EP4705343A1 - Anti-human m-cadherin (cdh15) antibodies, conjugates, and uses thereof for delivery of genetic payloads to muscle cells - Google Patents

Anti-human m-cadherin (cdh15) antibodies, conjugates, and uses thereof for delivery of genetic payloads to muscle cells

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Publication number
EP4705343A1
EP4705343A1 EP24728805.3A EP24728805A EP4705343A1 EP 4705343 A1 EP4705343 A1 EP 4705343A1 EP 24728805 A EP24728805 A EP 24728805A EP 4705343 A1 EP4705343 A1 EP 4705343A1
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EP
European Patent Office
Prior art keywords
protein
binding
antigen
capsid
antibody
Prior art date
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Pending
Application number
EP24728805.3A
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German (de)
French (fr)
Inventor
Trevor Stitt
Michael Stec
Mark W. Sleeman
Leah SABIN
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Regeneron Pharmaceuticals Inc
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Regeneron Pharmaceuticals Inc
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Publication of EP4705343A1 publication Critical patent/EP4705343A1/en
Pending legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
    • A61K51/1027—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against receptors, cell-surface antigens or cell-surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • This application is generally directed to human antibodies and antigen-binding fragments of human antibodies that bind human Cadherin 15 (hM-Cadherin, hCDH15), and methods of use thereof, e.g., in methods of treating a disorder in a patient in need thereof.
  • the application also relates to antigen-binding molecules comprising at least an antigen-binding fragment of an anti-hCDH15 antibody, wherein complexation of the antigen-binding molecule to CDH15 mediates internalization of the antigen-binding molecule/CDH15 complex, and/or blocks activity of CDH15.
  • the application further relates to conjugates comprising an anti-hCDH15 antibody (or antigen-binding molecules comprising an antigen-binding fragment of an anti- hCDH15 antibody) and a therapeutic agent, which conjugates may be useful in treating diseases.
  • the disclosure further relates to methods of making and using recombinant viral particles, e.g., recombinant AAV particles, comprising capsid proteins retargeted to Cadherin 15 (CDH15), useful for modification of muscle cells, such as muscle stem cells, in vitro or in vivo.
  • a gene delivery vehicle is able to stably introduce genetic material into desired cells and avoid introducing genetic material into non-target cells.
  • Viral particles particularly those based on adeno-associated virus (AAV), as gene delivery vehicles have been the focus of much research since AAVs are capable of transducing a wide range of primate species and tissues in vivo.
  • AAV safely transduces postmitotic tissues.
  • the virus can occasionally integrate into host chromosomes, it does so very infrequently into a safe-harbor locus in human chromosome 19, and only when the replication (Rep) proteins are supplied in trans.
  • Rep replication
  • a targeting ligand is directly inserted into, or coupled to, a viral capsid, i.e., protein viral capsid genes are modified to express capsid proteins comprising a heterologous targeting ligand.
  • the targeting ligand then redirects, e.g., binds, a receptor or marker preferentially or exclusively expressed on a target cell.
  • a viral capsid is modified with a heterologous “scaffold”, which then links to an adaptor that includes a targeting ligand.
  • the adaptor binds to the scaffold and the target cell.
  • Fc binding molecules e.g, Fc receptors, Protein A, etc.
  • (strept)avidin which binds to biotinylated adaptors
  • biotin which binds to adaptors fused with (strept)avidin
  • a detectable label which is useful for detection and/or isolation of viral particles, bound by a bispecific adaptor able to non-covalently bind the detectable label and target molecule
  • protein protein binding pairs that form isopeptide bonds have been described for a variety of viral particles. (See, e.g., Gigout et al.
  • Skeletal muscle is the largest organ in the body, comprising -40% of total body mass, and is one of the three significant muscle tissues in the human body.
  • Muscle stem cells MuSCs
  • MuSC-mediated muscle regeneration is delayed in aging subjects, which may be related, in part, to changes in muscle stem cell-specific markers.
  • anti -human antibodies capable of binding to muscle stem cellspecific markers could be helpful for therapy, e.g., stimulating muscle repair, particularly in aging subjects, and/or treating muscle-related cancers.
  • the anti-human antibodies as described herein may be used in conjunction with recombinant viral, e.g., AAV, particles for the targeted introduction of nucleic acids of interest into cells expressing muscle stem cell-specific markers.
  • recombinant viral e.g., AAV
  • some muscle-related cancers including rhabdomyosarcomas, may also benefit from therapeutics, e.g., antibody-drug conjugates, retargeted viral particles, etc., as described herein that target muscle stem cell-specific markers thereby.
  • the antigen-binding protein comprises a set of three heavy chain complementary determining region (HCDR1, HCDR2, and HCDR3) amino acid sequences selected from Table 1 below. In some embodiments, the antigen-binding protein comprises a set of three light chain complementary determining region (LCDR1, LCDR2 and LCDR3) amino acid sequences selected from Table 1 below. In some embodiments, the antigen-binding protein comprises a set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences selected from Table 1 below.
  • the antigen-binding protein comprises a set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences selected from the group consisting of SEQ ID NOs: 4-6-8-12-14-16, 24-26-28-32-34-36, 44-46-48-52-34-54, 62- 64-66-52-34-54, 72-74-76-52-34-54, 82-84-86-52-34-54, 92-94-96-100-34-102, 82-111-113- 117-34-119, 127-129-131-135-137-139, 147-149-151-155-157-159, 167-169-171-175-177-179, 187-189-191-52-34-196, 204-206-208-212-137-214, 222-224-226-52-34-54, 232-234-236-52- 34-54, 242-244-246-52-34-54, 82-253-255-52-34-54, 261-263-265-269
  • Table 1 sets forth the amino acid and nucleic acid sequence identifiers of the HCVRs and LCVRs, as well as HCDR1, HCDR2 and HCDR3 within each HCVR, and LCDR1, LCDR2 and LCDR3 within each LCVR of each of the example anti-hCDH15 antibodies of the present disclosure.
  • the antigen-binding protein comprises a heavy chain variable region (HCVR or VH).
  • the HCVR comprises a set of HCDR1- HCDR2-HCDR3 amino acid sequences selected from Table 1.
  • the HCVR comprises an amino acid sequence selected from any of the HCVR amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.
  • the antigen-binding protein comprises a light chain variable region (LCVR or VL).
  • the LCVR comprises a set of LCDR1- LCDR2-LCDR3 amino acid sequences selected from Table 1 below.
  • the LCVR comprises an amino acid sequence selected from any of the LCVR amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.
  • the antigen-binding protein comprises an anti-hCDH15 antibody or antigen-binding fragment thereof.
  • the anti-hCDH15 antibody or antigen-binding fragment thereof comprises a human or humanized antibody or antigen binding fragment thereof, a monovalent Fab’, a divalent Fab2, a F(ab)’3 fragment, a single-chain fragment variable (scFv), a bis-scFv, a (scFv)2, a diabody, a minibody, a nanobody, a triabody, a tetrabody, a disulfide stabilized Fv protein (dsFv), a single-domain antibody (sdAb), an Ig NAR, a bispecific antibody or binding fragment thereof, a bi-specific T-cell engager (BiTE), a trispecific antibody, or a chemically modified derivative thereof.
  • the scFv comprises variable regions arranged in the following orientation from N-terminus to C-terminus: HCVR-LCVR. In some embodiments, the scFv comprises variable regions arranged in the following orientation from N-terminus to C-terminus: LCVR-HCVR. In some embodiments, the scFv variable regions are connected by a linker. In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker is -(GGGGS)n- (SEQ ID NO: 789), wherein n is 1-10.
  • the antigen-binding protein binds to hCDH15 with a KD of about 1X10’ 7 M or a stronger affinity. In some embodiments, the antigen-binding protein binds to hCDH15 with a KD of about 10X10’ 8 to about 1X10' 10 . In some embodiments, the antigen-binding protein binds to hCDH15 with a KD of about 5X1 O’ 9 to about 1X1 O’ 10 .
  • the antigen-binding protein e.g, antibody or antigenbinding fragment thereof, comprises an HCVR and an LCVR amino acid sequence pair (HCVR/LCVR) comprising any of the HCVR amino acid sequences listed in Table 1 paired with any of the LCVR amino acid sequences listed in Table 1.
  • the antigenbinding protein e.g., antibody or antigen-binding fragment thereof, as described herein comprises an HCVR/LCVR amino acid sequence pair contained within any of the example anti- hCDH15 antibodies listed in Table 1.
  • the HCVR/LCVR amino acid sequence pair is selected from the group consisting of SEQ ID NOs: 2 and 10, 22 and 30, 42 and 50, 60 and 50, 70 and 50, 80 and 50, 90 and 98, 108 and 115, 125 and 133, 145 and 153, 165 and 173, 185 and 193, 202 and 210, 220 and 50, 230 and 50, 240 and 50, 250 and 50, 259 and 267, 279 and 287, 299 and 307, 319 and 327, 339 and 347, 358 and 366, 378 and 50, 386 and 394,
  • nucleic acid molecules i.e., polynucleotides, encoding the antigen-binding protein, e.g., anti-hCDH15 antibodies or antigen-binding fragments thereof, described herein.
  • the nucleic acid molecule as described herein comprises a nucleic acid sequence encoding a set of HCDR1-HCDR2-HCDR3 amino acid sequences listed in Table 1.
  • a nucleic acid molecule as described herein comprises a nucleic acid sequence encoding any of the HCVR amino acid sequences listed in Table 1.
  • the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.
  • the nucleic acid molecule as described herein comprises a nucleic acid sequence encoding a set of LCDR1-LCDR2-LCDR3 amino acid sequences listed in Table 1.
  • a nucleic acid molecule as described herein comprises a nucleic acid sequence encoding any of the LCVR amino acid sequences listed in Table 1.
  • the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCVR nucleic acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.
  • nucleic acid molecules encoding an HCVR wherein the HCVR comprises a set of three CDRs (i.e., HCDR1-HCDR2-HCDR3), wherein the HCDR1- HCDR2-HCDR3 amino acid sequence set is as defined by any of the example anti-hCDH15 antibodies listed in Table 1.
  • nucleic acid molecules encoding an LCVR wherein the LCVR comprises a set of three CDRs i.e., LCDR1-LCDR2-LCDR3), wherein the LCDR1- LCDR2-LCDR3 amino acid sequence set is as defined by any of the example anti- hCDH15 antibodies listed in Table 1.
  • nucleic acid molecules encoding both an HCVR and an LCVR, wherein the HCVR comprises an amino acid sequence of any of the HCVR amino acid sequences listed in Table 1, and wherein the LCVR comprises an amino acid sequence of any of the LCVR amino acid sequences listed in Table 1.
  • the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto, and a polynucleotide sequence selected from any of the LCVR nucleic acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.
  • the nucleic acid molecule encodes an HCVR and LCVR, wherein the HCVR and LCVR are both derived from the same anti-hCDH15 antibody listed in Table 1.
  • a pharmaceutical composition comprising the antigenbinding protein described herein, e.g., a recombinant human antibody or fragment thereof which binds human CDH15, and a pharmaceutically acceptable carrier.
  • a pharmaceutical composition as described herein comprises a combination of an anti-hCDH15 antibody and a second therapeutic agent.
  • the second therapeutic agent is any agent that is advantageously combined with an anti-hCDH15 antibody. Additional combination therapies and co-formulations involving the anti-hCDH15 antibodies are described herein.
  • Also described herein is a method of inhibiting the activity of CDH15 in a cell, e.g., in vivo, in vitro, or ex vivo, comprising contacting the cell expressing CDH15 with the antigen-binding protein that binds human CDH15, or the pharmaceutical composition thereof as described herein.
  • the cell expressing CDH15 is a muscle stem cell, a myoblast, or a myocyte.
  • Also described herein is a method of accelerating the transition from quiescence to activation of a muscle stem cell, e.g., in vivo, in vitro, or ex vivo, comprising contacting the muscle stem cell with the antigen-binding protein that binds human CDH15 or the pharmaceutical composition thereof as described herein.
  • a method of treating a condition in a subject in need thereof comprises administering a therapeutically effective amount of a pharmaceutical composition comprising the antigen-binding protein that binds human CDH15 as described herein to the subject.
  • the condition is muscle injury.
  • the condition is cancer, e.g., rhabdomyosarcoma.
  • the antigen-binding protein is conjugated to a therapeutic agent, e.g., an antibody-drug conjugate (ADC).
  • the therapeutic agent comprises a cytotoxic chemotherapeutic agent.
  • the therapeutic agent is Aflibercept, Amsacrine, Azacitidine, Azathioprine, Belantamab mafodotin, Bendamustine, Bleomycin, Bortezomib, Brentuximab vedotin, Busulfan, Cabazitaxel, Capecitabine, Carboplatin, Carfilzomib, Carmustine, Chlorambucil, Cisplatin, Cladribine, Clofarabine, Cyclophosphamide, Cytarabine, Cytarabine liposomal, dacarbazine, Dactinomycin (actinomycin D), Daunorubicin, Docetaxel, Doxorubicin, Doxorubicin liposomal, Epirubicin, Eribulin, Etoposide, Etoposide phosphate, Fludarabine, Fluorouracil, Fotemustine, Ganciclovir, Gemcitabine, Gemtuzumab ozo
  • the antigen binding protein is conjugated to the therapeutic agent via a valine-citrulline (VC). In some embodiments, the antigen binding protein is conjugated to the therapeutic agent via a para-aminobenzyl (PAB) linker. In some embodiments, the pharmaceutical composition is administered to the subject intravenously or subcutaneously.
  • VC valine-citrulline
  • PAB para-aminobenzyl
  • Also described herein is a method of restoring the muscle regenerative capacity of a subject, wherein the method comprises administering a therapeutically effective amount of a pharmaceutical composition comprising the antigen-binding protein that binds human CDH15 as described herein to the subject.
  • the subject is an aged subject.
  • the muscle regenerative capacity of the aged subject is restored to a functional state at or near that of a control subject.
  • the pharmaceutical composition is administered to the subject intravenously or subcutaneously.
  • a method of imaging a muscle cell in a subject in need thereof comprises administering a pharmaceutical composition comprising the antigen-binding protein that binds human CDH15 as described herein to the subject, wherein the antigen binding protein is conjugated to a detectable moiety.
  • the muscle cell comprises one or more selected from the group consisting of a muscle stem cell, a myoblast, and a myocyte.
  • the detectable moiety comprises a radionuclide.
  • the pharmaceutical composition is administered to the subject intravenously or subcutaneously.
  • antigen-binding protein that binds human CDH15 or a pharmaceutical composition thereof in the manufacture of a medicament, e.g., for the treatment of a condition as described herein.
  • an antigen-binding protein that binds human CDH15 or a pharmaceutical composition thereof for use in therapy, e.g., for treating a condition as described herein, and/or for use in restoring the muscle regenerative capacity of a subject.
  • an antibody or antigen-binding fragment that competes for binding to human CDH15 with a reference antibody comprising an HCVR/LCVR amino acid sequence pair as set forth in Table 1.
  • an antibody or antigen-binding fragment as described herein competes for binding to human CDH15 with a reference antibody comprising an HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 and 10, 22 and 30, 42 and 50, 60 and 50, 70 and 50, 80 and 50, 90 and 98, 108 and 115, 125 and 133, 145 and 153, 165 and 173, 185 and 193, 202 and 210, 220 and 50, 230 and 50, 240 and 50, 250 and 50, 259 and 267, 279 and 287, 299 and 307, 319 and 327, 339 and 347, 358 and 366, 378 and 50, 386 and 394, 404 and 412, 420 and 428, 436 and 444,
  • an antibody or antigen-binding fragment wherein the antibody or antigen-binding fragment thereof binds to the same epitope on human CDH15 as a reference antibody comprising an HCVR/LCVR amino acid sequence pair as set forth in Table 1.
  • an antibody or antigen-binding fragment as described herein binds to the same epitope on human CDH15 as a reference antibody comprising an HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 and 10, 22 and 30, 42 and 50, 60 and 50, 70 and 50, 80 and 50, 90 and 98, 108 and 115, 125 and 133, 145 and 153, 165 and 173, 185 and 193, 202 and 210, 220 and 50, 230 and 50, 240 and 50, 250 and 50, 259 and 267, 279 and 287, 299 and 307, 319 and 327, 339 and 347, 358 and 366, 378 and 50, 386 and 394,
  • an isolated antibody or antigen-binding fragment thereof that binds human CDH15 wherein the antibody or antigen-binding fragment comprises: the complementarity determining regions (CDRs) of a heavy chain variable region (HCVR) having an amino acid sequence as set forth in Table 1; and the CDRs of a light chain variable region (LCVR) having an amino acid sequence as set forth in Table 1.
  • CDRs complementarity determining regions
  • HCVR heavy chain variable region
  • LCVR light chain variable region
  • the isolated antibody or antigen-binding fragment comprises the heavy and light chain CDRs of a HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 and 10, 22 and 30, 42 and 50, 60 and 50, 70 and 50, 80 and 50, 90 and 98, 108 and 115, 125 and 133, 145 and 153, 165 and 173, 185 and 193, 202 and 210, 220 and 50, 230 and 50, 240 and 50, 250 and 50, 259 and 267, 279 and 287, 299 and 307, 319 and 327, 339 and 347, 358 and 366, 378 and 50, 386 and 394, 404 and 412, 420 and 428, 436 and 444, 452 and 460, 468 and 476, 484 and 492, 500 and 508, 516 and 524, 532 and 540, 548 and 556, 564 and 572, 580 and 588, 596 and 604, 612 and
  • the isolated antibody or antigen-binding fragment comprises HCDR1-HCDR2-HCDR3-LCDR1- LCDR2-LCDR3 domains, respectively, selected from the group consisting SEQ ID NOs: 4-6-8- 12-14-16, 24-26-28-32-34-36, 44-46-48-52-34-54, 62-64-66-52-34-54, 72-74-76-52-34-54, 82- 84-86-52-34-54, 92-94-96-100-34-102, 82-111-113-117-34-119, 127-129-131-135-137-139, 147-149-151-155-157-159, 167-169-171-175-177-179, 187-189-191-52-34-196, 204-206-208- 212-137-214, 222-224-226-52-34-54, 232-234-236-52-34-54, 242-244-246-52-34-54, 82-253- 255-52-34-54, 261-263-265-269
  • an isolated antibody or antigen-binding fragment thereof that binds human CDH15 wherein the antibody or antigen-binding fragment comprises: (a) a heavy chain variable region (HCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 22, 42, 60, 70, 80, 90, 108, 125, 145, 165, 185, 202, 220, 230, 240, 250, 259, 279, 299, 319, 339, 358, 378, 386, 404, 420, 436, 452, 468, 484,
  • HCVR heavy chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 22, 42, 60, 70, 80, 90, 108, 125, 145, 165, 185, 202, 220, 230, 240, 250, 259, 279, 299, 319, 339, 358, 378, 386, 404, 420, 436, 452, 468, 484,
  • LCVR light chain variable region
  • the isolated antibody or antigen-binding fragment comprises a HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 and 10, 22 and 30, 42 and 50, 60 and 50, 70 and 50, 80 and 50, 90 and 98, 108 and 115, 125 and 133, 145 and 153, 165 and 173, 185 and 193, 202 and 210, 220 and 50, 230 and 50, 240 and 50, 250 and 50, 259 and 267, 279 and 287, 299 and 307, 319 and 327, 339 and 347, 358 and 366, 378 and 50, 386 and 394, 404 and 412, 420 and 428, 436 and 444, 452 and 460, 468 and 476, 484 and 492,
  • viral particles that are particularly suited for the targeted introduction of a nucleotide of interest specifically to a muscle cell (e.g, a muscle stem cell) since the viral capsid or viral capsid protein(s) described herein comprise a targeting ligand that binds a muscle-cell specific surface protein (e.g, an antigen -binding protein as described herein that binds to human CDH15).
  • a targeting ligand that binds a muscle-cell specific surface protein (e.g, an antigen -binding protein as described herein that binds to human CDH15).
  • the viral capsid or viral capsid protein(s) described herein comprise(s) a direct insertion of the targeting ligand (e.g., the targeting ligand is directly coupled to, fused to, etc., optionally via a linker to the viral capsid or viral capsid proteins), e.g., viral capsid genes are modified to express capsid proteins comprising the targeting ligand.
  • a viral capsid or viral capsid protein comprises the targeting ligand via a scaffold or an adaptor, e.g., a first member of a protein: protein binding pair, which may be associated with its cognate second member of the protein: protein binding pair, wherein the second member is linked (e.g, fused to) a targeting ligand that binds a musclecell specific surface protein (e.g., an antigen-binding protein as described herein that binds to human CDH15).
  • the targeting ligand is operably linked to the second member, e.g, fused to the second member, optionally via a linker.
  • the targeting ligand may be a binding moiety, e.g, a natural ligand, antibody, a multispecific binding molecule, etc.
  • the targeting ligand is an antibody or portion thereof.
  • the targeting ligand is an antibody comprising a variable domain that binds a surface protein (e.g, a variable domain of an antigen-binding protein as described herein that binds to human CDH15) on a non-terminally differentiated muscle cell (e.g, a muscle stem cell, a myoblast, myocyte, any combination thereof, etc.) and a heavy chain constant domain.
  • a surface protein e.g, a variable domain of an antigen-binding protein as described herein that binds to human CDH15
  • a non-terminally differentiated muscle cell e.g, a muscle stem cell, a myoblast, myocyte, any combination thereof, etc.
  • the targeting ligand is an antibody comprising a variable domain that binds a non-terminally differentiated muscle cell surface protein (e.g, a variable domain of an antigenbinding protein as described herein that binds to human CDH15) on a target cell (e.g., a muscle stem cell, myoblast, myocyte, any combination thereof, e/c.), and optionally an IgG heavy chain constant domain.
  • a non-terminally differentiated muscle cell surface protein e.g, a variable domain of an antigenbinding protein as described herein that binds to human CDH15
  • a target cell e.g., a muscle stem cell, myoblast, myocyte, any combination thereof, e/c.
  • the targeting ligand is an antibody comprising a variable domain that binds a non-terminally differentiated muscle cell surface protein (e.g, a variable domain of an antigen-binding protein as described herein that binds to human CDH15) on a target cell (e.g, a muscle stem cell, myoblast, myocyte, any combination thereof, etc.) and an IgG heavy chain constant domain, wherein the IgG heavy chain constant domain is operably linked, e.g., directly or via a linker, to a capsid protein.
  • a non-terminally differentiated muscle cell surface protein e.g, a variable domain of an antigen-binding protein as described herein that binds to human CDH15
  • a target cell e.g, a muscle stem cell, myoblast, myocyte, any combination thereof, etc.
  • IgG heavy chain constant domain operably linked, e.g., directly or via a linker, to a capsid protein.
  • the targeting ligand is an antibody comprising (i) a variable domain that binds a non-terminally differentiated muscle cell surface protein (e.g., a variable domain of an antigen-binding protein as described herein that binds to human CDH15), and (ii) an IgG heavy chain constant domain, wherein the IgG heavy chain constant domain is operably linked (optionally via a linker) to a protein (e.g., second member of a protein: protein binding pair) that forms an isopeptide covalent bond with the cognate first member of the proteimprotein binding pair.
  • a non-terminally differentiated muscle cell surface protein e.g., a variable domain of an antigen-binding protein as described herein that binds to human CDH15
  • an IgG heavy chain constant domain wherein the IgG heavy chain constant domain is operably linked (optionally via a linker) to a protein (e.g., second member of a protein: protein binding pair) that forms an isopeptide co
  • a capsid protein described herein comprises a first member of the protein: protein binding pair, comprising, e.g., SpyTag (SEQ ID NO: 815) or a biologically equivalent variant thereof, operably linked to the viral capsid protein, wherein SpyTag or a biologically equivalent variant thereof is covalently linked (e.g., via an isopeptide bond) to the SpyTag to its second cognate protein: protein binding member, e.g., SpyCatcher (SEQ ID NO: 816) or a biologically equivalent variant thereof, which in turn may be linked to a targeting ligand comprising an antibody variable domain and an IgG heavy chain domain, wherein SpyCatcher and the IgG heavy chain domain are linked via an amino acid linker, e.g., GSGESG (SEQ ID NO: 828).
  • protein binding pair comprising, e.g., SpyTag (SEQ ID NO: 815) or a biologically equivalent variant thereof, operably linked to the viral capsid protein
  • the non-terminally differentiated muscle cell surface protein comprises CDH15.
  • the targeting ligand binds CDH15, e.g., human CDH15.
  • the targeting ligand comprises a variable domain of an antigen-binding protein as described herein that binds to human CDH15.
  • the targeting ligand comprises an antibody variable domain comprising a CDR, e.g., an HCDR1, an HCDR2, an HCDR3, an LCDR1, an LCDR2, and/or an LCDR3, of an HCVR and/or LCVR sequence as set forth in Table 1.
  • the targeting ligand comprises an antibody variable domain comprising a CDR, e.g., an HCDR1, an HCDR2, an HCDR3, an LCDR1, an LCDR2, and/or an LCDR3, as set forth in Table 1.
  • the targeting ligand comprises an antibody variable domain comprising a set of three CDRs, e.g., an HCDR1, an HCDR2, and an HCDR3, and/or an LCDR1, an LCDR2, and an LCDR3, of an HCVR and/or LCVR sequence as set forth in Table 1.
  • the targeting ligand comprises an antibody variable domain comprising a set of three CDRs, e.g., an HCDR1, an HCDR2, and an HCDR3, and/or an LCDR1, an LCDR2, and an LCDR3, as set forth in Table 1.
  • the targeting ligand comprises an antibody variable domain comprising an HCVR and/or LCVR as set forth in Table 1.
  • FIG. 1 depicts a schematic illustration of myogenesis of non-terminally differentiated muscle cells.
  • Muscle stem cells also known as satellite cells
  • MuSCs are mitotically quiescent and non-proliferative under steady-state conditions in adult muscle tissue.
  • MuSCs begin to actively divide, producing daughter cells. While some daughter cells become quiescent once more to replenish the pool of MuSCs, others continue to proliferate as myoblasts, which align and fuse together during differentiation into mature myotubes/myofibers that make up muscle fibers.
  • Quiescent muscle stem cells can be characterized by Pax7 expression
  • proliferating myoblasts can be characterized by Pax7 and myogenic differentiation factor 1 (MyoD) expression
  • myoblasts committing to terminal differentiation i.e., myocytes
  • myotubes can be characterized by myosin heavy chain (MyHC) expression.
  • MyoD myogenic differentiation factor 1
  • MyHC myosin heavy chain
  • FIGs 2A-2D demonstrate accelerated muscular regeneration in mice with CDH15 expression abolished.
  • Figure 2A depicts a schematic illustration of the example experiments. Wild-type (WT) or homozygous CDH15 knockout mice (CDH15-/-) were injected intramuscularly with cardiotoxin (CTX) at day 0 to induce muscle damage. Histology was then performed on samples of muscle retrieved 5-, 15-, and 25-days post-injury (dpi).
  • WT Wild-type
  • CDH15-/- homozygous CDH15 knockout mice
  • CTX cardiotoxin
  • Figure 2B depicts an example of immunohistochemical cross-sections of WT and CDH15-/- muscle samples stained with DAPI (blue; light grey in greyscale), laminin (white), and embryonic myosin heavy chain (eMyHC; green; dark grey in greyscale), which is transiently upregulated in immature myofibers but downregulated as myofibers mature (Rodgers, Growth Hormone & IGF Research, 2005, 15(6): 377-383). Cross-sections were visualized by fluorescence microscopy.
  • Figure 2C depicts the myofiber cross-section area (CSA) for WT and CDH15-/- mice at the indicated time points post-injury, as quantified from histological images, see, e.g., Figure 2B.
  • Figure 2D depicts the percentage of centrally nucleated myofibers for WT and CDH15-/- mice at 15 days post-injury, as quantified from histological images, see, e.g., Figure 2B. Data are reported as means +/- SEM, *p ⁇ 0.05, **p ⁇ 0.01.
  • Figures 3A-3B demonstrate improved functional recovery from injury in mice with CDH15 expression abolished.
  • Figure 3A depicts a schematic illustration of the example experiments. WT or CDH15-/- mice were injected with CTX into the extensor digitorum longus (EDL) muscle at day 0. At 15 dpi, the contractile force of the EDL muscle was measured ex vivo.
  • Figure 3B depicts the maximal tetanic force loss in WT or CDH15-/- muscle as compared to muscle isolated from uninjured EDL. Data are reported as means +/- SEM, *p ⁇ 0.05.
  • Figures 4A-4B demonstrate accelerated exit from quiescence ex vivo in MuSCs with CDH15 expression abolished.
  • Figure 4A depicts an example of immunohistochemical images of WT and CDH15-/- MuSCs following 48 hours of culture on single myofibers. Arrows in the Pax7 and Merge columns denote individual cells in a multi-cell cluster.
  • Figure 4B depicts the percentage of Pax7+ single-cell and multi-cell clusters (top) and the number of Pax7+ cells per cluster (bottom) for WT and CDH15-/- MuSCs, as quantified from histological images, see, e g., Figure 4A . Data are reported as means +/- SEM, **p ⁇ 0.01.
  • Figures 5A-5B demonstrate the upregulation of early response genes containing serum response factor (SRF) motifs.
  • Figure 5A depicts a volcano plot of genes that are downregulated or upregulated in FACS-isolated CDH15-/- MuSCs as compared to WT MuSCs.
  • Figure 5B depicts a transcription factor motif analysis, with several relevant transcription factors highlighted in red.
  • FIGs 6A-6D demonstrate that abolishing expression of CDH15 in aged mice rescues age-related declines in muscle regeneration.
  • Figure 6A depicts a schematic illustration of the example experiments. Aged (23 -month-old) wild-type (WT) or homozygous CDH15 knockout mice (CDH15-/-) were injected with cardiotoxin (CTX) at day 0 to induce muscle damage of the tibialis anterior muscle. Histology was then performed on samples of muscle retrieved 15 days post-injury (dpi).
  • CTR cardiotoxin
  • Figure 6B depicts an example of immunohistochemical cross-sections of aged WT and CDH15-/- muscle samples stained for nuclei (DAPI; blue; grey in greyscale) and laminin (white) and visualized by fluorescence microscopy.
  • Figure 6C depicts the myofiber cross-section area (CSA) for aged WT and CDH15-/- mice at 15 dpi, as quantified from histological images, see, e.g., Figure 6B.
  • the dotted line depicts the average CSA for young mice at 15 dpi.
  • Figure 6D depicts the percentage of centrally nucleated myofibers containing 3 or more central nuclei for aged WT and CDH15-/- mice at 15 dpi, as quantified from histological images, see, e.g., Figure 6B. Data are reported as means +/- SEM, *p ⁇ 0.05.
  • Figures 7A-7C demonstrate that CDH15 protein is focalized to the apical surface of the majority of wild-type (WT) muscle stem cells (MuSCs) and is undetectable in CDH15-/- MuSCs.
  • Figure 7A depicts an immunohistochemical cross-section of a WT single myofiber with an associated WT MuSC, with CDH15 protein present at the apical surface of the MuSC.
  • Anti- CDH15 staining is depicted as red and denoted with an arrow and dotted outline and DAPI staining is depicted as blue and denoted with an arrowhead and a dashed outline.
  • Figure 7B depicts an immunohistochemical cross-section of WT mouse muscle, with Pax7-positive MuSCs observed on the periphery of myofibers, again with CDH15 present at the apical surface of the MuSC.
  • Anti-CDH15 is depicted as red and denoted with an arrow and a dotted outline in the magnified inset image, DAPI-stained nuclei are depicted as blue, Pax7 is depicted as green, and laminin is depicted as white.
  • FIG. 7C depicts the proportion of Pax7-positive MuSCs that are also CDH15-positive in WT and CDH15-/- mouse muscle, as quantified from histological images, see, e.g., Figure 7A and 7B. While CDH15 protein is detected in the majority (-95%) of WT Pax7-positive muscle cells, CDH15 is undetectable in CDH15-/- Pax7- positive cells, as expected.
  • Figure 8 demonstrates the specific binding of anti-hCDH15 antibodies to human rhabdomyosarcoma cells, but not glioblastoma cells.
  • Alveolar, embryonal, and glioblastoma cells were live-stained with the indicated anti-hCDH15 antibodies (REGN8787 and REGN9295) for 30 minutes, washed, stained with anti-human IgG Alexa FluorTM 647-conjugated secondary antibody (red), washed again, fixed, and then stained for myogenin (green) and nuclei (DAPI; blue) prior to visualization by fluorescence microscopy.
  • Cells incubated with murine IgG2a and human IgG4 were also used as controls. The last row depicts a merge of the three markers.
  • Figure 9 demonstrates the specific binding of anti-hCDH15 antibodies to human rhabdospheres grown in 3D culture.
  • Embryonal rhabdomyosarcoma tumorspheres z.e., rhabdospheres
  • anti-hCDH15 or human IgG4 as a negative control for 30 minutes, washed, stained with anti-human IgG Alexa FluorTM 647-conjugated secondary antibody (white), washed again, fixed and stained for myogenin (red) and nuclei (DAPI; blue).
  • the last column depicts a merge of the three markers.
  • Figure 10 demonstrates anti-CDH15 antigen-binding domain-mediated retargeting of AAV9 to C2C12 mouse myoblasts. Depicted are representative immunofluorescence images demonstrating the transduction efficiency via GFP fluorescence of C2C12 myoblasts transduced with 2.5 x 10 5 vg/cell of AAV9 expressing eGFP +/- plasmids encoding anti-CDH15 antigen-binding domains or control antigen-binding domains (anti- ASGR1) and varying mosaic capsid ratios. DAPI-stained nuclei are depicted in blue (top row) and eGFP is depicted in green (bottom row).
  • the ratio provided indicates the ratio of quantities of transfected plasmid encoding SpyTag-conjugated AAV9 capsid versus non-conjugated N272A de-targeted AAV9 capsid.
  • an antigen-binding domain is indicated e.g., a mAb or a Fab
  • the AAV9 capsid comprises SpyTag inserted at position 453 and attached via a 10 amino acid linker
  • the antigen-binding domain comprises SpyCatcher fused to the C-terminus of the heavy chain construct.
  • Figure 11 demonstrates anti-CDH15 antigen-binding domain-mediated retargeting of AAV9 to human skeletal myoblasts. Depicted are representative immunofluorescence images demonstrating the transduction efficiency via GFP fluorescence of human skeletal myoblasts transduced with 2.5 x 10 5 vg/cell of AAV9 expressing eGFP +/- plasmids encoding anti-CDH15 antigen-binding domains or control antigen-binding domains (anti-ASGRl) and varying mosaic capsid ratios. DAPI-stained nuclei are depicted in blue (top row) and eGFP is depicted in green (bottom row).
  • the ratio provided indicates the ratio of quantities of transfected plasmid encoding SpyTag-conjugated AAV9 capsid versus nonconjugated N272A de-targeted AAV9 capsid.
  • an antigen-binding domain is indicated e.g., a mAb or a Fab
  • the AAV9 capsid comprises SpyTag inserted at position 453 and attached via a 10 amino acid linker
  • the antigen -binding domain comprises SpyCatcher fused to the C-terminus of the heavy chain construct.
  • Each individual skeletal muscle consists of thousands of muscle fibers wrapped together by connective tissue sheaths.
  • the individual bundles of muscle fibers in a skeletal muscle are known as fasciculi.
  • the outermost connective tissue sheath surrounding the entire muscle is known as epimysium.
  • the connective tissue sheath covering each fasciculus is known as perimysium, and the innermost sheath surrounding individual muscle fiber is known as endomysium.
  • Each muscle fiber comprises myofibrils containing multiple myofilaments.
  • the primary functions of the skeletal muscle take place via its intrinsic excitationcontraction coupling process. As the muscle is attached to the bone tendons, the contraction of the muscle leads to movement of that bone that allows for the performance of specific movements.
  • the skeletal muscle also provides structural support and helps in maintaining the posture of the body.
  • the skeletal muscle also acts as a storage source for amino acids that can be used by different organs of the body for synthesizing organ-specific proteins.
  • the skeletal muscle also acts as a site of glucose disposal in the form of muscle glycogen.
  • the skeletal muscle also plays a central role in maintaining thermostasis and acts as an energy source during starvation. Thus, skeletal muscle plays key roles in locomotion, thermoregulation, and in controlling whole body metabolism.
  • Treatments for muscle wasting and genetic muscle diseases typically consist of broad-acting therapies, such as testosterone therapy for muscle wasting, glucocorticoids for muscular dystrophies, and systemic AAV delivery for treatment of muscle diseases (e.g., X- linked myotubular myopathy (XLMTM), Duchenne muscular dystrophy (DMD), myotonic dystrophy (DM1), Facioscapulohumeral muscular dystrophy Type 1 (FSHD), congenital muscular dystrophy type 1A (MDC1A), Limb girdle muscular dystrophy, and dystroglycanopathy, etc.).
  • XLMTM X- linked myotubular myopathy
  • DMD Duchenne muscular dystrophy
  • DM1 myotonic dystrophy
  • FSHD Facioscapulohumeral muscular dystrophy Type 1
  • MDC1A congenital muscular dystrophy type 1A
  • Limb girdle muscular dystrophy and dystroglycanopathy, etc.
  • Muscle stem cells are important for skeletal muscle regeneration. While MuSCs are normally quiescent in adult muscle, upon injury, they activate, proliferate, and differentiate into functional myofibers. However, MuSCs in aging subjects display delayed activation and reduced motility in vitro, which translates to impaired MuSC mediated repair in vivo.
  • Mitotically quiescent muscle stem cells also called satellite cells
  • proliferating myoblasts are the embryonic precursors of myocytes (also called muscle cells) which have not yet fused together to form myotubes or myofibers that later become the muscle fibers.
  • Muscle stem cells and myoblasts differentiate into muscle cells through a process called myogenesis depicted schematically (not to scale) in Figure 1.
  • muscle stem cells In general, once exposed to signals from the damaged environment, muscle stem cells will leave their quiescent state, reenter the cell cycle, and start proliferating as myoblasts. Some daughter cells continue to differentiate, while others return to quiescence to replenish the reserve population of muscle stem cells. During the differentiation stage, certain genes (e.g., striated alpha-actin genes) are expressed and the myoblasts align with one another. The myoblasts then fuse to form myofibers with the recruitment of actin to the plasma membrane.
  • Muscle stem cells can be characterized by a combination of several genetic markers, such Pax7 and muscle regulatory proteins. Pax7 is a paired homeobox transcription factor, which specifies the myogenic properties of precursor muscle cells.
  • muscle stem cells whether quiescent or proliferating, may be characterized as Pax7+. See, e.g, Figure 1.
  • myogenic cellular lineage characterization is possible since myogenesis depends on the precise and dynamic integration of multiple muscle regulatory factors, such as myogenic factor 5 (MYF5), myogenic differentiation factor 1 (MYOD), myogenin (MYOG), and embryonic myosin heavy chain (MyHC).
  • MYOD myogenic factor 5
  • MYOD myogenic differentiation factor 1
  • MYOG myogenin
  • MyHC embryonic myosin heavy chain
  • MYOD is expressed in myogenic cells, but not expressed in stationary quiescent muscle stem cells, and thus, may be used to identify proliferating muscle stem cells, myoblasts, or other differentiating myocytes. See, e.g., Figure 1.
  • Myogenin appears to be expressed by myoblasts committed to differentiating into myofibers. See, e.g., Figure 1.
  • An additional marker that may be used to identify which stage of myogenesis a cell is undergoing includes, but is not limited to embryonic myosin heavy chain (eMyHC), which is transiently upregulated in immature myofibers but downregulated as myofibers mature. See, e.g., Figure 1.
  • eMyHC embryonic myosin heavy chain
  • Myoblasts may be classified as skeletal muscle myoblasts, smooth muscle myoblasts, and cardiac muscle myoblasts depending on the type of muscle cell that they will differentiate into. Thus, muscle stem cells, myoblasts, myocytes, and undifferentiated myotubes or myofibers may all be considered a non-terminally differentiated muscle cell.
  • Cadherin 15 An example cell surface protein found on non-terminally differentiated muscle cells Cadherin 15 (CDH15).
  • Cadherins are a class of calcium-dependent transmembrane proteins involved in cell-cell adhesion.
  • Classical cadherins consist of an extracellular domain comprising five repeats of an immunoglobulin-like cadherin domain, a single transmembrane region, and a cytoplasmic domain.
  • Cadherin 15 (also known as M-cadherin) is expressed at the apical surface of muscle stem cells and is believed to regulate adhesion of muscle stem cells to muscle myofibers.
  • Cadherin 15 is encoded by the CDH15 gene, located on the long arm of chromosome 16 (16q24.3).
  • CDH15 comprises 14 exons and is approximately 23,745 bases in length.
  • An exemplary sequence for human CDH15 gene is assigned NCBI Accession Number NM_004933.3 (SEQ ID NO: 787).
  • An exemplary human CDH15 protein is assigned NCBI Accession Number NP_004924.1 and/or UniProt Accession Number P55291 (SEQ ID NO: 788).
  • Genetically modified animal models may prove to be particularly useful for studying the function of CDH15 in muscle.
  • antagonistic antibodies, or CDH15 blocking antibodies may be useful in enhancing muscle regeneration in both young subjects and older adults following a damage stimulus (e.g., joint arthroplasty).
  • Rhabdomyosarcomas are characterized by the expression of myogenic genes, but multiple subtypes exist. For example, embryonal RMS is the most prevalent subtype, typically has a more favorable prognosis, and is believed to be driven by loss of tumor suppressor genes or gain-of-function of proto-oncogenes. Alveolar RMS is less common but has generally carries a much poorer prognosis. Alveolar RMS is believed to be caused by chromosomal translocation of Pax3 or Pax7 gene, or alternative gene fusions.
  • Rhabdomyosarcomas make up -3% of all childhood cancers, with -400-500 new cases per year in the U.S (American Cancer Society, 2021).
  • the prognosis is generally good in children (e.g., -70% survival), but depends on risk category. For example, 1/3 of patients with localized RMS and 2/3 of patients with metastatic RMS experience relapsed disease. If RMS is relapsed, the majority of patients have an estimated 5-year survival of -10%. Moreover, the prognosis is generally much poorer in adults (20-50% overall survival).
  • anti-CDH15 antibodies may be useful to deliver a therapeutic agent (e.g., a cytotoxic agent) to RMS tumors.
  • a therapeutic agent e.g., a cytotoxic agent
  • antigen-binding proteins e.g., antibodies and antigenbinding fragments thereof that bind to human CDH15.
  • the antibodies described herein may be useful, inter alia, for specifically directing the internalization of an agent, e.g., a drug conjugate, etc., to a muscle cell and/or a muscle-related cancer cell, for blocking activity of CDH15, and/or for specifically stimulating muscle regeneration.
  • antibodies, or antigen-binding fragments thereof, that bind human CDH15 include antibody-protein fusion constructs comprising an antibody, or antigen-binding fragment thereof, that bind human CDH15; and antibody drug conjugates comprising an antibody, or antigen-binding fragment thereof, that bind human CDH15.
  • viral particles e.g., AAV viral particles, that can target non-terminally differentiated muscle cell surface proteins, such as mammalian CDH15.
  • the “percent (%) identity” or the like may be readily determined for amino acid or nucleotide sequences, over the full-length of a protein, or a portion thereof. A portion may be at least about 5 amino acids or 24 nucleotides, respectively, in length, and may be up to about 700 amino acids or 2100 nucleotides, respectively. Generally, when referring to “identity”, “homology”, or “similarity” between two different adeno-associated viruses, “identity”, “homology” or “similarity” is determined in reference to “aligned” sequences. “Aligned” sequences or “alignments” refer to multiple nucleic acid sequences or protein (amino acids) sequences, often containing corrections for missing or additional bases or amino acids as compared to a reference sequence.
  • Alignments may be performed using any of a variety of publicly or commercially available Multiple Sequence Alignment Programs. Sequence alignment programs are available for amino acid sequences, e.g., the “Clustal X”, “MAP”, “PIMA”, “MSA”, “BLOCKMAKER”, “MEME”, and “Match-Box” programs. Generally, any of these programs are used at default settings, although one of skill in the art can alter these settings as needed. Alternatively, one of skill in the art can utilize another algorithm or computer program which provides at least the level of identity or alignment as that provided by the referenced algorithms and programs. See, e.g., J. D. Thomson et al, Nucl. Acids. Res., “A comprehensive comparison of multiple sequence alignments”, 27(13):2682-2690 (1999).
  • nucleic acid sequences are also available for nucleic acid sequences. Examples of such programs include, “Clustal W”, “CAP Sequence Assembly”, “MAP”, and “MEME”, which are accessible through Web Servers on the internet. Other sources for such programs are known to those of skill in the art. Alternatively, Vector NTI utilities are also used. There are also a number of algorithms known in the art that can be used to measure nucleotide sequence identity, including those contained in the programs described above. As another example, polynucleotide sequences can be compared using FASTATM, a program in GCG Version 6.1. FASTATM provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences. For instance, percent sequence identity between nucleic acid sequences can be determined using FASTATM with its default parameters (a word size of 6 and the NOPAM factor for the scoring matrix) as provided in GCG Version 6.1, herein incorporated by reference.
  • FASTATM provides alignments and percent sequence identity of the regions of the best overlap between the
  • “Significant identity” encompasses amino acid or nucleic acid sequences alignments that are at least 90%, e.g., at least 93%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99%, or e.g., at least 100% identical.
  • chimeric encompasses a functional gene or polypeptide comprising nucleic acid sequences or amino acid sequences, respectively, from at least two different AAV serotype, e.g., portions of a gene or polypeptide of at least a first and second AAV, wherein the at least first and second portions are operably linked to form a functional chimeric AAV nucleic acid that encodes a functional amino acid.
  • nucleotide sequences, genes, polypeptides, and amino acids are considered non-chimeric in that the nucleotide sequences, genes, polypeptides, and amino acids comprise a nucleic acid sequence or amino acid sequence having significant identity to a nucleic acid sequence or amino acid sequence, respectively, of a single AAV serotype.
  • operably linked includes a physical juxtaposition (e.g., in three-dimensional space) of components or elements that interact, directly or indirectly with one another, or otherwise coordinate with each other to participate in a biological event, which juxtaposition achieves or permits such interaction and/or coordination.
  • a regulatory element e.g., an expression control sequence
  • a regulatory element in a nucleic acid is said to be “operably linked” to a coding sequence when it is located relative to the coding sequence such that its presence or absence impacts expression and/or activity of the coding sequence.
  • operable linkage involves covalent linkage of relevant components or elements with one another.
  • covalent linkage is not required to achieve effective operable linkage.
  • proteins operably linked together may be associated with each other, e.g., via a covalent bond or a non- covalent bond.
  • a capsid protein as described herein may be operably linked to a targeting ligand, where the capsid protein is non-covalently bound to the targeting ligand, or covalently bound to the targeting ligand, optionally with or without a scaffold and/or adaptor between the capsid protein and the targeting ligand.
  • nucleic acid regulatory elements that are operably linked with coding sequences that they control are contiguous with the nucleotide of interest.
  • one or more such regulatory elements acts in trans or at a distance to control a coding sequence of interest.
  • regulatory element refers to polynucleotide sequences which are necessary and/or sufficient to effect the expression and processing of coding sequences to which they are ligated.
  • a regulatory element may be or comprise appropriate transcription initiation, termination, promoter and/or enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., Kozak consensus sequence); sequences that enhance protein stability; and/or, in some embodiments, sequences that enhance protein secretion.
  • one or more regulatory elements are preferentially or exclusively active in a particular host cell or organism, or type thereof.
  • regulatory elements may typically include promoter, ribosomal binding site, and transcription termination sequence; in eukaryotes, in many embodiments, regulatory elements may typically include promoters, enhancers, and/or transcription termination sequences.
  • regulatory elements refers to components whose presence is essential for expression and processing, and in some embodiments includes components whose presence is advantageous for expression (including, for example, leader sequences, targeting sequences, and/or fusion partner sequences).
  • an antibody that binds CDH15 includes an antibody and antigen-binding fragment thereof that specifically recognizes a single CDH15 molecule.
  • An antibody and antigen-binding fragment thereof as described herein may bind soluble CDH15 and/or cell surface expressed CDH15.
  • Soluble CDH15 includes natural CDH15 proteins as well as recombinant CDH15 protein variants that lack a transmembrane domain or are otherwise unassociated with a cell membrane.
  • cell surface-expressed CDH15 refers to one or more CDH15 protein(s) that is/are expressed on the surface of a cell in vitro or in vivo, such that at least a portion of a CDH15 protein is exposed to the extracellular side of the cell membrane and is accessible to an antigen-binding portion of an antibody.
  • a “cell surface-expressed CDH15” can comprise or consist of a CDH15 protein expressed on the surface of a cell which normally expresses CDH15 protein.
  • “cell surface-expressed CDH15” can comprise or consist of a CDH15 protein expressed on the surface of a cell that normally does not express human CDH15 on its surface but has been artificially engineered to express CDH15 on its surface.
  • antigen -binding molecule includes an antibody and an antigenbinding fragment of an antibody.
  • antibody refers to any antigen-binding molecule or molecular complex comprising at least one complementarity determining region (CDR) that specifically binds to or interacts with a particular antigen (e.g., CDH15).
  • CDR complementarity determining region
  • Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region.
  • the heavy chain constant region comprises three domains, CHI, CH2 and CH3.
  • Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region.
  • the light chain constant region comprises one domain, CL.
  • the VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy -terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (heavy chain CDRs may be abbreviated as HCDR1, HCDR2 and HCDR3; light chain CDRs may be abbreviated as LCDR1, LCDR2 and LCDR3.
  • the term “high affinity” antibody refers to those antibodies having a binding affinity to their target of at least 10' 9 M, at least IO' 10 M; at least 10' 11 M; or at least 10' 12 M, as measured by surface plasmon resonance, e.g., BIACORETM or solution-affinity ELISA.
  • antibody may encompass any type of antibody, such as e.g., monoclonal or polyclonal. Moreover, the antibody may be or any origin, such as e.g, mammalian or nonmammalian. In one embodiment, the antibody may be mammalian or avian. In a further embodiment, the antibody may be of human origin and may further be a human monoclonal antibody.
  • antibody also includes antigen-binding fragments of full antibody molecules.
  • antigen-binding portion of an antibody, “antigen-binding fragment” of an antibody, and the like include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex.
  • Antigen-binding fragments of an antibody may be derived, e.g., from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains.
  • DNA is known and/or is readily available from, e.g., commercial sources, DNA libraries (including, e.g., phage-antibody libraries), or can be synthesized.
  • the DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and/or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc.
  • Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab’)2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g, an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FR3- CDR3-FR4 peptide.
  • CDR complementarity determining region
  • engineered molecules such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalent nanobodies, bivalent nanobodies, efc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression “antigen-binding fragment”.
  • SMIPs small modular immunopharmaceuticals
  • shark variable IgNAR domains are also encompassed within the expression “antigen-binding fragment”.
  • An antigen-binding fragment of an antibody will typically comprise at least one variable domain.
  • the variable domain may be of any size or amino acid composition and will generally comprise at least one CDR which is adjacent to or in frame with one or more framework sequences.
  • the VH and VL domains may be situated relative to one another in any suitable arrangement.
  • the variable region may be dimeric and contain VH-VH, VH-VL or VL-VL dimers.
  • the antigen-binding fragment of an antibody may contain a monomeric VH or VL domain.
  • an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain.
  • variable and constant domains that may be found within an antigenbinding fragment of an antibody as described herein include: (i) VH-CH1; (ii) VH-CH2; (iii) VH- CH3; (iv) VH-CH1-CH2; (V) VH-CH1-CH2-CH3; (vi) VH-CH2-CH3; (vii) VH-CL; (viii) VL-CH1; (ix) VL-CH2; (X) VL-CH3; (xi) VL-C H 1-CH2; (xii) VL-CH1-C H 2-CH3; (xiii) VL-C H 2-CH3; and (xiv) V L - CL.
  • variable and constant domains may be either directly linked to one another or may be linked by a full or partial hinge or linker region.
  • a hinge region may consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in a flexible or semi -flexible linkage between adjacent variable and/or constant domains in a single polypeptide molecule.
  • an antigen-binding fragment of an antibody as described herein may comprise a homo-dimer or hetero-dimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with one another and/or with one or more monomeric VH or VL domain (e.g., by disulfide bond(s)).
  • antigen-binding fragments may be monospecific or multispecific (e.g., bispecific).
  • a multispecific antigen-binding fragment of an antibody will typically comprise at least two different variable domains, wherein each variable domain is capable of specifically binding to a separate antigen or to a different epitope on the same antigen.
  • Any multispecific antibody format including the example bispecific antibody formats disclosed herein, may be adapted for use in the context of an antigen-binding fragment of an antibody as described herein using routine techniques available in the art.
  • the anti-hCDH15 antibodies as described herein are human antibodies.
  • the term “human antibody” refers to antibodies having variable and constant regions derived from human germline immunoglobulin sequences.
  • the human antibodies as described herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3.
  • the term “human antibody” is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
  • the antibodies as described herein may, in some embodiments, be recombinant human antibodies.
  • the term “recombinant human antibody” is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described further below), antibodies isolated from a recombinant, combinatorial human antibody library (described further below), antibodies isolated from an animal e.g., a mouse) that is transgenic for human immunoglobulin genes (see e.g., Taylor et al. (1992) Nucl. Acids Res.
  • Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.
  • Human antibodies may exist in two general forms that are associated with hinge heterogeneity.
  • an immunoglobulin molecule comprises a stable four chain construct of approximately 150-160 kDa in which the dimers are held together by an interchain heavy chain disulfide bond.
  • the dimers are not linked via inter-chain disulfide bonds and a molecule of about 75-80 kDa is formed composed of a covalently coupled light and heavy chain (half-antibody). These forms have been extremely difficult to separate, even after affinity purification.
  • the frequency of appearance of the second form in various intact IgG isotypes is due to, but not limited to, structural differences associated with the hinge region isotype of the antibody.
  • a single amino acid substitution in the hinge region of the human IgG4 hinge can significantly reduce the appearance of the second form (Angal et al. (1993) Molecular Immunology 30: 105) to levels typically observed using a human IgGl hinge.
  • the antibodies as described herein may have one or more mutations in the hinge, CH2 or CH3 region which may be desirable, for example, in production, to improve the yield of the desired antibody form.
  • the antibodies as described herein may be isolated antibodies.
  • An “isolated antibody” refers to an antibody that has been identified and separated and/or recovered from at least one component of its natural environment. For example, an antibody that has been separated or removed from at least one component of an organism, or from a tissue or cell in which the antibody naturally exists or is naturally produced, may be considered an “isolated antibody.”
  • An isolated antibody also includes an antibody in situ within a recombinant cell. Isolated antibodies are antibodies that have been subjected to at least one purification or isolation step. According to certain embodiments, an isolated antibody may be substantially free of other cellular material and/or chemicals.
  • one-arm antibodies that bind CDH15.
  • the term “one- arm antibody” refers to an antigen-binding molecule comprising a single antibody heavy chain and a single antibody light chain.
  • the one-arm antibodies as described herein may comprise any of the HCVR/LCVR or CDR amino acid sequences as set forth in Table 1.
  • the anti-hCDH15 antibodies disclosed herein may comprise one or more amino acid substitutions, insertions and/or deletions in the framework and/or CDR regions of the heavy and light chain variable domains as compared to the corresponding germline sequences from which the antibodies were derived. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available from, for example, public antibody sequence databases.
  • antibodies, and antigen-binding fragments thereof which are derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework and/or CDR regions are mutated to the corresponding residue(s) of the germline sequence from which the antibody was derived, or to the corresponding residue(s) of another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are referred to herein collectively as “germline mutations”).
  • Germline mutations such sequence changes are referred to herein collectively as “germline mutations”.
  • all of the framework and/or CDR residues within the VH and/or VL domains are mutated back to the residues found in the original germline sequence from which the antibody was derived.
  • only certain residues are mutated back to the original germline sequence, e.g., only the mutated residues found within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only the mutated residues found within CDR1, CDR2 or CDR3.
  • one or more of the framework and/or CDR residue(s) are mutated to the corresponding residue(s) of a different germline sequence (z.e., a germline sequence that is different from the germline sequence from which the antibody was originally derived).
  • the antibodies as described herein may contain any combination of two or more germline mutations within the framework and/or CDR regions, e.g., wherein certain individual residues are mutated to the corresponding residue of a particular germline sequence while certain other residues that differ from the original germline sequence are maintained or are mutated to the corresponding residue of a different germline sequence.
  • an antibody and an antigen-binding fragment that contains one or more germline mutations can be easily tested for one or more desired property such as, improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc.
  • an antibody or an antigen-binding fragment as described herein is obtained in this general manner.
  • anti-hCDH15 antibodies comprising variants of any of the HCVR, LCVR, and/or CDR amino acid sequences disclosed herein having one or more conservative substitutions.
  • some embodiments include anti-hCDH15 antibodies having HCVR, LCVR, and/or CDR amino acid sequences with, e.g, 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc. conservative amino acid substitutions relative to any of the HCVR, LCVR, and/or CDR amino acid sequences set forth in Table 1 herein.
  • bispecific antibody includes an antibody capable of selectively binding two or more epitopes.
  • Bispecific antibodies generally comprise two different heavy chains, with each heavy chain specifically binding a different epitope — either on two different molecules (e.g., antigens) or on the same molecule (e.g., on the same antigen). If a bispecific antibody is capable of selectively binding two different epitopes (a first epitope and a second epitope), the affinity of the first heavy chain for the first epitope will generally be at least one to two or three or four orders of magnitude lower than the affinity of the first heavy chain for the second epitope, and vice versa.
  • the epitopes recognized by the bispecific antibody can be on the same or a different target (e.g., on the same or a different protein).
  • Bispecific antibodies can be made, for example, by combining heavy chains that recognize different epitopes of the same antigen.
  • nucleic acid sequences encoding heavy chain variable sequences that recognize different epitopes of the same antigen can be fused to nucleic acid sequences encoding different heavy chain constant regions, and such sequences can be expressed in a cell that expresses an immunoglobulin light chain.
  • a typical bispecific antibody has two heavy chains each having three heavy chain CDRs, followed by (N-terminal to C-terminal) a CHI domain, a hinge, a CH2 domain, and a CH3 domain, and an immunoglobulin light chain that either may not confer antigen-binding specificity but that can associate with each heavy chain, or that can associate with each heavy chain and that can bind one or more of the epitopes bound by the heavy chain antigen-binding regions, or that can associate with each heavy chain and participate in binding of one or both of the heavy chains to one or both epitopes.
  • heavy chain or “immunoglobulin heavy chain” includes an immunoglobulin heavy chain constant region sequence from any organism, and unless otherwise specified includes a heavy chain variable domain.
  • Heavy chain variable domains include three heavy chain CDRs and four FR regions, unless otherwise specified. Fragments of heavy chains include CDRs, CDRs and FRs, and combinations thereof.
  • a typical heavy chain has, following the variable domain (from N-terminal to C-terminal), a CHI domain, a hinge, a CH2 domain, and a CH3 domain.
  • a functional fragment of a heavy chain includes a fragment that is capable of specifically recognizing an antigen (e.g., recognizing the antigen with a KD in the micromolar, nanomolar, or picomolar range), that is capable of expressing and secreting from a cell, and that comprises at least one CDR.
  • an antigen e.g., recognizing the antigen with a KD in the micromolar, nanomolar, or picomolar range
  • the term “heavy chain only antibody,” “heavy chain only antigen binding protein,” “single domain antigen binding protein,” “single domain binding protein” or the like refers to a monomeric or homodimeric immunoglobulin molecule comprising an immunoglobulin-like chain comprising a variable domain operably linked to a heavy chain constant region, that is unable to associate with a light chain because the heavy chain constant region typically lacks a functional CHI domain.
  • the term “heavy chain only antibody,” “heavy chain only antigen binding protein,” “single domain antigen binding protein,” “single domain binding protein” or the like encompasses a both (i) a monomeric single domain antigen binding protein comprising one of the immunoglobulin-like chain comprising a variable domain operably linked to a heavy chain constant region lacking a functional CHI domain, or (ii) a homodimeric single domain antigen binding protein comprising two immunoglobulin-like chains, each of which comprising a variable domain operably linked to a heavy chain constant region lacking a functional CHI domain.
  • a homodimeric single domain antigen binding protein comprises two identical immunoglobulin-like chains, each of which comprising an identical variable domain operably linked to an identical heavy chain constant region lacking a functional CHI domain.
  • each immunoglobulin-like chain of a single domain antigen binding protein comprises a variable domain, which may be derived from heavy chain variable region gene segments (e.g., VH, DH, JH), light chain gene segments (e.g, VL, JL), or a combination thereof, linked to a heavy chain constant region (CH) gene sequence comprising a deletion or inactivating mutation in a CHI encoding sequence (and, optionally, a hinge region) of a heavy chain constant region gene, e.g., IgG, IgA, IgE, IgD, or a combination thereof.
  • CH heavy chain constant region
  • a single domain antigen binding protein comprising a variable domain derived from heavy chain gene segments may be referred to as a “VH- single domain antibody” or “Vn-single domain antigen binding protein”, see, e.g., U.S. Patent No. 8,754,287; U.S. Patent Publication Nos. 20140289876; 20150197553; 20150197554; 20150197555; 20150196015; 20150197556 and 20150197557, each of which is incorporated in its entirety by reference.
  • a single domain antigen binding protein comprising a variable domain derived from light chain gene segments may be referred to as a or “V -single domain antigen binding protein,” see, e.g., U.S. Publication No. 20150289489, incorporated in its entirety by reference.
  • light chain includes an immunoglobulin light chain constant region sequence from any organism, and unless otherwise specified includes human kappa and lambda light chains.
  • Light chain variable (VL) domains typically include three light chain CDRs and four framework (FR) regions, unless otherwise specified.
  • FR framework
  • a full-length light chain includes, from amino terminus to carboxyl terminus, a VL domain that includes FR1-CDR1- FR2-CDR2-FR3-CDR3-FR4, and a light chain constant domain.
  • Light chains that may be useful include e.g., those, that do not selectively bind either the first or second antigen selectively bound by the antigen-binding protein.
  • Suitable light chains include those that can be identified by screening for the most commonly employed light chains in existing antibody libraries (wet libraries or in silico), where the light chains do not substantially interfere with the affinity and/or selectivity of the antigen-binding domains of the antigen-binding proteins. Suitable light chains include those that can bind one or both epitopes that are bound by the antigen-binding regions of the antigen-binding protein.
  • variable domain includes an amino acid sequence of an immunoglobulin light or heavy chain (modified as desired) that comprises the following amino acid regions, in sequence from N-terminal to C-terminal (unless otherwise indicated): FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • a “variable domain” includes an amino acid sequence capable of folding into a canonical domain (VH or VL) having a dual beta sheet structure wherein the beta sheets are connected by a disulfide bond between a residue of a first beta sheet and a second beta sheet.
  • CDR complementarity determining region
  • a CDR includes an amino acid sequence encoded by a nucleic acid sequence of an organism’s immunoglobulin genes that normally (i.e., in a wildtype animal) appears between two framework regions in a variable region of a light or a heavy chain of an immunoglobulin molecule (e.g., an antibody or a T cell receptor).
  • a CDR can be encoded by, for example, a germline sequence or a rearranged or unrearranged sequence, and, for example, by a naive or a mature B cell or a T cell.
  • CDRs can be encoded by two or more sequences (e.g., germline sequences) that are not contiguous (e.g., in an unrearranged nucleic acid sequence) but are contiguous in a B cell nucleic acid sequence, e.g., as the result of splicing or connecting the sequences (e.g, V-D-J recombination to form a heavy chain CDR3).
  • sequences e.g., germline sequences
  • a B cell nucleic acid sequence e.g., as the result of splicing or connecting the sequences (e.g, V-D-J recombination to form a heavy chain CDR3).
  • Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs within the specified HCVR and/or LCVR amino acid sequences disclosed herein.
  • Examples of conventions that can be used to identify the boundaries of CDRs include, e.g., the Kabat definition, the Chothia definition, and the AbM definition.
  • the Kabat definition is based on sequence variability
  • the Chothia definition is based on the location of the structural loop regions
  • the AbM definition is a compromise between the Kabat and Chothia approaches.
  • antibody fragment refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen.
  • binding fragments encompassed within the term “antibody fragment” include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab’)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al.
  • antibody forms of single chain antibodies, such as diabodies are also encompassed under the term “antibody” (see e.g., Holliger el al. (1993) PNAS USA 90:6444-6448; Poljak et al. (1994) Structure 2: 1121-1123).
  • Fc-containing protein includes antibodies, bispecific antibodies, immunoadhesins, and other binding proteins that comprise at least a functional portion of an immunoglobulin CH2 and CH3 region.
  • a “functional portion” refers to a CH2 and CH3 region that can bind a Fc receptor (e.g., an FcyR; or an FcRn, i.e., a neonatal Fc receptor), and/or that can participate in the activation of complement. If the CH2 and CH3 region contains deletions, substitutions, and/or insertions or other modifications that render it unable to bind any Fc receptor and also unable to activate complement, the CH2 and CH3 region is not functional.
  • Fc-containing proteins can comprise modifications in immunoglobulin domains, including where the modifications affect one or more effector function of the binding protein (e.g, modifications that affect FcyR binding, FcRn binding and thus half-life, and/or CDC activity).
  • modifications include, but are not limited to, the following modifications and combinations thereof, with reference to EU numbering of an immunoglobulin constant region: 238, 239, 248, 249, 250, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280,
  • the binding protein is an Fc-containing protein and exhibits enhanced serum half-life (as compared with the same Fc-containing protein without the recited modification(s)) and have a modification at position 250 (e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L/Y/F/W or T), 254 (e.g, S or T), and 256 (e.g., S/R/Q/E/D or T); or a modification at 428 and/or 433 (e.g., L/R/SI/P/Q or K) and/or 434 (e.g, H/F or Y); or a modification at 250 and/or 428; or a modification at 307 or 308 (e.g., 308F, V308F), and 434.
  • 250 and 428 e.g., L or F
  • 252 e.g., L/Y/F/W or T
  • 254 e.g
  • the modification can comprise a 428L (e.g., M428L) and 434S (e.g., N434S) modification; a 428L, 2591 (e.g., V259I), and a 308F (e.g., V308F) modification; a 433K (e.g., H433K) and a 434 (e.g., 434Y) modification; a 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modification; a 250Q and 428L modification (e.g, T250Q and M428L); a 307 and/or 308 modification (e.g., 308F or 308P).
  • a 428L e.g., M428L
  • 434S e.g., N434S
  • a 428L, 2591 e.g., V259I
  • a 308F e.g., V308F
  • antigen-binding protein refers to a polypeptide or protein (one or more polypeptides complexed in a functional unit) that specifically recognizes an epitope on an antigen, such as a cell-specific antigen and/or a target antigen as described herein.
  • An antigen-binding protein may be multi-specific.
  • multi-specific with reference to an antigen-binding protein means that the protein recognizes different epitopes, either on the same antigen or on different antigens.
  • a multi-specific antigen-binding protein as described herein can be a single multifunctional polypeptide, or it can be a multimeric complex of two or more polypeptides that are covalently or non-covalently associated with one another.
  • the term “antigen-binding protein” includes antibodies or fragments thereof as described herein that may be linked to or co-expressed with another functional molecule, e.g, another peptide or protein.
  • an antibody or fragment thereof can be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent association or otherwise) to one or more other molecular entities, such as a protein or fragment thereof to produce a bispecific or a multi-specific antigenbinding molecule with a second binding specificity.
  • protein means any amino acid polymer having multiple amino acids covalently linked via amide bonds. Proteins contain one or more amino acid polymer chains, generally known in the art as “polypeptides”. Thus, a polypeptide may be a protein, and a protein may contain multiple polypeptides to form a single functioning biomolecule. Disulfide bridges (i.e., between cysteine residues to form cystine) may be present in some proteins. These covalent links may be within a single polypeptide chain, or between two individual polypeptide chains. For example, disulfide bridges are essential to proper structure and function of insulin, immunoglobulins, protamine, and the like. For a recent review of disulfide bond formation, see Oka and Bulleid, “Forming disulfides in the endoplasmic reticulum,” 1833(11) Biochim Biophys Acta 2425-9 (2013).
  • protein includes biotherapeutic proteins, recombinant proteins used in research or therapy, trap proteins and other Fc-fusion proteins, chimeric proteins, antibodies, monoclonal antibodies, human antibodies, bispecific antibodies, antibody fragments, nanobodies, recombinant antibody chimeras, scFv fusion proteins, cytokines, chemokines, peptide hormones, and the like. Proteins may be produced using recombinant cell-based production systems, such as the insect bacculovirus system, yeast systems (e.g., Pichia sp.), mammalian systems (e.g., CHO cells and CHO derivatives like CHO-K1 cells).
  • yeast systems e.g., Pichia sp.
  • mammalian systems e.g., CHO cells and CHO derivatives like CHO-K1 cells.
  • epitope refers to the portion of the antigen which is recognized by the multi-specific antigen-binding polypeptide.
  • a single antigen such as an antigenic polypeptide may have more than one epitope.
  • Epitopes may be defined as structural or functional. Functional epitopes are generally a subset of structural epitopes and are defined as those residues that directly contribute to the affinity of the interaction between the antigen- binding polypeptide and the antigen. Epitopes may also be conformational, that is, composed of non-linear amino acids.
  • epitopes may include determinants that are chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, in certain embodiments, may have specific three- dimensional structural characteristics, and/or specific charge characteristics. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents.
  • domain refers to any part of a protein or polypeptide having a particular function or structure.
  • domains as described herein bind to cell-specific or target antigens.
  • Cell-specific antigen- or target antigen-binding domains, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen.
  • half-body or “half-antibody”, which are used interchangeably, refers to half of an antibody, which essentially contains one heavy chain and one light chain. Antibody heavy chains can form dimers, thus the heavy chain of one half-body can associate with heavy chain associated with a different molecule (e.g., another half-body) or another Fc-containing polypeptide. Two slightly different Fc-domains may “heterodimerize” as in the formation of bispecific antibodies or other heterodimers, -trimers, -tetramers, and the like. See Vincent and Murini, “Current strategies in antibody engineering: Fc engineering and pH-dependent antigen binding, bispecific antibodies and antibody drug conjugates,” 7 Biotechnol. J. 1444-1450 (20912); and Shimamoto et al., “Peptibodies: A flexible alternative format to antibodies,” 4(5) MAbs 586-91 (2012).
  • single-chain variable fragment or “scFv” includes a single chain fusion polypeptide containing an immunoglobulin heavy chain variable region (VH) and an immunoglobulin light chain variable region (VL).
  • VH and VL are connected by a linker sequence of 10 to 25 amino acids.
  • ScFv polypeptides may also include other amino acid sequences, such as CL or CHI regions.
  • ScFv molecules can be manufactured by phage display or made by directly subcloning the heavy and light chains from a hybridoma or B-cell.
  • the term “muscle-related cancer” refers to any cancerous cell defined by the expression of myogenic genes e.g, expression of CDH15. “Muscle-related cancers” include, but are not limited to, rhabdomyosarcoma e.g., embryonal, alveolar, spindle cell/scl erosing, pleomorphic, etc.
  • target cells includes any cells in which expression of a nucleotide of interest is desired.
  • target cells exhibit a receptor on their surface that allows the cell to be targeted with a targeting ligand, as described below.
  • a target cell is a muscle cell.
  • a “muscle cell”, as used herein, refers to any cell expressing myogenic markers and/or involved in skeletal muscle regeneration, e.g., muscle stem cells (“MuSC”, also known as satellite cells) and which express Pax7 and thus are considered Pax7 + cells, e.g., Pax7 + /MyoD' when quiescent and Pax7 + /MyoD + when proliferating), myoblasts (e.g., MyoD + Myogenin + cells), myocytes, myotubes (also known as myofibers, e.g., MyHC + cells), etc.
  • Muscle cell refers to any cell expressing myogenic markers and/or involved in skeletal muscle regeneration, e.g., muscle stem cells (“MuSC”, also known as satellite cells) and which express Pax7 and thus are considered Pax7 + cells, e.g., Pax7 + /MyoD' when quiescent and Pax7 + /MyoD + when proliferating), myoblasts
  • “Retargeting” or “redirecting” may include a scenario in which the wildtype particle targets several cells within a tissue and/or several organs within an organism, and general targeting of the tissue or organs is reduced or abolished by insertion of the heterologous amino acid, and retargeting to more a specific cell in the tissue or a specific organ in the organism is achieved with the targeting ligand (e.g., via a targeting ligand) that binds a marker expressed by the specific cell.
  • the targeting ligand e.g., via a targeting ligand
  • Such retargeting or redirecting may also include a scenario in which the wildtype particle targets a tissue, and targeting of the tissue is reduced to or abolished by insertion of the heterologous amino acid, and retargeting to a completely different tissue is achieved with the targeting ligand.
  • “Specific binding pair,” “binding pair,” “protein:protein binding pair” and the like includes two members (e.g., a first member (e.g., a first polypeptide) and a second cognate member (e.g., a second polypeptide)) that interact to form a bond (e.g., a non-covalent bond between a first member epitope and a second member antigen-binding portion of an antibody that recognizes the epitope; a covalent bond between e.g., proteins capable of forming isopeptide bonds; split inteins that recognize each other and, through the process of protein trans-splicing, mediate ligation of the flanking proteins and their own removal).
  • a bond e.g., a non-covalent bond between a first member epitope and a second member antigen-binding portion of an antibody that recognizes the epitope
  • a covalent bond between e.g., proteins capable of forming isopeptide bonds e.g
  • cognate refers to components that function together.
  • Epitopes and cognate antibodies thereto, particularly epitopes that may also act as a detectable label (e.g., c-myc) are well-known in the art.
  • Specific proteimprotein binding pairs capable of interacting to form a covalent isopeptide bond are reviewed in Veggiani et al. (2014) Trends Biotechnol.
  • a first member of a protein: protein binding pair refers to member of a protein: protein binding pair, which is generally less than 30 amino acids in length, and which forms a spontaneous covalent isopeptide bond with the second cognate protein, wherein the second cognate protein is generally larger, but may also be less than 30 amino acids in length such as in the SpyTag:KTag system.
  • isopeptide bond refers to an amide bond between a carboxyl or carboxamide group and an amino group at least one of which is not derived from a protein main chain or alternatively viewed is not part of the protein backbone.
  • An isopeptide bond may form within a single protein or may occur between two peptides or a peptide and a protein.
  • an isopeptide bond may form intramolecularly within a single protein or intermolecularly i.e. between two peptide/protein molecules, e.g. between two peptide linkers.
  • an isopeptide bond may occur between a lysine residue and an asparagine, aspartic acid, glutamine, or glutamic acid residue or the terminal carboxyl group of the protein or peptide chain or may occur between the alpha-amino terminus of the protein or peptide chain and an asparagine, aspartic acid, glutamine or glutamic acid.
  • Each residue of the pair involved in the isopeptide bond is referred to herein as a reactive residue.
  • an isopeptide bond may form between a lysine residue and an asparagine residue or between a lysine residue and an aspartic acid residue.
  • isopeptide bonds can occur between the side chain amine of lysine and carboxamide group of asparagine or carboxyl group of an aspartate.
  • the SpyTag: SpyCatcher system is described in U.S. Patent No. 9,547,003 and Zaveri et al. (2012) PNAS 109:E690-E697, each of which is incorporated herein in its entirety by reference, and is derived from the CnaB2 domain of the Streptococcus pyogenes fibronectin- binding protein FbaB.
  • Zakeri et al. obtained a peptide “SpyTag” having the sequence AHIVMVDAYKPTK (SEQ ID NO: 815) which forms an amide bond to its cognate protein “SpyCatcher,” an 112 amino acid polypeptide having the amino acid sequence set forth in SEQ ID NO: 816.
  • SpyTag:KTag An additional specific binding pair derived from CnaB2 domain is SpyTag:KTag, which forms an isopeptide bond in the presence of SpyLigase.
  • SpyLigase was engineered by excising the P strand from SpyCatcher that contains a reactive lysine, resulting in KTag, a 10-residue first member of a protein: protein binding pair having the amino acid sequence ATHIKFSKRD (SEQ ID NO: 817).
  • SpyTag002 has the amino acid sequence VPTIVMVDAYKRYK, set forth as SEQ ID NO: 821, and binds SpyCatcher002.
  • SpyTag003 has the amino acid sequence RGVPHIVMVDAYKRYK, set forth as SEQ ID NO: 822, and binds SpyCatcher003.
  • SnoopTag SnoopCatcher system is described in Veggiani (2016) PNAS 113: 1202-07.
  • the D4 Ig-like domain of RrgA an adhesion from Streptococcus pneumoniae, was split to form SnoopTag (residues 734-745) and SnoopCatcher (residues 749-860).
  • the isopeptag:pilin-C specific binding pair was derived from the major pilin protein Spy0128 from Streptococcus pyogenes. (Zakeir and Howarth (2010) J. Am. Chem. Soc. 132:4526-27). Isopeptag has the amino acid sequence TDKDMTITFTNKKDAE, set forth as SEQ ID NO: 820, and binds pilin-C (residues 18-299 of SpyO 128). Incubation of isopeptag and pilin-C results in a spontaneous isopeptide bond that is specific between the complementary proteins. Zakeir and Howarth (2010), supra.
  • transduction or “infection” or the like refers to the introduction of a nucleic acid into a target cell e.g., a muscle stem cell, myoblast, myocyte, any combination thereof, etc.) nucleus by a viral particle.
  • efficiency in relation to transduction or the like e.g., “transduction efficiency” refers to the fraction e.g., percentage) of cells expressing a nucleotide of interest after incubation with a set number of viral particles comprising the nucleotide of interest.
  • Well-known methods of determining transduction efficiency include flow cytometry of cells transduced with a fluorescent reporter gene, RT-PCR for expression of the nucleotide of interest, etc.
  • “reference” viral capsid protein/capsid/particle are identical to test viral capsid protein/capsid/particle but for the change for which the effect is to be tested. For example, to determine the effect, e.g., on transduction efficiency, of inserting a first member of a specific binding pair into a test viral particle, the transduction efficiencies of the test viral particle (in the absence or presence of an appropriate targeting ligand) can be compared to the transduction efficiencies of a reference viral particle (in the absence or presence of an appropriate targeting ligand if necessary) which is identical to the test viral particle in every instance (e.g., additional point mutations, nucleotide of interest, numbers of viral particles and target cells, etc.) except for the presence of a first member of a specific binding pair.
  • a reference viral capsid protein is one that is able to form a capsid with a second viral capsid protein modified to comprise at least a first member of a proteimprotein binding pair, where the reference viral capsid protein does not comprise the first member of a protein: protein binding pair, preferably wherein the capsid formed by the reference viral capsid protein and the modified viral capsid protein is a mosaic capsid.
  • antigen-binding proteins e.g., antibodies, or antigen-binding fragments thereof, comprising an amino acid of an HCVR, HCDR1, HCDR2, HCDR3, LCVR, LCV1, LCVR2, and/or LCVR3 as set forth in Table 1.
  • an antigen-binding protein e.g., an antibody, or an antigen-binding fragment thereof, as described herein comprises a heavy chain CDR1 (HCDR1) comprising an amino acid sequence selected from any of the HCDR1 amino acid sequences listed in Table 1 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.
  • HCDR1 heavy chain CDR1
  • an antigen-binding protein e.g., an antibody, or an antigen-binding fragment thereof, as described herein comprises a heavy chain CDR2 (HCDR2) comprising an amino acid sequence selected from any of the HCDR2 amino acid sequences listed in Table 1 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.
  • HCDR2 heavy chain CDR2
  • an antigen-binding protein e.g., an antibody, or an antigen-binding fragment thereof, as described herein comprises a heavy chain CDR3 (HCDR3) comprising an amino acid sequence selected from any of the HCDR3 amino acid sequences listed in Table 1 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.
  • HCDR3 heavy chain CDR3
  • an antigen-binding protein e.g., an antibody, or an antigen-binding fragment thereof, as described herein comprises a light chain CDR1 (LCDR1) comprising an amino acid sequence selected from any of the LCDR1 amino acid sequences listed in Table 1 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.
  • LCDR1 light chain CDR1
  • an antigen-binding protein e.g., an antibody, or an antigen-binding fragment thereof, as described herein comprises a light chain CDR2 (LCDR2) comprising an amino acid sequence selected from any of the LCDR2 amino acid sequences listed in Table 1 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.
  • LCDR2 light chain CDR2
  • an antigen-binding protein e.g., an antibody, or an antigen-binding fragment thereof, as described herein comprises a light chain CDR3 (LCDR3) comprising an amino acid sequence selected from any of the LCDR3 amino acid sequences listed in Table lor a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.
  • LCDR3 light chain CDR3
  • an antibody, or an antigen-binding fragment thereof, as described herein comprises an HCDR3 and an LCDR3 amino acid sequence pair (HCDR3/LCDR3) comprising any of the HCDR3 amino acid sequences listed in Table 1 paired with any of the LCDR3 amino acid sequences listed in Table 1.
  • an antibody, or antigen-binding fragments thereof, as described herein comprises an HCDR3/LCDR3 amino acid sequence pair contained within any of the example anti-hCDH15 antibodies listed in Table 1.
  • the HCDR3/LCDR3 amino acid sequence pair is selected from the group consisting of SEQ ID NOs: 8 and 16, 28 and 36, 48 and 54, 66 and 54, 76 and 54, 86 and 54, 96 and 102, 113 and 119, 131 and 139, 151 and 159, 171 and 179, 191 and 196, 208 and 214, 226 and 54, 236 and 54, 246 and 54, 255 and 54, 265 and 273, 285 and 293, 305 and 313, 325 and 333, 345 and 352, 364 and 372, 382 and 54, 392 and 398, 410 and 414, 426 and 434, 442 and 450, 458 and 466, 474 and 482, 490 and 498, 506 and 514, 522 and 530, 538 and 546, 554 and 562, 570 and 578, 586 and 594, 602 and 610, 618 and 626, 634 and 642, 650 and 658, 6
  • antigen-binding proteins e.g., an antibody, or an antigenbinding fragment thereof, comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1- LCDR2-LCDR3) contained within any of the example anti-hCDH15 antibodies listed in Table 1.
  • the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences set is selected from the group consisting of SEQ ID NOs: 4-6-8-12-14-16, 24-26-28- 32-34-36, 44-46-48-52-34-54, 62-64-66-52-34-54, 72-74-76-52-34-54, 82-84-86-52-34-54, 92- 94-96-100-34-102, 82-111-113-117-34-119, 127-129-131-135-137-139, 147-149-151-155-157- 159, 167-169-171-175-177-179, 187-189-191-52-34-196, 204-206-208-212-137-214, 222-224- 226-52-34-54, 232-234-236-52-34-54, 242-244-246-52-34-54, 82-253-255-52-34-54, 261-263- 265-269-271-273, 281-283-
  • antibodies, or antigen-binding fragments thereof comprising a set of six CDRs (z.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within an HCVR/LCVR amino acid sequence pair as defined by any of the example anti-hCDH15 antibodies listed in Table 1.
  • an antibody, or antigenbinding fragments thereof, as described herein comprises the HCDR1-HCDR2-HCDR3-LCDR1- LCDR2-LCDR3 amino acid sequences set contained within an HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 and 10, 22 and 30, 42 and 50, 60 and 50, 70 and 50, 80 and 50, 90 and 98, 108 and 115, 125 and 133, 145 and 153, 165 and 173, 185 and 193, 202 and 210, 220 and 50, 230 and 50, 240 and 50, 250 and 50, 259 and 267, 279 and 287, 299 and 307, 319 and 327, 339 and 347, 358 and 366, 378 and 50, 386 and 394,
  • the antigen-binding protein comprises a heavy chain variable region (HCVR or VH).
  • the HCVR comprises a set of HCDR1- HCDR2-HCDR3 amino acid sequences selected from Table 1.
  • the HCVR comprises an amino acid sequence selected from any of the HCVR amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.
  • the antigen-binding protein comprises a light chain variable region (LCVR or VL).
  • the LCVR comprises a set of LCDR1- LCDR2-LCDR3 amino acid sequences selected from Table 1 below.
  • the LCVR comprises an amino acid sequence selected from any of the LCVR amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.
  • the antigen-binding protein e.g, antibody or antigenbinding fragment thereof, comprises an HCVR and an LCVR amino acid sequence pair (HCVR/LCVR) comprising any of the HCVR amino acid sequences listed in Table 1 paired with any of the LCVR amino acid sequences listed in Table 1.
  • the antigenbinding protein e.g., antibody or antigen-binding fragment thereof, as described herein comprises an HCVR/LCVR amino acid sequence pair contained within any of the example anti- hCDH15 antibodies listed in Table 1.
  • the HCVR/LCVR amino acid sequence pair is selected from the group consisting of SEQ ID NOs: 2 and 10, 22 and 30, 42 and 50, 60 and 50, 70 and 50, 80 and 50, 90 and 98, 108 and 115, 125 and 133, 145 and 153, 165 and 173, 185 and 193, 202 and 210, 220 and 50, 230 and 50, 240 and 50, 250 and 50, 259 and 267, 279 and 287, 299 and 307, 319 and 327, 339 and 347, 358 and 366, 378 and 50, 386 and 394,
  • nucleic acid molecules e.g., polynucleotides, encoding the antigen-binding proteins, e.g., antibodies or antigen-binding fragments thereof, as described herein.
  • nucleic acid molecules encoding any of the HCDR1 amino acid sequences listed in Table 1; in some embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR1 nucleic acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.
  • nucleic acid molecules encoding any of the HCDR2 amino acid sequences listed in Table 1; in some embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR2 nucleic acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.
  • nucleic acid molecules encoding any of the HCDR3 amino acid sequences listed in Table 1; in some embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR3 nucleic acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.
  • nucleic acid molecules encoding any of the LCDR1 amino acid sequences listed in Table 1; in some embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR1 nucleic acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.
  • nucleic acid molecules encoding any of the LCDR2 amino acid sequences listed in Table 1; in some embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR2 nucleic acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.
  • nucleic acid molecules encoding any of the LCDR3 amino acid sequences listed in Table 1; in some embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR3 nucleic acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto. Also described herein are nucleic acid molecules encoding both an HCVR and an LCVR, wherein the HCVR comprises an amino acid sequence of any of the HCVR amino acid sequences listed in Table 1, and wherein the LCVR comprises an amino acid sequence of any of the LCVR amino acid sequences listed in Table 1.
  • the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto, and a polynucleotide sequence selected from any of the LCVR nucleic acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.
  • the nucleic acid molecule encodes an HCVR and LCVR, wherein the HCVR and LCVR are both derived from the same anti-hCDH15 antibody listed in Table 1.
  • a recombinant expression vector capable of expressing a polypeptide comprising a heavy and/or light chain variable region of an anti-hCDH15 antibody.
  • a recombinant expression vector comprises any of the nucleic acid molecules mentioned above, i.e., nucleic acid molecules encoding any of the HCVR, LCVR, and/or CDR sequences as set forth in Table 1.
  • host cells into which such vectors have been introduced as well as methods of producing the antibodies or portions thereof by culturing the host cells under conditions permitting production of the antibodies or antibody fragments, and recovering the antibodies or antibody fragments so produced.
  • An anti-hCDH15 antibody and antigen-binding fragment thereof as described herein may be monospecific, bi-specific, or multispecific. Multispecific antibodies may be specific for different epitopes of one target polypeptide or may contain antigen-binding domains specific for more than one target polypeptide. See, e.g., Tutt et al., 1991, J. Immunol. 147:60-69; Kufer etal., 2004, Trends Biotechnol. 22:238-244.
  • An anti-hCDH15 antibody and antigenbinding fragment thereof as described herein can be linked to or co-expressed with another functional molecule, e.g, another peptide or protein.
  • an antibody or fragment thereof can be functionally linked e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment to produce a bi-specific or a multispecific antibody with a second or additional binding specificity.
  • Use of the expression “anti-hCDH15 antibody” herein is intended to include both monospecific anti-hCDH15 antibodies as well as bispecific antibodies comprising a CDH15- binding arm and a “target’ ’-binding arm.
  • the CDH15-binding arm can comprise any of the HCVR/LCVR or CDR amino acid sequences as set forth in Table 1 herein.
  • the CDH15-binding arm binds to human CDH15 and induces internalization of the CDH15 and antibody bound thereto. In certain embodiments, the CDH15-binding arm binds weakly to human CDH15 and induces internalization of CDH15 and antibody bound thereto. In certain embodiments, the CDH15-binding arm binds to human CDH15 and blocks the activity of CDH15.
  • the bispecific antigen-binding molecule is a bispecific antibody.
  • Each antigen-binding domain of a bispecific antibody comprises a heavy chain variable domain (HCVR) and a light chain variable domain (LCVR).
  • HCVR heavy chain variable domain
  • LCVR light chain variable domain
  • the CDRs of the first antigen-binding domain may be designated with the prefix “Al” and the CDRs of the second antigen-binding domain may be designated with the prefix “A2”.
  • the CDRs of the first antigen-binding domain may be referred to herein as A1-HCDR1, A1-HCDR2, and A1-HCDR3; and the CDRs of the second antigenbinding domain may be referred to herein as A2-HCDR1, A2-HCDR2, and A2-HCDR3.
  • the first antigen-binding domain and the second antigen-binding domain may be directly or indirectly connected to one another to form a bispecific antigen-binding molecule as described herein.
  • the first antigen-binding domain and the second antigen-binding domain may each be connected to a separate multimerizing domain.
  • the association of one multimerizing domain with another multimerizing domain facilitates the association between the two antigen-binding domains, thereby forming a bispecific antigen-binding molecule.
  • a “multimerizing domain” is any macromolecule, protein, polypeptide, peptide, or amino acid that has the ability to associate with a second multimerizing domain of the same or similar structure or constitution.
  • a multimerizing domain may be a polypeptide comprising an immunoglobulin CH3 domain.
  • a non-limiting example of a multimerizing component is an Fc portion of an immunoglobulin (comprising a CH2-CH3 domain), e.g., an Fc domain of an IgG selected from the isotypes IgGl, IgG2, IgG3, and IgG4, as well as any allotype within each isotype group.
  • Bispecific antigen-binding molecules as described herein will typically comprise two multimerizing domains, e.g, two Fc domains that are each individually part of a separate antibody heavy chain.
  • the first and second multimerizing domains may be of the same IgG isotype such as, e.g., IgGl/IgGl, IgG2/IgG2, IgG4/IgG4.
  • the first and second multimerizing domains may be of different IgG isotypes such as, e.g., IgGl/IgG2, IgGl/IgG4, IgG2/IgG4, etc.
  • the multimerizing domain is an Fc fragment or an amino acid sequence of from 1 to about 200 amino acids in length containing at least one cysteine residue. In other embodiments, the multimerizing domain is a cysteine residue, or a short cysteine-containing peptide.
