WO2013165972A2 - Anticorps anti-virus hépatite b et applications associées - Google Patents

Anticorps anti-virus hépatite b et applications associées Download PDF

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Publication number
WO2013165972A2
WO2013165972A2 PCT/US2013/038802 US2013038802W WO2013165972A2 WO 2013165972 A2 WO2013165972 A2 WO 2013165972A2 US 2013038802 W US2013038802 W US 2013038802W WO 2013165972 A2 WO2013165972 A2 WO 2013165972A2
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Prior art keywords
seq
amino acid
acid sequence
variable region
chain variable
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PCT/US2013/038802
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English (en)
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WO2013165972A3 (fr
Inventor
Shuji Sato
Sean Andre BEAUSOLEIL
Wan Cheung CHEUNG
Roberto D. POLAKIEWICZ
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Cell Signaling Technology, Inc.
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Publication of WO2013165972A2 publication Critical patent/WO2013165972A2/fr
Publication of WO2013165972A3 publication Critical patent/WO2013165972A3/fr

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    • 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
    • C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
    • C07K16/082—Hepadnaviridae, e.g. hepatitis B virus
    • 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
    • C07K2317/90—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

Definitions

  • Chronic HBV infection affects over 350 million people, a significant percentage of whom succumb to hepatic failure or have a high risk of developing hepatocellular carcinoma (Zuckerman et al, 2000, J. Infect., 41 : 130-136). Potently neutralizing monoclonal antibodies against hepatitis B virus that can be produced consistently in industrial scale batches would have clinical value.
  • This disclosure is based, at least in part, on the isolation of human anti-hepatitis B virus (HBV) antibodies.
  • HBV hepatitis B virus
  • anti-hepatitis B surface antigen binding agents e.g., antibodies
  • anti-hepatitis B surface antigen binding agents e.g., antibodies
  • (ix) (a) a heavy chain variable region having CDRs 1 , 2 and 3 having the amino acid sequences set forth in SEQ ID NOs: 119, 121 and 123, respectively, or SEQ ID NOs: 413, 67, and 415, respectively, and (b) a light chain variable region having CDRs 1, 2 and 3 having a set of amino acid sequences set forth in (i) SEQ ID NOs: 218, 220 and 222, respectively; (ii) SEQ ID NOs: 227, 229 and 231, respectively; (iii) SEQ ID NOs: 245, 247 and 249, respectively; (iv) SEQ ID NOs: 263, 265 and 267, respectively; (v) SEQ ID NOs: 290, 292 and 294, respectively; or (vi) SEQ ID NOs: 308, 310 and 312, respectively; (x) (a) a heavy chain variable region having CDRs 1 , 2 and 3 having the amino acid sequences set forth in SEQ ID NOs: 128,
  • SEQ ID NOs: 344, 346 and 348 respectively; (ii) SEQ ID NOs: 362, 364 and 366, respectively; (iii) SEQ ID NOs: 371, 373 and 375, respectively; or (iv) SEQ ID NOs: 380,
  • the disclosure features anti-hepatitis B surface antigen binding agents (e.g., antibodies) that include a heavy chain variable region having the sequence of SEQ ID NO: 45, 54, 63, 72, 81, 90, 99, 108, 117, 126, 135, 144, 153, 162, 171, 180, 189,
  • anti-hepatitis B surface antigen binding agents e.g., antibodies
  • a heavy chain variable region having the sequence of SEQ ID NO: 45, 54, 63, 72, 81, 90, 99, 108, 117, 126, 135, 144, 153, 162, 171, 180, 189,
  • anti-hepatitis B surface antigen binding agents e.g., antibodies
  • anti-hepatitis B surface antigen binding agents e.g., antibodies
  • a heavy chain variable region having an amino acid sequence at least 80% identical e.g., at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical
  • a light chain variable region having an amino acid sequence at least 80% identical e.g., at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical
  • a heavy chain variable region having an amino acid sequence at least 80% identical e.g., at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical
  • SEQ ID NO: 54 a heavy chain variable region having an amino acid sequence at least 80% identical (e.g., at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO: 54 and a light chain variable region having an amino acid sequence at least 80% identical (e.g., at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO: 234;
  • a heavy chain variable region having an amino acid sequence at least 80% identical e.g., at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical
  • SEQ ID NO: 63, 405, 409, or 412 a light chain variable region having an amino acid sequence at least 80% identical (e.g., at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO: 225, 243, 279, 288, 297, or 306;
  • a heavy chain variable region having an amino acid sequence at least 80% identical e.g., at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical
  • SEQ ID NO: 72 a heavy chain variable region having an amino acid sequence at least 80% identical (e.g., at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO: 72 and a light chain variable region having an amino acid sequence at least 80% identical (e.g., at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO: 279;
  • a heavy chain variable region having an amino acid sequence at least 80% identical e.g., at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical
  • SEQ ID NO: 81 a heavy chain variable region having an amino acid sequence at least 80% identical (e.g., at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO: 81 and a light chain variable region having an amino acid sequence at least 80% identical (e.g., at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO: 270;
  • a heavy chain variable region having an amino acid sequence at least 80% identical e.g., at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical
  • SEQ ID NO: 90 a heavy chain variable region having an amino acid sequence at least 80% identical (e.g., at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO: 90 and a light chain variable region having an amino acid sequence at least 80% identical (e.g., at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO: 279;
  • a heavy chain variable region having an amino acid sequence at least 80% identical e.g., at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical
  • a light chain variable region having an amino acid sequence at least 80% identical e.g., at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical
  • a heavy chain variable region having an amino acid sequence at least 80% identical e.g., at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical
  • SEQ ID NO: 108 a heavy chain variable region having an amino acid sequence at least 80% identical (e.g., at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO: 108 and a light chain variable region having an amino acid sequence at least 80% identical (e.g., at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO: 333;
  • (x) a heavy chain variable region having an amino acid sequence at least 80% identical (e.g., at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO: 126 and a light chain variable region having an amino acid sequence at least 80% identical (e.g., at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO: 234, 324, or 360;
  • a heavy chain variable region having an amino acid sequence at least 80% identical e.g., at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical
  • SEQ ID NO: 135 a heavy chain variable region having an amino acid sequence at least 80% identical (e.g., at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO: 135 and a light chain variable region having an amino acid sequence at least 80% identical (e.g., at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO: 216 or 252;
  • a heavy chain variable region having an amino acid sequence at least 80% identical e.g., at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical
  • SEQ ID NO: 144 a heavy chain variable region having an amino acid sequence at least 80% identical (e.g., at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO: 144 and a light chain variable region having an amino acid sequence at least 80% identical (e.g., at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO: 387;
  • xiii a heavy chain variable region having an amino acid sequence at least 80% identical (e.g., at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO: 153 and a light chain variable region having an amino acid sequence at least 80% identical (e.g., at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO: 360 or 378;
  • a heavy chain variable region having an amino acid sequence at least 80% identical e.g., at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical
  • SEQ ID NO: 162 a heavy chain variable region having an amino acid sequence at least 80% identical (e.g., at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO: 162 and a light chain variable region having an amino acid sequence at least 80% identical (e.g., at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO: 396;
  • xv a heavy chain variable region having an amino acid sequence at least 80% identical (e.g., at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO: 171 and a light chain variable region having an amino acid sequence at least 80% identical (e.g., at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO: 387;
  • a heavy chain variable region having an amino acid sequence at least 80% identical e.g., at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical
  • SEQ ID NO: 189 a heavy chain variable region having an amino acid sequence at least 80% identical (e.g., at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO: 189 and a light chain variable region having an amino acid sequence at least 80% identical (e.g., at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO: 342, 360, 369, or 378;
  • (xix) a heavy chain variable region having an amino acid sequence at least 80% identical (e.g., at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO: 207 and a light chain variable region having an amino acid sequence at least 80% identical (e.g., at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO: 315.
  • the disclosure features anti-hepatitis B surface antigen binding agents (e.g., antibodies) that bind to a polypeptide consisting of SEQ ID NO: 41, 42 or 43 with a dissociation constant of 2xl0 "8 M or less (e.g., lxlO "8 M, 5xl0 ⁇ 9 M or less, 2xl0 ⁇ 9 M or less, lxl 0 "9 M or less, or 5xlO "10 M or less).
  • anti-hepatitis B surface antigen binding agents e.g., antibodies
  • a polypeptide consisting of SEQ ID NO: 41, 42 or 43 with a dissociation constant of 2xl0 "8 M or less (e.g., lxlO "8 M, 5xl0 ⁇ 9 M or less, 2xl0 ⁇ 9 M or less, lxl 0 "9 M or less, or 5xlO "10 M or less).
  • the disclosure features anti-hepatitis B surface antigen binding agents (e.g., antibodies) that bind to a polypeptide consisting of SEQ ID NO: 41, 42 or 43 with an association rate of lxl 0 4 M ' V 1 or greater (e.g., 2xl0 4 M ' V 1 or greater, 5xl0 4 M ' V 1 or greater, lxlO 5 M ' V 1 or greater, 2xl0 5 M ' V 1 or greater, or 5xl0 5 M ' V 1 or greater) and/or a dissociation rate of 2xl0 "3 s "1 or less (e.g., lxlO "3 s "1 or less, 5xl0 "4 s "1 or less, 2xl0 "4 s "1 or less, lxl 0 “4 s "1 or less, 5x10 ⁇ 5 s "1 or less, or 2x10 ⁇ 5 s "1 or less).
  • the disclosure features anti-hepatitis B surface antigen binding agents (e.g., antibodies) that bind to the same epitope as an antibody selected from the group of:
  • anti-hepatitis B surface antigen binding agents e.g., antibodies
  • an antibody having a heavy chain amino acid sequence consisting of SEQ ID NO: 9, 411, 417, or 420 and a light chain amino acid sequence consisting of SEQ ID NO: 20, 21, 23, 25, 28, or 30;
  • the disclosure features anti-hepatitis B surface antigen binding agents (e.g., antibodies) that compete for binding to a polypeptide having an amino acid sequence consisting of SEQ ID NO: 41 with an antibody selected from the group consisting of:
  • anti-hepatitis B surface antigen binding agents e.g., antibodies
  • an antibody having a heavy chain amino acid sequence consisting of SEQ ID NO: 3, 404, 408, or 411 and a light chain amino acid sequence consisting of SEQ ID NO: 21, 23, 27, 28, 29, or 30;
  • the binding agent e.g., antibody
  • the binding agent is purified.
  • the binding agent e.g., antibody
  • the binding agent is human.
  • the disclosure features polynucleotides that encode a polypeptide chain (e.g., an antibody heavy or light chain) of any of the above binding agents.
  • the polynucleotide may include a sequence at least 80% identical (e.g., at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO: 44, 53, 62, 71, 80, 89, 98, 107, 116, 125, 134, 143, 152, 161, 170, 179, 188, 197, 206, 215, 224, 233, 242, 251, 260, 269, 278, 287, 296, 305, 314, 323, 332, 341, 350, 359, 368, 377, 386, or 395.
  • the disclosure also features vectors that include the above polynucleotides and isolated cells that include the above polynucleotides and/or vectors.
