Monoclonal antibodies targeting glypican-2 (GPC2) and use thereof
Inventors
Ho, Mitchell • Li, Nan • Dimitrov, Dimiter S.
Assignees
US Department of Health and Human Services
Publication Number
US-11066479-B2
Publication Date
2021-07-20
Expiration Date
2037-07-20
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Abstract
A panel of human variable heavy (VH) single domain monoclonal antibodies specific for cell-surface glypican-2 (GPC2) are described. Methods for the diagnosis and treatment and GPC2-positive cancer are also described. Recombinant immunotoxins comprised of a GPC2-specific VH domain antibody and a clinically used form of Pseudomonas exotoxin A (PE38) were generated and shown to inhibit GPC2-positive neuroblastoma tumor cell growth and inhibit neuroblastoma xenograft growth in nude mice, without significant toxicity. Chimeric antigen receptors comprising a GPC2-specific VH single domain antibody are also described. T cells expressing the GPC2-specific CARs potently killed GPC2-positive neuroblastoma cells in a dose-dependent manner.
Core Innovation
The invention provides a panel of human variable heavy (VH) single domain monoclonal antibodies that specifically bind cell-surface glypican-2 (GPC2). These antibodies include six antibodies named LH1, LH2, LH3, LH4, LH6, and LH7 and are isolated by phage display. The invention further includes conjugates comprising these VH single domain antibodies, such as immunotoxins composed of the antibody linked to Pseudomonas exotoxin A (PE38), chimeric antigen receptors (CARs) comprising the GPC2-specific VH domain antibodies, and antibody-drug conjugates, multi-specific antibodies, antibody-nanoparticle conjugates, and fusion proteins including these antibodies.
The invention addresses a critical need in pediatric cancer treatment, especially neuroblastoma, the most common extracranial solid tumor in children, characterized by poor survival in high-risk patients despite intensive therapies. There is an urgent unmet need for safe and effective treatments that can specifically target tumor cells. One challenge in treating neuroblastoma and other deadly solid tumors is the lack of tumor-specific targets. GPC2 mRNA is highly expressed in neuroblastoma and other pediatric cancers, and GPC2 protein is uniquely expressed in the nervous system. The invention discovers that GPC2 plays a role in neuroblastoma carcinogenesis and that targeting GPC2 with specific antibodies or conjugates can inhibit tumor growth and tumor cell survival.
The invention provides methods for the diagnosis and treatment of GPC2-positive cancers using these VH single domain antibodies and their conjugates. The recombinant immunotoxins comprising VH antibody domains and PE38 inhibit GPC2-positive neuroblastoma tumor cell growth in vitro and neuroblastoma xenograft growth in vivo without significant toxicity. CARs comprising GPC2-specific VH single domain antibodies can direct T cells to potently kill GPC2-positive neuroblastoma cells in a dose-dependent manner. Also disclosed are methods to detect GPC2 expression in samples using these VH single domain antibodies.
Claims Coverage
The patent contains multiple claims focusing on six VH single domain monoclonal antibodies that bind glypican-2 (GPC2) and their various conjugates and uses. The claims cover isolated antibodies, immunoconjugates, CARs, antibody-drug conjugates, multi-specific antibodies, antibody-nanoparticle conjugates, fusion proteins, compositions, nucleic acids encoding the antibodies, and methods of treating and detecting GPC2-positive cancers.
VH single domain monoclonal antibodies specific for GPC2
Isolated VH single domain monoclonal antibodies that bind glypican-2, comprising the CDR1, CDR2, and CDR3 sequences of any one of SEQ ID NOs: 2, 4, 6, 8, 10, or 12, with CDR sequences defined by the IMGT, Kabat, or Chothia numbering schemes, and including antibodies at least 90% identical to these sequences.
Immunoconjugates comprising VH single domain antibodies and effector molecules
Immunoconjugates comprising a VH single domain monoclonal antibody specific for GPC2 and an effector molecule, where the effector molecule can be a toxin, such as Pseudomonas exotoxin or variant thereof, including PE38, or a detectable label (fluorophore, enzyme, or radioisotope).
