WO2017205820A1 - Anticorps à large spectre à maturation affinée dirigés contre le virus de l'hépatite c - Google Patents

Anticorps à large spectre à maturation affinée dirigés contre le virus de l'hépatite c Download PDF

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WO2017205820A1
WO2017205820A1 PCT/US2017/034816 US2017034816W WO2017205820A1 WO 2017205820 A1 WO2017205820 A1 WO 2017205820A1 US 2017034816 W US2017034816 W US 2017034816W WO 2017205820 A1 WO2017205820 A1 WO 2017205820A1
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antibody
hcv
antibodies
virus
affinity
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Zhen-Yong Keck
Steven Foung
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The Board Of Trustees Of The Leland Stanford Junior University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1081—Togaviridae, e.g. flavivirus, rubella virus, hog cholera virus
    • C07K16/109—Hepatitis C virus; Hepatitis G virus
    • 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
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/515—Complete light chain, i.e. VL + CL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • 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
    • 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
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • HCV chronic hepatitis C virus
  • DAAs direct-acting antivirals
  • RAV Reliable virus virus
  • HCV-positive kidneys are transplanted in immunosuppressed HCV-negative recipients.
  • host-targeting agents that interfere with cellular factors involved in the viral life cycle. These include viral entry, translation, replication and assembly inhibitors, and biological response modifiers. Because these approaches target host proteins, a potential concern is interference with the normal functions of these proteins that can lead to host toxicity.
  • HCV can be classified into seven genetically distinct genotypes and further subdivided into a large number of subtypes, of which the seven major genotypes differ by approximately 30%, and the subtypes differ by 20%–25%, at the nucleotide level.
  • a significant challenge for immunotherapeutic development is the identification of protective epitopes conserved in the majority of viral genotypes and subtypes. This problem is compounded by the fact that the envelope E1E2 glycoproteins, the natural targets for the neutralizing response, are two of the most variable proteins.
  • the error-prone nature of the RNA-dependent RNA polymerase together with the high HCV replicative rate in vivo, results in the production of viral quasispecies leading to RAV.
  • HCV human monoclonal antibodies
  • HCV human monoclonal antibodies
  • HCV1 binds to a region of the E2 protein that is highly conserved, encompassing amino acids (aa) 412-423, RAV can occur with an N-glycan shift from the 417 to 415 position when there is an N417S/T mutation. Nonetheless, this study supports a therapeutic role for broadly neutralizing HMAbs to HCV to increase the barrier to viral resistance.
  • compositions and methods are provided relating to anti-HCV monoclonal antibodies.
  • the antibodies of the invention have been affinity matured for binding to a conformational epitope in a conserved and essential region of HCV E2 protein, and neutralize HCV influenza virus across multiple HCV genotypes.
  • Embodiments of the invention include isolated antibodies and derivatives and fragments thereof, pharmaceutical formulations comprising one or more of the human anti-HCV monoclonal antibodies; and cell lines that produce these monoclonal antibodies. Also provided are CDR amino acid sequences that confer the binding specificity of these monoclonal antibodies. These antibodies are used for immunotherapeutic methods for prevention of disease associated with HCV virus, including without limitation the neutralization of virus in association with organ transplantation, e.g. kidney, liver etc.
  • Therapies of interest include combination therapies with anti-HCV therapeutics such as monoclonal antibodies that specifically bind a different epitope than the antibodies of the invention, small molecule antivirals, interferon, and the like.
  • a combination of anti-HCV antibodies recognizing different epitopes which may be administered as a cocktail of two or more antibodies; or administered as single antibodies, e.g. sequentially, concomitantly, etc.
  • one of the antibodies is an affinity matured antibody of the antibody.
  • the second antibody binds to a different epitope on HCV E2, e.g. antibodies that bind to an epitope at residues 412-423 of E2. Included, without limitation is the antibody HC33.1 and variants thereof.
  • a combination of antibodies is administered comprising an affinity matured antibody of the invention in combination with one or more antibodies that specifically block the interaction of HCV with a host cell protein, e.g. one or more of glycosaminoglycans (GAGs), low density lipoprotein receptor (LDL-R), high density lipoprotein receptor scavenger receptor class B type I (SCARB1), tetraspanin CD81, claudin- 1 (CLDN1), and occludin (OCLN), which may be administered as a cocktail of two or more antibodies; or administered as single antibodies, e.g. sequentially, concomitantly, etc.
  • GAGs glycosaminoglycans
  • LDL-R low density lipoprotein receptor
  • SCARB1 high density lipoprotein receptor scavenger receptor class B type I
  • SCARB1 tetraspanin CD81
  • CLDN1 claudin- 1
  • OCLN occludin
  • Antibodies of the invention comprise, paired with a heavy chain as described below, a light chain variable region.
  • the affinity matured light chain variable region is HC84.26.5D (SEQ ID NO:4) or a set of CDR sequences derived therefrom in a variable region framework sequence, usually a human variable region framework sequence.
  • the anti-HCV antibody may have a light chain variable region comprising the amino acid sequence of CDR1 and/or CDR2 and/or CDR3 of the provided affinity matured monoclonal antibodies as provided herein.
  • Antibodies of the invention may comprise the heavy chain variable region of any one of HC84.26 (SEQ ID NO:1), HC84.26.5D (SEQ ID NO:2), HC84.26.5G (SEQ ID NO:3) or a set of CDR sequences derived therefrom in a variable region framework sequence, usually a human variable region framework sequence.
  • the anti-HCV antibody may have a heavy chain variable region comprising the amino acid sequence of CDR1 and/or CDR2 and/or CDR3 of the provided affinity matured monoclonal antibodies as provided herein.
  • the antibody comprises an amino acid sequence variant of one or more of the CDRs of the provided human antibodies, which variant comprises one or more amino acid insertion(s) within or adjacent to a CDR residue and/or deletion(s) within or adjacent to a CDR residue and/or substitution(s) of CDR residue(s) (with substitution(s) being the preferred type of amino acid alteration for generating such variants).
  • Such variants will normally having a binding affinity for HCV E2 of at least about 10 8 , and will bind to the same epitope as an antibody having the amino acid sequence of HC-84.26.5D.
  • the anti-HCV antibody may be a full length antibody, e.g. having a human immunoglobulin constant region of any isotope, e.g. IgG1, IgG2a, IgG2b, IgG3, IgG4, IgA, etc. or an antibody fragment, e.g. a F(ab') 2 fragment, and F(ab) fragment, etc.
  • the antibody may be labeled with a detectable label, immobilized on a solid phase and/or conjugated with a heterologous compound.
  • the invention provides a method for determining the presence of HCV virus comprising exposing a sample suspected of containing the HCV virus to the anti-HCV antibody and determining binding of the antibody to the sample.
  • an effective dose of the antibody or combination of antibodies is provided in a dose effective to neutralize HCV present in an individual, in a tissue source, etc.
  • the invention further provides: isolated nucleic acid encoding the antibodies and variants; a vector comprising that nucleic acid, optionally operably linked to control sequences recognized by a host cell transformed with the vector; a host cell comprising that vector; a process for producing the antibody comprising culturing the host cell so that the nucleic acid is expressed and, optionally, recovering the antibody from the host cell culture (e.g. from the host cell culture medium).
  • the invention also provides a composition comprising one or more of the human anti-HCV antibodies and a pharmaceutically acceptable carrier or diluent. This composition for therapeutic use is sterile and may be lyophilized, e.g. being provided as a pre-pack in a unit dose with diluent and delivery device, e.g. inhaler, syringe, etc.
  • HC84.26.5D protects humanized mice against HCV.
  • C Serum human IgG half-life measurements in mice treated with HC84.26.5D. Each timepoint was measured by ELISA in triplicates for HMAb in each mouse serum sample tested and the concentration was back-calculated based on a standard curve ( Figure 6).
  • FIG. 1 Structure of the HC84.26.5D–E2 434–446 complex.
  • A Ribbon diagram of the HC84.26.5D–E2 434–446 complex (side view). V L , green; V H , cyan; E2 434–446, orange. V L CDR loops are labeled L1–L3; V H CDR loops are labeled H1–H3. The side chains of epitope residues Trp437, Phe442, Tyr443, and Lys446 are drawn in stick representation.
  • the E2 434–446 epitope from the complex with HC84.26.5D (orange) was superposed onto E2 434–446 from the complex with HC84.1 (yellow) (Protein Data Bank accession code 4JZN) (16), and onto E2 434–446 from the structure of the E2 core glycoprotein (wheat) (4MWF) (14).
  • the E2 434–446 epitopes were superposed through residues 437–442.
  • the N- and C-termini are labeled.
  • the epitopes are in the same overall orientation as in (A).
  • FIG. 3 The HC84.26.5D–E2 434–446 binding interface.
  • A Close-up view of the interactions between Lys446 of E2 434–446 (orange) and and the V L (green) and V H (cyan) domains of HC84.26.5D.