  • the disclosure features methods of producing binding agents (e.g., antibodies) that include culturing the isolated cells under conditions where the binding agent is expressed and collecting the binding agent.
  • the disclosure features methods of detecting a hepatitis B virus or hepatitis B surface antigen in a sample that include contacting a sample with a binding agent (e.g., antibody) disclosed herein; and detecting binding of the binding agent to the sample, thereby detecting a hepatitis B virus or hepatitis B surface antigen in the sample.
  • a binding agent e.g., antibody
  • the disclosure features methods of inhibiting hepatitis B virus infection of a cell (e.g., a hepatocyte) that include contacting the cell with a binding agent (e.g., antibody) disclosed herein.
  • a binding agent e.g., antibody
  • the disclosure also features the binding agents (e.g., antibodies) disclosed herein and the use thereof for treatment, prophylaxis, or diagnosis of a hepatitis B infection.
  • binding agents e.g., antibodies
  • the disclosure features therapeutic, prophylactic, and/or diagnostic compositions for hepatitis B infection that include a binding agent (e.g., antibody) or polynucleotide disclosed herein.
  • the compositions can further include one or more antiviral compounds, e.g., lamivudine, adefovir, enofovir, elbivudine, or entecavir, and/or one or more immune modulators, e.g., an interferon (e.g., interferon alpha-2a or PEGylated interferon alpha-2a).
  • an interferon e.g., interferon alpha-2a or PEGylated interferon alpha-2a
  • the disclosure features methods for treatment of a hepatitis B infection (e.g., an acute or chronic hepatitis B infection) that include administering a binding agent (e.g., antibody) disclosed herein to a subject with a hepatitis B infection in a therapeutically effective amount.
  • a binding agent e.g., antibody
  • the methods further include administering to the subject one or more antiviral compounds, e.g., lamivudine, adefovir, enofovir, elbivudine, or entecavir, and/or one or more immune modulators, e.g., an interferon (e.g., interferon alpha-2a or PEGylated interferon alpha-2a).
  • one or more antiviral compounds e.g., lamivudine, adefovir, enofovir, elbivudine, or entecavir
  • an immune modulators e.g., an interferon (e.g., interferon alpha-2a or PEGylated interferon alpha-2a).
  • the disclosure features methods for hepatitis B prophylaxis that include administering a binding agent (e.g., antibody) disclosed herein to a subject in a prophylactically effective amount, wherein the administering results in inhibition or prevention of hepatitis B infection.
  • a binding agent e.g., antibody
  • the subject is a pregnant woman or a newborn.
  • the methods can further include administering to the subject one or more antiviral compounds, e.g., lamivudine, adefovir, enofovir, elbivudine, or entecavir, and/or one or more immune modulators, e.g., an interferon (e.g., interferon alpha-2a or PEGylated interferon alpha-2a).
  • antiviral compounds e.g., lamivudine, adefovir, enofovir, elbivudine, or entecavir
  • immune modulators e.g., an interferon (e.g., interferon alpha-2a or PEGylated interferon alpha-2a).
  • the methods include administering to the subject a hepatitis B vaccine.
  • the disclosure features methods of diagnosing hepatitis B infection that include contacting a sample from an individual with a binding agent (e.g., antibody) disclosed herein and detecting binding of the binding agent to hepatitis B virus, wherein detecting binding of the binding agent to hepatitis B virus in the sample is indicative of hepatitis B infection in the individual from whom the sample was obtained.
  • a binding agent e.g., antibody
  • the binding agent is linked to a detectable label.
  • compositions disclosed herein can provide monoclonal antibodies against hepatitis B virus (e.g., hepatitis B surface antigen (HBsAg)), which can be produced without the use of blood products. This can provide advantages in ease of production without the possibility of transmitting infectious agents (e.g., viruses or prions). Additionally, the use of monoclonal antibodies can allow for administration of less total active immunoglobulin in a smaller total volume than the use of intravenous immune globulin products, providing for fewer transfusion-related side effects.
  • hepatitis B virus e.g., hepatitis B surface antigen (HBsAg)
  • HBsAg hepatitis B surface antigen
  • Standard reference works setting forth the general principles of recombinant DNA technology include Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, New York (1989); Kaufman et al, Eds., Handbook of Molecular and Cellular Methods in Biology in Medicine, CRC Press, Boca Raton (1995); McPherson, Ed., Directed Mutagenesis: A Practical Approach, IRL Press, Oxford (1991). Standard reference works setting forth the general principles of pharmacology include Goodman and Gilman's The Pharmacological Basis of Therapeutics, 11th Ed., McGraw Hill Companies Inc., New York (2006).
  • anti-hepatitis B e.g., anti-HBsAg binding agents and compositions and methods utilizing the same.
  • binding agent is meant a molecule including, without limitation, an organic molecule such as a polypeptide (e.g., an antibody, as defined herein) or a polynucleotide, or an inorganic molecule such as a small chemical molecule or a synthetic polymer, that is capable of binding to a reference target molecule.
  • the binding agent specifically binds to the reference target molecule, where the phrase
  • binding agent can specifically bind to an epitope located anywhere on the target molecule.
  • a binding agent that binds to a fragment of a target molecule necessarily binds the larger target molecule (e.g., a binding agent that specifically binds SEQ ID NO: 42 or 43 within HBsAg also specifically binds to the entire (i.e., full length) HBsAg protein).
  • binding agent e.g., an antibody
  • target molecule e.g., HBsAg
  • binding fragment thereof means a fragment or portion of a binding reagent (e.g., an antigen binding domain of an antibody) that specifically binds the target molecule.
  • a binding agent that specifically binds to the target molecule may be referred to as a target-specific binding agent.
  • an antibody that specifically binds to a HBsAg molecule may be referred to as a HBsAg-specific antibody or an anti-HBsAg antibody.
  • purified refers to a molecule such as a nucleic acid sequence (e.g., a polynucleotide) or an amino acid sequence (e.g., a polypeptide) that is removed or separated from other components present in its natural environment.
  • an isolated antibody is one that is separated from other components of a eukaryotic cell (e.g., the endoplasmic reticulum or cytoplasmic proteins and RNA).
  • An isolated antibody-encoding polynucleotide is one that is separated from other nuclear components (e.g., histones) and/or from upstream or downstream nucleic acid sequences (e.g., an isolated antibody-encoding polynucleotide may be separated from the endogenous heavy chain or light chain promoter).
  • An isolated nucleic acid sequence or amino acid sequence may be at least 60% free, or at least 75% free, or at least 90% free, or at least 95% free from other components present in natural environment of the indicated nucleic acid sequence or amino acid sequence.
  • Naturally occurring antibodies are made up of two classes of polypeptide chains, light chains and heavy chains.
  • a non-limiting antibody of the disclosure can be an intact, four immunoglobulin chain antibody comprising two heavy chains and two light chains.
  • the heavy chain of the antibody can be of any isotype including IgM, IgG, IgE, IgA or IgD or sub-isotype including IgGl , IgG2, IgG2a, IgG2b, IgG3, IgG4, IgEl , IgE2, etc.
  • the light chain can be a kappa light chain or a lambda light chain.
  • a single naturally occurring antibody comprises two identical copies of a light chain and two identical copies of a heavy chain.
  • the heavy chains which each contain one variable domain (VH) and multiple constant domains, bind to one another via disulfide bonding within their constant domains to form the "stem" of the antibody.
  • the light chains which each contain one variable domain (VL) and one constant domain, each bind to one heavy chain via disulfide binding.
  • the variable domain of each light chain is aligned with the variable domain of the heavy chain to which it is bound.
  • the variable regions of both the light chains and heavy chains contain three hypervariable regions sandwiched between four more conserved framework regions (FR). These hypervariable regions, known as the
  • CDRs complementary determining regions
  • the four framework regions largely adopt a beta-sheet conformation and the CDRs form loops connecting, and in some cases forming part of, the beta-sheet structure.
  • the CDRs in each chain are held in close proximity by the framework regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding domain.
  • antibody as used herein is meant to include intact immunoglobulin molecules (e.g., IgGl , IgG2a, IgG2b, IgG3, IgM, IgD, IgE, IgA) for any species (e.g., human, rodent, camelid), as well as antigen binding domain fragments thereof, such as Fab, Fab', F(ab') 2 ; variants thereof such as scFv, Fv, Fd, dAb, bispecific scFvs, diabodies, linear antibodies (see U.S. Pat. No.
  • an antibody binding domain is meant any portion of an antibody that retains specific binding activity of the intact antibody (i.e., any portion of an antibody that is capable of specific binding to an epitope on the intact antibody's target molecule).
  • An "epitope” is smallest portion of a target molecule capable being specifically bound by the antigen binding domain of an antibody. The minimal size of an epitope may be about five or six to seven amino acids.
  • Non- limiting antigen binding domains include the heavy chain and/or light chain CDRs of an intact antibody, the heavy and/or light chain variable regions of an intact antibody, full length heavy or light chains of an intact antibody, or an individual CDR from either the heavy chain or the light chain of an intact antibody.
  • Antibodies disclosed herein include but are not limited to polyclonal, monoclonal, monospecific, polyspecific antibodies and fragments thereof and chimeric antibodies that include an immunoglobulin binding domain fused to another polypeptide.
  • an antibody that specifically binds to a target molecule provide a detection signal at least 5-, 10-, or 20-fold higher than a detection signal provided with other proteins when used in an immunochemical assay. In some embodiments, antibodies that specifically bind to a target molecule do not detect other proteins in immunochemical assays and can immunoprecipitate the target molecule from solution.
  • an immunoglobulin chain (e.g., a heavy chain or a light chain) may include in order from amino terminus to carboxy terminus a variable region and a constant region.
  • the variable region may include three complementarity determining regions (CDRs), with interspersed framework (FR) regions for a structure FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.
  • CDRs complementarity determining regions
  • FR interspersed framework
  • antibodies that include heavy or light chain variable regions, framework regions and CDRs.
  • the antibody may comprise a heavy chain constant region that comprises some or all of a CHI region, hinge, CH2 and/or CH3 region.
  • the antibody may comprise a light chain constant region that comprises some or all of a CL region.
  • Antibodies disclosed herein can be derived from any species of animal, including mammals.
  • Non-limiting exemplary natural antibodies include antibodies derived from human, camelids (e.g., camels and llamas), chicken, goats, and rodents (e.g., rats, mice, hamsters and rabbits), including transgenic rodents genetically engineered to produce human antibodies (see, e.g., Lonberg et al, WO 93/12227; U.S. Pat. No. 5,545,806; and
  • Natural antibodies are the antibodies produced by a host animal. "Genetically altered antibodies” refer to antibodies wherein the amino acid sequence has been varied from that of a native antibody. Because of the relevance of recombinant DNA techniques to this application, one need not be confined to the sequences of amino acids found in natural antibodies; antibodies can be redesigned to obtain desired
  • variable or constant region Changes in the constant region will, in general, be made in order to improve or alter characteristics, such as complement fixation, interaction with membranes and other effector functions. Changes in the variable region will be made in order to improve the antigen binding characteristics.