  • Other multimerizing domains include peptides or polypeptides comprising or consisting of a leucine zipper, a helix-loop motif, or a coiled-coil motif.
  • any bispecific antibody format or technology may be used to make the bispecific antigen-binding molecules as described herein.
  • an antibody or fragment thereof having a first antigen binding specificity can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment having a second antigen-binding specificity to produce a bispecific antigen-binding molecule.
  • bispecific formats include, without limitation, e.g., scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, Quadroma, knobs-into-holes, common light chain (e.g., common light chain with knobs-into-holes, etc.), CrossMab, CrossFab, (SEED)body, leucine zipper, Duobody, IgGl/IgG2, dual acting Fab (DAF)-IgG, and Mab 2 bispecific formats (see, e.g., Klein et al. 2012, mAbs 4:6, 1-11, and references cited therein, for a review of the foregoing formats).
  • the multimerizing domains e.g, Fc domains
  • the multimerizing domains may comprise one or more amino acid changes (e.g., insertions, deletions or substitutions) as compared to the wild-type, naturally occurring version of the Fc domain.
  • bispecific antigen-binding molecules may comprise one or more modifications in the Fc domain that results in a modified Fc domain having a modified binding interaction (e.g., enhanced or diminished) between Fc and FcRn.
  • the bispecific antigen-binding molecule comprises a modification in a CH2 or a CH3 region, wherein the modification increases the affinity of the Fc domain to FcRn in an acidic environment (e.g., in an endosome where pH ranges from about 5.5 to about 6.0).
  • Non-limiting examples of such Fc modifications include, e.g., a modification at position 250 e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L/Y/F/W or T), 254 (e.g., S or T), and 256 (e.g, S/R/Q/E/D or T); or a modification at position 428 and/or 433 (e.g., L/R/S/P/Q or K) and/or 434 (e.g., H/F or Y); or a modification at position 250 and/or 428; or a modification at position 307 or 308 (e.g., 308F, V308F), and 434.
  • a modification at position 250 e.g., E or Q e.g., E or Q
  • 250 and 428 e.g., L or F
  • 252 e.g., L/Y/F/W or T
  • 254
  • the modification comprises a 428L (e.g., M428L) and 434S (e.g., N434S) modification; a 428L, 2591 (e.g., V259I), and 308F (e.g., V308F) modification; a 433K (e.g., H433K) and a 434 (e.g, 434Y) modification; a 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modification; a 250Q and 428L modification (e.g., T250Q and M428L); and a 307 and/or 308 modification (e.g., 308F or 308P).
  • a 428L e.g., M428L
  • 434S e.g., N434S
  • 428L, 2591 e.g., V259I
  • 308F e.g., V308F
  • 433K
  • bispecific antigen-binding molecules comprising a first CH3 domain and a second Ig CH3 domain, wherein the first and second Ig CH3 domains differ from one another by at least one amino acid, and wherein at least one amino acid difference reduces binding of the bi specific antibody to Protein A as compared to a bi-specific antibody lacking the amino acid difference.
  • the first Ig CH3 domain binds Protein A and the second Ig CH3 domain contains a mutation that reduces or abolishes Protein A binding such as an H95R modification (by IMGT exon numbering; H435R by EU numbering).
  • the second CH3 may further comprise a Y96F modification (by IMGT; Y436F by EU).
  • the Fc domain may be chimeric, combining Fc sequences derived from more than one immunoglobulin isotype.
  • a chimeric Fc domain can comprise part or all of a CH2 sequence derived from a human IgGl, human IgG2 or human IgG4 CH2 region, and part or all of a CH3 sequence derived from a human IgGl, human IgG2 or human IgG4.
  • a chimeric Fc domain can also contain a chimeric hinge region.
  • a chimeric hinge may comprise an “upper hinge” sequence, derived from a human IgGl, a human IgG2 or a human IgG4 hinge region, combined with a “lower hinge” sequence, derived from a human IgGl, a human IgG2 or a human IgG4 hinge region.
  • a particular example of a chimeric Fc domain that can be included in any of the antigen-binding molecules set forth herein comprises, from N- to C-terminus: [IgG4 CHI] - [IgG4 upper hinge] - [IgG2 lower hinge] - [IgG4 CH2] - [IgG4 CH3],
  • Another example of a chimeric Fc domain that can be included in any of the antigen-binding molecules set forth herein comprises, from N- to C-terminus: [IgGl CHI] - [IgGl upper hinge] - [IgG2 lower hinge] - [IgG4 CH2] - [IgGl CH3]
  • These and other examples of chimeric Fc domains that can be included in any of the antigen -binding molecules as described herein are described in US Publication 2014/0243504, published August 28, 2014, which is herein incorporated in its entirety. Chimeric Fc domains having these general structural arrangements, and variants
  • an antibody heavy chain as described herein comprises a heavy chain constant (CH) region that comprises an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to any one of SEQ ID NOs: 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, or 802.
  • the heavy chain constant region (CH) region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, or 802.
  • an antibody heavy chain as described herein comprises an Fc domain that comprises an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to any one of SEQ ID NOs: 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, or 814.
  • the Fc domain comprises an amino acid sequence selected form the group consisting SEQ ID NOs: 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, or 814.
  • the anti-hCDH15 antibodies disclosed herein may comprise one or more amino acid substitutions, insertions and/or deletions in the framework and/or CDR regions of the heavy chain variable domains as compared to the corresponding germline sequences from which the antibodies were derived.
  • An anti-hCDH15 antibody and antigen-binding fragment thereof as disclosed herein may be derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework and/or CDR regions are mutated to the corresponding residue(s) of the germline sequence from which the antibody was derived, or to the corresponding residue(s) of another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are referred to herein collectively as “germline mutations”), and having weak or no detectable binding to a CDH15 antigen.
  • Germline mutations such sequence changes are referred to herein collectively as “germline mutations”
  • an anti-hCDH15 antibody and antigen-binding fragment thereof as disclosed herein may contain any combination of two or more germline mutations within the framework and/or CDR regions, e.g., wherein certain individual residues are mutated to the corresponding residue of a particular germline sequence while certain other residues that differ from the original germline sequence are maintained or are mutated to the corresponding residue of a different germline sequence.
  • an antibody or antigen-binding fragment that contains one or more germline mutations can be tested for one or more desired properties such as, improved binding specificity, weak or reduced binding affinity, improved or enhanced pharmacokinetic properties, reduced immunogenicity, etc.
  • an antibody or antigen-binding fragment as described herein is obtained in this general manner.
  • anti-hCDH15 antibodies and antigen-binding fragments thereof comprising variants of any of the HCVR, LCVR, and/or CDR amino acid sequences disclosed herein having one or more conservative substitutions.
  • an anti-hCDH15 antibody or antigen-binding fragment thereof as described herein may comprise HCVR, LCVR, and/or CDR amino acid sequences with, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc. conservative amino acid substitutions relative to any of the HCVR, LCVR, and/or CDR amino acid sequences set forth in Table 1 herein.
  • An antibody and antigen-binding fragment thereof as described herein may comprise one or more amino acid substitutions, insertions and/or deletions in the framework and/or CDR regions of the heavy and light chain variable domains as compared to the corresponding germline sequences from which the individual antigen-binding domains were derived, while maintaining or improving the desired weak-to-no detectable binding to, e.g., CDH15.
  • a “conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g, charge or hydrophobicity).
  • a conservative amino acid substitution will not substantially change the functional properties of a protein, i.e., the amino acid substitution maintains or improves the desired weak to no detectable binding affinity in the case of anti-hCDH15 binding molecules.
  • groups of amino acids that have side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide- containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartate and glutamate, and (7) sulfur-containing side chains are cysteine and methionine.
  • Preferred conservative amino acids substitution groups are: valine-leucine- isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine.
  • a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 1443- 1445.
  • a “moderately conservative” replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix.
  • anti-hCDH15 antibodies and antigen-binding fragments thereof comprising an antigen-binding domain with an HCVR and/or CDR amino acid sequence that is substantially identical to any of the HCVR and/or CDR amino acid sequences disclosed herein, while maintaining or improving the desired weak affinity to CDH15 antigen.
  • substantially identical when referring to an amino acid sequence means that two amino acid sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 95% sequence identity, even more preferably at least 98% or 99% sequence identity.
  • residue positions which are not identical differ by conservative amino acid substitutions.
  • the percent sequence identity or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well-known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24: 307-331.
  • Sequence similarity for polypeptides is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions.
  • GCG software contains programs such as Gap and Bestfit which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutein thereof. See, e.g, GCG Version 6.1. Polypeptide sequences also can be compared using FASTA using default or recommended parameters, a program in GCG Version 6.1.
  • FASTA e.g., FASTA2 and FASTA3
  • FASTA2 and FASTA3 provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson (2000) supra).
  • Another preferred algorithm when comparing a sequence as described herein to a database containing a large number of sequences from different organisms is the computer program BLAST, especially BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402.
  • antigen-binding domains that contain one or more germline mutations were tested for decreased binding affinity utilizing one or more in vitro assays.
  • antibodies that recognize a particular antigen are typically screened for their purpose by testing for high (i.e. strong) binding affinity to the antigen.
  • binding in the context of the binding of an antibody, immunoglobulin, antibody-binding fragment, or Fc-containing protein to either, e.g., a predetermined antigen, such as a cell surface protein or fragment thereof, typically refers to an interaction or association between a minimum of two entities or molecular structures, such as an antibody-antigen interaction.
  • binding affinity typically corresponds to a KD value of about 10' 7 M or less, such as about 10' 8 M or less, such as about 10' 9 M or less when determined by, for instance, surface plasmon resonance (SPR) technology in a BIAcore 3000 instrument using the antigen as the ligand and the antibody, Ig, antibody -binding fragment, or Fc-containing protein as the analyte (or antiligand).
  • SPR surface plasmon resonance
  • FACS fluorescent-activated cell sorting
  • an anti-hCDH15 antibody and antigen-binding fragment thereof as described herein bind to the predetermined antigen or cell surface molecule (receptor) having an affinity corresponding to a KD value that is at least ten-fold lower than its affinity for binding to a non-specific antigen (e.g., BSA, casein).
  • a non-specific antigen e.g., BSA, casein.
  • the affinity of an antibody corresponding to a KD value that is equal to or less than ten-fold lower than a non-specific antigen may be considered non- detectable binding, however such an antibody may be paired with a second antigen binding arm for the production of a bispecific antibody as described herein.
  • KD KD in molar
  • M the dissociation equilibrium constant of a particular antibody-antigen interaction, or the dissociation equilibrium constant of an antibody or antibody-binding fragment binding to an antigen.
  • KD KD
  • binding affinity There is an inverse relationship between KD and binding affinity, therefore the smaller the KD value, the higher, i.e. stronger, the affinity.
  • higher affinity or “stronger affinity” relate to a higher ability to form an interaction and therefore a smaller KD value
  • lower affinity or “weaker affinity” relate to a lower ability to form an interaction and therefore a larger KD value.
  • a higher binding affinity (or KD) of a particular molecule (e.g. antibody) to its interactive partner molecule (e.g. antigen X) compared to the binding affinity of the molecule (e.g. antibody) to another interactive partner molecule (e.g. antigen Y) may be expressed as a binding ratio determined by dividing the larger KD value (lower, or weaker, affinity) by the smaller KD (higher, or stronger, affinity), for example expressed as 5-fold or 10- fold greater binding affinity, as the case may be.
  • kd (sec -1 or 1/s) refers to the dissociation rate constant of a particular antibody-antigen interaction, or the dissociation rate constant of an antibody or antibody -binding fragment. Said value is also referred to as the k o ff value.
  • k a (M-l x sec-1 or 1/M) refers to the association rate constant of a particular antibody-antigen interaction, or the association rate constant of an antibody or antibody-binding fragment.
  • KA (M-l or 1/M) refers to the association equilibrium constant of a particular antibody-antigen interaction, or the association equilibrium constant of an antibody or antibody-binding fragment. The association equilibrium constant is obtained by dividing the k a by the ka.
  • EC50 refers to the half maximal effective concentration, which includes the concentration of an antibody which induces a response halfway between the baseline and maximum after a specified exposure time.
  • the EC50 essentially represents the concentration of an antibody where 50% of its maximal effect is observed.
  • the EC50 value equals the concentration of an antibody as described herein that gives half-maximal binding to cells expressing CDH15, as determined by e.g. a FACS binding assay or an androgen receptor activation luciferase assay. Thus, reduced or weaker binding is observed with an increased EC50, or half maximal effective concentration value.
  • decreased binding can be defined as an increased EC50 antibody concentration which may result in binding to the half-maximal amount of target cells.
  • an antigen-binding protein e.g., an antibody or antigenbinding fragment thereof, that binds CDH15-expressing cells with a single digit nM or triple digit pM KD, as measured by surface plasmon resonance, or equivalent assay.
  • an antigen-binding protein e.g., antibody or antigen-binding fragment thereof, that binds CDH15-expressing cells with an EC50 of greater than 100 nM as measured by FACS analysis.
  • an antigen-binding protein e.g., antibody or antigen-binding fragment thereof, that binds and is internalized into CDH15-expressing cells upon binding to CDH15.
  • an antigen-binding protein e.g., antibody or antigen-binding fragment thereof, that binds to and blocks the activity of CDH15.
  • An anti-hCACNGl antibody and antigen-binding fragment as described herein may comprise one or more amino acid substitutions, insertions and/or deletions in the framework and/or CDR regions of the heavy and light chain variable domains as compared to the corresponding germline sequences from which the individual antigen-binding domains were derived.
  • Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available from, for example, public antibody sequence databases.
  • the antigen-binding molecules as described herein may comprise antigen-binding domains which are derived from any of the example amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework and/or CDR regions are mutated to the corresponding residue(s) of the germline sequence from which the antibody was derived, or to the corresponding residue(s) of another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are referred to herein collectively as “germline mutations”).
  • germline mutations such sequence changes are referred to herein collectively as “germline mutations”.
  • all of the framework and/or CDR residues within the VH and/or VL domains are mutated back to the residues found in the original germline sequence from which the antigen-binding domain was originally derived.
  • only certain residues are mutated back to the original germline sequence, e.g., only the mutated residues found within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only the mutated residues found within CDR1, CDR2 or CDR3.
  • one or more of the framework and/or CDR residue(s) are mutated to the corresponding residue(s) of a different germline sequence (z.e., a germline sequence that is different from the germline sequence from which the antigen-binding domain was originally derived).
  • the antigen-binding domains may contain any combination of two or more germline mutations within the framework and/or CDR regions, e.g., wherein certain individual residues are mutated to the corresponding residue of a particular germline sequence while certain other residues that differ from the original germline sequence are maintained or are mutated to the corresponding residue of a different germline sequence.
  • antigenbinding domains that contain one or more germline mutations can be easily tested for one or more desired property such as, improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc.
  • desired property such as, improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc.
  • antigen-binding molecules comprising one or more antigen-binding domains obtained in this general manner.
  • antigen-binding molecules wherein one or both antigenbinding domains comprise variants of any of the HCVR, LCVR, and/or CDR amino acid sequences disclosed herein having one or more conservative substitutions.
  • antigenbinding molecules as described herein may comprise an antigen-binding domain having HCVR, LCVR, and/or CDR amino acid sequences with, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc. conservative amino acid substitutions relative to any of the HCVR, LCVR, and/or CDR amino acid sequences disclosed herein.
  • a “conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein.
  • Examples of groups of amino acids that have side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartate and glutamate, and (7) sulfur-containing side chains are cysteine and methionine.
  • Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine.
  • a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 1443-1445, herein incorporated by reference.
  • a “moderately conservative” replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix.
  • Antigen-binding molecules as described herein may comprise an antigen-binding domain with an HCVR, LCVR, and/or CDR amino acid sequence that is substantially identical to any of the HCVR, LCVR, and/or CDR amino acid sequences disclosed herein.
  • the term “substantial identity” or “substantially identical,” when referring to an amino acid sequence means that two amino acid sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 95% sequence identity, even more preferably at least 98% or 99% sequence identity.
  • residue positions which are not identical differ by conservative amino acid substitutions.
  • sequence identity or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well-known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24: 307-331, herein incorporated by reference.
  • Sequence similarity for polypeptides which is also referred to as sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions.
  • GCG software contains programs such as Gap and Bestfit which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutein thereof. See, e.g, GCG Version 6.1. Polypeptide sequences also can be compared using FASTA using default or recommended parameters, a program in GCG Version 6.1. FASTA (e.g, FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson (2000) supra).
  • BLAST Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402, each herein incorporated by reference. pH-Dependent Binding
  • anti-hCDH15 antibodies and antigen-binding fragments thereof with pH-dependent binding characteristics may exhibit reduced binding to CDH15 at acidic pH as compared to neutral pH.
  • anti-hCDH15 antibodies as described herein may exhibit enhanced binding to CDH15 at acidic pH as compared to neutral pH.
  • the expression “acidic pH” includes pH values less than about 6.2, e.g., about 6.0, 5.95, 5,9, 5.85, 5.8, 5.75, 5.7, 5.65, 5.6, 5.55, 5.5, 5.45, 5.4, 5.35, 5.3, 5.25, 5.2, 5.15, 5.1, 5.05, 5.0, or less.
  • neutral pH means a pH of about 7.0 to about 7.4.
  • neutral pH includes pH values of about 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35, and 7.4.
  • “reduced binding ... at acidic pH as compared to neutral pH” is expressed in terms of a ratio of the KD value of the antibody binding to its antigen at acidic pH to the KD value of the antibody binding to its antigen at neutral pH (or vice versa).
  • an antibody or antigen-binding fragment thereof may be regarded as exhibiting “reduced binding to CACNG1 at acidic pH as compared to neutral pH” for purposes of the description herein if the antibody or antigen-binding fragment thereof exhibits an acidic/neutral KD ratio of about 3.0 or greater.
  • the acidic/neutral KD ratio for an antibody or antigen-binding fragment as described herein can be about 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 20.0. 25.0, 30.0, 40.0, 50.0, 60.0, 70.0, 100.0 or greater.
  • Antibodies with pH-dependent binding characteristics may be obtained, e.g. , by screening a population of antibodies for reduced (or enhanced) binding to a particular antigen at acidic pH as compared to neutral pH. Additionally, modifications of the antigen-binding domain at the amino acid level may yield antibodies with pH-dependent characteristics. For example, by substituting one or more amino acids of an antigen-binding domain (e.g., within a CDR) with a histidine residue, an antibody with reduced antigen-binding at acidic pH relative to neutral pH may be obtained.
  • an anti-hCDH15 antibody and antigen-binding fragment thereof comprising an Fc domain comprising one or more mutations which enhance or diminish antibody binding to the FcRn receptor, e.g., at acidic pH as compared to neutral pH.
  • antibodies as described herein may comprise a mutation in the CH2 or a CH3 region of the Fc domain, wherein the mutation(s) increases the affinity of the Fc domain to FcRn in an acidic environment (e.g., in an endosome where pH ranges from about 5.5 to about 6.0).
  • Such mutations may result in an increase in serum half-life of the antibody when administered to an animal.
  • Fc modifications include, e.g., a modification at position 250 (e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L/Y/F/W or T), 254 (e.g., S or T), and 256 (e.g., S/R/Q/E/D or T); or a modification at position 428 and/or 433 (e.g., H/L/R/S/P/Q or K) and/or 434 (e.g., H/F or Y); or a modification at position 250 and/or 428; or a modification at position 307 or 308 (e.g., 308F, V308F), and 434.
  • a modification at position 250 e.g., E or Q
  • 250 and 428 e.g., L or F
  • 252 e.g., L/
  • the modification comprises a 428L (e.g., M428L) and 434S (e.g., N434S) modification; a 428L, 2591 (e.g., V259I), and 308F (e.g, V308F) modification; a 433K (e.g., H433K) and a 434 (e.g., 434Y) modification; a 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modification; a 250Q and 428L modification (e.g., T250Q and M428L); and a 307 and/or 308 modification (e.g, 308F or 308P).
  • a 428L e.g., M428L
  • 434S e.g., N434S
  • 428L, 2591 e.g., V259I
  • 308F e.g, V308F
  • 433K e.
  • an anti-hCDH15 antibody and antigen-binding fragment as described herein may comprise an Fc domain comprising one or more pairs or groups of mutations selected from the group consisting of: 250Q and 248L (e.g., T250Q and M248L);
  • 252Y, 254T and 256E e.g., M252Y, S254T and T256E
  • 428L and 434S e.g., M428L and N434S
  • 433K and 434F e.g, H433K and N434F
  • an antibody and antigen-binding fragment thereof that binds human CDH15 with high, medium or low affinity, depending on the therapeutic context and particular targeting properties that are desired.
  • a target antigen e.g., a tumor associated antigen
  • preferential targeting of the antigen-binding molecule to cells expressing the target antigen may be achieved while avoiding general/untargeted CDH15 binding and the consequent adverse side effects associated therewith.
  • antibodies, antigen-binding fragments, and bispecific antibodies thereof that bind human CDH15 with weak (i.e. low) or even no detectable affinity.
  • an antibody and antigen-binding fragment thereof as described herein binds human CDH15 (e.g., at 37°C) with a KD of greater than about 100 nM as measured by surface plasmon resonance.
  • an antibody or antigen-binding fragment as described herein binds CDH15 with a KD of greater than about greater than about 110 nM, at least 120 nM, greater than about 130 nM, greater than about 140 nM, greater than about 150 nM, at least 160 nM, greater than about 170 nM, greater than about 180 nM, greater than about 190 nM, greater than about 200 nM, greater than about 250 nM, greater than about 300 nM, greater than about 400 nM, greater than about 500 nM, greater than about 600 nM, greater than about 700 nM, greater than about 800 nM, greater than about 900 nM, or greater than about 1 pM, or with no detectable affinity, as measured by surface plasmon resonance (e.g., mAb-capture or antigencapture format), or a substantially similar assay.
  • surface plasmon resonance e.g., mAb-capture or antigencapture format
  • the epitope on CDH15 to which an anti-hCDH15 antibody and antigen-binding fragment thereof as described herein may consist of a single contiguous sequence of 3 or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids of a CDH15 protein.
  • the epitope may consist of a plurality of non-contiguous amino acids (or amino acid sequences) of CDH15.
  • epitope refers to an antigenic determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule known as a paratope.
  • a single antigen may have more than one epitope.
  • Epitopes may be either conformational or linear.
  • a conformational epitope is produced by spatially juxtaposed amino acids from different segments of the linear polypeptide chain.
  • a linear epitope is one produced by adjacent amino acid residues in a polypeptide chain.
  • an epitope may include moieties of saccharides, phosphoryl groups, or sulfonyl groups on the antigen.
  • Another method that can be used to identify the amino acids within a polypeptide with which an antigen-binding domain of an antibody interacts is hydrogen/deuterium exchange detected by mass spectrometry.
  • the hydrogen/deuterium exchange method involves deuterium-labeling the protein of interest, followed by binding the antibody to the deuterium-labeled protein. Next, the protein/antibody complex is transferred to water to allow hydrogen-deuterium exchange to occur at all residues except for the residues protected by the antibody (which remain deuterium- labeled).
  • the target protein After dissociation of the antibody, the target protein is subjected to protease cleavage and mass spectrometry analysis, thereby revealing the deuterium-labeled residues which correspond to the specific amino acids with which the antibody interacts. See, e.g., Ehring (1999) Analytical Biochemistry 262(2y.252-25 Engen and Smith (2001) Anal. Chem. 73:256A- 265A. X-ray crystallography of the antigen/antibody complex may also be used for epitope mapping purposes.
  • anti-hCDHl 5 antibodies that bind to the same epitope as any of the specific example antibodies described herein (e.g., antibodies comprising any of the amino acid sequences as set forth in Table 1 herein).
  • anti-hCDH15 antibodies that compete for binding to CDH15 with any of the specific example antibodies described herein (e.g., antibodies comprising any of the amino acid sequences as set forth in Table 1 herein).
  • One can easily determine whether a particular antigen-binding molecule e.g., antibody
  • antigen-binding domain thereof binds to the same epitope as, or competes for binding with, a reference antigen-binding molecule as described herein by using routine methods known in the art. For example, to determine if a test antibody binds to the same epitope on CDH15 as a reference bispecific antigen-binding molecule as described herein, the reference bispecific molecule is first allowed to bind to a CDH15 protein. Next, the ability of a test antibody to bind to the CDH15 molecule is assessed.
  • a particular antigen-binding molecule e.g., antibody
  • antigen-binding domain thereof binds to the same epitope as, or competes for binding with, a reference antigen-binding molecule as described herein by using routine methods known in the art. For example, to determine if a test antibody binds to the same epitope on CDH15 as a reference bi
  • test antibody If the test antibody is able to bind to CDH15 following saturation binding with the reference bispecific antigen-binding molecule, it can be concluded that the test antibody binds to a different epitope of CDH15 than the reference bispecific antigen-binding molecule. On the other hand, if the test antibody is not able to bind to the CDH15 molecule following saturation binding with the reference bispecific antigen-binding molecule, then the test antibody may bind to the same epitope of CDH15 as the epitope bound by the reference bispecific antigen-binding molecule as described herein.
  • Additional routine experimentation e.g., peptide mutation and binding analyses
  • peptide mutation and binding analyses can then be carried out to confirm whether the observed lack of binding of the test antibody is in fact due to binding to the same epitope as the reference bispecific antigen-binding molecule or if steric blocking (or another phenomenon) is responsible for the lack of observed binding.
  • steric blocking or another phenomenon
  • this sort can be performed using ELISA, RIA, Biacore, flow cytometry or any other quantitative or qualitative antibody-binding assay available in the art.
  • two antigen-binding proteins bind to the same (or overlapping) epitope if, e.g., a 1-, 5-, 10-, 20- or 100-fold excess of one antigen-binding protein inhibits binding of the other by at least 50% but preferably 75%, 90% or even 99% as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 1990:50: 1495-1502).
  • two antigen-binding proteins are deemed to bind to the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antigen-binding protein reduce or eliminate binding of the other.
  • Two antigen-binding proteins are deemed to have “overlapping epitopes” if only a subset of the amino acid mutations that reduce or eliminate binding of one antigenbinding protein reduce or eliminate binding of the other.
  • an antibody or antigen-binding domain thereof competes for binding with a reference antigen-binding molecule
  • the above-described binding methodology is performed in two orientations: In a first orientation, the reference antigen-binding molecule is allowed to bind to a CDH15 protein under saturating conditions followed by assessment of binding of the test antibody to the CDH15 molecule. In a second orientation, the test antibody is allowed to bind to a CDH15 molecule under saturating conditions followed by assessment of binding of the reference antigen-binding molecule to the CDH15 molecule.
  • an antibody that competes for binding with a reference antigen-binding molecule may not necessarily bind to the same epitope as the reference antibody, but may sterically block binding of the reference antibody by binding an overlapping or adjacent epitope.
  • Antigen-binding domains specific for particular antigens can be prepared by any antibody generating technology known in the art. Once obtained, two different antigen-binding domains, specific for two different antigens (e.g., CDH15 and a target antigen), can be appropriately arranged relative to one another to produce a bispecific antigen-binding molecule as described herein using routine methods. (A discussion of example bispecific antibody formats that can be used to construct the bispecific antigen-binding molecules as described herein is provided elsewhere herein).
  • one or more of the individual components (e.g., heavy and light chains) of the antigen-binding molecules as described herein are derived from chimeric, humanized or fully human antibodies. Methods for making such antibodies are well known in the art.
  • one or more of the heavy and/or light chains of the antigenbinding molecules as described herein can be prepared using VELOCIMMUNETM technology. Using VELOCIMMUNETM technology (or any other human antibody generating technology), high affinity chimeric antibodies to a particular antigen (e.g., CDH15) are initially isolated having a human variable region and a mouse constant region. The antibodies are characterized and selected for desirable characteristics, including affinity, selectivity, epitope, etc. The mouse constant regions are replaced with a desired human constant region to generate fully human heavy and/or light chains that can be incorporated into the antigen-binding molecules as described herein.
  • Genetically engineered animals may be used to make human bispecific antigenbinding molecules.
  • a genetically modified mouse can be used which is incapable of rearranging and expressing an endogenous mouse immunoglobulin light chain variable sequence, wherein the mouse expresses only one or two human light chain variable domains encoded by human immunoglobulin sequences operably linked to the mouse kappa constant gene at the endogenous mouse kappa locus.
  • Such genetically modified mice can be used to isolate heavy chain and light chain variable regions to produce fully human bispecific antigen-binding molecules.
  • the fully human bispecific antigen-binding molecules comprise two different heavy chains that associate with the same light chain. See, e.g., US 2011/0195454).
  • Fully human refers to an antibody, or antigen-binding fragment or immunoglobulin domain thereof, comprising an amino acid sequence encoded by a DNA derived from a human sequence over the entire length of each polypeptide of the antibody or antigen-binding fragment or immunoglobulin domain thereof.
  • the fully human sequence is derived from a protein endogenous to a human.
  • the fully human protein or protein sequence comprises a chimeric sequence wherein each component sequence is derived from human sequence. While not being bound by any one theory, chimeric proteins or chimeric sequences are generally designed to minimize the creation of immunogenic epitopes in the junctions of component sequences, e.g. compared to any wild-type human immunoglobulin regions or domains.
  • Bispecific antigen-binding molecules may be constructed with one heavy chain having a modified Fc domain that abrogates its binding to Protein A, thus enabling a purification method that yields a heterodimeric protein. See, for example, US Patent No. 8,586,713.
  • the bispecific antigen-binding molecules comprise a first CH3 domain and a second Ig CH3 domain, wherein the first and second Ig CH3 domains differ from one another by at least one amino acid, and wherein at least one amino acid difference reduces binding of the bispecific antibody to Protein A as compared to a bi-specific antibody lacking the amino acid difference.
  • the first Ig CH3 domain binds Protein A and the second Ig CH3 domain contains a mutation/modification that reduces or abolishes Protein A binding such as an H95R modification (by IMGT exon numbering; H435R by EU numbering).
  • the second CH3 may further comprise a Y96F modification (by IMGT; Y436F by EU).
  • Antigen-binding molecules having amino acid sequences that vary from those of the example molecules disclosed herein but that retain the ability to bind CDH15 are also described herein. Such variant molecules may comprise one or more additions, deletions, or substitutions of amino acids when compared to parent sequence, but exhibit biological activity that is essentially equivalent to that of the described bispecific antigen-binding molecules.
  • Antigen-binding molecules that are bioequivalent to any of the example antigenbinding molecules set forth herein are also described. Two antigen-binding proteins, or antibodies, are considered bioequivalent if, for example, they are pharmaceutical equivalents or pharmaceutical alternatives whose rate and extent of absorption do not show a significant difference when administered at the same molar dose under similar experimental conditions, either single dose or multiple doses.
  • antigen-binding proteins will be considered equivalents or pharmaceutical alternatives if they are equivalent in the extent of their absorption but not in their rate of absorption and yet may be considered bioequivalent because such differences in the rate of absorption are intentional and are reflected in the labeling, are not essential to the attainment of effective body drug concentrations on, e.g., chronic use, and are considered medically insignificant for the particular drug product studied.
  • two antigen-binding proteins are bioequivalent if there are no clinically meaningful differences in their safety, purity, and potency. [00191] In one embodiment, two antigen-binding proteins are bioequivalent if a patient can be switched one or more times between the reference product and the biological product without an expected increase in the risk of adverse effects, including a clinically significant change in immunogenicity, or diminished effectiveness, as compared to continued therapy without such switching.
  • two antigen-binding proteins are bioequivalent if they both act by a common mechanism or mechanisms of action for the condition or conditions of use, to the extent that such mechanisms are known.
  • Bioequivalence may be demonstrated by in vivo and in vitro methods.
  • Bioequivalence measures include, e.g., (a) an in vivo test in humans or other mammals, in which the concentration of the antibody or its metabolites is measured in blood, plasma, serum, or other biological fluid as a function of time; (b) an in vitro test that has been correlated with and is reasonably predictive of human in vivo bioavailability data; (c) an in vivo test in humans or other mammals in which the appropriate acute pharmacological effect of the antibody (or its target) is measured as a function of time; and (d) in a well-controlled clinical trial that establishes safety, efficacy, or bioavailability or bioequivalence of an antigen-binding protein.
  • Bioequivalent variants of the example bispecific antigen-binding molecules set forth herein may be constructed by, for example, making various substitutions of residues or sequences or deleting terminal or internal residues or sequences not needed for biological activity.
  • cysteine residues not essential for biological activity can be deleted or replaced with other amino acids to prevent formation of unnecessary or incorrect intramolecular disulfide bridges upon renaturation.
  • bioequivalent antigen-binding proteins may include variants of the example bispecific antigen-binding molecules set forth herein comprising amino acid changes which modify the glycosylation characteristics of the molecules, e.g., mutations which eliminate or remove glycosylation.
  • antigen-binding proteins e.g., antibodies or antigenbinding fragments thereof, such as anti-hCDH15 antibodies, comprising a modified glycosylation pattern.
  • modification to remove undesirable glycosylation sites may be useful, or an antibody lacking a fucose moiety present on the oligosaccharide chain, for example, to increase antibody dependent cellular cytotoxicity (ADCC) function (see Shields et al. (2002) JBC 277:26733), where cytotoxicity is desirable.
  • ADCC antibody dependent cellular cytotoxicity
  • modification of galactosylation can be made in order to modify complement dependent cytotoxicity (CDC).
  • antigen-binding molecules as described herein bind to human CDH15 but not to CDH15 from other species. Also described herein are antigen-binding molecules that bind to human CDH15 and to CDH15 from one or more non-human species.
  • antigen-binding molecules as described herein that bind to human CDH15 may bind, or not bind, as the case may be, to one or more of mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cow, horse, camel, cynomolgus, marmoset, rhesus or chimpanzee CDH15.
  • ADCs Antibody-Drug Conjugates
  • ADCs antibody-drug conjugates
  • a payload e.g., a drug or molecular cargo, (e.g., a small molecule and/or therapeutic moiety, etc.).
  • ADCs comprise: A - [L - P] y , in which A is an antigen-binding molecule, e.g.
  • an anti-hCDH15 antibody or a fragment thereof (e.g., a fragment comprising at least a HCDR3 selected from any of the HCDR3 amino acid sequences listed in Table 1)
  • L is a linker
  • P is the payload or molecular cargo
  • y is an integer from 1 to 30.
  • the ADC comprises an anti-hCDH15 antibody or antigen-binding fragment thereof that comprises the CDRs of a HCVR or a LCVR having the amino acid sequences of the SEQ ID NOs set forth in Table 1 (e.g., SEQ ID NOs: 2, 22, 42, 60, 70, 80, 90, 108, 125, 145, 165, 185, 202, 220, 230, 240, 250, 259, 279, 299, 319, 339, 358, 378, 386, 404, 420, 436, 452, 468, 484, 500, 516, 532, 548, 564, 580, 596, 612, 628, 644, 660, 676, 692, 708, 724, 740, 748, 764, and 780; or 10, 30, 50, 50, 50, 50, 50, 98, 115, 133, 153, 173, 193, 210, 50, 50, 50, 50, 267, 2
  • the anti-hCDH15 antibody or fragment comprises CDRs with the amino acid sequences of the SEQ ID NOs set forth in Table 1 e.g., SEQ ID NOs: 4-6-8-12-14-16, 24-26-28-32-34-36, 44-46-48-52-34-54, 62- 64-66-52-34-54, 72-74-76-52-34-54, 82-84-86-52-34-54, 92-94-96-100-34-102, 82-111-113- 117-34-119, 127-129-131-135-137-139, 147-149-151-155-157-159, 167-169-171-175-177-179, 187-189-191-52-34-196, 204-206-208-212-137-214, 222-224-226-52-34-54, 232-234-236-52- 34-54, 242-244-246-52-34-54, 82-253-255-52-34-54, 261-263-265-269-271-273, 281-283
  • the anti-hCDH15 antibody or fragment comprises a HCVR and a LCVR having the amino acid sequences of the SEQ ID NOs set forth in Table 1 (e.g., SEQ ID NOs: : 2, 22, 42, 60, 70, 80, 90, 108, 125, 145, 165, 185, 202, 220, 230, 240, 250, 259, 279, 299, 319, 339, 358, 378, 386, 404, 420, 436, 452, 468, 484, 500, 516, 532, 548, 564, 580, 596, 612, 628, 644, 660, 676, 692, 708, 724, 740, 748, 764, and 780; and 10, 30, 50, 50, 50, 50, 50, 98, 115, 133, 153, 173, 193, 210, 50, 50, 50, 50, 267, 287, 307, 327, 347, 366, 50, 394,
  • the payload or molecular cargo comprises a small molecule as a therapeutic agent, e.g., a therapeutic agent that may be useful for treating muscle wasting or genetic muscle diseases and/or muscle-related cancer.
  • a small molecule can enter cells easily because it has a low molecular weight (typically, up to about 1 kDa). Once inside the cells, the small molecule can affect other molecules, such as proteins, and may, for example, cause cancer cells to die. This is different from many large molecular weight molecules such as antibodies.
  • An example, of a small molecule may be conjugated to an anti- CDH15 antigen-binding protein, to form an anti-CDH15:SM conjugate.
  • Therapeutic agents that may be useful for treating muscle wasting or genetic muscle diseases include testosterone and biologically active variants thereof, (e.g., dihydrotestosterone (DHT)), 02-adrenergic receptor agonists (e.g., clenbuterol), rapamycin or its analogs, MAPK inhibitors, or histone deacetylase inhibitors, etc.
  • DHT dihydrotestosterone
  • clenbuterol e.g., clenbuterol
  • rapamycin or its analogs e.g., clenbuterol
  • MAPK inhibitors e.g., rapamycin or its analogs
  • histone deacetylase inhibitors e.g., histone deacetylase inhibitors, etc.
  • the therapeutic payload is testosterone, or a biologically active derivative and/or portion thereof, e.g., dihydrotestosterone.
  • the therapeutic payload is rapamycin or analogs thereof, a MAPK inhibitor, a histone deacetylase inhibitor, or a Notch ligand.
  • the payload is a chemotherapeutic e.g., a cytotoxic drug.
  • Therapeutic agents that may be useful for treating muscle-related cancer include any chemotherapeutic agents known to delay, halt and/or destroy cancerous cells.
  • therapeutic agents include, but are not limited to, Aflibercept, Amsacrine, Azacitidine, Azathioprine, Belantamab mafodotin, Bendamustine, Bleomycin, Bortezomib, Brentuximab vedotin, Busulfan, Cabazitaxel, Capecitabine, Carboplatin, Carfilzomib, Carmustine, Chlorambucil, Cisplatin, Cladribine, Clofarabine, Cyclophosphamide, Cytarabine, Cytarabine liposomal, dacarbazine, Dactinomycin (actinomycin D), Daunorubicin, Docetaxel, Doxorubicin, Doxorubicin liposomal, Epirubicin,
  • Inotuzumab ozogamicin Irinotecan, Ixazomib, Lomustine, Melphalan, Mercaptopurine, Methotrexate, Mitomycin, Mitotane, Mitozantrone, Nab-paclitaxel, Oxaliplatin, Paclitaxel, Pemetrexed, Pegaspargase, Polatuzumab vedotin, Pralatrexate, Procarbazine, Raltitrexed, Romidepsin, Sacituzumab govitecan, Temozolomide, Teniposide, Thiotepa, Tioguanine, Topotecan, Trabectedin, Trastuzumab deruxtecan, Trastuzumab emtansine, Trifluridine/tipiracil, Valganciclovir, Vinblastine, Vincristine, Vindesine, Vinflunine, Vinorelbine, or Vismodegib.
  • ARCs antibody-radionuclide conjugates
  • radionuclides that can be used in the context of this aspect of the disclosure include, but are not limited to, e.g, 225 Ac, 212 Bi, 213 Bi, 131 I, 186 Re, 227 Th, 222 Rn, 223 Ra, 224 Ra, and 90 Y.
  • ADCs comprising, e.g, an anti-hCDH15 antigen-binding protein conjugated to a therapeutic agent e.g., any of the therapeutic agents disclosed above) via a linker molecule.
  • Linkers are any group or moiety that links, connects, or bonds the antibody or antigen-binding proteins described herein with a therapeutic moiety, e.g. cytotoxic agent. Suitable linkers may be found, for example, in Antibody-Drug Conjugates and Immunotoxins,' Phillips, G.
  • suitable binding agent linkers for the antibody conjugates described herein are those that are sufficiently stable to exploit the circulating half-life of the antibody and, at the same time, capable of releasing its payload after antigen-mediated internalization of the conjugate.
  • Linkers can be cleavable or non-cleavable.
  • Cleavable linkers include linkers that are cleaved by intracellular metabolism following internalization, e.g., cleavage via hydrolysis, reduction, or enzymatic reaction.
  • Non-cleavable linkers include linkers that release an attached payload via lysosomal degradation of the antibody following internalization.
  • Suitable linkers include, but are not limited to, acid-labile linkers, hydrolysis-labile linkers, enzymatically cleavable linkers, reduction labile linkers, self-immolative linkers, and non-cleavable linkers.
  • Suitable linkers also include, but are not limited to, those that are or comprise peptides, glucuronides, succinimide-thioethers, polyethylene glycol (PEG) units, hydrazones, mal-caproyl units, dipeptide units, valine-citrulline units, and para-aminobenzyl (PAB) units.
  • PEG polyethylene glycol
  • PAB para-aminobenzyl
  • linker molecule or linker technology known in the art can be used to create or construct an ADC of the present disclosure.
  • the linker is a cleavable linker.
  • the linker is a non-cleavable linker.
  • linkers that can be used in the context of the present disclosure are provided, e.g., in US 7,754,681 and in Ducry, Bioconjugate Chem., 2010, 27:5-13, and the references cited therein, the contents of which are incorporated by reference herein in their entireties.
  • the linkers are stable in physiological conditions.
  • the linkers are cleavable, for instance, able to release at least the payload portion in the presence of an enzyme or at a particular pH range or value.
  • a linker comprises an enzyme-cleavable moiety.
  • Illustrative enzyme-cleavable moieties include, but are not limited to, peptide bonds, ester linkages, hydrazones, and disulfide linkages.
  • the linker comprises a cathepsin-cleavable linker.
  • the linker comprises a non-cleavable moiety.
  • Suitable linkers also include, but are not limited to, those that are chemically bonded to two cysteine residues of a single binding agent, e.g., antibody. Such linkers can serve to mimic the antibody’s disulfide bonds that are disrupted as a result of the conjugation process.
  • the linker comprises one or more amino acids. Suitable amino acids include natural, non-natural, standard, non-standard, proteinogenic, non- proteinogenic, and L- or D- a-amino acids.
  • the linker comprises alanine, valine, glycine, leucine, isoleucine, methionine, tryptophan, phenylalanine, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, or citrulline, a derivative thereof, or combination thereof.
  • one or more side chains of the amino acids is linked to a side chain group, described below.
  • the linker comprises valine and citrulline.
  • the linker comprises lysine, valine, and citrulline.
  • the linker comprises lysine, valine, and alanine. In some embodiments, the linker comprises valine and alanine. [00211] In some embodiments, the linker comprises a self-immolative group.
  • the self- immolative group can be any such group known to those of skill.
  • the self-immolative group is ?-aminobenzyl (PAB), or a derivative thereof. Useful derivatives include /?-aminobenzyloxycarbonyl (PABC).
  • PAB ?-aminobenzyl
  • PABC /?-aminobenzyloxycarbonyl
  • the linker is: wherein is a bond to the antibody or antigen-binding protein (e.g., via lysine residue) and is a bond to the therapeutic payload (e.g., testosterone or a biologically equivalent variant thereof).
  • the linker is: wherein is a bond to the antibody or antigen-binding protein (e.g., via lysine residue) and _ ⁇ P 4 is a bond to a therapeutic payload (e.g. , testosterone or a biologically equivalent variant thereof).
  • the linker is:
  • the linker is:
  • the linker is derived from maleimidylmethyl-4-trans- cyclohexanecarboxy succinate:
  • the linker is: wherein is a bond to the antibody or antigen-binding protein (e.g., via lysine residue) and is a bond to therapeutic payload (e.g. , testosterone or a biologically equivalent variant thereof).
  • the linker is: wherein is a bond to the antibody or antigen-binding protein (e.g, via lysine residue) and
  • the present disclosure comprises ADCs in which a linker connects an anti- hCDH15 antigen-binding protein as described herein to therapeutic agent through an attachment at a particular amino acid within the antibody or antigen-binding molecule.
  • amino acid attachments that can be used in the context of this aspect, e.g., lysine (see, e.g., US 5,208,020; US 2010/0129314; Hollander et al., Bioconjugate Chem., 2008, 19:358-361; WO 2005/089808; US 5,714,586; and US 2013/0101546), cysteine (see, e.g., US 2007/0258987; WO 2013/055993; WO 2013/055990; WO 2013/053873; WO 2013/053872; WO 2011/130598; US 2013/0101546; and US 7,750,116), selenocysteine (see, e.g., WO 2008/12
  • Linkers can also be conjugated to an antigen-binding protein via attachment to carbohydrates (see, e.g., US 2008/0305497, WO 2014/065661, and Ryan et al., Food & Agriculture Immunol., 2001, 13: 127-130) and disulfide linkers (see, e.g., WO 2013/085925, WO 2010/010324, WO 2011/018611, and Shaunak etal., Nat. Chem. Biol., 2006, 2:312-313).
  • Site specific conjugation techniques can also be employed to direct conjugation to particular residues of the antibody or antigen binding protein (see, e.g., Schumacher et al.
  • Site specific conjugation techniques include, but are not limited to glutamine conjugation via transglutaminase (see e.g., Schibli, Angew Chemie Inter Ed. 2010, 49 ,9995).
  • a residue of an antibody as described herein e.g., a residue in a heavy chain constant region of the antibody, may be substituted with a glutamine to further facilitate glutamine conjugation via transglutaminase.
  • a human heavy chain constant region may be modified with the N180Q substitution found in the sequence of the human IgGl heavy chain constant region. Such substitution provides for a total of 4 glutamines for conjugation by transglutaminase.
  • an anti-hCDH15 antigen-binding protein drug conjugate is prepared by contacting an anti-hCDH15 antigen-binding protein as described herein with a compound comprising the desired linker and therapeutic agent, wherein said linker possesses a moiety that is reactive with the antibody or antigen-binding protein, e.g., at the desired residue of the antibody or antigen-binding protein.
  • AAV Adeno-associated viruses
  • AAV is an abbreviation for adeno-associated virus and may be used to refer to the virus itself or derivatives thereof.
  • AAVs are small, non-enveloped, single-stranded DNA viruses.
  • ITR inverted terminal repeats
  • ORFs open reading frames
  • Rep and cap The wildtype rep reading frame encodes four proteins of molecular weight 78 kD (“Rep78”), 68 kD (“Rep68”), 52 kD (“Rep52”) and 40 kD (“Rep 40”).
  • Rep78 and Rep68 are transcribed from the p5 promoter, and Rep52 and Rep40 are transcribed from the pl 9 promoter. These proteins function mainly in regulating the transcription and replication of the AAV genome.
  • the wildtype cap reading frame encodes three structural (capsid) viral proteins (VPs) having molecular weights of 83-85 kD (VP1), 72-73 kD (VP2) and 61-62 kD (VP3). More than 80% of total proteins in an AAV virion (capsid) comprise VP3; in mature virions VP1, VP2 and VP3 are found at relative abundance of approximately 1 : 1 : 10, although ratios of 1 : 1 :8 have been reported. Padron et al. (2005) J. Virology 79:5047-58.
  • AAV encompasses all subtypes and both naturally occurring and modified forms that are well-known in the art.
  • AAV includes primate AAV (e.g, AAV type 1 (AAV1), primate AAV type 2 (AAV2), primate AAV type 3 (AAV3B), primate AAV type 4 (AAV4), primate AAV type 5 (AAV5), primate AAV type 6 (AAV6), primate AAV type 7 (AAV7), primate AAV type 8 (AAV8), primate AAV type 9 (AAV9), AAV10, AAV11, AAV12, AAV13, AAVDJ, Anc80L65, AAV2G9, AAV-LK03, primate AAV type rhlO (AAV rhlO), AAV type hlO (AAV hlO), AAV type hul 1 (AAV hul l), AAV type rh32.33 (AAV rh32.33), AAV retro (A
  • Prime AAV refers to AAV generally isolated from primates.
  • non-primate animal AAV refers to AAV isolated from non-primate animals.
  • “of a [specified] AAV” in relation to a gene e.g., rep, cap, etc.
  • capsid protein e.g., a VP1 capsid protein, a VP2 capsid protein, a VP3 capsid protein, etc.
  • region of a capsid protein of a specified AAV e.g., PLA2 region, VPl-u region, VP1/VP2 common region, VP3 region
  • nucleotide sequence e.g., ITR sequence
  • a cap gene or capsid protein of AAV etc. encompasses, in addition to the gene or the polypeptide respectively comprising a nucleic acid sequence or amino acid sequence set forth herein for the specified AAV, also variants of the gene or polypeptide,
  • a variant gene or a variant polypeptide comprises a nucleic acid sequence or amino acid sequence that differs from the nucleic acid sequence or amino acid sequence set forth herein for the gene or polypeptide of a specified AAV, wherein the difference(s) does not generally alter at least one biological function of the gene or polypeptide, and/or the phylogenetic characterization of the gene or polypeptide, e.g., where the difference(s) may be due to degeneracy of the genetic code, isolate variations, length of the sequence, etc.
  • rep gene and the cap gene as used here may encompass rep and cap genes that differ from the wildtype gene in that the genes may encode one or more Rep proteins and Cap proteins, respectively.
  • a Rep gene encodes at least Rep78 and/or Rep68.
  • cap gene includes those may differ from the wildtype in that one or more alternative start codons or sequences between one or more alternative start codons are removed such that the cap gene encodes only a single Cap protein, e.g, wherein the VP2 and/or VP3 start codons are removed or substituted such that the cap gene encodes a functional VP1 capsid protein but not a VP2 capsid protein or a VP3 capsid protein.
  • a rep gene encompasses any sequence that encodes a functional Rep protein.
  • a cap gene encompasses any sequence that encodes at least one functional cap gene.
  • the wildtype cap gene expresses all three VP1, VP2, and VP3 capsid proteins from a single open reading frame of the cap gene under control of the p40 promoter found in the rep ORF.
  • the term “capsid protein,” “Cap protein” and the like includes a protein that is part of the capsid of the virus.
  • the capsid proteins are generally referred to as VP1, VP2 and/or VP3, and may be encoded by the single cap gene.
  • the three AAV capsid proteins are produced in nature an overlapping fashion from the cap ORF alternative translational start codon usage, although all three proteins use a common stop codon.
  • the ORF of a wildtype cap gene encodes from 5’ to 3’ three alternative start codons: “the VP1 start codon,” “the VP2 start codon,” and “the VP3 start codon”; and one “common stop codon”.
  • the largest viral protein, VP1 is generally encoded from the VP1 start codon to the “common stop codon.”
  • VP2 is generally encoded from the VP2 start codon to the common stop codon.
  • VP3 is generally encoded from the VP3 start codon to the common stop codon.
  • VP1 comprises at its N-terminus sequence that it does not share with the VP2 or VP3, referred to as the VPl-unique region (VPl-u).
  • the VPl-u region is generally encoded by the sequence of a wildtype cap gene starting from the VP1 start codon to the “VP2 start codon.”
  • VPl-u comprises a phospholipase A2 domain (PLA2), which may be important for infection, as well as nuclear localization signals which may aid the virus in targeting to the nucleus for uncoating and genome release.
  • PHA2 phospholipase A2 domain
  • the VP1, VP2, and VP3 capsid proteins share the same C-terminal sequence that makes up the entirety of VP3, which may also be referred to herein as the VP3 region.
  • the VP3 region is encoded from the VP3 start codon to the common stop codon.
  • VP2 has an additional ⁇ 60 amino acids that it shares with the VP 1. This region is called the VP1/VP2 common region.
  • one or more of the Cap proteins of the invention may be encoded by one or more cap genes having one or more ORFs.
  • the VP proteins of the invention may be expressed from more than one ORF comprising nucleotide sequence encoding any combination of VP1, VP2, and/or VP3 by use of separate nucleotide sequences operably linked to at least one expression control sequence for expression in packaging cell, each producing one or more of VP1, VP2, and/or VP3 capsid proteins of the invention.
  • a VP capsid protein of the invention may be expressed individually from an ORF comprising nucleotide sequence encoding any one of VP1, VP2, or VP3 by use of separate nucleotide sequences operably linked to one expression control sequence for expression in a viral replication cell, each producing only one of VP1, VP2, or VP3 capsid protein.
  • VP proteins may be expressed from one ORF comprising nucleotide sequences encoding VP1, VP2, and VP3 capsid proteins operably linked to at least one expression control sequence for expression in a viral replication cell, each producing VP1, VP2, and VP3 capsid protein.
  • amino acid positions provided herein may be provided in relation to the VP1 capsid protein of the referenced AAV, a skilled artisan would be able to respectively and readily determine the position of that same amino acid within the VP2 and/or VP3 capsid protein of the AAV, and the corresponding position of amino acids among different AAV.
  • ITR Inverted Terminal Repeat
  • the phrase “Inverted Terminal Repeat” or “ITR” includes symmetrical nucleic acid sequences in the genome of adeno-associated viruses required for efficient replication. ITR sequences are located at each end of the AAV DNA genome. The ITRs serve as the origins of replication for viral DNA synthesis and are essential cis components for generating AAV particles, e.g., packaging into AAV particles.
  • AAV ITRs comprise recognition sites for replication proteins Rep78 or Rep68.
  • a “D” region of the ITR comprises the DNA nick site where DNA replication initiates and provides directionality to the nucleic acid replication step.
  • An AAV replicating in a mammalian cell typically comprises two ITR sequences.
  • a single ITR may be engineered with Rep binding sites on both strands of the “A” regions and two symmetrical D regions on each side of the ITR palindrome.
  • Such an engineered construct on a double-stranded circular DNA template allows Rep78 or Rep68 initiated nucleic acid replication that proceeds in both directions.
  • a single ITR is sufficient for AAV replication of a circular particle.
  • the rep encoding sequence encodes a Rep protein or Rep protein equivalent that is capable of binding an ITR comprised on the transfer plasmid.
  • the Cap proteins of the invention when expressed with appropriate Rep proteins by a packaging cell, may encapsidate a transfer plasmid comprising a nucleotide of interest and an even number of two or more ITR sequences.
  • a transfer plasmid comprises one ITR sequence.
  • a transfer plasmid comprises two ITR sequences.
  • Rep proteins may be expressed from more than one ORF comprising nucleotide sequence encoding any combination of Rep78, Rep68, Rep 52 and/or Rep40 by use of separate nucleotide sequences operably linked to at least one expression control sequence for expression in a viral replication cell, each producing one or more of Rep78, Rep68, Rep 52 and/or Rep40 Rep proteins.
  • Rep proteins may be expressed individually from an ORF comprising a nucleotide sequence encoding any one of Rep78, Rep68, Rep 52, or Rep40 by use of separate nucleotide sequences operably linked to one expression control sequence for expression in a packaging cell, each producing only one Rep78, Rep68, Rep 52, or Rep40 Rep protein.
  • Rep proteins may be expressed from one ORF comprising nucleotide sequences encoding Rep78 and Rep52 Rep proteins operably linked to at least one expression control sequence for expression in a viral replication cell each producing Rep78 and Rep52 Rep protein.
  • a rep encoding sequence and a cap gene of the invention may be provided a single packaging plasmid.
  • proviso is not necessary.
  • viral particles may or may not include a genome.
  • a “chimeric AAV capsid protein” includes an AAV capsid protein that comprises amino acid sequences, e.g., portions, from two or more different AAV and that is capable of forming and/or forms an AAV viral capsid/viral particle.
  • a chimeric AAV capsid protein is encoded by a chimeric AAV capsid gene, e.g., a chimeric nucleotide comprising a plurality, e.g., at least two, nucleic acid sequences, each of which plurality is identical to a portion of a capsid gene encoding a capsid protein of distinct AAV, and which plurality together encodes a functional chimeric AAV capsid protein.
  • a chimeric capsid protein comprises one or more portions from a capsid protein of that AAV and one or more portions from a capsid protein of a different AAV.
  • a chimeric AAV2 capsid protein includes a capsid protein comprising one or more portions of a VP1, VP2, and/or VP3 capsid protein of AAV2 and one or more portions of a VP1, VP2, and/or VP3 capsid protein of a different AAV.
  • portion refers to at least 5 amino acids or at least 15 nucleotides, but less than the full-length polypeptide or nucleic acid molecule, with 100% identity to a sequence from which the portion is derived, see Penzes (2015) J. General Virol. 2769.
  • a “portion” encompasses any contiguous segment of amino acids or nucleotides sufficient to determine that the polypeptide or nucleic acid molecule form which the portion is derived is “of a [specified] AAV” or has “significant identity” to a particular AAV, e.g., a non-primate animal AAV or remote AAV.
  • a portion comprises at least 5 amino acids or 15 nucleotides with 100% identity to a sequence associated with the specified AAV. In some embodiments, a portion comprises at least 10 amino acids or 30 nucleotides with 100% identity to a sequence associated with the specified AAV. In some embodiments, a portion comprises at least 15 amino acids or 45 nucleotides with 100% identity to a sequence associated with the specified AAV. In some embodiments, a portion comprises at least 20 amino acids or 60 nucleotides with 100% identity to a sequence associated with the specified AAV. In some embodiments, a portion comprises at least 25 amino acids or 75 nucleotides with 100% identity to a sequence associated with the specified AAV.
  • a portion comprises at least 30 amino acids or 90 nucleotides with 100% identity to a sequence associated with the specified AAV. In some embodiments, a portion comprises at least 35 amino acids or 105 nucleotides with 100% identity to a sequence associated with the specified AAV. In some embodiments, a portion comprises at least 40 amino acids or 120 nucleotides with 100% identity to a sequence associated with the specified AAV. In some embodiments, a portion comprises at least 45 amino acids or 135 nucleotides with 100% identity to a sequence associated with the specified AAV. In some embodiments, a portion comprises at least 50 amino acids or 150 nucleotides with 100% identity to a sequence associated with the specified AAV.
  • a portion comprises at least 60 amino acids or 180 nucleotides with 100% identity to a sequence associated with the specified AAV. In some embodiments, a portion comprises at least 70 amino acids or 210 nucleotides with 100% identity to a sequence associated with the specified AAV. In some embodiments, a portion comprises at least 80 amino acids or 240 nucleotides with 100% identity to a sequence associated with the specified AAV. In some embodiments, a portion comprises at least 90 amino acids or 270 nucleotides with 100% identity to a sequence associated with the specified AAV. In some embodiments, a portion comprises at least 100 amino acids or 300 nucleotides with 100% identity to a sequence associated with the specified AAV.
  • a Cap protein e.g., a VP1 capsid protein as described herein, a VP2 capsid protein as described herein, and/or a VP3 capsid protein as described herein, is modified to comprise any one or combination of e.g., insertion of a targeting ligand, a chemical modification, a first member of a binding pair, a detectable label, point mutation, etc.
  • modification of gene or a polypeptide of a specified AAV results in nucleic acid sequence or an amino acid sequence that differs from the nucleic acid sequence or amino acid sequence set forth herein for the specified AAV, wherein the modification alters, confers, or removes one or more biological functions, but does not change the phylogenetic characterization of, the gene or polypeptide as an AAV gene or AAV polypeptide.
  • Modifications may include any one or a combination of: substitution of sequences of a first AAV serotype with sequences of a second AAV serotype to create chimerism; chemical modification; an insertion of: a first member of a binding pair, and/or a point mutation; etc., such that the natural tropism of the capsid protein is reduced to abolished, the tropism of the capsid protein may be more easily redirected, and/or such that the capsid protein comprises a detectable label.
  • Modifications as described herein generally do not alter and preferably decrease the low to no recognition of the modified capsid by pre-existing antibodies found in the general population that were produced during the course of infection with another AAV, e.g., infection with serotypes such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVDJ, Anc80L65, AAV2G9, AAV-LK03, virions based on such serotypes, virions from currently used AAV gene therapy modalities, or a combination thereof.
  • serotypes such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVDJ, Anc80L65, AAV2G9, AAV-LK03
  • Modifications described herein may pertain to the association (e.g., display, operable linkage, binding) of a targeting ligand to a modified capsid protein and/or capsid comprising a modified capsid protein.
  • a targeting ligand as described herein binds a surface protein expressed by a mammalian muscle cell, e.g., a protein that is expressed on the surface of a mammalian muscle cell, e.g, a mammalian non-terminally differentiated muscle cell specific surface protein.
  • a modified capsid protein and/or modified capsid comprises a targeting ligand that binds mammalian CDH15, e.g., a human CDH15.
  • Table 1 provides a summary of the SEQ ID NO for each binding portion (e.g., heavy chain variable domain (HCVR), light chain variable domain (LCVR), and CDR1, CDR2, and CDR3) of non-limiting and exemplary anti-human-CDH15 monoclonal antibodies (mAb ID) that may be used to redirect an AAV capsid as described herein.
  • HCVR heavy chain variable domain
  • LCVR light chain variable domain
  • CDR1, CDR2, and CDR3 CDR1, CDR2, and CDR3
  • mAb ID anti-human-CDH15 monoclonal antibodies
  • an AAV capsid as described herein comprises a targeting ligand that binds human CDH15, wherein the targeting ligand comprises heavy chain variable domain, light chain variable domain, heavy chain variable domain/light chain variable domain pair, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and/or set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence(s) at least 90% identical to, respectively, an amino acid sequence of a heavy chain variable domain, light chain variable domain, heavy chain variable domain/light chain variable domain pair, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and/or set of HCDR1- HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 as set forth in any one of SEQ ID NOs: 1-786.
  • an AAV capsid as described herein comprises a targeting ligand that binds human CDH15, wherein the targeting ligand comprises a heavy chain variable domain, light chain variable domain, heavy chain variable domain/light chain variable domain pair, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and/or set of HCDR1-HCDR2-HCDR3-LCDR1- LCDR2-LCDR3 amino acid sequence at least 95% identical to, respectively, amino acid sequence(s) of a heavy chain variable domain, light chain variable domain, heavy chain variable domain/light chain variable domain pair, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and/or set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 set forth in any one of SEQ ID NOs: 1-786.
  • an AAV capsid as described herein comprises a targeting ligand that binds human CDH15, wherein the targeting ligand comprises a heavy chain variable domain, light chain variable domain, heavy chain variable domain/light chain variable domain pair, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and/or set of HCDR1-HCDR2- HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence at least 97% identical to amino acid sequence(s) of a heavy chain variable domain, light chain variable domain, heavy chain variable domain/light chain variable domain pair, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and/or set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 set forth in any one of SEQ ID NOs: 1-786.
  • an AAV capsid as described herein comprises a targeting ligand that binds human CDH15, wherein the targeting ligand comprises a heavy chain variable domain, light chain variable domain, heavy chain variable domain/light chain variable domain pair, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and/or set of HCDR1-HCDR2- HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence(s) at least 98% identical to amino acid sequence(s) of a heavy chain variable domain, light chain variable domain, heavy chain variable domain/light chain variable domain pair, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and/or set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 set forth in any one of SEQ ID NOs: 1-786.
  • an AAV capsid as described herein comprises a targeting ligand that binds human CDH15, wherein the targeting ligand comprises a heavy chain variable domain, light chain variable domain, heavy chain variable domain/light chain variable domain pair, HCDR1, HCDR2, CDR3, LCDR1, LCDR2, LCDR3, and/or set of HCDR1-HCDR2- HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences 99% identical to amino acid sequences of a heavy chain variable domain, light chain variable domain, heavy chain variable domain/light chain variable domain pair, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and/or set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 set forth in any one of SEQ ID NOs: 1-786.
  • antibodies, or antigen-binding fragments thereof comprising a set of six CDRs (z.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within an HCVR/LCVR amino acid sequence pair as defined by any of the exemplary anti-hCDH15 antibodies listed in Table 1.
  • a targeting ligand as described herein comprises the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences set contained within an HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2+10, 22+30, 42+50, 60+50, 70+50, 80+50, 90+98, 108+115, 125+133, 145+153, 165+173, 185+193, 202+210, 220+50, 230+50, 240+50, 250+50, 259+267, 279+287, 299+307, 319+327, 339+347, 358+366, 378+50, 386+394, 404+412, 420+428, 436+444, 452+460, 468+476, 484+492, 500+508, 516+524, 532+540, 548+556, 564+572, 580+588, 596+60
  • Non-limiting examples of targeting ligands that bind CDH15 include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FR3- CDR3-FR4 peptide.
  • CDR complementarity determining region
  • an anti-CDH15 targeting ligand that binds CDH15 useful for retargeting viral capsids as described herein comprises an scFv.
  • an scFv sequences in VL-(Gly4Ser)3-VH format useful for retargeting viral capsids as described herein may comprise a heavy chain variable domain, light chain variable domain, heavy chain variable domain/light chain variable domain pair, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and/or set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 that is 90%, 95%, 97%, 98%, 99% or 100% identical, respectively, to any one of the amino acid sequences of a heavy chain variable domain, light chain variable domain, heavy chain variable domain/light chain variable domain pair, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and/or set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 as set forth in any one of SEQ ID NOs: 1- 786.
  • a targeting ligand that binds a mammalian non-terminally differentiated muscle cell surface protein may be associated with (e.g., displayed by, operably linked to, bound to) a modified AAV capsid protein and resulting AAV capsids according to well-known methods, e.g., a direct approach in which the targeting ligand is directly inserted into (e.g., using recombinatorial methods) according to well-known methods. See, e.g., Stachler et al. (2006), supra, White et al. (2004), supra, Girod et al. (1999), supra, Grifman et al. (2001), supra, Shi et al.
  • a targeting ligand that binds a mammalian non-terminally differentiated muscle cell specific surface protein may be coupled to a modified AAV capsid protein and resulting AAV capsids using well-known chemical linkers, e.g., wherein the AAV capsid protein may be chemically modified to comprise a dibenzocycootyne group or an azide group, and optionally wherein a targeting ligand as described herein is attached to the dibenzocycootyne group or the azide group, see, e.g, U.S.
  • a modified capsid as described herein comprises a targeting ligand, e.g., an anti- CDH15 antibody or binding portion thereof, directly inserted into or coupled to it according to well-known direct recombinatorial methods.
  • a targeting ligand that binds a mammalian non-terminally differentiated muscle cell surface protein may be associated with (e.g., displayed by, operably linked to, bound to) a modified AAV capsid protein and resulting AAV capsids according to indirect recombinatorial approaches, wherein the AAV capsid protein is modified to comprise a first member of a binding pair (e.g., a heterologous scaffold), and optionally wherein the first member of the binding pair is linked to (e.g., covalently or non-covalently bound to) a second cognate member of the binding pair (e.g., an adaptor), further optionally wherein the second cognate member of the binding pair is fused to the targeting ligand.
  • a binding pair e.g., a heterologous scaffold
  • the first member of the binding pair is linked to (e.g., covalently or non-covalently bound to) a second cognate member of the binding pair (e.g., an adapt
  • modifications of a capsid protein as described herein include those that generally result from modifications at the genetic level, e.g., via modification of a cap gene, such as modifications that insert first member of a binding pair (e.g., a protein: protein binding pair, a proteimnucleic acid binding pair), a detectable label, etc., for display by the Cap protein.
  • modifications that insert first member of a binding pair e.g., a protein: protein binding pair, a proteimnucleic acid binding pair
  • a detectable label e.g., a detectable label, etc.
  • the first member forms a binding pair with an immunoglobulin constant domain.
  • the first member forms a binding pair with a metal ion, e.g., Ni 2+ , Co 2+ , Cu 2+ , Zn 2+ , Fe 3+ , etc.
  • the first member is selected from the group consisting of Streptavidin, Strep II, HA, L14, 4C-RGD, LH, and Protein A.
  • the binding pair comprises an enzyme ucleic acid binding pair.
  • the first member comprises a HUH-endonuclease or HUH-tag and the second member comprises a nucleic acid binding domain.
  • the first member comprises a HUH tag. See, e.g., U.S. 2021/0180082, incorporated herein in its entirety by reference.
  • a capsid protein of the invention comprises at least a first member of a peptide:peptide binding pair.
  • each of a first member and a second member of a peptide:peptide binding pair comprises an intein. See, e.g., Wagner et al., (2021) Adv. Sci. 8: 2004018 (1 of 22); Muik et al. (2017) Biomaterials 144: 84, each of which is incorporated herein in its entirety by reference.
  • a first member is a B cell epitope, e.g., is between about 1 amino acid and about 35 amino acids in length, and forms a binding pair with an antibody paratope, e.g., an immunoglobulin variable domain.
  • a capsid protein of the invention may be modified to comprise a detectable label as a first member of a binding pair. Many detectable labels are known in the art. (See, e.g. Nilsson et al. (1997) “Affinity fusion strategies for detection, purification, and immobilization of modified proteins” Protein Expression and Purification 11 : 1-16, Terpe et al.
  • Detectable labels include, but are not limited to, a polyhistidine detectable labels (e.g., a His-6, His-8, or His-10) that binds immobilized divalent cations (e.g, Ni 2+ ), a biotin moiety (e.g., on an in vivo biotinylated polypeptide sequence) that binds immobilized avidin, a GST (glutathione S -transferase) sequence that binds immobilized glutathione, an S tag that binds immobilized S protein, an antigen that binds an immobilized antibody or domain or fragment thereof (including, e.g., T7, myc, FLAG, and B tags that bind corresponding antibodies), a FLASH Tag (a high detectable label that couples to specific arsenic based moi
  • a polyhistidine detectable labels e.g., a His-6, His-8, or His-10
  • immobilized divalent cations e.g,
  • a detectable label is a SNAP -tag, commercially available from Covalys (www.covalys.com).
  • a detectable label disclosed herein comprises a detectable label recognized by an antibody paratope, wherein the detectable label and the antibody paratope form a proteimprotein binding pair.
  • a capsid protein of the invention comprises a first member of a protein: protein binding pair comprising a detectable label, which may also be used for the detection and/or isolation of the Cap protein and/or as a first member of a protein: protein binding pair.
  • a detectable label acts as a first member of a proteimprotein binding pair for the binding of a targeting ligand comprising a multispecific binding protein that may bind both the detectable label and a target expressed by a cell of interest.
  • a Cap protein of the invention comprises a first member of a proteimprotein binding pair comprising c-myc (SEQ ID NO: 818).
  • the first member comprises a Bl epitope (SEQ ID NO: 819).
  • a capsid protein is modified to comprise a Bl epitope in the VP3 region.
  • the first member is selected from the group consisting of FLAG, HA and c-myc (SEQ ID NO: 818).
  • a capsid protein comprises a first member of a protein: protein binding pair, wherein the protein: protein binding pair forms a covalent isopeptide bond.
  • the first member of a peptide:peptide binding pair is covalently bound via an isopeptide bond to a cognate second member of the peptide:peptide binding pair, and optionally wherein the cognate second member of the peptide:peptide binding pair is fused with a targeting ligand, which targeting ligand binds a target expressed by a cell of interest.
  • the protein: protein binding pair may be selected from the group consisting of SpyTag: SpyCatcher, SpyTag002:SpyCatcher002, SpyTag003:SpyCatcher003, SpyTag:KTag, Isopeptag:pilin-C, and SnoopTag: SnoopCatcher.
  • the first member is SpyTag (or a biologically active portion or variant thereof) and the protein (second cognate member) is SpyCatcher (or a biologically active portion or variant thereof).
  • the first member is SpyTag (or a biologically active portion or variant thereof) and the protein (second cognate member) is KTag (or a biologically active portion or variant thereof).
  • the first member is KTag (or a biologically active portion or variant thereof) and the protein (second cognate member) is SpyTag (or a biologically active portion or variant thereof).
  • the first member is SnoopTag (or a biologically active portion or variant thereof) and the protein (second cognate member) is SnoopCatcher (or a biologically active portion or variant thereof).
  • the first member is Isopeptag (or a biologically active portion or variant thereof) and the protein (second cognate member) is Pilin-C (or a biologically active portion or variant thereof).
  • a Cap protein of the invention comprises a SpyTag, or a biologically active portion or variant thereof.
  • a first member of a proteimprotein binding pair and/or detectable label is operably linked to (translated in frame with, chemically attached to, and/or displayed by) a Cap protein of the invention via a first and/or second linker, e.g, an amino acid spacer that is at least one amino acid in length.
  • the first member of a protein: protein binding pair is flanked by a first and/or second linker, e.g, a first and/or second amino acid spacer, each of which spacer is at least one amino acid in length.
  • the first and/or second linkers are not identical. In some embodiments, the first and/or second linker is each independently one or two amino acids in length. In some embodiments, the first and/or second linker is each independently one, two or three amino acids in length. In some embodiments, the first and/or second linker is each independently one, two, three, or four amino acids in length. In some embodiments, the first and/or second linker is each independently one, two, three, four, or five amino acids in length. In some embodiments, the first and/or second linker are each independently one, two, three, four, or five amino acids in length.
  • the first and/or second linker is each independently one, two, three, four, five, or six amino acids in length. In some embodiments, the first and/or second linker is each independently one, two, three, four, five, six, or seven amino acids in length. In some embodiments, the first and/or second linker is each independently one, two, three, four, five, six, seven, or eight amino acids in length. In some embodiments, the first and/or second linker is each independently one, two, three, four, five, six, seven, eight or nine amino acids in length. In some embodiments, the first and or second linker is each independently one, two, three, four, five, six, seven, eight, nine, or ten amino acids in length. In some embodiments, the first and or second linker is each independently one, two, three, four, five, six, seven, eight, nine, or ten amino acids in length. In some embodiments, the first and or second linker is each independently one, two, three, four, five, six, seven, eight, nine,
  • the first and second linkers are identical in sequence and/or in length and are each one amino acid in length. In some embodiments, the first and second linkers are identical in length, and are each one amino acid in length. In some embodiments, the first and second linkers are identical in length, and are each two amino acids in length. In some embodiments, the first and second linkers are identical in length, and are each three amino acids in length. In some embodiments, the first and second linkers are identical in length, and are each four amino acids in length, e.g, the linker is GLSG (SEQ ID NO: 823). In some embodiments, the first and second linkers are identical in length, and are each five amino acids in length.
  • the first and second linkers are identical in length, and are each six amino acids in length, e.g., the first and second linkers each comprise a sequence of GLSGSG (SEQ ID NO: 824) or GSGESG (SEQ ID NO: 828).
  • the first and second linkers are identical in length, and are each seven amino acids in length.
  • the first and second linkers are identical in length, and are each eight amino acids in length, e.g., the first and second linkers each comprise a sequence of GLSGLSGS (SEQ ID NO: 825).
  • the first and second linkers are identical in length, and are each nine amino acids in length.
  • the first and second linkers are identical in length, and are each ten amino acids in length, e.g., the first and second linkers each comprise a sequence of GLSGLSGLSG (SEQ ID NO: 826) or GLSGGSGLSG (SEQ ID NO: 827). In some embodiments, the first and second linkers are identical in length, and are each more than ten amino acids in length.
  • a first member of a protein protein binding pair amino acid sequence as described herein, e.g., comprising a first member of a specific binding pair by itself or in combination with one or more linkers, is between about 5 amino acids to about 50 amino acids in length.
  • the first member of a protein: protein binding pair amino acid sequence is at least 5 amino acids in length.
  • the first member of a protein: protein binding pair amino acid sequence is 6 amino acids in length.
  • the first member of a protein: protein binding pair amino acid sequence is 7 amino acids in length.
  • the first member of a proteimprotein binding pair amino acid sequence is 8 amino acids in length.
  • the first member of a protein: protein binding pair amino acid sequence is 9 amino acids in length. In some embodiments, the first member of a protein: protein binding pair amino acid sequence is 10 amino acids in length. In some embodiments, the first member of a protein :protein binding pair amino acid sequence is 11 amino acids in length. In some embodiments, the first member of a protein: protein binding pair amino acid sequence is 12 amino acids in length. In some embodiments, the first member of a protein: protein binding pair amino acid sequence is 13 amino acids in length. In some embodiments, the first member of a protein :protein binding pair amino acid sequence is 14 amino acids in length. In some embodiments, the first member of a protein: protein binding pair amino acid sequence is 15 amino acids in length.
  • the first member of a protein: protein binding pair amino acid sequence is 16 amino acids in length. In some embodiments, the first member of a protein :protein binding pair amino acid sequence is 17 amino acids in length. In some embodiments, the first member of a protein: protein binding pair amino acid sequence is 18 amino acids in length. In some embodiments, the first member of a protein: protein binding pair amino acid sequence is 19 amino acids in length. In some embodiments, the first member of a protein :protein binding pair amino acid sequence is 20 amino acids in length. In some embodiments, the first member of a protein: protein binding pair amino acid sequence is 21 amino acids in length. In some embodiments, the first member of a protein: protein binding pair amino acid sequence is 22 amino acids in length.
  • the first member of a protein :protein binding pair amino acid sequence is 23 amino acids in length. In some embodiments, the first member of a protein: protein binding pair amino acid sequence is 24 amino acids in length. In some embodiments, the first member of a protein: protein binding pair amino acid sequence is 25 amino acids in length. In some embodiments, the first member of a protein :protein binding pair amino acid sequence is 26 amino acids in length. In some embodiments, the first member of a protein: protein binding pair amino acid sequence is 27 amino acids in length. In some embodiments, the first member of a protein: protein binding pair amino acid sequence is 28 amino acids in length. In some embodiments, the first member of a protein :protein binding pair amino acid sequence is 29 amino acids in length.
  • the first member of a protein: protein binding pair amino acid sequence is 30 amino acids in length. In some embodiments, the first member of a protein: protein binding pair amino acid sequence is 31 amino acids in length. In some embodiments, the first member of a protein :protein binding pair amino acid sequence is 32 amino acids in length. In some embodiments, the first member of a protein: protein binding pair amino acid sequence is 33 amino acids in length. In some embodiments, the first member of a protein: protein binding pair amino acid sequence is 34 amino acids in length. In some embodiments, the first member of a protein :protein binding pair amino acid sequence is 35 amino acids in length. In some embodiments, the first member of a protein: protein binding pair amino acid sequence is 36 amino acids in length.
  • the first member of a protein: protein binding pair amino acid sequence is 37 amino acids in length. In some embodiments, the first member of a proteimprotein binding pair amino acid sequence is 38 amino acids in length. In some embodiments, the first member of a protein: protein binding pair amino acid sequence is 39 amino acids in length. In some embodiments, the first member of a protein: protein binding pair amino acid sequence is 40 amino acids in length. In some embodiments, the first member of a proteimprotein binding pair amino acid sequence is 41 amino acids in length. In some embodiments, the first member of a protein: protein binding pair amino acid sequence is 42 amino acids in length. In some embodiments, the first member of a protein: protein binding pair amino acid sequence is 43 amino acids in length.
  • the first member of a protein :protein binding pair amino acid sequence is 44 amino acids in length. In some embodiments, the first member of a protein: protein binding pair amino acid sequence is 45 amino acids in length. In some embodiments, the first member of a protein: protein binding pair amino acid sequence is 46 amino acids in length. In some embodiments, the first member of a proteimprotein binding pair amino acid sequence is 47 amino acids in length. In some embodiments, the first member of a protein: protein binding pair amino acid sequence is 48 amino acids in length. In some embodiments, the first member of a protein: protein binding pair amino acid sequence is 49 amino acids in length. In some embodiments, the first member of a proteimprotein binding pair amino acid sequence is 50 amino acids in length.
  • a targeting ligand comprises a multispecific binding molecule comprising (i) an antibody paratope that specifically binds the detectable label and (ii) a second binding domain that specifically binds a receptor, which may be conjugated to the surface of a bead (e.g, for purification) or expressed by a target cell (e.g., a muscle stem cell, myoblast, myocyte, any combination thereof, etc.).
  • a multispecific binding molecule comprising (i) an antibody paratope that specifically binds the detectable label and (ii) a second binding domain that specifically binds a receptor targets the viral particle.
  • Such “targeting” or “directing” may include a scenario in which the wildtype viral particle targets several cells within a tissue and/or several organs within an organism, which broad targeting of the tissue or organs is reduced to abolished by insertion of the detectable label, and which retargeting to more specific cells in the tissue or more specific organ in the organism is achieved with the multispecific binding molecule.
  • Such retargeting or redirecting may also include a scenario in which the wildtype viral particle targets a tissue, which targeting of the tissue is reduced to abolished by insertion of the detectable label, and which retargeting to a completely different tissue is achieved with the multispecific binding molecule.
  • An antibody paratope as described herein generally comprises at a minimum a complementarity determining region (CDR) that specifically recognizes the detectable label, e.g, a CDR3 region of a heavy and/or light chain variable domain.
  • a multispecific binding molecule comprises an antibody (or portion thereof) that comprises the antibody paratope that specifically binds the detectable label.
  • a multispecific binding molecule may comprise a single domain heavy chain variable region or a single domain light chain variable region, wherein the single domain heavy chain variable region or single domain light chain variable region comprises an antibody paratope that specifically binds the detectable label.
  • a multispecific binding molecule may comprise an Fv region, e.g., a multispecific binding molecule may comprise an scFv, that comprises an antibody paratope that specifically binds the detectable label.
  • a multispecific binding molecule as described herein comprises an antibody paratope that specifically binds c-myc (SEQ ID NO:818).
  • a viral capsid comprising a modified viral capsid protein as described herein is a mosaic capsid, e.g., comprises at least two sets of VP1, VP2, and/or VP3 proteins, each set of which is encoded by a different cap gene.
  • a mosaic capsid herein generally refers to a mosaic of a first viral capsid protein modified to comprise a first member of a binding pair and a second corresponding viral capsid protein lacking the first member of a binding pair.
  • the second viral capsid protein lacking the first member of a binding pair may be referred to as a reference capsid protein encoded by a reference cap gene.
  • a VP1, VP2, and/or VP3 reference capsid protein may comprise an amino acid sequence identical to that of the viral VP1, VP2, and/or VP3 capsid protein modified with a first member of a binding pair, except that the reference capsid protein lacks the first member of a binding pair.
  • a VP1, VP2, and/or VP3 reference capsid protein corresponds to the viral VP1, VP2, and/or VP3 capsid protein modified with a first member of a binding pair, except that the reference capsid protein lacks the first member of a binding pair.
  • a VP1 reference capsid protein corresponds to the viral VP1 capsid protein modified with a first member of a binding pair, except that the reference capsid protein lacks the first member of a binding pair.
  • a VP2 reference capsid protein corresponds to the viral VP2 capsid protein modified with a first member of a binding pair, except that the reference capsid protein lacks the first member of a binding pair.
  • a VP3 reference capsid protein corresponds to the viral VP3 capsid protein modified with a first member of a binding pair, except that the reference capsid protein lacks the first member of a binding pair.
  • a reference protein may be a corresponding capsid protein from which portions thereof form part of the chimeric capsid protein.
  • mosaic capsid comprising a chimeric AAV2/AAAV VP1 capsid protein modified to comprise a first member of a binding pair may further comprise as a reference capsid protein: an AAV2 VP1 capsid protein lacking the first member, an AAAV VP1 capsid protein lacking the first member, a chimeric AAV2/AAAV VP1 capsid protein lacking the first member.
  • a mosaic capsid comprising a chimeric AAV2/AAAV VP2 capsid protein modified to comprise a first member of a binding pair may further comprise as a reference capsid protein: an AAV2 VP2 capsid protein lacking the first member, an AAAV VP1 capsid protein lacking the first member, a chimeric AAV2/AAAV VP2 capsid protein lacking the first member.
  • a mosaic capsid comprising a chimeric AAV2/AAAV VP3 capsid protein modified to comprise a first member of a binding pair may further comprise as a reference capsid protein: an AAV2 VP2 capsid protein lacking the first member, an AAAV VP1 capsid protein lacking the first member, a chimeric AAV2/AAAV VP3 capsid protein lacking the first member.
  • a reference capsid protein may be any capsid protein so long as it that lacks the first member of the binding pair and is able to form a capsid with the first capsid protein modified with the first member of a binding pair.
  • mosaic particles may be generated by transfecting mixtures of the modified and reference Cap genes into production cells at the indicated ratios.
  • the protein subunit ratios e. , modified VP proteimunmodified VP protein ratios
  • the protein subunit ratios in the particle may, but do not necessarily, stoichiometrically reflect the ratios of the at least two species of the cap gene encoding the first capsid protein modified with a first member of a binding pair and the one or more reference cap genes, e.g., modified cap gene:reference cap gene(s) transfected into packaging cells.
  • the protein subunit ratios in the particle do not stoichiometrically reflect the modified cap gene reference cap gene(s) ratio transfected into packaging cells.
  • the protein subunit ratio ranges from about 1 :59 to about 59:1. In some mosaic viral particle embodiments, the protein subunit is at least about 1: 1 (e.g., the mosaic viral particle comprises about 30 modified capsid proteins and about 30 reference capsid protein). In some mosaic viral particle embodiments, the protein subunit ratio is at least about 1:2 e.g., the mosaic viral particle comprises about 20 modified capsid proteins and about 40 reference capsid proteins). In some mosaic viral particle embodiments, the protein subunit ratio is at least about 3:5. In some mosaic viral particle embodiments, the protein subunit ratio is at least about 1 :3 (e.g., the mosaic viral particle comprises about 15 modified capsid proteins and about 45 reference capsid proteins) .
  • the protein subunit ratio is at least about 1:4 (e.g., the mosaic viral particle comprises about 12 modified capsid proteins and 48 reference capsid proteins). In some mosaic viral particle embodiments, the protein subunit ratio is at least about 1:5 (e.g., the mosaic viral particle comprises 10 modified capsid proteins and 50 reference capsid proteins). In some mosaic viral particle embodiments, the protein subunit ratio is at least about 1:6. In some mosaic viral particle embodiments, the protein subunit ratio is at least about 1:7. In some mosaic viral particle embodiments, the protein subunit ratio is at least about 1 :8.
  • the protein subunit ratio is at least about 1 :9 (e.g., the mosaic viral particle comprises about 6 modified capsid proteins and about 54 reference capsid proteins). In some mosaic viral particle embodiments, the protein subunit ratio is at least about 1: 10. In some mosaic viral particle embodiments, the protein subunit ratio is at least about 1: 11 (e.g., the mosaic viral particle comprises about 5 modified capsid proteins and about 55 reference capsid proteins). In some mosaic viral particle embodiments, the protein subunit ratio is at least about 1 : 12. In some mosaic viral particle embodiments, the protein subunit ratio is at least about 1 : 13.
  • the protein subunit ratio is at least about 1 :14 (e.g., the mosaic viral particle comprises about 4 modified capsid proteins and about 56 reference capsid proteins). In some mosaic viral particle embodiments, the protein subunit ratio is at least about 1 : 15. In some mosaic viral particle embodiments, the protein subunit ratio is at least about 1 : 19 (e.g., the mosaic viral particle comprises about 3 modified capsid proteins and about 57 reference capsid proteins). In some mosaic viral particle embodiments, the protein subunit ratio is at least about 1 :29 (e.g, the mosaic viral particle comprises about 2 modified capsid proteins and about 58 reference capsid proteins). In some mosaic viral particle embodiments, the protein subunit ratio is at least about 1 :59.
  • the protein subunit ratio is at least about 2: 1 (e.g, the mosaic viral particle comprises about 40 modified capsid proteins and about 20 reference capsid proteins). In some mosaic viral particle embodiments, the protein subunit ratio is at least about 5:3. In some mosaic viral particle embodiments, the protein subunit ratio is at least about 3 : 1 (e.g, the mosaic viral particle comprises about 45 modified capsid proteins and about 15 reference capsid proteins). In some mosaic viral particle embodiments, the protein subunit ratio is at least about 4: 1 (e.g., the mosaic viral particle comprises about 48 modified capsid proteins and 12 reference capsid proteins).
  • the protein subunit ratio is at least about 5: 1 (e.g, the mosaic viral particle comprises 50 modified capsid proteins and 10 reference capsid proteins). In some mosaic viral particle embodiments, the protein subunit ratio is at least about 6: 1. In some mosaic viral particle embodiments, the protein subunit ratio is at least about 7: 1. In some mosaic viral particle embodiments, the protein subunit ratio is at least about 8:1. In some mosaic viral particle embodiments, the protein subunit ratio is at least about 9: 1 (e.g., the mosaic viral particle comprises about 54 modified capsid proteins and about 6 reference capsid proteins). In some mosaic viral particle embodiments, the protein subunit ratio is at least about 10:1.
  • the protein subunit ratio is at least about 11 :1 (e.g., the mosaic viral particle comprises about 55 modified capsid proteins and about 5 reference capsid proteins). In some mosaic viral particle embodiments, the protein subunit ratio is at least about 12:1. In some mosaic viral particle embodiments, the protein subunit ratio is at least about 13:1. In some mosaic viral particle embodiments, the protein subunit ratio is at least about 14: 1 (e.g., the mosaic viral particle comprises about 56 modified capsid proteins and about 4 reference capsid proteins). In some mosaic viral particle embodiments, the protein subunit ratio is at least about 15: 1.
  • the protein subunit ratio is at least about 19: 1 (e.g, the mosaic viral particle comprises about 57 modified capsid proteins and about 3 reference capsid proteins). In some mosaic viral particle embodiments, the protein subunit ratio is at least about 29: 1 (e.g, the mosaic viral particle comprises about 58 modified capsid proteins and about 2 reference capsid proteins). In some mosaic viral particle embodiments, the protein subunit ratio is at least about 59: 1.
  • the protein subunit ratio may be 1:0 wherein each capsid protein of the non-mosaic viral particle is modified with a first member of a binding pair. In some non-mosaic viral particle embodiments, the protein subunit ratio may be 0:1 wherein each capsid protein of the non-mosaic viral particle is not modified with a first member of a binding pair.
  • nucleic acids that encode a VP3 capsid protein of the invention may be, but are not necessarily, encoded by overlapping reading frames of the same gene with staggered start codons.
  • a nucleic acid that encodes a VP3 capsid protein of the invention does not also encode a VP2 capsid protein or VP1 capsid protein of the invention.
  • a nucleic acid that encodes a VP3 capsid protein of the invention may also encode a VP2 capsid protein of the invention but does not also encode a VP1 capsid of the invention.
  • a nucleic acid that encodes a VP3 capsid protein of the invention may also encode a VP2 capsid protein of the invention and a VP1 capsid of the invention.
  • One embodiment of the present invention is a multimeric structure comprising a modified viral capsid protein of the present invention.
  • a multimeric structure comprises at least 5, preferably at least 10, more preferably at least 30, most preferably at least 60 modified viral capsid proteins comprising a first member of a specific binding pair as described herein. They can form regular viral capsids (empty viral particles) or viral particles (capsids encapsidating a nucleotide of interest). The formation of viral particles comprising a viral genome is a highly preferred feature for use of the modified viral capsids described herein.