  • the side chains of contacting residues are shown in stick format with carbon atoms in orange (E2 434–446 ) or green (V L ); nitrogen atoms in blue, and oxygen atoms in red. Hydrogen bonds are drawn as dotted black lines.
  • the salt bridge linking Lys446 to V L Asp50 is represented as a solid red line.
  • FIG. 4 Affinity-matured residues in the HC84.26.5D-E2 434–446 interface and model of HC84.26.WH.5DL with E2 core protein.
  • A Amino acid sequences of the V H and V L regions of wild-type HC84.26 and affinity-matured HC84.26.5D were aligned. Bars show the position of CDRs. Amino acid differences between the two antibodies are highlighted in red. Contacting residues in the HC84.26.5D–E2 434–446 structure are marked with black stars.
  • Mutant antibody residues from affinity maturation are drawn in stick format, with carbon atoms in magenta, nitrogen atoms in blue, and oxygen atoms in red.
  • Three sites that were mutated in the HC84.26.5D-treated mouse with breakthrough infection (B.A818), and not inoculum or control mice, are shown as light blue sticks on the E2 core model and labeled.
  • Inset shows putative antibody V H hydrophobic contacts with E2 residue W616 based on the modeled complex (E2 residue W437 also shown for reference).
  • Two HC84.26.WH.5DL heavy chain CDR residue mutants proximal to E2 core in the modeled complex are labeled.
  • Serum hAAT was measured by a sandwich enzyme-linked immunosorbent assay employing a goat anti- hAAT primary capture antibody (#81902, Diasorin) and sheep anti-hAAT secondary antibody conjugated to horseradish peroxidase (#CL20000APHP, Cedarlane Laboratories).
  • FIG. 1 A standard curve for HC84.26.5D was generated starting at 100 ⁇ g/mL and serially diluted in semi-log steps to 0.001 ⁇ g/mL. Three separate concentrations of HC84.26.5D were spiked into neat normal mouse serum to generate the quality control samples.
  • B A 1:100 dilution of each sample was determined in the modified ELISA. The back-calculated concentrations for these samples were within an acceptable range for analytical recovery, indicating naive mouse sera did not have a significant effect on ELISA readout at a 1:100 dilution. DETAILED DESCRIPTION OF THE EMBODIMENTS
  • “Flaviviridae virus” or“flavivirus” is meant any virus from the Flaviviridae family, including those viruses that infect humans and non-human animals.
  • the polynucleotide and polypeptides sequences encoding these viruses are well known in the art, and may be found at NCBI’s GenBank database, e.g., as Genbank Accession nos.
  • the term“flavivirus” includes any member of the family Flaviviridae, including, but not limited to, Dengue virus, including Dengue virus 1, Dengue virus 2, Dengue virus 3, Dengue virus 4 (see, e.g., GenBank Accession Nos. M23027, M19197, A34774, and M14931); Yellow Fever Virus; West Nile Virus; Japanese Encephalitis Virus; St. Louis Encephalitis Virus; Bovine Viral Diarrhea Virus (BVDV); and Hepatitis C Virus (HCV); and any serotype, strain, genotype, subtype, quasispecies, or isolate of any of the foregoing.
  • Dengue virus including Dengue virus 1, Dengue virus 2, Dengue virus 3, Dengue virus 4 (see, e.g., GenBank Accession Nos. M23027, M19197, A34774, and M14931); Yellow Fever Virus; West Nile Virus; Japanese Encephalitis Virus; St. Louis Encephalitis Virus; Bovine
  • the HCV is any of a number of genotypes, subtypes, or quasispecies, including, e.g., genotype 1, including 1a and 1b, 2, 3, 4, 6, etc. and subtypes (e.g., 2a, 2b, 3a, 4a, 4c, etc.), and quasispecies.
  • hepatitis C virus “HCV,” “non- A non-B hepatitis,” or “NANBH” are used interchangeably herein, and include any “genotype” or “subgenotype” (also termed “subtype”) of the virion, or portion thereof (e.g., a portion of the E2 protein of genotype Ia of HCV), that is encoded by the RNA of hepatitis C virus or that occurs by natural allelic variation.
  • the HCV genome comprises a 5'-untranslated region that is followed by an open reading frame (ORF) that codes for about 3,010 amino acids.
  • ORF open reading frame
  • the amino acids are subdivided into ten proteins in the order from 5' to 3' as follows: C; El; E2; NSl; NS2; NS3; NS4 (a and b); and NS5 (a and b). These proteins are formed from the cleavage of the larger polyprotein by both host and viral proteases.
  • the C, El, and E2 proteins are structural and the NS1-NS5 proteins are nonstructural proteins.
  • the C region codes for the core nucleocapsid protein.
  • El and E2 are glycosylated envelope proteins that coat the virus.
  • NS2 may be a zinc metalloproteinase.
  • NS3 is a helicase.
  • NS4a functions as a serine protease cofactor involved in cleavage between NS4b and NS5a.
  • NS5a is a serine phosphoprotein whose function is unknown.
  • the NS5b region has both RNA-dependent RNA polymerase and terminal transferase activity.
  • HCV genotypes There are about six distinct HCV genotypes (e.g., genotypes 1, 2, 3, 4, 5, and 6) that are categorized by variations in the core protein and over 80 subgenotypes which exhibit further variation within each genotype, some of which include: Ia; Ib; Ic; 2a; 2b; 2c; 3a; 3b; 4a; 4b; 4c; 4d; 4e; 5a; and 6a.
  • the terms “neutralizes HCV,” “inhibits HCV,” and “blocks HCV” are used interchangeably to refer to the ability of an antibody of the invention to prevent HCV from infecting a given cell.
  • the term "effective dose” or “effective dosage” is defined as an amount sufficient to achieve or at least partially achieve the desired effect.
  • therapeutically effective dose is defined as an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. Amounts effective for this use will depend upon the severity of the disorder being treated and the general state of the patient's own immune system.
  • Polypeptide and “protein” as used interchangeably herein, can encompass peptides and oligopeptides. Where “polypeptide” is recited herein to refer to an amino acid sequence of a naturally-occurring protein molecule, "polypeptide” and like terms are not necessarily limited to the amino acid sequence to the complete, native amino acid sequence associated with the recited protein molecule, but instead can encompass biologically active variants or fragments, including polypeptides having substantial sequence similarity or sequence identify relative to the amino acid sequences provided herein. In general, fragments or variants retain a biological activity of the parent polypeptide from which their sequence is derived.
  • polypeptide refers to an amino acid sequence of a recombinant or non-recombinant polypeptide having an amino acid sequence of i) a native polypeptide, ii) a biologically active fragment of an polypeptide, or iii) a biologically active variant of an polypeptide.
  • Polypeptides suitable for use can be obtained from any species, e.g., mammalian or non-mammalian (e.g., reptiles, amphibians, avian (e.g., chicken)), particularly mammalian, including human, rodent (e.g., murine or rat), bovine, ovine, porcine, murine, or equine, particularly rat or human, from any source whether natural, synthetic, semi-synthetic or recombinant.
  • mammalian or non-mammalian e.g., reptiles, amphibians, avian (e.g., chicken)
  • rodent e.g., murine or rat
  • bovine, ovine, porcine, murine, or equine particularly rat or human
  • the term "derived from” indicates molecule that is obtained directly from the indicated source (e.g., when a protein directly purified from a cell, the protein is“derived from” the cell) or information is obtained from the source, e.g. nucleotide or amino acid sequence, from which the molecule can be synthesized from materials other than the source of information.
  • the term“isolated” indicates that the recited material (e.g, polypeptide, nucleic acid, etc.) is substantially separated from, or enriched relative to, other materials with which it occurs in nature (e.g., in a cell).
  • a material (e.g., polypeptide, nucleic acid, etc.) that is isolated constitutes at least about 0.1%, at least about 0.5%, at least about 1% or at least about 5% by weight of the total material of the same type (e.g., total protein, total nucleic acid) in a given sample.
  • subject and patient are used interchangeably herein to mean a member or members of any mammalian or non-mammalian species that may have a need for the pharmaceutical methods, compositions and treatments described herein.
  • Subjects and patients thus include, without limitation, primate (including humans), canine, feline, ungulate (e.g., equine, bovine, swine (e.g., pig)), avian, and other subjects.
  • Humans and non-human animals having commercial importance are of particular interest.
  • subject and patient refer to a subject or patient susceptible to infection by a Flaviviridae virus, particularly HCV.
  • “Mammal” means a member or members of any mammalian species, and includes, by way of example, canines; felines; equines; bovines; ovines; rodentia, etc. and primates, particularly humans. Non-human animal models, particularly mammals, e.g. primate, murine, lagomorpha, etc. may be used for experimental investigations.
  • the term“unit dosage form,” as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of compounds calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle.