  • oligoclonal antibodies refers to a predetermined mixture of distinct monoclonal antibodies. See, e.g., PCT publication WO 95/20401; U.S. Patent Nos.
  • oligoclonal antibodies consisting of a predetermined mixture of antibodies against one or more epitopes are generated in a single cell.
  • oligoclonal antibodies comprise a plurality of heavy chains capable of pairing with a common light chain to generate antibodies with multiple specificities (e.g., PCT publication WO 04/009618). Oligoclonal antibodies are particularly useful when it is desired to target multiple epitopes on a single target molecule.
  • those skilled in the art can generate or select antibodies or mixtures of antibodies that are applicable for an intended purpose and desired need.
  • Recombinant antibodies are also included in the present application. These recombinant antibodies are engineered to have the same amino acid sequence as the natural antibodies or to have altered amino acid sequences of the natural antibodies in the present application. They can be made in any expression systems including both prokaryotic and eukaryotic expression systems or using phage display methods (see, e.g., PCT Publication No. W091/17271, PCT Publication No. WO92/01047; U.S. Pat. No. 5,969,108; U.S. Pat. No. 6,331,415; US 7,498,024, and U.S. Pat. No. 7,485,291, which are herein incorporated by reference in their entirety).
  • Antibodies can be engineered in numerous ways. They can be made as single-chain antibodies (including small modular immunopharmaceuticals or SMIPs), Fab and F(ab') 2 fragments, etc. Antibodies can be humanized, chimerized, deimmunized, or fully human. Numerous publications set forth the many types of antibodies and the methods of engineering such antibodies. For example, see U.S. Patent Publication No. 20060099204; U.S. Patent Nos. 6,355,245; 6,180,370; 5,693,762; 6,407,213; 6,548,640; 5,565,332; 5,225,539;
  • modified antibodies provide improved stability or/and therapeutic efficacy.
  • modified antibodies include those with conservative substitutions of amino acid residues, and one or more deletions or additions of amino acids that do not significantly deleteriously alter the antigen binding utility. Substitutions can range from changing or modifying one or more amino acid residues to complete redesign of a region as long as the therapeutic utility is maintained.
  • Antibodies of this application can be modified post-translationally (e.g., acetylation, and/or
  • phosphorylation or can be modified synthetically (e.g., the attachment of a labeling group).
  • Antibodies with engineered or variant constant or Fc regions can be useful in modulating effector functions, such as, for example, antigen-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC).
  • ADCC antigen-dependent cytotoxicity
  • CDC complement-dependent cytotoxicity
  • genetically altered antibodies are chimeric antibodies and humanized antibodies.
  • the chimeric antibody is an antibody having portions derived from different antibodies.
  • a chimeric antibody may have a variable region and a constant region derived from two different antibodies.
  • the donor antibodies may be from different species.
  • the genetically altered antibodies disclosed herein include CDR grafted humanized antibodies.
  • the humanized antibody comprises heavy and/or light chain CDRs of a non-human donor immunoglobulin and heavy chain and light chain frameworks and constant regions of a human acceptor immunoglobulin.
  • Non-limiting methods for making humanized antibody are disclosed in U.S. Pat. Nos: 5,530,101; 5,585,089; 5,693,761; 5,693,762; and 6,180,370 each of which is incorporated herein by reference in its entirety.
  • an antibody disclosed herein will comprise substantially all of at least one, and typically two, variable domains (such as Fab, Fab', F(ab')2, Fabc, Fv) in which one or more of the CDR regions are synthetic amino acid sequences that specifically bind to the target molecule, and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence.
  • the framework regions can also be those of a native human immunoglobulin sequence.
  • Other CDR regions in the antibody can be selected to have human immunoglobulin consensus sequences for such CDRs or the sequence of a native human antibody.
  • the antibody can also comprise at least a portion of an immunoglobulin constant region (Fc) of a human immunoglobulin.
  • an antibody will contain both the light chain as well as at least the variable domain of a heavy chain.
  • the antibody also may include the CHI, hinge, CH2, CH3, and CH4 regions of the heavy chain.
  • the antibody fragments are truncated chains (truncated at the carboxyl end). In certain embodiments, these truncated chains possess one or more immunoglobulin activities (e.g., complement fixation activity). Examples of truncated chains include, but are not limited to, Fab fragments (consisting of the VL, VH, CL and CHI domains); Fd fragments (consisting of the VH and CHI domains); Fv fragments (consisting of VL and VH domains of a single chain of an antibody); dAb fragments
  • the truncated chains can be produced by conventional biochemical techniques, such as enzyme cleavage, or recombinant DNA techniques, each of which is known in the art.
  • These polypeptide fragments may be produced by proteolytic cleavage of intact antibodies by methods well known in the art, or by inserting stop codons at the desired locations in the vectors using site-directed mutagenesis, such as after CHI to produce Fab fragments or after the hinge region to produce (Fab') 2 fragments.
  • Single chain antibodies may be produced by joining VL- and VH-coding regions with a DNA that encodes a peptide linker connecting the VL and VH protein fragments
  • Fv usually refers to the minimum antibody fragment that contains a complete antigen-recognition and -binding site. This region consists of a dimer of one heavy- and one light-chain variable domain (i.e., a VL domain and a VH domain) in tight, non-covalent association. 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 V H -V L dimer. Collectively, the CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising three CDRs specific for an antigen) has the ability to recognize and bind antigen, although likely at a lower affinity than the entire binding site.
  • Single- chain Fv or “scFv” antibody fragments comprise the V H and V L domains of an antibody, wherein these domains are present in a single polypeptide chain.
  • the Fv polypeptide further comprises a polypeptide linker between the V H and V L domains that enables the scFv to form the desired structure for antigen binding.
  • Papain digestion of an intact antibody 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.
  • the Fab fragment contains the entire light chain (i.e., the constant domain (CL) and variable domain (VL) of the light chain) together with the first constant domain (CHI) and variable region (VH) 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 CHI 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 that have hinge cysteines between them. For example, pepsin treatment of an antibody yields an F(ab') 2 fragment that has two antigen-combining sites and is still capable of cross-linking antigen. In other words, an F(ab') 2 fragment comprises two disulfide linked Fab fragments. Other chemical couplings of antibody fragments are also known.
  • SMIPs are a class of single-chain peptides engineered to include a target binding region and effector domain (CH2 and CH3 domains).
  • the target-binding region may be derived from the variable region or CDRs of an antibody, e.g., an HBsAg-specific antibody disclosed herein.
  • the target-binding region is derived from a protein that binds the indicated target (e.g., a non-immunoglobulin molecule that binds to HBsAg).
  • Bispecific antibodies may be monoclonal, human or humanized antibodies that have binding specificities for at least two different antigens.
  • one of the binding specificities is for the indicated target (e.g., HBsAg)
  • the other one is for any other antigen, such as for example, a cell-surface protein or receptor or receptor subunit.
  • a therapeutic agent may be placed on one arm.
  • the therapeutic agent can be a drug, toxin, enzyme, DNA, radionuclide, etc.
  • the antigen-binding fragment can be a diabody.
  • the term "diabody” refers to a small antibody fragment with two antigen-binding sites, which fragment comprises a heavy-chain variable domain (V H ) connected to a light-chain variable domain (V L ) in the same polypeptide chain (V H -V L ).
  • Diabodies can be prepared by constructing scFv fragments with short linkers (about 5-10 residues) between the VH and VL domains such that inter-chain but not intra-chain pairing of the V domains is achieved, resulting in a multivalent fragment, i.e., a fragment having two antigen-binding sites.
  • Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et al, Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993).
  • Came lid antibodies refer to a unique type of antibodies that are devoid of light chain, initially discovered from animals of the camelid family.
  • the heavy chains of these so-called heavy-chain antibodies bind their antigen by one single domain, the variable domain of the heavy immunoglobulin chain, referred to as VHH.
  • VHHs show homology with the variable domain of heavy chains of the human VHIII family.
  • the VHHs obtained from an immunized camel, dromedary, or llama have a number of advantages, such as effective production in microorganisms such as Saccharomyces cerevisiae.
  • single chain antibodies, and chimeric, humanized or primatized (CDR-grafted) antibodies, as well as chimeric or CDR-grafted single chain antibodies, comprising portions derived from different species, are also encompassed by the present disclosure as antigen-binding fragments of an antibody.
  • the various portions of these antibodies can be joined together chemically by conventional techniques, or can be prepared as a contiguous protein using genetic engineering techniques.
  • nucleic acids encoding a chimeric or humanized chain can be expressed to produce a contiguous protein. See, e.g., U.S. Pat. Nos. 4,816,567 and 6,331,415; U.S. Pat. No. 4,816,397;
  • functional fragments of antibodies including fragments of chimeric, humanized, primatized or single chain antibodies, can also be produced.
  • Functional fragments of the subject antibodies retain at least one antigen binding domain function and/or modulation function of the full-length (i.e., intact) antibody from which they are derived. Since the immunoglobulin-related genes contain separate functional regions, each having one or more distinct biological activities, the genes of the antibody fragments may be fused to functional regions from other genes (e.g., enzymes, U.S. Pat. No. 5,004,692, which is incorporated by reference in its entirety) to produce fusion proteins or conjugates having novel properties.
  • Human antibodies may be made by means known in the art, e.g., by phage display using human antibody library sequences or by use of mice genetically engineered to produce antibodies from human gene sequences.
  • human antibodies may be derived from antibodies or cells in circulation, e.g., using the methods described in WO 2010/011337 and/or U.S. application serial no.
  • Non-immunoglobulin binding polypeptides are also contemplated.
  • CDRs from an antibody disclosed herein may be inserted into a suitable non-immunoglobulin scaffold to create a non-immunoglobulin binding polypeptide.
  • Suitable candidate scaffold structures may be derived from, for example, members of fibronectin type III and cadherin superfamilies.
  • non-antibody molecules such as protein binding domains or aptamers, which specifically bind to a target molecule described herein (e.g., HBsAg). See, e.g., Neuberger et al, Nature 312: 604 (1984). Aptamers are also contemplated.
  • DNA or RNA aptamers are typically short oligonucleotides, engineered through repeated rounds of selection to bind to a molecular target.
  • Peptide aptamers typically consist of a variable peptide loop attached at both ends to a protein scaffold. This double structural constraint generally increases the binding affinity of the peptide aptamer to levels comparable to an antibody (nanomolar range).
  • the binding agent is an antibody that specifically binds to an HBsAg molecule.
  • the binding agent is an antibody having one or more polypeptide sequences selected from any one of SEQ ID NOs: 1-40, 45-52, 54-61, 63- 70, 72-79, 81-88, 90-97, 99-106, 108-115, 117-124, 126-133, 135-142, 144-151, 153-160, 162-169, 171-178, 180-187, 189-196, 198-205, 207-214, 216-223, 225-232, 234-241, 243- 250, 252-259, 261-268, 270-277, 279-286, 288-295, 297-304, 306-313, 315-322, 324-331, 333-340, 342-349, 351-358, 360-367, 369-376, 378-385, 387-394, 396-403, and 404-422.
