including G10, F8, and IMR5. Low-antigen IMR32 and antigen-negative A431 cells showed no inhibitory effect.

[0030] Figures 12A-12C Characterization of sCT3-PE38 immunotoxin. Figure 12A-12B The sCT3-PE38 immunotoxin was purified using anion exchange and size exclusion chromatography. Tris-glycine 4-20% gels were used to assess the purity of the fractions during protein elution. Figure 12A This represents the fraction selected from the eluent of Qseph and MonoQ columns. Figure 12B This represents the fractions obtained from runs on two separate TSK columns. Figure 12CsCT3-PE38 immunotoxin selectively killed GPC2-expressing cell lines. Cells were incubated for three days with different concentrations of immunotoxin, and cell counts were determined using the WST-8 cell proliferation assay. Cell proliferation in untreated wells was set to 100% when calculating cell proliferation inhibition. sCT3-PE38 treatment resulted in inhibition of GPC2-positive cells, including G10, F8, IMR5, and SKNBE. Antigen-negative A431 and SKNAS cells showed no inhibition by sCT3-PE38.

[0031] Figure 13 Ten million F8 cells in a matrix gel were injected into the right dorsal side of nude mice. When the average tumor volume reached 100 mm... 3 Treatment with PBS, CT3-PE38 (0.25 mg / kg), or CT3-T20 (2 mg / kg) was initiated. Arrows indicate treatment days. Tumor volume significance was determined using a two-way ANOVA test (p < 0.0001, n = 5). Body weight was also measured in treated mice. The CT3-based immunotoxin was well tolerated in nude mice and significantly inhibited F8 subcutaneous xenografting.

[0032] Figures 14A-14B Twenty million G10 cells in a matrix gel were injected into the right dorsal side of nude mice. When the average tumor volume reached 100 mm², the tumor was successfully treated. 3 Treatment was initiated with PBS, CT3-PE38 (0.25 mg / kg), or CT3-T20 (6 mg / kg). Arrows indicate the number of days of treatment. Tumor volume was assessed. Figure 14A ),weight( Figure 14A ) and survival rate ( Figure 14B When the tumor exceeds 1500mm 3 Mice were euthanized when the tumor began to ulcerate. The mean survival time in the immunotoxin group was 28 days, compared to 21 days in the PBS-treated group. The significance of Kaplan-Meier survival curves was determined using the Mantel-Cox test (p < 0.05, n = 5). CT3-based immunotoxin treatment improved survival in nude mice in the G10 subcutaneous xenograft model. Mice at doses up to 6 mg / kg tolerated CT3-T20 well.

[0033] Figures 15A-15B Five million IMR5 cells were injected into nude mice via tail vein. Mice were treated with PBS or CT3-T20 (4 mg / kg). Arrows indicate treatment days. Emissivity was measured using IVIS Lumina series III after injection of 100 μl of xenolight D-fluorescein (30 mg / ml). Figure 15AIn the IMR5 metastasis model, treatment with CT3-T20 immunotoxin reduced tumor burden but did not significantly alter body weight. Figure 15B ).

[0034] sequence list

[0035] As defined in 37C.FR1.822, the nucleic acid and amino acid sequences listed in the accompanying sequence listing are displayed using standard alphabetical abbreviations of nucleotide bases and three-letter codes for amino acids. Only one strand is shown for each nucleotide sequence, however, complementary strands should be understood to include any references to the shown strand. The sequence listing is submitted as an ASCII text file, created on August 1, 2019, 26.6 Kb, and is incorporated herein by reference. In the accompanying sequence listing:

[0036] SEQ ID NO:1 is the nucleotide sequence of the CT3 VH domain.

[0037] SEQ ID NO:2 is the amino acid sequence of the CT3 VH domain.

[0038] SEQ ID NO:3 is the nucleotide sequence of the CT3 VL domain.

[0039] SEQ ID NO:4 is the amino acid sequence of the CT3 VL domain.

[0040] SEQ ID NO:5 is the nucleotide sequence of the CT5 VH domain.

[0041] SEQ ID NO:6 is the amino acid sequence of the CT5 VH domain.

[0042] SEQ ID NO:7 is the nucleotide sequence of the CT5 VL domain.

[0043] SEQ ID NO:8 is the amino acid sequence of the CT5 VL domain.

[0044] SEQ ID NO:9 is the amino acid sequence of CT3 scFv.

[0045] SEQ ID NO:10 is the amino acid sequence of humanized CT3 scFv (hCT3-1).

[0046] SEQ ID NO:11 is the amino acid sequence of humanized CT3 scFv(hCT3-2).

[0047] SEQ ID NO:12 is the amino acid sequence of sCT3 scFv, which contains the CDR sequence of CT3 and the frame sequence from mouse antibody SS1.

[0048] SEQ ID NO:13 is the amino acid sequence of a new peptide epitope.

[0049] SEQ ID NO:14 is the amino acid sequence of the GM-CSF receptor signaling sequence.

[0050] SEQ ID NO:15 is the amino acid sequence of the CD8α hinge region.

[0051] SEQ ID NO:16 is the amino acid sequence of the CD8α transmembrane domain.

[0052] SEQ ID NO:17 is the amino acid sequence of 4-1BB.

[0053] SEQ ID NO:18 is the amino acid sequence of CD3ζ.

[0054] SEQ ID NO:19 is the amino acid sequence of the T2A self-cleaving peptide sequence.

[0055] SEQ ID NO:20 is a truncated amino acid sequence of human epidermal growth factor receptor (huEGFRt).

[0056] SEQ ID NO:21 is the amino acid sequence of humanized CT3 scFv (hCT3-3).

[0057] SEQ ID NO:22 is the amino acid sequence of PE38.

[0058] SEQ ID NO:23 is the amino acid sequence of T20. Detailed Implementation

[0059] I. Abbreviations

[0060] ADC antibody-drug conjugate

[0061] ADCC antibody-dependent cell-mediated cytotoxicity

[0062] CAR chimeric antigen receptor

[0063] CDR Complementary Determinant Region

[0064] CTL cytotoxic T lymphocytes

[0065] EF1 extension factor 1

[0066] EGF (Epidermal Growth Factor)

[0067] EGFR epidermal growth factor receptor

[0068] ELISA (Enzyme-Linked Immunosorbent Assay)

[0069] FACS (Fluorescence-Activated Cell Sorting)

[0070] GMCSFRss Granulocyte-Macrophage Colony-Stimulating Factor Receptor Signaling

[0071] sequence

[0072] GPC2 Phosphatidylinositol Proteoglycan-2

[0073] GPI glycosylphosphatidylinositol

[0074] hFc person Fc

[0075] huEGFRt truncated human epidermal growth factor receptor

[0076] Ig Immunoglobulin

[0077] IL interleukin

[0078] Intraperitoneal IP

[0079] IV. Intravenous

[0080] mFc Mouse Fc

[0081] PE Pseudomonas exotoxin

[0082] sc subcutaneous

[0083] scFv single-chain variable fragment

[0084] VH or V H Variable heavy chain

[0085] VL or V L Variable light chain

[0086] II. Terminology and Methods

[0087] Unless otherwise stated, technical terms are used according to their usual usage. Definitions of commonly used terms in molecular biology can be found in Benjamin Lewin, Genes V, Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (eds.), Molecular Biology and Biotechnology: A Comprehensive Desk Reference, VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

[0088] To facilitate review of the various embodiments of this disclosure, the following explanations of specific terms are provided:

[0089] 4-1BB: A co-stimulatory molecule expressed by T-cell receptor (TCR)-activated lymphocytes and other cells, including natural killer cells. The connection of 4-1BB induces a signaling cascade that leads to cytokine production, expression of anti-apoptotic molecules, and an enhanced immune response. An exemplary amino acid sequence of 4-1BB is shown herein as SEQ ID NO:17.

[0090] Acute lymphoblastic leukemia (ALL): An acute form of leukemia characterized by an overproduction of lymphoblasts. ALL is most common in childhood, peaking between 2 and 5 years of age.

[0091] Antibody: A polypeptide ligand containing at least one variable region that recognizes and binds (e.g., specifically recognizes and specifically binds) an antigenic epitope. Mammalian immunoglobulin molecules consist of heavy (H) chains and light (L) chains, each possessing a variable region, referred to as the variable heavy chain (V). H ) region and variable light chain (V L ) area. V H District and V LThe antibody-binding antigen is jointly responsible for binding to the antibody. Mammalian immunoglobulins have five main heavy chain classes (or isotypes), which determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE. Antibody isotypes not found in mammals include IgX, IgY, IgW, and IgNAR. IgY is a 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.

[0092] The variable region of an antibody contains a "framework" region and a hypervariable region, called the "complementation-determining region" or "CDR". The CDR is primarily responsible for binding to the antigen epitope. The frame region of the antibody is used to locate and align the CDR in three-dimensional space. The amino acid sequence boundaries of a given CDR can be readily determined using any of many well-known numbering schemes, including Kabat et al. (Sequences of Proteins of Immunological Interest, USDapartment of Health and Human Services, 1991; “Kabat” numbering scheme), Chothia et al. (see Chothia and Lesk, JMol Biol 196:901-917, 1987; Chothia et al., Nature 342:877, 1989; and Al-Lazikanie et al., JMB273,927-948, 1997; “Chothia” numbering scheme), Kunik et al. (see Kunik et al., PLoS Comput Biol 8:e1002388, 2012; and Kunik et al., Nucleic Acids Res 40 (WebServer)). Issue: W521-524, 2012; “Paratome CDRs”) and the ImMunoGeneTics (IMGT) database (see Lefranc, Nucleic Acids Res 29:207-9, 2001; “IMGT” numbering scheme) described therein. The Kabat, Paratome, and IMGT databases are maintained online.

[0093] "Single-domain antibody" refers to an antibody possessing a single domain (variable domain) that can specifically bind to an antigen or epitope in the absence of other antibody domains. Single-domain antibodies include, for example, V... H domain antibodies, V NAR Antibody, Camelidae VH H antibody and V L Domain-specific antibodies. V NAR Antibodies are produced by cartilaginous fish, such as the harpoon shark, the spotted baleen shark, the white-spotted dogfish, and the bamboo shark. Camelidae V H H antibodies are produced by several species, including camels, llamas, alpacas, dromedary camels, and guanacos, which produce naturally occurring heavy chain antibodies lacking the light chain.

[0094] A monoclonal antibody is an antibody produced by a single clone of a lymphocyte or by a cell that has been transfected with the coding sequence of a single antibody. Monoclonal antibodies are produced by methods known to those skilled in the art. Monoclonal antibodies include humanized monoclonal antibodies.

[0095] "Chimeric antibodies" have framework residues from one species (such as humans) and CDRs from another species (which typically confer antigen binding).

[0096] "Humanized" antibodies are immunoglobulins that comprise a human framework region and one or more core receptors (CDRs) derived from a non-human immunoglobulin (e.g., mouse, rabbit, rat, shark, or synthetic). The non-human immunoglobulin providing the CDR is called the "donor," while the human immunoglobulin providing the framework is called the "receptor." In one embodiment, all CDRs are derived from the donor immunoglobulin within the humanized immunoglobulin. Constant regions are not required, but if present, they must be substantially identical to the constant regions of human immunoglobulins, i.e., at least about 85-90%, for example, about 95% or higher identity. Thus, except for possible CDRs, all portions of the humanized immunoglobulin are substantially identical to the corresponding portions of the native human immunoglobulin sequence. Humanized antibodies bind to the same antigen as the donor antibody providing the CDR. Humanized or other monoclonal antibodies may have other conserved amino acid substitutions that have substantially no effect on antigen binding or other immunoglobulin function.

[0097] Antibody-drug conjugates (ADCs): Molecules comprising antibodies (or antigen-binding fragments of antibodies) conjugated to a drug (e.g., a cytotoxic agent). ADCs can be used to specifically target drugs to cancer cells by the specific binding of antibodies to tumor antigens expressed on the cell surface. Exemplary drugs used with ADCs include antimicrotubule agents (e.g., maytansine compounds, auristatin E, and auristatin F) and interchain crosslinking agents (e.g., pyrrolobenzodiazepines; PDB).

[0098] Anti-microtubule agents: A type of drug that stops cell growth by halting mitosis. Also known as "antimitotic agents," they are used to treat cancer.

[0099] Binding affinity: The affinity of an antibody for an antigen. In one embodiment, affinity is calculated using a modified Scatchard method described in Frankel et al., Mol. Immunol., 16:101-106, 1979. In another embodiment, binding affinity is measured by the antigen / antibody dissociation rate. In another embodiment, high binding affinity is measured by a competitive radioimmunoassay. In another embodiment, binding affinity is measured by an ELISA. In other embodiments, antibody affinity is measured by flow cytometry or by a surface plasmon reference. An antibody that "specifically binds" an antigen (e.g., GPC2) is an antibody that binds the antigen with high affinity and does not significantly bind other unrelated antigens.

[0100] Bispecific antibodies are recombinant proteins that contain antigen-binding fragments of two different monoclonal antibodies, thus enabling them to bind to two different antigens. In some embodiments, bispecific antibodies are used in cancer immunotherapy by simultaneously targeting, for example, CTLs (e.g., CTL receptor components such as CD3) or effector natural killer (NK) cells and tumor antigens. Similarly, multispecific antibodies are recombinant proteins that comprise antigen-binding fragments of at least two different monoclonal antibodies, such as two, three, or four different monoclonal antibodies.

[0101] Chemotherapy agent: Any chemical agent that has a therapeutic effect in treating diseases characterized by abnormal cell growth. Such diseases include tumors, growths, and cancers, as well as diseases characterized by proliferative growth, such as psoriasis. In one embodiment, the chemotherapy agent is a drug for treating neuroblastoma. In one embodiment, the chemotherapy agent is a radioactive compound. Those skilled in the art can readily determine the chemotherapy agent used (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 2). nd ed., Churchill Livingstone, Inc.; Baltzer, L., Berkery, R. (eds.): Oncology Pocket Guide to Chemotherapy, 2nd ed. St. Louis, Mosby-Year Book, 1995; Fischer, DS, Knobf, MF, Durivage, HJ (eds.): The Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 1993). Combination chemotherapy involves administering more than one drug to treat cancer. One example is the combined use of antibodies against GPC2 with radioactive or chemical compounds.

[0102] Chimeric antigen receptors (CARs): Chimeric molecules that include an antigen-binding moiety (e.g., scFv) and a signal transduction domain, such as a signal transduction domain derived from a T-cell receptor (e.g., CD3ζ). Typically, a CAR consists of an antigen-binding moiety, a transmembrane domain, and an inner domain. The inner domain usually includes a signal transduction chain with an immune receptor tyrosine-based activation motif (ITAM), such as CD3ζ or FcεRIγ. In some cases, the inner domain also includes an intracellular portion of at least one additional co-stimulatory domain, such as CD28, 4-1BB (CD137), ICOS, OX40 (CD134), CD27, and / or DAP10.

[0103] Complementarity-determining regions (CDRs): These are highly variable amino acid sequence regions that define the binding affinity and specificity of antibodies. Mammalian immunoglobulins each have three CDRs in their light and heavy chains, designated L-CDR1, L-CDR2, L-CDR3 and H-CDR1, H-CDR2, H-CDR3, respectively.

[0104] Conjugate: In the context of this disclosure, a “conjugate” is an antibody or antibody fragment (e.g., an antigen-binding fragment) covalently linked to an effector molecule or a second protein (e.g., a second antibody). Effector molecules can be, for example, drugs, toxins, therapeutic agents, detectable tags, proteins, nucleic acids, lipids, nanoparticles, carbohydrates, or recombinant viruses. Antibody conjugates are generally referred to as “immunoconjugates.” When a conjugate contains an antibody linked to a drug (e.g., a cytotoxic agent), it is generally referred to as an “antibody-drug conjugate” or “ADC.” Other antibody conjugates include, for example, multispecific (e.g., bispecific or trispecific) antibodies and chimeric antigen receptors (CARs).

[0105] Conservative variants: "Conservative" amino acid substitutions are those that substantially do not affect or reduce the affinity of a protein (e.g., antibody to GPC2). For example, a monoclonal antibody that specifically binds to GPC2 may include up to about 1, up to about 2, up to about 5, and up to about 10 or up to about 15 conserved substitutions that specifically bind to the GPC2 polypeptide. The term "conservative variant" also includes the use of substituted amino acids in place of unsubstituted parent amino acids, provided that the antibody specifically binds to GPC2. Non-conservative substitutions are those that reduce activity or binding to GPC2.

[0106] It is well known to those skilled in the art to provide a table of conserved amino acid substitutions for functionally similar amino acids. The following six groups are examples of amino acids considered to be conserved substitutions for each other:

[0107] 1) Alanine (A), Serine (S), Threonine (T);

[0108] 2) Aspartic acid (D), glutamic acid (E);

[0109] 3) Asparagine (N), glutamine (Q);

[0110] 4) Arginine (R), Lysine (K);

[0111] 5) Isoleucine (I), leucine (L), methionine (M), valine (V); and

[0112] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0113] Contact: refers to direct physical association; including both solid and liquid forms.

[0114] Cytotoxic agents: Any drug or compound that kills cells.

[0115] Cytotoxicity: The toxicity of a molecule to cells intended for targeting (e.g., immunotoxins), as opposed to the toxicity to cells in the rest of an organism. Conversely, in one embodiment, the term "toxicity" refers to the toxicity of an immunotoxin to cells other than those intended to be targeted by the immunotoxin's target portion, while the term "animal toxicity" refers to the toxicity of an immunotoxin to an animal, not to the toxicity of an immunotoxin to cells other than those intended to be targeted by the immunotoxin.

[0116] Desmoplastic small round cell tumor (DRCT): This soft tissue sarcoma primarily occurs in childhood, especially in boys. DRCT is an aggressive and rare type of cancer that mainly presents as an abdominal mass, but can also be found in lymph nodes, abdominal lining, diaphragm, spleen, liver, chest wall, skull, spinal cord, intestines, bladder, brain, lungs, testes, ovaries, and pelvis.

[0117] Diagnosis: Identifying the presence or nature of a pathological condition (e.g., neuroblastoma). The sensitivity and specificity of diagnostic methods differ. The “sensitivity” of a diagnostic test is the percentage of diseased individuals who test positive (the percentage of true positives). The “specificity” of a diagnostic test is a difference between the false positive rate and the percentage of diseased individuals who test positive but do not have the disease. While a particular diagnostic method may not provide a definitive diagnosis of the condition, it is sufficient if the method provides a positive indication that aids in diagnosis. “Prognosis” is the likelihood (e.g., severity) of the progression of a pathological condition such as neuroblastoma.

[0118] Drug: Any compound used to treat, improve, or prevent a disease or condition in a subject. In some embodiments described herein, a drug is an anticancer agent, such as a cytotoxic agent, like an antimitotic agent or an antimicrotubule agent.

[0119] Effector molecules: A portion of a chimeric molecule designed to produce a desired effect on cells targeted by the chimeric molecule. Effector molecules are also known as effector moieties (EMs), therapeutic agents, or diagnostic agents, or similar terms. Therapeutic agents (or drugs) include compounds such as nucleic acids, proteins, peptides, amino acids or derivatives, glycoproteins, radioisotopes, lipids, carbohydrates, or recombinant viruses. Nucleic acid therapeutic and diagnostic moieties include antisense nucleic acids, derived oligonucleotides for covalent cross-linking with single-stranded or double-stranded DNA, and oligonucleotides that form triplet strands. Alternatively, the molecule linked to a targeting moieties, such as an anti-GPC2 antibody, can be an encapsulation system, such as liposomes or micelles containing a therapeutic composition, such as a drug, a nucleic acid (e.g., an antisense nucleic acid), or another therapeutic moieties that avoid direct exposure to the circulatory system. Methods for preparing liposomes attached to antibodies are well known to those skilled in the art (see, for example, U.S. Patent No. 4,957,735; and Connor et al., Pharm Ther 28:341-365, 1985). Diagnostic agents or moieties include radioisotopes and other detectable tags. Detectable tags suitable for such purposes are well known in the art, including radioactive isotopes, for example... 35 S, 11 C 13 N、 15 O、 18 F, 19 F, 99m Tc, 131 I, 3 H, 14 C 15 N、 90 Y、 99 Tc, 111 In and 125 I. Fluorescein, chemiluminescent agents, and enzymes.