Chimeric antigen receptors (CARs) comprising VH single domain antibodies
CARs containing a VH single domain antibody specific for GPC2, optionally including a hinge region (e.g., CD8α hinge), transmembrane domain (e.g., CD8α or CD28), costimulatory signaling moieties (e.g., 4-1BB and/or CD28), and signaling domains (e.g., CD3ζ). Also includes isolated cells expressing these CARs.
Antibody-drug conjugates (ADCs) comprising VH single domain antibodies
ADCs comprising a VH single domain monoclonal antibody specific for GPC2 conjugated to a drug, wherein the drug can be a small molecule, an anti-microtubule agent, an anti-mitotic agent, or other cytotoxic agent.
Multi-specific antibodies including VH single domain antibodies
Multi-specific antibodies, such as bispecific or trispecific antibodies, including the GPC2-specific VH single domain antibody and at least one additional monoclonal antibody or antigen-binding fragment thereof, which can specifically bind a T cell receptor component or a natural killer (NK) cell activating receptor.
Antibody-nanoparticle conjugates comprising VH single domain antibodies
Conjugates of VH single domain monoclonal antibodies specific for GPC2 with nanoparticles, including polymeric nanoparticles, nanospheres, nanocapsules, liposomes, dendrimers, polymeric micelles, or niosomes, optionally containing cytotoxic agents.
Fusion proteins comprising VH single domain antibodies and heterologous proteins or peptides
Fusion proteins comprising the VH single domain monoclonal antibody specific for GPC2 and a heterologous protein or peptide, such as an Fc protein (mouse or human), or a heterologous peptide not endogenous to humans, e.g., peptide NYHLENEVARLKKL (SEQ ID NO: 26).
Compositions comprising VH single domain antibodies and nucleic acids encoding them
Compositions comprising a pharmaceutically acceptable carrier and the VH single domain monoclonal antibodies or their conjugates; nucleic acid molecules encoding these antibodies or conjugates, operably linked to promoters; and vectors comprising these nucleic acids.
Methods of treating and detecting GPC2-positive cancers using VH single domain antibodies
Methods of treating GPC2-positive cancers, particularly pediatric cancers including neuroblastoma, acute lymphoblastic leukemia, embryonal and alveolar rhabdomyosarcoma, Ewing's sarcoma, desmoplastic small round cell tumor, and osteosarcoma, by administering immunoconjugates, CARs, or ADCs comprising the VH single domain antibody. Methods for detecting GPC2 expression in a sample by contacting the sample with the VH single domain antibody and detecting binding, optionally with labeled antibodies.
The claims collectively cover isolated human VH single domain monoclonal antibodies specific for GPC2 defined by specific CDR sequences, and various antibody-based therapeutics and diagnostics including immunoconjugates, CARs, ADCs, multi-specific antibodies, nanoparticle conjugates, fusion proteins, compositions and nucleic acids, as well as methods of treating and detecting GPC2-positive cancers using these antibodies and conjugates.
Stated Advantages
The disclosed antibodies and conjugates can specifically target GPC2, a tumor-specific antigen highly expressed in neuroblastoma and other pediatric cancers.
The recombinant immunotoxins inhibit GPC2-positive tumor cell growth and tumor xenograft growth in nude mice without significant toxicity.
Chimeric antigen receptors comprising GPC2-specific VH single domain antibodies induce potent, dose-dependent killing of GPC2-positive neuroblastoma cells.
The antibodies inhibit Wnt/β-catenin signaling and reduce expression of oncogenic drivers such as N-Myc, thereby suppressing tumor cell proliferation and inducing apoptosis.
Documented Applications
Treatment of GPC2-positive cancers, including pediatric cancers such as neuroblastoma, acute lymphoblastic leukemia, embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, Ewing's sarcoma, desmoplastic small round cell tumor, and osteosarcoma.
Diagnostic detection of GPC2 expression in biological samples, such as tumor biopsies, using the VH single domain monoclonal antibodies for confirming presence of GPC2-positive cancers.
Therapeutic applications using immunotoxins, CAR T cells, antibody-drug conjugates, and multi-specific antibodies comprising the VH single domain antibodies to inhibit tumor growth and metastasis in cell and animal models.
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