  • the binding agent includes at least one complementary determining region (CDR), wherein the CDR includes a sequence selected from any one of SEQ ID NOs: 47, 49, 51, 56, 58, 60, 65, 67, 69, 74, 76, 78, 83, 85, 87, 92, 94, 96, 101, 103, 105, 110, 112, 114, 119, 121, 123, 128, 130, 132, 137, 139, 141, 146, 148, 150, 155, 157, 159, 164, 166, 168, 173, 175, 177, 182, 184, 186, 191, 193, 195, 200, 202, 204, 209, 211, 213, 218, 220, 222, 227, 229, 231, 236, 238, 240, 245, 247, 249, 254, 256, 258, 263, 265, 267, 272, 274, 276, 281, 283, 285, 290, 292, 294,
  • CDR
  • the binding agent includes at least one variable region that includes a sequence selected from any one of SEQ ID NOs: 45, 54, 63, 72, 81, 90, 99, 108, 117, 126, 135, 144, 153, 162, 171, 180, 189, 198, 207, 216, 225, 234, 243, 252, 261, 270, 279, 288, 297, 306, 315, 324, 333, 342, 351, 360, 369, 378, 387, 396, 405, 409, 412, 418, and 421.
  • the binding agent specifically binds to an epitope within SEQ ID NO: 41, 42, or 43.
  • binding agents of the present disclosure include the antibodies having the amino acid sequences set forth herein (whether or not including a leader sequence), and binding agents that may include at least six contiguous amino acids encompassing the amino acid sequence of one or more CDR domains (either from the heavy chain or the light chain, or both) disclosed herein, as well as polypeptides that are at least 80% identical, or at least 85% identical, or at least 90%, 95%, 96%, 97%, 98% or 99% identical to those described above (e.g., at least 80%> identical, at least 85% identical, at least 90% identical, or at least 95% identical, or at least 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 1-40, 45, 54, 63, 72, 81, 90, 99, 108, 117, 126, 135, 144, 153, 162, 171, 180, 189, 198, 207, 216, 225, 234, 243, 252, 261, 270, 279, 288, 297
  • % identical for two polypeptides or two polynucleotides is intended a similarity score produced by comparing the amino acid sequences of the two polypeptides or by comparing the nucleotides sequences of the two polynucleotides using the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711) and the default settings for determining similarity. Bestfit uses the local homology algorithm of Smith and Waterman (Advances in Applied Mathematics 2: 482-489 (1981)) to find the best segment of similarity between two sequences.
  • polymers of amino acids of any length may be linear or branched, and it may comprise modified amino acids. Where the amino acid sequence is provided, unless otherwise specified, the sequence is in an N-terminal to C- terminal orientation. In some embodiments, the polymer may be interrupted by non-amino acids.
  • the terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component.
  • polypeptides containing one or more analogs of an amino acid including, for example, unnatural amino acids, etc.
  • polypeptides containing one or more analogs of an amino acid including, for example, unnatural amino acids, etc.
  • the polypeptides disclosed herein are based upon antibodies, the polypeptides can occur as single chains or associated chains.
  • nucleic acid molecule refers to polymers of nucleotides of any length, and include, without limitation, DNA, R A, DNA/R A hybrids, and modifications thereof. Unless otherwise specified, where the nucleotide sequence is provided, the nucleotides are set forth in a 5 ' to 3 ' orientation. Thus, the nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase.
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modification to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.
  • modifications include, for example, "caps", substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, cabamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as
  • any of the hydroxyl groups ordinarily present in the sugars may be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or may be conjugated to solid supports.
  • the 5' and 3' terminal OH can be phosphorylated or substituted with amines or organic capping group moieties of from 1 to 20 carbon atoms.
  • Other hydroxyls may also be derivatized to standard protecting groups.
  • Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars that are generally known in the art, including, for example, 2'-0-methyl-, 2'-0-allyl, 2'-fluoro- or 2'- azido-ribose, carbocyclic sugar analogs, alpha-anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs and abasic nucleoside analogs such as methyl riboside.
  • One or more phosphodiester linkages may be replaced by alternative linking groups.
  • linking groups include, but are not limited to, embodiments wherein phosphate is replaced by P(0)S ("thioate”), P(S)S ("dithioate”), "(0)NR2 ("amidate”), P(0)R, P(0)OR * , CO or CH2
  • each R or R is independently H or substituted or unsubstituted alkyl (1-20 C) optionally containing an ether ( ⁇ 0 ⁇ ) linkage, aryl, alkenyl, cycloalkyl,
  • the present application also provides the polynucleotide molecules encoding analogs of the binding agents (e.g., antibodies) described herein. Because of the degeneracy of the genetic code, a number of different nucleic acid sequences may encode each antibody amino acid sequence.
  • the desired nucleic acid sequences can be produced by de novo solid-phase DNA synthesis or by PCR mutagenesis of an earlier prepared variant of the desired polynucleotide.
  • the codons that are used comprise those that are typical for human, rabbit, or mouse (see, e.g., Nakamura, Y., Nucleic Acids Res. 28: 292 (2000)).
  • the present disclosure provides, in part, isolated polynucleotides that encode a binding agent disclosed herein, nucleotide probes that hybridize to such
  • nucleotide sequences and polypeptide sequences disclosed herein may have been determined using an automated peptide sequencer. As is known in the art for any DNA sequence determined by this automated approach, any nucleotide sequence determined herein may contain some errors. Nucleotide sequences determined by automation are typically at least about 90% identical, and more typically at least about 95% to about 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule. The actual sequence can be more precisely determined by other approaches including manual DNA sequencing methods well known in the art.
  • each nucleotide sequence set forth herein is presented as a sequence of deoxyribonucleotides (abbreviated A, G, C and T).
  • nucleotide sequence of a nucleic acid molecule or polynucleotide is intended, for a DNA molecule or polynucleotide, a sequence of deoxyribonucleotides, and for an RNA molecule or polynucleotide, the corresponding sequence of ribonucleotides (A, G, C and U), where each thymidine deoxyribonucleotide (T) in the specified deoxyribonucleotide sequence is replaced by the ribonucleotide uridine (U).
  • RNA molecule having a sequence disclosed herein is intended to indicate an RNA molecule having a sequence in which each deoxyribonucleotide A, G or C of the sequence has been replaced by the corresponding ribonucleotide A, G or C, and each deoxyribonucleotide T has been replaced by a ribonucleotide U.
  • the disclosure provides isolated polynucleotides (and isolated polynucleotides complementary thereto) that include a nucleotide sequence at least about 80% identical (e.g., at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical) to the sequence of any one of SEQ ID NOs: 44, 53, 62, 71, 80, 89, 98, 107, 116, 125, 134, 143, 152, 161, 170, 179, 188, 197, 206, 215, 224, 233, 242, 251, 260, 269, 278, 287, 296, 305, 314, 323, 332, 341, 350, 359, 368, 377, 386, and 395.
  • the disclosure provides an isolated polynucleotide (or an isolated polynucleotide complementary thereto) that includes a nucleotide sequence at least about 80%> identical (e.g., at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical) identical to nucleotide sequence encoding an antibody (or fragment thereof) comprising an amino acid sequence disclosed herein.
  • a nucleic acid molecule encoding a polypeptide binding agent e.g., an antibody
  • a polypeptide binding agent e.g., an antibody
  • the present disclosure provides, in part, full-length antibodies.
  • proteins secreted by mammalian cells have a signal or secretory leader sequence which is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated.
  • Most mammalian cells and even insect cells cleave secreted proteins with the same specificity.
  • cleavage of a secreted protein is not entirely uniform, which results in two or more mature species on the protein.
  • the cleavage specificity of a secreted protein is ultimately determined by the primary structure of the complete protein, that is, it is inherent in the amino acid sequence of the polypeptide.
  • nucleotide sequences encoding a heavy or light chain that includes any one of SEQ ID NOs: 3-20, with additional nucleic acid residues located 5' to the 5 '-terminal residues of the coding sequence.
  • the disclosure provides nucleotide sequences encoding CDRs, with additional nucleic acid residues located 5' to the 5 '-terminal residues of a polynucleotide that encodes a variable region disclosed herein (e.g., a variable region including the sequence set forth in any one of SEQ ID NOs: 45, 54, 63, 72, 81, 90, 99, 108, 117, 126, 135, 144, 153, 162, 171, 180, 189, 198, 207, 216, 225, 234, 243, 252, 261, 270, 279, 288, 297, 306, 315, 324, 333, 342, 351, 360, 369, 378,
  • a variable region including the sequence set forth in any one of SEQ ID NOs: 45, 54, 63, 72, 81, 90, 99, 108, 117, 126, 135, 144, 153, 162, 171, 180, 189, 198, 207, 216, 225
  • 387, 396, 405, 409, 412, 418, and 421) and/or CDR disclosed herein e.g., a CDR comprising the amino acid sequence set forth in any one of SEQ ID NOs: 47, 49, 51, 56, 58, 60, 65, 67, 69, 74, 76, 78, 83, 85, 87, 92, 94, 96, 101, 103, 105, 110, 112, 114, 119, 121, 123, 128, 130, 132, 137, 139, 141, 146, 148, 150, 155, 157, 159, 164, 166, 168, 173, 175, 177, 182, 184, 186, 191, 193, 195, 200, 202, 204, 209, 211, 213, 218, 220, 222, 227, 229, 231, 236, 238, 240, 245, 247, 249, 254, 256, 258, 263, 265, 267, 272,
  • polynucleotide comprises the nucleotide sequence set forth in SEQ ID NOs: 44, 53, 62, 71, 80, 89, 98, 107, 116, 125, 134, 143, 152, 161, 170, 179, 188, 197, 206, 215, 224, 233, 242, 251, 260, 269, 278, 287, 296, 305, 314, 323, 332, 341, 350, 359, 368, 377, 386, or 395.
  • the antibody-encoding or binding agent-encoding polynucleotide comprises a nucleotide sequence that encodes a variable region having the amino acid sequence set forth in any one of SEQ ID NOs: 45, 54, 63, 72, 81, 90, 99, 108, 117, 126, 135, 144, 153, 162, 171, 180, 189, 198, 207, 216, 225, 234, 243, 252, 261, 270, 279, 288, 297, 306, 315, 324, 333, 342, 351, 360, 369, 378, 387, 396, 405, 409, 412, 418, and 421 and/or a CDR having the amino acid sequence set forth in any one of SEQ ID NOs: 47, 49, 51, 56, 58, 60, 65, 67, 69, 74, 76, 78, 83, 85, 87, 92, 94, 96, 101, 103, 105, 110, 11
  • the polynucleotide encodes a polypeptide having the amino acid sequence set forth in any one of SEQ ID NOs: 1-40.
  • polynucleotides of the present disclosure may be in the form of RNA, such as mRNA, or in the form of DNA, including, for instance, cDNA and genomic DNA obtained by cloning or produced synthetically.
  • the DNA may be double-stranded or single- stranded.
  • Single-stranded DNA or RNA may be the coding strand, also known as the sense strand, or it may be the non-coding strand, also referred to as the anti-sense strand.
  • Isolated polynucleotides of the disclosure may be nucleic acid molecules, DNA or RNA, which have been removed from their native environment.