[0120] Epitopes: Antigenic determinants. These are specific chemical groups or peptide sequences on a molecule that possess antigenicity (i.e., elicit a specific immune response). Antibodies specifically bind to specific antigenic epitopes on polypeptides (e.g., GPC2).

[0121] Ewing's sarcoma: A rare type of malignant tumor found in bone or soft tissue. Ewing's sarcoma is a small, round, blue cell tumor.

[0122] Frame regions: Amino acid sequences inserted between CDRs. Frame regions include variable light and variable heavy frame regions. Frame regions are used to maintain the CDRs in the appropriate orientation for antigen binding.

[0123] Fusion protein: A protein that comprises parts of at least two different (heterologous) proteins.

[0124] Phosphatidylinositol proteoglycan-2 (GPC2): A member of the six-member phosphatidylinositol proteoglycan family, which is a heparan sulfate (HS) proteoglycan that attaches to the cell surface via 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), involved in cell adhesion, and is thought to regulate axonal growth and guidance. Additionally, GPC2 mRNA is highly expressed in neuroblastoma and other pediatric cancers (Orentas et al., Front Oncol 2:194, 2012). GPC2 is also known as brain proteoglycan and phosphatidylinositol proteoglycan-2. The GPC2 genome, mRNA, and protein sequences are publicly available (see, for example, NCBI Gene ID 221914).

[0125] GPC2-positive carcinoma: Cancer that overexpresses GPC2. Examples of GPC2-positive carcinoma include, but are not limited to, neuroblastoma, medulloblastoma, retinoblastoma, acute lymphoblastic leukemia, embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, Ewing's sarcoma, desmoplastic small round cell tumor, or osteosarcoma.

[0126] Heterogeneous: derived from a single genetic source or species.

[0127] Immune response: The response of immune system cells (e.g., B cells, T cells, or monocytes) to a stimulus. In one embodiment, the response is specific to a particular antigen (“antigen-specific response”). In one embodiment, the immune response is a T cell response, such as CD4+. + Response or CD8 +Response. In another implementation, the response is a B cell response, leading to the production of specific antibodies.

[0128] Immunoconjugates: Covalently linked effector molecules to antibodies or functional fragments thereof. Effector molecules can be detectable tags or immunotoxins. Specific, non-limiting examples of toxins include, but are not limited to, absinthecin, ricin, Pseudomonas exotoxins (PE, e.g., PE35, PE37, PE38, and PE40), diphtheria toxin (DT), botulinum toxin or modified toxins thereof, or other toxins that directly or indirectly inhibit cell growth or kill cells. For example, PE and DT are highly toxic compounds that typically cause death due to hepatotoxicity. However, PE and DT can be modified into a form that can be used as an immunotoxin by removing the natural targeting components of the toxin (e.g., domain Ia of PE and the B chain of DT) and replacing them with different targeting moieties (e.g., antibodies). A “chimeric molecule” is a targeting moieties, such as ligands or antibodies, conjugated to an effector molecule. The terms “conjugation” or “linking” refer to making two polypeptides a single, continuous polypeptide molecule. In one embodiment, an antibody is linked to an effector molecule. In another embodiment, the antibody linked to the effector molecule is further linked to a lipid or other molecule to form a protein or peptide to increase its half-life in vivo. The linking can be performed chemically or by recombination. In one embodiment, the linking is chemical, wherein a reaction between the antibody moiety and the effector molecule has generated a covalent bond between the two molecules to form a single molecule. A peptide linker (short peptide sequence) may optionally be included between the antibody and the effector molecule. Because immunoconjugates are originally prepared from two molecules with independent functions (e.g., an antibody and an effector molecule), they are sometimes referred to as “chimeric molecules.” Therefore, as used herein, the term “chimeric molecule” refers to a target moiety, such as a ligand or antibody, conjugated to an effector molecule.

[0129] Immunoliposomes: Liposomes whose surface is coupled with antibodies or antibody fragments. Immunoliposomes can deliver cytotoxic agents or other drugs to cells that target antibodies (such as tumor cells).

[0130] Interstrand crosslinkers: a type of cytotoxic drug that covalently binds between the two strands of DNA, thereby preventing DNA replication and / or transcription.

[0131] Isolated: "Isolated" biological components, such as nucleic acids, proteins (including antibodies), or organelles, have been substantially separated from or purified from other biological components naturally present in their environment (e.g., cells), namely other chromosomal and extrachromosomal DNA and RNA, proteins, and organelles. "Isolated" nucleic acid and protein molecules include those purified using standard purification methods. The term also covers nucleic acids and proteins prepared through recombinant expression in host cells, as well as chemically synthesized nucleic acids.

[0132] Tagging: A detectable compound or composition directly or indirectly coupled to another molecule (e.g., an antibody, such as one provided herein) or a protein) to facilitate the detection of that molecule. Specific, non-limiting examples of tagging include fluorescent tags, enzyme bonds, and radioisotopes. In one example, "tagged antibody" refers to the incorporation of another molecule into an antibody. For example, a tag is a detectable marker, such as the incorporation of a radiolabeled amino acid or the attachment of a peptide to a biotinylated moiety detectable by a labeled avidin (e.g., streptavidin containing a fluorescent marker or enzyme activity detectable by optical or colorimetric methods). A variety of methods for tagging peptides and glycoproteins can be used. Examples of tags for peptides include, but are not limited to, the following: radioisotopes or radioactive nucleotides (e.g., 5 S, 11 C 13 N、 15 O、 18 F, 19 F, 99m Tc, 131 I, 3 H, 14 C 15 N、 90 Y、 99 Tc, 111 In and 125 1) Fluorescent tags (e.g., FITC, rhodamine, lanthanide fluorescent powders), enzyme tags (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotin groups, predetermined peptide epitopes recognized by secondary reporter molecules (e.g., leucine zipper pairs, binding sites of secondary antibodies, metal-binding domains, epitope tags), or magnetic reagents, such as gadolinium chelates. In some embodiments, the tags are attached via spacer arms of various lengths to reduce potential steric hindrance.

[0133] Linker: In some cases, a linker is a peptide within an antibody-binding fragment (e.g., an Fv fragment) that is used to indirectly bind a variable heavy chain to a variable light chain. A “linker” can also refer to a peptide used to link a targeting moiety (e.g., an antibody) to an effector molecule (e.g., a cytotoxin or a detectable tag).

[0134] The terms “coupling,” “joining,” “binding,” or “linking” refer to the process of combining two polypeptides into a single, continuous polypeptide molecule, or the covalent attachment of a radionuclide or other molecule to a polypeptide, such as scFv. In a specific context, the term includes references to the attachment of a ligand (e.g., an antibody moiety) to an effector molecule. Linking can be achieved through chemical or recombination methods. “Chemical methods” refer to a reaction between the antibody moiety and the effector molecule that forms a covalent bond between the two molecules to form a single molecule.

[0135] Medulloblastoma: A rapidly growing cancer that forms in the cerebellum. Medulloblastomas tend to spread to the spinal cord or other parts of the brain via the cerebrospinal fluid. They can also spread to other parts of the body, but this is rare. Medulloblastomas are most common in children and young adults. They are a type of embryonic tumor of the central nervous system.

[0136] Tumor formation, malignancy, cancer, or tumor: A growth is an abnormal growth of tissue or cells caused by excessive cell division. Growth can develop into a tumor. The number of tumors in an individual is called the "tumor burden," which can be measured by the number, size, or weight of the tumors. Tumors that do not metastasize are called "benign." Tumors that invade surrounding tissues and / or can metastasize are called "malignant."

[0137] Neuroblastoma: A solid tumor caused by embryonic neural stem cells. Neuroblastomas typically occur in and around the adrenal glands, but can also occur anywhere sympathetic nerve tissue is found, such as in the abdomen, chest, neck, or nerve tissue near the spine. Neuroblastomas usually occur in children under 5 years of age.

[0138] Operable ligation: When the first and second nucleic acid sequences are functionally related, they are operably ligated. For example, if a promoter affects the transcription or expression of a coding sequence, the promoter is operably ligated to the coding sequence. Typically, the operably ligated DNA sequences are contiguous and, in cases where two protein-coding regions need to be ligated, are within the same reading frame.

[0139] Osteosarcoma: A type of cancerous tumor found in the bone. Osteosarcoma is an aggressive cancer that originates from primitive transformed cells derived from mesenchyme. This type of cancer is most common in children and young adults.

[0140] Pediatric cancers: Cancers that develop in children aged 0 to 14 years. 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 central nervous system tumors (such as medulloblastoma), Wilm's tumor, non-Hodgkin's lymphoma, and retinoblastoma.

[0141] Pharmaceutical: A chemical compound or composition which, when properly administered to a subject or cell, can induce a desired therapeutic or preventive effect.

[0142] Pharmaceutically acceptable delivery vehicles: The pharmaceutically acceptable delivery vehicles used are conventional. Remington's Pharmaceutical Sciences, by EW Martin, Mack Publishing Co., Easton, PA, 15th Edition, 1975, describes compositions and formulations suitable for drug delivery of the antibodies disclosed herein.

[0143] Typically, the properties of the delivery vehicle will depend on the specific route of administration employed. For example, parenteral preparations often contain injectable fluids, including pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, glucose solutions, glycerol, etc., as solvents. For solid compositions (e.g., in the form of powders, pills, tablets, or capsules), conventional non-toxic solid delivery vehicles may include, for example, pharmaceutical-grade mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral delivery vehicles, the pharmaceutical composition to be administered may also contain small amounts of non-toxic excipients, such as wetting agents or emulsifiers, preservatives, and pH buffers, such as sodium acetate or sorbitol monolaurate.

[0144] Prevention, treatment, or improvement of disease: "Prevention" of disease refers to suppressing the overall development of disease. "Treatment" refers to therapeutic interventions that improve the signs or symptoms of a disease or pathological condition after it has begun to develop, such as reducing the tumor burden or decreasing the size and number of metastases. "Improvement" refers to a reduction in the number or severity of signs or symptoms of a disease, such as cancer.

[0145] Purified: The term "purified" does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified peptide preparation is one in which the peptide or protein is more enriched than that present in cells in their natural environment. In one embodiment, the purified formulation comprises at least 50% of the total peptide or protein content of the preparation. Substantially purified means purified from other protein or cellular components. The purity of a substantially purified protein is at least 60%, 70%, 80%, 90%, 95%, or 98%. Thus, in a specific, non-limiting example, a substantially purified protein is 90% free of other protein or cellular components.

[0146] Pyrrolobenzodiazepines (PBDs): a class of sequence-selective DNA minor groove binding cross-linkers first discovered in species of the genus *Streptomyces*. PBDs are far more effective than systemic chemotherapy drugs. The mechanism of action of PBDs is related to their ability to form adducts in the DNA minor groove, thereby interfering with DNA processing. In the context of this disclosure, PBDs include naturally occurring and isolated PBDs, chemically synthesized naturally occurring PBDs, and chemically synthesized non-naturally occurring PBDs. PBDs also include monomeric, dimeric, and mixed PBDs (for a review, see Geratana, Med Res Rev 32(2):254-293, 2012).

[0147] Recombinant: Recombinant nucleic acids or proteins are recombinant nucleic acids or proteins that have sequences that are not naturally occurring or that are artificially combined from two otherwise separate sequence fragments. Such artificial combinations are usually achieved through chemical synthesis or by artificially manipulating isolated nucleic acid fragments, such as through genetic engineering techniques.

[0148] Retinoblastoma: A type of cancer that forms in the retinal tissue. Retinoblastoma usually occurs in children under the age of 5. It can be hereditary or non-hereditary (sporadic).

[0149] Rhabdomyosarcoma (RMS): A malignant soft tissue tumor originating from skeletal muscle. The most common primary sites of rhabdomyosarcoma are the head and neck (e.g., parameningeal, orbital, pharynx, etc.), genitourinary tract, and extremities. Other less common primary sites include the trunk, chest wall, abdomen (including the retroperitoneal space and bile ducts), and 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. This subtype is observed to be more frequent in adolescents and in patients with primary sites involving the extremities, trunk, and perineal / anal region. ARMS is associated with chromosomal translocations encoding fusion genes involving members of the FKHR and PAX family on chromosome 13. The embryonal subtype is the most frequently observed subtype in children, accounting for approximately 60–70% of childhood rhabdomyosarcomas. Tumors with embryonal histology commonly occur in the head and neck region or genitourinary tract, although they can occur at any primary site. ERMS is characterized by younger age at diagnosis, loss of heterozygosity, and altered genomic imprinting.

[0150] Sample (or biological sample): A biological sample obtained from a subject that contains genomic DNA, RNA (including mRNA), proteins, or combinations thereof. Examples include, but are not limited to, peripheral blood, tissue, cells, urine, saliva, tissue biopsy, fine-needle aspiration, surgical specimens, and autopsy materials. In one instance, the sample includes a tumor biopsy.

[0151] Sequence identity: The similarity between amino acid or nucleic acid sequences is expressed as sequence similarity; otherwise, it is called sequence identity. Sequence identity is often measured by percentage identity (or similarity or homology); the higher the percentage, the greater the similarity between the two sequences. When aligned using standard methods, homologs or variants of polypeptide or nucleic acid molecules will have a relatively high degree of sequence identity.

[0152] The sequence alignment methods used for comparison are well known in the art. Various procedures and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J. Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins and Sharp, Gene 73:237, 1988; Higgins and Sharp, CABIOS 5:151, 1989; Corpet et al., Nucleic Acids Research 16:10881, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988. Altschul et al., Nature Genet. 6:119, 1994, which present detailed considerations for sequence alignment methods and homology calculations.

[0153] The NCBI Local Sequence Alignment Retrieval Basic Tool (BLAST) (Altschul et al., J.Mol.Biol.215:403, 1990) is available from various resources, including the National Center for Biotechnology Information (NCBI, Bethesda, Md.), and is also available on the Internet. It is used in conjunction with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. Instructions on how to use this program to determine sequence identity are provided on the NCBI website.

[0154] V of antibodies that specifically bind to GPC2 peptide HHomologs and variants of the antibody are typically characterized by at least approximately 75% sequence identity calculated using NCBIBlast 2.0 (with gap blastp set as the default parameter) in a full-length alignment with the antibody's amino acid sequence, e.g., at least approximately 80%, 90%, 95%, 96%, 97%, 98%, or 99%. For comparisons of amino acid sequences longer than approximately 30 amino acids, the Blast 2 sequence function is used with the default BLOSUM62 matrix set as the default parameter (gap presence cost of 11, and a cost of 1 per residue gap). When aligning short peptides (less than approximately 30 amino acids), the Blast 2 sequence function should be used with the PAM30 matrix set as the default parameter (open gap 9, extended gap 1 penalty). When evaluated using this method, proteins with even higher similarity to the reference sequence will show an increased percentage of identity, e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%. When comparing sequence identity within a smaller range than the entire sequence, homologs and variants typically exhibit at least 80% sequence identity within a short window of 10–20 amino acids, and may have at least 85%, 90%, or 95% sequence identity depending on their similarity to the reference sequence. Methods for determining sequence identity within such short windows are available on the NCBI website on the Internet. Those skilled in the art will understand that these sequence identity ranges are provided for guidance only; it is entirely possible to obtain very important homologs beyond the provided ranges.

[0155] Small molecules: typically molecules with a molecular weight of less than about 1,000 Daltons, or in some embodiments less than about 500 Daltons, wherein the molecule is capable of modulating the activity of the target molecule to a measurable extent.

[0156] Subjects: Live multicellular vertebrate organisms, including human and veterinary subjects, including human and non-human mammals.

[0157] Synthetic: produced artificially in a laboratory, such as nucleic acids or proteins (e.g., antibodies) that can be chemically synthesized in a laboratory.

[0158] Therapeutic effective dose: A specific amount of a substance sufficient to achieve the desired effect in the treated subject. For example, this could be the amount needed to inhibit or suppress tumor growth. In one implementation, the therapeutic effective dose is the amount needed to eliminate, reduce tumor size, or prevent tumor metastasis. When administered to a subject, a dose that reaches a target tissue concentration (e.g., in the tumor) that has been proven to achieve the desired in vitro effect is typically used.

[0159] Toxins: Molecules that are cytotoxic to cells. Toxins include abrin, ricin, Pseudomonas exotoxin (PE), diphtheria toxin (DT), botulinum toxin, saponins, localized aspergillin, or white tree toxin or their modified forms. For example, PE and DT are highly toxic compounds that often lead to death due to hepatotoxicity. However, PE and DT can be modified to function as immunotoxins by removing their natural targeting components (e.g., domain Ia of PE or the B chain of DT) and replacing them with different targeting moieties (e.g., antibodies).

[0160] Vector: A nucleic acid molecule introduced into a host cell to produce a transformed host cell. The vector may include a nucleic acid sequence that allows it to replicate within the host cell, such as an origin of replication. The vector may also include one or more optional marker genes and other genetic factors known in the art. In some embodiments, the vector is a viral vector, such as a lentiviral vector.

[0161] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Unless the context clearly specifies otherwise, the singular terms “a,” “an,” and “the” include plural indicators. “Comprising A or B” means including A or B, or A and B. It should also be understood that all base sizes or amino acid sizes given for nucleic acids or polypeptides, and all molecular weights or molecular weight values, are approximate and provided for descriptive purposes. While similar or equivalent methods and materials to those described herein may be used in the practice or testing of this disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In the event of conflict, this specification (including the explanation of terms) shall prevail. Furthermore, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0162] III. Monoclonal antibodies specific to phosphatidylinositol proteoglycan-2 (GPC2)

[0163] This paper discloses high-affinity anti-GPC2 monoclonal antibodies. Antibodies CT3 and CT5, isolated from mouse hybridomas, were found to specifically bind to GPC2; these antibodies are not other proteins in the phosphatidylinositol proteoglycan family. The anti-GPC2 antibodies bind to GPC2-positive neuroblastoma, medulloblastoma, and retinoblastoma tumor cells, but not to normal (healthy) tissue. CARs based on GPC2-specific antibodies exhibit excellent killing activity against GPC2-expressing neuroblastoma tumor cells. Immunotoxins based on engineered CT3 antibodies inhibit the proliferation of various GPC2-expressing cell lines. The specificity of the disclosed antibodies to GPC2 and GPC2-expressing tumors makes these antibodies an excellent therapeutic option for pediatric cancers, including neuroblastoma, medulloblastoma, and retinoblastoma. The disclosed antibodies can also be used to detect GPC2-expressing cells and diagnose GPC2-positive tumors.

[0164] The nucleotide and amino acid sequences of the VH and VL domains of CT3 and CT4 are provided below and are shown in this document as SEQ ID NO: 1-8. Tables 1-4 list the amino acid positions of CDR1, CDR2, and CDR3 determined using Kabat, IMGT, or Paratome, or a combination of all three. Those skilled in the art can readily determine CDR boundaries using alternative numbering schemes, such as the Chothia numbering scheme.