  • a further embodiment of the present invention is the use of at least one modified viral capsid protein and/or a nucleic acid encoding same, preferably at least one multimeric structure (e.g., viral particle) for the manufacture of and use in transfer of a nucleotide of interest to a target cell e.g., a muscle stem cell, myoblast, myocyte, any combination thereof, etc.). Insertion sites
  • variable regions VR I to VR IX provide an overlay of ribbons from different dependoparvovirus at Figure 7, depicting the variable regions VR I to VR IX.
  • a skilled artisan may determine which amino acids within the variable region correspond to amino acid sequence of AAV that can accommodate the insertion of, e.g., a targeting ligand as described herein, a first member of a binding pair and/or detectable label.
  • the targeting ligand, first member of a binding pair, and/or detectable label may be inserted into a variable region or variable loop of an AAV capsid protein, a GH loop of an AAV capsid protein, etc.
  • the targeting ligand, first member of a binding pair, and/or detectable label may be inserted into a variable region or variable loop VRI of an AAV capsid protein.
  • the targeting ligand, first member of a binding pair, and/or detectable label may be inserted into a variable region or variable loop VRII of an AAV capsid protein.
  • the targeting ligand, first member of a binding pair, and/or detectable label may be inserted into a variable region or variable loop VRIII of an AAV capsid protein. In some embodiments, the targeting ligand, first member of a binding pair, and/or detectable label may be inserted into a variable region or variable loop VRIV of an AAV capsid protein. In some embodiments, the targeting ligand, first member of a binding pair, and/or detectable label may be inserted into a variable region or variable loop VRV of an AAV capsid protein. In some embodiments, the targeting ligand, first member of a binding pair, and/or detectable label may be inserted into a variable region or variable loop VRV of an AAV capsid protein.
  • the targeting ligand, first member of a binding pair, and/or detectable label may be inserted into a variable region or variable loop VRVI of an AAV capsid protein. In some embodiments, the targeting ligand, first member of a binding pair, and/or detectable label may be inserted into a variable region or variable loop VRVII of an AAV capsid protein. In some embodiments, the targeting ligand, first member of a binding pair, and/or detectable label may be inserted into a variable region or variable loop VRIII of an AAV capsid protein. In some embodiments, the targeting ligand, first member of a binding pair, and/or detectable label may be inserted into a variable region or variable loop VRIX of an AAV capsid protein.
  • the first member of a binding pair and/or detectable label is inserted in a VP 1 capsid protein of a non-primate animal AAV after an amino acid position corresponding with an amino acid position selected from the group consisting of G453 of AAV2 capsid protein VP1, N587 of AAV2 capsid protein VP1, G453 of AAV9 capsid protein VP1, and A589 of AAV9 capsid protein VP1.
  • the first member of a binding pair and/or detectable label is inserted in a VP 1 capsid protein of a non-primate animal AAV between amino acids that correspond with N587 and R588 of an AAV2 VP1 capsid.
  • the nomenclature I-###, I# or the like herein refers to the insertion site (I) with ### naming the amino acid number relative to the VP I protein of an AAV capsid protein, however such the insertion may be located directly N- or C-terminal, preferably C-terminal of one amino acid in the sequence of 5 amino acids N- or C-terminal of the given amino acid, preferably 3, more preferably 2, especially 1 amino acid(s) N- or C-terminal of the given amino acid.
  • positions referred to herein are relative to the VP1 protein encoded by an AAV capsid gene, and corresponding positions (and point mutations thereof) may be easily identified for the VP2 and VP3 capsid proteins encoding by the capsid gene by performing a sequence alignment of the VP 1, VP2 and VP3 proteins encoded by the appropriate AAV capsid gene.
  • Additional suitable insertion sites of a non-primate animal VP1 capsid protein include those corresponding to 1-1, 1-34, 1-138, 1-139, 1-161, 1-261, 1-266, 1-381, 1-447, 1-448, 1-
  • a modified virus capsid protein as described herein may be a non-primate animal capsid protein comprising a first member of a binding pair and/or detectable label inserted into a position corresponding with a position of an AAV2 capsid protein selected from the group consisting of 1-1, 1-34, 1-138, 1-139, 1-161, 1-261, 1-266, 1-381, 1-447, 1-448, 1-459, 1-471, 1-520, 1-534, 1-570, 1-573, 1-584, 1-587, 1-
  • a modified virus capsid protein as described herein may be a non-primate animal capsid protein comprising a targeting ligand, first member of a binding pair and/or detectable label inserted into a position corresponding with a position selected from the group consisting of 1-587 (AAV1), 1-589 (AAV1), 1-585 (AAV3), 1-585 (AAV4), 1-585 (AAV5), and a combination thereof.
  • AAV1 1-587
  • AAV1 1-589
  • AAV3 1-585
  • AAV4 1-585
  • AAV5 1-585
  • the first member of a binding pair and/or detectable label is inserted in a VP 1 capsid protein of a non-primate animal AAV after an amino acid position corresponding with an amino acid position selected from the group consisting of 1444 of an avian AAV capsid protein VP1, 1580 of an avian AAV capsid protein VP1, 1573 of a bearded dragon AAV capsid protein VP 1, 1436 of a bearded dragon AAV capsid protein VP1, 1429 of a sea lion AAV capsid protein VP1, 1430 of a sea lion AAV capsid protein VP1, 1431 of a sea lion AAV capsid protein VP1, 1432 of a sea lion AAV capsid protein VP1, 1433 of a sea lion AAV capsid protein VP1, 1434 of a sea lion AAV capsid protein VP1, 1436 of a sea lion AAV capsid protein VP1, 1437
  • insertion into the corresponding position of the coding nucleic acid of one of these sites of the cap gene leads to an insertion into VP1, VP2 and/or VP3, as the capsid proteins are encoded by overlapping reading frames of the same gene with staggered start codons. Therefore, for AAV2, for example, according to this nomenclature insertions between amino acids 1 and 138 are only inserted into VP1, insertions between 138 and 203 are inserted into VP1 and VP2, and insertions between 203 and the C-terminus are inserted into VP1, VP2 and VP3, which is of course also the case for the insertion site 1-587. Therefore, the present invention encompasses structural genes of AAV with corresponding insertions in the VP 1, VP2 and/or VP3 proteins.
  • a viral capsid comprising the modified viral capsid protein comprising the first and second members of a binding pair is able to infect a specific cell, e.g., has an enhanced capacity to target and bind a specific cell compared to that of a control viral capsid that is identical to the modified viral capsid protein except that it lacks either or both the first and second members of a binding pair, e.g., comprises a control capsid protein.
  • a viral capsid comprising a modified viral capsid protein as described herein bound to the first and second members of a binding pair linked to a targeting ligand exhibits a detectable transduction efficiency compared to the undetectable transduction efficiency of a control viral capsid.
  • a viral capsid comprising a modified viral capsid protein as described herein bound to the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 10% greater than the transduction efficiency of a control viral capsid.
  • a viral capsid comprising a modified viral capsid protein as described herein bound to the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 20% greater than the transduction efficiency of a control viral capsid.
  • a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 30% greater than the transduction efficiency of a control viral capsid.
  • a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 40% greater than the transduction efficiency of a control viral capsid.
  • a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 50% greater than the transduction efficiency of a control viral capsid.
  • a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 60% greater than the transduction efficiency of a control viral capsid.
  • a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 70% greater than the transduction efficiency of a control viral capsid.
  • a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 75% greater than the transduction efficiency of a control viral capsid.
  • a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 80% greater than the transduction efficiency of a control viral capsid.
  • a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 85% greater than the transduction efficiency of a control viral capsid.
  • a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 90% greater than the transduction efficiency of a control capsid.
  • a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 95% greater than the transduction efficiency of a control viral capsid.
  • a viral capsid comprising a modified viral capsid protein as described herein bound to the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 99% greater than the transduction efficiency of a control viral capsid.
  • a viral capsid comprising the modified viral capsid protein comprising the first and second members of a binding pair is able to infect a specific cell, e.g., has an enhanced capacity to target and bind a specific cell compared to that of a control viral capsid that is identical to the modified viral capsid protein except that it lacks either or both the first and second members of a binding pair, e.g., comprises a control capsid protein.
  • a viral capsid comprising a modified viral capsid protein as described herein bound to the first and second members of a binding pair linked to a targeting ligand exhibits a detectable transduction efficiency compared to the undetectable transduction efficiency of a control viral capsid.
  • a viral capsid comprising a modified viral capsid protein as described herein bound to the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 10% greater than the transduction efficiency of a control viral capsid.
  • a viral capsid comprising a modified viral capsid protein as described herein bound to the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 20% greater than the transduction efficiency of a control viral capsid.
  • a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 30% greater than the transduction efficiency of a control viral capsid.
  • a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 40% greater than the transduction efficiency of a control viral capsid.
  • a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 50% greater than the transduction efficiency of a control viral capsid.
  • a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 60% greater than the transduction efficiency of a control viral capsid.
  • a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 70% greater than the transduction efficiency of a control viral capsid.
  • a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 75% greater than the transduction efficiency of a control viral capsid.
  • a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 80% greater than the transduction efficiency of a control viral capsid.
  • a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 85% greater than the transduction efficiency of a control viral capsid.
  • a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 90% greater than the transduction efficiency of a control capsid.
  • a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 95% greater than the transduction efficiency of a control viral capsid.
  • a viral capsid comprising a modified viral capsid protein as described herein bound to the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 99% greater than the transduction efficiency of a control viral capsid.
  • a viral capsid comprising a modified viral capsid protein as described herein bound to the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at leastl.5-fold greater than the transduction efficiency of a control viral capsid.
  • a viral capsid comprising a modified viral capsid protein as described herein bound to the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 2-fold greater than the transduction efficiency of a control viral capsid.
  • a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 3-fold greater than the transduction efficiency of a control viral capsid.
  • a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 4-fold greater than the transduction efficiency of a control viral capsid.
  • a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 5-fold greater than the transduction efficiency of a control viral capsid.
  • a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 6-fold greater than the transduction efficiency of a control viral capsid.
  • a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 7-fold greater than the transduction efficiency of a control viral capsid.
  • a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 8-fold greater than the transduction efficiency of a control viral capsid.
  • a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 9-fold greater than the transduction efficiency of a control viral capsid.
  • a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 10-fold greater than the transduction efficiency of a control viral capsid.
  • a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 20-fold greater than the transduction efficiency of a control capsid.
  • a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 30-fold greater than the transduction efficiency of a control viral capsid.
  • a viral capsid comprising a modified viral capsid protein as described herein bound to the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 40-fold greater than the transduction efficiency of a control viral capsid.
  • a viral capsid comprising a modified viral capsid protein as described herein bound to the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 50-fold greater than the transduction efficiency of a control viral capsid.
  • a viral capsid comprising a modified viral capsid protein as described herein bound to the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 60-fold greater than the transduction efficiency of a control viral capsid.
  • a viral capsid comprising a modified viral capsid protein as described herein bound to the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 70-fold greater than the transduction efficiency of a control viral capsid.
  • a viral capsid comprising a modified viral capsid protein as described herein bound to the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 80-fold greater than the transduction efficiency of a control viral capsid.
  • a viral capsid comprising a modified viral capsid protein as described herein bound to the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 90-fold greater than the transduction efficiency of a control viral capsid.
  • a viral capsid comprising a modified viral capsid protein as described herein bound to the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 100-fold greater than the transduction efficiency of a control viral capsid
  • a viral particle of the invention comprising a viral capsid protein comprising an amino acid sequence of a capsid protein of a non-primate animal AAV, a remote AAV, or a combination thereof, and optionally comprising a first and second members of a binding pair (e.g., wherein the second member is operably linked to a targeting ligand, comprises a multispecific binding protein, etc.) is better able to evade neutralization by pre-existing antibodies in serum isolated from a human patient compared to an appropriate control viral particle (e.g, comprising a viral capsid of an AAV serotype from which a portion is included in the viral capsid of the invention, e.g, as
  • a viral particle of the invention comprising a viral capsid protein comprising an amino acid sequence of a capsid protein of a non-primate animal AAV, a remote AAV, or a combination thereof requires at least 2-fold more total IVIG or IgG for neutralization (e.g., 50% or more infection inhibition) compared to an appropriate control viral particle, e. , (e.g, a viral particle of the invention has an IC50 value that is at least 2-fold that of a control virus particle).
  • compositions comprising the antigenbinding molecules as described herein.
  • pharmaceutical compositions may be formulated with suitable carriers, excipients, and other agents that provide improved transfer, delivery, tolerance, and the like.
  • suitable carriers, excipients, and other agents that provide improved transfer, delivery, tolerance, and the like.
  • a multitude of appropriate formulations can be found in the formulary known to all pharmaceutical chemists: Remington’s Pharmaceutical Sciences, Mack Publishing Company, Easton, PA.
  • formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LIPOFECTINTM, Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax.
  • vesicles such as LIPOFECTINTM, Life Technologies, Carlsbad, CA
  • DNA conjugates such as LIPOFECTINTM, Life Technologies, Carlsbad, CA
  • DNA conjugates such as LIPOFECTINTM, Life Technologies, Carlsbad, CA
  • DNA conjugates such as LIPOFECTINTM, Life Technologies, Carlsbad, CA
  • DNA conjugates such as LIPOFECTINTM, Life Technologies, Carlsbad, CA
  • the dose of antigen-binding molecule administered to a patient may vary depending upon the age and the size of the patient, target disease, conditions, route of administration, and the like.
  • the preferred dose is typically calculated according to body weight or body surface area.
  • intravenously administer the antigen-binding molecule as described herein normally at a single dose of about 0.01 to about 20 mg/kg body weight, more preferably about 0.02 to about 7, about 0.03 to about 5, or about 0.05 to about 3 mg/kg body weight.
  • Effective dosages and schedules for administering a bispecific antigen-binding molecule may be determined empirically; for example, patient progress can be monitored by periodic assessment, and the dose adjusted accordingly.
  • interspecies scaling of dosages can be performed using well-known methods in the art (e.g., Mordenti ei ctL, 1991, Pharmaceut. Res. 5:1351).
  • Various delivery systems are known and can be used to administer the pharmaceutical composition as described herein, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the mutant viruses, receptor mediated endocytosis (see, e.g., Wu et al., 1987, J. Biol. Chem. 262:4429-4432).
  • Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes.
  • composition may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local.
  • epithelial or mucocutaneous linings e.g., oral mucosa, rectal and intestinal mucosa, etc.
  • Administration can be systemic or local.
  • a pharmaceutical composition as described herein can be delivered subcutaneously or intravenously with a standard needle and syringe.
  • a pen delivery device readily has applications in delivering a pharmaceutical composition as described herein.
  • Such a pen delivery device can be reusable or disposable.
  • a reusable pen delivery device generally utilizes a replaceable cartridge that contains a pharmaceutical composition. Once all of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can readily be discarded and replaced with a new cartridge that contains the pharmaceutical composition. The pen delivery device can then be reused.
  • a disposable pen delivery device there is no replaceable cartridge. Rather, the disposable pen delivery device comes prefilled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.
  • Numerous reusable pen and autoinjector delivery devices have applications in the subcutaneous delivery of a pharmaceutical composition as described herein. Examples include, but are not limited to AUTOPENTM (Owen Mumford, Inc., Woodstock, UK), DISETRONICTM pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75/25TM pen, HUMALOGTM pen, HUMALIN 70/30TM pen (Eli Lilly and Co., Indianapolis, IN), NOVOPENTM I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIORTM (Novo Nordisk, Copenhagen, Denmark), BDTM pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPENTM, OPTIPEN PROTM, OPTIPEN STARLETTM, and OPTICLIKTM (sanofi-aventis, Frankfurt, Germany), to name only a few.
  • Examples of disposable pen delivery devices having applications in subcutaneous delivery of a pharmaceutical composition as described herein include, but are not limited to the SOLOSTARTM pen (sanofi-aventis), the FLEXPENTM (Novo Nordisk), and the KWIKPENTM (Eli Lilly), the SURECLICKTM Autoinjector (Amgen, Thousand Oaks, CA), the PENLETTM (Haselmeier, Stuttgart, Germany), the EPIPEN (Dey, L.P.), and the HUMIRATM Pen (Abbott Labs, Abbott Park IL), to name only a few.
  • the pharmaceutical composition can be delivered in a controlled release system.
  • a pump may be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201).
  • polymeric materials can be used in another embodiment.
  • a controlled release system can be placed in proximity of the composition’s target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other controlled release systems are discussed in the review by Langer, 1990, Science 249: 1527- 1533.
  • the injectable preparations may include dosage forms for intravenous, subcutaneous, intracutaneous and intramuscular injections, drip infusions, etc. These injectable preparations may be prepared by methods publicly known. For example, the injectable preparations may be prepared, e.g., by dissolving, suspending or emulsifying the antibody or its salt described above in a sterile aqueous medium or an oily medium conventionally used for injections.
  • aqueous medium for injections there are, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents, etc., which may be used in combination with an appropriate solubilizing agent such as an alcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol, polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc.
  • an alcohol e.g., ethanol
  • a polyalcohol e.g., propylene glycol, polyethylene glycol
  • a nonionic surfactant e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil
  • oily medium there are employed, e.g., sesame oil, soybean oil, etc., which may be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc.
  • a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc.
  • the pharmaceutical compositions for oral or parenteral use described above are prepared into dosage forms in a unit dose suited to fit a dose of the active ingredients.
  • dosage forms in a unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc.
  • the amount of the aforesaid antibody contained is generally about 5 to about 500 mg per dosage form in a unit dose; especially in the form of injection, it is preferred that the aforesaid antibody is contained in about 5 to about 100 mg and in about 10 to about 250 mg for the other dosage forms.
  • a further embodiment provides a medicament comprising at least one modified viral capsid protein and appropriate targeting ligand according to this invention and/or a nucleic acid according to this invention.
  • a medicament is useful as a gene transfer particle.
  • compositions comprising the viral particles described herein and a pharmaceutically acceptable carrier and/or excipient.
  • pharmaceutical dosage forms comprising the viral particle described herein.
  • the viral particles described herein can be used for various therapeutic applications (in vivo and ex vivo) and as research tools.
  • compositions based on the viral particles disclosed herein can be formulated in any conventional manner using one or more physiologically acceptable carriers and/or excipients.
  • the viral particles may be formulated for administration by, for example, injection, inhalation or insulation (either through the mouth or the nose) or by oral, buccal, parenteral or rectal administration, or by administration directly to a tumor.
  • the pharmaceutical compositions can be formulated for a variety of modes of administration, including systemic, topical or localized administration. Techniques and formulations can be found in, for example, Remington’s Pharmaceutical Sciences, Meade Publishing Co., Easton, Pa. For systemic administration, injection is preferred, including intramuscular, intravenous, intraperitoneal, and subcutaneous.
  • the pharmaceutical compositions can be formulated in liquid solutions, preferably in physiologically compatible buffers, such as Hank's solution or Ringer's solution.
  • the pharmaceutical compositions may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms of the pharmaceutical composition are also suitable.
  • the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g. pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g. lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g. magnesium stearate, talc or silica); disintegrants (e.g. potato starch or sodium starch glycolate); or wetting agents (e.g. sodium lauryl sulfate).
  • binding agents e.g. pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose
  • fillers e.g. lactose, microcrystalline cellulose or calcium hydrogen phosphate
  • lubricants e.g. magnesium stearate, talc or silica
  • disintegrants e.g. potato starch or sodium starch glycolate
  • Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use.
  • Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g. sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g. lecithin or acacia); non-aqueous vehicles (e.g. oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g. methyl or propyl-p-hydroxybenzoates or sorbic acid).
  • the preparations can also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.
  • the pharmaceutical compositions can be formulated for parenteral administration by injection, e.g. by bolus injection or continuous infusion.
  • Formulations for injection can be presented in a unit dosage form, e.g. in ampoules or in multi -dose containers, with an optionally added preservative.
  • the pharmaceutical compositions can further be formulated as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain other agents including suspending, stabilizing and/or dispersing agents.
  • the pharmaceutical compositions can also be formulated as a depot preparation. These long acting formulations can be administered by implantation (e.g. subcutaneously or intramuscularly) or by intramuscular injection.
  • the compounds may be formulated with suitable polymeric or hydrophobic materials (e.g. as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • suitable delivery systems include microspheres, which offer the possibility of local noninvasive delivery of drugs over an extended period of time. This technology can include microspheres having a precapillary size, which can be injected via a coronary catheter into any selected part of an organ without causing inflammation or ischemia. The administered therapeutic is men slowly released from the microspheres and absorbed by the surrounding cells present in the selected tissue.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, bile salts, and fusidic acid derivatives.
  • detergents may be used to facilitate permeation.
  • Transmucosal administration can occur using nasal sprays or suppositories.
  • the viral particles described herein can be formulated into ointments, salves, gels, or creams as generally known in the art.
  • a wash solution can also be used locally to treat an injury or inflammation in order to accelerate healing.
  • Pharmaceutical forms suitable for injectable use can include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid. It must be stable under the conditions of manufacture and certain storage parameters (e.g. refrigeration and freezing) and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • a therapeutic agent can be formulated into a composition in a neutral or salt form.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
  • a carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents known in the art. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compounds or constructs in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • solutions can be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but slow release capsules or microparticles and microspheres and the like can also be employed.
  • aqueous solutions for parenteral administration in an aqueous solution
  • the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intratumorally, intramuscular, subcutaneous and intraperitoneal administration.
  • sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure.
  • one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion.
  • a subject may be administered viral particles described herein on a daily or weekly basis for a time period or on a monthly, bi-yearly or yearly basis depending on need or exposure to a pathogenic organism or to a condition in the subject (e.g., cancer).
  • parenteral administration such as intravenous, intratumorally, intradermal or intramuscular injection
  • other pharmaceutically acceptable forms include, e.g., tablets or other solids for oral administration; liposomal formulations; time release capsules; biodegradable and any other form currently used.
  • Nasal solutions can be aqueous solutions designed to be administered to the nasal passages in drops or sprays.
  • Nasal solutions can be prepared so that they are similar in many respects to nasal secretions.
  • the aqueous nasal solutions usually are isotonic and slightly buffered to maintain a pH of 5.5 to 7.5.
  • antimicrobial preservatives similar to those used in ophthalmic preparations, and appropriate drug stabilizers, if required, may be included in the formulation.
  • Various commercial nasal preparations are known and can include, for example, antibiotics and antihistamines and are used for asthma prophylaxis.
  • Oral formulations can include excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders.
  • oral pharmaceutical compositions will include an inert diluent or assimilable edible carrier, or they may be enclosed in hard or soft shell gelatin capsule, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet.
  • the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • the tablets, troches, pills, capsules and the like may also contain the following: a binder, as gum tragacanth, acacia, cornstarch, or gelatin; excipients, such as dicalcium phosphate; a disintegrating agent, such as com starch, potato starch, alginic acid and the like; a lubricant, such as magnesium stearate; and a sweetening agent, such as sucrose, lactose or saccharin may be added or a flavoring agent, such as peppermint, oil of wintergreen, or cherry flavoring.
  • a binder as gum tragacanth, acacia, cornstarch, or gelatin
  • excipients such as dicalcium phosphate
  • a disintegrating agent such as com starch, potato starch, alginic acid and the like
  • a lubricant such as magnesium stearate
  • a sweetening agent such as sucrose, lactose or saccharin may be added or
  • tablets, pills, or capsules may be coated with shellac, sugar or both.
  • a syrup of elixir may contain the active compounds sucrose as a sweetening agent methyl and propylparabens as preservatives, a dye and flavoring, such as cherry or orange flavor.
  • Kits can also include a suitable container, for example, vials, tubes, mini- or microfuge tubes, test tube, flask, bottle, syringe or other container. Where an additional component or agent is provided, the kit can contain one or more additional containers into which this agent or component may be placed. Kits herein will also typically include a means for containing the viral particles and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.
  • one or more additional active agents such as, e.g, anti-inflammatory agents, anti-viral agents, anti-fungal or anti-bacterial agents or anti -tumor agents may be needed for compositions described.
  • compositions disclosed herein may be administered by any means known in the art.
  • compositions may include administration to a subject intravenously, intratumorally, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, intrathecally, subcutaneously, subconjunctival, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularly, orally, locally, by inhalation, by injection, by infusion, by continuous infusion, by localized perfusion, via a catheter, via a lavage, in a cream, or in a lipid composition.
  • any method known to one skilled in the art maybe used for large scale production of viral particles, packaging cells and particle constructs described herein.
  • master and working seed stocks may be prepared under GMP conditions in qualified primary CEFs or by other methods.
  • Packaging cells may be plated on large surface area flasks, grown to near confluence and viral particles purified. Cells may be harvested and viral particles released into the culture media isolated and purified, or intracellular viral particles released by mechanical disruption (cell debris can be removed by large-pore depth filtration and host cell DNA digested with endonuclease). Virus particles may be subsequently purified and concentrated by tangential- flow filtration, followed by diafiltration.
  • the resulting concentrated bulk maybe formulated by dilution with a buffer containing stabilizers, filled into vials, and lyophilized. Compositions and formulations may be stored for later use. For use, lyophilized viral particles may be reconstituted by addition of diluent.
  • compositions as disclosed herein can also include adjuvants such as aluminum salts and other mineral adjuvants, tensoactive agents, bacterial derivatives, vehicles and cytokines.
  • adjuvants can also have antagonizing immunomodulating properties.
  • adjuvants can stimulate Thl or Th2 immunity.
  • Compositions and methods as disclosed herein can also include adjuvant therapy.
  • the method may comprise contacting the cell expressing CDH15 (e.g., in vitro, ex vivo, or in vivo) with an anti-hCDH15 antibody, antigen-binding fragment thereof, or pharmaceutical composition thereof of the present disclosure.
  • the activity of CDH15 includes, but is not limited to, inhibiting muscle regeneration e.g., by inhibiting activation of CDH15-expressing muscle stem cells.
  • the cell expressing CDH15 is a muscle cell.
  • the cell is a mammalian muscle cell.
  • the cell is a mammalian skeletal muscle cell.
  • the cell is a mammalian skeletal muscle cell that is not terminally differentiated. In some embodiments, the cell is a mammalian skeletal muscle cell that is terminally differentiated. In some embodiments, the cell is a muscle stem cell , a myoblast, or a myocyte. In some embodiments, the cell is a human rhabdomyosarcoma cell. [00305] Disclosed herein are also methods of accelerating the transition from quiescence to activation in a muscle stem cell, comprising contacting the muscle stem cell (e.g., in vitro, ex vivo, or in vivo) with an anti-hCDH15 antibody, antigen-binding fragment thereof, or pharmaceutical composition thereof of the present disclosure.
  • an anti-hCDH15 antibody, antigen-binding fragment thereof, or pharmaceutical composition thereof of the present disclosure comprising contacting the muscle stem cell (e.g., in vitro, ex vivo, or in vivo) with an anti-hCDH15 antibody, antigen-binding fragment
  • “Quiescence”, as used herein, refers to an inactive, non-proliferating state of a cell. For example, quiescent muscle stem cells do not proliferate or differentiate into myofibers. “Activation” as used herein in reference to muscle stem cells, refers to the ability of the muscle stem cells to proliferate and differentiate into myofibers e.g., to promote regrowth and/or repair of damaged muscle.
  • compositions comprising administering to a subject in need thereof a composition (e.g., a therapeutic composition) comprising an anti-hCDH15 antibody, antigen-binding fragment thereof or an antibody-drug conjugate comprising an anti-hCDH15 antibody (e.g., an anti-hCDH15 antibody, or ADC comprising any of the HCVR/LCVR or CDR sequences as set forth in Table 1 herein).
  • a composition e.g., a therapeutic composition
  • the therapeutic composition can comprise any of the anti-hCDH15 antibodies, antigen-binding fragments thereof, or ADCs disclosed herein, and a pharmaceutically acceptable carrier or diluent.
  • compositions e.g. , a therapeutic composition
  • a composition comprising an anti-hCDH15 antibody, antigen-binding fragment thereof, or an antibody-drug conjugate comprising an anti-hCDH15 antibody for the treatment of one or more condition as disclosed herein.
  • compositions comprising an anti-hCDH15 antibody, antigen-binding fragment thereof, or an antibody-drug conjugate comprising an anti-hCDH15 antibody for use as a therapy e.g., for use in the treatment of one or more condition as disclosed herein.
  • compositions e.g., a therapeutic compositions
  • an anti-hCDH15 antibody, antigen-binding fragment thereof, or an antibody-drug conjugate comprising an anti-hCDH15 antibody for use as a therapy e.g., for use in the treatment of one or more condition as disclosed herein.
  • the antibodies, antigen-binding fragment thereof, or an antibody-drug conjugate comprising an anti-hCDH15 antibody as described herein may be useful, inter alia, for the treatment, prevention and/or amelioration of one or more condition e.g., any disease or disorder associated with skeletal muscle tissue.
  • the antibodies and ADCs as described herein may be useful for the treatment of muscle wasting disorders (e.g., cachexia, glucocorticoid-induced muscle loss, heart failure induced muscle loss, HIV wasting, disuse, aging, etc ), muscular dystrophies/myopathies, and/or muscle-related cancers (e.g., rhabdomyosarcoma).
  • a method of treatment of a disease such as a muscle wasting disorder.
  • the method may include the step of providing an antibody or CDH15 antigen-binding fragment thereof, as described above, to a subject requiring said treatment.
  • a method of treatment of a muscle-related cancer may include the step of providing an antibody or CDH15 antigen-binding fragment thereof, as described above, to a subject requiring said treatment.
  • muscle-specific cancers include rhabdomyosarcomas, e.g, embryonal, alveolar, pleomorphic, botryoid and spindle/ sclerosing rhabdomyosarcomas.
  • a method of treatment of muscle injury may include the step of providing an antibody or CDH15 antigen-binding fragment thereof, as described above, to a subject requiring said treatment e.g., a subject that has experienced muscle injury and/or anticipates experiencing muscle injury. Accordingly, the antibody or CDH15 antigen-binding fragment thereof may be administered prior to injury e.g., prior to a muscle damaging surgery or after injury e.g., an unanticipated muscle injury.
  • a method of improving muscle regeneration in a subject following injury may include the step of providing an antibody or CDH15 antigen-binding fragment thereof, as described above, to a subject requiring said treatment.
  • “improved” in reference to muscle regeneration refers to any measurable increase in muscle repair.
  • muscle regeneration may be considered “improved” in a subject if it occurs at accelerated rate and/or to a greater degree as compared to a control subject e.g., a subject that has not been administered the anti-CDH15 antibody or antigen-binding fragment thereof.
  • “Improved” muscle regeneration may be an increase in rate or degree of at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, or at least 500%.
  • a method of restoring the muscle regenerative capacity of an aged subject may include the step of providing an antibody or CDH15 antigen-binding fragment thereof, as described above, to a subject requiring said treatment.
  • “restore” refers the ability to revert to a prior functional state.
  • the muscle regenerative capacity of an aged subject may be considered “restored” if it can be reverted to a muscle regenerative capacity at or near that of a control subject e.g, the same subject at a younger age and/or a separate subject that is representative of the aged subject at a younger age.
  • the functional state may be considered “restored” if it is comparable to (e.g., not statistically different from) the prior functional state or the functional state of a subject or subjects that is representative of the prior functional state.
  • the functional state may also be considered “restored” if it is no more than 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 45%, 50%, 60%, 70%, 80%, or 90% different from the prior functional state or the functional state of a subject or subjects that is representative of the prior functional state.
  • anti-hCDH15 antibodies as described herein have various utilities.
  • anti-hCDH15 antibodies as described herein may be used in diagnostic assays for CDH15, e.g., detecting its expression in specific cells, tissues, etc., e.g., as a reagent to identify /label skeletal muscle fibers and/or muscle stem cells.
  • diagnostic assays for CDH15 e.g., detecting its expression in specific cells, tissues, etc.
  • diagnostic and prognostic assay techniques known in the art may be used, such as competitive binding assays, direct or indirect sandwich assays and immunoprecipitation assays conducted in either heterogeneous or homogeneous phases (Zola (1987) Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc. pp. 147-1581).
  • the antibodies used in the assays can be labeled with a detectable moiety.
  • the detectable moiety should be capable of producing, either directly or indirectly, a detectable signal. Any method known in the art for conjugating the antibody to the detectable moiety may be employed. Methods of Use and Making Viral Particles
  • a further embodiment of the modified viral capsids described herein is the use of the modified viral capsids for delivering a nucleotide of interest, e.g., a reporter gene or a therapeutic gene, to a target cell (e.g., a muscle stem cell, myoblast, myocyte, any combination thereof, etc.).
  • a nucleotide of interest e.g., a reporter gene or a therapeutic gene
  • a target cell e.g., a muscle stem cell, myoblast, myocyte, any combination thereof, etc.
  • packaging of a nucleotide of interest comprises replacing an AAV genome between AAV ITR sequences with a gene of interest to create a transfer plasmid, which is then encapsulated in an AAV capsid according to well-known methods
  • a modified viral capsid as described herein may encapsulate a transfer plasmid and/or a nucleotide of interest, which may generally comprise 5' and 3' inverted terminal repeat (ITR) sequences flanking a gene of interest, e.g., reporter gene(s) or therapeutic gene(s), or a portion of the gene of interest (which may be under the control of a viral or non-viral promoter).
  • ITR inverted terminal repeat
  • a transfer plasmid and/or nucleotide of interest comprises from 5’ to 3’: a 5’ ITR, a promoter, a gene (e.g., a reporter and/or therapeutic gene) and a 3TTR.
  • a consideration for AAV transfer plasmid design is that a wildtype AAV genome is ⁇ 4.7kb.
  • strategies that provide for packaging nucleotides of interest that exceed the packaging capacity of an individual AAV.
  • Such strategies include, but are not limited to, dual-vector strategies that exploit ITR-mediated recombination to express genes of interest that are larger than a wildtype AAV genome by way of transcript splicing across intermolecularly recombined ITRs from two complementary vector genomes, vector recombination by homology, RNA trans-splicing, and/or protein “transsplicing” via split intein designs. See, e.g., Nakai, H. et al.
  • a trans-splicing approach takes advantage of the ability of AAV ITR sequences to concatemerized to reconstitute full-length genomes, wherein each of two or more viral capsids, respectively, encapsulate one of two or more transfer plasmids, each of which transfer plasmid comprises a portion of the gene of interest.
  • the two transfer plasmids may be designed as follows: the 5’-transfer plasmid comprises the promoter, the 5’ portion of the coding sequence of the gene of interest, and a splicing donor (SD) signal; the 3 ’-transfer plasmid comprises a splicing acceptor (SA) signal, the 3’ portion of the gene of interest, and the polyA signal.
  • SD splicing donor
  • SA splicing acceptor
  • a large gene of interest is also split when taking an overlapping region approach.
  • the 5’ and 3’ portions (and thus the 5’ transfer plasmid and 3’ transfer plasmid) share a recombinogenic sequence, e.g, region of homology, e.g., each portion comprises an overlapping sequence.
  • the gene of interest is made whole in a targeted cell via homologous recombination mediated by the recombinogenic sequence, e.g., homology/overlapping region.
  • the 5 ’-transfer plasmid and 3 ’-transfer plasmid each comprise a highly recombinogenic sequence, wherein the recombinogenic sequence is placed downstream of an SD signal of a 5’ portion of the coding sequence of the gene of interest and upstream of an SA signal of a 3’ portion of the coding sequence of the gene of interest.
  • the gene of interest may be made whole either via ITR-mediated concatemerization and splicing and/or by homologous recombination.
  • Trans-splicing at the RNA or protein levels may also be utilized.
  • two transfer plasmids may respectively encode for 5’ and 3’ fragments of the pre-mRNA of a large gene and share an intronic hybridization domain that can favor trans-splicing, leading to joining of the two half-transcripts into an intact full-length mRNA.
  • split-inteins Protein trans-splicing occurs post-translationally and is catalyzed by intervening proteins called split-inteins.
  • Split-inteins are expressed as two independent polypeptides (N- intein and C-intein) at the extremities of two host proteins.
  • the N-intein and C-intein polypeptides remain catalytically inactive until they encounter each other.
  • each intein precisely excises itself from the host protein while mediating ligation of the N- and C- host polypeptides via a peptide bond.
  • Split-intein use has been used in AAV- based delivery of therapeutic genes of interest in muscle, liver, and retinal diseases.
  • a modified viral capsid described herein encapsulates a nucleotide of interest, wherein the nucleotide of interest comprises a portion of a gene of interest.
  • a nucleotide of interest comprising a portion of a gene of interest further comprises a splicing donor signal or a splicing acceptor signal and/or a recombinogenic sequence.
  • a nucleotide of interest comprising a portion of a gene of interest comprises an intronic hybridization domain encoding sequence. In some embodiments, a nucleotide of interest comprising a portion of a gene of interest comprises a N-intein or C-intein encoding sequence.
  • Design of the transfer plasmid/nucleotide of interest includes including one or more regulatory elements, e.g., promoter and/or enhancer elements, that will control expression of the gene of interest.
  • useful promoters include, e.g., cytomegalovirus (CMV)-promoter, the spleen focus forming virus (SFFV)-promoter, the elongation factor 1 alpha (EFla)-promoter (the 1.2 kb EFla-promoter or the 0.2 kb EFla- promoter), the chimeric EF 1 a/IF4-promoter, and the phospho-glycerate kinase (PGK)- promoter.
  • CMV cytomegalovirus
  • SFFV spleen focus forming virus
  • EFla elongation factor 1 alpha
  • PGK phospho-glycerate kinase
  • An internal enhancer may also be present in the viral construct to increase expression of the gene of interest.
  • the CMV enhancer Karasuyama et al. 1989. J. Exp. Med. 169: 13, which is incorporated herein by reference in its entirety
  • the CMV enhancer can be used in combination with the chicken [3-actin promoter.
  • tissue specific regulatory elements e.g., a muscle specific promoter and/or regulatory element may be used to drive the expression of the gene of interest.
  • a transfer plasmid and/or nucleotide of interest herein comprises an enhancer and/or promoter of muscle creatine kinase (MCK), wherein the enhancer and/or promoter of MCK drives expression of the gene of interest.
  • MCK muscle creatine kinase
  • a transfer plasmid and/or nucleotide of interest herein comprises an enhancer and/or promoter element that recruits RNA Polymerase II, wherein the enhancer and/or promoter of MCK drives expression of the gene of interest.
  • a transfer plasmid and/or nucleotide of interest herein comprises an enhancer and/or promoter element that recruits RNA Polymerase III, wherein the enhancer and/or promoter of MCK drives expression of the gene of interest.
  • bidirectional promoter vectors have also been employed for delivery of dual therapeutic gene cassettes.
  • An example of this is the bidirectional chicken 0- actin ubiquitous promoter that drives the simultaneous expression of the hexosaminidase a- and 0-subunits of the HexA enzyme, the two respective genes involved in Tay-Sachs and Sandhoff diseases.
  • a transfer plasmid and/or nucleotide of interest herein comprises a bidirectional promoter, wherein the bidirectional promoter drives the expression of two different genes of interest.
  • reporter genes can be encapsidated in a multimeric structure comprising the modified viral capsid proteins described herein.
  • exemplary reporter genes include, for example, 0-galactosidase (encoded lacZ gene), Green Fluorescent Protein (GFP), enhanced Green Fluorescent Protein (eGFP), MmGFP, blue fluorescent protein (BFP), enhanced blue fluorescent protein (eBFP), mPlum, mCherry, tdTomato, mStrawberry, J- Red, DsRed, mOrange, mKO, mCitrine, Venus, YPet, yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (eYFP), Emerald, CyPet, cyan fluorescent protein (CFP), Cerulean, T-Sapphire, luciferase, alkaline phosphatase, or a combination thereof.
  • GFP Green Fluorescent Protein
  • eGFP enhanced Green Fluorescent Protein
  • MmGFP blue fluorescent protein
  • BFP enhanced
  • a variety of therapeutic genes can also be encapsidated in a multimeric structure comprising the modified viral capsid proteins described herein, e.g, as part of a transfer particle.
  • Non-limiting examples of a therapeutic gene include those that encode a toxin (e.g., a suicide gene), a therapeutic antibody or fragment thereof, a CRISPR/Cas system or portion(s) thereof, antisense RNA, siRNA, shRNA, etc.
  • a further embodiment of the present invention is a process for the preparation of a modified capsid protein, the method comprising the steps of: a) expressing a nucleic acid encoding the modified capsid protein under suitable conditions, and b) isolating the expressed capsid protein of step a).
  • a viral particle as described herein comprises a mosaic capsid, e.g., a capsid comprising capsid proteins genetically modified as described herein (in the absence or presence of a covalent bond with a targeting ligand) in a certain ratio with reference capsid proteins.
  • a method for making such a mosaic viral particle comprises: a) expressing a nucleic acid encoding the modified capsid protein and a nucleotide encoding a reference capsid protein at a ratio (wt/wt) of at least about 60: 1 to about 1 :60, e.g, 2: 1, 1 : 1, 3:5 ,1 :2, 1:3, 1 :8, etc. under suitable conditions, and b) isolating the expressed capsid protein of step a).
  • a composition described herein comprises, or a method described herein combines, a modified cap gene: reference cap gene (or combination of reference cap genes) at a ratio that ranges from at least about 1 :60 to about 60: 1, e.g., 2: 1, 1 :1, 3:5, 1 :2, 1 :3, 1 :8, etc.
  • the ratio is at least about 1 :2.
  • the ratio is at least about 1 :3.
  • the ratio is at least about 1 :4.
  • the ratio is at least about 1 :5.
  • the ratio is at least about 1 :6.
  • the ratio is at least about 1 :7.
  • the ratio is at least about 1 :8. In some embodiments, the ratio is at least about 1:9. In some embodiments, the ratio is at least about 1 : 10. In some embodiments, the ratio is at least about 1 : 11. In some embodiments, the ratio is at least about 1 : 12. In some embodiments, the ratio is at least about 1: 13. In some embodiments, the ratio is at least about 1 :14. In some embodiments, the ratio is at least about 1: 15. In some embodiments, the ratio is at least about 1 : 16. In some embodiments, the ratio is at least about 1 :17. In some embodiments, the ratio is at least about 1 : 18. In some embodiments, the ratio is at least about 1 : 19.
  • the ratio is at least about 1:20. In some embodiments, the ratio is at least about 1 :25. In some embodiments, the ratio is at least about 1:30. In some embodiments, the ratio is at least about 1 :35. In some embodiments, the ratio is at least about 1 :40. In some embodiments, the ratio is at least about 1 :45. In some embodiments, the ratio is at least about 1 :50. In some embodiments, the ratio is at least about 1:55. In some embodiments, the ratio is at least about 1 :60. In some embodiments, the ratio is at least about 2: 1. In some embodiments, the ratio is at least about 3 : 1. In some embodiments, the ratio is at least about 4:1.
  • the ratio is at least about 5: 1. In some embodiments, the ratio is at least about 6:1. In some embodiments, the ratio is at least about 7:1. In some embodiments, the ratio is at least about 8:1. In some embodiments, the ratio is at least about 9: 1. In some embodiments, the ratio is at least about 10: 1. In some embodiments, the ratio is at least about 11 :1. i some embodiments, the ratio is at least about 12: 1. In some embodiments, the ratio is at least about 13 : 1. In some embodiments, the ratio is at least about 14:1. In some embodiments, the ratio is at least about 15: 1. In some embodiments, the ratio is at least about 16: 1. In some embodiments, the ratio is at least about 17:1.
  • the ratio is at least about 18: 1. In some embodiments, the ratio is at least about 19: 1. In some embodiments, the ratio is at least about 20: 1. In some embodiments, the ratio is at least about 25:1. In some embodiments, the ratio is at least about 30: 1. In some embodiments, the ratio is at least about 35: 1. In some embodiments, the ratio is at least about 40:1. In some embodiments, the ratio is at least about 45 : 1. In some embodiments, the ratio is at least about 50: 1. In some embodiments, the ratio is at least about 55: 1. In some embodiments, the ratio is at least about 60:1.
  • VP protein subunit ratios in the mosaic viral particle may, but do not necessarily, stoichiometrically reflect the ratios of modified cap gene reference cap gene.
  • a mosaic capsid formed according to the method may be considered to, but does not necessarily, have a modified capsid proteimreference capsid protein ratio similar to the ratio (wt:wt) of nucleic acids encoding same used to produce the mosaic capsid.
  • a mosaic capsid comprises a protein subunit ratio of about 1 :59 to about 59:1.
  • a further embodiment of the present invention is a method for altering the tropism of a virus, the method comprising the steps of: (a) inserting a nucleic acid encoding an amino acid sequence into a nucleic acid sequence encoding an viral capsid protein to form a nucleotide sequence encoding a genetically modified capsid protein comprising the amino acid sequence and/or (b) culturing a packaging cell in conditions sufficient for the production of viral particles, wherein the packaging cell comprises the nucleic acid.
  • a further embodiment of the present invention is a method for displaying a targeting ligand on the surface of a capsid protein, the method comprising the steps of: (a) expressing a nucleic acid encoding a modified viral capsid protein as described herein (and optionally with a nucleotide encoding a reference capsid protein) under suitable conditions, wherein the nucleic acid encodes a capsid protein comprising a first member of a specific binding pair, (b) isolating the expressed capsid protein comprising a first member of a specific binding pair of step (a) or capsid comprising same, and (c) incubating the capsid protein or capsid with a second cognate member of the specific binding pair under conditions suitable for allowing the formation of an isopeptide bond between the first and second member, wherein the second cognate member of the specific binding pair is fused with a targeting ligand.
  • the packaging cell further comprises a helper plasmid and/or a transfer plasmid comprising a nucleotide of interest.
  • the methods further comprise isolating self-complementary adeno-associated viral particles from culture supernatant.
  • the methods further comprise lysing the packaging cell and isolating single-stranded adeno-associated viral particles from the cell lysate.
  • the methods further comprise (a) clearing cell debris, (b) treating the supernatant containing viral particles with nucleases, e.g, DNase I and MgCh, (c) concentrating viral particles, (d) purifying the viral particles, and (e) any combination of (a)-(d).
  • nucleases e.g, DNase I and MgCh
  • Packaging cells useful for production of the viral particles described herein include, e.g., animal cells permissive for the virus, or cells modified to be permissive for the virus; or the packaging cell construct, for example, with the use of a transformation agent such as calcium phosphate.
  • Non-limiting examples of packaging cell lines useful for producing viral particles described herein include, e.g., human embryonic kidney 293 (HEK-293) cells (e.g., American Type Culture Collection [ATCC] No.
  • HEK-293 cells that contain the SV40 Large T-antigen HEK-293 T or 293 T
  • HEK293T/17 cells human sarcoma cell line HT- 1080 (CCL-121), lymphoblast-like cell line Raji (CCL-86), glioblastoma-astrocytoma epithelial- like cell line U87-MG (HTB-14), T-lymphoma cell line HuT78 (TIB-161), NIH/3T3 cells, Chinese Hamster Ovary cells (CHO) (e.g, ATCC Nos. CRL9618, CCL61, CRL9096), HeLa cells (e.g, ATCC No.
  • Vero cells NIH 3T3 cells (e.g, ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g, ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS- 7 cells (ATCC No. CRL1651), RATI cells, mouse L cells (ATCC No. CCLI.3), HLHepG2 cells, CAP cells, CAP-T cells, and the like.
  • human amniocytic cells e.g, CAP, CAP-T
  • yeast cells including, but not limited to, S. cerevisiae, Pichia pastoris
  • plant cells including, but not limited to, Tobacco NT1 , BY-2
  • insect cells including but not limited to SF9, S2, SF21, Tni (e.g. High 5)
  • bacterial cells including, but not limited to, E. coli
  • packaging techniques and particles for packaging the nucleic acid genome into the pseudotyped viral particle see, for example, Polo, et al, Proc Natl Acad Sci USA, (1999) 96:4598-4603.
  • Methods of packaging include using packaging cells that permanently express the viral components, or by transiently transfecting cells with plasmids.
  • Further embodiments include methods comprising contacting a modified Cap protein as described herein with the targeting vector in conditions sufficient to operably link the modified Cap protein with the targeting vector, e.g., in conditions sufficient to promote association of the targeting vector to the modified Cap protein, e.g., via chemical linkage and/or association of first and second members of a specific binding pair, wherein the first member is inserted into the modified Cap protein the first member and the targeting vector is fused to the second member of the specific binding pair.
  • a wide variety of cells may be targeted in order to deliver a nucleotide of interest using a modified viral particle as disclosed herein.
  • the target cells will generally be chosen based upon the nucleotide of interest and the desired effect.
  • a nucleotide of interest may be delivered to a target cell (e.g., a muscle stem cell, myoblast, myocyte, any combination thereof, etc.) such that the target cell produces a protein that makes up for a deficiency in an organism, such as an enzymatic deficiency, or immune deficiency, such as X-linked severe combined immunodeficiency.
  • a target cell e.g., a muscle stem cell, myoblast, myocyte, any combination thereof, etc.
  • a nucleotide of interest such as a gene encoding an siRNA, may inhibit expression of a particular gene in a target cell (e.g., a muscle stem cell, myoblast, myocyte, any combination thereof, etc.).
  • the nucleotide of interest may, for example, inhibit expression of a gene involved in a pathogen life cycle.
  • a nucleotide of interest may inhibit expression of a gene that is responsible for production of a toxin in a target cell.
  • a nucleotide of interest may encode a toxic protein that kills cells in which it is expressed. In this case, tumor cells or other unwanted cells may be targeted.
  • a nucleotide of interest that encodes a therapeutic protein.
  • a nucleotide of interest encodes a therapeutic protein comprising an antigen binding protein, or an antigen binding portion thereof.
  • a nucleotide of interest encodes a therapeutic protein comprising a human or humanized antibody or antigen binding fragment thereof, a monovalent Fab’, a divalent Fab2, a F(ab)’3 fragment, a single-chain fragment variable (scFv), a bis-scFv, a (scFv)2, a diabody, a minibody, a nanobody, a triabody, a tetrabody, a disulfide stabilized Fv protein (dsFv), a single-domain antibody (sdAb), an Ig NAR, a bispecific antibody or binding fragment thereof, a bi-specific T-cell engager (BiTE), a trispecific antibody, or a chemical
  • the scFv comprises variable regions arranged in the following orientation from N- terminus to C-terminus: HCVR-LCVR. In some embodiments, the scFv comprises variable regions arranged in the following orientation from N-terminus to C-terminus: LCVR-HCVR. In some embodiments, the scFv variable regions are connected by a linker. In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker is -(GGGGS)n- (SEQ ID NO: 789), wherein n is 1-10.
  • a target is selected that is specifically expressed on that population of target cells.
  • the target may be expressed exclusively on that population of cells or to a greater extent on that population of cells than on other populations of cells.
  • the more specific the expression the more specifically delivery can be directed to the target cells.
  • the desired amount of specificity of the marker may vary. For example, for introduction of a toxic gene, a high specificity is most preferred to avoid killing non-targeted cells. For expression of a protein for harvest, or expression of a secreted product where a global impact is desired, less marker specificity may be needed.
  • the target may be any cell-surface moiety e.g, a protein, for which a targeting ligand can be identified or created.
  • the target is a peptide or polypeptide, such as a receptor.
  • the target may be a carbohydrate or other molecule that can be recognized by a binding partner. If a binding partner, e.g., ligand, for the target is already known, it may be used as the affinity molecule. However, if a binding molecule is not known, antibodies to the target may be generated using standard procedures. The antibodies can then be used as a targeting ligand.
  • target cells may be chosen based on a variety of factors, including, for example, (1) the application (e.g., therapy, expression of a protein to be collected, and conferring disease resistance) and (2) expression of a marker with the desired amount of specificity.
  • Target cells are not limited in any way and include both germline cells and cell lines and somatic cells and cell lines.
  • the target cells are germline cells, the target cells are preferably selected from the group consisting of single-cell embryos and embryonic stem cells (ES).
  • the target cell is a cell that expresses a non-terminally differentiated muscle cell surface protein. In some embodiments, the target cell is a cell that expresses a mammalian non-terminally differentiated muscle cell surface protein. In some embodiments, the target cell is a cell that expresses a human non-terminally differentiated muscle cell surface protein. In some embodiments, the target cell is a cell that expresses CDH15, e.g., mammalian CDH15, e.g., human CDH15.
  • CDH15 e.g., mammalian CDH15, e.g., human CDH15.
  • the target cell is a muscle cell. In some embodiments, the target cell is a mammalian muscle cell. In some embodiments, the target cell is a mammalian muscle cell. In some embodiments, the target cell is a mammalian muscle cell that is not terminally differentiated. In some embodiments, the target cell is a mammalian muscle cell that is terminally differentiated. In some embodiments, the target cell is a muscle stem cell (also known as a satellite cell), a myoblast, a myocyte, or a non-differentiated myofiber. In some embodiments, the target cell is a mammalian rhabdomyosarcoma cell.
  • a skeletal muscle related disorder e.g., a muscle wasting disease and/or a genetic muscle disease, e.g., X-linked myotubular myopathy (XLMTM), Duchenne muscular dystrophy (DMD), myotonic dystrophy (DM1), Facioscapulohumeral muscular dystrophy Type 1 (FSHD), congenital muscular dystrophy type 1A (MDC1A), Limb girdle muscular dystrophy, dystroglycanopathy, muscle atrophy conditions, metabolic diseases, etc.
  • XLMTM X-linked myotubular myopathy
  • DMD Duchenne muscular dystrophy
  • DM1 myotonic dystrophy
  • FSHD Facioscapulohumeral muscular dystrophy Type 1
  • MDC1A congenital muscular dystrophy type 1A
  • such methods comprise administering to a patient suffering from or at risk for such skeletal related disorder a viral particle or pharmaceutical composition as described herein, wherein the viral particle comprises:
  • a second member of the protein: protein binding pair wherein the second member of the proteimprotein binding pair comprises a targeting ligand that binds a non-terminally differentiated muscle cell surface protein that is expressed on the surface of a muscle cell (e.g., CDH15), wherein the first member of the protein: protein binding pair and the second member of the protein: protein binding pair are associated to direct the tropism of the viral capsid to the muscle cell in the patient thereof, and
  • nucleotide of interest encodes a therapeutic protein, a suicide gene, an antibody or a fragment thereof, a CRISPR/Cas system or a portion(s) thereof, an antisense oligonucleotide, a ribozyme, an RNAi molecule, or a shRNA molecule.
  • the nucleotide of interest may encode a growth factor, neurotrophic factor, a disease modifying muscle protein, a metabolic protein, e.g, for muscle atrophy conditions or metabolic diseases.
  • muscle-related cancer e.g., a cancer expressing a non-terminally differentiated muscle cell protein, e.g, CDH15.
  • muscle-specific cancers include rhabdomyosarcomas, e.g., embryonal, alveolar, pleomorphic, botryoid and spindle/ sclerosing rhabdomyosarcomas.
  • such methods comprise administering to a patient suffering from or at risk for such muscle-related cancer a viral particle or pharmaceutical composition as described herein, wherein the viral particle comprises:
  • a viral capsid modified to comprise the targeting ligand inserted directly into the viral capsid or via, e.g., a first member of a protein: protein binding pair and its cognate second member of the protein: protein binding pair, wherein the second member of the protein: protein binding pair comprises a targeting ligand that binds a muscle-specific surface protein that is expressed on the surface of a non-terminally differentiated muscle cell e.g., CDH15), wherein the first member of the protein: protein binding pair and the second member of the protein: protein binding pair are associated to direct the tropism of the viral capsid to the muscle cell in the patient thereof, and
  • the nucleotide of interest comprises or encodes a therapeutic protein, a suicide gene, an antibody or a fragment thereof, a CRISPR/Cas system or a portion(s) thereof, an antisense oligonucleotide, a ribozyme, an RNAi molecule, or a shRNA molecule capable of treating the muscle-related cancer cell e.g., slowing and/or arresting the growth of and/or destroying the muscle-related cancer cell.
  • Example non-limiting embodiments of said nucleotides of interest include a therapeutic protein that targets and inhibits an oncogenic intracellular signaling pathway, a suicide gene that directs the cell to undergo apoptosis, and an RNAi molecule that targets and inhibits an oncogenic transcript.
  • Table 2 Example sequences of non-primate adeno-associated viruses. [00358] Table 2 - continued [00359] Table 2 - continued [00360] Table 2 - continued [00361] Table 2 - continued
  • Muscle stem cells are mitotically quiescent and non-proliferative under steady-state conditions in adult muscle tissue. In response to injury, however, MuSCs begin to actively divide, producing daughter cells. While some daughter cells become quiescent once more to replenish the pool of MuSCs, others continue to proliferate as myoblasts, which align and fuse together during differentiation into mature myotubes/myofibers that make up muscle fibers.
  • Quiescent muscle stem cells can be characterized by Pax7 expression
  • proliferating myoblasts can be characterized by Pax7 and myogenic differentiation factor 1 (MyoD) expression
  • myoblasts committing to terminal differentiation z.e., myocytes
  • myotubes can be characterized by myosin heavy chain (MyHC) expression.
  • MyoD myogenic differentiation factor 1
  • MyHC myosin heavy chain
  • Cadherin 15 (CDH15) mRNA is highly expressed in MuSCs and expression increases following muscular injury.
  • CDH15 homozygous CDH15 knockout mice
  • CDH I 5 ' mice demonstrated accelerated muscle regeneration following injury.
  • CDH15" ' mice exhibited increased myofiber size 5- and 15-days post-injury via injection with cardiotoxin (CTX) into the tibialis anterior muscle, and an increased number of central myonuclei in myofibers 15-days post-injury.
  • CX cardiotoxin
  • CDH15 ' mice demonstrated improved functional recovery following CTX injury, as evidenced by normal tetanic force measurements in ex vivo isolated extensor digitorum longus muscles 15 days following CTX injury (see Figure 3B).
  • WT mice exhibited a tetanic force loss of approximately 40% in ex vivo isolated muscle 15-days post-injury.
  • MuSCs derived from WT or CDH15' ' mice were cultured ex vivo with their associated myofibers for 48 hours. As demonstrated in Figures 4A-4B, a greater percentage of multi-cell clusters and a greater number of Pax7 + cells per cluster was observed for cultured CDH15 /_ MuSCs, as compared to WT MuSCs. This result suggests that abolishing CDH15 expression in MuSCs accelerates the transition from quiescence to activation.
  • RNA-seq was performed on FACS- isolated MuSCs from uninjured, healthy mice. As shown in Figure 5A, 132 genes were downregulated and 270 genes were upregulated in CDH15 ’’ MuSCs as compared to WT MuSCs. Of those upregulated genes, a cluster of genes was identified that contain serum response factor (SRF) binding motifs (see Figure 5B), indicating that accelerated activation of MuSCs in the absence of CDH15 may be due to altered SRF signaling.
  • SRF serum response factor
  • Muscle stem cells (MuSCs) in aging subjects display delayed activation and reduced motility in vitro), which translates to impaired MuSC mediated repair in vivo. Accordingly, it was hypothesized that CDH15 may be involved in reduction of the muscle regenerative capacity for aged subjects. To test this, homozygous CDH15 knockout mice (CDH15 z ) were generated, aged to 23 months, and injected intramuscularly in the tibialis anterior muscle with cardiotoxin to induce muscular damage (day 0).
  • Anti-human CDH15 antibodies are obtained by immunizing a mouse (e.g., an engineered mouse comprising DNA encoding human immunoglobulin heavy and human kappa light chain variable regions, see, e.g., in US Patent No. 7,105,348; US Patent No. 8,642,835; and US 9,622,459, each of which is incorporated herein by reference), with human CDH15.
  • a mouse e.g., an engineered mouse comprising DNA encoding human immunoglobulin heavy and human kappa light chain variable regions
  • splenocytes are harvested from each mouse and either (1) fused with mouse myeloma cells to preserve their viability and form hybridoma cells and screened for human CDH15 specificity, or (2) B-cell sorted (as described in US 2007/0280945A1) using a either a human CDH15 fragment as the sorting reagent that binds and identifies reactive antibodies (antigen-positive B cells).
  • Chimeric antibodies to human CDH15 were initially isolated having a human variable region and a mouse constant region using, e.g., VELOCIMMUNE technology as described in US Patent No. 7,105,348; US Patent No. 8,642,835; and US 9,622,459, each of which is incorporated herein by reference.
  • mouse constant regions were replaced with a desired human constant region, for example wild-type human CH or modified human CH e.g., IgGl, IgG2 or IgG4 isotypes), and light chain constant region (CL), to generate a fully human anti-hCDH15 antibody, or antigen binding portion thereof.
  • a desired human constant region for example wild-type human CH or modified human CH e.g., IgGl, IgG2 or IgG4 isotypes
  • CL light chain constant region
  • Table 1 sets forth sequence identifiers of a nucleic acid (NA) sequence encoding, and in parentheses an amino acid (AA) sequence of, a heavy or light chain variable region
  • HCVR HCVR or LCVR, respectively
  • HCDR and LCDR heavy or light chain CDR

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Abstract

Antibodies, portions, and drug conjugates thereof to Cadherin (15) are provided herein. Also provided are nucleic acid sequences encoding same, viral particles comprising the same, e.g., for retargeting the viral particles to muscle cells, compositions comprising and methods of using the same, e.g., for treating a subject in need thereof, e.g., a subject having a skeletal muscle related disorder (e.g., X-linked myotubular myopathy (XLMTM), Duchenne muscular dystrophy (DMD), myotonic dystrophy (DM1), Facioscapulohumeral muscular dystrophy Type 1 (FSHD), congenital muscular dystrophy type 1A (MDC1A), Limb girdle muscular dystrophy, dystroglycanopathy, etc.) or a rhabdomyosarcoma.

Description

ANTI-HUMAN M-CADHERIN (CDH15) ANTIBODIES, CONJUGATES, AND USES
THEREOF FOR DELIVERY OF GENETIC PAYLOADS TO MUSCLE CELLS
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Application Nos. 63/499,531 and 63/499,527, both filed on May 2, 2023, the disclosures of which are hereby incorporated by reference in their entirety.
TECHNICAL FIELD
[0002] This application is generally directed to human antibodies and antigen-binding fragments of human antibodies that bind human Cadherin 15 (hM-Cadherin, hCDH15), and methods of use thereof, e.g., in methods of treating a disorder in a patient in need thereof. The application also relates to antigen-binding molecules comprising at least an antigen-binding fragment of an anti-hCDH15 antibody, wherein complexation of the antigen-binding molecule to CDH15 mediates internalization of the antigen-binding molecule/CDH15 complex, and/or blocks activity of CDH15. The application further relates to conjugates comprising an anti-hCDH15 antibody (or antigen-binding molecules comprising an antigen-binding fragment of an anti- hCDH15 antibody) and a therapeutic agent, which conjugates may be useful in treating diseases. The disclosure further relates to methods of making and using recombinant viral particles, e.g., recombinant AAV particles, comprising capsid proteins retargeted to Cadherin 15 (CDH15), useful for modification of muscle cells, such as muscle stem cells, in vitro or in vivo.
SEQUENCE LISTING
[0003] A Sequence Listing in XML format entitled “11486WO01 Sequence Listing XML. xml,” which was created April 30, 2024, and is 787 Kb, is incorporated herein by reference in its entirety. BACKGROUND
[0004] The delivery of genes into particular target cells has become one of the most important technologies in modern medicine for the potential treatment of a variety of chronic and genetic diseases. Ideally, a gene delivery vehicle is able to stably introduce genetic material into desired cells and avoid introducing genetic material into non-target cells.
[0005] Viral particles, particularly those based on adeno-associated virus (AAV), as gene delivery vehicles have been the focus of much research since AAVs are capable of transducing a wide range of primate species and tissues in vivo. (Muzyczka, et al. (1992) Current Topics in Microbiology and Immunology, 158:97-129). Moreover, AAV safely transduces postmitotic tissues. Although the virus can occasionally integrate into host chromosomes, it does so very infrequently into a safe-harbor locus in human chromosome 19, and only when the replication (Rep) proteins are supplied in trans. AAV genomes rapidly circularize and concatemerize in infected cells, and exist in a stable, episomal state in infected cells to provide long-term stable expression of their payloads.
[0006] Additionally, manipulating and redirecting AAV infection to specific cells has been achieved in recent years. Many of the advances in targeted gene therapy using viral particles may be summarized as non-recombinatorial (non-genetic) or recombinatorial (genetic) modification of the viral particle, which result in the pseudotyping, expanding, and/or retargeting of the natural tropism of the viral particle. (Reviewed in Nicklin and Baker (2002) Curr. Gene Ther. 2:273-93; Verheiji and Rottier (2012) Advances Virol 2012: 1-15).
[0007] In a direct recombinatorial targeting approach, a targeting ligand is directly inserted into, or coupled to, a viral capsid, i.e., protein viral capsid genes are modified to express capsid proteins comprising a heterologous targeting ligand. The targeting ligand than redirects, e.g., binds, a receptor or marker preferentially or exclusively expressed on a target cell. (Stachler et al. (2006) Gene Ther. 13:926-931; White et al. (2004) Circulation 109:513-519; see also Park et al., (2007) Frontiers in Bioscience 13 :2653— 59; Girod et al. (1999) Nature Medicine 5: 1052— 56; Grifman et al. (2001) Molecular Therapy 3:964-75; Shi et al. (2001) Human Gene Therapy 12:1697-1711; Shi and Bartlett (2003) Molecular Therapy 7:515-525).
[0008] In indirect recombinatorial approaches, a viral capsid is modified with a heterologous “scaffold”, which then links to an adaptor that includes a targeting ligand. The adaptor binds to the scaffold and the target cell. (Arnold et al. (2006) Mol. Ther. 5:125-132; Ponnazhagen et al. (2002) J. Virol. 76: 12900-907; see also WO 97/05266). Scaffolds such as (1) Fc binding molecules (e.g, Fc receptors, Protein A, etc.), which bind to the Fc of antibody adaptors, (2) (strept)avidin, which binds to biotinylated adaptors, (3) biotin, which binds to adaptors fused with (strept)avidin, (4) a detectable label, which is useful for detection and/or isolation of viral particles, bound by a bispecific adaptor able to non-covalently bind the detectable label and target molecule, and recently (5) protein: protein binding pairs that form isopeptide bonds have been described for a variety of viral particles. (See, e.g., Gigout et al. (2005) Molecular Therapy 11 :856-865; Stachler et al. (2008) Molecular Therapy 16:1467-1473; Quetglas et al. (2010) Virus Research 153: 179-196; Ohno et al. (1997) Nature Biotechnology 15:763-767; Klimstra et al. (2005) Virology 338:9-21).
[0009] Skeletal muscle is the largest organ in the body, comprising -40% of total body mass, and is one of the three significant muscle tissues in the human body. When skeletal muscle becomes damaged, muscle stem cells (MuSCs) activate, proliferate, and differentiate into functional myofibers to repair the damaged muscle. However, MuSC-mediated muscle regeneration is delayed in aging subjects, which may be related, in part, to changes in muscle stem cell-specific markers.
[0010] Accordingly, anti -human antibodies capable of binding to muscle stem cellspecific markers could be helpful for therapy, e.g., stimulating muscle repair, particularly in aging subjects, and/or treating muscle-related cancers. Moreover, the anti-human antibodies as described herein may be used in conjunction with recombinant viral, e.g., AAV, particles for the targeted introduction of nucleic acids of interest into cells expressing muscle stem cell-specific markers. Moreover, some muscle-related cancers, including rhabdomyosarcomas, may also benefit from therapeutics, e.g., antibody-drug conjugates, retargeted viral particles, etc., as described herein that target muscle stem cell-specific markers thereby.
SUMMARY
[0011] Described herein are antigen-binding proteins that bind to human Cadherin 15 (CDH15). In some embodiments, the antigen-binding protein comprises a set of three heavy chain complementary determining region (HCDR1, HCDR2, and HCDR3) amino acid sequences selected from Table 1 below. In some embodiments, the antigen-binding protein comprises a set of three light chain complementary determining region (LCDR1, LCDR2 and LCDR3) amino acid sequences selected from Table 1 below. In some embodiments, the antigen-binding protein comprises a set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences selected from Table 1 below. In some embodiments, the antigen-binding protein comprises a set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences selected from the group consisting of SEQ ID NOs: 4-6-8-12-14-16, 24-26-28-32-34-36, 44-46-48-52-34-54, 62- 64-66-52-34-54, 72-74-76-52-34-54, 82-84-86-52-34-54, 92-94-96-100-34-102, 82-111-113- 117-34-119, 127-129-131-135-137-139, 147-149-151-155-157-159, 167-169-171-175-177-179, 187-189-191-52-34-196, 204-206-208-212-137-214, 222-224-226-52-34-54, 232-234-236-52- 34-54, 242-244-246-52-34-54, 82-253-255-52-34-54, 261-263-265-269-271-273, 281-283-285- 289-291-293, 301-303-305-309-311-313, 321-323-325-329-331-333, 341-343-345-349-14-352, 360-362-364-368-370-372, 187-380-382-52-34-54, 388-390-392-396-14-398, 406-408-410-100- 34-414, 422-424-426-430-432-434, 438-440-442-446-448-450, 454-456-458-462-464-466, 470- 472-474-478-480-482, 486-488-490-494-496-498, 502-504-506-510-512-514, 518-520-522-526- 528-530, 534-536-538-542-544-546, 550-552-554-558-560-562, 566-568-570-574-576-578, 582-584-586-590-592-594, 598-600-602-606-608-610, 614-616-618-622-624-626, 630-632-634- 638-640-642, 646-648-650-654-656-658, 662-664-666-670-672-674, 678-680-682-686-688-690, 694-696-698-702-704-706, 710-712-714-718-720-722, 726-728-730-734-736-738, 742-744-746- 686-688-690, 750-752-754-758-760-762, and 766-768-770-774-776-778.
[0012] Table 1 sets forth the amino acid and nucleic acid sequence identifiers of the HCVRs and LCVRs, as well as HCDR1, HCDR2 and HCDR3 within each HCVR, and LCDR1, LCDR2 and LCDR3 within each LCVR of each of the example anti-hCDH15 antibodies of the present disclosure.
Table 1: anti-hCDH15 Sequence Identifiers [0013] In some embodiments, the antigen-binding protein comprises a heavy chain variable region (HCVR or VH). In some embodiments, the HCVR comprises a set of HCDR1- HCDR2-HCDR3 amino acid sequences selected from Table 1. In some embodiments, the HCVR comprises an amino acid sequence selected from any of the HCVR amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.
[0014] In some embodiments, the antigen-binding protein comprises a light chain variable region (LCVR or VL). In some embodiments, the LCVR comprises a set of LCDR1- LCDR2-LCDR3 amino acid sequences selected from Table 1 below. In some embodiments, the LCVR comprises an amino acid sequence selected from any of the LCVR amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.
[0015] In some embodiments, the antigen-binding protein comprises an anti-hCDH15 antibody or antigen-binding fragment thereof. In some embodiments, the anti-hCDH15 antibody or antigen-binding fragment thereof comprises a human or humanized antibody or antigen binding fragment thereof, a monovalent Fab’, a divalent Fab2, a F(ab)’3 fragment, a single-chain fragment variable (scFv), a bis-scFv, a (scFv)2, a diabody, a minibody, a nanobody, a triabody, a tetrabody, a disulfide stabilized Fv protein (dsFv), a single-domain antibody (sdAb), an Ig NAR, a bispecific antibody or binding fragment thereof, a bi-specific T-cell engager (BiTE), a trispecific antibody, or a chemically modified derivative thereof. In some embodiments, the scFv comprises variable regions arranged in the following orientation from N-terminus to C-terminus: HCVR-LCVR. In some embodiments, the scFv comprises variable regions arranged in the following orientation from N-terminus to C-terminus: LCVR-HCVR. In some embodiments, the scFv variable regions are connected by a linker. In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker is -(GGGGS)n- (SEQ ID NO: 789), wherein n is 1-10.
[0016] In some embodiments, the antigen-binding protein, e.g., antibody or antigenbinding fragment thereof, binds to hCDH15 with a KD of about 1X10’7 M or a stronger affinity. In some embodiments, the antigen-binding protein binds to hCDH15 with a KD of about 10X10’ 8 to about 1X10'10. In some embodiments, the antigen-binding protein binds to hCDH15 with a KD of about 5X1 O’9 to about 1X1 O’10. [0017] In some embodiments, the antigen-binding protein, e.g, antibody or antigenbinding fragment thereof, comprises an HCVR and an LCVR amino acid sequence pair (HCVR/LCVR) comprising any of the HCVR amino acid sequences listed in Table 1 paired with any of the LCVR amino acid sequences listed in Table 1. In some embodiments, the antigenbinding protein, e.g., antibody or antigen-binding fragment thereof, as described herein comprises an HCVR/LCVR amino acid sequence pair contained within any of the example anti- hCDH15 antibodies listed in Table 1. In certain embodiments, the HCVR/LCVR amino acid sequence pair is selected from the group consisting of SEQ ID NOs: 2 and 10, 22 and 30, 42 and 50, 60 and 50, 70 and 50, 80 and 50, 90 and 98, 108 and 115, 125 and 133, 145 and 153, 165 and 173, 185 and 193, 202 and 210, 220 and 50, 230 and 50, 240 and 50, 250 and 50, 259 and 267, 279 and 287, 299 and 307, 319 and 327, 339 and 347, 358 and 366, 378 and 50, 386 and 394,
404 and 412, 420 and 428, 436 and 444, 452 and 460, 468 and 476, 484 and 492, 500 and 508,
516 and 524, 532 and 540, 548 and 556, 564 and 572, 580 and 588, 596 and 604, 612 and 620,
628 and 636, 644 and 652, 660 and 668, 676 and 684, 692 and 700, 708 and 716, 724 and 732,
740 and 684, 748 and 756, and 764 and 772.
[0018] Also described herein are nucleic acid molecules, i.e., polynucleotides, encoding the antigen-binding protein, e.g., anti-hCDH15 antibodies or antigen-binding fragments thereof, described herein. In some embodiments, the nucleic acid molecule as described herein comprises a nucleic acid sequence encoding a set of HCDR1-HCDR2-HCDR3 amino acid sequences listed in Table 1. In some embodiments, a nucleic acid molecule as described herein comprises a nucleic acid sequence encoding any of the HCVR amino acid sequences listed in Table 1. In certain embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.
[0019] In some embodiments, the nucleic acid molecule as described herein comprises a nucleic acid sequence encoding a set of LCDR1-LCDR2-LCDR3 amino acid sequences listed in Table 1. In some embodiments, a nucleic acid molecule as described herein comprises a nucleic acid sequence encoding any of the LCVR amino acid sequences listed in Table 1. In some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCVR nucleic acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.
[0020] Also described herein are nucleic acid molecules encoding an HCVR, wherein the HCVR comprises a set of three CDRs (i.e., HCDR1-HCDR2-HCDR3), wherein the HCDR1- HCDR2-HCDR3 amino acid sequence set is as defined by any of the example anti-hCDH15 antibodies listed in Table 1.
[0021] Also described herein are nucleic acid molecules encoding an LCVR, wherein the LCVR comprises a set of three CDRs i.e., LCDR1-LCDR2-LCDR3), wherein the LCDR1- LCDR2-LCDR3 amino acid sequence set is as defined by any of the example anti- hCDH15 antibodies listed in Table 1.
[0022] Also described herein are nucleic acid molecules encoding both an HCVR and an LCVR, wherein the HCVR comprises an amino acid sequence of any of the HCVR amino acid sequences listed in Table 1, and wherein the LCVR comprises an amino acid sequence of any of the LCVR amino acid sequences listed in Table 1. In some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto, and a polynucleotide sequence selected from any of the LCVR nucleic acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto. In some embodiments, the nucleic acid molecule encodes an HCVR and LCVR, wherein the HCVR and LCVR are both derived from the same anti-hCDH15 antibody listed in Table 1.
[0023] Also described herein is a pharmaceutical composition comprising the antigenbinding protein described herein, e.g., a recombinant human antibody or fragment thereof which binds human CDH15, and a pharmaceutically acceptable carrier. In some embodiments, a pharmaceutical composition as described herein comprises a combination of an anti-hCDH15 antibody and a second therapeutic agent. In one embodiment, the second therapeutic agent is any agent that is advantageously combined with an anti-hCDH15 antibody. Additional combination therapies and co-formulations involving the anti-hCDH15 antibodies are described herein. [0024] Also described herein is a method of inhibiting the activity of CDH15 in a cell, e.g., in vivo, in vitro, or ex vivo, comprising contacting the cell expressing CDH15 with the antigen-binding protein that binds human CDH15, or the pharmaceutical composition thereof as described herein. In some embodiments, the cell expressing CDH15 is a muscle stem cell, a myoblast, or a myocyte.
[0025] Also described herein is a method of accelerating the transition from quiescence to activation of a muscle stem cell, e.g., in vivo, in vitro, or ex vivo, comprising contacting the muscle stem cell with the antigen-binding protein that binds human CDH15 or the pharmaceutical composition thereof as described herein.
[0026] Also described herein is a method of treating a condition in a subject in need thereof, wherein the method comprises administering a therapeutically effective amount of a pharmaceutical composition comprising the antigen-binding protein that binds human CDH15 as described herein to the subject. In some embodiments, the condition is muscle injury. In some embodiments, the condition is cancer, e.g., rhabdomyosarcoma. In some embodiments, the antigen-binding protein is conjugated to a therapeutic agent, e.g., an antibody-drug conjugate (ADC). In some embodiments, the therapeutic agent comprises a cytotoxic chemotherapeutic agent. In some embodiments, the therapeutic agent is Aflibercept, Amsacrine, Azacitidine, Azathioprine, Belantamab mafodotin, Bendamustine, Bleomycin, Bortezomib, Brentuximab vedotin, Busulfan, Cabazitaxel, Capecitabine, Carboplatin, Carfilzomib, Carmustine, Chlorambucil, Cisplatin, Cladribine, Clofarabine, Cyclophosphamide, Cytarabine, Cytarabine liposomal, Dacarbazine, Dactinomycin (actinomycin D), Daunorubicin, Docetaxel, Doxorubicin, Doxorubicin liposomal, Epirubicin, Eribulin, Etoposide, Etoposide phosphate, Fludarabine, Fluorouracil, Fotemustine, Ganciclovir, Gemcitabine, Gemtuzumab ozogamicin, Hydroxyurea, Idarubicin, Ifosfamide, Inotuzumab ozogamicin , Irinotecan, Ixazomib, Lomustine, Melphalan, Mercaptopurine, Methotrexate, Mitomycin, Mitotane, Mitozantrone, Nab-paclitaxel, Oxaliplatin, Paclitaxel, Pemetrexed, Pegaspargase, Polatuzumab vedotin, Pralatrexate, Procarbazine, Raltitrexed, Romidepsin, Sacituzumab govitecan, Temozolomide, Teniposide, Thiotepa, Tioguanine, Topotecan, Trabectedin, Trastuzumab deruxtecan, Trastuzumab emtansine, Trifluridine/tipiracil, Valganciclovir, Vinblastine, Vincristine, Vindesine, Vinflunine, Vinorelbine, or Vismodegib. In some embodiments, the antigen binding protein is conjugated to the therapeutic agent via a valine-citrulline (VC). In some embodiments, the antigen binding protein is conjugated to the therapeutic agent via a para-aminobenzyl (PAB) linker. In some embodiments, the pharmaceutical composition is administered to the subject intravenously or subcutaneously.
[0027] Also described herein is a method of restoring the muscle regenerative capacity of a subject, wherein the method comprises administering a therapeutically effective amount of a pharmaceutical composition comprising the antigen-binding protein that binds human CDH15 as described herein to the subject. In some embodiments, the subject is an aged subject. In some embodiments, the muscle regenerative capacity of the aged subject is restored to a functional state at or near that of a control subject. In some embodiments, the pharmaceutical composition is administered to the subject intravenously or subcutaneously.
[0028] Also described herein is a method of imaging a muscle cell in a subject in need thereof, wherein the method comprises administering a pharmaceutical composition comprising the antigen-binding protein that binds human CDH15 as described herein to the subject, wherein the antigen binding protein is conjugated to a detectable moiety. In some embodiments, the muscle cell comprises one or more selected from the group consisting of a muscle stem cell, a myoblast, and a myocyte. In some embodiments, the detectable moiety comprises a radionuclide. In some embodiments, the pharmaceutical composition is administered to the subject intravenously or subcutaneously.
[0029] Also described herein is the use of the antigen-binding protein that binds human CDH15 or a pharmaceutical composition thereof in the manufacture of a medicament, e.g., for the treatment of a condition as described herein. Further described herein is an antigen-binding protein that binds human CDH15 or a pharmaceutical composition thereof for use in therapy, e.g., for treating a condition as described herein, and/or for use in restoring the muscle regenerative capacity of a subject.
[0030] Also described herein is an antibody or antigen-binding fragment that competes for binding to human CDH15 with a reference antibody comprising an HCVR/LCVR amino acid sequence pair as set forth in Table 1. In some embodiments, an antibody or antigen-binding fragment as described herein competes for binding to human CDH15 with a reference antibody comprising an HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 and 10, 22 and 30, 42 and 50, 60 and 50, 70 and 50, 80 and 50, 90 and 98, 108 and 115, 125 and 133, 145 and 153, 165 and 173, 185 and 193, 202 and 210, 220 and 50, 230 and 50, 240 and 50, 250 and 50, 259 and 267, 279 and 287, 299 and 307, 319 and 327, 339 and 347, 358 and 366, 378 and 50, 386 and 394, 404 and 412, 420 and 428, 436 and 444, 452 and
460, 468 and 476, 484 and 492, 500 and 508, 516 and 524, 532 and 540, 548 and 556, 564 and
572, 580 and 588, 596 and 604, 612 and 620, 628 and 636, 644 and 652, 660 and 668, 676 and
684, 692 and 700, 708 and 716, 724 and 732, 740 and 684, 748 and 756, and 764 and 772.
[0031] Also described herein is an antibody or antigen-binding fragment, wherein the antibody or antigen-binding fragment thereof binds to the same epitope on human CDH15 as a reference antibody comprising an HCVR/LCVR amino acid sequence pair as set forth in Table 1. In some embodiments, an antibody or antigen-binding fragment as described herein binds to the same epitope on human CDH15 as a reference antibody comprising an HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 and 10, 22 and 30, 42 and 50, 60 and 50, 70 and 50, 80 and 50, 90 and 98, 108 and 115, 125 and 133, 145 and 153, 165 and 173, 185 and 193, 202 and 210, 220 and 50, 230 and 50, 240 and 50, 250 and 50, 259 and 267, 279 and 287, 299 and 307, 319 and 327, 339 and 347, 358 and 366, 378 and 50, 386 and 394,
404 and 412, 420 and 428, 436 and 444, 452 and 460, 468 and 476, 484 and 492, 500 and 508,
516 and 524, 532 and 540, 548 and 556, 564 and 572, 580 and 588, 596 and 604, 612 and 620,
628 and 636, 644 and 652, 660 and 668, 676 and 684, 692 and 700, 708 and 716, 724 and 732,
740 and 684, 748 and 756, and 764 and 772.
[0032] Also described herein is an isolated antibody or antigen-binding fragment thereof that binds human CDH15, wherein the antibody or antigen-binding fragment comprises: the complementarity determining regions (CDRs) of a heavy chain variable region (HCVR) having an amino acid sequence as set forth in Table 1; and the CDRs of a light chain variable region (LCVR) having an amino acid sequence as set forth in Table 1. In some embodiments, the isolated antibody or antigen-binding fragment comprises the heavy and light chain CDRs of a HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 and 10, 22 and 30, 42 and 50, 60 and 50, 70 and 50, 80 and 50, 90 and 98, 108 and 115, 125 and 133, 145 and 153, 165 and 173, 185 and 193, 202 and 210, 220 and 50, 230 and 50, 240 and 50, 250 and 50, 259 and 267, 279 and 287, 299 and 307, 319 and 327, 339 and 347, 358 and 366, 378 and 50, 386 and 394, 404 and 412, 420 and 428, 436 and 444, 452 and 460, 468 and 476, 484 and 492, 500 and 508, 516 and 524, 532 and 540, 548 and 556, 564 and 572, 580 and 588, 596 and 604, 612 and 620, 628 and 636, 644 and 652, 660 and 668, 676 and 684, 692 and 700, 708 and 716, 724 and 732, 740 and 684, 748 and 756, and 764 and 772. In yet another aspect, the isolated antibody or antigen-binding fragment comprises HCDR1-HCDR2-HCDR3-LCDR1- LCDR2-LCDR3 domains, respectively, selected from the group consisting SEQ ID NOs: 4-6-8- 12-14-16, 24-26-28-32-34-36, 44-46-48-52-34-54, 62-64-66-52-34-54, 72-74-76-52-34-54, 82- 84-86-52-34-54, 92-94-96-100-34-102, 82-111-113-117-34-119, 127-129-131-135-137-139, 147-149-151-155-157-159, 167-169-171-175-177-179, 187-189-191-52-34-196, 204-206-208- 212-137-214, 222-224-226-52-34-54, 232-234-236-52-34-54, 242-244-246-52-34-54, 82-253- 255-52-34-54, 261-263-265-269-271-273, 281-283-285-289-291-293, 301-303-305-309-311- 313, 321-323-325-329-331-333, 341-343-345-349-14-352, 360-362-364-368-370-372, 187-380- 382-52-34-54, 388-390-392-396-14-398, 406-408-410-100-34-414, 422-424-426-430-432-434, 438-440-442-446-448-450, 454-456-458-462-464-466, 470-472-474-478-480-482, 486-488-490- 494-496-498, 502-504-506-510-512-514, 518-520-522-526-528-530, 534-536-538-542-544-546, 550-552-554-558-560-562, 566-568-570-574-576-578, 582-584-586-590-592-594, 598-600-602- 606-608-610, 614-616-618-622-624-626, 630-632-634-638-640-642, 646-648-650-654-656-658, 662-664-666-670-672-674, 678-680-682-686-688-690, 694-696-698-702-704-706, 710-712-714- 718-720-722, 726-728-730-734-736-738, 742-744-746-686-688-690, 750-752-754-758-760-762, and 766-768-770-774-776-778. Also described herein is an isolated antibody or antigen-binding fragment thereof that binds human CDH15, wherein the antibody or antigen-binding fragment comprises: (a) a heavy chain variable region (HCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 22, 42, 60, 70, 80, 90, 108, 125, 145, 165, 185, 202, 220, 230, 240, 250, 259, 279, 299, 319, 339, 358, 378, 386, 404, 420, 436, 452, 468, 484,
500, 516, 532, 548, 564, 580, 596, 612, 628, 644, 660, 676, 692, 708, 724, 740, 748, 764, and
780; and/or (b) a light chain variable region (LCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 30, 50, 50, 50, 50, 98, 115, 133, 153, 173, 193, 210, 50, 50, 50, 50, 267, 287, 307, 327, 347, 366, 50, 394, 412, 428, 444, 460, 476, 492, 508,
524, 540, 556, 572, 588, 604, 620, 636, 652, 668, 684, 700, 716, 732, 756, and 772. In some embodiments, the isolated antibody or antigen-binding fragment comprises a HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 and 10, 22 and 30, 42 and 50, 60 and 50, 70 and 50, 80 and 50, 90 and 98, 108 and 115, 125 and 133, 145 and 153, 165 and 173, 185 and 193, 202 and 210, 220 and 50, 230 and 50, 240 and 50, 250 and 50, 259 and 267, 279 and 287, 299 and 307, 319 and 327, 339 and 347, 358 and 366, 378 and 50, 386 and 394, 404 and 412, 420 and 428, 436 and 444, 452 and 460, 468 and 476, 484 and 492,
500 and 508, 516 and 524, 532 and 540, 548 and 556, 564 and 572, 580 and 588, 596 and 604,
612 and 620, 628 and 636, 644 and 652, 660 and 668, 676 and 684, 692 and 700, 708 and 716,
724 and 732, 740 and 684, 748 and 756, and 764 and 772.
[0033] Further described herein are viral particles that are particularly suited for the targeted introduction of a nucleotide of interest specifically to a muscle cell (e.g, a muscle stem cell) since the viral capsid or viral capsid protein(s) described herein comprise a targeting ligand that binds a muscle-cell specific surface protein (e.g, an antigen -binding protein as described herein that binds to human CDH15). In some embodiments, the viral capsid or viral capsid protein(s) described herein comprise(s) a direct insertion of the targeting ligand (e.g., the targeting ligand is directly coupled to, fused to, etc., optionally via a linker to the viral capsid or viral capsid proteins), e.g., viral capsid genes are modified to express capsid proteins comprising the targeting ligand. In some embodiments, a viral capsid or viral capsid protein comprises the targeting ligand via a scaffold or an adaptor, e.g., a first member of a protein: protein binding pair, which may be associated with its cognate second member of the protein: protein binding pair, wherein the second member is linked (e.g, fused to) a targeting ligand that binds a musclecell specific surface protein (e.g., an antigen-binding protein as described herein that binds to human CDH15). In some embodiments, the targeting ligand is operably linked to the second member, e.g, fused to the second member, optionally via a linker. In some embodiments, the targeting ligand may be a binding moiety, e.g, a natural ligand, antibody, a multispecific binding molecule, etc. In some embodiments, the targeting ligand is an antibody or portion thereof. In some embodiments, the targeting ligand is an antibody comprising a variable domain that binds a surface protein (e.g, a variable domain of an antigen-binding protein as described herein that binds to human CDH15) on a non-terminally differentiated muscle cell (e.g, a muscle stem cell, a myoblast, myocyte, any combination thereof, etc.) and a heavy chain constant domain. In some embodiments, the targeting ligand is an antibody comprising a variable domain that binds a non-terminally differentiated muscle cell surface protein (e.g, a variable domain of an antigenbinding protein as described herein that binds to human CDH15) on a target cell (e.g., a muscle stem cell, myoblast, myocyte, any combination thereof, e/c.), and optionally an IgG heavy chain constant domain. In some embodiments, the targeting ligand is an antibody comprising a variable domain that binds a non-terminally differentiated muscle cell surface protein (e.g, a variable domain of an antigen-binding protein as described herein that binds to human CDH15) on a target cell (e.g, a muscle stem cell, myoblast, myocyte, any combination thereof, etc.) and an IgG heavy chain constant domain, wherein the IgG heavy chain constant domain is operably linked, e.g., directly or via a linker, to a capsid protein. In some embodiments, the targeting ligand is an antibody comprising (i) a variable domain that binds a non-terminally differentiated muscle cell surface protein (e.g., a variable domain of an antigen-binding protein as described herein that binds to human CDH15), and (ii) an IgG heavy chain constant domain, wherein the IgG heavy chain constant domain is operably linked (optionally via a linker) to a protein (e.g., second member of a protein: protein binding pair) that forms an isopeptide covalent bond with the cognate first member of the proteimprotein binding pair. In some embodiments, a capsid protein described herein comprises a first member of the protein: protein binding pair, comprising, e.g., SpyTag (SEQ ID NO: 815) or a biologically equivalent variant thereof, operably linked to the viral capsid protein, wherein SpyTag or a biologically equivalent variant thereof is covalently linked (e.g., via an isopeptide bond) to the SpyTag to its second cognate protein: protein binding member, e.g., SpyCatcher (SEQ ID NO: 816) or a biologically equivalent variant thereof, which in turn may be linked to a targeting ligand comprising an antibody variable domain and an IgG heavy chain domain, wherein SpyCatcher and the IgG heavy chain domain are linked via an amino acid linker, e.g., GSGESG (SEQ ID NO: 828). In some embodiments, the non-terminally differentiated muscle cell surface protein comprises CDH15. In some embodiments, the targeting ligand binds CDH15, e.g., human CDH15. In some embodiments, the targeting ligand comprises a variable domain of an antigen-binding protein as described herein that binds to human CDH15. In some embodiments, the targeting ligand comprises an antibody variable domain comprising a CDR, e.g., an HCDR1, an HCDR2, an HCDR3, an LCDR1, an LCDR2, and/or an LCDR3, of an HCVR and/or LCVR sequence as set forth in Table 1. In some embodiments, the targeting ligand comprises an antibody variable domain comprising a CDR, e.g., an HCDR1, an HCDR2, an HCDR3, an LCDR1, an LCDR2, and/or an LCDR3, as set forth in Table 1. In some embodiments, the targeting ligand comprises an antibody variable domain comprising a set of three CDRs, e.g., an HCDR1, an HCDR2, and an HCDR3, and/or an LCDR1, an LCDR2, and an LCDR3, of an HCVR and/or LCVR sequence as set forth in Table 1. In some embodiments, the targeting ligand comprises an antibody variable domain comprising a set of three CDRs, e.g., an HCDR1, an HCDR2, and an HCDR3, and/or an LCDR1, an LCDR2, and an LCDR3, as set forth in Table 1. In some embodiments, the targeting ligand comprises an antibody variable domain comprising an HCVR and/or LCVR as set forth in Table 1.