  • a "pharmaceutically acceptable excipient,” “pharmaceutically acceptable diluent,” “pharmaceutically acceptable carrier,” and “pharmaceutically acceptable adjuvant” means an excipient, diluent, carrier, and adjuvant that are useful in preparing a pharmaceutical composition that are generally safe, non-toxic and neither biologically nor otherwise undesirable, and include an excipient, diluent, carrier, and adjuvant that are acceptable for veterinary use as well as human pharmaceutical use.
  • “A pharmaceutically acceptable excipient, diluent, carrier and adjuvant” as used in the specification and claims includes both one and more than one such excipient, diluent, carrier, and adjuvant.
  • a “pharmaceutical composition” is meant to encompass a composition suitable for administration to a subject, such as a mammal, especially a human.
  • a“pharmaceutical composition” is sterile, and is usually free of contaminants that are capable of eliciting an undesirable response within the subject (e.g., the compound(s) in the pharmaceutical composition is pharmaceutical grade).
  • Pharmaceutical compositions can be designed for administration to subjects or patients in need thereof via a number of different routes of administration including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, intracheal and the like.
  • antibody is used in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity.
  • Antibodies (Abs) and “immunoglobulins” (Igs) are glycoproteins having the same structural characteristics. While antibodies exhibit binding specificity to a specific antigen, immunoglobulins include both antibodies and other antibody-like molecules which lack antigen specificity. Polypeptides of the latter kind are, for example, produced at low levels by the lymph system and at increased levels by myelomas.
  • epitopic determinants means any antigenic determinant on an antigen to which the paratope of an antibody binds.
  • Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.
  • “Native antibodies and immunoglobulins” are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (V H ) followed by a number of constant domains.
  • V H variable domain
  • Each light chain has a variable domain at one end (V L ) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain.
  • Particular amino acid residues are believed to form an interface between the light- and heavy-chain variable domains (Clothia et al., J. Mol. Biol. 186:651 (1985); Novotny and Haber, Proc. Natl. Acad. Sci. U.S.A.82:4592 (1985)).
  • variable refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions both in the light-chain and the heavy-chain variable domains. The more highly conserved portions of variable domains are called the framework (FR).
  • CDRs complementarity-determining regions
  • FR framework
  • the variable domains of native heavy and light chains each comprise four FR regions, largely adopting a ⁇ -sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the ⁇ -sheet structure.
  • the CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen- binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)).
  • the constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.
  • Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual "Fc” fragment, whose name reflects its ability to crystallize readily.
  • Pepsin treatment yields an F(ab') 2 fragment that has two antigen-combining sites and is still capable of cross-linking antigen.
  • Fv is the minimum antibody fragment which contains a complete antigen- recognition and -binding site.
  • this region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association.
  • scFv single-chain Fv species
  • one heavy- and one light-chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a "dimeric" structure analogous to that in a two-chain Fv species. It is in this configuration that the three CDRs of each variable domain interact to define an antigen- binding site on the surface of the VH-VL dimer.
  • the Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain.
  • Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region.
  • Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group.
  • F(ab') 2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
  • immunoglobulins There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), e.g., IgG 1 , IgG 2 , IgG 3 , IgG 4 , IgA 1 , IgA 2 .
  • the heavy-chain constant domains that correspond to the different classes of immunoglobulins are called respectively.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
  • human antibody includes antibodies having variable and constant regions (if present) of human germline immunoglobulin sequences.
  • Human antibodies can 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).
  • Human antibodies of the present invention may be affinity matured in vitro.
  • human antibody does not 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 (i.e., humanized antibodies).
  • Antibody fragment and all grammatical variants thereof, as used herein are defined as a portion of an intact antibody comprising the antigen binding site or variable region of the intact antibody, wherein the portion is free of the constant heavy chain domains (i.e. CH2, CH3, and CH4, depending on antibody isotype) of the Fc region of the intact antibody.
  • constant heavy chain domains i.e. CH2, CH3, and CH4, depending on antibody isotype
  • antibody fragments include Fab, Fab', Fab'-SH, F(ab') 2 , and Fv fragments; diabodies; any antibody fragment that is a polypeptide having a primary structure consisting of one uninterrupted sequence of contiguous amino acid residues (referred to herein as a "single-chain antibody fragment” or “single chain polypeptide"), including without limitation (1) single-chain Fv (scFv) molecules (2) single chain polypeptides containing only one light chain variable domain, or a fragment thereof that contains the three CDRs of the light chain variable domain, without an associated heavy chain moiety and (3) single chain polypeptides containing only one heavy chain variable region, or a fragment thereof containing the three CDRs of the heavy chain variable region, without an associated light chain moiety; and multispecific or multivalent structures formed from antibody fragments.
  • single-chain antibody fragment single-chain Fv
  • the heavy chain(s) can contain any constant domain sequence (e.g. CH1 in the IgG isotype) found in a non-Fc region of an intact antibody, and/or can contain any hinge region sequence found in an intact antibody, and/or can contain a leucine zipper sequence fused to or situated in the hinge region sequence or the constant domain sequence of the heavy chain(s).
  • any constant domain sequence e.g. CH1 in the IgG isotype
  • conjugate is defined as a heterogeneous molecule formed by the covalent attachment of one or more antibody fragment(s) to one or more polymer molecule(s), wherein the heterogeneous molecule is water soluble, i.e. soluble in physiological fluids such as blood, and wherein the heterogeneous molecule is free of any structured aggregate.
  • a conjugate of interest is PEG.
  • structured aggregate refers to (1) any aggregate of molecules in aqueous solution having a spheroid or spheroid shell structure, such that the heterogeneous molecule is not in a micelle or other emulsion structure, and is not anchored to a lipid bilayer, vesicle or liposome; and (2) any aggregate of molecules in solid or insolubilized form, such as a chromatography bead matrix, that does not release the heterogeneous molecule into solution upon contact with an aqueous phase.
  • conjugate encompasses the aforementioned heterogeneous molecule in a precipitate, sediment, bioerodible matrix or other solid capable of releasing the heterogeneous molecule into aqueous solution upon hydration of the solid.
  • mAb monoclonal antibody
  • monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts.
  • Monoclonal antibodies are highly specific, being directed against a single antigenic site. Each mAb is directed against a single determinant on the antigen.
  • the monoclonal antibodies are advantageous in that they can be synthesized by cell culture, uncontaminated by other immunoglobulins.
  • the modifier "monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the present invention may be made in an immortalized B cell or hybridoma thereof, may be made by recombinant DNA methods, including without limitation yeast display.
  • an "isolated" antibody is one which has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes.
  • the antibody will be purified (1) to greater than 75% by weight of antibody as determined by the Lowry method, and most preferably more than 80%, 90% or 99% by weight, or (2) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain.
  • Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.
  • label when used herein refers to a detectable compound or composition which is conjugated directly or indirectly to the antibody.
  • the label may itself be detectable by itself (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable.
  • solid phase is meant a non-aqueous matrix to which the antibody of the present invention can adhere.
  • solid phases encompassed herein include those formed partially or entirely of glass (e.g. controlled pore glass), polysaccharides (e.g., agarose), polyacrylamides, polystyrene, polyvinyl alcohol and silicones.
  • the solid phase can comprise the well of an assay plate; in others it is a purification column (e.g. an affinity chromatography column). This term also includes a discontinuous solid phase of discrete particles, such as those described in U.S. Pat. No. 4,275,149.
  • compositions and methods are provided relating to affinity matured anti-HCV monoclonal antibodies.
  • the antibodies of the invention bind to and neutralize HCV virus across multiple genotypes.
  • Embodiments of the invention include isolated antibodies and derivatives and fragments thereof, pharmaceutical formulations comprising one or more of the human anti-HCV monoclonal antibodies; cell lines that produce these monoclonal antibodies.
  • the present invention is directed to combinatorially derived human monoclonal antibodies which are specifically reactive with and neutralize HCV, and cell lines which produce such antibodies.
  • Antibodies of the invention comprise the light chain variable region of HC-84.26.5D (SEQ ID NO:4) or a set of CDR sequences derived therefrom in a variable region framework sequence, usually a human variable region framework sequence.
  • the anti-HCV antibody may have a light chain variable region comprising the amino acid sequence of CDR1 and/or CDR2 and/or CDR3 of the provided affinity matured monoclonal antibodies as provided herein.
  • Antibodies of the invention may comprise the heavy chain variable region of any one of HC-84.26 (SEQ ID NO:1), HC-84.26.5D (SEQ ID NO:2), HC-84.26.5G (SEQ ID NO:3) or a set of CDR sequences derived therefrom in a variable region framework sequence, usually a human variable region framework sequence.
  • the anti-HCV antibody may have a heavy chain variable region comprising the amino acid sequence of CDR1 and/or CDR2 and/or CDR3 of the provided affinity matured monoclonal antibodies as provided herein.