[0165] CT3 V H DNA (SEQ ID NO:1)

[0166] GAGGTCCAGCTGCAACAGTCTGGACCTGAACTGGTGAAGCCTGGGGCTTCAGTAAAGATGTCCTGCAAGGCTTCTAGATTCACATTCACTGACTACAACATACACTGGGTGAAGCAGAGCCCTGGAAAGACCCTTGAATGGATTGGATATATTAACCCTAACAATGGTGATATTTTCTACAAACAG AAGTTCAATGGCAAGGCCACATTGACTATAAACAAGTCCTCCAACACAGCCTACATGGAGCTCCGCAGCCTGACATCGGAGGATTCTGCAGTCTATTACTGTGTAAGATCCTCTAATATTCGTTATACTTTCGACAGGTTCTTCGATGTCTGGGGCACAGGGACCACGGTCACCGTCTCCTCACT3V H Protein (SEQ ID NO:2)

[0167] EVQLQQSGPELVKPGASVKMSCKASRFTFTDYNIHWVKQSPGKTLEWIGYINPNNGDIFYKQKFNGKATLTINKSSNTAYMELRSLTSEDSAVYYCVRSSNIRYTFDRFFDVWGTGTTVTVSS

[0168] Table 1. Positions of CDRs in the CT3 VH domain (SEQ ID NO: 2)

[0169] plan CDR1 CDR2 CDR3 Kabat 31-35 50-66 99-112 IMGT 26-33 51-58 97-112 Paratome 26-35 47-61 97-112 Combination 26-35 47-66 97-112

[0170] CT3 V L DNA (SEQ ID NO: 3)

[0171] GAAAATGTGCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCTAGGGGAGAAGGTCACCATGAGCTGCAGGGCCAGCTCAAGTGTAAATTACATTTACTGGTACCAGCAGAAGTCAGATGCCTCCCCCAAACTATGGATTTATTACACATCCAACCTGGCTCCTGGAGTCCCAGCTCGCTTCAGTGGCAGTGGGTCTGGGAACTCTTATTCTCTCACAATCAGCAGCATGGAGGGTGAAGATGCTGCCACTTATTACTGCCAGCAGTTTTCTAGTTCCCCATCCACGTTCGGTACTGGGACCAAGCTGGAGCTGAAA

[0172] CT3 V L Protein (SEQ ID NO: 4)

[0173] ENVLTQSPAIMSASLGEKVTMSCRASSSVNYIYWYQQKSDASPKLWIYYTSNLAPGVPARFSGSGSGNSYSLTISSMEGEDAATYYCQQFSSSPSTFGTGTKLELK

[0174] Table 2. Positions of CDRs in the CT3 VL domain (SEQ ID NO: 4)

[0175] plan CDR1 CDR2 CDR3 Kabat 24-33 49-55 88-96 IMGT 27-31 49-51 88-96 Paratome 27-33 45-55 88-95 Combination 24-33 45-55 88-96

[0176] CT5 V H DNA (SEQ ID NO: 5)

[0177] GAGGTGAAACTGGTGGAGTCTGGAGGAGGCTTGGTACAGTCTGGGCGTTCTCTGAGACTCTCCTGTGCAACTTCTGGATTCACCTTCAGTGATTTCTACATGGAGTGGGTCCGCCAAGCTCCAGGGAAGGGACTGGAGTGGATTGTTGCAAGTAGAGACAAAGCTAATGATTATACAACAGCGTATAGTGCATCTGTGAAGGGTCGGTTCATCGTCTCCAGAGACACTTCCCAAAGCATCCTCTACCTTCAGATGAATGCCCTGAGAGCTGAGGACACTGCCATTTATTACTGTGTAAGAGATTTCTATGATTACGACGAGGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCT

[0178] CT5 V H Protein (SEQ ID NO:6)

[0179] EVKLVESGGGLVQSGRSLRLSCATSGFTFSDFYMEWVRQAPGKGLEWIVASRDKANDYTTAYSASVKGRFIVSRDTSQSILYLQMNALRAEDTAIYYCVRDFYDYDEAYWGQGTLVTVS

[0180] Table 3. Positions of CDRs in the CT5 VH domain (SEQ ID NO:6)

[0181] plan CDR1 CDR2 CDR3 Kabat 31-35 50-68 101-109 IMGT 26-33 51-60 99-109 Paratome 27-35 47-62 99-109 Combination 26-35 47-68 99-109

[0182] CT5 V L DNA (SEQ ID NO:7)

[0183] GACATCCAGATGACTCAGTCTCCGTCCTCACTGTCTGCCTCTCTGGGAGGTACAGTCACCATCACTTGCAAGGCAAGCGAAGACATTAACAACTATATAGCTTGGTACCAACACAAGCCTGGAAAAGGTCCTCGGCTGCTCATACAATACACATCTACA TTACAGCCAGGCATCCCATCAAGGTTCAGTGGAAGTGGGTCTGGGCGAGATTATTCCCTCAGCATCAGCAACCTGGAGCCTGAAGATATTGCAACTTATTGTCTACAGTATGATATTCTGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA

[0184] CT5 V L Protein (SEQ ID NO:8)

[0185] DIQMTQSPSSLSASLGGTVTITCKASEDINNYIAWYQHKPGKGPRLLIQYTSTLQPGIPSRFSGSGSGRDYSLSISNLEPEDIATYYCLQYDILWTFGGGTKLEIK

[0186] Table 4. Location of CDR in the CT5 VL domain (SEQ ID NO:8)

[0187] plan CDR1 CDR2 CDR3 Kabat 24-34 50-56 89-96 IMGT 27-32 50-52 89-96 Paratome 27-34 46-56 89-95 Combination 24-34 46-56 89-96

[0188] This article also discloses CT3-based scFv molecules. In the amino acid sequences below, the combined Kabat / IMGT / Paratome CDR is shown in bold, the framework residues are shown in normal font, and the (G3S)4 linker sequence located between the VH and VL domains is underlined. The CT3 scFv contains the CDR and framework sequence from the original CT3 antibody. The hCT3-1, hCT3-2, and hCT3-3 scFv molecules contain the CDR sequence of CT3 and the human framework sequence. The sCT3 scFv contains the CDR sequence of CT3 and the framework sequence of the mouse antibody SS1 (see U.S. Patent No. 6,809,184; Chowdhury and Pastan, Nat Biotechnol 17:568-572,199; Pastan et al., Nat Rev Cancer 6:559-565,2006).

[0189] CT3 scFv (SEQ ID NO:9)

[0190]

[0191] Bold = Combined Kabat / IMGT / Paratome CDRs

[0192] = with underline = connector

[0193] Normal font = CT3 frame

[0194] hCT3-1 scFv (SEQ ID NO:10)

[0195]

[0196] Bold = Combined Kabat / IMGT / Paratome CDRs

[0197] = with underline = connector

[0198] Normal font = human frame

[0199] hCT3-2 scFv (SEQ ID NO:11)

[0200]

[0201] Bold = Combined Kabat / IMGT / Paratome CDRs

[0202] = with underline = connector

[0203] Normal font = human frame

[0204] hCT3-3 scFv (SEQ ID NO:21)

[0205]

[0206] Bold = Combined Kabat / IMGT / Paratome CDRs

[0207] = with underline = connector

[0208] Normal font = human frame

[0209] sCT3 (CT3 with SS1 frame; SEQ ID NO:12)

[0210]

[0211] Bold = Combined Kabat / IMGT / Paratome CDRs

[0212] = with underline = connector

[0213] Normal font = SS1 (mouse) frame

[0214] This document provides isolated monoclonal antibodies, or antigen-binding fragments thereof, that bind (e.g., specifically bind) to GPC2. The monoclonal antibody or antigen-binding fragment includes a variable heavy (VH) domain and a variable light (VL) domain. In some embodiments, the monoclonal antibody or antigen-binding fragment includes at least a portion of the amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:8, for example, one or more (e.g., all three) of the CDR sequence from SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:8. In some instances, the CDR position is determined by IMGT, Kabat, Paratome, Chothia, or a combination of one or more of them.

[0215] In some embodiments, the VH domain (or antigen-binding fragment) of the antibody contains the CDR sequence of SEQ ID NO:2, and the VL domain (or its antigen-binding fragment) contains the CDR sequence of SEQ ID NO:4. In some instances, the Kabat, IMGT, Paratome, or Chothia numbering schemes, or combinations thereof, are used to determine the CDR sequence. In specific instances, a combination of Kabat, IMGT, and Paratome is used to determine the CDR sequence.

[0216] In some embodiments, the VH domain (or antigen-binding fragment) of the antibody contains the CDR sequence of SEQ ID NO:6, and the VL domain (or its antigen-binding fragment) contains the CDR sequence of SEQ ID NO:8. In some instances, the Kabat, IMGT, Paratome, or Chothia numbering schemes, or combinations thereof, are used to determine the CDR sequence. In specific instances, a combination of Kabat, IMGT, and Paratome is used to determine the CDR sequence.

[0217] In some embodiments, the VH domain of the antibody or antigen-binding fragment comprises residues 31-35, 50-66, and 99-112 of SEQ ID NO:2. In some embodiments, the VL domain of the antibody or antigen-binding fragment comprises residues 24-33, 49-55, and 88-96 of SEQ ID NO:4. In some examples, the VH domain comprises residues 31-35, 50-66, and 99-112 of SEQ ID NO:2, and the VL domain comprises residues 24-33, 49-55, and 88-96 of SEQ ID NO:4.

[0218] In some embodiments, the VH domain of the antibody or antigen-binding fragment comprises residues 31-35, 50-68, and 101-109 of SEQ ID NO:6. In some embodiments, the VL domain of the antibody or antigen-binding fragment comprises residues 24-34, 50-56, and 89-96 of SEQ ID NO:8. In some instances, the VH domain comprises residues 31-35, 50-68, and 101-109 of SEQ ID NO:6, and the VL domain comprises residues 24-34, 50-56, and 89-96 of SEQ ID NO:8.

[0219] In some embodiments, the VH domain of the antibody or antigen-binding fragment comprises residues 26-33, 51-58, and 97-112 of SEQ ID NO:2. In some embodiments, the VL domain of the antibody or antigen-binding fragment comprises residues 27-31, 49-51, and 88-96 of SEQ ID NO:4. In some instances, the VH domain comprises residues 26-33, 51-58, and 97-112 of SEQ ID NO:2, and the VL domain comprises residues 27-31, 49-51, and 88-96 of SEQ ID NO:4.

[0220] In some embodiments, the VH domain of the antibody or antigen-binding fragment comprises residues 26-33, 51-60, and 99-109 of SEQ ID NO:6. In some embodiments, the VL domain of the antibody or antigen-binding fragment comprises residues 27-32, 50-52, and 89-96 of SEQ ID NO:8. In some examples, the VH domain comprises residues 26-33, 51-60, and 99-109 of SEQ ID NO:6, and the VL domain comprises residues 27-32, 50-52, and 89-96 of SEQ ID NO:8.

[0221] In some embodiments, the VH domain of the antibody or antigen-binding fragment comprises residues 26-35, 47-61, and 97-112 of SEQ ID NO:2. In some embodiments, the VL domain of the antibody or antigen-binding fragment comprises residues 27-33, 45-55, and 88-95 of SEQ ID NO:4. In some examples, the VH domain comprises residues 26-35, 47-61, and 97-112 of SEQ ID NO:2, and the VL domain comprises residues 27-33, 45-55, and 88-95 of SEQ ID NO:4.

[0222] In some embodiments, the VH domain of the antibody or antigen-binding fragment comprises residues 27-35, 47-62, and 99-109 of SEQ ID NO:6. In some embodiments, the VL domain of the antibody or antigen-binding fragment comprises residues 27-34, 46-56, and 89-95 of SEQ ID NO:8. In some instances, the VH domain comprises residues 27-35, 47-62, and 99-109 of SEQ ID NO:6, and the VL domain comprises residues 27-34, 46-56, and 89-95 of SEQ ID NO:8.

[0223] In some embodiments, the VH domain of the antibody or antigen-binding fragment comprises residues 26-35, 47-66, and 97-112 of SEQ ID NO:2. In some embodiments, the VL domain of the antibody or antigen-binding fragment comprises residues 24-33, 45-55, and 88-96 of SEQ ID NO:4. In some examples, the VH domain comprises residues 26-35, 47-66, and 97-112 of SEQ ID NO:2, and the VL domain comprises residues 24-33, 45-55, and 88-96 of SEQ ID NO:4.

[0224] In some embodiments, the VH domain of the antibody or antigen-binding fragment comprises residues 26-35, 47-68, and 99-109 of SEQ ID NO:6. In some embodiments, the VL domain of the antibody or antigen-binding fragment comprises residues 27-34, 46-56, and 89-96 of SEQ ID NO:8. In some instances, the VH domain comprises residues 26-35, 47-68, and 99-109 of SEQ ID NO:6, and the VL domain comprises residues 27-34, 46-56, and 89-96 of SEQ ID NO:8.

[0225] In some embodiments, the amino acid sequence of the VH domain has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:2 and / or the amino acid sequence of the VL domain has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:4. In some instances, the amino acid sequence of the VH domain comprises or is composed of SEQ ID NO:2 and / or the amino acid sequence of the VL domain comprises or is composed of SEQ ID NO:4.

[0226] In some embodiments, the amino acid sequence of the VH domain has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:6 and / or the amino acid sequence of the VL domain has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:8. In some instances, the amino acid sequence of the VH domain comprises or is composed of SEQ ID NO:6 and / or the amino acid sequence of the VL domain comprises or is composed of SEQ ID NO:8.

[0227] In some implementations, the monoclonal antibody or antigen-binding fragment is a humanized, chimeric, or synthetic monoclonal antibody or antigen-binding fragment.

[0228] In some instances, the monoclonal antibody is IgG. In other instances, the monoclonal antibody is IgA, IgD, IgE, or IgM.

[0229] In some embodiments, the antigen-binding fragment is a single-chain variable fragment (scFv), a Fab fragment, a Fab' fragment, an F(ab)'2 fragment, or a disulfide-bonded stable variable fragment (dsFv). In some instances, the antigen-binding fragment is scFv. In specific instances, the scFv comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:21. In non-limiting instances, the scFv comprises or consists of the amino acid sequence of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:21.

[0230] This document also provides chimeric antigen receptors (CARs) comprising the monoclonal antibodies or antigen-binding fragments disclosed herein. In some embodiments, the CAR further comprises a hinge region, a transmembrane domain, a co-stimulatory signaling portion, a signaling domain, or any combination thereof. Cells expressing GPC2-specific CARs are also provided. In some instances, the cells are T lymphocytes, such as CTLs. Section V further describes CARs and T cells expressing CARs.

[0231] This document also provides immunoconjugates comprising the monoclonal antibodies or antigen-binding fragments disclosed herein and effector molecules. In some embodiments, the effector molecule is a toxin, such as, but not limited to, Pseudomonas exotoxin or variants thereof, such as PE38. In other embodiments, the effector molecule is a detectable tag, such as, but not limited to, a fluorophore, an enzyme, or a radioisotope. Immunoconjugates are further described in Section IV.

[0232] This document also provides antibody-drug conjugates (ADCs), which comprise drugs conjugated to monoclonal antibodies or antigen-binding fragments disclosed herein. In some embodiments, the drug is a small molecule, such as an antimicrotubule agent, an antimitotic agent, and / or a cytotoxic agent. Section VI will further describe ADCs.

[0233] This document also provides multispecific antibodies, which include the monoclonal antibodies disclosed herein or antigen-binding fragments thereof, and at least one additional monoclonal antibody or antigen-binding fragment thereof. In some embodiments, the multispecific antibody is a bispecific antibody. In other embodiments, the multispecific antibody is a trispecific antibody. In some embodiments, at least one additional monoclonal antibody or antigen-binding fragment thereof specifically binds to components of the T-cell receptor or the natural killer (NK) cell activation receptor. Multispecific antibodies are further described in Section VII.

[0234] This document also provides antibody-nanoparticle conjugates comprising nanoparticles conjugated to monoclonal antibodies or antigen-binding fragments disclosed herein. In some embodiments, the nanoparticles include polymer nanoparticles, nanospheres, nanocapsules, liposomes, dendritic molecules, polymer micelles, or lipid vesicles. In some embodiments, the nanoparticles include cytotoxic agents. Antibody-nanoparticle conjugates are further described in Section VIII.

[0235] This document also provides fusion proteins, which comprise a monoclonal antibody or antigen-binding fragment disclosed herein and a heterologous protein or peptide. In some embodiments, the heterologous protein is an Fc protein. In some instances, the Fc protein is a mouse Fc or human Fc protein. In some embodiments, the heterologous peptide is not endogenous to humans (e.g., the heterologous peptide is a novel peptide epitope).

[0236] Compositions including pharmaceutically acceptable vectors and monoclonal antibodies or antigen-binding fragments, CARs, isolated cells (e.g., cells expressing CARs, such as CAR T cells), immunoconjugates, ADCs, multispecific antibodies, antibody-nanoparticle conjugates, or fusion proteins disclosed herein are further provided in this disclosure.

[0237] Nucleic acid molecules encoding the monoclonal antibodies or antigen-binding fragments disclosed herein are also provided. Nucleic acid molecules encoding the CARs, immunoconjugates, multispecific antibodies, or fusion proteins disclosed herein are also provided. In some embodiments, the nucleic acid molecules are operatively linked to a promoter. Vectors comprising the nucleic acid molecules are further provided herein.

[0238] In some embodiments, the nucleotide sequence of the VH domain has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:1. In some embodiments, the nucleotide sequence of the VL domain has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:3. In some instances, the nucleotide sequence of the VH domain comprises or is composed of SEQ ID NO:1, and the nucleotide sequence of the VL domain comprises or is composed of SEQ ID NO:3.

[0239] In some embodiments, the nucleotide sequence of the VH domain has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:5. In some embodiments, the nucleotide sequence of the VL domain has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:7. In some instances, the nucleotide sequence of the VH domain comprises or is composed of SEQ ID NO:5, and the nucleotide sequence of the VL domain comprises or is composed of SEQ ID NO:7.

[0240] In some embodiments, this document provides nucleic acid constructs expressing CAR and truncated human EGFR (huEGFRt). In some embodiments, the nucleic acid comprises, in the 5' to 3' directions: a nucleic acid encoding a first granulocyte-macrophage colony-stimulating factor receptor signaling sequence (GMCSFRss); a nucleic acid encoding a GPC2-specific monoclonal antibody or an antigen-binding fragment thereof disclosed herein; a nucleic acid encoding an extracellular hinge region; a nucleic acid encoding a transmembrane domain; a nucleic acid encoding an intracellular co-stimulatory domain; a nucleic acid encoding an intracellular signal transduction domain; a nucleic acid encoding a self-cleaving 2A peptide; a nucleic acid encoding a second GMCSFRss; and a nucleic acid encoding a truncated human epidermal growth factor receptor (huEGFRt). In some instances, the nucleic acid also includes a 5' portion of a human extension factor 1 (EF1α) promoter sequence encoding the first GMCSFRss. In some instances, the hinge region comprises a CD8α hinge region. In some instances, the transmembrane domain comprises a CD8α transmembrane domain. In some instances, the co-stimulatory signal transduction portion comprises a 4-1BB signal transduction portion. In some instances, the signal transduction domain includes the CD3ζ signal transduction domain. In some instances, the antigen-binding fragment is a single-stranded variable fragment (scFv). In certain non-limiting instances, the scFv comprises the amino acid sequence of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:21.

[0241] Isolated cells co-expressing the CPC2-specific CAR and huEGFRt disclosed herein were also provided. In some instances, the cells were cytotoxic T lymphocytes (CTLs).

[0242] IV. Immunoconjugates

[0243] The disclosed monoclonal antibodies can be conjugated to therapeutic agents or effector molecules. Immunoconjugates include, but are not limited to, molecules in which a therapeutic agent is covalently linked to an antibody. A therapeutic agent is an agent having specific biological activity against a specific target molecule or against cells carrying a target molecule. Those skilled in the art will understand that therapeutic agents can include various pharmaceuticals, such as vincristine, daunomycin, etc.; cytotoxins, such as natural or modified Pseudomonas exotoxins or diphtheria toxins; encapsulation agents (e.g., liposomes) comprising a pharmacological composition; and radioactive agents, such as… 125 I, 32 P, 14 C 3 H and 35 S and other tags, target parts, and ligands.

[0244] The choice of a specific therapeutic agent depends on the specific target molecule or cell and the desired biological effect. Thus, for example, a therapeutic agent could be a cytotoxin designed to induce death in specific target cells (e.g., tumor cells). Conversely, where a non-lethal biological response is required, the therapeutic agent can be conjugated to a non-lethal pharmacological agent or a liposome containing a non-lethal pharmacological agent.

[0245] Using the therapeutic agents and antibodies described herein, those skilled in the art can readily construct various clones containing functionally equivalent nucleic acids, such as nucleic acids with different sequences but encoding the same effector portion or antibody sequence. Therefore, this disclosure provides nucleic acids encoding antibodies, their conjugates, and fusion proteins.