BRIEF DESCRIPTION OF THE FIGURES
[0034] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
[0035] Figure 1 depicts a schematic illustration of myogenesis of non-terminally differentiated muscle cells. Muscle stem cells (MuSCs; also known as satellite cells) are mitotically quiescent and non-proliferative under steady-state conditions in adult muscle tissue. In response to injury, however, MuSCs begin to actively divide, producing daughter cells. While some daughter cells become quiescent once more to replenish the pool of MuSCs, others continue to proliferate as myoblasts, which align and fuse together during differentiation into mature myotubes/myofibers that make up muscle fibers. Quiescent muscle stem cells can be characterized by Pax7 expression, proliferating myoblasts can be characterized by Pax7 and myogenic differentiation factor 1 (MyoD) expression, myoblasts committing to terminal differentiation (i.e., myocytes) are characterized by MyoD and Myogenin expression, and myotubes can be characterized by myosin heavy chain (MyHC) expression.
[0036] Figures 2A-2D demonstrate accelerated muscular regeneration in mice with CDH15 expression abolished. Figure 2A depicts a schematic illustration of the example experiments. Wild-type (WT) or homozygous CDH15 knockout mice (CDH15-/-) were injected intramuscularly with cardiotoxin (CTX) at day 0 to induce muscle damage. Histology was then performed on samples of muscle retrieved 5-, 15-, and 25-days post-injury (dpi). Figure 2B depicts an example of immunohistochemical cross-sections of WT and CDH15-/- muscle samples stained with DAPI (blue; light grey in greyscale), laminin (white), and embryonic myosin heavy chain (eMyHC; green; dark grey in greyscale), which is transiently upregulated in immature myofibers but downregulated as myofibers mature (Rodgers, Growth Hormone & IGF Research, 2005, 15(6): 377-383). Cross-sections were visualized by fluorescence microscopy. Figure 2C depicts the myofiber cross-section area (CSA) for WT and CDH15-/- mice at the indicated time points post-injury, as quantified from histological images, see, e.g., Figure 2B. Figure 2D depicts the percentage of centrally nucleated myofibers for WT and CDH15-/- mice at 15 days post-injury, as quantified from histological images, see, e.g., Figure 2B. Data are reported as means +/- SEM, *p<0.05, **p<0.01.
[0037] Figures 3A-3B demonstrate improved functional recovery from injury in mice with CDH15 expression abolished. Figure 3A depicts a schematic illustration of the example experiments. WT or CDH15-/- mice were injected with CTX into the extensor digitorum longus (EDL) muscle at day 0. At 15 dpi, the contractile force of the EDL muscle was measured ex vivo. Figure 3B depicts the maximal tetanic force loss in WT or CDH15-/- muscle as compared to muscle isolated from uninjured EDL. Data are reported as means +/- SEM, *p<0.05.
[0038] Figures 4A-4B demonstrate accelerated exit from quiescence ex vivo in MuSCs with CDH15 expression abolished. Figure 4A depicts an example of immunohistochemical images of WT and CDH15-/- MuSCs following 48 hours of culture on single myofibers. Arrows in the Pax7 and Merge columns denote individual cells in a multi-cell cluster. Figure 4B depicts the percentage of Pax7+ single-cell and multi-cell clusters (top) and the number of Pax7+ cells per cluster (bottom) for WT and CDH15-/- MuSCs, as quantified from histological images, see, e g., Figure 4A . Data are reported as means +/- SEM, **p<0.01.
[0039] Figures 5A-5B demonstrate the upregulation of early response genes containing serum response factor (SRF) motifs. Figure 5A depicts a volcano plot of genes that are downregulated or upregulated in FACS-isolated CDH15-/- MuSCs as compared to WT MuSCs. Figure 5B depicts a transcription factor motif analysis, with several relevant transcription factors highlighted in red.
[0040] Figures 6A-6D demonstrate that abolishing expression of CDH15 in aged mice rescues age-related declines in muscle regeneration. Figure 6A depicts a schematic illustration of the example experiments. Aged (23 -month-old) wild-type (WT) or homozygous CDH15 knockout mice (CDH15-/-) were injected with cardiotoxin (CTX) at day 0 to induce muscle damage of the tibialis anterior muscle. Histology was then performed on samples of muscle retrieved 15 days post-injury (dpi). Figure 6B depicts an example of immunohistochemical cross-sections of aged WT and CDH15-/- muscle samples stained for nuclei (DAPI; blue; grey in greyscale) and laminin (white) and visualized by fluorescence microscopy. Figure 6C depicts the myofiber cross-section area (CSA) for aged WT and CDH15-/- mice at 15 dpi, as quantified from histological images, see, e.g., Figure 6B. The dotted line depicts the average CSA for young mice at 15 dpi. Figure 6D depicts the percentage of centrally nucleated myofibers containing 3 or more central nuclei for aged WT and CDH15-/- mice at 15 dpi, as quantified from histological images, see, e.g., Figure 6B. Data are reported as means +/- SEM, *p<0.05. [0041] Figures 7A-7C demonstrate that CDH15 protein is focalized to the apical surface of the majority of wild-type (WT) muscle stem cells (MuSCs) and is undetectable in CDH15-/- MuSCs. Figure 7A depicts an immunohistochemical cross-section of a WT single myofiber with an associated WT MuSC, with CDH15 protein present at the apical surface of the MuSC. Anti- CDH15 staining is depicted as red and denoted with an arrow and dotted outline and DAPI staining is depicted as blue and denoted with an arrowhead and a dashed outline. Figure 7B depicts an immunohistochemical cross-section of WT mouse muscle, with Pax7-positive MuSCs observed on the periphery of myofibers, again with CDH15 present at the apical surface of the MuSC. Anti-CDH15 is depicted as red and denoted with an arrow and a dotted outline in the magnified inset image, DAPI-stained nuclei are depicted as blue, Pax7 is depicted as green, and laminin is depicted as white. Overlap of DAPI and Pax7 is denoted with an arrowhead and a dashed outline in the magnified inset image. Figure 7C depicts the proportion of Pax7-positive MuSCs that are also CDH15-positive in WT and CDH15-/- mouse muscle, as quantified from histological images, see, e.g., Figure 7A and 7B. While CDH15 protein is detected in the majority (-95%) of WT Pax7-positive muscle cells, CDH15 is undetectable in CDH15-/- Pax7- positive cells, as expected.
[0042] Figure 8 demonstrates the specific binding of anti-hCDH15 antibodies to human rhabdomyosarcoma cells, but not glioblastoma cells. Alveolar, embryonal, and glioblastoma cells were live-stained with the indicated anti-hCDH15 antibodies (REGN8787 and REGN9295) for 30 minutes, washed, stained with anti-human IgG Alexa Fluor™ 647-conjugated secondary antibody (red), washed again, fixed, and then stained for myogenin (green) and nuclei (DAPI; blue) prior to visualization by fluorescence microscopy. Cells incubated with murine IgG2a and human IgG4 were also used as controls. The last row depicts a merge of the three markers.
[0043] Figure 9 demonstrates the specific binding of anti-hCDH15 antibodies to human rhabdospheres grown in 3D culture. Embryonal rhabdomyosarcoma tumorspheres (z.e., rhabdospheres) were incubated with anti-hCDH15 or human IgG4 as a negative control for 30 minutes, washed, stained with anti-human IgG Alexa Fluor™ 647-conjugated secondary antibody (white), washed again, fixed and stained for myogenin (red) and nuclei (DAPI; blue). The last column depicts a merge of the three markers. [0044] Figure 10 demonstrates anti-CDH15 antigen-binding domain-mediated retargeting of AAV9 to C2C12 mouse myoblasts. Depicted are representative immunofluorescence images demonstrating the transduction efficiency via GFP fluorescence of C2C12 myoblasts transduced with 2.5 x 105 vg/cell of AAV9 expressing eGFP +/- plasmids encoding anti-CDH15 antigen-binding domains or control antigen-binding domains (anti- ASGR1) and varying mosaic capsid ratios. DAPI-stained nuclei are depicted in blue (top row) and eGFP is depicted in green (bottom row). The ratio provided indicates the ratio of quantities of transfected plasmid encoding SpyTag-conjugated AAV9 capsid versus non-conjugated N272A de-targeted AAV9 capsid. Where an antigen-binding domain is indicated e.g., a mAb or a Fab, the AAV9 capsid comprises SpyTag inserted at position 453 and attached via a 10 amino acid linker, and the antigen-binding domain comprises SpyCatcher fused to the C-terminus of the heavy chain construct.
[0045] Figure 11 demonstrates anti-CDH15 antigen-binding domain-mediated retargeting of AAV9 to human skeletal myoblasts. Depicted are representative immunofluorescence images demonstrating the transduction efficiency via GFP fluorescence of human skeletal myoblasts transduced with 2.5 x 105 vg/cell of AAV9 expressing eGFP +/- plasmids encoding anti-CDH15 antigen-binding domains or control antigen-binding domains (anti-ASGRl) and varying mosaic capsid ratios. DAPI-stained nuclei are depicted in blue (top row) and eGFP is depicted in green (bottom row). The ratio provided indicates the ratio of quantities of transfected plasmid encoding SpyTag-conjugated AAV9 capsid versus nonconjugated N272A de-targeted AAV9 capsid. Where an antigen-binding domain is indicated e.g., a mAb or a Fab, the AAV9 capsid comprises SpyTag inserted at position 453 and attached via a 10 amino acid linker, and the antigen -binding domain comprises SpyCatcher fused to the C-terminus of the heavy chain construct.
DETAILED DESCRIPTION
[0046] Each individual skeletal muscle consists of thousands of muscle fibers wrapped together by connective tissue sheaths. The individual bundles of muscle fibers in a skeletal muscle are known as fasciculi. The outermost connective tissue sheath surrounding the entire muscle is known as epimysium. The connective tissue sheath covering each fasciculus is known as perimysium, and the innermost sheath surrounding individual muscle fiber is known as endomysium. Each muscle fiber comprises myofibrils containing multiple myofilaments.
[0047] When bundled together, all the myofibrils get arranged in a unique striated pattern forming sarcomeres which are the fundamental contractile unit of a skeletal muscle. The two most significant myofilaments are actin and myosin filaments arranged distinctively to form various bands on the skeletal muscle.
[0048] The primary functions of the skeletal muscle take place via its intrinsic excitationcontraction coupling process. As the muscle is attached to the bone tendons, the contraction of the muscle leads to movement of that bone that allows for the performance of specific movements. The skeletal muscle also provides structural support and helps in maintaining the posture of the body. The skeletal muscle also acts as a storage source for amino acids that can be used by different organs of the body for synthesizing organ-specific proteins. The skeletal muscle also acts as a site of glucose disposal in the form of muscle glycogen. The skeletal muscle also plays a central role in maintaining thermostasis and acts as an energy source during starvation. Thus, skeletal muscle plays key roles in locomotion, thermoregulation, and in controlling whole body metabolism.
[0049] In many muscle diseases as well as during normal aging, the size and function of skeletal muscle tissue is reduced, resulting in impaired functional mobility; and in the case of severe muscle diseases, long-term disability and early mortality.
[0050] Treatments for muscle wasting and genetic muscle diseases typically consist of broad-acting therapies, such as testosterone therapy for muscle wasting, glucocorticoids for muscular dystrophies, and systemic AAV delivery for treatment of muscle diseases (e.g., X- linked myotubular myopathy (XLMTM), Duchenne muscular dystrophy (DMD), myotonic dystrophy (DM1), Facioscapulohumeral muscular dystrophy Type 1 (FSHD), congenital muscular dystrophy type 1A (MDC1A), Limb girdle muscular dystrophy, and dystroglycanopathy, etc.). Untargeted delivery of these therapies reduces efficiency of specific muscle uptake, while also causing significant detrimental off-target effects on other organs. [0051] Muscle stem cells (MuSCs) are important for skeletal muscle regeneration. While MuSCs are normally quiescent in adult muscle, upon injury, they activate, proliferate, and differentiate into functional myofibers. However, MuSCs in aging subjects display delayed activation and reduced motility in vitro, which translates to impaired MuSC mediated repair in vivo.
[0052] Mitotically quiescent muscle stem cells (MuSCs, also called satellite cells) and proliferating myoblasts are the embryonic precursors of myocytes (also called muscle cells) which have not yet fused together to form myotubes or myofibers that later become the muscle fibers. Muscle stem cells and myoblasts differentiate into muscle cells through a process called myogenesis depicted schematically (not to scale) in Figure 1.
[0053] In general, once exposed to signals from the damaged environment, muscle stem cells will leave their quiescent state, reenter the cell cycle, and start proliferating as myoblasts. Some daughter cells continue to differentiate, while others return to quiescence to replenish the reserve population of muscle stem cells. During the differentiation stage, certain genes (e.g., striated alpha-actin genes) are expressed and the myoblasts align with one another. The myoblasts then fuse to form myofibers with the recruitment of actin to the plasma membrane. [0054] Muscle stem cells can be characterized by a combination of several genetic markers, such Pax7 and muscle regulatory proteins. Pax7 is a paired homeobox transcription factor, which specifies the myogenic properties of precursor muscle cells. Accordingly, muscle stem cells, whether quiescent or proliferating, may be characterized as Pax7+. See, e.g, Figure 1. Additionally, myogenic cellular lineage characterization is possible since myogenesis depends on the precise and dynamic integration of multiple muscle regulatory factors, such as myogenic factor 5 (MYF5), myogenic differentiation factor 1 (MYOD), myogenin (MYOG), and embryonic myosin heavy chain (MyHC). For example, MYOD is expressed in myogenic cells, but not expressed in stationary quiescent muscle stem cells, and thus, may be used to identify proliferating muscle stem cells, myoblasts, or other differentiating myocytes. See, e.g., Figure 1. Myogenin appears to be expressed by myoblasts committed to differentiating into myofibers. See, e.g., Figure 1. An additional marker that may be used to identify which stage of myogenesis a cell is undergoing includes, but is not limited to embryonic myosin heavy chain (eMyHC), which is transiently upregulated in immature myofibers but downregulated as myofibers mature. See, e.g., Figure 1.
[0055] Myoblasts may be classified as skeletal muscle myoblasts, smooth muscle myoblasts, and cardiac muscle myoblasts depending on the type of muscle cell that they will differentiate into. Thus, muscle stem cells, myoblasts, myocytes, and undifferentiated myotubes or myofibers may all be considered a non-terminally differentiated muscle cell.
[0056] Many pathological conditions, such as muscular dystrophies (MDs) or muscle wasting, cannot provide sufficient signals for muscle stem cells, or satellite cells may have inherent defects in these conditions, which will impair their regeneration potential. Accordingly, the ability to specifically target non-terminally differentiated muscle cells, e.g., muscle stem cells, myoblasts, myotubes, etc., may be useful in the therapy of skeletal muscle disorders.
[0057] An example cell surface protein found on non-terminally differentiated muscle cells Cadherin 15 (CDH15). Cadherins are a class of calcium-dependent transmembrane proteins involved in cell-cell adhesion. Classical cadherins consist of an extracellular domain comprising five repeats of an immunoglobulin-like cadherin domain, a single transmembrane region, and a cytoplasmic domain. Cadherin 15 (also known as M-cadherin) is expressed at the apical surface of muscle stem cells and is believed to regulate adhesion of muscle stem cells to muscle myofibers. Cadherin 15 is encoded by the CDH15 gene, located on the long arm of chromosome 16 (16q24.3). CDH15 comprises 14 exons and is approximately 23,745 bases in length. An exemplary sequence for human CDH15 gene is assigned NCBI Accession Number NM_004933.3 (SEQ ID NO: 787). An exemplary human CDH15 protein is assigned NCBI Accession Number NP_004924.1 and/or UniProt Accession Number P55291 (SEQ ID NO: 788). [0058] Genetically modified animal models may prove to be particularly useful for studying the function of CDH15 in muscle. For example, although deletion of Cdhl5 in genetically modified animals does not alter quiescence, proliferation, myogenic lineage progression, or differentiation capacity by MuSCs isolated therefrom, the deletion of Cdhl5 in these mice results in accelerated regeneration following injury (Figures 2A-2D, 3A-3B). This may be due, at least in part, to an accelerated exit from quiescence upon deletion of CDH15 expression (Figures 4A-4B), which is associated with an induction of SRF-regulated early response genes (Figures 5A-5B), which is in turn associated with a decrease in Rho/Rac signaling. Interestingly, Cdhl5~'~ MuSCs are slightly larger than wildtype control MuSC (data not shown), an indicator that these cells are more primed for activation. Similar to the effects seen in young mice, Cdhl5 deletion also rescues age-related deficits in muscle regeneration (Figures 6A-6D). Accordingly, antagonistic antibodies, or CDH15 blocking antibodies may be useful in enhancing muscle regeneration in both young subjects and older adults following a damage stimulus (e.g., joint arthroplasty).
[0059] Moreover, certain muscle-related cancers, including rhabdomyosarcomas, may benefit from therapeutics that target muscle-specific markers. Rhabdomyosarcomas (RMS) are characterized by the expression of myogenic genes, but multiple subtypes exist. For example, embryonal RMS is the most prevalent subtype, typically has a more favorable prognosis, and is believed to be driven by loss of tumor suppressor genes or gain-of-function of proto-oncogenes. Alveolar RMS is less common but has generally carries a much poorer prognosis. Alveolar RMS is believed to be caused by chromosomal translocation of Pax3 or Pax7 gene, or alternative gene fusions. Additional rarer subtypes include spindle cell/sclerosing RMS, and pleomorphic RMS. Rhabdomyosarcomas make up -3% of all childhood cancers, with -400-500 new cases per year in the U.S (American Cancer Society, 2021). The prognosis is generally good in children (e.g., -70% survival), but depends on risk category. For example, 1/3 of patients with localized RMS and 2/3 of patients with metastatic RMS experience relapsed disease. If RMS is relapsed, the majority of patients have an estimated 5-year survival of -10%. Moreover, the prognosis is generally much poorer in adults (20-50% overall survival).
[0060] Accordingly, since high CDH15 expression is observed in all rhabdomyosarcomas (RMS) and may be associated with chemoresistance and poor prognosis, anti-CDH15 antibodies may be useful to deliver a therapeutic agent (e.g., a cytotoxic agent) to RMS tumors.
[0061] Described herein are antigen-binding proteins, e.g., antibodies and antigenbinding fragments thereof that bind to human CDH15. The antibodies described herein may be useful, inter alia, for specifically directing the internalization of an agent, e.g., a drug conjugate, etc., to a muscle cell and/or a muscle-related cancer cell, for blocking activity of CDH15, and/or for specifically stimulating muscle regeneration. As such, also described herein are antibodies, or antigen-binding fragments thereof, that bind human CDH15; including antibody-protein fusion constructs comprising an antibody, or antigen-binding fragment thereof, that bind human CDH15; and antibody drug conjugates comprising an antibody, or antigen-binding fragment thereof, that bind human CDH15. Moreover, to enhance muscle delivery of therapeutic payloads and mitigate off-target effects, described herein are viral particles, e.g., AAV viral particles, that can target non-terminally differentiated muscle cell surface proteins, such as mammalian CDH15.
[0062] The description herein is not limited to particular embodiments, compositions, methods and experimental conditions described, as such embodiments, compositions, methods and conditions may vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
[0063] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing as described herein, some preferred methods and materials are now described. All publications cited herein are incorporated herein by reference to describe in their entirety. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.
[0064] Singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to “a method” includes one or more methods, and/or steps of the type described herein and/or which will become apparent to those persons skilled in the art upon reading this disclosure.
[0065] The term “about,” when used in reference to a particular recited numerical value, means that the value may vary from the recited value by no more than 1%. For example, the expression “about 100” includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).
[0066] The “percent (%) identity” or the like may be readily determined for amino acid or nucleotide sequences, over the full-length of a protein, or a portion thereof. A portion may be at least about 5 amino acids or 24 nucleotides, respectively, in length, and may be up to about 700 amino acids or 2100 nucleotides, respectively. Generally, when referring to “identity”, “homology”, or “similarity” between two different adeno-associated viruses, “identity”, “homology” or “similarity” is determined in reference to “aligned” sequences. “Aligned” sequences or “alignments” refer to multiple nucleic acid sequences or protein (amino acids) sequences, often containing corrections for missing or additional bases or amino acids as compared to a reference sequence.
[0067] Alignments may be performed using any of a variety of publicly or commercially available Multiple Sequence Alignment Programs. Sequence alignment programs are available for amino acid sequences, e.g., the “Clustal X”, “MAP”, “PIMA”, “MSA”, “BLOCKMAKER”, “MEME”, and “Match-Box” programs. Generally, any of these programs are used at default settings, although one of skill in the art can alter these settings as needed. Alternatively, one of skill in the art can utilize another algorithm or computer program which provides at least the level of identity or alignment as that provided by the referenced algorithms and programs. See, e.g., J. D. Thomson et al, Nucl. Acids. Res., “A comprehensive comparison of multiple sequence alignments”, 27(13):2682-2690 (1999).
[0068] Multiple sequence alignment programs are also available for nucleic acid sequences. Examples of such programs include, “Clustal W”, “CAP Sequence Assembly”, “MAP”, and “MEME”, which are accessible through Web Servers on the internet. Other sources for such programs are known to those of skill in the art. Alternatively, Vector NTI utilities are also used. There are also a number of algorithms known in the art that can be used to measure nucleotide sequence identity, including those contained in the programs described above. As another example, polynucleotide sequences can be compared using FASTA™, a program in GCG Version 6.1. FASTA™ provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences. For instance, percent sequence identity between nucleic acid sequences can be determined using FASTA™ with its default parameters (a word size of 6 and the NOPAM factor for the scoring matrix) as provided in GCG Version 6.1, herein incorporated by reference.
[0069] “Significant identity” encompasses amino acid or nucleic acid sequences alignments that are at least 90%, e.g., at least 93%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99%, or e.g., at least 100% identical.
[0070] The term “chimeric” encompasses a functional gene or polypeptide comprising nucleic acid sequences or amino acid sequences, respectively, from at least two different AAV serotype, e.g., portions of a gene or polypeptide of at least a first and second AAV, wherein the at least first and second portions are operably linked to form a functional chimeric AAV nucleic acid that encodes a functional amino acid. Unless specified as chimeric, nucleotide sequences, genes, polypeptides, and amino acids are considered non-chimeric in that the nucleotide sequences, genes, polypeptides, and amino acids comprise a nucleic acid sequence or amino acid sequence having significant identity to a nucleic acid sequence or amino acid sequence, respectively, of a single AAV serotype. [0071] The phrase “operably linked”, as used herein, includes a physical juxtaposition (e.g., in three-dimensional space) of components or elements that interact, directly or indirectly with one another, or otherwise coordinate with each other to participate in a biological event, which juxtaposition achieves or permits such interaction and/or coordination. To give but one example, a regulatory element (e.g., an expression control sequence) in a nucleic acid is said to be “operably linked” to a coding sequence when it is located relative to the coding sequence such that its presence or absence impacts expression and/or activity of the coding sequence. In many embodiments, “operable linkage” involves covalent linkage of relevant components or elements with one another. Those skilled in the art will readily appreciate that, in some embodiments, covalent linkage is not required to achieve effective operable linkage. For example, proteins operably linked together may be associated with each other, e.g., via a covalent bond or a non- covalent bond. As a non-limiting example, a capsid protein as described herein may be operably linked to a targeting ligand, where the capsid protein is non-covalently bound to the targeting ligand, or covalently bound to the targeting ligand, optionally with or without a scaffold and/or adaptor between the capsid protein and the targeting ligand. As another example, in some embodiments, nucleic acid regulatory elements that are operably linked with coding sequences that they control are contiguous with the nucleotide of interest. Alternatively, or additionally, in some embodiments, one or more such regulatory elements acts in trans or at a distance to control a coding sequence of interest. In some embodiments, the term “regulatory element” as used herein refers to polynucleotide sequences which are necessary and/or sufficient to effect the expression and processing of coding sequences to which they are ligated. In some embodiments, a regulatory element may be or comprise appropriate transcription initiation, termination, promoter and/or enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., Kozak consensus sequence); sequences that enhance protein stability; and/or, in some embodiments, sequences that enhance protein secretion. In some embodiments, one or more regulatory elements are preferentially or exclusively active in a particular host cell or organism, or type thereof. To give but one example, in prokaryotes, regulatory elements may typically include promoter, ribosomal binding site, and transcription termination sequence; in eukaryotes, in many embodiments, regulatory elements may typically include promoters, enhancers, and/or transcription termination sequences. Those of ordinary skill in the art will appreciate from context that, in many embodiments, the term “regulatory elements” refers to components whose presence is essential for expression and processing, and in some embodiments includes components whose presence is advantageous for expression (including, for example, leader sequences, targeting sequences, and/or fusion partner sequences).
[0072] The phrase “an antibody that binds CDH15,” an “anti-hCDHl 5 antibody,” and the like includes an antibody and antigen-binding fragment thereof that specifically recognizes a single CDH15 molecule. An antibody and antigen-binding fragment thereof as described herein may bind soluble CDH15 and/or cell surface expressed CDH15. Soluble CDH15 includes natural CDH15 proteins as well as recombinant CDH15 protein variants that lack a transmembrane domain or are otherwise unassociated with a cell membrane.
[0073] The expression “cell surface-expressed CDH15” refers to one or more CDH15 protein(s) that is/are expressed on the surface of a cell in vitro or in vivo, such that at least a portion of a CDH15 protein is exposed to the extracellular side of the cell membrane and is accessible to an antigen-binding portion of an antibody. A “cell surface-expressed CDH15” can comprise or consist of a CDH15 protein expressed on the surface of a cell which normally expresses CDH15 protein. Alternatively, “cell surface-expressed CDH15” can comprise or consist of a CDH15 protein expressed on the surface of a cell that normally does not express human CDH15 on its surface but has been artificially engineered to express CDH15 on its surface.
[0074] The term “antigen -binding molecule” includes an antibody and an antigenbinding fragment of an antibody.
[0075] The term “antibody” refers to any antigen-binding molecule or molecular complex comprising at least one complementarity determining region (CDR) that specifically binds to or interacts with a particular antigen (e.g., CDH15). The term “antibody”, as used herein, includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CHI, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy -terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (heavy chain CDRs may be abbreviated as HCDR1, HCDR2 and HCDR3; light chain CDRs may be abbreviated as LCDR1, LCDR2 and LCDR3. The term “high affinity” antibody refers to those antibodies having a binding affinity to their target of at least 10'9 M, at least IO'10 M; at least 10'11 M; or at least 10'12 M, as measured by surface plasmon resonance, e.g., BIACORE™ or solution-affinity ELISA. The term “antibody” may encompass any type of antibody, such as e.g., monoclonal or polyclonal. Moreover, the antibody may be or any origin, such as e.g, mammalian or nonmammalian. In one embodiment, the antibody may be mammalian or avian. In a further embodiment, the antibody may be of human origin and may further be a human monoclonal antibody.
[0076] The term “antibody” also includes antigen-binding fragments of full antibody molecules. The terms “antigen-binding portion” of an antibody, “antigen-binding fragment” of an antibody, and the like include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. Antigen-binding fragments of an antibody may be derived, e.g., from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains. Such DNA is known and/or is readily available from, e.g., commercial sources, DNA libraries (including, e.g., phage-antibody libraries), or can be synthesized. The DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and/or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc.
[0077] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab’)2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g, an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FR3- CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalent nanobodies, bivalent nanobodies, efc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression “antigen-binding fragment”.
[0078] An antigen-binding fragment of an antibody will typically comprise at least one variable domain. The variable domain may be of any size or amino acid composition and will generally comprise at least one CDR which is adjacent to or in frame with one or more framework sequences. In antigen-binding fragments having a VH domain associated with a VL domain, the VH and VL domains may be situated relative to one another in any suitable arrangement. For example, the variable region may be dimeric and contain VH-VH, VH-VL or VL-VL dimers. Alternatively, the antigen-binding fragment of an antibody may contain a monomeric VH or VL domain.
[0079] In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting, example configurations of variable and constant domains that may be found within an antigenbinding fragment of an antibody as described herein include: (i) VH-CH1; (ii) VH-CH2; (iii) VH- CH3; (iv) VH-CH1-CH2; (V) VH-CH1-CH2-CH3; (vi) VH-CH2-CH3; (vii) VH-CL; (viii) VL-CH1; (ix) VL-CH2; (X) VL-CH3; (xi) VL-CH1-CH2; (xii) VL-CH1-CH2-CH3; (xiii) VL-CH2-CH3; and (xiv) VL- CL. In any configuration of variable and constant domains, including any of the example configurations listed above, the variable and constant domains may be either directly linked to one another or may be linked by a full or partial hinge or linker region. A hinge region may consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in a flexible or semi -flexible linkage between adjacent variable and/or constant domains in a single polypeptide molecule. Moreover, an antigen-binding fragment of an antibody as described herein may comprise a homo-dimer or hetero-dimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with one another and/or with one or more monomeric VH or VL domain (e.g., by disulfide bond(s)).
[0080] As with full antibody molecules, antigen-binding fragments may be monospecific or multispecific (e.g., bispecific). A multispecific antigen-binding fragment of an antibody will typically comprise at least two different variable domains, wherein each variable domain is capable of specifically binding to a separate antigen or to a different epitope on the same antigen. Any multispecific antibody format, including the example bispecific antibody formats disclosed herein, may be adapted for use in the context of an antigen-binding fragment of an antibody as described herein using routine techniques available in the art.
[0081] In certain embodiments, the anti-hCDH15 antibodies as described herein are human antibodies. The term “human antibody” refers to antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies as described herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3. However, the term “human antibody” is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
[0082] The antibodies as described herein may, in some embodiments, be recombinant human antibodies. The term “recombinant human antibody” is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described further below), antibodies isolated from a recombinant, combinatorial human antibody library (described further below), antibodies isolated from an animal e.g., a mouse) that is transgenic for human immunoglobulin genes (see e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295) or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo. [0083] Human antibodies may exist in two general forms that are associated with hinge heterogeneity. In one general form, an immunoglobulin molecule comprises a stable four chain construct of approximately 150-160 kDa in which the dimers are held together by an interchain heavy chain disulfide bond. In a second general form, the dimers are not linked via inter-chain disulfide bonds and a molecule of about 75-80 kDa is formed composed of a covalently coupled light and heavy chain (half-antibody). These forms have been extremely difficult to separate, even after affinity purification.
[0084] The frequency of appearance of the second form in various intact IgG isotypes is due to, but not limited to, structural differences associated with the hinge region isotype of the antibody. A single amino acid substitution in the hinge region of the human IgG4 hinge can significantly reduce the appearance of the second form (Angal et al. (1993) Molecular Immunology 30: 105) to levels typically observed using a human IgGl hinge. The antibodies as described herein may have one or more mutations in the hinge, CH2 or CH3 region which may be desirable, for example, in production, to improve the yield of the desired antibody form.
[0085] The antibodies as described herein may be isolated antibodies. An “isolated antibody” refers to an antibody that has been identified and separated and/or recovered from at least one component of its natural environment. For example, an antibody that has been separated or removed from at least one component of an organism, or from a tissue or cell in which the antibody naturally exists or is naturally produced, may be considered an “isolated antibody.” An isolated antibody also includes an antibody in situ within a recombinant cell. Isolated antibodies are antibodies that have been subjected to at least one purification or isolation step. According to certain embodiments, an isolated antibody may be substantially free of other cellular material and/or chemicals.
[0086] Also described herein are one-arm antibodies that bind CDH15. The term “one- arm antibody” refers to an antigen-binding molecule comprising a single antibody heavy chain and a single antibody light chain. The one-arm antibodies as described herein may comprise any of the HCVR/LCVR or CDR amino acid sequences as set forth in Table 1.
[0087] The anti-hCDH15 antibodies disclosed herein may comprise one or more amino acid substitutions, insertions and/or deletions in the framework and/or CDR regions of the heavy and light chain variable domains as compared to the corresponding germline sequences from which the antibodies were derived. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available from, for example, public antibody sequence databases. Also described herein are antibodies, and antigen-binding fragments thereof, which are derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework and/or CDR regions are mutated to the corresponding residue(s) of the germline sequence from which the antibody was derived, or to the corresponding residue(s) of another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are referred to herein collectively as “germline mutations”). A person of ordinary skill in the art, starting with the heavy and light chain variable region sequences disclosed herein, can easily produce numerous antibodies and antigen-binding fragments which comprise one or more individual germline mutations or combinations thereof. In certain embodiments, all of the framework and/or CDR residues within the VH and/or VL domains are mutated back to the residues found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., only the mutated residues found within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only the mutated residues found within CDR1, CDR2 or CDR3. In other embodiments, one or more of the framework and/or CDR residue(s) are mutated to the corresponding residue(s) of a different germline sequence (z.e., a germline sequence that is different from the germline sequence from which the antibody was originally derived). Furthermore, the antibodies as described herein may contain any combination of two or more germline mutations within the framework and/or CDR regions, e.g., wherein certain individual residues are mutated to the corresponding residue of a particular germline sequence while certain other residues that differ from the original germline sequence are maintained or are mutated to the corresponding residue of a different germline sequence. Once obtained, an antibody and an antigen-binding fragment that contains one or more germline mutations can be easily tested for one or more desired property such as, improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc. In some embodiments, an antibody or an antigen-binding fragment as described herein is obtained in this general manner.
[0088] Also described herein are anti-hCDH15 antibodies comprising variants of any of the HCVR, LCVR, and/or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, some embodiments include anti-hCDH15 antibodies having HCVR, LCVR, and/or CDR amino acid sequences with, e.g, 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc. conservative amino acid substitutions relative to any of the HCVR, LCVR, and/or CDR amino acid sequences set forth in Table 1 herein.
[0089] The phrase “bispecific antibody” includes an antibody capable of selectively binding two or more epitopes. Bispecific antibodies generally comprise two different heavy chains, with each heavy chain specifically binding a different epitope — either on two different molecules (e.g., antigens) or on the same molecule (e.g., on the same antigen). If a bispecific antibody is capable of selectively binding two different epitopes (a first epitope and a second epitope), the affinity of the first heavy chain for the first epitope will generally be at least one to two or three or four orders of magnitude lower than the affinity of the first heavy chain for the second epitope, and vice versa. The epitopes recognized by the bispecific antibody can be on the same or a different target (e.g., on the same or a different protein). Bispecific antibodies can be made, for example, by combining heavy chains that recognize different epitopes of the same antigen. For example, nucleic acid sequences encoding heavy chain variable sequences that recognize different epitopes of the same antigen can be fused to nucleic acid sequences encoding different heavy chain constant regions, and such sequences can be expressed in a cell that expresses an immunoglobulin light chain. A typical bispecific antibody has two heavy chains each having three heavy chain CDRs, followed by (N-terminal to C-terminal) a CHI domain, a hinge, a CH2 domain, and a CH3 domain, and an immunoglobulin light chain that either may not confer antigen-binding specificity but that can associate with each heavy chain, or that can associate with each heavy chain and that can bind one or more of the epitopes bound by the heavy chain antigen-binding regions, or that can associate with each heavy chain and participate in binding of one or both of the heavy chains to one or both epitopes.
[0090] The phrase “heavy chain,” or “immunoglobulin heavy chain” includes an immunoglobulin heavy chain constant region sequence from any organism, and unless otherwise specified includes a heavy chain variable domain. Heavy chain variable domains include three heavy chain CDRs and four FR regions, unless otherwise specified. Fragments of heavy chains include CDRs, CDRs and FRs, and combinations thereof. A typical heavy chain has, following the variable domain (from N-terminal to C-terminal), a CHI domain, a hinge, a CH2 domain, and a CH3 domain. A functional fragment of a heavy chain includes a fragment that is capable of specifically recognizing an antigen (e.g., recognizing the antigen with a KD in the micromolar, nanomolar, or picomolar range), that is capable of expressing and secreting from a cell, and that comprises at least one CDR.
[0091] The term “heavy chain only antibody,” “heavy chain only antigen binding protein,” “single domain antigen binding protein,” “single domain binding protein” or the like refers to a monomeric or homodimeric immunoglobulin molecule comprising an immunoglobulin-like chain comprising a variable domain operably linked to a heavy chain constant region, that is unable to associate with a light chain because the heavy chain constant region typically lacks a functional CHI domain. Accordingly, the term “heavy chain only antibody,” “heavy chain only antigen binding protein,” “single domain antigen binding protein,” “single domain binding protein” or the like encompasses a both (i) a monomeric single domain antigen binding protein comprising one of the immunoglobulin-like chain comprising a variable domain operably linked to a heavy chain constant region lacking a functional CHI domain, or (ii) a homodimeric single domain antigen binding protein comprising two immunoglobulin-like chains, each of which comprising a variable domain operably linked to a heavy chain constant region lacking a functional CHI domain. In various aspects, a homodimeric single domain antigen binding protein comprises two identical immunoglobulin-like chains, each of which comprising an identical variable domain operably linked to an identical heavy chain constant region lacking a functional CHI domain. Additionally, each immunoglobulin-like chain of a single domain antigen binding protein comprises a variable domain, which may be derived from heavy chain variable region gene segments (e.g., VH, DH, JH), light chain gene segments (e.g, VL, JL), or a combination thereof, linked to a heavy chain constant region (CH) gene sequence comprising a deletion or inactivating mutation in a CHI encoding sequence (and, optionally, a hinge region) of a heavy chain constant region gene, e.g., IgG, IgA, IgE, IgD, or a combination thereof. A single domain antigen binding protein comprising a variable domain derived from heavy chain gene segments may be referred to as a “VH- single domain antibody” or “Vn-single domain antigen binding protein”, see, e.g., U.S. Patent No. 8,754,287; U.S. Patent Publication Nos. 20140289876; 20150197553; 20150197554; 20150197555; 20150196015; 20150197556 and 20150197557, each of which is incorporated in its entirety by reference. A single domain antigen binding protein comprising a variable domain derived from light chain gene segments may be referred to as a or “V -single domain antigen binding protein,” see, e.g., U.S. Publication No. 20150289489, incorporated in its entirety by reference.
[0092] The phrase “light chain” includes an immunoglobulin light chain constant region sequence from any organism, and unless otherwise specified includes human kappa and lambda light chains. Light chain variable (VL) domains typically include three light chain CDRs and four framework (FR) regions, unless otherwise specified. Generally, a full-length light chain includes, from amino terminus to carboxyl terminus, a VL domain that includes FR1-CDR1- FR2-CDR2-FR3-CDR3-FR4, and a light chain constant domain. Light chains that may be useful include e.g., those, that do not selectively bind either the first or second antigen selectively bound by the antigen-binding protein. Suitable light chains include those that can be identified by screening for the most commonly employed light chains in existing antibody libraries (wet libraries or in silico), where the light chains do not substantially interfere with the affinity and/or selectivity of the antigen-binding domains of the antigen-binding proteins. Suitable light chains include those that can bind one or both epitopes that are bound by the antigen-binding regions of the antigen-binding protein.
[0093] The phrase “variable domain” includes an amino acid sequence of an immunoglobulin light or heavy chain (modified as desired) that comprises the following amino acid regions, in sequence from N-terminal to C-terminal (unless otherwise indicated): FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. A “variable domain” includes an amino acid sequence capable of folding into a canonical domain (VH or VL) having a dual beta sheet structure wherein the beta sheets are connected by a disulfide bond between a residue of a first beta sheet and a second beta sheet.
[0094] The phrase “complementarity determining region,” or the term “CDR,” includes an amino acid sequence encoded by a nucleic acid sequence of an organism’s immunoglobulin genes that normally (i.e., in a wildtype animal) appears between two framework regions in a variable region of a light or a heavy chain of an immunoglobulin molecule (e.g., an antibody or a T cell receptor). A CDR can be encoded by, for example, a germline sequence or a rearranged or unrearranged sequence, and, for example, by a naive or a mature B cell or a T cell. In some circumstances (e.g, for a CDR3), CDRs can be encoded by two or more sequences (e.g., germline sequences) that are not contiguous (e.g., in an unrearranged nucleic acid sequence) but are contiguous in a B cell nucleic acid sequence, e.g., as the result of splicing or connecting the sequences (e.g, V-D-J recombination to form a heavy chain CDR3).
[0095] Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs within the specified HCVR and/or LCVR amino acid sequences disclosed herein. Examples of conventions that can be used to identify the boundaries of CDRs include, e.g., the Kabat definition, the Chothia definition, and the AbM definition. In general terms, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of the structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia approaches. See, e.g., Kabat, “Sequences of Proteins of Immunological Interest,” National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86.9268-9272 (1989). Public databases are also available for identifying CDR sequences within an antibody.
[0096] The term “antibody fragment”, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Examples of binding fragments encompassed within the term “antibody fragment” include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab’)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al. (1989) Nature 241 : 544-546), which consists of a VH domain, (vi) an isolated CDR, and (vii) an scFv, which consists of the two domains of the Fv fragment, VL and VH, joined by a synthetic linker to form a single protein chain in which the VL and VH regions pair to form monovalent molecules.
Other forms of single chain antibodies, such as diabodies are also encompassed under the term “antibody” (see e.g., Holliger el al. (1993) PNAS USA 90:6444-6448; Poljak et al. (1994) Structure 2: 1121-1123).
[0097] The phrase “Fc-containing protein” includes antibodies, bispecific antibodies, immunoadhesins, and other binding proteins that comprise at least a functional portion of an immunoglobulin CH2 and CH3 region. A “functional portion” refers to a CH2 and CH3 region that can bind a Fc receptor (e.g., an FcyR; or an FcRn, i.e., a neonatal Fc receptor), and/or that can participate in the activation of complement. If the CH2 and CH3 region contains deletions, substitutions, and/or insertions or other modifications that render it unable to bind any Fc receptor and also unable to activate complement, the CH2 and CH3 region is not functional. [0098] Fc-containing proteins can comprise modifications in immunoglobulin domains, including where the modifications affect one or more effector function of the binding protein (e.g, modifications that affect FcyR binding, FcRn binding and thus half-life, and/or CDC activity). Such modifications include, but are not limited to, the following modifications and combinations thereof, with reference to EU numbering of an immunoglobulin constant region: 238, 239, 248, 249, 250, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280,
283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 297, 298, 301, 303, 305, 307, 308, 309, 311,
312, 315, 318, 320, 322, 324, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 337, 338, 339,
340, 342, 344, 356, 358, 359, 360, 361, 362, 373, 375, 376, 378, 380, 382, 383, 384, 386, 388,
389, 398, 414, 416, 419, 428, 430, 433, 434, 435, 437, 438, and 439.
[0099] For example, and not by way of limitation, the binding protein is an Fc-containing protein and exhibits enhanced serum half-life (as compared with the same Fc-containing protein without the recited modification(s)) and have a modification at position 250 (e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L/Y/F/W or T), 254 (e.g, S or T), and 256 (e.g., S/R/Q/E/D or T); or a modification at 428 and/or 433 (e.g., L/R/SI/P/Q or K) and/or 434 (e.g, H/F or Y); or a modification at 250 and/or 428; or a modification at 307 or 308 (e.g., 308F, V308F), and 434. In another example, the modification can comprise a 428L (e.g., M428L) and 434S (e.g., N434S) modification; a 428L, 2591 (e.g., V259I), and a 308F (e.g., V308F) modification; a 433K (e.g., H433K) and a 434 (e.g., 434Y) modification; a 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modification; a 250Q and 428L modification (e.g, T250Q and M428L); a 307 and/or 308 modification (e.g., 308F or 308P).
[00100] The term “antigen-binding protein,” as used herein, refers to a polypeptide or protein (one or more polypeptides complexed in a functional unit) that specifically recognizes an epitope on an antigen, such as a cell-specific antigen and/or a target antigen as described herein. An antigen-binding protein may be multi-specific. The term “multi-specific” with reference to an antigen-binding protein means that the protein recognizes different epitopes, either on the same antigen or on different antigens. A multi-specific antigen-binding protein as described herein can be a single multifunctional polypeptide, or it can be a multimeric complex of two or more polypeptides that are covalently or non-covalently associated with one another. The term “antigen-binding protein” includes antibodies or fragments thereof as described herein that may be linked to or co-expressed with another functional molecule, e.g, another peptide or protein. For example, an antibody or fragment thereof can be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent association or otherwise) to one or more other molecular entities, such as a protein or fragment thereof to produce a bispecific or a multi-specific antigenbinding molecule with a second binding specificity.
[00101] The term “protein” means any amino acid polymer having multiple amino acids covalently linked via amide bonds. Proteins contain one or more amino acid polymer chains, generally known in the art as “polypeptides”. Thus, a polypeptide may be a protein, and a protein may contain multiple polypeptides to form a single functioning biomolecule. Disulfide bridges (i.e., between cysteine residues to form cystine) may be present in some proteins. These covalent links may be within a single polypeptide chain, or between two individual polypeptide chains. For example, disulfide bridges are essential to proper structure and function of insulin, immunoglobulins, protamine, and the like. For a recent review of disulfide bond formation, see Oka and Bulleid, “Forming disulfides in the endoplasmic reticulum,” 1833(11) Biochim Biophys Acta 2425-9 (2013).
[00102] As used herein, “protein” includes biotherapeutic proteins, recombinant proteins used in research or therapy, trap proteins and other Fc-fusion proteins, chimeric proteins, antibodies, monoclonal antibodies, human antibodies, bispecific antibodies, antibody fragments, nanobodies, recombinant antibody chimeras, scFv fusion proteins, cytokines, chemokines, peptide hormones, and the like. Proteins may be produced using recombinant cell-based production systems, such as the insect bacculovirus system, yeast systems (e.g., Pichia sp.), mammalian systems (e.g., CHO cells and CHO derivatives like CHO-K1 cells). For a recent review discussing biotherapeutic proteins and their production, see Ghaderi et al., “Production platforms for biotherapeutic glycoproteins. Occurrence, impact, and challenges of non-human sialylation,” 28 Biotechnol Genet Eng Rev. 147-75 (2012).
[00103] As used herein, the term “epitope” refers to the portion of the antigen which is recognized by the multi-specific antigen-binding polypeptide. A single antigen (such as an antigenic polypeptide) may have more than one epitope. Epitopes may be defined as structural or functional. Functional epitopes are generally a subset of structural epitopes and are defined as those residues that directly contribute to the affinity of the interaction between the antigen- binding polypeptide and the antigen. Epitopes may also be conformational, that is, composed of non-linear amino acids. In certain embodiments, epitopes may include determinants that are chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, in certain embodiments, may have specific three- dimensional structural characteristics, and/or specific charge characteristics. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents.
[00104] The term “domain” refers to any part of a protein or polypeptide having a particular function or structure. Preferably, domains as described herein bind to cell-specific or target antigens. Cell-specific antigen- or target antigen-binding domains, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen.
[00105] The term “half-body” or “half-antibody”, which are used interchangeably, refers to half of an antibody, which essentially contains one heavy chain and one light chain. Antibody heavy chains can form dimers, thus the heavy chain of one half-body can associate with heavy chain associated with a different molecule (e.g., another half-body) or another Fc-containing polypeptide. Two slightly different Fc-domains may “heterodimerize” as in the formation of bispecific antibodies or other heterodimers, -trimers, -tetramers, and the like. See Vincent and Murini, “Current strategies in antibody engineering: Fc engineering and pH-dependent antigen binding, bispecific antibodies and antibody drug conjugates,” 7 Biotechnol. J. 1444-1450 (20912); and Shimamoto et al., “Peptibodies: A flexible alternative format to antibodies,” 4(5) MAbs 586-91 (2012).
[00106] The term “single-chain variable fragment” or “scFv” includes a single chain fusion polypeptide containing an immunoglobulin heavy chain variable region (VH) and an immunoglobulin light chain variable region (VL). In some embodiments, the VH and VL are connected by a linker sequence of 10 to 25 amino acids. ScFv polypeptides may also include other amino acid sequences, such as CL or CHI regions. ScFv molecules can be manufactured by phage display or made by directly subcloning the heavy and light chains from a hybridoma or B-cell. Ahmad et al., Clinical and Developmental Immunology, volume 2012, article ID 98025 is incorporated herein by reference for methods of making scFv fragments by phage display and antibody domain cloning. [00107] As used herein, the term “muscle-related cancer” refers to any cancerous cell defined by the expression of myogenic genes e.g, expression of CDH15. “Muscle-related cancers” include, but are not limited to, rhabdomyosarcoma e.g., embryonal, alveolar, spindle cell/scl erosing, pleomorphic, etc.
[00108] The term “target cells” includes any cells in which expression of a nucleotide of interest is desired. Preferably, target cells exhibit a receptor on their surface that allows the cell to be targeted with a targeting ligand, as described below. In certain embodiments, a target cell is a muscle cell. A “muscle cell”, as used herein, refers to any cell expressing myogenic markers and/or involved in skeletal muscle regeneration, e.g., muscle stem cells (“MuSC”, also known as satellite cells) and which express Pax7 and thus are considered Pax7+ cells, e.g., Pax7+/MyoD' when quiescent and Pax7+/MyoD+ when proliferating), myoblasts (e.g., MyoD+Myogenin+ cells), myocytes, myotubes (also known as myofibers, e.g., MyHC+ cells), etc.
[00109] “Retargeting” or “redirecting” may include a scenario in which the wildtype particle targets several cells within a tissue and/or several organs within an organism, and general targeting of the tissue or organs is reduced or abolished by insertion of the heterologous amino acid, and retargeting to more a specific cell in the tissue or a specific organ in the organism is achieved with the targeting ligand (e.g., via a targeting ligand) that binds a marker expressed by the specific cell. Such retargeting or redirecting may also include a scenario in which the wildtype particle targets a tissue, and targeting of the tissue is reduced to or abolished by insertion of the heterologous amino acid, and retargeting to a completely different tissue is achieved with the targeting ligand.
[00110] “Specific binding pair,” “binding pair,” “protein:protein binding pair” and the like includes two members (e.g., a first member (e.g., a first polypeptide) and a second cognate member (e.g., a second polypeptide)) that interact to form a bond (e.g., a non-covalent bond between a first member epitope and a second member antigen-binding portion of an antibody that recognizes the epitope; a covalent bond between e.g., proteins capable of forming isopeptide bonds; split inteins that recognize each other and, through the process of protein trans-splicing, mediate ligation of the flanking proteins and their own removal). In some embodiments, the term “cognate” refers to components that function together. Epitopes and cognate antibodies thereto, particularly epitopes that may also act as a detectable label (e.g., c-myc) are well-known in the art. Specific proteimprotein binding pairs capable of interacting to form a covalent isopeptide bond are reviewed in Veggiani et al. (2014) Trends Biotechnol. 32:506, and include peptide:peptide binding pairs such as SpyTag: SpyCatcher, SpyTag002:SpyCatcher002; SpyTag:KTag; isopeptag:pilin C, SnoopTag: SnoopCatcher, etc., and biologically equivalent variants thereof, e.g. , SpyTag003 : SpyCatcher003. Generally, a first member of a protein: protein binding pair refers to member of a protein: protein binding pair, which is generally less than 30 amino acids in length, and which forms a spontaneous covalent isopeptide bond with the second cognate protein, wherein the second cognate protein is generally larger, but may also be less than 30 amino acids in length such as in the SpyTag:KTag system.
[00111] The term “isopeptide bond” refers to an amide bond between a carboxyl or carboxamide group and an amino group at least one of which is not derived from a protein main chain or alternatively viewed is not part of the protein backbone. An isopeptide bond may form within a single protein or may occur between two peptides or a peptide and a protein. Thus, an isopeptide bond may form intramolecularly within a single protein or intermolecularly i.e. between two peptide/protein molecules, e.g. between two peptide linkers. Typically, an isopeptide bond may occur between a lysine residue and an asparagine, aspartic acid, glutamine, or glutamic acid residue or the terminal carboxyl group of the protein or peptide chain or may occur between the alpha-amino terminus of the protein or peptide chain and an asparagine, aspartic acid, glutamine or glutamic acid. Each residue of the pair involved in the isopeptide bond is referred to herein as a reactive residue. In preferred embodiments of the invention, an isopeptide bond may form between a lysine residue and an asparagine residue or between a lysine residue and an aspartic acid residue. Particularly, isopeptide bonds can occur between the side chain amine of lysine and carboxamide group of asparagine or carboxyl group of an aspartate.
[00112] The SpyTag: SpyCatcher system is described in U.S. Patent No. 9,547,003 and Zaveri et al. (2012) PNAS 109:E690-E697, each of which is incorporated herein in its entirety by reference, and is derived from the CnaB2 domain of the Streptococcus pyogenes fibronectin- binding protein FbaB. By splitting the domain, Zakeri et al. obtained a peptide “SpyTag” having the sequence AHIVMVDAYKPTK (SEQ ID NO: 815) which forms an amide bond to its cognate protein “SpyCatcher,” an 112 amino acid polypeptide having the amino acid sequence set forth in SEQ ID NO: 816. (Zakeri (2012), supra). An additional specific binding pair derived from CnaB2 domain is SpyTag:KTag, which forms an isopeptide bond in the presence of SpyLigase. (Fierer (2014) PNAS 111 :E1176-1181) SpyLigase was engineered by excising the P strand from SpyCatcher that contains a reactive lysine, resulting in KTag, a 10-residue first member of a protein: protein binding pair having the amino acid sequence ATHIKFSKRD (SEQ ID NO: 817). The SpyTag002:SpyCatcher002 system is described in Keeble et al (2017) Angew Chem Int Ed Engl 56: 16521-25, incorporated herein in its entirety by reference. SpyTag002 has the amino acid sequence VPTIVMVDAYKRYK, set forth as SEQ ID NO: 821, and binds SpyCatcher002. SpyTag003 has the amino acid sequence RGVPHIVMVDAYKRYK, set forth as SEQ ID NO: 822, and binds SpyCatcher003.
[00113] The SnoopTag: SnoopCatcher system is described in Veggiani (2016) PNAS 113: 1202-07. The D4 Ig-like domain of RrgA, an adhesion from Streptococcus pneumoniae, was split to form SnoopTag (residues 734-745) and SnoopCatcher (residues 749-860).
Incubation of SnoopTag and SnoopCatcher results in a spontaneous isopeptide bond that is specific between the complementary proteins. Veggiani (2016)), supra.
[00114] The isopeptag:pilin-C specific binding pair was derived from the major pilin protein Spy0128 from Streptococcus pyogenes. (Zakeir and Howarth (2010) J. Am. Chem. Soc. 132:4526-27). Isopeptag has the amino acid sequence TDKDMTITFTNKKDAE, set forth as SEQ ID NO: 820, and binds pilin-C (residues 18-299 of SpyO 128). Incubation of isopeptag and pilin-C results in a spontaneous isopeptide bond that is specific between the complementary proteins. Zakeir and Howarth (2010), supra.
[00115] The term “transduction” or “infection” or the like refers to the introduction of a nucleic acid into a target cell e.g., a muscle stem cell, myoblast, myocyte, any combination thereof, etc.) nucleus by a viral particle. The term efficiency in relation to transduction or the like, e.g., “transduction efficiency” refers to the fraction e.g., percentage) of cells expressing a nucleotide of interest after incubation with a set number of viral particles comprising the nucleotide of interest. Well-known methods of determining transduction efficiency include flow cytometry of cells transduced with a fluorescent reporter gene, RT-PCR for expression of the nucleotide of interest, etc.
[00116] Generally, “reference” viral capsid protein/capsid/particle are identical to test viral capsid protein/capsid/particle but for the change for which the effect is to be tested. For example, to determine the effect, e.g., on transduction efficiency, of inserting a first member of a specific binding pair into a test viral particle, the transduction efficiencies of the test viral particle (in the absence or presence of an appropriate targeting ligand) can be compared to the transduction efficiencies of a reference viral particle (in the absence or presence of an appropriate targeting ligand if necessary) which is identical to the test viral particle in every instance (e.g., additional point mutations, nucleotide of interest, numbers of viral particles and target cells, etc.) except for the presence of a first member of a specific binding pair. In some embodiments, a reference viral capsid protein is one that is able to form a capsid with a second viral capsid protein modified to comprise at least a first member of a proteimprotein binding pair, where the reference viral capsid protein does not comprise the first member of a protein: protein binding pair, preferably wherein the capsid formed by the reference viral capsid protein and the modified viral capsid protein is a mosaic capsid.
Antigen-Binding Proteins
[00117] Described herein are antigen-binding proteins, e.g., antibodies, or antigen-binding fragments thereof, comprising an amino acid of an HCVR, HCDR1, HCDR2, HCDR3, LCVR, LCV1, LCVR2, and/or LCVR3 as set forth in Table 1.
[00118] In certain embodiments, an antigen-binding protein, e.g., an antibody, or an antigen-binding fragment thereof, as described herein comprises a heavy chain CDR1 (HCDR1) comprising an amino acid sequence selected from any of the HCDR1 amino acid sequences listed in Table 1 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.
[00119] In certain embodiments, an antigen-binding protein, e.g., an antibody, or an antigen-binding fragment thereof, as described herein comprises a heavy chain CDR2 (HCDR2) comprising an amino acid sequence selected from any of the HCDR2 amino acid sequences listed in Table 1 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.
[00120] In certain embodiments, an antigen-binding protein, e.g., an antibody, or an antigen-binding fragment thereof, as described herein comprises a heavy chain CDR3 (HCDR3) comprising an amino acid sequence selected from any of the HCDR3 amino acid sequences listed in Table 1 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.
[00121] In certain embodiments, an antigen-binding protein, e.g., an antibody, or an antigen-binding fragment thereof, as described herein comprises a light chain CDR1 (LCDR1) comprising an amino acid sequence selected from any of the LCDR1 amino acid sequences listed in Table 1 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.
[00122] In certain embodiments, an antigen-binding protein, e.g., an antibody, or an antigen-binding fragment thereof, as described herein comprises a light chain CDR2 (LCDR2) comprising an amino acid sequence selected from any of the LCDR2 amino acid sequences listed in Table 1 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.
[00123] In certain embodiments, an antigen-binding protein, e.g., an antibody, or an antigen-binding fragment thereof, as described herein comprises a light chain CDR3 (LCDR3) comprising an amino acid sequence selected from any of the LCDR3 amino acid sequences listed in Table lor a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.
[00124] In some embodiments, an antibody, or an antigen-binding fragment thereof, as described herein comprises an HCDR3 and an LCDR3 amino acid sequence pair (HCDR3/LCDR3) comprising any of the HCDR3 amino acid sequences listed in Table 1 paired with any of the LCDR3 amino acid sequences listed in Table 1. In some embodiments, an antibody, or antigen-binding fragments thereof, as described herein comprises an HCDR3/LCDR3 amino acid sequence pair contained within any of the example anti-hCDH15 antibodies listed in Table 1. In some embodiments, the HCDR3/LCDR3 amino acid sequence pair is selected from the group consisting of SEQ ID NOs: 8 and 16, 28 and 36, 48 and 54, 66 and 54, 76 and 54, 86 and 54, 96 and 102, 113 and 119, 131 and 139, 151 and 159, 171 and 179, 191 and 196, 208 and 214, 226 and 54, 236 and 54, 246 and 54, 255 and 54, 265 and 273, 285 and 293, 305 and 313, 325 and 333, 345 and 352, 364 and 372, 382 and 54, 392 and 398, 410 and 414, 426 and 434, 442 and 450, 458 and 466, 474 and 482, 490 and 498, 506 and 514, 522 and 530, 538 and 546, 554 and 562, 570 and 578, 586 and 594, 602 and 610, 618 and 626, 634 and 642, 650 and 658, 666 and 674, 682 and 690, 698 and 706, 714 and 722, 730 and 738, 746 and 690, 754 and 762, and 770 and 778.
[00125] Described herein are antigen-binding proteins, e.g., an antibody, or an antigenbinding fragment thereof, comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1- LCDR2-LCDR3) contained within any of the example anti-hCDH15 antibodies listed in Table 1. In some embodiments, the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences set is selected from the group consisting of SEQ ID NOs: 4-6-8-12-14-16, 24-26-28- 32-34-36, 44-46-48-52-34-54, 62-64-66-52-34-54, 72-74-76-52-34-54, 82-84-86-52-34-54, 92- 94-96-100-34-102, 82-111-113-117-34-119, 127-129-131-135-137-139, 147-149-151-155-157- 159, 167-169-171-175-177-179, 187-189-191-52-34-196, 204-206-208-212-137-214, 222-224- 226-52-34-54, 232-234-236-52-34-54, 242-244-246-52-34-54, 82-253-255-52-34-54, 261-263- 265-269-271-273, 281-283-285-289-291-293, 301-303-305-309-311-313, 321-323-325-329-331- 333, 341-343-345-349-14-352, 360-362-364-368-370-372, 187-380-382-52-34-54, 388-390-392- 396-14-398, 406-408-410-100-34-414, 422-424-426-430-432-434, 438-440-442-446-448-450, 454-456-458-462-464-466, 470-472-474-478-480-482, 486-488-490-494-496-498, 502-504-506- 510-512-514, 518-520-522-526-528-530, 534-536-538-542-544-546, 550-552-554-558-560-562, 566-568-570-574-576-578, 582-584-586-590-592-594, 598-600-602-606-608-610, 614-616-618- 622-624-626, 630-632-634-638-640-642, 646-648-650-654-656-658, 662-664-666-670-672-674, 678-680-682-686-688-690, 694-696-698-702-704-706, 710-712-714-718-720-722, 726-728-730- 734-736-738, 742-744-746-686-688-690, 750-752-754-758-760-762, and 766-768-770-774-776- 778.
[00126] Also described herein are antibodies, or antigen-binding fragments thereof, comprising a set of six CDRs (z.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within an HCVR/LCVR amino acid sequence pair as defined by any of the example anti-hCDH15 antibodies listed in Table 1. In some embodiments, an antibody, or antigenbinding fragments thereof, as described herein comprises the HCDR1-HCDR2-HCDR3-LCDR1- LCDR2-LCDR3 amino acid sequences set contained within an HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 and 10, 22 and 30, 42 and 50, 60 and 50, 70 and 50, 80 and 50, 90 and 98, 108 and 115, 125 and 133, 145 and 153, 165 and 173, 185 and 193, 202 and 210, 220 and 50, 230 and 50, 240 and 50, 250 and 50, 259 and 267, 279 and 287, 299 and 307, 319 and 327, 339 and 347, 358 and 366, 378 and 50, 386 and 394,
404 and 412, 420 and 428, 436 and 444, 452 and 460, 468 and 476, 484 and 492, 500 and 508,
516 and 524, 532 and 540, 548 and 556, 564 and 572, 580 and 588, 596 and 604, 612 and 620,
628 and 636, 644 and 652, 660 and 668, 676 and 684, 692 and 700, 708 and 716, 724 and 732,
740 and 684, 748 and 756, and 764 and 772. [00127] In some embodiments, the antigen-binding protein comprises a heavy chain variable region (HCVR or VH). In some embodiments, the HCVR comprises a set of HCDR1- HCDR2-HCDR3 amino acid sequences selected from Table 1. In some embodiments, the HCVR comprises an amino acid sequence selected from any of the HCVR amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.
[00128] In some embodiments, the antigen-binding protein comprises a light chain variable region (LCVR or VL). In some embodiments, the LCVR comprises a set of LCDR1- LCDR2-LCDR3 amino acid sequences selected from Table 1 below. In some embodiments, the LCVR comprises an amino acid sequence selected from any of the LCVR amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.
[00129] In some embodiments, the antigen-binding protein, e.g, antibody or antigenbinding fragment thereof, comprises an HCVR and an LCVR amino acid sequence pair (HCVR/LCVR) comprising any of the HCVR amino acid sequences listed in Table 1 paired with any of the LCVR amino acid sequences listed in Table 1. In some embodiments, the antigenbinding protein, e.g., antibody or antigen-binding fragment thereof, as described herein comprises an HCVR/LCVR amino acid sequence pair contained within any of the example anti- hCDH15 antibodies listed in Table 1. In certain embodiments, the HCVR/LCVR amino acid sequence pair is selected from the group consisting of SEQ ID NOs: 2 and 10, 22 and 30, 42 and 50, 60 and 50, 70 and 50, 80 and 50, 90 and 98, 108 and 115, 125 and 133, 145 and 153, 165 and 173, 185 and 193, 202 and 210, 220 and 50, 230 and 50, 240 and 50, 250 and 50, 259 and 267, 279 and 287, 299 and 307, 319 and 327, 339 and 347, 358 and 366, 378 and 50, 386 and 394,
404 and 412, 420 and 428, 436 and 444, 452 and 460, 468 and 476, 484 and 492, 500 and 508,
516 and 524, 532 and 540, 548 and 556, 564 and 572, 580 and 588, 596 and 604, 612 and 620,
628 and 636, 644 and 652, 660 and 668, 676 and 684, 692 and 700, 708 and 716, 724 and 732,
740 and 684, 748 and 756, and 764 and 772.
Nucleic Acid Molecules
[00130] Described herein are also nucleic acid molecules, e.g., polynucleotides, encoding the antigen-binding proteins, e.g., antibodies or antigen-binding fragments thereof, as described herein. [00131] Also described herein are nucleic acid molecules encoding any of the HCDR1 amino acid sequences listed in Table 1; in some embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR1 nucleic acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.
[00132] Also described herein are nucleic acid molecules encoding any of the HCDR2 amino acid sequences listed in Table 1; in some embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR2 nucleic acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.
[00133] Also described herein are nucleic acid molecules encoding any of the HCDR3 amino acid sequences listed in Table 1; in some embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR3 nucleic acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.
[00134] Also described herein are nucleic acid molecules encoding any of the LCDR1 amino acid sequences listed in Table 1; in some embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR1 nucleic acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.
[00135] Also described herein are nucleic acid molecules encoding any of the LCDR2 amino acid sequences listed in Table 1; in some embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR2 nucleic acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.
[00136] Also described herein are nucleic acid molecules encoding any of the LCDR3 amino acid sequences listed in Table 1; in some embodiments the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR3 nucleic acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto. Also described herein are nucleic acid molecules encoding both an HCVR and an LCVR, wherein the HCVR comprises an amino acid sequence of any of the HCVR amino acid sequences listed in Table 1, and wherein the LCVR comprises an amino acid sequence of any of the LCVR amino acid sequences listed in Table 1. In some embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto, and a polynucleotide sequence selected from any of the LCVR nucleic acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto. In some embodiments, the nucleic acid molecule encodes an HCVR and LCVR, wherein the HCVR and LCVR are both derived from the same anti-hCDH15 antibody listed in Table 1.
[00137] Also described herein are recombinant expression vectors capable of expressing a polypeptide comprising a heavy and/or light chain variable region of an anti-hCDH15 antibody. In some embodiments, a recombinant expression vector comprises any of the nucleic acid molecules mentioned above, i.e., nucleic acid molecules encoding any of the HCVR, LCVR, and/or CDR sequences as set forth in Table 1. Also described are host cells into which such vectors have been introduced, as well as methods of producing the antibodies or portions thereof by culturing the host cells under conditions permitting production of the antibodies or antibody fragments, and recovering the antibodies or antibody fragments so produced.
Multispecific Antigen-Binding Molecules
[00138] An anti-hCDH15 antibody and antigen-binding fragment thereof as described herein may be monospecific, bi-specific, or multispecific. Multispecific antibodies may be specific for different epitopes of one target polypeptide or may contain antigen-binding domains specific for more than one target polypeptide. See, e.g., Tutt et al., 1991, J. Immunol. 147:60-69; Kufer etal., 2004, Trends Biotechnol. 22:238-244. An anti-hCDH15 antibody and antigenbinding fragment thereof as described herein can be linked to or co-expressed with another functional molecule, e.g, another peptide or protein. For example, an antibody or fragment thereof can be functionally linked e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment to produce a bi-specific or a multispecific antibody with a second or additional binding specificity. [00139] Use of the expression “anti-hCDH15 antibody” herein is intended to include both monospecific anti-hCDH15 antibodies as well as bispecific antibodies comprising a CDH15- binding arm and a “target’ ’-binding arm. Thus, described herein are bispecific antibodies wherein one arm of an immunoglobulin binds human CDH15, and the other arm of the immunoglobulin is specific for another target molecule. The CDH15-binding arm can comprise any of the HCVR/LCVR or CDR amino acid sequences as set forth in Table 1 herein.
[00140] In certain embodiments, the CDH15-binding arm binds to human CDH15 and induces internalization of the CDH15 and antibody bound thereto. In certain embodiments, the CDH15-binding arm binds weakly to human CDH15 and induces internalization of CDH15 and antibody bound thereto. In certain embodiments, the CDH15-binding arm binds to human CDH15 and blocks the activity of CDH15.
[00141] In certain embodiments, the bispecific antigen-binding molecule is a bispecific antibody. Each antigen-binding domain of a bispecific antibody comprises a heavy chain variable domain (HCVR) and a light chain variable domain (LCVR). In the context of a bispecific antigen-binding molecule comprising a first and a second antigen-binding domain (e.g., a bi specific antibody), the CDRs of the first antigen-binding domain may be designated with the prefix “Al” and the CDRs of the second antigen-binding domain may be designated with the prefix “A2”. Thus, the CDRs of the first antigen-binding domain may be referred to herein as A1-HCDR1, A1-HCDR2, and A1-HCDR3; and the CDRs of the second antigenbinding domain may be referred to herein as A2-HCDR1, A2-HCDR2, and A2-HCDR3.
[00142] The first antigen-binding domain and the second antigen-binding domain may be directly or indirectly connected to one another to form a bispecific antigen-binding molecule as described herein. Alternatively, the first antigen-binding domain and the second antigen-binding domain may each be connected to a separate multimerizing domain. The association of one multimerizing domain with another multimerizing domain facilitates the association between the two antigen-binding domains, thereby forming a bispecific antigen-binding molecule. A “multimerizing domain” is any macromolecule, protein, polypeptide, peptide, or amino acid that has the ability to associate with a second multimerizing domain of the same or similar structure or constitution. For example, a multimerizing domain may be a polypeptide comprising an immunoglobulin CH3 domain. A non-limiting example of a multimerizing component is an Fc portion of an immunoglobulin (comprising a CH2-CH3 domain), e.g., an Fc domain of an IgG selected from the isotypes IgGl, IgG2, IgG3, and IgG4, as well as any allotype within each isotype group.
[00143] Bispecific antigen-binding molecules as described herein will typically comprise two multimerizing domains, e.g, two Fc domains that are each individually part of a separate antibody heavy chain. The first and second multimerizing domains may be of the same IgG isotype such as, e.g., IgGl/IgGl, IgG2/IgG2, IgG4/IgG4. Alternatively, the first and second multimerizing domains may be of different IgG isotypes such as, e.g., IgGl/IgG2, IgGl/IgG4, IgG2/IgG4, etc.
[00144] In certain embodiments, the multimerizing domain is an Fc fragment or an amino acid sequence of from 1 to about 200 amino acids in length containing at least one cysteine residue. In other embodiments, the multimerizing domain is a cysteine residue, or a short cysteine-containing peptide. Other multimerizing domains include peptides or polypeptides comprising or consisting of a leucine zipper, a helix-loop motif, or a coiled-coil motif.
[00145] Any bispecific antibody format or technology may be used to make the bispecific antigen-binding molecules as described herein. For example, an antibody or fragment thereof having a first antigen binding specificity can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment having a second antigen-binding specificity to produce a bispecific antigen-binding molecule. Specific examples of bispecific formats include, without limitation, e.g., scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, Quadroma, knobs-into-holes, common light chain (e.g., common light chain with knobs-into-holes, etc.), CrossMab, CrossFab, (SEED)body, leucine zipper, Duobody, IgGl/IgG2, dual acting Fab (DAF)-IgG, and Mab2 bispecific formats (see, e.g., Klein et al. 2012, mAbs 4:6, 1-11, and references cited therein, for a review of the foregoing formats).
[00146] In the context of bispecific antigen-binding molecules as described herein, the multimerizing domains, e.g, Fc domains, may comprise one or more amino acid changes (e.g., insertions, deletions or substitutions) as compared to the wild-type, naturally occurring version of the Fc domain. For example, bispecific antigen-binding molecules may comprise one or more modifications in the Fc domain that results in a modified Fc domain having a modified binding interaction (e.g., enhanced or diminished) between Fc and FcRn. In one embodiment, the bispecific antigen-binding molecule comprises a modification in a CH2 or a CH3 region, wherein the modification increases the affinity of the Fc domain to FcRn in an acidic environment (e.g., in an endosome where pH ranges from about 5.5 to about 6.0). Non-limiting examples of such Fc modifications include, e.g., a modification at position 250 e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L/Y/F/W or T), 254 (e.g., S or T), and 256 (e.g, S/R/Q/E/D or T); or a modification at position 428 and/or 433 (e.g., L/R/S/P/Q or K) and/or 434 (e.g., H/F or Y); or a modification at position 250 and/or 428; or a modification at position 307 or 308 (e.g., 308F, V308F), and 434. In one embodiment, the modification comprises a 428L (e.g., M428L) and 434S (e.g., N434S) modification; a 428L, 2591 (e.g., V259I), and 308F (e.g., V308F) modification; a 433K (e.g., H433K) and a 434 (e.g, 434Y) modification; a 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modification; a 250Q and 428L modification (e.g., T250Q and M428L); and a 307 and/or 308 modification (e.g., 308F or 308P).
[00147] Also described herein are bispecific antigen-binding molecules comprising a first CH3 domain and a second Ig CH3 domain, wherein the first and second Ig CH3 domains differ from one another by at least one amino acid, and wherein at least one amino acid difference reduces binding of the bi specific antibody to Protein A as compared to a bi-specific antibody lacking the amino acid difference. In one embodiment, the first Ig CH3 domain binds Protein A and the second Ig CH3 domain contains a mutation that reduces or abolishes Protein A binding such as an H95R modification (by IMGT exon numbering; H435R by EU numbering). The second CH3 may further comprise a Y96F modification (by IMGT; Y436F by EU). See, for example, US Patent No. 8,586,713. Further modifications that may be found within the second CH3 include: D16E, L18M, N44S, K52N, V57M, and V82I (by IMGT; D356E, L358M, N384S, K392N, V397M, and V422I by EU) in the case of IgGl antibodies; N44S, K52N, and V82I (IMGT; N384S, K392N, and V422I by EU) in the case of IgG2 antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I by EU) in the case of IgG4 antibodies.
[00148] In certain embodiments, the Fc domain may be chimeric, combining Fc sequences derived from more than one immunoglobulin isotype. For example, a chimeric Fc domain can comprise part or all of a CH2 sequence derived from a human IgGl, human IgG2 or human IgG4 CH2 region, and part or all of a CH3 sequence derived from a human IgGl, human IgG2 or human IgG4. A chimeric Fc domain can also contain a chimeric hinge region. For example, a chimeric hinge may comprise an “upper hinge” sequence, derived from a human IgGl, a human IgG2 or a human IgG4 hinge region, combined with a “lower hinge” sequence, derived from a human IgGl, a human IgG2 or a human IgG4 hinge region. A particular example of a chimeric Fc domain that can be included in any of the antigen-binding molecules set forth herein comprises, from N- to C-terminus: [IgG4 CHI] - [IgG4 upper hinge] - [IgG2 lower hinge] - [IgG4 CH2] - [IgG4 CH3], Another example of a chimeric Fc domain that can be included in any of the antigen-binding molecules set forth herein comprises, from N- to C-terminus: [IgGl CHI] - [IgGl upper hinge] - [IgG2 lower hinge] - [IgG4 CH2] - [IgGl CH3], These and other examples of chimeric Fc domains that can be included in any of the antigen -binding molecules as described herein are described in US Publication 2014/0243504, published August 28, 2014, which is herein incorporated in its entirety. Chimeric Fc domains having these general structural arrangements, and variants thereof, can have altered Fc receptor binding, which in turn affects Fc effector function.
[00149] In certain embodiments, an antibody heavy chain as described herein comprises a heavy chain constant (CH) region that comprises an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to any one of SEQ ID NOs: 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, or 802. In some embodiments, the heavy chain constant region (CH) region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, or 802. [00150] In some embodiments, an antibody heavy chain as described herein comprises an Fc domain that comprises an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to any one of SEQ ID NOs: 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, or 814. In some embodiments, the Fc domain comprises an amino acid sequence selected form the group consisting SEQ ID NOs: 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, or 814.
Germline Mutations
[00151] The anti-hCDH15 antibodies disclosed herein may comprise one or more amino acid substitutions, insertions and/or deletions in the framework and/or CDR regions of the heavy chain variable domains as compared to the corresponding germline sequences from which the antibodies were derived. [00152] An anti-hCDH15 antibody and antigen-binding fragment thereof as disclosed herein may be derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework and/or CDR regions are mutated to the corresponding residue(s) of the germline sequence from which the antibody was derived, or to the corresponding residue(s) of another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are referred to herein collectively as “germline mutations”), and having weak or no detectable binding to a CDH15 antigen. Several such example antibodies that recognize CDH15 are described in Table 1 herein.
[00153] Furthermore, an anti-hCDH15 antibody and antigen-binding fragment thereof as disclosed herein may contain any combination of two or more germline mutations within the framework and/or CDR regions, e.g., wherein certain individual residues are mutated to the corresponding residue of a particular germline sequence while certain other residues that differ from the original germline sequence are maintained or are mutated to the corresponding residue of a different germline sequence. Once obtained, an antibody or antigen-binding fragment that contains one or more germline mutations can be tested for one or more desired properties such as, improved binding specificity, weak or reduced binding affinity, improved or enhanced pharmacokinetic properties, reduced immunogenicity, etc. In some embodiments, an antibody or antigen-binding fragment as described herein is obtained in this general manner.
[00154] Also described herein are anti-hCDH15 antibodies and antigen-binding fragments thereof comprising variants of any of the HCVR, LCVR, and/or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, an anti-hCDH15 antibody or antigen-binding fragment thereof as described herein may comprise HCVR, LCVR, and/or CDR amino acid sequences with, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc. conservative amino acid substitutions relative to any of the HCVR, LCVR, and/or CDR amino acid sequences set forth in Table 1 herein. An antibody and antigen-binding fragment thereof as described herein may comprise one or more amino acid substitutions, insertions and/or deletions in the framework and/or CDR regions of the heavy and light chain variable domains as compared to the corresponding germline sequences from which the individual antigen-binding domains were derived, while maintaining or improving the desired weak-to-no detectable binding to, e.g., CDH15. A “conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g, charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein, i.e., the amino acid substitution maintains or improves the desired weak to no detectable binding affinity in the case of anti-hCDH15 binding molecules. Examples of groups of amino acids that have side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide- containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartate and glutamate, and (7) sulfur-containing side chains are cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine- isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 1443- 1445. A “moderately conservative” replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix.
[00155] Also described herein are anti-hCDH15 antibodies and antigen-binding fragments thereof comprising an antigen-binding domain with an HCVR and/or CDR amino acid sequence that is substantially identical to any of the HCVR and/or CDR amino acid sequences disclosed herein, while maintaining or improving the desired weak affinity to CDH15 antigen. The term “substantial identity” or “substantially identical,” when referring to an amino acid sequence means that two amino acid sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 95% sequence identity, even more preferably at least 98% or 99% sequence identity. Preferably, residue positions which are not identical differ by conservative amino acid substitutions. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well-known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24: 307-331.
[00156] Sequence similarity for polypeptides, which is also referred to as sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, GCG software contains programs such as Gap and Bestfit which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutein thereof. See, e.g, GCG Version 6.1. Polypeptide sequences also can be compared using FASTA using default or recommended parameters, a program in GCG Version 6.1. FASTA e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson (2000) supra). Another preferred algorithm when comparing a sequence as described herein to a database containing a large number of sequences from different organisms is the computer program BLAST, especially BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402.
[00157] Once obtained, antigen-binding domains that contain one or more germline mutations were tested for decreased binding affinity utilizing one or more in vitro assays. Generally, antibodies that recognize a particular antigen are typically screened for their purpose by testing for high (i.e. strong) binding affinity to the antigen.
[00158] Unexpected benefits, for example, improved pharmacokinetic properties and low toxicity to the patient may be realized from further modifying the antibodies as described herein by the methods described herein.
Binding Properties of the Antibodies
[00159] The term “binding” in the context of the binding of an antibody, immunoglobulin, antibody-binding fragment, or Fc-containing protein to either, e.g., a predetermined antigen, such as a cell surface protein or fragment thereof, typically refers to an interaction or association between a minimum of two entities or molecular structures, such as an antibody-antigen interaction.
[00160] For instance, binding affinity typically corresponds to a KD value of about 10'7 M or less, such as about 10'8 M or less, such as about 10'9 M or less when determined by, for instance, surface plasmon resonance (SPR) technology in a BIAcore 3000 instrument using the antigen as the ligand and the antibody, Ig, antibody -binding fragment, or Fc-containing protein as the analyte (or antiligand). Cell-based binding strategies, such as fluorescent-activated cell sorting (FACS) binding assays, are also routinely used and provide binding characterization data with respect to cell-surface expressed proteins. FACS data correlates well with other methods such as radioligand competition binding and SPR (Benedict, CA, J Immunol Methods . 1997, 201(2):223-31 ; Geuijen, CA, et al. J Immunol Methods . 2005, 302(l-2):68-77).
[00161] Accordingly, an anti-hCDH15 antibody and antigen-binding fragment thereof as described herein bind to the predetermined antigen or cell surface molecule (receptor) having an affinity corresponding to a KD value that is at least ten-fold lower than its affinity for binding to a non-specific antigen (e.g., BSA, casein). The affinity of an antibody corresponding to a KD value that is equal to or less than ten-fold lower than a non-specific antigen may be considered non- detectable binding, however such an antibody may be paired with a second antigen binding arm for the production of a bispecific antibody as described herein.
[00162] The term “KD” or “KD” in molar (M) refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, or the dissociation equilibrium constant of an antibody or antibody-binding fragment binding to an antigen. There is an inverse relationship between KD and binding affinity, therefore the smaller the KD value, the higher, i.e. stronger, the affinity. Thus, the terms “higher affinity” or “stronger affinity” relate to a higher ability to form an interaction and therefore a smaller KD value, and conversely the terms “lower affinity” or “weaker affinity” relate to a lower ability to form an interaction and therefore a larger KD value. In some circumstances, a higher binding affinity (or KD) of a particular molecule (e.g. antibody) to its interactive partner molecule (e.g. antigen X) compared to the binding affinity of the molecule (e.g. antibody) to another interactive partner molecule (e.g. antigen Y) may be expressed as a binding ratio determined by dividing the larger KD value (lower, or weaker, affinity) by the smaller KD (higher, or stronger, affinity), for example expressed as 5-fold or 10- fold greater binding affinity, as the case may be.
[00163] The term “kd” (sec -1 or 1/s) refers to the dissociation rate constant of a particular antibody-antigen interaction, or the dissociation rate constant of an antibody or antibody -binding fragment. Said value is also referred to as the koff value.
[00164] The term “ka” (M-l x sec-1 or 1/M) refers to the association rate constant of a particular antibody-antigen interaction, or the association rate constant of an antibody or antibody-binding fragment. [00165] The term “KA” (M-l or 1/M) refers to the association equilibrium constant of a particular antibody-antigen interaction, or the association equilibrium constant of an antibody or antibody-binding fragment. The association equilibrium constant is obtained by dividing the ka by the ka.
[00166] The term “EC50” or “EC50” refers to the half maximal effective concentration, which includes the concentration of an antibody which induces a response halfway between the baseline and maximum after a specified exposure time. The EC50 essentially represents the concentration of an antibody where 50% of its maximal effect is observed. In certain embodiments, the EC50 value equals the concentration of an antibody as described herein that gives half-maximal binding to cells expressing CDH15, as determined by e.g. a FACS binding assay or an androgen receptor activation luciferase assay. Thus, reduced or weaker binding is observed with an increased EC50, or half maximal effective concentration value.
[00167] In one embodiment, decreased binding can be defined as an increased EC50 antibody concentration which may result in binding to the half-maximal amount of target cells. [00168] Also described herein is an antigen-binding protein, e.g., an antibody or antigenbinding fragment thereof, that binds CDH15-expressing cells with a single digit nM or triple digit pM KD, as measured by surface plasmon resonance, or equivalent assay. Also described herein is an antigen-binding protein, e.g., antibody or antigen-binding fragment thereof, that binds CDH15-expressing cells with an EC50 of greater than 100 nM as measured by FACS analysis. Also described herein is an antigen-binding protein, e.g., antibody or antigen-binding fragment thereof, that binds and is internalized into CDH15-expressing cells upon binding to CDH15. Also described herein is an antigen-binding protein, e.g., antibody or antigen-binding fragment thereof, that binds to and blocks the activity of CDH15.
Sequence Variants
[00169] An anti-hCACNGl antibody and antigen-binding fragment as described herein may comprise one or more amino acid substitutions, insertions and/or deletions in the framework and/or CDR regions of the heavy and light chain variable domains as compared to the corresponding germline sequences from which the individual antigen-binding domains were derived. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available from, for example, public antibody sequence databases. The antigen-binding molecules as described herein may comprise antigen-binding domains which are derived from any of the example amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework and/or CDR regions are mutated to the corresponding residue(s) of the germline sequence from which the antibody was derived, or to the corresponding residue(s) of another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are referred to herein collectively as “germline mutations”). A person of ordinary skill in the art, starting with the heavy and light chain variable region sequences disclosed herein, can easily produce numerous antibodies and antigen-binding fragments which comprise one or more individual germline mutations or combinations thereof. In certain embodiments, all of the framework and/or CDR residues within the VH and/or VL domains are mutated back to the residues found in the original germline sequence from which the antigen-binding domain was originally derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., only the mutated residues found within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only the mutated residues found within CDR1, CDR2 or CDR3. In other embodiments, one or more of the framework and/or CDR residue(s) are mutated to the corresponding residue(s) of a different germline sequence (z.e., a germline sequence that is different from the germline sequence from which the antigen-binding domain was originally derived). Furthermore, the antigen-binding domains may contain any combination of two or more germline mutations within the framework and/or CDR regions, e.g., wherein certain individual residues are mutated to the corresponding residue of a particular germline sequence while certain other residues that differ from the original germline sequence are maintained or are mutated to the corresponding residue of a different germline sequence. Once obtained, antigenbinding domains that contain one or more germline mutations can be easily tested for one or more desired property such as, improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc. Described herein are antigen-binding molecules comprising one or more antigen-binding domains obtained in this general manner.
[00170] Also described herein are antigen-binding molecules wherein one or both antigenbinding domains comprise variants of any of the HCVR, LCVR, and/or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, antigenbinding molecules as described herein may comprise an antigen-binding domain having HCVR, LCVR, and/or CDR amino acid sequences with, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc. conservative amino acid substitutions relative to any of the HCVR, LCVR, and/or CDR amino acid sequences disclosed herein. A “conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. Examples of groups of amino acids that have side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartate and glutamate, and (7) sulfur-containing side chains are cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 1443-1445, herein incorporated by reference. A “moderately conservative” replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix.
[00171] Antigen-binding molecules as described herein may comprise an antigen-binding domain with an HCVR, LCVR, and/or CDR amino acid sequence that is substantially identical to any of the HCVR, LCVR, and/or CDR amino acid sequences disclosed herein. The term “substantial identity” or “substantially identical,” when referring to an amino acid sequence means that two amino acid sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 95% sequence identity, even more preferably at least 98% or 99% sequence identity. Preferably, residue positions which are not identical differ by conservative amino acid substitutions. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well-known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24: 307-331, herein incorporated by reference. [00172] Sequence similarity for polypeptides, which is also referred to as sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, GCG software contains programs such as Gap and Bestfit which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutein thereof. See, e.g, GCG Version 6.1. Polypeptide sequences also can be compared using FASTA using default or recommended parameters, a program in GCG Version 6.1. FASTA (e.g, FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson (2000) supra). Another preferred algorithm when comparing a sequence to a database containing a large number of sequences from different organisms is the computer program BLAST, especially BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402, each herein incorporated by reference. pH-Dependent Binding
[00173] Also described herein are anti-hCDH15 antibodies and antigen-binding fragments thereof with pH-dependent binding characteristics. For example, an anti-hCDH15 as described herein may exhibit reduced binding to CDH15 at acidic pH as compared to neutral pH. Alternatively, anti-hCDH15 antibodies as described herein may exhibit enhanced binding to CDH15 at acidic pH as compared to neutral pH. The expression “acidic pH” includes pH values less than about 6.2, e.g., about 6.0, 5.95, 5,9, 5.85, 5.8, 5.75, 5.7, 5.65, 5.6, 5.55, 5.5, 5.45, 5.4, 5.35, 5.3, 5.25, 5.2, 5.15, 5.1, 5.05, 5.0, or less. The expression “neutral pH” means a pH of about 7.0 to about 7.4. The expression “neutral pH” includes pH values of about 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35, and 7.4.
[00174] In certain instances, “reduced binding ... at acidic pH as compared to neutral pH” is expressed in terms of a ratio of the KD value of the antibody binding to its antigen at acidic pH to the KD value of the antibody binding to its antigen at neutral pH (or vice versa). For example, an antibody or antigen-binding fragment thereof may be regarded as exhibiting “reduced binding to CACNG1 at acidic pH as compared to neutral pH” for purposes of the description herein if the antibody or antigen-binding fragment thereof exhibits an acidic/neutral KD ratio of about 3.0 or greater. In certain embodiments, the acidic/neutral KD ratio for an antibody or antigen-binding fragment as described herein can be about 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 20.0. 25.0, 30.0, 40.0, 50.0, 60.0, 70.0, 100.0 or greater.