  • Antibodies of interest include these provided combinations, as well as fusions of the variable regions to appropriate constant regions or fragments of constant regions, e.g. to generate F(ab)’ antibodies.
  • Exemplary sequences include, for example:
  • Variable regions of interest include at least one CDR sequence from the variable regions provided herein, usually at least 2 CDR sequences, and more usually 3 CDR sequences.
  • An exemplary CDR designation is shown in Figure 4, corresponding to the underlined residues above, however one of skill in the art will understand that a number of definitions of the CDRs are commonly in use, including the Kabat definition (see“Zhao et al. A germline knowledge based computational approach for determining antibody complementarity determining regions.” Mol Immunol. 2010;47:694–700), which is based on sequence variability and is the most commonly used.
  • the Chothia definition is based on the location of the structural loop regions (Chothia et al.“Conformations of immunoglobulin hypervariable regions.” Nature. 1989;342:877–883).
  • Alternative CDR definitions of interest include, without limitation, those disclosed by Honegger,“Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool.” J Mol Biol. 2001;309:657–670; Ofran et al.“Automated identification of complementarity determining regions (CDRs) reveals peculiar characteristics of CDRs and B cell epitopes.” J Immunol.
  • antibodies of interest include a pair of variable regions as set forth in SEQ ID NO:1, 4; SEQ ID NO:2, 4; SEQ ID NO:3, 4.
  • a CDR set comprises a heavy and light chain comprising, respectively, the CDR sequences within SEQ ID NO:1 and SEQ ID NO:4; the CDR sequences within SEQ ID NO:2 and SEQ ID NO:4; the CDR sequences within SEQ ID NO:3 and SEQ ID NO:1.
  • Antibodies of the invention bind to HCV E2 proteins of different HCV genotypes, including 1A, 2A, 2B, 4A, 5A and 6A.
  • Epitope mapping of antibody binding to HCV E2 protein shows that the antibodies recognize a conformational epitope, which may include residues 434-446.
  • Contact residues may comprise L441, F442, Y443, and W616.
  • the antibodies, relative to the non-affinity-matured version may have increased binding with at least a 5-fold increase in affinity, at least a 10 fold increase in affinity, and may have at least a 20-fold increase in affinity or more.
  • One or more residues of a CDR may be altered to modify binding to achieve a more favored on-rate of binding, a more favored off-rate of binding, or both, such that an optimized binding constant is achieved.
  • Affinity maturation techniques are well known in the art and can be used to alter the CDR region(s), followed by screening of the resultant binding molecules for the desired change in binding.
  • modifications can also be made within one or more of the framework regions, FRl, FR2, FR3 and FR4, of the heavy and/or the light chain variable regions of a human antibody, so long as these modifications do not eliminate the binding affinity of the human antibody.
  • the framework regions of human antibodies are usually substantially identical, and more usually, identical to the framework regions of the human germline sequences from which they were derived.
  • many of the amino acids in the framework region make little or no direct contribution to the specificity or affinity of an antibody.
  • many individual conservative substitutions of framework residues can be tolerated without appreciable change of the specificity or affinity of the resulting human immunoglobulin.
  • the variable framework region of the human antibody shares at least 85% sequence identity to a human germline variable framework region sequence or consensus of such sequences.
  • the variable framework region of the human antibody shares at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a human germline variable framework region sequence or consensus of such sequences.
  • a monoclonal antibody may be selected for its retention of other functional properties of antibodies of the invention, such as binding to multiple genotypes of HVC E2 and/or binding with an ultra-high affinity such as, for example, a K D of 10 "9 M or lower.
  • a polypeptide of interest has a contiguous sequence of at least about 10 amino acids as set forth in any one of SEQ ID NO:1-4, at least about 15 amino acids, at least about 20 amino acids, at least about 25 amino acids, at least about 30 amino acids, up to the complete provided variable region.
  • Polypeptides of interest also include variable regions sequences that differ by up to one, up to two, up to 3, up to 4, up to 5, up to 6 or more amino acids as compared to the amino acids sequence set forth in any one of SEQ ID NO:1-4.
  • a polypeptide of interest is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% identical to the amino acid sequence set forth in any one of SEQ ID NO:1-4.
  • the isolation of cells producing monoclonal antibodies of the invention can be accomplished using routine screening techniques, which permit determination of the elementary reaction pattern of the monoclonal antibody of interest.
  • routine screening techniques which permit determination of the elementary reaction pattern of the monoclonal antibody of interest.
  • a human monoclonal antibody has the same specificity as a human monoclonal antibody of the invention by ascertaining whether the former prevents the latter from binding to or neutralizing HCV, including without limitation an ability to neutralize an HCV virus comprising a panel of mutations in the E2 protein, as exemplified in the Examples herein. If the human monoclonal antibody being tested competes with the human monoclonal antibody of the invention, as shown by a decrease in binding by the human monoclonal antibody of the invention, then the two monoclonal antibodies bind to the same, or a closely related, epitope.
  • Still another way to determine whether a human monoclonal antibody has the specificity of a human monoclonal antibody of the invention is to pre-incubate the human monoclonal antibody of the invention with HCV with which it is normally reactive, and then add the human monoclonal antibody being tested to determine if the human monoclonal antibody being tested is inhibited in its ability to bind HCV. If the human monoclonal antibody being tested is inhibited then, in all likelihood, it has the same, or functionally equivalent, epitopic specificity as the monoclonal antibody of the invention. Screening of human monoclonal antibodies of the invention can be also carried out utilizing HCV and determining whether the monoclonal antibody neutralizes HCV.
  • single chain antibodies can be constructed according to the method of U.S. Pat. No. 4,946,778 to Ladner et al, which is incorporated herein by reference in its entirety. Single chain antibodies comprise the variable regions of the light and heavy chains joined by a flexible linker moiety. Yet smaller is the antibody fragment known as the single domain antibody, which comprises an isolate VH single domain. Techniques for obtaining a single domain antibody with at least some of the binding specificity of the intact antibody from which they are derived are known in the art.
  • the invention includes methods of treating or preventing an HCV-mediated disease in a subject by administering to the subject an isolated human monoclonal antibody or antigen binding portion thereof as described herein (i.e., that specifically binds to HCV) in an amount effective to inhibit the virus, e.g., HCV infection, HCV-mediated symptoms or morbidity.
  • diseases may include various liver conditions associated with HCV infection.
  • Treatment of patients before, during and/or after liver or kidney transplant. Treatment may include the use of the monoclonal antibodies of the invention as a single agent, or as an agent in combination with additional antiviral agents, including drugs, additional antibodies, vaccines, and the like.
  • Another treatment modality comprises infusing the neutralizing antibody into an HCV- positive donor organ to neutralize HCV prior to transplantation to decrease or eliminate the virus.
  • HCV- positive donor organ to neutralize HCV prior to transplantation to decrease or eliminate the virus.
  • other tissues such as heart, lung, etc can be treated in a similar manner.
  • a combination of an affinity matured antibody of the invention with a second antibody e.g. HC33.1 or a variant thereof, can provide for substantially complete protection across a variety of mutations.
  • a second antibody e.g. HC33.1 or a variant thereof.
  • Description of the HC33.1 antibodies may be found, for example, at Li et al. (2015) J. Biol. Chem.290(16):10117-25, herein specifically incorporated by reference.
  • An effective dose of an antibody or combination of antibodies according to the present invention can be used in vitro and/or in vivo during transplantation to prevent infection of the graft recipient from virus present in the graft.
  • Subjects suspected of having an HCV infection can be screened prior to therapy. Further, subjects receiving therapy may be tested in order to assay the activity and efficacy of the treatment. Significant improvements in one or more parameters is indicative of efficacy. It is well within the skill of the ordinary healthcare worker (e.g., clinician) to adjust dosage regimen and dose amounts to provide for optimal benefit to the patient according to a variety of factors (e.g., patient-dependent factors such as the severity of the disease and the like, the compound administered, and the like). For example, HCV infection in an individual can be detected and/or monitored by the presence of HCV RNA in blood, and/or having anti-HCV antibody in their serum. Other clinical signs and symptoms that can be useful in diagnosis and/or monitoring of therapy include assessment of liver function and assessment of liver fibrosis (e.g., which may accompany chronic viral infection).
  • Subjects for whom the therapy described herein can be administered include na ⁇ ve individuals (e.g., individuals who are diagnosed with HCV infection, but who have not been previously treated for HCV) and individuals who have failed prior treatment for HCV (“treatment failure” patients).
  • Previous HCV therapy includes, for example, treatment with IFN- ⁇ monotherapy (e.g., IFN- ⁇ and/or PEGylated IFN- ⁇ ) or IFN- ⁇ combination therapy, where the combination therapy may include administration of IFN- ⁇ and an antiviral agent such as ribavirin.