  • recombinant DNA molecules contained in a vector are considered isolated for the purposes of the present disclosure.
  • Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution.
  • Isolated RNA molecules include in vivo or in vitro RNA transcripts of the DNA molecules of the present disclosure.
  • Isolated nucleic acid molecules according to the present disclosure further include such molecules produced synthetically.
  • Polynucleotides of the disclosure include the nucleic acid molecules having the sequences set forth in SEQ ID NOs: 44, 53, 62, 71, 80, 89, 98, 107, 116, 125, 134, 143, 152, 161, 170, 179, 188, 197, 206, 215, 224, 233, 242, 251, 260, 269, 278, 287, 296, 305, 314, 323, 332, 341, 350, 359, 368, 377, 386, or 395, nucleic acid molecules comprising the coding sequence for the antibodies and binding agents of the disclosure that include a sequence different from those described above but which, due to the degeneracy of the genetic code, still encode an antibody or binding agent disclosed herein.
  • the genetic code is well known in the art. Thus, it would be routine for one skilled in the art to generate such degenerate variants.
  • the disclosure further provides isolated polynucleotides comprising nucleotide sequences having a sequence complementary to one of the binding agent-encoding or antibody-encoding polynucleotides disclosed herein.
  • isolated molecules particularly DNA molecules, are useful as probes for gene mapping, by in situ hybridization with chromosomes, and for detecting expression of the antibody in tissue (e.g., human tissue), for instance, by northern blot analysis.
  • the binding agents (e.g., antibodies) of the disclosure are encoded by at least a portion of the nucleotide sequences set forth herein.
  • a "portion" or “fragment” means a sequence fragment comprising a number of contiguous amino acid residues (if a polypeptide fragment (which may also be referred to herein a peptide)) or a sequence fragment comprising a number of nucleotide residues (if a polynucleotide fragment) that is less than the number of such residues in the whole sequence ⁇ e.g., a 50 nucleotide sequence is a portion of a 100 nucleotide long sequence).
  • fragment of an indicated molecule that is smaller than the indicated molecule.
  • the binding agent-encoding polynucleotides and/or the antibody-encoding polynucleotides disclosed herein may comprise portions of intron sequences that do not encode any amino acids in the resulting binding agent or antibody.
  • a fragment of a polynucleotide may be at least about 15 nucleotides, or at least about 20 nucleotides, or at least about 30 nucleotides, or at least about 40 nucleotides in length, which are useful as diagnostic probes and primers as discussed herein.
  • fragments of about 50- 1500 nucleotides in length are also useful according to the present disclosure, as are fragments corresponding to most, if not all, of the antibody-encoding or binding agent- encoding nucleotide sequence of the cDNAs having sequences set forth in SEQ ID NOs: 44, 53, 62, 71 , 80, 89, 98, 107, 1 16, 125, 134, 143, 152, 161 , 170, 179, 188, 197, 206, 215, 224, 233, 242, 251 , 260, 269, 278, 287, 296, 305, 314, 323, 332, 341 , 350, 359, 368, 377, 386, or 395.
  • a fragment at least 20 nucleotides in length is meant fragments that include 20 or more contiguous nucleotides from the respective nucleotide sequences from which the fragments are derived.
  • Polynucleotide fragments are useful as nucleotide probes for use diagnostically according to conventional DNA hybridization techniques or for use as primers for
  • PCR polymerase chain reaction
  • a polynucleotide which hybridizes only to a poly A sequence or to a complementary stretch of T (or U) resides would not be included in a polynucleotide of the disclosure used to hybridize to a portion of a nucleic acid disclosed herein, since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any double-stranded cDNA clone).
  • DNA fragments are routine to the skilled artisan, and may be accomplished, by way of example, by restriction endonuclease cleavage or shearing by sonication of DNA obtainable from the cDNA clone described herein or synthesized according to the sequence disclosed herein. Alternatively, such fragments can be directly generated synthetically.
  • the disclosure provides an isolated polynucleotide (e.g., a nucleotide probe) that hybridizes under stringent conditions to a binding agent-encoding or a antibody-encoding polynucleotide disclosed herein (e.g., any one of SEQ ID NOs: 44, 53, 62, 71 , 80, 89, 98, 107, 1 16, 125, 134, 143, 152, 161 , 170, 179, 188, 197, 206, 215, 224, 233, 242, 251 , 260, 269, 278, 287, 296, 305, 314, 323, 332, 341 , 350, 359, 368, 377, 386, or 395).
  • a binding agent-encoding or a antibody-encoding polynucleotide disclosed herein e.g., any one of SEQ ID NOs: 44, 53, 62, 71 , 80, 89, 98, 107, 1 16,
  • stringent conditions with respect to nucleotide sequence or nucleotide probe hybridization conditions is the “stringency” that occurs within a range from about T m minus 5 °C (i.e., 5 °C below the melting temperature (T m ) of the probe or sequence) to about 20 °C to 25 °C below T m .
  • Typical stringent conditions are: overnight incubation at 42 °C in a solution comprising: 50% formamide, 5 X.SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5X Denhardt's solution, 10%> dextran sulfate, and 20 micrograms/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1X SSC at about 65 °C.
  • the stringency of hybridization may be altered in order to identify or detect identical or related polynucleotide sequences.
  • a polynucleotide or nucleotide probe that hybridizes to a reference polynucleotide is intended that the polynucleotide or nucleotide probe (e.g., DNA, RNA, or a DNA-RNA hybrid) hybridizes along the entire length of the reference polynucleotide or hybridizes to a portion of the reference polynucleotide that is at least about 15 nucleotides (nt), or to at least about 20 nt, or to at least about 30 nt, or to about 30-70 nt of the reference polynucleotide.
  • polynucleotide for instance, a portion 50-750 nt in length, or even to the entire length of the reference polynucleotide, are useful as probes according to the present disclosure, as are polynucleotides corresponding to most, if not all, of the nucleotide sequence of the cDNAs described herein or the nucleotide sequences set forth in SEQ ID NOs: 44, 53, 62, 71 , 80, 89, 98, 107, 1 16, 125, 134, 143, 152, 161 , 170, 179, 188, 197, 206, 215, 224, 233, 242, 251 , 260, 269, 278, 287, 296, 305, 314, 323, 332, 341 , 350, 359, 368, 377, 386, or 395.
  • nucleic acid molecules of the present disclosure which encode binding agents disclosed herein, may include but are not limited to those encoding the amino acid sequence of the mature intact polypeptide, by itself; fragments thereof; the coding sequence for the mature polypeptide and additional sequences, such as those encoding the leader or secretory sequence, such as a pre-, or pro- or pre-pro-protein sequence; the coding sequence of the mature polypeptide, with or without the aforementioned additional coding sequences, together with additional, non-coding sequences, including for example, but not limited to introns and non-coding 5' and 3' sequences, such as the transcribed, non-translated sequences that play a role in transcription, mR A processing, including splicing and polyadenylation signals, for example—ribosome binding and stability of mRNA; an additional coding sequence which codes for additional amino acids, such as those which provide additional functionalities.
  • the sequence encoding the polypeptide may be fused to a marker sequence, such as a sequence encoding a peptide that facilitates purification of the fused polypeptide.
  • the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (Qiagen, Inc.), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86: 821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein.
  • the "HA” tag is another peptide useful for purification which corresponds to an epitope derived from the influenza hemagglutinin protein, which has been described by Wilson et al., Cell 37: 767 (1984).
  • other such fusion proteins include the binding agents and/or antibodies of the disclosure fused to an Fc domain at the N- or C-terminus.
  • the present disclosure further relates to variants of the nucleic acid molecules disclosed herein, which encode portions, analogs or derivatives of a binding agent or antibody disclosed herein.
  • Variants may occur naturally, such as a natural allelic variant.
  • allelic variant is intended one of several alternate forms of a gene occupying a given locus on a chromosome of an organism. See, e.g. GENES II, Lewin, B., ed., John Wiley &
  • Non-naturally occurring variants may be produced using art-known mutagenesis techniques.
  • variants include those produced by nucleotide substitutions, deletions or additions.
  • the substitutions, deletions or additions may involve one or more nucleotides.
  • the variants may be altered in coding regions, non-coding regions, or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions. Some alterations included in the disclosure are silent substitutions, additions and deletions, which do not alter the properties and activities (e.g. specific binding activity) of the binding agent and/or antibody disclosed herein.
  • inventions of the disclosure include isolated polynucleotides comprising a nucleotide sequence at least 80% identical, e.g., at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical, to a binding agent-encoding or antibody-encoding polynucleotide of the disclosure.
  • nucleotide sequences set forth in SEQ ID NOs: 44, 53, 62, 71, 80, 89, 98, 107, 116, 125, 134, 143, 152, 161, 170, 179, 188, 197, 206, 215, 224, 233, 242, 251, 260, 269, 278, 287, 296, 305, 314, 323, 332, 341, 350, 359, 368, 377, 386, or 395 or to a nucleotide sequence that encodes a polypeptide disclosed herein can be determined conventionally using known computer programs such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711.
  • degenerate variants of these nucleotide sequences all encode the same polypeptide, this will be clear to the skilled artisan even without performing the above described comparison assay. It will be further recognized in the art that, for such nucleic acid molecules that are not degenerate variants, a reasonable number will also encode a polypeptide that retains the specific binding activity of the reference binding agent or antibody of the disclosure. This is because the skilled artisan is fully aware of amino acid substitutions that are either less likely or not likely to significantly effect protein function (e.g., replacing one aliphatic amino acid with a second aliphatic amino acid).
  • Methods for DNA sequencing may be used to practice any polynucleotide embodiments of the disclosure.
  • the methods may employ such enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE (US Biochemical Corp, Cleveland, Ohio), Taq polymerase (Invitrogen), thermostable T7 polymerase (Amersham, Chicago, 111.), or combinations of recombinant polymerases and proofreading exonucleases such as the ELONGASE Amplification System marketed by
  • Gibco BRL Gibco BRL (Gaithersburg, Md.). The process may be automated with machines such as the Hamilton Micro Lab 2200 (Hamilton, Reno, Nev.), Peltier Thermal Cycler (PTC200; MJ Research, Watertown, Mass.), ABI 377 DNA sequencers (Applied Biosystems), and 454 sequencers (Roche).
  • Polynucleotide sequences encoding a binding agent or antibody disclosed herein may be extended utilizing a partial nucleotide sequence and employing various methods known in the art to detect upstream sequences such as promoters and regulatory elements.
  • one method that may be employed "restriction-site" PCR, uses universal primers to retrieve unknown sequence adjacent to a known locus (Sarkar, G., PCR Methods Applic. 2: 318-322 (1993)).
  • genomic DNA is first amplified in the presence of primer to linker sequence and a primer specific to the known region.
  • Exemplary primers are those described in Example 4 herein.
  • amplified sequences are then subjected to a second round of PCR with the same linker primer and another specific primer internal to the first one.
  • Products of each round of PCR are transcribed with an appropriate RNA polymerase and sequenced using reverse transcriptase.
  • Inverse PCR may also be used to amplify or extend sequences using divergent primers based on a known region (Triglia et ah, Nucleic Acids Res. 16: 8186 (1988)).