[00175] Antibodies with pH-dependent binding characteristics may be obtained, e.g. , by screening a population of antibodies for reduced (or enhanced) binding to a particular antigen at acidic pH as compared to neutral pH. Additionally, modifications of the antigen-binding domain at the amino acid level may yield antibodies with pH-dependent characteristics. For example, by substituting one or more amino acids of an antigen-binding domain (e.g., within a CDR) with a histidine residue, an antibody with reduced antigen-binding at acidic pH relative to neutral pH may be obtained.
Antibodies Comprising Fc Variants
[00176] In some embodiments, an anti-hCDH15 antibody and antigen-binding fragment thereof (including a multispecific antigen-binding molecule and a multidomain therapeutic protein comprising an anti-hCDH15 antibody or an antigen-binding fragment thereof) is provided comprising an Fc domain comprising one or more mutations which enhance or diminish antibody binding to the FcRn receptor, e.g., at acidic pH as compared to neutral pH. For example, antibodies as described herein may comprise a mutation in the CH2 or a CH3 region of the Fc domain, wherein the mutation(s) increases the affinity of the Fc domain to FcRn in an acidic environment (e.g., in an endosome where pH ranges from about 5.5 to about 6.0). Such mutations may result in an increase in serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications include, e.g., a modification at position 250 (e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L/Y/F/W or T), 254 (e.g., S or T), and 256 (e.g., S/R/Q/E/D or T); or a modification at position 428 and/or 433 (e.g., H/L/R/S/P/Q or K) and/or 434 (e.g., H/F or Y); or a modification at position 250 and/or 428; or a modification at position 307 or 308 (e.g., 308F, V308F), and 434. In one embodiment, the modification comprises a 428L (e.g., M428L) and 434S (e.g., N434S) modification; a 428L, 2591 (e.g., V259I), and 308F (e.g, V308F) modification; a 433K (e.g., H433K) and a 434 (e.g., 434Y) modification; a 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modification; a 250Q and 428L modification (e.g., T250Q and M428L); and a 307 and/or 308 modification (e.g, 308F or 308P). [00177] For example, an anti-hCDH15 antibody and antigen-binding fragment as described herein may comprise an Fc domain comprising one or more pairs or groups of mutations selected from the group consisting of: 250Q and 248L (e.g., T250Q and M248L);
252Y, 254T and 256E (e.g., M252Y, S254T and T256E); 428L and 434S (e.g., M428L and N434S); and 433K and 434F (e.g, H433K and N434F). All possible combinations of the foregoing Fc domain mutations, and other mutations within the antibody variable domains disclosed herein, are contemplated within the description herein.
Biological Characteristics of the Antibodies and Bispecific Antigen-Binding Molecules [00178] Also described herein is an antibody and antigen-binding fragment thereof that binds human CDH15 with high, medium or low affinity, depending on the therapeutic context and particular targeting properties that are desired. For example, in the context of a bispecific antigen-binding molecule, wherein one arm binds CDH15 and another arm binds a target antigen (e.g., a tumor associated antigen), it may be desirable for the target antigen-binding arm to bind the target antigen with high affinity while the anti-hCDH15 arm binds CDH15 with only moderate or low affinity. In this manner, preferential targeting of the antigen-binding molecule to cells expressing the target antigen may be achieved while avoiding general/untargeted CDH15 binding and the consequent adverse side effects associated therewith.
[00179] Also described herein are antibodies, antigen-binding fragments, and bispecific antibodies thereof that bind human CDH15 with weak (i.e. low) or even no detectable affinity. In some embodiments, an antibody and antigen-binding fragment thereof as described herein binds human CDH15 (e.g., at 37°C) with a KD of greater than about 100 nM as measured by surface plasmon resonance. In some embodiments, an antibody or antigen-binding fragment as described herein binds CDH15 with a KD of greater than about greater than about 110 nM, at least 120 nM, greater than about 130 nM, greater than about 140 nM, greater than about 150 nM, at least 160 nM, greater than about 170 nM, greater than about 180 nM, greater than about 190 nM, greater than about 200 nM, greater than about 250 nM, greater than about 300 nM, greater than about 400 nM, greater than about 500 nM, greater than about 600 nM, greater than about 700 nM, greater than about 800 nM, greater than about 900 nM, or greater than about 1 pM, or with no detectable affinity, as measured by surface plasmon resonance (e.g., mAb-capture or antigencapture format), or a substantially similar assay.
Epitope Mapping and Related Technologies
[00180] The epitope on CDH15 to which an anti-hCDH15 antibody and antigen-binding fragment thereof as described herein may consist of a single contiguous sequence of 3 or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids of a CDH15 protein. Alternatively, the epitope may consist of a plurality of non-contiguous amino acids (or amino acid sequences) of CDH15. The term “epitope” refers to an antigenic determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule known as a paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind to different areas on an antigen and may have different biological effects. Epitopes may be either conformational or linear. A conformational epitope is produced by spatially juxtaposed amino acids from different segments of the linear polypeptide chain. A linear epitope is one produced by adjacent amino acid residues in a polypeptide chain. In certain circumstances, an epitope may include moieties of saccharides, phosphoryl groups, or sulfonyl groups on the antigen.
[00181] Various techniques known to persons of ordinary skill in the art can be used to determine whether an antigen-binding domain of an antibody “interacts with one or more amino acids” within a polypeptide or protein. Examples of techniques include, e.g., routine crossblocking assay such as that described Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY), alanine scanning mutational analysis, peptide blots analysis (Reineke, 2004, Methods Mol Biol 248:443-463), and peptide cleavage analysis. In addition, methods such as epitope excision, epitope extraction and chemical modification of antigens can be employed (Tomer, 2000, Protein Science 9:487-496). Another method that can be used to identify the amino acids within a polypeptide with which an antigen-binding domain of an antibody interacts is hydrogen/deuterium exchange detected by mass spectrometry. In general terms, the hydrogen/deuterium exchange method involves deuterium-labeling the protein of interest, followed by binding the antibody to the deuterium-labeled protein. Next, the protein/antibody complex is transferred to water to allow hydrogen-deuterium exchange to occur at all residues except for the residues protected by the antibody (which remain deuterium- labeled). After dissociation of the antibody, the target protein is subjected to protease cleavage and mass spectrometry analysis, thereby revealing the deuterium-labeled residues which correspond to the specific amino acids with which the antibody interacts. See, e.g., Ehring (1999) Analytical Biochemistry 262(2y.252-25 Engen and Smith (2001) Anal. Chem. 73:256A- 265A. X-ray crystallography of the antigen/antibody complex may also be used for epitope mapping purposes.
[00182] Also described herein are anti-hCDHl 5 antibodies that bind to the same epitope as any of the specific example antibodies described herein (e.g., antibodies comprising any of the amino acid sequences as set forth in Table 1 herein). Likewise, also described herein are anti- hCDH15 antibodies that compete for binding to CDH15 with any of the specific example antibodies described herein (e.g., antibodies comprising any of the amino acid sequences as set forth in Table 1 herein).
[00183] One can easily determine whether a particular antigen-binding molecule (e.g., antibody) or antigen-binding domain thereof binds to the same epitope as, or competes for binding with, a reference antigen-binding molecule as described herein by using routine methods known in the art. For example, to determine if a test antibody binds to the same epitope on CDH15 as a reference bispecific antigen-binding molecule as described herein, the reference bispecific molecule is first allowed to bind to a CDH15 protein. Next, the ability of a test antibody to bind to the CDH15 molecule is assessed. If the test antibody is able to bind to CDH15 following saturation binding with the reference bispecific antigen-binding molecule, it can be concluded that the test antibody binds to a different epitope of CDH15 than the reference bispecific antigen-binding molecule. On the other hand, if the test antibody is not able to bind to the CDH15 molecule following saturation binding with the reference bispecific antigen-binding molecule, then the test antibody may bind to the same epitope of CDH15 as the epitope bound by the reference bispecific antigen-binding molecule as described herein. Additional routine experimentation (e.g., peptide mutation and binding analyses) can then be carried out to confirm whether the observed lack of binding of the test antibody is in fact due to binding to the same epitope as the reference bispecific antigen-binding molecule or if steric blocking (or another phenomenon) is responsible for the lack of observed binding. Experiments of this sort can be performed using ELISA, RIA, Biacore, flow cytometry or any other quantitative or qualitative antibody-binding assay available in the art. In accordance with some embodiments described herein, two antigen-binding proteins bind to the same (or overlapping) epitope if, e.g., a 1-, 5-, 10-, 20- or 100-fold excess of one antigen-binding protein inhibits binding of the other by at least 50% but preferably 75%, 90% or even 99% as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 1990:50: 1495-1502). Alternatively, two antigen-binding proteins are deemed to bind to the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antigen-binding protein reduce or eliminate binding of the other. Two antigen-binding proteins are deemed to have “overlapping epitopes” if only a subset of the amino acid mutations that reduce or eliminate binding of one antigenbinding protein reduce or eliminate binding of the other.
[00184] To determine if an antibody or antigen-binding domain thereof competes for binding with a reference antigen-binding molecule, the above-described binding methodology is performed in two orientations: In a first orientation, the reference antigen-binding molecule is allowed to bind to a CDH15 protein under saturating conditions followed by assessment of binding of the test antibody to the CDH15 molecule. In a second orientation, the test antibody is allowed to bind to a CDH15 molecule under saturating conditions followed by assessment of binding of the reference antigen-binding molecule to the CDH15 molecule. If, in both orientations, only the first (saturating) antigen-binding molecule is capable of binding to the CDH15 molecule, then it is concluded that the test antibody and the reference antigen-binding molecule compete for binding to CDH15. As will be appreciated by a person of ordinary skill in the art, an antibody that competes for binding with a reference antigen-binding molecule may not necessarily bind to the same epitope as the reference antibody, but may sterically block binding of the reference antibody by binding an overlapping or adjacent epitope.
Preparation of Antigen-Binding Domains and Construction of Bispecific Molecules [00185] Antigen-binding domains specific for particular antigens can be prepared by any antibody generating technology known in the art. Once obtained, two different antigen-binding domains, specific for two different antigens (e.g., CDH15 and a target antigen), can be appropriately arranged relative to one another to produce a bispecific antigen-binding molecule as described herein using routine methods. (A discussion of example bispecific antibody formats that can be used to construct the bispecific antigen-binding molecules as described herein is provided elsewhere herein). In certain embodiments, one or more of the individual components (e.g., heavy and light chains) of the antigen-binding molecules as described herein are derived from chimeric, humanized or fully human antibodies. Methods for making such antibodies are well known in the art. For example, one or more of the heavy and/or light chains of the antigenbinding molecules as described herein can be prepared using VELOCIMMUNE™ technology. Using VELOCIMMUNE™ technology (or any other human antibody generating technology), high affinity chimeric antibodies to a particular antigen (e.g., CDH15) are initially isolated having a human variable region and a mouse constant region. The antibodies are characterized and selected for desirable characteristics, including affinity, selectivity, epitope, etc. The mouse constant regions are replaced with a desired human constant region to generate fully human heavy and/or light chains that can be incorporated into the antigen-binding molecules as described herein.
[00186] Genetically engineered animals may be used to make human bispecific antigenbinding molecules. For example, a genetically modified mouse can be used which is incapable of rearranging and expressing an endogenous mouse immunoglobulin light chain variable sequence, wherein the mouse expresses only one or two human light chain variable domains encoded by human immunoglobulin sequences operably linked to the mouse kappa constant gene at the endogenous mouse kappa locus. Such genetically modified mice can be used to isolate heavy chain and light chain variable regions to produce fully human bispecific antigen-binding molecules. As such, the fully human bispecific antigen-binding molecules comprise two different heavy chains that associate with the same light chain. See, e.g., US 2011/0195454). Fully human refers to an antibody, or antigen-binding fragment or immunoglobulin domain thereof, comprising an amino acid sequence encoded by a DNA derived from a human sequence over the entire length of each polypeptide of the antibody or antigen-binding fragment or immunoglobulin domain thereof. In some instances, the fully human sequence is derived from a protein endogenous to a human. In other instances, the fully human protein or protein sequence comprises a chimeric sequence wherein each component sequence is derived from human sequence. While not being bound by any one theory, chimeric proteins or chimeric sequences are generally designed to minimize the creation of immunogenic epitopes in the junctions of component sequences, e.g. compared to any wild-type human immunoglobulin regions or domains. [00187] Bispecific antigen-binding molecules may be constructed with one heavy chain having a modified Fc domain that abrogates its binding to Protein A, thus enabling a purification method that yields a heterodimeric protein. See, for example, US Patent No. 8,586,713. As such, the bispecific antigen-binding molecules comprise a first CH3 domain and a second Ig CH3 domain, wherein the first and second Ig CH3 domains differ from one another by at least one amino acid, and wherein at least one amino acid difference reduces binding of the bispecific antibody to Protein A as compared to a bi-specific antibody lacking the amino acid difference. In one embodiment, the first Ig CH3 domain binds Protein A and the second Ig CH3 domain contains a mutation/modification that reduces or abolishes Protein A binding such as an H95R modification (by IMGT exon numbering; H435R by EU numbering). The second CH3 may further comprise a Y96F modification (by IMGT; Y436F by EU).
Bioequivalents
[00188] Antigen-binding molecules having amino acid sequences that vary from those of the example molecules disclosed herein but that retain the ability to bind CDH15 are also described herein. Such variant molecules may comprise one or more additions, deletions, or substitutions of amino acids when compared to parent sequence, but exhibit biological activity that is essentially equivalent to that of the described bispecific antigen-binding molecules.
[00189] Antigen-binding molecules that are bioequivalent to any of the example antigenbinding molecules set forth herein are also described. Two antigen-binding proteins, or antibodies, are considered bioequivalent if, for example, they are pharmaceutical equivalents or pharmaceutical alternatives whose rate and extent of absorption do not show a significant difference when administered at the same molar dose under similar experimental conditions, either single dose or multiple doses. Some antigen-binding proteins will be considered equivalents or pharmaceutical alternatives if they are equivalent in the extent of their absorption but not in their rate of absorption and yet may be considered bioequivalent because such differences in the rate of absorption are intentional and are reflected in the labeling, are not essential to the attainment of effective body drug concentrations on, e.g., chronic use, and are considered medically insignificant for the particular drug product studied.
[00190] In one embodiment, two antigen-binding proteins are bioequivalent if there are no clinically meaningful differences in their safety, purity, and potency. [00191] In one embodiment, two antigen-binding proteins are bioequivalent if a patient can be switched one or more times between the reference product and the biological product without an expected increase in the risk of adverse effects, including a clinically significant change in immunogenicity, or diminished effectiveness, as compared to continued therapy without such switching.
[00192] In one embodiment, two antigen-binding proteins are bioequivalent if they both act by a common mechanism or mechanisms of action for the condition or conditions of use, to the extent that such mechanisms are known.
[00193] Bioequivalence may be demonstrated by in vivo and in vitro methods. Bioequivalence measures include, e.g., (a) an in vivo test in humans or other mammals, in which the concentration of the antibody or its metabolites is measured in blood, plasma, serum, or other biological fluid as a function of time; (b) an in vitro test that has been correlated with and is reasonably predictive of human in vivo bioavailability data; (c) an in vivo test in humans or other mammals in which the appropriate acute pharmacological effect of the antibody (or its target) is measured as a function of time; and (d) in a well-controlled clinical trial that establishes safety, efficacy, or bioavailability or bioequivalence of an antigen-binding protein.
[00194] Bioequivalent variants of the example bispecific antigen-binding molecules set forth herein may be constructed by, for example, making various substitutions of residues or sequences or deleting terminal or internal residues or sequences not needed for biological activity. For example, cysteine residues not essential for biological activity can be deleted or replaced with other amino acids to prevent formation of unnecessary or incorrect intramolecular disulfide bridges upon renaturation. In other contexts, bioequivalent antigen-binding proteins may include variants of the example bispecific antigen-binding molecules set forth herein comprising amino acid changes which modify the glycosylation characteristics of the molecules, e.g., mutations which eliminate or remove glycosylation.
[00195] Also described herein are antigen-binding proteins, e.g., antibodies or antigenbinding fragments thereof, such as anti-hCDH15 antibodies, comprising a modified glycosylation pattern. In some embodiments, modification to remove undesirable glycosylation sites may be useful, or an antibody lacking a fucose moiety present on the oligosaccharide chain, for example, to increase antibody dependent cellular cytotoxicity (ADCC) function (see Shields et al. (2002) JBC 277:26733), where cytotoxicity is desirable. In other applications, modification of galactosylation can be made in order to modify complement dependent cytotoxicity (CDC).
Species Selectivity and Species Cross-Reactivity
[00196] In some embodiments, antigen-binding molecules as described herein bind to human CDH15 but not to CDH15 from other species. Also described herein are antigen-binding molecules that bind to human CDH15 and to CDH15 from one or more non-human species.
[00197] In some embodiments, antigen-binding molecules as described herein that bind to human CDH15 may bind, or not bind, as the case may be, to one or more of mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cow, horse, camel, cynomolgus, marmoset, rhesus or chimpanzee CDH15.
Antibody-Drug Conjugates (ADCs)
[00198] Also described herein are antibody-drug conjugates (ADCs) comprising an anti- hCDH15 antibody or an antigen-binding fragment thereof conjugated to a payload, e.g., a drug or molecular cargo, (e.g., a small molecule and/or therapeutic moiety, etc.). Anti-hCDH15 antibodies, antigen-binding fragments thereof, and/or multispecific antigen-binding molecules thereof, conjugated to a therapeutic moiety are also provided. In general terms, the ADCs comprise: A - [L - P]y, in which A is an antigen-binding molecule, e.g. an anti-hCDH15 antibody, or a fragment thereof (e.g., a fragment comprising at least a HCDR3 selected from any of the HCDR3 amino acid sequences listed in Table 1), L is a linker, P is the payload or molecular cargo, and y is an integer from 1 to 30.
[00199] In various embodiments, the ADC comprises an anti-hCDH15 antibody or antigen-binding fragment thereof that comprises the CDRs of a HCVR or a LCVR having the amino acid sequences of the SEQ ID NOs set forth in Table 1 (e.g., SEQ ID NOs: 2, 22, 42, 60, 70, 80, 90, 108, 125, 145, 165, 185, 202, 220, 230, 240, 250, 259, 279, 299, 319, 339, 358, 378, 386, 404, 420, 436, 452, 468, 484, 500, 516, 532, 548, 564, 580, 596, 612, 628, 644, 660, 676, 692, 708, 724, 740, 748, 764, and 780; or 10, 30, 50, 50, 50, 50, 98, 115, 133, 153, 173, 193, 210, 50, 50, 50, 50, 267, 287, 307, 327, 347, 366, 50, 394, 412, 428, 444, 460, 476, 492, 508, 524, 540, 556, 572, 588, 604, 620, 636, 652, 668, 684, 700, 716, 732, 756, and 772), or specific HCVR/LCVR pairs (e.g., SEQ ID NOs: 2+10, 22+30, 42+50, 60+50, 70+50, 80+50, 90+98, 108+115, 125+133, 145+153, 165+173, 185+193, 202+210, 220+50, 230+50, 240+50, 250+50, 259+267, 279+287, 299+307, 319+327, 339+347, 358+366, 378+50, 386+394, 404+412, 420+428, 436+444, 452+460, 468+476, 484+492, 500+508, 516+524, 532+540, 548+556, 564+572, 580+588, 596+604, 612+620, 628+636, 644+652, 660+668, 676+684, 692+700, 708+716, 724+732, 740+684, 748+756, and 764+772). In some cases, the anti-hCDH15 antibody or fragment comprises CDRs with the amino acid sequences of the SEQ ID NOs set forth in Table 1 e.g., SEQ ID NOs: 4-6-8-12-14-16, 24-26-28-32-34-36, 44-46-48-52-34-54, 62- 64-66-52-34-54, 72-74-76-52-34-54, 82-84-86-52-34-54, 92-94-96-100-34-102, 82-111-113- 117-34-119, 127-129-131-135-137-139, 147-149-151-155-157-159, 167-169-171-175-177-179, 187-189-191-52-34-196, 204-206-208-212-137-214, 222-224-226-52-34-54, 232-234-236-52- 34-54, 242-244-246-52-34-54, 82-253-255-52-34-54, 261-263-265-269-271-273, 281-283-285- 289-291-293, 301-303-305-309-311-313, 321-323-325-329-331-333, 341-343-345-349-14-352, 360-362-364-368-370-372, 187-380-382-52-34-54, 388-390-392-396-14-398, 406-408-410-100- 34-414, 422-424-426-430-432-434, 438-440-442-446-448-450, 454-456-458-462-464-466, 470- 472-474-478-480-482, 486-488-490-494-496-498, 502-504-506-510-512-514, 518-520-522-526- 528-530, 534-536-538-542-544-546, 550-552-554-558-560-562, 566-568-570-574-576-578, 582-584-586-590-592-594, 598-600-602-606-608-610, 614-616-618-622-624-626, 630-632-634- 638-640-642, 646-648-650-654-656-658, 662-664-666-670-672-674, 678-680-682-686-688-690, 694-696-698-702-704-706, 710-712-714-718-720-722, 726-728-730-734-736-738, 742-744-746- 686-688-690, 750-752-754-758-760-762, and 766-768-770-774-776-778). In some cases, the anti-hCDH15 antibody or fragment comprises a HCVR and a LCVR having the amino acid sequences of the SEQ ID NOs set forth in Table 1 (e.g., SEQ ID NOs: : 2, 22, 42, 60, 70, 80, 90, 108, 125, 145, 165, 185, 202, 220, 230, 240, 250, 259, 279, 299, 319, 339, 358, 378, 386, 404, 420, 436, 452, 468, 484, 500, 516, 532, 548, 564, 580, 596, 612, 628, 644, 660, 676, 692, 708, 724, 740, 748, 764, and 780; and 10, 30, 50, 50, 50, 50, 98, 115, 133, 153, 173, 193, 210, 50, 50, 50, 50, 267, 287, 307, 327, 347, 366, 50, 394, 412, 428, 444, 460, 476, 492, 508, 524, 540, 556, 572, 588, 604, 620, 636, 652, 668, 684, 700, 716, 732, 756, and 772), or specific amino acid sequence pairs (e.g., SEQ ID NOs: 2+10, 22+30, 42+50, 60+50, 70+50, 80+50, 90+98, 108+115, 125+133, 145+153, 165+173, 185+193, 202+210, 220+50, 230+50, 240+50, 250+50, 259+267, 279+287, 299+307, 319+327, 339+347, 358+366, 378+50, 386+394, 404+412, 420+428, 436+444, 452+460, 468+476, 484+492, 500+508, 516+524, 532+540, 548+556, 564+572, 580+588, 596+604, 612+620, 628+636, 644+652, 660+668, 676+684, 692+700, 708+716, 724+732, 740+684, 748+756, and 764+772).
[00200] In some embodiments, the payload or molecular cargo comprises a small molecule as a therapeutic agent, e.g., a therapeutic agent that may be useful for treating muscle wasting or genetic muscle diseases and/or muscle-related cancer. A small molecule (SM) can enter cells easily because it has a low molecular weight (typically, up to about 1 kDa). Once inside the cells, the small molecule can affect other molecules, such as proteins, and may, for example, cause cancer cells to die. This is different from many large molecular weight molecules such as antibodies. An example, of a small molecule may be conjugated to an anti- CDH15 antigen-binding protein, to form an anti-CDH15:SM conjugate.
[00201] Therapeutic agents that may be useful for treating muscle wasting or genetic muscle diseases include testosterone and biologically active variants thereof, (e.g., dihydrotestosterone (DHT)), 02-adrenergic receptor agonists (e.g., clenbuterol), rapamycin or its analogs, MAPK inhibitors, or histone deacetylase inhibitors, etc.
[00202] In some embodiments, the therapeutic payload is testosterone, or a biologically active derivative and/or portion thereof, e.g., dihydrotestosterone. In some embodiments, the therapeutic payload is rapamycin or analogs thereof, a MAPK inhibitor, a histone deacetylase inhibitor, or a Notch ligand. In some embodiments the payload is a chemotherapeutic e.g., a cytotoxic drug.
[00203] Therapeutic agents that may be useful for treating muscle-related cancer include any chemotherapeutic agents known to delay, halt and/or destroy cancerous cells. Examples of therapeutic agents that may be useful for treating muscle-related cancer include, but are not limited to, Aflibercept, Amsacrine, Azacitidine, Azathioprine, Belantamab mafodotin, Bendamustine, Bleomycin, Bortezomib, Brentuximab vedotin, Busulfan, Cabazitaxel, Capecitabine, Carboplatin, Carfilzomib, Carmustine, Chlorambucil, Cisplatin, Cladribine, Clofarabine, Cyclophosphamide, Cytarabine, Cytarabine liposomal, Dacarbazine, Dactinomycin (actinomycin D), Daunorubicin, Docetaxel, Doxorubicin, Doxorubicin liposomal, Epirubicin, Eribulin, Etoposide, Etoposide phosphate, Fludarabine, Fluorouracil, Fotemustine, Ganciclovir, Gemcitabine, Gemtuzumab ozogamicin, Hydroxyurea, Idarubicin, Ifosfamide,
Inotuzumab ozogamicin , Irinotecan, Ixazomib, Lomustine, Melphalan, Mercaptopurine, Methotrexate, Mitomycin, Mitotane, Mitozantrone, Nab-paclitaxel, Oxaliplatin, Paclitaxel, Pemetrexed, Pegaspargase, Polatuzumab vedotin, Pralatrexate, Procarbazine, Raltitrexed, Romidepsin, Sacituzumab govitecan, Temozolomide, Teniposide, Thiotepa, Tioguanine, Topotecan, Trabectedin, Trastuzumab deruxtecan, Trastuzumab emtansine, Trifluridine/tipiracil, Valganciclovir, Vinblastine, Vincristine, Vindesine, Vinflunine, Vinorelbine, or Vismodegib. [00204] Also provided herein are antibody-radionuclide conjugates (ARCs) comprising anti-hCDH15 antibodies conjugated to one or more radionuclides. Examples of radionuclides that can be used in the context of this aspect of the disclosure include, but are not limited to, e.g, 225 Ac, 212Bi, 213Bi, 131I, 186Re, 227Th, 222Rn, 223Ra, 224Ra, and 90Y.
[00205] In certain embodiments provided herein, ADCs are provided comprising, e.g, an anti-hCDH15 antigen-binding protein conjugated to a therapeutic agent e.g., any of the therapeutic agents disclosed above) via a linker molecule. Linkers are any group or moiety that links, connects, or bonds the antibody or antigen-binding proteins described herein with a therapeutic moiety, e.g. cytotoxic agent. Suitable linkers may be found, for example, in Antibody-Drug Conjugates and Immunotoxins,' Phillips, G. L., Ed.; Springer Verlag: New York, 2013; Antibody-Drug Conjugates,' Ducry, L., Ed.; Humana Press, 2013; Antibody-Drug Conjugates,' Wang, J., Shen, W.-C., and Zaro, J. L., Eds.; Springer International Publishing, 2015, the contents of each incorporated herein in their entirety by reference. Generally, suitable binding agent linkers for the antibody conjugates described herein are those that are sufficiently stable to exploit the circulating half-life of the antibody and, at the same time, capable of releasing its payload after antigen-mediated internalization of the conjugate. Linkers can be cleavable or non-cleavable. Cleavable linkers include linkers that are cleaved by intracellular metabolism following internalization, e.g., cleavage via hydrolysis, reduction, or enzymatic reaction. Non-cleavable linkers include linkers that release an attached payload via lysosomal degradation of the antibody following internalization. Suitable linkers include, but are not limited to, acid-labile linkers, hydrolysis-labile linkers, enzymatically cleavable linkers, reduction labile linkers, self-immolative linkers, and non-cleavable linkers. Suitable linkers also include, but are not limited to, those that are or comprise peptides, glucuronides, succinimide-thioethers, polyethylene glycol (PEG) units, hydrazones, mal-caproyl units, dipeptide units, valine-citrulline units, and para-aminobenzyl (PAB) units.
[00206] Any linker molecule or linker technology known in the art can be used to create or construct an ADC of the present disclosure. In certain embodiments, the linker is a cleavable linker. According to other embodiments, the linker is a non-cleavable linker. Examples of linkers that can be used in the context of the present disclosure include, linkers that comprise or consist of e.g., MC (6-maleimidocaproyl), MP (maleimidopropanoyl), val-cit (valine-citrulline), val-ala (valine-alanine), val-gly (valine-glycine), dipeptide site in protease-cleavable linker, ala-phe (alanine-phenylalanine), dipeptide site in protease-cleavable linker, PAB (p- aminobenzyloxycarbonyl), SPP (N-Succinimidyl 4-(2-pyridylthio) pentanoate), SMCC (N- Succinimidyl 4-(N-maleimidomethyl)cyclohexane-l carboxylate), SLAB (N-Succinimidyl (4- iodo-acetyl)aminobenzoate), and variants and combinations thereof. Additional examples of linkers that can be used in the context of the present disclosure are provided, e.g., in US 7,754,681 and in Ducry, Bioconjugate Chem., 2010, 27:5-13, and the references cited therein, the contents of which are incorporated by reference herein in their entireties.
[00207] In certain embodiments, the linkers are stable in physiological conditions. In certain embodiments, the linkers are cleavable, for instance, able to release at least the payload portion in the presence of an enzyme or at a particular pH range or value. In some embodiments, a linker comprises an enzyme-cleavable moiety. Illustrative enzyme-cleavable moieties include, but are not limited to, peptide bonds, ester linkages, hydrazones, and disulfide linkages. In some embodiments, the linker comprises a cathepsin-cleavable linker.
[00208] In some embodiments, the linker comprises a non-cleavable moiety.
[00209] Suitable linkers also include, but are not limited to, those that are chemically bonded to two cysteine residues of a single binding agent, e.g., antibody. Such linkers can serve to mimic the antibody’s disulfide bonds that are disrupted as a result of the conjugation process. [00210] In some embodiments, the linker comprises one or more amino acids. Suitable amino acids include natural, non-natural, standard, non-standard, proteinogenic, non- proteinogenic, and L- or D- a-amino acids. In some embodiments, the linker comprises alanine, valine, glycine, leucine, isoleucine, methionine, tryptophan, phenylalanine, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, or citrulline, a derivative thereof, or combination thereof. In certain embodiments, one or more side chains of the amino acids is linked to a side chain group, described below. In some embodiments, the linker comprises valine and citrulline. In some embodiments, the linker comprises lysine, valine, and citrulline. In some embodiments, the linker comprises lysine, valine, and alanine. In some embodiments, the linker comprises valine and alanine. [00211] In some embodiments, the linker comprises a self-immolative group. The self- immolative group can be any such group known to those of skill. In particular embodiments, the self-immolative group is ?-aminobenzyl (PAB), or a derivative thereof. Useful derivatives include /?-aminobenzyloxycarbonyl (PABC). Those of skill will recognize that a self-immolative group is capable of carrying out a chemical reaction which releases the remaining atoms of a linker from a payload.
[00212] In some embodiments, the linker is: wherein is a bond to the antibody or antigen-binding protein (e.g., via lysine residue) and is a bond to the therapeutic payload (e.g., testosterone or a biologically equivalent variant thereof). In some embodiments, the linker is: wherein is a bond to the antibody or antigen-binding protein (e.g., via lysine residue) and _ ^ P 4 is a bond to a therapeutic payload (e.g. , testosterone or a biologically equivalent variant thereof). In certain embodiments, the linker is:
[00213] In certain embodiments, the linker is:
[00214] In some embodiments, the linker is derived from maleimidylmethyl-4-trans- cyclohexanecarboxy succinate:
[00215] In some embodiments, the linker is: wherein is a bond to the antibody or antigen-binding protein (e.g., via lysine residue) and is a bond to therapeutic payload (e.g. , testosterone or a biologically equivalent variant thereof).
[00216] In some embodiments, the linker is: wherein is a bond to the antibody or antigen-binding protein (e.g, via lysine residue) and
4 is a bond to therapeutic payload (e.g. , testosterone or a biologically equivalent variant thereof).
[00217] The present disclosure comprises ADCs in which a linker connects an anti- hCDH15 antigen-binding protein as described herein to therapeutic agent through an attachment at a particular amino acid within the antibody or antigen-binding molecule. Examples of amino acid attachments that can be used in the context of this aspect, e.g., lysine (see, e.g., US 5,208,020; US 2010/0129314; Hollander et al., Bioconjugate Chem., 2008, 19:358-361; WO 2005/089808; US 5,714,586; and US 2013/0101546), cysteine (see, e.g., US 2007/0258987; WO 2013/055993; WO 2013/055990; WO 2013/053873; WO 2013/053872; WO 2011/130598; US 2013/0101546; and US 7,750,116), selenocysteine (see, e.g., WO 2008/122039; and Hofer et al., Proc. Natl. Acad. Set, USA, 2008, 705:12451-12456), formyl glycine (see, e.g., Carrico etal., Nat. Chem. BioL, 2007, 3:321-322; Agarwal et al., Proc. Natl. Acad. Set, USA, 2013, 110A6-5 , and Rabuka et al., Nat. Protocols, 2012, 10: 1052-1067), non-natural amino acids (see, e.g., WO 2013/068874, and WO 2012/166559), and acidic amino acids (see, e.g., WO 2012/05982).
Linkers can also be conjugated to an antigen-binding protein via attachment to carbohydrates (see, e.g., US 2008/0305497, WO 2014/065661, and Ryan et al., Food & Agriculture Immunol., 2001, 13: 127-130) and disulfide linkers (see, e.g., WO 2013/085925, WO 2010/010324, WO 2011/018611, and Shaunak etal., Nat. Chem. Biol., 2006, 2:312-313). Site specific conjugation techniques can also be employed to direct conjugation to particular residues of the antibody or antigen binding protein (see, e.g., Schumacher et al. J Clin Immunol (2016) 36(Suppl 1): 100). Site specific conjugation techniques, include, but are not limited to glutamine conjugation via transglutaminase (see e.g., Schibli, Angew Chemie Inter Ed. 2010, 49 ,9995). In some embodiments, a residue of an antibody as described herein, e.g., a residue in a heavy chain constant region of the antibody, may be substituted with a glutamine to further facilitate glutamine conjugation via transglutaminase. As a non-limiting example, a human heavy chain constant region may be modified with the N180Q substitution found in the sequence of the human IgGl heavy chain constant region. Such substitution provides for a total of 4 glutamines for conjugation by transglutaminase.
[00218] The antibody drug conjugates described herein can be prepared using conjugation conditions known to those of ordinary skill in the art, (see, e.g. , Doronina et al. Nature Biotechnology 2003, 21, 7, 778, which is incorporated herein by reference in its entirety). In some embodiments an anti-hCDH15 antigen-binding protein drug conjugate is prepared by contacting an anti-hCDH15 antigen-binding protein as described herein with a compound comprising the desired linker and therapeutic agent, wherein said linker possesses a moiety that is reactive with the antibody or antigen-binding protein, e.g., at the desired residue of the antibody or antigen-binding protein.
Adeno-associated viruses (AAV)
[00219] “AAV” is an abbreviation for adeno-associated virus and may be used to refer to the virus itself or derivatives thereof. AAVs are small, non-enveloped, single-stranded DNA viruses. Generally, a wildtype AAV genome is 4.7 kb and is characterized by two inverted terminal repeats (ITR) and two open reading frames (ORFs), rep and cap. The wildtype rep reading frame encodes four proteins of molecular weight 78 kD (“Rep78”), 68 kD (“Rep68”), 52 kD (“Rep52”) and 40 kD (“Rep 40”). Rep78 and Rep68 are transcribed from the p5 promoter, and Rep52 and Rep40 are transcribed from the pl 9 promoter. These proteins function mainly in regulating the transcription and replication of the AAV genome. The wildtype cap reading frame encodes three structural (capsid) viral proteins (VPs) having molecular weights of 83-85 kD (VP1), 72-73 kD (VP2) and 61-62 kD (VP3). More than 80% of total proteins in an AAV virion (capsid) comprise VP3; in mature virions VP1, VP2 and VP3 are found at relative abundance of approximately 1 : 1 : 10, although ratios of 1 : 1 :8 have been reported. Padron et al. (2005) J. Virology 79:5047-58.
[00220] The genomic sequences of various serotypes of AAV, as well as the sequences of the native inverted terminal repeats (ITRs), Rep proteins, and capsid subunits are known in the art. Such sequences may be found in the literature or in public databases such as GenBank. See, e.g., GenBank Accession Numbers NC_002077 (AAV1), AF063497 (AAV1), NC001401 (AAV-2), AF043303 (AAV2), NC_001729 (AAV3), NC_001829 (AAV4), U89790 (AAV4), NC_006152 (AAV5), AF513851 (AAV7), AF513852 (AAV8), and NC_006261 (AAV8); the disclosures of which are incorporated by reference herein for teaching AAV nucleic acid and amino acid sequences. See also, e.g, Srivistava et al. (1983) J. Virology 45:555; Chiorini et al. (1998) J. Virology 71:6823; Chiorini et al. (1999) J. Virology 73: 1309; Bantel-Schaal et al. (1999) J. Virology 73:939; Xiao et al. (1999) J. Virology 73:3994; Muramatsu et al. (1996) Virology 221 :208; Shade et al. ,(1986) J. Virol. 58:921; Gao et al. (2002) Proc. Nat. Acad. Sci. USA 99: 11854; Moris et al. (2004) Virology 33:375-383; US Patent Publication 20170130245; international patent publications WO 00/28061, WO 99/61601, WO 98/11244; and U.S. Pat. No. 6,156,303, each of which is incorporated by reference in its entirety by reference. Table 2 herein provides sequences of various non-primate AAV.
[00221] “AAV” encompasses all subtypes and both naturally occurring and modified forms that are well-known in the art. AAV includes primate AAV (e.g, AAV type 1 (AAV1), primate AAV type 2 (AAV2), primate AAV type 3 (AAV3B), primate AAV type 4 (AAV4), primate AAV type 5 (AAV5), primate AAV type 6 (AAV6), primate AAV type 7 (AAV7), primate AAV type 8 (AAV8), primate AAV type 9 (AAV9), AAV10, AAV11, AAV12, AAV13, AAVDJ, Anc80L65, AAV2G9, AAV-LK03, primate AAV type rhlO (AAV rhlO), AAV type hlO (AAV hlO), AAV type hul 1 (AAV hul l), AAV type rh32.33 (AAV rh32.33), AAV retro (AAV retro), AAV PHP.B, AAV PHP.eB, AAV PHP.S, AAV2/8, etc., non-primate animal AAV (e.g, avian AAV (AAAV)) and other non-primate animal AAV such as mammalian AAV (e.g, bat AAV, sea lion AAV, bovine AAV, canine AAV, equine AAV, caprine AAV, and ovine AAV etc.), squamate AAV (e.g., snake AAV, bearded dragon AAV), etc. “Primate AAV” refers to AAV generally isolated from primates. Similarly, “non-primate animal AAV” refers to AAV isolated from non-primate animals. [00222] As used herein, “of a [specified] AAV” in relation to a gene (e.g., rep, cap, etc.), capsid protein (e.g., a VP1 capsid protein, a VP2 capsid protein, a VP3 capsid protein, etc.), region of a capsid protein of a specified AAV (e.g., PLA2 region, VPl-u region, VP1/VP2 common region, VP3 region), nucleotide sequence (e.g., ITR sequence), e.g., a cap gene or capsid protein of AAV etc., encompasses, in addition to the gene or the polypeptide respectively comprising a nucleic acid sequence or amino acid sequence set forth herein for the specified AAV, also variants of the gene or polypeptide, including variants comprising the least number of nucleotides or amino acids required to retain one or more biological functions. As used herein, a variant gene or a variant polypeptide comprises a nucleic acid sequence or amino acid sequence that differs from the nucleic acid sequence or amino acid sequence set forth herein for the gene or polypeptide of a specified AAV, wherein the difference(s) does not generally alter at least one biological function of the gene or polypeptide, and/or the phylogenetic characterization of the gene or polypeptide, e.g., where the difference(s) may be due to degeneracy of the genetic code, isolate variations, length of the sequence, etc. For example, rep gene and the cap gene as used here may encompass rep and cap genes that differ from the wildtype gene in that the genes may encode one or more Rep proteins and Cap proteins, respectively. In some embodiments, a Rep gene encodes at least Rep78 and/or Rep68. In some embodiments, cap gene includes those may differ from the wildtype in that one or more alternative start codons or sequences between one or more alternative start codons are removed such that the cap gene encodes only a single Cap protein, e.g, wherein the VP2 and/or VP3 start codons are removed or substituted such that the cap gene encodes a functional VP1 capsid protein but not a VP2 capsid protein or a VP3 capsid protein. Accordingly, as used herein, a rep gene encompasses any sequence that encodes a functional Rep protein. A cap gene encompasses any sequence that encodes at least one functional cap gene.
[00223] It is well-known that the wildtype cap gene expresses all three VP1, VP2, and VP3 capsid proteins from a single open reading frame of the cap gene under control of the p40 promoter found in the rep ORF. The term “capsid protein,” “Cap protein” and the like includes a protein that is part of the capsid of the virus. For adeno-associated viruses, the capsid proteins are generally referred to as VP1, VP2 and/or VP3, and may be encoded by the single cap gene. For AAV, the three AAV capsid proteins are produced in nature an overlapping fashion from the cap ORF alternative translational start codon usage, although all three proteins use a common stop codon. The ORF of a wildtype cap gene encodes from 5’ to 3’ three alternative start codons: “the VP1 start codon,” “the VP2 start codon,” and “the VP3 start codon”; and one “common stop codon”. The largest viral protein, VP1, is generally encoded from the VP1 start codon to the “common stop codon.” VP2 is generally encoded from the VP2 start codon to the common stop codon. VP3 is generally encoded from the VP3 start codon to the common stop codon. Accordingly, VP1 comprises at its N-terminus sequence that it does not share with the VP2 or VP3, referred to as the VPl-unique region (VPl-u). The VPl-u region is generally encoded by the sequence of a wildtype cap gene starting from the VP1 start codon to the “VP2 start codon.” VPl-u comprises a phospholipase A2 domain (PLA2), which may be important for infection, as well as nuclear localization signals which may aid the virus in targeting to the nucleus for uncoating and genome release. The VP1, VP2, and VP3 capsid proteins share the same C-terminal sequence that makes up the entirety of VP3, which may also be referred to herein as the VP3 region. The VP3 region is encoded from the VP3 start codon to the common stop codon. VP2 has an additional ~ 60 amino acids that it shares with the VP 1. This region is called the VP1/VP2 common region.
[00224] In some embodiments, one or more of the Cap proteins of the invention may be encoded by one or more cap genes having one or more ORFs. In some embodiments, the VP proteins of the invention may be expressed from more than one ORF comprising nucleotide sequence encoding any combination of VP1, VP2, and/or VP3 by use of separate nucleotide sequences operably linked to at least one expression control sequence for expression in packaging cell, each producing one or more of VP1, VP2, and/or VP3 capsid proteins of the invention. In some embodiments, a VP capsid protein of the invention may be expressed individually from an ORF comprising nucleotide sequence encoding any one of VP1, VP2, or VP3 by use of separate nucleotide sequences operably linked to one expression control sequence for expression in a viral replication cell, each producing only one of VP1, VP2, or VP3 capsid protein. In another embodiment, VP proteins may be expressed from one ORF comprising nucleotide sequences encoding VP1, VP2, and VP3 capsid proteins operably linked to at least one expression control sequence for expression in a viral replication cell, each producing VP1, VP2, and VP3 capsid protein. Accordingly, although amino acid positions provided herein may be provided in relation to the VP1 capsid protein of the referenced AAV, a skilled artisan would be able to respectively and readily determine the position of that same amino acid within the VP2 and/or VP3 capsid protein of the AAV, and the corresponding position of amino acids among different AAV.
[00225] The phrase “Inverted Terminal Repeat” or “ITR” includes symmetrical nucleic acid sequences in the genome of adeno-associated viruses required for efficient replication. ITR sequences are located at each end of the AAV DNA genome. The ITRs serve as the origins of replication for viral DNA synthesis and are essential cis components for generating AAV particles, e.g., packaging into AAV particles.
[00226] AAV ITRs comprise recognition sites for replication proteins Rep78 or Rep68. A “D” region of the ITR comprises the DNA nick site where DNA replication initiates and provides directionality to the nucleic acid replication step. An AAV replicating in a mammalian cell typically comprises two ITR sequences.
[00227] A single ITR may be engineered with Rep binding sites on both strands of the “A” regions and two symmetrical D regions on each side of the ITR palindrome. Such an engineered construct on a double-stranded circular DNA template allows Rep78 or Rep68 initiated nucleic acid replication that proceeds in both directions. A single ITR is sufficient for AAV replication of a circular particle. In methods of producing an AAV viral particle of the invention, the rep encoding sequence encodes a Rep protein or Rep protein equivalent that is capable of binding an ITR comprised on the transfer plasmid.
[00228] The Cap proteins of the invention, when expressed with appropriate Rep proteins by a packaging cell, may encapsidate a transfer plasmid comprising a nucleotide of interest and an even number of two or more ITR sequences. In some embodiments, a transfer plasmid comprises one ITR sequence. In some embodiments, a transfer plasmid comprises two ITR sequences.
[00229] Either Rep78 and/or Rep68 bind to unique and known sites on the sequence of the ITR hairpin, and act to break and unwind the hairpin structures on the end of an AAV genome, thereby providing access to replication machinery of the viral replication cell. As is well-known, Rep proteins may be expressed from more than one ORF comprising nucleotide sequence encoding any combination of Rep78, Rep68, Rep 52 and/or Rep40 by use of separate nucleotide sequences operably linked to at least one expression control sequence for expression in a viral replication cell, each producing one or more of Rep78, Rep68, Rep 52 and/or Rep40 Rep proteins. Alternatively, Rep proteins may be expressed individually from an ORF comprising a nucleotide sequence encoding any one of Rep78, Rep68, Rep 52, or Rep40 by use of separate nucleotide sequences operably linked to one expression control sequence for expression in a packaging cell, each producing only one Rep78, Rep68, Rep 52, or Rep40 Rep protein. In another embodiment, Rep proteins may be expressed from one ORF comprising nucleotide sequences encoding Rep78 and Rep52 Rep proteins operably linked to at least one expression control sequence for expression in a viral replication cell each producing Rep78 and Rep52 Rep protein.
[00230] In a method of producing an AAV virion, e.g., viral particle, of the invention, a rep encoding sequence and a cap gene of the invention may be provided a single packaging plasmid. However, a skilled artisan will recognize that such proviso is not necessary. Such viral particles may or may not include a genome.
[00231] A “chimeric AAV capsid protein” includes an AAV capsid protein that comprises amino acid sequences, e.g., portions, from two or more different AAV and that is capable of forming and/or forms an AAV viral capsid/viral particle. A chimeric AAV capsid protein is encoded by a chimeric AAV capsid gene, e.g., a chimeric nucleotide comprising a plurality, e.g., at least two, nucleic acid sequences, each of which plurality is identical to a portion of a capsid gene encoding a capsid protein of distinct AAV, and which plurality together encodes a functional chimeric AAV capsid protein. Association of a chimeric capsid protein to a specific AAV indicates that the capsid protein comprises one or more portions from a capsid protein of that AAV and one or more portions from a capsid protein of a different AAV. For example, a chimeric AAV2 capsid protein includes a capsid protein comprising one or more portions of a VP1, VP2, and/or VP3 capsid protein of AAV2 and one or more portions of a VP1, VP2, and/or VP3 capsid protein of a different AAV.
[00232] The term “portion” refers to at least 5 amino acids or at least 15 nucleotides, but less than the full-length polypeptide or nucleic acid molecule, with 100% identity to a sequence from which the portion is derived, see Penzes (2015) J. General Virol. 2769. A “portion” encompasses any contiguous segment of amino acids or nucleotides sufficient to determine that the polypeptide or nucleic acid molecule form which the portion is derived is “of a [specified] AAV” or has “significant identity” to a particular AAV, e.g., a non-primate animal AAV or remote AAV. In some embodiments, a portion comprises at least 5 amino acids or 15 nucleotides with 100% identity to a sequence associated with the specified AAV. In some embodiments, a portion comprises at least 10 amino acids or 30 nucleotides with 100% identity to a sequence associated with the specified AAV. In some embodiments, a portion comprises at least 15 amino acids or 45 nucleotides with 100% identity to a sequence associated with the specified AAV. In some embodiments, a portion comprises at least 20 amino acids or 60 nucleotides with 100% identity to a sequence associated with the specified AAV. In some embodiments, a portion comprises at least 25 amino acids or 75 nucleotides with 100% identity to a sequence associated with the specified AAV. In some embodiments, a portion comprises at least 30 amino acids or 90 nucleotides with 100% identity to a sequence associated with the specified AAV. In some embodiments, a portion comprises at least 35 amino acids or 105 nucleotides with 100% identity to a sequence associated with the specified AAV. In some embodiments, a portion comprises at least 40 amino acids or 120 nucleotides with 100% identity to a sequence associated with the specified AAV. In some embodiments, a portion comprises at least 45 amino acids or 135 nucleotides with 100% identity to a sequence associated with the specified AAV. In some embodiments, a portion comprises at least 50 amino acids or 150 nucleotides with 100% identity to a sequence associated with the specified AAV. In some embodiments, a portion comprises at least 60 amino acids or 180 nucleotides with 100% identity to a sequence associated with the specified AAV. In some embodiments, a portion comprises at least 70 amino acids or 210 nucleotides with 100% identity to a sequence associated with the specified AAV. In some embodiments, a portion comprises at least 80 amino acids or 240 nucleotides with 100% identity to a sequence associated with the specified AAV. In some embodiments, a portion comprises at least 90 amino acids or 270 nucleotides with 100% identity to a sequence associated with the specified AAV. In some embodiments, a portion comprises at least 100 amino acids or 300 nucleotides with 100% identity to a sequence associated with the specified AAV.
Modified virus capsid proteins, viral particles, nucleic acids
[00233] In some embodiments, a Cap protein, e.g., a VP1 capsid protein as described herein, a VP2 capsid protein as described herein, and/or a VP3 capsid protein as described herein, is modified to comprise any one or combination of e.g., insertion of a targeting ligand, a chemical modification, a first member of a binding pair, a detectable label, point mutation, etc. [00234] Generally, modification of gene or a polypeptide of a specified AAV, or variants thereof, results in nucleic acid sequence or an amino acid sequence that differs from the nucleic acid sequence or amino acid sequence set forth herein for the specified AAV, wherein the modification alters, confers, or removes one or more biological functions, but does not change the phylogenetic characterization of, the gene or polypeptide as an AAV gene or AAV polypeptide. Modifications may include any one or a combination of: substitution of sequences of a first AAV serotype with sequences of a second AAV serotype to create chimerism; chemical modification; an insertion of: a first member of a binding pair, and/or a point mutation; etc., such that the natural tropism of the capsid protein is reduced to abolished, the tropism of the capsid protein may be more easily redirected, and/or such that the capsid protein comprises a detectable label. Modifications as described herein generally do not alter and preferably decrease the low to no recognition of the modified capsid by pre-existing antibodies found in the general population that were produced during the course of infection with another AAV, e.g., infection with serotypes such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVDJ, Anc80L65, AAV2G9, AAV-LK03, virions based on such serotypes, virions from currently used AAV gene therapy modalities, or a combination thereof.
Targeting ligand
[00235] Modifications described herein may pertain to the association (e.g., display, operable linkage, binding) of a targeting ligand to a modified capsid protein and/or capsid comprising a modified capsid protein. Generally, a targeting ligand as described herein binds a surface protein expressed by a mammalian muscle cell, e.g., a protein that is expressed on the surface of a mammalian muscle cell, e.g, a mammalian non-terminally differentiated muscle cell specific surface protein. In some embodiments, a modified capsid protein and/or modified capsid comprises a targeting ligand that binds mammalian CDH15, e.g., a human CDH15. [00236] Table 1 provides a summary of the SEQ ID NO for each binding portion (e.g., heavy chain variable domain (HCVR), light chain variable domain (LCVR), and CDR1, CDR2, and CDR3) of non-limiting and exemplary anti-human-CDH15 monoclonal antibodies (mAb ID) that may be used to redirect an AAV capsid as described herein. In some embodiments, an AAV capsid as described herein comprises a targeting ligand that binds human CDH15, wherein the targeting ligand comprises heavy chain variable domain, light chain variable domain, heavy chain variable domain/light chain variable domain pair, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and/or set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence(s) at least 90% identical to, respectively, an amino acid sequence of a heavy chain variable domain, light chain variable domain, heavy chain variable domain/light chain variable domain pair, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and/or set of HCDR1- HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 as set forth in any one of SEQ ID NOs: 1-786. In some embodiments, an AAV capsid as described herein comprises a targeting ligand that binds human CDH15, wherein the targeting ligand comprises a heavy chain variable domain, light chain variable domain, heavy chain variable domain/light chain variable domain pair, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and/or set of HCDR1-HCDR2-HCDR3-LCDR1- LCDR2-LCDR3 amino acid sequence at least 95% identical to, respectively, amino acid sequence(s) of a heavy chain variable domain, light chain variable domain, heavy chain variable domain/light chain variable domain pair, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and/or set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 set forth in any one of SEQ ID NOs: 1-786. In some embodiments, an AAV capsid as described herein comprises a targeting ligand that binds human CDH15, wherein the targeting ligand comprises a heavy chain variable domain, light chain variable domain, heavy chain variable domain/light chain variable domain pair, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and/or set of HCDR1-HCDR2- HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence at least 97% identical to amino acid sequence(s) of a heavy chain variable domain, light chain variable domain, heavy chain variable domain/light chain variable domain pair, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and/or set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 set forth in any one of SEQ ID NOs: 1-786. In some embodiments, an AAV capsid as described herein comprises a targeting ligand that binds human CDH15, wherein the targeting ligand comprises a heavy chain variable domain, light chain variable domain, heavy chain variable domain/light chain variable domain pair, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and/or set of HCDR1-HCDR2- HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence(s) at least 98% identical to amino acid sequence(s) of a heavy chain variable domain, light chain variable domain, heavy chain variable domain/light chain variable domain pair, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and/or set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 set forth in any one of SEQ ID NOs: 1-786. In some embodiments, an AAV capsid as described herein comprises a targeting ligand that binds human CDH15, wherein the targeting ligand comprises a heavy chain variable domain, light chain variable domain, heavy chain variable domain/light chain variable domain pair, HCDR1, HCDR2, CDR3, LCDR1, LCDR2, LCDR3, and/or set of HCDR1-HCDR2- HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences 99% identical to amino acid sequences of a heavy chain variable domain, light chain variable domain, heavy chain variable domain/light chain variable domain pair, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and/or set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 set forth in any one of SEQ ID NOs: 1-786. Also described herein are antibodies, or antigen-binding fragments thereof, comprising a set of six CDRs (z.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within an HCVR/LCVR amino acid sequence pair as defined by any of the exemplary anti-hCDH15 antibodies listed in Table 1. In some embodiments, a targeting ligand as described herein comprises the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences set contained within an HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2+10, 22+30, 42+50, 60+50, 70+50, 80+50, 90+98, 108+115, 125+133, 145+153, 165+173, 185+193, 202+210, 220+50, 230+50, 240+50, 250+50, 259+267, 279+287, 299+307, 319+327, 339+347, 358+366, 378+50, 386+394, 404+412, 420+428, 436+444, 452+460, 468+476, 484+492, 500+508, 516+524, 532+540, 548+556, 564+572, 580+588, 596+604, 612+620, 628+636, 644+652, 660+668, 676+684, 692+700, 708+716, 724+732, 740+684, 748+756, and 764+772.
[00237] Non-limiting examples of targeting ligands that bind CDH15 include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FR3- CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression “targeting ligand,” as used herein. In non-limiting embodiments, an anti-CDH15 targeting ligand that binds CDH15 useful for retargeting viral capsids as described herein comprises an scFv. As a non-limiting example, an scFv sequences in VL-(Gly4Ser)3-VH format useful for retargeting viral capsids as described herein may comprise a heavy chain variable domain, light chain variable domain, heavy chain variable domain/light chain variable domain pair, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and/or set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 that is 90%, 95%, 97%, 98%, 99% or 100% identical, respectively, to any one of the amino acid sequences of a heavy chain variable domain, light chain variable domain, heavy chain variable domain/light chain variable domain pair, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and/or set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 as set forth in any one of SEQ ID NOs: 1- 786.
[00238] A targeting ligand that binds a mammalian non-terminally differentiated muscle cell surface protein may be associated with (e.g., displayed by, operably linked to, bound to) a modified AAV capsid protein and resulting AAV capsids according to well-known methods, e.g., a direct approach in which the targeting ligand is directly inserted into (e.g., using recombinatorial methods) according to well-known methods. See, e.g., Stachler et al. (2006), supra, White et al. (2004), supra, Girod et al. (1999), supra, Grifman et al. (2001), supra, Shi et al. (2001), supra, Shi and Bartlett (2003), supra. A targeting ligand that binds a mammalian non-terminally differentiated muscle cell specific surface protein may be coupled to a modified AAV capsid protein and resulting AAV capsids using well-known chemical linkers, e.g., wherein the AAV capsid protein may be chemically modified to comprise a dibenzocycootyne group or an azide group, and optionally wherein a targeting ligand as described herein is attached to the dibenzocycootyne group or the azide group, see, e.g, U.S. 2022/028234, incorporated herein by reference in its entirety; wherein targeting ligand is covalently linked to a primary amino acid group of an AAV capsid protein, e.g., via a -CSNH- bond, etc. In some embodiments, a modified capsid as described herein comprises a targeting ligand, e.g., an anti- CDH15 antibody or binding portion thereof, directly inserted into or coupled to it according to well-known direct recombinatorial methods.
Binding pairs
[00239] In some embodiments, a targeting ligand that binds a mammalian non-terminally differentiated muscle cell surface protein may be associated with (e.g., displayed by, operably linked to, bound to) a modified AAV capsid protein and resulting AAV capsids according to indirect recombinatorial approaches, wherein the AAV capsid protein is modified to comprise a first member of a binding pair (e.g., a heterologous scaffold), and optionally wherein the first member of the binding pair is linked to (e.g., covalently or non-covalently bound to) a second cognate member of the binding pair (e.g., an adaptor), further optionally wherein the second cognate member of the binding pair is fused to the targeting ligand. Non-limiting and exemplary binding pairs are listed in Buning and Srivastava (2019) Mol. Ther. Methods Clin Dev 12:248- 265.
[00240] Accordingly, in some embodiments, modifications of a capsid protein as described herein include those that generally result from modifications at the genetic level, e.g., via modification of a cap gene, such as modifications that insert first member of a binding pair (e.g., a protein: protein binding pair, a proteimnucleic acid binding pair), a detectable label, etc., for display by the Cap protein.
[00241] In some embodiments, the first member forms a binding pair with an immunoglobulin constant domain. In some embodiments, the first member forms a binding pair with a metal ion, e.g., Ni2+, Co2+, Cu2+, Zn2+, Fe3+, etc. In some embodiments, the first member is selected from the group consisting of Streptavidin, Strep II, HA, L14, 4C-RGD, LH, and Protein A.
[00242] In some embodiments, the binding pair comprises an enzyme ucleic acid binding pair. In some embodiments, the first member comprises a HUH-endonuclease or HUH-tag and the second member comprises a nucleic acid binding domain. In some embodiments, the first member comprises a HUH tag. See, e.g., U.S. 2021/0180082, incorporated herein in its entirety by reference.
[00243] In some embodiments, a capsid protein of the invention comprises at least a first member of a peptide:peptide binding pair.
[00244] In some embodiments, each of a first member and a second member of a peptide:peptide binding pair comprises an intein. See, e.g., Wagner et al., (2021) Adv. Sci. 8: 2004018 (1 of 22); Muik et al. (2017) Biomaterials 144: 84, each of which is incorporated herein in its entirety by reference.
[00245] In some embodiments, a first member is a B cell epitope, e.g., is between about 1 amino acid and about 35 amino acids in length, and forms a binding pair with an antibody paratope, e.g., an immunoglobulin variable domain. In some embodiments, a capsid protein of the invention may be modified to comprise a detectable label as a first member of a binding pair. Many detectable labels are known in the art. (See, e.g. Nilsson et al. (1997) “Affinity fusion strategies for detection, purification, and immobilization of modified proteins” Protein Expression and Purification 11 : 1-16, Terpe et al. (2003) “Overview of tag protein fusions: From molecular and biochemical fundamentals to commercial systems” Applied Microbiology and Biotechnology 60:523-533, and references therein). Detectable labels include, but are not limited to, a polyhistidine detectable labels (e.g., a His-6, His-8, or His-10) that binds immobilized divalent cations (e.g, Ni2+), a biotin moiety (e.g., on an in vivo biotinylated polypeptide sequence) that binds immobilized avidin, a GST (glutathione S -transferase) sequence that binds immobilized glutathione, an S tag that binds immobilized S protein, an antigen that binds an immobilized antibody or domain or fragment thereof (including, e.g., T7, myc, FLAG, and B tags that bind corresponding antibodies), a FLASH Tag (a high detectable label that couples to specific arsenic based moieties), a receptor or receptor domain that binds an immobilized ligand (or vice versa), protein A or a derivative thereof (e.g., Z) that binds immobilized IgG, maltose- binding protein (MBP) that binds immobilized amylose, an albumin-binding protein that binds immobilized albumin, a chitin binding domain that binds immobilized chitin, a calmodulin binding peptide that binds immobilized calmodulin, and a cellulose binding domain that binds immobilized cellulose. Another exemplary detectable label is a SNAP -tag, commercially available from Covalys (www.covalys.com). In some embodiments, a detectable label disclosed herein comprises a detectable label recognized by an antibody paratope, wherein the detectable label and the antibody paratope form a proteimprotein binding pair.
[00246] In some embodiments, a capsid protein of the invention comprises a first member of a protein: protein binding pair comprising a detectable label, which may also be used for the detection and/or isolation of the Cap protein and/or as a first member of a protein: protein binding pair. In some embodiments, a detectable label acts as a first member of a proteimprotein binding pair for the binding of a targeting ligand comprising a multispecific binding protein that may bind both the detectable label and a target expressed by a cell of interest. In some embodiments, a Cap protein of the invention comprises a first member of a proteimprotein binding pair comprising c-myc (SEQ ID NO: 818). Use of a detectable label as a first member of a protein: protein binding pair is described in, e.g., W02019006043, incorporated herein in its entirety by reference.
[00247] In some embodiments, the first member comprises a Bl epitope (SEQ ID NO: 819). In some embodiments, a capsid protein is modified to comprise a Bl epitope in the VP3 region. In some embodiments, the first member is selected from the group consisting of FLAG, HA and c-myc (SEQ ID NO: 818).
[00248] In some embodiments, a capsid protein comprises a first member of a protein: protein binding pair, wherein the protein: protein binding pair forms a covalent isopeptide bond. In some embodiments, the first member of a peptide:peptide binding pair is covalently bound via an isopeptide bond to a cognate second member of the peptide:peptide binding pair, and optionally wherein the cognate second member of the peptide:peptide binding pair is fused with a targeting ligand, which targeting ligand binds a target expressed by a cell of interest. In some embodiments, the protein: protein binding pair may be selected from the group consisting of SpyTag: SpyCatcher, SpyTag002:SpyCatcher002, SpyTag003:SpyCatcher003, SpyTag:KTag, Isopeptag:pilin-C, and SnoopTag: SnoopCatcher. In some embodiments, wherein the first member is SpyTag (or a biologically active portion or variant thereof) and the protein (second cognate member) is SpyCatcher (or a biologically active portion or variant thereof). In some embodiments, wherein the first member is SpyTag (or a biologically active portion or variant thereof) and the protein (second cognate member) is KTag (or a biologically active portion or variant thereof). In some embodiments, wherein the first member is KTag (or a biologically active portion or variant thereof) and the protein (second cognate member) is SpyTag (or a biologically active portion or variant thereof). In some embodiments, wherein the first member is SnoopTag (or a biologically active portion or variant thereof) and the protein (second cognate member) is SnoopCatcher (or a biologically active portion or variant thereof). In some embodiments, wherein the first member is Isopeptag (or a biologically active portion or variant thereof) and the protein (second cognate member) is Pilin-C (or a biologically active portion or variant thereof). In some embodiments, wherein the first member is SpyTag002 (or a biologically active portion or variant thereof) and the protein (second cognate member) is SpyCatcher002 (or a biologically active portion or variant thereof). In some embodiments, wherein the first member is SpyTag003 (or a biologically active portion or variant thereof) and the protein (second cognate member) is SpyCatcher003 (or a biologically active portion or variant thereof). In some embodiments, a Cap protein of the invention comprises a SpyTag, or a biologically active portion or variant thereof. Use of a first member of a protein: protein binding pair is described in WO2019006046, incorporated herein in its entirety.
[00249] In some embodiments, a first member of a proteimprotein binding pair and/or detectable label is operably linked to (translated in frame with, chemically attached to, and/or displayed by) a Cap protein of the invention via a first and/or second linker, e.g, an amino acid spacer that is at least one amino acid in length. In some embodiments, the first member of a protein: protein binding pair is flanked by a first and/or second linker, e.g, a first and/or second amino acid spacer, each of which spacer is at least one amino acid in length.
[00250] In some embodiments, the first and/or second linkers are not identical. In some embodiments, the first and/or second linker is each independently one or two amino acids in length. In some embodiments, the first and/or second linker is each independently one, two or three amino acids in length. In some embodiments, the first and/or second linker is each independently one, two, three, or four amino acids in length. In some embodiments, the first and/or second linker is each independently one, two, three, four, or five amino acids in length. In some embodiments, the first and/or second linker are each independently one, two, three, four, or five amino acids in length. In some embodiments, the first and/or second linker is each independently one, two, three, four, five, or six amino acids in length. In some embodiments, the first and/or second linker is each independently one, two, three, four, five, six, or seven amino acids in length. In some embodiments, the first and/or second linker is each independently one, two, three, four, five, six, seven, or eight amino acids in length. In some embodiments, the first and/or second linker is each independently one, two, three, four, five, six, seven, eight or nine amino acids in length. In some embodiments, the first and or second linker is each independently one, two, three, four, five, six, seven, eight, nine, or ten amino acids in length. In some embodiments, the first and or second linker is each independently one, two, three, four, five, six, seven, eight, nine, ten, or more amino acids in length.
[00251] In some embodiments, the first and second linkers are identical in sequence and/or in length and are each one amino acid in length. In some embodiments, the first and second linkers are identical in length, and are each one amino acid in length. In some embodiments, the first and second linkers are identical in length, and are each two amino acids in length. In some embodiments, the first and second linkers are identical in length, and are each three amino acids in length. In some embodiments, the first and second linkers are identical in length, and are each four amino acids in length, e.g, the linker is GLSG (SEQ ID NO: 823). In some embodiments, the first and second linkers are identical in length, and are each five amino acids in length. In some embodiments, the first and second linkers are identical in length, and are each six amino acids in length, e.g., the first and second linkers each comprise a sequence of GLSGSG (SEQ ID NO: 824) or GSGESG (SEQ ID NO: 828). In some embodiments, the first and second linkers are identical in length, and are each seven amino acids in length. In some embodiments, the first and second linkers are identical in length, and are each eight amino acids in length, e.g., the first and second linkers each comprise a sequence of GLSGLSGS (SEQ ID NO: 825). In some embodiments, the first and second linkers are identical in length, and are each nine amino acids in length. In some embodiments, the first and second linkers are identical in length, and are each ten amino acids in length, e.g., the first and second linkers each comprise a sequence of GLSGLSGLSG (SEQ ID NO: 826) or GLSGGSGLSG (SEQ ID NO: 827). In some embodiments, the first and second linkers are identical in length, and are each more than ten amino acids in length.
[00252] Generally, a first member of a protein: protein binding pair amino acid sequence as described herein, e.g., comprising a first member of a specific binding pair by itself or in combination with one or more linkers, is between about 5 amino acids to about 50 amino acids in length. In some embodiments, the first member of a protein: protein binding pair amino acid sequence is at least 5 amino acids in length. In some embodiments, the first member of a protein: protein binding pair amino acid sequence is 6 amino acids in length. In some embodiments, the first member of a protein: protein binding pair amino acid sequence is 7 amino acids in length. In some embodiments, the first member of a proteimprotein binding pair amino acid sequence is 8 amino acids in length. In some embodiments, the first member of a protein: protein binding pair amino acid sequence is 9 amino acids in length. In some embodiments, the first member of a protein: protein binding pair amino acid sequence is 10 amino acids in length. In some embodiments, the first member of a protein :protein binding pair amino acid sequence is 11 amino acids in length. In some embodiments, the first member of a protein: protein binding pair amino acid sequence is 12 amino acids in length. In some embodiments, the first member of a protein: protein binding pair amino acid sequence is 13 amino acids in length. In some embodiments, the first member of a protein :protein binding pair amino acid sequence is 14 amino acids in length. In some embodiments, the first member of a protein: protein binding pair amino acid sequence is 15 amino acids in length. In some embodiments, the first member of a protein: protein binding pair amino acid sequence is 16 amino acids in length. In some embodiments, the first member of a protein :protein binding pair amino acid sequence is 17 amino acids in length. In some embodiments, the first member of a protein: protein binding pair amino acid sequence is 18 amino acids in length. In some embodiments, the first member of a protein: protein binding pair amino acid sequence is 19 amino acids in length. In some embodiments, the first member of a protein :protein binding pair amino acid sequence is 20 amino acids in length. In some embodiments, the first member of a protein: protein binding pair amino acid sequence is 21 amino acids in length. In some embodiments, the first member of a protein: protein binding pair amino acid sequence is 22 amino acids in length. In some embodiments, the first member of a protein :protein binding pair amino acid sequence is 23 amino acids in length. In some embodiments, the first member of a protein: protein binding pair amino acid sequence is 24 amino acids in length. In some embodiments, the first member of a protein: protein binding pair amino acid sequence is 25 amino acids in length. In some embodiments, the first member of a protein :protein binding pair amino acid sequence is 26 amino acids in length. In some embodiments, the first member of a protein: protein binding pair amino acid sequence is 27 amino acids in length. In some embodiments, the first member of a protein: protein binding pair amino acid sequence is 28 amino acids in length. In some embodiments, the first member of a protein :protein binding pair amino acid sequence is 29 amino acids in length. In some embodiments, the first member of a protein: protein binding pair amino acid sequence is 30 amino acids in length. In some embodiments, the first member of a protein: protein binding pair amino acid sequence is 31 amino acids in length. In some embodiments, the first member of a protein :protein binding pair amino acid sequence is 32 amino acids in length. In some embodiments, the first member of a protein: protein binding pair amino acid sequence is 33 amino acids in length. In some embodiments, the first member of a protein: protein binding pair amino acid sequence is 34 amino acids in length. In some embodiments, the first member of a protein :protein binding pair amino acid sequence is 35 amino acids in length. In some embodiments, the first member of a protein: protein binding pair amino acid sequence is 36 amino acids in length. In some embodiments, the first member of a protein: protein binding pair amino acid sequence is 37 amino acids in length. In some embodiments, the first member of a proteimprotein binding pair amino acid sequence is 38 amino acids in length. In some embodiments, the first member of a protein: protein binding pair amino acid sequence is 39 amino acids in length. In some embodiments, the first member of a protein: protein binding pair amino acid sequence is 40 amino acids in length. In some embodiments, the first member of a proteimprotein binding pair amino acid sequence is 41 amino acids in length. In some embodiments, the first member of a protein: protein binding pair amino acid sequence is 42 amino acids in length. In some embodiments, the first member of a protein: protein binding pair amino acid sequence is 43 amino acids in length. In some embodiments, the first member of a protein :protein binding pair amino acid sequence is 44 amino acids in length. In some embodiments, the first member of a protein: protein binding pair amino acid sequence is 45 amino acids in length. In some embodiments, the first member of a protein: protein binding pair amino acid sequence is 46 amino acids in length. In some embodiments, the first member of a proteimprotein binding pair amino acid sequence is 47 amino acids in length. In some embodiments, the first member of a protein: protein binding pair amino acid sequence is 48 amino acids in length. In some embodiments, the first member of a protein: protein binding pair amino acid sequence is 49 amino acids in length. In some embodiments, the first member of a proteimprotein binding pair amino acid sequence is 50 amino acids in length.
[00253] In some embodiments of the invention comprising a detectable label, a targeting ligand comprises a multispecific binding molecule comprising (i) an antibody paratope that specifically binds the detectable label and (ii) a second binding domain that specifically binds a receptor, which may be conjugated to the surface of a bead (e.g, for purification) or expressed by a target cell (e.g., a muscle stem cell, myoblast, myocyte, any combination thereof, etc.). Accordingly, a multispecific binding molecule comprising (i) an antibody paratope that specifically binds the detectable label and (ii) a second binding domain that specifically binds a receptor targets the viral particle. Such “targeting” or “directing” may include a scenario in which the wildtype viral particle targets several cells within a tissue and/or several organs within an organism, which broad targeting of the tissue or organs is reduced to abolished by insertion of the detectable label, and which retargeting to more specific cells in the tissue or more specific organ in the organism is achieved with the multispecific binding molecule. Such retargeting or redirecting may also include a scenario in which the wildtype viral particle targets a tissue, which targeting of the tissue is reduced to abolished by insertion of the detectable label, and which retargeting to a completely different tissue is achieved with the multispecific binding molecule. An antibody paratope as described herein generally comprises at a minimum a complementarity determining region (CDR) that specifically recognizes the detectable label, e.g, a CDR3 region of a heavy and/or light chain variable domain. In some embodiments, a multispecific binding molecule comprises an antibody (or portion thereof) that comprises the antibody paratope that specifically binds the detectable label. For example, a multispecific binding molecule may comprise a single domain heavy chain variable region or a single domain light chain variable region, wherein the single domain heavy chain variable region or single domain light chain variable region comprises an antibody paratope that specifically binds the detectable label. In some embodiments, a multispecific binding molecule may comprise an Fv region, e.g., a multispecific binding molecule may comprise an scFv, that comprises an antibody paratope that specifically binds the detectable label. In some embodiments, a multispecific binding molecule as described herein comprises an antibody paratope that specifically binds c-myc (SEQ ID NO:818).
Modified Capsids Comprising Modified Capsid Proteins
[00254] In some embodiments a viral capsid comprising a modified viral capsid protein as described herein is a mosaic capsid, e.g., comprises at least two sets of VP1, VP2, and/or VP3 proteins, each set of which is encoded by a different cap gene. A mosaic capsid herein generally refers to a mosaic of a first viral capsid protein modified to comprise a first member of a binding pair and a second corresponding viral capsid protein lacking the first member of a binding pair. In relation to a mosaic capsid, the second viral capsid protein lacking the first member of a binding pair may be referred to as a reference capsid protein encoded by a reference cap gene. In some mosaic capsid embodiments, preferably when the VP 1, VP2, and/or VP3 capsid proteins modified with a first member of protein: protein pair is not a chimeric capsid protein, a VP1, VP2, and/or VP3 reference capsid protein may comprise an amino acid sequence identical to that of the viral VP1, VP2, and/or VP3 capsid protein modified with a first member of a binding pair, except that the reference capsid protein lacks the first member of a binding pair. In some mosaic capsid embodiments, a VP1, VP2, and/or VP3 reference capsid protein corresponds to the viral VP1, VP2, and/or VP3 capsid protein modified with a first member of a binding pair, except that the reference capsid protein lacks the first member of a binding pair. In some embodiments, a VP1 reference capsid protein corresponds to the viral VP1 capsid protein modified with a first member of a binding pair, except that the reference capsid protein lacks the first member of a binding pair. In some embodiments, a VP2 reference capsid protein corresponds to the viral VP2 capsid protein modified with a first member of a binding pair, except that the reference capsid protein lacks the first member of a binding pair. In some embodiments, a VP3 reference capsid protein corresponds to the viral VP3 capsid protein modified with a first member of a binding pair, except that the reference capsid protein lacks the first member of a binding pair. In some mosaic capsid embodiments comprising a chimeric VP1, VP2, and/or VP3 capsid protein further modified to comprise a first member of a binding pair, a reference protein may be a corresponding capsid protein from which portions thereof form part of the chimeric capsid protein. As a non-limiting example in some embodiments, mosaic capsid comprising a chimeric AAV2/AAAV VP1 capsid protein modified to comprise a first member of a binding pair may further comprise as a reference capsid protein: an AAV2 VP1 capsid protein lacking the first member, an AAAV VP1 capsid protein lacking the first member, a chimeric AAV2/AAAV VP1 capsid protein lacking the first member. Similarly, in some embodiments, a mosaic capsid comprising a chimeric AAV2/AAAV VP2 capsid protein modified to comprise a first member of a binding pair may further comprise as a reference capsid protein: an AAV2 VP2 capsid protein lacking the first member, an AAAV VP1 capsid protein lacking the first member, a chimeric AAV2/AAAV VP2 capsid protein lacking the first member. In some embodiments, a mosaic capsid comprising a chimeric AAV2/AAAV VP3 capsid protein modified to comprise a first member of a binding pair may further comprise as a reference capsid protein: an AAV2 VP2 capsid protein lacking the first member, an AAAV VP1 capsid protein lacking the first member, a chimeric AAV2/AAAV VP3 capsid protein lacking the first member. In some mosaic capsid embodiments, a reference capsid protein may be any capsid protein so long as it that lacks the first member of the binding pair and is able to form a capsid with the first capsid protein modified with the first member of a binding pair.
[00255] Generally, mosaic particles may be generated by transfecting mixtures of the modified and reference Cap genes into production cells at the indicated ratios. The protein subunit ratios, e. , modified VP proteimunmodified VP protein ratios, in the particle may, but do not necessarily, stoichiometrically reflect the ratios of the at least two species of the cap gene encoding the first capsid protein modified with a first member of a binding pair and the one or more reference cap genes, e.g., modified cap gene:reference cap gene(s) transfected into packaging cells. In some embodiments, the protein subunit ratios in the particle do not stoichiometrically reflect the modified cap gene reference cap gene(s) ratio transfected into packaging cells.
[00256] In some mosaic viral particle embodiments, the protein subunit ratio ranges from about 1 :59 to about 59:1. In some mosaic viral particle embodiments, the protein subunit is at least about 1: 1 (e.g., the mosaic viral particle comprises about 30 modified capsid proteins and about 30 reference capsid protein). In some mosaic viral particle embodiments, the protein subunit ratio is at least about 1:2 e.g., the mosaic viral particle comprises about 20 modified capsid proteins and about 40 reference capsid proteins). In some mosaic viral particle embodiments, the protein subunit ratio is at least about 3:5. In some mosaic viral particle embodiments, the protein subunit ratio is at least about 1 :3 (e.g., the mosaic viral particle comprises about 15 modified capsid proteins and about 45 reference capsid proteins) . In some mosaic viral particle embodiments, the protein subunit ratio is at least about 1:4 (e.g., the mosaic viral particle comprises about 12 modified capsid proteins and 48 reference capsid proteins). In some mosaic viral particle embodiments, the protein subunit ratio is at least about 1:5 (e.g., the mosaic viral particle comprises 10 modified capsid proteins and 50 reference capsid proteins). In some mosaic viral particle embodiments, the protein subunit ratio is at least about 1:6. In some mosaic viral particle embodiments, the protein subunit ratio is at least about 1:7. In some mosaic viral particle embodiments, the protein subunit ratio is at least about 1 :8. In some mosaic viral particle embodiments, the protein subunit ratio is at least about 1 :9 (e.g., the mosaic viral particle comprises about 6 modified capsid proteins and about 54 reference capsid proteins). In some mosaic viral particle embodiments, the protein subunit ratio is at least about 1: 10. In some mosaic viral particle embodiments, the protein subunit ratio is at least about 1: 11 (e.g., the mosaic viral particle comprises about 5 modified capsid proteins and about 55 reference capsid proteins). In some mosaic viral particle embodiments, the protein subunit ratio is at least about 1 : 12. In some mosaic viral particle embodiments, the protein subunit ratio is at least about 1 : 13. In some mosaic viral particle embodiments, the protein subunit ratio is at least about 1 :14 (e.g., the mosaic viral particle comprises about 4 modified capsid proteins and about 56 reference capsid proteins). In some mosaic viral particle embodiments, the protein subunit ratio is at least about 1 : 15. In some mosaic viral particle embodiments, the protein subunit ratio is at least about 1 : 19 (e.g., the mosaic viral particle comprises about 3 modified capsid proteins and about 57 reference capsid proteins). In some mosaic viral particle embodiments, the protein subunit ratio is at least about 1 :29 (e.g, the mosaic viral particle comprises about 2 modified capsid proteins and about 58 reference capsid proteins). In some mosaic viral particle embodiments, the protein subunit ratio is at least about 1 :59. In some mosaic viral particle embodiments, the protein subunit ratio is at least about 2: 1 (e.g, the mosaic viral particle comprises about 40 modified capsid proteins and about 20 reference capsid proteins). In some mosaic viral particle embodiments, the protein subunit ratio is at least about 5:3. In some mosaic viral particle embodiments, the protein subunit ratio is at least about 3 : 1 (e.g, the mosaic viral particle comprises about 45 modified capsid proteins and about 15 reference capsid proteins). In some mosaic viral particle embodiments, the protein subunit ratio is at least about 4: 1 (e.g., the mosaic viral particle comprises about 48 modified capsid proteins and 12 reference capsid proteins). In some mosaic viral particle embodiments, the protein subunit ratio is at least about 5: 1 (e.g, the mosaic viral particle comprises 50 modified capsid proteins and 10 reference capsid proteins). In some mosaic viral particle embodiments, the protein subunit ratio is at least about 6: 1. In some mosaic viral particle embodiments, the protein subunit ratio is at least about 7: 1. In some mosaic viral particle embodiments, the protein subunit ratio is at least about 8:1. In some mosaic viral particle embodiments, the protein subunit ratio is at least about 9: 1 (e.g., the mosaic viral particle comprises about 54 modified capsid proteins and about 6 reference capsid proteins). In some mosaic viral particle embodiments, the protein subunit ratio is at least about 10:1. In some mosaic viral particle embodiments, the protein subunit ratio is at least about 11 :1 (e.g., the mosaic viral particle comprises about 55 modified capsid proteins and about 5 reference capsid proteins). In some mosaic viral particle embodiments, the protein subunit ratio is at least about 12:1. In some mosaic viral particle embodiments, the protein subunit ratio is at least about 13:1. In some mosaic viral particle embodiments, the protein subunit ratio is at least about 14: 1 (e.g., the mosaic viral particle comprises about 56 modified capsid proteins and about 4 reference capsid proteins). In some mosaic viral particle embodiments, the protein subunit ratio is at least about 15: 1. In some mosaic viral particle embodiments, the protein subunit ratio is at least about 19: 1 (e.g, the mosaic viral particle comprises about 57 modified capsid proteins and about 3 reference capsid proteins). In some mosaic viral particle embodiments, the protein subunit ratio is at least about 29: 1 (e.g, the mosaic viral particle comprises about 58 modified capsid proteins and about 2 reference capsid proteins). In some mosaic viral particle embodiments, the protein subunit ratio is at least about 59: 1.
[00257] In some non-mosaic viral particle embodiments, the protein subunit ratio may be 1:0 wherein each capsid protein of the non-mosaic viral particle is modified with a first member of a binding pair. In some non-mosaic viral particle embodiments, the protein subunit ratio may be 0:1 wherein each capsid protein of the non-mosaic viral particle is not modified with a first member of a binding pair.
[00258] Also provided herein are nucleic acids that encode a VP3 capsid protein of the invention. AAV capsid proteins may be, but are not necessarily, encoded by overlapping reading frames of the same gene with staggered start codons. In some embodiments, a nucleic acid that encodes a VP3 capsid protein of the invention does not also encode a VP2 capsid protein or VP1 capsid protein of the invention. In some embodiments, a nucleic acid that encodes a VP3 capsid protein of the invention may also encode a VP2 capsid protein of the invention but does not also encode a VP1 capsid of the invention. In some embodiments, a nucleic acid that encodes a VP3 capsid protein of the invention may also encode a VP2 capsid protein of the invention and a VP1 capsid of the invention.
[00259] One embodiment of the present invention is a multimeric structure comprising a modified viral capsid protein of the present invention. A multimeric structure comprises at least 5, preferably at least 10, more preferably at least 30, most preferably at least 60 modified viral capsid proteins comprising a first member of a specific binding pair as described herein. They can form regular viral capsids (empty viral particles) or viral particles (capsids encapsidating a nucleotide of interest). The formation of viral particles comprising a viral genome is a highly preferred feature for use of the modified viral capsids described herein.
[00260] A further embodiment of the present invention is the use of at least one modified viral capsid protein and/or a nucleic acid encoding same, preferably at least one multimeric structure (e.g., viral particle) for the manufacture of and use in transfer of a nucleotide of interest to a target cell e.g., a muscle stem cell, myoblast, myocyte, any combination thereof, etc.). Insertion sites
[00261] Due to the high conservation of at least large stretches and the large member of closely related family members, the corresponding insertion sites for AAV other than the enumerated AAV can be identified by performing an amino acid alignment or by comparison of the capsid structures. See, e.g., Rutledge et al. (1998) J. Virol. 72:309-19; Mietzsch et al. (2019) Viruses 11, 362, 1-34, and U.S. Patent No. 9,624,274 for exemplary alignments of different AAV capsid proteins, each of which is incorporated herein by reference in its entirety. For example, Mietzcsh et al. (2019) provide an overlay of ribbons from different dependoparvovirus at Figure 7, depicting the variable regions VR I to VR IX. Using such structural analysis as described therein, and sequence analysis, a skilled artisan may determine which amino acids within the variable region correspond to amino acid sequence of AAV that can accommodate the insertion of, e.g., a targeting ligand as described herein, a first member of a binding pair and/or detectable label.
[00262] Generally, the targeting ligand, first member of a binding pair, and/or detectable label may be inserted into a variable region or variable loop of an AAV capsid protein, a GH loop of an AAV capsid protein, etc. In some embodiments, the targeting ligand, first member of a binding pair, and/or detectable label may be inserted into a variable region or variable loop VRI of an AAV capsid protein. In some embodiments, the targeting ligand, first member of a binding pair, and/or detectable label may be inserted into a variable region or variable loop VRII of an AAV capsid protein. In some embodiments, the targeting ligand, first member of a binding pair, and/or detectable label may be inserted into a variable region or variable loop VRIII of an AAV capsid protein. In some embodiments, the targeting ligand, first member of a binding pair, and/or detectable label may be inserted into a variable region or variable loop VRIV of an AAV capsid protein. In some embodiments, the targeting ligand, first member of a binding pair, and/or detectable label may be inserted into a variable region or variable loop VRV of an AAV capsid protein. In some embodiments, the targeting ligand, first member of a binding pair, and/or detectable label may be inserted into a variable region or variable loop VRV of an AAV capsid protein. In some embodiments, the targeting ligand, first member of a binding pair, and/or detectable label may be inserted into a variable region or variable loop VRVI of an AAV capsid protein. In some embodiments, the targeting ligand, first member of a binding pair, and/or detectable label may be inserted into a variable region or variable loop VRVII of an AAV capsid protein. In some embodiments, the targeting ligand, first member of a binding pair, and/or detectable label may be inserted into a variable region or variable loop VRIII of an AAV capsid protein. In some embodiments, the targeting ligand, first member of a binding pair, and/or detectable label may be inserted into a variable region or variable loop VRIX of an AAV capsid protein.
[00263] In some embodiments, the first member of a binding pair and/or detectable label is inserted in a VP 1 capsid protein of a non-primate animal AAV after an amino acid position corresponding with an amino acid position selected from the group consisting of G453 of AAV2 capsid protein VP1, N587 of AAV2 capsid protein VP1, G453 of AAV9 capsid protein VP1, and A589 of AAV9 capsid protein VP1. In some embodiments, the first member of a binding pair and/or detectable label is inserted in a VP 1 capsid protein of a non-primate animal AAV between amino acids that correspond with N587 and R588 of an AAV2 VP1 capsid.
[00264] The nomenclature I-###, I# or the like herein refers to the insertion site (I) with ### naming the amino acid number relative to the VP I protein of an AAV capsid protein, however such the insertion may be located directly N- or C-terminal, preferably C-terminal of one amino acid in the sequence of 5 amino acids N- or C-terminal of the given amino acid, preferably 3, more preferably 2, especially 1 amino acid(s) N- or C-terminal of the given amino acid. Additionally, the positions referred to herein are relative to the VP1 protein encoded by an AAV capsid gene, and corresponding positions (and point mutations thereof) may be easily identified for the VP2 and VP3 capsid proteins encoding by the capsid gene by performing a sequence alignment of the VP 1, VP2 and VP3 proteins encoded by the appropriate AAV capsid gene.
[00265] Additional suitable insertion sites of a non-primate animal VP1 capsid protein include those corresponding to 1-1, 1-34, 1-138, 1-139, 1-161, 1-261, 1-266, 1-381, 1-447, 1-448, 1-
459. 1-471, 1-520, 1-534, 1-570, 1-573, 1-584, 1-587, 1-588, 1-591, 1-657, 1-664, 1-713 and 1-716 of the VP1 capsid protein of AAV2 (Wu et al. (2000) J. Virol. 74:8635-8647). A modified virus capsid protein as described herein may be a non-primate animal capsid protein comprising a first member of a binding pair and/or detectable label inserted into a position corresponding with a position of an AAV2 capsid protein selected from the group consisting of 1-1, 1-34, 1-138, 1-139, 1-161, 1-261, 1-266, 1-381, 1-447, 1-448, 1-459, 1-471, 1-520, 1-534, 1-570, 1-573, 1-584, 1-587, 1-
588. 1-591, 1-657, 1-664, 1-713, 1-716, and a combination thereof. Additional suitable insertion sites of a non-primate animal AAV that include those corresponding to 1-587 or 1-590 of AAV1, 1-589 of AAV1, 1-585 of AAV3, 1-584 or 1-585 of AAV4, and 1-575 or 1-585 of AAV5. In some embodiments, a modified virus capsid protein as described herein may be a non-primate animal capsid protein comprising a targeting ligand, first member of a binding pair and/or detectable label inserted into a position corresponding with a position selected from the group consisting of 1-587 (AAV1), 1-589 (AAV1), 1-585 (AAV3), 1-585 (AAV4), 1-585 (AAV5), and a combination thereof.