  • Treatment failure patients include non-responders (i.e., individuals in whom the HCV titer was not significantly or sufficiently reduced by a previous treatment for HCV to provide a clinically significant response, e.g., a previous IFN- ⁇ monotherapy, a previous IFN- ⁇ and ribavirin combination therapy, or a previous pegylated IFN- ⁇ and ribavirin combination therapy); and relapsers (i.e., individuals who were previously treated for HCV (e.g., who received a previous IFN- ⁇ monotherapy, a previous IFN- ⁇ and ribavirin combination therapy, or a previous pegylated IFN- ⁇ and ribavirin combination therapy), in whom the HCV titer decreased to provide a clinically significant response, but in whom the decreased HCV titer was not maintained due to a subsequent increase in HCV titer).
  • non-responders i.e., individuals in whom the HCV titer was not significantly or sufficiently reduced by
  • HCV titer HCV infection which is normally defined as an HCV titer of at least about 10 5 , at least about 5 x 10 5 , or at least about 10 6 or more genome copies of HCV per milliliter of serum, 2) are infected with HCV of a genotype that is recognized in the field as being associated with treatment failure (e.g. HCV genotype 1, subtypes thereof (e.g., 1a, 1b, etc.), and quasispecies thereof or 3) both.
  • HCV genotype 1, subtypes thereof e.g., 1a, 1b, etc.
  • Antibodies of the invention or portions thereof can be administered in a variety of suitable fashions, e.g., intravenously (IV), subcutaneously (SC), or, intramuscularly (IM) to the subject.
  • the antibody or antigen-binding portion thereof can be administered alone or in combination with another therapeutic agent, e.g., a second human monoclonal antibody or antigen binding portion thereof.
  • the second human monoclonal antibody or antigen binding portion thereof specifically binds to a second HCV isolate that differs from the isolate bound to the first antibody.
  • the antibody is administered together with another agent, for example, an antiviral agent.
  • Antiviral agents includes pegylated interferon ⁇ , ribivarin, etc.
  • the antibody is administered together with a polyclonal gamma- globulin (e.g., human gammaglobulin).
  • the antibody is administered before, after, or contemporaneously with a HCV vaccine.
  • the monoclonal antibodies of the invention can be used in vitro and in vivo to monitor the course of HCV disease therapy.
  • a particular therapeutic regimen aimed at ameliorating the HCV disease is effective.
  • the monoclonal antibodies of the invention may be used in vitro in immunoassays in which they can be utilized in liquid phase or bound to a solid phase carrier.
  • the monoclonal antibodies in these immunoassays can be detectably labeled in various ways.
  • types of immunoassays which can utilize monoclonal antibodies of the invention are competitive and non-competitive immunoassays in either a direct or indirect format.
  • Examples of such immunoassays are the radioimmunoassay (RIA) and the sandwich (immunometric) assay.
  • Detection of the antigens using the monoclonal antibodies of the invention can be done utilizing immunoassays which are run in either the forward, reverse, or simultaneous modes, including immunohistochemical assays on physiological samples. Those of skill in the art will know, or can readily discern, other immunoassay formats without undue experimentation.
  • the monoclonal antibodies of the invention can be bound to many different carriers and used to detect the presence of HCV.
  • carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agaroses and magnetite.
  • the nature of the carrier can be either soluble or insoluble for purposes of the invention. Those skilled in the art will know of other suitable carriers for binding monoclonal antibodies, or will be able to ascertain such, using routine experimentation.
  • labels and methods of labeling known to those of ordinary skill in the art.
  • Examples of the types of labels which can be used in the present invention include enzymes, radioisotopes, fluorescent compounds, colloidal metals, chemiluminescent compounds, and bio-luminescent compounds.
  • Those of ordinary skill in the art will know of other suitable labels for binding to the monoclonal antibodies of the invention, or will be able to ascertain such, using routine experimentation.
  • the binding of these labels to the monoclonal antibodies of the invention can be done using standard techniques common to those of ordinary skill in the art.
  • HCV may be detected by the monoclonal antibodies of the invention when present in biological fluids and tissues.
  • Any sample containing a detectable amount of HCV can be used.
  • a sample can be a liquid such as urine, saliva, cerebrospinal fluid, blood, serum and the like, or a solid or semi-solid such as tissues, feces, and the like, or, alternatively, a solid tissue such as those commonly used in histological diagnosis.
  • Another labeling technique which may result in greater sensitivity consists of coupling the antibodies to low molecular weight haptens. These haptens can then be specifically detected by means of a second reaction. For example, it is common to use haptens such as biotin, which reacts with avidin, or dinitrophenol, pyridoxal, or fluorescein, which can react with specific anti-hapten antibodies.
  • the antibody of the present invention can be provided in a kit, i.e., a packaged combination of reagents in predetermined amounts with instructions for performing the diagnostic assay.
  • the kit will include substrates and cofactors required by the enzyme (e.g., a substrate precursor which provides the detectable chromophore or fluorophore).
  • substrates and cofactors required by the enzyme e.g., a substrate precursor which provides the detectable chromophore or fluorophore
  • other additives may be included such as stabilizers, buffers (e.g., a block buffer or lysis buffer) and the like.
  • the relative amounts of the various reagents may be varied widely to provide for concentrations in solution of the reagents which substantially optimize the sensitivity of the assay.
  • the reagents may be provided as dry powders, usually lyophilized, including excipients which on dissolution will provide a reagent solution having the appropriate concentration.
  • the invention also provides isolated nucleic acids encoding the human anti-HCV antibodies, vectors and host cells comprising the nucleic acid, and recombinant techniques for the production of the antibody.
  • Exemplary polynucleotides encode the heavy or light chain variable region sequences set forth herein, e.g. SEQ ID NO:1-4.
  • Nucleic acids of interest may be at least about 80% identical to a sequence that encodes SEQ ID NO:1-4, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or identical.
  • a contiguous nucleotide sequence is at least about 20 nt., at least about 25 nt, at least about 50 nt., at least about 75 nt, at least about 100 nt, and up to the complete coding sequence may be used.
  • Such contiguous sequences may encode a CDR sequence or may encode a complete variable region. As is known in the art, a variable region sequence may be fused to any appropriate constant region sequence.
  • the nucleic acid encoding it is inserted into a replicable vector for further cloning (amplification of the DNA) or for expression.
  • DNA encoding the monoclonal antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody).
  • Many vectors are available.
  • the vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.
  • the anti-HCV antibody of this invention may be produced recombinantly not only directly, but also as a fusion polypeptide with a heterologous or homologous polypeptide, which include a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide, an immunoglobulin constant region sequence, and the like.
  • a heterologous signal sequence selected preferably may be one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell.
  • the signal sequence is substituted by a prokaryotic signal sequence selected.
  • An "isolated" nucleic acid molecule is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the antibody nucleic acid.
  • An isolated nucleic acid molecule is other than in the form or setting in which it is found in nature. Isolated nucleic acid molecules therefore are distinguished from the nucleic acid molecule as it exists in natural cells.
  • an isolated nucleic acid molecule includes a nucleic acid molecule contained in cells that ordinarily express the antibody where, for example, the nucleic acid molecule is in a chromosomal location different from that of natural cells.
  • control sequences refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism.
  • the control sequences that are suitable for prokaryotes include a promoter, optionally an operator sequence, and a ribosome binding site.
  • Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.
  • Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence.
  • DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide;
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or
  • a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
  • "operably linked” means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.
  • the expressions "cell,” “cell line,” and “cell culture” are used interchangeably and all such designations include progeny.
  • the words “transformants” and “transformed cells” include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where distinct designations are intended, it will be clear from the context.
  • Suitable host cells for cloning or expressing the DNA are the prokaryote, yeast, or higher eukaryote cells.
  • useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR(CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol.
  • monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TR1 cells (Mather et al., Annals N.Y. Acad. Sci.383:44-68 (1.982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).
  • Host cells are transformed with the above-described expression or cloning vectors for anti-HCV antibody production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
  • the provided human antibody variable regions and/or CDR regions are used in a screening method to select for antibodies optimized for affinity, specificity, and the like.
  • random or directed mutagenesis is utilized to generate changes in the amino acid structure of the variable region or regions, where such variable regions will initially comprise one or more of the provided CDR sequences, e.g. a framework variable region comprising CDR1, CDR2, CDR3 from the heavy and light chain sequences provided in SEQ ID NO:1-4.
  • variable region sequences which are optionally combined with a second variable region sequence, i.e. V H VL, with constant regions, as a fusion protein to provide for display, etc., as known in the art.
  • Methods for selection of antibodies with optimized specificity, affinity, etc. are known and practiced in the art, e.g. including methods described by Presta (2006) Adv Drug Deliv Rev. 58(5-6):640-56; Levin and Weiss (2006) Mol Biosyst.2(1):49-57; Rothe et al. (2006) Expert Opin Biol Ther.6(2):177-87; Ladner et al. (2001) Curr Opin Biotechnol.