  • the primers may be designed using OLIGO 4.06 Primer Analysis software (National Biosciences Inc., Madison, Minn.), or another appropriate program, to be 22-30 nucleotides in length, to have a GC content of 50% or more, and to anneal to the target sequence at temperatures about 68-72 °C.
  • the method uses several restriction enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template.
  • capture PCR which involves PCR
  • libraries that have been size-selected to include larger cDNAs may be used or random-primed libraries, which contain more sequences that contain the 5' regions of genes.
  • a randomly primed library is useful for situations in which an oligo d(T) library does not yield a full-length cDNA.
  • Genomic libraries may be useful for extension of sequence into the 5' and 3' non-transcribed regulatory regions.
  • Capillary electrophoresis systems which are commercially available, may be used to analyze the size or confirm the nucleotide sequence of sequencing or PCR products.
  • capillary sequencing may employ flowable polymers for electrophoretic separation, four different fluorescent dyes (one for each nucleotide) that are laser activated, and detection of the emitted wavelengths by a charge coupled device camera.
  • Output/light intensity may be converted to electrical signal using appropriate software (e.g.,
  • Capillary electrophoresis is useful for the sequencing of small pieces of DNA that might be present in limited amounts in a particular sample.
  • the present disclosure also provides recombinant vectors (e.g., an expression vectors) that include an isolated polynucleotide disclosed herein (e.g., a polynucleotide that encodes a polypeptide disclosed herein), host cells into which are introduced the recombinant vectors (i.e., such that the host cells contain the polynucleotide and/or a vector comprising the polynucleotide), and the production of recombinant binding agent polypeptides (e.g., antibodies) or fragments thereof by recombinant techniques.
  • recombinant vectors e.g., an expression vectors
  • an isolated polynucleotide disclosed herein e.g., a polynucleotide that encodes a polypeptide disclosed herein
  • host cells i.e., such that the host cells contain the polynucleotide and/or a vector comprising the polynucleotide
  • a "vector” is any construct capable of delivering one or more polynucleotide(s) of interest to a host cell when the vector is introduced to the host cell.
  • An "expression vector” is capable of delivering and expressing the one or more
  • polynucleotide(s) of interest as encoded polypeptide in a host cell introduced with the expression vector.
  • the polynucleotide of interest is positioned for expression in the vector by being operably linked with regulatory elements such as a promoter, enhancer, poly-A tail, etc., either within the vector or in the genome of the host cell at or near or flanking the integration site of the polynucleotide of interest such that the polynucleotide of interest will be translated in the host cell introduced with the expression vector.
  • vectors are inserted into the host cell by any means including, without limitation, electroporation, fusion with a vector-containing liposomes, chemical transfection (e.g., DEAE-dextran), transformation, transvection, and infection and/or transduction (e.g., with recombinant virus).
  • vectors include viral vectors (which can be used to generate recombinant virus), naked DNA or R A, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.
  • a polynucleotide disclosed herein may be introduced using a viral expression system (e.g., vaccinia or other pox virus, retrovirus, or adenovirus), which may involve the use of a non-pathogenic (defective), replication competent virus, or may use a replication defective virus. In the latter case, viral propagation generally will occur only in complementing virus packaging cells. Suitable systems are disclosed, for example, in Fisher-Hoch et al, 1989, Proc. Natl. Acad. Sci. USA 86:317-321; Flexner et al, 1989, Ann. N.Y. Acad Sci.
  • a viral expression system e.g., vaccinia or other pox virus, retrovirus, or adenovirus
  • the polynucleotides may be joined to a vector containing a selectable marker for propagation in a host.
  • a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid.
  • the vector may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.
  • the methods disclosed herein may be practiced with vectors comprising cis- acting control regions to the polynucleotide of interest. Appropriate trans-acting factors may be supplied by the host, supplied by a complementing vector or supplied by the vector itself upon introduction into the host.
  • the vectors provide for specific expression, which may be inducible and/or cell type-specific (e.g., those inducible by environmental factors that are easy to manipulate, such as temperature and nutrient additives).
  • DNA insert comprising an antibody-encoding or polypeptide- encoding polynucleotide disclosed herein may be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few. Other suitable promoters are known to the skilled artisan.
  • the expression constructs can further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation.
  • the coding portion of the mature transcripts expressed by the constructs may include a translation initiating at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.
  • the expression vectors may include at least one selectable marker.
  • markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture and tetracycline or ampicillin resistance genes for culturing in E. coli and other bacteria.
  • bacterial cells such as E. coli, Streptomyces and Salmonella typhimurium cells
  • fungal cells such as yeast cells
  • insect cells such as Drosophila S2 and Spodoptera Sf9 cells
  • animal cells such as CHO, COS, Bowes melanoma, and HK 293 cells
  • plant cells Appropriate culture mediums and conditions for the above-described host cells are known in the art.
  • Non-limiting vectors for use in bacteria include pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia.
  • Non-limiting eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Other suitable vectors will be readily apparent to the skilled artisan.
  • Non-limiting bacterial promoters suitable for use include the E. coli lacl and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR and PL promoters and the trp promoter.
  • Suitable eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the promoters of retroviral LTRs, such as those of the Rous sarcoma virus (RSV), and metallothionein promoters, such as the mouse metallothionein-I promoter.
  • yeast Saccharomyces cerevisiae a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH may be used.
  • constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH.
  • Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY (1986).
  • Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act to increase transcriptional activity of a promoter in a given host cell-type.
  • enhancers include the SV40 enhancer, which is located on the late side of the replication origin at basepairs 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
  • secretion signals may be incorporated into the expressed polypeptide.
  • the signals may be endogenous to the polypeptide or they may be heterologous signals.
  • the polypeptide may be expressed in a modified form, such as a fusion protein (e.g., a GST-fusion), and may include not only secretion signals, but also additional heterologous functional regions. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, peptide moieties may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide. The addition of peptide moieties to polypeptides to engender secretion or excretion, to improve stability and to facilitate purification, among others, are familiar and routine techniques in the art.
  • a binding agent or antibody of the disclosure may comprise a heterologous region from an immunoglobulin that is useful to solubilize proteins.
  • an immunoglobulin that is useful to solubilize proteins.
  • US 7,253,264 discloses fusion proteins comprising various portions of constant region of immunoglobulin molecules together with another human protein or part thereof.
  • the Fc part in a fusion protein is thoroughly advantageous for use in therapy and diagnosis and thus results, for example, in improved pharmacokinetic properties.
  • binding agents and antibodies can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose
  • Polypeptides of the present disclosure include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells. Depending upon the host employed in a recombinant production procedure, the polypeptides of the present disclosure may be glycosylated or may be non-glycosylated. In addition, polypeptides of the disclosure may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.
  • the disclosure provides a method for producing a recombinant binding agent or antibody by culturing a recombinant host cell (as described above) under conditions suitable for the expression of the fusion polypeptide and recovering the polypeptide.
  • Culture conditions suitable for the growth of host cells and the expression of recombinant polypeptides from such cells are well known to those of skill in the art. See, e.g., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel FM et al, eds., Volume 2, Chapter 16, Wiley Interscience.
  • the disclosure also provides immortalized cell lines that produce an antibody disclosed herein.
  • hybridoma clones constructed as described above, that produce monoclonal antibodies to the target molecule (e.g., HBsAg) disclosed herein are also provided.
  • the disclosure includes recombinant cells producing an antibody as disclosed herein, which cells may be constructed by well known techniques; for example the antigen combining site of the monoclonal antibody can be cloned by PCR and single-chain antibodies produced as phage-displayed recombinant antibodies or soluble antibodies in E. coli (see, e.g., ANTIBODY ENGINEERING PROTOCOLS, 1995, Humana Press, Sudhir Paul editor.).
  • the disclosure also provides binding agents, particularly antibodies that specifically bind to an epitope on a target molecule.
  • the disclosure provides epitopes useful for identifying the binding agents that specifically bind to a target molecule comprising the epitope.
  • phage display is an in vitro selection technique in which a peptide is genetically fused to a coat protein of a bacteriophage resulting in display of a fused protein on the exterior of the virion. Biopanning of these virions by incubating the pool of phage displayed variants with a specific antibody of interest, which has been immobilized on a plate. The unbound phage is then washed away and the specifically bound phage is then eluted. The eluted phage is then amplified in E. coli and the process is repeated, resulting in enrichment of the phage pool in favor of the tightest binding sequences.
  • An advantage of this technology is that it allows for the screening of greater than 10 9 sequences in an unbiased way. Phage display is especially useful if the immunogen is unknown or a large protein fragment.
  • phage display includes cross contamination between phage particles.
  • Cross contamination between phage particles may enrich for sequences that do not specifically bind the antibody.
  • sequences that are not found in nature will be present in the phage displayed peptide library. These sequences may not resemble the immunizing peptide at all and may bind tightly to the antibody of interest. Retrieving sequences that do not resemble the immunizing peptide can be very confounding and it is difficult to decipher whether these peptides are contamination or unnatural peptides with high binding affinity to the antibody of interest.
  • binding agents of the present disclosure may be employed in various methods.
  • the binding agents of the disclosure may be used in any known assay method, such competitive binding assays, direct and indirect sandwich assays, and
  • the binding agents may be detectably labeled (e.g., with a fluorophore such as FITC or phycoerythrin or with an enzyme substrate, such as a substrate for horse radish peroxidase) for easy detection.
  • a fluorophore such as FITC or phycoerythrin
  • an enzyme substrate such as a substrate for horse radish peroxidase
  • the binding agents of the disclosure may be used for in vivo diagnostic assays, such as in vivo imaging.
  • the antibody is
  • radionucleotide such as H, In, C, P, or I
  • a binding agent such as an antibody
  • binding agents disclosed herein need not be labeled, and the presence thereof can be detected using a labeled antibody, which binds to the binding agent.
  • the methods of detection and diagnosis may be performed on any biological sample, e.g., a sample from an individual infected with hepatitis B virus or suspected of being infected with hepatitis B virus.
  • the biological sample can be a bodily fluid (e.g., urine, saliva, blood, tears, semen, or breast milk) or a sample that includes cells (e.g., hepatocytes).
  • the antibody may also be used as staining reagent in pathology, following techniques well known in the art.
  • the disclosure provides methods for detecting hepatitis B virus.
  • the methods include contacting a sample suspected of containing hepatitis B virus with a binding agent disclosed herein and detecting specific binding of the binding agent to the sample, wherein presence of specific binding of the binding agent to the sample identifies the sample as containing hepatitis B virus.
  • Such detection of specific binding by the binding agent to the sample may be made by any known method including, without limitation, western blotting analysis, immunohistochemistry (IHC) analysis,
  • IF immunofluorescence
  • an "individual,” also referred to herein as a "subject,” or “patient” is a vertebrate animal, such a mammal (e.g., a human). Mammals include, without limitation, to, farm animals (such as cows, pigs, and chicken), domestic animals (such as cats, parrots, turtles, lizards, dogs, and horses), primates (such as chimpanzees and gorillas), and rodents (such as mice and rats).