[00266] In some embodiments, the first member of a binding pair and/or detectable label is inserted in a VP 1 capsid protein of a non-primate animal AAV after an amino acid position corresponding with an amino acid position selected from the group consisting of 1444 of an avian AAV capsid protein VP1, 1580 of an avian AAV capsid protein VP1, 1573 of a bearded dragon AAV capsid protein VP 1, 1436 of a bearded dragon AAV capsid protein VP1, 1429 of a sea lion AAV capsid protein VP1, 1430 of a sea lion AAV capsid protein VP1, 1431 of a sea lion AAV capsid protein VP1, 1432 of a sea lion AAV capsid protein VP1, 1433 of a sea lion AAV capsid protein VP1, 1434 of a sea lion AAV capsid protein VP1, 1436 of a sea lion AAV capsid protein VP1, 1437 of a sea lion AAV capsid protein VP1, and 1565 of a sea lion AAV capsid protein VP1.
[00267] An insertion into the corresponding position of the coding nucleic acid of one of these sites of the cap gene leads to an insertion into VP1, VP2 and/or VP3, as the capsid proteins are encoded by overlapping reading frames of the same gene with staggered start codons. Therefore, for AAV2, for example, according to this nomenclature insertions between amino acids 1 and 138 are only inserted into VP1, insertions between 138 and 203 are inserted into VP1 and VP2, and insertions between 203 and the C-terminus are inserted into VP1, VP2 and VP3, which is of course also the case for the insertion site 1-587. Therefore, the present invention encompasses structural genes of AAV with corresponding insertions in the VP 1, VP2 and/or VP3 proteins.
Transduction Efficiency
[00268] In some embodiments, a viral capsid comprising the modified viral capsid protein comprising the first and second members of a binding pair (e.g, wherein the second member is operably linked to a targeting ligand, comprises a multispecific binding protein, etcf is able to infect a specific cell, e.g., has an enhanced capacity to target and bind a specific cell compared to that of a control viral capsid that is identical to the modified viral capsid protein except that it lacks either or both the first and second members of a binding pair, e.g., comprises a control capsid protein. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to the first and second members of a binding pair linked to a targeting ligand exhibits a detectable transduction efficiency compared to the undetectable transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 10% greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 20% greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 30% greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 40% greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 50% greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 60% greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 70% greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 75% greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 80% greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 85% greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 90% greater than the transduction efficiency of a control capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 95% greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 99% greater than the transduction efficiency of a control viral capsid.
[00269] In some embodiments, a viral capsid comprising the modified viral capsid protein comprising the first and second members of a binding pair (e.g, wherein the second member is operably linked to a targeting ligand, comprises a multispecific binding protein, etc.) is able to infect a specific cell, e.g., has an enhanced capacity to target and bind a specific cell compared to that of a control viral capsid that is identical to the modified viral capsid protein except that it lacks either or both the first and second members of a binding pair, e.g., comprises a control capsid protein. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to the first and second members of a binding pair linked to a targeting ligand exhibits a detectable transduction efficiency compared to the undetectable transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 10% greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 20% greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 30% greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 40% greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 50% greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 60% greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 70% greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 75% greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 80% greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 85% greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 90% greater than the transduction efficiency of a control capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 95% greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is 99% greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at leastl.5-fold greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 2-fold greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 3-fold greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 4-fold greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 5-fold greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 6-fold greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 7-fold greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 8-fold greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 9-fold greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 10-fold greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 20-fold greater than the transduction efficiency of a control capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to an appropriate the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 30-fold greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 40-fold greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 50-fold greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 60-fold greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 70-fold greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 80-fold greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 90-fold greater than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein bound to the first and second members of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 100-fold greater than the transduction efficiency of a control viral capsid In some embodiments, a viral particle of the invention comprising a viral capsid protein comprising an amino acid sequence of a capsid protein of a non-primate animal AAV, a remote AAV, or a combination thereof, and optionally comprising a first and second members of a binding pair (e.g., wherein the second member is operably linked to a targeting ligand, comprises a multispecific binding protein, etc.) is better able to evade neutralization by pre-existing antibodies in serum isolated from a human patient compared to an appropriate control viral particle (e.g, comprising a viral capsid of an AAV serotype from which a portion is included in the viral capsid of the invention, e.g, as part of the viral capsid protein comprising an amino acid sequence of a capsid protein of a non-primate animal AAV, a remote AAV, or a combination thereof), which also optionally comprises a first and second members of a binding pair (e.g., wherein the second member is operably linked to a targeting ligand, comprises a multispecific binding protein, etc.). In some embodiments, a viral particle of the invention comprising a viral capsid protein comprising an amino acid sequence of a capsid protein of a non-primate animal AAV, a remote AAV, or a combination thereof requires at least 2-fold more total IVIG or IgG for neutralization (e.g., 50% or more infection inhibition) compared to an appropriate control viral particle, e. , (e.g, a viral particle of the invention has an IC50 value that is at least 2-fold that of a control virus particle).
Therapeutic Formulation and Administration of Antigen-binding Molecules
[00270] Also described herein are pharmaceutical compositions comprising the antigenbinding molecules as described herein. In some embodiments, pharmaceutical compositions may be formulated with suitable carriers, excipients, and other agents that provide improved transfer, delivery, tolerance, and the like. A multitude of appropriate formulations can be found in the formulary known to all pharmaceutical chemists: Remington’s Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LIPOFECTIN™, Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al. “Compendium of excipients for parenteral formulations” PDA (1998) J Pharm Sci Technol 52:238-311.
[00271] The dose of antigen-binding molecule administered to a patient may vary depending upon the age and the size of the patient, target disease, conditions, route of administration, and the like. The preferred dose is typically calculated according to body weight or body surface area. When an antigen-binding molecule as described herein is used for therapeutic purposes in an adult patient, it may be advantageous to intravenously administer the antigen-binding molecule as described herein normally at a single dose of about 0.01 to about 20 mg/kg body weight, more preferably about 0.02 to about 7, about 0.03 to about 5, or about 0.05 to about 3 mg/kg body weight. Depending on the severity of the condition, the frequency and the duration of the treatment can be adjusted. Effective dosages and schedules for administering a bispecific antigen-binding molecule may be determined empirically; for example, patient progress can be monitored by periodic assessment, and the dose adjusted accordingly.
Moreover, interspecies scaling of dosages can be performed using well-known methods in the art (e.g., Mordenti ei ctL, 1991, Pharmaceut. Res. 5:1351).
[00272] Various delivery systems are known and can be used to administer the pharmaceutical composition as described herein, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the mutant viruses, receptor mediated endocytosis (see, e.g., Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local.
[00273] A pharmaceutical composition as described herein can be delivered subcutaneously or intravenously with a standard needle and syringe. In addition, with respect to subcutaneous delivery, a pen delivery device readily has applications in delivering a pharmaceutical composition as described herein. Such a pen delivery device can be reusable or disposable. A reusable pen delivery device generally utilizes a replaceable cartridge that contains a pharmaceutical composition. Once all of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can readily be discarded and replaced with a new cartridge that contains the pharmaceutical composition. The pen delivery device can then be reused. In a disposable pen delivery device, there is no replaceable cartridge. Rather, the disposable pen delivery device comes prefilled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.
[00274] Numerous reusable pen and autoinjector delivery devices have applications in the subcutaneous delivery of a pharmaceutical composition as described herein. Examples include, but are not limited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75/25™ pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIK™ (sanofi-aventis, Frankfurt, Germany), to name only a few. Examples of disposable pen delivery devices having applications in subcutaneous delivery of a pharmaceutical composition as described herein include, but are not limited to the SOLOSTAR™ pen (sanofi-aventis), the FLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (Eli Lilly), the SURECLICK™ Autoinjector (Amgen, Thousand Oaks, CA), the PENLET™ (Haselmeier, Stuttgart, Germany), the EPIPEN (Dey, L.P.), and the HUMIRA™ Pen (Abbott Labs, Abbott Park IL), to name only a few.
[00275] In certain situations, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, polymeric materials can be
I l l used; see, Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida. In yet another embodiment, a controlled release system can be placed in proximity of the composition’s target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other controlled release systems are discussed in the review by Langer, 1990, Science 249: 1527- 1533.
[00276] The injectable preparations may include dosage forms for intravenous, subcutaneous, intracutaneous and intramuscular injections, drip infusions, etc. These injectable preparations may be prepared by methods publicly known. For example, the injectable preparations may be prepared, e.g., by dissolving, suspending or emulsifying the antibody or its salt described above in a sterile aqueous medium or an oily medium conventionally used for injections. As the aqueous medium for injections, there are, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents, etc., which may be used in combination with an appropriate solubilizing agent such as an alcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol, polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc. As the oily medium, there are employed, e.g., sesame oil, soybean oil, etc., which may be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection thus prepared is preferably filled in an appropriate ampoule.
[00277] Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared into dosage forms in a unit dose suited to fit a dose of the active ingredients. Such dosage forms in a unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the aforesaid antibody contained is generally about 5 to about 500 mg per dosage form in a unit dose; especially in the form of injection, it is preferred that the aforesaid antibody is contained in about 5 to about 100 mg and in about 10 to about 250 mg for the other dosage forms.
Pharmaceutical compositions, dosage forms and administration for viral particles [00278] A further embodiment provides a medicament comprising at least one modified viral capsid protein and appropriate targeting ligand according to this invention and/or a nucleic acid according to this invention. Preferably such medicament is useful as a gene transfer particle.
[00279] Also disclosed herein are pharmaceutical compositions comprising the viral particles described herein and a pharmaceutically acceptable carrier and/or excipient. In addition, disclosed herein are pharmaceutical dosage forms comprising the viral particle described herein.
[00280] As discussed herein, the viral particles described herein can be used for various therapeutic applications (in vivo and ex vivo) and as research tools.
[00281] Pharmaceutical compositions based on the viral particles disclosed herein can be formulated in any conventional manner using one or more physiologically acceptable carriers and/or excipients. The viral particles may be formulated for administration by, for example, injection, inhalation or insulation (either through the mouth or the nose) or by oral, buccal, parenteral or rectal administration, or by administration directly to a tumor.
[00282] The pharmaceutical compositions can be formulated for a variety of modes of administration, including systemic, topical or localized administration. Techniques and formulations can be found in, for example, Remington’s Pharmaceutical Sciences, Meade Publishing Co., Easton, Pa. For systemic administration, injection is preferred, including intramuscular, intravenous, intraperitoneal, and subcutaneous. For the purposes of injection, the pharmaceutical compositions can be formulated in liquid solutions, preferably in physiologically compatible buffers, such as Hank's solution or Ringer's solution. In addition, the pharmaceutical compositions may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms of the pharmaceutical composition are also suitable.
[00283] For oral administration, the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g. pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g. lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g. magnesium stearate, talc or silica); disintegrants (e.g. potato starch or sodium starch glycolate); or wetting agents (e.g. sodium lauryl sulfate). The tablets can also be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g. sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g. lecithin or acacia); non-aqueous vehicles (e.g. oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g. methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations can also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.
[00284] The pharmaceutical compositions can be formulated for parenteral administration by injection, e.g. by bolus injection or continuous infusion. Formulations for injection can be presented in a unit dosage form, e.g. in ampoules or in multi -dose containers, with an optionally added preservative. The pharmaceutical compositions can further be formulated as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain other agents including suspending, stabilizing and/or dispersing agents.
[00285] Additionally, the pharmaceutical compositions can also be formulated as a depot preparation. These long acting formulations can be administered by implantation (e.g. subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (e.g. as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt. Other suitable delivery systems include microspheres, which offer the possibility of local noninvasive delivery of drugs over an extended period of time. This technology can include microspheres having a precapillary size, which can be injected via a coronary catheter into any selected part of an organ without causing inflammation or ischemia. The administered therapeutic is men slowly released from the microspheres and absorbed by the surrounding cells present in the selected tissue.
[00286] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, bile salts, and fusidic acid derivatives. In addition, detergents may be used to facilitate permeation. Transmucosal administration can occur using nasal sprays or suppositories. For topical administration, the viral particles described herein can be formulated into ointments, salves, gels, or creams as generally known in the art. A wash solution can also be used locally to treat an injury or inflammation in order to accelerate healing. [00287] Pharmaceutical forms suitable for injectable use can include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid. It must be stable under the conditions of manufacture and certain storage parameters (e.g. refrigeration and freezing) and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
[00288] If formulations disclosed herein are used as a therapeutic to boost an immune response in a subject, a therapeutic agent can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
[00289] A carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents known in the art. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
[00290] Sterile injectable solutions can be prepared by incorporating the active compounds or constructs in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
[00291] Upon formulation, solutions can be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but slow release capsules or microparticles and microspheres and the like can also be employed.
[00292] For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intratumorally, intramuscular, subcutaneous and intraperitoneal administration. In this context, sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion.
[00293] The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. For example, a subject may be administered viral particles described herein on a daily or weekly basis for a time period or on a monthly, bi-yearly or yearly basis depending on need or exposure to a pathogenic organism or to a condition in the subject (e.g., cancer).
[00294] In addition to the compounds formulated for parenteral administration, such as intravenous, intratumorally, intradermal or intramuscular injection, other pharmaceutically acceptable forms include, e.g., tablets or other solids for oral administration; liposomal formulations; time release capsules; biodegradable and any other form currently used.
[00295] One may also use intranasal or inhalable solutions or sprays, aerosols or inhalants. Nasal solutions can be aqueous solutions designed to be administered to the nasal passages in drops or sprays. Nasal solutions can be prepared so that they are similar in many respects to nasal secretions. Thus, the aqueous nasal solutions usually are isotonic and slightly buffered to maintain a pH of 5.5 to 7.5. In addition, antimicrobial preservatives, similar to those used in ophthalmic preparations, and appropriate drug stabilizers, if required, may be included in the formulation. Various commercial nasal preparations are known and can include, for example, antibiotics and antihistamines and are used for asthma prophylaxis.
[00296] Oral formulations can include excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders. In certain defined embodiments, oral pharmaceutical compositions will include an inert diluent or assimilable edible carrier, or they may be enclosed in hard or soft shell gelatin capsule, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet. For oral therapeutic administration, the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
[00297] The tablets, troches, pills, capsules and the like may also contain the following: a binder, as gum tragacanth, acacia, cornstarch, or gelatin; excipients, such as dicalcium phosphate; a disintegrating agent, such as com starch, potato starch, alginic acid and the like; a lubricant, such as magnesium stearate; and a sweetening agent, such as sucrose, lactose or saccharin may be added or a flavoring agent, such as peppermint, oil of wintergreen, or cherry flavoring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup of elixir may contain the active compounds sucrose as a sweetening agent methyl and propylparabens as preservatives, a dye and flavoring, such as cherry or orange flavor.
[00298] Further embodiments disclosed herein can concern kits for use with methods and compositions. Kits can also include a suitable container, for example, vials, tubes, mini- or microfuge tubes, test tube, flask, bottle, syringe or other container. Where an additional component or agent is provided, the kit can contain one or more additional containers into which this agent or component may be placed. Kits herein will also typically include a means for containing the viral particles and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained. Optionally, one or more additional active agents such as, e.g, anti-inflammatory agents, anti-viral agents, anti-fungal or anti-bacterial agents or anti -tumor agents may be needed for compositions described.
[00299] Compositions disclosed herein may be administered by any means known in the art. For example, compositions may include administration to a subject intravenously, intratumorally, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostaticaly, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, intrathecally, subcutaneously, subconjunctival, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularly, orally, locally, by inhalation, by injection, by infusion, by continuous infusion, by localized perfusion, via a catheter, via a lavage, in a cream, or in a lipid composition.
[00300] Any method known to one skilled in the art maybe used for large scale production of viral particles, packaging cells and particle constructs described herein. For example, master and working seed stocks may be prepared under GMP conditions in qualified primary CEFs or by other methods. Packaging cells may be plated on large surface area flasks, grown to near confluence and viral particles purified. Cells may be harvested and viral particles released into the culture media isolated and purified, or intracellular viral particles released by mechanical disruption (cell debris can be removed by large-pore depth filtration and host cell DNA digested with endonuclease). Virus particles may be subsequently purified and concentrated by tangential- flow filtration, followed by diafiltration. The resulting concentrated bulk maybe formulated by dilution with a buffer containing stabilizers, filled into vials, and lyophilized. Compositions and formulations may be stored for later use. For use, lyophilized viral particles may be reconstituted by addition of diluent.
[00301] Certain additional agents used in the combination therapies can be formulated and administered by any means known in the art.
[00302] Compositions as disclosed herein can also include adjuvants such as aluminum salts and other mineral adjuvants, tensoactive agents, bacterial derivatives, vehicles and cytokines. Adjuvants can also have antagonizing immunomodulating properties. For example, adjuvants can stimulate Thl or Th2 immunity. Compositions and methods as disclosed herein can also include adjuvant therapy.
Therapeutic and Diagnostic Uses for Antigen-binding Molecules
[00303] Disclosed herein are methods of, for example, inhibiting the activity of CDH15 in a cell. The method may comprise contacting the cell expressing CDH15 (e.g., in vitro, ex vivo, or in vivo) with an anti-hCDH15 antibody, antigen-binding fragment thereof, or pharmaceutical composition thereof of the present disclosure. The activity of CDH15 includes, but is not limited to, inhibiting muscle regeneration e.g., by inhibiting activation of CDH15-expressing muscle stem cells. [00304] In various embodiments, the cell expressing CDH15 is a muscle cell. In some embodiments, the cell is a mammalian muscle cell. In some embodiments, the cell is a mammalian skeletal muscle cell. In some embodiments, the cell is a mammalian skeletal muscle cell that is not terminally differentiated. In some embodiments, the cell is a mammalian skeletal muscle cell that is terminally differentiated. In some embodiments, the cell is a muscle stem cell , a myoblast, or a myocyte. In some embodiments, the cell is a human rhabdomyosarcoma cell. [00305] Disclosed herein are also methods of accelerating the transition from quiescence to activation in a muscle stem cell, comprising contacting the muscle stem cell (e.g., in vitro, ex vivo, or in vivo) with an anti-hCDH15 antibody, antigen-binding fragment thereof, or pharmaceutical composition thereof of the present disclosure. “Quiescence”, as used herein, refers to an inactive, non-proliferating state of a cell. For example, quiescent muscle stem cells do not proliferate or differentiate into myofibers. “Activation” as used herein in reference to muscle stem cells, refers to the ability of the muscle stem cells to proliferate and differentiate into myofibers e.g., to promote regrowth and/or repair of damaged muscle.
[00306] Disclosed herein are also methods comprising administering to a subject in need thereof a composition (e.g., a therapeutic composition) comprising an anti-hCDH15 antibody, antigen-binding fragment thereof or an antibody-drug conjugate comprising an anti-hCDH15 antibody (e.g., an anti-hCDH15 antibody, or ADC comprising any of the HCVR/LCVR or CDR sequences as set forth in Table 1 herein). The therapeutic composition can comprise any of the anti-hCDH15 antibodies, antigen-binding fragments thereof, or ADCs disclosed herein, and a pharmaceutically acceptable carrier or diluent.
[00307] Disclosed herein are also uses of a composition (e.g. , a therapeutic composition) comprising an anti-hCDH15 antibody, antigen-binding fragment thereof, or an antibody-drug conjugate comprising an anti-hCDH15 antibody for the treatment of one or more condition as disclosed herein.
[00308] Disclosed herein are also compositions (e.g., a therapeutic compositions) comprising an anti-hCDH15 antibody, antigen-binding fragment thereof, or an antibody-drug conjugate comprising an anti-hCDH15 antibody for use as a therapy e.g., for use in the treatment of one or more condition as disclosed herein. [00309] Disclosed herein is also the use of an anti-hCDH15 antibody, antigen-binding fragment thereof, or an antibody-drug conjugate comprising an anti-hCDH15 antibody in the manufacture of a medicament for the treatment of one or more condition as disclosed herein. [00310] The antibodies, antigen-binding fragment thereof, or an antibody-drug conjugate comprising an anti-hCDH15 antibody as described herein may be useful, inter alia, for the treatment, prevention and/or amelioration of one or more condition e.g., any disease or disorder associated with skeletal muscle tissue. For example, the antibodies and ADCs as described herein may be useful for the treatment of muscle wasting disorders (e.g., cachexia, glucocorticoid-induced muscle loss, heart failure induced muscle loss, HIV wasting, disuse, aging, etc ), muscular dystrophies/myopathies, and/or muscle-related cancers (e.g., rhabdomyosarcoma).
[00311] In another embodiment, provided is a method of treatment of a disease, such as a muscle wasting disorder. The method may include the step of providing an antibody or CDH15 antigen-binding fragment thereof, as described above, to a subject requiring said treatment.
[00312] In another embodiment, provided is a method of treatment of a muscle-related cancer. The method may include the step of providing an antibody or CDH15 antigen-binding fragment thereof, as described above, to a subject requiring said treatment. Non-limiting examples of muscle-specific cancers include rhabdomyosarcomas, e.g, embryonal, alveolar, pleomorphic, botryoid and spindle/ sclerosing rhabdomyosarcomas.
[00313] In another embodiment, provided is a method of treatment of muscle injury. The method may include the step of providing an antibody or CDH15 antigen-binding fragment thereof, as described above, to a subject requiring said treatment e.g., a subject that has experienced muscle injury and/or anticipates experiencing muscle injury. Accordingly, the antibody or CDH15 antigen-binding fragment thereof may be administered prior to injury e.g., prior to a muscle damaging surgery or after injury e.g., an unanticipated muscle injury.
[00314] In another embodiment, provided is a method of improving muscle regeneration in a subject following injury. The method may include the step of providing an antibody or CDH15 antigen-binding fragment thereof, as described above, to a subject requiring said treatment. As used herein, “improved” in reference to muscle regeneration refers to any measurable increase in muscle repair. For example, muscle regeneration may be considered “improved” in a subject if it occurs at accelerated rate and/or to a greater degree as compared to a control subject e.g., a subject that has not been administered the anti-CDH15 antibody or antigen-binding fragment thereof. “Improved” muscle regeneration may be an increase in rate or degree of at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, or at least 500%.
[00315] In another embodiment, provided is a method of restoring the muscle regenerative capacity of an aged subject. The method may include the step of providing an antibody or CDH15 antigen-binding fragment thereof, as described above, to a subject requiring said treatment. As used herein, “restore” refers the ability to revert to a prior functional state. For example, the muscle regenerative capacity of an aged subject may be considered “restored” if it can be reverted to a muscle regenerative capacity at or near that of a control subject e.g, the same subject at a younger age and/or a separate subject that is representative of the aged subject at a younger age. The functional state may be considered “restored” if it is comparable to (e.g., not statistically different from) the prior functional state or the functional state of a subject or subjects that is representative of the prior functional state. The functional state may also be considered “restored” if it is no more than 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 45%, 50%, 60%, 70%, 80%, or 90% different from the prior functional state or the functional state of a subject or subjects that is representative of the prior functional state.
[00316] The anti-hCDH15 antibodies as described herein have various utilities. For example, in some embodiments, anti-hCDH15 antibodies as described herein may be used in diagnostic assays for CDH15, e.g., detecting its expression in specific cells, tissues, etc., e.g., as a reagent to identify /label skeletal muscle fibers and/or muscle stem cells. Various diagnostic and prognostic assay techniques known in the art may be used, such as competitive binding assays, direct or indirect sandwich assays and immunoprecipitation assays conducted in either heterogeneous or homogeneous phases (Zola (1987) Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc. pp. 147-1581). The antibodies used in the assays can be labeled with a detectable moiety. The detectable moiety should be capable of producing, either directly or indirectly, a detectable signal. Any method known in the art for conjugating the antibody to the detectable moiety may be employed. Methods of Use and Making Viral Particles
[00317] A further embodiment of the modified viral capsids described herein is the use of the modified viral capsids for delivering a nucleotide of interest, e.g., a reporter gene or a therapeutic gene, to a target cell (e.g., a muscle stem cell, myoblast, myocyte, any combination thereof, etc.). Generally, packaging of a nucleotide of interest comprises replacing an AAV genome between AAV ITR sequences with a gene of interest to create a transfer plasmid, which is then encapsulated in an AAV capsid according to well-known methods Thus, a modified viral capsid as described herein may encapsulate a transfer plasmid and/or a nucleotide of interest, which may generally comprise 5' and 3' inverted terminal repeat (ITR) sequences flanking a gene of interest, e.g., reporter gene(s) or therapeutic gene(s), or a portion of the gene of interest (which may be under the control of a viral or non-viral promoter). According to well-known methods of packaging AAV viral particles, the modified viral capsids, the 5’ ITR, and the 3’ ITR need not be of the same AAV serotype. In one embodiment, a transfer plasmid and/or nucleotide of interest comprises from 5’ to 3’: a 5’ ITR, a promoter, a gene (e.g., a reporter and/or therapeutic gene) and a 3TTR.
[00318] A consideration for AAV transfer plasmid design is that a wildtype AAV genome is ~4.7kb. Thus, included herein are the well-known strategies that provide for packaging nucleotides of interest that exceed the packaging capacity of an individual AAV. Such strategies include, but are not limited to, dual-vector strategies that exploit ITR-mediated recombination to express genes of interest that are larger than a wildtype AAV genome by way of transcript splicing across intermolecularly recombined ITRs from two complementary vector genomes, vector recombination by homology, RNA trans-splicing, and/or protein “transsplicing” via split intein designs. See, e.g., Nakai, H. et al. (2000) Nat. Biotechnol. 18:527-532; Sun, L. (2000) Nat. Med. 6: 599-602 (2000); Ghosh, A., et al. (2008) Mol. Ther. 16: 124-130 (2008); Lai, Y (2005) Nat. Biotechnol. 23: 1435-1439; Chew, W. L. et al. (2016) Nat. Methods 13:868-874; Li, J. (2008) Hum. Gene Ther. 19:958-964, each of which reference is incorporated herein in its entirety by reference.
[00319] Dual AAV vector strategies to transfer a large gene into target cells have been described, which rely on different mechanisms including, but are not limited to, trans-splicing, including overlapping regions in the dual vectors, and a hybrid of the two. Tornabene and Trapani (2020) Human Gene Ther. 31 :47-56; see also U.S. Patent No. 8,236,557, each of which is incorporated herein by reference in its entirety.
[00320] A trans-splicing approach takes advantage of the ability of AAV ITR sequences to concatemerized to reconstitute full-length genomes, wherein each of two or more viral capsids, respectively, encapsulate one of two or more transfer plasmids, each of which transfer plasmid comprises a portion of the gene of interest. For example, in a dual vector approach, the two transfer plasmids may be designed as follows: the 5’-transfer plasmid comprises the promoter, the 5’ portion of the coding sequence of the gene of interest, and a splicing donor (SD) signal; the 3 ’-transfer plasmid comprises a splicing acceptor (SA) signal, the 3’ portion of the gene of interest, and the polyA signal. Upon tail-to-head ITR-mediated concatemerization of the two AAV genomes, the SD and SA signals will allow splicing of the recombined genome.
[00321] A large gene of interest is also split when taking an overlapping region approach. In the overlapping region approach, the 5’ and 3’ portions (and thus the 5’ transfer plasmid and 3’ transfer plasmid) share a recombinogenic sequence, e.g, region of homology, e.g., each portion comprises an overlapping sequence. The gene of interest is made whole in a targeted cell via homologous recombination mediated by the recombinogenic sequence, e.g., homology/overlapping region.
[00322] In a hybrid approach, the 5 ’-transfer plasmid and 3 ’-transfer plasmid each comprise a highly recombinogenic sequence, wherein the recombinogenic sequence is placed downstream of an SD signal of a 5’ portion of the coding sequence of the gene of interest and upstream of an SA signal of a 3’ portion of the coding sequence of the gene of interest. In this hybrid system, the gene of interest may be made whole either via ITR-mediated concatemerization and splicing and/or by homologous recombination.
[00323] Trans-splicing at the RNA or protein levels may also be utilized. In an RNA trans-splicing approach, two transfer plasmids may respectively encode for 5’ and 3’ fragments of the pre-mRNA of a large gene and share an intronic hybridization domain that can favor trans-splicing, leading to joining of the two half-transcripts into an intact full-length mRNA.
[00324] Protein trans-splicing occurs post-translationally and is catalyzed by intervening proteins called split-inteins. Split-inteins are expressed as two independent polypeptides (N- intein and C-intein) at the extremities of two host proteins. The N-intein and C-intein polypeptides remain catalytically inactive until they encounter each other. Upon encountering each other, each intein precisely excises itself from the host protein while mediating ligation of the N- and C- host polypeptides via a peptide bond. Split-intein use has been used in AAV- based delivery of therapeutic genes of interest in muscle, liver, and retinal diseases. For example, on co-delivery of two halves of the mini -dystrophin cDNA fused to N- and C-intein coding sequences, efficient production of the two polypeptides was shown. Li et al. (2008) Hum Gene Ther 19:958-64. Similarly, AAV-split-inteins have been widely used for the expression and ligation of the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 nuclease.
[00325] The above dual vector approaches are well-known in the art. See, e.g, Tornabene and Trapani (2020), supra, U.S. Patent No. 8,236,557. Thus, in some embodiments, a modified viral capsid described herein encapsulates a nucleotide of interest, wherein the nucleotide of interest comprises a portion of a gene of interest. In some embodiments, a nucleotide of interest comprising a portion of a gene of interest further comprises a splicing donor signal or a splicing acceptor signal and/or a recombinogenic sequence. In some embodiments, a nucleotide of interest comprising a portion of a gene of interest comprises an intronic hybridization domain encoding sequence. In some embodiments, a nucleotide of interest comprising a portion of a gene of interest comprises a N-intein or C-intein encoding sequence.
[00326] Design of the transfer plasmid/nucleotide of interest includes including one or more regulatory elements, e.g., promoter and/or enhancer elements, that will control expression of the gene of interest. Non-limiting examples of useful promoters include, e.g., cytomegalovirus (CMV)-promoter, the spleen focus forming virus (SFFV)-promoter, the elongation factor 1 alpha (EFla)-promoter (the 1.2 kb EFla-promoter or the 0.2 kb EFla- promoter), the chimeric EF 1 a/IF4-promoter, and the phospho-glycerate kinase (PGK)- promoter. An internal enhancer may also be present in the viral construct to increase expression of the gene of interest. For example, the CMV enhancer (Karasuyama et al. 1989. J. Exp. Med. 169: 13, which is incorporated herein by reference in its entirety) may be used. In some embodiments, the CMV enhancer can be used in combination with the chicken [3-actin promoter. In some embodiments, tissue specific regulatory elements, e.g., a muscle specific promoter and/or regulatory element may be used to drive the expression of the gene of interest. For example, the use of muscle-specific regulatory elements based on the muscle creatine kinase gene has been employed for muscle gene therapy treatments, such as Duchenne muscular dystrophy (DMD) and limb-girdle muscular dystrophy (LGMD). See, e.g., Salva, M. Z. et al. (2007) Mol. Ther. 15: 320-329, incorporated herein in its entirety by reference. In some embodiments, a transfer plasmid and/or nucleotide of interest herein comprises an enhancer and/or promoter of muscle creatine kinase (MCK), wherein the enhancer and/or promoter of MCK drives expression of the gene of interest. In some embodiments, a transfer plasmid and/or nucleotide of interest herein comprises an enhancer and/or promoter element that recruits RNA Polymerase II, wherein the enhancer and/or promoter of MCK drives expression of the gene of interest. In some embodiments, a transfer plasmid and/or nucleotide of interest herein comprises an enhancer and/or promoter element that recruits RNA Polymerase III, wherein the enhancer and/or promoter of MCK drives expression of the gene of interest.
[00327] In some embodiments, bidirectional promoter vectors have also been employed for delivery of dual therapeutic gene cassettes. An example of this is the bidirectional chicken 0- actin ubiquitous promoter that drives the simultaneous expression of the hexosaminidase a- and 0-subunits of the HexA enzyme, the two respective genes involved in Tay-Sachs and Sandhoff diseases. Lahey, et al. (2020) Mol. Ther. 28: 2150-2160, incorporated herein in its entirety by reference. In some embodiments, a transfer plasmid and/or nucleotide of interest herein comprises a bidirectional promoter, wherein the bidirectional promoter drives the expression of two different genes of interest.
[00328] A variety of reporter genes (or detectable moieties) can be encapsidated in a multimeric structure comprising the modified viral capsid proteins described herein. Exemplary reporter genes include, for example, 0-galactosidase (encoded lacZ gene), Green Fluorescent Protein (GFP), enhanced Green Fluorescent Protein (eGFP), MmGFP, blue fluorescent protein (BFP), enhanced blue fluorescent protein (eBFP), mPlum, mCherry, tdTomato, mStrawberry, J- Red, DsRed, mOrange, mKO, mCitrine, Venus, YPet, yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (eYFP), Emerald, CyPet, cyan fluorescent protein (CFP), Cerulean, T-Sapphire, luciferase, alkaline phosphatase, or a combination thereof. The methods described herein demonstrate the construction of targeting particles that employ the use of a reporter gene that encodes green fluorescent protein, however, persons of skill upon reading this disclosure will understand that the viral capsids described herein can be generated in the absence of a reporter gene or with any reporter gene known in the art. [00329] A variety of therapeutic genes can also be encapsidated in a multimeric structure comprising the modified viral capsid proteins described herein, e.g, as part of a transfer particle. Non-limiting examples of a therapeutic gene include those that encode a toxin (e.g., a suicide gene), a therapeutic antibody or fragment thereof, a CRISPR/Cas system or portion(s) thereof, antisense RNA, siRNA, shRNA, etc.
[00330] A further embodiment of the present invention is a process for the preparation of a modified capsid protein, the method comprising the steps of: a) expressing a nucleic acid encoding the modified capsid protein under suitable conditions, and b) isolating the expressed capsid protein of step a).
[00331] In some embodiments, a viral particle as described herein comprises a mosaic capsid, e.g., a capsid comprising capsid proteins genetically modified as described herein (in the absence or presence of a covalent bond with a targeting ligand) in a certain ratio with reference capsid proteins. A method for making such a mosaic viral particle comprises: a) expressing a nucleic acid encoding the modified capsid protein and a nucleotide encoding a reference capsid protein at a ratio (wt/wt) of at least about 60: 1 to about 1 :60, e.g, 2: 1, 1 : 1, 3:5 ,1 :2, 1:3, 1 :8, etc. under suitable conditions, and b) isolating the expressed capsid protein of step a).
[00332] In some embodiments, a composition described herein comprises, or a method described herein combines, a modified cap gene: reference cap gene (or combination of reference cap genes) at a ratio that ranges from at least about 1 :60 to about 60: 1, e.g., 2: 1, 1 :1, 3:5, 1 :2, 1 :3, 1 :8, etc. In some embodiments, the ratio is at least about 1 :2. In some embodiments, the ratio is at least about 1 :3. In some embodiments, the ratio is at least about 1 :4. In some embodiments, the ratio is at least about 1 :5. In some embodiments, the ratio is at least about 1 :6. In some embodiments, the ratio is at least about 1 :7. In some embodiments, the ratio is at least about 1 :8. In some embodiments, the ratio is at least about 1:9. In some embodiments, the ratio is at least about 1 : 10. In some embodiments, the ratio is at least about 1 : 11. In some embodiments, the ratio is at least about 1 : 12. In some embodiments, the ratio is at least about 1: 13. In some embodiments, the ratio is at least about 1 :14. In some embodiments, the ratio is at least about 1: 15. In some embodiments, the ratio is at least about 1 : 16. In some embodiments, the ratio is at least about 1 :17. In some embodiments, the ratio is at least about 1 : 18. In some embodiments, the ratio is at least about 1 : 19. In some embodiments, the ratio is at least about 1:20. In some embodiments, the ratio is at least about 1 :25. In some embodiments, the ratio is at least about 1:30. In some embodiments, the ratio is at least about 1 :35. In some embodiments, the ratio is at least about 1 :40. In some embodiments, the ratio is at least about 1 :45. In some embodiments, the ratio is at least about 1 :50. In some embodiments, the ratio is at least about 1:55. In some embodiments, the ratio is at least about 1 :60. In some embodiments, the ratio is at least about 2: 1. In some embodiments, the ratio is at least about 3 : 1. In some embodiments, the ratio is at least about 4:1. In some embodiments, the ratio is at least about 5: 1. In some embodiments, the ratio is at least about 6:1. In some embodiments, the ratio is at least about 7:1. In some embodiments, the ratio is at least about 8:1. In some embodiments, the ratio is at least about 9: 1. In some embodiments, the ratio is at least about 10: 1. In some embodiments, the ratio is at least about 11 :1. i some embodiments, the ratio is at least about 12: 1. In some embodiments, the ratio is at least about 13 : 1. In some embodiments, the ratio is at least about 14:1. In some embodiments, the ratio is at least about 15: 1. In some embodiments, the ratio is at least about 16: 1. In some embodiments, the ratio is at least about 17:1. In some embodiments, the ratio is at least about 18: 1. In some embodiments, the ratio is at least about 19: 1. In some embodiments, the ratio is at least about 20: 1. In some embodiments, the ratio is at least about 25:1. In some embodiments, the ratio is at least about 30: 1. In some embodiments, the ratio is at least about 35: 1. In some embodiments, the ratio is at least about 40:1. In some embodiments, the ratio is at least about 45 : 1. In some embodiments, the ratio is at least about 50: 1. In some embodiments, the ratio is at least about 55: 1. In some embodiments, the ratio is at least about 60:1.
[00333] In some embodiments, VP protein subunit ratios in the mosaic viral particle may, but do not necessarily, stoichiometrically reflect the ratios of modified cap gene reference cap gene. As a non-limiting exemplary embodiment, a mosaic capsid formed according to the method may be considered to, but does not necessarily, have a modified capsid proteimreference capsid protein ratio similar to the ratio (wt:wt) of nucleic acids encoding same used to produce the mosaic capsid. In some embodiments, a mosaic capsid comprises a protein subunit ratio of about 1 :59 to about 59:1.
[00334] A further embodiment of the present invention is a method for altering the tropism of a virus, the method comprising the steps of: (a) inserting a nucleic acid encoding an amino acid sequence into a nucleic acid sequence encoding an viral capsid protein to form a nucleotide sequence encoding a genetically modified capsid protein comprising the amino acid sequence and/or (b) culturing a packaging cell in conditions sufficient for the production of viral particles, wherein the packaging cell comprises the nucleic acid. A further embodiment of the present invention is a method for displaying a targeting ligand on the surface of a capsid protein, the method comprising the steps of: (a) expressing a nucleic acid encoding a modified viral capsid protein as described herein (and optionally with a nucleotide encoding a reference capsid protein) under suitable conditions, wherein the nucleic acid encodes a capsid protein comprising a first member of a specific binding pair, (b) isolating the expressed capsid protein comprising a first member of a specific binding pair of step (a) or capsid comprising same, and (c) incubating the capsid protein or capsid with a second cognate member of the specific binding pair under conditions suitable for allowing the formation of an isopeptide bond between the first and second member, wherein the second cognate member of the specific binding pair is fused with a targeting ligand.
[00335] In some embodiments, the packaging cell further comprises a helper plasmid and/or a transfer plasmid comprising a nucleotide of interest. In some embodiments, the methods further comprise isolating self-complementary adeno-associated viral particles from culture supernatant. In some embodiments, the methods further comprise lysing the packaging cell and isolating single-stranded adeno-associated viral particles from the cell lysate. In some embodiments, the methods further comprise (a) clearing cell debris, (b) treating the supernatant containing viral particles with nucleases, e.g, DNase I and MgCh, (c) concentrating viral particles, (d) purifying the viral particles, and (e) any combination of (a)-(d).
[00336] Packaging cells useful for production of the viral particles described herein include, e.g., animal cells permissive for the virus, or cells modified to be permissive for the virus; or the packaging cell construct, for example, with the use of a transformation agent such as calcium phosphate. Non-limiting examples of packaging cell lines useful for producing viral particles described herein include, e.g., human embryonic kidney 293 (HEK-293) cells (e.g., American Type Culture Collection [ATCC] No. CRL-1573), HEK-293 cells that contain the SV40 Large T-antigen (HEK-293 T or 293 T), HEK293T/17 cells, human sarcoma cell line HT- 1080 (CCL-121), lymphoblast-like cell line Raji (CCL-86), glioblastoma-astrocytoma epithelial- like cell line U87-MG (HTB-14), T-lymphoma cell line HuT78 (TIB-161), NIH/3T3 cells, Chinese Hamster Ovary cells (CHO) (e.g, ATCC Nos. CRL9618, CCL61, CRL9096), HeLa cells (e.g, ATCC No. CCL-2), Vero cells, NIH 3T3 cells (e.g, ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g, ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS- 7 cells (ATCC No. CRL1651), RATI cells, mouse L cells (ATCC No. CCLI.3), HLHepG2 cells, CAP cells, CAP-T cells, and the like.
[00337] L929 cells, the FLY viral packaging cell system outlined in Cosset et al (1995) J
Virol 69,7430-7436, NSO (murine myeloma) cells, human amniocytic cells (e.g, CAP, CAP-T), yeast cells (including, but not limited to, S. cerevisiae, Pichia pastoris), plant cells (including, but not limited to, Tobacco NT1 , BY-2), insect cells (including but not limited to SF9, S2, SF21, Tni (e.g. High 5)) or bacterial cells (including, but not limited to, E. coli).
[00338] For additional packaging cells and systems, packaging techniques and particles for packaging the nucleic acid genome into the pseudotyped viral particle see, for example, Polo, et al, Proc Natl Acad Sci USA, (1999) 96:4598-4603. Methods of packaging include using packaging cells that permanently express the viral components, or by transiently transfecting cells with plasmids.
[00339] Further embodiments include methods comprising contacting a modified Cap protein as described herein with the targeting vector in conditions sufficient to operably link the modified Cap protein with the targeting vector, e.g., in conditions sufficient to promote association of the targeting vector to the modified Cap protein, e.g., via chemical linkage and/or association of first and second members of a specific binding pair, wherein the first member is inserted into the modified Cap protein the first member and the targeting vector is fused to the second member of the specific binding pair.
[00340] Further embodiments include methods of redirecting a virus and/or delivering a reporter or therapeutic gene to a target cell (e.g., a muscle stem cell, myoblast, myocyte, any combination thereof, etc.), the method comprising a method for transducing a target cell in vitro (e.g., ex vivo) or in vivo, the method comprising the steps of: contacting the target cell with a viral particle comprising a capsid described herein, wherein the capsid comprises a targeting ligand that specifically binds a receptor expressed by the target cell. In some embodiments, the target cell is in vitro (e.g, ex vivo). In other embodiments, the target cell is in vivo in a subject, e.g, a human. Target Cells
[00341] A wide variety of cells may be targeted in order to deliver a nucleotide of interest using a modified viral particle as disclosed herein. The target cells will generally be chosen based upon the nucleotide of interest and the desired effect.
[00342] In some embodiments, a nucleotide of interest may be delivered to a target cell (e.g., a muscle stem cell, myoblast, myocyte, any combination thereof, etc.) such that the target cell produces a protein that makes up for a deficiency in an organism, such as an enzymatic deficiency, or immune deficiency, such as X-linked severe combined immunodeficiency. Thus, in some embodiments, cells that would normally produce the protein in the animal are targeted. In other embodiments, cells in the area in which a protein would be most beneficial are targeted. [00343] In other embodiments, a nucleotide of interest, such as a gene encoding an siRNA, may inhibit expression of a particular gene in a target cell (e.g., a muscle stem cell, myoblast, myocyte, any combination thereof, etc.). The nucleotide of interest may, for example, inhibit expression of a gene involved in a pathogen life cycle. Thus, cells susceptible to infection from the pathogen or infected with the pathogen may be targeted. In other embodiments, a nucleotide of interest may inhibit expression of a gene that is responsible for production of a toxin in a target cell.
[00344] In other embodiments, a nucleotide of interest may encode a toxic protein that kills cells in which it is expressed. In this case, tumor cells or other unwanted cells may be targeted.
[00345] In still other embodiments a nucleotide of interest that encodes a therapeutic protein. In some embodiments, a nucleotide of interest encodes a therapeutic protein comprising an antigen binding protein, or an antigen binding portion thereof. In some embodiments, a nucleotide of interest encodes a therapeutic protein comprising a human or humanized antibody or antigen binding fragment thereof, a monovalent Fab’, a divalent Fab2, a F(ab)’3 fragment, a single-chain fragment variable (scFv), a bis-scFv, a (scFv)2, a diabody, a minibody, a nanobody, a triabody, a tetrabody, a disulfide stabilized Fv protein (dsFv), a single-domain antibody (sdAb), an Ig NAR, a bispecific antibody or binding fragment thereof, a bi-specific T-cell engager (BiTE), a trispecific antibody, or a chemically modified derivative thereof. In some embodiments, the scFv comprises variable regions arranged in the following orientation from N- terminus to C-terminus: HCVR-LCVR. In some embodiments, the scFv comprises variable regions arranged in the following orientation from N-terminus to C-terminus: LCVR-HCVR. In some embodiments, the scFv variable regions are connected by a linker. In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker is -(GGGGS)n- (SEQ ID NO: 789), wherein n is 1-10.
[00346] Once a particular population of target cells is identified in which expression of a nucleotide of interest is desired, a target is selected that is specifically expressed on that population of target cells. The target may be expressed exclusively on that population of cells or to a greater extent on that population of cells than on other populations of cells. The more specific the expression, the more specifically delivery can be directed to the target cells. Depending on the context, the desired amount of specificity of the marker (and thus of the gene delivery) may vary. For example, for introduction of a toxic gene, a high specificity is most preferred to avoid killing non-targeted cells. For expression of a protein for harvest, or expression of a secreted product where a global impact is desired, less marker specificity may be needed.
[00347] As discussed above, the target may be any cell-surface moiety e.g, a protein, for which a targeting ligand can be identified or created. Preferably the target is a peptide or polypeptide, such as a receptor. However, in other embodiments the target may be a carbohydrate or other molecule that can be recognized by a binding partner. If a binding partner, e.g., ligand, for the target is already known, it may be used as the affinity molecule. However, if a binding molecule is not known, antibodies to the target may be generated using standard procedures. The antibodies can then be used as a targeting ligand.
[00348] Thus, target cells may be chosen based on a variety of factors, including, for example, (1) the application (e.g., therapy, expression of a protein to be collected, and conferring disease resistance) and (2) expression of a marker with the desired amount of specificity.
[00349] Target cells are not limited in any way and include both germline cells and cell lines and somatic cells and cell lines. When the target cells are germline cells, the target cells are preferably selected from the group consisting of single-cell embryos and embryonic stem cells (ES).
[00350] In various embodiments, the target cell is a cell that expresses a non-terminally differentiated muscle cell surface protein. In some embodiments, the target cell is a cell that expresses a mammalian non-terminally differentiated muscle cell surface protein. In some embodiments, the target cell is a cell that expresses a human non-terminally differentiated muscle cell surface protein. In some embodiments, the target cell is a cell that expresses CDH15, e.g., mammalian CDH15, e.g., human CDH15.
[00351] In various embodiments, the target cell is a muscle cell. In some embodiments, the target cell is a mammalian muscle cell. In some embodiments, the target cell is a mammalian muscle cell. In some embodiments, the target cell is a mammalian muscle cell that is not terminally differentiated. In some embodiments, the target cell is a mammalian muscle cell that is terminally differentiated. In some embodiments, the target cell is a muscle stem cell (also known as a satellite cell), a myoblast, a myocyte, or a non-differentiated myofiber. In some embodiments, the target cell is a mammalian rhabdomyosarcoma cell.
Skeletal Muscle Related Disorders
[00352] Also provided herein are methods of treating a skeletal muscle related disorder, e.g., a muscle wasting disease and/or a genetic muscle disease, e.g., X-linked myotubular myopathy (XLMTM), Duchenne muscular dystrophy (DMD), myotonic dystrophy (DM1), Facioscapulohumeral muscular dystrophy Type 1 (FSHD), congenital muscular dystrophy type 1A (MDC1A), Limb girdle muscular dystrophy, dystroglycanopathy, muscle atrophy conditions, metabolic diseases, etc.
[00353] Generally, such methods comprise administering to a patient suffering from or at risk for such skeletal related disorder a viral particle or pharmaceutical composition as described herein, wherein the viral particle comprises:
(i) a viral capsid modified to comprise a first member of a protein: protein binding pair,
(ii) a second member of the protein: protein binding pair, wherein the second member of the proteimprotein binding pair comprises a targeting ligand that binds a non-terminally differentiated muscle cell surface protein that is expressed on the surface of a muscle cell (e.g., CDH15), wherein the first member of the protein: protein binding pair and the second member of the protein: protein binding pair are associated to direct the tropism of the viral capsid to the muscle cell in the patient thereof, and
(iii) a nucleotide of interest encapsidated within the viral capsid. [00354] In some embodiments, the nucleotide of interest encodes a therapeutic protein, a suicide gene, an antibody or a fragment thereof, a CRISPR/Cas system or a portion(s) thereof, an antisense oligonucleotide, a ribozyme, an RNAi molecule, or a shRNA molecule. For example, in some embodiments, the nucleotide of interest may encode a growth factor, neurotrophic factor, a disease modifying muscle protein, a metabolic protein, e.g, for muscle atrophy conditions or metabolic diseases.
Muscle-Related Cancer
[00355] Also provided herein are methods of treating muscle-related cancer, e.g., a cancer expressing a non-terminally differentiated muscle cell protein, e.g, CDH15. Non-limiting examples of muscle-specific cancers include rhabdomyosarcomas, e.g., embryonal, alveolar, pleomorphic, botryoid and spindle/ sclerosing rhabdomyosarcomas.
[00356] Generally, such methods comprise administering to a patient suffering from or at risk for such muscle-related cancer a viral particle or pharmaceutical composition as described herein, wherein the viral particle comprises:
(i) a viral capsid modified to comprise the targeting ligand, inserted directly into the viral capsid or via, e.g., a first member of a protein: protein binding pair and its cognate second member of the protein: protein binding pair, wherein the second member of the protein: protein binding pair comprises a targeting ligand that binds a muscle-specific surface protein that is expressed on the surface of a non-terminally differentiated muscle cell e.g., CDH15), wherein the first member of the protein: protein binding pair and the second member of the protein: protein binding pair are associated to direct the tropism of the viral capsid to the muscle cell in the patient thereof, and
(ii) a nucleotide of interest encapsidated within the viral capsid.
[00357] In some embodiments, the nucleotide of interest comprises or encodes a therapeutic protein, a suicide gene, an antibody or a fragment thereof, a CRISPR/Cas system or a portion(s) thereof, an antisense oligonucleotide, a ribozyme, an RNAi molecule, or a shRNA molecule capable of treating the muscle-related cancer cell e.g., slowing and/or arresting the growth of and/or destroying the muscle-related cancer cell. Example non-limiting embodiments of said nucleotides of interest include a therapeutic protein that targets and inhibits an oncogenic intracellular signaling pathway, a suicide gene that directs the cell to undergo apoptosis, and an RNAi molecule that targets and inhibits an oncogenic transcript. Table 2: Example sequences of non-primate adeno-associated viruses. [00358] Table 2 - continued [00359] Table 2 - continued [00360] Table 2 - continued [00361] Table 2 - continued
Table 3: Brief description of the sequences in the sequence listing
EXAMPLES
Example 1: Cadherin 15 regulates the quiescence-to-activation transition of MuSCs
[00362] Muscle stem cells (MuSCs) are mitotically quiescent and non-proliferative under steady-state conditions in adult muscle tissue. In response to injury, however, MuSCs begin to actively divide, producing daughter cells. While some daughter cells become quiescent once more to replenish the pool of MuSCs, others continue to proliferate as myoblasts, which align and fuse together during differentiation into mature myotubes/myofibers that make up muscle fibers. Quiescent muscle stem cells can be characterized by Pax7 expression, proliferating myoblasts can be characterized by Pax7 and myogenic differentiation factor 1 (MyoD) expression, myoblasts committing to terminal differentiation (z.e., myocytes) are characterized by MyoD and Myogenin expression, and myotubes can be characterized by myosin heavy chain (MyHC) expression.
[00363] Cadherin 15 (CDH15) mRNA is highly expressed in MuSCs and expression increases following muscular injury. To determine the role that CDH15 plays in skeletal muscle biology, homozygous CDH15 knockout mice (CDH15") were generated. While abolishing CDH15 expression did not appear to affect MuSC quiescence, proliferation, or differentiation, CDH I 5 ' mice demonstrated accelerated muscle regeneration following injury. As shown in Figures 2C-2D, CDH15" ' mice exhibited increased myofiber size 5- and 15-days post-injury via injection with cardiotoxin (CTX) into the tibialis anterior muscle, and an increased number of central myonuclei in myofibers 15-days post-injury. Moreover, CDH15 ' mice demonstrated improved functional recovery following CTX injury, as evidenced by normal tetanic force measurements in ex vivo isolated extensor digitorum longus muscles 15 days following CTX injury (see Figure 3B). Notably, WT mice exhibited a tetanic force loss of approximately 40% in ex vivo isolated muscle 15-days post-injury.
[00364] To determine how abolishing CDH15 in mice results in improved skeletal muscle regeneration, MuSCs derived from WT or CDH15' ' mice were cultured ex vivo with their associated myofibers for 48 hours. As demonstrated in Figures 4A-4B, a greater percentage of multi-cell clusters and a greater number of Pax7+ cells per cluster was observed for cultured CDH15 /_ MuSCs, as compared to WT MuSCs. This result suggests that abolishing CDH15 expression in MuSCs accelerates the transition from quiescence to activation.
[00365] To further explore how abolishing CDH15 expression in MuSCs impacts the expression of other genes that affect muscle regeneration, RNA-seq was performed on FACS- isolated MuSCs from uninjured, healthy mice. As shown in Figure 5A, 132 genes were downregulated and 270 genes were upregulated in CDH15 ’’ MuSCs as compared to WT MuSCs. Of those upregulated genes, a cluster of genes was identified that contain serum response factor (SRF) binding motifs (see Figure 5B), indicating that accelerated activation of MuSCs in the absence of CDH15 may be due to altered SRF signaling.
Example 2: Abolishing CDH15 expression in aged mice restores muscle regeneration
[00366] Muscle stem cells (MuSCs) in aging subjects display delayed activation and reduced motility in vitro), which translates to impaired MuSC mediated repair in vivo. Accordingly, it was hypothesized that CDH15 may be involved in reduction of the muscle regenerative capacity for aged subjects. To test this, homozygous CDH15 knockout mice (CDH15 z ) were generated, aged to 23 months, and injected intramuscularly in the tibialis anterior muscle with cardiotoxin to induce muscular damage (day 0). Fifteen days post-injury (dpi), histology was performed on tibialis anterior muscle samples from the aged (23 month) CDH15 /_ mice and aged (23 month) WT controls (see Figures 6A-6B). As shown in Figures 6C-6D, at 15 dpi aged CDH15'/_ mice exhibited increased myofiber size (Figure 6C) and an increased number of myofibers with 3 or more central nuclei (Figure 6D) as compared to WT mice of the same age. Notably, the myofiber size for aged CDH15’’ mice was comparable to that of younger WT mice (see Figure 6C, dotted line), suggesting that abolishing CDH15 expression may restore the muscle regenerative capacity of aged mice to that of younger mice.
Example 3: Examples of CDH15 antibodies
[00367] Generation of anti-human CDH15 antibodies
[00368] Anti-human CDH15 antibodies are obtained by immunizing a mouse (e.g., an engineered mouse comprising DNA encoding human immunoglobulin heavy and human kappa light chain variable regions, see, e.g., in US Patent No. 7,105,348; US Patent No. 8,642,835; and US 9,622,459, each of which is incorporated herein by reference), with human CDH15.
[00369] Following immunization, splenocytes are harvested from each mouse and either (1) fused with mouse myeloma cells to preserve their viability and form hybridoma cells and screened for human CDH15 specificity, or (2) B-cell sorted (as described in US 2007/0280945A1) using a either a human CDH15 fragment as the sorting reagent that binds and identifies reactive antibodies (antigen-positive B cells).
[00370] Chimeric antibodies to human CDH15 were initially isolated having a human variable region and a mouse constant region using, e.g., VELOCIMMUNE technology as described in US Patent No. 7,105,348; US Patent No. 8,642,835; and US 9,622,459, each of which is incorporated herein by reference.
[00371] In some antibodies, for testing purposes, mouse constant regions were replaced with a desired human constant region, for example wild-type human CH or modified human CH e.g., IgGl, IgG2 or IgG4 isotypes), and light chain constant region (CL), to generate a fully human anti-hCDH15 antibody, or antigen binding portion thereof. While the constant region selected may vary according to specific use, high affinity antigen-binding and target specificity characteristics reside in the variable region.
[00372] Certain biological properties of the example anti-human CDH15 antibodies generated in accordance with the methods of this Example are described in detail in the
Examples set forth below.
[00373] Heavy and Light Chain Variable Region Amino Acid and Nucleic Acid Sequences of anti-hCDH15 antibodies
[00374] Table 1 sets forth sequence identifiers of a nucleic acid (NA) sequence encoding, and in parentheses an amino acid (AA) sequence of, a heavy or light chain variable region
(HCVR or LCVR, respectively), or a heavy or light chain CDR (HCDR and LCDR, respectively) of selected anti-hCDH15 antibodies used to generate the therapeutic anti-hCDH15 proteins disclosed herein. Such sequences are also included below.
SEQ ID NO: 1
CAGGTGCAGCTGGTGGAGTCGGGGGGAGGCGTGGTCCAGCCTGGGAG GTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTATCTAT GTCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGACTGGAGTGGGT GGCAGTTATATCACATGATGGAAATAATAGATACTATGCAGACTCCGT GAAGGGCCGATTCACCATCTCCAGAGACAACTCCAAGAACACGCTGT ATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCTGTATATTACT GTGCGAAAGGGGTAGCAGTGACCCCCTTTGACCACTGGGGCCAGGGA ACCCTGGTCACCGTCTCCTCA
SEQ ID NO: 2
QVQLVESGGGVVQPGRSLRLSCAASGFTFSIYVMHWVRQAPGKGLEWV AVISHDGNNRYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA KGVAVTPFDHWGQGTLVT VS S
SEQ ID NO: 3
GGATTCACCTTCAGTATCTATGTC
SEQ ID NO: 4
GFTFSIYV
SEQ ID NO: 5 ATATCACATGATGGAAATAATAGA
SEQ ID NO: 6
ISHDGNNR
SEQ ID NO: 7
GCGAAAGGGGTAGCAGTGACCCCCTTTGACCAC
SEQ ID NO: 8
AKGVAVTPFDH
SEQ ID NO: 9
GATATTGTGATGACCCAGACTCCACTCTCCTCACCTGTCACCCTTGGAC
AGCCGGCCTCCATCTCCTGCAGGTCTAGTCAAAGCCTCCTACACAGTG
ATGGAAACACCTACTTGAGTTGGCTTCAGCAGCGGCCAGGCCAGCCTC
CAAGACTCCTAATTTATAAGACTTCTAACCGGTTCTCTGGGGTCCCAG
ACAGATTCAGTGGCAGTGGGGCAGGGACAGATTTCACACTGAAAATC
AGCAGGTTGGAAGCTGAGGATGTCGGCATTTATTACTGCATGCAAGCT
ACACAATTTCCGTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAA
A
SEQ ID NO: 10
DIVMTQTPLSSPVTLGQPASISCRSSQSLLHSDGNTYLSWLQQRPGQPPRL
LIYKTSNRFSGVPDRFSGSGAGTDFTLKISRLEAEDVGIYYCMQATQFPWT
FGQGTKVEIK
SEQ ID NO: 11
CAAAGCCTCCTACACAGTGATGGAAACACCTAC
SEQ ID NO: 12
QSLLHSDGNTY
SEQ ID NO: 13
AAGACTTCT SEQ ID NO: 14
KTS
SEQ ID NO: 15
ATGCAAGCTACACAATTTCCGTGGACG
SEQ ID NO: 16
MQATQFPWT
SEQ ID NO: 17
CAGGTGCAGCTGGTGGAGTCGGGGGGAGGCGTGGTCCAGCCTGGGAG
GTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTATCTAT
GTCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGACTGGAGTGGGT
GGCAGTTATATCACATGATGGAAATAATAGATACTATGCAGACTCCGT
GAAGGGCCGATTCACCATCTCCAGAGACAACTCCAAGAACACGCTGT
ATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCTGTATATTACT
GTGCGAAAGGGGTAGCAGTGACCCCCTTTGACCACTGGGGCCAGGGA
ACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTC
CCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTG
GGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGG
AACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTA
CAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC
AGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCC
CAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCC
CATGCCCACCCTGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCT
TCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCC
CTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAG
GTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAG
ACAAAGCCGCGGGAGGAGCAGTTCCAAAGCACGTACCGTGTGGTCAG
CGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACA
AGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCA
TCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTG
CCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTG
CCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGA
GCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTG
GACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTCACCGTGGACAAG
AGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAG
GCTCTGCACAACCACTACACACAGAAGTCCCTCTCCCTGTCTCTGGGT
AAATGA
SEQ ID NO: 18 QVQLVESGGGVVQPGRSLRLSCAASGFTFSIYVMHWVRQAPGKGLEWV
AVISHDGNNRYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA
KGVAVTPFDHWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLV
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKT
YTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTL
MISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYT
LPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS
DGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK*
SEQ ID NO: 19
GATATTGTGATGACCCAGACTCCACTCTCCTCACCTGTCACCCTTGGAC
AGCCGGCCTCCATCTCCTGCAGGTCTAGTCAAAGCCTCCTACACAGTG
ATGGAAACACCTACTTGAGTTGGCTTCAGCAGCGGCCAGGCCAGCCTC
CAAGACTCCTAATTTATAAGACTTCTAACCGGTTCTCTGGGGTCCCAG
ACAGATTCAGTGGCAGTGGGGCAGGGACAGATTTCACACTGAAAATC
AGCAGGTTGGAAGCTGAGGATGTCGGCATTTATTACTGCATGCAAGCT
ACACAATTTCCGTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAA
ACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAG
CAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCT
ATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAA
TCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAG
CACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACG
AGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGC TCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG
SEQ ID NO: 20
DIVMTQTPLSSPVTLGQPASISCRSSQSLLHSDGNTYLSWLQQRPGQPPRL
LIYKTSNRFSGVPDRFSGSGAGTDFTLKISRLEAEDVGIYYCMQATQFPWT
FGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ
WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV
THQGLSSPVTKSFNRGEC*
SEQ ID NO: 21
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAG
GTCCCTGAGACTCTCCTGTGAAGCCTCTGGATTCATCTTCAGTAGTTAT
GGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGATTGGGT
GGCAGTTATTGCATATGACGGAAACAGTAAATACTATGCAGACTCCGT
GAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTAT
ATCTACAAATGAACGGCCTGAGAGCTGAGGACACGGCTCTGTATTACT
GTGCGAGAATCTATTATGGGTCGGGACTTATCTATACGGACTTCTGGG GCCAGGGAACCCTGGTCACCGTCTCCTCA SEQ ID NO: 22
QVQLVESGGGVVQPGRSLRLSCEASGFIFSSYGMHWVRQAPGKGLDWV
AVIAYDGNSKYYADSVKGRFTISRDNSKNTLYLQMNGLRAEDTALYYCA
RIYYGSGLIYTDFWGQGTLVTVSS
SEQ ID NO: 23
GGATTCATCTTCAGTAGTTATGGC
SEQ ID NO: 24
GFIFSSYG
SEQ ID NO: 25
ATTGCATATGACGGAAACAGTAAA
SEQ ID NO: 26
IAYDGNSK
SEQ ID NO: 27
GCGAGAATCTATTATGGGTCGGGACTTATCTATACGGACTTC
SEQ ID NO: 28
ARIYYGSGLIYTDF
SEQ ID NO: 29
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGTATCTGTAGGA
GACAGAGTCACCATCACTTGCCGGGCGAGTCAGGACATTAGCAATTAT
TTAGCCTGGTATCAACAGAAACCAGGGAAAGTTCCTAAGCTCCTGATC
TATGCTGCATCCACTTTGCAATCAGGGGTCCCATCTCGGATCAGTGGC
AGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCT
GAAGATGTTGCAACTTATTACTGTCAAAAGTTTAACAGTGCCCCATTC
ACTTTCGGCCCTGGGACCAAAGTGGATATCAAA SEQ ID NO: 30
DIQMTQSPSSLSVSVGDRVTITCRASQDISNYLAWYQQKPGKVPKLLIYA
ASTLQSGVPSRISGSGSGTDFTLTISSLQPEDVATYYCQKFNSAPFTFGPGT KVDIK
SEQ ID NO: 31
CAGGACATTAGCAATTAT
SEQ ID NO: 32
QDISNY
SEQ ID NO: 33
GCTGCATCC
SEQ ID NO: 34
AAS
SEQ ID NO: 35
CAAAAGTTTAACAGTGCCCCATTCACT
SEQ ID NO: 36
QKFNSAPFT
SEQ ID NO: 37
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAG
GTCCCTGAGACTCTCCTGTGAAGCCTCTGGATTCATCTTCAGTAGTTAT
GGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGATTGGGT
GGCAGTTATTGCATATGACGGAAACAGTAAATACTATGCAGACTCCGT
GAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTAT
ATCTACAAATGAACGGCCTGAGAGCTGAGGACACGGCTCTGTATTACT
GTGCGAGAATCTATTATGGGTCGGGACTTATCTATACGGACTTCTGGG
GCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCAT
CGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAG
CCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGG
TGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGG
CTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCG TGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATC
ACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATAT
GGTCCCCCATGCCCACCCTGCCCAGCACCTGAGTTCCTGGGGGGACCA
TCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCC
GGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGAC
CCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAAT
GCCAAGACAAAGCCGCGGGAGGAGCAGTTCCAAAGCACGTACCGTGT
GGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGG
AGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGA
AAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTAC
ACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCT
GACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTG
GGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCG
TGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTCACCGTGG
ACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGC
ATGAGGCTCTGCACAACCACTACACACAGAAGTCCCTCTCCCTGTCTC
TGGGTAAATGA
SEQ ID NO: 38
QVQLVESGGGVVQPGRSLRLSCEASGFIFSSYGMHWVRQAPGKGLDWV AVIAYDGNSKYYADSVKGRFTISRDNSKNTLYLQMNGLRAEDTALYYCA RIYYGSGLIYTDFWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT KTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPK
DTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQ STYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQ VYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG K*
SEQ ID NO: 39
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGTATCTGTAGGA
GACAGAGTCACCATCACTTGCCGGGCGAGTCAGGACATTAGCAATTAT
TTAGCCTGGTATCAACAGAAACCAGGGAAAGTTCCTAAGCTCCTGATC
TATGCTGCATCCACTTTGCAATCAGGGGTCCCATCTCGGATCAGTGGC
AGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCT
GAAGATGTTGCAACTTATTACTGTCAAAAGTTTAACAGTGCCCCATTC
ACTTTCGGCCCTGGGACCAAAGTGGATATCAAACGAACTGTGGCTGCA
CCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAA
CTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCA
AAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAG
GAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAG
CAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCT ACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAG
AGCTTCAACAGGGGAGAGTGTTAG
SEQ ID NO: 40
DIQMTQSPSSLSVSVGDRVTITCRASQDISNYLAWYQQKPGKVPKLLIYA
ASTLQSGVPSRISGSGSGTDFTLTISSLQPEDVATYYCQKFNSAPFTFGPGT
KVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDN
ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS
SPVTKSFNRGEC*
SEQ ID NO: 41
CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGA
GACCCTGTCCCTCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGTTTAC
TACTGGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGAT
TGGGGAGATCAGTCATACTGGAAGGACCAACTACAACCCGTCCCTCA
AGAGTCGAGTCACCATTTCAGTAGACACGTCCAAGAACCAGTTCTCCC
TGAAGCTGACCTCTGTGACCGCCGCGGACACGGCTGTGTATTACTGTG
CGAGAGGCCGTGTGGCAGCAGCGGGGTTGGACGTCTGGGGCCTAGGG
ACCACGGTCACCGTCTCCTCA
SEQ ID NO: 42
QVQLQQWGAGLLKPSETLSLTCAVYGGSFSVYYWSWIRQPPGKGLEWIG
EISHTGRTNYNPSLKSRVTISVDTSKNQFSLKLTSVTAADTAVYYCARGR
VAAAGLD VWGLGTTVT VS S
SEQ ID NO: 43
GGTGGGTCCTTCAGTGTTTACTAC
SEQ ID NO: 44
GGSFSVYY
SEQ ID NO: 45
ATCAGTCATACTGGAAGGACC
SEQ ID NO: 46
ISHTGRT SEQ ID NO: 47
GCGAGAGGCCGTGTGGCAGCAGCGGGGTTGGACGTC
SEQ ID NO: 48
ARGRVAAAGLDV
SEQ ID NO: 49
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGA
GACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTAT
TTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATC
TATGCTGCATCCAGTTTGCAAAGTGGGGTCCCGTCAAGGTTCAGTGGC
AGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCT
GAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTCCG
ATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA
SEQ ID NO: 50
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAA
SSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTR
LEIK
SEQ ID NO: 51
CAGAGCATTAGCAGCTAT
SEQ ID NO: 52
QSISSY
SEQ ID NO: 53
CAACAGAGTTACAGTACCCCTCCGATCACC
SEQ ID NO: 54
QQSYSTPPIT
SEQ ID NO: 55
CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGA
GACCCTGTCCCTCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGTTTAC TACTGGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGAT
TGGGGAGATCAGTCATACTGGAAGGACCAACTACAACCCGTCCCTCA
AGAGTCGAGTCACCATTTCAGTAGACACGTCCAAGAACCAGTTCTCCC
TGAAGCTGACCTCTGTGACCGCCGCGGACACGGCTGTGTATTACTGTG
CGAGAGGCCGTGTGGCAGCAGCGGGGTTGGACGTCTGGGGCCTAGGG
ACCACGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTC
CCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTG
GGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGG
AACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTA
CAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC
AGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCC
CAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCC
CATGCCCACCCTGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCT
TCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCC
CTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAG
GTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAG
ACAAAGCCGCGGGAGGAGCAGTTCCAAAGCACGTACCGTGTGGTCAG
CGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACA
AGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCA
TCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTG
CCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTG
CCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGA
GCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTG
GACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTCACCGTGGACAAG
AGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAG
GCTCTGCACAACCACTACACACAGAAGTCCCTCTCCCTGTCTCTGGGT
AAATGA
SEQ ID NO: 56
QVQLQQWGAGLLKPSETLSLTCAVYGGSFSVYYWSWIRQPPGKGLEWIG EISHTGRTNYNPSLKSRVTISVDTSKNQFSLKLTSVTAADTAVYYCARGR
VAAAGLDVWGLGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLS S VVTVPS S SLGTKTYT CNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRV VSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLP PSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK*
SEQ ID NO: 57
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGA GACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTAT
TTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATC TATGCTGCATCCAGTTTGCAAAGTGGGGTCCCGTCAAGGTTCAGTGGC AGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCT
GAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTCCG
ATCACCTTCGGCCAAGGGACACGACTGGAGATTAAACGAACTGTGGCT
GCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTG
GAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGG
CCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCC
CAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCT
CAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAG
TCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAA
AGAGCTTCAACAGGGGAGAGTGTTAG
SEQ ID NO: 58
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAA
SSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTR
LEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA
LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS
PVTKSFNRGEC*
SEQ ID NO: 59
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGG
GTCCCTGAAAATCTCCTGTGCAACTTCTGGATTCACCTTCGGTGGCTCT
GATATGCACTGGGTCCGCCAGGCTTCCGGGAAAGGGCTGGAGTGGGTT
GGCCGCATTTCAAAGAATGCAAATACTCACGCGGCAGAATATGGTGC
GTCGGTGAAAGGCAGATTCACCATCTCCAGAGATGATTCAAAGAACA
CGGCGTTTCTGCAATTGAGCAGCCTGAAAACCGAGGACACGGCCGTCT
ATTATTGTACTAGAGAATATAGTGGGAGTTACCCCCTGTACTTCTACG
GTATGGACTTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA
SEQ ID NO: 60
EVQLVESGGGLVQPGGSLKISCATSGFTFGGSDMHWVRQASGKGLEWV GRISKNANTHAAEYGASVKGRFTISRDDSKNTAFLQLSSLKTEDTAVYYC TREYSGS YPL YF YGMDF WGQGTTVT VS S
SEQ ID NO: 61
GGATTCACCTTCGGTGGCTCTGAT
SEQ ID NO: 62
GFTFGGSD SEQ ID NO: 63
ATTTCAAAGAATGCAAATACTCACGCGGCA
SEQ ID NO: 64
ISKNANTHAA
SEQ ID NO: 65
ACTAGAGAATATAGTGGGAGTTACCCCCTGTACTTCTACGGTATGGAC
TTC
SEQ ID NO: 66
TREYSGSYPLYFYGMDF
SEQ ID NO: 67
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGG
GTCCCTGAAAATCTCCTGTGCAACTTCTGGATTCACCTTCGGTGGCTCT
GATATGCACTGGGTCCGCCAGGCTTCCGGGAAAGGGCTGGAGTGGGTT
GGCCGCATTTCAAAGAATGCAAATACTCACGCGGCAGAATATGGTGC
GTCGGTGAAAGGCAGATTCACCATCTCCAGAGATGATTCAAAGAACA
CGGCGTTTCTGCAATTGAGCAGCCTGAAAACCGAGGACACGGCCGTCT
ATTATTGTACTAGAGAATATAGTGGGAGTTACCCCCTGTACTTCTACG
GTATGGACTTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCCT
CCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCA
CCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCC
CCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGC
GTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCA
GCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTAC
ACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAG
AGTTGAGTCCAAATATGGTCCCCCATGCCCACCCTGCCCAGCACCTGA
GTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGA
CACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGA
CGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATG
GCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTC
CAAAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGAC
TGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCT
CCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCC
GAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACC
AAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGC
GACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTA
CAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTAC AGCAGGCTCACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTT
CTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAA
GTCCCTCTCCCTGTCTCTGGGTAAATGA
SEQ ID NO: 68
EVQLVESGGGLVQPGGSLKISCATSGFTFGGSDMHWVRQASGKGLEWV
GRISKNANTHAAEYGASVKGRFTISRDDSKNTAFLQLSSLKTEDTAVYYC
TREYSGSYPLYFYGMDFWGQGTTVTVSSASTKGPSVFPLAPCSRSTSEST
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP
SSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLF
PPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPR
EEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQ
PREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK
TTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLS
LSLGK*
SEQ ID NO: 69
GAGGTGCAGCTGGTGGAGTCTGGGGGAGTCGTGGTCCAGCCTGGGGG
ATCCCTGCGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGATAT
GCTATGCACTGGGTCCGCCAGGCTCCAGGGAAGGGACTGGATTATGTT
TCAGCTATTACTACTGATGGGGGAAGTACTTTTTATGCAGACTCTGTG
AAGGGCAGATTCACCATTTCCAGAGACAATTCCAAGAATATGGTATAT
CTTCAAATGGGCAGCCTGAAACCTGAAGACATGGCTGTGTATTATTGT
GCGAGAGATCGGCACTGGGGCTACTGGTACTTCGATCTCTGGGGCCGT
GGCACCCTGGTCACCGTCTCCTCA
SEQ ID NO: 70
EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYAMHWVRQAPGKGLDYV
SAITTDGGSTFYADSVKGRFTISRDNSKNMVYLQMGSLKPEDMAVYYCA
RDRHWGYWYFDLWGRGTLVTVSS
SEQ ID NO: 71
GGATTCACCTTCAGTAGATATGCT
SEQ ID NO: 72
GFTFSRYA
SEQ ID NO: 73
ATTACTACTGATGGGGGAAGTACT SEQ ID NO: 74
ITTDGGST
SEQ ID NO: 75
GCGAGAGATCGGCACTGGGGCTACTGGTACTTCGATCTC
SEQ ID NO: 76
ARDRHWGYWYFDL
SEQ ID NO: 77
GAGGTGCAGCTGGTGGAGTCTGGGGGAGTCGTGGTCCAGCCTGGGGG
ATCCCTGCGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGATAT
GCTATGCACTGGGTCCGCCAGGCTCCAGGGAAGGGACTGGATTATGTT
TCAGCTATTACTACTGATGGGGGAAGTACTTTTTATGCAGACTCTGTG
AAGGGCAGATTCACCATTTCCAGAGACAATTCCAAGAATATGGTATAT
CTTCAAATGGGCAGCCTGAAACCTGAAGACATGGCTGTGTATTATTGT
GCGAGAGATCGGCACTGGGGCTACTGGTACTTCGATCTCTGGGGCCGT
GGCACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTC
TTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCC
CTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCG
TGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTC
CTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCC
TCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAA
GCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTC
CCCCATGCCCACCCTGCCCAGCACCTGAGTTCCTGGGGGGACCATCAG
TCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGA
CCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCC
GAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCC
AAGACAAAGCCGCGGGAGGAGCAGTTCCAAAGCACGTACCGTGTGGT
CAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGT
ACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAA
ACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACAC
CCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGA
CCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGG
AGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTG
CTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTCACCGTGGAC
AAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCAT
GAGGCTCTGCACAACCACTACACACAGAAGTCCCTCTCCCTGTCTCTG
GGTAAATGA SEQ ID NO: 78
EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYAMHWVRQAPGKGLDYV SAITTDGGSTFYADSVKGRFTISRDNSKNMVYLQMGSLKPEDMAVYYCA RDRHWGYWYFDLWGRGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALG CLVKD YFPEP VTVS WNSGALT SGVHTFP AVLQ S SGL YSLS S VVT VPS S SLG TKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQ STYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQ VYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV
LDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG K*
SEQ ID NO: 79
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTCGGTACAGCCTGGGGG
GTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGTAGCTAT
GGCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTAAAATGGGT
CTCAATTATTAGTGCTAGTGGTGGTAGAACATACTACGCAGACTCCGT
GAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGT
ATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACT
GTGCGGGGAATATAGTGGCCTTTGACTACTGGGGCCAGGGAACCCTGG
TCACTGTCTCCTCA
SEQ ID NO: 80
EVQLVESGGGSVQPGGSLRLSCAASGFTFSSYGMSWVRQAPGKGLKWVS
IISASGGRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGN IVAFD YWGQGTL VT VS S
SEQ ID NO: 81
GGATTCACCTTTAGTAGCTATGGC
SEQ ID NO: 82
GFTFSSYG
SEQ ID NO: 83
ATTAGTGCTAGTGGTGGTAGAACA
SEQ ID NO: 84
ISASGGRT SEQ ID NO: 85
GCGGGGAATATAGTGGCCTTTGACTAC
SEQ ID NO: 86
AGNIVAFDY
SEQ ID NO: 87
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTCGGTACAGCCTGGGGG
GTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGTAGCTAT
GGCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTAAAATGGGT
CTCAATTATTAGTGCTAGTGGTGGTAGAACATACTACGCAGACTCCGT
GAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGT
ATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACT
GTGCGGGGAATATAGTGGCCTTTGACTACTGGGGCCAGGGAACCCTGG
TCACTGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGC
GCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCT
GGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGG
CGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTC
AGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTT
GGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACA
CCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCA
CCCTGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTC
CCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTC
ACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTT
CAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGC
CGCGGGAGGAGCAGTTCCAAAGCACGTACCGTGTGGTCAGCGTCCTC
ACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAA
GGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAA
AGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCAT
CCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTC
AAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGG
GCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCG
ACGGCTCCTTCTTCCTCTACAGCAGGCTCACCGTGGACAAGAGCAGGT
GGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGC
ACAACCACTACACACAGAAGTCCCTCTCCCTGTCTCTGGGTAAATGA
SEQ ID NO: 88
EVQLVESGGGSVQPGGSLRLSCAASGFTFSSYGMSWVRQAPGKGLKWVS
IISASGGRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGN
IVAFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFP EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCN VDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISR TPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVV
SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPP
SQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS
FFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK*
SEQ ID NO: 89
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGA
GTCCCTAAAACTCTCCTGTGCAGCCTCTGGGTTCGCCTTCAGTGTCTCT
ATTATACACTGGGTCCGCCAGAGTTCCGGGAAAGGGCTGGAGTGGATT
GGCCGTATCAGAACCAAAACTAATAATTACGCGACAGGATATAGTGA
GTCGATGAAGGGCAGGTTCACCATTTCCAGAGATGATTCAGAGAACAC
GGCGTATCTGCAAATGAACAGCCTCAAAACCGAAGACACGGCCGTAT ATTATTGTACTAGACATTATTATAGCAGCACCTTTTACTACTACTACGG AATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA
SEQ ID NO: 90
EVQLVESGGGLVQPGESLKLSCAASGFAFSVSIIHWVRQSSGKGLEWIGRI
RTKTNNYATGYSESMKGRFTISRDDSENTAYLQMNSLKTEDTAVYYCTR
HYYS STF YYYYGMD VWGQGTT VTVS S
SEQ ID NO: 91
GGGTTCGCCTTCAGTGTCTCTATT
SEQ ID NO: 92
GFAFSVSI
SEQ ID NO: 93
ATCAGAACCAAAACTAATAATTACGCGACA
SEQ ID NO: 94
IRTKTNNYAT
SEQ ID NO: 95
ACTAGACATTATTATAGCAGCACCTTTTACTACTACTACGGAATGGAC
GTC SEQ ID NO: 96
TRHYYSSTFYYYYGMDV
SEQ ID NO: 97
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTTGGA
GACAGAGTCATCATCACTTGCCGGGCAAGTCAGGGCATTAGAAATGAT
TTAGGCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCGCCTGAT
CTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGG
CAGTGGATCTGGGACAGAATTCACTCTCACAATCAGCAGCCTGCAGCC
TGAAGATTTTGCAACTTATTACTGTCTACAGCATAATAGTTACCCGATC
ACCTTCGGCCAAGGGACACGACTGGAGATTAAA
SEQ ID NO: 98
DIQMTQSPSSLSASVGDRVIITCRASQGIRNDLGWYQQKPGKAPKRLIYAA
SSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPITFGQGTR
LEIK
SEQ ID NO: 99
CAGGGCATTAGAAATGAT
SEQ ID NO: 100
QGIRND
SEQ ID NO: 101
CTACAGCATAATAGTTACCCGATCACC
SEQ ID NO: 102
LQHNSYPIT
SEQ ID NO: 103
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGA
GTCCCTAAAACTCTCCTGTGCAGCCTCTGGGTTCGCCTTCAGTGTCTCT
ATTATACACTGGGTCCGCCAGAGTTCCGGGAAAGGGCTGGAGTGGATT
GGCCGTATCAGAACCAAAACTAATAATTACGCGACAGGATATAGTGA
GTCGATGAAGGGCAGGTTCACCATTTCCAGAGATGATTCAGAGAACAC GGCGTATCTGCAAATGAACAGCCTCAAAACCGAAGACACGGCCGTAT
ATTATTGTACTAGACATTATTATAGCAGCACCTTTTACTACTACTACGG
AATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCCTC
CACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCAC
CTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCC
CGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCG
TGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAG
CAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACAC
CTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAG
TTGAGTCCAAATATGGTCCCCCATGCCCACCCTGCCCAGCACCTGAGT
TCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACA
CTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACG
TGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGC
GTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCCA
AAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTG
GCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCC
CGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGA
GAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAA
GAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGA
CATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACA
AGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACA
GCAGGCTCACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTC
TCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAG
TCCCTCTCCCTGTCTCTGGGTAAATGA
SEQ ID NO: 104
EVQLVESGGGLVQPGESLKLSCAASGFAFSVSIIHWVRQSSGKGLEWIGRI
RTKTNNYATGYSESMKGRFTISRDDSENTAYLQMNSLKTEDTAVYYCTR
HYYSSTFYYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAA
LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS
LGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPK
PKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQ
FQSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPRE
PQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
PVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSL
GK*
SEQ ID NO: 105
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTTGGA
GACAGAGTCATCATCACTTGCCGGGCAAGTCAGGGCATTAGAAATGAT
TTAGGCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCGCCTGAT
CTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGG
CAGTGGATCTGGGACAGAATTCACTCTCACAATCAGCAGCCTGCAGCC
TGAAGATTTTGCAACTTATTACTGTCTACAGCATAATAGTTACCCGATC ACCTTCGGCCAAGGGACACGACTGGAGATTAAACGAACTGTGGCTGC
ACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGA
ACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCC
AAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCA
GGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCA
GCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTC
TACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAG
AGCTTCAACAGGGGAGAGTGTTAG
SEQ ID NO: 106
DIQMTQSPSSLSASVGDRVIITCRASQGIRNDLGWYQQKPGKAPKRLIYAA
SSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPITFGQGTR
LEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA
LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS
PVTKSFNRGEC*
SEQ ID NO: 107
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAC
GTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGCAGCTAT
GGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTAGAATGGGT
GGCAGTTACATCATATGATGGAGGTTATAAATTCTATGCAGACTCCGT
GAAGGACCGATTCATCATTTCCAGAGACAATTCCAAGAATACGCTGTA
TCTGCAAATGAACAGCCTGAGACCTGATGATACGGCTGTGTATTTCTG
TGCGAAAGATCTGTGGATAAAGTTATGGTTACTTGACTACTGGGGCCA
GGGAACCCTGGTCACCGTCTCCTCA
SEQ ID NO: 108
QVQLVESGGGVVQPGTSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWV
AVTSYDGGYKFYADSVKDRFIISRDNSKNTLYLQMNSLRPDDTAVYFCA
KDLWKLWLLD YWGQGTL VTVS S
SEQ ID NO: 109
GGATTCACCTTCAGCAGCTATGGC
SEQ ID NO: 110
ACATCATATGATGGAGGTTATAAA
SEQ ID NO: 111
TSYDGGYK SEQIDNO: 112
GCGAAAGATCTGTGGATAAAGTTATGGTTACTTGACTAC
SEQIDNO: 113
AKDLWKLWLLDY
SEQIDNO: 114
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGA
GACAGAGTCACCATCACTTGCCGGGCGAGTCAGGGCATTAACAATTAT
TTAGCCTGGTATCAGCAGAAACCAGGGAAAGTTACTAAGCTCCTGATC
TACGCTGCATCCACTTTGCATTCAGGGGTCCCATCTCGGTTCAGTGGCA
GTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTG
AAGATGTTGCAGCTTATTACTGTCAAAAGTATAACAGTGCCCCGCTCA
CTTTCGGCGGAGGGACCAAGGTGGAGATCAAA
SEQIDNO: 115
DIQMTQSPSSLSASVGDRVTITCRASQGINNYLAWYQQKPGKVTKLLIYA
ASTLHSGVPSRFSGSGSGTDFTLTISSLQPEDVAAYYCQKYNSAPLTFGGG TKVEIK
SEQIDNO: 116
CAGGGCATTAACAATTAT
SEQIDNO: 117
QGINNY
SEQIDNO: 118
CAAAAGTATAACAGTGCCCCGCTCACT
SEQIDNO: 119
QKYNSAPLT SEQ ID NO: 120
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAC
GTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGCAGCTAT
GGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTAGAATGGGT
GGCAGTTACATCATATGATGGAGGTTATAAATTCTATGCAGACTCCGT
GAAGGACCGATTCATCATTTCCAGAGACAATTCCAAGAATACGCTGTA
TCTGCAAATGAACAGCCTGAGACCTGATGATACGGCTGTGTATTTCTG
TGCGAAAGATCTGTGGATAAAGTTATGGTTACTTGACTACTGGGGCCA
GGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGT
CTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGC
CCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTC
GTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGT
CCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCC
CTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACA
AGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGT
CCCCCATGCCCACCCTGCCCAGCACCTGAGTTCCTGGGGGGACCATCA
GTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGG
ACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCC
CGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGC
CAAGACAAAGCCGCGGGAGGAGCAGTTCCAAAGCACGTACCGTGTGG
TCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGT
ACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAA
ACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACAC
CCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGA
CCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGG
AGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTG
CTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTCACCGTGGAC
AAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCAT
GAGGCTCTGCACAACCACTACACACAGAAGTCCCTCTCCCTGTCTCTG
GGTAAATGA
SEQ ID NO: 121
QVQLVESGGGVVQPGTSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWV
AVTSYDGGYKFYADSVKDRFIISRDNSKNTLYLQMNSLRPDDTAVYFCA
KDLWIKLWLLDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGC
LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
KTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPK
DTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQ
STYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQ
VYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV
LDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG K* SEQ ID NO: 122
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGA
GACAGAGTCACCATCACTTGCCGGGCGAGTCAGGGCATTAACAATTAT
TTAGCCTGGTATCAGCAGAAACCAGGGAAAGTTACTAAGCTCCTGATC
TACGCTGCATCCACTTTGCATTCAGGGGTCCCATCTCGGTTCAGTGGCA
GTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTG
AAGATGTTGCAGCTTATTACTGTCAAAAGTATAACAGTGCCCCGCTCA
CTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGAACTGTGGCTGCA
CCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAA
CTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCA
AAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAG
GAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAG
CAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCT
ACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAG
AGCTTCAACAGGGGAGAGTGTTAG
SEQ ID NO: 123
DIQMTQSPSSLSASVGDRVTITCRASQGINNYLAWYQQKPGKVTKLLIYA
ASTLHSGVPSRFSGSGSGTDFTLTISSLQPEDVAAYYCQKYNSAPLTFGGG
TKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD
NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG
LSSPVTKSFNRGEC*
SEQ ID NO: 124
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTAAAGCCTGGGGG
GTCCCTTAGACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAACGCC
TGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGT
TGGCCGTATTAAAAGCAAAACTGATGGTGGGACAACAGACTACGCTG
CACCCGTGAAAGGCAGATTCACCATCTCAAGAGATGATTCAAAAAAC
ACGCTGTATCTGCAAATGAACAGCCTGAAAACCGAGGACACAGCCGT
GTATTACTGTACCACGGGTATAGGGGACTACTGGGGCCAGGGAACCCT
GGTCACCGTCTCCTCA
SEQ ID NO: 125
EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWV
GRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYY
CTTGIGDYWGQGTLVTVSS
SEQ ID NO: 126
GGATTCACTTTCAGTAACGCCTGG SEQIDNO: 127
GFTFSNAW
SEQIDNO: 128
ATTAAAAGCAAAACTGATGGTGGGACAACA
SEQIDNO: 129
IKSKTDGGTT
SEQIDNO: 130
ACCACGGGTATAGGGGACTAC
SEQIDNO: 131
TTGIGDY
SEQIDNO: 132
GACATCGTGAEGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGC
GAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTATATAGG
TCCAACAATAAGAACAACTTAGCTTGGTACCAGCAGAAACCAGGACA
GCCTCCTAAGATGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGT
CCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCAC
CATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAGCA
ATATTATAGTACTCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGAT
CAAA
SEQIDNO: 133
DIVMTQSPDSLAVSLGERATINCKSSQSVLYRSNNKNNLAWYQQKPGQPP
KMLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYST
PLTFGGGTKVEIK
SEQIDNO: 134
CAGAGTGTTTTATATAGGTCCAACAATAAGAACAAC
SEQIDNO: 135
QSVLYRSNNKNN SEQ ID NO: 136
TGGGCATCT
SEQ ID NO: 137
WAS
SEQ ID NO: 138
CAGCAATATTATAGTACTCCGCTCACT
SEQ ID NO: 139
QQYYSTPLT
SEQ ID NO: 140
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTAAAGCCTGGGGG
GTCCCTTAGACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAACGCC
TGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGT
TGGCCGTATTAAAAGCAAAACTGATGGTGGGACAACAGACTACGCTG
CACCCGTGAAAGGCAGATTCACCATCTCAAGAGATGATTCAAAAAAC
ACGCTGTATCTGCAAATGAACAGCCTGAAAACCGAGGACACAGCCGT
GTATTACTGTACCACGGGTATAGGGGACTACTGGGGCCAGGGAACCCT
GGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCT
GGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCT
GCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACT
CAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGT
CCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCA
GCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGC
AACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATG
CCCACCCTGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCT
GTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGA
GGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCC
AGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACA
AAGCCGCGGGAGGAGCAGTTCCAAAGCACGTACCGTGTGGTCAGCGT
CCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGT
GCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCT
CCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCC
CCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCT
GGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCA
ATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGAC TCCGACGGCTCCTTCTTCCTCTACAGCAGGCTCACCGTGGACAAGAGC
AGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCT
CTGCACAACCACTACACACAGAAGTCCCTCTCCCTGTCTCTGGGTAAA TGA
SEQ ID NO: 141
EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWV
GRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYY
CTTGIGDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKD
YFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLS S VVTVPS S SLGTKTYT CNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMI
SRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRV
VSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLP
PSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK*
SEQ ID NO: 142
GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGC
GAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTATATAGG
TCCAACAATAAGAACAACTTAGCTTGGTACCAGCAGAAACCAGGACA
GCCTCCTAAGATGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGT
CCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCAC
CATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAGCA
ATATTATAGTACTCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGAT
CAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGAT
GAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAAC
TTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTC
CAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGA
CAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACT
ACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTG AGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG
SEQ ID NO: 143
DIVMTQSPDSLAVSLGERATINCKSSQSVLYRSNNKNNLAWYQQKPGQPP
KMLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYST
PLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK
VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC
EVTHQGLSSPVTKSFNRGEC*
SEQ ID NO: 144
CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGA
GACCCTGTCCCTCACCTGCACTGTCTCTGGAGGCTCCATCAGTAGTTAC TACTGGAGCTGGATCCGGCAGTCCCCAGGGAAGGGACTGGAATGGAT
TGGGTACATCTATTACAGTGGGAGCACCAACTACAACCCCTCCCTCAA
GAGTCGAGTCACCATATCAGTCGACACGTCCAAGAACCAGTTCTCCCT
GAAGCTGACCTCTGTGACCGCCGCCGACACGGCCGTGTATTACTGTGC
GCGAAATGGTGGATATAGTGGCTACGATGACTACTGGGGCCAGGGAA
CCCTGGTCACCGTCTCCTCA
SEQ ID NO: 145
QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQSPGKGLEWIGYI
YYSGSTNYNPSLKSRVTISVDTSKNQFSLKLTSVTAADTAVYYCARNGGY
SGYDDYWGQGTLVTVS S
SEQ ID NO: 146
GGAGGCTCCATCAGTAGTTACTAC
SEQ ID NO: 147
GGSISSYY
SEQ ID NO: 148
ATCTATTACAGTGGGAGCACC
SEQ ID NO: 149
IYYSGST
SEQ ID NO: 150
GCGCGAAATGGTGGATATAGTGGCTACGATGACTAC
SEQ ID NO: 151
ARNGGYSGYDDY
SEQ ID NO: 152
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTCGGA
GACAGAGTCACCATCACTTGCCGGGCAAGTCAGACCATTAGTAGTTAT
TTAAATTGGTATCAGCAGAAAGCAGGGAAGGCCCCTAAGCTCCTAATC
TATTCTGCATCCAGTTTGCAAACTGGGGTCCCATCAAGGTTCAGTGGC AGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCT
GAAGATTTCTCAACTTACTACTGTCAACAGACTTACAGTGTCCCTCGG
ACGTTCGGCCAGGGGACCAAGGTGGAAATCAAA
SEQ ID NO: 153
DIQMTQSPSSLSASVGDRVTITCRASQTISSYLNWYQQKAGKAPKLLIYSA SSLQTGVPSRFSGSGSGTDFTLTISSLQPEDFSTYYCQQTYSVPRTFGQGTK VEIK
SEQ ID NO: 154
CAGACCATTAGTAGTTAT
SEQ ID NO: 155
QTISSY
SEQ ID NO: 156
TCTGCATCC
SEQ ID NO: 157
SAS
SEQ ID NO: 158
CAACAGACTTACAGTGTCCCTCGGACG
SEQ ID NO: 159
QQTYSVPRT
SEQ ID NO: 160
CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGA
GACCCTGTCCCTCACCTGCACTGTCTCTGGAGGCTCCATCAGTAGTTAC
TACTGGAGCTGGATCCGGCAGTCCCCAGGGAAGGGACTGGAATGGAT
TGGGTACATCTATTACAGTGGGAGCACCAACTACAACCCCTCCCTCAA
GAGTCGAGTCACCATATCAGTCGACACGTCCAAGAACCAGTTCTCCCT
GAAGCTGACCTCTGTGACCGCCGCCGACACGGCCGTGTATTACTGTGC GCGAAATGGTGGATATAGTGGCTACGATGACTACTGGGGCCAGGGAA CCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCC
CCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGG
CTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAA
CTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACA
GTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAG
CAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCA
GCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCA
TGCCCACCCTGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTC
CTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCT
GAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGT
CCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGAC
AAAGCCGCGGGAGGAGCAGTTCCAAAGCACGTACCGTGTGGTCAGCG
TCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGT
GCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCT
CCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCC
CCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCT
GGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCA
ATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGAC
TCCGACGGCTCCTTCTTCCTCTACAGCAGGCTCACCGTGGACAAGAGC
AGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCT
CTGCACAACCACTACACACAGAAGTCCCTCTCCCTGTCTCTGGGTAAA
TGA
SEQ ID NO: 161
QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQSPGKGLEWIGYI
YYSGSTNYNPSLKSRVTISVDTSKNQFSLKLTSVTAADTAVYYCARNGGY
SGYDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYF
PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCN
VDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISR
TPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVV
SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPP
SQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTEPPVLDSDGS
FFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK*
SEQ ID NO: 162
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTCGGA
GACAGAGTCACCATCACTTGCCGGGCAAGTCAGACCATTAGTAGTTAT
TTAAATTGGTATCAGCAGAAAGCAGGGAAGGCCCCTAAGCTCCTAATC
TATTCFGCATCCAGTTTGCAAACTGGGGTCCCATCAAGGTTCAGTGGC
AGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCT
GAAGATTTCTCAACTTACTACFGTCAACAGACTTACAGTGTCCCTCGG
ACGTTCGGCCAGGGGACCAAGGTGGAAATCAAACGAACTGTGGCTGC
ACCATCTGFCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCFGGA
ACTGCCTCTGTTGTGTGCCTGCTGAATAACTFCTATCCCAGAGAGGCC AAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCA
GGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCA
GCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTC
TACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAG
AGCTTCAACAGGGGAGAGTGTTAG
SEQ ID NO: 163
DIQMTQSPSSLSASVGDRVTITCRASQTISSYLNWYQQKAGKAPKLLIYSA SSLQTGVPSRFSGSGSGTDFTLTISSLQPEDFSTYYCQQTYSVPRTFGQGTK VEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC*
SEQ ID NO: 164
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAG
GTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTACCTTT
GGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGT
GGCACTTATATCATATGATGGAAGTTATAAATACTATGCAGGCTCCGT
GAAGGGCCGATTCACCATCTCCAGAGACACTTCCAAGAACACACTGTT
TCTGCAAATGGACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTG
TGCGAAAGATCGGGACAGTAACTACTTTGACTACTGGGGCCAGGGAA
CCCTGGTCACTGTCTCCTCA
SEQ ID NO: 165
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTFGMHWVRQAPGKGLEWV
ALISYDGSYKYYAGSVKGRFTISRDTSKNTLFLQMDSLRAEDTAVYYCAK
DRDSNYFDYWGQGTLVTVSS
SEQ ID NO: 166
GGATTCACCTTCAGTACCTTTGGC
SEQ ID NO: 167
GFTFSTFG
SEQ ID NO: 168
ATATCATATGATGGAAGTTATAAA SEQIDNO: 169
ISYDGSYK
SEQIDNO: 170
GCGAAAGATCGGGACAGTAACTACTTTGACTAC
SEQIDNO: 171
AKDRDSNYFDY
SEQIDNO: 172
GATATTGTGATGACCCAGACTCCACTCTCCTCACCTGTCACCCTTGGAC
AGCCGGCCTCCATCTCCTGCCGGTCTAGTCAAAGCCTCGTACACAGTA
ATGGAAACACCTACTTGAGTTGGCTTCAGCAGAGGCCAGGCCAGCCTC
CAAGACTCCTGATTTATAAGATCTCTAACCGGTTCTCTGGGGTCCCAG
ACAGATTCAGTGGCAGTGGGACAGGGACAGATTTCACACTGAAAATC
AGCAGGGTGGAACCTGAGGATGTCGGGACTTATTACTGCATGCAAGCT
ACACATTTTCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA
SEQIDNO: 173
DIVMTQTPLSSPVTLGQPASISCRSSQSLVHSNGNTYLSWLQQRPGQPPRL
LIYKISNRFSGVPDRFSGSGTGTDFTLKISRVEPEDVGTYYCMQATHFPLTF
GGGTKVEIK
SEQIDNO: 174
CAAAGCCTCGTACACAGTAATGGAAACACCTAC
SEQIDNO: 175
QSLVHSNGNTY
SEQIDNO: 176
AAGATCTCT
SEQIDNO: 177
KIS SEQ ID NO: 178
ATGCAAGCTACACATTTTCCTCTCACT
SEQ ID NO: 179
MQATHFPLT
SEQ ID NO: 180
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAG
GTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTACCTTT
GGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGT
GGCACTTATATCATATGATGGAAGTTATAAATACTATGCAGGCTCCGT
GAAGGGCCGATTCACCATCTCCAGAGACACTTCCAAGAACACACTGTT
TCTGCAAATGGACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTG
TGCGAAAGATCGGGACAGTAACTACTTTGACTACTGGGGCCAGGGAA
CCCTGGTCACTGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCC
CCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGG
CTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAA
CTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACA
GTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAG
CAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCA
GCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCA
TGCCCACCCTGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTC
CTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCT
GAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGT
CCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGAC
AAAGCCGCGGGAGGAGCAGTTCCAAAGCACGTACCGTGTGGTCAGCG
TCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGT
GCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCT
CCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCC
CCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCT
GGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCA
ATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGAC
TCCGACGGCTCCTTCTTCCTCTACAGCAGGCTCACCGTGGACAAGAGC
AGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCT
CTGCACAACCACTACACACAGAAGTCCCTCTCCCTGTCTCTGGGTAAA TGA
SEQ ID NO: 181
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTFGMHWVRQAPGKGLEWV
ALISYDGSYKYYAGSVKGRFTISRDTSKNTLFLQMDSLRAEDTAVYYCAK DRDSNYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVK
DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTY
TCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTL
MISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYE
LPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS
DGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK*
SEQ ID NO: 182
GATATTGTGATGACCCAGACTCCACTCTCCTCACCTGTCACCCTTGGAC
AGCCGGCCTCCATCTCCTGCCGGTCTAGTCAAAGCCTCGTACACAGTA
ATGGAAACACCTACTTGAGTTGGCTTCAGCAGAGGCCAGGCCAGCCTC
CAAGACTCCTGATTTATAAGATCTCTAACCGGTTCTCTGGGGTCCCAG
ACAGATTCAGFGGCAGTGGGACAGGGACAGATTECACACTGAAAATC
AGCAGGGTGGAACCTGAGGATGTCGGGACTTATTACTGCATGCAAGCT
ACACATTTTCCTCTCACFTTCGGCGGAGGGACCAAGGTGGAGATCAAA
CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGC
AGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTA
TCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAAT
CGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGC
ACCTACAGCCFCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGA
GAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCT
CGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG
SEQ ID NO: 183
DIVMTQTPLSSPVTLGQPASISCRSSQSLVHSNGNTYLSWLQQRPGQPPRL LIYKISNRFSGVPDRFSGSGTGTDFTLKISRVEPEDVGTYYCMQAFHFPLTF GGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT HQGLSSPVTKSFNRGEC*
SEQ ID NO: 184
CAGGTGCAGCTGGTGGAGTCGGGGGGGGGCGTGGTCCAGCCTGGGAG GTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTACCTAT GGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGT GGCAGTTATCTGGTATGATGGAAGTAATAAATACTATGTAGACTCCGT GAAGGGCCGATTCACCATCTCCAGAGAAAATTCCAAGAACACGCTGT ATCTGCAAAFGAATAGCCTGAGAGCCGAGGACACGGCTGTGTATTACT GTGCGAGAGACGGAGTAGCAGCCCAATACTTFGAGTACTGGGGCCAG GGAACCCTGGTCACTGTCTCCFCA SEQIDNO: 185
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWV
AVIWYDGSNKYYVDSVKGRFTISRENSKNTLYLQMNSLRAEDTAVYYCA RDGVAAQYFEYWGQGTLVTVS S
SEQIDNO: 186
GGATTCACCTTCAGTACCTATGGC
SEQIDNO: 187
GFTFSTYG
SEQIDNO: 188
ATCTGGTATGATGGAAGTAATAAA
SEQIDNO: 189
IWYDGSNK
SEQIDNO: 190
GCGAGAGACGGAGTAGCAGCCCAATACTTTGAGTAC
SEQIDNO: 191
ARDGVAAQYFEY
SEQIDNO: 192
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTGGGA
GACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGTTAT
TTAAATTGGTATCAGCAGAAACTAGGGAAAGCCCCTAAGCTCCTGATC
TCTGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGC
AGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAGCCT
GAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTCGG ACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA SEQ ID NO: 193
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKLGKAPKLLISAA
SSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPRTFGQGTK VEIK
SEQ ID NO: 194
CAGAGCATTAGCAGTTAT
SEQ ID NO: 195
CAACAGAGTTACAGTACCCCTCGGACG
SEQ ID NO: 196
QQSYSTPRT
SEQ ID NO: 197
CAGGTGCAGCTGGTGGAGTCGGGGGGGGGCGTGGTCCAGCCTGGGAG GTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTACCTAT GGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGT GGCAGTTATCTGGTATGATGGAAGTAATAAATACTATGTAGACTCCGT GAAGGGCCGATTCACCATCTCCAGAGAAAATTCCAAGAACACGCTGT ATCTGCAAATGAATAGCCTGAGAGCCGAGGACACGGCTGTGTATTACT GTGCGAGAGACGGAGTAGCAGCCCAATACTTTGAGTACTGGGGCCAG GGAACCCTGGTCACTGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTC TTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCC CTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCG TGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTC CTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCC TCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAA GCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTC CCCCATGCCCACCCTGCCCAGCACCTGAGTTCCTGGGGGGACCATCAG TCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGA CCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCC GAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCC AAGACAAAGCCGCGGGAGGAGCAGTTCCAAAGCACGTACCGTGTGGT CAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGT ACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAA ACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACAC CCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGA CCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGG AGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTG CTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTCACCGTGGAC
AAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCAT
GAGGCTCTGCACAACCACTACACACAGAAGTCCCTCTCCCTGTCTCTG GGTAAATGA
SEQ ID NO: 198
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWV AVIWYDGSNKYYVDSVKGRFTISRENSKNTLYLQMNSLRAEDTAVYYCA RDGVAAQYFEYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT KTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPK
DTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQ STYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQ VYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG K*
SEQ ID NO: 199
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTGGGA
GACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGTTAT
TTAAATTGGTATCAGCAGAAACTAGGGAAAGCCCCTAAGCTCCTGATC
TCTGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGC
AGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAGCCT
GAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTCGG
ACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGAACTGTGGCTGC
ACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGA
ACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCC
AAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCA
GGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCA
GCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTC
TACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAG
AGCTTCAACAGGGGAGAGTGTTAG
SEQ ID NO: 200
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKLGKAPKLLISAA SSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPRTFGQGTK VEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC* SEQ ID NO: 201
CAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCA GACCCTCTCACTCACCTGTGCCATTTCCGGGGACAGTGTCTCTAGCAA CAGTGCTGCTTGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGA GTGGCTGGGAAGGACATACTACATGTCCAAGTGGTATAATGATTATGC AGTATCTGTGAAGAGTCGAATAACCATCAACCCAGATACATCCAAGA
ACCATTTCTCCCTGCAGTTGAACTCTGTGACTCCCGAGGACACGGCTG TGTATTACTGTGCAAGAGATGGGGGGTGGGAGGTATTCTTTGACTACT GGGGCCAGGGAACCCTGGTCACCGTCTCCTCA
SEQ ID NO: 202
QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWL
GRTYYMSKWYNDYAVSVKSRITINPDTSKNHFSLQLNSVTPEDTAVYYC
ARDGGWEVFFDYWGQGTL VTVS S
SEQ ID NO: 203
GGGGACAGTGTCTCTAGCAACAGTGCTGCT
SEQ ID NO: 204
GDSVSSNSAA
SEQ ID NO: 205
ACATACTACATGTCCAAGTGGTATAAT
SEQ ID NO: 206
TYYMSKWYN
SEQ ID NO: 207
GCAAGAGATGGGGGGTGGGAGGTATTCTTTGACTAC
SEQ ID NO: 208
ARDGGWEVFFDY SEQ ID NO: 209
GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGC
GAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTATACAGC
TCCAAGAATCAGAACTACTTAGCTTGGTACCAGCAGAAACCAGGACA
GCCTCCTAAACTACTCATTTACTGGGCATCTACCCGGGAATCCGGGGT
CCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCAC
CATCAGCGGCCTGCAGCCTGAAGATGTGGCAGTTTATTCCTGTCAGCA
ATATTATAGTATTCCGTACACTTTTGGCCAGGGGACCAAGCTGGAGAT CAAA
SEQ ID NO: 210
DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSKNQNYLAWYQQKPGQPP
KLLIYWASTRESGVPDRFSGSGSGTDFTLTISGLQPEDVAVYSCQQYYSIP
YTFGQGTKLEIK
SEQ ID NO: 211
CAGAGTGTTTTATACAGCTCCAAGAATCAGAACTAC
SEQ ID NO: 212
QSVLYSSKNQNY
SEQ ID NO: 213
CAGCAATATTATAGTATTCCGTACACT
SEQ ID NO: 214
QQYYSIPYT
SEQ ID NO: 215
CAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCA
GACCCTCTCACTCACCTGTGCCATTTCCGGGGACAGTGTCTCTAGCAA
CAGTGCTGCTTGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGA
GTGGCTGGGAAGGACATACTACATGTCCAAGTGGTATAATGATTATGC
AGTATCTGTGAAGAGTCGAATAACCATCAACCCAGATACATCCAAGA
ACCATTTCTCCCTGCAGTTGAACTCTGTGACTCCCGAGGACACGGCTG
TGTATTACTGTGCAAGAGATGGGGGGTGGGAGGTATTCTTTGACTACT
GGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCC
CATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCA
CAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGA CGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCC
CGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGA
CCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAG
ATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAA
TATGGTCCCCCATGCCCACCCTGCCCAGCACCTGAGTTCCTGGGGGGA
CCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCT
CCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAA
GACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCAT
AATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCCAAAGCACGTACCG
TGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAA
GGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGA
GAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGT
ACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGC
CTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAG
TGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCC
CGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTCACCGT
GGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGA
TGCATGAGGCTCTGCACAACCACTACACACAGAAGTCCCTCTCCCTGT
CTCTGGGTAAATGA
SEQ ID NO: 216
QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWL GRTYYMSKWYNDYAVSVKSRITINPDTSKNHFSLQLNSVTPEDTAVYYC ARDGGWEVFFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT KTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPK
DTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQ STYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQ VYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG K*
SEQ ID NO: 217
GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGC
GAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTATACAGC
TCCAAGAATCAGAACTACTTAGCTTGGTACCAGCAGAAACCAGGACA
GCCTCCTAAACTACTCATTTACTGGGCATCTACCCGGGAATCCGGGGT
CCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCAC
CATCAGCGGCCTGCAGCCTGAAGATGTGGCAGTTTATTCCTGTCAGCA
ATATTATAGTATTCCGTACACTTTTGGCCAGGGGACCAAGCTGGAGAT
CAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGAT
GAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAAC
TTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTC
CAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGA CAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACT
ACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTG
AGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG
SEQ ID NO: 218
DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSKNQNYLAWYQQKPGQPP
KLLIYWASTRESGVPDRFSGSGSGTDFTLTISGLQPEDVAVYSCQQYYSIP
YTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV
QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLS SPVTKSFNRGEC *
SEQ ID NO: 219
CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCCTCACA
GACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGTAGTGGT
TATTACTACTGGAGTTGGATCCGCCAGCACCCAGGGAAGGGCCTGGAG
TGGATTGGGTTCATCTATTTTAGAGGGAGCACTTACTATGACCCGTCCC
TCAAGAGTCGACTTACCATATCAGTAGACACGTCTAAGAACCAGTTCT
CCCTGAAACTGAGCTCTGTGACTGCCGCGGACACGGCCGTATATTACT
GTGCGAGACGGAGCAGCTCGCCGGGGGCTTTCGATATCTGGGGCCAA GGGACAATGGTCACCGTCTCTTCA
SEQ ID NO: 220
QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGYYYWSWIRQHPGKGLEWI
GFIYFRGSTYYDPSLKSRLTISVDTSKNQFSLKLSSVTAADTAVYYCARRS
S SPGAFDIWGQGTMVTVS S
SEQ ID NO: 221
GGTGGCTCCATCAGTAGTGGTTATTACTAC
SEQ ID NO: 222
GGSISSGYYY
SEQ ID NO: 223
ATCTATTTTAGAGGGAGCACT
SEQ ID NO: 224
IYFRGST SEQ ID NO: 225
GCGAGACGGAGCAGCTCGCCGGGGGCTTTCGATATC
SEQ ID NO: 226
ARRSSSPGAFDI
SEQ ID NO: 227
CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCCTCACA
GACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGTAGTGGT
TATTACTACTGGAGTTGGATCCGCCAGCACCCAGGGAAGGGCCTGGAG
TGGATTGGGTTCATCTATTTTAGAGGGAGCACTTACTATGACCCGTCCC
TCAAGAGTCGACTTACCATATCAGTAGACACGTCTAAGAACCAGTTCT
CCCTGAAACTGAGCTCTGTGACTGCCGCGGACACGGCCGTATATTACT
GTGCGAGACGGAGCAGCTCGCCGGGGGCTTTCGATATCTGGGGCCAA
GGGACAATGGTCACCGTCTCTTCAGCCTCCACCAAGGGCCCATCGGTC
TTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCC
CTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCG
TGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTC
CTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCC
TCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAA
GCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTC
CCCCATGCCCACCCTGCCCAGCACCTGAGTTCCTGGGGGGACCATCAG
TCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGA
CCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCC
GAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCC
AAGACAAAGCCGCGGGAGGAGCAGTTCCAAAGCACGTACCGTGTGGT
CAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGT
ACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAA
ACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACAC
CCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGA
CCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGG
AGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTG
CTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTCACCGTGGAC
AAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCAT
GAGGCTCTGCACAACCACTACACACAGAAGTCCCTCTCCCTGTCTCTG
GGTAAATGA
SEQ ID NO: 228
QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGYYYWSWIRQHPGKGLEWI
GFIYFRGSTYYDPSLKSRLTISVDTSKNQFSLKLSSVTAADTAVYYCARRS SSPGAFDIWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLS S VVTVPS S SLGTKTYT CNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRV VSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLP PSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK*
SEQ ID NO: 229
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTTCAGCCTGGAGG GTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAATTAT GAAATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGACTGGAGTGGGT TTCATACATTGATAATAGTGGTAGTGCCATATATTACGCAGACTCTGT GAAGGGCCGATTCACCCTCTCCAGAGACAACGCCAAGAACTCACTGT ATCTGCAAATGAGCAGCCTGAGAGCCGAGGACACGGCTATTTTTTTCT GTGTGAGAGAAGGAGGATATGACAGCTCGTCCCGGACCTGGAGGGAT
GCTTTTGATATCTGGGGCCAGGGGACAATGGTCACCGTCTCTTCA
SEQ ID NO: 230
EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYEMNWVRQAPGKGLEWV
SYIDNSGSAIYYADSVKGRFTLSRDNAKNSLYLQMSSLRAEDTAIFFCVRE
GGYDSSSRTWRDAFDIWGQGTMVTVSS
SEQ ID NO: 231
GGATTCACCTTCAGTAATTAFGAA
SEQ ID NO: 232
GFTFSNYE
SEQ ID NO: 233
ATTGATAAFAGTGGTAGTGCCATA
SEQ ID NO: 234
IDNSGSAI
SEQ ID NO: 235 GTGAGAGAAGGAGGATATGACAGCTCGTCCCGGACCTGGAGGGATGC
TTTTGATATC
SEQ ID NO: 236
VREGGYDSSSRTWRDAFDI
SEQ ID NO: 237
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTTCAGCCTGGAGG
GTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAATTAT
GAAATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGACTGGAGTGGGT
TTCATACATTGATAATAGTGGTAGTGCCATATATTACGCAGACTCTGT
GAAGGGCCGATTCACCCTCTCCAGAGACAACGCCAAGAACTCACTGT
ATCTGCAAATGAGCAGCCTGAGAGCCGAGGACACGGCTATTTTTTTCT
GTGTGAGAGAAGGAGGATATGACAGCTCGTCCCGGACCTGGAGGGAT
GCTTTTGATATCTGGGGCCAGGGGACAATGGTCACCGTCTCTTCAGCC
TCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGC
ACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTC
CCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGG
CGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTC
AGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTA
CACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGA
GAGTTGAGTCCAAATATGGTCCCCCATGCCCACCCTGCCCAGCACCTG
AGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGG
ACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGG
ACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGAT
GGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTT
CCAAAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGA
CTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCC
TCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCC
GAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACC
AAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGC
GACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTA
CAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTAC
AGCAGGCTCACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTT
CTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAA
GTCCCTCTCCCTGTCTCTGGGTAAATGA
SEQ ID NO: 238
EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYEMNWVRQAPGKGLEWV
SYIDNSGSAIYYADSVKGRFTLSRDNAKNSLYLQMSSLRAEDTAIFFCVRE
GGYDSSSRTWRDAFDIWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTA
ALGOL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS SSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFP
PKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPRE
EQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQP
REPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT
TPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSL SLGK*
SEQ ID NO: 239
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCGGGGGG
GTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCCTCAGTAGGTA
CGACATGTACTGGGTCCGCCAAACTACAGGAAAAGGTCTGGAGTGGG
TCTCAGCTATCAATACTGCTGGTGACACATACTATCCAGGCTCCGTGA
AGGGCCGATTCACCATCTCCAGAGAAAATGCCCAGAACTCCTTATATC
TTCAAATGAACAGCCTGAGAGCCGGGGACACGGCTGTGTATTACTGTG
TAAGAGAGGGGTACATTCGTACAACTGGAACGACGGGATTCTCCTTTG
ACTACTGGGGCCAGGGAACCCTGGTCACTGTCTCCTCA
SEQ ID NO: 240
EVQLVESGGGLVQPGGSLRLSCAASGFTLSRYDMYWVRQTTGKGLEWV
SAINTAGDTYYPGSVKGRFTISRENAQNSLYLQMNSLRAGDTAVYYCVR
EGYIRTTGTTGFSFDYWGQGTLVTVSS
SEQ ID NO: 241
GGATTCACCCTCAGTAGGTACGAC
SEQ ID NO: 242
GFTLSRYD
SEQ ID NO: 243
ATCAATACTGCTGGTGACACA
SEQ ID NO: 244
INTAGDT
SEQ ID NO: 245
GTAAGAGAGGGGTACATTCGTACAACTGGAACGACGGGATTCTCCTTT
GACTAC SEQ ID NO: 246
VREGYIRTTGTTGF SFD Y
SEQ ID NO: 247
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCGGGGGG
GTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCCTCAGTAGGTA
CGACATGTACTGGGTCCGCCAAACTACAGGAAAAGGTCTGGAGTGGG
TCTCAGCTATCAATACTGCTGGTGACACATACTATCCAGGCTCCGTGA
AGGGCCGATTCACCATCTCCAGAGAAAATGCCCAGAACTCCTTATATC
TTCAAATGAACAGCCTGAGAGCCGGGGACACGGCTGTGTATTACTGTG
TAAGAGAGGGGTACATTCGTACAACTGGAACGACGGGATTCTCCTTTG
ACTACTGGGGCCAGGGAACCCTGGTCACTGTCTCCTCAGCCTCCACCA
AGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCG
AGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAAC
CGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCAC
ACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGC
GTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGC
AACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGA
GTCCAAATATGGTCCCCCATGCCCACCCTGCCCAGCACCTGAGTTCCT
GGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCT
CATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAG
CCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGG
AGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCCAAAGC
ACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTG
AACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTC
CTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGC
CACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAAC
CAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATC
GCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGAC
CACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAG
GCTCACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCAT
GCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGTCCC
TCTCCCTGTCTCTGGGTAAATGA
SEQ ID NO: 248
EVQLVESGGGLVQPGGSLRLSCAASGFTLSRYDMYWVRQTTGKGLEWV
SAINTAGDTYYPGSVKGRFTISRENAQNSLYLQMNSLRAGDTAVYYCVR
EGYIRTTGTTGFSFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAA
LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS
LGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPK
PKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQ
FQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPRE PQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
PVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSL GK*
SEQ ID NO: 249
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAG
GTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGTTAT
GGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGAT
GTCAGTTATATCATATGATGGAAGTAATAAATACTATGCAGACTCCGT
GAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGT
ATCTGCAAATGAACAGCTTGAGACCTGAGGACACGGCTGTGTATTACT
GTTCAATAATGAGACGGAACTCTGGTCTATACAGCTCGTTCGACCCCT
GGGGCCAGGGAACCCTGGTCACCGTCTCCTCA
SEQ ID NO: 250
QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWM
SVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCSI MRRNSGLYS SFDPWGQGTLVTVS S
SEQ ID NO: 251
GGATTCACCTTCAGTAGTTATGGC
SEQ ID NO: 252
ATATCATATGATGGAAGTAATAAA
SEQ ID NO: 253
ISYDGSNK
SEQ ID NO: 254
TCAATAATGAGACGGAACTCTGGTCTATACAGCTCGTTCGACCCC
SEQ ID NO: 255
SIMRRNSGLYSSFDP SEQ ID NO: 256
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAG
GTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGTTAT
GGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGAT
GTCAGTTATATCATATGATGGAAGTAATAAATACTATGCAGACTCCGT
GAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGT
ATCTGCAAATGAACAGCTTGAGACCTGAGGACACGGCTGTGTATTACT
GTTCAATAATGAGACGGAACTCTGGTCTATACAGCTCGTTCGACCCCT
GGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCC
CATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCA
CAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGA
CGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCC
CGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGA
CCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAG
ATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAA
TATGGTCCCCCATGCCCACCCTGCCCAGCACCTGAGTTCCTGGGGGGA
CCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCT
CCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAA
GACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCAT
AATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCCAAAGCACGTACCG
TGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAA
GGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGA
GAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGT
ACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGC
CTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAG
TGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCC
CGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTCACCGT
GGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGA
TGCATGAGGCTCTGCACAACCACTACACACAGAAGTCCCTCTCCCTGT
CTCTGGGTAAATGA
SEQ ID NO: 257
QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWM SVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCSI MRRNSGLYSSFDPWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALG CLVKD YFPEP VTVS WNSGALT SGVHTFP AVLQ S SGL YSLS S VVT VPS S SLG TKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQ STYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQ VYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV
LDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG K* SEQ ID NO: 258
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTAAAGTCGGGGGG
GTCCCTTAGACTCTCCTGTGCAGCCTCTGGATTCACTTTCACTAACACC
TGGATGAGCTGGGTCCGCCAGTCTCCAGGGAAGGGGCTGGAGTGGGT
TGGCCGTATTAAAACCAAAACTGATGGTGGGACATCGTACTACGGTTC
ACCCGTGAAAGACAGATTCACCATCTCAAGAGATGATTCAAAAACCA
CGCTGTATCTGCAAATGAACAGTCTGAGAGCCGAAGACACAGCCGTCT
ATTACTGTCTTACAGGACTGGGGGACTACTGGGGCCAGGGAACCCTGG TCACCGTCTCCTCA
SEQ ID NO: 259
EVQLVESGGGLVKSGGSLRLSCAASGFTFTNTWMSWVRQSPGKGLEWV
GRIKTKTDGGTSYYGSPVKDRFTISRDDSKTTLYLQMNSLRAEDTAVYYC
LTGLGD YWGQGTL VTVS S
SEQ ID NO: 260
GGATTCACTTTCACTAACACCTGG
SEQ ID NO: 261
GFTFTNTW
SEQ ID NO: 262
ATTAAAACCAAAACTGATGGTGGGACATCG
SEQ ID NO: 263
IKTKTDGGTS
SEQ ID NO: 264
CTTACAGGACTGGGGGACTAC
SEQ ID NO: 265
LTGLGDY SEQIDNO: 266
GAAATAGTTTTGACACAGAGTCCCGGCACACTGTCACTCTCTCCCGGG
GAAAGAGCCACCTTGTCATGTAGAGCAAGTCAGTCAGTCTCTAGCTCT
TATCTCGCCTGGTACCAGCAGAAGCCGGGACAGGCCCCTAGACTGCTG
ATCTACGGGGCAAGTTCCAGGGCCACCGGAATCCCCGACCGGTTCAGT
GGAAGCGGAAGCGGAACCGATTTTACTTTGACGATTTCTAGACTGGAG
CCAGAGGATTTCGCCGTTTACTATTGTCAACAGTACGGAAGCAGCCCG
TGGACGTTTGGCCAGGGCACGAAGGTAGAAATCAAG
SEQIDNO: 267
EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYG
AS SRATGIPDRF SGSGSGTDFTLTISRLEPEDF AVYYCQQYGS SPWTFGQG
TKVEIK
SEQIDNO: 268
CAGTCAGTCTCTAGCTCTTAT
SEQIDNO: 269
QSVSSSY
SEQIDNO: 270
GGGGCAAGT
SEQIDNO: 271
GAS
SEQIDNO: 272
CAACAGTACGGAAGCAGCCCGTGGACG
SEQIDNO: 273
QQYGSSPWT
SEQIDNO: 274
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTAAAGTCGGGGGG
GTCCCTTAGACTCTCCTGTGCAGCCTCTGGATTCACTTTCACTAACACC TGGATGAGCTGGGTCCGCCAGTCTCCAGGGAAGGGGCTGGAGTGGGT
TGGCCGTATTAAAACCAAAACTGATGGTGGGACATCGTACTACGGTTC
ACCCGTGAAAGACAGATTCACCATCTCAAGAGATGATTCAAAAACCA
CGCTGTATCTGCAAATGAACAGTCTGAGAGCCGAAGACACAGCCGTCT
ATTACTGTCTTACAGGACTGGGGGACTACTGGGGCCAGGGAACCCTGG
TCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGC
GCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCT
GGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGG
CGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTC
AGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTT
GGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACA
CCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCA
CCCTGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTC
CCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTC
ACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTT
CAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGC
CGCGGGAGGAGCAGTTCCAAAGCACGTACCGTGTGGTCAGCGTCCTC
ACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAA
GGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAA
AGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCAT
CCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTC
AAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGG
GCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCG
ACGGCTCCTTCTTCCTCTACAGCAGGCTCACCGTGGACAAGAGCAGGT
GGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGC
ACAACCACTACACACAGAAGTCCCTCTCCCTGTCTCTGGGTAAATGA
SEQ ID NO: 275
EVQLVESGGGLVKSGGSLRLSCAASGFTFTNTWMSWVRQSPGKGLEWV GRIKTKTDGGTSYYGSPVKDRFTISRDDSKTTLYLQMNSLRAEDTAVYYC LTGLGDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLS S VVTVPS S SLGTKTYTC
NVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMIS
RTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRV VSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLP PSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK*
SEQ ID NO: 276
GAAATAGTTTTGACACAGAGTCCCGGCACACTGTCACTCTCTCCCGGG GAAAGAGCCACCTTGTCATGTAGAGCAAGTCAGTCAGTCTCTAGCTCT TATCTCGCCTGGTACCAGCAGAAGCCGGGACAGGCCCCTAGACTGCTG ATCTACGGGGCAAGTTCCAGGGCCACCGGAATCCCCGACCGGTTCAGT GGAAGCGGAAGCGGAACCGATTTTACTTTGACGATTTCTAGACTGGAG CCAGAGGATTTCGCCGTTTACTATTGTCAACAGTACGGAAGCAGCCCG
TGGACGTTTGGCCAGGGCACGAAGGTAGAAATCAAGCGAACTGTGGC
TGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCT
GGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAG
GCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTC
CCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCC
TCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAA
GTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACA
AAGAGCTTCAACAGGGGAGAGTGTTAG
SEQ ID NO: 277
EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYG AS SRATGIPDRF SGSGSGTDFTLTISRLEPEDF AVYYCQQYGS SPWTFGQG TKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC*
SEQ ID NO: 278
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGATACAGCCTGGGGG
GTCCCTGAGACTCTCCTGTGCAGCCTCTGGCTTCACCTTTAGCAGCTAT
GCCATGGGCTGGGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGT
CTCAACTATTACTAGTAGTGGTGGTAGCTCATACTTCGCACACTCCGTG
AAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTAT
CTGCACATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTTCTGT
GCGAAAGATGGCCCTAATTACGATGTTTGGAGTGGTTATTACTGGGGC
CAGGGAACCCTGGTCACCGTCTCCTCA
SEQ ID NO: 279
EVQLVESGGGLIQPGGSLRLSCAASGFTFSSYAMGWVRQAPGKGLEWVS TITSSGGSSYFAHSVKGRFTISRDNSKNTLYLHMNSLRAEDTAVYFCAKD GPNYDVWSGYYWGQGTLVTVS S
SEQ ID NO: 280
GGCTTCACCTTTAGCAGCTATGCC
SEQ ID NO: 281
GFTFSSYA SEQIDNO: 282
ATTACTAGTAGTGGTGGTAGCTCA
SEQIDNO: 283
ITSSGGSS
SEQIDNO: 284
GCGAAAGATGGCCCTAATTACGATGTTTGGAGTGGTTATTAC
SEQIDNO: 285
AKDGPNYDVWSGYY
SEQIDNO: 286
GATATTGTGATGACCCAGTCTCCACTCTCCCTGCCCGTCACACCTGGA
GAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGTATAGT
AATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGACAGTCT
CCACAACTCCTGATCTATCTGGGTTCTAATCGGGCCTCCGGGGTCCCT
GACAGGTTCAGAGGCAGTGGATCAGGCACAGATTTTACACTGAAAAT
CAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGC
TCTACAAACTCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCA
AA
SEQIDNO: 287
DIVMTQSPLSLPVTPGEPASISCRSSQSLLYSNGYNYLDWYLQKPGQSPQL
LIYLGSNRASGVPDRFRGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPR
TFGQGTKVEIK
SEQIDNO: 288
CAGAGCCTCCTGTATAGTAATGGATACAACTAT
SEQIDNO: 289
QSLLYSNGYNY
SEQIDNO: 290
CTGGGTTCT SEQ ID NO: 291
LGS
SEQ ID NO: 292
ATGCAAGCTCTACAAACTCCTCGGACG
SEQ ID NO: 293
MQALQTPRT
SEQ ID NO: 294
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGATACAGCCTGGGGG
GTCCCTGAGACTCTCCTGTGCAGCCTCTGGCTTCACCTTTAGCAGCTAT
GCCATGGGCTGGGTCCGTCAGGCTCCAGGGAAGGGGCTGGAGTGGGT
CTCAACTATTACTAGTAGTGGTGGTAGCTCATACTTCGCACACTCCGTG
AAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTAT
CTGCACATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTTCTGT
GCGAAAGATGGCCCTAATTACGATGTTTGGAGTGGTTATTACTGGGGC
CAGGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCG
GTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCC
GCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTG
TCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCT
GTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTG
CCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCAC
AAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGG
TCCCCCATGCCCACCCTGCCCAGCACCTGAGTTCCTGGGGGGACCATC
AGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCG
GACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACC
CCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATG
CCAAGACAAAGCCGCGGGAGGAGCAGTTCCAAAGCACGTACCGTGTG
GTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGA
GTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAA
AACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACA
CCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTG
ACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGG
GAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGT
GCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTCACCGTGGA
CAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCA
TGAGGCTCTGCACAACCACTACACACAGAAGTCCCTCTCCCTGTCTCT GGGTAAATGA SEQ ID NO: 295
EVQLVESGGGLIQPGGSLRLSCAASGFTFSSYAMGWVRQAPGKGLEWVS
TITSSGGSSYFAHSVKGRFTISRDNSKNTLYLHMNSLRAEDTAVYFCAKD
GPNYDVWSGYYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCL
VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
KTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPK
DTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQ
STYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQ
VYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV
LDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG
K*
SEQ ID NO: 296
GATATTGTGATGACCCAGTCTCCACTCTCCCTGCCCGTCACACCTGGA
GAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGTATAGT
AATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGACAGTCT
CCACAACTCCTGATCTATCTGGGTTCTAATCGGGCCTCCGGGGTCCCT
GACAGGTTCAGAGGCAGTGGATCAGGCACAGATTTTACACTGAAAAT
CAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGC
TCTACAAACTCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCA
AACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGA
GCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTC
TATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCA
ATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACA
GCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTAC
GAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAG
CTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG
SEQ ID NO: 297
DIVMTQSPLSLPVTPGEPASISCRSSQSLLYSNGYNYLDWYLQKPGQSPQL
LIYLGSNRASGVPDRFRGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPR
TFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ
WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC*
SEQ ID NO: 298
CAGATCACCTTGAAGGAGTCTGGTCCTACGCTGGTGAAACCCACACAG
ACCCTCACGCTGACCTGCACCTTCTCTGGGTTCTCACTCAGCACTAGTG
GAGTGGGTGTGGGCTGGATCCGTCAGCCCCCAGGAAAGGCCCTTGAGT
GGCTTGCACTCATTTATTGGAATGATGTTCAGCGCTTCAGCCCATCTCT GAAGAGCAGGCTCACCATCACCAAGGACACCTCCAAAAACCAGGTTG TCCTTTCAATGACCAATATGGACCCTCTAGACACAGCCACATATTACT GTGCACACACATTAACTAACTTTTTCGACCCCTGGGGCCAGGGAACCC
TGGTCACCGTCTCCTCA
SEQ ID NO: 299
QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGVGVGWIRQPPGKALEWLA
LIYWNDVQRFSPSLKSRLTITKDTSKNQVVLSMTNMDPLDTATYYCAHTL
TNFFDPWGQGTLVT VS S
SEQ ID NO: 300
GGGTTCTCACTCAGCACTAGTGGAGTGGGT
SEQ ID NO: 301
GFSLSTSGVG
SEQ ID NO: 302
ATTTATTGGAATGATGTTCAG
SEQ ID NO: 303
IYWNDVQ
SEQ ID NO: 304
GCACACACATTAACTAACTTTTTCGACCCC
SEQ ID NO: 305
AHTLTNFFDP
SEQ ID NO: 306
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGA
GACAGAGTCACCATCACTTGCCGGGCGAGTCAGGGCATTAGCAATTAT
TTAGCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAACTCCTGATC
TATGCTGTTTCCACTTTGCAATCAGGGGTCCCTTCTCGGTTCAGTGGCC
GTGGATCTGGGGCAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTG
AGGATGTTGCAACTTATTACTGTCAAAAGTATTCCAGTACCCCGCTCA
CTTTCGGCGGAGGGACCAAGGTGGAGATCAAA SEQ ID NO: 307
DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKVPKLLIYA
VSTLQSGVPSRFSGRGSGADFTLTISSLQPEDVATYYCQKYSSTPLTFGGG TKVEIK
SEQ ID NO: 308
CAGGGCATTAGCAATTAT
SEQ ID NO: 309
QGISNY
SEQ ID NO: 310
GCTGTTTCC
SEQ ID NO: 311
AVS
SEQ ID NO: 312
CAAAAGTATTCCAGTACCCCGCTCACT
SEQ ID NO: 313
QKYSSTPLT
SEQ ID NO: 314
CAGATCACCTTGAAGGAGTCTGGTCCTACGCTGGTGAAACCCACACAG
ACCCTCACGCTGACCTGCACCTTCTCTGGGTTCTCACTCAGCACTAGTG
GAGTGGGTGTGGGCTGGATCCGTCAGCCCCCAGGAAAGGCCCTTGAGT
GGCTTGCACTCATTTATTGGAATGATGTTCAGCGCTTCAGCCCATCTCT
GAAGAGCAGGCTCACCATCACCAAGGACACCTCCAAAAACCAGGTTG
TCCTTTCAATGACCAATATGGACCCTCTAGACACAGCCACATATTACT
GTGCACACACATTAACTAACTTTTTCGACCCCTGGGGCCAGGGAACCC
TGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCT
GGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCT
GCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACT
CAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGT
CCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCA GCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGC AACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATG CCCACCCTGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCT GTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGA
GGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCC
AGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACA AAGCCGCGGGAGGAGCAGTTCCAAAGCACGTACCGTGTGGTCAGCGT CCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGT GCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCT
CCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCC CCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCT GGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCA ATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGAC
TCCGACGGCTCCTTCTTCCTCTACAGCAGGCTCACCGTGGACAAGAGC AGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCT CTGCACAACCACTACACACAGAAGTCCCTCTCCCTGTCTCTGGGTAAA TGA
SEQ ID NO: 315
QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGVGVGWIRQPPGKALEWLA LIYWNDVQRFSPSLKSRLTITKDTSKNQVVLSMTNMDPLDTATYYCAHTL
TNFFDPWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFP
EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCN VDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISR TPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVV SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPP SQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK*
SEQ ID NO: 316
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGA GACAGAGTCACCATCACTTGCCGGGCGAGTCAGGGCATTAGCAATTAT TTAGCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAACTCCTGATC TATGCTGTTTCCACTTTGCAATCAGGGGTCCCTTCTCGGTTCAGTGGCC GTGGATCTGGGGCAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTG AGGATGTTGCAACTTATTACTGTCAAAAGTATTCCAGTACCCCGCTCA CTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGAACTGTGGCTGCA CCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAA CTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCA AAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAG GAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAG CAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCT
ACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAG AGCTTCAACAGGGGAGAGTGTTAG SEQ ID NO: 317
DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKVPKLLIYA
VSTLQSGVPSRFSGRGSGADFTLTISSLQPEDVATYYCQKYSSTPLTFGGG
TKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD
NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC*
SEQ ID NO: 318
CAGGTTCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGC
CTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACCAGCTAT
GGTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGCCTTGAGTGGAT
GGGATGGATCAGCACTTACAATGGTAACACAAACTATGCACAGAAAC
TCCAGGGCAGAGTCACCATGACCACAGACACATCCACGAGTACAGCC
TCCATGGAGCTGAGGAGCCTGAGATCTGACGACACGGCCGTTTATTAC
TGCGCGAGAGACTCGGGTATTGCAGCTCGTCCTAGGTGGTTCGACCCC
TGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA
SEQ ID NO: 319
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWM
GWISTYNGNTNYAQKLQGRVTMTTDTSTSTASMELRSLRSDDTAVYYCA
RDSGIAARPRWFDPWGQGTLVTVSS
SEQ ID NO: 320
GGTTACACCTTTACCAGCTATGGT
SEQ ID NO: 321
GYTFTSYG
SEQ ID NO: 322
ATCAGCACTTACAATGGTAACACA
SEQ ID NO: 323
ISTYNGNT SEQIDNO: 324
GCGAGAGACTCGGGTATTGCAGCTCGTCCTAGGTGGTTCGACCCC
SEQIDNO: 325
ARDSGIAARPRWFDP
SEQIDNO: 326
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGA
GACAGAGTCACCATCACTTGCCAGACGAGTCAGGACATTAGCAACGA
TTTAACTTGGTATCAGCAGAAACCAGGAAAAGCCCCTAAACTCCTGAT
CTTCGATGCTTCCAATTTGTTAACAGGGGTCCCATCAAGGGTCAGTGG
AAGTGGATCTGGGACAGATTTTACTCTCACCATCACCAGCCTGCAGCC
TGAAGATGTTTCAACTTATTACTGTCAACACTATCATAGTCTCCCGATC
ACCTTCGGCCAAGGGACACGACTGGAGATTAAA
SEQIDNO: 327
DIQMTQSPSSLSASVGDRVTITCQTSQDISNDLTWYQQKPGKAPKLLIFDA
SNLLTGVPSRVSGSGSGTDFTLTITSLQPEDVSTYYCQHYHSLPITFGQGTR LEIK
SEQIDNO: 328
CAGGACATTAGCAACGAT
SEQIDNO: 329
QDISND
SEQIDNO: 330
GATGCTTCC
SEQIDNO: 331
DAS
SEQIDNO: 332
CAACACTATCATAGTCTCCCGAECACC SEQ ID NO: 333
QHYHSLPIT
SEQ ID NO: 334
CAGGTTCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGC CTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACCAGCTAT GGTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGCCTTGAGTGGAT GGGATGGATCAGCACTTACAATGGTAACACAAACTATGCACAGAAAC
TCCAGGGCAGAGTCACCATGACCACAGACACATCCACGAGTACAGCC
TCCATGGAGCTGAGGAGCCTGAGATCTGACGACACGGCCGTTTATTAC
TGCGCGAGAGACTCGGGTATTGCAGCTCGTCCTAGGTGGTTCGACCCC
TGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGC
CCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGC
ACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTG
ACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTC
CCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTG
ACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTA
GATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAA
ATATGGTCCCCCATGCCCACCCTGCCCAGCACCTGAGTTCCTGGGGGG
ACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGAT
CTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGG
AAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTG
CATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCCAAAGCACGTA
CCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGG CAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCAT CGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGG TGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTC
AGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTG
GAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCC TCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTCACC GTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGT GATGCATGAGGCTCTGCACAACCACTACACACAGAAGTCCCTCTCCCT
GTCTCTGGGTAAATGA
SEQ ID NO: 335
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWM GWISTYNGNTNYAQKLQGRVTMTTDTSTSTASMELRSLRSDDTAVYYCA RDSGIAARPRWFDPWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALG CLVKD YFPEP VTVS WNSGALT SGVHTFP AVLQ S SGL YSLS S VVT VPS S SLG TKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQ STYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQ VYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV
LDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG K*
SEQ ID NO: 336
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGA
GACAGAGTCACCATCACTTGCCAGACGAGTCAGGACATTAGCAACGA
TTTAACTTGGTATCAGCAGAAACCAGGAAAAGCCCCTAAACTCCTGAT
CTTCGATGCTTCCAATTTGTTAACAGGGGTCCCATCAAGGGTCAGTGG
AAGTGGATCTGGGACAGATTTTACTCTCACCATCACCAGCCTGCAGCC
TGAAGATGTTTCAACTTATTACTGTCAACACTATCATAGTCTCCCGATC
ACCTTCGGCCAAGGGACACGACTGGAGATTAAACGAACTGTGGCTGC
ACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGA
ACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCC
AAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCA
GGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCA
GCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTC
TACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAG
AGCTTCAACAGGGGAGAGTGTTAG
SEQ ID NO: 337
DIQMTQSPSSLSASVGDRVTITCQTSQDISNDLTWYQQKPGKAPKLLIFDA
SNLLTGVPSRVSGSGSGTDFTLTITSLQPEDVSTYYCQHYHSLPITFGQGTR
LEHCRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA
LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC*
SEQ ID NO: 338
CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGC
CTCAGTGAGGGTCTCCTGTAAGGCTTCTGGATACACCTTCATCGCCTAC
TATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATG
GGTTGGATCAACCCTAACAGTGGTGGCACAGACTATGCACAGAAGTTT
CAGGGCAGGGTCTCCATGACCAGGGACACGTCCATCACCACAGCCTA
CATGGACCTGAGCAGCCTGAGATCTGACGACACGGCCGTATATTACTG
TGCGAGAGGGGGTAGAGGCTATACTTTTGATATCTGGGGCCAAGGGA
CAATGGTCACCGTCTCTTCA
SEQ ID NO: 339
QVQLVQSGAEVKKPGASVRVSCKASGYTFIAYYMHWVRQAPGQGLEW
MGWINPNSGGTDYAQKFQGRVSMTRDTSITTAYMDLSSLRSDDTAVYYC
ARGGRGYTFDIWGQGTMVTVS S SEQIDNO: 340
GGATACACCTTCATCGCCTACTAT
SEQIDNO: 341
GYTFIAYY
SEQIDNO: 342
ATCAACCCTAACAGTGGTGGCACA
SEQIDNO: 343
INPNSGGT
SEQIDNO: 344
GCGAGAGGGGGTAGAGGCTATACTTTTGATATC
SEQIDNO: 345
ARGGRGYTFDI
SEQIDNO: 346
GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGA
GACAGAGTCACCATCACTTGCCGGGCCAGTCAGAGTGTTAGTAGCTGG
TTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATC
TATAAGACGTCTAGTTTAGAAAGTGGGGTCCCATCAAGGTTCAGCGGC
AGTGGATCTGGGACAGAATCCACTCTCACCGTCAGCGGCCTGCAGCCT
GATGATTTTGCAACTTATTACTGCCAACAGTATGATAGTTATTCGTGGA
CGTTCGGCCAAGGGACCAAGGTGGAAATCAAA
SEQIDNO: 347
DIQMTQSPSTLSASVGDRVTITCRASQSVSSWLAWYQQKPGKAPKLLIYK
TSSLESGVPSRFSGSGSGTESTLTVSGLQPDDFATYYCQQYDSYSWTFGQ
GTKVEIK
SEQIDNO: 348
CAGAGTGTTAGTAGCTGG SEQ ID NO: 349
QSVSSW
SEQ ID NO: 350
AAGACGTCT
SEQ ID NO: 351
CAACAGTATGATAGTTATTCGTGGACG
SEQ ID NO: 352
QQYDSYSWT
SEQ ID NO: 353
CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGC
CTCAGTGAGGGTCTCCTGTAAGGCTTCTGGATACACCTTCATCGCCTAC
TATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATG
GGTTGGATCAACCCTAACAGTGGTGGCACAGACTATGCACAGAAGTTT
CAGGGCAGGGTCTCCATGACCAGGGACACGTCCATCACCACAGCCTA
CATGGACCTGAGCAGCCTGAGATCTGACGACACGGCCGTATATTACTG
TGCGAGAGGGGGTAGAGGCTATACTTTTGATATCTGGGGCCAAGGGA
CAATGGTCACCGTCTCTTCAGCCTCCACCAAGGGCCCATCGGTCTTCC
CCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGG
GCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGA
ACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTAC
AGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCA
GCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCC
AGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCC
ATGCCCACCCTGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTT
CCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCC
TGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGG
TCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGA
CAAAGCCGCGGGAGGAGCAGTTCCAAAGCACGTACCGTGTGGTCAGC
GTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAA
GTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCAT
CTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGC
CCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGC
CTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAG
CAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGG ACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTCACCGTGGACAAGA
GCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGG
CTCTGCACAACCACTACACACAGAAGTCCCTCTCCCTGTCTCTGGGTA AATGA
SEQ ID NO: 354
QVQLVQSGAEVKKPGASVRVSCKASGYTFIAYYMHWVRQAPGQGLEW MGWINPNSGGTDYAQKFQGRVSMTRDTSITTAYMDLSSLRSDDTAVYYC ARGGRGYTFDIWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT KTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPK
DTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQ STYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQ VYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG K*
SEQ ID NO: 355
GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGA
GACAGAGTCACCATCACTTGCCGGGCCAGTCAGAGTGTTAGTAGCTGG
TTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATC
TATAAGACGTCTAGTTTAGAAAGTGGGGTCCCATCAAGGTTCAGCGGC
AGTGGATCTGGGACAGAATCCACTCTCACCGTCAGCGGCCTGCAGCCT
GATGATTTTGCAACTTATTACTGCCAACAGTATGATAGTTATTCGTGGA
CGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGAACTGTGGCTGCA
CCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAA
CTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCA
AAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAG
GAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAG
CAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCT
ACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAG
AGCTTCAACAGGGGAGAGTGTTAG
SEQ ID NO: 356
DIQMTQSPSTLSASVGDRVTITCRASQSVSSWLAWYQQKPGKAPKLLIYK
TSSLESGVPSRFSGSGSGTESTLTVSGLQPDDFATYYCQQYDSYSWTFGQ
GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ
GLSSPVTKSFNRGEC* SEQ ID NO: 357
GAGGTGCAGCTGGTGGAGTCGGGGGGAGGCTTGGTACAGCCTGGGGG
GTCCCTGAGACTCTCCTGTGCAGCCGCTGGATTTAGTTTTAGTACCTAT
GCCATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGT
CTCAACTATTAGTGGGAGAGCTGGTTCCACATTCTACGGTGACTCCGT
GAAGGGCCGGTTCACCCTCTCCAGAGACACTTCCAGTAACACGGTGCA
TCTGCAAATGAATAGTCTGAGAGCCGAGGATACGGCCGTATATTACTG
TGCGAAAGGAAATGGAGTCTTTGACTACTGGGGCCAGGGAACCCTGG
TCACCGTCTCCTCA
SEQ ID NO: 358
EVQLVESGGGLVQPGGSLRLSCAAAGFSFSTYAMNWVRQAPGKGLEWV
STISGRAGSTFYGDSVKGRFTLSRDTSSNTVHLQMNSLRAEDTAVYYCAK
GNGVFD YWGQGTLVTVS S
SEQ ID NO: 359
GGATTTAGTTTTAGTACCTATGCC
SEQ ID NO: 360
GFSFSTYA
SEQ ID NO: 361
ATTAGTGGGAGAGCTGGTTCCACA
SEQ ID NO: 362
ISGRAGST
SEQ ID NO: 363
GCGAAAGGAAATGGAGTCTTTGACTAC
SEQ ID NO: 364
AKGNGVFDY SEQIDNO: 365
GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGA
GACAGAGTCACCATCACTTGCCGGGCCAGTCAGACTATTACTAACTAC
TTGGCCTGGTATCAACAGAACCCAGGGAAACCCCCTAAGTTACTGATC
TATAAGGCGTCTAGTTTAGAAAGTGGGGTCCCATCAAGGTTCAGTGGC
AGTGGATCTGGGACAGAATTCACTCTAACCATCAGCAGCCTGCAGCCT
GATGATTTTGCAACTTATTACTGCCAACAGTATAATAATTTTTACACTT
TTGGCCAGGGGACCAAGCTGGAGATCAAA
SEQIDNO: 366
DIQMTQSPSTLSASVGDRVTITCRASQTITNYLAWYQQNPGKPPKLLIYKA
SSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNNFYTFGQGTK
LEIK
SEQIDNO: 367
CAGACTATTACTAACTAC
SEQIDNO: 368
QTITNY
SEQIDNO: 369
AAGGCGTCT
SEQIDNO: 370
KAS
SEQIDNO: 371
CAACAGTATAATAATTTTTACACT
SEQIDNO: 372
QQYNNFYT
SEQIDNO: 373
GAGGTGCAGCTGGTGGAGTCGGGGGGAGGCTTGGTACAGCCTGGGGG
GTCCCTGAGACTCTCCTGTGCAGCCGCTGGATTTAGTTTTAGTACCTAT GCCATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGT
CTCAACTATTAGTGGGAGAGCTGGTTCCACATTCTACGGTGACTCCGT
GAAGGGCCGGTTCACCCTCTCCAGAGACACTTCCAGTAACACGGTGCA
TCTGCAAATGAATAGTCTGAGAGCCGAGGATACGGCCGTATATTACTG
TGCGAAAGGAAATGGAGTCTTTGACTACTGGGGCCAGGGAACCCTGG
TCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGC
GCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCT
GGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGG
CGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTC
AGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTT
GGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACA
CCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCA
CCCTGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTC
CCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTC
ACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTT
CAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGC
CGCGGGAGGAGCAGTTCCAAAGCACGTACCGTGTGGTCAGCGTCCTC
ACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAA
GGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAA
AGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCAT
CCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTC
AAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGG
GCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCG
ACGGCTCCTTCTTCCTCTACAGCAGGCTCACCGTGGACAAGAGCAGGT
GGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGC
ACAACCACTACACACAGAAGTCCCTCTCCCTGTCTCTGGGTAAATGA
SEQ ID NO: 374
EVQLVESGGGLVQPGGSLRLSCAAAGFSFSTYAMNWVRQAPGKGLEWV
STISGRAGSTFYGDSVKGRFTLSRDTSSNTVHLQMNSLRAEDTAVYYCAK
GNGVFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDY
FPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLS S VVTVPS S SLGTKTYTC
NVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMIS
RTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRV
VSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLP
PSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK*
SEQ ID NO: 375
GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGA
GACAGAGTCACCATCACTTGCCGGGCCAGTCAGACTATTACTAACTAC
TTGGCCTGGTATCAACAGAACCCAGGGAAACCCCCTAAGTTACTGATC
TATAAGGCGTCTAGTTTAGAAAGTGGGGTCCCATCAAGGTTCAGTGGC
AGTGGATCTGGGACAGAATTCACTCTAACCATCAGCAGCCTGCAGCCT GATGATTTTGCAACTTATTACTGCCAACAGTATAATAATTTTTACACTT
TTGGCCAGGGGACCAAGCTGGAGATCAAACGAACTGTGGCTGCACCA
TCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTG
CCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAG
TACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAG
AGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAG
CACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACG
CCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCT
TCAACAGGGGAGAGTGTTAG
SEQ ID NO: 376
DIQMTQSPSTLSASVGDRVTITCRASQTITNYLAWYQQNPGKPPKLLIYKA
SSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNNFYTFGQGTK
LEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA
LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS
PVTKSFNRGEC*
SEQ ID NO: 377
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAG
GTCCCTGAGACTCTCCTGTGAAGCCTCTGGATTCACCTTCAGTACCTAT
GGCATGTTCTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGT
GGCAGTTATATCATATGATGGAAGTGATAAATATTATACAGACTCTAT
TAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAAAATACACTGTT
TCTCCAAATGAACAGCCTGAGAGTAGAGGACACGGCTGTGTATTGGTG
TGCGAGGATGAAGGGGTATAGAAGTTCGTCCGCCCACCACTACTACAG
TATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA
SEQ ID NO: 378
QVQLVESGGGVVQPGRSLRLSCEASGFTFSTYGMFWVRQAPGKGLEWV
AVISYDGSDKYYTDSIKGRFTISRDNSKNTLFLQMNSLRVEDTAVYWCAR
MKGYRS S S AHHYYSMD VWGQGTT VTVS S
SEQ ID NO: 379
ATATCATATGATGGAAGTGATAAA
SEQ ID NO: 380
ISYDGSDK SEQ ID NO: 381
GCGAGGATGAAGGGGTATAGAAGTTCGTCCGCCCACCACTACTACAGT
ATGGACGTC
SEQ ID NO: 382
ARMKGYRSSSAHHYYSMDV
SEQ ID NO: 383
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAG
GTCCCTGAGACTCTCCTGTGAAGCCTCTGGATTCACCTTCAGTACCTAT
GGCATGTTCTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGT
GGCAGTTATATCATATGATGGAAGTGATAAATATTATACAGACTCTAT
TAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAAAATACACTGTT
TCTCCAAATGAACAGCCTGAGAGTAGAGGACACGGCTGTGTATTGGTG
TGCGAGGATGAAGGGGTATAGAAGTTCGTCCGCCCACCACTACTACAG
TATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCCTC
CACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCAC
CTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCC
CGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCG
TGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAG
CAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACAC
CTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAG
TTGAGTCCAAATATGGTCCCCCATGCCCACCCTGCCCAGCACCTGAGT
TCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACA
CTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACG
TGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGC
GTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCCA
AAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTG
GCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCC
CGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGA
GAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAA
GAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGA
CATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACA
AGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACA
GCAGGCTCACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTC
TCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAG TCCCTCTCCCTGTCTCTGGGTAAATGA
SEQ ID NO: 384
QVQLVESGGGVVQPGRSLRLSCEASGFTFSTYGMFWVRQAPGKGLEWV
AVISYDGSDKYYTDSIKGRFTISRDNSKNTLFLQMNSLRVEDTAVYWCAR MKGYRSSSAHHYYSMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSEST
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP
SSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLF
PPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPR
EEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQ
PREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK
TTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLS
LSLGK*
SEQ ID NO: 385
CAGGTGCAGCTGGTGCAGTCTGGGGCTGAAGTGAAGAAGCCTGGGGC
CTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACTCCTTCACCAACTA
CTATATACACTGGGTGCGACAGGCCCCTAGACAAGGACTTGAGTGGAT
GGGATGGATCAACCCTAACAGTGGTGACACAAACTATGTACAGAACTT
TCAGGGCAGGGTCACCATGACCAGGGACACGTCCATCAGCACAGTCT
ACATGGAGCTGAGCAGGCTGAGATCTGACGACACGGCCGTGTATTATT
GTGCGAGACTGGAACTTGGCTACTATTACAACGATATGGACGTCTGGG
GCCAAGGGACCACGGTCACCGTCTCCTCA
SEQ ID NO: 386
QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYYIHWVRQAPRQGLEWM
GWINPNSGDTNYVQNFQGRVTMTRDTSISTVYMELSRLRSDDTAVYYCA RLELGYYYNDMD VWGQGTTVT VS S
SEQ ID NO: 387
GGATACTCCTTCACCAACTACTAT
SEQ ID NO: 388
GYSFTNYY
SEQ ID NO: 389
ATCAACCCTAACAGTGGTGACACA
SEQ ID NO: 390
INPNSGDT SEQ ID NO: 391
GCGAGACTGGAACTTGGCTACTATTACAACGATATGGACGTC
SEQ ID NO: 392
ARLELGYYYNDMDV
SEQ ID NO: 393
GATATTGTGATGACCCAGACTCCACTCTCCTCACCTGTCACCCTTGGAC
AGCCGGCCTCCATCTCCTGCAGGTCTAGTCAAAGCCTCGTACACAGTG
ATGGAAACACCTACTTGAGTTGGCTTCAGCAGACGCCAGGCCAGCCTC
CAAGACTCCTAATTTATAAGACTTCTAACCGATTCTCTGGGGTCCCAG
ACAGATTCAGTGGCAGTGGGGCAGGGACAGATTTCACACTGAAAATC
AGCAGGGTGGAAGCTGAGGATGTCGGGGTTTATTACTGCATGCAAGCT
ACACAATTTCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA
SEQ ID NO: 394
DIVMTQTPLSSPVTLGQPASISCRSSQSLVHSDGNTYLSWLQQTPGQPPRL
LIYKTSNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCMQATQFPL
TFGGGTKVEIK
SEQ ID NO: 395
CAAAGCCTCGTACACAGTGATGGAAACACCTAC
SEQ ID NO: 396
QSLVHSDGNTY
SEQ ID NO: 397
ATGCAAGCTACACAATTTCCTCTCACT
SEQ ID NO: 398
MQATQFPLT
SEQ ID NO: 399
CAGGTGCAGCTGGTGCAGTCTGGGGCTGAAGTGAAGAAGCCTGGGGC
CTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACTCCTTCACCAACTA CTATATACACTGGGTGCGACAGGCCCCTAGACAAGGACTTGAGTGGAT
GGGATGGATCAACCCTAACAGTGGTGACACAAACTATGTACAGAACTT
TCAGGGCAGGGTCACCATGACCAGGGACACGTCCATCAGCACAGTCT
ACATGGAGCTGAGCAGGCTGAGATCTGACGACACGGCCGTGTATTATT
GTGCGAGACTGGAACTTGGCTACTATTACAACGATATGGACGTCTGGG
GCCAAGGGACCACGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCAT
CGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAG
CCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGG
TGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGG
CTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCG
TGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATC
ACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATAT
GGTCCCCCATGCCCACCCTGCCCAGCACCTGAGTTCCTGGGGGGACCA
TCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCC
GGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGAC
CCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAAT
GCCAAGACAAAGCCGCGGGAGGAGCAGTTCCAAAGCACGTACCGTGT
GGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGG
AGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGA
AAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTAC
ACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCT
GACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTG
GGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCG
TGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTCACCGTGG
ACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGC
ATGAGGCTCTGCACAACCACTACACACAGAAGTCCCTCTCCCTGTCTC
TGGGTAAATGA
SEQ ID NO: 400
QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYYIHWVRQAPRQGLEWM GWINPNSGDTNYVQNFQGRVTMTRDTSISTVYMELSRLRSDDTAVYYCA RLELGYYYNDMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAAL GCLVKD YFPEP VT VS WNSGALT SGVHTFP A VLQ S SGL YSLS S VVT VP S S SL GTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQF QSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREP QVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSL GK*
SEQ ID NO: 401
GATATTGTGATGACCCAGACTCCACTCTCCTCACCTGTCACCCTTGGAC
AGCCGGCCTCCATCTCCTGCAGGTCTAGTCAAAGCCTCGTACACAGTG
ATGGAAACACCTACTTGAGTTGGCTTCAGCAGACGCCAGGCCAGCCTC CAAGACTCCTAATTTATAAGACTTCTAACCGATTCTCTGGGGTCCCAG
ACAGATTCAGTGGCAGTGGGGCAGGGACAGATTTCACACTGAAAATC
AGCAGGGTGGAAGCTGAGGATGTCGGGGTTTATTACTGCATGCAAGCT
ACACAATTTCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA
CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGC
AGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTA
TCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAAT
CGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGC
ACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGA
GAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCT CGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG
SEQ ID NO: 402
DIVMTQTPLSSPVTLGQPASISCRSSQSLVHSDGNTYLSWLQQTPGQPPRL
LIYKTSNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCMQATQFPL
TFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ
WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV
THQGLSSPVTKSFNRGEC*
SEQ ID NO: 403
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGG
GTCCCTGAAACTCTCCTGTGCAGCCTCTGGGTTCACCTTCAGTGACTCT
GCTATGCACTGGGTCCGCCAGGCTTCCGGGAAAGGGCTGGAGTGGGTT
GGCCGTATTAGAAGCAAAACTAACAGTTACGCGACAGCATATACTGC
GTCGGTGAAAGACAGGTTCACCATCTCCAGAGATGATTCAAAGAACA
CGGCGTATCTGCAAATGAACAGCCTGAAAACCGAGGACACGGCCGTA
TATTACTGTACTAGACTGGCAGTAACTGACTACTGGGGCCAGGGAACC
CTGGTCACCGTCTCCTCA
SEQ ID NO: 404
EVQLVESGGGLVQPGGSLKLSCAASGFTFSDSAMHWVRQASGKGLEWV
GRIRSKTNSYATAYTASVKDRFTISRDDSKNTAYLQMNSLKTEDTAVYYC
TRL AVTD YWGQGTL VTVS S
SEQ ID NO: 405
GGGTTCACCTTCAGTGACTCTGCT
SEQ ID NO: 406
GFTFSDSA SEQIDNO: 407
ATTAGAAGCAAAACTAACAGTTACGCGACA
SEQIDNO: 408
IRSKTNSYAT
SEQIDNO: 409
ACTAGACTGGCAGTAACTGACTAC
SEQIDNO: 410
TRLAVTDY
SEQIDNO: 411
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTTTGCATCTGTAGGA
GACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGAAATGA
TTTAGGCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCGCCTGAT
CTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGG
CAGTGGATCTGGGACTGAATTCACTCTCACAATCAGCAGCCTGCAGCC
TGAAGATTTTGCAACTTATTACTGTCTACAGCATAAAACTTACACGTTC
GGCCAAGGGACCAAGGTGGAAATCAAA
SEQIDNO: 412
DIQMTQSPSSLFASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYA
ASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQHKTYTFGQGTK
VEIK
SEQIDNO: 413
CTACAGCATAAAACTTACACG
SEQIDNO: 414
LQHKTYT
SEQIDNO: 415
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGG
GTCCCTGAAACTCTCCTGTGCAGCCTCTGGGTTCACCTTCAGTGACTCT GCTATGCACTGGGTCCGCCAGGCTTCCGGGAAAGGGCTGGAGTGGGTT
GGCCGTATTAGAAGCAAAACTAACAGTTACGCGACAGCATATACTGC
GTCGGTGAAAGACAGGTTCACCATCTCCAGAGATGATTCAAAGAACA
CGGCGTATCTGCAAATGAACAGCCTGAAAACCGAGGACACGGCCGTA
TATTACTGTACTAGACTGGCAGTAACTGACTACTGGGGCCAGGGAACC
CTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCC
TGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCT
GCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACT
CAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGT
CCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCA
GCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGC
AACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATG
CCCACCCTGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCT
GTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGA
GGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCC
AGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACA
AAGCCGCGGGAGGAGCAGTTCCAAAGCACGTACCGTGTGGTCAGCGT
CCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGT
GCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCT
CCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCC
CCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCT
GGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCA
ATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGAC
TCCGACGGCTCCTTCTTCCTCTACAGCAGGCTCACCGTGGACAAGAGC
AGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCT
CTGCACAACCACTACACACAGAAGTCCCTCTCCCTGTCTCTGGGTAAA
TGA
SEQ ID NO: 416
EVQLVESGGGLVQPGGSLKLSCAASGFTFSDSAMHWVRQASGKGLEWV GRIRSKTNSYATAYTASVKDRFTISRDDSKNTAYLQMNSLKTEDTAVYYC
TRLAVTDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKD
YFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLS S VVTVPS S SLGTKTYT CNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRV VSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLP PSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK*
SEQ ID NO: 417
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTTTGCATCTGTAGGA GACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGAAATGA TTTAGGCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCGCCTGAT CTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGG CAGTGGATCTGGGACTGAATTCACTCTCACAATCAGCAGCCTGCAGCC
TGAAGATTTTGCAACTTATTACTGTCTACAGCATAAAACTTACACGTTC
GGCCAAGGGACCAAGGTGGAAATCAAACGAACTGTGGCTGCACCATC
TGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCC
TCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTA
CAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAG
TGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCA
CCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCC
TGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTC
AACAGGGGAGAGTGTTAG
SEQ ID NO: 418
DIQMTQSPSSLFASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYA
ASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQHKTYTFGQGTK
VEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA
LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS
PVTKSFNRGEC*
SEQ ID NO: 419 caggcgcacctgcaggagtcgggcccaggactggtgaagccttcggagaccctgtccctcacctgcactgtctctg gtggctccctcagtaattactactggagctggatccggcagcccccagggaagggactggaatggattggctatatc cattacagtgggcataccaagtacaattcctccctcaagagtcgagtcaccatatcagtagacacgtccaaaaaccag ttctccctgatgctgacctctgtgaccgccgcagacacggccctgtattattgtgcgagacatggtataactggaacta cgtggttcgacccctggggccagggagccctggtcaccgtctcctca
SEQ ID NO: 420
QAHLQESGPGLVKPSETLSLTCTVSGGSLSNYYWSWIRQPPGKGLEWIGY IHYSGHTKYNSSLKSRVTISVDTSKNQFSLMLTSVTAADTALYYCARHGIT GTTWFDPWGQGAL VT VS S
SEQ ID NO: 421 ggtggctccctcagtaattactac
SEQ ID NO: 422
GGSLSNYY
SEQ ID NO: 423 atccattacagtgggcatacc SEQIDNO: 424
IHYSGHT
SEQIDNO: 425 gcgagacatggtataactggaactacgtggttcgacccc
SEQIDNO: 426
ARHGITGTTWFDP
SEQIDNO: 427 gacatccagatgacccagtctccatcttccgtgtctgcatctgtaggggacagagtcaccatcacttgtcgggcgcgt cagggcattaactactggttagcctggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcatcc agtttgcaaagtggggtcccatcaaggttcagcggcagtggatctgggacagatttcactctcaccatcagcagcctg cagcctgaagattttgcaacttactattgtcaacaggctaacagtttccctccgacgttcggacaagggaccaaggtgg aaatcaag
SEQIDNO: 428
DIQMTQSPSSVSASVGDRVTITCRARQGINYWLAWYQQKPGKAPKLLIYA ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPPTFGQGT KVEIK
SEQIDNO: 429 cagggcattaactactgg
SEQIDNO: 430
QGINYW
SEQIDNO: 431 gctgcatcc
SEQIDNO: 432
AAS SEQIDNO: 433 caacaggctaacagtttccctccgacg
SEQIDNO: 434
QQANSFPPT
SEQIDNO: 435 gaggtgcagctgttggagtctgggggaggcgtggtacagcctggggagtccctgagactctcctgtgcggcctctg aattttcctttagtagttatgccatgggctgggtccgccaggctccagggaaggggctggagtgggtctcagctattag tggtagtggtggtaggacatactacgcagactccgtgaagggccggttcaccatctccagagacaattccaagaaca cgctgtttctgcaaatgaacagcctgagagccgaggacacggccgtatattactgtgtgggggttatagtggcctttga ctactggggccagggaaccctggtcaccgtctcctca
SEQIDNO: 436
EVQLLESGGGVVQPGESLRLSCAASEFSFSSYAMGWVRQAPGKGLEWVS AISGSGGRTYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCVG VIVAFD YWGQGTL VT VS S
SEQIDNO: 437 gaattttcctttagtagttatgcc
SEQIDNO: 438
EFSFSSYA
SEQIDNO: 439 attagtggtagtggtggtaggaca
SEQIDNO: 440
ISGSGGRT
SEQIDNO: 441 gtgggggttatagtggcctttgactac SEQIDNO: 442
VGVIVAFDY