  • Such screening methods may involve mutagenizing a variable region sequence comprising one or more CDR sequences set forth herein; expressing the mutagenized sequence to provide a polypeptide product; contacting the polypeptide with an HCV antigen; identifying those polypeptide having the desired antigen affinity or specificity.
  • the antibody composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique.
  • affinity chromatography is the preferred purification technique.
  • the suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody.
  • Protein A can be used to purify antibodies that are based on human ⁇ 1, ⁇ 2, or ⁇ 4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for human ⁇ 3 (Guss et al., EMBO J.5:15671575 (1986)).
  • the matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Where the antibody comprises a CH 3 domain, the Bakerbond ABX TM resin (J. T. Baker, Phillipsburg, N.J.) is useful for purification.
  • the mixture comprising the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, preferably performed at low salt concentrations (e.g., from about 0-0.25M salt).
  • the antibody formulations of the present invention may be used to treat the various HCV associated diseases, including before or after organ transplantation, as described herein.
  • the recipient is at a high risk of infection.
  • Antibody formulations may comprise one or more antibodies as set forth here, and may comprise a cocktail of antibodies, i.e. two, three, four or more antibodies that recognize different epitopes.
  • the antibody formulation is administered by any suitable means, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal.
  • Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.
  • the antibody formulation is suitably administered by pulse infusion, particularly with declining doses of the antibody.
  • the appropriate dosage of antibody will depend on the type of disease to be treated, the severity and course of the disease, whether the antibody is administered for preventive purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician.
  • the antibody is suitably administered to the patient at one time or over a series of treatments.
  • an article of manufacture containing materials useful for the treatment of the disorders described above comprises a container and a label.
  • Suitable containers include, for example, bottles, vials, syringes, and test tubes.
  • the containers may be formed from a variety of materials such as glass or plastic.
  • the container holds a composition which is effective for treating the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • the active agent in the composition is one or more antibodies in a formulation of the invention as described above.
  • the label on, or associated with, the container indicates that the composition is used for treating the condition of choice.
  • the article of manufacture may further comprise a second container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
  • a pharmaceutically-acceptable buffer such as phosphate-buffered saline, Ringer's solution and dextrose solution.
  • It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
  • Therapeutic formulations comprising one or more antibodies of the invention are prepared for storage by mixing the antibody having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions.
  • the antibody composition will be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners.
  • the "therapeutically effective amount" of the antibody to be administered will be governed by such considerations, and is the minimum amount necessary to reduce virus titer in an infected individual.
  • the therapeutic dose may be at least about 0.01 ⁇ g/kg body weight, at least about 0.05 ⁇ g/kg body weight; at least about 0.1 ⁇ g/kg body weight, at least about 0.5 ⁇ g/kg body weight, at least about 1 ⁇ g/kg body weight, at least about 2.5 ⁇ g/kg body weight, at least about 5 ⁇ g/kg body weight, and not more than about 100 ⁇ g/kg body weight. It will be understood by one of skill in the art that such guidelines will be adjusted for the molecular weight of the active agent, e.g. in the use of antibody fragments, or in the use of antibody conjugates.
  • the dosage may also be varied for localized administration, or for systemic administration, e.g. i.m., i.p., i.v., and the like.
  • the antibody need not be, but is optionally formulated with one or more agents currently used to prevent or treat HCV infection. These are generally used in the same dosages and with administration routes as used hereinbefore or about from 1 to 99% of the heretofore employed dosages.
  • Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyidimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, his
  • the formulation herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other.
  • active compound preferably those with complementary activities that do not adversely affect each other.
  • it may be desirable to further provide an antiviral agent.
  • Such molecules are suitably present in combination in amounts that are effective for the purpose intended.
  • the active ingredients may also be entrapped in microcapsule prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsule and poly-(methylmethacylate) microcapsule, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
  • a pharmaceutically effective dose is that dose required to prevent, inhibit the occurrence, or treat (alleviate a symptom at least to some extent) of a disease state, e.g. to reduce virus titer in an infected individual.
  • the pharmaceutically effective dose depends on the type of disease, the composition used, the route of administration, the type of subject being treated, subject-dependent characteristics under consideration, concurrent medication, and other factors that those skilled in the medical arts will recognize. Generally, an amount between 0.1 mg/kg and 100 mg/kg body weight/day of active ingredients is administered.
  • Formulations and methods of delivery of agents to the liver are known in the art, see, e.g., Wen et al., 2004, World J. Gastroenterol. 10:244-9; Murao et al., 2002, Pharm. Res. 19:1808-14; Liu et al., 2003, Gene Ther.10:180-7; Hong et al., 2003, J. Pharm. Pharmacol. 54;51-8; Herrmann et al., 2004, Arch. Virol. 149:1611-7; and Matsuno et al., 2003, Gene. Ther.10:1559-66.
  • Formulations and methods of delivery of agents to the skin or mucosa are known in the art.
  • Such delivery systems include, e.g., aqueous and nonaqueous gels, creams, multiple emulsions, microemulsions, liposomes, ointments, aqueous and nonaqueous solutions, lotions, patches, suppositories, and tablets, and can contain excipients such as solubilizers, permeation enhancers (e.g., fatty acids, fatty acid esters, fatty alcohols and amino acids), and hydrophilic polymers (e.g., polycarbophil and polyvinylpyrolidone).
  • Oral administration can be accomplished using pharmaceutical compositions containing an agent of interest formulated as tablets, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, or syrups or elixirs.
  • Such oral compositions can contain one or more such sweetening agents, flavoring agents, coloring agents or preservative agents in order to provide pharmaceutically elegant and palatable preparations.
  • Tablets which can be coated or uncoated, can be formulated to contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients, e.g., inert diluents; such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, e.g., starch, gelatin or acacia; and lubricating agents, for example magnesium stearate, stearic acid or talc. Where a coating is used, the coating delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.
  • non-toxic pharmaceutically acceptable excipients e.g., inert diluents; such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate
  • granulating and disintegrating agents for example, corn starch, or algin
  • the formulation is an aqueous suspension
  • such can contain the active agent in a mixture with a suitable excipient(s).
  • excipients can be, as appropriate, suspending agents (e.g., sodium carboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia); dispersing or wetting agents; preservatives; coloring agents; and/or flavoring agents.
  • Suppositories e.g., for rectal administration of agents, can be prepared by mixing the agent with a suitable non-irritating excipient that is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug.
  • suitable non-irritating excipient include cocoa butter and polyethylene glycols.
  • Dosage levels can be readily determined by the ordinarily skilled clinician, and can be modified as required, e.g., as required to modify a subject's response to therapy. In general dosage levels are on the order of from about 0.1 mg to about 140 mg per kilogram of body weight per day. The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form varies depending upon the host treated and the particular mode of administration. Dosage unit forms generally contain between from about 1 mg to about 500 mg of an active ingredient. [00121]
  • DAAs Direct acting antivirals
  • HCV chronic hepatitis C virus
  • RAV resistance-associated variants
  • HMAbs human monoclonal antibodies
  • HC84.26 an IgG 1 HMAb, is member of a panel of neutralizing antibodies directed at a cluster of overlapping conformational epitopes on HCV E2, designated as antigenic domain D. These antibodies neutralize infectious cell culture derived genotype 1-6 HCV isolates (HCVcc). When a 2a HCVcc isolate is grown under increasing concentrations of HC84.26, complete viral elimination occurs at a critical antibody concentration.
  • Affinity maturation was undertaken by a yeast display approach.
  • the advantages of yeast display over phage display include avoiding expression, purification and characterization of a large number of different phage-display scFv, the ability to measure K D directly on the yeast surface, and the ability to enrich for higher affinity clones with decreasing antigen concentration by flow cytometry.
  • employing this approach we isolated affinity-matured HC84.26 clones that have improved binding and neutralization activities against HCV isolates that were neutralized poorly by wild type (wt) HC84.26.
  • HC84.26 neutralizes a panel of genotype 1-6 HCVcc; this and other related antigenic domain D HMAbs are against epitopes on E2 that include critical residues at 441, 442 and 443, and 616 by alanine substitution studies. While the residues at L441, W443 and W616 are absolutely conserved among all HCV isolates, F442 is only 60% conserved with the remaining 40% having either F442I or F442L mutations. Yet, no viral escape was noted when a 2a HCVcc was co-cultured with HC84.26. The explanation is provided by structural studies.
  • the first step was to optimize the pairing of the heavy chain variable gene regions (VH) of HC84.26 with different light chain variable gene regions (VL) for binding to a mutant H77C 1a F442I E2 recombinant protein.
  • HC84.26 wild-type VH was cloned into a yeast vector pYD2 library that contained VL. This VL library was generated from the B cells of the donor in which we isolated the HC84.26 HMAb.