[0246] A variety of methods known to those skilled in the art can be used to attach effector molecules to target antibodies. Covalent and non-covalent attachment methods can be used. The procedure for attaching effector molecules to antibodies varies depending on the chemical structure of the effector. Peptides typically contain various functional groups; for example, carboxylic acid (COOH), free amine (-NH2), or thiol (-SH) groups, which can be used to react with suitable functional groups on the antibody to result in the binding of the effector molecule. Alternatively, the antibody can be derivatized to expose or attach other reactive functional groups. Derivatization can involve the attachment of any of many known linker molecules. The linker can be any molecule used to link the antibody to the effector molecule. The linker is capable of forming covalent bonds with both the antibody and the effector molecule. Suitable linkers are well known to those skilled in the art and include, but are not limited to, straight-chain or branched carbon linkers, heterocyclic carbon linkers, or peptide linkers. When the antibody and effector molecules are peptides, the linker can be attached to the constituent amino acid or to the α-carbon amino and carboxyl groups attached to the terminal amino acid via their side groups (e.g., via disulfide bonds with cysteine).

[0247] In some cases, when an immunoconjugate reaches its target site, it is desirable to release effector molecules from the antibody. Therefore, in these situations, the immunoconjugate will contain a cleavable linker near the target site. The cleavage of the linker to release the effector molecules from the antibody can be facilitated by enzymatic activity or by the conditions experienced by the immunoconjugate inside or near the target cell.

[0248] Given that numerous methods have been reported for attaching various radiodiagnostic compounds, radiotherapy compounds, tags (such as enzymes or fluorescent molecules), drugs, toxins, and other reagents to antibodies, those skilled in the art can determine suitable methods for attaching a given reagent to an antibody or other peptide.

[0249] The antibodies disclosed herein can be derivatized or linked to another molecule (e.g., another peptide or protein). Typically, antibodies or portions thereof are derivatized such that binding to the target antigen is not adversely affected by derivatization or labeling. For example, antibodies can be functionally linked (through chemical coupling, genetic fusion, non-covalent binding, or other means) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or a biantibody), a detection agent, a pharmaceutical agent and / or a protein or peptide that can mediate the binding of an antibody or a portion thereof to another molecule (e.g., a streptavidin nucleus region or a polyhistidine tag).

[0250] One type of derivatized antibody is produced by crosslinking two or more antibodies (of the same or different types, for example, to generate bispecific antibodies). Suitable crosslinking agents include those that are heterobifunctional, having two distinct reactive groups separated by a suitable spacer group (e.g., m-maleimide benzoyl-N-hydroxysuccinimide ester), or those that are homobifunctional (e.g., disuccinimide octanoate). Such linkers are commercially available.

[0251] Antibodies can be conjugated to detectable biomarkers; for example, detectable biomarkers that can be detected by ELISA, spectrophotometry, flow cytometry, microscopy, or diagnostic imaging techniques (e.g., computed tomography (CT), computed axial computed tomography (CAT), magnetic resonance imaging (MRI), nuclear magnetic resonance imaging (NMRI), magnetic resonance imaging (MTR), ultrasound, fiber optic examination, and laparoscopy). Specific, non-limiting examples of detectable biomarkers include fluorophores, chemiluminescent agents, enzyme bonds, radioisotopes, and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for detection by MRI). For example, useful detectable biomarkers include fluorescent compounds, including luciferin, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors, etc. Bioluminescent biomarkers, such as luciferase, green fluorescent protein (GFP), and yellow fluorescent protein (YFP), can also be used. Antibody or antigen-binding fragments can also be conjugated to enzymes that can be used for detection, such as horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, and glucose oxidase. When an antibody or antigen-binding fragment is conjugated to a detectable enzyme, detection can be achieved by adding other reagents to produce a recognizable reaction product. For example, in the presence of horseradish peroxidase reagent, the addition of hydrogen peroxide and diaminobenzidine results in a colored reaction product that is visually detectable. Antibody or antigen-binding fragments can also be conjugated to biotin and detected indirectly by measuring the binding of avidin or streptavidin. It should be noted that avidin itself can be conjugated to enzymes or fluorescent tags.

[0252] Antibodies can be labeled with magnetic reagents such as gadolinium. Antibodies can also be labeled with lanthanides (e.g., europium and dysprosium) and manganese. Paramagnetic particles such as superparamagnetic iron oxide can also be used as tags. Antibodies can also be labeled with predetermined peptide epitopes recognized by secondary reporter molecules (e.g., leucine zipper pairs, binding sites of secondary antibodies, metal-binding domains, epitope tags). In some embodiments, the tag is attached via spacer arms of various lengths to reduce potential steric hindrance.

[0253] Antibodies can also be labeled with radioactively labeled amino acids. Radiolabeling can be used for diagnostic and therapeutic purposes. For example, radiolabels can be used to detect the expression of target antigens by X-rays, emission spectroscopy, or other diagnostic techniques. Examples of peptide labels include, but are not limited to, the following radioisotopes or radionucleotides: 3 H, 14 C 15 N、 35 S, 90 Y、 99 Tc, 111 In、 125 I, 131 I.

[0254] Antibodies can also be derived from chemical groups such as polyethylene glycol (PEG), methyl or ethyl, or carbohydrate groups. These groups can be used to improve the biological properties of antibodies, such as increasing serum half-life or increasing tissue binding.

[0255] The toxin can be used in conjunction with the monoclonal antibodies described herein to produce an immunotoxin. Exemplary toxins include ricin, absinthecin, diphtheria toxin and its subunits, and botulinum toxins A through F. These toxins are readily available from commercial sources (e.g., Sigma Chemical Company, St. Louis, MO). Contemplated toxins also include variants of the toxins described herein (see, for example, U.S. Patent Nos. 5,079,163 and 4,689,401). In one embodiment, the toxin is a pseudomonad exotoxin (PE) (U.S. Patent No. 5,602,095). As used herein, “pseudomonas exotoxin” refers to full-length natural (naturally occurring) PE or modified PE. Such modifications may include, but are not limited to, the elimination of domain Ia, the deletion of various amino acids in domains Ib, II, and III, the substitution of a single amino acid, and the addition of one or more sequences at the carboxyl terminus (e.g., see, Siegall et al., J. Biol. Chem. 264:14256-14261, 1989).

[0256] PEs used with the monoclonal antibodies described herein may include natural sequences, cytotoxic fragments of natural sequences, and conserved modified variants of natural PEs and their cytotoxic fragments. Cytotoxic fragments of PEs include those that exhibit cytotoxicity in target cells, with or without subsequent proteolytic or other processing. Cytotoxic fragments of PEs include PE40, PE38, and PE35. For additional descriptions of PE and its variants, see, for example, U.S. Patent Nos. 4,892,827; 5,512,658; 5,602,095; 5,608,039; 5,821,238; and 5,854,044; U.S. Patent Application Publication No. 2015 / 0099707; PCT Publications Nos. WO 99 / 51643 and WO 2014 / 052064; Pai et al., Proc. Natl. Acad. Sci. USA 88:3358-3362, 1991; Kondo et al., J. Biol. Chem. 263:9470-9475, 1988; Pastan et al., Biochim. Biophys. Acta 1333: C1-C6, 1997.

[0257] This article also considers protease-resistant PE variants and immunogenic PE variants, such as, but not limited to, PE-LR, PE-6X, PE-8X, PE-LR / 6X, and PE-LR / 8X (see, for example, Weldon et al., Blood 113(16):3792-3800, 2009; Onda et al., Proc Natl Acad Sci USA 105(32):11311-11316, 2008; and PCTP Publication Nos. WO 2007 / 016150, WO 2009 / 032954 and WO 2011 / 032022, which are incorporated herein by reference).

[0258] In some instances, PE is a variant resistant to lysosomal degradation, such as PE-LR (Weldon et al., Blood 113(16):3792-3800, 2009; PCT Publication No. WO 2009 / 032954). In other instances, PE is a variant named PE-LR / 6X (PCT Publication No. WO 2011 / 032022). In other instances, PE variants are PE with reduced immunogenicity. In other instances, PE is a variant named PE-LR / 8M (PCT Publication No. WO2011 / 032022).

[0259] Modifications to PE may occur in any of the previously described variants, including cytotoxic fragments of PE (e.g., PE38, PE-LR, and PE-LR / 8M). Modified PE may include any substitutions (multiple), such as one or more amino acid residues within one or more T-cell epitopes and / or B-cell epitopes of PE, or deletions of one or more T-cell and / or B-cell epitopes (see, for example, U.S. Patent Application Publication No. 2015 / 0099707).

[0260] The anticipated forms of PE also include deimmunized forms of PE, such as versions lacking domain II (e.g., PE24). Deimmunized forms of PE are described, for example, in PCT publications WO 2005 / 052006, WO 2007 / 016150, WO 2007 / 014743, WO 2007 / 031741, WO 2009 / 32954, WO 2011 / 32022, WO 2012 / 154530 and WO 2012 / 170617.

[0261] The antibodies described herein can also be used to target a variety of different diagnostic or therapeutic compounds to cells that express tumor or viral antigens on their surface. Therefore, the antibodies of this disclosure can attach directly or via a linker to a drug that will be delivered directly to the cell surface antigens of the expressing cells. This can be done for therapeutic, diagnostic, or research purposes. Therapeutic agents include compounds such as nucleic acids, proteins, peptides, amino acids or derivatives, glycoproteins, radioisotopes, lipids, carbohydrates, or recombinant viruses. Nucleic acid therapeutic and diagnostic components include antisense nucleic acids, derived oligonucleotides for covalent cross-linking with single-stranded or double-stranded DNA, and oligonucleotides that form triplet strands.

[0262] Alternatively, the molecule attached to the antibody can be an encapsulation system, such as nanoparticles, liposomes, or micelles containing a therapeutic composition such as a drug, a nucleic acid (e.g., an antisense nucleic acid), or another preferably therapeutic component that avoids direct exposure to the circulatory system. Methods for preparing antibody-attached liposomes are well known to those skilled in the art (see, for example, U.S. Patent No. 4,957,735; Connor et al., Pharm. Ther. 28:341-365, 1985).

[0263] The antibodies described herein can also be covalently or non-covalently linked to detectable tags. Detectable tags suitable for this purpose include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electronic, optical, or chemical means. Useful tags include magnetic beads, fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, green fluorescent protein, etc.), and radioactive tags (e.g., ...). 3 H, 125 I,35 S, 14 C or 32 P), enzymes (such as horseradish peroxidase, alkaline phosphatase, and other enzymes commonly used in ELISA), and colorimetric labels (such as colloidal gold or colored glass or plastic beads (such as polystyrene, polypropylene, latex, etc.).

[0264] Methods for detecting such labels are well known to those skilled in the art. Therefore, for example, photographic film or a scintillation counter can be used to detect radioactive labels, and a photodetector can be used to detect emitted illumination to detect fluorescent markers. Enzyme labels are typically detected by providing a substrate to an enzyme and detecting the reaction product produced due to the enzyme's action on the substrate, and colorimetric labels are detected by simply visualizing the colored label.

[0265] V. Chimeric antigen receptor (CAR)

[0266] The disclosed monoclonal antibodies can also be used to generate CARs (also known as chimeric T-cell receptors, artificial T-cell receptors, or chimeric immunoreceptors) and / or engineered cytotoxic T lymphocytes (CTLs) to express CARs. Typically, a CAR comprises a binding moiety, extracellular hinge and spacer elements, a transmembrane region, and an internal domain that performs signal transduction functions (Cartellierie et al., J Biomed Biotechnol 2010:956304, 2010; Dai et al., J Natl Cancer Inst 108(7):djv439, 2016). In many cases, the binding moiety is an antigen-binding fragment of a monoclonal antibody such as scFv or a single-domain antibody. The spacer / hinge region typically includes sequences derived from IgG subclasses, such as IgG1, IgG4, IgD, and CD8 domains. The transmembrane domain can be derived from a variety of different T-cell proteins, such as CD3ζ, CD4, CD8, or CD28. Several different internal domains have been used to generate CARs. For example, the inner domain may consist of a signaling chain with ITAM, such as CD3ζ or FcεRIγ. In some cases, the inner domain may also include an intracellular portion of at least one additional costimulatory domain, such as CD28, 4-1BB (CD137, TNFRSF9), OX-40 (CD134), ICOS, CD27, and / or DAP10.

[0267] CAR-expressing CTLs can be used to target specific cell types, such as GPC2-positive tumor cells. Therefore, the monoclonal antibody disclosed herein can be used to engineer CTLs expressing CARs containing a GPC2-specific monoclonal antibody (or a fragment thereof), thereby enabling the engineered CTLs to target GPC2-expressing tumor cells. Engineered T cells have previously been used for adoptive therapy in certain types of cancer (see, for example, Park et al., Mol Ther 15(4):825-833, 2007). The use of CAR-expressing T cells is more widespread than standard CTL-based immunotherapy because CAR-expressing CTLs are not HLA-restricted and can therefore be used in any patient with tumors expressing the target antigen.

[0268] Therefore, this document provides CARs comprising GPC2-specific antibodies. Also provided are isolated nucleic acid molecules and vectors encoding the CAR, as well as host cells, such as CTLs, expressing the CAR. CTLs expressing CARs composed of GPC2-specific monoclonal antibodies can be used to treat cancers expressing GPC2. In some embodiments described herein, the CAR is a bispecific CAR.

[0269] In some cases, it is desirable to modulate the activation and expansion of CAR-expressing T cells after infusion into patients. As described by Rodgers et al. (Proc Natl Acad Sci USA 113(4):E459-E468,2016), several strategies have been developed to modularize CAR-expressing T cells in vivo, including the use of antibody-based switches that mediate the interaction between CAR-expressing T cells and targeted tumor cells. The antibody-based switches consist of tumor antigen-specific antibodies grafted with peptide neoepitaxies (PNEs). Switchable CAR T (sCAR-T) cells are designed to bind specifically to PNEs. Since sCAR-T cells do not bind to endogenous antigens, a switch is necessary for their activation.

[0270] Therefore, this document provides antibody-based switches comprising the disclosed GPC2-specific antibodies fused with a heteropeptide (e.g., PNE). In some embodiments, the heteropeptide is not endogenous to humans (e.g., it is a peptide not found in the human proteome). In some instances, the heteropeptide is about 8 amino acids to about 20 amino acids in length, for example, about 10 to about 18 amino acids in length, for example, about 12 to about 16 amino acids in length, for example, about 14 amino acids in length. In specific instances, the heteropeptide is about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. In specific non-limiting instances, the PNE comprises or is composed of NYHLENEVARLKKL (SEQ ID NO:13).

[0271] In some embodiments, the CAR includes a signal peptide sequence, for example, at the N-terminus of the antigen-binding domain. The signal peptide sequence can be any suitable signal peptide sequence, such as those derived from the granulocyte-macrophage colony-stimulating factor receptor (GMCSFR), immunoglobulin light chain κ, or IL-2. Although the signal peptide sequence can promote CAR expression on the cell surface, its presence in the expressed CAR is not essential for the CAR to function. The signal peptide sequence can be cleaved from the CAR when it is expressed on the cell surface. Therefore, in some embodiments, the CAR lacks a signal peptide sequence.

[0272] In some implementations, the CAR disclosed herein is expressed from a truncated construct that also expresses human EGFR (huEGFRt) (e.g., from a lentiviral vector). The CAR and huEGFRt are separated by a self-cleaving peptide sequence (e.g., T2A) such that the CAR is cleaved from huEGFRt after expression in transduced cells.

[0273] In some embodiments disclosed herein, the CAR construct encodes the following amino acid sequence in the N-terminal to C-terminal direction:

[0274] GMCSFRss:MLLVTSLLCELPHPAFLLIP(SEQ ID NO:14)

[0275] NdeI:HM

[0276] Antigen binding: GPC2-specific scFv (e.g., any one of SEQ ID NO: 9-12)

[0277] SpeI:TS

[0278] CD8α hinge: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO:15)

[0279] CD8α TM: IYIWAPLAGTCGVLLLSLVIT (SEQ ID NO:16)

[0280] 4-1BB: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO:17)

[0281] CD3ζ:

[0282] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:18)

[0283] T2A: EGRGSLLTCGDVEENPGP (SEQ ID NO:19)

[0284] GMCSFRss: MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO:14) ​​​​​​​The human epidermal growth factor receptor (EGFR) consists of four extracellular domains, a transmembrane domain, and three intracellular domains. The EGFR domains are found in the following N-terminal to C-terminal sequence: Domain I - Domain II - Domain III - Domain IV - Transmembrane (TM) domain - Juxtamembrane domain - Tyrosine kinase domain - C-terminal tail. Domains I and III are leucine-rich domains that participate in ligand binding. Domains II and IV are cysteine-rich domains and do not contact EGFR ligands. Domain II mediates the formation of homodimers or heterodimers with domains similar to other EGFR family members, and Domain IV can form disulfide bonds with Domain II. The EGFR TM domain crosses the cell membrane only once and may play a role in protein dimerization. Intracellular domains include the juxtamembrane domain, tyrosine kinase domain, and C-terminal tail that mediate EGFR signal transduction (Wee and Wang, Cancers 9(52), doi:10.3390 / cancers9050052; Ferguson, AnnuRev Biophys 37:353-373,2008; Wang et al., Blood 118(5):1255-1263,2011).

[0288] The truncated form of human EGFR, referred to herein as "huEGFRt," includes only domains III, IV, and the TM domain. Therefore, huEGFRt lacks domains I, II, and all three intracellular domains. huEGFRt cannot bind to EGF and lacks signaling activity. However, this molecule retains the ability to bind to specific EGFR-specific monoclonal antibodies, such as the FDA-approved cetuximab (PCT Publication No. WO 2011 / 056894, which is incorporated herein by reference).

[0289] T cells can be transduced using constructs (e.g., lentiviral vectors) encoding huEGFRt and tumor antigen-specific CARs disclosed in this paper, and can be treated with the labeled EGFR monoclonal antibody cetuximab (ERBITUX). TM Selective transduced T cells. For example, cetuximab can be biotin-labeled, and commercially available avidin-converting magnetic beads (e.g., from Miltenyi Biotec) can be used to select transduced T cells. Co-expression of huEGFRt also allows for the tracking of adoptively transferred CAR-expressing T cells in vivo. Furthermore, the binding of cetuximab to huEGFRt-expressing T cells induces cytotoxicity of ADCC effector cells, thereby providing a mechanism for the elimination of transduced T cells in vivo (Wang et al., Blood 118(5):1255-1263,2011), for example, at the end of treatment.

[0290] VI. Antibody-Drug Conjugates (ADCs)

[0291] Antibody-addictive drugs (ADCs) are compounds composed of tumor antigen-specific antibodies (or their antigen-binding fragments) and drugs, typically cytotoxic agents such as anti-microtubule agents or cross-linking agents. Because ADCs can specifically target cancer cells, their efficacy may be greater than that of drugs used in standard chemotherapy. The most common cytotoxic drugs currently used in conjunction with ADCs have an IC50 of... 50 These cytotoxic agents are 100 to 1000 times more potent than traditional chemotherapy drugs. Common cytotoxic agents include antimicrotubule agents, such as maytansine compounds and auristatins (e.g., auristatin E and auristatin F). Other cytotoxic agents used with ADCs include pyrrolobenzodiazepines (PDB), which covalently binds to the minor groove of DNA to form interstrand crosslinks. In many cases, ADCs contain an antibody to drug ratio of 1:2 to 1:4 (Bander, Clinical Advances in Hematology & Oncology 10(8; suppl 10):3-7, 2012).

[0292] Antibodies and drugs can be linked via cleavable or non-cleavable linkers. However, in some cases, it is desirable to have linkers that are stable in circulation to prevent systemic release of cytotoxic drugs, which could lead to significant off-target toxicity. Non-cleavable linkers prevent the release of cytotoxic agents before the ADC is internalized by the target cells. Once in the lysosome, the digestion of the antibody by lysosomal proteases leads to the release of the cytotoxic agent (Bander, Clinical Advances in Hematology & Oncology 10(8; suppl 10):3-7, 2012).