  • the patient may or may not be afflicted with a condition (e.g., a hepatitis B infection) and/or may or may not presently show symptoms.
  • a condition e.g., a hepatitis B infection
  • the subject is infected with hepatitis B virus. In some embodiments, the subject is at risk for hepatitis B virus infection. In some embodiments, the subject is undergoing or has undergone additional treatment (e.g., treatment with an intravenous immunoglobulin preparation, a different anti-hepatitis B virus antibody, an antiviral compound, an immune modulator (e.g., an interferon) or a hepatitis B vaccine).
  • additional treatment e.g., treatment with an intravenous immunoglobulin preparation, a different anti-hepatitis B virus antibody, an antiviral compound, an immune modulator (e.g., an interferon) or a hepatitis B vaccine.
  • biological sample is used in its broadest sense, and means any biological sample suspected of containing a molecule of interest, and may comprise a cell, an extract from cells, blood, urine, marrow, or a tissue, and the like.
  • the biological sample can be a bodily fluid (e.g., urine, saliva, blood, tears, semen, or breast milk) or a sample that includes cells (e.g., peripheral blood leukocytes).
  • Biological samples useful in the practice of the methods of the disclosure may be obtained from any mammal in which hepatitis B infection might be present.
  • Cellular extracts of the foregoing biological samples may be prepared, either crude or partially (or entirely) purified, in accordance with standard techniques, and used in the methods of the disclosure.
  • biological samples comprising whole cells may be utilized in assay formats such as immunohistochemistry (IHC), flow cytometry (FC), and immunofluorescence (IF).
  • IHC immunohistochemistry
  • FC flow cytometry
  • IF immunofluorescence
  • the neutralization activity of binding agents can be characterized using the cell and/or animal models available, e.g., as described in Tajiri et al., 2010, Antiviral Res. 87:40- 49.
  • an "effective amount” is an amount or dosage sufficient to effect beneficial or desired results including halting, slowing, halting, retarding, or inhibiting progression of an hepatitis B infection in a patient or preventing or inhibiting development of hepatitis B infection in a patient.
  • An effective amount will vary depending upon, e.g., an age and a body weight of a subject to which the a binding agent, binding agent-encoding polynucleotide, vector comprising the polynucleotide and/or compositions thereof is to be administered, a severity of symptoms and a route of administration, and thus administration is determined on an individual basis.
  • the daily adult dosage for oral administration is about 0.1 to 1000 mg, given as a single dose or in divided doses.
  • the compositions can be administered in the range of 0.01 ⁇ g/kg/min to 1.0 ⁇ g/kg/min, desirably 0.025 ⁇ g/kg/min to 0.1 ⁇ g/kg/min.
  • an effective amount can be administered in one or more administrations.
  • an effective amount of a binding agent is an amount sufficient to ameliorate, stop, stabilize, reverse, inhibit, slow and/or delay progression of a hepatitis B infection or a hepatitis B-related disease in a patient or is an amount sufficient to ameliorate, stop, stabilize, reverse, slow and/or delay hepatitis B infection of a cell (e.g., a biospsied cell or
  • an effective amount of a binding agent may vary, depending on, inter alia, patient history as well as other factors such as the type (and/or dosage) of binding agent used.
  • Effective amounts and schedules for administering the binding agents, binding agent- encoding polynucleotides, and/or compositions disclosed herein may be determined empirically, and making such determinations is within the skill in the art. Those skilled in the art will understand that the dosage that must be administered will vary depending on, for example, the mammal that will receive the binding agents, binding agent-encoding polynucleotides, and/or compositions disclosed herein, the route of administration, the particular type of binding agents, binding agent-encoding polynucleotides, and/or compositions disclosed herein used and other drugs being administered to the mammal.
  • a typical daily dosage of an effective amount of a binding agent used alone might range from about 1 ⁇ g/kg to up to 100 mg/kg of body weight or more per day, depending on the factors mentioned above.
  • any of the following doses may be used: a dose of at least about 50 mg/kg body weight; at least about 10 mg/kg body weight; at least about 3 mg/kg body weight; at least about 1 mg/kg body weight; at least about 750 ⁇ g/kg body weight; at least about 500 ⁇ g/kg body weight; at least about 250 ⁇ g/kg body weight; at least about 100 ⁇ g /kg body weight; at least about 50 ⁇ g/kg body weight; at least about 10 ⁇ g /kg body weight; at least about 1 ⁇ g/kg body weight, or more, is administered.
  • a dose of a binding agent (e.g., antibody) provided herein is between about 0.01 mg/kg and about 50 mg/kg, between about 0.05 mg/kg and about 40 mg/kg, between about 0.1 mg and about 30 mg/kg, between about 0.1 mg and about 20 mg/kg, between about 0.5 mg and about 15 mg, or between about 1 mg and 10 mg.
  • the dose is between about 1 mg and 5 mg. In some alternative embodiments, the dose is between about 5 mg and 10 mg.
  • the methods described herein further comprise the step of treating the subject with an additional form of therapy.
  • the additional form of therapy is an additional anti-viral composition.
  • the methods described herein further comprise the step of treating the subject with an intravenous immunoglobulin preparation, a different anti-hepatitis B protein antibody (e.g., an antibody against hepatitis B virus HBc, HBs, HBx, or HBp protein), an antiviral compound, and/or an immune modulator (e.g., an interferon).
  • the antiviral compound is lamivudine, adefovir, enofovir, elbivudine, or entecavir.
  • the interferon is interferon alpha-2a or PEGylated interferon alpha-2a.
  • the methods described herein can be accomplished by a single direct injection or infusion at a single time point or multiple time points to a single or multiple sites. Administration can also be nearly simultaneous to multiple sites.
  • Frequency of administration may be determined and adjusted over the course of therapy, and is base on accomplishing desired results.
  • sustained continuous release formulations of binding agents (including antibodies), polynucleotides, and pharmaceutical compositions disclosed herein may be appropriate.
  • Various formulations and devices for achieving sustained release are known in the art.
  • binding agent e.g., an antibody
  • binding agent-encoding polynucleotide e.g., an antibody
  • vector containing such a polynucleotide may be administered to the patient in a carrier;
  • composition e.g., a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier and (a) a binding agent disclosed herein, (b) a binding agent-encoding polynucleotide disclosed herein and/or (c) a vector comprising a binding agent-encoding polynucleotide.
  • pharmaceutically acceptable carrier or “pharmaceutically acceptable excipient” includes any material which, when combined with an active ingredient, allows the ingredient to retain biological activity and is non-reactive with the subject's immune system and non-toxic to the subject when delivered.
  • examples include, but are not limited to, any of the standard pharmaceutical carriers such as a phosphate buffered saline solution, water, emulsions such as oil/water emulsion, and various types of wetting agents.
  • diluents for aerosol or parenteral administration are phosphate buffered saline, normal (0.9%) saline, Ringer's solution and dextrose solution.
  • the pH of the solution may be from about 5 to about 8, or from about 7 to about 7.5.
  • Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of antibody being administered. Compositions comprising such carriers are formulated by well-known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990; and Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing, 2000).
  • compositions of the disclosure i.e., containing a binding agent or a binding agent-encoding polynucleotide
  • a binding agent or a binding agent-encoding polynucleotide are well known in the art, such as those described by
  • compositions that include the binding agent and/or binding agent-encoding polynucleotide disclosed herein may be formulated for any appropriate manner of
  • compositions may also be administered by isolated perfusion techniques, such as isolated tissue perfusion, to exert local therapeutic effects.
  • parenteral administration such as subcutaneous injection
  • the carrier preferably comprises water, saline, alcohol, a fat, a wax or a buffer.
  • any of the above carriers or a solid carrier such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, and magnesium carbonate, may be employed.
  • the formulation of the compositions is resistant to decomposition in the digestive tract, for example, as microcapsules encapsulating the binding agent (or binding agent-encoding polynucleotide or vector comprising such a polynucleotide) within liposomes.
  • Biodegradable microspheres e.g., polylactate polyglycolate
  • Suitable biodegradable microspheres are disclosed, for example, in U.S. Pat. Nos. 4,897,268 and 5,075,109.
  • compositions may also comprise buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose or dextran), mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide) and/or preservatives.
  • buffers e.g., neutral buffered saline or phosphate buffered saline
  • carbohydrates e.g., glucose, mannose, sucrose or dextran
  • mannitol e.g., proteins, polypeptides or amino acids
  • proteins e.glycine
  • antioxidants e.g., glycine
  • chelating agents such as EDTA or glutathione
  • adjuvants e.g., aluminum hydroxide
  • preservatives e.g., aluminum
  • polynucleotide also may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (for example,
  • microcapsules respectively
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
  • the term "salvage receptor binding epitope” refers to an epitope of the Fc region of an IgG molecule (e.g., IgGl, IgG2, IgG3, and IgG4) that is responsible for increasing the in vivo serum half-life of the IgG molecule.
  • the binding agents (and/or binding agent-encoding polynucleotides) disclosed herein may also be formulated as liposomes.
  • Liposomes containing the binding agents (and/or binding agent-encoding polynucleotides) are prepared by methods known in the art, such as described in Epstein et al, 1985, Proc. Natl. Acad. Sci. USA 82:3688; Hwang et al, 1980, Proc. Natl Acad. Sci. USA 77:4030; and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556. Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid
  • composition comprising phosphatidylcholine, cholesterol and PEG-derivatized
  • Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter.
  • the binding agent is an antibody
  • antibodies including antigen binding domain fragments such as Fab' fragments
  • Administration of expression vectors includes local or systemic administration, including injection, oral administration, particle gun or catheterized administration, and topical administration.
  • One skilled in the art is familiar with administration of expression vectors to obtain expression of an exogenous protein in vivo. See, e.g., U.S. Pat. Nos. 6,436,908; 6,413,942; and 6,376,471.
  • Targeted delivery of therapeutic compositions comprising a polynucleotide encoding a polypeptide or antibody disclosed herein can also be used.
  • Receptor-mediated DNA delivery techniques are described in, for example, Findeis et al, Trends Biotechnol. (1993) 11 :202; Chiou et al., Gene Therapeutics: Methods And Applications Of Direct Gene Transfer (J. A. Wolff, ed.) (1994); Wu et al, J. Biol. Chem. (1988) 263:621; Wu et al, J. Biol. Chem. (1994) 269:542; Zenke et al, Proc. Natl. Acad. Sci.
  • compositions containing a polynucleotide are administered in a range of about 100 ng to about 200 mg of DNA for local administration in a gene therapy protocol. Concentration ranges of about 500 ng to about 50 mg, about 1 ⁇ g to about 2 mg, about 5 ⁇ g to about 500 ⁇ g, and about 20 ⁇ g to about 100 ⁇ g of DNA can also be used during a gene therapy protocol.
  • the therapeutic polynucleotides and polypeptides of the present disclosure can be delivered using gene delivery vehicles.
  • the gene delivery vehicle can be of viral or non- viral origin (see generally, Jolly, Cancer Gene Therapy (1994) 1 :51; Kimura, Human Gene Therapy (1994) 5:845; Connelly, Human Gene Therapy (1995) 1 : 185; and Kaplitt, Nature Genetics (1994) 6: 148). Expression of such coding sequences can be induced using endogenous mammalian or heterologous promoters. Expression of the coding sequence can be either constitutive or regulated.