  • the constructed HC84.26 VH/VL library was screened for scFv displayed clones that bound to F442I E2 by magnetic immunobead selection and by fluorescence-activated cell sorting (FACS).
  • HC84.26.5 A number of these clones were selected for further maturation by random mutagenesis of their VH and VL fragment genes. Additional rounds of selection led to the isolation of a scFv clone designated as HC84.26.5 with significantly greater binding than wt HC84.26 to F442I E2 by FACS analysis. Targeted mutagenesis on VH CDR1 and CDR2 of HC84.26.5 resulted in two scFv clones, designated as HC84.26.5D and HC84.26.5G having additional increase in binding to F442I E2. Combining the observed mutations in these two clones into a new clone, HC84.26.5DG, however, showed no further improvements by flow analysis.
  • K D Total improvements in K D ranged between 25-34 fold differences for these three affinity mature clones. Against F442L E2, more moderate K on , K off and K D improvements were observed. K D improvements ranged between 3.2-6.3 folds. We next assessed whether improved affinities led to improved virus neutralization (Table 2). Against 1a H77C HCVpp, the IC 50 values were in the same range for wt HC84.26, HC84.26.5D, HC84.26.5G and HC84.26.5DG.
  • IC 50 values for the three matured clones ranged between 0.37-1.95 ⁇ g/ml, with HC84.26.5D having the lowest IC 50 that also has the most improved K D .
  • the most improved clone was again HC84.26.5D with a two log reduction in IC 50 value of 0.14 ⁇ g/ml.
  • HC84.26.5D prevents acute HCV infection in humanized mice.
  • In vivo protection of acute HCV infection was tested in the human liver chimeric alb-uPA/SCID mouse model over a 72 day study.
  • the test group received HC84.26.5D antibody and the control group received R04, an isotype-matched IgG 1 HMAb to human cytomegalovirus that exhibited no activity in vitro against HCV.
  • Antibodies were administrated just once on day -1 at 250 mg/kg by intraperitoneal injection, based in part on earlier studies with other antibodies.
  • each mouse in both groups was challenged by intravenous injection of 10 5 IU of genotype 1b HCV infected human serum. No further treatment was given to the animals.
  • the mice were followed at weekly intervals for 10 weeks after challenge and monitored for hAAT and HCV RNA titers.
  • the serum hAAT values remained high for each mouse in both groups, which confirmed the viability of the human hepatocyte grafts for the duration of the study (Figs.5A; 5B).
  • the E2 434–446 peptide represents a portion of the domain D epitope of E2 recognized by wt HC84.26, which is conformational in nature. Electron density difference maps revealed well-defined density for the E2 434–446 peptide in which the peptide adopts an ⁇ -helical turn spanning aa437–442, while the C-terminus exhibits an extended conformation comprising aa443–446 (Fig. 2A). The conformation of E2 434–446 bound to HC84.26.5D is very similar to that of this same peptide bound to the related HMAbs HC84.1 and HC84.27.
  • Y443 forms stacking interactions with VL CDR3Trp90, as well as a main-chain hydrogen bond with VL CDR3Ser93 (Table 5; Fig. 3A).
  • K446 whose mutation to Glu or Asn in HCV escape variants abolished (Glu) or substantially reduced (Asn) neutralization by HC84.26.5D (see below, Table 3).
  • the positively charged side chain of K446 lies in an electronegative depression on the VL domain, between CDR1 and CDR2, where it forms a salt bridge with VL CDR2Asp50 (Fig.3A).
  • the predicted energetic impact of the K446E and K446N mutations on HC84.26.5D binding was assessed by modeling these substitutions and evaluating their ⁇ ⁇ Gs, starting with the HC84.26.5D– E2 434–446 crystal structure. In silico mutagenesis was performed using Rosetta, a protein modeling program, and ZAFFI, an energy-based scoring function.
  • the K446A substitution was predicted to lower antibody affinity by 6-fold, which is comparable to the 3–5-fold affinity loss observed for E2 K446A binding to the related HMAbs HC84.26, HC84.1 and HC84.27, the latter two featuring very similar electrostatically-driven interactions with K446 in their complexes with E2 434–446 .
  • HMAb, wt HC84.26, HC84.26.5D and HC84.26.5G, and a control HC33.1 at 20 ⁇ g/ml was tested against the genotype 1 panel that also included H77C (Table 3).
  • HC33.1 is a neutralizing HMAb directed against aa412-423 on E2. Neutralizing activity ⁇ 40% was viewed as no activity; 41-60% as modest; 61-80% as moderate; and >80% as strong.
  • Wt HC84.26 did not neutralize 1a80 and 1a142, and neutralized modestly 1a09 and 1a157 HCV isolates.
  • Both 1a80 and 1a142 have F442I, and 1a09 has F442L mutations, which account for the lost or modest activities by wt HC84.26.
  • the drop in activity against 1a157 is not due to a mutation at 442 but is likely due to a K446N mutation.
  • This is supported by the observation that a K446A mutation significantly reduces binding by wt HC84.26 by 60%.
  • a deleterious effect of the K446N mutation on antibody binding is also supported by the structure of the HC84.26.5D–E2 434–446 complex.
  • both HC84.26.5D and HC84.26.5G performed worse than wt HC84.26.
  • HC84.26.WH.5DL Two isolates, 1a80 and 1a129, not neutralized by HC33.1 are neutralized by HC84.26.WH.5DL.
  • the single isolate not neutralized by HC84.26.WH.5DL is neutralized by HC33.1. While >90% of the panel is neutralized by HC84.26.WH.5DL alone, a second antibody, e.g. HC33.1, is required to achieve 100% breadth of protection coverage.
  • HCV variants with a mutation at F442 and/or at K446: 1a80 and 1a142 have F442I; 1a09 has F442L; 1a142, K446E; 1a157, K446N; and 1b21, K446H.
  • An antibody was selected that is less likely to induced RAV for affinity maturation in an effort to increase neutralization potency and breadth of protection. This was achieved with HC84.26, an IgG 1 HMAb that earlier studies showed broad neutralization against genotype 1-6 HCVcc. In addition, complete elimination of virus occurs at a critical concentration when this antibody is co-cultured with infectious virions.
  • HC84.26.5D Affinity maturation by a yeast display approach led to the isolation of a matured HC84.26 clone, HC84.26.5D that neutralized an E2 F442I mutant HCVpp that was not neutralized by the parental antibody.
  • HC84.26.5D protected three of four mice over a prolonged period. The fourth mouse showed breakthrough infection at a timepoint when the serum antibody concentration was reduced by 100-fold. Sequence analysis of the breakthrough infection revealed no viral escape mutation.
  • a second broadly neutralizing antibody to non-competing epitope is required and more likely a third antibody will be necessary since wt HC84.26 and HC33.1 failed to neutralized one of the genotype 1 isolates, 1a80. Only the affinity matured HC84.26.WH.5DL neutralized this isolate.
  • HEK-293T cells were obtained from the ATCC.
  • Huh7.5 cells (generously provided by Dr. C. Rice, Rockefeller University) were grown in Dulbecco's modified minimal essential medium (Invitrogen, Carlsbad, CA), supplemented with 10% fetal calf serum (Sigma-Aldrich Co., St. Louis, MO) and 2 mM glutamine.
  • HMAbs CBH-4G, HC- 84.26 and HC33.1 against HCV E2 glycoprotein were produced as described.
  • Yeast Saccharomyces cerevisiae strain EBY-100 (GAL1-AGA1:URA3 ura3-52 trp1 leu2 ⁇ 1 his3 ⁇ 200 pep4::HIS2 prb1 ⁇ 1.6R can1 GAL) (Invitrogen, Carlsbad, CA) was maintained in YPD broth (Difco).
  • the yeast display vector pYD2 was kindly provided by Dr. J. D. Marks (UCSF).
  • IgG 1 -Abvec for full-length IgG 1 expression was kindly provided by Dr. P. Wilson (University of Chicago).
  • the VH fragment of the pYD2.HC84.26 was amplified by primer extension with forward primer GAP5 (5'-TTA AGC TTC TGC AGG CTA GTG-3') and reverse primer HuJHR: (5'-ACC TCC GGA GCC ACC TCC GCC TGA ACC GCC TCC ACC TGT CGA CCC TGA-3').
  • the HC84.26 light chain shuffle library was created by ligating amplified VH fragments (10 ⁇ g) to pre-digested pYD2.HC84-VK/VL (NcoI/SalI, 50 ⁇ g) in Saccharomyces cerevisiae strain EBY100 through gap repairing. Library characterization and induction has been described previously.
  • C For site-directed mutagenesis scFv yeast display libraries: To further increase the binding affinity and/or to increase breadth of neutralization, amino acid change was sequentially introduced in VH-CDR1/CDR2 using a QuikChange II site-directed mutagenesis kit with degenerated primers (NNS) as described. Library construction and growth was carried out as described above.