[0293] One approach to site-specific and stable conjugation of drugs to monoclonal antibodies is through glycan engineering. Monoclonal antibodies possess a conserved N-linked oligosaccharide chain at the Asn297 residue of the CH2 domain in each heavy chain (Qasba et al., Biotechnol Prog 24:520-526, 2008). Using a mutant β1,4-galactosyltransferase (Y289L-Gal-T1; U.S. Patent Application Publications 2007 / 0258986 and 2006 / 0084162, incorporated herein by reference), 2-keto-galactose is transferred to free GlcNAc residues on the antibody heavy chain to provide a chemical treatment for conjugation.

[0294] Based on the terminal galactose residue, oligosaccharide chains attached to monoclonal antibodies can be classified into three categories: fully galactosylated (two galactose residues; IgG-G2), one galactose residue (IgG-G1), or fully degalactosylated (IgG-G0). Treatment of monoclonal antibodies with β1,4-galactosidase converts them to the IgG-G0 glycoform. Mutant β1,4-galactosyltransferases can transfer 2-keto-galactose or 2-azido-galactose from their respective UDP derivatives to the GlcNAc residues on the IgG-G1 and IgG-G0 glycoforms. Chemical treatment of the transferred sugars allows for the conjugation of various molecules to monoclonal antibodies via glycan residues (Qasba et al., Biotechnol Prog24:520-526, 2008).

[0295] The ADCs described herein include drugs (e.g., cytotoxic agents) conjugated to monoclonal antibodies that bind (e.g., specifically bind) to GPC2. In some embodiments, the drug is a small molecule. In some instances, the drug is a cross-linking agent, an anti-microtubule agent and / or an anti-mitotic agent, or any cytotoxic agent suitable for mediating the killing of tumor cells. Exemplary cytotoxic agents include, but are not limited to, PDB, auristatin, maytansine compounds, sulphoside, calichimycin, nemorubicin and its derivatives, PNU-159682, anthracyclines, vinca alkaloids, taxanes, trichothecene, CC1065, camptothecin, elinefad, compressin, sulphoside, duocarmycin, enediyne, gerdemycin, pyrrolo-benzodiazepine dimer, puromycin, tubulysin, hammetrine, spliceostatin or pladienolide, as well as cytotoxic stereoisomers, isosteres, analogs and their derivatives.

[0296] In some embodiments, the ADC includes pyrrolobenzodiazepine (PBD). The natural product aprotinin (PBD) was first reported in 1965 (Leimgruber et al., JAM Chem Soc, 87:5793-5795, 1965; Leimgruber et al., JAM Chem Soc, 87:5791-5793, 1965). Since then, many PBDs have been reported, both naturally occurring and synthetic analogs (Gerratana, MedRes Rev 32(2):254-293, 2012; and US Patent Nos. 6,884,799; 7,049,311; 7,067,511; 7,265,105; 7,511,032; 7,528,126; and 7,557,099). As an example, PBD dimers recognize and bind to specific DNA sequences and have been demonstrated as cytotoxic agents. PBD dimers have been conjugated with antibodies, and the resulting ADCs have shown anticancer properties (see, for example, US 2010 / 0203007). Exemplary linker sites on PBD dimers include a five-membered pyrrole ring, a chain between PBD units, and an N10-C11 imine group (see WO2009 / 016516; US 2009 / 304710; US 2010 / 047257; US 2009 / 036431; US ​​2011 / 0256157; and WO2011 / 130598).

[0297] In some embodiments, the ADC comprises an antibody conjugated to one or more maytansine-like compound molecules. Maytansine-like compounds are derivatives of maytansine and are mitotic inhibitors that act by inhibiting tubulin polymerization. Maytansine was originally isolated from the East African shrub *Maytanthes dentata* (US Patent No. 3,896,111). Subsequently, it was discovered that certain microorganisms also produce maytansine-like compounds, such as maytanol and C-3 maytanol ester (US Patent No. 4,151,042). Synthetic maytansine compounds are disclosed, for example, in U.S. Patent Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348; 4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and 4,371,533.

[0298] In some embodiments, the ADC includes an antibody conjugated to scabrain or auristatin or their analogues or derivatives (see U.S. Patent Nos. 5,635,483; 5,780,588; 5,767,237; and 6,124,431). Auristatins are derivatives of the marine mollusc compound scabrain-10. Scabrain and auristatin have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cell division (Woyke et al., Antimicrob Agents and Chemother 45(12):3580-3584, 2001) and have anticancer activity (U.S. Patent No. 5,663,149) and antifungal activity (Pettit et al., Antimicrob Agents Chemother 42:2961-2965, 1998). Exemplary aurestatins include, but are not limited to, aurestatin 10, aurestatin E, aurestatin F, aurestatin EB (AEB), aurestatin EFP (AEFP), MMAD (monomethylaurestatin D or monomethylaurestatin 10), MMAF (monomethylaurestatin F or N-methylvaline-valine-dolaisoleuine-dolaproine-phenylalanine), MMAE (monomethylaurestatin E or N-methylvaline-valine-dolaisoleuine-dolaproine-norephedrine), 5-benzoylvaleric acid-AE ester (AEVB), and other aurestatins (see, for example, U.S. Publication No. 2013 / 0129753).

[0299] In some embodiments, the ADC comprises an antibody conjugated to one or more calicheamicin molecules. The calicheamicin antibiotic family and its analogues are capable of producing double-strand DNA breaks at subpicomolar concentrations (Hinman et al., Cancer Res 53:3336-3342, 1993; Lode et al., Cancer Res 58:2925-2928, 1998). Exemplary methods for preparing ADCs having a calicheamicin pharmaceutical moiety are described in U.S. Patent Nos. 5,712,374; 5,714,586; 5,739,116; and 5,767,285.

[0300] In some implementations, the ADC includes anthracyclines. Anthracyclines are antibiotic compounds with cytotoxic activity. Anthracyclines are believed to kill cells through a variety of different mechanisms, including inserting drug molecules into the DNA of cells, thereby inhibiting DNA-dependent nucleic acid synthesis; inducing the generation of free radicals, which then react with cellular macromolecules, thereby damaging cells; and / or the interaction of drug molecules with the cell membrane. Non-limiting exemplary anthracyclines include doxorubicin, epirubicin, idarubicin, doxorubicin, epirubicin, nemorubicin, pentorubicin, and mitoxantrone and their derivatives. For example, PNU-159682 is an effective metabolite (or derivative) of nemorubicin (Quintieri et al., Clin Cancer Res 11(4):1608-1617, 2005). Nemorubicin is a semi-synthetic analogue of doxorubicin, with a 2-methoxymorpholino group on the glycosidic amino group of doxorubicin (Grandi et al., Cancer Treat Rev 17:133, 1990; Ripamonti et al., BrJ Cancer 65:703-707, 1992).

[0301] In some embodiments, the ADC may further include a linker. In some instances, the linker is a bifunctional or multifunctional portion that can be used to link one or more drug moieties to an antibody to form an ADC. In some embodiments, the ADC is prepared using a linker having reactive functional groups for covalent attachment to the drug and antibody. For example, a cysteine ​​thiol of an antibody may form a bond with a reactive functional group of the linker or drug linker intermediate to form an ADC.

[0302] In some instances, the linker has a functional group capable of reacting with free cysteine ​​present on the antibody to form a covalent bond. Exemplary linkers having such reactive functional groups include maleimide, haloacetamide, β-haloacetyl, activated esters such as succinimide ester, 4-nitrophenyl ester, pentafluorophenyl ester, tetrafluorophenyl ester, acid anhydride, acyl chloride, sulfonyl chloride, isocyanate, and isothiocyanate.

[0303] In some instances, the linker has a functional group capable of reacting with an electrophilic group present on the antibody. Examples of such an electrophilic group include, but are not limited to, aldehyde and ketone carbonyl groups. In some cases, the heteroatom of the reactive functional group of the linker can react with the electrophilic group on the antibody and form a covalent bond with the antibody unit. Non-limiting examples include acylhydrazides, oximes, amino groups, hydrazides, thioureas, hydrazine esters, and aromatic acylhydrazides.

[0304] In some instances, the linker is a cleavable linker that facilitates drug release. Examples of cleavable linkers include acid-labile linkers (e.g., those containing hydrazones), protease-sensitive linkers (e.g., peptidase-sensitive linkers), light-labile linkers, and disulfide-containing linkers (Chari et al., Cancer Res 52:127-131, 1992; US Patent No. 5, 208, 020).

[0305] The ADCs disclosed herein can be used alone or in combination with another therapeutic agent and / or in combination with any standard therapy for treating cancer (e.g., surgical resection of the tumor, chemotherapy, or radiation therapy).

[0306] VII. Multispecific antibodies

[0307] Multispecific antibodies are recombinant proteins composed of antigen-binding fragments of two or more different monoclonal antibodies. For example, a bispecific antibody consists of antigen-binding fragments of two different monoclonal antibodies. Thus, a bispecific antibody binds to two different antigens, and a trispecific antibody binds to three different antigens. Multispecific antibodies are used in cancer immunotherapy by simultaneously targeting, for example, CTLs (e.g., CTL receptor components such as CD3) or effector natural killer (NK) cells and at least one tumor antigen. The GPC2-specific monoclonal antibody disclosed herein can be used to generate multispecific (e.g., bispecific or trispecific) antibodies targeting both GPC2 and CTLs, or targeting GPC2 and NK cells, thereby providing a means of treating cancers expressing GPC2.

[0308] Bispecific T-cell adaptor (BiTE) is a type of bispecific monoclonal antibody that is a fusion of a first single-chain variable fragment (scFv) targeting a tumor antigen and a second scFv that binds to T cells (e.g., binds to CD3 on T cells). In some embodiments herein, one of the binding portions of the BiTE (e.g., one of the scFv molecules) is specific for GPC2.

[0309] Bispecific killer cell adaptor (BiKE) is a type of bispecific monoclonal antibody that is a fusion of a first scFv that targets a tumor antigen and a second scFv that binds to an NK cell activation receptor (e.g., CD16).

[0310] This document provides multispecific (e.g., trispecific or bispecific) monoclonal antibodies comprising GPC2-specific monoclonal antibodies. In some embodiments, the multispecific monoclonal antibody further comprises a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to a component of a T-cell receptor (e.g., CD3). In other embodiments, the multispecific monoclonal antibody further comprises a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to an NK-cell activation receptor (e.g., CD16, Ly49, or CD94). Isolated nucleic acid molecules and vectors encoding the multispecific antibody are also provided, as well as host cells comprising the nucleic acid molecules or vectors. Multispecific antibodies comprising GPC2-specific antibodies can be used to treat cancers expressing GPC2. Therefore, this document provides a method for treating a subject with cancer by selecting a subject with GPC2-expressing cancer and administering a therapeutically effective amount of a GPC2-targeting multispecific antibody to that subject.

[0311] VIII. Antibody-Nanoparticle Conjugates

[0312] The monoclonal antibodies disclosed in this paper can be conjugated to a variety of different types of nanoparticles to deliver cytotoxic agents or other anticancer agents directly to tumor cells via antibody binding to tumor-specific antigens (e.g., GPC2) expressed on the tumor surface. The use of nanoparticles reduces off-target side effects and can also improve drug bioavailability and reduce the drug dosage required to achieve therapeutic effects. Nanoparticle formulations can be tailored to suit the drugs to be carried or encapsulated within the nanoparticles. For example, hydrophobic molecules can be incorporated into the core of the nanoparticle, while hydrophilic drugs are carried in an aqueous core protected by a polymer or lipid shell. Examples of nanoparticles include, but are not limited to, nanospheres, nanocapsules, liposomes, dendritic molecules, polymer micelles, lipovesicles, and polymer nanoparticles (Fay and Scott, Immunotherapy 3(3):381-394, 2011).

[0313] Liposomes are currently one of the most common types of nanoparticles used for drug delivery. Antibodies conjugated to liposomes are often referred to as "immunoliposomes." The liposomal component of an immunoliposome typically consists of one or more concentric phospholipid bilayers of lipid vesicles. In some cases, the phospholipids are composed of a hydrophilic head group and two hydrophobic chains to encapsulate both hydrophobic and hydrophilic drugs. Conventional liposomes are rapidly removed from circulation by macrophages in the reticuloendothelial system (RES). To produce long-circulating liposomes, the composition, size, and charge of the liposomes can be modulated. The surface of the liposomes can also be modified, for example, with glycolipids or sialic acid. For example, the inclusion of polyethylene glycol (PEG) can significantly increase the circulating half-life. Liposomes used as drug delivery agents have been described in the art, including liposomes for the preparation of immunoliposomes (see, for example, Paszko and Senge, CurrMed Chem 19(31)5239-5277, 2012; Immordino et al., Int J Nanomedicine 1(3):297-315, 2006; U.S. Patent Application Publication No. 2011 / 0268655; 2010 / 00329981).

[0314] Liposome vesicles are nonionic surfactant-based vesicles with a structure similar to liposomes. The membrane of a liposome consists solely of a nonionic surfactant, such as polyglycerol alkyl ethers or N-palmitoylglucosamine. Liposome vesicles range from small monolayer particles to large multilayer particles. These nanoparticles are monodisperse, water-soluble, chemically stable, low in toxicity, biodegradable, and non-immunogenic, increasing the bioavailability of encapsulated drugs.

[0315] Dendritic molecules comprise a series of branched polymer complexes. These nanoparticles are water-soluble, biocompatible, and sufficiently non-immunogenic for human use. Typically, dendritic molecules consist of an initiator core surrounded by selected polymer layers grafted onto that core, forming a branched macromolecular complex. Dendritic molecules are commonly produced using polymers such as poly(amidoamine) or poly(L-lysine). Dendritic molecules have been used in a variety of therapeutic and diagnostic applications, including for the delivery of DNA, RNA, bioimaging contrast agents, and chemotherapeutic agents.

[0316] Polymer micelles consist of aggregates of amphiphilic copolymers (composed of hydrophilic and hydrophobic monomer units) that assemble into a hydrophobic core and are surrounded by a corona discharge of hydrophilic polymer chains exposed to an aqueous environment. In many cases, the polymers used to prepare polymer micelles are heterobifunctional copolymers composed of hydrophilic PEG blocks, poly(vinylpyrrolidone), and hydrophobic poly(L-lactide) or poly(L-lysine) forming the particle core. Polymer micelles can be used to transport poorly soluble drugs. These nanoparticles have been used to encapsulate many anticancer drugs, including doxorubicin and camptothecin. Cationic micelles have also been developed to carry DNA or RNA molecules.

[0317] Polymer nanoparticles include nanospheres and nanocapsules. Nanospheres consist of a solid polymer matrix, while nanocapsules contain an aqueous core. The choice of formulation typically depends on the solubility of the therapeutic agent to be carried / encapsulated; drugs with poor water solubility are more easily encapsulated in nanospheres, while water-soluble and unstable drugs (such as DNA and proteins) are more easily encapsulated in nanocapsules. Polymers used to produce these nanoparticles include, for example, poly(acrylamide), poly(ester), poly(alkyl cyanoacrylate), poly(lactic acid) (PLA), poly(glycolic acid) (PGA), and poly(D,L-lactic-co-glycolic acid) (PLGA).

[0318] Antibodies (or fragments thereof) can be conjugated to suitable nanoparticles according to standard methods known in the art. For example, the conjugation can be covalent or non-covalent. In some embodiments where the nanoparticles are liposomes, the antibody is attached to a spatially stable long-circulating liposome via a PEG chain. The conjugation of the antibody or antibody fragment to the liposome can also involve thioester bonds, for example, via a reaction of a thiol and a maleimide group. Crosslinking agents can be used to generate thiol groups to allow the antibody to attach to the nanoparticle (Paszko and Senge, Curr Med Chem 19(31)5239-5277, 2012).

[0319] IX. Composition and Method of Use

[0320] Compositions comprising one or more disclosed monoclonal antibodies, said monoclonal antibodies binding (e.g., specifically binding) to GPC2 in a vector are provided. Compositions comprising ADCs, CARs (and CAR-containing CTLs), multispecific (e.g., bispecific or trispecific) antibodies, antibody-nanoparticle conjugates, immunoliposomes, and immunoconjugates are also provided. The compositions can be prepared in unit dosage forms for administration to a subject. The amount and timing of administration are determined by the clinician to determine whether the desired outcome is achieved. Antibodies, ADCs, CARs, CTLs, multispecific antibodies, antibody-nanoparticle conjugates, immunoliposomes, or immunoconjugates can be formulated for systemic or local (e.g., intratumoral) administration. In one example, the antibody is formulated for parenteral administration, such as intravenous administration.

[0321] Compositions for administration may include solutions of antibodies, ADCs, CARs, CTLs, multispecific (e.g., bispecific or trispecific) antibodies, antibody-nanoparticle conjugates, immunoliposomes, or immunoconjugates in pharmaceutically acceptable carriers (e.g., aqueous carriers). Various aqueous carriers may be used, such as buffered saline. These solutions are sterile and generally free of undesirable substances. These compositions may be sterilized using conventional, well-known sterilization techniques. The compositions may contain pharmaceutically acceptable excipients required to approximate physiological conditions, such as pH adjusters and buffers, toxicity modifiers, etc., such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of antibodies in these formulations can vary widely and will be selected based primarily on fluid volume, viscosity, weight, etc., depending on the specific route of administration chosen and the needs of the subject.

[0322] Typical pharmaceutical compositions for intravenous administration contain approximately 0.1 to 10 mg of antibody (or ADC, CAR, multispecific antibody, antibody-nanoparticle conjugate, or immunoconjugate) per subject per day. Doses of approximately 0.1 to 100 mg per subject per day may be used, particularly if the agent is administered to a concealed site rather than into the circulatory or lymphatic system, such as into the cavities or lumens of body cavities or organs. Practical methods for preparing administerable compositions are known or obvious to those skilled in the art and are described in more detail in publications such as Remington's Pharmaceutical Science, 19th ed., Mack Publishing Company, Easton, PA (1995).

[0323] Antibodies (or other therapeutic molecules) may be provided in lyophilized form and rehydrated with sterile water prior to administration, although they are also provided as sterile solutions of known concentrations. The antibody solution is then added to an infusion bag containing 0.9% sodium chloride, USP, and administered in some cases at doses of 0.5 to 15 mg / kg body weight. Since 1997, RITUXAN TM Since their approval, antibody drugs have accumulated considerable experience in administration, and these drugs are already marketed in the United States. Antibodies, ADCs, CARs, multispecific (e.g., bispecific or trispecific) antibodies, antibody-nanoparticle conjugates, immunoliposomes, or immunoconjugates can be administered via slow infusion rather than via intravenous bolus or bolus injection. In one instance, a higher loading dose is administered, followed by a maintenance dose at a lower level. For example, an initial loading dose of 4 mg / kg can be infused over approximately 90 minutes, followed by a weekly maintenance dose of 2 mg / kg infused over 30 minutes for 4–8 weeks, provided the initial dose is well tolerated.

[0324] Controlled-release parenteral formulations can be formulated as implants, oily injectables, or particulate systems. For a broad overview of protein delivery systems, see Banga, AJ, 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 a therapeutic protein, such as a cytotoxin or drug, as a central core. In microspheres, the therapeutic agent is dispersed throughout the particle. Particles, microspheres, and microcapsules smaller than about 1 μm are generally referred to as nanoparticles, nanospheres, and nanocapsules, respectively. Capillary diameters are about 5 μm, making only nanoparticles suitable for intravenous administration. Microparticles typically have diameters of about 100 μm and can be 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).