SEQIDNO: 443 gacatccacatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagt cagagtattagcagctatttaaattggtatcagcagaagccagggaaagcccctaaattcctgatctatactgcatcca gtttgcaaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactctcaccatcagcagtctgca acctgtagattttgctacttactactgtcaacagagttacactaccccgtacacttttggccaggggaccaagctggaga tcaca
SEQIDNO: 444
DIHMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKFLIYTA SSLQSGVPSRFSGSGSGTDFTLTISSLQPVDFATYYCQQSYTTPYTFGQGT KLEIT
SEQIDNO: 445 cagagtattagcagctat
SEQIDNO: 446
QSISSY
SEQIDNO: 447 actgcatcc
SEQIDNO: 448
TAS
SEQIDNO: 449 caacagagttacactaccccgtacact
SEQIDNO: 450
QQSYTTPYT SEQ ID NO: 451 caggtgcagctgcaggagtcgggcccaggactggtgaacccctcggagaccctgtccctcacctgcactgtctctg gtggctccatcaatggttactactggaattggatccggcagtccccaggaaaggaacttgagtggattggctatatcttt tatagtgggagcaccacctacagcccttccctcaggggtcgagtcaccatgtcggtagactcgtccaagaaccaattc tctctgaggttggacgctgtgaccgccgcagactcggccctatattactgtgcgagacgctattacgatactttcactg gttggggttactttgactcctggggccagggaaccctggtcaccgtctcctca
SEQ ID NO: 452
QVQLQESGPGLVNPSETLSLTCTVSGGSINGYYWNWIRQSPGKELEWIGYI FYSGSTTYSPSLRGRVTMSVDSSKNQFSLRLDAVTAADSALYYCARRYY DTFTGWGYFDSWGQGTLVTVS S
SEQ ID NO: 453 ggtggctccatcaatggttactac
SEQ ID NO: 454
GGSINGYY
SEQ ID NO: 455 atcttttatagtgggagcacc
SEQ ID NO: 456
IFYSGST
SEQ ID NO: 457 gcgagacgctattacgatactttcactggttggggttactttgactcc
SEQ ID NO: 458
ARRYYDTFTGWGYFDS
SEQ ID NO: 459 gacatccagatgacccagtctccatcctccgtgtctgcatctgtgggagacagagtcaccgtcacttgtcgggcgagt caggttattagcagatggttagcctggtatcagcagaagccagggaaagcccctaaactcctgatctatgctgcgtcc agtttacaaagtggggtcccatcacggttcagcggcagtggatctgggacagattttactctcaccatcagcggcctg cagcctgaagattttgcaatttacttttgtcaacaggctaacagtttcccattcactttcggccctgggaccaaagtggat agcaag
SEQ ID NO: 460
DIQMTQSPSSVSASVGDRVTVTCRASQVISRWLAWYQQKPGKAPKLLIY AASSLQSGVPSRFSGSGSGTDFTLTISGLQPEDFAIYFCQQANSFPFTFGPG TKVDSK
SEQ ID NO: 461 caggttattagcagatgg
SEQ ID NO: 462
QVISRW
SEQ ID NO: 463 gctgcgtcc
SEQ ID NO: 464
AAS
SEQ ID NO: 465 caacaggctaacagtttcccattcact
SEQ ID NO: 466
QQANSFPFT
SEQ ID NO: 467 catgtgcagttggtgcagtctggggctgaggtgaagaagcctggggcctcagtgaaggtttcctgcaaggcatctgg ctacaccttcaccagctactatctgcactgggtgcgacaggcccctggacaagggcttgagtggatgggagtcatca aacctagtggtggtaacacaggttacgcacagcagttccgggacagagtcaccatgaccagggacacgtccacga gcacagtctccatggaactgagcagcctgagatatgaagacacggccgtgtattattgtgtgagagataatgtggaac tggaactagggtattggggccagggaaccctggtcaccgtctcctca SEQ ID NO: 468
HVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYLHWVRQAPGQGLEW MGVIKPSGGNTGYAQQFRDRVTMTRDTSTSTVSMELSSLRYEDTAVYYC VRDNVELELGYWGQGTLVT VS S
SEQ ID NO: 469 ggctacaccttcaccagctactat
SEQ ID NO: 470
GYTFTSYY
SEQ ID NO: 471 atcaaacctagtggtggtaacaca
SEQ ID NO: 472
IKPSGGNT
SEQ ID NO: 473 gtgagagataatgtggaactggaactagggtat
SEQ ID NO: 474
VRDNVELELGY
SEQ ID NO: 475 gacatcgtgatgacccagtctccagactccctgtctatgtctctgggcgagaggaccaccatcaactgcaagtccagc cagagtgttttatacagctccaacaataggaactacttagcttggttccaacagaaaccaggacagcctcctaaacttct catttactgggcatctacccgggagtccggggtccctgaccgattccgtggcagcgggtctgggacagatttcactct caccatcagcagcctgcaggctgatgatgtggcagtttattactgtcagcaatattttgatactccgtacacttttggcct ggggaccaagctggagatcaaa
SEQ ID NO: 476
DIVMTQSPDSLSMSLGERTTINCKSSQSVLYSSNNRNYLAWFQQKPGQPP KLLIYWASTRESGVPDRFRGSGSGTDFTLTISSLQADDVAVYYCQQYFDT
PYTFGLGTKLEIK SEQIDNO: 477 cagagtgttttatacagctccaacaataggaactac
SEQIDNO: 478
QSVLYSSNNRNY
SEQIDNO: 479 tgggcatct
SEQIDNO: 480
WAS
SEQIDNO: 481 cagcaatattttgatactccgtacact
SEQIDNO: 482
QQYFDTPYT
SEQIDNO: 483 gaggtacatctgttggagtctgggggaggcttagtacagcctggggggtccctgagactctcctgtgcagcctctgg actcacctttagtaactatgccatgagctgggtccgccaggttccagggaaggggctggcgtgggtctcaggtattaa tgatcgtggtagtggcacatactacgcagactccgtgaagggccggttcaccatctccagagacaattccaagaaca cactgtatctgcaaatgaacagcctgagagccgaggacacggccgtatattactgtgcgaaagaggactacggtaa ctttgactattggggccagggaaccctggtcaccgtctcctca
SEQIDNO: 484
EVHLLESGGGLVQPGGSLRLSCAASGLTFSNYAMSWVRQVPGKGLAWV SGINDRGSGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA KED YGNFDYWGQGTLVT VS S
SEQIDNO: 485 ggactcacctttagtaactatgcc SEQIDNO: 486
GLTFSNYA
SEQIDNO: 487 attaatgatcgtggtagtggcaca
SEQIDNO: 488
INDRGSGT
SEQIDNO: 489 gcgaaagaggactacggtaactttgactat
SEQIDNO: 490
AKEDYGNFDY
SEQIDNO: 491 gacatccagatgacccagtctccttccaccctgtctgcatctgtaggagacagagtcaccatcacttgccgggccagt cagaatattagtaactggttggcctggtatcagcagaaaccagggaaagcccctaaactcctgatctataaggcgtct agtttagaaagtggggtcccatcaaggttcagcggcagtggatctgggacagaattcactctcaccatcagcagcct gcagcctgatgattttgcaacttattactgccaacagtataatagttattctatgtacacttttggccaggggaccaagct ggagatcaaa
SEQIDNO: 492
DIQMTQSPSTLSASVGDRVTITCRASQNISNWLAWYQQKPGKAPKLLIYK ASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYSMYTFGQ GTKLEIK
SEQIDNO: 493 cagaatattagtaactgg
SEQIDNO: 494
QNISNW SEQIDNO: 495 aaggcgtct
SEQIDNO: 496
KAS
SEQIDNO: 497 caacagtataatagttattctatgtacact
SEQIDNO: 498
QQYNSYSMYT
SEQIDNO: 499 gaggtacagctggtggagtctgggggaggcttggtacagcctggagggtccctgacactttcctgtgcagcctctgg attcaccttcagtagttatgaaatgaattgggtccgccaggctccagggaaggggctggagtgggtttcatacattagt agtagtggtagtaccatatactacgcagactctgtgaagggccgattcaccatttatagagacaacgccaagaactca ctgtatctgcaaatgaatagcctgagagccgaggacacggctgtttattactgtgcgagagatgaggattttggagtg gcccactactacggtatggacgtctggggccaagggaccacggtcaccgtctcctca
SEQIDNO: 500
EVQLVESGGGLVQPGGSLTLSCAASGFTFSSYEMNWVRQAPGKGLEWVS YIS S SGSTIYYADSVKGRFTIYRDNAKNSLYLQMNSLRAEDTAVYYC ARD EDFGVAHYYGMD VWGQGTT VTVS S
SEQIDNO: 501 ggattcaccttcagtagttatgaa
SEQIDNO: 502
GFTFSSYE
SEQIDNO: 503 attagtagtagtggtagtaccata SEQIDNO: 504
ISSSGSTI
SEQIDNO: 505 gcgagagatgaggattttggagtggcccactactacggtatggacgtc
SEQIDNO: 506
ARDEDFGVAHYYGMDV
SEQIDNO: 507 gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagt cagagcattagaagctatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcaaac agtttgcaaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactctcaccatcagcagtctgc aacctgaagattttgcaacttactactgtcaacagagttacagtacccatccgatcaccttcggccaagggacacgact ggagattaaa
SEQIDNO: 508
DIQMTQSPSSLSASVGDRVTITCRASQSIRSYLNWYQQKPGKAPKLLIYAA NSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTHPITFGQGT RLEIK
SEQIDNO: 509 cagagcattagaagctat
SEQIDNO: 510
QSIRSY
SEQIDNO: 511 gctgcaaac
SEQIDNO: 512
AAN SEQIDNO: 513 caacagagttacagtacccatccgatcacc
SEQIDNO: 514
QQSYSTHPIT
SEQIDNO: 515 gaggtgcaactgttggagtctgggggaggcttggtgcagccgggggggtccctgagactctcctgtgcagcctctg ggttcatctttagtagttatgccatgtcctgggtccgccaggctccagggaaggggctggagtgggtctcaattattagt ggaagtggtggtagaatatactacgcagagtccgtgaagggccggttcaccatctccagagacaattccaagaaca cgttgtatctgcaaatgaaaagtgtgcgagccgaggacacggccggatattactgtgcggggggaatagttacgctc tttgactattggggccagggaaccctggtcaccgtctcctca
SEQIDNO: 516
EVQLLESGGGLVQPGGSLRLSCAASGFIFSSYAMSWVRQAPGKGLEWVSI ISGSGGRIYYAESVKGRFTISRDNSKNTLYLQMKSVRAEDTAGYYCAGGI VTLFD YWGQGTLVT VS S
SEQIDNO: 517 gggttcatctttagtagttatgcc
SEQIDNO: 518
GFIFSSYA
SEQIDNO: 519 attagtggaagtggtggtagaata
SEQIDNO: 520
ISGSGGRI
SEQIDNO: 521 gcggggggaatagttacgctctttgactat SEQIDNO: 522
AGGIVTLFDY
SEQIDNO: 523 gacatccagatgacccagtctccatcctccctgtctgcatctgtaggcgacagagtcaccatcacttgccgggcaagt cagagcattagcagttatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcatcca gtttgcatagtggggtcccatcaaggttcagtggcagtggatctgggacagagttcactctcaccatcagcagtctgc aacctgatgattttgcaacttactactgtcatcagagttacagtagtcctccgatcaccttcggccaagggacacgagtg gacattaaa
SEQIDNO: 524
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAA SSLHSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCHQSYSSPPITFGQGTR VDIK
SEQIDNO: 525 cagagcattagcagttat
SEQIDNO: 526
QSISSY
SEQIDNO: 527 gctgcatcc
SEQIDNO: 528
AAS
SEQIDNO: 529 catcagagttacagtagtcctccgatcacc
SEQIDNO: 530
HQSYSSPPIT SEQ ID NO: 531 gaggtgcagctggtggagtctgggggaggcctggtcaagcctggggggtccctgagactctcctgtgcagcctctg gattcaccttcagtagctatagcatgaactgggtccgccaggctccagggaaggggctggagtgggtctcatccatta gtagtagtagtacttacatatactacgcagactcagtgaagggccgattcaccatctccagagacaacgccaagaact cactgtatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtgcgagagaccggggctggaa ctttgactactggggccagggaaccctggtcaccgtctcctcc
SEQ ID NO: 532
EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVS SISSSSTYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARD RGWNFDYWGQGTLVTVSS
SEQ ID NO: 533 ggattcaccttcagtagctatagc
SEQ ID NO: 534
GFTFSSYS
SEQ ID NO: 535 attagtagtagtagtacttacata
SEQ ID NO: 536
ISSSSTYI
SEQ ID NO: 537 gcgagagaccggggctggaactttgactac
SEQ ID NO: 538
ARDRGWNFDY
SEQ ID NO: 539 gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagt cagaccattaacagctatttaaattggtttcagcagaaaccagggaaagcccctaagctcctgatctatactgcatcca gtttgcaaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactctcaccatcagcagtctgca acctgaagattttgcaacttactactgtcaacagagttacagtacccctccgacgttcggccaagggaccaaggtgga aatcaaa
SEQ ID NO: 540
DIQMTQSPSSLSASVGDRVTITCRASQTINSYLNWFQQKPGKAPKLLIYTA SSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTK VEIK
SEQ ID NO: 541 cagaccattaacagctat
SEQ ID NO: 542
QTINSY
SEQ ID NO: 543 actgcatcc
SEQ ID NO: 544
TAS
SEQ ID NO: 545 caacagagttacagtacccctccgacg
SEQ ID NO: 546
QQSYSTPPT
SEQ ID NO: 547 gaggtgcagttggtggaatttgggggagacctggtcaagccgggggggtccctgagactctcctgtgcagcctctg gattcgccttcagttcctatagcatgaactgggtccgccaggctccagggaagagcctggagtgggtctcctctatca ctaatagtggtagtttcatatactacgcagactcagtgaagggccgattcaccatttccagagacaacgccaagagttc actgtttctgcaaatgaacagcctgagagtcgaggacacggctgtgtatttttgtgcgagtaagacttcacttgactattg gggccagggaaccctggtcaccgtctcctca SEQ ID NO: 548
EVQLVEFGGDLVKPGGSLRLSC AASGFAF S S YSMNWVRQAPGKSLEWVS SITNSGSFIYYADSVKGRFTISRDNAKSSLFLQMNSLRVEDTAVYFCASKT SLDYWGQGTLVTVSS
SEQ ID NO: 549 ggattcgccttcagttcctatagc
SEQ ID NO: 550
GFAFSSYS
SEQ ID NO: 551 atcactaatagtggtagtttcata
SEQ ID NO: 552
ITNSGSFI
SEQ ID NO: 553 gcgagtaagacttcacttgactat
SEQ ID NO: 554
ASKTSLDY
SEQ ID NO: 555 ggtattgtgatgacccagactccactctcctcacctgtcacccttggacagccggcctccatctcctgcaggtctagtc aaagcctcgtgcacagtgatggaaacacctacttgagttggcttcagcagaggccaggccagcctcctagactccta atttctaagatttctgaccggttatctggggtcccagacagattcactggcagtgggacagggacagatttcacactga agatcagcagggtggaagttgaggatgtcggggtttattactgcatgcaaactacacaatttccgacgttcggccaag ggaccaaggtggaaatcaaa
SEQ ID NO: 556
GIVMTQTPLSSPVTLGQPASISCRSSQSLVHSDGNTYLSWLQQRPGQPPRL LISKISDRLSGVPDRFTGSGTGTDFTLKISRVEVEDVGVYYCMQTTQFPTF GQGTKVEIK SEQIDNO: 557 caaagcctcgtgcacagtgatggaaacacctac
SEQIDNO: 558
QSLVHSDGNTY
SEQIDNO: 559 aagatttct
SEQIDNO: 560
KIS
SEQIDNO: 561 atgcaaactacacaatttccgacg
SEQIDNO: 562
MQTTQFPT
SEQIDNO: 563 gaggtgcagctggtggagtctgggggaggcttggtaaagcctggggggtcccttagactctcctgtgtagcctctgg attcaatttcactaacacctggatgagctgggtccgccaggttccagggagggggctggagtgggttggccgtattaa aagcgaaactgatggtgggacaacagactacgctgcacccgtgaaaggcagattcaccatctcaagagatgattca aaaaactcgctgtatctgcaaatgaacagcctgaaaaccgaggacacagccgtgtattcctgtaccacagggatcgg ggactactggggccagggaaccctggtcaccgtctcctca
SEQIDNO: 564
EVQLVESGGGLVKPGGSLRLSCVASGFNFTNTWMSWVRQVPGRGLEWV GRIKSETDGGTTDYAAPVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYSC TTGIGD YWGQGTLVT VS S
SEQIDNO: 565 ggattcaatttcactaacacctgg SEQIDNO: 566
GFNFTNTW
SEQIDNO: 567 attaaaagcgaaactgatggtgggacaaca
SEQIDNO: 568
IKSETDGGTT
SEQIDNO: 569 accacagggatcggggactac
SEQIDNO: 570
TTGIGDY
SEQIDNO: 571 gacatcgtgatgacccagtctccagactccctggctgtgtctctgggcgagagggccaccatcaactgcaagtccag ccagagtgttttatacagctccaacaataagaactacttagcttggtaccagcagaaaccaggacagcctcctaagct gctcatttactgggcatctacccgggaatccggggtccctgaccgattcagtggcagcgggtctgggacagatttcac tctcaccatcagcagcctgcaggctgaagatgtggcagtttattactgtcaacaatattatagtactccgctcactttcgg cggagggaccaaggtggagatcaaa
SEQIDNO: 572
DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPP KLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTP LTFGGGTKVEIK
SEQIDNO: 573 cagagtgttttatacagctccaacaataagaactac
SEQIDNO: 574
QSVLYSSNNKNY SEQIDNO: 575 tgggcatct
SEQIDNO: 576
WAS
SEQIDNO: 577 caacaatattatagtactccgctcact
SEQIDNO: 578
QQYYSTPLT
SEQIDNO: 579 gaggtgcaactggtggagtctgggggaggcctggtcaagcctggggggtccctaagactctcctgtgcagcctctg gattcaccttcagttcttatagcatgaactgggtccgccagtctccagggaaggggctggagtgggtctcatccattag tagtagcagtaatttcatatactacgcagactcagtgaagggccgcttcaccatctccagagacaacgccagaaactc actgtatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtgtgagaagtatcgcaacaactg gctttgactattggggccagggaaccctggtcaccgtctcctcg
SEQIDNO: 580
EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQSPGKGLEWVS SISSSSNFIYYADSVKGRFTISRDNARNSLYLQMNSLRAEDTAVYYCVRSI ATTGFD YWGQGTLVTVS S
SEQIDNO: 581 ggattcaccttcagttcttatagc
SEQIDNO: 582
GFTFSSYS
SEQIDNO: 583 attagtagtagcagtaatttcata SEQIDNO: 584
ISSSSNFI
SEQIDNO: 585 gtgagaagtatcgcaacaactggctttgactat
SEQIDNO: 586
VRSIATTGFDY
SEQIDNO: 587 gatattgtgatgacccagactccactctcctcacctgtcacccttggacagccggcctccatctcctgcaggtctagtc aaagcctcgtacacagtgatggaaatacgtacttgagttggcttcatcagaggccaggccagcctccaagactcctca tttataagatttctaaccggttttctggggtcccagacagattcagtggcagtggggcaggggcagacttcacactgaa aatcagcagggtggaaactgaggatgtcggggtttattactgcgtgcaagctacacaatttccgacgttcggccaagg gaccaaggtggaaatcaaa
SEQIDNO: 588
DIVMTQTPLSSPVTLGQPASISCRSSQSLVHSDGNTYLSWLHQRPGQPPRL LIYKISNRFSGVPDRFSGSGAGADFTLKISRVETEDVGVYYCVQATQFPTF GQGTKVEIK
SEQIDNO: 589 caaagcctcgtacacagtgatggaaatacgtac
SEQIDNO: 590
QSLVHSDGNTY
SEQIDNO: 591 aagatttct
SEQIDNO: 592
KIS SEQIDNO: 593 gtgcaagctacacaatttccgacg
SEQIDNO: 594
VQATQFPT
SEQIDNO: 595 gaggtgcagctggtggagtctgggggaggcctggtcaagcctggggggtccctgagactctcctgtgcagcctctg gattcaccttcagtagctatagcatgaactgggtccgccaggctccagggaaggggctggagtgggtctcatacatta gtagtagtagtagtttcatatatcacgcagactcagtgaagggccgattcaccatttccagagacaacgccaagaact cactgtatctgcaaatgaacagcctgagagtcgaggacacggctgtgtattcctgtgcgagattccgaaccgggaga gatgcttttgatatctggggccaagggacaatggtcaccgtctcttca
SEQIDNO: 596
EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVS YIS S S SSFIYHADS VKGRFTISRDNAKNSLYLQMNSLRVEDTAVYSC ARFR TGRDAFDIWGQGTMVTVS S
SEQIDNO: 597 ggattcaccttcagtagctatagc
SEQIDNO: 598
GFTFSSYS
SEQIDNO: 599 attagtagtagtagtagtttcata
SEQIDNO: 600
ISSSSSFI
SEQIDNO: 601 gcgagattccgaaccgggagagatgcttttgatatc SEQIDNO: 602
ARFRTGRDAFDI
SEQIDNO: 603 gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagt cagagcattaacagctatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcttcca gtttgcaaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactctcaccatcagcagtctgca acctgaagattttgcaacttactactgtcaacagagttacagtacccctccgatcaccttcggccaagggacacgactg gagattaaa
SEQIDNO: 604
DIQMTQSPSSLSASVGDRVTITCRASQSINSYLNWYQQKPGKAPKLLIYAA SSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTR LEIK
SEQIDNO: 605 cagagcattaacagctat
SEQIDNO: 606
QSINSY
SEQIDNO: 607 gctgcttcc
SEQIDNO: 608
AAS
SEQIDNO: 609 caacagagttacagtacccctccgatcacc
SEQIDNO: 610
QQSYSTPPIT SEQ ID NO: 611 gaggtgcagctggtggagtctgggggaggcctggtcaagcctggggggtccctgagactctcctgtgcagcctctg gattcaccttcagtagttatagcatgaattgggtccgccaggctccagggaaggggctggagtgggtctcatacatta gtagtagtagtagtttcatatatcacgcagactcagtgaagggccgattcaccatttccagagacaacgccaagaact cactgtatctgcaaatgaacagtctgagagtcgaggacacggctgtgtattcctgtgcgagattccgaaccgggaga gatgcttttgatatctggggccaagggacaatggtcaccgtctcttca
SEQ ID NO: 612
EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVS
YIS S S SSFIYHADS VKGRFTISRDNAKNSLYLQMNSLRVEDTAVYSC ARFR
TGRDAFDIWGQGTMVTVS S
SEQ ID NO: 613 ggattcaccttcagtagttatagc
SEQ ID NO: 614
GFTFSSYS
SEQ ID NO: 615 attagtagtagtagtagtttcata
SEQ ID NO: 616
ISSSSSFI
SEQ ID NO: 617 gcgagattccgaaccgggagagatgcttttgatatc
SEQ ID NO: 618
ARFRTGRDAFDI
SEQ ID NO: 619 gacatccagatgacccagtctccatcctccctgtctgcatttgtaggagacagagtcaccatcacttgccgggcaagt cagagcattaacagctatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcttcca gtttgcaaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactctcaccatcagcagtctgca acctgaagattttgcaacttactactgtcaacagagttacagtacccctccgatcaccttcggccaagggacacgactg gacattaaa
SEQ ID NO: 620
DIQMTQSPSSLSAFVGDRVTITCRASQSINSYLNWYQQKPGKAPKLLIYAA SSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTR LDIK
SEQ ID NO: 621 cagagcattaacagctat
SEQ ID NO: 622
QSINSY
SEQ ID NO: 623 gctgcttcc
SEQ ID NO: 624
AAS
SEQ ID NO: 625 caacagagttacagtacccctccgatcacc
SEQ ID NO: 626
QQSYSTPPIT
SEQ ID NO: 627 cagctgcaactacaggagtcgggcccaggactggtgaagccttcggagaccctgtccctcacttgcactgtctctgg tggctccatcaccaataataattactactggggctggatccgccagcccccagggaagggactggaatggataggg attatctattatggtgggatcacctactacaatccgtccctcaagagtcgagtcagcatatccgtggacacgtccaaga atcagttctccctgaatctgagttctgtcaccgccgcagacacggctgtctattactgtgcgagactcgatttttggagtg gttatctctttgactactggggccagggagccccggtcaccgtctcctca SEQ ID NO: 628
QLQLQESGPGLVKPSETLSLTCTVSGGSITNNNYYWGWIRQPPGKGLEWI GIIYYGGITYYNPSLKSRVSISVDTSKNQFSLNLSSVTAADTAVYYCARLD FWSGYLFDYWGQGAPVTVS S
SEQ ID NO: 629 ggtggctccatcaccaataataattactac
SEQ ID NO: 630
GGSITNNNYY
SEQ ID NO: 631 atctattatggtgggatcacc
SEQ ID NO: 632
IYYGGIT
SEQ ID NO: 633 gcgagactcgatttttggagtggttatctctttgactac
SEQ ID NO: 634
ARLDFWSGYLFDY
SEQ ID NO: 635 gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagggtcaccatcacttgccgggcgagt cagagcattagcagctatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcatcca gtttgcaaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactctcaccatcaacagtctgca acctgaagattttgcaacttactactgtcaacagagttacagtagtcctccgatcaccttcggccaagggacacgactg gagattaaa
SEQ ID NO: 636
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAA SSLQSGVPSRFSGSGSGTDFTLTINSLQPEDFATYYCQQSYSSPPITFGQGT RLEIK SEQIDNO: 637 cagagcattagcagctat
SEQIDNO: 638
QSISSY
SEQIDNO: 639 gctgcatcc
SEQIDNO: 640
AAS
SEQIDNO: 641 caacagagttacagtagtcctccgatcacc
SEQIDNO: 642
QQSYSSPPIT
SEQIDNO: 643 gaggtgcagctggtggagtctgggggaggcttggaacagcctggagggtccctgagactctcctgtgcagcctctg gattcattttcagtagttatgaaatgaactgggtccgccaggctccagggaaggggctggaatgggtttcatatattagt agtagtggtgataccataaactccgcagactctgtgaagggccgattcaccatctccagagacaacgccaagaaatc actgtatcttcaaatgaacagcctgagagccgaggacacggcagtctattattgtgtgagagggggatattgttctagt agcagttgctttatggcctactattacggcatggacgtctggggccaagggaccacggtcaccgtctcctca
SEQIDNO: 644
EVQLVESGGGLEQPGGSLRLSCAASGFIFSSYEMNWVRQAPGKGLEWVS YISSSGDTINSADSVKGRFTISRDNAKKSLYLQMNSLRAEDTAVYYCVRG GYCSSSSCFMAYYYGMDVWGQGTTVTVSS
SEQIDNO: 645 ggattcattttcagtagttatgaa SEQIDNO: 646
GFIFSSYE
SEQIDNO: 647 attagtagtagtggtgataccata
SEQIDNO: 648
ISSSGDTI
SEQIDNO: 649 gtgagagggggatattgttctagtagcagttgctttatggcctactattacggcatggacgtc
SEQIDNO: 650
VRGGYCSSSSCFMAYYYGMDV
SEQIDNO: 651 gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccggacaagt cagagcattaacagctatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcatcca gtttgcaaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactctcaccatcagcagtctgca acctgaagattttgcaacttactactgtcaacagagttacagtacccctccgatcaccttcggccaagggacacgactg gagattaaa
SEQIDNO: 652
DIQMTQSPSSLSASVGDRVTITCRTSQSINSYLNWYQQKPGKAPKLLIYAA SSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTR LEIK
SEQIDNO: 653 cagagcattaacagctat
SEQIDNO: 654
QSINSY SEQIDNO: 655 gctgcatcc
SEQIDNO: 656
AAS
SEQIDNO: 657 caacagagttacagtacccctccgatcacc
SEQIDNO: 658
QQSYSTPPIT
SEQIDNO: 659 gaggtgcaactggtggagtctgggggaggcccggtcaagcctggggggtccctgagactctcctgtgcaaccgct ggattcaccttcagtagttatagcatgaactgggtccgccaggctccagggaaggggctggagtgggtctcatccatt agtagtcgtagtagtttcatttactacgcagactcagtgaagggccgattcaccatctccagagacaacgccaagaatt cactgtttctgcaaatgaacagcctgagagtcgaggacacggctgtatatttctgtgtgagattccgaatcgggaggg atgcttttgacatctggggccaagggacaatggtcaccgtctcttca
SEQIDNO: 660
EVQLVESGGGPVKPGGSLRLSC ATAGFTF S S YSMNWVRQAPGKGLEWVS SISSRSSFIYYADSVKGRFTISRDNAKNSLFLQMNSLRVEDTAVYFCVRFRI GRD AFDIWGQGTMVTVS S
SEQIDNO: 661 ggattcaccttcagtagttatagc
SEQIDNO: 662
GFTFSSYS
SEQIDNO: 663 attagtagtcgtagtagtttcatt SEQIDNO: 664
ISSRSSFI
SEQIDNO: 665 gtgagattccgaatcgggagggatgcttttgacatc
SEQIDNO: 666
VRFRIGRDAFDI
SEQIDNO: 667 gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacgtgccgggcaagt cagagcattaacagctatttaaattggtatcagcagaaaccagggaaagcccctaagctccttatctattctgcatccag tttgcaaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactctcatcatcagcagtctgcaa cctgaagattttgcaacttactactgtcaacagagtgacagtacccctccgatcaccttcggccaagggacacgactg gagattaaa
SEQIDNO: 668
DIQMTQSPSSLSASVGDRVTITCRASQSINSYLNWYQQKPGKAPKLLIYSA SSLQSGVPSRFSGSGSGTDFTLIISSLQPEDFATYYCQQSDSTPPITFGQGTR LEIK
SEQIDNO: 669 cagagcattaacagctat
SEQIDNO: 670
QSINSY
SEQIDNO: 671 tctgcatcc
SEQIDNO: 672
SAS SEQ ID NO: 673 caacagagtgacagtacccctccgatcacc
SEQ ID NO: 674
QQSDSTPPIT
SEQ ID NO: 675 caggttcagctggtgcagtctgaagctgtggtgaagaagcctggggcctcagtgaaggtctcttgcaaggcttctggt tacaccttaccaactatggtatcggctgggtgcgacaggcccctggacaagggcttgagtggatgggatggatcag cggttacaatggtaacacaaactatgtacagaaactccagggcagagttaccatgaccacagacacatccacgagc acagcctacatggagctgaggagcctgagatctgacgacacggccgtatattattgtgtgagaaataactgggaatat gacttccactattacggtatggacgtctggggccaagggaccacggtcaccgtctcctca
SEQ ID NO: 676
QVQLVQSEAVVKKPGASVKVSCKASGYTFTNYGIGWVRQAPGQGLEWM
GWISGYNGNTNYVQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYC
VRNNWEYDFHYYGMDVWGQGTTVTVSS
SEQ ID NO: 677 ggttacacctttaccaactatggt
SEQ ID NO: 678
GYTFTNYG
SEQ ID NO: 679 atcagcggttacaatggtaacaca
SEQ ID NO: 680
ISGYNGNT
SEQ ID NO: 681 gtgagaaataactgggaatatgacttccactattacggtatggacgtc SEQIDNO: 682
VRNNWEYDFHYYGMDV
SEQIDNO: 683 gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagt cagagcattagcagttatataaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgttgcatcca gtttgcaaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactctcaccatcagcagtctgca acctgaagattttgcaacttattactgtcaacagagttacagtacccctgcgatcaccttcggccaagggacacgactg gagattaaa
SEQIDNO: 684
DIQMTQSPSSLSASVGDRVTITCRASQSISSYINWYQQKPGKAPKLLIYVA SSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPAITFGQGT RLEIK
SEQIDNO: 685 cagagcattagcagttat
SEQIDNO: 686
QSISSY
SEQIDNO: 687 gttgcatcc
SEQIDNO: 688
VAS
SEQIDNO: 689 caacagagttacagtacccctgcgatcacc
SEQIDNO: 690
QQSYSTPAIT SEQ ID NO: 691 caggtccatctggtggagtctgggggaggcttggtcaagcctggagggtccctgagactctcctgtgcagcctctgg attcaccttcagtgactactatatgacctggatccgccaggctccagggaagggactggactgggtttcatatattagt ggtggtggtagtaccgtaaactacgcagactctgtgaagggccgcttcaccatctccagggacaacgccaagaattt aatgtctctgcaaatgaacagcctgggagtcgaggacacggccgtgtattattgtgcgagagaagggggacacagtt atggtcgcttttccttcggtttggacgtctggggccaagggaccacggtcaccgtctcctca
SEQ ID NO: 692
QVHLVESGGGLVKPGGSLRLSCAASGFTFSDYYMTWIRQAPGKGLDWVS YISGGGSTVNYADSVKGRFTISRDNAKNLMSLQMNSLGVEDTAVYYCAR EGGHSYGRFSFGLDVWGQGTTVTVSS
SEQ ID NO: 693 ggattcaccttcagtgactactat
SEQ ID NO: 694
GFTFSDYY
SEQ ID NO: 695 attagtggtggtggtagtaccgta
SEQ ID NO: 696
ISGGGSTV
SEQ ID NO: 697 gcgagagaagggggacacagttatggtcgcttttccttcggtttggacgtc
SEQ ID NO: 698
AREGGHSYGRFSFGLDV
SEQ ID NO: 699 gacatccagatggcccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagt cagagcattagcagatatttaaattggtatcagcacaaagcagggaaagcccctaaactcctgatctatgctgcatcca gtttacaaagtggggtcccatcaaggttcagcggcagtggatctgggacagatttcactctcaccatcagcagtctgc agcctgaagattttgcaacttactactgtcaacagagttacagtaaccctccgatcaccttcggccaagggacacgact ggagattaaa
SEQ ID NO: 700
DIQMAQSPSSLSASVGDRVTITCRASQSISRYLNWYQHKAGKAPKLLIYA ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSNPPITFGQG TRLEIK
SEQ ID NO: 701 cagagcattagcagatat
SEQ ID NO: 702
QSISRY
SEQ ID NO: 703 gctgcatcc
SEQ ID NO: 704
AAS
SEQ ID NO: 705 caacagagttacagtaaccctccgatcacc
SEQ ID NO: 706
QQSYSNPPIT
SEQ ID NO: 707 caggtacagctgcaggagtcgggcccaggactggtgaagccttcacagaccctgtccctcacctgcactgtctctgg tggctccatcagcagtggtaattattactggagctggatccgccagcacccagggaagggcctggagtggattgggt acatcaactacagtggatacacctactacaatccgtccctcaagagtcgaattaacatatcagcagacacgtctaaga atcagttctccctgaaggtgagctctgtgacggccgcggacacggccgtgtattactgtgcgagacataactcgaatt atgaaatggactactggggccagggaaccctggtcaccgtctcttca SEQ ID NO: 708
QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGNYYWSWIRQHPGKGLEWI GYINYSGYTYYNPSLKSRINISADTSKNQFSLKVSSVTAADTAVYYCARH NSNYEMD YWGQGTL VTVS S
SEQ ID NO: 709 ggtggctccatcagcagtggtaattattac
SEQ ID NO: 710
GGSISSGNYY
SEQ ID NO: 711 atcaactacagtggatacacc
SEQ ID NO: 712
INYSGYT
SEQ ID NO: 713 gcgagacataactcgaattatgaaatggactac
SEQ ID NO: 714
ARHNSNYEMDY
SEQ ID NO: 715 gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagt cagagcattagcagctatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgctgcatcca gtttgcaaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactctcaccatcagcagtctgca acctgaagattttgcaacttactactgtcaacagagttacagtacccctccgatcaccttcggccaagggacacgactg gagattaaa
SEQ ID NO: 716
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAA SSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTR LEIK SEQIDNO: 717 cagagcattagcagctat
SEQIDNO: 718
QSISSY
SEQIDNO: 719 gctgcatcc
SEQIDNO: 720
AAS
SEQIDNO: 721 caacagagttacagtacccctccgatcacc
SEQIDNO: 722
QQSYSTPPIT
SEQIDNO: 723 gaggtgcagctggtggagtctgggggaggcctggtcaagcctggggagtccctgaaactctcctgtgcagcctctg gattcaccttcagtaattataacatgcactgggtccggcaggctccagggaaggggctgaagtgggtctcatccatta gtggtagtagtagttacatatactacgcagactcagtgaagggccgattcaccatctccagagacaacaccaataaat cactatatctccaaatgaacagcctgagagccgaggacacggctgtctattactgtgcgagactctatagggggggt atggacgtctggggccaggggaccacggtcaccgtctcctca
SEQIDNO: 724
EVQLVESGGGLVKPGESLKLSCAASGFTFSNYNMHWVRQAPGKGLKWV SSISGSSSYIYYADSVKGRFTISRDNTNKSLYLQMNSLRAEDTAVYYCARL YRGGMD VWGQGTTVTVS S
SEQIDNO: 725 ggattcaccttcagtaattataac SEQIDNO: 726
GFTFSNYN
SEQIDNO: 727 attagtggtagtagtagttacata
SEQIDNO: 728
ISGSSSYI
SEQIDNO: 729 gcgagactctatagggggggtatggacgtc
SEQIDNO: 730
ARLYRGGMDV
SEQIDNO: 731 caggtgcagctacaacagtggggcgcaggactgttgaagccttcggagaccctgtccctcacctgcgctgtctatgg tgggtccttcagtggttactactggagctggatccgccagcccccagggaaggggctggagtggattggggaaatc agtcatcgtggaagcaccaactacaatccgtccctcaagagtcgagtcaccatatcagtagacacgtccaggaacca gttctccctgaagctgagctctgtgaccgccgcggacacggctgtgtatttctgtgcgaattattttgggagatcctactt tgacgactggggcccgggaaccctggtcaccgtctcctca
SEQIDNO: 732
QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIG EISHRGSTNYNPSLKSRVTISVDTSRNQFSLKLSSVTAADTAVYFCANYFG RS YFDDWGPGFL VT VS S
SEQIDNO: 733 ggtgggtccttcagtggttactac
SEQIDNO: 734
GGSFSGYY SEQIDNO: 735 atcagtcatcgtggaagcacc
SEQIDNO: 736
ISHRGST
SEQIDNO: 737 gcgaattattttgggagatcctactttgacgac
SEQIDNO: 738
ANYFGRSYFDD
SEQIDNO: 739 gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagactcaccatcacttgccgggcaagt cagagcattagcagctatttaaattggtatcagcagaaaccagggaaagcccctaatctcctgatctatgctgcatcca gtttgcaaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactctcaccatcagcagtctgca acctgaagattttgcaacttactactgtcaacagagtttcagtagccctccgatcaccttcggccaagggacacgactg gagattaaa
SEQIDNO: 740
DIQMTQSPSSLSASVGDRLTITCRASQSISSYLNWYQQKPGKAPNLLIYAA
SSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSFSSPPITFGQGTR
LEIK
SEQIDNO: 741 cagagcattagcagctat
SEQIDNO: 742
QSISSY
SEQIDNO: 743 gctgcatcc SEQIDNO: 744
AAS
SEQIDNO: 745 caacagagtttcagtagccctccgatcacc
SEQIDNO: 746
QQSFSSPPIT
SEQIDNO: 747 caggtgcagctacagcagtggggcgcaggactgttgaagccttcggataccctgtccctcacctgcgctgtctatggt gggtccttcagtgattactactggagttggatccgccagcccccagggaagggactggaatggattggggaaggca gtcatactggaagaacaaactacaacccgtccctcaagagtcgaatcaccatatcagtagacacgtccaagaaccag ttctccctgaagctgaactctgtgaccgccgcggacacggctttatattactgtgcgagaggagcaccagctggtaag ggcttcgacccctggggccagggaaccctggtcaccgtctcctca
SEQIDNO: 748
QVQLQQWGAGLLKPSDTLSLTCAVYGGSFSDYYWSWIRQPPGKGLEWIG EGSHTGRTNYNPSLKSRITISVDTSKNQFSLKLNSVTAADTALYYCARGAP AGKGFDPWGQGTLVTVS S
SEQIDNO: 749 ggtgggtccttcagtgattactac
SEQIDNO: 750
GGSFSDYY
SEQIDNO: 751 ggcagtcatactggaagaaca
SEQIDNO: 752
GSHTGRT SEQIDNO: 753 gcgagaggagcaccagctggtaagggcttcgacccc
SEQIDNO: 754
ARGAPAGKGFDP
SEQIDNO: 755 gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagt cagagcattagcaggtatttaaattggtatcagcagaaaccagggaaagcccctaagctcctcatctatgctgcatcca gtttgcaaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactctcaccatcagcagtctgca acctgaagattttgcaacttactactgtcaacagagttacagtacccctccgatcaccttcggccaagggacacgactg gagattaaa
SEQIDNO: 756
DIQMTQSPSSLSASVGDRVTITCRASQSISRYLNWYQQKPGKAPKLLIYAA SSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTR LEIK
SEQIDNO: 757 cagagcattagcaggtat
SEQIDNO: 758
QSISRY
SEQIDNO: 759 gctgcatcc
SEQIDNO: 760
AAS
SEQIDNO: 761 caacagagttacagtacccctccgatcacc SEQ ID NO: 762
QQSYSTPPIT
SEQ ID NO: 763 caggtacaactggtggagtctgggggaggcgtggtccagcctgggaggtccctgagactctcctgtgcagcgtctg gattcaccttcactaactatggcatacactgggtccgccaggctccaggcaaggggctggagtgggtggcagttatat ggtatgatgaaacttatagattcagtgcagactccgtgaagggccgattcaccatctccagagacaactccaagaaca cggtgtatctgcaaatgaacagcctgagagccgaggacacggctgtttattactgtgcgcgccatagtgggaattact tacgtgacttttacggtttggacgtctggggccaagggactacggtcaccgtctcctca
SEQ ID NO: 764
QVQLVESGGGVVQPGRSLRLSCAASGFTFTNYGIHWVRQAPGKGLEWV AVIWYDETYRFSADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCA RHSGNYLRDFYGLDVWGQGTTVTVSS
SEQ ID NO: 765 ggattcaccttcactaactatggc
SEQ ID NO: 766
GFTFTNYG
SEQ ID NO: 767 atatggtatgatgaaacttataga
SEQ ID NO: 768
IWYDETYR
SEQ ID NO: 769 gcgcgccatagtgggaattacttacgtgacttttacggtttggacgtc
SEQ ID NO: 770
ARHSGNYLRDFYGLDV SEQ ID NO: 771 gacatccagatgacccagtctccttcctccctttctgcatctgttggagacagagtcaccatcacttgccgggcaagtc agagcattagcagctatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgttgcatccag tttgcaaagtggggtcccatcaaggttcagtggcagtggatctgggacagatttcactctcaccatcagcagtctgcaa cctgaagattttgcaacttactactgtcaacagagttacaatacccctccgatcaccttcggccaagggacacgactgg agattaaa
SEQ ID NO: 772
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYVA SSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYNTPPITFGQGT RLEIK
SEQ ID NO: 773 cagagcattagcagctat
SEQ ID NO: 774
QSISSY
SEQ ID NO: 775 gttgcatcc
SEQ ID NO: 776
VAS
SEQ ID NO: 777 caacagagttacaatacccctccgatcacc
SEQ ID NO: 778
QQSYNTPPIT
SEQ ID NO: 779 caggtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtccctgagactctcctgtgcagcgtctg gattcaccttcagtttctatggcatgcactgggtccgccagactcctggcaaggggctggagtgggtggcagtcatat ggtatgatggaagttctaaatactatgcagactccgtgaagggccgattcaccatctccagagacaattccaagaact cgctgtatctgcaaatgagcagcctgagagccgaggacacggctctatattactgtgtcgctcacggcaccaaccctt ttgagtactggggccagggaaccctggtcacc tctcctca
SEQ ID NO: 780
QVQLVESGGGVVQPGRSLRLSCAASGFTFSFYGMHWVRQTPGKGLEWV AVIWYDGSSKYYADSVKGRFTISRDNSKNSLYLQMSSLRAEDTALYYCV AHGTNPFEYWGQGTL VTVS S
SEQ ID NO: 781 ggattcaccttcagtttctatggc
SEQ ID NO: 782
GFTFSFYG
SEQ ID NO: 783 atatggtatgatggaagttctaaa
SEQ ID NO: 784
IWYDGSSK
SEQ ID NO: 785 gtcgctcacggcaccaacccttttgagtac
SEQ ID NO: 786 VAHGTNPFEY
Example 4: Binding Parameters of example CDH15 antibodies
[00375] Equilibrium dissociation constants (KD) for CADH15 binding to purified anti- CDH15 monoclonal antibodies were determined using a real-time surface plasmon resonance (SPR) based Biacore 4000 biosensor. All binding studies were performed in lOmM HEPES, 150mM NaCl, 1 mM CaCh, 0.5 mM MgCh, 0.05% and 0.05% v/v surfactant Tween-20, pH 7.4 (HBS-P) running buffer at 25 °C and 37 °C. The Biacore CM5 sensor surface was first derivatized by amine coupling with a monoclonal mouse anti-human Fc antibody (REGN2567) or anti -mouse Fc specific antibody (GE Healthcare, # BRI 00838) to capture anti-CDH15 monoclonal antibodies. Different concentrations of CDH15 reagents, human CDH15 extracellular domain expressed with a C-terminal myc-myc-hexahistidine tag (hCDH15-MMH; REGN4409), human CDH15(K584Q) extracellular domain expressed with a C-terminal myc- myc-hexahistidine tag (hCDH15_K584Q -MMH; REGN5943), monkey CDH15 extracellular domain expressed with a C-terminal myc-myc-hexahistidine tag (mfCDH15-MMH;
REGN4586), and mouse CDH15 extracellular domain expressed with a C-terminal myc-myc- hexahistidine tag (mCDH15-MMH; REGN4410), at concentrations ranging from 6.25 nM to 100 nM in a series of 4-fold dilutions prepared in HBS-P running buffer were injected at a flow rate of 30 pL/min for 2.5 or 4 minutes. The dissociation of different CDH15 reagents bound to anti- CDH15 monoclonal antibodies was monitored for 7 or 10 minutes in HBS-P running buffer. At the end of each cycle, the anti-CDH15 monoclonal antibodies capture surface was regenerated using a 12 or 60 sec injection of 20mM H3PO4 or Glycine, pH 1.5.
[00376] The association rate (ka) and dissociation rate (kd) were determined by fitting the real-time binding sensorgrams to a 1 : 1 binding model with mass transport limitation using Scrubber 2.0c curve-fitting software. Binding dissociation equilibrium constant (KD) and dissociative half-life (t’A) were calculated from the kinetic rates as: kd
KD (M) = — , and fl/2 (min) v ’ ka v ’
[00377] The equilibrium and kinetic binding parameters for anti-CDH15 monoclonal antibodies binding to hCDH15-MMH, hCDH15(K584Q).MMH, mfCDH15-MMH, and mCDH15-MMH, at 25 °C and 37 °C are shown in Table 4 through Table 11, respectively.
Results
[00378] At 25 °C, anti-CDH15 monoclonal antibodies bound to hCDH15-MMH with KD values ranging from 27.6pM to 125 nM, as shown in Table 4 below. Table 4: Kinetic binding parameters for the interaction of hCDH15-MMH with anti-
CDH15 monoclonal antibodies at 25 °C.
[00379] At 37 °C, anti-CDH15 monoclonal antibodies bound to hCDH15-MMH with KD values ranging from 218 pM to 234 nM, as shown in Table 5 below.
Table 5: Kinetic binding parameters for the interaction of hCDH15-MMH with anti- CDH15 monoclonal antibodies at 37 °C.
[00380] At 25 °C, anti-CDH15 monoclonal antibodies bound to hCDH15(K584Q)-MMH with KD values ranging from 65.6 pM to 235 nM, as shown in Table 6 below.
Table 6: Kinetic binding parameters for the interaction of hCDH15(K584Q)-MMH with anti-CDH15 monoclonal antibodies at 25 °C.
[00381] At 37 °C, anti-CDH15 monoclonal antibodies bound to hCDH15(K584Q)-MMH with KD values ranging from 375pM to 334nM, as shown in Table 7 below. Table 7: Kinetic binding parameters for the interaction of hCDH15(K584Q)-MMH with anti-CDH15 monoclonal antibodies at 37 °C.
[00382] At 25 °C, anti-CDH15 monoclonal antibodies bound to mfCDH15-MMH with KD values ranging from 86.7 pM to 1.33 pM, as shown in Table 8 below.
Table 8: Kinetic binding parameters for the interaction of mfCDH15-MMH with anti-
CDH15 monoclonal antibodies at 25°C.
[00383] At 37 °C, anti-CDH15 monoclonal antibodies bound to mfCDH15-MMH with
KD values ranging from 151 pM to 539 nM, as shown in Table 9 below.
Table 9: Kinetic binding parameters for the interaction of mfCDH15-MMH with anti-
CDH15 monoclonal antibodies at 37 °C.
[00384] At 25 °C, anti-CDH15 monoclonal antibodies bound to mCDH15-MMH with KD values ranging from 129 pM to 323 nM, as shown in Table 10 below. Table 10: Kinetic binding parameters for the interaction of mCDH15-MMH with anti-
CDH15 monoclonal antibodies at 25°C.
[00385] At 37 °C, anti-CDH15 monoclonal antibodies bound to mCDH15-MMH with KD values ranging from 637 pM to 391 nM, as shown in Table 11 below.
Table 11: Kinetic binding parameters for the interaction of mCDH15-MMH with anti-
CDH15 monoclonal antibodies at 37 °C.
Example 5: Cross-competition between example CDH15 antibodies
[00386] Binding competition between different anti-CDH15 monoclonal antibodies (mAbs) was determined using a real time, label-free bio-layer interferometry (BLI) assay on the Octet HTX biosensor platform (Sartorius ForteBio Corp.). The entire experiment was performed at 25 °C in lOmM HEPES buffer containing 150mM NaCl, 0.5mM MgCh, ImM CaCh, Img/mL BSA, 0.02% NaNs, and 0.05% v/v Surfactant Tween-20 at pH7.4 (HBS-P) with the plate shaking at a speed of 1000 rpm. To assess the ability of one antibody to compete with another antibody for binding to CDH15, around 0.3 nM of recombinant human CDH15 extracellular domain expressed with a C-terminal myc-myc-hexahistidine (hCDH15-MMH) was first captured onto anti-Penta-His antibody coated Octet biosensor tips (Fortebio Inc, # 18-5122) by submerging the biosensor tips in wells containing 20pg/mL solution of the hCDH15-MMH for 180 seconds. The antigen captured biosensor tips were then saturated with a first anti-CDH15 monoclonal antibody (subsequently referred to as mAb-1) by dipping into wells containing 50pg/mL solution of mAb-1 for 4 minutes. The biosensor tips were then dipped into wells containing 50pg/mL solution of a second anti-CDH15 monoclonal antibody (subsequently referred to as mAb-2) for 3 minutes. The biosensor tips were washed in HBS-P buffer in between every step. The real-time binding response was monitored and the binding response at the end of every step was recorded. The response of mAb-2 binding to hCDH15-MMH pre-complexed with mAb-1 was compared to the binding response hCDH15-MMH alone (mAb-1 = isotype control), and if pre-bound mAb-1 reduced binding of mAb-2 by more than 60%, mAb-1 is categorized as a competitor to mAb-2. Competitors of each antibody tested are summarized in Table 12 below. Table 12. Cross-competition between different anti-CDH15 monoclonal antibodies for binding to hCDH15-MMH.
Example 6: Binding of purified CDH15 antibodies to mouse muscle stem cells
[00387] As described above, CDH15 mRNA is highly expressed in MuSCs. As shown in Figure 7A, the example anti-hCDH15 antibody, 8787, was able to bind to mouse MuSCs associated with individual myofibers. Accordingly, this demonstrates that anti-hCDH15 antibodies of the present disclosure are capable of specific binding to mCDH15 on muscle stem cells that are located within their native niche (i.e., attached to a single myofiber).
[00388] Moreover, as shown in Figure 7B, an example commercial anti-mCDH15 antibody was also able to bind to MuSCs adjacent to muscle fibers in muscle cross sections from WT mice. Furthermore, Figure 7C demonstrates that, in wildtype mice, almost all Pax7+ MuSCs (-95%) displayed some degree of CDH15 protein expressed on the cell surface, while CDH15 protein was absent from Pax7+ MuSCs in CDH15-/- mice. Accordingly, this demonstrates that commercial anti-mouse CDH15 antibodies bind to mCDH15 in patterns that are comparable to the anti-hCDH15 antibodies of the disclosure, while no binding is detected in CDH15-/- mice, as expected.
Example 7: Binding of purified CDH15 antibodies to rhabdomyosarcoma cells.
[00389] CDH15 is highly expressed in RMS and can specifically distinguish RMS from other sarcomas. Moreover, high CDH15 expression is associated with chemoresistance and a poorer prognosis for soft tissue sarcomas. The ability of the anti-hCDH15 antibodies of the present disclosure to bind to RMS cells was examined. As shown in Figure 8, example anti- hCDH15 antibodies 8787 and 9295 bound specifically to alveolar and embryonal RMS cells, but not to glioblastoma cells used as a control. Moreover, 9295 was able to specifically bind to RMS tumorspheres (see Figure 9). Accordingly, this demonstrates that anti-hCDH15 antibodies of the present disclosure are capable of specific binding to hCDH15 on RMS cells and tumorspheres.
Methods
In vivo muscle injury and histological analysis
[00390] For assessing muscle regenerative capacity in vivo (Figure 2A-C, Figure 6A-D), WT and CDH15-/- mice were anesthetized via isoflurane and leg shaved and the tibialis anterior (TA) muscle was subsequently injected with 50uL of lOuM cardiotoxin (CTX, Sigma #217503, lot #3123340 diluted in 0.9% saline). At either 5, 15, or 25 days post injury (dpi), mice were sacrificed and TA muscles were dissected and cryopreserved in O.C.T. compound (Tissue-Tek Catalog # 4583) by freezing in liquid nitrogen-cooled isopentane. Tissues were stored at -80°C and subsequently sectioned onto SuperFrost Plus charged glass slides (ThermoFisher Scientific Catalog # 12-550-15) at 12pm thickness. Sections were fixed with 4% PFA for 15 minutes.
Slides were then washed 3 times for 5 minutes each with PBS. Tissue sections were covered with blocking buffer (20% Goat Serum, 0.3% Triton, in PBS) for 1 hour at RT. Primary antibodies were added (diluted 1 : 100 in blocking buffer for Laminin: Sigma L9393) and incubated overnight. Slides were then washed with PBS and incubated for 1 hour with secondary antibody (diluted 1 :250 in blocking buffer, Invitrogen A32733). Slides were washed with PBS, counterstained with Hoechst 33342 (ThermoFisher, catalog # 00-4958-02, 1 : 1000) for 5 minutes, and mounted in Fluoromount G (ThermoFisher Cat #00-4958-02). Slides were dried overnight, and subsequently imaged using a Zeiss Axioscan microscope. The cross sectional area and number of nuclei per myofiber were quantified using HALO imaging software (Indica Labs) and Python.
Ex vivo muscle function analysis
[00391] For assessing functional regeneration (Figure 3A-B), WT and CDH15-/- mice were anesthetized and tibialis anterior and extensor digitorum longus (EDL) muscles were injected with 50uL of lOuM cardiotoxin (Sigma #217503, lot #3123340 diluted in 0.9% saline). 15 days after injury, mice were sacrificed and EDL muscles were carefully dissected and muscle force production was assessed using the Muscle Strip System from MyoDynamics (840MD). Sutures were tied around the EDL tendon, and the EDL was subsequently locked into place into the 840MD system. The media used in the 840MD system was Tyrode’s Solution (Modified II) from Boston BioProducts BSS-375. The EDL was stretched to optimal length, and subsequently stimulated with a range of tetanic stimuli (20V for 2 ms with pulse frequency starting at 25 Hz and going up to 150 Hz with 2 minutes in between each bout). Before removing the EDL from the 840MD system, the length of the EDL was recorded. Then the EDL was removed and weighed, and these data were used to calculate specific force. The maximal tetanic force produced by the EDL 15 days post injury was then plotted in relation to the maximal tetanic force of uninjured mice of the same respective genotype.
Muscle stem cell activation on isolated single myofibers
[00392] To assess the quiescence-to-activation transition of MuSCs ex vivo within their native myofiber-associated niche (Figure 4A-B), the gastrocnemius muscle of WT and CDH15- /- mice was removed, collagenase digested, and individual myofibers were isolated, washed, and incubated for 48 hours at 37°C at 5% CO2, in DMEM + 20% FBS. After 48 hours, myofibers were fixed with 4% paraformaldehyde and stained for Pax7 (Developmental Studies Hybridoma Bank: PAX7), Hoechst (Invitrogen: H3570), and actin (Invitrogen: A22287). Myofibers were then transferred to microscope slides, coverslipped, and imaged using a Zeiss LSM 710 confocal microscope. Using Zen software (Zeiss), the total number of Pax7+ MuSCs as well as the number of Pax7+ MuSCs within each clonal cluster were manually quantified (from n = 3 WT and 3 CDH15-/- mice), and the percent of clones containing only one Pax7+ MuSC or more than one MuSC, as well as the total number of MuSCs per cluster were plotted.
RNAseq of FACS isolated MuSCs
[00393] To determine whether CDH15 deletion in quiescent MuSCs affects their transcriptional profile, MuSCs from healthy, uninjured WT and CDH15-/- mice were isolated via FACS (Figure 5A-B). Mice were anesthetized and sacrificed, and the gastrocnemius, soleus, quadriceps, tibialis anterior, and extensor digitorum longus muscles were isolated and minced. Minced muscles were incubated in a rocking shaker at 37°C in 700U/mL of collagenase, type II (Gibco, 16050-122) in Ham’s F10 buffer (Life Technologies) for 1.5 hours, and then switched to a digestion media containing lOOU/mL collagenase type II and 2U/mL dispase II (Roche). Following enzymatic digestion, the muscle slurry was pipetted up and down through a lOmL syringe with 20G needle ten times, and then passed through a 70um cell strainer. Satellite Cell Isolation Beads (Miltenyi Biotec.) were used to purify the samples, and then samples were labeled with fluorophore conjugated antibodies against a7-integrin (R&D Systems, FAB3518G) and CD34 (BD Biosciences, 560230) to stain MuSCs on ice for 20 minutes. Samples were washed and suspended in FACS buffer, and a7-integrin+/CD34+ MuSCs were sorted on a FACSAria Fusion. Sorted MuSCs were lysed in Trizol and RNA isolated with miRNeasy Micro kit (Qiagen).
[00394] Strand-specific RNA-seq libraries were prepared using KAPA mRNA HyperPrep (Roche Sequencing Solutions). Twelve-cycle PCR was performed to amplify libraries.
Sequencing was performed on Illumina HiSeq 2500 (Illumina) by multiplexed single-read run with 33 cycles. Raw BCL sequence data were converted to FASTQ format via Illumina Casava vl .8.2. Reads were decoded based on their barcodes, and read quality was evaluated with FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Reads were mapped to the mouse transcriptome (NCBI GRCm38) using Array Studio (QIAGEN OmicSoft) allowing two mismatches. Reads mapped to the exons of a gene were summed at the gene level.
[00395] Transcriptomic analysis was performed using custom R code. The DESeq2 package was used for differential gene expression analysis, and significantly perturbed genes were defined as those with fold changes no less than 1.5 in either up or down direction and with Benjamini and Hochberg multiple-comparison adjusted P < .05. Genome Browser PWM analysis within the Enrichr application was used to identify enrichment of genes with specific transcription factor binding motifs that were differentially regulated in WT compared to CDH15- /- MuSCs.
CDH15 antibody binding to single myofiber associated MuSCs
[00396] For individual myofiber isolation (Figure 7A), the gastrocnemius muscle was removed, collagenase digested, and individual myofibers were isolated, washed, and incubated overnight at 37°C at 5% CO2, in DMEM + 10% horse serum. Following overnight incubation, single myofibers were incubated with lOOnM of either anti-hCDH15 antibody (REGN8787) or an isotype control antibody for 30 minutes. Myofibers were then washed twice in DMEM + 10% horse serum, and subsequently incubated with lOug/mL of fluorescent-conjugated secondary antibodies for 30 minutes, washed twice in DMEM + 10% horse serum, and then fixed with 4% paraformaldehyde (PF A) for 15 minutes at room temperature. Individual fibers were then washed twice with PBS, stained with Hoechst for 5 minutes, washed once more with PBS, then transferred to microscope slides, coverslipped, and imaged using a Zeiss LSM 710 confocal microscope.
Quantification of the percentage ofMuSCs expressing CDH15 protein at the cell surface
[00397] For detection of CDH15 protein expression and localization in tissue sections (Figures 7B-C), the gastrocnemius muscle of wildtype or CDH15-/- mice was removed and cryopreserved in O.C.T. compound (Tissue-Tek Catalog # 4583) by freezing in liquid nitrogen- cooled isopentane. Tissues were stored at -80°C and subsequently sectioned onto SuperFrost Plus charged glass slides (ThermoFisher Scientific Catalog # 12-550-15) at 12pm thickness. Sections were fixed with 4% PFA for 15 minutes. Slides were then washed 3 times for 5 minutes each with PBS. Tissue sections were covered with blocking buffer (20% Goat Serum, 0.3% Triton, in PBS) for 1 hour at RT. Primary antibodies were added (diluted 1 : 100 in blocking buffer for Laminin: Sigma L9393, CDH15: BD 611101, and Pax7 DSHB: Pax7) and incubated overnight. Slides were then washed with PBS and incubated for 1 hour with secondary antibody (diluted 1 :250 in blocking buffer, Invitrogen). Slides were washed with PBS, counterstained with Hoechst 33342 (ThermoFisher, catalog # 00-4958-02, 1: 1000) for 5 minutes, and mounted in Fluoromount G (ThermoFisher Cat #00-4958-02). Slides were dried overnight, and subsequently imaged using a Zeiss Axioscan microscope. The total number of Pax7+ cells, as well as the number of Pax7+ cells that were positive for CDH15 staining were manually counted and quantified.
Binding of CDH15 antibodies to rhabdomyosarcoma cells
[00398] To determine whether CDH15 antibodies can bind specifically to rhabdomyosarcoma cells but not other tumor cells (e.g., glioblastoma), in vitro binding assays were performed (Figure 8, Figure 9). The rhabdomyosarcoma cell lines that were tested were RH30 (ATCC) and RD (ATCC), and a glioblastoma cell line U87 (ATCC) was used as a negative control. For adherent cell culturing media, the RH30 cell line used RPMI-1640 with 10% FBS and the RD and U87 used DMEM (11995-065) with 10% FBS.
[00399] For live staining adherent cells, the cells lines were seeded in collagen coated 96 well plates and stained 72 hours after plating. Cells were incubated in 100 nM of CDH15 antibody or isotype control antibody for 30 minutes, washed twice with media, then incubated for 30 minutes in fluorescent conjugated secondary at 10 ug/mL (Invitrogen A-21445 for human antibodies and Invitrogen A-21235 for mouse antibodies). Cells were washed two more times with media and then fixed for 15 minutes with 4% PFA. Cells then were washed with PBS, stained with Hoescht (1 : 1000) for 5 minutes, and washed two more times with PBS. Cells were then incubated in blocking buffer (20% Goat Serum, 0.3% Triton, in PBS) for 1 hour at RT. Primary antibody was then added (diluted 1 : 100 in blocking buffer with myogenin: Abeam AB 124800) and incubated overnight. Cells were washed twice with PBS then secondaries were added for 1 hour (diluted 1 :200 in blocking buffer, Invitrogen Al 1035). Cells were washed twice with PBS and imaged on Zeiss Axio observer microscope.
[00400] For tumorsphere culturing, the RD cells were seeded in a low attachment 6 well plate (Corning 3471) and cultured in tumorsphere media (1 :1 DMEM (11995-065) & Ham's F12 (11765-054) + 2X B27 supplement (17504004) + lOng/mL human bFGF (Thermo, RP-8628) + 10 ng/mL human EGF (Thermo, PHG0311) + 1% Penicillin Streptomycin (Catalog # 15140148)) until tumorspheres were formed. Once tumorspheres developed, cells were transferred into a 15 mL conical tube and let settle by gravity for 10 minutes (every wash step cells were allowed to settle by gravity for 10 minutes). Cells were incubated in lOOnM of CDH15 or isotype antibody for 30 minutes, washed twice with media the incubated for 30 minutes in fluorescent conjugated secondary at 10 ug/mL (Invitrogen A-21445). Cells were washed two more times with media and then fixed for 15 minutes with 4% PFA. Cells were thenwashed with PBS, stained with Hoescht (1 : 1000) for 5 minutes, and washed two more times with PBS. Cells were then incubated in blocking buffer (20% Goat Serum, 0.3% Triton, in PBS) for 1 hour at RT. Primary antibody was then added (diluted 1 : 100 in blocking buffer with myogenin: Abeam AB 124800) and incubated overnight. Cells were washed twice with PBS then secondaries were added for 1 hour (Diluted 1:200 in blocking buffer, Invitrogen Al 1035). Cells were washed twice with PBS, mounted on a slide in fluoromount G (Invitrogen 00-4958-02), cover slipped and imaged using a Zeiss LSM 710 confocal microscope.
Example 8: Retargeting AAV with anti-cadherin 15 antigen-binding domains
[00401] Figure 10 shows AAV9-based retargeted virus transduction of C2C12 mouse myoblasts after 3 days of transduction with 2.5 x 105 vg/cell of AAV9 expressing eGFP under the control of the CAG promoter. Provided are representative immunofluorescence images depicting transduction efficiency via GFP fluorescence of C2C12 myoblasts transduced with WT AAV9 particles, N272A de-targeted AAV9 particles, N272A de-targeted AAV9 particles conjugated to an anti-ASGRl mAb at a 1:8 mosaic decoration, N272A de-targeted AAV9 particles conjugated to an anti-CDH15 mAb (9295 mAb; HC = SEQ ID NOs: 832 [DNA] and 833 [Protein]; LC = SEQ ID NOs: 834 [DNA] and 835 [Protein]) at a 1 :8 mosaic decoration, N272A de-targeted AAV9 particles conjugated to an anti-ASGRl Fab at a 1:8 mosaic decoration, N272A de-targeted AAV9 particles conjugated to an anti-CDH15 Fab (9295 Fab; HC = SEQ ID NOs: 836 [DNA] and 837 [Protein]; LC = SEQ ID NOs: 834 [DNA] and 835 [Protein]) at a 1:8 mosaic decoration, N272A de-targeted AAV9 particles conjugated to an anti- ASGRl Fab at a 1 :2 mosaic decoration, and N272A de-targeted AAV9 particles conjugated to 9295 Fab at a 1 :2 mosaic decoration. As shown in Figure 10, conjugation of REGN9295-based anti-CDH15 mAb and Fab, when conjugated at a 1 :8 mosaic, improves transduction of C2C12 myoblasts compared to WT AAV9.
[00402] Figure 11 shows AAV9-based retargeted virus transduction of human skeletal myoblasts after 3 days of transduction with 2.5 x 105 vg/cell of AAV9 expressing eGFP under the control of the CAG promoter. Provided are representative immunofluorescence images depicting transduction efficiency via GFP fluorescence of human myoblasts transduced with WT AAV9 particles, N272A de-targeted AAV9 particles, N272A de-targeted AAV9 particles conjugated to an anti-ASGRl mAb at a 1:8 mosaic decoration, N272A de-targeted AAV9 particles conjugated to 9295 mAb at a 1:8 mosaic decoration, N272A de-targeted AAV9 particles conjugated to an anti-ASGRl Fab at a 1 :8 mosaic decoration, N272A de-targeted AAV9 particles conjugated to 9295 Fab at a 1 :8 mosaic decoration, N272A de-targeted AAV9 particles conjugated to an anti-ASGRl Fab at a 1 :2 mosaic decoration, and N272A de-targeted AAV9 particles conjugated to 9295 Fab at a 1 :2 mosaic decoration. As shown in Figure 4, conjugation of 9295-based anti-CDH15 mAb and Fab, when conjugated at a 1 :8 mosaic, improves transduction of human myoblasts compared to WT AAV9.
[00403] The heavy chain (HC) for 9295 mAb described above comprises the heavy chain variable region (HCVR) of 9295, three heavy chain constant regions (CH1-CH3), and SpyCatcher e.g., SEQ ID NO: 833 = SEQ ID NO: 202 + CH1-CH3 + SEQ ID NO: 816. The HC for 9295 Fab described above comprises the HCVR of 9295, CHI, and SpyCatcher, e.g., SEQ ID NO: 837 = SEQ ID NO: 202 + CHI + SEQ ID NO: 816. The light chain (LC) for 9295 mAb and Fab described above comprises the light chain variable region of 9295 and a light chain constant region (CL), e.g, SEQ ID NO: 835 = SEQ ID NO: 210 + CL.
Materials & Methods
A A V capsid protein constructs
[00404] GeneBlocks encoding the desired SpyTag insertions, flanking linker amino acids, and additional mutations were purchased from IDT and cloned into BsiWI and XcmLdigested pAAVR2C9 (AAV9 wildtype RepCap plasmid) using Gibson Assembly according to the manufacturer’s protocol (NEB).
Cloning SpyCatcher into antibodies
[00405] GeneBlocks encoding antibody heavy chains were purchased from IDT, and cloned into appropriate SpyCatcher-containing backbone plasmids using Gibson assembly. Heavy chains were cloned as full Fes and as Fabs. Light chain was cloned into a standard human light chain expression plasmid.
Preparation of AAV viral vectors
[00406] Virus was generated by transfecting 293 T packaging cells using PEI Pro with the following plasmids: pAd Helper, an AAV2 ITR-containing genome plasmid encoding eGFP under the control of CAG, and a pAAV-CAP plasmid encoding AAV Rep and Cap genes (with or without sequences encoding a 10 amino acid linker and SpyTag inserted at position 453 of the encoded capsid protein). Under certain conditions, the 293T cells were further transfected with additional plasmids encoding an antigen-binding domain, e.g, a complete monoclonal antibody (mAb) or a Fab fragment thereof. The antibody heavy chain constructs are all fused to SpyCatcher at their C-terminus as described in WO2019006046, incorporated herein by reference in its entirety, such that upon contacting AAV9 capsid proteins comprising SpyTag, a covalent isopeptide bond is formed between the capsid protein and the antigen-binding domain. Transfection complexes were prepared in incomplete DMEM (no additional supplements) and incubated at room temperature for 10 minutes.
[00407] Each virus was generated by transfecting 15 cm plates of 293 T packaging cells with the following plasmids and quantities: WT AAV9 / AAV9 N272A with eGFP pAd Helper 16 pg pAAV-CAG eGFP 8 pg pAAV9-CAP WT or pAAV9-CAP N272A 8 pg
AAV9 Anti-human ASGR1 or Anti-CDH15 (REGN9295) mAb and Fab with eGFP (1:8 ratio) pAd Helper 16 pg pAAV-CAG eGFP 8 pg pAAV9-CAP G453 LinkerlO SpyTag 1 pg pAAV9-CAP N272A 7 pg
With: pAnti -human ASGR1 or Anti -human CDH15 h!gG4US SpyCatcher Vh 1.5 pg
ULC1-39 or pAnti -human CDH15 Vk 3 pg or pAnti -human ASGR1 or pAnti -human CDH15 Fab SpyCatcher Vh 1.5 pg
ULC1-39 or pAnti -human CDH15 Vk 3 pg
AAV9 Anti -human ASGR1 or Anti-CDH15 (REGN9295) Fab with eGFP (1 :2 ratio) pAd Helper 16 pg pAAV-CAG eGFP 8 pg pAAV9-CAP G453 LinkerlO SpyTag 4 pg pAAV9-CAP N272A 4 pg
With: pAnti -human ASGR1 or pAnti -human CDH15 Fab SpyCatcher Vh 1.5 pg
ULC1-39 or pAnti -human CDH15 Vk 3 pg
[00408] Post incubation, complexes were added to DMEM supplemented with 10% FBS, 1XNEAA, l% Pen/Strep, and 1% L-Glutamine.
[00409] Transfected packaging cells were incubated for 3 days at 37°C, then virus was collected from cell lysates using a standard freeze-thaw protocol. Packaging cells were lifted by scraping and pelleted. Supernatant was removed, and cells were resuspended in a solution of 50mM Tris-HCl, 150mM NaCl, and 2 mM MgC12, pH 8.0. Intracellular virus particles were released by inducing cell lysis via three consecutive freeze-thaw cycles, consisting of shuttling cell suspension between dry ice/ethanol bath and 37°C water bath with vigorous vortexing. Viscosity was reduced by treating lysate with EMD Millipore Benzonase (50 U/ml of cell lysate) for 60 min at 37°C, with occasional mixing. Debris was then pelleted by centrifugation, and the resulting supernatant was filtered through a 0.22 pm PVDF Millex-GV Filter. For crude virus to be tested in vitro, the filtered lysate is added directly to an Amicon Ultra-15 Centrifugal Filter Unit with Ultracel-100 membrane (100 KDa MWCO) filter cartridge. The filter unit was centrifuged at 5-10 minute intervals until desired volume was reached in the upper chamber, then concentrated crude virus was pipetted into a low-protein-binding tube and stored at 4 °C.
[00410] Titer (viral genomes per milliliter vg/mL) was determined by qPCR using a standard curve of a virus of known concentration.
Cells
[00411] C2C12 myoblast cell lines were maintained in DMEM supplemented with 10%
FBS, 1% Pen/Strep, and 1% L-glutamine and were grown in a 37°C incubator with 5% CO2. C2C12 cell lines were obtained from ATCC (American Type Culture Collection, Manassas, VA).
[00412] Human skeletal myoblast cells were purchased from Cook Myosite (Pittsburgh, PA; SkMDC; Lot # P01059-14M) and maintained in MyoTonic Basal Media (MB-2222) supplemented with MyoTonic Growth Supplement (MS-3333) and grown in a 37°C incubator with 5% CO2.
Cell Transduction and Analysis
[00413] C2C12 myoblasts and human myoblasts were seeded in collagen-coated 96 well plates with a clear base and black walls at a density of 8,000 cells/well. To infect cells, viral particles were added directly to the media of cells in culture at 2.5 x 105 vg/cell, and the mixture was incubated at 37°C. Three days post-infection, cells fixed with 4% PFA for 15 minutes, washed, and stored in PBS until analysis.
[00414] Following washing, cells were stained with Hoescht (1 : 1,000) and imaged on an Axio Observer microscope (Zeiss, Oberkochen, Germany) for GFP detection. Single myofiber and muscle cross section immunostaining
[00415] For single myofiber immunostaining, wildtype C57BL/6 mice were sacrificed and gastrocnemius muscle was carefully dissected and digested for 90 minutes at 37°C in 700 units/mL of collagenase II (Gibco, 17101-015) in DMEM (high glucose, +glutamine,
+1 lOmg/mL sodium pyruvate, Fisher 11995-065). The muscles were placed on a horse serum coated plate and undamaged single fibers were picked and transferred to a plate with wash media (DMEM, 10% horse serum, 1% pen/strep). The single fibers were washed 3 times in fresh wash media in horse serum coated plates and then transferred to a plate with DMEM only and incubated for 1 hour. Single fibers were transferred to low-adherence 6-well plates (Corning, 3474) and incubated overnight in DMEM with 2% horse serum. Single fibers were live stained with lOOnM anti-CDH15 Ab for 30 minutes, washed twice, and stained with 1 :200 APC- conjugated human secondary (Jackson Cat# 109-136-170). The single fibers were then washed twice, fixed with 4% PF A, and then incubated overnight in Pax7 (1 TOO, DSHB). Single fibers were then incubated for 5 min with Hoescht (1 : 1000), washed with PBS, and then mounted on to a coverslip and imaged on a Zeiss LSM 710 confocal microscope.
[00416] For muscle cross section immunostaining, wildtype C57BL/6 mice were sacrificed and gastrocnemius muscle was embedded in optical coherence tomography (OCT) compound, frozen in liquid nitrogen-cooled isopentane, and subsequently cryo-sectioned at lOum onto microscope slides. Tissue sections were fixed with 4% PF A, blocked and permeabilized with 0.1% Triton-XlOO in 20% goat serum, and stained overnight with anti- CDH15 Ab, anti-Pax7 Ab, and anti -laminin Ab, followed by detection with Alexa-fluor conjugated secondary antibodies (1 :250). Tissues were then washed, counterstained with Hoescht (1 : 1000), coverslipped, and imaged on an Axio Observer microscope.
[00417] While the invention has been particularly shown and described with reference to a number of embodiments, it would be understood by those skilled in the art that changes in the form and details may be made to the various embodiments disclosed herein without departing from the spirit and scope of the invention and that the various embodiments disclosed herein are not intended to act as limitations on the scope of the claims. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some preferred methods and materials are now described. All publications cited herein are incorporated herein by reference to describe in their entirety. Unless defined otherwise, 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 invention belongs.

Claims

CLAIMS WHAT IS CLAIMED:
1. An antigen-binding protein that binds to human Cadherin 15 (CDH15), wherein the antigen-binding protein comprises a set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences selected from the group consisting of SEQ ID NOs: 4-6-8-12-14-16, 24-26- 28-32-34-36, 44-46-48-52-34-54, 62-64-66-52-34-54, 72-74-76-52-34-54, 82-84-86-52-34-54, 92-94-96-100-34-102, 82-111-113-117-34-119, 127-129-131-135-137-139, 147-149-151-155- 157-159, 167-169-171-175-177-179, 187-189-191-52-34-196, 204-206-208-212-137-214, 222- 224-226-52-34-54, 232-234-236-52-34-54, 242-244-246-52-34-54, 82-253-255-52-34-54, 261- 263-265-269-271-273, 281-283-285-289-291-293, 301-303-305-309-311-313, 321-323-325-329- 331-333, 341-343-345-349-14-352, 360-362-364-368-370-372, 187-380-382-52-34-54, 388-390- 392-396-14-398, 406-408-410-100-34-414, 422-424-426-430-432-434, 438-440-442-446-448- 450, 454-456-458-462-464-466, 470-472-474-478-480-482, 486-488-490-494-496-498, 502-504- 506-510-512-514, 518-520-522-526-528-530, 534-536-538-542-544-546, 550-552-554-558-560- 562, 566-568-570-574-576-578, 582-584-586-590-592-594, 598-600-602-606-608-610, 614-616- 618-622-624-626, 630-632-634-638-640-642, 646-648-650-654-656-658, 662-664-666-670-672- 674, 678-680-682-686-688-690, 694-696-698-702-704-706, 710-712-714-718-720-722, 726-728- 730-734-736-738, 742-744-746-686-688-690, 750-752-754-758-760-762, and 766-768-770-774- 776-778.
2. The antigen-binding protein of claim 1, wherein the antigen-binding protein comprises a heavy chain variable region (HCVR or VH).
3. The antigen-binding protein of claim 1, wherein the antigen-binding protein comprises a light chain variable region (LCVR or VL).
4. The antigen -binding protein of any one of claims 1-3, wherein the antigen-binding protein comprises an anti-hCDH15 antibody or antigen-binding fragment thereof.
5. The antigen-binding protein of claim 4, wherein the anti-hCDH15 antibody or antigenbinding fragment thereof is selected from the group consisting of a human or humanized antibody or antigen binding fragment thereof, a monovalent Fab’, a divalent Fab2, a F(ab)’3 fragment, a single-chain fragment variable (scFv), a bis-scFv, a (scFv)2, a diabody, a minibody, a nanobody, a triabody, a tetrabody, a disulfide stabilized Fv protein (dsFv), a single-domain antibody (sdAb), an IgNAR, a bispecific antibody or binding fragment thereof, abi-specific T-cell engager (BiTE), a trispecific antibody, and a chemically modified derivative thereof.
6. The antigen-binding protein of claim 5, wherein the anti-hCDH15 antibody or antigenbinding fragment thereof comprises a fragment antigen-binding region (Fab).
7. The antigen-binding protein of claim 5, wherein the anti-hCDH15 antibody or antigenbinding fragment thereof comprises a single chain fragment variable (scFv).
8. The antigen-binding protein of claim 7, wherein the scFv comprises variable regions arranged in the following orientation from N-terminus to C-terminus: HCVR-LCVR.
9. The antigen-binding protein of claim 7, wherein the scFv comprises variable regions arranged in the following orientation from N-terminus to C-terminus: LCVR-HCVR.
10. The antigen-binding protein of any one of claims 7-8, wherein the scFv variable regions are connected by a linker.
11. The antigen-binding protein of claim 10, wherein the linker is a peptide linker.
12. The antigen-binding protein of claim 11, wherein the peptide linker is -(GGGGS)n- (SEQ ID NO: 789), wherein n is 1-10.
13. The antigen-binding protein of any one of claims 1-12, wherein the antigen-binding protein binds to hCDH15 with a KD of about 1X10'7 M or a stronger affinity.
14. The antigen-binding protein of any one of claims 1-13, wherein the antigen-binding protein binds to hCDH15 with a KD of about 10X10'8 to about 1X1O'10.
15. The antigen-binding protein of any one of claims 1-14, wherein the antigen-binding protein binds to hCDH15 with a KD of about 5X10’9 to about IXIO’10.
16. The antigen-binding protein of any one of claims 1-15, wherein the antigen-binding protein comprises an HCVR/LCVR amino acid sequence pair having at least 90% sequence identity to an HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 and 10, 22 and 30, 42 and 50, 60 and 50, 70 and 50, 80 and 50, 90 and 98, 108 and 115, 125 and 133,
145 and 153, 165 and 173, 185 and 193, 202 and 210, 220 and 50, 230 and 50, 240 and 50, 250 and 50, 259 and 267, 279 and 287, 299 and 307, 319 and 327, 339 and 347, 358 and 366, 378 and 50, 386 and 394, 404 and 412, 420 and 428, 436 and 444, 452 and 460, 468 and 476, 484 and 492, 500 and 508, 516 and 524, 532 and 540, 548 and 556, 564 and 572, 580 and 588, 596 and 604,
612 and 620, 628 and 636, 644 and 652, 660 and 668, 676 and 684, 692 and 700, 708 and 716,
724 and 732, 740 and 684, 748 and 756, and 764 and 772.
17. A polynucleotide comprising a sequence encoding the antigen-binding protein of any one of claims 1-16.
18. A pharmaceutical composition comprising the antigen binding protein of any one of claims 1-16, and a pharmaceutically acceptable carrier.
19. A method of inhibiting the activity of CDH15 in a cell, comprising contacting the cell expressing CDH15 with the antigen-binding protein of any one of claims 1-16 or the pharmaceutical composition of claim 18.
20. The method of claim 19, wherein the cell expressing CDH15 is a muscle stem cell, a myoblast, or a myocyte.
21. A method of accelerating the transition from quiescence to activation of a muscle stem cell, comprising contacting the muscle stem cell with the antigen-binding protein of any one of claims 1-16 or the pharmaceutical composition of claim 18.
22. A method of treating a condition in a subject in need thereof, wherein the method comprises administering a therapeutically effective amount of the pharmaceutical composition of claim 18 to the subject.
23. The method of claim 22, wherein the condition is muscle injury.
24. A method of improving muscle regeneration following a muscle injury in a subject, wherein the method comprises administering a therapeutically effective amount of the pharmaceutical composition of claim 18 to the subject.
25. A method of restoring the muscle regenerative capacity of a subject, wherein the method comprises administering a therapeutically effective amount of the pharmaceutical composition of claim 18 to the subject.
26. The method of claim 25, wherein the muscle regenerative capacity of the subj ect is restored to a functional state at or near that of a control subject.
27. A method of treating a condition in a subject in need thereof, wherein the method comprises administering a therapeutically effective amount of the pharmaceutical composition of claim 18 to the subject, wherein the antigen-binding protein is conjugated to a therapeutic agent.
28. The method of claim 27, wherein the condition is cancer.
29. The method of claim 28, wherein the cancer is rhabdomyosarcoma.
30. The method of any one of claims 27-29, wherein the therapeutic agent comprises a cytotoxic chemotherapeutic agent.
31. The method of any one of claims 27-30, wherein the therapeutic agent comprises Aflibercept, Amsacrine, Azacitidine, Azathioprine, Belantamab mafodotin, Bendamustine, Bleomycin, Bortezomib, Brentuximab vedotin, Busulfan, Cabazitaxel, Capecitabine, Carboplatin, Carfilzomib, Carmustine, Chlorambucil, Cisplatin, Cladribine, Clofarabine, Cyclophosphamide, Cytarabine, Cytarabine liposomal, Dacarbazine, Dactinomycin (actinomycin D), Daunorubicin, Docetaxel, Doxorubicin, Doxorubicin liposomal, Epirubicin, Eribulin, Etoposide, Etoposide phosphate, Fludarabine, Fluorouracil, Fotemustine, Ganciclovir, Gemcitabine, Gemtuzumab ozogamicin, Hydroxyurea, Idarubicin, Ifosfamide, Inotuzumab ozogamicin , Irinotecan, Ixazomib, Lomustine, Melphalan, Mercaptopurine, Methotrexate, Mitomycin, Mitotane, Mitozantrone, Nab-paclitaxel, Oxaliplatin, Paclitaxel, Pemetrexed, Pegaspargase, Polatuzumab vedotin, Pralatrexate, Procarbazine, Raltitrexed, Romidepsin, Sacituzumab govitecan, Temozolomide, Teniposide, Thiotepa, Tioguanine, Topotecan, Trabectedin, Trastuzumab deruxtecan, Trastuzumab emtansine, Trifluridine/tipiracil, Valganciclovir, Vinblastine, Vincristine, Vindesine, Vinflunine, Vinorelbine, or Vismodegib.
32. The method of any one of claims 27-31, wherein the antigen binding protein is conjugated to the therapeutic agent via a valine-citrulline (VC) and/or a para-aminobenzyl (PAB) linker.
33. A method of imaging a muscle cell in a subject in need thereof, wherein the method comprises administering the pharmaceutical composition of claim 18 to the subject, wherein the antigen binding protein is conjugated to a detectable moiety.
34. The method of claim 33, wherein the muscle cell comprises one or more cell selected from the group consisting of a muscle stem cell, a myoblast, and a myocyte.
35. The method of claim 33 or 34, wherein the detectable moiety comprises a radionuclide.
36. The method of any one of claims 22-35, wherein the administering comprises administering the pharmaceutical composition to the subject intravenously or subcutaneously.
37. A recombinant adeno-associated virus (AAV) particle comprising:
(i) an AAV capsid comprising a modified AAV capsid protein, and
(ii) a targeting ligand that binds a surface protein of a non-terminally differentiated muscle cell, wherein the AAV capsid is operably linked with the targeting ligand.
38. The recombinant AAV particle of claim 37, wherein:
(a) the modified AAV capsid protein comprises a first member of a protein: protein binding pair, wherein the first member of the proteimprotein binding pair is operably linked to a second member of the protein: protein binding pair; and
(b) the second member of the protein: protein binding pair comprises the targeting ligand that binds the surface protein of the non-terminally differentiated muscle cell, wherein the first member of the protein: protein binding pair and the second member of the protein: protein binding pair are associated to direct the tropism of the recombinant AAV particle to the non-terminally differentiated muscle cell.
39. The recombinant AAV particle of claim 37 or claim 38, wherein the non-terminally differentiated muscle cell comprises one or more cell selected from the group consisting of a muscle stem cell, a myoblast, a myocyte, a myotube, and a combination thereof.
40. The recombinant AAV particle of any one of claims 37-39, comprising the AAV capsid bound to the surface protein of the non-terminally differentiated muscle cell, which is expressed on the surface of a mammalian cell.
41. The recombinant AAV particle of any one of claims 37-40, comprising the AAV capsid bound to the surface protein of the non-terminally differentiated muscle cell, which is expressed on the surface of a mammalian muscle cell, wherein the surface protein of the non-terminally differentiated muscle cell is a human surface protein of the non-terminally differentiated muscle cell, and wherein the mammalian muscle cell is a non-human animal muscle cell genetically modified to express the human surface protein of the non-terminally differentiated muscle cell.
42. The recombinant AAV particle of any one of claims 37-41, comprising the AAV capsid bound to the surface protein of a non-terminally differentiated muscle cell, which is expressed on the surface of a mammalian muscle cell, wherein the surface protein of the non-terminally differentiated muscle cell is a human surface protein of the non-terminally differentiated muscle cell, and wherein the mammalian muscle cell is a rodent muscle cell genetically modified to express the human surface protein of the non-terminally differentiated muscle cell.
43. The recombinant AAV particle of any one of claims 37-42, comprising the AAV capsid bound to the surface protein of the non-terminally differentiated muscle cell, which is expressed on the surface of a mammalian muscle cell, wherein the surface protein of the non-terminally differentiated muscle cell is a human surface protein of the non-terminally differentiated muscle cell, and wherein the mammalian muscle cell is a rat or mouse muscle cell genetically modified to express the human surface protein of the non-terminally differentiated muscle cell.
44. The recombinant AAV particle of any one of claims 37-43, comprising the AAV capsid bound to the surface protein of the non-terminally differentiated muscle cell, which is expressed on the surface of a mammalian muscle cell, wherein the surface protein of the non-terminally differentiated muscle cell is a human cellspecific surface protein of the non-terminally differentiated muscle cell, and wherein the mammalian muscle cell is a mouse muscle cell genetically modified to express the human cell-specific surface protein of the non-terminally differentiated muscle cell.
45. The recombinant AAV particle of any one of claims 37-41, comprising the AAV capsid bound to the surface protein of the non-terminally differentiated muscle cell, which is expressed by a mammalian muscle cell, wherein the surface protein of the non-terminally differentiated muscle cell is a human surface protein of the non-terminally differentiated muscle cell, and wherein the mammalian muscle cell is a human muscle cell.
46. The recombinant AAV particle of any one of claims 37-45, wherein the recombinant AAV particle is in vitro.
47. The recombinant AAV particle of any one of claims 37-45, wherein the recombinant AAV particle is in vivo.
48. The recombinant AAV particle of any one of claims 37-42, wherein the surface protein of the non-terminally differentiated muscle cell is mammalian Cadherin 15 (CDH15).
49. The recombinant AAV particle of any one of claims 37-48, wherein the surface protein of the non-terminally differentiated muscle cell is human CDH15.
50. The recombinant AAV particle of any one of claims 38-49, wherein:
(a) the first member of the protein: protein binding pair comprises SpyTag, Isopeptag, SnoopTag, or SpyTag002,
(b) the second member of the proteimprotein binding pair comprises
(i) SpyCatcher, KTag, pilin-C, SnoopCatcher, or SpyCatcher002, and
(ii) the targeting ligand that binds a mammalian muscle-specific surface protein, and
(c) the first member of the protein: protein binding pair and the second member of the protein: protein binding pair are associated by an isopeptide bond.
51. The recombinant AAV particle of any one of claims 38-50, wherein:
(a) the first member of the protein: protein binding pair comprises SpyTag, and
(b) the second member of the proteimprotein binding pair comprises SpyCatcher fused to the targeting ligand that binds a mammalian muscle-specific surface protein.
52. The recombinant AAV particle of any one of claims 38-49, wherein:
(a) the first member of the protein: protein binding pair comprises a c-myc amino acid sequence as set forth in SEQ ID NO: 818, and
(b) the second member of the protein: protein binding pair comprises a bispecific binding protein comprising an anti-c-myc antibody and the targeting ligand that binds a mammalian muscle-specific surface protein.
53. The recombinant AAV particle of any one of claims 38-52, comprising a first and/or second linker operably linking the first member of the protein: protein binding pair to a capsid protein of the recombinant AAV particle.
54. The recombinant AAV particle of claim 53, wherein the first and second linker are not identical.
55. The recombinant AAV particle of claim 53, wherein the first and second linker are identical.
56. The recombinant AAV particle of any one of claims 53-55, wherein the first linker is 10 amino acids in length and/or the second linker is 10 amino acids in length, optionally wherein the amino acid sequence of the first linker and/or the amino acid sequence of the second linker comprises an amino acid sequence as set forth in SEQ ID NO: 826 or SEQ ID NO:827.
57. The recombinant AAV particle of any one of claims 38-56, wherein:
(a) the AAV capsid comprises modified VP1 capsid proteins, modified VP2 capsid proteins, and/or modified VP3 capsid proteins encoded by a mutated cap gene, and
(b) the mutated cap gene or a portion thereof comprises a sequence at least 90% identical to a cap gene of an AAV or portion thereof and is genetically modified to comprise an insertion of a nucleotide sequence encoding the first member of the proteimprotein binding pair such that the modified VP1 capsid proteins, the modified VP2 capsid proteins, and/or the modified VP3 capsid proteins comprise the first member of the protein: protein binding pair.
58. The recombinant AAV particle of claim 57, wherein the mutated cap gene is genetically modified to comprise one or more additional mutations such that the modified VP 1 capsid proteins, the modified VP2 capsid proteins, and/or the modified VP3 capsid proteins comprise, in addition to the first member of the protein: protein binding pair:
(i) a substitution, insertion, or deletion of an amino acid,
(ii) a chimeric amino acid sequence, or
(iii) both a point mutation and a chimeric amino acid sequence.
59. The recombinant AAV particle of claim 58, wherein the substitution, insertion, or deletion of an amino acid reduces the natural tropism of the AAV capsid and/or creates a detectable label.
60. The recombinant AAV particle of any one of claims 38-59, wherein:
(a) the AAV capsid comprises modified VP1 capsid proteins, modified VP2 capsid proteins, and/or modified VP3 capsid proteins encoded by a mutated cap gene, and
(b) the mutated cap gene or a portion thereof comprises a sequence at least 90% identical to a cap gene of an AAV or portion thereof and is genetically modified to comprise an insertion of a nucleotide sequence encoding the first member of the proteimprotein binding pair such that the modified VP1 capsid proteins, the modified VP2 capsid proteins, and/or the modified VP3 capsid proteins comprise the first member of the protein: protein binding pair, and
(c) the AAV is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, a non-primate animal AAV listed in Table 2, and a combination thereof.
61. The recombinant AAV particle of any one of claims 57-60, wherein the AAV is AAV2.
62. The recombinant AAV particle of any one of claims 57-61, wherein the AAV capsid comprises modified AAV2 VP1 capsid proteins that comprise the first member of the protein: protein binding pair linked, optionally via a linker, to an amino acid at position 1453 and/or 1587.
63. The recombinant AAV particle of any one of claims 57-62, wherein the AAV capsid comprises modified AAV2 VP1 capsid proteins that comprise the first member of the protein: protein binding pair displayed, via a linker, at position G453, optionally wherein the modified AAV2 VP1 capsid proteins further comprise an R585A modification, an R588A modification, or both the R585A modification and the R588A modification, and optionally wherein the modified AAV2 VP1 capsid proteins further comprise an R484A modification, an R487A modification, an R585A modification, an R588A modification, and an K532A modification, or any combination of an R484A modification, an R487A modification, an R585A modification, an R588A modification, and an K532A modification.
64. The recombinant AAV particle of claim 62 or claim 63, wherein the AAV capsid is a mosaic AAV capsid comprising a second set of AAV2 VP1 capsid proteins lacking the first member of the protein: protein binding pair, optionally wherein the second set of AAV2 VP1 capsid proteins comprise an R585A modification, an R588A modification, or both the R585A modification and the R588A modification.
65. The recombinant AAV particle of any one of claims 57-60, wherein the AAV is AAV9.
66. The recombinant AAV particle of any one of claims 57-60 and claim 65, wherein the AAV capsid comprises modified AAV9 VP1 capsid proteins that comprise the first member of the protein: protein binding pair linked, optionally via a linker, to an amino acid at position 1453 or 1589.
67. The recombinant AAV particle of any one of claims 57-60 and 65-66, wherein the AAV capsid comprises modified AAV9 VP1 capsid proteins that comprise the first member of the protein: protein binding pair displayed, via a linker, at G453, optionally wherein the modified AAV9 VP1 capsid proteins further comprise an N272A modification, a W503A modification, or both the N272A modification and the W503A modification.
68. The recombinant AAV particle of claim 66 or claim 67, wherein the AAV capsid is a mosaic AAV capsid comprising a second set of AAV9 VP1 capsid proteins lacking the first member of the protein: protein binding pair, optionally wherein the second set of AAV2 VP1 capsid proteins comprise an N272A modification, a W503A modification, or both the N272A modification and the W503A modification.
69. The recombinant AAV particle of claim 60, wherein the non-primate animal AAV is an avian AAV (AAAV), a non-human mammalian AAV or a squamate AAV.
70. The recombinant AAV particle of claim 60 or claim 69, wherein the non-primate animal AAV is an AAAV.
71. The recombinant AAV particle of claim 60, 69, or 70, wherein the AAV capsid comprises modified AAAV VP 1 capsid proteins that comprise the first member of the protein: protein binding pair linked, optionally via a linker, to an amino acid at position 1444 or 1580.
72. The recombinant AAV particle of claim 60 or claim 69, wherein the non-primate animal AAV is a squamate AAV.
73. The recombinant AAV particle of claim 60, 69 or claim 72, wherein the non-primate animal AAV is a bearded dragon AAV.
74. The recombinant AAV particle of any one of claim 60, 69, 72, or 73, wherein the AAV capsid comprises modified bearded dragon VP1 capsid proteins that comprise the first member of the protein: protein binding pair linked, optionally via a linker, to an amino acid at position 1573 or 1436.
75. The recombinant AAV particle of claim 60 or claim 69, wherein the non-primate animal
AAV is a non-human mammalian AAV.
76. The recombinant AAV particle of any one of claims 60, 69 or 75, wherein the non-primate animal AAV is a sea lion AAV.
77. The recombinant AAV particle of any one of claims 60, 69, 75, or 76, wherein the AAV capsid comprises modified AAAV VP1 capsid proteins that comprise the first member of the protein: protein binding pair linked, optionally via a linker, to an amino acid at a position selected from the group consisting of 1429, 1430, 1431, 1432, 1433, 1434, 1436, 1437, and 1565.
78. The recombinant AAV particle of any one of claims 37-77, wherein the AAV capsid is a mosaic AAV capsid, optionally wherein the mosaic AAV capsid comprises a first plurality of reference capsid proteins each of which do not comprise the first member of the protein: protein binding pair and a second plurality of capsid proteins each of which comprise the first member of the protein: protein binding pair, optionally wherein the mosaic AAV capsid comprises the first plurality of reference capsid proteins and the second plurality of capsid proteins at a ratio of 1 :7.
79. The recombinant AAV particle of any one of claims 37-78, wherein the targeting ligand is an antibody or a portion thereof.
80. The recombinant AAV particle of any one of claims 37-79, further comprising a nucleotide of interest encapsidated within the AAV capsid.
81. The recombinant AAV particle of claim 80, wherein the nucleotide of interest is a reporter gene.
82. The recombinant AAV particle of claim 80 or claim 81, wherein the nucleotide of interest encodes P-galactosidase, green fluorescent protein (GFP), enhanced Green Fluorescent Protein (eGFP), MmGFP, blue fluorescent protein (BFP), enhanced blue fluorescent protein (eBFP), mPlum, mCherry, tdTomato, mStrawberry, J-Red, DsRed, mOrange, mKO, mCitrine, Venus, YPet, yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (eYFP), Emerald, CyPet, cyan fluorescent protein (CFP), Cerulean, T-Sapphire, luciferase, alkaline phosphatase, or a combination thereof.
83. The recombinant AAV particle of claim 80, wherein the nucleotide of interest encodes a therapeutic protein, a suicide gene, an antibody or a fragment thereof, a CRISPR/Cas system or a portion(s) thereof, an antisense oligonucleotide, a ribozyme, an RNAi molecule, or a shRNA molecule.
84. The recombinant AAV particle of any one of claims 37-83, wherein the targeting ligand comprises the antigen-binding protein of any one of claims 1-16, optionally wherein the targeting ligand comprises a sequence selected from the group consisting of SEQ ID NO: 833, SEQ ID NO: 835, SEQ ID NO: 837, and any combination thereof.
85. A pharmaceutical composition comprising (a) the recombinant AAV particle according to any one of claims 37-84 and (b) a pharmaceutically acceptable carrier or excipient.
86. A method of delivering a nucleotide of interest to a mammalian muscle cell comprising contacting the mammalian muscle cell with (a) the recombinant AAV particle according to any one of claims 37-84 or (b) the pharmaceutical composition of claim 85, wherein the mammalian muscle cell expresses the mammalian muscle-specific surface protein.
87. The method of claim 86, wherein the contacting is performed ex vivo.
88. The method of claim 86, wherein the contacting is performed in a subject, optionally wherein the subject is modified to express the targeting ligand, e.g., from a safe harbor locus.
89. The method of claim 88, wherein the subject is a primate animal, preferably a human.
90. The method of any one of claims 86-89, wherein the mammalian muscle cell is a mammalian skeletal muscle cell, optionally wherein the mammalian skeletal muscle cell is not terminally differentiated, optionally wherein the mammalian skeletal muscle cell comprises one or more cell selected from the group consisting of a muscle stem cell, a myoblast, and a myocyte.
91. The method of any one of claims 86-90, wherein the mammalian muscle-specific surface protein is CDH15.
92. The method of any one of claims 86-91, wherein the nucleotide of interest encodes a therapeutic protein, a suicide gene, an antibody or a fragment thereof, a CRISPR/Cas system or a portion(s) thereof, an antisense oligonucleotide, a ribozyme, an RNAi molecule, or a shRNA molecule.
93. A method of treating a muscle wasting or genetic muscle disease in a patient in need thereof comprising administering to the patient (a) the recombinant AAV particle according to any one of claims 37-84 or (b) the pharmaceutical composition of claim 85, wherein the recombinant AAV particle comprises a nucleotide of interest encapsidated within the AAV capsid, and wherein the nucleotide of interest encodes a therapeutic protein, a suicide gene, an antibody or a fragment thereof, a CRISPR/Cas system or a portion(s) thereof, an antisense oligonucleotide, a ribozyme, an RNAi molecule, or a shRNA molecule.
94. Use of the recombinant AAV particle according to any one of claims 37-84 or the pharmaceutical composition of claim 85 for the manufacture of a medicament for the treatment of muscle wasting or a genetic muscle disease.
95. The method of claim 93 or use of claim 94, wherein the muscle wasting or genetic muscle disease is selected from the group consisting of X-linked myotubular myopathy (XLMTM), Duchenne muscular dystrophy (DMD), myotonic dystrophy (DM1), Facioscapulohumeral muscular dystrophy Type 1 (FSHD), congenital muscular dystrophy type 1A (MDC1A), Limb girdle muscular dystrophy, and dystroglycanopathy.
96. A method of treating a rhabdomyosarcoma in a patient in need thereof comprising administering to the patient (a) the recombinant AAV particle according to any one of claims 37-84 or (b) the pharmaceutical composition of claim 85, wherein the recombinant AAV particle comprises a nucleotide of interest encapsidated within the AAV capsid, and wherein the nucleotide of interest encodes a therapeutic protein, a suicide gene, an antibody or a fragment thereof, a CRISPR/Cas system or a portion(s) thereof, an antisense oligonucleotide, a ribozyme, an RNAi molecule, or a shRNA molecule.
97. Use of the recombinant AAV particle according to any one of claims 37-84 or the pharmaceutical composition of claim 85 for the manufacture of a medicament for the treatment of a rhabdomyosarcoma.
98. The method of claim 96 or the use of claim 97, wherein the rhabdomyosarcoma is selected from the group consisting of embryonal, alveolar, pleomorphic, botryoid and spindle/sclerosing rhabdomyosarcoma.
EP24728805.3A 2023-05-02 2024-05-01 Anti-human m-cadherin (cdh15) antibodies, conjugates, and uses thereof for delivery of genetic payloads to muscle cells Pending EP4705343A1 (en)

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