  • D Isolation of candidate clones: MACS and FACS sorting of antigen-specific affinity-improved scFv clones and cloning of immunoglobulin genes were carried out using previously described protocol.
  • Binding affinity and specificity measurement of selected clones Affinity of scFv clones during screening was approximated by flow cytometry and binding kinetics were measured by surface plasmon resonance (BIAcore 3000, Pharmacia Biosensor), as described. Antibody specificity was evaluated in a Galanthus nivalis agglutinin (GNA)- captured E1E2 ELISA also as described.
  • GAA Galanthus nivalis agglutinin
  • In vivo protection study Twelve Alb-uPA/SCID mice with high engraftment of human hepatocytes were allocated to the two study groups. Six mice received the negative control R04 antibody and six received HC84.26.5D. Baseline blood draws were obtained on Day -6 for measurement of serum hAAT levels and allocation to the study groups such that the group average hAAT values were equivalent. Animals were administered their respective antibodies on Day -1 by intraperitoneal injection of a 250 mg/kg dose. On Day 0 the animals received a single intravenous challenge dose of 10 5 IU gt1b HCV. Blood samples were taken weekly from day 7 to 70 for measurement of hAAT and HCV titers.
  • IgG half-life measurement A modification of the standard enzyme-linked immunosorbent assay (ELISA) was used to measure serum HC84.26.5D levels in treated mice. This required the establishment of a standard curve for this antibody, as outlined in Fig. 6.
  • the ELISA was performed as follows. Plates were coated with GNA and blocked with 2.5% nonfat dry milk and 2.5% normal goat serum in 0.1% Tween-phosphate-buffered saline (PBS).
  • Lysates of cells expressing recombinant HCV E2 antigens were captured by GNA onto a microtiter plate and followed by incubation with different dilutions of known concentrations of HC84.25.5D or R04, an isotype-matched negative control HMAb that does not bind to HCV E2, or test mouse sera at various dilutions. Bound HMAb was detected as described. The serum concentrations for HC84.26.5D at different serum dilutions were back-calculated based on the standard curve and the half-life determinations were determined using a nonlinear regression curve fit (Graphpad Prism, Graphpad software, Inc., CA).
  • HCV-pseudotype retroviral particle (HCVpp) production and neutralization HCVpp expressing genotype 1 HCV E1E2 glycoproteins were produced and tested in neutralization assay as described. All assays were performed in triplicates and antibody concentrations resulting in 50% neutralization (IC 50 ) were calculated by nonlinear regression analysis (Graphpad Prism).
  • RNA from the test sample was extracted using commercial kits (Qiagen, Valenica, CA).
  • cDNA of the E2 glycoprotein was synthesized with SuperScript III reverse transcriptase (Thermo Fisher Scientific, Waltham, MA) by using Random Hexamer primer in a 20- ⁇ l reaction of the manufacturer’s recommended buffer.
  • PCR products were ligated into the TOPO cloning vector (Invitrogen, Carlsbad, CA), and 10 individual clones containing an insert of the expected size were sequenced in both sense and antisense strands using vector primers T7F and M13Rev. (ElimBiopharm, Hayward, CA).
  • the HC84.26.5D antibody was expressed as a single-chain Fv fragment (scFv) by in vitro folding from inclusion bodies produced in Escherichia coli.
  • the scFv construct consisted of the heavy chain variable (V H ) region (residues Glu1–Ser127) connected to the light chain variable (V L ) region (residues Gln1– Leu110) by an 18-residue linker (GSTGGGGSGGGGSGGGGS).
  • V H heavy chain variable
  • V L light chain variable
  • GSTGGGGSGGGGSGGGGS 18-residue linker
  • Bacteria were grown at 37 o C in LB medium to an absorbance of 0.6–0.8 at 600 nm, and induced with 1 mM isopropyl- ⁇ -D-thiogalactoside. After incubation for 3 h, the bacteria were harvested by centrifugation and resuspended in 50 mM Tris-HCl (pH 8.0) containing 0.1 M NaCl and 2 mM EDTA; cells were disrupted by sonication.
  • Tris-HCl pH 8.0
  • Inclusion bodies were washed extensively with 50 mM Tris-HCl (pH 8.0) and 2% (v/v) Triton X-100, then dissolved in 8 M urea, 50 mM Tris-HCl (pH 8.0), and 10 mM DTT.
  • inclusion bodies were diluted into ice-cold folding buffer containing 1 M L- arginine-HCl, 50 mM Tris-HCl (pH 8.0), 1 mM EDTA, 3 mM reduced glutathione, and 0.9 mM oxidized glutathione, to a final protein concentration of 60 mg/l.
  • the folding mixture was concentrated 50-fold, dialyzed against 50 mM MES (pH 6.0), and centrifuged to remove aggregates. Correctly folded HC84.26.5D scFv was then purified using sequential Superdex 75 HR and MonoQ columns (GE Healthcare).
  • Protein Data Bank accession code Coordinates and structure factors for the HC84.26.5D–E2 434–446 complex have been deposited in the Protein Data Bank under accession code 4Z0X.
  • Rosetta Floppytail protocol to generate 2400 minimized models of the HC84.26.5D complex with E2, via flexible backbone modeling of E2 residues 429–452, while keeping the epitope region rigid and fixed to the bound antibody using pairwise distance constraints.
  • Residues 429–452 were selected for flexible modeling as this region is bounded at either end by disulfide bonds, but is on the surface of E2 core and thought to adopt multiple conformations based on structural data.
  • the top model from the set of 2400 was selected based on ZRANK2 score.
  • Fafi-Kremer S Fofana I, Soulier E, Carolla P, Meuleman P, Leroux-Roels G, Patel AH, Cosset FL, Pessaux P , Doffoel M, et al. Viral entry and escape from antibody-mediated neutralization influence hepatitis C virus reinfection in liver transplantation. J Exp Med. 2010;207(9):2019-31.
  • Boder ET and Wittrup KD. Yeast surface display for screening combinatorial polypeptide libraries. Nat Biotechnol.1997;15(6):553-7.
  • Boder ET Midelfort KS, and Wittrup KD. Directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity. Proc Natl Acad Sci U S A. 2000;97(20):10701-5.
  • Kidney Disease Improving Global Outcomes (KDIGO). KDIGO clinical practice guidelines for the prevention, diagnosis, evaluation, and treatment of hepatitis C in chronic kidney disease. Kidney Int 2008;73(Suppl.109):S1-S99.

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Abstract

L'invention concerne des compositions et des procédés relatives à des anticorps monoclonaux anti-VHC E2 à maturation affinée. Les anticorps de l'invention se lient à une région conservée de la protéine HCV E2 et neutralisent le virus de la grippe HCV à travers de multiples génotypes de HCV. Des modes de réalisation de l'invention comprennent des anticorps isolés et des dérivés et des fragments de ceux-ci, des formulations pharmaceutiques comprenant un ou plusieurs parmi les anticorps monoclonaux anti-HCV ; et des lignes cellulaires qui produisent ces anticorps monoclonaux.
PCT/US2017/034816 2016-05-27 2017-05-26 Anticorps à large spectre à maturation affinée dirigés contre le virus de l'hépatite c WO2017205820A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060036076A1 (en) * 2003-11-19 2006-02-16 Dyax Corp. Metalloproteinase-binding proteins
US20080095775A1 (en) * 2006-06-13 2008-04-24 Lewis Katherine E Il-17 and il-23 antagonists and methods of using the same
US20100183625A1 (en) * 2009-01-06 2010-07-22 Dyax Corp. Treatment of Mucositis with Kallikrein Inhibitors
US20110200611A1 (en) * 2010-01-06 2011-08-18 Dyax Corp. Plasma kallikrein binding proteins
US20110207917A1 (en) * 2009-02-09 2011-08-25 Patrys Limited Sam-6 variants, target and methods of use
WO2014053634A1 (fr) * 2012-10-04 2014-04-10 Institut Pasteur Nouveaux anticorps neutralisants dirigés contre le virus de l'hépatite c

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060036076A1 (en) * 2003-11-19 2006-02-16 Dyax Corp. Metalloproteinase-binding proteins
US20080095775A1 (en) * 2006-06-13 2008-04-24 Lewis Katherine E Il-17 and il-23 antagonists and methods of using the same
US20100183625A1 (en) * 2009-01-06 2010-07-22 Dyax Corp. Treatment of Mucositis with Kallikrein Inhibitors
US20110207917A1 (en) * 2009-02-09 2011-08-25 Patrys Limited Sam-6 variants, target and methods of use
US20110200611A1 (en) * 2010-01-06 2011-08-18 Dyax Corp. Plasma kallikrein binding proteins
WO2014053634A1 (fr) * 2012-10-04 2014-04-10 Institut Pasteur Nouveaux anticorps neutralisants dirigés contre le virus de l'hépatite c

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