[0325] Polymers can be used for the ion-controlled release of the antibody-based compositions disclosed herein. A variety of degradable and non-degradable polymer matrices for controlled drug delivery are known in the art (Langer, Accounts Chem. Res. 26:537-542, 1993). For example, the block copolymer poloxamer 407 exists as a viscous but flowable liquid at low temperatures, but forms a semi-solid gel at body temperature. It has proven to be an effective solvent for the formulation and sustained delivery of recombinant interleukin-2 and urease (Johnston et al., Pharm. Res. 9:425-434, 1992; and Pec et al., J. Parent. Sci. Tech. 44(2):58-65, 1990). Additionally, hydroxyapatite has been used as a microcarrier for the controlled release of proteins (Ijntema et al., Int. J. Pharm. 112:215-224, 1994). On the other hand, liposomes can be used for the controlled release of drugs encapsulated in lipid capsules and for drug targeting (Betageri et al., Liposome Drug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, PA (1993)). Many other systems for the 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).

[0326] A. Treatment methods

[0327] The antibodies, compositions, CARs (and CAR-expressing CTLs), ADCs, multispecific (e.g., bispecific or trispecific) antibodies, antibody-nanoparticle conjugates, immunoliposomes, and immunoconjugates disclosed herein can be administered to slow or inhibit the growth or metastasis of tumor cells, such as in GPC2-positive cancers. In these applications, a therapeutically effective amount of the composition is administered to the subject in an amount sufficient to inhibit the growth, replication, or metastasis of cancer cells, or to suppress signs or symptoms of cancer. Thus, in some instances, the disclosed methods reduce the size, weight, and / or volume of the primary tumor and / or metastases by at least 20%, at least 25%, at least 50%, at least 75%, at least 80%, at least 90%, at least 95%, at least 99%, or even 100%. Thus, in some instances, the disclosed methods reduce the number of metastases by at least 20%, at least 25%, at least 50%, at least 75%, at least 80%, at least 90%, at least 95%, at least 99%, or even 100%. Suitable subjects may include those diagnosed with cancers that express GPC2, such as, but not limited to, neuroblastoma, medulloblastoma, retinoblastoma, acute lymphoblastic leukemia, embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, Ewing's sarcoma, desmoplastic small round cell tumor, or osteosarcoma.

[0328] This document provides methods for treating GPC2-positive cancer in a subject by administering a therapeutically effective amount of a GPC2-specific antibody, immunodrug conjugate, CAR (e.g., a CTL expressing a CAR), ADC, multispecific (e.g., bispecific or trispecific) antibody, antibody-nanoparticle conjugate, immunoliposome, or a composition disclosed herein. This document also provides methods for inhibiting tumor growth or metastasis of GPC2-positive cancer in a subject by administering a therapeutically effective amount of a GPC2-specific antibody, immunodrug conjugate, CAR (e.g., a CTL expressing a CAR), ADC, multispecific (e.g., bispecific or trispecific) antibody, antibody-nanoparticle conjugate, immunoliposome, or a composition disclosed herein. In some embodiments, the GPC2-positive cancer is neuroblastoma, medulloblastoma, retinoblastoma, acute lymphoblastic leukemia, embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, Ewing's sarcoma, desmoplastic small round cell tumor, or osteosarcoma.

[0329] The therapeutically effective amount of a GPC2-specific monoclonal antibody (or fragment thereof), ADC, CAR (e.g., a CTL expressing a CAR), multispecific (e.g., bispecific or trispecific) antibody, immunoconjugate, immunoliposome, or composition disclosed herein will depend on the severity of the disease, the type of disease, and the patient's general health status. The therapeutically effective amount of an antibody-based composition is the amount that provides subjective relief of symptoms(s) or objectively identifiable improvement as indicated by a clinician or other qualified observer.

[0330] The administration of the GPC2-specific antibodies, ADCs, CARs, immunoconjugates, multispecific (e.g., bispecific or trispecific) antibodies, antibody-nanoparticle conjugates, immunoliposomes, and compositions disclosed herein may also be administered concurrently with other anticancer agents or therapeutic treatments (e.g., surgical resection of tumors). Any suitable anticancer agent may be administered in combination with the antibodies, compositions, and immunoconjugates disclosed herein (e.g., sequentially or simultaneously). Exemplary anticancer agents include, but are not limited to, chemotherapeutic agents, such as, for example, mitotic inhibitors, alkylating agents, antimetabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, anti-survival agents, biological response modifiers, antihormones (e.g., antiandrogens), and antiangiogenic agents. Other anticancer treatments include radiotherapy and other antibodies that specifically target cancer cells.

[0331] Non-limiting examples of alkylating agents include nitrogen mustard (e.g., dichloromethyldiethylamine, cyclophosphamide, melphalan, uracil nitrogen mustard, or phenylbutyrate nitrogen mustard), alkyl sulfonates (e.g., busulfan), and nitrosoureas (e.g., carmustine, lomustine, semustine, streptozotocin, or dacarbazine).

[0332] Non-limiting examples of antimetabolites include folic acid analogs (e.g., methotrexate), pyrimidine analogs (e.g., 5-FU or cytarabine), and purine analogs such as mercaptopurine or thioguanine.

[0333] Non-limiting examples of natural products include vinca alkaloids (e.g., vincristine, vinblastine, or vincristine), epipodophyllotoxins (e.g., etoposide or teniposide), antibiotics (e.g., actinomycin, daunorubicin, doxorubicin, bleomycin, procainomic acid, or mitomycin C), and enzymes (e.g., L-asparaginase).

[0334] Other non-limiting examples of reagents include platinum coordination complexes (e.g., cis-dichloroplatinum II, also known as cisplatin), substituted urea (e.g., hydroxyurea), methylhydrazine derivatives (e.g., procarbazine), and adrenocortical hormone inhibitors (e.g., mitotane and ammoniaglutide).

[0335] Non-limiting examples of hormones and antagonists include corticosteroids (e.g., prednisone), progestins (e.g., hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrol acetate), estrogens (e.g., diethylstilbestrol and ethinylestradiol), anti-estrogens (e.g., tamoxifen), and androgens (e.g., testosterone propionate and fluoromethesterone). Examples of commonly used chemotherapy drugs include adriamycin, melphalan, cytarabine (Ara-C), BiCNU, busulfan, CCNU, carboplatin, cisplatin, cyclophosphamide, daunorubicin, DTIC, 5-FU, fludarabine, hydroxyurea, idarubicin, ifosfamide, methotrexate, mithramycin, mitomycin, mitoxantrone, and nitrogen mustard. Mustard, Taxol (or other taxanes such as docetaxel), Velban, Vincristine, VP-16, and some newer drugs include Gemcitabine (Gemzar), Herceptin, Irinotecan (Camptosar, CPT-11), Leustatin, Navelbine, Rituxan STI-571, Taxotere, Topotecan (Hycamtin), Xeloda (Capecitabine), Zevelin, and calcitriol.

[0336] Non-limiting examples of immunomodulators that may be used include AS-101 (Wyeth-Ayerst Labs.), brompiridamine (Upjohn), interferon-gamma (Genentech), GM-CSF (granulocyte-macrophage colony-stimulating factor; Genetics Institute), IL-2 (Cetus or Hoffman-LaRoche), human immunoglobulin (Cutter Biological), IMREG (Imreg from New Orleans, La.), SK&F 106528, and TNF (tumor necrosis factor; Genentech).

[0337] Non-limiting examples of other biologics (e.g., monoclonal antibodies) that can be used with the antibodies, compositions, and immunoconjugates disclosed herein include antibodies that antagonize PD-1 or PD-L1, such as atezolizumab, MPDL3280A, BNS-936558 (nivolumab), pembrolizumab, pitilizumab, CT011, AMP-224, AMP-514, MEDI-0680, BMS- 936559, BMS935559, MEDI-4736, MPDL-3280A, MSB-0010718C, MGA-271, Indoximod, Epacadostat, BMS-986016, MEDI-4736, MEDI-4737, MK-4166, BMS-663513, PF-05082566 (PF-2566), Lirilumab, and Durvalumab. In some instances, other therapeutic agents administered included 3F8, abagovomab, adecatumumab, afutuzumab, alacizumab, alemtuzumab, atumomab penetate, anatumomab mafenatox, apolizumab, arcitumomab, bavituximab, beectumomab, belimumab, besilesomab, bevacizumab, and bivatuzumab. Mertansine, Blinatumomab, Brentuximab vedotin, Cantuzumab mertansine, Capromab pendetide, Catuxomab, CC49, Cetuximab, CitatuzumabBogatox, Cixutumumab, Clivatuzumabtetraxetan, Conatumumab, Dacetuzumab, Detumomab, Ecromeximab, Eculizumab, Edrecolomab, Epratuzumab, Ertumaxomab, Etaracizumab, Farletuzumab, Figitumumab, Galiximab, Gemtuzumab ozogamicum, Girentuximab, Glembatumumab vedotin, Ibritumomab tiuxetan, Igovomab, Imciromab, Intetumumab, Inotuzumabozogamicin, Ipilimumab, Iratumumab, Labetuzumab, Lexatumumab, Lintuzumab, Lorvotuzumab mertansine, Lucarumumab, Lumiliximab, Mapatumumab, Matuzumab, Mepolizumab, Metelimumab, Milatuzumab, Mitumomab, Morolimumab, Nacolomab tafenatox, Naptumomabestafenatox, Necitumumab, Nimotuzumab, Nofetumomab merpentan, Ofatumumab, Olaratumab, OportuzumabMonatox, Oregovomab, Panitumumab, Pemtumomab, Pertuzumab, Pintumomab, Pritumumab, Ramucirumab, Rilotumumab, Rituximab, Robatumumab, Satumomab pendetide, Sibrotuzumab, Sonepizumab, Tacatuzumab tetraxetan, Taplitumomab One or more of the following: paptox, tenatumomab, TGN1412, ticilimumab, tremelimumab, tigatuzumab, TNX-650, trastuzumab, tremelimumab, tucotuzumab celmoleukin, vetuzumab, volociximab, votumumab, and zalutumumab. Another exemplary treatment for some types of cancer is surgical treatment, such as surgical removal of the cancer or a portion thereof. Another example of treatment is radiation therapy, such as the application of radioactive material or energy (e.g., external beam therapy) to the tumor site prior to surgical removal to help eradicate or shrink the tumor.

[0338] This document also provides kits comprising GPC2-specific monoclonal antibodies or antigen-binding fragments, CARs, isolated cells, immunoconjugates, ADCs, multispecific antibodies, antibody-nanoparticle conjugates, fusion proteins, or compositions disclosed herein, and a second therapeutic agent for the treatment of cancer. In some instances, the second therapeutic agent is a radioactive or chemical compound.

[0339] B. Diagnostic and Detection Methods

[0340] This article provides methods for detecting GPC2 protein in vitro or in vivo. In some cases, GPC2 expression is detected in biological samples. Samples can be any type of sample, including but not limited to tissues used for biopsies, autopsies, and pathological specimens. Biological samples also include tissue sections, such as frozen sections taken for histological purposes. Biological samples also include bodily fluids, such as blood, serum, plasma, sputum, cerebrospinal fluid, or urine. Biological samples are typically obtained from mammals, such as humans or non-human primates.

[0341] This article provides a method for determining whether a subject has GPC2-positive cancer by contacting a subject's sample with a GPC2-specific monoclonal antibody disclosed herein and detecting antibody binding to the sample. Increased antibody binding to the sample, compared to antibody binding to a control sample, identifies the subject as having GPC2-positive cancer.

[0342] In another embodiment, a method is provided to confirm the diagnosis of GPC2-positive cancer in a subject by contacting a sample from a subject diagnosed with GPC2-positive cancer with a GPC2-specific monoclonal antibody disclosed herein and detecting the binding of the antibody to the sample. Increased binding of the antibody to the sample, compared to binding to a control sample, confirms the diagnosis of GPC2-positive cancer in the subject.

[0343] In some instances of the disclosed methods, monoclonal antibodies are directly labeled.

[0344] In other instances, the method further includes contacting a sample with a second antibody that specifically binds to a monoclonal antibody; and detecting the binding of the second antibody. Increased binding of the second antibody to the sample, compared to binding to a control sample, detects or confirms a diagnosis of GPC2-positive cancer in the subject.

[0345] In some cases, cancer can be neuroblastoma, medulloblastoma, retinoblastoma, acute lymphoblastic leukemia, embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, Ewing's sarcoma, desmoplastic small round cell tumor, or osteosarcoma.

[0346] In some instances, the control sample is a sample from a subject without cancer. In specific instances, the sample is a blood or tissue sample.

[0347] In some embodiments of the diagnostic and detection methods, the antibody that binds to (e.g., specifically binds to) GPC2 is directly labeled with a detectable marker. In another embodiment, the antibody that binds to (e.g., specifically binds to) GPC2 (the primary antibody) is not labeled, and a secondary antibody or other molecule that can bind to the antibody that specifically binds to GPC2 is labeled. As is known to those skilled in the art, a specific type and class of secondary antibody capable of specifically binding to the primary antibody is selected. For example, if the primary antibody is human IgG, the secondary antibody may be anti-human IgG. Other molecules that can bind to the antibody include, but are not limited to, protein A and protein G, both of which are commercially available.

[0348] Suitable labeling for antibodies or secondary antibodies includes various enzymes, prosthetic groups, fluorescent materials, luminescent materials, magnetic reagents, and radioactive substances. Non-limiting examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase. Non-limiting examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin. Non-limiting examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazineamine fluorescein, dansyl chloride, or phycoerythrin. A non-limiting exemplary luminescent material is luminol; a non-limiting exemplary magnetic agent is gadolinium; and a non-limiting exemplary radiolabeling includes… 125 I, 131 I, 35 S or 3 H.

[0349] In an alternative implementation, GPC2 can be measured in a biological sample using a competitive immunoassay, employing a GPC2 protein standard labeled with a detectable substance and an unlabeled antibody that specifically binds to GPC2. In this assay, the biological sample, the labeled GPC2 protein standard, and the antibody that specifically binds to GPC2 are combined, and the amount of labeled GPC2 protein standard binding to the unlabeled antibody is determined. The amount of GPC2 in the biological sample is inversely proportional to the amount of labeled GPC2 protein standard bound by the antibody that specifically binds to GPC2.

[0350] The immunoassays and methods disclosed herein can be used for a variety of purposes. In one embodiment, a specifically binding antibody can be used to detect the production of GPC2 in cells within a cell culture. In another embodiment, the antibody can be used to detect the amount of GPC2 in a biological sample such as a tissue sample or a blood or serum sample. In some instances, GPC2 is cell surface GPC2. In other instances, the GPC2 protein is soluble (e.g., in cell culture supernatant or in bodily fluid samples, such as blood or serum samples).

[0351] In one embodiment, a kit is provided for detecting GPC2 in a biological sample, such as a blood sample or tissue sample. For example, to confirm a cancer diagnosis in a subject, a biopsy may be performed to obtain a tissue sample for histological examination. The kit for detecting the peptide may include a monoclonal antibody that specifically binds to GPC2, such as any monoclonal antibody disclosed herein. In another embodiment, the antibody is labeled (e.g., with a fluorescent, radioactive, or enzyme-labeled substance).

[0352] In one embodiment, the kit includes instructional material disclosing methods for using an antibody that binds to GPC2. The instructional material may be written in electronic form (e.g., computer floppy disk or optical disc) or visual (e.g., video file). The kit may also include additional components to facilitate kit design for a specific application. Thus, for example, the kit may additionally include means for detecting markers (e.g., enzyme substrates for enzyme markers, filter banks for detecting fluorescent markers, suitable second markers, such as second antibodies, etc.). The kit may additionally include buffers and other reagents conventionally used to perform the specific method. Such kits and suitable contents are well known to those skilled in the art.

[0353] In one embodiment, the diagnostic kit includes an immunoassay. While the details of the immunoassay may vary depending on the specific form employed, methods for detecting GPC2 in biological samples generally include the following steps: contacting the biological sample with an antibody that specifically reacts with GPC2 under immunoreactive conditions; allowing the antibody to specifically bind under immunoreactive conditions to form an immune complex; and detecting the presence of the immune complex (bound antibody) directly or indirectly.

[0354] The antibodies disclosed herein can also be used in immunoassays, such as, but not limited to, radioimmunoassay (RIA), ELISA, or immunohistochemistry. The antibodies can also be used in fluorescence-activated cell sorting (FACS). FACS employs multiple color channels, low-angle and obtuse-angle light scattering detection channels, impedance channels, and other more sophisticated detection levels to separate or classify cells (see U.S. Patent No. 5,061,620). Any monoclonal antibody that binds to GPC2, as disclosed herein, can be used in these assays. Therefore, this antibody can be used in routine immunoassays, including but not limited to ELISA, RIA, FACS, immunohistochemistry, Western blotting, or immunoprecipitation.

[0355] The following examples are provided to illustrate certain features and / or implementations. These examples should not be construed as limiting this disclosure to the specific features or implementations described.

[0356] Example

[0357] Example 1: Isolation and characterization of high-affinity GPC2-specific antibodies

[0358] GPC2-specific monoclonal antibodies were isolated using mouse hybridoma technology. For immunization, a 50-mer peptide (residues 504-553) corresponding to the C-terminus of the cell surface protein GPC2 was synthesized. Additional cysteine ​​residues were introduced to the N-terminus of the peptide. Therefore, it was assumed that the monoclonal antibody (mAb) generated against this peptide binds to the membrane-bound C-terminus of GPC2. Three C57BL / 6 mice were immunized three times and boosted with a peptide-KLH conjugate. Spleen cell fusion was performed using two of the best-performing mice. Two antibodies, CT3 and CT5, were isolated from ten clones and isotyped to IgG1κ. The nucleotide and amino acid sequences of the VH and VL domains of CT3 and CT5 are shown in this paper as SEQ ID NO: 1-8. The positions of the CDR sequences in each domain are shown in Tables 1-4 (see Section III above).

[0359] ELISA was performed using recombinant human and / or mouse phosphatidylinositol proteoglycan to determine the binding specificity of the two antibodies. For example... Figure 1A As shown, CT3 and CT5 bind only to human GPC2, but not to human GPC3, GPC5, or GPC6. Further evaluation of the binding ability of CT3 to human GPC proteins and mouse GPC2 is needed. Figure 1B As shown, the CT3 antibody specifically binds to human GPC2. Octet body kinetics analysis was performed to measure the binding affinity of the CT3 antibody to human GPC2. Figure 1C As shown, CT3 exhibits high affinity (K... D =0.5nM) combined with GPC2.

[0360] Flow cytometry was used to further evaluate the CT3 and CT5 antibodies to determine their ability to bind to GPC2 expressed on the cell surface. In the first study, the binding of CT5 to two neuroblastoma cell lines expressing GPC2, LAN1 and IMR5, was tested. The results demonstrated that the CT5 antibody bound to GPC2 in both neuroblastoma cell lines. Figure 2A The second study measured the binding of CT3 to GPC2-negative A431 cells, GPC2-positive G10 cells (A431 cell lines overexpressing GPC2), IMR5 cells, and F8 cells (IMR5 cell lines overexpressing GPC2). Figure 2B As shown, the CT3 antibody specifically recognizes GPC2 expressed on the cell surface of all three GPC2-positive cell lines.

[0361] To evaluate the application of CT3 and CT5 antibodies in immunohistochemistry, CT3 antibody was used to examine GPC2 expression in human tissues from patients with neuroblastoma or non-malignant diseases. In all experiments, tissues were labeled with 1 μg / ml CT3 antibody. Figure 3 As shown, GPC2 markers were evident in samples from patients with neuroblastoma (i-iv), but were essentially undetectable in normal peripheral nerves from patients with non-malignant diseases (v and vi). Neuroblastoma tumor tissue showed strong GPC2 staining in 23 out of 25 cases (92%). GPC2 expression in other pediatric cancers, including medulloblastoma and retinoblastoma, was also assessed using the CT3 antibody. Figure 4 As shown, strong GPC2 expression was found in 10 out of 20 medulloblastoma cases (i to iv), while no staining was detected in normal brain samples (v and vi). Nearly 80% of retinoblastoma samples (11 out of 14 cases) showed high levels of GPC2 expression compared to adjacent normal corneal and retinal tissue. Figure 5 To further analyze GPC2 expression in normal human tissues, an FDA-recommended array of normal human tissues was probed using the CT3 antibody. No significant GPC2 staining was observed in normal tissues including vital organs such as the brain, heart, lungs, and liver. Figure 6 These results indicate tumor-specific expression of GPC2 and the potential use of these antibodies as diagnostic tools for neuroblastoma, medulloblastoma, retinoblastoma, and other cancers.