  • Viral-based vectors for delivery of a desired polynucleotide and expression in a desired cell are well known in the art.
  • Exemplary viral-based vehicles include, but are not limited to, recombinant retroviruses (see, e.g., PCT Publication Nos. WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; WO 93/11230; WO 93/10218; WO 91/02805; U.S. Pat. Nos. 5,219,740; 4,777,127; GB Patent No.
  • alphavirus- based vectors e.g., Sindbis virus vectors, Semliki forest virus (ATCC VR-67; ATCC VR- 1247), Ross River virus (ATCC VR-373; ATCC VR-1246) and Venezuelan equine encephalitis virus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532)
  • AAV adeno-associated virus
  • Non- viral delivery vehicles and methods can also be employed, including, but not limited to, polycationic condensed DNA linked or unlinked to killed adenovirus alone (see, e.g., Curiel, Hum. Gene Ther. (1992) 3: 147); ligand-linked DNA (see, e.g., Wu, J. Biol. Chem. (1989) 264: 16985); eukaryotic cell delivery vehicles cells (see, e.g., U.S. Pat. No. 5,814,482; PCT Publication Nos. WO 95/07994; WO 96/17072; WO 95/30763; and WO 97/42338) and nucleic charge neutralization or fusion with cell membranes. Naked DNA can also be employed.
  • Exemplary naked DNA introduction methods are described in PCT Publication No. WO 90/11092 and U.S. Pat. No. 5,580,859.
  • Liposomes that can act as gene delivery vehicles are described in U.S. Pat. No. 5,422,120; PCT Publication Nos. WO 95/13796; WO 94/23697; WO 91/14445; and EP 0 524 968. Additional approaches are described in Philip, Mol. Cell Biol. (1994) 14:2411, and in Woffendin, Proc. Natl. Acad. Sci. (1994) 91 : 1581.
  • compositions described herein may be administered as part of a sustained release formulation (i.e., a formulation such as a capsule or sponge that effects a slow release of compound following administration).
  • a sustained release formulation i.e., a formulation such as a capsule or sponge that effects a slow release of compound following administration.
  • Such formulations may generally be prepared using well known technology and administered by, for example, oral, rectal or subcutaneous implantation, or by implantation at the desired target site.
  • Sustained-release formulations may contain a polypeptide, polynucleotide or antibody dispersed in a carrier matrix and/or contained within a reservoir surrounded by a rate controlling membrane.
  • Carriers for use within such formulations are biocompatible, and may also be biodegradable; preferably the formulation provides a relatively constant level of active component release.
  • the amount of active compound contained within a sustained release formulation depends upon the site of implantation, the rate and expected duration of release and the nature of the condition to be treated.
  • compositions of the disclosure include bulk drug compositions useful in the manufacture of non-pharmaceutical compositions (e.g., impure or non-sterile compositions) and pharmaceutical compositions (i.e., compositions that are suitable for administration to a subject or patient) that can be used in the preparation of unit dosage forms.
  • the compositions and methods disclosed herein can be used for treatment of patients having or at risk for hepatitis B infection, e.g., acute or chronic hepatitis B infection.
  • the compositions and methods disclosed herein can be used to for hepatitis B prophylaxis in newborns of hepatitis B-positive women.
  • compositions and methods disclosed herein can be used for treatment of hepatitis
  • B-related disease e.g., hepatitis (e.g., chronic hepatitis), cirrhosis, hepatocellular carcinoma, serum-sickness-like syndrome, acute necrotizing vasculitis (polyarteritis nodosa), membranous glomerulonephritis, or papular acrodermatitis of childhood (Gianotti-Crosti syndrome).
  • hepatitis e.g., chronic hepatitis
  • cirrhosis e.g., cirrhosis
  • hepatocellular carcinoma e.g., hepatocellular carcinoma
  • serum-sickness-like syndrome e.g., acute necrotizing vasculitis (polyarteritis nodosa), membranous glomerulonephritis, or papular acrodermatitis of childhood (Gianotti-Crosti syndrome).
  • Anti-HBV antibodies were isolated from the serum of a volunteer collected seven days after a second dose of immunization with ENGERIX-B hepatitis B vaccine.
  • This vaccine includes a recombinant form of the HBsAg protein.
  • the vaccine-specific response of the donor was confirmed by antigen-specific ELISA using total IgG purified with
  • Binding kinetics were assessed on a CM5 chip coated with HBsAg (SEQ ID NO: 41) as the ligand at low density ( ⁇ 50 RU) to minimize avidity effects of the antibody.
  • Kinetics analysis was performed with the Biacore kinetics analysis software where the fitted curves for binding of the ligand to analyte were modeled as a 1 : 1 interaction.
  • the assayed antibodies had affinities with K D values ranging from 517 pM to 613 nM (Table 1). Table 1. Characteristics of anti-HBV antibodies
  • the epitope of antibody C14 was mapped to within 10 amino acids (TKPTFGNCTC; SEQ ID NO: 42) with and the epitope of antibody C 19 was mapped to within 20 amino acids (PCKTCTTPAQGNSKFPSCCC; SEQ ID NO: 43) in the neutralization-sensitive, antigenic loop of HBsAg.
  • This example demonstrates the isolation and production of high affinity human antibodies against HBV HBsAg from immunized subjects.
  • the full-length antibody chain (HC or LC), variable region nucleic acid sequence (Nuc), variable region (VH or VL), framework regions (FR1-FR4), and complementarity determining regions (CDR1-CDR3) are provided.
  • HC or LC variable region nucleic acid sequence
  • Nuc variable region
  • VH or VL variable region
  • FR1-FR4 framework regions
  • CDR1-CDR3 complementarity determining regions
  • YIGTSRGTIFYADSVEGRFS I SRDNAKNSVYLQMSSLRAEDTATYYCAG PGCWGGSCYPFHYWGQGALVTVSSASTKGPSVRPLAPSSKSTSGGTAAL GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSS SLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMI SRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTI SK AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSPGK
  • VL 279 E IVLTQSPPTLSLSPGERATLSCRASQS IGSYLAWYQQKPGQAPRLLIY
  • VL 315 E IVLTQSPGTLSLSPGESATLSCRASQSVGRTYLAWYQQRPGRAPRLLI
  • VL 360 SYVLTQPTSVSVAPGQTARITCGGSDIGSKSVNWYQQKPGQAPWWYD
  • VL 369 SYVLTQPPSTSVAPGQTAKI SCGGNNIGSESVNWYQQKSGQAPVLWHD

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Abstract

La présente invention concerne des anticorps anti-virus hépatite B et des méthodes de traitement, de prophylaxie, de détection et de diagnostic associées.
PCT/US2013/038802 2012-04-30 2013-04-30 Anticorps anti-virus hépatite b et applications associées WO2013165972A2 (fr)

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WO2017015622A3 (fr) * 2015-07-22 2017-03-02 Scholar Rock, Inc Protéines de liaison à gdf11 et leurs utilisations
WO2018039514A1 (fr) * 2016-08-25 2018-03-01 Vanderbilt University Anticorps pour la neutralisaton du virus ebola.
US10167333B2 (en) * 2014-01-16 2019-01-01 Mario Umberto Francesco Mondelli Neutralizing human monoclonal antibodies against hepatitis B virus surface antigen
US10751413B2 (en) 2015-09-15 2020-08-25 Scholar Rock, Inc. Anti-pro/latent-Myostatin antibodies and uses thereof
US10882904B2 (en) 2016-01-08 2021-01-05 Scholar Rock, Inc. Methods for inhibiting myostatin activation by administering anti-pro/latent myostatin antibodies
US10946036B2 (en) 2016-06-13 2021-03-16 Scholar Rock, Inc. Use of myostatin inhibitors and combination therapies
US11135291B2 (en) 2014-11-06 2021-10-05 Scholar Rock, Inc. Methods for making and using anti-myostatin antibodies
US11155611B2 (en) 2017-01-06 2021-10-26 Scholar Rock, Inc. Compositions and methods for making and using anti-myostatin antibodies
US11542332B2 (en) 2016-03-26 2023-01-03 Bioatla, Inc. Anti-CTLA4 antibodies, antibody fragments, their immunoconjugates and uses thereof

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WO2004050683A2 (fr) * 2002-12-02 2004-06-17 Abgenix, Inc. Anticorps agissant sur le facteur de necrose des tumeurs (tnf) et leurs utilisation
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WO1994011495A1 (fr) * 1992-11-06 1994-05-26 Sandoz, Ltd. Production d'anticorps monoclonaux humains actifs contre l'antigene de surface de l'hepatite b
WO2004050683A2 (fr) * 2002-12-02 2004-06-17 Abgenix, Inc. Anticorps agissant sur le facteur de necrose des tumeurs (tnf) et leurs utilisation
US20080152658A1 (en) * 2005-04-18 2008-06-26 Shlomo Dagan Stabilized Anti-Hepatitis B (Hbv) Antibody Formulations
WO2012017003A1 (fr) * 2010-08-05 2012-02-09 F. Hoffmann-La Roche Ag Protéine de fusion anticorps anti-cmh cytokine antivirale

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10167333B2 (en) * 2014-01-16 2019-01-01 Mario Umberto Francesco Mondelli Neutralizing human monoclonal antibodies against hepatitis B virus surface antigen
US11135291B2 (en) 2014-11-06 2021-10-05 Scholar Rock, Inc. Methods for making and using anti-myostatin antibodies
US11925683B2 (en) 2014-11-06 2024-03-12 Scholar Rock, Inc. Compositions for making and using anti-Myostatin antibodies
WO2017015622A3 (fr) * 2015-07-22 2017-03-02 Scholar Rock, Inc Protéines de liaison à gdf11 et leurs utilisations
US10751413B2 (en) 2015-09-15 2020-08-25 Scholar Rock, Inc. Anti-pro/latent-Myostatin antibodies and uses thereof
US11439704B2 (en) 2015-09-15 2022-09-13 Scholar Rock, Inc. Methods for using anti-myostatin antibodies
US10882904B2 (en) 2016-01-08 2021-01-05 Scholar Rock, Inc. Methods for inhibiting myostatin activation by administering anti-pro/latent myostatin antibodies
US12006359B2 (en) 2016-01-08 2024-06-11 Scholar Rock, Inc. Methods for altering body composition by administering anti-pro/latent myostatin antibodies
US11542332B2 (en) 2016-03-26 2023-01-03 Bioatla, Inc. Anti-CTLA4 antibodies, antibody fragments, their immunoconjugates and uses thereof
US10946036B2 (en) 2016-06-13 2021-03-16 Scholar Rock, Inc. Use of myostatin inhibitors and combination therapies
WO2018039514A1 (fr) * 2016-08-25 2018-03-01 Vanderbilt University Anticorps pour la neutralisaton du virus ebola.
US11155611B2 (en) 2017-01-06 2021-10-26 Scholar Rock, Inc. Compositions and methods for making and using anti-myostatin antibodies

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