[0362] To explore the therapeutic applications of anti-GPC2 mAb, a chimeric antigen receptor (CAR) was constructed using a single-chain variable fragment (scFv) of the CT3 antibody. The CAR nucleic acid construct contained in the 5' to 3' directions included: a nucleic acid encoding the first GMCSFR signaling sequence; a nucleic acid encoding the CT3 scFv; a nucleic acid encoding the CD8α extracellular hinge region; a nucleic acid encoding the CD8 transmembrane (TM) domain; a nucleic acid encoding the 4-1BB intracellular co-stimulatory domain; a nucleic acid encoding the CD3ζ intracellular signal transduction domain; a nucleic acid encoding the self-cleaving T2A peptide; a nucleic acid encoding the second GMCSFR signaling sequence; and a nucleic acid encoding huEGFRt. Figure 7A ).like Figure 7B As shown, the transduction efficiency of CT3 CAR in human T cells was 56%. Even with an effector:target ratio (E:T) as low as 5.5:1, CT3CAR T cells effectively lysed three GPC2-positive neuroblastoma cell lines (SKNBE2, NBEB, and LAN1). Figures 7C-7E In contrast, T-cell-mediated killing, which mimics transduction, has the least killing effect in neuroblastoma cells.

[0363] To humanize the CT3 antibody for clinical application in treating cancer patients, the CT3 CDR was transplanted into a human frame sequence. The humanized scFv construct contains a (Kabat / IMGT / Paratome) CDR combining CT3 (see Tables 1 and 2) and a human frame sequence. The VH and VL domains are separated by a (G3S)4 linker. Three clones were generated: hCT3-1 (SEQ ID NO:10), hCT3 (SEQ ID NO:11), and hCT3-3 (SEQ ID NO:21). CAR T cells expressing the humanized CT3 antibody showed efficacy against GPC2-positive tumor cells (G10, IMR5, and F8). Figure 8B-8D The original CT3-based CAR T cells showed considerable killing activity, but not against GPC2-negative A431 cells. Figure 8A The data were not normalized according to their transduction efficiency. If normalized by their transduction efficiency, humanized CT3-derived CAR T cells are more active than original CT3 CAR T cells because humanized CT3 CARs have reduced transduction efficiency compared to original CT3 CARs.

[0364] CT3 CAR T cells were also tested in a mouse neuroblastoma model. On day 0, NSG mice were administered 7 million IMR5-luc tumor cells. On day 35, IMR5-bearing NSG mice were administered blank control T cells, or 2.5 million, 5 million, or 10 million CT3 CAR T cells via tail vein injection. Figure 9A Tumor burden was monitored by bioluminescence imaging on the day of CAR T-cell infusion and on days 3, 11, 19, and 28 post-CAR T-cell infusion. Figure 9B CT3 CAR T cells significantly inhibited neuroblastoma growth in mice at higher doses (5 million and 10 million), while a low dose (2.5 million) inhibited tumor growth. The persistence of GPC2-targeting CAR T cells in vivo was assessed by measuring CAR vector-positive cells in the spleen of mice after 2–4 weeks of treatment. Specifically, the spleens of mice treated with 2.5 million CAR T cells were harvested on day 34, the spleens of mice treated with 5 million CAR T cells were harvested on day 14, and the spleens of mice treated with 10 million CAR T cells were harvested on day 28. Figure 10 As shown, 41.2%, 26.54%, and 21.2% of the cells harvested from mice treated with 2.5 million, 5 million, and 10 million CAR T cells, respectively, were identified as CAR T cells, indicating that CAR T cells remained in the spleen of mice for at least one month after treatment.

[0365] To test the immunotoxin form of the CT3 antibody, the CT3 CDR was grafted onto the framework sequence of the mouse antibody SS1 (see Chowdhury and Pastan, Nat Biotechnol 17:568-572, 1999; Pastan et al., Nat RevCancer 6:559-565, 2006). The SS1P immunotoxin has been generated and tested in clinical trials for the treatment of mesothelioma, ovarian cancer, and pancreatic cancer. The scFv contains a combined (Kabat / IMGT / Paratome) CDR of CT3, framework residues of SS1, and a (G3S)4 linker between the VH and VL domains. The resulting scFv is designated sCT3 (SEQ ID NO:12). An immunotoxin containing sCT3scFv and a truncated Pseudomonas exotoxin (PE38) was generated. Figure 12A-12B The production of sCT3-PE38 immunotoxin was demonstrated. Its purity and yield were similar to SS1P. The ability of sCT3-PE38 to kill GPC2-expressing cells was tested. Cells were incubated with different concentrations of immunotoxin for three days, and cell counts were determined using the WST-8 cell proliferation assay. Cell proliferation in untreated wells was set to 100% when calculating inhibition of cell proliferation. sCT3-PE38 immunotoxin killed GPC2-positive tumor cell lines but did not kill GPC2-negative cells. Figure 12C These results demonstrate that the cell inhibition induced by sCT3-PE38 is antigen-dependent.

[0366] Two additional GPC2-targeting immunotoxins were developed using CT3 scFv (SEQ ID NO:9), along with either PE38 (SEQ ID NO:22) or T20 (SEQ ID NO:23) as the toxins. Octet body kinetics were performed to evaluate the binding of the GPC2-targeting immunotoxins to human GPC2. Human GPC2-His (250 ng / ml) was loaded onto a Ni-NTA biosensor using the Octet Red96e system. Immunotoxins (CT3-PE38 and CT3-T20) were added at 100 nM to determine binding affinity. Association occurred at 600 sec, and dissociation at 1800 sec. The affinity (K0.05) of CT3-PE38 and CT3-T20 for human GPC2 was determined. D The values ​​were determined to be 0.22 nM and 0.16 nM, respectively. Figure 11A Due to its high affinity for human GPC2, CT3 is well-suited for immunotoxin development. Tris-glycine 4-20% gel was used to evaluate the fraction purity during the CT3-PE38 elution process. Figure 11B-11C ) and CT3-T20 ( Figure 11D-11EImmunotoxins. The ability of two immunotoxins to kill GPC2-expressing cell lines was then analyzed. Cells were incubated for three days with different concentrations of immunotoxins, and cell numbers were determined using the WST-8 cell proliferation assay. CT3-PE38 ( Figure 11F ) and CT3-T20 ( Figure 11G Treatment resulted in inhibition of GPC2-positive G10, F8, and IMR5 cells. In contrast, low-antigen IMR32 and antigen-negative A431 cells showed no inhibitory effect.

[0367] Next, experiments were conducted to evaluate GPC2-targeting immunotoxins in GPC2-expressing animal tumor models. Ten million F8 cells in a matrix gel were injected into the right dorsal side of nude mice. When the average tumor volume reached 100 mm, the immunotoxin was used to evaluate the tumor. 3 Treatment was initiated with PBS, CT3-PE38 (0.25 mg / kg), or CT3-T20 (2 mg / kg). Treatment with any of the immunotoxins resulted in a significant reduction in tumor volume (p < 0.0001), and the immunotoxins were well-tolerated. Figure 13 ).

[0368] In another study, two million G10 cells in a matrix gel were injected into the right dorsal side of nude mice. When the average tumor volume reached 100 mm... 3 Treatment was initiated with PBS, CT3-PE38 (0.25 mg / kg), or CT3-T20 (6 mg / kg). Treatment was initiated when the tumor exceeded 1500 mm. 3 Mice were euthanized when the tumor began to ulcerate. The mean survival time in the immunotoxin group was 28 days, compared to 21 days in the PBS treatment group. CT3-based immunotoxin treatment significantly improved the survival rate of nude mice in the G10 subcutaneous xenograft model (p<0.05). Figure 14B Mice at doses up to 6 mg / kg tolerated CT3-T20 well. Figure 14A ).

[0369] In the third study, five million IMR5 cells were injected into nude mice via the tail vein. Mice were treated with either PBS or CT3-T20 (4 mg / kg). Emissivity was measured using IVIS Lumina series III after injection of 100 μl of xenolight D-fluorescein (30 mg / ml). Figure 15A Treatment with CT3-T20 immunotoxin resulted in a reduction in tumor burden in the IMR5 metastasis model. Figure 15B ).

[0370] Given the many possible embodiments to which the principles of the disclosed subject matter can be applied, it should be recognized that the illustrated embodiments are merely examples of the disclosure and should not be considered as limiting the scope of the disclosure. Rather, the scope of the disclosure is defined by the following claims. Therefore, we claim all that falls within the scope and spirit of these claims. sequence list <110> The U.S. government (represented by the Secretary of Health and Human Services) <120> High-affinity monoclonal antibodies targeting phosphatidylinositol proteoglycan-2 and their applications <130> 4239-101087-02 <150> US 62 / 716,169 <151> 2018-08-08 <160> twenty three <170> PatentIn version 3.5 <210> 1 <211> 369 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotides <400> 1 gaggtccagc tgcaacagtc tggacctgaa ctggtgaagc ctggggcttc agtaaagatg 60 tcctgcaagg cttctagatt cacattcact gactacaaca tacactgggt gaagcagagc 120 cctggaaaga cccttgaatg gattggatat attaacccta acaatggtga tattttctac 180 aaacagaagt tcaatggcaa ggccacattg actataaaca agtcctccaa cacagcctac 240 atggagctcc gcagcctgac atcggaggat tctgcagtct attactgtgt aagatcctct 300 aatattcgtt atactttcga caggttcttc gatgtctggg gcacagggac cacggtcacc 360 gtctcctca 369 <210> 2 <211> 123 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide<00!1024><400> 2 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Arg Phe Thr Phe Thr Asp Tyr 20 25 30 Asn Ile His Trp Val Lys Gln Ser Pro Gly Lys Thr Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Asn Asn Gly Asp Ile Phe Tyr Lys Gln Lys Phe 50 55 60<00!1033>Asn Gly Lys Ala Thr Leu Thr Ile Asn Lys Ser Ser Asn Thr Ala Tyr[[ID=!5]] 65 70 75 80 Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95[[ID=!1]] Val Arg Ser Ser Asn Ile Arg Tyr Thr Phe Asp Arg Phe Phe Asp Val 100 105 110 Trp Gly Thr Gly Thr Thr Val Thr Val Ser Ser [[ID=!7]] (注:原文中“<5001024>”疑似应为“ ”;“<00!1033>”疑似应为“ ”;“<00!1037>”疑似应为“ ”,已按正确格式翻译。)115 120 <210> 3 <211> 318 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotide <400> 3 gaaaatgtgc tcacccagtc tccagcaatc atgtctgcat ctctagggga gaaggtcacc 60 atgagctgca gggccagctc aagtgtaaat tacatttact ggtaccagca gaagtcagat 120 gcctccccca aactatggat ttattacaca tccaacctgg ctcctggagt cccagctcgc 180 ttcagtggca gtgggtctgg gaactcttat tctctcacaa tcagcagcat ggagggtgaa 240 gatgctgcca cttattactg ccagcagttt tctagttccc catccacgtt cggtactggg 300 accaagctgg agctgaaa 318 <210> 4 <211> 106 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <400> 4 Glu Asn Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Leu Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Arg Ala Ser Ser Ser Val Asn Tyr Ile 20 25 30 Tyr Trp Tyr Gln Gln Lys Ser Asp Ala Ser Pro Lys Leu Trp Ile Tyr 35 40 45 Tyr Thr Ser Asn Leu Ala Pro Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Asn Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Gly Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Phe Ser Ser Ser Pro Ser Thr 85 90 95 Phe Gly Thr Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 5 <211> 357 <212> DNA <213> Artificial Sequence <220> <223> Synthetic polynucleotide <400> 5 gaggtgaaac tggtggagtc tggaggaggc ttggtacagt ctgggcgttc tctgagactc 60 tcctgtgcaa cttctggatt caccttcagt gatttctaca tggagtgggt ccgccaagct 120 ccagggaagg gactggagtg gattgttgca agtagagaca aagctaatga ttatacaaca 180 gcgtatagtg catctgtgaa gggtcggttc atcgtctcca gagacacttc ccaaagcatc 240 ctctaccttc agatgaatgc cctgagagct gaggacactg ccatttatta ctgtgtaaga 300 gatttctatg attacgacga ggcttactgg ggccaaggga ctctggtcac tgtctct 357 <210> 6 <211> 119 [[ID=⑧]]<212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 6 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ser Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Ser Asp Phe 20 25 30 Tyr Met Glu Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Val Ala Ser Arg Asp Lys Ala Asn Asp Tyr Thr Thr Ala Tyr Ser Ala 50 55 60 Ser Val Lys Gly Arg Phe Ile Val Ser Arg Asp Thr Ser Gln Ser Ile 65 70 75 80 Leu Tyr Leu Gln Met Asn Ala Leu Arg Ala Glu Asp Thr Ala Ile Tyr 85 90 95 Tyr Cys Val Arg Asp Phe Tyr Asp Tyr Asp Glu Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser 115 <210> 7 <211> 318 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotide <400> 7 gacatccaga tgactcagtc tccgtcctca ctgtctgcct ctctgggagg tacagtcacc 60 atcacttgca aggcaagcga agacattaac aactatatag cttggtacca acacaagcct 120 ggaaaaggtc ctcggctgct catacaatac acatctacat tacagccagg catcccatca 180 aggttcagtg gaagtgggtc tgggcgagat tattccctca gcatcagcaa cctggagcct 240 gaagatattg caacttatta ttgtctacag tatgatattc tgtggacgtt cggtggaggc 300 accaagctgg aaatcaaa 318 <210> 8 <211> 106 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <400> 8 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Gly Thr Val Thr Ile Thr Cys Lys Ala Ser Glu Asp Ile Asn Asn Tyr 20 25 30 Ile Ala Trp Tyr Gln His Lys Pro Gly Lys Gly Pro Arg Leu Leu Ile 35 40 45 Gln Tyr Thr Ser Thr Leu Gln Pro Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Arg Asp Tyr Ser Leu Ser Ile Ser Asn Leu Glu Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Ile Leu Trp Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 9 <211> 244 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 9 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Arg Phe Thr Phe Thr Asp Tyr 20 25 30 Asn Ile His Trp Val Lys Gln Ser Pro Gly Lys Thr Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Asn Asn Gly Asp Ile Phe Tyr Lys Gln Lys Phe 50 55 60 Asn Gly Lys Ala Thr Leu Thr Ile Asn Lys Ser Ser Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Val Arg Ser Ser Asn Ile Arg Tyr Thr Phe Asp Arg Phe Phe Asp Val 100 105 110 Trp Gly Thr Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Asn Val Leu Thr Gln 130 135 140 Ser Pro Ala Ile Met Ser Ala Ser Leu Gly Glu Lys Val Thr Met Ser 145 150 155 160 Cys Arg Ala Ser Ser Ser Val Asn Tyr Ile Tyr Trp Tyr Gln Gln Lys 165 170 175 Ser Asp Ala Ser Pro Lys Leu Trp Ile Tyr Tyr Thr Ser Asn Leu Ala 180 185 190 Pro Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Asn Ser Tyr 195 200 205 Ser Leu Thr Ile Ser Ser Met Glu Gly Glu Asp Ala Ala Thr Tyr Tyr 210 215 220 Cys Gln Gln Phe Ser Ser Ser Pro Ser Thr Phe Gly Thr Gly Thr Lys 225 230 235 240 Leu Glu Leu Lys <210> 10 <211> 243 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 10 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Arg Phe Thr Phe Thr Asp Tyr 20 25 30 Asn Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Asn Asn Gly Asp Ile Phe Tyr Lys Gln Lys Phe 50 55 60 Asn Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Arg Ser Ser Asn Ile Arg Tyr Thr Phe Asp Arg Phe Phe Asp Val 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Gly Gly Gly Gly Ser Gly 115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Val Met Thr Gln Ser 130 135 140 Pro Leu Ser Leu Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys 145 150 155 160 Arg Ala Ser Ser Ser Val Asn Tyr Ile Tyr Trp Tyr Leu Gln Lys Pro 165 170 175 Gly Gln Ser Pro Gln Leu Trp Ile Tyr Tyr Thr Ser Asn Leu Ala Pro 180 185 190 Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 195 200 205 Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys 210 215 220 Gln Gln Phe Ser Ser Ser Pro Ser Thr Phe Gly Gln Gly Thr Lys Leu 225 230 235 240 Glu Ile Lys <210> 11 <211> 243 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 11 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Arg Phe Thr Phe Thr Asp Tyr 20 25 30 Asn Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Asn Asn Gly Asp Ile Phe Tyr Lys Gln Lys Phe 50 55 60 Asn Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Arg Ser Ser Asn Ile Arg Tyr Thr Phe Asp Arg Phe Phe Asp Val 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Gly Gly Gly Gly Ser Gly 115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Val Met Thr Gln Ser 130 135 140 Pro Ala Phe Leu Ser Val Thr Pro Gly Glu Lys Val Thr Ile Thr Cys 145 150 155 160 Arg Ala Ser Ser Ser Val Asn Tyr Ile Tyr Trp Tyr Gln Gln Lys Pro 165 170 175 Asp Gln Ala Pro Lys Leu Trp Ile Tyr Tyr Thr Ser Asn Leu Ala Pro 180 185 190 Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 195 200 205 Phe Thr Ile Ser Ser Leu Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys 210 215 220 Gln Gln Phe Ser Ser Ser Pro Ser Thr Phe Gly Gln Gly Thr Lys Leu 225 230 235 240 Glu Ile Lys <210> 12 <211> 245 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 12 Met Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Glu Lys Pro Gly 1 5 10 15 Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Arg Phe Thr Phe Thr Asp 20 25 30 Tyr Asn Ile His Trp Val Lys Gln Ser His Gly Lys Cys Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Asn Pro Asn Asn Gly Asp Ile Phe Tyr Lys Gln Lys 50 55 60 Phe Asn Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala 65 70 75 80 Tyr Met Asp Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe 85 90 95 Cys Val Arg Ser Ser Asn Ile Arg Tyr Thr Phe Asp Arg Phe Phe Asp 100 105 110 Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Glu Leu Thr 130 135 140 Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met 145 150 155 160 Thr Cys Arg Ala Ser Ser Ser Val Asn Tyr Ile Tyr Trp Tyr Gln Gln 165 170 175 Lys Ser Gly Thr Ser Pro Lys Leu Trp Ile Tyr Tyr Thr Ser Asn Leu 180 185 190 Ala Pro Gly Val Pro Gly Arg Phe Ser Gly Ser Gly Ser Gly Asn Ser 195 200 205 Tyr Ser Leu Thr Ile Ser Ser Val Glu Ala Glu Asp Asp Ala Thr Tyr 210 215 220 Tyr Cys Gln Gln Phe Ser Ser Ser Pro Ser Thr Phe Gly Cys Gly Thr 225 230 235 240 Lys Leu Glu Ile Lys 245 <210> 13 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 13 Asn Tyr His Leu Glu Asn Glu Val Ala Arg Leu Lys Lys Leu 1 5 10 <210> 14 <211> twenty two <212> PRT <213> Homo sapiens <400> 14 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro 20 <210> 15 <211> 45 <212> PRT <213> Homo sapiens <400> 15 Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 1 5 10 15 Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 20 25 30 Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp 35 40 45 <210> 16 <211> twenty one <212> PRT <213> Homo sapiens <400> 16 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu 1 5 10 15 Ser Leu Val Ile Thr 20 <210> 17 <211> 42 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 17 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met 1 5 10 15 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 20 25 30 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 35 40 <210> 18 <211> 112 <212> PRT <213> Homo sapiens <400> 18 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 1 5 10 15 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 50 55 60 [[ID=十六]]Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 65 70 75 80 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 85 90 95 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 110 <210> 19 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 19 Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro 1 5 10 15 Gly Pro <210> 20 <211> 335 It should be noted that there is an error in the original text where "[[ID=十六]]" should be corrected to a proper ID number or name. Also, the translation of some terms like "人工序列" as "Artificial Sequence" and "合成多肽" as "Synthetic Polypeptide" is for reference only and may need to be adjusted according to specific patent context and relevant terminologies.<212> PRT <213> Homo sapiens <400> 20 Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu 1 5 10 15 Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile 20 25 30 Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe 35 40 45 Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr 50 55 60 Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn 65 70 75 80 Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg 85 90 95 Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile 100 105 110 Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val 115 120 125 Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp 130 135 140 Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn 145 150 155 160 Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu 165 170 175 Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser 180 185 190 Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu 195 200 205 Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln 210 215 220 Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly 225 230 235 240 Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro 245 250 255 His Cys Val Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr 260 265 270 Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His 275 280 285 Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro 290 295 300 Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly Ala 305 310 315 320 Leu Leu Leu Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met 325 330 335 <210> 21 <211> 243 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 21 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Arg Phe Thr Phe Thr Asp Tyr 20 25 30 Asn Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Asn Asn Gly Asp Ile Phe Tyr Lys Gln Lys Phe 50 55 60 Asn Gly Lys Ala Thr Met Thr Val Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Arg Ser Ser Asn Ile Arg Tyr Thr Phe Asp Arg Phe Phe Asp Val 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Gly Gly Gly Gly Ser Gly 115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser 130 135 140 Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys 145 150 155 160 Arg Ala Ser Ser Ser Val Asn Tyr Ile Tyr Trp Tyr Gln Gln Lys Ser 165 170 175 Gly Lys Ala Pro Lys Leu Trp Ile Tyr Tyr Thr Ser Asn Leu Ala Pro 180 185 190 Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 195 200 205 Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys 210 215 220 Gln Gln Phe Ser Ser Ser Pro Ser Thr Phe Gly Gln Gly Thr Lys Leu 225 230 235 240 Glu Ile Lys <210> 22 <211> 355 <212> PRT <213> artificial sequence <220> <223> Synthesized multiple peptides <400> 22 Ser Ala Ala Lys Ala Ser Gly Gly Pro Glu Gly Gly Ser Leu Ala Ala 1 5 10 15 Leu Thr Ala His Gln Ala Cys His Leu Pro Leu Glu Thr Phe Thr Arg 20 25 30 His Arg Gln Pro Arg Gly Trp Glu Gln Leu Glu Gln Cys Gly Tyr Pro 35 40 45 Val Gln Arg Leu Val Ala Leu Tyr Leu Ala Ala Arg Leu Ser Trp Asn 50 55 60 Gln Val Asp Gln Val Ile Arg Asn Ala Leu Ala Ser Pro Gly Ser Gly 65 70 75 80 Gly Asp Leu Gly Glu Ala Ile Arg Glu Gln Pro Glu Gln Ala Arg Leu 85 90 95 Ala Leu Thr Leu Ala Ala Ala Glu Ser Glu Arg Phe Val Arg Gln Gly 100 105 110 Thr Gly Asn Asp Glu Ala Gly Ala Ala Asn Gly Pro Ala Asp Ser Gly 115 120 125 Asp Ala Leu Leu Glu Arg Asn Tyr Pro Thr Gly Ala Glu Phe Leu Gly 130 135 140 Asp Gly Gly Asp Val Ser Phe Ser Thr Arg Gly Thr Gln Asn Trp Thr 145 150 155 160 Val Glu Arg Leu Leu Gln Ala His Arg Gln Leu Glu Glu Arg Gly Tyr 165 170 175 Val Phe Val Gly Tyr His Gly Thr Phe Leu Glu Ala Ala Gln Ser Ile 180 185 190 Val Phe Gly Gly Val Arg Ala Arg Ser Gln Asp Leu Asp Ala Ile Trp 195 200 205 Arg Gly Phe Tyr Ile Ala Gly Asp Pro Ala Leu Ala Tyr Gly Tyr Ala 210 215 220 Gln Asp Gln Glu Pro Asp Ala Arg Gly Arg Ile Arg Asn Gly Ala Leu 225 230 235 240 Leu Arg Val Tyr Val Pro Arg Ser Ser Leu Pro Gly Phe Tyr Arg Thr 245 250 255 Ser Leu Thr Leu Ala Ala Pro Glu Ala Ala Gly Glu Val Glu Arg Leu 260 265 270 Ile Gly His Pro Leu Pro Leu Arg Leu Asp Ala Ile Thr Gly Pro Glu 275 280 285 Glu Glu Gly Gly Arg Leu Glu Thr Ile Leu Gly Trp Pro Leu Ala Glu 290 295 300 Arg Thr Val Val Ile Pro Ser Ala Ile Pro Thr Asp Pro Arg Asn Val 305 310 315 320 Gly Gly Asp Leu Asp Pro Ser Ser Ile Pro Asp Lys Glu Gln Ala Ile 325 330 335 Ser Ala Leu Pro Asp Tyr Ala Ser Gln Pro Gly Lys Pro Pro Arg Glu 340 345 350 Asp Leu Lys 355 <210> 23 <211> 243 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 23 Lys Leu Lys Ala Ser Gly Gly Arg His Arg Gln Pro Arg Gly Trp Glu 1 5 10 15 Gln Leu Gly Gly Gly Gly Gly Ser Pro Thr Gly Ala Glu Phe Leu Gly 20 25 30 Asp Gly Gly Asp Val Ser Phe Ser Thr Arg Gly Thr Gln Asn Trp Thr 35 40 45 Val Glu Arg Leu Leu Gln Ala His Ala Gln Leu Glu Glu Arg Gly Tyr 50 55 60 Val Phe Val Gly Tyr His Gly Thr Ala Leu Glu Ala Ala Gln Ser Ile 65 70 75 80 Val Phe Gly Gly Val Arg Ala Arg Ser Gln Asp Leu Asp Ala Ile Trp 85 90 95 Arg Gly Phe Tyr Ile Ala Gly Asp Pro Ala His Ala Tyr Gly Tyr Ala 100 105 110 Gln Asp Gln Glu Pro Asp Ala Arg Gly Arg Ile Ala Asn Gly Ala Leu 115 120 125 Leu Arg Val Tyr Val Pro Ala Ser Ser Leu Pro Gly Phe Tyr Arg Thr 130 135 140 Ser Leu Thr Leu Ala Ala Pro Glu Ala Ala Gly Glu Val Glu Arg Leu 145 150 155 160 Ile Gly His Pro Leu Pro Leu Arg Leu Asp Ala Ile Thr Gly Pro Glu 165 170 175 Glu Glu Gly Gly Arg Glu Glu Thr Ile Leu Gly Trp Pro Leu Ala Glu 180 185 190 Arg Thr Val Val Ile Pro Ser Ala Ile Pro Thr Asp Pro Arg Asn Val 195 200 205 Gly Gly Asp Leu Asp Pro Ser Ser Ile Pro Asp Lys Glu Gln Ala Ile 210 215 220 Ser Ala Leu Pro Asp Tyr Ala Ser Gln Pro Gly Lys Pro Pro Arg Glu 225 230 235 240 Asp Leu Lys

Claims

1. A monoclonal antibody or antigen-binding fragment thereof that binds to GPC2, comprising a variable heavy chain (VH) domain and a variable light chain (VL) domain, wherein: The CDR1, CDR2 and CDR3 sequences of the VH domain are the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 2, respectively, and the CDR1, CDR2 and CDR3 sequences of the VL domain are the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 4, respectively; or The CDR1, CDR2 and CDR3 sequences of the VH domain are respectively the CDR1, CDR2 and CDR3 sequences of SEQ ID NO:6, and the CDR1, CDR2 and CDR3 sequences of the VL domain are respectively the CDR1, CDR2 and CDR3 sequences of SEQ ID NO:8, The CDR sequences are defined using the Kabat, IMGT or Paratome numbering schemes.

2. The monoclonal antibody or antigen-binding fragment according to claim 1, wherein: The CDR1, CDR2 and CDR3 sequences of the VH domain consist of residues 31-35, 50-66 and 99-112 of SEQ ID NO:2, respectively, and the CDR1, CDR2 and CDR3 sequences of the VL domain consist of residues 24-33, 49-55 and 88-96 of SEQ ID NO:4, respectively; or The CDR1, CDR2 and CDR3 sequences of the VH domain consist of residues 31-35, 50-68 and 101-109 of SEQ ID NO:6, respectively, and the CDR1, CDR2 and CDR3 sequences of the VL domain consist of residues 24-34, 50-56 and 89-96 of SEQ ID NO:8, respectively; The CDR sequences are defined using the Kabat numbering scheme.

3. The monoclonal antibody or antigen-binding fragment according to claim 1, wherein: The CDR1, CDR2 and CDR3 sequences of the VH domain consist of residues 26-33, 51-58 and 97-112 of SEQ ID NO:2, respectively, and the CDR1, CDR2 and CDR3 sequences of the VL domain consist of residues 27-31, 49-51 and 88-96 of SEQ ID NO:4, respectively; or The CDR1, CDR2 and CDR3 sequences of the VH domain consist of residues 26-33, 51-60 and 99-109 of SEQ ID NO:6, respectively, and the CDR1, CDR2 and CDR3 sequences of the VL domain consist of residues 27-32, 50-52 and 89-96 of SEQ ID NO:8, respectively; The CDR sequences are defined using the IMGT numbering scheme.

4. The monoclonal antibody or antigen-binding fragment according to claim 1, wherein: The CDR1, CDR2 and CDR3 sequences of the VH domain consist of residues 26-35, 47-61 and 97-112 of SEQ ID NO:2, respectively, and the CDR1, CDR2 and CDR3 sequences of the VL domain consist of residues 27-33, 45-55 and 88-95 of SEQ ID NO:4, respectively; or The CDR1, CDR2 and CDR3 sequences of the VH domain consist of residues 27-35, 47-62 and 99-109 of SEQ ID NO:6, respectively, and the CDR1, CDR2 and CDR3 sequences of the VL domain consist of residues 27-34, 46-56 and 89-95 of SEQ ID NO:8, respectively; The CDR sequences are defined using the Paratome numbering scheme.

5. The monoclonal antibody or antigen-binding fragment according to any one of claims 2 to 4, wherein: The VH domain comprises residues 26-35, 47-66 and 97-112 of SEQ ID NO:2 and the VL domain comprises residues 24-33, 45-55 and 88-96 of SEQ ID NO:4; or The VH domain comprises residues 26-35, 47-68 and 99-109 of SEQ ID NO:6 and the VL domain comprises residues 27-34, 46-56 and 89-96 of SEQ ID NO:

8.

6. The monoclonal antibody or antigen-binding fragment according to any one of claims 1 to 4, wherein the amino acid sequence of the VH domain consists of SEQ ID NO: 2 and / or the amino acid sequence of the VL domain consists of SEQ ID NO:

4.

7. The monoclonal antibody or antigen-binding fragment according to any one of claims 1 to 4, wherein the amino acid sequence of the VH domain consists of SEQ ID NO: 6 and / or the amino acid sequence of the VL domain consists of SEQ ID NO:

8.

8. The monoclonal antibody or antigen-binding fragment according to any one of claims 1 to 4, which is a humanized monoclonal antibody or antigen-binding fragment.

9. The antigen binding fragment of any one of claims 1-4, wherein the antigen binding fragment is a single-chain variable fragment (scFv), a Fab fragment, a Fab' fragment, a F(ab)'2 fragment, or a disulfide-stabilized variable fragment (dsFv). The antigen-binding fragment according to claim 9 , which is a scFv.

11. The antigen-binding fragment of claim 10, wherein the scFv consists of the amino acid sequence of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:

21.

12. A chimeric antigen receptor (CAR) comprising the monoclonal antibody or antigen binding fragment according to any one of claims 1-11.

13. The CAR of claim 12, further comprising a hinge region, a transmembrane domain, a co-stimulatory signaling portion, a signaling domain, or any combination thereof.

14. The CAR of claim 13, wherein the hinge region comprises a CD8α hinge region.

15. The CAR of claim 13, wherein the transmembrane domain comprises a CD8α transmembrane domain.

16. The CAR of claim 13, wherein the co-stimulatory signaling moiety comprises a 4-1BB signaling moiety.

17. The CAR of claim 13, wherein the signaling domain comprises a CD3 zeta signaling domain.

18. The CAR according to any one of claims 12-17, wherein the antigen binding fragment is a scFv consisting of the amino acid sequence of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:

21.

19. An isolated cell expressing the CAR of any one of claims 12-18.

20. The isolated cell according to claim 19, which is a cytotoxic T lymphocyte (CTL).

21. An immunoconjugate comprising the monoclonal antibody or antigen-binding fragment according to any one of claims 1-11 and an effector molecule.

22. The immunoconjugate of claim 21, wherein the effector molecule is a toxin.

23. The immunoconjugate of claim 22, wherein the toxin is Pseudomonas exotoxin or a variant thereof.

24. The immunoconjugate of claim 23, wherein the Pseudomonas exotoxin variant is PE38.

25. The immunoconjugate of claim 21, wherein the effector molecule is a detectable label.

26. The immunoconjugate of claim 25, wherein the detectable label comprises a fluorophore, an enzyme, or a radioisotope.

27. An antibody-drug conjugate (ADC) comprising a drug conjugated to the monoclonal antibody or antigen-binding fragment of any one of claims 1-11.

28. The ADC of claim 27, wherein the drug is a small molecule.

29. The ADC of claim 27 or claim 28, wherein the drug is an anti-microtubule agent, an anti-mitotic agent and / or a cytotoxic agent.

30. A multispecific antibody comprising the monoclonal antibody or antigen-binding fragment of any one of claims 1-11 and at least one additional monoclonal antibody or antigen-binding fragment thereof. The multispecific antibody according to claim 30 , which is a bispecific antibody.

32. The multispecific antibody of claim 30, which is a trispecific antibody.

33. The multispecific antibody of any one of claims 30-32, wherein the at least one additional monoclonal antibody or antigen-binding fragment thereof specifically binds to a component of a T cell receptor or a natural killer (NK) cell activating receptor.

34. An antibody-nanoparticle conjugate comprising a nanoparticle conjugated to the monoclonal antibody or antigen-binding fragment of any one of claims 1-11.

35. The antibody-nanoparticle conjugate of claim 34, wherein the nanoparticle comprises a polymer nanoparticle, a nanosphere, a nanocapsule, a liposome, a dendrimer, a polymer micelle or a lipid vesicle.

36. The antibody-nanoparticle conjugate of claim 34 or claim 35, wherein the nanoparticle comprises a cytotoxic agent.

37. A fusion protein comprising the monoclonal antibody or antigen-binding fragment according to any one of claims 1 to 11 and a heterologous protein or peptide.

38. The fusion protein of claim 37, wherein the heterologous protein is an Fc protein.

39. A nucleic acid molecule encoding a monoclonal antibody or antigen-binding fragment according to any one of claims 1-11, a CAR according to any one of claims 12-18, an immunoconjugate according to any one of claims 21-26, a multispecific antibody according to any one of claims 30-33, or a fusion protein according to claim 37 or claim 38.

40. The nucleic acid molecule of claim 39, operably linked to a promoter.

41. A vector comprising the nucleic acid molecule of claim 39 or claim 40.

42. A nucleic acid molecule encoding a chimeric antigen receptor (CAR), comprising in the 5' to 3' direction: a nucleic acid encoding the first granulocyte-macrophage colony stimulating factor receptor signal sequence (GMCSFRss); A nucleic acid encoding the monoclonal antibody or antigen-binding fragment according to any one of claims 1 to 11; a nucleic acid encoding an extracellular hinge region; a nucleic acid encoding a transmembrane domain; a nucleic acid encoding an intracellular costimulatory domain; a nucleic acid encoding an intracellular signaling domain; a nucleic acid encoding a self-cleaving 2A peptide; a nucleic acid encoding a second GMCSFRss; and Nucleic acid encoding truncated human epidermal growth factor receptor (huEGFRt).

43. The nucleic acid molecule of claim 42, further comprising a human elongation factor 1 alpha (EF1 alpha) promoter sequence 5' of the nucleic acid encoding the first GMCSFRss.

44. The nucleic acid molecule of claim 42 or claim 43, wherein the antigen binding fragment is a single chain variable fragment (scFv).

45. A vector comprising the nucleic acid molecule according to any one of claims 42-44.

46. ​​The vector of claim 45, wherein the vector is a lentiviral vector.

47. An isolated host cell comprising the nucleic acid molecule of any one of claims 42-44 or the vector of claim 45 or claim 46.

48. A composition comprising a pharmaceutically acceptable carrier and a monoclonal antibody or antigen-binding fragment according to any one of claims 1-11, a CAR according to any one of claims 12-18, an isolated cell according to claim 19 or claim 20, an immunoconjugate according to any one of claims 21-26, an ADC according to any one of claims 27-29, a multispecific antibody according to any one of claims 30-33, an antibody-nanoparticle conjugate according to any one of claims 34-36, or a fusion protein according to claim 37 or claim 38.

49. Use of the isolated cell of claim 20, the immunoconjugate of any one of claims 22-24, the ADC of any one of claims 27-29, the multispecific antibody of claim 33, the antibody-nanoparticle conjugate of claim 36, or the fusion protein of claim 38 in the preparation of a medicament for treating a GPC2-positive cancer in a subject, wherein the GPC2-positive cancer is neuroblastoma, medulloblastoma, retinoblastoma, acute lymphoblastic leukemia, embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, Ewing's sarcoma, desmoplastic small round cell tumor, or osteosarcoma.

50. Use of the isolated cell of claim 20, the immunoconjugate of any one of claims 22-24, the ADC of any one of claims 27-29, the multispecific antibody of claim 33, the antibody-nanoparticle conjugate of claim 36, or the fusion protein of claim 38 in the preparation of a medicament for inhibiting tumor growth or metastasis of a GPC2-positive cancer in a subject, wherein the GPC2-positive cancer is neuroblastoma, medulloblastoma, retinoblastoma, acute lymphoblastic leukemia, embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, Ewing's sarcoma, desmoplastic small round cell tumor, or osteosarcoma.

51. Use of the monoclonal antibody or antigen-binding fragment according to any one of claims 1 to 11 in the preparation of a reagent for detecting the expression of GPC2 in a sample, wherein the detection comprises: contacting the sample with the monoclonal antibody or antigen-binding fragment according to any one of claims 1 to 11; and The binding of the antibody to the sample is detected, thereby detecting the expression of GPC2 in the sample.

52. The use according to claim 51, wherein the monoclonal antibody or antigen-binding fragment is directly labeled.

53. The use according to claim 51, wherein the reagent further comprises a second antibody, and the detection further comprises: contacting the monoclonal antibody or antigen-binding fragment with a second antibody, and The binding of the second antibody to the monoclonal antibody or antigen-binding fragment is detected, thereby detecting the expression of GPC2 in the sample.

54. The use according to any one of claims 51-53, wherein the sample is obtained from a subject suspected of having a GPC2-positive cancer.

55. The use according to any one of claims 51-53, wherein the sample is a tumor biopsy.

56. Use of the monoclonal antibody or antigen-binding fragment according to any one of claims 1 to 11 in the preparation of a reagent for diagnosing a subject with a GPC2-positive cancer, wherein the diagnosis comprises: contacting a sample obtained from the subject with the monoclonal antibody or antigen-binding fragment according to any one of claims 1 to 11; and Binding of the antibody to the sample is detected, thereby diagnosing the subject as having a GPC2-positive cancer.

57. The use according to claim 56, wherein the monoclonal antibody or antigen-binding fragment is directly labeled.

58. The use according to claim 56, wherein the reagent further comprises a second antibody, and the diagnosis further comprises: contacting the monoclonal antibody or antigen-binding fragment with a second antibody, and Binding of the second antibody to the monoclonal antibody or antigen-binding fragment is detected, thereby diagnosing the subject as having a GPC2-positive cancer.

59. The use according to any one of claims 56-58, wherein the sample is a tumor biopsy.

60. The use according to any one of claims 56-58, wherein the GPC2-positive cancer is neuroblastoma, medulloblastoma, retinoblastoma, acute lymphoblastic leukemia, embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, Ewing's sarcoma, desmoplastic small round cell tumor, or osteosarcoma.

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